Glycerin/water ratio for freezing yeast

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MalFet

MalFet

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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
 
Few comments:

MalFet said:
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.
Click to expand...

Correct.


MalFet said:
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.
Click to expand...

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?
 
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:

Chris White said:
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.
Click to expand...

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.
-MalFet
 
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.
 
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
 
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.
 
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.
 
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.
 
BBL_Brewer said:
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.
Click to expand...

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.
 
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.
 
BBL_Brewer said:
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.
Click to expand...

I think yeast population dynamics aren't terribly well understood in the context of brewing. I had a long conversation with a yeast geneticist about this a while back, and his basic message was, "Mutation isn't an issue on a homebrew scale, but genetic drift in response to selective pressure definitely is." I don't completely understand it. I've noticed very quick change in yeast performance after improperly cropping, but I've never had any problem with similar changes after repeatedly reculturing from ten single-cell derived colonies on a plate. With numbers that small, I would expect probability to catch up to me and to see some significant drift, but so far so good. Plating yeast is a tried and true standard for a reason, I guess. Anybody know why plating doesn't lead to strong (random) selection?
 
MalFet said:
Anybody know why plating doesn't lead to strong (random) selection?
Click to expand...

You pose an interesting question when it comes to "random" selection. I would think (I have nothing to back this up) that what you propose would be more of a worry when sampling from a higher population density. If you start with say a commercial sample of yeast that already contains billions of cells and you pick just one of those cells to isolate the strain......what are the chances that you will isolate a cell that is representative of the population as a whole?

However, once you isolate a single cell and start culturing from that colony, it stands to reason that your chances of staying consistent from that point on are considerably higher. Reproduction is ocuring at a much slower rate and you are picking from a much smaller population when you do subsequent plate cultures.
 
BBL_Brewer said:
You pose an interesting question when it comes to "random" selection. I would think (I have nothing to back this up) that what you propose would be more of a worry when sampling from a higher population density. If you start with say a commercial sample of yeast that already contains billions of cells and you pick just one of those cells to isolate the strain......what are the chances that you will isolate a cell that is representative of the population as a whole?

However, once you isolate a single cell and start culturing from that colony, it stands to reason that your chances of staying consistent from that point on are considerably higher. Reproduction is ocuring at a much slower rate and you are picking from a much smaller population when you do subsequent plate cultures.
Click to expand...

Hmm...I'm not sure I follow you. I am wondering why plating doesn't sometimes lead to an undesirable reduction in genetic diversity in the way that, say, poor cropping technique can. Are you saying that it's because the reduction has already happened, and therefore it can't get worse? I have always gotten consistent yeast performance from just ten colonies, so my anecdotal experience doesn't jibe with the idea that the diversity has already been flattened.

Perhaps it is just that the raw probabilities are sufficient to keep all alleles represented, but that doesn't seem intuitive to me. If you start seeding desert islands with randomly selected humans, I've got to imagine that it wouldn't take too long before you started getting all redheads or no redheads. Anybody have a gene sequencer I could borrow for a few days?
 
Mal, I believe there is a distinction to be made between selecting a yeast culture based on poor flocculation and large krausening (top cropping) or any other specific observed behavior in the brewing application versus selecting smallscale colonies from a plate.

First of all, plate selection is done as a way of visibly ensuring 1) you do not have an infected culture and 2) There is no abnormal visible growth characteristics.

In yeast, according to SWMBO's experience, the vast majority of mutations will not result in a viable candidate. These cells die and do not reproduce properly. Other mutations will often change the habit of colony growth so that they are obviously visibly different. However there are only a very small number of genetic elements within the overall genetic code that manifest as brewing-specific or that the define the characteristics of a given strain, so the likelihood of a brewing-relevant mutation is quite minute. However, as you multiply the size of the overall colony, the chances of having mutations increases, since it is a function of random probability for the most part.

So growing on a plate with small cell numbers is still the best means of ensuring you have no infection, that you grab viable and visibly consistent yeast colonies and in generally small enough numbers where the chance of a mutation up to that point is as close to zero as is reasonably possible.

Overall, this is why it is preferable to take from frozen "dormant" stocks and grow up versus repitching or harvesting yeast slurry or slants...

That said, the vast majority of commercial breweries re-used and harvest yeast a good number of times (5 to 12 is common) before starting over from a mother culture (maintained themselves or purchased from one of the big yeast labs).
 
Randar said:
Mal, I believe there is a distinction to be made between selecting a yeast culture based on poor flocculation and large krausening (top cropping) or any other specific observed behavior in the brewing application versus selecting smallscale colonies from a plate.

