frozen bacteria in glycerol - quickly shock in liquid nitrogen? (Jun/24/2009 )

A silly question:
To keep frozen bacteria in glycerol (to preserve a plasmid), which is better:
- add glycerol, mix put them at the revco, or
- add glycerol, put them quickly in liquid nitrogen and then keep them at the revco.

I think the quick shock (from amb tem to liquid nitrogen) would be helpful to get more competent frozen cells, but just to keep bacteria with plasmid this would be not better.
But I’m not sure.

I simply mix and put it to -80°C. I never had problems with my glycerol-stocks, but I also don't know if there is any better procedure...

I prepare my bacteria stock in a final concentration of around 33% glycerol and mix well before leaving it in the -80 freezer. Never tried putting it in liquid nitrogen at all. SInce liquid nitrogen is so cold, the freezing process should be fast, but it is always better to have something to prefer water crystals that can kill the bacteria cells.

For making glycerol stocks of bacteria transformed with plasmids, we generally mix 100 µl 80% glycerol + 1 ml bacterial culture before putting them in the -80 freezer. For competant cell aliquots, snap freeze (liquid nitrogen shock) before putting them in the -80. The buffer our competant cells are resuspended in contains 50% glycerol. We have never had a problem with either.

Interestingly enough, several years ago when our -80 went down, we didn't know it for several days because it was a holiday weekend. Upon opening the door, I found lots of water and thawed samples. I decided to test the glycerol stocks before discarding them. Much to my surprise, all of the vials I tested grew whatever bacteria they contained and I have since isolated DNA from their cultures. I wouldn't recommend storing them long term at warmer temperatures, but it's good to know that a broken freezer may not kill your stocks.

Glycerol readily passes through the cell membrane and provides both intracellular and extracellular protection against freezing. It is thus a so-called "penetrating cryoprotectant", and must be allowed time to enter the cells to be effective.

Just add glycerol and put them at -80.

Roo on Jul 8 2009, 06:15 AM said:

Roo on Jul 7 2009, 06:15 PM said:

You're right, the competant cells were toast. We just kept the glycerol stocks containing plasmids.