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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
 
Awesome article! Thanks for posting! This looks like something I need to try.
Do you need to decant off the glycerin off the top of the vial or the starter before pitching in to the fermentor?
 
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  • #3
Thanks. You should use food or pharmaceutical grade glycerine. There is no need to pour off the glycerine since there is very little present and yeast can metabolize glycerine via the same pathways that it uses for glucose.
 
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  • #4
Two updates to the article (both suggested by Forkhead):
First, the cell number for 25 mls of yeast may be on the order of 25 billion rather that 50-100 billion. I will confirm this soon but for now it seems reasonable that my numbers are off. That should be without consequence if an overnight starter is prepared but my be low for pitching directly.
Next, Forkhead suggests that I put a notice in the article "DO NOT USE STYROFOAM BOXES THAT DIRECTLY CONTACT ISOPROPYL ALCOHOL".
I will be updating the article soon but since it requires effort on the part of the moderator to do so I will refrain until I have all the data.
 
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  • #5
I have now confirmed that a thick slurry of WLP090 settled from a starter contains about 40-50 billion cells/25 mls. So, 100 billion cells in a vial is achievable if you are careful to let your cells settle well and then pour off most of the wort leaving just enough to resuspend and pour. 40 mls of that slurry should contain around 100 billion cells. Adjusting that to 7.5%-15% using concentrated glycerine will leave enough space for freezing.
 
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  • #6
Forkhead has determined that the optimal glycerine concentration is 15% (not 7.5%) for freezing at -20C. This is the same as both of us had observed for -80C. I have recommended a range from 7.5% - 15%. I will revise sometime soon.
 
I live in Tucson, AZ and my chest freezer is outdoors. In the summer temperature swings can be upwards of -20 to 0 degrees, is this an issue if proper insulation is used?
 
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  • #8
@Schnitzengiggle I would think that, if you are giving me temperatures in degrees F, you should be fine. The temp is below freezing at all times and you will have insulation to buffer against the change.
 
Can you just use a salt water solution instead of alcohol? There'd be a lot of thermal mass; and the ability to remove the tubes from the solution (since they won't be frozen in a block!)
 
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  • #11
I presume if the salt is high enough to not freeze it should work fine. I used to use the gel from non-freezing gel packs for a similar application. The nice thing about that is that it forms a semi solid mass with holes in it for the tubes. The gel packs are expensive though. There are lots of solutions to this problem. The key is something that has sufficient thermal mass to stay below freezing and is not too nasty to work with.
 
does the yeast really metabolizes the glicerin in the solution? or what effect would glicering have on the wort or finished beer?
 
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  • #13
The yeast takes up some of the glycerine and it acts to prevent the formation of ice crystals that are destructive to the yeast. The amount of glycerine in the yeast is tiny compared to the volume of the wort, especially if you make a starter and cold crash it. It would add very little in any case. Besides glycerine is essentially tasteless.
 
Was thinking about giving it a try and I found these on Ebay. Do they look good? http://www.ebay.com/itm/50-mL-Elkay-Printed-Pro-Centrifuge-Tubes-Bag-of-50-/370481849006?pt=LH_DefaultDomain_0&hash=item56427062ae#ht_641wt_893
 
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Funktavious, those appear to be essentially the same tubes in my picture. They should work fine. That is a great price too.
 
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@funktavious - The rack came with the tubes from a scientific supply company. Not the most economical way to buy them. Perhaps some of the suppliers online have them in racks but they will undoubtedly be expensive.
 
Is this the same Glycerin you were using? Or is this the correct kind?
http://www.amazon.com/Starwest-Botanicals-Vegetable-Glycerine-16-Ounce/dp/B000VUZXF6/ref=sr_1_13?ie=UTF8&qid=1369925574&sr=8-13&keywords=glycerin
 
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  • #19
Coff,
That is an appropriate glycerin for the job. I think you can find other vegetable glycerins for cheaper in larger quantities but that one will work.
 
First of all, great post, this got me really interested in freezing yeast and it's very easy to follow. After reading this, I also read a lot of the original yeast freezing thread and I have a couple questions as I didn't read the entire thing. I take it you still want to sterilize the Glycerin in the pressure cooker? Also, many people were talking about sterilizing the preforms in the pressure cooker, but theirs were glass. Do you just use star san to sanitize in the plastic preforms or am I missing something?
 
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  • #22
@anico4704 Yes, glycerine should be sterilized. It can be done before or after dilution to a working concentration. It is fine to use starsan for sterilizing the plastic. Heating to boiling for any period of time is not a good idea for the preforms. Many forms of plastic can, of course, be heated in boiling water and some even stand up to the pressure cooker.
Hope that helps.
 
