The following points highlight the top nine methods used for controlling Microbial Growth. The Methods are: 1. Sterilization by Heat 2. Sterilization by Dry Heat 3. Sterilization by Moist Heat 4. Steam Under Pressure: Autoclave 5. Tyndallization (Fractional Sterilization) 6. Pasteurization 7. Sterilization by Filtration 8. Radiation 9. Sterilization by Chemicals.
Control of Microbial Growth: Method # 1.
Sterilization by Heat:
Heat is most widely used lethal agent for purpose of sterilization. Articles may be sterilized by either dry heat, applied hot air oven or by moist heat, provided by steam. Moist heat is the most effective and efficient. It kills the microorganism by coagulating and denaturing their enzymes and proteins, this helps a lot by presence of water.
Spores are killed by exposure to moist heat at 121°C for 10-30 minutes. Dry heat kills micro-organism by oxidative destruction of the cell constituents. Spores are killed by exposure to dry heat at 160°C for 1-2 hours.
Control of Microbial Growth: Method # 2.
Sterilization by Dry Heat:
Dry heat is glass-wares or other heat stable solid material used to sterilize many object/articles in the absence of water. The items to be sterilized are placed in an oven at 160 to 170°C for 2 to 3 hours. Microbial death apparently results from oxidation of cell constituents and denaturation of protein.
Dry air heat is less effective than moist heat. Clostridium botulinum spores are killed in 5 minutes at 121°C by moist heat but only after 2 hours at 160°C with dry heat.
It does not corrode glassware and metal instruments as moist heat does and it can be used to sterilize powders, oils and similar items. Most laboratories sterilize glass, petridishes and pipettes with dry heat. Despite these above advantages dry heat sterilization is slow and not suitable for heat sensitive materials like many plastics and rubber items.
(I) Hot Air oven or Sterilizer:
Hot air oven can be used for heat sterilization, which is thermostatically controlled. The materials which can be sterilized by dry heat include glassware such as petridishes, pipettes, assembled glass syringes and metal instruments such as forceps, scalpels at temperature of 160 to 180°C for 1.5 to 3.0 hours. It sterilizes the materials by dry heating without air and takes longer time in comparison to moist heat.
(II) Incineration:
This process is used for destruction of microorganisms by burning in the laboratory when transfer needle is introduced into the flame of the Bunsen Burner. Incineration is used for the destruction of carcasses, infected laboratory animals, and other injected materials to be disposed of.
Special precautions need to be taken to ensure that exhaust fumes do not carry particulate matter containing viable micro-organisms into the atmosphere.
Control of Microbial Growth: Method # 3.
Sterilization by Moist Heat:
Micro-organism and their spores are destroyed at lower temperature by moist heat sterilization than by dry heat. However, Moist heat cannot be used to destroy microorganisms from water-proof materials such as oils and greases and to dry material in sealed containers.
Moist heat is used in sterilization of culture media and other liquids required to retain their water content. It can be achieved by autoclaving Pasteurization and Tyndallization.
Control of Microbial Growth: Method # 4.
Steam Under Pressure: Autoclave:
Microorganisms and their spores are destroyed by the use of saturated steam under pressure which provide temperatures above those attainable by boiling (Table). In addition, it has advantage of rapid heating, penetration and moisture in abundance the coagulation of protein.
Treatment is usually carried out in a metal vessel known as an autoclave, a device somewhat like a fancy Pressure cooker, which can be filled with saturated steam and maintained at desired temperature and pressure for any period of time.
Autoclaving provides moist heat at temperature higher than 100°C. Many media, solution, discarded cultures, and contaminated material are routinely sterilized with this apparatus. Generally, but not always, the autoclave is operated at a pressure of approximately 15 lb/in2 (at 121°C).
The time of operation depends on the nature of material being sterilized, type of container and the volume for example small volume of liquid can be sterilized by exposure for 15-20 minutes while when larger volumes are to be sterilized, the time of treatment must be extended.
The Fig. 6.2 Schematic diagram of a laboratory autoclave in use to sterilize microbial culture medium.
Control of Microbial Growth: Method # 5.
Tyndallization (Fractional Sterilization):
This method involves heating the object at 100oC for three successive days with incubation periods in between. Resistant spore germinates during incubation periods. The objects are steamed for 30-45 minutes on each of three successive days.
On the first occasion, vegetative bacterial cell, are killed. Resistant spore germinates during the incubation period in the medium over-night, producing vegetative form that is killed in second or third steaming. An apparatus known as steam arod is used for this technique.
