Autoclave Validation

1. Biological Indicator Tests

Biological indicator systems are designed to demonstrate that an autoclave is capable of killing microorganisms.  Only Geobacillus stearothermophilus spores can be used to monitor the effectiveness of steam autoclaves once a month.

Typical biological indicator systems consist of a vial with spore strips or a small glass ampule of growth medium with spores and indicator dye.  Refer to manufacturers’ instructions for use.  The biological indicator is removed from a load after it has been autoclaved.  Then, the biological indicator is incubated at 56oC for three days.  Your control vial, that was not autoclaved, should be turbid after incubation; the successfully decontaminated test vial should remain clear without evidence of turbidity (no growth) and the pH should not change.  If the autoclaved biological indicator is turbid (cloudy, indicating growth) the autoclave did not function properly.  Notify your area supervisor when this happens.

Where to purchase biological indicators:

Some manufacturers of biological indicators have incubators available to fit their vials.  Some examples of biological indicators are: “Duo-Spore Biological Indicator; VWR Catalog # 15425-032.

2. Chemical Color Change Indicator Tests

Chemical indicators for steam autoclaves change color after being exposed to normal autoclave operating temperatures of 121oC (250oF) at 15 psi.  Hence, chemical indicators can give you a quick visual reference for heat penetration inside the load.  Chemical indicators should be positioned near the center of each load, and toward the bottom front of the autoclave.


Most chemical indicators can only be used to verify that your autoclave reaches normal operating temperature for decontamination, 121oC (250oF); they have no time factor.
Chemical indicators alone are not designed nor intended to prove that organisms are actually killed during a decontamination cycle.

Where to Purchase Chemical Indicators:

Chemical indicators are manufactured by many companies and come in a wide variety of sizes, shapes, and colors.  Some examples are: “Dualchek and Single Indicators” - BAXTER, “Test-A-Clave” - BAXTER,  “ATI Indicator Tape” - Thomas, “ATI Sterilometer-Plus 250” - Thomas, “Sterikon-Bioindicator” from EM - VWR, and “TSI Time Indicator” – VWR

3. Tape Indicators

Tape indicators are adhesive backed paper tape with heat sensitive, chemical indicator markings.  Commonly used heat sensitive markings include diagonal stripes (autoclave tape), and/or the word “sterile”.  These markings only appear when the tape has been exposed for a few minutes to normal autoclave decontamination temperatures.  Tape indicators should be used on all material decontaminated by autoclaving to show that the material has been processed.  A three to four inch strip of autoclave tape placed on the outside of the autoclave pan, bag, or individual container is sufficient. If the temperature sensitive tape does not indicate a temperature of at least 250° F (121° C) was reached during the sterilization process, the biomedical waste is not considered decontaminated.


Tape indicators can only be used to verify that the autoclave is reaching normal operating temperatures for decontamination, 121oC (250oF).  Tape indicators alone are not designed nor intended to prove that organisms have actually been killed.