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. :-)
