Sterilization (microbiology)

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Sterilization (or sterilisation) is the elimination of all transmissible agents (such as bacteria, prions and viruses) from a surface, a piece of equipment, food or biological culture medium. This is different from disinfection, where only organisms that can cause disease are removed by a disinfectant.

In general, any instrument that enters an already sterile part of the body (such as the blood, or beneath the skin) should be sterilized. This includes equipment like scalpels, hypodermic needles and artificial pacemakers.

Heat sterilization is known to have been in used in Ancient Rome, but it mostly disappeared throughout the Middle Ages where sanitation was not usually a concern.

The preferred principle for sterilization is through heat. There are also chemical methods of sterilization.

Image:Autoclave Front Loading composition.jpg

Contents

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Heat sterilization

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Autoclaves

A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to 121°C (250°F), at 103 kPa (15 psi) above atmospheric pressure, for 15 minutes. The steam and pressure transfer sufficient heat into organisms to kill them.

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions (discussed later).

To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of times.

Indicator tape is often taped onto packages of products to be autoclaved. The tape contains a chemical that will change color when the appropriate conditions have been met. Some types of packaging have built-in indicators on them. Biological indicators, (such as the Attests), can also be used. These contain Bacillus stearothermophilus spores, which are among the toughest organisms an autoclave will have to destroy.

After a run in an autoclave, the internal glass in the Attest vial is shattered, allowing the spores into a differential liquid medium. If the autoclave destroyed the spores, the medium will remain a blue color. If autoclaving was unsuccessful the B. sterothermophilus will metabolize, causing a yellow color change after two days of incubation at 56°C (132°F).

For effective autoclaving, the steam needs to be able to penetrate everywhere. For this reason, an autoclave must not be overcrowded, and the lids of bottles and containers must be ajar. Indicators should be placed in the most difficult place sterilization is wanted; for instance, if you are sterilizing the contents of universals (a type of small glass jar), the Attest vial should be placed in a universal, to ensure that steam actually penetrates these areas.

For autoclaving, as for all disinfection of sterilization methods, the cleaning off of any biological material is also critical. Biological matter or any grime may shield organisms from the property intended to kill them, whether it physical or chemical. Cleaning can also remove a large number of organisms at once. Proper cleaning can be achieved by physical scrubbing to remove dirt; this should be done with detergent and warm water to get the best results. Where it is not feasible, ultrasound or pulsed air can be used to remove debris.

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Food

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.

It is generally recommended Template:Fact that cooked food be heated to between 74 and 83°C (165 and 180°F) during preparation, as this is the range which actually kills most bacteria; 4 to 60°C (40 to 140°F) is considered the "danger range," in which bacteria actually grow faster, not slower. 60 to 74°C (140 to 165°F) is a range considered safe for "holding" food, but does not sterilize enough living bacteria to be a safe cooking temperature.

The UK Food Standards Agency http://www.food.gov.uk/ publishes recommendations as part of its Hazard Analysis and Critical Control Points (HACCP) programme. The relevant guidelines are at: http://www.food.gov.uk/multimedia/pdfs/csctcooking.pdf. They state that:

"Cooking food until the CORE TEMPERATURE is 75°C or above will ensure that harmful bacteria are destroyed.
However, it should be noted that lower cooking temperatures are acceptable provided that the CORE TEMPERATURE is maintained for a specified period of time as follows :
  • 60°C for a minimum of 45 minutes
  • 65°C for a minimum of 10 minutes
  • 70°C for a minimum of 2 minutes"

Previous guidance from a leaflet produced by the UK Department Of Health “Handling Cooked Meats Safely A Ten Point Plan” also allowed for:

  • 75°C for a minimum of 30 seconds
  • 80°C for a minimum of 6 seconds

as well as the above. Secondary references for the above may be found at:

Note that the above advice is open to critique. For example, spore forming bacteria will survive cooking until the CORE TEMPERATURE is 75°C or above - and may in fact be stimulated to grow. If food is cooked to a core temperature of 75°C It must be kept out of the "danger zone" (5 to 60°C) thereafter to prevent spore formers from multiplying. Spore formers like Clostridium perfringens can cause serious gastroenteritis. Another problem is that although a core temperature of 75°C will kill most dangerous vegetative bacteria it does not inactivate some toxins (eg staphylococcal enterotoxin). So people can still get sick after eating well cooked food.

Recommended cooking conditions are only appropriate if bacterial numbers in food are small. Sterilization does not replace poor hygiene.

For more information, see Foodborne illness.

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Food Utensils

Dishwashers often only use hot tap water or heat the water to between 49 and 60°C (120 and 140°F), and thus provide temperatures that could promote bacterial growth. That is to say, they do not effectively sterilize utensils. Some dishwashers do actually heat water up to 74°C (165°F) or higher; those often are specifically described as having sterilization modes of some sort, but this is not a substitute for autoclaving. Note that dishwashers remove food traces from the utensils by a combination of mechanical action (the action of water hitting the plates and cutlery) and the action of detergents and enzymes on fats and proteins - this removes one of the factors required for bacterial growth (food), and explains why items with cracks and crevices should either be washed by hand or disposed of, as if the water cannot get to the area needing cleaning, the conditions in the dishwasher will promote bacterial growth.

