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

My question is pointed to starting concentration (B/ml) before freezing.

What I want to say is if you want 180 B cells in 2 L starter, you"ll need ~37 B to start with (according you yeastcalc.com).
So, with 25% vitality there has to be 150 B or 3 B/ml cells in 50 ml tube that you thawed, since 25% of 150 B is 37 B.

Am I missing something?

Well, I hadn't seen yeastcalc before. The graphs are a little deceptive if you don't realize that the limiting factor is the amount of wort which limits the total available nutrient. It is not the inoculation rate that is limiting the total amount of cells generated in the starter but the volume of the wort. If this plotted a two liter starter inoculated at different densities rather than a given amount of cells inoculated into decreasing amounts of wort, the outcome would be very different. I'm not sure whether the calculator is correct for static volume with different pitch sizes. It seems a little off to me but I'm judging from empirical data. The author says it will generate the data from the graphs with the appropriate input.

With fixed volume, I expect that the total amount of cells achievable in the starter would be about 1-3x10e8/ml with stirring unless the inoculation of cells was at a density higher than the maximum density generally achievable with the wort, for example 8x10e11 cells inoculated into 2 liters would give you a little more than 8x10e8 cells per ml because the cells would exhaust the available sugar with very little growth. That is why the yeastcalc graphs flatten out above a certain density (in that case, without aeration or stirring).

So, that said, when I inoculate 2 liters with 40B cells to get 2x10e7/ml, my starter will grow to about 2x10e8/ml or 200B cells after a little more than three generations. When I inoculate with 100B to get 5x10e7/ml, I will still end with a bit more than 200B cells but it will only require two generations. Both of those will only take overnight with aeration or stirring. Alternatively, if I inoculate with 5x10e6/ml, it will take longer but I will ultimately get there. It is best to inoculate higher to avoid contamination but that is another issue. There are other influences on maximum cell density but at the accuracy that is needed for brewing, this is a good approximation.

What you don't want to do is drastically under pitch. So it is worthwhile figuring out about how many doublings it takes to get to maximal density and then give it the appropriate amount of time. Or, better yet, do it in steps if you are too low. I do mine with a large inoculum and give it from overnight to 24 hours with stirring or shaking.

Does that make sense?

Brewitt said:

…
Does that make sense?

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Sorry to jump into the conversation, but on behalf of the Eejit section of the bleachers, I have a question:

On Monday I pitched a WLP051 vial into a 1.2L/90g DME starter. On Tuesday I pitched another WLP051 vial (same mfg. date and handling) into an identical batch of starter wort – but – the second day I didn’t shake the vial too well and there was maybe 1/8th of the yeast cake stuck inside the vial that didn’t make it into solution and thus didn’t make it out of the vial and into the starter. (True story.)

What I’m hearing here is that both yeast populations will grow into the 90g of DME over 24 hours and result in about the same total cell counts. That is not to say the yeast populations will be the exact same “taste”/health wise, because of slight generational/stress issue, etc., but you’re saying the actual cell count will be very similar – relative to a homebrewers level of accuracy.

So by extension, an older vial - say 70% viability - should result in about the same pitching rate as a newer 80% viable vial, given a 24hr stir-plate starter (but perhaps with slightly different results in yeast performance: health/attenuation/ester reabsorption/etc./etc.

True?

Without having all the data (volumes, etc) it Is hard to say precisely but to a first approximation I would say that you are correct. As long as the time is sufficient to get the starter growing and to grow it to density. The first phase is affected by the health of the stock (not just the viability).

DiS, I hope I didn't completely miss the point of your post. Yes, you are right in your calculations based upon yeastcalc. I think I start with somewhere in the 25-50B viable cells in my vial. Certainly not less, perhaps more. However, the point of my post was to discuss the accuracy and relevance of the numbers derived from the calculator.

From reading this and other threads, I get the impression that the frozen yeast that I am thawing relatively rapidly in warm water seems to get growing rapidly after inoculation. Although I haven't monitored it precisely, I am generally able to detect active growth within 4 hours or so after pitching. After growing 1.5-2 liters to full density overnight with shaking/stirring, I generally cold crash in the frig overnight and pour off the spent wort. I pitch that directly after warming to room temperature. I have had very quick starts to my fermentations with airlock activity within 2-3 hours. That is why I say I have found my sweet spot. There is certainly much more to learn about this but I feel like this is near optimal behavior of the yeast.

I should probably start plating some of the timepoints for my test!

ScoRas said:

I should probably start plating some of the timepoints for my test!

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I wish you were closer. I have some jars that are approaching 3 years in the freezer (strains I don't use that often anymore) that would be great to throw in on the mix.

