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Chapter wd 3, MICROBIOLOGICAL ASSAYS, , + LEARNING OBJECTIVES «, After completing this chapter, reader should be able to understand:, , © Techniques for Standardization of Pharmaceuticals by Microbiological Assays, © The Importance of Assessment of new Antibiotic by MIC, , , , , , , , , , , , 43.4 INTRODUCTION ©, , , , , , A microbiological assay may be defined as qualitative or quantitative determination of, any chemical compound from a simple or even complex material with the use of, microorganisms. It is necessary to assay antimicrobial agents for determination of potency,, for determining the pharmacokinetics of a drug in animals or man and for monitoring and, controlling antimicrobial chemotherapy. Quantitative chemical or physical methods can, assay most of the currently employed therapeutic agents. Many therapeutic agents, which, either inhibit the growth of microorganisms (antibiotics) or are essential for their growth, (vitamins and amino acids) can be standardized by microbiological assays., , Microbiological assays are relatively as accurate as chemical methods. It is a simple,, specific, inexpensive and convenient method. Compared with biological assay methods, using animals, microbiological techniques possess the advantages of minimal requirements, , of space, labour, materials and time. Microbiological assays are very useful for detecting, , changes in potency of antibiotics and their preparations. Microbial assays are more difficult, , to perform as compared to physical and chemical assays and also require proper calibration., They are less reproducible and have greater error as compared to other assays., Microbiological assay are not used if a good alternative physical or chemical assay is, , available,, , [13.2 MICROBIOLOGICAL ASSAY OF ANTIBIOTICS, h under standardized conditions may be utilised for, , antibiotics. The microbiological assay is based, of growth of microorganisms by measured, be examined with that produced by known, tibiotic having a known activity., , , , , , The inhibition of microbial growt, demonstrating the therapeutic efficacy of, “pon a comparison of the inhibition, ‘oncentrations of the antibiotics to, Concentrations of a standard preparation of the an, , (13.1)

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Pharmaceutical Microbiology (B.Pharm. Sem. 13.2 Microbiological, , 43.2.1 Media used for Antiblotic Assay, The media required for the preparation of test microorganisms, inoculums are made, from the ingredients listed in Table 13.1. Dissolve the ingredients (as per Table 13.1) in, sufficient water to produce 1000 ml and add sufficient 1M sodium hydroxide or 1M, hydrochloric acid, as required, so that after sterilization the pH is adjusted as given in Table, , 13.1., Table 13.1: Composition of media: Quantities in gm per 1000 mi medium, , , , , , , , , , , , , , , , , , , , , , , , , , Ingredient ~>lelelolelricocintiu J, , Peptone so | 60 | so | 60 | 60 | 60 | 4 | - | 100) =, Yeast extract 30 | 30 | 18 | 30 | 30 | 30 | a? | - - Bee! ertract 16 | 15 | 1 | 48 | 15 | 18 | 26 - | to] =, Pancreatic digest of casein 40 | - - | 40] - - - | 170 | - | 150, Destose ao | - | 10 | wo] - - | 0] 2] - Papaic digest! of toyabean ull :<? fia - - - - 390 - 50, Agar sso | 130 | = | 150 | 150 | 130 | 235 | 120 | 170 | 150, Glycerin - - - - - - - - wo] Polysorbate 20 - - - - - - - | wo] - Sodium chloride - jj - | as] - - - | 10 | 50 | 30 |] so, Oppolassium hydrogen phosphate - | - 388 - - = = 25 = *, Potassium dtiydogen phosphate - - 4.32 i is | - = ° = we, Fira ph 66 | 65 | 69s-| 78-|78- | se | 60 | 74 | 69 | 72, -66 | -56! 