Guide to Making a Frozen Yeast Bank

Just a data point for all you bankers out there:

I did 2 cultures, stepped up from wyeast packs, into autoclaved starters. 2308 and 1098. After the ferment, I mized 5050 glycerine to slurry. Mixed once in the tubes, and froze immediately to -20F. After a month or two, neither culture woke up well. I added a bit or fresh wort to the tubes, after decanting the glycerine portion, and neither of the cultures really woke up. They were barely fermenting 6 days after the wort addition, and this was only noticeable by shaking the tubes and noting the co2 release. So, this could have been a contaminate I introduced, or just terrible viability. So, the viability was either terrible or zero. Either way it didn't work. Not sure if it was from too much glycerine, or the rapid freeze. It was enough glycerine that the solution was still a liquid after freezing to -20F.

Non-frost-free freezer, and the tubes were inside a small cooler inside with blueice packs.

I might try this again. From what I've read, I should use less glycerine, as well as let the culture sit with the glycerine present for some time (room T and/or fridge T?). Maybe I should try both, and each without the other, as a sort of experiment.

The yeast should have been extremely healthy going into the tubes, so it must have been my procedure/process....

I am sure this in in here some were, but how do you know that the glycerin you buy is safe to ingest? May be a dumb question, but I want to make sure, mine says skin protectant, but so far I don't read any warnings that if u ingest u dieor anything so I think it's safe...

Active ingredient is 95.5% glycerin Anhydrous. .... Purpose is skin protectant.

If I picked up the wrong one, I just would like to make sure before I use.. Thanks:rockin:

kennywd said:

I am sure this in in here some were, but how do you know that the glycerin you buy is safe to ingest? May be a dumb question, but I want to make sure, mine says skin protectant, but so far I don't read any warnings that if u ingest u dieor anything so I think it's safe...

Active ingredient is 95.5% glycerin Anhydrous. .... Purpose is skin protectant.

If I picked up the wrong one, I just would like to make sure before I use.. Thanks:rockin:

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Yes, it is also used in that fashion, as well as to make soaps and as a food sweetener. But I made sure MINE was by buying from a place that labeled it as 'food grade'..
In all honesty, even if it WEREN'T, you are talking about a very miniscule amount in a (relatively) large amount of beer (grin) -and it will have been further diluted if you get the freshly defrosted yeast 'ready' by pitching it into a starter.
You should be fine though.

Nightbiker said:

Yes, it is also used in that fashion, as well as to make soaps and as a food sweetener. But I made sure MINE was by buying from a place that labeled it as 'food grade'..
In all honesty, even if it WEREN'T, you are talking about a very miniscule amount in a (relatively) large amount of beer (grin) -and it will have been further diluted if you get the freshly defrosted yeast 'ready' by pitching it into a starter.
You should be fine though.

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Thanks, I will be back at the store and they had one more I will just check it to make sure.

Where is everyone getting their glassware for this? I've been hunting for autoclavable screw tops in the 15-20mL range, but I can't seem to find them for under $2/unit and that's in orders of 144. Anything else I find I can't seem to confirm that it will survive in the autoclave. Thoughts?

uberg33k said:

Where is everyone getting their glassware for this? I've been hunting for autoclavable screw tops in the 15-20mL range, but I can't seem to find them for under $2/unit and that's in orders of 144. Anything else I find I can't seem to confirm that it will survive in the autoclave. Thoughts?

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Cynmar.com is a pretty popular place to source from. Also, I've had good luck with discountvials.com.

uberg33k said:

Where is everyone getting their glassware for this? I've been hunting for autoclavable screw tops in the 15-20mL range, but I can't seem to find them for under $2/unit and that's in orders of 144. Anything else I find I can't seem to confirm that it will survive in the autoclave. Thoughts?

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I bought my at my LHBS, but they had to special order them it wasn't something they carried.


Full disclosure. I work at my LHBS part time ... and I do the ordering

The wholesaler was Brewmaster Inc. out of San Leandro, California. They don't list the cap as being autoclaveable, but it is. Item number TE011P1. If your LHBS has an account with them, they may be able to order for you.

