Setting up a yeast lab?

[McMullan]

@McMullan - I went to freezing because my attempts at keeping a yeast cake from a batch with the hopes of repitching at a later date proved unsuccessful. Basically, my brew schedule was too drawn out for the yeast to survive just under some wort in a mason jar at fridge temps. I have heard that two weeks is the cutoff for keeping a large amount of cells alive for repitching.

So can you add some more detail about your method? Does it only apply to yeast poured directly from the package as it is in a special solution for storage? So far I have a had good luck with all of my ale strains. Lager has been complicated as I also started pressure fermenting at normal lager temps which requires more yeast than normal. So I think my attempts from frozen have been underpitches. I am working on a solution of freezing bottles of 75ml of slurry from a batch and then starting with two and growing up for a batch.

While very little homebrewers do is "optimal", it can still be an improvement for schedules and not being a business etc... Thanks,

Yes, 1.5-2.0ml aliquots direct from a fresh yeast pack. That’s the simplest way. Next would be making a small sterile culture/starter to aliquot. No additives. I’m experimenting with trehalose, but not enough data yet. Scaling things down regardless, to 1-2ml is one of the tricks, fridge or freezer. It forces us to start from little and step up multiple times, promoting a better pitchable population. For lagers, my final ‘starter’ (stepped up from 2L) is actually a half batch lager 10L) with harvested yeast re pitched into a full batch or another half batch, which ferments out within 5 days or so. I find it the best way to build up a decent quantity of really health yeast, with the benefit of getting a beer out of it. Better than wasting 4-5L on a starter.

 

[McMullan]

He does mention to leave in the fridge for a few days before freezing to increase vitality but doesn’t say exactly why…

It doesn’t take that long to replace a significant amount of H2O, but it is advisable to allow the cells to condition themselves (increase tolerance) before prepping them for freezing. Hours rather than days, for glycerol to work its way in. And trehalose is much better than glycerol, if it’s easier to get hold of.

 

[beren]

It doesn’t take that long to replace a significant amount of H2O, but it is advisable to allow the cells to condition themselves (increase tolerance) before prepping them for freezing. Hours rather than days, for glycerol to work its way in. And trehalose is much better than glycerol, if it’s easier to get hold of.

So usp glycerin doesn’t work? And I have been freezing about 5-6ml of slurry per vial.

 

[McMullan]

So usp glycerin doesn’t work? And I have been freezing about 5-6ml of slurry per vial.

Not as well as trehalose, which has been recognised as a much better cryoprotectant for years. I think once samples go above about 2ml, controlling freeze and thaw rates starts to get a bit sketchy with diminishing returns, wasting space and resources. When it comes to propagating from a frozen sample, ideally, only a tiny scrape with a needle is required to inoculate a 10ml mini prep/starter. There seems to be a deeply flawed approach recommended by home-brew protocols where the whole vial, up to 50ml or more gets tossed into an oversized starter, or even pitched directly. These freshly-thawed yeast are pretty much knackered mainly and are much better cultured up from something small. It might well ‘work’ according to an individual’s expectations, and that’s absolutely fine, but compared to what? Wouldn’t you want to make it work noticeably better, possibly with less effort and lower cost? I’m not trying to sell you anything, just offering better advice for free. It gives me a buzz to help people with what I know. If I charged lots of money, would it convince you more?

 

[beren]

Not as well as trehalose, which has been recognised as a much better cryoprotectant for years. I think once samples go above about 2ml, controlling freeze and thaw rates starts to get a bit sketchy with diminishing returns, wasting space and resources. When it comes to propagating from a frozen sample, ideally, only a tiny scrape with a needle is required to inoculate a 10ml mini prep/starter. There seems to be a deeply flawed approach recommended by home-brew protocols where the whole vial, up to 50ml or more gets tossed into an oversized starter, or even pitched directly. These freshly-thawed yeast are pretty much knackered mainly and are much better cultured up from something small. It might well ‘work’ according to an individual’s expectations, and that’s absolutely fine, but compared to what? Wouldn’t you want to make it work noticeably better, possibly with less effort and lower cost? I’m not trying to sell you anything, just offering better advice for free. It gives me a buzz to help people with what I know. If I charged lots of money, would it convince you more?

