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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Yeast banking – #3 Isotonic sodium chloride

Eureka, today I proceed with the second technique to bank yeast at home (or in a lab). I am sorry for the delay of this post. I am currently very busy and writing such a post is very time-consuming. The introduction to the banking yeast series can be found here. The post about the first technique (bank with agar plates) can be found here. Lets proceed with another technique, isotonic sodium chloride solutions (or any other sterile solutions).

Description of the technique

One way to store yeasts over a period of time is storing them in sterile solutions such as isotonic sodium chloride solutions. Isotonic in this case refers to solutions which have the same osmotic pressure as the cells itself. The osmotic pressure mainly depends on the salt concentration of a liquid. If you have a liquid with a high salt concentration, the osmotic pressure of this solution is high. On the other hand, distilled water (low to no salt concentration) has a low osmotic pressure. If two liquids with different osmotic pressures are connected by a membrane, the two pressures can equalize: Water from the low pressure solution passes the membrane and flows into the high pressure solution. The water flows until the two pressure potentials of the two solutions are equalized.

Lets get back to the topic. Yeast cells have a membrane as well. It basically surrounds the whole cell. If you store your yeasts in a solution with a higher osmotic pressure than the yeast cells (lot of salts and other organic compounds are in a cell), your yeast cells will eventually die due to dehydration (loss of water). Losing water is not ideal for a cell. Think about humans, losing water can lead to severe health problems as well. On the other hand, storing yeast in distilled water could eventually burst the cells since water thrives into the yeast cell (into the high osmotic pressure solution). This is not ideal as well. The key is to prevent an osmotic pressure difference between the yeast cell and the surrounding liquid. You do so by using liquids with the same salt concentration as in the cell. You therefore have the same osmotic pressures in the cell and the surrounding liquid. And this is called isotonic. If you dissolve 9 g of sodium chloride (a.k.a table salt) in one liter of distilled water, the solution is isotonic. For other salts/compounds, there are lists are available to look up the amounts you need to get an isotonic solution. Sodium chloride seems to be in every kitchen and why not use it to store your yeasts. I will therefore talk only about isotonic sodium chloride from now on.

Fig 1: Glass tube for yeast banking

First of all, you have to think about a containment for your yeast-sodium chloride solution. I used small glass tubes with a screw cap first (volume around 5 mL) as shown in Fig 1. Any other tube will do the job as well. Just keep the sterility in mind. It is best to have a sealed containment to avoid any contaminations. In addition, any containment which can be sterilized in a pressure cooker is even better.

Fig 2: Isotonic chloride ampule

Another way to go is using ampules filled with sterile sodium chloride solution (Fig 2). These ampules are used in hospitals very frequently and can be bought in many pharmacies in Europe. Can’t tell it this is true for any other country though. You only need a sterile syringe and a cannula and inject the yeasts in the ampule. Done!

I used the tubes (Fig 1) first but went with the ampule technique later on because of the volume. I first thought 5 mL of yeast solution are not enough for my purposes. Now I know, most of the ampules still contain about 95% of the original liquid… If you have the same concerns, just fill additional tubes with the same strain you use more often.

If I would consider going into the isotonic solution method again, I would probably use the glass tubes with a volume of approximately 5 mL, fill them with 4- 5 mL of isotonic sodium chloride solution and sterilize them in a pressure cooker. With the ampules you need syringes and cannulae in addition.

Material

– Containment for the yeast-sodium chloride solution (i.e. glass tubes with screw cap)

– Sodium chloride. Common table salt will do the job.

– Distilled water

– Sterile syringes and cannulae to transfer the yeast into the containment

Preparation

Isotonic sodium chloride solution: Dissolve 9 g of sodium chloride (a.k.a table salt) in 1 L of distilled water. Depending on the water quality, even tap water would work it is low in salts. However, get yourself one liter of distilled water if you can. 1 L of the sodium chloride solution will last for many, many tubes.

Depending on your containment:

– If you can sterilize your containments, fill them with the isotonic sodium chloride solution and sterilize them in a pressure cooker or in boiling water for approximately 15 min. They are ready to go. You can even store the sterilized tubes at room temperature for nearly forever. Just make yourself a batch of tubes and the yeast banking can begin.

