STERILIZATION / CONTROL OF MICROORGANISMS

One should remember that microorganisms are universally present and if given the opportunity they will contaminate everything, each and every equipments used for study. While isolating microorganisms for detailed study one must take utmost care to avoid contaminants.

One must remove or kill all microorganisms from equipments and media used for microbiological works to eliminate or reduce the possibility of unwarranted contaminants entering subsequently in them. All the equipments such as glassware, scalpels, needles, forceps, etc., and the media for culturing microorganisms must be sterilized thoroughly using principles of aseptic technique.

Definition of Terms in Sterilization

Sterilization: means complete destruction of all-forms of unwanted microbial life. A sterile object, in the microbiological sense, is free of all living microorganisms. The meaning of these terms, sterile and sterilization is absolute. There is no such thing as a "practically sterile" or "nearly sterile"; it is either sterile or it is not sterile.

Disinfection: means the destruction (killing) of pathogenic microorganisms.

Disinfectant: represents an agent, usually a chemical, which is used to destroy (kill) disease producing agents.
Germicide (microbicide): an agent that kills living microorganisms but not necessarily their resistant spores. In practice, a germicide is almost the same thing as a disinfectant but germicides are generally used for all kinds of microorganisms and at any place.

Bactericide (microbicide): an agent that kills bacteria.

Fungicide (adj. fungicidal): an agent that kills fungi.

Virucide (adj. virucidal): an agent that kills viruses.

Sporicide (adj. sporicidal): an agent that kills spores.

Sanitizer: is an agent that reduces the microbial population to safe levels. Usually it is a chemical agent that kills 99.9% of growing bacteria. They are generally applied to inanimate objects and are commonly used in the daily care of equipment and utensils etc.

Antiseptic: is an agent that opposes sepsis. The latter is a word derived from the Greek and means 'rotting, putrefaction, decay'. An antiseptic must have the property of preventing the multiplication of microorganisms (i.e., stops growth). It may be a much weaker agent than a disinfectant because the latter actually destroys microorganisms.

Bacteriostat (adj. bacteriostatic): an agent that stops growth of bacteria. Fungistat (adj. fungistatic): an agent that stops growth of fungi.

Inhibitor: an agent which merely slows, but does not necessarily prevents growth of the microorganisms.

Antimicrobial: an agent that interferes with the growth, i.e., slows the growth of microorganisms.

Antibacterial: an agent that slows the growth of bacteria.

Antifungal: an agent that slows the growth of fungi.

Antiviral: an agent that slows the growth of viruses.

Antispores: an agent that slows the growth of spores.

Antipathogen: an agent that slows the growth of pathogens.

METHODS OF STERILIZATION

There are many methods of sterilization but most of them fall under the broad classification of physical, chemical and gaseous ones.

A. Physical Methods of Sterilization

The physical method of sterilization includes high temperature, low temperature, dessication, radiation, filtration and osmotic pressure etc.

1. Heat / high temperature

1. 1. Dry Heating

(a) Flaming

Small objects, e.g., inoculating loops and needles that are not easily injured by heat are sterilized by thrusting them into the flame dull-red heating to a temperature high enough to destroy any organisms present upon the surface. In sterilizing the transfer needle, care should be taken to prevent 'spattering' (splashing), since the droplet which fly off are likely to carry viable organisms, Spattering can be greatly reduced or eliminated by drying the needle outside the flame before it is plunged into the flame.

(b) Hot-air sterilization

Autoclaving is not recommended for glassware sterilization since glassware is left wet after such sterilization. They are first dried properly, wrapped in brown paper and then exposed to hot-air in electric or gas oven to a temperature of 160°C for two hours. This treatment is sufficient for complete sterilization because at this temperature destruction of all living cells and viable spores due to destructive oxidation of the cell contents takes place. Uniform heating depends upon proper loading of the oven. A further rise in temperature may char the paper or cotton plug.