Thanks guys

I was curious about discountvials.com products since it doesn't mention if the caps are autoclavable, so I sent an email. Got a reply back in less than 10 min!

Hi,

No ... the liner wouldn't hold up. You would need either Teflon or 14B Rubber to withstand an autoclave. Thanks!

Best Regards,

Rhett Roeth
Discount Vials
4462 Robertson Rd.
Madison WI 53714
608 442-8061 FAX 608 204-9509
http://www.discountvials.com

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Looks like for the 4 dram (15ml) vials teflon lined caps are available for an extra $3 on a package of 25. Still a pretty good deal at $0.65 a vial

camiller said:

I was curious about discountvials.com products since it doesn't mention if the caps are autoclavable, so I sent an email. Got a reply back in less than 10 min!



Looks like for the 4 dram (15ml) vials teflon lined caps are available for an extra $3 on a package of 25. Still a pretty good deal at $0.65 a vial

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I wouldn't hesitate to order from Rhett. I received non-autoclavable caps on one out of 4 separate orders that I placed with him, and he straightened out my order by the end of the same week. Great customer service, and that is what I like paying for.

Just thought I would throw a pic in here....

mountainman619 said:

Just thought I would throw a pic in here....

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Great looking samples and labels!

ditto! Nice example of a bank. We lost our deep freezer a few months ago -and with it, all of my yeast samples, hops, venison, and beef steaks (and home-smoked sausages). Made me sick just thinking about it. Now I'm just getting back to repopulating the bank.

Just pitched Whitbread vial into a started today...the vial was frozen 18 months ago...I'll let ya know how it goes.

Hey Flyguy (OP) are you still posting in this thread? I tried to send you a PM but I think you may have the option to receive them turned off.

-TK

mountainman619- What program/template are you using to label your vials? I used the mailing label and hand writing solution and realizing quickly that I hate it. The mailing labels aren't sticking worth a crap.
tom

Moose777 said:

Just pitched Whitbread vial into a started today...the vial was frozen 18 months ago...I'll let ya know how it goes.

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how'd it go?

TomRep said:

mountainman619- What program/template are you using to label your vials? I used the mailing label and hand writing solution and realizing quickly that I hate it. The mailing labels aren't sticking worth a crap.
tom

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Masking tape and pencil...I put the tape on once clean and dry. Rubbing alcohol will help dry them faster.

I've been freezing yeast for several months now.
For vials I have these: http://www.sks-bottle.com/340c/fin59a.html

I got my Glycerin at Rite-aid. It is the skin treatment kind. But with no additives, I guess it is OK.

The first step I take is to make a starter a little bigger that what I need for brew day. I then make 4 vials for generation 1 to freeze. If I made 4 more each time I could go just 4 generations and make 256 batches from the one original vial/pack of yeast! That is a significant $$ savings!

6 varieties now and 21 vials in storage.

I mix 10ml yeast, 10ml glycerin and 20ml water. I have revived 2 so far, 1056 and WLP023 Burton Ale Yeast, with now problems at all.

I do steps of 250 ml decant, 500 ml decant, Then it looks like the same amount as a vial/pack. I then make a starter of the recommended size for the gravity brew planned.

The Burton's is in a Porter and filled my blow off jar after a day!

I use Avery address labels.. they have a template on their site that you just import to ms word and type away. Works great - I'm at work or I'd take a picture.

I'm not using a sticky label at all. I created it in Word, printed them on regular paper, cut them out and then taped them on with scotch tape. Vials have to be super clean and even then it helps to wrap the tape around and back onto its self. Otherwise they just slide off.

Good to know. thanks for the reply mountainman (and all of you, really).
tom

mountainman619 said:

I'm not using a sticky label at all. I created it in Word, printed them on regular paper, cut them out and then taped them on with scotch tape. Vials have to be super clean and even then it helps to wrap the tape around and back onto its self. Otherwise they just slide off.

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Nice work on the labels... Any chance you'd be willing to post a copy of your Word doc?