No I love Quality free advice. I have about 15 strains in the freezer and would love to know how I can do things better in the future. Would you consider doing an updated post with your recommendations?

 

[Bassman2003]

Thanks for the reply. I can't argue that smaller is better. New cells can't be beat. But, how many stages are required for a build up? 2ml - 20ml - 200ml - 2000ml It is effort vs time. Right now I go from 25ml - 200ml to 2000ml into the batch. So I guess only one extra early stage step. That is for an ale. Lager is a lot more as you stated.

I am going to experiment with freezing 75ml slurries which would be grown up in parallel for a lager build up. I know it is not optimal but if it can work, it is much less time compared to brewing a light batch just for the final starter step. I am just trying to find a balance between very effective and not being too much of a time constraint. One slurry from a batch (300ml) would yield two full pitches. IF it is a viable process.

 

[McMullan]

No I love Quality free advice. I have about 15 strains in the freezer and would love to know how I can do things better in the future. Would you consider doing an updated post with your recommendations?

Thanks for the reply. I can't argue that smaller is better. New cells can't be beat. But, how many stages are required for a build up? 2ml - 20ml - 200ml - 2000ml It is effort vs time. Right now I go from 25ml - 200ml to 2000ml into the batch. So I guess only one extra early stage step. That is for an ale. Lager is a lot more as you stated.

I am going to experiment with freezing 75ml slurries which would be grown up in parallel for a lager build up. I know it is not optimal but if it can work, it is much less time compared to brewing a light batch just for the final starter step. I am just trying to find a balance between very effective and not being too much of a time constraint. One slurry from a batch (300ml) would yield two full pitches. IF it is a viable process.

From frozen, I’ll add a scratch to a 10ml overnight culture, plate a loop then add loop, collected from a few healthy-looking colonies, into 10ml, 100ml, 500ml, 2.5L then 10L (half batch of lager) then start repitching. Adding more frozen slurry to a bigger 1st step starter is increasing the chance of problems and could promote under pitching, even if the recommended number of cells are being pitched, because so many are potentially aberrant/knackered. It might work better simply due to Lady Luck, but it won’t be a reliable strategy. Otherwise White Labs and Wyeast, etc., would be selling packs of frozen wet yeast.

 

[Bassman2003]

Thanks for sharing your process. I will keep this in mind as a barometer to judge my experiment. The task of a cold ferment pressure lager is daunting from a yeast point of view. So many cells are needed from such a small starting point. Which leads me back to the OP's reason for posting...

What is the definition of a yeast lab? Looking at McMullan's process, I think at least a safe space to operate in with some microbiology protection comes to mind. All of the steps need some sort of sanitizing/sterilization which is quite a lot to keep track of and execute in the home.

@matt_m - what amount of participation are looking to have with yeast ranching and what are thinking when you envision your yeast lab?

 

[RM-MN]

With all this discussion of the requirements of a yeast lab, would you not be farther ahead to keep the original yeast, building it up as needed before pitching?

 

[matt_m]

Been reading through all the great replies, could get really complicated it sounds. I don’t think I want to take it nearly as far as freezing yeast long term or anything like that. My thoughts at this time are more along the lines of buying fresh yeast when I can and propagating that forward for a few to several months until I can get more fresh yeast. Probably a mix of overbuilding starters and harvesting

Further, I was just thinking along the lines of storing yeast in PET jars in the refrigerator. Probably could go way overboard and buy a few 2” sight glasses and end caps to match my new yeast brink and use those to avoid handling.

Is this a workable plan to have fresh yeast?

How valuable doing counts is for my use case?

 

[McMullan]

Storing yeast, regardless, requires a little effort (very little, imo) building up step cultures occasionally. It takes minutes with stored vials of wort, etc., ready to hand. Very little hands-on time, in reality. Ideally with at least some repitched generations, to appreciate the reward of pitching something that can’t be bought. The best pitchable yeast possible. There’s really no need for home brewers to make it much more complicated than it needs to be.

 

[Bassman2003]

Yeast brinks seem to be the best way to store yeast. They need to be 100% oxygen free to be most effective.

