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Untitled-1 1, , 09/03/2019 19:35
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Provide New Learning Pathways, to Understand the Why Behind, the Science, C ASE ST U DY, HAND WASHING AND ASEPTIC TECHNIQUE: A CASE STUDY, You are presented with an eosin–methylene blue, (EMB) agar plate that has bacterial colonies with, a slight greenish, metallic sheen. Your laboratory, manager explains the background for the culture, you are observing on the plate: An unknown contaminate was found in a meat processing machine,, and the in-house pathogen control office performed, a swab and a streak on an EMB plate. After incubation and observation of the weak reaction, the, manufacturers concluded that the contaminate was, not E. coli and that no further tests were required., Upper management decided that to protect the, company from potential lawsuits, they would hire, the laboratory you work for to ensure that their, laboratory technicians concluded correctly., , Due to cost and time restrictions, your lab, is limited regarding how many assays can be, performed. Using a series of biochemical tests to, confirm or refute the analysis of the processing, plant, you will need to determine whether the, bacteria is an enteric and then whether it is an, E. coli isolate., , Questions to Consider:, 1. Does the lack of a strong reaction on the EMB, plate refute the determination that the isolated, bacteria are an E. coli culture?, 2. Which series of assays would best be used to, prove/disprove the E. coli determination?, , NEW! Clinical Case Studies now within each section of the lab, manual bring career relevance to the lab experiments. These open-ended, cases can be used to fuel class discussion and group work about the topics, covered in lab., , Susceptibility Testing published by the American, Society for Microbiology (ASM)., , NEW! Further, Reading Sections, help students know, where to look in their, textbook if they need, more background, information to, understand the, science behind the, experiment., , F U RT H E R RE A D I N G, Refer to the section on antimicrobial compounds, in your textbook for further information on the, compounds that have an effect on bacterial cells., In your textbook’s index, search under “Chemotherapy,” “Antibiotics,” and “Analog.”, , ❏, ❏, ❏, ❏, ❏, ❏, ❏, , Penicillin G, 10 mg, Streptomycin, 10 mg, Tetracycline, 30 mg, Chloramphenicol, 30 mg, Gentamicin, 10 mg, Vancomycin, 30 mg, Sulfanilamide, 300 mg, , Equipment, C L I N I C A L A P P L I C AT I O N, Selection of Effective Antibiotics, Upon isolation of an infectious agent, a chemotherapeutic agent is selected and its effectiveness, must be determined. This can be done using the, Kirby-Bauer Antibiotic Sensitivity Test. This is the, essential tool used in clinical laboratories to select, the best agent with which to treat patients with bacterial infections., , A01_CAPP8996_12_SE_VWT.indd 1, , Antimicrobial-Sensitivity Discs, , ❏, ❏, ❏, ❏, ❏, ❏, , Sensi-Disc™ dispensers or forceps, Microincinerator or Bunsen burner, Sterile cotton swabs, Glassware marking pencil, 70% ethyl alcohol, Millimeter ruler, , Procedure Lab One, 1., , Place agar plates right-side-up in an inc, heated to 37°C for 10 to 20 minutes wit, 30/11/18 10:12 PM, covers adjusted so, that the plates are s
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Connect Lecture and Lab with, Mastering Microbiology, MicroLab Practical, Activities assess, students’ observation, skills and give them, extra practice to, analyze important lab, tests, procedures, and, results., , Prepare for lab with pre-lab quizzes for each of the 72 experiments in, Microbiology: A Laboratory Manual Twelfth Edition, and then follow up to, measure comprehension with post-lab quizzes in Mastering Microbiology™., , A01_CAPP8996_12_SE_VWT.indd 2, , 30/11/18 10:12 PM
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And Come to Class Better, Prepared for Lab, Videos and Coaching, Activities help instructors, , and students get the most out of, lab time. Students can practice, their lab skills virtually reviewing, proper lab techniques with realworld applications. Live action, video combined with molecular, animation with assessment and, feedback coach students how to, interpret and analyze different, lab results., , Lab Technique Videos give students an opportunity to see techniques performed correctly, and quiz themselves on lab procedures both before and after lab time, improving confidence and, proficiency. Assign as pre-lab quizzes in Mastering Microbiology and include coaching and feedback, on a wide range of lab techniques., , A01_CAPP8996_12_SE_VWT.indd 3, , 30/11/18 10:12 PM
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Additional Instructor Support to, Customize Your Course Your Way, Easy-to-adapt Lab Reports include, , blank spaces for individual course customization., Instructors can select their preferred organism., , EXPER IMENT, , 11, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, E. coli, , B. cereus, , S. aureus, , Mixture, , Draw a, representative field., , Cell morphology:, Shape, Arrangement, Cell color, Gram reaction, , Review Questions, 1. Why must you use heat or a surface-active agent when applying the primary stain during acid-fast, staining?, , To create the perfect lab manual, visit, www.pearsoncustomlibrary.com., , 2. Why do you use acid-alcohol rather than ethyl alcohol as a decolorizing agent?, , Experiment 11: Lab Report, , M11_CAPP8996_12_SE_C11.indd 83, , 83, , 09/11/2018 19:24, , Pearson Collections, www.pearsoncollections., com Your course materials should match your, course, not the other way around. We offer a, comprehensive catalog linked to easy-to-use, curation tools. Everything is set up so you can, easily design your custom content and then share, it with your students., , Instructor’s Guide for Microbiology: A, Laboratory Manual by James G. Cappuccino,, , Chad T. Welsh (© 2019 0-13-520429-1 / 978-0-13520429-0) is a valuable teaching aid for instructors., Tools include: recommended readings, detailed lists, of required materials, tables for calculating the, amount of media and equipment needed for your, class, procedural points to emphasize, helpful tips, for preparing and implementing each experiment,, answers to review questions in the lab manual, and, information on lab safety protocol., , A01_CAPP8996_12_SE_VWT.indd 4, , 30/11/18 10:12 PM
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Microbiology, A Laboratory Manual, Twelfth Edition, , James G. Cappuccino, Emeritus, SUNY Rockland Community College, , Chad Welsh, Lindenwood University
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Courseware Portfolio Manager:, Jennifer McGill Walker, Director of Portfolio Management:, Serina Beauparlant, Content Producer: Norine Strang, Managing Producer: Nancy Tabor, Courseware Director, Content Development:, Barbara Yien, Courseware Analyst: Coleen Morrison, Courseware Editorial Assistant: Katrina Taylor, Senior Media Producer: Tod Regan, Rich Media Content Producer: Lucinda Bingham, Full-Service Vendor: Pearson CSC, , Art Coordinator: Courtney Coffman, Design Manager: Mark Ong, Interior Designer: Preston Thomas, Cover Designer: Preston Thomas, Rights & Permissions Project Manager:, Grace Annalyn Subito, Rights & Permissions Management: Eric Schrader, Manufacturing Buyer: Stacey Weinberger, Director of Field Marketing: Tim Galligan, Director of Product Marketing: Allison Rona, Field Marketing Manager: Kelli Galli, , Cover Photo Credit: Tek image/science photo library, Copyright © 2020, 2017, 2014 by Pearson Education, Inc. 221 River Street, Hoboken, NJ 07030., Printed in the United States of America. This publication is protected by copyright, and, permission should be obtained from the publisher prior to any prohibited reproduction, storage, in a retrieval system, or transmission in any form or by any means, electronic, mechanical,, photocopying, recording, or otherwise. For information regarding permissions, request forms, and the appropriate contacts within the Pearson Education Global Rights & Permissions, department., Attributions of third party content appear on page 529, which constitutes an extension of this, copyright page., Unless otherwise indicated herein, any third-party trademarks that may appear in this work, are the property of their respective owners and any references to third-party trademarks, logos, or other trade dress are for demonstrative or descriptive purposes only. Such references are, not intended to imply any sponsorship, endorsement, authorization, or promotion of Pearson's, products by the owners of such marks, or any relationship between the owner and Pearson, Education, Inc. or its affiliates, authors, licensees or distributors., Library of Congress Cataloging-in-Publication Data, Names: Cappuccino, James G., author. | Welsh, Chad, author., Title: Microbiology : a laboratory manual / James G. Cappuccino, Chad Welsh., Description: Twelfth edition. | New York : Pearson, 2019. |, Includes bibliographical references and index., Identifiers: LCCN 2018048736| ISBN 9780135188996 (student edition : alk. paper), | ISBN 0135188997 (student edition : alk. paper), | ISBN 0135203996 (instructor's review copy : alk. paper), Subjects: | MESH: Microbiology | Laboratory Manuals, Classification: LCC QR63 | NLM QW 25 | DDC 579--dc23, LC record available at https://urldefense.proofpoint.com/v2/url?u=https-3A__lccn.loc.gov_20180, 48736&d=DwIFAg&c=0YLnzTkWOdJlub_y7qAx8Q&r=0iwwgabCT73eU7_y8BJiDD-U-Xt2puPiRSv, wtn3PwE0&m=xuqtMqIWFQm4BUO9EyRhAQNt8--Jvw4qOW528-2wOW0&s=zaHPcnvprJfdn6IbZ, jIDk18X_HXY9CX-ppSX788_M9s&e=, , (Student edition), ISBN 10: 0-13-518899-7;, ISBN 13: 978-0-13-518899-6, , www.pearson.com, , (Instructor’s Review Copy), ISBN 10: 0-13-520399-6;, ISBN 13: 978-0-13-520399-6
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Contents, , Preface vi, Laboratory Safety ix, Laboratory Protocol xi, , PART 1 Basic Laboratory Techniques, for Isolation, Cultivation, and, Cultural Characterization of, Microorganisms 1, Introduction 1, Experiment 1: Effectiveness of Hand, Washing 7, Experiment 2: Culture Transfer, Techniques 13, Experiment 3: Techniques for, Isolation of Pure Cultures 19, Part A: Isolation of Discrete, , Colonies from a Mixed Culture 19, Part B: Isolation of Pure Cultures, from a Spread-Plate or StreakPlate Preparation 22, Experiment 4: Cultural, Characteristics of Microorganisms 29, , PART 2 Microscopy 35, Introduction 35, Experiment 5: Microscopic, Examination of Stained Cell, Preparations 37, Experiment 6: Microscopic, Examination of Living, Microorganisms Using a HangingDrop Preparation or a Wet Mount 45, , PART 3 Bacterial Staining 51, Introduction 51, Experiment 7: Preparation of, Bacterial Smears 55, Experiment 8: Simple Staining 61, Experiment 9: Negative Staining 67, Experiment 10: Gram Stain 71, Experiment 11: Acid-Fast Stain 79, , Experiment 12: Differential Staining, for Visualization of Bacterial Cell, Structures 85, Part A: Spore Stain, , (Schaeffer-Fulton Method) 85, Part B: Capsule Stain (Anthony, Method) 88, , PART 4 Cultivation of Microorganisms:, Nutritional and Physical, Requirements, and, Enumeration of Microbial, Populations 93, Introduction 93, Experiment 13: Nutritional, Requirements: Media for the Routine, Cultivation of Bacteria 97, Experiment 14: Using Differential,, Selective, and Enriched Media 103, Experiment 15: Physical Factors:, Temperature 113, Experiment 16: Physical Factors: pH, of the Extracellular Environment 119, Experiment 17: Physical, Factors: Atmospheric Oxygen, Requirements 123, Experiment 18: Techniques, for Cultivating Anaerobic, Microorganisms 129, Experiment 19: Serial Dilution—Agar, Plate Procedure to Quantitate Viable, Cells 135, Experiment 20: The Bacterial Growth, Curve 143, , PART 5 Biochemical Activities of, Microorganisms 151, Introduction 151, Experiment 21: Extracellular, Enzymatic Activities of, Microorganisms 155, Experiment 22: Carbohydrate, Fermentation 161, , iii
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Experiment 23: Triple Sugar–Iron, Agar Test 167, Experiment 24: IMViC Test 173, , Part A: Indole Production, Test 174, Part B: Methyl Red Test V, ogesProskauer Test (MR-VP) 175, Part C: Citrate Utilization, Test 177, Experiment 25: Hydrogen Sulfide, Test 185, Experiment 26: Urease Test 189, Experiment 27: Litmus–Milk, Reactions 193, Experiment 28: Nitrate Reduction, Test 199, Experiment 29: Catalase Test 203, Experiment 30: Oxidase Test 207, Experiment 31: Utilization of Amino, Acids 211, , Part A: Decarboxylase Test 211, Part B: Phenylalanine, Deaminase Test 213, Experiment 32: Genus Identification, of Unknown Bacterial Cultures 217, , PART 6 The Protozoa 223, Introduction 223, Experiment 33: Free-Living, Protozoa 225, Experiment 34: Parasitic, Protozoa 231, , PART 7 The Fungi 239, Introduction 239, Experiment 35: Cultivation and, Morphology of Molds 241, , Part A: Slide Culture, Technique 241, Part B: Mold Cultivation on, Solid Surfaces 243, Experiment 36: Isolation of a Soil, Fungal Species 253, Experiment 37: Yeast Morphology,, Cultural Characteristics, and, Reproduction 257, , PART 8 The Viruses 265, Introduction 265, Experiment 38: Cultivation and, Enumeration of Bacteriophages 269, Experiment 39: Isolation of, Coliphages from Raw Sewage 275, , iv, , Contents, , Experiment 40: Propagation of, Isolated Bacteriophage Cultures 281, , PART 9 Physical and Chemical Agents, for the Control of Microbial, Growth 285, Introduction 285, Experiment 41: Physical Agents of, Control: Moist Heat 289, Experiment 42: Chemical Agents of, Control: Chemotherapeutic Agents 295, , Part A: The Kirby-Bauer, Antibiotic Sensitivity Test, Procedure 296, Part B: Synergistic Effect of, Drug Combinations 299, Experiment 43: Determination of, Penicillin Activity in the Presence and, Absence of Penicillinase 305, , Part A: MIC Determination, Using a Spectrophotometer 306, Part B: MIC Determination, Using a Plate Reader 307, Experiment 44: Chemical Agents, of Control: Disinfectants and, Antiseptics 311, , Part A: Disc Diffusion Testing, of Disinfectants and, Antiseptics 314, Part B: Modified-Use Dilution, Testing of Disinfectants and, Antiseptics 315, , PART 10 Microbiology of Food 319, Introduction 319, Experiment 45: Microbiological, Analysis of Food Products: Bacterial, Count 321, Experiment 46: Isolation of, Salmonella from Raw Meat 325, Experiment 47: Microbial, Fermentation 329, , Part A: Alcohol, Fermentation 329, Part B: Lactic Acid, Fermentation 331, , PART 11 Microbiology of Water 335, Introduction 335, Experiment 48: Standard Qualitative, Analysis of Water 337, Experiment 49: Quantitative, Analysis of Water: Membrane Filter, Method 345
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PART 12 Microbiology of Soil 351, Introduction 351, Experiment 50: Microbial, Populations in Soil: Enumeration 355, Experiment 51: Isolation of, Antibiotic-Producing Microorganisms, and Determination of Antimicrobial, Spectrum of Isolates 361, , Part A: Isolation of AntibioticProducing Microorganisms 363, Part B: Determination of, Antimicrobial Spectrum of, Isolates 363, Experiment 52: Isolation of, Pseudomonas Species by Means of the, Enrichment Culture Technique 367, , PART 13 Bacterial Genetics 373, Introduction 373, Experiment 53: Enzyme, Induction 375, Experiment 54: Bacterial, Conjugation 381, Experiment 55: Isolation of a, Streptomycin-Resistant Mutant 387, Experiment 56: The Ames Test: A, Bacterial Test System for Chemical, Carcinogenicity 391, Experiment 57: Utilization of, Bacterial Plasmids 397, Experiment 58: Restriction Analysis, and Electrophoretic Separation of, Bacteriophage Lambda DNA 409, , PART 14 Medical Microbiology 419, Introduction 419, Experiment 59: Microbial Flora of the, Mouth: Determination of Susceptibility, to Dental Caries 421, Experiment 60: Normal Microbial, Flora of the Throat and Skin 425, Experiment 61: Identification of, Human Staphylococcal Pathogens 433, Experiment 62: Identification of, Human Streptococcal Pathogens 441, Experiment 63: Identification of, Streptococcus Pneumoniae 449, Experiment 64: Identification, of Enteric Microorganisms Using, Computer-Assisted Multitest, Microsystems 455, , Experiment 65: Isolation and, Presumptive Identification of, Campylobacter 465, Experiment 66: Microbiological, Analysis of Urine Specimens 469, Experiment 67: Microbiological, Analysis of Blood Specimens 475, Experiment 68: Species Identification, of Unknown Bacterial Cultures 481, , PART 15 Immunology 489, Introduction 489, Experiment 69: Precipitin Reaction:, The Ring Test 491, Experiment 70: Agglutination, Reaction: The Febrile Antibody, Test 495, Experiment 71: Enzyme-Linked, Immunosorbent Assay 501, Experiment 72: Sexually Transmitted, Diseases: Rapid Immunodiagnostic, Procedures 505, , Part A: Rapid Plasma Reagin, Test for Syphilis 505, Part B: Genital Herpes:, Isolation and Identification of, Herpes Simplex Virus 507, Part C: Detection of Sexually, Transmitted Chlamydial, Diseases 508, , Appendices, APPENDIX 1: Scientific Notation 513, APPENDIX 2: Methods for the, Preparation of Dilutions 515, APPENDIX 3: Microbiological, Media 517, APPENDIX 4: Biochemical Test, Reagents 523, APPENDIX 5: Staining Reagents 526, APPENDIX 6: Experimental, Microorganisms 527, Credits 529, Index 531, , Contents, , v
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Preface, , Microbiology is a dynamic science. It constantly, evolves as more information is added to the, continuum of knowledge, and as microbiological techniques are rapidly modified and refined., The twelfth edition of Microbiology: A Laboratory Manual continues to provide a blend of, traditional methodologies with more contemporary procedures to meet the pedagogical needs, of all students studying microbiology. As in, previous editions, this laboratory manual provides a wide variety of critically selected and, tested experiments suitable for undergraduate, students in allied health programs, as well as, elementary and advanced general microbiology, courses., , Our Approach, This laboratory manual helps students develop, manipulative skills and techniques essential, for understanding the biochemical structure, and function of a single cell. Its main goal is to, encourage students to apply these laboratory, skills in the vocational field of applied microbiology and allied health or to study life at the molecular level., In this manual, we begin each major area of, study with comprehensive introductory material, then specific explanations and detailed, directions precede each experiment. This, approach augments, enhances, and reinforces, course lectures, enabling students to comprehend more readily the concepts and purposes, of each experiment. This also provides a review, aid if the laboratory and lecture sections are, not taught concurrently. The manual should, also reduce the time required for explanations, at the beginning of each laboratory session, and thus allow more time for performing the, experiments. Finally, the supplies, equipment,, and instrumentation for the experimental procedures can be commonly found in undergraduate, institutions., , vi, , Organization, This manual consists of 72 experiments arranged, into 15 parts. The experiments progress from, basic and introductory, which require minimal, manipulations, to more complex, which require, more sophisticated skills. The format of each, experiment is intended to facilitate presentation, of the material by the instructor and to maximize, the learning experience. To this end, each experiment is designed with the following components:, , Learning Objectives, This introductory section defines the specific, principles and/or techniques students will master., , Principle, This is an in-depth discussion of the microbiological concept or technique and the specific -experimental procedure., , Further Reading, This section aids the student in identifying the key, terms and concepts within the textbook for continued reading on the topic., , Clinical Application, Clinical or medical applications that appear, within each experiment help students connect, what they are learning in lecture with what they, are doing in the lab. For students who intend to, have careers as nurses or in other allied health, fields, Clinical Applications explain the relevance, of each lab technique to their career plans., , At the Bench, This section signals the beginning of the experiment, and includes the materials, notes of caution, and procedural instructions—all of the, things students will need to know at the bench, throughout the experiment.
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Materials, This comprehensive checklist helps students, and instructors prepare for each laboratory session. Materials appear under one of the following, headings:, Cultures These are the selected test organisms, that have been chosen to demonstrate effectively, the experimental principle or technique under, study. The choice is also based on their ease of, cultivation and maintenance in stock culture., Appendix 6 gives a complete listing of the experimental cultures and prepared slides., Media These are the specific media and their, quantities per designated student group. Appendix, 3 lists the composition and method of preparation, of all the media used in this manual., Reagents These include biological stains as well, as test reagents. Appendices 4 and 5 present the, chemical composition and preparation of the, reagents., Equipment Listed under this heading are the supplies and instrumentation that students need for, the laboratory session. The suggested equipment, was selected to minimize expense while reflecting, current laboratory technique., , Procedure, This section provides explicit instructions, augmented by diagrams, that aid in the execution and, interpretation of the experiment., A caution icon has been placed in experiments that may use potentially pathogenic, materials. The instructor may wish to perform, some of these experiments as demonstrations., , Lab Report, These tear-out sheets, located at the end of each, experiment, facilitate interpretation of data and, subsequent review by the instructor. The Observations and Results portion of the report provides, tables for recording observations and results, and, helps the students draw conclusions from and, interpret their data. The Review Questions aid the, instructor in determining the student’s ability to, understand the experimental concepts and techniques. Questions that call for more critical thinking are indicated by the brain icon., , New to the Twelfth Edition, For this twelfth edition, the primary aim was to, build upon and enrich the student experience., The changes described below impart the relevance of microbiological lab techniques to published standard protocols, and enhance student, understanding in the validity of each of the microbiological procedures as they apply laboratories, in both the educational and industrial setting., , Clinical Case Studies, Included with each section of the laboratory manual is a Clinical Case Study, which reviews a fictitious case that illustrates the laboratory science, addressed in one or more experiments within that, part. These open-ended cases have accompanying, questions to facilitate class discussions about the, topics covered in lab., , Further Reading, This new section, found in the introductory material for each part in the manual and within each, experiment, instructs students on where to look, in their textbook for more background information concerning the science behind the experiment. Worded in a general manner, this section, is not specific for a singular textbook but utilizes, common textbook section descriptions and the, nomenclature that is found in most indexes., , Check Lists, With the lengthy lists of materials, media, and, organisms required in some experiments, many, students have found the preparation for the, experiment daunting. To aid the students in ensuring that they have acquired all of the needed, materials, these lists have been converted to, check-lists., , New Experiment 36: Isolation of, Fungal Species, A newly designed experiment that illustrates a, method for the isolation of a singular or multiple, fungal species from an environmental sample., This is a generic protocol that will allow for individual customization by Instructors to fit their, labs or interests., , Preface, , vii
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New Experiment 46: Detection of, Enteric Bacteria on Raw Meat, Loosely based on the published protocols of the, United States Department of Agriculture (USDA), and Food Safety and Inspection Service (FSIS), for the cultivation, isolation, and identification of, enteric bacteria on commercially prepared meat, and meat products, this laboratory experiment, is based on government guidelines published in, MLG 4.09., , Information Concerning Governing, Bodies, Where appropriate, information concerning, governing bodies, such as the USDA and its, regulatory agency FSIS, has been included in the, introductory material for some experiments. By, drawing attention to governing bodies beyond the, American Society for Microbiology (ASM) that, have published laboratory standards, students are, introduced to the various industry standards that, regulate microbiology laboratories., , Updates and Revisions, Throughout the manual, updates and revisions, were made to background information, terminology, equipment, and procedural techniques,, including the following:, • Experiment 1 Handwashing was added back, to this edition, by popular demand., • New or updated artwork in some experiments., • Experiment 24 now has a combined laboratory, procedure for the Methyl Red (MR) and VogesProskauer (VP) tests to minimize student, • Experiments 56 and 57 were combined into, one new Experiment 57 that is now a multiweek bacterial Isolation and Transformation, lab., • Experiment 64 now also introduces the commercially available EnteroPleuri test for identifying enteric bacteria., , Instructor Resources, The Instructor Guide (ISBN 978-0-134-29869-6), is a valuable teaching aid for instructors. It was, updated to reflect changes in the main text, and, provides:, • Laboratory safety protocol for the instructional staff, • Laboratory safety protocol for the technical, staff, viii, , Preface, , • New Additional Reading research articles for, each experiment, • Detailed lists of required materials, procedural, points to emphasize, suggestions for optional, procedural additions or modifications, helpful, tips for preparing or implementing each experiment, and answers to the Review Questions in, the student manual, • Appendices with the formulas for the, preparation of all media, test reagents, and, microbiological stains, as well as the microorganisms required for the performance of each, procedure, , Acknowledgments, I wish to express my sincere gratitude to the following instructors for their manuscript reviews of, the eleventh edition. Their comments and direction contributed greatly to the twelfth edition., Mohannad AL-Saghir, Ohio University, Rachelle Bassen, Western Nevada College, Maria Carles, Northern Essex Community, College, Stella M. Doyungan, Texas A&M University –, Corpus Christi, Eric Ford, East Mississippi Community, College, James Hutcherson, Southeastern Community, College, Chris T. McAllister, Eastern Oklahoma State, College, James L. McEvoy, Saginaw Valley State, University, Laura D. Meder, Averett University, Amee Mehta, Seminole State College of Florida, Oluwatoyin Osunsanya, Muskingum, University, I would like to express my sincere condolences to the family of Dr. James Cappuccino., I appreciate their allowing the continued publication of this laboratory manual that has been his, work for the past 20-plus years., I also wish to extend my appreciation to the, staff at Pearson who helped me through the creation of this manual. Specifically, I would like to, thank Jennifer McGill, Coleen Morrison, Norine, Strang, and Sonsy Matthews for stewarding this, revision., Chad Welsh
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Laboratory Safety, , General Rules and Regulations, A rewarding laboratory experience demands, strict adherence to prescribed rules for personal, and environmental safety. The former reflects, concern for your personal safety in terms of, avoiding laboratory accidents. The latter requires, that you maintain a scrupulously clean laboratory, setting to prevent contamination of experimental, procedures by microorganisms from exogenous, sources., Because most microbiological laboratory, procedures require the use of living organisms,, an integral part of all laboratory sessions is the, use of aseptic techniques. Although the virulence, of microorganisms used in the academic laboratory environment has been greatly diminished, because of their long-term maintenance on artificial media, all microorganisms should be treated, as potential pathogens (organisms capable of, producing disease). Thus, microbiology students, must develop aseptic techniques (free of contaminating organisms) in the preparation of pure, cultures that are essential in the industrial and, clinical marketplaces., You should observe the following basic steps, at all times to reduce the ever-present microbial, flora of the laboratory environment., 1. Upon entering the laboratory, place coats,, books, and other paraphernalia in specified, locations—never on bench tops., 2. Keep doors and windows closed during the, laboratory session to prevent contamination, from air currents., 3. At the beginning and termination of each, laboratory session, wipe bench tops with, a disinfectant solution provided by the, instructor., 4. Do not place contaminated instruments,, such as inoculating loops, needles, and, pipettes, on bench tops. Loops and needles, should be sterilized by incineration, and, pipettes should be disposed of in designated, receptacles., , 5. On completion of the laboratory session,, place all cultures and materials in the disposal area as designated by the instructor., 6. Rapid and efficient manipulation of fungal, cultures is required to prevent the dissemination of their reproductive spores in the, laboratory environment., , To prevent accidental injury and infection, of yourself and others, observe the following, regulations:, 1. Wash your hands with liquid detergent, rinse, with 95% ethyl alcohol, and dry them with, paper towels upon entering and prior to, leaving the laboratory., 2. Always use the appropriate safety equipment as determined by your instructor:, a. A laboratory coat or apron may be necessary while working in the laboratory. Lab, coats protect clothing from contamination or accidental discoloration by staining solutions., b. You may be required to wear gloves while, performing the lab exercises. Gloves, shield your hands from contamination by, microorganisms. They also prevent the, hands from coming in direct contact with, stains and other reagents., c. Masks and safety goggles may be required, to prevent materials from coming in contact with your eyes., 3. Wear a paper cap or tie back long hair to, minimize its exposure to open flames., 4. Wear closed shoes at all times in the laboratory setting., 5. Never apply cosmetics or insert contact, lenses in the laboratory., 6. Do not smoke, eat, or drink in the laboratory. These activities are absolutely, prohibited., , ix
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7. Carry cultures in a test-tube rack when moving around the laboratory. Likewise, keep, cultures in a test-tube rack on the bench, tops when not in use. This serves a dual purpose: to prevent accidents and to avoid contamination of yourself and the environment., 8. Never remove media, equipment, or especially, microbial cultures from the laboratory. Doing so is absolutely prohibited., 9. Immediately cover spilled cultures or broken culture tubes with paper towels and, then saturate them with disinfectant solution. After 15 minutes of reaction time,, remove the towels and dispose of them in a, manner indicated by the instructor., 10. Report accidental cuts or burns to the, instructor immediately., 11. Never pipette by mouth any broth cultures, or chemical reagents. Doing so is strictly, prohibited. Pipetting is to be carried out, with the aid of a mechanical pipetting device, only., 12. Do not lick labels. Use only self-stick labels, for the identification of experimental, cultures., , 13. Speak quietly and avoid unnecessary movement around the laboratory to prevent distractions that may cause accidents., , The following specific precautions must be, observed when handling body fluids of unknown, origin due to the possible transmission of human, immunodeficiency virus (HIV) and hepatitis B, virus in these test specimens., 1. Wear disposable gloves during the manipulation of test materials such as blood, serum,, and other body fluids., 2. Immediately wash hands if contact with any, of these fluids occurs and also on removal of, the gloves., 3. Wear masks, safety goggles, and laboratory, coats if an aerosol might be formed or splattering of these fluids is likely to occur., 4. Decontaminate spilled body fluids with a, 1:10 dilution of household bleach, covered, with paper toweling, and allowed to react, for 10 minutes before removal., 5. Place test specimens and supplies in contact, with these fluids into a container of disinfectant prior to autoclaving., , I have read the above laboratory safety rules and regulations and agree to abide by them., , Name:, , x, , Date:, , Laboratory Safety
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Laboratory Protocol, , Student Preparation for, Laboratory Sessions, , identification of the culture should be written, on the cover of the Petri dish., , The efficient performance of laboratory exercises, mandates that you attend each session fully prepared to execute the required procedures. Read, the assigned experimental protocols to effectively, plan and organize the related activities. This will, allow you to maximize use of laboratory time., , Inoculation Procedures, , Preparation of, Experimental Materials, Microscope Slides: Meticulously clean slides, are essential for microscopic work. Use commercially pre-cleaned slides for each microscopic, slide preparation. However, wipe these slides with, dry lens paper to remove dust and finger marks, prior to their use. With a glassware marking pencil, label one end of each slide with the abbreviated name of the organism to be viewed., Labeling of Culture Vessels: Generally,, microbiological experiments require the use, of a number of different test organisms and, a variety of culture media. To ensure the successful completion of experiments, organize all, experimental cultures and sterile media at the, start of each experiment. Label culture vessels, with non–water-soluble glassware markers and/, or self-stick labels prior to their inoculation., The labeling on each of the experimental vessels should include the name of the test organism, the name of the medium, the dilution of, sample (if any), your name or initials, and the, date. Place labeling directly below the cap of, the culture tube. When labeling Petri dish cultures, only the name of the organism(s) should, be written on the bottom of the plate, close to, its periphery, to prevent obscuring observation, of the results. The additional information for the, , Part 1 of this manual fully describes aseptic techniques for the transfer or isolation of microorganisms, using the necessary transfer instruments., You will acquire technical skill through repetitive, practice., Inoculating Loops and Needles: It is, imperative that you incinerate the entire wire, to ensure absolute sterilization. You should, also briefly pass the shaft through the flame to, remove any dust or possible contaminants. To, avoid killing the cells and splattering the culture,, cool the inoculating wire by tapping the inner, surface of the culture tube or the Petri dish cover, prior to obtaining the inoculum, or touch the, edge of the medium in the plate., When performing an aseptic transfer of, microorganisms, a minute amount of inoculum, is required. If an agar culture is used, touch only, a single area of growth with the inoculating wire, to obtain the inoculum. Never drag the loop or, needle over the entire surface, and take care not, to dig into the solid medium. If a broth medium is, used, first tap the bottom of the tube against the, palm of your hand to suspend the microorganisms. Caution: Do not tap the culture vigorously, as this may cause spills or excessive foaming of, the culture, which may denature the proteins in, the medium., Pipettes: Use only sterile, disposable, pipettes or glass pipettes sterilized in a canister., The practice of pipetting by mouth has been, discontinued to eliminate the possibility of autoinfection by accidentally imbibing the culture or, infectious body fluids. Instead, use a mechanical, pipetting device to obtain and deliver the material to be inoculated., , xi
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Incubation Procedure, , Review Questions, , Microorganisms exhibit a wide temperature range, for growth. However, for most used in this manual, optimum growth occurs at 37°C over a period, of 18 to 24 hours. Unless otherwise indicated in, specific exercises, incubate all cultures under the, conditions cited above. Place culture tubes in a, rack for incubation. Petri dishes may be stacked;, however, they must always be incubated in an, inverted position (top down) to prevent water, condensation from dropping onto the surface of, the culture medium. This excess moisture could, allow the spread of the microorganisms on the, surface of the culture medium, producing confluent rather than discrete microbial growth., , The review questions are designed to evaluate, the student’s understanding of the principles, and the interpretations of observations in each, experiment. Completion of these questions will, also serve to reinforce many of the concepts that, are discussed in the lectures. At times, this will, require the use of ancillary sources such as textbooks, microbiological reviews, or abstracts. The, designated critical-thinking questions stimulate, further refinement of cognitive skills., , Procedure for Recording, Observations and Results, , 1. Return all equipment, supplies, and chemical reagents to their original locations., , The accurate accumulation of experimental data, is essential for the critical interpretation of the, observations upon which the final results will be, based. To achieve this end, it is imperative that, you complete all the preparatory readings that, are necessary for your understanding of the basic, principles underlying each experiment. Meticulously record all the observed data in the Lab, Report of each experiment., In the experiments that require drawings to, illustrate microbial morphology, it will be advantageous to depict shapes, arrangements, and cellular structures enlarged to five to ten times their, actual microscopic size, as indicated by the following illustrations. For this purpose, a number, two pencil is preferable. Stippling may be used, to depict different aspects of cell structure (e.g.,, endospores or differences in staining density)., , Microscopic drawing, , xii, , Enlarged drawing, , Laboratory Protocol, , Procedure for Termination, of Laboratory Sessions, 2. Neatly place all capped test tube cultures, and closed Petri dishes in a designated collection area in the laboratory for subsequent, autoclaving., 3. Place contaminated materials, such as, swabs, disposable pipettes, and paper, towels, in a biohazard receptacle prior to, autoclaving., 4. Carefully place hazardous biochemicals,, such as potential carcinogens, into a sealed, container and store in a fume hood prior to, their disposal according to the institutional, policy., 5. Wipe down table tops with recommended, disinfectant., 6. Wash hands before leaving the laboratory.
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Basic laboratory Techniques, for Isolation, Cultivation, and, Cultural Characterization of, Microorganis111s, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, , 1. Identify the laboratory equipment and culture media needed to develop and, maintain pure cultures., , 2. Identify the types of microbial flora that live on the skin and exr;,lail'.I how, hand washing affects them., 3. Describe the concept of aseptic technique and the procedu es necessary for, successful subculturing of microorganisms., 4. Explain streak-plate and spread-plate isolation of mitroorganisms from a, mixed microbial population for subsequent pure cult re isolation., , 5. Identify cultural and morphological characteristics of liTiicroorganisms grown, in pure culture., , Introduction, Microorganisms are ubiquitous. We find them in, soil, air, water, food, and sewage, ano on body, surfaces. In short, every area of our environment, is replete with them. Microbiologists separate, these mixed populations into individual species, for study. A culture containing a single, unadulterated species of cells is called a pure culture. To, isolate and study microorganisms in pure culture,, microbiologists require basic laboratory equipment and apply specific techniques, as illustrated, in Figure P1.1 ., , Media, The survival and continued growth of microorganisms depend on an adequate supply of nutrients, and a favorable growth environment. For survival,, most microbes must use soluble, low-molecularweight substances that are frequently derived, from the enzymatic degradation of complex, nutrients. A solution containing these nutrients, , is a culture medium. All culture media are liquid, semisolid, or solid. A liquid medium lacks a, solidifying agent and is called a broth medium., A broth medium is useful for cultivating high, numbers of bacterial cells in a small volume of, medium, which is particularly helpful when an, assay requires a high number of healthy bacterial cells. A broth medium supplemented with a, solidifying agent called agar results in a solid or, semisolid medium. Agar, an extract of seaweed,, is a complex carbohydrate composed mainly of, galactose, and is without nutritional value. Agar, serves as an excellent solidifying agent because it, liquefies at l00°C and solidifies at 40°C. Because, of these properties, we can cultivate organisms,, especially pathogens, at temperatures of 37.5°C or, slightly higher without fear of the medium liquefying. A completely solid medium requires an agar, concentration of 1.5% to 1.8%. A concentration of, less than 1% agar results in a semisolid medium., A semisolid medium is useful for testing a cell's, ability to grow within the agar at lower oxygen, , 1
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Media, , Broth, Semisolid, Solid, , Autoclave, Bunsen burner, Microincinerator, Culture tubes, Petri dishes, Wire loops and needles, Pipettes, Waterbaths, Incubators, Refrigerators, , Equipment, , Pure culture techniques, , Streak plate, Pour plate–loop dilution, Spread plate, , Agar slant, Agar deep, Agar plate, , Transfer instruments, Cultivation chambers, , Isolation of pure cultures, , Figure P1.1 Laboratory apparatus and culture techniques, , levels and for testing the species’ motility. A solid, medium is advantageous because it presents a, hardened surface on which microorganisms can, be grown using specialized techniques for the, isolation of discrete colonies. Each colony is a, cluster of cells that originates from the multiplication of a single cell and represents the growth of, a single species of microorganism. Such a defined, and well-isolated colony is a pure culture. Also,, while in the liquefied state, we can place solid, media in test tubes, which then cool and harden, in a slanted position, producing agar slants., These are useful for maintaining pure cultures., The slanted surface of the agar maximizes the, , Side view, , Front view, , (a) Agar slants, , Figure P1.2 Forms of solid (agar) media, 2, , Part 1, , available surface area for microorganism growth, while minimizing the amount of medium required., Similar tubes that, following preparation, harden, in the upright position are designated as agar, deep tubes. Agar deep tubes are used primarily, for studying gaseous requirements of microorganisms, since gas exchange between the agar at the, butt of the test tube and the external environment, is impeded by the height of the agar. Liquid agar, medium can also be poured into Petri dishes, producing agar plates, which provide large surface, areas for the isolation and study of microorganisms. The various forms of solid media are illustrated in Figure P1.2., , (b) Agar deep tube, , (c) Agar plate
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Dry (hot air), , 1605 to 1805C for 11/2 to 3 hours; for, empty glassware, glass pipettes, and glass syringes, , Heat, , Moist (wet heat), , Free-flowing steam at 1005C (intermittent, sterilization); for thermolabile solutions (e.g.,, sugars, milk), Autoclave, steam under pressure, temperatures, above 1005C; for culture media, syringes,, thermostable solutions, etc., , Filtration, , Cellulose-acetate membrane filters, with pore sizes in the range of 8.0 mm, to less than 0.05 mm, , Removal of organisms from thermolabile solutions, by passage through filters that retain bacteria; note,, viruses are not removed by this procedure, , Chemicals, , Ethylene oxide, Beta-propiolactone, , Plastic dishes and pipettes, Living tissues, , Radiation, , Ionizing, , Plastic pipettes and Petri dishes, , Figure P1.3 Sterilization techniques, , In addition to nutritional needs, we must regulate environmental factors, including proper pH,, temperature, gaseous requirements, and osmotic, pressure. You can read a more detailed explanation about the cultivation of microorganisms, in Part 4; for now, you should simply note that, numerous types of media are available., , Aseptic Technique, Sterility is the hallmark of successful work in the, microbiology laboratory, and sterilization is the, process of rendering a medium or material free of, all forms of life. To achieve sterility, it is mandatory, that you use sterile equipment and employ aseptic techniques when handling bacterial cultures., Using correct aseptic techniques minimizes the, likelihood that bacterial cultures will be contaminated, and reduces the opportunity that you will, be exposed to potential pathogens. Figure P1.3 is a, brief outline of the routine techniques used in the, microbiology laboratory, and you will learn more, about the control of microorganisms in Part 9., , Culture Tubes and Petri Dishes, We use glass test tubes and glass or plastic Petri, dishes to cultivate microorganisms. We can add a, suitable nutrient medium in the form of broth or, agar to the tubes, while we use only a solid medium, in Petri dishes. We maintain a sterile environment in, culture tubes by various types of closures. Historically, the first type, a cotton plug, was developed by, Heinrich G. F Schröeder and Theodor von Dusch in, the nineteenth century. Today most laboratories use, sleeve-like caps (Morton closures) made of metal,, , such as stainless steel, or heat-resistant plastics., The advantage of these closures over the cotton, plug is that they are labor-saving and, most of all,, that they slip on and off the test tubes easily., Petri dishes provide a larger surface area for, growth and cultivation. They consist of a bottom, dish portion that contains the medium and a larger, top portion that serves as a loose cover. Petri, dishes are manufactured in various sizes to meet, different experimental requirements. For routine, purposes, we use dishes approximately 15 cm in, diameter. The sterile agar medium is dispensed, to previously sterilized dishes from molten agar, deep tubes containing 15 ml to 20 ml of medium,, or from a molten sterile medium prepared in bulk, and contained in 250-, 500-, and 1000-ml flasks,, depending on the volume of medium required., When cooled to 40°C, the medium will solidify., Remember that after inoculation, Petri dishes are, incubated in an inverted position (top down) to, prevent condensation formed on the cover during solidification from dropping down onto the, surface of the hardened agar. For this reason, we, should label Petri dishes on the bottom of the, dish. This makes it easier to read the label and, minimizes confusion if two Petri dish covers are, interchanged. Figure P1.4 illustrates some of the, culture vessels used in the laboratory. Built-in, ridges on tube closures and Petri dishes provide, small gaps necessary for the exchange of air., , Transfer Instruments, Microorganisms must be transferred from one vessel to another, or from stock cultures to various, media, for maintenance and study. This transfer, Part 1, , 3
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A, , B, , C, , D, , E, , (b) Petri dish, , A. Bacteriological tube, B. Screw cap, C. Plastic closure, , D. Metal closure, E. Nonabsorbent cotton, , (a) Test tube rack with tubes showing various closures, , (c) DeLong shaker flask with closure, , Figure P1.4 Culture vessels, , is called subculturing, and must be carried out, under aseptic conditions to prevent possible, contamination., Wire loops and needles are made from inert, metals such as Nichrome or platinum and are, inserted into metal shafts that serve as handles., They are extremely durable instruments and are, easily sterilized by incineration in the blue (hottest) portion of the Bunsen burner flame. A wire, loop is useful for transferring a small volume of, bacteria onto the surface of an agar plate or slant., We use a needle to inoculate a culture into a broth, medium or into an agar deep tube., A pipette is another instrument used for, aseptic transfers. Pipettes are similar in function, to straws; that is, they draw up liquids. They are, glass or plastic, and drawn out to a tip at one end,, with a mouthpiece forming the other end. They are, calibrated to deliver different volumes depending, on requirements. Pipettes may be sterilized in bulk, inside canisters, or they may be wrapped individually in brown paper and sterilized in an autoclave, or dry-heat oven. A micropipette (commonly, 4, , Part 1, , called a “pipetter”) with a disposable, single-use, plastic tip is useful for transferring small volumes, of liquid (less than 1 ml)., Figure P1.5 illustrates these transfer instruments. Your instructor will demonstrate the proper, procedure for using pipettes., , Pipetting by mouth is not permissible!, Pipetting must be performed with mechanical, pipette aspirators., , Cultivation Chambers, Part 4 discusses specific temperature requirements for growth; however, a prime requirement, for the cultivation of microorganisms is that they, be grown at their optimum temperature. We use, an incubator to maintain optimum temperature, during the necessary growth period. It resembles, an oven, and is thermostatically controlled so
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TD 1 IN 1/100 ml, , Etched ring, on mouthpiece, (blow out), , Needle, , Identification, and graduations, , 10 IN 1/10 ml, , Loop, No etched ring, on mouthpiece, (to deliver), , TD, 205 C, 10 ml, , 0.1 ml: major, division, 0.01 ml each:, minor divisions, , Shaft, , Handle, , Final few drops, must be blown, out to deliver, indicated volume, (a) Transfer, needle, , (b) Transfer, loop, , (c) Blow-out, pipette, , (d) To-deliver, pipette, , Mechanical Pipette Aspirators, , (e) Micropipette, , (f) Plastic, pump, , (g) Rubber, bulb, , Figure P1.5 Transfer instruments, Part 1, , 5
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that temperature can be varied depending on the, requirements of specific microorganisms. Most, incubators use dry heat. Moisture is supplied by, placing a beaker of water in the incubator during, the growth period. A moist environment retards, dehydration of the medium and thereby helps, avoids misleading experimental results., A thermostatically controlled shaking, waterbath is another piece of apparatus used to, cultivate microorganisms. Its advantage is that, it provides a rapid and uniform transfer of heat, to the culture vessel, and its agitation provides, increased aeration, resulting in acceleration of, growth. The primary disadvantage of this instrument is that it can be used only for cultivation of, organisms in a broth medium., Many laboratories also use shaking incubators that utilize dry air incubation to promote, aeration of the broth medium. This method has, a distinct advantage over a shaking waterbath,, , since there is no chance of cross contamination, from microorganisms that might grow in the, waterbath., , Refrigerator, We use a refrigerator for a wide variety of purposes, such as maintaining and storing stock, cultures between subculturing periods, and, storing sterile media to prevent dehydration., It is also used as a repository for thermolabile, solutions, antibiotics, serums, and biochemical, reagents., , F U RT H E R RE A D I N G, Refer to the section on microbial growth in your, textbook for more information on materials and, techniques utilized in the cultivation of bacteria., Search the index for the specific terms “Agar,”, “Colony,” and “Sterile.”, , C ASE STUDY, HAND WASHING AND ASEPTIC TECHNIQUE, A local microbiological testing laboratory service,, Aureus Systems, notified its regional headquarters, about a possible contamination issue in either its, Quality Assurance/Quality Control (QA/QC) lab or, in its testing center proper. As an outside adviser,, you have been hired to investigate the situation, and to monitor the laboratory procedures of this, local branch. Upon your arrival, a senior lab technician (John Doe) allows you to shadow him and, answers your questions for the week of your visit., During your week, you notice some instances of, gross indifference to standard laboratory practices, concerning personal hygiene and personal protection practices., On numerous instances you have recorded, Mr. Doe removing his latex gloves and continuing, to handle specimens and laboratory media without, washing his hands. Many times, Mr. Doe has been, reprimanded for this practice, as well as for failure to wash his hands before leaving the lab room, itself. Mr. Doe argues that his aseptic technique, practices are at a high enough standard that he is, , 6, , Part 1, , incapable of contaminating any specimens that he, is working on in the lab. On numerous occasions, his supervisors have recorded that stock media, preparations used by Mr. Doe and other laboratory technicians have been contaminated with, unknown microbes., The regional headquarters requires laboratory proof that Mr. Doe—and not the equipment, or the lab environment—is the source of the, contamination., , Questions to Consider:, 1. Why is it important to wash your hands, BEFORE and AFTER using bacterial cultures?, 2. How would you isolate the contaminating, microbes from the contaminated stocks to, determine what species they are?, 3. Why would the use of “aseptic technique” be, important in a testing lab, or any microbiology, lab?
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E XP E R IMENT, , Effectiveness of Hand Washing, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to:, 1. Differentiate between the residential flora, and transient flora found on skin surfaces, 2. Determine the effect of hand washing on, the reduction of organisms on the skin, 3. Explain the effectiveness of using soap, alone or soap accompanied by surgical, brushing, , Principle, Each day our hands come in contact with numerous objects and surfaces that are contaminated, with microorganisms. These may include door, handles, light switches, shopping carts, sinks, toilet seats, books, or even things like compost piles, or body fluids, to name a few. The lack of adequate, hand washing is a major vehicle in the transmission of microbial infection and disease., Our skin is sterile while in utero and first, becomes colonized by a normal microbial flora at, birth as it is passed through the birth canal. By the, time you reach adulthood, your skin is calculated, to contain 1012 (1,000,000,000,000), or one trillion,, bacteria, most of which are found in the superficial, layers of the epidermis and upper hair follicles., This normal flora of microorganisms is called the, resident flora, the presence of which does not, cause negative effects in healthy individuals. In fact,, it forms a symbiotic relationship with your skin,, which is vital to your health. This beneficial relationship can change in patients who are immunocompromised, or when residential flora accidentally, gains entrance to the host via inoculating needles,, indwelling catheters, lacerations, and the like., Microorganisms that are less permanent, present, for only short periods, are termed transient flora., This latter flora can be removed with good hand, washing techniques. Resident flora is more difficult, to remove because it is found in the hair follicles, and is covered by hair, oil, and dead skin cells that, obstruct its removal by simple hand washing with, , 1, , soap. Surgical scrubbing is the best means for, removal of these organisms from the skin., Surgical hand washing was introduced into, medical practice in the mid-nineteenth century, by the Hungarian physician Ignaz Semmelweis, while working at an obstetric hospital in Vienna., He observed that the incidence of puerperal fever, (childbirth fever) was very high, with a death rate, of about 20%. He further observed that medical, students examining patients and assisting in deliveries came directly from cadaver (autopsy) laboratories without stopping to wash their hands. Upon, his insistence, medical students and all medical, personnel were required to wash their hands in a, chloride of lime (bleach) solution before and after, all patient contact. The incidence of death from, puerperal fever dropped drastically to around 1%., Semmelweis’s effort led to the development of, routine surgical scrubbing by surgeons, which has, become essential practice for all surgical procedures in modern medicine., , F U RT H E R RE A D I N G, Refer to the sections on hand washing and, laboratory hygiene to review proper laboratory, protocols and microbe handling safety. In, your textbook’s index, search under the terms, “Hygiene” and “Aseptic Technique.”, , C L I N I C A L A P P L I C AT I O N, Preventing Nosocomial Infections, Nosocomial (hospital-acquired) infections are, mainly transmitted from the unwashed hands of, healthcare providers. Transient and residential flora, on healthcare providers’ skin can infect hospital, patients whose immune systems are compromised., The cornerstone for the prevention of nosocomial, infections is meticulous hand washing and scrubbing by healthcare personnel. In the laboratory, setting, your normal flora may contaminate patient, samples and skew your results, leading to a misdiagnosis. It is important for everyone in the lab to, correctly wash their hands before and after handling biological materials., 7
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AT THE B E N C H, , Materials, Media, ❏❏ 4 nutrient agar plates per student pair, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Liquid antibacterial soap, 8 sterile cotton swabs, 2 test tubes of sterile saline, Microincinerator, Glass marking pencil, Surgical hand brush, Quebec colony counter, Stopwatch, , Procedure Lab One, , R1 R2, , 8, , Experiment 1, , at, , er, , er, , Figure 1.2 Plate inoculation., , L1 L2, , oa, , p, , L3 L4, , oa, , S, , Figure 1.1 Plate labeling, , at, , S, , er, , R1 R2, , R3 R4, , W, , W, , at, , The assistant then rubs the moistened cotton, swab on the pad of the washer’s right thumb., 4. The assistant then aseptically inoculates the, half of the nutrient agar plate labeled R1 by, streaking the far edge of the plate several, times, then making a zigzag streak on only the, half labeled R1. See Figure 1.2. Caution: Do, not gouge the surface of the agar plate., 5. The assistant turns on the tap on the lab sink,, so that the washer can wash the right hand, under warm running water, without soap,, concentrating on the thumb (rubbing the, thumb over the right index and middle finger), for one minute. The assistant turns off the tap., The washer shakes off the excess water from, the hand, but does not blot dry. The assistant,, using a new dry (not moistened with saline), sterile cotton swab, obtains a sample from the, right thumb pad and inoculates the section of, , W, , 1. One student becomes the washer and the, other student the assistant. The washer must, not wash hands before coming to the lab., 2. The assistant uses the glass marking pencil to, label the bottoms of the nutrient agar plates., The assistant marks two plates as “Water” and, two plates as “Soap,” and draws a line down, the middle of each plate to divide each plate in, half. For the “Water” plates, label the halves as, R1, R2, R3, and R4. For the “Soap” plates, label, the halves as L1, L2, L3, and L4. See Figure 1.1., 3. The assistant aseptically dips a sterile cotton, swab into the first test tube of sterile saline. To, do this, complete the following steps., a. First, light the Bunsen burner., b. Uncap the test tube; after removing the cap,, keep the cap in your hand with the inner, , aspect of the cap pointed away from your, palm. The cap must never be placed on the, laboratory bench, because doing so would, compromise the aseptic procedure., c. Flame the neck of the tube by briefly passing it through the flame of the Bunsen, burner., d. Remove the tube from the flame and dip, the swab in the tube, soaking it with saline., Avoid touching the sides of the tube with, the swab., , p
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the nutrient agar plate labeled R2 in the same, way that R1 was inoculated., 6. Repeat step 5 two more times, washing the, thumb for 2 minutes and then 3 minutes, respectively. The assistant uses a new dry sterile cotton, swab each time, and aseptically inoculates, R3 and R4, respectively. See Table 1.1., 7. The assistant and washer now move to the left, hand. The assistant aseptically dips the sterile, cotton swab into the second test tube of sterile saline (following the process from step 3),, rubs the moistened cotton swab over the pad, of the left thumb, and aseptically inoculates L1, as shown in Figure 1.2., 8. The assistant turns on the tap of the lab’s sink, so that the washer can wet the thumb and, index finger of the left hand under warm running water. The assistant applies one or two, drops of liquid soap to the thumb and index, finger and the washer washes for 1 minute by, rubbing the thumb over the index finger. Rinse, well. Shake off water from the hand but do not, blot dry. The assistant turns off the tap. The, assistant then uses a dry sterile cotton swab to, obtain a sample from the washed thumb pad, and inoculates L2., TABLE 1.1 , , Inoculation of Nutrient, Agar Plates, , WATER—RIGHT, THUMB, , SOAP—LEFT, THUMB, , R1, , No wash, damp, cotton swab, , L1, , No wash, damp, cotton swab, , R2, , Wash 1 minute,, dry cotton swab, , L2, , Wash with soap 1, minute, dry cotton, swab, , R3, , Wash 2 minutes,, dry cotton swab, , L3, , Soap and surgical, brush 2 minutes,, dry cotton swab, , R4, , Wash 3 minutes,, dry cotton swab, , L4, , Soap and surgical, brush 3 minutes,, dry cotton swab, , 9. Repeat step 8 two more times, not only using, soap but also scrubbing the thumb with a surgical brush, for 2 minutes and then 3 minutes,, respectively. The washer holds the surgical, brush and the assistant adds saline to the, brush to dampen it, and then adds one or two, drops of soap to the thumb and also to the, brush. Caution: Place the brush bristles-up, on a dry paper towel between washings. The, assistant uses a new dry sterile cotton swab, each time, and aseptically inoculates L3 and, L4, respectively. Refer back to Table 1.1., 10. Incubate all plates in an inverted position at, 37°C for 24 to 48 hours., , Procedure Lab Two, Examine and record the amount of growth found, on each nutrient agar plate. Results may be determined by two methods., 1. Macroscopically. Visually observe the, presence of growth on the surface of, each agar plate in each section. Record, your results in your Lab Report as, 0 = no growth, 1+ = slight growth, 2+, = moderate growth, 3+ = heavy growth,, and 4+ = maximum growth., 2. Percent Growth Reduction., a. Count the colonies that appear in each section of the agar plates using a Quebec colony counter. If more than 300 colonies are, present, label it as “too numerous to count, (TNTC)”; if fewer than 30 colonies are present, label it as “too few to count (TFTC).”, b. For sections R2, R3, R4, L2, L3, and L4,, calculate the percent growth reduction, from the first section, using the following, equation:, Percent reduction = [Colonies (section 1), - Colonies (section x)] , Colonies (section 1), X = sections 2, 3, 4 for each hand, , Experiment 1, , 9
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E XP E R IMENT, , 1, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Record the macroscopic observations in the chart below., , Section, (Water—, Right, Thumb), , Time (min), , Growth, (0 = none,, 1 + = slight,, 2 + = moderate,, 3 + = heavy,, 4 + = maximum), , Section, (Soap—, Left, Thumb), , Time, (min), , R1, , 0, , L1, , 0, , R2, , 1, , L2, , 1, , R3, , 2, , L3, , 2, , R4, , 3, , L4, , 3, , Growth, (0 = none,, 1 + = slight,, 2 + = moderate,, 3 + = heavy,, 4 + = maximum), , 2. Record the percent growth reduction in the following chart., Section, (Water—, Right, Thumb), , Percent, Reduction, , Section, (Soap—, Left, Thumb), , Time, (Min), , Time, (Min), , R1, , 0, , —, , L1, , 0, , R2, , 1, , L2, , 1, , R3, , 2, , L3, , 2, , R4, , 3, , L4, , 3, , Number of, Colonies, , Number of, Colonies, , Percent, Reduction, —, , Review Questions, 1. Compare the effectiveness of hand washing with water, with soap, and with, soap and surgical scrubbing., , 2. How does the presence of residential flora influence the infectious process?, , Experiment 1: Lab Report, , 11
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3. How does hand washing affect residential versus transient flora?, , 4. Why do you think hand washing is necessary when medical and surgical personnel wear gloves during surgery and when examining patients?, , 12, , Experiment 1: Lab Report
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E XP ER IME NT, , Culture Transfer Techniques, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Perform the technique for aseptic removal, and transfer of microorganisms for, subculturing., , 5., , 2. Correctly sterilize inoculating instruments, in a microincinerator or the flame of a, Bunsen burner., 3. Correctly remove and replace the test, tube closure., , 6., , Principle, We transfer microorganisms from one medium, to another by subculturing. This technique, is used routinely in preparing and maintaining, stock cultures, as well as in microbiological test, procedures., Microorganisms are always present in the, air and on laboratory surfaces, benches, and, equipment. These ambient microorganisms can, serve as a source of external contamination and, interfere with experimental results unless proper, aseptic techniques are used during subculturing., Described below are essential steps that you must, follow for aseptic transfer of microorganisms., Figure 2.1 illustrates the complete procedure., 1. Label the tube you will inoculate with the, name of the organism and your initials., 2. Hold the stock culture tube and the tube you, will inoculate in the palm of your hand, secure, with your thumb, and separate the two tubes, to form a V in your hand., 3. Sterilize an inoculating needle or loop by holding it in the microincinerator or the hottest, portion of the Bunsen burner flame until the, wire becomes red hot. Once sterilized, hold, the loop in your hand and allow it to cool for, 10 to 20 seconds; never put it down., 4. Uncap each tube by grasping the first cap with, your little finger and the second cap with your, next finger and lifting the closure upward., Note: Once removed, these caps must be kept, , 7., 8., 9., , 2, , in the hand that holds the sterile inoculating loop or needle; the inner aspects of the, caps point away from the palm of the hand., Never place the caps on the laboratory bench,, because that would compromise the aseptic, procedure., After removing the caps, flame the necks and, mouths of the tubes by briefly passing them, through the opening of the microincinerator, or through the Bunsen burner flame two to, three times rapidly. Cool the sterile transfer, instrument further by touching it to the sterile, inside wall of the culture tube before removing, a small sample of the inoculum., Depending on the culture medium, a loop or, needle is used for removal of the inoculum., Loops are commonly used to obtain a sample, from a broth culture. Either instrument can be, used to obtain the inoculum from an agar slant, culture by carefully touching the surface of, the solid medium in an area exhibiting growth, so as not to gouge the agar. A straight needle, is always used when transferring microorganisms to an agar deep tube from both solid and, liquid cultures., a. For a slant-to-broth transfer, obtain inoculum from the slant and lightly shake the, loop or needle in the broth culture to, dislodge the microorganisms., b. For a broth-to-slant transfer, obtain a loopful of broth and place at the base of an agar, slant medium. Lightly draw the loop over, the hardened surface in a straight or zigzag line, from the base of the agar slant to, the top., c. For a slant-to-agar deep tube transfer,, obtain the inoculum from the agar slant., Insert a straight needle to the bottom of the, tube in a straight line and rapidly withdraw, along the line of insertion. This is called a, stab inoculation., Following inoculation, remove the instrument, and reheat or reflame the necks of the tubes., Replace the caps on the same tubes from, which they were removed., Resterilize the loop or needle to destroy any, remaining organisms., 13
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PROCEDURE, , 1 Label the tube to be inoculated with the, name of the organism and your initials., , 3 Flame the needle or loop, until the wire is red., , 6 Slant-to-broth transfer: Obtain, inoculum from slant and dislodge, inoculum in the broth with a slight, agitation., , 7 Flame the necks of the tubes by, rapidly passing them through, the flame once., , Figure 2.1 Subculturing procedure, , 14, , Experiment 2, , 2 Place the tubes in the palm of your hand, secure, with your thumb, and separate to form a V., , 4 With the sterile loop or needle, in hand, uncap the tubes., , Broth-to-slant transfer: Obtain a loopful, of broth and place at base of slant., Withdraw the loop in a zigzag motion., , 8 Recap the tubes., , 5 Flame the necks of the tubes by, rapidly passing them through, the flame once., , Slant-to-agar deep transfer: Obtain, inoculum from slant. Insert the needle to, the bottom of the tube and withdraw, along the line of insertion., , 9 Reflame the loop or needle.
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In this experiment, you will master the manipulations required for aseptic transfer of microorganisms in broth-to-slant, slant-to-broth, and, slant-to-agar deep tubes. You will use a positive, and a negative control to test your ability to maintain aseptic techniques while transferring cultures., Experiment 3 discusses the technique for transfer, to and from agar plates., , FUR T HE R R E AD I N G, Refer to the section on aseptic culture techniques, in your textbook; more information on culturing, technique practices in the microbiological laboratory will be reviewed. In your textbook’s index,, search for the terms “Aseptic Technique” and, “Sterile.”, , C L I N I C A L A P P L I C AT I O N, Aseptic Inoculation and Transfer, It is mandatory that microbiology laboratory workers learn and perfect the skill of inoculating bacterial specimens on agar plates, in liquid broth, or in, semisolid medium, and be able to subculture the, organism from one medium to another. A sterile, inoculating needle or loop is the basic instrument, of transfer. Keep in mind that transferring bacterial, cultures requires aseptic or sterile techniques at all, times, especially if you are working with pathogens., Do not contaminate what you are working with and, do not contaminate yourself., , AT T H E B E N C H, , Materials, Cultures, ❏❏ Twenty-four–hour nutrient broth and nutrient, agar slant cultures of Serratia marcescens, and a sterile tube of nutrient broth. The nutrient broth tubes will be labeled “A” and “B,”, and the contents will be known only by the, instructor., , Equipment, ❏❏ Microincinerator or Bunsen burner, ❏❏ Inoculating loop and needle, ❏❏ Glassware marking pencil, , Procedure Lab One, 1. Label all tubes of sterile media as described in, the Laboratory Protocol section on page xv., 2. Following the procedure outlined and illustrated previously (Figure 2.1), perform the, following transfers., a. Broth culture “A” to a nutrient agar slant,, nutrient agar deep tube, and nutrient, broth., b. Broth culture “B” to a nutrient agar slant,, nutrient agar deep tube, and nutrient, broth., c. S. marcescens agar slant culture to a, nutrient agar slant, nutrient agar deep, tube, and nutrient broth., 3. Incubate all cultures at 25°C for 24 to 48 hours., , Procedure Lab Two, 1. Examine all cultures for the appearance of, growth, which is indicated by turbidity in, the broth culture and the appearance of an, orange-red growth on the surface of the slant, and along the line of inoculation in the agar, deep tube., 2. Record your observations in the chart provided in the Lab Report., 3. Confirm your results with the instructor to, determine the negative control tube., , TIPS FOR SUCCESS, 1. It is imperative that you maintain sterility and, utilize aseptic techniques at all times. If you, allow a contaminating organism into your bacterial culture, you will see a positive growth in, media that was inoculated with the negative, control., , Media, Per student:, ❏❏ Three nutrient broth tubes, ❏❏ Three nutrient agar slants, ❏❏ Three nutrient agar deep tubes, Experiment 2, , 15
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E XP E R IMENT, , 2, , Name:, Date:, , Lab Report, , Section:, , Observations and Results Culture “A”, Nutrient, Broth, , Nutrient Agar, Slant, , Nutrient Agar, Deep, , Growth, (+) or (–), Orange-red, pigmentation, (+) or (–), , Draw the, distribution of, growth., , Observations and Results Culture “B”, Nutrient, Broth, , Nutrient Agar, Slant, , Nutrient Agar, Deep, , Growth, (+) or (–), Orange-red, pigmentation, (+) or (–), , Draw the, distribution of, growth., , Experiment 2: Lab Report, , 17
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Observations and Results S. marcescens, Nutrient, Broth, , Nutrient Agar, Slant, , Nutrient Agar, Deep, , Growth, (+) or (–), Orange-red, pigmentation, (+) or (–), , Draw the, distribution of, growth., , 1. Explain why the following steps are essential during subculturing:, a. Flaming the inoculating instrument prior to and after each inoculation, , b. Holding the test tube caps in the hand as illustrated in Figure 2.1 on page 14, , c. Cooling the inoculating instrument prior to obtaining the inoculum, , d. Flaming the neck of the tubes immediately after uncapping and before recapping, , 2. Describe the purposes of the subculturing procedure., , 3. Explain why a straight inoculating needle is used to inoculate an agar deep tube., , 4. There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled, S. marcescens. Does this necessarily indicate the presence of a contaminant? Explain., , 5. U, pon observation of the nutrient agar slant culture, you strongly suspect that the culture is, contaminated. Outline the method you would follow to ascertain whether your suspicion is, justified., , 18, , Experiment 2: Lab Report
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E XP E R IMENT, , 3, , Techniques for Isolation, of Pure Cultures, , In nature, microbial populations do not segregate, themselves by species, but exist with a mixture of, many other cell types. In the laboratory, we can, separate these populations into pure cultures., These cultures contain only one type of organism, and allow us to study their cultural, morphological, and biochemical properties., In this experiment, you will first use one of the, techniques designed to produce discrete colonies., Colonies are individual, macroscopically visible, masses of microbial growth on a solid medium, surface, each representing the multiplication of, a single organism. Once you have obtained these, discrete colonies, you will make an aseptic transfer onto nutrient agar slants for the isolation of, pure cultures., , Isolation of Discrete, Colonies from a Mixed Culture, PA R T A, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Perform the streak-plate and/or the spreadplate inoculation procedure to separate, the cells of a mixed culture so that discrete, colonies can be isolated., , Turn, plate 905., Flame, loop., , 1, , The techniques commonly used for isolation of, discrete colonies initially require that the number, of organisms in the inoculum be reduced. The, resulting diminution of the population size ensures, that, following inoculation, individual cells will, be sufficiently far apart on the surface of the, agar medium to separate the different species., The f ollowing are techniques that we can use to, accomplish this necessary dilution., 1. The streak-plate method is a rapid qualitative, isolation method. It is a dilution technique that, spreads a loopful of culture over the surface of, an agar plate as a means to separate and dilute, the microbes and ensure individual colony, growth. There are many different procedures, for preparing a streak plate; the four-way, or, quadrant, streak will be described. Figure 3.1, illustrates this technique., a. Place a loopful of culture on the agar, surface in Area 1. Flame the loop, cool it, by touching it to an unused part of the agar, surface close to the periphery of the plate,, and then drag it rapidly several times across, the surface of Area 1., b. Reflame and cool the loop, and turn the, Petri dish 90°. Then touch the loop to a, corner of the culture in Area 1 and drag it, several times across the agar in Area 2. The, loop should never enter Area 1 again., c. Reflame and cool the loop and again; turn, the dish 90°. Streak Area 3 in the same, manner as Area 2., Turn, plate 905., , 2, , Flame, loop., , Principle, , Flame, loop., , Turn, plate 905., , 3, 2, , 4, 3, , 1, 1, , 1, 2, , Figure 3.1 Four-way streak-plate technique, 19
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yet to master the necessary lab skills that, would allow them to use the rapid method, listed above. This alternative method involves, spreading a loopful of culture over the surface, of an agar plate that has the quadrants laid, out visibly for quick reference. Figure 3.3, illustrates this technique., a. Using a marker, draw two bisecting lines, on the bottom of the Petri dish to divide the, plate into 4 equal parts. Label each quadrant 1 through 4, starting with the top right, quadrant and labeling counterclockwise., • When we sterilize the loop at the indicated points, the culture is diluted, because fewer organisms are available to, streak into each area. This gives us the, final desired separation., b. Turn the Petri dish over and place a loopful, of culture on the agar surface in quadrant, 1. Using the edge of the loop and holding, the loop at a shallow angle so as not to, gouge the agar, quickly spread the bacteria, throughout the quadrant., c. Reflame and cool the loop, and turn the, Petri dish 90°. Then touch the loop into, an area that has been streaked in quadrant 1 and drag it across the agar into, quadrant 2. Repeat this twice without, flaming the loop., , Heavy confluent, growth, Heavy growth, Discrete colonies, , Light growth, , Figure 3.2 Four-way streak-plate inoculation with, Serratia marcescens, , d. Without reflaming the loop, again turn the, dish 90° and then drag the culture from, a corner of Area 3 across Area 4, using, a wider streak. Don’t let the loop touch, any of the previously streaked areas. The, purpose of flaming of the loop at the points, indicated is to dilute the culture so that, fewer organisms are streaked in each area,, resulting in the final desired separation., Figure 3.2 shows a photograph of a streakplate inoculation., 2. An alternative streak-plate method is for, students new to the laboratory who have, , (a), Label bottom of dish, 2, 3, , View through agar, , 1, 4, , 1, 4, , 2, 3, , 2, , 4, , 3, , 4, , 3, , Figure 3.3 Alternate streak-plate method, , 20, , Experiment 3, , 3, , 1, , 3, , 4, , 4, , 2, , 1, , 1, , 2, , (b), , 2, , 1
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d. Reflame and cool the loop and again turn the, dish 90°. Streak the bacteria into quadrant 3, in the same manner used for quadrant 2., e. Reflame and cool the loop and again, turn the dish 90°. Streak the bacteria into, quadrant 4 in the same manner used for, quadrant 3., 3. The spread-plate technique requires that we, use a previously diluted mixture of microorganisms. During inoculation, the cells are, spread over the surface of a solid agar medium, with a sterile, L-shaped bent glass rod while, the Petri dish is spun on a “lazy Susan” turntable. The step-by-step procedure for this technique is as follows:, a. Place the bent glass rod into a beaker and, add a sufficient amount of 95% ethyl alcohol, to cover the lower, bent portion., b. Place an appropriately labeled nutrient, agar plate on the turntable. With a sterile, pipette, place one drop of sterile water on, the center of the plate, followed by a sterile, loopful of Micrococcus luteus. Mix gently, with the loop and replace the cover., c. Remove the glass rod from the beaker, and, pass it through the Bunsen burner flame, with the bent portion of the rod pointing, downward to prevent the burning alcohol, from running down your arm. Allow the, alcohol to burn off the rod completely. Cool, the rod for 10 to 15 seconds., d. Remove the Petri dish cover and spin the, turntable., e. While the turntable is spinning, lightly touch, the sterile bent rod to the surface of the, agar and move it back and forth. This will, spread the culture over the agar surface., f. When the turntable comes to a stop, replace, the cover. Immerse the rod in alcohol and, reflame., g. In the absence of a turntable, turn the Petri, dish manually and spread the culture with, the sterile bent glass rod., 4. The pour-plate technique requires a serial, dilution of the mixed culture by means of, a loop or pipette. The diluted inoculum is, then added to a molten agar medium in, a Petri dish, mixed, and allowed to solidify. Experiment 19 outlines the serial dilution, and pour-plate procedures., , F U RT H E R RE A D I N G, Refer to the section on colony isolation to review, other methods beyond the streak-plate technique, to isolate microbes and to study colony formation. Use the index to search for the terms “Streak, plate” and “Colony.”, , C L I N I C A L A P P L I C AT I O N, Culture Isolation as a Diagnostic Technique, The isolation of pure cultures is the most important, diagnostic tool used in a clinical or research laboratory to uncover the cause of an infection or disease., Before any biochemical or molecular techniques, may be used to identify or characterize the causative organism, an individual bacterial colony must, be isolated for testing. The isolation of Staphylococcus aureus from cultures taken from abscesses, or Streptococcus pyogenes from a throat culture, are two examples of clinical applications of this, technique., , AT T HE BE NCH, , Materials, Cultures, 24- to 48-hour nutrient broth cultures of, ❏❏ Mixture of one part Serratia marcescens and, three parts M. luteus, ❏❏ Mixture of one part Escherichia coli and ten, parts M. luteus, Sources of mixed cultures from the environment could include cultures from a tabletop, a, bathroom sink, a water fountain, or the inside of, an incubator. Each student should obtain a mixed, culture from one of the environmental sources, listed above., , Media, ❏❏ Three Trypticase® soy agar plates per designated student group for each inoculation technique to be performed, , Experiment 3, , 21
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Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Turntable, Glassware marking pencil or Sharpie, Culture tubes containing 1 ml of sterile water, Test tube rack, Sterile cotton swabs, , Procedure Lab One, 1. Following the procedures previously, described, prepare a spread-plate and/or, streak-plate inoculation of each test culture on, an appropriately labeled plate., 2. Prepare an environmental mixed culture., a. Dampen a sterile cotton swab with sterile water. Wring out the excess water by, pressing the wet swab against the walls of, the tube., b. With the moistened cotton swab, obtain, your mixed-culture specimen from one of, the selected environmental sources listed in, the section on cultures., c. Place the contaminated swab back into, the tube of sterile water. Mix gently and let, stand for 5 minutes., d. Perform spread-plate and/or streak-plate, inoculation on an appropriately labeled, plate., 3. Incubate all plates in an inverted position for, 48 to 72 hours at 25°C., , Procedure Lab Two, 1. Examine all agar plate cultures to identify the, distribution of colonies. In the charts provided, in Part A of the Lab Report, complete the, following:, a. Draw the distribution of colonies appearing, on each of the agar plate cultures., b. On each of the agar plate cultures, select, two discrete colonies that differ in appearance. Using Figure 4.1 on page 30 as a, reference, describe each colony’s, • Form: circular, irregular, or spreading, • Pigmentation, • Size: pinpoint, small, medium, or large., , 22, , Experiment 3, , 2. Retain the mixed-culture plates to perform, Part B of this experiment., , TIPS FOR SUCCESS, 1. An isolation plate has isolated distinct, individual colonies. If your technique results in, isolated colonies in a quadrant that was not the, last one to be streaked, that is okay. The point of, using this method is to get those individual colonies somewhere on the plate., 2. Pay attention to how well you sterilize your, loop and maintain your aseptic technique. If, you do not properly sterilize your loop between, streaks, or you do not maintain your aseptic, technique, the resulting plate will not exhibit, a decrease in bacteria leading to individual, colonies. With that in mind, if a plate you have, streaked or poured does not exhibit a decrease, in bacterial colonies area-to-area, you may, want to re-examine your technique for maintaining sterilization., , Isolation of Pure, Cultures from a Spread-Plate or, Streak-Plate Preparation, PA R T B, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Prepare a stock culture of an organism, using isolates from mixed cultures, prepared on an agar streak plate and/or, spread plate., , Principle, Once discrete, well-separated colonies develop, on the surface of a nutrient agar plate culture,, each may be picked up with a sterile needle and, transferred to separate nutrient agar slants. Each, of these new slant cultures represents the growth, of a single bacterial species and is designated as a, pure culture or stock culture.
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C L I N I C A L A P P L I C AT I O N, Transferring a Colony of Bacteria, Daughter Cells, To identify a bacterial pathogen, a discrete bacterial, colony must be transferred from a streak or spread, plate to the new testing media. This new culture, will consist of daughter cells that are genetic and, metabolic clones of the original bacterial cells that, were transferred to the plate. This will allow us to, identify the unknown bacterial species through its, biochemical and molecular characteristics., , AT T H E B E N C H, , Materials, Cultures, Mixed-culture, nutrient agar streak-plate and/or, spread-plate preparations of, ❏❏ S. marcescens and M. luteus, ❏❏ M. luteus and E. coli, ❏❏ Environmental specimen plate from Part A, , Equipment, ❏❏ Microincinerator or Bunsen burner, ❏❏ Inoculating needle, ❏❏ Glassware marking pencil, , Procedure Lab One, 1. Aseptically transfer, from visibly discrete colonies, the yellow M. luteus, the white E. coli,, the red S. marcescens, and a discrete colony, from the environmental agar plate specimen to, the appropriately labeled agar slants as shown, in Figure 3.4., 2. Incubate all slants at 37°C for 18 to 24 hours., , Procedure Lab Two, 1. In the chart provided in Part B of the Lab, Report, complete the following:, a. Draw and indicate the type of growth of, each pure-culture isolate, using Figure 4.1, on page 30 as a reference., b. Observe the color of the growth and record, its pigmentation., c. Indicate the name of the isolated organisms., , Media, ❏❏ Four Trypticase soy agar slants per designated, student group, , Experiment 3, , 23
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PROCEDURE, , 1 Flame the straight needle until the entire, wire is red., , 2 After isolating a discrete colony on the agar streak, plate, touch the straight needle to the surface of, the selected colony., , 3 Uncap the agar slant and pass the neck of the, tube rapidly over the Bunsen burner flame., , 4 Inoculate the slant by drawing the needle upward, in a zigzag motion along the surface of the agar., Do not dig into the agar., , 5 Flame the neck of the tube and recap., , 6 Flame the inoculating needle., , Figure 3.4 Procedure for the preparation of a pure culture, , 24, , Experiment 3
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E XP ER IME NT, , 3, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, PART A: Isolation of Discrete Colonies, from a Mixed Culture, STREAK-PLATE TECHNIQUE, S. marcescens and M. Iuteus, , M. Iuteus and E. coli, , Draw the colonies that, appear on each agar plate., , Colony description:, , Isolate 1, , Isolate 2, , Isolate 3, , Isolate 4, , Form, Elevation, Pigmentation, Size, , Experiment 3: Lab Report, , 25
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ENVIRONMENTAL SPECIMEN, Spread-Plate Technique, , Streak-Plate Technique, , Draw the colonies that, appear on each agar plate., , Colony description:, Form, Elevation, Pigmentation, Size, , PART B: Isolation of Pure Cultures from a Spread-Plate, or Streak-Plate Preparation, , Draw the distribution of, growth on the slant, surface., , Type of growth, Pigmentation, Name of organism, , 26, , Experiment 3: Lab Report
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Review Questions, 1. Can you prepare a pure culture from a mixed-broth or a mixed-agar-slant, culture? Explain., , 2. Observation of a streak-plate culture shows more growth in quadrant 4 than, in quadrant 3. Account for this observation., , 3. Why is a needle used to isolate individual colonies from a spread plate or, streak plate?, , 4. How can you determine if the colony that you chose to isolate is a pure, culture?, , Experiment 3: Lab Report, , 27
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E XP E R IMENT, , 4, , Cultural Characteristics, of Microorganisms, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Determine the cultural characteristics of, microorganisms as an aid to identify and, classify organisms into taxonomic, groups., , Principle, When grown on a variety of media, microorganisms exhibit differences in the macroscopic, appearance of their growth. We use these, differences, called cultural characteristics, to, separate microorganisms into taxonomic groups., The Bergey’s Manual of Systematic Bacteriology, outlines the cultural characteristics for all known, microorganisms. They are determined by culturing, the organisms on nutrient agar slants and plates, in, nutrient broth, and in nutrient gelatin. The patterns, of growth in each of these media are described, below, and some are illustrated in Figure 4.1., , Nutrient Agar Slants, These have a single straight line of inoculation on, the surface and are evaluated by, 1. Abundance of growth: The amount of, growth is designated as none, slight, moderate,, or large., 2. Pigmentation: Chromogenic microorganisms, may produce intracellular pigments that are, responsible for the coloration of the organisms as seen in surface colonies. Other organisms produce extracellular soluble pigments, that are excreted into the medium and also, produce a color. Most organisms, however,, are nonchromogenic and will appear white, to gray., 3. Optical characteristics: Optical characteristics may be evaluated by the amount of, light transmitted through the growth. These, characteristics are opaque (no light transmission), translucent (partial transmission), or, transparent (full transmission)., , 4. Form: The appearance of the single-line, streak of growth on the agar surface is designated as, a. Filiform: continuous, threadlike growth, with smooth edges, b. Echinulate: continuous, threadlike growth, with irregular edges, c. Beaded: nonconfluent to semiconfluent, colonies, d. Effuse: thin, spreading growth, e. Arborescent: treelike growth, f. Rhizoid: rootlike growth., 5. Consistency:, a. Dry: free from moisture, b. Buttery: moist and shiny, c. Mucoid: slimy and glistening, , Nutrient Agar Plates, These demonstrate well-isolated colonies and are, evaluated by, 1. Size: pinpoint, small, moderate, or large, 2. Pigmentation: color of colony, 3. Form: The shape of the colony is described as, follows:, a. Circular: unbroken, peripheral edge, b. Irregular: indented, peripheral edge, c. Rhizoid: rootlike, spreading growth, 4. Margin: The appearance of the outer edge of, the colony is described as follows:, a. Entire: sharply defined, even, b. Lobate: marked indentations, c. Undulate: wavy indentations, d. Serrate: toothlike appearance, e. Filamentous: threadlike, spreading edge, 5. Elevation: The degree to which colony growth, is raised on the agar surface is described as:, a. Flat: elevation not discernible, b. Raised: slightly elevated, c. Convex: dome-shaped elevation, d. Umbonate: raised, with elevated convex, central region, , 29
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Nutrient Broth Cultures, These are evaluated by the distribution and, appearance of the growth as, 1. Uniform fine turbidity: finely dispersed, growth throughout, 2. Flocculent: flaky aggregates dispersed, throughout, 3. Pellicle: thick, padlike growth on surface, 4. Sediment: Concentration of growth at the, bottom of broth culture may be granular, flaky,, or flocculent., , Nutrient Gelatin, This solid medium may be liquefied by the enzymatic action of gelatinase. Liquefaction occurs in a, variety of patterns:, 1. Crateriform: Liquefied surface area is, saucer-shaped., 2. Napiform: Bulbous-shaped liquefaction at, surface, 3. Infundibuliform: Funnel-shaped, 4. Saccate: Elongated, tubular, 5. Stratiform: Complete liquefaction of the, upper half of the medium, , FUR T HE R R E AD I N G, Refer to your textbook for description and explanations of growth characteristics that will lead, to the different colony morphologies seen in, this experiment. In your textbook’s index, use, the search terms “Colony,” “Pigmentation,” and, “Growth Curve.”, , C L I N I C A L A P P L I C AT I O N, Examining Colony Growth Characteristics, to Aid Identification, Bacterial species each have a characteristic, pattern of colony growth in a liquid culture or on, a solid medium. While not truly a diagnostic tool,, our recognition of these characteristic patterns in, a clinical lab setting helps us minimize the list of, potential bacterial species to test for., , AT T HE BE NCH, , Materials, Cultures, Twenty-four–hour nutrient broth cultures of, ❏❏ Pseudomonas aeruginosa BSL -2, ❏❏ Bacillus cereus, ❏❏ Micrococcus luteus, ❏❏ Escherichia coli, ❏❏ 72-to-96-hour Trypticase® soy broth culture of, Mycobacterium smegmatis, , Media, Per designated student group, ❏❏ Five each of nutrient agar slants, ❏❏ Nutrient agar plates, ❏❏ Nutrient broth tubes, ❏❏ Nutrient gelatin tubes, , Equipment, ❏❏ Microincinerator or Bunsen burner, ❏❏ Inoculating loop and needle, ❏❏ Glassware marking pencil, , Procedure Lab One, 1. Using aseptic technique, inoculate each of the, appropriately labeled media in the following list:, a. Nutrient agar slants: With a sterile needle,, make a single-line streak of each of the, cultures provided, starting at the butt and, drawing the needle up the center of the, slanted agar surface., b. Nutrient agar plates: With a sterile loop,, prepare a streak-plate inoculation of each, of the cultures for the isolation of discrete, colonies., c. Nutrient broth cultures: Using a sterile, loop, inoculate each organism into a tube of, nutrient broth. Shake the loop a few times, to dislodge the inoculum., d. Nutrient gelatin: Using a sterile needle,, prepare a stab inoculation of each culture, provided., 2. Incubate all cultures at 37°C for 24 to 48 hours., , Experiment 4, , 31
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Procedure Lab Two, 1. Before beginning observation of all the, cultures, place the gelatin cultures in a refrigerator for 30 minutes or in a beaker of crushed, ice for 5 to 10 minutes. Observe the gelatin, culture last., 2. Refer to Figure 4.1 on page 30 and this, Experiment’s introduction while observing the, following:, a. Nutrient agar slants: Observe each of the, nutrient agar slant cultures for the amount,, pigmentation, form, and consistency of the, growth. Record your observations in the, chart provided in the Lab Report., b. Nutrient agar plates: Observe a single,, well-isolated colony on each of the nutrient, , 32, , Experiment 4, , agar plate cultures and identify its size,, elevation, margin, form, and pigmentation. Record your observations in the chart, provided in the Lab Report., c. Nutrient broth cultures: Observe each of the, nutrient broth cultures for the appearance, of growth (flocculation, turbidity, sediment,, or pellicle). Record your observations in the, chart provided in the Lab Report., d. Nutrient gelatin: Remove gelatin cultures, from the refrigerator or beaker of crushed, ice, and observe whether liquefaction of, the medium has developed and whether the, organism has produced gelatinase. Record, your observations in the chart provided in, the Lab Report.
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E XP E R IMENT, , 4, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Nutrient Agar Slants, NUTRIENT AGAR SLANT CULTURES, M. Iuteus, , P. aeruginosa, , M. smegmatis, , E. coli, , B. cereus, , Draw the distribution of, growth on the slant, surface., , Amount of growth, Pigmentation, Form, Consistency, , Nutrient Agar Plates, NUTRIENT AGAR PLATES, M. Iuteus, , P. aeruginosa, , M. smegmatis, , E. coli, , B. cereus, , Draw distribution, of colonies., , Size, Elevation, Margin, Form, Pigmentation, , Experiment 4: Lab Report, , 33
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Nutrient Broth Cultures, NUTRIENT BROTH CULTURES, M. Iuteus, , P. aeruginosa, , M. smegmatis, , E. coli, , B. cereus, , Draw the distribution of, growth., , Appearance of growth, , Nutrient Gelatin, NUTRIENT GELATIN CULTURES, M. Iuteus, , Draw liquefaction, patterns., , Liquefaction (+) or (–), Type of liquefaction, , 34, , Experiment 4: Lab Report, , P. aeruginosa, , M. smegmatis, , E. coli, , B. cereus
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Microscopy, , LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, , 1. Discuss the history and diversity of microscopic instruments., 2. Identify the components of and demonstrate the proper use and care of the, brightfield microscope., 3. Correctly use the microscope to observe and measure microo, , Introduction, Microbiology, the branch of science that has so, vastly extended and expanded our knowledge, of the living world, owes its existence to Antoni, van Leeuwenhoek. In 1673, with the aid of a, crude microscope consisting of a biconcave lens, enclosed in two metal plates, Leeuwenhoek: introduced the world to the existenf!e of microl5fal, forms of life. Over the years, mieroscopes have, evolved from the simple, single-lens instrument, of Leeuwenhoek, with a mag ification of 300X, to, the present-day electron m· croscopes capable of, magnifications greater than 250,000X., Microscopes are designated as either light, microscopes or electron microscopes. The former, use visible light or ultraviolet rays to illuminate, specimens. They include brightfield, darkfield,, phase-contrast, and fluorescent instruments., Fluorescent microscopes use ultraviolet radiations whose wavelengths are shorter than those, of visible light and are not directly perceptible to, the human eye. Electron microscopes use electron beams (instead of light rays) and magnets, (instead of lenses) to observe submicroscopic, particles., , Essential Features of Various, i roscopes, Brightfield Microscope: This instrument contains, two lens systems for magnifying specimens: the, ocular lens in the eyepiece and the objective lens, located in the nosepiece. The specimen is illuminated by a beam of tungsten light focused on it, by a substage lens called a condenser; the result, is a specimen that appears dark against a bright, background. A major limitation of this system is, the absence of contrast between the specimen and, the surrounding medium, which makes it difficult, to observe living cells. Therefore, most brightfield, observations are performed on nonviable, stained, preparations., Darkfield Microscope: This is similar to the ordinary light microscope; however, the condenser, system is modified so that the specimen is not illuminated directly. The condenser directs the light, obliquely so that the light is deflected or scattered, from the specimen, which then appears bright, against a dark background. Living specimens may, be observed more readily with darkfield than with, brightfield microscopy., , 35
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Phase-Contrast Microscope: This microscope, allows us to observe microorganisms in an, unstained state. The optics include special objectives and a condenser that make visible cellular, components that differ only slightly in their refractive indexes. As light is transmitted through a, specimen with a refractive index different from, that of the surrounding medium, a portion of the, light is refracted (bent) due to slight variations in, density and thickness of the cellular components., The special optics convert the difference between, transmitted light and refracted rays, resulting in, a significant variation in the intensity of light and, thereby producing a discernible image of the structure under study. The image appears dark against a, light background., Fluorescent Microscope: This microscope is, used most frequently to visualize specimens that, are chemically tagged with a fluorescent dye. The, source of illumination is an ultraviolet (UV) light, obtained from a high-pressure mercury lamp or, hydrogen quartz lamp. The ocular lens is fitted, with a filter that permits the longer ultraviolet, wavelengths to pass, while the shorter wavelengths are blocked or eliminated. Ultraviolet, radiations are absorbed by the fluorescent label,, and the energy is re-emitted in the form of a different wavelength in the visible light range. The, fluorescent dyes absorb at wavelengths between, 230 and 350 nanometers (nm) and emit orange,, yellow, or greenish light. This microscope is used, primarily to detect antigen–antibody reactions., Antibodies are conjugated with a fluorescent dye, that becomes excited in the presence of ultraviolet light, and the fluorescent portion of the dye, becomes visible against a black background., Electron Microscope: This instrument provides a, revolutionary method of microscopy, with magnifications up to 1 million* . This permits visualization, , 36, , Part 2, , of submicroscopic cellular particles as well as, viral agents. In the electron microscope, the specimen is illuminated by a beam of electrons rather, than by light, and electromagnets— instead of a, set of optics—focus on the specimen. These components are sealed in a tube in which a complete, vacuum is established. Transmission electron, microscopes require specimens that are prepared, as thin filaments, fixed and dehydrated for the, electron beam to pass freely through them. As the, electrons pass through the specimen, images are, formed by directing the electrons onto photographic film, thus making internal cellular structures visible. Scanning electron microscopes are, used for visualizing surface characteristics rather, than intracellular structures. A narrow beam of, electrons scans back and forth, producing a threedimensional image as the electrons are reflected, off the specimen’s surface., While scientists have a variety of optical, instruments with which to perform routine laboratory procedures and sophisticated research, the, compound brightfield microscope is the “workhorse” and is commonly found in all biological, laboratories. Although you should be familiar, with the basic principles of microscopy, you probably have not been exposed to this diverse array, of complex and expensive equipment. Therefore, only the compound brightfield microscope, will be discussed in depth and used to examine, specimens., , F U RT H E R RE A D I N G, Refer to the section on the importance of microscopy in your textbook and focus on the uses of, light and fluorescent microscopy. In the index, use, the search terms “Microscope,” “Magnification,”, and “Focal Point.”
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Microscopic Examination, of Stained Cell Preparations, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. List the theoretical principles of brightfield, microscopy., 2. Identify the component parts of a compound microscope., 3. Demonstrate proper use and care of a, compound microscope., 4. Use a compound microscope to visualize the cellular morphology from stained, slide preparations., , Principle, Microbiologists study living organisms that are too, small to see with the naked eye by using a good, compound microscope. Although there are many, types and variations, compound microscopes all, consist of a two-lens system, a variable but controllable light source, and mechanical adjustable, parts for determining focal length between the, lenses and specimen (Figure 5.1)., , Components of the Microscope, Stage A fixed platform with an opening in the, center allows light to pass from an illumination, source below to the lens system above. This, platform provides a surface on which to place a, specimen slide over the central opening. In addition to the fixed stage, most microscopes have a, mechanical stage that can be moved vertically or, horizontally by means of adjustment controls. Less, sophisticated microscopes have clips on the fixed, stage, and the slide must be positioned manually, over the central opening., Illumination The light source is positioned in the, base of the instrument. Some microscopes have, a built-in light source to provide direct illumination. Others have a reversible mirror that has one, side flat and the other concave. An external light, , E XP E R IMENT, , 5, , source, such as a lamp, is placed in front of the, mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial, light, and the concave side for sunlight., Abbé condenser This component is found, directly under the stage and contains two sets, of lenses that collect and concentrate light as it, passes upward from the light source into the lens, systems. The condenser is equipped with an iris, diaphragm, a shutter controlled by a lever that is, used to regulate the amount of light entering the, lens system., Body tube Above the stage and attached to the, arm of the microscope is the body tube. This structure houses the lens system that magnifies the, specimen. The upper end of the tube contains the, ocular lens or eyepiece lens lens. The lower portion consists of a movable nosepiece containing, the objective lenses. Rotation of the nosepiece, positions objectives above the stage opening. The, body tube may be raised or lowered with the aid of, coarse-adjustment and fine-adjustment knobs, that are located above or below the stage, depending on the type and make of the instrument., , Theoretical Principles of Microscopy, To use the microscope efficiently and with, minimal frustration, you should understand the, basic principles of microscopy: magnification,, resolution, numerical aperture, illumination, and, focusing., Magnification Enlargement, or magnification, of, a specimen is the function of a two-lens system;, the ocular lens is found in the eyepiece, and the, objective lens is situated in a revolving nosepiece., These lenses are separated by the body tube. The, objective lens is nearer the specimen and magnifies it, producing the real image that is projected, up into the focal plane and then magnified by the, ocular lens to produce the final image., The most commonly used microscopes are, equipped with a revolving nosepiece containing, four objective lenses, each possessing a different, degree of magnification. When these are combined, 37
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Eyeguard, Eyepiece, Binocular tube, , Stand, , Objective lens, , Mechanical stage, , Coarse focus control, Fine focus control, , Condenser lens, Slide holder, Light exit, , Y-Axis stage control, X-Axis stage control, , Figure 5.1 A compound microscope, , with the magnification of the ocular lens, the total, or overall linear magnification of the specimen is, obtained, as shown in Table 5.1., Resolving power or resolution Although magnification is important, you must be aware that, unlimited enlargement is not possible by merely, increasing the magnifying power of the lenses, or by using additional lenses, because lenses are, limited by a property called resolving power., By definition, resolving power is how far apart, two adjacent objects must be before a given lens, shows them as discrete entities. When a lens cannot discriminate—that is, when the two objects, appear as one—it has lost resolution. Increased, , 38, , Experiment 5, , magnification will not rectify the loss, and will blur, the object. The resolving power of a lens is dependent on the wavelength of light used and on the, numerical aperture, which is a characteristic of, each lens and is imprinted on each objective. The, numerical aperture is a function of the diameter, of the objective lens in relation to its focal length., It is doubled by use of the substage condenser,, which illuminates the object with rays of light that, pass through the specimen obliquely as well as, directly. Thus, resolving power is expressed mathematically as follows:, resolving power =, , wavelength of light, 2 * numerical aperture
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TABLE 5.1, , Overall Linear Magnification, MAGNIFICATION, , OBJECTIVE LENSES, , TOTAL MAGNIFICATION, OCULAR LENS, , OBJECTIVE MULTIPLIED BY OCULAR, , Scanning 4*, , 10*, , 40*, , Low-power 10*, , 10*, , 100*, , High-power 40*, , 10*, , 400*, , Oil-immersion 100*, , 10*, , 1000*, , Based on this formula, the shorter the wavelength, the greater the resolving power of the, lens. Thus, for the same numerical aperture, short, wavelengths of the electromagnetic spectrum, are better suited for higher resolution than are, longer wavelengths. However, as with magnification, resolving power also has limits. Decreasing, the wavelength will not automatically increase, the resolving power of a lens, because the visible, portion of the electromagnetic spectrum is very, narrow and b, orders on the very short wavelengths, found in the ultraviolet portion of the spectrum., This relationship between wavelength and numerical aperture is valid only for increased resolving, power when light rays are parallel. Therefore,, the resolving power is also dependent on another, factor, the refractive index. This is the bending, power of light passing through air from the glass, slide to the objective lens. The refractive index of, air is lower than that of glass; as light rays pass, from the glass slide into the air, they are bent or, refracted so that they do not pass into the objective lens. This would cause a loss of light, which, would reduce the numerical aperture and diminish, the resolving power of the objective lens. We can, compensate for loss of refracted light by interposing mineral oil, which has the same refractive, index as glass, between the slide and the objective, lens. In this way, decreased light refraction occurs, and more light rays enter directly into the objective lens, producing a vivid image with high resolution (Figure 5.2)., Illumination Effective illumination is required, for efficient magnification and resolving power., Since the intensity of daylight is an uncontrolled, variable, artificial light from a tungsten lamp is the, most commonly used light source in microscopy., The light is passed through the condenser located, beneath the stage. The condenser contains two, lenses that are necessary to produce a maximum, numerical aperture. The height of the condenser, can be adjusted with the condenser knob. Always, keep the condenser close to the stage, especially, when using the oil-immersion objective., , Between the light source and the condenser, is the iris diaphragm, which can be opened and, closed by means of a lever, thereby regulating the, amount of light entering the condenser. Excessive, illumination may actually obscure the specimen, because of lack of contrast. The amount of light, entering the microscope differs with each objective, lens used. A rule of thumb is that as the magnification of the lens increases, the distance between, the objective lens and slide—called working distance—decreases, whereas the numerical aperture of the objective lens increases (Figure 5.3)., Use and Care of the Microscope You are responsible for the proper care and use of microscopes, using the following best practices., Often, you must move microscopes to your, laboratory bench. The correct way to do this is to, , Objective, lens, Refracted (lost), light rays, , Immersion oil, Slide, , Air, , Condenser, , Light source, , Figure 5.2 Refractive index in air and in mineral oil, , Experiment 5, , 39
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Objective, , Working Distance, , Diaphragm Opening, , Scanning, 4:, , 4:, Reduced, , 9–10 mm, , Slide, Low power, 10:, , 10:, Not fully, opened, , 5–8 mm, Slide, , High power, 40:, , 40:, Not fully, opened, , 0.5–0.7 mm, Slide, , Oil immersion, 100:, , 100:, Fully, opened, , 0.13–0.18 mm, Slide, , Figure 5.3 Relationship between working distance, objective, and diaphragm opening, 40, , Experiment 5
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grip the microscope arm firmly with one hand and, the base with your other hand to lift the instrument. Carry it close to the body and gently place, it on the laboratory bench. This will prevent collision with furniture or coworkers and will protect, the instrument against damage., Once the microscope is placed on the laboratory bench, observe the following rules:, 1. Remove all unnecessary materials (including books, papers, purses, and hats) from the, laboratory bench., 2. Uncoil the microscope’s electric cord and plug, it into an electrical outlet., 3. Clean all lens systems; the smallest bit of dust,, oil, lint, or eyelash will decrease the efficiency, of the microscope. The ocular, scanning, lowpower, and high-power lenses may be cleaned, by wiping several times with acceptable lens, tissue. Never use a paper towel or cloth on, a lens surface. If the oil-immersion lens is, gummy or tacky, ask your instructor to use a, piece of lens paper moistened with xylol to, wipe it clean. Your instructor should immediately remove the xylol with a tissue moistened, with 95% alcohol, and wipe the lens dry with, lens paper. Note: This xylol cleansing procedure should be performed only by the instructor and only if necessary; consistent use of, xylol may loosen the lens., The following routine procedures must be followed to ensure correct and efficient use of the, microscope., 1. Place the microscope slide with the specimen, within the stage clips on the fixed stage. Move, the slide to center the specimen over the opening in the stage directly over the light source., 2. Raise the microscope stage up as far as it will, go. Rotate the scanning lens or low-power lens, into position. Lower the body tube with the, coarse-adjustment knob to its lowest position., Note: Never lower the body tube while looking, through the ocular lens; this may allow for an, impact with the slide and damage to the slide, or the microscope., 3. While looking through the ocular lens, use, the fine-adjustment knob, rotating it back and, forth slightly, to bring the specimen into sharp, focus., 4. Adjust the substage condenser to achieve optimal focus., 5. Routinely adjust the light source by means of, the light-source transformer setting, and/or, the iris diaphragm, for optimum illumination, , for each new slide and for each change in, magnification., 6. Most microscopes are parfocal, which means, that when one lens is in focus, other lenses, will also have the same focal length and can, be rotated into position without further major, adjustment. In practice, however, usually a, half-turn of the fine-adjustment knob in one, direction or the other is necessary for sharp, focus., 7. Once you have brought the specimen into, sharp focus with a low-powered lens, prepare, to visualize the specimen under oil immersion., Place a drop of oil on the slide directly over, the viewing area. Rotate the nosepiece until, the oil-immersion objective locks into position., Note: Care should be taken not to allow the, high-power objective to touch the drop of oil., Observe the slide from the side as the objective, is rotated slowly into position. This will ensure, that the objective will be properly immersed in, the oil. Readjust the fine-adjustment knob to, bring the image into sharp focus., 8. During microscopic examination of microbial, organisms, it is always necessary to observe, several areas of the preparation. To do so,, scan the slide without the application of additional immersion oil. Note: This will require, continuous, very fine adjustments by the, slow, back-and-forth rotation of the fineadjustment knob only., On completion of the laboratory exercise,, return the microscope to its cabinet in its original, condition. The following steps are recommended:, 1. Clean all lenses with dry, clean lens paper., Note: Use xylol to remove oil from the, stage only., 2. Place the low-power objective in position and, lower the body tube completely., 3. Center the mechanical stage., 4. Coil the electric cord around the body tube, and the stage., 5. Carry the microscope to its position in its cabinet carefully, as previously described., , F U RT H E R RE A D I N G, Refer to the section on microscopy in your textbook for further information on visual light and, microscopy and the uses and procedures for, microscopy using UV and non-visual wavelengths., In your textbook’s index, use the search terms, “Microscope,” “Magnification,” and “Focal Point.”, Experiment 5, , 41
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C L I N I C A L A P P L I C AT I O N, Using Microscopic Examination to Diagnose, Tuberculosis, Visualizing stained bacterial cells using a compound, light microscope can be the first step in diagnosing, microbial infections. For example, a rapid diagnosis, for tuberculosis can be made by identifying the, unique characteristics of Mycobacterium tuberculosis in a stained sample of patient sputum., , AT THE B E N C H, , Materials, Slides, Commercially prepared slides of, ❏❏ Staphylococcus aureus, ❏❏ Bacillus subtilis, , 42, , Experiment 5, , ❏❏ Aquaspirillum itersonii, ❏❏ Alternate slides, , Equipment, ❏❏ Compound microscope, ❏❏ Lens paper, ❏❏ Immersion oil, , Procedure, 1. Review the parts of the microscope, making, sure you know their names and understand the, function of each of these components., 2. Review instructions for the use of the microscope, giving special attention to the use of the, oil-immersion objective., 3. Examine the prepared slides, noting the shapes, and the relative sizes of the cells under the, high-power (also called high-dry, because it is, the highest power that does not use oil) and oilimmersion objectives., 4. Record your observations in the Lab Report.
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E XP ER IME NT, , 5, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Draw several cells from a typical microscopic field as viewed under each, magnification, and give the total magnification for each objective., High Power, , Oil Immersion, , S. aureus, , Magnification, , B. subtilis, , Magnification, , S. cerevisiae, , Magnification, , Magnification, , Magnification, , Experiment 5: Lab Report, , 43
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Review Questions, 1. Explain why you should not lower the body tube of the microscope while looking through the, ocular lens., , 2. For what purpose would you adjust each of the following microscope components during a microscopy exercise?, a. Iris diaphragm, , b. Coarse-adjustment knob, , c. Fine-adjustment knob, , d. Condenser, , e. Mechanical stage control, , 3., , As a beginning student in the microbiology laboratory, you experience some difficulties in, using the oil-immersion lens. Describe the steps you would take to correct the following, problems:, a. Inability to bring the specimen into sharp focus, , b. Insufficient light while viewing the specimen, , c. Artifacts in the microscopic field, , 44, , Experiment 5: Lab Report
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Microscopic Examination of Living, Microorganisms Using a HangingDrop Preparation or a Wet Mount, , E XP E R IMENT, , 6, , Examination of living microorganisms is useful,, however, to do the following:, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, , 1. Observe cell activities such as motility and, binary fission., 2. Observe the natural sizes and shapes of the, cells, considering that heat fixation (the, rapid passage of the smear over the Bunsen, burner flame) and exposure to chemicals during staining cause some degree of distortion., , 1. Microscopically examine living, microorganisms., 2. Make a hanging-drop preparation or wet, mount to view living microorganisms., , In this experiment, you will use individual, cultures of Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, and Proteus, vulgaris for a hanging-drop preparation or a wet, mount. You may substitute hay infusion or pond, water for the above organisms. Figure 6.1 illustrates several organisms commonly found in pond, water and hay infusions., , Principle, Bacteria, because of their small size and a refractive index that closely approximates that of, water, do not lend themselves readily to microscopic examination in a living, unstained state., , Algae, , Euglena, , Diatoms, , Spirogyra, , Scenedesmus, , Chlamydomonas, , Volvox, , Protozoa, , Paramecium, , Stylonychia, , Amoeba, , Vorticella, , Heteronema, , Figure 6.1 Algae and protozoa commonly found in natural infusions and pond water (drawings are not, to scale), , 45
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You will observe the preparation(s) microscopically for differences in the sizes and shapes, of the cells, as well as for motility, a self-directed, movement. It is essential to differentiate between, actual motility and Brownian movement, a, vibratory movement of the cells due to their bombardment by water molecules in the suspension., Hanging-drop preparations and wet mounts make, the movement of microorganisms easier to see, because they slow down the movement of water, molecules., , C L I N I C A L A P P L I C AT I O N, Observation of Living Bacteria and the, Diagnosis of Syphilis, Some microorganisms are difficult or impossible to, stain. One of these bacteria is Treponema pallidum,, the causative agent for syphilis. Special stains must, be used to stain this bacterium; however, it can be, viewed unstained and alive using a darkfield microscope. Under those conditions, you can observe its, characteristic shape and motility, leading to a diagnosis of syphilis., , AT THE B E N C H, , Twenty-four–hour broth cultures of, ❏❏ P. aeruginosa BSL -2, ❏❏ B. cereus, ❏❏ S. aureus BSL -2, ❏❏ P. vulgaris, ❏❏ Hay infusion broth cultures or pond water (see, Appendix 3 for the preparation of hay infusion, broth), , Equipment, , Experiment 6, , Perform the following steps for each culture, provided in this experiment. Figure 6.2 illustrates, steps 1 to 4., 1. With a cotton swab, apply a ring of petroleum, jelly around the concavity of the depression, slide., 2. Using aseptic technique, place a loopful of the, culture in the center of a clean coverslip., 3. Place the depression slide, with the concave, surface facing down, over the coverslip so, that the depression covers the drop of culture., Press the slide gently to form a seal between, the slide and the coverslip., 4. Quickly turn the slide right side up so that the, drop continues to adhere to the inner surface, of the coverslip., 5. For microscopic examination, first focus on, the drop culture under the low-power objective (10* ) and reduce the light source by, adjusting the Abbé condenser. Repeat using, the high-power objective (40* )., 6. Examine the hanging-drop preparation and, record your observations in the Lab Report., , A wet mount may be substituted for the hangingdrop preparation using a similar procedure:, , Cultures, , 46, , Hanging-Drop Preparation, , Wet Mount, , Materials, , ❏❏ Microincinerator or, Bunsen burner, ❏❏ Inoculating loop, ❏❏ Depression slides, ❏❏ Glass slides, , Procedure, , ❏❏, ❏❏, ❏❏, ❏❏, , Coverslips, Microscope, Petroleum jelly, Cotton swabs, , 1. With a cotton swab, apply a thin layer of petroleum jelly along the four edges of a coverslip., 2. Using aseptic technique, place a loopful of the, culture in the center of a clean coverslip., 3. Place a clean glass slide over the coverslip and, press the slide gently to form a seal between, the slide and the coverslip., 4. Follow steps 4 and 5 in the hanging-drop, procedure., 5. Examine the wet-mount preparation and, record your observations in the Lab Report.
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PROCEDURE, Slide, concavity, , Petroleum jelly, ring, , Loopful, of bacterial, culture, , Depression slide, , Coverslip, , 1 Spread a ring of petroleum jelly around the concavity, of the depression slide., , 2 Place a loopful of the bacterial culture in the center, of the coverslip., , Culture drop, Coverslip, , Depression, slide, , Coverslip, , Culture drop, , 3 Lower the depression slide, with the concavity, facing down, onto the coverslip. Press gently, to form a seal., , Petroleum jelly, , Hanging-drop preparation, , 4 Turn the hanging-drop preparation over so that, the culture drop adheres to the coverslip., , Figure 6.2 Hanging-drop preparation, , Experiment 6, , 47
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E XP ER IM E NT, , 6, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Examine the hanging-drop or wet-mount preparation to determine shape, and motility of the different bacteria present. Record your results in the, chart below., Organisms, , True Motility or, Brownian Movement?, , Shape, , S. aureus, P. aeruginosa, B. cereus, P. vulgaris, , 2. Draw a representative field of each of the above organisms., , S. aureus, , P. aeruginosa, , B. cereus, , P. vulgaris, , Experiment 6: Lab Report, , 49
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3. Draw representative fields of pond water and hay infusion, if you used them. Try to identify some, of the organisms that you see by referring to Figure 6.1. Note the shape and type of movement in, the chart below., , Pond water, , Hay infusion, , Pond Water, , Hay Infusion, , Shape, True motility or Brownian movement?, Organism, , Review Questions, 1. Why are living, unstained bacterial preparations more difficult to observe microscopically than, stained preparations?, , 2., , What is the major advantage of using living cell preparations, (hanging-drop or wet-mount) rather than stained preparations?, , 3. How do you distinguish between true motility and Brownian movement?, , 4., , 50, , During the microscopic observation of a drop of stagnant, pond water, what criteria would you use to distinguish viable organisms, from nonviable suspended debris?, , Experiment 6: Lab Report
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Bacterial Staining, , LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, , 1. Explain the chemical and theoretical basis of biological staining., 2. Manipulate techniques of smear preparation., , 3. Perform procedures for simple staining and negative staining., 4. Describe the method for performing differential staining proce, , uch as, , the Gram, acid-fast, capsule, and spore stains., , Introduction, Visualizing microorganisms in the living state, is quite difficult, not only because they are, minute, but also because they are transparent, and practically colorless when suspended in an, aqueous medium. To study their properties and, to divide microorganisms into specifie groups, for diagnostic purposes, biological stains, and staining procedures in conjunction with, light microscopy haveE,tCome major tools in, ~, microbiology., Chemically, a stain (dye) is an organic, compound containing a benzene ring plus a chromophore and an auxochrome group ( Figure P3.1 )., The stain picric acid illustrates this definition, as, seen in Figure P3.2., The ability of a stain to bind to macromolecular cellular components such as proteins or, nucleic acids depends on the electrical charge, , 1, , Benzene:, , ±Qnd on the chromogen portion, as well as on the, cellular component to be stained., Acidic stains are anionic, which means that,, on ionization of the stain, the chromogen portion, exhibits a negative charge and therefore has a, strong affinity for the positive constituents of the, cell. Proteins, positively charged cellular components, will readily bind to and accept the color of, the negatively charged, anionic chromogen of an, acidic stain. Structurally, picric acid is an example, of an acidic stain that produces an anionic chromogen, as illustrated in Figure P3.3., Basic stains are cationic, because on ionization the chromogen portion exhibits a positive, charge and therefore has a strong affinity for the, negative constituents of the cell. Nucleic acids,, negatively charged cellular components, will readily bind to and accept the color of the positively, charged, cationic chromogen of a basic stain., , Organic colorless solvent, , +, Chromophore:, , Stain, , +, Auxochrome:, , Chemical group that imparts, color to benzene, , Chromogen:, } - Colored compound,, not a stain, , Chemical group that conveys the, property of ionization to the chromogen,, enabling it to form salts and bind to, fibers or tissues, , Figure P3.1 Chemical composition of a stain, , 51
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H, , H, , H, , OH, NO2, , O2N, , H, , + 3NO2–, H, , + OH–, H, , H, , H, , H, , NO2, , H, Nitro groups, chromophore, , Benzene, colorless, , NO2, , O2N, , H, NO2, , Trinitrobenzene, chromogen, yellow, in color due to the, presence of, chromophores, , Auxochrome, , Trinitrohydroxybenzene, (picric acid) yellow stain, , Figure P3.2 Chemical formation of picric acid, , –, O, , OH, , NO2, , O2N, , NO2, , O2N, , +, , Ionization, H, , H, , H, , +, H, , H, NO2, , NO2, , Anionic chromogen, , Picric acid, , Figure P3.3 Picric acid: an acidic stain, , N, , +, , N, Ionization, , (CH3 )2 N, , S, , N(CH3 )2 Cl, , Methylene blue, , –, , +, (CH3 )2 N, , S, , Cl, , N(CH3 )2, , Cationic chromogen, , Figure P3.4 Methylene blue: a basic stain, , Structurally, methylene blue is a basic stain that, produces a cationic chromogen, as illustrated in, Figure P3.4., Figure P3.5 summarizes acidic and basic, stains. Basic stains are more commonly used for, bacterial staining. The presence of a negative, charge on the bacterial surface acts to repel most, acidic stains and thus prevent their penetration, into the cell. Through the use of a series of simple, stains, many attributes of microbes may be determined. The morphological characteristics and, cellular structures of bacteria can be visualized,, differentiated, and separated through numerous, , 52, , Part 3, , staining techniques. Figure P3.6 outlines a summary of commonly used procedures and their, purposes., , F U RT H E R RE A D I N G, Refer to the section on staining in your textbook,, and pay close attention to the uses of the different, stains as a means of differentiating the different, microbes you will be working with in a lab. In the, index of your textbook, use the following search, terms: “Gram’s Stain”, “Differential Stains,” and, “Simple Stains.”
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Acidic, , Sodium, potassium, calcium, or ammonium salts of colored acids, ionize to give a negatively charged chromogen, , Basic, , The chloride or sulfate salts of colored bases ionize to give a, positively charged chromogen, , Stains, , Figure P3.5 Acidic and basic stains, , Simple staining:, Use of single stain, Types of, staining, techniques, , For visualization of morphological shape (cocci,, bacilli, and spirilli) and arrangement (chains,, clusters, pairs, and tetrads), Gram stain, Separation into groups, Acid-fast stain, , Differential staining:, Use of two contrasting, stains, , Visualization of, structures, , Flagella stain, Capsule stain, Spore stain, Nuclear stain, , Figure P3.6 Staining techniques, , C AS E STUDY, INITIAL STEPS IN THE MICROBIOLOGY LABORATORY, During a late-night blizzard, a patient presents at, a rural medical facility complaining of a p, ossible, infected leg injury. Upon examination, the attending Emergency Room physician notes a deep, laceration to the left lateral thigh. The wound is, approximately 60 mm in length and approximately, 15 mm deep. The assumed infected purulent, wound has a greenish pus discharge, and the, surrounding flesh is raised, warm to the touch,, and showing signs of red streaking extending in, toward the left groin. The edges of the wound, have begun to exhibit a dark discoloration that is, indicative of early stages of necrosis., Due to the time of night and prevailing, weather conditions, the medical center’s diagnostic laboratory has been closed and left unattended. According to the patient’s medical history,, the patient has shown signs of hypersensitivity to, , most broad-spectrum antibiotics, which precludes, their use initially to treat the infection and arrest, the potential spread of necrosis. The attending, physician is left with two choices for antibiotics, that will treat the patient while not running the, risk of inducing anaphylaxis: Vancomycin for a, gram-positive infection, or Ciprofloxacin for a, gram-negative microbial infection. The patient, has been treated with both antibiotics in the past., Which antibiotic should the physician use?, , Questions to Consider:, 1. What can the physician do to quickly and, easily determine which antibiotic would be the, correct one to use for this patient?, 2. Would a simple stain be enough to determine, which antibiotic to use? Why or why not?, , Part 3, , 53
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E XP E R IMENT, , Preparation of Bacterial Smears, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Prepare bacterial smears to microscopically visualize bacteria., , Principle, Bacterial smears must be prepared prior to executing of any of the staining techniques listed in, Figure P3.6 on page 53. Although not difficult, the, preparation requires adequate care. Be sure to, carefully follow the rules listed below., 1. Prepare the glass microscope slide: Clean, slides are essential to prepare microbial, smears. Grease or oil from the fingers on, slides must be removed by washing the slides, with soap and water or scouring powders such, as Bon Ami®, followed by a water rinse and, a rinse of 95% alcohol. After cleaning, dry the, slides and place them on laboratory towels, until ready for use. Note: Remember to hold, the clean slides by their edges., 2. Label slides: Proper labelling of the slide is, essential. Write the initials of the organism on, either end of the slide with a glassware marking pencil on the surface on which the smear, is to be made. Ensure that the label does not, come into contact with staining reagents., 3. Prepare the smear: It is crucial to avoid, thick, dense smears. A thick or dense smear, occurs when too much of the culture is used, in its preparation, which concentrates a large, number of cells on the slide. This type of preparation diminishes the amount of light that can, pass through and makes it difficult to visualize, the morphology of single cells., Note: Smears require only a small amount, of the bacterial culture. A good smear is, one that, when dried, appears as a thin whitish layer or film. The print of your textbook, should be legible through the smear. Different, , 7, , techniques are used depending on whether the, smear is made from a broth or solid-medium, culture., a. Broth cultures: Resuspend the culture, by tapping the tube with your finger., Depending on the size of the loop and the, amount of culture growth, apply one or two, loopfuls to the center of the slide with a, sterile inoculating loop and spread evenly, over an area about the size of a dime. Set, the smears on the laboratory table and, allow to air-dry., b. Cultures from solid medium: Organisms, cultured in a solid medium produce thick,, dense surface growth and are not amenable, to direct transfer to the glass slide. These, cultures must be diluted by placing one or, two loopfuls of water on the center of the, slide, in which the cells will be emulsified., Transfer the cells using a sterile inoculating loop or a needle. Only the tip of the, loop or needle should touch the culture, to prevent the transfer of too many cells., Suspension is accomplished by spreading, the cells in a circular motion in the drop of, water with the loop or needle. This helps, to avoid cell clumping. The finished smear, should occupy an area about the size of a, nickel and should appear as a translucent,, or semitransparent, confluent whitish film, (Figure 7.1). Allow the smear to dry completely. Note: Do not blow on slide or wave, it in the air., , Figure 7.1 A bacterial smear following fixation, 55
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4. Heat fixation: Unless fixed on the glass slide,, the bacterial smear will wash away during the, staining procedure. This is avoided by heat, fixation, during which the bacterial proteins, are coagulated and fixed to the glass surface., Heat fixation is performed by the rapid passage of the air-dried smear two or three times, over the flame of the Bunsen burner or in, front of a microincinerator. While many texts, discuss using a Bunsen burner for sterilization and heat fixation, the American Society, for Microbiology (ASM)—one of the governing bodies that determines safe laboratory, procedures—changed the proscribed methods, for heat fixation and benchtop sterilization to, utilize a microincinerator instead of a Bunsen, burner to reduce the possibility of aerosolization of bacteria on the slide or loop. Figure 7.2, illustrates the preparation of a bacterial smear., , C L I N I C A L A P P L I C AT I O N, Proper Slide Preparation, Before any staining or visualization of a bacterial, sample can take place, you must prepare a proper, smear. A smear that is too thick may give a false, result due to retention of dye that should have been, rinsed away or because the thickness may prevent, dye penetration. A smear that is too thin may have, too few cells, increasing the time and energy, required to find the bacteria under magnification., Inconclusive results due to improperly prepared, slides may have an impact on patient treatment and, outcomes. Good smears are those that allow newsprint to be read through the smear., , FU RT HER R E ADING, Refer to the section in your textbook regarding, slide preparation and bacterial smears for further, information on the process for making slides suitable for staining. In your textbook’s index, use the, search terms “Smear,” “Stains,” and “Microscopes.”, , AT THE B E N C H, , Materials, Cultures, ❏❏ T wenty-four–hour nutrient agar slant culture, of Bacillus cereus, 56, , Experiment 7, , ❏❏ T wenty-four–hour nutrient broth culture of, Staphylococcus aureus BSL -2, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Glass microscope slides, Microincinerator or Bunsen burner, Inoculating loop and needle, Glassware marking pencil, , Procedure, Smears from a Broth Medium, Label three clean slides with the initials of the, organism, and number them 1, 2, and 3. Resuspend the sedimented cells in the broth culture, by tapping the culture tube with your finger. The, next four steps of this procedure are illustrated in, Figure 7.2a and c:, 1. With a sterile loop, place one loopful of culture, on slide 1, two loopfuls on slide 2, and three, loopfuls on slide 3., 2. With a circular movement of the loop, spread, the cell suspension into an area approximately, the size of a dime., 3. Allow the slide to air-dry completely. This may, be done by placing the slide on a drying tray, attached to a microincinerator or by placing, the slide on the bench., 4. Heat fix the preparation. Note: Pass the, air-dried slide in front of the entrance, to the microincinerator or pass the slide, through the outer portion of the Bunsen, flame to prevent overheating, which can, distort the morphology through plasmolysis, of the cell wall., Examine each slide for the confluent, whitish film,, or haze and record your results in the Lab Report., , Smears from a Solid Medium, Label four clean slides with the initials of the, organism. Label slides 1 and 2 with an L for loop,, and slides 3 and 4 with an N for needle. The next, four steps of this procedure are illustrated in, Figure 7.2b and c:, 1. Using a loop, place one to two loops of water, on each slide.
