4 Main Methods of Sterilization | Organisms | Microbiology

Among the various methods followed for controlling microbial activity, the best by far is sterilization as it eliminates all the microbes. Sterilization is achieved by the following methods: 1. Physical Methods 2. Radiation Methods 3. Ultrasonic Methods 4. Chemical Methods.

1. Physical Methods:

Physical methods of sterilization include killing of microbes by applying moist heat as in steaming or dry heat as in a hot air oven or by various methods of filtration to free the medium of microbes. We shall study each one of them.

i. Physical Control with Heat:

The Citadel is novel by A.J. Cronin that follows the life of a young British physician, beginning in the 1920s. Early in the story the physician, Andrew Manson, begins his practice in a small coal­mining town in Wales. Almost immediately, he encounters an epidemic of typhoid fever.

When his first patient dies of the disease, Manson becomes terribly distraught. However, he realizes that the epidemic can be halted, and in the next scene, he is tossing all of the patient’s bed-sheets, clothing, and personal effects into a huge bonfire.

The killing effect of heat on microorganisms has long been known. Heat is fast, reliable, and relatively inexpensive, and it does not introduce chemicals to a substance, as disinfectants sometimes do. Above maximum growth temperatures, biochemical changes in the cell’s organic molecules result in its death.

These changes arise from alterations in enzyme molecules or chemical breakdowns of structural molecules, especially in the cell membranes. Heat also drives off water, and since all organisms depend on water, this loss may be lethal.

The killing rate of heat may be expressed as a function of time and temperature. For example, tubercle bacilli are destroyed in 30 minutes at 58°C, but in only 2 minutes at 65°C, and in a few seconds at 72°C. Each microbial species has a thermal death time (TDT), the time necessary for killing it at a given temperature. Each species also has a thermal death point (TDT), the temperature at which it dies in a given time.

In this method temperature is kept constant and time necessary to kill the cells is determined. The term thermal death point is no more practice. Since a particular temperature cannot be lethal all the times and also for all kinds of microorganisms.

Recently the heat sensitivity is defined using the term D value. D value is the exposure time at a given temperature required to reduce the number of viable organisms by 90%. Mathematically, it is equal to the reciprocal of the slope of the survivor curve or survivor curve to traverse one log cycle. D values can be used to determine the relative heat sensitivity of a microorganism to different temperature by calculation.

The z value is the temperature change needed to reduce the D value by one log cycle when log D is plotted against temperature. The F value is the D value at 250°F. These measurements are particularly important in the food industry, where heat is used for preservation.

In determining the time and temperature for microbial destruction with heat, certain factors bear consideration. One factor is the type of organism to be killed. For example, if materials are to be sterilized, the physical method must be directed at bacterial spores. Milk, however, need not be sterile for consumption, and heat is therefore aimed at the most resistant vegetative cells of pathogens.

Another factor is the type of material to be treated. Powder is subjected to dry heat rather than moist heat, because moist heat will leave it soggy. Saline solutions, by contrast, can be sterilized with moist heat but are not easily treated with dry heat.

Other factors are the presence of organic matter and the acidic or basic nature of the material. Organic matter may prevent heat from reaching microorganisms, while acidity or alkalinity may encourage the lethal action of heat.

ii. Direct Flame:

Perhaps the most rapid sterilization method is the direct flame method used in the process of incineration. The flame of the Bunsen burner is employed to sterilize the bacteriological loop before removing a sample from a culture tube and after preparing a smear. Flaming the tip of the tube also destroys organisms that happen to contact the tip, while burning away lint and dust.

In general, objects must be disposable if a flame is used for sterilization. Disposable hospital gowns and certain plastic apparatus are examples of materials that may be incinerated. In past centuries, the bodies of disease victims were burned to prevent spread of the pestilence.

