Do you know how to make a yeast starter? Then why not farm yeast and freeze it?

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Wow, 2 weeks in a defrost freezer? I would assume adding freezer packs for stabilizing would help for longer term storage, but that is still interesting to know.

My thoughts are that the day in the fridge will help with glycogen build up storage in the cell, and not glycerol diffusion, that should happen rather quickly.

This is good stuff. Should drive the point home how easy it is to make your own ready for starter yeast library. I've been using vials, maybe 3/4 inch compact slurry in the bottom (2-3 mls worth) and stepping up starting at 200ml. They're ready in 1-1.5 L within 48 hours.
 
Was only two weeks because I got impatient (that and we were limited on freezer space :lol ) and I've been toying with the process. Additional to those was a vial I placed in some steaks I have in the freezer with the same mix but a lot less cells and I anticipate very VERY long term storage out of it. Once I have a bit more freezer space to work with I'm going to pick up one of those insulated lunchboxes and go the frozen coldpack route. I can't speak for the super-longterm currently but there are enough people above saying it works pretty well.

It does seem to make a very noticeable difference to at the very least give it some fridge time before freezing it. I wonder, however, if you were to chill it in ice water (or just ice) until super cold before freezing it if you'd get the same results or if it's the time the yeast has to either build up glycerine inside the cell and/or it's glycogen stores as you mention above.
 
Oh, and as a sidenote I did have a brain fart previously a couple years ago and placed my washed yeast in the freezer and it seemed to have killed the yeast quite efficiently (all slurry combined after thaw and no activity in 5 days in a starter). And it was only in there for two days before I realized my stupidity and took it out. If any did manage to survive it couldn't have been that much.
 
Accidic said:
Oh, and as a sidenote I did have a brain fart previously a couple years ago and placed my washed yeast in the freezer and it seemed to have killed the yeast quite efficiently (all slurry combined after thaw and no activity in 5 days in a starter). And it was only in there for two days before I realized my stupidity and took it out. If any did manage to survive it couldn't have been that much.
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Well, I guess you just proved the importance of glycerine, in case anyone asks! Again, it's interesting to know the limits of these things. If I had to guess, I would have thought there would be at least some fraction of superhero cells that made it through the freezing process. Though freeze/thaw cycles are a normal cell lysis protocol for cell work, so I guess there's some validity there.
 
seriously awesome! this is exactly how we will have to do things here in El salvador with our micro brewery. We gotta be yeast farmers!:tank:
 
There may have been a small number of cells but in the timeframe I gave them they did not multiply enough to have visible viability. I had an old wl vial that had gotten packed (forgot I had it honestly) when I escaped Cali and shipped ups in the summer. High heat + ~20 month old vial behaved the same way. There were probably some living cells and maybe I could have nursed enough back to use it but made more sense to just but another vial. If I couldn't have gotten the strain again tho I probably would have pushed harder.
 
This one of the most fascinating threads I've ever seen here! +1
Two questions:
1 We seem to be referring interchangeably to glycerine and glycerol. I just wanted to know if it really is the same product, and if any kind of glycerine would do, or do you need "Food grade" stuff.
2. Has anyone tried this with any other anti-freezing agent? Agar, Gelatine, Alcohol? Rockey-Road ice cream? I'm just curious. :)
 
Glycerin, Glycerine, Glycerol = same stuff. I would use pharmaceutical grade because it will be sterile when you buy it. I don't know what the price is in the drug store but it should be readily available. If you don't see it ask the pharmacist. You can save on the volume if you let your yeast settle out first and pour off the wort. Only about 5% of the total volume of the starter is yeast. Bring that up in a 15-30% solution made up in boiled water and you should be fine. Doing all this at refrigerator temps should work well.
 
Seeing as right now my kitchen feels about as cold as the inside of my fridge that shouldn't be a problem. :) Thanks for the info. What about using other stuff? Anyone try?

Also, if I can't get my hands of sterile glycerin, can I sterilize it just by boiling the mixture, or would that ruin it somehow?
 
You can sterilize it in a pressure cooker just like wort. I use USP grade, but drug store glycerine works just fine. I'll dilute it with water to about 40%, sterilize it in a pressure cooker, store and dilute that 1:1 with yeast slurry before cooling/freezing. Never tried agar, etc, but you really need a cryoprotector, something that penetrates the cell to allow it to stretch and not fracture while freezing.
 