– If you can’t sterilize your containments (either because they are made out of plastic and melt during the sterilization process or you do not have an opportunity to sterilize them) you need to sterilize the sodium chloride solution by boiling it and then transfer it into the containments later on. However, don’t forget the sterility of the containment itself. If you buy them as pre-sterilized, well good enough. If you buy something not sterile, disinfect it somehow. Either use diluted Javelle water or Vodka. Just remember a disinfection is not the same as a sterilization. Some microorganisms will survive the disinfection process. Either way, I would not recommend this method. Get yourself some sterilisable tubes and all the worries are gone.

Bank the yeast

Now as the tubes are filled and sterilized, its time to bank the yeast. The easiest way in my opinion is to get the yeast directly from the source such as a fresh Activator package from Wyeast or a vial from White Labs. Use a sterile syringe and get yourself approximately 1 mL of yeast slurry for 5 mL of isotonic sodium chloride solution from the package or vial respectively and transfer the volume into the sodium chloride solution. You are basically done. You could even pour some of the yeast slurry from the package or vial into the sodium chloride solutions directly. Whatever works.

Fig 3: Wyeast’s 3942 Belgian Wheat yeast in sodium chloride solution

On the other hand, you could harvest yeast from Kräusen and transfer them into the isotonic solution. In this stage the yeast cells are very viable and vital. Using harvested yeast could work as well though the viability/vitality could be an issue here. I would recommend doing a small starter with the harvested yeast first (around 10- 50 mL), decant as much of the supernatant off the yeast as possible and transfer the yeast slurry into the isotonic sodium chloride solutions.

In addition, you could transfer a colony from an agar plate into a sodium chloride solution.

To summarize, you could basically use every source of yeast possible. Just keep the vitality/viability in mind. You do not want to bank unhealthy yeasts.

Storage

If possible, store all the yeasts in the sterile solutions at around 6°C (43°F). In general, cold temperature would be fine. Just don’t freeze them. They probably won’t survive. If cold storage is no option, store them at a cool and dark place. After a short time, the yeast forms a nice sediment at the bottom of the tube/ampule etc.

Reanimation

To get from the banked yeast to a yeast starter. Collect 1 mL of the liquid from the vial with a sterile syringe (shake before removal to get the yeast back in solution) and transfer to around 100 mL of a 10°P sterile starter wort made with dry malt extract. To get a 10°P starter just add 10 g of any dry malt extract, some yeast nutrients, dissolve in 100 mL of water and sterilize it with a pressure cooker if possible. I use 500 mL Schott bottles for this purpose. Any mason jar will do just fine as well. Just sterilize it in either a pressure cooker or in some boiling water. This is a very crucial step because the yeast cells from the isotonic sodium chloride solutions might be quite slow growers at the beginning. Any contamination in the starter will outgrow the yeast for sure. And don’t use too large starters for this step.

Let the fermentation go for some days (up to seven days if necessary). A small layer of yeast will form. Then increase the volume up to 1 L in total (add 900 mL of freshly sterilized 10°P wort on top or transfer the 100 mL starter with the yeast to 900 mL of fresh wort). After the 1 L starter, there should be roughly the same amount of cells as in a fresh Wyeast activator package (100E9 cells). This may vary between yeast strains. Use a counting chamber to determine the exact cell concentration and cell amount if possible or estimate the cell count from the yeasts volume.

My experiences with this method

I use(d) this method for quite a while and all my yeast from my yeast library are/were in sodium chloride solutions at one point. My oldest strains are in the ampules since Summer 2010 and I could reanimate them successfully in Summer 2012. In my experience, the yeasts can be stored this way for at least two years without any problems (refrigerated at 6°C). This holds true for several different yeast strains. However, I lost some of the strains due to an infection.

 Advantage Disadvantage
Rather easy method  Needs space
No maintenance work  Contaminations invisible
Not a lot of equipment necessary  Long term storage?
Viable > 2 years (@ 6°C)  Storing yeast/bacteria mixtures

All in all, this is a really easy and cheap method in my point of view. I had a hard time to find disadvantages for this method. I do not bank my yeasts with this method anymore because of the space it needed. Please consider that we are talking about 40 different strains in my case. My refrigerator is basically filled with ampules… Some of them in two ampules… Another disadvantage is the observation of contamination. Other techniques (such as the agar plate method) are easier to identify any contamination. However, I would not expect any contaminations to occur if you work cleanly and with sterile equipment. The only unsolved question remaining is “how long can you store the yeast with this method”. From my experience, yeasts can be banked for two years at least. Without any maintenance work.

