Maintaining A Healthy Yeast Bank Long Term

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One of the most important things you can do to brew great beer is ensure you have a healthy, unstressed yeast population. From pitching rates, fermentation temperature, avoiding contamination by competitive organisms, all the way through bottle conditioning; an unhappy yeast culture will kill a batch faster than you can drop a 5 gallon glass carboy on a concrete patio.
As long as your clean, you can do prepare your yeast for storage outside of a lab.
The best way to ensure you have the happiest yeast possible is to completely control the cold-chain of your yeast supply. By that I mean yeast ranching, and doing it the right way to ensure you have a real, pitchable amount on brew day. Add in the ability to archive rare strains, as well as save you a few bucks per batch, and you’re now wondering “why haven’t I done this sooner?” You don’t have to own a certified lab to do this either (ie, my “laboratory”, an unfinished plywood tabletop in a storage room). All you need is a little patience and a penchant for cleanliness and detail.
Table 1: Overview comparing 3 different yeast banking techniques
The table outlines some of the various advantages and disadvantages of each type of storage outlined in this article. Generally, there are 2 ways to keep a yeast bank: at 4°C or at -20°C. Refrigerator temperatures are good for short term storage, but true banking will require a freezer stock to essentially halt biological activity. Since yeast cell walls will rupture due to the formation of ice crystals at freezer temperatures, you’ll need to add a cryo-protectant to reduce the crystal formation. We’ll go into that later. First, the easy way.

Storing Under Beer

Keeping yeast under beer is effective if you have a fast turnaround
Let’s say you brew a beer using a sachet or liquid pack of yeast from the homebrew store. At the end of primary fermentation, you’re left with a boat load of cells (usually around 2 trillion in a 5 gallon batch) in a cake after racking. Using this trub is the easiest way to keep a fresh stock of a favorite yeast strain. Fresh is the key word here. Yeast will continue to metabolize and be otherwise fairly biologically active in any environment above -20°C, so the term “fresh” is time dependent. A good rule of thumb is to store yeast in the fridge under beer no longer than 2 months before taking a portion of it and making a new starter to restart the clock.
Take the cake, swirl it around in the bucket or carboy in the little beer left at the bottom to re-suspend, and pour into mason jars sanitized with your favorite solution. Keep the caps loosened for a few days and store them in the refrigerator. If used within a couple of weeks, 50ml of this compacted slurry at the bottom of the jar is good enough to inoculate a new 5 gallon batch. Viability will be reduced, and sluggishness will increase with time, so take that into consideration or use one of many online calculators (MrMalty, BrewersFriend, etc) to estimate the volume to use.
This is a very quick and dirty method for maintaining a yeast bank. While being quite easy and well suited for brewing many beers back-to-back with the same strain, it will quickly become difficult to maintain many strains or keep mutations to a minimum. That being said, there are also some requirements to keep yeast cultures this way. The batch used to create the initial cake must be very low impact to the yeast. It should be low gravity, reasonably low temperature (no hot and fast ferments), must be free from bacterial and wild yeast contamination, and it must not be a blend of strains that are used in many commercially available cultures. Also, it seems as if there has been a shift in the opinion in “water washing” the yeast prior to storage in a jar. Though logic suggests that a yeast population absent of hop debris and coagulated proteins would be beneficial for storage, the general thinking these days is that more collection steps would only increase the possibility of infection. I agree with this argument, and I suggest just dumping the whole lot into a sanitized jar for collection. K.I.S.S., always a good mantra in brewing.

