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Cryoprotectants --> Freezing Bacterial Cells - Help with cryoprotectants in freezing E. coli. (Feb/06/2006 )

I'm freezing E. coli cells and I was wondering a couple thing about the cryoprotectants that I will need. I believe that 10% glycerol and 5% DMSO are common however these dilute the colour, dilute the culture and potentially change the nutrient condition nutrient broth. These problems will affect my analysis by changing the background readings, changing the culture concentration or changing the growth itself. How can I control these problems?

I appreciate any help you could provide.


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I've only ever preserved my freezer stocks in 10% glycerol + whatever media I cultured in. The cyroprotectants really shouldn't matter, since they are usually used for the purpose of seeding a new overnight culture. This effectively dilutes whatever cyroprotectants you use into negligible amounts, depending on your new culture volume. Background readings and culture concentrations are all obsolete once you begin a new culture from your freezer stock.

Even when I go to transform CaCl-competent cells, the glycerol has no noticeable impact on the transformation process.

Hope that helps. I guess it depends on what you're using your freezer stocks for, but those are the uses that I am most familiar with.



Thanks so much. Any other opinions?



I agree with Haringsh, frozen stocks should only be used for seeding new cultures or plates, not used directly in any experiment.



I agree with them.
you should culture your bacteria for few hours before to do your experiments.
What I do is an overnight culture, them dilute 50 times (because I know that in these conditions I have an OD600 of 0.1) and let them grow until OD600 reaches 0.5 which corresponds to exponential growth. by this way I have reproducible conditions.