Webinar Q&A — Microbiological Monitoring of Germ-Free and Gnotobiotic Colonies

Dr. Paula Roesch recently presented a webinar on microbiological monitoring of germ-free and gnotobiotic colonies. Taconic Biosciences has decades of experience in this area and Dr. Roesch presented a thorough look at all aspects of the process, starting from tools and approaches to process validation and monitoring and moving into isolator monitoring techniques.

She even included lessons learned from a case study where things didn't go as expected.

We present the full webinar Q&A here:

Sterilization and Validations

• Please comment on the use of hydrogen peroxide as a cold sterilant.

Dr. Paula Roesch: We have not ever used it for germ-free or gnotobiotic work. The contact time for spores is really long for hydrogen peroxide, which is why we don't use it in the context of germ-free and gnotobiotic production.

• Q: Have you tried validating isolator sterilants via spore discs within the isolator?

PR: No, but we do use spore discs in another way. When validating new sterilants, we use an immersion test first. We have a disc with spores on it that we submerge into the sterilant for 2 minutes and 5 minutes. If the sterilant is going to work at all, it should pass this immersion test. We have never done this in an isolator, though.

• Q: What type of sterilization approach would you take for heat-sensitive materials, such as live mammalian cells, entering an isolator?

PR: Hopefully this would be a sterile liquid with mammalian cells in it. Prepare your tube in a biosafety cabinet using a tube with a gasket on the top so you can dip it in sterilant — completely submerge it — because it is very hard to get sterilization around the rings of a tube like that. Do it in a biosafety cabinet with a sterile tube, dip it and bring it into the isolator.

That's the best you can do and it should work.

Water Supply Sterilization

• Please discuss entry of water bottles into isolators using overnight sterilization.

PR: When we add water bottles to the port of an isolator, we wipe them all down with sterilant very thoroughly. Wiping is much more effective than spraying because it gets off more of the contaminants.

We spray it thoroughly to make sure it's really soaked. We close up the port and it sits there overnight so it has a maximum contact time.

• Have you ever tried irradiated water for mice?

PR: We have not. We have always used autoclaved.

Contaminants

• What is the most common contaminant found in isolators?

PR: It varies by season. In warmer months, contaminants are almost always fungal mold. We see a lot of Bacillus, which is a spore former found in soil; this is likely coming from feed.

16S PCR Screening for Contaminants

• Have you ever had a 16S PCR positive and not found anything via culture?

PR: We have not, so far. We have had instances where we got the 16S result back on Monday and found the culture positive later that week or the following week.

• Do you recommend using 16S PCR as part of a primary screening regimen or to confirm and/or identify a culture positive result?

PR: Both. It's good to use to back up your microbial monitoring you are doing by culture. This can give you peace of mind that you haven't missed anything.

In particular, some fastidious anaerobes are tricky. Doing the 16S helps you know that you are being as stringent as possible. It's also good to confirm a finding.

• Can you offer any thoughts on the use of qPCR versus endpoint PCR for 16S detection?

PR: We use qPCR. We spent a lot of time working with IDEXX BioAnalytics on this PCR. We took a published assay and then refined it.

The shape of the curve is actually very helpful to see. It will help you determine if it's background contamination versus a real contaminant. The PCR we do with IDEXX has a larger PCR product than a standard qPCR. It's almost 200 bases, compared to 100 bases for most assays. This also helps because the DNA fragments from feed and bedding are very small, so a larger fragment helps ensure that positive results are real.

Culture Monitoring

• Have you ever used Rodac plates to monitor contamination?

PR: We use Rodac plates a lot. It depends on what your use is. We use Rodac plates to monitor the environment outside of an isolator. They are rich media and almost anything will grow on them.

I do believe they are a little more expensive than TSA with blood, but they are very effective. We just don't use them routinely for gnotobiotic monitoring; we use them more for other types of environmental monitoring.

• For researchers who do not have access to an in-house microbiology lab, what recommendations do you have for how to accomplish microbiological monitoring?

PR: I would highly recommend IDEXX BioAnalytics. They have a whole program now for germ-free microbial monitoring. They will do culture and 16S and it's set up as a package. It's very good.

We've worked with them extensively over the years. I asked them to set up the 16S PCR specifically for us. They are great to work with.