Do you know how to make a yeast starter? Then why not farm yeast and freeze it?

IsItBeerYet said:

I believe that I read somewhere that a good "cool" storage alerts the yeast that "hard times are a'comin!" and triggers a glycogen uptake, which would increase long-term viability. Althought I am no bio-chemist. Don't even play one on TV.

I've found that a quick reheat is pretty vital to recoverability of the strain as well. I'll usually give a vial to my wife straight from the freezer, who will warm it up in her pocket for 30 minutes or so. Her contribution to my brewing.....

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That's good stuff. Found you a cheap incubator

For those of you following this thread, I have just updated the original post to include some of the new information that we have been gathering. The edits are in red.

EDITED: It isn't at all surprising that there is significant variability between yeast strains. However, keep in mind that there is a lot of variability between preparations of yeast in the home lab (usually known as a kitchen ;-)), as well. Nevertheless, even anecdotal information can be useful if taken for what it is. Unfortunately I cannot comment on the viability commercial strains since I am using yeast recovered from bottles. I have successfully frozen and reused yeast from Racer 5 IPA and from Ballast point Sculpin IPA. The former is supposed to be a Scottish ale yeast derivative (WLP028?) and the latter a California ale yeast like WLP001.

OK boys and girls,

I'm putting up a Belgian wit yeast this evening. We'll see how she turns out! I've still got 2 ferments in "cleanup" that I need to put up afterwards.

Awesome thread. I am a biologist/biochemist and will try to replicate some of these results in my hands I am especially interested in testing better storage formulations for -20C vs. -80C storage.

I just did this with a washed population of yeast from 6 gallons of IPA. After washing I did a quick and dirty check in the microscope for the condition of the yeast. They were abundant, healthy and clean (no visible bacteria, of course a very inaccurate test but....). Surprisingly, there was not a lot of debris (trub) visible in the washed cells. I brought up the cell slurry in 1/4 volume of 60% glycerol, split it up into 16 x 50 ml and froze it at -80 C using the stepwise cooling method I have described earlier. I'll use these tubes of yeast to innoculate overnight starters. This saves me from having to buy or grow up stepwise from small samples I have put away.

ScoRas said:

Awesome thread. I am a biologist/biochemist and will try to replicate some of these results in my hands I am especially interested in testing better storage formulations for -20C vs. -80C storage.

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Great!
Please post results from -20 vs -80 storage.

Don't be shy about posting results. We welcome any and all data.

diS said:

Great!
Please post results from -20 vs -80 storage.

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Will do, probably about 4 weeks out.

BBL_Brewer said:

Don't be shy about posting results. We welcome any and all data.

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Definitely. I wasn't trying to step on any toes, as this has been an extremely informative thread. I really want to try to help come up with a good -20C storage solution. -80C is pretty easy, mix your yeast (near saturation), with an equal volume of 40% glycerol (final 20%), and you are good.

-20C is tough, because 20% glycerol is on the border of freezing at this temperature. I'm thinking of trying to optimize a lower glycerol percentage, but use other formulations that will protect viability (all food grade of course!)

This is intriguing (and a protein family my lab studies), but I worry about introducing undesirable mutations to the strains: http://www.ncbi.nlm.nih.gov/pubmed/1571943

ScoRas said:

This is intriguing (and a protein family my lab studies), but I worry about introducing undesirable mutations to the strains: http://www.ncbi.nlm.nih.gov/pubmed/1571943

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I'm not sure you read back to seem my quick and dirty analysis of 30% vs 15% glycerol but that clearly showed an advantage of 15%. I should point out that I did my analysis at -30C since that is where we have a frost free freezer set that had room available for the test. As you point out, that may be somewhat different from -20C. I also found that extended stepwise cooling of the culture was advantageous. I'll be curious to see what you find. It may be 10% is better than 15% glycerol.

With regard to the induction of HSPs, I think this might be a useful step prior to cooling. I don't know how much one has to worry about induced mutation with a short thermal stress, although 2 hours does seem to be pushing it. Of course, any stress has some potential for mutation associated with it but we routinely use short heat shocks in the laboratory during transformation protocols without undue concern. I, for one, would be interested in the results.

