An autoclave uses saturated steam under high pressure to achieve sterilization, eliminating all forms of microbial life, including highly resistant spores. To confirm that the intense conditions inside the chamber were sufficient for decontamination, professionals rely on biological indicators (BIs). These indicators represent the only direct measure of the process’s lethality, moving beyond simple physical readings of time and temperature. By challenging the sterilization cycle with the most resilient organisms, BIs serve as the definitive quality assurance tool for ensuring safety.
Selecting the Correct Biological Indicator
The selection of the appropriate biological indicator must be highly specific to the sterilization method being monitored. For steam sterilization, the universally accepted test organism is the spore of Geobacillus stearothermophilus. This bacterium is chosen because its spores exhibit a high degree of natural resistance to moist heat, making it an excellent challenge organism for the autoclave cycle. BIs are available in several physical formats, most commonly as self-contained vials or as spore strips placed inside a process challenge device (PCD).
The self-contained vial is the most convenient format, consisting of a spore-coated carrier and a sealed ampoule of growth medium, all within a small plastic case. A control indicator is necessary to validate the test, regardless of the format used. This control, which is from the same lot as the test BI but is not processed, confirms that the spores were viable and that the incubation medium supports growth. Without a viable control that shows growth, a negative result from the test BI cannot be trusted as confirmation of successful sterilization.
Placement and Monitoring During the Autoclave Cycle
Correct placement of the biological indicator within the autoclave chamber is a procedural step that determines the validity of the entire test. The BI must be situated in the “cold spot,” which is the location within the sterilizer load that is the most difficult for the steam to penetrate and where conditions are the least favorable for sterilization. This location is typically found near the drain or on the lowest shelf, often positioned in the center of the largest package or load. Placing the BI in this worst-case scenario ensures that if the indicator is successfully sterilized, all other items in the load have also been processed effectively.
Many facilities utilize a Process Challenge Device (PCD), a standardized package designed to present a significant challenge to steam penetration, housing the BI within it. While the BI provides biological proof of lethality, physical parameters must still be monitored. Pressure gauges and temperature recorders provide real-time data on the time, temperature, and pressure achieved during the run. The BI then validates that these physical readings translated into the actual destruction of microorganisms, confirming the steam was saturated and able to penetrate the load.
Retrieval, Incubation, and Interpretation of Results
Once the autoclave cycle is complete, the items must be allowed to cool before the biological indicator is retrieved. Immediate handling of the items or the BI can pose a burn risk and may affect the integrity of the indicator. The processed test BI and the unprocessed control BI are then transferred for incubation, which allows any surviving spores to grow. For G. stearothermophilus, the required incubation temperature is maintained between 55°C and 60°C, which is optimal for the thermophilic organism.
The incubation time can vary depending on the product, ranging from rapid-read systems that provide results in 20 minutes to traditional systems requiring 24 to 48 hours for a definitive result. The growth medium within the BI is checked for signs of spore germination and growth. Self-contained BIs often use a pH-sensitive color indicator in the medium, which changes color if the growing bacteria produce acid. A successful sterilization cycle is confirmed if the test BI shows no evidence of growth, meaning the medium remains the original color and is not turbid.
The control BI must demonstrate growth, typically indicated by a color change or cloudiness, confirming the spores were viable under incubation conditions. A sterilization failure is indicated if the test BI shows the same signs of growth as the control (e.g., a color change from purple to yellow), signifying that spores survived the cycle. This interpretation provides the final assurance of sterility.
Troubleshooting Failed Sterilization Cycles
Upon confirmation of a biological indicator failure—where the test BI shows microbial growth—immediate action is necessary to prevent the release of non-sterile materials. The entire load associated with the failed cycle must be immediately quarantined and recalled if any items were already distributed. The first investigative step involves retesting the autoclave using a new BI to determine if the failure was an isolated incident or a recurring problem. If the subsequent retest also indicates a failure, the autoclave must be promptly removed from service until a maintenance technician can address the underlying issue.
Common reasons for sterilization failure often relate to operational errors or mechanical deficiencies. Overloading the chamber, which prevents proper steam circulation and penetration, is a frequent cause. Insufficient steam quality, such as an excessive amount of non-condensable gases or superheated steam, can also compromise the process by failing to transfer the required latent heat. Other issues include mechanical malfunctions, such as clogged filters, faulty temperature sensors, or problems with the chamber door’s seal, all of which compromise the precise conditions required for microbial inactivation.