Ranching/Freezing yeast: Has Anyone Tried This Technique?

[Brooothru]

I've been harvesting, storing, freezing, propagating, etc., yeasts for a few years now with generally very good results. When it comes specifically to freezing yeast, I've followed the general process of harvesting directly from a fresh starter and/or mixing a fresh vial with equal volumes of 30:70 ratio of glycol to distilled water prior to chilling and freezing. Recently it occurred to me that when I decant the supernatant from the settled yeast culture I might be dumping out a portion of the most viable yeast cells, literally throwing out the baby with the bathwater, since the healthiest cells are the last ones to flocculate out of suspension.

My question is, would it make more sense to use a portion (or all 70%) of the supernatant in place of distilled water in the 30% glycol/water mix? I currently have five propagations going using the SNS "Shaken, Not Stirred" (my stir plate shot craps) method in gallon jugs that have put out an abundance of fresh yeast cells. I'm not likely to even come close to using all this yeast within the next year, so refrigeration storage won't cut it. Therefore, freezing will buy me some extra time. But the thought of dumping a couple of gallons of supernatant that might contain some of the healthiest cells just seems absurdly shortsighted. I know that a popular process for storing refrigerated harvested yeast from a batch fermentation uses beer as a cover medium to protect the yeast, and I do that as a matter of course. It seems that, likewise, supernatant from a propagation used instead of distilled water might offer some protection as well as supply a few random but very healthy cells to the mix.

So, has anyone tried this or have any thoughts suggesting that this might not be such a good idea? The downsides I've considered are an imprecise ratio of glycol to "water" due to the unknown proportion of additional "gunk" volume in the supernatant, and the increased risk (slight) of infection since the "water" is not "pure." BTW, has anybody else noted how it's gotten nearly impossible to buy distilled water lately? (another rant for a different thread).

I'm getting good stratification now in each of the propagations with a significant layer of at least ¼"~⅜" of solid yeast deposit in each gallon jug, topped by an opaque layer of liquid and a much clearer 2"~3" layer of supernatant above that. It's clearly time to harvest and prep for freezing. Any advice or suggestions?

 

[Knkbrand]

surprised no one has answered your question. I was looking for the answer as well.

 

[IslandLizard]

surprised no one has answered your question. I was looking for the answer as well.

Yeah, this thread was missed somehow, for no good reason. It's not that this specific forum is very busy.

Recently it occurred to me that when I decant the supernatant from the settled yeast culture I might be dumping out a portion of the most viable yeast cells, literally throwing out the baby with the bathwater, since the healthiest cells are the last ones to flocculate out of suspension.

Do you thoroughly cold crash your starters (and for how long) before freezing the slurries? Some yeasts take longer to completely flocculate, we should take that into consideration.
I think it's important to let the supernatant clear completely or as clear as can be before decanting and harvesting and subsequently freezing the slurry.

As long as the supernatant is clear I doubt there's a ton of good healthy cells left in there, the majority will have crashed out and ended up in the top layer of the slurry.

Maybe someone has counted leftover cells in supernatants and has some concrete numbers for us?

It seems that, likewise, supernatant from a propagation used instead of distilled water might offer some protection as well as supply a few random but very healthy cells to the mix.

I don't see any problems using using the supernatant as part of the medium, instead of distilled water. On the other side, those 3 ml of (well cleared) supernatant in the vial with frozen slurry doesn't add all that many more yeast cells. But more than none.

 

[Brooothru]

Yep. Pretty much agree with everything you said. I'm about half way through freezing my propagations, having chickened out and defaulted to distilled water/glycerin. I decanted and dumped the supernatant, just leaving enough behind to re-suspend the settled yeast cake, then blending with the 30/70% glycerin/water.