Looking forward to your report.

Very interesting. Heat shock huh? So here's a question. If chilling the yeast for a period time helps to build trehalose (which also helps as a cryoprotectant I assume) should this step still be incorporated or skip it in lieu of heat shock? And if you were going to do both, which one should you do first?

Brewitt said:

I'm not sure you read back to seem my quick and dirty analysis of 30% vs 15% glycerol but that clearly showed an advantage of 15%. I should point out that I did my analysis at -30C since that is where we have a frost free freezer set that had room available for the test. As you point out, that may be somewhat different from -20C. I also found that extended stepwise cooling of the culture was advantageous. I'll be curious to see what you find. It may be 10% is better than 15% glycerol.

With regard to the induction of HSPs, I think this might be a useful step prior to cooling. I don't know how much one has to worry about induced mutation with a short thermal stress, although 2 hours does seem to be pushing it. Of course, any stress has some potential for mutation associated with it but we routinely use short heat shocks in the laboratory during transformation protocols without undue concern. I, for one, would be interested in the results.

Looking forward to your report.

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I saw your analysis, and definitely want to use the lower glycerol concentrations for the lower temp storage, per your results. I think I would keep any heat shock under 30m, which is about what we use for yeast plasmid transformations. Basically, I'll just test +/- heatshock, I'm not going to do a whole range at this point.

I can try and test the frost-free lunch freezer vs. a lab non-cycling -20 as well.

BBL_Brewer said:

Very interesting. Heat shock huh? So here's a question. If chilling the yeast for a period time helps to build trehalose (which also helps as a cryoprotectant I assume) should this step still be incorporated or skip it in lieu of heat shock? And if you were going to do both, which one should you do first?

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I think these effects could potentially both help, so my hunch is to heat shock and then use the step-wise cooling method Brewitt tested with good results.

Update: I just received a new vial of Whitelabs WLP001 california ale yeast. I'll be starting my own viability freezing tests soon.

ScoRas said:

Update: I just received a new vial of Whitelabs WLP001 california ale yeast. I'll be starting my own viability freezing tests soon.

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Sweet. I'm still scrounging up some funds for a microscope and a hemo.

I made up my starter in autoclaved 10% DME last night, and put in a shaking incubator at 20º C (68º F) for 24 hours. I counted the cells with a hemocytometer, and they were at 50 billion cells/Liter after 24 hours. I was surprised, as this was about 4-fold lower than I expected based on the mr. malty constant aeration calculator (no O2 infusion.

Anyways, I set up a comparison of -80 vs. -20 storage at 20, 10, and 5% glycerol, both with and without heat-shock at 42º C for 20 minutes.

I will normalize the different concentrations of cells before I plate them on DME/Agar, and assay viability by counting colonies. I figure I will wait at least a month for the first plating. I'll report then. Suggestions are welcome.

ScoRas said:

I made up my starter in autoclaved 10% DME last night, and put in a shaking incubator at 20º C (68º F) for 24 hours. I counted the cells with a hemocytometer, and they were at 50 billion cells/Liter after 24 hours. I was surprised, as this was about 4-fold lower than I expected based on the mr. malty constant aeration calculator (no O2 infusion.

Anyways, I set up a comparison of -80 vs. -20 storage at 20, 10, and 5% glycerol, both with and without heat-shock at 42º C for 20 minutes.

I will normalize the different concentrations of cells before I plate them on DME/Agar, and assay viability by counting colonies. I figure I will wait at least a month for the first plating. I'll report then. Suggestions are welcome.

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So what did you start from?

One tube of whitelabs WLP001. It really won't make much of a difference for this test, and the cells should be pretty healthy having not grown to complete saturation.

ScoRas, It will be interesting to see where you end up with this one. I have a couple questions/comments.

1. Did you precool your culture? If so, how was it done?
2. Was the whitelabs vial fresh, if you have any way of knowing? I would expect it to grow to density in 24 hours if it was. I think reaching the post diauxic phase is useful because cells tend to be more stress resistant in that state.
3. What size were your aliquots? What was the concentration of packed cells?

