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Old 04-13-2012, 04:58 AM   #151
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Yes I now, and I was afraid of this answer
Since we know about glycerine solution percentage I hope someone will do cell counting with different slurry/solution ratio...
gotta get myself a microscope

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Old 04-13-2012, 06:39 AM   #152
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Quote:
Originally Posted by diS
Yes I now, and I was afraid of this answer
Since we know about glycerine solution percentage I hope someone will do cell counting with different slurry/solution ratio...
gotta get myself a microscope
I generally consider the final volume when adding glycerine. That is, if we want 15% final and start with 60%, add 10 ml glycerine to 30 mls cells for a total of 40mls. The cells are included in the volume. Generally there is a slurry with a significant volume of wort. That is fine. I don't think it matters much if the final solution is 30% cells, 50% cells or 75% cells, the final concentration of glycerine is 15% of the total.

I always do a starter. In my experience a 40-50 ml tube of cells overnight in 1.5-2 liters of wort with stirring is great.

Hope that helps.
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Old 04-13-2012, 08:40 AM   #153
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Actually it does help. I like the way you dilute "major" glycerine solution (60%), so it will end with 15% when mixed at 1:4 ratio, easy and precisely, thanks!

These days I"m going to wash wlp029 and I"ll try this route.

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Old 04-13-2012, 06:07 PM   #154
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The cell percentage does matter. The cells are not dissolved in the solution, so you would have very different concentrations of glycerine.

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Old 04-13-2012, 06:59 PM   #155
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Quote:
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The cell percentage does matter. The cells are not dissolved in the solution, so you would have very different concentrations of glycerine.
Formally, this is true. However, a large percentage of the volume is the liquid in between the cells and, in addition, it is my understanding that the cells themselves take up the glycerine. What the actual intracellular concentration of glycerol becomes, I don't know but there is some equilibration which contributes to the cryoprotection of the cells. It is probably a known value, I just don't know it.

Regardless, my empirical experience is that, when I adjust a slurry that I would guess is 50% or more packed cells to 15% final concentration of glycerol, it works well. Similarly, when I preserve small samples that are less than 5% cells, they also retain relatively high viability.
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Old 04-13-2012, 07:08 PM   #156
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In my lab we generally mix a saturated culture of yeast 1:1 with 40% glycerol (w/v) for a final concentration of 20%.

In my experiment, I am testing -80 v. -20C storage with 5,10,15, and 20% glycerol, with and without 42C heat shock. I haven't thawed any to test for viability yet, because I'd like to go out qt least a couple of months.

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Old 06-01-2012, 10:50 PM   #157
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2 liter starters made with a 50 ml vial of a very thick slurry of washed yeast from a fermentation frozen in a final concentration of 15% glycerol is working great! I have found my sweet spot.

Still interested to hear what ScoRas learns.

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Old 06-08-2012, 02:05 PM   #158
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I froze some wild yeast (along with other strains) back in 2010 in my parents chest freezer following the instructions in this thread. I took a vial from their house on mother's day and it's been sitting in my fridge for a few weeks. Yesterday I made a starter and I can already tell there is some definite yeast activity. You can't miss the smell of this wild yeast. I know originally there was both some primary fermentation strain and brett and some unknown bacteria. There definitely seems to be yeast activity in the beer but I'll have to wait and see if any of the bacteria survived the freezing process. I guess I'll check back in a year when the beer has had time to age and the bacteria have had a chance to work.

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Old 06-12-2012, 08:19 AM   #159
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Quote:
Originally Posted by Brewitt View Post
2 liter starters made with a 50 ml vial of a very thick slurry of washed yeast from a fermentation frozen in a final concentration of 15% glycerol is working great! I have found my sweet spot.

Still interested to hear what ScoRas learns.
What concentration of yeast (B/ml) do you think you have in that slurry?

I mean, if vitality is aprox. 25% and for 5 gallon of 1.048 beer you need 170 billions of cells, the you"ll need 3B/ml (3 B/ml x 50 ml = 150 B; 150 B x 0.25 = 37 B in 2 L starter gives ~184 B cells).

In "Yeast", C. White says that washed yeast have between 0.8 and 2 B/ml.
I usually stick with middle range (1.5 B/ml) just to be on safe side, even I rinse it to the point where the is no or very little trub in slurry.
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Old 06-13-2012, 05:51 AM   #160
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Quote:
Originally Posted by diS View Post
In "Yeast", C. White says that washed yeast have between 0.8 and 2 B/ml.
I usually stick with middle range (1.5 B/ml) just to be on safe side, even I rinse it to the point where the is no or very little trub in slurry.
Dis, I think you missed my point. What I did was to thaw my 50 ml tube of yeast, pitch into 2 liters of 1.040 wort and grow it overnight with stirring. The viable cells grow up to density overnight. Even with 25% viable cells in my slurry the viable ones should grow to maximum density of about 1-2x10e8/ml or 200 B cells total after overnight. Pitched into about 5.5 gallons that gives me a final concentration of about 2x10e7/ml which is about optimal. Are we on the same page here?
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