Sterilization (microbiology) 414144 224839082 2008-07-10T17:23:06Z Arrataz 1207510 /* Chemical sterilization */ corrected subject / verb number mismatch. {{articleissues|article=y|restructure=March 2008|expert=Microbiology}} {{otheruses3|Sterilization}} '''Sterilization''' (or '''sterilisation''', see [[American and British English spelling differences|spelling differences]]) refers to any process that effectively kills or eliminates transmissible agents (such as [[fungi]], [[bacterium|bacteria]], [[virus]]es, [[prion]]s and spore forms etc.) from a surface, equipment, foods, medications, or biological culture medium. Sterilization can be achieved through application of [[heat]], [[chemical]]s, [[irradiation]], [[High pressure food preservation | high pressure]] or [[filtration]]. It may be defined as: * ''the process of freeing an article of all living microorganisms, viz. virus, bacteria, fungi, or their spores or parasites completely''<ref>www.who.int/reproductive-health/publications/MSM_98_1/MSM_98_1_glossary.en.html</ref><ref>www.ph.ucla.edu/epi/bioter/anthapha_def_a.html</ref> ==Classification== There are two types of sterilization: physical and chemical. Physical sterilization includes: * heat sterilization * radiation sterilization Chemical sterilization includes: * [[ethylene oxide]] * [[ozone]] * [[chlorine bleach]] * [[glutaraldehyde]] * [[formaldehyde]] * [[hydrogen peroxide]] * [[peracetic acid]] ==Applications == ===Foods=== 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. ===Medicine and surgery=== 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 [[scalpel]]s, [[hypodermic needle]]s and [[artificial pacemaker]]s. 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 [[Infection|adventitious agent]]s after initial sterilization. Food sterilization is usually considered a harsher form of [[Pasteurization]]<ref>{{cite web |url=http://www.ncfst.iit.edu/Media/mahpp.html |title=National Center installs world's first high pressure food sterilization unit |accessdate=2008-04-15}}</ref>, and is carried out through heating, though other methods are available. Food sterilization is commonly a part of [[canning]] and is used in combination with or instead of [[preservative]]s, refrigeration, and other ways to preserve food. ==Heat sterilization== {{See also|Dry heat sterilization}} {{See also|Moist heat sterilization}} ===Steam sterilization=== [[Image:Autoclave Front Loading composition.jpg|thumb|400px|right|Front-loading autoclaves are very common.]] A widely-used method for heat sterilization is the [[autoclave]]. Autoclaves commonly use steam heated to 121 °C or 134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C 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. 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 [[spore]]s, which can be quite resistant. It will not necessarily eliminate all [[prions]]. For prion elimination, various recommendations state 121&ndash;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 [[Bovine spongiform encephalopathy|BSE]] are more resistant. Using [[mouse|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 [[Logarithm|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 ''[[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 (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 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. 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 of 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. === Food === {{See also|Food safety}} 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]]. === Food utensils === [[Dishwasher]]s 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), it clearly 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 === [[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 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. '''[[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 viruses, but boiling is ineffective against [[prion]]s and many bacterial and fungal [[spore]]s; 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]]'''<ref>{{cite journal | last = Mesquita | first = J. A. M. | authorlink = | coauthors = Teixeira, M.A. and Brandao, S. C. C. | title = Tindalization of goats' milk in glass bottles | journal =J. Anim. Sci. /J. Dairy Sci. | volume =Vol. 76, Suppl. 1 / Vol. 81, Suppl. 1/ | pages = 21 | publisher = | date = 1998 | url = http://www.asas.org/jas/98meet/98df.pdf | accessdate = 2007-03-06 }}</ref> /'''Tyndallization'''<ref>{{cite web |url=http://www.umsl.edu/~microbes/pdf/tyndallization.pdf |title=http://www.umsl.edu/~microbes/pdf/tyndallization.pdf |date = 1999 |last = Thiel |first = Theresa |publisher = Science in the Real World |format=pdf |accessdate=2007-03-06 }}</ref> 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.[http://www.health.gov.ab.ca/resources/publications/PersonalServicesPt1.pdf][http://www.tpub.com/content/medical/14274/css/14274_146.htm] 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). '''[[Prion]]s''' can be inactivated by immersion in [[sodium hydroxide]] (NaOH 0.09N) for two hours plus one hour autoclaving (121 °C/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. ==Chemical sterilization== 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 optic]]s, electronics, and many [[plastic]]s. '''[[Ethylene oxide]]''' (EO or EtO) 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 and 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 sterilizers are used to process sensitive instruments which cannot be adequately sterilized by other methods. EtO can kill all known viruses, bacteria and fungi, including bacterial spores and is satisfactory for most medical materials, even with repeated use. However it is highly flammable, and requires a longer time to sterilize than any heat treatment. The process also requires a period of post-sterilization aeration to remove toxic residues. 