 

[user 341102]

Check out Sui Genesis Brewing (link below). Brian is a microbiologist by day and has some useful info on how to bring some of these techniques to the home scale without breaking the bank. Wild yeast collection and isolation, cell count and viability, clean workspace, etc. Lots of other interesting stuff on his site and YouTube videos, too.

https://suigenerisbrewing.com/

 

[McMullan]

Yeast brinks seem to be the best way to store yeast. They need to be 100% oxygen free to be most effective.

Yes, can’t beat it, but it’s a vessel for storing harvested fresh yeast slurry for days, on the verge of being repitched, while the yeast cells are still at the highest levels of viability and vitality. Here’s a simple scaled-down home brew version:

 

[hottpeper13]

I do overbuild starters and save them in 250 ml Erlenmeyer flasks. The different yeasts seem to have the same amount of sediment and since it's an overbuild the trub is minimal. So I deem them to have the same cell count,and make starters accordingly. Every procedure needs an SOP in my brewery.

 

[McMullan]

So I deem them to have the same cell count,and make starters accordingly.

The potential problem here, with large volumes of stored slurry, as the slurry ages, is cell counts and assumptions ignore a crucial part of the puzzle, vitality, which declines too over time. This can result in under pitching even if enough cells have been pitched. A case where pitching fewer healthier cells ferments better than more unhealthy cells. By starting at very small volumes, like 10ml, you force things in your favour.

 

[Bassman2003]

Thanks for sharing. I think my approach for lager yeast will be to stick with my 50ml vials (25ml slurry + 25ml glycerin/wort) but just use more. Take out six vials, thaw them out and put into 2 parallel starter routines. I know this is not the same as starting with one 10ml vial and growing it up but it is for convenience which has to have some sacrifices. I see 50ml vials as better than 150ml vials for freezing. If it all does not work well, it is just yeast, my time and some learning. Thanks for your perspective.

 

[McMullan]

Thanks for sharing. I think my approach for lager yeast will be to stick with my 50ml vials (25ml slurry + 25ml glycerin/wort) but just use more. Take out six vials, thaw them out and put into 2 parallel starter routines. I know this is not the same as starting with one 10ml vial and growing it up but it is for convenience which has to have some sacrifices. I see 50ml vials as better than 150ml vials for freezing. If it all does not work well, it is just yeast, my time and some learning. Thanks for your perspective.

Bear in mind that freezing, regardless, is going to kill and knacker yeast cells at a high rate determined by a number of factors and that they won’t be conditioned well for fermenting wort. Adding more won’t change much and most likely promote an extended ‘lag phase’ with associated risks, especially for a lager, in terms of partial infection and yeast stress leaving subtle off flavours. Try to do a meaningful comparison with repitching freshly harvested yeast. I find a lager yeast cake kept cool under beer in primary is good for repitching directly for at least 2 weeks after fermentation finishes.

 

[Bassman2003]

My problem is that I only brew lager a few times a year. And some ale styles months apart. So repitching is not a viable option for me. I see freezing the yeast as a 'long term repitch' in some way. It is my only way to have a chance at keeping yeast and using them further down the line. So far, the ales have been acting well. I have done a few lagers that have had long ferments, but I think there was a severe underpitch involved as I only started with two vials and fermented the beers at 48F with 2 bar of pressure. Pretty stupid looking back! Which is why I am thinking to start with six vials out of the freezer and see if my outcome is improved.

Pitfalls would be that I am moving around and pitching more dead yeast cells. But I also gain more live yeast cells. This is a frustrating part of the hobby for me as I do not have the time or inclination to properly prop up a yeast cake for lagers. It is still a hobby and I do not follow lab procedure like you do with a microscope etc... Just trying to find the best way forward that still has a percentage of success.

So given all of that, can you think of a better way for homebrewers to achieve long term repitching? Thanks.

 

[McMullan]

So given all of that, can you think of a better way for homebrewers to achieve long term repitching?

