The following points highlight the top four methods adopted for sterilization of bacteria. The methods are: 1. Heat Sterilization 2. Sterilization by Filtration 3. Sterilization by Radiation 4. Sterilization by Chemicals.
Method # 1. Heat Sterilization:
One of the common methods of sterilization is by application of heat. Bacteriological media, both liquid and solid, are generally sterilized by moist heat in an instrument called an autoclave which works in the same principle as a domestic pressure cooker.
The instrument is provided with a boiler for generation of steam which can be superheated to a temperature above. 100°C by increasing the inside pressure above the atmospheric level. A temperature of 120°C is reached when the pressure is about 2 atmospheres (15 lbs/sq. inch). In general, this temperature is adequate for killing all microorganisms in 15 to 20 min.
The higher pressure is necessary for raising the boiling point of water in the boiler. Pressure of this magnitude has no lethal power itself. The duration of autoclaving depends on the volume of the liquid to be sterilized. Normally, 15 min prove adequate if the volume does not exceed 200-250 ml. For larger volumes, a longer exposure becomes necessary.
Sometimes, culture media contain substances that are destroyed at 120°C and for such media autoclaving is not possible. An alternative to moist heat is the so-called Fractional sterilization or Tyndallization. In this method, the medium is exposed to steam at 100°C in a steamer for 30 min on three consecutive days. After each exposure, the flasks are incubated at 30°-37°C.
The basis of this procedure is that most common bacteria are killed at 100°C, except bacterial endospores, certain fungal spores and some yeast. During the intervening incubations these spores germinate and become vulnerable during the next exposure.
The flasks, tubes or any other vessels containing culture medium are plugged with absorbent cotton wool and the mouth is covered with brown paper before inserting them into an autoclave or steamer in order to protect the contents against contamination following sterilization.
Dry heat is used for sterilization of heat-stable laboratory glass-wares, like tubes, flasks, petridishes, pipettes etc. in a temperature-controlled hot-air oven. The articles are wrapped with brown paper and when necessary cotton plugged before sterilization at a temperature of 160°C for 2 hr. Microorganisms can withstand dry heat better than moist heat and that is why in a hot-air oven a higher temperature and longer exposure become necessary for complete sterilization.
Method # 2. Sterilization by Filtration:
Some culture media may contain substances which are extremely heat-labile. For sterilization of such media the sterile-filtration method has to be adopted. Different filtering materials are used for this purpose e.g. unglazed porcelain (Chamberland filter), diatomaceous earth (Berkefeld filter), asbestos (Seitz filter), cellulose (membrane filter), sintered glass etc.
The critical factor of any of these filters is the porosity. The pore size should be such as to prevent the passage of microbial cells of any type, though virus particles are too small to be arrested by such filters. Also, mycoplasmas which are cell- wall less bacteria can pass through filters because of their plasticity. In Chamberland filter a candle of unglazed porcelain, which is a thin tube with one end closed, is placed within a glass tube.
In Berkefeld filter a similar candle of diatomaceous earth is used. In Seitz filter (Fig. 7.14A) a disc of compressed shreds of asbestos is placed between the base and upper part of a stainless steel funnel. More popular and handy is a sintered glass filter (Fig. 7.14B) which is a glass funnel with a fused sintered glass disc.
Whatever may be the type of filter, it has to be fitted to a suction flask. The filtration unit is sterilized by heat before use. The liquid to be filtered is either sucked in or forced through the filter and transferred aseptically to a sterile container capped with cotton plug.
Method # 3. Sterilization by Radiation:
Both ionizing and non-ionizing radiations possess bactericidal properties. Among the ionizing radiations are X-rays, gamma rays, as well as the particulate radiations like alpha, beta and cathode rays. The non-ionizing radiation includes ultraviolet light. The lethal action of different kinds of radiations has been attributed to their interaction with nucleic acids and proteins.
The molecules of these important groups of biological polymers undergo drastic photochemical changes leading to derangement in structure and function. The lethality depends on the wave length of radiations. The shorter the wave length, more quantum energy they possess and greater is their devastating power.
Ultraviolet radiations (UV) having wavelengths below 300 nm have bactericidal effect. Due to the presence of aromatic amino acids — like tyrosine, phenylalanine and tryptophan — proteins absorb UV at 280 nm. A much stronger absorption peak between 250-265 nm is due to nucleic acids. Maximum germicidal effect is shown between 260-270 nm. The lethal action has been attributed to prevention of DNA replication mainly due to formation of thymine dimers.
UV is strongly absorbed by glass and liquids. Its germicidal efficacy is mainly restricted to the surface. UV is employed for minimizing airborne organisms in closed chambers, laminar air flows, operation theatres, hospital wards, animal rooms etc. While vegetative cells are killed rapidly, fungal and bacterial spores are more resistant. Low pressure mercury vapour lamps used as germicidal lamps emit nearly 90% of UV having wavelength of 254 nm.
The high-energy electromagnetic waves like X-rays and gamma rays ionize whatever molecules come on their way. They have much shorter wavelength and, therefore, are more penetrative and destructive than UV. However, the practical usefulness of these radiations is limited, mainly because their action depends on direct hits on molecules.