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. Because of these problems a micro-dose sterilization method was 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.<ref>[http://anpro.com/articles/eto%20benefits.htm Micro-dose sterilization method]</ref> [[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 the objects treated can be quarantined until the test results are available. '''[[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. '''[[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. The dilution factor must take into account the volume of any liquid waste that it is being used to sterilize.<ref>[[Beth Israel Deaconess Medical Center]] Biosafety Manual ([[2004]] edition)</ref> 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 [[fixation (histology)|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 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 vaccine|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. '''[[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. '''Low Temperature Plasma''' sterilization chambers use hydrogen peroxide vapor to sterilize heat-sensitive equipment such as rigid [[endoscope]]s. 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 (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]]''' (0.2%) is used to sterilize instruments in the Steris system. '''[[Prion]]s''' are highly resistant to chemical sterilization. Treatment with [[aldehyde]]s (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. ==Radiation sterilization== Methods exist to sterilize using [[radiation]] such as [[electron beam]]s, [[X-rays]], [[gamma rays]], or [[subatomic particle]]s. *[[Gamma rays]] are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, [[cannula]]s and IV sets. Gamma radiation requires bulky shielding for the safety of the operators; they also require storage of a [[radioisotope]] (usually [[Cobalt-60]]), which continuously emits gamma rays (it cannot be turned off, and therefore always presents a hazard in the area of the facility). *[[Electron beam processing]] is also commonly used for medical device 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. *[[X-rays]] are less penetrating than gamma rays and tend to require longer exposure times, but require less shielding, and are generated by an [[X-ray machine]] that can be turned off for servicing and when not in use. *[[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. UV irradiation is routinely used to sterilize the interiors of [[biological safety cabinet]]s 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 many plastics, such as [[polystyrene]] foam. {{further|[[Ultraviolet Germicidal Irradiation]]}} *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. 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== 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 [[micrometre|µm]] will effectively remove [[bacteria]]. If [[virus]]es must also be removed, a much smaller pore size around 20 [[nanometre|nm]] is needed. Solutions filter slowly through membranes with smaller pore diameters. Prions are not removed by filtration. The filtration equipment and the filters themselves may be purchased as presterilized 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 sterility, the filtration system must be tested to ensure that the membranes have not been punctured prior to or during use. To ensure the best results, pharmaceutical sterile filtration is performed in a room with highly filtered air ([[HEPA]] filtration) or in a [[laminar flow cabinet]] or "flowbox", a device which produces a [[laminar]] stream of HEPA filtered air. ==Notes== <references/> ==See also== *[[Antibacterial soap]] *[[Contamination control]] *[[Electron irradiation]] *[[Pasteurization]] ==References== ===Notes=== <references/> ===General references=== *Central Service Technical Manual, 6th Edition, Jack Ninemeier, PhD, Editor , International Association of Healthcare Central Service Materiel Management <!-- Note 'Materiel' is *NOT* a misspelling --> *[http://www.bact.wisc.edu/Microtextbook/modules.php?op=modload&name=Sections&file=index&req=viewarticle&artid=13&page=1 Control of microbes] *Raju, G.K. & Cooney, C.L., 1993. Media and air sterilization. in Biotechnology, ed. Stephanopoulos, G., Vol 3., pp 157-184. ==External links== *[http://www.instrumentcleaning.com/chemical_disinfection.html Chemical Disinfection] [[Category:Hygiene]] [[Category:Microbiology]] [[bs:Sterilizacija]] [[cs:Sterilizace (mikrobiologie)]] [[da:steril]] [[de:Sterilisation]] [[fr:Stérilisation (microbiologie)]] [[it:Sterilizzazione (igiene)]] [[he:עיקור (מיקרוביולוגיה)]] [[nl:Steriliseren (micro-organismen)]] [[ja:殺菌]] [[pl:Sterylizacja (technologia)]] [[pt:Esterilização (materiais)]] [[ru:Стерилизация (микробиология)]] [[sh:Sterilizacija (mikrobiologija)]] [[zh:殺菌]]