Purpose and Scope
This standard operating procedure for high pressure steam sterilizers is established to standardize operator procedures and ensure personnel safety and normal equipment operation. These procedures apply to verification of high pressure steam sterilizer sterilization effectiveness in laboratory settings.
Responsibilities
- Quality Manager: Approves the verification procedure.
- Quality Supervisor: Responsible for supervising implementation of this procedure.
- Inspection Personnel: Responsible for implementing this procedure and maintaining relevant records.
Experimental Validation Steps
High pressure steam sterilization ensures items reach sterility requirements, which is a fundamental guarantee in pharmaceutical testing. Whether sterilization effectiveness of the autoclave is qualified is the most basic and critical primary step in experimental success.
Most culture media require 121 degrees Celsius high-pressure sterilization for 15 to 30 minutes. Incomplete sterilization of sterile test culture media directly affects drug sterility test results. Therefore, sterilization effectiveness validation of the autoclave must be conducted conscientiously.
Test Materials for Validation
- Bacillus stearothermophilus spores test strips: ATCC7053 is the commonly used biological indicator strain in this method, with spore count of 10 to the 5th power CFU per strip.
- 121 degrees Celsius pressure steam sterilization chemical indicator card.
- Bromocresol purple peptone water culture medium: Prepared and sterilized at 116 degrees Celsius for 20 minutes before use.
- 0 to 150 degrees Celsius temperature recording thermometer.
Method and Results
Place Bacillus stearothermophilus spore test strips (hereinafter referred to as test strips) into sealed test tubes using sterile forceps. Place chemical indicator cards and temperature recording thermometers into open test tubes. Prepare 5 to 10 copies each of these two types of test tubes. Place them respectively at the steam inlet, bottom exhaust port, and bottom water outlet, or at 5 positions including upper, lower, left, right, and center. If the sterilizer has two layers, place at 10 positions.
After sterilization, test strips should be placed into sterilized bromocresol purple peptone water culture medium under strict aseptic conditions and cultured at 56 to 60 degrees Celsius for 24 to 48 hours to observe color changes. If the culture medium turns yellow, it indicates that Bacillus stearothermophilus spores in the test strip have not been completely inactivated and bacteria can still grow in the culture medium, decomposing glucose and producing acid that turns it yellow. If the culture medium color does not change and remains purple, it indicates the spores have been inactivated.
Chemical indicator card indicator color blocks change from light yellow to black during high pressure steam sterilization. According to the depth of color change and comparison with the control color, one can judge whether the sterilization effect meets requirements. Chemical indicator cards should be stored in dry locations, as moisture exposure causes color change and affects observation of sterilization effectiveness.
Key Validation Requirement
High pressure steam sterilization must allow steam to smoothly enter the sterilizer, contact sterilization items, and expel original cold air to achieve sterilization effectiveness. Empty heat distribution and loaded heat penetration validation must be conducted (load should not exceed two-thirds of total volume). Each of the two validations should be repeated three times, for a total of six times.
Results from five points across six tests showing temperatures at 121 degrees Celsius, chemical indicator cards turning black with degree consistent with the control color, and culture media showing no color change all indicate qualified high pressure steam sterilization effectiveness.
Daily Maintenance
For high pressure sterilization pots, daily and periodic maintenance is required. For easily aged components such as sealing rings, frequent inspection is necessary, and damaged parts should be replaced promptly. When using the high pressure steam sterilizer, first ensure that cold air inside the sterilizer is completely expelled before closing the exhaust hole. If some air remains inside the sterilizer, although the pressure gauge may show a certain pressure value, the internal temperature will not reach the corresponding degree. The more retained air there is, the greater the difference between pressure and temperature, resulting in insufficient internal temperature and incomplete sterilization.
