Guide to Making a Frozen Yeast Bank

jdoiv said:

Ok, so I went by the LHBS this morning and picked up what I hope is the right stuff. If was Glycerin USP Finishing Formula from JD Carlson. Is this the right stuff?

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Yep. padding

jdoiv said:

If was Glycerin USP Finishing Formula from JD Carlson. ...

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I Think that is LD Carlson. Since they sell it as an additive for wines and liqueurs it certainly should be safe for consumption.

Wow, this sounds like something I'd like to try. I don't have any of the glassware needed, but I was looking around on that Cynmar company's site, and it looks like the whole "kit" could be pieced together pretty cheap. Maybe I'll go for it.

For how many generations is a given yeast supposed to be good for? I'm sure this process shouldn't be repeated forever, right? It seems like I remember the magic number of 4 generations from somewhere...

Thanks for the info. I'm just starting to get into yeast banking. Is that a Corning PC-353? I got the same one at the university surplus store for $5. Its already paid for itself after making just a couple of starters, and the amount of yeast it produces per volume is pretty sweet. Also a little off topic but how hard was it to tap a valve into the top of the pressure cooker(pretty thick)? I'd hate to ruin the nice new $90 pressure cooker I'm looking at.

PintOfBitter said:

Wow, this sounds like something I'd like to try. I don't have any of the glassware needed, but I was looking around on that Cynmar company's site, and it looks like the whole "kit" could be pieced together pretty cheap. Maybe I'll go for it.

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Scientific glassware is nice, but you can get everything you need at the grocery, just remove the food and wash. Of all the pieces I have, the two I like best came filled with apple juice and Ragu...

left field brewer said:

Thanks for the info. I'm just starting to get into yeast banking. Is that a Corning PC-353? I got the same one at the university surplus store for $5. Its already paid for itself after making just a couple of starters, and the amount of yeast it produces per volume is pretty sweet. Also a little off topic but how hard was it to tap a valve into the top of the pressure cooker(pretty thick)? I'd hate to ruin the nice new $90 pressure cooker I'm looking at.

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Yep, I got mine surplus at my university, too! Works great still.

Regarding tapping the valve, it was dead easy. But I would recommend to anyone to be very cautious when you do it. If it isn't threaded properly, you are just asking for serious trouble. To anyone buying a new pressure cooker, I would HIGHLY recommend finding one that has a pressure relief valve that unscrews -- then you can add a tee fitting, put the pressure relief valve back on, and then add your own needle valve for the steam outlet. Brewman ! has pics of his system (see top of post for links).

One may also look at getting a separatory Funnel with stopcock, After making your starter and removing from the stirplate, transfer your slurry into the stopcock, seal let settle then sterilize the stopcock tip before pouring into the vials.


1000ml

250ml

Just FYI, you can get anything you need from a scientific supply house like Fisher Scientific or VWR, I plan on making my frozen stock soon, you can get sterile tubes, anything you need.

MadWeezel said:

One may also look at getting a separatory Funnel with stopcock, After making your starter and removing from the stirplate, transfer your slurry into the stopcock, seal let settle then sterilize the stopcock tip before pouring into the vials.


1000ml

250ml

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I can't believe I didn't think of this. Well done. Really, good form.

Quick question / addition:

I've seen these baby soda bottles online for a fairly low price. They're preforms for 2 l soda bottles.



It's a bit hard to tell from the photos, but they look to be about the size of the White Labs pitchable liquid yeasts. I'm planning on saving and freezing some yeast from my next batch. Anyhow, I'm thinking these might be a good size for both storing/freezing yeast, and possibly building a mini-starter in as well.

Has anybody used these for yeast storage or starters?

MadWeezel said:

One may also look at getting a separatory Funnel with stopcock, After making your starter and removing from the stirplate, transfer your slurry into the stopcock, seal let settle then sterilize the stopcock tip before pouring into the vials.

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That was my thought too, but a 2L separatory funnel is big $$$.

I like the baby bottles! I'm using 35ml glass, and every so often one fails under the stress of freezing.

Actually, after using a White Labs liquid yeast today, I'm pretty sure the soda bottle preforms are exactly what WL sells for their 'pitchable liquid yeast'.

jds said:

Actually, after using a White Labs liquid yeast today, I'm pretty sure the soda bottle preforms are exactly what WL sells for their 'pitchable liquid yeast'.

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My only concern with those would be sterilizing them. If you're making a yeast bank, you're going to be propagating from the cultures time after time. It would be prudent to go beyond simply sanitizing and fully sterilizing those suckers. It's not worth the risk of contamination, to me. I don't trust it if I can't autoclave it

I do like those tubes....very very cool

mrkristofo said:

It would be prudent to go beyond simply sanitizing and fully sterilizing those suckers. It's not worth the risk of contamination, to me. I don't trust it if I can't autoclave it

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That's certainly the most conservative method, but it raises the question; Do you actually autoclave your vials? I know that I certainly do not. I just drop them in boiling water for 5 min, cap, and cool.

pldoolittle said:

That's certainly the most conservative method, but it raises the question; Do you actually autoclave your vials? I know that I certainly do not. I just drop them in boiling water for 5 min, cap, and cool.

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Yes, I do in fact.

When you work in a lab, you can get the vials in a sterile pack

Professor Frink said:

When you work in a lab, you can get the vials in a sterile pack

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This is also true. 15mL falcon tubes work great for saving a smidge of yeast in the fridge that's pitched into a starter. Get about 10 of those off a 1L culture.

mrkristofo said:

Yes, I do in fact.

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Very cool. No easy access here. Anyway, I meant "you" in a broad sense. I wasn't trying to insinuate you specifically didn't, and I apologize if it read that way.