Agar Plates and Slants

Keeping yeast in slants is more viable, but will require you to build up a healthy starter.
A better way, though fairly more involved, of keep a yeast bank is through agar gel slants and plates. This is the method of choice for microbiologists for keeping viable strains actively growing, so why not for us homebrewers? Once you have this method down, it opens up a whole new powerful set of possibilities. Some examples include culturing wild yeast from your backyard or garden, removing bacterial contamination from a rare strain, and selecting a high growth or high alcohol tolerant strain.
There are as many ways out there to accomplish agar slanting as there are styles of brew, so I will very roughly walk through the general procedure. Google “agar slants” and a multitude of great articles will come up outlining the details. There is a general list of equipment needed for agar slanting: agar powder, pressure cooker, petri dishes or small vials, stainless wire or inoculating loop, and mason jars. Ok, so not a long list, and certainly doable without a large budget. The general steps are fairly straightforward:
  1. Prepare a 2% (2g in 100ml wort) agar solution and pressure cook for 10 minutes in a jar
  2. Allow the agar to cool to around 70°C, and pour 10ml into each 100mm petri dish, or fill the vial halfway, and keep at a slant (“agar slants”, get it?) while the agar gels.
  3. Allow the dishes and vials to cool.
  4. Take the sterilized stainless wire or inoculating loop and dip a sample into the smackpack or yeast vial and spread on the surface of the agar gel
  5. Cover and allow the yeast to grow into a nice thick layer on the gel at room temperature
  6. Seal the plates or screw the lid of the vials and keep at 4°C
Since the culture has not been through a true growth phase in the stress of making a normal beer, you can keep reculturing these plates without too much worry about mutations, as opposed to keeping jars of slurry noted above. The plates can be kept in the fridge for 6 months to a year before having to replate, and replating is as easy as taking the inoculating loop and smearing a sample from the old plate into a new one and repeating the growth/storage process. To grow a starter from the plate, take the sterilized loop and scoop a sample from the slant, twist it around in 2-5 ml of wort. Grow the wort for a couple of days, and incrementally build that volume into your target starter volume, not exceeding a 1:10 dilution (for example, take the 5ml starter, dump it into a 50ml starter and grow, and dump that into a 500ml starter, etc).
Without describing every single detail along the way, use common sense in preparing the cultures. If you're using glass petri dishes and vials, sterilize them in the pressure cooker. If using plastic, make sure you buy pre-sterilized packs, keep them sealed, and don’t reuse them. Beyond just keeping a master stock of yeast, this method can be used to clean up a contaminated culture by smearing a sample of the batch, and picking and growing individual colonies of yeast from a single cell. The yeast are white, foamy looking, while bacteria are usually shiny. Mold is obvious, and the whole plate should be thrown out if seen, as the hyphae underneath the gel will no doubt pop up somewhere else.

