Sterilization (microbiology)

Sterilization (or Sterilisation) is the elimination of all transmissible agents (such as bacteria, prions and viruses) from a surface or piece of equipment. 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 things 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 some chemical methods capable of sterilization.

Heat sterilization

Probably the most widely used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to 121°C, at 103 kPa (15 psi), for 15 minutes. The steam and pressure allow sufficient heat is transferred into the organism to kill them.

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

To ensure the autoclaving process was able to cause sterilization, most autoclaves haves 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 if 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 sterothermophilus spores, which are amongst 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.

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 universal is 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. 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. Heating small metal or glass objects is really the extent to which this method is used.

Incineration will also burn any organism to ash. It obviously is only used to sanitize unwanted medical and other waste before its ash goes to the tip. Boiling in water for 15 minutes is unsuitable for sterilization. It’s 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 required, must be increased. The standard setting for a hot air oven is at least two hours at 160°C. A rapid method heats air to 190°C 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. (E.g. it won’t cause rust.)

In terms of prion elimination there is no agreed ideal time and temperature. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by standard sterilization procedures. Other strains of scrapie, as well as strains of CJD and BSE have shown much more resistance.

Using mice as a test animal, one experiment showed that heating BSE positive brain tissue at 134-138°C 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 infectively 4 logs, and the sterilization method reduce it a further 5 logs. The recommendations state 121-132°C for 60 minutes, and 134°C for at least 18 minutes. There is little conclusive evidence to prove this is either sufficient or insufficient, though the BSE example above does not seem to suggest it's appropriate.

By combining immersion in sodium hydroxide (NaOH 0.09N) for two hours, with one hour autoclaving (121°C) several investigators have shown complete (>7.4 logs) inactivation. (Sodium hydroxide may, though, be deleterious to surgical instruments.)

Chemical sterilization

Chemicals also have a place in sterilization. Heating provides the most effective ways to rid and object of all transmissible agents, though it is not always appropriate, because of the material an object is made of. (Such as plastics, fiber optics, and electronics.)

Ethylene oxide (EO) gas is a commonly used sterilization procedure for objects that cannot survive temperatures greater than 60°C. Ethylene oxide treatment is generally carried out at 54-60°C in 60-75% humidity for at least 12 hours. Ethylene oxide penetrates very well, moving through paper, cloth, and same plastic films. It is however highly flammable and requires a much longer time than any heat treatment. It also needs time to aerate completely, as it is a potential carcinogen.

There is a new rapid biological indicator for use in EO sterilizers. These indicators contain 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 have occurred.

The fluorescence is emitted when a particular (EO resistant) enzyme is present, which means 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.

Glutaraldehyde is an accepted liquid antiseptic, provided the immersion time is long enough – it can take up to 12 hours to kill all spores. It is not a particularly nice chemical to deal with, it is toxic and skin contact should be avoided. It has quite a short shelf-left (<2 weeks), and is expensive.

A new chemical comparable to gluteraldehyde is ortho-phthalaldehyde (OPA), which got FDA clearance in late 1999. Typically used in a 0.55% solution, OPA shows better myco-bactericidal activity than gluteraldehyde. It also is effective against gluteraldehyde-resistant spores.

OPA has superior stability, has less odor and does not irritate skin or eyes, and it acts more quickly than gluteraldehyde. It is more expensive though, and will stain proteins (including skin) gray in color.

Another chemical antiseptic used is hydrogen peroxide. Hydrogen peroxide is made of two atoms of hydrogen and two of oxygen, because of this it is non-toxic and leaves no residue.

The Sterrad 50 (as well as other Sterrad sterilization chambers) use hydrogen peroxide vapor to sterilize heat-sensitive equipment such an endoscapes. The Sterrad 50 sterilizes in 45 minutes, and it sterilizes as effectively as ethylene oxide treatment, also penetrating small lumened devices.

Endoclens is also a device 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 sports showed this machine achieved sterilization effectively.

Another 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, gluteraldehyde and peracetic acid also fail this test (one hour treatment).

Only chlorine, a phenolic, guanidine thiocyanate and sodium hydroxide (NaOH) reduce the titre by more than 4 logs.

Chlorine and NaOH are the most consistent of these. Chlorine though is sometimes 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.

It must be remembered that prion elimination is not important in most cases, as CJD and BSE are not common.

Radiation sterilization

Methods exist to sterilize using radiation such as X-rays or gamma rays. Devices to do so are used, for example, by the United States Postal Service to sterilize mail in the DC area. Also, some foods are irradiated for sterilization. Irradiation does not make things radioactive.

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