There hasn't been any activity in this thread for a couple months, so I don't know if anyone is still working on this, but I thought I might be able to offer some help on determining the ideal freezing conditions to maximize yeast viability after thawing.

A previous post had a link to this paper: http://aem.asm.org/content/72/2/1330.full.pdf which shows that glycerol concentration and freezing rate and temperature dramatically effect the viability after thawing. I'll skip going into the science of why for now. It's not clear how their methods of freezing would actually translate to your average homebrewer. I contacted the authors for more advice, and hopefully they will respond.

Meanwhile, I work in a lab and have access to all sorts of goodies. -20 freezers, -80 freezers, liquid nitrogen, hemocytometers, viability measuring machines, etc. I'm going to be growing up a big starter next week and then freezing down identical tubes of yeast using various concentrations of glycerol and freezing rates and temperatures. Then I will thaw them all after a few days and count the number of yeast cells and viability. Hopefully this will help optimize the procedure.

I'll post my results in a couple weeks.

Have you had any results Katy_bug?

Forkhead said:

Have you had any results Katy_bug?

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Ha, to be honest ... I got swamped and haven't started yet.

Hi Forkhead. Thanks for the link. When I get some time I'll thumb through it. I'm still trying to rustle up some funds for a microscope. I almost bought one recently, but opted for an RO unit instead. Since we're on the subject though, I brewed up a couple fivers yesterday with 3 year old yeast. They are bubbling away happily at the moment. Feel free to post any findings that you come up with.

Cheers

I went with this method and switched over my 5 yeast strains to frozen vials w/ the glycerin. I've had a really hard time getting them going again in starters. Probably not freezing enough yeast, the vials are maybe 20 mL or so. Do I need to increase vials up to 100 mL, or what is the reco for volume?

If I can't improve I will go back to washed yeast jars in the fridge again. Wife would not be happy with that decision.

solbes said:

I went with this method and switched over my 5 yeast strains to frozen vials w/ the glycerin. I've had a really hard time getting them going again in starters. Probably not freezing enough yeast, the vials are maybe 20 mL or so. Do I need to increase vials up to 100 mL, or what is the reco for volume?

If I can't improve I will go back to washed yeast jars in the fridge again. Wife would not be happy with that decision.

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Hopefully someone more knowledgeable can give you a better answer than I. I freeze an estimated 100 billion cells. I've had plenty of starters that were slow to take off, but I've never had a single jar that didn't turn into a big pile of yeast after a few days on the stir plate.

What kind of freezer are you using? What is the volume of your first step?

I start with 1 pint and one jar of yeast. Normally takes 36-48 hours to ferment out the first step. Then it's business as usual. If you are starting with a very low cell count, you should be using a very small starter for the first step and slowly stepping up.

In order to take the guess work out I freeze half pint jelly jars roughly half full of washed yeast slurry.
That gives me over 100ml of yeasties to start with, no problem so far with waking them up after months in the freezer.
Thanks to everyone who have contributed to this thread, my yeast handling has improved as well as my starter preparations.

I am hoping we are going to hear back from the couple microbiology types that have offered to do viability tests under different freezing and thawing conditions. I realize that my test ended up being less thorough than I had planned (science got in the way), but I want to report back that after about 10 mos, my slow chill, rapid thaw method posted earlier in the thread continues to yield yeast stocks with high viability that can be pitched directly for an overnight start or, more appropriately, used for an overnight starter for a vigorous start with airlock activity starting within a few hours. Again, one caveat relative to most homebrewers is that I have a -80C freezer. However, I expect my experience would not be dramatically different in a home freezer if the sample was protected from frost free cycles.

Looking forward to those follow up reports.

Regular old freezer with a thaw cycle. But I put the vials in an insulated cooler with ice, buried in the middle of the freezer. Also did the slow chill, rapid thaw technique.

When I step it up I usually do 1 pint at 1.030 OG. Maybe I will try a larger volume of yeast next.

BBL_Brewer said:

I'm still trying to rustle up some funds for a microscope. I almost bought one recently, but opted for an RO unit instead.

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I bought a $24 microscope on Amazon. To be sure, you get what you pay for, but it's actually good enough to focus in on a hemocytometer. I haven't tried counting yeast yet, but I think it'll work OK.

http://www.amazon.com/dp/B0001FPRC0/?tag=skimlinks_replacement-20

Forkhead said:

I bought a $24 microscope on Amazon. To be sure, you get what you pay for, but it's actually good enough to focus in on a hemocytometer. I haven't tried counting yeast yet, but I think it'll work OK.

http://www.amazon.com/dp/B0001FPRC0/?tag=skimlinks_replacement-20

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Sweet! Let me know how it works out when you take it for a test drive.

