Yeast banking – #5 Frozen yeasts

Eureka, today is the last post in the series about yeast banking at home (or in a lab). Please refer to the yeast basics page for links to all the other posts. The three previous methods (agar plates, isotonic sodium chloride solutions and agar slants) work all at room temperatures or colder. But not below 0°C (32°F) since the yeasts would probably die and the media (agar and sodium chloride solution) would freeze. Storing your yeasts at colder temperatures prevents some of the growth. If the yeasts do not grow during the storage time, the chances are high to have the same exact strain after you revive them. If you store your yeasts in a refrigerator your yeast can grow (even slowly) and might mutate and try to adapt to the colder temperatures. The yeasts could therefore change and maybe lose specific characteristics. This could lead to loss of flocculation or even loss of your most loved aroma profile (banana or clove aroma in wheat yeasts for example). However, such a conversion does not have to happen. It might. And this is why a lot of labs store their microorganisms or cells at lower temperatures such as -80°C (-112°F). At this low temperature no growth occurs. Even the whole metabolism of the cells arrest. The cells kind of stops entirely. You can store your cells at this temperature for nearly forever.

I am not sure how many of you out there have a -80°C freezer at home. Most of you might have a freezer at around -20°C (-4°F). And you can store your yeasts at -20°C as well. Just don’t use a freezer with thaw-cycles. The only disadvantage here is the metabolism of the yeasts might still work and some changes could occur as well. In comparison to a storage at room temperature or colder temperatures, far fewer changes can/do happen at -20°C. And this is why freezing your yeasts is as far as I know the only method to bank your yeasts over a longer period of time (years) at home.

Description of the technique

As already described, this method here is about freezing your yeasts at -20°C (-4°F) or lower/higher if you want. For this purpose you use a special media which consists of a cryoprotectant (antifreezer) such as glycerin. Please don’t use antifreezer you use for your car. If you have your storage media ready you just add some yeasts to the media and put it in your freezer and leave it there until you want to use the yeast for a future batch. Please notice, this is about banking yeasts and not yeast storage. Only small amounts of yeasts will be frozen here. Not pitchable amounts.

I would like to mention already, this is the most sophisticated method of all the four described already (agar plates, isotonic sodium chloride solution and agar slants). I do not recommend to go with this method if you haven’t tried one of the previous ones before. If you are new to yeast banking try to bank your yeast with another technique than this one before and get some experience. I recommend the isotonic yeast storage method for beginners. If you manage to revive the yeasts without an infection you might step forward to this method. If infections occur regularly, try to find the source for the infection and work on that. This method here does not work if you have troubles with your sterility and cleanliness… It just does not. In addition, the technique below is just one way to do it. I am certain there are other ways to freeze your yeasts.

Material

Fig 1: Tube filled with storage media and yeast

– Vial, tube or any other containment you can heat sterilize to store your yeast in a freezer. I use 1.5 mL reaction tubes for this purpose (Fig 1). They are small and easy to sterilize

– Food grade glycerin. Glycerin solutions work as well as long as the glycerin concentration is above 60%. I use a 85% food grade glycerin solution I bought at a local pharmacy

– Pressure cooker or any other source to heat sterilize your tubes and the food grade glycerin

– Media. I guess dried malt extract solution or even an isotonic sodium chloride solution could work as well. I use a lab media (called YPD) as a storage media. And add some ascorbic acid as well. More about my storage media later on.

– Sterile pipettes, micro pipette with sterile tips or a sterile syringes. You need sterile devices to add the storage media to your containments before freezing and get the yeast out of the containment for reviving. See “bank the yeast” description below for further information.

Preparation

First, get the freshest, purest yeast you can get. This could be from a starter or from a fresh yeast package or vial. This is very crucial. If you freeze unhealthy yeast you could risk to either loose them entirely or have problems during reviving them. Or a different outcome of a batch of beer (attenuation, taste, flocculation etc.). Please do not freeze or bank any unhealthy yeasts. And don’t expect the yeasts to come around during banking. If you have problems during a fermentation (stuck or whatever) don’t bank the yeast afterwards and hope the yeasts will be fine. They probably won’t.

What yeast sources could you use?