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PROCEDURE, (a) From broth medium, , (b) From solid medium, , 1 Place one to two loopfuls of the cell suspension, on the clean slide., , 1 Place one to two loopfuls of water on, the center of the slide., , 2 With a circular movement of the loop, spread the, suspension into a thin area approximately the size, of a dime., , 2 Transfer a small amount of the bacterial inoculum, from the slant culture into the drop of water. Spread, both into a thin area approximately the size of a nickel., , (c) Fixation for solid and broth media, , 3 Allow the smear to air-dry., , 4 While holding the slide at one end, quickly pass the, smear over the flame of the Bunsen burner two to, three times., , Figure 7.2 Bacterial smear preparation, , Experiment 7, , 57
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2. With a sterile loop, touch the entire loop to, the culture and emulsify the cells in water on, slide 1. Then, with a sterile loop, just touch, the tip of the loop to the culture and emulsify, it in the water on slide 2. Repeat steps 1 and 2, using a sterile inoculating needle on slides 3, and 4., 3. Allow all slides to air-dry completely. This may, be done by placing the slides on a drying tray, attached to a microincinerator or by placing, the slides on the bench., 4. Heat fix the preparation. Note: Pass the, air-dried slide in front of the entrance to the, microincinerator or pass the slide through, the outer portion of the Bunsen flame to, prevent overheating, which can distort the, morphology through plasmolysis of the, cell wall., Examine each slide for the confluent, whitish film,, or haze and record your results in the Lab Report., , 58, , Experiment 7, , TIPS FOR SUCCESS, 1. The bacterial smear should be heavy enough to, leave a slight film but not so heavy that you can, plainly see the bacteria without a microscope., Students sometimes err on the side of adding, too much bacteria to a slide to make sure there, will be “enough” bacteria there for later visualization. This has the potential to interfere with, later staining procedures and produce false, results., 2. Heat fixing should warm the slide until it is, hot to the touch but not to the point of burning., Overheating the slide during this step increases, the potential for damaging the cells. Damaged, cells do not retain stains, and produce inconclusive staining results. Underheating of the, slide does not allow the cells to affix to the, glass. Resulting washes or stains will rinse the, bacteria off the glass, leaving few if any bacteria present for later viewing.
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E XP E R IMENT, , 7, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Select the preparation that you think is best and ask your instructor to comment, on your choice. Remember that printed material should be legible through a, good smear. Indicate by slide number the consistency of smears from both broth, and solid cultures that you considered best., Broth culture ___________ Solid culture: Loop ___________ Needle ___________, , Review Questions, 1. Why are thick or dense smears less likely to provide a good smear preparation for microscopic evaluation?, , 2. Why is it essential that smears be air-dried? Why can’t they be gently heated, over a flame to speed up the drying process?, , 3. Why should you be careful not to overheat the smear during the heat-fixing, process?, , 4., , Why do you think the presence of grease or dirt on a glass slide, will result in a poor smear preparation? Cite two or three reasons., , Experiment 7: Lab Report, , 59
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E XP E R IMENT, , 8, , Simple Staining, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Perform a simple staining procedure., 2. Compare the morphological shapes and, arrangements of bacterial cells., , Cocci are spherical in shape., (a) Diplococcus, , Diplo = pair, , (b) Streptococcus, , Strepto = chain, , (c) Staphylococcus, , Staphlyo = cluster, , (d) Tetrad, , Tetrad = packet of 4, , (e) Sarcina, , Sarcina = packet of 8, , Principle, In simple staining, we stain the bacterial smear, with a single reagent, which produces a distinctive contrast between the organism and its background. Basic stains with a positively charged, chromogen are preferred because bacterial, nucleic acids and certain cell wall components, carry a negative charge that strongly attracts and, binds to the cationic chromogen. The purpose of, simple staining is to elucidate the morphology and, arrangement of bacterial cells (Figure 8.1). The, most commonly used basic stains are methylene, blue, crystal violet, and carbol fuchsin., , Bacilli are rod-shaped., (a) Diplobacillus, , Diplo = pair, , (b) Streptobacillus, , Strepto = chain, , Spiral bacteria are rigid or flexible., (a) Vibrios are curved rods., (b) Spirilla are helical and rigid., (c) Spirochetes are helical and flexible., , C L I N I C A L A P P L I C AT I O N, Quick and Simple Stain, Simple stains are relatively quick and useful methods of testing for the presence of, determining the, shape of, or determining the numbers of bacteria, present in a sample. Generally involving only a, single staining step, simple staining methods are not, considered differential or diagnostic, and will have, limited uses. However, this is a quick procedure for, determining whether a clinical sample has the presence of a foreign bacterial pathogen., , Figure 8.1 Bacterial shapes and arrangements, , F U RT H E R RE A D I N G, Refer to the section on stain in your textbook, for background information on the use of simple, stains and their limitations in a laboratory setting., In your textbook’s index, use the search terms, “Simple Stains” and “Differential Stains.”, , 61
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AT THE B E N C H, , PROCEDURE, , Materials, Cultures, T wenty-four–hour nutrient agar slant cultures of, ❏❏ Escherichia coli, ❏❏ Bacillus cereus, T wenty-four–hour nutrient broth culture of, ❏❏ Staphylococcus aureus BSL -2, Alternatively, use the smears prepared in, Experiment 7., , Reagents, , 1 Place slide on the staining tray and flood the smear, with methylene blue. Allow 1 to 2 minutes of, exposure to the stain., , ❏❏ Methylene blue, ❏❏ Crystal violet, ❏❏ Carbol fuchsin, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Staining tray, Microscope, Lens paper, Bibulous (highly absorbent) paper, Glass slides, 2 Gently wash the smear with tap water., , Procedure, 1. Prepare separate bacterial smears of the, organisms following the procedure described, in Experiment 7. Note: All smears must be, heat fixed prior to staining., , Simple Staining, Figure 8.2 illustrates the following steps:, , 1. Place a slide on the staining tray and flood, the smear with one of the indicated stains,, using the appropriate exposure time: carbol, fuchsin, 15 to 30 seconds; crystal violet, 20, to 60 seconds; methylene blue (shown in, Figure 8.2), 1 to 2 minutes., , 62, , Experiment 8, , 3 Blot the slide dry with bibulous paper., , Figure 8.2 Simple staining procedure
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2. Gently wash the smear with tap water to, remove excess stain. During this step, hold, the slide parallel to the stream of water; in this, way, you reduce the loss of organisms from, the preparation., 3. Using bibulous paper, blot dry, but do not wipe, the slide., 4. Repeat this procedure with the remaining two, organisms, using a different stain for each., 5. Examine all stained slides under oil, immersion., 6. In the chart provided in the Lab Report,, complete the following:, a. Draw a representative field for each organism. Refer to page xvi for proper drawing, procedure., b. Describe the morphology of the organisms, with reference to their shapes (e.g., bacilli,, cocci, or spirilla) and arrangements (e.g.,, chains, clusters, or pairs). Refer to the, photographs in Figure 8.3., , Diplobacilli, , (a) Bacilli and diplobacilli (rod-shaped) bacteria, , (b) Spirilla (spiral-shaped) bacteria, , (c) Cocci (spherical-shaped) bacteria: Staphylococcus, , Figure 8.3 Micrographs showing bacteria, morphology, , Experiment 8, , 63
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E XP E R IMENT, , 8, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Methylene Blue, , Crystal Violet, , Carbol Fuchsin, , Draw a representative field., , Organism, Cell morphology:, Shape, Arrangement, Cell color, , Review Questions, 1. Why are basic dyes more effective for bacterial staining than acidic dyes?, , 2. Can simple staining techniques be used to identify more than the morphological characteristics of, microorganisms? Explain., , Experiment 8: Lab Report, , 65
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3., , During the performance of the simple staining procedure, you failed to heat fix your E. coli, smear preparation. On microscopic examination, how would you expect this slide to differ, from the correctly prepared slides?, , 4., , During a coffee break, your friend spills coffee on your lab coat and the fabric is discolored., Is this a true biological stain or simply a compound capable of imparting color? Explain, your rationale., , 66, , Experiment 8: Lab Report
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E XP E R IMENT, , 9, , Negative Staining, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Perform a negative staining procedure., 2. Explain the benefits obtained from visualizing unstained microorganisms., , F U RT H E R RE A D I N G, Refer to the section discussing simple stains in, your textbook for further information on the, differences between negative and positive stains, such as the simple. In your textbook’s index, use, the search terms “Simple Stains,” “Positive Stains,”, “Negative Stains,” and “Differential Stains.”, , C L I N I C A L A P P L I C AT I O N, , Principle, Negative staining requires the use of an acidic, stain such as India ink or nigrosin. The acidic, stain, with its negatively charged chromogen, will, not penetrate the cells, because of the negative, charge on the surface of bacteria. Therefore, the, unstained cells are easily discernible against the, colored background., The practical application of negative staining, is twofold. First, since heat fixation is not required, and the cells are not subjected to the distorting, effects of chemicals and heat, we can see their, natural size and shape. Second, we can observe, bacteria that are difficult to stain, such as some, spirilla. Because heat fixation is not done during the, staining process, keep in mind that the organisms, are not killed and slides should be handled with, care. Figure 9.1 shows a negative stain of bacilli., , Detecting Encapsulated Invaders, The principle application of negative staining is, to determine if an organism possesses a capsule, (a gelatinous outer layer that makes the microorganism more virulent), although it can also be used, to demonstrate spore formation. The technique is, frequently used in the identification of fungi such, as Cryptococcus neoformans, an important infectious agent found in bird droppings that is linked to, meningeal and lung infections in humans., , AT T HE BE NCH, , Materials, Cultures, Twenty-four–hour agar slant cultures of, ❏❏ Micrococcus luteus, ❏❏ Bacillus cereus, ❏❏ Other alternate bacterial cultures, , Reagent, ❏❏ Nigrosin, , Figure 9.1 Negative staining with nigrosin: bacilli, (1000 : ), , 67
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Equipment, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Inoculating loop, ❏❏ Staining tray, , 4. Push the slide away from the drop of, suspended organisms to form a thin smear., Air-dry. Note: Do not heat fix the slide., 5. Repeat Steps 1 to 4 for slide preparations of, the remaining cultures., 6. Examine the slides under oil immersion,, and record your observations in the Lab, Report., , ❏❏ Glass slides, ❏❏ Lens paper, ❏❏ Microscope, , Procedure, Figure 9.2 illustrates Steps 1 to 4., , 1. Place a small drop of nigrosin close to one end, of a clean slide., 2. Using aseptic technique, place a loopful of, inoculum from the M. luteus culture in the, drop of nigrosin and mix., 3. Place a slide against the drop of suspended, organisms at a 45° angle and allow the drop to, spread along the edge of the applied slide., , TIPS FOR SUCCESS, 1. Allow the slide to dry completely before, attempting to observe using the microscope., Wet smears will continue to move due to microcurrents, making finding and getting a cell in, focus much more difficult., , PROCEDURE, , 1 Place a drop of nigrosin at one end of the slide., , 2 Place a loopful of the inoculum into the drop of stain, and mix with the loop., , 455, , 3 Place a slide against the drop of suspended organisms, at a 455 angle and allow the drop to spread along the, edge of the applied slide., , Figure 9.2 Negative staining procedure, , 68, , Experiment 9, , 4 Push the slide away from the previously spread drop, of suspended organisms, forming a thin smear. Air-dry, the slide.
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E XP E R IMENT, , 9, , Name:, Date:, , Section:, , Lab Report, , Observations and Results, 1. Draw representative fields of your microscopic observations., , M. luteus, , 2. Describe the microscopic appearance of the different bacteria using the chart below., Organism, , M. luteus, , Shape, Arrangement, Magnification, , Experiment 9: Lab Report, , 69
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Review Questions, 1. Why can’t methylene blue be used in place of nigrosin for negative staining? Explain., , 2. What are the practical advantages of negative staining?, , 3. Why doesn’t nigrosin penetrate bacterial cells?, , 70, , Experiment 9: Lab Report
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E XP E R IMENT, , Gram Stain, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Explain the chemical and theoretical basis, for differential staining procedures., 2. Describe the chemical basis for the Gram, stain., 3. Perform the procedure to differentiate, between two principal groups of bacteria:, gram positive and gram negative., , Principle, Differential staining requires the use of at least, four chemical reagents that are applied sequentially to a heat-fixed smear. The first reagent is, called the primary stain. Its function is to impart, its color to all cells. The second stain is a mordant, used to intensify the color of the primary stain., In order to establish a color contrast, the third, reagent used is the decolorizing agent. Based on, the chemical composition of cellular components,, the decolorizing agent may remove the primary, stain from the entire cell or only from certain cell, structures. The final reagent, the counterstain,, has a contrasting color to that of the primary, stain. Following decolorization, if the primary, stain is not washed out, the counterstain cannot, be absorbed, and the cell or its components will, retain the color of the primary stain. If the primary, stain is removed, the decolorized cellular components will accept and assume the contrasting color, of the counterstain. In this way, cell types or their, structures can be distinguished from each other on, the basis of the stain that is retained., The most important differential stain used, in bacteriology is the Gram stain, named after, Dr. Hans Christian Gram. It divides bacterial cells, into two major groups, gram positive and gram, negative, which makes it an essential tool for, classification and differentiation of microorganisms. Figure 10.1 shows gram-positive and gramnegative cells. The Gram stain reaction is based, on the difference in the chemical composition of, , 10, , bacterial cell walls. Gram-positive cells have a, thick peptidoglycan layer, whereas the peptidoglycan layer in gram-negative cells is much thinner, and surrounded by outer lipid-containing layers., Peptidoglycan is a polysaccharide composed of, two chemical subunits found only in the bacterial, cell wall. These subunits are N-acetylglucosamine, and N-acetylmuramic acid. With some organisms, as the adjacent layers of peptidoglycan are, formed, they are cross-linked by short chains of, peptides by means of a transpeptidase enzyme,, resulting in the shape and rigidity of the cell wall., In the case of gram-negative bacteria and several, of the gram-positive, such as the Bacillus, the, cross-linking of the peptidoglycan layer is direct, because the bacteria do not have short peptide, , (a) Gram-positive stain of streptococci, , (b) Gram-negative stain of E. coli, , Figure 10.1 Gram-stained cells, 71
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tails. Early experiments have shown that a grampositive cell denuded of its cell wall by the action, of lysozyme or penicillin will stain gram-negative., The Gram stain uses four different reagents., Descriptions of these reagents and their mechanisms of action follow. Figure 10.2 shows the, microscopic appearance of cells at each step of, the Gram staining procedure., , Primary Stain, Crystal Violet (Hucker’s): This violet stain is used, first and stains all cells purple., , Mordant, Gram’s Iodine: This reagent serves not only as, a killing agent but also as a mordant, a substance, that increases the cells’ affinity for a stain. The, reagent does this by binding to the primary stain,, thus forming an insoluble complex. The resultant, crystal–violet–iodine (CV-I) complex serves to, intensify the color of the stain. At this point, all, cells will appear purple-black., , Decolorizing Agent, Ethyl Alcohol, 95%: This reagent serves a dual, function as a protein-dehydrating agent and as a, lipid solvent. Its action is determined by two factors, the concentration of lipids and the thickness, Primary stain, , (a) Application, of crystal violet:, All cells are purple., , Mordant, , (b) Application of, Gram’s iodine:, All cells are purple-black., , of the peptidoglycan layer in bacterial cell walls., In gram-negative cells, the alcohol increases the, porosity of the cell wall by dissolving the lipids in, the outer layers. Thus, the CV-I complex can be, more easily removed from the thinner and less, highly cross-linked peptidoglycan layer. Therefore,, the washing-out effect of the alcohol facilitates the, release of the unbound CV-I complex, leaving the, cells colorless or unstained. The much thicker, peptidoglycan layer in gram-positive cells is, responsible for the more stringent retention of the, CV-I complex, as the pores are made smaller due, to the dehydrating effect of the alcohol. Thus, the, tightly bound primary stain complex is difficult to, remove, and the cells remain purple. Note: Be careful not to over-decolorize the smear with alcohol., , Counterstain, Safranin: This is the final reagent, used to stain, pink those cells that have been previously decolorized. Since only gram-negative cells undergo decolorization, they may now absorb the counterstain., Gram-positive cells retain the purple color of the, primary stain., Preparing adequately stained smears requires, the following precautions:, , 1. The most critical phase of the procedure is the, decolorization step, which is based on the ease, Decolorizing agent, , (c) 95% alcohol wash:, Gram-positive cells are, purple; gram-negative, cells are colorless., , Counterstain, , (d) Application of, safranin:, Gram-positive cells are, purple; gram-negative, cells are pink., , Figure 10.2 Microscopic observation of cells following steps in the Gram staining procedure, , 72, , Experiment 10
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with which the CV-I complex is released from, the cell. Remember that over-decolorization, will result in loss of the primary stain, causing, gram-positive organisms to appear gram negative. Under-decolorization, however, will not, completely remove the CV-I complex, causing gram-negative organisms to appear gram, positive. Strict adherence to all instructions, will help remedy part of the difficulty, but individual experience and practice are the keys to, correct decolorization., 2. It is imperative that, between applications, of the reagents, we thoroughly wash slides, under running water or water applied with an, eyedropper. This removes excess reagent and, prepares the slide for application of the subsequent reagent., 3. The best Gram-stained preparations are, made with fresh cultures (i.e., not older than, 24 hours). As cultures age, especially in the, case of gram-positive cells, the organisms tend, to lose their ability to retain the primary stain, and may appear to be gram-variable; that is,, some cells will appear purple, while others, will appear pink., , FUR T HE R R E AD I N G, Refer to the section in your textbook on slide preparation and the differences between simple and, complex stains for bacterial smears in the microbiology laboratory and their uses in research. In your, textbook’s index, use the search terms “Gram’s, Stain,” “Differential Stains,” and “Cell Wall.”, , C L I N I C A L A P P L I C AT I O N, Gram Staining: The First Diagnostic Test, The Gram stain is a diagnostic staining procedure, that can be done on body fluids, tissue biopsies,, throat cultures, samples from abscesses when, infection is suspected, and more. Clinically, important results are obtained much more rapidly, from staining than from culturing the specimen. The, results of the Gram stain will aid a clinical lab in, determining which additional tests may be required, for identifying the bacterial strain in question. Once, the bacterial gram type, shape, and orientation are, determined, a physician can choose the appropriate, antibiotic to treat the patient., , AT T HE BE NCH, , Materials, Cultures, Twenty-four–hour nutrient agar slant cultures of, ❏❏ Escherichia coli, ❏❏ Staphylococcus aureus BSL-2, ❏❏ Bacillus cereus, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, , Crystal violet, Gram’s iodine, 95% ethyl alcohol, Safranin, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop or needle, Staining tray, Glass slides, Bibulous paper, Lens paper, Microscope, , Procedure, Smear Preparation, 1. Obtain four clean glass slides., 2. Using aseptic technique, prepare a smear, of each of the three organisms, and on the, remaining slide, prepare a smear consisting, of a mixture of S. aureus BSL -2 , and E. coli., Do this by placing a drop of water on the, slide, and then transferring each organism, separately to the drop of water with a sterile,, cooled loop. Mix and spread both organisms, by means of a circular motion of the inoculating loop. Note: If bacteria are taken from, a broth culture, the drop of water is not, required. Place a loop of bacterial suspension, directly on the glass slide., , Experiment 10, , 73
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3. Allow smears to air-dry and then heat fix in the, usual manner., , Gram Staining, Figure 10.3 shows the following steps:, , 1. Gently flood smears with crystal violet and let, stand for 1 minute., 2. Gently wash with tap water., 3. Gently flood smears with the Gram’s iodine, mordant and let stand for 1 minute., 4. Gently wash with tap water., 5. Decolorize with 95% ethyl alcohol. Note:, Do not over-decolorize. Add reagent drop by, drop until the alcohol runs almost clear, showing only a blue tinge., 6. Gently wash with tap water., 7. Counterstain with safranin for 45 seconds., 8. Gently wash with tap water., 9. Blot dry with bibulous paper and examine, under oil immersion., 10. As you observe each slide under oil immersion, complete the chart provided in the Lab, Report., a. Draw a representative microscopic field., b. Describe the cells according to their morphology and arrangement., c. Describe the color of the stained cells., d. Classify the organism as to the Gram reaction: gram positive or gram negative., , 74, , Experiment 10, , TIPS FOR SUCCESS, 1. Proper slide preparation is key to successful, staining. Incorrect heat fixation will affect the, number of bacteria that will be present during, staining. Fixation that was not hot enough or, was too short will not allow the cells to adhere, to the glass slide properly, and the cells will be, rinsed away during the multiple stain and rinse, steps. Conversely, overheating will result in the, destruction of the cells and in cell debris adhering to the cells. Few, if any, cells will remain, intact for the staining process., 2. Timing of the decolorizing step may be the, most important aspect of the procedure. Overdecolorizing with an incorrect alcohol solution,, or allowing the slide to decolorize too long, will, remove the CV-I complex by causing extensive, damage to the cell membrane and cell wall,, even on a gram-positive cell. Alternatively,, decolorizing for too short a time period will not, remove enough CV-I complexes. The safraninstained cells will appear to be darker in color,, and could be mistaken for a light purple, grampositive stained cell., 3. The age of the culture or colony being stained, may impact the Gram stain results. The best, Gram-stained preparations are made with, fresh cultures that are no more than 24-hours, old. As cultures age, especially in the case, of gram-positive cells, the organisms tend to, lose their ability to retain the primary stain, and may appear to be gram variable; that is,, some cells will appear purple, while others will, appear pink.
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PROCEDURE, , 1 Gently stain with crystal violet for, 1 minute., , 2 Gently wash off the stain with, tap water., , 3 Gently apply Gram's iodine for, 1 minute., , 4 Gently wash off the Gram's iodine, with tap water., , 5 Add 95% alcohol drop by drop, until the alcohol runs almost clear., , 6 Gently wash off the 95% alcohol, with tap water., , 7 Counterstain with safranin for, 45 seconds., , 8 Gently wash off the safranin with, tap water., , 9 Blot dry with bibulous paper., , Figure 10.3 Gram staining procedure, , Experiment 10, , 75
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E XP E R IMENT, , 10, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, E. coli, , B. cereus, , S. aureus, , Mixture, , Draw a, representative field., , Cell morphology:, Shape, Arrangement, Cell color, Gram reaction, , Review Questions, 1. What are the advantages of differential staining procedures over the simple staining technique?, , 2. Cite the purpose of each of the following reagents in a differential staining procedure., a. Primary stain, , b. Mordant, , c. Decolorizing agent, , d. Counterstain, Experiment 10: Lab Report, , 77
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3. Why is it essential that the primary stain and the counterstain be contrasting colors?, , 4. Which is the most crucial step in performing the Gram staining procedures? Explain., , 5., , 78, , Because of a snowstorm, your regular laboratory session was canceled and the Gram staining procedure was performed on cultures incubated for a longer period of time. Examination of the stained B. cereus slides revealed a great deal of color variability, ranging from an intense, blue to shades of pink. Account for this result., , Experiment 10: Lab Report
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E XP E R IMENT, , 11, , Acid-Fast Stain, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Explain the chemical basis of the acid-fast, stain., 2. Perform the procedure to differentiate, bacteria into acid-fast and non–acid-fast, groups., , Principle, While the majority of bacterial organisms are stainable by either simple or Gram staining procedures,, a few genera, particularly the members of the genus, Mycobacterium, are visualized more clearly by the, acid-fast method. Since Mycobacterium tuberculosis and Mycobacterium leprae represent bacteria, that are pathogenic to humans, the stain is of diagnostic value in identifying these organisms., The characteristic difference between mycobacteria and other microorganisms is the presence of a, thick, waxy (lipoidal) wall that makes penetration, by stains extremely difficult. Mycobacteria tend to, clump together, and it is difficult to identify individual cells in stained preparations if this clumping, effect occurs. Avoiding or minimizing this phenomenon requires careful preparation of the smear. Place, a small drop of water on the slide, suspend the culture in the water, and mix the suspension thoroughly, to dislodge and disperse some of the cells. Once the, stain has penetrated, however, it cannot be readily, removed even with the vigorous use of acid-alcohol, as a decolorizing agent (unlike the 95% ethyl alcohol, used in the Gram stain). Because of this property,, these organisms are called acid-fast, while all other, microorganisms, which are easily decolorized by, acid-alcohol, are non–acid-fast. The acid-fast stain, uses the three different reagents listed below along, with a description of their purpose., , Primary Stain, Carbol fuchsin: Unlike cells that are easily stained, by ordinary aqueous stains, most species of, , mycobacteria are not stainable with common, dyes such as methylene blue and crystal violet., Carbol fuchsin, a dark red stain in 5% phenol that, is soluble in the lipoidal materials that constitute, most of the mycobacterial cell wall, does penetrate, these bacteria, and is retained. Applying heat, enhances penetration further, which drives the, carbol fuchsin through the lipoidal wall and into the, cytoplasm. This application of heat is used in the, Ziehl-Neelsen method. The Kinyoun method, a, modification of the Ziehl-Neelsen method, circumvents the use of heat by adding a wetting agent, (Tergitol®), which reduces surface tension between, the cell wall of the mycobacteria and the stain., Following application of the primary stain, all cells, appear red., , Decolorizing Agent, Acid-alcohol (3% HCl + 95% ethanol): Prior, to decolorization, the smear is cooled, which, allows the waxy cell substances to harden. On, application of acid-alcohol, acid-fast cells are, resistant to decolorization, since the primary stain, is more soluble in the cellular waxes than in the, decolorizing agent. In this event, the primary stain, is retained and the mycobacteria will stay red., This is not the case with non–acid-fast organisms,, which lack cellular waxes. The primary stain is, more easily removed during decolorization,, leaving these cells colorless or unstained., , Counterstain, Methylene blue: This is used as the final reagent, to stain previously decolorized cells. As only non–, acid-fast cells undergo decolorization, they may, now absorb the counterstain and take on its blue, color, while acid-fast cells retain the red of the, primary stain., , F U RT H E R RE A DI N G, Refer to the section in your textbook discussing, bacterial stains for further information on when, to use the acid-fast stain instead of the widely, used Gram stain. In your textbook’s index, search, under “Acid-Fast Stain,” “Mycolic Acid,” and “Soil, Microbes.”, 79
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PROCEDURE, , or, , 1a Heat method: Apply carbol fuchsin and steam over a beaker of, boiling water that is placed on a hot plate for 5 minutes. Do not, allow the stain to evaporate., , 2 Cool and wash off stain with, tap water., , 3 Add acid-alcohol drop by drop, until the alcohol runs almost clear., , 4 Wash off the acid-alcohol with, tap water., , 5 Counterstain with methylene blue, for 2 minutes., , 6 Wash off the methylene blue with, tap water., , 7 Blot the slide dry with bibulous, paper., , Figure 11.1 Acid-fast staining procedure, , 80, , 1b Heatless method: Apply carbol fuchsin, with Tergitol for 5 to 10 minutes., , Experiment 11
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C L I N I C A L A P P L I C AT I O N, Diagnosing Leprosy and Lung Infections, The cell walls of bacteria belonging to the genera, Mycobacterium and Nocardia contain mycolic acid, and are resistant to penetration by water-soluble, stains such as the Gram stain, which can lead to a, false gram-positive result. Acid-fast stains are medically important in diagnosing the Mycobacterium, species, which cause tuberculosis, leprosy, and, other infections. The genus Nocardia, which is the, causative agent for lung infections, is also identified, by the acid-fast staining method., , AT T H E B E N CH, , Materials, Cultures, ❏❏ 72- to 96-hour Trypticase soy broth culture of, Mycobacterium smegmatis, ❏❏ 18- to 24-hour culture of Staphylococcus, aureus BSL -2, , Reagents, ❏❏ Carbol fuchsin, ❏❏ Acid-alcohol, , ❏❏ Methylene blue, , Equipment, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Hot plate, ❏❏ 250-ml beaker, ❏❏ Inoculating loop, , ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Glass slides, Bibulous paper, Lens paper, Staining tray, Microscope, , Acid-Fast Staining, Figure 11.1 shows steps 1–7., , 1. a. Flood smears with carbol fuchsin and, place over a beaker of water on a warm hot, plate, allowing the preparation to steam, for 5_minutes. Note: Do not allow stain to, evaporate; replenish stain as needed. Also,, prevent stain from boiling by adjusting, the hot-plate temperature., b. For a heatless method, flood the smear with, carbol fuchsin containing Tergitol for 5 to, 10 minutes., 2. Wash with tap water. Heated slides must be, cooled prior to washing., 3. Decolorize with acid-alcohol, adding the, reagent drop by drop until the alcohol runs, almost clear with a slight red tinge., 4. Wash with tap water., 5. Counterstain with methylene blue for, 2 minutes., 6. Wash the smear with tap water., 7. Blot dry with bibulous paper and examine, under oil immersion., 8. In the chart provided in the Lab Report,, complete the following:, a. Draw a representative microscopic field for, each preparation., b. Describe the cells according to their shapes, and arrangements., c. Describe the color of the stained cells., d. Classify the organisms as to reaction: acidfast or non–acid-fast., Refer to Figure 11.2 for a photograph of an acidfast stain., , Procedure, Smear Preparation, 1. Obtain three clean glass slides., 2. Using aseptic technique, prepare a bacterial, smear of each organism plus a third mixed, smear of M. smegmatis and S. aureus BSL -2 ., 3. Allow smears to air-dry and then heat fix in the, usual manner., , Figure 11.2 Acid-fast stain of mycobacteria, , Experiment 11, , 81
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EX PE RIME NT, , 11, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, E. coli, , B. cereus, , S. aureus, , Mixture, , Draw a, representative field., , Cell morphology:, Shape, Arrangement, Cell color, Gram reaction, , Review Questions, 1. Why must you use heat or a surface-active agent when applying the primary stain during acid-fast, staining?, , 2. Why do you use acid-alcohol rather than ethyl alcohol as a decolorizing agent?, , Experiment 11: Lab Report, , 83
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3. What is the specific diagnostic value of this staining procedure?, , 4., , 5., , 84, , Why is the application of heat or a surface-active agent not required during the application, of the counterstain in acid-fast staining?, , A child presents symptoms suggestive of tuberculosis, namely a respiratory infection with a, productive cough. Microscopic examination of the child’s sputum reveals no acid-fast rods., However, examination of gastric washings reveals the presence of both acid-fast and non–acid-fast, bacilli. Do you think the child has active tuberculosis? Explain., , Experiment 11: Lab Report
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E XP E R IMENT, , Differential Staining, for Visualization of, Bacterial Cell Structures, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Explain the chemical basis for the spore, and capsule stains., 2. Perform the procedure to differentiate, between bacterial spore and vegetative, cell forms., 3. Perform the procedure to distinguish capsular material from the bacterial cell., , Spore Stain (SchaefferFulton Method), PA RT A, , Principle, Members of the anaerobic genera Clostridium, and Desulfotomaculum and the aerobic genus, Bacillus are examples of organisms that have the, capacity to exist either as metabolically active, vegetative cells or as highly resistant, metabolically inactive cell types called spores. When, environmental conditions become unfavorable for, continuing vegetative cellular activities, particularly with the exhaustion of a nutritional carbon, source, these cells have the capacity to undergo, sporogenesis and give rise to a new intracellular, structure called the endospore, which is surrounded by impervious layers called spore coats., As conditions continue to worsen, the endospore, is released from the degenerating vegetative cell, and becomes an independent cell called a free, spore. Because of the chemical composition of, spore layers, the spore is resistant to the damaging effects of excessive heat, freezing, radiation,, desiccation, and chemical agents, as well as to, the commonly employed microbiological stains., With the return of favorable environmental conditions, the free spore may revert to a metabolically, active and less resistant vegetative cell through, , 12, , germination (see Figure 12.1). Note that sporogenesis and germination are not means of reproduction but merely mechanisms that ensure cell, survival under all environmental conditions., In practice, the spore stain uses two different reagents. An alternative method known as the, Dorner method is widely published, and utilizes, nigrosin—which may be found on websites like, www.microbelibrary.org—as the counterstain., , Primary Stain, Malachite green: Unlike most vegetative cell, types that stain by common procedures, the free, spore, because of its impervious coats, will not, accept the primary stain easily. For further penetration, we must apply heat. After we apply the, primary stain and heat the smear, both the vegetative cell and spore appear green., , Decolorizing Agent, Water: Once the spore accepts the malachite green,, it cannot be decolorized by tap water, which, removes only the excess primary stain. The spore, remains green. On the other hand, the stain does, not demonstrate a strong affinity for vegetative, cell components; the water removes it, and these, cells will be colorless., , Counterstain, Safranin: This contrasting red stain is used as the, second reagent to color the decolorized vegetative cells, which will absorb the counterstain and, appear red. The spores retain the green of the, primary stain. A micrograph of spore-stained cells, appears in Figure 12.2., , F U RT H E R RE A D I N G, Refer to the section on differential stains in your, textbook for further information on the uses of, gram stain, acid-fast staining, and spore stains., In your textbook’s index, search under “Stains,”, “Spore Stains,” and “Endospores.”, , 85
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Binary fission, , Sporogenesis, , Favorable environmental, conditions, , Unfavorable environmental, conditions, Vegetative cell, , Forespore, , Endospore, Spore coat, rupture, , Degenerating, vegetative cell, and developing, spore coat, , Germination, Favorable, environmental, conditions, , Free spore, , Figure 12.1 Life cycle of a spore-forming bacterium, , C L I N I C A L A P P L I C AT I O N, , Free spore, , Identification of Dangerous Spore-Forming, Bacteria, Some spore-forming bacteria can have extremely, negative health effects. These bacteria include Bacillus anthracis, which causes anthrax, and certain, Clostridia bacteria, which are the causative agents, for tetanus, gas gangrene, food poisoning, and pseudomembranous colitis. Differential stains can stain, endospores inside bacterial cells, as well as free, spores, to identify these pathogenic bacteria., , Vegetative, cells, , Endospores, , AT T HE BE NCH, , Materials, Free spore, , Figure 12.2 Spore stain showing free spores and, vegetative bacilli, , 86, , Experiment 12, , Cultures, ❏❏ 48- to 72-hour nutrient agar slant culture of, Bacillus cereus, ❏❏ Thioglycollate culture of Clostridium, sporogenes
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Reagents, , Procedure, , ❏❏ Malachite green, ❏❏ Safranin, , Smear Preparation, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Hot plate, Staining tray, Inoculating loop, Glass slides, Bibulous paper, Lens paper, Microscope, , 1. Obtain two clean glass slides., 2. Make individual smears in the usual manner, using aseptic technique., 3. Allow smear to air-dry, and heat fix in the usual, manner., , Spore Staining, Figure 12.3 illustrates steps 1 to 5., , 1. Flood the smears with malachite green and, place on top of a beaker of water sitting on a, warm hot plate, allowing the preparation to, , PROCEDURE, , 1 Flood smears with malachite green and steam over, a beaker of water placed on a hot plate., , 3 Counterstain with safranin for, 30 seconds., , 2 Cool and wash off stain with tap water. The water, also serves as the decolorizing agent., , 4 Wash off the safranin with, tap water., , 5 Blot the slide dry with bibulous, paper., , Figure 12.3 Spore-staining procedure, , Experiment 12, , 87
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2., 3., 4., 5., 6., , steam for 2 to 3 minutes. Note: Do not allow, stain to evaporate; replenish stain as needed., Prevent the stain from boiling by adjusting the, hot plate temperature., Remove the slides from the hot plate, cool, and, wash under running tap water., Counterstain with safranin for 30 seconds., Wash with tap water., Blot dry with bibulous paper and examine, under oil immersion., In the chart provided in the Lab Report, complete the following:, a. Draw a representative microscopic field of, each preparation., b. Describe the location of the endospore, within the vegetative cell as central, subterminal, or terminal on each preparation., c. Indicate the color of the spore and vegetative cell on each preparation., , PART B, , Method), , Capsule Stain (Anthony, , Principle, A capsule is a gelatinous outer layer that is secreted, by the cell and surrounds and adheres to the cell, wall. It is not common to all organisms. Cells that, have a heavy capsule are generally virulent and capable of producing disease, since the structure protects, bacteria against the normal phagocytic activities of, host cells. Chemically, the capsular material is composed mainly of complex polysaccharides such as, levans, dextrans, and celluloses., Capsule staining is more difficult than other, types of differential staining procedures because, the capsular materials are water-soluble and may, be dislodged and removed with vigorous washing. We should not heat smears, because the, resultant cell shrinkage may create a clear zone, around the organism that is an artifact that can, be mistaken for the capsule. The capsule stain, uses two reagents: a primary stain and a decolorizing agent., , Primary Stain, Crystal violet (1% aqueous): A violet stain is applied to a non–heat-fixed smear. At this point, the cell, and the capsular material take on the dark color., , 88, , Experiment 12, , Decolorizing Agent, Copper sulfate (20%): Because the capsule is, nonionic, unlike the bacterial cell, the primary, stain adheres to the capsule but does not bind to, it. In the capsule staining method, copper sulfate is, used as a decolorizing agent rather than water. The, copper sulfate washes the purple primary stain, out of the capsular material without removing the, stain bound to the cell wall. At the same time, the, decolorized capsule absorbs the copper sulfate,, and the capsule appears blue in contrast to the, deep purple color of the cell. Figure 12.4 shows, the presence of a capsule as a clear zone surrounding the darker-stained cell., , C L I N I C A L A P P L I C AT I O N, Encapsulated Bacterial Pneumonia, The virulence of an organism is increased by the, presence of a capsule, since the capsule protects, the organism from phagocytosis by white blood, cells and inhibits antibody or complement fixation., The water-soluble polysaccharide and/or the polypeptide composition of the bacterial capsule makes, staining difficult. Gram-negative bacteria that form, capsules include Haemophilus influenzae and Klebsiella pneumoniae. Gram-positive bacteria that form, capsules include Bacillus anthracis and Streptococcus pneumoniae. If a bacterial infection is not, being cleared or responding to antibiotic therapy as, expected, staining of isolated organisms to determine the presence of a capsule may be warranted., , AT T HE BE NCH, , Materials, Cultures, ❏❏ Skimmed milk cultured for 48 hours with, Alcaligenes viscolactis, Leuconostoc mesenteroides, and Enterobacter aerogenes, , Reagents, ❏❏ 1% crystal violet, ❏❏ 20% copper sulfate (CuSO4 # 5H2O)
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Cell, Capsule, , (a) Enlarged illustration of a, completed capsule stain, , (b) Capsule stain: capsulated diplococci, , Figure 12.4 Capsule stain, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop or needle, Staining tray, Bibulous paper, Lens paper, Glass slides, Microscope, , Procedure, Figure 12.5 illustrates steps 1 to 5., , 1. Obtain one clean glass slide. Place several, drops of crystal violet stain on the slide., 2. Using aseptic technique, add three loopfuls of, a culture to the stain and gently mix with the, inoculating loop., 3. With a clean glass slide, spread the mixture over, the entire surface of the slide to create a very, , 4., 5., 6., 7., , thin smear. Let stand for 5 to 7 minutes. Allow, smears to air-dry. Note: Do not heat fix., Wash smears with 20% copper sulfate solution., Gently blot dry and examine under oil, immersion., Repeat steps 1 to 5 for each of the remaining, test cultures., In the chart provided in the Lab Report, complete the following:, a. Draw a representative microscopic field of, each preparation., b. Record the comparative size of the capsule;, that is, small, moderate, or large., c. Indicate the color of the capsule and of the, cell on each preparation., , Experiment 12, , 89
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PROCEDURE, , 1 Place several drops of crystal violet stain on a clean, glass slide., , 3 With a clean glass slide, spread, mixture to form a thin smear., Air-dry., , 4 Wash smear with 20% copper, sulfate solution., , Figure 12.5 Capsule staining procedure, , 90, , Experiment 12, , 2 Aseptically transfer 3 loopfuls of culture to the stain, and gently mix with the loop., , 5 Gently blot dry with bibulous paper.
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E XP E R IMENT, , 12, , Name:, , , Date:, , Lab Report, , Section:, , Observations and Results, PART A: Spore Stain, C. sporogenes, , B. cereus, , Draw a representative field., , Color of spores, Color of vegetative cells, Location of endospore, , PART B: Capsule Stain, A. viscolactis, , L. mesenteroides, , E. aerogenes, , Draw a representative field., , Capsule size, Color of capsule, Color of cell, , Experiment 12: Lab Report, , 91
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Review Questions, 1. Why is heat necessary in spore staining?, , 2. Explain the function of water in spore staining., , 3., , Assume that, during the performance of this exercise, you made several errors in your sporestaining procedure. In each of the following cases, indicate how your microscopic observations would differ from those observed when the slides were prepared correctly., a. You used acid-alcohol as the decolorizing agent., , b. You used safranin as the primary stain and malachite green as the counterstain., , c. You did not apply heat during the application of the primary stain., , 4. Explain the medical significance of a capsule., , 5. Explain the function of copper sulfate in this procedure., , 92, , Experiment 12: Lab Report
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PART 4, , Cultivation of Microorganisms:, Nutritional and Physical, Requirements, and Enumeration, of Microbial Populations, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Explain the nutritional and environmental requirements for the cellular activities of all forms of life., 2. Explain the principles associated with the use of routine and special-purpose, media for microbial cultivation., 3. Describe the diversified physical factors essential for microbial cultivation., 4. Utilize the specialized techniques for the cultivation of anaerobic, microorganisms., 5. Perform the serial dilution–agar plate technique for enumeration of viable, microorganisms., 6. Describe growth dynamics of bacterial populations., , Introduction, As do all living organisms, microorganisms require, certain basic nutrients and physical factors for, the sustenance of life. However, their particular, requirements vary greatly. Understanding these, needs is necessary to successfully cultivate microorganisms in the laboratory., , Nutritional Needs, In the laboratory, a variety of media supply the, nutritional needs of microbial cells. The following, list illustrates the nutritional diversity that exists, among microbes., 1. Carbon: This is the most essential and central, atom common to all cellular structures and, functions. Among microbial cells, two carbondependent types are noted:, a. Autotrophs: These organisms can be cultivated in a medium consisting solely of inorganic compounds; specifically, they use inorganic carbon in the form of carbon dioxide., b. Heterotrophs: These organisms cannot, be cultivated in a medium consisting solely, , of inorganic compounds; they must be, supplied with organic nutrients, primarily, glucose., 2. Nitrogen: This is also an essential atom in, many cellular macromolecules, particularly, proteins and nucleic acids. Proteins serve, as the structural molecules forming the socalled skeleton of the cell and as functional, molecules, enzymes, that are responsible for, the metabolic activities of the cell. Nucleic, acids include DNA—the genetic basis of, cell life—and RNA, which plays an active, role in protein synthesis within the cell., Some microbes use atmospheric nitrogen;, others rely on inorganic compounds,, including ammonium or nitrate salts; and still, others require nitrogen-containing organic, compounds such as amino acids., 3. Nonmetallic elements: Two major, nonmetallic ions are used for cellular nutrition:, a. Sulfur is integral to some amino acids, and is therefore a component of proteins., Sources include organic compounds, such, as sulfur-containing amino acids; inorganic, 93
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4., , 5., , 6., , 7., , compounds, such as sulfates; and elementary, sulfur., b. Phosphorus is necessary for the formation, of the nucleic acids DNA and RNA and also, for synthesis of the high-energy organic, compound adenosine triphosphate (ATP)., Phosphorus is supplied in the form of phosphate salts for use by all microbial cells., Metallic elements: Ca21, Zn21, Na1, K1,, Cu21, Mn21, Mg21, Fe21, and Fe3+ are some, of the metallic ions necessary for continued, efficient performance of varied cellular, activities. Some of these activities are, osmoregulation, regulation of enzyme activity,, and electron transport during biooxidation., Remember that these ions are micronutrients, and are required in trace concentrations only., Inorganic salts supply these materials., Vitamins: These organic substances, contribute to cellular growth and are essential, in minute concentrations for cell activities., They are also sources of coenzymes, which are, required for the formation of active enzyme, systems. Some microbes require vitamins to, be supplied in a preformed state for normal, metabolic activities. Some possess extensive, vitamin-synthesizing pathways, whereas, others can synthesize only a limited number, from other compounds present in the medium., Water: All cells require distilled water in the, medium so that the low-molecular-weight, nutrients can cross the cell membrane., Energy: Active transport, biosynthesis, and, biodegradation of macromolecules are the, metabolic activities of cellular life. These, activities can be sustained only if there is a, constant availability of energy within the cell., Two bioenergetic types of microorganisms, exist:, a. Phototrophs use radiant energy as their, sole energy source., b. Chemotrophs depend on oxidation of, chemical compounds as their energy, source. Some microbes use organic molecules, such as glucose; others utilize inorganic compounds, such as H2S or NaNO2., , Physical Factors, Three of the most important physical factors that, influence the growth and survival of cells are, temperature, pH, and the gaseous environment., , 94, , Part 4, , Understanding the roles they play in cell metabolism is essential., 1. Temperature influences the rate of chemical, reactions through its action on cellular, enzymes. Bacteria, as a group of organisms,, exist over a wide range of temperatures., However, individual species can exist only, within a narrower spectrum of temperatures., Low temperatures slow down or inhibit enzyme, activity, thereby slowing down or inhibiting, cell metabolism and, consequently, cell, growth. High temperatures cause coagulation, and thus irreversibly denature thermolabile, enzymes. Although enzymes differ in, their degree of heat sensitivity, generally, temperatures in the range of 70°C destroy, most essential enzymes and cause cell death., 2. The pH of the extracellular environment, greatly affects cells’ enzymatic activities., Most commonly, the optimum pH for cell, metabolism is in the neutral range of 7. An, increase in the hydrogen ion concentration,, resulting in an acidic pH (below 7), or a, decrease in the hydrogen ion concentration,, resulting in an alkaline pH (above 7), is often, detrimental. Either increase or decrease will, slow down the rate of chemical reactions, because of the destruction of cellular, enzymes, thereby affecting the rate of growth, and, ultimately, survival., 3. The gaseous requirement in most cells, is atmospheric oxygen, which is necessary, for the biooxidative process of respiration., Atmospheric oxygen plays a vital role in ATP, formation and the availability of energy in a, utilizable form for cell activities. Other cell, types, however, lack the enzyme systems for, respiration in the presence of oxygen, and, therefore must use an anaerobic form of respiration or fermentation., The following exercises will demonstrate the, diversity of nutritional and environmental requirements among microorganisms., , F U RT H E R RE A D I N G, Refer to the section on microbiological media in, your textbook, paying close attention to the uses, of the differential and selective media for the, cultivation of bacteria. In your textbook’s index,, use the search terms “Agar,” “Selective,” and, “Differential.”
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C AS E STUDY, HAND WASHING AND ASEPTIC TECHNIQUE: A CASE STUDY, Researchers have found indications of new species, of bacteria that may have antibiotic properties in a, water sample from a Rocky Mountain hot spring in, a northern North Dakota state park. A researcher, who was out mountain climbing located what she, considered to be a pristine sulfur hot spring, and, decided to take a collection of samples. She discovered that the spring had a distinct coloration., Using a field microscope and a field staining kit, the, researcher determined that the sample was composed primarily of a singularly shaped and Gram, staining microbe. The researcher concluded that, either this was due to the water conditions, or some, compound produced by the microbe is altering the, microbe competition in the spring. The samples, were taken at the water surface (minimal growth),, 20 cm below the surface (some growth), and 1 m, down (maximal growth). The samples were transported to the laboratory for bacterial isolation., , Laboratory technicians attempted to culture the microbe in the lab but failed to do so., Attempts were made using nutrient agar at different temperatures. With every attempt, no growth, was evident on the agar plates. Laboratory staff, wondered if the growth conditions or nutrient, compositions of the agars were restricting microbial growth., Questions to Consider:, 1. What nutrients or minerals are in a sulfur hot, spring that may be required in the agar for, maximum microbial growth?, 2. What environmental variable should, the lab manipulate based on apparent growth in the hot spring? Hint:, temperature-oxygen-nutrients., , Part 4, , 95
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Nutritional Requirements: Media for, the Routine Cultivation of Bacteria, , Once you have completed this experiment,, you should be able to, 1. Explain the abilities of several types of, media to support the growth of different, bacterial species., 2. Determine the nutritional needs of the, bacteria under study., , The exact chemical composition of these media, is not known. They are made of extracts of plant, and animal tissue and are variable in their chemical composition. Most contain abundant amino, acids, sugars, vitamins, and minerals; however, the, quantities of these constituents are not known., They are capable of supporting the growth of most, heterotrophs. We use the following two complex, media in this exercise., 1. Nutrient broth: This basic complex medium, is prepared by incorporating the following, ingredients per 1000 ml of distilled water:, , Principle, To satisfy the diverse nutritional needs of bacteria,, bacteriologists employ two major categories of, media for routine cultivation: chemically defined, media and complex media., , Chemically Defined Media, These are composed of known quantities of, chemically pure, specific organic and/or inorganic, compounds. Their use requires knowledge of the, organism’s specific nutritional needs. We use the, following two chemically defined media in this, exercise:, 1. Inorganic synthetic broth: This completely, inorganic medium is prepared by incorporating the following salts per 1000 ml of water:, 5.0 g, 0.2 g, 1.0 g, , (NH4H2PO4), , Dipotassium hydrogen phosphate, , 13, , Complex Media, , LEARNING OBJECTIVES, , Sodium chloride (NaCl), Magnesium sulfate (MgSO4), Ammonium dihydrogen phosphate, , E XP E R IMENT, , 1.0 g, , (K2HPO4), , Atmospheric carbon dioxide (CO2), 2. Glucose salts broth: This medium is composed of salts incorporated into the inorganic, synthetic broth medium plus glucose, 5 g per, liter, which serves as the sole organic carbon, source., , Peptone, Beef extract, , 5.0 g, 3.0 g, , Peptone, a semi-digested protein, is primarily, a nitrogen source. The beef extract, a beef, derivative, is a source of organic carbon, nitrogen, vitamins, and inorganic salts., 2. Yeast extract broth: This is composed of, the basic artificial medium ingredients used in, the nutrient broth plus yeast extract, 5 g per, liter, which is a rich source of vitamin B and, provides additional organic nitrogen and carbon compounds., The yeast extract broth is an example of an, enriched medium and is used for the cultivation of fastidious microorganisms—organisms, that have highly elaborate and specific nutritional, needs. These bacteria do not grow—or grow, poorly—on a basic artificial medium, and require, the addition of one or more growth-supporting, substances, enrichments such as additional plant, or animal extracts, vitamins, or blood., , Measuring Turbidity, In this experiment, you will evaluate (1) the abilities of media to support the growth of different, species of bacteria, and (2) the nutritional needs, of the bacteria. You will observe the amount of, growth, measured by turbidity, present in each culture following incubation. To evaluate the amount, , 97
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Tungsten lamp source, Galvanometer, % T scale, Entrance slit, A scale, , Objective lens, , Sample, cuvette, , Exit, slit, , Phototube, photomultiplier, , Wavelength, cam, , Diffraction, grating, , Figure 13.1 Schematic diagram of a spectrophotometer, , of growth accurately, a Bausch & Lomb Spectronic, 20 spectrophotometer will be used., This instrument measures the amount of light, transmitted (T) or absorbed (A). It transmits a, beam of light at a single wavelength (monochromatic light) through a liquid culture. The cells suspended in the culture interrupt the passage of light,, and the amount of light energy transmitted through, the suspension is measured on a photoelectric cell, and converted into electrical energy. The electrical, energy is then recorded on a galvanometer using a, range between 0% to 100% T. Figure 13.1 shows a, schematic representation of a spectrophotometer., In practice, the density of a cell suspension is expressed as absorbance (A) rather than, as percent T, since A is directly proportional to, , the concentration of cells, whereas percent T is, inversely proportional to the concentration of, suspended cells. Therefore, as the turbidity of a, culture increases, the A increases and percent T, decreases, indicating growth of the cell population, in the culture. For example, in comparing three, cultures with A readings of 0.10 (percent T = 78),, 0.30 (percent T = 49), and 0.50 (percent T = 30),, the A reading of 0.50 would be indicative of the, most abundant growth, and the 0.10 reading, would be indicative of the least amount of growth., Figure 13.2 shows the Bausch & Lomb Spectronic, 20 spectrophotometer, as well as a Unico RS1100, model. For the purpose of this experiment, we will, be discussing the procedure using the Busch &, Lomb Spectronic 20., , Pilot lamp, , Pilot lamp, Sample holder, , Wavelength, , Power switch/, zero control, , Sample holder, , Wavelength, , 100% control, (a), , (b), , Figure 13.2 (a) The Bausch & Lomb Spectronic 20 (b) and the Unico 1100RS spectrophotometer, , 98, , Experiment 13
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You will also use a colorimetric plate reader, to measure turbidity in small-volume cultures., Plate readers are generally used in a laboratory, setting to quantify the degree of color development at specified wavelengths in individual wells, of a 96-well plate. When using a 600-nm filter, a, plate reader can be used to determine the increase, in turbidity in a culture with less than a 200@mL, volume., , FUR T HE R R E AD I N G, Refer to the section on bacterial growth in your, textbook for further information on the use of, different growth media. In your textbook’s index,, search under “Complex Media,” “Defined Media,”, and “Turbidity.”, , C L I N I C A L A P P L I C AT I O N, The Purpose of Specialized Media, The successful cultivation of bacteria requires the, use of culture media containing the nutritional and, biochemical requirements capable of supporting, growth. There is no single medium that can support, the growth of all microorganisms. The development of a variety of specialized media overcomes, this challenge. For example, the streptococci require media supplemented with blood in order to, determine certain properties that are necessary, for isolation and species identification. Another example is the thioglycollate medium, which contains, thioglycolic acid that removes oxygen from the medium to encourage the growth of certain anaerobic, bacteria., Scientists are developing methods of using a set, standard to measure growth. Many procedures utilize sterile broth as a “blank” to set the spectrophotometer at zero and then measure the absorbance, above that zero setting as a means of quantifying, cell density. Commercially available standards such, as the McFarland Standards utilize microscopic, plastic particles to simulate cells in suspension and, allow for standardization in preparation of bacterial, suspensions. The 0.5 McFarland Standard has been, shown to correlate to 108 cells per ml in Escherichia, coli cultures., , AT T HE BE NCH, , Materials, Cultures, Saline suspension of 24-hour Trypticase soy broth, cultures, adjusted to 0.05 absorbance at a wavelength of 600 nm (or equilibrated to the 0.5 McFarland Standard), of, ❏❏ E. coli, ❏❏ Alcaligenes faecalis, ❏❏ Streptococcus mitis BSL -2 ., , Media, Per designated student group: three test tubes, (13 * 100 mm) of each type of broth:, ❏❏ Inorganic synthetic broth, ❏❏ Glucose salts broth, ❏❏ Nutrient broth, ❏❏ Yeast extract broth, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Sterile 1-ml serological pipettes, Mechanical pipetting device, Micropipette and tips, Glassware marking pencil, Test tube rack, 96-well clear plastic culture plate, Bausch & Lomb Spectronic 20 (or, comparable) spectrophotometer, ❏❏ Colorimetric plate reader, , Procedure Lab One, 1. Using sterile tips and a micropipette, add 100 ml, of the E. coli culture to one test tube of each of, the appropriately labeled media., 2. Using a micropipette, add 100 ml of broth and, 50 ml of the E. coli culture made in Step 1 to, designated wells in the 96-well plate., 3. Repeat steps 1 and 2 for inoculation with, A. faecalis and S. mitis BSL -2 ., 4. Follow manufacturer’s guidelines for the plate, reader to measure the turbidity of the wells at, 600 nm, and record the preliminary readings., 5. Incubate the test cultures for 24 to 48 hours at, 37°C., , Experiment 13, , 99
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Procedure Lab Two, Follow the instructions below and refer to, Figure 13.2 for a guide to the use of the Bausch &, Lomb Spectronic 20 spectrophotometer to obtain, the absorbance readings of all your cultures. Follow the instructions provided by the manufacturer or your instructor for all other spectrophotometers., 1. Use the plate reader to measure change in turbidity readings at 600 nm wavelength. Subtract, initial readings measured on Day One readings, from readings measured on Day Two. Record, the results., 2. Turn the spectrophotometer on 10 to 15 minutes, prior to use., 3. Set wavelength at 600 nm., 4. Set percent transmittance to 0% (A to 2) by turning the knob on the left., 5. Read the four yeast extract broth cultures as, follows:, a. Wipe the provided test tube of sterile yeast, broth that will serve as the blank for the, yeast broth culture readings clean. Fingerprints on the test tube will obscure the light, path of the spectrophotometer., b. Insert the yeast extract broth blank into the, tube holder, close the cover, and set the A, to 0 (percent T = 100) by turning the knob, on the right., c. Shake lightly or tap one of the tubes of, yeast extract broth culture to resuspend the, , 100, , Experiment 13, , bacteria, wipe the test tube clean, and allow, it to sit for several seconds for the equilibration of the bacterial suspension., d. Remove the yeast extract broth blank from, the tube holder., e. Insert a yeast extract broth culture into the, tube holder, close the cover, and read and, record the optical density reading in the, chart provided in the Lab Report., f. Remove the yeast extract broth culture, from the tube holder., g. Reset the spectrophotometer to an A of 2, with the tube holder empty and to an A of 0, with the yeast extract broth blank., h. Repeat steps c through g to read and record, the absorbance of the remaining yeast, extract broth cultures., 6. Repeat step 4 (a–h) to read and record the, absorbance of the nutrient broth cultures. Use, the provided nutrient broth blank to set the, spectrophotometer to an A of 0., 7. Repeat step 4 (a–h) to read and record the, absorbance of the glucose salts broth cultures., Use the provided glucose salts broth blank to, set the spectrophotometer to an A of 0., 8. Repeat step 4 (a–h) to read and record the, absorbance of the inorganic synthetic broth, cultures. Use the provided inorganic synthetic, broth blank to set the spectrophotometer to an, A of 0., 9. At the end of the experiment, return all cultures to the area designated for their disposal., 10. Complete the Lab Report.
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EXPERIMENT, , 13, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Optical Density Readings Using the Spectrophotometer, , Yeast Extract Broth, , Nutrient Broth, , Glucose Broth, , Inorganic, Synthetic Broth, , E. coli, A. faecalis, S. mitis, , Optical Density Readings Using a Colorimetric Plate Reader, , Yeast Extract Broth, , Nutrient Broth, , Glucose Broth, , Inorganic, Synthetic Broth, , E. coli, A. faecalis, S. mitis, , 1. On the basis of the previous data, list the media in order (from best to worst) according to their ability, to support the growth of bacteria., , 2. List the three bacterial species in order of their increasing fastidiousness., , 3. Why did the most fastidious organism grow poorly in the chemically defined medium?, , Experiment 13: Lab Report, , 101
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Review Questions, 1. Explain the advantages of using A readings rather than percent T as a means of estimating microbial, growth., , 2. Explain the reason for the use of different medium blanks in adjusting the spectrophotometer prior to, obtaining A readings., , 3. Why are complex media preferable to chemically defined media for routine cultivation of, microorganisms?, , 4. Would you expect a heterotrophic organism to grow in an inorganic synthetic medium? Explain., , 5., , A soil isolate is found to grow poorly in a basic artificial medium. You suspect that a vitamin, supplement is required., a. What supplement would you use to enrich the medium to support and maintain the growth of the, organism? Explain., , b. Outline the procedure you would follow to determine the specific vitamins required by the organism, to produce a more abundant growth., , 102, , Experiment 13: Lab Report
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Using Differential, Selective,, and Enriched Media, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the use and function of specialized media for the selection and differentiation of microorganisms., 2. Explain how an enriched medium like, blood agar can also function as both a, selective and differential medium., , Principle, Numerous special-purpose media are available for, functions including the following:, 1. Isolating bacterial types from a mixed population of organisms, 2. Differentiation among closely related groups, of bacteria by the colonies’ macroscopic, appearance and biochemical reactions within, the medium, 3. Enumeration of bacteria in sanitary microbiology, such as in water and sewage, and also in, food and dairy products, 4. Assay of naturally occurring substances,, including antibiotics, vitamins, and products, of industrial fermentation, 5. Characterization and identification of bacteria, by their abilities to produce chemical changes, in different media, In addition to nutrients necessary for the, growth of all bacteria, special-purpose media, contain both nutrients and chemical compounds, important for specific metabolic pathways in different types of bacteria. In this exercise, three, types of media will be studied and evaluated:, selective media, differential/selective media, and, enriched media., , Selective Media, These media are used to select (isolate) specific, groups of bacteria. They incorporate chemical, substances that inhibit the growth of one type of, , E XP E R IMENT, , 14, , bacteria while permitting growth of another, thus, facilitating bacterial isolation., 1. Phenylethyl alcohol agar: This medium is, used for the isolation of most gram-positive, organisms. The phenylethyl alcohol is partially, inhibitory to gram-negative organisms, which, may form visible colonies whose size and, number are much smaller than those on other, media., 2. Crystal violet agar: This medium is selective for most gram-negative microorganisms., Crystal violet dye exerts an inhibitory effect, on most gram-positive organisms., 3. 7.5% sodium chloride agar: This medium, is inhibitory to most organisms other than, halophilic (salt-loving) microorganisms. It is, most useful in the detection of members of the, genus Staphylococcus., Figure 14.1 illustrates the selective effect of, phenylethyl alcohol agar, which inhibits the, gram-negative organism Escherichia coli and, selects for the gram-positive organism Staphylococcus aureus., , Differential/Selective Media, These media can distinguish among morphologically and biochemically related groups of organisms. They incorporate chemical compounds that,, following inoculation and incubation, produce a, characteristic change in the appearance of bacterial growth and/or the medium surrounding the, colonies, which permits differentiation., Sometimes differential and selective characteristics are combined in a single medium. MacConkey agar is a good example of this because it, contains bile salts and crystal violet, which inhibit, gram-positive organisms and allow gram-negative, organisms to grow. In addition, it contains the, substrate lactose and the pH indicator neutral red,, which differentiates the red lactose-fermenting, colonies from the translucent non-fermenting, colonies. The following are examples of this type, of media:, 1. Mannitol salt agar: This medium contains, a high salt concentration, 7.5% NaCl, which, 103
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E. coli, , S. aureus, , (a) Nutrient agar, , E. coli, , S. aureus, , (b) Phenylethyl alcohol agar, , Figure 14.1 Selective effect of phenylethyl alcohol agar reduces the growth of E. coli, and selects for S. aureus, , is inhibitory to the growth of most but not all, bacteria other than the staphylococci. The, medium also performs a differential function:, It contains the carbohydrate mannitol, which, some staphylococci are capable of fermenting,, and phenol red, a pH indicator for detecting, acid produced by mannitol-fermenting staphylococci. These staphylococci exhibit a yellow, zone surrounding their growth; staphylococci, that do not ferment mannitol will not produce, a change in coloration., 2. MacConkey agar: The inhibitory action of, crystal violet on the growth of gram-positive, organisms allows the isolation of gram-negative bacteria. Incorporation of the carbohydrate lactose, bile salts, and the pH indicator, neutral red permits differentiation of enteric, bacteria on the basis of their ability to ferment, lactose. On this basis, enteric bacteria are, separated into two groups:, a. Coliform bacilli produce acid as a result of, lactose fermentation. The bacteria exhibit a, red coloration on their surface. E. coli produce greater quantities of acid from lactose, than do other coliform species. When this, occurs, the medium surrounding the growth, also becomes pink, because of the action of, the acid that precipitates the bile salts, followed by absorption of the neutral red., b. Dysentery, typhoid, and paratyphoid, bacilli are not lactose fermenters and, therefore do not produce acid. The colonies, appear tan and frequently transparent when, grown in MacConkey agar., , 104, , Experiment 14, , 3. Eosin–methylene blue agar (Levine):, Lactose and the dyes eosin and methylene, blue permit differentiation between enteric, lactose fermenters and non-fermenters as, well as identification of the colon bacillus,, E. coli. The E. coli colonies are blue–black, with a metallic green sheen caused by the, large quantity of acid that is produced and, that precipitates the dyes onto the growth’s, surface. Other coliform bacteria, such as, Enterobacter aerogenes, produce thick,, mucoid, pink colonies on this medium., Enteric bacteria that do not ferment lactose, produce colorless colonies, which because of, their transparency appear to take on the purple color of the medium. This medium is also, partially inhibitory to the growth of grampositive organisms, and thus gram-negative, growth is more abundant., A photographic representation of the effects, of selective/differential media is presented in, Figure 14.2., , Enriched Media, Enriched media are media that have been supplemented with highly nutritious materials, such as, blood, serum, or yeast extract, for the purpose of, cultivating fastidious organisms., For example, in blood agar, the blood incorporated into the medium is an enrichment ingredient for the cultivation of fastidious organisms,, such as the Streptococcus spp. The blood also, permits demonstration of the hemolytic properties of some microorganisms, particularly the
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Fermenter, , (a) Mannitol salt agar, , Nonfermenter, , Fermenter, , Nonfermenter, , (b) MacConkey agar, , (c) Eosin–methylene blue agar, , Figure 14.2 Effects of selective/differential media, , streptococci, whose hemolytic activities are classified as follows:, 1. Gamma hemolysis: No lysis of red blood, cells results in no significant change in the, appearance of the medium surrounding the, colonies., 2. Alpha hemolysis: Incomplete lysis of red, blood cells, with reduction of hemoglobin, to methemoglobin, results in a greenish halo, around the bacterial growth., 3. Beta hemolysis: Lysis of red blood cells with, complete destruction and use of hemoglobin by the organism results in a clear zone, surrounding the colonies. This hemolysis is, , (a) Gamma hemolysis, , produced by two types of beta hemolysins,, namely streptolysin O—an antigenic, oxygen-labile enzyme—and streptolysin S,, a nonantigenic, oxygen-stable lysin. The, hemolytic reaction is enhanced when blood, agar plates are streaked and simultaneously, stabbed to show subsurface hemolysis by, streptolysin O in an environment with reduced, oxygen tension. Based on the hemolytic, patterns on blood agar, the pathogenic betahemolytic streptococci may be differentiated, from other streptococci., Figure 14.3 shows the different types of hemolysis exhibited by different species of the genus, Streptococcus on blood agar., , (b) Alpha hemolysis, , (c) Beta hemolysis, , Figure 14.3 Types of hemolysis exhibited on a blood agar plate, , Experiment 14, , 105
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FU RT HER R E ADING, Refer to the section on selective and differential, media in your textbook for further information, on the use of different media for the isolation or, identification of bacteria. In your textbook’s index,, search under “Blood Agar,” “Enteric Bacteria,” and, “Bile Salts.”, , C L I N I C A L A P P L I C AT I O N, First Steps in Infected Wound Diagnosis, Wounds that have become infected may be, swabbed or surgically processed to remove tissue., Once stained samples have revealed infectious, agents, cultures are typically made on (1) blood, agar for isolation of staphylococci and streptococci, bacteria, (2) MacConkey agar for gram-negative, rods, and (3) enriched media that can support aerobes or anaerobes, such as thioglycollate broth., Additional media may be used, depending on what, was observed microscopically, including Sabouraud, dextrose agar for fungi and Löwenstein-Jensen, medium for acid-fast rods. Once the microbes are, isolated, further tests (which you will learn soon!), would likely be needed for complete identification., , AT THE B E N C H, , Materials, Cultures, 24- to 48-hour Trypticase® soy broth cultures of:, ❏❏ E. aerogenes, ❏❏ E. coli, ❏❏ Streptococcus var. Lancefield Group E, ❏❏ Streptococcus mitis BSL -2, ❏❏ Enterococcus faecalis BSL -2, ❏❏ Staphylococcus aureus BSL -2, ❏❏ Staphylococcus epidermidis, ❏❏ Salmonella typhimurium BSL -2, , Media, One of each per designated student group:, ❏❏ Phenylethyl alcohol agar, ❏❏ Crystal violet agar, ❏❏ 7.5% sodium chloride agar, , 106, , Experiment 14, , ❏❏, ❏❏, ❏❏, ❏❏, , Mannitol salt agar, MacConkey agar, Eosin–methylene blue agar, Blood agar, , Equipment, ❏❏ Microincinerator or Bunsen burner, ❏❏ Inoculating loop, ❏❏ Glassware marking pencil, , Procedure Lab One, 1. Using the bacterial organisms listed in the table,, prepare and inoculate each of the plates in the, following manner:, Agar Plate, , Organisms, , Phenylethyl, alcohol agar, , E. coli, S. aureus BSL -2 , and E. faecalis BSL -2 BSL -2, , Crystal violet, agar, , E. coli, S. aureus BSL -2 , and E. faecalis BSL -2 ., , 7.5% sodium, chloride agar, , S. aureus BSL -2 , S. epidermidis,, and E. coli, , Mannitol salt, agar, , S. aureus BSL -2 , S. epidermidis, E., aerogenes, and E. coli, , MacConkey, agar, , E. coli, E. aerogenes, S. typhimurium, BSL -2 , and S. aureus BSL -2, , Eosin–methylene blue agar, , E. coli, E. aerogenes, S. typhimurium, BSL -2 , and S. aureus BSL -2, , Blood agar, , E. faecalis BSL -2 , S. mitis, BSL -2 , and Streptococcus var., Lancefield Group E, , a. Label the cover of each plate appropriately,, as indicated in the Laboratory Protocol section on page xi., b. Divide each of the Petri dishes into the, required number of sections (one section, for each different organism) by marking the, bottom of the dish. Label each section with, the name of the organism to be inoculated,, as illustrated in Figure 14.4a., c. Using aseptic technique, inoculate all, plates, except the blood agar plate, with the, designated organisms by making a single, line of inoculation of each organism in its, appropriate section (Figure 14.4b). Be sure, to close the Petri dish and flame the inoculating needle between inoculations of the, different organisms.
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Fermenter, , Nonfermenter, , (a) Mannitol salt agar, , Fermenter, , (b) MacConkey agar, , Nonfermenter, , (c) Eosin–methylene blue agar, , Figure 14.4 Mannitol salt agar plate preparation and inoculation procedure, , d. Using aseptic technique, inoculate the blood, agar plate as described in step 1c. On completion of each single line of inoculation,, use the inoculating loop and make three or, four stabs at a 45° angle across the streak., 2. Incubate the phenylethyl alcohol agar plate in, an inverted position for 48 to 72 hours at 37°C., Incubate the remaining plates in an inverted, position for 24 to 48 hours at 37°C., , a. Amount of growth along line of inoculation as follows: 0 = none; 1+ = scant; and, 2 + = moderate to abundant, b. Appearance of the growth: coloration and, transparency, c. Change in the appearance of the medium, surrounding the growth: coloration and, transparency indicative of hemolysis, , Procedure Lab Two, 1. Carefully examine each of the plates. Note and, record the following in the chart provided in the, Lab Report:, , Experiment 14, , 107
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E XP E R IMENT, , 14, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Type of, Medium, Selective, , Medium, , Bacterial Species, , Amount of Growth, , Appearance, of Growth, , Appearance, of Medium, , Phenylethyl, alcohol agar, , Crystal violet agar, , 7.5% sodium, chloride agar, , Differential/, Selective, , Mannitol salt agar, , MacConkey agar, , Eosin–methylene, blue agar, , Enriched, , Blood agar, , Experiment 14: Lab Report, , 109
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Indicate the specific selective and/or differential purpose of each of the following media:, a. Phenylethyl alcohol agar, , b. Crystal violet agar, , c. 7.5% sodium chloride agar, , d. Mannitol salt agar, , e. MacConkey agar, , 110, , Experiment 14: Lab Report
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f. Eosin–methylene blue agar (Levine), , g. Blood agar, , Review Questions, 1. Explain the purpose of the following:, a. Crystal violet in the MacConkey agar medium, , b. Blood in the blood agar medium, , c. Eosin and methylene blue dyes in the eosin–methylene blue agar medium, , Experiment 14: Lab Report, , 111
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d. High salt concentration in the mannitol salt agar medium, , e. Lactose in the MacConkey agar medium, , f. Phenylethyl alcohol in the phenylethyl alcohol agar medium, , 2. Why are crystal violet agar and 7.5% sodium chloride agar considered selective media?, , 3., , 112, , A patient exhibits a boil on his neck. You, as a microbiology technician, are asked to identify the causative organism and determine whether it is pathogenic. Describe the procedure, that you would follow to make this determination., , Experiment 14: Lab Report
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E XP E R IMENT, , 15, , Physical Factors: Temperature, , requires a narrower range that is determined by the, heat sensitivity of its enzyme systems. Specific temperature ranges consist of the following cardinal, (significant) temperature points (Figure 15.1):, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Determine the diverse temperature requirements for bacteria growth., , 1. Minimum growth temperature: The lowest temperature at which growth will occur., Below this temperature, enzyme activity is, inhibited and the cells are metabolically inactive so that growth is negligible or absent., 2. Maximum growth temperature: The highest temperature at which growth will occur., Above this temperature, most cell enzymes are, destroyed and the organism dies., 3. Optimum growth temperature: The temperature at which the rate of reproduction is most, rapid; however, it is not necessarily optimum, or ideal for all enzymatic activities of the cell., , 2. Determine whether the optimum growth, temperature is also the ideal temperature, for enzyme-regulated cell activities, such, as pigment production and carbohydrate, fermentation., , Principle, Microbial growth is directly dependent on how temperature affects cellular enzymes. With increasing, temperatures, enzyme activity increases until the, three-dimensional configuration of these molecules, is lost because of denaturation of their protein, structure. As the temperature is lowered toward, the freezing point, enzyme inactivation occurs and, cellular metabolism gradually diminishes. At 0°C,, biochemical reactions cease in most cells., Bacteria, as a group of living organisms, are, capable of growth within an overall temperature, range of - 5°C to 80°C. Each species, however,, , 1. Psychrophiles: Bacterial species that will, grow within a temperature range of 5°C - 5°C, to 20°C. The distinguishing characteristic of all, psychrophiles is that they will grow between, 0°C and 5°C., , Mesophile, , Thermophile, , Population growth rate, , Psychrophile, , Figure 15.2 shows the effects of temperature, on bacterial growth and pigment production., All bacteria can be classified into one of three, major groups, depending on their temperature, requirements:, , 0, , 5, , 10, , 15, , 20, , Optimum, temperature, for psychrophile, , 25, , 30, , 35, , 40, , 45, , 50, , Optimum, temperature, for mesophile, , 55, , 60, , 65, , 70, , Optimum, temperature, for thermophile, , Temperature (°C), , Figure 15.1 Effect of temperature on the growth of microorganisms, , 113
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5°C, , 22°C, , 35°C, , 45°C, , Figure 15.2 Effect of temperature on bacterial growth and pigmentation, , 2. Mesophiles: Bacterial species that will grow, within a temperature range of 20°C to 45°C., The distinguishing characteristics of all mesophiles are their ability to grow at human body, temperature (37°C) and their inability to grow, at temperatures above 45°C. Included among, the mesophiles are two distinct groups:, a. Mesophiles with optimum growth temperature between 20°C and 30°C are plant, saprophytes., b. Mesophiles with optimum growth temperature between 35°C to 40°C are organisms, that prefer to grow in the bodies of warmblooded hosts., 3. Thermophiles: Bacterial species that will, grow at 35°C and above. Two groups of thermophiles exist:, a. Facultative thermophiles: organisms that, will grow at 37°C, with an optimum growth, temperature of 45°C to 60°C, b. Obligate thermophiles: organisms that, will grow only at temperatures above 50°C,, with optimum growth temperatures above, 60°C, The ideal temperature for specific enzymatic, activities may not coincide with the optimum, growth temperature for a given organism. To, understand this concept, you will investigate pigment production and carbohydrate fermentation, by selected organisms at a variety of incubation, temperatures., 1. Serratia marcescens produces an endogenous, red or magenta pigment, depending on the, presence of an orange to deep red coloration, on the surface of the colonial growth., , 114, , Experiment 15, , 2. Carbohydrate fermentation by Saccharomyces, cerevisiae is indicated by the presence of gas,, one of the end products of this fermentative, process. Detection of this accumulated gas, may be noted as an air pocket, of varying size,, in an inverted inner vial (Durham tube) within, the culture tube.Refer to Experiment 21 for, a more extensive discussion of carbohydrate, fermentation., , F U RT H E R RE A D I N G, Refer to the section on bacterial growth in your, textbook for further information on the different, growth characteristics of bacteria. In your textbook’s index, search under “Growth Curve,” “Temperature,” and “Psychrophile.”, , C L I N I C A L A P P L I C AT I O N, Cold-Resistant Killers, Food science is highly concerned with the temperature-related growth patterns of bacteria. Refrigeration temperatures below 4.4°C are generally, considered safe for the short-term storage of food,, since most pathogenic bacteria grow very slowly, below that temperature. However, some dangerous, bacteria are resistant to cold. Listeria monocytogenes, which causes a flu-like illness and can be, deadly, is capable of doubling its population every, 36 hours, even at 4.2°C, and can still attain slow, growth below 2°C. The cold tolerance of Listeria, may be due to adaptive genes, prompting research, into novel methods of controlling its growth at low, temperatures.
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AT T H E B E N C H, , Materials, Cultures, 24- to 48-hour nutrient broth cultures of, ❏❏ Escherichia coli, ❏❏ Bacillus stearothermophilus, ❏❏ Pseudomonas savastanoi, ❏❏ S. marcescens, ❏❏ Sabouraud broth culture of S. cerevisiae, , Media, Four of each per designated student group:, ❏❏ Trypticase soy agar plates, ❏❏ Sabouraud broth tubes containing inverted, Durham tubes, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Refrigerator set at 4°C, Two incubators set at 37°C and 60°C, Sterile Pasteur pipette, Test tube rack, Glassware marking pencil, , Procedure Lab One, 1. Score the underside of each plate into four, quadrants with a glassware marker. Label each, section with the name of the test organism to, be inoculated. When labeling the cover of each, plate, include the temperature of incubation, (4°C, 20°C, 37°C, or 60°C)., 2. Aseptically inoculate each of the plates with, E. coli, B. stearothermophilus, P. savastanoi,, and S. marcescens by means of a single line of, , inoculation of each organism in its appropriately labeled section., 3. Appropriately label the four Sabouraud broth, tubes, including the temperatures of incubation, as indicated above., 4. Gently shake the S. cerevisiae culture to suspend the organisms. Using a sterile Pasteur, pipette, aseptically add one drop of the culture, into each of the four tubes of broth media., 5. Incubate all plates in an inverted position and, the broth cultures at each of the four experimental temperatures (4°C, 20°C, 37°C, and, 60°C) for 24 to 48 hours., , Procedure Lab Two, 1. In the chart provided in the Lab Report, complete the following:, a. Observe all the cultures for the presence of, growth. Record your observations: (1+ ) for, scant growth; (2 + ) for moderate growth;, (3 + ) for abundant growth; and (- ) for the, absence of growth. Evaluate the amount of, growth in the S. cerevisiae cultures by noting the degree of developed turbidity., b. Observe the S. marcescens growth on, all the plate cultures for the presence or, absence of orange to deep red pigmentation. Record the presence of pigment on, a scale of 1 + to 3+ , and enter (- ) for the, absence of pigmentation., c. Observe the S. cerevisiae cultures for the, presence of a gas pocket in the Durham, tube, which is indicative of carbohydrate, fermentation. Record your observations, using the following designations: (1+ ) for a, minimal amount of gas; (2+ ) for a moderate, amount of gas; (3+ ) for a large amount of, gas; and (- ) for the absence of gas., d. Record and classify the cultures as psychrophiles, mesophiles, facultative thermophiles, or obligate thermophiles., , Experiment 15, , 115
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E XP E R IMENT, , 15, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, , S. marcescens, Temperature, , Pigment, , Growth, , P. savastanoi, , E. coli, , B. stearothermophilus, , Growth, , Growth, , Growth, , S. cerevisiae, Growth, , Gas, , 4°C (refrigerator), 20°C (room temp.), 37°C (body temp.), 60°C, Classification, , Based on your observations of the S. marcescens and S. cerevisiae cultures, is the optimum growth temperature the ideal temperature for all cell activities? Explain., , Review Questions, 1. In the following chart, indicate the types of organisms that would grow preferentially in or on various, environments, and indicate the optimum temperature for their growth., Environment, , Type of Organism, , Optimum Temperature, , Ocean bottom near shore, Ocean bottom near hot vent, Hot sulfur spring, Compost pile (middle), High mountain lake, Center of an abscess, Antarctic ice, , Experiment 15: Lab Report, , 117
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2. Explain the effects of temperatures above the maximum and below the minimum growth temperatures on cellular enzymes., , 3., , If an organism grew at 20°C, explain how you would determine experimentally whether the, organism was a psychrophile or a mesophile., , 4., , Is it possible for thermophilic organisms to induce infections in warm-blooded animals?, Explain., , 118, , Experiment 15: Lab Report
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E XP E R IME NT, , 16, , Physical Factors: pH of the, Extracellular Environment, , Despite this diversity and the fact that certain, organisms can grow at extremes of the pH scale,, generalities can be made. The specific range for, bacteria is between 4 and 9, with the optimum, being 6.5 to 7.5. Fungi (molds and yeasts) prefer, an acidic environment, with optimum activities at, a pH of 4 to 6., Because a neutral or nearly neutral environment is generally advantageous to the growth of, microorganisms, the pH of the laboratory medium, is frequently adjusted to approximately 7. Metabolic activities of the microorganism will result, in the production of wastes, such as acids from, carbohydrate degradation and alkali from protein, breakdown, and these will cause shifts in pH that, can be detrimental to growth., To retard this shift, chemical substances, that act as buffers are frequently incorporated, when the medium is prepared. A commonly, used buffering system involves the addition of, equimolar concentrations of K2HPO4, a salt of, a weak base, and KH2PO4, a salt of a weak acid., In a medium that has become acidic, the K2HPO4, absorbs excess H+ to form a weakly acidic salt and, a potassium salt with the anion of the strong acid., , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Describe pH requirements of, microorganisms., , Principle, Microorganisms’ growth and survival are greatly, influenced by the pH of the environment, and, all bacteria and other microorganisms differ as to their requirements. Based on their, optimal pH, microorganisms may be classified, as acidophiles, neutrophiles, or alkalophiles, (Figure 16.1). Each species has the ability to, grow within a specific pH range; the range may, be broad or limited, with the most rapid growth, occurring within a narrow, optimum range., These specific pH needs reflect the organisms’, adaptations to their natural environment. For, example, enteric bacteria are capable of survival, within a broad pH range, which is characteristic, of their natural habitat, the digestive system., Bacterial blood parasites, on the other hand,, can tolerate only a narrow range; the pH of the, circulatory system remains fairly constant at, approximately 7.4., , S KH2PO4 + KCL, K2HPO4 + HCL, Salt of a, Strong, Salt of a, Potassium, weak acid acid, weak acid chloride, salt, , Neutrophile, , Optimum pH, for acidophile, , Optimum pH, for neutrophile, , Alkalophile, , Population growth rate, , Acidophile, , 0, , 7, , 14, Optimum pH, for alkalophile, , pH, , Figure 16.1 Effect of pH on the growth of microorganisms, , 119
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In a medium that has become alkaline, KH2PO4, releases H+, which combines with the excess OHto form water, and the remaining anionic portion, of the weakly acidic salt combines with the cation, of the alkali., S K2HPO4, KH2PO4, + KOH, + H2O, Strong, Salt of a, water, Salt of a, weak acid, base, weak base, , Most media contain amino acids, peptones,, and proteins, which can act as natural buffers, because of their amphoteric nature. For example,, amino acids are zwitterions, molecules in which, the amino group and the carboxyl group ionize to, form dipolar ions. These behave in the following, manner:, Acidic, medium H+, , H+, , Basic, medium, , H, H, , N, , RCH, , COO, , H, , H, , NH3+, , Materials, Cultures, Saline suspensions of 24-hour nutrient broth, cultures, adjusted to an absorbance (A) of 0.05, or equilibrated to a 0.5 McFarland Standard at a, wavelength of 600 nm, of the following:, ❏❏ Alcaligenes faecalis ❏❏ Saccharomyces, ❏❏ Escherichia coli, cerevisiae, , Media, 12 total Trypticase® soy broth (TSB) tubes per, designated student group, with 3 tubes each of the, following pH designations:, ❏❏ pH 3.0, ❏❏ pH 7.0, ❏❏ ph 6.0, ❏❏ pH 9.0, The pH should be adjusted with 1N sodium, hydroxide or 1N hydrochloric acid., , Equipment, RCH, , COO–, , FU RT HER R E ADING, Refer to the section on bacterial growth in your, textbook for further information on the use, of different growth media. In your textbook’s, index, search under “Buffered Media,” “pH,” and, “Acidophile.”, , C L I N I C A L A P P L I C AT I O N, pH as a Defense Against Infection, Most bacteria grow best at a pH between 6.5 and, 7.5, and fungi show optimal growth between a pH of, 4 and 6. Many microorganisms are not able to cause, stomach infections because the pH of the stomach, is 2.0, resembling that of hydrochloric acid. In this, way, the acid of the stomach acts as a defense, against infection. By the same token, the pH of the, skin varies between 4 and 7, with lower ranges, (around 5) being the most common, helping prevent, many infections of the skin., , 120, , AT T HE B EN CH, , Experiment 16, , ❏❏ Microincinerator, or Bunsen burner, ❏❏ Sterile 1-ml, pipettes, ❏❏ Mechanical pipetting device, , ❏❏ Bausch & Lomb, Spectronic 20 spectrophotometer, ❏❏ Test tube rack, ❏❏ Glassware marking, pencil, , Procedure Lab One, 1. Using a sterile pipette, inoculate a series of the, appropriately labeled TSB tubes of media, pH, values of 3, 6, 7, and 9, with E. coli by adding, 0.1 ml of the saline culture to each., 2. Repeat step 1 for the inoculation of A. faecalis, and then of S. cerevisiae, using a new sterile, pipette each time., 3. Incubate the A. faecalis and E. coli cultures, for 24 to 48 hours at 37°C and the S. cerevisiae, cultures for 48 to 72 hours at 25°C., , Procedure Lab Two, 1. Using the spectrophotometer as described in, Experiment 14, determine the absorbance of, all cultures. Record the readings in the chart, provided in the Lab Report., 2. In the second chart provided in the Lab, Report, summarize your findings as to the, overall range and optimum pH of each organism studied.
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E XP E R IMENT, , 16, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Absorbance Readings, ABSORBANCE READINGS, Microbial Species, , pH 3, , pH 6, , pH 7, , pH 9, , pH Summary, Microbial Species, , pH Range, , Optimum pH, , Review Questions, 1. Explain the mechanism by which buffers prevent radical shifts in pH., , 2. Explain why it is necessary to incorporate buffers into media in which, microorganisms are grown., , Experiment 16: Lab Report, , 121
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3. Why are proteins and amino acids considered to be natural buffers?, , 4. Explain why microorganisms differ in their pH requirements., , 5. Will all microorganisms grow optimally at a neutral pH? Explain., , 6., , You are instructed to grow E. coli in a chemically defined medium, containing glucose and NH4Cl as the carbon and nitrogen sources, and also in nutrient broth that contains beef extract and peptone. Both, media are adjusted to a pH of 7. With turbidity as an index for the amount of, growth in each of the cultures, the following spectrophotometric readings, are obtained following incubation:, ABSORBANCE READINGS, Time (Hours), , Chemically Defined Medium, , Nutrient Broth Medium, , 6, , 0.100, , 0.100, , 12, , 0.300, , 0.500, , 18, , 0.275, , 0.900, , 24, , 0.125, , 1.500, , Based on the previously given data, explain why E. coli ceased growing in, the chemically defined medium but continued to grow in the nutrient broth., , 122, , Experiment 16: Lab Report
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E XP E R IMENT, , Physical Factors: Atmospheric, Oxygen Requirements, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Describe the diverse atmospheric oxygen, requirements of microorganisms., , Principle, Microorganisms exhibit great diversity in their, ability to use free oxygen (O2) for cellular respiration. These variations in O2 requirements reflect, the differences in biooxidative enzyme systems, present in the various species. Microorganisms, can be classified into one of five major groups, according to their O2 needs:, 1. Aerobes require the presence of atmospheric, oxygen for growth. Their enzyme system, necessitates use of O2 as the final hydrogen, (electron) acceptor in the complete oxidative, degradation of high-energy molecules, such as, glucose., 2. Microaerophiles require limited amounts, of atmospheric oxygen for growth. Oxygen, in excess of the required amount appears to, block the activities of their oxidative enzymes, and results in death., 3. Obligate anaerobes require the absence of, free oxygen for growth because their oxidative enzyme system requires the presence of, molecules other than O2 to act as the final, hydrogen (electron) acceptor. In these organisms, as in aerobes, the presence of atmospheric oxygen results in the formation of, toxic metabolic end products, such as superoxide, O2, a free radical of oxygen. However,, these organisms lack the enzymes superoxide, dismutase and c atalase, whose function is to, degrade the superoxide to water and oxygen, as follows:, , 17, , 2O2- + 2H +, , Superoxide, , ¡, , dismutase, , H2O2 + O2, , Catalase, , 2H2O2 ¡ 2H2O + O2, , In the absence of these enzymes, small, amounts of atmospheric oxygen are lethal, and, these organisms are justifiably called obligate, anaerobes., 4. Aerotolerant anaerobes are fermentative, organisms, and therefore do not use O2 as a, final electron acceptor. Unlike the obligate, anaerobes, they produce catalase and/or, superoxide dismutase, and thus are not killed, by the presence of O2. Hence, these organisms, are anaerobes that are termed aerotolerant., 5. Facultative anaerobes can grow in the, presence or absence of free oxygen. They preferentially use oxygen for aerobic respiration., However, in an oxygen-poor environment,, cellular respiration may occur anaerobically—, utilizing such compounds as nitrates (NO3-), or sulfates (SO42-) as final hydrogen acceptors—or via a fermentative pathway. (Refer to, Experiment 23.), The oxygen needs of microorganisms can be, determined by noting their growth distributions, following a shake-tube inoculation. This procedure requires introduction of the inoculum into, a melted agar medium, shaking of the test tube, to disperse the microorganisms throughout the, agar, and rapid solidification of the medium to, ensure that the cells remain dispersed. Following, incubation, the growth distribution indicates the, organisms’ oxygen requirements. Aerobes exhibit, surface growth, whereas anaerobic growth is limited to the bottom of the deep tube. Facultative, anaerobes, because of their indifference to the, presence or absence of oxygen, exhibit growth, throughout the medium. Microaerophiles grow in, a zone slightly below the surface. Figure 17.1 illustrates the shake-tube inoculation procedure and, the distribution of growth following an appropriate incubation period., , 123
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PROCEDURE, , 1 Transfer two drops of inoculum from the test culture into, a melted agar deep tube., , 2 Disperse the organisms throughout the molten agar, medium by rapidly rotating the tube between the palms, of your hands., , 3 Cool rapidly by immersion in an iced waterbath., , 4 Incubate at 37°C., , Distribution of Growth, , Oxygen, concentration, High, , Low, , Aerobic, , Microaerophilic, , Facultative, anaerobic, , Aerotolerant, , Figure 17.1 Procedure for determination of oxygen requirements, , 124, , Experiment 17, , Anaerobic
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FUR T HE R R E AD I N G, , Equipment, , Refer to the section on bacterial growth in your, textbook for further information on atmospheric, conditions required for bacterial growth. In your, textbook’s index, search under “Aerobe,” “Final, Electron Acceptor,” and “Anaerobe.”, , ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , C L I N I C A L A P P L I C AT I O N, Differentiating Aerobes and Anaerobes, Samples suspected of containing anaerobes need, to be handled carefully and transported promptly, to a lab, where they are typically inoculated onto, anaerobic blood agar plates and anaerobic broth, as, well as onto MacConkey agar and an aerobic blood, plates. Growth on aerobic or anaerobic agars will, determine oxygen requirements, while comparable, growth on both aerobic and anaerobic media suggests a facultative anaerobe., , AT T H E B E N CH, , Materials, Cultures, 24- to 48-hour nutrient broth cultures of, ❏❏ Staphylococcus aureus BSL -2, ❏❏ Corynebacterium xerosis, ❏❏ Enterococcus faecalis BSL -2, 48- to 72-hour cultures of, ❏❏ Saccharomyces cerevisiae in Sabouraud broth, ❏❏ Aspergillus niger in Sabouraud broth, ❏❏ Clostridium sporogenes in Thioglycollate, broth, , Media, ❏❏ Six brain heart infusion agar deep tubes per, designated student group, , Microincinerator or Bunsen burner, Waterbath, Iced waterbath, Thermometer, Sterile Pasteur pipettes, Test tube rack, Glassware marking pencil, , Procedure Lab One, 1. Liquefy the sterile brain heart infusion agar by, boiling in a waterbath at 100°C., 2. Cool molten agar to 45°C; check the, temperature with a thermometer inserted into, the waterbath., , Determining Oxygen Requirements, 1. Using aseptic technique, inoculate each, experimental organism by introducing two, drops of the culture from a sterile Pasteur, pipette into the appropriately labeled tubes of, molten agar., 2. Vigorously rotate the freshly inoculated molten infusion agar between the palms of the, hands to distribute the organisms., 3. Place inoculated test tubes in an upright, position in the iced waterbath to solidify the, medium rapidly., 4. Incubate the S. aureus BSL -2 , C. xerosis,, E. faecalis BSL -2 , and C. sporogenes cultures, for 24 to 48 hours at 37°C, and the A. niger, and S. cerevisiae cultures for 48 to 72 hours, at 25°C., , Procedure Lab Two, 1. Observe each of the experimental cultures for, the distribution of growth in each tube., 2. Record your observations and your determination of the oxygen requirements for each of, the experimental species in the chart provided, in the Lab Report., , Experiment 17, , 125
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E XP ER IME NT, , 17, , Name:, Date:, , Section:, , Lab Report, , Observations and Results, Species, , Distribution of Growth, , Classification According to, Oxygen Requirement, , S. aureus, C. xerosis, E. faecalis, A. niger, S. cerevisiae, C. sporogenes, , Review Questions, 1. Why is it necessary to place the inoculated molten agar cultures in an iced waterbath, for rapid solidification?, , 2. As indicated by its oxygen requirements, which group of microorganisms has the, most extensive bioenergetic enzyme system? Explain., , Experiment 17: Lab Report, , 127
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3. Account for the inability of aerobes to grow in the absence of O2., , 4. Account for the subsurface growth of microaerophiles in a shake-tube culture., , 5., , Consider the culture type in which growth was distributed throughout the, entire medium and explain why the growth was more abundant toward the, surface of the medium in some c ultures, whereas other cultures showed an equal distribution of growth throughout the tubes., , 6., , Account for the fact that the C. sporogenes culture showed a separation, within the medium or an elevation of the medium from the bottom of the, test tube., , 7., , 128, , Your instructor asks you to explain why the Streptococcus species that are, catalase-negative are capable of growth in the presence of oxygen. How, would you respond?, , Experiment 17: Lab Report
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E XP E R IMENT, , 18, , Techniques for Cultivating, Anaerobic Microorganisms, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Describe the methods for cultivation of, anaerobic organisms., , Redox, potential, High, Free exchange, of oxygen, , Principle, Microorganisms differ in their abilities to use, oxygen for cellular respiration. Respiration, involves the oxidation of substrates for energy, necessary to life. A substrate is oxidized when it, loses a hydrogen ion and its electron (H+e-). Since, the H+e- cannot remain free in the cell, it must, immediately be picked up by an electron acceptor, which becomes reduced. Therefore, reduction means gaining the H+e-. These reactions are, termed oxidation-reduction (redox) reactions., Some microorganisms have enzyme systems in, which oxygen can serve as an electron acceptor,, thereby being reduced to water. These cells have, high oxidation-reduction potentials; others have, low potentials and must use other substances as, electron acceptors., The enzymatic differences in microorganisms, are explained more fully in the section dealing, with metabolism (see Part 5). This discussion, is limited to cultivation of the strict anaerobes,, which cannot be cultivated in the presence of, atmospheric oxygen (Figure 18.1). The procedure, is somewhat more difficult because it involves, sophisticated equipment and media enriched, with substances that lower the redox potential., Figure 18.2 shows some of the methods available, for anaerobic cultivation., The following experiment uses fluid thioglycollate medium and the GasPak anaerobic system., , Aerobic cells, 2H+e– + O2, H 2O, Reduction: oxygen final, electron acceptor, Facultatively anaerobic cells, , Low, , Decreased, exchange, of oxygen, , 2H+e–, , Complete, absence, of oxygen, , Strictly anaerobic cells, Electron acceptors, 2H+e–, other than oxygen, , Electron acceptors, other than oxygen, , Figure 18.1 Illustration of redox potentials in an, agar deep tube, , C L I N I C A L A P P L I C AT I O N, Oxygen as a Treatment?, The causative agent of gas gangrene, Clostridium, perfringens, is an anaerobic bacterium that thrives, in wounds deprived of circulation and oxygen and, that can cause limb loss and death. Treatment may, involve amputation or surgical removal of infected, tissue. Doctors may also prescribe therapy using, enriched oxygen delivered to the patient in a, hyperbaric chamber. This allows the blood to carry, more oxygen to the wounds, slowing the growth of, anaerobic microbes. Patients typically undergo five, 90-minute sessions lying in a chamber pressurized, to 2.5 atmospheres, possibly alleviating the need, for surgery., , 129
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Evacuation and replacement, of oxygen atmosphere in, sealed jars, , Solid, medium, , Specialized, methods not, requiring the, use of, sealed jars, , Brewer jar:, , High-vacuum pump evacuates O2 , which is, replaced with a mixture of 95% N2 + 5%, CO2 ., Platinum catalyst in jar lid results in binding, of residual O2 with H2 , causing formation of H2O., , GasPak, system:, , Disposable H2 + CO2 envelope generator., Requires no evacuation of jar, no high-vacuum, pumping equipment. Room-temperature catalyst, that requires no electrical activation is used., Evolved H2 reacts with O2 to yield H2O. (See, Figure 18.4.), , Chromium–, sulfuric acid, method:, , H2 is generated in a desiccator jar following the, reaction of 15% H2SO4 with chromium powder., H2SO4 + Cr2+, CrSO4 + H2 . As H2 is, evolved, O2 is forced out of desiccator jar and, replaced with H2 ., , Shake-culture, technique:, , Molten and cooled nutrient agar is inoculated with, a loopful of organism. The tube is shaken, cooled, rapidly, and incubated. Position of growth in tube, is an index of gaseous requirement of organism., (See Figure 17.1.), , Pyrogallic, acid, technique:, , Streak cultures on nutrient agar slants. Push a, cotton plug into tube until it nearly touches slant., Fill space above cotton with pyrogallic acid crystals and add sodium hydroxide. Insert stopper, tightly. Invert and incubate. Chemicals absorb, O2 , producing anaerobic environment., , Paraffin plug, technique:, , Any medium containing reducing substances, (such as brain heart infusion, liver veal, cystine,, or ascorbic acid) may be used. The medium is, heated to drive off O2 , rapidly cooled, and inoculated with a loopful of culture. This is immediately, sealed with a half-inch of melted paraffin and, incubated., , Broth, medium, , Fluid, This medium contains sodium thioglycollate,, thioglycollate: which binds to O2 , thus acting as a reducing, compound. Also present is a redox potential indicator, such as resazurin, that produces a pink, coloration in an oxidized environment., Figure 18.2 Methods for the cultivation of anaerobic microorganisms, , FU RT HER R E ADING, Refer to the section on bacterial growth in your, textbook for further information on the anaerobic, 130, , Experiment 18, , metabolism in microbial cells. In your textbook’s, index, search under “Cellular Respiration,” “Redox, Reactions,” and “Anaerobic.”
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pink color in the upper one-third of the medium. If this coloration is present, loosen the, screw caps and place the tubes in a boiling, water bath for 10 minutes to drive off the dissolved O2 from the medium. Cool the tubes to, 45°C before inoculation., 2. Aseptically inoculate the appropriately labeled, tubes of thioglycollate with their respective, test organisms by means of loop inoculations, to the depths of the media., 3. Incubate the cultures for 24 to 48 hours, at 37°C., The appearance of the growth of organisms, according to their gaseous requirements in, thioglycollate medium is shown in Figure 18.3., , AT T H E B E N C H, , Materials, Cultures, 24- to 48-hour nutrient broth cultures of, ❏❏ Bacillus cereus, ❏❏ Escherichia coli, ❏❏ Micrococcus luteus, 48-hour thioglycollate broth culture of, ❏❏ Clostridium sporogenes, , Media, , GasPak Anaerobic Technique, , Per designated student group:, ❏❏ Four screw-cap tubes of fluid thioglycollate, medium, ❏❏ Four nutrient agar plates, , The GasPak system, shown in Figure 18.4, is a, contemporary method for the exclusion of oxygen, from a sealed jar used for incubation of anaerobic, cultures in a nonreducing medium. This system, uses a GasPak generator that consists of a foil, package that generates hydrogen and carbon, dioxide upon the addition of water. A palladium, catalyst in the lid of the jar combines the evolved, hydrogen with residual oxygen to form water,, thereby creating a carbon dioxide environment, within the jar that is conducive for anaerobic, growth. The establishment of anaerobic conditions, is verified by the color change of a methylene, blue indicator strip in the jar. This blue indicator, becomes colorless in the absence of oxygen., 1. With a glassware marking pencil, divide the, bottom of each nutrient agar plate into two, sections., , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, GasPak anaerobic system, Test tube rack, Glassware marking pencil, , Procedure Lab One, Fluid Thioglycollate Medium, 1. For the performance of this procedure, the, fluid thioglycollate medium must be fresh., Freshness is indicated by the absence of a, , (a), , (b), , (c), , (d), , (e), , Figure 18.3 Bacterial growth patterns in thioglycollate broth tubes., (a) Uninoculated control. (b, c) Uniform growth indicates facultative anaerobic bacteria., (d) Bubbles indicate gas-producing bacteria. (e) Bottom growth indicates anaerobic bacteria., , Experiment 18, , 131
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Screw clamp, , Gasket, , Palladium, catalyst and, holder, Hydrogen, GasPak, generator, Inverted, inoculated, plates, Anaerobic, indicator strip, (methylene blue), , 5. Tear off the corner of the hydrogen and carbon, dioxide gas generator and insert this inside the, GasPak jar., 6. Place one set of plate cultures in an inverted, position inside the GasPak chamber., 7. Expose the anaerobic indicator strip and place, it inside the anaerobic jar so that the wick is, visible from the outside., 8. With a pipette, add the required 10 ml of water, to the gas generator, and quickly seal the, chamber with its lid., 9. Place the sealed jar in an incubator at 37°C for, 24 to 48 hours. After several hours of incubation, observe the indicator strip for a color, change to colorless, which is indicative of, anaerobic conditions., 10. Incubate the duplicate set of plates in an, inverted position for 24 to 48 hours at, 37°C under aerobic conditions., , Procedure Lab One, Figure 18.4 GasPak system, , 2. Label each section on two plates with the name, of the organism to be inoculated., 3. Repeat step 2 to prepare a duplicate set of, cultures., 4. Using aseptic technique, make a single-line, streak inoculation of each test organism in its, respectively labeled section on each set of plates., , 132, , Experiment 18, , 1. Observe the fluid thioglycollate cultures,, GasPak system, and aerobically incubated, plate cultures for the presence of growth., Record your results in the chart provided in, the Lab Report., 2. Based on your observation, record the oxygen requirement classification of each test, organism as anaerobe, facultative anaerobe,, or aerobe.
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E XP E R IMENT, , 18, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Bacterial, Species, , Fluid, Thioglycollate, , GasPak, Anaerobic, Incubation, , Aerobic, Incubation, , Oxygen, Requirement, Classification, , M. luteus, B. cereus, E. coli, C. sporogenes, , Review Questions, 1. Why can media such as brain heart infusion and thioglycollate be used for, the cultivation of anaerobes?, , 2. What are the purposes of the indicator strip and the gas generator in the, GasPak system?, , Experiment 18: Lab Report, , 133
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3., , Heroin addicts have a high incidence of Clostridium tetani, infections. Discuss the reasons for the development of this type, of infection in these IV drug users., , 4., , While you are working in your garden, a tine of the pitchfork, accidentally produces a deep puncture wound in the calf of your, leg. Discuss the type of infectious process you would be primarily concerned about and why., , 5., , The physician who treats your puncture wound opts to insert, a drain before applying the dressing. What is the rationale for, the insertion of the drain?, , 134, , Experiment 18: Lab Report
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Serial Dilution—Agar Plate, Procedure to Quantitate Viable Cells, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the diverse methods used to, determine the number of cells in a, bacterial culture., 2. Explain how to determine quantitatively, the number of viable cells in a bacterial, culture., , Principle, , E XP E R IMENT, , 19, , (cubic mm). All cells are counted in this square, millimeter., The number of cells counted is calculated as, follows:, number of cells per mm =, number of cells counted * dilution * 50,000, , The factor of 50,000 is used in order to determine, the cell count for 1 ml : 1 ml = 1000 mm3 =, (50 times the chamber depth of 0.02 mm) * 1000., Although rapid, a direct count has the disadvantages that both living and dead cells are counted, and that it is not sensitive to populations of fewer, than 1 million cells., Breed smears are used mainly to quantitate bacterial cells in milk. Using stained smears, , Studies involving the analysis of materials, including food, water, milk, and—in some cases—air,, require quantitative enumeration of microorganisms in the substances. Many methods have been, devised to accomplish this, including direct microscopic counts, use of an electronic cell counter, such as the Coulter Counter, chemical methods for, estimating cell mass or cellular constituents, turbidimetric measurements for increases in cell mass,, and the serial dilution–agar plate method., , Counting chamber, , Direct Microscopic Counts, Direct microscopic counts require the use of a, specialized slide called the Petroff-Hausser, counting chamber, in which an aliquot of a, eukaryotic cell suspension is counted and the total, number of cells is determined mathematically. The, Petroff-Hausser counting chamber is a thick glass, microscope slide with a chamber 0.02 mm (1/50, mm) deep in the center. The chamber contains an, etched grid and has improved Neubauer rulings, (1/400 square mm). Figure 19.1 illustrates the slide, and the counting chamber., The rulings cover 9 mm2. The boundary lines, (Neubauer rulings) are the center lines of the, groups of three. The center square millimeter is, ruled into groups of 16 small squares, and each, group is separated by triple lines, the middle, one of which is the boundary. The ruled surface, is 0.02 mm below the cover glass, which makes, the volume over a square millimeter 0.02 mm3, , (a) Petroff-Hausser counting chamber, , 1.00 mm, , 0.05 mm, , 0.25 mm, , 1.00 mm, , 1.00 mm, , (b) Petroff-Hausser counting chamber grid, , Figure 19.1 The Petroff-Hausser counting chamber, 135
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Figure 19.2 Pour-plate technique, , confined to a 1-square-millimeter ruled area of, the slide, the total population is determined mathematically. This method also fails to discriminate, between viable and dead cells., , Electronic Cell Counters, The Coulter Counter is an example of an instrument capable of rapidly counting the number of, cells suspended in a conducting fluid that passes, through a minute orifice through which an electric, current is flowing. Cells, which are nonconductors, increase the electrical resistance of the conducting fluid, and the resistance is electronically, recorded, enumerating the number of organisms, flowing through the orifice. In addition to its, inability to distinguish between living and dead, cells, the apparatus is also unable to differentiate, inert particulate matter from cellular material., , Chemical Methods, While not considered means of direct quantitative analysis, chemical methods may be used to, indirectly measure increases both in protein concentration and in DNA production. In addition, cell, mass can be estimated by dry weight determination of a specific aliquot of the culture. Measurement of certain metabolic parameters may also, be used to quantitate bacterial populations. The, amount of oxygen consumed (oxygen uptake) is, directly proportional to the increasing number of, vigorously growing aerobic cells, and the rate of, carbon dioxide production is related to increased, growth of anaerobic organisms., , Spectrophotometric Analysis, Increased turbidity in a culture is another index, of growth. With turbidimetric instruments, the, 136, , Experiment 19, , amount of transmitted light decreases as the cell, population increases, and the decrease in radiant, energy is converted to electrical energy and indicated on a galvanometer. This method is rapid but, limited, because sensitivity is restricted to microbial suspensions of 10 million cells or greater., , Serial Dilution–Agar Plate Analysis, While all these methods may be used to enumerate the number of cells in a bacterial culture,, the major disadvantage common to all is that, the total count includes dead as well as living, cells. Sanitary and medical microbiology, at, times, require determination of viable cells. To, accomplish this, the serial dilution–agar plate, technique is used. Briefly, this method involves, serial dilution of a bacterial suspension in sterile water blanks, which serve as a diluent of, known volume. Once diluted, the suspensions, are placed on suitable nutrient media. The pourplate technique, illustrated in Figure 19.2, is, usually employed. Molten agar, cooled to 45°C,, is poured into a Petri dish containing a specified, amount of the diluted sample. Following addition of the molten-then-cooled agar, the cover, is replaced, and the plate is gently rotated in a, circular motion to achieve uniform distribution, of microorganisms. This procedure is repeated, for all dilutions to be plated. Dilutions should, be plated in duplicate for greater accuracy,, incubated overnight, and counted on a Quebec, colony counter either by hand or by an electronically modified version of this instrument., Figure 19.3 is an illustration of this apparatus for, counting colonies., Plates suitable for counting must contain neither fewer than 30 nor more than 300 colonies., See Figure 19.4. The total count of the suspension
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F U RT H E R RE A D I N G, Refer to the section on bacterial growth in your, textbook for further information on the quantification of bacterial cultures. In your textbook’s index,, search under “Colony Forming Units,” “Serial Dilution,” and “Binary Fission.”, , C L I N I C A L A P P L I C AT I O N, , Figure 19.3 Quebec colony counter for the, enumeration of bacterial colonies, , is obtained by multiplying the number of cells per, plate by the dilution factor, which is the reciprocal, of the dilution., Advantages of the serial dilution–agar plate, technique are as follows:, 1. Only viable cells are counted., 2. It allows isolation of discrete colonies that can, be subcultured into pure cultures, which may, then be easily studied and identified., Disadvantages of this method are as follows:, 1. Overnight incubation is necessary before colonies develop on the agar surface., 2. More glassware is used in this procedure., 3. The need for greater manipulation may result, in erroneous counts due to errors in dilution, or plating., The following experiment uses the pourplate technique for plating serially diluted culture, samples., , 105, , 106, , The Multiple Uses of Cell Counts, Determining how many cells are present in a sample, is important in the food and dairy industries, which, monitor the number and types of bacteria in their, products. Elevated bacteria counts can indicate a, sick animal, inadequate sanitation, or improper storage. Viable cell counts are also used in water treatment facilities as well as in wineries and breweries,, where the number of yeast cells is monitored. In, medical laboratories, sometimes the number of cells, and growth rates are used to determine antimicrobial sensitivity as well as the course of infection., , AT T HE BE NCH, , Materials, Culture, ❏❏ 24- to 48-hour nutrient broth culture of, Escherichia coli, , Media, Per designated student group:, ❏❏ Six 20-ml nutrient agar deep tubes, ❏❏ Seven sterile 0.9-ml water blanks, , 107, , 108, , Figure 19.4 Agar plating method for viable cell counts using dilutions, 1 : 105, 1 : 106, 1 : 107, and 1 : 108, , Experiment 19, , 137
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Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Hot plate, Waterbath, Thermometer, Test tube rack, Microincinerator or Bunsen burner, Vortex mixer, Micropipette tips, Mechanical pipetting device, Sterile Petri dishes, Quebec colony counter, Manual hand counter, Disinfectant solution in a 500-ml beaker, Glassware marking pencil, Turntable, Bent glass rod, Beaker with 95% alcohol, , Procedure Lab One, Figure 19.5 illustrates the following pour-plate, , procedure. A photograph of the dilutions is shown, in Figure 19.6., 1. Liquefy six agar deep tubes in an autoclave, or by boiling. Cool the molten agar tubes and, maintain in a waterbath at 45°C., 2. Label the E. coli culture tube with the number, 1 and the seven 9-ml water blanks as numbers 2, through 8. Place the labeled tubes in a test tube, rack. Label the Petri dishes 1A, 1B, 2A, 2B, 3A,, and 3B., 3. Mix the E. coli culture (Tube 1) by rolling the, tube between the palms of your hands to ensure, even dispersal of cells in the culture., 4. With a sterile pipette, aseptically transfer 1 ml, from the bacterial suspension, Tube 1, to water, blank Tube 2. Discard the pipette in the beaker, of disinfectant. The culture has been diluted 10, times to 10-1., 5. Mix Tube 2 and, with a fresh pipette, transfer, 1 ml from Tube 2 to Tube 3. Discard the pipette., The culture has been diluted 100 times to 10-2., 6. Mix Tube 3 and, with a fresh pipette, transfer, 1 ml from Tube 3 to Tube 4. Discard the pipette., The culture has been diluted 1000 times to 10-3., 7. Mix Tube 4 and, with a fresh pipette, transfer, 1 ml from Tube 4 to Tube 5. Discard the pipette., The culture has been diluted 10,000 times to 10-4., 8. Mix Tube 5 and, with a fresh pipette, transfer, 0.1 ml of this suspension from Tube 5 to Plate 1A., , 138, , Experiment 19, , Return the pipette to Tube 5 and transfer 1 ml, from Tube 5 to Tube 6. Discard the pipette. The, culture has been diluted 100,000 times to 10-5., 9. Mix Tube 6 and, with a fresh pipette, transfer, 1 ml of this suspension from Tube 6 to Plate, 1B. Return the pipette to Tube 6 and transfer, 0.1 ml from Tube 6 to Plate 2A. Return the, pipette to Tube 6 and transfer 1 ml from Tube, 6 to Tube 7. Discard the pipette. The culture, has been diluted 1,000,000 times to 10-6., 10. Mix Tube 7 and, with a fresh pipette, transfer, 1 ml of this suspension from Tube 7 to Plate, 2B. Return the pipette to Tube 7 and transfer 0.1 ml from Tube 7 to Plate 3A. Return, the pipette to Tube 7 and transfer 1 ml from, Tube 7 to Tube 8. Discard the pipette. The, culture has been diluted 10,000,000 times, to 10-7., 11. Mix Tube 8 and, with a fresh pipette, transfer, 1 ml of this suspension from Tube 8 to Plate, 3B. Discard the pipette. The dilution procedure is now complete., 12. Check the temperature of the molten agar, medium to be sure the temperature is 45°C., Remove a tube from the waterbath and wipe, the outside surface dry with a paper towel., Using the pour-plate technique, pour the agar, into Plate 1A as shown in Figure 19.2 and, rotate the plate gently to ensure uniform distribution of the cells in the medium., 13. Repeat step 12 for the addition of molten nutrient agar to Plates 1B, 2A, 2B, 3A, and 3B., 14. Once the agar has solidified, incubate the, plates in an inverted position for 24 hours at, 37°C., The spread-plate technique requires that a previously diluted mixture of microorganisms be used., During inoculation, the cells are spread over the, surface of a solid agar medium with a sterile,, L-shaped bent glass rod while the Petri dish is, spun on a lazy Susan/turntable. The step-by-step, procedure for this technique is as follows:, 1. Prepare bacterial suspensions as described, above and label agar plates accordingly., 2. Place the bent glass rod into a beaker and add a, sufficient amount of 95% ethyl alcohol to cover, the lower, bent portion., 3. Place an appropriately labeled nutrient agar, plate on the turntable. With a sterile pipette,, place 0.1 ml of bacterial suspension on the center of the plate.
