Frozen yeast bank question

[brumer0]

Hi all,

A few weeks ago I finished adding my first yeast to my frozen yeast bank. I isolated a yeast colony and grew it, stepping it up to a 500ml starter before washing the yeast. I have 25ml vials that I filled with 25% glycerin, 25% water, and the other 50% concentrated yeast from my 500ml flask (I left the flask in the fridge for 2 days to let the yeasties build up their energy reserves).

So the question is, today I opened my container in the freezer to add more vials, and I noticed that the previous vials are frozen. This is bad, right? I thought the glycerin made the water not freeze.... Did I just kill the yeast and waste my time to date?

Thanks,

 

[JimTheHick]

Take one out and see if it grows. When we freeze cells in culture the rule is to always thaw out one of the aliquots you made to verify viability post-freeze.

I would guess some yeast survived. But that's just a guess.

 

[Joemama474]

I'm not a micro guy, but I don't think the glycerin will keep the water from freezing, I think it changes something about the way the ice crystals form so that fewer yeast cells die as a result of having their cell walls pierced by freezing crystals. I think. Maybe.

 

[JimTheHick]

I'm not a micro guy, but I don't think the glycerin will keep the water from freezing, I think it changes something about the way the ice crystals form so that fewer yeast cells die as a result of having their cell walls pierced by freezing crystals. I think. Maybe.

+1. Right. We use DMSO which does a better job with the keeping crystal formation down. A glycerol solution of something like 30%-50% won't freeze at -20, but your was diluted down to 12.5%.

Any type of freezing will kill lots of cells- its just keeping the ones that survive in good enough shape for taking off in a starter later on.

 

[brumer0]

Thanks everybody! I appreciate your help. Ill check the viability of one of the samples I have. In the future Ill make sure I increase the amount of glycerin in the vial.

 

[JimTheHick]

In the future Ill make sure I increase the amount of glycerin in the vial.

Be sure to double check on the glycerin. It might be providing enough cryoprotection even though your solution froze. I know this sounds weird but it's the length of the crystals that form on a micro level that is the key, not whether the solution is liquid or solid. 50% glycerin might be harder on yeast than freezing.... My experience is with human cell culture, not yeasties.

Even with DMSO the solutions still freeze. You may have done nothing wrong.

 

[passedpawn]

The tubes will freeze, and this is OK. I've frozen a lot of yeast. With glycerine it'll be OK.

I know why you're questioning this. I read some stuff about the glycerine preventing the stuff from freezing, and that is just not true. But with glycerine, when the yeast freezes the cell walls will be protected in some way from the ice crystals that form.

 

[Brewitt]

The point of the glycerin is not to prevent freezing. The point is to allow freezing with less damage. 15% glycerin at -80C (much colder than the -20C of a standard freezer) is standard preservation conditions for yeast in the laboratory. I have recently learned that slow cooling and rapid thawing is optimal.

That said, I am in the midst of checking a bunch of conditions for viability but have not had time to finish the experiment. However, I can tell you that freezing the yeast that settle out from a half liter culture in the fridge overnight and adjusting them to a 15% final glycerin concentration, chilling at refrigerator temperature for a few hours and then put in the freezer will maintain sufficient viability for at least several weeks and probably much longer to inoculate an overnight 1.5 liter starter very effectively. It seems to be best to thaw the yeast quickly in about 100 F water and immediately add to wort. Remember, frost free refrigerators will cause problems so put your yeast in a box with a bunch of cold packs to prevent thawing on the defrost cycle.

I have decided to put away a bunch of similar sized samples and use them as needed.

 

[brumer0]

As always I am impressed with the knowledge on this board. Thanks for the help!!

I very recently bought a chest freezer and insured to pick one that had a manual defrost, not an automatic. I have also wrapped my cultures in ice to be safe in case there is a power loss.