Sterilization (microbiology)

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One of the first steps toward sterilization was made by Nicolas Appert who discovered that thorough application of heat over a suitable period 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[3] and pascalization (the use of high pressure to kill microorganisms).[4]

Medicine and surgery[edit]


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.


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 terminate 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.


There are strict international rules to protect the contamination of Solar System bodies from biological material from Earth. Standards vary depending on both the type of mission and its 'destiny'; the more likely a planet is considered to bear life, the stricter the requirements are.

Many of the instruments' components are very delicate and would not withstand very high temperatures, so scientists use different techniques: heating to 120 °C, chemical sterilization, oxidization, and irradiation with ultraviolet light and other kinds of radiation.[5]


The aim of sterilization is the reduction of initially present microorganisms or other potential pathogens. The degree of sterilization is commonly expressed by multiples 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.[citation needed] Then the number of microorganisms N after sterilization time t is given by

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

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

For sterilization 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 microorganism is never zero.



Steam sterilization[edit]

Front-loading autoclaves

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.







Heat sterilization of foods[edit]

See also: Food safety



Other heat sterilization methods[edit]







Dry heat sterilizer

Dry Heat Sterilizer


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]

Bunsen burner is a common sterilization method employed in laboratory that produces a single open gas flame, which is used for heating, sterilization, and combustion. The laboratory equipment is named after Robert Bunsen. The gas used in Bunsen burner can be natural gas (methane) or a liquefied petroleum gas such as propane, butane, or a mixture of both.

Chemical sterilization[edit]



Ethylene oxide[edit]

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.


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[edit]

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.[21]




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)[23] 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 decomposes over time when exposed to air, so fresh solutions should be made daily.[25]

Glutaraldehyde and formaldehyde[edit]



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[edit]





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

Dry sterilization process[edit]

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 by 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[edit]

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


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.[29] In the antique Greek Hippocratic Corpus it is written that silver has beneficial healing and anti-disease properties,[30] 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.[31] 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),[32] which was generally the "standard of care" for the antibacterial and antibiotic treatment of serious burns until the late 1990s.[33] 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.[34] Consequently a major systematic review by the Cochrane Collaboration found insufficient evidence to recommend the use of silver-treated dressings to treat infected wounds.[34]

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.[35] 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.[36] In addition, Samsung has introduced washing machines with a final rinse containing silver ions to provide several days of antibacterial protection in the clothes.[37] 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."[38] 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.[39]


Potential for chemical sterilization of prions[edit]


Radiation sterilization[edit]


Non-ionizing radiation sterilization[edit]

  • Ultraviolet light irradiation (UV, from a germicidal lamp) is useful 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. UV light is also used to disinfect water and wastewater.[44]

Ionizing radiation sterilization[edit]

Illustration of the penetration properties of the different radiation technologies (electron beam, X-ray, gamma rays)

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.

  • Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food. The gamma radiation is emitted from a radioisotope (usually Cobalt-60 or caesium-137). Caesium-137 is used in small hospital units to treat blood before transfusion to prevent Graft-versus-host disease. Use of a radioisotope requires shielding for the safety of the operators while in use and in storage as these radioisotopes continuously emit gamma rays (cannot be turned off). With most designs the radioisotope is lowered into a water-filled source storage pool (the water in the pool absorbs the radiation) to allow maintenance personnel to enter the radiation shield. One variant of gamma irradiators keeps the radioisotope under water at all times and lowers the product to be irradiated under water in hermetic bells. No further shielding is required for such designs. Other uncommonly used designs feature dry storage by providing movable shields that reduce radiation levels in areas of the irradiation chamber. An incident in Decatur, Georgia where water soluble caesium-137 leaked into the source storage pool requiring NRC intervention[45] has led to near elimination of this radioisotope; it has been replaced by the more costly, non-water soluble cobalt-60. Cobalt-60 gammas also has about twice the energy and therefore normally greater penetrating range than Caesium-137 gammas.
  • 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.


