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Home Brew Forums > Home Brewing Beer > Fermentation & Yeast > Do you know how to make a yeast starter? Then why not farm yeast and freeze it?
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Old 01-11-2012, 08:57 PM   #101
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Katy_bug

Let me start by saying welcome to homebrewing and the forum community

As for your observations, you've pretty much got it covered. The ultimate goal is to have a frozen aliquot which upon thawing has a viable cell count similar to that of a store bought vial or smack pack (roughly 100 billion viable cells). However, if vialbility after thaw is really low (like the 25% in brewitts initial findings, then that may not be practical.

If you have the time and are willing to do some experiments, we welcome your input. The more data the merrier. With that said, I think a lot of us would like to see more data at the home level. Since the vast majority of us do not have access to a lab or -80 freezers, etc, evaluating those parameters won't really help us any. If you could conduct your experiments with an ordinary home deep freeze and self-defrosting freezer, that would be ideal. For any self-defrosting freezer experiments, putting the aliquots in a small cooler with some ice packs is the accepted practice for homebrewers that do not have a deep freeze.

Here are my thoughts on your parameters to evaluate.

Ideal % of glycerol as a cryoprotectant - Definitely
Ideal frozen aliquot size - Definitely. It would be intersting to see if the viablity upon thaw is dramatically affected by aliqot size. For instance, a lot of people yeast bank by freezing small amounts of yeast in vials. Do smaller samples fair better than larger ones?

Freezing protocol that maximizes viability
-Flash freeze - yes, or at least at the home level, putting the aliquot strait into the freezer without a refridgeration step.
-Cold crash - yes, and also optimal cold crash time needed. It seems that cold crashing the yeast helps build trehalose reserves but how much time is really needed to accomplish this effectively?
-Controlled rate freeze - not really of interest since it cannot be acurately controlled at the home level. EDIT: I somehow missed your post above about freezing in propanol. Could this be accomplished with OTC isopropyl alcahol? If so, then that would be a parameter of interest
-Sequential freeze (room temp, fridge for a while, freezer) - I believe this was a parameter that brewitt threw in on the mix. He was experimenting with what temperature to acclimate the yeast to the glycerine solution. I think the idea here is: Is it better to mix cold crashed yeast with cold glycerine solution or is it acceptable to mix the yeast and glycerine solution at room temperature and then refridgerate. This is actually of interest to me as well as some of the strains I use settle great at room temp and some I have to cold crash to settle out, so I actually do both. If I misinterpreted your goal here brewitt, please chime in.

Ideal freezing protocol - I think you meant to say "Thawing Protocol"
-fast thaw at 37C - yes, ideal fast thaw temp
-slow thaw in the fridge - yes

Thank you for your interest, and we look forward to hearing any results that you can contribute.

Cheers

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Old 01-12-2012, 05:25 AM   #102
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The ultimate goal is to have a frozen aliquot which upon thawing has a viable cell count similar to that of a store bought vial or smack pack (roughly 100 billion viable cells). However, if vialbility after thaw is really low (like the 25% in brewitts initial findings, then that may not be practical.
My impression is that you will not achieve a pitchable population of yeast without some period of recovery and, probably, growth in wort prior to pitching. I do think that you could pitch a large quantity of glycerol frozen cells but the time for strong fermentation would be relatively long (guess: 12 hours). That said, 8 hours of pregrowth may be sufficient.

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Definitely. It would be intersting to see if the viablity upon thaw is dramatically affected by aliqot size. For instance, a lot of people yeast bank by freezing small amounts of yeast in vials. Do smaller samples fair better than larger ones?
At small vial size, viability is not a real issue (even several logs of killing is OK) since the goal is to be able to recover enough live cells to step up to large starters, but not to generate and quick starter. This is the way we preserve cells for banking and it works just fine.

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-Sequential freeze (room temp, fridge for a while, freezer): Is it better to mix cold crashed yeast with cold glycerine solution or is it acceptable to mix the yeast and glycerine solution at room temperature and then refridgerate. This is actually of interest to me as well as some of the strains I use settle great at room temp and some I have to cold crash to settle out, so I actually do both. If I misinterpreted your goal here brewitt, please chime in.
It is correct that there are two components to moving to the cold before adding cold glycerine; first, cold crashing concentrates the cells, second, the slow cooling from room temp to freezing seems to be advantageous (whether due to trehalose or some other form of protection). I would like to see more data but this clearly seems like the way to go.

Based on my own experience and the data I have seen in research papers on the topic, I believe slow cooling and quick thawing are optimal. As you say, the home freezer is the wildcard.
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Old 01-12-2012, 06:24 PM   #103
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My impression is that you will not achieve a pitchable population of yeast without some period of recovery and, probably, growth in wort prior to pitching. I do think that you could pitch a large quantity of glycerol frozen cells but the time for strong fermentation would be relatively long (guess: 12 hours). That said, 8 hours of pregrowth may be sufficient.



At small vial size, viability is not a real issue (even several logs of killing is OK) since the goal is to be able to recover enough live cells to step up to large starters, but not to generate and quick starter. This is the way we preserve cells for banking and it works just fine.



It is correct that there are two components to moving to the cold before adding cold glycerine; first, cold crashing concentrates the cells, second, the slow cooling from room temp to freezing seems to be advantageous (whether due to trehalose or some other form of protection). I would like to see more data but this clearly seems like the way to go.

