Guide to Making a Frozen Yeast Bank

for the 1 ml vials you pitch 1 vial into 20-50ml of sterlized wort "step up" and let it ferment out. Next pitch that whole 50 ml into a larger 200ml-1L starter and ferment out. At this point for a low gravity beer there should be plenty of yeast if using a stir plate. For high OG beer or no stir plate an additional step up is needed. Yeast grow like bacteria. It only take a VERY small amount and due to exponetial growth a "step up" method will yeld tons of yeast

 

I see, thanks. Just by stepping up. It makes sense.

So, after you are out of vials, do you start your yeast bank from a new fresh smart pack or taken yeast samples from the starter? I just wonder how many generations of yeast I can have from a single smack pack.

 

1 ml -> 20 ml -> 1000ml

I have never gone through a full set of vials as i brew only once a month at most. However, I would probably just buy a new smack pack. I have some german ale on its way which i have already calculated i can get about 15 ml from without make a huge starter. The yeast cost me $6 with about $1 worth of dme for each starter from a 1 ml vial. About $1.50 for yeast starter (75% each starter)

 

So I am gathering supplies to start my own yeast bank, and I had a quick question.

I am looking for glycerine since my local walmart didn't have any. Found this on ebay and I want to make sure it is fine for this application.

http://cgi.ebay.com/12-OZ-GLYCERINE...aultDomain_0&hash=item2305452113#ht_871wt_907

Just want to make sure that is the one I need. If not, if someone could direct me to the correct one that would be great.

 

My Walmart had it in the pharmacuetical aisle, with the hand lotions etc.

The one you posted looks fine.

 

Yea I found one in my walmart, but it was only 10ml, so doing 30ml of glycerine wouldn't work if I could only buy 10.

Ok. If it looks fine then so be it, I guess I will order it now.

 

World of Warcraft :-O I can not believe it but I finally made it through all 45 pages of this thread. Judging from the recent posts it appears FlyGuy has checked out, can't say that I blame him.

My question, how long can I store my vials after they have been pressure cooked? (Keep in mind I have not added any yeast yet.) So far I have kept the foil a top of the beaker, and have the beaker chilling in the fridge.

 

once pressure cooked and sealed air tight indefently

 

I've read through most of this thread and have a few questions.

1) Do you put any water(how much?) in the pressure cooker to sterilize the vials?

Some of the pictures look like about an inch of water is in the bottom of the pressure cooker but some showed them empty and I missed where it says how much to use or any at all.

2) Glycerine from Walmart that says its 95.5% anhydrous is this the right stuff or too concentrated?

3) Slow freeze/Fast Thaw?

There were some posts talking about the best way to Freeze(slow) and Thaw(fast),but the original post doesn't appear to be updated to reflect that because it still talks about slow thawing. Was this ever resolved and what is the best method?

jm

 

My old pressure cooker will melt the fuse if there is no water in the thing. You need water to build the pressure. You need enough that the thing won't run dry during the process.

 

1 inch is what is rec. I however add about 2 cause i dont want to burn the bottom. nothing is hurt by adding more as long as it doesnt boil up into your stuff. DO NOT RUN IT DRY!

 

I have been thinking about doing this for a while and just found this thread the other day. Finally got through reading it and I have some ideas for possible improvements.

I was thinking that instead of aloquoting into glass vials and pressure cooking them that I would make up a solution to be added to the decanted starter. I found these 4 oz canning jars that seem to be about the right size. Then when you are making your starter, all you would have to do is sanitize your equipment and pull out a jar of your glycerol solution. After you have decanted the spent wort, just flame your jar lip and dump your solution and let your stir bar do all the mixing. At this point you could aliquot the entire contents of the flask to each vial you are going to freeze. This would save you the time of having to break out the pressure cooker every time you wanted to make a starter. You could even borrow one just to can your glycerol solution. It would also save you alot of time pipetting into each container, which I would think increases the chance of infection. It would also keep you from having to shake each vial by hand as it would be pretty well mixed in the flask.

 

...so from reading this thread it seems that a starter in a 2L flask would yeild approximately 100mL of yeast slurry? If you figure the 4oz canning jar would hold abou 100mL a 50/50 solution of glycerin and water would give you a final 25% glycerin in your stock which seems to work for most people. Somebody said earlier that Jamil recommends adding ascorbic acid to the stocks as well. This could possibly be added to this canned glycerin freeze media as well. Would it be beneficial to use wort instead of water, maybe add some chlorine dioxide and yeast nutrient?

 

I don't think I would use wort, at this point the yeast are room temp and will immediately start eating it when they should be getting ready for their long cold nap. I know chlorine dioxide is used in "real" yeast washing to kill bacteria but I don't know enough on the subject to know if it is OK to freeze the yeast in the presence of it. I'm thinking (dangerous ground here) that as the yeast freeze and later thaw they might be more vulnerable to the chlorine dioxide than when they are at room temp.

 

With a proper solution (Final % is roughly 25% glycerol, 25% water, and 50% dense yeast slurry) it will not freeze at all unless you are freezing at temps below the typical home freezer's -20C capability.

 

Use water, not wort. You are trying to keep the yeast in a deep sleep, so to speak. Don't wake them up with added nutrients that will restart metabolism.

Add the ascorbic acid to the glycerol solution to give yourself one simple solution to maintain and use in the process. I keep mine in a media storage jar and re-sterilize when sterilizing the vials before use.

 

What is the purpose of the Ascorbic Acid?