705 | 89 | 80 | -60 | -62 | -73 | -71 | -78, , , , , , 13.2.2 Preparation of Standard, Test Solution and innoculums, , Preparation of standard solution: A standard preparation is an authentic sample of the, appropriate antibiotic for which the potency has been precisely determined by reference to, the appropriate international standard. The potency of the standard preparation may be, expressed in International Units or in ug per mg of the pure antibiotic., , Dissolve a quantity of the standard preparation of a given antibiotic in the solvent, specified in Table 13.2. Dilute the preparation to get the required concentration as stated, and store in a refrigerator. On the day of assay, prepare from stock solution five or more test, dilutions, the successive solutions increasing stepwise in concentration, usually in the ratio, 1:1.25 for Method A or for Method B., , Preparation of sample solution: From the information available for the substance, under examination (test sample), assign to it an assumed potency per unit weight or volume,, and on this assumption prepare on the day of assay a stock solution and test dilution as, specified for each antibiotic in Table 13.2,, , Preparation of buffer solution: Prepare the buffer solutions of given quantities in, Table 13.3 of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in, sufficient water to produce 1000 ml after sterilization, adjusting the pH with 8 M phosphoric, acid or 10 M potassium hydroxide,

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Pharmaceutical Microblology (B.Pharm, Sem. II) 13.4 Microbiological Assays, , 1 Bacillus pumitus ATCC 14884 as test organism; 2 —» Stophylococcus auveus ATCC 9144 as, test organism; 3 ~» Staphyococcus aureus ATCC 29737 at test organism; 4 — Bacillus cereus, var mycoides ATCC 11778 as test organism; 5 — Bacillus subtilis ATCC 6635 as test organism,, 6 — Klebsiella pneumoniae ATCC 10031 as test organism; B denotes buffer solution and, number following refers to the buffer number., , Table 13.3: Buffer solutions, , , , , , , , , , Buffer | Dipotassium hydrogen Potassium dihydrogen pH adjusted after, No. | phosphate, K,HPO, (g) phosphate, KH;POs (g) sterilization to, es 2.0 8.0 6.0401, 2 16.73 0.523 8.0401, 3. = 13.61 45+01, 4. 20.0 80.0 6.0401, 5. 35.0 - 10.5+0.1, 6. 136 40 7.0402, , , , , , , , , , , , , , Test microorganisms: The test microorganism for each antibiotic is listed in Table 234, with its identification number in the American Type Culture Collection (ATCC). Maintain a, culture on slants of the medium under the incubation conditions (Table 13.5) and transfer, weekly to fresh slants., , Preparation of inoculum: Prepare the microbial suspensions for the inoculum for the, assay as given in Table 13.5. Test microorganism suspensions are Prepared by any one of the, following metheds:, , (i) Maintain the test microorganism on slants of medium A and transfer to a fresh slant, , once a week. Incubate the slants at the specified temperature for 24 hours. Using 3, ml of saline solution, wash the microorganisms from the agar slant onto a large, agar surface of medium A such as a Roux bottle containing 250 ml of agar, Incubate, for 24 hours at the appropriate temperature. Wash the growth from the nutrient, surface using SO ml of saline solution. Store the test Microorganisms under, refrigeration. Determine the dilution factor which will give 25% light transmission at, about 530 nm, Determine the amount of suspensions to be added to each 100 mi, , of agar of nutrient broth by use of test plates or test broth. Store the Suspension, under refrigeration., , (ii) Proceed as described in method 1 but incubate the Roux bottle for S days., , Centrifuge and decant the supernatant liquid. Resuspend the sediment with 50 to, 70 ml of saline solution and heat the Suspension for 30 minutes at 70°C. Wash the, Spore suspension three times with 50 to 70 ml of saline solution and heat shock, again for 30 minutes. Use test plates to determine the amount of the suspension, required for 100 ml agar. Store the Suspension under refrigeration,, , (in) Maintain the test microorganisms on 10 mi agar slant of medium G (Table 13.1)., Incubate at 32 to 35°C for 24 hours. Inoculate 100 mi of Nutrient broth. Incubate for, 18 to 24 hours at 37°C and proceed as described in method 1., , (iv) Proceed as described in method 1 but wash the growth from the nutrient surface, , using 50 ml of medium 1 (prepared without agar) in place of saline solution,