Yeast Bank Question- will it actually freeze solid?

[SenorWanderer]

I prepared my first batch of yeast for my yeast bank on monday evening. everything went well, no mishaps. i followed FlyGuy's instructions pretty much to the letter. the only place i varied is that instead of putting the glycerine in each vial before autoclaving in the pressure cooker, i put 50ml of glycerine in a beaker covered with foil and put that in the pressure cooker. after that was cool i decanted the beer off the yeast and then used a pipette and bulb to transfer 100ml of yeast slurry into the beaker, giving me a 33% glycerine solution. this afternoon when i got home i took the vials out of the fridge, and put them in the freezer. 6 hours later they're not frozen. the glycerine was separating while they were in the fridge, so i'd shake them every time i opened the fridge. is this something to be concerned about? i feel like i did a really good job of getting just yeast out of the starter. should i have included a little beer/water. so far they're not separating like they did in the fridge, but still definitely not frozen. the glycerine i used is NOW brand vegetable glycerine. i got it at a health foods store, it's 100% pure and is "a USP and food grade, all natural product derived from vegetable oil". someone please tell me to not freak out!

 

[FlyGuy]

Hey SW -- don't freak out.

It sounds to me like your technique should work. Although, that seems like a lot of glycerin to me, especially if you are not diluting with a bit of water. Most authoritative references on yeast freezing seem to recommend using between 15 and 25% glycerol. Dilute this down with another 25% volume of sterilized water and mix it REALLY well. If the glycerol solution is too thick, it will be hard to get the yeast into suspension. That might be your problem. And it might not matter, but I would suggest using sterile water instead of beer (from the starter) in the vials because the alcohol could harm the yeast.

(Note: I see that my instructions for preparing the glycerin dilution are not very clear in my yeast bank thread. I cleaned it up a bit, but will update it properly sometime soon.)

Regarding freezing, yes if it is mixed correctly you should see freezing. If you had too much glycerol in the solution, it might not freeze solid, especially if it wasn't mixing well. But everything may turn out still. Just in case, on your first use of your frozen yeast, I suggest pitching two vials (instead of one) when you make your starter, and have a packet of good dry yeast on hand just in case.

Best of luck!