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Freezing Yeast

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Freezing Starter-Sized Samples of Yeast for Long-Term Storage
Written by Brewitt with contributions from HBT members


WHAT: The purpose of this article is to summarize what has been learned about freezing yeast by a number of contributors to the thread "Do you know how to make a yeast starter? Then why not farm yeast and freeze it?" (https://www.homebrewtalk.com/f163/do-you-know-how-make-yeast-starter-then-why-not-farm-yeast-freeze-269488/) started by, and with extensive contributions from, BBL_Brewer. Other major contributors include Brewitt (me), Forkhead, katy bug, ScoRas, and IsItBeerYet. Many others have made contributions that can be read in their original form in the thread. Together, through reading and experimentation, we have come up with an effective and relatively simple method for long term storage of pitchable quantities of yeast in either standard frost-free freezers or, better yet, non-frost-free freezers often used for food storage. Keep in mind that some of the recommended procedures have not been fully tested and will undoubtedly be undergoing some modifications but the method outlined below works well. Some modifications and additional considerations are listed at the end of the article.

Figure 1: Tubes of frozen yeast. These were prepared from a starter of yeast from a bottle of Sculpin IPA (Ballast Point Brewery) and the yeast from a fermentation started with a bottle of Pranqster Belgian Golden Ale (North Coast Brewery). These have been stored in a non-frost-free freezer for 7 and 9 months, respectively.
WHY: The method(s) described below is sufficient for storing cells in portions large enough to pitch directly (50-200 billion cells, similar to the amount in a smack pack from Wyeast or a tube from White Labs) (See Figure 1). Nevertheless, we recommend an overnight starter to ensure a large number of viable cells and rapid initiation of fermentation. The cells to be frozen can be from a large starter culture made from a tube or pack of commercial yeast. A single large starter can be broken up and saved for multiple future brewing days. A similar approach can be taken with a small starter made from yeast recovered from commercial bottle-conditioned beer or home brews that you would like to save for later. Finally, the yeast can be washed cells from a recently completed fermentation (a single 5 gallon fermentation can provide starter samples for as many as 10 future brews). Keep in mind that you will save $7-$10 per 5 gallon batch of beer each time you pull a tube out of the freezer.
GOALS: The goals of the method that we have devised are three-fold:
  • To allow the preservation of large samples of cells in freezers in common use by homebrewers
  • To use materials available to homebrewers at a reasonable cost
  • To achieve high viability of stored cells for weeks, months, or perhaps years

Figure 2: Examples of tubes useful for freezing yeast.Sanitized used White Labs tubes and 50 ml screw cap conical tubes (see Resources).
SUPPLIES: Materials required for preparation of frozen cells:
  • A large quantity of yeast culture that you want to store for future use. Any "clean" yeast culture can be used (for example: one or more quarts/liters of aerated or stirred starter culture; washed cells from a 5 gallon fermentation (see "Sticky: Yeast Washing Illustrated") (https://www.homebrewtalk.com/f163/yeast-washing-illustrated-41768/)).
  • Sanitized tubes for freezing samples of yeast. This could be plastic White Labs tubes saved from prior purchases (these are apparently also called "baby soda bottles"); 50 ml plastic screw top tubes commonly used in laboratories (see Resource section at the end of this article), any other small freezable container that can be sanitized and tightly sealed. See Figure 2.
  • Some form of tube holder.
  • Glycerin, food grade (must be diluted in sanitized water before use; a rate of 1 part glycerin to 4 parts water gives a 20% final concentration). Glycerin, glycerine and glycerol are all the same thing. See Resource section at the end of this article. See Figure 3.
  • Isopropyl alcohol (also called "rubbing alcohol", available at any drug store), freezable gel packs, other good insulator for tubes of yeast.
  • Container large enough to hold multiple tubes of yeast that will hold alcohol or gel packs. An insulated container is best (small Styrofoam ice chest or discarded shipping box for frozen goods).
  • Sanitized water

