Normally freeze-dried or frozen cultures require more time for grow and recovery than normal cultures. You should wait longer for be sure that no grow.

Better to use liquid broth media for culture recovery than use solid plate for cultivation. If you taking part of your freeze dried culture and you use Pt loop, be sure that your loop not so hot when you taking powder and also check Everything ok with medias used.

Normally freeze-dried or frozen cultures require more time for grow and recovery than normal cultures. You should wait longer for be sure that no grow.

Better to use liquid broth media for culture recovery than use solid plate for cultivation. If you taking part of your freeze dried culture and you use Pt loop, be sure that your loop not so hot when you taking powder and also check Everything ok with medias used.

Which media (would assume 507 and/or alike but not the 508 or alike) did you use for this revival? After you transfer the lyophilized bacteria to your culture media, you need to give the microbe some time to acclimatize at around 65 deg. C and then raise the temp. to 78 deg. C (optimum for their growth). Also, while resuscitating and maintaining, you need to maintain strict anaerobic condition (I am sure you do so as oxygen is your enemy here). While purging the bottle with oxygen-free gas, hold the bottle in inverted position to avoid any kind of oxygen escaping the degassing procedure. Last but not the least (might sound silly), remember to adjust the ingredients and volume of your media according to the amount of lyophilized stuff you are using for this revival purpose.

I agree with Dr. Karen's answer. Liquid broth is better option as compared to solid medium.

Jaysankar De Thank you for the answer! I am using 507, for autotrophic growth on carbon monoxide. I incubated once at 65C and the second time at 72C. Both times I could not revive the culture. One problem I noticed, is the precipitation of the media components upon incubation, which makes it difficult to deduce the presence of any kind of growth. I have been confirming only by microscopy, and gas chromatography. GC analysis shows no hydrogen, or reduction in carbon monoxide levels, indicating that revival did not occur. Could you please elaborate on the degassing procedure? I can't visualise what you mean by purging the gas with the bottle held inverted.

Also, how should I adjust the ingredients of the medium according to the inoculum? The composition of the medium remains the same right?

Pavan Kumar, please look at the attached pdf and check the pages 4 and 8. It shows how to work with anaerobes with bottles in inverted (upside down) position. Is it the sulfur getting precipitated out of your media? Media composition must remain the same...you just scale it down according to the amount of inoculum (lyophilized powder). Since you have run into problem by trying to use the DSMZ culture in installments, you can probably try to revive using the whole stuff from the ampule and later store some portion of the revived culture (either as lyophilized powder or as liquid culture in crimp-sealed bottles in anaerobic condition) for future use. I guess you followed this strictly " Inoculated vessels are pressurized with sterile carbon monoxide gas to 2 bar overpressure. " Do you use anaerobic chamber or gas pak jar for working with anaerobes? Also check this link if you haven't done so. https://www.dsmz.de/catalogues/catalogue-microorganisms/culture-technology.html. Good luck.

Jaysankar De Oh. You meant the overpressurisation. I have been following this same procedure. I believe it is the trace metal carbonates, phosphates, or sulphates that are being precipitated out, because the precipitate forms even when I use sulphite(Na2SO3) instead of sulphide(Na2S).

Pavan Kumar It's Sod. thiosufate if your intended formula is Na2S2O3. Sod. thiosulfite is Na2S2O2. Difference is in their oxidation status (-1 vs -2). Anyway, Na2S2O3 is a reductant as well. We use it for dechlorination.

Jaysankar De No, It is sodium sulphite (Na2SO3). As recommended by Rothe et.al (2000), Sulphite is an excellent alternative to Sulphide even for cultures of extremely oxygen-sensitive archaea. It also increases ease of handling the cultures as it eliminates the need for an anaerobic chamber, due to its low reactivity!! It is a pretty good reducing agent for microbial cultures as its reduction potential is lower than that of sulphide.

Hi - I used to work with Carboxydothermus hydrogenoformans quite a while ago and published some work on it. Here some random comments that I hope will help solve your problem. Good luck.

C. hydrogenoformans is extremely sensitive to oxygen - I would not recommend your method of using a loop to take just part of the freeze dried material. But then I only have experience with our method of reviving freeze dried stocks of strict anaerobes which was as follows (you need syringes, needles and serum flasks with proper butyl rubber stoppers, ~1cm thick black or blue stoppers, not the thin grey stoppers that you can also get). Prepare anaerobic medium in butyl-rubber stoppered serum flasks. At least an hour before innoculation pre-reduce the medium of 4 or 5 bottles by adding sodium sulfide solution. Better, if you use resazurin as indicator wait until the medium is colourless and remains colourless after vigorously shaking the bottles. If it doesn't turn colourless, wait longer (overnight if you must), if that doesn't help you need to find out where in your medium making oxygen is not removed sufficiently or re-enters the bottles. The following needs to happen pretty quick, no coffee breaks!! You can do this at the bench at room temperature with room temperature medium. If you medium is properly reduced you prepare an 1mL or 2.5mL anaerobic syringe+needle by first flushing it with N2 and then taking up 1mL of prereduced medium. Then you can break the glass vial (heat one end, away from the pellet, in a flame, then drop cold sterile water on the hot glass to crack it, pry open with sterilised tweezers), resuspend all of the freeze dried pellet in pre-reduced medium and take up by syringe and inject into one pre-reduced medium serum bottle. With the same syringe take up and expell liquid a couple of times and then take 1 or 2mL up in the syringe and inject the next pre-reduced medium serum bottle. Repeat 2 or three more times. Incubate at 65 degrees Celsius. It may happen that the first bottle turns pink, but the second and later bottles should be still ok. Wait there are signs of growth, it may take a bit longer than normal (give it a few days if you must). An actively growing culture will start to show clear signs of growth the next morning.

Since this is a thermophile, you can keep liquid cultures fairly well on the bench at room temperature for several weeks, but at growth temperature when they run out of food they lyse pretty quick.

In my experience growth at 65 degrees Celsius is preferable, it was better than at 70 degrees Celcius. At 70 degrees Celsius you are very close too the maximum temperature that it tolerates, so slight fluctuations or inaccuracies of your incubator may push it over the maximum.

Sulfite (SO32-) is toxic to C. hydrogenoformans above 5mM, so be careful if you use it, or better don't use it at all. There are no instability issues with sodium sulfide if you use it in sealed/stoppered serum bottles, it will equilibrate with H2S in the head space (please note, H2S is more hazardous than CO, get properly informed of the risks of working with both). You could also try cysteine instead.

Precipitations of the medium are likely related to phosphate, calcium and magnesium. Try adding them from separately sterilised stock solutions (phosphate separate from the others), if that is not enough, use less calcium.

Anne M Henstra Thank you for taking the time to give such a detailed and informative solution, sir. All the more significant for me because it was your doctoral thesis work on the same organism that motivated me to pursue this line of research, in the first place!!

I tried implementing all your suggestions including pre-reduction and the other handling techniques you have mentioned. I was finally able to achieve perfect anaerobicity using Cysteine-HCl which was stable for a long time. However, even after rectifying all apparent problems, we were not able to revive the organism successfully. The only explanation that remains is that the original freeze-dried culture was non-viable for some reason.

In any case, I have learnt a lot of valuable lessons from your answer. Thanks a lot once again!!

With Cysteine-HCl you need to be mindful that it is very acidic and may drop the pH of your medium, if you do not neutralise the acid. Alternatively you can use Cysteine free base which does not have this issue.