yeast freezing

[barneyfife]

Hello all,
I am going to try my first attempt at yeast freezing following directions found on this site. My preform tubes are in the mail, but in the mean time,my wife wast throwing out her old plastic beading tubes. I was curious if anyone has used these for freezing yeast. I realize they cant be boiled in water to sterilize, but then neither can the preforms (I assume so anyway). They screw cap seems to seal tight enough, because i have filled one with water, and had it upside down for an hour, with no leaks.

Obviously they are not food grade plastic, but if soaked in Iodophor, would that be enough to sanitize? I will probably try one for the hell of it anyway, but some input would certainly help

Thanks
Barney

 

[stpug]

Lots of little concerns about using that, but who cares - give it a shot. If nothing else, freeze up some yeast with some glycerin at a specific percentage. Give it 1-2 weeks, and thaw it out to make a starter just for kicks. If you feel good about it at that time then you can even try pitching in a batch of beer, or toss it and call it purely an experiment. It feels pretty awesome to pull yeast from your freezer and then make beer with it - it just doesn't feel right (but I love it!)

I'm using some centrifugal 50ml plastic containers with blue lids and I boil them no problem.

Have fun and practice with that little tube. If you're using harvested slurry from a batch of beer then you're not wasting much as all. I don't imagine you'll be able to get more than 25ml of slurry in there and still have room for glycerin.

Play, play, play

 

[barneyfife]

Hi,
I just boiled 1 of the tubes for 3 mins, and seems good. The screw cap still screws on tight, no distortion. I will be harvesting Dennys favorite from an ale in about a week, so no big loss there if things dont turn out right. I think if I boil a tube for 5 mins, then soak in iodophor, i should be good to go.

The tube hold about 30 mls filled to the top, so should my mixture be 10 mls yeast, and 10 mls glycerin, and leave 10 mls headspace?

Thanks

 

[stpug]

Definitely leave a little headspace; 5ml should be enough.

However you decide to mix your ratios is fine, but you'll want to keep the glycerin percentage in the 15-25% range. So if you're filling each vial with 25ml then 5ml of pure glycerin is 20%, the remainder of the space can be yeast slurry (and water). You'll want to use sterile glycerin.

There are lots of varying opinions on percentages, how sterile things needs to be, diluted glycerin or not, how quickly to freeze, how quickly to thaw, etc, etc, etc... You'll have to decide what works for you and whose advice to heed. I think there's truth in everyone opinion; if your process works for your system then it's a good process. Since everyone's system is different, their processes are frequently different too.

I personally use a 50% glycerin solution (half glycerin, half filtered water). I boil in microwave for 1-2 minutes in a small glass jar and then cover with plastic wrap and aluminum foil, and allow to cool on counter to under 100F, and then into the fridge. In your case that would mean using 10ml 50% glycerin solution, 15ml yeast slurry, 5ml headspace. After filling a vial, be sure to shake it up really well to ensure very good mixing of the the glycerin with the yeast.

After I fill my vials with yeast and glycerin solution, I'll shake really well and put in fridge for a couple hours. Then I'll shake again and put them in my deep freeze.

When I want to use one, I'll prep my starter wort and get it in the flask. When I'm ready for yeast, I'll pull one from the deep freeze and put it in a container with 100F water for a few minutes making sure to stay below the lid. Shake. Add new 100F water for a few minutes. Shake. By this point (6 minutes maybe) the yeast is usually liquid again and I'll pitch into my starter wort immediately.

The first step-up out of the freezer moves pretty slow for me (1-3 days for activity). After the first step-up has finished, the next steps move fairly quickly (regular pace).

At least this how my experience has been so far, but I've only used 4 of my frozen yeasts so far.

 

[EarlyAmateurZymurgist]

Soaking the tubes in 63C (145F) water for 30 minutes will kill live microflora. One must autoclave (pressure cook) storage containers @ 121C (250F) for at least 15 minutes to render them absolutely sterile. Moist heat below 121C does not kill spores.