Hot-Air Sterilizer (Hot-Air Oven)

Hot-air sterilizer is an oven operated by electric power and used for sterilizing glasswares, e.g., Petri dishes, flasks, tubes, pipettes in microbiological laboratories. The walls of the sterilizer are made of stainless steel or aluminium and are so devised that the heat conduction from inside to outside is completely prevented.
The oven consists of a big chamber into which the materials to be sterilized are kept. A fan is set at the bottom of the oven which forces steam of hot dry air circulating through the chamber resulting in rise in chamber-temperature to sterilize the materials. A thermometer is fitted for recording the temperature of the oven. Temperature at 160°C sterilizes the glasswares in a period of two hours.

Hot air Sterilizer

1.	Exhaust	4.	Air Flow Sampler
2.	Diffusion Wall	5.	Turbo Blower
3.	Glass Wool Insulation	6.	Motor

1. 2. Moist Heating

Moist heating is more efficient in sterilization than is the dry heating because heat conduction is less rapid, the process takes much longer time and the death rate is lower in dry heat as compared to the moist heat.
Culture media, aqueous solutions, cloths, rubber and other materials that would be destroyed by dry heating are sterilized by moist heating. Following are the commonly used methods of moist heat sterilization.

(a) Streaming steam

Streaming steam or live steam is ordinarily accomplished in an Arnold Steam Sterilizer or some similar apparatus which allows the live steam to come in contact with the material to be sterilized. An Arnold sterilizer consists of a pan partially filled with water, a portion of which is between the layers of the double bottom. This water between the bottoms soon reaches boiling temperature when placed over a flame and is replaced through suitable openings in the pan above as rapidly as it evaporates.

The steam arises through the large opening in the centre and escapes finally through the opening in the top or around the doors. Live steam has a temperature of 100°C and a single exposure for 90 minutes can satisfactorily sterilize the materials.

(b) Tyndallization or Intermittent sterilization

Certain media containing gelatin, milk and sugars get adversely affected by heating at high temperature and it would, therefore, be prudent to use intermittent (also called 'fractional') sterilization with the help of earlier described Arnold Steam Sterilizer. This method involves heating the material at 100°C for 30 minutes on three successive days with incubation periods in between.

Resistant spores germinate during the incubation periods; the newly formed vegetative cells get destroyed on subsequent exposure to heat. The disadvantage of this process is that it is time consuming and non-nutrient solution can not be sterilized by this method because resistant spores may not germinate but remain dormant in such solution.

(c) Steam under Pressure

This method is useful for sterilization of media as well as apparatus. The laboratory apparatus designed to use steam under regulated pressure is called an autoclave. The autoclave is an essential unit of equipment in every microbiological laboratory.

Autoclave
Autoclave is laboratory equipment used to sterilize media and apparatus by steam under pressure technique. There are various types of autoclaves; the principle of the operation is same in every kind. The steam is allowed to form in the inner cylinder by hearing water. The steam pressure inside the cylinder increases with heating time. The rise in pressure is indicated by a pressure gauge.

The steam pressure increases the temperature inside to a desired level. The pressure most commonly allowed to develop inside is 15 pound (lb)/inch² in excess of the atmospheric pressure which is equivalent to a temperature of 121 °C (250°F) at sea level.

Exposure for 15 minutes at this temperature is sufficient to sterilize any medium if all the precautionary measures are taken. However, the time of operation at a temperature or pressure level to achieve complete sterility depends upon the nature of materials being sterilized, the type of container and the volume of the materials.

Autoclave

Principle
The steam is allowed to form in the inner cylinder of the autoclave by heating water. The steam pressure inside the cylinder increases with the time of heating and the steam value is shut off. The pressure developing inside can be read out by a pressure gauge.

Generally the autoclave is operated at a pressure of approximately 15 Ib/in2 (121°C). The time of operation to achieve sterility depends on the nature of the material being sterilized, the type of container, and the volume. For example, test tubes of liquid media can be sterilized in 10-15 minutes at 15 lb/in2 (121°C) the same media in 10 liter quantities would require an hour or more at the same temperature for complete sterilization.

After the desired time of exposure to steam under pressure, the supply of heat is cut of and steam pressure in the autoclave allowed to come down to zero before opening the lid for removal of sterilized materials.

