Sterilization Efficacy Verification of Autoclaves

One of the most essential instruments in a microbiology laboratory is the sterilizer, with the autoclave (high-pressure steam sterilizer) being the most commonly used. According to GB 4789.1-2016, laboratory equipment should be regularly inspected and/or calibrated (with inspection tags), maintained, and serviced to ensure proper performance and operational safety. But has your autoclave undergone such inspections? And if you want to perform verification, how should you do it? Today, we'll summarize the key methods for verifying the sterilization efficacy of autoclaves.

Verification of autoclave sterilization efficacy generally includes methods such as chemical indicator, maximum thermometer, self-made temperature tube, and biological indicator. Although these methods share similar principles—mainly confirming whether the sterilizer reaches the required temperature during sterilization—you can choose one or more based on your lab’s specific conditions.

1. Chemical Indicator Method

Principle: Chemical indicators undergo a color or shape change under specific temperature and time conditions. This change is used to determine whether the sterilization parameters have been met.

A commonly used item in laboratories is the 3M autoclave indicator tape, which changes color after sterilization. The tape is made from thermosensitive chemicals, color developers, and paint materials, printed in a stripe pattern on adhesive tape. It is applied directly to the outside of the sterilization package, with a minimum length of 5 cm. Press the tape lightly to ensure good adhesion and sealing. After being exposed to 121°C for 20 minutes or 130°C for 4 minutes, the diagonal white stripes on the tape should turn completely black. If the color change is uneven or incomplete, the package is considered not properly sterilized.

2. Maximum Thermometer Method

Principle: This method uses a mercury thermometer that does not return to a lower temperature once heated, similar to a traditional medical thermometer. It indicates the maximum temperature reached during sterilization.

For verification, place the mercury thermometer inside a water-filled Erlenmeyer flask. During sterilization, position the flask at the upper and lower sections of the autoclave. After the process, check whether the thermometer's reading matches the required temperature. This method can only verify the temperature and cannot confirm whether the sterilization time requirement was met, so it represents the minimum standard for autoclave verification.

3. Self-Made Temperature Tube Method

Principle: This method utilizes certain chemicals that melt and recrystallize at specific temperatures, with distinctive crystal forms post-cooling. These chemicals are sealed in small glass tubes and placed inside the autoclave. After sterilization, the crystal shape is examined to determine if the correct temperature was reached.

Benzoic acid is commonly used, with a melting point of 121–123°C, which aligns with the autoclave’s standard sterilization temperature. Solid benzoic acid is sealed in small glass tubes and placed in the autoclave. After sterilization, the state of the benzoic acid is observed to confirm if the target temperature was achieved. Like the maximum thermometer method, this approach only verifies temperature, not the duration of sterilization.

4. Biological Indicator Method

Principle: This method uses non-pathogenic spores of Geobacillus stearothermophilus as indicator organisms to assess the effectiveness of thermal sterilization. These spores are highly heat-resistant, with resistance similar to that of pathogenic Clostridium botulinum spores, making them suitable indicators for verifying sterilization efficacy.

Biological indicators are available in three forms: spore suspensions, spore strips, and integrated spore-and-medium tubes. These are typically placed at five locations in the sterilization container: the front, middle, and back of the lower layer, as well as the center points of the upper and middle layers. After sterilization, the indicators are inoculated into bromocresol purple-glucose-peptone water and incubated at 55–60°C for 2–7 days. If the medium remains clear and unchanged in color, the spores were killed, indicating good sterilization. If the medium becomes yellow and turbid, the spores survived, suggesting poor sterilization performance. Both spore suspensions and spore strips follow the same validation process.

Commercial biological indicator tubes are also commonly used in laboratories. They contain Geobacillus stearothermophilus spores and a sealed glass ampoule with growth medium. The tubes are placed throughout the sterilization container. After autoclaving, the glass ampoule is crushed to release the medium, and the tube is incubated at 56°C, alongside a positive control. If sterilization is inadequate, surviving spores will grow and turn the broth yellow. If sterilization is effective, the spores are inactivated, and the broth remains purple.

Verification Frequency

Currently, there is no strict national standard regarding the frequency of autoclave efficacy verification. Laboratories are expected to establish their own verification schedules and adhere strictly to them. Considering both ease of use and reliability of results, we recommend using indicator tape in combination with biological indicator tubes, as they offer simple operation and comprehensive verification of sterilization effectiveness.

Notes on Sterilization (not required for some imported fully automatic autoclaves)
When using a high-pressure steam sterilizer, it is crucial to expel all cold air inside the chamber when steam is released. Only after all the cold air is removed should the exhaust valve be closed. If any air remains, the pressure gauge may indicate the correct pressure, but the actual temperature inside the chamber will fall short. The more air retained, the greater the discrepancy between pressure and temperature, potentially resulting in incomplete sterilization. (This is often observed during the sterilization of fermentation media, where air bubbles remain in small guide tubes—try exhausting the cold air more thoroughly.)