For information on how to safely operate an autoclave see Autoclave Safety and Operation.
Although minimum autoclave parameters of 15 minutes (excluding exhaust time) at 121°C/250°F at 15 psi may achieve sterilization, it is recommended that biological waste run times are extended to at least 60 minutes to assure that the waste has been appropriately decontaminated. Studies have shown that the processing time necessary to achieve decontamination of biological material depends on several factors: load size, type of container, and moisture content. If the waste is packed too tightly or it is a very large load, the length of the sterilization time may need to be increased to allow the steam to penetrate the center of the load. The tighter the autoclave is packed, the longer it will take to reach 121°C in the center of the load. Proper cycle parameters for effective decontamination of infectious waste are done using autoclave indicators and performing validation procedures.
Autoclave indicators are devices used to check the performance of an autoclave and confirm or validate that certain performance standards have been met. The three basic types of indicators are:
Most autoclave users rely on visual and mechanical indicators as routine verification for each autoclave run. However, to assure that the entire biological load has been effectively treated prior to disposal into the regular waste stream, periodic validations using biological indicators should be performed.
According to the Illinois Environmental Protection Agency (IEPA), Title 35 Illinois Administrative Code; Subtitle M, anyone generating and treating infectious biological waste by autoclave shall perform and record validation results at least monthly. To be in compliance with state regulations, all campus laboratories or clinical units who generate and autoclave biohazardous waste under Biosafety Level 2 (BSL-2) containment are required to perform and record monthly validations. Validation testing with biological indicators is also recommended for labs generating Risk Group 1 biological waste such as non-infectious recombinant or synthetic nucleic acids and for other non-waste material to ensure proper autoclave performance.
To assist people on campus who are generating waste, the Division of Research Safety (DRS) developed an autoclave validation program that provides the following services at no charge:
The validation process consists of placing a spore vial (a spore disc inoculated with Geobacillus stearothermophilus spores and a culture medium encased in a glass ampoule with a pH indicator) in the center of representative bags of waste. The vial is retrieved after treatment and incubated to check for cell growth, indicated by a color change. The procedure is as follows:
Control vial: Because the control vial was not autoclaved, the spores remain viable and therefore, will grow when exposed to media. Turbidity and/or media color change are signs indicating growth. Acid production associated with growth lowers the pH, causing the media to change color toward yellow. If the control vial does not exhibit growth via a color change or turbidity, the spores are not viable and the test is considered invalid.
Test vial: The autoclaved vial should exhibit no growth, and the media should retain the original color prior to processing. If spore growth is exhibited–the autoclaved vial changes color or the liquid becomes turbid–the autoclave has failed to sterilize the load and the test must be repeated.
Monthly validation results should be entered on the DRS website for each PI working at BSL-2. If a PIs lab has not entered a result for a month, a reminder email is automatically sent to his/her lab safety contacts. If no results are entered after six months the PI of the lab is notified.
To report results, login to the DRS website by clicking on "My Campus User Login" using your net ID and password and follow the steps below.
For shared lab spaces, only one validation needs to be performed monthly for waste generated from multiple labs. However, results should be entered individually for each PIs lab so that automatic reminder will not be sent.
If test results indicate that the autoclave failed to kill the spores, first check the recorder chart to make sure the correct temperature and cycle time were reached. If the correct time and temperature were achieved in your initial test and the autoclaved spore vial exhibited growth, you will need to change one or more of these parameters when you re-autoclave:
Once enrolled in the autoclave waste validation program, you will be able to request spore vials online by logging into the DRS website and using the link under Profile --> Request Materials--> Spore Vials. Select the Responsible Person, location where spore vials will be delivered, number of vials, and any comments. Save in the upper right hand corner.
If an autoclave in your building is not functioning correctly, it is important that you contact the facilities manager or department safety contact to report the issue as soon as possible. For an autoclave in your laboratory, contact the manufacturer for information on service/repair.