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Friday, 27 November 2015

Easy Home Yeast Banking - and a Video!

Wow, two videos in as many weeks - that has to be a new production record for me! This time around the video is on my most requested topic - an easy to implement home yeast banking system. The video outlines a method, based on the use of slants, to store yeast for future use. Managed carefully this method will allow the average home brewer to easily maintain stocks of up to 2 dozen strains with minimal cost or effort.

Because of the length of the video, and the presence of multiple separate methods, I have provided written instructions, below, to complement the video.



Overview

Storing yeast on agar slants is an easy and inexpensive way to maintain a yeast bank. Practically, at home, you can maintain 1-2 dozen strains this way; more than that can become laborious to manage. It is important to perform all steps in an appropriate work environment, and using the best aseptic techniques you can manage. If you are not experienced with these methods, I'd recommend you watch the relevant videos in my Your Home Yeast Lab Made Easy video series

Preparing Slants


Equipment:
  • 16 mm x 100 mm (or similar size) screw-cap glass culture tubes
  • Suitable growth media such as 1.005 gravity wort + 2% agar
  • Measuring cup
  • Small funnel (optional)
  • Pressure cooker
  • Cookie sheet
  • "Wedge"
Procedure:
  1. Pre-fill the pressure cooker to half the depth of the tubes and begin heating on the stove.
  2. While the pressure cooker begins to heat, dissolve the agar into the media by microwaving in short (20-30 second) bursts. Stir the media between bursts. It is not necessary to boil the media for any length of time; stop heating once the agar is completely dissolved.
  3. Fill the culture tubes half-full with the hot media, cap loosely, and place in a tube rack
  4. Once all tubes are filled, place the tube rack & tubes into the now-hot pressure cooker.
  5. Seal the pressure cooker and turn up the heat. Steam at maximum pressure for 15 minutes.
  6. Let the pressure cooker cool, while still sealed, until it is below 80C (generally 30-40 minutes). Remove tubes & tighten the caps.
  7. Place the tubes on a cookie sheet wedged 10-15 degrees above horizontal, with the cap-side "uphill".
  8. Let the media solidify; as soon as it is solid, but while still warm, invert the tubes into your tube rack. Let cool in the fridge over night.
  9. The next morning, working near a flame, remove the caps and flick out the condensation which has collected overnight. Re-seal the tubes and keep refrigerated until needed.

Inoculating & Storing Yeast on a Slant

Equipment:
  • Slants, prepared above
  • A source of yeast (tube of yeast, bottle sediment, top- or bottom-cropped yeast, etc)
  • Bacteriological loop
  • Alcohol lamp or Bunsen burner
  • Vinyl (electrical) tape or parafilm
  • Optional: Distilled water or pharmaceutical-grade mineral oil, sterilized using your pressure cooker.
Procedure:
  1. Flame your loop and the opening of your yeast-source container.
  2. Cool the loop on the inside of the yeast source container (not on the yeast itself), then grab a loopful of yeast.
  3. Open the slant, flame the opening (but not your loop!), and starting at the bottom of the slant, wipe the yeast off of the loop onto the slant media, using a zig-zag pattern to cover as much of the slant surface as possible.
  4. Re-flame the slant tube opening and cap, then close the tube loosely (so that gas can escape) with the cap.
  5. Let sit in a dark, warm location (20C/68F to 28C/82F) for 24 to 72 hours, or until large (3-6 mm diameter) colonies have formed.
  6. Optional: Once growth is complete, open and flame the opening to the yeast slant, and open & flame the opening of the sterile mineral oil (or distilled water). Fill the slant with the mineral oil (or distilled water), and recap the slant tightly.
  7. Tightly seal the cap with vinyl (electrical) tape, or with parafilm.
  8. Store in a refrigerator between 3C and 6C (37-42 F).
Notes:
  • Yeast stored in a tape-sealed slant should be stable for 8 - 24 months. Reculturing is recommended every 6 months if storing yeast in this manner.
  • Yeast stored under sterile distilled water should be stable for 18 to 24 months. Reculturing is recommended every 12 months if storing yeast in this manner.
  • Yeast stored under sterile mineral oil should be stable for 3 to 30+ years. Reculturing is recommended every 18 to 24 months if storing yeast in this manner.

