Autoclave sterilization effect verification

One of the essential laboratory instruments in a microbiology laboratory is the sterilizer, with autoclaves being the most commonly used type. According to GB 4789.1-2016, laboratory equipment should be regularly inspected and/or calibrated (with appropriate labeling), maintained, and serviced to ensure operational performance and safety. But does your sterilizer undergo similar inspections? If such validation is required, how should it be conducted?

Today, we will summarize the key aspects of sterilization efficacy validation for autoclaves.

The common methods for validating autoclave sterilization efficacy include the chemical indicator method, the fixed-point thermometer method, the self-made temperature probe method, and the biological indicator method. The principles behind these methods are similar, primarily focusing on verifying whether the temperature inside the sterilizer reaches the required level during sterilization. Depending on the specific conditions of the laboratory, one or multiple methods can be selected for validation.

1. Chemical Indicator Method
Principle: Chemical indicators undergo color change or deformation when exposed to a specific temperature and duration, allowing assessment of whether sterilization parameters have been met.

A commonly used indicator in laboratories is the 3M autoclave indicator tape, which changes color before and after sterilization to indicate effectiveness. This tape is made with heat-sensitive chemicals and color-developing agents printed in stripes on a special adhesive tape. The tape should be applied to the outside of the package, with a minimum length of 5 cm, and pressed firmly for better adhesion and sealing. After sterilization at 121°C for 20 minutes or 130°C for 4 minutes, the diagonal white stripes on the tape will turn completely black. If the color change is uneven or incomplete, the package may not have met sterilization conditions.

2. Fixed-Point Thermometer Method
Principle: This method uses a mercury thermometer that retains the highest temperature reached, similar to a traditional clinical thermometer. It helps determine the maximum temperature achieved inside the autoclave during sterilization.

For validation, a mercury thermometer is placed inside a large conical flask filled with water. During sterilization, the flask is positioned at both the upper and lower sections of the autoclave. After sterilization, the thermometer reading is checked against the required temperature. However, this method only verifies temperature and does not confirm whether the sterilization duration was sufficient, making it the most basic standard for autoclave validation.

3. Self-Made Temperature Probe Method
Principle: This method leverages the melting and recrystallization characteristics of certain chemicals when exposed to heat. By sealing these chemicals inside small glass tubes and placing them in the autoclave, the crystal formation after sterilization can indicate whether the required temperature was reached.

A commonly used reagent is benzoic acid, which has a melting point of 121–123°C, closely matching the required sterilization temperature. During sterilization, solid benzoic acid is sealed in a small glass tube and placed inside the autoclave. After the process, the crystal structure of the benzoic acid is examined to determine if the required temperature was achieved.

Like the fixed-point thermometer method, this approach only indicates temperature and cannot confirm whether the sterilization duration was sufficient.

4. Biological Indicator Method
Principle: This method uses non-pathogenic Geobacillus stearothermophilus spores as indicator organisms to assess the effectiveness of heat sterilization. These spores are highly resistant to heat and have a thermal resistance similar to Clostridium botulinum spores, making them a reliable reference for evaluating whether the autoclave meets sterilization requirements.

Biological indicators come in three forms:

Spore suspensions
Spore strips
Spore strips combined with a culture medium (biological indicator tubes)
The biological indicators are typically placed at five locations inside the sterilization chamber:

Lower level: front, middle, and back
Upper level: center
After sterilization, the indicators are inoculated into bromocresol purple-glucose peptone water and incubated at 55–60°C for 2–7 days:

If the culture medium remains clear and unchanged in color, the spores have been killed, indicating effective sterilization.
If the medium turns yellow and turbid, the spores have survived, meaning the sterilization process was ineffective.
The same validation method applies to both spore suspensions and spore strips.

Many laboratories also use commercial biological indicator tubes, which work similarly to spore suspensions and strips. These tubes contain G. stearothermophilus spores along with a glass ampoule of culture medium. After autoclaving, the glass ampoule inside the tube is crushed to release the culture medium, and the tube is incubated at 56°C, with a positive control included.

If sterilization was ineffective, viable spores will grow, turning the broth yellow.
If sterilization was successful, the spores are inactivated, and the broth remains purple.
Frequency of Autoclave Validation
Currently, there are no strict regulatory standards defining how frequently autoclaves must be validated. However, laboratories should establish their own validation schedule and strictly adhere to it.

For ease of operation and reliable validation, chemical indicator tape and biological indicator tubes are highly recommended. These methods are user-friendly and provide a comprehensive assessment of sterilization effectiveness.

Key Considerations for Autoclaving
(Some fully automated imported autoclaves may not require manual venting)

When using an autoclave, it is essential to remove cold air from the chamber while introducing steam. The exhaust vent should remain open until all cold air is expelled, ensuring an even temperature distribution inside.

If any air remains inside the chamber, the pressure gauge may indicate the correct pressure, but the actual temperature will be lower than expected. The more residual air, the greater the discrepancy, leading to incomplete sterilization.

(For those encountering air bubbles in small tubes when sterilizing fermentation-based media, try increasing air evacuation to improve results.)