Fig 4: Part of Eureka Brewing’s yeast library

Another disadvantage of this method is storing yeast or yeast/bacteria mixtures. Storing mixtures with this method might change the ratio between the yeasts or yeasts and bacteria strains. On the other hand, there is no useful method in storing mixtures after all. Even if you manage to keep the ratio between the different strains constant during the storage, during the reanimation the ratios might change again… If you want to store mixtures, the only way would be to first determine the ratio of the different strains present and then separate them and bank them separately as well. Then create the mixture from the banked cultures again. Very labor intensive and not easy (been there). This even goes way beyond the topic of this post.

To summarize, banking yeasts in sterile solutions is a rather easy and cheap method. Not only is it less labor intensive than banking with agar plates but less expensive as well. From my point of view, this is a method where you can bank yourself yeasts for at least two years in a rather easy way and rather low in maintenance work. If I have to recommend a banking technique, I would recommend banking yeasts with sterile isotonic sodium chloride solutions. In addition, you can even easily trade your yeasts with other homebrewers.

The next post will be about banking yeasts with agar slants. 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!

#9 Agar plates (Wyeast’s Brettanomyces lambicus)

Eureka, this is yet another post about Brettanomyces and agar plates. Todays post is about the results of streaking a pure Wyeast’s Brettanomyces lambicus culture on Sabouraud agar plates. I first streaked some liquid from the Brett package on a plate and let it incubate for six days.

Fig 1: Wyeast's Brettanomyces lambicus on Sabouraud agar after six days of incubation

There are some bigger circular colonies and smaller ones visible (Fig 1). The bigger ones are circular, off-white, glossy, even and convex. To check if there are two different morphologies (big and small colonies) as seen in Fig 1, I picked a small colony and streaked it on a new plate.

Fig 2: Wyeast's Brettanomyces lambicus on Sabouraud agar after eleven days of incubation

And there is just one kind of colony visible (Fig 2). These colonies are off-white, glossy, more or less even, circular or wavy and convex. So very similar to the bigger ones as seen in Fig 1. Therefore both colonies (big and small in Fig 1) are the same. Some margins of the colonies were not even anymore and had a wavy margin. But this can even be observed in pure Saccharomyces cerevisiae colonies.

To summarize, Brettanomyces lambicus from Wyeast form off-white, glossy colonies on Sabouraud much like Saccharomyces cerevisiae. So no way to differ between Brettanomyces and Saccharomyces. I already observed the same with the colonies from Brettanomyces bruxellensis. And even the colonies from both Brettanomyces (B. bruxellensis and B. lambicus) look very similar as well. Similar observations were made by Jason from sciencebrewer. This concludes, Sabouraud agar is really not useful to differ between Saccharomyces and Brettanomyces. But this was not the goal of these plates in the first place. I just wanted to see how the different Brettanomyces look like on the plates. Further experiments are necessary to find an agar to differ between Brettanomyces and Saccharomyces. Nevertheless, for the upcoming experiments, it is useful to know how the colonies could look like in the first place.

I am still planning my next experiments concerning Brettanomyces so it could take a while until further results are available. But there will be other posts about Brettanomyces in the future. Some of them will focus on the morphology, some about techniques how to differ between them such as using PCR, another post will be about the taxonomy of Brettanomyces and other posts will be more about the genetics of Brettanomyces. Basically more about science behind Brettanomyces rather than experimental data. I try to make them as easy to read as possible and hope these posts will be interesting to some of you as well.

#5 Agar plates (Kombucha)

Eureka, today ends the pentade about agar plates with a short post about kombucha. The other posts can be found here. I plated some kombucha on a Sabouraud agar plate and incubated it for three days. Here is what I got.

Kombucha:

Kombucha culture on Sabouraud agar after three days of incubation

I could observe at least three different colonies:
– Off-white color, even, circular, glossy, convex, 2 mm diameter.
– Transparent, circular, less than 1 mm diameter
– wavy, white, 1 mm diameter

I expect the off-white colonies to be yeasts. The other colonies must be bacteria of some kind. The plate has a distinct vinegar smell. My microscopy analysis revealed that the off-white colonies were again typical Saccharomyces cerevisia yeast cells. And the other colonies are bacteria of different shapes. I did not take any photographs due to lack of time. I am very sorry about that.

Lets summarize, there are several bacteria strains in kombucha as well as yeasts. I am not that surprised about the presence of bacteria as the kombucha had a vinegary smell as I tasted it. There should be some Acetobacter in there I guess. And some other bacteria as well. As well as yeast(s). Can’t tell if there are more than one strain(s).