Brewitt said:
I haven't done anything toward plating out the cells and counting yet. Probably be next month now given the way time is going. However, I did freeze up about 1/3 liter worth of cells in 30 ml 15% glycerol by chilling to refrigerator, then freezer, then ultracold freezer (-80C) temperature. I thawed that out last night and used it to start a 1.5 liter starter. This morning it is full density and cold crashing. I will keep that cold, dump the slurry of settled cells into 1.5 liter of fresh wort Sunday morning and use it to start my fermentation.

I think this is probably an equally good plan for the general user. Grow up a gallon of starter, split it into 8 half liter portions, cold crash, freeze the cells in 15% glycerol and use one of those to start an overnight starter of up to a gallon as needed. I will do the same without the ultracold step next time and see whether I get equally satisfying results for the home brewer. As long as there is not a significant lag, it should be great. Always good to wake up your cells for a bit before pitching anyway so the overnight is a good idea.
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Glad to see some results starting to come in. I've been crazy busy lately and haven't been keeping up. Keep it coming. Thanks Brewitt.
 
Small update. Finally getting around to brewing after a busy couple months.

I thawed out 6 samples on Wed. All Wyeast 1272. 2 of them were just over a year old, frozen immediately with no refridgeration step and 35% glycerine. The other 4 were just frozen a couple months ago. 1 @ 35% glycerine, no refridgeration, 1 @ 35% glycerine with refridgeration, 1 @ 50% glycerine no refridgeration and 1 @ 50% with refridgeration. All samples were thawed quickly in a 70F water bath and swirled occasionally. Took about an hour and a few water changes to bring them up to temp.

All of the samples showed signs of fermentation after 12 hours. Color change and or condensation in the starter vessel. The younger samples finished quicker. The samples that were refridgerated finsihed the quickest. No significant difference noticed in the samples with 50% glycerince vs 35%.

So, refridgeration before freezing and quick thaw appear to help with viability. Next, I will try lowering the glycerine level down to 15% as Brewitt has been doing. If it works just as well, why waste glycerine.
 
Sweet! Nice to know the amount of glycerine really doesn't matter that much. Were all the samples the same amount of slurry?
 
BBL Brewer, thanks for the report. Sounds like what I learned from the literature is proving correct. Sorry I have not gotten to my analysis yet. I have been drowning in work. Hopefully get back to it soon. I did 30 and 15% glycerol so we should have the whole range covered.
 
All samples were 100 ml with an estimated cell count of 100 billion. Just pitched them all tonight, got plenty of beer coming down the pipeline now.
 
If I had a White Labs vial and didn't expect to use it for a while, could I pour off some of the liquid in the vial, top it off with gylcerine, shake to mix well, and then just freeze it?
 
Nice... just need to figure out the most sanitary way to dispense the glycerine then. I have 10ml pipettes, but I feel like there's got to be a better way.
 
You could star san the pipette and rinse with sterile water if you have any. Also, depending on how much glycerine you use, make sure you leave enough head space in the vial for expansion upon freezing.
 
I also have a gallon of 85% phosphoric acid (used for yeast washing, pH adjustments), and an alcohol lamp that may be somehow useful.

This is the one area where I feel like "sanitize" is not enough.
 
Sterilize is a better word. Try to pressure cook everything, including pipettes if possible. I just pour the glycerine from the sterilized jar.
 
Thanks everyone for a fantastic thread! I followed the steps and this is what I've done:

I made a 4.5 liter starter of Denny's Favorite (Wyeast 1450), cold crashed it for 2 days, decanted almost all the wort, and then froze 4 30ml (8 dram) vials with a 15% glycerin solution. I made only 4 vials because I wanted the rest of the slurry for my next batch of beer.

So, my question is this: how big a starter can I pitch one of my vials into? According to: the yeast propagation guide it is recommended to use less than a 200 fold increase in starter size. But I'm confused, what is the original starter concentration?

Here's my concentration: When I decanted the 4.5L of wort, the slurry was pretty compact. Maybe not as compact as a White Labs vial, but pretty stiff. After letting the 15% glycerol / slurry solution sit in the fridge for two days (before freezing, like you all suggested above) I saw separation between the glycerol, wort, and slurry. I estimated about 15 - 20 ml of slurry (including wort), but maybe about 7 ml of compact slurry (without wort).

So, can I pitch one vial into 5 liters of wort again to make more vials? Or can I pitch it into 2 L of wort for a beer starter? Or should i pitch into 500 ml and step up to the next level?