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PROCEDURE, Transfer, 1 ml, with, pipette, 1., , Transfer, 1 ml, with, pipette, 2., , Transfer, 1 ml, with, pipette, 5., , Transfer, 1 ml, with, pipette, 6., , Transfer, 1 ml, with, pipette, 7., , Tube 2, , Tube 3, , Tube 4, , Tube 5, , Tube 6, , Tube 7, , Tube 8, , E. coli, culure, , H2O, 9 ml, , H2O, 9 ml, , H2O, 9 ml, , H2O, 9 ml, , H2O, 9 ml, , H2O, 9 ml, , H2O, 9 ml, , 10-1, , 10-2, , 10-3, , 10-4, , 10-5, , 10-6, , 10-7, , Transfer, 1.0 ml, with, pipette 6., , Transfer, 0.1 ml, with, pipette 6., , Transfer, 1.0 ml, with, pipette 7., , Transfer, 0.1 ml, with, pipette 5., , 2. Addition of sample, of suspension to, plates, , ** 4. Dilution factor, , Transfer, 1 ml, with, pipette, 4., , Tube 1, , * 1. Dilutions, , 3. Pour nutrient agar,, 455C, into each plate., Mix by rotation of, plate for the serial, dilution–agar plate, method or use the, spread-plate method., , Transfer, 1 ml, with, pipette, 3., , 1A, 0.1 ml, 105, , 1B, 1.0 ml, 105, , 2A, 0.1 ml, 106, , 2B, 1.0 ml, 106, , Transfer, 0.1 ml, with, pipette 7., , 3A, 0.1 ml, 107, , Transfer, 1.0 ml, with, pipette 8., , 3B, 1.0 ml, 107, , 5. Incubate 24 hr at 375C., 6. Enumerate using Quebec colony counter., * Dilution refers to varying the concentration of the substance., ** Dilution factor is expressed mathematically as the reciprocal of the dilution., For example, a dilution of 10-3 has a dilution factor of 103., , Figure 19.5 Serial dilution–agar plate procedure, , Experiment 19, , 139
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10-1, , 10-2, , 10-3, , 10-4, , 10-5, , 10-6, , Figure 19.6 Serial dilution of bacterial culture for quantitation of viable cell numbers, , 4. Remove the glass rod from the beaker, and pass, it through the Bunsen burner flame with the, bent portion of the rod pointing downward to, prevent the burning alcohol from running down, your arm. Allow the alcohol to burn off the rod, completely. Cool the rod for 10 to 15 seconds., 5. Remove the Petri dish cover and spin the, turntable., 6. While the turntable is spinning, lightly touch the, sterile bent rod to the surface of the agar and, move it back and forth. This will spread the culture over the agar surface., 7. When the turntable comes to a stop, replace the, cover. Immerse the rod in alcohol and reflame., 8. In the absence of a turntable, turn the Petri dish, manually and spread the culture with the sterile, bent glass rod., , Procedure Lab Two, 1. Using a Quebec colony counter and a mechanical hand counter, observe all colonies on plates., Statistically valid plate counts are only obtained, from bacterial cell dilutions that yield between, 30 and 300 colonies. Plates with more than 300, colonies cannot be counted and are designated, as too numerous to count—TNTC; plates, with fewer than 30 colonies are designated, as too few to count—TFTC. Count only, plates containing between 30 and 300 colonies., Remember to count all subsurface as well as, surface colonies., , 140, , Experiment 19, , 2. The number of organisms per ml of original culture is calculated by multiplying the number of, colonies counted by the dilution factor:, number of cells per ml =, number of colonies * dilution factor, Examples:, a. Colonies per plate = 50, Dilution factor = 1 : 1 * 106 (1 : 1,000,000), Volume of dilution added to plate = 1 ml, 50 * 1,000,000 = 50,000,000 or, (5 * 107) CFUs/ml, (colony-forming units), b. Colonies per plate = 50, Dilution factor = 1 : 1 * 105 (1 : 1,00,000), Volume of dilution added to plate = 0.1 ml, 50 * 100,000 = 50,000,000 (5 * 106), cells/0.1 ml, 5,000,000 * 10 = 50,000,000, (5 * 107) CFUs/ml, 3. Record your observations and calculated bacterial counts per ml of sample in the Lab Report., 4. Since the dilutions plated are replicates of each, other, determine the average of the duplicate, bacterial counts per ml of sample and record in, the chart provided in the Lab Report.
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E XP E R IMENT, , 19, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, , Plate, , Dilution, Factor, , ml of, Dilution, Plated, , Final, Dilution, on Plate, , Number, of, Colonies, , Bacterial, Count per ml, of Sample, (CFU/ml), , Average, Count per ml, of Sample, (CFU/ml), , 1A, 1B, 2A, 2B, 3A, 3B, , Review Questions, 1. What is the major disadvantage of microbial counts performed by methods other than the serial, dilution–agar plate procedure?, , 2. Distinguish between dilution and dilution factor., , Experiment 19: Lab Report, , 141
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3. What are the advantages and disadvantages of the serial dilution–agar plate, procedure?, , 4. If 0.1 ml of a 1 * 10-6 dilution plate contains 56 colonies, calculate the number of, cells per ml of the original culture., , 5. How would you record your observation of a plate containing 305 colonies? A plate, with 15 colonies?, , 6. Explain the chemical methods for measuring cell growth., , 7., , 142, , Your instructor asks you to determine the number of organisms in a water, sample. Observation of your dilution plates reveals the presence of spreading colonial forms on some of the culture plates. What is the rationale for the elimination of these plate counts from your experimental data?, , Experiment 19: Lab Report
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E XP E R IMENT, , 20, , The Bacterial Growth Curve, , Log, , e, , 3. Determine the generation time of a, bacterial culture from the bacterial, growth curve., , clin, De, , 2. Plot a bacterial growth curve., , 4:, , 1. Explain the population growth dynamics, of bacterial cultures., , 3: Stationary, , 2:, , Once you have completed this experiment,, you should be able to, , Log of the number of cells per ml, , LEARNING OBJECTIVES, , 1: Lag, Time, , Principle, Bacterial population growth studies require, inoculation of viable cells into a sterile broth, medium and incubation of the culture under, optimum temperature, pH, and gaseous conditions., Under these conditions, the cells will reproduce, rapidly and the dynamics of the microbial growth, can be charted in a population growth curve, which, is constructed by plotting the increase in cell numbers, versus time of incubation. The curve can be used to, delineate stages of the growth cycle. It also facilitates measurement of cell numbers and the rate of, growth of a particular organism under standardized, conditions as expressed by its generation time, the, time required for a microbial population to double., The stages of a typical growth curve, (Figure 20.1) are as follows:, 1. Lag phase: During this stage, the cells are, adjusting to their new environment. Cellular, metabolism is accelerated, resulting in rapid, biosynthesis of cellular macromolecules, primarily enzymes, in preparation for the next, phase of the cycle. Although the cells are, increasing in size, there is no cell division and, therefore no increase in numbers., 2. Logarithmic (log) phase: Under optimum, nutritional and physical conditions, the physiologically robust cells reproduce at a uniform, and rapid rate by binary fission. Thus, there, , Figure 20.1 Population growth curve, , is a rapid exponential increase in population,, which doubles regularly until a maximum, number of cells is reached. The time required, for the population to double is the generation time. The length of the log phase varies,, depending on the organisms and the composition of the medium. The average may be estimated to last 6 to 12 hours., 3. Stationary phase: During this stage, the, number of cells undergoing division is equal to, the number of cells that are dying. Therefore,, there is no further increase in cell number, and, the population is maintained at its maximum, level for a period of time. The primary factors, responsible for this phase are the depletion of, some essential metabolites and the accumulation of toxic acidic or alkaline end products in, the medium., 4. Decline, or death, phase: Because of the, continuing depletion of nutrients and buildup of, metabolic wastes, the microorganisms die at a, rapid and uniform rate. The decrease in population closely parallels its increase during the, log phase. Theoretically, the entire population, should die during a time interval equal to that, of the log phase. This does not occur, however,, since a small number of highly resistant organisms persist for an indeterminate length of time., , 143
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3.0, , 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, , Generation time, , Log 10 cell numbers per ml, , Absorbance at 660 nm, , 2.0, , 0.2, , CO = number of cells at time zero, CE = number of cells at end of a specified time (t), N = number of generations (doublings), , To describe logarithmic growth, the following, equation is used:, N = (log CE - log CO)/log 2, , Using this formula, the logarithmic tables to, the base 10, and the following supplied information, we may now solve for the generation time:, , 0.1, 0, , 30, , 60, , 90, , 120, , 150, , 180, , 210, , Time (in minutes), , Figure 20.2 Indirect method of determining, generation time, , Construction of a complete bacterial growth, curve requires that aliquots of a 24-hour, shakeflask culture be measured for population size at, intervals during the incubation period. Such a, procedure does not lend itself to a regular laboratory session. Therefore, this experiment follows a, modified procedure designed to demonstrate only, the lag and log phases. You will plot the curve on, semilog paper by using two values for the measurement of growth. The direct method requires, enumeration of viable cells in serially diluted samples of the test culture taken at 30-minute intervals as described in Experiment 19. The indirect, method uses spectrophotometric measurement, of the developing turbidity at the same 30-minute, intervals, as an index of increasing cellular mass., You will determine generation time with, indirect and direct methods by using data on the, growth curve. Indirect determination is made by, simple extrapolation from the log phase as illustrated in Figure 20.2. Select two points on the, absorbance scale that represent a doubling of turbidity, such as 0.2 and 0.4. Using a ruler, extrapolate by drawing a line between each of the selected, absorbances on the ordinate (y-axis) and the plotted line of the growth curve. Then draw perpendicular lines from these endpoints on the plotted line, of the growth curve to their respective time intervals on the abscissa (x-axis). With this information,, determine the generation time (GT) as follows:, GT = t(A 0.4) - t(A 0.2), GT = 90 minutes - 60 minutes = 30 minutes, , 144, , The generation time may be calculated directly, using the log of cell numbers scale on a growth, curve. The following example uses information, from a hypothetical growth curve to calculate the, generation time directly., , Experiment 20, , CE = 52,000,000 cells log CE = 7.7218, CO = 25,000 cells, , log CO = 4.4048, , , , log 2 = 0.301, N = (7.7218 - 4.4048)/0.301 = 11 generations, generation time (GT) =, , , , , the specified time (t), number of generations (N), t = 180 minutes, , GT = 180/11 = 16 minutes, , F U RT H E R RE A D I N G, Refer to the section on bacterial growth in your, textbook for further information on the calculation of bacterial growth. In your textbook’s index,, search under “Growth Curve,” “Generation Time,”, and “Logarithmic Plot.”, , C L I N I C A L A P P L I C AT I O N, Using Growth Curves to Determine, Antimicrobial Resistance, In medical laboratories, growth curves are being, mathematically modeled to quickly determine antimicrobial susceptibility. By monitoring turbidity in, a series of wells, each containing a test bacterium, and a dilution of an antimicrobial agent, the entire, growth curve of the bacterium can be determined, from early measurements, greatly speeding up, the testing process for drugs as well as assessing, newly resistant bacteria.
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AT T H E B E N C H, , Materials, Cultures, ❏❏ 5- to 10-hour (log phase) brain heart infusion, broth culture of Escherichia coli with A of, 0.08 to 0.10 or equilibrated to a 0.5 McFarland, Standard at 600 nm, , Media, Per designated student group, ❏❏ 100 ml of brain heart infusion in a 250-ml, Erlenmeyer flask, ❏❏ 42 sterile 9-ml water blanks, ❏❏ 24 nutrient agar plates, , Equipment, ❏❏ 37°C waterbath shaker incubator, ❏❏ Bausch & Lomb Spectronic 20, spectrophotometer, ❏❏ 13 * 100@mm cuvettes, ❏❏ Quebec colony counter, ❏❏ 1-ml sterile pipettes, ❏❏ Mechanical pipetting device, ❏❏ Glassware marking pencil, ❏❏ 1000-ml beaker, ❏❏ L-shaped bent glass rod, ❏❏ 95% ethyl alcohol, ❏❏ Microincinerator or Bunsen burner, , Procedure Lab One, 1. Separate the 42 sterile 9-ml water blanks into, six sets of seven water blanks each. Label, each set as to time of inoculation, (t 0, t 30, t 60, t 90, t 120, t 150) and the dilution, to be effected in each water blank, (10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7)., 2. Label six sets of four nutrient agar plates as to, time of inoculation and dilution to be plated, (10-4, 10-5, 10-6, 10-7)., , 3. With a sterile pipette, add approximately 5 ml, of the log phase E. coli culture to the flask containing 100 ml of brain heart infusion broth. The, approximate initial A (t 0) should be 0.08 to 0.1, at 600 nm. Refer to Experiment 12 for proper, use of the spectrophotometer., 4. After the (t 0) A has been determined, shake the, culture flask and aseptically transfer 1 ml to the, 9-ml water blank labeled t 0 10-1, and continue, to dilute serially to 10-2 through 10-7. Note: A, new pipette must be used for each subsequent, dilution., 5. Place the culture flask in a waterbath shaker, set at 120 rpm at 37°C, and time for the required, 30-minute intervals., 6. Place 1 ml of bacterial suspension from tubes, labeled 10-4, 10-5, 10-6, and 10-7 on appropriately labeled nutrient agar plates. Use a sterile, L-shaped rod to spread bacteria and allow, the plates to dry, covered with their lids, for, 15 minutes. Refer to Experiment 19 for proper, spread plate techniques., 7. Thereafter, at each 30-minute interval, shake, and aseptically transfer a 5-ml aliquot of the, culture to a cuvette and determine its absorbance. Also, aseptically transfer a 1-ml aliquot, of the culture into the 10-1 water blank of the, set labeled with the appropriate time, complete, the serial dilution, and plate in the respectively, labeled Petri dishes as shown in Figure 20.3., Note: A new pipette must be used for each subsequent dilution., 8. Incubate plates in an inverted position for, 24 hours at 37°C., , Procedure Lab Two, 1. Perform cell counts on all plates as described, in Experiment 19. Cell counts are often referred, to as colony-forming units (CFUs) because, each single cell in the plate becomes visible as a, colony, which can then be counted., 2. Record your results in the Lab Report., , Experiment 20, , 145
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PROCEDURE, , Shake culture and, transfer 5.0-ml, sample to cuvette., , Determine absorbance, at 600 nm., , Cuvette, , Transfer 1 ml Transfer 1 ml Transfer 1 ml Transfer 1 ml Transfer 1 ml Transfer 1 ml, (serial dilution) (serial dilution) (serial dilution) (serial dilution) (serial dilution) (serial dilution), , Brain heart infusion, broth culture of E. coli, , Shake, culture and, transfer 1 ml., , 10-, , 1, , 10-2, , 10-3, , 10-4, , 10-5, , 10-6, , 10-7, , 9-ml sterile, water blank, , 9-ml sterile, water blank, , 9-ml sterile, water blank, , 9-ml sterile, water blank, , 9-ml sterile, water blank, , 9-ml sterile, water blank, , 9-ml sterile, water blank, , Plate 1 ml, of dilution., , 10-4, , Plate 1 ml, of dilution., , 10-5, , Plate 1 ml, of dilution., , 10-6, , Plate 1 ml, of dilution., , 10-7, , Figure 20.3 Spectrophotometric and dilution-plating procedure for use in bacterial growth curves, , 146, , Experiment 20
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E XP E R IMENT, , 20, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Record the absorbances and corresponding cell counts in the following, chart., Incubation Time (minutes), , Absorbance at 600 nm, , Plate Counts (CFU/ml), , Log of CFU/ml, , 0, 30, 60, 90, 120, 150, , 2. On the semilog paper provided on pages 149 and 150:, a. Plot a curve relating the absorbances on the ordinate versus incubation, time on the abscissa as shown in Figure 20.2., b. Plot a population curve with the log of the viable cells/ml on the ordinate, and the incubation time on the abscissa. On both graphs, use a ruler to, draw the best line connecting the plotted points. The straight-line portion, of the curve represents the log phase., 3. Calculate the generation time for this culture by the direct method (using, the mathematical formula) and by the indirect method (extrapolating from, the A scale on the plotted curve). Show calculations, and record the generation time., a. Direct method:, , b. Indirect method:, , Experiment 20: Lab Report, , 147
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Review Questions, 1. Does the term growth convey the same meaning when applied to bacteria and to, multicellular organisms? Explain., , 2. Why do variations in generation time exist, a. Among different species of microorganisms?, , b. Within a single microbial species?, , 3., , The generation time and growth rate of an organism grown in the laboratory, can be easily determined by constructing a typical growth curve., a. Would you expect the growth rate of the infectious organisms found in an abscess, that developed from a wound to mimic the growth curve obtained in the laboratory? Explain., , b. Would you expect antibiotic therapy to be effective without any other concurrent, treatment of the abscess?, , 4., , 148, , Is generation time a useful parameter to indicate the types of media best, suited to support the growth of a specific organism? Explain., , Experiment 20: Lab Report
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10, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, Absorbance versus Incubation Time, , Experiment 20: Lab Report, , 149
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10, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, Colony-Forming Units (CFUs) versus Incubation Time, , 150, , Experiment 20: Lab Report
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PART 5, , Biochemical Activities, of Microorganisms, LEARNING OBJECTIVES, When you have completed the experiments in this section, you should be able to, 1. Explain the nature and activities of exoenzymes and endoenzymes., 2. Perform the experimental procedures for differentiation of enteric microorganisms., 3. Describe the biochemical test procedures for identification of microorganisms., , Introduction, Microorganisms must be separated and identified, for a wide variety of reasons, including, 1. Determining pathogens responsible for, infectious diseases, 2. Selecting and isolating strains of fermentative, microorganisms necessary for the industrial, production of alcohols, solvents, vitamins,, organic acids, antibiotics, and industrial, enzymes, 3. Isolating and developing suitable microbial, strains necessary for manufacturing and, enhancing the quality and flavor in certain, food materials, including yogurt, cheeses, and, other milk products, 4. Comparing biochemical activities for taxonomic purposes, To accomplish these tasks, microbiologists, utilize the fact that microorganisms all have their, own identifying biochemical characteristics. These, so-called biochemical fingerprints are the properties controlled by the cells’ enzymatic activity, and, they are responsible for bioenergetics, biosynthesis, and biodegradation., The sum of all these chemical reactions is, defined as cellular metabolism, and the biochemical transformations that occur both outside, , and inside the cell are governed by biological catalysts called enzymes., , Extracellular Enzymes (Exoenzymes), Exoenzymes act on substances outside of the cell., Most high-molecular-weight substances are not, able to pass through cell membranes, and therefore these raw materials—food-related substances,, including polysaccharides, lipids, and proteins—, must be degraded to low-molecular-weight materials (nutrients) before they can be transported, into the cell. Because of the reactions involved,, exoenzymes are mainly hydrolytic enzymes that, reduce high-molecular-weight materials into their, building blocks by introducing water into the molecule. This liberates smaller molecules, which may, then be transported into the cell and assimilated., , Intracellular Enzymes (Endoenzymes), Endoenzymes function inside the cell and are, mainly responsible for synthesis of new protoplasmic requirements and production of cellular, energy from assimilated materials. The ability, of cells to act on nutritional substrates permeating cell membranes indicates the presence, of many endoenzymes capable of transforming, the chemically specific substrates into essential, materials., , 151
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Extracellular Enzymes, , Intracellular Enzymes, , Starch hydrolysis, Lipid hydrolysis, Casein hydrolysis, Gelatin hydrolysis, Carbohydrate fermentation, Litmus milk reactions, Hydrogen sulfide production, Nitrate reduction, Catalase reactions, Urease test, Oxidase test, , IMViC, test, , Indole, Methyl red, Voges-Proskauer, Citrate utilization, , Special tests for, the separation, of enteric, microorganisms, , Triple sugar–iron test, , Figure P5.1 Biochemical activities of microorganisms, , This transformation is necessary for cellular, survival and function, and it is the basis of cellular metabolism. As a result of these metabolic, processes, metabolic products are formed and, excreted by the cell into the environment. Assay, of these end products not only aids in identification of specific enzyme systems but also serves to, identify, separate, and classify microorganisms., Figure P5.1 represents a simplified schema of, experimental procedures used to acquaint students with the intracellular and extracellular enzymatic activities of microorganisms., The experiments you will carry out in this, section can be performed in either of two ways., A short version uses a limited number of organisms to illustrate the possible end product(s) that, may result from enzyme action on a substrate. The, organisms for this version are designated in the, individual exercises., The alternative, or long, version involves the, use of 13 microorganisms. This version provides a, complete overview of the biochemical fingerprints, of the organisms and supplies the format for their, separation and identification. These organisms, were chosen to serve as a basis for identification, of an unknown microorganism in Experiment 31., If this alternative version is selected, the following, organisms are recommended for use:, Escherichia coli, Enterobacter aerogenes, , 152, , Part 5, , Klebsiella pneumoniae BSL-2, Shigella dysenteriae BSL-2, Salmonella typhimurium BSL-2, Proteus vulgaris, Pseudomonas aeruginosa BSL-2, Alcaligenes faecalis, Micrococcus luteus, Lactococcus lactis, Staphylococcus aureus BSL-2, Bacillus cereus, Corynebacterium xerosis, Bacteria have been designated as “BSL-2”-level, organisms using guidelines published by the American Society for Microbiology (www.asm.org) and, the Centers for Disease Control and Prevention, (www.cdc.gov) covering organisms to be utilized, in an undergraduate teaching laboratory., , F U RT H E R RE A D I N G, Refer to the section on bacterial metabolism in, your textbook, paying close attention to the uses, of the differential and selective media for the cultivation of bacteria. In your textbook’s index, use, the search terms “Nitrogen Metabolism,” “Indole,”, and “Fermentation.”
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C AS E STUDY, HAND WASHING AND ASEPTIC TECHNIQUE: A CASE STUDY, You are presented with an eosin–methylene blue, (EMB) agar plate that has bacterial colonies with, a slight greenish, metallic sheen. Your laboratory, manager explains the background for the culture, you are observing on the plate: An unknown contaminate was found in a meat processing machine,, and the in-house pathogen control office performed, a swab and a streak on an EMB plate. After incubation and observation of the weak reaction, the, manufacturers concluded that the contaminate was, not E. coli and that no further tests were required., Upper management decided that to protect the, company from potential lawsuits, they would hire, the laboratory you work for to ensure that their, laboratory technicians concluded correctly., , Due to cost and time restrictions, your lab, is limited regarding how many assays can be, performed. Using a series of biochemical tests to, confirm or refute the analysis of the processing, plant, you will need to determine whether the, bacteria is an enteric and then whether it is an, E. coli isolate., , Questions to Consider:, 1. Does the lack of a strong reaction on the EMB, plate refute the determination that the isolated, bacteria are an E. coli culture?, 2. Which series of assays would best be used to, prove/disprove the E. coli determination?, , Part 5, , 153
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Extracellular Enzymatic Activities, of Microorganisms, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the function of microbial, extracellular enzymes., 2. Determine microorganisms’ ability to, excrete hydrolytic extracellular enzymes, capable of degrading the polysaccharide, starch, the lipid tributyrin, and the proteins, casein and gelatin., , E XP E R IMENT, , 21, , of nutrient agar supplemented with starch, which, serves as the polysaccharide substrate. The detection of the hydrolytic activity following the growth, period is made by performing the starch test to, determine the presence or absence of starch in, the medium. Starch in the presence of iodine will, impart a blue-black color to the medium, indicating the absence of starch-splitting enzymes and, representing a negative result. If the starch has, been hydrolyzed, a clear zone of hydrolysis will, surround the growth of the organism. This is a, positive result. Positive and negative results are, shown in Figure 21.1., , Lipid Hydrolysis, , Principle, Because of their large sizes, high-molecularweight nutrients such as polysaccharides, lipids,, and proteins are not capable of permeating the, cell membrane. These macromolecules must first, be hydrolyzed by specific extracellular enzymes, into their respective basic building blocks. These, low-molecular-weight substances can then be, transported into the cells and used for the synthesis of protoplasmic requirements and energy production. The following procedures are designed to, investigate the exoenzymatic activities of different, microorganisms., , Lipids are high-molecular-weight compounds possessing large amounts of energy. The degradation, of lipids such as triglycerides is accomplished, by extracellular hydrolyzing enzymes, called, lipases (esterases), that cleave the ester bonds, in this molecule by the addition of water to form, the building blocks glycerol (an alcohol) and, fatty acids. Figure 21.2 shows this reaction. Once, assimilated into the cell, these basic components, can be further metabolized through aerobic respiration to produce cellular energy, adenosine, triphosphate (ATP). The components may also, , Starch Hydrolysis, Starch is a high-molecular-weight, branching, polymer composed of glucose molecules linked, together by glycosidic bonds. The degradation of, this macromolecule first requires the presence of, the extracellular enzyme amylase for its hydrolysis into shorter polysaccharides, namely dextrins,, and ultimately into maltose molecules. The final, hydrolysis of this disaccharide, which is catalyzed, by maltase, yields low-molecular-weight, soluble, glucose molecules that can be transported into, the cell and used for energy production through, the process of glycolysis., In this experimental procedure, starch agar, is used to demonstrate the hydrolytic activities, of these exoenzymes. The medium is composed, , Figure 21.1 Starch agar plate. Starch hydrolysis, on left; no starch hydrolysis on right., , 155
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Casein Hydrolysis, , O, CH2, , O, , C, , CH2OH + RCOOH, , R, O, , CH, , O, , C, , R¿, O, , CH2, , O, , C, , + 3H2O, , CHOH + R¿COOH, , Lipase, , R–, , Triglyceride, , CH2OH + R–COOH, Glycerol Fatty acids, , Figure 21.2 Lipid hydrolysis, , enter other metabolic pathways for the synthesis, of other cellular protoplasmic requirements., In this experimental procedure, we use tributyrin agar to demonstrate the hydrolytic activities, of the exoenzyme lipase. The medium is composed, of nutrient agar supplemented with the triglyceride tributyrin as the lipid substrate. Tributyrin, forms an emulsion when dispersed in the agar,, producing an opaque medium that is necessary for, observing exoenzymatic activity., Following inoculation and incubation of the, agar plate cultures, organisms excreting lipase will, show a zone of lipolysis, which is demonstrated, by a clear area surrounding the bacterial growth., This loss of opacity is the result of the hydrolytic reaction yielding soluble glycerol and fatty, acids, and represents a positive reaction for lipid, hydrolysis. In the absence of lipolytic enzymes,, the medium retains its opacity. This is a negative, reaction. Figure 21.3 shows positive and negative, results., , Casein, the major milk protein, is a macromolecule composed of amino acid subunits linked, together by peptide bonds (CO—NH). Before, their assimilation into the cell, proteins must, undergo step-by-step degradation into peptones,, polypeptides, dipeptides, and ultimately into, their building blocks, amino acids. This process, is called peptonization, or proteolysis, and, it is mediated by extracellular enzymes called, proteases. The function of these proteases is, to cleave the peptide bond CO—NH by introducing water into the molecule. The reaction, then liberates the amino acids, as illustrated in, Figure 21.4., The low-molecular-weight soluble amino acids, can now be transported through the cell membrane into the intracellular amino acid pool for, use in the synthesis of structural and functional, cellular proteins., In this experimental procedure, milk agar, is used to demonstrate the hydrolytic activity, of these exoenzymes. The medium is composed, of nutrient agar supplemented with milk that, contains the protein substrate casein. Similar to, other proteins, milk protein is a colloidal suspension that gives the medium its color and opacity, because it deflects light rays rather than transmitting them., Following inoculation and incubation of the, agar plate cultures, organisms secreting proteases, will exhibit a zone of proteolysis, which is demonstrated by a clear area surrounding the bacterial growth. This loss of opacity is the result of a, hydrolytic reaction yielding soluble, noncolloidal, amino acids, and it represents a positive reaction., In the absence of protease activity, the medium, surrounding the growth of the organism remains, opaque, which is a negative reaction., , Polypeptide chain, NH2 RCH, Protease, + HOH, , Figure 21.3 Tributyrin agar plate. Lipid hydrolysis, on left; no lipid hydrolysis on right., , 156, , Experiment 21, , CO NH, Peptide, bond, , OH-, , NH2 RCH, , COOH, , RCH, , CO, , NH, , RCH, HOH, , H+, +, , NH2 RCH, , Figure 21.4 Protein hydrolysis, , COOH, , etc. . .
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F U RT H E R RE A D I N G, Refer to the section on bacterial metabolism in, your textbook for further information on the use of, different growth media to test for enzymatic activities. In your textbook’s index, search under “Lipid, Metabolism,” “Enzymatic Activity,” and “Hydrolysis.”, (a) Positive for gelatin liquefaction, , C L I N I C A L A P P L I C AT I O N, , (b) Negative for gelatin liquefaction, , Figure 21.5 Nutrient gelatin hydrolysis, , Gelatin Hydrolysis, Although the value of gelatin as a nutritional source, is questionable (it is an incomplete protein, lacking, the essential amino acid tryptophan), its value in, identifying bacterial species is undeniable. Gelatin, is a protein produced by hydrolysis of collagen, a, major component of connective tissue and tendons, in humans and other animals. Below temperatures, of 25°C, gelatin will maintain its gel properties and, exist as a solid; at temperatures above 25°C, gelatin, is liquid. Figure 21.5 shows gelatin hydrolysis., Liquefaction is accomplished by some microorganisms capable of producing a proteolytic extracellular enzyme called gelatinase, which acts to, hydrolyze this protein to amino acids. Once this, degradation occurs, even the very low temperature, of 4°C will not restore the gel characteristic., In this experimental procedure, you will use, nutrient gelatin deep tubes to demonstrate the, hydrolytic activity of gelatinase. The medium, consists of nutrient broth supplemented with 12%, gelatin. This high gelatin concentration results in, a stiff medium and also serves as the substrate for, the activity of gelatinase., Following inoculation and incubation for, 48 hours, we place cultures in a refrigerator at, 4°C for 30 minutes. Cultures that remain liquefied, produce gelatinase and demonstrate rapid gelatin, hydrolysis. Re-incubate all solidified cultures for, an additional 5 days. Refrigerate for 30 minutes, and observe for liquefaction. Cultures that remain, liquefied are indicative of slow gelatin hydrolysis., , Pathogens and Extracellular Enzymes, Bacteria use enzymes to alter their environments, and to gain new sources of nutrients. When, known bacterial pathogens are causing symptoms, or damage not normally associated with that, species, laboratories may test for newly acquired, extracellular enzymes. Most known pathogens, have been characterized by their abilities to, digest proteins (fibronectin and collagen) as, well as lipids and starches (glycolipids and, glycoproteins)., , AT T HE BE NCH, , Materials, Cultures, 24- to 48-hour Trypticase™ soy broth cultures of, Short Version, ❏❏ Escherichia coli, ❏❏ Bacillus cereus, ❏❏ Pseudomonas aeruginosa BSL -2, ❏❏ Staphylococcus aureus BSL -2, 24- to 48-hour brain heart infusion broth cultures, of the 13 organisms listed on page 152 for the long, version, , Media, Short version: two plates each per group, ❏❏ Starch agar, ❏❏ Tributyrin agar, ❏❏ Milk agar, ❏❏ Three nutrient gelatin deep tubes, Long version: four plates each per group, ❏❏ Starch agar, ❏❏ Tributyrin agar, , Experiment 21, , 157
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❏❏ Milk agar, ❏❏ 14 nutrient gelatin deep tubes, , Procedure Lab Two, , Reagent, , 1. Flood the starch agar plate cultures with, Gram’s iodine solution, allow the iodine, to remain in contact with the medium for, 30 seconds, and pour off the excess., 2. Examine the cultures for the presence or, absence of a blue-black color surrounding, the growth of each test organism. Record, your results in the chart provided in the Lab, Report., 3. Based on your observations, determine and, record the organisms that were capable of, hydrolyzing the starch., , Starch Hydrolysis, , Gram’s iodine solution, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop and needle, Glassware marking pencil, Test tube rack, Refrigerator, , Controls, Test, , Positive, Control, , Negative, Control, , Starch hydrolysis, , B. cereus, , E. coli, , Lipid hydrolysis, , S. aureus, , E. coli, , Casein hydrolysis, , B. cereus, , E. coli, , Gelatin hydrolysis, , B. cereus, , E. coli, , Procedure Lab One, 1. Prepare the starch agar, tributyrin agar, and, milk agar plates for inoculation as follows:, a. Short procedure: Using two plates per, medium, divide the bottom of each Petri, dish into two sections. Label the sections, as E. coli, B. cereus, P. aeruginosa, and, S. aureus BSL -2 , respectively., b. Long procedure: Repeat step 1a, dividing, three plate bottoms into three sections and, one plate bottom into four sections for each, of the required media, to accommodate the, 13 test organisms., 2. Using aseptic technique, make a single-line, streak inoculation of each test organism on, the agar surface of its appropriately labeled, section on the agar plates., 3. Using aseptic technique, inoculate each, experimental organism in its appropriately, labeled gelatin deep tube by means of a stab, inoculation., 4. Incubate all plates in an inverted position for, 24 to 48 hours at 37°C. Incubate the gelatin, deep tube cultures for 48 hours. Re-incubate, all negative cultures for an additional 5 days., , 158, , Experiment 21, , Lipid Hydrolysis, 1. Examine the tributyrin agar plate cultures for, the presence or absence of a clear area, or, zone of lipolysis, surrounding the growth of, each of the organisms. Record your results in, the chart provided in the Lab Report., 2. Based on your observations, determine and, record which organisms were capable of, hydrolyzing the lipid., , Casein Hydrolysis, 1. Examine the milk agar plate cultures for the, presence or absence of a clear area, or zone, of proteolysis, surrounding the growth of, each of the bacterial test organisms. Record, your results in the chart provided in the Lab, Report., 2. Based on your observations, determine and, record which of the organisms were capable, of hydrolyzing the milk protein casein., , Gelatin Hydrolysis, 1. Place all gelatin deep tube cultures into a, refrigerator at 4°C for 30 minutes., 2. Examine all the cultures to determine, whether the medium is solid or liquid. Record, your results in the chart provided in the Lab, Report., 3. Based on your observations following the, 2-day and 7-day incubation periods, determine, and record in the Lab Report (a) which organisms were capable of hydrolyzing gelatin and, (b) the rate of hydrolysis.
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E XP E R IMENT, , 21, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Starch and Lipid Hydrolysis, STARCH HYDROLYSIS, Bacterial, Species, , Appearance, of Medium, , Result, , ( +) or ( −), , LIPID HYDROLYSIS, Appearance, of Medium, , Result, , ( +) or ( −), , E. coli, B. cereus, P. aeruginosa, S. aureus, , Experiment 21: Lab Report, , 159
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Casein and Gelatin Hydrolysis, CASEIN HYDROLYSIS, Bacterial, Species, , Appearance, of Medium, , Result, ( +) or ( −), , GELATIN HYDROLYSIS, Liquefaction ( +) or ( −), 2 days, , 7 days, , Rate of Hydrolysis, (Slow or Rapid), , E. coli, B. cereus, P. aeruginosa, S. aureus, , Review Questions, 1. Why is the catalytic activity of enzymes essential to ensure and regulate cellular metabolism?, , 2. Why are microorganisms able to cause dairy products, such as milk, to sour or curdle?, , 3. Give a reason why it is necessary for polysaccharides, such as starch or cellulose, to be digested outside of the cell even though disaccharides, such as lactose or sucrose, are digestible inside the cell., , 160, , Experiment 21: Lab Report
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E XP E R IMENT, , 22, , Carbohydrate Fermentation, , LEARNING OBJECTIVES, , Aerobic: Biooxidations in, which molecular oxygen, can serve as the final, electron acceptor., , Once you have completed this experiment,, you should be able to, 1. Distinguish between cellular respiration, and fermentation., , Cellular, respiration, , 2. Determine how microorganisms degrade, and ferment carbohydrates by producing, acid and gas., , Principle, Most microorganisms obtain their energy through, a series of orderly and integrated enzymatic reactions leading to the biooxidation of a substrate,, frequently a carbohydrate. The major pathways, involved are shown in Figure 22.1., Organisms use carbohydrates differently, depending on their enzyme complement. Some, organisms are capable of fermenting sugars such, as glucose anaerobically, while others use the aerobic pathway. Still others, facultative anaerobes,, are enzymatically competent to use both aerobic, and anaerobic pathways, and some organisms, lack the ability to oxidize glucose by either pathway. In this exercise, we’ll focus on fermentative, pathways., In fermentation, substrates such as carbohydrates and alcohols undergo anaerobic dissimilation and produce an organic acid (for example,, lactic, formic, or acetic acid) that may be accompanied by gases such as hydrogen or carbon dioxide. Facultative anaerobes are usually the so-called, fermenters of carbohydrates. Fermentation is best, described by the degradation of glucose by way of, the Embden-Meyerhof pathway, also known as, the glycolytic pathway, illustrated in Figure 22.2., As the diagram shows, one mole of glucose is, converted into two moles of pyruvic acid, which, is the major intermediate compound produced by, glucose degradation. Subsequent metabolism of, pyruvate is not the same for all organisms, and a, variety of end products result that define their different fermentative capabilities. This can be seen, in Figure 22.3., , Fermentation, , Anaerobic: Biooxidations, in which inorganic ions, other than oxygen, such as, NO3– or SO42–, can serve as, the final electron acceptors., A biooxidative process not, requiring oxygen in which, an organic substrate serves, as the final electron, acceptor., , Figure 22.1 Biooxidative pathways, , Fermentative degradation under anaerobic, conditions is carried out in a fermentation broth, tube containing a Durham tube, an inverted inner, vial for the detection of gas production, as illustrated in Figure 22.4. A typical carbohydrate fermentation medium contains, 1. Nutrient broth ingredients for the support of, the growth of all organisms, 2. A specific carbohydrate that serves as the substrate for determining the organism’s fermentative capabilities, 3. The pH indicator phenol red, which is red at, a neutral pH (7) and changes to yellow at a, slightly acidic pH of 6.8, indicating that slight, amounts of acid will cause a color change., Because of the critical nature of the fermentation reaction and the activity of the indicator,, all cultures should be observed within 48 hours., Extended incubation may mask acid-producing, reactions by production of alkali because of, enzymatic action on substrates other than the, carbohydrate., Following incubation, carbohydrates that, have been fermented with the production of acidic, wastes will cause the phenol red (Figure 22.5a) to, turn yellow, thereby indicating a positive reaction, (Figure 22.5b and Figure 22.5c). In some cases,, 161
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Fermentation, broth, , Incubation, , Durham tube, , Gas, , No gas, , Figure 22.4 Detection of gas production, , evolution of gas. Figure 22.5d illustrates this negative reaction., The lack of carbohydrate fermentation by some, organisms should not be construed as absence of, growth. The organisms use other nutrients in the, medium as energy sources. Among these nutrients, are peptones present in nutrient broth. Peptones, can be degraded by microbial enzymes to amino, acids that are in turn enzymatically converted by, oxidative deamination to ketoamino acids. These, are then metabolized through the Krebs cycle for, energy production. These reactions liberate ammonia, which accumulates in the medium, forming, ammonium hydroxide (NH4OH) and producing an, alkaline environment. When this occurs, the phenol, red turns to a deep red in the now basic medium., Figure 22.6 illustrates this alternative pathway of, aerobic respiration., , C L I N I C A L A P P L I C AT I O N, Using Fermentation Products to, Identify Bacteria, The fermentation of carbohydrates allows, microbiologists to identify some bacteria by, determining what nutrients they are using and, what products they produce. The pattern of sugars, fermented may be unique to a particular genus,, species, or strain. Lactose fermentation is one test, that distinguishes between enteric and non-enteric, bacteria. Dextrose fermentation allows for the, differentiation between the oxidase ( + ) Vibrio and, Pseudomonads species in patients suffering from, septicemia after eating contaminated fish., , (a), , (b), , (c), , (d), , Figure 21.5 Carbohydrate fermentation test., (a) Uninoculated, (b) acid and gas, (c) acid, and, (d) negative., , F U RT H E R RE A DI N G, Refer to the section on cellular metabolism and, metabolic pathways in your textbook for further, information on bacterial metabolism and growth., In your textbook’s index, search under “Fermentation,” “Embden-Meyerhof Pathway,” and “Cellular, Respiration.”, , AT T HE BE NCH, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase soy, broth cultures of, ❏❏ Escherichia coli, ❏❏ Alcaligenes faecalis, ❏❏ Salmonella typhimurium BSL -2, ❏❏ Staphylococcus aureus BSL -2, For the long version, 24- to 48-hour Trypticase soy, broth cultures of the 13 organisms listed on page 152., , Media, For the short version, per designated student, group 5ml tubes (each with Durham tubes):, ❏❏ Phenol red lactose broth, ❏❏ Dextrose (glucose) broth, ❏❏ Sucrose broth, For the long version, 14 of each., , Experiment 22, , 163
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Glucose, NH3, , CH3 C COOH (Pyruvic acid), O, CH3 C CoA (Acetyl-CoA), , COOH, , O, , CH NH2, CH3, , (Alanine), COOH, C O, CH2, , NH3, Oxidative, deamination, , Krebs, cycle, , Citric acid, , COOH, (Oxaloacetic acid), , COOH, CH NH2, , COOH, C O, CH2, CH2, , CH2, , COOH, (Aspartic acid), , COOH, (α-ketoglutaric acid), , Amino, acids, Oxidative, deamination, Proteins, , NH3, COOH, , CH NH2, CH2, CH2, Amino acids, COOH, (Glutamic acid), , Figure 22.6 Proteins as energy sources for microbes, , Equipment, ❏❏ Microincinerator or Bunsen burner, ❏❏ Inoculating loop, ❏❏ Glassware marking pencil, , Controls, Sugar, , Acid, , Dextrose, Sucrose, Lactose, , S. aureus, S. aureus, S. aureus, , REACTION, Acid w/Gas, E. coli, K. pneumoniae, E. coli, , Procedure Lab One, 1. Using aseptic technique, inoculate each, experimental organism into its appropriately, labeled medium by means of loop inoculation., 164, , Experiment 22, , Note: Take care during this step not to shake, the fermentation tube; shaking the tube may, accidentally force a bubble of air into the, inverted gas vial, displacing the medium and, possibly rendering a false-positive result., The last tube will serve as a control., 2. Incubate all tubes for 24 hours at 37°C., , Procedure Lab Two, 1. Examine all carbohydrate broth cultures for, color and the presence or absence of a gas, bubble. Record your results in the chart provided in the Lab Report., 2. Based on your observations, determine and, record whether each organism was capable of, fermenting the carbohydrate substrate with, the production of acid or acid and gas.
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E XP E R IMENT, , 22, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, , Bacterial, Species, , Lactose, Observation (color, of medium,, bubble in, fermentation tube), , Dextrose, Observation (color, Result, of medium, bubble, (A), (A/G),, in fermentation, or (-), tube), , Result, (A),, (A/G),, or (- ), , Sucrose, Observation (color, of medium, bubble, in fermentation, tube), , Result, (A),, (A/G),, or (- ), , E. coli, A. faecalis, S. typhimurium, S. aureus, K. pneumoniae, P. vulgaris, P. aeruginosa, E. aerogenes, M. luteus, L. lactis, S. dysenteriae, B. cereus, C. xerosis, Alternate, organism, Control, , Review Questions, 1. Distinguish between respiration and fermentation., , Experiment 22: Lab Report, , 165
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2. Do all microorganisms use pyruvic acid in the same way? Explain., , 3. Describe a pathway used for the degradation of carbohydrates by strict anaerobes., , 4., , From your experimental data, you know that P. aeruginosa did not utilize any of the carbohydrates in the test media. In view of this, how do these organisms generate energy to sustain their viability?, , 5., , Clostridium perfringens, an obligate anaerobe, is capable of utilizing the carbohydrates, released from injured tissues as an energy source. During the infectious process, large, amounts of gas accumulate in the infected tissues. Would you expect this gas to be CO2? Explain., , 166, , Experiment 22: Lab Report
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E XP E R IMENT, , Triple Sugar–Iron Agar Test, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Differentiate between and describe, members of the Enterobacteriaceae., 2. Distinguish between the Enterobacteriaceae and other groups of intestinal bacilli., , Principle, The triple sugar–iron (TSI) agar test is, designed to differentiate among the groups or genera of the Enterobacteriaceae, which are all gramnegative bacilli capable of fermenting glucose with, the production of acid, and to distinguish Enterobacteriaceae from other gram-negative intestinal, bacilli. This differentiation is made through differences in carbohydrate fermentation patterns and, hydrogen sulfide production by the various groups, of intestinal organisms., To observe carbohydrate utilization patterns,, the TSI agar slants contain lactose and sucrose, in 1% concentrations and glucose (dextrose) in a, concentration of 0.1%, which permits detection of, the utilization of this substrate only. The acid-base, indicator phenol red is also used to detect carbohydrate fermentation that is indicated by a change, in color of the medium from orange-red to yellow, in the presence of acids. The slant is inoculated, by means of a stab-and-streak procedure. This, requires the insertion of a sterile, straight needle, from the base of the slant into the butt. Upon, withdrawal of the needle, the slanted surface of, the medium is streaked. Following incubation, you, will determine the fermentative activities of the, organisms as follows., 1. Alkaline slant (red) and acid butt, (yellow) with or without gas production, (breaks in the agar butt): Only glucose, fermentation has occurred. The organisms, , 23, , preferentially degrade glucose first. Since this, substrate is present in minimal concentration,, the small amount of acid produced on the, slant surface is oxidized rapidly. The peptones, in the medium are also used in the production, of alkali. In the butt, the acid reaction is maintained because of reduced oxygen tension and, slower growth of the organisms., 2. Acid slant (yellow) and acid butt (yellow), with or without gas production. Lactose, and/or sucrose fermentation has occurred., Since these substances are present in higher, concentrations, they serve as substrates for, continued fermentative activities with maintenance of an acid reaction in both slant and, butt., 3. Alkaline slant (red) and alkaline butt, (red) or no change (orange-red) butt., No carbohydrate fermentation has occurred., Instead, peptones are catabolized under anaerobic and/or aerobic conditions, resulting in an, alkaline pH due to production of ammonia. If, only aerobic degradation of peptones occurs,, the alkaline reaction is evidenced only on the, slant surface. If there are aerobic and anaerobic utilization of peptone, the alkaline reaction, is present on the slant and the butt., For you to obtain accurate results, it is absolutely essential to observe the cultures within, 18 to 24 hours following incubation. Doing so will, ensure that the carbohydrate substrates have not, been depleted and that degradation of peptones, yielding alkaline end products has not taken place., The TSI agar medium also contains sodium, thiosulfate, a substrate for hydrogen sulfide (H2S), production, and ferrous sulfate for detection of, this colorless end product. Following incubation,, only cultures of organisms capable of producing H2S will show an extensive blackening in the, butt, because of the precipitation of the insoluble, ferrous sulfide. (Refer to Experiment 25 for a, more detailed biochemical explanation of H2S, production.), , 167
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Acid slant, Acid butt, No H2S, , Acid slant, Acid butt, H2S produced, , Alkaline slant, Acid butt, No H2S, , Alkaline slant, Acid butt, H2S produced, , Alkaline slant, Alkaline or no, change butt, , Escherichia, Klebsiella, Enterobacter, , Citrobacter, Arizona, Some Proteus spp., , Shigella, Some Proteus spp., , Most Salmonella, Arizona, Citrobacter, , Alcaligenes, Pseudomonas, Acinetobacter, , Figure 23.1 TSI reactions for differentiation of enteric microorganisms, , FU RT HER R E ADING, Refer to the section covering bacterial metabolism, in your textbook for further information on the, carbohydrate fermentation process. In your textbook’s index, search under “Fermentation,” “H2S, Production,” and “Phenol Red.”, , ❏❏ Salmonella typhimurium BSL -2, ❏❏ Shigella dysenteriae BSL -2, ❏❏ P. vulgaris, For the long version, use 24-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , Media, C L I N I C A L A P P L I C AT I O N, Differentiating Between Proteus Species, The TSI test can differentiate enteric organisms based on their abilities to reduce sulfur, and ferment carbohydrates. It can be used, to separate the three species of Proteus—, P. vulgaris, P. mirabilis, and P. penneri—all of, which are human opportunistic pathogens., P. mirabilis causes urinary tract infections and is, sensitive to treatment with ampicillin and cephalosporins. P. vulgaris, a less common cause of, urinary tract infections, is not sensitive to these, antibiotics, and is found as a nosocomial infectious agent among immunocompromised patients., , Per designated student group: triple sugar–iron, agar slants, ❏❏ 7 for the short version, ❏❏ 14 for the long version, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating needle, Test tube rack, Glassware marking pencil, , Controls, Refer to Figure 23.1 for a description of positive, controls for the different results exhibited when, using a triple sugar–iron agar slant., , Procedure Lab One, AT THE B E N C H, , Materials, Cultures, For the short version, use 24-hour Trypticase soy, broth cultures of, ❏❏ Alcaligenes faecalis, ❏❏ Escherichia coli, ❏❏ Pseudomonas aeruginosa BSL -2, , 168, , Experiment 23, , 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube by means of a stab-and-streak inoculation. Note: Do not fully tighten screw cap., The last tube will serve as a control., 2. Incubate for 18 to 24 hours at 37°C., , Procedure Lab Two, 1. Examine the color of both the butt and slant, of all agar slant cultures (Figure 23.2). Based, on your observations, determine the type of, reaction that has taken place (acid, alkaline,
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or none) and the carbohydrate that has been, fermented (dextrose, lactose, sucrose, all, or, none) in each culture. Record your observations and results in the chart provided in the, Lab Report., 2. Examine all cultures for the presence or, absence of blackening within the medium., Based on your observations, determine, whether each organism was capable of H2S, production. Record your observations and, results in the chart provided in the Lab Report., , (a), , (b), , (c), , (d), , (e), , Figure 23.2 Reactions in triple sugar–iron agar., (a) Uninoculated; (b) alkaline slant/acid butt, H2S;, (c) alkaline slant/acid butt; (d) acid slant/acid butt,, gas; and (e) acid slant/acid butt., , Experiment 23, , 169
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2. Explain why the TSI medium contains a lower concentration of glucose, than of lactose or sucrose., , 3. Explain the purpose of the phenol red in the medium., , 4. Explain the purpose of thiosulfate in the medium., , 5. Explain why the test observations must be made between 18 and 24 hours, after inoculation., , 172, , Experiment 23: Lab Report
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E XP ER IME NT, , 24, , IMViC Test, , Identifying enteric bacilli is important in controlling intestinal infections by preventing contamination of food and water supplies. The groups of, bacteria that will be isolated from the intestinal, tract of humans and lower mammals are classified, as members of the family Enterobacteriaceae., They are short, gram-negative, non–spore-forming, bacilli. Included in this family are, 1. Pathogens, such as members of the genera, Salmonella and Shigella, 2. Occasional pathogens, such as members of, the genera Proteus and Klebsiella, 3. Normal intestinal flora, such as members, of the genera Escherichia and Enterobacter,, which are saprophytic inhabitants of the intestinal tract, We can differentiate between the principal groups, of Enterobacteriaceae by their biochemical properties and enzymatic reactions in the presence, of specific substrates using the IMViC series of, tests (indole, methyl red, Voges-Proskauer,, and citrate utilization). Figure 24.11 on page 179, shows the biochemical reactions that occur during, the IMViC tests., The following experiments are designed for, either a short or long version. The short version, uses selected members of the enteric family. The, long procedure uses bacterial species that do not, belong solely to the Enterobacteriaceae. Nonenteric forms are included to acquaint you with the, biochemical activities of other organisms grown, in these media and to enable you to use these data, for further comparisons of both types of bacteria., Selected organisms to use in the long-version procedures are listed on 152. The enteric organisms, , may be subdivided as lactose fermenters and non–, lactose fermenters., Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, , Lactose, fermenters, Enteric, , Salmonella typhimurium, Shigella dysenteriae, Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenes faecalis, , Non–lactose, fermenters, , Corynebacterium xerosis, Micrococcus luteus, Lactococcus lactis, Staphylococcus aureus, Bacillus cereus, , Nonenteric, , C L I N I C A L A P P L I C AT I O N, Identification of Enteric Bacteria, Microbiologists use the IMViC test to identify members of the Enterobacteriaceae, some of which are, powerful pathogens such as members of the genera, Shigella and Salmonella, which cause intestinal, infections. Identification of the causative agent, may lead to the source of the infection, such as, raw food (Salmonella) or fecal contamination of, food (Shigella). This aids healthcare workers in, determining the possible number of individuals who, have been exposed and who may require medical, attention. This test uses the organisms’ biochemical, properties and enzymatic reactions on specific substrates as a means of identification., , 173
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FU RT HER R E ADING, Refer to the section on bacterial metabolism in, your textbook for further information on the metabolic pathways to be tested. In your textbook’s, index, search under “Indole,” “Methyl Red,” and, “Tryptophan Metabolism.”, , Indole Production Test, , PART A, , LEARNING OBJECTIVE, Once you have completed this test, you, should be able to, , of indole is detectable by adding Kovac’s reagent,, which produces a cherry red reagent layer. The, reagent produces this color, which is composed, of p-dimethylaminobenzaldehyde, butanol, and, hydrochloric acid. Indole is extracted from the, medium into the reagent layer by the acidified, butyl alcohol component and forms a complex, with the p-dimethylaminobenzaldehyde, yielding, the cherry red color. Figure 24.2 illustrates this, chemical reaction., Cultures producing a red reagent layer following addition of Kovac’s reagent are indole-positive;, an example of this is E. coli. The absence of red, coloration demonstrates that the substrate tryptophan was not hydrolyzed and indicates an indolenegative reaction., , 1. Determine the ability of microorganisms, to degrade the amino acid tryptophan., , Principle, Tryptophan is an essential amino acid that can, undergo oxidation by way of the enzymatic activities of some bacteria. The enzyme tryptophanase mediates the conversion of tryptophan into, metabolic products. Figure 24.1 illustrates the, chemistry of this reaction. This ability to hydrolyze, tryptophan with the production of indole is not a, characteristic of all microorganisms and therefore, serves as a biochemical marker., In this experiment, SIM agar, which contains, the substrate tryptophan, is used. The presence, , CH2, , CH, , COOH, , AT T HE BE NCH, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase™, soy broth cultures of, ❏❏ E. coli, ❏❏ Proteus vulgaris, ❏❏ Enterobacter aerogenes, For the long version, 24- to 48-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , NH2, , N, H, , CH3, , Tryptophanase, , + C, N, H, , Tryptophan, , O + NH3, , COOH, , Indole, , Pyruvic, acid, , Ammonia, , Figure 24.1 Enzymatic degradation of tryptophan, H, , CHO, HCI, Alcohol, , +, N, H, , N(CH3)2, p-dimethylaminobenzaldehyde, , Indole, , Figure 24.2 Indole reaction with Kovac’s reagent, 174, , Experiment 24, , C, , N+(CH3)2, , NH, , Dehydration, reduction, Quinoidal red-violet compound
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3. Based on your observations, determine and, record whether each organism was capable of, hydrolyzing the tryptophan., , PART B Methyl Red Test, Voges-Proskauer Test (MR-VP), LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, (a), , (b), , (c), , Figure 24.3 Indole production test., (a) Uninoculated, (b) negative, and (c) positive., , 1. Determine the ability of microorganisms to, ferment glucose with the production and, stabilization of high concentrations of acid, end products., , Media, , 2. Differentiate between all glucose-, fermenting enteric organisms, particularly, E. coli and E. aerogenes., , SIM agar deep tubes per designated student group, ❏❏ 4 for the short version, ❏❏ 14 for the long version, , 3. Differentiate further between enteric, organisms such as E. coli, E. aerogenes,, and K. pneumoniae., , Reagent, Kovac’s reagent, , Principle, , Equipment, , The hexose monosaccharide glucose is the major, substrate utilized by all enteric organisms for, energy production. The end products of this process will vary depending on the specific enzymatic, pathways present in the bacteria. In this test, the pH, indicator methyl red detects the presence of large, concentrations of acid end products. Although most, enteric microorganisms ferment glucose with the, production of organic acids, this test is of value in, the separation of E. coli and E. aerogenes., Both of these organisms initially produce, organic acid end products during the early incubation period. E. coli stabilizes and maintains, the low acidic pH (4) at the end of incubation., During the later incubation period, E. aerogenes, enzymatically converts these acids to nonacidic, end products, such as 2,3-butanediol and acetoin, (acetylmethylcarbinol), resulting in an elevated pH, of approximately 6. Figure 24.4 illustrates the glucose fermentation reaction generated by E. coli., As shown, at a pH of 4.4 or lower, the methyl red, indicator in the pH range of 4 will turn red, which, is indicative of a positive test. At a pH of 6.2 or, higher, still indicating the presence of acid but, , ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating needle, Test tube rack, Glassware marking pencil, , Procedure Lab One, 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled, deep tube by means of a stab inoculation. The, last tube will serve as a control., 2. Incubate tubes for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Add 10 drops of Kovac’s reagent to all deep tube, cultures and agitate the cultures gently., 2. Examine the color of the reagent layer in each, culture. (Refer to Figure 24.3). Record your, results in the chart in the Lab Report., , Experiment 24, , 175
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Media, , with a lower hydrogen ion concentration, the indicator turns yellow and is a negative test., The Voges-Proskauer test determines the capability of some organisms to produce nonacidic, or neutral end products, such as acetylmethylcarbinol, from the organic acids that result from, glucose metabolism. Figure 24.5 illustrates this, glucose fermentation, which is characteristic of, E. aerogenes., The reagent used in this test, Barritt’s reagent,, consists of a mixture of alcoholic a@naphthol and, 40% potassium hydroxide solution. Detection of, acetylmethylcarbinol requires this end product to, be oxidized to a diacetyl compound. This reaction, will occur in the presence of the a@naphthol catalyst and a guanidine group that is present in the, peptone of the MR-VP medium. As a result, a pink, complex is formed, imparting a rose color to the, medium. Figure 24.6 illustrates the chemistry of, this reaction., Development of a deep rose color in the culture 15 minutes following the addition of Barritt’s, reagent is indicative of the presence of acetylmethylcarbinol and represents a positive result. The, absence of rose coloration is a negative result., , MR-VP broth per designated student group, ❏❏ 4 for the short version, ❏❏ 14 for the long version, , Reagent, ❏❏ Methyl red indicator, ❏❏ Barritt’s reagents A and B, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Test tubes, Glassware marking pencil, , Procedure Lab One, 1. Using aseptic technique, inoculate each, experimental organism into its appropriately, labeled tube of medium by means of a loop, inoculation. The last tube will serve as a, control., 2. Incubate all cultures for 24 to 48 hours at 37°C., , AT THE B E N C H, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase, soy broth cultures of, ❏❏ E. coli, ❏❏ E. aerogenes, ❏❏ K. pneumoniae BSL -2, , (a), , For the long version, 24- to 48-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , Glucose + H2O, , Lactic acid, Acetic acid, Formic acid, , +, , CO2, , Experiment 24, , (c), , Figure 24.5 Methyl red test. (a) Uninoculated,, (b) positive, and (c) negative, , +, , H2 (pH 4.0), , Methyl red indicator turns red color, , Figure 24.4 Glucose fermentation reaction with methyl red pH reagent, , 176, , (b)
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Glucose, , +, , O2, , Acetic, acid, , 2,3-butanediol, acetylmethylcarbinol, , +, , CO2, , +, , H2 (pH 6.0), , Figure 24.6 Methyl Red Test. (a) Uninoculated, (b) positive, and (c) negative, CH3, C, CH, , CH3, , OH, O, OH, , +, , 40% KOH, Oxidation, , CH3, Acetylmethylcarbinol, , C, , O, , C, , O, , CH3, , a-naphthol, , Diacetyl, , NH2, , +, , C, , NH, , Pink, complex, , NH R, Guanidine, group of, peptone, , Figure 24.7 Acetylmethylcarbinol reaction with Barritt’s reagent, , Procedure Lab Two, 1. Transfer approximately one-third of each culture into an empty test tube and label these, tubes for the Voges-Proskauer test. Label the, original tube “MR.”, 2. Add five drops of the methyl red indicator to the, remaining aliquot of each culture (MR tube)., 3. Examine the color of all cultures (refer to, Figure 24.7). Record the results in the chart in, the Lab Report., 4. Based on your observations, determine and, record whether each organism was capable of, fermenting glucose with the production and, maintenance of a high concentration of acid., 5. To the aliquots of each broth culture separated, from step 1, add 10 drops of Barritt’s reagent, A and shake the cultures. Immediately add, 10 drops of Barritt’s reagent B and shake., Reshake the cultures every 3 to 4 minutes., 6. Examine the color of the cultures 15 minutes, after the addition of Barritt’s reagent. Refer to, Figure 24.8. Record your results in the, Lab Report., 7. Based on your observations, determine and, record whether each organism was capable, of fermenting glucose with ultimate production of acetylmethylcarbinol., , (a), , (b), , (c), , Figure 24.8 Voges-Proskauer test., (a) Uninoculated, (b) negative, and (c) positive, , PART C, , Citrate Utilization Test, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Differentiate between enteric organisms, by their ability to ferment citrate as a sole, source of carbon., , Experiment 24, , 177
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1., , HO, , COOH, , COOH, , CH2, , C, , C, , COOH, , CH2, COOH, Citrate, , 2. CO2, , +, , 2Na+, , Citrase, , CH2, , C, , COOH, , CH3, , Oxaloacetic, acid, , +, , H2O, , Na2CO3, , CH3, , COOH, , O, , O, , +, , Pyruvic, acid, , Alkaline pH, , COOH, , Acetic acid, , +, , CO2, , Excess, carbon, dioxide, , Color change from green to blue, , Figure 24.9 Enzymatic degradation of citrate, , Principle, In the absence of fermentable glucose or lactose,, some microorganisms are capable of using citrate, as a carbon source for their energy. This ability, depends on the presence of a citrate permease that facilitates the transport of citrate in the, cell. Citrate is the first major intermediate in the, Krebs cycle, and is produced by the condensation, of active acetyl with oxaloacetic acid. Citrate is, acted on by the enzyme citrase, which produces, oxaloacetic acid and acetate. These products are, then enzymatically converted to pyruvic acid and, carbon dioxide. During this reaction, the medium, becomes alkaline—the carbon dioxide that is generated combines with sodium and water to form, sodium carbonate, an alkaline product. The presence of sodium carbonate changes the bromthymol blue indicator incorporated into the medium, from green to deep Prussian blue. Figure 24.9 illustrates the chemistry of this reaction., Following incubation, citrate-positive cultures, are identified by the presence of growth on the, surface of the slant, which is accompanied by blue, coloration, as seen with E. aerogenes. Citratenegative cultures will show no growth, and the, medium will remain green., , For the long version, 24- to 48-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , Media, Simmons citrate agar slants per designated, student group, ❏❏ 4 for the short version, ❏❏ 14 for the long version, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating needle, Test tube rack, Glassware marking pencil, , Procedure Lab One, 1. Using aseptic technique, inoculate each organism into its appropriately labeled tube by means, , AT THE B E N C H, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase soy, broth cultures of, ❏❏ E. coli, ❏❏ E. aerogenes, ❏❏ K. pneumoniae BSL-2, 178, , Experiment 24, , (a), , (b), , Figure 24.10 Citrate utilization test. (a) Tube is, negative, showing no growth on slant surface., (b) Tube is positive, showing growth on slant, surface.
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of streak inoculation. The last tube will serve as, a control., 2. Incubate all cultures for 24 to 48 hours at 37°C., , the medium. Refer to Figure 24.10. Record your, results in the chart in the Lab Report., 2. Based on your observations, determine and, record whether each organism was capable of, using citrate as its sole source of carbon., , Procedure Lab Two, 1. Examine all agar slant cultures for the presence or absence of growth and coloration of, , Reagents, , Indole Test, , Kovac's, , Medium: SIM agar, Substrate: Tryptophan, CH2, , CH, , COOH, , CH3, , Tryptophanase, , +, N, H, , NH2, , N, H, , C, , O, , +, , NH3, , COOH, , Indole, , Pyruvic, acid, Indole (+), , Methyl Red Test, , Indole (–), , Methyl red, , Medium: MR-VP broth, Substrate: Glucose, , E. coli, , Acids, , +, , Lactic, Acetic, Formic, , +, , CO2, , +, , H2 (pH = 4.0), , H2O, E. aerogenes, , Lactic, , Ethanol, , Acetic, , Acetylmethylcarbinol, , Acids, , +, H2O, , +, , CO2 (pH = 6.5), , pH = 4.0, MR (+), , Voges-Proskauer Test, , Barritt's, , Medium: MR-VP broth, Substrate: Glucose, , +, , / O2, , Acetic acid, , 1 2, , pH = 6.5, MR (-), , 2,3-Butanediol, Acetylmethylcarbinol, , +, , H2O, , +, , CO2 (pH = 6.0), , VP (+), , VP (–), , Growth,, blue medium, Citrate (+), , No growth,, green medium, Citrate (-), , Citrate Test, Medium: Simmons citrate, Substrate: Citrate, Citrate, permease, Citrase, , Pyruvate, , +, , Oxaloacetic acid, , CO2, , +, , Excess, sodium, , Na2CO3, Alkaline pH, , Figure 24.11 Summary of IMViC reactions, Experiment 24, , 179
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3. Account for the development of alkalinity in cultures capable of using, citrate as their sole carbon source., , 4., , In the carbohydrate fermentation test, we found that both E. coli, and E. aerogenes produced the end products acid and gas., Account for the fact that E. coli is methyl red–positive and E, . aerogenes is, methyl red–negative., , 5., , The end products of tryptophan degradation are indole and pyruvic acid. Why do we test for the presence of indole rather than, pyruvic acid as the indicator of tryptophanase activity?, , 6., , Simmons citrate medium contains primarily inorganic ammonium,, potassium, and sodium salts, plus organic citrate. What is the rationale for using a medium with this type of composition for the p, erformance, of the citrate utilization test?, , 184, , Experiment 24: Lab Report
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E XP E R IMENT, , 25, , Hydrogen Sulfide Test, , LEARNING OBJECTIVES, , 2S2O32-, , Once you have completed this experiment,, you will be able to, 1. Explain how microorganisms produce, hydrogen sulfide from sulfur-containing, amino acids or inorganic sulfur, compounds., 2. Determine the mobility of microorganisms in SIM or TTC agar., , Principle, There are two major fermentative pathways by, which some microorganisms are able to produce, hydrogen sulfide (H2S)., Pathway 1: Gaseous H2S may be produced, by the reduction (hydrogenation) of organic, sulfur present in the amino acid cysteine, which, is a component of peptones contained in the, medium. These peptones are degraded by microbial enzymes to amino acids, including the sulfurcontaining amino acid cysteine. This amino acid in, the presence of a cysteine desulfurase loses the, sulfur atom, which is then reduced by the addition, of hydrogen from water to form bubbles of hydrogen sulfide gas (H2S c ) as illustrated:, CH2, , SH, , CH, , NH2, , Cysteine, desulfurase, , CH3, C, , O, , COOH, , COOH, , Cysteine, , Pyruvic, acid, , +, , H2S, , +, , NH3, , +, , 4H+, , +, , 4e-, , Thiosulfate, reductase, , Thiosulfate, , Ammonia, , Pathway 2: Gaseous H2S may also be, roduced by the reduction of inorganic sulfur comp, pounds such as the thiosulfates (S2O32-), sulfates, (SO42-), or sulfites (SO32-). The medium contains, sodium thiosulfate, which certain microorganisms, are capable of reducing to sulfite with the liberation of hydrogen sulfide. The sulfur atoms act as, hydrogen acceptors during oxidation of the inorganic compound as illustrated in the following:, , +, , Sulfite, , 2H2S, Hydrogen, Sulfide, gas, , In this experiment, the SIM medium contains, peptone and sodium thiosulfate as the sulfur, substrates; ferrous sulfate (FeSO4), which behaves, as the H2S indicator; and sufficient agar to make, the medium semisolid and thus enhance anaerobic, respiration. Regardless of which pathway is used,, the hydrogen sulfide gas is colorless and therefore not visible. Ferrous ammonium sulfate in the, medium serves as an indicator by combining with, the gas, forming an insoluble black ferrous sulfide, precipitate that is seen along the line of the stab, inoculation and is indicative of H2S production., Absence of the precipitate is evidence of a negative, reaction. Figure 25.1 illustrates the overall reactions, for both pathways and their interpretation., CH2, , SH, , CH, , NH2, , CH3, , Cysteine, , C, , desulfurase, , O, , +, , +, , NH3, , H2S, , COOH, , COOH, Cysteine, , Pyruvic, acid, , Ammonia, , H2S gas, (colorless), , Sodium thiosulfate (Na2S2O3) + Bacterial acids, , H2S (gas), , +, , Fe2+ (ions), Hydrogen, sulfide, gas, , 2SO32-, , Black precipitate, FeS, , H2S (+), , No H2S, , +, , (gas), , Fe2+ (ions), No FeS, , H2S (–), , Figure 25.1 Detectivhydrogen sulphide, 185
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Motility, SIM agar may also be used to detect motile organisms. Motility is recognized when culture growth, (turbidity) of flagellated organisms is not restricted, to the line of inoculation. Growth of nonmotile, organisms is confined to the line of inoculation. A, gelatin-based complex agar with a colorless dye,, 2,3,5-triphenyltetrazolium chloride (TTC), may also, be used to determine bacterial motility. This test is, based on the reduction of TTC by motile bacteria, to form formazan, an insoluble red pigment., , F U RTHER R EA D ING, Refer to the section on the structure of bacterial, flagella in your textbook for further information on, bacterial motility. In your textbook’s index, search, under “Flagella,” “TTC,” and “SIM Agar.”, , C L I N I C A L A P P L I C AT I O N, Identifying Intestinal Pathogens, While generally considered a self-limiting symptom,, diarrhea due to Proteus is initially difficult to differentiate, from early stages of the more severe bloody diarrhea, (dysentery) associated with some Shigella or Salmonella species. Bacteria belonging to the genera Salmonella and Proteus enzymatically metabolize inorganic, sulfur compounds and sulfur-containing amino acids,, producing H2S. The hydrogen sulfide test is one way to, separate and identify Shigella dysentariae, which does, not produce H2S, from Proteus and Salmonella., , TTC agar deep tubes per designated student group, ❏❏ 5 for the short, ❏❏ 14 for the long, version, version, , Equipment, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Inoculating needle, , ❏❏ Test tube rack, ❏❏ Glassware marking, pencil, , Procedure Lab One, 1. Aseptically inoculate each experimental, organism into its appropriately labeled tube by, means of stab inoculation. The last tube will, serve as a control., 2. Incubate all cultures for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Examine all SIM cultures for the presence or, absence of black coloration along the line of, the stab inoculation. Refer to Figure 25.2, and, record your results in the chart provided in the, Lab Report., 2. Examine all TTC cultures for the presence or, absence of black coloration along the line of, the stab inoculation. Refer to Figure 25.2, and, record your results in the chart provided in the, Lab Report., 3. Based on your observations, determine and, record whether each organism was capable of, producing hydrogen sulfide., 4. Observe all cultures for the presence (+ ) or, absence (- ) of motility. Record your results in, the chart in the Lab Report., , AT THE B E N C H, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase soy, broth cultures of, ❏❏ Enterobacter aerogenes, ❏❏ Shigella dysenteriae BSL -2, ❏❏ Proteus vulgaris, ❏❏ Salmonella typhimurium BSL -2, For the long version, 24- to 48-hour Trypticase soy, broth cultures of the 13 organisms listed on page 152., , Media, SIM agar deep tubes per designated student group, ❏❏ 5 for the short, ❏❏ 14 for the long, version, version, 186, Experiment 25, , (a), , (b), , (c), , Figure 25.2 Hydrogen sulfide production test., (a) Negative, (b) positive with motility, and, (c) positive with no motility.
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2. Explain how SIM medium is used to detect motility., , 3. Explain the function of the ferrous ammonium sulfate in SIM agar., , 4., , 5., , 188, , Why is P. vulgaris H2S-positive and E. aerogenes H2S-negative?, , A stool specimen of a patient with severe diarrhea was cultured in, a series of specialized media for isolation of enteric organisms., The cultures yielded three isolates that were species of Salmonella,, Shigella, and Escherichia. Explain why the H2S production test would be, diagnostically significant., , Experiment 25: Lab Report
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E XP E R IMENT, , 26, , Urease Test, , F U RT HE R R E ADI N G, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Determine how microorganisms degrade, urea by means of the enzyme urease., , Refer to the section on urea metabolism in, your textbook for further information on the, utilization of urea by bacteria. In your textbook’s, index, search under “Urea Cycle,” “Proteus,” and, “Urease.”, , Principle, , C L I N I C A L A P P L I C AT I O N, , Urease, which is produced by some microorganisms, is an enzyme that is especially helpful in, the identification of Proteus vulgaris. Although, other organisms may produce urease, their action, on the substrate urea tends to be slower than, that seen with Proteus species. Therefore, this, test serves to rapidly distinguish members of this, genus from other non–lactose-fermenting enteric, microorganisms., Urease is a hydrolytic enzyme that attacks the, nitrogen–carbon bond in amide compounds such, as urea and forms the alkaline end product ammonia. Figure 26.1 illustrates this chemical reaction., The presence of urease is detectable when, the organisms are grown in a urea broth medium, containing the pH indicator phenol red. As the substrate urea is split into its products, the presence, of ammonia creates an alkaline environment that, causes the phenol red to turn to a deep pink. This, is a positive reaction for the presence of urease., Failure of a deep pink color to develop is evidence, of a negative reaction., , Pathogens and the Urease Test, The urease test is primarily used to distinguish, the small number of urease-positive enterics from, other non–lactose-fermenting enteric bacteria., Many enterics can degrade urea, but only a few are, termed rapid urease-positive organisms. While part, of the normal flora, these commensals have been, identified as opportunistic pathogens. Members, of the gastroduodenal commensals are included, among this group of organisms., , H2N, , NH2, C, , +, , 2H2O Urease, , CO2, , +, , H2O, , +, , 2NH3, , O, Urea, , Carbon, dioxide, , Water, , Ammonia, , Figure 26.1 Enzymatic degradation of urea, , AT T HE BE NCH, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase, soy broth cultures of, ❏❏ Escherichia coli, ❏❏ Proteus vulgaris, ❏❏ Klebsiella pneumoniae BSL -2, ❏❏ Salmonella typhimurium BSL -2, For the long version, 24- to 48-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , 189
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Media, Urea broth per designated student group, ❏❏ 5 for the short version, ❏❏ 14 for the long version, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Test tube rack, Glassware marking pencil, (a), , Procedure Lab One, 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube by means of loop inoculation. The last, tube will serve as a control., 2. Incubate cultures 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Examine all urea broth cultures for color., (Refer to Figure 26.2). Record your results in, the chart in the Lab Report., , 190, , Experiment 26, , (b), , Figure 26.2 Urease test. (a) Negative and, (b) positive., , 2. Based on your observations, determine and, record whether each organism was capable of, hydrolyzing the substrate urea.
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2. Explain the function of phenol red in the urea broth medium., , 3. Explain how the urease test is useful for identifying members of the genus, Proteus., , 4., , 192, , A swollen can of chicken soup is examined by the public health, laboratory and found to contain large numbers of gram-negative,, H2S-positive bacilli. Which biochemical tests would you perform to identify, the genus of the contaminant? Justify your test choices., , Experiment 26: Lab Report
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E XP E R IMENT, , 27, , Litmus–Milk Reactions, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Differentiate between microorganisms, that enzymatically transform different, milk substrates into varied metabolic, end products., , Principle, The major milk substrates capable of transformation are the milk sugar lactose and the milk, proteins casein, lactalbumin, and lactoglobulin., To distinguish among the metabolic changes, produced in milk, a pH indicator, the oxidationreduction indicator litmus, is incorporated into, the medium. Litmus milk now forms an excellent, differential medium in which microorganisms can, metabolize milk substrates depending on their, enzymatic complement. A variety of different biochemical changes result, as follows:, , Litmus milk, , Lactose fermentation, Gas production, Litmus reduction, Curd formation, Proteolysis, Alkaline reaction, , Lactose Fermentation, Organisms capable of using lactose as a carbon, source for energy production utilize the inducible, enzyme B-galactosidase and degrade lactose as, follows:, Lactose, , B-galactosidase, , Glucose + Galactose, Embden-Meyerhof, pathway, Pyruvic acid, , The presence of lactic acid is easily detected, because litmus is purple at a neutral pH and turns, pink when the medium is acidified to an approximate pH of 4., , Gas Formation, The end products of the microbial fermentation of lactose are likely to include thegases, CO2 c + H2 c . The presence of gas may be seen, in separations of the curd or by the development, of tracks or fissures within the curd as gas rises to, the surface., , Litmus Reduction, Fermentation is an anaerobic process involving biooxidations that occur in the absence of, molecular oxygen. These oxidations may be, visualized as the removal of hydrogen (dehydrogenation) from a substrate. Since hydrogen ions, cannot exist in the free state, there must be an, immediate and concomitant electron acceptor, available to bind these hydrogen ions, or else, oxidation-reduction reactions are not possible, and cells cannot manufacture energy. In the litmus milk test, litmus acts as such an acceptor., While in the oxidized state, the litmus is purple;, when it accepts hydrogen from a substrate, it, will become reduced and turn white or milk-, colored. This oxidation of lactose, which produces lactic acid, butyric acid, CO2 c , and H2 c , is, as follows:, Lactose, , Glucose, , Pyruvic acid, , Lactic acid, Butyric acid, CO2 + H2, , The excess hydrogen is now accepted by the, hydrogen acceptor litmus, which turns white and, is said to be reduced., , Lactic acid, , 193
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Curd Formation, The biochemical activities of different microorganisms grown in litmus milk may result in the, production of two distinct types of curds (clots)., Curds are designated as either acid or rennet,, depending on the biochemical mechanism responsible for their formation., , Acid, curd, , Lactic acid or other organic, acids cause precipitation of, the milk protein casein as, calcium caseinate to form an, insoluble clot. The clot is hard, and will not retract from the, walls of the test tube. An acid, curd is easily identified if the, tube is inverted and the clot, remains immoble., , (a), , (b), , (c), , (d), , (e), , Figure 27.1 Litmus milk reactions., (a) Uninoculated, (b) acid, (c) acid with reduction, and curd, (d) alkaline, and (e) proteolysis., , Curd, , Rennet, curd, , Some organisms produce, rennin, an enzyme that acts on, casein to form paracasein,, which in the presence of, calcium ions is converted to, calcium paracaseinate and, forms an insoluble clot. Unlike, the acid curd, this is a soft, semisolid clot that will flow, slowly when the tube is tilted., , Proteolysis (Peptonization), The inability of some microorganisms to obtain, their energy by way of lactose fermentation means, they must use other nutritional sources, such as, proteins, for this purpose.(See Figure 21.4.) By, means of proteolytic enzymes, these organisms, hydrolyze the milk proteins, primarily casein, into, their basic building blocks, namely amino acids., This digestion of proteins is accompanied by the, evolution of large quantities of ammonia, resulting, in an alkaline pH in the medium. The litmus turns, deep purple in the upper portion of the tube, while, the medium begins to lose body and produces a, translucent, brown, whey-like appearance as the, protein is hydrolyzed to amino acids., , Alkaline Reaction, An alkaline reaction is evident when the color, of the medium remains unchanged or changes, to a deeper blue. This reaction is indicative of, , 194, , Experiment 27, , the partial degradation of casein into shorter, polypeptide chains, with the simultaneous, release of alkaline end products that are responsible for the observable color change., Figure 27.1 and Figure 27.2 show the possible, litmus milk reactions and their appearance following the appropriate incubation of the cultures., , F U RT H E R RE A D I N G, Refer to the section on fermentation in your, textbook for further information on the bacterial, process of fermenting milk sugars and proteins. In, your textbook’s index, search under “Proteolysis,”, “Lactose Fermentation,” and “Litmus Reduction.”, , C L I N I C A L A P P L I C AT I O N, Differentiating Enterobacteriaceae and, Clostridium, The litmus milk test differentiates members of the, Enterobacteriaceae from other gram-negative, bacilli based on the enterics’ ability to reduce, litmus. It is also used to differentiate members, within the genus Clostridium. Watery diarrhea, caused by C. perfringes (contaminated food) is generally considered self-limiting. But diarrhea caused, by C. difficle may be associated with antibiotic use, that has removed the normal flora of the colon.
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Pink, , Pink band, , Pink band, , Pink band, , White, , White, , White, Fissures, in curd (gas), , Solid, Lactose, fermentation, (acid), , Acid followed, by reduction, , Acid, reduction,, and curd, , Solid, Acid, reduction,, curd, and gas, , Purple band, White, , Litmus reduction, , Deep purple band, Whey-like brownish, translucent medium, Litmus milk, , Proteolysis, , Unchanged or deep blue, , Alkaline reaction, , Figure 27.2 Summary of possible litmus milk reactions, , Experiment 27, , 195
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AT THE B E N C H, , Materials, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Test tube rack, Glassware marking pencil, , Cultures, , Procedure Lab One, , For the short version, 24- to 48-hour Trypticase soy, broth cultures of, ❏❏ Escherichia coli, ❏❏ Alcaligenes faecalis, ❏❏ Lactococcus lactis, ❏❏ Pseudomonas aeruginosa BSL -2, For the long version, 24- to 48-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , 1., , Media, Litmus milk broth per designated student group, ❏❏ 5 for the short version, ❏❏ 14 for the long version, , 196, , Experiment 27, , Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube by means of a loop inoculation. The last, tube will serve as a control., 2. Incubate all cultures for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Examine all the litmus milk cultures for color, and consistency of the medium. Record your, results in the chart in the Lab Report., 2. Based on your observations, determine and, record the type(s) of reaction(s) that have, taken place in each culture.
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2. Describe the litmus milk reactions that may occur when proteins are metabolized as an energy source., , 3. Explain how the litmus in the litmus milk acts as a redox indicator., , 4., , Can a litmus milk culture show a pink band at the top and a, brownish translucent layer at the bottom? Explain., , 5., , Explain why litmus milk is considered a good differential medium., , 198, , Experiment 27: Lab Report
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E XP E R IMENT, , 28, , Nitrate Reduction Test, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Determine how some microorganisms, reduce nitrates (NO3–) to nitrites (NO2–) or, beyond the nitrite stage., , Principle, The reduction of nitrates by some aerobic and, facultative anaerobic microorganisms occurs in, the absence of molecular oxygen, an anaerobic, process. In these organisms, anaerobic respiration is an oxidative process whereby the cell uses, inorganic substances such as nitrates (NO3−), or sulfates (SO42−) to supply oxygen that is subsequently utilized as a final hydrogen acceptor, during energy formation. The biochemical transformation may be visualized as follows:, Partial Reduction, NO3-, , +, , Nitrate, , +, , 2H+, , Nitrate, , 2e-, , Hydrogen, electrons, , +, , NO2-, , reductase, , Nitrite, , H2O, Water, , Some organisms possess the enzymatic capacity to, act further on nitrites to reduce them to ammonia, (NO3+) or molecular nitrogen (N2). These reactions may be described as follows:, Complete reduction, NO2-, , NH3+, , Nitrite, , Ammonia, , with 0.1% potassium nitrate (KNO3) as the nitrate, substrate. In addition, the medium is made into a, semisolid by the addition of 0.1% agar. The semisolidity impedes the diffusion of oxygen into the, medium, thereby favoring the anaerobic requirement for nitrate reduction., Following incubation of the cultures, an organism’s ability to reduce nitrates to nitrites is determined by the addition of two reagents: Solution, A, which is sulfanilic acid, followed by Solution, B, which is a-naphthylamine. Note: This should, not be confused with Barritt’s reagent. Following, reduction, the addition of Solutions A and B will, produce an immediate cherry red color., NO3-, , Nitrate, , NO2- 1Red color on addition of, reductase, , Solutions A and B2, , Cultures that do not produce a color change, suggest one of two possibilities: (1) Nitrates were, not reduced by the organism, or (2) the organism possessed such potent nitrate reductase, enzymes that nitrates were rapidly reduced, beyond nitrites to ammonia or even molecular, nitrogen. To determine whether nitrates were, reduced past the nitrite stage, a small amount of, zinc powder is added to the basically colorless, cultures already containing Solutions A and B., Zinc reduces nitrates to nitrites. The development, of red color therefore verifies that nitrates were, not reduced to nitrites by the organism. If nitrates, were not reduced, a negative nitrate reduction, reaction has occurred. If the addition of zinc does, not produce a color change, the nitrates in the, medium were reduced beyond nitrites to ammonia or nitrogen gas. This is a positive reaction, as, shown in Figure 28.1. Results of nitrate reduction, tests are shown in Figure 28.2., , or, 2NO3Nitrate, , +, , 12H+, , +, , 10e-, , N2, , +, , 6H2O, , Molecular, nitrogen, , Nitrate reduction can be determined by cultivating organisms in a nitrate broth medium., The medium is a nutrient broth supplemented, , F U RT H E R RE A D I N G, Refer to the section on nitrogen metabolism in, your textbook for further information on the utilization of nitrogen by bacteria. In your textbook’s, index, search under “Nitrate Reduction,” “Nitrite,”, and “Sulfanilic Acid.”, , 199
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NH2, , NH2, , +, , N, , +, , N, , NH2, , +, , HNO2, , SO3H, , H2O, , SO2H, , A-naphthylamine, , Sulfanilic acid, (colorless), , (colorless), , Nitrous, acid, , Sulfobenzene azoA-naphthylamine, (red), , Water, , Figure 28.1 Formation of colored complex indicative of NO3- reduction, , ❏❏ Escherichia coli, ❏❏ Pseudomonas ae, BSL -2, ❏❏ Alcaligenes faecalis, For the long version, 24- to 48-hour Trypticase soy, broth cultures of the 13 organisms listed on page 152., , Media, Trypticase nitrate broth per designated student group, ❏❏ 4 for the short, ❏❏ 14 for the long, version, version, , Reagents, (a), , (b), , (c), , (d), , Figure 28.2 Nitrate reduction tests. (a) Uninoculated,, (b) positive with Solutions A + B, (c) positive with, Solutions A + B + zinc powder, and (d) negative, with Solutions A + B + zinc powder., , C L I N I C A L A P P L I C AT I O N, Differentiating Mycobacterium Tuberculosis, from Non-tubercle Mycobacterium, This test identifies intestinal bacteria that are able, to reduce nitrates to nitrites. When presented with, a patient who exhibits the symptoms of tuberculosis and is positive for tubercles on an x-ray, test, a sputum sample for Mycobacterium. To distinguish between Mycobacterium tuberculosis and, other Mycobacterium species, a nitrate reduction test is used, since M. tuberculosis is the only, Mycobacterium species with this capacity., , AT THE B E N C H, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase soy, broth cultures of, 200, , Experiment 28, , ❏❏ Solution A, (sulfanilic acid), , ❏❏ Solution B, (a-naphthylamine), ❏❏ Zinc powder, , Equipment, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Inoculating loop, , ❏❏ Test tube rack, ❏❏ Glassware marking, pencil, , Procedure Lab One, 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube by means of a loop inoculation. The last, tube will serve as a control., 2. Incubate all cultures for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Add five drops of Solution A and then five, drops of Solution B to all nitrate broth cultures. Observe and record in the Lab Report, chart whether a red coloration develops in, each of the cultures., 2. Add a minute quantity of zinc to the cultures, in which no red color developed. Observe and, record whether red coloration develops in, each of the cultures., 3. On the basis of your observations, determine and record in the Lab Report chart, whether each organism was capable of, nitrate reduction. Identify the end product, (NO2− or NH3+/N2), if any, that is present.
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E XP E R IMENT, , 28, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Bacterial, Species, , Red Coloration with, Solutions A and, B ( + ) or ( −), , Red Coloration with, Zinc ( + ) or ( −), , Nitrate Reductions, (plus) or ( −), , End Products, , E. coli, E. aerogenes, K. pneumoniae, S. dysenteriae, S. typhimurium, P. vulgaris, P. aeruginosa, A. faecalis, M. luteus, L. lactis, S. aureus, B. cereus, C. xerosis, Alternate organism, Control, , Review Questions, 1. Explain the function of the 0.1% agar in the nitrate medium., , Experiment 28: Lab Report, , 201
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2. Explain the functions of Solutions A and B., , 3. If a culture did not undergo a color change on the addition of Solutions, A and B, explain how you would interpret this result., , 4. Explain why the development of a red color on the addition of zinc is a, negative test., , 5., , 202, , Discuss the relationship between an organism’s ability to reduce, nitrate past the nitrite stage and that organism’s proteolytic activity., , Experiment 28: Lab Report
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E XP E R IMENT, , 29, , Catalase Test, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Determine how some microorganisms, degrade hydrogen peroxide by producing, the enzyme catalase., , Principle, During aerobic respiration, microorganisms produce hydrogen peroxide and, in some cases, an, extremely toxic superoxide. Accumulation of, these substances will result in death of the organism unless they can be enzymatically degraded., These substances are produced when aerobes,, facultative anaerobes, and microaerophiles use the, aerobic respiratory pathway, in which oxygen is, the final electron acceptor, during degradation of, carbohydrates for energy production. Organisms, capable of producing catalase rapidly degrade, hydrogen peroxide as illustrated:, 2H2O2, , Catalase, , Hydrogen, peroxide, , 2H2O, Water, , +, , O2, Free, oxygen, , Aerobic organisms that lack catalase can degrade, especially toxic superoxides using the enzyme, superoxide dismutase; the end product of a, superoxide dismutase is H2O2, but this is less toxic, to the bacterial cells than are the superoxides., The inability of strict anaerobes to synthesize, catalase, peroxidase, or superoxide dismutase may, explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the, toxic concentration of H2O2 cannot be degraded, when these organisms are cultivated in the presence of oxygen., Catalase production can be determined by, adding the substrate H2O2 to an appropriately, incubated Trypticase soy agar slant culture., If catalase is present, the chemical reaction, mentioned is indicated by bubbles of free, , oxygen gas O2 c . This is a positive catalase test;, the absence of bubble formation is a negative catalase test. Figure 29.1 shows the results of the catalase test using (a) the tube method, (b) the plate, method, and (c) slide method., , F U RT H E R RE A DI N G, Refer to the section on oxygen radicals in your, textbook for further information on the degradation of superoxides by bacteria. In your textbook’s, index, search under “Catalase,” “Superoxide, Dismutase (SOD),” and “Hydrogen Peroxide.”, , C L I N I C A L A P P L I C AT I O N, Differentiation of Staphylococci,, Streptococci, and Enterobacteriaceae, The catalase test is used for the biochemical differentiation of catalase-positive Staphylococci, and catalase-negative Streptococci, as well as, members of the Enterobacteriaceae. With the increasing worry about methicillin-resistant strains of, Staphylococcus in hospitals, the catalase test is a, quick and easy way to differentiate S. aureus, which, may be methicillin-resistant S. aureus (MRSA), from, other Staphylococcus species that have exhibited, lower incidences of methicillin resistance., , AT T HE BE NCH, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase, soy broth cultures of, ❏❏ Staphylococcus aureus BSL -2, ❏❏ Micrococcus luteus, ❏❏ Lactococcus lactis, For the long version, 24- to 48-hour Trypticase soy, broth cultures of the 13 organisms listed on page 152., 203
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Figure 29.1, Catalase test., Negative results, are shown on the, left and positive, results on the, right in the, (a) tube method, and (b) plate, method. Negative, results are shown, on the top and, positive results, on the bottom, in the (c) slide, method., , (b) Plate method, , (a) Tube method, , (c) Slide method, , Media, , Procedure Lab Two, , Trypticase soy agar slants per designated student, group, ❏❏ 4 for the short version, ❏❏ 14 for the long version, , Tube Method, , Reagent, ❏❏ 3% hydrogen peroxide, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Test tube rack, Glassware marking pencil, Glass microscope slides, Petri dish and cover, , Procedure Lab One, Tube Method, 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube by means of a streak inoculation. The last, tube will serve as a control., 2. Incubate all cultures for 24 to 48 hours, at 37°C., 204, , Experiment 29, , 1. Allow three or four drops of the 3% hydrogen, peroxide to flow over the entire surface of, each slant culture., 2. Examine each culture for the presence or, absence of bubbling or foaming. Record your, results in the chart in the Lab Report., 3. Based on your observations, determine and, record whether each organism was capable of, catalase activity., , Slide Method, 1. Label slides with the names of the organisms., 2. Using a sterile loop, collect a small sample of, the first organism from the culture tube and, transfer it to the appropriately labeled slide., 3. Place the slide in the Petri dish., 4. Place one drop of 3% hydrogen peroxide on, the sample. Do not mix. Place the cover on the, Petri dish to contain any aerosols., 5. Observe for immediate presence of bubble formation. Record your results in the chart in the, Lab Report., 6. Repeat steps 2 through 5 for the remaining test, organisms.
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EX PE RIME NT, , 29, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, PRESENCE OR ABSENCE OF BUBBLING, Bacterial Species, , Tube, , Slide, , CATALASE PRODUCTION ( -) OR ( +), Tube, , Slide, , E. coli, E. aerogenes, K. pneumoniae, S. dysenteriae, S. typhimurium, P. vulgaris, P. aeruginosa, A. faecalis, M. luteus, L. lactis, S. aureus, B. cereus, C. xerosis, Alternate organism, Control, , Review Questions, 1. Explain the toxic effect of O2 on strict anaerobes., , Experiment 29: Lab Report, , 205
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2. Illustrate the chemical reaction involved in the degradation of hydrogen, peroxide in the presence of catalase., , 3., , Would catalase be classified as an endoenzyme or an exoenzyme?, Explain., , 4., , Account for the ability of streptococci to tolerate O2 in the absence, of catalase activity., , 206, , Experiment 29: Lab Report
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E XP E R IMENT, , 30, , Oxidase Test, , LEARNING OBJECTIVES, , F U RT H E R RE A DI N G, , Once you have completed this experiment,, you should be able to, , Refer to the section on aerobic respiration in your, textbook for further information on the electron, transport system during bacterial ATP synthesis. In, your textbook’s index, search under “Cytochrome, Oxidase” and “Oxidase.”, , 1. Perform an experimental procedure that, differentiates between groups of bacteria, on the basis of cytochrome oxidase activity., , C L I N I C A L A P P L I C AT I O N, , Principle, Oxidase enzymes play a vital role in the operation, of the electron transport system during aerobic, respiration. Cytochrome oxidase catalyzes the, oxidation of a reduced cytochrome by molecular, oxygen (O2), resulting in the formation of H2O or, H2O2. Aerobic bacteria, as well as some facultative, anaerobes and microaerophiles, exhibit oxidase, activity. The oxidase test aids in differentiation, among members of the genera Neisseria and, Pseudomonas, which are oxidase-positive, and, Enterobacteriaceae, which are oxidase-negative., The ability of bacteria to produce cytochrome, oxidase can be determined by the addition of, the test reagent p-aminodimethylaniline oxalate, to colonies grown on a plate medium. This light, pink reagent serves as an artificial substrate,, donating electrons and thereby becoming oxidized to a blackish compound in the presence of, the oxidase and free oxygen. Following the addition of the test reagent, the development of pink,, then maroon, and finally dark purple coloration, on the surface of the colonies is indicative of, cytochrome oxidase production and represents, a positive test. No color change, or a light pink, coloration on the colonies, is indicative of the, absence of oxidase activity and is a negative test., The filter paper method may also be used, and is, described in this experiment., , Test to Distinguish Family Enterobacteriaceae, from Non-Enterobacteriaceae, Enterobacteriaceae are cytochrome oxidase–, negative, while Neisseria and Pseudomonas are, cytochrome oxidase–positive. The oxidase test is, an important tool in identifying N. meningitis, the, causative agent of bacterial meningitis, which has, significant morbidity and mortality rates. In addition, yeast, such as Candida, can be separated from, Saccharomyces and Torulopsis by this test., , AT T HE BE NCH, , Materials, Cultures, For the short version, 24- to 48-hour Trypticase soy, broth cultures of, ❏❏ Escherichia coli, ❏❏ Pseudomonas aeruginosa BSL -2, ❏❏ Alcaligenes faecalis, For the long version, 24- to 48-hour Trypticase, soy broth cultures of the 13 organisms listed on, page 152., , 207
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Media, Trypticase soy agar plates per designated student, group, ❏❏ 1 for the short version, ❏❏ 4 for the long version, , Reagent, ❏❏ p-Aminodimethylaniline oxalate , (Difco 0329-13-9), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Glassware marking pencil, Filter paper, , Procedure Lab One, Plate Method, 1. Prepare the Trypticase soy agar plate(s) for, inoculation as follows:, a. Short procedure: With a glassware marking pencil, divide the bottom of a Petri dish, into three sections and label each section, with the name of the test organism to be, inoculated., b. Long procedure: Follow step 1a, dividing, three plates into three sections and one, plate into four sections to accommodate the, 13 test organisms., 2. Using aseptic technique, make a single-line, streak inoculation of each test organism on, the agar surface of its appropriate section of, the plate(s)., 3. Incubate the plate(s) in an inverted position, for 24 to 48 hours at 37°C., , Procedure Lab Two, Plate Method, 1. Add two or three drops of the p-aminodimethylaniline oxalate to the surface of the growth, of each test organism., , 208, , Experiment 30, , Figure 30.1 Oxidase test. Negative test, on left,, results in no color change, and positive test, on, right, results in a color change to purple., , 2. Observe the growth for the presence or, absence of a color change from pink, to, maroon, and finally to purple. A positive test, result (+ ) will exhibit a color change in 10 to, 30 seconds; while a negative test result (- ), would exhibit no color change, or a light pink, color. Refer to Figure 30.1. Record the results, on the chart in the Lab Report., 3. Based on your observations, determine and, record whether each organism was capable of, producing cytochrome oxidase., , Filter Paper Method, 1. Prepare Petri dishes as described in Lab One, steps 1a and 1b., 2. Place filter paper in Petri dishes., 3. With a sterile loop, obtain a heavy loopful of, the first test organism and gently smear it on, the filter paper., 4. Drop one or two drops of p-aminodimethylaniline, oxalate reagent on the test organism., 5. Observe the organism for the appearance of a, purple color within 30 seconds of contact with, the oxidase reagent, indicating a positive test., 6. Repeat steps 3 to 5 for the remaining test, organisms., 7. Record your results in the chart in the Lab, Report.
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2. Why are strict aerobes oxidase-positive?, , 3. The oxidase test is used to differentiate among which groups of bacteria?, , 4. What is the function of the test reagent in this procedure?, , 5., , 210, , Your instructor asks you to isolate and identify the organisms in, an unknown culture. You find that the culture contains two gramnegative bacilli that produce swarming colonies. What biochemical test, would you use to identify the bacilli? Justify your answer., , Experiment 30: Lab Report
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E XP E R IMENT, , 31, , Utilization of Amino Acids, , FUR T HE R R E AD I N G, Refer to the section on amino acid metabolism in, your textbook for further information on the utilization of amino acids by bacteria during normal, growth. In your textbook’s index, search under, “Decarboxylase,” “Deaminase,” and “Amino Acid.”, , PA RT A, , Decarboxylase Test, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Identify and differentiate between organisms based on their ability to enzymatically, degrade amino acid substrates., , Principle, Every biologically active protein is composed of, the 20 essential amino acids. Structurally, amino, acids are composed of an alpha carbon ( ¬ C ¬ ),, an amino group ( ¬ NH2), a carboxyl group, ( ¬ COOH), and a hydrogen atom ( ¬ H). Also, attached to the alpha carbon is a side group or, an atom designated by an ( ¬ R), which differs in, each of the amino acids., —, , R, NH2 — C — COOH, —, , The study of amino acid metabolism began in the, early part of the twentieth century. Some scientists found that the enteric microorganisms, such, as Proteus and the so-called Providence species,, were able to deaminate a variety of amino acids, that provided a vehicle for distinguishing these, microorganisms from other members of the large, family of the Enterobacteriaceae. Researchers, determined that 11 of the 22 amino acids were, deaminated by amino acid oxidases, and it was, phenylalanine deaminase that produced the most, rapid enzymatic activity. Thus, phenylalanine, deaminase became the most widely studied deaminase used to differentiate enteric organisms., Likewise, researchers found that some, organisms are capable of decarboxylating amino, acids, providing a way to differentiate between, the enteric genera and species. For instance,, lysine decarboxylase is capable of differentiating, between Salmonella and Citrobacter. Ornithine, decarboxylase separates Enterobacter from, Klebsiella. Decarboxylase enzymes are numerous,, and each is specific for a particular substrate., Decarboxylases and deaminases play a vital role in, the utilization of amino acids and the metabolism, of nitrogen compounds., , H, , Decarboxylation is a process whereby some, microorganisms that possess decarboxylase, enzymes remove the carboxyl group to yield end, products consisting of an amine or diamine plus, carbon dioxide. Decarboxylated amino acids play, an essential role in cellular metabolism, since the, amines produced may serve as end products for the, synthesis of other molecules required by the cell., Decarboxylase enzymes are designated as adaptive, (or induced) enzymes and are produced in the presence of specific amino acid substrates upon which, they act. These amino acid substrates must possess, at least one chemical group other than an amine, ( ¬ NH2) or a carboxyl group ( ¬ COOH). In the, process of decarboxylation, organisms are cultivated in an acid environment and in the presence of, a specific substrate. The decarboxylation end product (amines) results in a shift to a more alkaline pH., In a clinical or diagnostic microbiology, laboratory, three decarboxylase enzymes are, used to differentiate between members of, the Enterobacteriaceae: lysine, ornithine, and, arginine. Decarboxylase activity is determined, by cultivating the organism in a nutrient, medium containing glucose, the specific amino, acid substrate, and bromthymol blue (the pH, indicator). If decarboxylation occurs, the pH, of the medium becomes alkaline despite the, fermentation of glucose, since the end products, (amines or diamines) are alkaline. The f unction, of the glucose in the medium is to ensure good, microbial growth and thus more reliable results, 211
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—, , H, , H, , —, , —, , NH2, , NH2 — C — C — C — C — C — H + CO2, —, , —, , H, , H, , —, , —, , —, , H, , H, —, , —, , —, , H, , decarboxylase, , H, , —, , —, , —, , NH2 — C — C — C — C — C — COOH, , H, Lysine, , —, , —, , NH2, , —, , H, , —, , H, , —, , H, , —, , H, , H, , H, , H, , H, , H, , Cadaverine, , Lysine, , Carbon, dioxide, , Figure 31.1 Degradation of lysine, , in the presence of the pH indicator. The presence, of each decarboxylase enzyme can be tested for, by supplementing decarboxylase broth with the, specific amino acid substrate, namely lysine,, arginine, or ornithine. For example, lysine, decarboxylase degrades l-lysine, forming the, diamine end product cadaverine plus carbon, dioxide as illustrated in Figure 31.1., In the experiment that follows, the decarboxy, lation of L-lysine will be studied. Note that, decarboxylation reactions occur under anaerobic, conditions that are satisfied by sealing the culture, tubes with sterile mineral oil. In the sealed tubes,, all of the unbound oxygen is utilized during the, organisms’ initial growth phase, and the pH of the, medium becomes alkaline as carbon dioxide (CO2), is produced in the culture tube. A pH indicator,, such as bromcresol purple, is usually incorporated into the medium for the easy detection of, pH changes. The production of acid end products, will cause the bromcresol purple to change color, from purple to yellow, indicating that acid has, formed, the medium has been acidified, and the, decarboxylase enzymes have been activated. The, activated enzyme responds with the production of, the alkalinizing diamine (cadaverine) and carbon, dioxide, which will produce a final color change, from yellow back to purple, thereby indicating that, L-lysine has been decarboxylated. The development of a turbid purple color verifies a positive, test for amino acid decarboxylation. The absence, of a purple color indicates a negative result., , C L I N I C A L A P P L I C AT I O N, Distinguishing between Enterobacter Species, The decarboxylase test identifies bacteria based, on the production of ammonia from the amino acids, lysine, ornithine, and arginine. The decarboxylase, test can be used to differentiate the causative, agent in many nosocomial infections of immunocompromised patients. The bacterium Enterobacter, aerogenes is lysine decarboxylase–positive while, other Enterobacter species are negative. The decarboxylase test is used primarily to identify bacteria, within the Enterobacteriaceae family., , 212, , Experiment 31, , AT T HE BE NCH, , Materials, Cultures, For the short version, 24-hour nutrient broth, cultures of, ❏❏ Proteus vulgaris, ❏❏ Citrobacter freundii, ❏❏ Escherichia coli, For the long version, 24- to 48-hour nutrient broth, cultures of the 13 organisms listed on page 152., , Media, Per designated student group, ❏❏ Three tubes of Moeller’s decarboxylase broth, supplemented with L-lysine (10 g/l), (labeled LD + ), ❏❏ Three tubes of Moeller’s decarboxylase broth, without lysine (labeled LD - ), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Glassware marking pencil, Inoculating loop and needle, Sterile Pasteur pipettes, Rubber bulbs, Test tube rack, Sterile mineral oil, , Controls, A positive control organism for this test is E. coli., , Procedure Lab One, 1. With a glassware marking pencil, label three, tubes of the LD + medium with the name of, the organism to be inoculated. Similarly label, three tubes of LD - medium. The use of (LD - ), control tubes is essential, since some bacterial, strains are capable of turning substrate-free, media positive. Note: Control tubes should
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2., , 3., , 4., 5., , remain yellow after incubation, denoting that, only glucose was fermented. The presence of a, positive control tube invalidates the test, and no, interpretation is possible., Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube using a loop inoculation., Place a rubber bulb onto a sterile Pasteur, pipette and overlay the surface of the inoculated culture tubes with 1 ml of sterile mineral, oil. Hold the tubes in a slanted position while, adding the mineral oil. Note: Do not let the tip, of the pipette touch the inoculated medium or, the sides of the test tube walls., Repeat the above procedure for the remaining, test cultures., Incubate all tubes at 37°C for 24 to 48 hours., , Procedure Lab Two, 1. Examine each culture tube for the presence of a, color change. Refer to Figure 31.2., 2. Based on your observations, determine whether, each organism was capable of performing, decarboxylation of lysine., 3. Record your results in the chart in the Lab Report., , (a), , (b), , (c), , Figure 31.2 Decarboxylase test (a) uninoculated,, (b) negative, and (c) positive, , CH2, , CH, , COOH, , Phenylalanine, deaminase, , Phenylalanine, Deaminase Test, PART B, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Demonstrate how some organisms remove, the amino group ( -NH2) from amino acids., , Principle, Microorganisms that contain deaminase, enzymes can remove the amino group ( ¬ NH2), from amino acids and other NH2-containing, chemical compounds. During this process the, amino acid, under the auspices of its specific, deaminase, will produce keto acids and ammonia, end products. In the experiment to follow, the, amino acid phenylalanine will be deaminated by, phenylalanine deaminase and converted to the, keto acid phenylpyruvic acid and ammonia., The organisms are cultured on a medium, incorporating phenylalanine as the substrate., Figure 31.3 illustrates this chemical reaction., If the organism possesses phenylalanine, deaminase, phenylpyruvic acid will be released, into the medium and can be detected by the, addition of a 10 percent to 12 percent ferric, chloride solution to the surface of the medium. If, a green color develops, the enzymatic deamination, of the substrate has occurred and is indicative, of a positive result. The absence of any color, change indicates a negative result. The resultant, green color produced upon the addition of ferric, chloride (FeCl 3) is due to the formation of a keto, acid (phenylpyruvic acid). It has been shown that, a@ and b@keto acids give a positive color reaction, with either alcoholic or aqueous solutions of, FeCl 3. Phenylpyruvic acid is an a@keto acid. The, results should be read immediately following the, addition of the reagent, since the color produced, fades quickly. When not in use, the ferric chloride, , CH2, , C, , COOH + NH3, , NH2, , O, , Phenylalanine, , Phenylpyruvic, acid, , Ammonia, , Figure 31.3 Deamination of phenylalanine, Experiment 31, , 213
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reagent should be refrigerated and kept in a dark, bottle to avoid exposure to light. The stability of, this reagent varies and should be checked weekly, with known positive cultures., , C L I N I C A L A P P L I C AT I O N, Differentiating Intestinal Bacteria, The phenylalanine deaminase test uses the differential medium phenylalanine agar to detect, bacteria containing the enzyme phenylalanine, deaminase, and is used to differentiate the genera, Proteus, Morganella, and Providencia from other, gram-negative intestinal bacilli. These genera of, enteric and environmental bacteria are known to, cause UTIs and gastroenteritis. Distinguishing them, from other enteric bacteria is clinically important, because of their high level of antibiotic resistance., , AT THE B E N C H, , Materials, Cultures, , Controls, A positive-control organism for this test is E. coli., , Procedure Lab One, 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled, tube using a streak inoculation., 2. Incubate cultures at 37°C for 24 to 48 hours., , Procedure Lab Two, 1. Add 5 to 10 drops of the ferric chloride solution to each agar slant and mix gently. Ferric, chloride is a chelating agent and binds to the, phenylpyruvic acid to produce a green color on, the slant (Figure 31.4)., 2. Based on your observations, determine whether, each organism was capable of amino acid, deamination. Note: Results should be read, immediately following the addition of, ferric chloride because the green color, fades rapidly., 3. Record your results in the Lab Report., , For the short version, 24-hour nutrient broth cultures of, ❏❏ E. coli, ❏❏ P. vulgaris, For the long version, 24-hour nutrient broth cultures of the 13 organisms listed on page 152., , Media, ❏❏ Two phenylalanine agar slants, , Reagents, ❏❏ 10 percent to 12 percent ferric chloride, solution, , Equipment, ❏❏ Microincinerator, or Bunsen burner, ❏❏ Glassware, marking pencil, , 214, , Experiment 31, , ❏❏, ❏❏, ❏❏, ❏❏, , Pasteur pipettes, Rubber bulbs, Test tube racks, Inoculating loop, , (a), , (b), , (c), , Figure 31.4 Phenylalanine deaminase test, (a) uninoculated, (b) negative, and (c) positive
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Review Questions, 1. A negative decarboxylase test is indicated by the production of a yellow color in the medium. Explain, the reason for the development of this color., , 2. Explain why deaminase activity must be determined immediately following the addition of ferric, chloride., , 3. What is the function of ferric chloride in the detection of deaminase activity?, , 4. Explain why the anaerobic environment is essential for decarboxylation of the substrate to occur., , 5., , Following a normal delivery, a nurse observes that an infant’s urine has a peculiar odor, resembling that of burnt sugar or maple syrup. Subsequent examination by the pediatrician, reveals that this child has maple syrup urine disease., a. What is this disease?, , b. How is it treated?, , 216, , Experiment 31: Lab Report
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Genus Identification of Unknown, Bacterial Cultures, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Use previously studied staining, cultural, characteristics, and biochemical procedures to identify the independent genus of, an unknown bacterial culture., , Principle, Identifying unknown bacterial cultures is one of the, major responsibilities of a microbiologist. Samples, of blood, tissue, food, water, and cosmetics are, examined daily in laboratories throughout the, world for the presence of contaminants. Additionally, industrial organizations are constantly screening materials to isolate new antibiotic-producing, organisms or organisms that will increase the yield, of marketable products, such as vitamins, solvents,, and enzymes. Once isolated, these unknown organisms must be identified and classified., The science of classification is called, taxonomy and deals with the separation of living, organisms into interrelated groups. Bergey’s Manual has been the official, internationally accepted, reference for bacterial classification since 1923., The current edition, Bergey’s Manual of Systematic Bacteriology, arranges related bacteria into 33, groups called sections rather than into the classical taxonomic groupings of phylum, class, order,, and family. The interrelationship of the organisms, in each section are based on characteristics such, as morphology, staining reactions, nutrition, cultural characteristics, physiology, cellular chemistry, and biochemical test results for specific, metabolic end products., You have developed sufficient knowledge of, staining methods, isolation techniques, microbial, nutrition, biochemical activities, and characteristics of microorganisms to work independently in, attempting to identify the genus of an unknown, culture. Table 32.1 gives characteristics of the major, organisms that we have used in experiments thus, far. You can use this table to identify unknown, , E XP E R IMENT, , 32, , cultures. The observations and results obtained following the experimental procedures are the basis of, your identification. However, you should note that, your biochemical results may not be identical to, those shown in Table 32.1; they may vary because, of variations in bacterial strains (subgroups of a, species). Therefore, it becomes imperative to recall, the specific biochemical tests that differentiate, among the different genera of the test organisms., Experiment 68 illustrates how to identify an, unknown culture using a more extensive procedure to differentiate bacterial species., , F U RT H E R RE A D I N G, Refer to the section in your textbook on metabolic, assays and the differences between these assays, for use in identification of gram-positive or -negative bacteria. In your textbook’s index, search, under “IMVIC,” “Triple Sugar Iron,” and “Durham, Tube.”, , C L I N I C A L A P P L I C AT I O N, Application of Learned Assays to Identify an, Unknown Bacterial Pathogen, The role of the clinical laboratory in a hospital is to, quickly and efficiently identify the causative agent, of a patient’s infection. This will entail choosing the, correct assays and performing them in the correct, order to logically identify the genus and species of, the agent., , TIPS FOR SUCCESS, • Gram stain your unknown culture first, then, determine which tests would be useful in identifying your bacteria. For example, the oxidase, test and the citrate test would be of no use, in identifying a gram-positive cocci bacterial, species., • Since many of the tests utilize agars that are, similar in appearance, be sure to label all tubes, and plates to ensure that results are collected, for the correct test., , 217
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Table 32.1 , , Cultural and Biochemical Characteristics of Unknown Organisms, , NO3 REDUCTION, , INDOLE PRODUCTION, , MR REACTION, , VP REACTION, , CITRATE USE, , UREASE ACTIVITY, , CATALASE ACTIVITY, , OXIDASE ACTIVITY, , GELATIN LIQUEFACTION, , STARCH HYDROLYSIS, , LIPID HYDROLYSIS, , AG, , A {, , -, , +, , +, , +, , -, , -, , -, , +, , -, , -, , -, , -, , AG, , AG, , -, , +, , -, , -, , +, , +, , -, , +, , -, , -, , -, , -, , -, , { { +, , +, , +, , -, , -, , -, , -, , DEXTROSE, , LACTOSE, ORGANISM, Escherichia coli, , AGAR SLANT, CULTURAL, LITMUS MILK, GRAM, STAIN CHARACTERISTICS, REACTION, Rod White, moist,, Acid, curd { , AG, glistening growth, gas { ,, reduction{, , AG, , SUCROSES, , H2S PRODUCTION, , FERMENTATION, , Enterobacter, aerogenes, , Rod, -, , Abundant, thick,, white, glistening, growth, , Acid, , Klebsiella, pneumoniae, , Rod, -, , Slimy, white,, somewhat, translucent, raised, growth, , Acid, gas,, curd{, , AG, , AG, , AG, , -, , +, , Shigella, dysenteriae, , Rod, -, , Thin, even, grayish, growth, , Alkaline, , -, , A, , A {, , -, , { { +, , -, , -, , -, , +, , -, , -, , -, , -, , Salmonella, typhimurium, , Rod, -, , Thin, even, grayish, growth, , Alkaline, , -, , AG ; A {, , +, , +, , -, , +, , -, , +, , -, , +, , -, , -, , -, , -, , Proteus vulgaris, , Rod, -, , Thin, blue-gray,, spreading growth, , Alkaline, , -, , AG, , AG { +, , +, , +, , +, , -, , { +, , +, , -, , +, , -, , -, , Pseudomonas, aeruginosa, , Rod, -, , Abundant, thin, white, growth, with medium, turning green, , Rapid, peptonization, , -, , -, , -, , -, , +, , -, , -, , -, , +, , -, , +, , +, , +, Rapid, , -, , +, , Alcaligenes, faecalis, , Rod*, , Thin, white, spreading, Alkaline, viscous growth, , -, , -, , -, , -, , -, , -, , -, , -, , { -, , +, , +, , -, , -, , -, , Staphylococcus, aureus, , Cocci, +, , Abundant, opaque,, golden growth, , Acid, reduction{, , A, , A, , A, , -, , +, , -, , +, , { -, , -, , +, , -, , +, , -, , +, , Lactococcus, lactis, , Cocci, +, , Thin, even growth, , Acid, rapid, reduction with, curd, , A, , A, , A, , -, , -, , -, , +, , -, , -, , -, , -, , -, , -, , -, , -, , Micrococcus, luteus, , Cocci, +, , Soft, smooth, yellow, growth, , Alkaline, , -, , { -, , -, , -, , -, , +, , +, , -, , +, Slow, , -, , -, , -, , +, , -, , -, , -, , -, , -, , +, , -, , -, , -, , -, , -, , +, , -, , -, , { -, , -, , +, , -, , +, Rapid, , +, , {, , AG{, , -, , -, , Corynebacterium, xerosis, , Rod, +, , Grayish, granular,, limited growth, , Alkaline, , -, , Bacillus cereus, , Rod, +, , Abundant, opaque,, white, waxy growth, , Peptonization, , -, , Note: AG = acid and gas; { = variable reaction; Rod* = coccobacillus, , 218, , Experiment 32, , -, , A { A {, , A, , A
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AT T H E B E N C H, , Materials, Cultures, Number-coded 24- to 48-hour Trypticase soy agar, slant cultures of the 13 bacterial species listed on, page 152. You will be provided with one unknown, pure culture., , Media, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Two Trypticase soy agar slants, Phenol red sucrose broth, Phenol red lactose broth, Phenol red dextrose broth, SIM agar deep tube, MR-VP broth, Tryptic nitrate broth, Simmons citrate agar slant, Urea broth, litmus milk, Trypticase soy agar plate, Nutrient gelatin deep tube, Starch agar plate, Tributyrin agar plate, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Crystal violet, Gram’s iodine, 95 percent ethyl alcohol, Safranin, Methyl red, 3 percent hydrogen peroxide, Barritt’s reagent, Solutions A and B, Kovac’s reagent, Zinc powder, p-aminodimethylaniline oxalate, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop and needle, Staining tray, Immersion oil, Lens paper, Bibulous paper, Microscope, Glassware marking pencil, , Procedure Lab One, 1. Perform a Gram stain of the unknown, organism. Observe and record in the Lab, Report chart the reaction and the morphology, and arrangement of the cells., 2. Using aseptic inoculating technique, inoculate, two Trypticase soy agar slants by means of a, streak inoculation. Following incubation, you will, use one slant culture to determine the cultural, characteristics of the unknown microorganism., You will use the second as a stock subculture, should it be necessary to repeat any of the tests., 3. Exercising care in aseptic technique so as not, to contaminate cultures and thereby obtain, spurious results, inoculate the media for the, following biochemical tests:, a., b., c., d., e., , Medium, Phenol red lactose broth, Phenol red dextrose broth, Phenol red sucrose broth, Litmus milk, SIM medium, , f. Tryptic nitrate broth, g. MR-VP broth, h., i., j., k., l., m., n., , Simmons citrate agar slant, Urea broth, Trypticase soy agar slant, Starch agar plate, Tributyrin agar plate, Nutrient gelatin deep tube, Trypticase soy agar plate, , Test, Carbohydrate, fermentation, Litmus milk reactions, Indole production, H2S production, Nitrate reduction, Methyl red test, Voges-Proskauer test, Citrate utilization, Urease activity, Catalase activity, Starch hydrolysis, Lipid hydrolysis, Gelatin liquefaction, Oxidase test, , 4. Incubate all cultures for 24 to 72 hours at 37°C., , Procedure Lab Two, 1. Examine a Trypticase soy agar slant culture, and determine the cultural characteristics of, your unknown organism. Record your results, in the Lab Report., 2. Perform biochemical tests on the remaining, cultures, making reference to the specific, laboratory exercise for each test. Record your, observations and results., 3. Based on your results, identify the genus, and species of the unknown organism. Note:, Results may vary depending on the strains of, each species used and the length of time the, organism has been maintained in stock culture. The observed results may not be identical to the expected results. Therefore, choose, the organism that best fits the results summarized in Table 32.1., Experiment 32, , 219
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EX PE RIME NT, , 32, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Description of Unknown’s Characteristics, , , Student ______________, , Culture No. ___________, Organism ____________, , Experimental Procedure, , Observations, , Results, , Gram stain, Acid-fast stain, Shape and arrangement, Cultural characteristics, Litmus milk reactions, Carbohydrate fermentations:, Lactose, Dextrose, Sucrose, H2S production, Nitrate reduction, Indole production, Methyl red test, Voges-Proskauer test, Citrate utilization, Urease activity, Catalase activity, Starch hydrolysis, Lipid hydrolysis, Gelatin liquefaction, Oxidase test, , Experiment 32: Lab Report, , 221
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PART 6, , The Protozoa, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Describe the distinguishing characteristics of protozoans., 2. Identify free-living and parasitic protozoans in microscopic views., , Introduction, The protozoa are a large and diverse group of, unicellular, eukaryotic organisms. Most are freeliving, but some are parasites. Their major distinguishing characteristics are, 1. The absence of a cell wall; some, however,, possess a flexible layer, a pellicle, or a rigid, shell of inorganic materials outside of the cell, membrane, 2. The ability to move by locomotor organelles or, by a gliding mechanism during their entire life, cycle or part of it, 3. Heterotrophic nutrition whereby the free-living, forms ingest particulates such as bacteria, yeast,, and algae, while the parasitic forms derive nutrients from the body fluids of their hosts, 4. Primarily asexual means of reproduction,, although sexual modes occur in some groups, Protozoan taxonomy is being continually, updated as new technology enables classification based on molecular characteristics. For our, discussion of protozoans, we follow a more traditional taxonomic scheme, dividing them into four, groups based on means of locomotion., 1. Sarcodina: Motility results from the streaming, of ectoplasm, producing protoplasmic projections called pseudopods (false feet). Prototypic, amoebas include the free-living Amoeba proteus and the parasite Entamoeba histolytica., , 2. Mastigophora: One or more whiplike, thin, structures called flagella effects locomotion., Free-living members include the genera Cercomonas, Heteronema, and Euglena, which, are photosynthetic protists that may be classified as flagellated algae. The parasitic forms, include Trichomonas vaginalis, Giardia, intestinalis (formerly called Giardia lamblia), and the Trypanosoma species., 3. Ciliophora: Short, hairlike projections called, cilia, whose synchronous beating propels the, organisms, carry out locomotion. The characteristic example of free-living members of, this group is Paramecium caudatum, and the, parasitic example is Balantidium coli., 4. Sporozoa: Unlike other members of this phylum, sporozoa do not have locomotor organelles in their mature stage; however, immature, forms exhibit some type of movement. All the, members of this group are parasites. The most, significant members belong to the genus Plasmodium, the malarial parasites of animals and, humans., , F U RT H E R RE A D I N G, Refer to the section on eukaryotes in your textbook, paying close attention to the sections on, cellular metabolism and single-celled organisms., In your textbook’s index, search for terms such as, “Protozoa,” “Algae,” and “Plasmodium.”, , 223
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C ASE STUDY, A CASE OF STOMACH CRAMPS, A hiker enters the Emergency Department, complaining of severe stomach cramps and, loose, smelly diarrhea. During the initial physical, examination, the patient mentions that he has been, having the pain and bowel issues for about two, weeks with no lessening in severity. Through further questioning, you are able to fill in the patient’s, medical history and current travel/diet history. The, hiker says that four weeks ago, he went on a backpacking trip into the mountains to obtain fresh, spring water from the source. Unable to locate the, spring source for a stream, he decided to collect, some water from a clean stream and transport it, home. He drank the water daily, and to ensure that, no minerals were removed, he chose not to purify, , 224, , Part 6, , or sterilize the water before drinking. Two weeks, after returning from the trip, he began to experience some abdominal discomfort that progressed, to severe pain and the loose stools., , Questions to Consider:, 1. Even though the stream was clean-looking, and, because of its location in the mountains,, was probably too cold to support most bacterial growth, should it be considered sterile?, 2. To determine the causative agent of the, patient’s intestinal issues, should you examine, the water, the patient’s stool, or both? Explain, your reasoning.
