20. Autoclaving Procedures

Autoclaves are the most dependable system available for the decontamination of laboratory waste and the sterilization of laboratory glassware and reagents. Autoclaves use saturated steam, which contains the maximum amount of water vapor at a given temperature and pressure.

Moist heat will destroy microorganisms at lower temperatures and shorter contact times than dry heat.

For maximum heat transfer, steam must flush out the air in the autoclave chamber. Before using the autoclave, empty the drain screen at the bottom of the chamber. If the sieve is blocked with tape or debris, an air layer may be formed at the bottom of the autoclave where optimum temperatures will not be reached.

Preparation of Materials

• Fill liquid containers only half full.
• Loosen caps.
• Use only vented closures.

Loading

• Remember that steam must make contact with all surfaces; the temperature within an air pocket is much lower than that of the saturated steam surrounding it.
• Areas where steam cannot penetrate, or from which air cannot escape may not get sterilized.
• Air in closed or upright containers (animal cages, erlenmeyer flasks, etc.) is not readily replaced with steam and, consequently, the temperature in these containers may remain sublethal.

To Facilitate Air Removal / Steam Penetration

• Loosen the lids of containers.
• Do not seal biohazard bags.
• Leave space between bags.
• Use shallow trays and low-sided containers to catch spills.

Cycle Selection

Remember to use the liquid cycle when autoclaving liquid waste. This allows for slow exhaust and prevents boiling over of the superheated liquids.

Time Selection

• The operator must take into account the size of the articles to be autoclaved. A two-liter flask containing one liter of broth takes longer to sterilize than four 500 ml bottles each containing 250 ml of broth.
• Material with a high insulating capacity (animal bedding, high-sided polyethylene containers) will require longer autoclave cycles for the load to reach sterilizing temperatures.
• Biohazard bags should be autoclaved at least 60 minutes to assure decontamination.

Removing the Load

• Check that the chamber pressure is zero.
• Wear goggles, lab coat, and heat insulating gloves.
• Open the autoclave door only a crack; do not throw it wide open. Let residual steam escape and allow time for gradual cooling of the load.

Monitoring

Autoclaving techniques should be tested to prove effectiveness.