I 'double-decanted' the supernatant, emptying the bulk of the cleared liquid from the gallon jugs down the drain. Then I re-suspended the remaining slurry and poured it into the tall slender 500 ml Mason jars to refrigerate for 3~4 days and settle again before decanting the second layer of supernatant. This concentrated the dense slurry into roughly 100+ ml. cakes in the bottom of the jars. Since the storage media for this freezing is 180 ml sterilized breast milk freezing bags, after combing 80~90 ml of thick slurry with 80~90 ml of the glycerin/water blend, so I'm freezing about 170 ml total in each bag. That leaves behind about 20~25 ml that I'm propagating again with 1.040 SG/9.4 Plato wort that I will refrigerate for use in the next 4 to 6 months. Hopefully the frozen samples will survive for the next 2-3 years.

This whole exercise got me looking into my frozen samples from several years ago. They were stored in sterile 'test tube' containers, stored in an insulated box and surrounded with frozen chill packs to prevent thawing in the frost-free freezer compartment. The 50 ml capacity tubes only allow for only about 25 ml of actual yeast. I pulled out two frozen vials that I'm going to attempt to revitalize after more than three years of suspended animation. One is WLP840 American Lager and the other is Wyeast 1217-PC, both 1st generation propagations from the original packaged pitches. The Wy 1217 is a seasonal release that I haven't seen in a while, and for some reason the 840 American lager is hard to find even though it's a core offering. It's about the longest I've gone without reviving a sample (or replacing it with fresher) since I started 'ranching' yeast. Anxious to see how it works out.

 

[IslandLizard]

Since the storage media for this freezing is 180 ml sterilized breast milk freezing bags, after combing 80~90 ml of thick slurry with 80~90 ml of the glycerin/water blend, so I'm freezing about 170 ml total in each bag.

Are you sure it's OK to freeze such a relatively large volume?
Instructions I've read use smaller, 15-30 ml glass vials, or those plastic centrifuge tubes.

https://www.homebrewtalk.com/threads/guide-to-making-a-frozen-yeast-bank.35891/

 

[bracconiere]

why not just add the 30% glycerine to the starter?

 

[mac_1103]

Recently it occurred to me that when I decant the supernatant from the settled yeast culture I might be dumping out a portion of the most viable yeast cells, literally throwing out the baby with the bathwater, since the healthiest cells are the last ones to flocculate out of suspension.

I would think that nearly all of the cells in a well-prepared starter should be very healthy, so the babies in the bathwater aren't going to be much different than the ones stuck to the bottom of the bath tub.

It seems that, likewise, supernatant from a propagation used instead of distilled water might offer some protection

The glycerine should be all the protection you need.

 

[Brooothru]

Are you sure it's OK to freeze such a relatively large volume?
Instructions I've read use smaller, 15-30 ml glass vials, or those plastic centrifuge tubes.

https://www.homebrewtalk.com/threads/guide-to-making-a-frozen-yeast-bank.35891/

In the past I’ve always used centrifuge tubes or old White Labs vials. They’re about 50 ml capacity, so 25ml slurry to 25 ml glycerol 30% solution. The ratios for my bagged samples are the same even though the volumes are 3x greater. The samples are pre-chilled to 38F, then frozen lying flat rather than upright to increase surface area and reduce vertical stratification. I’m hopeful that it will result in positive outcomes, but won’t really know for sure until I thaw one and attempt to propagate.

The backup plan is to reserve ~25 ml from each propagation before freezing to build an additional propagation for refrigeration. When ready to brew, I’ll prop a fresh 1~2L starter, save some dregs for the next brew, and pitch the majority of the propagation at high Krausen.

 

[IslandLizard]

In the past I’ve always used centrifuge tubes or old White Labs vials. They’re about 50 ml capacity, so 25ml slurry to 25 ml glycerol 30% solution. The ratios for my bagged samples are the same even though the volumes are 3x greater. The samples are pre-chilled to 38F, then frozen lying flat rather than upright to increase surface area and reduce vertical stratification. I’m hopeful that it will result in positive outcomes, but won’t really know for sure until I thaw one and attempt to propagate.

Yes, please keep us updated on how well the yeast fares after being frozen in larger volumes.
I know people have had success freezing in WLP tubes, which are about twice the width of centrifuge tubes, and much deeper. Using the breast milk pouches is a decent step up size-wise, holding 3-4 times the useful volume of WLP tubes, and 10-20x that of 15 ml centrifuge tubes.