Physical methods of sterilization
Physical methods of sterilization involves the control of microbial growth by using Sunlight, Heat, Filtration, Radiation, etc.
Physical methods of sterilization include the following:
- Temperature (high and low).
- Osmotic pressure.
1. Heat or Temperature
- Microorganisms can grow over a wide range of temperatures, from very low temperatures characteristic of psychrophiles to the very high growth temperatures characteristic of thermophiles.
- Every type of microorganism has an optimum, minimum, and maximum growth temperature.
- Temperatures above the maximum generally kill, while those below the minimum usually produce stasis (inhibition of metabolism) and may even be considered preservative.
- In this method basically moist heat and dry heat is used to kill or inhibit the microorganisms.
- Moist heat kills microorganisms by coagulating their proteins and is much more rapid and effective than dry heat.
- Dry heat destroys microorganisms by oxidizing their chemical constituents.
- Two examples will illustrate the difference between moist heat and dry heat, such as; Spores of Clostridium botulinum are killed in 4 to 20 min by moist heat at 120°C, whereas a 2hour exposure to dry heat at the same temperature is required. Spores of B. anthracis are destroyed in 2 to 15 min by moist heat at 100°C, but with dry heat, 1 to 2 hours at 150°C is required to achieve the same result.
- Vegetative cells are much more sensitive to heat than are spores; the higher level of “water activity” in the vegetative cells accounts for this. Cells of most bacteria are killed in 5 to 10 min at 60 to 70°C (moist heat). Vegetative cells of yeasts and other fungi are usually killed in 5 to 10 min by moist heat at 50 to 60°C; their spores are killed in the same time but at temperatures of 70 to 80°C.
Destruction of Microorganisms by Heat or Temperature
In the laboratory, the Destruction of Microorganisms by heat is accomplished in these two following methods;
- Destruction of Microorganisms by Applying High Temperature.
- Destruction of Microorganisms by Applying Low Temperature.
A. Destruction of Microorganisms by Applying High Temperature.
Practical procedures by which heat is employed are conveniently divided into two categories:
- Sterilization by Moist Heat
- Sterilization by Dry Heat
a. Sterilization by Moist Heat
- Moist heat occurs in the form of hot water, boiling water, or steam (vaporized water).
- The temperature of moist heat usually ranges from 60 to 135°C.
- Moist heat destroys cells and viruses by degrading nucleic acids, denaturing proteins, and disrupting cell membranes.
- Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eukaryotic spores.
Sterilization by moist heat can be classified in the following groups:
- Sterilization at a temperature < 100°C
- Sterilization at a temperature of 100°C
- Sterilization at a temperature > 100°C
- Intermittent sterilization
(i) Sterilization at a temperature < 100°C / Pasteurization
Pasteurization is an example of sterilisation at a temperature 100°C.
- Pasteurization is a technique in which heat is applied to liquids to kill potential agents of infection and spoilage, while at the same time retaining the liquid’s flavor and food value.
- This technique is named after Louis Pasteur who devised this method.
- This method is extensively used for sterilization of milk and other fresh beverages, such as fruit juices, beer, and wine which are easily contaminated during collection and processing.
- Two methods of pasteurization are followed: flash method and holder method.
- In the flash method, milk is exposed to heat at 72°C for 15–20 seconds followed by a sudden cooling to 13°C or lower.
- In the holder method, milk is exposed to a temperature of 63°C for 30 minutes followed by cooling to 13°C or lower, but not less than 6°C.
- The flash method is preferable for sterilization of milk because it is less likely to change the flavor and nutrient content, and it is more effective against certain resistant pathogens, such as Coxiella and Mycobacterium.
- Although pasteurization inactivates most viruses and destroys the vegetative stages of 97–99% of bacteria and fungi, it does not kill endospores or thermoduric species (mostly nonpathogenic lactobacilli, micrococci, and yeasts).
- Newer techniques have now been used to produce sterile milk that has a storage life of 3 months. In this method, milk is processed with ultrahigh temperature (UHT) of 134°C for 1–2 seconds.
