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Pseudomonas preservation

Yersinia yersinia at CYBERNEX.NET
Wed Jun 11 12:45:36 EST 1997


Rob Harling writes,

<Does anyone know the best means of long-term storage for Pseudomonas 
fluorescens?  I have a large collection of these organisms from plant 
leaf surfaces and I would like them to remain viable and free of 
mutations.>

I'd suggest making freeze cultures of each strain. BTW, this method works 
with all kinds of bacteria - I never made a freeze culture for P. 
fluorescens, but it works for P. aeruginosa and several other bacteria:

"Freeze culture medium" consists of TSB without dextrose (27.5 g/L) and 
glycerin (15% v/v). Dispense in 100 mL screwcap milk dilution bottles and 
autoclave for 15 min 121C. Prepare this medium, then isolate your 
organisms. Then inoculate two TSA slants worth of each organism you 
desire to preserve (presuming you have more than one strain of your P. 
fluorescens, that is). When the slants have 48 hours growth, wash the one 
with the best growth off with 2 mL of plain TSB (gently rub off growth 
with a sterile culture loop),  vortex the tubes for homogeneity and put 
the harvests in one bottle of the freeze culture medium - two slants and 
one bottle of freeze culture medium for each 50 cryovials of each strain 
of organism you want to keep. Shake the bottle gently to mix, and 
dispense 2 mL into each of 50 sterile cryovials. Cap the vials, label, 
and freeze at -20C if that's the best you can do, or preferably -70 to 
-80C.  One to two weeks later, you should do a plate count population 
check - thaw a vial from each strain collection, dilute in peptone,  pH 
7.2 buffer or saline to 10^-9. Plate out the -7, -8 and -9 dilutions in 
duplicate.  Also, after 6 months, you should do another population check. 
Cultures prepared in this fashion are viable for at least a year. And 
since they're frozen, they don't grow in storage and therefore become 
subject to mutation. If you need to store your collection for longer than 
that, then every year I'd recommend repeating this process - thaw a vial, 
grow it out on slants, etc.

Good luck!

Infectionately,
Yersinia.

Mycelium is Yourcelium.  :-)




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