Guide to Making a Frozen Yeast Bank

You'll be fine. The slurry will still have the glycerine mixed in for when you freeze them. Mine always settle out in the freezer.

I've had my starter going for 48 hours now (forgot about it last night). When is the best time to crash cool?

how long would these last if stored in a 35F keg fridge instead? half as long as if frozen? and what are some average storage terms you guys get in the freezer?

I crash cool them the night before brewing usually. A starter will normally finish in 24 hours.

Frozen I've been getting a year or more out of stored yeast. For the fridge you shouldn't use glycerin and instead just follow the yeast washing procedures and store in the mason jars. Look into the yeast washing thread for the storage time for those.

thanks. i can't wait to try it.

What about baby food jars? I mean the price is right and I have plenty of those.

I am sure it can stand up to autoclave temps.
So, why not save the cash for grain?

why not just save one bottle of each beer you make, and culture the yeast from that whenever you want to use that yeast strain again.

RBlagojevich said:

why not just save one bottle of each beer you make, and culture the yeast from that whenever you want to use that yeast strain again.

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While that is certainly one way to do it, if your planning ahead you can use a sanitized funnel and put a cup of yeast slurry into a beer bottle as well. I know at least one guy locally that does that.

RedOctober said:

What about baby food jars? I mean the price is right and I have plenty of those.

I am sure it can stand up to autoclave temps.
So, why not save the cash for grain?

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I'm not sure if the lid (specifically the lining in the lid) will take autoclaving. You might also use a empty clean beer bottle, they are not hard to find.

RedOctober said:

What about baby food jars? I mean the price is right and I have plenty of those.

I am sure it can stand up to autoclave temps.
So, why not save the cash for grain?

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They are a bit big for freezing yeast. You want to preserve a small sample when freezing.

But baby food jars are excellent for preserving small quantities of yeast in the fridge, however. And yes, I believe you could autoclave the lids.

RBlagojevich said:

why not just save one bottle of each beer you make, and culture the yeast from that whenever you want to use that yeast strain again.

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It can be done, but there is a very big risk of yeast mutation and/or selecting the least flocculent yeast. Also the yeast in the dregs of your bottles isn't the healthiest stuff, so it can be finicky to work with.

If you want to preserve a pure strain of yeast that you can bank and have on hand whenever you want it, freezing yeast is the method I prefer. You can also bank slants or small quantities of yeast in your fridge.

can i use Vegetable Glycerine ( used for making suger dough)?

Great article,

the only thing i wonder is
What else can I freeze like this? blood, semen?

subsribed...this is a great write up and I'll definitely give this a go!

How vigorous do you spin the stir bar? Do you keep a vortex going overnight while it ferments?

I spin it just fast enough to get a small vortex appearing. That should be all it needs.

And by doing this, what is the average time to peak fermentation, at which you make the stocks? I assume there is tremendous variability in different strains....


Thanks for the info!

gitapaynts said:

And by doing this, what is the average time to peak fermentation, at which you make the stocks? I assume there is tremendous variability in different strains....


Thanks for the info!

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Yes, lots of variability, but typically they are fermented out in 12 - 24 hours. I usually give it about 18 hours (i.e. make up the starter the evening before my afternoon brew session).

Read through the first couple pages of this thread and here are my questions on yours or an alternative process:

1) Why store a large heap of yeast in a liquid vial as opposed to streaking them to plates and storing them in a similar manner?
2) Won't streaking to plates filled with wort agar be a lower cost option?


My wife is in yeast genetics (relating to cancer biology) and stores her yeast in something along the following process:

1) Dip inocculation loop in ethanol and then flame it to sterilize
2) streak several plates from primary culture (in this case our starter) (this process can be described for folks here later if needed)
3) incubate cultures (in the case of beer yeast this will be at roughly room temp)
4) examine yeast cultures to ensure single colonies have been produced free of infection (and you will know it when looking if it is infected)
5) Freeze cultured plates (wrapped in parafilm) in the freezer (they have a -80 C freezer but she seems to think the home freezer would be fine if you are careful to avoid big temp swings or power outages).


In order to regrow yeast, you take plate and with a sterilized loop or stick, scrape off a single colony into a tube or small flask containing cooled wort. Grow out a small sized starter (10-50 ml) and then build it up once you have verified you have gtood desired yeast activity

It seems from first glance at your list of materials that this would be a cheaper method (no tubes, glycerine, smaller storage requirements, etc).

