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The following points highlight the top three physical methods used to kill microorganisms. The physical methods are: 1. Heat (Temperature) Sterilization 2. Filtration 3. Radiation.
1. Heat (Temperature) Sterilization:
Fire and boiling water have been used for sterilization and disaffection since the time of the Greeks, and heating is still one of the most popular ways to kill microorganisms. Microorganisms grow over a wide range of temperatures, and every type of them has an optimum, minimum and maximum growth temperature.
Temperatures above the maximum generally kill microorganisms subject to the fact that the parameters that influence heat (temperature) sterilization are favourable. However, heat is employed either in dry state (dry heat sterilization) or in moist state (moist heat sterilization). High heat combined with high moisture is one of the most effective methods of killing microorganisms.
Parameters that Influence Heat (Temperature) Sterilization:
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Exposure to heat (temperature) is one of the ways to kill microorganisms and sterilize the inanimate substances. But heat (temperature) sterilization is dependent on certain parameters such as sensitivity of microorganisms to heat, effectiveness of heat to microorganisms, and role of water present in environment.
1. Sensitivity of microorganisms to heat:
When a microbial population is exposed to elevated temperature, all the cells do not die instantly but follow a constant exponential rate (i.e. inverse of exponential growth rate). Let us, examine a hypothetical case. Let the initial number of microorganisms be 106 in a product.
When this product is exposed to a high temperature for a minute, the reduction in the population number is 10-fold (105 survivors). After exposure to another minute, further 90% reduces (104 survivors). In this way the exposure to heat is continued and reduction in population number by 10-fold continues (Table 21.1).
Critical evaluation of the data reveals that, when the population of microbes is high, death occurs at relatively rapid rate. As the number of survivor decrease the death rate slows down. From the example, initially 90,000 organisms died after a minute exposure and to kill last 10 organisms it took another minute.
Thus the thermal death of microorganisms follows first order kinetics. It means that at any instant the rate of death is proportional to the concentration of organisms at that instant. If N0 is the initial number of microorganisms and alter me t, Nt is the number of organisms surviving, then death rate constant or inactivation constant K can be calculated as.
2. Effectiveness of heat to microorganisms:
Effectiveness of heat (or any lethal agent) to microorganisms can be studied by assessing the death of microorganism. The death of microorganisms in a microbial population is determined by assessing the reduction in the number of viable microorganisms. The microorganism is declared dead if it does not grow when inoculated into a medium that would normally support its growth.
Earlier, the effectiveness of heat to microorganisms was expressed in terms of thermal death point (TDP), which is the temperature necessary to kill a given number of microorganisms in a fixed time.
Since a particular temperature cannot be lethal all the times and also for all kinds of microorganisms, the thermal death point is no more in practice and has been replaced by a new method in which D, Z and F values are used.
D value denotes the exposure time at a given temperature to reduce the number of viable microorganisms by 90%, Z value is the temperature change needed to reduce the D value by one log cycle when log D is plotted against temperature, and the F value denotes the D value at 250°F. The D, Z and F values are useful in determining the total exposure time of temperature required for killing the microorganisms.
3. Role of water present in environment:
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Water plays very important role in the survival of microorganisms since most of their activities are performed in the aqueous environment. Elevated temperature combined with high humidity, therefore, becomes one of the most effective methods of killing microorganisms. Bacterial endospores are the most heat resistant and are able to survive heat that would otherwise kill vegetative cells of the same species.
High concentrations of sugars or salts decreases the water content of the medium and thus, increases the exposure time. Similarly, sterilization of dry objects requires longer time and high temperature than the wet objects (Table 21.2).
Moist Heat Sterilization:
Moist heat sterilization, i.e., sterilization by heat combined with moisture is one of the most effective methods of killing microorganisms. Moist heat kills microorganisms by coagulating their proteins and sterilizes the equipment’s, etc. quite rapidly.
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However, the moist heat sterilization can be done at temperature below 100°C, at 100°C (boiling water or free steam), and above 100°C under increased pressure at saturated steam (e.g., autoclave). The first two are used as disinfection devices and only the third one is suitable for sterilization and killing of bacterial endospores.