Preforms are usually sterilized using vaporized H2O2 (a.k.a. Vaporized Hydrogen Peroxide). Vaporized H2O2 kills microflora via oxidation.

 

[barneyfife]

Definitely leave a little headspace; 5ml should be enough.

However you decide to mix your ratios is fine, but you'll want to keep the glycerin percentage in the 15-25% range. So if you're filling each vial with 25ml then 5ml of pure glycerin is 20%, the remainder of the space can be yeast slurry (and water). You'll want to use sterile glycerin.

There are lots of varying opinions on percentages, how sterile things needs to be, diluted glycerin or not, how quickly to freeze, how quickly to thaw, etc, etc, etc... You'll have to decide what works for you and whose advice to heed. I think there's truth in everyone opinion; if your process works for your system then it's a good process. Since everyone's system is different, their processes are frequently different too.

I personally use a 50% glycerin solution (half glycerin, half filtered water). I boil in microwave for 1-2 minutes in a small glass jar and then cover with plastic wrap and aluminum foil, and allow to cool on counter to under 100F, and then into the fridge. In your case that would mean using 10ml 50% glycerin solution, 15ml yeast slurry, 5ml headspace. After filling a vial, be sure to shake it up really well to ensure very good mixing of the the glycerin with the yeast.

After I fill my vials with yeast and glycerin solution, I'll shake really well and put in fridge for a couple hours. Then I'll shake again and put them in my deep freeze.

When I want to use one, I'll prep my starter wort and get it in the flask. When I'm ready for yeast, I'll pull one from the deep freeze and put it in a container with 100F water for a few minutes making sure to stay below the lid. Shake. Add new 100F water for a few minutes. Shake. By this point (6 minutes maybe) the yeast is usually liquid again and I'll pitch into my starter wort immediately.

The first step-up out of the freezer moves pretty slow for me (1-3 days for activity). After the first step-up has finished, the next steps move fairly quickly (regular pace).

At least this how my experience has been so far, but I've only used 4 of my frozen yeasts so far.

If all goes well, i will post my results

Thank you

 

[barneyfife]

Hey all,

Just tried my first attempt at freezing a wyeast 1450, from a 2nd gen cream ale. Made a 1:4 solution glycerin/boiled H20. Then 10mls washed yeast and 10 mls glycerin solution. Put in fridge for 24 hours, then wrapped in ice pack, and put in cooler, then in freezer.

My curiosity got the better of me, so pulled a vial out after 5 days, thawed in 30C glass of water, and then pitched in a 500 ml/1.020 wort. Put on stir plate for 24 hours, then into a 2000 ml/1.030 wort and stir plate for 36 hours.

My stir plate flask is a 1 gallon apple juice jug.

Pitched into a dry irish stout, and bubbling away after 14 hrs

1. frozen yeast
2. 500 ml starter
3. 2000 ml starter

 

[stpug]

That's awesome!! Good job!! Now you can store yeast for year(s). Careful though, it starts to build up quickly in a freezer (but not nearly as quickly as jars in a fridge )

 

[barneyfife]

That's awesome!! Good job!! Now you can store yeast for year(s). Careful though, it starts to build up quickly in a freezer (but not nearly as quickly as jars in a fridge )

Thanks Pug,

I went to a brewpub the other day, which is a 4 hour round trip, and the brew master there was nice enough to give me a quart of his lager yeast and ale yeast. After washing it down a bit, i have about 200 mls of solid yeast that i am going to split up and freeze a few vials, and pitch some. I asked what strains of yeast they were, but he said it was so long ago, and about a 1000 generations old, he couldn't say. I sampled all their brews whilst I was there, and it still makes good beer:cross:

I'm stoked

Barney

 

[stpug]

Very nice!

I've heard similar stories from folks getting yeast from breweries. It seems that some breweries don't overly worry about things like "strain of yeast" like us homebrewers . Really, who cares as long as the beer tastes good, right?

