For information on how to safely operate an autoclave see Autoclave Safety and Operation.

Autoclaving Biological Waste

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

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: 

  1. Visual indicators (chemical/tape) measure one or more physical conditions of the autoclave cycle. The most commonly used visual indicator is autoclave tape, which contains a heat-sensitive ink that changes color from white to a visible pattern following processing. This indicates that a temperature of 121°C has been achieved. If the tape fails to change color, this alerts the user that there is a problem. Note that the color change does not assure decontamination effectiveness; it only verifies that the outside of the container came to temperature and does not reflect conditions inside the load.
  2. Mechanical indicators are integrated into the autoclave and record the time-temperature-pressure profile attained during a cycle.
  3. Biological indicators are composed of a standardized population of heat-resistant bacterial spores such as Geobacillus stearothermophilus, most commonly in the form of spore vials. They are used to determine if the sterilization cycle parameters were sufficient to kill the test microorganisms. 

Autoclave Waste Validations

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:

  • Providing initial basic autoclave safety and validation orientation to designated safety contacts. A starter kit containing spore vials, vial crushing tool, and a wood dowel rod for vial retrieval is provided.
  • Delivering additional spore vials to safety contacts.
  • Collecting and disposing of expired spore vials.
  • Hosting an online log for safety contacts to record monthly validation results.
  • Providing technical support.


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:

  1. Don PPE (gloves, lab coat, and safety glasses).
  2. Check spore vials to assure that they are not expired.
  3. Securely tape the spore vial to one end of the dowel rod using lab tape or autoclave tape for easy retrieval after processing.
  4. Position the end of the dowel rod with the taped spore vial into the center of the waste load, allowing the opposite end of the dowel rod to stick out of the bag.
  5. Autoclave material as usual (see instructions for autoclave use).
  6. After the cycle is complete, remove waste from autoclave and cool.
  7. Retrieve the vial and cool for 10 additional minutes. 
    Caution: The vial is hot and under pressure. Failure to allow sufficient cooling time may result in an injury if the vial bursts.
  8. Do not discard the test load until the validation test is completed and passed.
  9. Using the crushing tool provided by DRS, break the media ampule inside the autoclaved vial and an untreated vial (positive control), exposing the spores to media.
  10. Incubate both vials in a vertical position in a heat block, incubator, or water bath at 55°C overnight.
  11. After the appropriate incubation time, compare the color of the media in the vials. 

Interpreting the Results

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.

Reporting Results

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.

  1. Click "Profile" then select "Autoclave Waste Validations" located underneath "Facility Info".
  2. Click "Add New" located in the blue bar in the upper right hand corner.
  3. Select the Responsible Person/PI from the dropdown menu.
  4. Select the Reporting Month for the validation result being entered from the dropdown menu.
  5. Indicate status of waste validation.
  6. If prompted - select the autoclave used and fill out Date Tested, Cycle Name, Temperature (C), Duration (Minutes), Pass or Fail (if needed).
  7. Click "Add Attempt".
  8. Click "Save" located in the blue bar in the upper right hand corner.

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.

Test Failure

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:

  • Time: Increase the cycle time by repeating validation procedure. 
    Reminder: The cycle time required will vary with the composition of the load.
  • Density: Load the bag to 75% of the holding capacity or less. Do not compress waste to fit more in the bag because steam cannot penetrate completely through densely packed waste bags.
  • Steam: Add approximately one half cup of water to your dry waste loads to facilitate steam generation.
  • Loading techniques: change how you load the autoclave to allow steam to better move from the top of the chamber to the bottom and penetrate the load.
  • Place bags in the back of the autoclave rather than in the front of the autoclave, which is the coldest part of the chamber.
  • Avoid crowding or stacking.
  • Check that the bags are not touching the top or sides of the autoclave as you push the tray inside.
  • Try using shallower trays (but make sure the tray will still contain any spills).

Request Spore Vials

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.

Autoclave Repair

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.

    Last Updated: 4/28/2023