Well, this is it. The pentade is over. I had a lot of fun with the different bugs and learned a lot of new stuff concerning yeasts and bacteria. I hope you enjoyed it as well. I will post a summary about the different plates by the end of February 2012 with new results as well.

What’s my next project concerning wild bugs? I haven’t planned a new experiment yet but I might try out the potato agar mentioned by BKYeast. If you do not already know BKYeast’s page, please check it out. There are some pretty neat results and pictures of different bugs. Cheers to everyone!

#4 Agar plates (#3763 Roeselare Blend)

Eureka, today another post about yeast hunting. The other posts of the pentade about agar plates can be found here. This time about plating Wyeast’s #3763 Roeselare Blend isolated from the liquid of my Flanders Red. I took a sample of the fermenter and plated it on a Sabouraud plate. I then picked some colonies from the first plate and transferred them on separate Sabouraud plates. Lets get into the results.

Roeselare the First:

Plated the liquid from the fermenter and incubated it for nearly three days (Fig 1).

Fig 1: #3763 Roeselare Blend on Sabouraud agar after three days of incubation

I could observe at least two different microorganisms: (and three mold colonies as well)

– Off-white color, even, circular, glossy, convex, 2- 4 mm diameter.

– White, wavy, irregular, convex, not glossy, 2- 3 mm diameter.

The white colonies seem to grow on top of the off-white colonies. I guess the off-white colonies are yeasts. The microscopy will tell. And I have no clue about what the other colonies might be. Wyeast states on their page that they blend a Belgian yeast strain with a sherry strain, two Brettanomyces strains, a Lactobacillus and Pediococcus strain. Assumed that the off-white colonies are the Belgian yeasts, then the white colonies could be the sherry strain or some sort of Brettanomyces. I can exclude the Lactobacillus due to the results from the post about Lactobacillus where I concluded that Lactobacillus do not grow on this agar. I exclude Pediococcus as well as Pediococcus do not easily grow in presence of oxygen.

As there were some molds on the agar, I picked one of the colonies each and streaked them on separate plates.

After incubating for nearly four days, the following colonies appeared on the plate where I streaked the off-white colonies (Fig 2):

Fig 2: Off-white colony of Roeselare blend

Off-white color, even, circular, glossy, convex, 2- 4 mm diameter

No surprise. The colonies still have the same morphology.

I picked a colony and prepared it for a microscopy analysis (Fig 3).

Fig 3: Microscopy picture Roeselare blend off-white colony

My guess seemed to be spot on again. The off-white colonies are indeed a sort of yeasts. The fun part: I isolated the yeast strain(s) from the Roeselare blend with this plate. But as already mentioned, Wyeast states, that there are two different yeast strains in the blend: one Belgian Ale and a Sherry strain. So I can’t say whether strain this is or if it is a blend of both of them. Something is a bit unexpected here. The yeasts do not aggregate as top fermenting yeasts do in general. And a Belgian yeast should be a top fermenting one. Maybe just a coincidence. I could not find any picture of a sherry strain. Maybe this is the sherry strain? How could I find out whether this is a Belgian yeast or a sherry strain? And how could I determine if this is a mixture of both? Well, scientists need questions to find an answer. And I got myself a few new ones…

Lets move on to the other colonies. I could observe the following colonies (Fig 4):

Fig 4: White colony of Roeselare blend

Off-white color, even, circular, not glossy, 2- 3 mm diameter.

The morphology is very similar to the one in the first plate. The colonies seemed to be flat, even and circular this time. These colonies look quite different compared to the yeasts in Fig 2.

Nevertheless, I took a sample for a microscopy analysis to get further information about the colonies.

. Microscopy picture Roeselare blend white colony (Take 1)

Fig 5: Microscopy picture Roeselare blend white colony (Take 1)

Lets go step by step. The cells (Fig 5) have the same size as the yeasts above (Fig 3). So these could be yeasts again. The white colonies (Fig 5) are definitely no normal yeast strains as the off-white colony in Fig 3. But what do I got myself here? The only other, different yeasts in the blend are the sherry strain and Brettanomyces.