 

[Biobrewer]

Hey all,

Just tried my first attempt at freezing a wyeast 1450, from a 2nd gen cream ale. Made a 1:4 solution glycerin/boiled H20. Then 10mls washed yeast and 10 mls glycerin solution. Put in fridge for 24 hours, then wrapped in ice pack, and put in cooler, then in freezer.

My curiosity got the better of me, so pulled a vial out after 5 days, thawed in 30C glass of water, and then pitched in a 500 ml/1.020 wort. Put on stir plate for 24 hours, then into a 2000 ml/1.030 wort and stir plate for 36 hours.

My stir plate flask is a 1 gallon apple juice jug.

Pitched into a dry irish stout, and bubbling away after 14 hrs

1. frozen yeast
2. 500 ml starter
3. 2000 ml starter

It appears as though you are getting yeast settling in your vial. How are you storing these? In your normal freezer? Just a word of advice: most freezers are auto-defrost and go through warming and cooling cycles to prevent ice buildup. If you want to freeze yeast and maintain good quality and viability, you will need to buy a manual defrost freezer.

I use this one:
http://www.homedepot.com/p/t/202766...talogId=10053&productId=202766304&R=202766304

Woks great.

 

[kh54s10]

I use small 20 ml plastic vials. I need to go to the website to see about the composition. I have been freezing yeast for about 2 years and have some Neobrittania from Northern Brewer that I saved from a second generation. I froze it on 11/27/11 with 5 ml yeast, 5 ml glycerin and 10 ml water. I did a 250 ml starter step last week then a 1 liter step and a 1.5 liter step. I will be pitching it in my Oatmeal Stout later tonight. Didn't plan for the cold crash so the whole 1.5 liters is going in!

I have about 10 varieties of yeast in my bank.

 

[barneyfife]

It appears as though you are getting yeast settling in your vial. How are you storing these? In your normal freezer? Just a word of advice: most freezers are auto-defrost and go through warming and cooling cycles to prevent ice buildup. If you want to freeze yeast and maintain good quality and viability, you will need to buy a manual defrost freezer.

I use this one:
http://www.homedepot.com/p/t/202766...talogId=10053&productId=202766304&R=202766304

Woks great.

Hi,
I have this one that i just purchased a month or so ago.

http://www1.thebrick.com/brickb2c/jsp/catalog/product.jsp?id=FFU13M3HW&navAction=jump&navCount=2

Told the better half we should have a good dedicated freezer. As for the settling yeast, after refrigerating, i swirled it up,(should have shook the beejeezus out of it), and put it in the freezer, with the intention of letting it "thicken up" a bit, then shake the **** out of it.
After 30 mins, the buggers were froze solid, so made mental note (shaken,not stirred)

Barney

 

[Biobrewer]

Hi,
I have this one that i just purchased a month or so ago.

http://www1.thebrick.com/brickb2c/jsp/catalog/product.jsp?id=FFU13M3HW&navAction=jump&navCount=2

Told the better half we should have a good dedicated freezer. As for the settling yeast, after refrigerating, i swirled it up,(should have shook the beejeezus out of it), and put it in the freezer, with the intention of letting it "thicken up" a bit, then shake the **** out of it.
After 30 mins, the buggers were froze solid, so made mental note (shaken,not stirred)

Barney

It appears as though it is a manual defrost. Interesting. I usually have no issues keeping my cell stocks in suspension before they freeze. Maybe a good shake will keep everything in suspension.

Cheers!

 

[stpug]

I have separation in my frozen stock as well and I shake the holy living sh*t out of it - TWICE - before it lands in a manual defrost freezer. After filling vials I shake really well and put into fridge for a little while. Then as I'm walking them to the freezer I'm shaking them really well again. The different strains tend to separate to different extents for me. However, I've been successful reviving them regardless of the separation, but ultimately I think a more homogeneous mixture would be best.

