Validation of sterilization effect of high-pressure sterilizer

Validation of sterilization effect of high-pressure sterilizer

One of the essential experimental instruments in microbiology laboratories is a sterilizer, and high-pressure steam sterilizers are commonly used in laboratories. The regulations require that experimental equipment should be regularly inspected and/or calibrated (marked), maintained and serviced to ensure work performance and operational safety. Has your sterilizer undergone similar calibration or validation? What is the specific method? Let's take a look at the verification of the sterilization effect of high-pressure steam sterilizers together.

Validation of sterilization effect of high-pressure sterilizerThere are generally chemical indicator method, point thermometer method, self-made temperature measuring tube method, and biological indicator method. The principle of each method is similar, mainly by verifying whether the temperature in the sterilizer can meet the requirements during sterilization. We can choose one or more methods for validation based on the specific situation of our laboratory.

1、 Principle of chemical indicator method:

Chemical indicators will change color or deform due to heat at a certain temperature and exposure time. Based on this characteristic, it is determined whether the required sterilization parameters have been achieved. The commonly used indicator tape in laboratories is 3M pressure sterilization tape, which uses the color change of the tape before and after sterilization to determine the sterilization effect. It is made of thermosensitive chemicals, colorants, and paint additives to create ink, which is then printed in stripes on a specially designed adhesive tape. The indicator tape can be directly attached to the outside of the package, with a length of not less than 5cm, and lightly pressed to increase adhesion and packaging effect; After 20 minutes at 121 ℃ or 4 minutes at 130 ℃, the diagonal white indicator lines printed on the tape will turn black and become black lines; If the color change is uneven, it can be considered that the package does not meet the sterilization conditions.

2、 Principle of leaving a temperature gauge method:

The retention thermometer method utilizes the non reflux characteristic of mercury thermometers, and its principle is similar to traditional thermometers. It can indicate the highest temperature reached by the sterilizer during the sterilization process. When verifying, place the mercury thermometer in a large triangular flask filled with water. When sterilizing, place the triangular flask on the upper and lower parts of the sterilizer. After sterilization, check whether the temperature of the mercury thermometer is consistent with the required temperature. This method can only verify temperature and cannot indicate whether the sterilization time meets the requirements, so it is a relatively low standard for sterilizer validation.

3、 Principle of self-made temperature measuring tube method:

By utilizing the different characteristics of crystal shapes caused by the melting and cooling of certain chemicals, the chemicals are sealed in small glass tubes and placed in sterilizers during sterilization. After sterilization, the shape of the crystals can be observed to determine whether the temperature meets the standard. The commonly used reagent is benzoic acid, which has a melting point of 121-123 ℃, which is basically consistent with the sterilization temperature required by our sterilizer. Therefore, during sterilization, solid benzoic acid is sealed in a small glass tube and placed in the sterilizer. After sterilization, the state of benzoic acid can be observed to verify whether the sterilizer has reached the required temperature. The limitations of this method are the same as the point thermometer method, which can only indicate the temperature during sterilization and cannot determine whether the sterilization time meets the requirements.

4、 Principle of biological indicator method:

Using non pathogenic thermophilic Bacillus spores as indicator bacteria to determine the effectiveness of thermal sterilization. The spores of thermophilic fat bacteria have strong resistance to heat, and their heat resistance is similar to that of pathogenic microorganism Clostridium botulinum spores. This is used as an indicator bacteria to verify whether the sterilizer can meet the sterilization requirements. There are three types of biological indicators: spore suspension, spore bacterial slices, and mixed indicator tubes of bacterial slices and culture medium. Generally placed at five points on the sterilization container: the front, middle, and back of the lower layer, and the central point of the upper and middle layers. After sterilization, inoculate the indicator into bromocresol purple glucose peptone water and incubate at 55-60 ℃ for 2-7 days. If the culture medium is clear and the color does not change, it indicates that the spores have been killed, and the sterilization effect of the sterilizer is good; If the culture medium is yellow and turbid, it indicates that the spores have not been killed and the sterilization effect of the sterilizer is not qualified. The validation methods for spore suspension and spore pellet are the same.

At present, commercial biological indicator tubes are commonly used in laboratories, which operate on the same principle as spore suspensions and spore sheets. The indicator tube contains thermophilic fat loving Bacillus spores and a small glass tube containing culture medium. The indicator tube is placed at various points in a sterilization container, and after high-pressure sterilization, the glass tube containing the culture medium is crushed. The culture medium is released from the small tube inside and placed in a 56 ℃ incubator for cultivation, while also serving as a positive control. If the sterilization effect of the sterilizer is not qualified, the growth of spores in the indicator tube after resurrection will change the color of the meat soup, which will turn yellow; If the sterilization effect of the sterilizer is good, the spores inside the tube will be inactivated and no longer grow, and the meat soup will still be purple as before. There is currently no strict standard for the frequency of verification of the effectiveness of sterilizers, but laboratories should establish their own verification frequency regulations and strictly follow the requirements. From the perspectives of operability and verification conclusions, the editor recommends using indicator tape and biological indicator tube because these two methods are easy to operate and can comprehensively verify the sterilization effect.

Attachment: Precautions for sterilization (some imported fully automatic sterilization pots do not need to consider exhaust)

When using a high-pressure steam sterilizer, the first thing to pay attention to is to remove the cold air inside the sterilizer while opening the steam. The exhaust vent can only be closed after all the cold air inside the device has been expelled. If there is still some air remaining in the sterilizer, although the pressure gauge has reached a certain pressure value, the temperature inside the sterilizer has not yet reached the corresponding degree. The more air remains, the greater the difference between the two, and the insufficient temperature inside the device will affect the sterilization effect.