Frozen Stocks

I am a microbiologist by education and trade, and in my opinion, keeping frozen stocks of yeast cultures is the way to go for a homebrewer (and also quite simple to do). While you don’t get that warm, fuzzy feeling of watching a single cell grow into a colony and knowing exactly how pure your culture is, with careful aseptic technique you can keep dozens of strains ready to go for pitching in a couple of days, and it doesn’t take a lot of maintenance or equipment (or valuable refrigerated space). The addition of a cryopreservative (food grade glycerin) is required as mentioned above to keep ice crystals from rupturing the cells, but everything else is pretty much the same as keeping slurry jars. An added benefit of storing cells at such a low temperature is that biological activity slows way down, essentially halting the chance of spontaneous mutations that may occur from fridge temperatures.
You can store a lot of yeast for a lot of time if you're able to freeze your cultures effectively.
My method for keeping frozen stocks of yeast is focused on creating single-pitch tubes to make fresh starters for 5 gallon batches. You can also make much smaller tubes to steal a small quantity without thawing the stock and step the culture up for a starter. That takes more time, and time is valuable, so I don’t do it, but it is perfectly viable. The equipment needed for frozen banking is simple: pressure cooker, mason jars, sterile tubes, and glycerin (drug store).
  1. Make a 25% v/v stock solution of glycerin in water (30ml glycerin, 70ml water) and pressure cook for 10 minutes.
  2. Make a 1000ml starter from a fresh yeast culture and allow to settle or cold crash in the fridge
  3. Decant the beer off the top and swirl the yeast up
  4. Divide the yeast into 10 vials
  5. Double the volume of each vial using the cooled 25% glycerin solution (for example, after decanting you are left with 50ml of yeast slurry. Add 5 ml slurry to each vial and add 5ml 25% glycerin to each vial. This will give you a final concentration of 12.5% glycerin).
  6. Mix the tubes well and place in a container filled with isopropyl alcohol to the liquid level in the tubes to reduce the freezing rate in the freezer (this can be skipped, but cell viability will be reduced if placed directly into the freezer. Cells prefer to freeze at a rate of 1°C per minute).
  7. After 24 hours the frozen tubes can be removed from the alcohol and stored for several years.
  8. To inoculate a 1000ml starter for a brew, quickly unfreeze the tube to the starter wort temperature and dump it in. That will create a 1:10 dilution from the earlier split. Using a stir plate will have the starter ready and settled usually within 48 hours.
  9. Once you are down to one tube in the freezer, make a starter as in Step 8 using the tube, and freeze into more tubes as in Step 3. So you theoretically from one smack pack and some DME have made enough cells for almost 30 batches in just 3 controlled generations of culture (or over 700 batches if you expanded each tube out 3 generations).
If you increase the final concentration of glycerin in the frozen culture to around 50%, viability will be reduced, but the cells will not freeze solid, and you can dip a sterilized loop in there to inoculate a small starter, very much like inoculating from a slant. This way you can cut down the storage volume of your stocks to a few 1.5ml tubes instead of larger vials. But keep in mind it takes a week or more to grow a starter from that instead of a couple of days.
Regarding the storage time, it is also assumed that a chest freezer or manual defrost freezer is used. If a fridge/freezer is used that has an automatic defrost cycle, the tubes should be placed in a styrofoam box with ice packs inside to maintain temperature while the defrost cycle works.
Keep Records
Whichever method you choose to maintain your yeast bank, keeping detailed records is mandatory. Typical details are yeast ID, strain, source, date commercially manufactured, storage date, number of generations from packaging, parameters of beer obtained from (if slurry), date to re-plate or refreeze, among others. These records will be the living document that tells you how viable the stocks may be, when routine culturing is necessary, and depending on how detailed you get, how changes in your protocol can increase cell viability. These are all good things, and just as necessary as keeping a brewing log book to note how a beer turned out. If you’re a yeast freak like myself and eventually build up a stock of over 40 different strains, you’ll definitely need a spreadsheet with tasting notes, fermentation parameters, and ideal beer styles in order to plan your next brew.

