May have incorrectly thawed a bacterial culture from -80 C - (Jun/28/2012 )

Hi guys,

So I'm trying to create a fresh LB culture (in order to later maxiprep) from a stock frozen at -80. Initially I had the culture thawing at room temperate for maybe 3 to 4 minutes (which I no know to be wrong) before I placed it back into the -80. I put it back so I can first figure out what the hell I should do in order to not kill the bacterial stock (if I haven't already).

So now my PI has told me the best way to do this is to pull the culture out stick a torched inoculating loop in there, scrap up some gunk and quickly transfer to the warmed LB broth. Then throw back into -80 immediately.

In your guys' experience, is that 3 to 4 minute initial room temp period enough time to destroy a stock like this? For the most part I saw that the culture still remained solid as I was placing it back to -80.

Thanks!

The solid part should be fine. As you should now have a culture growing up, make a fresh glycerol stock from it and check that the culture still has what it is supposed to (usually a plasmid), then replace the old one with your fresh glycerol.

Ok, thanks for the advice. Now I get to learn how to make glycerol cultures!

Even if you completely thaw a glycerol stock and then re-freeze it, some cells will survive. I don't recommend making a practice of it.