It is still common practice to incinerate the carcasses of cattle that have died of anthrax and to put the contaminated field to the torch because anthrax spores cannot adequately be destroyed by other means. British law even stipulates that anthrax-contaminated animals may not be autopsied before burning.

iii. Hot-Air Sterilizer:

The hot-air-sterilizer utilizes radiating dry heat for sterilization. It is also called hot air oven. It is constructed with three walls and two air spaces. The outer walls are covered with thick asbestos to reduce the radiation of heat. A burner manifold runs along both sides and rear between the outside and the intermediate walls. Convection currents travel a complete circuit through the wall space and interior of the oven, and the products of combustion escape through an opening in the top.

The hot-air sterilizer is operated at a temperature of 160 to 180°C. (320 to 356°F.) for a period of 1½ hr. If the temperature goes above 180°C., there will be danger of the cotton stoppers charring. Therefore, the thermometer must be watched closely at first until the sterilizer is regulated to the desired temperature. The necessity of watching the sterilizer may be avoided by having the oven equipped with a temperature regulator.

The effect of dry heat on microorganisms is equivalent to that of baking. The heat changes microbial proteins by oxidation reactions and creates an arid internal environment, thereby burning microorganisms slowly. It is essential that organic matter such as oil or grease films be removed from the materials, because organic matter insulates against dry heat. Moreover, the time required for heat to reach sterilizing temperatures varies among materials. Thus this factor must be considered in determining the total exposure time.

The hot-air sterilizer is used for sterilizing all kinds of laboratory glassware, such as test tubes, pipettes, Petri dishes, and flasks. In addition, it may be used to sterilize other laboratory materials and equipment that are not burned by the high temperature of the sterilizer. Under no conditions should the hot-air sterilizer be used to sterilize culture media, as the liquids would boil to dryness.

iv. Arnold Sterilizer (Boiling Water):

Immersion in boiling water is the first of several moist-heat methods that we shall consider. Moist heat penetrates materials much more rapidly than dry heat because water molecules conduct heat better than air. Lower temperatures and less time of exposure are therefore required than for dry heat.

The Arnold makes use of streaming steam as the sterilizing agent. The sterilizer is built with a quick-steaming base that is automatically supplied with water from an open reservoir. The water passes from the open reservoir, through small apertures, into the steaming base, to which the heat is applied. Since the base contains only a thin layer of water, steam is produced very rapidly. The steam rises through a funnel in the center of the apparatus and passes into the sterilizing chamber.

Moist heat kills microorganisms by denaturing their proteins. Denaturation involves changes in the chemical or physical properties of proteins. It includes structural alterations due to destruction of the chemical bonds holding proteins in a three-dimensional form.

As proteins revert to a two-dimensional structure, they coagulate (denature) and become nonfunctional. Egg protein undergoes a similar transformation when it is boiled. You might find a review of the chemical structure of proteins, helpful to your understanding of this process. The coagulation and denaturing of proteins require less energy than oxidation, and therefore, less heat need be applied.

Sterilization is effected by employing streaming steam at a temperature of approximately 100°C. (212°F.) for a period of 20 min or longer on three consecutive days. The length of the heating period will depend upon the nature of the materials to be treated and the size of the container. Agar, for example, must be first completely melted before recording the beginning of the heating period.

It must be remembered that a temperature of 100°C. for 20 min. is not sufficient to destroy spores. A much higher temperature is required to effect a complete sterilization in one operation over a relatively short exposure period.

The principle underlying this method is that the first heating period kills all the vegetative cells present. After a-lapse of 24 hr. in a favourable medium and at a warm temperature, the spores, if present, will germinate into vegetative cells. The second heating will again destroy all vegetative cells.

It sometimes happens that all spores do not pass into vegetative forms before the second heating period. Therefore, an additional 24-hr. period is allowed to elapse to make sure that all spores have germinated into vegetative cells. It may be seen that unless the spores germinate the method will fail to sterilize.

v. Fractional Sterilization:

In the years before development of the autoclave, liquids and other objects were sterilized by exposure to free-flowing steam at 100°C for 30 minutes on each of three successive days. The method was called fractional sterilization because a fraction was accomplished on each day. It was also called tyndallization after its developer, John Tyndall and intermittent sterilization because it was a stop-and-start operation.

