Freezing Yeast: something went wrong (?)

[SamBrewer]

Hi !
yesterday I've tried for the first time to freezing yeast using Glycerin solution; I basically followed this procedure:

1)Mix glycerin and water in the ratio of 30 ml glycerin to 70 ml water. This will yield a 30% glycerine solution.
2) Boiled the solution in a pressure cooker ( I've put the solution into a jar )
3)Allow to mixture cool to room temp.
4) Add equal amounts of the 30% glycerin solution and the yeast slurry to the test tube ( 9ml of yeast slurry + 9ml of solution into a 20ml tube so at the end it should be a 15% glycerine solution)
5) Cap the tube and shake.
6) Mark a piece of masking tape with the yeast type and label the tube and Freeze it


Now here is my question (maybe it's just a stupid one...)
Why after one day the liquid in my test tube is iced???? I thought that by using glycerine the solution would never turned into ice, but just a thick liquid.
Is the glycerine only preventing the damaging of yeast cells in the iced solution or I've ruined my yeast?

Should I use more glycerin the next time?

thanks!!

 

[Polboy]

you will be ok, it should turn in to ice, glycerine prevents water icicle formation in the cells so by having glycerin you increase survival of frozen cells. Btw its better to cool it slowly than putting it right away in the freezer, i freeze in -80 and use this:
http://www.nalgenunc.com/products/p...ware&category_name=Cryoware&subcategory_name=

 

[Revelator]

Could your stock glycerol solution potentially be a diluted mixture? That might result in a lower than expected concentration

 

[SamBrewer]

Could your stock glycerol solution potentially be a diluted mixture? That might result in a lower than expected concentration

I thought this too, But it was so viscous that I couldn't use my graduated pipette to measure it ( that's why I've use a syringe to diluate in water before mixing with yeast) so I think it's pure glycerine.

I didn't put the tube directly in the freezer, they passed all the night in my fridge, but anyway if you say that is OK if the solution is tourned into ice I think that I'm worrying too much

 

[Polboy]

Im sure its fine but let us know when you make a starter, for freezing im using 60% glycerol solution and i use 1/4 of it so i have more yeast cells per freezing but im freezing smaller than you amounts (1.5ml). I only defrosted 6months old samples and yeasts grew just fine (all my my vials are frozen solid )

 

[gyst]

It depends on how cold your freezer is. In lab, glycerol solutions don't freeze at -20C, but do freeze at -70C (note this is Celcius!). If it makes you feel better, we normally store lab stock yeast in 15% glycerol in the -70, so its frozen - as Polboy says, the glycerin is to prevent ice crystal formation that would puncture the cell wall, not to totally prevent freezing.

 

[SamBrewer]

It depends on how cold your freezer is. In lab, glycerol solutions don't freeze at -20C, but do freeze at -70C (note this is Celcius!). If it makes you feel better, we normally store lab stock yeast in 15% glycerol in the -70, so its frozen - as Polboy says, the glycerin is to prevent ice crystal formation that would puncture the cell wall, not to totally prevent freezing.

this defenativelly makes me feel better! thanks!

Anyway I'm going to test it with a starter before trying this procedure again.

I usually buy yeast on internet; my goal is to have at least the 4 strain of yeast that I use for almost every brew I make so I don't have to worry about storing temp. of the yeast during the road travelling from the homebrew shop to my house

 

[BBL_Brewer]

Yeah, you're good. That's not enough glycerine to keep the mixture from freezing. I use more glycerine than that and my aliquots always freeze solid. You have to use around 50% or over to keep it from freezing, which some people do.