I had good success with freezing but one thing is still bothering me- what slurry/glycerine solution ratio to use, and is it that important (as it is glycerine/water ratio).
My process is:
- make 15% glycerine solution (eg. 25ml of glycerine and 75ml of water)
- mix that with slurry in ratio of 75% slurry and 25% glycerine solution
Beside long lag phase (36-48h) on 1st step, after stepping up starter fermented pretty quick.
We know that it is best to go with 15% glycerine solution, since greater % can decrease viability due to poisonous effect on yeast, but what ratio of slurry/glycerine solution is most optimal.
On this and other threads I found several ratios:
You want less than one-third of the solution in the test tubes to be glycerinee, so I shoot for about 2-3 ml in each tube (15%).
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Now, one at a time, uncap your vial and extract a sample of yeast slurry from the bottom of the flask. Inject enough into the vial to fill it about 80%.
When I started with freezing I used FlyGuy"s thread as guide, besides that it is somewhat greater glycerine solution it is also greater slurry/glycerine solution ratio.
I use a 35% weight by volume glycerine solution at a rate of about 70% solution and 30% yeast slurry
I setlle out the yeast, siphon off the starter beer and add enough glycerinee/water solution to bring the volume up to 500 milliliters.
This is pretty bigger ratio..
I harvested about 60 mls of packed cells (1/4 cup containing approximately 4x10e11 cells based upon past experience) and resuspended them in an 100 mls (a bit less than half a cup of 15% glycerol, split them into four 40 ml tubes and cooled them stepwise to -112F for later use.
Brewitt uses similar scale..
My next doubt is that if we are going to make big glycerine solution/slurry ratio (eg. 70/30) we will need pretty big flask for eg. 25B viable cells after thawing.
Math would be:
According to Brewitt"s test, viability after freezing is close to 25% (at best) so we need 100B cells for freezing. If we make starter, or wash yeast from previous batch we will get somewhere between 1 and 1.5 B/ml.
That is 100ml of slurry (for 1B/ml) that we need to mix with 225ml of glycerine solution (to achieve 70/30 solution/slurry ratio).
At the end we will have 325 ml flask ready for freezing which is not small, especially if we want to make couple of them..
I know that this seems like over-complicating, but if I am doing it I would like to know why is it on this or that way...