Moist Heat Sterilization below 100°C Temperature (Pasteurization):
Pasteurization is the process by which milk, cream, certain alcoholic beverages, vaccines, and eating utensils are sterilized using moist heat below 100°C temperature. Pasteurization kills microorganisms of certain types (usually pathogens and spoilage causing microorganisms) but does not kill all the microorganisms hence called ‘partial sterilization’.
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In the 1860s the French wine industry was plagued by the problem of wine spoilage, which made wine storage and shipping difficult. Pasteur examined spoiled wine under the microscope and detected microorganisms that appeared like the bacteria for lactic acid and acetic acid fermentations.
He then discovered that a brief heating at 55-60°C would destroy these microorganisms and preserve wine for long periods. In 1886 the German Chemists V.H. and F. Soxhler adapted the technique of pasteurization for preserving milk and reducing milk-transmissible diseases.
Moist Heat Sterilization at 100°C Temperature:
1. Boiling at 100°C:
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Boiling at 100°C for 5-10 minutes is supposed to kill non-spore forming and few spore forming microorganisms and is widely used in hospitals to sterilize objects such as needles, syringes, blades, etc. Boiled objects removed from the boiler should be allowed to dry before handling to avoid contamination by bacteria from the skin of the handler’s fingers.
Contaminated materials or objects exposed to boiling water cannot be sterilized with certainty. It is true that all vegetative cells will be destroyed within minutes by exposure to boiling water, but some bacterial spores can withstand this condition for many hours.
The practice of exposing instruments for short periods of time in boiling water is more likely to bring about disinfection (destruction of vegetative cells of disease-producing microorganisms) —Steam chamber rather than sterilization. Boiling water cannot be (and is not) used in the laboratory as a method of sterilization.
2. Steaming at 100°C:
Generally, the steaming at 100°C is carried out in a sterilizer called Arnold sterilizer —Water or some similar apparatus which allows the live steam to come in contact with the material to be sterilized. An Arnold sterilizer (Fig. 21.1) consists of a pan filled with water, a portion of which also fills the space between the layers of the double bottom.
This water between the bottom layers soon reaches boiling temperature (100°C) at normal atmospheric pressure when placed over a flame and is replaced through the suitable openings in the pan above as rapidly as it evaporates. The live steam (steam at temp, of 100°C) arises through the large opening in the centre and escapes finally in the top or around the doors.
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Two techniques of sterilization are employed using steam at 100°C:
(i) A single exposure at 100°C for 90 min and
(ii) The method of tyndallization (intermittent heating or fractional sterilization).
(i) Single exposure:
A single exposure of steam at 100°C for 90 minutes (including the heating up time) is used to sterilize culture media containing sugars. This treatment allows the survival of endospores of thermophilic and mesophilic bacteria but, practically, it rarely fails to sterilize materials due to the rare presence of endospores of these bacteria.
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(ii) Tyndallization:
Some microbiological media, solution of chemicals, and biological materials cannot be heated above 100°C without being damaged. If, however, they can withstand the temperature of free- flowing steam at 100°C temperature, it is possible to sterilize them employing tyndallization (also called fractional sterilization ox intermittent boiling). Tyndallization was discovered by John Tyndall, an English physicist, in the year 1877.
It is explained for tyndallization that bacteria exist in two forms: heat-labile forms (thermolabile) which could be killed by exposure to high temperatures, and heat-resistant forms which could not be killed by continuous boiling of the broth (liquid) and, after the broth cools, they result in microbial growth in such broths.
If such broths are subjected to intermittent boiling (discontinuous boiling) on 3 successive days for 20-45 minutes accompanied with incubation period in between two subsequent boiling’s, the process now popular as tyndallization or fractional sterilization, the heat-resistant forms of bacteria are killed and the broths become completely free of them, and do not show any microbial growth.
It so happens because the first boiling kills vegetative cells of bacteria but endospores remain as such. The endospores now germinate in cooled broth and produce new bacterial cells which are killed during further boiling, and so on.
However, the drawback of tyndallization is that thermophilic and anaerobic bacteria, whose endospores do not germinate in the broth during incubation period, generally escape killing during this process.
