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!

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34 thoughts on “Yeast banking – #3 Isotonic sodium chloride

  1. Another great post Sam.

    The process is very well defined and easy to follow, even for the non-scientific. I think you were right, if I’m ever going to venture into yeast banking, this looks like the method I would take. It might be a few years in the future (with the young family and such), but hopefully I’ll get to give this a try… seems straight forward enough and worthwhile.

    • Darryl, thank you very much for your comment. Really appreciate your feedback. In addition, I am glad you could follow the process. Yeast banking is fun. Let me know how it works for you. Cheers Sam

  2. Pingback: PSA: Monitor your Yeast Bank | DC Yeast Lab

  3. I am wondering if it might help to include 20-30% glycerol in the salt solution so that the cultures can be stored at -20 C (or even -80) for longer than 2 years. In other words, why bother with the YPD media in a glycerol stock? I would think this would work nicely for banking yeast directly from smack packs and vials (e.g. Wyeast and White Labs strains).

    • Hi John, I cannot tell if it works without YPD since I do not have any experience here. I stick to the protocol storing yeasts in YPD, ascorbic acid and glycerol. Simply because it works and I do not want to change a working protocol. Cheers, Sam

  4. I’m considering finally getting into yeast banking after nearly 15 years of brewing! This method looks the easiest of all option you reviewed. Would you still recommend doing a few slants or plates along with the isotonic solution vials when banking a particular strain? Also I’ve read you can use plain distilled water instead of a salt solution. Which is preferable? Thanks!

    • Hi Walter, I don’t have a lot of long term experiences with the isotonic sodium chloride method because I switched to a frozen yeast bank couple of years ago. I never encountered problems reviving strains from isotonic solutions though. If you are worried, store your yeasts in slants as well. To be absolutely sure.

      Concerning distilled water. I never tried distilled water myself and cannot tell if it works better/equal/worse than salt solutions. From a theoretical standpoint, I would go with salt solutions as these seem to mimic the natural osmotic conditions.

      Happy yeast banking

  5. And Sam thanks a lot you have saved me almost hundreds of dollars already. In India we do not have an easy access for yeasts. I have brewed nearly eight batches of beer using yeast from NaCl solutions.

  6. Sam, thanks for the great articles. Have you measured the viability of some of your older samples? Looking at another of your posts on yeast viability, the graphs all look to be fairly linear and tending towards zero viability after a few months. You obviously have better results than this, since your isotonic saline samples are lasting for a year or two.
    Also, do you know if the yeast population density has any effect on the long term viability? I am planning to use the isotonic saline method with larger populations by diluting a new smack-pack to 500mL, and then storing five 100mL samples.
    Cheers, Rob.

    • Hi Rob, haven’t measured viability in a while and actually do not use the isotonic NaCl method any more due to lack of space. Should make another post about that reviving all my left over NaCl samples though. Concerning the linear models you mention, I guess at some point, the function gets asymptotic. Meaning there are still some yeast cells alive even at pretty low levels. Do not have empirical evidence for this assumption though.

      Yeast population density and storage. I actually don’t know if there is any effect of density on viability. And I do not dare to make any guesses here. Never stored big volumes of yeast before myself.
      Thanks for reading and cheers, Sam

  7. Hi Sam
    Would it be possible to use yeast from the bottom of the fermentor (after washing them with cooled hot water) and then saving it in bulk in a larger container with increased isotonic sodium chloride ?

  8. Hi Sam,
    and thanks for a very inspiring post (and blog)!

    Just a couple of newbie thoughts and questionmarks:
    When reanimating the “saline yeast” you mention 1 mL in a 100 mL starter; is the starter volume correlated to the live yeast concentration in the bank, or rather to the number of cells in the 1 mL?
    My thought is that depending on harvest method/source the bank concentration can vary quite a lot. Even when harvesting from a commersial smack-pack or vial my impression is that the cell count can vary with at least a factor of 4-5 depending on the age.
    Or is the way to think that reanimating in 100 mL of 10°P wort results in [a certain number] of happy yeast cells and that the initial number of cells only affects the time it takes to reach that count?
    Would 2 mL in 200mL result in twice as many cells (in the same amount of time)? I assume there are scaling factors to not make it so…

    On a correlated thought, would it be feasable to put a higher ratio of harvested yeast in a higher concentration saline solution to end up with a higher concentration of yeast in the isotonic solution?
    For instance a 1:1 mix of yeast slurry and 1,8% saline, 3:1 in 3,6%, or even 9:1 in 9%?
    Or would some of the yeast cells have a high risk of dying due to a “saline shock”?