Control of Microbial Growth: Method # 6.
Pasteurization:
Many substances such as milk, cream and certain alcoholic beverages (beer and wines are subjected with controlled heating at temperature below boiling, a process known as pasteurization which kills micro-organisms of certain types but does not destroy all organisms.
Pasteurization does not sterilize a beverage, milk and cream but it does kill any pathogens present and drastically slow spoilage by reducing the level of non-pathogenic microorganism. Table 6.1 shows the recommended use of heat to control bacterial growth.
Control of Microbial Growth: Method # 7.
Sterilization by Filtration:
Filtration is the process to reduce the microbial population in a solution. Standard bacteriological membrane filters are composed of porous membrane and made up of Cellulose acetate, Cellulose nitrate, poly carbonate or other synthetic material having pores.
Membrane with pore size about 0.2 pm in diameter are used to remove most vegetative cells but not viruses from solution. These filters are used to sterilize pharmaceuticals, opthalmic solutions, culture media, oils, antibiotics and other heat sensitive solutions.
Microorganisms such as bacteria, algae, yeast and molds can be separated from fluids, but viruses and mycoplasma cannot be eliminated. It is never possible that filtration procedure that renders a solution of bacterium free will also be free of viruses.
Control of Microbial Growth: Method # 8.
Radiation:
Energy transmitted through space in a variety of forms is called radiation. UV radiation and ionization radiations are most significant to sterilize or disinfect objects.
Ultraviolet radiation around 2650 Å is quite lethal but does not penetrate glass, dirt films, water and other substances very effectively. Many lamps are available which emit a high concentration of ultraviolet light in the most effective region 2600 to 2700 Å. These are used for disinfection of ultra surfaces such as inoculation chambers after cleaning with disinfectants.
UV radiations are harmful to mankind burn the skin and damage eye. People working in such area must be certain that the UV lamps are off when the area is in use. The application of ultraviolet radiations has been made in irradiation of air with the wavelength of 2650 Å which is microbicidal but not so irritating. UV rays act by inducing thimine dimers in DNA and the interference with DNA replication of an organism.
Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. It will destroy bacterial endospores and vegetative cells, both procaryotic and eucaryotic. Gamma radiation from a radioactive element Cobalt-60 source is used in sterilization of antibiotics, hormones and plastic disposable supplies such as syringes. It is also used to sterilize and pasteurize meat and other foods.
Control of Microbial Growth: Method # 9.
Sterilization by Chemicals:
Chemical agents are used in disinfection antisepsis. Chemical agents are used to prevent microbial growth in food and certain chemicals are used to treat infectious diseases.
A disinfectant is used to free a substance from infections and is usually a chemical agent which destroys disease germs or other harmful microorganisms or inactivate viruses for example 95% of ethanole is germicides and 0.1% of HgCI2 solution is used for surface sterilization.
1. Ethylene Oxide:
(ETO) is one of the most commonly used forms of chemical sterilization. Due to its low boiling point 10.4°C at atmospheric pressure ETO behaves as a gas at room temperature. It chemically reacts with amino acids, proteins and DNA to prevent microbial reproduction.
ETO is used for cellulose and plastic irradiation and can be used for a wide range of plastics (e.g., Petridishes, pipettes, syringes and medical devices etc.) and other materials without affecting their integrity.
Ethylene oxide (C2H4O) however; should be used in special cases as the gas is explosive and toxic to man. It has been employed to disinfect viruses, bacterial, fungal cells etc. It, however, finds useful application in medical microbiology as it is also being used to disinfect, chemical respirators and several other medical equipment’s.
2. Ozone Sterilization:
It is being used as a disinfectant for water and food. In U.S. many municipalities are using ozone technology to purify water and sewage. The O3 is used in both gaseous and liquid forms as an antimicrobial agent in the treatment, storage and processing of foods, including meat, poultry and eggs.
3. Low Temperature Gas Plasma:
(LTGP) is used as on alternative to ethylene oxide. It uses a small amount of liquid hydrogen peroxide (H2O2) which is energized with radio frequency waves into gas plasma. This leads to the generation of free radicals and other chemical species, which destroy organisms.
Betapropiolactane
BPL also kills a wide variety of bacteria and fungi. It is used in a gaseous form (becomes liquid at 20°C), and in liquid form it can sterilize sera and vaccines.
It should, however, be noted that the disinfectants and antiseptics are distinguished on the basis whether they are safe for application to mucous membrane and safety often depends on the concentration of the compound.
