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Discussion on Sterility Verification in Microbial Testing

2023-12-19 17:26

Sterility verification is divided into eleven steps: equipment inspection, smoke test and dust particle test, staining test, auxiliary system test, positive pressure hood environment pre test, patch test, bottle in and out challenge test, lid in and out challenge test, LG culture medium pre test, product test and LG culture medium test. This article will focus on three main parts: bottle in and out challenge testing, cap in and out challenge testing, and LG culture medium testing.

The preparation work before the experiment needs to ensure that the initial bacterial content of the packaging and product meets the requirements:

Bottle (newly blown):<3CFU/inside and outside the bottle;

Bottle cap:<20 CFU/cap;

Product:<100 CFU/ml Nutrient bacteria;

<10 CFU/ml heat-resistant spores;

Process water:<50 CFU/250 ml.

Packaging container

Empty bottle: Ensure an average sterilization rate of log6, which refers to inoculating rod-shaped bacteria inside the bottle and on the bottle cap as the initial carrier of bacteria. The entire process is as follows: Inoculate Bacillus subtilis ATCC 9372 spore suspension (load: 0.1ml/107CFU per milliliter) into 130 bottles using a pipette and dry for 8 to 24 hours. Note that the number of bacterial cells and spores may decrease over time and temperature.

120 bottles were filled with sterile water by a filling machine and capped by a capping machine (hereinafter referred to as the "test sample"), while 10 bottles were used to detect the initial bacterial load (hereinafter referred to as the "positive control sample") (to prevent excessive loss of bacterial count, it is recommended to increase the inoculation concentration by 1 log). Evaluate Bacillus subtilis spores using endpoint method calculation membrane filtration method. The results are only affected by the target bacteria.

Bottle Challenge Test:

① Select more than 260 intact empty bottles;

② Inoculate the spore suspension of Bacillus subtilis ATCC 9372 into empty bottles: 130 bottles for 105 and 106, each. The inoculation position depends on the bottle type, but try to choose a sunken area inside the bottle that is not easy to sterilize and shake it thoroughly;

③ After the empty bottle is air dried normally, it is ready for testing to ensure that the sterilization requirements can be met in the shortest possible time at the highest production speed. The concentration should be low first and then high. The system needs to be pre debugged, and sterile water should be prepared in the sterile tank;

④ Before the test, 10 empty bottles of 105 were randomly selected and sent to the laboratory for positive control testing. The method was to fill the empty bottles with 100ml of sterile water (pre added Tween 80 to assist in elution), cover them with sterile bottle caps (pre removed anti-theft rings and wrapped in aluminum foil, sterilized at 121 ℃ * 15 minutes), shake and clean thoroughly, then perform gradient dilution, dilute at least 5 gradients, and take two gradient samples of appropriate concentration, Take 1ml each for inverted plates, and make 2-3 parallel runs for each gradient sample according to GB 4789.2-201

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