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Application Note – Sterilization N.3 – Good Sterilization Practice

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Application Note – Sterilization N.3

Good Sterilization Practice


Sterilization by steam autoclaving is not correctly performed if only time and temperature parameters are considered. The following notes has the purpose to help the staff to perform a correct sterilization cycle.

  1. Air trapping. Steam displaces air downward. Therefore, long, thin containers should not be placed in the autoclave in an upright position. Sterilization of materials in plastic bags will likewise fail, unless the bags are wide open and only partly filled to allow access of steam and escape of air.
  2. Heat penetration. A cold object placed in an autoclave takes time (over and above the time it takes for the chamber to reach sterilizing temperature) to heat adequately.  For example, a 1 liter container of fluid must be autoclaved for 25 minutes after the chamber reaches 121°C, but a 4-liter container may require an hour for sterilization.
  3. Contact with steam or water. Dry microorganisms protected from contact with steam (for example, with oil, plastic wrappings, or containers of talcum powder) cannot reliably be killed by autoclaving.
  4. Heat indicators. Test tubes and tapes containing a heat-sensitive chemical indicator are often included along with objects when they are autoclaved. The indicator changes color during the autoclaving and thus gives a visual means of checking whether the objects have been subjected to adequate heat. A changed indicator does not always indicate that the object is sterile, because heating may not have been uniform, and even stored wrapped objects may have organisms reintroduced (see No. 7). Tubes or envelopes containing large numbers of heat-resistant spores of the nonpathogenic bacterium Bacillus stearothermophilus, which acts as a biological indicator, are also frequently included with packs of materials being autoclaved.  Death of these spores indicates adequate killing at the point where the tube or envelope was placed, which should be near the center of the material being autoclaved.
  5. Elevated boiling points under pressure. Fluids in autoclaves are prevented from boiling, even though well above their normal boiling point, by the elevated pressure. If at the end of the period of autoclaving, valves are opened to release the pressure, these fluids will immediately boil and may even explode their containers. The pressure must be maintained to prevent boiling until the temperature has dropped below the boiling point at atmospheric pressure.
  6. Negative effects of heat on some materials. Most autoclaves can be adjusted to operate at temperatures lower than 121°C, the usual autoclaving temperature. The use of’ lower temperatures is satisfactory for materials that can be tested for successful sterilization before being used. For example, certain heat-sensitive bacteriological media are sterilized at 115°C. This temperature is usually effective because heat-resistant microbial contaminants are rarely present in the media. Moreover, samples of the autoclaved media are easily tested for sterility before being used.
  7. Prevention of recontamination. Objects to be autoclaved are usually wrapped in paper or cloth to allow penetration of steam during sterilization and to prevent recontamination thereafter. When wet, these coverings are readily permeable to bacteria and should therefore be allowed to dry before removal from the autoclave chamber. After that they should be stored in a closed cupboard or drawer to prevent the reintroduction of contaminants by ants, moths, or other means.

Font: E. Nester, E. Roberts, M. Lidstrom, N. Pearsall, M. Nester: Practical Aspects of Autoclave Use – Microbiology,  Saunders College Publishing. pg 802-803.