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Old 01-03-2011, 06:14 PM   #1
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Default Glycerin/water ratio for freezing yeast

Hello hello,

I am in the process of gearing up to do some frozen yeast banking and -- thanks to the kind work of FlyGuy, Kaiser, and many others -- I've put together a procedure I feel comfortable with.

The conventional wisdom seems to be that using glycerin as a cryoprotectant in a ~25% solution is the best way to do things in a home freezer. If I understand correctly, the cold temperature slows down the yeast's metabolism significantly, and the glycerin prevents ice crystals that form in the suspension from tearing the yeast to bits.

But, if cold=good and freeze=bad, is there any reason not to use a higher proportion of glycerin (50%-60%) to prevent the suspension from freezing entirely (cf. freezing points)? It seems this would get us the cold temperatures we want in a gentler environment.

I'm sure I could be missing something here. Perhaps that level of glycerin is toxic to the yeast, or perhaps the freezing is actually necessary for some reason. I'm going to be doing some comparative viability tests and will post the results here when they start coming in, but it's a long term experiment. In the meantime, do any ScienceGuys have thoughts? I've always been impressed with the level of expertise here.

-malfet

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Old 01-03-2011, 07:28 PM   #2
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Few comments:

Quote:
Originally Posted by MalFet View Post
The conventional wisdom seems to be that using glycerin as a cryoprotectant in a ~25% solution is the best way to do things in a home freezer. If I understand correctly, the cold temperature slows down the yeast's metabolism significantly, and the glycerin prevents ice crystals that form in the suspension from tearing the yeast to bits.
Correct.


Quote:
Originally Posted by MalFet View Post
But, if cold=good and freeze=bad, is there any reason not to use a higher proportion of glycerin (50%-60%) to prevent the suspension from freezing entirely (cf. freezing points)? It seems this would get us the cold temperatures we want in a gentler environment.


I'm sure I could be missing something here. Perhaps that level of glycerin is toxic to the yeast, or perhaps the freezing is actually necessary for some reason. I'm going to be doing some comparative viability tests and will post the results here when they start coming in, but it's a long term experiment. In the meantime, do any ScienceGuys have thoughts? I've always been impressed with the level of expertise here.
OK, what the glycerol does is protect the yeast cells as you noted, but in typical -80 storage, the 25% glycerin media does in fact freeze solid (and when flash-frozen in liquid nitrogen... well, you get the idea). But the glycerol acts as an "insulator" to the yeast cells to reduce the effect of H20 expansion (and promotes "small crystal" formation in the H20 for -80C storage) and cleaving of the cells. So freezing is not inherently bad.

The method described for -20 storage by Chris White in Yeast is different than typical bio lab storage since it is geared toward the home yeast rancher and their -20C freezer. This method includes forcing yeast into stationary phase and building trehalose reserve (to make them more durable to storage techniques) and also including (IIRC) 1% ascorbic acid in the 50% glycerol solution (which is combined with 50% dense yeast slurry).

If you are going to try your hand at yeast ranching, i think the book is a must-read.


As for the effect of the strength of the glycerol solution, I am not sure what the real reasoning is. Glycerol will modify the freezing point, so this may be part of the reasoning. Also, as long as your yeast are in stationary phase and are not left at room temp with higher percentage glycerol solutions, I would expect they would be similarly resilient to being "thawed". I have read that people have had success with as high as 50/50 glycerol/yeast-slurry solutions, but why not stick with the proven methods with scientific backing?
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Old 01-05-2011, 04:48 AM   #3
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Good eye Randar, and thanks for the info.

I do indeed have White's yeast book. It's a great read. I even remember him mentioning the ascorbic acid for home freezers, but somehow managed to miss his suggestion of a higher glycerin content. Here's the relevant passage:

Quote:
Originally Posted by Chris White
Using a frost-free freezer results in temperature swings that will freeze and thaw the culture, causing repeated damage to the cells. When storing at -20* C it may prove beneficial to increase the amount of cryoprotectant used, so that the culture does not freeze solid. This provides the benefits of the low temperature, but avoids loss of viability from freezing and repeated freeze/thaw cycles.
That's pretty much the answer I was looking for; I've just got to read a bit more carefully. I'm with you on sticking to what has scientific backing, but I was thinking that the 25% thing was another holdover from professional practices that may or may not apply to home brewers. Certainly, all of my microbiology friends told me to go with 25%, but step one in their protocols was always "Top off the liquid nitrogen in your $100k lab-grade freezer".

I'd be curious to hear if anyone has experimented with any of this. It'd be nice to get longer viable storage times in a standard home freezer.
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Old 01-05-2011, 04:59 AM   #4
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If you have a non self defroster you won't get the freeze/thaw cycle. Another method is to use a small foam cooler filled with gel packs to avoid the fluctuation.

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Old 01-05-2011, 01:27 PM   #5
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As Hermit pointed out, if you have a chest or upright freezer you can avoid the freeze/thaw cycles and if you use a good insulated stryofoam container you will avoid fluctuations from opening/closing the freezer.

If you are storing in a standard fridge/freezer with auto-defrost then you will definitely need to take some additional steps to try to keep the solution from freeze/thaw cycles.