Inquiring minds....

Looking forward to your results.

Brewitt said:

ScoRas, It will be interesting to see where you end up with this one. I have a couple questions/comments.

1. Did you precool your culture? If so, how was it done?
2. Was the whitelabs vial fresh, if you have any way of knowing? I would expect it to grow to density in 24 hours if it was. I think reaching the post diauxic phase is useful because cells tend to be more stress resistant in that state.
3. What size were your aliquots? What was the concentration of packed cells?

Inquiring minds....

Looking forward to your results.

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1) I cooled on ice after heatshock, I did not do incremental cooling. I have seen the data that shows slow cooling helps, but decided to leave out that variable.

2) The whitelabs vial was fresh, it was 2 weeks before its use-by date.

3) I the cell concentration was 5^6 cells/ml in 1 ml aliquots. I basically kept my highest concentration a 1:1 mix with 40% glycerol.

Explanation for possible lowish cell count- I did spill a lot of yeast due to blow out of the tube! With all my experience growing cultures from single colonies, I've never had them shipped to me in a tube before. Lol, lab experience doesn't make one not a brewing noob.

I pitched 900ml of the culture to my 1.08 SG IIPA on wednesday, and it was fermenting vigorously by 12 hours, so the cells were pretty happy.

ScoRas said:

1)
With all my experience growing cultures from single colonies, I've never had them shipped to me in a tube before. Lol, lab experience doesn't make one not a brewing noob.

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No kidding! I've experienced the same thing. But I'm learning fast. I grow my starters 1.5 liter at a time in Fernbach flasks on a lab shaker. They haven't blown over yet but gotten pretty close. I've been trying to do my tests at about 50% packed cells (way more that 5^6/ml, more like 5^9/ml).

ScoRas said:

I pitched 900ml of the culture to my 1.08 SG IIPA on wednesday, and it was fermenting vigorously by 12 hours, so the cells were pretty happy.

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Sounds great. Just about to taste 4 different single hopped IPAs I made last month with frozen yeast from Ballast Point Sculpin IPA. Tasted great going into the bottle. Hop test, here I come.

Hey guys,

Just a thought. When I would make BIG starters (and before I got my big honkin' 5000 ml flask), I would put FermCap-S into the starters to keep from bubbling over. Surface tension is surface tension, whether from a boil or a ferment. Plus, it helped keep the SWMBO happy. Which allows ME to partake in my favorite hobby......brewing.

Stupid me. I decided to email my micro instructor from college, Dr. Mitchell. I remember him as being a great guy and thought he might actually take some interest in this or maybe point me to a grad student who wouldn't mind doing a little research in the interest of knowledge. I ran some of the techniques discussed in this thread by him and got a pretty useless, almost condescending response. The only potentially useful information that I got from him was the following: "Prep 40% v/v glycerol and sterilize. Prep 10% w/v non-fat dry milk; filter through cheesecloth and sterilize. Mix 1:1….this is best cryoprotectant I have tested that will work for bacteria and fungi….so final glycerol = 20% v/v in whatever nutrient medium you choose."

BBL_Brewer said:

Stupid me. I decided to email my micro instructor from college, Dr. Mitchell. I remember him as being a great guy and thought he might actually take some interest in this or maybe point me to a grad student who wouldn't mind doing a little research in the interest of knowledge. I ran some of the techniques discussed in this thread by him and got a pretty useless, almost condescending response. The only potentially useful information that I got from him was the following: "Prep 40% v/v glycerol and sterilize. Prep 10% w/v non-fat dry milk; filter through cheesecloth and sterilize. Mix 1:1….this is best cryoprotectant I have tested that will work for bacteria and fungi….so final glycerol = 20% v/v in whatever nutrient medium you choose."

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I wouldn't call that stupid. I've done the same in the past with a few professors across a couple of disciplines, sometimes you get extremely helpful responses (I got a full chapter from an unreleased textbook) to no response at all. Professors are supposed to be helpful!