    I hope these questions aren’t too stupid, I’ve read your posts and Googled around but haven’t been able to get a grip on this.

    • Hi Daniel,
      thank you very much for reading and commenting.
      > When reanimating the “saline yeast” you mention 1 mL in a 100 mL starter; is the starter volume correlated to the live yeast concentration in the bank, or rather to the number of cells in the 1 mL?
      Number of cells in 1 mL which however correlates to the amount of living cells. The crucial point here is to consider ratio. Not to start a 2 L starter with the 1 mL yeast sample.
      > My thought is that depending on harvest method/source the bank concentration can vary quite a lot. Even when harvesting from a commersial smack-pack or vial my impression is that the cell count can vary with at least a factor of 4-5 depending on the age.
      Absolutely true.
      > Or is the way to think that reanimating in 100 mL of 10°P wort results in [a certain number] of happy yeast cells and that the initial number of cells only affects the time it takes to reach that count?
      That’s how I like to think about it.
      > Would 2 mL in 200mL result in twice as many cells (in the same amount of time)? I assume there are scaling factors to not make it so…
      Yes. But not because of the 2 mL but because of the 200 mL starter. Because, if you would start with 1 mL it only takes one duplication to get the same amount as you would have with 2 mL. The amount of yeast depends on the amount of sugar and other molecules. Not the initial pitch. I think about it this way: The amount of yeast you get within a certain volume of starter is constant. And therefore independent of the initial amount you start with. The only thing that makes a difference is the time to get there. Pitch less, takes more time. And vice versa.
      > On a correlated thought, would it be feasable to put a higher ratio of harvested yeast in a higher concentration saline solution to end up with a higher concentration of yeast in the isotonic solution? For instance a 1:1 mix of yeast slurry and 1,8% saline, 3:1 in 3,6%, or even 9:1 in 9%?
      That should all work. Crucial is the 0.9% salt concentration in the end. I would keep that a constant.
      > Or would some of the yeast cells have a high risk of dying due to a “saline shock”?
      This might indeed be a problem. Especially for a mix with a 9% NaCl solution. The easiest solution here would be to let the yeast sediment, discard the supernatant and then mix with 0.9% NaCl solution. Or use a centrifuge if you have one. By doing this, you concentrate the cells as well and would not risk a saline shock.

      Feel free to ask any further questions. I see from your questions that you are on the right track.

      • Thanks for that Sam, and for helping me to be on the right track.

        So I have some tests to perform; just received a nice Austrailian stirplate, sterile syringes and trying to find a pressure cooker that will reach 1bar/121°C…

        However, I’m still wondering what your experience is on the actual numbers of the initial pitch ratio.
        If you are harvesting from a banked yeast cake with some ~1.4e9 cells/mL (source Wyeast), then dilluted to 20% of that in the saline solution, the growth ratio for 1 mL in 100 mL10°P would be around 16e9/0.3e9 ~= 55x. (Braukaiser / Brewersfriend)
        Harvesting a 4 month old smackpack you would perhaps end up with a ratio of 16e9/24e6 ~= 650x (Brewersfriend). But looking at fig 4 it seems you have much more dilluted solutions than that…
        Do you have any feedback on this? Raines-Casselman mention 200x, but that seems to be for bacterial overtake reasons,is not for saline yeast and again your fig 4…
        What is in your experience the danger with a too high ratio? You mention an infection, but I don’t see how that could have been correlated to the ratio?
        I did a quick and dirty test a couple of days ago when I got my stirplate and made a 250mL starter from approx 15..20e6 cells (est. by Wyeast method) from a two week old starter (not saline), resulting in an approx. 2000x growth factor. Grew well, smells fine but I don’t really know how to evaluate it… Nevertheless, the point is that the very high dillution was not in it self the limiting factor here.

        As you see, I’m tring to find data in order to have a sound process of using your isotonic yeast bank method for my homebrewing. Since my previous comment here I’ve done some homework and have collected figures for the later steps in the fermentation process.
        The main idea is not to push the limits but to know where/why they are and to have a reasonable margin to them.

        On that note, do you know why / how your bank was infected?

        My plan is to have one work + one backup 20mL tube of harvested yeast (is that what you call Gen0?)
        Then grow a tube of Gen1work from the Gen0work, which will be used for making starters, perhaps 10 of them before making a new Gen1work from the Gen0work (if making more than 10 starters in two years…).
        What to do after two years I don’t know, maybe just let the bank step up from Gen0bu to Gen1bu + Gen1work?

        Still confused, but on a higher(?) level
        /Daniel

        • Hi Daniel,
          to make it easier to read my answers, just shoot your questions to contacteurekabrewing@gmail.com and I can answer your questions directly.