First of all, plate selection is done as a way of visibly ensuring 1) you do not have an infected culture and 2) There is no abnormal visible growth characteristics.

In yeast, according to SWMBO's experience, the vast majority of mutations will not result in a viable candidate. These cells die and do not reproduce properly. Other mutations will often change the habit of colony growth so that they are obviously visibly different. However there are only a very small number of genetic elements within the overall genetic code that manifest as brewing-specific or that the define the characteristics of a given strain, so the likelihood of a brewing-relevant mutation is quite minute. However, as you multiply the size of the overall colony, the chances of having mutations increases, since it is a function of random probability for the most part.

So growing on a plate with small cell numbers is still the best means of ensuring you have no infection, that you grab viable and visibly consistent yeast colonies and in generally small enough numbers where the chance of a mutation up to that point is as close to zero as is reasonably possible.

Overall, this is why it is preferable to take from frozen "dormant" stocks and grow up versus repitching or harvesting yeast slurry or slants...

That said, the vast majority of commercial breweries re-used and harvest yeast a good number of times (5 to 12 is common) before starting over from a mother culture (maintained themselves or purchased from one of the big yeast labs).
Click to expand...

Indeed. I'm not really concerned about mutation, but more about emphasizing or eliminating already existent phenotypic traits. My understanding matches what your wife describes: that the chances are extremely small of stumbling upon a mutation that (a) doesn't kill the yeast outright, (b) is favored enough to out-compete other gene variants, and (most importantly, c) is actually consequential to beer production. It's nice to have somebody with credentials support it :mug:

Still, I have to think that selective pressures can impact the relative frequency of already existing alleles. This is, after all, what's happening with bad cropping technique, no? A good yeast pitch has a good mix of early and late floccers, but harvesting from secondary (for example) takes only the late floccers. It's probably simplistic to think that this is all controlled by a single gene, and most of the time you'd expect ten colonies to represent a relatively even spread the majority of the time.

But, anytime you are doing probabilistic operations on small populations, you're bound to get anomalies. Do those happen? What's to prevent me from unknowingly harvesting ten colonies that are all early floccers, for example?
 
Randar said:
Mal, I believe there is a distinction to be made between selecting a yeast culture based on poor flocculation and large krausening (top cropping) or any other specific observed behavior in the brewing application versus selecting smallscale colonies from a plate.

First of all, plate selection is done as a way of visibly ensuring 1) you do not have an infected culture and 2) There is no abnormal visible growth characteristics.

In yeast, according to SWMBO's experience, the vast majority of mutations will not result in a viable candidate. These cells die and do not reproduce properly. Other mutations will often change the habit of colony growth so that they are obviously visibly different. However there are only a very small number of genetic elements within the overall genetic code that manifest as brewing-specific or that the define the characteristics of a given strain, so the likelihood of a brewing-relevant mutation is quite minute. However, as you multiply the size of the overall colony, the chances of having mutations increases, since it is a function of random probability for the most part.

So growing on a plate with small cell numbers is still the best means of ensuring you have no infection, that you grab viable and visibly consistent yeast colonies and in generally small enough numbers where the chance of a mutation up to that point is as close to zero as is reasonably possible.

Overall, this is why it is preferable to take from frozen "dormant" stocks and grow up versus repitching or harvesting yeast slurry or slants...

That said, the vast majority of commercial breweries re-used and harvest yeast a good number of times (5 to 12 is common) before starting over from a mother culture (maintained themselves or purchased from one of the big yeast labs).
Click to expand...

Indeed. I'm not really concerned about mutation, but more about emphasizing or eliminating already existent phenotypic traits. My understanding matches what your wife describes: that the chances are extremely small of stumbling upon a mutation that (a) doesn't kill the yeast outright, (b) is favored enough to out-compete other gene variants, and (most importantly, c) is actually consequential to beer production. It's nice to have somebody with credentials support it :mug:

Still, I have to think that selective pressures can impact the relative frequency of already existing alleles. This is, after all, what's happening with bad cropping technique, no? A good yeast pitch has a good mix of early and late floccers, but harvesting from secondary (for example) takes only the late floccers. It's probably simplistic to think that this is all controlled by a single gene, and most of the time you'd expect ten colonies to represent a relatively even spread the majority of the time.

But, anytime you are doing probabilistic operations on small populations, you're bound to get anomalies. Do those happen? What's to prevent me from unknowingly harvesting ten colonies that are all early floccers, for example?