I think you would have to get to at least 50% to avoid the freeze/thaw cycles according to the freezing point tables for glycerol solutions:
http://www.dow.com/glycerine/resources/table8.htm

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Old 01-13-2011, 11:51 AM   #6
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Thanks guys. Just to update that I've got a dozen micro tubes in the freezer now at 50% glycerin and 50% slurry, none of which are freezing.

I'd like a dedicated non-defrost freezer, but the limited space in my manhattan apartment means it isn't going to happen anytime soon. Beyond that, though, I think I'd use this approach with any non-lab freezer.

I'll do some comparative viability tests in a few months and post back here.

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Old 03-26-2011, 03:27 AM   #7
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Hey. I put some lager yeast in 25%glyc and in styrofoam box. Did not freeze. (I guess my freezer is not -20). I just tried reviving it 5 months later. Some tubes had a few ice crystals in, some none. Yeast settled to bottom not affected by ice. I just poured the liquid away and pitched the sludge into non stirred DME starter at room temp (17-20c). It took maybe 4 days but it looks ok now. I'll smell it and make sure it's not just contamination. But the quantity of the sediment has gone up so good sign. I think freezing prolly better for v long term but glyc is ok for yeast. At -12 or whatever my freezer is they won't metabolize much. I'm interested in these slants but if this works then why not. I'll report back if yeast attenuates properly or not. It was out of my initial starter so not old. Zurich wyelabs.

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Old 03-26-2011, 06:11 PM   #8
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I'm very interested in hearing about your results Malfet.

I've been freezing for two years now. Not so much for yeast banking purposes.....although maybe not a bad idea. I like to start with a smack pack, grow up huge starters and then split em up into small 4 oz jars. I shoot for an estimated 100 billion cells in each jar. My theory is that I can treat each frozen jar as if it were a Wyeast smack pack equivalent. The method has been working. I get great beer with normal attenuations. I use a 35% weight by volume glycerin solution at a rate of about 70% solution and 30% yeast slurry. My jars freeze solid. I use a chest freezer and keep the yeast jars at the bottom to protect from temp flucts. However, I often wonder what my % viability is after the freeze/thaw. The thing is, I have been experiencing unusally long lag times from the moment I pitch the thawed yeast until noticeable activity begins in the starter. It has been like this from the very begining. This leads to me to believe that either the yeast are just "sluggish" coming out of the freeze and take a little more time to regain full activity......or......a significant amount of cells have perished during the freeze and it takes longer for the yeast to reach a population size capable of exhibiting visual activity.

Only viability analysis would tell the real story. Again...I'd love to hear the results of your experiment.

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Old 03-26-2011, 08:35 PM   #9
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Quote:
Originally Posted by BBL_Brewer
I'm very interested in hearing about your results Malfet.

I've been freezing for two years now. Not so much for yeast banking purposes.....although maybe not a bad idea. I like to start with a smack pack, grow up huge starters and then split em up into small 4 oz jars. I shoot for an estimated 100 billion cells in each jar. My theory is that I can treat each frozen jar as if it were a Wyeast smack pack equivalent. The method has been working. I get great beer with normal attenuations. I use a 35% weight by volume glycerin solution at a rate of about 70% solution and 30% yeast slurry. My jars freeze solid. I use a chest freezer and keep the yeast jars at the bottom to protect from temp flucts. However, I often wonder what my % viability is after the freeze/thaw. The thing is, I have been experiencing unusally long lag times from the moment I pitch the thawed yeast until noticeable activity begins in the starter. It has been like this from the very begining. This leads to me to believe that either the yeast are just "sluggish" coming out of the freeze and take a little more time to regain full activity......or......a significant amount of cells have perished during the freeze and it takes longer for the yeast to reach a population size capable of exhibiting visual activity.

Only viability analysis would tell the real story. Again...I'd love to hear the results of your experiment.
Are you pitching directly into beer, or building a starter?

I haven't done any formal testing yet, thanks to a broken hemacytometer. Anecdotally, though, I'm quite pleased with how it has been working. I generally culture from 1.5mL micro centrifuge tubes onto plates, so it is hard to do real comparisons. But, when I've pitched them directly into 20mL wort starters I've been noticing growth within 4-6 hours.
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Old 03-26-2011, 11:04 PM   #10
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Before I begin here........I normally brew higher gravity beers. Between 1.060 and 1.070. I thaw the yeast in the fridge for 24 hours and then acclimate to room temp for 6-8 hours. I pitch the jars of yeast to a 1 pint starter (with plenty O2) and then step up another pint or quart before pitching to a 5 gallon batch (again, I have been treating each jar as if there are 100 billion viable cells in it). When visible activity begins in the first pint of starter (usually about 24 hours) I give the yeast another shot of O2. It usually takes another 12-16 hours after this point for the first pint of starter to ferm out. When the second step is added....it ferms out rather quickly (12-16 hours).

I'm just a little unsure as to what is actually happening during those first 24 hours. Are the majority of the yeast cells slowly regaining metabolic activity..... or are the remianing few cells that survived the freeze reproducing like crazy until they reach sufficient numbers to dig in on a whole pint of starter?

I'd like to start culturing on plates and doing some yeast banking like you are doing. If nothing else, I'm always leary about commercial strains of yeast mutating somewhat and changing in flavor profile. It's just so much easier for me to buy a smack pack once a year. Split it up, freeze it and forget about it.

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