I am conducting in vitro fermentation of gut microbiota, but due to equipment limitations, some steps have to be performed outside the anaerobic chamber. For example, during 10% fecal slurry preparation and homogenization, can I perform these steps with N2 flushing over the open-lid solution? The entire homogenization and aliquoting process takes about 10 minutes before transferring to -80°C storage. I am unsure how much short-term oxygen exposure might affect bacterial viability.
Similarly, for media pre-reduction, I usually place media or PBS in the anaerobic chamber with the lid open overnight before use. However, I know some labs flush or bubble the solution with N2 for 30 minutes before placing it in the chamber.
For those with experience in similar setups, could you share your insights on whether my current approach (brief O2 exposure) is viable, or if I should take additional precautions?Thanks!
Hi Qin,
We do it a bit differently. We do something similar but with a bit of modification.
1. We prepare the media (we have A and B and then purge 8 hrs or overnight with N2; if PBS is to be used, better do that too, so to have very sure ananeoribc things). "lid open overnight before use", it wont' help as concern is dissolved O2 and so add a magnet bar and stirr while N2 purging or you cal it bubble help a lot.
2. I don't know why you need to prepare the fecal slurry outside. It's easy. As you mentioned, 10%, just add PBS inside. That is it. When it's thawed inside the chamber, which is already anaerobic, mix gas and N2 at least five times, based on your equipment, and vacuum removed each time.
We usually use slurry from a company, and we do the 10% when we are ready to start fermentation. We have stored samples in -80.
Let me know if you need more clarification on what I mentioned.
Good luck
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