Biobrewer, can you describe your process from the point of just finished filling vials? In terms of shaking, time in fridge, how you freeze them, etc.

I shake really well and put in fridge for at least 30 minutes or up to about 8 hours. I then remove and shake really well again on the way to the freezer, where I put them in a "vial holder" (i.e. styrofoam with holes cut into it to hold the vials). That's it. The door is almost always closed until they're frozen solid.

 

[Biobrewer]

I have separation in my frozen stock as well and I shake the holy living sh*t out of it - TWICE - before it lands in a manual defrost freezer. After filling vials I shake really well and put into fridge for a little while. Then as I'm walking them to the freezer I'm shaking them really well again. The different strains tend to separate to different extents for me. However, I've been successful reviving them regardless of the separation, but ultimately I think a more homogeneous mixture would be best.

Biobrewer, can you describe your process from the point of just finished filling vials? In terms of shaking, time in fridge, how you freeze them, etc.

I shake really well and put in fridge for at least 30 minutes or up to about 8 hours. I then remove and shake really well again on the way to the freezer, where I put them in a "vial holder" (i.e. styrofoam with holes cut into it to hold the vials). That's it. The door is almost always closed until they're frozen solid.

Well, I do 1 mL stocks in which I take colonies off of a plate and mix it with 1 mL sterile filtered 20% glycerol in a sterile cryovial. When I put them in the freezer, I invert back and forth briefly once or twice in the first hour, and they are nice and uniform, which I need to be able to quickly open the top, scrape a little bit, and close it up and get it back in the freezer before thawing. I'm sure size has something to do with it.

However, putting the tubes in Styrofoam is not a great idea when trying to get the fluid inside to freeze, as it will insulate the tube really well and cause it to freeze much slowler.

Maybe try a wire rack.

 

[stpug]

Well, I do 1 mL stocks in which I take colonies off of a plate and mix it with 1 mL sterile filtered 20% glycerol in a sterile cryovial. When I put them in the freezer, I invert back and forth briefly once or twice in the first hour, and they are nice and uniform, which I need to be able to quickly open the top, scrape a little bit, and close it up and get it back in the freezer before thawing. I'm sure size has something to do with it.

However, putting the tubes in Styrofoam is not a great idea when trying to get the fluid inside to freeze, as it will insulate the tube really well and cause it to freeze much slowler.

Maybe try a wire rack.

Very good point on the styrofoam, plus I like the idea of inverting a couple times during the first hour. I think those two suggestions will help a lot.

 

[barneyfife]

I thought I read in this forum, that slower freezing is better for viability down the road, although mine froze very quickly, and was still viable a week later.

Barney

 

[stpug]

I thought I read in this forum, that slower freezing is better for viability down the road, although mine froze very quickly, and was still viable a week later.

Barney

I've read that BOTH ways are better

Same goes for thawing; BOTH ways are better

I've picked the speed of my freezer for freezing , and a quick thaw in 90-100F water. It's worked a couple times. I'll change my process and the outcome dictates

 

[barneyfife]

I've read that BOTH ways are better

Same goes for thawing; BOTH ways are better

I've picked the speed of my freezer for freezing , and a quick thaw in 90-100F water. It's worked a couple times. I'll change my process and the outcome dictates

Hmm, better pop the top on a Shaftsbury Cream Ale clone, and give this some hard thinnin Baba Looey

Quick Draw

 

[Biobrewer]

I thought I read in this forum, that slower freezing is better for viability down the road, although mine froze very quickly, and was still viable a week later.

Barney

Placing them at -20 C will give you a relatively slow freeze, certainly slower than a "rapid" freeze in something like liquid nitrogen, but not too slow as to cause cell damage. Freezing by placing at -20 C will prevent the freezing of intracellular water and allow the cells to dehydrate (via aquaporins allowing water to move across it's concentration gradient and out of the cell) as the medium around the cell freezes. Freeze too slow though, and I believe the cells get damaged by both the slow narrowing of channels of unfrozen medium and extended exposure to high solute concentrations (solute concentration increases as water begins to freeze). Freezing by placing at -20 C is completely acceptable though, and I have had no issues.