Any thoughts on my method?

 

I honestly can't recall the reasoning given in the book off the top of my head. I did talk it over with the wife who is in yeast genetics and it passed the sanity test. She doesn't use it in -80C storage stocks and White/Jamil only suggest it for -20 storage methods, not -80 methods.

I can look it up if I remember tonight when home, but if you are seriously considering yeast ranching, it's best to buy the book yourself.

 

It's on the way, should be here tomorrow.

.... just to be sure, this is the one you are talking about right?

On a side note, I picked up First Steps in Yeast Culture before i found this thread. It has an interesting section on long-term storage in sucrose. With this method you want a very small number of yeast cells. I personally like the idea of this method alot better. It leaves with more of a working stock that will survive long-term storage.

 

Ascorbic acid is an anti-oxidant that helps keep the cell membrane healthy during storage.

I'm not sure I completely understand your method, but wouldn't you still need to sterilize your vials, pipette, etc.?

 

Some of what is valid with other yeast may not be valid with brewer's yeast. I remember a guy posting that said someone that does research on yeast said not to use plain water because it will force the little critters into sexual reproduction. In White's book he says the brewers yeast has pretty much had that capability selected out of them.

 

Yes.

I have not read through or considered that method, but I personally would stick to the brewery yeast management methods. Lots of yeast (species/strains) researchers out there using all kinds of methods that are suited to varying applications...

 

They still obviously reproduce. They simply do it asexually, not sexually. This means mutations during the replication process are the only source of colony variation as opposed to sexual reproduction in which each successive generation is unique.

 

I am planning on using 15 ml conicals that come presterilized. The flask, baster and stir bar I would just soak in one step. When I do get a pressure cooker, I might get some of those autoclave bags and a few more glass basters. I'd autoclave 4 or 5 at a time so I would always have a sterile baster on hand.

 

There is also a method of storing in distilled water with a very small number of cells at room temperature that supposedly keeps them viable for years. I don't think I'd use sucrose since the yeast will still consume and reproduce and suppress creation of the enzymes that allow them to break down maltose and maltotriose, successive generations will lose the ability.

 

I do like the idea of mixing the storage solution with the yeast before portioning to the vials, it would be more uniform of a mixture. I know my first attempts some vials ended up with more yeast than others.

 

You might want to consider plating a few of those samples sometime. I think you'll be surprised by what you find, speaking from experience (unfortunately). The Brew Strong show about sanitation has an interview with one of the chemists at five star chemicals explaining why you shouldn't use chemical sanitizers for yeast storage work.

 

Thanks for the heads up. I found the podcast you were referring to and will have to listen to it over lunch. If that is the case I will just go ahead and pick up my own pressure cooker. I still might look into those autoclave bags and possibly do the aluminum foil/autoclave tape thing on my flasks so I can do all my equipment ahead of time and have it ready to go.

 

Ok so I have my tubes filled and have been in the fidge for 48 hours and they have seperated so do I shake them up again before putting in the freezer? Or just leave them alone and just put them in the freezer as they are?

 

Shake em up and re-suspend.

 

Do you have to use yeast from the bottom of a starter? Could you use some of the yeast cake straight off the bottom of the primary, or maybe washed yeast?

 

It probably could work if you did it right, but it seems like a lot more work than just building up proper starter.

Your two concerns are sterility and genetic diversity. Most standard washing protocols don't get the level of sterility you need for long term storage, but might be adapted to work. Likewise, a lot of cake harvesting technique I see makes me nervous about genetic drift, and freezing would only compound that.

 

Help me oh-so-wise ones. I have all the equipment to get this project off the ground, sans the pressure cooker, and I do not intend to get one. What's my best route for sterilization of the glycerin/water solution and the vials?

 

Other than a pressure cooker? Nothing. You can research Tyndallization if you are absolutely positively dead set on not buying one, but it's extremely tedious and less effective. If you can't buy a pressure cooker, I'd recommend not freezing yeast.

 

I have 50ml culture tubes that I previously used for slants. Are there any issues with leaving headspace in the tubes or should I use a larger volume of soultion/slurry?

 

I can't soak the tubes in Star-san and boil the water/glycerin solution? Or put it all in the oven? Having to buy a pressure cooker essentially negates the financial benefit of creating a yeast bank for a college student with a limited budget, and no foreseeable use of the pressure cooker.

 

Starsan doesn't cut it. Sanitatization typically has a kill rate much below what is necessary for longterm yeast freezing. To freeze, you need true sterilization. The BrewStrong episode about sanitization goes into some of the specifics of this distinction.

In theory, the oven would be feasible if you could keep things at the right temps long enough. But, I can't think of how to make it work. If you seal your vials up, I suspect they would crack. If you don't seal them, the water will boil off. Give it a shot, though. If you can figure out how to keep water and glycerine at 350F for an hour, that should be sufficient for most purposes.

If you're only doing this to save money, though, consider yeast washing. The threshold for acceptable error is a lot wider with washing.

 

I can't think of any reason this would be a problem, other than being bulkier to store. That said, if you've already got the big tubes, why not use more solution and slurry?

 

Alright so I baked the vials with the glycerin/water solution in them and lost about 1/3 the volume, not a problem as I overfilled them (in the proper ratio) to accomodate for this. My only issue is that it appears the white seal under the caps has disfigured under the heat, will this present a program for long term storage? Also, is a freezer-safe thick Rubbermain container satisfactory for freezer storage?

 

If the caps don't seal, it will be a problem.