1. Can laser-punched positive samples be reused and how should they be preserved?

Since the pore size of positive samples is small and easily clogged, it is generally not recommended to reuse them;

If the sample is used for the development and verification of a deterministic method and is not destroyed, it can be used for probabilistic methods (colored water, microbial challenge). If it is used for probabilistic destructive methods, it is forbidden to reuse the leak after it contacts the aqueous solution;

The samples cannot be exposed to the air for a long time. It is strictly forbidden to expose them to dusty environments or high-pressure flow environments. It is recommended to store them in sealed clean packaging.

It is strictly forbidden to directly touch the positive sample with your hands during use, especially the leaking hole. It is recommended that operators wear clean gloves before touching the positive sample;

If the sample has not been used for a long time, the recommended re-test period is 6 months.

2. Why do the laser-perforated positive bottles used in microbial challenge experiments not change during the sterilization process?

Laser drilling technology was used to prepare a 5μm pore size for the glass ampoule sample. After normal sterilization procedures, it was observed under a microscope (see the figure below) that there was a significant change before and after the treatment, and there was pore blockage.

3. When comparing physical methods and microbiological methods, a capillary tube (10 μm inner diameter, 5 mm length) was used to prepare a positive sample, which was negative (no bacterial growth) in the microbiological method. Is there any relevant experience that can be used as a reference?

When using the capillary (microtube) method to prepare positive samples, the length of the capillary and whether there is gas in the tube will have a great impact on the invasion of microorganisms and may produce false negative results; therefore, the preparation of positive samples is very important. On the one hand, it is necessary to try to imitate the actual leakage of the product, and on the other hand, it is necessary to avoid factors that may produce false negative results, such as avoiding the capillary being too long (as close to the thickness of the packaging material itself) or avoiding gas in the tube. In addition, a certain pressure can be applied externally to ensure the probability of detecting microorganisms.

4. What is the reference basis for the number of positive samples?

Regarding the packaging integrity test of sterile preparations, different methods require different numbers of positive samples, and there is currently no unified quantity requirement. Different methods should set different sampling standards based on batch and packaging specifications. For the microbial challenge method, please refer to the USP literature for at least 10 samples for each leakage level.