Guide to Making a Frozen Yeast Bank

I don't know how helpful this will be for you, because I keep my frozen yeast library in a -80C non-defrosting freezer, but it might give you some idea of the range of conditions that can be used to store your frozen stocks. My method is to grow up a roughly 2inch x 2inch patch of yeast on a malt agar plate, then scrape it off and resuspend it in 1.0ml of 100% glycerol. This doesn't freeze solid at these temps -- it is easy to scrape a chunk out with a sterile toothpick when I want to make a new starter. I have not tested my viability in terms of cell count, but it is good enough for me. When I spread a chunk of the frozen stock onto a malt agar plate I get a solid patch unless I deliberately streak it out to separate individual colonies.

Sardoman said:

I don't know how helpful this will be for you, because I keep my frozen yeast library in a -80C non-defrosting freezer, but it might give you some idea of the range of conditions that can be used to store your frozen stocks. My method is to grow up a roughly 2inch x 2inch patch of yeast on a malt agar plate, then scrape it off and resuspend it in 1.0ml of 100% glycerol. This doesn't freeze solid at these temps -- it is easy to scrape a chunk out with a sterile toothpick when I want to make a new starter. I have not tested my viability in terms of cell count, but it is good enough for me. When I spread a chunk of the frozen stock onto a malt agar plate I get a solid patch unless I deliberately streak it out to separate individual colonies.

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How do you manage that? According to dow, there is no dilution of glycerol that will stay liquid at -80ºC.

MalFet said:

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.

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I think there is something that I am missing here.
Why would water be more important when we harvest from batch..?
Isn't composition of slurry same in starter as it is in washed yeast, assuming that we wash yeast few times and get nice more or less clean layer of yeasts (without too much trub)..

diS said:

I think there is something that I am missing here.
Why would water be more important when we harvest from batch..?
Isn't composition of slurry same in starter as it is in washed yeast, assuming that we wash yeast few times and get nice more or less clean layer of yeasts (without too much trub)..

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I just mean that I get the stocks I freeze from a white labs vial. Harvested yeast is typically thicker than that. To get equivalent proportions, you'll need to add some water.

MalFet said:

How do you manage that? According to dow, there is no dilution of glycerol that will stay liquid at -80ºC.

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I don't know if Dow is using the traditional scientific definition of "liquid" or the conventional understanding of the term. I can best describe my frozen stocks as a very viscous liquid. Its hardness is closer to cheddar cheese than it is to parmesan. Easy enough to scrape a chunk out with a wooden toothpick.

Sardoman said:

I don't know if Dow is using the traditional scientific definition of "liquid" or the conventional understanding of the term. I can best describe my frozen stocks as a very viscous liquid. Its hardness is closer to cheddar cheese than it is to parmesan. Easy enough to scrape a chunk out with a wooden toothpick.

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Hmm...I just chucked a vial of my glycerol in my conventional freezer and it has already frozen quite solid. Are you sure your glycerol stock isn't already significantly diluted somehow?

I would think the diluted stuff would freeze before the concentrated stuff (since water would freeze more readily).

Nightbiker said:

I would think the diluted stuff would freeze before the concentrated stuff (since water would freeze more readily).

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Nope

As Sardoman points out, too, freezing is a bit different for glycol since there's more of a graded increase in viscosity than you'd see in, say, your ice cube tray.

Sardoman said:

I don't know how helpful this will be for you, because I keep my frozen yeast library in a -80C non-defrosting freezer, but it might give you some idea of the range of conditions that can be used to store your frozen stocks. My method is to grow up a roughly 2inch x 2inch patch of yeast on a malt agar plate, then scrape it off and resuspend it in 1.0ml of 100% glycerol. This doesn't freeze solid at these temps -- it is easy to scrape a chunk out with a sterile toothpick when I want to make a new starter. I have not tested my viability in terms of cell count, but it is good enough for me. When I spread a chunk of the frozen stock onto a malt agar plate I get a solid patch unless I deliberately streak it out to separate individual colonies.

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Where did you get this freezer, and how much did it run you? I would absolutely love to get a freezer capable of maintaining -80° temps, but I had been under the impression that they are prohibitively expensive for individuals to purchase for household, private use.

emjay said:

Where did you get this freezer, and how much did it run you? I would absolutely love to get a freezer capable of maintaining -80° temps, but I had been under the impression that they are prohibitively expensive for individuals to purchase for household, private use.