Based on my own experience and the data I have seen in research papers on the topic, I believe slow cooling and quick thawing are optimal. As you say, the home freezer is the wildcard.
Ok, maybe I should refrain from using the word "pitchable." The only reason I used that is because that is how commercial yeast companies advertise thier product. However, my goal is not to pitch strait from thaw to fermenter, it is just to have a viable cell count similar to commercial packs or vials so that a large starter can be made from the onset without small steps.

As for the cold crashing, I'm just curious as to whether it makes a difference if you cold crash first then add glycerine vs mixing settled yeast at room temp with glycerine and then refridgerating. I realize that the refridgeration step seems to help viability, but like you said, cold crashing and refridgeration are two different things. Cold crashing generally means chilling the starter with the goal of settling out yeast. Refridgerating before freezing for a period of time is done to build trehalose. The strain I use the most settles very well at room temp. So I have been decanting and adding the glycerine solution then refridgerating for a couple days before freezing. So basically, the question is should you refridgerate to build trehalose before or after adding the glycerine or does it even make a difference?

I realize with small vials viability is less of issue because enough cells will survive that a new population can be built back up. I'm just curious as to whether viability is affected by aliquot size. If I freeze a vial with 1 ml of slurry vs a jar with 20-30 ml of slurry is the survival rate the same?
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Old 01-13-2012, 06:32 PM   #104
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BBL Brewer, I did the comparison of settling at room temperature, pouring off wort, adding room temperature glycerol, then moving to the cold, as compared to settling in the cold and then adding cold glycerol. The cold cells fared better than the room temperature ones. However, it is true that the cold cells had been crashed overnight and then after glycerol addition left for the same amount of time that the room temperature cells were chilled for. Therefore, the cold crashed cells got more time in the cold. Whether that is the difference or the addition of cold glycerol is the difference, or both, I can't say. I will guess both contribute.

With regard to freezing small versus large volumes, I haven't tested that but the large volumes did "well enough" for the application you are interested in. If I was publishing a paper on this I would have to do it more carefully and better controlled but since we are looking for "useful" outcomes, I think we know enough. That said, I would be happy to know the answer.

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Old 01-20-2012, 03:58 PM   #105
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I've been trying to search for the question I have, but can't find the answer. Is there any advantage to having both the glycerine frozen yeast as well as some slants in the fridge, or should I just stick with one or the other?
Tom

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Old 01-20-2012, 07:06 PM   #106
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I've been trying to search for the question I have, but can't find the answer. Is there any advantage to having both the glycerine frozen yeast as well as some slants in the fridge, or should I just stick with one or the other?
Tom
Well, that kind of depends on your situation and how often you want to reculture. You don't have to freeze yeast to maintain it long term. You can use slants or agar plates as well. If you plan to work only from your own stock and stop buying yeast altogether, then you should do a streak plate to isolate a pure culture before making a new slant. You can maintain a yeast strain indefinitely this way. The draw back is that the yeast will lose viability more quickly storing at fridge temps and you will have to reculture more often. At the same time, freezing some vials for yeast banking purposes would also be a good idea in case your yeast mutates on you or you somehow lose your unfrozen cultures. If you're not planning on long term maintenance and are still going to buy yeast once in a while, then I would stick with freezing. This way you can grow up what you need from a fresh sample and put it strait into long term storage.
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Old 01-20-2012, 08:57 PM   #107
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A few comments. First off very cool thread! I have some vials at work I was just going to throw away. Quick stop at the store tonight to pickup some glycerine and I'll be in business. I hope to freeze some of the Munich Lager yeast I have going, and will also freeze some of the Chimay I have in a starter (if it takes off). My wife was starting to complain about all of the yeast jars in the fridge, so this should help!

Second, nice avatar BBL_Brewer. And lastly, nice quote you have there. I got problems.

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Old 01-20-2012, 10:01 PM   #108
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A few comments. First off very cool thread! I have some vials at work I was just going to throw away. Quick stop at the store tonight to pickup some glycerine and I'll be in business. I hope to freeze some of the Munich Lager yeast I have going, and will also freeze some of the Chimay I have in a starter (if it takes off). My wife was starting to complain about all of the yeast jars in the fridge, so this should help!
Now she'll be complaining about all the jars in the freezer

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Second, nice avatar BBL_Brewer
Thanks

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And lastly, nice quote you have there. I got problems.
I'm right there with ya man
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Old 01-28-2012, 07:04 PM   #109
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Love the thread.

I've been farming for a little over two years. I'm glad to see some guys that know what they are doing generating some metrics on this. I've spent some time changing my variables around like you guys (slurry%, glycerin%, temps) and also have to mention a variable that I have encountered as well. As much as I hate to admit it, some yeast just don't freeze well. I've had issues with several strains, but on the other had have strains that I am still using from an initial freeze 2 years ago. Go figure.

Do love the thread, though. Just be prepared guys. Every other week or so, I get somebody from the brew club asking if I have some Wyeast/Whitelabs <<insert random strain here>>. But on the plus side, I haven't paid for yeast (with the exception of adding to the Zoo) in almost 18 months!

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Old 01-28-2012, 07:16 PM   #110
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As much as I hate to admit it, some yeast just don't freeze well. I've had issues with several strains, but on the other had have strains that I am still using from an initial freeze 2 years ago. Go figure.
If you think you can make that claim and not name names, think again.

Would you mind elaborating, perhaps making an ordered list?
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