Moist Heat Sterilization above 100°C Temperature:
1. Moist heat under pressure—the autoclaving:
(i) Pressure-temperature relationship of steam under pressure:
Heat moisted with saturated steam under pressure destroys bacterial endospores. Steam is described as saturated when it is at a temperature corresponding, to the boiling point (100°C) and when it is subjected to pressure the temperature goes above 100°C. A relationship between pressure and temperature is shown in Table 21.3.
(ii) The heat energy:
The heat energy in the saturated steam is of two forms-sensible heat and latent heat. Total heat energy present in the saturated steam is sum of sensitive heat and latent heat. Sensible heat is the heat required to raise the temperature of water from 0°C to its boiling point (100°C), whereas the latent heat is the additional heat needed to convert water as its boiling point to steam at the same temperature.
The amount of energy required to raise the temperature of 1 lb. of water by 1°F is equal to 1 Btu (British thermal unit). Thus, sensible heat in 1b. of steam at 100°C is about 180 Btu and that of latent heat is 971 Btu. The latent heat is much higher than the sensible heat. A comparative data of temperature and total heat energy is given in Table 21.4.
(iii) Autoclave:
Autoclave (Fig. 21.2) is an equipment used to sterilize objects by steam under regulated pressure. It is essentially a double-jacketed steam chamber with devices which permit the chamber to be filled with saturated steam and maintained at a designated temperature and pressure for any period of time.
In autoclave, when the saturated steam strikes the comparatively cooler object put for sterilization, it immediately liberates all the latent heat and condenses into small volume (1ml of water from 865 ml steam at 121°C).
This not only increases the temperature of the striked surface but the condensates also retains the sensible heat preventing the fall in the temperature. Additional steam flows rapidly into the region of low pressure created due to condensation. Again the condensation of steam will occur to give its latent heat.
This cycle continues until the temperature of the object raises to that of the steam. After the desired time of exposure to steam under pressure the supply of heat is cut off and steam pressure in the autoclave allowed to come down to zero before opening the door to remove the sterilized articles.
The autoclave is an essential unit of equipment in every microbiology laboratory. Many media, solutions, discarded cultures, and contaminated materials are routinely sterilized using 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 to achieve sterility depends on the nature of material being sterilized, the type of the container, and the volume.
For instance, 1000 test tubes filled with 10 ml each of a liquid medium can be sterilized in 10 to 15 minute at 12PC; 10 litres of the same medium contained in a single container would require 1 hour or more at the same temperature to ensure sterilization. Table 21.5 shows the exposure time for aqueous solutions or liquids in various containers affording a reasonable factor of safety for sterilization by autoclaving.
In the operation of an autoclave the following precautions must be taken into account:
1. All air in the chamber of autoclave should be completely replaced by saturated steam by keeping the steam outlet (in some autoclaves ‘exhaust valve’) open unit such time when saturated steam starts going out.
It is absolutely essential because of the following reasons:
(i) The mixture of steam + air will lower the temperature,
(ii) Air hinders penetration of steam in the articles, and
(iii) Air being denser than steam will sink down and make a covering layer over the articles.
2. Too much loading must be avoided because this prevents proper circulation of steam.
3. When the pressure or temperature reaches the required level, time counting should be started and the same should be maintained constantly for the required period of time.
Dry Heat Sterilization:
Dry heat (or hot air) sterilization is recommended where it is either undesirable or unlikely that steam under pressure will make direct and complete contact with the objects to be sterilized.
This is true of certain items of laboratory glassware (e.g., Petridishes, pipettes) and objects like oils, powders, and similar substances. Many a time, the surfaces of some articles like inoculating needle, scalpel or mouth of culture tubes are contaminated with the microorganisms. Direct flaming or heating of such objects can be one of the methods of dry heat sterilization.
Similarly, if the objects arc of disposable type, such as dressings, fomites of patients, they can be directly incinerated and sterilization can be achieved. However, hot-air oven is the most widely used apparatus to carry out dry-heat sterilization.
Incineration:
Destruction of microorganisms by burning is practiced routinely in the laboratory when the inoculation (transfer) needle is introduced into the flame. When the inoculation (transfer) needle is placed in a flame, spattering may occur during which droplets fly off and spread viable microorganisms in the atmosphere.