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Probably works in a lab or something similar. I'd LOVE to have that capability as well, and also figured they were simply out of my financial reach.

Experiment with S-04 continues...
After stepping up starter to 1L (from 250 ml and yeast harvested and stored in baby food jars) I got 8 vials (15 ml)- composed from 15% glycerin, 15% water and 70% slurry.
They are in fridge right now, its been 24 hours and I"ll keep them in fridge 48 hrs before putting them in freezer (still have to make styrofoam box to prevent thawing damage).

One thing keeps tickling me: yeast slurry is separated on bottom of vials, despite that I shake them vigorously more times during last 24 hrs.
I know that it shouldn't be a big problem but I am wondering have anyone succeed in mixing solution without centrifuge..

Mine separate and I've successfully cultured the yeast

What size vials and how much yeast in each so that I don't have to make the mini starter? Like a wyeast pack or white labs vial ready to go.

thanks,

hopfen

hopfen said:

What size vials and how much yeast in each so that I don't have to make the mini starter? Like a wyeast pack or white labs vial ready to go.

thanks,

hopfen

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That will depend on too many factors to predict reliably, but...off the top of my head...100mL? You'll still need to make a starter, but this might let you skip a few of the steps.

hopfen said:

What size vials and how much yeast in each so that I don't have to make the mini starter? Like a wyeast pack or white labs vial ready to go.

thanks,

hopfen

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I personally have used 24ml vials for storing yeast. I use 50% glycerine & 50% slurry.

I don't think you can avoid making a starter, but I'm sure someone else can help out/comment.

I personally would recommend making a starter everytime regardless, even if i was just using a fresh vial or smack pack. According to mr. Malty a typical 1.060 wort (5 gal batch) requires 208 billion cells which is a little over two vials.

emjay said:

Where did you get this freezer, and how much did it run you? I would absolutely love to get a freezer capable of maintaining -80° temps, but I had been under the impression that they are prohibitively expensive for individuals to purchase for household, private use.

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Here is a cheap one:
http://www.cooler-store.com/sanyo_ultra_low_temperature_vip_plus_freezer_mdf_c8v1_8720_prd1.htm

The -40C freezers are a little more reasonable.
Still a bit much to save a few dollars on yeast.

I don't have a problem with making a starter and plan on it every time. The directions in the first post said to make a mini starter then make a regular 2 liter starter. I'm trying to skip the first step.

I want to save yeast in vials that I can make a regular starter from.

thanks,

hopfen

emjay said:

Where did you get this freezer, and how much did it run you? I would absolutely love to get a freezer capable of maintaining -80° temps, but I had been under the impression that they are prohibitively expensive for individuals to purchase for household, private use.

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I don't own any such freezer, I just have access to one. The owner allows me to store my strain library in it.

You are right, these things start at several thousand dollars for the smallest, least expensive units. They also put out a lot of heat and probably use a lot of electricity. -80C is not necessary for storage of a strain collection. Any non-defrosting freezer would work just fine.

Sardoman said:

I don't own any such freezer, I just have access to one. The owner allows me to store my strain library in it.

You are right, these things start at several thousand dollars for the smallest, least expensive units. They also put out a lot of heat and probably use a lot of electricity. -80C is not necessary for storage of a strain collection. Any non-defrosting freezer would work just fine.

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Yeah, but the -80° temps are capable of sustaining yeast almost indefinitely.

Looks like I need to befriend somebody similar

So I found out that While Labs vials are 35mL

How much yeast do I need to put in them to allow me to make a 2 liter starter?

thanks,

hopfen

hopfen said:

So I found out that While Labs vials are 35mL

How much yeast do I need to put in them to allow me to make a 2 liter starter?

thanks,

hopfen

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Like I said, there are a lot of assumptions going into any number, but (based on my playings with a hemocytometer) 100mL sounds about right to me based on good quality, average storage times.

How is 100mL going to fit in a 35mL vial?

hopfen said:

How is 100mL going to fit in a 35mL vial?

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It's not going to. Your frozen sample is going to have low viability, and it will be half-glycerin. You're simply going to need more volume of the frozen stuff than of the fresh stuff.

hopfen said:

How is 100mL going to fit in a 35mL vial?

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White Labs uses a process that highly concentrates the yeast to a much higher density than typical slurry.

Thanks,

That's the part I was missing.

