As the title suggests, I would like to know if cells kept in room temperature glycerol for >1 hour are at risk of degradation. I usually have numerous samples that I culture from glycerol stocks and find myself rushing to streak the plates because I'm concerned that the liquified glycerol will damage the stored cells ( I go back to the glycerol stocks for other tests).
Is there a safe amount of time that cells can remain in liquid glycerol before cryopreservation?
Thank you.
Update: I was able to find a document stating that glycerol is toxic to bacterial cells and should not be kept at room temp beyond the initial inoculation.
I've attached the document in case others had similar concerns.
How are you streaking out your cells? You shouldn't need to defrost your stocks at any point: the glycerol is a cryoprotectant, but it also means the final frozen stock isn't completely rock solid: just use a sterile toothpick or innoculating loop and scrape a bit directly from the surface as soon as you get the stock pot open.
If you need to streak out multiple cultures, just pop them all on dry ice, take them to your work area, lid off, scrape, streak, lid back on, back on to dry ice, repeat. No need to let them thaw at all.
Even in a few microlitres of slush scrapings from the surface you'll have tens of thousands of cells.
To my knowledge, glycerol is not toxic to cells (bacterial or eukaryotic). However, it does inhibit growth. The thing that is indeed toxic to bacteria is the repeated freeze/thaw cycles.
A glycerol stock from -80C should never be completely thawed. You should scrape the surface of the frozen stock and streak it directly. What we do to avoid excessive thawing when handling multiple samples is we put the tubes in a box full of dry ice.
The same trick could be used when you prepare new glycerol stocks. Mix your cells in 15-25% glycerol (sterile of course), and place the tube in dry ice while you take care of the next ones. Even if you did leave them in liquid glycerol for too long, all you need is like 0.000001% survival to still recover confluant growth, given that your initial inoculum is important. I usually take a loopfull of fresh bacteria to prepare a stock, which is like 10^9 CFUs.
Bottom line is : Don't worry about glycerol toxicity. Even if you left your cells an entire day in liquid glycerol, chances are you would still get abundant growth when plated on fresh agar.
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