Sterilization by the fractional method is achieved by an interesting series of events. During the first day’s exposure, steam kills virtually all organisms except bacterial spores, and it stimulates spores to germinate to vegetative cells. During overnight incubation the cells multiply and are killed on the second day.

Again, the material is cooled and the few remaining spores germinate, only to be killed on the third day. Although the method usually results in sterilization, occasions arise when several spores fail to germinate. The method also requires that spores be in a suitable medium for germination, such as a broth.

Fractional sterilization has assumed importance in modern microbiology with the development of high-technology instrumentation and new chemical substances. Often, these materials cannot be sterilized at autoclave temperatures, or by long periods of boiling or baking, or with chemicals. An instrument that generates free-flowing steam, such as the Arnold sterilizer, is used in these instances.

vi. Pasteurization:

Pasteurization is not the same as sterilization. Its purpose is to reduce the bacterial population of a liquid such as milk and to destroy organisms that may cause spoilage and human disease. Spores are not affected by pasteurization.

One method for milk pasteurization, called the holding method, involves heating at 62.9°C for 30 minutes. Although thermophilic bacteria thrive at this temperature, they are of little consequence because they cannot grow at body temperature.

For decades, pasteurization has been aimed at destroying Mycobacterium tuberculosis, long considered the most heat-resistance bacterium. More recently, however, attention has shifted to destruction of Coxiella burnetii, the agent of Q fever, because this organism has a higher resistance to heat.

Two other methods of pasteurization are the flash pasteurization method at 71.6°C for 15 seconds, and the ultra-pasteurization method at 82°C for 3 seconds.

vii. Desiccation:

In addition to freezing, many foods are preserved by desiccation. Water is required for microbial growth. Although lack of available water prevents microbial growth, it does not necessarily accelerate the death rate of microorganisms. Some microorganisms, therefore, can be preserved by drying.

One can readily purchase active dried yeast for baking purposes and after the addition of water, the yeasts begin to carry out active metabolism. Freeze-drying or lyophilization is a common means of removing water that can be used for preserving microbial cultures. During freeze-drying, water is removed by sublimation. This process generally eliminates damage to microbial cells from the expansion of ice crystals.

Whereas some microorganisms are relatively resistant to drying, other microorganisms are unable to survive desiccating conditions for even a short period of time. For example, Treponema pallidum, the bacterium that causes syphilis, is extremely sensitive to drying and dies almost instantly in the air or on a dry surface.

The fact that microorganisms are unable to grow at low water activities can be used for the preservation of many products. Salting was one of the early means of preserving foods and is still employed today. By adding high concentrations of salt, the A_w is lowered sufficiently to prevent the growth of most microorganisms.

Canvas and other textiles are preserved in temperate zones by the lack of water in the air, but in tropical zones these same materials are subject to bio-deterioration because the humidity is sufficiently high to permit microbial growth. Exposed wood surfaces are often painted to keep the wood dry enough to preclude microbial growth. Many food products are also preserved by drying.

This preservation method depends on maintaining the product in a dry state, and exposure to high humidity can negate the factor limiting microbial growth and promote microbial spoilage of food products preserved in this manner. If food can be maintained at an A_w value of 0.65 or less, spoilage is unlikely for several years. Products preserved by drying include fruits, vegetables, eggs, cereals, grains, meat, and milk.

Physical Control by Other Methods:

Filtration:

Heat is valuable physical agent for controlling microorganisms but sometimes it is important to use. For example, no one would suggest removing the microbial population from a tabletop by using a Bunsen burner, nor can heat-sensitive solutions be subjected to an autoclave. In instances such as these and numerous others, a heat-free method must be used. This section describes some examples.

Filters came into prominent use in microbiology as interest in viruses grew in the 1890s. Previous to that time, filters had been utilized to trap airborne organisms and sterilize bacteriological media, but now they became essential of filter technology was Charles Chamberland, an associate of Pasteur. His porcelain filter was important to early virus research. Another pioneer was Julius Petri (inventor of the Petri dish), who developed a sand filter to separate bacteria from the air.