Reculturing Yeast

Equipment:
  • Slant(s) in need of reculturing
  • Fresh slants
  • Bacteriological loop
  • Alcohol lamp or Bunsen burner
  • Vinyl (electrical) tape or parafilm
  • Optional: Distilled water or pharmaceutical-grade mineral oil, sterilized using your pressure cooker.
Procedure:
  1. Flame your loop and the opening of the old yeast slant.
  2. Cool your loop by dipping it into the mineral oil (or water) filling the old yeast slant. If water or oil was not used, cool the loop on the inside glass surface of the tube (not on the agar).
  3. Insert the loop to the bottom of the old slant and drag it outwards, across the slant surface. You want to grab yeast from as many colonies as possible.
  4. Open & flame the new slant. Insert the loop to the bottom of the slant and inoculate as per step 3 of "Inoculating & Storing Yeast on a Slant", above.
  5. Grow, seal and store the new slant as per steps 4-8 of "Inoculating & Storing Yeast on a Slant", above.

Beginning a Starter from a Slant

Equipment:
  • Slant containing the desired yeast
  • Bacteriological loop
  • Alcohol lamp or Bunsen burner
  • Tube containing 5 to 10 mL of sterile 1.040 wort
Procedure:
  1. Flame your loop and the opening of the yeast slant.
  2. Cool your loop by dipping it into the mineral oil (or water) filling the old yeast slant. If water or oil was not used, cool the loop on the inside glass surface of the tube (not on the agar).
  3. Grab several colonies with your loop, but leave enough behind for future use of the yeast and reculturing. Flame the lip of the slant and seal with the tube cap.
  4. Open and flame the tube of wort (but not the loop).
  5. Vigorously swirl the loop in the wort; remove and flame the loop.
  6. Flame the lip of the container of wort and the cap; recap tightly.
  7. Shake the tube of wort vigorously to oxygenate, then loosen the cap and place in a warm location  (20C/68F to 28C/82F). 
  8. Re-seal the slant with vinyl tape or parafilm, and return to the fridge for future use.
  9. A minimum of two times each day tighten the lid on the tube of wort and shake vigorously; then loosen the cap and return to the warm location.
In 2 to 4 days the yeast should grow, providing you with a tube of yeast at ~10 million cells/mL. A 5 to 10 mL starter tube can be used to pitch a 250 mL (1 cup) to 500 mL starter, providing ~35 or 75 billion cells. This can then be stepped upto pitchable amounts following conventional starter calculators.


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48 comments:

  1. Unknown28 November 2015 at 22:41

    Do these slants work good for both Saccharomyces and Brettanomyces? Great video, B!

    ReplyDelete
    Replies
    1. Bryan30 November 2015 at 08:21

      Yep, and lacto and pedio and pretty much any other microorganism that'll grow on wort.

      Delete
  2. test blogger1 December 2015 at 14:55

    Thank you for sharing. You are truly a gentleman and a scholar.

    ReplyDelete
  3. Brad4 December 2015 at 10:54

    You and others have mentioned that some yeast strains don't keep as well as others. Do you know of any specific strains from your experience? For example I've heard wheat strains don't store well. Thank you!

    ReplyDelete
    Replies
    1. Bryan4 December 2015 at 11:31

      I cannot think of any singular strain off-hand (and my notes are not that good), but it certainty happens. To my recollection there is no real pattern either - i.e. its not like lager yeasts do better than ales, or saccs better than bretts. Some strains just don't seem to last as long as others.