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E XP E R IMENT, , 33, , Free-Living Protozoa, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Describe the protozoa found in pond water., , Principle, There are more than 20,000 known species of, free-living protozoa. This manual does not p, resent, an in-depth study of this large and diverse population. Therefore, in this procedure, you will use, Table 33.1, Figure 33.1, and Figure 33.2 to become, familiar with the general structural characteristics, of representative protozoa, and you will identify, these in a sample of pond water., , FUR T HE R R E A D I N G, Refer to the section in your textbook on protozoa, and the differences between d, ifferent recognized, groups. In your textbook’s index, search under, “Amoebae,” “Euglena,” and “Pseudopodia.”, , Reagent, ❏❏ Methyl cellulose, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microscope, Glass slides, Coverslips, Pasteur pipettes, , Procedure, 1. Obtain a drop of pond water from the bottom, of the culture and place it in the center of a, clean slide., 2. Add a drop of methyl cellulose to the culture, to slow down the movement of the protozoa., 3. Apply a coverslip in the following manner to, prevent formation of air bubbles:, a. Place one edge of the coverslip against the, outer edge of the drop of culture., , C L I N I C A L A P P L I C AT I O N, Wet Mounts for Diagnosis, Wet mount slides, often utilizing stains, are routinely, used in the examination of stool samples for infectious protozoans, such as Entamoeba histolytica., This organism causes amoebic dysentery and, has been known to lead to severe liver damage., Although most infections are asymptomatic, carriers, can still spread the disease. Diagnosis may require, examination of several slide preparations., , b. After the drop of culture spreads along the, inner aspect of the edge of the coverslip,, gently lower the coverslip onto the slide., , AT T H E B E N C H, , Materials, Cultures, ❏❏ Stagnant pond water and prepared slides of, amoebas, paramecia, euglenas, and stentors, , 4. Examine your slide preparation under scanning, low-power, and high-power objectives, with diminished light, and observe for the different protozoa present. Record your results in, the Lab Report., 225
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TABLE 33.1, , Structural Characteristics of Free-Living Protozoa, , Sarcodina, Pseudopod, Ectoplasm, Contractile vacuole, Endoplasm, Food vacuole, Nucleus, , Amoeba, , Mastigophora, , 1. Flagella: One to several long whiplike, structures that function for locomotion, 2. Pellicle: Elastic layer outside of cell, membrane, 3. Mouth: Present but indistinct, 4. Chloroplast: Organelles containing, chlorophyll; present in photosynthetic, forms only, 5. Eye spot: Light-sensitive pigmented spot, 6. Nucleus: One present, , Flagellum, Mouth, Eye spot, Chloroplast, Pellicle, Nucleus, , Euglena, , Cercomonas, , Heteronema, , Ciliophora, , Cilia, Pellicle, Food vacuole, Oral groove, Micronucleus, Macronucleus, Contractile, vacuole, , Paramecium, , 1. Pseudopods: Protoplasmic projections that, function for locomotion, 2. Ectoplasm: Outer layer of cytoplasm; clear, in appearance, 3. Endoplasm: Inner cytoplasmic region;, granular in appearance, 4. Nucleus: One present, 5. Food vacuoles: Contain engulfed food, undergoing digestion, 6. Contractile vacuole: Large, clear circular, structure that regulates internal water, pressure, , Stentor, , Vorticella, , 1. Cilia: Numerous, short, hairlike structures that, function for locomotion, 2. Pellicle: Outermost flexible layer, 3. Contractile vacuole with radiating canals;, regulates osmotic pressure, 4. Oral groove: Indentation that leads to the, mouth and gullet, 5. Food vacuoles: Sites of digestion of, ingested food, 6. Macronucleus: A large nucleus that, functions to control the cell's activities; one, to several may be present, 7. Micronucleus: A small nucleus that, functions in conjugation, a mode of sexual, reproduction, , 1., , 1., , (a) Euglena viridis, , Figure 33.1 Amoeba, 226, , Experiment 33, , (b) Paramecium caudatum, , Figure 33.2 Euglena and Paramecium
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E XP E R IME NT, , 33, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. In the space provided, draw a representative sketch of several of the, observed protozoa in stagnant pond water, indicate the magnifications used,, and label their structural components. Identify each organism according to, its class based on its mode of locomotion and its genus., , Magnification:, Organelles of locomotion:, Class:, Genus:, , Magnification:, Organelles of locomotion:, Class:, Genus:, , Experiment 33: Lab Report, , 227
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2. Draw representative sketches, indicate magnification, and label the structural, components. Identify each organism according to its class based on locomotion and genus., , Amoeba, , Paramecium, , Euglena, , Stentor, , Magnification:, Organelles of locomotion:, Class:, Genus:, , Magnification:, Organelles of locomotion:, Class:, Genus:, , 228, , Experiment 33: Lab Report
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Review Questions, 1. What are the distinguishing characteristics of the free-living members of, Sarcodina, Mastigophora, and Ciliophora?, , 2. Identify and give the function of the following:, a. Pseudopods, , b. Contractile vacuole, , c. Eye spot, , d. Micronucleus, , e. Pellicle, , f. Oral groove, , Experiment 33: Lab Report, , 229
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3., , 230, , People with AIDS are vulnerable to toxoplasmosis caused by the, protozoan Toxoplasma gondii, resulting in infection of lungs, liver,, heart, and brain, and often leading to death. About 25% of the world’s, population is infected, usually without developing symptoms. Why then are, people with AIDS so susceptible to this disease?, , Experiment 33: Lab Report
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E XP E R IMENT, , Parasitic Protozoa, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Describe parasitic protozoan forms., , Principle, Unlike the life cycles of the free-living forms, the, life cycles of parasitic protozoa vary greatly in, complexity. Knowing the various developmental, stages in these life cycles is essential in the diagnosis, clinical management, and use of chemotherapy, to treat parasitic infections., The following parasites have the simplest or, most direct life cycles not requiring an intermediate host:, 1. Entamoeba histolytica: a pseudopodian parasite of the class Sarcodina that causes amebic, dysentery. Infective, resistant cysts are released, from the lumen of the intestine through the, feces and are deposited in water, in soil, or on, vegetation. Upon ingestion, the mature quadrinucleated cyst wall disintegrates and the nuclei, divide, producing eight active trophozoites, (metabolically active cells) that move to the, colon, where they establish infection., 2. Balantidium coli: The ciliated parasitic protozoan exhibits a life cycle similar to that of, Entamoeba histolytica except that no multiplication occurs within the cyst. This organism, resides primarily in the lumen and submucosa, of the large intestine. It causes intestinal, ulceration and alternating constipation and, diarrhea., 3. Giardia intestinalis: The intestinal mastigophoric flagellate exhibits a life cycle comparable to those of the above parasites. This, organism is responsible for abdominal discomfort and severe diarrhea. Diagnosis is made, , 34, , by finding cysts in the formed stool and both, cysts and trophozoites in the diarrhetic stool., The mastigophoric hemoflagellate responsible for various forms of African sleeping, sickness has a more complex life cycle. The, Trypanosoma must have two hosts to complete its cyclic development: a vertebrate, and an invertebrate, blood-sucking insect, host. Humans are the definitive hosts harboring the sexually mature forms; the tsetse, fly (Glossina) and the reduviid bug are the, invertebrate hosts in which the developmental, forms occur., Table 34.1 illustrates the morphological characteristics of prototypic members of the parasitic, protozoa except the Sporozoa., Protozoa demonstrating the greatest degree of, cyclic complexity are found in the class Sporozoa., They are composed of exclusively obligate parasitic, forms, such as members of the genus Plasmodium,, and are responsible for malaria in both humans, and animals. The life cycle requires two hosts, a, human being and the female Anopheles mosquito., It is significant to note that in this life cycle, the, mosquito—and not the human—is the definitive, host harboring the sexually mature parasite., Malaria is initiated when a person is bitten by, an infected mosquito, during which time infective, sexually mature sporozoites are injected along with, the insect’s saliva. These parasites pass rapidly, from the blood into the liver, where they infect the, parenchymal cells. This is the pre-erythrocytic, stage. The parasites develop asexually within the, liver cells by a process called schizogony, producing merozoites. This cycle may be repeated, or the merozoites that are released from the ruptured liver cells may now infect red blood cells, and initiate the erythrocytic stage. During this, asexual development, the parasite undergoes a, series of morphological changes that are of diagnostic value. These forms are designated as signet, rings, trophozoites, schizonts, segmenters,, merozoites, and gametocytes. The merozoites, , 231
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TABLE 34.1, , Structural Characteristics of Free-Living Protozoa, , CLASS, ORGANISM, AND, INFECTION, SARCODINA, Entamoeba histolytica, Infection: Amebic, dysentery, Peripheral chromatin, Central karyosome, Uniform cytoplasm, Red blood cell, , Nuclei, , Early, , Chromatoid, body, , Late, , MASTIGOPHORA, Trypanosoma gambiense, Infection: African, sleeping sickness, Flagellum, Undulating, membrane, , STRUCTURAL, CHARACTERISTICS, , LOCOMOTOR, ORGANELLES, , SITE OF, INFECTION, , Trophozoite:, Shape: Variable, Nucleus: Discrete nuclear, membrane with central, karyosome and peripheral, chromatin granules, Cytoplasm: Clear, red blood, cells may be present, Cyst:, Shape: Round to oval with, thick wall, Nuclei: 1–4 present; mature, cyst is quadrinucleated, Chromatoid bodies: Sausageshaped with rounded ends,, present in young cysts only, , Pseudopods, , Large intestine by, ingestion of mature, cysts, , Trophozoite:, Shape: Crescent, Nucleus: Large, central, and, polymorphic, Cytoplasm: Granular, Cyst: None, , Single, flagellum, along, undulating, membrane, , None, , ISOLATION OF, PARASITIC FORM, , Diarrhetic stool, , Formed stool, , Peripheral bloodstream by means, of tsetse fly vector, , Peripheral, blood, , Nucleus, Volutin granules, Kinetoplast, , are capable of reinfecting other blood cells or liver, cells. Ingestion of the microgametocytes (♂) and, macrogametocytes (♀) by another mosquito during a blood meal initiates the sexual cycle called, sporogamy. Male and female gametes give rise to a, zygote in the insect’s gut. The zygote is then transformed into an ookinete that burrows through the, gut wall to form an oocyst in which the sexually, mature sporozoites develop, thereby completing, the life cycle., In this experiment, you will study the parasitic, protozoa using prepared slides and the diagnostic, , 232, , Experiment 34, , characteristics shown in Figure 34.1 on page 232, and Table 34.1. The purpose of the experiment, is to help you understand life cycles of parasitic, protozoa., , F U RT H E R RE A D I N G, Refer to the section on protozoa in your textbook, for further information on the species that are, parasites of humans. In your textbook’s index,, search under “Plasmodium,” “Parasites,” and, “Trichomonas.”
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TABLE 34.1, , Characteristics of Representative Parasitic Protozoa (continued), , CLASS, ORGANISM, AND, INFECTION, MASTIGOPHORA, Giardia intestinalis, Infection: Dysentery, Nucleus, Karyosome, Median bodies, Axonemes, , Nuclei, Median bodies, Retracted, protoplasm, Axonemes, CILIOPHORA, Balantidium coli, Infection: Dysentery, Cytostome, Cilia, Micronucleus, Macronucleus, Cyst wall, Macronucleus, , STRUCTURAL, CHARACTERISTICS, , LOCOMOTOR, ORGANELLES, , SITE OF, INFECTION, , ISOLATION OF, PARASITIC FORM, , Trophozoite:, Shape: Pear-shaped with, concave sucking disc, Nuclei: 2 bilaterally located, with central karyosome and, no peripheral chromatin, Cytoplasm: Uniform and, clear, Cyst:, Shape: Oval to ellipsoidal, Nuclei: 2–4 present and, protoplasm retracted from, cyst wall, Axostyle, Parabasal body, , 4 pairs of, flagella, , Small intestine, through ingestion, of cysts, , Diarrhetic stool, , Trophozoite:, Shape: Oval, Nuclei: Kidney-shaped, macronucleus and a, micronucleus, Cytoplasm: Vacuolated, Cyst:, Shape: Round and thickwalled, Nuclei: 1 macronucleus and, a micronucleus that is not, visible, , Cilia, , C L I N I C A L A P P L I C AT I O N, Understanding Parasitic Protozoa, Parasitic protozoa can exist extracellularly or, intracellularly, and possess diverse morphologies., They rapidly reproduce, asexually or sexually, with, short generation times. They are highly organ-,, tissue-, or cell-specific organisms. Examples are, Plasmodium species, which colonize red blood, cells (malaria); Trichomonas, which colonize the, urinary tract (vaginal infections); and Entamoeba,, which colonizes the large intestine (severe, diarrhea)., , 4 pairs of, flagella, within cyst, , Formed stool, , Large intestine by, the ingestion of, cysts, , Cilia within, cyst, , Diarrhetic stool, , Formed stool, , AT T HE BE NCH, , Materials, Prepared Slides, ❏❏ E. histolytica trophozoite and cyst, ❏❏ Giardia intestinalis trophozoite and cyst, (formerly G. lamblia), ❏❏ Balantidium coli trophozoite and cyst, ❏❏ Trypanosoma gambiense, ❏❏ Plasmodium vivax in human blood smears, , Experiment 34, , 233
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Host: Mosquito, Sexual Reproduction, , Host: Human, Asexual Reproduction, Blood Meal, , 1. Sporozoites infect, live cells, , 2. Mature into, Oocysts, , 2. Mature into, Schizonts, , 3. Oocysts release, Sporozoites, , 3. Merozoites, released, , 1. Micro-and, Macrogametocytes, are ingested by Anopheles, Anopheles mosquito, , 1. Merozoites infect, RBC, , Hind Gut (lumen), , 2. Ring stage, Trophozoites, mature, , 2. Gametocytes fuse, from zygotes, , 3. Merozoites released, Ookinetes, , 3. Zygotes mature into, Ookinetes, , 4. Some Trophozoites, mature into, Gametocytes, &, , Red Blood Cell, , Mid Gut, , 1. Ookinetes invade, the midgut wall, , Live, , Sporozites, , Gametocytes, , Blood Meal, , Figure 34.1 Life cycle of Plasmodium vivax, , Equipment, Microscope, immersion oil, and lens paper, , Procedure, 1. Examine all available slides under the oilimmersion objective. Use Table 34.1, Figure, 34.1, and the photographs in Figure 34.2, , 234, , Experiment 34, , through Figure 34.6 to identify the distinguishing microscopic characteristics of each parasite studied., 2. Record your observations in the Lab Report.
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E XP E R IMENT, , 34, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Draw representative sketches of the parasitic organisms that you studied, and, label the distinguishing structural characteristics you were able to observe., , E. histolytica, , G. intestinalis (G. lamblia), , T. gambiense, , B. coli, , P. vivax: Erythrocytic stages, , Experiment 34: Lab Report, , 237
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Review Questions, 1. Describe the developmental stages of the malarial parasite during sporogamy and schizogony., , 2. What role does the invertebrate host play in the life cycle of the trypanosomes? Explain., , 3. Distinguish between the pre-erythrocytic and erythrocytic stages in the life, cycle of the malarial parasite., , 4., , In malarial infections, the sexually mature parasite is found in, which host? Is this true for all other protozoan parasitic, infections? Explain., , 5., , On returning from a trip overseas, an individual with persistent, diarrhea is diagnosed as having an E. histolytica infection. Fecal, examination reveals the presence of blood in the stool, suggesting damage, to the intestinal mucosa. Explain why and how the mucosa was compromised by this parasite., , 238, , Experiment 34: Lab Report
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PART 7, , The Fungi, LEARNING OBJECTIVES, When you have completed the experiments in this section, you should be able to, 1. Describe macroscopic and microscopic structures of yeast and molds., 2. Perform basic mycological culturing and staining procedures., 3. Identify selected common fungal organisms., , Introduction, The branch of microbiology that deals with, the study of fungi (yeasts and molds) is called, mycology. True fungi are separated into the, following four groups on the basis of their sexual, modes of reproduction:, 1. Zygomycetes: Bread and terrestrial molds., Reproductive spores are external and uncovered. Sexual spores are zygospores, and asexual spores are sporangiospores., 2. Ascomycetes: Yeasts and molds. Sexual, spores, called ascospores, are produced in a, saclike structure called an ascus. Conidia are, asexual spores produced on a conidiophore., 3. Basidiomycetes: Fleshy fungi, toadstools,, mushrooms, puffballs, and bracket fungi., Reproductive spores, basidiospores, are separate from specialized stalks called basidia., 4. Deuteromycetes: Also called Fungi, Imperfecti because no sexual reproductive, phase has been observed., Table P7.1 shows the major characteristics, of these four groups of fungi. Nutritionally, fungi, are heterotrophic, eukaryotic microorganisms, that are enzymatically capable of metabolizing a, wide variety of organic substrates. Fungi can have, , beneficial or detrimental effects on humans. Fungi, that inhabit the soil play a vital role in decomposing, dead plant and animal tissues, thereby maintaining, a fertile soil environment. The fermentative fungi, are industrially important in producing beer and, wine, bakery products, cheeses, industrial enzymes,, and antibiotics. The detrimental activities of some, fungi include spoiling foods by rots, mildews, and, rusts found on fruit, vegetables, and grains. Some, species are capable of producing toxins (for example, aflatoxin) and hallucinogens. A few fungal, species are medically significant because of their, capacities to produce diseases in humans. Many of, the pathogenic fungi are deuteromycetes and can, be divided into two groups based on site of infection. The superficial mycoses cause skin, hair,, and nail infections (for example, ringworm infections). The systemic mycoses cause subcutaneous, and deeper tissue infections such as those of the, lungs, genital areas, and nervous system., , F U RT H E R RE A D I N G, Refer to the sections on eukaryotes in your textbook, paying close attention to the sections on, cellular metabolism and fungal organisms. In your, textbook’s index, search for terms such as “Mycelium,” “Septate,” and “Mycosis.”, , 239
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TABLE P7.1, , Major Characteristics of the Four Groups of Fungi, GROUP, ZYGOMYCETES, , CHARACTERISTICS, , ASCOMYCETES, , BASIDIOMYCETES, , DEUTEROMYCETES, , Mycelium, , Nonseptate, , Septate, , Septate, , Septate, , Asexual spores, , Found in sporangium;, sporangiospores, (nonmotile), , Formed on tip of, conidiophore; conidia, (nonmotile), , Same as the, ascomycetes, , Same as the, ascomycetes, , Sexual spores, , Zygospores (motile),, found in terrestrial, forms; oospores, found, in aquatic forms, , Ascospores, contained, in a saclike structure, called the ascus, , Basidiospores, carried, on the outer surface, of a club-shaped cell, called the basidium, , Fungi Imperfecti—no, sexual reproductive, phase observed; some, members of the, ascomycetes and, basidiomycetes are, Fungi Imperfecti, , Common species, , Bread molds; mildews;, potato blight; Rhizopus, species, , Cup fungi; ergot; Dutch, elm; yeast species, , Smuts; rusts; puffballs;, toadstools; mushrooms, , Aspergillus; Candida;, Trichophyton; Cryptococcus; Blastomyces;, Histoplasma; Microsporum; Sporothrix, , C ASE STUDY, ANOTHER CASE OF STOMACH CRAMPS, A student goes to the campus clinic complaining, of abdominal cramps, difficulty swallowing, and, the beginnings of a rash on her neck and face., You quickly recognize the symptoms of an allergic reaction to an ingested allergen. The student, relates that she has a known fungi allergy and, makes a concerted effort to refrain from coming, into contact with molds or fungi. Further questioning reveals that 30 minutes before coming to the, clinic, the student had been eating at one of the, campus cafeterias but was diligent to not come, into contact with any mushrooms. For breakfast,, she had chosen to eat only toast and milk to limit, any exposure. She chose to toast her own bread, , 240, , Part 7, , from a sealed, commercially bought loaf to ensure, that no mushrooms came into contact with the, bread. She mentions that the loaf of bread did, have a few slices with a couple of blue spots on, them, but she made sure not to eat those slices., , Questions to Consider:, 1. What do you know about how fungi and molds, spread via asexual reproduction?, 2. How would this knowledge impact your diagnosis and treatment, considering the student's, choice to use bread from a sealed bag with visible reproductive hyphae showing?
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E XP E R IMENT, , 35, , Cultivation and Morphology, of Molds, , Molds are the major fungal organisms that can, be seen by the naked eye. We have all seen them, growing on foods such as bread or citrus fruit as, a cottony, fuzzy, black, green, or orange growth,, or as a mushroom with a visible cap attached to a, stalk, depending on the mold. Examination with a, simple hand lens shows that these organisms are, composed of an intertwining branching mat called, a mycelium. The filaments that make up this mycelial mat are called hyphae. Most of the mat grows, on or in the surface of the nutrient medium so that, it can extract nutrients; the mat is therefore called, vegetative mycelium. Some of the mycelium mat, rises upward from the mat and is referred to as, aerial mycelium. Specialized hyphae are produced, from the aerial mycelium and give rise to spores, that are the reproductive elements of the mold., Figure 35.1, Figure 35.2, Figure 35.5, and Figure 35.6, show the reproductive structures of some fungi., The cultivation, growth, and observation of, molds require techniques that differ from those used, for bacteria. Mold cultivation requires the use of a, selective medium such as Sabouraud agar or potato, dextrose agar. These media favor mold growth, because their low acidity (pH 4.5 to 5.6) discourages, the growth of bacteria, which favor a neutral (pH, 7.0) environment. The temperature requirements of, molds are also different from those of bacteria, in, that molds grow best at room temperature (25°C)., In addition, molds grow at a much slower rate than, , bacteria do, requiring several days to weeks before, visible colonies appear on a solid agar surface., Figure 35.3 and Figure 35.4 show colony growth., , PART A, , Slide Culture Technique, , LEARNING OBJECTIVES, When you have completed this experiment,, you should be able to, 1. Perform mold cultivation using glass, slides., 2. Visualize and identify the structural, components of molds., , Principle, Because the structural components of molds are, very delicate, even simple handling with an inoculating loop may result in mechanical disruption of their, components. The following slide culture technique, is used to avoid such disruption. A deep concave, slide containing a suitable nutrient medium with an, acidic pH, such as Sabouraud agar, is covered by, a removable coverslip. Mold spores are deposited, in the surface of the agar and incubated in a moist, , Spores, , Sporangiospores, , Conidia, , Oidia, , Chlamydospore, , Ascospores, , Basidiospores, , Vegetative, hyphae, , Sporangiophore, , Conidiophore, , Oidiophore, , Spores forming, in hyphae, , Ascus, , Basidium, , Figure 35.1 Spore and sporangia types, , 241
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Conidia, Sporangiophore, , Conidiophore, , Figure 35.2 Conidiophore and conidia of mold, Aspergillus niger, , Figure 35.5 Mucor mucedo, , Sporangiospore, Sporangiophore, , Figure 35.6 Rhizopus stolonifer, , Figure 35.3 Colony of Penicillium chrysogenum, , chamber at room temperature. Direct microscopic, observation is then possible without fear of disruption or damage to anatomical components. Molds, can be identified as to spore type and shape, type, of sporangia, and type of mycelium, as shown in, Figure 35.1 and Table 35.1 on pages 245–247., , F U RT H E R RE A D I N G, Refer to the section on fungi in your textbook for, further information on the species that are environmentally important. In your textbook’s index, search, under “Sporangium,” “Mycology,” and “Condidia.”, , C L I N I C A L A P P L I C AT I O N, , Figure 35.4 Colony of A. niger on a Sabouraud, agar plate, , 242, , Experiment 35, , Cultivation of Fungi on Glass Slides, Since sporangia may be damaged during transfer to, a glass slide, the slide culture technique prevents the, disturbance and damage of the sporangia and other, spore structures required for fungi identification. Intact, samples can be used to distinguish a fungi like A., niger, which causes the most common fungal infection, of the ear, from Aspergillus flavus, a fungal pathogen, that may result in disseminating infection of the lungs.
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AT T H E B E N C H, , Materials, Cultures, , 6. With a sterile Pasteur pipette, add one or two, drops of cooled Sabouraud agar to the concavity of each slide., 7. Place a coverslip over the concave portion of, each slide so that it is completely sealed., 8. With forceps, stand each slide upright inside, its respective Petri dish until the agar solidifies, as illustrated below:, , 7- to 10-day Sabouraud agar cultures of, ❏❏ P. chrysogenum, ❏❏ A. niger, (formerly called, ❏❏ M. mucedo, Penicillium, ❏❏ R. stolonifer, notatum), , 1., , Media, , 9. When the agar is fully hardened, slide the, coverslips downward with forceps, and with, a sterile needle inoculate each prepared slide, with the spores from the test cultures., 10. Push the coverslips to their original positions,, thereby sealing off the slide., 11. With a Pasteur pipette, moisten the filter paper, with sterile water to provide a moist atmosphere. Remoisten filter paper when necessary, during the incubation period., 12. Place the slide on the U-shaped bent rod,, replace the Petri dish cover, and label with the, names of the organism and your initials., 13. Incubate the preparations for 7 days at 25°C., , ❏❏ One Sabouraud agar deep tube (per group), , Equipment, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Waterbath, ❏❏ Four concave glass, slides, ❏❏ Four coverslips, ❏❏ Petroleum jelly, ❏❏ Sterile Pasteur, pipettes, ❏❏ Toothpicks, ❏❏ Four sterile Petri, dishes, , ❏❏ Filter paper, ❏❏ Forceps, ❏❏ Inoculating loop and, needle, ❏❏ Four sterile, U-shaped bent glass, rods, ❏❏ Thermometer, ❏❏ Dissecting, microscope, ❏❏ Beaker with 95%, ethyl alcohol, , Procedure Lab One, 1. Melt the deep tube of Sabouraud agar in a boiling water bath and cool to 45°C., 2. Place a piece of filter paper in the bottom of, each Petri dish, lay a sterile bent glass rod in, each dish, and replace the covers., 3. Using forceps, dip the concave slides and coverslips in a beaker of 95% ethyl alcohol, pass, through Bunsen burner flame, remove from, flame, and hold until all the alcohol has burned, off the slides and coverslips., 4. Cool the slides and coverslips. Place a slide, concave side up, with a coverslip to one side of the, concavity, on the glass rod inside each Petri dish., 5. With a toothpick, add petroleum jelly to three, sides surrounding the concavity of each slide., The fourth side will serve as a vent for air., 1., , Space above, top of agar, Agar, Coverslip, Slide, , Procedure Lab Two, 1. Examine each mycological slide preparation, under the low and high power of a dissecting, microscope. Identify the mycelial mat, vegetative and reproductive hyphae, and spores. Use, Table 35.1 on pages 245–247 to aid with your, identification of mold structures., 2. Record your observations in the Lab Report., , Mold Cultivation on, Solid Surfaces, PART B, , LEARNING OBJECTIVES, When you have completed this experiment,, you should be able to, 1. Describe the technique of mold cultivation, on agar plates., 2. Observe and identify colonial characteristics, such as growth rate, texture, pigmentation on the surface and reverse side, and, folds or ridges on the surface., Experiment 35, , 243
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Principle, Cultivating molds on solid surfaces allows you, to observe the variations in gross colonial morphology among different genera of molds. These, variations in colonial appearance play a major, role in the identification of the filamentous fungi., Most microbiologists are familiar with the gross, appearance of multicellular fungi, but even to the, untrained, the macroscopic differences in colonial growths are obvious and recognizable. For, example, most people have seen rotting citrus, fruits (lemons and oranges) produce a blue-green, velvety growth characteristic of Penicillium species. It is also common for stale cheese to show a, grayish-white furry growth of Mucor species, and, the black, stalklike appearance of Rhizopus molds, growing on bread is familiar to many., In this part of the experiment, you will be able, to visualize the gross appearance of the colonial, growth of four different molds., , C L I N I C A L A P P L I C AT I O N, Isolation of Fungi on Solid Media, Before a fungal species may be identified or studied, it must first be isolated. Similar to using an agar, plate for isolating a distinct bacterial species, agar, plating may be used as a growth medium for the, isolation of fungi spores. Once spores have been, isolated from individual sporangia, subculturing on, solid agar or slides will allow for characterization, and genetic studies of the fungus., , Media, Per designated student group, ❏❏ Three Sabouraud agar plates, ❏❏ One potato dextrose agar plate, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Four test tubes containing 2 ml of sterile saline, Dissecting microscope, Inoculating loop, , Procedure Lab One, 1. Label the three Sabouraud agar plates as, A. niger, P. chrysogenum, and M. mucedo, and, label the fourth plate containing potato dextrose agar as R. stolonifer., 2. Prepare a saline suspension of each mold culture., Label each of the four tubes of saline with the, name of the organism. Using a sterile inoculating, loop, scrape two loopfuls of mold culture into the, corresponding tube of 2 ml of sterile saline, and, mix well by tapping the tube with your finger., 3. Using aseptic technique, inoculate each of, the plates by placing a single loopful of mold, suspension in the center of its respective agar, plate. Note: Do not spread the inoculum and do, not shake or jostle the plates., 4. Incubate all plates at room temperature, 25°C,, for 7 to 10 days. Note: Do not invert the plates., , Procedure Lab Two, AT THE B E N C H, , Materials, Cultures, 7- to 10-day Sabouraud agar cultures of, ❏❏ A. niger, ❏❏ P. chrysogenum (formerly called P. notatum), ❏❏ M. mucedo, ❏❏ R. stolonifer, , 244, , Experiment 35, , 1. Examine each mold plate under the low and, high power of a dissecting microscope. Refer to, Table 37.1 for your identification of mold structures. Note: Do not remove Petri dish covers., 2. Record your observations in the Lab Report.
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TABLE 35.1, , Identification of Fungi (continued), , DIAGRAM, , COLONIAL MORPHOLOGY, , Conidia, , MICROSCOPIC APPEARANCE, , Rapidly growing compact and, moist colonies becoming, cottony with aerial hyphae, that are gray or rose-colored, , Single-celled conical or, elliptical spores (conidia) held, together in clusters at the tips, of the conidiophores by a, mucoid substance; erect,, unbranched conidiophores, arise from a septate mycelium, , Colonies are pink, moist, with, unbroken, even edges, , Cells are oval, colorless, and, reproduce by budding, , Colonies are small, round,, moist, and colorless, with, unbroken, even edges, , Yeastlike fungus produces, pseudomycelium, , Conidiophore, Mycelium, Cephalosporium:, Antibiotic production, Yeast, , Bud, , Torula:, Cheese and food contaminant, Chlamydoconidium, Pseudomycelium, Blastospores, , Candida:, Human pathogen, , Experiment 35, , 247
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EXPERIMENT, , 35, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Part A: Slide Culture Technique, Draw a representative microscopic field under low-power and high-power magnification and label the structural components of each test organism., , Low Power, , High Power, , Low Power, , Penicillium chrysogenum (P. notatum), , Low Power, , High Power, Rhizopus stolonifer, , High Power, Aspergillus niger, , Low Power, , High Power, Mucor mucedo, , Experiment 35: Lab Report, , 249
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Part B: Mold Cultivation on Solid Surfaces, Draw sketches of the mold colonies under low power, indicating the extent of, growth (diameter in mm), pigmentation, and the presence or absence of aerial, hyphae. Refer to Table 37.1 to aid with your identification of mold structures., , Penicillium chrysogenum (P. notatum), , Aspergillus niger, , Rhizopus stolonifer, , Mucor mucedo, , Colony diameter (mm):, Pigmentation:, Aerial hyphae (+ or -):, , Colony diameter (mm):, Pigmentation:, Aerial hyphae (+ or -):, , 250, , Experiment 35: Lab Report
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Review Questions, 1. Cite some beneficial and harmful aspects of molds., , 2. What is the advantage of using Sabouraud agar?, , 3. In the slide culture technique, what is the purpose of the following?, a. Moistened filter paper in the Petri dish, , b. A U-shaped glass rod in the Petri dish, , Experiment 35: Lab Report, , 251
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4., , What is the advantage of the slide culture technique over that of a simple loop inoculation, onto an agar plate (as in Part B)?, , 5. Why would it be advantageous to observe mold colonies on an agar plate?, , 6., , 252, , Since dimorphism is a property of fungi, how do you account for the fact that molds grow, preferentially in vitro rather than in vivo?, , Experiment 35: Lab Report
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E XP E R IMENT, , Isolation of a Soil Fungal Species, , 36, , LEARNING OBJECTIVES, , C L I N I C A L A P P L I C AT I O N, , Once you have completed this experiment,, you should be able to, , In an attempt to find new sources of the next class, of antibiotics to treat bacterial infections, scientists screen environmental samples for potential, antibacterial activities. While screening different, solvent extracts may identify an extract sample, with antibacterial activity, it does not identify the, new compound, or allow for characterization of the, microbes or harvesting of the compound. Before, this may occur, all of the potential species present, in that sample must be isolated and to be identified, as the source for the compound., , 1. Isolate or enrich a single fungal species, from an environmental sample., , Principle, Before a fungal specimen may be characterized or, described, it must be isolated from other bacteria, or fungal species that are present. C, omplicating, this is the fact that estimates have placed the, number of species per gram of soil to be between, one thousand and tens of thousands depending, on the source of the sample. With each species, represented by an unknown number of organisms,, trying to isolate an individual colonial growth of, fungal mycelium could be a daunting task. Microbiologists use numerous methods to isolate fungal, growths for later study and cultivation. A direct, transfer, also referred to as direct plating,, involves taking a small sample from a visible, growth that is relatively ubiquitous and transferring it directly to an agar plate for growth in a lab., This will allow for growth of all organisms present, but may not allow for individual species to be, directly isolated. For samples that may be more, complex in the potential number of species present, dilution plating may allow isolated mycelium to grow and be transferred for pure cultures., Similar to the bacterial dilution plating method in, Experiment 18, a prepared sample solution of soil, and water will be serially diluted to yield fewer, and fewer colonies until individual mycelium, growths result., , FUR T HE R R E AD I N G, Refer to the section on fungi in your textbook for, further information on the species that have been, shown to be of environmental importance. In your, textbook’s index, search under “Soil,” “Mycelium,”, and “Nitrogen.”, , AT T HE BE NCH, , Materials, Cultures, ❏❏ 10 grams of soil per group, , Media, Per lab group, ❏❏ 6 Potato Dextrose Agar plates, PDA (antibiotics optional), ❏❏ 2 Potato Dextrose Agar slants, ❏❏ Three 50 mL tube, ❏❏ 150 mL sterile water, ❏❏ Two microcentrifuge tubes, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Dissecting scope, Loop, Disposable Pasteur pipettes (sterile), L-spreaders (sterile), Vortex, Balance, Parafilm, , 253
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Procedure Lab One, 1. Transfer 1g of soil to one of the 50 mL tubes, and add 45 mL of sterile water., 2. Vortex the sample to evenly suspend soil particles in water for about 3 minutes., 3. Allow the soil suspension to sit for 15 minutes, so the debris and large soil particles settle out., 4. While the solution is settling, label the PDA, plates as #1 through #4 and add 50 mL sterile, water to the second tube., 5. Using a sterile Pasteur pipette, transfer 1 mL, of soil suspension to plate #1 and 100 mL to, plate #2., 6. Transfer 1 mL soil suspension to the second, tube with 50 mL and vortex for 1 minute to, mix., 7. From the second tube, transfer 1 mL of suspension to plate #3 and 100 mL to plate #4., 8. Using a sterile L-spreader, start with plate #4, and spread the soil suspension evenly across, the plate surface, then continue to plates #3,, #2 and finally #1 in that order., • NOTE: This dilution series is not for calculation of the number of organisms but for, isolation of distinct colonies., 9. Seal the plates with Parafilm and incubate at, room temperature for 7 to 10 days., , 2. Using a sterile loop, transfer a sample from a, singular mycelium growth to a tube with 20 mL, of sterile water, and vortex to release fungal, spores., 3. Transfer 1 mL to a new PDA plate labeled as, #5 and 100 mL to plate #6., 4. Seal the plates with Parafilm and incubate at, room temperature for 7 to 10 days., , Procedure Lab Three, 1. Determine which plate has the lowest growth, and potentially a mycelium that is segregated, from other fungal growths., 2. Using a sterile loop, transfer a sample from, a singular mycelium growth to a microcentrifuge tube with 1 mL of sterile water, and, vortex to release fungal spores. Repeat for the, second microcentrifuge tube., 3. Transfer 100 mL of spore suspension from the, microcentrifuge tube to the PDA agar slant., 4. Seal the plates with Parafilm and incubate at, room temperature for 7 to 10 days., 5. Compare the growth on the agar slants with, the tubes in Figure 36.2 to determine relative, isolation of a single species of fungi., 6. Use the dissecting scope to locate any, identifying microscopic characteristics or, structures., , Procedure Lab Two, 1. Determine which plate has the lowest growth, and potentially a mycelium that is segregated, from other fungal growths. See Figure 36.1 for, an example of a low-growth fungal plate., , Figure 36.2 Potato Dextrose Slant with, potentially isolated fungal growth due to dilution, plating, , Figure 36.1 Potato Dextrose Plate with minimal, fungal growth due to dilution plating, , 254, , Experiment 36
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E XP E R IMENT, , 36, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Draw representative sketches of the Potato Dextrose Agar plate with minimal, fungal growth and agar slant that you studied, and label any structures you are, able to observe under magnification., , PDA Plates, , PDA Slants, , Colors:, Structures:, , Review Questions, 1. Why is it important to isolate an organism before trying to study its cellular, biology?, , 2. Were you able to isolate a single fungal species? How would you confirm this?, , Experiment 36: Lab Report, , 255
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Yeast Morphology, Cultural, Characteristics, and Reproduction, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the morphology of different genera of yeast., 2. Explain the growth and fermentative, properties of yeast cells., 3. Describe the sexual and asexual modes of, reproduction in yeast cells., , Principle, Yeasts are nonfilamentous unicellular fungi. Yeast, cultures resemble bacteria when grown on the surface of artificial laboratory media; however, they, are 5 to 10 times larger than bacteria. Figure 37.1, illustrates yeast colonies. Microscopically, yeast, cells may be ellipsoidal, spherical, or in some, cases cylindrical (Figure 37.2). Unlike molds, yeast, do not have aerial hyphae or supporting sporangia., Yeast reproduce asexually by budding or by, fission. In budding, an outgrowth from the parent, cell (a bud) pinches off, producing a daughter cell, (Figure 37.3a and Figure 37.4). Fission occurs in, certain species of yeast, such as those in the genus, Schizosaccharomyces. During fission, the parent, cell elongates, its nucleus divides, and it splits, evenly into two daughter cells., , E XP E R IMENT, , 37, , Some yeast may also undergo sexual reproduction when two sexual spores conjugate, giving, rise to a zygote, or diploid cell. The nucleus of, this cell divides by meiosis, producing four new, haploid nuclei (sexual spores), called ascospores,, contained within a structure called the ascus, (Figure 37.3b). When the ascus ruptures, the ascospores are released and conjugate, starting the, cycle again., Yeasts are important for many reasons. Saccharomyces cerevisiae is referred to as baker’s, yeast and is used as the leavening agent in dough., Two major strains of yeast, Saccharomyces carlsbergensis and S. cerevisiae, are used for brewing., The wine industry relies on wild yeast (present, on the grape) for the fermentation of grape juice,, which is supplemented with Saccharomyces, ellipsoideus to begin the fermentation. Also, the, high vitamin content of yeasts makes them particularly valuable as food supplements. As useful, as some yeasts are, there are a few species that, can create problems in the food industry or that, are harmful to humans. Undesired yeast must be, excluded from the manufacture of fruit juices,, such as grape juice or apple cider, to prevent the, fermentation of fruit sugars to alcohol. The contamination of soft cheese by some forms of yeast, will destroy the product. Finally, some yeast, such as Candida albicans are pathogenic and, responsible for urinary tract and vaginal infections, known as moniliasis, and infections of the, mouth called thrush., , Figure 37.1 Colonies of yeast cells, 257
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Buds, , Figure 37.2 Yeast cells S. octosporus, , Figure 37.4 Asexual yeast reproduction by, budding S. cerevisiae, , Bud, , C L I N I C A L A P P L I C AT I O N, Parent cell, , (a) Asexual reproductive yeast structures, Ascus, , Ascospore, , Opportunistic Yeast, Opportunistic yeast may cause serious or lifethreatening infections in immunocompromised, patients, such as people with cancer or AIDS. The, yeast C. albicans can cause relatively minor infections in healthy people, such as thrush or vaginal, yeast infections; however, it can cause a dangerous, bloodstream infection called invasive candidiasis, in those with weakened immune systems. Similarly,, the yeast Cryptococcus neoformans can cause a, pulmonary infection that can lead to meningitis,, most often in immunocompromised people., , AT T HE BE NCH, , (b) Sexual reproductive yeast structures, , Figure 37.3 Reproductive structures of yeast, , We’ll study the cultural characteristics, the, type of reproduction, and the fermentative activities used to identify the different genera in this, experiment., , FU RT HER R E ADING, Refer to the section on fungi in your textbook for, further information on the species that are clinically important. In your textbook’s index, search, under “Candida,” “Mycology,” and “Thrush.”, , 258, , Experiment 37, , Materials, Cultures, 7-day Sabouraud agar cultures of, ❏❏ S. cerevisiae, ❏❏ C. albicans, ❏❏ Rhodotorula rubra, ❏❏ Selenotila intestinalis, ❏❏ Schizosaccharomyces octosporus, , Media, Five tubes of each per student group, ❏❏ Bromcresol purple glucose broth (Durham, tube)
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❏❏ Bromcresol purple maltose broth (Durham, tube), ❏❏ Bromcresol purple lactose broth (Durham, tube), ❏❏ Bromcresol purple sucrose broth (Durham, tube), ❏❏ Two glucose-acetate agar plates, ❏❏ Five test tubes (13 * 100mm) containing 2 ml, of sterile saline, , Reagents, ❏❏ Water–iodine solution, ❏❏ Lactophenol–cotton-blue solution, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop and needle, 10 glass slides, 10 coverslips, 5 sterile Pasteur pipettes, Glassware marking pencil, Microscope, , Procedure Lab One, Morphological Characteristics, Prepare a wet mount of each yeast culture in the, following manner:, 1. Suspend a loopful of yeast culture in a few, drops of lactophenol–cotton-blue solution, on a microscope slide and cover with a, coverslip., 2. Examine all yeast wet-mount slide preparations under low and high power, noting the, shape and the presence or absence of budding. Record your observations in the Lab, Report., , Sexual Reproduction, 1. With a glassware marking pencil, divide the, bottom of a glucose-acetate agar plate into, three sections, and divide another glucoseacetate agar plate in half., 2. Label each section with the name of a test, organism., 3. Label each tube of sterile saline with the name, of a test organism., 4. With a sterile inoculating loop, suspend a, heavy loopful of each test organism into its, appropriately labeled tube of saline. Tap the, tube with your finger to obtain a uniform cell, suspension., 5. With a sterile Pasteur pipette, inoculate one, drop of each test organism onto the surface of, the appropriately labeled section on an agar, plate. Note: Allow the inoculum to diffuse, into the agar for a few minutes. Do not swirl, or rotate the plates., 6. Incubate all plates at 25°C for 7 days. Note: Visit, the laboratory, if possible, during the incubation period and note when sporulation begins., , Procedure Lab Two, Fermentation Studies, 1. Examine all fermentation tubes for the presence of growth (turbidity), the presence, or absence of acid (change in the color of, medium), and the presence or absence of gas, (bubble in Durham tube)., 2. Record your results in the chart provided in, the Lab Report., , Procedure Lab Three, , Fermentation Studies, , Sexual Reproduction, , 1. With a sterile loop, inoculate each experimental organism into appropriately labeled, tubes of bromcresol purple glucose,, maltose, lactose, and sucrose fermentation, broths., 2. Incubate all cultures at 25°C for, 4 to 5 days., , 1. Examine the glucose-acetate agar plates for, the presence or absence of sporulation., 2. Prepare a water–iodine wet mount using a, loopful of culture from each respective section, on the glucose-acetate agar plate., 3. Observe the cells using the high-dry objective, and record your observations in the Lab Report., , Experiment 37, , 259
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EXPERIMENT, , 37, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Morphological Characteristics, Draw a representative field for each organism in the chart below. Note the, shape and presence or absence of budding ( + or - )., , Saccharomyces cerevisiae, , Candida albicans, , Rhodotorula rubra, , Selenotila intestinalis, , Shape:, Budding (+ or –):, , Shape:, Budding (+ or –):, , Schizosaccharomyces octosporus, Shape:, Budding (+ or –):, , Experiment 37: Lab Report, , 261
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Fermentation Studies, Use a plus (+ ) or minus (- ) in the chart below to record your results., Glucose, Organism, , T, , A, , Maltose, G, , T, , A, , Lactose, G, , T, , A, , Sucrose, G, , T, , A, , G, , S. cerevisiae, C. albicans, R. rubra, S. intestinalis, S. octosporus, Note: T = turbidity, A = acid, and G = gas, , Sexual Reproduction, In the circles below, draw representative reproductive structures and label the, parts., , Saccharomyces cerevisiae, , Candida albicans, , Rhodotorula rubra, , 262, , Experiment 37: Lab Report, , Schizosaccharomyces octosporus, , Selenotila intestinalis
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Review Questions, 1. Indicate the significance of the following structures in the reproductive, activities of yeast cells., a. Buds, , b. Ascus, , c. Ascospores, , 2. Why are yeast cells classified as fungi, and how do they differ from other, fungi?, , 3. Why is yeast of industrial importance?, , Experiment 37: Lab Report, , 263
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4. Why are yeasts significant from a medical perspective?, , 5. Why is it necessary to pasteurize fruit juices?, , 6., , 7., , 264, , A female patient develops candidiasis (moniliasis) following prolonged antibiotic therapy for a bladder infection caused by Pseudomonas aeruginosa. How can you account for the development of this, concurrent vaginal infection?, , With regard to the fermentation of wine, what kind of wine would, be produced if you washed the grapes prior to crushing them?, , Experiment 37: Lab Report
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PART 8, , The Viruses, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Explain chemical structures, morphologies, and replicative activities of bacterial, viruses (bacteriophages)., 2. Perform a phage dilution procedure for the cultivation and, enumeration of bacterial viruses., 3. Isolate bacteriophages from sewage., , Introduction, , Viruses are noncellular biological entities composed solely of a single type of nucleic acid, surrounded by a protein coat called the capsid., Because of their limited and simplistic structures,, viruses can be chemically defined as nucleoproteins. They are devoid of the sophisticated enzymatic and biosynthetic machinery essential for, independent activities of cellular life. This lack of, metabolic machinery mandates that they exist as, parasites, and they cannot be cultivated outside of, a susceptible living cell. Viruses are differentiated, from cellular forms of life on the following bases:, 1. They are ultramicroscopic and can only be, visualized with the electron microscope., 2. They are filterable: They are able to pass, through bacteria-retaining filters., 3. They do not increase in size., 4. They must replicate within a susceptible cell., 5. Replication occurs because the viral nucleic, acid subverts the synthetic machinery of the, host cell (namely, common host cell components and enzyme systems involved in, decomposition, synthesis, and bioenergetics) for the purpose of producing new viral, components., 6. Viruses are designated either RNA or DNA, viruses because they contain one of the, nucleic acids but never both., , Much of our knowledge of the mechanism of, animal viral infection and replication has been, based on our understanding of infection in bacteria by bacterial viruses, called bacteriophages, or, phages. The bacteriophages were first described, in 1915 almost simultaneously by Frederick Twort, and Felix d’Herelle. The name bacteriophage,, which in Greek means “to eat bacteria,” was, coined by d’Herelle because of the destruction, through lysis of the infected cell. Bacteriophages, exhibit notable variability in their sizes, shapes,, and complexities of structure. The T-even (T2, T4,, and T6) phages illustrated in Figure P8.1 demonstrate the greatest morphological complexity., Phage replication depends on the ability of the, phage particle to infect a suitable bacterial host cell., Infection consists of the following sequential events:, 1. Adsorption: Tail fibers of the phage particle, bind to receptor sites on the host’s cell wall., 2. Penetration (infection): The spiral protein, sheath retracts, and an enzyme, early muramidase, perforates the bacterial cell wall, enabling, the phage nucleic acid to pass through the, hollow core into the host cell’s cytoplasm. The, empty protein shell remains attached to the cell, wall and is called the protein ghost., 3. Replication: The phage genome subverts, the cell’s synthetic machinery, which is, then used for the production of new phage, components., 265
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Polyhedral head (capsid), Nucleic acid core, , Collar, Spiral protein sheath, Central hollow core, , End plate, , Tail fibers, , The functions of these structural components are as follows:, , Component, , Function, , Capsid (protein coat), , Protection of nucleic acid from destruction by DNases, , Nucleic acid core, , Phage genome carrying genetic information necessary for, replication of new phage particles, , Spiral protein sheath, , Retracts so that nucleic acid can pass from capsid into host cell's, cytoplasm, , End plate and tail fibers, , Attachment of phage to specific receptor sites on a susceptible, host's cell wall, , Figure P8.1 Bacteriophage: Structural components and their functions, , 4. Maturation: During this period, the new, phage components are assembled and form, complete, mature virulent phage particles., 5. Release: Late muramidase (lysozyme) lyses, the cell wall, liberating infectious phage particles that are now capable of infecting new, susceptible host cells, thereby starting the, cycle over again., Virulent phage particles that infect susceptible host cells always initiate the lytic cycle as, described above. Other phage particles, called, temperate phages or lambda 1 L 2 phages, incorporate their nucleic acid into the host’s chromosome. Lysis of the host cell does not occur until, it is induced by exogenous physical agents, such, 266, , Part 8, , as ultraviolet or ionizing radiation, or chemical, mutagenic agents. Bacterial cells containing the, incorporated phage nucleic acid, the prophage,, are called lysogenic cells. Lysogenic cells appear, and function as normal cells, and they reproduce, by fission. When induced by physical or chemical, agents, these cells will release a virulent prophage, from the host’s genome, which then initiates the, lytic cycle. Figure P8.2 illustrates the lytic and, lysogenic life cycles of a bacteriophage., Animal viruses differ structurally from bacteriophages in that they lack the spiral protein, sheath, end plate, and tail fibers. Their shapes may, be helical or cuboidal (icosahedral, containing 20, triangular facets). Some animal viruses are designated as naked viruses because they are composed
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Lysogenic, cycle, , Adsorption, , Lytic, cycle, , Release or burst, , Penetration, (infection), , Maturation, , Incorporation of, viral DNA prophage, state, , Replication, , Activation of prophage, (induction), Normal bacterial, cell activity, , Disruption, of bacterial DNA, , Figure P8.2 The lytic and lysogenic life cycles of a bacteriophage, , solely of nucleocapsids. In others, referred to as, enveloped viruses, the nucleocapsid is surrounded, by a lipid bilayer that may have glycoproteins, associated with it., The infectious process of the animal virus is, very similar to bacteriophage infection. However,, there are some notable differences:, 1. Adsorption of the virus is to receptor sites that, are located on the cell membrane of the host, cell instead of the cell wall, as in the bacterial, host., 2. Viral penetration is accomplished by endocytosis, an energy-requiring, receptor-mediated, process in which the entire virus enters the, host cell., , 3. The uncoating of the animal virus, removal of, the capsid, occurs within the host cell; with, bacteriophage infection, the phage capsid, remains on the outside of the host., 4. The latent period, the time between adsorption and the release of virulent viral particles,, is considerably longer—hours to days rather, than minutes, as in bacteriophage infection., , F U RT H E R RE A DI N G, Refer to the section on viruses in your textbook,, paying close attention to the sections on viral genomes and viral reproduction. In your textbook’s, index, search for terms such as “Prophage,” “Bacteriophage,” and “Latency.”, Part 8, , 267
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C ASE STUDY, A NEW ANTIBACTERIAL TREATMENT, As a lead researcher for Acme BioChemicals and, Anvils, you have been tasked with developing the, next antibacterial treatments that are not related, to current antibiotics on the market. You have chosen to look into the use of viruses as a means of, decreasing bacterial pathogens during an intestinal, infection. You are examining bacteriophages that, have shown to have a level of tropism for Escherichia coli in particular over other gram-negative, bacteria. This area of medical research is not new,, but your choice to utilize a strain of phage that, has a higher rate of lysogenic life cycle is a new, approach. The isolated strain is stable in bacterial, cultures and is able to handle the acidic environment of the stomach with minimal damage. This, , 268, , Part 8, , means that this potential treatment may be taken, orally and will begin to show a reduction in E. coli, numbers within hours., , Questions to Consider:, 1. What allows viruses to be tropic or selective, for which cell types to infect?, 2. Knowing the life cycle of a lysogenic, virus, will this result in the death of an, infected cell? Will all cells infected die at, the same time?, 3. What are the potential long-term effects of, having a patient’s GI tract colonized with, E. coli–carrying prophages?
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Cultivation and Enumeration, of Bacteriophages, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Perform techniques for cultivation and, enumeration of bacteriophages., , Principle, This exercise demonstrates how viruses replicate inside a susceptible host cell. For this, purpose, you will be provided with a virulent, phage and a susceptible host cell culture. This, technique also enables you to enumerate phage, particles on the basis of plaque formation in, a solid agar medium. Plaques are clear areas, in an agar medium previously seeded with a, diluted phage sample and a host cell culture., Each plaque represents the lysis of a phageinfected bacterial cell., The procedure requires the use of a doublelayered culture technique in which the hard agar, serves as a base layer, and a mixture of phage and, host cells in a soft agar forms the upper overlay., Susceptible E. coli cells multiply rapidly and produce a lawn of confluent growth on the medium., When one phage particle adsorbs to a susceptible, cell, penetrates the cell, replicates, and goes on, to lyse other host cells, the destroyed cells produce a single plaque in the bacterial lawn. (See, Figure 38.1). Each plaque can be designated as a, plaque-forming unit (PFU) and used to quantitate the number of infective phage particles in the, culture., The number of phage particles contained in, the original stock phage culture is determined by, counting the number of plaques formed on the, seeded agar plate and multiplying this by the dilution factor. For a valid phage count, the number of, plaques per plate should not exceed 300 or be less, than 30., Example: 200 PFUs are counted in a 10-6, dilution., , E XP E R IMENT, , 38, , 200 * 106 = 200 * 106 or 2 * 108, Plates showing greater than 300 PFUs are, too numerous to count (TNTC); plates showing fewer than 30 PFUs are too few to count, (TFTC)., The procedure covered in this experiment is, based on protocols published by The American, Society for Microbiology (www.asm.org) and is, an example of the numerous procedures that can, be found for the propagation and enumeration of, bacteriophages. Refer to online sources such as, ASM MicrobeLibrary (www.microbelibrary.org) or, American Type Culture Collection (www.atcc.org), for alternate methods based on your needs or, available laboratory equipment., , F U RT H E R RE A D I N G, Refer to the section on viruses in your textbook, for further information on the viruses that are, infective for bacteria. In your textbook’s index,, search under “Bacteriophage,” “T-even,” and, “Lysogenic.”, , Figure 38.1 Plaque-forming units (PFUs)., , 269
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C L I N I C A L A P P L I C AT I O N, Identifying Pathogenic Bacteria, Bacterial viruses (bacteriophages) are very common, in all natural environments, and are directly related, to the number of bacteria present. They are most, prevalent in soil, animal intestines, sewage, and seawater. These viral particles have played an important, role in the development of all types of viruses. Since, many phages are specific about which bacteria they, attack, a process called phage typing is used in clinical and diagnostic laboratories to identify pathogenic, bacteria., , AT THE B E N C H, , Materials, Cultures, 24-hour nutrient broth cultures of, ❏❏ E. coli B, ❏❏ T2 coliphage, , Media, Five each of the following per designated student, group, ❏❏ 1.5% tryptone agar plates, ❏❏ 0.7% tryptone soft agar (2 ml per tube), ❏❏ Nine tryptone broth tubes (900 ml per tube), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Waterbaths, Thermometer, Micropipetter and tips, Test tube rack, Glassware marking pencil, , Procedure Lab One, To perform the dilution procedure as illustrated in, Figure 38.2, do the following:, , 270, , Experiment 38, , 1. Label all dilution tubes and media as follows:, a. Five tryptone soft agar tubes:, 10-5, 10-6, 10-7, 10-8, 10-9, b. Five tryptone hard agar plates:, 10-5, 10-6, 10-7, 10-8, 10-9, c. Nine tryptone broth tubes: 10-1 through 10-9, 2. Place the five labeled soft tryptone agar tubes, into a waterbath. Water should be of a depth, just slightly above that of the agar in the tubes., Bring the waterbath to 100°C to melt the agar., Transfer the agar tubes to the second waterbath and maintain the melted agar at 45°C., 3. With the micropipetter, aseptically perform a, 10-fold serial dilution of the provided phage, culture using the nine 900@μl tubes of tryptone, broth., 4. To the tryptone soft agar tube labeled 10-5,, aseptically add 200 μl of the E. coli B culture, and 100 μl of the 10-4 tryptone broth phage, dilution. Rapidly mix by rotating the tube, between the palms of your hands, and pour, the contents over the hard tryptone agar plate, labeled 10-5, thereby forming a double-layered, plate culture preparation. Swirl the plate gently and allow to harden., 5. Using separate sterile micropipette tips, repeat, step 4 for the tryptone broth phage dilution, tubes labeled 10-5 through 10-8 to effect the, 10-6 through 10-9 tryptone soft agar overlays., 6. Following solidification of the soft agar overlay, incubate all plate cultures in an inverted, position for 24 hours at 37°C., , Procedure Lab Two, 1. Observe all plates for the presence of plaqueforming units that develop on the bacterial, lawn., 2. Count the number of PFUs in the range of, 30 to 300 on each plate., 3. Calculate the number of phage particles per ml, of the stock phage culture based on your PFU, count., 4. Record your results in the chart in the Lab, Report.
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PROCEDURE, Perform, a 10-fold, serial dilution., , 100 ml, , Phage, stock, , 100 ml, , 10-1, , 100 ml, , 10-2, , 100 ml, , 100 ml, , -3, , 10-4, , 10, , 100 ml, , -5, , 10, , 100 ml, , 10-6, , 100 ml, , 100 ml, , 10-7, , 10-8, , 10-9, , Tryptone broth tubes (900 ml each), 100 ml, , 100 ml, , 100 ml, , 100 ml, , 100 ml, , 10-5, , 10-6, , 10-7, , 10-8, , 10-9, , Add 200 ml, of E. coli B culture, to each tube., , Tryptone soft-agar tubes, Mix and pour., Overlay of tryptone soft agar, Tryptone hard agar, , 10-5, , 10-6, , 10-7, , 10-8, , 10-9, , Figure 38.2 Dilution procedure for cultivation and enumeration of bacteriophages, , Experiment 38, , 271
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E XP E R IMENT, , 38, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Phage Dilution, , Number of PFUs, , Calculation:, PFUs * Dilution Factor, , PFUs/ml of Stock Phage, Culture, , 10-5, 10-6, 10-7, 10-8, 10-9, , Review Questions, 1. Discuss the effects of lytic and lysogenic infections on the life cycle of the, host cell., , 2. Discuss the factors responsible for the transformation of a lysogenic infection to one that is lytic., , 3. Distinguish between the replicative and maturation stages of a lytic phage, infection., , Experiment 38: Lab Report, , 273
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4. In this experimental procedure, why is it important to use a hard agar with a, soft agar overlay technique to demonstrate plaque formation?, , 5. Explain what is meant by plaque-forming units., , 6. Determine the number of PFUs per ml in a 10-9 dilution of a phage culture, that shows 204 PFUs in the agar lawn., , 7., , 274, , The release of phage particles from the host bacterium always occurs, by lysis of the cell and results in the death of the host. A, nimal viruses, are released by either the lysis of the host cell or exocytosis, a reverse pinocytosis. Regardless of the mechanism of release, most infected cells die, while, other viruses may escape the cell without damaging the host cell. Explain., , Experiment 38: Lab Report
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E XP E R IMENT, , 39, , Isolation of Coliphages, from Raw Sewage, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Isolate virulent coliphages from sewage., , Principle, Isolates of bacterial viruses (bacteriophages) can, be obtained from a variety of natural sources,, including soil, intestinal contents, raw sewage,, and some insects, such as cockroaches and flies., Their isolation from these environments is not an, easy task, because the phage particles are usually, present in low concentrations. Therefore, isolation, requires a series of steps:, 1. Collection of the phage-containing sample at, its source, 2. Addition of an enriched susceptible host–cell, culture to the sample to increase the number, of phage particles for subsequent isolation, 3. Following incubation, centrifugation of the, enriched sample for the removal of gross, particles, 4. Filtration of the supernatant liquid through a, bacteria-retaining membrane filter, 5. Inoculation of the bacteria-free filtrate onto a, lawn of susceptible host cells grown on a soft, agar plate medium, 6. Incubation and observation of the culture, for the presence of phage particles, which is, indicated by plaque formation in the bacterial, lawn, In the following experiment, you will use, this procedure, as illustrated in Figure 39.1, for, the isolation of Escherichia coli phage particles, from raw sewage. Most bacteriophages that infect, E. coli (coliphages) are designated by the letter T,, indicating types. Seven types have been identified, and are labeled T1 through T7. The T-even phages, , (T2, T4, and T6) differ from the T-odd phages in, that the former vary in size, form, and chemical, composition. All of the T phages are capable of, infecting the susceptible E. coli B host cell., , F U RT H E R RE A D I N G, Refer to the section on viruses in your textbook for, further information on the viruses that are infective for bacteria. In your textbook’s index, search, under “Bacteriophage,” “T-even,” and “Lysogenic.”, , C L I N I C A L A P P L I C AT I O N, Phage Therapy, Phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. It is, mainly used in Russia and the Republic of Georgia,, and is not universally approved elsewhere. In Western cultures, no phage therapies are authorized for, use on humans, although phages for killing foodpoisoning bacteria (Listeria) are now in use. They, may also be used as a possible therapy against, many strains of drug-resistant bacteria., , AT T HE BE NCH, , Materials, Cultures, Lab One, ❏❏ 5-ml, 24-hour broth cultures of E. coli B, ❏❏ 45-ml samples of fresh sewage collected in, screw-capped bottles, Lab Two, ❏❏ 10-ml, 24-hour broth cultures of E. coli B, , 275
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PROCEDURE, , Pour in several, centrifuge tubes, and centrifuge, , Remove tubes, and decant, supernatant, into a 125-ml beaker., , sewage sample, at 2500 rpm, for 20 minutes., , Filter supernatant, through membrane, filter into vacuum flask., , E. coli B enriched sewage, sample in 250-ml flask, , Flask and membrane, filter apparatus, , Centrifuge, , Bacteria-free phage filtrate in vacuum flask, , 1 drop, , 2 drops, , 3 drops, , 4 drops, , 5 drops, , 1, , 2, , 3, , 4, , 5, , Add 0.1 ml, to tubes 1–5, of molten, soft tryptone, agar., , E. coli B, culture, , 1, , 2, , 3, Plates of hard tryptone agar, , Figure 39.1 Procedure for isolation of coliphages from raw sewage, 276, , Experiment 39, , 4, , 5
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Media, Per designated student group, Lab One, ❏❏ One 5-ml tube of bacteriophage nutrient broth, (ten times normal concentration), Lab Two, ❏❏ Five tryptone agar plates, ❏❏ Five 3-ml tubes of tryptone soft agar, , Equipment, Lab One, ❏❏ Sterile 250-ml Erlenmeyer flask and stopper, Lab Two, ❏❏ Sterile membrane filter apparatus, ❏❏ Sterile 125-ml Erlenmeyer flask and stopper, ❏❏ 125-ml flask, ❏❏ 1000-ml beaker, ❏❏ Centrifuge, ❏❏ Microincinerator or Bunsen burner, ❏❏ Forceps, ❏❏ 1-ml sterile disposable pipettes, ❏❏ Sterile Pasteur pipette, ❏❏ Mechanical pipetting device, ❏❏ Test tube rack, ❏❏ Glassware marking pencil, , Procedure Lab One, Use disposable gloves. It is essential to handle, raw sewage with extreme caution because it may, serve as a vehicle for the transmission of human, pathogens., , Enrichment of Sewage Sample, 1. Aseptically add 5 ml of bacteriophage nutrient, broth, 5 ml of the E. coli B broth culture, and, 45 ml of the raw sewage sample to an appropriately labeled sterile 250-ml Erlenmeyer, flask., 2. Incubate the culture for 24 hours at 37°C., , Procedure Lab Two, Filtration and Seeding, 1. Following incubation, pour the phage-infected, culture into a 100-ml centrifuge bottle or several centrifuge tubes, and centrifuge at 2500, rpm for 20 minutes., 2. Remove the centrifuge bottle or tubes, being, careful not to stir up the sediment, and carefully decant the supernatant into a 125-ml, beaker., 3. Pour the supernatant solution through a sterile membrane filter apparatus to collect the, bacteria-free, phage-containing filtrate in the, vacuum flask below. Refer to Experiment 48, for the procedure in assembling the filter, membrane apparatus., 4. Melt the soft tryptone agar by placing the five, tubes in a boiling waterbath, and cool to 45°C., 5. Label the five tryptone agar plates and the, five tryptone agar tubes 1, 2, 3, 4, and 5,, respectively., 6. Using a sterile 1-ml pipette, aseptically add, 0.1 ml of the E. coli B culture to all the molten, soft agar tubes., 7. Using a sterile Pasteur pipette, aseptically, add 1, 2, 3, 4, and 5 drops of the filtrate to the, respectively labeled molten soft agar tubes., Mix and pour each tube of soft agar into its, appropriately labeled agar plate., 8. Allow the agar to harden., 9. Incubate all the plates in an inverted position, for 24 hours at 37°C., , Procedure Lab Three, 1. Examine all the culture plates for plaque formation, which is indicative of the presence of, coliphages in the culture., 2. Indicate the presence ( + ) or absence ( - ) of, plaques in each of the cultures in the chart in, the Lab Report., , Experiment 39, , 277
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E XP ER IM E NT, , 39, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Drops of Phage, Filtrate, , 1, , 2, , 3, , 4, , 5, , Plaque formation, (+) or (- ), , Based on your observations, what is the relationship between the number of, plaques observed and the number of drops of filtrate in each culture?, , Review Questions, 1. Why is enrichment of the sewage sample necessary for the isolation of, phage?, , 2. How is enrichment of the sewage sample accomplished?, , Experiment 39: Lab Report, , 279
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3. How are bacteria-free phage particles obtained?, , 4., , 280, , Why must you exercise caution when handling raw sewage, samples?, , Experiment 39: Lab Report
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E XP E R IMENT, , 40, , Propagation of Isolated, Bacteriophage Cultures, , LEARNING OBJECTIVES, , C L I N I C A L A P P L I C AT I O N, , Once you have completed this experiment,, you should be able to, , With the increase in the rates of antibiotic, resistance in clinically relevant bacteria,, pharmaceutical companies and researchers are, looking for new therapeutic treatments in unlikely, places. They are now looking at the possibility, of treating a resistant bacterial infection with, a virus. Current research is examining the, clinical uses of bacteriophages as a means of, treating bacterial infections in the absence of, antibiotics., , 1. Isolate bacteriophages from a plaque, culture for later genetic studies or, manipulations., 2. Enumerate the plaque-forming units isolated from an individual plaque., , Principle, This exercise will demonstrate the procedure, for isolating and propagating a specific bacteriophage species from a single plaque picked, from a lawn plate. Before a microbiologist or, virologist may begin studying a new bacteriophage or begin genetic recombination studies,, an individual strain must be isolated. This is, similar to what must be done before performing, assays on bacterial species; a single colony must, be chosen so that all the bacteria present will, be genetic and metabolic clones of each other., These same practices will be followed when, studying viruses., What begins as a single virus infecting a single, bacterium will eventually spread to neighboring, cells. With the release of phage particles from, an infected cell, the phages will spread via diffusion to neighboring cells. Since the viruses have, no mechanisms for propulsion, such as a flagella, or fimbriae, the particles must rely on diffusion, through the soft agar medium to spread from cell, to cell. This exercise will use that occurrence, to remove the phage particles from an isolated, plaque., , FUR T HE R R E AD I N G, Refer to the section on viruses in your textbook for, further information on the viruses that are infective, for bacteria. In your textbook’s index, search under, “Prophage,” “Transformation,” and “Lytic.”, , AT T HE BE NCH, , Materials, Cultures, ❏❏ Agar plates reserved from Experiment 38 or, Experiment 39 that have individual plaques, ❏❏ 24-hour nutrient broth culture of Escherichia, coli B, , Media, Per designated student group, ❏❏ 10 ml of TRIS-buffered saline (TBS), ❏❏ Tryptone agar plates, ❏❏ Tryptone soft agar (2 ml per tube), ❏❏ Nine tryptone broth tubes (0.9 ml per tube), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Waterbath, Thermometer, 1.5-ml centrifuge tubes, 1-ml sterile pipettes, Sterile glass Pasteur pipettes, Rubber bulb, Mechanical pipetting device, Test tube rack, Glassware marking pencil, , 281
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Procedure Lab One, Utilizing one or more plates reserved from Experiment 38 and Experiment 39, use the following procedure to isolate bacteriophages:, 1. Place a rubber bulb on the end of a glass Pasteur pipette and use the end of the pipette to, remove the plaque-containing agar from the, selected plate as follows:, a. Following the procedure illustrated in, Figure 40.1, use the tapered end of the, glass Pasteur pipette and plunge the pipette, through the agar that surrounds the plaque., b. Give the pipette a few turns to reduce contact between the agar and the bottom of the, petri dish., c. Lift up the pipette and the agar plug that, contains the plaque., 2. Gently depress the bulb to dislodge the agar, plug into a 1.5-ml centrifuge tube., 3. Add 1 ml of TRIS-buffered saline (TBS) to the, tube and incubate at 4°C overnight or up to, one week., , Procedure Lab Two, 1. Label all dilution tubes and media as follows:, a. Five tryptone soft agar tubes:, 10-5, 10-6, 10-7, 10-8, 10-9, , 2., , 3., , 4., , 5., , 6., , b. Five tryptone hard agar plates:, 10-5, 10-6, 10-7, 10-8, 10-9, c. Nine tryptone broth tubes: 10-1 through 10-9, Place the five labeled soft tryptone agar tubes, into a waterbath. Water should be of a depth, just slightly above that of the agar in the tubes., Bring the waterbath to 100°C to melt the agar., Transfer the agar tubes to the second waterbath and maintain the melted agar at 45°C., With micropipetter, aseptically perform a, tenfold serial dilution of the provided phage, culture using the nine 900@ml tubes of tryptone, broth., To the tryptone soft agar tube labeled 10-5,, aseptically add 200 ml of the E. coli B culture, and 100 ml of the 10-4 tryptone broth phage, dilution. Rapidly mix by rotating the tube, between the palms of your hands, and pour, the contents over the hard tryptone agar plate, labeled 10-5, thereby forming a double-layered, plate culture preparation. Swirl the plate gently and allow it to harden., Using separate sterile micropipette tips, repeat, step 4 for the tryptone broth phage dilution, tubes labeled 10-5 through 10-8 to effect the, 10-6 through 10-9 tryptone soft agar overlays., Following solidification of the soft agar overlay, incubate all plate cultures in an inverted, position for 24 hours at 37°c., , Procedure Lab Three, 1. Observe all plates for the presence of plaqueforming units that develop on the bacterial, lawn., 2. Count the number of CFUs per plate in the, range of 30 to 300 on each plate., 3. Calculate the number of phage particles and, record your results., , Petri dish, with agar, , Figure 40.1 Use of a glass Pasteur pipette to, remove an agar plug from a petri dish that, contains a plaque of interest, , 282, , Experiment 40
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E XP E R IMENT, , 40, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Phage Dilution, , Number of PFUs, , Calculation: PFUs : Dilution Factor, , PFUs/mL of Recovered, Phage Culture, , 10-5, 10-6, 10-7, , Review Questions, 1. How many bacteriophage particles were isolated from a single plaque? How many different strains of, phage would be present?, , You have tested a sewage sample for the presence of bacteriophages and have several plates, with plaques present. Will all of these plaques be due to the same type or strain of virus? How, would you go about answering this question?, , Experiment 40: Lab Report, , 283
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PART 9, , Physical and Chemical, Agents for the Control, of Microbial Growth, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Explain the basic methods for inhibiting microbial growth and the modes of, antimicrobial action., 2. Describe the effects of physical agents, moist heat, osmotic pressure, and, ultraviolet radiation on selected microbial populations., 3. Explain the effects on selected microbial populations of chemical agents, used as disinfectants, antiseptics, and antibiotics., , Introduction, Control of microorganisms is essential in the, home, industry, and medical fields to prevent and, treat diseases and to inhibit the spoilage of foods, and other industrial products. Common methods, of control involve chemical and physical agents, that adversely affect microbial structures and, functions, thereby producing a microbicidal or, microbistatic effect. A microbicidal effect is one, that kills the microbes immediately; a microbistatic effect inhibits the reproductive capacities, of the cells and maintains the microbial population, at a constant size., , Chemical Methods for Control, of Microbial Growth, 1. Antiseptics: chemical substances used on, living tissue that kill or inhibit the growth of, vegetative microbial forms, 2. Disinfectants: chemical substances that kill, or inhibit the growth of vegetative microbial, forms on nonliving materials, 3. Chemotherapeutic agents: chemical, substances that destroy or inhibit the, growth of microorganisms in living, tissues, , Physical Methods for Control, of Microbial Growth, The modes of action of the different chemical, and physical agents of control vary, although, they all produce damaging effects to one or more, essential cellular structures or molecules in order, to cause cell death or inhibition of growth. Sites, of damage that can result in malfunction are the, cell wall, cell membrane, cytoplasm, enzymes,, and nucleic acids. The adverse effects manifest, themselves in the following ways., 1. Cell-wall injury: This can occur in one of two, ways. First, lysis of the cell wall will leave the, wall-less cell, called a protoplast, susceptible, to osmotic damage, and a hypotonic environment may cause lysis of the vulnerable protoplast. Second, certain agents inhibit cell wall, synthesis, which is essential during microbial, cell reproduction. Failure to synthesize a, missing segment of the cell wall results in an, unprotected protoplast., 2. Cell-membrane damage: This may be the, result of lysis of the membrane, which will, cause immediate cell death. Also, the selective nature of the membrane may be affected, without causing its complete disruption. As a, result, there may be a loss of essential cellular, 285
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Heat, , Temperature, Cold, , Dry: Gas and electric ovens, Moist:, 1. Steam under pressure, a. Autoclave, 2. Free-flowing steam, a. Boiling, b. Intermittent sterilization (tyndallization), 3. Pasteurization, Refrigerator: Microbistatic, Freezing, Lyophilization, , Osmotic pressure, , Hypertonicity: Increased salt and sugar concentrations, Hypotonicity: Increased water concentration, , Radiation, , X-ray, Gamma ray, Ultraviolet, , Desiccation, Sonic vibrations, Filtration, , Ionizing radiation, Thymine dimerization, , Removal of cellular water, High-frequency sound waves, Removal of organisms or particulates from thermolabile solutions using microbial filters, , Figure P9.1 Physical methods used for the control of microbial growth, , molecules or interference with the uptake of, nutrients. In both cases, metabolic processes, will be adversely affected., 3. Alteration of the colloidal state of cytoplasm: Certain agents cause denaturing of, cytoplasmic proteins. Denaturing processes, are responsible for enzyme inactivation and, cellular death by irreversibly rupturing the, molecular bonds of these proteins and rendering them biologically inactive., 4. Inactivation of cellular enzymes: Enzymes, may be inactivated competitively or noncompetitively. Noncompetitive inhibition is irreversible and occurs following the application, of some physical agent, such as mercuric chloride (HgCl 2), that results in the uncoiling of, the protein molecule, rendering it biologically, inactive. Competitive inhibition occurs when a, natural substrate is forced to compete for the, active site on an enzyme surface with a chemically similar molecular substrate, which can, block the enzyme’s ability to create end products. Competitive inhibitions are reversible., 5. Interference with the structure and, function of the DNA molecule: The DNA, molecule is the control center of the cell and, may also represent a cellular target area for, destruction or inhibition. Some agents have an, affinity for DNA and cause breakage or distortion of the molecule, thereby interfering with, its replication and role in protein synthesis., Figure P9.1 illustrates the acceptable physical, methods used for the control of microbial growth., , 286, , Part 9, , Awareness of the mode of action of the physical and chemical agents is absolutely essential for, their proper selection and application in microbial, control. The exercises in this section are designed, to acquaint you more fully with several commonly, employed agents and their uses., , Governing Bodies for Laboratory, Procedures, Numerous groups consisting of individuals involved, in academics, microbiological research, industry, and, government agencies have developed accepted procedures and practices for the research and development of new antibiotics and anti-microbial chemical, agents. Groups that include Clinical and Laboratory, Standards Institute (CLSI), the Association of Analytical Communities (AOAC), and the American Society, for Microbiology (ASM) partner with government, agencies, such as the U.S. Environmental Protection, Agency (EPA) and the Centers for Disease Control, and Prevention (CDC), to determine best practices, for use in clinical and laboratory research that many, of the procedures presented in Part 9 will be based, on. Where possible, a formal agency guideline will be, included for future reference., , F U RT HE R R E ADI N G, Refer to the section on chemical control of microbial growth in your textbook, paying close, attention to the sections on the differing levels of, chemical and physical sterilization. In your textbook’s index, search for terms such as “Sterilize,”, “Sanitize,” and “Disinfect.”
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C AS E STUDY, HOW CLEAN IS CLEAN?, A food-processing plant faces a recall of three, months’ worth of food production. A bacterial, contaminant was found in the packaged, ready-toeat food. Independent investigating laboratories, have identified the bacterial pathogen that led to, the hospitalization of six people so far. The bacterial pathogen is associated with raw chicken and, is easily killed upon heating the food products, containing chicken to their target cooking temperature. Your job as an investigator for one of, the state health agencies is to identify the source, of the contamination and advise the company on, how to fix the procedural issues that allowed the, contamination to occur., On your third day in the processing plant, you, notice that during peak periods during the second, shift, when the processing line is at maximum, output, some of the local supervisors will pull in, workers from other areas to help with the final, processing steps of ensuring a clean cut of food, products before packaging. One of the workers who, , has been cross-trained to do this step when needed, is one of the workers from the beginning of the processing line who is responsible for initial cutting of, the chicken carcass. This worker elects to supply, his own cutting implements that fits his hand better, to increase his own production numbers. You document that this worker will wipe his knife with a wet, cloth before transferring over to the final stage and, cutting cooked and cooled food for packaging., , Questions to Consider:, 1. What separates the levels of bacterial contamination when we sterilize, sanitize, decontaminate, or disinfect something?, 2. What level of cleanliness or of decreasing the, bacterial counts do you think this worker has, achieved with a damp cloth?, 3. What level should he achieve before working with cooked foods destined for human, consumption?, , Part 9, , 287
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Physical Agents of Control:, Moist Heat, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Explain the susceptibility of microbial, species to destruction by the application, of moist heat., , E XP E R IMENT, , 41, , jacket. (See Figure 41.1.) At a designated pressure,, the saturated steam is released into the inner chamber, from which all the air has been evacuated. The, steam under pressure in the vacuumed inner chamber is now capable of achieving temperatures in, excess of 100°C. The temperature is determined by, the pounds of pressure applied per square inch:, PRESSURE (pounds/inch2), , TEMPERATURE (°C), , 0 (free-flowing steam), , 100, , 10, , 115, , Principle, , 15, , 121, , Temperature has an effect on cellular enzyme, systems and therefore a marked influence on the, rate of chemical reactions, and thus the life and, death of microorganisms. Despite the diversity, among microorganisms’ temperature requirements, for growth, extremes in temperature can be used, in microbial growth control. Sufficiently low temperatures will inactivate enzymes and produce a, static effect. High temperatures destroy cellular, enzymes, which become irreversibly denatured., The application of heat is a common means of, destroying microorganisms. Both dry and moist, heat are effective. However, moist heat—which,, because of the hydrolyzing effect of water and its, greater penetrating ability, causes coagulation of, proteins—kills cells more rapidly and at lower, temperatures than does dry heat. Sterilization,, the destruction of all forms of life, is accomplished, in 15 minutes at 121°C with moist heat (steam), under pressure; dry heat requires a temperature of, 160°C to 180°C for 1 1 2 to 3 hours., Microbes exhibit differences in their resistance, to moist heat. As a general rule, bacterial spores, require temperatures above 100°C for destruction,, whereas most bacterial vegetative cells are killed at, temperatures of 60°C to 70°C in 10 minutes. Fungi, can be killed at 50°C to 60°C, and fungal spores, require 70°C to 80°C for 10 minutes for destruction., Because of this variability, moist heat can either, sterilize or disinfect. Common applications include, free-flowing steam under pressure (autoclaving),, free-flowing steam at 100°C (tyndallization), and the, use of lower temperatures (pasteurization)., Free-flowing steam under pressure requires, the use of an autoclave, a double-walled metal vessel that allows steam to be pressurized in the outer, , 20, , 126, , 25, , 130, , A pressure of 15 pounds/inch2 achieves a temperature of 121°C and sterilizes in 15 minutes. This is, the usual procedure; however, depending on the heat, sensitivity of the material to be sterilized, the operating pressure and time conditions can be adjusted., Application of free-flowing steam requires, exposure of the contaminated substance to a temperature of 100°C, which is achieved by boiling water., Exposures to boiling water for 30 minutes will result, in disinfection only; all vegetative cells will be killed,, but not necessarily the more heat-resistant spores., Another procedure is tyndallization, also, referred to as intermittent or fractional sterilization., This procedure requires exposure of the material to, free-flowing steam at 100°C for 20 minutes on 3 consecutive days with intermittent incubation at 37°C., The steaming kills all vegetative cells. Any spores, that may be present germinate during the period, of incubation and are destroyed during subsequent, exposure to a temperature of 100°C. Repeating this, procedure for 3 days ensures germination of all, spores and their destruction in the vegetative form., Because tyndallization requires so much time, it is, used only for sterilization of materials that are composed of thermolabile chemicals and that might be, subject to decomposition at higher temperatures., Pasteurization exposes fairly thermolabile, products, such as milk, wine, and beer, for a given, period of time to a temperature that is high enough, to destroy pathogens and some spoilage-causing, microorganisms that may be present, without, necessarily destroying all vegetative cells. There are, three types of pasteurization. The high-temperature,, short-time (HTST) procedure requires a, 289
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Chamber, pressure, gauge, Steam exhaust, valve, , Steam, exhaust, , Deflector, plate, Steam, jacket, , Door, , Chamber, Temperature, gauge, , Air vent, , Steam, supply, valve, Steam, supply, , (a) An autoclave, , (b) Schematic representation, , Figure 41.1 The autoclave, , temperature of 71°C for 15 seconds. The lowtemperature, long-time (LTLT) method requires, 63°C for 30 minutes, and the ultra–high temperature, (UHT) approach occurs at 138°C for 2 seconds., , FU RT HER R E ADING, Refer to the section on sterilization in your textbook for further information on the methods that, are utilized to sterilize or disinfect laboratory, equipment. In your textbook’s index, search under, “Autolclave,” “Sporicidal,” and “Heat Sterilization.”, , C L I N I C A L A P P L I C AT I O N, Autoclave Performance Testing, While the original “autoclave” was invented as a, pressure cooker for food, modern autoclaves are, precision instruments and require maintenance, and periodic testing, especially if control of human, pathogens is involved. Commonly, a sample of, spores of the bacterium Bacillus stearothermophilus, is sterilized in the chamber with a normal load, and, then the sample is allowed to incubate—any growth, indicates that the autoclave needs to be serviced., 290, , Experiment 41, , AT T HE BE NCH, , Materials, Cultures, 48- to 72-hour nutrient broth cultures (50 ml per, 250-ml Erlenmeyer flask) of, ❏❏ Staphylococcus aureus BSL-2, ❏❏ Bacillus cereus, 72- to 96-hour Sabouraud broth cultures (50 ml per, 250-ml Erlenmeyer flask) of, ❏❏ Aspergillus niger, ❏❏ Saccharomyces cerevisiae, , Media, Per designated student group (pairs or groups, of four), ❏❏ Five nutrient agar plates, ❏❏ Five Sabouraud agar plates, ❏❏ One 10-ml tube of nutrient broth
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Equipment, ❏❏ Microincinerator or Bunsen burner, ❏❏ 800-ml beaker (waterbath), ❏❏ Tripod and wire gauze screen with heat-resistant pad, ❏❏ Thermometer, ❏❏ Sterile test tubes, ❏❏ Glassware marking pencil, ❏❏ Inoculating loop, , Procedure Lab One, 1. Label the covers of each of the nutrient agar, and Sabouraud agar plates, indicating the, experimental heat temperatures to be used:, 25°C (control), 40°C, 60°C, 80°C, and 100°C., 2. Score the underside of each plate with a, glassware marking pencil into two sections., On the nutrient agar plates, label one section, S. aureus and the other B. cereus. On the Sabouraud agar plates, label one section A. niger, and the second S. cerevisiae., 3. Using aseptic technique, inoculate the nutrient, agar and Sabouraud agar plates labeled 25°C, by making a single-line loop inoculation of, each test organism in its respective section of, the plate., 4. Using a sterile pipette and mechanical pipetter, transfer 10 ml of each culture to four, sterile test tubes labeled with the name of the, organism and the temperature (40°C, 60°C,, 80°C, and 100°C)., 5. Set up the waterbath as illustrated in, Figure 41.2, inserting the thermometer in an, uncapped tube of nutrient broth., 6. Slowly heat the water to 40°C; check the, thermometer frequently to ensure that it, does not exceed the desired temperature., Place the four cultures of the experimental, organisms into the beaker and maintain the, temperature at 40°C for 10 minutes. Remove, the cultures and aseptically inoculate each, organism in its appropriate section on the, two plates labeled 40°C., , Thermometer, , Beaker with water, , 10-ml test tube, of nutrient broth, Wire gauze, , Bunsen burner, , Figure 41.2 Waterbath for moist heat experiment, , 7. Raise the waterbath temperature to 60°C and, repeat step 6 for the inoculation of the two, plates labeled 60°C., 8. Raise the waterbath temperature to 80°C and, repeat step 6 for the inoculation of the two, plates labeled 80°C., 9. Raise the waterbath temperature to 100°C and, repeat step 6 for the inoculation of the two, plates labeled 100°C., 10. Incubate the nutrient agar plate cultures in an, inverted position for 24 to 48 hours at 37°C, and the Sabouraud agar plate cultures for 4 to, 5 days at 25°C in a moist chamber., , Procedure Lab Two, 1. Observe all plates for the amount of growth of, the test organisms at each of the temperatures., 2. Record your results in the chart provided in, the Lab Report., , Experiment 41, , 291
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E XP ER IME NT, , 41, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Record your results in the chart as 0 = none; 1+ = slight; 2+ = moderate; 3+ = abundant., AMOUNT OF GROWTH, MICROBIAL SPECIES, , 25°C, , 40°C, , 60°C, , 80°C, , 100°C, , B. cereus, S. aureus, A. niger, S. cerevisiae, , 2. List the microbial organisms in order of increasing heat resistance., , _______________________________________, _______________________________________, _______________________________________, _______________________________________, , Review Questions, 1. Account for the microbistatic effect produced by low temperatures as compared with the, microbicidal effect produced by high temperatures., , 2. Cite the advantages of each of the modes of sterilization: tyndallization and autoclaving., , Experiment 41: Lab Report, , 293
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3. Discuss the detrimental effects of control agents on the following: the cytoplasm, the cell wall,, nucleic acids, and the cell membrane., , 4. Explain why milk is subjected to pasteurization rather than sterilization., , 5., , 6., , 294, , A. niger and B. cereus cultures used in this experiment contained spores. Why is B. cereus, more heat-resistant?, , Account for the fact that aerobic and anaerobic bacterial spore-formers are more heatresistant than is the tubercle bacillus, which is also known to tolerate elevated, temperatures., , Experiment 41: Lab Report
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Chemical Agents of Control:, Chemotherapeutic Agents, , Chemotherapeutic agents are chemical substances, used to treat infectious diseases. Their mode of, action is to interfere with microbial metabolism,, thereby producing a bacteriostatic or bactericidal, effect on the microorganisms, without producing a, like effect in host cells. Chemotherapeutic agents, act on a number of cellular targets. Their mechanisms of action include inhibition of cell-wall synthesis, inhibition of protein synthesis, inhibition of, nucleic acid synthesis, disruption of the cell membrane, and inhibition of folic acid synthesis. These, drugs can be separated into two categories:, 1. Antibiotics are synthesized and secreted by, some true bacteria, actinomycetes, and fungi, that destroy or inhibit the growth of other, microorganisms. Today, some antibiotics are, laboratory synthesized or modified; however,, their origins are living cells., 2. Synthetic drugs are synthesized in the, laboratory., To determine a therapeutic drug of choice,, it is important to determine its mode of action,, , TABLE 42.1 , ANTIBIOTIC, , E XP E R IMENT, , 42, , possible adverse side effects in the host, and the, scope of its antimicrobial activity. The specific, mechanism of action varies among different drugs,, and the short-term or long-term use of many drugs, can produce systemic side effects in the host., These vary in severity from mild and temporary, upsets to permanent tissue damage (Table 42.1)., , Synthetic Agents, Sulfadiazine (a sulfonamide) produces a static, effect on a wide range of microorganisms by, a mechanism of action called competitive, inhibition. The active component of the drug,, sulfanilamide, acts as an antimetabolite that, competes with the essential metabolite,, p-aminobenzoic acid (PABA), during the synthesis, of folic acid in the microbial cell. Folic acid is an, essential cellular coenzyme involved in the synthesis of amino acids and purines. Many microorganisms possess enzymatic pathways for folic acid, synthesis and can be adversely affected by sulfonamides. Human cells lack these enzymes, and the, , Prototypic Antibiotics, MODE OF ACTION, , POSSIBLE SIDE EFFECTS, , Penicillin, , Prevents transpeptidation of the N-acetylmuramic acids,, producing a weakened peptidoglycan structure., , Penicillin resistance; sensitivity (allergic, reaction), , Streptomycin, , Has an affinity for bacterial ribosomes, causing misreading of codons on mRNA, thereby interfering with protein, synthesis., , May produce damage to auditory nerve,, causing deafness., , Chloramphenicol, , Has an affinity for bacterial ribosomes, preventing, peptide bond formation between amino acids during, protein synthesis., , May cause aplastic anemia, which is fatal, because of destruction of RBC-forming and, WBC-forming tissues., , Tetracyclines, , Have an affinity for bacterial ribosomes; prevent hydrogen bonding between the anticodon on the tRNA–amino, acid complex and the codon on mRNA during protein, synthesis., , Permanent discoloration of teeth in young, children, , Bacitracin, , Inhibits cell-wall synthesis., , Nephrotoxic if taken internally; used for topical application only, , Polymyxin, , Destroy cell membrane., , Toxic if taken internally; used for topical, application only, , Rifampin, , Inhibits RNA synthesis., , Appearance of orange–red urine, feces,, saliva, sweat, and tears, , Quinolone, , Inhibits DNA synthesis., , Affects the development of cartilage., , 295
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Sulfadiazine (sulfonamide), , p-Aminobenzoic acid, COOH, , H, N, O2S, , N, , C, , CH, CH, , N, C, , NH2, , H, NH2, , Pyrimidine, component, , PABA, (essential metabolite), , Sulfanilamide, component, (antimetabolite), , Figure 42.1 Chemical similarity of sulfanilamide, and PABA, , essential folic acid enters the cells in a preformed, state. Therefore, these drugs have no competitive, effect on human cells. Figure 42.1 illustrates the, similarity between the chemical structure of the, antimetabolite sulfanilamide and the structure of, the essential metabolite PABA., , The Kirby-Bauer, Antibiotic Sensitivity Test, Procedure, PART A, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, , susceptibility of microorganisms isolated from, infectious processes. This method allows the, rapid determination of the efficacy of a drug by, measuring the diameter of the zone of inhibition, that results from diffusion of the agent into the, medium surrounding the disc. In this procedure,, filter-paper discs of uniform size are impregnated, with specified concentrations of different antibiotics and then placed on the surface of an agar, plate that has been seeded with the organism to, be tested. The medium of choice is Mueller-Hinton, agar, with a pH of 7.2 to 7.4, which is poured into, plates to a uniform depth of 5 mm and refrigerated after solidification. Prior to use, the plates are, transferred to an incubator at 37°C for 10 to, 20 minutes to dry off the moisture that develops, on the agar surface. The plates are then heavily, inoculated with a standardized inoculum by means, of a cotton swab to ensure the confluent growth of, the organism. The discs are aseptically applied to, the surface of the agar plate at well-spaced intervals. Once applied, each disc is gently touched, with a sterile applicator stick to ensure its firm, contact with the agar surface., Following incubation, the plates are examined, for the presence of growth inhibition, which is, indicated by a clear zone surrounding each disc, (Figure 42.2). The susceptibility of an organism to, a drug is assessed by the size of this zone, which is, affected by other variables such as the following:, 1. The ability and rate of diffusion of the antibiotic into the medium and its interaction with, the test organism, 2. The number of organisms inoculated, 3. The growth rate of the organism, , 1. Perform the Kirby-Bauer procedure and, evaluate the antimicrobial activity of chemotherapeutic agents., , Principle, The available chemotherapeutic agents vary in, their scope of antimicrobial activity. Some have, a limited spectrum of activity, effective against, only one group of microorganisms. Others exhibit, broad-spectrum activity against a range of microorganisms. The drug susceptibilities of many, pathogenic microorganisms are known, but it is, sometimes necessary to test several agents to, determine the drug of choice., A standardized diffusion procedure with filterpaper discs on agar, known as the Kirby-Bauer, method, is frequently used to determine the drug, 296, , Experiment 42, , Figure 42.2 Kirby-Bauer antibiotic sensitivity test
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Table 42.2 , , one Diameter Interpretive Standards for Organisms Other Than, Z, Haemophilus and Neisseria gonorrhoeae, ZONE DIAMETER, NEAREST WHOLE MM, , ANTIMICROBIAL AGENT, , DISC CONCENTRATION, , RESISTANT, , INTERMEDIATE, , SUSCEPTIBLE, , Ampicillin, when testing gram-negative, bacteria, , 10 mg, , …13, , 14–16, , Ú17, , when testing gram-positive, bacteria, , 10 mg, , …28, , —, , Ú29, , when testing Pseudomonas, , 100 mg, , …13, , 14–16, , Ú17, , when testing other gram-negative, organisms, , 100 mg, , …19, , 20–22, , Ú23, , Cefoxitin, , 30 mg, , …14, , 15–17, , Ú18, , Cephalothin, , 30 mg, , …14, , 16–17, , Ú18, , Chloramphenicol, , 30 mg, , …12, , 13–17, , Ú18, , Clindamycin, , 2 mg, , …14, , 15–20, , Ú21, , Erythromycin, , 15 mg, , …13, , 14–22, , Ú23, , Gentamicin, , 10 mg, , …12, , 13–14, , Ú15, , Kanamycin, , 30 mg, , …13, , 14–17, , Ú18, , Methicillin when testing, staphylococci, , 5 mg, , …9, , 10–13, , Ú14, , Novobiocin, , 30 mg, , …17, , 18–21, , Ú22, , when testing staphylococci, , 10 units, , …28, , —, , Ú29, , when testing other bacteria, , 10 units, , …14, , —, , Ú15, , Rifampin, , 5 mg, , …16, , 17–19, , Ú20, , Streptomycin, , 10 mg, , …11, , 12–14, , Ú15, , Tetracycline, , 30 mg, , …14, , 15–18, , Ú19, , Carbenicillin, , Penicillin G, , Tobramycin, , 10 mg, , …12, , 13–14, , Ú15, , 1.25/23.75 mg, , …10, , 11–15, , Ú16, , when testing enterococci, , 30 mg, , …14, , 15–16, , Ú17, , when testing Staphylococcus spp., , 30 mg, , —, , —, , Ú15, , Sulfonamides, , 250 or 300 mg, , …12, , —, , Ú17, , Trimethoprim, , 5 mg, , …10, , —, , Ú16, , Trimethoprim/sulfamethoxazole, Vancomycin, , Source: Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests, Tenth E dition, 2008., , A measurement of the diameter of the zone, of inhibition in millimeters is made, and its size is, compared with that contained in a standardized, chart, which is shown in Table 42.2. Based on this, comparison, the test organism is determined to, be resistant, intermediate, or susceptible to the, antibiotic., , The procedure given in this section is, an approximation of the industry-accepted, Performance Standards published by the Clinical, and Laboratory Standards Institute (CLSI) in, published standards documents M02-A12 and, M07-A10, as well as the Manual of Antimicrobial, , Experiment 42, , 297
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Susceptibility Testing published by the American, Society for Microbiology (ASM)., , FU RT HER R E ADING, Refer to the section on antimicrobial compounds, in your textbook for further information on the, compounds that have an effect on bacterial cells., In your textbook’s index, search under “Chemotherapy,” “Antibiotics,” and “Analog.”, , Antimicrobial-Sensitivity Discs, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Penicillin G, 10 mg, Streptomycin, 10 mg, Tetracycline, 30 mg, Chloramphenicol, 30 mg, Gentamicin, 10 mg, Vancomycin, 30 mg, Sulfanilamide, 300 mg, , Equipment, C L I N I C A L A P P L I C AT I O N, Selection of Effective Antibiotics, Upon isolation of an infectious agent, a chemotherapeutic agent is selected and its effectiveness, must be determined. This can be done using the, Kirby-Bauer Antibiotic Sensitivity Test. This is the, essential tool used in clinical laboratories to select, the best agent with which to treat patients with bacterial infections., , AT THE B E N C H, , Materials, Cultures, 0.85% saline suspensions adjusted to an absorbance of 0.1 at 600 nanometer (nm) or equilibrated, to a 0.5 McFarland Standard, ❏❏ Escherichia coli, ❏❏ Staphylococcus aureus BSL -2, ❏❏ Pseudomonas aeruginosa BSL -2, ❏❏ Proteus vulgaris, ❏❏ Mycobacterium smegmatis, ❏❏ Bacillus cereus, ❏❏ Enterococcus faecalis BSL -2, Note: For enhanced growth of M. smegmatis, add, Tween™ 80 (1 ml/liter of broth medium) and, incubate for 3 to 5 days in a shaking waterbath,, if available., , Media, Per designated student group, ❏❏ Seven Mueller-Hinton agar plates, , 298, , Experiment 42, , ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Sensi-Disc™ dispensers or forceps, Microincinerator or Bunsen burner, Sterile cotton swabs, Glassware marking pencil, 70% ethyl alcohol, Millimeter ruler, , Procedure Lab One, 1. Place agar plates right-side-up in an incubator, heated to 37°C for 10 to 20 minutes with the, covers adjusted so that the plates are slightly, opened, allowing the plates to warm up and, the surface to dry., 2. Label the bottom of each of the agar plates, with the name of the test organism to be, inoculated., 3. Using aseptic technique, inoculate all agar, plates with their respective test organisms as, follows:, a. Dip a sterile cotton swab into a well-mixed, saline test culture and remove excess inoculum by pressing the saturated swab against, the inner wall of the culture tube., b. Using the swab, streak the entire agar surface horizontally, vertically, and around the, outer edge of the plate to ensure a heavy, growth over the entire surface., 4. Allow all culture plates to dry for about, 5 minutes., 5. Using the Sensi-Disc dispenser, apply the antibiotic discs by placing the dispenser over the, agar surface and pressing the plunger, depositing the discs simultaneously onto the agar, surface (Figure 42.3, Step 1a). Or, if dispensers are not available, distribute the individual, discs at equal distances with forceps dipped in, alcohol and flamed (Figure 42.3, Step 1b).
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PROCEDURE, , Antibiotic disc, dispenser, , OR, , Antibiotic discs, , Inoculated, agar plate, , 1a Dispense antibiotic discs with the dispenser., , 1b Space antibiotic discs equidistant from each other on, the inoculated plate with a sterile forceps., Zone of inhibition, Confluent bacterial growth, Millimeter ruler, , 2 Gently touch each disc with a sterile, applicator or forceps., , 3 Following incubation, measure the diameter of each, zone of inhibition with a millimeter ruler., , Figure 42.3 Kirby-Bauer antibiotic sensitivity procedure, , 6. Gently press each disc down with the wooden, end of a cotton swab or with sterile forceps, to ensure that the discs adhere to the surface, of the agar (Figure 42.3, Step 2). Note: Do not, press the discs into the agar., 7. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Examine all plate cultures for the presence or, absence of a zone of inhibition surrounding, each disc., 2. Using a ruler graduated in millimeters, carefully measure each zone of inhibition to, the nearest millimeter (Figure 42.3, Step 3)., Record your results in the chart provided in, the Lab Report., , 3. Compare your results with Table 42.2 and, determine the susceptibility of each test organism to the chemotherapeutic agent. Record, your results in the Lab Report., , Synergistic Effect of, Drug Combinations, PART B, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Perform the disc–agar diffusion technique, for determination of synergistic combinations of chemotherapeutic agents., , Experiment 42, , 299
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Principle, Combination chemotherapy, the use of two or, more antimicrobial or antineoplastic agents, is, being employed in medical practice with everincreasing frequency. The rationale for using drug, combinations is the expectation that effective, combinations might lower the incidence of bacterial resistance, reduce host toxicity of the antimicrobial agents (because of decreased dosage, requirements), or enhance the agents’ bactericidal, activity. Enhanced bactericidal activity is known, as synergism. Synergistic activity is evident when, the sum of the effects of the chemotherapeutic, agents used in combination is significantly greater, than the sum of their effects when used individually. This result is readily differentiated from an, additive (indifferent) effect, which is evident, when the interaction of two drugs produces a, combined effect that is no greater than the sum of, their separately measured individual effects., A variety of in vitro methods are available to, demonstrate synergistic activity. In this experiment, a disc–agar diffusion technique will be, performed to demonstrate this phenomenon. This, technique uses the Kirby-Bauer antibiotic susceptibility test procedure, as described in Part A of, this experiment, and requires both Mueller-Hinton, agar plates previously seeded with the test organisms and commercially prepared, antimicrobialimpregnated discs. The two discs, representing the, drug combination, are placed on the inoculated, agar plate and separated by a distance (measured, in mm) that is equal to or slightly greater than onehalf the sum of their individual zones of inhibition, when obtained separately. Following the incubation period, an additive effect is exhibited by the, presence of two distinctly separate circles of inhibition. If the drug combination is synergistic, the, two inhibitory zones merge to form a “bridge” at, their juncture, as illustrated in Figure 42.4., The following drug combinations will be used, in this experimental procedure:, 1. Sulfisoxazole, 150 Mg, and trimethoprim,, 5 Mg. Both antimicrobial agents are enzyme, inhibitors that act sequentially in the metabolic pathway, leading to folic acid synthesis. The antimicrobial effect of each drug is, enhanced when the two drugs are used in, combination. The pathway thus exemplifies, synergism., , 300, , Experiment 42, , (a) Synergistic effect, , (b) Additive effect, , Figure 42.4 Synergistic and additive effects of, drug combinations, , 2. Trimethoprim, 5 Mg, and tetracycline,, 30 Mg. The modes of antimicrobial activity, of these two chemotherapeutic agents differ;, tetracycline acts to interfere with protein synthesis at the ribosomes. Thus, when used in, combination, these drugs produce an additive, effect., , F U RT H E R RE A D I N G, Refer to the section on antimicrobial compounds, in your textbook for further information on the, compounds that have an effect on bacterial cells., In your textbook’s index, search under “Chemotherapy,” “Antibiotics,” and “Analog.”, , C L I N I C A L A P P L I C AT I O N, Multiple Drug Therapy, In antimicrobial therapy for drug-resistant bacteria,, such as the opportunistic pathogen P. aeruginosa,, multiple drugs may be used to take advantage of, synergistic effects. Research has shown that use, of ampicillin to degrade gram-negative cell walls, allows easier entry of kanamycin, which then inhibits protein synthesis. Combination therapies taking, advantage of synergism also allow use of lower, doses of each drug, which reduces overall toxic, effects on the patient.
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AT T H E B E N C H, , Materials, Cultures, 0.85% saline suspensions adjusted to an absorbance of 0.1 at 600 nm or equilibrated to a 0.5, McFarland Standard, ❏❏ E. coli, ❏❏ S. aureus BSL -2, , Media, Per designated student group, ❏❏ Four Mueller-Hinton agar plates, , Antimicrobial-Sensitivity Discs, ❏❏ Tetracycline, 30 mg, ❏❏ Trimethoprim, 5 mg, ❏❏ Sulfisoxazole, 150 mg, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Forceps, Sterile cotton swabs, Millimeter ruler, Glassware marking pencil, , Procedure Lab One, 1. To inoculate the Mueller-Hinton agar plates,, follow Steps 1 through 4 as described under, the procedure in Part A of this experiment., 2. Using the millimeter ruler, determine the center of the underside of each plate and mark, with a glassware marking pencil., , 3. Using the glassware marking pencil, mark the, underside of each agar plate culture on both, sides from the center mark at the distances, specified below:, a. E. coli–inoculated plate for trimethoprim, and sulfisoxazole combination sensitivity:, 12.5 mm on each side of center mark, BSL -2 –inoculated plate for trib. S. aureus BSL-2, methoprim and sulfisoxazole combination, sensitivity: 14.5 mm on each side of center, mark, c. E. coli–inoculated plate for trimethoprim, and tetracycline combination sensitivity:, 14.0 mm on each side of center mark, BSL -2 –inoculated plate for trimd. S. aureus BSL-2, ethoprim and tetracycline combination:, 14.0 mm on each side of center mark, 4. Using sterile forceps, place the antimicrobial, discs, in the combinations specified in Step 3,, onto the surface of each agar plate culture at, the previously marked positions. Gently press, each disc down with the sterile forceps to, ensure that it adheres to the agar surface., 5. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Examine all agar plate cultures to determine, the zone of inhibition patterns exhibited. Distinctly separate zones of inhibition are indicative of an additive effect, whereas a merging of, the inhibitory zones is indicative of synergism., 2. Record your observations and results in the, chart provided in the Lab Report., , Experiment 42, , 301
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E XP E R IMENT, , 42, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Part A: Kirby-Bauer Antibiotic Sensitivity Test Procedure, 1. Record the zone size and the susceptibility of each test organism to the chemotherapeutic agent as resistant (R), intermediate (I), or sensitive (S) in the, charts below., GRAM-NEGATIVE, E. coli, Chemotherapeutic, Agent, , Zone, Size, , Susceptibility, , P. aeruginosa, , ACID-FAST, P. vulgaris, , Zone, Size Susceptibility, , Zone, Size, , M. smegmatis, , Susceptibility, , Zone, Size, , Susceptibility, , Penicillin, Streptomycin, Tetracycline, Chloramphenicol, Gentamicin, Vancomycin, Sulfanilamide, GRAM-POSITIVE, S. aureus, Chemotherapeutic, Agent, , Zone, Size, , Susceptibility, , E. faecalis, Zone, Size, , Susceptibility, , B. cereus, Zone, Size, , Susceptibility, , Penicillin, Streptomycin, Tetracycline, Chloramphenicol, Gentamicin, Vancomycin, Sulfanilamide, , Experiment 42: Lab Report, , 303
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2. For each of the chemotherapeutic agents, indicate the following:, a. The spectrum of its activity as broad or limited, b. The type or types of organisms it is effective against as gram-positive,, gram-negative, or acid-fast, Chemotherapeutic Agent, , Spectrum of Activity, , Type(s) of Microorganisms, , Penicillin, Streptomycin, Tetracycline, Chloramphenicol, Gentamicin, Vancomycin, Sulfanilamide, , Part B: Synergistic Effect of Drug Combinations, Cultures, , Appearance of Zone Inhibition, , Synergistic or Additive Effect, , E. coli:, trimethoprim and sulfisoxazole, , ______________________, , ______________________, , trimethoprim and tetracycline, , ______________________, , ______________________, , trimethoprim and sulfisoxazole, , ______________________, , ______________________, , trimethoprim and tetracycline, , ______________________, , ______________________, , S. aureus:, , Review Questions, 1., , 304, , Your experimental results indicate that antibiotics, such as tetracycline, streptomycin, and chloramphenicol, have a broad spectrum of activity against prokaryotic cells. Why do these antibiotics lack, inhibitory activity against eukaryotic cells such as fungi?, , Experiment 42: Lab Report
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E XP E R IMENT, , Determination of Penicillin Activity, in the Presence and Absence, of Penicillinase, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Employ a broth culture system to determine the minimal inhibitory concentration, (MIC) of penicillin., 2. Demonstrate the reversal of penicillin, inhibition against the test organism in the, presence of penicillinase (b-lactamase)., , 43, , CH2, , C, , CH3, , S, , O, NH, , O, , CH, , CH, , C, , C, , N, , CH, , Beta-lactamase–, susceptible portion, , CH3, COOH, , Beta-lactam ring, , Figure 43.1 Molecular structure of, benzylpenicillin (penicillin G), , Principle, In addition to the Kirby-Bauer paper disc–agar dif, fusion procedure, the broth tube dilution method, may be used to determine the susceptibility of an, organism to an antibiotic. The latter procedure,, in which dilutions of the antibiotic are prepared, in the broth medium, also permits the minimal, inhibitory concentration (MIC) to be determined for the antibiotic under investigation. The, MIC is the lowest concentration of an antimicrobial, agent that inhibits the growth of the test microorganism. Quantitative data of this nature may be, used by a clinician to establish effective antimi, crobial regimens for the treatment of a bacterial, infection in a host. These data are of particular, significance when the toxicity of the antibiotic is, known to produce major adverse effects in host, tissues., Penicillin is a potent antibiotic produced by, the mold Penicillium chrysogenum (formerly, called P. notatum). Sir Alexander Fleming’s discovery of penicillin in 1928 provided the world, with the first clinically useful antibiotic in the, fight to control human infection. The activity, of this antibiotic, as illustrated in Figure 43.1,, is associated with the b-lactam ring within its, molecular structure. Shortly after the clinical, introduction of benzylpenicillin (penicillin G),, pathogenic organisms, such as Staphylococcus, aureus, were found to be resistant to this “wonder, drug.” Research revealed that some organisms, were genetically capable of producing b-lactamase, (penicillinase), an enzyme that breaks a bond in, , Figure 43.2 Penicillinase activity. Penicillin, sensitivity is shown on the left; penicillin, resistance is shown on the right., , the b-lactam ring portion of the molecule. When, the integrity of this ring is compromised, the inhibitory activity of the antibiotic is lost. Figure 43.2, illustrates penicillinase activity., In this experiment, the MIC of penicillin will, be determined against penicillin-sensitive and, penicillinase-producing strains of Staphylococcus, aureus. The procedure involves specific concentrations of the penicillin prepared by means of a, twofold serial dilution technique in an enriched, broth medium. The tubes containing the antibiotic, dilutions are then inoculated with a standardized, concentration of the test organism Figure 43.3., 305
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C L I N I C A L A P P L I C AT I O N, Wider Capability Seen in B-lactamases, Penicillinases are b-lactam ring breakers with specific activity against penicillin, while cephalosporins, are generally not affected by them. New gene variants in gram-negative bacteria such as Klebsiella, pneumoniae and Neisseria gonorrhoeae are now, producing extended-spectrum b-lactamases, (ESBLs), which hydrolyze not only penicillin but also, many cephalosporins and monobactams. These, variants have been reported worldwide and now, pose significant challenges in infection control., Figure 43.3 Minimal inhibitory concentration, tube setup, , AT T HE BE NCH, , Table 43.1 illustrates the protocol for the antibiotic, , serial dilution–broth medium setup., Following incubation, spectrophotometric, absorbance readings will be used to determine the, presence or absence of growth in the cultures. The, culture that shows no growth in the presence of, the lowest concentration of penicillin represents, the minimal inhibitory concentration of this antibiotic against S. aureus., , MIC Determination, Using a Spectrophotometer, , FU RT HER R E ADING, , Cultures, , Refer to the section on antimicrobial compounds, in your textbook for further information on the, compounds that have an effect on bacterial cells., In your textbook’s index, search under “Resistance,” “Penicillinase,” and “Cell Wall.”, , 1:1000 brain heart infusion (BHI) broth dilutions of, 24-hour BHI broth cultures of, BSL -2, ❏❏ Staphylococcus aureus ATCC® 27661™ BSL-2, (penicillin-sensitive strain), BSL -2, ❏❏ Staphylococcus aureus ATCC 27659 BSL-2, (penicillinase-producing strain), , TABLE 43.1 Antibiotic, , PART A, , Materials, , Serial Dilution–Broth Medium Setup, TUBE NUMBER, , ADDITIONS (ML) TO:, , 1, , 2, , 3, , 4, , 5, , 6, , 7, , 8, , 9, , 10, , Medium, , 0, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , Penicillin, , 2, , 2, , Test culture, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , 2, , Total volume, , 4, , 4, , 4, , 4, , 4, , 4, , 4, , 4, , 4*, , 4, , Penicillin (mg/ml), , 50, , 50, , 25, , 12.5, , 6.25, , 3.12, , 1.56, , 0.78, , 0.39, , 0, , Control, , (-), , * After 2 ml discarded, , 306, , Experiment 43, , Serial dilution (See protocol), , 0, , (+ )
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Media, Per designated student group, ❏❏ 40 ml of brain heart infusion broth in a 100-ml, Erlenmeyer flask, ❏❏ 10 ml of sterile aqueous crystalline penicillin G, solution (100 mg/ml), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Sterile 13 * 100@mm test tubes, Test tube racks, Sterile 2-ml and 10-ml pipettes, Mechanical pipetting device, Microincinerator or Bunsen burner, Spectrophotometer, Glassware marking pencil, Disinfectant solution in a 500-ml beaker, , 7. Repeat Step 6 to inoculate all the tubes in Set, II with the 1:1000 dilution of S. aureus ATCC, 27659 BSL -2 (penicillinase-producing strain)., Discard the pipette., 8. Incubate both sets of tubes for 12 to 18 hours, at 37°C., , Procedure Lab Two, 1. Follow the instructions for the use of the spectrophotometer as outlined in Experiment 13 to, determine the absorbance readings for Tubes, 2 through 10 in Sets I and II. Use the Number, 1 tubes, the negative controls, as your blanks, to adjust the spectrophotometer., 2. Record your absorbance readings in the chart, in the Lab Report., , Procedure Lab One, 1. Into each of two test tube racks, place a set, of 10 sterile 13 * 100@mm test tubes labeled, 1 through 10. Label one rack Set I—penicillinsensitive, and the other rack Set II—penicillin-resistant. Refer to Table 43.1 for Steps 2, through 7., 2. Using a sterile 10-ml pipette and mechanical, pipetting device, add 2 ml of BHI broth to the, tubes labeled 2 through 10 in Sets I and II., Note: Discard the pipette into the beaker of, disinfectant., 3. With a 2-ml sterile pipette, add 2 ml of the penicillin solution to Tubes 1 and 2 in Sets I and II., Discard the pipette. Note: Mix the contents of, the tubes well., 4. Set I Serial Dilution: Using a sterile 2-ml, pipette, transfer 2 ml from Tube 2 to Tube 3., Mix well and transfer 2 ml from Tube 3 to Tube, 4. Continue this procedure through Tube 9 into, the beaker. Discard 2 ml from Tube 9. Tube 10, receives no antibiotic and serves as a positive, control. Discard the pipette. Note: Remember, to mix the contents of each tube well between, transfers., 5. Set II Serial Dilution: Using a sterile 2-ml, pipette, repeat Step 4., 6. Using a sterile 2-ml pipette, add 2 ml of the, 1:1000 dilution of the S. aureus ATCC 27661, BSL -2 (penicillin-sensitive strain) to all tubes, in Set I. Discard the pipette., , MIC Determination, Using a Plate Reader, PART B, , Materials, Cultures, 1:1000 brain heart infusion (BHI) broth dilutions of, 24-hour BHI broth cultures of, ❏❏ Staphylococcus aureus ATCC® 27661 BSL-2, BSL -2, (penicillin-sensitive strain), ❏❏ Staphylococcus aureus ATCC 27659 BSL-2, BSL -2, (penicillinase-producing strain), , Media, Per designated student group, ❏❏ 40 ml of brain heart infusion, ❏❏ 10 ml of sterile aqueous crystalline penicillin G, solution (100 mg/ml), , Equipment, Sterile 96-well plate with cover, micropipette with, sterile tips, and a colorimetric plate reader, , Experiment 43, , 307
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TABLE 43.2 Antibiotic, , Serial Dilution-Plate Setup, , WELL, , 1, , 2, , 3, , 4, , 5, , 6, , 7, , 8, , 9, , 10, , 11, , 12, , Medium, , 0, , 0, , 100, , 100, , 100, , 100, , 100, , 100, , 100, , 100, , 100, , 100, , Penicillin (ml), , 100, , 200, , Test culture (ml), , 100, , 100, , 100, , Final volume, , 200, , 200, , 200, , 200, , 200, , 200, , 200, , 200, , Penicillin (mg/ml), , 50, , 50, , 25, , 12.5, , 6.25, , 3.12, , 1.56, , 0.78, , Control, , (- ), , Serial dilution (See protocol), 100, , 100, , 100, , 100, , 100, , 0, 100, , 100, , 100, , 100, , 200, , 200, , 200*, , 200, , 0.39, , 0.19, , 0.09, , 0, (+), , * After 200 ml discarded, , Procedure Lab One, 1. For each organism to be tested, prepare a row, of wells by adding 100 ml of BHI broth to wells, 3 through 12 using a micropipette and sterile, tips. Refer to Table 43.2 on the following page, for the remaining steps., 2. Add 100 ml of penicillin G solution to well 1, and 200 ml to well 2 using a micropipette and, sterile tips., 3. Perform a serial dilution of the penicillin G, solution by transferring 100 ml of solution, from well 2 into well 3 (which has 100 ml of, BHI broth already added). Transfer 100 ml of, the BHI/penicillin solution from well 3 into, well 4, and repeat this procedure until well, 11, when the 100 ml taken from well 11 will be, discarded., , 308, , Experiment 43, , 4. Using a micropipette and a sterile tip, add, 100 ml of bacterial suspension to each well,, starting at well 12 and continuing to well 1., Discard the tip before the addition of new bacterial suspension to each row., 5. Cover plate and incubate at 37°C for 12 to 18, hours., , Procedure Lab Two, 1. Follow the instructions for the use of the plate, reader, as discussed in Experiment 13, to, determine the absorbance readings for each, well at 600 nanometers (nm). Wells 1 and 12, should be used as the negative and positive, controls, respectively, for this experiment to, determine growth in each well., 2. Record your absorbance readings in the chart, in the Lab Report.