Figure 3: Glycerine for freezing yeast.One example of glycerine suitable for freezing brewing yeast (see Resources).
HOW: A single method is outlined. Alternatives are discussed in below.
Freezing:

1. Collect yeast by chilling the culture or washed cells in a refrigerator without disturbing for 4 hours or more (preferably overnight). Pour off spent wort or washing water leaving just enough to allow cells to be suspended as a very dense slurry. The goal is to get sufficient cells for an overnight starter culture in a volume that is convenient to freeze. A good sample size is about 50-200 billion yeast cells (0.5-2 x 1011 cells). One quart of starter culture can usually be reduced to 1/40th the volume or less giving you about 100 billion cells in 25 milliliters (about 1 1/2 Tablespoons).
2. Split up the slurry of settled yeast into samples of about 100 billion cells (cells from about 1 quart of starter or a half gallon of fermented beer) in sanitized freezable screw top containers. That amount of slurry should fill the container less than half way.
3. Add an equal volume of chilled 20% glycerol to each of the samples of yeast and cap tube tightly. Swirl or stir until fully mixed without frothing. This will lead to a final concentration of glycerin of 10%. Concentrations of glycerin higher than 15% appear to be detrimental to cells when stored at home freezer temperature (20C, -3F). Make sure there is sufficient room for expansion during freezing.
4. Loosen the caps of the tubes and place into the large container, standing up to avoid spilling, and add enough isopropyl alcohol to the large container submerge the tubes to the level of the glycerin solution but not to the level of the cap. You don't want isopropyl alcohol mixing with your yeast. If using gel packs, make sure tubes are completely surrounded by the gel.
5. Place the container in the freezer. The isopropyl alcohol or the gel packs will slow the freezing of the cells, which is an advantage for maintenance of viability, and will avoid thawing during frost-free cycles.
6. After freezing is complete, tighten the caps of the tubes. If you are in a non-frost-free freezer, you can remove them from the alcohol and save it for a future use.
Thawing:

1. Remove tube of yeast from the large freezing/storage container.
2. Immediately submerge tube in water at approximately body temperature (37C, 98F). This can be running warm water or a large volume of water for submersion of the tube. Swirl continuously until completely thawed.
3. Remove tube of cells from the water, wipe dry with sanitizer and pitch into 1-4 liters of wort to prepare an overnight starter. In general that inoculation of the starter will grow to maximum density within 12-24 hours with appropriate shaking or aeration.
Other useful considerations and information:
Keep in mind that the cleanliness of your starter culture, washed yeast, and freezing supplies is key. Your subsequent beers will only be as good as your starting yeast strain and any contamination in your starter culture will be perpetuated in brews made with the frozen yeast prepared from it. If you have a beer with flaws due to contamination, don't freeze the yeast for later use. Also, yeast blends and blends of yeast and bugs will not necessarily be present at the same ratios after growing starters or using them for a fermentation. They can be frozen but results may vary.
Isopropyl alcohol for freezing and for storage can be cumbersome. The goal is to freeze slowly and avoid thawing during frost-free cycling in home freezers. Another approach to slow freezing is to chill in a refrigerator overnight and then to place in an insulated container like a Styrofoam ice chest or box and place in the freezer. It is useful to use gel packs or some similar frozen insulator when storing in a frost-free refrigerator.
Glycerin is a cryoprotectant. It helps maintain the viability of frozen cells. We have tested final concentrations from 7.5% to 50% and found 7.5%-15% to be optimal for maintaining viability (upwards of 75% viability after several months of freezing). Lower final concentrations may also work but higher concentrations are detrimental.
Glycerin can be added to the yeast slurry before or after splitting into smaller portions. If you want a more concentrated slurry, you can pour off more spent wort and/or resuspend your slurry in more concentrated glycerin to achieve a final concentration of 7.5%-15%.
If you have yeast that have been settled at room temperature, you can add glycerin solution that is at room temperature and then chill.
Although yeast can be used directly, this is likely to lead to under-pitching and a slow start to fermentation.
Resources: (The following are meant as examples, not necessarily recommended vendors or products)
Tubes for freezing yeast: Some examples from Amazon
http://www.amazon.com/Centrifuge-Tubes-Sterile-50ml-disposable/dp/B0018MQ9TK/ref=sr_1_13?ie=UTF8&qid=1351877946&sr=8-13&keywords=plastic+tubes+50+ml
http://www.amazon.com/Karter-Scientific-Centrifuge-30x115mm-Conical/dp/B005Z4S3ZU/ref=sr_1_1?s=industrial&ie=UTF8&qid=1351880358&sr=1-1&keywords=centrifuge+tubes+50+ml
http://www.amazon.com/150MM-5-75-POLYCARBONATE-TUBES-SCREW/dp/B005CBFATS/ref=sr_1_1?ie=UTF8&qid=1352237539&sr=8-1&keywords=baby+soda+bottles
Glycerin:
http://www.dudadiesel.com/search.php?query=glycerin
http://www.starwest-botanicals.com/category/vegetable-glycerin/
http://www.bulkapothecary.com/glycerin.htm
Gel Packs:
http://www.amazon.com/Hot-Cold-Pack-Microwave-Freeze/dp/B001EOJ0MU/ref=sr_1_cc_1?s=aps&ie=UTF8&qid=1351880412&sr=1-1-catcorr&keywords=freezable+gel+packs
http://www.amazon.com/Vesture-Pack-Replacement-Microcore-Casserole-Carriers/dp/B001B16Z0G/ref=sr_1_1?s=home-garden&ie=UTF8&qid=1351880619&sr=1-1&keywords=hot+cold+gel+pack
 
