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Chapter ... /, STERILIZATION, , ¢ LEARNING OBJECTIVES ¢, After completing this chapter, reader should be able to understand:, © ‘The different methods of Sterilization with Principle, Advantages, Disadvantages and Applications, © Different Equipments Employed in Large Scale Sterilization, © ©The technique used for Evaluation of Efficiency of Sterilization Methods, , , , , , INTRODUCTION, , , , , , , , , , Sterilization is an essential stage in the processing of any product used for parenteral, administration, broken skin, mucosal surfaces or internal organs. Sterilization of, microbiological materials, surgical dressings and equipments and other contaminated items, is necessary to minimize the health hazard associated with these articles., , The main reasons for controlling microorganisms are:, , 1. To prevent contamination in sterile products., , 2. To prevent transmission of pathogenic microorganisms which are responsible for, , causing diseases in plants, animals and human beings., , 3. To prevent decomposition and spoilage of food and food products., , 4. To prevent the contamination of unwanted microbes in pure cultures and other, , microbiological experiments performed for research studies., , 5. To prevent unwanted microbial contamination in antibiotic, enzyme, vitamin, , fermentation and other industrial processes., , 6 To prevent contamination in aseptic areas which are used for the preparation of, , sterile dosage forms and sterility testing., , Microorganisms can be killed, eliminated or inhibited by various physical and chemical, agents. Several terms used to describe the physical processes and chemical agents, Employed in controlling microorganisms are:, , Sterilization: Sterilization is a process by which an article, surface or medium is freed of, , all Microorganisms either in the vegetative or spore state., , (7.1)

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iY, , Pharmaceutical Microbiology (B.Pharm. Sem. I) 7.2 Sterilizaty, , , , .7.2 STERILIZATION METHODS., , Disinfection: Disinfection means the destruction of all pathogenic organisms tr, organisms capable of giving rise to infections. In this process, vegetative cells are killeg, but not heat-resistant spores. Disinfection is usually accomplished by chemical agent,, called disinfectants. A disinfectant is normally applied to in- animate objects such %, floors, buildings, equipment, laundry etc., , Sanitization: Sanitization is the process of disinfection including cleansing action,, Sanitizers are commonly applied to inanimate objects., , Antiseptic: The term antiseptic is used to designate any substance which would Prevent, sepsis, either by killing microorganisms or by inhibiting their growth. An antiseptic can, be applied to body tissues without causing injury to the tissue., , Germicide: A germicide is an agent that kills vegetative cells but not necessarily the, resistant spore forms of germs. The terms bactericide, fungicide, virucide and sporicide, refer to agents that kill bacteria, fungi, viruses and spores respectively., , Microbiostasis: Microbiostasis is the Process of preventing the growth, reproduction, and multiplication of microorganisms but not of killing them., , Preservative: A preservative is a substance that Prevents the growth of microorganisms., , These substances are mainly added in food and pharmaceuticals to prevent microbial, growth. Preservatives are not harmful to living tissues., , , , , The various methods used in sterilization can be classified as follows:, Physical methods:, , (a) Dry heat sterilization:, e.g. Incineration, direct flame, red heat, hot air etc., (b) Moist heat sterilization/steam sterilization, , e.g. Pasteurization, tyndallization, autoclave etc., (c) Radlation/cold sterilization:, , (i) —_Use of ultra-violet rays: UV light., , (ii) Ionising radiations: X-rays, gamma rays, beta rays,, (d) Filtration / mechanical methods:, , (i) Asbestos filter (seitz filter)., , (ii) Sintered glass filter (morton filter),, , (iii) Filter candles (ceramic/Berkefeld filter), , (iv) Membrane filter (millipore/ultra filter).

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eS, , pha rmaceutical Microbiology (B.Pharm. Sem. Il) 7.3 Sterilization, , 1 chemical methods:, (a) Gaseous sterilization:, eg. Formaldehyde, ethylene oxide etc., (b) Byusing disinfectants:, eg. Cresol, phenol etc., L Physical Methods, , These methods involve processes by the use of physical means. These may involve the, , utilisation of heat in the presence or in the absence of moisture or the applications of, radiations or mechanical filtration., , (a) Dry heat sterilization:, , Heat is the most reliable and rapid method of sterilization. The killing effect of dry heat, is due to protein denaturation, oxidative damage and the toxic effect of elevated levels of, electrolytes. The factors influencing sterilization by heat are nature of heat, number of, microorganisms present, temperature and time, characteristics of the microorganisms etc., , The time required for sterilization is inversely proportional to the temperature of, exposure. This can be expressed as thermal death time, which is the minimum time required, to kill a suspension of microorganisms at a prescribed temperature and under specific, conditions. Sterilization time is directly related to the number of microorganisms in the, suspension, presence and nature of spores, the strain and characteristics of the, microorganisms. Microorganisms are more resistant to dry heat as compared to moist heat, and therefore, this process requires higher temperatures and longer exposure times., , 1. Sunlight and drying: Sunlight possesses ultraviolet rays which along with heat, fays are responsible for appreciable germicidal activity. These rays cannot, penetrate through glass. This is a natural method for sterilization of water in, tanks, rivers and lakes. Drying in air has deleterious effect on many bacteria., Spores are unaffected by drying. Hence it is a very unreliable method., , 2. Red heat: It is used to sterilize metallic objects by holding them on a flame till, they are red hot e.g. inoculating wires, needles, forceps etc., , 3. Flaming: The article is passed over flame without allowing it to become red hot, e.g. mouth of culture tubes, glass slides, scalpels, needles, coverslips etc. These, items may be sometimes immersed in spirit in a tray and the spirit is then burnt, off. However, it does not produce sufficient heat and destroys only vegetative, microorganisms., , 4. Incineration: This is an excellent method for rapidly destroying materials e.g., Pathological material, bedding, animal's carcasses, soiled dressing etc., Polystyrene types of materials emit clouds of dense black smoke and hence, should be autoclaved in appropriate containers.