      Delete
    2. Brad4 December 2015 at 12:42

      Thanks Bryan. I look forward to your Freezer Bank post/video. I work in a research lab so I do have access to some of the things you do. I'm just on the fence about dipping into it. I've got several strains under isotonic water in the fridge and honestly I've neglected them so I can't say how viable they are.

      Delete
    3. Bryan4 December 2015 at 12:46

      If you have access to a lab than I'd suggest going the freezing route; its far easier and requires less on-going maintenance than slants.

      Since that origonal freezing video I've altered my method somewhat:

      1) Grow up yeast in 5ml of 1.040 wort
      2) Pellet by centrifugation
      3) Suspend in 2 mL of 1.020 wort + 20% glycerol (autoclaved)
      4) Divide between two 1.5 mL cryogenic tubes

      One of those tubes goes into a -80C freezer for long term storage; the second into a -20C freezer. I make up yeast for pitching from the -20C stock, and use the -80C stock to replenish the -20C stock (either when it runs out, or viability drops too low). This keeps generation numbers low, and ensures that a failiure in either freezer down't result in a loss of yeast.

      Delete
  4. Anonymous7 December 2015 at 13:05

    Hello.
    First of all thank you for great article.
    I tried you method at home. And all my colonies on slants washed away after adding water. I ended with no colonies on agar, and all my yeast was dissolved in water. I tried both PDA and wort agar slants with same results. Any suggestions why it happened? Thank you.

    ReplyDelete
    Replies
    1. Bryan7 December 2015 at 13:09

      Are you sure the washed away? Due to the refractive index of the water, it is often hard to see the colonies once the tubes are filled. The colonies are pretty strongly bound to each other, and the agar, and should stay put (assuming you are being gentle with filling, etc).

      You can see this effect at 13:25 in my video; the colonies are still there, but are much harder to see.

      Delete
    2. Anonymous7 December 2015 at 13:22

      Yep. I gently pull out water from the tube with pipet and can't see any colonies. Maybe should I add more agar when preparing my slants?

      Delete
    3. Anonymous8 December 2015 at 04:50

      This comment has been removed by the author.

      Delete
    4. Anonymous8 December 2015 at 04:55

      Here photos of one of my slants after 12 hours in fridge: https://imgur.com/a/4t7d6. You can clearly see all yeast dropped down and no colonies on agar.

      I understand you can't remotely say what wrong with my process, but maybe you can give me direction. Like add more agar, or maybe try another yeast satin (this one is WLP800), or maybe something else to try.

      Thanks.

      Delete
    5. Bryan8 December 2015 at 09:28

      Is the white stuff at the bottom the yeast?

      Delete
    6. Anonymous8 December 2015 at 10:30

      Yes.

      Delete
    7. Bryan8 December 2015 at 11:33

      What percentage of agar are you using in your gels? They may be too hard, thus preventing proper adhesion by the yeast. Your gels should be in the 1.5 - 2% agar (weight/volume) range - i.e. 1.5g to 2g per 100 mL of medium.

      Delete
  5. Anonymous9 December 2015 at 04:27

    I used 2% agar. Will try to use 1.5% on this weekend. Thanks.

    ReplyDelete
  6. pinyhost18 February 2016 at 16:51

    Great videos! A question popped up while watching this last one though. Why is a pressure cooker needed when creating slants, but simple 5 minutes boil is sufficient for preparing medium for plates? Thanks!

    ReplyDelete
    Replies
    1. Bryan19 February 2016 at 09:08

      Ideally you want to use a pressure cooker in both cases, as that is the only way to guarantee sterility. If boiling the advantage of boiling for more than 5 min is negligable - anything that survives boiling for 5 min will likely survive 10, 15 or 20 min of boiling as well.

      In theory you can simply boil slants as well, but its not the best of ideas as you run the risk of contaminating your stored yeast. In contrast, even if a plate has some contamination you can pick clean colonies off of it.