Anyone here have an opinion on preference between the 2 methods or if they are basically equivalent? I think a good plate culture would keep nearly indefinitely and could supply 10-20 starters worth of colonies per plate.

well, this process seems to work, and I can find glycerine at Walmart. No local source for agar. i think plating is usually used to preserve the purity of a culture rather than maximize viability.

I'm interested in trying out the plating method, but I do like the glycerine vials.

Well, Agar-agar is dirt cheap at asian or health food places and you simply combine with wort (haven't thought about proportions yet though). Just need to acquire some sterile plastic petri dishes or some reusable glass ones.

Had SWMBO streak out some WLP001 for me that had been in the fridge a bit too long after I built the starter up. Between the culture and the scope examination, everything looked good. I will post a picture of the plate tonight if I remember.

PintOfBitter said:

to preserve the purity of a culture rather than maximize viability.

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Oh, and isn't this basically the #1 objective here to ensure we continue to produce predictable yeast behavior and characteristics? Might have to work a little harder to build up your starter slowly, but I think keeping the purity intact is priority #1.

I have done all the methods you suggest, both as a homebrewer and in the micro lab. At the end of the day, I find the frozen yeast bank to be much more convenient, that's all. Streak plating, maintaining agar plates/slants, and building up yeast starters from single cells is a lot of work. Thawing and pitching some frozen yeast into a starter is pretty easy. If you like the microbiology approach, go for it. If you are pressed for time like me and want the shortcut method, frozen yeast banking works very well too. There is no 'better' or 'worse' method, really -- it just depends on what you enjoy.

FlyGuy said:

I have done all the methods you suggest, both as a homebrewer and in the micro lab. At the end of the day, I find the frozen yeast bank to be much more convenient, that's all. Streak plating, maintaining agar plates/slants, and building up yeast starters from single cells is a lot of work. Thawing and pitching some frozen yeast into a starter is pretty easy. If you like the microbiology approach, go for it. If you are pressed for time like me and want the shortcut method, frozen yeast banking works very well too. There is no 'better' or 'worse' method, really -- it just depends on what you enjoy.

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Cool. Thanks for the quick response. 2 more short questions:

1) Would you expect any different in viability between the 2 methods
2) Have you been able to adequately identify infection or mutation using the slant method? Do you see any difference between the 2 in that regard?

Randar said:

Cool. Thanks for the quick response. 2 more short questions:

1) Would you expect any different in viability between the 2 methods
2) Have you been able to adequately identify infection or mutation using the slant method? Do you see any difference between the 2 in that regard?

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I don't have great answers to these questions as I have never compared the two methods directly. Regarding viability, well-maintained slants in the fridge are probably better than frozen vials in a regular home freezer (although if you had access to a lab-grade freezer then the frozen method is probably better). Regarding infection/mutatation, that doesn't depend on the method of storage. You have to plate your yeast to detect that (which one could do from yeast stored in slants or frozen).

If you want to be really anal (like a lab) and grow yeast from a single cell to ensure purity, then slants and plating your yeast is the method of choice. But if you want something convenient for the purpose of easy homebrewing, I advocate the frozen method (but YMMV).

Cheers!

FlyGuy said:

I don't have great answers to these questions as I have never compared the two methods directly. Regarding viability, well-maintained slants in the fridge are probably better than frozen vials in a regular home freezer (although if you had access to a lab-grade freezer then the frozen method is probably better). Regarding infection/mutatation, that doesn't depend on the method of storage. You have to plate your yeast to detect that (which one could do from yeast stored in slants or frozen).

If you want to be really anal (like a lab) and grow yeast from a single cell to ensure purity, then slants and plating your yeast is the method of choice. But if you want something convenient for the purpose of easy homebrewing, I advocate the frozen method (but YMMV).

Cheers!

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Hey, this is great fun! I do have access to a -80, but the thought of my yeast-bank being in a lab outside of my daily control does make me more nervous.

I didn't mean to imply the method of storage being an issue for detection of infection/mutation. I was wondering if it was harder to detect such a mutation. With the plated culture you would be able to only select the viable colonies individually that showed no evidence of infection or mutation (color shift, shape variation, etc). It seems intuitive to me that it is harder to detect mutation in a slant (infection should be obvious in either method but in a plate it is generally more localized and viable colonies usually still exist free of infection). But, my intuition relating to biology is not something I trust!

