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4.15 Verifying Autoclave Performance

4.15.1 Verification Requirement

Autoclaves used for decontamination of biohazardous waste must be regularly tested to verify their performance in reaching proper decontamination conditions. This is necessary to ensure that infectious agents in waste are inactivated prior to leaving the university for final disposal.


4.15.2 Overview of Testing Methods

Approved testing devices include Biological Indicators (BIs), and Chemical Integrators (CIs). So-called “autoclave tape” reacts to exposure to heat with a light-to-dark color reaction, but it is NOT a definitive indicator of decontamination conditions having been met. This tape must not be used as a verification testing method.

Location of the test BI or CI in a waste load is important. Placement of the test device on the outside of an autoclave bag of solid biological waste will not yield the information needed about conditions on the inside of the bag. Instead, the test device must be placed within the bag, preferably in the center of the waste where steam will have the greatest penetration challenge, but not so deeply buried that it cannot be safely and successfully retrieved after decontamination in the autoclave.

Depending on the size of your autoclave chamber, a “load” of waste in need of decontamination may consist of multiple items (e.g., several bags of waste, or a bag and several sharps containers, etc.). You only need to use one test device per load, not in every bag or container in the load. NOTE: The density and volume of your load must determine the length of the cycle you use; i.e., denser, larger loads require longer process times.

When decontaminating liquid biological waste, place the test device in an empty vessel similar or identical in size to the vessel(s) containing the liquid waste (e.g., bottle, flask, etc.) and cap the vessel with the same type of closure as used on the waste container(s). Prior to autoclaving, place the waste vessels and the vessel with the test device in a secondary container (e.g., Nalgene pan, stainless steel pan), spacing them as evenly as possible and leaving room between vessels to allow for good air flow around them.

BIs are used to test autoclaves for:

  • Routine performance verification
  • Following autoclave installation and repair
  • When new autoclave cycles are added, or when cycle parameters are changed
  • When a new load configuration or packaging material is introduced

BIs require incubation to determine results, but they are the more accurate testing method. CIs are not as absolutely accurate as BIs; however, they are generally quite reliable, and provide immediate results (no incubation required). They also are less expensive. Thus, an autoclave verification program which includes both testing methods can make use of each to its best advantage:

  • Verify autoclave performance monthly with BIs and record results in BI log.
  • Verify decontamination of each waste load with CIs and record results on autoclave use log.


4.15.3 Standard Procedure for BI and CI Verification Testing

Biological Indicators (BI)

  1. Label each BI with date, autoclave identification and the cycle type you are testing.
  2. Place the BI inside a bag of solid biological waste.
  3. Label one extra BI as a positive control for the lot number being used. This BI will not be autoclaved; it will be activated and incubated to confirm that the bacterial spores of the test organism will germinate under favorable conditions.
  4. Run the autoclave cycle.
  5. Fill out the monthly autoclave verification log.
  6. Upon cycle completion and cooling of the load, retrieve the BI.
  7. Wearing appropriate PPE, follow product instructions to release the culture media in all test vials to put it into contact with the bacterial spores.
  8. Make sure the vial incubator is turned on and set at the recommended temperature. Place vials in incubator.
  9. Observe and record any color changes at 24 and 48 hours. The positive control should grow (and vial liquid should change color); test vials that have been exposed to successful decontamination conditions in the autoclave should not grow (and vial liquid will not change color).

Chemical Integrators (CI)

  1. Check expiration date on CI before use.
  2. Wearing appropriate PPE, place a CI within a bag of waste, then adjust the closure of the bag to approximately a one inch opening. If autoclaving liquid waste, place CI in a like-sized container.
  3. Run the load on an appropriate cycle for the waste type.
  4. When cycle is complete and load has been removed and cooled, retrieve the CI, interpret the reaction according to information supplied with the CI product.
  5. Record results on Autoclave Use log.


4.15.4 BI or CI Failure to Verify

When BIs or CIs indicate decontamination conditions were not met, an investigation of autoclave performance must take place; the machine must be taken out of service until its status can be determined, and malfunction corrected.

Waste autoclaved with a verification device that failed to validate kill conditions must not be discarded, but re-autoclaved in a different autoclave, or held until the cause of the problem is identified and it can be determined that the waste was in fact sufficiently decontaminated.

Possible causes of BI or CI failure to verify (other than autoclave malfunction):

  • Test devices “Use By” dates have expired.
  • The wrong kind of test device was used (e.g., a test device designed to test dry heat or gas sterilization equipment); the correct test device will specify that it is meant to be used for steam autoclaves.
  • CIs which have been overexposed to light, completely soaked with water, or crimped/bent during use in an autoclave run can result in their performance being compromised.
  • Steam did not fully penetrate the load because 1) the bag was packed too densely with material; 2) the bag was not opened at least one inch before the decontamination cycle was run; 3) the autoclave chamber itself was packed too densely which prohibited steam penetration.