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

[diS]

This experiment sounds great, I am highly interested about the results.
Great job Brewitt!

 

[Kurisu]

Wow, I can't wait for the results Brewitt! This is the kind of experiment that homebrewers will quote to each other for years.

Science: it works.

 

[Brewitt]

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.

 

[dannedry]

Wow... one of the coolest threads I've seen on here... as an engineer, this is very fascinating! Great work guys, I can't wait to see the final results! AWESOME!!!

 

[Accidic]

I don't have the -80 degree freezer to work with but I did tests with some of the extra washed yeast (washed twice, separated out, pouring off the excess, and resuspending for division into glasses which was done by weight) I knew I wasn't going to use. With ~30% by volume glycerine I did three batches (WLP001). One went into the Freezer immediately after mixing and shaking, the other two were placed into the fridge and one was shaken vigorously every day for three days.

They were left in the freezer for 2 weeks (+ 3 days for the straight to freezer one) and all three brought out and added to Malta based starters (I had extra bottles, sue me). The one put straight into the freezer was actively started within just under 60 hours. The other two were active within ~30 hours. The shaken daily while refrigerated before freezing one was active *MAYBE* 4-6 hours faster, but just over 30 hours later they seemed equal so I don't know if that was coincidental or not.

All three were done in 1 cup freezer safe jars (Ball) and pitched to 1L flasks for starting (@~750ml of wort) and ran on stirplates with a 1.5" stirbar until my curiousity was satisfied. All three seem to have washable yeast that looked quite healthy and the starters tasted pretty much identical to my tastebuds. I know the above is far from scientific but I didn't have fancy things like microscopes and cyclotrons (centrifuge but cyclotron sounds so much cooler!) to work with.

 

[PoppinCaps]

All three seem to have washable yeast that looked quite healthy and the starters tasted pretty much identical to my tastebuds. I know the above is far from scientific but I didn't have fancy things like microscopes and cyclotrons (centrifuge but cyclotron sounds so much cooler!) to work with.

That's awesome, thanks for the results! So refrigerate for a few days before going to the freezer? Sounds like they need some time to get their parkas on.

And hey, if they taste and look good (well as good as a starter can), who needs fancy microscopes?

 

[Brewitt]

The chilling at refrigerator temperatures for several hours seems to make a significant difference. Not sure if giving them several days should make a difference. My culture grew very well and was at full density after overnight with shaking. Remember, if you start with a good dense slurry of yeast you are not asking for very much growth to reach a maximum density starter. The cells only need to go through a few rounds of division and they are there. I do think there is a very substantial advantage to getting your starter going overnight rather than just pitching it. My fermentation that is just finishing up was actively bubbling away in 4 hours after pitching with couple liter overnight starter made with my tube of glycerol frozen yeast, thawed in 100 F water with shaking until just thawed. Based upon this experience I think this is a very good approach and the one I will be using. I am going to make up half a dozen tubes of frozen cells of a couple different yeasts over the next couple weeks and keep them for exactly this purpose.

 

[Accidic]

I agree but my question is does the refrigeration thing only chill it to reduce the size of the resulting ice crystals or do the yeast cells have more time to imbibe more glycerine into their cells preventing even further cell damage? At least that was my impression of how the glycerine aids it if it isn't in high enough volume to completely prevent freezing...

 

[Accidic]

Btw, per mr malty I was around only 40-45b cells. I wasn't holding them for a batch after all but just for testing.

 

[Accidic]

Oh, and I should probably mention mine was from a self defrosting freezer. If I go library this route I will use icepacks but did not this time.

 

[PoppinCaps]

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.

 

[Accidic]

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.

 

[Accidic]

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.

 

[PoppinCaps]

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.

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.

 

[Andynewbom]

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!

 

[Accidic]

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.

 

[boist]

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.

 

[Brewitt]

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.

 

[boist]

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?

 

[PoppinCaps]

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.

 

[BBL_Brewer]

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.

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.

 

[BBL_Brewer]

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.

 

[PoppinCaps]

Sweet! Nice to know the amount of glycerine really doesn't matter that much. Were all the samples the same amount of slurry?

 

[Brewitt]

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.

 

[Brewitt]

Forgot to ask, what was the volume of the samples are used.

 

[BBL_Brewer]

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.

 

[emjay]

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?

 

[BBL_Brewer]

You definitely could.

 

[emjay]

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.

 

[BBL_Brewer]

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.