Appendix C: Autoclave Quality Assurance Program

Autoclaving is an accepted procedure for the decontamination of certain biohazardous waste. Biological cultures and stocks, contaminated solid waste, and liquid waste can be sterilized through autoclaving. After sterilization in a steam autoclave, these materials are considered non-infectious. All autoclaved waste is placed into the solid biohazard waste stream. Wastes from the BSL4 and BSL3 containment laboratories are first autoclaved before being disposed as biological waste. Materials that contain hazardous chemicals or radioisotopes are not to be autoclaved. To ensure that biohazardous waste is properly decontaminated during autoclaving, the following procedures should be followed by laboratory personnel.

1. Processing Time

Infectious waste must be treated in an autoclave for a minimum of 30 minutes at 121°C (250°F); however, the total processing time required to decontaminate infectious waste depends on the specific loading factors (container type, water content, quantity, etc.). A total processing time of 60 minutes is recommended for gravity displacement autoclaves and 10 minutes for vacuum-type autoclaves (132° C).

Sterilization by autoclaving is accomplished through exposure and penetration of the contaminated material by superheated steam for an adequate amount of time. Because steam will not penetrate a sealed plastic autoclave bag, the bags containing dry loads must not be tightly sealed (rubber band closures will allow bags to “breathe”) to allow the steam to enter the bag or an adequate amount of water must be added to the load. Consult the manufacturer’s instructions for sterilizing materials inside plastic autoclave bags. Liquid waste may also be autoclaved in lieu of adding an appropriate chemical disinfectant and disposed of in the sink. Animal carcasses from the ABSL3 and ABLS4 will be autoclaved inside an autoclavable bag.

2. Steam Sterilization Indicator

All autoclaved waste must include a chemical sterilization indicator (the use of biohazard bags with a “built-in” indicator is recommended).

3. Minimum Temperature

Steam autoclaves used to treat infectious waste must operate at a minimum temperature of 121° C. The operating temperature of the autoclave must be verified for each run by maintaining a record of the temperature either as a chart or paper tape recording or a manual recording in a logbook.

4. Confirm Adequate Sterilization Conditions

On a monthly basis, confirm that adequate sterilization conditions are being met through the use of Biological Indicators (“BI”) containing heat-resistant spores (e.g. Geobacillus stearothermophilus) placed in the center of an autoclave load. In conjunction with the BI testing, measure and record the maximum temperature achieved during the autoclave cycle through the use of a maximum registering (or “holding”) thermometer or calibrated data logger for full cycle.

5. Maintain Records

Maintain records of BI testing and maximum autoclave temperature recordings for a minimum of one year (see Autoclave QC Log at end of appendix).

Monthly Spore Testing Procedure

1. Place BI spores and holding thermometer or data logger in the center of an autoclave load.
2. Process the load under normal operating procedures.
3. The highest temperature indicated on the holding thermometer is entered on the Autoclave QC Log.
   • If this temperature is less than 121°C, the autoclave is not to be used to treat infectious waste until it has been repaired and passes retesting. In the interim, tag the autoclave as “Not Approved for Infectious Waste.”
4. Incubate the autoclaved BI and a non-autoclaved, control BI according to the manufacturer’s instructions (normally 55°-60°C overnight).
5. If a color change (cell growth) occurs, the sterilization process was unsuccessful. Discontinue use of the autoclave until it is checked, repaired as needed. and passes retesting. Tag the autoclave as “Not Approved for Infectious Waste” until the autoclave passes retesting.
6. Indicate test results on Autoclave QC Log available from EHS and retain for at least three years.