The filter is a mechanical device for removing microorganisms from a solution. As fluid passes through the filter, organisms are trapped in the pores of the filtering material. The solution that drips into the receiving container is decontaminated or, in some cases, sterilized. Filters are used to purify such things as intravenous solutions, bacteriological media, many pharmaceuticals, and beverages.

Several types of filters are available for use in the microbiology laboratory.

i. Porcelain or Chamberland Filters:

Porcelain filters are hollow, unglazed cylinders, closed at one end. They are composed of hydrous aluminium silicate or kaolin with the addition of quartz sand and are heated to a temperature sufficiently low to avoid sintering. These filters are prepared in graduated degrees of porosity, from LI to LI3.

Cylinders having the largest pores are marked LI; those having the smallest pores are designated LI3. The finer the pores, the slower will be the rate of filtration. The LI and L2 cylinders are preliminary filters intended for the removal of coarse particles and large bacteria. The L3 filter is probably satisfactory for all types of bacterial filtrations.

ii. Berkefeld Filters:

Kieselguhr is a deposit of fine, usually white siliceous powder composed chiefly or wholly of the remains of diatoms. It is also called diatomaceous earth and infusorial earth.

Berkefeld filters are manufactured in Germany. They are prepared by mixing carefully purified diatomaceous earth with asbestos and organic matter, pressing into cylinder form, and drying. The dried cylinders are heated in an oven to a temperature of about 2000°C. to bind the materials together. The burned cylinders are then machined into the desired shapes and sizes.

The cylinders are graded as W (dense), N (normal), and V (coarse), depending upon the sizes of the pores. The grading depends upon the rate of flow of pure filtered water under a certain constant pressure.

iii. Mandler Filters:

These filters are similar to the Berkefeld type but are manufactured in this country. They are composed of 60 to 80 per cent diatomaceous earth, 10 to 30 per cent asbestos, and 10 to 15 per cent plaster of Paris. The proportions vary, depending upon the sizes of the pores desired. The ingredients are mixed with water, subjected to high pressure, and then baked in ovens to a temperature of 980 to 1650°C. to bind the materials together.

The finished cylinders are tested by connecting a tube to the nipple of the filter, submerging in water, and passing compressed air to the inside. A gauge records the pressure when air bubbles first appear on the outside of the cylinder in the water. Each cylinder is marked with the air pressure obtained in actual test.

A convenient arrangement of apparatus for filtering liquids through a Mandler or Berkefeld filter is shown in Fig.3.10. The reduced pressure is indicated by the manometer. The liquid to be filtered is poured into the mantle, and the filtrate is collected in a graduated vessel, from which it may be withdrawn aseptically. Filtration may be interrupted at any time by stopping the vacuum pump and opening the stopcock on the trap bottle to equalize the pressure.

iv. Fritted-Glass Filters:

Filters of this type are prepared by fritting finely pulverized glass into disk form in a suitable mold. The pulverized glass is heated to a temperature just high enough to cause the particles to become a coherent solid mass, without thoroughly melting, and leaving the disk porous.

The disk is then carefully fused into a glass funnel and the whole assembled into a filter flask by means of a rubber stopper. Another arrangement is the coupling of the filter to the flask through a ground-glass joint thus eliminating the use of a rubber stopper.

The filters are marketed in five degrees of porosity as follows: EC (extra coarse), C (coarse), M (medium), F (fine), and UF (ultrafine).

Bacteriological filters are generally employed under conditions of reduced pressure; Bush (1946) recommended filtration through glass filters by the use of positive pressure. Positive pressure not only reduces or eliminates evaporation of the filtrate, but greatly facilitates the interchange of receivers— particularly important in bacteriological filtrations which must be handled aseptically.

A convenient arrangement is shown in Fig. 3.13. The main body of the filter B contains a fritted-glass disk. A shield A protects the receiver from dust, and a pressure head carries a stopcock. An alternate pressure permits the retention of pressure head C’ contains a built-in mercury manometer. The stopcock on C (C’) permits the retention of pressure after the apparatus is detached from the source of compressed air.