Fig 2: Small yeast starter with yeast at bottom

1. Yeast starter

Get yeast directly from a fresh yeast starter. Wait until no more growth occurs. Then mix up the whole yeast starter to get the yeasts back into solution. Remove some of the volume from the yeast starter and fill your pre-sterilized containments. If you want to cool down the yeast starter to let the yeasts settle to the bottom of the flask and discard as much of the yeast starter as possible (and only pitch the yeast sediment), remove the yeasts for banking before cooling down the starter. Store the filled containments for 48 h in a refrigerator. During this step the yeast build up some important molecules they need to survive and settle to the bottom (Fig 2). Remove as much of the supernatant as possible (Fig 3). This can mostly be done by just inverting the tubes, vials etc. The yeast sediment at the bottom should stay at the bottom. Just don’t turn the tubes too fast and too slow. You now should have a nice yeast sediment at the bottom (Fig 3). The volume of the yeast sediment should be below 10% of the volume of the containment. If it is a bit more or less don’t worry. However, discard some of the sediment if it is more than 20% of the volume.

Then proceed with the steps described as “bank the yeast” below.

2. Yeast package from manufacturer

Use yeast from the manufacturer directly. Make a small yeast starter and add a few mL of yeast slurry from the package or vial (Fig 2). I use glass tubes for this purpose which are filled with 4 mL of a malt extract yeast starter media (10 g of dried malt extract dissolved in 100 mL of water) and sterilized them in a pressure cooker.

Leave the starter at room temperature for 48 h. Then proceed with the steps described as “yeast starter” above. If your yeast is very fresh, you might skip the whole starter-step and bank the yeasts directly. Therefore fill your containments with the yeasts and let them settle down in a refrigerator for 48 h then discard the supernatant. Then proceed with method described as “bank the yeast” below.

3. Yeast sediment from fermenter

I would not recommend to directly bank yeasts from a slurry. At least wash them first to get rid of trub and any dead cells and do a small yeast starter. Just harvest a small amount of the sediment (like 100 mL) and wash them with sterile water for a few times until only the viable cells remain. Discard as much of the supernatant as possible. Then make a small yeast starter (100 to 200 mL), add the washed yeast cells and leave the starter at room temperature for 48 h. Then proceed with steps described as “yeast starter” above.

4. Yeast sediment from bottles

Procedure is similar to “yeast package from manufacturer” above. Make a small yeast starter and add some of the bottle sediment. Leave the starter at room temperature for 48 h. Then proceed with steps described as “yeast starter” above.

Bank the yeast

Fig 3: Tube with yeast sediment at bottom

The yeast sediment you now have in your containments should consist of very healthy and pure yeast cells (Fig 3). Now its time to add the storage media (see below) and freeze the yeasts. I add about ten times the volume of storage media for every volume of yeast. In my case I have about 0.05 to 0.1 mL of yeast sediment (Fig 3). I therefore add 0.5 mL of storage media. To add the storage media you need a sterile device such as a pipette, micro pipette with sterile tips or sterile syringes. Please pre-sterilize the storage media in a pressure cooker for 15 min if possible. Let the storage media cool down to room temperature first before proceeding. Then add the media and either gently shake the tubes, vials or use the pipette, syringe, micro pipette for a thoroughly mix. You are basically done. Just label your containments very well and put them in your freezer. Done!

Storage media:

1. Malt extract based (haven’t try this one): For 100 mL of storage media use 10 g of dried malt extract, 50 g of glycerin and fill up to 100 mL with water. Add 0.1 g ascorbic acid (aka vitamin C) if possible. The ascorbic acid helps to stabilize the membranes of the yeasts. If you have a glycerin solution for example a 85% glycerin solution calculate the amount you need as following: 50 g divided by percentage of solution (divided by 100). In this example 50 divided by 0.85 equals 58.8 g. You therefore have to add 58.8 g of your 85% glycerin media. Sterilize the storage media in a pressure cooker for 15 min if possible.

2. YPD storage media (I use this one). The recipe for this YPD-media based storage media is from the book “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff. For 100 mL you need: 5 g YPD bouillon, 50 g of glycerin and 0.1 g ascorbic acid. Add up with water to 100 mL. Sterilize the storage media in a pressure cooker for 15 min if possible.

Storage

Put your containments in your freezer. Nothing to do more. I use a rack for my tubes to have some organizing system (Fig 4).