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E XP ER IME NT, , 43, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Part A: MIC Determination Using a Spectrophotometer, Absorbance Readings at 600 nm, TUBE NUMBER, , 2, , 3, , 4, , 5, , 6, , 7, , 8, , 9, , 10, , Penicillin concentration (mg/ml), , 50, , 25, , 12.5, , 6.25, , 3.12, , 1.56, , 0.78, , 0.39, , 0, , Set I ______________________________, Set II ______________________________, , Set I: Minimal inhibitory concentration: ______________________________, Set II: Minimal inhibitory concentration: ______________________________, , Part B: MIC Determination Using a Plate Reader, WELL NUMBER, , 1, , Penicillin concentration (mg/ml), , 2, , 3, , 4, , 5, , 6, , 7, , 8, , 9, , 10, , 11, , 12, , 50, , 25, , 12.5, , 6.25, , 3.12, , 1.56, , 0.78, , 0.39, , .19, , .09, , 0, , Organism 1:, Organism II:, , Organism I: Minimal inhibitory concentration: ______________________________, Organism II: Minimal inhibitory concentration: ______________________________, , Review Questions, 1., , Was the ability of some microorganisms to produce b-lactamase, present prior to their exposure to the antibiotic penicillin? Explain., , Experiment 43: Lab Report, , 309
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2., , 310, , Can the results of an MIC test be used to determine whether an, antibiotic is bactericidal or bacteriostatic? If not, set up an, experimental procedure to determine whether the effect is bactericidal or, bacteriostatic., , Experiment 43: Lab Report
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E XP E R IMENT, , 44, , Chemical Agents of Control:, Disinfectants and Antiseptics, , Antiseptics and disinfectants are chemical, substances used to prevent contamination and, infection. Many are available commercially for, disinfection and asepsis., TABLE 44.1 , , Table 44.1 shows the major groups of antimicrobial agents, their modes and ranges of action,, and their practical uses., , Chemical Agents—Disinfectants and Antiseptics, , AGENT, Phenolic Compounds, Phenol, , MECHANISM OF ACTION, 1. Germicidal effect caused by alteration of protein structure resulting in protein denaturation, 2. Surface-active agent (surfactant) precipitates, cellular proteins and disrupts cell membranes., (Phenol has been replaced by better disinfectants that are less irritating, less toxic to tissues,, and better inhibitors of microorganisms.), , Cresols, , Hexachlorophene, , 1. Similar to phenol, 2. Poisonous and must be used externally, 3. 50% solution of cresols in vegetable oil, (sold, as Lysol®), Germicidal activity similar to phenol, (This agent is to be used with care, especially, on infants, because after absorption it may, have neurotoxic effects.), , Resorcinol, , Hexylresorcinol, , 1. Germicidal activity similar to that of phenol, 2. Acts by precipitating cell protein., Germicidal activity similar to that of phenol, , Thymol, , 1. Related to the cresols, 2. More effective than phenol, , Alcohols, , 1. Lipid solvent, 2. Denaturation and coagulation of proteins, 3. Wetting agent used in tinctures to increase, the wetting ability of other chemicals, 4. Germicidal activity increases with increasing, molecular weight., , Ethyl: CH3CH2OH, Isopropyl: (CH3)2CHOH, , 1. Germicidal effect resulting from rapid combination with proteins, Chlorine compounds:, 2. Chlorine reacts with water to form hypochlorous acid, which is bactericidal., Sodium hypochlorite, 3. Oxidizing agent, (Dakin’s fluid): NaOCl, Chloramine: CH3C6H4SO2NNaCl 4. Noncompetitively inhibits enzymes, especially, those dealing with glucose metabolism, by, reacting with SH and NH2 groups on the, enzyme molecule., Halogens, , USE, 1. 5% solution: disinfection, 2. 0.5% to 1% solutions: antiseptic, effect and relief of itching as it exerts, a local anesthetic effect on sensory, nerve endings, , 2% to 5% Lysol solutions used as, disinfectants, , 1. Reduction of pathogenic organisms, on skin; added to detergents, soaps,, lotions, and creams, 2. Effective against gram-positive, organisms, 3. An antiseptic used topically, 1. Antiseptic, 2. Keratolytic agent for softening or, dissolving keratin in epidermis, 1. Treatment of worm infections, 2. Urinary antiseptic, 1. Antifungal activity, 2. Treatment of hookworm infections, 3. Mouthwashes and gargle solutions, Skin antiseptics:, Ethyl—50% to 70%, Isopropyl—60% to 70%, , 1. Water purification, 2. Sanitation of utensils in dairy and, restaurant industries, 3. Chloramine, 0.1% to 2% solutions, for, wound irrigation and dressings, 4. Microbicidal, , 311
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TABLE 44.1 (Continued), AGENT, , MECHANISM OF ACTION, , Iodine compounds:, Tincture of iodine, Povidone-iodine solution, (Betadine®), Heavy Metals, Mercury compounds:, Inorganic:, Mercury bichloride, Mercurial ointments, Organic mercurials:, Mercurochrome (merbromin), Merthiolate (thimerosal), Metaphen (nitromersol), Merbak (acetomeroctol), Silver compounds:, Silver nitrate, Surface-Active Agents, Wetting agents:, Emulsifiers, soaps, and, detergents, , Cationic agents:, Quaternary ammonium, compounds, Benzalkonium chloride, Anionic agents:, Tincture of green soap, Sodium tetradecyl sulfate, , Acids (H +), -, , Alkali (OH ), , 312, , Experiment 44, , USE, , 1. Mechanism of action is not entirely known,, but it is believed that it precipitates proteins., 2. Surface-active agent, , 1. Tinctures of iodine are used for skin, antisepsis., 2. Treatment of goiter, 3. Effective against spores, fungi, and, viruses, , 1. Mercuric ion brings about precipitation of cellular proteins., 2. Noncompetitive inhibition of specific enzymes, caused by reaction with sulfhydryl group (SH), on enzymes of bacterial cells, , 1. Inorganic mercurials are irritating to, tissues, toxic systemically, adversely, affected by organic matter, and incapable of acting on spores., 2. Mercury compounds are mainly used, as disinfectants of laboratory materials., , 1. Similar to those of inorganic mercurials, but in, proper concentrations are useful antiseptics., 2. Much less irritating than inorganic mercurials, , 1. Less toxic, less irritating; used mainly, for skin asepsis, 2. Do not kill spores., , 1. Precipitate cellular proteins., 2. Interfere with metabolic activities of microbial, cells., 3. Inorganic salts are germicidal., , 1. Asepsis of mucous membrane of throat, and eyes, , 1. Lower surface tension and aid in mechanical, removal of bacteria and soil., 2. If active portion of the agent carries a negative electric charge, it is called an anionic, surface-active agent. If active portion of the, agent carries a positive electric charge, it is, called a cationic surface-active agent., 3. Exert bactericidal activity by interfering, with or by depressing metabolic activities of, microorganisms., 4. Disrupt cell membranes., 5. Alter cell permeability., , Weak action against fungi, acid-fast, microorganisms, spores, and viruses, , 1. Lower surface tension because of keratolytic,, detergent, and emulsifying properties., 2. Their germicidal activities are reduced by, soaps., , 1. Bactericidal, fungicidal; inactive, against spores and viruses, 2. Asepsis of intact skin, 3. Disinfectant for operating-room, equipment, 4. Dairy and restaurant sanitization, , 1. Neutral or alkaline salts of high-molecularweight acids. Common soaps included in this, group., 2. Exert their maximum activity in an acid, medium and are most effective against grampositive cells., 3. Same as all surface-active agents, , 1. Cleansing agent, 2. Sclerosing agent in treatment of varicose veins and internal hemorrhoids, , 1. Destruction of cell wall and cell membrane, 2. Coagulation of proteins, , Disinfection; however, of little practical, value
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TABLE 44.1 (Continued), AGENT, , MECHANISM OF ACTION, , USE, , Formaldehyde (liquid or gas), , Alkylating agent causes reduction of enzymes., , 1. Room disinfection, 2. Alcoholic solution for instrument, disinfection, 3. Specimen preservation, , Ethylene Oxide, , Alkylating agent causes reduction of enzymes., , Sterilization of heat-labile material, , B@Propiolactone (liquid or gas), , Alkylating agent causes reduction of enzymes., , 1. Sterilization of tissue for grafting, 2. Destruction of hepatitis virus, 3. Room disinfection, , Basic Dyes, , Affinity for nucleic acids; interfere with reproduction in gram-positive organisms., , 1. Skin antiseptic, 2. Laboratory isolation of gram-negative, bacteria, , Crystal violet, , The efficiency of all disinfectants and antiseptics is influenced by a variety of factors, including, the following:, 1. Concentration: The concentration of a, chemical substance markedly influences its, effect on microorganisms, with higher concentrations producing a more rapid death., Concentration cannot be arbitrarily determined; the toxicity of the chemical to the tissues being treated and the damaging effect on, nonliving materials must also be considered., 2. Length of exposure: All microbes are not, destroyed within the same exposure time., Sensitive forms are destroyed more rapidly, than resistant ones. The longer the exposure, to the agent, the greater its antimicrobial, activity. The toxicity of the chemical and environmental conditions must be considered in, assessing the length of time necessary for disinfection or asepsis., 3. Type of microbial population to be, destroyed: Microorganisms vary in their, susceptibility to destruction by chemicals., Bacterial spores are the most resistant forms., Capsulated bacteria are more resistant than, noncapsulated forms; acid-fast bacteria are, more resistant than non–acid-fast; and older,, metabolically less-active cells are more resistant than younger cells. Awareness of the, types of microorganisms that may be present, will influence the choice of agent., 4. Environmental conditions: Conditions, under which a disinfectant or antiseptic, affects the chemical agent are as follows:, a. Temperature: Cells are killed as the result, of a chemical reaction between the agent, , and cellular component. As increasing, temperatures increase the rate of chemical reactions, application of heat during, disinfection markedly increases the rate, at which the microbial population is, destroyed., b. pH: The pH conditions during disinfection, may affect not only the microorganisms, but also the compound. Extremes in pH, are harmful to many microorganisms and, may enhance the antimicrobial action of a, chemical. Deviation from a neutral pH may, cause ionization of the disinfectant; depending on the chemical agent, this may serve to, increase or decrease the chemical’s microbicidal action., c. Type of material on which the microorganisms exist: The destructive power of, the compound on cells is due to its combination with organic cellular molecules. If, the material on which the microorganisms, are found is primarily organic, such as, blood, pus, or tissue fluids, the agent will, combine with these extracellular organic, molecules, and the agent’s antimicrobial, activity will be reduced., Numerous laboratory procedures are available, for evaluating the antimicrobial efficiency of disinfectants or antiseptics. These procedures provide, a general rather than an absolute measure of the, effectiveness of any agent because test conditions, frequently differ considerably from those seen, during practical use. The agar plate-sensitivity, method, a commonly employed procedure, is, presented., , Experiment 44, , 313
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PART A Disc Diffusion Testing, of Disinfectants and Antiseptics, LEARNING OBJECTIVE, , F U RT H E R RE A D I N G, Refer to the section on antimicrobial compounds, in your textbook for further information on the, compounds that have an effect on bacterial cells., In your textbook’s index, search under “Disinfection,” “Susceptibility,” and “Antiseptic.”, , Once you have completed this experiment,, you should be able to, 1. Evaluate the effectiveness of antiseptic, agents against selected test organisms., , Principle, This procedure requires the heavy inoculation of, an agar plate with the test organism. Sterile, colorcoded filter-paper discs are impregnated with, different antiseptic and equally spaced on the inoculated agar plate. Following incubation, the agar, plate is examined for zones of inhibition (areas, of no microbial growth) surrounding the discs., A zone of inhibition is indicative of microbicidal, activity against the organism. Absence of a zone of, inhibition indicates that the chemical was ineffective against the test organism. Note: The size of the, zone of inhibition is not indicative of the degree, of effectiveness of the chemical agent. Antiseptic, susceptibility is represented in Figure 44.1., , C L I N I C A L A P P L I C AT I O N, Mrsa and Disinfection, Methicillin-resistant Staphylococcus aureus, (MRSA) is notorious for causing infections that are, difficult to treat with conventional antimicrobials,, but these strains have also demonstrated resistance, to disinfection. One study showed that resistance to, methicillin is directly related to lack of susceptibility, to benzalkonium chloride and other disinfectants. It, may be that adjusted contact times are necessary, to adequately kill these troublesome strains., , AT T HE BE NCH, , Materials, Cultures, , I, Uninoculated, control, H2O2, A, CL, Zone of, inhibition, , 24- to 48-hour Trypticase soy broth cultures of, ❏❏ Escherichia coli, ❏❏ Bacillus cereus, ❏❏ Staphylococcus aureus BSL-2, ❏❏ Mycobacterium smegmatis, 7-day-old Trypticase soy broth culture of Bacillus, cereus, , Media, Per designated student group, ❏❏ Five Trypticase soy agar plates, labeled with, organism names, , Figure 44.1 Antiseptic susceptibility test. Discs, , Antiseptics/Disinfectants, , are saturated with chlorine bleach (CL), hydrogen, peroxide (H2O2), isopropyl alcohol (A), and tincture, of iodine (I)., , 10 ml of each of the following dispensed in 25-ml, beakers per designated group, ❏❏ Tincture of iodine, ❏❏ 3% hydrogen peroxide, ❏❏ 70% isopropyl alcohol, ❏❏ 5% chlorine bleach, , 314, , Experiment 44
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Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Four different-colored sterile Sensi-Discs, Forceps, Sterile cotton swabs, Microincinerator or Bunsen burner, Glassware marking pencil, , PART B Modified-Use Dilution, Testing of Disinfectants and, Antiseptics, LEARNING OBJECTIVE, , Procedure Lab One, 1. Aseptically inoculate the appropriately labeled, agar plates with their respective test organisms by streaking each plate in horizontal and, vertical directions and around the edge with a, sterile swab., 2. Color-code the Sensi-Discs according, to the chemical agents to be used (e.g.,, red = chlorine bleach)., 3. Using forceps dipped in alcohol and flamed,, expose five discs of the same color by placing, them into the solution of one of the chemical, agents. Drain the saturated discs on absorbent, paper immediately prior to placing one on, each of the inoculated agar plates. Place each, disc approximately 2 cm in from the edge of, the plate. Gently press the discs down with the, forceps so that they adhere to the surface of, the agar., 4. Impregnate the remaining discs as described, in Step 3. Place one of each of the three, remaining colored discs on the surface of each, of the five inoculated agar plates equidistant, from each other around the periphery of the, plate., 5. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C., , Procedure Lab Two, 1. Observe all the plates for the presence of a, zone of inhibition surrounding each of the, impregnated discs., 2. Record your observations in the chart provided in the Lab Report., , Once you have completed this experiment,, you should be able to, 1. Evaluate the effectiveness of antiseptic, agents against selected test organisms., , Principle, This procedure requires the adherence of dried, bacterial cells to a treatable surface that can, withstand exposure to the disinfectant being, tested. The United States Environmental Protection Agency (EPA)–accepted protocol recognizes, guidelines published by the Association of Analytical Communities (AOAC) for S. aureus (Method, 955.15) and P. aeruginosa (Method 964.02), which, utilize a stainless steel carrier that will be dipped, in the disinfectant or antiseptic to be tested. For, this procedure, the carrier will be a glass slide that, has the bacteria dried on its surface before being, submerged in the test solution. The heated carrier, is placed in a tube of broth and allowed to incubate up to 48 hours to determine if any cells have, remained viable., , F U RT H E R RE A D I N G, Refer to the section on antimicrobial compounds, in your textbook for further information on, the compounds that have an effect on bacterial, cells. In your textbook’s index, search under, “Disinfection,” “Susceptibility,” and “Antiseptic.”, , Experiment 44, , 315
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AT THE B E N C H, , Materials, Cultures, 24- to 48-hour Trypticase soy broth cultures of, ❏❏ Escherichia coli, ❏❏ Bacillus cereus, ❏❏ Staphylococcus aureus BSL -2 BSL-2, ❏❏ Mycobacterium smegmatis, 7-day-old Trypticase soy broth culture of Bacillus, cereus, , Media, Per designated student group, ❏❏ Twenty-five 50-ml tubes containing 20 ml of, tryptic soy broth each, , Antiseptics/Disinfectants, 10 ml of each of the following dispensed in 25-ml, beakers per designated student group, ❏❏ Tincture of iodine, ❏❏ 3% hydrogen peroxide, ❏❏ 70% isopropyl alcohol, ❏❏ 5% chlorine bleach, , Equipment, ❏❏ Sterile glass slides or cover slips, ❏❏ Forceps, , 316, , Experiment 44, , ❏❏ Microincinerator or Bunsen burner, ❏❏ Glassware marking pencil, ❏❏ 70% ethyl alcohol, , Procedure Lab One, 1. Aseptically add E. coli to five sterile glass, slides or cover slips and allow to air-dry for, 10 minutes., 2. Separate the broth tubes into five sets, and, label each set for a different bacteria being, tested. Also label each tube with the antiseptic, or disinfectant treatment, reserving one tube, per set as the untreated control., 3. Once the E. coli slides have dried, submerge, each slide in one of the antiseptic/disinfectant, solutions for 30 to 60 seconds., 4. Place each slide on a paper towel to dry before, placing the treated slide in an appropriately, labelled broth tube., 5. Repeat Steps 1 through 4 for each bacterial, culture to be tested., 6. Incubate all tubes, loosely capped, at 37°C for, 24 to 48 hours., , Procedure Lab Two, 1. Observe all tubes for the presence of bacterial, growth signified by a cloudy appearance., 2. Record your observations in the chart provided in the Lab Report.
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E XP ER IM E NT, , 44, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, PART A: Disc Diffusion Testing of Disinfectants and, Antiseptics, 1. Indicate the absence of a zone of inhibition as (0), and the presence of a, zone of inhibition as (+ )., ANTIMICROBIAL AGENT, Bacterial Species, , Tincture of Iodine, , 3% Hydrogen, Peroxide, , 70% Isopropyl, Alcohol, , 5% Chlorine, Bleach, , E. coli gram-negative, S. aureus gram-positive, M. smegmatis acid-fast, B. cereus spore-former, gram-positive, B. cereus spore-former, gram-positive 7-day-old, , 2. Indicate which of the antiseptics exhibited microbicidal activity against, each of the following groups of microorganisms., Bacterial Group, , Tincture of Iodine, , 3% Hydrogen, Peroxide, , 70% Isopropyl, Alcohol, , 5% Chlorine, Bleach, , Gram-negative, Gram-positive, Acid-fast, Spore-former, , 3. Which of the experimental chemical compounds appears to have the broadest range of microbicidal activity? The narrowest range of microbicidal, activity?, , Experiment 44: Lab Report, , 317
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PART B: Modified-Use Dilution Testing of Disinfectants, and Antiseptics, 1. Indicate the absence of a bacterial growth in each tube as (0) and the presence of growth as (+ )., ANTIMICROBIAL AGENT, Bacterial Species, , Tincture of Iodine, , 3% Hydrogen, Peroxide, , 70% Isopropyl, Alcohol, , 5% Chlorine, Bleach, , E. coli gram-negative, S. aureus gram-positive, M. smegmatis acid-fast, B. cereus spore-former, gram-positive, B. cereus spore-former, gram-positive 7-day-old, , 2. Indicate which of the antiseptics exhibited microbicidal activity against, each of the following groups of microorganisms., Bacterial Group, , Tincture of Iodine, , 3% Hydrogen, Peroxide, , 70% Isopropyl, Alcohol, , Gram-negative, Gram-positive, Acid-fast, Spore-former, , 3. Which of the experimental chemical compounds appears to have the broadest, range of microbicidal activity? The narrowest range of microbicidal activity?, , Review Questions, 1. Can the disinfection period (exposure time) be arbitrarily increased? Explain., , 2., , 318, , A household cleanser is labeled germicidal. Explain what this, means to you., , Experiment 44: Lab Report, , 5% Chlorine, Bleach
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PART 10, , Microbiology of Food, , LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Describe the endogenous and exogenous organisms found in food products., 2. Analyze food products to determine their quality from the public health point, of view., 3. Utilize the process of microbiological production of wine., , Introduction, Microbiologists have always been aware that, foods, especially milk, have served as important, inanimate vectors in the transmission of disease., Foods contain the organic nutrients that provide, an excellent medium to support the growth and, multiplication of microorganisms under suitable, temperatures., Food and dairy products may be contaminated, in a variety of ways and from a variety of sources:, 1. Soil and water: Food-borne organisms that, may be found in soil and water and that may, contaminate food are members of the genera, Alcaligenes, Bacillus, Citrobacter, Clostridium, Pseudomonas, Serratia, Proteus,, Enterobacter, and Micrococcus. The common soil and water molds include Rhizopus, Penicillium, Botrytis, Fusarium, and, Trichothecium., 2. Food utensils: The type of microorganism, found on utensils depends on the type of food, and the manner in which the utensils were, handled., , 3. Enteric microorganisms of humans and, animals: The major members of this group, are Bacteroides, Lactobacillus, Clostridium,, Escherichia, Salmonella, Proteus, Shigella,, Staphylococcus, and Streptococcus. These, organisms find their way into the soil and, water, from which they contaminate plants, and are carried by wind currents onto utensils, or prepared and exposed foods., 4. Food handlers: People who handle foods are, especially likely to contaminate them because, microorganisms on hands and clothing are, easily transmitted. A major offending organism, is Staphylococcus, which is generally found, on hands and skin, and in the upper respiratory tract. Food handlers with poor personal, hygiene and unsanitary habits are most likely, to contaminate foods with enteric organisms., 5. Animal hides and feeds: Microorganisms, found in water, soil, feed, dust, and fecal, debris can be found on animal hides. Infected, hides may serve as a source of infection for, workers, or the microorganisms may migrate, , 319
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into the musculature of the animal and remain, viable following its slaughter., By enumerating microorganisms in milk and, foods, the quality of a particular sample can be, determined. Although the microorganisms cannot be identified, the presence of a high number, suggests a good possibility that pathogens are, present. Even if a sample contains a low microbial, count, it can still transmit infection., In the laboratory procedures that follow, you, will have an opportunity to directly and indirectly, , enumerate the number of microorganisms present, in milk and other food products and to thereby, determine the quality of the samples., , F U RT H E R RE A D I N G, Refer to the section on food microbiology in your, textbook, paying close attention to the uses of, selective media and metabolic assays for determining bacterial contamination. In your textbook’s, index, use the search terms “Triple Sugar Iron,”, “Selective,” and “Fermentation.”, , C ASE STUDY, FERMENTATION PRESERVATION, Your lab company has been contracted to develop, new commercial methods for preserving food, items destined for long-term storage. Instead of, developing new preservative compounds, you, decide to examine microbial by-products as a preservation means. Fermentation by native bacterial, and fungal species has been used by humans for, thousands of years to preserve food items. A byproduct of microbial metabolism in an anaerobic, environment is lactic acid 1C3H6O3 2. Lactic acid, is produced during fermentation by non–alcoholproducing mechanisms. Numerous microbes,, such as Lactobacillus, can utilize the glycocytic, pathways and produce lactic acid in the absence, of oxygen. While lactic acid is a good preservative, , 320, , Part 10, , itself, other microbes that may be present, such, as Eubacterium, have been shown to utilize lactic, acid as a carbon source. Your current project is to, develop an assay to test for lactic acid degradation, in the presence of a fermenter., , Questions to Consider:, 1. Why would the degrading of lactic acid by, another bacterial species be a problem during, food preservation?, 2. Would a bacterial species that can utilize lactate be a problem if the lab used an alcohol, fermenter to preserve the food?
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Microbiological Analysis of Food, Products: Bacterial Count, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Determine the total number of micro, organisms present in food products., 2. Calculate the number of coliform bacteria, in the selected food products., , Principle, Microorganisms in food may be harmful in some, cases, while in other cases, they are beneficial., Certain microorganisms are necessary in preparation of foods, including cheese, pickles, yogurt,, and sausage. However, other microorganisms are, responsible for serious and sometimes fatal food, poisoning and spoilage., , FUR T HE R R E AD I N G, Refer to the section on food safety in your textbook for further information on the microbes, responsible for food spoilage. In your textbook’s, index, search under “Coliform,” “Lactic Acid Fermentation,” and “Spoilage.”, , C L I N I C A L A P P L I C AT I O N, Microorganisms in Food, Some of the pathogens that are tested for in food, include: Escherichia coli, Listeria monocytogenes,, Salmonella species, and Aspergillus fungi., , AT T H E B E N C H, , Materials, Cultures, ❏❏ Samples of fresh vegetables, ground beef, and, dried fruit, , E XP E R IMENT, , 45, , Media, Per designated student group, ❏❏ Nine brain heart infusion agar deep tubes, ❏❏ Three eosin–methylene blue (EMB) agar plates, ❏❏ Three 99-ml sterile water blanks, ❏❏ Three 180-ml sterile water blanks, , Equipment, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Waterbath, ❏❏ Quebec or, electronic colony, counter, ❏❏ Balance, ❏❏ Sterile glassine, weighing paper, , ❏❏ Blender with three, sterile jars, ❏❏ Sterile Petri dishes, ❏❏ 1-ml pipettes, ❏❏ Mechanical, pipetting device, ❏❏ Inoculation loop, ❏❏ Glassware marking, pencil, , Procedure Lab One, Refer to Figure 45.1, which illustrates the procedure., 1. Label three sets of three Petri dishes for each, of the food samples to be tested and their dilutions (10-2, 10-3, and 10-4). Label the three, EMB agar plates with the names of the food., 2. Melt the brain heart infusion agar deep tubes, in a waterbath, cool, and maintain at 45°C., 3. Place 20 g of each food sample, weighed on, sterile glassine paper, into its labeled blender, jar. Add 180 ml of sterile water to each of, the blender jars and blend each mixture for, 5m, inutes. You will have made a 1:10 (10-1), dilution of each food sample., 4. Transfer 1 ml of the 10-1 ground beef suspension into its labeled 99-ml sterile water blank,, thereby effecting a 10-3 dilution, and 0.1 ml, to the appropriately labeled 10-2 Petri dish., Shake the 10-3 sample dilution, and using a, different pipette, transfer 1 ml to the plate, labeled 10-3 and 0.1 ml to the plate labeled, 10-4. Add a 15-ml aliquot of the molten and, cooled agar to each of the three plates. Swirl, the plates gently to obtain a uniform distribution, and allow the plates to solidify., 5. Repeat Step 4 for the remaining two 10-1 test, food sample dilutions., 6. Aseptically prepare a four-way streak plate, as, described in Experiment 3, and inoculate each, 321
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10-1 food sample dilution on its appropriately, labeled EMB agar plate., 7. Incubate all plates in an inverted position for, 24 to 48 hours at 37°C., , PROCEDURE, , Procedure Lab Two, 1. Following the instructions in the Lab Report,, count and record the number of colonies on, each plate., , Figure 45.1 Preparation of a food sample for analysis, , Sterile glassine paper, Food sample, , Weigh 20 g, of sample., , g, Place sample in, blender jar. Add, 180 ml of sterile, water and blend, for 5 minutes., , Transfer 1.0 ml., , 99 ml, , 10–3 dilution, 10–1 dilution, Prepare a, four-way, streak., , EMB, , 322, , Experiment 45, , Transfer, 0.1 ml., , 10– 2, , Transfer, 1.0 ml., , 10– 3, , Transfer, 0.1 ml., , 10– 4
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E XP E R IMENT, , 45, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Using either the Quebec or electronic colony counter, count the number, of colonies on each plate. Count only statistically valid plates that contain, between 30 and 300 colonies. Designate plates with fewer than 30 colonies, as too few to count (TFTC) and plates with more than 300 colonies as, too numerous to count (TNTC)., 2. Determine the number of organisms per ml of each food sample on plates, not designated as TFTC or TNTC by multiplying the number of colonies, counted by the dilution factor., 3. Record in the chart below the number of colonies per plate and the number, of organisms per milliliter of each food sample., Type of Food, , Dilution, , Number of Colonies per Plate, , Number of Organisms per ml, , 10-2, Ground beef, , 10-3, 10-4, 10-2, , Fresh vegetables, , 10-3, 10-4, 10-2, , Dried fruits, , 10-3, 10-4, , 4. Examine the eosin–methylene blue agar plate cultures for colonies with a, metallic green sheen on their surfaces, which is indicative of E. coli. Indicate in the chart below the presence or absence of E. coli growth and the, possibility of fecal contamination of the food., Sample, , E.coli ( + ) or ( − ), , Fecal Contamination ( + ) or ( − ), , Ground beef, , Fresh vegetables, , Dried fruit, , Experiment 45: Lab Report, , 323
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Review Questions, 1. Indicate some possible ways in which foods may become contaminated, with enteric organisms., , 2., , 3., , 324, , Explain why it is not advisable to thaw and then refreeze food, products without having cooked them., , Following a Fourth of July picnic lunch of ham, sour pickles,, potato salad, and cream puffs, a group of students were admitted, to the hospital with severe gastrointestinal distress. A diagnosis of staphylococcal food poisoning was made. Explain how the staphylococci can multiply in these foods and produce severe abdominal distress., , Experiment 45: Lab Report
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E XP E R IMENT, , 46, , Isolation of Salmonella, from Raw Meat, , LEARNING OBJECTIVE, , C L I N I C A L A P P L I C AT I O N, , Once you have completed this experiment,, you should be able to, , Yearly, there are over a million reported cases of, Salmonella-associated food poisoning. The government, through federal agencies such as the USDA,, has enacted laws that require testing of all commercially produced food items for the presence of, Salmonella in an attempt to reduce the number of, cases and ultimately reduce the mortality rate., , 1. Isolate and identify Salmonella species, from commercially available raw meat., , Principle, The procedure that you will use in this lab is a, reduced laboratory process used by federal agencies such as the United States Department of, Agriculture (USDA) and its regulatory agency,, the Food Safety and Inspection Service (FSIS)., This procedure will introduce you to the methods, utilized for the analysis of various meat, poultry,, and Siluriformes (fish) products; sponge and rinse, samples; and egg products for Salmonella. Your, success in enriching and isolation of Salmonella, will be related to a number of factors, including, food preparation procedures, the number of organisms present, sample handling after collection, and, your aseptic technique., FSIS published a laboratory guidebook containing the guidelines referenced here (MLG 4.09)., They include the necessary procedures for initial, enrichment, isolation, media testing, and immunological testing for identification. For this experiment, you will only complete the procedures that, deal with bacterial enrichment (increasing total, numbers of cells present for later testing) and, preliminary identification based on the use of, selective media and metabolic assays learned in, previous experiments., , FUR T HE R R E AD I N G, Refer to the section on food safety in your textbook for further information on the microbes, responsible for food spoilage. In your textbook’s, index, search under “Coliform,” “Salmonellosis,”, and “Spoilage.”, , AT T HE BE NCH, , Materials, Cultures, ❏❏ Sample of fresh ground meat, , Media, Per designated student group, ❏❏ 100 mL Modified Tryptone Soya Broth (mTSB), ❏❏ Brilliant green sulfa agar plate (BGS; contains, 0.1% sodium sulfapyridine), ❏❏ Double modified lysine iron agar plate, (DMLIA), ❏❏ Four tubes of triple sugar iron agar (TSI), ❏❏ Four tubes of lysine iron agar (LIA), ❏❏ Four tubes of Trypticase soy broth, , Equipment, ❏❏ Sterile spoon, ❏❏ Sterile plain, clear polypropylene bags, (ca. 24″ * 30< to 36″), ❏❏ Balance, ❏❏ Sterile loops, ❏❏ Pipettes, ❏❏ Vortex mixer, , 325
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Procedure Lab One:, Enrichment Procedure, , 3. Incubate at 35 { 2°C for 18 to 24 hours., 4. Select typical colonies., , Refer to Figure 46.1, which illustrates this, procedure., , Procedure Lab Three: Picking, Colonies from Plated Media, , 1. Using the provided sterile bag, weigh out 10 g, of the ground meat provided., 2. To the bag containing the meat sample, add 40, mL of Modified Tryptone Soya Broth., 3. Blend or hand-massage the bag contents until, the clumps are dispersed., 4. Incubate at 35 { 2°C for 20 to 24 hours., , 1. After the recommended incubation interval,, examine the selective-differential agar plates, for the presence of colonies meeting the, description for suspect Salmonella colonies., 2. Identify well-isolated colonies with the correct, morphological appearance., • BGS: Select colonies that are pink and, opaque with a smooth appearance and an, entire edge surrounded by a red color in the, Procedure Lab Two:, medium. On very crowded plates, look for, Selective Enrichment and, colonies that give a tan appearance against, a green background., Media Plating, • DMLIA: Select purple colonies with (H2S1. Streak the BGS and DMLIA plates using a loop, positive) or without (H2S-negative) black, of inoculum for each plate., centers. Since Salmonella typically decar2. Streak for isolation using the entire agar plate., boxylate lysine and ferment neither lactose nor sucrose, the color of the medium, reverts to purple., Check, 3. Choose at least one typical, when, complete, Stage, Procedures, isolated colony from any of, the plates., 10g of meat and 40mL of mTSB, 4. Transfer the chosen colony into, Enrichment, should be kneaded in a sealed bag, a TSB tube and vortex to mix., and incubated overnight at 355C, 5. Inoculate TSI and LIA slants, in tandem with a single colony, suspension by stabbing the, butts and streaking the slants, in one operation., Streak, Streak for, 6. Incubate at 35 { 2°C for, for isolation, isolation on BGS, 24 { 2 hours., Isolation, on DMLIA, and incubate, and incubate, ❏❏ If screw-cap tubes are used,, overnight at 355C, overnight at 355C, the caps must be loosened., , Identification, , Inoculate TSI, and LIA slants, and incubate, overnight at 355C, , Inoculate TSI, and LIA slants, and incubate, overnight at 355C, , Refer to Part 5 for further assays, to confirm Salmonella identification, , Figure 46.1 Preparation of a food sample for analysis, 326, , Experiment 46, , Procedure Lab, Four: Media, Screening, 1. E, xamine TSI and LIA slants as, a set., 2. Note the colors of butts and, slants, blackening of the media,, and for TSI slants the presence, of gas as indicated by gas pockets or cracking of the agar., 3. Note also the appearance of the, growth on the slants along the, line of streak.
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E XP E R IMENT, , 46, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Describe the growth characteristics within the overnight bag incubation. Was, there little or a high level of growth? Culture coloration?, , 2. Based on previous experiments, did the results of your TSI and LSI slants, indicate Salmonella growth?, , Review Questions, 1. Indicate some possible ways in which the foods tested may become, contaminated with a Salmonella species., , Experiment 46: Lab Report, , 327
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E XP E R IMENT, , 47, , Microbial Fermentation, , PA RT A, , are acids and minerals whose concentrations are, increased in the finished product and that are, responsible for the characteristic tastes and bouquets of different wines. For red wine, the crushed, grapes must be fermented with their skins to allow, extraction of their color into the juice. White wine, is produced from the juice of most different colored grapes without their skins., The commercial production of wine is a long, and exacting process. First, the grapes are crushed, or pressed to express the juice, which is called, must. Potassium metabisulfite is added to the, must to retard the growth of acetic acid bacteria,, molds, and wild yeast that are endogenous to, grapes in the vineyard. A wine-producing strain of, yeast, Saccharomyces cerevisiae var. ellipsoideus,, is used to inoculate the must, which is then, incubated for 3 to 5 days under aerobic conditions, at 21°C to 32°C. This is followed by an anaerobic incubation period. The wine is then aged for, 1 year to 5 years in aging tanks or wooden barrels., During this time, the wine is clarified of any, turbidity, thereby producing volatile esters that are, responsible for characteristic flavors. The clarified, product is then filtered, pasteurized at 60°C for, 30 minutes, and bottled., , Alcohol Fermentation, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Produce wine using the fermentative, activities of yeast cells., , Principle, Wine is a product of the natural fermentation of, the juices of grapes and other fruits, including, peaches, pears, plums, and apples, by the action of, yeast cells. This biochemical conversion of juice, to wine occurs when the yeast cells enzymatically, degrade the fruit sugars, fructose, and glucose,, first to acetaldehyde and then to alcohol, as illustrated in Figure 47.1., Grapes containing 20% to 30% sugar concentration will yield wines with an alcohol content of, approximately 10% to 15%. Also present in grapes, , CH2OH, O, , OH, H, , OH, H, , H, , OH, , O, Glycolytic, enzymes, , 2CH3, , C, , COOH, , H, , Decarboxylation, , 2CH3CHO, Acetaldehyde, , OH, , 4H+, 2CH3, , Glucose, , +, , Pyruvic acid, , reduction, CH2, , CO2, Carbon, dioxide, , OH, , Ethyl alcohol, , Figure 47.1 Biochemical pathway for alcohol production, , 329
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This experiment utilizes a modified method in, which white wine is produced from white grape, juice. You will examine the fermenting wine at, 1-week intervals during the incubation period for, the following:, 1. Total acidity (expressed as % tartaric acid): To, a 10-ml aliquot of the fermenting wine, add, 10 ml of distilled water and 5 drops of 1% phenolphthalein solution. Mix and titrate to the, first persistent pink color with 0.1N sodium, hydroxide. Calculate total acidity using the, following formula:, % tartaric acid =, , ml alkali * normality of alkali * 7.5, weight of sample in g*, , *1 ml = 1 g, 2. Volatile acidity (expressed as % acetic acid):, Following titration, calculate volatile acidity, using the following formula:, ml alkali * normality of alkali * 6.0, % acetic acid =, weight of sample in g*, , *1 ml = 1 g, 3. Alcohol (expressed as volume %): optional;, can be determined by means of an, ebulliometer., 4. Aroma: fruity, yeast-like, sweet, or none, 5. Clarity: clear or turbid, , FU RT HER R E ADING, Refer to the section on microbial metabolism in, your textbook for further information on the metabolic activities associated with fermentation in, microbial cells. In your textbook’s index, search, under “Fermentation,” “Saccharomyces,” and, “Anaerobic.”, , C L I N I C A L A P P L I C AT I O N, Drinking Wine Instead of Water for Better, Health, For thousands of years mankind has allowed, crushed fruits and boiled grains to ferment, creating wine. Wild yeasts and bacteria metabolize and, break down the inherent sugars in these liquids, and, the fermentation byproduct of alcohol kills all bacteria and protozoa present. Early civilizations drank, wine instead of water to protect against diseases., Poorer subjects and young children would drink, watered-down wine. By replacing water with wine, in their daily diet, early civilizations were able to, limit their exposure to pathogens., 330, , Experiment 47, , AT T HE BE NCH, , Materials, Cultures, ❏❏ 50 ml of white grape juice broth culture of, Saccharomyces cerevisiae var. ellipsoideus, incubated for 48 hours at 25°C, , Media, Per designated student group, ❏❏ 500 ml of pasteurized Welch’s® commercial, white grape juice, , Reagents, ❏❏ 1% phenolphthalein solution, ❏❏ 0.1N sodium hydroxide, ❏❏ Sucrose, , Equipment, ❏❏ 1-liter Erlenmeyer flask, ❏❏ One-holed rubber stopper containing a 2-inch, glass tube plugged with cotton, ❏❏ Pan balance, ❏❏ Spatula, ❏❏ Glassine paper, ❏❏ 10-ml graduated cylinder, ❏❏ Ebulliometer (optional), ❏❏ Burette or pipette for titration, , Procedure, 1. Pour 500 ml of the white grape juice into the, 1-liter Erlenmeyer flask. Add 20 g of sucrose and, the 50 ml of S. cerevisiae grape juice broth culture (10% starter culture). Close the flask with, the stopper containing a cotton-plugged air vent., 2. Add 20 g of sucrose to the fermenting wine on, days 2 and 4 during the incubation., 3. Incubate the fermenting wine for 21 days at 25°C., 4. Using uninoculated white grape juice:, a. Perform a titration to determine total acidity and volatile acidity., b. Note aroma and clarity., c. Determine volume % alcohol (optional)., 5. Record your results in the chart in the Lab, Report., 6. At 7-day intervals, using samples of the fermenting wine, repeat Steps 4a though 4c and record, your results in the Lab Report.
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PA RT B Lactic Acid Fermentation, , AT T HE B EN CH, , LEARNING OBJECTIVE, , Materials, , Once you have completed this experiment, you should be able to, , Cultures, , 1. Produce yogurt using the fermentative, activities of bacterial cells., , 24-hours old bacterial cultures (50 mL) of each of, the following:, ❏❏ Lactobacillus delbrueckii subsp. bulgaricus, ❏❏ Streptococcus thermophiles, , Media, , Principle, , Per designated student group, ❏❏ 400 ml of pasteurized heavy cream, , Yogurt is produced when bacterial species such as, Lactobacillus bulgarius and Streptococcus thermophiles consume the sugars found in dairy products and produce lactic acid. The primary sugar, utilized in this form of fermentation is the milk, sugar lactose, a disaccharide sugar derived from, galactose and glucose. Lactic acid fermentation, occurs when sugars are broken down during glycolysis into lactate and release energy that powers, the cell. Single sugar molecules are broken down, during glycolysis into two molecules of pyruvate., As seen in Figure 47.2, in an anaerobic environment the enzyme lactate dehydrogenase converts, pyruvate into lactic acid and allows for the oxidation of NADH back into NAD +. This oxidation step, frees up NAD + for the cell to continue glycolysis, in the absence of oxygen. A decrease in pH due to, the buildup of lactic acid causes the milk to clot,, or form a soft gel that is characteristic of yogurt., The fermentation of lactose also produces the flavor compounds that are characteristic of yogurt., , Equipment, Per designated student group, ❏❏ Four 400-mL stoppered graduated Erlenmeyer, flasks, ❏❏ Sterile 10-mL serological pipettes, ❏❏ Hot plates, ❏❏ Varying ranges of pH paper, ❏❏ Glass markers, , Procedure, 1. Aliquot 100 mL of heavy cream into four Erlenmeyer flasks labeled as follows:, a. Control (no bacteria), b. Lactobacillus, c. Streptococcus, d. Both, , CH2OH, O, , OH, H, , OH, , H, , H, , OH, , Glucose, , OH, , O, Glycolytic, enzymes, , 2CH3, , C, , COOH, , H, , Oxidation, , 2CH3CH(OH)COOH, Lactic Acid, , Pyruvic acid, , Figure 47.2 Biochemical pathway for lactic acid fermentation, , Experiment 47, , 331
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2. Measure the pH of untreated cream and, record., 3. Heat the cream on a hot plate to a pproximately, 45°C, with stoppers placed loosely. C, aution:, The high fat and high sugar content of the, cream will easily burn, so monitor the, temperature closely., , 332, , Experiment 47, , 4. After warming, remove flasks from the hot, plate and add 10 mL of bacterial cultures to the, appropriate flasks., 5. Incubate overnight at 42°C with loosened, stoppers., 6. Store flasks at 4°C for 3 days while recording, pH and cream consistency every 24 hours.
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E XP E R IMENT, , 47, , Name:, Date:, , Lab Report, , Section:, , Observations and Results for Alcohol Fermentation, FERMENTING WINE, Grape Juice, , 7 Days, , 14 Days, , 21 Days, , % Tartaric acid, % Acetic acid, Volume % alcohol, Aroma, Clarity, , Observations and Results for Lactic Acid Fermentation, Day, , Flask, , Ph, , Consistency, , Lactobacillus, Streptococcus, 0, Both, Control, Lactobacillus, Streptococcus, 1, Both, Control, Lactobacillus, Streptococcus, 2, Both, Control, Lactobacillus, Streptococcus, 3, Both, Control, , Experiment 47: Lab Report, , 333
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Review Questions, 1. What is the purpose of adding sulfite to the must?, , 2. Explain what occurs during the aging process in the commercial preparation of wine., , 3. What are the chemical end products of fermentation?, , 4., , Why is wine pasteurized? Would it be preferable to sterilize the wine? Explain., , 5., , What is the effect of lowered pH on the proteins found in dairy products during lactic acid, fermentation?, , 334, , Experiment 47: Lab Report
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PART 11, , Microbiology of Water, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Identify the types of microorganisms present in water., 2. Utilize laboratory methods to determine the potability of water using, standard qualitative and quantitative procedures., , Introduction, The importance of potable (drinking) water supplies cannot be overemphasized. With increasing, industrialization, water sources available for consumption and recreation have been adulterated, with both industrial waste and animal and human, wastes. As a result, water has become a formidable factor in disease transmission. Polluted waters, contain vast amounts of organic matter that serve, as excellent nutritional sources for the growth and, multiplication of microorganisms. The presence of, nonpathogenic organisms is not of major concern,, but intestinal contaminants of fecal origin are, important. These pathogens are responsible for, intestinal infections such as bacillary dysentery,, typhoid fever, cholera, and paratyphoid fever., The World Health Organization (WHO) estimates that 1.7 million deaths per year result from, unsafe water supplies. Most of these are from diarrheal diseases, and 90% of these deaths are of children living in developing countries where sanitary, facilities and potable water are at a minimum. The, WHO indicates that about 3.4 million deaths annually are caused by dangerous waterborne enteric, bacterial pathogens such as Shigella dysenteriae,, Campylobacter jejuni, Salmonella typhi, and Vibrio cholerae., , In addition to bacterial infections, unsafe, water supplies are responsible for numerous parasitological infections, including helminth diseases, such as schistosomiasis and especially guinea, worm (Dracunculus medinensis), which infects, about 200 million people worldwide each year., Intestinal, hepatic, and pulmonary flukes, including Fasciolopsis buski, Clonorchis sinensis,, and Paragonimus westermani, are responsible, for human infection and are all associated with, unsafe water and sanitation. The parasitic protozoa Entamoeba histolytica, Giardia intestinalis, (formerly called G. lamblia), and Balantidium, coli are just a few of the protozoa responsible for, major diarrheal disease in humans., Although water-borne infections occur in the, United States, their incidence in comparison with, the rest of the world is much lower, and they occur, sporadically. This can be attributed to the diligent, attention given to our water supplies and sewage, disposal systems., Analysis of water samples on a routine basis, would not be possible if each pathogen required, detection. Therefore, water is examined to detect, Escherichia coli, the bacterium that indicates, fecal pollution. Since E. coli is always present in, the human intestine, its presence in water alerts, public health officials to the possible presence, , 335
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of other human or animal intestinal pathogens., However, in the tropics and subtropics it is not, considered a reliable indicator of fecal pollution,, because the soil in these regions naturally contains, high levels of E. coli. Therefore, E. coli is present, in the water anytime there is surface runoff. Both, qualitative and quantitative methods are used to, determine the sanitary condition of water., , F U RT H E R RE A D I N G, Refer to the section on environmental microbiology in your textbook for further information on, the enteric bacteria that may be found in waterways. In your textbook’s index, search under, “Enteric,” “Coliforms,” and “Potable water.”, , C ASE STUDY, WHAT ARE YOU DRINKING?, It is your second day as a new team member who, specializes in identification of the source of waterborne epidemics for the World Health Organization, (WHO). Potable or drinkable water is one of the, scarcest necessities in many third world countries., Doctors in the country where you are currently, stationed have tentatively identified a potential, cholera outbreak. As the number of patients exhibiting cholera-like symptoms increases, it is now, up to your team to identify the water source that, all the patients may have in common and to begin, testing for the presence of coliform bacteria., , 336, , Part 11, , Questions to Consider:, 1. Why are you testing for a general list of bacteria that fall under “coliform?” Why is your, team not testing just for Vibrio cholera if the, doctors are already thinking that is what may, be causing the disease?, 2. Are there other enteric-associated bacteria, that may cause cholera-like symptoms during, an infection?
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Standard Qualitative Analysis, of Water, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Determine the presence of coliform, bacteria in a water sample., 2. Obtain an index indicating the possible, number of organisms present in the, sample under analysis., 3. Confirm the presence of coliform bacteria, in a water sample for which the presumptive test was positive., 4. Confirm a suspicious or doubtful result, from a positive presumptive coliform test., , Principle, The three basic tests to detect coliform bacteria in, water are presumptive, confirmed, and completed, (Figure 48.1). The tests are performed sequentially, on each sample under analysis. They detect the, presence of coliform bacteria (indicators of fecal, contamination), through the fermentation of lactose that will produce acid and gas that is detectable following a 24-hour incubation period at 37°C., , The Presumptive Test, The presumptive test is specific for detection of, coliform bacteria. Measured aliquots of the water, to be tested are added to a lactose fermentation, broth containing an inverted gas vial. Because, these bacteria are capable of using lactose as a, carbon source (the other enteric organisms are, not), their detection is facilitated by the use of this, medium. In this experiment, you will use lactose, fermentation broth containing an inverted Durham, tube for gas collection. Tubes of this lactose, medium are inoculated with 10-ml, 1-ml, and 0.1-ml, aliquots of the water sample. The series consists of, at least three groups, each composed of five tubes, of the specified medium. The tubes in each group, are then inoculated with the designated volume of, , E XP E R IMENT, , 48, , the water sample, as described under “Procedure:, Lab One.” The greater the number of tubes per, group, the greater the sensitivity of the test. Development of gas in any of the tubes is presumptive, evidence of the presence of coliform bacteria in, the sample. The presumptive test also enables the, microbiologist to obtain some idea of the number, of coliform organisms present by means of the, most probable number (MPN) test. The MPN is, estimated by determining the number of tubes in, each group that show gas following the incubation, period (Table 48.1 on page 339)., , The Confirmed Test, The presence of a positive or doubtful presumptive, test immediately suggests that the water sample is, nonpotable. Confirmation of these results is necessary because positive presumptive tests may be the, result of organisms of noncoliform origin that are, not recognized as indicators of fecal pollution., The confirmed test requires that selective, and differential media (e.g., eosin–methylene blue, (EMB) or Endo agar) be streaked from a positive, lactose broth tube obtained from the presumptive test. The nature of the differential and selective media was discussed in Experiment 14 but, is reviewed briefly here. Eosin–methylene blue, contains the dye methylene blue, which inhibits the, growth of gram-positive organisms. In the presence, of an acid environment, EMB forms a complex that, precipitates out onto the coliform colonies, producing dark centers and a green metallic sheen. The, reaction is characteristic for Escherichia coli, the, major indicator of fecal pollution. Endo agar is a, nutrient medium containing the dye fuchsin, which, is present in the decolorized state. In the presence, of acid produced by the coliform bacteria, fuchsin, forms a dark pink complex that turns the E. coli, colonies and the surrounding medium pink., , The Completed Test, The completed test is the final analysis of the, water sample. It is used to examine the coliform, colonies that appeared on the EMB or Endo agar, plates used in the confirmed test. An isolated, colony is picked up from the confirmatory test, 337
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PROCEDURE, , Water, sample, , 10, , 10, , Presumptive, Test, , 10, , 10, , 10, , 1.0, , 1.0, , 1.0, , 1.0, , Double-strength, lactose broth, , 1.0, , 0.1, , 0.1, , 0.1, , 0.1, , 0.1, , Single-strength, lactose broth, Incubate 48 hr at 375C., , Positive result:, Gas in fermentation tube, , Negative result:, No gas in fermentation tube, , Confirmed, Test, , 24 hr at 375C., Streak EMB agar plate from, positive tube., , Positive, confirmed test:, Typical coliform, colonies, water, nonpotable, Incubate 24 hr, at 375C., , Completed, Test, Lactose broth, If gas is produced, , Figure 48.1 Standard method for bacteriological water analysis, 338, , Negative, confirmed test:, No coliform, colonies, water, potable, , Incubate, , Experiment 48, , Nutrient agar slant, Prepare Gram stain from slant. Gram-negative, short bacilli indicate positive completed test.
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TABLE 48.1, , The MPN Index per 100 ml for Combinations of Positive and Negative, Presumptive Test Results When Five 10-ml, Five 1-ml, and Five 0.1-ml, Portions of Sample Are Used, , NUMBER OF TUBES WITH POSITIVE RESULTS, FIVE OF FIVE OF FIVE OF, MPN, 10 ML, 1 ML, 0.1 ML INDEX PER, EACH, EACH, EACH, 100 ML, 0, , 0, , 0, , <2, , NUMBER OF TUBES WITH POSITIVE RESULTS, , 95%, CONFIDENCE, LIMITS, LOWER UPPER, 0, , 6, , FIVE OF FIVE OF FIVE OF, MPN, 10 ML, 1 ML, 0.1 ML INDEX PER, EACH, EACH, EACH, 100 ML, 4, , 2, , 1, , 26, , 95%, CONFIDENCE, LIMITS, LOWER UPPER, 7, , 67, , 0, , 0, , 1, , 2, , <0.5, , 7, , 4, , 3, , 0, , 27, , 9, , 78, , 0, , 1, , 0, , 2, , <0.5, , 7, , 4, , 3, , 1, , 33, , 9, , 78, , 0, , 2, , 0, , 4, , <0.5, , 11, , 4, , 4, , 0, , 34, , 11, , 93, , 1, , 0, , 0, , 2, , 0.1, , 10, , 5, , 0, , 0, , 23, , 7, , 70, , 1, , 0, , 1, , 4, , 0.7, , 10, , 5, , 0, , 1, , 31, , 11, , 89, , 1, , 1, , 0, , 4, , 0.7, , 12, , 5, , 0, , 2, , 43, , 14, , 100, , 1, , 1, , 1, , 6, , 1.8, , 15, , 5, , 1, , 0, , 33, , 10, , 100, , 1, , 2, , 0, , 6, , 1.8, , 15, , 5, , 1, , 1, , 46, , 14, , 120, , 2, , 0, , 0, , 5, , <0.5, , 13, , 5, , 1, , 2, , 63, , 22, , 150, , 2, , 0, , 1, , 7, , 1, , 17, , 5, , 2, , 0, , 49, , 15, , 150, , 2, , 1, , 0, , 7, , 1, , 17, , 5, , 2, , 1, , 70, , 22, , 170, , 2, , 1, , 1, , 9, , 2, , 21, , 5, , 2, , 2, , 94, , 34, , 230, , 2, , 2, , 0, , 9, , 2, , 21, , 5, , 3, , 0, , 79, , 22, , 220, , 2, , 3, , 0, , 12, , 3, , 28, , 5, , 3, , 1, , 110, , 34, , 250, , 3, , 0, , 0, , 8, , 2, , 22, , 5, , 3, , 2, , 140, , 52, , 400, , 3, , 0, , 1, , 11, , 4, , 23, , 5, , 3, , 3, , 180, , 70, , 400, , 3, , 1, , 0, , 11, , 5, , 35, , 5, , 4, , 0, , 130, , 36, , 400, , 3, , 1, , 1, , 14, , 6, , 36, , 5, , 4, , 1, , 170, , 58, , 400, , 3, , 2, , 0, , 14, , 6, , 36, , 5, , 4, , 2, , 220, , 70, , 440, , 3, , 2, , 1, , 17, , 7, , 40, , 5, , 4, , 3, , 280, , 100, , 710, , 3, , 3, , 0, , 17, , 7, , 40, , 5, , 4, , 4, , 350, , 100, , 710, , 4, , 0, , 0, , 13, , 4, , 35, , 5, , 5, , 0, , 240, , 70, , 710, , 4, , 0, , 1, , 17, , 6, , 36, , 5, , 5, , 1, , 350, , 100, , 1100, , 4, , 1, , 0, , 17, , 6, , 40, , 5, , 5, , 2, , 540, , 150, , 1700, , 4, , 1, , 1, , 21, , 7, , 42, , 5, , 5, , 3, , 920, , 220, , 2600, , 4, , 1, , 2, , 26, , 10, , 70, , 5, , 5, , 4, , 1600, , 400, , 4600, , 4, , 2, , 0, , 22, , 7, , 50, , 5, , 5, , 5, , ≥2400, , 700, , –––, , th, , Sources: pp 9–51, Standard Methods for the Examination of Water and Wastewater, 20 Edition (1998). M. J. Taras, A. E. Greenberg, R. D. Hoak,, and M. C. Rand, eds. American Public Health Association, Washington, D.C. Copyright 1998, American Public Health Association, and Bacteriological Analytical Manual (BAM), 8th Edition, Food and Drug Administration, 1998., , Experiment 48, , 339
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plate, inoculated into a tube of lactose broth, and, streaked on a nutrient agar slant to perform a, Gram stain. Following inoculation and incubation,, tubes showing acid and gas in the lactose broth, and presence of gram-negative bacilli on microscopic examination are further confirmation of, the presence of E. coli, and they are indicative of a, positive completed test., , Environmental Protection Agency, This step-wise method to determine the presence, of coliform bacteria, indicating fecal contamination of sludge or treated water, is similar to one of, the approved methods published by the U.S. Environmental Protection Agency (EPA). The EPA has, the government mandate to protect the nation’s, waterways and terrestrial environments from, human contamination or damage. The published, “Method 1681: Fecal Coliforms in Sewage Sludge, (Biosolids) by Multiple-Tube Fermentation using, A-1 Medium” utilizes the process of presumptive, tests followed by confirmed tests to determine, the amount of fecal contamination in collected, samples., , FU RT HER R E ADING, Refer to the section on environmental microbiology in your textbook for further information on, the enteric bacteria that may be found in waterways. In your textbook’s index, search under, “Enteric,” “Coliforms,” and “Potable water.”, , C L I N I C A L A P P L I C AT I O N, Testing for Safe Water, Water used for human consumption and recreational use is routinely analyzed for safety. Water, sources are regularly tested for the presence of, Escherichia coli to determine the quality and safety, of municipal water supplies. Several testing methods are available for this purpose, including most, probable numbers (MPN), ATP testing, membrane, filtration, and the use of pour plates., , 340, , Experiment 48, , AT T HE BE NCH, , Materials, Cultures, Lab One, ❏❏ Water samples from sewage plant, pond, and, tap, Lab Two, ❏❏ One 24-hour-old positive lactose broth culture, from each of the three series from the presumptive test, Lab Three, ❏❏ One 24-hour, coliform-positive EMB or Endo, agar culture from each of the three series of, the confirmed test, , Media, Lab One (per designated student group), ❏❏ 15 double-strength lactose fermentation broths, (LB2X), ❏❏ 30 single-strength lactose fermentation broths, (LB1X)., Lab Two (three each per designated student group), ❏❏ Eosin–methylene blue agar plates or Endo, agar plates, Lab Three (three each per designated student group), ❏❏ Nutrient agar slants, ❏❏ Lactose fermentation broths, , Reagents, Lab Three, ❏❏ Crystal violet, ❏❏ Gram’s iodine, ❏❏ 95% ethyl alcohol, ❏❏ Safranin, , Equipment, Lab One, ❏❏ Microincinerator or Bunsen burner, ❏❏ 45 test tubes with Durham tubes, ❏❏ Test tube rack, ❏❏ Sterile 10-ml pipettes, ❏❏ Sterile 1-ml pipettes, ❏❏ Sterile 0.1-ml pipettes, ❏❏ Mechanical pipetting device, ❏❏ Glassware marking pencil
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Lab Two, ❏❏ Microincinerator or Bunsen burner, ❏❏ Glassware marking pencil, ❏❏ Inoculating loop, Lab Three, ❏❏ Microincinerator or Bunsen burner, ❏❏ Staining tray, ❏❏ Inoculating loop, ❏❏ Lens paper, ❏❏ Bibulous paper, ❏❏ Microscope, ❏❏ Glassware marking pencil, , Procedure Lab One, Presumptive Test, Exercise care in handling the sewage waste, water sample, because enteric pathogens may be, present., , 1. Set up three separate series consisting of three, groups each, a total of 15 tubes per series, in, a test tube rack; for each tube, label the water, source and volume of sample inoculated as, illustrated., 5 tubes of LB2X-10 ml, Series 1: Sewage water, , 5 tubes of LB1X-1 ml, , 6. Repeat Steps 2 through 5 for the tap and pond, water samples., 7. Incubate all tubes for 48 hours at 37°C., , Procedure Lab Two, Presumptive Test, 1. Examine the tubes from your presumptive, test after 24 and 48 hours of incubation. Your, results are positive if the Durham tube fills, 10% or more with gas in 24 hours, doubtful if, gas develops in the tube after 48 hours, and, negative if there is no gas in the tube after, 48 hours. Refer to Figure 48.2 for a summary, of possible MPN presumptive test results., Figure 48.3 shows actual results from an MPN, presumptive test for a water sample. Record, your results in the Lab Report., 2. Determine the MPN using Table 47.1, and, record your results in the Lab Report., , Confirmed Test, 1. Label the covers of the three EMB plates or, the three Endo agar plates with the source of, the water sample (sewage, pond, and tap)., 2. Using a positive 24-hour lactose broth culture, from the sewage water series from the presumptive test, streak the surface of one EMB, , 5 tubes of LB1X-0.1 ml, 5 tubes of LB2X-10 ml, Series 2: Pond water, , 5 tubes of LB1X-1 ml, 5 tubes of LB1X-0.1 ml, 5 tubes of LB2X-10 ml, , Series 3: Tap water, , 5 tubes of LB1X-1 ml, 5 tubes of LB1X-0.1 ml, , 2. Mix the sewage plant water sample by shaking, thoroughly., 3. Flame bottle and then, using a 10-ml pipette,, transfer 10-ml aliquots of water sample to the, five tubes labeled LB2X-10 ml., 4. Flame bottle and then, using a 1-ml pipette,, transfer 1-ml aliquots of water sample to the, five tubes labeled LB1X-1 ml., 5. Flame bottle and then, using a 0.1-ml pipette,, transfer 0.1-ml aliquots of water sample to the, five tubes labeled LB1X-0.1 ml., , (a), , (b), , (c), , (d), , (e), , Figure 48.2 Possible MPN presumptive test, results. (a) Uninoculated control tube, (b, c), inoculated tubes with no change, (d) inoculated, tube with acid production only, and (e) inoculated, tube with acid and gas production—the only, positive result of the five tubes, , Experiment 48, , 341
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Procedure Lab Three, Confirmed Test, , (a) Positive results (acid and gas) in five 10-ml tubes, , (b) Positive results (acid and gas) in five 1-ml tubes, , (c) Negative results (acid only) in five 0.1-ml tubes, , Figure 48.3 MPN presumptive test results for a, water sample. The results of this test (5 positive, 5, positive, and 5 negative) indicate 240 coliforms per, 100 ml of water. (See Table 47.1.) This represents, a positive presumptive test for the presence of, coliforms in the tested water sample, , or one Endo agar plate, as described in Experiment 3, to obtain discrete colonies., 3. Repeat Step 2 using the positive lactose broth, cultures from the pond and tap water series, from the presumptive test to inoculate the, remaining plates., 4. Incubate all plate cultures in an inverted position for 24 hours at 37°C., , 342, , Experiment 48, , 1. Examine all the plates from your confirmed, test for the presence or absence of E. coli, colonies. (Refer to the description of the confirmed test in the experiment introduction,, and see Figure 13.2 for an illustration of E. coli, growth on EMB agar.) Record your results in, the Lab Report., 2. Based on your results, determine whether, each of the samples is potable or nonpotable., The presence of E. coli is a positive confirmed, test, indicating that the water is nonpotable., The absence of E. coli is a negative test, indicating that the water is not contaminated with, fecal wastes and is therefore potable. Record, your results in the Lab Report., , Completed Test, 1. Label each tube of nutrient agar slants and lactose fermentation broths with the source of its, water sample., 2. Inoculate one lactose broth and one nutrient, agar slant with a positive isolated E. coli colony obtained from each of the experimental, water samples during the confirmed test., 3. Incubate all tubes for 24 hours at 37°C., , Procedure Lab Four, Completed Test, 1. Examine all lactose fermentation broth cultures for the presence or absence of acid and, gas. Record your results in the Lab Report., 2. Prepare a Gram stain, using the nutrient agar, slant cultures of the organisms that showed, a positive result in the lactose fermentation, broth. (Refer to Experiment 10 for the staining, procedure.), 3. Examine the slides microscopically for the, presence of gram-negative short bacilli, which, are indicative of E. coli and thus nonpotable, water. In the Lab Report, record your results, for Gram stain reaction and morphology of the, cells.
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E XP E R IMENT, , 48, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Presumptive Test, Using Table 48.1, determine and record the MPN., Example: If gas appeared in all five tubes labeled LB2X-10, in two of the tubes, labeled LB1X-1, and in one labeled LB1X-0.1, the series would be read as 5-2-1., From the MPN table, such a reading would indicate approximately 70 microorganisms per 100 ml of water, with a 95% probability that between 22 and 170, microorganisms are present., GAS, , Water, Sample, , 1, , LB2X-10, , LB1X-1, , LB1X-0.1, , Tube, , Tube, , Tube, , 2, , 3, , 4, , 5, , 1, , 2, , 3 4, , 5, , 1, , 2, , 3, , 4, , 5 Reading, , MPN, , 95%, Probability, Range, , Sewage, Pond, Tap, , Confirmed Test, COLIFORMS, Water Sample, , EMB Plate, , Endo Agar Plate, , Potable, , Nonpotable, , Sewage, Pond, Tap, , Completed Test, GRAM STAIN, , Water Source, , Lactose Broth, A/G ( + ) or, ( − ), , Reaction/, Morphology, , POTABILITY, , Potable, , Nonpotable, , Sewage, Pond, Tap, , Experiment 48: Lab Report, , 343
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Review Questions, 1. What is the rationale for selecting E. coli as the indicator of water potability?, , 2. Why is this procedure qualitative rather than quantitative?, , 3. Explain why it is of prime importance to analyze water supplies that serve, industrialized communities., , 4., , 344, , Account for the presence of microorganisms in natural bodies of, water and sewage systems. What is their function? Explain., , Experiment 48: Lab Report
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Quantitative Analysis of Water:, Membrane Filter Method, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Determine the quality of water samples, using the membrane filter method., , Principle, Bacteria-tight membrane filters capable of, retaining microorganisms larger than 0.45 micrometer (mm) are frequently used for analysis of, water. These filters offer several advantages over, the conventional, multiple-tube method of water, analysis: (1) Results are available in a shorter, period of time, (2) larger volumes of sample can, be processed, and (3) because of the high accuracy of this method, the results are readily reproducible. A disadvantage involves the processing of, turbid specimens that contain large quantities of, suspended materials; particulate matter clogs the, pores and inhibits passage of the specific volume, of water., A water sample is passed through a sterile, membrane filter that is housed in a special filter, apparatus contained in a suction flask. Following, filtration, the filter disc that contains the trapped, microorganisms is aseptically transferred to a, sterile Petri dish containing an absorbent pad saturated with a selective, differential liquid medium., Following incubation, the colonies present on the, filter are counted with the aid of a microscope., This experiment is used to analyze a series, of dilutions of water samples collected upstream, and downstream from an outlet of a sewage treatment plant. EPA-approved guidelines for the, determination of fecal contaminating organisms, (EPA Method 1103.1), similar to what are seen in, this experiment, are routinely utilized worldwide, to examine water samples before treated water is, released into a nation’s waterways. A total count, of coliform bacteria determines the potability of, the water sources. Also, the types of fecal pollution, if any, are established by means of a fecal, coliform count, indicative of human pollution,, , E XP E R IMENT, , 49, , and a fecal streptococcal count, indicative of pollution from other animal origins. The ratio of the, fecal coliforms to fecal streptococci per milliliter, of sample is interpreted as follows: Between 2 and, 4 indicates human and animal pollution; 74 indicates human pollution; and 60.7 indicates poultry, and livestock pollution., , F U RT H E R RE A D I N G, Refer to the section on environmental microbiology in your textbook for further information on, the enteric bacteria that may be found in waterways. In your textbook’s index, search under, “Enteric,” “Coliforms,” and “Potable water.”, , C L I N I C A L A P P L I C AT I O N, Rapid Water Analysis, In the late 1950s, the membrane filter method was, introduced as an alternative to the most probable, number (MPN) method. Microbiological analysis of, water by the membrane filter procedure is a rapid, method that isolates discrete bacteria that are able, to be accurately counted, whereas the MPN method, only allows for the approximate determination of the, number of organisms and does not separate species, without further testing., , AT T HE BE NCH, , Materials, Cultures, Water samples collected near the outlet of, a sewage treatment plant, ❏❏ Upstream (labeled U), ❏❏ Downstream (labeled D), , 345
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Media, Per designated student group for analysis of one, water sample, ❏❏ One 20-ml tube of m-Endo broth, ❏❏ One 20-ml tube of m-FC broth, ❏❏ One 20-ml tube of KF broth, ❏❏ Four 90-ml sterile water blanks, ❏❏ One 300-ml flask of sterile water, , Equipment, ❏❏ Sterile membrane filtration apparatus, (e.g., Millipore®; Pall® Gelman; sterile, plastic,, disposable membrane filters), ❏❏ 1-liter suction flask, ❏❏ 15 sterile membrane filters and absorbent, pads, ❏❏ 15 sterile 50-mm Petri dishes, ❏❏ 12 10-ml pipettes, ❏❏ Mechanical pipetting device, ❏❏ Small beaker of 95% alcohol, ❏❏ Membrane forceps, ❏❏ Waterproof tape, ❏❏ Watertight plastic bags, ❏❏ 44.5°C waterbath, ❏❏ Dissecting microscope, ❏❏ Glassware marking pencil, , Procedure Lab One, The following instructions are for analysis of one, of the provided water samples using the Millipore, system. Different samples may be assigned to individual groups., Use disposable gloves when handling the, water samples in this experiment., , 1. Label the four 90-ml water blanks with the, source of the water sample and dilution, (10-1, 10-2, 10-3, and 10-4)., 2. Using 10-ml pipettes, aseptically perform a, 10-fold serial dilution of the assigned undiluted water sample, using the four 90-ml water, blanks to effect the 10-1, 10-2, 10-3, and 10-4, dilutions., 3. Arrange the 15 Petri dishes into three sets of, five plates. Label each set as follows:, a. For total coliform count (TCC) and, dilutions (undiluted, 10-1, 10-2, 10-3,, and 10-4), , 346, , Experiment 49, , b. For fecal coliform count (FCC) and dilutions as in Step 3a, c. For fecal streptococcal count (FSC) and, dilutions as in Step 3a, , Membrane Filter Technique, Refer to Figure 49.1 as you read the instructions, following., 1. Using sterile forceps dipped in 95% alcohol, and flamed, add a sterile absorbent pad to all, Petri dishes., 2. With sterile 10-ml pipettes, aseptically add the, following:, a. To each pad in the plates labeled TCC, 2 ml, of m-Endo broth, b. To each pad in the plates labeled FCC, 2 ml, of m-FC broth, c. To each pad in the plates labeled FSC, 2 ml, of KF broth, 3. Aseptically assemble the sterile paperwrapped membrane filter unit as follows:, a. Unwrap and insert the sintered glass-filter, base into the neck of a 1-liter side-arm suction flask., b. With sterile forceps, place a sterile membrane filter disc, grid side up, on the sintered glass platform., c. Unwrap and carefully place the funnel, section of the apparatus on top of the filter disc.Using the filter clamp, secure the, funnel to the filter base., d. Attach a rubber hose from the side-arm on, the vacuum flask to a vacuum source., 4. Using the highest sample dilution (10-4) and, a pipette, place 20 ml of the dilution into the, funnel and start the vacuum., a. When the entire sample has been filtered,, wash the inner surface of the funnel with 20, ml of sterile water., 5. Disconnect the vacuum, unclamp the filter, assembly, and with sterile forceps, remove the, membrane filter., 6. Place the filter on the medium-saturated pad, in the Petri dish labeled TCC, 10-4., 7. Aseptically place a new membrane on the, platform, reassemble the filtration apparatus,, and repeat Steps 4 through 6 twice, adding the, filter discs to the 10-4 dilution plates labeled, FCC and FSC.
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PROCEDURE, , 1 Aseptically place an absorbent pad in a, 50-mm petri dish., , 2 Saturate the absorbent pad with the specified, selective broth medium., , Funnel, , Membrane filter, Cotton plug, Base, , Fused, porous glass platform, , Rubber, , Holding clamp, , To vacuum, Sterile flask, , 3 Assemble the filter apparatus and insert, membrane filter., , 4 Pour test sample into funnel, filter under vacuum,, and rinse with sterile water., , 5 Aseptically remove filter., , 6 Place filter in Petri dish on top of medium-saturated, pad and incubate., , Figure 49.1 Membrane filter technique, , Experiment 49, , 347
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8. Repeat Steps 4a through 7, using 20 ml of the, 10-3, 10-2, and 10-1 dilutions and the undiluted, samples., 9. Incubate the plates in an inverted position as, follows:, a. TCC and FSC plates for 24 hours at 37°C, b. FCC plates sealed with waterproof tape and, placed in a weighted watertight plastic bag,, which is then submerged in a 44.5°C waterbath for 24 hours, , Procedure Lab Two, 1. Using sterile membrane forceps, remove the, filter discs from the Petri dishes and allow to, dry on absorbent paper for 1 hour., 2. Using membrane forceps, place each dry filter, disc into its Petri dish cover. Keep the discs, within the covers at all times for further, observation., 3. Examine all filter discs under a dissecting, microscope. Refer to Figure 49.2, which shows, colonies developing on the membrane filter., Perform colony counts on each set of discs as, follows:, , Figure 49.2 Development of colonies on a, membrane filter following incubation, , 348, , Experiment 49, , a. TCC: Count colonies on m-Endo agar, that present a golden metallic sheen (performed on a disc showing 20 to 80 of these, colonies)., b. FCC: Count colonies on m-FC agar that are, blue (performed on a disc showing 20 to 60, of these colonies)., c. FSC: Count colonies on KF agar that are, pink to red (performed on a disc showing, 20 to 100 of these colonies)., Dilution samples that show fewer colonies, than indicated are designated as TFTC, and, those showing a greater number of colonies, are designated as TNTC., 4. For each of the three counts, determine the, number of fecal organisms present in 100 ml of, the water sample, using the following formula:, colony count * dilution factor, * 100, ml of sample used, 5. Record your results in the Lab Report.
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EXPERIMENT, , 49, , Name:, , Lab Report, , Section:, , Date:, , Observations and Results, UPSTREAM WATER, TCC, Dilution, , FCC, Cells/100, ml, , Count, , FSC, Cells/100, ml, , Count, , Cells/100, ml, , Count, , Undiluted, 10-1, 10-2, 10-3, 10-4, , DOWNSTREAM WATER, TCC, Dilution, , Count, , FCC, Cells/100, mL, , Count, , FSC, Cells/100, ml, , Count, , Cells/100, ml, , Undiluted, 10-1, 10-2, 10-3, 10-4, , Determine the fecal coliform–to–fecal streptococcal (FC:FS) ratio. Record your results, in the chart below., UPSTREAM WATER, , DOWNSTREAM WATER, , Cells/ml*, Dilution, , FCC, , Cells/ml*, FSC, , FC:FS Ratio, , FCC, , FSC, , FC:FS RatiO, , Undiluted, 1021, 1022, 1023, 1024, *Cells/ml =, , Cells/100 ml, 100, , Experiment 49: Lab Report, , 349
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Based on your FC:FS ratio, indicate the type of fecal pollution, if any, in the, two samples:, a. Upstream water sample:, , b. Downstream water sample:, , Review Questions, 1. What are the advantages of the membrane filter method in the analysis of, water samples?, , 2. What are the disadvantages of the membrane filter method?, , 3. What is the purpose of determining the FC:FS ratio?, , 4. Cite some other microbiological applications of the membrane filter technique in environmental studies., , 350, , Experiment 49: Lab Report
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PART 12, , Microbiology of Soil, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Understand the characteristics and activities of soil microorganisms., 2. Enumerate soil microorganisms., 3. Demonstrate the ability of some soil microorganisms to produce antibiotics., 4. Demonstrate the use of enrichment cultures for the isolation of specific soil, microorganisms., , Introduction, Soil is often thought of as an inert substance by, the average layperson. However, contrary to this, belief, it serves as a repository for many life forms,, including a huge and diverse microbial population., The beneficial activities of these soil inhabitants, far outweigh their detrimental effects., Life on this planet could not be sustained, in the absence of microorganisms that inhabit, the soil. This flora is essential for degradation, of organic matter deposited in the soil, such as, dead plant and animal tissues and animal wastes., Hydrolysis of these macromolecules by microbial, enzymes supplies and replenishes the soil with, basic elemental nutrients. By means of enzymatic, transformations, plants assimilate these nutrients, into organic compounds essential for their growth, and reproduction. In turn, these plants serve as, a source of nutrition for animals and man. Thus,, many soil microorganisms play a vital role in a, number of elemental cycles, such as the nitrogen, cycle, the carbon cycle, and the sulfur cycle., , Nitrogen Cycle, The nitrogen cycle is concerned with the enzymatic conversion of complex nitrogenous compounds in the soil and atmosphere into nitrogen, , compounds that plants are able to use for the, synthesis of essential macromolecules, including, nucleic acids, amino acids, and proteins. The four, distinct phases in this cycle are as follows:, 1. Ammonification: Soil microorganisms, sequentially degrade nitrogenous organic, compounds derived from dead plants and, animals deposited in the soil. The degraded, nitrogenous organic compounds are converted, to inorganic nitrogen compounds and then to, ammonia., 2. Nitrification: In this two-step process, (1), ammonia is oxidized to nitrite ions (NO2-), by an aerobic species of Nitrosomonas, and, then (2) nitrites are converted to nitrate ions, (NO3-) by another aerobic species, Nitrobacter. Nitrates are released into the soil and are, assimilated as a nutritional source by plants., 3. Denitrification: Nitrates (NO3-) that are not, used by plants are reduced to gaseous nitrogen, (N2 c ) and are liberated back into the atmosphere by certain groups of microorganisms., 4. Nitrogen fixation: This vital process involves, the chemical combination of gaseous nitrogen, (N2 c ) with other elements to form fixed nitrogen (nitrogen-containing compounds), which, is useful for plant growth. The two types of, , 351
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microorganisms involved in this process are, free-living and symbiotic. Free-living microorganisms include Azotobacter, Pseudomonas,, Clostridium, and Bacillus, as well as some, species of yeast. Symbiotic microorganisms,, such as Rhizobium, grow in tumor-like nodules in the roots of leguminous plants, and use, nutrients in the plant sap to fix gaseous nitrogen as ammonia for its subsequent assimilation into plant proteins. Animals then consume, the leguminous plants and convert plant protein to animal protein, completing the process., The nitrogen cycle is shown in Figure P12.1., , Carbon Cycle, Carbon dioxide is the major carbon source for, the synthesis of organic compounds. The carbon, cycle is basically represented by the following two, steps:, 1. Oxidation of organic compounds to carbon, dioxide with the production of energy and, heat by heterotrophs, 2. Fixation of carbon dioxide into organic compounds by green plants and some bacteria, the, autotrophic soil flora, , Ingested, by animals, , Animals, protein and, nucleic acid, synthesis, , Degradation by, microorganisms, , Symbiotic, Plants, and free-living, protein, organisms, Ni, synthesis, t, (A fix rog, zo at en, to ion, ba, cte, Nitrate, Atmospheric, r), utilization, nitrogen, (N2), n, catio, itrifi y, n, e, NO3– D by manms, nis, orga, , Amino acids, , Ammonification, (Pseudomonas, and Bacillus), , NH3, Nitrification, (Nitrosomonas), , Nitrification, (Nitrobacter), NO2–, , Figure P12.1 The nitrogen cycle, , 352, , Part 12, , Sulfur Cycle, Elemental sulfur and proteins cannot be utilized, by plants for growth. They must first undergo, enzymatic conversions into inorganic sulfur-containing compounds. The basic steps in the sulfur, cycle are the following:, 1. Degradation of proteins into hydrogen sulfide, (H2S) by many heterotrophic microorganisms, 2. Oxidation of H2S to sulfur (S) by a number of, bacterial genera, such as Beggiatoa, 3. Oxidation of sulfur to utilizable sulfate (SO24 ), by several chemoautotrophic genera, such as, Thiobacillus, Some soil microorganisms also play a role in, the enzymatic transformation of other elements,, such as phosphorus, iron, potassium, zinc, manganese, and selenium. These biochemical changes, make the minerals available to plants in a soluble, form., Many members of the soil flora, because of, their fermentative and synthetic capabilities, play, an important role in the synthesis of a variety of, industrial products:, 1. Food. Penicillium spp. are used in the production of cheeses, including Camembert,, Roquefort, and Brie., 2. Beverages. Saccharomyces spp. are utilized, in the wine, beer, and ale industries., 3. Vitamins. Eremothecium ashbyii and Pseudomonas denitrificans synthesize riboflavin, (vitamin B2) and cobalamin (vitamin B12),, respectively., 4. Enzymes. Amylases, pectinases, and proteases are produced by Aspergillus spp., 5. Antibiotics. Penicillium spp. (penicillin),, Streptomyces spp. (kanamycins and tetracyclines), and Bacillus spp. (bacitracin), 6. Steroids. Rhizopus, Streptomyces, and Curvularia are microorganisms that are used, to carry out specific reactions, bioconversions, to aid in the manufacture of these lipid, compounds., 7. Industrial chemicals. Clostridium acetobutylicum is used in the production of acetone, and butanol, and Aspergillus niger is used in, the synthesis of citric acid.
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The major adverse effect of soil organisms is, the ability of some species to produce disease in, plants and animals. Soil-borne human pathogens, include members of the spore-forming bacterial, genera Clostridium and Bacillus, and some fungal, genera, such as Cryptococcus and Coccidioides., , F U RT H E R RE A D I N G, Refer to the section on soil microbiology in your, textbook, paying close attention to the alternate, energy pathways that may be utilized by soil, associated microbes. In your textbook’s index,, search under “Nitrogen metabolism,” “Sulfur,”, and “Fixation.”, , C AS E STUDY, A BITE WORSE THEN A BARK, During a late-night shift the Emergency Department in the hospital where you are the lead infectious disease lab technician, you are informed that, a fight broke out at a wedding rehearsal party and, numerous attendees have come to the hospital for, a wide range of bite wounds and head contusions., A few days later, during your shift, the ED doctors, send you a swab from one of the Saturday-night, rehearsal patients, who returned to the ED with, an infected bite on his arm. The area around the, wound is red, warm, and raised, signifying inflammation and possible infection. Within the puncture, wounds caused by the teeth, a rotten egg smell, is evident and you see a black precipitate in the, swabs. As a microbiologist, you are aware that, , both black precipitate and the smell indicate that, hydrogen sulfide (H2S) production is occurring., After doing some research, you find that there are, some bacteria that can utilize the amino acid cysteine and produce hydrogen sulfide. How do you, determine if these bacteria, which can utilize the, cysteine synthesis pathway, are the bacteria present in the wound tracts?, , Questions to Consider:, 1. What could be the source of the cysteine?, 2. Cysteine utilizations are generally found in soil, microbes. How could it have gotten into a bite, wound?, , Part 12, , 353
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Microbial Populations in Soil:, Enumeration, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the microbial soil flora., 2. Determine the number of bacteria and, fungi present in a soil sample., , 50, , serial dilution–agar plate procedure described in, Experiment 19. Different media are employed to, support the growth of these three types of microorganisms: glycerol yeast agar for the isolation of, actinomycetes, Sabouraud agar for the isolation, of fungi, and nutrient agar for the isolation of bacteria. The glycerol yeast agar and Sabouraud agar, are supplemented with 10 μg of chlortetracycline, (Aureomycin) per ml of medium to inhibit the, growth of bacteria., , F U RT H E R RE A D I N G, , Principle, Soil contains myriads of microorganisms, including bacteria, fungi, protozoa, algae, and viruses., The most prevalent are bacteria, including the, mold-like actinomycetes, and fungi:, Simple bacteria, , E XP E R IMENT, , Predominantly members of the, orders Pseudomonadales and, Eubacteriales, , Actinomycetes, (moldlike bacteria), , Predominantly members of the, genus Streptomyces; characterized, by pleomorphism and filamentous, structure, , Fungi, , Predominantly members of the, zygomycetes (Rhizopus, Mucor,, and Absidia) and deuteromycetes, (Penicillium, Aspergillus, Alternaria,, Stemphylium, and Cladosporium), , Keep in mind that the soil environment differs, from one location to another and from one period, of time to another. Therefore, factors including, moisture, pH, temperature, gaseous oxygen content, and organic and inorganic composition of soil, are crucial in determining the specific microbial, flora of a particular sample., Just as the soil differs, microbiological methods used to analyze soil also vary. A single technique cannot be used to count all the different, types of microorganisms present in a given soil, sample, because no one laboratory cultivation procedure can provide all the physical and nutritional, requirements for the growth of a greatly diverse, microbial population. In this experiment, only, the relative numbers of bacteria, actinomycetes,, and fungi are determined. The method used is the, , Refer to the section on environmental microbiology in your textbook for further information on the, bacterial and fungal cells generally found in the soil., In your textbook’s index, search under “Actinomycetes,” “Sabouraud agar,” and “Enumeration.”, , C L I N I C A L A P P L I C AT I O N, Soil Testing, The enumeration of organisms in soil helps to, establish the level of soil fertility as well as the, types and kinds of pathogens it contains. From a, clinical view, many bacterial pathogens originate, from a soil environment. Current thought is that the, ability of Bacillus species (for example, B. subtilis, and B. anthracis) and Mycobacterium species (for, example, M. tuberculosis) to survive in a soil environment—one that contains low nutrients and low, moisture, and that necessitates sporulation or slow, growth—aids the bacteria in infecting human tissues and surviving the immune response., , AT T HE BE NCH, , Materials, Soil, ❏❏ 1-g sample of finely pulverized, rich garden, soil in a flask containing 99 ml of sterile water;, flask labeled 1:100 dilution (10-2), 355
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Media, Per designated student group, ❏❏ Four glycerol yeast agar deep tubes, ❏❏ Four Sabouraud agar deep tubes, ❏❏ Four nutrient agar deep tubes, ❏❏ Two 99-ml flasks of sterile water, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, 12 Petri dishes, Quebec colony counter, Mechanical hand counter, Sterile 1-ml pipettes, Mechanical pipetting device, L-shaped bent glass rod, Turntable (optional), 95% alcohol in a 500-ml beaker, Glassware marking pencil, , Procedure Lab One, Refer to Figure 50.1 as you read the following, instructions., 1. Liquefy the glycerol yeast, Sabouraud, and, nutrient agar deep tubes in an autoclave or by, boiling. Cool the molten agar tubes and maintain in a waterbath at 45°C., 2. Divide the Petri dishes into three groups of, four; using a glassware marking pencil, label, the groups as nutrient agar, glycerol yeast, extract agar, and Sabouraud agar. Then, label, each set of Petri dishes as follows:, • Nutrient agar: 10-4, 10-5, 10-6, and 10-7 (to, be used for enumeration of bacteria), • Glycerol yeast extract agar: 10-3, 10-4, 10-5,, and 10-6 (to be used for enumeration of, actinomycetes), • Sabouraud agar: 10-2, 10-3, 10-4, and 10-5, (to be used for enumeration of fungi), 3. With a glassware marking pencil, label the soil, sample flask as Flask 1, and label the 99-ml, sterile water flasks as Flasks 2 and 3., 4. Vigorously shake the provided soil sample, dilution of 1:100 (10-2) approximately 30, times, with your elbow resting on the table., 5. With a sterile 1-ml pipette, transfer 1 ml of the, provided soil sample dilution to Flask 2 and, shake vigorously as before. The final dilution, is 1:10,000 (10-4)., 6. Using another sterile 1-ml pipette, transfer 1 ml of Dilution 2 to Flask 3 and shake, , 356, , Experiment 50, , vigorously as before. The final dilution is, 1:1,000,000 (10-6)., 7. Using sterile 1-ml pipettes and aseptic technique, add the proper amount of each dilution, into each Petri dish as indicated in a–c and, shown in Figure 50.1., a. For actinomycetes—in plates labeled, glycerol yeast extract agar:, Transfer 0.1 ml of Dilution 1 into plate to, effect a 10-3 dilution., Transfer 1 ml of Dilution 2 into plate to, effect a 10-4 dilution., Transfer 0.1 ml of Dilution 2 into plate to, effect a 10-5 dilution., Transfer 1 ml of Dilution 3 into plate to, effect a 10-6 dilution., b. For molds—in plates labeled Sabouraud, agar:, Transfer 1 ml of Dilution 1 into plate to, effect a 10-2 dilution., Transfer 0.1 ml of Dilution 1 into plate to, effect a 10-3 dilution., Transfer 1 ml of Dilution 2 into plate to, effect a 10-4 dilution., Transfer 0.1 ml of Dilution 2 into plate to, effect a 10-5 dilution., c. For bacteria—in plates labeled nutrient, agar:, Transfer 1 ml of Dilution 2 into plate to, effect a 10-4 dilution., Transfer 0.1 ml of Dilution 2 into plate to, effect a 10-5 dilution., Transfer 1 ml of Dilution 3 into plate to, effect a 10-6 dilution., Transfer 0.1 ml of Dilution 3 into plate to, effect a 10-7 dilution., 8. Check the temperature of the molten agar, medium to be sure that the temperature is, 45°C. Remove the tubes from the waterbath, and wipe the outside surface dry with a paper, towel. Using the pour-plate technique, pour, the liquefied agar into the plates as shown in, Figure 18.2 on page 130 and rotate gently to, ensure uniform distribution of the cells in the, medium., 9. Incubate the plates in an inverted position at, 25°C. Perform colony counts on nutrient agar, plate cultures in 2 to 3 days and on the remaining agar plate cultures in 4 to 7 days.
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PROCEDURE, , –3, , (a) For actinomycetes:, Pour 455C glycerol yeast, agar into Petri plates. Mix, by rotation of plate for, serial dilution–agar, plate method., , 10–4, , 10, , Transfer, 0.1 ml., , (c) For bacteria:, Pour 455C nutrient agar, into Petri plates. Mix by, rotation of plate for serial, dilution–agar plate method., , Transfer, 0.1 ml., , Flask, 2, , Transfer 1.0 ml, into Flask 2, and shake., , 10–6, , Transfer, 1.0 ml., , Transfer 1.0 ml, into Flask 3, and shake., , Flask, 3, , 1.0 g of soil, in 99 ml, of H2O, , H 2O, 99 ml, , H2O, 99 ml, , –2, 1:100 (10 ) dilution, , 1:10,000 (10–4 ) dilution, , 1:1,000,000 (10–6 ) dilution, , Transfer, 1.0 ml., , (b) For molds:, Pour 455C Sabouraud, agar into Petri plates., Mix by rotation of plate, for serial dilution–agar, plate method., , Transfer, 1.0 ml., , Flask, 1, , Shake Flask 1, 30 times., , 10–5, , 10, , Transfer, 0.1 ml., , –2, , Transfer, 1.0 ml., , 10–3, , 10–4, , Transfer, 1.0 ml., , 10–4, , Transfer, 0.1 ml., , 10–5, , Transfer, 0.1 ml., , 10–5, , Transfer, 1.0 ml., , 10–6, , Transfer, 0.1 ml., , 10–7, , Figure 50.1 Procedure for enumeration of soil microorganisms, , Experiment 50, , 357
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Procedure Lab Two, 1. Using an electronic colony counter or a Quebec colony counter and a mechanical hand, counter, observe all the colonies on each, nutrient agar plate 2 to 3 days after incubation, begins. Plates with more than 300 colonies, cannot be counted and should be designated, as too numerous to count (TNTC); plates, with fewer than 30 colonies should be designated as too few to count (TFTC). Count, only plates with between 30 and 300 colonies., 2. Determine the number of organisms per milliliter of original culture on all plates other, , 358, , Experiment 50, , than those designated as TFTC or TNTC by, multiplying the number of colonies counted by, the dilution factor. Refer to Experiment 20 for, examples of the calculation of cell counts., 3. Record your observations and calculated cell, counts per gram of sample in the Lab Report, chart., , Procedure Lab Three, 1. Repeat Steps 1 through 3 from Lab Two for the, Sabouraud agar and glycerol yeast extract agar, plates 4 to 7 days after incubation begins.
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E XP E R IMENT, , 50, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Organism, , Dilution, , Number of Colonies, , Organisms per Gram of Soil, , 10-4, 10-5, Bacteria, 10-6, 10-7, 10-3, 10-4, Actinomycetes, 10-5, 10-6, 10-2, , Molds, , 10-3, 10-4, 10-5, , Based on your results, which of the three types of soil organisms was most abundant in your, sample? Least abundant?, , Review Questions, 1. Would you expect to be able to duplicate your results if a soil sample were taken from the, same location at a different time of the year? Explain., , Experiment 50: Lab Report, , 359
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2. In the experiment performed, why wasn’t the same medium used for enumeration of all three types of soil organisms?, , 3. Would you expect to be able to isolate an anaerobic organism from any of, your cultures? Explain., , 4. Explain why most microorganisms are present in the upper layers of the, soil., , 5., , 360, , Following the nuclear disaster at Chernobyl, the regional microbial, flora was destroyed. What impact did this have on higher forms of, plant and animal life in this area?, , Experiment 50: Lab Report
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Isolation of Antibiotic-Producing, Microorganisms and Determination, of Antimicrobial Spectrum of Isolates, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Isolate antibiotic-producing, microorganisms., 2. Determine the spectrum of antimicrobial, activity of the isolated antibiotic., , Principle, Soil is the major repository of microorganisms, that produce antibiotics capable of inhibiting the, growth of other microorganisms. Clinically useful, antibiotics have been isolated from five groups of, soil microorganisms—Streptomyces, Amycolatopsis (including some species formerly classified as, Streptomyces), Bacillus, Penicillium, and Acremonium—that represent three microbial types,, namely, actinomycetes, true bacteria, and molds., Although soils from all parts of the world are, continually screened in industrial laboratories for, the isolation of new antibiotic-producing microorganisms, industrial microbiology is directing its, energies toward chemical modification of existing, antibiotic substances. This is accomplished by, adding or replacing chemical side chains, reorganizing intramolecular bonding, or producing, mutant microbial strains capable of excreting a, more potent form of the antibiotic. The establishment of chemical congeners has been responsible, for the circumvention of antibiotic resistance,, minimizing adverse side effects in the host and, increasing the effective spectrum of a given, antibiotic., In Part A of this experiment, you will use the, crowded-plate technique for isolation of antibiotic-producing microorganisms from two soil samples, one of which is seeded with Streptomyces, griseus to serve as a positive control. Figure 51.1, illustrates the procedure to be followed. In Part, B, isolates exhibiting antibiotic activity will be, , E XP E R IMENT, , 51, , screened against several different microorganisms, to establish their effectiveness., , F U RT H E R RE A D I N G, Refer to the section on environmental microbiology, in your textbook for further information on the bacterial and fungal cells generally found in the soil., In your textbook’s index, search under “Actinomycetes,” “Sabouraud agar,” and “Antibiotics.”, , C L I N I C A L A P P L I C AT I O N, Testing New Antibiotics, Soil is the major reservoir housing microorganisms, that produce antibiotics, which are used offensively, to reduce competition for available nutrients. The, most prolific antibiotic producers are within the, phylum Actinobacteria. The genus Streptomyces, are the major producers of currently used antibiotics (for example, neomycin, streptomycin, and, tetracycline) along with the genus Actinomycetes, (for example, erythromycin). Interestingly, hundreds, of new antibiotics are isolated annually using the, crowded plate technique and other techniques, but, most have a limited spectrum and only a few are, found to be clinically acceptable., , AT T HE BE NCH, , Materials, Cultures, For Part B: 24-hour Trypticase soy broth cultures, of the following:, ❏❏ Escherichia coli, ❏❏ Staphylococcus aureus BSL -2, ❏❏ Mycobacterium smegmatis, ❏❏ Pseudomonas aeruginosa, , 361
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PROCEDURE, Transfer 5.0 ml and mix., , 0.1 g soil in, 50 ml H2O, , 1:500 Dilution, , Transfer 5.0 ml and mix., , Transfer 5.0 ml and mix., , 1, , 2, , 3, , 5.0 ml, H2O, , 5.0 ml, H 2O, , 5.0 ml, H 2O, , 1:1000 Dilution, , 1:2000 Dilution, , 1:4000 Dilution, , Transfer, 1.0 ml., , Pour molten Trypticase, soy agar, cooled to 455C,, into each plate, and mix by, gentle rotation. Incubate at, 255C for 2 to 4 days., , 1:1000, , Transfer, 1.0 ml., , 1:2000, , Figure 51.1 Crowded-plate technique for isolation of antibiotic-producing microorganisms, , Soil Suspensions, For Part A, ❏❏ 1:500 dilution of soil sample suspension (0.1 g, of soil per 50 ml of tap water) to serve as an, unknown, ❏❏ 1:500 dilution of soil sample seeded with, S. griseus (0.1 g of soil per 50 ml of tap water), to serve as a positive control, , Media, Per designated student group, Part A, ❏❏ Six 15-ml Trypticase soy agar deep tubes, ❏❏ Two Trypticase soy agar slants, , 362, , Experiment 51, , Part B, ❏❏ Two Trypticase soy agar plates, , Equipment, Part A, ❏❏ 500-ml beaker, ❏❏ 15 Test tubes, ❏❏ Test tube rack, ❏❏ Sterile Petri dishes, ❏❏ Inoculating needle, ❏❏ Hot plate, ❏❏ Thermometer, ❏❏ 1-ml and 5-ml pipettes, ❏❏ Mechanical pipetting device, ❏❏ Magnifying hand lens, , Transfer, 1.0 ml., , 1:4000
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Part B, ❏❏ Microincinerator or Bunsen burner, ❏❏ Inoculating loop, ❏❏ Glassware marking pencil, , Isolation of AntibioticProducing Microorganisms, PA RT A, , Procedure Lab One, 1. Label two sets of three sterile Petri dishes with, the types of soil samples being used and dilutions (1:1000, 1:2000, and 1:4000)., 2. Place six Trypticase soy agar deep tubes into, a beaker of water and bring to 100°C on a hot, plate. Once agar is liquefied, add cool water to, the waterbath. Cool to 45°C, checking the temperature with a thermometer., 3. Prepare a serial dilution of the unknown and, positive control 1:500 soil samples as follows, (refer to Figure 51.1):, a. Label three test tubes 1, 2, and 3. With a, pipette, add 5 ml of tap water to each tube., b. Shake the provided 1:500 soil sample thoroughly for 5 minutes to effect a uniform, soil–water suspension., c. Using a 5-ml pipette, transfer 5 ml from the, 1:500 dilution to Tube 1 and mix. The final, dilution is 1:1000., d. Using another pipette, transfer 5 ml from, Tube 1 to Tube 2 and mix. The final dilution, is 1:2000., e. Using another pipette, transfer 5 ml from, Tube 2 to Tube 3 and mix. The final dilution, is 1:4000., f. Using separate 1-ml pipettes, transfer 1 ml, from of the 1:1000, 1:2000, and 1:4000 dilutions to their appropriately labeled Petri, dishes., g. Pour one tube of molten Trypticase soy, agar, cooled to 45°C, into each plate and, mix by gentle rotation., h. Allow all plates to solidify., 4. Incubate all plates in an inverted position for 2, to 4 days at 25°C., , Procedure Lab Two, 1. Examine all crowded-plate dilutions for colonies exhibiting zones of growth inhibition. Use, a hand magnifying lens, if necessary. Record in, the Lab Report the number of colonies showing zones of inhibition., 2. Aseptically isolate one colony showing a zone, of growth inhibition from each soil culture, with an inoculating needle and streak onto, Trypticase soy agar slants labeled with the soil, sample from which the isolate was obtained., 3. Incubate the slants for 2 to 4 days at 25°C., These will serve as stock cultures of antibiotic-producing isolates to be used in Part B., , Determination of, Antimicrobial Spectrum of, Isolates, PART B, , Procedure Lab One, 1. Label the Trypticase soy agar plates with the, soil sample source of the isolate., 2. Using the aseptic technique, make a single-line, streak inoculation of each isolate on the surface of an agar plate so as to divide the plate in, half as shown:, , Inoculum, , 3. Incubate the plates in an inverted position for, 3 to 5 days at 25°C., , Experiment 51, , 363
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Procedure Lab Two, 1. Following incubation, on the bottom of each, plate draw four lines perpendicular to the, growth of the antibiotic-producing isolate as, shown:, , Perpendicular, lines, 1, , 2, , Growth, 3, , 364, , Experiment 51, , 4, , 2. Aseptically make a single-line streak inoculation of each of the four test cultures following, the inoculation template on each plate. Start, close to—but not touching—the growth of the, antibiotic-producing isolate and streak toward, the edge of the plate., 3. Incubate the plates in an inverted position for, 24 hours at 37°C., , Procedure Lab Three, 1. Examine all plates for inhibition of test organisms, and record your observations in the Lab, Report.
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E XP E R IMENT, , 51, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, PART A: Isolation of Antibiotic-Producing Microorganisms, NUMBER OF COLONIES SHOWING INHIBITION ZONE, Dilutions, Soil Sample, , 1:1000, , 1:2000, , 1:4000, , Unknown, Positive control, , PART B: Determination of Antimicrobial Spectrum of, Isolates, 1. Draw a representation of the observed antibiotic activity against the test, organisms., , Antibiotic-Producing Isolate 1, , Antibiotic-Producing Isolate 2, , 2. Based on your observations, record in the chart the presence (+ )] or, absence (- ) of antibiotic activity against each of the test organisms and the, spectrum of antimicrobial activity (broad or narrow)., TEST ORGANISMS, Soil Sample, , E. coli, S. aureus, P. aeruginosa M. smegmatis, Gram-negative Gram-positive Gram-negative, Acid-fast, , Spectrum, , Unknown, Positive control, , Experiment 51: Lab Report, , 365
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Review Questions, 1. Why is it frequently advantageous to modify antibiotics in industrial laboratories?, , 2. Is the ability to produce antibiotics limited only to bacterial species? Explain., , 3. Do you feel that sufficient test organisms were used in Part B to determine fully the spectrum of, activity of each isolated antibiotic? Explain., , 366, , Experiment 51: Lab Report
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Isolation of Pseudomonas, Species by Means of the, Enrichment Culture Technique, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Explain the enrichment culture technique, for the isolation of a specific microbial cell, type., , Principle, The enrichment culture technique is used for the, isolation of a specific type of microorganism from, an environment that is replete with different types, of microbes. In such an environment, the desired, organism may be present only in very small numbers because of the competitive activities of this, diverse microbial population. Under these circumstances, the use of conventional enriched media is, not suitable for the selection of a specific cell type., These special-purpose media are supplemented, with a variety of enriching nutrients capable of, supporting the growth of many organisms rather, than a single cell type in the test sample. Enrichment broths, on the other hand, are designed to, contain a limited number of specific substrates, that will preferentially promote the growth of the, desired microorganisms., The enrichment culture technique employs, a specifically designed enrichment broth for the, initial inoculation of the test sample. Once growth, occurs in the primary culture, it is sequentially, transferred into a fresh medium of the same, composition until the desired microorganisms, are predominant in the culture. These organisms, are capable of exponential growth because of, their ability to adapt to the medium and to enzymatically use the incorporated substrate(s) as an, energy source. Most of the competitors, however,, are incapable of utilizing the substrate(s) and, therefore remain in the lag phase of the growth, curve. In some instances, the organisms to be, , E XP E R IMENT, , 52, , isolated do not grow more rapidly than their competitors. Instead, they produce a growth inhibitor, that greatly suppresses the growth of the competing population. After the serial transfer through, the broth medium, the culture is streaked on an, agar plate of the same composition as the enrichment broth for the isolation and subsequent identification of the discrete colonies., The use of the enrichment culture technique, has a wide range of applications in clinical, industrial, and environmental microbiology. Enrichment, methods may be used to isolate and cultivate, specific soil microorganisms for the production, of industrial products such as steroids, enzymes,, and vitamins. Likewise, a beneficial environmental, application may involve the isolation by enrichment of petroleum-utilizing microorganisms,, such as Pseudomonas, that would be capable of, degrading environmentally destructive oil spills in, waterways., In this experimental procedure, we will use, a compost or a rich garden soil sample to isolate, Pseudomonas species by means of the enrichment culture procedure. Members of the genus, Pseudomonas can utilize mandelic acid aerobically as their sole carbon and energy source., Therefore, this compound is the most important, factor in the enrichment broth, which also contains a number of inorganic salts. The pseudomonads are gram-negative, motile organisms that generally produce a diffusible yellow–green pigment., In addition, they commonly reduce nitrates (NO3-), and produce an alkaline or proteolytic reaction in, litmus milk. Figure 52.1 illustrates the schema for, the experimental procedure on the following page., , F U RT H E R RE A D I N G, Refer to the section in your textbook on environmental microbiology and the differences between, soil microbes. In your textbook’s index, use the, search terms “Isolation,” “Enrichment,” and, “Pseudomonas.”, , 367
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Soil sample, Primary broth culture, , Gram stain, , Secondary broth culture, , Gram stain, , Primary agar plate culture, , Gram stain, , Secondary agar plate culture, , Gram stain, , Trypticase soy agar slant culture, , Gram stain, , Litmus milk, culture, , Trypticase nitrate, culture, , Figure 52.1 Enrichment culture procedure schema, , C L I N I C A L A P P L I C AT I O N, Medical Use for the Enrichment Culture, Technique, Medically, microbiologists use the enrichment, culture technique to isolate intestinal pathogens, from fecal samples when these organisms may be, present only in low concentrations during the infectious process. With hundreds of different bacterial, species composing our intestinal flora, identifying, a new bacterial pathogen, such as Salmonella or a, new strain of E. coli, within that population through, normal plating techniques may not be possible., By increasing the number of bacteria present in a, medium that is enriched, thus lowering competition,, bacterial species with low numbers may increase, their percentage of the population and increase the, chances of their identification., , AT THE B E N C H, , Materials, Cultures, ❏❏ Rich garden soil or compost sample, , 368, , Experiment 52, , Media, Per designated student group, ❏❏ Two Erlenmeyer flasks containing 20 ml of, basal salts broth supplemented with 2 ml of, 2.5% mandelic acid, ❏❏ Two agar plates of the same composition as, the broth, ❏❏ One Trypticase nitrate broth, ❏❏ One litmus milk, ❏❏ One Trypticase soy agar slant, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Crystal violet, Gram’s iodine, 95% ethanol, Safranin, Solution A (sulfanilic acid) Note: Solutions A, and B are not Barritt’s reagent., ❏❏ Solution B (alpha-naphthylamine) Note: Solutions A and B are not Barritt’s reagent., ❏❏ Zinc powder, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Sterile 10-ml, 5-ml, and 1-ml pipettes, Mechanical pipetting device, Microspatula, Microincinerator or Bunsen burner, Staining tray, Glass slides
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❏❏, ❏❏, ❏❏, ❏❏, , Lens paper, Bibulous paper, Inoculating loop, Glassware marking pencil, , 3. Prepare and examine a Gram-stained smear of, the secondary broth culture. Record your observations of cellular morphology and Gram reaction in the Lab Report., 4. Refrigerate the secondary broth culture., , Procedure Lab One, Primary Broth Culture Preparation, 1. Inoculate an appropriately labeled Erlenmeyer, flask containing the enrichment broth by adding, an amount of the soil sample equivalent to the, size of a pea with a microspatula. Gently swirl, the flask to mix the culture., 2. Incubate the primary broth culture for 24 hours, at 30°C., , Procedure Lab Two, Secondary Broth Culture Preparation, 1. Examine the primary culture for presence of, growth. If growth is not present, return the flask, to the incubator for an additional 24 hours., 2. If growth is present, aseptically transfer 1 ml of, the primary culture to an appropriately labeled, Erlenmeyer flask containing fresh enrichment, medium. Swirl the flask., 3. Incubate the secondary broth culture for 24, hours at 30°C., 4. Prepare and examine a Gram-stained smear, from the primary culture. Record your observations of cellular morphology and Gram reaction, in the Lab Report., 5. Refrigerate the primary broth culture., , Procedure Lab Three, Primary Agar Plate Preparation, 1. If growth is present in the secondary broth, culture, aseptically perform a four-way streak, inoculation on the appropriately labeled agar, plate of the enrichment medium. (Refer to, Experiment 2.), 2. Incubate the agar plate culture in an inverted, position for 24 hours at 30°C., , Procedure Lab Four, Secondary Agar Plate Preparation, 1. Examine the primary plate culture for the presence of discrete colonies. Record your observations of the cultural characteristics of these, colonies in the Lab Report. Using a discrete, colony:, a. Aseptically prepare and examine a Gramstained smear. Record your observations of, cellular morphology and Gram reaction in, the Lab Report., b. Aseptically perform a four-way streak, inoculation on an appropriately labeled agar, plate of the enrichment medium., 2. Incubate the secondary agar plate culture in an, inverted position for 24 hours at 30°C., 3. Refrigerate the primary agar plate culture., , Procedure Lab Five, Pure Culture Isolation, 1. Examine the secondary agar plate culture., Record your observations of the cultural characteristics of these colonies in the Lab Report., If the cultural characteristics of discrete colonies appear to be similar:, a. Prepare and examine a Gram-stained smear, from a discrete colony. Record your observations of cellular morphology and Gram, reaction in the Lab Report., b. Pick a discrete colony and aseptically inoculate a Trypticase soy agar slant by means, of a streak inoculation., 2. Incubate the agar slant culture for 24 to 48, hours at 30°C., 3. Refrigerate the secondary agar plate culture., , Experiment 52, , 369
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Procedure Lab Six, , Procedure Lab Seven, , Genus Identification of Isolate, , 1. Observe the litmus milk culture. Determine, the type of reaction that has taken place, (refer to Experiment 27), and record in the, Lab Report., 2. Perform the nitrate reduction test on the Trypticase nitrate broth culture. (Refer to Experiment, 28.) Record your results in the Lab Report., , 1. Prepare and examine a Gram-stained smear, from the Trypticase agar slant culture. Record, your observations of cellular morphology and, Gram reaction in the Lab Report., 2. Using the Trypticase agar slant culture, aseptically inoculate the appropriately labeled tubes, of Trypticase nitrate broth and litmus milk by, means of a loop inoculation., 3. Incubate the litmus milk and Trypticase nitrate, broth cultures for 24 to 48 hours at 30°C., , 370, , Experiment 52
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E XP E R IME NT, , 52, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Gram Reactions and Colony Characteristics, Culture, , Gram Stain; Cellular, Morphology, , Cultural, Characteristics, , Primary broth culture, , Secondary broth culture, , Primary agar plate culture, , Secondary agar plate culture, , Trypticase soy agar slant culture, , Litmus Milk Reaction, Record the type of reaction below., , Nitrate Reduction Test, Record whether or not the organism was capable of nitrate reduction (+ or −) below., , Experiment 52: Lab Report, , 371
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Review Questions, 1., , A child is hospitalized with severe gastroenteritis that is suspected to be symptomatic of food, poisoning caused by a Salmonella species. Explain why the hospital laboratory supervisor, uses an enrichment broth technique rather than selective media to confirm her suspicions., , 2., , A patient is afflicted with a disease that generates a large volume of gelatinous abdominal ascites. Drainage by surgical means is not successful. The use of a microbial enzyme capable of, degrading this viscous ascites is suggested. Explain how you would go about isolating an organism that, is enzymatically competent to act on this unusual substrate., , 372, , Experiment 52: Lab Report
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PART 13, , Bacterial Genetics, , LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Demonstrate enzyme induction systems., 2. Transfer genetic material through conjugation., 3. Isolate a streptomycin-resistant mutant., 4. Detect potential chemical carcinogens., , Introduction, In recent years, bacteria have proved to be essential organisms in research into the structure and, function of DNA, the universal genetic material., Their use is predicated on the following:, 1. Their haploid genetic state, which allows the, phenotypic, observable expression of a genetic, trait in the presence of a single mutant gene, 2. Their rapid rate of growth, which permits, observation of transmission of a trait through, many generations, 3. The availability of large test populations,, which allows isolation of spontaneous, mutants and their induction by chemical and, physical mutagenic agents, 4. Their low cost of maintenance and propagation, which makes it possible to perform a, large number of experimental procedures, In the following experiments, bacterial test, systems are used to demonstrate enzyme induction, screening for chemical carcinogens, and, the genetic phenomena of mutation and genetic, transfer. The last two mechanisms introduce, genetic variability, which is essential for evolutionary survival in asexually reproducing bacterial, populations., Point mutations are permanent, sudden, qualitative alterations in genetic material that, , arise as a result of the addition, deletion, or substitution of one or more bases in the region of a, single gene. As a result, one or more amino acid, substitutions occur during translation, and a protein that may be inactive, reduced in activity, or, entirely different is synthesized. Spontaneous, mutations are the result of the chemical and, physical components in the organism’s natural, environment. The rate at which they occur is, extremely low in all organisms. For example,, in Escherichia coli, the spontaneous mutation, rate at a single locus (specific site on the DNA), is estimated to be about 1 * 10-7, and the possibility of a mutation at any locus in the genome, is approximately 1 * 10-4. Induced mutations, are genetic changes resulting from the organism’s, exposure to an artificial physical or chemical, mutagen—that is, an agent capable of inducing, a mutation. The resultant mutations are of the, same type that occur spontaneously; however,, their rate is increased, and in some cases dramatically so., Transfer of genetic material and its subsequent, incorporation into the bacterial genome are also a, source of genetic variation in some bacteria. This, transfer may occur by means of the following:, 1. Conjugation: a mating process between, “sexually” differentiated bacterial strains, that allows unidirectional transfer of genetic, material, 373
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2. Transduction: a bacteriophage-mediated, transfer of genetic material from one cell to, another, 3. Transformation: a genetic alteration in a, cell, resulting from the introduction of free, DNA from the environment across the cell, membrane, , F U RT H E R RE A D I N G, Refer to the section in your textbook on, bacterial genetics. In your textbook’s index, use, the search terms “Operon,” “Transformation,”, and “Mutation.”, , C ASE STUDY, BACTERIAL EXPRESSION OF A SPECIAL PROTEIN, As the new lab technician at Big Pharm, your supervisor has decided to give you a new project that, shows some potential centered around treatment, for athlete’s foot. The lead researcher has identified a bacterial protein that inhibits the growth of, the causative agent, Tinea pedis. Unfortunately,, the bacterium that normally makes this protein, is an extremely slow grower and produces small, concentrations of the protein. Big Pharm decided, to express the gene that codes for this protein in, E. coli and attempt to produce high concentrations of proteins for later isolation and packaging., , 374, , Part 13, , A commercial plasmid has been chosen, and now, your job is to insert the gene into the plasmid and, then put the new plasmid into E. coli for expression, of the gene., , Questions to Consider:, , 1. Why did the lead researcher warn you to “pay, attention to reading frame” when choosing, where to insert the gene into the plasmid?, 2. Can a bacteria express any gene that is, inserted into it? What could be the limitations?
