Long term yeast storage: agar vs. water

I'm trying to decide on the method for storage for my yeast library. I'm thinking about going with water instead of agar slants. From what I understand, water let's you store your cultures longer. Another benefit is that you do not have to refrigerate the culture as with an agar slant. The water slants can also be stored in smaller vials from what I read off one link off of Google. l Am I understanding this correctly?

I have been researching this and have switched to using only distilled boiled water. According to the research of Dr. Emil Christian Hansen, he was able to store yeast in distilled de-oxygenized water for 14 years and have it still be viable.

Viability will certainly be an issue that you will have to learn how to deal with, however, ample time and a stir plate should allow you to culture up sufficient yeast to brew with. As in any long term yeast storage you can either use the craps shoot method and just culture up a starter and pray for the best or you can make a mini batch with the yeast.

I use yeast nutrients and a stir plate to get my yeast up and running.

m.

My plan is to streak a plate, make a 10ml starter from spores off the plate. Then step it up to 50, 250, 500, 1000ml etc as needd for my cell count. I figure the plate will make it easy to get the best chance of a good. 10ml starter from the get go.

Sounds like you have a good plan. Flame everything that could contaminate it.

I starsan spray everything, then flame the loop and the bottle/flask opening. Cover everything with aluminum foil that has been in starsan.

Seems like a lot of work to some people, but to save 8 dollars every brew is a great. I calculate it out to 15 cents a beer. I should have my yeast culturing equipment paid off in about 10 brews. After that is money in the bank!

But really its the fun of culturing the yeast.

m.

I just bought a hemacytometer for $15 new on ebay. I'm getting a cheap Mead 900x microscope for $7. A local guy has shipping container full of lab glass from a shutdown refinery that I'm going to check out. He said he'll sell for 30% of new cost, so I'll get a lot of my beakers and erlenmeyer flasks off him. I still need to find a cheap place to get the culture tubes and a few glass petrie dishes.I'll need to buy an inoculation loop, and I need the pressure cooker too. I'll check craigslist for that. Not bad for a start. Pretty cheap really for the cost of a smack-pack or tube of yeast. Besides that, it's fun to be geeky and watch our cultures make bubbles.

Do you think a microscope with 43x is enough to count my yeast?

This is the type of scope I use. I only look at colonies on plates. Mine does 20x. So 43x should show a lot.

http://www.amazon.com/dp/B000Z4NYX6/?tag=skimlinks_replacement-20

m.

Mateo said:

Sounds like you have a good plan. Flame everything that could contaminate it.

I starsan spray everything, then flame the loop and the bottle/flask opening. Cover everything with aluminum foil that has been in starsan.

Seems like a lot of work to some people, but to save 8 dollars every brew is a great. I calculate it out to 15 cents a beer. I should have my yeast culturing equipment paid off in about 10 brews. After that is money in the bank!

But really its the fun of culturing the yeast.

m.

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In addition I think that beer from re-pitched yeast is actually better....

Well, washing and storing that much yeast would take up too much space for my purpose. Plus I think after a few batches (from what I've read) your yeast mutates anyway. If I culture new yeast each time I'll avoid those problems. Plus, if I also understand yeast washing and storage, it's only viable for short term storage if you don't repitch it right off the bat. I hope I got all of this right.

Mateo, what size vials do you use? And how many culture do you keep of a same yeast?

I have 3 different sizes. I am pretty sure they are 1, 2 and 3 dram vials. I like the long ones with round bottoms because they can go in a canning jar and will not fall over. I use an inoculation loop to pull a culture and then put it in a 1 dram vial to start my culture for a starter.

I try to keep a minimum of three vials of each of my yeasts, which are only 2. Kölsch and Budvar. I also have canning jars of washed yeast from brews. This is where I get 99% of brewing stock yeast and only go back to the vials if needed.

When I do a starter from a vial I work over a flame, flame the flame the vials opening and inoculate a 1 dram vial of wort that was 17 minutes in the pressure cooker.