So if I have yeast that isn't frozen, (been in fridge in a mason jar from last week's brew), I can take 100mL and make a new 2 liter starter?

How long will it be ok in the fridge before I should freeze it?

hopfen said:

Thanks,

That's the part I was missing.

So if I have yeast that isn't frozen, (been in fridge in a mason jar from last week's brew), I can take 100mL and make a new 2 liter starter?

How long will it be ok in the fridge before I should freeze it?

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If it hasn't been frozen, then you want to use something like the Mr. Malty pitching rate calculator to figure out how much slurry you need. That's largely a separate issue.

48h in the fridge before freezing is fairly standard.

I'm using Mr Malty and now all of the pieces are fitting together.

So, if I don't have enough slurry for the batch that Mr Malty says I need, it sounds like I can take 100mL of slurry and make a new 2 liter starter, correct?

Wow, 48 hours - I thought you could go 2 weeks after washing yeast and storing it in the fridge.

thanks for all of the info.

hopfen said:

I'm using Mr Malty and now all of the pieces are fitting together.

So, if I don't have enough slurry for the batch that Mr Malty says I need, it sounds like I can take 100mL of slurry and make a new 2 liter starter, correct?

Wow, 48 hours - I thought you could go 2 weeks after washing yeast and storing it in the fridge.

thanks for all of the info.

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Yep. You can make a starter out of slurry. In fact, it's a good idea.

Re: time in fridge. I misunderstood your question before. Many people stick their samples in the fridge for 48 hours before transferring to the freezer to let the yeast build up trehalose reserves. I now understand that you were asking about the maximum possible time. Slurry, if properly stored, can stay in the fridge for a long time. Months even. But, if you generally want very, very healthy yeast going into the freezer, since the long storage process is generally pretty hard on the cells. Generally, I would build a starter up and then freeze that slurry if I had a stock that had been sitting around for a couple of weeks.

this is probably a stupid question, however i still feel inclined to ask. would it be possible to store the yeast sample in pre-sterilized transfer pipettes, and heat seal the end? I've any disadvantages?

Greetings frozen yeast ranchers. I will be starting my own bank very shortly. I have all the equipment but I was waiting on a few items before I really got into this.

I will be using the cynmar 16ml glass vials that are mentioned. A thing of note to anyone using these vials, I have a semi cheap way to hold them individually without a PITA DIY. I believe and will confirm in a few days, that a 12/10 gauge shotgun shell case will hold 25 vials with no issue. I thought a 16/12 gauge would work but it is just a bit much to get them in and then back out. So I will use those for their intended purpose...the 12/10's are on the way.

My plan is to use the case to hold 10, gen 1 vials with 1 strain stretched between 2 boxes. I ordered 4 boxes from Midway, the shipping was a bit nasty but I require a good way to store these in my freezer, so I can currently have 10 strains in my bank.

What is the longest time someone has frozen and then revived a specimen?

I'm in the planning stages of yeast farming/freezing. I think this is an excellent solution to managing yeast needs. I'm trying to get a handle on numbers and I'd like opinions on my very rough calculations and assumptions. Bear in mind I'm still a brewing rookie (only a year and about 25 batches). I'm looking for practical numbers to be used for everyday brewing.

Start with 1 Wyeast pack = 100B
2L Starter @ 1.040 OG = 311B yield (from Mr. Malty)
# needed for 5gl batch @ 1.050 OG = 175B (again Mr. Malty)
# needed for 1L starter for 5 gl batch = 58B
# needed for 1L starter if stepped up once = 19B
Assume 60% viability after freezing of the 19B = 11.6B

So, if I plan on only stepping up my starter only once (I don't want to babysit a starter for a week) I need to freeze 11.6B cells initially. So from my initial starter yield of 311B cells, I can potentially harvest enough cells for 11 5gl batches, call it 10 to be conservative. So if I allow my initial 2L starter to settle, then separate into 10 equal vials, I'll have sufficient quantity to step up once and pitch a 5gl batch.

So, what do you think? Any major problems with my assumptions? Thanks.

You are not going to have anywhere near 60% viability after freezing. If I remember correctly it will be more along the lines of 1-10%. Ideally you should baby it up from the freezing state. My first step from 10 mL frozen yeast is 200 mL of sterile 1.020 wort with a small amount of nutrients in order to reduce osmotic stress on the yeast, but a 50 ml step before that would be even better.

