How do make a glycerol stock of bacteria for future use?
I need valuable suggestions for that.Please put here and share your experience.
Grow your bacteria overnight. Then add sterile glycerol to a final concentration of 15-20% then freeze at -80c
Hi Ramiz Raza
As an add on to Hanna Alalam 's instructions, which are standard protocol for conserving strains, I would recommend filter sterilizing the glycerol stock with a 0.2 um pore size syringe filter (you really need that pressure to get it through). You do not want to autoclave glycerol. Furthermore, ensure that you glycerol mixture is very well mixed prior to pipetting and condition the pipette tips by pipetting up and down several times as glycerol will simply leak out of the tip if this is not done. This approach will also ensure you are truly adding glycerol to a final concentration of 20% (v/v), which will ensure the long term preservation of your stock. Afterwards the stocks are fine to go into a freezer.
As an additional tip, if you're working with obligate anaerobes, you need to speed up the freezing process to avoid the infiltration of oxygen which will render your cryostock culture useless. In this case, you need liquid nitrogen and a specialized container that can hold it to flash freeze cryostock tubes. The tubes can handle it but even still, be careful when removing the tubes with tongs as they can crack.
We used this approach in my paper with a variety of obligate anaerobes and it worked extremely well:
Thesis Photosynthetic and Fermentative Bacteria Reveal New Pathways...
Article Heliobacteria Reveal Fermentation As a Key Pathway for Mercu...
Some people use alternative preservation agents such as DMSO, or sometimes methanol, but glycerol is really the standard.
I hope this helps.
Dear Ramiz,
grow the bacteria first on a liquid culture overnight, then add equal volumes from your culture and 50% glycerol. for example 1 ml of the bacterial culture and 1 ml of 50% glycerol , so the final glycerol concentration would be 25%. you can then store it at -80 . Hope it helps!
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