Once it hits high barm I start stepping up on the stir plate. If you start on Monday you can have a good starter going for Saturday. If you have slow starting yeast its good to start on Friday and have the starter fully geared up by the following weekend. Its a little slow going from the loop to 1 liter, but little careful steps and you will have no problem.

Just remember you cannot compromise sanitation.

You will need to only do single colony selection once the ratio of bacteria to yeast starts to produce undesirable results in the beer. People repeat what they hear about mutations, however, the biggest threat is bacteria ratio. The mutations you will experience in your culturing might be beneficial to your brewing practices. The yeast will adapt to your techniques and environment, whether that means good beer or bad, fast or slow fermentation.

Lots of opinions have been given on this. I can only speak to the method used at SWMBO's yeast genetics lab. It is to store in a glycerol solution in a -80c freezer. Yeast will keep "forever" in this environment. This can be replicated by a chest freezer, styrofoam cooler (bio-med quality aka THICK preferred) and some freeze-packs.

Thought only the abstract's available for free viewing from this 1991 article, it looks like water will work fine for a few years (just not at 37 C) and it backs up Hansen's data:
http://informahealthcare.com/doi/abs/10.1080/02681219180000651

Stabs (don't slant the agar in a tube, let it solidify and literally stab the inoculating needle or loop into it) with a sterile mineral oil overlay also work well.

The abstract:

A total of 1583 yeast isolates, mostly Candida albicans, has been maintained under sterile distilled water for periods ranging from 1 to 18 years. Overall, 71 (4·5%) of the isolates were not recoverable at 37°C. Survival of the yeasts was 97% in the first 5 years and 96% after 10 years. Isolates of Candida krusei and Saccharomyces cerevisiae were particularly unlikely to survive storage in water and these species should be preserved by other means if they are to be kept for more than a few years. All of the Candida guilliermondii (n = 19) and Candida parapsilosis isolates (n = 51) survived water storage. The water preservation method is inexpensive and simple and is recommended for maintenance of most yeasts isolated from clinical material.

Perhaps you are doing something really novel, or just want to preserve the pathogen from a good case of oral thrush or vaginitis? (SWMBO will NOT thank you!) Otherwise, preserving Candida species won't help you. Saccharomyces cerevesiae is our friend in brewing, making our wort into beer. Unless there are other articles to the contrary, it looks like you are swimming against the tide with your water preservation plans.

From my reading of most of the yeast ranching threads, the deoxygenated distilled water approach (stored at room temp) works very well for up to a year's storage. Beyond that, simple loop streaking isn't enough to culture it up again; during at least the 1-3 year period, putting several drops on the culture medium seems to work, though. It is a bit more work bringing it back to viability than a frozen yeast bank is. But not needing refrigeration can be a big win (frozen yeast banks or refrigerated agar plates can lose huge amounts of viability if you suffer a power outage).

To me, ease of use is as important as long-term storage (I save yeast to save money by not buying Wyeast/White Labs for every brew that needs those strains, not to keep strains around long-term that aren't easily available), so I wouldn't do it, but if your needs are different it's not a totally outlandish idea.

I've not tried it personally, so hopefully some of the people who actually did can chime in.

You're best bet for long term storage is freezing with a cell permeable freezing agent, such as glycerol (15%). You can also use a non-permeable agent such as 1M sorbitol for shorter periods. Ideally you want to store at -80C, but -20C in a non-defrost freezer is sufficient for many years.

I have no doubt you may get viable yeast after many years in distilled water, but odds are you are bottlenecking for a population of yeast that does not resemble your original population.

ThreedogsNE, yeah, I saw that and, yeah, I know the difference between the various genera of yeast. The critical point is this one:

if they are to be kept for more than a few years.

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Not much beats a -80 glycerol stock or freeze drying, but most folks don't have access to -80 freezers or lyophilizers. Water's cheap, and apparently effective enough. This guy seems to make it work: http://www.alsand.com/beer/yeast/store_E.html He has some references as well.

