How do you rehydrate freeze-dried Fusobacterium nucleatum? ATCC says that anaerobic bacteria need to be re-hydrated under anaerobic conditions, however, is it OK to re-hydrate Fuso in aerobic conditions using growth medium stored in aerobic conditions, then transfer the culture to an anaerobic chamber for further culturing?
Use a fresh culture broth containing a Redox agent may be FeS, and add this sterile medium to the freeze dried cells in an anaerobic chamber. Incubate the cells and see they grow over a period of time.
My recommendation having revived obligate anaerobes such as Heliobacterium modesticaldum Ice1, Heliobacillus mobilis, and Clostridium acetobutylicum ATCC 824 is to do everything in the anaerobic chamber.
Reviving freeze-dried cultures is already a bit of a challenge even aerobically. If this strain is sensitive to oxygen you want to make sure you work in glass as much as possible (plastic can store trace O2 levels) and you want all medium to be bubbled with N2 or argon and stored in an anaerobic chamber for at least 24 hours. Usually anaerobic growth medium has some type of color indicator as well included to let you know when redox conditions are suitable (typically resazurin which goes from blue to pink, then pink to clear).
Once you handle everything in the anaerobic chamber (doing your best to maintain sterility in the absence of a flame) I would quickly transfer them to an anaerobic tube that can be crimped, sealed shut, and I would flush the headspace again with N2 or argon just to make sure any trace levels of O2 have been eliminated. Anaerobes can be fickle, so be prepared to wait a while for growth to resume as well (although I am not familiar with this strain).
Another reason for avoiding using medium that has been stored on the bench top for too long is many essential trace elements, namely metals, will change their chemical speciation depending on the prevailing redox conditions (iron is a particular one to be mindful of). Sometimes, this can result in that trace nutrient precipitating (although you won't see it) and becoming unavailable.
All of this to say, reviving anaerobes can be a challenge, but I would follow ATCC or DSMZ's instructions to the letter (I prefer DSMZ because they tend to be more detailed). In this way, if something happens to stunt growth you can at least troubleshoot and draw on the expertise of the culture collection if possible.
I hope this help and good luck reviving your strain!
Hi, Dear Robin Mjelle,
Please try to follow the ATCC directions as much as possible to revive the the culture you bought. Then try to chanllenge the ATCC procedure to establish your own one. Make a medium anerobic buy adding cysteine. good luck.
In addition, you better add a redox indicator like Resazurin or any others as required by pH change of your medium to assess the anerobicity of your medium.
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