Thanks;
Steve

mountainman619- Another question. what is the "1002PT1SKYA1" on your label? I assume it's some kind of code for you or the production code on the yeast vial? Thanks!
tom

I have regenerated a vial of Kolsh yeast that has been frozen for 6 months. It is aggressively chewing through an Oktoberfest and a Kolsh.

I really want to do this to save money but I'm driving myself crazy trying to find a good source of autoclavable vials.

What I've been doing up to this point is saving samples of each strain in small glass bottles. These bottles were bottles of tonic water that I salvaged from work. They cleaned up nicely and don't take up tons of space. Here's a shot:



I'm wondering if I can do the same thing but add glycerine and freeze them. Maybe I could have a small bottle full of yeast/glycerine mixture and just pour some out when I need to.

Thoughts?

Heads up for anyone looking for glycerin online.

http://www.mountainroseherbs.com/bulkmisc/bulkmisc.php#bu_gly



16oz for 8.75+ shipping. They also carry bitter orange peel, irish moss, and many other brewing spices and goodies mostly organic if anyone cares.

SteveCO2 said:

Nice work on the labels... Any chance you'd be willing to post a copy of your Word doc?

Thanks;
Steve

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Steve - I am willing, but can't from this particular computer. However you can open a new blank document then create a table with 2 columns and 5 or more rows. That gave me 10 spaces to fill. I will try to remember to make one and post it tomorrow (Sunday).

TomRep said:

mountainman619- Another question. what is the "1002PT1SKYA1" on your label? I assume it's some kind of code for you or the production code on the yeast vial? Thanks!
tom

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Tom - It is a production code from the White Labs vial. It allows me to track strains that are the same but from different original sources. If I end up with a problem I can back track and know which vials could be suspect.

Yeast freezing is great. Has anyone here froze yeast with a full pitchable rate? Wouldn't it be a good idea to freeze the whole volume of yeast that you're going to pitch using the glycerin method? Then 24 hours before brewday you just take it out of the freezer, put it in the fridge, and pitch without a starter. Of course if you brew often then you'd probably run out of fridge space pretty fast.

Does this look like the right amount of yeast to have (16ml vials)? Too much liquid? I have had them in the fridge for 24 hrs, tomorrow they'll go into the freezer. Do I shake them up again before freezing?

I'm sorry if these questions were already answered at some point, but I don't have time to read all 400000 posts. I am VERY interested in anything that can contribute to the lowering of brewing costs.

1. How long can the frozen yeast be kept?Indefinitely?

2. Wouldn't you only want to take yeast right out of the pack/vial instead of from the fermenter yeast cake? Seems to me there would be hop trub and other particulate in that stuff.

3. Can you do this with rehydrated dry yeast? I realize dry yeast can keep for much longer, and is already cheaper, but free is even better.

Yes that's plenty of yeast. In fact that's a lot more than I've put in. Shake them before you freeze just to be sure the glycerin is well mixed in.

Thank you!

This thread seems as good of a place to get answers as any, so... I plan to start freezing yeast, primarily because of the limited availability of WY3864, which I'm getting my hands on while it's around right now. I do not have a pressure cooker, however.

I plan to use sterile 50 ml conicals, so sterilizing vials is not a concern. Thus, my only concern is sterilizing my glycerol stock solution (and any equipment that will touch the yeast). There is of course no household way of sterilizing other than a pressure cooker (the efficacy of bleach solutions on surfaces/equipment is debatable), but boiling will kill anything except endospores. Tyndallization is another possible option for solutions, but is also of inconsistent efficacy (as well as being a pain in the butt).

My question is this, though. Is complete sterilization (i.e. more than what can be achieved by boiling) actually necessary for freezing yeast, given that the source of the yeast itself isn't sterile. I haven't seen anyone recommend autoclaving/pressure cooking one's Erlenmeyer and starter wort, just boiling it, in which case the starter itself will never be truly sterile and will be a far greater source of potential contamination than the glycerol/equipment.

Anyways, thoughts? Am I fine just with boiling everything, or do I really need to purchase a pressure cooker? I don't plan to passage yeast more than maybe twice, so there won't be repeated expansion of contaminants.

Thanks!

