Freezing yeast % of Glycerin... HELP!

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mikescooling

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Hey guys, I'm trying to freeze some yeast. I've read a lot of stuff about % of Glycerin and I'm confused. I made a mix of 75% H20 with 25% Glycerin, for the purpose of keeping this simple so I can understand lets call this the "mix" I added 50% mix to 50% crashed yeast slurry (we all know how to do starters and crash). How far off am I from doing this correctly? Please microbiologist keep words small, so this pipe fitter can understand.
 
Personally I think you're reversed in your ratio. Just FYI, my mix ratios are all by volume, not by weight - for me it's easier to measure a cup, vs. getting out a scale and weighing. I've done some at 50-50 and they work OK, but it took some effort to get good yeast activity after the thaw. My 75% glycerin with 25% water with the yeast slurry added (which has some additional water) has produced faster responding yeast after thawing.
 
Back when I was in lab, we had a stock solution of 30% glycerol: 70% water and made a 50:50 mixture of yeast slurry to glycerol solution. In other words, the final glycerol concentration was 15%.
 
One thing I noticed with the frozen yeast I have (%50 slurry %50 glycerol) is that the particles of yeast would settle to the bottom eventually. This means that unless agitated the very bottom of the tube is gonna be 98% yeast.
 
Thanks for the responses guys, to recap we have 70% glycerol, 30%glycerol and 100% glycerol, mixed with slurry. You can see how I am confused? Are their that many ways to do this? What's the down side of one way working better than another? Is a larger size vial better via more yeast?
I did notice everything settled to the bottom.
 
It's a fairly new technique. I used 5ml vials and all my yeast are dead in 2 years.

Might I ask your goals? And also how much storage space you have and at what temperatures you plan on keeping the yeast. I would probably just try to sway you towards keeping yeast in the fridge instead of the freezer regardless of your answers.
 
Grathan, my goal is to only pay for yeast once a year. I'd like to take new yeast and make a big starter, then store that new yeast in vials in my big chest freezer I think it's a -20F, so I have lots of space. The vials I am using now are glass 25ml, but I ordered some bigger sterile plastic vials that are 50ml; I was thinking bigger is better? Are you referring to slanting yeast and keeping it in the fridge? I have room in there too.
 
No not slanting, just washing. I would think it would last a year either way (freezing or fridge), though you will probably have to revive the yeast after a year. Bigger is better. My 5ml vials never had a chance. I didn't autoclave everything, just simply spray with starsan.

If I didn't have a LHBS I would try it again with bigger vials. But since I have every yeast I could need stored by local shop it just comes down to cost savings. For me it's cheapest just to reuse the yeast for all the recipes that use it and then move on and just buy a vial again down the road.
 
You want your total volume of slurry to contain 10% glycerol/glycerin. My technique is to make a 2L starter, decant off the wort when its finished, then add 100% glycerol so it comprises 10% of the volume that remains. You can still do the same thing with diluted mixes of glycerol, you just need to do some calculations so the end result is 10%.

It is obviously easier with graduated flasks. Make sure it is mixed very well (glycerol is very viscous) then freeze as cold as you can get it.
 
ColoHox I was thinking "why am I diluting my slurry with water" adding straight glycerol to slurry sounds good (thanks). Do you guys pressure can your glycerol before use?
 
One way to prevent yeast settling in the tubes is by flash freezing. In the lab, the tubes would be dipped in liquid nitrogen and stored at -80 degrees Celsius -- neither of which is practical at home. If you have access to dry ice you can do something similar by crushing some dry ice and pouring in some denatured alcohol and dipping the tube in the mixture (being mindful that the alcohol will remove any marker labeling you may have done). Besides preventing yeast settling, flash freezing vastly improves long term viability of the yeast. To some extent, slow freezing causes formation and thawing of ice crystals that can puncture yeast cells.
 
It is a good idea to pressure cook-sterilize your glycerol before use. It is easy to do, and eliminates a potential source of contamination of your seed stocks.

@Ishaner, lab conditions are obviously ideal, although impractical, like you mentioned. The glycerol is added as a cryoprotectant, so the detrimental effects of ice crystals is reduced if used correctly.
 
I don't know if this is the "proper" ratio but I use 20 ml vials and using a medicine syringe put 5ml yeast, 5ml glycerin and 10ml water in each vial. I cool them overnight in the refrigerator then shake and freeze.

I have 9 or 10 varieties (starting with 4 vials each). I have some that are almost 1.5 years frozen and have "resurrected" several so far that had been frozen for more than a year.

It takes me a week of step up starters to revive one.
 
May as well throw my novice approach into the mix as well :D

-50 ml non-sterile centrifugal plastic tubes boiled for 5 minutes and left in water until moments before they're filled
-Using well washed yeast (minimal trub); clean looking yeast; decanted prior to mixing
-80% glycerin solution for mixing with yeast; mixed and boiled in microwave for 1 minute; covered and cooled
-Each tube gets 31 ml washed yeast and 14 ml glycerin solution for 25% glycerin content; mixed well
-Refrigerated overnight 12-24 hours
-Given one last gentle mixing and moved to upright frosting freezer for storage

I've chosen the glycerin content based some things I read in the following blog:
http://suigenerisbrewing.blogspot.ca/2012/11/yeast-banking-i-managing-large-bank.html

Here's the real catch though..... I haven't used any yet :D so I can't really comment on how well they've stored or how long they'll take to revive.

I've estimated anywhere from 60-120 billion yeast cells based on how well the yeast was washed and how compact the yeast layer. The better the wash and the more compact the layer, the higher I estimate (S-04 is a good example). I think I'm usually sitting in the 80-100 billion cell range so I basically look at it like I'll be starting with a white labs vial prior to use. This gives me the opportunity to make up a starter and build up the cell population prior to use AS WELL AS see how long it takes to revive.
 
How important is it to keep the slurry from any settlement during freezing? I was under the assumption that, once mixed well with the glycerin solution, it really doesn't matter if the yeast settle out some. It's not like all of the glycerin is going to float to the top, is it?
 
The settled gunk from washing is not ideal to include when freezing stocks. As long as the glycerol is well mixed (ie all cells are very well suspended in the mixed solution), some settling of the yeast in the tube doesn't really matter. Without mixing, the added glycerol will actually sink.
 
One way to prevent yeast settling in the tubes is by flash freezing. In the lab, the tubes would be dipped in liquid nitrogen and stored at -80 degrees Celsius -- neither of which is practical at home. If you have access to dry ice you can do something similar by crushing some dry ice and pouring in some denatured alcohol and dipping the tube in the mixture (being mindful that the alcohol will remove any marker labeling you may have done). Besides preventing yeast settling, flash freezing vastly improves long term viability of the yeast. To some extent, slow freezing causes formation and thawing of ice crystals that can puncture yeast cells.

I thought you were supposed to freeze slowly. It was thawing that was to be done quickly. Seems to me what I remembered from the yeast freezing thread.
 
I thought you were supposed to freeze slowly. It was thawing that was to be done quickly. Seems to me what I remembered from the yeast freezing thread.

I know that we just dipped our cryogenic tubes straight into the liquid nitrogen but I did some more googling and found some information from Boulton and Quain's Brewing Yeast and Fermentation text book that supports both of us. First, the yeast are grown oxidatively (i.e., with something like glycerol or ethanol as a sole carbon source). Next, with the yeast in a 5% glycerol solution, the temperature is brought down to -30C over a two hour time period. Finally, the tube is immersed in liquid nitrogen.

As you noted, thawing is done quickly by immersing the tube in 25 to 37C water.
 

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