# Sterilization (microbiology)

Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.[1][2] Sterilization can be achieved by applying heat, chemicals, irradiation, high pressure, and filtration or combinations thereof.

The term has evolved to include the disabling or destruction of infectious proteins such as prions related to Transmissible Spongiform Encephalopathies (TSE).[3]

## Applications

### Foods

One of the first steps toward sterilization made by Nicolas Appert.

He learned that thorough cooking (applying a suitable amount of heat over a suitable period of time) slowed the decay of foods and various liquids, preserving them for safe consumption for a longer time than was typical. Canning of foods is an extension of the same principle, and has helped to reduce food borne illness ("food poisoning"). Other methods of sterilizing foods include food irradiation[4] and pascalization (the use of high pressure to kill microorganisms).[5]

### Medicine and surgery

In general, surgical instruments and medications that enter an already aseptic part of the body (such as the bloodstream, or penetrating the skin) must be sterilized to a high sterility assurance level, or SAL. Examples of such instruments include scalpels, hypodermic needles and artificial pacemakers. This is also essential in the manufacture of parenteral pharmaceuticals.

Heat (flame) 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 also well-designed containers to prevent entry of adventitious agents after initial product sterilization.

Sterilization as a definition terminates all life; whereas sanitization and disinfection terminates selectively and partially. Both sanitization and disinfection reduce the number of targeted pathogenic organisms to what are considered "acceptable" levels - levels that a reasonably healthy, intact, body can deal with. An example of this class of process is Pasteurization.

## Quantification

The aim of sterilization is the reduction of initially present microorganisms or other potential pathogens. It is commonly expressed by multiplies of the decimal reduction time $D$ denoting the time needed to reduce the initial number $N_0$ to one tenth ($10^{-1}$) of its original value. Then the number of microorganisms $N$ after sterilisation time $t$ is given by

$\frac{N}{N_0} = 10^{\left(-\frac{t}{D}\right)}$.

$D$ is a function of sterilisation conditions and varies with the type of microorganism, temperature, water activity, pH etc.. For steam sterilisation (see below) typically the temperature (in °Celsius) is given as index.

For sterilisation a reduction by one million ($10^{-6}$) is minimally required with six times $D$. For transfusion or other venous injections $10^{-7}$ is typically required to reduce infection risks. For Disinfection $10^{-5}$ is sufficient. Theoretically, the likelihood of survival of an individual microrganism is never zero.

## Methods

### Heat

#### Steam sterilization

A widely used method for heat sterilization is the autoclave, sometimes called a converter. Autoclaves commonly use steam heated to 121–134 °C (250–273 °F). To achieve sterility, a holding time of at least 15 minutes at 121 °C (250 °F) at 100 kPa (15 psi), or 3 minutes at 134 °C (273 °F) at 100 kPa (15 psi) is required. Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature (unnecessary in machines that grind the contents prior to sterilization). Following sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released. Modern converters operate around this problem by gradually depressing the sterilization chamber and allowing liquids to evaporate under a negative pressure, while cooling the contents.

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.

For prion elimination, various recommendations state 121–132 °C (250–270 °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 are more resistant. 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). For a significant margin of safety, cleaning should reduce infectivity by 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 time. Indicator tape is often placed on packages of products prior to autoclaving. A chemical in the tape 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. Simple bioindicator devices are commercially available based on microbial spores. Most contain spores of the heat resistant microbe Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), among the toughest organisms for an autoclave to destroy. Typically these devices have a self-contained liquid growth medium and a growth indicator. After autoclaving an internal glass ampule is shattered, releasing the spores into the growth medium. The vial is then incubated (typically at 56 °C (133 °F)) for 24 hours. If the autoclave destroyed the spores, the medium will retain its original color. If autoclaving was unsuccessful the B. sterothermophilus will metabolize during incubation, causing a color change during the incubation.

For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar. Alternatively steam penetration can be achieved by shredding the waste in some Autoclave models that also render the end product unrecognizable. During the initial heating of the chamber, residual air must be removed. 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 or sterilization methods, cleaning is critical. Extraneous biological matter or 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. Proper cleaning can be achieved by physical scrubbing. This should be done with detergent and warm water to get the best results. Cleaning instruments or utensils with organic matter, cool water must be used because warm or hot water may cause organic debris to coagulate. Treatment with ultrasound or pulsed air can also be used to remove debris.