Fig 4: Yeast library part 1

Reanimation

1. Make yourself a yeast starter. I recommend 100 mL for the first step. Therefore dissolve 10 g of dried malt extract in 100 mL of water, add some yeast nutrients if possible and sterilize the starter for 15 min with a pressure cooker. Cool down the starter to room temperature.

2. Get your tube, vial (or whatever containment you use for yeast banking) out of your freezer and increase the temperature as fast as possible. I let the tubes warm up in my hands. Then gently mix the yeast and storage media and add the whole content to your yeast starter. I use a micro pipette for this step. Then wait a few days until signs of fermentation arise (cloudiness, white foam, yeast sediment at bottom, bubbling etc.). Wait until a yeast sediment formed at the bottom. You can either stir your yeast starter the whole time or just leave it unstirred.

3. Prepare your next yeast starter. I normally do a 1 L stirred yeast starter as a second starter here. Therefore dissolve 100 g of dried malt extract in 1000 mL of water and sterilize it. Discard the supernatant from the first yeast starter and only transfer the yeast sediment to your next 1 L yeast starter. I recommend to taste the supernatant (before discarding) to check if the starter is okay. If the starter tastes bad probably an infection occurred. If the yeast starter tastes good, congratulations!

4. Repeat the yeast starter steps until you have the amount of yeast you need. It is hard to tell how many yeast starters you need and what volume you should choose. There are way too many different way on how to bank the yeasts. The only way to tell how many yeast cells you have would be to count the cells (have a look at this post concerning this topic).

From my experience and with the amount of yeast I bank (about 0.1 mL as it can be seen in Fig 3), I need a 100 mL yeast starter first, followed by a 1 L yeast starter, followed by another 1 L yeast starter afterwards to have approximately 100E9 cells. This would be equal to the amount of yeast you get in a Wyeast’s Activator package or White Labs vial.

My experiences with this method

My procedure looks as following. As already mentioned, I use a YPD-based storage media to bank the yeasts. And I use the tubes shown in Fig 3 for banking. After discarding the supernatant after storing the tubes 48 h in the refrigerator, I add approximately 0.5 mL of the storage media to the tubes with a micro pipette and a sterile tip (Fig 5).

Fig 5: Equipment for yeast banking. YPD-based storage media (left), yeast sediment in tube (right) and micro pipette (1000 uL) with sterile tip

Then use the micro pipette to mix the yeast and the storage media. After that the tube look like shown in Fig 1. I then put the tubes in a box (shown in Fig 4) and store them in my freezer (at -20°C).

What are the advantages and disadvantages for this method compared to the others?

Advantage Disadvantage
Long term storage method Lot of equipment necessary (freezer, lab equipment etc.)
No maintenance work Contaminations not visible
Does not require a lot of space Can’t store yeast mixtures, blends
Rather complicated method

This is for sure one of the least labor intensive methods. And the only one to store your yeast over a longer period of time. On the other hand, you do need some extra equipment such as a freezer and some lab equipment (syringe or pipette or micro pipette, containments, chemicals (ascorbic acid)). I think this method is only for the people really interested in yeast banking. And I would not recommend to go with this method if you haven’t tried one of the previous ones before. Sure the long-term storage seems very advantageous. However, do not underestimate the time and equipment you need to prepare the yeast for this banking method. On the other hand, your equipment has to be very clean and mostly sterile.

As with other methods, it is not easily visible whether your yeast is infected or not by just looking at your vial, tube etc. You will know after the first yeast starter. And you can’t bank yeast blends and other mixtures with this method as well. The ratio of the different microorganisms will eventually change during the reviving. If you do want to store a blend you might have to separate the blend before…

For all of you still interested in freezing your yeasts, I would like to mention the book “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff again. In there are further information on how to freeze your yeasts.

This is the end of the yeast banking posts. I hope I could give you some information about the topic. Please feel free to comment and ask questions if something is not clear enough. The next posts will be about some recipes, tasting notes and yeast hunting stories again. Stay tuned!

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18 thoughts on “Yeast banking – #5 Frozen yeasts

  1. Extremely informative, as the other techniques and description. For me, it’s just pure awesomeness 🙂
    May I ask, what kind of pressure cooker are you using (model, brand?) and what is your average sterilization time (20 min or more) ?
    I know that most of european models (therefore mine too 😦 ) can’t reach 15 psi (1 bar @ 121C).

