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Sterilization
Procedures -
Sterilization involves either inactivation or removal of living organisms. This may be achieved by
(i) heating,
(ii) irradiation.
(iii) Chemicals or
(iv) filtration; these are briefly discussed below.
Heating. It is the most commonly used and the least expensive sterilizing agent. Dry heat is used in ovens and is suitable for sterilization of solids, which can withstand the high temperatures needed for sterilization, e.g., laboratory glassware, talc, etc. Steam, i.e., moist or wet heat, is used for sterilization of media and fermenter vessels.
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An autoclave uses steam for sterilization (at 121°C and 15p.s.i.). the period of time at this temperature pressure depending on medium volume, e.g., 12-15 min for 200 mi. 17-22 min for 500 ml, 20-25 min for 1 I and 30-35 min for 2 l. But sterilization of oils will require a few hours, and concentrated media (10-20% solid) must be agitated for effective sterilization.
Autoclaves can also be used to sterilize laboratory vessels, small volumes of media and even small fermenters.Large fermenters are sterilized by either a direct injection of steam or by indirect heating by passing steam through heat exchange coils or a jacket. The steam should always be saturated.
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Media sterilization may be achieved in a continuous flow sterilization system either by direct steam injection or by indirect steam heating, and then filled in a sterile fermenter. Alternatively, the medium may be filled in the fermenter and steam-sterilized with the latter.
Heat killing in most part is due to protein inactivation. In general, moist heat is far superior to dry heat. Bacterial spores are the most heat resistant, e.g., spores of thermophilic bacteria can survive steam at 30p.s.i. at 134°C for 1-10 min and dry heat at 180°C for up to 15 min.
Radiation. High energy X-rays are used for sterilization of a variety of labware and of food. In general, vegetative cells are much more susceptible than bacterial spores (Clostridium spores can resist nearly 0.5 M rad). But Deinococcus radiodurans vegetative cells can survive 6 M rad.
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Viruses are usually similar to bacterial spores but some viruses, e.g., encephalitis virus require up to 4.5 M rad. In practice, 2.5 M rad is used for sterilizing pharmaceutical and medical products. X-rays cause inactivation by inducing single and doublestrand DNA breaks, and by producing free radicals and peroxides, to which -SH enzymes are particularly susceptible.
Chemicals. The chemicals used for sterilization cause inactivation by oxidation or alkylation; these are formaldehyde, H2O2, ethylene
oxide, propylene oxide etc. H2O2 (10-25% w/v) is being increasingly used in the sterilization of milk and of containers for food products.
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It is a powerful oxidizing agent, kills both vegetative cells and spores and is very safe. Ethylene oxide is used for sterilizing equipments, which are likely to be damaged by heat, and is very effective, but highly toxic and violently explosive if mixed with air.
Filtration. Aerobic fermentation requires a very high rate of air supply often equaling 1 vol of air (equal to medium volume) every minute. Air contains both fungal spores and bacteria, which are ordinarily removed by filtration using either a depth filter or a screen filter.
Depth filters are made from fibrous or powdered materials pressed or bonded together in a relatively thick layer; the materials used are fiberglass, cotton, mineral wool, cellulose fibers, etc. in form of mats, wads or cylinders.
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Modem depth filters are cylinders of bonded borosilicate microfibers. Depth filters allow higher filtration rates and efficiencies than screen filters, but are not suitable for filtration of moist air.
Screen filters are membranes of cellulose esters or other polymers with pores of 0.45 µm or smaller (bacterial contaminants are 0.5 µm or larger). Usually, a microfibers profiler is used with such filters to remove gross contamination.
All filters themselves must be sterilized before they can be used to sterilize the air. Filters are also used to sterilize the effluent gases from fermenters, especially in case of pathogenic microorganisms.
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