#### Heat sterilization of foods

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.

#### Other heat sterilization methods

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 inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave residues on the object, e.g. carbon, if the object is not heated enough.

A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses, and has the advantage that it leaves less residue than a gas flame. This method works well for the glass "hockey stick"-shaped bacteria spreaders.

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 fifteen minutes will kill most vegetative bacteria and inactivate 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 people, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.

Tindalization[6] /Tyndallization[7] 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 sterilizer

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.[8][9] 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).

Prions can be inactivated by immersion in sodium hydroxide (NaOH 0.09N) for two hours plus one hour autoclaving (121 °C or 250 °F). Several investigators have shown complete (>7.4 logs) inactivation with this combined treatment. However, sodium hydroxide may corrode surgical instruments, especially at the elevated temperatures of the autoclave.

Glass bead sterilizer, once a common sterilization method employed in dental offices as well as biologic laboratories,[10] is not approved by the U.S. Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) to be used as inter-patients sterilizer since 1997.[11] Still it is popular in European as well as Israeli dental practice although there are no current evidence-based guidelines for using this sterilizer.[10]

### Chemical sterilization

Chemiclav

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. Low temperature gas sterilizers function by exposing the articles to be sterilized to high concentrations (typically 5 - 10% v/v) of very reactive gases (alkylating agents such as ethylene oxide, and oxidizing agents such as hydrogen peroxide and ozone). Liquid sterilants and high disinfectants typically include oxidizing agents such as hydrogen peroxide and peracetic acid and aldehydes such as glutaraldehyde and more recently o-phthalaldehyde. While the use of gas and liquid chemical sterilants/high level disinfectants avoids the problem of heat damage, users must ensure that article to be sterilized is chemically compatible with the sterilant being used. The manufacturer of the article can provide specific information regarding compatible sterilants. In addition, the use of chemical sterilants poses new challenges for workplace safety. The chemicals used as sterilants are designed to destroy a wide range of pathogens and typically the same properties that make them good sterilants makes them harmful to humans. American employers have a duty to ensure a safe work environment (Occupational Safety and Health Act of 1970, section 5 for United States) and work practices, engineering controls and monitoring should be employed appropriately.

#### Ethylene oxide

Ethylene oxide

Chemical formula:C2H4O

(EO or EtO) gas is commonly used to sterilize objects sensitive to temperatures greater than 60 °C and / or radiation 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 and 800 mg/l, and typically lasts for at least three hours. Ethylene oxide penetrates well, moving through paper, cloth, and some plastic films and is highly effective. EtO can kill all known viruses, bacteria and fungi, including bacterial spores and is compatible with most materials (e.g. of medical devices), even when repeatedly applied. However, it is highly flammable, toxic and carcinogenic with a potential to cause adverse reproductive effects. Ethylene oxide sterilizers requires biological validation and testing of every load, after sterilization installation, repairs or process failure. Biological testing or spore testing are paper filter saturated in millions of Bacillus atropheus known as Bacillus subtilis.

A typical process consists of a preconditioning phase, the actual sterilization run and a period of post-sterilization aeration to remove toxic residues, such as ethylene oxide residues and by-products such ethylene glycol (formed out of EtO and ambient humidity) and ethylene chlorohydrine (formed out of EtO and materials containing chlorine, such as PVC). Besides moist heat and irradiation, ethylene oxide is the most common sterilization method, used for over 70% of total sterilizations, and for 50% of all disposable medical devices.

The two most important ethylene oxide sterilization methods are: (1) the gas chamber method and (2) the micro-dose method. To benefit from economies of scale, EtO has traditionally been delivered by flooding a large chamber with a combination of EtO and other gases used as dilutants (usually CFCs or carbon dioxide). This method has drawbacks inherent to the use of large amounts of sterilant being released into a large space, including air contamination produced by CFCs and/or large amounts of EtO residuals, flammability and storage issues calling for special handling and storage, operator exposure risk and training costs.