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Bathing

Likewise, no shower or bath you can take, no matter how hot, sterilizes bacteria on your skin. You would die from being scalded long before the water was actually hot enough to do anything but increase the rate of bacterial growth, again ignoring the effects of any detergents or soaps. 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), all of which is considered a prime range for increasing bacterial growth, rather than sterilization, and most human beings shower in water considerably cooler, though it feels scalding hot to them. Humans find water painful at a mere 41 to 42°C (106 to 108°F), which to many bacteria is just starting to get warm enough for them to grow really fast; they will keep growing faster, rather than being killed, up to 55°C (130°F) or more.

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Other Methods

Other heat methods include using dry heat, boiling, flaming and incineration.

Flaming is done to loops and straight-wires in microbiology labs. Leaving the loop in a Bunsen burner until it glows red ensures that any infectious agent gets oxidized completely into small molecules. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below).

Incineration will also burn any organism to ash. It is used to sanitize medical and other biohazardous waste before its ash goes to the tip.

Boiling in water for 15 minutes is unsuitable for sterilization. It is a simple and familiar enough process for anyone to do, though is hazardous and cumbersome. Boiling will kill bacteria and viruses, but it is ineffective against many bacterial spores and 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 be increased. 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 6–12 hours. 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).

For prion elimination, various recommendations state 121–132°C(270°F) for 60 minutes or 134°C (273°F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, as well as strains of CJD and BSE have shown much more resistance. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134-138°C (273-280°F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity. (The initial BSE concentration in the tissue was relatively low). To have a significant margin of safety, cleaning should reduce infectivity 4 logs, and the sterilization method should reduce it a further 5 logs.

By combining immersion in sodium hydroxide (NaOH 0.09N) for two hours with one hour autoclaving (121°C / 250°F), several investigators have shown complete (>7.4 logs) inactivation. (Note that sodium hydroxide may corrode surgical instruments, especially if the sodium hydroxide immersion and autoclaving steps are combined.)

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Chemical sterilization

Chemicals are also used for sterilization. Although heating provides the most effective way to rid an object of all transmissible agents, it is not always appropriate, because it destroys objects such as most fiber optics, most electronics, and some plastics.

Ethylene oxide (EO) gas is commonly used to sterilize objects that cannot survive 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 200mg/l and 800mg/l for at least 3 hours. Ethylene oxide penetrates very well, moving through paper, cloth, and some plastic films and is highly effective. Ethylene oxide however is highly flammable, and requires a longer time to sterilize than any heat treatment. The process also requires time for aeration post sterilization to remove toxic residues. Ethylene oxide is widely used and sterilizes around 50% of all disposable medical devices.

A rapid biological indicator is available for use in EO sterilizers. This indicator contains Bacillus subtilis, which is a very resistant organism. If sterilization fails, incubation at 37°C will cause a fluorescent change within four hours, which is read by an auto-reader. After 96 hours, a visible color change will occur. The fluorescence is emitted when a particular (EO resistant) enzyme is present, which means that spores are still active. The color change is brought on by a pH shift due to bacterial metabolism. The test is suitable for most types of ethylene oxide cycles. The rapid results mean that if a cycle was found to be ineffective, the objects treated can be quarantined and physicians quickly advised of possible contamination.

Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. It has the benefit of being able to oxidize most organic matter. On the other hand, it is a toxic and unstable gas that must be produced on-site, so it is not practical to use in many settings.

Bleach is another accepted liquid sterilizing agent. Household bleach, also used in hospitals and biological research laboratories, consists of 5.25% sodium hypochlorite. At this concentration it is most stable for storage, but not most active. According to the Beth Israel Deaconess Medical Center Biosafety Manual (2004 edition), in most cases, it should be diluted to 1/10 of its storage concentration immediately before use; however, it should be diluted only to 1/5 of the storage concentration to kill Mycobacterium tuberculosis. This dilution factor must take into account the volume of any liquid waste that it is being used to sterilize. Bleach will kill many organisms immediately, but should be allowed to incubate for 20 minutes for full sterilization. Bleach will kill many spores, but is ineffective against certain extremely resistant spores. It is highly corrosive, even causing rust of stainless steel surgical implements.

Glutaraldehyde and formaldehyde solutions (also used as fixatives) are additional accepted liquid sterilizing agents, provided that the immersion time is long enough – it can take up to 12 hours for glutaraldehyde to kill all spores, and even longer for formaldehyde. (This assumes that a liquid not containing large solid particles is being sterilized. Sterilization of large 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 quite 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.

Ortho-phthalaldehyde (OPA) is a sterilizing chemical which received 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.

Another chemical sterilizing agent is hydrogen peroxide. It is relatively non-toxic once diluted to low concentrations (although a dangerous oxidizer at high concentrations), and leaves no residue.