      In other words, if you have a pressure cooker, use it for both. If you don't you can prepare both by boiling for 5 minutes; but you run a higher risk of loosing your cultures as a result.

      Delete
    2. pinyhost19 February 2016 at 14:19

      Thank you. That makes sense.

      Delete
  7. pinyhost27 February 2016 at 12:33

    Hi Bryan! One more question popped up and I wasn't able to find the answer online. I ordered some vials, but the caps they came with are foam lined (I believe it's F217). Will I be able to boil those or there is no way they can be sterilized? Thanks!

    ReplyDelete
    Replies
    1. Bryan29 February 2016 at 09:33

      I don't know; try one and find out! If it doesn't work you can always pull the foams out and use the tubes without it

      Delete
  8. pinyhost27 February 2016 at 12:43

    This comment has been removed by the author.

    ReplyDelete
  9. bytemyfoot15 March 2016 at 21:04

    I can't seem to find mineral oil that doesn't have tocopherols added. Is that an issue?

    ReplyDelete
    Replies
    1. Bryan16 March 2016 at 10:25

      Probably not - in fact, adding vitamin E family compounds (i.e. tocopherols) is done in some storage media as an antioxident.

      Delete
  10. Anonymous15 July 2016 at 15:34

    You are a good teacher!
    I think I have seen all of your videos; I am working with yeast so I am familiar with those basic techniques, but I wish to do the same work with lactobacillus. Is it correct that you have not addressed lacto directly in your videos? If I have overlooked something I am sorry but I feel
    like I am flying blind. Could you point out where the procedures might differ? To give you an idea of what I mean, here are some of the questions I have: Yeast are microscopic, but they grow into very visable colonies... will bacteria like lacto also? When I culture lacto on to a slant, will I know that it is there and that it is ready to be stored, simply by looking at it? In what way will it differ from a yeast slant or plate?

    From reading various source material, I gather MRS is the best media, followed perhaps by a wort/apple juice nutrient +/- a buffer + agar media. Time and Temperature to be determined by specfic species used... So just adapting the previously learned procedures should get where I want to go?
    Again, are there differences to be aware of?

    Generally speaking, wouldn't stabs be better for storage of lacto than slants?

    Would the culturing up procedure be the same... eg same quanty of innoculant, same step-up volumes, but with perhaps different temps?

    I might be able to find more questions somewhere in my head but this should give you an idea of what I need to know. In the short run, I want to use a lacto culture to brew a Berliner Weisse, but I want to bank the pure culture while I have it in its clean state, before I pitch the majority of it into the wort, and don't want blunder it. Thanks for being a teacher!

    ReplyDelete
    Replies
    1. Bryan17 July 2016 at 14:28

      I have not covered lacto, although most of the methods I described work equally well for bacteria as they do for yeast. Lacto will form visible colonies on plates and slants, although they may take longer to become visible (in my hands, at room temp, most lacto take about a week to form visible colonies).

      For growing lacto, potato-dextrose-agar or wort-agar is fine; I use both with good success. MRS is idea, but expensive. Stabs or slants for storage doesn't matter; lacto is non-motile, so stabs may be difficult to recover bacteria from.

      I have a few posts on culturing lacto (search my blog). Smaller volumes can be used, as you get more cells/volume, and you need smaller pitch rates. Lacto culturing is best done at 35-45C (exact temp is strain dependent), as lacto is most active at these temps.

      Delete
  11. Øystein24 July 2016 at 17:54

    Hi,

    I'm about to start banking yeast, inspired by your excellent articles.
    I'm planning to store using sterile water or mineral oil as I gain more experience.
    But one question: Is using Potato-dextrose-Agar as good as wort-agar?

    Also, do you have any experiences in storing Brett on slants?
    From Milk The Funk there seems to be a lot of info that storing refrigerated isn't very good, but I don't know if that applies to storage with mineral oil, or alternatively if storing on slants in room temperature is better?

