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Sterilization (or sterilisation) refers to any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses and prions) from a surface, equipment, foods, medications, or biological culture medium. Sterilization can be achieved through application of heat, chemicals, irradiation, or filtration.
The first application of sterilization was thorough cooking to effect the partial heat sterilization of foods and water. Cultures that practice heat sterilization of food and water have longer life expectancy and lower rates of disability. Canning of foods by heat sterilization was an extension of the same principle. Ingestion of contaminated food and water remains a leading cause of illness and death in the developing world, particularly for children.
In general, surgical instruments and medications that enter an already sterile part of the body (such as the blood, or beneath the skin) must have a high sterility assurance level. Examples of such instruments include scalpels, hypodermic needles and artificial pacemakers. This is also essential in the manufacture of parenteral pharmaceuticals.
Heat sterilization of medical instruments is known to have been used in Ancient Rome, but it mostly disappeared throughout the Middle Ages resulting in significant increases in disability and death following surgical procedures.
Preparation of injectable medications and intravenous solutions for fluid replacement therapy requires not only a high sterility assurance level, but well-designed containers to prevent entry of adventitious agents after initial sterilization.
A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to 121°C (250°F), at 103 kPa (15 psi) above atmospheric pressure. Solid surfaces are effectively sterilized when heated this temperature for at least 15 minutes or to 134°C for a minimum of 3 minutes. However, liquids and instruments packed in layers of cloth require a much longer time to reach a sterilizing temperature. After sterilization, autoclaved liquids must be cooled slowly to avoid boiling over when the pressure is released.
Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions (discussed later).
For prion elimination, various recommendations state 121–132°C(270°F) for 60 minutes or 134°C (273°F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, as well as strains of CJD and BSE have shown much more resistance. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134-138°C (273-280°F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity. (The initial BSE concentration in the tissue was relatively low). To have a significant margin of safety, cleaning should reduce infectivity 4 logs, and the sterilization method should reduce it a further 5 logs.
To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of times. Indicator tape is often taped onto packages of products to be autoclaved. The tape contains a chemical that will change color when the appropriate conditions have been met. Some types of packaging have built-in indicators on them.
Biological indicators ("bioindicators") can also be used to independently confirm autoclave performance. Several simple bioindicator devices are commercially available based on microbial spores. Most contain pure strains of the heat resistant microbe Bacillus stearothermophilus which are among the toughest organisms an autoclave will have to destroy. Several of these devices have a self-contained growth medium (with or separate to the spores) and a growth indicator.
After a run in an autoclave, the internal glass ampule in the biological indicator vial is shattered, allowing the spores into a differential liquid medium. The vial is then incubated (typically at at 56°C (132°F)) for 48 hours. If the autoclave destroyed the spores, the medium will remain its original color. If autoclaving was unsuccessful the B. sterothermophilus will metabolize during incubation, causing a color change during the prescribed period of incubation.
For effective autoclaving, the steam needs to be able to penetrate uniformly. For this reason, an autoclave must not be overcrowded, and the lids of bottles and containers must be ajar. During the initial heating of the chamber, residual air must be allowed to escape as steam enters the autoclave chamber; otherwise the final temperature will be less than that of the entering steam. Indicators should be placed in the most difficult places for the steam to reach to ensure that steam actually penetrates there.
For autoclaving, as for all disinfection of sterilization methods, the cleaning off of any biological material is also critical. Biological matter or any grime may shield organisms from the property intended to kill them, whether it physical or chemical. Cleaning can also remove a large number of organisms at once. Proper cleaning can be achieved by physical scrubbing to remove dirt; this should be done with detergent and warm water to get the best results. Manual cleaning works through agitation, where the organisms are literally brushed off using detergent. When manual cleaning instruments or utensils that have organic matter on them, cool water must be used because warm or hot water may cause organic debris to coagulate. Where it is not feasible, ultrasound or pulsed air can be used to remove debris. Ultrasound works by a process called cavitation, in which sound waves are pulsed through a water/detergent medium, causing tiny bubbles to form. When these bubbles become unstable, they implode, causing organic debris to be pulled off. Ultrasonic machines must be used with the lid in the closed position because of the creation of aerosols which can be harmful to the operator.
Although imperfect, cooking and canning are the most common applications of heat sterilization. Boiling water kills the vegetative stage of all common microbes. Roasting meat until it is well done typically completely sterilizes the surface. Since the surface is also the part of food most likely to be contaminated by microbes, roasting usually prevents food poisoning. Note that the common methods of cooking food do not sterilize food - they simply reduce the number of disease-causing micro-organisms to a level that is not dangerous for people with normal digestive and immune systems. Pressure cooking is analogous to autoclaving and when performed correctly renders food sterile. However, some foods are notoriously difficult to sterilize with home canning equipment, so expert recommendations should be followed for home processing to avoid food poisoning.