My (poorly delivered) point was that so often us techies tend to engineer things beyond what reality calls for. And for the majority of us who consider 'sterile' a 3 min dunk in a pot of boiling water, there's probably no perceivable benefit to paying extra for 'glassware' rated beyond 212*F

Now for you guys that do have access, light it up!

nothing to say but.... thanks

I use baby food jars.

Question for anybody who has done this:

I made the samples up and let them sit in the fridge, but the yeast separated and went to the bottom after a day. So I shook it back up and put in in the freezer. Does that sound right, or should I have frozen it in the separated state?

mr x said:

Question for anybody who has done this:

I made the samples up and let them sit in the fridge, but the yeast separated and went to the bottom after a day. So I shook it back up and put in in the freezer. Does that sound right, or should I have frozen it in the separated state?

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You were right to shake them up. Also, if you store the vials on their side, they won't separate as much/as quickly.

I just came across this and have join the other by saying: nice write-up.

I have been building a yeast bank myself. But mostly on agar. Recently I started running into viabiliy issues with a culture that was 9 month old. It took much longer to get a pitch of yeast for a brew that I actually decided to keep at 34*F for a 2 days before I was able to pitch the yeast. I didn't want to go to the store or postpone the brew day.

Freezing yeast seems to be a nice way to make sure you always have back-up. I like to see that you can autoclave the glcerin, not sure why I never thought of that.

When I get around, I'd like to add your write-up to the Wiki unless you want to do it yourself or someone else want's to do this.

BTW, what about exchanging yeast? I have yeast on agar now and soon I should have some on glycerin.

Kai

So if the power goes out for a couple of days, you should take the yeast vials out and shake them up soon after the power kicks back on?

Guess what, after shaking up the yeast into suspension, I put it in the freezer. Three days later I checked it, and it has separated again. Not quite so well defined in all tubes, but it has separated. I wonder what is going on here?

Mine all do that. They stratify faster than they freeze.

yeah, not good when they layer out like that. You're not getting all the protection from the glycerol. Eevery freeze/thaw cycle makes them less and less viable, in case you're thinking about pulling them out, thawing them, shaking them up, and putting them back in the freezer. My recommendation is to flash-freeze them, but for those of you using glass vials you need to use your own judgement.

mrkristofo said:

yeah, not good when they layer out like that. You're not getting all the protection from the glycerol.

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It is my understanding from all the micro-biologists here that the cells are not being protected by being suspended in the glycerine, but rather that the glycerol is absorbed into the cell itself. If that is correct, is seems that once the cell has absorbed the glycerol, dropping out of suspension is of little consequence to the protection process.

FWIW, my test tubes are stored vertically and have all dropped out completely. The incidence of dead (brown) versus live (beige) cells appears to have little to do with settling as some are brown over beige, others inverted, and some stratified but swirled like fudge ripple. Based only on a visual examination, I expect that the bulk of the necrosis happens well before the yeast settle out. Also, I only have two cells that are totally brown, and they were refrigerated for 1 week prior to freezing. They looked much better going into the freezer, but now look worse than many who have spent several months longer frozen.

As for freezing my test tubes, I find that about 1:10 break the glass and about 1:5 crack the plastic tops but do not break the glass.

hmmm, I'm finding the same thing, quite a hodgepodge of final results. Some frozen and mottled, some light beige over brown, still in liquid form, with the water stratified over the top of that

So the glycerine does not prevent freezing the liquid, rather it protects the cell walls from damage, correct?

Correct. It helps prevent the crystallization of water within the cell walls of the yeast.

You can get the vials at the link below they have all type of bottle and vials.....

http://www.specialtybottle.com/

D

Bellybuster said:

Not that Flyguys write up isn't good enough, it's excellent.
CD Pritchard put out another great article on freezing yeast. This is how I got started and the kit linked to on that site is a great cheap way to get started without layin out much cash.

http://home.chattanooga.net/~cdp/freezyea/freezyea.htm#Sources-maybe

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An update to this. I went to the linked site and found the country wines link to buy a pre-made yeast bank but could not find the item on their online catalog. I emailed them and got the following response:

Hi Dave,

Country Wines was bought by South Hills Brewing Supply in September. We are in the process of straightening things out. Yes we have Yeast Bank, but it just hasn't made it's way onto the website yet. I hope to do it this afternoon.

Sincerely,
Jon Benedict, Owner
South Hills Brewing Supply
Country Wines

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so are you just pitching the entire vial including the glycerin or is it possible to just swirl a sterile loop in the vial and transfer that to a small container to start growth?

scottfro said:

so are you just pitching the entire vial including the glycerin or is it possible to just swirl a sterile loop in the vial and transfer that to a small container to start growth?

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I pitch the whole vial. You could just grab a smaller amount, but then you would have to refreeze the remainder and that would hurt the yeast.

yeah i'm curious how much it would hurt them to refreeze. maybe i'll give it a test run sometime. would be nice to be able to have just a few vials of each strain and be able to dip into each one 2 or 3 times before tossing the remainder.

i have yet to save yeast this way, up till now i've just done slants in the fridge. so that will be the first step, then some experimenting can happen

Wow. This is great info. But I'm wondering -- is it possible to chemically sterilize? For instance, could a feller drop a bunch of salvaged White Labs vials into a strong bleach solution for 20 minutes and then rinse them by dropping them into a pan of boiling water?

Yes, you don't NEED to autoclave/pressure-cook. It is just a precaution. Thorough cleaning and proper use of a good sanitizer is probably just fine. But I would be tempted to use something like Star San that is rinseless.

Great write-up on this, flyguy, you made me want to try it out! Do you know how long the yeast will stay viable if properly stored? We don't brew nearly as much as I would like and I'm afraid the hard work would be wasted by not being able to use it in time.