The danger from spattering can be greatly reduced or eliminated by using a Bunsen burner (Fig. 21.3) or an electric heat coil. Bunsen burner is equipped with a tube and the inoculation needle is exposed to a flame within tubular space.
Incineration is used for the destruction of carcasses, infected laboratory animals, and other infected materials to be disposed of. Special precautions need to be taken to ensure that the exhaust fumes do not carry particulate matter containing viable microorganisms into the atmosphere.
Hot Air Oven:
The hot-air ovens (hot air sterilizers) are made up of stainless steel or aluminum chamber (Fig. 21.4). The chamber is enclosed within thick layer of glass- insulation that makes up the outer wall.
The door consists of similar type of insulation and generally of flanged type. The door has a lining of asbestos to ensure tight seal while closed. Thermostatically controlled heaters are fixed on the outer surface of the chamber (from three sides).
At the back of the chamber, fan is fitted to circulate the air inside the chamber. Generally, a baffle is present in front of the fan (Fig. 21.5). The hot-air oven selected for sterilization purpose must take care of two factors, every article inside must receive the correct exposure and the sterilizing temperature must be reached quickly and be maintained with little variation.
Sterilization in hot-air oven can be achieved by transfer of heat from the source to the articles by way of conduction, convection, and radiation. Not much of the heat gets transferred by conduction since the contact between the shelves and articles is limited (only bottom part of article has indirect contact with the hot shelves).
Though heat transfer by convection is achieved but the transferred heat is unable to increase and maintain the temperature of oven to the required extent because air is poor conductor of heat (low specific heat) and it takes longer time to heat up. Also air gives up its heat and cools down, when it strikes the cooler surface.
Thus, the majority of heat in oven is transferred by radiation. Heaters are fitted all over the chamber to produce radiation heat. Also, a fan is fitted on the wall that forms the back of the chamber. In this way, air is heated quickly and heated air is circulated with the fan. Baffle allows uniform circulation of heated air and helps to remove cool air packets present in the chamber (usually at the corners).
The exposure time and temperature to achieve sterilization in hot-air oven vary depending on the type of microorganisms present in the load and nature of the load to be sterilized.
For instance, Clostridium titani, the cause of tetanus, is considered to be the pathogen most resistant to dry heat. Typically the thermal death times temperatures (TDT) are determined for this resistant microbial pathogen at a temperature range from 150°C to 190°C (Table 21.6).
In order to calculate total exposure time for sterilization cycle, a safety factor of 50% and the time for the temperature 10°C lower are added to the TDT. For example, if sterilization has to be carried out at 160°C then the exposure time will be 45 min and thus 1 hr exposure is sufficient to ensure sterilization. However, one must consider the stability of the product and should not expose it to the conditions greatly in excess of those needed to produce sterility.
Dry-air oven is suitable for sterilizing most of the glassware used in the microbiology laboratory such as flasks, beakers, tubes, and pipettes. Similarly, dry-air oven is useful for sterilization of materials, which are very sensitive to moisture such as dry powders, paraffin, wool alcohol, and other fat bases. But the method is highly unsuitable for materials that are thermolabile such as rubber, plastic, cotton, etc.
2. Filtration:
Many of the biological fluids (liquids) or gases that need to be sterilized cannot be done so by the application of heat. Their sterilization is achieved by filtration.
The following are the methods of filtration of biological fluids and air:
(i) Filtration of Biological Fluids (Biological Filters):
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.
Following are some important biological filters:
Seitz Filter (Asbestos filter):
This filter (Fig. 21.6) consists of 2-6 mm compressed asbestos fibre filter sheet. A variety of filter sheets containing different pore sizes are available in discs or squares ready for use and work satisfactorily only for a few hours. The medium to be filtered (sterilized) is poured into the funnel-like structure and drawn through filter sheet by vacuum. When the filtration is complete the filter sheet is discarded and the filtrate is obtained.
A modified Seitz filter in which vacuum-drawn filtrate technique has been replaced by centrifugal technique is also used now-a-days where the filter is mounted on a centrifuge which forces the filtrate into the tube.
