Guide to Making a Frozen Yeast Bank

Homebrew Talk - Beer, Wine, Mead, & Cider Brewing Discussion Forum

Help Support Homebrew Talk - Beer, Wine, Mead, & Cider Brewing Discussion Forum:

This site may earn a commission from merchant affiliate links, including eBay, Amazon, and others.
Are there any larger autoclavable vials than in the op? I'd rather not have to do two starters to get a frozen vial ready to pitch.

Or would a well stirred starter on a stirplate with one of these vials allow for good propagation?

You would need a pretty huge vial of frozen culture to get by with just a single step. Based on some vaguely scientific experiments with a hemocytometer, I think just two steps is even pushing it.
 
I was thinking it'd be possible to freeze a good amount in a vial like White Labs sells theirs in (a good 30ml? whatever the amount is they sell). However, these aren't autoclavable.
 
No, but they're made of plastic, as they're just 2L pre forms. So I would assume that they aren't.

How does White Labs sterilize them?

I read several years ago (I think on another forum) that they could be autoclaved.
 
This post was also in that thread:

"by oneal66 » Wed Feb 06, 2008 5:01 pm

In the interest of science I went ahead and used our autoclave here in the lab to check. White labs vials are autoclavable. I did a cycle @ 121C for 20 min, and the tube and cap both came out fine. However, the tube did come out discolored. I'm not sure if this would cause a problem with yeast health or not. The tube did not come out disfigured though.
oneal66"

White Labs may have their own (financial) reasons for saying that they're not autoclavable.
 
This post was also in that thread:

"by oneal66 » Wed Feb 06, 2008 5:01 pm

In the interest of science I went ahead and used our autoclave here in the lab to check. White labs vials are autoclavable. I did a cycle @ 121C for 20 min, and the tube and cap both came out fine. However, the tube did come out discolored. I'm not sure if this would cause a problem with yeast health or not. The tube did not come out disfigured though.
oneal66"

White Labs may have their own (financial) reasons for saying that they're not autoclavable.

No need to be cynical about Chris White's motives here, especially considering how incredibly positive a figure he has been in the HB community. I assure you, he is not worried about how your reusing his vials will affect his bottom line.

If a plastic vial changes color during autoclaving, it has undergone some kind of chemical or structural change. That means it is not autoclavable. Maybe it matters, maybe it doesn't (and maybe it won't matter until you've done it six or eight times), but for something to be properly called "autoclavable" it needs to remain stable through the process.
 
I wish they had 2 oz jars, but maybe ~50-60ml of yeast slurry and the rest glycerin/water solution would do too.
 
There are 2 methods of true sterilization used on the commercial level. The first is autoclaving (kill curves rated at 121 Celsius). The second is exposure to ethylene oxide gas (EtO). This compound is so reactive and poisonous it will permeate through packaging/bags/vials/etc and kill EVERY microorganism present. EtO is more expensive and difficult to work with obviously, but it is the method used to sterilize items that can't be heated to autoclave temps safely (eg. medical equipment/supplies, plastics, etc).

If I had to guess I would say the vials are either treated with EtO or UV (but UV light is not true sterilization, just sanitization... there is a recognizable difference).
 
Laboratory grade germicidal UV exposure will sterilize if done for the correct amount of time and direct line of sight exposure. It works by mutating the host DNA. The UV lamps you buy from skymal and the ones on the vaccum cleaners that say they sterilize after a quick 5 sec exposure do little to nothing
 
malfet said:
no need to be cynical about chris white's motives here, especially considering how incredibly positive a figure he has been in the hb community. I assure you, he is not worried about how your reusing his vials will affect his bottom line.

If a plastic vial changes color during autoclaving, it has undergone some kind of chemical or structural change. That means it is not autoclavable. Maybe it matters, maybe it doesn't (and maybe it won't matter until you've done it six or eight times), but for something to be properly called "autoclavable" it needs to remain stable through the process.

+1
 
There are 2 methods of true sterilization used on the commercial level. The first is autoclaving (kill curves rated at 121 Celsius). The second is exposure to ethylene oxide gas (EtO). This compound is so reactive and poisonous it will permeate through packaging/bags/vials/etc and kill EVERY microorganism present. EtO is more expensive and difficult to work with obviously, but it is the method used to sterilize items that can't be heated to autoclave temps safely (eg. medical equipment/supplies, plastics, etc).

Cobalt Irradiation is also still used by some businesses. I would avoid that and the EtO, as well. I don't think anyone here would try to use EtO, but just as a warning, it is also toxic, flammable, explosive, mutagenic, carcinogenic and has a nasty habit of undergoing a violent exothermic reaction once you get it above about about 125F.
 