Excellent article! Keeping your own yeast bank is yet another way of keeping good home-brewed beer cheaper than a case of Keystone Lite. Lol
 
Maybe I am missing something very simple here, but what is the actual quantity of glycerine being added to the vials? Maybe I'm confused by the fact that we are already watering the glycerine down to a 20% concentration (also, based on the 1/4 ratio, aren't we really starting out with a 25% concentration?), so to achieve that optimal 15% concentration overall, how do we figure that? Thanks.
 
@keesimps
It's all in the math :D
I have 50ml vials.
I create a 50% glycerin solution because it's easy to mix.
I want 15% glycerin concentration in my vials.
15% of 50ml is 7.5 ml of GLYCERIN.
Double that to 15ml because my stock solution is only 50% glycerin.
(50ml * 0.15 / 0.50 = 15 ml of 50% glycerin solution to 35 ml yeast slurry)
I can then fill the vial up with any amount of anything (except glycerin) and maintain a 15% glycerin concentration in my vial. Regardless of how thick/thin my yeast slurry is, as long as I fill up to 50ml the 15% glycerin concentration is maintained. If I'm a little short in one vial then I top up with sterile water to the 50ml mark - no problem.
The hard/questionable part is estimating how many billion cells you have put into each vial AND it's viability once thaw.
On a side note: I'm also showing sluggish yeast after freezing. I'm seeing an easy 24-48 lag from pitching in starter to it actually starting. I suspect that freezing will affect different strains differently. I had a US05 come back after about 24 hours (frozen 2 months), but my 3787 trappish high gravity has taken 2-3 days just to awaken and it was only frozen for about a week.
Cheers!
 
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stpug: Your calculation summary is right on. On the other issue, I have belgian and american ale yeast as well as several lager yeasts frozen. I have used most of them after many months in the freezer but I am freezing at -80C in a non-frost free refrigerator. I always grow a starter. I see increase in cells in my starter after 4-8 hours. When I grow an overnight starter I have airlock activity within a couple hours of pitching. I don't have any experience with the frost free freezer method but would be interested in hearing what others have seen.
 