It seems you don't have a strong feeling one way or another on that topic, true?


Off topic, did you read the BYO article a few months back (middle of the summer?) that talked about this very topic? I saw quite a few inaccuracies in that article and my wife plain cringed at times... flame your innocculation loop and THEN put it in the ethanol???? Curious if you'd seen that and had any impressions on it.

I guess what I meant was that you can plate yeast from any source (frozen or slants), and build up yeast stock from an isolated colony. But obviously if you want to to go that route, slants are more convenient (simply because you don't want to thaw then re-freeze your yeast each time you want to plate a sample). I am not sure how you would accurately check for infection/mutation in frozen yeast without plating, however.

In homebrewing, people often use slants or frozen yeast banks as a yeast source, but never plate their yeast. They just pull a sample and use that to inocculate sterile wort (that's my method). I guess the idea here is that brewers are generally able to detect yeast problems at the point when the beer doesn't turn out the way the expected. We just have a different method of 'quality control' than a lab would.

And yes, I did read that article. Did you see that letter to the editor criticizing their techniques in the latest issue? Apparently you weren't the only one to notice!

Bumping this thread up, I prepared a frozen yeast bank of three strains, S05, Safbrew S06, and Pacman. I used a 20%-30% glycerin, 70% yeast slurry, and put them in my freezer. I went to look at them about a week later and noticed that the glycerin liquid in my vials did not freeze. I'm not too sure if I now have 12 vials of garbage or if everything will work out. I plan to use the pac man again so I'll know when I make my starter. Has anyone run into this problem before or did I screw this one up all on my own?

FlyGuy said:

And yes, I did read that article. Did you see that letter to the editor criticizing their techniques in the latest issue? Apparently you weren't the only one to notice!

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I just went back to the October issue to see the complaint (only the complaint about bacteria vs yeast)... weak-sauce. I still question the man's sanity if he thinks it is proper to flame an inoculation loop before dipping it in ethanol, risking creating an ethanol bomb or flame-out in the vapor.

Annnnnnyways, took a pic of my plate... talked some more with SWMBO and I think she was speaking Greek and I was listening in Spanish last time we talked about this.

So, confirming...

1 - plates dry out faster and colonies will die in a matter of a few months
2 - Slants as described in the other thread aren't really used in her work but she feels they'd be subject to the same effect as plates, but it may simply take longer
3 - she reviewed your process and generally agrees with it. She questioned why you'd use 10ml vials instead of the smaller ones to maximize storage space as well as to simply scrape a sample from the top and grow up from there, but thawing the whole thing and pitching directly is a choice of simplicity requiring less building up, so that's a choice of preference here...


Plated out some WLP001 to check for infection during grow up of some old yeast... infection top left, good colonies the top white puffs...


Her frozen stocks are in a mix of 0.5ml of sterile 50% glycerol and 0.5ml dense yeast slurry. Then, to regrow, she will scrape off a small amount of the frozen slush from the top of the ~1ml of stock and regrow in a small starter. Now, in her work she then plates out to single colonies to check for purity and to ensure the integrity of the colony, but that is not needed here since we have the steps in making our larger starter to detect infection or other issues. In her work that 1ml stock will last indefinitely. It really requires only saving off and keeping one vial (although you could keep a couple for backup) and keeping only a couple ml total of frozen stock per yeast strain.

Just a smaller alternative (but requires more extensive stepping up from a smaller initial cell count)...

flananuts said:

Bumping this thread up, I prepared a frozen yeast bank of three strains, S05, Safbrew S06, and Pacman. I used a 20%-30% glycerin, 70% yeast slurry, and put them in my freezer. I went to look at them about a week later and noticed that the glycerin liquid in my vials did not freeze. I'm not too sure if I now have 12 vials of garbage or if everything will work out. I plan to use the pac man again so I'll know when I make my starter. Has anyone run into this problem before or did I screw this one up all on my own?

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Did you shake the **** out of them when you first put them in the freezer? Definitely want the result to be a combined solution of glycerol/yeast slurry.

one drawback to freezing is to maintain constant temp.

Randar-

Was the infection on your plate present in the vial? Is there any way to tell. I am hopefully entering the yeast management world after Christmas and am trying to get a handle on all this stuff.

I love these threads.

