Autoclave/Dry Heat Monitoring

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Autoclave/Dry Heat Monitoring

Postby russ » Thu Jun 02, 2011 12:58 pm

Upon requests, this is some info that may help get started: Biological Indicator usage for Steam Autoclaves and Dry Heat Ovens to meet NELAC Certification Requirements
Author: Russ Nyberg BS, BSEd., MAM Technical Support for Raven Biological Laboratories`

An increasing number of water testing laboratories are seeking NELAC (ELAP) Certification. Laboratories seeking this accreditation under NELAP must assure implementation of all the NELAP QA policies, procedures and methods specified by NELAP for quality systems.
The use of Biological Indicators (BIs) to check the efficacy of the laboratories steam autoclaves and dry heat ovens is part of the requirements for meeting NELAC certification of equipment. For many laboratories this is something that has not been done before and thus many questions are now being brought to the forefront. Hopefully the following information may help to answer many of the questions being asked.
Chapter 4 of the ‘Virus Methods Manual’ states on page 6, section 6.11 and 6.13 that “autoclave operation must also be checked weekly with maximum-minimum thermometers and spore strips or suspensions”, and “hot air ovens must also be checked weekly with spore strips.” Other regulations, states, etc. require monthly testing. This requirement may be something very new to many laboratory facilities. NELAC and ELAP may require similar testing requirements on a weekly or monthly basis. NELAC (on-site laboratory assessment checklist), D.3.8(b)(2)(ii) looks for monthly use of biological indicators for sterilizer effectiveness.
Looking at the steam autoclave requirement first, we have a choice of BIs to use. They can be either spore strips or spore suspensions. Both the spore strips and suspension BIs will contain the same species of bacterial spores for use. Both must contain Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) spores as listed in USP as the organism of choice for use in steam sterilization. This organism was chosen due to its high resistance to moist heat sterilization. The spore strips will have the spores in a dry form impregnated throughout the fiber content of the filter paper strip. The spore strips then are normally packaged in a glassine envelope. This envelope makes it easy to use and handle the strip without contaminating the actual paper strip containing the spores. Prior to the start of a steam cycle that is to be checked, several spore strips are randomly placed in different areas of the chamber and in the chamber load itself. These areas should include the most difficult to sterilize areas of the chamber including the area near the chamber drain.
It is important to remember that a spore strip placed into the chamber is checking the chamber sterilization conditions only. This means that if you are running a ‘liquid load’, spore strips placed into the chamber will not be checking sterilization conditions in the actual liquid flasks you would be trying to sterilize. Due to a long delay in the actual time needed for a liquid load to come up to temperature as compared to the short time needed for the chamber to come up to temperature, the exposure time needed for the two different areas to reach sterilization conditions will differ greatly. Your chamber may hit 121°C within the first 30 seconds of your cycle while the liquid in a flask that you are running may not hit 121°C for another 10 to 15 minutes. To actually sterilize your media ‘in the flask’ or follow manufacturers instructions of ‘sterilize at 121C for 15 minutes’, you would need to extend your come-up time by 15 minutes. If the liquid took 15 minutes to come up to temperature, then the total cycle set time would need to be at 30 minutes (15 min come-up and 15min at 121C). For use in liquid loads, the use of a suspension type BI would provide more accurate information as to the cycle’s efficacy in liquid sterilization. The use of such suspension BIs will be addressed later.
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Continuing with the use of spore strips (Photo #1 shows typical spore strip packaging), once the spore strips have been placed about in the chamber or load, the sterilization cycle is started. Once the cycle is complete, the spore strips can now be removed. To provide information as to whether the spore strips were killed or not during the sterilization cycle, the spore strips must now be transferred ‘aseptically’ to a suitable tube containing growth media. This would typically be a 10ml tube containing Tryptic Soy Broth (TSB). Sterile tubes of TSB that have been tested and certified for ‘growth promotion’ according to USP can be purchased commercially. The important part of the transfer of the spore strips into TSB is the word ‘aseptic’. Aseptic transfers must be made to avoid the possibility of post-sterilization contamination. The use of a laminar flow hood may be needed to achieve an aseptic transfer. The tubes are then incubated at 55-60°C and monitored for signs of ‘Growth/No Growth’. The ‘Growth/No Growth’ testing results should be documented for each cycle tested and these documents retained as verification that testing was done. If your cycle was successful in killing the resistant spores of the BI and ‘No Growth’ occurred with incubation, it is then likely that all other types of bacteria were also killed in the cycle and that your load has a high sterility assurance level.
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If one is using a suspension type BI for checking the steam autoclave, the ‘aseptic transfer’ step is eliminated. This is due to the suspension BI ampoule being completely sealed to avoid any possibility of post sterilization contamination (Photo #2 shows various volume sizes of suspension ampoules). You will note that it is important to have the ampoules sink within the liquid rather than float on the top of the liquid. We are not testing the surface conditions of the liquid which heats up faster than the bottom. The lower portion of the liquid is the proper location for suspending the ampoules. The suspension ampoule will contain bacterial spores and it also contains the TSB growth media already in the ampoule. No aseptic transfer to growth media is needed as with the spore strips. With the suspension ampoule, it is placed in the autoclave, the cycle is run and when the ampoule is removed, it is just placed directly into the incubator at 55-60°C and monitored for ‘Growth / No Growth’.
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The suspension ampoule is much easier to use and the equipment needed to perform aseptic transfers is not required. Another benefit of the suspension ampoule is that since it is a sealed glass ampoule suspension, it can be dropped directly into a flask of liquid being sterilized (Photo #3 shows suspension ampoules placed into liquids to be sterilized). This way one is checking the sterilization conditions of the actual liquid being sterilized rather than just the chamber conditions or surface of the liquid.
With dry heat ovens, spore strips must be used. With dry heat, spore strips that contain spores of the bacteria Bacillus atrophaeus (formerly Bacillus subtilis) are used. The placement of spore strips within the dry heat oven is similar as to the use and placement within the steam autoclave as mentioned above. Following an exposure cycle, the strips are removed from the dry heat oven and aseptically transferred to tubes containing TSB. The tubes are then incubated at 30-35°C and again ‘Growth / No Growth’ results are documented and retained. Small table top incubators as shown on the right are available for incubating your Biological indicators. You can get these incubators with temperatures pre-set for use with Steam or dry heat ovens. Small table top incubators are much less expensive than the large lab incubators usually used to incubate media plates.
When choosing a vendor for your supply of BIs it is important that you choose one that is registered as a medical device manufacturer with the FDA and who has demonstrated through 510k submissions the reliability of the BIs made. This is extremely important so that your documentation can show that acceptable BIs were used in your testing. This issue could certainly come up during a certification audit.
Implementing the use of biological indicators to meet NELAC requirements for certification is not a difficult task. Most manufacturers of BIs would be happy to answer any questions you may have for getting started with BIs.


Russ Nyberg is currently a member of AAMI, PDA, ASM, APHL, TNI, ABSA and AWWA. Russ works as Technical Support Director for Raven Labs in Omaha, Nebraska at the Biological Indicator manufacturing site. Russ can be contacted at russ@ravenlabs.com
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