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E XP E R IMENT, , Enzyme Induction, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the mechanism of the lactose, operon., 2. Identify the factors that affect the expression of the b@galactosidase gene., , Principle, Although bacteria possess a single chromosome,, each cell is capable of synthesizing hundreds of, different enzymes. Studies have shown that these, enzymes are not present within the cells in equal, concentrations. Some enzymes, called constitutive enzymes, are synthesized at a constant rate, regardless of conditions in the cell’s environment., Synthesis of other enzymes, called adaptive, enzymes, occurs only when necessary, and it is, subject to regulatory mechanisms that are dependent on the environment. One such mechanism,, induction, requires the presence of a substrate,, the inducer, in the environment to initiate synthesis of its specific enzyme, called an inducible enzyme. An extensively studied inducible, enzyme in E. coli is 𝛃-galactosidase, which acts, on the disaccharide lactose to yield the monosaccharides glucose and galactose. The gene for, β-@galactosidase is a member of a cluster of genes,, called an operon, which is involved in the, metabolism of lactose. The member genes of, the lactose (lac) operon function as a unit, all, being transcribed only when the inducer, lactose, is present in the surrounding medium. See, Figure 53.1., , 53, , To illustrate β-@galactosidase induction, we, will use two test strains of E. coli: a prototrophic, (wild type) strain (lactose-positive) and an auxotrophic (mutant) strain (lactose-negative), which, carries a mutation in the gene for β@galactosidase, as well as a mutation in the lactose operon regulatory gene. We will grow both test strains in the following media:, 1. Inorganic synthetic medium lacking an organic, carbon and energy source that is required by, the heterotrophic E. coli, 2. Inorganic synthetic medium plus glucose,, which can be utilized by both strains as a carbon and energy source, 3. Inorganic synthetic medium plus lactose, which, can be utilized only by the prototrophic strain, Orthonitrophenyl-β@d@galactoside (ONPG), a, colorless analog of lactose, can serve as the substrate for the induction of β@galactosidase synthesis. As the inducer, it is hydrolyzed to galactose, and a yellow nitrophenolate ion. Following a short, incubation period, growth in all the cultures will, be determined by spectrophotometry. Induction, of β@galactosidase synthesis and activity will be, indicated by the appearance of a yellow color in, the medium following addition of ONPG, which, occurs only in the presence of the nitrophenolate, ion. Absence of this macroscopically visible color, change indicates that enzyme induction in the, lactose-negative strain did not occur., , F U RT H E R RE A D I N G, Refer to the section in your textbook on bacterial, genetics and the gene expression. In your textbook’s index, use the search terms “Transcription,”, “Inducer,” and “Operon.”, , 375
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Regulator, gene, Lac I, , Promoter, gene, , Operator, gene, , Lac Z, , Structural genes, Lac Y, Lac A, , Promoter, gene, , Operator, gene, , Lac Z, , Structural genes, Lac Y, Lac A, , Genes of lactose operon, Regulator gene (Lac I):, produces repressor protein., Promoter gene:, binding site for RNA polymerase, Operator gene:, binding site for repressor protein, Structural genes:, Lac Z: codes for b-galactosidase, Lac Y: codes for galactoside permease, Lac A: codes for galactoside transacetylase, , (a) Lactose operon., , Regulator, gene, Lac I, Expresses, repressor, protein, , RNAP, , mRNA transcription of, structural genes stopped, , (b) No lactose present. The regulator gene (Lac I) expresses the repressor protein., Because no lactose is present, the repressor protein binds to the operator gene,, blocking the RNA polymerase and stopping mRNA trascription of structural genes., Regulator, gene, Lac I, Expresses, repressor, protein, , Promoter, gene, , Operator, gene, , Lac Z, , Structural genes, Lac Y, Lac A, , RNAP, , Repressor protein, inactivated, , mRNA transcription of, structural genes, b-galactosidase, Translation, , (c) Lactose present. Lactose acts as an inducer, binding to the repressor protein and, inactivating it. The repressor protein cannot bind to the operator gene; therefore, mRNA transcription of the structural genes can proceed., , galactoside permease, galactoside transacetylase, , Figure 53.1 Enzyme induction: The mechanism of operation of the lactose operon, , C L I N I C A L A P P L I C AT I O N, Enzyme Inducers and Cancer, Inducer molecules can include hormones produced, by the body as well as toxins and drugs. Both, enzyme induction and inhibition are used by the, body to control a number of interactions that play a, role in many cellular reactions, from digestion to cell, death. One important type of current research is the, deliberate induction of human enzymes that protect, against environmental carcinogens. Such intervention may provide advance protection against cell, damage., , 376, , Experiment 53, , AT T HE BE NCH, , Materials, Cultures, 25-ml inorganic synthetic broth suspensions of, 12-hour nutrient agar cultures (adjusted to an, absorbance of 0.1 at 600 nm) of, ❏❏ Lactose-positive E. coli strain (ATCC e 23725), ❏❏ Lactose-negative E. coli strain (ATCC e 23735)
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Media, Per designated student group, ❏❏ Dropper bottles of sterile 10% glucose, ❏❏ 10% lactose, ❏❏ Water, , Reagents, , 3. Using a sterile 1-ml pipette, aseptically add, 0.5 ml of the glucose and lactose solutions and, 0.5 ml of sterile distilled water to the appropriately labeled tubes., 4. Determine the absorbance of all cultures at a, wavelength of 600 nm. Record your results in, the Lab Report., , Dropper bottles of, ❏❏ Toluene, ❏❏ Orthonitrophenyl -b@D@galactoside (ONPG), , 5. Aseptically transfer each culture to its appropriately labeled flask. (Note: If side-arm flasks, are available, additions and absorbance, readings may be made directly.), , Equipment, , 6. Incubate all flasks for 2 hours in a shaking, waterbath at 37°C and 100 strokes per minute., , ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , 1-ml and 5-ml sterile pipettes, Mechanical pipetting device, Six sterile 13@ * 100@mm test tubes, Test tube racks, Six sterile 25-ml Erlenmeyer flasks, Spectrophotometer, Shaking waterbath incubator, Glassware marking pencil, , Procedure, 1. Label three sterile test tubes and three sterile, 25-ml Erlenmeyer flasks as “Lac +< (lactosepositive), with the name of the substrate to be, added (glucose, lactose, or water). Similarly, label three sterile tubes and flasks “Lac -<, (lactose-negative) for each test organism., 2. Using sterile 5-ml pipettes, aseptically transfer, 5 ml of the Lac + and Lac - inorganic synthetic, broth cultures to their respectively labeled test, tubes., , 7. Following incubation, transfer all cultures, back to their appropriately labeled test, tubes., 8. Determine and record in the Lab Report the, absorbance for each culture at a wavelength of, 600 nm. Based on your observations, indicate, whether growth has occurred in each of the, cultures., 9. To each culture, add 5 drops of toluene and, shake vigorously. (Toluene ruptures the cells,, releasing intact enzymes.), 10. To each culture, add 5 drops of ONPG, solution., 11. Incubate all cultures for 40 minutes at 37°C., 12. Following the addition of ONPG, observe the, cultures for the presence of yellow coloration, indicative of b@galactosidase synthesis and, activity. In the Lab Report, record the colors of, your cultures and the presence (+ ) or absence, (- ) of the b@galactosidase activity., , Experiment 53, , 377
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E XP E R IME NT, , 53, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, ABSORBANCE AT 600 NM, Cultures, , Prior to, Incubation, , Following, Incubation, , Growth, ( + ) or ( − ), , Color of Culture, with ONPG, , B@Galactosidase, ( + ) or ( − ), , Lac + E. coli, Glucose, Lactose, Water, Lac − E. coli, Glucose, Lactose, Water, , Explain the absence of growth in some of the cultures., , Review Questions, 1. Distinguish between constitutive enzymes and inducible enzymes., , Experiment 53: Lab Report, , 379
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2. Explain what is meant by an operon., , 3. Explain the purpose of the ONPG in the procedure., , 4., , Compare and contrast the methods for DNA transfer in microbial, cells., , 5., , How can you explain why Staphylococcus aureus, which was, initially sensitive to penicillin, is now resistant to this antibiotic?, , 380, , Experiment 53: Lab Report
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E XP E R IMENT, , 54, , Bacterial Conjugation, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Demonstrate genetic recombination in, bacteria by the process of conjugation., , Principle, Genetic variability is essential for the evolutionary, success of all organisms. In diploid eukaryotes, the, processes of crossing over (exchange of genetic, material between homologous chromosomes) and, meiosis contribute to this variability. In haploid,, asexually reproducing prokaryotic organisms,, genetic recombination may occur by conjugation, transduction, and transformation. In this, experiment, only the process of conjugation is, considered., Conjugation is a mating process during, which a unidirectional transfer of genetic material, occurs at physical contact between two “sexually”, differentiated cell types. This differentiation, or, existence of different mating strains in some bacteria, is determined by the presence of a fertility, factor, or F factor, within the cell. Cells that lack, the F factor are recipients (females) of the genetic, material during conjugation and are designated as, F − . Cells possessing the F factor have the ability, to act as genetic donors (males) during mating. If, this F factor is extrachromosomal (a plasmid or, episome), the cells are designated as F + ; most, commonly, only the F factor is transferred during, conjugation. If this factor becomes incorporated, into the bacterial chromosome, there is a transfer, of chromosomal genes, although generally not, involving the entire chromosome or the F factor., The resulting cells are designated Hfr, for highfrequency recombinants., In this experiment, you will prepare a mixed, culture representing a cross between an Hfr, prototrophic (wild-type) strain of E. coli that is, , streptomycin-sensitive and an F - auxotrophic, (mutant) E. coli strain that requires threonine, (thr), leucine (leu), and thiamine (thi), and that, is streptomycin-resistant (Str-r). Following a, short incubation period, you will isolate only the, threonine and leucine recombinants by plating, the mixed culture on a minimal medium containing streptomycin and thiamine. The streptomycin, is incorporated into the medium to inhibit the, growth of the wild-type, streptomycin-sensitive, (Str-s) parental Hfr cells. The thiamine is required, as a growth factor for the thiamine-negative (thi-), recombinant cells. Because of its distant location, on the chromosome, this marker will not be transferred during the short mating period. A genetic, map denotes the time in minutes required for the, transfer of a marker (operon) from the donor cell, to the recipient cell. Figure 54.1 depicts the genetic, map showing the site of origin of transfer and locations of relevant markers in this experiment., , Start and order of transfer, THR, LEU, THI, 0, 80, , 10, , HFR, , STR-R, 20, , 70, , 30, , 60, , 40, , 50, minutes, , Figure 54.1 Genetic map of Escherichia coli, , 381
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FU RT HER R E ADING, Refer to the section in your textbook on bacterial, genetics and gene expression. In your textbook’s, index, use the search terms “Cloning,” “Transformation,” and “Vector.”, , C L I N I C A L A P P L I C AT I O N, Antibiotic Resistance, Conjugation is a major cause of the spread of antibiotic resistance, and represents a serious problem, in antibiotic therapy of immunosuppressed patients., Bacteria that carry several resistant genes are, called multi-drug-resistant superbugs. The indiscriminate use of antibiotics within the healthcare, profession and the illegal use of drugs without prescriptions are largely responsible for the increased, spread of antibiotic resistance., , AT THE B E N C H, , Materials, Cultures, 12-hour nutrient broth cultures of, ❏❏ F - E. coli strain thr -, leu-, thi-, and Str-r, (ATCC e 23724), ❏❏ Hfr E. coli strain Str-s (ATCC e 23740), , Media, Per designated student group, ❏❏ Three plates of minimal medium plus streptomycin and thiamine, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , 382, , Microincinerator or Bunsen burner, Beaker with 95% ethyl alcohol, L-shaped bent glass rod, 1-ml sterile pipettes, Mechanical pipetting device, , Experiment 54, , ❏❏ Sterile 13@ * 100@mm test tube, ❏❏ Waterbath shaker, ❏❏ Glassware marking pencil, , Procedure Lab One, 1. With separate sterile 1-ml pipettes, aseptically, transfer 1 ml of the F - E. coli culture and, 0.3_ml of the Hfr E. coli culture into the sterile, 13@ * 100@mm test tube., 2. Mix by gently rotating the culture between the, palms of your hands., 3. Incubate the culture for 30 minutes at 37°C in, a waterbath shaker at the lowest speed setting., 4. Appropriately label two minimal plus streptomycin and thiamine agar plates, to be used for, the control plates in Step 5., 5. To prepare control plates of the parental Hfr, and F - E. coli strains, aseptically add 0.1 ml of, each E. coli strain to its appropriately labeled, agar plate., 6. Use the spread-plate technique as shown in, Figure 54.2 and as follows:, a. Dip the bent glass rod into the beaker of, 95% ethyl alcohol., b. Sterilize the glass rod by flaming with a, Bunsen burner., c. Remove the glass rod from the Bunsen, burner, allow flame to extinguish, and cool, the glass rod., d. Spread the inoculum over the agar surface, by rotating the plate., 7. Following incubation of the mixed culture,, vigorously agitate it to terminate the genetic, transfer., 8. Appropriately label a minimal plus streptomycin and thiamine plate. Aseptically add 0.1 ml, of the mixed culture. Spread the inoculum, over the entire surface with a sterile glass rod., 9. Incubate all plates in an inverted position for, 48 hours at 37°C., , Procedure Lab Two, 1. Observe all plates for growth of colonies., 2. Record your observations in the Lab Report.
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PROCEDURE, , a Dip the bent glass rod into the beaker, of 95% ethyl alcohol., , b Sterilize the glass rod by flaming with, a Bunsen burner., , c Remove from Bunsen burner, allow flame, to extinguish, and cool the glass rod., , d Spread the inoculum over the agar surface, by rotating the plate., , Figure 54.2 Spread-plate technique, , Experiment 54, , 383
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E XP E R IMENT, , 54, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Observe all plates for the presence (+ ) or absence (- ) of colonies. Record, your results in the chart., Hfr E. coli Plate, , F − E. coli Plate, , Mixed-Culture Plate, , Growth ( + ) or ( - ), , 2. Do you expect any growth to be present on the two parental E. coli minimal, agar plates? Explain., , 3. Did genetic recombination occur? Explain how your observations support, your answer., , Review Questions, 1. Explain how genetic variations may be introduced in eukaryotic and, prokaryotic cells., , Experiment 54: Lab Report, , 385
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2. Explain the significance of the F factor., , 3. Distinguish between F + and Hfr bacterial strains., , 4., , 386, , Explain the importance of the streptomycin marker in the parental, E. coli strain., , Experiment 54: Lab Report
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E XP E R IMENT, , 55, , Isolation of a StreptomycinResistant Mutant, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Isolate a streptomycin-resistant mutant in a, prototrophic bacterial population using the, gradient-plate technique., , Principle, Mutation, a change in the base sequence of a single gene, although infrequent, is one of the sources, of genetic variability in cells. In some instances,, these changes enable the cell to survive in an, otherwise deleterious environment. An example, of such a genetic adaptation is the development, of antibiotic resistance in a small population of, microorganisms prior to the advent and large-scale, use of these agents. This microbial characteristic, of antibiotic resistance is of major clinical importance because the number of drug-resistant microbial strains continues to increase. This resistance, has occured due to antibiotics’ extensive use and, frequent misuse over the years and the selection, for the drug-resistant strains by their microbicidal effects on the less sensitive cell forms. These, drugs select for the resistant mutant and do not, act as inducers of the mutation., In a drug-resistant organism, the mutated gene, enables the cell to circumvent the antimicrobial, effect of the drug by any of a variety of mechanisms, including the following:, , 2. A change in the selective permeability of the, cell membrane, as in streptomycin resistance, 3. A decrease in the sensitivity of the organism’s, enzymes to inhibiting mechanisms, as in the, resistance to streptomycin, which interferes, with the translation process at the ribosomes, 4. An overproduction of a natural substrate, (metabolite) to compete effectively with the, drug (antimetabolite), as in the resistance to, sulfonamides, which produce their antimicrobial effect by competitive inhibition, The following procedure is designed to allow, you to isolate a streptomycin-resistant mutant, from a prototrophic (wild-type, streptomycinsensitive) Escherichia coli culture by means of the, gradient-plate technique. This requires preparation of a double-layered agar plate as illustrated in, Figure 55.1. The lower, slanted agar-medium layer, lacks streptomycin. When poured over the lower, slanted layer, the molten agar medium containing, the antibiotic will produce a streptomycin concentration gradient in the surface layer. Following a, spread-plate inoculation of the prototrophic test, culture and incubation, the appearance of colonies, in a region of high streptomycin concentration is, indicative of streptomycin-resistant mutants., , F U RT H E R RE A D I N G, Refer to the section in your textbook on environmental microbiology and the use of antimicrobial, compounds by soil microbes. In your textbook’s, index, use the search terms “Streptomyces,”, “Antibiotic,” and “Resistance.”, , 1. The production of an enzyme that alters the, chemical structure of the antibiotic, as in, penicillin resistance, Trypticase soy agar, , Trypticase soy agar, Glassware, marking, pencil, , Streptomycin agar, , LSC, , HSC, , LSC = Low streptomycin concentration, HSC = High streptomycin concentration, , Figure 55.1 Preparation of a streptomycin gradient plate, 387
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C L I N I C A L A P P L I C AT I O N, Searching for Resistance Mutations, Once resistant strains of bacteria are isolated,, microbiologists attempt to find the gene or genes, responsible. The sasX gene, found in methicillinresistant Staphylococcus aureus (MRSA), has, almost doubled in frequency over the past decade., It is located in a mobile genetic element that allows, its easy transfer to other bacteria. This gene helps, the bacterium to more effectively colonize nasal tissues and evade the host’s immune system. Targeting this gene may provide a route for highly effective, therapies., , AT THE B E N C H, , Materials, Cultures, ❏❏ 24-hour nutrient broth culture of E. coli, , Media, Per designated student group, ❏❏ Two 10-ml Trypticase soy agar deep tubes, , Reagent, ❏❏ Stock streptomycin solution (10 mg per 100 ml, of sterile distilled water), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , 388, , Sterile Petri dish (100 * 15 mm), Sterile 1-ml pipettes, Mechanical pipetting device, Inoculating loop, Bent glass rod, Beaker with 70% ethanol, Waterbath, Glassware marking pencil, , Experiment 55, , Procedure Lab One, 1. In a hot waterbath, melt two Trypticase soy, agar tubes. Cool and maintain at 45°C., 2. Place a pencil under one end of a sterile Petri, dish, pour in a sufficient amount of the molten, agar medium to cover the entire bottom surface,, and allow to solidify in the slanted position., 3. Using a sterile 1-ml pipette, add 0.1 ml of the, streptomycin solution to a second tube of molten Trypticase soy agar. Mix by rotating the, tube between the palms of your hands., 4. Place the dish in a horizontal position, pour in, a sufficient amount of the molten agar medium, containing streptomycin to cover the gradient, agar layer, and allow to solidify., 5. With a sterile 1-ml pipette, add 0.2 ml of the, E. coli test culture. With an alcohol-dipped and, flamed bent glass rod, spread the culture over, the entire agar surface as illustrated in, Figure 54.2 on page 393., 6. Incubate the appropriately labeled culture in, an inverted position for 48 hours at 37°C., , Procedure Lab Two, 1. Observe the plate for the appearance of discrete colonies and indicate their positions in, the “Initial Incubation” diagram in the Lab, Report., 2. Select one or two isolated colonies present in, the middle of the streptomycin concentration, gradient. With a sterile inoculating loop, streak, the selected colonies toward the high-concentration end of the plate., 3. Incubate the plate in an inverted position for, 48 hours at 37°C., , Procedure Lab Three, 1. Observe the plate for a line of growth from the, streaked colonies into the area of high streptomycin concentration. Growth in this area is, indicative of streptomycin-resistant mutants., 2. Indicate the observed line(s) of growth in, the “Second Incubation” diagram in the Lab, Report.
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E XP E R IMENT, , 55, , Name:, Date:, , , , Lab Report, , Section:, , Observations and Results, 1. Indicate the positions of discrete colonies in the diagram below., (LSC = low streptomycin concentration; HSC = high streptomycin concentration.), , LSC, , HSC, , Initial incubation, , 2. Indicate the observed line(s) of growth in the diagram below., , LSC, , HSC, , Second incubation, , Review Questions, 1. What mechanisms are responsible for antibiotic resistance?, , Experiment 55: Lab Report, , 389
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2. Why is it necessary to use an antibiotic gradient-plate preparation for isolation of mutants?, , 3., , Why has there been an increase in drug-resistant bacterial strains in, recent years?, , 4., , Does the streptomycin in the medium cause the mutations? Explain., , 390, , Experiment 55: Lab Report
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The Ames Test: A Bacterial, Test System for Chemical, Carcinogenicity, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Screen for potential chemical carcinogens, using a bacterial test system., , Principle, Our exposure to a wide variety of chemical compounds has increased markedly over the past, decades. Oncological epidemiologists strongly, suspect that the intrusion of these chemicals in, the form of industrial pollutants, pesticides, food, additives, hair dyes, cigarette smoke, and the like, plays a significant role in the induction of malignant transformations in humans. From a genetic, aspect there is strong evidence linking carcinogenicity to mutagenicity. Research indicates that, approximately 90% of the chemicals proved to be, carcinogens are mutagens; they cause cancer by, inducing mutations in somatic cells. These mutations are most frequently a result of base substitutions—substitutions of one base for another in the, DNA molecule—and frameshift mutations, which, are shifts in the reading frame of a gene resulting, from the addition or deletion of a base., In view of the rapid advent of new products, and new industrial processes with their resultant, pollutants, it is essential to determine their potential genetic hazards. Despite the fact that mammalian cell structure and human enzymatic pathways, differ from those in bacteria, the chemical nature, of DNA is common to all organisms; this permits, the use of bacterial test systems for the rapid, detection of possible mutagens and therefore possible carcinogens., The Ames test is a simple and inexpensive procedure that uses a bacterial test organism to screen, for mutagens. The test organism is a histidinenegative 1his- 2 and biotin-negative 1bio- 2 auxotrophic strain of Salmonella typhimurium that will, not grow on a medium deficient in histidine unless, a back mutation to his+ (histidine-positive) has, , E XP E R IMENT, , 56, , occurred. It is recognized that the mutagenic effect, of a chemical is frequently influenced by the enzymatic pathways of an organism, whereby nonmutagens are transformed into mutagens and vice versa, when introduced into human systems. In mammals,, this toxification or detoxification frequently occurs, in the liver. The Ames test generally requires the, addition of a liver homogenate, S-9, which serves as, a source of activating enzymes, to make this bacterial system more comparable to a mammalian test, system., In the Ames test, by means of the spot method,, molten agar containing the test organism, S-9 mix,, and a trace of histidine to allow the bacteria to, undergo the several cell divisions necessary for, mutation to occur is poured on a minimal agar, plate. A disc impregnated with the test chemical, is then placed in the center of the test plate. Following diffusion of the test compound from the, disc, a concentration gradient of the chemical is, established. Following incubation, a qualitative, indication of the mutagenicity of the test chemical, can be determined by noting the number of colonies present on the plate. Each colony represents, a his- S his+ revertant. A positive result, indicating mutagenicity, is obtained when an obvious, increase in the number of colonies is evident as, compared with the number of spontaneous revertants on the negative control plate., The current protocol utilized in most testing, laboratories is the Ames II test, involving small, aliquots of bacterial suspension and microliter, volumes. This assay is performed in a 96-well plate, and allows for testing of numerous dilutions of a, potential carcinogen simultaneously. The 96-well, plate assay uses similar strains of Salmonella and, operates under the same theory of induced mutation’s leading to regaining a lost ability. The industry, switch from the agar plate method to the 96-well, plate is due to efficiency and increased testing abilities. As previously mentioned, the well plate allows, for greater flexibility in testing protocols and automated determination of growth. A colored growth, indicator measured in a plate reader, as opposed to, the detection of a colony by the human eye, means, that there will be a level of precision and speed, beyond what can be done by hand., 391
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In the following procedure, you will perform, a modified Ames test; you will not use the S-9 mix, to test for the mutagenicity of nitro compounds,, which, as in humans, are activated by the bacterial nitroreductases. Four minimal agar plates are, inoculated with the S. typhimurium test organism., One plate, the negative control, is not exposed to, a test chemical. Any colonies developing on this, plate are representative of spontaneous his- S his+, mutations. The second plate, the positive control,, is exposed to a known nitrocarcinogen, 2-nitrofluorene. The remaining two plates are used to determine the mutagenicity of two commercial hair dyes., , FU RT HER R E ADING, Refer to the section in your textbook on bacterial, genetics and gene expression. In your textbook’s, index, use the search terms “Transcription,”, “Mutation,” and “DNA repair.”, , C L I N I C A L A P P L I C AT I O N, Testing for Cancer-Causing Chemicals, The Ames test is a procedure used for the identification of mutagenic chemical and physical, agents. The test was named after Bruce Ames, who, invented the test in the 1970s. While the Ames test, does not detect all mutagenic chemicals, it is used, in the pharmaceutical industry to test drugs prior to, use in clinical trials, and also in the cosmetic industry, to check on the mutagenic potential of makeup., A positive Ames test results in the rejection of the, drug or agent for further development and testing., , AT THE B E N C H, , Materials, Cultures, ❏❏ 24-hour nutrient broth cultures of, S. typhimurium, ❏❏ Strain TA 1538 (ATCC e 29631), , Media, Per designated student group, ❏❏ Four minimal agar plates, ❏❏ Four 2-ml top agar tubes, 392, , Experiment 56, , Reagents, ❏❏ Sterile biotin–histidine solution, ❏❏ 2-nitrofluorene dissolved in alcohol, ❏❏ Two commercial hair dyes, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , 1-ml sterile pipettes, Mechanical pipetting device, Sterile discs, Forceps, Waterbath, Microincinerator or Bunsen burner, Glassware marking pencil, , Procedure Lab One, Wear disposable gloves and a laboratory coat, when handling 2-nitrofluorene. For disposal of, this chemical, place excess into a sealable container and put it inside a fume hood for subsequent, removal according to your institution’s policy for, disposal of hazardous materials., , Refer to Figure 56.1 as you read the following, instructions., 1. Label three minimal agar plates with the name, of the test chemical to be used. Label the, fourth plate as a negative control., 2. Melt four tubes of top agar in a hot waterbath, and maintain the molten agar at 45°C., 3. To each molten top agar tube, aseptically add, 0.2 ml of the sterile biotin–histidine solution, and 0.1 ml of the S. typhimurium test culture., Mix by rotating the test tube between the, palms of your hands., 4. Aseptically pour the top agar cultures onto the, minimal agar plates and allow to solidify., 5. Using sterile forceps, dip each disc into, its respective test chemical solution and, drain by touching the disc to the side of the, container., 6. Place the chemical-impregnated discs in the, centers of the respectively labeled minimal, agar plates. Place a sterile disc on the plate, labeled ‘negative control.’ With the sterile forceps, gently press down on the discs so that, they adhere to the surface of the agar., 7. Incubate all plates in an inverted position for, 24 hours at 37°C.
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PROCEDURE, , 0.2 ml of, biotin-histidine, solution, , 0.1 ml of Salmonella, typhimurium culture, , 1 Add test culture and reagent, to molten top agar tube, and mix by rotation., Molten top agar, , 2 Pour onto minimal agar plates, and let solidify., , Minimal medium, , Minimal medium, , Minimal medium, , 3 Place chemical-impregnated discs in the center of the minimal agar plates., , Sterile disc, , 2-nitrofluoreneimpregnated disc, , Hair dye–, impregnated disc, , 4 Incubate for 24 hours at 375C, Control plate, shows rate of, spontaneous, mutation, , Nonmutagen, , Strong mutagen, , Slight mutagen, , Figure 56.1 The Ames test, , Experiment 56, , 393
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Procedure Lab Two, 1. Count the number of large colonies present on, each plate and record on the chart in the Lab, Report., 2. Determine and record the number of chemically induced mutations by subtracting the, number of colonies on the negative control plate, representative of spontaneous, , 394, , Experiment 56, , mutations, from the number of colonies on, each test plate., 3. Determine and record in the Lab Report the, relative mutagenicity of the test compounds, on the basis of the number of induced mutations: If below 10, (- ); if greater than 10, (1+ );, if greater than 100, 12 + 2; and if greater than, 500, 13 + 2.
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E XP E R IMENT, , 56, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Test Chemical, , Number of Colonies, , Number of Induced, Mutations, , Negative Control, , Degree of Mutagenicity, 1 − 2 , 11+ 2, 1 2+ 2, or 1 3+ 2 *, , 2-Nitrofluorene, Hair Dye 1, Hair Dye 2, *If below 10, 1− 2; if greater than 10, 11 + 2; if greater than 100, 12 + 2; and if greater than 500, 13 + 2., , Review Questions, 1. What is the purpose of the S-9 in the Ames test?, , 2. What is the purpose of the biotin–histidine solution in the Ames test?, , Experiment 56: Lab Report, , 395
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3., , What is the relationship between chemical carcinogenicity and mutagenicity?, , 4., , What are the advantages of using bacterial systems instead of mammalian systems to test for, chemical carcinogenicity? What are the disadvantages?, , 396, , Experiment 56: Lab Report
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E XP E R IMENT, , Utilization of Bacterial Plasmids, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Isolate plasmids from plasmid-bearing, bacteria., 2. Separate plasmids using agarose gel, electrophoresis., 3. Compare electrophoretic mobilities of, plasmids., 4. Transform a competent ampicillinsusceptible strain of Escherichia coli into, one that is ampicillin-resistant by means, of a DNA plasmid., , Principle, Isolating pure DNA is a necessary step in studies, that incorporate cloning, gene sequencing, gene, mapping, or any other recombinant DNA technique. Many microorganisms contain small pieces, of circular DNA called plasmids that exist separately from the host-cell genome. In studies that, use recombinant DNA techniques, plasmid DNA is, often preferred over chromosomal DNA because it, is smaller and easier to manipulate. Plasmids that, are commonly used as cloning vectors possess, three important elements: an origin of replication,, which allows the plasmid to be replicated independently of the cell’s chromosome; a selectable, marker, so the presence of the plasmid in the cell, can be detected; and a cloning site into which a, gene can be inserted. Although a selectable marker, is not a required element of a plasmid, it can be, useful in order to signal that the plasmid has been, incorporated into the host cell. Plasmid genes, that code for resistance to antibiotics are able to, confer this resistance to the host cell, and a test of, the host cell reveals that the transfer has occurred., In fact, antibiotic-resistance genes are among the, most commonly used selectable markers., , 57, , Because plasmids are circular and capable of, self-replication, they are able to serve as vectors, for transportation of cloned fragments of DNA, into other cells for genetic engineering purposes., To do this, plasmids must have a multiple cloning, site, or polylinker, which is a DNA segment with, several unique insertion sites for restriction endonucleases located next to each other, as shown in, Figure 57.1., Gel electrophoresis is a technique used to separate different sizes of DNA fragments from a sample of DNA. Because DNA is negatively charged,, when a sample is loaded into a porous agarose gel, and subjected to an electric current with a positive, charge at one end of the gel and a negative charge, at the other, DNA fragments will migrate through, the pores in the gel, toward the positively charged, end. Different-sized fragments of linear and circular DNA move through the gel at different speeds,, thus traveling different distances in the gel over a, set time period. Larger, longer pieces snake their, way through the pores more slowly, while shorter,, smaller pieces move more quickly and travel farther toward the positively charged end of the gel., There are two types of circular DNA: closed, and nicked. Closed circular DNA has all of its, nucleotides linked with phosphodiester bonds and, is supercoiled. Nicked circular DNA has at least, , SalI, , EcoRI, , SmaI, , BamHI, , KpnI, , HindIII, Polylinker, (insertion site), , ampr, (selectable, marker), , Origin of, replication, , Figure 57.1 Illustration of a plasmid, showing a, selectable marker and a multiple cloning site, , 397
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one broken phosphodiester linkage. Nicked DNA, is sometimes referred to as “relaxed” because, some of the tension present in covalently coiled, and twisted DNA has been released. Figure 57.2, illustrates the relative distance that each type of, DNA described previously (linear, closed circular,, and nicked circular) travels in an electrophoresed, agarose gel., Isolating plasmids is a multistep process,, which involves rupturing a plasmid-bearing bacterium, using a variety of reagents to remove cellular components, and suspending plasmid DNA, in an aqueous solution. After a plasmid-bearing, organism is cultured, cells are lysed using alkali, to release the plasmid DNA. The cellular debris is, then precipitated by using a detergent and potassium acetate. Following centrifugation, the pellet, that forms is removed, and alcohol is added to the, supernatant to precipitate the DNA. The DNA precipitate is resuspended in Tris-EDTA buffer., During the transformation process, the, donor cells forcibly lyse, releasing small segments, of DNA containing 10 to 20 genes. These small segments have the ability to pass through the cell wall, and cell membrane of a competent cell (a cell, that is able to take up DNA from its environment)., During naturally occurring transformations, a, double-stranded DNA segment passes through the, cell wall and into the cell’s cytoplasm, and if there, is sufficient sequence similarity, the foreign DNA, undergoes homologous recombination with the, recipient chromosome. The genome of the recipient cell has been modified to contain DNA with, genetic characteristics of the donor cell. Not all, bacteria are naturally transformable, however, and, methods have been developed to produce competency in various types of cells and transform those, cells artificially. This process was initiated in the, 1970s when it was shown that treating a recipient, , cell with a cold calcium chloride 1CaCl 2 2 solution, allows the passage of donor DNA into the cell., The porosity of the cell wall is already almost sufficient to allow the passage of intact DNA; it is the, cell membrane that is the true barrier, and its permeability is altered by this drastic treatment with, CaCl 2, allowing DNA to pass through the membrane and into the cell. With our rapidly advancing, knowledge in the field of molecular genetics, it is, now possible to artificially induce transformations, by the use of plasmids., Plasmids are small, circular pieces of extrachromosomal DNA with a length of 5,000 to, 100,000 base pairs (bp), capable of autonomous, replication in the bacterial cytoplasm. Another, membrane-altering method is electroporation. In, this method, cells are suspended in a DNA solution, and subjected to high-voltage electric impulses, that destabilize the cell membrane, resulting in, increased permeability and enabling DNA to pass, into the cells. Transduction is a method of horizontal passage of genetic material from one bacterial cell to another by means of a bacteriophage., Conjugation occurs when bacterial DNA is transferred from one cell to another via the formation, of a protoplasmic bridge, called a conjugative, or, sex, pilus., In the following experiment, we will use two, different strains of plasmid-bearing Escherichia, coli—E. coli-1 and E. coli-2. Half of the class will, isolate E. coli-1 plasmid DNA, and the other half, will isolate E. coli-2 plasmid DNA. The class will, then utilize the isolated plasmids for digestion,, electrophoresis, and transformation of E. coli., , F U RT H E R RE A D I N G, Refer to the section in your textbook on bacterial genetics and plasmids. In your textbook’s, index, use the search terms “Resistance Plasmids,”, “Transformation,” and “Digestion Sites.”, , Nicked circular, Linear, , Closed circular, , +, Figure 57.2 Gel migration pattern for linear, nicked, circular, and closed circular DNA, , 398, , Experiment 57, , C L I N I C A L A P P L I C AT I O N, Plasmids and Genetic Engineering, Plasmids are mostly found in bacteria and are, used in recombinant DNA research to transfer, genes between cells. Plasmids that confer antibiotic resistance (R plasmids) have been of special, interest because of their medical importance, and, also because of their significant role in genetic, engineering.
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AT T H E B E N C H, , Materials, Cultures, 18- to 24-hour Luria-Bertani (LB) agar base streak, plate cultures of, ❏❏ Escherichia coli, 24-hour Luria-Bertani broth, 50 mg/ml of ampicillin, cultures of plasmid-bearing, ❏❏ Escherichia coli ATCC 39991 (plasmid designation pIVEV), ❏❏ Escherichia coli ATCC 53100 (plasmid designation pDGR-2), , Media, Per designated student group, ❏❏ Two LB agar base plates, ❏❏ Three LB agar base plates plus ampicillin, 1Amp+ 2, ❏❏ One tube of LB broth, , Reagents, ❏❏ 50 mM CaCl 2 solution, ❏❏ Glucose-Tris-ethylenediaminetetraacetic acid, (EDTA) buffer, ❏❏ Tris-EDTA buffer, ❏❏ Tris-acetate-EDTA buffer, ❏❏ 5M potassium acetate (KOAc), ❏❏ Sodium hydroxide containing 1% sodium, dodecyl-sulfate (NaOH/SDS), ❏❏ 95% ethanol at 0°C, ❏❏ 70% ethanol, ❏❏ Molten agarose at 55°C, ❏❏ Gel electrophoresis running dye, ❏❏ Carolina Blu stain or 0.025% methylene blue, stain, ❏❏ HindIII-cut bacteriophage lambda 1l2 DNA, (used as the standard for comparing fragment, sizes), Note: The formulations for some of these buffers and reagents may be found in Appendix 4., Although it is cheaper to prepare your own solutions, your instructor may have ordered a kit, containing premade solutions., , Equipment, ❏❏ Microcentrifuge, ❏❏ 2-ml microcentrifuge tubes, ❏❏ Digital micropipette—10, 100, and 200 ml, , ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Small and large micropipette tips, Waterbath, Rubber micro test tube racks capable of floating, Glassware marking pencil, Ice bucket, Crushed ice, Light box or overhead projector, Millimeter ruler, Agarose gel casting tray, Staining tray, Plastic sandwich-size bags, Electrophoretic apparatus, Sterile plastic 13@ * 100@mm test tubes, Glass beads (6-mm diameter), Disposable plastic inoculating loops (standard, wire loops may be used), ❏❏ Microincinerator or Bunsen burner, ❏❏ Beaker- labeled as “Waste”, , Procedure, Using a Micropipette, Before the start of the experiment, familiarize, yourself with the use of a micropipette, the function of which is to accurately deliver microliter, volumes of solution. Not all micropipettes work, the same way. Some are designed to deliver a fixed, volume, while others can deliver variable volumes., Your instructor will demonstrate the proper handling and use of these expensive instruments., Using samples of colored water, practice using, a micropipette, attaching different-sized micropipette tips and delivering various sample volumes, to digestion tubes., 1. Set the scale on the pipette to the volume you, wish to deliver., 2. Place a tip on the micropipette by pushing it, firmly onto the pipette., 3. Depress the plunger to the first stop. This is, necessary to remove all of the air from the tip., 4. To load the pipette, dip the pipette tip into, the solution and release the plunger slowly to, draw up the solution., 5. Touch the end of the tip to the side of the tube, to remove any excess solution., 6. To deliver the solution, touch the side of the, micropipette tip to the inside of the tube, receiving the solution to produce a capillary., 7. Depress the plunger to the first stop and then, continue depressing the plunger to the second, stop to deliver the full volume of sample, blowing out the last bit in the tip., Experiment 57, , 399
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8. Continue depressing the plunger while you, remove the pipette tip from the tube., Note: Releasing the plunger before removing, the tip of the pipette from the tube will cause, fluid to suck back into the tip., Before the laboratory session, E. coli-1 and, E. coli-2 were inoculated in their medium and, grown overnight. Before the start of the lab, your, instructor dispensed 1 ml of culture into a microcentrifuge tube and spun it for 1 minute in a centrifuge. The supernatant was discarded, and the, pellet retained. Another 1 ml of culture was added, to the tube, and the process was repeated., , PROCEDURE, Prior to the lab, your instructor centrifuged 1 ml of E. coli, culture for 1 minute. The supernatant was discarded and the, pellet retained. Another 1 ml of E. coli culture was added to, the pellet. It was centrifuged for 1 minute. The supernatant, was discarded and the pellet retained for this experiment., Add 100 ml of GTE buffer., Resuspend pellet., Incubate at room temperature, for 5 min., , 1, Add 200 ml of NaOH/SDS., Mix gently by inversion., Incubate in ice bucket for 5 min., , Isolating the Plasmid, Obtain a microcentrifuge tube from your instructor with a retained pellet labeled “EC-1” or “EC-2.”, With a glassware marking pencil, label the tube, with your group name or number to identify it, later. Refer to Figure 57.3 as you complete the following steps., 1. Add 100 ml of GTE (glucose, Tris, and EDTA), buffer to your tube and resuspend the pelleted, cells by tapping with your finger or mixing by, vortex. Note: The EDTA in the buffer chelates, the divalent metal ions, Ca2+ and Mg2+, which, destabilizes the cell membrane and inhibits, the activity of DNases. The glucose maintains the osmolarity, preventing the buffer, from bursting the cell., 2. Add 200 ml of NaOH/SDS solution and mix, gently by inversion four or five times. Incubate, the tube in an ice bucket for 5 minutes. Note:, This is a highly alkaline solution that lyses, the cell, releasing the cytoplasm into the buffer, and separates the chromosomal DNA into, single strands (ssDNA) and complexes with, cellular proteins., 3. Remove the tube from the ice bucket. Then, add 500 ml of potassium acetate (KOAc) and, mix thoroughly by gentle inversion. Note: The, KOAc promotes the precipitation of chromosomal ssDNA and large RNA molecules,, which are insoluble in this salt., 4. Reincubate the tube in the ice bucket for, another 5 minutes., 5. Remove the tube from the ice bucket and, centrifuge for 5 minutes. Be sure the tubes are, balanced in the centrifuge. Note: In this step,, pellets form from all of the cellular debris and, organic molecules precipitated in the previous steps., 400, , Experiment 57, , 2, Remove from ice bucket., Add 500 ml of KOAc., Mix gently by inversion., Incubate in ice bucket for 5 min., , 3 – 4, , Supernatant, Pellet, , Remove from ice, bucket. Centrifuge, for 5 min. Decant, supernatant to a, new tube., , 5 – 6, Add 1 ml 95% ethanol at 05C, to the new tube containing, supernatant. Incubate in ice, bucket for 15 min. Centrifuge, for 15 min. Decant and discard, supernatant., , 7 – 10, , 11 – 14, , Plasmid, , Add 500 ml of cold 70% ethanol., Mix gently., Centrifuge for 5 min., Decant and discard supernatant., Allow pellet to air dry for 15 min., , Add 100 ml of TAE buffer., Resuspend pellet., Store in freezer, if necessary., , 15 – 16, , Figure 57.3 Procedure for isolating bacterial, plasmid DNA
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6. Carefully decant the supernatant solution into, a new microcentrifuge tube. Note: The plasmid remains in the supernatant solution., The pellet and the tube are discarded., 7. Add 1 ml of 95% ethanol at 0°C to the supernatant fluid in the new tube. Note: The ethanol, precipitates the plasmid., 8. Incubate the plasmid in the ice bucket for, 15 minutes., 9. Centrifuge the tube for 15 minutes to make the, precipitated plasmid form a pellet., 10. Decant and discard the supernatant. Note:, Care must be taken not to shake the tube, before or after decanting the supernatant. Do, not be concerned if you do not see a pellet. It, is there, provided that you were careful during the decanting step., 11. Add 500 ml of cold 70% ethanol to the pellet, and gently tap the tube with your finger or, rock the tube back and forth. Note: This step, washes the plasmid by removing the excess, salt. The plasmid is insoluble in ethanol., 12. Centrifuge the tube for 5 minutes., 13. Decant and discard the supernatant fluid., 14. Allow the pellet to dry for about 15 minutes,, until you no longer smell alcohol., 15. Add 100 ml of TAE (Tris-acetate-EDTA) buffer, to resuspend the pellet., 16. The plasmid may be placed in the freezer until, the next lab class, or you may proceed to the, electrophoresis step. Note: If the electrophoresis is to be done during this class period,, practice loading and casting the gel, which, are described next., , 2. Close off the ends of the tray with the rubber, dams by tightening the knob on the top of the, casting tray box., 3. Place a well-forming comb in the first notch at, the end of the casting tray., 4. Pour 60 ml to 70 ml of agarose solution that, has been cooled to 55°C into the tray. Use a, toothpick or applicator stick to move the bubbles to the edge of the gel before it solidifies., 5. Allow the gel to solidify completely. It should, be firm to the touch after 20 minutes., 6. Slowly remove the rubber dams and very gently remove the well-forming comb by pulling, it straight up. Note: Use extreme care not to, damage or tear the wells., 7. Place the gel on the platform in the electrophoresis box so that the formed wells are, properly oriented toward the anode (negative pole with black cord). Because DNA is, negatively charged, the cut DNA fragments, will migrate to the cathode (positive pole with, the red cord). Refer to Figure 57.4 to see the, proper setup of an electrophoretic apparatus., 8. Fill the electrophoresis box with TAE buffer, to a level that just covers the gel, about 2 mm., Make sure that all of the wells are filled with, the buffer., , Casting the Agarose Gel, Note: Not all casting trays are the same. Your, instructor will indicate which type will be used, and whether there are special considerations, during the setup., Your instructor prepared the 0.8% agarose gel, in a 1X TAE buffer solution before class and maintained it at 55°C in a waterbath. It is ready to pour., One or two drops of Carolina Blu stain were added, to the agarose buffer solution to give a small tinge, of blue to the gel. At this concentration, the pores, that form the gel lattice are such that they allow, the free migration of the cut DNA fragments between 0.5 and 10 kilobases (kb)., Figure 57.4 Setup of agarose gel unit for DNA, , 1. Place the casting tray inside the casting tray, box on a level surface., , electrophoresis, , Experiment 57, , 401
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Practicing Gel Loading, Before loading your sample into the wells of the agarose gel, practice this challenging technique. Your, instructor will demonstrate the proper method for, loading the wells. Each student should practice on a, gel that has been prepared earlier by the instructor,, not on the gel to be used for running the samples., 1. Load the pipette with 22 ml of loading gel., 2. Hold the pipette with both hands and dip the, tip slightly through the buffer covering the gel,, with the tip barely in the well., 3. Slowly discharge the contents of the pipette., Note: The loading gel contains sucrose, which, is heavier than the DNA and will pull the, sample into the well., 4. Practice the technique until you are comfortable with it., , Electrophoresing of the Plasmids, 1. Add 18 ml of plasmid in the 1X TAE buffer to a, new microcentrifuge tube. Then add 4 ml of gel, electrophoresis running dye to the tube., 2. Add 18 ml of HindIII-cut lambda 1l2 DNA and, 4 ml of the gel electrophoresis running dye., With your glass marking pencil, label this tube, HindIII. This DNA has been cut into six linear, fragments with the HindIII restriction enzyme., The fragments (bands) are various sizes: 23, kb, 9.4 kb, 6.6 kb, 4.4 kb, 2.3 kb, and 2.0 kb., 3. When the wells are ready to be loaded, make a, diagram so that you will know the position of, your sample in the agarose gel., 4. Fill the wells by designating the EC-1 samples, as odd-numbered groups and EC-2 samples, as even-numbered groups, as shown in, Figure 57.5., 5. After the samples are loaded into the wells,, place the lid on the electrophoresis gel box., 1, , 2, , 3, , 4, , 5, , lDNA, , EC-2, , EC-1, , EC-2, , EC-1, , Figure 57.5 Gel loading scheme, 402, , Experiment 57, , Check that the power switch is turned to the, “off” position and then attach the electrical, leads (red to red and black to black) from the, power supply to the box., 6. Turn the power pack on and adjust the rheostat dial to 110V., 7. Electrophorese the gel for 30 to 40 minutes or, until the leading edge of the bromphenol blue, dye (the dye in the loading gel) has traveled, roughly three-fourths of the distance to the, edge of the gel., 8. Turn the rheostat to zero and turn off the, power. Disconnect the leads and remove the, cover from the gel box., , Staining the Gel, 1. Put on a pair of disposable laboratory gloves., 2. Lift the gel tray out of the electrophoresis box,, and slide the gel into a staining tray containing, approximately 100 ml of Carolina Blu stain or, 0.025% of methylene blue stain., 3. Allow the gel to remain in the stain for 30 to 40, minutes., 4. Pour off the stain into a waste beaker. Transfer, the gel to a staining tray containing 100 ml of, distilled water and allow the gel to decolorize, (destain) for another 30 minutes. Frequent dest, aining with fresh distilled water for 2 minutes, increases the intensity of the bands. For best, results, let the gel destain overnight in a small volume of water. Note: If the gel is left overnight in a, large volume of water, it may destain too much., 5. Pour off the water, carefully remove the gel, from the staining tray, and place it in a plastic, sandwich-size bag or wrap it in a piece of clear, plastic wrap. Note: Be careful to keep the gel, flat as you place it in the bag or plastic wrap., 6. The gel can be placed in the refrigerator until, the next lab period., , Transformation, Refer to Figure 57.7 for steps involved in transforming a competent bacterial cell line., 1. With a glassware marking pencil, label two, 13@ * 100@mm test tubes, one as “DNA + <, and the other as “DNA-.” The DNA + tube will, receive the plasmid from the section above., 2. Using a sterile pipette, transfer 250 ml (0.25 ml), of ice-cold CaCl 2 solution into each tube., 3. Place both tubes in a 500-ml beaker of, crushed ice., 4. Using a sterile inoculation loop, obtain a, large mass of cells approximately 5 mm, in size (about the size of a pencil eraser)
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11. Return both tubes to the crushed-ice beaker, for 1 minute., 12. With a sterile pipette, add 250 ml (0.25 ml) of, LB broth to both the DNA + and DNA - tubes., Tap the tubes with your finger to achieve uniform cell suspension. (These are the transformation tubes.), 13. Incubate both tubes in a test tube rack for 10, minutes. Note: This is the recovery period,, when the cells convert their newly modified, genotype into a functionally ampicillinresistant phenotype., 14. Using a new plastic micropipette tip or disposable plastic transfer pipette for each inoculation, inoculate 100 ml (0.1 ml) of cells from the, DNA + transformation tube onto the surface of, LB plates 1–4, and inoculate 100 ml (0.1 ml) of, cells from the DNA- transformation tube onto, plates 5–8., 15. Place six sterile 6-mm glass beads on the, Figure 57.6 shows an agarose gel stain with methyl, surface of each inoculated plate. Replace the, ene blue following electrophoresis of plasmid DNA, cover and spread the cell suspension by gently, moving the plate up and down and then side to, from the E. coli starter plate, and inoculate, side a few times. Note: Do not swirl or rotate, the tube labeled “DNA + .< Note: Be sure to, the plate. This step may be eliminated if the, immerse the loop directly into the CaCl2 and, spread-plate method is used., shake the loop vigorously to dislodge the, 16. Repeat Steps 14 and 15 for the remaining plates., inoculum. Discard the plastic loops in the, 17. Allow the plates to set for a few minutes so the, beaker labeled “waste” or sterilize the wire, inoculum may be absorbed by the agar., loop by flaming it., 18. Remove the glass beads from the plate by lift5. Disperse the cells by gently tapping the tube, ing the cover slightly while holding the plate, with your finger until a uniform milky-white, vertically over a beaker of disinfectant, allowtranslucent cell suspension is obtained., ing the beads to leave the plate. Note: This, 6. Repeat Steps 4 and 5 to inoculate the tube, step may be eliminated if the spread-plate, marked “DNA-,” using an equal amount of, procedure is used., inoculum and a sterile inoculating loop., 19. Incubate all plates at 37°C for 24 to 36 hours, 7. Using a sterile pipette, deliver 10 ml (0.01 ml), or at room temperature for 48 to 72 hours., of isolated plasmid into the DNA + tube. Tap, 20. In the Lab Report, predict whether each plate, the tube several times with your finger to, will experience growth or no growth. Use a, ensure complete mixing of the plasmid and, plus (+ ) sign for growth and a minus (- ) for, cell suspension. Note: Discard the plastic tip, no growth., or disposable pipette into a beaker contain21. Without removing the cover of the Petri plates,, ing disinfectant solution., observe the colonies through the bottom of, 8. Return the DNA + tube to the crushed-ice beaker, each plate., and incubate for 15 minutes. During this time,, 22., Perform a colony count on each plate using, label your agar plates as described in Step 9., a permanent marker to mark each colony on, 9. Label the eight LB agar plates as follows (two, the bottom of the plate as it is counted. Plates, sets, DNA + and DNA - ):, with more than 300 colonies should be desigPlate 3: LB Plate 1: LB +, nated as TNTC (too numerous to count);, Plate 2: LB/Amp+, Plate 4: LB/Ampplates with fewer than 30 colonies are designated as TFTC (too few to count). Record, 10. Remove both tubes from ice after 15 minutes;, your results in the Lab Report., place them in a test tube rack and immediately, into a 42°C waterbath with gentle agitation, 23. For each plate, did transformation occur?, for 90 seconds (heat shocking)., Record your results in the Lab Report., Experiment 57, , 403
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PROCEDURE, , Add 10 ml plasmid., , DNA, +, , DNA, -, , Inoculate with E. coli, from E. coli starter plate., , Inoculate with E. coli., , Add 250 ml cold CaCl2., , Add 250 ml cold CaCl2., , DNA, , +, , DNA, , –, , Incubate for, 15 minutes in bucket, of crushed ice., , Plate Preparation, Plate, 1, , Plate, 2, , Plate, 3, , Plate, 4, , LB+, , LB/Amp+, , LB-, , LB/Amp-, , DNA, , Test tube rack, , +, , DNA, , -, , Place in 42°C waterbath, and agitate gently for, 90 seconds (heat shocking)., , Waterbath, , Add 250ml, LB broth., , Add 250 ml, LB broth., , Transfer, 100 ml., , DNA, , DNA, , +, , -, , Transfer, 100 ml., , Transfer, 100 ml., , Plate, 1, , Plate, 2, , Plate, 3, , Plate, 4, , LB+, , LB/Amp+, , LB-, , LB/Amp-, , Figure 57.7 Procedure for transforming a bacterial cell, 404, , Transfer, 100 ml., , Experiment 57
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E XP E R IMENT, , 57, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Tape a millimeter ruler to your light box or to the glass on your overhead, projector. If you prefer, you may use a millimeter ruler to measure the, migration of the plasmid., 2. Align your gel so that the front end of the well is set at the zero point on the, ruler., 3. Measure the migration distances from the front of the well to the front edge, of the band, and record the distances in the following chart., 4. Prepare a standard curve on the semilog paper provided on page 407 by, plotting the distance traveled in millimeters on the x-axis versus the size of, the fragment of HindIII-cut l DNA in kilobases. Record your results in the, following chart., Note: 0.8% agarose gel has pore sizes that will allow the free movement of, nucleic acids between 0.5 and 10 kb. Therefore, draw the best-fit straight, line for all bands except the 23-kb band., Migration Distances of Lambda DNA, Kilobases, , 23, , 9.4, , 6.6, , 4.4, , 2.3, , 2.0, , Millimeters, , 5. Determine the number of bands in each plasmid, and use a ruler to measure, the migration distance in centimeters., Band #, , 1, , 2, , 3, , 4, , 5, , Total, , E. coli-1, E. coli-2, , 6. Draw a diagram of your agarose gel and indicate which bands are linear,, closed circular, or nicked circular., 1, , 2, , 3, , 4, , 5, , lDNA, , EC-2, , EC-1, , EC-2, , EC-1, , Experiment 57: Lab Report, , 405
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7. Determine the size of linear DNA segments that would migrate the same distance as the various forms of the plasmids, using the standard curve. Record, your results in the following chart:, PLASMID DNA, Linear DNA, , E. coli-1, , E. coli-2, , 8. Record the results of your transformation experiment in the chart following:, Plate Number, , Designation, , 1, , LB +, , 2, , LB/Amp +, , 3, , LB -, , 4, , LB/Amp -, , Growth + or −, , Transformation, Yes or No, , Review Questions, 1. Why is plasmid DNA preferred for genetic engineering studies?, , 2. What are selectable markers, and why are they important to cloning, vectors?, , 3. What is the rationale for using each of the following solutions for the isolation of plasmids?, a. EDTA:, , 406, , Experiment 57: Lab Report, , Number of Colonies
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10, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, Migration Distance (mm) versus Fragment Size (kb), , Experiment 57: Lab Report, , 407
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b. Sodium dodecylsulfate (SDS):, , c. Potassium acetate:, , 4. Alcohol is considered to be a significant reagent for the isolation of nucleic, acids (RNA and DNA). Why is this so?, , 5. What is nicked circular DNA, and why is it termed “relaxed”?, , 6., , When might you not be able to use a standard curve to determine, the size of a plasmid?, , 7., , When plasmids are isolated from bacterial cells, they may exist in, a number of forms., a. List the different forms that may be found., , b. Which do you think would migrate the fastest and farthest in an electrophoresis experiment and why?, , 408, , Experiment 57: Lab Report
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E XP E R IMENT, , Restriction Analysis and, Electrophoretic Separation of, Bacteriophage Lambda DNA, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Cut DNA into small fragments by using, restriction endonucleases., 2. Separate DNA fragments through agarose, gel electrophoresis., 3. Determine the length of DNA fragments, in kilobases., , Principle, Through their pioneering work, Werner Arber, and Hamilton Smith discovered that bacteria possess enzymes that can act as molecular scissors, and cut DNA molecules into smaller fragments., These enzymes, called endonucleases, are able, to differentiate between DNA endogenous to an, organism and foreign DNA, including infecting, bacteriophages. Endonucleases can cut foreign, DNA, rendering it nonfunctional, which spares, the bacterium from infection. For scientists, the, discovery of restriction endonucleases has been, vital in advancing research over the past 40 years, because small DNA fragments are much easier, to manipulate than longer DNA strands. Now scientists can accurately map a gene’s location on a, chromosome and describe its base-pair sequence., Restriction endonucleases are also being used, to develop DNA recombinants for commercial use,, detect genetic defects, map restriction sites on, plasmids, and create DNA profiles for use in medicine and forensics., Endonucleases recognize palindromic, sequences, four to six base pairs long, on DNA, molecules. In everyday usage, a palindrome is a, word that is spelled the same way forward and, backward. For example, the word “racecar” is a, common palindrome. On a double-stranded DNA, molecule, a palindrome is a sequence of base pairs, that reads the same on one strand 5′ to 3′ as it, does on the other strand 5′ to 3′. Keep in mind, , 58, , that reading 5′ to 3′ on one strand is done in the, opposite direction of reading 5′ to 3′ on the other., Each endonuclease has its own unique restriction, site. Figure 58.1 shows an example of a palindromic base-pair sequence and the cutting site for, the restriction enzyme EcoRI. In the figure, EcoRI, cuts the molecule between guanine and adenine,, producing two fragments with staggered ends., The key property of endonucleases is that they, recognize and digest, or cut, one specific sequence, of nucleotides on a DNA molecule and cut this, same sequence every time. Several endonucleases make staggered cuts in the double-stranded, molecule, producing single strands of DNA with, cohesive, or sticky, ends that allow them to combine with complementary single-stranded DNA., Other endonucleases cut DNA sequences straight, through both strands, producing blunt ends., Figure 58.2 illustrates the restriction sites of some, commonly used endonucleases. The arrows indicate the cutting sites on each strand. The endonucleases that produce sticky, staggered ends are, clearly distinguishable in Figure 58.2 from those, that produce blunt ends., DNA fragments cut with the same restriction, enzyme can pair with one another. The sticky ends, of different strands will join together because of, the formation of hydrogen bonds between complementary bases. However, joined fragments lack, Palindrome, , 5¿, , GTAGAATTCATTCACGCA, , 3¿, , 3¿, , CATCTTAAGTAAGTGCGT, , 5¿, , GTAG, CATCTTAA, Fragment 1, , +, , AATTCATTCACGCA, GTAAGTGCGT, Fragment 2, , Figure 58.1 Palindrome for EcoRI endonuclease, 409
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F U RT H E R RE A D I N G, EcoRI, , BamHI, , SmaI, , 5¿, , GAATTC, , 3¿, , 3¿, , CTTAAG, , 5¿, , 5¿, , GGATCC, , 3¿, , 3¿, , CCTAGG, , 5¿, , 5¿, , CCCGGG, , 3¿, , 3¿, , GGGCCC, , 5¿, , HindIII, , AluI, , HbaI, , 5¿, , AAGCTT, , 3¿, , 3¿, , TTCGAA, , 5¿, , 5¿, , AGCT, , 3¿, , C L I N I C A L A P P L I C AT I O N, , 3¿, , TCGA, , 5¿, , 5¿, , GCGC, , 3¿, , 3¿, , CGCG, , 5¿, , Enzyme Digestion to Isolate Genes of Interest, It was quickly recognized that restriction enzymes, would provide a new tool for the investigation of, gene organization, function, and expression. Current medical research is examining how restriction, enzymes can be used to insert the genes required, for insulin production into diabetic patients., , Refer to the section in your textbook on bacterial, genetics and plasmids. In your textbook’s index,, use the search terms “HindIII,” “Transformation,”, and “Restriction Digestion.”, , Figure 58.2 Illustration of restriction sites for, common endonucleases that leave blunt and, staggered ends, , phosphodiester bonds between guanine and adenine, and nicks form as a result. These nicks are, annealed by DNA ligase enzymes. Under optimum, environmental conditions (salt concentration, pH,, and temperature), restriction endonucleases will, cut a strand of DNA into a number of varying-sized, fragments. The exact number and sizes of the, fragments depend on the location and number of, restriction sites for the enzyme., Restriction enzymes are named based on the, genus and species of bacteria from which they are, obtained. The first letter of the genus name is followed by the first two letters of the species name., For example, an endonuclease from Escherichia, coli is named Eco. If a bacterium produces more, than one restriction enzyme, each endonuclease is, differentiated by Roman numerals. If the enzyme, is coded for on a resistance factor, it is further designated with an “R.” Thus EcoRI is one of several, endonucleases produced by E. coli and is coded, for on a restriction site. Other widely used endonucleases are obtained from Haemophilus influenzae D (HindIII, which cuts between adenine, bases) and Bacillus amyloliquefaciens H (BamHI,, which cuts between two guanine bases)., In the following experiment, you will use, endonucleases to cut bacteriophage lambda 1l2, DNA, containing 48,502 base pairs (48.5 kb), into, fragments. You will separate the fragments by, using agarose gel electrophoresis, and you will, determine the size of each., , 410, , Experiment 58, , AT T HE BE NCH, , Materials, DNA Source, ❏❏ Bacteriophage l 1200 ml2, , Restriction Endonucleases, ❏❏ EcoRI, ❏❏ HindIII, ❏❏ BamHI, , Reagents, ❏❏ Tris-acetate buffer, type-specific buffers for, EcoRI, HindIII, and BamHI, ❏❏ Electrophoresis loading dye, ❏❏ Carolina Blu or 0.025% methylene blue stain, ❏❏ 0.8% agarose in 1X TAE buffer, Note: Formulas for the preparation of type-specific buffers may be found in Appendix 4., , Equipment, ❏❏ Plastic 1.5-ml microcentrifuge tubes, ❏❏ Microcentrifuge, ❏❏ Adjustable micropipettes 10.5 ml to 10 ml2,, 15 ml to 10 ml2, and 110 ml to 100 ml2, ❏❏ Large and small fine-point micropipette tips, ❏❏ Waterbath, ❏❏ Ice bucket
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❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Crushed ice, Staining tray, Disposable gloves, Glassware marking pencil, Hot plate, 250-ml Erlenmeyer flask, Beaker for waste, Micro test tube racks, Electrophoretic apparatus, Millimeter ruler, Light box or overhead projector, , Procedure, Note: In the following procedure, the steps for, practicing micropipette use, casting a gel, and, loading samples were covered in Experiment 57:, Isolation of Bacterial Plasmids. If you completed, these practice steps in Experiment 57, you may, want to skip them here. If you are uncomfortable, with any of the techniques, practice them again., , Using a Micropipette, Before the start of the experiment, familiarize, yourself with the use of a micropipette, the function of which is to accurately deliver microliter, volumes of solution. Not all micropipettes work, the same way. Some are designed to deliver a fixed, volume, while others can deliver variable volumes., Your instructor will demonstrate the proper handling and use of these expensive instruments., Using samples of colored water, practice using a, micropipette, attaching different-sized micropipette tips and delivering various sample volumes, to digestion tubes., 1. Set the scale on the pipette to the volume you, wish to deliver., 2. Place a tip on the micropipette by pushing it, firmly onto the pipette., 3. Depress the plunger to the first stop. This is, necessary to remove all of the air from the tip., 4. To load the pipette, dip the pipette tip into, the solution and release the plunger slowly to, draw up the solution., 5. Touch the end of the tip to the side of the tube, to remove any excess solution., 6. To deliver the solution, touch the side of the, micropipette tip to the inside of the tube, receiving the solution to produce a capillary., , 7. Depress the plunger to the first stop and then, continue depressing the plunger to the second, stop to deliver the full volume of sample, blowing out the last bit in the tip., 8. Continue depressing the plunger while you, remove the pipette tip from the tube. Note:, Releasing the plunger before removing the tip, of the pipette from the tube will cause fluid to, suck back into the tip., , Digesting of Lambda 1L 2 DNA, , Be sure to wear gloves, as enzymes on your, skin degrade DNA in the experiment., , 1. Obtain a sample of lambda DNA from the, instructor., 2. With a glassware marking pencil, label four, microcentrifuge tubes with your name or, group number followed by an “L” for the uncut, DNA, “E” for EcoRI, “H” for HindIII, and “B”, for BamHI., 3. Using a new pipette tip for each reagent, add, the reagents to the digestion tubes in the following order:, a. Lambda DNA, b. Deionized or distilled water 1dH2O2, c. Restriction enzyme buffer 10X, d. Restriction endonucleases 110 units/ml2, Note: The restriction enzyme must be added, last to the digestion tubes. Addition of the, endonucleases before DNA or buffer may, inactivate the endonuclease. Each reagent is, added with a new pipette tip to avoid contaminating the digestion tubes., 4. The addition of the reagents to each tube may, be made following the scheme in Table 58.1., 5. Pulse centrifuge or tap your finger on each, tube several times to mix the reagents., 6. Place all tubes in a foam rubber test tube, rack or a suitable microcentrifuge rack and, incubate them in the water bath at 37°C for 60, minutes., 7. The digestion tubes may be stored in the, refrigerator until the next class period. If you, are continuing with the experiment now, place, the tubes in an ice bucket and proceed to the, next step., , Experiment 58, , 411
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Microliters 1Ml2per Digestion Tube, , TABLE 58.1, , TUBE LAMBDA DNA, , DH2O, , 10X RESTRICTION BUFFER, , ENZYMES, ECORI, , HINDIII, , BAMHI, , TOTAL, , B, , 6, , 10, , 2, , 0, , 0, , 2, , 20, , E, , 6, , 10, , 2, , 2, , 0, , 0, , 20, , H, , 6, , 10, , 2, , 0, , 2, , 0, , 20, , L, , 6, , 12, , 2, , 0, , 0, , 0, , 20, , Casting the Agarose Gel, Note: Not all casting trays are the same. Your, instructor will indicate which type will be used, and whether there are special considerations, during the setup., Your instructor prepared the 0.8% agarose gel, in a 1X TAE buffer solution before class and maintained it at 55°C in a waterbath. It is ready to pour., One or two drops of Carolina Blu stain were added, to the agarose buffer solution to give a small tinge, of blue to the gel. At this concentration, the pores, that form the gel lattice are such that they allow, the free migration of the cut DNA fragments between 0.5 and 10 kb., Refer to Figure 57.4 on page 411 to see the, proper setup of an electrophoretic apparatus., 1. Place the casting tray inside the casting tray, box on a level surface., 2. Close off the ends of the tray with the rubber, dams by tightening the knob on the top of the, casting tray box., 3. Place a well-forming comb in the first notch at, the end of the casting tray., 4. Pour 60 ml to 70 ml of agarose solution that, has been cooled to 55°C into the tray. Use a, toothpick or applicator stick to move the bubbles to the edge of the gel before it solidifies., 5. Allow the gel to solidify completely. It should, be firm to the touch after 20 minutes., 6. Slowly remove the rubber dams and very gently remove the well-forming comb by pulling, it straight up. Note: Use extreme care not to, damage or tear the wells., 7. Place the gel on the platform in the electrophoresis box so that the formed wells are properly, oriented toward the anode (negative pole, with black cord). Because DNA is negatively, charged, the cut DNA fragments will migrate, , 412, , Experiment 58, , to the cathode (positive pole with the, red cord)., 8. Fill the electrophoresis box with TAE buffer, to a level that just covers the gel, about 2 mm., Make sure that all of the wells are filled with, the buffer., , Practicing Gel Loading, Before loading your sample into the wells of the, agarose gel, practice this challenging technique., Your instructor will demonstrate the proper, method for loading the wells. Each student should, practice on a gel that has been prepared earlier by, the instructor, not on the gel to be used for running the samples., 1. Load the pipette with 22 ml of loading gel., 2. Hold the pipette with both hands and dip the, tip slightly through the buffer covering the gel,, with the tip barely in the well., 3. Slowly discharge the contents of the pipette., Note: The loading gel contains sucrose, which, is heavier than the DNA and will pull the, sample into the well., 4. Practice the technique until you are comfortable with it., , Loading the DNA Digests into the, Wells and Electrophoresing the, Samples, 1. Remove the digestion tubes from the ice bucket, and add 4 ml of 6X loading dye to each tube., 2. Pulse centrifuge or tap your finger on each, tube several times so that the contents of the, tube move to the bottom., 3. Set the dial on the micropipette to deliver, 24 ml (20 ml of restriction digests plus 4 ml of, loading dye).
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4. Deliver each of the four enzyme digests to a, separate well in the agarose gel., 5. Remember the order of your samples and the, position of each in the agarose gel. Because, the gel cannot be marked, you should draw, a diagram of the gel and label the position of, your samples as shown in Figure 58.3., 6. After the samples are loaded into the wells,, place the lid on the electrophoresis gel box., Check that the power switch is turned to the, “off” position and then attach the electrical, leads (red to red and black to black) from the, power supply to the box., 7. Turn the power pack on and adjust the rheostat dial to 110V., 8. Electrophorese the gel for 30 to 40 minutes or, until the leading edge of the bromphenol blue, dye (the dye in the loading gel) has traveled, , B, , E, , H, , L, , Figure 58.3 Example of gel loading scheme, , roughly three-fourths of the distance to the, edge of the gel., 9. Turn the rheostat to zero and turn off the, power. Disconnect the leads and remove the, cover from the gel box., , Staining the Gel, 1. Put on a pair of disposable laboratory gloves., 2. Lift the gel tray out of the electrophoresis box,, and slide the gel into a staining tray containing, approximately 100 ml of Carolina Blu stain., 3. Allow the gel to remain in the stain for 30 to 40, minutes., 4. Pour off the stain into a waste beaker. Transfer, the gel to a staining tray containing 100 ml of, distilled water and allow the gel to decolorize, (destain) for another 30 minutes. Frequent, destaining with fresh distilled water for 2 minutes increases the intensity of the bands. For, best results, let the gel destain overnight in a, small volume of water. Note: If the gel is left, overnight in a large volume of water, it may, destain too much., 5. Pour off the water, carefully remove the gel, from the staining tray, and place it in a plastic, sandwich-size bag or wrap it in a piece of clear, plastic wrap. Note: Be very careful to keep the, gel flat as you place it in the bag or plastic, wrap., 6. The gel can be placed in the refrigerator until, the next lab period. Refer to Figure 57.6, on, page 403, for a photo of an electrophoresed, and stained gel., , Experiment 58, , 413
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E XP E R IMENT, , 58, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, 1. Tape a millimeter ruler to the surface of a light box or to the glass surface of, an overhead projector., 2. Place the stained gel (inside plastic bag) next to the zero point on the ruler, and measure the distances that each fragment (band) migrated. Measure, the distance from the front of the well to the front of the band. Record your, results in the following chart., Migration Distances of Fragments (mm), Uncut l DNA, , BamHI cut l DNA, , EcoRI cut l DNA, , HindIII cut l DNA, , Linear DNA fragments migrate at rates inversely proportional to the log10 of, their molecular weight and base-pair length., 3. A graph (standard curve) can be constructed by plotting known kilobase-pair, fragments versus distances migrated from the wells to the front of the fragment. The six kilobase-pair fragment sizes for HindIII are well established, and can be used to plot a standard curve., 4. Once the fragment sizes are measured and distances traveled are plotted on, semilog paper found on page 417, a best-fit straight line can be drawn. The, size of each unknown fragment can be determined by drawing a vertical line, from the migration distance (mm) on the x-axis up to the point on the curve, that intersects that straight line. From there, draw a horizontal line to the, fragment size on the y-axis., , Experiment 58: Lab Report, , 415
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5. In the following table, the kilobase lengths of HindIII are provided. From the standard curve, use the migration distance you have measured to determine the basepair lengths for the three restriction enzymes. Record your results in the table., HindIII*, Distance, (mm), , BamHI, , Actual kb, , Distance, (mm), , EcoRI, , Calculated, kb, , Distance, (mm), , l DNA, , Calculated, kb, , Distance, (mm), , Calculated, kb, , 27.4*, 23.1*, 9.4, 6.6, 4.4, 2.3, 2.0, * Note: Remember that 0.8% agarose allows the free migration of DNA in the range of 0.5 to 10 kb. Therefore, the 27.4- and 23.1-kb fragments will not be detected., , 6. Calculate the fragment lengths of EcoRI and BamHI from the standard curve, and compare them with the actual kilobase lengths listed in the following chart., HindIII, Actual kb, 27.4*, , Distance, (mm), , BamHI, Actual kb, , Distance, (mm), , 16.8*, , EcoRI, Calculated, Length, , Actual kb, 24.6*, , 23.1*, , 12.3, , 21.2*, , 9.4, , 7.2, , 7.4, , 6.6, , 6.7*, , 5.8*, , 4.3, , 6.5*, , 5.6*, , 2.3, , 5.6*, , 4.9, , 2.0, , 5.5*, , 3.5, , * Note: These fragments appear as a single band., , 7. Compare and contrast your calculated kilobase pair from the standard curve, with the actual kilobase pair for the restriction endonucleases., a. List those that were most accurate., b. List those that were least accurate., , Review Questions, 1. Why were the DNA digestions carried out at 37°C?, , 416, , Experiment 58: Lab Report, , Distance, (mm), , Calculated, Length
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10, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, 9, 8, 7, 6, 5, , 4, , 3, , 2, , 1, Migration Distance (mm) versus Fragment Size (kb), , Experiment 58: Lab Report, , 417
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2. Would any or all of these endonucleases cut the DNA of another bacteriophage or bacterium?, , 3. Why were specific restriction buffers needed for each of the restriction, enzyme digests?, , 4. What could account for low endonuclease activity?, , 5. Why are the restriction enzymes added last to the digestion mixtures?, , 6., , 7., , 418, , Assume you have one organism with a gene for ampicillin resistance and another organism with a gene for luciferinase. How, would you isolate the gene from one organism and connect it with the gene, of the other organism?, , How would restriction enzymes play a role in developing an organism produce a protein that it normally doesn’t make?, , Experiment 58: Lab Report
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PART 14, , Medical Microbiology, , LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be to, 1. Explain the methodology for isolating and identifying selected pathogenic microorganisms., 2. Describe the indigenous microbial flora of selected human anatomical sites., , Introduction, Although microorganisms are ubiquitous and their, benefits to humans have been recorded, a small, group of organisms remains a focus of concern:, They are the pathogens, whose existence makes, medical or clinical microbiology an especially, important science., That living agents are capable of inducing, infections (contagium vivum) was first put forward by the monk Fracastoro in Verona about 500, years ago. In 1659, Athanasius Kircher reported, the presence of minute motile organisms in the, blood of plague victims. Two hundred years, after Fracastoro developed his initial concept,, the germ theory of disease was formulated by, Marcus Antonius von Plenciz based on Antonie, van Leeuwenhoek’s revolutionary microscopic, observation of microorganisms. Perhaps the most, important contributions to microbiology were, made by Louis Pasteur, Robert Koch, and Joseph, Lister during the Golden Era of Microbiology,, from 1870 to 1920. These investigators and their, students recorded the observations and discoveries that cemented the cornerstone of medical, microbiology. The body of knowledge that has, accrued since these early years has made clinical, microbiology a major component of laboratory or, , diagnostic medicine. The major responsibility of, this science is isolating and identifying infectious, pathogens to enable physicians to treat patients, with infectious disease prudently, intelligently,, and rapidly., Many of the experiments described so far, have application in the field of clinical microbiology. Among these are isolation and identification, of unknown cultures, the use of selective and, differential media, and biochemical tests used to, separate and identify various microorganisms., Although studying all of the bacterial pathogens, responsible for human illness is not possible here,, routine experiments for isolating and identifying, some of the most frequently encountered infectious organisms and microorganisms that constitute the indigenous flora of the human body are, included. The pathogens chosen are pyogenic, cocci, members of the genera Staphylococcus and, Streptococcus, the Enterobacteriaceae, and the, organisms suspected in formation of dental caries., Experimental procedures designed for the detection and presumptive identification of microorganisms in blood and urine, which are normally sterile, body fluids, have also been incorporated into this, section. Organisms that naturally reside in or on, body surfaces and constitute the body’s normal, flora are also examined., , 419
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The need for the expeditious detection and, identification of pathogens has led to the development of rapid testing methods. These are microbiologically and immunologically based and can, be performed quickly and without the need for, sophisticated and expensive equipment. Some prototypic experiments using these rapid methods are, included along with the traditional procedures., , Many of the organisms that are used, although, attenuated by having been subcultured on artificial complex media for many generations, must, be viewed as potential pathogens and therefore, handled with respect. At this point in your training, your manipulative skills should be sufficiently, developed, allowing you to perform aseptically in, any medical, hospital, or clinical laboratory setting, to prevent infection of yourself and others., , FU RT HER R E ADING, Refer to the section in your textbook on the resident biota and oral bacteria. In your textbook’s, index, use the search terms “Oral Bacteria,”, “Normal Flora,” and “Dental Carries.”, , C ASE STUDY, SORE THROATS AND INFECTED CUTS, A patient is presented to you in the hospital’s, Infectious Diseases wing. This young patient complains of a sore throat that has been bothering, him for over two weeks and an infected cut on the, back of his right hand. The infected cut exhibits, a distinct swollen appearance with angry, raised, red skin and the beginning of streaking up the, wrist. Upon closer examination, you notice that, the areas directly around the cut are starting to, turn dark, a classic sign that necrosis is beginning, in the underlying tissues. The patient’s parents are, , 420, , Part 14, , confused when you order a throat swab while trying to determine the cause of the infected wound, on the hand., , Questions to Consider:, 1. What bacterial species is the most common, cause of sore throats?, 2. What implication could this have on determining which bacteria may be causing a necrotic, wound on the hand?
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Microbial Flora of the Mouth:, Determination of Susceptibility to, Dental Caries, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the organisms responsible for, dental caries., 2. Perform experiments that demonstrate the, host’s susceptibility to formation of caries., , Principle, A variety of microorganisms are known to be, involved in the formation of dental caries, including Lactobacillus acidophilus, Streptococcus, mutans, and Actinomyces odontolyticus. These, organisms in the oral flora produce organic acids,, particularly lactic acid, by fermenting carbohydrates that adhere to the surface of the teeth., In the continued presence of lactic acid, dental, enamel undergoes decalcification and softening,, which result in the formation of tiny perforations, called dental caries., The actual mechanism of action of these, organisms is still unclear. However, it has been, noted that S. mutans excretes an enzyme called, dextransucrase (glycosyl transferase), which is, capable of polymerizing sucroses into a large polymer, dextran, plus the monosaccharide fructose., This polysaccharide clings tenaciously to the teeth, and forms dental plaque, in which streptococci, reside and ferment fructose with the formation of, lactic acid (Figure 59.1)., Similarly, L. acidophilus produces lactic acid, as an end product of carbohydrate fermentation., Oral lactobacilli are capable of metabolizing glucose found in the mouth, producing organic acids, that reduce the oral acid concentration to a pH of, less than 5. At this pH, decalcification occurs and, dental decay begins., One of the best microbiological methods for, determining susceptibility to dental caries is the, Snyder test. This test measures the amount of, acid produced by the action of the lactobacilli on, glucose. The test employs a differential medium,, Snyder agar (pH 4.7), which contains glucose and, , E XP E R IMENT, , 59, , the pH indicator bromcresol green, which gives, the medium a green color., Following incubation, Snyder agar cultures, containing lactobacilli from the saliva will show, glucose fermentation with the production of acid,, which tends to lower the pH to 4.4, the level of, acidity at which dental caries form. At this pH the, green medium turns yellow. A culture demonstrating a yellow color within 24 to 48 hours is suggestive of the host’s susceptibility to the formation of, dental caries. A culture that does not change color, is indicative of lower susceptibility., , F U RT H E R RE A D I N G, Refer to the section in your textbook on the resident biota and oral bacteria. In your textbook’s, index, use the search terms “Oral Bacteria,” “Normal Flora,” and “Dental Carries.”, , C L I N I C A L A P P L I C AT I O N, Preventing Dental Caries, Factors that help control the development of dental, caries are proper oral hygiene, consumption of adequate fluoride, and moderation in the consumption, of foods that cause decay. Foods likely to lead to, decay are sticky, highly processed, and high in fermentable carbohydrates, such as breads, muffins,, and dried fruits. Also, the use of products to control, oral pH might help ensure that bacteria that cause, caries will not flourish., , AT T HE BE NCH, , Materials, Cultures, ❏❏ Organisms of the normal oral flora present in, saliva, , Media, Per designated student group, ❏❏ Two Snyder test agar deep tubes, 421
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H, , CH2OH, O, , HOCH2, , H, , H, , O, , Sucrase, , H, OH, , H, , HO, H, , O, , OH, , H, OH, , H, , +, , H, , HO, , CH2OH, , HOCH2, O, , H, , O, H, OH, , HO, H, , CH2OH, , CH2OH, , OH, , OH, , Glucose, , Sucrose, , HO, , H, , OH, H, , H, , H, , H, , Fructose, , CH2, O, , Glycosyl, transferase, O, CH2, , CH3, , O, H, O, , C, , OH, , COOH, Lactic acid, , CH2, O, , O, , Figure 59.1 Degradation of sucrose and, subsequent conversion of glucose into, dextran by Streptococcus mutans, , Equipment, ❏❏ Microincinerator or ❏❏ Mechanical pipetBunsen burner, ting device, ❏❏ Ice-water bath, ❏❏ Sterile test tubes, ❏❏ 1-in. square blocks, ❏❏ Glassware marking, of paraffin, pencil, ❏❏ Sterile 1-ml pipettes, , Procedure Lab One, 1. Melt two appropriately labeled Snyder agar, deep tubes and cool to 45°C., 2. Chew one square of paraffin for 3 minutes, without swallowing the saliva. As saliva, develops, collect it in a sterile test tube., 3. Vigorously shake the collected saliva sample, and transfer 0.2 ml of saliva with a sterile, pipette into one of the Snyder test medium, tubes that have been cooled to 45°C. Note:, Don’t let the pipette touch the sides of the, tubes or the agar., 4. Mix the contents of the tube thoroughly by, rolling the tube between the palms of your, hands or by tapping it with your finger., 5. Rapidly cool the inoculated tube of Snyder, agar in an ice-water bath., 6. Repeat Steps 3 through 5 to inoculate the second tube., 422, , Experiment 59, , Dextran, , 7. Incubate both tubes for 72 hours at 37°C., Observe cultures at 24, 48, and 72 hours., , Procedure Lab Two, 1. Examine the Snyder test cultures daily during, the 72-hour incubation period for a change in the, color of the culture medium. Use an uninoculated tube of the medium as a control. Figure 59.2, shows positive and negative Snyder tests., 2. Record the color of the cultures in the Lab, Report., , (a), , (b), , Figure 59.2 Snyder test. (a) No change in the color, indicates a negative result. (b) The color change to, yellow indicates a positive result.
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E XP E R IMENT, , 59, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Using Table 59.1 to interpret your observations, record your findings about susceptibility to caries in the chart below., TABLE 59.1, , Assessment of Susceptibility to Dental Caries, HOURS OF INCUBATION, , CARIES ACTIVITY, , 24, , 48, , 72, , Marked, , Positive, , ..., , ..., , Moderate, , Negative, , Positive, , ..., , Slight, , Negative, , Negative, , Positive, , Negative, , Negative, , Negative, , Negative, , Source: Courtesy of Difco Laboratories, Inc., Detroit, Michigan., Positive: Complete color change; green is no longer dominant., Negative: No color change or a slight color change; medium retains green color throughout., , COLOR OF SNYDER TEST CULTURES, Tube Number, , 24 hr, , 48 hr, , Caries Susceptibility, (Yes or No), , 72 hr, , Review Questions, 1. How would you explain the differential nature of the Snyder agar medium, as used for the detection of dental caries?, , Experiment 59: Lab Report, , 423
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2. How would you explain the mechanism responsible for the formation of, dental caries by resident microorganisms?, , 3. What is the function of the paraffin in this procedure?, , 4. Based on your results, what is your tendency to form dental caries? Is this, result consistent with your dental history?, , 5., , Are all members of the resident flora of the mouth capable of initiating dental caries? Explain., , 6., , What is the ideal time of day to perform this procedure? Why?, , 424, , Experiment 59: Lab Report
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Normal Microbial Flora, of the Throat and Skin, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Identify microorganisms that normally, reside in the throat and skin., , Principle, Normal flora are regularly found in specific areas, of the body. This specificity is far from arbitrary, and depends on environmental factors such as pH,, oxygen concentration, amount of moisture present, and types of secretions associated with each, anatomical site. Native microbial flora are broadly, located as follows:, 1. Skin: staphylococci (predominantly, Staphylococcus epidermidis), streptococci, (alpha-hemolytic, nonhemolytic), enterococci,, diphtheroid bacilli, yeasts, and fungi, 2. Eye conjunctiva: staphylococci, streptococci, diphtheroids, and neisseriae, 3. Upper respiratory tract: staphylococci;, streptococci (alpha-hemolytic, nonhemolytic,, and Streptococcus pneumoniae); enterococci;, diphtheroids; spirochetes; and members, of the genera Moraxella (formerly called, Branhamella), Neisseria, and Haemophilus, 4. Mouth and teeth: anaerobic spirochetes and, vibrios, fusiform bacteria, staphylococci, and, anaerobic levan-producing and dextran-producing streptococci responsible for dental caries, 5. Intestinal tract: in the upper intestine, predominantly lactobacilli and enterococci. In, the lower intestine and colon, 96% to 99% is, composed of anaerobes, such as members, of the genera Bacteroides, Lactobacillus,, Clostridium, and Streptococcus, and 1% to 4%, is composed of aerobes, including coliforms;, enterococci; and a small number of Proteus,, Pseudomonas, and Candida species., 6. Genitourinary tract: staphylococci, streptococci, lactobacilli, gram-negative enteric, , E XP E R IMENT, , 60, , bacilli, clostridia, spirochetes, yeasts, and protozoa, such as Trichomonas species, , Isolation of Microbial Flora, In this exercise, you will study the resident flora, of the throat and skin. Since these sites represent, sources of mixed microbial populations, you will, perform streak-plate inoculations, as outlined in, Experiment 2, to effect their separations. The discrete colonies thus formed can be studied morphologically, biochemically, and microscopically, to identify the individual genera of these mixed, flora., The procedure used to identify the native flora, of the throat involves the following steps:, 1. A blood agar plate is inoculated to demonstrate the alpha-hemolytic and beta-hemolytic, reactions of some streptococci and staphylococci. Hemolytic reactions on blood agar are, shown in Figure 60.1. A distinction between, these two genera can be made based on their, colonial and microscopic appearances. The, streptococci typically form pinpoint colonies, on blood agar, whereas the staphylococci, form larger pinhead colonies that might show, a golden coloration. When viewed under a, microscope, the streptococcal cells form, chains of varying lengths, whereas the staphylococci are arranged in clusters., 2. A chocolate agar plate is inoculated to detect, Neisseria spp. by means of the oxidase test., Members of this genus are recognized when, the colonies develop coloration that is pink to, dark purple on addition of p-aminodimethylaniline oxalate following incubation. Figure 60.2, shows colonies growing on chocolate agar, from a throat culture., 3. A Mueller-Hinton tellurite or Tinsdale, agar plate is inoculated to demonstrate the, presence of diphtheroids, which appear as, black, pinpoint colonies on this medium, (Figure 60.3). This coloration is due to the diffusion of the tellurite ions into the bacterial, cells and their subsequent reduction to tellurium metal, which precipitates inside the cells., 425
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(a) Beta hemolysis, , (b) Alpha hemolysis, , Figure 60.1 Beta- and alpha-hemolytic reactions on blood agar, , Figure 60.2 Colony growth on chocolate agar, , Figure 60.3 Mueller-Hinton tellurite agar plate., , from a throat culture, , Growth of black, pinpoint colonies indicates the, presence of diphtheroids., , The procedure used to identify the native flora, of the skin involves the following steps:, , elevated, moist, and glistening. Mold colonies, will appear as fuzzy, powdery growths arising from a mycelial mat in the agar medium., Figure 60.5 shows yeast colonies and a mold, colony., 4. A chocolate agar plate is inoculated to detect, Neisseria spp. The presence of Neisseria spp., produces pink-to-purple-to-black colonies on, this medium., 5. Mueller-Hinton tellurite or Tinsdale media, is inoculated to detect Corynebacterium spp., (diphtheroids). These colonies are black in, appearance., , 1. A blood agar plate is inoculated to determine, the presence of hemolytic microorganisms,, specifically the staphylococci and streptococci. Differentiation between these two genera may be made as previously described., 2. A mannitol salt agar plate is inoculated for the, isolation of the staphylococci. The generally, avirulent staphylococcal species can be differentiated from the pathogenic Staphylococcus, aureus because the latter is able to ferment, mannitol, causing yellow coloration of this, medium surrounding the growth. Figure 60.4, shows fermenter and a nonfermenter organisms on a mannitol salt agar plate., 3. A Sabouraud agar plate is inoculated to detect, yeasts and molds. Yeast cells will develop, pigmented or nonpigmented colonies that are, 426, , Experiment 60, , F U RT H E R RE A D I N G, Refer to the section in your textbook on the resident biota and transient bacteria. In your textbook’s, index, use the search terms “Transient Bacteria,”, “Normal Flora,” and “Opportunistic Bacteria.”
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C L I N I C A L A P P L I C AT I O N, Skin Flora and Acne, The bacterial population on a single human’s skin is, about 1012 organisms. A normal flora of microorganisms colonizes the human skin at birth as it passes, through the birth canal, and typically inhabits the, superficial layers of the epidermis and upper parts, of the hair follicles. They consist mainly of Staphylococcus epidermidis, Micrococcus, and corynebacteria such as Propionibacterium. Propionibacterium, acnes is normally found in low concentrations,, but overgrows in the anaerobic environment of a, blocked hair follicle, producing acne., , Fermenter, , Nonfermenter, , AT T HE BE NCH, , Materials, Media, Per designated student group, ❏❏ Two blood agar plates, ❏❏ Two mannitol salt agar plates, ❏❏ One chocolate agar plate, ❏❏ One Mueller-Hinton tellurite or Tinsdale agar, plate, ❏❏ One Sabouraud agar plate, ❏❏ Two 5-ml sterile saline tubes, , Reagents, ❏❏ Crystal violet, ❏❏ Gram’s iodine, ❏❏ Safranin, , ❏❏ 1% p-aminodimethylaniline oxalate, ❏❏ lactophenol–, cotton-blue, , Equipment, ❏❏ Sterile cotton swabs ❏❏ Microincinerator or, ❏❏ Tongue depressors, Bunsen burner, ❏❏ Desiccator jar with ❏❏ Glassware marking, candle, pencil, ❏❏ Microscope, ❏❏ Disposable gloves, ❏❏ Glass slides, Figure 60.4 Mannitol salt agar plate showing a, fermenter and a nonfermenter organism, , (a) Yeast colonies, , (b) Mold colony, , Figure 60.5 Sabouraud agar plate. (a) Yeast colonies have an elevated, moist, and, glistening appearance. (b) A mold colony shows fuzzy, powdery growth., , Experiment 60, , 427
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Procedure Lab One, , 8. Incubate the inverted Sabouraud agar plate for, 48 hours at 25°C and the remaining plates for, 48 hours at 37°C., , You must wear disposable gloves in Steps 1–3., , 1. Place a tongue depressor on the extended, tongue of the volunteer and, with a sterile cotton swab, obtain a specimen from the palatine, tonsil by rotating the swab vigorously over, its surface without touching the tongue, as, illustrated., , Palatine, tonsil, , Cotton swab, Tongue depressor, , 2. Inoculate a tube of sterile saline with the swab, and mix until you have a uniform suspension., 3. Using a sterile inoculating loop, inoculate one, plate each of blood agar, chocolate agar, mannitol salt agar, and Mueller-Hinton tellurite or Tins, dale agar, all previously labeled with the source, of the specimen, by means of a four-way streak, inoculation as described in Experiment 2., 4. Using a sterile cotton swab moistened in sterile saline, obtain a specimen from the skin by, rubbing the swab vigorously against the palm, of your hand., 5. Inoculate a tube of sterile saline with the swab, and mix the solution., 6. Inoculate one plate each of blood agar, mannitol salt agar, and Sabouraud agar, as described, in Step 3., 7. Incubate the inverted chocolate agar plate in a, CO2 incubator, in a CO2 incubation bag, or in, a candle jar. If you use the candle jar, place a, lighted candle in a desiccator jar and cover the, jar tightly to effect the 5% to 10% CO2 environment required for the growth of the Neisseria., Incubate the jar for 48 hours at 37°C., , 428, , Experiment 60, , Procedure Lab Two, Selection and Differentiation of Skin, and Throat Isolates, 1. Examine the blood agar plate cultures for, zones of hemolysis. (Refer to Figure 60.1 and, Experiment 14.), 2. Add the p-aminodimethylaniline oxalate to the, surface of the growth on the chocolate agar, plate. Observe for the appearance of a pink-topurple-to-black color on the surface of any of, the colonies (Figure 60.2)., 3. Examine the Mueller-Hinton tellurite or, Tinsdale agar plate for the presence of black, colonies (Figure 60.3)., 4. Examine the Sabouraud agar plate for the, appearance of mold-like growth (Figure 60.5)., 5. Examine the mannitol salt agar plate for, the presence of growth that is indicative of, staphylococci. Then examine the color of the, medium surrounding the growth. A yellow, color is indicative of S. aureus BSL -2 (Refer, to Figure 60.4 and Experiment 13.), 6. Record your observations in the Lab Report, and indicate the types of organisms that may, be present in each specimen., , Staining and Morphological, Characteristics of Skin and Throat, Isolates, 1. Prepare two Gram-stained smears from each, of the blood agar cultures, choosing wellisolated colonies that differ in their cultural, appearances and demonstrate hemolytic activity. Observe microscopically for the Gram, reaction and the size, shape, and arrangement, of the cells. Record your observations in, the Lab Report and attempt to identify each, isolate., 2. Prepare two lactophenol–cotton-blue–stained, smears of organisms obtained from discrete, colonies that differ in appearance on the Sabouraud agar culture. (Refer to Experiment 35.), Observe microscopically, draw a representative, field in the Lab Report, and attempt to identify, the fungi by referring to Experiment 35.
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E XP E R IMENT, , 60, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Isolation of Microbial Flora, Selection and Differentiation of Skin and Throat Isolates, Cultures, , Throat Specimen, , Skin Specimen, , Blood agar:, Staphylococcus spp., Streptococcus spp., Type of hemolysis: alpha, beta, , Chocolate agar:, Neisseria spp., 1 + 2 or 1 - 2 pink to purple to black, , Mueller-Hinton tellurite or Tinsdale:, Corynebacterium spp., 1 + 2 or 1 - 2 black colonies, , Sabouraud agar:, Fungal colonies, 1 + 2 or 1 - 2, , Mannitol salt agar:, Staphylococcus aureus, Other Staphylococcus spp., (S. epidermidis, S saprophyticus), 1 + 2 or 1 - 2 growth, , Color of medium, , Types of organisms present, , Experiment 60: Lab Report, , 429
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Staining and Morphological Characteristics of Skin and Throat Isolates, Skin Specimen, , Isolate 1, , Isolate 2, , Isolate 1, , Isolate 2, , Isolate 1, , Isolate 2, , Draw a representative field., , Gram reaction, Morphology, Organism, , Throat Specimen, , Draw a representative field., , Gram reaction, Morphology, Organism, , Sabouraud Agar Colonies Specimen, , Draw a representative field., , Morphology, Organism, , 430, , Experiment 60: Lab Report
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Review Questions, 1. How does the presence of residential flora influence the infectious process?, , 2. Why are some microorganisms termed “normal flora,” and of what value are, they to the well-being of the host?, , 3. A 6-year-old female is taken to her pediatrician for a checkup. As the doctor, takes the child’s history, her mother reports that the child had a severe sore, throat several weeks earlier that regressed without treatment. Upon examination, the pediatrician notes that the child has a systolic heart murmur consistent with mitral insufficiency and suspects that she has rheumatic fever., a. How was the earlier pharyngitis related to the subsequent development, of rheumatic fever?, , b. Rheumatic fever is diagnosed on clinical and serological findings. What, test should be done to diagnose rheumatic fever?, , c. How are patients with rheumatic fever treated?, , Experiment 60: Lab Report, , 431
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4. A 35-year-old female underwent serious abdominal surgery involving extensive bowel resection. She was maintained postoperatively on a regimen, of intravenous broad-spectrum antibiotics. Three days postoperative, she, spiked a fever without a clear source. She complains of vaginal discomfort. Blood cultures reveal the presence of an ovoid cell that reproduces by, budding., a. Based on this observation, what do you think this organism is?, , b. Is it part of the normal flora in humans?, , c. How did the treatment with broad-spectrum antibiotics predispose the, patient to infection with this organism?, , d. Compare the effectiveness of handwashing with water, with soap, and, with soap and surgical scrubbing., , 432, , Experiment 60: Lab Report
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Identification of Human, Staphylococcal Pathogens, , E XP E R IMENT, , 61, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the medical significance of the, staphylococci pathogen., 2. Use selected laboratory procedures to, differentiate between the major staphylococcal species., , Principle, The genus Staphylococcus is composed of both, pathogenic and nonpathogenic organisms. The, three major species are S. aureus, S. saprophyticus,, and S. epidermidis. Strains of the last two species, are generally avirulent; however, under special, circumstances in which a suitable portal of entry, is provided, S. epidermidis may be the etiological, agent for skin lesions and endocarditis, and S. saprophyticus may be the cause of some urinary tract, infections. Figure 61.1 is a streak-plate culture of, Staphylococcus aureus., Infections are primarily associated with, S. aureus pathogenic strains that are often, responsible for the formation of abscesses, localized pus-producing lesions. These lesions most, commonly occur in the skin and its associated, structures, resulting in boils, carbuncles, acne, and, impetigo. Infections of internal organs and tissues, are not uncommon, however, and include pneumonia, osteomyelitis (abscesses in bone and bone, marrow), endocarditis (inflammation of the endocardium), cystitis (inflammation of the urinary bladder), pyelonephritis (inflammation of the kidneys),, staphylococcal enteritis due to enterotoxin contamination of foods, and, on occasion, septicemia., Strains of S. aureus produce a variety of metabolic end products, some of which may play roles, in the organisms’ pathogenicity. Included among, these are coagulase, which causes clot formation;, leukocidin, which lyses white blood cells; hemolysins, which are active against red blood cells; and, enterotoxin, which is responsible for a type of gastroenteritis. Additional metabolites of a nontoxic, , Figure 61.1 Streak-plate culture of Staphylococcus, aureus. Produces colonies that are circular,, convex, smooth, and cream-colored to golden, yellow in appearance, , nature are DNase, lipase, gelatinase, and the fibrinolysin staphylokinase., When there is a possibility of staphylococcal, infection, isolation of S. aureus is of clinical, importance. These virulent strains can be differentiated from other staphylococci and identified, by a variety of laboratory tests, some of which are, illustrated in Table 61.1., In this exercise, you will distinguish among the, Staphylococcus species by performing traditional, test procedures, a computer-assisted multi-test procedure, or a newer rapid latex agglutination test., , Traditional Procedures, The traditional procedures involve the following, steps:, 1. Mannitol salt agar: This medium is selective, for salt-tolerant organisms such as staphylococci. Differentiation among the staphylococci, is predicated on their ability to ferment mannitol. Following incubation, mannitol-fermenting, organisms, typically S. aureus strains, exhibit, a yellow halo surrounding their growth, and, nonfermenting strains do not. (Refer to, 433
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TABLE 61.1, , Laboratory Tests for Differentiation of Staphylococcal Species, , TEST, , S. AUREUS, , S. EPIDERMIDIS, , S. SAPROPHYTICUS, , Growth, , +, , +, , +, , Fermentation, , +, , -, , -, , Generally golden yellow, , White, , White, , Coagulase, , +, , -, , -, , DNase, , +, , -, , -, , Generally beta, , -, , -, , Sensitive, , Sensitive, , Resistant, , Mannitol salt agar, , Colonial pigmentation, , Hemolysis, Novobiocin sensitivity, , Figure 60.4.) Note that other salt-tolerant, microorganisms, such as enterococci, are, capable of growth on mannitol salt agar. These, two genera are easily differentiated by performing a catalase test. Staphylococcus will, grow in the presence of catalase, while the, enterococci will not., 2. Coagulase test: Production of coagulase is, indicative of an S. aureus strain. The enzyme, acts within host tissues to convert fibrinogen to fibrin. Microbiologists theorize that, the fibrin meshwork that is formed by this, conversion surrounds the bacterial cells or, infected tissues, protecting the organism, from nonspecific host resistance mechanisms such as phagocytosis and the antistaphylococcal activity of normal serum. In, the coagulase tube test for bound and free, coagulase, a suspension of the test organism, in citrated plasma is prepared and the inoculated plasma is then periodically examined, for fibrin formation, or coagulation. Clot, formation within four hours is interpreted as, a positive result and indicative of a virulent, S. aureus strain. The absence of coagulation after 24 hours of incubation is a negative result, indicative of an avirulent strain, (Figure 61.2)., 3. Deoxyribonuclease (DNase) test: Generally,, coagulase-positive staphylococci also produce, the hydrolytic enzyme DNase; thus this test, can be used to reconfirm the identification, of S. aureus. The test organism is grown on, an agar medium containing DNA. Following, incubation, DNase activity is determined by the, addition of 0.1% toluidine blue to the surface, of the agar. DNase-positive cultures capable, , 434, , Experiment 61, , of DNA hydrolysis will show a rose-pink halo, around the area of growth. The absence of this, halo is indicative of a negative result and the, inability of the organism to produce DNase, (Figure 61.3)., 4. Novobiocin sensitivity: This test is used to, distinguish S. epidermidis from, S. saprophyticus. The Mueller-Hinton agar, plate is heavily seeded with the test organism, to produce a confluent growth on the agar, surface. After the seeding, a 30-μg, novobiocin antibiotic disc is applied to the, agar surface. Following incubation, the, sensitivity of an organism to the antibiotic, is determined by the Kirby-Bauer method, as described in Experiment 42 and as shown, in Figure 61.4., , STAPH-IDENT® System Procedure, A computer-assisted procedure is the API®, (Analytical Profile Index) STAPH-IDENT system (developed by Analytab Products, Division, of Sherwood Medical, Plainview, New York)., STAPH-IDENT is a rapid, computer-based micromethod for the separation and identification of, the newly proposed 13 species of staphylococci., The system consists of ten microcupules containing dehydrated substrates for the performance, of conventional and chromogenic tests. The, addition of a suspension of the test organism, serves to hydrate the media and to initiate the, biochemical reactions. The identification of the, staphylococcal species is made with the aid of, the differential charts or the STAPH-IDENT Profile Register that is part of the system (Table 61.2, on page 437), or both.
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(a) Positive coagulase test, , (b) Negative coagulase test, , Figure 61.2 Coagulase test. (a) Clot formation, indicates a positive result; (b) the absence of, coagulation is a negative result., , Latex Agglutination Procedure, The latex agglutination test is a rapid diagnostic, slide test for Staphylococcus aureus. The Remel, BactiStaph® diagnostic kit (Fisher Health Care) uses, protein-coated latex particles that are able to detect, the clumping factor (bound coagulase and protein, A) that causes the S. aureus to adhere to the black, latex particles, producing a visible agglutination., , Figure 61.4 Novobiocin test. Staphylococcus, aureus and Staphylococcus epidermidis (on top), are sensitive to the antibiotic, while Staphylococcus, saprophyticus (on bottom) is resistant., , F U RT H E R RE A D I N G, Refer to the section on bacterial metabolism, and virulence factors in your textbook for further information on the use of different enzyme, activities to further the infection process. In, your textbook’s index, search under “DNase,”, “Enzymatic Activity,” and “Coagulase.”, , C L I N I C A L A P P L I C AT I O N, Staphyloxanthin, Staphylococcus aureus is one of the most common, species of staphylococci to cause human disease,, producing many types of skin infections as well as, life-threatening diseases like meningitis, osteomyelitis, endocarditis, and toxic shock syndrome. Its, pathogenic success is due to its immune-evasive, properties, mainly through the production of its yellow pigment staphyloxanthin. This pigment behaves, as a virulence factor that helps the organism evade, the immune system of the host. Blocking the synthesis of staphyloxanthin may present a unique and, vital target for antimicrobials., , Figure 61.3 DNase test. A rose-pink halo around, the area of growth on the left side of the plate, indicates a positive result, while the absence of a, halo on the right is a negative result., , Experiment 61, , 435
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AT THE B E N C H, , Materials, Cultures, 24-hour Trypticase soy agar slant cultures of, ❏❏ Staphylococcus epidermidis, ❏❏ Staphylococcus saprophyticus (ATCC 15305), ❏❏ Staphylococcus aureus (ATCC 27660) BSL -2, Number-coded, 24-hour blood agar cultures of the, above organisms for the STAPH-IDENT system., , Media, Per designated student group, ❏❏ Three mannitol salt agar plates, ❏❏ One DNA agar plate, ❏❏ Three Mueller-Hinton agar plates, ❏❏ STAPH-IDENT system, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Citrated human or rabbit plasma, 0.1% toluidine blue, 0.85% saline (pH 5.5–7.0), McFarland barium sulfate standards, BactiStaph diagnostic kit (latex agglutination, test), , Equipment, ❏❏ Microincinerator, ❏❏ Inoculating loop, ❏❏ 13 * 100@mm test, tubes, ❏❏ 15 * 150@mm test, tubes, ❏❏ Sterile Pasteur, pipettes, ❏❏ 1-ml sterile pipettes, ❏❏ Mechanical, pipetting device, , ❏❏ Sterile cotton swabs, ❏❏ 30@mg novobiocin, antibiotic discs, ❏❏ Glassware marking, pencil, ❏❏ Metric ruler, ❏❏ Forceps, ❏❏ Beaker with 95%, ethyl alcohol, , Procedure Lab One, Traditional Procedures, 1. Preparation of DNA agar plate culture:, a. With a glassware marking pencil, divide, the bottom of the plate into three sections., Label each section with the name of the, organism to be inoculated., b. Aseptically make a single line of inoculation, of each test organism in its respective, sector on the agar plate., 2. Preparation of agar plate cultures for novobiocin sensitivity determination:, 436, , Experiment 61, , a. Label the three Mueller-Hinton agar plates, with the name of the test organism to be inoculated. Inoculate each plate with its respective organism according to the Kirby-Bauer, procedure as outlined in Experiment 42., b. Using alcohol-dipped and flamed forceps,, aseptically apply a novobiocin antibiotic disc, to the surface of each inoculated plate. Gently press the discs down with sterile forceps, to ensure that they adhere to the agar surface., 3. Preparation of mannitol salt agar plate cultures: Aseptically make a single line of inoculation of each test organism in the center of the, appropriately labeled agar plates., 4. Incubation of all plate cultures: Incubate them in, an inverted position for 24 to 48 hours at 37°C., 5. Coagulase test procedure:, a. Label three 13 * 100@mm test tubes with, the name of the organism to be inoculated., b. Aseptically add 0.5 ml of a 1:4 dilution of, citrated rabbit or human plasma and 0.1, ml of each test culture to its appropriately, labeled test tube., c. Examine the bacterial plasma suspensions for, clot formation at 5 minutes, 20 minutes, 1 hour,, and 4 hours after inoculation by holding the, test tubes in a slanted position. Record your, observations and results in the Lab Report., d. At the end of the laboratory session, place, all tubes that are coagulase-negative in an, incubator for 20 hours at 37°C., , STAPH-IDENT System Procedure, 1. Prepare strip:, a. Dispense 5 ml of tap water into incubation tray., b. Place API strip in incubation tray., 2. Prepare inoculum:, a. Add 2 ml of 0.85% saline (pH 5.5–7.0) to a, sterile 15 × 150-mm test tube., b. Using a sterile swab, pick up a sufficient, amount of inoculum to prepare a saline suspension with a final turbidity that is equivalent to a No. 3 McFarland (BaSO4) turbidity, standard. Note: be sure to use suspension, within 15 minutes of preparation., 3. With a sterile Pasteur pipette, add 2 or 3 drops, of the inoculum to each microcupule., 4. Place plastic lid on tray and incubate for 5, hours at 37°C., , Latex Agglutination Procedure, 1. Label three of the provided slides (cards) with, the name of the organism to be inoculated., 2. Place one drop of Staphylococcus latex reagent, in the center of the circle on the provided slide.