Randar said:

Oh, and isn't this basically the #1 objective here to ensure we continue to produce predictable yeast behavior and characteristics? Might have to work a little harder to build up your starter slowly, but I think keeping the purity intact is priority #1.

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And how are you going to determine whether that colony you picked doesn't have any mutations from the common strain? I would think you're more likely to get results similar to the original yeast if you save as much as possible.


Guess my point is, if you're saving 100 million cells, mutations in one isn't a big deal.

Synovia said:

, if you're saving 100 million cells, mutations in one isn't a big deal.

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Unless that mutation becomes the dominant strain in the culture, making it impossible to isolate, which you;re essentially taking the same chance on.

According to the resident expert, in the >millions of possible genetic mutations, it's likely only a handful could/would affect the finished product, while most will actually trigger apoptosis (programmed cell death). Those that survive often show visible colony differences in color, shape, growth rate, etc. Therefore, the probability is such that any surviving mutation that also affects growth/regulation/flavor carries an extremely remote probability.

Infection is the thing to be careful of.

As for the question about the infection on the plate in the picture, I believe this likely came from the initial starter lag time of using an older WLP001 vial (straight from White Labs, so I don't think the infection was present prior to my opening the vial) and perhaps some laziness in my sterilization (sample was taken from slow starting culture). But, I grew it out, decanted, and regrew and it has been fine and showed no signs of infection in the slurry prior to pitching and the fermenting batch smells spot-on. You could certainly streak right from yeast bank vial to a plate to determine presence of infection.

mordantly said:

one drawback to freezing is to maintain constant temp.

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Yes, most home freezers are auto-defrost so you have to take steps to keep temps somewhat regulated. Using a small styrofoam cooler (best if you can find one of the thick biological grade ones) with some gel-ice packs will keep things frozen and in good condition even through defrost cycles, although you'll still be at risk if you have any sort of extended power outage. I thinkt he ease of doing this at home is the key, and even in my case where I have access to a lot of equipment and whatnot, the though of keeping my samples in a freezer 35 min from home in some shared minus-80 C freezer is not as appealing as always having control and access to it.

mordantly said:

one drawback to freezing is to maintain constant temp.

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It's actually not a big deal:

https://www.homebrewtalk.com/f13/guide-making-frozen-yeast-bank-35891/#post351738

Hi Randar

Yeah, I definitely did. I just looked this week and some are froze, but others are not. My freezer temp gauge is well into the sub zero range. I'm about to make Jamil's robust porter using the pacman so I'll know a couple days in advance if I still have good a good viable yeast bank.

Thanks for the inspiring thread!

I just tried using a 25ML vial of Wyeast 1056 that I froze a week or so ago using the procedure in the OP. I had it 25% full of glycerin and topped it off with fresh yeast slurry from a 1st gen starter.

I was only 48 hours from brew day so I thought I'd give it a shot and had US-05 dry yeast on standby just in case it didn't work out.

I took the vial out of the cooler packed with ice packs in the freezer and let it warm up on the counter for a few hours. I pitched into a 625ML starter made with 1 gram DME per 10ML of water plus a pinch of yeast nutrient prepared in pressure cooker onto the stir plate. Room temp was in the mid 60s.

Next morning there appeared to be a krausen ring so I put 1200ML more starter wort into the flask for a total of 1800ML. I let that sit on the stir plate for 24 hours and I crash cooled in the fridge for 12 hours.

There are very few to almost no yeast at the bottom. Even after 36 hours there is no additional matter besides some trub from the starters. I hydrometer tested a sample of this and it was at 1.047. Looks like I didn't get any attenuation.

What do you think went wrong? Is there anything glaring in what I posted above? I'm guessing my initial starter was too big or that particular vial didn't have enough viable yeast to get going. I'm going to try another vial in a 250ML starter on the stir plate and see how that goes.

I have a tangential question.

I have been following this method for making my yeast bank but I was wondering about storing wort as well.

Right now I make my starters using dme but I was wondering if anyone had tried making some wort from all grain and then boiling it to sterilize and then tried storing/freezing it in mason jars or something similar that had been sterilized in a pressure cooker.

I realize that this isn't a huge issue and that dme isn't that expensive.

Groosh, I really recommend this article. It's been working really well on my regular starters. Using these with the frozen yeast, as you can see above, I'm still working out the details

http://www.maltosefalcons.com/tech/starter-made-easy-pressure-cooking-your-starter-wort-ahead-time