    • Hi Mike, thank you very much for your comments. Always appreciate such comments. I inherited my pressure cooker made by Kuhn Rikon from my grandmother and it is a very standard pressure cooker for cooking potatoes and such. I have no information about the model. It just comes with a standard pressure releasing valve. That’s it. No pressure gauge or anything fancy. I therefore have no idea about the pressure in the pot during the sterilization process or the temperature. Concerning the time, I heat up the pot until it releases pressure. Just to get rid of the air inside and replace it by water vapour. After releasing the pressure for some seconds, I decrease the temperature a bit to stop the valve from releasing further pressure. Then hold the temperature for 10 to 15 min. If you want to sterilize bigger liquid volumes (> 500 mL) such as starter media etc. increase the time to 20 to 30 min. However, I sterilize my yeast starters (around 100- 200 mL) for 10 to 15 min and never had any contaminations so far. After that simply turn off the heat and let the whole pot cool down on the hot cooking plate. Hope I could give you some information here. I guess the best way would be to start with a defined procedure and if it works, stick to it. Thats what I did here.
      Cheers, Sam

      • Thank you very much Sam! I really appreciate your time and effort, dedicated to the implementation and placing the effects of your work, in the form of this blog 🙂 Easily accessible to all interested – sharing means caring 🙂

        After that, when I bought and learned that my pressure cooker, unfortunately, does not maintain 15 psi. I conducted a small “research” on the subject 🙂 You may find yourself lucky, because apparently yours – Kuhn Rikon and maybe 2 other products (in EU), can produce and maintain such pressure.
        As I mentioned earlier, the majority of ‘european’ pressure cookers holds lower pressures & temperatures.
        Of course, in US they got no such issues, all PC have as a standard 15 psi – damn … those lucky Yanks 🙂
        What I did, was that, I just extended the time of ‘test drive’ sterilization for 45 min (~100 ml wort / agar medium and few petri dishes). The result was indeed very pleasing. After one week, of storing in ambient temps, I didn’t noticed any contamination 🙂

        Once again, thanks for sharing your knowledge and the effects of homebrewing /microbiologics ‘experiments’.
        Cheers, and keep ’em coming!

        • Thanks for sharing your information as well. I haven’t put many thoughts into pressure cookers so far 🙂 And I don’t know how important it is to reach a pressure of 15 psi. Sure there is the golden rule of +1 bar (2 bar in total) and 121°C and somewhat keeping this temperature for 20 and 30 min. That’s how it is done in most of the labs I have been working so far. However, your test drive gave you satisfactory results. And I manage to do the same with my pressure cooker without measuring any temperatures or pressures.

          Good luck with your yeast ranching. And I will keep writing further posts about yeast ranching and stuff for sure. Promised! Cheers, Sam

  2. Thank you for such an inspiring and informative blog. Just started ranching myself (3711, 1450, 3944 and 1768) and will be harvesting from several bottles soon!

    Thank you for your time and effort,
    Dan

    • Hi, actually I have not measured any viabilities (yet). The only evidence that all works is from all the successfully revived yeasts. Even Brettanomyces survive a freezing period (check out the post from 2nd of February 2013). Time will tell how this all works on a larger timescale. Let me know if you have experience with a frozen yeast library over a longer period of time.
      Thanks for commenting, cheers Sam

      • I’ve started with a frozen library around half year ago. My very first strains were successfully propagated a few times. Initially I took a sample with the inoculation loop from the eppendorf tube kept in the fridge and plate it. Then inoculated 10ml wort and propagated it up to a liter that I use in my batchs. Recently I started inoculating the 10ml tube directly from the bank (same result and less time) and only plate to check viability or make new mother cultures. Well.. the thing is that last week I plated that very first strain and I found few that colonies grew and it took it more time than usual.. So.. my mother culture is dying after 6 months.. Unfortunatelly, I only have a frost free freezer available right now (with thaw-cycles). I keep my tubes in a styrofoam box with ice to amortiguate the temp variations.. but it doesn’t seem to be working that well.. So, I’m moving to an isotonic backup until I get a decent freezer for my yeast.. and I’m starting to check viability on all my strains.. and that’s why I wanted to know your experience with it.. 🙂

        • Thanks for sharing your experiences. Your procedure is very similar to mine. I am sorry to hear that you observe a decrease of viability of your yeasts. I don’t know if there might be a connection to your freezer with thaw-cycles. Periodical thawing is for sure not great for the yeasts and stresses them a lot. Still, there might be other reasons such as strain dependency etc. The isotonic sodium chloride solution worked very well in my opinion. A good choice for a backup.