    ReplyDelete
    Replies
    1. Bryan25 July 2016 at 08:22

      I've not tried long-term slants with PDA, but given how well yeast grow on it, I'd suspect it is as good as wort-agar for storage.

      I freeze all my stored yeasts, and all of them do fine frozen. I've not tried long-term storage of brett on slants.

      Delete
    2. Øystein25 July 2016 at 13:57

      Thanks for the reply.
      I'll try some with PDA and some with wort agar then, to be sure. I was planning on making one 1. gen and use that to "seed" second gen slants that ibessnplanning to use for building my starter when needed, but then I can rather have one PDA and one wort agar first gen as backup.

      Thanks a lot for the inspiration to start this.

      Delete
    3. Bryan3 August 2016 at 13:51

      Good luck, and let us know how well it works!

      Delete
  12. Øystein30 September 2016 at 08:22

    Hi,
    I was about to start my next step into my capturing and banking adventures.
    I have several captures now, from fruit, berries and flowers, and are going through some miniature test-fermetations to check for attunation, smell and avatually taste.
    I've also been able to create plates from what I've learnt from your videos, and have now streaked the first of these captures out on a couple of plates. I have quite a way to go before I'm good at this, but at least my second try looked a lot better than my first.

    BUT:
    At the same time as I created the first two plates I also tried creating some slants that I'm planning to use for banking isolated yeast on in the future.
    When I created them they looked reasonably good. Not a perfect fill, but usable as far as I could see.
    I've stored them in the fridge since I created them 2-3 weeks ago, but when I looked at them today most, if not all, of the content in every tube had liquified again. I'm 100% sure that it had solidified into a "jelly" before I put them in the fridge, and all the plates I created at the same time seems to still be solid, or at least not liquid. The plates are stored upside down in the fridge right next to the slants, so temperature should be pretty similar if that matters.
    Have you ever experienced this and/or have a tip on what I should try to change?
    Could it be that I'm just on the edge of the correct amount of agar, and that adding more would help?

    ReplyDelete
    Replies
    1. Bryan30 September 2016 at 08:50

      I've never had agar re-liquefy in the fridge; if anything it tends to dry out and crack. Are you storing the slants under water or mineral oil? How much agar did you use?

      Delete
    2. Øystein30 September 2016 at 13:21

      Hi,
      This is before I've grown yeast on the slants, I've just created them for future use. So for now it's only the wort agar there. I have a few test tubes with pressure cooked mineral oil as well, but was planning to add that first when I had yeast on the slant.
      Unless I did something wrong I used 2% (6g in 300ml of wort).

      Delete
    3. Bryan30 September 2016 at 14:12

      Sounds like you are using the right amount of agar. What is the gravity of the wort you are using? Anything above 1.020 may cause issues with the agar - I typically use 1.006 to 1.010.

      Delete
    4. Øystein30 September 2016 at 15:30

      If I remember correctly I used 3g per 100ml, as that was the average of your receipe.

      But, since it looks like I haven't done any obvious mistakes I will just give it another try. Probably some with wort and some with PDA just to get two versions.
      Thanks for your feedback :-)

      Delete
  13. FerventBrewer30 September 2016 at 08:59

    I know it's best to get your inoculation from a pure source like a new pitch from a reliable lab. What about bottle dregs. Is that just a waste of time and effort?

    ReplyDelete
    Replies
    1. Bryan30 September 2016 at 09:02

      Dregs are great, but you need to approach them with care. Some breweries will filter out the yeast for fermentation and use a special bottling yeast. Likewise, bottle dregs need to be run through a starter prior to use, and you need to be careful to avoid contamination.

      Many breweries also use yeasts available from wyeast/white labs, so IMO its often not worth it unless the brewery uses a truly unique yeast.

      About 1/3rd of the yeast I have banked are bottle dregs, and they make great beer.