(ii) Sterilization at a temperature of 100°C
Sterilsation at a temperature of 100ºC is accomplished by these two following methods;
- Steam sterilizer at 100°C
- Sterilization by boiling is facilitated by addition of 2% sodium bicarbonate to water.
- Simple boiling of water for 10–30 minutes kills most of the vegetative forms of bacteria but not bacterial spores.
- Exposing materials to boiling water for 30 minutes kills most nonspore-forming pathogens including resistant species, such as the tubercle bacillus and staphylococci.
- Since boiling only once at 100°C does not kill all spores, this method cannot be used for sterilization but only for disinfection.
- Hence, it is not recommended for sterilizing instruments used for surgical procedure.
- The greatest disadvantage of this method is that the items sterilized by boiling can be easily recontaminated when removed from water after boiling.
Steam sterilizer at 100°C:
- Usually, Koch’s or Arnold’s steam sterilizer is used for heat-labile substances that tend to degrade at higher temperatures and pressure, such as during the process of autoclaving.
- These substances are exposed to steam at atmospheric pressure for 90 minutes during which most vegetative forms of the bacteria except for the thermophiles are killed by the moist heat.
(iii) Sterilization at a temperature > 100°C / steam under pressure
- This method is also known as sterilization by steam under pressure.
- A temperature of 100°C is the highest that steam can reach under normal atmospheric pressure at sea level. This pressure is measured at 15 pounds per square inch ( psi), or 1 atmosphere.
- In order to raise the temperature of steam above this point, it must be pressurized in a closed chamber. This phenomenon is explained by the physical principle that governs the behavior of gases under pressure. When a gas is compressed, its temperature rises in direct relation to the amount of pressure. So, when the pressure is increased to 5 psi above normal atmospheric pressure, the temperature of steam rises to 109°C. When the pressure is increased to 10 psi above normal, its temperature will be 115°C and at 15 psi (a total of 2 atmospheres), it will be 121°C.
- It is not the pressure by itself that is killing microbes, but the increased temperature it produces.
- Such pressure–temperature combinations can be achieved only with a special device that can subject pure steam to pressures greater than 1 atmosphere.
- Health and commercial industries use an autoclave for this purpose and a comparable home appliance is the pressure cooker.
- It is a good method to sterilize heat-resistant materials, such as glassware, cloth (surgical dressings), rubber (gloves), metallic instruments, liquids, paper, some media, and some heat-resistant plastics.
- It is also useful for sterilization of heat-sensitive items, such as plastic Petri plates that need to be discarded.
- It is useful for sterilization of materials that cannot withstand the higher temperature of the hot-air oven.
(iv) Intermittent sterilization
Certain heat-labile substances (e.g., serum, sugar, egg, etc.) that cannot withstand the high temperature of the autoclave can be sterilized by a process of intermittent sterilization, known as tyndallization.
- It is carried out over a period of 3 days and requires a chamber to hold the materials and a reservoir for boiling water.
- Items to be sterilized are kept in the chamber and are exposed to free-fl owing steam at 100°C for 20 minutes, for each of the three consecutive days.
- On the first day, the temperature is adequate to kill all the vegetative forms of the bacteria, yeasts, and molds but not sufficient to kill spores.
- The surviving spores are allowed to germinate to vegetative forms on the second day and are killed on re-exposure to steam.
- The third-day re-ensures killing of all the spores by their germination to vegetative forms.
- Intermittent sterilization is used most often to sterilize heat-sensitive culture media, such as those containing sera (e.g., Loeffl er’s serum slope), egg (e.g., Lowenstein–Jensen’s medium), or carbohydrates (e.g., serum sugars) and some canned foods.
Applications of Moist Heat Sterilization
- Used for sterilization of milk and other fresh beverages, such as fruit juices, beer, and wine which are easily contaminated during collection and processing.
- Used for disinfection of hospital’s utilities.
- Used to sterilize heat-resistant materials, such as glassware, cloth (surgical dressings), rubber (gloves), metallic instruments, liquids, paper, some media, and some heat-resistant plastics.
