Autoclave Validation

Chemical and biological indicators are used to validate whether an autoclave is working properly. Chemical indicators are quick to read, but generally, only demonstrate that the autoclave has reached a certain minimum temperature.

They do not give an indication of how long that high temperature was held or whether the procedure was sufficient to provide adequate sterilization. Biological indicators, in contrast, take more time to read but measure the growth or inhibition of bacterial spores generally known to have high resistance to steam and vaporized hydrogen peroxide.

Note that autoclave tape only indicates that a certain temperature threshold has been met and therefore is not sufficient as the only indicator of sterilization. 

Biological Indicators

Biological indicator systems are designed to validate that an autoclave run was successful at killing microorganisms. This is achieved by demonstrating actual effectiveness on bacterial spores, which are generally regarded as difficult to kill. Specifically, Geobacillus stearothermophilus spores are highly resistant to steam and vaporized hydrogen peroxide and are therefore most commonly used in biological indicator tests. G. stearothermophilus spores are inactivated when exposed to temperatures of 121.1oC for at least 20 minutes and therefore can be used as an indicator both of temperature reached as well as the length of time the minimum temperature was held. 

Typical biological indicator systems consist of spore strips that must be plated on growth media for incubation or small glass ampules of growth medium with spores and indicator dye that act as self-contained biological indicators.  The indicator should be placed in the most challenging location to sterilize, which is commonly on the bottom shelf near the drain. After the autoclave load has been run, the indicator is incubated at the manufacturer-specified temperature for 2-3 days and evaluated for spore growth. A control vial, which was not run in the load, should be incubated at the same time and compared against the test vial. If spores survived and proliferated in the test vial, rendering the contents turbid and cloudy, the validation has failed as bacterial spores were still viable. If no spores survived, the test vial will remain clear and validation was successful. If indicator dyes are included in the growth media, there may be a color change in the media with relation to spore growth, due to a change in pH. The control tube, which was not autoclaved, should be turbid, as spores would be expected to grow. If there is no growth in the control tube, the test is invalid and must be repeated. If validation has failed, notify your supervisor or floor administrator, as the autoclave may require servicing and/or settings may need to be changed.

Examples of biological indicators include the Duo-Spore Biological Indicator (VWR Catalog # 15425-032), Sterikon-Bioindicator EM (VWR ), and the Mesa Labs Prospore Self-Contained Biological Indicator (Fisher Scientific Catalog #12-001-1). Whichever product you purchase, follow manufacturers’ instructions for use. 

Chemical Indicators

Chemical indicators for steam autoclaves irreversibly change color after being exposed to normal autoclave operating temperatures of 121oC (250oF) at 15 psi. Note that there is no time factor that causes this color change. Chemical indicators can give you a quick visual reference for heat penetration inside the load.  They are frequently sold as paper or card strips that are positioned near the center of each load, and toward the bottom front of the autoclave. Some indicators may be multi-parameter, and change color with temperature and ethylene oxide, while other indicators may be manufactured for other temperature and time ranges (eg. 275oC, 3min) than standard. Ensure that you are using the appropriate indicator for your sterilization technique.

Chemical indicators alone are not designed nor intended to prove that organisms were actually killed during a decontamination cycle.

Chemical indicators are manufactured by many companies and come in a wide variety of sizes, shapes, and colors.  Some examples include Verify Steam indicator strips and 3M Comply Steam Chemical Indicator strips.

Chemical Tape Indicators

Chemical Tape Indicators commonly appear on adhesive-backed paper tape or on sterilization pouches, where the areas darken upon reaching a specified temperature to form dark diagonal lines and may display words, such as “sterile” or “autoclaved”. 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 that a temperature of at least 250° F (121° C) was reached during the sterilization process, the biomedical waste is not considered decontaminated.