Ethylene oxide is still widely used by medical device manufacturers for larger scale sterilization (e.g. by the pallet), but while still used, EtO is becoming less popular in hospitals. Since EtO is explosive from its lower explosive limit of 3% all the way to 100%, EtO was traditionally supplied with an inert carrier gas such as a CFC or halogenated hydrocarbon. The use of CFCs as the carrier gas was banned because of concerns of ozone depletion [12] and halogenated hydrocarbons are being replaced by so-called 100% EtO systems because of the much greater cost of the blends. In hospitals, most EtO sterilizers use single use cartridges (e.g. 3M's Steri-Vac line,[13] or Steris Corporation's Stericert sterilizers[14]) because of the convenience and ease of use compared to the former plumbed gas cylinders of EtO blends. Another 100% method is the so-called micro-dose sterilization method, developed in the late 1950s, using a specially designed bag to eliminate the need to flood a larger chamber with EtO. This method is also known as gas diffusion sterilization, or bag sterilization. This method minimizes the use of gas.[15]

Another reason for the decrease in use of EtO are the well known health effects. In addition to being a primary irritant, EtO is now classified by the IARC as a known human carcinogen.[16] The US OSHA has set the permissible exposure limit (PEL) at 1 ppm calculated as an eight hour time weighted average (TWA) [29 CFR 1910.1047] and 5 ppm as a 15 minute TWA. The NIOSH Immediately dangerous to life and health limit for EtO is 800 ppm.[17] The odor threshold is around 500 ppm[18] and so EtO is imperceptible until concentrations well above the OSHA PEL. Therefore, OSHA recommends that some kind of continuous gas monitoring system be used to protect workers using EtO for sterilization.[19] While the hazards of EtO are generally well known, it should be noted that all chemical sterilants are designed to kill a broad spectrum of organisms, by exposing them to high concentrations of reactive chemicals. Therefore, it is no surprise that all the common chemical gas sterilants are toxic and adequate protective measures must be taken to protect workers using these materials.

Employees health records must be maintained during employment and after termination of employment for 30 years.

#### Nitrogen dioxide

Nitrogen dioxide

Chemical Formula: NO2

Nitrogen Dioxide NO2 gas is a rapid and effective sterilant for use against a wide range of microorganisms, including common bacteria, viruses, and spores. The unique physical properties of NO2 gas allow for sterilant dispersion in an enclosed environment at room temperature and ambient pressure. The mechanism for lethality is the degradation of DNA in the spore core through nitration of the phosphate backbone, which kills the exposed organism as it absorbs NO2. This degradation occurs at even very low concentrations of the gas.[20] NO2 has a boiling point of 21°C at sea level, which results in a relatively high saturated vapor pressure at ambient temperature. Because of this, liquid NO2 may be used as a convenient source for the sterilant gas. Liquid NO2 is often referred to by the name of its dimer, dinitrogen tetroxide (N2O4). Additionally, the low levels of concentration required, coupled with the high vapor pressure, assures that no condensation occurs on the devices being sterilized. This means that no aeration of the devices is required immediately following the sterilization cycle.[21] NO2 is also less corrosive than other sterilant gases, and is compatible with most medical materials and adhesives.[22]

The most-resistant organism (MRO) to sterilization with NO2 gas is the spore of Geobacillus stearothermophilus, which is the same MRO for both steam and hydrogen peroxide sterilization processes. The spore form of G. stearothermophilus has been well characterized over the years as a biological indicator in sterilization applications. Microbial inactivation of G. stearothermophilus with NO2 gas proceeds rapidly in a log-linear fashion, as is typical of other sterilization processes. Noxilizer, Inc. has commercialized this technology to offer contract sterilization services for medical devices at its Baltimore, MD facility.[23] This has been demonstrated in Noxilizer’s lab in multiple studies and is supported by published reports from other labs. These same properties also allow for quicker removal of the sterilant and residuals through aeration of the enclosed environment. The combination of rapid lethality and easy removal of the gas allows for shorter overall cycle times during the sterilization (or decontamination) process and a lower level of sterilant residuals than are found with other sterilization methods.[24]

#### Ozone

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.

