Storing E. coli -20 or -70

Bryan J. Maloney cavaggione at sbcglobal.net.spam.must.die.com
Mon Nov 25 23:07:47 EST 2002


Jim Kami <jakami at ucdavis.edu> wrote in
news:3DE27AD8.26AFACFD at ucdavis.edu: 

> Hi,
>     A small question; is it really necessary to store frozen,
>     permanent 
> E. coli cultures at -70C or will they do just as well at -20C ? The
> cultures are frozen at -70 in a 4% glycerol "freezing media" (LB +
> some extra potassium salts) and, so long as they don't thaw, is there
> any real benefit to keeping them that cold.

A couple of years ago, a visiting scientist thought that he could store 
his E. coli cultures at -20C in about 10% glycerol in LB.  I managed to 
revive 1/5 of those cultures when we discovered them after he left.

>     Also, if anyone knows if there is a way to keep permanent cultures
> at room temp., that would solve all our problems. But I've not yet
> heard of a practical method.

That depends upon how practical you consider lyophilization and storage of 
those cultures in hard-vacuum vials.  I worked with a culture collection 
that had 20-year old bacterial cultures stored at 4C in those 
circumstances.  The cultures were first spun down gently to collect the 
cells and resuspended in sterile skim milk.  They were then dried under 
low vacuum (at room temperature, believe it or not) in that matrix and 
then put under hard vacuum, then sealed under hard vacuum.  Every once in 
a while, I would break out an old culture just to check viability.  I 
believe the method was formally written up in John Tuite's methods manual 
(actually an instructor's supplement to his plant pathology classroom 
text).

Unfortunately, this was essentially self-published, and came out very 
shortly before Dr. Tuite's demise.  Purdue University's Department of 
Botany and Plant Pathology might be able to direct you to a copy.




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