The procedure one should follow would be to stock them and occasionally plate out a subsample to check for viability. When it starts to drop, bulk back up and put them back in storage.

Professor Frink's point

but odds are you are bottlenecking for a population of yeast that does not resemble your original population.

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is a good one. IMHO, it's valid for any method other than -80 stocks.

I have ~60 strains in glycerol stocks in a -80 freezer. Because I don't have to, I won't go near water storage. But if I didn't have access to deep freezers, I'd think long and hard about what to do. "Liquid Drying" might work, though one still needs access to a vacuum source, just no freezing needed.

My main goal is to have these yeast cultures on hand so I don't have to go buy yeast al the time. Where can I get glycerol?

It's sold in drug stores and the health sections of mega stores as glycerin

JohnMc said:

The procedure one should follow would be to stock them and occasionally plate out a subsample to check for viability. When it starts to drop, bulk back up and put them back in storage.

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Yet this is creating a new generation and each successive generation is less likely to be a match to the original. Again, it is introducing a point for mutation to become the dominant strain.

Islandboy85 said:

Do you think a microscope with 43x is enough to count my yeast?

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You'll have a hard time counting at ~40x, you should really have 100x.

Again, it is introducing a point for mutation to become the dominant strain.

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That would be why I said Professor Frink's point holds for everything except -80 stocks (aka, cryopreservation). However, if someone doesn't have access to cryoperservation, then the choices are limited.

As far as this goes, though, one could isolate individual colonies and prepare test beers from each. Re-preserve only those that perform and taste the same as the parent (good notes will help here).

FWIW, if someone's going to try to keep yeast for years, then they should learn to recognize key features (growth rate, flocculation, colony morphology, etc) and select for those that are the "type" they're after, to attempt to keep genetic drift to a minimum.

JohnMc said:

FWIW, if someone's going to try to keep yeast for years, then they should learn to recognize key features (growth rate, flocculation, colony morphology, etc) and select for those that are the "type" they're after, to attempt to keep genetic drift to a minimum.

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So, can I see yeast morphing with a microscope? Or, is it more of how the yeast actually performs? I guess what I'm asking is is it a physical change that you can see rather than having to do all kinds of tests?

Ok, after checking my freezer, I found out it IS a self defrosting freezer. It goes to -4 degrees Fahrenheit. Now that we all know my freezer is less than adequate to do the job of freezing yeast decently, would you recommend slants or water storage?

I've been using slants for almost 2 years now. I have 8 strains and start with 6-8 slants per strain. When I get down to 2, I reculture and make new slants.

Two days ago I pulled out a slant of WLP011 that was well over 1-1/2 years old, added a couple ml of wort, and within 24 hours had a small krausen.

My problem with all this is the amount of time and planning it takes to pull it off. I'm starting to wonder if it's really worth it.

So, can I see yeast morphing with a microscope? Or, is it more of how the yeast actually performs? I guess what I'm asking is is it a physical change that you can see rather than having to do all kinds of tests?

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It isn't the yeast that morph for this purpose, thought they do while they grow. Morphology is the shape and form at the time of observation. Basically, you let the colonies on a petri plate get really big, like dime-size. (I wouldn't propagate from large colonies, FWIW). It's a technique that used to be used before DNA technology and biochemical analysis became cheap enough to distinguish strains of things. These are still cost prohibitive for home brewers, and large colony morphology is cheap; you just leave it out of the fridge longer. Basically, you let your initial plate go long (after banking), record notes & maybe pix. When bulking up again, do it in aliquots/isolated colonies, then let some go long again and compare to the originals. Discard those that don't match.

Cool, thanks JohnMc.