An ordinary rubber pressure bulb is satisfactory for producing pressures up to at least 450 mm. Mercury. If the ground-glass joints are well lubricated and the parts held together with strong rubber bands or springs, the apparatus should hold this pressure for days.

v. Asbestos Filters:

The best-known filter employing asbestos as the filtering medium is the Seitz filter. The asbestos is pressed together into thin disks and tightly clamped between two smooth metal rims by means of three screw clamps. The liquid to be filtered is poured into the metal apparatus, in which the asbestos disk is clamped, and the solution drawn through by vaccum, the filtering disks are capable of effectively retaining bacteria and other particulate matter.

At the end of the operation, the asbestos disk is removed, a new one inserted, and the assembled filter sterilized. This feature makes the Seitz filter very convenient to use, since no preliminary cleaning is necessary.

A modification of the Seitz filter, utilizing centrifugal force instead of suction or pressure, has been suggested by Boerner. The filter consists of a cylinder and a funnel-shaped part with stem, which holds the filter pad supported on a wire gauze disk. The cylinder screws into the funnel with the filter disk pressed between them.

The assembled filter fits closely into the top of a 15-ml. metal centrifuge tube, with the knurled collar of the funnel portion resting on the top of the metal tube. The filtrate is collected in a glass tube inside the cup. The filter can also be used for vacuum filtration in the conventional manner by inserting the stem through a rubber stopper fitted to a filter flask.

vi. Jenkins Filter:

This filter consists of a metal mantle holding a soft rubber sleeve and a porcelain filter block. The porcelain block is held in the rubber sleeve and made watertight by screwing together two metal parts. The filter block is not fragile. It is washed after each use, dried, and inserted in the mantle. The mantle is fitted with a rubber stopper, wrapped in paper, and sterilized in an autoclave.

The filter is designed to be used for the sterilization of small quantities of liquids.

vii. Ultrafilter:

Ultrafiltration generally means the separation of colloidal particles from their solvents and from crystalloids by means of jelly filters known a ultrafilter.

The early jelly filters were composed of gelatin and of silicic acid, but these have been replaced by collodion in membrane or sac form, or collodion deposited in a porous supporting structure. The supporting structure may be filter paper in sheet and thimble form, unglazed porcelain dishes and crucibles, Buchner funnels, filter cylinders, etc.

viii. Membrane Filter:

The membrane filter is a third type of filter that has received broad acceptance. It consists of a pad of organic compounds such as cellulose acetate or polycarbonate, mounted in a holding device. This filter is particularly valuable because bacteria multiply and form colonies on the filter pad when the pad is placed on a plate of culture medium.

Microbiologists can then count the colonies to determine the number of bacteria originally present. For example, if a 100-ml sample of liquid were filtered and 59 colonies appeared on the pad after incubation, it could be assumed that 59 bacteria were in the sample.

ix. Cleaning Filters:

Some filters are discarded after each use and new ones employed; others are intended to be cleaned after each filtration and, with proper care, may be used repeatedly. Collodion membranes are easily prepared, and the Seitz asbestos disks are relatively low in cost. These filters are intended to be used once, then discarded. On the other hand, porcelain, diatomaceous earth, and fritted-glass filters are too expensive to be used only once, but are easily cleaned.

Porcelain filters are cleaned by placing them in a muffle furnace and raising the temperatures to a red heat. This burns the organic matter in the pores and restores the filters to their original condition.

Filters of the Berkefeld and Mandler types are cleaned by placing the cylinders in a special metal holder connected to a faucet. The flow of water is reversed by passing through the cylinder from within outward. This should be continued until all foreign matter has been washed away from the filter pores.

Albuminous or similar materials remaining in the pores of the filters are likely to be coagulated by heat during the process of sterilization, with the result that the filters will be clogged. Filters in this condition are useless for further work.

Clogged filters may be cleaned in various ways but probably most conveniently by continuous suction of full-strength Clorox, or similar solution, for 5 to 15 min. This treatment quickly dissolves the coagulated material and restores the usefulness of the filter. Thorough washing is necessary to remove the last traces of the oxidizing solution.

