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Home Brew Forums > Home Brewing Beer > Brew Science > Glycerin/water ratio for freezing yeast
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Old 03-27-2011, 01:16 PM   #11
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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.
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?
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Old 03-28-2011, 02:52 PM   #12
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Anybody know why plating doesn't lead to strong (random) selection?
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.
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Old 03-28-2011, 03:08 PM   #13
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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.
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?
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Old 03-28-2011, 03:39 PM   #14
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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).

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Old 03-28-2011, 04:23 PM   #15
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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).
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

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?
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Old 03-28-2011, 04:26 PM   #16
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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).
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

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.
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Old 03-28-2011, 04:35 PM   #17
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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?
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.
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Old 03-28-2011, 04:40 PM   #18
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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.
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.
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Old 03-28-2011, 11:54 PM   #19
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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?

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Old 04-05-2011, 05:00 AM   #20
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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.

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