Brettanomyces Project has some cool pictures of Brettanomyces available to compare with. And my cells look very similar to Brettanomyces. What kind is difficult to say. The only way to be sure about this is to have a look at pure Brettanomyces cultures myself and compare them. I already ordered some Brettanomyces cultures for some experimenting and I will definitely take some pictures of the different strains. And I end this post with another picture of the same cells:

Microscopy picture Roeselare blend white colony (Take 2)

Microscopy picture Roeselare blend white colony (Take 2)

The results are very cool in my understanding. I might have isolated some Brettanomyces out of a bunch of bugs, right? Another cool thing are the plates with the dregs from the first post. They are still incubating and they look very different now. Will post about these plates in another post soon. Could be the sixth post in my pentade…

The end of the pentade about agar plates is near. It ends with the fifth post about cultivating kombucha bugs on Sabouraud plates. I might publish it tomorrow night. But don’t rely on it: I can pick up my case of Westvleteren 12 tomorrow evening…

#3 Agar plates (#1056, #3787)

Eureka, this is the third post in the pentade about some agar plates. The other two posts about dregs and Lactobacillus can be found here. Today, some pictures of brewers yeast Saccaromyces cerevisiae. I plated Wyeast’s #1056 American Ale and #3787 Trappist High Gravity yeasts a while ago on Sabouraud agar and now its time to have a look at it.

#1056 American Ale

#1056 American Ale yeast on Sabouraud agar after three days of incubation.

Could observe just one sort of colonies after three days of incubation: Off-white color, even, circular, glossy, convex, 2 mm diameter. Typical yeast morphology.

Microscopy picture #1056 American Ale yeast colony

And the microscopy observation confirms that the colonies are indeed yeast cells. This is a very typical picture of the slightly oval yeast cells. But let me just add some further information that can be derived from the picture. Not only is it possible to identify these cells as yeasts, but it is further possible to identify this particular yeast strain to be top fermenting. Top fermenting yeasts tend to stick together, bottom fermenting ones don’t (or less). Unfortunately, I do not have a picture of bottom fermenting yeast cells available right now to show the difference.

#3787 Trappist High Gravity from 2010

I have a sample of this yeast strain in my yeast library since 2010 and I originally isolated this strain from an agar plate. As I was not sure if the yeast was really pure (had no microscope yet), I plated it and hoped to get an answer.

#3787 Trappist High Gravity yeast on Sabouraud agar after three days of incubation.

And there were just one sort of colonies, so far so good: Off-white color, serrated, irregular, glossy, flat, 2 mm diameter. Some look like snow flakes. Well, this is a bit strange. I expected the colonies to look like those in the picture of #3787 below. The colonies look very similar to those from the 3 Fonteinen dregs.

And the microscopy analysis identified the colonies to be bacteria not yeasts. I have no picture of the microscopy sample as it is really difficult to get a good picture due to the really small size of bacteria. This means that my #3787 from 2010 was indeed not pure.

#3787 Trappist High Gravity from 2011

I brewed a batch of Belgian style Ale in 2011 and as I was not really sure about the quality of the yeast strain in the yeast library (see above), I got myself a fresh culture of #3787 Trappist High Gravity to be sure.

#3787 Trappist High Gravity yeast on Sabouraud agar after three days of incubation.

Just one sort of colonies on the agar: Off-white color, even, circular, glossy, convex, 2 mm diameter. Typical yeast morphology again.

Microscopy picture #3787 Trappist High Gravity yeast colony

And again the typical yeast cells in the microscopy sample. I observed something interesting as well. Look at the colony in the middle of the left side, the one which looks like a club. I already observed a cell like this before in the BFM dregs analysis a while ago. See Fig5 in this thread. This would mean that there could be some yeasts in the dregs as well. And this is of no surprise to me as I took the dregs out of a fermenter and there could certainly be yeast cells in the bottom of the fermenter.

So far so good. Because I had some cool results in the past two posts and not in this one yet, I kind of tried to shoot a cool picture of yeasts. I already mentioned, my microscope and camera are not the best ones. But they do their job. One thing that is really difficult is to look at the samples with the highest magnification of 2000x using the immersion oil objective. And to take a photo of such a magnification is even harder. But I did my best to get the following picture.

Microscopy picture #3787 Trappist High Gravity yeast colony

I think it is a quite good picture. And you can even see the club-formed cell as well. I like this picture very much.

Well, that’s all about Saccaromyces cerevisiae so far. I hope some of you enjoyed the pictures as I did and maybe learned something new. If someone noticed that I did not mentioned anything about the Heidelberger Kellerhefe, although mentioned in the first post, well, the yeast seems to have finally given up on me. I could not observe any colonies, even after further incubation…

The next post about agar will be about Wyeast’s #3763 Roeselare Blend culture(s). And there will be some cool results, I promise. And I just decided to post some pictures of top and bottom fermenting yeasts in the future as well to show the differences. Feel free to comment below anytime. I really appreciate it.