Microbiology List: FW: Yeast...Long-Term Storage

Yersinia yersinia at cybernex.net
Wed Oct 4 22:32:55 EST 2000

Max writes,

<Maybe this is a stupid question but....someone could tell me what is the 
best way for have long-term storage yeast stocks ?  I've found discordant 
answers to it....>

No question is stupid when you don't know the answer. The only yeast I've 
done long term storage of is Candida albicans, and for that I 
successfully used -20C freeze cultures. Hope it works on your yeast too. 
Note: this method will also keep bacteria. Here's how:

(1) Prepare a sufficient amount of freeze culture medium, which consists 
of TSB without dextrose plus 15% glycerin. One milk dilution bottle will 
yield 50 2-mL cryovials for storage. Make enough so that you have not 
only enough for your cultures, but to pull small amounts for harvesting 
(see step 3)
(2) Grow up your yeast on PDA or SDA slants or plates. For each milk 
dilution bottle, grow two slants, or one plates. If you use plates, do a 
spread streak all over the plates so you get maximum surface area growth. 
Incubate at 25C for 48-72 hours.
(3) Carefully harvest the growth with a sterile streaking loop and 2 mL 
of additional freeze culture medium. The harvest from two slants or one 
plate should go into one milk dilution bottle of freeze culture media.
(4) Tightly cap and shake the milk dilution bottle to produce a 
homogenous mixture.
(5) Pipette 2 mL aliquots of the above mixture into cryovials, then 
freeze. Don't forget to label the box and include the date of 
preparation. Go ahead and stick the box in the freezer.

A week after freezing, you should enumerate your cultures. Defrost one 
vial and make serial dilutions to 10e-8. Plate out in duplicate and count 
the -6, -7 and -8 dilutions. Average the counts and use the dilution 
which yields 10-100 colonies to multiply against the dilution factor: 
this gives you the population per mL of your stock freeze culture. Do it 
again after 6 months. Unless you use up all your vials and have to make 
more, these are good for a year frozen, or for a week after defrosting, 
if you keep them refrigerated.

Have fun!



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