Sterilization of test tubes containing liquid media

Exposure periods required for aqueous solution or liquids in various containers affording a reasonable factor of safety for sterilization by autoclaving

Contain Container	Diameter	Minutes exposure at 250-254°F(121-123°C)
Test tubes	18 × 150 mm 32 × 200 mm 38 × 200 mm	13-17 12-14 15-20
Erlenmeyer flask	50ml 500ml 1000ml 2000 ml	12-14 17-22 20-25 30-35
Fenwal flask	500 ml 1000 ml 2000 ml	24-28 25-30 40-45
Milk-dilution bottle	200 ml	13-17
Serum bottle	9000 ml	50-55

Source: J.J, Perkins, Principles and Methods of Sterilization. Charles C. Thomas. Springfield. III., 1956.

Precautions
1.It is important to note that it is not the pressure rather it is the high temperature of the steam that kills the organisms. Therefore, the air in the chamber of autoclave must be completely replaced by the pure steam. If air is present, it will reduce the temperature of the steam attained within the chamber substantially below. This can be done by keeping the steam outlet (in some autoclaves 'exhaust valve') open until pure steam starts going out.

2. Too much loading must be avoided because this would prevent proper circulation of steam.

3. When the pressure or temperature reaches the required level, i.e., 15 Ib/in2 (121°C temp.) time counting should be started. The required 15 Ib/in2 pressure (121°C temp.) must be maintained constantly for the required period of time.

2. Low temperature

Mode of Action: The effect of low temperatures on microorganisms depends on the particular microbe and the intensity of application. For example, at temperatures of ordinary refrigerators (0 ˚ C), the metabolic rate of some microbes is so reduced that they cannot reproduce or synthesize toxins. In other words, ordinary refrigeration has a bacteriostatic effect, but does not kill many microbes. Heat is much more effective than cold at killing microorganism.

Disadvantage:

Yet psychrotrophs do grow slowly at refrigerator temperatures and will alter the appearance and taste of foods after a time. For example, a single microbe reproducing only three times a day would reach a population of more than 2 million within a week.

Advantage by Medical Point of View:

Pathogenic bacteria generally will not grow at refrigerator temperature.

Uses of Cold temperature: Refrigeration is used to prevent food spoilage. Freezing, drying, and freeze-drying are used to preserve both foods and microorganism, but these methods do not achieve sterilization.

Optimum Conditions: Surprisingly, some bacteria can grow at temperatures several degrees below freezing. Most foods remain unfrozen until -2oC or lower. Rapidly attained subfreezing temperatures tend to render microbes dormant but do not necessarily kill them. Slow freezing is more harmful to bacteria; the ice crystals that form and grow disrupt the cellular and molecular structure of the bacteria. Thawing, being inherently slower is actually the more damaging part of a freeze-thaw cycle. Once frozen, one third of the population of some vegetative bacteria might survive a year, whereas other species might have very few survivors after this time.

Results of Low Temperature Treatment: Many eukaryotic parasites, such as the roundworms that cause trichinosis, are killed by several days of freezing temperatures.

Conditions: Many fresh foods can be prevented from spoiling by keeping them at 5 ° C (ordinary refrigerator temperature).

Limitations: However, storage should be limited to a few days because some bacteria and molds continue to grow at this temperature. To convince yourself of this, recall some of the strange things you have found growing on left over of the back of your refrigerator. In rare instances strains of Clostridium botulinum have been found growing and producing lethal toxins in a refrigerator when the organism were deep within a container of food, where anaerobic conditions exist.

2. 1. Refrigeration

Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.

2. 2. Cryopreservation

Cryopreservation (i.e., freezing in liquid nitrogen at -196°C) helps survival of pure cultures for long storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol that prevent the formation of ice crystals and promote cell survival.