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J, , Pharmaceutical Microbiology (B.Pharm. Sem. 11) __7.4 Steriizay,, , 5. Hot air oven: This is the most widely used method of sterilization by dry hey, The modern hot air ovens consist of a double walled chamber of aluminium ,, stainless steel separated from the outer case by a thick layer of insulation Magy, of fiberglass (Fig. 7.1). Insulation is also filled in the hollow flanged door, Which, carries an asbestos jacket that provides a tight seal. Heating is affecteg, electrical heating elements and thermostats automatically control temperatus, The material should be arranged in a manner which allows free circulation of g,, between the objects and it should not be overloaded. Glassware should t,, perfectly dry and wrapped in Kraft paper before being placed in the oven. Th, oven must be allowed to cool slowly for about two hours before the door,, opened, since the glassware may get cracked by sudden cooling., , , , , , Outer case containing glass fibre, , , , Asbestos gasket, , , , , , , , Perforated shelf, , Regulator, , , , , , Fig. 7.1: Hot air oven, , Substances that are not heat-labile and can tolerate temperature upto 250°C may be, sterilized by hot air oven. Normally the spores as well as the vegetative forms of all, microorganisms are killed in two hours at a temperature of 160°C. The relation between the, temperature and relative time required for sterilization in hot air oven is given in Table 7.1., , Table 7.1: Temperature and time relationship for hot air oven, , , , , , , , , , Temperature (°C) Time (minutes), 170 60, 160 120, 150 150, 140 180

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pharmaceutical Microbiology (B.Pharm. Sem. I) 7.5 Sterilization, , Hot air oven is used to sterilize glassware forceps, scalpels, scissors, spatula, swabs, some, harmaceutical substances such as glycerin, fixed oil, liquid paraffin, propylene glycol,, sulphonamides and dusting powders such as kaolin, talc, zinc oxide, starch etc. It is not, suitable for surgical dressings, rubbers, plastics, volatile and heat labile substances, Hot air is, a bad conductor of heat and its penetration power is low as compared to moist heat. Hot air, ovens are commonly available in pharmaceutical laboratories for drying and sterilization., (b) Moist heat sterilization:, , Sterilization by moist heat means killing of microorganisms with hot water or steam. The, lethal effect of moist heat is due to the denaturation and coagulation of proteins., , Moist heat sterilization is divided into three forms., , (i) Temperature below 100°C., (ii) Temperature at 100°C., , (ii) Temperature above 100°C., , (i) Temperature below 100°C: Heat-labile fluids may be disinfected by heating at, temperatures below 100°C. The temperature employed is either 63°C for 30 minutes (holder, method) or 72°C for 20 seconds (flash method) followed by rapid cooling to 13°C or lower., This method is known as pasteurization and it is widely applied in dairy products like milk, and butter. By this method non-sporing microorganism such as mycobacteria, brucella and, salmonellae are destroyed. Heat-labile fluids such as serum may be disinfected by heating at, 56°C for one hour. Vaccines prepared from non-sporing bacteria may be inactivated in a, water bath at 60°C for one hour. Among the most heat resistant cells are the spores of, , Clostridium botulinum which require 120°C for four minutes., , (ii) Temperature at 100°C: Boiling at 100°C for 10 to 30 minutes kills all vegetative, bacteria and some bacterial spores. Therefore, it is not recommended for sterilization of, instruments for surgical procedures. Addition of small quantity of acid, alkali or washing, , soda markedly increases the sterilizing power of boiling water., An atmosphere of free steam is used to sterilize culture media which may decompose if, , Subjected to higher temperatures. A Koch or Arnold steam sterilizer is usually used. This, , steam sterilizer consists of a vertical metal cylinder with a removable conical lid having a, small opening for the escaping steam. Water is added on the bottom and a perforated shelf, , above the water level is present. Single exposure to steam for 90 minutes ensures complete, Sterilization but media containing sugar and gelatin, which may get decompose on long, heating. Hence, such materials may be exposed at 100°C for 20 minutes on three successive, days. This is known as ‘tyndallization’ or ‘intermittent’ or ‘fractional sterilization’. First, €xPosure to steam kills all vegetative bacteria and at second exposure all spores germinate, 'N @ favourable medium and are killed on subsequent occasions. Therefore, non-nutrient, , Media cannot be sterilized by this method., , (ill) Temperature above 100°C: Heat in the form of saturated steam under pressure is, the most practical and dependable agent for sterilization. The laboratory apparatus, designed to use steam under regulated pressure is called an autoclave. Saturated steam isa