Chamberland-Pasteur Filter. (Porcelain Filter):
These filters (Fig. 21.7), consists of hollow unglazed cylinders having a short open end. The cylinders are composed of oxides of silicon, aluminium, potassium and sodium with traces of oxides of iron, calcium and magnesium (the mixture commonly called ‘porcelain’).
The cylinders are baked at a temperature as high as possible without sintering the porcelain. These filters are prepared to various degrees of porosity from 0.65 to 15 µ and are used to remove bacteria and other coarse materials.
Berkefeld or Mandler Filter. (Diatomaceous earth filter):
These filters are widely used for keeping bacteria and other particles off the fluid. Cylindrical candles made up of ‘diatomaceous earth’ are used in it. Diatomaceous earth is a fine, usually white, silicious powder composed chiefly or wholly of the remains of diatoms. Actually, the cylindrical candles are prepared by mixing diatomaceous earth with asbestos and organic matter.
The mixture is subjected to high pressure and then baked in an oven at 1,000° to 2,000°C. The liquid to be filtered is poured into the mantle and it is filtered by applying vacuum. When filtration is complete the vacuum is released, filter is removed from the flask, and the filtrate is collected and sterilized.
Membrane or Molecular Filter:
A new type of filter (Fig. 21.8) called ‘membrane’ or ‘molecular’ filter has been developed in recent years. Unlike bacteriological filters which retain only bacteria, membrane filter retains all forms of microorganisms whatever their size be.
These filters, are made up of biologically inert cellulose esters, and are prepared as circular membranes of about 150 µm diameter consisting of millions of pores of an uniform and specifically predetermined size. Membrane filters were originally manufactured by the Millipore Filter Corporation (USA) and therefore they are also known as “Millipore” or ‘Ultra filters’.
At the time of filtration, membrane filters of various porosity are used on the principle of ‘graded filtration’ (Fig. 21.9) and finally fluid free of all organisms larger than 10 µm is obtained.
(ii) Filtration of Air:
The microbial population in the air becomes generally high in microbiological laboratories due to activities such as opening the culture plates, observing them under microscope, transfer, and inoculation of culture, etc. Air in such laboratories cannot always be made microbe free. To make air free from unwanted microbes, a device called ‘laminar air flow’ is used at appropriate times in the laboratories.
The laminar air flow system (Fig. 21.10) is used for reducing the danger of infection while working with pathogenic microbes. It works on the principle of application of fibrous filters in air filtration. In this system, air of a closed cabinet or room is made to pass through high efficiency particulate air filters (HEPA).
The HEPA filters the air and does not allow any suspended particle above 0.3 mm dimension to go out and as such the air is free of all suspended particles (above 0.3 mm size). The laminar air flow apparatus sucks the air in the room continuously and blows out-the air through a pack of filters. The air is blown out uniform velocity and in parallel flow line.
There are two models of laminar air flow hoods (system), horizontal and vertical. Both are same in principle and construction except for their orientation. Laminar air flow systems are an integral part of microbiological laboratory, hospital rooms, pharmaceutical laboratories, electronic industries (computer rooms), etc.
In microbiological laboratories, all operations involving inoculation and transfer of cultures, opening of lyophilized cultures, etc. can be performed in open without necessitating a closed chamber. The working platform of a laminar air flow system always has a particle free (above 0.3 µm) air providing a microbe free environment.
3. Radiations:
Radiation refers to the transmission of energy in a variety of forms through space or through a medium. The most effective type of radiation to sterilize or reduce the microbial burden in almost any substance is through the use of electromagnetic radiation. Various types of electromagnetic radiations are separated within the electromagnetic spectrum and such types are shown in Fig. 21.11 on the basis of their wavelengths.
The radiations of shorter wavelengths are more damaging to microorganisms. Thus, two types of radiations of primary interest in sterilization are—electromagnetic waves and streams of minute particles. The electromagnetic waves in decreasing orders of wavelengths are—infrared, ultra-violet light, X-rays and gamma rays, whereas the streams of minute particles of matter are alpha and beta radiation.
However, the sterilization by electromagnetic radiation is commonly called ‘cold sterilization’ and is ideal for disposable materials made up of plastics, wool, cotton, etc., which can be sterilized using a high dose of irradiation without altering the material. For others, complete sterilization is difficult without causing changes in colour and flavour of the materials which occur at higher doses of radiations.