      Delete
    2. FerventBrewer14 October 2016 at 08:27

      I made my first slant on Tuesday of this week. Are you supposed to use any special streak pattern on a slant (seems impossible to do so but...)? I'm not sure I ended up with any single colonies

      Here are a couple of poor photos of the results.

      https://goo.gl/photos/1ZWxRVTbv74YMERUA
      https://goo.gl/photos/HU3VBFncNNVNFUmD7

      I also made extra agar slants I want to use whenever I brew with different yeasts, will they last in the refrigerator indefinitely? I used the pressure cooking method from your videos to create and sanitize them along with mineral oil and starter wort.

      Thanks so much for the information on this blog!

      Delete
    3. Bryan14 October 2016 at 08:42

      Your slants look good!

      There is no specific streak patter to use - usually you try to start as deep as possible in the tube, and streak side-to-side while moving upwards. The goal is to cover as much of the surface of the slant as possible - which you seem to have achieved.

      If your slants are sterile, and you seal the caps with vinyl (electrical) tape, they should last in the fridge for at least a year.

      Delete
  14. Eric Smith30 October 2016 at 20:56

    I attempted my first try at making several yeast slants last week. I ran into a issue with the slants not solidifying, could it be the type of agar is used (malt extract agar(MEA))? I ordered some regular agar online just in case this is the issue. I followed your video step-by-step which is a great video by the way! My problem could be I did not mix the water, dme and mea thoroughly enough? Thanks

    ReplyDelete
    Replies
    1. Bryan31 October 2016 at 08:08

      You want to use plain agar; if you used MEA + DME the agar probably did not solidify as there was too much sugar. MEA is essentially a commercial version of what I make here, so if you've bought that you should be able to use it without additional DME. Just make it up as per the manufactures' instructions

      Delete
  15. Unknown31 October 2016 at 10:01

    Ok thanks, I'll give it a shot!

    ReplyDelete
  16. Unknown11 January 2017 at 14:10

    I am trying to identify different yeasts from a can of my favorite beer. I see the video of what to avoid but I was wondering if the video of identification is up. Thanks

    ReplyDelete
    Replies
    1. Bryan11 January 2017 at 14:42

      Unfortuantly, it is very difficult to identify what specie(s) of yeast/bugs you have in your beer using a home lab. This type of identification usually involves sequencing a portion of the organisms DNA, which requires proper lab facilities and a fair bit of money.

      I've done a few blog posts on these methods, if you're interested:
      http://suigenerisbrewing.blogspot.ca/2013/04/identifying-yeasts-using-ribosomal.html

      http://suigenerisbrewing.blogspot.ca/2013/05/yeast-identification-test.html

      http://suigenerisbrewing.blogspot.ca/2013/05/identifying-wild-yeast-part-deux.html

      Delete
  17. Tom10 April 2017 at 14:29

    Maybe I did something wrong, when I went in to add distilled water to my slants a lot of the yeast was pulled off the agar and into the water. Did I screw it up or will it be fine??

    ReplyDelete
    Replies
    1. Bryan13 April 2017 at 09:53

      A few people have reported this problem; I do not know why it is occurring. The yeast will settle to the bottom of the tube and can be recovered from there.

      People using mineral oil have not reported this issue.

      Delete
  18. Sneakygosch7 July 2017 at 05:58

    Hi Bryan. Thanks for the great articles and videos.I did my first agar slants after watching your videos. I have one question. How many times would you say you could scrape off a colony from your slants to propagate before you discard the vial?

    ReplyDelete
    Replies
    1. Bryan14 July 2017 at 07:54

      So long as their is yeast, and it is viable, you can keep reusing the vial. The biggest concern with re-use is the risk of contamination; be sure to use proper aspectic techniques to avoid contamination and you should be fine. I indicate the "average" lifespan of a slant in the video, I'd recommend following those guidelines in terms of timing your re-propagation onto new slants.

      Delete

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