3. Filtration

When ingredients of a culture medium are thermolabile. i.e., destroyed by heat, the use of heat sterilization is not practicable. For instance, biological fluids such as solutions of antibiotics, vitamins, tissue extracts, animal serum, etc. come under this category. In such cases, however, the process of filtration is used. The filters suitable for the purpose are Seize filter (Asbestos filter), Chamberland-­Pasteur filter (Porcelain filter), Berkefeld filter (Diatomaceous earth filter) and Membrane or Molecular filter. The first three filters are bacteriological filters. i.e., they allow liquid to pass but retain bacteria.

Contrary to this, the membrane filters retain all forms of organisms whatever small they may be (even viruses). The mean pore diameter in these filters ranges from one to several micrometers. These filters do not merely serve the mechanical prevention but other factors such as electric charges of the filter, electric charge of the microorganisms and the nature of the fluid being filtered. HEPA (High Efficient Particulate Air) filter and laminar air flow are commonly used in lab for filtering the incoming air and outgoing air respectively. HEPA filter prevents the income of 0.3 microns and large size particles to enter.

4. Radiations

Energy is transmitted through space in a variety of forms generally called 'radiations'. Some of the radiations viz., ultraviolet light, X-rays, gamma rays are used in sterilizing microorganisms particularly heat-sensitive ones. This method of sterilization is referred to as "cold sterilization" and is ideal for disposable materials made up of plastics, wool, cotton, etc which can be sterilized using a high dose or irradiation without altering the material. For others, complete sterilization is difficult without causing changes in color and flavor of the materials which occur at higher doses of radiations. Radiations like UV rays are non-ionizing radiation and those of X-rays and gamma rays are ionizing radiations.

4. 1. Ultraviolet Lamps

The ultraviolet portion of spectrum includes all radiations from 150 to 3900 A wavelengths. Wavelengths around 2650 A have the highest bactericidal efficiency. Many lamps such as 'germicidal lamps' are now available which emit a high concentration of ultraviolet light in the most effective wavelength range of 2600 to 2700 A.
The germicidal lamps arc widely used to kill or reduce the number of viable microorganisms to sterilize microbiological laboratories hospital operating rooms, and especfic filling rooms in various industries. Ultraviolet light has very little ability to penetrate matter; it cannot penetrate even into liquid. Therefore, only the microorganisms present on the surface of an object are destroyed by intense and prolonged exposure to it.

4. 2 Gamma rays and X-rays

Production of Gamma rays: Gamma rays are emitted by certain radioactive elements such as cobalt, and electron beams are produced by accelerating electrons to high energies in special machines.

Production of X rays: X rays, which are produced by machines in a manner similar to the production of electron beams, are similar in nature to gamma rays.

Mode of Action: Both, X rays and Gamma rays have wavelength shorter than the wavelength of ultraviolet light. X rays, which have wavelength of 0.1 to 40 nm, and gamma rays, which have even shorter wavelength, are forms of ionizing radiation, so named because it can dislodge electrons from atoms, creating ions. (Longer wavelengths comprise nonionizing radiation.) These forms of radiation also kill microorganisms and viruses and ionizing radiation damages DNA and produces peroxides, which act as powerful oxidizing agents in cells. This radiation can also kill or cause mutations in human cells if it reaches them.

Advantage of Gamma rays:

Gamma rays penetrate deeply but may requite hours to sterilize large masses.

High energy Electron Beams:

Effectiveness of Electron Beam:

High energy electron beams have much lower penetration power but usually require only a few seconds of exposure.

Application of Method: The food industry has recently renewed it interest in the use of radiation for food preservation. It can be used to prevent spoilage in seafood by doses of 100 to 250 kilorads, in meats and poultry by doses of 50 to 100 kilorads, and in fruits by doses of 200 to 300 kilorads. (one kilorad equals 1000 rads) many consumers in the United States reject irradiation foods for fear o receiving radiation, but such foods are quite safe free of both pathogens and radiation. In Europe, mil and other foods are often irradiated to achieve sterility. Especially high energy electron beams, is used for the sterilization pharmaceuticals and disposable dental and medical supplies, such as plastic syringes, surgical gloves, suturing materials, and catheters. As a protection against Bioterrorism, the postal service often uses electron beam radiation to sterilize certain classes of mail. These radiations can be used to differentiate between Gram positive and negative bacteria. Gram-positive bacteria are more sensitive to ionizing radiations than gram-negative bacteria. Ionizing radiations are currently used to sterilize such heat sensitive pharmaceuticals as vitamins, hormones, and antibiotics, as well as certain plastics and suture materials.