- It is also used for the sterilization of heat-sensitive items, such as plastic Petri plates that need to be discarded.
- Used to sterilize heat-sensitive culture media, such as those containing sera (e.g., Loeffl er’s serum slope), egg (e.g., Lowenstein–Jensen’s medium), or carbohydrates (e.g., serum sugars) and some canned foods.
|Autoclave||Sterilizing instruments, linens. utensils, and treatment trays. media and other liquids||Ineffective against organisms in materials impervious to steam; cannot be used for heat-sensitive articles|
|Free-flowing steam or boiling water||Destruction of nonsporeforming pathogens, sanitizes bedding. clothing, and dishes||Cannot be guaranteed to produce sterilization on one exposure|
|Pasteurization||used for sterilization of milk and other fresh beverages, such as fruit juices, beer, and wine||Doesn’t kill heat resistant pathogens. Reduction in the nutrition content, It kills pathogens. Enhances storage period.|
|Steam sterilizer||used for heat-labile substances||Deleterious for heat-sensitive instruments, Microsurgical instruments damaged by repeated exposure, May leave instruments wet, causing them to rust. Potential for burns.|
|Tyndallization||used most often to sterilize heat-sensitive culture media, such as those containing sera (e.g., Loeffl er’s serum slope), egg (e.g., Lowenstein–Jensen’s medium), or carbohydrates (e.g., serum sugars) and some canned foods.||–|
Approximate Conditions for Moist Heat Inactivation
|Yeasts||5 minutes at 50-60°C||5 minutes at 70-80°C|
|Molds||30 minutes at 62°C||30 minutes at 80°C|
|Bacteria||10 minutes at 60-70°C||2 to over 800 minutes at 100°C |
0.5-12 minutes at 121C
|Viruses||30 minutes at 60°C||–|
|Prions||90 minutes at 134°C||–|
b. Sterilization by dry heat
- Sterilization by dry heat makes use of air with a low moisture content that has been heated by a flame or electric heating coil.
- The temperature of dry heat ranges from 160°C to several thousand degrees Celsius.
- The dry heat kills microorganisms by protein denaturation, oxidative damage, and the toxic effect of increased level of electrolytes.
- Dry heat is not as versatile or as widely used as moist heat, but it has several important sterilization applications.
- The temperature and time employed in dry heat vary according to the particular method, but in general they are greater than with moist heat.
Sterilization by dry heat is accomplished by the following methods;
- Hot air oven.
- Sterilization of inoculating loop or wire, the tip of forceps, searing spatulas, etc., is carried out by holding them in the flame of the Bunsen burner till they become red hot.
- Glass slides, scalpels, and mouths of culture tubes are sterilized by passing them through the Bunsen flame without allowing them to become red hot.
- Incineration is an excellent method for safely destroying infective materials by burning them to ashes.
- Incinerators are used to carry out this process and are regularly employed in hospitals and research labs to destroy hospital and laboratory wastes.
- The method is used for complete destruction and disposal of infectious material, such as syringes, needles, culture material, dressings, bandages, bedding, animal carcasses, and pathology samples.
- This method is fast and effective for most hospital wastes, but not for metals and heat-resistant glass materials.
(iii) Hot-air oven:
- The hot-air oven is electrically heated and is fitted with a fan to ensure adequate and even distribution of hot air in the chamber.
- It is also fitted with a thermostat that ensures circulation of hot air of desired temperature in the chamber.
- Heated, circulated air transfers its heat to the materials inside the chamber.
- While sterilizing by hot-air oven, it should be ensured that the oven is not overloaded.
- The materials should be dry and arranged in a manner which allows free circulation of air inside the chamber.
- It is essential to fit the test tubes, flasks, etc., with cotton plugs and to wrap Petri dishes and pipettes in a paper.
- Sterilization by hot-air oven requires exposure to 160–180°C for 2 hours and 30 minutes, which ensures thorough heating of the objects and destruction of spores.
- Thermocouples, chemical indicators, and bacteriological spores of Bacillus subtilis are used as sterilization controls to determine the effi cacy of sterilization by hot-air oven.