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3. Using an applicator stick or sterile needle,, spread one colony of each organism in the, reagent of its respective slide., 4. Spread the mixture over the entire circle., 5. Rotate the slide in a circular motion for 60, seconds., 6. Observe all slides for the presence or absence, of agglutination. A positive agglutination, reaction usually occurs in 15 seconds and is, indicated by a clumping together of the black, latex suspension, followed by the loss of the, black background. A negative reaction results, in little or no agglutination and no loss of the, black background within 60 seconds., 7. Record your results as positive (+ ) or as negative (- ) in the chart provided in the Lab Report., , Procedure Lab Two, Traditional Procedures, 1. Examine the coagulase-negative tubes, and, record your observations in the Lab Report., 2. Examine the mannitol salt agar plate. Note and, record the following in the Lab Report:, a. Presence (+ ) or absence (- ) of growth of, each test organism., b. Color of the medium surrounding the, growth of each test organism., TABLE 61.2 API, , c. Whether each test organism is a mannitol, fermenter (+ ) or non–mannitol fermenter (- )., 3. Flood the DNA agar plate with 0.1% toluidine, blue. Observe for the delayed development of a, rose-pink coloration surrounding the growth of, each test organism. Record your color observation and indicate the presence (+ ) or absence, (- ) of DNase activity in the Lab Report., 4. With a metric ruler, measure the size of the zone, of inhibition, if present, surrounding each of, the novobiocin discs on the agar plates. A zone, of inhibition of 17 mm or less is indicative of, novobiocin resistance, whereas a zone greater, than 17 mm indicates that the organism is sensitive to this antibiotic. Record the susceptibility, of each test organism to novobiocin as sensitive, (S) or resistant (R) in the Lab Report., , STAPH-IDENT System Procedure, 1. Interpret your STAPH-IDENT system reactions, on the basis of the observed color changes, in each of the microcupules described in the, chart in the Lab Report. Report your color, observations and results as (+ ) or (- ) for, each test in the Lab Report., 2. Construct a four-digit profile for your unknown, organisms using the guidelines provided in the, Lab Report., , STAPH-IDENT Profile Register, , PROFILE, 0 040, 0 060, 0 100, 0 140, 0 200, 0 240, 0 300, 0 340, 0 440, 0 460, 0 600, 0 620, 0 640, 0 660, , IDENTIFICATION, STAPH CAPITIS, STAPH HAEMOLYTICUS, STAPH CAPITIS, STAPH CAPITIS, STAPH COHNII, STAPH CAPITIS, STAPH CAPITIS, STAPH CAPITIS, STAPH HAEMOLYTICUS, STAPH HAEMOLYTICUS, STAPH COHNII, STAPH HAEMOLYTICUS, STAPH HAEMOLYTICUS, STAPH HAEMOLYTICUS, , 1 000, 1 040, 1 300, 1 540, 1 560, 2 541, 2 561, , STAPH EPIDERMIDIS, STAPH EPIDERMIDIS, STAPH AUREUS, STAPH HYICUS (An), STAPH HYICUS (An), STAPH SIMULANS, STAPH SIMULANS, , PROFILE, 2 000, 2 001, 2 040, 2 041, 2 061, 2 141, 2 161, 2 201, 2 241, 2 261, 2 341, 2 361, 2 400, 2 401, 2 421, 2 441, 2 461, 6 101, 6 121, , IDENTIFICATION, STAPH SAPROPHYTICUS, STAPH HOMINIS, STAPH SAPROPHYTICUS, STAPH SAPROPHYTICUS, STAPH HOMINIS, STAPH SIMULANS, STAPH SIMULANS, STAPH SIMULANS, STAPH SIMULANS, STAPH SAPROPHYTICUS, STAPH SIMULANS, STAPH SIMULANS, STAPH SIMULANS, STAPH SIMULANS, STAPH HOMINIS, STAPH SAPROPHYTICUS, STAPH SAPROPHYTICUS, STAPH SIMULANS, STAPH SIMULANS, STAPH SIMULANS, STAPH XYLOSUS, STAPH XYLOSUS, , NOVO R, NOVO S, NOVO R, NOVO S, , NOVO S, NOVO R, , Experiment 61, , 437
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E XP E R IMENT, , 61, , Name:, , Lab Report, , Section:, , Date:, , Observations and Results, Traditional Procedures, APPEARANCE OF PLASMA: CLOTTED ( + ) OR UNCLOTTED ( - ), Staphylococcal Species, , 5 min., , 20 min., , 1 hr., , 4 hr., , 24 hr., , Coagulase, (+ ) Or (−), , S. aureus, S. epidermidis, S. saprophyticus, , Procedure, , S. aureus, , S. epidermidis, , S. saprophyticus, , Mannitol salt agar:, Growth, Color of medium, Fermentation, DNA agar:, Color of medium, DNase activity, Novobiocin sensitivity:, Growth inhibition in mm, Susceptibility—(R) or (S), , STAPH-IDENT System Procedure, MICROCUPULE, , INTERPRETATION OF REACTIONS, , No., , Substrate, , Positive, , Negative, , 1, , PHS, , p-Nitrophenyl-phosphate,, disodium salt, , Yellow, , Clear or, , REACTION RESULTS, Color, , (+ ) Or (−), , straw-colored, , 2, , URE, , Urea, , Purple to red-orange, , 3, , GLS, , p-Nitrophenyl- b-dglucopyranoside, , Yellow, , Yellow or, yellow-orange, , 4, , MNE, , Mannose, , 5, , MAN, , Mannitol, , 6, , TRE, , Trehalose, , 7, , SAL, , Salicin, , 8, , GLC, , p-Nitrophenyl-b-dglucuronide, , Clear or, straw-colored, , Yellow or, yellow-orange, , Red or, , Yellow, , Clear or, straw-colored, , orange, , Experiment 61: Lab Report, , 439
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STAPH-IDENT System Procedure (continued), MICROCUPULE, , INTERPRETATION OF REACTIONS, , No., , Substrate, , Positive, , Negative, , 9, , ARG, , Purple to, , Yellow or, yellow-orange, , Arginine, , red-orange, 10, , NGP, , REACTION RESULTS, Color, , (∙ ) Or (∙), , Add 1–2 drops of STAPH-IDENT reagent, , 2@Naphthyl@β -dgalactopyranoside, , Plum-purple, , Yellow or, , (mauve), , colorless, , Construct a four-digit profile for your unknown organism as follows: A four-digit profile is derived from, the results obtained with STAPH-IDENT. The ten biochemical tests are divided into four groups, as, follows:, PHS, , MNE, , SAL, , URE, , MAN, , GLC, , GLS, , TRE, , ARG, , NGP, , Only positive reactions are assigned a numerical value. The value depends on the location within, the group., A value of 1 for the first biochemical in each group (e.g., PHS, MNE), A value of 2 for the second biochemical in each group (e.g., URE, MAN), A value of 4 for the third biochemical in each group (e.g., GLS, TRE), A value of 0 for all negative reactions, A four-digit number is obtained by totaling the values of each of the groups., , PHS, , URE, , GLS, , MNE, , MAN, , TRE, , SAL, , GLC, , ARG, , NGP, , Using Table 61.2 and your four-digit profile number, identify your organism., Unknown organism: ______________________________________________, , Latex Agglutination Procedure, Record the presence of agglutination as (+ ), and the absence of agglutination as (- )., S. aureus, Agglutination, No agglutination, , 440, , Experiment 61: Lab Report, , S. epidermidis, , S. saprophyticus
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E XP E R IMENT, , 62, , Identification of Human, Streptococcal Pathogens, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Discuss the medical significance of, streptococci pathogens., 2. Use selected laboratory procedures to, differentiate streptococci on the basis of, hemolytic activity and biochemical , patterns associated with the Lancefield, group classifications., , Principle, Members of the genus Streptococcus are perhaps, responsible for a greater number of infectious, diseases than any other group of microorganisms., Morphologically, they are cocci that divide in a, single plane, forming chains. They form circular,, translucent-to-opaque, pinpoint colonies on solid, media. All members of this group are gram-positive,, and many are nutritionally fastidious, requiring, enriched media such as blood for growth., The streptococci are classified by means of, two major methods: (1) their hemolytic activity,, and (2) the serologic classification of Lancefield. The observed hemolytic reactions on blood, agar are of the following three types:, 1. (𝛂) Alpha hemolysis, an incomplete form, of hemolysis, produces a green zone around, the colony. α@Hemolytic streptococci, the, Streptococcus viridans species, are u, sually, nonpathogenic opportunists. In some, instances, however, they are capable of, inducing human infections such as s ubacute, endocarditis, which may precipitate valvular damage and heart failure if untreated., Streptococcus pneumoniae, the causative, agent of lobar pneumonia, will be studied in, a separate experiment., 2. (b) Beta hemolysis, a complete destruction, of red blood cells, exhibits a clear zone of, approximately two to four times the diameter, , of the colony. The streptococci capable of, producing β@hemolysins are most frequently, associated with pathogenicity., 3. (γ) Gamma hemolysis is indicative of the, absence of any hemolysis around the colony., Most commonly, γ@hemolytic streptococci are, avirulent., These hemolytic reactions are shown in, , Figure 62.1., , Lancefield classified the streptococci into 20, serogroups, designated A through V, omitting, I and J, based on the presence of an antigenic, group-specific hapten called the C-substance., This method of classification generally implicates, the members of Groups A, B, C, and D in human, infectious processes., β@Hemolytic streptococci belonging to Group, A, and collectively referred to as Streptococcus, pyogenes, are the human pathogens of prime, importance. Members of this group are the main, etiological agents of human respiratory infections such as tonsillitis, bronchopneumonia,, and scarlet fever, as well as skin disorders such, as erysipelas and cellulitis. In addition, these, organisms are responsible for the development of, complicating infections, namely glomerulonephritis and rheumatic fever, which may surface, when primary streptococcal infections either go, untreated or are not completely eradicated by, antibiotics. The β-hemolytic streptococci found, in Group B are indigenous to the vaginal mucosa, and have been shown to be responsible for puerperal fever (childbirth fever), a sometimes-fatal, neonatal meningitis, and endocarditis. Members of Group C are also be-hemolytic and have, been implicated in erysipelas, puerperal fever,, and throat infections. The enterococci formerly, classified as Group D streptococci have been, reclassified and are now considered a separate, genus. The enterococci differ significantly from, other members of Group D, such as S. bovis,, which may be the etiological agent of urinary tract, infections. Enterococci such as Enterococcus faecalis may cause infections to the lungs, urinary, tract, or bloodstream through an intestinal laceration or poor personal hygiene. The enterococci, , 441
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(a) Alpha hemolysis, , (b) Beta hemolysis, , (c) Gamma hemolysis, , Figure 62.1 Types of hemolytic reactions on blood agar, , tend to be antibiotic-resistant, particularly to penicillin and more recently to vancomycin., The virulence of the streptococci is associated with their ability to produce a wide variety of, extracellular metabolites. Included among these, are the hemolysins (α and β), leukocidins that, destroy phagocytes, and the erythrogenic toxin, responsible for the rash of scarlet fever. Also of, medical significance are three metabolic end products that facilitate the spread of the organisms,, thereby initiating secondary sites of streptococcal, infection. These metabolites are hyaluronidase, (the spreading factor), which hydrolyzes the tissue, cement hyaluronic acid; streptokinase, a fibrinolysin; and the nucleases, ribonuclease and deoxyribonuclease, which destroy viscous tissue debris., Although the different groups of streptococci, have similar colonial morphology and microscopic, appearance, they can be separated and identified, , TABLE 62.1 , , by the performance of a variety of laboratory, tests. Toward this end, you will perform laboratory procedures to differentiate among the medically significant streptococci on the basis of their, Lancefield group classification and their hemolytic patterns. Table 62.1 will aid in this separation., Identification of Group A streptococci, involves the following procedures:, 1. Bacitracin sensitivity test: A filter-paper, disc impregnated with 0.04 unit of bacitracin is applied to the surface of a blood agar, plate previously streaked with the organism, to be identified. Following incubation, the, appearance of a zone of growth inhibition, surrounding the disc is indicative of Group A, streptococci. Absence of this zone suggests a, non–Group A organism. Figure 62.2 shows the, result of a bacitracin sensitivity test., , Laboratory Differentiation of Streptococci, , GROUP:, , A, , B, , C, , D, , K,H,N, , S. bovis, NON-ENTEROCOCCI, , S. salivarius, S. sanguis, S. mitis, , ORGANISMS:, , S. pyogenes, , S. agalactiae, , S. equi, , Hemolysis, , β, , αSγ, , β, , Bacitracin sensitivity, , R, , R, , R, , CAMP test, , -, , +, , -, , E. faecalis, ENTEROCOCCI, , A, , αSγ, , R, , R, , R, , -, , -, , -, , Bile esculin hydrolysis, , -, , -, , -, , +, , -, , +, , 6.5% NaCl medium, , NG, , NG, , NG, , NG, , NG, , G, , Growth at 10°C, , NG, , NG, , NG, , NG, , NG, , G, , Growth at 45°C, , NG, , NG, , NG, , NG or G, , NG, , G, , NG = no growth; G = growth; S = sensitive; R = resistant, , 442, , Experiment 62
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CAMP-positive test, Group B streptococci, with arrowhead zone, of increased hemolysis, , Beta-hemolytic, Staphylococcus, aureus, CAMP-negative test, Group A streptococci, , Figure 62.2 Bacitracin sensitivity test. Positive for, , Figure 62.3 CAMP reactions, , beta-hemolytic Group A streptococci on the left;, negative on the right, , 2. Directigen™ test: A rapid, non–growthdependent immunological procedure for the, detection of the Group A antigen, developed, by Becton Dickinson and Company. In this, test, a clinical specimen is subjected to, reagents designed to extract the Group A, antigen, which is then mixed with a reactive, and a negative control latex. Agglutination, with the reactive latex is indicative of Group A, streptococci., Group B streptococci are identified with the, CAMP test (named for Christie, Atkins, and, Munch-Petersen). Group B streptococci produce a, peptide, the CAMP substance, that acts in concert, with the β-hemolysins produced by some strains, of Staphylococcus aureus, causing an increased, hemolytic effect. Following inoculation and incubation, the resultant effect appears as an arrowshaped zone of hemolysis adjacent to the central, streak of S. aureus growth. The non–Group, B streptococci do not produce this reaction., Figure 62.3 illustrates the CAMP reactions., Identification of Group D streptococci, involves the following:, 1. Bile esculin test: In the presence of bile,, Group D streptococci hydrolyze the glycoside, esculin to 6,7-dihydroxy coumarin that reacts, with the iron salts in the medium to produce a, brown-to-black coloration of the medium following incubation (Figure 62.4). Lack of this, dark coloration is indicative of a non–Group D, organism., 2. 6.5% sodium chloride broth: The enterococci can be separated from the non-enterococci by the ability of the former to grow, , Figure 62.4 Positive bile esculin test. A brown-toblack coloration of the medium indicates positive, identification of Group D streptococci, , in this medium. This reaction is shown in, Figure 62.5., Hemolytic activity is identified with a blood, agar medium. The pathogenic streptococci, primarily the β@hemolytic, can be separated from, the generally avirulent α- and γ@hemolytic streptococci by the type of hemolysis produced on blood, agar, as previously described., , F U RT H E R RE A D I N G, Refer to the section on bacterial metabolism, and virulence factors in your textbook for further information on the use of different enzyme, activities to further the infection process. In, your textbook’s index, search under “Hemolysins,”, “Streptokinase,” and “Leukocidins.”, , Experiment 62, , 443
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C L I N I C A L A P P L I C AT I O N, Streptococci Infections, Medically, the streptococci are of significant importance because they are responsible for a wide, variety of infections, many of which are pyogenic, (pus-producing). Streptococcus agalactiae (Lancefield group B) may colonize the vagina as well as, the upper respiratory tract of humans, and is the, most frequent cause of neonatal pneumonia in the, United States. Meanwhile, Streptococcus pyogenes, (Lancefield group A) Causes necrotizing fasciitis,, a rare but devastating infection that destroys skin,, muscle, and underlying tissue. The CAMP test is, used to identify Group A Streptococcus pyogenes, from Group B Streptococcus agalatiae., , Media, Per designated student group, ❏❏ Five blood agar plates, ❏❏ Three bile esculin agar plates, ❏❏ Three 6.5% sodium chloride broths, , Reagents, Directigen Rapid Group A Strep Test (Becton, Dickinson and Company), ❏❏ Crystal violet, ❏❏ Gram’s iodine, ❏❏ Ethyl alcohol, ❏❏ Safranin, ❏❏ Taxo™ A discs (0.04 unit of bacitracin), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, (a), , (b), , Figure 62.5 65% sodium chloride test. (a) Growth, indicates the presence of Group D enterococci., (b) The absence of growth indicates the presence, of Group D non-enterococci., , AT THE B E N C H, , Materials, Cultures, 24-hour blood agar slant cultures of, ❏❏ Streptococcus pyogenes (ATCC 12385) BSL -2, ❏❏ Enterococcus faecalis BSL -2, ❏❏ Streptococcus bovis, ❏❏ Streptococcus agalactiae BSL -2, ❏❏ Streptococcus mitis BSL -2, ❏❏ Staphylococcus aureus (ATCC 25923) BSL -2, , 444, , Experiment 62, , Microincinerator, Inoculating loop, Staining tray, Lens paper, Bibulous paper, Microscope, Sterile cotton swabs, Glassware marking pencil, Sterile 12 * 75@mm test tubes, Sterile Pasteur pipettes, Sterile applicators, 95% ethyl alcohol in beaker, Forceps, Mechanical rotator, , Procedure Lab One, 1. Prepare a Gram-stained preparation of each, streptococcal culture and observe under oil, immersion. Record in the Lab Report your, observations of cell morphology and Gram, reaction., 2. Prepare the blood agar plate cultures to identify the type of hemolysis as follows:, a. With a glassware marking pencil, divide the, bottoms of two blood agar plates to accommodate the five test organisms. Label each, section with the name of the culture to be, inoculated., b. Using aseptic inoculating technique, make, a single line streak of inoculation of each, organism in its respective sector on the, blood plates., 3. Prepare the blood agar plate cultures for the, bacitracin test as follows:
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4., , 5., , 6., , 7., , a. With a glassware marking pencil, label the, covers of two blood agar plates with the, names of the organisms to be inoculated,, S. pyogenes BSL -2 and S. agalactiae BSL -2 ., b. Using a sterile cotton swab, inoculate the, agar surface of each plate with its respective test organism by streaking first in a horizontal direction, then vertically to ensure a, heavy growth over the entire surface., c. Using alcohol-dipped and flamed forceps,, apply a single 0.04-unit bacitracin disc to, the surface of each plate. Gently touch each, disc to ensure its adherence to the agar, surface., Prepare a blood agar plate culture for the, CAMP test as follows:, a. Using a sterile inoculating loop, make a, single line of inoculation along the center of, the plate using the S. aureus BSL -2 culture., b. With a sterile loop, inoculate S. pyogenes, BSL -2 on one side and perpendicular to, the central S. aureus BSL -2 streak, s tarting, about 5 mm from the central streak and, extending toward the periphery of the agar, plate., c. On the opposite side of the central streak,, but not directly opposite the S. pyogenes, BSL -2 line of inoculation, repeat Step 4b, using S. agalactiae BSL -2 ., Prepare the bile esculin agar plate cultures as, follows:, a. Label the three bile esculin plates with the, names of the organisms to be inoculated,, S. bovis, S. mitis BSL -2 , and E. faecalis, BSL -2 ., b. Aseptically inoculate each plate with its test, organism by making several lines of inoculation on the agar surface., Prepare 6.5% sodium chloride broth cultures, as follows:, a. Label three tubes of 6.5% sodium chloride, broth with the names of the organisms to be, inoculated, S. bovis, E. faecalis BSL -2 , and, S. mitis BSL -2 ., b. With a sterile loop, inoculate each tube with, its organism., Conduct the Directigen test procedure as, follows:, a. Label two sterile 12 * 75@mm test tubes, as S. pyogenes BSL -2 and S. agalactiae, BSL -2 ., , b. A, dd 0.3 ml of Reagent 1 to both test tubes., c. Using a sterile cotton swab, transfer the, test organisms into their respectively, labeled test tubes. Note: These samples, will emulate the throat swabs obtained in, a clinical solution., d. Add 1 drop of Reagent 2 to each test tube., Mix by rotating the swab against the side of, the tube. Allow the swabs to remain in the, test tubes for 3 minutes., e. Add 1 drop of Reagent 3 to both tubes and, mix., f. Remove swabs after extracting as much liquid as possible by rolling them against the, sides of the tubes., g. Place 1 drop of negative antigen control on, both circles in Column A of test slide., h. Place 1 drop of positive antigen control on, both circles in Column B of test slide., i. Dispense 1 drop of each streptococcal, sample on both circles in Columns C and, D, respectively., j. Using a new sterile applicator for each, specimen, spread each specimen within the, confines of both circles in Columns A, B, C,, and D., k. Add 1 drop of reactive latex to the top row, of circles., l. Add 1 drop of control latex to the bottom, row of circles., m. Place the slide on a mechanical rotator for, 4 minutes under a moistened humidifying, cover., n. Compare the agglutination seen in the, upper “reactive latex” circles with the consistency of the latex in the bottom “control, latex” circles. Any agglutination in the top, circles distinct from any background granules seen in the bottom circles indicates, Group A streptococci., 8. Incubate all tubes and plates in an inverted, position for 24 hours at 37°C., , Procedure Lab Two, 1. Examine the two blood agar plates for bacitracin activity. Record in the Lab Report your, observations of the presence (+ ) or absence, (- ) of a zone of inhibition of any size surrounding the discs., , Experiment 62, , 445
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2. Examine the blood agar plate for the CAMP, reaction. Record your observations of the, presence (+ ) or absence (- ) of increased, arrow-shaped hemolysis., 3. Examine the bile esculin plates for the presence (+ ) or absence (- ) of a brown-black, coloration in the medium and record your, observations., 4. Observe the 6.5% sodium chloride broth cultures for the presence (+ ) or absence (- ) of, growth and record your observations., 5. Examine the two blood agar plates for the, presence and type of hemolysis produced by, , 446, , Experiment 62, , each of the test organisms. Record your observations of the appearance of the medium surrounding the growth and the type of hemolytic, reaction that has occurred—α, β, or γ., 6. Observe the Directigen test slide for the presence (+ ) or absence (- ) of agglutination in, the reactive and control latex circles., 7. Based on your observations, classify each test, organism according to its Lancefield group., Record your results., 8. Check that all of your observations have been, recorded in the Lab Report.
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E XP E R IMENT, , 62, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Procedure, , S. pyogenes, , S. agalactiae, , S. bovis, , E. faecalis, , S. mitis, , Gram stain:, Morphology, Reaction, Bacitracin test:, Zone of inhibition, CAMP test:, Increased hemolysis, Bile esculin test:, Color of medium, Result: ( + ) or ( - ), 6.5% NaCl broth:, Growth, Hemolytic activity:, Appearance of medium, Type of hemolysis, Directigen test:, Agglutination ( + ) or ( - ) in:, Reactive circle, Control circle, Lancefield group, Group classification, , Review Questions, 1. How do the purposes of the bacitracin and CAMP tests differ?, , Experiment 62: Lab Report, , 447
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2. What is the mechanism of the bile esculin test?, , 3. Why is it important medically to distinguish between the enterococci and the non-enterococci?, , 4., , 5., , 448, , Why can some streptococci produce secondary sites of infection?, , The streptococci are known to be fastidious organisms that require an enriched medium for, growth. How would you account for the fact that a medium enriched with blood (blood, agar) is the medium of preference for growth of these organisms?, , Experiment 62: Lab Report
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Identification of Streptococcus, Pneumoniae, , Once you have completed this experiment,, you should be able to, 1. Use the laboratory procedures required, to differentiate between Streptococcus, pneumoniae and other α@hemolytic, streptococci., , Principle, The pneumococcus Streptococcus pneumoniae is, the major α@hemolytic, streptococcal pathogen in, humans. It serves as an etiological agent of lobar, pneumonia, an infection characterized by acute, inflammation of the bronchial and alveolar membranes. These organisms are gram-positive cocci,, tapered or lancet-shaped at their edges, and occur, in pairs or as short, tight chains. The large, thick, capsules formed in vivo are responsible for antiphagocytic activity, which is believed to enhance, the organisms’ virulence. In addition, the pneumococci produce -A@hemolysis on blood agar plates., Figure 63.1 shows the effects of Streptococcus, pneumoniae on blood agar. Because of these properties (short-chain formation, α@hemolysis, and, failure of the capsule to stain on Gram staining),, the organisms closely resemble Streptococcus viridans species. The S. pneumoniae can be differentiated from other α@hemolytic, streptococci on the, basis of the following laboratory tests:, S. pneumoniae, , 63, , Brief descriptions of the tests and their mechanisms follow:, , LEARNING OBJECTIVE, , Test, , E XP E R IMENT, , 1. Bile solubility test: In the presence of, surface-active agents such as bile and bile, salts (sodium desoxycholate or sodium, dodecyl sulfate), the cell wall of the pneumococcus undergoes lysis. Other members of the, a@hemolytic streptococci will not be lysed by, these agents and are bile-insoluble. Following, incubation, bile-soluble cultures will appear, clear, and bile-insoluble cultures will be turbid., 2. Optochin sensitivity test: This is a, growth inhibition test in which 6-mm, filter-paper discs impregnated with 5 mg, of ethylhydrocupreine hydrochloride, (optochin) and called Taxo P discs are applied, to the surface of a blood agar plate streaked, with the test organisms. The S. pneumoniae,, being sensitive to this surface-active agent, are, lysed with the resultant formation of a zone of, inhibition greater than 15 mm surrounding the, P disc. Nonpneumococcal α@hemolytic streptococci are resistant to optochin and either fail, to show a zone of inhibition or produce a zone, smaller than 15 mm. Sensitivity to optochin is, illustrated in Figure 63.2., , S. mitis, , Hemolysis, , α, , α, , Bile solubility, , +, , -, , Optochin sensitivity, , +, , -, , Inulin fermentation, , +, , -, , Quellung reaction, , +, , -, , Mouse virulence, , +, , -, , Figure 63.1 Streptococcus pnuemoniae forms, large thick capsules and produces alpha hemolysis, on blood agar, , 449
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F U RT H E R RE A D I N G, Refer to the section on gram-positive bacteria, and virulence factors in your textbook for further, information on the use of different enzyme activities to further the infection process. In your textbook’s index, search under “Optochin,” “Inulin,”, and “Hemolysis.”, , C L I N I C A L A P P L I C AT I O N, , Figure 63.2 Optochin sensitivity test. The, formation of a zone of inhibition greater than 15, mm on the left indicates the presence of alphahemolytic S. pneumoniae. The absence of a zone, of inhibition on the right indicates the presence of, other alpha-hemolytic streptococcal species, , 3. Inulin fermentation: The pneumococci, are capable of fermenting inulin, while most, other α@hemolytic streptococci are non–inulin, fermenters. Following incubation, the acid, resulting from inulin fermentation will change, the color of the culture from red to yellow., Cultures that are not capable of fermenting, inulin will not exhibit a color change, which is, a negative test result., 4. Quellung (Neufeld) reaction: This, capsular swelling reaction is a sensitive and, accurate method of determining the presence, of S. pneumoniae in sputum. The reaction of, the pneumococcal capsular polysaccharide, a, hapten antigen, with an omnivalent capsular, antiserum (Abcam, Inc.) produces a microscopically visible swollen capsule surrounding, the S. pneumoniae organisms., 5. Mouse virulence test: Laboratory white, mice are highly susceptible to infection by, S. pneumoniae and resistant to other streptococcal infections. Intraperitoneal injection of, 0.1 ml of pneumococcus-infected sputum will, kill the mouse. Examination of the peritoneal, fluid by Gram stain and culture will reveal the, presence of S. pneumoniae., In the following experiment, you will use, hemolytic patterns, bile solubility, the Quellung, reaction, the optochin test, and the inulin fermentation test for laboratory differentiation of, S. pneumoniae from other α@hemolytic, streptococci., , 450, , Experiment 63, , Pneumococcus Infections, Streptococcus pneumoniae, formerly called Diplococcus pneumoniae, appears as a lancet-shaped, diplococcus and is unlike all other cocci. The pneumococcus, as it is called, is the causative agent of lobar, pneumonia (lung), otitis media (middle ear), and meningitis (meninges) infections. It is currently the leading, invasive bacterial disease in children and the elderly., Presently a vaccine is available for people who are, designated as high-risk for infection with this organism., , AT T HE BE NCH, , Materials, Cultures, 24-hour blood agar slant cultures of, ❏❏ Streptococcus pneumoniae BLS -2, ❏❏ Streptococcus mitis BLS -2, , Media, Per designated student group, ❏❏ One blood agar plate, ❏❏ Two phenol red inulin broth tubes, ❏❏ Four 13 * 75-mm tubes containing 1 ml of, nutrient broth, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Crystal violet, Gram’s iodine, Ethyl alcohol, Safranin, Methylene blue, 10% sodium desoxycholate, Commercially available Taxo P discs (5 mg of, optochin), ❏❏ Omnivalent pneumococcal antiserum
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Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator, Waterbath, Inoculating loop, Glass slides, Coverslips, Sterile cotton swabs, Sterile 1-ml serological pipettes, Mechanical pipetting device, 95% ethyl alcohol in beaker, Forceps, Glassware marking pencil, , Procedure Lab One, 1. Bile solubility test, a. Label two nutrient broth tubes S. pneumoniae BLS -2 and two other tubes, S. mitis BLS -2 ., b. Aseptically add 2 loopfuls of the test organisms to the appropriately labeled sterile test, tubes to effect a heavy suspension., c. Aseptically add 0.5 ml of sodium desoxycholate to one tube of each test culture., The remaining two cultures will serve as, controls., d. Incubate the tubes in a waterbath at 37°C, for 1 hour., e. After incubation, examine the tubes for, the presence or absence of turbidity in, each culture. Record your observations of, the appearance (clear or turbid) and bile, solubility of each test organism in the Lab, Report., 2. Optochin test, a. With a glassware marking pencil, divide the, bottom of a blood agar plate into two equal, sections, and label one section, S. pneumoniae BLS -2 and the other S., mitis BLS -2 ., b. Using a sterile cotton swab, heavily inoculate the surface of each section with its, respective test organism in a horizontal and, then vertical direction, being careful to stay, within the limits of each section., c. Using alcohol-dipped and flamed forceps,, apply a single Taxo P disc (optochin) to the, , surface of the agar in each section of the, inoculated plate. Touch each disc slightly to, ensure its adherence to the agar surface., d. Incubate the plate in an inverted position, for 24 to 48 hours at 37°C., 3. Inulin fermentation test, a. Label two phenol red inulin broth tubes, with the name of each test organism to be, inoculated., b. Using aseptic technique and loop inoculation, inoculate each experimental organism, in its appropriately labeled tube of medium., c. Incubate the tube cultures for 24 to 48, hours at 37°C., 4. Quellung reaction, a. Spread a loopful of each test culture on a, separate labeled clean glass slide and allow, the slides to air-dry., b. Place a loopful of the omnivalent capsular, antiserum and a loopful of methylene blue, on each of two coverslips., c. Place the coverslips over the dried bacterial, smears. Prepare a Gram-stained preparation, of each test organism and observe under, oil immersion. Record your observations of, cell morphology and Gram reaction in the, Lab Report., , Procedure Lab Two, 1. Examine blood agar plates for the presence, of hemolysis and optochin activity by measuring the zone of inhibition, if any, surrounding, the disc. Record the measurement in the Lab, Report and indicate whether each organism is, optochin-sensitive (zone of inhibition greater, than 15 mm) or optochin-resistant (no zone, or, zone less than 15 mm)., 2. Observe the inulin fermentation broth cultures, containing phenol red, and record the color of, each culture and whether it is indicative of a, positive (+ ) or negative (- ) result in the Lab, Report., 3. Examine slides of the Quellung reaction under, oil immersion and indicate in the Lab Report, the presence (+ ) or absence (- ) of capsular, swelling surrounding the blue-stained cells., , Experiment 63, , 451
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2. How would you separate S. pneumoniae from other α@hemolytic, streptococci?, , 3., , What are secondary pneumonias? Why do they develop most, frequently following viral infections?, , 4., , Why did it require many years of research to develop an effective,, long-term pneumococcal vaccine?, , 454, , Experiment 63: Lab Report
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Identification of Enteric, Microorganisms Using ComputerAssisted Multitest Microsystems, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Discuss the members of the family, Enterobacteriaceae., 2. Use laboratory procedures to identify, enteric pathogens using commercial, multi-test microsystems., , Principle, Enterobacteriaceae is a significant group of bacteria that is endogenous to the intestinal tract or that, may gain access to this site via a host’s ingestion, of contaminated food and water. The family consists of a number of genera whose members vary, in their capacity to produce disease. Salmonella, and Shigella are considered to be pathogenic., Members of other genera, particularly Escherichia, and Enterobacter, and to a lesser extent Klebsiella, and Proteus, constitute the natural flora of the, intestines and are generally considered to be avirulent. Remember, however, that all can produce, disease under appropriate conditions., The Enterobacteriaceae are gram-negative,, short rods. They are mesophilic, nonfastidious, organisms that multiply in many foods and water, sources. They are all non–spore-formers and susceptible to destruction by common physical and, chemical agents. They are resistant to destruction, by low temperatures and can therefore frequently, survive in soil, sewage, water, and many foods for, extended periods., From a medical point of view, the pathogenic, Enterobacteriaceae are salmonellae and shigellae., Salmonellae are responsible for enteric fevers,, typhoid, the milder paratyphoid, and gastroenteritis. In typhoid, Salmonella typhi penetrates, the intestinal mucosa and enters the bloodstream,, thus infecting organs such as the gallbladder, intestines, liver, kidney, spleen, and heart. Ulceration, , E XP E R IMENT, , 64, , of the intestinal wall, caused by the release of the, lipopolysaccharide endotoxin into the blood over, a long febrile period, and enteric symptoms are, common. Gastroenteritis is caused by a number of Salmonella species. Symptoms associated, with this type of food poisoning include abdominal pain, nausea, vomiting, and diarrhea, which, develop within 24 hours of ingestion of contaminated food and last for several days., Several shigellae are responsible for shigellosis, a bacillary dysentery that varies in severity., Ulceration of the large intestine, explosive diarrhea, fever, and dehydration occur in the more, severe cases., Isolation and identification of enteric bacteria, from feces, urine, blood, and fecally contaminated, materials are of major importance in the diagnosis, of enteric infections. Although the Enterobacteriaceae are morphologically alike and in many ways, metabolically similar, laboratory procedures for, the identification of these bacteria are based on, differences in biochemical activities (Figure 64.1)., There are several multitest systems that have, been developed for differentiation and identification of members of the Enterobacteriaceae. They, use microtechniques that incorporate a number, of media in a single unit. Many of these multitest, systems are currently commercially available and, widely used. The obvious advantages of these units, are the need for minimal storage space, the use of, less media, the rapidity with which results may be, obtained, and the applicability of the results to a, computerized system for identification of organisms. There are also certain disadvantages with, these systems, including difficulty in obtaining the, proper inoculum size, since some media require, heavy inoculation while others need to be lightly, inoculated; the possibility of media carryover from, one compartment to another; and the possibility of, using inoculum of improper age. Despite these difficulties, when properly correlated with other properties such as Gram stain and colonial morphology on, specialized solid media, these systems are acceptable for the identification of Enterobacteriaceae., The most frequently used systems are discussed., , 455
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Fecal specimen, (stool or rectal swab), Enrichment broth, Selinite broth inhibits gram-positives and selects, Salmonella and Shigella species, , Direct plating, Differential media such as MacConkey agar,, EMB, and Hektoen enteric agar may also be, used (see Exp. 14)., , Subculture, Differential media such as MacConkey agar,, EMB, or Hektoen enteric agar may also be used, (see Exp. 14)., , Isolated colonies, Lactose+, Lactose-, , Isolated colonies, Lactose+, Lactose-, , Triple sugar–iron agar (see Exp. 23), Acid slant, Acid butt, H2S-, , Acid slant, Acid butt, H2S+, , Alkaline slant, Acid butt, H2S+, , Alkaline slant, Alkaline or no, change butt, , Escherichia, Klebsiella, Enterobacter, , Citrobacter, Arizona, Some Proteus spp., , Most Salmonella, Arizona, Citrobacter, , Alcaligenes, Pseudomonas, Acinetobacter, , Biochemical tests for positive identification, , IMViC (see Exp. 24), Carbohydrate fermentation (see Exp. 22), Motility (see Exp. 25), Urease (see Exp. 26), , Figure 64.1 Conventional laboratory procedures for isolation and identification of enteric microorganisms, , Enterotube II Multitest System, and ENCISE II, The Enterotube™ II Multitest System (Roche, Diagnostics, Division of Hoffmann-La Roche,, Inc.) consists of a single tube containing 12 compartments (Figure 64.2a) and a self-enclosed, inoculating needle. This needle can touch a, single isolated colony and then in one operation, be drawn through all 12 compartments, thereby, inoculating all of the test media. In this manner,, , 456, , Experiment 64, , 15 standard biochemical tests can be performed, in one inoculating procedure. Following incubation, the color changes that occur in each of the, compartments are interpreted according to the, manufacturer’s instructions to identify the organisms (Figure 64.2b). This method has been further, refined to permit identification of the enteric, bacteria by means of a computer-assisted system, called ENCISE (Enterobacteriaceae numerical, coding and identification system for Enterotube).
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Cap, , Handle, , Individual compartments, , Inoculating needle, , (a) Diagram of Enterotube, , Inoculated, with test, organism, Uninoculated, control, , (b) Inoculated and uninoculated control, , Figure 64.2 Enterotube II Multitest System, , Plastic strip, Cupule, Microtube, Dehydrated, media, (a) Uninoculated control, , (b) Inoculated with test organism, , Figure 64.3 The API 20-E system, , API (Analytical Profile Index) System, ®, , The API 20-E employs a plastic strip composed, of 20 individual microtubes, each containing a, dehydrated medium in the bottom and an upper, cupule as shown in Figure 64.3. The media, become hydrated during inoculation of a suspension of the test organism, and the strip is then, incubated in a plastic-covered tray to prevent, evaporation. In this manner, 22 biochemical tests, are performed. Following incubation, identification of the organism is made by using differential, charts supplied by the manufacturer or by means, , of a computer-assisted system called PRS (Profile, Recognition System). PRS includes an API coder,, profile register, and selector., In the following experiment, you will inoculate, an Enterotube and an API strip with an unknown, enteric organism. Following incubation, you will, make your identification by two methods: (1) the, traditional method of noting the characteristic, color changes and interpreting them according to, manufacturer’s instructions, and (2) the computerassisted methods illustrated in Figure 64.4., , Experiment 64, , 457
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L, Y, S, , 2+1, , 4, , ID value =, , O, R, N, , 2 + 1, , +, , 3, , H2S, , G G, L A, U S, , I, N, D, , A, D, O, , L, A, C, , A, R, B, , S, O, R, , 4 + 2, , + 1, , 4, , + 2, , 4, , 3, , V, P, , D, U, L, +1, , P, A, , U, R, E, , C, I, T, , 4 + 2 +, , 6, , 1, , 3, , = Klebsiella, pneumoniae, , 1. Each positive reaction is indicated by circling the number directly below its compartment., 2. The circled numbers in each bracket are added together, and the sum is placed in the box below., 3. The resultant 5-digit number (ID value) is then located in the computer coding manual to identify the organism., (a) The Enterotube® II, , ONPG AHD, , +, 1, , 0, , LDC, , ODC, , CIT, , H2S, , URE, , TDA, , IND, , VP, , GEL, , GLU, , MAN, , INO, , SOR, , RHA, , SAC, , MEL, , +, , +, , -, , -, , -, , -, , +, , -, , -, , +, , +, , -, , +, , +, , +, , +, , 4, , 1, , 5, , 0, , 0, , 1, , 7-digit profile number =, , 0, , 0, , 4, , 0, , 0, , 4, , 5 1 4 4 5 7 2, , 4, , 1, , 4, , 0, , 5, , 4, , 1, , 2, , 7, , 4, , AMY ARA, , 0, , +, 2, , OXI, , 0, , 2, , = E. coli, , 1. The 21 tests are divided into seven groups of three each., 2. A value of 1 is assigned to the first positive test in each group., 3. A value of 2 is assigned to the second positive test in each group., 4. A value of 4 is assigned to the third positive test in each group., 5. A 7-digit number is obtained by totaling the positive values of each of the seven groups of three. This number is located, in the analytical profile index to identify the organism., (b) The API® strip, , Figure 64.4 Computer-assisted techniques for the identification of Enterobacteriaceae, , EnteroPluri-Test System, The basis of the EnteroPluri-Test system as shown, in Figure 64.5 is quite similar to what is seen in the, Enterotube II and API testing systems. An enclosed, container with numerous compartments, each, containing a different testing media to determine, the biochemical profile of an unknown bacteria, species. The EnteroPluri-Test System was designed, , Figure 64.5 The EnteroPluri-Test System, , for use in identifying oxidase-negative, gramnegative bacteria. Fifteen tests are conducted in, twelve compartments. After an incubation period,, reagents will be added to specific compartments to, test for enzymatic activities or metabolic products., A scoring system is used to generate a unique fivedigit code that signifies a specific genus and species of bacteria., , (Source: Liofilchem®, EnteroPluri-Test Systems brochure, (www.liofilchem.com/images/prodottievidenza/enteropluritest.pdf)), , 458, , Experiment 64
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FUR T HE R R E AD I N G, Refer to the section on enteric bacteria and cytochrome oxidases in your textbook for further, information on the use of enzyme activities to, generate energy. In your textbook’s index, search, under “Cytochrome c,” “Oxidase,” and “Enteric.”, , C L I N I C A L A P P L I C AT I O N, Enterobacteriacae Infections, The Enterobacteriacae are a very diverse group of, bacteria that commonly inhabit the human colon,, but can cause a variety of infections throughout the, body. In the hospital environment these often result, from colonization of intravascular catheters, leading, to bacteremia that can progress rapidly to sepsis, and septic shock. Once identification of the infectious agent has been made, treatment with effective, antimicrobials may be used., , ❏❏ Barritt’s reagent (VP test reagent for, Enterotube II system), ❏❏ 1.5% hydrogen peroxide, ❏❏ 1% p-aminodimethylaniline oxalate (oxidase, reagent), , Equipment, Microincinerator, ❏❏ Inoculating loop, ❏❏ 5-ml pipette, ❏❏ Mechanical pipetting device, ❏❏ Sterile Pasteur pipettes, ❏❏ Glassware marking pencil, ❏❏ API Profile Recognition System and differential identification charts, ❏❏ Enterotube II ENCISE pads and color reaction, charts, , Procedure Lab One, Enterotube II System, , AT T H E B E N C H, , Materials, Cultures, Number-coded, 24-hour Trypticase soy agar streak, plates of, ❏❏ Escherichia coli, ❏❏ Salmonella typhimurium BLS -2, ❏❏ Klebsiella pneumoniae BLS -2, ❏❏ Enterobacter aerogenes, ❏❏ Shigella dysenteriae BLS -2, ❏❏ Proteus vulgaris, , Media, One of each per designated student group, ❏❏ Enterotube II, ❏❏ API 20-E strip, ❏❏ 5-ml tube of 0.85% sterile saline, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Sterile mineral oil, 10% ferric chloride, Kovac’s reagent, VP reagent for API system, Nitrate reduction reagents, , 1. Familiarize yourself with the components of, the system: screw caps at both ends, mediumcontaining compartments, self-enclosed inoculating needle, plastic side bar, and blue-taped, section., 2. Label the Enterotube II with your name and, the number of the unknown culture supplied, by the instructor., 3. Remove the screw caps from both ends of the, Enterotube II. Using the inoculating needle, contained in the Enterotube II, aseptically pick, some inoculum from an isolated colony on the, provided streak-plate culture., 4. Inoculate the Enterotube II as follows:, a. Twist the needle in a rotary motion and withdraw it slowly through all 12 compartments., b. Replace the needle in the tube and, with a, rotary motion, push the needle into the first, three compartments (GLU/GAS, LYS, and, ORN). The point of the needle should be, visible in the H2S>IND compartment., c. Break the needle at the exposed notch by, bending, discard the needle remnant, and, replace the caps at both ends. The presence, of the needle in the three compartments, maintains anaerobiosis, which is necessary, for glucose fermentation, CO2 production, and the decarboxylation of lysine and, ornithine., , Experiment 64, , 459
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5. Remove the blue tape covering the ADO,, LAC, ARB, SOR, VP, DUL/PA, URE, and CIT, compartments. Beneath this tape are tiny air, vents that provide aerobic conditions in these, compartments., 6. Place the clear plastic slide band over the, GLU/GAS compartment to contain the wax,, which may be spilled by the excessive gas production of some organisms., 7. Incubate the tube on a flat surface for 24 hours, at 37°C., , API 20-E System, 1. Familiarize yourself with the components of, the system: incubation tray, lid, and the strip, with 20 microtubes., 2. Label the elongated flap on the incubation, tray with your name and the number of the, unknown culture supplied by the instructor., 3. With a pipette, add approximately 5 ml of tap, water to the incubation tray., 4. Using a sterilized loop, touch an isolated, colony on the provided streak-plate culture,, transfer the inoculum to a 5-ml tube of sterile saline, and mix well to effect a uniform, suspension., 5. Remove the API strip from its sterile envelope, and place it in the incubation tray., 6. Tilt the incubation tray. Using a sterile Pasteur, pipette containing the bacterial saline suspension, fill the tube section of each compartment, by placing the tip of the pipette against the side, of the cupule. Fill the cupules in the CIT, VP, and, GEL microtubes with the bacterial suspension., 7. Using a sterile Pasteur pipette, fill the cupules, of the AHD, LDC, ODC, and URE microtubes, with sterile mineral oil to provide an anaerobic, environment., 8. Cover the inoculated strip with the tray lid and, incubate for 18 to 24 hours at 37°C., , Procedure Lab Two, Enterotube II System, 1. Observe all reactions in the Enterotube II except IND and VP, and interpret your observations using the manufacturer’s instructions., , 460, , Experiment 64, , Record your observations and results in the, Lab Report., 2. Perform the IND and VP tests as follows:, a. Place the Enterotube II in a rack with, the GLU and VP compartments facing, downward., b. With a needle and a syringe, gently pierce, the plastic film of the H2S>IND compartment and add 2 or 3 drops of Kovac’s, reagent. Read the results after 1 minute., c. As in Step 2b, add 2 drops of Barritt’s, reagent to the VP compartment and read, the results after 20 minutes., d. Record your IND and VP observations and, results in the Lab Report., 3. Based on your results, identify your unknown, organism using the manufacturer’s color identification charts., 4. Determine and record in the Lab Report the, five-digit ID value as described in Figure 64.4a, on page 458. Identify your unknown organism, by referring to the computer coding manual., , API 20-E System, 1. Observe all reactions in the API strip that do, not require addition of a test reagent, and interpret your observations using the manufacturer’s instructions. Record your observations, and results in the Lab Report., 2. Add the required test reagents in the following, order: Kovac’s reagent to IND, VP reagent to VP, (read the result after 15 minutes), ferric chloride, to TDA, nitrate reagents to GLU, and oxidase, reagent to OXI. Note color changes and interpret, your observations according to the manufacturer’s instructions. Record your observations and, results in the Lab Report., 3. Based on your results, identify your unknown, organism using the differential identification, chart., 4. Determine and record in the Lab Report, the seven-digit profile number as described, in Figure 64.4b on page 458. Identify your, unknown organism by referring to the Profile, Recognition System.
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L, Y, S, , 2+1, , 4, , O, R, N, +, , H2S, , G G, L A, U S, , 2 + 1, , I, N, D, , A, D, O, , L, A, C, , A, R, B, , S, O, R, , 4 + 2, , + 1, , 4, , + 2, , V, P, , P, A, , D, U, L, , U, R, E, , C, I, T, , 4 + 2 +, , +1, , 1, , ID value =, , Organism, , Determination of Enterotube II five-digit identification number, , ONPG, , AHD, , LDC, , ODC, , CIT, , H2S, , URE, , TDA, , IND, , VP, , GEL, , GLU, , MAN, , INO, , SOR, , RHA, , Organism, , Determination of API 20-E seven-digit profile number, , Review Questions, 1. What are the advantages of multitest systems? Disadvantages?, , 462, , Experiment 64: Lab Report, , SAC, , MEL, , AMY, , ARA, , OXI
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2. What Enterobacteriaceae are of medical significance? List and describe the infections caused by, these organisms., , 3. Would similar results be obtained by use of the computer-assisted method and the traditional, color-change method?, , 4., , hat is the clinical justification for the use of a rapid test procedure such as the Enterotube, W, II System for the identification of enteric microorganisms?, , Experiment 64: Lab Report, , 463
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Isolation and Presumptive, Identification of Campylobacter, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Use the laboratory procedures required to, isolate, cultivate, and identify the genus, Campylobacter., , Principle, Clinicians are aware of the medical significance of, Campylobacter strains as the etiological agents of, enteric infections. The incidence of enteritis caused, by Campylobacter jejuni equals or exceeds that of, salmonellosis or shigellosis. The clinical syndrome,, although varying in severity, is generally characterized by acute gastroenteritis accompanied by the, rapid onset of fever, headache, muscular pain, malaise, nausea, and vomiting. Twenty-four hours following this acute phase, diarrhea develops that may, be mucoid, bloody, bile-stained, and watery. The, precise epidemiology of the infection is not clear;, however, contact with animals, waterborne organisms, and fecal–oral transmission remain suspect., The organisms (campylo, curved; bacter, rod), were formerly called vibrios because of their curved, and spiral morphology. In the early 1980s they were, reclassified in the genus Campylobacter. They are, gram-negative and curved or spiral, with a single, flagellum located at one or both poles of the cell. In, pure culture, two types of colonies have been recognized and designated as Types I and II. The more, commonly observed Type I colonies are large, flat,, and spread with uneven margins. They are nonhemolytic, watery, and grayish. Type II colonies are also, nonhemolytic, but they are smaller (1 to 2 mm), with, unbroken edges. They are convex and glistening., Initially, the isolation of Campylobacter organisms from fecal specimens was difficult because of, their microaerophilic nature and their 42°C optimal, growth temperature. Furthermore, in the absence, of selective media, their growth was masked by the, overgrowth of other enteric organisms, and they, were often overlooked on primary isolation. This, situation has been rectified with the development, , E XP E R IMENT, , 65, , of selective media that are designed specifically for, isolating Campylobacter species and that inhibit, the growth of other enteric organisms. These, media are nutritionally enriched and supplemented, with 5% to 10% sheep or horse blood. In addition,, they contain three to five antimicrobial agents,, depending on the medium. For example, cephalosporins, one of the antimicrobial agents present in, the Campy-BAP medium, is selective for C. jejuni, and inhibits the species C. intestinalis, which is, rarely responsible for enteric infections., The most essential requirement for cultivating, Campylobacter is a microaerophilic incubation, atmosphere. High concentrations of oxygen are, toxic to these organisms, and an atmosphere of, 3% to 10% carbon dioxide and 5% to 10% oxygen is, optimal for their growth. The incubation temperature for C. jejuni is 42°C. At this temperature the, organism grows optimally, while growth of, C. intestinalis is inhibited., In the experiment to follow, a simulated fecal, specimen (a culture containing an attenuated strain, of C. jejuni and other enteric organisms) is used., You will attempt to isolate the Campylobacter, organisms by using the following two procedures:, 1. A conventional method uses MacConkey agar, directly, circumventing enrichment procedures, using a mixed simulated fecal population as the test culture., 2. A special method employs Campy-BAP agar, and the CampyPak® and GasPak® jars, which, are illustrated in Figure 65.1., Presumptive identification is made on the, basis of colonial morphology and the microscopic, appearance of the organisms obtained from a typical isolated colony. You may perform the catalase, and oxidase tests as described in Experiment 28 and, Experiment 29 for further presumptive identification., In the case of C. jejuni, both tests should be positive., , F U RT H E R RE A D I N G, Refer to the section on enteric bacteria and anaerobic metabolism in your textbook for further information on the use of enzymes to overcome the effects, of oxygen. In your textbook’s index, search under, “Anaerobic,” “Microaerophilic,” and “Oxidase.”, 465
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❏❏ Salmonella typhimurium, ❏❏ Escherichia coli, , BLS-2, , Media, ❏❏ Per designated student group, ❏❏ One Campy-BAP agar plate, ❏❏ One MacConkey agar plate, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, , Crystal violet, Gram’s iodine, 95% ethyl alcohol, 0.8% carbol fuchsin, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator, Inoculating loop, Glassware marking pencil, CampyPak and GasPak jars, 10-ml pipettes, , Procedure Lab One, Figure 65.1 CampyPak and GasPak jar, , C L I N I C A L A P P L I C AT I O N, Traveler’s Diarrhea, Campylobacter is the most common cause of bacterial diarrheal diseases worldwide. It is also referred to, as “Traveler’s Diarrhea.” Poultry and poultry products, have been associated with Campylobacter infections., Other foods have also been implicated in its transmission. Campylobacter jejuni and Campylobacter coli, are the two most clinically significant, and may be, isolated from the intestinal tract of poultry. They are, slow-growing organisms and are identified by biochemical, immunological, and molecular techniques., , AT THE B E N C H, , Materials, Cultures, Mixed saline suspensions of, ❏❏ Campylobacter jejuni BLS -2 (cultured on a, sheep blood–enriched medium), 466, , Experiment 65, , 1. Aseptically perform a four-way streak inoculation as described in Experiment 2 for the isolation of discrete colonies on both appropriately, labeled agar plates., 2. Place the inoculated Campy-BAP agar plate, in the GasPak jar in an inverted position. Following the manufacturer’s instructions, open, the CampyPak envelope and place it in the, jar. With a pipette, add 10 ml of water to each, envelope and immediately seal the jar to establish a microaerophilic environment., 3. Incubate the jar for 48 hours at 42°C., 4. Incubate the MacConkey agar plate culture in, an inverted position for 48 hours at 37°C., , Procedure Lab Two, 1. Observe both plate cultures for the presence, of discrete colonies. Record your observations, in the chart provided in the Lab Report., 2. Prepare a Gram stain, using 0.8% carbol fuchsin as the counterstain, of a representative, colony agar plate culture. Observe microscopically and record in the Lab Report the microscopic morphology and Gram reaction of each, preparation., 3. Based on your observations, identify your isolates and record in the Lab Report., 4. Optional: Perform the catalase and oxidase, tests on the representative isolates.
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E XP E R IMENT, , 65, , Name:_______________________________________________, Date:________________________, , Lab Report, , Section: ______________, , Observations and Results, 1. In the chart below, diagram the appearance of representative colonies, on both plates and describe their colonial characteristics. Also, note and, record the color of the medium surrounding the representative colonies on, the MacConkey plate. (Refer to Experiment 14 for an explanation of the, selective and differential nature of MacConkey agar.), Plate Culture, , Diagram of Colonies, , Colonial Characteristics, , Color of Medium, , Campy-BAP agar, , MacConkey agar, , 2. Record your observations of the Gram reactions in the chart below., , Gram Stain, Preparation, , Campy-BAP, Plate Isolate, , MACCONKEY AGAR PLATE, Isolate 1, , Isolate 2, , Draw a representative field., , Microscopic morphology, Gram reaction, , Experiment 65: Lab Report, , 467
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3. Based on your observations, identify your isolates:, Campy-BAP agar culture isolate: _________________, MacConkey agar culture isolate 1: _________________, MacConkey agar culture isolate 2: _________________, , Review Questions, 1. How would you describe the clinical syndrome induced by C. jejuni?, , 2. What are the purposes of the antimicrobial agents present in the selective, media used for the isolation of Campylobacter?, , 3. How may C. jejuni be separated from C. intestinalis?, , 4., , 468, , hy might members of Campylobacter not be isolated from a, W, stool specimen in a diagnostic laboratory?, , Experiment 65: Lab Report
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Microbiological Analysis of Urine, Specimens, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Discuss the organisms responsible for, infections of the genitourinary tract., , Grampositive, Bacteria, , 2. Use laboratory methods to detect bacteriuria and identify microorganisms associated with the urinary tract., , The anatomical structure of the mammalian urinary system is such that the external genitalia and, the lower aspects of the urethra are normally contaminated with a diverse population of microorganisms. The tissues and organs that compose the, remainder of the urinary system, the bladder, ureters, and kidneys, are sterile, and therefore urine, that passes through these structures is also sterile., When pathogens gain access to this system, they, can establish infection. Some etiological agents, of urinary tract diseases are illustrated on this, page., Urinary tract infections may be limited to a, single tissue or organ, or they may spread upward, and involve the entire system. Infections such as, cystitis involve the bladder but may spread through, the ureters to the kidneys. Infections limited to the, ureters and kidneys are called pyelitis. Glomerulonephritis is an inflammation that results in the, destruction of renal corpuscles; pyelonephritis, results in the destruction of renal tubules. Organisms other than bacteria may also act as etiological, agents of urogenital infections. Trichomonas vaginalis, a pathogenic flagellated protozoan, is commonly found in the vagina, and under appropriate, conditions, it is responsible for a severe inflammatory vaginitis. Candida albicans, a pathogenic, yeast, is normally found in low numbers in the, intestines. Under suitable conditions, such as the, use of antibacterial antibiotics, which disrupt the, normal intestinal flora and allow Candida to proliferate, it can enter the urogenital systems, where, it gives rise to vaginal infections. Schistosoma, , 66, , Staphylococcus aureus, Streptococcus pyogenes, Enterococcus, faecalis, Enterococci, , Enterococcus, faecium, , Escherichia coli, Pseudomonas aeruginosa, Gramnegative Proteus vulgaris, Klebsiella pneumoniae, Viruses, , Principle, , E XP E R IMENT, , Herpes hominus (type II), , Fungi, , Candida albicans, Blastomyces dermatitidis, Coccidioides immitis, , Helminths, , Schistosoma haematobium, Wuchereria bancrofti, , Protozoa, , Trichomonas vaginalis, Entamoeba histolytica, , haematobium is a pathogenic fluke, a helminth,, responsible for severe bladder infections., The initial step in diagnosing a possible urinary tract infection is laboratory examination, of a urine specimen. The sample must be collected midstream in a sterile container following, adequate cleansing of the external genitalia. It is, imperative to culture the freshly voided, unrefrigerated urine sample immediately to avoid growth, of normal indigenous organisms, which may overtake the growth of the more slowly growing pathogens. In this event, the infectious organism might, be overlooked, resulting in an erroneous diagnosis., Clinical evaluation of the specimen requires a, quantitative determination of the microorganisms, per ml of urine. Urine in which the bacterial count, per ml exceeds 100,000 1105 2 denotes significant, bacteriuria and is indicative of a urinary tract, infection. Urine in which counts range from 0 to, 1000 per ml are generally normal., In the conventional method, a urine sample is, streaked over the surface of an agar medium with, a special loop calibrated to deliver a known volume. Following incubation, the number of isolated, colonies present on the plate is determined and, multiplied by a factor that converts the volume of, 469
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Midstream urine sample, Streak for bacterial counts and isolation of colonies, and incubate for 48 hours at 245C, , Blood agar plate (see Exp. 14), Significant growth, , MacConkey or EMB agar plate (see Exp. 14), Abundant growth, , Abundant growth, , Gram stain, , Gram stain, , Gram-positive cocci, , Gram-negative bacilli, , Catalase (see Exp. 29), , -, , +, , Streptococcus spp., , Staphylococcus spp., , Differentiation of, Streptococcus spp., (see Exp. 63), , Differentiation of, Staphylococcus spp., (see Exp. 62), , Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, TSI agar (see Exp. 23), A/A*, H2S+, , A/A*, H2SE. coli, Klebsiella spp., , Urease test (see Exp. 26), , *A/A = Acid slant/Acid butt, , Proteus spp., , E. coli, , No change, , Pseudomonas spp., , +, , Klebsiella spp., , Figure 66.1 Laboratory procedures for the isolation and identification of urinary tract pathogens, , urine to 1 ml. The final calculation is then equal to, the number of organisms per ml of sample., Example: Twenty-five colonies were present on a, plate inoculated with a loop calibrated to deliver, 0.01 ml of a urine specimen., factor that, organisms, =, converts, per ml, 0.01 ml to 1 ml, *, 100, = 2500 organisms, per ml, , number of, *, colonies, 25, , If the specimen is turbid, dilution is necessary, prior to culturing. In this case, conventional, 10-fold dilutions are prepared in physiological, saline to effect a final dilution of 1:1000., (See Experiment 21.) Each of the dilutions, 110-1, 10-2, and 10-3 2 is then streaked on the, 470, , Experiment 66, , surface of a suitable agar plate medium for isolation of colonies. Following incubation, the number, of microorganisms per ml of sample is determined, by the following formula:, organisms per ml = number of colonies, * factor that converts the, volume of urine to 1 ml, * dilution factor, Example: Twenty-five colonies were counted on, a 10-2 dilution plate inoculated with a loop calibrated to deliver 0.01 ml of urine., Calculation:, 25 * 100 * 100 = 250,000 organisms per ml, On determination of bacteriuria, identification, of the infectious organism can be accomplished by, the laboratory procedures outlined in Figure 66.1.
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A newer, less conventional, and less timeconsuming method uses a diagnostic urine-culture, tube, Bacturcult®, devised by Wampole Laboratories (Figure 66.2). Bacturcult is a sterile, disposable plastic tube coated on the interior with a, special medium that allows detection of the bacteriuria and a presumptive class identification of, urinary bacteria., Following incubation of the Bacturcult urine, culture, bacteriuria can be detected with a bacterial count. This is performed by placing the, counting strip around the Bacturcult tube over an, area of even colony distribution and counting the, number of colonies within the circle. The average, number of colonies counted is interpreted in, Table 66.1., For the presumptive identification of bacteria,, the medium contains two substrates, lactose and, urea, and the pH indicator phenol red. Depending on the organism’s enzymatic action on these, substrates, differentiation of urinary bacteria into, three groups following incubation is possible, based on observable color changes that occur in, the culture:, , Figure 66.2 Bacturcult culture tube, , Group I: E. coli and Enterococcus—yellow., Group II: Klebsiella, Staphylococcus, and Streptococcus—rose to orange., Group III: Proteus and Pseudomonas—purplishred., Mixed cultures do not always produce clearcut color changes, however. Therefore, if additional testing is required, the discrete colonies that, develop on the medium can be used as the source, for subculturing into other media., In this experiment, you will use seeded saline, cultures to simulate urine specimens. This is done, to minimize the risk of using a potentially infectious body fluid, urine, as the test sample. You will, use the conventional procedure performed with, the calibrated loop to determine the number of, , TABLE 66.1 Bacturcult:, AVERAGE NUMBER OF, COLONIES WITHIN CIRCLE, , cells in the specimens. The Bacturcult tube will, be used for enumeration and presumptive group, identification. If your instructor desires to emulate, more closely a clinical evaluation of urine, then a, mixed seeded culture must be used. Representative colonies isolated from the blood agar streakplate culture for detection of bacteriuria can then, be identified following the schema in Figure 66.1., , F U RT H E R RE A D I N G, Refer to the section on bacterial infections of the, genital-urinary tract system in your textbook for, further information on the microbes that are part, of the microflora or are pathogens of the system., In your textbook’s index, search under “UTI,”, “Vaginitis,” and “Nephritis.”, , Interpretation of Colony Counts, APPROXIMATE NUMBER, OF BACTERIA PER ML, , 6 25, , 6 25,000, , 25 to 50, , 25,000 to 100,000, , 6 50, , 6 100,000, , DIAGNOSTIC, SIGNIFICANCE, Negative bacteriuria, Suspicious*, Positive bacteriuria, , Source: Wampole Laboratories Division, Carter-Wallace, Inc., Cranbury, NJ, 08512. Reprinted with permission., * Additional testing recommended., , Experiment 66, , 471
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C L I N I C A L A P P L I C AT I O N, The Oldest Clinical Test, Urinary tract infections are among the most frequently occurring problems in clinical medicine., Urine is composed of 95% water, with the remainder, consisting mainly of urea, uric acid, ammonia, hormones, sloughed squamous cells, proteins, salts,, and minerals. Urinalysis is performed for the diagnosis of metabolic or systemic diseases that affect, kidney function, for disorders of the kidney and urinary tract, screening for drug abuse, and monitoring, patients with diabetes. Urinalysis is considered to, be the oldest clinical test, with physical examination of urine for diagnosis having been performed, as much as 6000 years ago. Hippocrates, in the 4th, century BCE, first realized that urine was a filtrate, from the kidneys., , AT THE B E N C H, , Materials, Cultures, Six saline cultures, each seeded with one of the, following 24-hour cultures, ❏❏ Enterococcus faecalis BLS -2, ❏❏ Staphylococcus aureus BLS -2, ❏❏ Proteus vulgaris, ❏❏ Escherichia coli, ❏❏ Pseudomonas aeruginosa BLS -2, ❏❏ Klebsiella pneumoniae BLS -2, Optional: Saline culture seeded with a gram-, positive and a gram-negative organism., , Procedure Lab One, Bacturcult, 1. Label each Bacturcult tube with the name of, the bacterial organism present in the urine, sample., 2. Fill each tube almost to the top with urine., 3. Immediately pour the urine out of each tube,, allowing all the fluid to drain for several seconds. Replace the screw cap securely., 4. Immediately prior to incubation, loosen the, cap on each tube by turning the screw cap, counterclockwise for one-half turn., 5. Incubate the tubes with the caps down for 24, hours at 37°C., , Calibrated Loop for Bacterial Counts, 1. Label the three 9-ml sterile saline tubes and, the three blood agar plates 10-1, 10-2, and 10-3,, respectively., 2. Using the three 9-ml saline blanks, aseptically, prepare a 10-fold dilution of the urine sample, to effect 10-1, 10-2, and 10-3 dilutions., 3. With a calibrated loop, aseptically add 0.01 ml, of the 10-1 urine dilution to the appropriately, labeled blood agar plate and streak for isolation of colonies as illustrated., 4. Repeat Step 3 to inoculate the remaining urine, sample dilutions., 5. Incubate all plates in an inverted position for, 24 hours at 37°C., , Media, Per designated student group, ❏❏ Three blood agar plates, ❏❏ Three sterile 9-ml tubes of saline, ❏❏ Six Bacturcult culture tubes, , Equipment, Microincinerator or Bunsen burner, ❏❏ Calibrated 0.01-ml platinum loop, ❏❏ Glassware marking pencil, ❏❏ Sterile 1-ml pipettes, ❏❏ Mechanical pipetting device, 472, , Experiment 66, , Procedure Lab Two, 1. Determine the number of colonies in each of, the Bacturcult urine cultures. (Refer to Lab, Report for further instructions.), 2. Record your results in the Lab Report.
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E XP E R IMENT, , 66, , Name: _______________________________________________, Date: ________________________, , Lab Report, , Section: ______________, , Observations and Results, Bacturcult Procedure, 1. Determine the number of colonies in each of the Bacturcult urine cultures as, follows:, a. Place the counting strip around the tube over an area of even colony distribution and count the number of colonies within the circle., b. Repeat the count in another area of the tube., c. Average the two counts., d. Record in the Lab Report the average number of colonies counted within, the circle., 2. Based on your colony count, determine and record in the Lab Report the, approximate number of bacteria per ml of each sample and its diagnostic significance as negative bacteriuria, suspicious, or positive bacteriuria., 3. Observe and record in the Lab Report the color of the medium in each of the, urine cultures and the presumptive bacterial group., Urine Culture, , Number of, Colonies, , Number of, Bacteria Per Ml, , Diagnostic, Significance, , Color of, Medium, , Presumptive, Group, , E. faecalis, S. aureus, K. pneumoniae, P. vulgaris, P. aeruginosa, E. coli, , Calibrated Loop Procedure, Determine the number of colonies on each blood agar culture plate and calculate the number of organisms per ml of the urine. Record your results in the Lab, Report., Urine Sample Dilution, , Number of Colonies, , Organisms Per Ml of Sample, , Bacteriuria (+) or (−), , 10 -1, 10 -2, 10 -3, , Experiment 66: Lab Report, , 473
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Review Questions, 1. What types of urinary infections may be caused by different, microorganisms?, , 2. How is a clinical diagnosis of a bacteriuria established?, , 3. If five colonies were counted on a 10-3 dilution plate streaked with 0.01 ml, of urine, what was the number of organisms per ml of the original specimen,, and is this count indicative of bacteriuria? Explain., , 4., , 5., , 474, , How accurate is a laboratory analysis of a 24-hour, unrefrigerated,, non-midstream urine sample? Explain., , A male patient is diagnosed as having a urinary tract infection. A, urine culture is ordered by his physician. She requests that a, voided specimen be used rather than a catheterized sample. Why does she, make this request?, , Experiment 66: Lab Report
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Microbiological Analysis of Blood, Specimens, , LEARNING OBJECTIVES, Once you have completed this experiment,, you should be able to, 1. Describe the microorganisms most, frequently associated with septicemia., 2. Use laboratory methods to isolate, and identify the etiological agents of, septicemia., , Principle, Blood is normally a sterile body fluid. This sterility, may be breached, however, when microorganisms, gain entry into the bloodstream during the course, of an infectious process. The transient occurrence, of bacteria in the blood is designated as bacteremia and implies the presence of nonmultiplying, organisms in this body fluid., Bacteremias may be encountered in the course, of some bacterial infections such as pneumonia,, meningitis, typhoid fever, and urinary tract infections. A bacteremia of this nature does not present, a life-threatening situation, because the bacteria, are present in low numbers and the activity of the, host’s innate (nonspecific) immune system is generally capable of preventing further systemic invasion of tissues. A more dangerous and clinically, alarming syndrome is septicemia, a condition, characterized by the rapid multiplication of microorganisms, with the possible elaboration of their, toxins into the bloodstream. The clinical picture, frequently present in septicemia is that of septic, shock, which is recognized by a severe febrile episode with chills, prostration, and a drop in blood, pressure., A large and diverse microbial population has, been implicated in septicemia. The major offenders include the following:, 1. Gram-negative bacteria, because of their, endotoxic properties, are the most frequently, encountered etiological agents of the serious complications of septicemia. Among, these agents are Haemophilus influenzae,, , E XP E R IMENT, , 67, , Neisseria meningitidis, Serratia marcescens,, Escherichia coli, Pseudomonas aeruginosa,, and Salmonella spp. Less frequently implicated are Francisella tularensis and members, of the genera Campylobacter and Brucella., 2. Gram-positive bacteria that generally do not, produce the presenting signs of septic shock, include primarily members of the genera, Streptococcus and Staphylococcus., 3. Candida albicans is the major fungal invader, of the bloodstream., In the clinical setting, to facilitate the rapid initiation of effective chemotherapy, a culture of the, suspect blood sample is required for the isolation, and identification of the offending organisms. A, blood sample is drawn and cultured in an appropriate medium under both aerobic and anaerobic, conditions. Over a period of three to seven days,, the cultures are observed for turbidity and Gramstained smears are prepared to ascertain the presence of microorganisms in the blood. On detection, of microbial growth in the cultures, transfers, onto a variety of specialized agar media are made, for the identification of the infectious agent. The, schema for this protocol is shown in Figure 67.1., This exercise outlines two methods. Either, method or both methods may be used for the, isolation and presumptive identification of the, microorganisms in the experimental culture. Both, procedures use a simulated blood specimen: a prepared culture containing blood previously seeded, with selected microorganisms. The traditional, method is a modification of the schema shown in, Figure 67.1. This procedure requires the preparation of Gram-stained smears for the morphological study of the organisms and the inoculation of, selected agar media for their isolation and preliminary identification. The alternative method uses, the commercially available BBL Septi-Chek™, System, a single unit composed of the Septi-Chek, culture bottle and the Septi-Chek slide, as illustrated in Figure 67.2. The culture-bottle component permits the qualitative determination of the, presence of microorganisms in the blood sample,, and the slide component is designed for the simultaneous subculturing of the organisms onto a, plastic slide containing three differential media, 475
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476, , Experiment 67, Staphylococcus spp., (for differentiation,, see Exp. 62), , Streptococcus spp., (for differentiation,, see Exp. 63), , Hemolysis, , Blood agar (stab and, streak inoculation), , P. aeruginosa, , C. albicans, , Neisseria spp., , Salmonella spp., , Lactophenol–, cotton-blue stain, (see Exp. 36), , Sabouraud agar, , Ovoid bodies, , Oxidase test, (see Exp. 30), , Chocolate agar, and CO2, , E. coli, , H2S production, (-) (see Exp. 25) (+), , P. aeruginosa, Salmonella spp., , Lactose fermentation, (-) (see Exp. 22) (+), , Enteric bacteria, , MacConkey agar, , Diplococci, , Figure 67.1 Schema for the isolation and identification of the etiological agents of septicemia, , Brucella spp., , Brucella medium, , Bacilli, , Gram stain, , (-), , Gram stain, (+), Cocci, , Observe for turbidity, , Observe for turbidity, , (-), Bacilli, , 3- to 7-day Trypticase soy, broth culture, vented, , 3- to 7-day Trypticase soy, broth culture, unvented, , Blood sample, , Hemolysis, , Blood agar, , Cocci, , Streptococcus spp., (for differentiation,, see Exp. 63), Staphylococcus spp., (for differentiation,, see Exp. 62), , (+)
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(chocolate, MacConkey, and malt agar). Differential growth on these media provides preliminary, information as to the nature of the infectious agent, and isolated colonies for further study., , FUR T HE R R E AD I N G, Refer to the section on bacterial infections of the, blood system in your textbook for further information on the microbes that are commonly found, to be pathogens of the system. In your textbook’s, index, search under “Bacteremia,” “Septic,” and, “Endocarditis.”, , C L I N I C A L A P P L I C AT I O N, Drawing Blood for Cultures, Normally, drawing blood for hematological analysis, simply requires cleansing of the skin with an alcohol, pad, but those draws intended for microbiological, testing require a different protocol. The area of the, draw is cleaned thoroughly with alcohol followed, by a disinfectant such as Chloraprep® One-Step. All, palpation after cleansing of the skin must be done, with sterile gloves, and the phlebotomist wears a, face shield. While it is nearly impossible to eliminate, all bacteria from the skin, these techniques attempt, to minimize contamination of drawn blood from contact with the skin flora, which could produce false, positive blood cultures., , AT T H E B E N CH, , Materials, Culture, 48- to 72-hour simulated blood culture prepared, as follows: 10 ml of citrated blood, obtained from, a blood bank, or 10 ml of saline seeded with 2, drops each of bacteria that has been adjusted to an, absorbance of 0.1 at 600 nm, in 90 ml of Trypticase, soy broth containing 0.05% of sodium polyanetholesulfonate (SPS) used to prevent clotting of, the blood sample, ❏❏ Escherichia coli, ❏❏ Neisseria perflava, ❏❏ Saccharomyces cerevisiae, , Figure 67.2 Septi-Chek System, , Media, One each per designated student group, ❏❏ Blood agar plate, ❏❏ Sabouraud agar, ❏❏ MacConkey agar, plate, plate, ❏❏ Septi-Chek System, ❏❏ Chocolate agar plate, , Reagents, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Crystal violet, ❏❏ 70% isopropyl, Gram’s iodine, alcohol, 95% ethyl alcohol, ❏❏ 1% p-aminodimethylSafranin, aniline oxalate, Lactophenol–cottonblue stain, , Equipment, ❏❏ Sterile 20-gauge,, 11 2@inch needles, ❏❏ Sterile 1-ml and, 10-ml syringes, ❏❏ Microincinerator or, Bunsen burner, ❏❏ Staining tray, ❏❏ Inoculating loop, , ❏❏ Glass microscope, slide, ❏❏ Lens paper, ❏❏ Bibulous paper, ❏❏ Microscope, ❏❏ Glassware marking, pencil, ❏❏ Disposable gloves, , Procedure Lab One, Use gloves throughout the procedure., , 1. Swab the rubber stopper of the blood-culture, bottle with 70% isopropyl alcohol and allow to, air-dry., Experiment 67, , 477
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2. Using a sterile needle and 1-ml syringe, aseptically remove 0.5 ml of the blood culture by, penetrating the rubber stopper., Dispose of the needle and syringe, as a single, unit, into the provided puncture-proof receptacle., , 3. To prepare a smear, place a small drop of, the culture on a clean glass slide and spread, evenly with an inoculating loop., 4. Place 1 drop of culture in one corner of the, appropriately labeled blood agar plate and, prepare a four-way streak inoculation as, described in Experiment 2., 5. Repeat Step 4 to inoculate the MacConkey,, chocolate, and Sabouraud agar plates., 6. Incubate the agar plate cultures in an inverted, position for 24 to 48 hours as follows: Sabouraud agar culture at 25°C, chocolate agar, culture in a 10% CO2 atmosphere at 37°C, and, the remaining cultures at 37°C., 7. Follow the Septi-Chek System procedure, as follows:, a. Remove the protective top of the screw cap, of the culture bottle, disinfect the rubber, stopper with 70% isopropyl alcohol, and, allow to air-dry., b. Using the 10-ml syringe, aseptically transfer, 10 ml of the experimental culture to the, appropriately labeled Septi-Chek culture, bottle., c. Aseptically vent the bottle for aerobic, incubation., d. Replace the protective top of the screw cap, on the bottle., e. Gently invert the bottle two or three times, to disperse the blood evenly throughout the, medium., f. Incubate the culture for four to six hours at, 37°C., g. Attach the Septi-Chek slide according to the, manufacturer’s instructions., h. Tilt the combined system to a horizontal, position and hold until the liquid medium, enters the slide chamber and floods the, agar surfaces. While maintaining this position, rotate the entire system one complete, turn to ensure that all agar surfaces have, come in contact with the liquid medium., Return the system to an upright position., , 478, , Experiment 67, , i. Incubate the system in an upright position, at 37°C., j. Check the culture bottle daily for turbidity, and the slide for visible colony formation., , Procedure Lab Two, 1. Examine the blood agar plate culture for the, presence (+ ) or absence (- ) of hemolytic, activity. (Refer to Figure 60.1.) If hemolysis is, present, determine the type observed. Record, your observations in the Lab Report., 2. For the performance of the oxidase test, add, p-aminodimethylaniline oxalate to the surface, of the growth on the chocolate agar plate. The, presence of pink-to-purple colonies is indicative of Neisseria spp. (Refer to Figure 60.2.), Record your observations and the oxidase test, results in the Lab Report., 3. Examine the MacConkey agar plate culture, for determination of lactose fermentation., Lactose fermenters exhibit a pink-to-red halo, in the medium, a red coloration on the surface of their growth, or both a halo and red, coloration. (Refer to Figure 14.2b.) Record, your observations and indicate the presence, or absence of lactose fermenters in the Lab, Report., 4. Examine the Sabouraud agar plate culture, for the presence of growth. Prepare a lactophenol–cotton-blue–stained smear from an, isolated colony. (See Experiment 35.) Examine, the smear microscopically for the presence of, large ovoid bodies indicative of the yeast cells., Record your morphological observations in, the Lab Report., 5. Observe the Septi-Chek slide system for the, presence of growth on the three agar surfaces., If growth is present on:, a. Medium 1 (MacConkey agar), examine for, fermentative patterns as described in Step, 3 and record your observations in the Lab, Report., b. Medium 2 (chocolate agar), perform the, oxidase test as described in Step 2 and, record your observations in the, Lab Report., c. Medium 3 (malt agar), prepare and examine, microscopically a lactophenol–cotton-blue–, stained smear as described in Step 4. Record, your observations in the Lab Report.
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2. Why are blood samples cultured in both vented and unvented systems?, , 3., , A 15-year-old boy is admitted to the hospital and presents the following symptoms: chills, fever, increased pulse rate, and a drop in, blood pressure. The patient indicates that these symptoms have occurred, intermittently. The physician suspects a bacteremia and orders a series of, three blood cultures over a 24-hour period. Explain the following:, a. Why did the physician order more than one blood culture?, , b. Why does blood culture medium contain an anticoagulant?, , 4., , 480, , Prior to the introduction of antibiotic therapy, what was the prognosis for patients with septicemia? What significant factors played, roles in recovery in the absence of antibiotics?, , Experiment 67: Lab Report
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Species Identification of Unknown, Bacterial Cultures, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Identify an unknown bacterial species by, using dichotomous keys and Bergey’s, Manual of Systematic Bacteriology., , Principle, At this point in the course, you have developed the, manipulative skills and the cognitive microbiological knowledge to identify microorganisms beyond, their genus classification to the level of their, species identification. Therefore, in this experiment, you will use dichotomous keys, Bergey’s, Manual of Systematic Bacteriology, and information accrued from previously performed laboratory procedures to help identify the species of an, unknown culture., In Experiment 32, “Genus Identification of, Unknown Bacterial Cultures,” you were required, to use a variety of biochemical tests to successfully, accomplish the experimental purpose. Your review, of the required procedures and ensuing results, should indicate that only a few of these tests were, actually necessary, in most instances for the identification of the unknown culture. Similarly, species identification can be accomplished by using, a limited number of carefully selected laboratory, procedures. Notice that what appears to be a spurious result in some cases, one that departs from, the expected norm for a particular species, may be, attributable to strain differences within the given, species. These nonconforming results may be verified by the use of Bergey’s Manual to ascertain the, existence of variable biochemical test results for, the particular species being studied., In this experimental procedure, you will, receive a mixed culture containing a gram-positive, and a gram-negative organism. The protocol will, require (1) Gram staining, (2) streak plating for, observation of colonial characteristics, (3) use, of selective media for the preparation of pure, cultures, (4) the performance of appropriate, , E XP E R IMENT, , 68, , biochemical tests as indicated in the dichotomous, keys outlined in Figure 68.1 and Figure 68.2, and, (5) information in Bergey’s Manual., , F U RT H E R RE A DI N G, Refer to the section on bacterial metabolism and, cellular components in your textbook for further, information on the identification characteristics, of prokaryotes. In your textbook’s index, search, under “Cell wall,” “Bergey’s,” and “Selective media.”, , C L I N I C A L A P P L I C AT I O N, New Molecular Techniques for Rapid Species, Identification, Once bacteria from blood or other tissues has been, cultured, the organisms must be positively identified. While biochemical and serological tests are, the norm for such identification, a recently developed technique of mass spectrometry using matrixassisted laser desorption/ionization (MALDI) offers, a quicker (less than 1 hour after detection in blood), way to identify organisms. This technique releases, key molecules from the organisms in question, and—using analysis of the size-to-charge ratios, of the molecules and specialized computer software—provides accurate identification of infectious, organisms, and may provide a future alternative or, addition to both biochemical and genomic identification schemes., , AT T HE BE NCH, , Materials, Cultures, ❏❏ Per student: number-coded, 24- to 48-hour, mixed Trypticase soy broth cultures each containing a gram-positive and a gram-negative, 481
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Gram-Positive Bacteria, Bacilli, Corynebacterium spp., Lactobacillus spp., Spore Formation, (+), Bacillus spp., , (-), Corynebacterium spp., Lactobacillus spp., , Mannitol, , Catalase, , (+), B. megaterium, B. subtilis, , (-), B. cereus, , (+), Corynebacterium spp., , Nitrate Reduction, , Voges-Proskauer, (+), B. subtilis, , (-), B. megaterium, , (-), Lactobacillus spp., , (+), C. xerosis, , Glucose, , (-), C. kutscheri, , (A/G), L. fermentum, , (A), L. casei, L. delbrueckii, Mannitol, , (+), L. casei, , (-), L. delbrueckii, , NG= No growth; G= Growth; A/G= Acid and gas; A= Acid only, , Figure 68.1 (continued) Schema for the identification of gram-positive bacteria, , Procedure Lab One, Separation of the Bacteria in Mixed, Unknown Culture, 1. Prepare a Trypticase soy agar broth subculture, of the unknown bacterial species and refrigerate following incubation. You will use this, culture if contamination of the test culture is, suspected during the identification procedure., 2. Prepare a Gram-stained smear of the original, unknown culture. Examine the smear and, record your observations in the Lab Report., 3. Prepare four-way streak inoculations(see, Experiment 2) on the following media for the, separation of the microorganisms in the mixed, cultures:, • Trypticase soy agar for observation of colonial characteristics., , • Phenylethyl alcohol agar for isolation of, gram-positive bacteria., • MacConkey agar for isolation of gram-, negative bacteria., 4. Incubate all the plates in an inverted position, and then subculture for 24 to 48 hours at 37°C., , Procedure Lab Two, Preparation of Pure Cultures, 1. Isolate a discrete colony on both the, phenylethyl alcohol agar plate and the, MacConkey agar plate, and aseptically transfer each onto a Trypticase soy agar slant.(See, Experiment 2.), 2. Incubate the Trypticase soy agar slants for 24, to 48 hours at 37°C., , Experiment 68, , 483
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Gram-Negative Bacteria, Cocci, Moraxella spp., Neisseria spp., Glucose, (+), Neisseria spp., , (-), Moraxella spp., , Nitrate, Reduction, , Nitrate, Reduction, , (+), N. mucosa, , (-), N. sicca, , (+), M. catarrhalis, , Lactose, (+), Citrobacter spp., Enterobacter spp., Escherichia spp., Klebsiella spp., (see Experiments 24 and 65), , (-), M. bovis, , Indole, (+), C. intermedius, E. coli, Citrate, (+), C. intermedius, , (-), C. freundii, Enterobacter spp., Klebsiella spp., , Methyl Red, (-), E. coli, (+), (-), C. freundii, E. aerogenes, K. ozaenae, K. pneumoniae, Urea, , H2S Production, (+), C. freundii, , (-), K. ozaenae, , (+), K. pneumoniae, , (-), E. aerogenes, , NG=No growth; G=Growth; A/G=Acid and gas; A=Acid only, , Figure 68.2 Schema for the identification of gram-negative bacteria, , Procedure Lab Three, Preparation of Pure Cultures, 1. Examine the Trypticase soy agar plate for the, appearance of discrete colonies. Select two, colonies that differ in appearance and record, their colonial morphologies in the Lab Report., 2. Examine the phenylethyl alcohol and MacConkey, agar plates. Record your observations in the, Lab Report., , 484, , Experiment 68, , Identification of Unknown Bacterial, Species, 1. Prepare a Gram-stained smear from each of, the Trypticase soy agar slant cultures to verify, its purity by means of the Gram reaction and, cellular morphology. Examine the smears and, record your observations in the Lab Report., 2. If each Gram-stained preparation is not solely, gram-positive or gram-negative, repeat the, steps in Labs One and Two, using the refrigerated Trypticase soy agar subculture as the test, culture.
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Gram-Negative Bacteria, Bacilli, Citrobacter spp., Enterobacter spp., Escherichia spp., Klebsiella spp., Proteus spp., Pseudomonas spp., Lactose, (-), Proteus spp., Pseudomonas spp., Glucose, (+), Proteus spp., , (-), Pseudomonas spp., , Indole, , Nitrate Reduction, , (+), P. vulgaris, P. rettgeri, , (-), P. mirabilis, P. inconstans, , (+), P. aeruginosa, P. fluorescens, , H2S Production, , Urea, , Litmus Milk, , (+), P. vulgaris, , (-), P. rettgeri, , (+), P. mirabilis, , (-), P. inconstans, , Peptonization, P. aeruginosa, , (-), P. mallei, , Alkaline, P. fluorescens, , NG= No growth; G= Growth; A/G= Acid and gas; A= Acid only, , Figure 68.2 (continued) Schema for the identification of gram-negative bacteria, , 3. If the isolates are deemed to be pure on the, basis of their cultural and cellular morphologies, continue with the identification procedure. During this period and in subsequent, sessions, use the dichotomous keys in, Figure 68.1 and Figure 68.2 to select and perform the necessary biochemical tests on each, of your isolates for identification of its species., Incubate all cultures for 24 to 48 hours at 37°C., , Procedure Lab Four, Identification of Unknown Bacterial, Species, 1. Examine all the biochemical test cultures and, record your observation and results in the Lab, Report., , Experiment 68, , 485
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E XP ER IM E NT, , 68, , Name: _______________________________________________, Date: ________________________, , Lab Report, , Section: ______________, , Observations and Results, Separation of the Bacteria in Mixed Unknown Culture, Record your observations of the Gram-stained smear of your mixed unknown, culture in the chart below., Organism, , Cellular Morphology, , Gram Reaction, , 1, 2, , Preparation of Pure Cultures, 1. Select from the Trypticase soy agar plates two colonies that differ in appearance, and record their colonial morphologies., Isolate 1:, , Isolate 2:, , 2. Record your observations of the phenylethyl alcohol and MacConkey agar, plates in the chart below., Medium, , Growth (+) or (−), , Colonial Morphology, , Coloration of Medium, , Phenylethyl alcohol agar, MacConkey agar, , Identification of Unknown Bacterial Species, 1. Record your observations of the Gram-stained smears of the Trypticase soy, agar cultures obtained from the phenylethyl alcohol and MacConkey agar, plates in the chart below., Agar Slant From, , Cellular Morphology, , Gram Reaction, , Phenylethyl alcohol agar plate culture, MacConkey agar plate culture, , Experiment 68: Lab Report, , 487
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2. Record your observations and results of all the biochemical tests in the chart, below., Gram-Positive Isolate, Biochemical Test, , Observation, , Unknown gram-negative organism: ________________________________, , 488, , Experiment 68: Lab Report, , Result
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PART 15, , Immunology, LEARNING OBJECTIVES, Once you have completed the experiments in this section, you should be able to, 1. Explain the basic principles of nonspecific (innate) and specific (acquired), immunity., 2. Perform serological procedures that demonstrate immunological reactions of, agglutination and precipitin formation., 3. Perform rapid immunodiagnostic screening procedures., , Introduction, Immunity, or resistance, is a state in which, a person, either naturally or by some acquired, mechanism, is protected from contracting certain, diseases or infections. The ability to resist disease, may be innate (nonspecific), or it may be adaptive, (also called acquired, or specific), when the disease state is stimulated in the host., Innate immunity is native or natural. It is, inborn and provides the basic mechanisms that, defend the host against intrusion of foreign substances or agents of disease. This defense is not, restricted to a single or specific foreign agent, but, provides the body with the ability to resist many, pathological conditions. The mechanisms responsible for this native immunity include the mechanical barriers, such as the skin and mucous, membranes; biochemical factors, such as antimicrobial substances present in the body fluids; and, the more sophisticated process of phagocytosis, and action of the reticuloendothelial system., Adaptive immunity, either cell-mediated, or humoral, is acquired by the host in response, to the presence of a single or particular foreign, substance, usually protein, called an antigen, , (immunogen). In humoral immunity, antigens that, penetrate the mechanical barriers of the host,, namely the skin and mucous membranes, stimulate formation of antibodies. The function of, the antibodies is to bind to the specific antigens, that are responsible for their production and to, inactivate or destroy them. Antibodies are a group, of homologous proteins called immunoglobulins, which are found in serum and represent five, distinct classes: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM),, immunoglobulin D (IgD), and immunoglobulin E, (IgE)., The primary immunological complexes, (antigen + antibody) are as follows:, 1. Agglutination: This type of reaction uses specific antibodies, agglutinins, that are formed, in response to the introduction of particulate, antigens into host tissues. When these particulate antigens combine with a homologous antiserum, a three-dimensional mosaic complex, occurs. This is called an agglutination reaction, and can be visualized microscopically and in, some cases macroscopically., , 489
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2. Precipitin formation: This reaction requires, specific antibodies, precipitins, that are, formed in response to the introduction of soluble, nonparticulate antigens into host tissues., These antibodies, when present in serum,, form a complex with the specific homologous, nonparticulate antigen and result in a visible, precipitate., Advances in chemistry, especially immunochemistry, have enabled us to study the interaction of antigens and immunoglobulins outside the, body, in a laboratory setting. These advances have, provided an immunological discipline known as, serology, which studies these in vitro reactions, that have diagnostic, therapeutic, and epidemiological implications., In the experiments to follow, you will study, several serological procedures based on the principles of agglutination and precipitin formation for, the detection of serum antibodies or antigens. The, techniques presented in these experiments span a, spectrum of methods, ranging from basic reactions, to more sophisticated forms of antigen and antibody interactions., It is further suggested that your instructor, present experiments that use positive and negative control test kits as laboratory demonstrations., , Note that some of the experimental protocols, use positive and negative controls provided in the, test kits to demonstrate the desired immunological reactions. These controls do not represent the, source of potential pathogens capable of inducing, infection in students and instructional staff. The, rationale for this design is that body fluids, particularly blood of unknown origin, may serve as, a major vehicle for the transmission of infectious, viral agents. Thus, our concern with the spread of, AIDS and hepatitis precludes the use of blood as a, test specimen in a college laboratory., , This will reduce the cost of the required materials,, which may otherwise be prohibitive at many academic institutions, but will still allow you and your, fellow students to observe the advances in immunological serology., , F U RT H E R RE A D I N G, Refer to the section on the immune system in your, textbook for further information on the acquired, immune immunity responses. In your textbook’s, index, search under “Antibody,” “B cells,” and, “Serology.”, , C ASE STUDY, PAST EXPOSURE, A young child was brought to the Emergency, Department by her parents, who were worried, about a fever and a rash she had for the past two, weeks. The ER nurse took a history of the child, and learned that a month before, the family had, been on a camping trip deep in the woods. The, parents are sure that they checked their child each, day for ticks, but they admit that they were less, than thorough some days in their search. Upon, inspection, the attending physician notices a rash, has developed on the child’s inner thigh and that, it has a red streaking pattern that resembles a, bull’s-eye pattern. The child also complains of, feeling joint aches and tenderness. The attending, , 490, , Part 16, , physician surmises the possible disease the child, has contracted based on the few clues presented., The physician orders a blood draw for a serological test to determine potential exposures and confirm diagnosis., , Questions to Consider:, 1. With the symptoms given, which bacterial, pathogen could be causing this infection?, What is the common name for this infection?, 2. Which serological test would the Infectious, Diseases laboratory perform to confirm this, infection?
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E XP E R IMENT, , Precipitin Reaction: The Ring Test, , LEARNING OBJECTIVE, , = antigen, , Once you have completed this experiment,, you should be able to, , = antibody, Zone of, antibody excess, , 1. Demonstrate a precipitin reaction by, means of the ring test., , 69, , Zone of, equivalence, , Zone of, antigen excess, , The ring test or interfacial test is a simple, serological technique that illustrates the precipitin, reaction in solution. This antigen–antibody reaction can be demonstrated by the formation of a, visible precipitate, a flocculent or granular turbidity, in the test fluid. Antiserum is introduced into, a small-diameter test tube, and the antigen is then, carefully added to form a distinct upper layer. Following a period of incubation of up to four hours,, a ring of precipitate forms at the point of contact, (interface) in the presence of the antigen–antibody, reaction. The rate at which the visible ring forms, depends on the concentration of antibodies in the, serum and the concentration of the antigen., To detect the precipitin reaction, a series, of dilutions of the antigen is used because both, insufficient (zone of antibody excess) and excessive (zone of antigen excess) amounts of antigen, will prevent the formation of a visible precipitate, (zone of equivalence), as shown in Figure 69.1. In, addition, you will be able to determine the optimal, antibody:antigen ratio by the presence of a pronounced layer of granulation at the interface of the, antiserum and antigen solution. Figure 69.2 illustrates this immunological reaction., , FUR T HE R R E AD I N G, Refer to the section on the immune system in your, textbook for further information on the acquired, immune immunity responses. In your textbook's, index, search under “Antibody,” “B cells,” and, “Serology.”, , Amount of antibody precipitated, , Principle, , Increasing concentration of antigen, , Figure 69.1 The precipitin reaction, , C L I N I C A L A P P L I C AT I O N, Criminology, The precipitin reaction is a serological test in which, an antibody reacts with a specific soluble antigen to, form a visible precipitate ring in the tube. This test is, mainly used today in criminology for the identification of human blood or other bloodstains, in cases of, disputed parentage, and for the determination of the, cause of death., , 491
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Antiserum, Precipitin ring, Bovine serum, Saline, , Bovine serum dilutions:, , 1:25, , 1:50, , 1:75, , Control, , Figure 69.2 Ring test: Precipitin reactions, , AT THE B E N C H, , Materials, Reagents, ❏❏ Physiological saline (0.85% NaCl), ❏❏ Commercially available bovine globulin, antiserum, ❏❏ Normal bovine serum diluted to 1:25, 1:50, and, 1:75 with physiological saline, The normal bovine serum contains the antigen, (bovine globulin), to which antibodies were made, commercially in another animal species and provided as antiserum to bovine globulin., , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , 492, , Serological test tubes (8 * 75 mm), 0.5-ml pipettes, Serological test tube rack, Mechanical pipetting device, Glassware marking pencil, 37°C incubator, , Experiment 69, , Procedure, 1. Label three serological test tubes according, to the antigen dilution to be used (1:25, 1:50,, and 1:75) and the fourth test tube as a saline, control., 2. Using a different 0.5-ml pipette each time, transfer 0.3 ml of each of the normal bovine serum, dilutions into its appropriately labeled test tube., 3. Using a clean 0.5-ml pipette, transfer 0.3 ml of, saline into the test tube labeled as control., 4. Carefully overlay all four test tubes with 0.3 ml, of bovine globulin antiserum. To prevent mixing of the sera, tilt the test tube and allow the, antiserum to run down the side of the test tube., 5. Incubate all test tubes for 30 minutes at 37°C., 6. Examine all test tubes for the development of, a ring of precipitation at the interface. Indicate, the presence or absence of a ring in the Lab, Report., 7. Determine and record the antigen dilution, that produced the greatest degree of precipitation; this is indicative of the optimal, antibody:antigen ratio.
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E XP E R IMENT, , 69, , Name: _______________________________________________, Date: ________________________, , Lab Report, , Section: ______________, , Observations and Results, ANTIGEN DILUTIONS, 1:25, , 1:50, , 1:75, , Saline Control, , Presence of interfacial, ring: (+ ) or (-), , Dilution showing optimal antibody:antigen ratio is ___________., , Review Questions, 1. How do precipitin and agglutination reactions differ?, , 2. How would you determine the optimal antigen:antibody ratio by means of the, ring test?, , Experiment 69: Lab Report, , 493
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3., , Why is it essential to use a series of antigen dilutions in this, procedure?, , 4., , How would you explain the absence of visible precipitate?, , 494, , Experiment 69: Lab Report
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E XP E R IMENT, , 70, , Agglutination Reaction:, The Febrile Antibody Test, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Demonstrate the agglutination reaction, using the febrile antibody test and an antibody titer determination., , Principle, The febrile antibody test is used in the diagnosis, of diseases that produce febrile (fever) symptoms., Some of the microorganisms responsible for, febrile conditions are salmonellae, brucellae, and, rickettsiae. Febrile antigens—such as endotoxins, enzymes, and other toxic end products—, elaborated by these organisms are used specifically, to detect or exclude the homologous antibodies, that develop in response to these antigens during, infection., In this procedure, the antigen is mixed on a, slide while the serum is observed. Cellular clumping indicates the presence of homologous antibodies in the serum; no visible clumping indicates, the absence of homologous antibodies. Only the, febrile antigens and antibodies of Salmonella spp., will be used. Figure 70.1 shows a positive and a, negative agglutination reaction., The second part of this experiment is designed, to illustrate that agglutination reactions, such as, the febrile antibody test, can be used to identify, an unknown microorganism through serotyping., A specific antiserum prepared in a susceptible,, immunologically competent laboratory animal is, mixed with a variety of unknown bacterial antigen, preparations on slides. The bacterial antigen that, is agglutinated by the antiserum is identified and, confirmed to be the agent of infection., These tests are strictly qualitative. A quantitative result can be obtained by performing, the antibody titer test, which measures the, concentration of an antibody in the serum and, allows the physician to follow the course of an, infection. The patient’s serum is titrated (diluted),, and the decreasing concentrations of the antiserum are mixed with a constant concentration of, , (a), , (b), , Figure 70.1 Agglutination reaction. (a) Visible, , clumping indicates the presence of homologous, antibodies in the serum, and a positive reaction., (b) The lack of visible clumping indicates the, absence of homologous antibodies, and a, negative reaction., , homologous antigen. The test ends when the test, tube that contains the serum with the highest dilution shows agglutination., , F U RT H E R RE A D I N G, Refer to the section on the immune system in your, textbook for further information on the acquired, immunity responses. In your textbook’s index,, search under “Antibody,” “B cells,” and “Serology.”, , C L I N I C A L A P P L I C AT I O N, Febrile Disease Diagnosis, Febrile antigens are standardized suspensions of, bacteria or bacterial antigens used to qualify or, quantify specific serum antibodies that develop, during some febrile infections. Based on the Widal, Agglutination test for the diagnosis of typhoid fever,, serum from patients can be tested for the presence, of antibodies correlating to infectious diseases such, as brucellosis, salmonellosis, and some rickettsial, infections., 495
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AT THE B E N C H, , Materials, Cultures, Number-coded, washed saline suspensions of, ❏❏ Escherichia coli, ❏❏ Proteus vulgaris, ❏❏ Salmonella typhimurium BSL -2, ❏❏ Shigella dysenteriae BSL -2, , Reagents, ❏❏ Physiological saline (0.85% NaCl), ❏❏ Commercial preparations of Salmonella, typhimurium H antigen, ❏❏ Commercial preparations of Salmonella, typhimurium H antiserum (Abcam, Inc.), , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Microincinerator or Bunsen burner, Inoculating loop, Glass microscope slides, 13 * 100@mm test tubes, Sterile 1-ml pipettes, Mechanical pipetting device, Applicator sticks, Glassware marking pencil, Microscope, Waterbath, , Procedure, Febrile Antibody Test, 1. With a glassware marking pencil, make two, circular areas about 1 2 inch in diameter on a, microscope slide. Label the circles A and B., 2. To Area A, add 1 drop of S. typhimurium H, antigen and 1 drop of 0.85% saline. Mix the two, with an applicator stick., 3. To Area B, add 1 drop of S. typhimurium H, antigen and 1 drop of S. typhimurium H antiserum. Mix the two with a clean applicator stick., 4. Pick up the slide and, with two fingers of one, hand, rock the slide back and forth., 5. Observe the slide both macroscopically and, microscopically, under low power, for cellular, clumping (agglutination)., 6. Indicate the presence or absence of macroscopic and microscopic agglutination, and, 496, , Experiment 70, , draw a representative field of Areas A and B in, the Lab Report., , Serological Identification, of an Unknown Organism, 1. Prepare two microscope slides as in the previous procedure. Label the four areas on the, slides with the numbers of your four unknown, cultures., 2. Into each area on both slides, place 1 drop of, S. typhimurium H antiserum., 3. With a sterile inoculating loop, suspend a loopful of each number-coded unknown culture, in the drop of antiserum in its appropriately, labeled area on the slides., 4. Pick up the slides and slowly rock them back, and forth., 5. Observe both slides macroscopically and, microscopically, under low power, for, agglutination., 6. In the Lab Report, indicate the presence or, absence of macroscopic and microscopic, agglutination in each of the suspensions. Also,, indicate the suspension that is indicative of a, homologous antigen–antibody reaction., , Determination of Antibody Titer, Refer to Figure 70.2 when reading the following, instructions., 1. Place a row of 10 test tubes (13 * 100@mm) in, a rack and number the tubes 1 through 10., 2. Pipette 1.8 ml of 0.85% saline into the first tube, and 1 ml into each of the remaining nine tubes., 3. Into Tube 1, pipette 0.2 ml of Salmonella, typhimurium H antiserum. Mix thoroughly by, pulling the fluid up and down in the pipette., Note: Avoid vigorous washing. The antiserum, has now been diluted 10 times (1:10)., 4. Using a clean pipette, transfer 1 ml from Tube, 1 to Tube 2 and mix thoroughly as described., Using the same pipette, transfer 1 ml from, Tube 2 to Tube 3. Continue this procedure, through Tube 9., 5. Discard 1 ml from Tube 9. Tube 10 will serve, as the antigen control and therefore will not, contain antiserum., 6. The antiserum has been diluted during this, twofold dilution to give final dilutions of 1:10,, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, and, 1:2560.
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PROCEDURE, Transfer 1.0 ml from tube to tube., , Discard., , 6, , 7, , 8, , 9, , 10, , Test tube 10, will serve as the, antigen control., , 1:, 60, , 80, , 0, , 0, , 0, , 25, , 12, 1:, , 40, 1:, , 5, , 64, 1:, , 20, 1:, , 4, , 32, 1:, , 3, , 16, 1:, , 2, , 80, 1:, , 1, , 10, 1:, , Pipette into test tube1:, 1.8 ml of saline, 0.2 ml of antiserum, , Pipette 1.0 ml of saline into tubes 2–10., , Figure 70.2 Antibody titer test. Serial dilution of Salmonella typhimurium H antibody, , 7. Add 1 ml of the Salmonella typhimurium H, antigen suspension adjusted to an absorbance, of 0.5 at 600 nm to all tubes., 8. Mix the contents of the test tubes by shaking, the rack vigorously., , 9. Incubate the test tubes in a 55°C waterbath for, 2 to 3 hours., 10. In the Lab Report, indicate the presence or, absence of agglutination in each of the antiserum dilutions. Also, indicate the end point of, the reaction., , Experiment 70, , 497
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E XP E R IMENT, , 70, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Febrile Antibody Test, Area A, , Area B, , Draw the appearance of the, mixture and of the control., , Saline, S. typhimurium H antigen, , S. typhimurium H. antiserum, S. typhimurium H antigen, , Macroscopic agglutination ( ) or ( ), Microscopic agglutination ( ) or ( ), , Serological Identification of an Unknown Organism, AGGLUTINATION, , Cell Antigen, , Antiserum, , Unknown No: ___, , S. typhimurium H, , Unknown No: ___, , S. typhimurium H, , Unknown No: ___, , S. typhimurium H, , Unknown No: ___, , S. typhimurium H, , Macroscopic, (+) or (−), , Microscopic, (+) or (−), , Homologous, Antigen–Antibody, Reaction, , Experiment 70: Lab Report, , 499
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Determination of Antibody Titer, Tube, , Dilution, , 1, , 1:10, , 2, , 1:20, , 3, , 1:40, , 4, , 1:80, , 5, , 1:160, , 6, , 1:320, , 7, , 1:640, , 8, , 1:1280, , 9, , 1:2560, , 10, , Agglutination, , Titer, , Antigen control, , Review Questions, 1. What are febrile antibodies?, , What is their clinical significance?, , 2. What is the purpose of determining an antibody titer?, , 3., , 500, , hy does the antibody titer determination use twofold dilutions of the antiserum rather than, W, 10-fold dilutions?, , Experiment 70: Lab Report
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Enzyme-Linked Immunosorbent, Assay, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Identify either an antigen or an antibody, using an enzyme-labeled antibody test, procedure., , Principle, The enzyme-linked immunosorbent assay, (ELISA) procedure is used for the detection of, specific antigens or antibodies. The procedure is, predicated on the use of an enzyme-linked (labeled), specific antibody to demonstrate the agglutination, reaction for the interpretation of the test result., This test can be performed as a double-antibody, technique or as an indirect immunosorbent assay., The former method is used for the detection of test, antigens; the latter is used for the detection of the, test antibodies. In both methods the reactions are, carried out in a well of a plastic microtiter plate., The double-antibody system requires that the, unlabeled antibody be allowed to adsorb to the inner, surface of the plastic well in the microtiter plate., Any unbound antibody is then washed away, and a, specific test antigen is added to the well. If the antigen binds with the antibody adhering to the walls of, the well, this immunocomplex will not be removed, by the subsequent washing for the removal of any, unbound antigen. An enzyme-linked antibody, specific for the antigen, is now added. If the antigen is, present in the well, this labeled antibody binds to the, antigen, forming an antibody–antigen–antibody complex. Any unbound enzyme-linked antibody is again, removed by washing. This is followed by the addition of a substrate that is capable of producing a colored end product upon its reaction with the enzyme., The resultant enzymatically produced color change, may be observed by eye or spectrophotometrically., The indirect immunosorbent test procedure, is similar to the double-antibody technique in that, it requires the use of an enzyme-linked antibody., However, an antigen, rather than an antibody, is, adsorbed onto the inner surface of the well., Enzyme-linked immunosorbent assays are, used extensively for the diagnosis of human, , E XP E R IMENT, , 71, , infectious diseases. Included among these are, viral infections, such as AIDS, influenza, respiratory syncytial viral infection, and rubella. Bacterial, infections such as syphilis, brucellosis, salmonellosis, and cholera can also be ascertained by means, of this technique. This procedure also can be used, for the detection of drugs in blood or tissues., In this experiment, you will use the Directigen™, Flu A Test (Becton, Dickinson and Company), to demonstrate the application of an in vitro, enzyme immunoassay. This rapid, qualitative test, employs an enzyme immuno-membrane filter, assay to detect influenza A antigen extracted, from nasopharyngeal or pharyngeal specimens of, symptomatic patients. These specimens are added, to a ColorPAC™ test device, and any influenza A, antigen present is nonspecifically bound to the, membrane surface. Detector enzyme conjugated, to monoclonal antibodies specific for the influenza, A nucleoprotein antigen is bound to the trapped, antigen following its addition to the ColorPAC, membrane. Two substrates are then added sequentially and allowed to incubate for 5 to 30 minutes, prior to determination of the result., In the experimental procedure to be followed,, the positive control will simulate the nasopharyngeal specimen of a symptomatic patient and will, be indicative of a positive result. A pharyngeal, swab sample of an asymptomatic individual will be, used to illustrate a negative result., , F U RT H E R RE A D I N G, Refer to the section on the immune system in your, textbook for further information on the acquired, immune responses. In your textbook’s index, search, under “Antibody,” “Precipitate,” and “Peroxidase.”, , C L I N I C A L A P P L I C AT I O N, Lyme Disease, The ELISA test is commonly used in the diagnosis of, Lyme disease for the detection of antibodies to Borrelia burgdorferi. Because of the test’s sensitivity, it, can sometimes produce false positive results, and, it is not used as the sole basis for diagnosis of Lyme, disease. It is generally followed up by a Western, blot test to confirm the diagnosis., 501
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AT THE B E N C H, , Materials, Cultures, ❏❏ Directigen Flu A positive control, ❏❏ Pharyngeal swab specimen from an asymptomatic individual, , Media, Per designated student group or demonstration, ❏❏ One test tube with 2 ml of sterile saline, , Equipment, ❏❏, ❏❏, ❏❏, ❏❏, ❏❏, , Directigen Flu A Test kit, Sterile cotton swabs, Sterile 0.2-ml 1200@ml2 pipette, Mechanical pipetting device, Disposable gloves, , Procedure, Note: This test may be performed as a demonstration for economic reasons or conservation of, laboratory time., , Wear disposable gloves during the procedure., , Preparation of Negative Result by, Use of a Pharyngeal Specimen, 1. Using a sterile cotton swab, obtain a specimen, from the palatine tonsil by rotating the swab, vigorously over its surface., 2. Immerse the cotton swab into a test tube containing 2 ml of sterile saline. Mix well. Remove, as much liquid from the swab as possible by, pressing the swab against the inner surface of, the tube. Discard the swab into a container of, disinfectant., 3. Using a 0.2-ml 1200@ml2 pipette and a mechanical pipetting device, transfer 124ml of the, pharyngeal specimen into a DispensTube™, provided in the kit., , 502, , Experiment 71, , 4. Gently mix and add 8 drops of Reagent A into, the DispensTube. Mix well., 5. Insert a tip, provided in the kit, into the DispensTube. Dispense all of the extracted specimen into the ColorPAC test well in drops with, the sealed flow controller in position. Allow, for complete adsorption., 6. Gently mix and rapidly add drops of Reagent, 1 until the test well is filled. Allow sufficient, time for complete adsorption., 7. Remove the flow controller from the ColorPAC well and discard it into a container of, disinfectant., 8. Gently mix and add 4 drops of Reagent 2 onto, the ColorPAC membrane. Allow sufficient time, for complete adsorption., 9. Gently mix and add 4 drops of Reagent 3 onto, the ColorPAC membrane. Allow sufficient, time for complete adsorption. Let stand for 2, minutes., 10. Rapidly add enough drops of Reagent 4 to fill, the ColorPAC well. Allow sufficient time for, complete adsorption., 11. Gently mix and add 4 drops of Reagent 5 onto, the ColorPAC membrane. Allow sufficient time, for complete adsorption., 12. Gently mix and add 4 drops of Reagent 6 onto, the ColorPAC membrane. Allow sufficient time, for complete adsorption. Note: The membrane, will turn yellow., 13. Gently mix and add 4 drops of Reagent 7 onto, the ColorPAC membrane. Allow sufficient time, for complete adsorption., 14. Wait at least 5 minutes, but no longer than 30, minutes, and then read the results in a welllighted area., 15. Observe the appearance of the inner surface, of the ColorPAC test wells and record your, results in the Lab Report., , Preparation of Positive Result by Use, of Positive Control, 1. Dispense 4 drops of the positive control, provided in the test kit, into a DispensTube., 2. Repeat Steps 4 through 15 as outlined previously for the preparation of the negative pharyngeal specimen.
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E XP E R IMENT, , 71, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, Record your results below based on the following interpretations of your, observations:, Positive test (antigen present): The appearance of a purple triangle (of any, intensity) on the ColorPAC membrane indicates the presence of the influenza A, antigen in the specimen. A purple dot may be evident in the center of the triangle., The background area should be grayish white., Negative test (no antigen detected): The appearance of a purple dot on the, ColorPAC membrane indicates the absence of the influenza A antigen in the, specimen. The background area should be grayish white., , Uninterpretable test: The absence of a purple dot, a purple triangle, or an, incomplete purple triangle indicates an uninterpretable test., , Negative pharyngeal specimen:, , Positive control specimen:, , Result, , Result, , Review Question, 1., , Why is the ELISA test used to screen human serum for the AIDS, virus, while the Western blot procedure is used only as the confirmation test?, , Experiment 71: Lab Report, , 503
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Sexually Transmitted Diseases: Rapid, Immunodiagnostic Procedures, , Sexually transmitted diseases (STDs) represent a, diverse group of infectious syndromes that share, the same mode of transmission, direct sexual, contact. Their etiological agents represent a broad, spectrum of pathogenic microorganisms that, include bacteria, viruses, yeasts, and protozoa. The, bacterial STDs include gonorrhea, syphilis, nongonococcal urethritis, and lymphogranuloma, venereum. The representative viral infections are, genital herpes, genital warts, hepatitis B, and, the latest member of this group, AIDS. The protozoal and fungal infections, namely trichomoniasis, and candidiasis, are diseases of lesser magnitude, on the spectrum of STDs., The experimental procedures that follow were, chosen to demonstrate some of the rapid tests, that are currently available for the diagnosis of, selected STDs, specifically syphilis, genital herpes,, and the chlamydial infections. In the methods that, follow, you will perform modified procedures in, the absence of clinical specimens. Commercially, available positive and negative controls will be, used to simulate clinical materials. It is suggested, that any of these tests, if performed, should be, done as demonstrations., , Rapid Plasma Reagin, Test for Syphilis, PA RT A, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Perform a rapid screening procedure for, diagnosis of syphilis., , Principle, Treponema pallidum, the causative agent of, syphilis, is a tightly coiled, highly motile, delicate, spirochete that can be cultivated only in rabbit, tissue cultures or rabbit testes. The organisms are, , E XP E R IMENT, , 72, , resistant to common staining procedures and are, best observed under darkfield microscopy., Syphilis is a systemic infection that, if, untreated, progresses through three clinical, stages. The first stage, primary syphilis, is characterized by the formation of a painless papule, called a chancre, at the site of infection., Secondary syphilis represents the systemic extension of the infection and presents itself in the form, of a maculopapular rash, malaise, and lymphadenopathy. Following this stage, the disease becomes, self-limiting, and the patient appears asymptomatic until the development of tertiary syphilis. In, this final stage, life-threatening complications may, develop as a result of the extensive cardiovascular, and nervous tissue damage that has ensued., The rapid plasma reagin (RPR) test, which, has to a large extent replaced the VDRL (Venereal, Disease Research Laboratory) agglutination test,, determines the presence of reagin, the nonspecific antibody present in the plasma of individuals, with a syphilitic infection. The reagin appears in, the plasma within two weeks of infection and will, remain at high concentrations until the disease is, eradicated. In the RPR test, if the reagin is present, in the blood, it will react with a soluble antigen, bound to carbon particles to produce a macroscopically visible antigen, or carbon–antibody, complex. This procedure has several advantages, over the VDRL test:, 1. The serum does not require inactivation by, heat for 30 minutes., 2. The serum may be obtained from a finger, puncture, unlike the VDRL test, which requires, a venous blood sample., 3. The required materials, which include the, antigen suspension with a dispensing bottle,, diagnostic cards, and capillary pipettes, are all, contained in individual kits that do not require, additional equipment and are disposable., In the qualitative form of the RPR test, the, patient’s blood serum and the carbon-bound, antigen suspension are mixed within a circle on, the diagnostic card. In the presence of a positive, (reactive) serum, the antigen–antibody complex, , 505
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AT T HE BE NCH, , Materials, Reactive, , Nonreactive, , Figure 72.1 Test card showing results of the rapid, plasma reagin test, , will produce a macroscopically visible black agglutination reaction. The macroscopic appearance, of a light-gray suspension, devoid of any form of, agglutination, is indicative of a negative (nonreactive) serum (Figure 72.1)., Since this is a nonspecific test, false-positive, results may be obtained. It is believed that the, reagin is an antibody against tissue lipids in general. Therefore, it may be present in uninfected, individuals due to the release of lipids resulting, from normally occurring wear and tear of body, tissues. It has also been found that serum levels, of reagin are elevated during the course of other, infectious diseases such as viral pneumonia, lupus, erythematosus, infectious mononucleosis, yaws,, and pinta. The serum of patients with a reactive, RPR result is subjected to additional serological, testing, such as the FTA-ABS (fluorescent treponemal antibody-absorption) test, or the TPI, (Treponema pallidum immobilization) test, using, the Treponema pallidum bacterium as an antigen, to detect specific antibodies that are also present, in the serum during syphilitic infection., , C L I N I C A L A P P L I C AT I O N, Spirochetes, The rapid plasma regain test detects nonspecific, antibodies in the blood of patients that may indicate, the spirochete Treponema pallidum that causes, syphilis. This test is used to screen asymptomatic, patients, diagnose symptomatic infection, and track, the progress of disease over the treatment period., High incidence of false positives due to cross-reactivity and false negatives due to low antibody titers, requires further testing using the Venereal Disease, Research Laboratory (VDRL) test in many clinical, labs., , 506, , Experiment 72, , Reagents, ❏❏ Commercially prepared syphilitic serum 4+, and nonsyphilitic serum, , Equipment, ❏❏ RPR test kit (Inverness Medical Professional, Diagnostics), ❏❏ Disposable gloves, ❏❏ Rotating machine (optional), , Procedure, Wear disposable gloves, , 1. Label circles on the diagnostic plastic card as, reactive and nonreactive., 2. Use a capillary pipette with an attached rubber, bulb; draw the reactive serum up to the indicated mark (0.05 ml)., 3. Expel the serum directly onto the card in the, circle labeled reactive serum. With a clean, applicator stick, spread the serum to fill the, entire circle., 4. Repeat Steps 2 and 3 for the nonreactive serum., 5. Shake the dispensing bottle to mix the suspension. Hold the bottle with attached 20-gauge, needle in a vertical position and dispense 1 drop, onto each circle containing the test serum., 6. If a mechanical rotator is available, place the, card on the rotator set at 100 rpm, or rotate the, card back and forth manually for 8 minutes., 7. In the presence of direct light, while tilting the, card back and forth, determine the presence, or absence of black clumping in each of the, serum–antigen mixtures. Record your observations and the reaction as (+ ) or (- ) in the Lab, Report.
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Genital Herpes:, Isolation and Identification of, Herpes Simplex Virus, PA RT B, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Perform a tissue culture procedure to grow, and identify the herpes simplex virus., , Principle, The double-stranded DNA herpes simplex virus, (HSV) is the etiological agent of a variety of human, infections. Included among these are herpes, labialis, fever blisters around the lips; keratoconjunctivitis, infection of the eyes; herpes genitalis, eruptions on the genitalia; herpes encephalitis, a severe infection of the brain; and neonatal, herpes. The herpes simplex virus is divided into, two antigenically distinct groups, HSV-1 and HSV2. The former is most frequently implicated with, infections above the waist, whereas the latter is, predominantly responsible for genital infections., Primary infection with HSV-2 manifests itself, with the appearance of vesicular lesions, characterized by itching, tingling, or burning sensations, on or within the male and female genitalia. These, vesicles regress spontaneously within two weeks., Following this symptomatic phase, the virus, reverts to a latent state in the sacral nerves and, remains quiescent until exacerbated by some environmental factor. With no chemotherapeutic cure, presently available, recurrent genital herpes with, subclinical symptoms is common., Detection of the herpes simplex virus requires, the use of tissue culture techniques. The presence, of the virus is then determined by the development, of cytopathogenic effects in these cultures, such, as the detection of intranuclear inclusion bodies., In recent years, these time-consuming, specialized, procedures have been greatly facilitated by the, availability of immunoenzymatic reagents for the, identification of this clinically significant virus., The Cellmatics™ HSV Detection System, is a self-contained system providing for both the, growth and the identification of the virus from, clinical specimens. In this procedure, the provided tissue culture tubes are inoculated with the, clinical sample. Following a 24-hour incubation, , period and fixation, the presence of HSV antigens, is determined by the addition of anti-HSV antibodies, which specifically bind to the HSV antigens., To demonstrate this antigen–antibody complex,, a secondary antibody, substrate, and chromogen, are added. Following this staining process, HSVpositive cultures viewed microscopically will, exhibit brown–black areas of viral infection on a, clear background of unstained cells., In this exercise, you will perform a modified, procedure. In the absence of a clinical specimen,, the actual culturing and fixation process will not, be performed. Instead, the positive and negative, commercially available controls will be used to, simulate the clinical samples., , C L I N I C A L A P P L I C AT I O N, Culturing HSV, Genital herpes is caused by herpes simplex-2 virus, (HSV-2). It is spread from person to person during sexual contact. The infection is transmitted by, means of viral shedding, which may occur even, when no signs or symptoms appear. A swab sample, of a vesicular lesion or from the site of a previous, lesion is taken from the patient and sent to the clinical or infectious disease lab for identification. This, virus can only be grown in tissue culture and not, cultivated on or in other laboratory media., , AT T HE BE NCH, , Materials, Cultures, ❏❏ Cellmatics HSV Positive and Negative Control, Tubes, , Reagents, ❏❏ Cellmatics Immunodiagnostic Reagents Kit,, distilled water (Difco Labs), , Equipment, ❏❏ 5-ml pipettes, ❏❏ Mechanical pipetting device, ❏❏ Microscope, , Experiment 72, , 507
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Procedure, 1. Warm immunodiagnostic reagents to room, temperature., 2. Drain all fluid from the positive and negative, control tubes., 3. Using a 5-ml pipette, wash the culture tubes, twice with 5 ml of distilled water and drain., Note: When washing, exercise care to prevent, disruption of the monolayer., 4. Add 10 drops of primary antiserum (Vial 1)., Note: When adding reagents, hold the vials, vertically to ensure proper delivery., 5. Incubate the tightly capped tubes in a horizontal position for 15 minutes at 37°C. To, ensure complete coverage of the monolayer,, occasionally rock the tubes gently during, incubation., 6. Wash three times with 5 ml of distilled water, and drain., 7. Add 10 drops of secondary antibody (Vial 2)., 8. Incubate for 15 minutes at 37°C as described, in Step 5., 9. Wash three times with 5 ml of distilled water, and drain., 10. Add 10 drops of substrate (Vial 3) and 2 drops, of chromogen (Vial 4). Mix gently., 11. Incubate for 15 minutes at 37°C as described, in Step 5., 12. Wash three times with 5 ml of distilled water, and drain., 13. Examine microscopically for the presence of, stained cells at 40 * and 100* magnifications., Scan the entire stained monolayer of both, culture tubes for the presence of brown to, blackish-brown stained cells. HSV infection is, indicated by the presence of dark-colored cells, when viewed against an unstained background, of normal cells., 14. Record your observations in the Lab Report., , 508, , Experiment 72, , Detection of Sexually, Transmitted Chlamydial Diseases, PART C, , LEARNING OBJECTIVE, Once you have completed this experiment,, you should be able to, 1. Perform an immunofluorescent procedure, for diagnosis of Chlamydia infections., , Principle, Members of the genus Chlamydia are a group, of obligate intracellular parasites. Although they, were once believed to be viruses, their morphological and physiological characteristics more closely, resemble bacteria, so they are now considered, small bacteria. Chlamydiae are gram-negative,, nonmotile, thick-walled, spherical organisms, possessing both DNA and RNA that reproduce, by means of binary fission. Their dependence on, living tissues for cultivation and their lack of an, ATP-generating system emulate the characteristics of viruses, but their bacterial nature is further, affirmed by their sensitivity to antibiotic therapy., Chlamydia trachomatis, the human pathogen, is, now recognized to be responsible for two sexually, transmitted diseases, nongonococcal urethritis, (NGU) and lymphogranuloma venereum (LGV)., The incidence of both diseases in contemporary, society is increasing dramatically., NGU is a urethritis (inflammation of the urethra) with symptoms similar to, but less severe, than, those of gonorrhea. Undiagnosed and, untreated infections may lead to epididymitis, and proctitis in men and cervicitis, salpingitis,, and pelvic inflammatory disease in women., Nongonococcal urethritis is also caused by other, bacteria, such as Ureaplasma urealyticum and, Mycoplasma hominis, as well as the protozoan
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Trichomonas vaginalis. LGV, the most severe of, the genital chlamydial infections, initially develops with a painless lesion at the portal of entry,, the genitalia. Systemic involvement is evidenced, by swelling of the regional lymph nodes, which, become tender and suppurative before disseminating the organisms to other tissues. In the absence, of chemotherapeutic intervention, scarring of the, lymphatic vessels can cause their obstruction,, leading to elephantiasis, enlargement of the, external genitalia, in men and narrowing of the, rectum in women., The MicroTrak® Direct Specimen Test, is a rapid, immunofluorescent procedure for the, detection of C. trachomatis. The procedure circumvents the need to culture these organisms in, susceptible tissues prior to their identification., This slide test is designed to detect elementary, bodies, the infectious particles produced during the life cycle of this organism, by the use of a, staining reagent, a fluorescein-labeled monoclonal, antibody specific for the principal protein of the, C. trachomatis outer membrane. In this procedure, a slide smear is prepared from the clinical, specimen. Following fixation, when the slide is, exposed to the Direct Specimen Reagent, the antibody binds to the organisms. Their presence is, then determined by the appearance of apple-green, chlamydiae against a red background of counterstained cells when viewed under a fluorescent, microscope., , C L I N I C A L A P P L I C AT I O N, Treating Chlamydia, Chlamydial infections are the most commonly, reported sexually transmitted diseases. More than, 50 million infections occur worldwide, with 3 million, cases occurring in the United States annually. Any, sexually active person can contract Chlamydia, but, it most frequently occurs in teenagers and young, adults. The incidence appears higher in females, than in males. Chlamydia may be transmitted by an, infected mother to her newborn during birth. If a, mother’s medical or sexual history indicates possible exposure, a urogenital swab will be used to, collect a sample for testing., , AT T HE BE NCH, , Materials, Cultures, ❏❏ Commercially prepared positive and negative, control slides, , Reagents, ❏❏ MicroTrak Direct Specimen Test for Chlamydia trachomatis (VWR Scientific), , Equipment, ❏❏ Fluorescent microscope, , Procedure, 1. Stain the positive and negative control slides, with the MicroTrak reagent for 15 minutes., 2. Incubate slides for 15 minutes., 3. Rinse the slides with distilled water., 4. Air-dry the slides., 5. Examine the slides under a fluorescent microscope for the presence of apple-green particles, indicative of chlamydiae. The particles are, evident against a reddish background of counterstained cells., 6. Record your results in the Lab Report., , Experiment 72, , 509
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E XP ER IM E NT, , 72, , Name:, Date:, , Lab Report, , Section:, , Observations and Results, PART A: Rapid Plasma Reagin Test for Syphilis, Reactive Serum, , Nonreactive Serum, , Appearance of serum-antigen mixture, Reaction (+) or (-), , Draw the observed reaction., , PART B: Genital Herpes: Isolation and Identification, of Herpes Simplex Virus, Indicate in the chart below the presence (+ ) or absence (- ) of dark-stained, patches., CONTROL CULTURES, Negative, 40 *, , Positive, 100 *, , 40 *, , 100 *, , PART C: Detection of Sexually Transmitted Chlamydial, Diseases, Record your results below, indicating the presence (+ ) or absence (- ) of the, apple-green chlamydiae on each of the slides., Positive control slide: _____________________________________________, Negative control slide: ____________________________________________, , Experiment 72: Lab Report, , 511
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Review Questions, 1., , 2., , 512, , hy is an adult who has a high-antibody titer to herpes simplex-2 virus (HSV-2) subject to, W, recurrent genital herpes infections?, , A 20-year-old college student was informed following a physical examination that her blood, test for syphilis was reactive. She indicated that she was a virgin and had never received, a blood transfusion. A repeat RPR test was positive, but the TPI test was negative. How would you, explain these seemingly bizarre results, and what is the clinical status of this patient?, , Experiment 72: Lab Report
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AP PENDIX, , 1, , Scientific Notation, , Microbiologists are required to perform a variety, of laboratory techniques, including preparing and, diluting solutions; expressing concentrations of, chemicals, antibiotics, and antiseptics in solution; making quantitative determinations of cell, populations based on the standard method for, plate counting; and making serial dilutions to, accommodate the latter procedure. These techniques commonly involve the use of very large, or very small numbers (e.g., 9,000,000,000 or, 0.0000000009), which can be so cumbersome to, manipulate that errors may result. Therefore, it is, essential for microbiologists to have a good command of scientific exponential notation known as, scientific notation., , Appendix Table 1.1, , The basis for this system is predicated on the, fact that all numbers can be expressed as the product, of two numbers, one of which is the power of the, number 10. In scientific notation, the small superscript number next to the 10 is called the exponent., Positive exponents tell us how many times the, number must be multiplied by 10, while negative, exponents indicate how many times a number must, be divided by 10 (that is, multiplied by one-tenth)., For example, a number written using the, exponential form designated as scientific notation would appear as 7.5 * 103, meaning that, 7.5 * 10 * 10 * 10 = 7500. Appendix Table 1.1, shows both large and small numbers written in the, exponential form., , Scientific (Exponential) Notation, , NUMBERS GREATER THAN ONE, 9, , NUMBERS LESS THAN ONE, , 1,000,000,000 = 1 * 10, , 0.000 000 001 = 1 * 10-9, , 100,000,000 = 1 * 108, , 0.000 000 01 = 1 * 10-8, , 10,000,000 = 1 * 107, , 0.000 000 1 = 1 * 10-7, , 1,000,000 = 1 * 106, , 0.000 00 1 = 1 * 10-6, , 100,000 = 1 * 105, , 0.000 01 = 1 * 10-5, , 10,000 = 1 * 104, , 0.000 1 = 1 * 10-4, , 1000 = 1 * 103, , 0.001 = 1 * 10-3, , 100 = 1 * 102, , 0.01 = 1 * 10-2, , 10 = 1 * 101, , 0.1 = 1 * 10-1, , 1 = 1 * 100, , 1 = 1 * 100, , Note: The exponent to which the power of 10 is raised is, equal to the number of zeros to the right of 1., , Note: The exponent to which the power of 10 is raised is, equal to the number of zeros to the left of 1 plus 1., , , , 513
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Multiplication, , Division, , Rule: To multiply two numbers that are written in, scientific notation (exponential form) you must, add the exponents., , Rule: To divide two numbers in scientific notation,, you must subtract the exponents., , Using numbers larger than 1:, 75 * 1200 = 90,000, Scientific notation: 17.5 * 101 2 * 11.2 * 103 2 = 9 * 104, Addition of exponents: 1 + 3 = 4, , Using numbers less than 1:, 0.75 * 1200 = 900, Scientific notation: 17.5 * 10-1 2 * 11.200 * 103 2 = 9 * 102, Addition of exponents: 1 - 1 + 3 = 22, , 0.75 * 0.12 = 0.09, , Scientific notation: 17.5 * 10-1 2 * 11.2 * 10-1 2 = 9 * 10-2, Addition of exponents: 1 - 12 + 1 - 12 = -2, , 514, , Appendix 1, , 75,000 , 1,200,000 = 0.0625, Scientific notation: 17.5 * 104 2, 11.2 * 106 2 = 6.25 * 10-2, Subtraction of exponents: 14 - 6 = -22, , 7,500 , 012 = 625,000, , Scientific notation: 17.5 * 103 2, 11.2 * 10-2 2 = 6.25 * 105, Subtraction of exponents: 3 - 1 - 22 = 5, , As you practice the use of scientific notation with, large and small numbers, you will become more, proficient and more comfortable with this system, of scientific calculation.