5. Dessication

Mode of Action: In the absence of water, a condition known as desiccation, microorganisms cannot grow or reproduce but can remain viable for years. Then, when water is made available to them, they can resume their growth and division. This ability is used in the laboratory when microbes are preserved by lyophilization, or freeze-drying. Certain foods are also freeze-dried (for example, coffee and some fruit additives for dry cereals).

Resistance and Desiccation: The resistance of vegetative cells to desiccation varies with the species and the organism's environment. For example, the gonorrhea bacterium can withstand dryness for only about an hour, but tuberculosis bacterium can remain viable for months. Viruses are generally resistant to desiccation, but they are not as resistant as bacterial endospores, some of which have survived for centuries. This ability of certain dried microbes and endospores to remain viable is important in a hospital setting. Dust, clothing, bedding, and dressing might contain infectious microbes in dried mucus, urine, pus, and feces.

5. 1. Lyophilization (Freeze-Drying)

In this method, the culture is rapidly frozen at a very low temperature (-70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators. Freeze-drying method is the most frequently used technique by culture collection centers.

6. Osmotic Pressure

Mode of Action: The use of high concentrations of salts and sugars to preserve food is based on the effects of osmotic pressure. High concentrations of these substances create a hypertonic environment that causes water to leave the microbial cell; this effect is also called as plasmolysis. Loss of water severely interferes with cell function and eventually leads to cell death. This process resembles preservation by desiccation, in that both methods deny the cell the moisture it needs for growth.

Application of Osmotic Pressure: The use of sugar jellies, jams, and syrups or salts solution in curing meat and making pickles plasmolysis most organisms present and prevents growth of new organisms. A few halophilic organisms, however, thrive in these conditions and cause spoilage, especially of pickles, and some fungi can live on the surface of jams.

Limitations: As a general rule, molds and yeasts are much more capable than bacteria of growing in materials with low moisture or high osmotic pressures. This property of molds, sometimes combine with their ability to grow under acidic conditions, is the reason fruits and grains are spoiled by molds rather than by bacteria. It is also part of the reason molds are able to form mildew on a damp wall or a shower curtain.

7. Electricity

High and low frequency electric currents are used for the process of sterilization. The passage of a current through liquid containing microorganisms kills a considerable portion of the microbial flora of the liquid. Since no complete sterilization is achieved, the application of electricity in the process of sterilization has been limited in pasteurization of milk and fruit juices and disinfection of water.

B. Chemical Methods of Sterilization

A variety of non-volatile chemicals are generally used in the laboratory to sterilize discarded glassware, desk, hand gloves, etc. The primary objectives of the use of such chemicals are to kill potentially dangerous microorganisms present on such articles, and also to reduce the laboratory atmosphere from fungal spores. These are wide varieties of disinfectants; some important ones are as follows:

Halogens Chlorin Chlorine and its compounds Work as general disinfectant and sanitizer Iodine and Iodophores Antiseptic Alcohols Ethyl and Isopropyl Skin antiseptic Heavy metals Mercuric chloride and organomercurials Useful disinfectant for surface sterilization of bench tops, inanimate objects; orgnanomercurials as antiseptics for skin. Detergents Soap Roccal and Zephiran Disinfectants for utensils and glasswares; skin antisepetic. Phenolic compounds Lysol, Cresol etc. Germicidal agents, effective against wide range of microorganisms. Quarternary ammonium compounds Alkyl-dimethyl-benzyl-ammonium chloride etc. Skin antiseptic, disinfectant for utensils. Nitrates Silver nitrate Disinfectant for the surface of test materials Aldehydes Formaldehyde Use as microbiostatic; it is less commonly used because of its irritating vapour.