Forkhead said:

I bought a $24 microscope on Amazon. To be sure, you get what you pay for, but it's actually good enough to focus in on a hemocytometer. I haven't tried counting yeast yet, but I think it'll work OK.

http://www.amazon.com/dp/B0001FPRC0/?tag=skimlinks_replacement-20

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One other question. What stains would I need to check for viability and or contamination and where is a good place to get them?

I use trypan blue to measure viability, but I don't know where you can buy it other than scientific supply stores like Sigma-Aldrich. I don't know if they'll sell to an individual user not affiliated with an organization. I think you can also use methylene blue, which you may have an easier time finding.

If you want to do it, you take a small sample of your yeast and dilute it in trypan (methylene) blue and place on the microscope. Live cells exclude trypan (methylene), dead cells will look dark blue.

Forkhead said:

I use trypan blue to measure viability, but I don't know where you can buy it other than scientific supply stores like Sigma-Aldrich. I don't know if they'll sell to an individual user not affiliated with an organization. I think you can also use methylene blue, which you may have an easier time finding.

If you want to do it, you take a small sample of your yeast and dilute it in trypan (methylene) blue and place on the microscope. Live cells exclude trypan (methylene), dead cells will look dark blue.

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Cool. That sounds easier than the way we did it in micro back in the day. Thanks.

Colony formation is a lot more accurate.

Brewitt said:

Colony formation is a lot more accurate.

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I agree colony forming units (CFU) is the most accurate way to measure viability. The problem is that it takes 24-48 hrs for colonies to grow up. Meanwhile, what do you do with your starter? It'd be nice to have a real time measurement of viability. As far as I can find, no one has directly compared viability measurements by CFU or trypan/methylene blue to see if they have similar accuracy.

If I have time, I'll pour some plates and grow up colonies to compare CFU with my trypan blue measurements. My yeast got delivered today, I'll have some results in just over a week.

I really don't want to get into plating. Although it would save some money to streak once in a while. Even if staining is less accurate, it's still better than flying bilnd and a lot less hassle.

Well, the experiment has begun. I made my starter last night. I decided to use California Ale yeast from White Labs (WLP001). Before pitching the yeast, I took out a small aliquot to count on a microscope and hemocytomter, and measured viability by Trypan Blue exclusion. The total # of viable yeast cells in the vial from White Labs was ~75 billion and this correlated to ~85% viability.

I was expecting the total # to be closer to 100 billion, but I guess White Labs doesn't actually put that many in each vial. I also plated the cells on YPD plates to compare the numbers from the hemocytometer/Trypan with actual colonies. It'll take ~24 hrs for the colonies to grow up. I'll post again when I get those results. Meanwhile, I'll keep growing up more yeast in the starter to test various freezing conditions.

Forkhead said:

Well, the experiment has begun. I made my starter last night. I decided to use California Ale yeast from White Labs (WLP001). Before pitching the yeast, I took out a small aliquot to count on a microscope and hemocytomter, and measured viability by Trypan Blue exclusion. The total # of viable yeast cells in the vial from White Labs was ~75 billion and this correlated to ~85% viability.

I was expecting the total # to be closer to 100 billion, but I guess White Labs doesn't actually put that many in each vial. I also plated the cells on YPD plates to compare the numbers from the hemocytometer/Trypan with actual colonies. It'll take ~24 hrs for the colonies to grow up. I'll post again when I get those results. Meanwhile, I'll keep growing up more yeast in the starter to test various freezing conditions.

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Did you use the $24 amazon scope to view the cells with the hemo?

I first counted at home using the $24 microscope from Amazon, then I took the cells into work to count using a real microscope. I was able to get pretty close using the $24 scope, but it was pretty difficult, and I have many years of experience counting cells on microscopes. There's no way you could do a viability measurement on the $24 scope because it was impossible to distinguish live from dead cells.

Another tip for anyone wanting to repeat this. I pitched a 35ml vial of White Lab yeast into 1.4L of wort, which is a 40-fold dilution. I had to dilute the aliquot that I took out of the starter another 20-fold before they were dilute enough to count on the microscope, which is a 800-fold total dillution. For plating the cells, I diluted the aliquot taken from the starter 100,000-fold so that hopefully there will be ~100 colonies per plate.

Well, so much for a cheap scope. Thanks for the info.

One concern less. Thanks for info.