The Sterrad 50 and other Sterrad sterilization chambers use hydrogen peroxide vapor to sterilize heat-sensitive equipment such as rigid endoscopes. The Sterrad 50 sterilizes in 45 minutes and also penetrates some lumen devices. The most recent Sterrad model, Sterrad NX, can sterilize most hospital loads in as little as 20 minutes and has greatly expanded lumen claims compared to earlier models. The Sterrad has limitations with processing certain materials such as paper and long thin lumens.

Endoclens is another device used to sterilize endoscopes. It mixes two chemicals (hydrogen peroxide and formic acid) together to make its antiseptic as needed. The machine has two independent asynchronous bays, and cleans (in warm detergent with pulsed air), sterilizes and dries the endoscopes automatically. All air and water inlets are filtered, and the machine handles temperature, timing and chemical concentration. The total time for the whole process is 30 minutes, and a hard-copy report of the cycle is printed (as well as being stored electronically). Studies with synthetic soil containing bacterial spores showed this machine achieved sterilization effectively.

The Dry Sterilisation Process, DSP, is a process originally designed for the sterilisation of plastic bottles in the beverage industry. It uses hydrogen peroxide with a concentration of 30-35% and runs under vacuum conditions. Using the common reference germs for hydrogen peroxide sterilisation processes, endospores of different strains of bacillus subtilis and bacillus stearothermophilus, the Dry Sterilisation Process achieves a germ reduction of 106...108. The complete cycle time of the process is 6 seconds. The surface temperature of the sterilised items is only slightly increased during the process by 10°-15°. Particularly due to the high germ reduction and the slight temperature increase the Dry Sterilisation Process is also useful for medical and pharmaceutical applications.

NB #1: Unfortunately it is common diction to say "the kill rate is log6" or "the germ reduction is log6", which strictly speaking is not only wrong but nonsensical. By saying this one means that the germ reduction is 6 orders of magnitude or the survival probability of each single germ is 10-6. (This wrong diction originates from a misunderstanding of the mathematical expression log 106 = 6)

NB #2: Strictly speaking it is also wrong to talk about single germs or the like. It's correct to use the item cfu or colony forming unit. The main problem is not inevitably the presence of germs (bacteria, spores, ...) but their ability of fast fissiparous, which gives an exponential increase of the number of the germs with time. If one tries to count "a number of germs" one has to, simply spoken, cultivate them on an agar plate, let them grow for a few days and count the macroscopic colonies which have formed. Each of these colonies is resulting from 1 cfu (= 1 "augmentable germ").

Example #1: One item which has to be sterilized carries a contamination of 107 germs prior to sterilisation. The germ reduction capability of the sterilisation process is 6 orders of magnitude (=106 or "log 6"), which means the survival probability of the germs is 10-6. If such items are sterilised the average number of "surviving germs" or, correctly spoken, cfu's which are found on the items after sterilisation is: 107 / 106 = 10 or 107 * 10-6 = 10.

Example #2 (statistically equivalent to #1): A lot of items which have to be sterilized are carrying a contamination of 10 germs each prior to sterilisation. The germ reduction capability of the sterilisation process is 6 orders of magnitude (=106 or "log 6"), which means the survival probability of the germs is 10-6. If one sterilises a statistically significant number of these items the average number of cfu's which is found on the items after sterilisation is: 10 / 106 = 10-5 or 10 * 10-6 = 10-5. This means that, in average 1 cfu is found per 105 = 100.000 items.


A similar chemical used to sterilize instruments is peracetic acid (0.2%), which is used in the Steris system.

Prions show a great deal of resistance to these sterilization chemicals. Hydrogen peroxide (3%) for one hour was shown to be ineffective in that it had a less than 3 log reduction. 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 the titre by more than 4 logs.

Chlorine and NaOH are the most consistent of these. Chlorine is too corrosive to use on certain objects. Sodium hydroxide has had many studies showing its effectiveness.

In all, the chemical antiseptics do not work well against prions. An interesting note is that treatment with aldehydes (e.g., formaldehyde) have been shown to increase prion resistance.

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Radiation sterilization

Methods exist to sterilize using radiation such as X-rays, gamma rays, or subatomic particles. Gamma rays are very penetrating, but as a result require bulky shielding for the safety of the operators of the gamma irradiation facility; they also require storage of a radioisotope, which continuously emits gamma rays (it cannot be turned off, and therefore always presents a hazard in the area of the facility). X-rays are less penetrating 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. 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.

Devices to irradiate objects are used, for example, by the United States Postal Service to sterilize mail in the Washington, DC area. Also, some foods are irradiated for sterilization (see food irradiation).

Ultraviolet light (UV, from a germicidal lamp) can also be used for irradiation, but only on surfaces and some transparent objects (note that many objects that are transparent to visible light actually absorb UV). It 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 many plastics, as can be seen if one forgets a polystyrene foam object in the cabinet with the germicidal lamp turned on overnight.cs:Sterilizace de:Sterilisation it:Sterilizzazione (medicina) he:עיקור (מיקרוביולוגיה) nl:Sterilisatie (micro-organismen) ja:殺菌 pl:Sterylizacja (technologia) pt:Esterilização (materiais)

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