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Old 07-05-2013, 01:11 PM   #11
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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?

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Old 07-05-2013, 02:07 PM   #12
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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.

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Old 07-05-2013, 03:19 PM   #13
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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.

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Old 07-05-2013, 03:35 PM   #14
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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.

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Old 07-05-2013, 04:09 PM   #15
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May as well throw my novice approach into the mix as well

-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...arge-bank.html

Here's the real catch though..... I haven't used any yet 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.

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Old 07-05-2013, 04:31 PM   #16
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Here is an instruction that Pappers put together for his Club.

I think people have been pretty successful following it.

File Type: pdf Banking Yeast 3.pdf (29.1 KB, 64 views)
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Old 07-05-2013, 05:03 PM   #17
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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?

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Old 07-05-2013, 05:17 PM   #18
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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.

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Old 07-05-2013, 05:21 PM   #19
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Quote:
Originally Posted by lshaner View Post
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.
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Old 07-06-2013, 01:00 PM   #20
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Quote:
Originally Posted by StMarcos View Post
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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