I am developing my ability to have a yeast bank and sorting out questions that enter my mind. I have my microscope, hemocytometer, rather than a stir plate, a rotating flask apparatus, a refrigerator/freezer kegerator conversion and a good deal of hope. I have about 100 lbs of trehalose from a extraction/purification project with trehalose a decade ago. Since the buildup of trehalose within the cell is important for cell protection, does anybody know if the addition of trehalose at the point of refrigeration will assist the yeast and improve viability? Does it just make slackers out of the yeast and result in them expecting assistance and from then on they don't do a lick of work?

A comment on sterility and the use of pressure cookers. Sterility can be gotten by just putting the glassware in an oven at about 250F for a couple hours. The oxidation of the organics is very thorough. When I was working on trehalose purification, I was able to produce non-pyrogenic material using lab glassware that had been "cleaned" for pyrogenic material in this fashion.

(Since the word pyrogenic is rather obscure and probably not used outside medical circles, I should explain. It refers to the ability to induce a fever in an immune system. Basically, if you do the oven thing, there will not be enough biologically active organics present for your immune system to detect. My guess is that will be great for yeast. Just for clarity, I'm a Chem. E. with a sugar background. I had never heard of the word before either.)

I actually knew what a pyrogen is

But that's coming from more of a medical background, heh.

I use a few yeast strains for most of my beers, and have been pretty successful at keeping them around by simply washing or planning brewdays for transfer days and repitching a portion of the yeast cake. I use a commercially harvested strain a lot (Bell's) and my last batch of washed yeast was used up without ever being replaced because I am the one who washes and my brew friends use some and also transfer stuff for me when I can't get to it, so they dump the yeast.

Anyway, I just harvest a bunch of Bell's again, so I am going to freeze some this time! I got a pressure canner for my birthday last week, and found the glycerin at WallyMarts. I have several years experience growing/saving bacteria from aliquots in sterile conditions, so I have the process down just fine.

I was thinking of going with larger vials (16mL) so that the I have a larger yeast volume to go into the first starter step of ~100mL. Is anyone having success with this size tube? And is there any reason that the smaller centrifuge tubes would be better? (Space is not a problem since we have a dedicated brewing freezer).

Also, what is everyone using to transfer yeast to the vials? I think I have one of those green plastic auto pipettes, so I was thing of just getting a few glass pipette tips in the 10 mL volume.

Thanks in advance for the help,

I went with the 16mL vials.... Just finished filling 12 vials with some Bell's yeast I grew up from a bottle.

Uhm, I know I missed it, but where are you getting those vials? I'd love to have a batch of them -would really prefer larger ones (16 is such a small amount) -I've BEEN using sterile sample containers like we use in the hospital (110ml). I've never tried to harvest from a commercial bottle -and didn't know Bells bottle-conditioned. I love their brown ale -had it out of state once. Will have to see if I can find it and harvest some.
-thanks. Those vials look GREAT (love the tan look of yeast!)

Nightbiker said:

Uhm, I know I missed it, but where are you getting those vials? I'd love to have a batch of them -would really prefer larger ones (16 is such a small amount) -I've BEEN using sterile sample containers like we use in the hospital (110ml). I've never tried to harvest from a commercial bottle -and didn't know Bells bottle-conditioned. I love their brown ale -had it out of state once. Will have to see if I can find it and harvest some.
-thanks. Those vials look GREAT (love the tan look of yeast!)

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I got mine through my LHBS, but it was a special order, not something they normally carry. One source might be Cynmar $7.50 for 12 vials. IIRC they have a minimum order quantity of $25, but you might pick up some other things as well. Otherwise ebay/amazon/etc.

Nightbiker said:

Uhm, I know I missed it, but where are you getting those vials? I'd love to have a batch of them -would really prefer larger ones (16 is such a small amount) -I've BEEN using sterile sample containers like we use in the hospital (110ml). I've never tried to harvest from a commercial bottle -and didn't know Bells bottle-conditioned. I love their brown ale -had it out of state once. Will have to see if I can find it and harvest some.
-thanks. Those vials look GREAT (love the tan look of yeast!)

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I ordered mine from Cynmar. The $25 minimum order is easy to surpass with just a few sets of vials, and they have the bigger ones you seek.

Thanks! I'll give 'em a look-see. I'm assuming that borosilicate glass (it says that the cap is even autoclavable -which is excellent) is also freezer safe?