Sterilization by irradiation with gamma rays may however in some cases affect material properties.[46]


Sterile filtration[edit]

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.[47] 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[edit]

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.

Preservation of sterility[edit]

A curette in sterile packaging.

Instruments that have undergone steriliztion can be maintained in such condition by containment in sealed packaging until use.

Aseptic technique is the act of maintaining sterility during procedures.

See also[edit]


  1. ^ WHO Glossary
  2. ^ UCLA Dept. Epidemiology: Definitions
  6. ^ Mesquita, J. a. m.; Teixeira, M.A. and Brandao, S. C. C. (1998). "Tindalization of goats' milk in glass bottles". J. Anim. Sci. /J. Dairy Sci. 76, Suppl. 1 / Vol. 81, Suppl. 1/: 21. Retrieved 2007-03-06. 
  7. ^ Thiel, Theresa (1999). "" (PDF). Science in the Real World. Retrieved 2007-03-06. 
  8. ^ "– Alberta Health and Wellness". Retrieved 2010-06-25. 
  9. ^ Dental Volume 1 - Dentist training manual for military dentists
  11. ^ 2008-09-11
  13. ^ "Sterilization/Aeration Equipment". Retrieved 2010-06-25. 
  14. ^ "Unique solutions for infection control, sterile processing, SPD, and sterilization: EtO Sterilization Solutions". SteriCert. Retrieved 2010-06-25. 
  15. ^ Micro-dose sterilization method
  16. ^ "IARC Vol 60" (PDF). Retrieved 2010-06-25. 
  21. ^ a b c "Mechanism Overview, June 2012". Noxilizer, Inc. Retrieved 2 July 2013. 
  23. ^
  24. ^ Beth Israel Deaconess Medical Center Biosafety Manual (2004 edition)
  30. ^ "Hippocrates on Ulcers". 
  31. ^ "Antibacterial effects of silver". 
  32. ^ Chang TW, Weinstein L (December 1975). "Prevention of Herpes Keratoconjunctivitis in Rabbits By Silver Sulfadiazine". Antimicrob. Agents Chemother. 8 (6): 677–8. doi:10.1128/AAC.8.6.677. PMC 429446. PMID 1211919. 
  37. ^ "Samsung laundry featuring SilverCare Technology". Samsung. Archived from the original on 2006-05-31. Retrieved 2007-08-06. 
  38. ^ "Ultra-Fresh technology is based on the power of silver to fight bacteria". Express Textile. 
  40. ^ Akhavan, O.; Ghaderi, E. (2009). "Enhancement of antibacterial properties of Ag nanorods by electric field". Sci. Technol. Adv. Mater 10: 015003. 
  41. ^ Padmavathy, N. et al. (2007). "Enhanced bioactivity of ZnO nanoparticles—an antimicrobial study". Sci. Technol. Adv. Mater 9: 035004. Bibcode:2008STAdM...9c5004P. doi:10.1088/1468-6996/9/3/035004. 
  42. ^ Bauman, PA; Lawrence LA, Biesert L, Dichtelmüller H, Fabbrizzi F, Gajardo R, Gröner A, Jorquera JI, Kempf C, Kreil TR, von Hoegen I, Pifat DY, Petteway SR Jr, Cai K (July 2006). "Critical factors influencing prion inactivation by sodium hydroxide.". Vox Sang 91 (1): 34–40. doi:10.1111/j.1423-0410.2006.00790.x. PMID 16756599. 
  44. ^ Chang (1985). "UV Inactivation of pathogenic and indicator organisms". Applied Environmental Microbiology 49 (6): 1361. 
  46. ^ Bharati, S; Soundrapandian C; Basu D; Datta S (2009). "Studies on a novel bioactive glass and composite coating with hydroxyapatite on titanium based alloys: Effect of γ-sterilization on coating". J Eur. Ceram. Soc. 29 (12): 2527–35. doi:10.1016/j.jeurceramsoc.2009.02.013. 
  47. ^ Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice. U.S. Department of Health and Human Services. 2004 

Other references[edit]

  • “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