- Hot-air oven are used for sterilization of: Glasswares (syringes, Petri dishes, flasks, pipettes, test tubes, etc.), Surgical instruments (scalpels, scissors, forceps, etc.), Chemicals (liquid paraffin, sulfonamide powders, etc.); and Oils that are not penetrated well by steam used in moist heat sterilization.
Applications of Dry heat Sterilization
- Used to sterilize Glass slides, scalpels, and mouths of culture tubes.
- Used to sterilize inoculating loop or wire, the tip of forceps, searing spatulas, etc.
- Used for complete destruction and disposal of infectious material, such as syringes, needles, culture material, dressings, bandages, bedding, animal carcasses, and pathology samples.
- Used for sterilization of Glasswares (syringes, Petri dishes, flasks, pipettes, test tubes, etc.), Surgical instruments (scalpels, scissors, forceps, etc.), Chemicals (liquid paraffin, sulfonamide powders, etc.); and Oils that are not penetrated well by steam used in moist heat sterilization.
|Hot-air oven||Sterilizing materials impermeable to or damaged by moisture, e.g., oils, glass, sharp instruments, metals||Destructive to materials which cannot withstand high temperatures for long periods|
|Incineration||Recomitended Uses Disposal of contaminated objects that cannot be reused.||Size of Incinerator must be ade quate to burn largest load promptly and completely: potential of air pollution.|
|Flaming||Sterilization of inoculating loop or wire, the tip of forceps, searing spatulas, etc.,||–|
|Incineration||used for complete destruction and disposal of infectious material, such as syringes, needles, culture material, dressings, bandages, bedding, animal carcasses, and pathology samples||not effective for metals and heat-resistant glass materials.|
B. Destruction of Microorganisms by Applying Low Temperature
- Temperatures below the optimum for growth depress the rate of metabolism, and if the temperature is sufficiently low, growth and metabolism cease.
- Low temperatures are useful for preservation of cultures, since microorganisms have a unique capacity for surviving extreme cold.
- Microorganisms maintained at freezing or subfreezing temperatures may be considered dormant; they perform no detectable metabolic activity.
- Preservation by Freeze Drying or Byophilization is an example of Destruction of Microorganisms at a Low Temperature.
Applications of Low Temperature Sterilization
- Agar-slant cultures of some bacteria, yeasts, and molds are customarily stored for long periods of time at refrigeration temperatures of about 4 to 7°C.
- Many bacteria and viruses can be maintained in a deep-freeze unit at temperatures from -20 to – 70°C.
- Liquid nitrogen, at a temperature of -196°C, is used for preserving cultures of many viruses and microorganisms, as well as stocks of mammalian tissue cells used in animal virology and many other types of research.
- Desiccation of the microbial cell causes a cessation of metabolic activity, followed by a decline in the total viable population.
- In general, the time of survival of microorganisms after desiccation varies, depending on the following factors: The kind of microorganism, The material in or on which the organisms are dried, The completeness of the drying process, and The physical conditions to which the dried organisms are exposed.
- Species of Gram-negative cocci such as gonococci and meningococci are very sensitive to desiccation; they die in a matter of hours. Streptococci are much more resistant: some survive weeks after being dried.
- The tubercle bacillus (Mycobacterium tuberculosis) dried in sputum remains viable for even longer periods of time. Dried spores of microorganisms are known to remain viable indefinitely.
- Lyophilization is one of the methods of Desiccation.
- In the process of lyophilization, organisms are subjected to extreme dehydration in the frozen state and then sealed in a vacuum. In this condition, desiccated (lyophilized) cultures of microorganisms remain viable for many years.
- In Pharmaceutical and biotechnology, it is used to increase the shelf life of products, such as vaccines and other injectables.
3. Osmotic Pressure
- When two solutions with differing concentrations of solute are separated by a semipermeable membrane, there will occur a passage of water, through the membrane, in the direction of the higher concentration.
- The trend is toward equalizing the concentration of solute on both sides of a membrane.