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Methods for the Preparation, of Dilutions, , In microbiology laboratories as in other science, laboratories, solutions must be diluted to achieve, a desired final concentration of the active material, contained in that solution. A solution may be, defined as a mixture of two or more substances, (solute) in which the molecules of the solute are, evenly distributed and will not separate on standing, or precipitate from the solution. Solutes are, dissolved in a solvent or diluent, such as water,, alcohol, or some other vehicle in which the solute is soluble. Solutions are usually referred to as, stock solutions and may be diluted by a variety of, methods, depending upon the experimental requirements. Some of these methods are listed as follows:, 1. A dilution factor must be determined first in, order to dilute a solution. This dilution factor, tells us how many times a solution must be, diluted and is calculated by dividing the, initial concentration (IC) of the solution by, the final concentration (FC) desired., , 10% , 2% = 5(dilution factor), , Take 1.0 ml of the 10% stock solution plus, 4.0 ml of diluent (solvent), which equals a total, of 5.0 ml. Thus each ml of the final solution, will contain 2% solute., 2. Another method is used when a specific volume composed of a specific concentration is, required., a., , IC, = 10(dilution factor), FC, , b., , volume needed, amount of initial, =, concentration required, solution needed, , c. volume needed - amount of initial solution, = amount of diluent, , Example: You have a 50% concentrated solution and you need 200 ml of a 5% solution., a., , IC, 50%, =, = 10(dilution factor), FC, 5%, , b., , volume needed, 200 ml, =, = 40 ml, concentration required, 5%, , 2, , c. 40 ml of 50% IC + 160 ml of diluent = 200 ml, of a solution, such that each ml will contain, 5% solute rather than the original 50% in the, stock solution, , 3. The ability to prepare large dilutions is absolutely essential for work in the microbiology, laboratory. This method requires that large, dilutions be prepared in two steps., Example: A solution contains 1.0 g per ml, of an active material and needs to be diluted, to a final concentration of 1.0 μg per ml. A, 1,000,000 1 1 * 106 2 -fold dilution must be, made. It is not practical to make such a dilution in one step since 999,999 ml of diluent, would be required. This type of dilution is, made as follows:, a. Dilute 1 ml of the stock solution 1000 times:, 1.0 ml + 999 ml of diluent = 1000 µg/ml, , b. Dilute the solution containing 1000 µg/ml, another 1000 times:, 1 ml of 1000 µg/ml + 999 ml diluent = 1.0 µg/ml, , IC , FC = dilution factor, , Example: You wish to dilute a 10% stock solution to a final concentration of 2%., , AP PENDIX, , 4. When working with large molecules, such as, proteins, there will be times when you will be, required to make large dilutions of the sample, to be contained in a specific volume., Example: You need to make 50 ml of a, 1/20,000 dilution of albumin., 20,000, final dilution, =, = 400(dilution factor), volume needed, 50, 1.0 ml of, 399 ml of, +, = 1>400 dilution, albumin, diluent, 50 ml of a, 1.0 ml of, 49 ml of, soution; each ml, b. a 1/400 +, =, diluent, contains 1>20,000, dilution, of albumin., 501volume2 * 4001dilution factor2 = 20,000, , a., , 5. Perhaps the most useful type of dilution, used in microbiology and immunology is the, serial dilution. This is essential when small, volumes of material are needed. This type, of dilution procedure has many uses in the, microbiology laboratory, especially for the, determination of the total number of cells, , , , 515
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in culture (Experiments 19 and 20, the number of viral plaques found in suspensions of, viruses (Experiment 38), the antibody titer, (Experiment 72), and in other immunological studies. The procedure requires the use of, dilution blanks containing a known volume of, diluent (distilled water, saline, etc.) to which, a specific volume of the sample is added. To, facilitate the ease of calculations, dilutions are, usually made in multiples of 10. For example:, 1.0 ml of a sample is added to a 9.0-ml dilution, blank 1 1.0 ml + 9.0 ml = 10 2 and is recorded, as a 1:10 dilution., , dilution has been explained and illustrated in, Experiment 19. For the convenience of the student, it is illustrated in Appendix Figure 2.1:, 1. All dilution blanks contain 9.0 ml of diluent., 2. A fresh pipette is used for each dilution, and, the used pipettes are placed in a beaker of, disinfectant., 3. After delivery of the sample, the tubes are, mixed thoroughly before the next dilution is, made., 4. Pippetting by mouth is not allowed. Only, mechanical pipette aspirators may be used., , It has been statistically determined that, greater accuracy is achieved with very large dilutions made from a series of smaller dilutions., The procedure for the performance of a serial, , Transfer, 1 ml., , Transfer, 1 ml., , Transfer, 1 ml., , Transfer, 1 ml., , Transfer, 1 ml., , Stock, solution, , Tube 1, , Tube 2, , Tube 3, , Tube 4, , Tube 5, , Tube 6, , Dilution, , 10-1, , 10-2, , 10-3, , 10-4, , 10-5, , 10-6, , Final, dilution, , 1:10, , 1:100, , 1:1000, , 1:10,000, , 1:100,000, , 1:1,000,000, , 1 * 10-2, , 1 * 10-3, , 1 * 10-4, , 1 * 10-5, , 1 * 10-6, , Scientific, notation, , 1 * 10-1, , Appendix Figure 2.1, , 516, , Transfer, 1 ml., , The stock solution in Appendix Figure 2.1 has, been diluted 1 million times. In other words, 1.0 ml, from Tube 6 will contain 1>1,000,000 of the sample, contained in the stock solution., , Appendix 2, , Serial dilution
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Microbiological Media, , The formulas of the media used in the exercises, in this manual are listed alphabetically in grams, per liter of distilled water unless otherwise specified. Sterilization of the media is accomplished by, autoclaving at 15 lb pressure for 15 minutes unless, otherwise specified. Most of the media are available commercially in powdered form, with specific, instructions for their preparation and sterilization., Ammonium sulfate broth (pH 7.3), Ammonium sulfate, Magnesium sulfate • 7H2O, Ferric sulfate • 7H2O, Sodium chloride, Magnesium carbonate, Dipotassium hydrogen phosphate, , 2.0, 0.5, 0.03, 0.3, 10.0, 1.0, , Bacteriophage broth 10: (pH 7.6), Peptone, Beef extract, Yeast extract, Sodium chloride, Potassium dihydrogen phosphate, , 100.0, 30.0, 50.0, 25.0, 80.0, , Basal salts agar* and broth (pH 7.0), 0.5 M sodium diphosphate, 100.0 ml, 1.0 M potassium dihydrogen, 100.0 ml, phosphate, Distilled water, 800.0 ml, 0.1 M calcium chloride, 1.0 ml, 1.0 M magnesium sulfate, 1.0 ml, Ammonium sulfate, 2.0, *Agar, 15.0, Note: Swirl until completely dissolved, autoclave, and cool. Aseptically add 10.0 ml of 1%, sterile glucose., Bile esculin (pH 6.6), Beef extract, Peptone, Esculin, Oxgall, Ferric citrate, Agar, , 3.0, 5.0, 1.0, 40.0, 0.5, 15.0, , AP PENDIX, , 3, , Blood agar (pH 7.3), Infusion from beef heart, 500.0, Tryptose, 10.0, Sodium chloride, 5.0, Agar, 15.0, Note: Dissolve the above ingredients and, autoclave. Cool the sterile blood agar base to, 45°C to 50°C. Aseptically add 50 ml of sterile, defibrinated blood. Mix thoroughly, avoiding accumulation of air bubbles. Dispense into sterile, tubes or plates while liquid., Brain heart infusion (pH 7.4), Infusion from calf brain, Infusion from beef heart, Peptone, Dextrose, Sodium chloride, Disodium phosphate, Agar, , 200.0, 250.0, 10.0, 2.0, 5.0, 2.5, 1.0, , Brilliant green sulfa agar plate (BGS;, contains 0.1% sodium sulfapyridine), Approximate Formula* Per Liter, Yeast Extract, Proteose Peptone No. 3, Lactose, Saccharose, Sodium Sulfapyridine, Sodium Chloride, Agar, Brilliant Green, Phenol Red, , 3.0, 10.0, 10.0, 10.0, 1.0, 5.0, 20.0, 12.5 mg, 0.08, , Bromcresol purple dextrose fermentation, broth (pH 7.2), Bacto® casitone, 10, Dextrose, 5, Bromcresol purple (0.2%), 0.01, Bromcresol purple (0.2%) is made separately and, filter sterilized. 5 ml is aseptically added to the, medium., Note: Autoclave at 12 lb pressure for 15 minutes., , , , 517
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Bromcresol purple lactose fermentation, broth (pH 7.2), Bacto casitone, 10, Lactose, 5, Bromcresol purple (0.2%), 0.01, Bromcresol purple (0.2%) is made separately and, filter sterilized. 5 ml is aseptically added to the, above medium., Note: Autoclave at 12 lb pressure for 15 minutes., , Crystal violet agar (pH 7.0), Bacto beef extract, 3, Bacto peptone, 5, Bacto crystal violet, 0.00014, Bacto agar, 15, Note: 1.0 ml of a crystal violet stock solution, may be added to the base medium. Stock, solution: 14 mg of crystal violet dye dissolved, in 100 ml of distilled water., , Bromcresol purple maltose fermentation, broth (pH 7.2), Bacto casitone, 10, Maltose, 5, Bromcresol purple (0.2%), 0.01, Bromcresol purple (0.2%) is made separately and, filter sterilized. 5 ml is aseptically added to the, medium., Note: Autoclave at 12 lb pressure for 15 minutes., , Decarboxylase broth (Moeller) (pH 6.0), Peptone, 5.0, Beef extract, 5.0, Dextrose, 0.5, Bromcresol purple, 0.01, Cresol red, 0.005, Pyridoxal, 0.005, Distilled water, 1000.0 ml, , Bromcresol purple sucrose fermentation, broth (pH 7.2), Bacto casitone, 10, Sucrose, 5, Bromcresol purple (0.2%), 0.01, Bromcresol purple (0.2%) is made separately, and filter sterilized. 5 ml is added to the medium, aseptically., Note: Autoclave at 12 lb pressure for 15 minutes., Campy-BAP agar (pH 7.0), Trypticase peptone, 10.0, Thiotone™, 10.0, Dextrose, 1.0, Yeast extract, 2.0, Sodium chloride, 5.0, Sodium bisulfide, 0.1, Agar, 15.0, Vancomycin, 10.0 mg, Trimethoprim lactate, 5.0 mg, Polymyxin B sulfate, 2500.0 IU, Amphotericin B, 2.0 mg, Cephalothin, 15.0 mg, Defibrinated sheep blood, 10.0%, Note: Aseptically add the antibiotics and, defibrinated sheep blood to the sterile, molten,, and cooled agar., Chocolate agar (pH 7.0), Proteose peptone, 20.0, Dextrose, 0.5, Sodium chloride, 5.0, Disodium phosphate, 5.0, Agar, 15.0, Note: Aseptically add 5.0% defibrinated sheep, blood to the sterile and molten agar. Heat at, 80°C until a chocolate color develops., , 518, , Appendix 3, , To make amino acid–specific medium, add, one of the amino acids below; dispense in, 3- to 4-ml amounts and autoclave at 121°C, for 10 minutes., l-lysine dihydrochloride or, l-arginine monohydrochloride or, l-ornithine dihydrochloride, 10 g/l, Deoxyribonuclease (DNase) agar (pH 7.3), Deoxyribonucleic acid, Phytane, Sodium chloride, Trypticase, Agar, Endo agar (pH 7.5), Peptone, Lactose, Dipotassium phosphate, Sodium sulfite, Basic fuchsin, Agar, , 2.0, 5.0, 5.0, 15.0, 15.0, 10.0, 10.0, 3.5, 2.5, 0.4, 15.0, , Double modified lysine iron agar plate (DMLIA), Peptic digest of animal tissue, 5.0, Yeast extract, 3.0, Dextrose, 1.0, L-Lysine, 10.0, Ferric ammonium citrate, 0.8, Sodium thiosulphate, 6.8, Bile salt, 1.5, Lactose, 10.0, Sucrose, 10.0, Bromocresol purple, 0.02, Agar, 15.0, Final pH (at 25°C) 6.7 { 0.2, , **Formula adjusted, standardized to suit, performance parameters
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Eosin–methylene blue agar (Levine) (pH 7.2), Peptone, 10.0, Lactose, 5.0, Dipotassium phosphate, 2.0, Agar, 13.5, Eosin Y, 0.4, Methylene blue, 0.065, , Salicin, Sodium chloride, Sodium thiosulfate, Ferric ammonium citrate, Bromthymol blue, Acid fuchsin, Agar, , Gel diffusion agar, Sodium barbital buffer, Noble agar, , Inorganic synthetic broth (pH 7.2), Sodium chloride, Magnesium sulfate, Ammonium dihydrogen phosphate, Dipotassium hydrogen phosphate, , 100.0 ml, 0.8, , Glucose acetate yeast sporulation agar (pH 5.5), Glucose, 1, Yeast extract, 2, 5, Sodium acetate (with 3H2O), Bacto agar, 15, Glucose salts broth (pH 7.2), Dextrose, Sodium chloride, Magnesium sulfate, Ammonium dihydrogen phosphate, Dipotassium hydrogen phosphate, , 5.0, 5.0, 0.2, 1.0, 1.0, , Glycerol yeast extract agar supplemented with, aureomycin (pH 7.0), Glycerol, 5.0 ml, Yeast extract, 2.0, Dipotassium phosphate, 1.0, Agar, 15.0, Note: Aseptically add aureomycin, 10 mg per ml,, to the sterile, molten, and cooled agar., Grape juice broth, Commercial grape or apple juice, Ammonium biphosphate, 0.25%, Note: Sterilization not required when using a, large yeast inoculum., Hay infusion broth, Hay infusion broth preparations are prepared, 1 week ahead of the laboratory session in which, they will be used. Into a 2000-ml beaker place, about 800 ml of water and two to three handfuls of, dry grass or hay (obtained from a farm or storage, barn). During the incubation period, the infusion, should be aerated by passing air through a rubber, tube attached to an air supply. This preparation, is sufficient for a class and can be dispensed in, 50-ml beakers., Hektoen enteric agar (pH 7.1), Peptic digest of animal tissue, Yeast extract, Bile salt, Lactose, Sucrose, , 12.0, 3.0, 9.0, 12.0, 12.0, , 2.0, 5.0, 5.0, 1.5, 0.064, 0.5, 13.5, , KF broth (pH 7.2), Polypeptone, Yeast extract, Sodium chloride, Sodium glycerophosphate, Sodium carbonate, Maltose, Lactose, Sodium azide, Phenol red, , 5.0, 0.2, 1.0, 1.0, 10.0, 10.0, 5.0, 10.0, 0.636, 20.0, 1.0, 0.4, 0.018, , Lactobacilli MRS Broth Composition, Proteose peptone, 10.0 g, Beef extract, 10.0 g, Yeast extract, 5.0 g, Dextrose, 20.0 g, Sorbitan monooleate, 1.0 g, Ammonium citrate, 2.0 g, Sodium acetate, 5.0 g, 0.05 g, MnSO4 * H2O, 2.0 g, Na 2HPO4, Deionized water, 1000 ml, Note: Final pH of 6.5, autoclave 121°C to sterilize., Lactose fermentation broth 1:: and 2 :*, (pH 6.9), Beef extract, 3.0, Peptone, 5.0, Lactose, 5.0, *For 2 * broth use twice the concentration of the, ingredients., Litmus milk (pH 6.8), Skim milk powder, 100.0, Litmus, 0.075, Note: Autoclave at 12 lb pressure for 15 minutes., Luria-Bertani (Miller) agar base (pH 7.0), Pancreatic digest of casein, Yeast extract, Sodium chloride, Agar, , Appendix 3, , 10.0, 5.0, 0.5, 15.0, , 519
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Luria-Bertani (Miller) broth (pH 7.0), Tryptone, Yeast extract, Sodium chloride, , 10.0, 5.0, 10.0, , Lysine Iron Agar (LIA), L-Lysine Hydrochloride, Peptone, Yeast Extract, Dextrose, Ferric Ammonium Citrate, Sodium Thiosulfate, Bromcresol Purple, Agar, , 10.0, 5.0, 3.0, 1.0, 0.5, 0.04, 0.02, 15.0, , MacConkey agar (pH 7.1), Bacto peptone, Proteose peptone, Lactose, Bile salts mixture, Sodium chloride, Agar, Neutral red, Crystal violet, , 17.0, 3.0, 10.0, 1.5, 5.0, 13.5, 0.03, 0.001, , Mannitol salt agar (pH 7.4), Beef extract, Peptone, Sodium chloride, d-Mannitol, Agar, Phenol red, , 1.0, 10.0, 75.0, 10.0, 15.0, 0.025, , m-Endo broth (pH 7.5), Yeast extract, 6.0, Thiotone peptone, 20.0, Lactose, 25.0, Dipotassium phosphate, 7.0, Sodium sulfite, 2.5, Basic fuchsin, 1.0, Note: Heat until boiling; do not autoclave., m-FC broth (pH 7.4), Biosate™ peptone, 10.0, Polypeptone peptone, 5.0, Yeast extract, 3.0, Sodium chloride, 5.0, Lactose, 12.5, Bile salts, 1.5, Aniline blue, 0.1, Note: Add 10 ml of rosolic acid (1% in 0.2N, sodium hydroxide). Heat to boiling with, agitation; do not autoclave., Milk agar (pH 7.2), Skim-milk powder, 100.0, Peptone, 5.0, Agar, 15.0, Note: Autoclave at 12 lb pressure for 15 minutes., 520, , Appendix 3, , Minimal agar (pH 7.0) Minimal agar,, supplemented with streptomycin and, thiamine*, Solution A (pH 7.0), Potassium dihydrogen phosphate, 3.0, Disodium hydrogen phosphate, 6.0, Ammonium chloride, 2.0, Sodium chloride, 5.0, Distilled water, 800.0 ml, Solution B (pH 7.0), Glucose, 8.0, 0.1, Magnesium sulfate • 7H2O, Agar, 15.0, Distilled water, 200.0 ml, Note: Autoclave Solutions A and B separately, and combine., *To Solution B, add 0.001 g of thiamine prior to, autoclaving. To the combined sterile and molten, medium, add 50 mg (1 ml of 50 mg per ml) sterile, streptomycin solution before pouring agar plates., Modified Tryptone Soys Broth (mTSB), Pancreatic digest of casein, Papaic digest of soybean meal, Sodium chloride, Dipotassium hydrogen phosphate, Glucose, Bile Salts, pH 7.4 { 0.2 @ 25°C, MR-VP broth (pH 6.9), Peptone, Dextrose, Potassium phosphate, Mueller-Hinton agar (pH 7.4), Beef, infusion, Casamino acids, Starch, Agar, , 17, 3.0, 5.0, 4.0, 2.5, 1.5, , 7.0, 5.0, 5.0, 300.0, 17.5, 1.5, 17.0, , Mueller-Hinton tellurite agar (pH 7.4), Casamino acids, 20.0, Casein, 5.0, l-tryptophan, 0.05, Potassium dihydrogen phosphate, 0.3, Magnesium sulfate, 0.1, Agar, 20.0, Note: Aseptically add 12.5 ml of tellurite serum, to the sterile, 50°C molten agar., Nitrate broth (pH 7.2), Peptone, Beef extract, Potassium nitrate, , 5.0, 3.0, 5.0
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Nitrite broth (pH 7.3), Sodium nitrite, Magnesium sulfate • 7H2O, Ferric sulfate • 7H2O, Sodium chloride, Sodium carbonate, Dipotassium hydrogen sulfate, , 2.0, 0.5, 0.03, 0.3, 1.0, 1.0, , Nitrogen-free mannitol agar* and broth (pH 7.3), Mannitol, 15.0, Dipotassium hydrogen phosphate, 0.5, Magnesium sulfate, 0.2, Calcium sulfate, 0.1, Sodium chloride, 0.2, Calcium carbonate, 5.0, *Agar, 15.0, Nutrient agar :: and broth (pH 7.0), Peptone, Beef extract, *Agar, Nutrient gelatin (pH 6.8), Peptone, Beef extract, Gelatin, Peptone broth (pH 7.2), Peptone, , 5.0, 3.0, 15.0, 5.0, 3.0, 120.0, 4.0, , Phenol red dextrose broth (pH 7.3), Trypticase, 10.0, Dextrose, 5.0, Sodium chloride, 5.0, Phenol red, 0.018, Note: Autoclave at 12 lb pressure for 15 minutes., Phenol red inulin broth (pH 7.3), Trypticase, 10.0, Inulin, 5.0, Sodium chloride, 5.0, Phenol red, 0.018, Note: Autoclave at 12 lb pressure for 15 minutes., Phenol red lactose broth (pH 7.3), Trypticase, 10.0, Lactose, 5.0, Sodium chloride, 5.0, Phenol red, 0.018, Note: Autoclave at 12 lb pressure for 15 minutes., Phenol red sucrose broth (pH 7.3), Trypticase, 10.0, Sucrose, 5.0, Sodium chloride, 5.0, Phenol red, 0.018, Note: Autoclave at 12 lb pressure for 15 minutes., , Phenylalanine agar (pH 7.3), Yeast extract, 3.0, Dipotassium phosphate, 1.0, Sodium chloride, 5.0, dl-phenylalanine, 2.0, Bacto agar, 12.0, Distilled water, 1000.0 ml, Note: Completely dissolve ingredients in boiling, water. Dispense in tubes, autoclave, and cool in, slanted position., Phenylethyl alcohol agar (pH 7.3), Trypticase, Phytane, Sodium chloride, b-Phenylethyl alcohol, Agar, Potato dextrose agar (pH 5.6), Infusion from potatoes, Bacto dextrose, Bacto agar, , 15.0, 5.0, 5.0, 2.0, 15.0, 200.0, 20.0, 15.0, , Sabouraud agar (pH 5.6) Sabouraud agar, supplemented with chlortetracycline, (Aureomycin)*, Peptone, 10.0, Dextrose, 40.0, Agar, 15.0, *Aseptically add Aureomycin, 10 µg per ml, to the, sterile, molten, and cooled medium., Salt medium—Halobacterium, Sodium chloride, 250.0, 10.0, Magnesium sulfate • 7H2O, Potassium chloride, 5.0, 0.2, Calcium chloride • 6H2O, Yeast extract, 10.0, Tryptone, 2.5, Agar, 20.0, Note: The quantities given are for preparation, of 1-liter final volume of the medium. In preparation, make up two solutions, one involving, the yeast extract and tryptone and the other, the salts. Adjust the pH of the nutrient solution to 7. Sterilize separately. Mix and dispense, aseptically., SIM agar (pH 7.3), Peptone, Beef extract, Ferrous ammonium sulfate, Sodium thiosulfate, Agar, , 30.0, 3.0, 0.2, 0.025, 3.0, , Appendix 3, , 521
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Simmons citrate agar (pH 6.9), Ammonium dihydrogen phosphate, Dipotassium phosphate, Sodium chloride, Sodium citrate, Magnesium sulfate, Agar, Bromthymol blue, , 1.0, 1.0, 5.0, 2.0, 0.2, 15.0, 0.08, , Snyder test agar (pH 4.8), Tryptone, Dextrose, Sodium chloride, Bromcresol green, Agar, , 20.0, 20.0, 5.0, 0.02, 20.0, , Sodium chloride agar, 7.5% (pH 7.0), Bacto beef extract, Bacto peptone, Sodium chloride, Bacto agar, , 3.0, 5.0, 7.5, 15.0, , Sodium chloride broth, 6.5% (pH 7.0), Brain heart infusion broth, Sodium chloride, Starch agar (pH 7.0), Peptone, Beef extract, Starch (soluble), Agar, Thioglycollate, fluid (pH 7.1), Peptone, Yeast extract, Dextrose, l-cystine, Thioglycollic acid, Agar, Sodium chloride, Resazurin, , 100.0 ml, 6.5, 5.0, 3.0, 2.0, 15.0, 15.0, 5.0, 5.0, 0.75, 0.3 ml, 0.75, 2.5, 0.001, , Tinsdale agar (pH 7.4), Proteose peptone, No. 3, 20.0, Sodium chloride, 5.0, Agar, 20.0, Note: Following boiling, distribute in 100-ml, flasks. Autoclave, cool to 55°C, add 15 ml of, rehydrated Tinsdale enrichment to each 100 ml,, and mix thoroughly before dispensing., Top agar (for Ames test), , Sodium chloride, Agar, , 522, , Appendix 3, , 5.0, 6.0, , Tributyrin agar (pH 7.2), Peptone, 5.0, Beef extract, 3.0, Agar, 15.0, Tributyrin, 10.0, Note: Dissolve peptone, beef extract, and agar, while heating. Cool to 90°C, add the tributyrin,, and emulsify in a blender., Triple sugar–iron agar (pH 7.4), Beef extract, Yeast extract, Peptone, Proteose peptone, Lactose, Saccharose, Dextrose, Ferrous sulphate, Sodium chloride, Sodium thiosulfate, Phenol red, Agar, , 3.0, 3.0, 15.0, 5.0, 10.0, 10.0, 1.0, 0.2, 5.0, 0.3, 0.024, 12.0, , Trypticase nitrate broth (pH 7.2), Trypticase, Disodium phosphate, Dextrose, Agar, Potassium nitrate, , 20.0, 2.0, 1.0, 1.0, 1.0, , Trypticase soy agar (pH 7.3), Trypticase, Phytane, Sodium chloride, Agar, Tryptone agar* and broth, Tryptone, Calcium chloride (reagent), Sodium chloride, *Agar, Tryptone soft agar, Tryptone, Potassium chloride (reagent), Agar, , 15.0, 5.0, 5.0, 15.0, 10.0, 0.01 - 0.03 M, 5.0, 11.0, 10.0, 5.0 ml, 9.0, , Urea broth, Urea broth concentrate (filter-sterilized 10.0 ml, solution), Sterile distilled water, 90.0 ml, Note: Aseptically add the urea broth concentrate to the sterilized and cooled distilled, water. Under aseptic conditions, dispense 3-ml, amounts into sterile tubes., Yeast extract broth (pH 7.0), Peptone, Beef extract, Sodium chloride, Yeast extract, , 5.0, 3.0, 5.0, 5.0
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Biochemical Test Reagents, , Agarose gel, for Electrophoresis, , Agarose, , 1g, 125 ml, , Tris-borate buffer 11 * 2, Note: Melt agarose, being careful not to overboil., Cover loosely with foil and hold at room temperature, or place in 60°C waterbath until ready, for use., Barritt’s reagent, for detection of, acetylmethylcarbinol, , Solution A, Alpha-naphthol, 5.0 g, Ethanol, absolute, 95.0 ml, Note: Dissolve the alpha-naphthol in the ethanol, with constant stirring., Solution B, Potassium hydroxide, 40.0 g, Creatine, 0.3 g, Distilled water, 100.0 ml, Note: Dissolve the potassium hydroxide in 75 ml, of distilled water. The solution will become, warm. Allow to cool to room temperature., Add the creatine and stir to dissolve. Add the, remaining water. Store in a refrigerator., Biotin-histidine solution, for Ames test, l-Histidine HCl, Biotin, Distilled water, , 0.5 mM, 0.5 mM, 10.0 ml, , Buffered glycerol (pH 7.2), for, immunofluorescence, , Glycerin, Phosphate buffered saline, , 90.0 ml, 10.0 ml, , Diphenylamine reagent, for detection of nitrates, Dissolve 0.7 g diphenylamine in a mixture of 60 ml, concentrated sulfuric acid and 28.8 ml of distilled, water. Cool and slowly add 11.3 ml of concentrated, hydrochloric acid. Allow to stand for 12 hours., Sedimentation indicates that the-reagent is saturated., , AP PENDIX, , 4, , Endonuclease buffers, , Buffer 1: EcoRI buffer, Tris-HCl (pH 7.5), , MgCl 2, NaCl, Triton® X-100, BSA, Buffer 2: Hind III buffer, Tris-HCl (pH 8.5), MgCl 2, KCl, BSA, Buffer 3: Bam HI buffer, Tris-HCl (pH 8.0), MgCl 2, KCl, 2-Mercaptoethanol, Triton X-100, BSA, , 50 mM, 10 mM, 100 mM, 0.02%, 0.1 mg/ml, 10 mM, 10 mM, 100 mM, 0.1 mg/ml, 10 mM, 5 mM, 100 mM, 1 mM, 0.02%, 0.1 mg/ml, , Ferric chloride reagent, , Ferric chloride, Distilled water, , 10.0 g, 100.0 ml, , Gram’s iodine, for detection of starch, As in Gram’s stain, Hydrogen peroxide, 3%, for detection of, catalase activity, Note: Refrigerate when not in use., Kovac’s reagent, for detection of indole, , p-Dimethylaminobenzaldehyde, 5.0 g, Amyl alcohol, 75.0 ml, Hydrochloric acid (concentrated), 25.0 ml, Note: Dissolve the p-dimethylaminobenzaldehyde in the amyl alcohol. Add the hydrochloric, acid., , , , 523
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Loading Dye 6:, for gel electrophoresis, , Nessler’s reagent, for detection of ammonia, , Glycerol (50%), 6 ml, Bromphenol blue (2%), 1 ml, Xylene cyanol (2%), 1 ml, Distilled water, 1000 ml, Note: This can be stored in the refrigerator, indefinitely., , Potassium iodide, 50.0 g, Distilled water (ammonia-free), 35.0 ml, Add saturated aqueous solution of mercuric chloride until a slight precipitate persists., Potassium hydroxide (50% aqueous), 400.0 ml, Note: Dilute to 1000 ml with ammonia-free, distilled water. Let stand for 1 week, decant, supernatant liquid, and store in a tightly, capped amber bottle., , McFarland Barium Sulfate Standards, for API, Staph-Ident procedure, Prepare 1% aqueous barium chloride and, 1%-aqueous sulfuric acid solutions. Using the following table, add the amounts of barium chloride, and sulfuric acid to clean 15@ * 150@mm screwcapped test tubes. Label the tubes 1 through 10., Preparation of McFarland Standards, , Tube, , Barium Sulfuric, Chloride, Acid, 1% (ml) 1% (ml), , Corresponding, Approximate Density of, Bacteria (million/ml), , 1, , 0.1, , 9.9, , 300, , 2, , 0.2, , 9.8, , 600, , 3, , 0.3, , 9.7, , 900, , 4, , 0.4, , 9.6, , 1200, , 5, , 0.5, , 9.5, , 1500, , 6, , 0.6, , 9.4, , 1800, , 7, , 0.7, , 9.3, , 2100, , 8, , 0.8, , 9.2, , 2400, , 9, , 0.9, , 9.1, , 2700, , 10, , 1.0, , 9.0, , 3000, , Tetramethyl-p-phenylene diamine, dihydrochloride, Distilled water, , 10.0 g, 90.0 ml, , 10 ml, 390 ml, , Methyl red solution, for detection of acid, , Methyl red, 0.1 g, Ethyl alcohol, 300.0 ml, Distilled water, 200.0 ml, Note: Dissolve the methyl red in the 95% ethylalcohol. Dilute to 500 ml with distilled water., , 524, , Appendix 4, , 8.0 g, 1000.0 ml, , 5.0 g, 1000.0 ml, , 1.0 g, 100 ml, , Orthonitrophenyl-b -D-galactoside (ONPG), for, enzyme induction, , Methylene blue stain (0.025%), , Methylene blue 1% stock solution, (1 g + 99 ml distilled H2O), Distilled water, , Solution A, Sulfanilic acid, Sulfanilic acid, Acetic acid, 5 N: 1 part glacial acetic acid to 2.5 parts distilled water, Solution B, Alpha-naphthylamine, Alpha-naphthylamine, Acetic acid, 5 N, Oxidase Reagent, , Methyl cellulose, for microscopic observation, of protozoa, , Methyl cellulose, Distilled water, , Nitrate test solution, for detection of nitrites, , 0.1 M sodium phosphate buffer, (pH 7.0), , 50.0 ml, , ONPG 18 * 10 -4M2, , 12.5 mg, , p-Aminodimethylaniline oxalate, for detection, of oxidase activity, , p-Aminodimethylaniline oxalate, 0.5 g, Distilled water, 50.0 ml, Note: To dissolve fully, gently warm the solution., Phosphate-buffered saline, 1% (pH 7.2–7.4), for, immunofluorescence, , Solution A, Disodium phosphate, 1.4 g, Distilled water, 100.0 ml, Solution B, Sodium dihydrogen phosphate, 1.4 g, Distilled water, 100.0 ml, Note: Add 84.1 ml of Solution A to 15.9 ml of, Solution B. Add 8.5 g of sodium chloride and, enough distilled water to make 1 liter.
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Rabbit plasma, for detection of coagulase, activity, Note: Store vials at 2°C to 8°C. Reconstitute by, the addition of 7.5 ml of sterile water., Sodium barbital buffer, for immunofluorescence, , Sodium barbital, Sodium chloride, 1 N hydrochloric acid, Distilled water, to fill to 1000 ml, , 6.98 g, 6.0 g, 27.0 ml, , Tris-acetate buffer 1 :, , Tris-acetate buffer 10 *, (see previous entry), 100 ml, Distilled water, 900 ml, Note: Buffer can be stored indefinitely at room, temperature., Tris-borate buffer 5 :, , Toluidine blue solution, 0.1%, for detection of, DNase activity, , Tris base, Boric acid, EDTA (0.5M, pH 8.0), Distilled water, , 1% toluidine blue solution, Distilled water, , Tris-borate buffer 1 : working solution, , 0.1 ml, 99.9 ml, , Tris-acetate buffer 10 :, , Tris base, 48.4 g, Glacial acetic acid, 11 g, EDTA (0.5 M), 20 ml, Distilled water, 1000 ml, Note: Add ingredients to 1 liter volumetric flask, and then add water to volume., , Tris-borate buffer 5 *, (see previous entry), Distilled water, , 54 g, 27.5 g, 20 ml, 1000 ml, , 200 ml, 800 ml, , Appendix 4, , 525
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A P P E ND I X, , 5, , Staining Reagents, , Acid-Fast Stain, , Gram Stain, , Carbol fuchsin (Ziehl’s), , Crystal violet (Hucker’s), , Solution A, Basic fuchsin (90% dye content), 0.3 g, Ethtyl alcohol (95%), 10.0 ml, Solution B, Phenol, 5.0 g, Distilled water, 95.0 ml, Note: Mix Solutions A and B. Add 2 drops of, Triton X per 100 ml of stain for use in heatless, method., , Solution A, Crystal violet (90% dye content), Ethyl alcohol (95%), Solution B, Ammonium oxalate, Distilled water, Note: Mix Solutions A and B., , Acid Alcohol, , Iodine, Potassium iodide, Distilled water, , Ethyl alcohol (95%), Hydrochloric acid, , 97.0 ml, 3.0 ml, , Methylene blue, Methylene blue, Distilled water, , 0.3 g, 100.0 ml, , Capsule Stain, Crystal violet (1%), Crystal violet (85% dye content), Distilled water, , 1.0 g, 100.0 ml, , Copper sulfate solution (20%), Copper sulfate 1 CuSO4 ~ 5H2O2, Distilled water, , 20.0 g, 80.0 ml, , Fungal Stains, , Lactophenol–cotton-blue solution, Lactic acid, 20.0 ml, Phenol, 20.0 g, Glycerol, 40.0 ml, Distilled water, 20.0 ml, Aniline blue, 0.05 g, Note: Heat gently in hot water (double, boiler) to dissolve; then add aniline blue dye., , Water-iodine solution, Gram’s iodine (as in Gram’s stain), Distilled water, 526, , 10.0 ml, 30.0 ml, , 2.0 g, 20.0 ml, 0.8 g, 80.0 ml, , Gram’s iodine, 1.0 g, 2.0 g, 300.0 ml, , Ethyl alcohol (95%), Ethyl alcohol (100%), Distilled water, , 95.0 ml, 5.0 ml, , Safranin, Safranin O, Ethyl alcohol (95%), Distilled water, , 0.25 ml, 10.0 ml, 100.0 ml, , Negative Stain, Nigrosin, Nigrosin, water-soluble, Distilled water, Note: Immerse in boiling waterbath, for 30 minutes., Formalin, Note: Filter twice through double, filter paper., , 10.0 g, 100.0 ml, , 0.5 ml, , Spore Stain, Malachite green, Malachite green, Distilled water, , Safranin, Same as in Gram stain, , 5.0 g, 100.0 ml
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Credits, , Photo Credits, 03.02:, 05.01:, 07.01:, 08.03a:, 08.03b:, 08.03c:, 09.01:, 10.01a:, 10.01b:, 10.02a:, 10.02b:, 10.02c:, 10.02d:, 11.02:, 12.02:, 12.04:, 13.02a:, 13.02b:, 14.01a:, 14.01b:, 14.02a:, 14.02b:, 14.02c:, 14.03a:, 14.03b:, 14.03c:, 15.01:, 18.03:, 19.01:, 19.03:, 19.04:, 19.06:, 20.03:, 21.01:, 21.03:, 21.05a:, 21.05b:, 22.05:, 23.02:, 24.03:, 24.05:, 24.08:, , James Cappuccino, Leica Microsystems, Inc., L. Brent Selinger/Pearson Education, Inc., L. Brent Selinger/Pearson Education, Inc., Jennifer Warner, Michael Abbey/Science Source, Chad Welsh, James Cappuccino, Centers for Disease Control and Prevention, David B. Alexander, David B. Alexander, David B. Alexander, David B. Alexander, James Cappuccino, Steven R. Spilatro, James Cappuccino, L. Brent Selinger/Pearson Education, Inc., James Cappuccino, L. Brent Selinger/Pearson Education, Inc., L. Brent Selinger/Pearson Education, Inc., James Cappuccino, L. Brent Selinger/Pearson Education, Inc., James Cappuccino, L. Brent Selinger/Pearson Education, Inc., James Cappuccino, James Cappuccino, David B. Alexander, L. Brent Selinger/Pearson Education, Inc., Courtesy of Hausser Scientific, L. Brent Selinger/Pearson Education, Inc., L. Brent Selinger/Pearson Education, Inc., L. Brent Selinger/Pearson Education, Inc., L. Brent Selinger/Pearson Education, Inc., David B. Alexander, James Cappuccino, L. Brent Selinger/Pearson Education, Inc., L. Brent Selinger/Pearson Education, Inc., James Cappuccino, James Cappuccino, James Cappuccino, James Cappuccino, James Cappuccino, , 24.10: James Cappuccino, 25.02: James Cappuccino, 26.02: James Cappuccino, 27.01: James Cappuccino, 28.02: James Cappuccino, 29.01a: David B. Alexander, 29.01b: David B. Alexander, 29.01c: Brenda G Wellmeyer, 30.01: David B. Alexander, 31.02: L. Brent Selinger/Pearson Education, Inc., 31.04: L. Brent Selinger/Pearson Education, Inc., 33.01: Eric V. Grave/Science Source, 33.02a: Biophoto Associates/Science Source, 33.02b: M. I. Walker/Science Source, 34.02a: Centers for Disease Control and Prevention, (CDC), 34.02b: Centers for Disease Control and Prevention, (CDC), 34.03a: Centers for Disease Control and Prevention, (CDC), 34.03b: Biophoto Associates/Science Source, 34.04: Eric V. Grave/Science Source, 34.05: Dr. Marilise B. Rott, 34.06: Centers for Disease Control, Office on Smoking, and Health, 35.02: Jared Martin, 35.03: Leonard Lessin/FBPA/Science Source, 35.04: James Cappuccino, 35.05: Perennou Nuridsany/Science Source, 35.06: Biophoto Associates/Science Source, 36.01: ggw/Shutterstock, 36.02: Chad Welsh, 37.01a: James Cappuccino, 37.01b: Biophoto Associates/Science Source, 37.01c: John Durham/Science Source, 37.02: Nhu Nguyen, 37.04: James Cappuccino, 38.01: L. Brent Selinger/Pearson Education, Inc., 41.01: STERIS Corporation, 42.02: James Cappuccino, 42.04: James Cappuccino, 43.02: James Cappuccino, 43.03: Microchem Laboratory, 44.01: L. Brent Selinger, , , , 529
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48.02:, 48.03a:, 48.03b:, 48.03c:, 49.02:, 57.04:, 57.06:, 59.02:, 60.01a:, 60.01b:, 60.02:, 60.03:, 60.04:, 60.05a:, 60.05b:, 61.01:, 61.02a:, 61.02b:, 61.03:, 61.04:, 62.01a:, 62.01b:, 62.01c:, 62.02:, 62.03:, 62.04:, 62.05:, 63.01:, , 530, , L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, James Cappuccino, James Cappuccino, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, James Cappuccino, James Cappuccino, James Cappuccino, James Cappuccino, L. Brent Selinger, L. Brent Selinger, L. Brent Selinger, James Cappuccino, L. Brent Selinger, James Cappuccino, James Cappuccino, American Society for Microbiology Archives, , Credits, , 63.01: MyFavoriteTime/Shutterstock, 63.03a: L. Brent Selinger, 64.02: L. Brent Selinger, 64.03b: L. Brent Selinger, 64.05: Liofilchem, Inc., 65.01: L. Brent Selinger, 67.02: Courtesy and © Becton, Dickinson and, Company, 70.01: LeBeau/Custom Medical Stock Photo/Newscom, CVR TEK IMAGE/SCIENCE PHOTO LIBRARY, , Text Credits, Table 42.02: Clinical and Laboratory Standards, Institute. Performance Standards for Antimicrobial Disk, Susceptibility Tests, Tenth Edition, 2008., Table 48.01: Standard Methods for the Examination, of Water and Wastewater, 20th Edition (1998). M. J., Taras, A. E. Greenberg, R. D. Hoak, and M. C. Rand, eds., American Public Health Association, Washington, D.C., Copyright 1998, American Public Health Association,, and Bacteriological Analytical Manual (BAM), 8th, Edition, Food and Drug Administration, 1998., Table 59.01: Courtesy of Difco Laboratories, Inc.,, Detroit, Michigan., Table 61.02: STAPH-IDENT, Analytab Products,, Division of Sherwood Medical, Plainview, New York., Table 66.01: Wampole Laboratories Division, CarterWallace, Inc., Cranbury, NJ 08512.
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Index, , A, Abbé condenser, 37, Abdominal cramps, 224, 240, Abscesses, 21, 73, 433, Absidia, 355, Absorbance (A), 98, 120, Absorbent, bacteria growth, 30f, Abundance of growth (cultural, characteristic), 29, Acetate, 178, Acetic acid bacteria, 162f, Acetobacter, 162f, Acetoin, 175, Acetone, 162f, 352, Acetyl, 178, Acetylmethylcarbinol, 175–177, Acid, pH indicator, 167–168, Acid curd, litmus milk reactions, 195, Acid slant/acid butt, pH indicator, 167–168, reactions in TSI agar test, 168–169, Acid-alcohol, in acid-fast staining, 79–80, Acid-fast stain, 79–84, of mycobacteria, 81f, principle, 79, procedure, 80–81, 80f, Acidic salts, 119–120, Acidic stains, 52–53f, 67f, Acidophiles, pH impacting growth of, 119f, Acid-producing reactions, 161, 163, Acinetobacter, 168f, 456f, Acne, 427, Acquired immunity, 489, Acquired immunodeficiency syndrome, (AIDS), 505, Acremonium, 361, Actinobacteria, 361, Actinomyces odontolyticus, 421, Actinomycetes, antibiotics, 295, 361, enumeration of, 355–356, Adaptive enzymes, 375, Adaptive immunity, 489, Additive (indifferent) effect, 300, 300f, Adenosine triphosphate (ATP), 155–156, Adsorption, in viral infections, 265, Aerial mycelium, 241, Aerobes, aerobic respiration, 203, classification of microorganisms,, 123–124, differentiating from anaerobes, 125, oxidase test and, 207, Aerobic microorganisms, 123–124, 124f, 199, Aerobic respiration, lipid hydrolysis and, 155–156, oxidase enzymes role in, 207, oxidase test and, 203, Aerotolerant anaerobes, 123–124, African sleeping sickness, 231–232, 236f, Agar, .1% agar in nitrate medium, 199, in broth medium, 1, differential/selective media, 103–104, , enriched media, 104–105, plaques in, 269, preparing pure culture, 24f, selective media, 103, solid media, 2f, Agar deep tubes, 2, 14, Agar plates, bacteria growth in, 30f, inoculation of nutrient agar plates, 29, labeling and inoculation, 8f, 9t, preparing pure culture, 24f, serial dilution–agar plate technique,, 135–136, solid media, 2f, starch agar plate, 155, 155f, Agar slants, bacteria growth in, 30f, culture media, 2, culture transfer, 14–15, inoculation of, 29, preparing pure culture, 24f, Agarose gel, casting, 401–404, 403f, gel loading scheme, 413f, separating DNA fragments using electrophoresis, 412–413, Agglutination, febrile antibody test, 495–500, immunological reactions, 489, 495f, VDRL agglutination test, 505, Agglutinins, antibodies, 489, AIDS (acquired immunodeficiency syndrome),, 505, Alcaligenes, 319, 456f, Alcaligenes faecalis, cultural and biochemical characteristics,, 218t, non-lactose fermenters, 173, Alcaligenes viscolactis, 88, Alcohol, biochemical pathway, 329f, disinfectants and antiseptics, 311t, microbial fermentation, 329–330, Algae, 45f, Alkaline, enzymatic degradation of citrate, 178, litmus milk reaction, 193–195, pH indicator, 167–168, Alkaline slant/acid butt, pH indicator, 167–168, TSI reactions, 168–169, Alkalophile, 119f, Alpha hemolysis, hemolytic reactions on blood agar, 105f,, 441, 442f, overview of, 105, Streptococcus pneumoniae on blood agar,, 449, a-naphthol, MR-VP test, 176, Alternaria, 246t, Amebic dysentery, 235f, Ames test, 391–394, 391–396, 393f, Amine, 211, Amino acids, 211–216, in casein, 156, , gelatin hydrolysis and, 157, metabolism, 211–214, peptone degraded into, 163, principle, 211–214, procedure, 212–214, sulfur-containing, 185, tryptophan, 174, Ammonia, ammonification in nitrogen cycle, 351, indole production test, 174, phenylalanine converted to, 213–214,, 213f, Ammonification, nitrogen cycle, 351, Ammonium hydroxide, 163, Amoeba, 45f, 223, Amycolatopsis, 361, Amylase, 155, Anaerobes, 123, 125, Anaerobic microorganisms, 129–134, cultivation of, 130f, determining oxygen requirements,, 123, 124f, nitrate reduction test, 199, principle, 129–130, procedure, 131–132, Anaerobic respiration, 199, Analytical Profile Index (API) system, 457,, 457f, 460, Animal feed/hides, 319, Animal viruses, 266–267, Anionic chromogen, basic stains, 52f, Anopheles mosquito, 231, 234f, Antibiotic resistance, conjugation factor in, 382, genetic adaptation and, 387, increase in, 281, Antibiotics, 361–366, antimicrobial spectrum of isolates, 363–364,, 363–364f, chemotherapeutic agents, 295, developing, 268, diarrhea and, 194, isolation of, 361–363, laboratory regulations, 286, minimal inhibitory concentration, 305, prototypic, 295t, search for, 253, 298, serial dilution plate setup, 308t, soil flora in, 352, testing new, 361, treating Proteus species, 168, Antibodies, adaptive immunity, 489, agglutination reaction, 495f, detecting, 501–502, precipitin reaction, 491, 491f, Antibody titer test, 495–496, 497f, Antigens (immunogens), adaptive immunity, 489, detecting, 501–502, febrile antigens, 495, herpes simplex virus (HSV), 507, precipitin reaction, 491, 491f, Treponema pallidum as, 506, Antimetabolites, 295–296, , Index, , 531
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Antimicrobial agents, antimicrobial spectrum of isolates, 363–364,, 363–364f, disinfectants and antiseptics, 311–313t,, 311–315, isolation of, 361–363, 362f, measuring zone of inhibition, 297t, minimal inhibitory concentration, 305, MRSA and, 314, overview of, 285–287, synergistic effects, 300, Antimicrobial resistance/susceptibility, 144, Antimicrobial spectrum of isolates, 361–366, Antimicrobial-sensitivity discs, 298, Antiseptics, 311–318, controlling microbial growth, 285, disc diffusion test, 314–315, efficiency of, 313, modified-use dilution test, 315, susceptibility test, 314f, types of, 311–313t, AOAC (Association of Analytical, Communities), 286, 303, Aperture, microscope, 38, API (Analytical Profile Index) system, 457,, 457f, 460, API STAPH-IDENT Profile Register, 437–438t, Apple fermentation, 257, 329, Aquaspirillum itersonii, 42, Arizona, 168, Ascomycetes, 239, 240t, Ascospores, 257, Ascus, 257, Aseptic techniques, 3, 9, Aspergillus, enzymes produced by, 352, food microorganisms, 321, identification of fungi, 246t, soil microorganisms, 355, Aspergillus flavus, 242, Aspergillus niger, conidiophore and conidia of, 242f, in Sabouraud broth, 125, synthesis of citric acid, 352, Association of Analytical Communities, (AOAC), 286, 303, Athlete’s foot, 374, Atmospheric oxygen. see Oxygen requirements,, microbial, ATP (Adenosine triphosphate), 155–156, Autoclave, 4, Autotroph, 93, Auxochrome, 51, 52f, Azotobacter, 352, , B, Bacillary dysentery, 335, Bacillus, as antibiotic, 352, 361, Enterobacteriaceae and, 167, free-living microorganisms, 352, gram-positive bacteria, 71, identification of gram-positive bacteria, 483f, rod-shaped, 61f, soil-borne pathogens, 353, 355, as vegetative cells or spores, 85, Bacillus amyloliquefaciens H, 410, Bacillus anthracis, 86, 88, Bacillus cereus, biochemical activities, 152, cultural and biochemical characteristics,, 218t, culture, 31, 56, examination of, 45, nonenteric, 173, Bacillus subtilis, 42, Bacitracin, 295t, 352, Bacitracin test, 442, 443f, Bacteremia, 475, , 532, , Index, , Bacteria, in blood (bacteremia), 475, conjugation, 381–386, contamination, 287, cultivation, 97–100, cultural characteristics, 30f, diagnosis of syphilis, 46, differentiation of enteric, 168, differentiation of mycobacterium tuberculosis from non-tubercle mycobacterium,, 200, enumeration of soil population, 355–356, fermentation in identification of, 163, genetics and, 373–374, genus identification, 217–221, 218t, growth in thioglycollate broth, 131, identification of, 481–488, identifying intestinal pathogens, 186, life cycle of spore-forming, 86, 86f, morphology, 63f, oxidase test, 207, producing cytochrome oxidase, 207, refrigeration and, 114, shapes and arrangements, 61f, temperature impacting growth of, 113–114,, 114f, Bacterial cell counts. see also Cell counts, agar plating method, 137f, Bacturcult®, 471t, calibrated loop for, 472, clinical applications, 137, Coulter counter, 135–136, food analysis, 321–322, 322f, procedure, 145, 358, Bacterial conjugation, 381–386, Bacterial genetics, bacterial conjugation, 381–386, detecting potential carcinogens, 391–396, enzyme induction, 375–380, isolation of streptomycin-resistant mutant,, 387–390, overview of, 373–374, Bacterial growth curve, 143–150, determining generation time, 144f, phases of, 143f, principle, 143–144, procedure, 144–146, techniques, 146f, Bacterial plasmids, 397–408. see also Plasmids, principle, 397–398, procedure, 399–404, Bacterial pneumonia, 88. see also Pneumonia, Bacterial population growth studies. see, Bacterial growth curve, Bacterial smears, 51–60, acid-fast staining, 81, following fixation, 55f, gram staining, 73–74, preparing, 57f, principle, 55–56, procedure, 56–58, spore staining, 87, Bacterial staining, introduction to, 51–52, staining techniques, 53f, Bacterial test system. see Ames test, Bacterial transformation, 381, 391–396. see also, Transformation, Bacteriophages (phages), 269–274, bacterial viruses, 265, components and functions, 266f, cultivation and enumeration, 269–271, 271f, identification, 270, isolation, 282, isolation of coliphages from raw sewage,, 275–277, 276f, lytic cycle, 267f, phage replication, 265–266, propagation, 281–283, restriction endonucleases, 409–411, , separating DNA fragments, 411–413, Bacteriuria, 469, Bacteroides, 319, 425, Bacturcult®, interpretation of colony counts, 471t, urine analysis, 471–472, 471f, Baker’s yeast, 257, Balantidium coli, 231, 236f, Barritt’s reagent, 176–177, Basic stains, 51–53, 52f, 313t, Basidiomycetes, 239, 240t, Basidiospores, 239, 240t, 241, BBL Septi-Chek System, 475, 478, Beaded (cultural characteristics), 30f, Beef extract, 97, Benzalkonium chloride, 312t, 314, Benzene, 51, Benzylpenicillin, 305, Bergey’s Manual of Systematic Bacteriology,, 29, 217, 481, Beta hemolysis, 105, 105f, 441, 442f, Beta-galactosidase, 193, 375, Beta-lactamases, 306f, BGS (Brilliant green sulfa) agar plate, 326, BHI (Brain heart infusion), 130–131, Bile, 449, Bile esculin hydrolysis (or test), 443, 443f, Bile salts, 449, Bile solubility test, 449, Binary fission, 86, Biochemical activities of microorganisms, amino acid utilization, 211–216, carbohydrate fermentation, 161–166, catalase test, 203–206, 204f, enzymatic activities, 155–160, genus identification, 217–221, 218t, hydrogen sulfide production test, 185–188, 186f, IMViC test, 173–184, litmus milk reaction, 193–198, 194–195f, nitrate reduction test, 199–202, 200f, overview of, 151–153, 152f, oxidase test, 207–210, 208f, TSI agar test, 167–172, 168–169f, urease test, 189–192, 190f, Biochemical characteristics of bacteria, 218t, Biochemical factors, in native immunity, 489, Biochemical test reagents, 207–208. see also, Reagents, Biooxidative pathways, 161, 161f, Bladder infections, 469, Blood, drawing for cultures, 477, microbiological analysis, 475–480, Blood agar, enriched media, 104–105, hemolytic reactions, 105f, 441, 442f, isolation of microbial flora, 425–426, 426f, Streptococcus pneumoniae on, 449, Blow-out pipette, 5f, Body tube, microscope, 37, Boiling heat, 277, Borrelia burgdorferi, 501, Botrytis, 319, Brain heart infusion (BHI), 130–131, Breed smears, 135, Brewer jar, 130, Brightfield microscope, 35, Brilliant green sulfa (BGS) agar plate, 326, Bronchopneumonia, 441, Broth medium, bacteria growth, 30f, 31, 34, inorganic synthetic broth, 97, nutrient broth, 97, 101, preparing, 55–57, soy. see Trypticase soy broth (TSB), subculturing for culture transfer, 14, types of media, 1, Broth-to-slant transfer, 13, Brownian movement, 46, 49, Budding, yeast reproduction, 257, 258f
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Buffers, pH, 119–120, Bunsen burner, 2f, Butanol, 174, 352, , C, Cadaverine, 212, Calcium caseinate, 194, Calibrated loop, for bacterial counts, 472, CAMP test, 443–444, 443f, Campylobacter, 465–468, Campylobacter coli, 466, Campylobacter jejuni, 465–466, CampyPak®, 466, Candida, 207, 245t, Candida albicans, fungal invasion of bloodstream, 475, invasive candidiasis, 258, pathogenic yeasts, 257, urinary tract infections, 469, Candidiasis, 258, 505, Capsular swelling, 450, Capsule stain (Anthony method), illustration of, 89f, principle, 88, procedure, 89–90, 90f, Capsules, 449, 449f, Carbenicillin, 297t, Carbohydrate, substrate, 161, Carbohydrate fermentation, 161–166, detection of gas production, 163f, principle, 161–164, procedure, 164, test for, 163f, 167, 171, Carbol fuchsin, 79–81, Carbon, 193, Carbon cycle, 352, Carbon dioxide, carbon cycle, 352, citrate utilization test, 178, decarboxylation of amino acids, 211–212, Carcinogenicity, 391, Carcinogens, Ames test for identifying, 391–396, Cardinal (significant) temperature points,, 113–114, Caries (dental), 421–422, Casein, 193–194, Casein hydrolysis, 156, 158, 160, Catalase, 203, Catalase test, 203–206, 204, 204f, 207–208, principle, 203–204, procedure, 204, Cationic agents, 312t, Cationic chromogen, 52f, CDC (Centers for Disease Control and, Prevention), 152, 286, Cefoxitin, 297t, Cell counts. see also Bacterial cell counts, colony-forming units (CFUs) and, 145, Coulter counter, 135–137, food analysis, 321–322, 322f, interpretation of colony counts, 471t, Quebec colony counter, 136–137f, TNTC/TFTC, 140, 269, for viable cells, 140f, Cellmatics™ HSV Detection System, 507, Cell-mediated immunity, 489, Cell-membrane damage, from antimicrobial, agents, 285–286, Cellular enzymes, 113–114, 286, Cellular respiration, 129, 161, Cellulitis, 441, Cell-wall injury, from antimicrobial agents, 285, Centers for Disease Control and Prevention, (CDC), 152, 286, Cephalosporin, 245t, Cervicitis, NGU and, 508, CFUs (Colony-forming units), 145, Chancre, 505, Chemical agents, 311–313t, 311–315, , Chemical method, 136, Chemicals, role of soil flora in, 352, test for carcinogens, 391–394, Chemotherapeutic agents, 285–304, controlling microbial growth, 285, drug categories, 295, Kirby-Bauer antibiotic sensitivity test,, 296–299, synergistic effects of drug combinations,, 299–300, synthetic drugs, 295–296, Chlamydia, 508–509, Chlamydia trachomatis, 508–509, Chlamydomonas, 45f, Chocolate agar plate, 425–426, 426f, Cholera, 335, Chromium–sulfuric acid method, 130, Ciliated, classification of protozoa, 223, 231, Ciliophora, classification of protozoa, 223, Circular, colony form on agar plates, 30f, Citrase, 178, Citrate, as source of carbon, 177–178, Citrate permease, 178, Citrate utilization test, IMViC test, 173, overview of, 177–178, principle, 178–179, procedure, 179, reactions, 178f, reagents, 179, Citrobacter, differentiating from Salmonella, 211, food-borne organisms in soil and water, 319, identification of gram-negative bacteria,, 484–485f, isolation and identification, 456, TSI reactions, 168f, Citrobacter freundii, 212, Cladosporium, 245t, 355, Clinical and Laboratory Standards Institute, (CLSI), 286, 297, Clostridium, clinical applications, 194, differentiating Enterobacteriaceae from, 194, flora of intestinal tract, 425, food-borne organisms in soil and water, 319, free-living microorganisms, 352, soil-borne pathogens, 353, spore stain, 85, Clostridium acetobutylicum, 352, Clostridium difficile, 194, Clostridium perfringens, 129, 194, Clostridium sporogenes, 125, 131, CLSI (Clinical and Laboratory Standards, Institute), 286, 297, Coagulase test, 434, 435f, Coarse-adjustment knob, microscope, 37, Cocci, spherical shape, 61f, 63f, Coccidioides, 353, Coccidioides immitis, 469, Coliform bacteria, 104, Coliphages, isolation from raw sewage, 275–, 277, 275–280, 276f, Colloidal state, of cytoplasm, 286, Colonies, of cells, 2, Colony-forming units (CFUs), 145, Colored complex, indicating nitrate reduction,, 200f, Commensals, 189, Competent cell, 398, Competitive inhibition, 295–296, 297t, Complex media, 97, Computer-assisted systems, 455–462, Condenser knob, microscopes, 39, Conidia, of Aspergillus niger mold, 242f, Conidiophore, of Aspergillus niger mold, 242f, Conjugation, 381–386, bacterial, 381–384, as cause of antibiotic resistance, 382, , of genetic material, 398, genetic recombination, 381, in transfer of genetic material, 373, Constitutive enzymes, 375, Contamination, bacterial, 287, of food, 319–320, Convex, colony form on agar plate, 30f, Copper sulfate, as decolorizing agent, 88, 90, Corynebacterium xerosis, 173, 218t, Coulter counter, 135–136, Counterstain, acid-fast staining, 79–81, gram staining, 75, overview of, 72–73, spore staining, 85, 88, Crateriform, cultural characteristics of, bacteria, 30f, Criminology, precipitin reaction in, 491, Cross over, genetic variability and, 381, Crowded-plate technique, isolating, antibiotic-producing microorganisms,, 361, 362f, Cryptococcus, 353, Cryptococcus neoformans, 258, Crystal violet, capsule staining, 88, 90, gram staining, 75, primary stain, 71–72, as selective media, 103, C-substance, Streptococci classification, 441, Cultivation chambers, 4, 6, Cultivation of microorganisms, anaerobes, 129–132, 130f, bacterial growth curve, 143–148, bacteriophages (phages), 269–271, 271f, counting viable cells, 135–142, differential, selective, and enriched media,, 103–112, enumeration of microbial population, 136–, 137f, 269–271, 271f, 355–358, fungi (molds), 242, nutritional requirements, 97–102, overview of, 93–95, oxygen requirements, 123–128, pH requirements, 119–122, techniques for, 129–134, temperature requirements, 113–118, Cultural characteristics, of microorganisms,, 29–34, Culture, age impacting stain results, 74, bacterial smear, 55–57, capsule staining experiments, 88, for cultivation of bacteria, 99, drawing blood for, 477, enumeration of cells, 136f, genus identification, 217–221, 218t, identifying unknown bacteria, 481–488, isolating pure culture, 21, 24f, media, 1, propagation of bacteriophages, 281–283, pure culture. see Pure culture, selective/differential/enriched media, 106, spore staining, 86, sterilization vessels, 4f, techniques, 2f, Culture transfer, 13–18, aseptic techniques, 15, of bacteria daughter cells, 23, experiment, 13–15, instruments, 3–4, Curd formation, litmus milk reaction,, 193–195, Cysteine, 185, Cysteine desulfurase, 185, Cystitis, 469, Cytochrome, 207, Cytochrome oxidase, 207, Cytoplasm, 286, , Index, , 533
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D, Darkfield microscope, 35, Deamination, of amino acids, 211, 213f, Decarboxylation, of amino acids, 211–213f, 215, Decline (or death) phase, bacterial population,, 143, Decolorizing agent, in acid-fast stain, 79, in capsule staining, 88, in differential staining, 71–72, in spore staining, 85, timing of, 74, Degradation, casein into polypeptide chains, 194, of citrate, 178, of glucose, 161, of hydrogen peroxide and superoxide, 203, of lactic acid, 320, of lysine, 212f, of sucrose, 422f, of tryptophan, 174, of urea, 189, Dehydration reaction, indole production test,, 174, DeLong shaker flask, 4f, Denitrification, nitrogen cycle, 351, Dental caries, 421–422, Deoxyribonuclease (DNase) test, 434, 435f, Deoxyribonucleic acid. see DNA (deoxyribonucleic acid), Deuteromycetes, 239, 240t, Dextran, converting glucose to, 422f, Dextransucrase, 421, Dextrins, 155, Dextrose, carbohydrate fermentation, 163–165, differentiation of Enterobacteriaceae, 167, D’Herelle, Felix, 265, Diagnosis, culture isolation in, 21, gram stains in, 73, of leprosy and lung infections, 79, of syphilis, 46, of wounds, 106, Diamine, 211–212, Diaphragm opening, microscope, 40f, Diarrhea, Campylobacter causing, 466, differentiating Enterobacteriaceae from, Clostridium, 194, identifying intestinal pathogens, 186, Diatoms, 45f, Differential media, 103–112, principle, 103–104, procedure, 106–107, Differential staining, 85–92, capsule stain, 88–89, chemical reagents in, 71, gram stains. see Gram stains, principle, 85–87, procedure, 87–90, spore stain, 85–88, techniques, 53f, visualizing bacterial cell structures, 85, Dilution-plating technique. see also Serial, dilution–agar plate technique, antibiotic setup, 308t, for bacterial growth, 146, 146f, cultivation and enumeration of bacteriophages, 271f, for fungal growth, 253, 254f, Dipeptides, 156, Diplobacilli, rod-shaped bacteria, 61f, 63f, Diplococcus, 61f, Diplococcus pneumoniae. see Streptococcus, pneumoniae, Direct transfer (plating), isolating fungal, growth, 253, Directigen test, 443, 501, , 534, , Index, , Disc diffusion test, for disinfectants and antiseptics, 314–315, Disinfectants, 311–318, controlling microbial growth, 285, disc diffusion test, 314–315, efficiency of, 313, modified-use dilution test, 315, types of, 311–313t, Dismutase, 203, DMLIA (double modified lysine iron) agar, plate, 326, DNA (deoxyribonucleic acid), bacterial genetics, 373–374, electrophoresis, 411–413, gel migration pattern, 398f, herpes simplex virus and, 507, interference with DNA molecule, 286, plasmids and polylinkers, 397–398, DNase (Deoxyribonuclease) test, 434, 435f, Double modified lysine iron (DMLIA) agar, plate, 326, Dracunculus medinensis (guinea worm), 335, Drug-resistance, mutations and, 387, Durham tube, fermentation broth, 161, Dyes, 313t. see also Stains, Dysentery, amebic dysentery, 235f, bacillary dysentery, 335, identifying intestinal pathogens, 186, MacConkey agar and, 104, , E, E. aerogenes. see Enterobacter aerogenes, E. coli. see Escherichia coli, Echinulate, bacteria growth, 30f, Effuse, bacteria growth, 30f, Electron microscope, 36, Electrophoresis, separating DNA fragments,, 412–413, Elephantiasis, 509, ELISA (Enzyme-linked immunosorbent assay),, 501–503, Embden-Meyerhof pathway, 161–162, 162f, Endocarditis, 441, Endonucleases, 409, 409f, Endospore, in spore staining, 85, Energy, sources for microbes, 164f, Enriched media, 103–112, principle, 104–105, procedure, 106–107, yeast extract broth as, 97, Enrichment culture technique, 367–372, isolation of Pseudomonas species, 367–370,, 368f, isolation of Salmonella, 326, medical uses, 368, Entamoeba, 233, Entamoeba histolytica, in amebic dysentery, 235f, motility, 223, parasitic protozoa, 231, 335, 469, structural characteristics, 232f, Enteric bacteria. see also Enterobacteriaceae, ability to ferment glucose, 175–176, citrate utilization test, 177–179, differentiation using TSI agar test, 168, 168f, food microbiology, 319, identification of, 173, 455–463, identification of Campylobacter, 465–468, lactose fermenters and nonlactose fermenters, 173, urease test, 189, Enterobacter, differentiating from Clostridium, 194, differentiating from Klebsiella, 211, food-borne organisms in soil and water, 319, gram-negative bacteria, 484–485f, identifying, 212, identifying using TSI agar test, 167, 168f, , normal intestinal flora, 173, 455, Enterobacter aerogenes, biochemical characteristics, 152, 218t, citrate utilization test, 178, on eosin-methylene blue agar, 104, glucose fermentation, 175, identifying, 212, lactose fermentation and, 173, pyruvic acid and, 162f, Enterobacteriaceae, computer-assisted identification, 458, defined, 167, differentiating from Clostridium, 194, identifying, 203, identifying enteric microorganisms, 455–463,, 456f, IMViC test, 173, infections, 459, oxidase negative, 207, principle, 167–168, procedure, 168–169, Enterococcus faecalis, 441, 469, EnteroPluri-Test system, 458, Enterotube Multitest System, 456, 457f, 459–460, Entire, colony form on agar plates, 30f, Enumeration, of Actinomycetes, 357f, of bacterial colonies, 137f, of bacteriophages, 269–271, 271f, of microbial population of soil, 355–358, pour-plate technique in, 136f, Environmental Protection Agency (EPA), 345, Enzyme induction, 375–380, Enzyme-linked immunosorbent assay (ELISA),, 501–503, Enzymes, 375–380. see also Extracellular enzymatic activities, cardinal (significant) temperature points,, 113–114, citrase, 178, converting amino acids to ketoimine acids,, 163, degradation of citrate, 178, endonucleases, 409, hydrolytic, 189, induction, 375–377, 376f, oxidase, 207, in oxidation of tryptophan, 174, role of soil flora in, 352, Eosin-methylene blue agar, as differential/selective media, 104, 107, effects of, 105f, plate preparation and inoculation, 107f, EPA (Environmental Protection Agency), 345, Epididymitis, 508, Episome, 381, Erysipelas, 441, Erythrocytic stage, of parasitic development,, 231, Erythrogenic toxin, streptococcal pathogens,, 442, ESBLs (Extended-spectrum ®-lactamases),, 306f, Escherichia, 484–485f, Escherichia coli, ampicillin resistance, 397–398, antibacterial treatment, 268, bacterial expression of special protein, 374, biochemical characteristics, 218t, blood analysis, 475, differentiation of enteric microorganisms,, 168f, endonucleases, 410, enteric microorganisms, 319, food pathogens, 321, genetic map of, 381f, glucose fermentation, 175, gram stains, 71, 71f, as gram-negative organism, 103, 484–485f, indole production test, 174
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intestinal flora, 173, 455, isolating coliphages from raw sewage,, 275–277, 276f, isolation and identification, 456f, McFarland standard, 99, pyruvic acid and, 162f, quantifying infective phage particles, 269, selective effects of phenylethyl alcohol agar,, 104f, spontaneous mutation rate, 373, urine analysis and, 469, 470f, water quality and, 335–336, Essential metabolite, p-aminobenzoic acid, (PABA), 295–296, Ester bonds, 155, Ethyl alcohol, as decolorizing agent, 72, Ethylene oxide, disinfectants and antiseptics,, 313t, Ethylhydrocupreine hydrochloride, 449, Euglena, 45f, Extended-spectrum ®-lactamases (ESBLs),, 306f, Extracellular enzymatic activities, 155–160, principle, 155–157, procedure, 158, Extracellular metabolites, hemolysins, 442, Eye conjunctiva, 425, Eyepiece lens, microscope, 37, , F, Facultative anaerobes, aerobic respiratory pathway, 203, classification by oxygen needs, 123–124, oxidase enzymes and, 207, Fastidious microorganisms, cultivation of, 97, Fatty acids, 155, Febrile antibody test, 495–500, Febrile antigens, 495, Fermentation, 329–334, alcohol fermentation, 329–330, biooxidative pathways, 161, carbohydrate fermentation. see, Carbohydrate fermentation, citrate utilization test, 177–179, food preservation and, 320, glucose fermentation, 175–176, hydrogen sulfide production test, 185, identification of bacteria, 163, lactic acid fermentation, 331–332, lactose fermenters and nonlactose, fermenters, 173, litmus milk reaction, 193, soil flora and, 352, Ferrous sulfate, 167-168, 185, Fertility factor (F), 381, Filamentous, colony form on agar, plates, 30f, Filiform, bacteria growth in agar slants, 30f, Filter paper method, oxidase test, 207–208, Filtration, filter paper method, 207–208, isolating coliphages from raw sewage, 275,, 277, membrane filters, 345–348, 348f, sterilization techniques, 3, Fine-adjustment knob, microscope, 37, Fission, yeast reproduction, 257, Flagellated algae, 223, Flat, colony form on agar plates, 30f, Fleming, Sir Alexander, 305, Flora, identifying enteric microorganisms, 455, intestinal, 173, of mouth, 421–424, normal, 419, of soil, 352, of throat and skin, 425–432, Fluid thioglycollate, 130–131, 131f, Fluorescent microscope, 36, , Fluorescent treponemal antibody-absorption, test (FTA-ABS), 506, Food microbiology, 319–324, bacterial counts, 321–322, 322f, isolating Salmonella from raw meat, 325–, 326, 326f, overview of, 319–320, role of soil flora in, 352, Food Safety and Inspection Service (FSIS), 325, Forespore, 86, Formaldehyde, 313t, Four-way streak-plate inoculation, 19f, 20f, Fracastoro, monk, 419, Free spore, in spore staining, 85, Free-living microorganisms, 352, FSIS (Food Safety and Inspection Service), 325, FTA-ABS (Fluorescent treponemal antibodyabsorption test), 506, Fungi, 239–254. see also Yeasts, characteristics, 240t, cultivation, 242, dilution-plating technique, 254f, enumeration, 355, identification, 245–247t, isolation, 244, 253–254, molds, 241, pH and growth, 120, sexual reproduction, 239, yeast morphology, 257–259, Fungi Imperfecti, 239, Fusarium, 246t, , G, Gametocyte, 231, Gamma hemolysis, classification of streptococcus, 441, media, 105f, 442f, overview of, 105, Gangrene, 129, Gas, carbohydrate fermentation, 163, litmus milk reactions, 193, 195, Gas gangrene, 129, GasPak system (or generator), anaerobic technique, 132f, cultivating anaerobic microorganisms,, 131–132, identifying Campylobacter, 466, Gastroenteritis, 455, Gelatin, bacteria growth in, 31, 34, Gelatin hydrolysis, 157–158, 157f, 160, Gelatin liquefication, cultural characteristics of, bacteria, 30f, Gelatinase, 157, Gelatin-based agar, 185, Generation time, bacterial population growth,, 143, Genetics, bacterial, 373–374, conjugation, 381–386, detecting carcinogens, 391–396, enzyme induction, 375–380, isolating genes of interest, 410, isolating MRSA mutant, 387–390, operon genes, 375, 376f, Genital herpes, causative agent, 508, isolating/identifying, 507–508, viral STDs, 505, Genital warts, 505, Genitourinary tract, 425, Genus, 217–221, identifying bacteria cultures, 217–221, 218t, of isolate, 370, principle, 217–219, procedure, 219, Germination, 85–86, Giardia intestinalis, 231, 236f, Glass slide, cultivation of fungi, 242, , Glassware marking pencil, 168, Glomerulonephritis, causative agent, 441, urinary tract infections, 469, Glossina (tsetse fly), 231, Glucose, converting to dextran, 422f, degradation, 161, determining ability to ferment, 175–176, differentiation of Enterobacteriaceae, 167, starch hydrolysis and, 155, Glucose salts broth, 97, 101, Glycerol, 155, Glycolytic pathway, 161, Glycosidic bonds, 155, Golden Era of microbiology, 419, Gonorrhea, 505, Gradient-plate technique, isolation of MRSA, mutant, 387, 387f, Gram, Dr. Hans Christian, 71, Gram stains, 71–78, diagnosing infections, 79, Enterobacteriaceae and, 167, examination of cells, 72f, examples, 71f, principle, 71–73, procedure, 73–75, of unknown culture, 219, Gram-negative bacteria, differentiating Enterobacteriaceae from, Clostridium, 194, identifying, 71–72, 484–485f, septicemia and, 475, Gram-positive bacteria, identifying, 71–72, 482–483f, types of, 475, Gram’s iodine, gram staining procedure, 75, mordant stain, 72, as reagent, 158, Gram-variable, 74, Group A, Streptococci classification, 441, 444, Group B, Streptococci classification, 441,, 443–444, Group C, Streptococci classification, 441, 444, Group D, Streptococci classification, 441, 443, Guinea worm (Dracunculus medinensis), 335, , H, Haemophilus, 425, Haemophilus influenzae, 88, 410, 475, Halogens, 311–312t, Hand washing, 7–12, aseptic techniques, 6, effectiveness, 7–11, Hanging-drop procedure, observing bacteria,, 46–47, 47f, Heat fixation, for bacterial smear, 56–58, in spore staining, 87, Heat/heatless methods, in acid-fast staining,, 80–81, Heavy metals, 312t, Helminth, 335, 469, Hemolysins, extracellular metabolites, 442, Hemolysis, blood analysis, 476f, differentiation of streptococci, 442t, identifying streptococci, 442–444, 449, 449f, identifying gram-negative bacteria, 482f, reactions on blood agar, 105f, 426f, test, 434t, types, 441, 442f, Hemolytic activities, 105, 441, Hepatitis B, 505, Herpes encephalitis, 507, Herpes genitalis, 507, Herpes labialis, 507, Herpes simplex virus (HSV), 507–508, , Index, , 535
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Heteronema, 45f, High-frequency recombinants (Hfr), 381, High-molecular-weight substances, 155, HSV (Herpes simplex virus), 507–508, HSV-1 (herpes simplex virus Type 1), 507, HSV-2 (herpes simplex virus Type 2), 507, Humoral immunity, 489, Hydrochloric acid, 120, Hydrogen peroxide, 203, Hydrogen sulfide, detecting, 185f, producing, 171, substrate, 167–168, Hydrogen sulfide production test, 185–189, detecting hydrogen sulfide, 185f, principle, 185, procedure, 186, reactions, 186f, Hydrolysis, casein hydrolysis, 156, 158, 160, gelatin hydrolysis, 157, 158, 160, indole production test, 174, lipid hydrolysis, 155–156, 156f, 158–159, nutrient gelatin hydrolysis, 157f, protein hydrolysis, 156f, starch hydrolysis, 155, 155f, 158–159, Hydrolytic enzymes, 189, Hyphae, of mycelium, 241, , I, Identification of microorganisms, bacteriophages, 270, Campylobacter, 465–468, carcinogens, 391–394, enteric microorganisms, 173, 455–463, 456f,, 457f, fermentation in, 163, fungi, 245–247t, gram-positive bacteria, 482–483f, 484–485f, herpes simplex virus (HSV), 507–508, intestinal pathogens, 186, spore-forming, 86, staphylococcal pathogens, 433–440, 441–448, streptococcus pneumoniae, 449–454, 449f,, 450f, unknown cultures, 481–488, urease test, 189, Illumination, microscope, 37, 39, Immunity, 489, Immunocompromised patients, 168, Immunogens. see Antigens (immunogens), Immunoglobulins, 489, Immunology, adaptive and native, 489–490, agglutination reaction, 495–500, 495f, case study, 490, ELISA, 501–503, precipitin reaction (ring test), 491–494, sexually transmitted diseases, 505–509, IMViC test, 173–184, overview of, 173–174, principle, 174–175, 178, procedure, 175–179, reactions, 179f, Incubation, of agar plate cultures, 156, carbohydrate fermentation, 161, Incubators, 6, India ink, negative staining, 67, Indole production test, IMViC tests, 173–175, reactions in, 175f, reagents, 179, Induced mutations, 373, Inducible enzyme, 375, Induction, enzyme, 375–377, 376f, Infection, Enterobacteriaceae, 459, herpes simplex virus, 507, , 536, , Index, , leprosy and lung infections, 79, living agents inducing, 419, nosocomial, 7, pH as defense, 120, pneumococcus, 450, proteus, 168, respiratory, 441, site of, 239, sore throats and infected cuts, 420, streptococci, 444, urinary, 469, 470f, 472, viral, 265–266, water-borne, 335–336, yeast, 258, Infundibuliform, 30f, Inoculating loop, 190, Inoculation, agar plate, 8f, 9t, 156, agar slants, 29, aseptic, 15, 164, loop inoculation, 164, plate preparation and, 107f, streak-plate, 19f, 20, 20f, 475, TSI agar test, 171, Inorganic sulfur compounds, 185, Inorganic synthetic broth, 97, Intestinal flora, 173, 425, Intestinal infections, 335, Intestinal pathogens, 186, Inulin fermentation, 450, Invasive candidiasis, 258, Inverted inner vial, 161, Ionizing radiation, 3f, Iris diaphragm, microscope, 37, Irregular, colony form, 30f, Isolation of microorganisms, antibiotic-producing, 361–365, 363–364f, bacteriophages (phages), 282, Campylobacter, 465–466, coliphages from raw sewage, 275–277, 276f, DNA and, 397–398, 397f, E. coli, 456f, flora, 426f, 427f, herpes simplex virus, 507–508, isolation techniques, 19–25, microbial flora, 425–426, MRSA, 387–390, 387f, plasmids, 400–401, 400f, Pseudomonas species, 367–370, Salmonella from raw meat, 325–326, 326f, Isolation techniques, determining antimicrobial spectrum of isolates, 361–363, for diagnostics, 21, discrete colonies from mixed culture, 25, pure culture, 19–23, , K, Keratoconjunctivitis, 507, Keto acids, 213–214, 213f, Ketoamino acids, 163, Kinyoun method, acid-fast stain, 79, Kirby-Bauer test, antibiotic sensitivity test, 296–299, 296f, 299f, synergistic effects of drugs, 300, Kircher, Athanasius, 419, Klebsiella, 173, Klebsiella pneumoniae, 175, Koch, Robert, 419, Kovac’s reagent, 174–175, 174f, Krebs cycle, citrate in, 178, metabolization of ketoamino acids, 163, , L, Laboratory techniques, 1–27, apparatus and culture, 2f, , aseptic, 3, 3f, 6, cultivation chambers, 4, 6, culture vessels, 3, 4f, hand washing, 6–12, isolation, 19–20f, 19–27, 24f, media, 1–3, 2f, plate labeling and inoculation, 8f, 9t, transfer instruments, 3–4, 5f, transfer, 13–18, 14f, Lactalbumin, 193, Lactic acid, biochemical pathway, 331f, degradation, 320, litmus milk reaction, 193, microbial fermentation, 331–332, Lactobacillus, enteric microorganisms, 319–320, identifying gram-positive bacteria, 483f, intestinal tract flora, 425, pyruvic acid uses, 162f, Lactobacillus acidophilus, 421, Lactobacillus bulgaricus, 331, Lactococcus lactis, 173, 218t, Lactoglobulin, 193, Lactose, ®-galactosidase and, 375, 376f, carbohydrate fermentation, 163–165, differentiation of Enterobacteriaceae, 167, fermenters, 173, litmus milk reactions, 193, 195, Lag phase, bacterial population growth, 143,, 147, Lambda A:B DNA, 411, 412t, Lambda A:B phages, 266, Lancefield group classifications, 441, Latex agglutination test, 436–437, Leprosy, 79, Leuconostoc mesenteroides, 88, Leukocidin, 442, LGV (Lymphogranuloma venereum), 505,, 508–509, Lipase, 155, Lipid hydrolysis, extracellular enzymatic activities, 155–156, illustration, 156f, procedure, 158, tributyrin agar plate, 156f, Lipids, 155, Liquefaction, bacteria growth in gelatin, 34, Lister, Joseph, 419, Listeria, 275, Listeria monocytogenes, 114, 321, Litmus milk reaction, 193–198, principle, 193–196, procedure, 196, reactions, 194–195f, Litmus reduction, 193–194, Lobar pneumonia, 449, 449f, Lobate, colony form on agar plates, 30f, Logarithmic (log) phase, bacterial population, growth, 143, Loop inoculation, 164, Low-molecular-weight substances (or nutrients), 155–156, Lung infections, 79, Lyme disease, 501, Lymphogranuloma venereum (LGV), 505,, 508–509, Lysine, 212f, Lysine decarboxylase, 211–212, Lysine iron agar (LIA), 325–326, Lysogenic cells (or cycle), of bacteriophage,, 266, 267f, Lytic cycle, of viral infection, 266, 267f, , M, MacConkey agar, as differential/selective media, 104, 107, effects of, 105f
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plate preparation and inoculation, 107f, Macrogametocyte, in sporogamy, 232, Maculopapular rash, syphilis, 505, Magnification, microscope, 37, 39f, 43, Malachite green, in spore staining, 85, 87, Malaria, 231, 235f, MALDI (matrix-assisted laser desorption/ionization), 481, Maltose, 155, Mannitol salt agar, as differential/selective media, 103–104, 107, effects of, 105f, identifying staphylococcal pathogens,, 433–434, isolating microbial flora, 426, 427f, plate preparation and inoculation, 107f, Mastigophora, 223, 231, Matrix-assisted laser desorption/ionization, (MALDI), 481, Maturation, in viral infections, 266, McFarland barium sulfate standards, 120, Mechanical barriers, in native immunity, 489, Media, chemically defined, 97, complex, 97, for cultivation of bacteria, 99, differential. see Differential media, enriched. see Enriched media, experiment isolating pure culture, 21, in handwashing experiment, 8–9, preparing for bacterial smear, 55–57, principle, 97–99, procedure, 100, purpose of specialized, 99, selective. see Selective media, solid, 56–57, types of, 1–3, Media screening, isolation of Salmonella, 326, Medical microbiology, API STAPH-IDENT Profile Register, 435–438t, blood analysis, 475–480, 476f, 477f, conversion of glucose to dextran, 422f, flora of mouth, 421–424, flora of throat and skin, 425–432, growth and reactions on agar, 426–427f, identification of Campylobacter, 465–468,, 466f, identification of enteric microorganisms,, 455–463, 456f, 457f, 458f, identification of staphylococcal pathogens,, 433–440, 433f, 434t, 435f, identification of streptococcal pathogens,, 441–448, 442f, 442t, 443f, 444f, identification of streptococcus pneumoniae,, 449–454, 449f, 450f, identification of unknown bacterial cultures,, 481–488, 482f, 483f, 484f, 485f, overview of, 419–420, Snyder test (or agar), 422f, urine analysis, 469–474, 470f, 471t, 472f, Meiosis, genetic variability and, 381, Membrane filter method, colony development, 348f, quantitative water analysis, 345–348, 347f, Merozoites, 231, 231–232, Mesophiles, 112, Metabolism, of ketoamino acids, 163, Methicillin-resistant S. aureus (MRSA), anti-microbial agents, 314, isolating MRSA mutant, 387–388, resistance, 203, Methyl red test, IMViC test, 173, principle, 175–176, procedure, 176–177, reactions, 176f, reagents, 179, Methylene blue, basic stains, 52f, counterstain, 79–81, , MIC. see Minimal inhibitory concentration, (MIC), Microaerophiles, aerobic respiratory pathway, 203, classifying microorganisms by oxygen needs,, 123–124, oxidase enzymes and, 207, Microbes, proteins as energy sources for, 164f, types of soil microbes, 355, Microbial fermentation, alcohol, 329–330, lactic acid, 331–332, Microbial flora. see Flora, Microbial growth, chemotherapeutic controls, 295–301, 295t,, 296f, 297t, 299f, controls, 285–287, 286f, disinfectants and antiseptics, 311–313t,, 311–318, 314f, glucose ensuring, 211, growth curve, 143–148, 143f, 144f, penicillin inhibiting, 305–310, 305f, 306f,, 306t, 308t, on solid media, 19, temperature impacting, 113, 113–114, Microbicidal effect, 285, Micrococcus, 319, 427, 482, Micrococcus luteus, 21, 173, 218, Microgametocyte, in sporogamy, 232, Micrograph, of bacteria morphology, 63f, Microincinerator, in atmospheric oxygen experiment, 125, citrate utilization test, 178, cultivating anaerobic microorganisms, 131, extracellular enzymatic activities, 158, hydrogen sulfide production test, 186, indole production test, 175, litmus milk reactions, 196, oxidase test, 207–208, TSI agar test, 168, urease test, 190, Micropipette, transfer instruments, 5f, 399–400,, 411, Microscope slide, preparing for bacterial, smear, 55, Microscopy, direct microscopic counts, 135, Microscopy, examining living microorganisms,, 45–50, hanging-drop procedure, 47f, principle, 45–46, 45f, procedure, 46, Microscopy, examining stained cells, 37–44, compound microscope, 38f, linear magnification, 39t, principle, 37–41, procedure, 42, refractive indexes, 39f, working distance, objective, and diaphragm, opening, 40f, MicroTrak® Direct Specimen Test, 509, Milk, litmus milk reaction, 193, microbiology of food, 319–320, Millipore® system, water analysis, 346, Minimal inhibitory concentration (MIC), defined, 305, determination with plate reader,, 307–308, determination with spectrophotometer, 305, tube setup, 306f, Mixed culture, isolation techniques, 19–20, Modified-use dilution test, 315, Molds, 241–252, cultivation and morphology, 241–244, 242f, cultivation on solid surface, 244, enumeration of microbial population, 356, identification of fungi, 245–247t, Penicillium chrysogenum, 305, spore and sporangia types, 241f, , Molecular techniques, for species, identification, 481, Moniliasis, 257, Mordant stain, 71–72, 72f, Morganella, 214, Mosquito (Anopheles), 231, 234f, Most probable number (MPN), water analysis,, 345, Motility, hydrogen sulfide production test, 185, observation of living bacteria, 49, Mouse virulence test, 450, Mouth, flora of, 425, MPN (Most probable number), water analysis,, 345, Mucor, 244, 247t, 355, Mucor mucedo, 242f, Mucous membrane, in native immunity, 489, Mueller-Hinton tellurite agar, 296, 425–426, 426f, Multitest systems, API system, 457, 457f, 460, EnteroPluri-Test system, 458, Enterotube Multitest System, 456, 457f,, 459–460, identification of enteric microorganisms, 455, Mutagenicity, 391, Mutation, bacterial genetics, 373, defined, 387, searching for resistant mutations, 388, Mycelium, fungal, 241, Mycobacteria, acid-fast stain, 79, 81, 81f, Mycobacterium, 355, Mycobacterium leprae, 79, Mycobacterium tuberculosis, 79, 200, Mycology, 239, Mycoplasma hominis, 508, Mycoses, 239, , N, Napiform, 30f, Native immunity, 489, Negative reactions, indole production test, 175, MR-VP test, 176–177, Negative staining, 67–70, principle, 67f, procedure, 68f, Neisseria, flora of throat and skin, 425–426, 428, 478, identifying gram-negative bacteria, 484, oxidase test for differentiation, 207, septicemia and, 476f, Neisseria gonorrhoeae, 306, Neisseria meningitis, 207, 475, Neonatal herpes, 507, Neonatal meningitis, 441, Neonatal pneumonia, 444, Neutrophile, 119f, NGU (Nongonococcal urethritis), 505, 508, Nigrosin, negative staining, 67f, 68f, Nitrate reductase, 199, Nitrate reduction test, 199–202, indicators of nitrate reduction, 200f, principle, 199, procedure, 200, reactions, 200f, Nitrates, denitrification, 351, Nitrification, nitrogen cycle, 351, Nitrite, 199, Nitrite ions, 351, Nitrobacter, 351–352, Nitrogen cycle, 351–352, 352f, Nitrogen fixation, 351–352, Nongonococcal urethritis (NGU), 505, 508, Non-lactose fermenters, 173, Non-tubercle mycobacterium, 200, Normal flora, 419, Nosepiece, microscope, 37, , Index, , 537
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Nosocomial infections, 7, 168, Novobiocin sensitivity, 434, 435f, Nucleoproteins, 265, Numerical aperture, microscope, 38, Nutrient agar plates, 8–9, 9t, 29, Nutrient agar slants, 15, 19, 22, 29, Nutrient broth, 31, , O, Objective, microscope, 40f, Obligate anaerobes, 123–124, Ocular lens, microscope, 37, 39t, Oil-immersion lens, microscope, 40f, 43, ONPG (ortho-nitrophenyl-®-d-galactosidase),, 375, Oocyst, mosquito life cycle, 232, Ookinete, mosquito life cycle, 232, Operon genes, 375, 376f, Optochin sensitivity test, 449, 450f, Ortho-nitrophenyl-®-d-galactosidase (ONPG),, 375, Oxaloacetic acid, citrate utilization test, 178, Oxidase enzymes, 207, Oxidase test, 207–210, principle, 207–208, procedure, 208, reactions, 208f, Oxidation, of tryptophan, 174, Oxidation-reduction (redox) reaction, litmus milk reaction, 193, redox potentials shown in agar tube, 129,, 129f, Oxidative deamination, 163, Oxidative processes, anaerobic respiration, 199, Oxidized, substrate, 129, Oxygen, in treatment of gas gangrene, 129, Oxygen bubbles, catalase test, 203, Oxygen requirements, microbial, 123–128, determining, 124f, principle, 123, procedure, 124–125, , P, P. aeruginosa, see Pseudomonas aeruginosa, P-aminobenzoic acid (PABA), 295–296, 296f, P-aminodimethylaniline oxalate, 207–208, Paraffin plug technique, 130, Paragonimus westermani, 335, Paramecium, 45f, Paramecium caudatum, 223, Parasitic protozoa, as causative agents, 235–236f, characteristics, 233t, classification, 223, life cycles, 231–232, 234f, water quality and, 335, Paratyphoid fever, enteric fevers, 455, intestinal infections related to water, 335, MacConkey agar and, 104, Parfocal, microscope, 41, Pasteur, Louis, 419, Pasteur pipette, 282f, Pathogens, extracellular enzymes and, 157, identifying intestinal, 186, identifying staphylococcal, 433–440, identifying streptococcal, 441–448, IMViC test, 173, soil-borne, 353, TSI test, 168, urease test, 189, Pathway 1, hydrogen sulfide production, 185, Pathway 2, hydrogen sulfide production, 185, P-dimethylaminobenzaldehyde, 174, Pellicle, bacteria growth, 30f, Pelvic inflammatory disease, 508, , 538, , Index, , Penetration, in viral infections, 265, Penicillin, 305–310, activity of, 305–306, antibiotic serial dilution plate setup, 308t, MIC with plate reader, 307–308, MIC with spectrophotometer, 306–307, resistance/sensitivity, 305, 305f, Penicillinase activity, penicillin resistance and,, 305f, Penicillium, antibiotic-producing organisms in soil, 361, cultivation, 244, enumeration of microbial population, 355, identification, 246t, in production of cheese, 352, soil and water molds, 319, Penicillium chrysogenum, 242f, 305, Peptide bond, in casein, 156, Peptone, broth medium, 97, casein hydrolysis and, 156, degrading into amino acids, 163, hydrogen sulfide production test, 185, Peptonization. see Proteolysis (peptonization), Petri dish, cultivation of microorganisms, 3, sterilization vessels, 4f, streak-plate inoculation, 20, Petroff-Hausser chamber, 135, 135f, PFU (Plaque-forming unit), 269, 269f, pH, 119–122, acid base indicator, 167–168, carbohydrate fermentation, 161, citrate utilization test, 178, impacting growth of microorganisms, 119f, MR-VP test, 175–176, oxidation-reduction indicator litmus,, 193–194, phenol red indicator, 189, principle, 119–120, procedure, 120, urease test, 189, Phagocytosis, 489, Phase-contrast microscope, 36, Phenol red, 189, Phenol red insulin broth, 167, 171, Phenolic compounds, 311t, Phenylalanine, 211, 213f, Phenylalanine deaminase test, deamination of amino acids, 213–215, 213f, differentiation of intestinal bacteria, 214, Phenylethyl alcohol agar, 103, 104f, Phenylpyruvic acid, 213–214, 213f, Physical controls, microbial growth, 285–286, Physical factors in experiments, atmospheric oxygen requirements. see, Oxygen requirements, microbial, pH. see pH, temperature. see Temperature, impact on, experiments, Picric acid, 52f, Pipette, transfer instruments, 4–5, Plaque-forming unit (PFU), 269, 269f, Plaques, in agar, 269, Plasmids, electrophoresing, 402, F factor and, 381, genetic engineering and, 398, isolating, 400f, isolation of DNA, 397–398, 397f, Plasmodium, 233, Plasmodium vivax, 234f, 235f, Plastic pump, transfer instruments, 5f, Plate reader, for MIC determination, 307–308, Pneumococcus infection, 450, Pneumonia, bacterial pneumonia, 88, bacteremias and, 475, Klebsiella pneumoniae, 175, lobar pneumonia, 441, , neonatal, 444, Staphylococcus pneumoniae, 433, 449–454, viral pneumonia, 506, Point mutations, 373, Polylinker, DNA segment, 397, Polypeptides, casein degradation into polypeptide chains,, 194, casein hydrolysis and, 156, Positive reactions, indole production test, 175, MR-VP test, 176–177, Potassium chloride salt, 119–120, Potassium hydroxide solution, 176, Potato dextrose sugar, cultivation of molds, 241, dilution-plating technique, 254f, Pour-plate technique, 136–138, 136f, Precipitin formation, 490, Precipitins, immunological reactions, 490, ring test, 491–494, 491f, 492f, Pre-erythrocytic stage, of parasitic, development, 231, Prepared slides, 56, Primary stain, acid-fast staining, 79, capsule staining, 92, differential staining, 71–72, spore staining, 85, 88, Proctitis, 508, Prophage, 266, Protein hydrolysis, 156, 156f, Proteins, as energy sources for microbes, 164f, Proteolysis (peptonization), casein hydrolysis and, 156, litmus milk reaction, 194-195, Proteus, deamination of amino acids, 211, diarrhea from, 186, differentiating enteric microorganisms, 168,, 168f, differentiating intestinal bacteria, 214, enteric microorganisms, 319, 455, identifying, 186, 456f, 471, identifying gram-negative bacteria, 485f, intestinal bacteria, 214, 425, pathogens, 173, TSI test, 168, urease test, 189, Proteus mirabilis, 168, Proteus penneri, 168, Proteus vulgaris, cultural and biochemical characteristics,, 218t, identifying, 189, IMViC test, 173, microscopic examination, 45, urinary tract infections, 470f, urine analysis, 469, Protozoa, 223–240, characteristics and means of locomotion,, 223–224, common types, 45f, free-living protozoa, 232t, parasitic protozoa, 231–232, 233t, Providencia, 214, Pseudomonads, 207, Pseudomonas, differentiating enteric microorganisms, 168, flora of throat and skin, 425, food-borne organisms in soil and water,, 319, free-living microorganisms, 352, identifying, 471, identifying enteric microorganisms, 456, identifying gram-negative bacteria, 485f, isolating, 367–370, oxidase test, 207, urinary tract infections, 470f
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Pseudomonas aeruginosa, antimicrobial therapy, 300, blood analysis, 475, cultural and biochemical characteristics,, 218t, IMViC test, 173, microscopic examination, 45, urinary tract infections, 470f, urine analysis, 469, Pseudomonas denitrificans, 352, Pseudopods, 223, 231, Psychrophiles, 113, 113f, Puerperal fever, 441, Pulmonary infection, 258, Pure culture, 19–27, defined, 1, isolation techniques, 19–23, 369, preparation, 483–484, Pyelitis, 469, Pyelonephritis, 469, Pyrogallic acid technique, 130, Pyruvic acid, carbohydrate fermentation, 162, citrate utilization test, 178, indole production test, 174, variations in use of, 162f, , Q, Quantitative water analysis, 345–348, 347f, Quebec colony counter, 136–137, 137f, Quellung (Neufeld) reaction, 450, Quinoidal red-violet compound, 174, , R, R Plasmids, 398, Radiation, inducing lysis, 266, 285, ionizing radiation, 3f, spore resistance to, 85, ultraviolet, 35–36, Raised, colony form, 30f, Rapid plasma reagin (RPR) test, for syphilis,, 505–506, 506f, Rapid species identification, 481, Rapid testing methods, 505–506, 506f, Rapid urease positive organisms, 189, Reactions, indole production test, 174–175, MR-VP test, 176–177, oxidation-reduction (redox) reaction, 129, TSI agar test, 168–169, Reagents, Barritt’s reagent, 176–177, capsule staining, 88, differential staining, 71, Gram’s iodine, 158, Kovac’s reagent, 174–175, p-aminodimethylaniline oxalate, 207–208, spore staining, 87, Real image, principles of microscopy, 37, Redox reaction. see Oxidation-reduction, (redox) reaction, Reduction, nitrate, 199, Refractive indexes, microscope, 38f, 39f, Refrigerator, bacterial growth and, 114, Release, in viral infections, 266, Replication, in viral infections, 265, Resistance, antibiotic, 281, antimicrobial, 144, benzylpenicillin, 305, conjugation factor in, 382, defined, 489, MRSA, 203, 314, penicillin, 305, 305–306f, searching for resistance mutations, 388, Resolution, microscope, 38, , Respiration, cellular, 129, comparing cellular respiration and fermentation, 161–162, Respiratory tract (upper), flora, 425, infections, 441, Restriction analysis, of bacteriophage Lambda, DNA, 409–418, principle, 409–410, procedure, 411–413, Restriction endonucleases, 409–411, Reticuloendothelial system, 489, Rheumatic fever, 441, Rhizobium, 352, Rhizoid, 30f, Rhizopus stolonifer, 242f, Rhodotorula rubra, 258, Ring (interfacial) test, precipitin reaction,, 491–494, 491f, 492f, Rod-shaped, bacilli, 61, Rubber bulb, transfer instruments, 5f, , S, S. aureus, see Staphylococcus aureus, Sabouraud broth culture, cultivation of molds, 241, 242f, isolation of microbial flora, 426, 427f, Saccate, 30f, Saccharomyces, fission process in yeast, 257, identifying, 207, oxidase test, 207, pyruvic acid uses, 162f, use in beverages, 352, Saccharomyces carlsbergensis, 257, Saccharomyces cerevisiae, carbohydrate fermentation, 114, as leavening agent, 257, wine production and, 329, yeast reproduction, 258f, Saccharomyces ellipsoideus, 257, 329, Safranin, as counterstain, 72, 85, gram staining, 75, spore staining, 88, Salmonella, 325–327, in febrile conditions, 495, identifying enteric microorganisms, 455, identifying intestinal pathogens, 186, isolation from raw meat, 325–326, 326f, microorganisms in food, 321, pathogens of Enterobacteriaceae family, 173, septicemia and, 475, 476f, Salmonella typhimurium, Ames test, 391–392, 393f, antibody titer test, 497f, cultural and biochemical characteristics,, 218t, IMViC test, 173, Salpingitis, 508, Sarcina, spherically shaped bacteria, 61f, Sarcodina, 223, 231, Scarlet fever, 441, Scenedesmus, 45f, Schistosoma haematobium, 469, Schizogony stage, of parasitic development, 231, Schizonts, 231, Schizosaccharomyces, 257, Sediment, in broth media, 30f, Segmenters, parasitic, 231, Selective media, 103–112, principle, 103, procedure, 106–107, Semisolid media, 1, Septicemia, 475, 476f, Serial dilution–agar plate technique, 135–142, principle, 135–136, procedure, 137–140, 139f, , for viable cell count, 140f, Serogroup, classification by, 441, Serologic classification of Lancefield, 441, Serrate, colony form, 30f, Serratia, 319, Serratia marcescens, 20f, 114, Sewage, isolation of coliphages, 275–277, 276f, Sexually transmitted diseases (STDs), 505–512, detecting chlamydial diseases, 508–509, isolating and identifying herpes simplex, virus, 507–508, rapid plasma reagin test, 505–506, 506f, types of, 505, Shake-culture technique, 130, Shake-tube inoculation (or culture), 123–124, Shigella, enteric microorganisms, 319, identifying enteric microorganisms, 455, identifying intestinal pathogens, 186, isolating and identifying, 456f, pathogens, 173, 186, TSI reactions, 168f, Shigella dysenteriae, cultural and biochemical characteristics, 218t, pathogens, 173, 186, waterborne pathogens, 335, Shigellosis, 455, Signet ring, parasitic, 231, SIM agar, 185–186, Simple staining, 61–66, principle, 61–62, procedure, 62–63f, techniques, 53f, Skin, flora of, 425–427, mechanical immunity barrier, 489, selecting and differentiating isolates, 428, staining and morphological characteristics,, 428, Slide preparation, 55, Smear. see Bacterial smears, Snyder test (or agar), 422, 422f, SOD (Superoxide dismutase), 203, Sodium chloride agar (7.5%), 103, Sodium chloride test, 6.5% sodium chloride broth, 443, 444f, identifying streptococcal pathogens, 443,, 444f, Sodium thiosulfate, 167–168, 185, Soil microbiology, 351–360, antibiotic-producing organisms, 361, enumerating microbial populations, 355–358, overview of, 351–353, Solid medium, 55–57, Sore throats, 420, Species, identifying unknown bacterial, cultures, 481–488, Spectronic 20 spectrophotometer, 98f, 120, Spectrophotometer, analyzing cell population, 136, for bacterial growth curve, 146, 146f, MIC determination, 306–307, in pH experiment, 120, schematic diagram, 98f, Spherical shape, cocci, 61, Spiral bacteria, 61f, Spirilla, 61f, 63f, Spirochetes, 61f, 506, Spirogyra, 45f, Spontaneous mutations, 373, Sporangia, 241f, Spore stain (Schaeffer-Fulton Method), 85, principle, 85–87, procedure, 87–88, 87f, showing free spores and vegetative bacilli,, 86f, Spore-forming bacterium, 86, Spores, staining techniques, 85–88, 86f, 87f, types of, 241f, , Index, , 539
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Sporogamy, sexual cycle of mosquitos, 232, Sporogenesis, 85, 86, Sporozoa, 223, Sporozoites, life cycle of mosquitos, 232, Spread-plate, culture isolation experiment, 22, 24f, serial dilution–agar plate technique, 138, using, 383f, Stage, microscope, 37, Stained cells, microscopic examination, 37–42, Stains, acid fast, 79–84, basic, 51–53, chemical composition, 51f, gram stains, 71–75, negative staining, 67–70, simple staining, 61–66, slide as key to success, 74, staining techniques, 53f, STAPH-IDENT, 434, 436–437, 437–438t, Staphylococcus, contamination of food, 319, differentiating staphylococcal species, 434t, differentiating Staphylococci, Streptococci,, and Enterobacteriaceae, 203, flora of skin, 425, identifying staphylococcal pathogens,, 433–440, spherically shaped, 61f, Staphylococcus aureus, CAMP reactions, 443f, common cause of human disease, 435, cultural and biochemical characteristics,, 218t, gram-positive bacteria, 103, identifying staphylococcal pathogens, 433, isolating, 21, mannitol salt agar, 426, methicillin-resistant. see Methicillinresistant S. aureus (MRSA), microscopic examination, 45, nonenteric, 173, novobiocin test, 435f, penicillin resistant, 305, selective effects of phenylethyl alcohol agar,, 104f, streak-plate culture of, 433f, Staphylococcus epidermis, 433, Staphylococcus saprophyticus, 433, Staphyloxanthin, 435, Starch hydrolysis, extracellular enzymatic activities, 155, procedure, 158, on starch agar plate, 155f, Stationary phase, of bacterial population, growth, 143, STDs. see Sexually transmitted diseases (STDs), Sterilization techniques, 3, 3f, Steroids, 352, Stock culture, 22, Stomach cramps. see Abdominal cramps, Stomach pH, 120, Stratiform, cultural characteristics of, bacteria, 30f, Streak-plate, alternative method, 20f, culture isolation, 22–24, four-way streak-plate inoculation, 19f, isolation techniques, 19–20, of Staphylococcus aureus, 433f, Streptobacillus, rod-shaped bacteria, 61f, Streptococcus, on blood agar, 105, cultivating, 104, differentiating types, 203, 442t, enteric microorganisms, 319, gram stains, 71, 71f, identifying gram-positive bacteria, 482f, identifying streptococcal pathogens,, 441–448, , 540, , Index, , infectious diseases, 441, isolating urinary tract pathogens, 470f, 471, pyogenic cocci, 419, pyruvic acid uses, 162f, septicemia and, 475, 476f, spherically shaped bacteria, 61f, Streptococcus agalactiae, 444, Streptococcus mitis, 442t, Streptococcus mutans, 421, 422f, Streptococcus pneumoniae, causative agent of lobar pneumonia, 441, encapsulated bacterial pneumonia, 88, flora of respiratory and intestinal tracts, 425, identifying, 449–454, 449f, pneumococcus infections, 450, Streptococcus pyogenes, 21, 441, 444, Streptococcus salivarius, 442t, Streptococcus sanguis, 442t, Streptococcus thermophiles, 331, Streptococcus viridans, 441, 449, Streptolysin O, 105, Streptolysin S, 105, Streptomyces, 361, Streptomycin, 387f, Streptomycin-resistant mutant, 387–390, Strong acids, buffering system, 119–120, Stylonychia, 45f, Subacute endocarditis, 441, Subculturing, for culture transfer, 13–14, 14f, overview of, 4, Substrates, carbohydrate, 161, for hydrogen sulfide production, 167–168, litmus milk reaction, 193, oxidized, 129, Sucrose, carbohydrate fermentation, 163–165, degradation of, 422f, differentiation of Enterobacteriaceae, species, 167, Sulfadiazine, 295, Sulfanilamide, 295–296, 296f, Sulfates, in oxidative processes, 199, Sulfisoxazole, 300, Sulfur, 185, Sulfur cycle, 352–353, Superficial mycoses, site of infection, 239, Superoxide, in aerobic respiration, 203, Superoxide dismutase (SOD), 203, Surface-active agent, disinfectants and antiseptics, 312t, Symbiotic microorganisms, in nitrogen fixation, 352, Synergistic effects, of drug combinations,, 299–300, 300f, Synthetic drugs, 295–296, Syphilis, bacterial STDs, 505, causative agent, 505, diagnosis, 46, Systemic mycoses, site of infection, 239, , T, Taxonomy, 217, Teeth, flora of, 425, Temperate phages, 266, Temperature, impact on experiments, 113–118, growth and coloration of bacteria, 113f, 114f, principle, 113–114, procedure, 115, Test tubes, 3, 14, Tetracycline, 300, Tetrads, spherically shaped bacteria, 61f, TFTC (Too few to count) plates, 140, 269, Thermophiles, in classification of bacteria,, 113f, 114, Thioglycollate broth tube, 131f, Thiosulfate, , hydrogen sulfide production test, 185, TSI agar test, 167–168, 171, Throat, flora of, 425–427, infections, 441, selecting/differentiating isolates, 428, staining and morphological characteristics,, 428, Thrush, 257, Tinea pedis, 374, Tinsdale agar plate, 425, Tissue culture, 507, TNTC (Too numerous to count) plates, 140, 269, To-deliver pipette, transfer instruments, 5f, Tonsillitis, 441, Too few to count (TFTC) plates, 140, 269, Too numerous to count (TNTC) plates, 140, 269, Torula, 245t, Torulopsis, 207, Toxins, 203, TPI (Treponema pallidum immobilization), test, 506, Transduction, of genetic material, 398, genetic recombination, 381, transfer of genetic material, 374, Transfer instruments, 3–4, 5f, Transfer loop, 5f, Transfer needle, 5f, Transformation, DNA segments release during, 398, genetic recombination, 381, transfer of genetic material, 374, Transmitted light (T), measuring, 98, Traveler’s Diarrhea, 466, Treponema pallidum, 505–506, Treponema pallidum immobilization (TPI), test, 506, Tributyrin agar, extracellular enzymatic activities, 158, lipid hydrolysis, 156, 156f, Trichomonas, 233, Trichomonas vaginalis, 469, 508, Trichomoniasis, 505, Triglycerides, 155, Trimethoprim, 300, Triple sugar–iron (TSI) agar test, 167–172, differentiating enteric microorganisms, 168f, isolating Salmonella from raw meat, 325–326, principle, 167–168, procedure, 168–169, reactions, 169f, Trophozoites, 231, Trypanosoma gambiense, 236f, Trypticase soy broth (TSB), in biochemical tests, 203, 219, media, 120, Tryptophan, enzymatic degradation, 174f, indole reaction, 174, 174f, Tryptophanase, 174, Tsetse fly (Glossina), 231, TSI agar test. see Triple sugar–iron (TSI) agar, test, TTC (2,3,5-triphenyltetrazolium chloride), 186, Tuberculosis, 200, Turbidity, hydrogen sulfide production test, 185, measuring, 97–99, Twort, Frederick, 265, Typhoid fever, enteric fevers, 455, intestinal infections related to water, 335, MacConkey agar and, 104, , U, Ultraviolet (UV), 35–36, 266, 285, Umbonate, colony form, 30f, Undulate, colony form, 30f
Page 558 :
Unico 1100RS spectrophotometer, 98f, Uninoculated, reactions in indole production test, 175, reactions in MR-VP test, 176–177, reactions in TSI agar test, 168–169, United States Department of Agriculture, (USDA), 325, Urea, 189, 189f, Ureaplasma urealyticum, 508, Urease test, 189–192, enzymatic degradation of urea, 189f, principle, 189–190, procedure, 190, reactions, 190f, Urethritis, 508, Urinary tract infections, history of, 472, treating, 168, urine analysis, 469, 470f, Urine analysis, 469–474, 470f, USDA (United States Department of, Agriculture), 325, UV (Ultraviolet), 35–36, 266, 285, , V, Vaginal infections, 469, Vaginitis, 469, Van Leeuwenhoek, Antoni, 35, VDRL (Venereal Disease Research Laboratory),, 505–506, Vegetative bacillus, 86, Vegetative cells, 85, , Vegetative mycelium, 241, Venereal Disease Research Laboratory (VDRL),, 505–506, Vibrio, dental caries and, 425, fermentation in identification of, 163, rigid or flexible bacteria, 61f, Viral pneumonia, 506, Virulent phage, in lytic cycle, 266, Viruses, 265–268, alternative to antibiotics, 281, comparing human with animal viruses,, 266–267, cultivation and enumeration, 269–271, differentiating from cellular life forms, 265, herpes simplex. see Herpes simplex virus, (HSV), isolating coliphages from raw sewage,, 275–277, 276f, sequential events in infections, 265–266, Voges-Proskauer test, IMViC test, 173, principle, 175–176, procedure, 176–177, reactions, 177f, reagents, 179, Volvox, algae types, 45f, Vorticella, protozoa types, 45f, , W, Water, 335–350, case study, 336, , as decolorizing agent, 85, microbiology of, 335–336, quantitative analysis, 345–348, Waterbath, 2f, 6, 124f, Water-borne epidemics, 336, Weak acids, buffering system, 119–120, Wet mount, 46, WHO (World Health Organization), 336, Widal Agglutination test, 495, Wine, 330, Wire loops, transfer instruments, 4–5, Working distance, microscope, 40f, World Health Organization (WHO), 336, Wounds, 106, Wuchereria bancrofti, 469f, , Y, Yeast extract broth, 97, 101, Yeasts, 257–264, alcohol fermentation and, 329, colonies, 257f, infections, 258, morphology, characteristics, and, reproduction, 257–259, 258f, , Z, Ziehl-Neelsen method, 79, Zygomycetes, characteristics, 240t, types of fungi, 239, 428, , Index, , 541
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