A Final Note

Yeast are incredibly resilient little buggers. Just a few years ago a professor at California Polytechnic State brewed beer with a yeast strain found to be 45 million years old that was stuck in a chunk of amber, inside the digestive tract of a bee. That’s one tough cookie of a cell that’s been through a lot over the millennia. In contrast, the methods given above are aimed at reducing exactly this kind of stress, but are by no means set in stone or an all-inclusive list. Variations in almost every step will likely give great results. Keep in mind the few critical rules: reduce contamination, reduce stress and mutations, and control the growth cycle. No special tools are required to keep a healthy yeast bank long-term; use some common sense, the guidelines above, and you’ll have a yeast population ready for any brew you can throw at them.
I have been using the "harvesting from the starter" method for the past year or so, thanks brulosophy. I really only use Conan, since I rarely brew anything other than pale ale or IPAs, and I love the character it brings to the brews. I use 4 oz jelly jars, so it's a perfect size. It doesn't use up much of my starter, and when I'm ready to brew again, I just pull out a jar and make a new starter with it. Then I repeat the cycle.
Very nice. Looks like I might start a frozen yeast bank, this write up really demystified it.
But the important question I have from this article: Where do we get a vial/smack pack of that amber bee yeast?
Any size will do, but get one that you can put a few mason jars in. And when I say pressure cook it, I mean in glass jars that you can then seal shut once the cook is done. Sorry if I made it seem like the cooker was full of solution, that's not what you want to do. I use a cheap aluminum one for the stove, my wife has the electric one :)
Yeah, I don't think this guy is down with spreading the love. From an article on him and Fossil Fuels Brewing Co, "His only worry is that the unfiltered nature of this beer means that some of his yeast will invariably settle to the bottom of the glass or bottle, and an unscrupulous brewer could collect that and use it in another beer. The microbiologist has applied for a patent on his strains and has sequenced the genomes so he can tell if someone else has stolen it. "I am the keeper of the family jewels," Cano says."
Yikes. Here is the patent: http://www.google.com/patents/WO2010042896A1?cl=en
It's all about standardizing your own protocol, and it looks like it works great for you! I've got some Conan in the freezer that I need to bring out for a brew, haven't had a chance to try it. I need to see what everyone is raving about...
Do these methods work for Lactobacillius? Also, I'm making a cider with Nottingham. How would that hold up to these methods. Washing Nottingham from a cider then storing it? I know Nottingham is inexpensive, it's more about availability for me. Banking yeast means I don't have to wait, or more importantly pay for overseas shipping rates.
Yep, bacteria will do fine with all of these procedures. Nottingham will work fine as well. If you're banking to save some money on shipping, I would make a master batch right from the package and freeze. After a run through a stressful batch (most ciders/high gravity due to lacking nutrients), the yeast may not be as pure or viable. If you're already got the cider going, take a sample after racking and make a starter, tasting and smelling, making sure they're still healthy.
Thank you for this, I found some sterile tubes but wondering is there a certain type of glycerin needed for this? or any will do?
So you make 100 mL of the solution glycerin to water, sterilize it by Pressure cooker, then split into the tubes? 30% gly to 70% water ratio? then add your yeast after its all cooled down?
Any USP glycerin from the drug store will work. I would make a stock of the glycerin/water solution in a mason jar so you always have it on hand, or just put it into the tubes after it's cooled a bit, either works. You can do 30% glycerin or 25% glycerin and water (typo in the protocol), they both will work. Which ever way you do it, you'll want a final glycerin concentration of 12.5-15% in the frozen slurry, which is why you add 5ml each of the glycerin stock and slurry to the tube before freezing.
Nice article! Working with cells I always was curious how it translates to homebrewing. I usually use DMSO as a cyro-preservative in lab instead of glycerol. My question is though that we usually spin down the cells and decant/aspirate the supernatant to remove the cryo-preservative prior to seeding. Obviously centrifuges are not a common household item and would be costly for the average homebrewer. Your instructions just have pitching the thawed tube straight into the starter, my question is if there is any worry in the glycerol affecting the flavor or anything of the beer? I guess for a 5 gal batch you will be at a 1:3800 dilution but just curious
Even though DMSO would work as well, definitely not the right application here, and in case anyone else is reading this, DON'T USE DMSO!
But as far as glycerin, I'm not too concerned. If you take that 5ml of 25% glycerin (1.25ml pure glycerin), and dump it all in the 1000ml starter, you would have a 1:800 dilution. If you then dump that whole starter in the 5 gal beer (19000ml), it would be a 1:15,000 dilution. And if you first decanted the starter for pitching and retained 50ml of slurry, it would be around 1:300,000 dilution. At this dilution, I wouldn't have a problem drinking a FDA appointed GRAS component, and I doubt anyone would detect any flavor addition to the beer.
OK cool I will definitely try this. One more question, what kind of isopropanol box are you using for 15 mL tubes? I only know of the Mr Frosty boxes for the 1-2 mL cryovials