For stepping up I was thinking about these. They are Lab bottles so they could be pressure cooker safe and they can be easily stored or fitted with an airlock or simple tin foil. Filling the 1 oz and 8 oz bottle halfway full would give you from a 1 ml frozen vial a 1:15:10:10 step up to 1L. I know they say the first couple should not exceed 1:4 but if everything has been sterilized it should be fine right? I am sure canning jars would work as well...

1 oz = 30 ml
2 oz = 59 ml
4 oz = 118 ml
8 oz = 237 ml
 
with promotional code EDU133591V you can get 3 - 1 oz bottles and 3 - 8 oz bottles for $7.08. Add a little extra wort after brew day, pressure cook, and set away for next time.
 
ekjohns said:
For stepping up I was thinking about these. They are Lab bottles so they could be pressure cooker safe

I would read that again it says it has lab seals. Not that they are lab bottles they are just plastic and may melt in a pressure cooker.
 
I would read that again it says it has lab seals. Not that they are lab bottles they are just plastic and may melt in a pressure cooker.

I believe they are polypropylene bottles which are autoclavable. Before I buy them later today I will call nalgene to confirm this.
 
Yeah the more I look the more I think they are polyethylene which will melt in a pressure cooker
 
I found a supplier of polypropylene bottles (1 oz and 8 oz) that come in 12 pks each which is more than I need. Would people be interested in getting some as well. Price wise it would be $7.00 for 1 - 1 oz and 1 - 8 oz. I would ship the cheapest way possible.
 
1st of all thanks to OP and other members for great information's in thread.
I read all 55 pages and decided to give this a shoot.

One thing I didnt noticed in previous posts- is other equipment than standard 2000 ml Erlenmeyer flask required for stepping up starter?
If I understood correctly, we are stepping from 250 ml to 1-2 quarts in two steps (15 ml vials), can it all be done in 2000 ml flask or we need some additional flask?
 
Has anyone ever made Nottingham with a starter and then banked it in vials for freezing? I want to use this yeast ...but it is 4 dollars a package. If i am paying almost liquid prices for yeast I want to keep it!
 
1st of all thanks to OP and other members for great information's in thread.
I read all 55 pages and decided to give this a shoot.

One thing I didnt noticed in previous posts- is other equipment than standard 2000 ml Erlenmeyer flask required for stepping up starter?
If I understood correctly, we are stepping from 250 ml to 1-2 quarts in two steps (15 ml vials), can it all be done in 2000 ml flask or we need some additional flask?

It can be done in anything that is seal-able (airlock) -I think most of us use the 2L flask because its handy to use -you can boil your wort in it, chill it, aerate and pitch in it without having to transfer the wort -less chance of contamination. But I know brewers who use their growlers as 'flasks' to make starters -and before I got my 2L flasks, I was using mason jars with a hole and grommet in the lid. So long as you keep it sanitized, you can use pretty much anything you want. Honestly, I get fine results without using stir bars (just give the flask a shake from time to time) -so equipment needs are basically none more than a container that can be sealed and air-locked.
 
It can be done in anything that is seal-able (airlock) -I think most of us use the 2L flask because its handy to use -you can boil your wort in it, chill it, aerate and pitch in it without having to transfer the wort -less chance of contamination. But I know brewers who use their growlers as 'flasks' to make starters -and before I got my 2L flasks, I was using mason jars with a hole and grommet in the lid. So long as you keep it sanitized, you can use pretty much anything you want. Honestly, I get fine results without using stir bars (just give the flask a shake from time to time) -so equipment needs are basically none more than a container that can be sealed and air-locked.

2 days ago I"ve finished my stirplate and decided to give it test run with some washed S-04, stored in small baby food jars for 4 months.
I used 0.75 L jar for 250 ml step and it works great (according to nice vortex), next would be 1L step in Erlenmeyer. After that I"m going to try to divide yeast cake in 15 ml plastic vials (polypropylene centrifuge tubes), and put it into freezer.

I am not concerned about spending some yeast and throwing it away if something goes wrong, purpose of test is to introduce in stepping starter as well as making frozen yeast bank, so what ever comes from it I"ll be richer for one good experience.

After that I"ll try with WLP300.
Do you usually make starter and then fill vials or just divide brought yeast tube in vials?

Picked up a 16qt canning pressure cooker today to replace the small one I had.
I'm wondering if anyone has bothered to get glycerin in bulk (I think I recall reading that it should be cut 70% water and 30% glycerin) ?
http://www.bulknaturaloils.com/Products/15779-bulk-glycerin-usp-wholesale.aspx
http://www.aaa-chemicals.com/glveusp1ga.html
Would this be the right stuff?