Partial sterilization of frozen food has been tried by irradiation with ionizing radiations. But the food often becomes unacceptable due to rancidity of fats, bleaching of meat, destruction of vitamins, degradation of carbohydrates and loss of characteristic flavour. Gamma rays have been used for sterilization of objects like disposable plastic goods, rubber materials and certain foodstuff which cannot be sterilized by application of heat. Cobalt 60 is used as source of gamma rays.
Method # 4. Sterilization by Chemicals:
Many different classes of chemical compounds possess antimicrobial property. The use of common salt and cane sugar for preservation of food by stopping microbial deterioration is known since antiquity. They prevent microbial growth by creating high osmotic pressure.
The chemicals used as sterilizing agents are variously termed disinfectants, antiseptics, and germicides. The mechanism of their bio-cidal activity depends on their chemical nature. An ideal agent would be expected to kill all microorganisms at a low concentration with the least harmful effects on animals and man.
Phenol and phenolic compounds are among the most well-known disinfectants. Phenol itself is a highly irritating and hazardous chemical. Its bactericidal property rests on its ability to denature proteins and to act as a detergent. More acceptable as antiseptics are the aliphatic homologues of phenol, like cresol, xylenol and thymol.
Thymol (5-methyl-2-isopropyl phenol) and eugenol (4-allyl-2-methoxy phenol) possess pleasant odour and they find use in mouth-washes. A halogenated derivative of phenol, hexachlorophene is widely known as a disinfectant. Alkyl esters of p-hydroxy-benzoic acid are used as food preservatives, because they are non-toxic due to their rapid hydrolysability.
Phenol is taken as a standard disinfectant for comparison of the germicidal activity of other compounds. The relative potency of other agents is expressed as phenol-coefficient. A standard procedure has been prescribed for determination of this coefficient. A standard inoculum of a standard strain of Staphylococcus aureus or Salmonella typhosa is introduced in a series of tubes containing different dilutions of phenol in a standard medium.
Same quantities of inoculum are also added to a series of tubes containing dilutions of the compound to be tested. All tubes are incubated at 20°C. After 5, 10 and 15 min, standard samples are withdrawn from each tube and inoculated into fresh medium without any disinfectant.
After incubation, it is determined which dilutions of phenol and the test compound have killed all organisms in 10 min, but not in 5 min. Supposing that the end-point dilution of phenol is 1:100 and that of the test compound 1:500, then the phenol-coefficient of that compound is 5 which would mean that the particular compound is five times more effective as a disinfectant than phenol.
Among halogens, iodine and chlorine are well known for their germicidal activity. Tincture of iodine (2% I2, 2.4% KI and 49% ethyl alcohol: USP) is widely used as an antiseptic for skin wound and mucous membrane. Iodine acts by combining with proteins through iodination of tyrosine residues.
It is also an oxidizing agent. Chlorine is a more powerful oxidant. It reacts with water forming hypochlorous acid which is responsible for its oxidizing activity. Cl2 + H2O —> HOCl + HCl. Chlorination of .drinking water is a common practice in many cities and towns. Chlorine reacts with NaOH to form sodium hypochlorite which is used for surface sterilization and also as a bleach. The disinfecting property of bleaching powder (CaOCl.Cl) in public sanitation is well known.
Alkylating agents, like formaldehyde (HCHO) and ethylene oxide, possess strong antimicrobial activity by virtue of their ability to react with the amino (NH2~) and hydroxyl (-OH) groups of proteins and nucleic acids causing their irreversible changes. Ethylene oxide is a useful sterilizing agent of foodstuff, pharmacological products, surgical instruments etc. Ethylene oxide is an explosive gas and, to reduce hazards, it is mixed with nitrogen or carbon dioxide.
Formaldehyde and its 37% aqueous solution, known as formalin, are well-known in biological laboratories as preservative and disinfectant. Formaldehyde gas is sometimes used as a fumigant for disinfection of closed chambers, like incubators, inoculation cubicles etc. As a germicide, it is effective on both vegetative cells as well as spores.
Detergents which may be anionic, non-ionic or cationic are surface-active agents. Cationic detergents possess germicidal property exerted by their ability to interact with cell membrane making it lose its osmotic control. As a result the cell loses its integrity.
The quaternary ammonium compounds among cationic detergents are well-known for their germicidal property, particularly those having alkyl and aryl substitutions. They are non-toxic to man and other animals and are effective at very low concentration (~1ppm). Some anionic detergents like sodium dodecyl sulfate or sodium tetradecyl sulfate are also effective bactericidal agents. Detergents are widely used for disinfecting skin and kitchen utensils.
Salts of some heavy metals, like HgCl2 and AgNO3, are strong germicides, but they are poisonous and irritating to man and other animals. Their antibacterial action is due to their great affinity for proteins, particularly for the sulfhydryl (-SH) groups of proteins.
Their direct use as disinfecting agents is practically obsolete, though use of some organic mercury compounds, e.g. mercurochrome, is still popular as they are non-toxic. Eye drops containing AgNO3 were used for disinfecting eyes of newborns to prevent gonococcal infections until the antibiotics appeared.