Hi,
I would like to prepare anaerobic medium for cultivation of gut microflora. I have an anaerobic chamber. How should I remove oxygen from the medium? Is it enough to leave it for 48 hours in the chamber? Or maybe I should flush the medium before sterilization with N2?
Thank you in advance.
If your medium flask has a wide-bore neck, overnight should be OK, particularly if you shake it periodically. Non sterilized medium left for 48 h will likely become contaminated. Anaerobic media usually contain a reducing agent, such as cysteine. Dissolve the reducing agent into deoxygenated water and add it to medium after deoxygenating in the anaerobic chamber, then sterilize. The usual way to check for reduction of the medium is to add resazurin, which turns from purple to colorless when the medium is reduced. Regarding flushing with N2, it should also be OK, except if your medium contains bicarbonate: the CO2 will be flushed out and the medium will turn alkaline.
I agree with @Pierre Beguin, we generally flush the medium after being prepared by nitrogen gas for several minutes. We can control the end of this process by measuring the redox potential which decreases due to the removal of dissolved oxygen from the medium. The redox potential value will be stable after several minutes when the dissolved oxygen is totally removed. The medium then could be sterilized.
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