I've been following this thread closely as I have the same questions regarding storage, specifically storage of the yeast I've captured off of juniper berries: https://www.homebrewtalk.com/f163/can-i-culture-yeast-juniper-berries-169156/ I have saved a yeast vial of the "mother" yeast, (that is to say the initial yeast that grew from the first starter) washed and stored in the refrigerator. I am seeing that yeast is alot tougher than alot of people give it credit for, I mean doesn't/didn't all yeast initially exist in nature, in some cases freezing for months, thawing, freezing again numerous times then surviving through 100F temps for months etc, etc only to repeat the process again all the while reproducing more cells and new generations.

Which brings up another question; If you store a certain yeast in freezing temperatures that naturally existed in a tropical environment, Aren't all the surviving yeast likely to be mutated is some way from the original? We have no way of knowing where or when most of the commercially available yeast comes/came from, nor how much mutation it's gone through, nor what generation it is, etc, etc. We only know what properties/effects it's suposed to impart to our brew.

My point is this; Whenever a human uses or stores yeast, it will likely mutate in some way. I keep trying to find some inexpensive way to store my juniper yeast dry and in a manner which will not allow it to mutate, after a few days of deduction/procrastination I have realized that it (Or some form of it) is already stored on all the juniper berries on all the trees in the hundreds of acres of BLM land in Colorado and I'm certain other states. I should stop worrying about it and stop trying to control it, when I run out or it becomes mutated or unviable I will simply trek out and pick some more berries.

I will still use commercial yeast for certain brews assuming the juniper yeast doesn't work in some manner for certain styles (still testing). But when I do find it has failed ( I already know it's alcohol tollerance is ~15%) in some way, that allows me to search for another naturally occuring yeast that will not fail under those circumstances, that's what I find exciting, in the yeast world anyway.

Wow, long post, I guess you all know me now . . . . in a yeast-shell.

Keep on yeasting my friends

I looked in to the water storage a while back. There's several labs down the hall the work with S. Cervisiae for various research including bio fuels.

Basically for the water storage you are creating a nitrogen free enviornment and this causes the yeast to sporalate. The yeast spores can survive for a long time in a spore state. The lab manager I spoke with recommended using a microscope to check for sporalation and if you have spores then your sample should last for years. She also said to expect a long lag time to wake up the yeast. Not much if anything can penetrate their cell walls in the spore state so it takes cells a while to realize when they are back in ideal media to start propogating.

I washed and stored some WLP001 about 6 months ago. At the 3 month mark it cultured up just fine. When I hit a year I'll try again and see how it goes.

Sort of related...about a year and a half ago I made some stabs. Those are similar to slants in that you put agar media in to a test tube. But instead of resting on the side to make a slant you let it cool in an upright position. Once cool you stab the yest deep in to agar several times. Supposedly yeast can survive for several years in the refrigerator this way. I'm going to try to culture some when it reaches the two year mark.

I downloaded a bunch of pdfs (which are all on my work computer right now) that dealt with this topic area. While water storage can't be beat for cheap, I now think that slants stored under sterile mineral oil are better. There's a 1940's report of wine yeast being kept unchanged (as far as their fermentation qualities go) for 9 years.

Basically for the water storage you are creating a nitrogen free enviornment and this causes the yeast to sporalate.

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IMHO, sporulation isn't the best outcome. For S. cerevisiae, spores are gametes; for sexual reproduction. That means re-arrangement of things like genes. That'll accelerate population drift, not that it doesn't happen anyway.
FWIW, I've read most beer yeast are aneuploid and don't sporulate very well.

about a year and a half ago I made some stabs.

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Stabs work well, particularly with a mineral oil overlay.

Thanks for the mineral oil suggestion. I can see how that would help and I never heard of it before (or never paid attention for whatever reason)

Does mineral oil need to be sterilized?

Does mineral oil need to be sterilized?

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Good question, yes, it does. You can buy it sterile or sterilize it in the oven. It can be sterilized by dry heat, 1-2 hours at 350 deg F in your oven.