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stpug: Your calculation summary is right on. On the other issue, I have belgian and american ale yeast as well as several lager yeasts frozen. I have used most of them after many months in the freezer but I am freezing at -80C in a non-frost free refrigerator. I always grow a starter. I see increase in cells in my starter after 4-8 hours. When I grow an overnight starter I have airlock activity within a couple hours of pitching. I don't have any experience with the frost free freezer method but would be interested in hearing what others have seen.
 
Hey, getting back into brewing and I'll definitely be trying this. One dumb-guy question here, perhaps. Is there any reason why I wouldn't use cheap vodka instead of isopropyl alcohol to slow down freezing? Seems like it would do the same thing that you suggest, wouldn't it? Also, it's cheap, easy to locate and replace, food-safe in case of an accident, and comes in a convenient container with a handle.
 
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@beernardo I see no reason why Vodka wouldn't be perfectly fine. It won't freeze at typical home freezer temperatures and you can drink it when your done ;) Here's an idea. Partially fill a plastic bag and then just push your tubes or bottles into a container with the plastic bag so it conforms to the tubes keeping them cold and your vodka pure.
 
Hi guys.
This is some interesting work so thanks for sharing. I do have one question though. You say you have to wait extra time for the yeast to become active. Has anyone tried a control test where there control wort is exposed to the same environment as the wort with freshly thawed pitched yeast (no yeast added to the control)? I see fermentation start in wort after 2 to 3 days that I have pitched no yeast in, ie it has come from the surrounding air.
 
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tonyfelloni, If your wort undergoes fermentation after 2-3 days you have contamination. That is not entirely unexpected but suggests you aren't being as clean as you should be. The contaminants are probably kept at bay by the ongoing yeast fermentation if it gets a good start before they do. When I say the lag is longer before fermentation starts, I am talking about hours not days. Usually it is well underway within 12 hours but sometimes it takes somewhat more. Never 2 days. When I do a big fresh starter using that yeast it gets my airlock going within 2-4 hours. I don't think the side by side is necessary.
 
Great thread!
I used to have 10 or 12 test tubes in the freezer at any given time, used agar (kinda like jello?) and inoculated it with yeast cells. Just a dab in each tube but enough to make a starter. Stretched one pack of liquid yeast to many batches.
Already read the yeast washing thread mentioned above, would make for a higher concentration to work with.
A discussion on yeast starters for bread got me here, thinking of a frozen "backup" in case the starter got compromised. I'll explore the site, was thinking about brewing again. My last batch was a mead, fermented for 10 months and I couldn't wait, bottled it up.
Could have gone longer but was tasty.
 
Reviving an older thread here but this looks very interesting. A few questions.
1) If I were to purchase new vials of yeast and separate into multiples, these would all be considered 1st gen, correct?
2) Could you use the RV antifreeze as a medium to slow the freezing process of the yeast.
 
One could also probably use breast milk tubes. They have a flat wider base and will stand on their own. They are also intended to be frozen. Like these: http://www.amazon.com/gp/aw/d/B0009XBX4K/ref=mp_s_a_1_1?qid=1422290329&sr=8-1&pi=AC_SY200_QL40&dpPl=1&dpID=313lrM9glnL&ref=plSrch
 