- The solute concentration within microbial cells is approximately 0.95 percent. Thus if cells are exposed to solutions with higher solute concentrations, water will be drawn out of the cell. The process is called plasmolysis.
- The reverse process, that is, the passage of water from a low solute concentration into the cell, is termed plasmoptysis.
- The pressure built up within the cell as a result of this water intake is termed osmotic pressure.
- These phenomena can be observed more conveniently with animal cells since they do not have rigid cell walls.
- Plasmolysis results in dehydration of the cell, and as a consequence metabolic processes are retarded partially or completely.
- The antimicrobial effect is similar to that caused by desiccation. Because of the great rigidity of microbial cell walls (except for protozoa), the cell-wall structure does not exhibit distortions as a result of plasmolysis or plastmoptysis. However, changes in the cytoplasmic membrane, and particularly shrinkage of the proloplast from the cell wall, can be observed during plasmolysis.
Mainly two types of radiation are used for sterilization such as ionizing and nonionizing radiation;
A. Ionizing radiations
- Ionizing radiation is a form of energy that acts by removing electrons from atoms and molecules of materials that include air, water, and living tissue.
- Ionizing radiation is an excellent sterilizing agent with very high penetrating power.
- These radiations penetrate deep into objects and destroy bacterial endospores and vegetative cells, both prokaryotic and eukaryotic.
- These are, however, not that effective against viruses.
- Ionizing radiations include (a) X-rays, (b) gamma rays, and (c) cosmic rays.
- X-rays are lethal to microorganisms and highet forms of life.
- They have considerable energy and penetration ability.
- X-rays have been widely employed experimentally to produce microbial mutants.
- However, they are impractical for purposes of controlling microbial populations because (1) they are very expensive to produce in quantity and (2) they are difficult to utilize efficiently, since radiations are given off in all directions from their point of origin.
(b) Gamma rays
- Gamma radiations are high-energy radiations emitted from certain radioactive isotopes.
- Gamma rays are similar to X-rays but are of shorter wavelength and higher energy.
- They are capable of great penetration into matter, and they are lethal to all life, including microorganisms.
- Because of their great penetrating power and their microbicidal effect, gamma rays are attractive for use in commercial sterilization of materials of considerable thickness or volume, e.g packaged foods and medical devices.
(c) Cosmic rays
- Cosmic rays are high-energy protons and atomic nuclei that move through space at nearly the speed of light.
- They originate from the Sun, from outside of the Solar System in our own galaxy, and from distant galaxies.
- Direct sunlight is a natural method of sterilization of water in tanks, rivers, and lakes.
- Direct sunlight has an active germicidal effect due to its content of ultraviolet and heat rays.
- Bacteria present in natural water sources are rapidly destroyed by exposure to sunlight.
B. Nonionizing radiations
- Non-ionizing radiation refers to any type of electromagnetic radiation that does not carry enough energy per quantum (photon energy) to ionize atoms or molecules.
- Some examples of Nonionizing radiations are infrared and ultraviolet radiations.
(a) Ultraviolet radiations
- Ultraviolet (UV) radiation with wavelength of 240–280 nm is quite lethal and has a marked bactericidal activity.
- It acts by denaturation of bacterial protein and also interferes with replication of bacterial DNA.
- UV radiation is used primarily for disinfection of closed areas in microbiology laboratory, inoculation hoods, laminar flow, and operating theaters.
- It kills most vegetative bacteria but not spores, which are highly resistant to these radiations.
- It does not penetrate glass, dirt films, water, and other substances very effectively.
- Since UV radiations on prolonged exposure tend to burn the skin and cause damage to the eyes, UV lamps should be switched off while people are working in such areas.
(b) Infrared radiation
- Infrared, sometimes called infrared light, is electromagnetic radiation with wavelengths longer than of visible light. It is therefore invisible to the human eye.