TriangleIL said:

I wouldn't call that stupid. I've done the same in the past with a few professors across a couple of disciplines, sometimes you get extremely helpful responses (I got a full chapter from an unreleased textbook) to no response at all. Professors are supposed to be helpful!

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Unfortunately Profs are human. Most of my college prof would neber respond because they aren't getting paid. It's a crummy way to be, but I guess it works for them. My chem prof was a droll, monotone kind of guy who basically used his class as a weeder course to eliminate as many students as he could. The U of F admissions and grade requirements made him a super start in their eyes. Then you had to pay a re-admit fee and go to the board for a decision whether they would allow you to continue your studies. All this while the tuition was sky rocketing even for in state student and the Lottery was supposed to be adding extra income to schools. All they really did was change the surrent level of funding from the state to the lottery. Ever time the lottery put in a dollar, the state cut their funding the same amount. So much for better or cheaper advanced education. If your state doesn't have Lottery, vote no if they ask. It's just a scam on the residents.
Bob

Rbeckett said:

If your state doesn't have Lottery, vote no if they ask. It's just a scam on the residents.
Bob

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Pretty much.

I guess you guys didn't win the most recent MegaMillions
My problems was: you have to play to win. I didn't and don't. I'm with you on this one.

I had good success with freezing but one thing is still bothering me- what slurry/glycerine solution ratio to use, and is it that important (as it is glycerine/water ratio).

My process is:
- make 15% glycerine solution (eg. 25ml of glycerine and 75ml of water)
- mix that with slurry in ratio of 75% slurry and 25% glycerine solution

Beside long lag phase (36-48h) on 1st step, after stepping up starter fermented pretty quick.
We know that it is best to go with 15% glycerine solution, since greater % can decrease viability due to poisonous effect on yeast, but what ratio of slurry/glycerine solution is most optimal.

On this and other threads I found several ratios:

FlyGuy said:

You want less than one-third of the solution in the test tubes to be glycerinee, so I shoot for about 2-3 ml in each tube (15%).
........
Now, one at a time, uncap your vial and extract a sample of yeast slurry from the bottom of the flask. Inject enough into the vial to fill it about 80%.

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When I started with freezing I used FlyGuy"s thread as guide, besides that it is somewhat greater glycerine solution it is also greater slurry/glycerine solution ratio.

BBL_Brewer said:

I use a 35% weight by volume glycerine solution at a rate of about 70% solution and 30% yeast slurry

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BBL_Brewer said:

I setlle out the yeast, siphon off the starter beer and add enough glycerinee/water solution to bring the volume up to 500 milliliters.

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This is pretty bigger ratio..

Brewitt said:

I harvested about 60 mls of packed cells (1/4 cup containing approximately 4x10e11 cells based upon past experience) and resuspended them in an 100 mls (a bit less than half a cup of 15% glycerol, split them into four 40 ml tubes and cooled them stepwise to -112F for later use.

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Brewitt uses similar scale..

My next doubt is that if we are going to make big glycerine solution/slurry ratio (eg. 70/30) we will need pretty big flask for eg. 25B viable cells after thawing.
Math would be:
According to Brewitt"s test, viability after freezing is close to 25% (at best) so we need 100B cells for freezing. If we make starter, or wash yeast from previous batch we will get somewhere between 1 and 1.5 B/ml.
That is 100ml of slurry (for 1B/ml) that we need to mix with 225ml of glycerine solution (to achieve 70/30 solution/slurry ratio).
At the end we will have 325 ml flask ready for freezing which is not small, especially if we want to make couple of them..

I know that this seems like over-complicating, but if I am doing it I would like to know why is it on this or that way...

diS said:

I know that this seems like over-complicating, but if I am doing it I would like to know why is it on this or that way...

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That's the magic question. We all know it works, but no one has a definitive answer as to what the best method for success is. The only concrete advice I have at the moment is if you have a method works for you, stick with it.