Ozone offers many advantages as a sterilant gas; ozone is a very efficient sterilant because of its strong oxidizing properties (E = 2.076 vs SHE, CRC Handbook of Chemistry and Physics, 76th Ed, 1995–1996) capable of destroying a wide range of pathogens, including prions without the need for handling hazardous chemicals since the ozone is generated within the sterilizer from medical grade oxygen. The high reactivity of ozone means that waste ozone can be destroyed by passing over a simple catalyst that reverts it back to oxygen and also means that the cycle time is relatively short. The downside of using ozone is that the gas is very reactive and very hazardous. The NIOSH immediately dangerous to life and health limit for ozone is 5 ppm, 160 times smaller than the 800 ppm IDLH for ethylene oxide. Documentation for Immediately Dangerous to Life or Health Concentrations (IDLH): NIOSH Chemical Listing and Documentation of Revised IDLH Values (as of 3/1/95)[25] and OSHA has set the PEL for ozone at 0.1 ppm calculated as an 8 hour time weighted average (29 CFR 1910.1000, Table Z-1). The Canadian Center for Occupation Health and Safety provides an excellent summary of the health effects of exposure to ozone. The sterilant gas manufacturers include many safety features in their products but prudent practice is to provide continuous monitoring to below the OSHA PEL to provide a rapid warning in the event of a leak and monitors for determining workplace exposure to ozone are commercially available.

#### Bleach

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, and 1/2.5 (1 part bleach and 1.5 parts water) to inactivate prions. The dilution factor must take into account the volume of any liquid waste that it is being used to sterilize.[26] 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 also highly corrosive.

Bleach decomposes over time when exposed to air, so fresh solutions should be made daily.[27]

#### Glutaraldehyde and formaldehyde

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 22 hours with glutaraldehyde and even longer with formaldehyde. The presence of solid particles may lengthen 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.

#### Phthalaldehyde

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. Some side effects from equipment sterilized using this reagent have been reported. For example, two cases of anaphylaxis following cystoscopy with endoscopes sterilized with OPA were reported by Cooper, et al., (J Endourol. 2008 Sep;22(9):2181-4), and four cases of ortho-phthalaldehyde-induced anaphylaxis after laryngoscopy with the detection of specific IgE in serum were reported by Suzukawa, et al., (Allergol Int. 2007 Sep;56(3):313-6. Epub 2007 Jul 1; J Allergy Clin Immunol. 2006 Jun;117(6):1500-1. Epub 2006 Mar 31).

#### Hydrogen peroxide

Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic when diluted to low concentrations, such as the familiar 3% retail solutions although hydrogen peroxide is a dangerous oxidizer at high concentrations (> 10% w/w). Hydrogen peroxide is strong oxidant and these oxidizing properties allow it to destroy a wide range of pathogens and it is used to sterilize heat or temperature sensitive articles such as rigid endoscopes. In medical sterilization hydrogen peroxide is used at higher concentrations, ranging from around 35% up to 90%. The biggest advantage of hydrogen peroxide as a sterilant is the short cycle time. Whereas the cycle time for ethylene oxide (discussed above) may be 10 to 15 hours, the use of very high concentrations of hydrogen peroxide allows much shorter cycle times. Some hydrogen peroxide modern sterilizers, such as the Sterrad NX have a cycle time as short as 28 minutes.

Hydrogen peroxide sterilizers have their drawbacks. Since hydrogen peroxide is a strong oxidant, there are material compatibility issues and users should consult the manufacturer of the article to be sterilized to ensure that it is compatible with this method of sterilization. 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. The penetrating ability of hydrogen peroxide is not as good as ethylene oxide and so there are limitations on the length and diameter of lumens that can be effectively sterilized and guidance is available from the sterilizer manufacturers.

While hydrogen peroxide offers significant advantages in terms of throughput, as with all sterilant gases, sterility is achieved through the use of high concentrations of reactive gases. Hydrogen peroxide is primary irritant and the contact of the liquid solution with skin will cause bleaching or ulceration depending on the concentration and contact time. The vapor is also hazardous with the target organs being the eyes and respiratory system. Even short term exposures can be hazardous and NIOSH has set the Immediately Dangerous to Life and Health Level (IDLH) at 75 ppm.[17] less than one tenth the IDLH for ethylene oxide (800 ppm). Prolonged exposure to even low ppm concentrations can cause permanent lung damage and consequently OSHA has set the permissible exposure limit to 1.0 ppm, calculated as an 8 hour time weighted average (29 CFR 1910.1000 Table Z-1). Employers thus have a legal duty to ensure that their personnel are not exposed to concentrations exceeding this PEL. Even though the sterilizer manufacturers go to great lengths to make their products safe through careful design and incorporation of many safety features, workplace exposures of hydrogen peroxide from gas sterilizers are documented in the FDA MAUDE database.[28] When using any type of gas sterilizer, prudent work practices will include good ventilation (10 air exchanges per hour), a continuous gas monitor for hydrogen peroxide as well as good work practices and training. Further information about the health effects of hydrogen peroxide and good work practices is available from OSHA[29] and the ATSDR.[30]