I'm basically confused about how the 200-fold increase was determined -- what concentration of yeast were they talking about?

Thanks for any help! This is probably my favorite thread on the whole board. :) Playing with yeasties is almost more fun than making the beer itself. hehe.
 
200-fold seems pretty steep. I usually dump around 7-10ml of compacted slurry in the vial into 0.5L wort, and it's going good the next day. I wouldn't stress them too much right out of the freezer by underpitching the starter, and you could easily step that up to 2L the next day.
 
Kurisu said:
So, my question is this: how big a starter can I pitch one of my vials into? According to: the yeast propagation guide it is recommended to use less than a 200 fold increase in starter size. But I'm confused, what is the original starter concentration?

Here's my concentration: When I decanted the 4.5L of wort, the slurry was pretty compact. Maybe not as compact as a White Labs vial, but pretty stiff. After letting the 15% glycerol / slurry solution sit in the fridge for two days (before freezing, like you all suggested above) I saw separation between the glycerol, wort, and slurry. I estimated about 15 - 20 ml of slurry (including wort), but maybe about 7 ml of compact slurry (without wort).

So, can I pitch one vial into 5 liters of wort again to make more vials? Or can I pitch it into 2 L of wort for a beer starter? Or should i pitch into 500 ml and step up to the next level?

I'm basically confused about how the 200-fold increase was determined -- what concentration of yeast were they talking about?

Thanks for any help! This is probably my favorite thread on the whole board. :) Playing with yeasties is almost more fun than making the beer itself. hehe.
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I would try and focus more on pitching rates/cell counts and worry less about your step up ratios. Just use a reliable pitching rate calculator like Mr. Malty or Wyeast and grow up a known, estimated amount of cells. Then split up your slurry into equal volumes before freezing. It's kind of hard to estimate cell counts and pitching rates by slurry volume alone. This is why I use a pitching rate calculator to grow an estimated 500 billion cells and split the slurry into 5 equal portions. This way I have an estimated 100 billion cells in each frozen jar of yeast (the same amount as a smack pack) and can easily use the pitching rate calculator to determine what size starter to make for the gravity beer that I'm brewing. Once you brew enough batches, you'll get a feel for how big of starter to make and you won't have to rely as much on the calculators, but that's where I would start.
 
I just found this thread and wanted to add some info from expirience
I have been freezing yeast for few months, i have around 100 2ml size vials of 5 or 6 different strains frozen. I use 60% glycerol solution at 1:4 volume ratio (4 of yeast slurry) so only around 15% glycerol at final concentration. I harvest yeast from the starter im making for brewing, basically i leave some of the starter behind in the flask and i add fresh dme solution or wort of low SG and let it go for around 48h or until fermentation significantly slow down, then i cold crash it for a day or 2 and dump liquid part, then transfer to smaller container, let it sit in the fridge for few hours to settle down to get really dense slurry remove liquid part again and mix the rest with glycerol. I tried centrifugation once to get even higher concentration but it was very hard to mix with glycerol so i dont do it anymore.
I freeze 1.5ml size aliquots and i use this: fisherSci.com - Research Products International Cryo Freezing Container
this container ensures that cooling rate is around 1 degree per minute. Im lucky to have an access to -80 freezer so i freeze and store my vials in it.
I only used one frozen vial from my stock, it was 1+ month old freezing, i warmed it up in my hand and when all ice melted i sanitize it and drop the content in the flask with around 500ml cooled DME solution and let it go on the stir plate, i dont remember exactly how long i had to wait for signs of fermentation but i remember i was surprised how fast it took off (im almost positive it was way below 4h to see nice airlock activity), but next time i will probably do the step starter with 100ml volum at the begining.
This year i was brewing all different recipes and using different strains so i was building my collection of yeast in the same time but starting in spring i will repeat some of my brews and in the same time i will check how the freezing is working
 
BBL_Brewer said:
I would try and focus more on pitching rates/cell counts and worry less about your step up ratios. Just use a reliable pitching rate calculator like Mr. Malty or Wyeast and grow up a known, estimated amount of cells. Then split up your slurry into equal volumes before freezing. It's kind of hard to estimate cell counts and pitching rates by slurry volume alone. This is why I use a pitching rate calculator to grow an estimated 500 billion cells and split the slurry into 5 equal portions. This way I have an estimated 100 billion cells in each frozen jar of yeast (the same amount as a smack pack) and can easily use the pitching rate calculator to determine what size starter to make for the gravity beer that I'm brewing. Once you brew enough batches, you'll get a feel for how big of starter to make and you won't have to rely as much on the calculators, but that's where I would start.
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That's fine, except that how much yeast you have after X litres is completely dependent on how many yeasts you start with. So if you start with frozen slurry (that you didn't split up equally, or even if you did and you weren't perfect to the .1 ml) you need some way to estimate the slurry. Else you'll be off on everything else by a _very_ large margin.