Thanks BrewiTT for a very detailed and educational article. It, by far, has been the best one that I have ran across - and thanks to the folks who have posted comments as well. ALL great stuff, that is for sure!!
I am going to take a stab at freezing yeast, I've washed some leftover yeast from a couple 5 gallon carboy beer fermentations (primary fermenter) with good success thanks to Bernie's article which y'all can find here: https://www.homebrewtalk.com/f163/yeast-washing-illustrated-41768/
I was wondering if I could get away from using rubbing alcohol or gel packs as insulators by using an insulated lunch bag. So my idea is to mix the yeast and the glycerine into 50ml centrifuge tubes as indicated in Brewitt's "Freezing Yeast" article, place them in a test tube rack, and then cool the vials in the fridge overnight.
The next day, take the test tubes and the rack that holds them and place them directly into the insulated lunch bag, zip it up, and place it into the freezer. If this works the lunch bag will hold 2 test tube racks, with each test tube rack holding 24 - 50ml tubes, which would be pretty nice!
Do you think the insulated lunch bag would provide adequate protection against the defrost cycle on my "home grade frost-free" freezer? I also have a solution for handling storing of the yeast in alcohol, but if you think the freezer bag would insulate well enough - that would be a better solution as it simplifies the storage process. What do you think? Would it be recommended / suggested to place ice packs in the lunch bag? I guess it wouldn't hurt and it would help keep the yeast cold for a longer period of time in the event of a power outage.
Oh this may be handy for some folks who are looking for supplies. It is a link to a list of items that I am planning on using to freeze yeast:
http://www.amazon.com/gp/registry/wishlist/110QWN1UI24JN/ref=cm_wl_rlist_go_o?
The plastic boxes are the closest thing I could find to accommodate the test tube rack dimensions. Which if the lunch bag idea works, I won't need the plastic boxes.
Thoughts?
Thanks !!
 
I have missed something obviously.
What on earth is the isopropyl alcohol used for?
Its not low cost & its toxic.
I'd appreciate a bit of help here.
Thanks
 
Maybe I missed this. But it's the solution (10% glycol, 40% Distilled water, 50% yeast slurry) going to freeze solid in the freezer?
 
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  • #58
Use "glycerol" or "glycerine", not "glycol". Yes, the slurry is going to freeze solid. The glycerine reduces damage to the cells on freezing.
 
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Isopropyl alcohol is rubbing alcohol. You can buy it at the drug store or Target or many other places. It is not being used in the yeast slurry, it is being used as a non-freezing solution to keep the temperature more constant during the freeze-thaw cycles of a self-defrosting freezer. The bath is just made up once and reused. I prefer the gel from freezer gel packs but the alcohol will work.
 
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For some reason I have not been getting notifications of comments here until getting one today. I will try to go back and answer some of the questions when time allows.
 
Hi,
I have been reading and watching various methods for home freezing yeast. This looks great. I have a question.
The method and thread talks about sanitizing everything. Some times the word sterilize is used in the thread, but I get the idea that you are only sanitizing (e.g. you can't use Starsan to sterilize plastic components.
Other sites and videos have stressed that equipment should be sterilized (using a pressure cooker or autoclave if you are so lucky). Have you had any issues with infection with sanitizing versus sterilizing?
This method seems a whole lot easier than others I have seen.
Thanks!
 
How did you come to the conclusion that 25ml of yeast slurry will have 40-50 Billion yeast cells? Shouldn't the density of cells in the slurry depend on how many cells were create in the starter? I usually build a 1.5L starter and with a fresh pack of 100 billion yeast cells, and with a SG of 1.036, the brewersfriend starter-calculator
(https://www.brewersfriend.com/yeast-pitch-rate-and-starter-calculator/) says that you can get up to 316 billion cells. I typically pour one liter of the starter with yeast in suspension into a 1L jar and crash for a few days prior to brew-day, and the other half-liter into a half-liter jar for future starters. If I did get the full potential of my starter, that .5L jar should contain 105 Billion yeast cells. I have measured the yeast cake in my .5L jar and it is exactly 25ml, so to me it seems odd that you say its half to sixty percent fewer cells than I reckoned. Is it that the missing cells are still in suspension at time of decanting? It seems unlikely that 50-60 billion yeast cells would refuse to settle out after a few days cold-crash.
 
First of all, thank you very much for your time with this write up. I have only just recently found it. Quick question for anyone that has successfully used this method. What kind of viability have you found after thawing these yeast vials? In general, for example, if you were to purchase a pack of say White Labs liquid yeast form your local home-brew shop that was produced even just 1 month before you bought it, that 100 Billion cells is already down to approx 75 billion.
I'm sure that, even under the best of conditions, the little yeast cell walls are going through some tremendous stress in the freezing and thawing processes. I wouldn't be at all surprised by less than 50% viability after thawing, but who knows? Anyone's input on this is much appreciated.
 
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