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!

Yeast banking – #1 Introduction

Eureka, today is the beginning of another post-series in the yeast basic area. The topic of this one is all about yeast banking at home (or in a lab). Today is all about the basics of yeast banking, what to consider and what methods there are. The next posts will be about the different methods. Lets begin.

What is yeast banking?

Yeast banking is a technique to store your yeast strains over a longer period of time to either reuse it later on or maintain a collection of strains. Storing freshly harvested yeast for a short time is yeast storage in my opinion and not the topic of these posts. Yeast banking is a long-time storage of your yeast to have them ready again in a year or maybe more. Another difference between yeast banking and storage is the amount of yeast. In yeast banking you only store a small amount of yeast.

Reasons to consider yeast banking?

Yeast banking can have different advantages such as lower prizes for your yeasts (use for multiple times or use them again in a year or more), having different strains available or even maintain yeast strains which are not commercially available. In addition, you can trade yeast strains with others. Let me add, if you brew only with dried yeasts, yeast banking is not a technique you should consider. It is not worth the money.

What are the basics of yeast banking?

Fig 1: Banking with agar plates

The goal of yeast banking is to have the yeast in a state where it can survive for a long time. Dormant yeast cells are very suited for this job. First, the yeast cells have to be very vital before you bank them.

Viability and vitality. Viability describes the live/dead ratio of a yeast population. A very viable yeast sample has a high amount of live yeast cells and only a few dead ones. Typically, healthy yeast samples have viabilities greater than 95%. Meaning 95% of the yeast cells are viable, 5% aren’t. Vitality on the other hand describes the physiological state of a yeast cell. It more or less describes how fit the yeast cells are. Storing yeasts with a low viability will only cause troubles sooner or later. The first thing to consider in yeast banking is the cells viability, second the vitality.

To summarize, the yeast for the banking process should be very viable and vital. More about this later on.

The next thing to consider is the environment you put your yeast in for the storage. I call it media from now on. Any media which can stress the yeast cells is not suitable for the job. Factors causing stress are alcohol or the osmotic pressure for example. Storing yeast in distilled water will stress the yeast cells since the distilled water will lead to a swelling of the yeast cells (water flows into the yeast cells) and the yeast cells will probably burst. What are suitable media for your yeast cells? One way to go is use isotonic sodium chloride solutions. The sodium chloride prevents any osmotic pressure difference between the water surrounding the cell and the cell’s interior. So no swelling and bursting of the yeast cells. Further information about the media will follow in future posts about the different techniques.

Fig 2: Yeast banking with liquids

Third, depending on the period of time you want to store your yeast, a different kind of technique has to be used. Therefore consider how long you want to keep your yeasts in general and how much maintenance work you want to put into your yeast banking system.

To summarize, important things to remember before heading into yeast banking are the vitality/viability of your yeast, the media you put your yeast into and the time frame you want to store the yeasts. For example, banking yeast which was in the refrigerator for four weeks is probably not the best source of yeast since the vitality/viability will have suffered over time for sure. Do a starter first to increase the vitality/viability.

Another important thing is sterility. You have to work very cleanly to avoid any contaminations in your yeasts. The yeast cells might have suffered a bit over the period of time and any contamination may have an easy game to overgrow the yeasts.

What are the different techniques for yeast banking?

Four different techniques come into my mind when I think about yeast banking. I am sure there are different ones as well but many of them are derivatives of the following four:

– Yeast banking with agar plates
– Yeast banking with agar slants
– Yeast banking with isotonic sodium chloride solutions (or in any other liquids)
– Frozen yeast banking

The next four posts in this series will be about one of these techniques each.

Why am I yeast banking?

I am yeast banking due to several reasons. First, to have different yeast strains available and save some money because I do not want to buy yeast for every single batch. Second and most importantly store my special yeasts. These are yeast I isolated from different bottles of beer and are not commercially available. In addition some seasonal yeasts releases from the private collection from Wyeast’s. And last because it is fun and you can do yeast trades.

Fig 3: Frozen yeast library

What is the best method for yeast banking?

I can already answer this question: There is none. Every method has its advantages and disadvantages. Only you can decide which method is best for you. However, I am really fond of the isotonic chloride solutions because I have a lot of good experience with this method. Some yeasts can survive up to two years in there without doing any maintenance work. Unfortunately, not every strain behaves the same. Some yeast strains will be dead sooner than others.

What method do I use?

As already mentioned, I use(d) the sterile liquids approach. I store(d) my yeasts in isotonic sodium chloride solutions and had no troubles so far. At the moment, I am switching to a frozen library because of the amount of strains I have. However, I keep the sodium chloride liquids until they are either gone or the yeasts are dead.

Are there any other sources to get information about yeast banking?

Sure, yeast banking is not my invention. These techniques are very common in the labs to store nearly any kind of microorganisms for later uses. Therefore a lot of different protocols exist about banking. Since we are in the homebrewers area here, the protocols should be rather easy to follow. Very often in labs the microorganisms are stored in -80°C (-112°F) fridges or even colder. I wonder if anyone of you has such a fridge at home… Therefore other techniques have to be used.

Fig 4: Banking with agar slants

A book I recommend is “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff. Everyone of you out there interested in getting into yeast banking and such should buy a copy. I am not related to any of the authors so no personal benefit for me in there. The whole chapter six is dedicated to “Your Own Yeast Lab Made Easy”. An excellent source in my opinion.

Other sources can be found with your search engine of choice.

What will the next posts be about?

The next four posts will be about each of the four techniques mentioned above. I will try to explain the method how they work, the material you need and tell you some advantages and disadvantages. In addition, my experiences with the technique if I have some. Stay tuned!