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Old 10-03-2011, 07:26 PM   #21
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Sorry for the delay in doing some experimentation on this. Work has been quite busy and I have had no time for side projects. I will prep a starter this week and set up some frozen preparations at a few glycerin concentrations from 15 to 60% and at -30C and -80C. It will probably be a couple weeks before I know anything about viability since it requires cell counts and colony counts.
No worries! Take your time. Very cool of you to go to all that trouble in the first place. Can't wait to see the results though
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Never Ending Liquid Yeast - How to Farm Yeast and Freeze it.

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Old 10-04-2011, 12:14 AM   #22
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Although I haven't had time to prep cells for this analysis, I did do some reading regarding yeast preservation conditions and found some results that appear reliable but were surprising to me. The results seem to indicate that freezing yeast at a rate of less than 1 degree C per minute in 10% glycerol and then thawing at 37 degree C yielded viabilities on the order of 50% whereas faster freezing and slower thawing led to significantly low viabilities. The one thing that did not replicate home freezing methods was that the storage was in liquid nitrogen (-196C) after freezing to -80C. However, I presume that the slow freezing and rapid thawing will still be advantageous. So, cooling by putting a large volume in the refrigerator and then moving to a standard freezer sounds like it might be a good approach and then thawing at around 100F with agitation (to prevent heating of the thawed portion) until it is completely thawed sounds like a good idea. Let's see.

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Old 10-04-2011, 12:38 AM   #23
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Should say, all of this was done with small volumes (probably 1 ml or less).

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Old 10-04-2011, 12:47 AM   #24
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You wouldn't think more glycerol would be a bad thing. Slow freezing I can see. Fast thawing seems odd, but if it works all the better (cuts down on lead time). Your results will be very interesting to see. Thanks Brewitt!

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Old 10-04-2011, 01:04 AM   #25
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BBL Brewer - great post. I need to set up some time and farm some yeast for the year as you did.

Brewitt - you have me excited to see the results of the testing. This is a thread worth saving.

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Old 10-05-2011, 02:56 PM   #26
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My mother always chilled ice cream before mixing in the cranker claiming it made better Ice Cream. I've seen more of this as I've gotten older and the explanation typically used is smaller ice crystals. Smaller crystals would be less likely to rupture the yeast cells so that makes sense. I can also buy the fast cooling thing but the extra agitation part doesn't quite make sense to me. Even with glycerine absorption, there will still be some small amount of water inside the cells that crystalized as you can't have a perfect mix inside the cells. That being the case it seems to me at least logically that it would increase the chance of damaging the cell. On the other hand it would also make more sense that it would be quicker to get up to speed (aka cell agitation already out of the way to get rid of the lethargy).

That said, I can't help but wonder if jars might not be the best medium to store them in at least in the traditional sense. While the vacuum isn't really necessary, I wonder if vacuum bags wouldn't function better as you could still store them inside a jar filled with water preventing a lot of the need for the cooler setup, and the surface area would be greater for thawing quicker with less potential cellular damage.

That said if you want to thaw them extremely quickly with agitation turn the jar sideways (I've not done it with 1/2 cup jars but 1c work fabulously) in a bowl and run cool tap water over it. It'll thaw in a matter of minutes. We do the same thing with our babies bottles (with a bit of hotter water added into the mix) and you can go from frozen to warmed enough for consumption in about 3 minutes depending on volume. The agitation (spinning in the water filled bowl) will keep the temperature smooth throughout as well.

That said I still think I'm going to give this a try. I'll probably go the vial route though and lean towards the higher end of the spectrum glycerine-wise to be safe as I have far less freezer space to work with.

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Old 10-05-2011, 10:26 PM   #27
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My mother always chilled ice cream before mixing in the cranker claiming it made better Ice Cream. I've seen more of this as I've gotten older and the explanation typically used is smaller ice crystals. Smaller crystals would be less likely to rupture the yeast cells so that makes sense. I can also buy the fast cooling thing but the extra agitation part doesn't quite make sense to me. Even with glycerine absorption, there will still be some small amount of water inside the cells that crystalized as you can't have a perfect mix inside the cells. That being the case it seems to me at least logically that it would increase the chance of damaging the cell. On the other hand it would also make more sense that it would be quicker to get up to speed (aka cell agitation already out of the way to get rid of the lethargy).