Microbial cells possess various vital molecules, which are made up of atoms. An atom consists of a small nucleus surrounded by planetary electrons. When the electromagnetic rays and radiation particles pass through the matter, they give energy called radiant energy to the electrons of constituent atoms.
In this condition, two types of effects are seen:
(i) The electrons acquire a high energy to tear themselves free from the atoms in a process called ionization and
(ii) If the energy acquired is not high enough to cause ionization it raises the electrons to excessive energetic states and this process is called as excitation.
Both these effects can, however, cause death of microbial cell as destruction of vital molecules occur. The high-speed electron beams generated from machines, X-rays, and the gamma rays from radioactive isotopes cause ionization, whereas UV-rays cause excitation (non-ionization).
Several factors are found to be involved when one considers the effects of radiation on microorganisms. It has been reported that gram-positive bacteria are more resistant to the radiation than gram-negative bacteria. Spore formers are in general more resistant than non- spore formers. The number of organisms have the same effect upon the efficiency of radiation as it was in the case of heat.
The larger the number of cells, the less effective is the given dose. Similarly, microorganisms become less sensitive when suspended in medium containing proteins.
Proteins exert a protective effect against radiation. At the same time, presence of nitrites in the medium makes bacterial endospore more sensitive to radiation. The microorganisms show greater resistance to radiation in the absence of oxygen. Also, bacteria are most resistant to radiation in the lag phase and more sensitive in log phase.
The radiation suitable for commercial sterilization must have good penetrating power, high sterilizing efficiency, should cause minimum damage to irradiated materials and capable of being produced efficiently and continuously.
Ionizing radiation usually satisfies all these properties and thus, ionizing radiation are most powerful and widely used sterilizing agents. Two types of radiations are used in the process of sterilization, ionizing radiation and nonionizing (excitation) radiation.
These two radiations are the following:
(i) Ionizing Radiation:
Ionizing radiation is the radiation of very short wavelength or high energy which can cause atoms to loss electrons or ionize. Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. It destroys bacterial endospores and vegetative cells of both prokaryotes and eukaryotes. It is also effective sometimes against viruses. Ionizing radiation causes a variety of changes in the cells.
It breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, and polymerizes some molecules. Oxygen enhances these destructive effects, probably through the generation of hydroxyl radicals (OH).
Although many types of cell constituents can be affected by ionizing radiation, it is strongly advocated that destruction of DNA is the major cause of death. However, there are three forms of ionizing radiation that are often used in sterilization.
These forms are:
(i) Gamma rays,
(ii) X-rays, and
(iii) Electron beam radiation (cathode rays).
Gamma rays are emitted during radioisotope decay, the X-rays are artificially produced, and the electron beams are emitted by cathode.
1. Gamma Rays:
Gamma rays are generated from the radioactive isotope of Cobalt, 60CO. When the isotope disintegrates, it emits two gamma rays in succession (as cascade) each of which has mean energy of 1.25 MeV. The gamma rays cause both excitation and ionization by targeting vital molecules present in the cell. The death of a cell takes place because of ionization of target molecules (like water) present either inside or outside the cell.
The absorption of radiation by the water produces free radicals such as H2O+, OH–, H2O2 and HO2 (Fig. 21.12). All these radicals are powerful oxidizing agents, capable of destroying the other vital molecules and thus causing the death of the cell. The unit of sterilizing dose of gamma rays is Kilo Gray (1 Gray = 100 rad; 1000 Gray = 1 K Gray). The sterilizing dose is between 18-25 K Gray and the exposure time is about few seconds.
Application:
Several articles are regularly sterilized by gamma rays on a commercial scale. These include antibiotics, hormones, sutures, plastic syringes, cathetars, and other surgical instruments. Gamma rays have also been used to sterilize and pasteurize meat and other food and various agencies have approved food irradiation and declared it safe.
The U.S. government has currently approved the use of gamma rays radiation to treat poultry, beef, pork, lamb, fruits, vegetables, and spices. It is hoped that this will be more extensively employed in days to come.
Advantages and disadvantages:
The major advantage of gamma rays radiation is that all types of thermolabile substance can be easily sterilized. Moreover, aseptic handling is not necessary in the entire manufacturing process.