Just breeching this article but man! Great information. You should get some money or something for writing it up! Thanks!
 
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Babybatch - Please send freshly minted unmarked $100 bills to ..... No, really, it was fun to write up and I got a free year of membership. Plenty of pay for the job.
 
Yeah, huge thanks. I just made a successful starter with my first thawed vial. I'm not even brewing with it right now, I just could wait to see if it worked. I tried freezing once before without this tutorial and the yeast was completely dead. This time, SUCCESS!
It did take an extra 24 hours or so to show signs of healthy yeast activity compared to the fresh smack pack in the starter next to it. So if someone is trying this out, don't lose hope.
 
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  • #26
I generally split one 40 ml tube of about 50% yeast suspension into three liters of wort and grow overnight with stirring and it is completely saturated and ready to go by the next day. I almost always have airlock activity within a few hours after pitching.
 
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I generally clean everything I use with oxiclean and then an iodophore rinse. Works fine on most of the tubes.
 
Ok..... A few questions. Ph.D. in Molecular Biology here and work for one of the largest scientific product suppliers in the world so I have access to pretty much everything one could need for this. I have tried freezing yeast a couple times based off of some threads I saw here on HBT. I swear that the threads I read suggested using a 50/50 mix of yeast slurry and straight glycerine, not the 20% solution you recommend. First time I tried it the wouldn't free because of the high glycerine concentration (I assume anyways). So next time I got them to freeze in a dry ice/ethanol bath, however after I transferred them to my freezer they then melted.
Also, since I'm assuming it takes several hours for these to freeze, does the yeast drop out of suspension and form a pellet in the bottom of the tubes?
 
This is a great article thanks for sharing, I want to give this a try now. Ive been washing yeast for some time now, but dont always get to use it in a timely manner and it goes to waste. I am very interested in this process and am going to order supplies. Would you be able to put together a video of your process?
 
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  • #31
everty2007: If you wash yeast, there are only a few more steps to the process. Settle the yeast and pour off the chilled water. Then I would add cold glycerine diluted to give the appropriate final concentration when combined with the yeast slurry. Finally, freeze. Use some form of protection from thawing if you are putting them in a frost free freezer. I don't think there is much to video.
 
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  • #32
Jersh: Sounds like we have the same training and resources. Let me clarify. The goal here is not to prevent freezing. 50% glycerol will not freeze at home freezer temperatures. 10-20% will. That is the range that we are shooting for. I used 20% but others say between 10-15% is optimal for recovery of cell viability using this approach. I freeze mine in an ultracold (-80C) which, of course, has even better preservation properties. Use 10-20%. Even in an ultra cold, where 50% will freeze, 20% yields better viability. I hope that answers your question.
With regard to settling of the yeast: Yes, if you freeze in a home freezer the yeast is going to settle some before it freezes. This is not a problem. The point of the glycerol is not to suspend the yeast, it is to affect the osmolarity of the yeast and the medium. That happens before the yeast freeze. So, no problem.
 
@Brewitt
I am going to be trying this tomorrow. I have a few questions.
When sanitizing water should I add glycerol to the boil or add after? Will boiling effect the glycerol?
I am using a home freezer and don't have room for a styrofoam cooler. I am using the 50ml Centrifuges like you have. I set up a jar with a tin foil lid and pushed the centrifuge through and plan to fill jar with rubbing alcohol. Will this be sufficient to keep it from thawing/re-freezing or would buying some gel packs and tightly wrapping be a better idea?
 
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  • #36
You want to sanitize the glycerol so you can put dilute it to working concentration in a bottle and put the bottle in boiling water for 15 minutes. A canning Jar is good for that.
The bottle sounds like a great solution for storing the tubes. Or just a wide mouth bottle you can throw a couple tubes in and put something that doesn't freeze like alcohol or gel pack. Close the tubes tightly.
You might get more ideas from the thread on the forums.
 
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  • #37
You want to sanitize the glycerol so you can put dilute it to working concentration in a bottle and put the bottle in boiling water for 15 minutes. A canning Jar is good for that.
The bottle sounds like a great solution for storing the tubes. Or just a wide mouth bottle you can throw a couple tubes in and put something that doesn't freeze like alcohol or gel pack. Close the tubes tightly.
You might get more ideas from the thread on the forums.
 
Ok thanks so I think what I will do will be incredibly rigged but totally work. The bottom half of a 2 litter bottle with cardboard cut to fit the top, and about 6-7 hoes cut to fit 50ml Centrifuge tubes. I will fill with alcohol, put cardboard over and tape loosely on edge with electric tape. Then drop tubes of yeast in through hols and lay 2 gel packs on top of them. This will all be set inside of my large ice tray filled with ice.
 
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