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!