How do I identify stored yeast (frozen) that has gone bad?

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timsch

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I've had a number of batches brewed over the recent past that have not had the good taste that I was expecting. I had been attributing it to my 1/6 barrel SS keg that I use for fermenting that I never could be sure I was getting fully clean due to the small opening on top. I've recently made a tool that should be able to clean it well. Now I'm wondering if there might have been another reason for the off-tastes.

I just made a DME starter from a batch of WLP001 that I had grown, split and then frozen in 30ml vials with glycerine. The yeast did propagate well in the starter, but the smell was not too appealing. I don't recall smelling my starters much before, so maybe they're all like that. These frozen samples had been in the deep freezer (manual) for probably 9 months now, and there was some separation in the vials.

Even though the yeast propagated, what are the odds that it's not something I want to use for brewing, and how would I be able to tell?
 

bionicbelly

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I am very new to freezing yeast, and brewing in general, but the starters I make from my frozen vials all smell great/as they should. I actually make a point to smell them, just to make sure I am not ramping up something that is infected or compromised.
As far as that particular yeast, I have never used it, so maybe it just smells like that?
 

deuc224

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Maybe the glycerin isnt completely pasteurized? I clean all my everything and make sure i dont leave the top open too long for the glycerin when im making my vials of yeast, but i do 50ml total, 25ml yeast and 25 glycerin mix, that way its around 100b in one tube. Ive made small vials and they all look brown as if its not enough glycerin to freeze them evenly, also do you freeze them straight up or do you freeze them in iso alcohol for 48 hours?
 
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timsch

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I follow the procedure in the Banking Yeast attachment.

I'll have to check, but I think mine may have a brownish tint to them as well. I do boil a glycerine water mix and am careful about sanitization.

I have always frozen them straight up.

Edit: I have kept my freezer around -10F
 

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timsch

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Definitely a lot of good information in that thread. Thanks.

Too bad about the original directions I was following saying that the yeast should be frozen as fast as possible. Very different from the directions in that thread.
 
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timsch

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So what are the ramifications of me having used the quick freeze method on all of my frozen yeast? Is it likely that all will have bad characteristics like off flavor?
 

deuc224

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I dont think itll cause the yeast to go bad but you could possibly be stressing the yeast out when you grow it because a fast freeze will rupture the yeast cell wall and basically kill the yeast. So the ones that survive are trying to multiply and eat all the sugars that is way too much for them. Basically underpitching and the yeast releasing a bunch of esters. Sorry i dont know why i just didnt start with that lol.
 

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You shouldn't be getting any noticeable separation between water and glycerol once frozen. It's possible if they weren't mixed throughly, I guess. Are you using a frost-free freezer? 30mL is a lot to be freezing slowly and thawing rapidly. I use 1.8ml cryovials and usually 'scratch' a needle's worth to seed a 10ml starter then step up.

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Glycerol (also called glycerine) is actually a pretty poor cryoprotectant, to be honest. Trehalose is much better. Regardless, freeze slowly after incubating in the fridge for several hours. I use an insulated food box to slow the freezing rate.

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If you want to use the whole frozen aliquot, thaw it rapidly at 40℃ (104℉) for a minute or two.

Yeast turn a darker shade when they die. It's quite obvious once you recognise it. Although usually recoverable, viability falls of a cliff. This is one good reason to store at much smaller volumes and just step up from an initial 10ml culture/starter. But you shouldn't be freezing so much that you could tell. Yeast for long-term storage need to be stored at quite low cell density.
 
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timsch

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The freezer is a large manual chest type, but it has been kept at -10F rather than -20, so that might have contributed. I'd shake the vials about 3 times 10 minutes apart as they were freezing. Initially they looked fairly well mixed. Only later did I find separation. I have started keeping them in an insulated tote down lower in the freezer than I had been. That should help somewhat.

I mistakenly said I was using 30ml vials. I am actually using 15ml. I'd like to try to make this size work before trying a different size. It seems like I've done several things if not wrong, at least not good. I'll correct those before making other changes. Thanks for the suggestions though.

IMG_20211210_174218261.jpgIMG_20211210_174152807.jpgIMG_20211210_174106163.jpg

I've attached pictures of the 3 strains I've stored. WLP001 is the longest stored and the one subjected to the most abuse. Wyeast 3068 was the one most recently stored, and I was hoping it would look significantly better, but it looks pretty rough as well. Even taking the samples out of the freezer for just a minute for pictures, the separated glycerine became liquid, if it was not already.
 

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Tom R

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So what are the ramifications of me having used the quick freeze method on all of my frozen yeast? Is it likely that all will have bad characteristics like off flavor?
I really don't know. That's above my pay grade.
But I've sometimes been surprised at how resilient yeast can be.
If it looks and smells like yeast should, I'd use it.

Regarding separation:
I use a 25% glycerin/75% water solution, mixed 50-50 with the yeast.
The yeast has always settled to the bottom of the centrifuge tubes.
I've frozen over a hundred, and built up about nine into starters so far.
They have all worked well.
IMG_0710.JPG
 

McMullan

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So what are the ramifications of me having used the quick freeze method on all of my frozen yeast? Is it likely that all will have bad characteristics like off flavor?
Significantly lower viability and probably days (rather than overnight) before anything cultures after thawing. It's possible by pushing viability off a cliff you increase the chance of randomly selecting deviation from expected characteristics, especially as the cells probably haven't been conditioned for freezing too. The logic of simply adding more slurry to the vial is flawed and just makes things even worse due to problems associated with larger volumes and higher cell density. E.g. lack of control over glycerol replacing water in the cell membranes, steady freezing rate and rapid thaw. Scaling up doesn't work. The correct logic is actually more like smaller is bigger 🤪
 

Tom R

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Perhaps I should have been more specific.

All aspects of the fermentation (including attentuation, fermentation rate, and flavor of the beer) had not degraded in any noticable manner from the first pitch of the yeast.

My point was that separation doesn't necessarily mean failure.

I have no doubt that there are some who would dump my beer :barf:, . Some taste-impaired fools pretend to enjoy it, and come back for more. 😝
 
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timsch

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Turns out that I didn't have -10F in the freezer after all. I checked last night, and with my external temperature controller showing -12F, the actual temperature where I was keeping the yeast was actually at 2F. I bumped the controller down to -20F and measured again this morning and actual temperature was -9F. The chest freezer is a 24 cu.ft GE. With it being that size, I suppose I shouldn't be surprised that it is not uniform throughout, with it being a little warmer at the ends near the walls.
 
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timsch

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I really don't know. That's above my pay grade.
But I've sometimes been surprised at how resilient yeast can be.
If it looks and smells like yeast should, I'd use it.

Regarding separation:
I use a 25% glycerin/75% water solution, mixed 50-50 with the yeast.
The yeast has always settled to the bottom of the centrifuge tubes.
I've frozen over a hundred, and built up about nine into starters so far.
They have all worked well.View attachment 751897
When is that picture from? After freezing? thawed?
 

deuc224

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To thaw yeast i just throw the vial in a cup of water, it thaws in about 5 minutes. Never had a problem with that either, try the glycerin mix cuz it seems like you have a lot of brown cells vs glycerin.
 
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timsch

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McMullan said "You shouldn't be getting any noticeable separation between water and glycerol once frozen". Tom, I guess what I see in your frozen vials is not actually separation, but settling. Compared to my pictures above which looks to show ice crystals having formed, which is probably the actual separation of the glycerol & water.
 
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