The revival of microbial culture from Glycerol stock has always been a paradox of sort. Recently, I was told that during the thaw period of the stocks the cells have a probability of loosing their plasmids(if any).
Instead it is a good practice to take the culture out using a sterile loop as 'crystals' and allow it to thaw on an agar plate.
Is this true?
Is it a bad practice to allow the stock to thaw completely before taking out the cells using a sterile loop?
What are the chances that the cells will undergo mutation during this freeze-thaw cycle?
Sounds odd... If you move the bacteria from a frozen stock to a selective solid medium, whic allows you to avoid non resistant bacteria from piggybacking on your plasmid carriers than I can't see any problem with either method, if you don't than both methods are unreliable
The reason you don't want the stock to thaw completely is if you intend to refreeze again. Repeated freeze-thaw cycles will tend to kill bacteria from ice crystal formation. So by keeping the stock frozen the entire time, there is only one cycle and not repeated cycle. That is why most people use a sterile loop or applicator to gather a little bit of the frozen material and apply directly to a selection plate. The problem is not about causing mutations, but just killing the stock.
Don’t let the stock thaw. I just scrape off a bit of frozen stock ( I transport the frozen vial to the hood in a pre-chilled (-20 degrees) mini-cooler for restriction enzymes) with a sterile pipette tip and streak on my selective plate. Then stock back to the -80 ASAP.
I agree with Michael Frederick Dunn. Carry your glycerol stock in a EP tube cooler and Work as quickly as possible. Scrape the surface of the glycerol stock with a pipette tip and inoculate on plates and keep the stock immediately back into the freezer. I only thaw them completely if I do not intend to keep the stock back into the freezer. For your info, I have stored my glycerol stocks at -20 too. They can be stored up to 6 months at -20, from my experience.
Thank you Michael J. Benedik and Michael Frederick Dunn for your responses to my queries. Now I have started to just take crystals instead of thawing the culture completely.
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