I got 200ml glycerin and that would be plenty for ~100 vials, so I dont bother with buying in bulk.
In one of above links it says that vegetable glycerin is sweet, I am not sure is "ordinary" glycerin sweet and where this "sweetness" in vegetable glyc. comes from.
 
Vegetable glycerin is sweet -when purchased as 'pure' (or 'refined') I believe its supposed to be cut with water 70%water 30% glycerin. I usually just wash the yeast if it is being harvested from the fermenter -but if I'm using fresh yeast, I make a starter because I like to have a larger sample to store (I use 100ml sterile containers and like to have two stored samples of each -one gets used while the other gets 'saved' for later use -no real reason other than that is what I like to do). If you have enough of a sample, no real reason to make a starter just to put 'em in hibernation.
 
Buddy in 250 ml starter is live!! SG is 1.010 (OG was 1.030) so 65% attenuation seems fine for yeast harvested and stored in jars for 4 months, so experiment continues...

I am going to step it up to 1L today, leave it on stirplate for next two days, cold crash and then mix it with water/glycerin and pour in 15 ml vials.
To expand the experiment, I will take 2 vials out from freezer after couple of days, from one I"ll try to make starter (to define viability), and other I"ll keep in room temperature for 3-4 days and then make starter (to simulate shipping conditions and then see what is viability after that).

Has anyone shipped any vial from freezing bank jet and what are results in making starter with that sample?
I know that keeping yeast in cold environment (e.g. dry ice) during shipping will increase viability and reduce shock, but if I can I"ll rather keep it simpler.

Also, one thing bothers me, what ratio of glycerin/water do you use (in ~30% of overall mixture)?
 
Very informative thread.
According to information I've read in that thread, concentration of glycerin in solution depends on environment where vials will be stored- whether freezer is with freezing/thaw cycles or is it freezing/thaw free.
If understood correctly, since higher concentration will increase freezing point, in standard fridge/freezer with defrost cycle there si benefit from using more concentrated solution since we are looking to avoid freezing. Reason is that because freeze/thaw cycles are going to hurt yeasty viability, so it is better to avoid freezing while still keeping it in low temps (-20° C).

From this table (http://www.dow.com/glycerine/resources/table8.htm) we can see that concentration of 50/50 have freezing point at -23° C, which will keep us in safe area from freezing our vials.

This leads me to next question: does the vials have to be frozen to preserve the vitality of yeast?
I mean, if yeast is stored at -20° C and that temperature is enough to keep yeast suspension for long time, will it actually benefit from being frozen in term of viability?
 
According to the Yeast book http://www.amazon.com/dp/0937381969/?tag=skimlinks_replacement-20 by Chris White and Jamil Z., the colder the better, but it will still help preserve them if they don't 'freeze solid'. I may start using a higher percentage (50/50) to help ward against the thaw/freeze/thaw cycles -right now I have them wrapped up in those freezer gel packs (ice packs?) to help ward against fluctuations, but the extra protection of a higher concentration of glycerin (the higher concentration helps 'soften' the shock to the cells from repeated cycles) is starting to sound like a good idea. Gonna order a gallon of glycerin later today.
 
Last edited by a moderator:
I think it is definitely worth to try with higher concentration (especially with auto-defrost freezer), but any advise from experienced users would be highly helpful.
One print of the book is on its way to me, and I hope it will introduce me closely to world of yeasts, and solve some questions I got.
 
I think it is definitely worth to try with higher concentration (especially with auto-defrost freezer), but any advise from experienced users would be highly helpful.
One print of the book is on its way to me, and I hope it will introduce me closely to world of yeasts, and solve some questions I got.

I switched over to a 1:1 (glycerine:slurry) solution about eight months ago and have been getting much higher viability rates in my auto-defrost freezer. The solution never actually freezes. It's hardly been a controlled experiment, but I'm happy enough with the results that I plan to stay with this concentration.
 
That is glycerin/water:yeast slurry ratio?
How much glycerin do you mix with water?
 
diS said:
That is glycerin/water:yeast slurry ratio?
How much glycerin do you mix with water?

Nope, that's pure glycerine:slurry. Based on some semi-scientific futzing with a centrifuge, I figure that gives me about a 70:30 glycerine:water. This is for the contents of a vial. If you are harvesting, you'll probably need to add some water.
 
I've got a lot of frozen yeast. I am growing a 2.5 yr old frozen lager yeast right now (stepping up from a 15mL tube). I used 30% glycerine solution that was added to equal part yeast slurry, resulting in a final glycerine % of 15%.

Something is fermenting in there. It has krausened. Spun for almost a week, then stepped up, now fermenting again (3 liters). I'm probably growing a nice crop of bacteria.
 
Back
Top