Professor Frink said:

You're best bet for long term storage is freezing with a cell permeable freezing agent, such as glycerol (15%). You can also use a non-permeable agent such as 1M sorbitol for shorter periods. Ideally you want to store at -80C, but -20C in a non-defrost freezer is sufficient for many years.

I have no doubt you may get viable yeast after many years in distilled water, but odds are you are bottlenecking for a population of yeast that does not resemble your original population.

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Professor,

I'll concede that -80C storage is the best option here for long-term storage if you have access to the equipment. I don't want to argue this point. But, I don't have access to this equipment, so I'm considering sterile water storage.

I'm familiar with the concept of bottlenecking and the affects it can have on populations (B.S. in microbiology), but would appreciate a little more explanation on your thinking.

Consider this situation: you get a yeast strain and prepare a master sterile water sample that goes into long-term, archival storage. You also prepare a working sterile water sample(s) from which you culture and prepare starters. Then, every year or so you use the master sample to rebuild the working sample(s).

I realize that the viability of the master sample will degrade over time and there may be occasions (I'm not exactly sure how long the master will stay viable, but let's say 5 years) that you have to create a culture from the master and then rebuild a new master. This could create a bottleneck that could alter the yeast and some testing would be in order, but I wouldn't expect to see a huge drift in the strain between these, essentially, two generations. I guess what I'm getting at is, assuming you rebuild the master every 5 years, over a 20 year brewing career your final yeast strain would only be the 4th generation and any genetic drift would be minimal.

Any thoughts?

Aaron

Long term storage of yeast needs to not only keep the yeast viable (which is what most information here and elsewhere focuses on) but also ensure the yeast does not mutate and change. Colder temperatures mean less cell mutation, change and genetic drift, if you don't have -80C, then a standard -20C freezer is the next best thing. Using a 50% glycerin mix means that the samples stay liquid and do not actually freeze solid at -20, so it should be easier to maintain them even with freeze/thaw cycles in a normal freezer.

I guess what I'm getting at is, assuming you rebuild the master every 5 years, over a 20 year brewing career your final yeast strain would only be the 4th generation and any genetic drift would be minimal.

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Plus, if one kept good notes and made a few submasters each time, tested them, and discarded any off types and kept a good one, it would probably be even more minimal. Unless one selected for drift by taste preference.

Using a 50% glycerin mix means that the samples stay liquid and do not actually freeze solid at -20, so it should be easier to maintain them even with freeze/thaw cycles in a normal freezer.

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that jives with my vague memory on -20C storage, though I've this little feeling there's a caveat that I can't remember. Oh well, I'm on the down side of middle age, so maybe I have an excuse.

JohnMc said:

. . . . .that jives with my vague memory on -20C storage, though I've this little feeling there's a caveat that I can't remember. Oh well, I'm on the down side of middle age, so maybe I have an excuse.

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I've read somewhere that glycerin is toxic to yeast but I think it's minimized as long as the freeze/thaw times are kept short and dilution is sought imediately after thaw?

But then the reason to use glycerin is so that the solution is kept in a "liquid" state, so I'm confused on that point if glycerin is toxic to yeast.

But then there are those that say to refrigerate the yeast/water/glycerin samples for two days prior to freezing to stimulate the production of trehalose in the yeast cells (which protects them from the cold better)? So I'm confused on the quick freeze time, etc . . . . . .

I'm just a big ball of confusion and am leaning toward sterile water storage in the fridge? . . . . Because I'm lazy. Does sterile mineral oil keep yeast from sporulating?

Wolfy,

You make an important distinction between viability and mutation. I think we've been muddling the waters by mixing these two in the discussion. Let's concede that freezing yields the longest anticipated viability, and that sterile water probably yields viability for as long as we need as homebrewers. I mean, we're not preserving strains for decades here. We're just trying to save yeast at home so we can reduce the cost of brewing.