I just wish I could find some way to estimate slurry density. On Jamil's site he says 1 b/ml is an "easy to pour slurry" while 4.5 b/ml is the yeast packed into the bottom of a white labs vial. Now, it that the yeast sludge only, or the yeast + the half vial of clear wort in the white labs vial? And is the 1 b/ml the yeast sludge only, once the "easy to pour" slurry has settled overnight in the fridge, or the yeast and wort together which are easy to pour?

See, very confusing.
 
Kurisu said:
That's fine, except that how much yeast you have after X litres is completely dependent on how many yeasts you start with. So if you start with frozen slurry (that you didn't split up equally, or even if you did and you weren't perfect to the .1 ml) you need some way to estimate the slurry. Else you'll be off on everything else by a _very_ large margin.

I just wish I could find some way to estimate slurry density. On Jamil's site he says 1 b/ml is an "easy to pour slurry" while 4.5 b/ml is the yeast packed into the bottom of a white labs vial. Now, it that the yeast sludge only, or the yeast + the half vial of clear wort in the white labs vial? And is the 1 b/ml the yeast sludge only, once the "easy to pour" slurry has settled overnight in the fridge, or the yeast and wort together which are easy to pour?

See, very confusing.
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In that case, for your current vials, just use good judgement and make a slightly bigger starter than you think you'll need and brew away. Even if you slightly over or under pitch it won't have a severe impact. We're merely estimating here anyway, even with a starter made from a store bought vial or smack pack. The only way to determine exactly how many viable cells you're pitching would be to do cell counts like with a hemacytometer. My advise is not to worry so much and brew a few batches with your frozen yeast. Experience is the best teacher.
 
Really this is a great thread but I got tired eyes trying to read & absorb all the info & opinions. It became a bit difficult to know who said what by th end.
I'd like to ask if the original poster might be prepared to sum up his current beliefs taking into consideration the other contributions & cover the following specific points:
1) Initial harvesting - simple procedure how to with washing or without, what's meant by oxygen shots etc.
2) Individual sample size - never mind the cell count, what is a realistic sample size to start a 5 gallon brew & what is a practical container for such samples.
3) Nutrient & other "desirable additions to enhance yeast survivability what is practical How to achive.
4) Glycerol/glycerine recommended simple mix by volume suggestions.
5) Freeze & thaw rates - important or not, suggested procedure if control is needed.

As I said great post but I believe a simple reliable summary would help further.
Thanks again
 
peterlonz said:
Really this is a great thread but I got tired eyes trying to read & absorb all the info & opinions. It became a bit difficult to know who said what by th end.
I'd like to ask if the original poster might be prepared to sum up his current beliefs taking into consideration the other contributions & cover the following specific points:
1) Initial harvesting - simple procedure how to with washing or without, what's meant by oxygen shots etc.
2) Individual sample size - never mind the cell count, what is a realistic sample size to start a 5 gallon brew & what is a practical container for such samples.
3) Nutrient & other "desirable additions to enhance yeast survivability what is practical How to achive.
4) Glycerol/glycerine recommended simple mix by volume suggestions.
5) Freeze & thaw rates - important or not, suggested procedure if control is needed.

As I said great post but I believe a simple reliable summary would help further.
Thanks again
Click to expand...

I was actually planning on revising the original post but I was waiting for Brewitt to complete some bioassays first so that I would have some actual data to back up my conclusions. In the mean time, I will try and address your concerns.

1. Where you get the yeast is completely up to you. I prefer to start from a fresh smack pack so I know it is contaminant free. If you want to harvest, go for it, just be as sanitary as possible. As for yeast washing, I've never done it and don't plan to. My frozen yeast is inexpensive enough that I use it once and don't do any additional harvesting. As for shots of oxygen, I use a stainless steel airstone with an oxygen tank. I use pure oxygen to aerate the wort.

