GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

6. MICROBIAL GROWTH

6.1. EFFECT OF EXTERNAL FACTORS ON MICROORGANISMS

6.1.3. Methods of Sterilization

Microbial death is the irreversible loss of The ability to grow and reproduce. Many types of damage that typically result in Cell death can be reversible under certain conditions. For example, The phenomenon of photoreactivation occurs after a culture is exposed to ultraviolet light.

Sterilization is the elimination of all living microorganisms, including their dormant forms (spores), from any given material. Sterilization should be distinguished from partial decontamination (pasteurization) as well as preservation. If foreign microorganisms inadvertently enter a sterile medium or microbial culture, this is referred to contamination (infection).

The effectiveness of various agents used to destroy microorganisms is quantified by the D_t0 value (the time required to kill 90% of the Cells in a given population under specific conditions).

Complete or partial sterilization is achieved using moist heat, dry heat, filtration, irradiation, or various chemical agents.

Moist heat. Vegetative cells of most Bacteria and Fungi are destroyed within 5-10 minutes at 60 C, Yeast and fungal spores at temperatures above 80 C, and bacterial spores above 120 C (15 minutes). To attain temperatures exceeding the boiling point of Water, an autoclave is used. When air is present, a specific pressure corresponds to a significantly lower Temperature. The destruction of microorganisms by moist heat depends on temperature rather than pressure. Therefore, temperature rather than pressure should be monitored during autoclaving, although pressure is commonly measured due to its simplicity and safety. The required temperature and duration of sterilization depend on the COMPOSITION OF THE nutrient medium. For instance, milk, gelatin media, and media containing sugars, Vitamins, etc., are sterilized at 112–115 C and a pressure of 0.05 MPa for 20-30 minutes; meat-peptone media at 120 C (0.075-0.1 MPa) for 20-30 minutes; and salt solutions at 131 C (0.15 MPa) for 40-60 minutes.

Dry heat. During dry heat sterilization, bacterial spores withstand higher temperatures for longer periods compared to moist heat sterilization. Therefore, heat-resistant glassware, powders, and oils are sterilized for 2 hours at 160 C in a hot-air oven. Heat sterilization is based on the coagulation of cellular Proteins.

Filtration. Solutions containing thermolabile substances are sterilized by filtration (so-called cold sterilization). This method utilizes membrane filters with various pore diameters, making it possible to retain organisms of different Sizes and Shapes.

Irradiation. Ultraviolet, X-rays, and gamma rays are employed for this purpose. In laboratory practice, UV sterilization is the most widely used method. It is commonly applied to sterilize rooms and plasticware that cannot be autoclaved.

Chemical agents. Ethylene oxide is used for the sterilization of food products, Pharmaceuticals, and various instruments. It destroys both vegetative cells and spores, but is effective only when the Materials being sterilized contain a certain amount of moisture (5-15%). Ethylene oxide is typically applied as a gas mixture with nitrogen or carbon dioxide, in which its concentration ranges from 2 to 50%.

To preserve thermolabile substances present in nutrient media, beta-propiolactone sterilization was introduced into practice. It is significantly more active than propylene oxide, but it also exhibits carcinogenic properties. Beta-propiolactone at a concentration of 0.2% is added to prepared nutrient media, which are then incubated for 2 hours at 37 C. Beverages are also sterilized using diethyl pyrocarbonate (0.003-0.020%).

For the sterilization of seeds intended for the cultivation of axenic plants, standard antimicrobial agents are suitable, such as bromine water (1%), corrosive sublimate (1% solution of HgCl₂ in alcohol), a 0.05% silver nitrate solution, and others.

Methods of preservation. Organic materials are decomposed by microorganisms unless special precautions are taken. Methods ensuring the preservation of food products are of paramount importance. These issues are addressed by food microbiology.

Food products spoil not only due to microbial decomposition (resulting from aerobic oxidation or anaerobic putrefaction), but also through contamination with bacteria and fungi that produce toxins. Notable toxin producers include Clostridium botulinum and various staphylococcal species. Certain fungi produce mycotoxins, the most well-known being aflatoxin (a metabolite of the fungus Aspergillus flavus).

Both Physical and Chemical methods are used for preservation.

Physical methods. These primarily involve high-temperature sterilization. Metal cans are processed in an autoclave. For the preservation of acidic fruit juices, pasteurization is sufficient, as it destroys only vegetative cells while spores remain viable (endospores do not germinate in an acidic environment).

Fruit juices, mineral waters, and pharmaceutical preparations are sterilized by passing them through fine-pored asbestos or Cellulose filters. An old and widespread method of food preservation—drying—is based on the fact that Microbial growth requires a certain level of moisture (above 10%). Oatmeal, dried fruits, hay, and grain are preserved precisely because of their dry state.

Irradiation for food preservation is not yet widely adopted. UV rays are primarily used for air sterilization in dairies, bakeries, and other facilities. A reliable method that competes with salting even in domestic settings is low-temperature storage. In deep-freezing chambers, products are stored at minus 20 C and below.

Chemical methods. Acid-based preservation relies on the fact that very few microorganisms can grow at low pH in the absence of air. Heat-resistant spores do not germinate at a pH below 5.0. For the preparation of sauerkraut, silage, pickled cucumbers, and dry-cured sausages, natural acidification resulting from Lactic acid Fermentation is utilized. Acetic, citric, and lactic acids are also frequently added to products for preservation.

Meat and fish products are preserved by smoking. Smokehouse smoke contains products of thermal decomposition, such as phenols, cresols, and aldehydes, which possess inherent antiseptic properties.

For salting, products are submerged in a 14-25% salt solution. High concentrations of sugar (up to 50%) also inhibit microbial growth, which is the principle behind the preparation of marmalades, jams, and syrups.

Chemical preservatives are also used for food preservation. Sulfurous acid, diethyl pyrocarbonate, sorbic, benzoic, and formic acids are added to wine. Citrus fruits are treated with diphenyl and o-phenylphenol. Attempts are also being made to use Antibiotics for preservation.