          > If you are harvesting from a banked yeast cake with some ~1.4e9 cells/mL (source Wyeast), then dilluted to 20% of that in the saline solution, the growth ratio
          > for 1 mL in 100 mL10°P would be around 16e9/0.3e9 ~= 55x. (Braukaiser / Brewersfriend)
          > Harvesting a 4 month old smackpack you would perhaps end up with a ratio of 16e9/24e6 ~= 650x (Brewersfriend). But looking at fig 4 it seems you have much more
          > dilluted solutions than that…

          Just a quick information about the yeast calculators and small amounts first. As far as I know, most of the current yeast calculators interpolate the growth patterns for smaller initial pitches (like the 0.3E9 cells you mention above) and might not rightfully describe the growth patterns there. I would therefore be careful with the numbers you get from the calculators. Furthermore, don’t let you distract from the big doubling numbers. As mentioned in my last reply, the amount of yeast you get (independent of the initial pitch) depends on the amount of sugars you have in your starter. Easy to check by playing around with the yeast calculators. And for the doubling numbers, imagine the doubling number if you would start with one single cell…

          Unfortunately I cannot check the cell counts in my NaCl vials since I don’t use this technique anymore. But you might be right that I used a smaller amount of yeast than the amount mentioned in my post. Looks like the vials contain less yeast than described. I actually don’t care about the amount of banked yeast that much as I always get more or less the same amount after a 100 mL starter. Only variable here is the starter time when starting with a lower initial pitch. In case you want to standardize your yeast bank and starters, better bank the same amount of yeast for each strain which would give you more control about the necessary starter times (due to the same initial pitches; leaving the viability aside here).

          > Do you have any feedback on this? Raines-Casselman mention 200x, but that seems to be for bacterial overtake reasons,is not for saline yeast and again your fig 4…

          200x in what context (pitching rates)? I am not a big fan of doubling numbers (as well as generations) since they don’t mean anything on their own (see above). And I don’t understand the connection to bacterial contaminants.

          > What is in your experience the danger with a too high ratio? You mention an infection, but I don’t see how that could have been correlated to the ratio

          High ratio in terms of banking or starter? In case of starter, high ratio (yeast per volume) lowers the change of a contamination due to the high amount of initial yeast ratio. As for banking, I don’t see any reasons why not to bank more yeast.

          > I did a quick and dirty test a couple of days ago when I got my stirplate and made a 250mL starter from approx 15..20e6 cells (est. by Wyeast method) from a two week old starter (not saline), resulting in an approx. 2000x growth factor. Grew well, smells fine but I don’t really know how to evaluate it… Nevertheless, the point is that the very high dillution was not in it self the limiting factor here.

          How did you get the 2000x growth factor (yeast calculator)? Based on the sediment volume (& estimate the cell count)?

          > On that note, do you know why / how your bank was infected?

          Unfortunately not. But contaminations are a common business when you handle yeast. Especially doing it at home.

          > My plan is to have one work + one backup 20mL tube of harvested yeast (is that what you call Gen0?)
          > Then grow a tube of Gen1work from the Gen0work, which will be used for making starters, perhaps 10 of them before making a new Gen1work from the Gen0work (if making more than 10 starters in two years…).
          > What to do after two years I don’t know, maybe just let the bank step up from Gen0bu to Gen1bu + Gen1work?

          Gen0 would represent the cells you harvested. (generation zero). And Gen1 would represent your current working bank (with Gen0 representing your master bank & gen01 your first generation of gen0). So far for the theory. Ideally you would have multiple Gen0 stocks used to make new working stocks (Gen1). Simply because each and every handling of your Gen0 stocks might introduce contaminations or thawing/re-freezing or any temperature shifting might destroy your cells as well. This is all a bit complicated with isotonic NaCl solutions due to the space you need. This is just one reason why I switched to a frozen library with multiple aliquots of Gen0 and Gen1 tubes. Ensuring I always have a Gen0 aliquot and working with a Gen2 working bank (made from Gen1).

          I would suggest you make yourself a Gen0 and make a Gen1 out of it. Depending on the space you have either make yourself a Gen2 out of Gen 1 (which would leave the Gen0 as a backup) and work with Gen2 as your working stock. If you encounter problems or run out of Gen2, make a fresh one from Gen1. Same for Gen1, in case of problems etc you can always go back to Gen0. This would however make it necessary to have at least three vials of each strain (Gen0, 1 & 2) but with the advantage of having a double-backup system. If this is no option, better leave it at Gen1 and Gen0. And when you run out of Gen0 you either buy yourself a fresh sample or shift one generation forward.

