Much to my horror, recent attempts to revive four(!) different banked yeasts from my library have been unsuccessful. I fortunately have “backups” of the ones I consider most important, the “DCY” yeasts (which reminds me that I need to post an update about the DCambic yeasts, which are still fermenting one half-gallon each) and was able to work from that. But what I had considered my primary source of the two DCambic strains appears to be totally dead after just a few months.
I made a switch about six months ago to banking in isotonic NaCl solution after reading Samuel’s post on the matter. My previous attempts at reviving strains have succeeded, even with vials that were a few months older at the time. So, I’m suspecting operator error at this point and am very unwilling to cast this as a fault of the method. After all, Samuel used it for a while, and this is not a novel method. Possible problems include:
- Improper solution: I used sea salt, because I couldn’t find a pure salt without additives. A lot contain minute amounts of dextrose, and I was trying to limit any sort of metabolism. Due to unknown levels of other salts in the mix it could require a different amount to be isotonic. I could also just have measured it wrong.
- Storage temperatures at the back of the fridge were too low. Perhaps they dipped into freezing at times, and the small volume cooled off quickly.
- I just moved a few weeks ago and somehow they got damaged in transit. Not likely, I don’t think, since I just moved within the same building and they were back in the fridge in just minutes.
- Or, perhaps, I simply killed the sample while transferring into the salt solution. I had already revised my transfer method to avoid shocking yeast with a hot loop but it’s still a possibility.
The other two attempts were from slants. I have not had any issues in the past even with old slants, but these two for whatever reason opened slightly in storage and the tape around the cap wasn’t enough to hold the air in. They dried out as a result. I have successfully recovered yeast from such a slant before, but I am not so lucky this time–not yet, at least. As a last-ditch effort I will add sterile wort directly to the slant vial instead of picking off with a loop.
In either case, though, this highlights the need for monitoring things and perhaps something as simple as periodically tightening the caps on my vials. I may switch back to slants, at least temporarily, while I figure out what could be going wrong with the salt solution method.
Update: I was able to wake the two slanted strains up from their dead, dried, reddish state by adding sterile wort directly to the slant and shaking vigorously throughout the evening. In addition to having dried off, they were also slanted on media I made very early on that wasn’t ideal. I am surprised, frankly, that I was able to do this.
Update #2: Correcting update #1, it looks like only one of the slants actually yielded live yeast. As for the two DCambic yeasts from the NaCl storage medium, colonies finally are visible a week after plating. This is not unusual for Brettanomyces left at 20-22C/68-72F so this may have been much ado about nothing.