I have glycerol stocks of M.tb in -80°C freezer. But it is not reviving properly; shall I rapidly thaw it? or Shall I keep in -30°C for one hour , 4°C for one hour and in room temperature (25 °C ) for one hour and use for inoculation?
Which one is correct and what is optimum percentage of glycerol should be used for preparation of glycerol stock for Mycobacterium tuberculosis i.e. final concentration
Thanks in advance
For making bacterial glycerol stocks and the suggested percentage of glycerol please check the attachment.
Good luck 👍
I don't know about M.tb but with most bacteria you just need to take a little of the frozen stock (like with a loop or a sterile wooden applicator stick) and put into broth or onto a plate and streak.
Note that you should never thaw the tube out and then put back in the freezer, this will certainly kill all the cells. So only if you have multiple single use aliquots should you thaw out the tube.
In order to revive your isolates correctly, you have to fill a bucket of ice and place your isolates box on the ice. try to plate/inoculate your isolates as quickly as possible. DO NOT THAW.
I have used the above method and haven't had any problems. Regarding Glycerol concentration, most researchers use between 15% to 25% of Glycerol. I hope that helps. Good luck
Hi dear Krisna
sometimes bacteria doesn't grow properly if they suffer from an osmotic shock, you need an enrichment step, so try to cultivate an aliquot of your -80 store (keep in 4°C for 15 min before use) in a liquid medium for 24h or 48h in an adequate temperature, after that plate from this liquid medium culture in a petri dish solid medium, you should take in the consideration that Mycobacterium tuberculosis colonies grow slowly.
the optimum percentage of glycerol that I used in general for bacterial conservation is 25% and it works well.
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