I just use a mason jar and fill the alcohol to the level of the liquid in the tubes. The number of tubes and volume of alcohol don't seem to affect the viability noticeably. Some day I'll actually track the temperature drop in the tube to see how far off I am from the 1degC/min.
Ok, I looked for some tubes that were sterile, I saw the ones you posted up above... but 300 seems like a lot.. I did find some but they were 50ml I guess thats probably too big?
So you make the 70/30 solution in a mason jar, in pressure cooker, and you seal it and use it as needed? or everytime you make a new set of yeast banks?
Here are some more, smaller pack: http://amzn.com/B00ES3TIRG
I make a 300ml stock solution of 25-30% glycerin in a pint size jar, pressure cook and just use it as needed until empty. It's enough for 6 batches (60 tubes).
I've never had a problem with very old pitches of estery strains as long as I build up an appropriate starter. I just did a WLP-550 from over a year ago and it's tasting great so far (in the fermenter anyway). I might be more inclined to fret over an American style strain that old, where stress flavors are something to fret over.
I have been reusing yeast for a couple of years. I use a simple bleach solution on jars, counter, general area I'm working in (galley kitchen). I have had no problem. Beer is great. I have also reused yeast many times. I will let the yeast stay in the primary for a few days under remaining beer, then, wash the yeast before storing in fridge. I also save bleach solution for reuse.
Top cropping is a great way to collect a selection of highly active yeast. Collecting from the bottom after fermentation essentially gets you the same result, but cropping ensures an active population.
could you combine methods 1 and 3 by adding glycerin to pitch-able quantity of slurry then you could skip making a starter? would the freeze/thaw be too slow? too much glycerin going in the beer?
is there a way to test yeast slurry for bacteria levels. Can you grow a sample of slurry to see what else aside from yeast is in it? or can you see bacteria through a microscope.
Awesome write up again!
Thanks for the earlier response, and detailed follow up regarding cell counts.
I typically brew ten gallon batches of various OG. Was curious about the aforementioned density you a finding in your starters, if it would benefit me to do a larger sized starter, 2L instead of the suggested 1L in this case for ten gallons of beer.
Also, regarding sterilizing the tubes you are using. Can these tolerate being sterilized in a pressure cooker? An Autoclave (I have access to one at work)?
Do they come with those cardboard holders when you purchase the tubes?
Thanks for this great article. I have received my test tubes, rack and pressure cooker to go the frozen route, however I have a question. When you sterilize your water/glycerin solution in the pressure cooker, is the solution in a mason jar with the lid sealed tight, as if you were canning preserves or jam? Can you enlighten me on this aspect?
Pete thank you for your post, so far I've been using
slants for my small bank and wanted to try frozen stock
with a White Labs (WLP002) which is very flocculant.
In steps 2. and 3. the decanted yeast was very clumped
and it was impossible to divide the decanted yeast into
the tubes 5 ml each.
What can I do to 'unflocculate' this decanted yeast into
a more liquid mixture?
I just did some tubes this afternoon with Wyeast 1098 and it's very flocculant also. I left the stir bar in and, after decanting, I put it on the stir plate while I was getting everything else set up. Seemed to work pretty well, but was still a little clumpy.
Don't seal it tight. You want there to be enough wiggle in the lid to let pressure escape or else...boom. I bought some autoclavable media bottles with screw on caps a while back and love them. I use them for making wort agar media and more recently for glycerin solution. If you're using a Mason jar, I would probably just put some foil over the top when sterilizing in the pressure cooker. If you're making a big batch and aren't going to use it all, I would Starsan a lid and ring and then put that on after you're done using it.
For danthebugman. Link for those media bottles please?
For OP, poppincaps. When you refer to a "fresh yeast culture" which we begin with, I am wondering how I might translate that into using wyeast/white labs liquid yeast or dry yeast sachets when brewing.
For liquid yeast, I would typically make a 2 L starter with two smack packs or two vials (sometimes three or more vials, step up starters, or larger volume starters depending on what I'm brewing). What might we do when making a starter to collect a bit of the culture for a subsequent 1 L starter in order to generate ten vials for storage?
With dry yeast I do not make a starter. I do rehydrate in 10x the weight (or mass actually) in sterile water of the yeast. Seems to be the best spot for harvesting the yeast for storage, or stealing a portion for making a starter to collect enough yeast for ten vials for storage. What do you recommend in this situation?
I assume that in both instances, that it is better to use the fresh from commercial producer cultures to follow your method, rather then collect yeast from a finished batch of beer and attempt the same.
Did anyone else get way more yeast than expected? Ended up making a 1200ml starter instead of 1000, yeast was on stir plate for 48 hrs. I got 30 tubes of yeast & probably dumped another 100 ml of yeast down the drain. I crashed & dumped off the stale beer twice before filling the tubes. Could of stored some yeast in jar for another brew.
Can these tubes be cleaned, sanitized & reused?
Great article. I got my frozen bank underway a few weeks ago with four strains in the freezer as of now, with two more strains cold crashing.
Question - is there any reason why I shouldn't use my glycerin solution at a later date, after it's been pressure cooked, opened and used once? As long as the jar is closed back up when done, it should be fine, correct?