          I hope I could answer some of your questions there.
          Sam

  9. Sam, first of all many thanks for taking the time to write great posts, plenty of useful information. You have saved many of us from wasting money and time with your detailed explanations!

    As I don’t have local access in my country to liquid yeasts and I will soon have the chance to bring some Wyeast Smack Packs from Europe, I will probably use the isotonic solution method to start a bank so I can have good strains all year round.

    The question is: would this method work fine for Wyeast Brettanomyces/Lactobacillus/Pediococcus directly from Smack Packs? I am considering pure cultures of course, not mixtures. I know you are not using this method anymore but it would be useful if you have had any experience with them or have any suggestions of the storage/reanimation method applied to this strains.

    Many thanks! Cheers!!
    Esteban

    • Hi Esteban, thank you for your lines.
      > Would this method work fine for Wyeast Brettanomyces/Lactobacillus/Pediococcus directly from Smack Packs?
      I used the NaCl method for Bretts and this worked very well. Never for bacteria though as these are a bit trickier to store. Furthermore, Wyeast’s Pediococcus is a very hardy strain to propagate in my opinion. Since I am not intentionally brewing with Pedio, I don’t have any in my current library. All the lactos I have are in my frozen bank though. Maybe try to keep the bacteria at room temperature and some kind of unhopped wort with subsequently replacing the media.

  10. Hello and thank you for this very informative post. I will be trying storing yeast in 100 ml ampules I found at the local pharmacy here in Italy. You suggest adding about 20 ml of yeast slurry to each ampule by using a sterile syringe and storing in fridge until needed. When time comes I can supposedly syringe around 1ml of yeast in suspension and use in a 100 ml starter. I have a few questions regarding this process. Will the ampule remain sterile over time even after being poked with the sterile syringes? If I use more than 1ml of yeast for the starter can I make a bigger starter? Thank you for your help with this

    • Hi Luca,
      I will give you a quick answer and post the reply on my blog later on. Just too busy to keep the blog up to date right now.

      > Will the ampule remain sterile over time even after being poked with the sterile syringes?
      They should. As long as you use a sterile needle and don’t push any non-sterile liquid into the ampule.

      > If I use more than 1ml of yeast for the starter can I make a bigger starter?
      Absolutely. Just make sure your bigger starter volume is really sterile. Best option would be to use a pressure cooker with the containment you are going to use for the propagation (flask, bottle etc). So you just have to open the containment after heat treatment and add the yeast. No liquid handling etc going on. The main problem people face with bigger starter volumes for initial yeast propagation is sterility. Bigger volumes are harder to handle and it is therefore more likely to catch contaminations. And since the yeasts take a while to get to full growth, the contaminants have enough time to establish themselves before the yeasts are in full growth mode.

  11. Hi Sam,

    Thank you for this great post.
    I read it carefully before begin my first yeast bank 10 days ago 🙂

    So, I’ve already store with sucess 3 strains, but I’ve still one question.
    I’ve filled little 15ml sterile plexiglass tubes with sterial isotonic solution (my mother is a nurse … :)), but I was not precise during my manipulations. (too big syringe)
    Some tubes has ~6, 6,5ml of isotonic solution.

    Will it decrease yeast viability over time, due to lack of osmotic presure ?
    (my aim is to sucessfully woke up yeast in 2years).

    Best Regards,

    Guillaume

      • Hi sam,
        Thanks for the reply !

        I alwyas keep my yeast (banked and yeast in White Labs package) in my fride, @6°c.
        Anyway, I’ve already begin my bank (http://bit.ly/1GUPFfn).
        I’ll know in the future if it will be a good solution.
        If not, I think to migrate to freezed yeast @-80°c, but a “hyper-freezer” is quite expensive.

        Best Regards,

        Guillaume

        • Hi Guillaume,
          I guess time will tell. As far as I know, yeast can even be stored in distilled water. And as far as I can follow your descriptions you are more likely at the lower end of sodium chloride concentration, right? Meaning lower than the expected 0.9%. If that’s the case, I would not be too worried.

          If you have access to a -80°C freezer, then take full advantage of it 🙂 Otherwise, -20°C is the only non-expensive alternative. Sure you could buy yourself a -80°C freezer. But beside the initial costs to buy one, don’t forget the running costs…
          Kind regards, Samuel

          • Hi again sam,

            Okay, thank you for advice.
            I’ll notice you on my future works, but I hope my yeast will still wake up after two years.

            Anyway, yesterday, I succes to wake up a WLP099 put in boiled water 4month ago.
            It was my first banked yeast (no isotonic solution at this time).
            And no noticeable infection (smell, visual and taste).

            Thank you again for your amazing blog…

            Guillaume

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