If we, temporarily at least, leave the viability discussion let's talk about mutation. Are there any microbiologists in the house that can provide some background on the topic? How do yeast mutate and how do you think the different storage methods would affect the mutation rate? What sort of rate would we expect to see?

It's been a long time since I took microbiology, so my understanding of the topic is poor at best, but I always understood mutation to occur during reproduction. In other words, we don't observe mutation until genes are passed to successive generations. If we store a sample, either frozen or under sterile water, we are attempting to prevent reproduction and would not expect to see mutation? How far off base am I here? Can we see mutation within the same generation?

Science Daily

The article linked to above makes it seem that the mutuation takes place in the DNA of the cells when they split. It also provides a very key piece of information as to why the mutations cause bad beer: "the weak survive." So, if we take that at face value, we still have a good deal of perfect yeast. They're just mixed in with just as much bad (mutated) yeast. At least that's how I deciphered it.

<--not a microbiologist (but I did stay at a Holiday Inn last night).

Most mutations don't happen at any one particular point, although certain sorts (ie, non-homologous recombinations) only occur doing division.

That experiment was designed to create an environment free of selection so that all mutations would be visible. This allowed "weak" (whatever that is supposed to mean) yeast to live. Agar slants, etc, are definitely not absent selection pressure, although it's almost entirely irrelevant to the question at hand.

I have big doubts that mutation actually plays a serious role in yeast strain deterioration as seen by most homebrewerers. Genetic drift might play a bigger role, but in homogenous populations (as nearly any yeast strain will be) it, too, is likely pretty minor.

In cases where strain deterioration occurs (and while it undoubtedly does, I think it is talked about far more often than it is observed) it is, I am fairly certain, almost solely because of contamination.

"Mutation" is treated by homebrewers as a sort of boogeyman, in a way no one in biology would take seriously. It really probably isn't a problem.

Not sure who is still left reading this thread but let me go down the line..

COLObrewer said:

I've read somewhere that glycerin is toxic to yeast but I think it's minimized as long as the freeze/thaw times are kept short and dilution is sought imediately after thaw?

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I think the definition of toxic is required here. Concentration is always important in this discussion and the extremely wide usage of glycerin in storage at either -20 or -80 is a viable data point that contradicts the assertion of being toxic. These methods generally result in glycerin being 15-25% concentration of the final solution.

COLObrewer said:

But then the reason to use glycerin is so that the solution is kept in a "liquid" state, so I'm confused on that point if glycerin is toxic to yeast.

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Not totally true. In -20 storage it will not freeze, but at -80 it does in fact freeze. The glycerin is meant to prevent the yeast cells themselves from rupturing due to the expansion of the water molecules (remember that water expands when frozen, glycerol does not). So even though the -80 stocks are "frozen", the 25% concentration of glycerin prevents enough of the cells from being compromised that the original culture is recoverable.

COLObrewer said:

But then there are those that say to refrigerate the yeast/water/glycerin samples for two days prior to freezing to stimulate the production of trehalose in the yeast cells (which protects them from the cold better)? So I'm confused on the quick freeze time, etc . . . . . .

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You can do this before you collect the yeast into the storage tubes. Jamil/White mention to put yeast in fridge for 48 hrs prior to storing. Lab protocol is to freeze in liquid nitrogen and/or dump straight into the -80. There is no tempering of the solution. Again, if you have a sterile storage of the yeast (a WL vial or WYeast smack pack that has been smacked, grown, then chilled for 48 hrs) there is no need for resting it in the fridge after creating the storage solution.

COLObrewer said:

I'm just a big ball of confusion and am leaning toward sterile water storage in the fridge? . . . . Because I'm lazy. Does sterile mineral oil keep yeast from sporulating?

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Storing at -20 is REALLY REALLY easy. Can you get yourself all confused? Sure, but when it comes down to it, there are only a couple of basic steps and a much lower chance of sanitation being compromised compared to liquid/fridge storage techniques (never mind all the other drawbacks to storing at room temp or fridge temps)