Fritted-glass filters may be cleaned by treatment with concentrated sulfuric acid containing sodium nitrate. The strong acid quickly oxidizes and dissolves the organic matter. Thorough washing is necessary to remove the last traces of acid.

2. Radiation Method:

i. Sterilization by Ultraviolet Light:

Visible light is a type of radiant energy detected by the sensitive cells of the eye. The wavelength of this energy is between 400 and 800 nanometers (nm). Other types of radiations have wavelengths longer or shorter than that of visible light and therefore, they cannot be detected by the human eye.

One type of radiant energy, ultraviolet light, is useful for controlling microorganisms. Ultraviolet light has a wavelength between 100 and 400 nm, with the energy at about 265 nm most destructive to bacteria. When microorganisms are subjected to ultraviolet light, cellular DNA absorbs the energy, and adjacent thymine molecules link together.

Linked thymine molecules are unable to position adenine on messenger RNA molecules during the process of protein synthesis. Moreover, replication of the chromosome in binary fission is impaired. The damaged organism can no longer produce critical proteins or reproduce, and it dies quickly.

Ultraviolet light effectively reduces the microbial population where direct exposure takes place. It is used to limit airborne or surface contamination in a hospital room, morgue, pharmacy, toilet facility, or food service operation. It is noteworthy that ultraviolet light from the sun may be an important factor in controlling microorganisms in the air and upper layers of the soil, but it may not be effective against all bacterial spores. Ultraviolet light does not penetrate liquids or solids, and it may cause damage in human skin cells.

ii. Ionizing Radiation:

High-energy, short wavelength radiation disrupts DNA molecules, and exposure to short wavelength radiations may cause mutations, many of which are lethal. Exposure to gamma radiation (short wavelengths of 10^-3  – 10^-1 nanometers), X ray (wavelengths of 10^-3– 10² nanometers), and ultraviolet radiation (ultraviolet light with wavelengths of 100-400 nanometers) increases the death rate of microorganisms and is used in various sterilization procedures to kill microorganisms. Viruses as well as other microorganisms are inactivated by exposure to ionizing radiation.

Sensitivities to ionizing radiation vary. Resistance to ionizing radiation is based on the biochemical constituents of a given microorganism. Non-reproducing (dormant) stages of microorganisms tend to be more resistant to radiation than are growing organisms. For example, endospores are more resistant than are the vegetative cells of many bacterial species.

Exposure to 0.3-0.4 Mrads (million units of radiation) is necessary to cause a tenfold reduction in the number of viable bacterial endospores. An exception is the bacterium Micrococcus radiodurans, which is particularly resistant to exposure to ionizing radiation.

Vegetative cells of M. radiodurans tolerate as much as 1 Mrad of exposure to ionizing radiation with no reduction in viable count. It appears that efficient DNA repair mechanisms are responsible for the high degree of resistance to radiation exhibited by this bacterium.

Ionizing radiation is used to pasteurize or sterilize some products. Some commercially produced plastic petri plates are sterilized by exposure to gamma radiation. Most sterilization procedures involving exposure to radiation employ gamma radiation from cobalt-60 or cesium-137.

Bacon, for example, can be sterilized by radappertization, a process of sterilizing foods by exposure to radiation, using radiation doses of 4.5-5.6 Mrads. Hadurization, functionally equivalent to pasteurization, is used to kill non-spore-forming human pathogens that may be present in food. Radurization can be used to increase the shelf life of sea-foods, vegetables, and fruits.

Unlike gamma radiation, ultraviolet light does not have high penetrating power and is useful for killing microorganisms only on or near the surface of clear solutions. The strongest germicidal wavelength of 260 nanometers coincides with the absorption maxima of DNA, suggesting that the principle mechanism by which ultraviolet light exerts its lethal effect is through the disruption of the DNA. In fact, ultraviolet light causes the formation of covalently linked thymine dimers within the DNA in place of the normal thymine-adenine hydrogen bonded base pairs.

Microorganisms have enzymes that can repair the alterations in the DNA caused by exposure to ultraviolet light. The photo-reactivation enzymes require exposure to light in the visible spectrum. Exposure to ultraviolet light sometimes is used to maintain the sterility of some surfaces. In some hospitals bench-tops are maintained bacteria-free when not in use by using an ultraviolet lamp.

