Hi Tuba.

I think it would be better to do a recovery culture for a little while.

But I'm too lazy to do that, so I'll just suspend a small amount in medium and streak it directly.

Best,

After your culture retain to its normal state transfer small quantity from this culture to brain heart infusion broth and incubate culture tubes overnight for activation then subculture on plate agar.

I have stocks at -80 that are 20 years old and they work just fine. Use a sterile loop or toothpick and just streak out on a plate like normal. It should be fine.

Dear Tuba Siddiqa many thanks for asking this interesting technical question. In addition to the valuable answers which have already been provided by experts, I suggest that you also have a look at the answers given to the following closely related question which has been posted earlier on RG:

Which is the better procedure to revive a glycerol stock of bacterial culture stored at -80C?

https://www.researchgate.net/post/Which_is_the_better_procedure_to_revive_a_glycerol_stock_of_bacterial_culture_stored_at-80C

(79 answers)

Good luck with your work and best wishes!

I agree with Michael J. Benedik. I use glycerol stocks very often, and I just place them on ice while i scrap it with a inoculation loop and then, proceed to streak on a previously warmed plate.

Although I did not freeze the bacterial culture to such a degree, I think the principle is the same...First, the thawing should be gradual so that it is not left at room temperature, but first in the freezer of the refrigerator for 1 hour and then transferred to the refrigerator 4 ° C overnight, with the preparation of fresh media, preferably the same media that was used in isolation, and if the media that was used previously was a selective media, do not add the selective supplement, because the bacteria are stressed and then after thewing transferred from the broth, a 2 loup full, streaked on agar and incubated with the same previous conditions ...And then if you want to freeze again, it is preferable to prepare double-strength broth to preserve these bacteria for a longer period, and medical glycerol is added to it by 20%. I have used this method many times with Campylobacter and it was very satisfactory although Campylo is a very sensitive and fastidious microorganism... As for putting culture in the water bath, I do not advise that because you may expose it to shock due to the great difference in temperature...I wish you good luck