Sterilization can be performed after packing the material in final container or packing. However, the disadvantages of this method are as follows. Its capital and replacement costs are high. Also the method requires elaborate and expensive precautions to protect the operators.
2. X-rays:
X-rays, which are artificially produced by X-ray machines, possess considerable energy and penetration power and are lethal to microorganisms and higher forms of life (Table 21.8).
However, the use of X-rays is impractical for purposes of controlling populations of microorganisms because of two main reasons:
(1) They are very expensive to produce in quantity and
(2) Their efficient use is difficult as they are given off in all directions from their production sites.
In contrast to sterilization, the X-rays are widely used experimentally to cause mutations to generate microbial mutants.
3. Electron beam radiation (cathode rays):
When a high voltage is established between a cathode and an anode in an evacuated tube, the cathode emits beams of electrons, called electron beams or cathode rays. For this purpose specially designed equipment called ‘electron accelerator is used. This equipment produces electrons of very high intensities (millions of volts).
These high intensity electrons are accelerated to extremely high velocities. These beams of accelerated electrons of very high intensities produced by electron accelerator possess ability to kill microorganisms as well as they have other effects on biological and non-biological materials.
Electron accelerator is now used for sterilization of many surgical equipment’s, drugs, and other materials. The unique feature of electron beam radiation in that this process can sterilize the object at room temperature even after it has been packaged because the beams of electrons penetrate the wrappings.
Although the electron beam radiation possesses limited penetration power, it sterilizes the object on very brief exposure. Lethal doses of electron beams for different microorganisms in shown in Table 21.9.
(ii) Nonionizing Radiation:
1. Ultraviolet radiation:
The ultraviolet portion of the electromagnetic spectrum (Fig. 21.11) includes all radiations from 15-390 nm (150 – 3900 Å) and the UV-radiation around 260 nm (2600 Å) is the most lethal to microorganisms.
Although the radiant energy of sunlight is partly composed of ultraviolet light, most of the shorter wavelengths of this type are filtered out by the earth’s atmosphere (ozone layer, clouds and smoke) and, consequently, the ultraviolet radiation that reaches the earth’s surface is restricted to the span from about 260- 390 nm (2600-3900 Å).
Since the short wavelength and high energy UV radiation has very little penetrating ability, only the microorganisms present on the surface of an object where they are exposed directly to the ultraviolet radiation are susceptible to destruction.
Ultraviolet lamps, which emit ultraviolet radiations, are widely used for sterilization. In an ultraviolet lamp the ultraviolet radiation is generated by passing a low current at high voltage through mercury vapour in an evacuated tube. The tube is made up of borosilicate glass called as Vycor (quartz-like substance). The planetary electrons of the outer orbit of mercury atoms become excited and discharge their excess energy as UV radiation.
However, due to their non-penetrating ability, the UV radiation is often used as sterilizing agent only in a few specific situations. UV lamps are sometimes placed on the ceilings of rooms or in biological safety cabinets to sterilize the air and any exposed surfaces. Because UV radiation burns the skin and damages eyes, people working in such areas must be certain that the UV lamps are off when the areas are in use.
Mode of action:
Ultraviolet radiation is absorbed by many cellular materials but most significantly by the nucleic acids, where it does the most damage. The purine and pyrimidine bases of the nucleic acids absorb UV radiation around 260 nm (2600 Å) strongly. Although several effects of this absorption on purine and pyrimidine bases of nucleic acids are known, one well- established effect is the production in DNA of pyrimidine dimers.
This is a state in which two adjacent pyrimidine bases (cytosine or thymine) on the same strand of DNA become covalently bonded in such a way that the probability of DNA polymerase misreading the sequence during DNA replication is greatly increased thus resulting in inhibition of DNA replication and function.
Application:
Ultraviolet radiation from UV lamps is used extensively in hospital operating rooms, in aseptic filling rooms, in the pharmaceutical industry where sterile products are dispensed into vials or ampules, and in the food and dairy industries for treatment of contaminated surfaces. Commercial units containing UV lamps are available for water treatment. Pathogens and other microorganisms are destroyed when a thin layer of water is passed under the lamps.