2. Honestly, it doesn't matter how much yeast you freeze. As long as enough cells survive the freeze, you can make a starter and build back up a sufficient population to brew with. You expressed that you are not concerned with cell counts. Well, without knowing a rough estimation of what your cell counts are, you won't be able to determine what size starter you need to hit your pitching rates for a 5 gallon batch. With that said, I freeze 100ml of slurry/glycerine solution in 4oz jelly jars. I do not plan to deviate from this practice as this works well for me for 5 gallon batches. However, some people like the vial method, so again, it's up to you to decide which method you like the best.

3. This is just basic starter methodology. Make sure the yeast have plenty of oxygen, use a starter wort with gravity between 1.030 and 1.040, and add a little bit of yeast nutrient to the wort.

4. As for the glycerine mix, I'm still experimenting with that myself so, it's kind of hard to give you a concrete "here's exactly how much you should use" answer because it's debatable. I have consistently used a 35% solution and it works. So, if you would like to try this concentration, put 131ml of glycerine in a pint jar and fill the rest of the way with water. Shake it up to mix evenly.

5. Again, it's hard for me to give you exact advice on this stuff becasue I don't have any hard data to back it up. I've been doing my own experiments, but the results are qualitative not quantititative. Refridgerate the yeast for 24-48 hrs before putting in the freezer. At home, I doubt you will able to accurately control freeze rate. However, thaw rate can be controlled. How you thaw it depends on the container you decide to freeze in. Last time I brewed I thawed my 4oz jelly jars in a 70F water bath and it worked pretty well.
 
HolyGhostBrew said:
great Thread.....one question, do you pressure cook the water/glycol mix before you mix it with the yeast?
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Yes. I pressure cook everything that I can. This includes the glycerine solution, starter wort and the jars I freeze the yeast in.
 
HolyGhostBrew said:
Thanks for clearing that up....i wasnt sure if it affected the glycol if it were pressure cooked.
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I noticed you are asking about "glycol" I hadn't noticed that in your first post. You need to use food grade Glycerine (glycerol) not glycol. And especially not ethylene glycol (antifreeze) as it is toxic and could kill you. Although propylene glycol is non toxic I haven't heard of anyone using it to freeze yeast.
 
I just wrote a fairly lengthy novel, then lost it because my session timed out... frustrating... anyway, this is my first post here, I have been searching for a good homebrew forum for quite some time now, that has active members that are interested in not only following directions but innovating the hobby as well. After reading this thread, I joined.

I propagate yeast using yeast slants. Avoiding the lengthy background I wrote with my lost post, due to a lack of time, I skip to my question.

I am concerned with propagating yeast blends. If one yeast strain in the blend is more aggressive than the other, it will not take long for the more aggressive strain to completely take over, this could even happen in the starter culture.

What are your thoughts on this? Do you take this into consideration? (BBL this isn't necessarily directed at you since you mentioned you typically use fresh yeast each time)

Thanks!

-Max
 
I don't know why you would want to culture (propagate) yeast blends. Why not just keep the two strains seperate? If you want to blend them to brew with, just do that right before you pitch. If you are worried about contamination from wild yeast eventually taking over after repeated re-culturing, then work from stock cultures. Use a streak plate to isolate a pure colony every time you make a stock culture and you should never have any problems provided you technique is sound.
 
BBL_Brewer said:
1. Where you get the yeast is completely up to you. I prefer to start from a fresh smack pack so I know it is contaminant free. If you want to harvest, go for it, just be as sanitary as possible. As for yeast washing, I've never done it and don't plan to. My frozen yeast is inexpensive enough that I use it once and don't do any additional harvesting.
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You should consider yeast washing if you repeat a recipe. It isn't just for cost benefit, the first pitch is not necessarily going to perform the best.
 
Bbl I'm speaking about using pre made blends like White labs 080 cream ale blend, where I do not know what exact yeast strains are used in the blend. I do use petri dishes, but find better success in using yeast slants. Hard to isolate strains but less contamination.
 
mliptack said:
Bbl I'm speaking about using pre made blends like White labs 080 cream ale blend, where I do not know what exact yeast strains are used in the blend. I do use petri dishes, but find better success in using yeast slants. Hard to isolate strains but less contamination.
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For your purposes, streaking plates is absolutely necessary. BBL was right that the only way to do what you want is to keep the strains separate.

And since they come to you blended, the only option you have is to painstakingly identify and separate the individual strains out on plates. And then store them individually.
 

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