          Great blog of yours by the way. I can use my Spanish again… 🙂
          Cheers, Sam

  3. Hello! Excellent post even for those of us experienced in microbio. I work in a TB lab, so this is all very familiar to me 🙂 I am wondering if there are growth log charts available for different strains somewhere? I am used to being able to take an OD and it makes me slightly uncomfortable to just wing it while doing upscales. Also, is there any practical way to get an OD reading without buying a spectrophotometer or using a counting chamber? (I kind of hate counting chambers with a passion…I blame all the class labs I’ve taken!)

    • Hi, I am not aware of any source concerning log charts. I imagine these are strain dependent anyway. Concerning the OD. I estimate the cell counts from the yeast volumes. To get the connection between volume and cell count I use the yeast calculator from mrmalty (http://www.mrmalty.com/calc/calc.html) and the re-pitching from slurry tab. This is a very easy way to go. Cheers, Sam

  4. Thank you. But I wish I had found this about 4 years ago. I got interested in yeast ranching for the fun of it and just because I could (though I told the SO is was to save money). Really it was about being able to have the yeast I wanted when I wanted it and not have to depend on mailing liquid yeast in the middle of the summer. It was a long road of mis-starts and mistakes. I pasted through the yeast washing and storing slurry in bottles (very short term) and freezing with glycerin. I finally found some information on sterile distilled water. Oh, by the way I don’t have any Microbiology background and it took weeks of googling to understand what streaking was (as opposed to the running around naked of the late 60’s and 70’s) or what a slant was. The last time I did any kind of chem lab work was about 40 years ago! Now I am storing yeast in sterile distilled water. I had not seen anything concern the .9% saline and osmotic pressure. It is time to re-master my stores and I will definitely use the isotonic solution. I use the petri dish to isolate the original colony and use the inoculation loop to pick up a few drops from the yeast bank to streak on a slant. I will pull all of the colonies from the slant and pitch that to 150ml or so and then step up to a full starter.

    I am planning to experiment with pitching directly from the slant to a full starter. I don’t know of any down side except for the possible risk of contamination due to the very low concentration of yeast in the 600-1000 ml starter.

    Again, thank you for your research and for sharing it. I now have some confirmation (and more information) that I stumbled onto the right path.

    David

    • David, thanks for reading and your feedback. Always appreciated and keeps me motivated to write up such methods and such.

      Good luck with re-mastering your current bank. I hope everything goes according to plan. By the way, your way of doing it sounds very reasonable to me. You did your research very well.

      Directly pitching from slants to starters. I agree that contaminations are the most important factor to consider. I do not feel comfortable pitching from slants directly to >600 mL starters. Simply because of the risk of contaminations. I go with 100 – 200 mL starters first and then go from there. Simply because I can sterilize the 200 mL starters in my pressure cooker and kill everything. But I would not be surprised either if it works.
      Cheers, Sam

  5. Thank you for this post, this is way better than any of the forum posts I have encountered so far!
    Buying small quantities of YPD is very, very expensive.
    There are some guides on making your own YPD. Almost all of them use lab chemicals, which are equally expensive in small quantities for a private individual. For yeast extract, a few suggest simply autoclaving dry yeast, which would seem like a good way to make your own yeast extract. Dextrose/glucose is usually sold in food stores. This leaves the peptone, again very expensive in small quantities.
    The peptone is simply a nitrogen source in the form of partially hydrolysed protein. Could you simply use protein powder form a training store as the nitrogen source?
    I would love to see a guide on making a good YPD substitute. Sure you can use malt extract, but I prefer the empiric approach and eliminating variables.

    • Cheers. Don’t know if protein from a training store would work as a substitute for peptone. Never tried that myself. And since my time is very limited lately, I don’t even care to look for MRS (or YPD) substitutes. Its cheaper to just use the more expensive material if one considers the time one needs to find cheaper alternatives. I am therefore sorry that I cannot help you out here. Better have a look at bkyeast.wordpress.com. Dmitri is looking for media alternatives.

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