Hydrogen peroxide can also be mixed with formic acid 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.

Vaporized hydrogen peroxide (VHP) is used to sterilize large enclosed and sealed areas such as entire rooms and aircraft interiors.

#### Dry sterilization process

Dry sterilization process (DSP) uses hydrogen peroxide at a concentration of 30-35% under low pressure conditions. This process achieves bacterial reduction of 10−6...10−8. The complete process cycle time is just 6 seconds, and the surface temperature is increased only 10-15 °C (18 to 27 °F). 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

Peracetic acid (0.2%) is used to sterilize instruments in some STERIS Corporation systems.

#### Silver

Silver ions and silver compounds show a toxic effect on some bacteria, viruses, algae and fungi, typical of heavy metals like lead or mercury, but without the high toxicity to humans that is normally associated with these other metals. Its germicidal effects kill many microbial organisms in vitro, but testing and standardization of silver products is yet difficult.[31] In the antique Greek Hippocratic Corpus it is written that silver has beneficial healing and anti-disease properties,[32] and the Phoenicians used to store water, wine, and vinegar in silver bottles to prevent spoiling. In the early 1900s people would put silver dollars in milk bottles to prolong the milk's freshness.[33] The exact process of silver's germicidal effect is still not well understood. One of the explanations is the oligodynamic effect, which accounts for the effect on microorganisms but not on viruses.

Silver compounds were used to prevent infection in World War I before the advent of antibiotics. Silver nitrate solution was a standard of care but was largely replaced by silver sulfadiazine cream (SSD Cream),[34] which was generally the "standard of care" for the antibacterial and antibiotic treatment of serious burns until the late 1990s.[35] Now, other options, such as silver-coated dressings (activated silver dressings), are used in addition to SSD cream. However, the evidence for the use of such silver-treated dressings is mixed and although the evidence on if they are effective is promising, it is marred by the poor quality of the trials used to assess these products.[36] Consequently a major systematic review by the Cochrane Collaboration found insufficient evidence to recommend the use of silver-treated dressings to treat infected wounds.[36]

The widespread use of silver went out of fashion with the development of antibiotics. However, recently there has been renewed interest in silver as a broad-spectrum antimicrobial. In particular, silver is being used with alginate, a naturally occurring biopolymer derived from seaweed, in a range of products designed to prevent infections as part of wound management procedures, particularly applicable to burn victims.[37] In 2007, AGC Flat Glass Europe introduced the first antibacterial glass to fight hospital-caught infection: it is covered with a thin layer of silver.[38] In addition, Samsung has introduced washing machines with a final rinse containing silver ions to provide several days of antibacterial protection in the clothes.[39] Kohler has introduced a line of toilet seats that have silver ions embedded to kill germs. A company called Thomson Research Associates has begun treating products with Ultra Fresh, an anti-microbial technology involving "proprietary nano-technology to produce the ultra-fine silver particles essential to ease of application and long-term protection."[40] The U.S. Food and Drug Administration (FDA) has recently approved an endotracheal breathing tube with a fine coat of silver for use in mechanical ventilation, after studies found it reduced the risk of ventilator-associated pneumonia.[41]

It has long been known that antibacterial action of silver is enhanced by the presence of an electric field. Applying a few volts of electricity across silver electrodes drastically enhances the rate that bacteria in solution are killed. It was found recently that the antibacterial action of silver electrodes is greatly improved if the electrodes are covered with silver nanorods.[42] Note that enhanced antibacterial properties of nanoparticles compared to bulk material is not limited to silver, but has also been demonstrated on other materials such as ZnO[43]

#### Potential for chemical sterilization of prions

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 (10−3) reduction in contamination. Iodine, formaldehyde, glutaraldehyde and peracetic acid also fail this test (one hour treatment). Only chlorine, phenolic compounds, 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.[44]

Methods of sterilization exist using radiation such as electron beams, X-rays, gamma rays, or subatomic particles.[45]

• Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light absorb UV, glass for example completely absorbs all UV light. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under dirt (which may become polymerized after prolonged irradiation, so that it is very difficult to remove). It also damages some plastics, such as polystyrene foam if exposed for prolonged periods of time.