That said, I can't help but wonder if jars might not be the best medium to store them in at least in the traditional sense. While the vacuum isn't really necessary, I wonder if vacuum bags wouldn't function better as you could still store them inside a jar filled with water preventing a lot of the need for the cooler setup, and the surface area would be greater for thawing quicker with less potential cellular damage.

That said if you want to thaw them extremely quickly with agitation turn the jar sideways (I've not done it with 1/2 cup jars but 1c work fabulously) in a bowl and run cool tap water over it. It'll thaw in a matter of minutes. We do the same thing with our babies bottles (with a bit of hotter water added into the mix) and you can go from frozen to warmed enough for consumption in about 3 minutes depending on volume. The agitation (spinning in the water filled bowl) will keep the temperature smooth throughout as well.

That said I still think I'm going to give this a try. I'll probably go the vial route though and lean towards the higher end of the spectrum glycerine-wise to be safe as I have far less freezer space to work with.
Great points! The vacume bag inside a jar of water is a novel idea for those who have a self-defrosting freezer. It would also take up less space or at least be easier to work with functionally in a smaller freezer.

As for thawing, if a quick thaw is the ticket, you're right, water would be the way to go. That's how I thaw my frozen meat. Works like a charm and quick to boot. However, using my existing method, I would have to put the jars in a zip lock since the jars are not sealed upon freezing and the agitation from spinning in the bowl would not be an option. I don't plan to use the bag in a jar method becasue the 4 oz jars are so small and full boxes of them stack nicely in my chest freezer while taking up a relatively small amount of space. Personally, I think just water bathing period would be better than any other heating method even without agitation.

Thanks for your input
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Old 10-13-2011, 11:58 PM   #28
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EDITED FOR CLARITY (hopefully):

I have initiated the freezing experiment using a blend of the approaches that have been discussed here. I started with half a liter and ended with samples of about 5 ml (much smaller volumes than you would have for a full starter) but I presume the results will be about the same. The goal was to try different concentrations of glycerol (15% and 30%) and different cooling regimens (Add glycerol at room temp then transfer to refrigerator temp and then to freezer temp or cool first, add cold glycerol and then go straight to freezer). Finally, I will thaw in refrigerator overnight or in the warm. Here is basic outline.

1. Grow half a liter of starter using 10X dilutions to full density in 1.040 wort with nutrient. Don't have a number yet but around 2x10e8 per ml.

2. Split, chill 1/2 to 4C (refrigerator temp) and let half settle at room temp overnight (assuming that none of you have centrifuges so gravity settle to concentrate).

3. Pour off wort, and resuspend cells in 15 ml of either 15% glycerol at room temperature or 4C depending on the state of the starting cells.

4. Split each into three portions and adjust one portion of each to 30% glycerol (cold or room temp).

5. Room temperature culture was moved to 4C for an hour after adding glycerol and then to freezer. Culture that was in cold before adding glycerol was simply moved to freezer after glycerol was added. A single sample of the later was also later moved to an ultracold freezer where we usually store yeast.

6. After a week or two (depending on my schedule) I will remove from freezer, either thaw in refrigerator or at 37 (body temp) and then do a viability assay (count cells and plate and see how many grow up). I will also try to put some in wort and see how long they take to recover. This is a lot of work so it's going to take me a bit.

Hopefully this will be informative.

I should say that for a scaled up process this would be the same as growing three liters of starter, letting it settle out, and bringing all the cells up in about 2/3 cup of glycerol. That could then be split into thirds to make three starters of about 2x10e11 cells each in about 1/3 cup total volume. Just a bit more dilute than a tube of White Labs yeast. Also, this will tell us about freezing and thawing but not about long term storage. If you have a non-frost-free freezer or a frost-free freezer with some ice packs around your tubes I would think the results I report should hold for at least several months to a year.

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Old 10-14-2011, 03:38 AM   #29
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If you have any luck with the Brett group pls post it. I was thinking of harvesting from one and I'd swear I read someone saying it doesn't work. TMF maybe? Will have to go try and dig that link up. He did use equipment like a centrifuge tho iirc along with other niceties.

Froze a s05 (just cause I plan on using it relatively soon and have a backup) and wlp001 which I don't have a need for for at least 9mos. Have read the 05 has no problems usually anyway tho.

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Old 10-14-2011, 04:10 AM   #30
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I have not done anything with Brett. I have never used it and don't have any to work with.

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