So - does anyone have a recommendation on an "affordable" microscope that would allow me to do viability counts? I'd like it to be functional, but I don't wanna have to take out loan papers to get it, y'know? If we're gonna start testing our hypotheses, I wanna play too!

IsItBeerYet said:

So - does anyone have a recommendation on an "affordable" microscope that would allow me to do viability counts? I'd like it to be functional, but I don't wanna have to take out loan papers to get it, y'know? If we're gonna start testing our hypotheses, I wanna play too!

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Keep an eye out on E-bay. That's the best I can tell ya.

I've seen some "class room kits" out there in the $70 - 80 range, but am hesitant to buy if the microscope is subpar.

OK, so my colonies have grown up so I can compare counting on a hemocytomter with CFU by plating. I want to revise my previous calculation based on hemocytomter counts to say that the original vial had 66 billion yeast. The CFU on the plates say there were 44 billion. It's pretty close, but not exactly the same. One thing to keep in mind though, is that to plate the cells, I had to dilute them 100,000-fold in order to get a countable number/plate. It's possible that such a large dilution introduces some error into the count.

I'd say my conclusion so far is that both methods give pretty close results. I'll keep going with trying to grow up enough yeast to experiment with the freezing method. My starter isn't growing as fast as I thought it would. I only had 115 billion total cells this morning. I'm trying to get to 1 trillion so that I can freeze down 9 different aliquots of 100 billion cells. This may take longer than I anticipated.

As far as microscopes, I don't know, but this one looks pretty good:
http://www.amazon.com/dp/B005TJ5CEG/?tag=skimlinks_replacement-20

I'd consider this one if you want a cheaper alternative though:
http://www.amazon.com/dp/B000NOU54O/?tag=skimlinks_replacement-20

If you end up getting one, please post on here an let us know which one you got and how well it works. Thanks!

Forkhead said:

OK, so my colonies have grown up so I can compare counting on a hemocytomter with CFU by plating. I want to revise my previous calculation based on hemocytomter counts to say that the original vial had 66 billion yeast. The CFU on the plates say there were 44 billion. It's pretty close, but not exactly the same. One thing to keep in mind though, is that to plate the cells, I had to dilute them 100,000-fold in order to get a countable number/plate. It's possible that such a large dilution introduces some error into the count.

I'd say my conclusion so far is that both methods give pretty close results. I'll keep going with trying to grow up enough yeast to experiment with the freezing method. My starter isn't growing as fast as I thought it would. I only had 115 billion total cells this morning. I'm trying to get to 1 trillion so that I can freeze down 9 different aliquots of 100 billion cells. This may take longer than I anticipated.

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Did you get 115 billion from the 1.4L starter or have you stepped it up since then? Just wondering what your growth rate was so I can compare that to the yeast calculators.

You're doing great man. If it takes a little longer, no worries as long as it's not inconveniencing you. You're posting results much faster than the rest of us so keep the momentum going if you can

One more question for ya Forkhead, are you using a stir plate or simple starter?

Nice write up.

I just started freezing my yeast and have some 3711 and 3068 in the chest freezer in the basement. The great thing is that I don't make wheats and saisons often (maybe once or twice a year) so I can store the yeast a long time and not take up the space that mason jars use. I haven't used any of my frozen yeasts yet so I'll have to see how that goes.

The vial from White Labs was ~50 billion (depending on how you count), which grew up to 115 billion in the 1.4L starter. Don't try to factor in time for the growth rate on my starter. I had it at room temp, but then cold crashed it for a few hours thinking it was done, but then I put it back at room temp for another day when the numbers weren't higher. Yada yada yada... I'm learning as I go.

I just cold crashed it for a few hours this afternoon, took of the wort, and added back fresh wort. My starter is up to a total volume of 1.8L now. I'm using a stir plate. I'm also putting it at 30oC (86oF) to speed up the growth now too. I'll let it go over the weekend and see what the count is on Monday.

Forkhead said:

The vial from White Labs was ~50 billion (depending on how you count), which grew up to 115 billion in the 1.4L starter. Don't try to factor in time for the growth rate on my starter. I had it at room temp, but then cold crashed it for a few hours thinking it was done, but then I put it back at room temp for another day when the numbers weren't higher. Yada yada yada... I'm learning as I go.

I just cold crashed it for a few hours this afternoon, took of the wort, and added back fresh wort. My starter is up to a total volume of 1.8L now. I'm using a stir plate. I'm also putting it at 30oC (86oF) to speed up the growth now too. I'll let it go over the weekend and see what the count is on Monday.

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Those results are a little disturbing. That's ~1/3 less yeast cells than Mr. Malty predicts and 1/2 as much as Wyeasts calculator predicts.