If you have good sanitary brewing technique you will be fine. You're right that you will be able to use it for fewer batches. The level of contamination isn't necessarily either or for ruining a beer...you may just see a gradual degradation in flavor quality and not something that is necessarily detectable until you've reused the yeast several times.

Best practice is of course to keep everything as sterile and sanitary as possible. I sterilize my starter vessel in the oven (I use 4L wine jugs and place them in the oven at 320F for 2 hours and let it cool slowly overnight) and use precanned sterile starter wort that I prepare in batches in my pressure cooker.

orangehero said:

If you have good sanitary brewing technique you will be fine. You're right that you will be able to use it for fewer batches. The level of contamination isn't necessarily either or for ruining a beer...you may just see a gradual degradation in flavor quality and not something that is necessarily detectable until you've reused the yeast several times.

Best practice is of course to keep everything as sterile and sanitary as possible. I sterilize my starter vessel in the oven (I use 4L wine jugs and place them in the oven at 320F for 2 hours and let it cool slowly overnight) and use precanned sterile starter wort that I prepare in batches in my pressure cooker.

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This is not what I need to be hearing. XD I'm a biologist by profession, so I'm already paranoid about sanitization/sterility. I guess buying a pressure cooker is unavoidable for me at this point...

Let me add a data point here. I had about 20 yeast lines stored in cryotubes in 15% (v/v) glycerol and sterile water (I just removed pure colonies from a plate and put them into the water/glycerol mix). I stored the tubes at -20C in a frost freezer, and after about 18 months of this I started to notice a dropoff in viability. I couldn't get them to grow when I streaked a little bit on a plate.

Now that I'm restarting my library, I'm planning on doing 40% glycerol, in an attempt to avoid freezing (15% froze at -20).

Furthermore I'd say that a pressure cooker is an absolute necessity - things need to be sterile if you're stepping up from a small amount of yeast, there is no room for error here.

passedpawn said:

FYI, you can buy glycerin locally. I don't bank anymore (not pure yeast, anyway) but I used to get glycerin at Walmart, in the pharma aisles. I probably posted this info about 100 pages back in this thread, but thought I'd throw it out there again.

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So would something like this work ?? --> http://www.walmart.com/ip/Humco-Skin-Protectant-Glycerin-Usp-6-oz/10417582

Also - I have been reading up and down this post regarding the glycerin and pressure cooker...

I use star-san and LD Carlson 1-step sanitizer to sanitize EVERYTHING I use... If I boil small mason jars and then star-san them afterwards (in essence doubling the sterilization for insurance pts) will this suffice to add the glycerin and yeast slurry and then freeze?

Or is it that the glycerin itself is what needs to be sterlized, hence the pressure cooker?

samandbekah said:

So would something like this work ?? --> http://www.walmart.com/ip/Humco-Skin-Protectant-Glycerin-Usp-6-oz/10417582

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Yes, that glycerin will work. I use the Walgreen's variety. USP grade Glycerin is USP glycerin is USP glycerol...all the same (99.7%).

samandbekah said:

I use star-san and LD Carlson 1-step sanitizer to sanitize EVERYTHING I use... If I boil small mason jars and then star-san them afterwards (in essence doubling the sterilization for insurance pts) will this suffice to add the glycerin and yeast slurry and then freeze?

Or is it that the glycerin itself is what needs to be sterlized, hence the pressure cooker?

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None of these actions will sterilize. This means that, compared to using a pressure canner as an autoclave to actually sterilize, you will have higher counts of all living contaminants. This higher count will multiply and amplify at each starter step, and your results could suffer negatively as a result of the contamination.

Ex: 4-step starter from frozen vials, with either 1 contaminant or 1000:


Pony up $100 for the presto 23qt. canner, make you yeast library properly, and then learn to make and can wort (at different ° plato) for making starters without any prep work. If you want to cook or can other things (veggies, meat, etc.) get a 3-piece regulator too.

Okay but what is actually not being sanitized, the yeast or the glycerol or both etc?

And are you implying that the Star-San and 1-step cleaners are never really sanitizing the brewing equipment?