And just to clarify: I don't mean to compare plating and bad harvesting technique. Bad harvesting will ensure that you have a poorly representative all of the time, whereas I suspect that plating ten colonies actually gets a good representation most of the time. But, I am curious about why you don't occasionally see anomalies.
 
MalFet said:
But, anytime you are doing probabilistic operations on small populations, you're bound to get anomalies. Do those happen? What's to prevent me from unknowingly harvesting ten colonies that are all early floccers, for example?
Click to expand...

Statistics. If you select one colony alone, you are taking your chances, yes. 10 colonies? I think you're diverse enough to eliminate reasonable opportunity for singular behavior among the 10 colonies.
 
Randar said:
Statistics. If you select one colony alone, you are taking your chances, yes. 10 colonies? I think you're diverse enough to eliminate reasonable opportunity for singular behavior among the 10 colonies.
Click to expand...

Makes sense for evenly distributed alleles (.5^10 is mighty small, after all), but I've still got to think that this has the tendency to eliminate relatively less frequent traits (.9^10 isn't quite so tiny). If we did this with people, we'd run out of redheads pretty quick.
 
Fwiw my non-frozen, yet freezer stored, revived Zurich white labs yeast tasted ok in starter. Bit sweet. It didn't look very lively ...so so. Although lager yeast I figured a blast of heat would help kick it off after pitching. 24h later at 23C nice 5" froth and bucket lid about to blow off. I'll drop temp slowly down to 12c for rest of ferment. I read elsewhere Zurich not robust to freezing so I was mildly concerned.

edit:OK so we're down to 18C which is till high I know for a lager yeast but it stinks very sulfery. The bucket lid is bulging out so plenty of CO2 coming off. Perhaps what I resurrected was a nasty zombie version of what lived before? I read yeast flavors change with temp but this is retch inducing. Beery undertones but mainly farts. Not had this before in my limited experience but then I read the Apfelwein stinks awful too but tastes great, so will the farty smell go away from the brew or is it doooomed?
 
Mildly concerned. The farty smell replaced by something a bit more solid and more stinky. I'm hoping that's the gunge dried on sides of primary. Beer itself tasted ok once smell gone. Bit bland ( munston bock). Sg 1.015. I guess a month will tell if I've brewed a self defense agent or a pleasant bock.
 
MalFet said:
Makes sense for evenly distributed alleles (.5^10 is mighty small, after all), but I've still got to think that this has the tendency to eliminate relatively less frequent traits (.9^10 isn't quite so tiny). If we did this with people, we'd run out of redheads pretty quick.
Click to expand...

Well, the idea is obviously that genetic diversity isn't even ideal with yeast. You're taking 10 colonies of (virtually) genetically identical yeast cells. The aim is to keep the yeast the same, with minimal change. So not only is the disappearance of redheads not a problem, but you don't want them pop up, and when they do, you WANT them to be stamped out. The point is, that with good practice, the traits you want should *never* be relatively infrequent within your sample.

You can't really compare yeast with people like that, because there's an enormous, key difference - genetic diversity is completely undesirable when you're trying to maintain a pure yeast culture.
 
emjay said:
Well, the idea is obviously that genetic diversity isn't even ideal with yeast. You're taking 10 colonies of (virtually) genetically identical yeast cells. The aim is to keep the yeast the same, with minimal change. So not only is the disappearance of redheads not a problem, but you don't want them pop up, and when they do, you WANT them to be stamped out. The point is, that with good practice, the traits you want should *never* be relatively infrequent within your sample.

You can't really compare yeast with people like that, because there's an enormous, key difference - genetic diversity is completely undesirable when you're trying to maintain a pure yeast culture.
Click to expand...

That's never been my understanding, based on hearing yeast people talk. Certainly some key features, particularly flocculation, are desirable in distribution.
 
From what I've read on yeast culturing, there is no substitution to tasting some of a trial beer prior to pitching the main tank. There IS a limit to what can be done with with scoping and plating etc. This was very frustrating to me when I first started culturing. Not that I've ever noticed a problem (or done trial batches for that matter), but I was hoping for a set of rules, that if followed, would guarantee a perfect # of healthy cells that were always mostly the same yeast I bought from the lab....
 
Greater than 10% glycerin leads to plasmid instability. ( plasmid instability = mutations )
10 % glycerine solution = 29.12 F degrees freezing point.
You just need to slow yeast metabolism to just under freezing. :mug:
Common misconceptions are that the colder the better.
Cryogenic temperatures can be used for gene modification on cells. Working on cells require them to stay still.
Others may know better but this is my protocols of how to keep yeast cultures dormant until needed.

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Good beer should be Good AND Cheap
 

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