Friday, July 24, 2009

Frozen Yeast Banking

After reading a thread on beeradvocate's homebrewing forum, I decided it was time to do a little reading on freezing aliquots of yeast slurry for long(er) term yeast storage. I would love to be able to buy any given yeast strain once, and then have a way to keep them stored, unharmed, in relative perpetuity, like Walt Disney's head, only with a slightly better chance of reanimation.

But, what were my other options?

Pitching on an existing yeast cake sounds great, but I haven't brewed sequential similar batches of increasing gravity (ie. APA-IPA-IIPA) and can't imagine being able to plan brewdays to line up properly.

Storing yeast slurry in mason jars in the fridge is ok, if you know you are going to use it in a month or so, but aside from maybe US05, I rarely will use the same yeast with such frequency. And Esther rolls her eyes when I have more than a couple samples in the fridge.

Everytime I dump the yeast down the drain, it just feels like such a waste.

So, freezing yeast slurry is, what appears to be my solution.

Being able to go to my yeast bank (clever name yet to be imagineered) and (eventually) pull from 15-20 different strains to make a starter without having to pay close to 10 bucks, and not have to either make a trip to the LHBS or wait for shipping, well, that's just a pretty attractive option for the homebrewer.

For freezing yeast, you really don't need much:
  • some plastic screw top tubes (a fellow Boston wort, thanks Byron, donated some 50ml sterile centrifuge tubes to my cause). Otherwise, you can find them in bulk (100 tubes/pack) for about a buck a tube, or in smaller quantities on ebay. You could certainly use smaller tubes, and if you don't get the sterile tubes, you can sanitize them before you put your yeast/glycerine solution in to them. If you choose to boil sanitize, make sure the tops can be boiled, some are made from a lower temp compliant HDPE than the tubes themselves. Screw tops are important to ensure a good seal.
  • 100% glycerin. This is the key to freezing yeast. It acts as a cryoprotectant, which means that it helps to prevent large ice crystals from puncturing the yeast cell walls. That's what would otherwise kill your slumbering yeast cells. With some internet shopping, and some trips to 3 local (massachusetts) pharmacies and the health food supplement section at whole foods, the best option I found was a double-16oz.bottle pack at amazon.com for ~16bucks, free shipping, no tax. tough to beat amazon, for price and convinience, once again. I understand that once opened, the glycerine doesn't 'go bad' either, so no worries about getting such a large quantity. If the link goes down, just search amazon for Now Foods Vegetable Glycerine. Found some glycerine at whole foods, but it was 10 bucks for 4ozs. uh... yeah.
  • Turkey baster
  • Santizer
  • Freezer
You want to make aliquots with ~15% glycerine 85% water/yeast slurry. A thick yeast slurry is preferable, more cells=better cell count upon reanination. I plan to still make a starter to ensure good health and descent cell count prior to pitching.

  • Save yeast slurry from a starter or from a primary, and wash the yeast, if desired. I didn't wash, which isn't ideal, but sometimes extra steps are just another chance to introduce contamination. At least that's what I tell myself sometimes. Store the slurry in the fridge. I can't quickly find the reference, but storing the yeast in chilling temps causes the yeast to build up trehalose in the cell wall, further increasing its ability to survive the freezing/thawing cycle.
  • Make a 50/50 solution of glycerine with water (glycerine is soluble in water), and microwave to boil to sanitize for a few minutes, cover with plastic wrap and cool in the fridge.
  • In order to make 15% glycerine solution, I'd need 7.5ml of glycerine in my 50ml tube (50 x .15 = 7.5), if I fill them. I don't plan to completely fill them, in order to leave some headspace to allow for expansion when freezing, but I'll still shoot for 15ml of the sanitized cryoprotectant solution. I made 1/4 cup + 1/4 cup, way more than what I needed for this small batch of tubes. Those 16oz bottles will last me a while, which is great.
  • Insert your santized turkey baster in to your yeast slurry, holding down on the bulb, then when you hit the top of the slurry, try to suck up the top most part (highest yeast cell concentration, if you didn't wash).
  • Store the mixed yeast slurry/glycerine in the fridge for a couple days, before freezing. Rationale here.
  • Ideally, you'd then store in a chest freezer vs. a regular home freezer to prevent damaging multiple freeze/thaw cycles, but I don't have one. I've read anecdotally that other homebrewers store their frozen yeast in the 'coldest' part of their freezer with very acceptable results, so I feel pretty good about trying this.
I'm making 5 aliquots of 3rd generation Wyeast 3944 for the start to the yeast bank.
I'll be sure to report back when I re-animate for the first time.


7 comments:

  1. Nice work JC.

    Here's a tip for working with glycerol - cut the concentration to 70% with sterile water (and good sterile technique). It is much easier to handle. Just add 15/.7 to make the final conc 15%.

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  2. Oops - just noticed you are cutting to 50%. That works too...

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  3. Hi, thank you for the "how to".
    I have been trying to figure out why 15% glycerin is recommendet, and did not find any answeres. Do you know ?

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  4. I imagine the 15% number was arrived at over the course of serial experiments, testing different concentrations. I suspect that the 15% number provided the most CFUs (colony forming units), without any significant increases as the concentration of glycerin went up beyond that. I don't think it would be harmful to add more, but I also don't believe it would add increased viability, either. I don't have any specific published citations, but look through the references section of this presentation for possible papers that would describe it in more detail:
    http://www.ahaconference.org/presentations/2007/TSPresentation.pdf

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  5. Thanks for the reply JCTetreault. I have looked at some 'scientific articles' and there they even use as little as 10% Glycerin/Glycerol at liquid nitrogen temperatures with out much damage to the cells. So I assume 15-20% is a good amount to get the most CFU's. According to wiki 50% glycerol will prevent the solution from freezing at about -18C but I expect that even if the solution freezes with 20% glycerol the yeast are still protected.

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  6. Thanks for your input Bjorn.
    I agree in that more glycerin will yield more viable CFUs, but there has to be a diminishing yield as you increase the glycerin concentration as you subsequently dilute the yeast slurry.

    I still have several aliquots of the frozen Chico and Belgian Wit yeast in the freezer, and will make a few more reanimation attempts and post updates here. Cheers, JC

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  7. does it has to be vegetable glycerin, or I could use normal farmacy-shopped Glycerine? thanks

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