To thaw (if you are using a stock that is not for streaking a plate, but rather for dumping into a starter), thaw quickly in 37 C water to prevent similar issues related to cell exposure to high solute concentration. I keep all my stocks frozen, dunk them in 70% ethanol, take a little out with a sterile loop and streak on a plate, and put the vial back in the fridge ASAP. I then work up the colonies from plate to starter to fermentor.

 

[barneyfife]

Placing them at -20 C will give you a relatively slow freeze, certainly slower than a "rapid" freeze in something like liquid nitrogen, but not too slow as to cause cell damage. Freezing by placing at -20 C will prevent the freezing of intracellular water and allow the cells to dehydrate (via aquaporins allowing water to move across it's concentration gradient and out of the cell) as the medium around the cell freezes. Freeze too slow though, and I believe the cells get damaged by both the slow narrowing of channels of unfrozen medium and extended exposure to high solute concentrations (solute concentration increases as water begins to freeze). Freezing by placing at -20 C is completely acceptable though, and I have had no issues.

To thaw (if you are using a stock that is not for streaking a plate, but rather for dumping into a starter), thaw quickly in 37 C water to prevent similar issues related to cell exposure to high solute concentration. I keep all my stocks frozen, dunk them in 70% ethanol, take a little out with a sterile loop and streak on a plate, and put the vial back in the fridge ASAP. I then work up the colonies from plate to starter to fermentor.

Do you agree with keeping them in the fridge for 24 hours before freezing?

I will be more vigilant on the shaking as they are freezing to keep in suspension.

Thank you
Barney

 

[Biobrewer]

Do you agree with keeping them in the fridge for 24 hours before freezing?

Personally, I'm not sure what this achieves. Do you have a source for why this might be done? If I'm freezing something, I'll go straight to the freezer. I know a lot of people who do cryo, and they typically do -20 C for 3 hours, -80 over night, and then into cryo (-196 C) the next day. I think this is done to ensure that the cells don't freeze to fast. If you're just going to -20C, I'd just stick 'em in there!

 

[kh54s10]

I have some that I put into the freezer directly but now I refrigerate first. I shake them well then freeze. I have also had some that settled out. They still fermented well in the starter.

I guess I have done 6-8 with frozen yeast and all of them were successful. I did forget to make more from my 1056 though, so I will have to get another pack at some point.
Today's was Neobritannia that was frozen on 11-27-11

 

[Biobrewer]

I have some that I put into the freezer directly but now I refrigerate first.

Just out of curiosity, what is supposed to be the benefit of refrigerating first? Do you have a source that says it's beneficial? I only ask because I am a biological scientist in my day job and have done a lot of cell stocking in my day, and have never heard of anyone doing that. I know some people that cryo go from -20 C for 3 hours, to -80 over night, and then into cryo (-196 C) the next day to ensure that the cells don't freeze to fast. But, not sure I understand the benefit of refrigeration first.

 

[Biobrewer]

I have some that I put into the freezer directly but now I refrigerate first.

Just out of curiosity, what is supposed to be the benefit of refrigerating first? Do you have a source that says it's beneficial? I only ask because I am a biological scientist in my day job and have done a lot of cell stocking in my day, and have never heard of anyone doing that. I know some people that cryo go from -20 C for 3 hours, to -80 over night, and then into cryo (-196 C) the next day to ensure that the cells don't freeze to fast. But, not sure I understand the benefit of refrigeration first.

 

[stpug]

Just out of curiosity, what is supposed to be the benefit of refrigerating first? Do you have a source that says it's beneficial? I only ask because I am a biological scientist in my day job and have done a lot of cell stocking in my day, and have never heard of anyone doing that. I know some people that cryo go from -20 C for 3 hours, to -80 over night, and then into cryo (-196 C) the next day to ensure that the cells don't freeze to fast. But, not sure I understand the benefit of refrigeration first.