See also Food safety.
Dishwashers often only use hot tap water or heat the water to between 49 and 60°C (120 and 140°F), and thus provide temperatures that could promote bacterial growth. That is to say, they do not effectively sterilize utensils. Some dishwashers do actually heat water up to 74°C (165°F) or higher; those often are specifically described as having sterilization modes of some sort, but this is not a substitute for autoclaving. Note that dishwashers remove food traces from the utensils by a combination of mechanical action (the action of water hitting the plates and cutlery) and the action of detergents and enzymes on fats and proteins. This removal of food particles thus removes one of the factors required for bacterial growth (food), and explains why items with cracks and crevices should either be washed by hand or disposed of: if the water cannot get to the area needing cleaning, the warm, moist, dark conditions in the dishwasher can actually promote bacterial growth.
Bathing and washing are not hot enough to sterilize bacteria without scalding the skin. Most hot tap water is between 43 and 49°C (110 and 120°F), though some people set theirs as high as 55°C (130°F). Humans begin to find water painful at 41 to 42°C (106 to 108°F), which to many bacteria is just starting to get warm enough for them to grow quickly; they will grow faster, rather than be killed at temperatures up to 55°C (130°F) or more.
Other heat methods include flaming, incineration, boiling, tindalization, and using dry heat.
Flaming is done to loops and straight-wires in microbiology labs. Leaving the loop in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be "sprayed" from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the innoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area.
Incineration will also burn any organism to ash. It is used to sanitize medical and other biohazardous waste before it is discarded with non-hazardous waste.
Boiling in water for 15 minutes will kill most vegetative bacteria and viruses, but boiling is ineffective against prions and many bacterial and fungal spores; therefore boiling is unsuitable for sterilization. However, since boiling does kill most vegetative microbes and viruses, it is useful for reducing viable levels if no better method is available. Boiling is a simple process, and is an option available to most anyone most anywhere, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.
Tindalization[1] /Tyndallization[2] named after John Tyndall is a lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method. The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again. The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is effective because many spores are stimulated to grow by the heat shock. The procedure only works for media that can support bacterial growth - it will not sterilize plain water. Tindalization/tyndallization is ineffective against prions.
Dry heat can be used to sterilize items, but as the heat takes much longer to be transferred to the organism, both the time and the temperature must usually be increased, unless forced ventilation of the hot air is used. The standard setting for a hot air oven is at least two hours at 160°C (320°F). A rapid method heats air to 190°C (374°F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects [1] [2]. Dry heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected by steam (for instance, it does not cause rusting of steel objects).
By combining immersion in sodium hydroxide (NaOH 0.09N) for two hours with one hour autoclaving (121°C / 250°F), several investigators have shown complete (>7.4 logs) inactivation. (Note that sodium hydroxide may corrode surgical instruments, especially if the sodium hydroxide immersion and autoclaving steps are combined.)
Chemicals are also used for sterilization. Although heating provides the most reliable way to rid objects of all transmissible agents, it is not always appropriate, because it will damage heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics.
Ethylene oxide (EO) gas is commonly used to sterilize objects sensitive to temperatures greater than 60°C such as plastics, optics and electrics. Ethylene oxide treatment is generally carried out between 30°C and 60°C with relative humidity above 30% and a gas concentration between 200 - 800 mg/L for at least three hours. Ethylene oxide penetrates well, moving through paper, cloth, and some plastic films and is highly effective. Ethylene oxide however is highly flammable, and requires a longer time to sterilize than any heat treatment. The process also requires time for aeration post sterilization to remove toxic residues. Ethylene oxide is used to sterilize around 50% of all disposable medical devices.
Bacillus subtilis, a very resistant organism, is used as a rapid biological indicator for EO sterilizers. If sterilization fails, incubation at 37°C causes a fluorescent change within four hours, which is read by an auto-reader. After 96 hours, a visible color change occurs. Fluorescence is emitted if a particular (EO resistant) enzyme is present, which means that spores are still active. The color change indicates a pH shift due to bacterial metabolism. The rapid results mean that if a cycle was found to be ineffective, the objects treated can be quarantined and physicians quickly advised of possible contamination.
Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. It has the benefit of being able to oxidize most organic matter. On the other hand, it is a toxic and unstable gas that must be produced on-site, so it is not practical to use in many settings.
Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists of 5.25% sodium hypochlorite. It is usually diluted to 1/10 immediately before use; however to kill Mycobacterium tuberculosis it should be diluted only 1/5. The dilution factor must take into account the volume of any liquid waste that it is being used to sterilize.[3] Bleach will kill many organisms immediately, but for full sterilization it should be allowed to react for 20 minutes. Bleach will kill many, but not all spores. It is highly corrosive and may corrode even stainless steel surgical instruments.
Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores in a clear liquid can take up to 12 hours with glutaraldehyde and even longer with formaldehyde. The presence of solid particles may lenghthen the required period or render the treatment ineffective. Sterilization of blocks of tissue can take much longer, due to the time required for the fixative to penetrate. Glutaraldehyde and formaldehyde are volatile, and toxic by both skin contact and inhalation. Glutaraldehyde has a short shelf life (<2 weeks), and is expensive. Formaldehyde is less expensive and has a much longer shelf life if some methanol is added to inhibit polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also used as a gaseous sterilizing agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.
Ortho-phthalaldehyde (OPA) is a chemical sterilizing agent that received Food and Drug Administration (FDA) clearance in late 1999. Typically used in a 0.55% solution, OPA shows better myco-bactericidal activity than glutaraldehyde. It also is effective against glutaraldehyde-resistant spores. OPA has superior stability, is less volatile, and does not irritate skin or eyes, and it acts more quickly than glutaraldehyde. On the other hand, it is more expensive, and will stain proteins (including skin) gray in color.
Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic once diluted to low concentrations (although a dangerous oxidizer at high concentrations), and leaves no residue.
Sterrad sterilization chambers use hydrogen peroxide vapor to sterilize heat-sensitive equipment such as rigid endoscopes. A recent model can sterilize most hospital loads in as little as 20 minutes. The Sterrad has limitations with processing certain materials such as paper/linens and long thin lumens. Paper products cannot be sterilized in the Sterrad system because of a process called cellulostics, in which the hydrogen peroxide would be completely absorbed by the paper product.
Hydrogen peroxide and formic acid are mixed as needed in the Endoclens device for sterilization of endoscopes. This device has two independent asynchronous bays, and cleans (in warm detergent with pulsed air), sterilizes and dries endoscopes automatically in 30 minutes. Studies with synthetic soil with bacterial spores showed the effectiveness of this device.
Dry Sterilization Process (DSP) uses hydrogen peroxide at a concentration of 30-35% under low pressure conditions. This process achieves bacterial reduction of 10<sup>-6</sup>...10<sup>-8</sup>. The complete process cycle time is just 6 seconds, and the surface temperature is increased only 10°-15°C. Originally designed for the sterilization of plastic bottles in the beverage industry, because of the high germ reduction and the slight temperature increase the Dry Sterilization Process is also useful for medical and pharmaceutical applications.
Peracetic acid (0.2%) is used to sterilize instruments in the Steris system.
Prions are highly resistant to chemical sterilization. Treatment with aldehydes (e.g., formaldehyde) have actually been shown to increase prion resistance. Hydrogen peroxide (3%) for one hour was shown to be ineffective, providing less than 3 logs (10<sup>-3</sup>) reduction in contamination. Iodine, formaldehyde, glutaraldehyde and peracetic acid also fail this test (one hour treatment). Only chlorine, a phenolic compound, guanidinium thiocyanate, and sodium hydroxide (NaOH) reduce prion levels by more than 4 logs. Chlorine and NaOH are the most consistent agents for prions. Chlorine is too corrosive to use on certain objects. Sodium hydroxide has had many studies showing its effectiveness.
Methods exist to sterilize using radiation such as X-rays, gamma rays, or subatomic particles.
Irradiation with X-rays or gamma rays does not make materials radioactive. Irradiation with particles may make materials radioactive, depending upon the type of particles and their energy, and the type of target material: neutrons and very high-energy particles can make materials radioactive, but have good penetration, whereas lower energy particles (other than neutrons) cannot make materials radioactive, but have poorer penetration.
Irradiation is used by the United States Postal Service to sterilize mail in the Washington, DC area. Some foods (e.g. spices, ground meats) are irradiated for sterilization (see food irradiation).
Clear liquids that would be damaged by heat, irradiation or chemical sterilization can be sterilized by mechanical filtration. This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research. A filter with pore size 0.2 µm will effectively remove bacteria. If viruses must also be removed, a much smaller pore size around 20 nm is needed. Prions are not removed by filtration.