My Pichia pastoris culture is not growing in shake-flask culture, however, they grew well on my YPD agar plate (same cells) at 30 C. I use Minimal Glycerol media (MGY) to grow my cells. It contains:
- yeast nitrogen base (YNB) without amino acids and ammonium sulfate.
- ammonium sulfate
- glycerol
- biotin
I am using new YNB and biotin powders, however, the ammonium sulfate and glycerol were available in the lab.
For media preparation, I filter-sterilize it (no autoclave)
Can ammonium sulfate or glycerol be the reason the culture is not growing? (if the chemicals are old and were open in 2007?)
What else can be the problem if not the reagents? Aeration?
(PS: I have used the same media before and it was growing fine until recently when I prepared new media using different chemicals)
Thanks!
Hi Toka,
I'm interested in this question too. I have grown Pichia without problem on agar plates using yeast nitrogen base with various carbon sources and even in Nitrogen base without a carbon source added. I have yet to go back and investigate further on these one-off trials.
I did have trouble growing it in liquid media using disaccharides such as maltose or sucrose, but no problem growing in dextrose.
Hi there,
There is no aminoacid supplement in your recipe... As your YNB is without aa there should be supplement. Unless it's just a mistake.
Hi Everyone,
Thank you Dominique Liger for your answer, yes my YNB doesn't contain amino acids, but my cultures did grow before with the same YNB and without adding amino acids.
Matthew Winans I prepared all the reagents fresh and ordered new chemicals, the problem was in the glycerol, it seems like because I was using an older bottle, it affected its quality as a carbon source for my yeast cells. The cultures with the new glycerol worked fine. I hope this helps!
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