IIRC, the benefit of fridge then freezer was basically to more slowly bring them to their final freezing temperature. I think the idea was less temperature shock. But I've wondered if it accomplished anything (aside from yeast stress ).

I've found my patience lacking during this fridge stage and have only allowed about 10 minutes on my last couple freezer batches, and after hearing your process I'm going straight from filling the vials to the freezer.

 

[ldave]

Regarding the 'why store in the fridge for some time' question. I believe it is to give the yeast time to uptake the glycerol into the cell. Remember, since the vial contents is freezing, despite having the cryoprotectant in it, what saves the cell is having the cryoprotectant inside of it. Then, when the cell excretes water (dehydrates) as a response to the lower vapor pressure of the ice outside the cell, it effectively concentrates the cryoprotectant inside the cell, producing a lower freeze temp, protecting the cell. The underlying assumption is that it does not also excrete cryoprotectant. But this uptake of glycerol takes time. The more you give it, the more it uptakes. But. Experiment indicates too much contact time is also detrimental. Concensus is around 2 days contact time at 4 deg C. In another discussion relevant to this topic I found a patent relevant to the very question you've asked. Experiment results? 3 days contact time optimum for very low glycerol concentration (3%). Here's a link to that post:

https://www.homebrewtalk.com/f163/do-you-know-how-make-yeast-starter-then-why-not-farm-yeast-freeze-269488/index48.html#post5820998

You're obviously getting acceptable results with what you're doing, so this wouldn't be a game changer for you or anything. But it might increase viability leading to quicker starters. If viability is good enough, you don't need small starters. Just jump right up to final starter size.

 

[IvanTheTerribrew]

Just learning about yeast freezing. I have two questions:

Where do you get glycerin from?

How long does yeast that is frozen with glycerin last compared to just washing it?

Thanks ahead of time


- ISM NRP

 

[stpug]

Where do you get glycerin from?

Amazon or Walmart or Walgreens.

How long does yeast that is frozen with glycerin last compared to just washing it?

Months maybe for washed, refrigerated yeast.
Years for frozen yeast.

 

[ldave]

Glycerin:
No find at the Walgreens where I live. Walmart has it. I assume it's the animal fat sourced version. Nutrition stores have the vegetable sourced version, which is slightly more pure, and more expensive. They're chemically identical, so I doubt the source matters. Interestingly, the nutrition store in my area also had packaged pipettes (lab version of an eyedropper, easier to sanitize), which are handy for transfering fluids into little vials.

 

[barneyfife]

Hey all,

Just tried my first attempt at freezing a wyeast 1450, from a 2nd gen cream ale. Made a 1:4 solution glycerin/boiled H20. Then 10mls washed yeast and 10 mls glycerin solution. Put in fridge for 24 hours, then wrapped in ice pack, and put in cooler, then in freezer.

My curiosity got the better of me, so pulled a vial out after 5 days, thawed in 30C glass of water, and then pitched in a 500 ml/1.020 wort. Put on stir plate for 24 hours, then into a 2000 ml/1.030 wort and stir plate for 36 hours.

My stir plate flask is a 1 gallon apple juice jug.

Pitched into a dry irish stout, and bubbling away after 14 hrs


1. frozen yeast
2. 500 ml starter
3. 2000 ml starter

Just a follow up to my frozen yeast. Thawed a wyeast 1450 that i froze back in Oct/13, and did a 500 ml starter for 24 hours, and now a 2 liter which has been on the stir plate for 24 hours and it is a nice creamy color. The starter wort was from the tail runnings from a previous mash, which was about a 1.020, which I boiled, then froze. Will be pitching into a cream ale after cold crashing. Will post pictures after cold crashing.

Very pleased with the results

barney