The safety of irradiation facilities is regulated by the United Nations International Atomic Energy Agency and monitored by the different national Nuclear Regulatory Commissions. The incidents that have occurred in the past are documented by the agency and thoroughly analyzed to determine root cause and improvement potential. Such improvements are then mandated to retrofit existing facilities and future design.

Illustration of the penetration properties of the different radiation technologies (electron beam, X-ray, gamma rays)
Efficiency illustration of the different radiation technologies (electron beam, X-ray, gamma rays)
• Electron beam processing is also commonly used for sterilization. Electron beams use an on-off technology and provide a much higher dosing rate than gamma or x-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. A limitation is that electron beams are less penetrating than either gamma or x-rays. Facilities rely on substantial concrete shields to protect workers and the environment from radiation exposure.
• X-rays, High-energy X-rays (bremsstrahlung) are a form of ionizing energy allowing to irradiate large packages and pallet loads of medical devices. Their penetration is sufficient to treat multiple pallet loads of low-density packages with very good dose uniformity ratios. X-ray sterilization is an electricity based process not requiring chemical nor radio-active material. High energy and high power X-rays are generated by an X-ray machine that can be turned off for when not in use, and therefore does not require any shielding when in storage. X-rays are generated by colliding accelerated electrons with a dense material (target) such as tantalum or tungsten in a process known as bremsstrahlung-conversion. These systems generally have low energetic efficiency during the conversion of electron energy to photon radiation requiring much more electrical energy than other systems.
• Subatomic particles may be more or less penetrating, and may be generated by a radioisotope or a device, depending upon the type of particle.

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.

Sterlization by irradiation with gamma rays may however in some cases affect material properties.[47]

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).

### Sterile filtration

Fluids that would be damaged by heat (such as those containing proteins like large molecule drug products, but also wine and beer) irradiation or chemical sterilization, can be only sterilized by Microfiltration using membrane filters[citation needed]. This method is commonly used for heat labile pharmaceuticals and protein solutions in medicinal drug processing. Usually, a filter with pore size 0.2 µm (microfiltration) will effectively remove microorganisms.[48] In the processing of Biologics, viruses must be removed or inactivated. Nanofilters with a smaller pore size of 20 -50 nm (nanofiltration) are used. The smaller the pore size the lower the flow rate. To achieve higher total throughput or to avoid premature blockage, pre-filters might be used to protect small pore membrane filters.

Membrane filters used in production processes are commonly made from materials such as mixester cellulose or polyethersulfone (PES). The filtration equipment and the filters themselves may be purchased as pre-sterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure proper functioning of the filter, the membrane filters are integrity tested post-use and in occasions pre-use. The non-destructive integrity test assures the filter is undamaged, it also is a regulatory requirement enforced by agencies like FDA, EMA etc. For best results, final or terminal pharmaceutical sterile filtration is performed in cleanroom classes A.

## Cleaning methods that do not achieve sterilization

This is a brief list of cleaning methods that may be thought to "kill germs" but do not achieve sterilization.

• Washing in a dishwasher: Dishwashers often only use hot tap water or heat the water to between 49 and 60 °C (120 and 140 °F)[citation needed], which is not hot enough to kill some bacteria on cooking or eating utensils.
• Bathing can not sterilize skin, even using antibacterial soap.
• Disinfectants (for non-living objects) or antiseptics (for living objects such as skin) can kill or remove bacteria and viruses, but not all.
• Pasteurization of food also kills some bacteria and viruses, but not all.

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## Other references

• “US Department of Labor, Occupational Safety and Health Administration.29 CFR 1910.1020. Access to Employee Medical Records." .October 26, 2007.
• Perioperative Standards and Recommended Practices, AORN 2013,ISBN 978-1-888460-77-3