- Infrared radiations are used for rapid and mass sterilization of disposable syringes and catheters.
|Ultraviolet light||Control of airborne infection: disinfection of surfaces||Must be absorbed to be effective (does not pass through transparent glass or opaque objects): irritating to eyes and skin; low penetration|
|X-ray, gamma, and cathode radiation||Sterilization of heat-sensitive surgical materials and other medical devices.||Expensive and requires special facilities for use|
- Filters are used to sterilize these heat-labile solutions.
- Filters simply remove contaminating microorganisms from solutions rather than directly destroying them.
Types of Filtration
There are present two types of filters such as;
- depth filters.
- membrane filters.
(a) Depth filters
- Depth filters consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter.
- The solution containing microorganisms is sucked in through this layer under vacuum and microbial cells are removed by physical screening or entrapment and also by adsorption to the surface of the filter material.
Types of Depth filters
There are present different types of Depth filters, such as;
- These are made up of diatomaceous earth (e.g., Berkefeld filters) or unglazed porcelain (e.g., Chamberlain filters).
- They are available in different grades of porosity and are used widely for purification of water for drinking and industrial uses.
- These are made up of asbestos such as magnesium silicate.
- Seitz and Sterimat filters are examples of such filters.
- These are disposable and single-use discs available in different grades.
- They have high adsorbing capacity and tend to alkalinize the filtered fluid.
- Their use is limited by the carcinogenic potential of asbestos.
Sintered glass filters
- These are made up of finely powdered glass particles, which are fused together.
- They have low absorbing properties and are available in different pore sizes.
- These filters, although can be cleaned easily, are brittle and expensive.
(b) Membrane filters
- Membrane filters are made up of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, or other synthetic materials.
- These filters are circular porous membranes and are usually 0.1 mm thick.
- Although a wide variety of pore sizes (0.015–12 m) are available, membranes with pores about 0.2 m are used, because the pore size is smaller than the size of bacteria.
- These filters are used to remove most vegetative cells, but not viruses, from solutions to be filtered.
- In the process of filtration, the membranes are held in special holders and often preceded by depth filters made of glass fibers to remove larger particles that might clog the membrane filter.
- The solution is then pushed or forced through the filter with a vacuum or with pressure from a syringe, peristaltic pump, or nitrogen gas bottle, and collected in previously sterilized containers.
|Membrane filters||Sterilization of heat-sensitive biological fluids||Fluid must be relatively free of sus. pended particulate matter|
6. Sound (sonic) waves
- High-frequency sound (sonic) waves beyond the sensitivity of the human ear are known to disrupt cells.
- Gram-negative rods are most sensitive to ultrasonic vibrations, while Gram-positive cocci, fungal spores, and bacterial spores are resistant to them.
- Ultrasonic devices are used in dental and some medical offices to clear debris and saliva from instruments before sterilization and to clean dental restorations.
Mode of action
- Sonication transmits vibrations through a water-filled chamber (sonicator) to induce pressure changes and create intense points of turbulence that can stress and burst cells in the vicinity.
- Sonication also forcefully dislodges foreign matter from objects.
- Heat generated by the sonic waves (up to 80°C) also appears to contribute to the antimicrobial action.
|Ultrasonics||Effective in decontaminating delicate cleaning instruments||Not effective alone, but as adjunct procedure enhances effectiveness of other methods|
What are Thermal Death Time and Decimal Reduction Time?
- Thermal death time refers to the shortest period of time to kill a suspension of bacteria (or spores) at a prescribed temperature and under specific conditions.
- While Decimal reduction time is another unit of measurement of the destruction of microorganisms by heat.
- From the definition of these terms, it is clear that they express a time-temperature relationship to killing. In thermal death time, the temperature is selected as the fixed point and the time varied.
- Decimal reduction time is a modification of thermal death time which measures a 90 percent rather than 100 percent kill rate.
- Thermal-death-time data and decimal-reduction-time data are extremely important in many applications of microbiology.
- The canning industry, for example, carries out extensive studies on this subject to establish satisfactory processing temperatures for the preservation of canned foods.
- Microbiology by Michael J. Pelczar, JR., E.C.S. Chan, Noel R. Krieg
- Prescott’s Microbiology
- Textbook of Microbiology and Immunology