It's impossible to maintain sufficient viability and vitality of wet yeast for direct pitching without feeding them regularly, which is a lot more hassle than stepping up from small volumes. Note if a procedure causes high rates of mortality in yeast cells, imagine what it does the the cells which survive. I'll only go to the effort when I plan to brew at least a few batches back-to-back, so I can repitch the yeast at their best. Binge brewing. But, as I've noted, if you already have the required sterile wort step ups frozen in bottles ready to go, it's just a little time management with minimal hands-on. I don't even bother with a stirplate anymore, just leave starters on the countertop in my brew room and swirl them whenever I pass.

 

[Bassman2003]

I take the frozen 50ml vials out to thaw for 24 hours then put them in 200ml of 1.020 wort for a few days then add 2L of 1.035 wort and let that ferment out. Crash, decant then pitch. So not really direct pitching and hopefully a fair amount of new growth. For the lager example I would do two of these at the same time but starting with three vails each instead of one. Proportionality is off but hopefully more cells in the end.

Maybe I need to invent an autofeeder that sits in the fridge and keeps the yeast fed on a schedule?

 

[McMullan]

When I used to keep a sourdough starter and feed it daily, the thought of occasionally adding a tablespoon or two of LME to a pitchable amount of brewer's yeast slurry did cross my mind. It might at least keep them pitchable for longer than weeks.

 

[beren]

When I used to keep a sourdough starter and feed it daily, the thought of occasionally adding a tablespoon or two of LME to a pitchable amount of brewer's yeast slurry did cross my mind. It might at least keep them pitchable for longer than weeks.

I feed my starter every week or 2 when it’s in the fridge
Sorry, sourdough.

 

[user 341102]

Has anyone actually measured cell count and viability in their frozen cultures? Pretty easy with a relatively small financial investment. I've never bothered because I get pretty good results from stepping up a single 50ml conical for a 2L starter (lagers, mostly).

To me, a home yeast lab means: microscope, hemocytometer, glycerine, and 50ml conicals. Not much else needed at this level (unless you plan to ranch wild yeasts).

 

[McMullan]

Yes and, judging from the extended “lag phase”, vitality was pretty poor too. Presumably why commercial yeast suppliers don’t sell yeast in this frozen format. I suspect my lager fermentations are done by the time yours get started. My fermentations are predictable so I can plan ahead with confidence. I’m in control of the process with my method. Yours is more “wait and see” with fingers crossed. If you disagree, contact Chris White and tell him about your exciting discovery.

 

[user 341102]

There are a lot of technical considerations when freezing and thawing yeast such as osmotic pressure of solution and speed of freezing and thawing. I have decent wet-lab education, training, and experience which carries over well to the home brewing. I haven't specifically measured my cell counts and viability, but get decent enough fermentation timecourses and flavor profiles in beer.

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So, it's likely not as simple as "freezing yeast doesn't work." There will be a heavy influence of the technical aspects of the lab procedures that one implements. I think one key aspect everyone could do is to freeze yeast slowly (about -1C/min) by placing in an insulated container in the freezer and thawing rapidly about about 30-35C.

 

[mac_1103]

In a previous life, I did QC/QA/product documentation for a company that sold frozen micro-organisms, and I can assure you that our methods were a lot closer to what McMullan describes than almost any of the "how to reuse your yeast" advice you will find on the internet. If you're happy with the results you get with one of the various shortcuts that homebrewers use, then by all means keep doing that. But if you're going to go to the trouble of setting up something worthy of being called a lab then you might as well do things right. And "right" means preserving a population of healthy cells and scaling up from a single one of those healthy cells for each use.

 

[mac_1103]

I think one key aspect everyone could do is to freeze yeast slowly (about -1C/min) by placing in an insulated container in the freezer and thawing rapidly about about 30-35C.

Mr. Frosty. Pretty simple to DIY too. But you still have to start with a healthy yeast population.

 

[hottpeper13]

I don't know about yeast but in the food industry the faster(colder) the food is frozen the less ice crystals form. I installed doors in these 20 below 50 mph wind freezers.

 

[DBhomebrew]

I don't know about yeast but in the food industry the faster(colder) the food is frozen the less ice crystals form. I installed doors in these 20 below 50 mph wind freezers.

Pretty standard. Same goes for igneous rock.