The dangers involved in human exposure to excess ultraviolet radiation, which include blindness if an ultraviolet light is viewed directly, have led to the use of alternative methods for maintaining the sterility of such areas.

Like ultraviolet radiation, long wavelength infrared radiation (103-105 nanometers) and microwave radiations (wavelengths greater than 106 nanometers) have poor penetrating power. Infrared and microwave radiations do not appear to kill microorganisms directly. Absorption of such long wavelength radiation, however, results in increased temperature.

Exposure to infrared or microwave radiations can thus indirectly kill microorganisms by exposing them to temperatures that are higher than their maximal growth temperatures. Because microwaves generally do not kill microorganisms directly, there is some concern in the food industry that cooking with microwave ovens may not adequately kill microorganisms contaminating food products.

3. Ultrasonic Method:

Ultrasonic Vibrations:

Ultrasonic vibrations are high-frequency sound waves beyond the range of the human ear. When directed against environment surfaces, they have little value because air particles deflect and disperse the vibrations. However, when propagated in fluids, ultrasonic vibrations cause the formation of microscopic bubbles, or cavities, and the water appears to boil. Some observers call this “cold boiling.”

The cavities rapidly collapse, and send out shock waves. Microorganisms in the fluid are quickly disintegrated by the external pressures. The formation and implosion of the cavities is known as cavitation.

Ultrasonic vibrations are valuable in research for breaking open tissue cells and obtaining their parts for study. A device called the cavitron is used by dentists to clean teeth, and ultrasonic machines are available for cleaning dental plates, jewelry, and coins. A major appliance company has also experimented with an ultrasonic washing machine.

As a sterilizing agent, ultrasonic vibrations have received minimal attention because liquid is required and other methods are more efficient. However, many research laboratories use ultrasonic probes for cell disruption and hospitals use ultrasonic devices to clean their instruments. When used with an effective germicide, an ultrasonic device may achieve sterilization, but the current trend is to use ultrasonic vibrations as a cleaning agent and follow the process by sterilization in autoclave.

4. Chemical Method:

i. Preservation Methods:

Over the course of many centuries, various physical methods have evolved for controlling microorganisms in food. Though valuable for preventing the spread of infectious agents, these procedures are used principally to retard spoilage and prolong the shelf life of foods, rather than for sterilization.

Drying is useful in the preservation of various metals, fish, cereals, and other foods. Since water is a necessary requisite for life, it follows that where there is no water, there is virtually no life. Many of the foods in the kitchen pantry typify this principle. One example is discussed in MicroFocus.

Preservation by salting is based upon the principle of osmotic pressure. When food is salted, water diffuses out of microorganisms to the higher salt concentration and lower water concentration in the surrounding environment. This flow of water, called osmosis, leaves microorganisms to shrivel and die.

The same phenomenon occurs in highly sugared foods such as syrups, jams, and jellies. However, fungal contamination may remain at the surface because aerobic molds tolerate high sugar concentrations.

Low temperatures found in the refrigerator and freezer retard spoilage by reducing the rate of metabolism in microorganisms and, consequently, reducing their rate of growth. Spoilage is not totally eliminated in cold foods, however, and many microorganisms remain alive, even at freezer temperatures. These organisms multiply rapidly when food thaws, which is why prompt cooking is recommended.

Note in these examples that there are significant differences between killing microorganisms, holding them in check, and reducing their numbers. The preservation methods are described as bacteriostatic because they prevent the further multiplication of bacteria.

ii. Gaseous Sterilization:

Heat sterilization is mostly unstable for thermolabile solid medicament and thermolabile equipment including articles of plastics, delicate rubber items. Because of high capital cost and use of elaborate precautionary measures, the radiation method which is one of the methods of sterilization has become unpopular.

Thus the sterilization of such materials with a chemical in gaseous state finds a greater application. Previously formaldehyde was widely used, but at present ethylene oxide is the only compound of outstanding importance in pharmaceutical and medical fields.