Purpose
Establish standard operating procedures for autoclave operation and maintenance, standardize operator practices, thereby ensuring personnel safety and normal equipment operation.
Scope
Applicable to the verification of sterilization effectiveness of high-pressure steam sterilizers.
Test Procedure
High-pressure steam sterilizers are used to achieve sterility of items, which is a fundamental requirement in laboratory testing. Whether the sterilization effect is qualified is the most basic and critical first step in the success or failure of experiments. Most culture media require sterilization at 121 degrees Celsius for 15 to 30 minutes under high pressure. In particular, incomplete sterilization of sterility test media directly affects the results of sterility testing of pharmaceutical products. Therefore, it is essential to carefully verify the sterilization effectiveness of the autoclave.
Test Materials
- Bacillus stearothermophilus paper strips (ATCC 7053) as the biological indicator, containing 5 x 10^5 CFU per strip.
- 121 degrees Celsius pressure steam sterilization chemical indicator cards.
- Bromocresol purple peptone water medium, sterilized at 116 degrees Celsius for 20 minutes before use.
- 0 to 150 degrees Celsius maximum-indicating thermometer.
Methods and Results
Using sterile forceps, place the Bacillus stearothermophilus paper strips into sealed test tubes. Place chemical indicator cards and maximum-indicating thermometers into open test tubes. Prepare 5 to 10 copies of each type of tube.
Position these tubes at five locations throughout the sterilizer: the steam inlet, the bottom exhaust port, the bottom water outlet, and the top, bottom, left, right, and center positions. For two-tier sterilizers, use 10 placement locations.
The maximum-indicating thermometer must be calibrated and qualified before use in verification testing. Before testing, the mercury column must be shaken down to below 40 degrees Celsius. After each monitoring cycle, the temperature difference between maximum-indicating thermometers should be within 1 degree Celsius, indicating uniform temperature distribution within the sterilizer.
After sterilization, under strict aseptic conditions, transfer the sterilized paper strips into sterilized bromocresol purple peptone water medium and incubate at 56 to 60 degrees Celsius for 24 to 48 hours, observing color changes.
- If the medium turns yellow, it indicates that the Bacillus stearothermophilus on the paper strip was not completely inactivated, and bacteria are still growing in the medium, decomposing glucose and producing acid.
- If the medium remains purple, it indicates that the spores have been inactivated.
Simultaneously, use unsterilized paper strips placed in medium as a positive control, and blank medium without paper strips as a negative control.
The indicator color block on the chemical indicator card changes from light yellow to black during high-pressure steam sterilization. By comparing the depth of color change with the reference color, the sterilization effect can be determined.
High-pressure steam sterilization must allow steam to enter the sterilizer smoothly, make contact with the items being sterilized, and expel the original cold air to achieve effective sterilization. Perform empty-load heat distribution and full-load heat penetration verification, with each type of verification repeated three times for a total of six tests.
Routine Periodic Maintenance
Regular periodic maintenance must be performed on the autoclave. For components prone to aging such as the sealing ring, frequent inspection is required, and damaged parts should be replaced promptly.
Operating Precautions
When using a high-pressure steam sterilizer, the first important note is to expel the cold air from within the sterilizer while releasing steam. The cold air within the chamber must be completely expelled before closing the exhaust port.
If residual air remains in the sterilizer, even if the pressure gauge has reached a certain value, the temperature inside may not have reached the corresponding level. The more air that remains, the greater the discrepancy between the pressure reading and actual temperature. If the internal temperature is insufficient, sterilization will be incomplete.