Yes I now, and I was afraid of this answer
Since we know about glycerine solution percentage I hope someone will do cell counting with different slurry/solution ratio...
gotta get myself a microscope

diS said:

Yes I now, and I was afraid of this answer
Since we know about glycerine solution percentage I hope someone will do cell counting with different slurry/solution ratio...
gotta get myself a microscope

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I generally consider the final volume when adding glycerine. That is, if we want 15% final and start with 60%, add 10 ml glycerine to 30 mls cells for a total of 40mls. The cells are included in the volume. Generally there is a slurry with a significant volume of wort. That is fine. I don't think it matters much if the final solution is 30% cells, 50% cells or 75% cells, the final concentration of glycerine is 15% of the total.

I always do a starter. In my experience a 40-50 ml tube of cells overnight in 1.5-2 liters of wort with stirring is great.

Hope that helps.

Actually it does help. I like the way you dilute "major" glycerine solution (60%), so it will end with 15% when mixed at 1:4 ratio, easy and precisely, thanks!

These days I"m going to wash wlp029 and I"ll try this route.

The cell percentage does matter. The cells are not dissolved in the solution, so you would have very different concentrations of glycerine.

orangehero said:

The cell percentage does matter. The cells are not dissolved in the solution, so you would have very different concentrations of glycerine.

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Formally, this is true. However, a large percentage of the volume is the liquid in between the cells and, in addition, it is my understanding that the cells themselves take up the glycerine. What the actual intracellular concentration of glycerol becomes, I don't know but there is some equilibration which contributes to the cryoprotection of the cells. It is probably a known value, I just don't know it.

Regardless, my empirical experience is that, when I adjust a slurry that I would guess is 50% or more packed cells to 15% final concentration of glycerol, it works well. Similarly, when I preserve small samples that are less than 5% cells, they also retain relatively high viability.

In my lab we generally mix a saturated culture of yeast 1:1 with 40% glycerol (w/v) for a final concentration of 20%.

In my experiment, I am testing -80 v. -20C storage with 5,10,15, and 20% glycerol, with and without 42C heat shock. I haven't thawed any to test for viability yet, because I'd like to go out qt least a couple of months.

2 liter starters made with a 50 ml vial of a very thick slurry of washed yeast from a fermentation frozen in a final concentration of 15% glycerol is working great! I have found my sweet spot.

Still interested to hear what ScoRas learns.

I froze some wild yeast (along with other strains) back in 2010 in my parents chest freezer following the instructions in this thread. I took a vial from their house on mother's day and it's been sitting in my fridge for a few weeks. Yesterday I made a starter and I can already tell there is some definite yeast activity. You can't miss the smell of this wild yeast. I know originally there was both some primary fermentation strain and brett and some unknown bacteria. There definitely seems to be yeast activity in the beer but I'll have to wait and see if any of the bacteria survived the freezing process. I guess I'll check back in a year when the beer has had time to age and the bacteria have had a chance to work.

Brewitt said:

2 liter starters made with a 50 ml vial of a very thick slurry of washed yeast from a fermentation frozen in a final concentration of 15% glycerol is working great! I have found my sweet spot.

Still interested to hear what ScoRas learns.

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What concentration of yeast (B/ml) do you think you have in that slurry?

I mean, if vitality is aprox. 25% and for 5 gallon of 1.048 beer you need 170 billions of cells, the you"ll need 3B/ml (3 B/ml x 50 ml = 150 B; 150 B x 0.25 = 37 B in 2 L starter gives ~184 B cells).

In "Yeast", C. White says that washed yeast have between 0.8 and 2 B/ml.
I usually stick with middle range (1.5 B/ml) just to be on safe side, even I rinse it to the point where the is no or very little trub in slurry.

diS said:

In "Yeast", C. White says that washed yeast have between 0.8 and 2 B/ml.
I usually stick with middle range (1.5 B/ml) just to be on safe side, even I rinse it to the point where the is no or very little trub in slurry.

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Dis, I think you missed my point. What I did was to thaw my 50 ml tube of yeast, pitch into 2 liters of 1.040 wort and grow it overnight with stirring. The viable cells grow up to density overnight. Even with 25% viable cells in my slurry the viable ones should grow to maximum density of about 1-2x10e8/ml or 200 B cells total after overnight. Pitched into about 5.5 gallons that gives me a final concentration of about 2x10e7/ml which is about optimal. Are we on the same page here?