Freezing Yeast

[mike77]

A few years back someone on here claimed to be freezing yeast and then successfully using it to make starters with. Can't remember who it was. Anyway their process is simple. They were mixing the slurry from the yeast cake and mixing it with glycerin and then freezing. I bought some glycerine and some vials and never got around to trying it. Mid last week I collected several jars of WLP090 after doing a brew and tonight I decided to give it a go.

I decanted the wort from the slurry, put the slurry into the sanitized vials and added about 30% glycerin. Then into the freezer. No idea if it will work or not. I'll try and make a starter off one fairly soon to check if any viable yeast re-animates after being defrosted. If that works then I guess it may be possible to store it longer term.

 

[TheRedDarren]

Excellent, be sure to keep us posted

 

[Covrich]

Interesting to see how it works out

 

[mike77]

Checked it this morning and it looks like the amount of glycerin I have added is preventing it from freezing. Not sure if this is good or bad. The glycerin is supposed to protect the yeast but not sure it's supposed to be enough to prevent it from freezing. I recall that it was important to get it to freeze as quickly as possible.

 

[McMullan]

Checked it this morning and it looks like the amount of glycerin I have added is preventing it from freezing. Not sure if this is good or bad. The glycerin is supposed to protect the yeast but not sure it's supposed to be enough to prevent it from freezing. I recall that it was important to get it to freeze as quickly as possible.

At minus 20, they probably won’t freeze. It’s important to mix the glycerol and yeast/slurry thoroughly (shake the jesus out of it) before storing in a non frost-free freezer. Chilling them on ice before freezing and 'thawing' them on ice when they come out of the freezer helps reduce stress. How long are you planning to store before the first test? Will you use all 50mL or just a part of it?

 

[mike77]

Wasn't going to wait long to do the first test and planned to use the 50ml. First test was just to see if the concept worked rather than try and determine how long term you could store it for. I'll still do one with the batch I have. I may do a second batch with a little less glycerin. I have a brew fermenting just now so will be able to collect more yeast shortly.

 

[shuggie159]

I co-incidentally am also trying this process.
Perhaps I should have looked on-line earlier....
I have just used thick slurry and about a 50% glycerin mix, in glass tubes with screw lids (previously used for slants).
What's the point of the additional water?
I'm storing at about -17degC and have no freezing issues.
I would guess the length of time in the freezer is not the issue here.
I haven't tried using/defrosting any yet, but guess the thawing process is the critical part. Do you do it very slowly, or give them their ideal environment ASAP?
Seems stupid waiting until I need to use them... Time to suck it and see!

 

[McMullan]

I use a sterile 50% glycerol solution (1:1 with water) in 1.5mL tubes. I haven't tried 50mL TBH. You could add about 5-10mL slurry to about 40mL glycerol solution. That should give you some reliable long term storage. Wake them with about 300mL 1.020 wort and step up to 1L at 1.040. I know it's drifting away from what you're attempting here, but have you considered doing it with a yeast starter from a fresh yeast pack? The yeast would be purer and healthier, which is essential if they are going to be stored in the freezer.

 

[McMullan]

I co-incidentally am also trying this process.
Perhaps I should have looked on-line earlier....
I have just used thick slurry and about a 50% glycerin mix, in glass tubes with screw lids (previously used for slants).
What's the point of the additional water?
I'm storing at about -17degC and have no freezing issues.
I would guess the length of time in the freezer is not the issue here.
I haven't tried using/defrosting any yet, but guess the thawing process is the critical part. Do you do it very slowly, or give them their ideal environment ASAP?
Seems stupid waiting until I need to use them... Time to suck it and see!

Yes, thaw slowly on ice or at least in a cold fridge. Rapid thawing stresses and even kills many of the cells. A 50mL vial would take hours. I'd store it in the fridge overnight then bring it up to room temperature.

 

[mike77]

I use a sterile 50% glycerol solution (1:1 with water) in 1.5mL tubes. I haven't tried 50mL TBH. You could add about 5-10mL slurry to about 40mL glycerol solution. That should give you some reliable long term storage. Wake them with about 300mL 1.020 wort and step up to 1L at 1.040. I know it's drifting away from what you're attempting here, but have you considered doing it with a yeast starter from a fresh yeast pack? The yeast would be purer and healthier, which is essential if they are going to be stored in the freezer.

For the moment it is really just about proof of concept. If I have some success then I'll maybe go down the route of creating starters. Then again if this works well I may be thinking why bother and just take samples from the fermenter.

 

[shuggie159]

OK cryogenic thaw underway.
1 phil under ice in fridge. I guess it will take the rest of the day to reach 5degC.
I have saved sweet wort stored from autoclave (pressure cooker) also put in the fridge. I plan to unite chilled yeast and wort and then bring slowly to room temp. Any advice/observations?
I don't want to mutate a Frankenyeast....

 

[McMullan]

OK cryogenic thaw underway.
1 phil under ice in fridge. I guess it will take the rest of the day to reach 5degC.
I have saved sweet wort stored from autoclave (pressure cooker) also put in the fridge. I plan to unite chilled yeast and wort and then bring slowly to room temp. Any advice/observations?
I don't want to mutate a Frankenyeast....

+1 for the pressure cooker :thumb:

I'd combine the wort and yeast when they're at room temperature. Depends how pure the yeast is I'd say.

 

[mike77]

http://www.homebrewtalk.com/freezing-yeast.html

This post here seems to suggest thawing the sample quickly in warm water. Also suggests lower concentrations of glycerin

 

[shuggie159]

Yup! Just read all that... I've got too high a glycerine ratio, and I flash froze. So by all accounts my chances a iffy.
It's important to learn by mistakes so I'll keep you posted.

I should note that my frozen sample had separated into approximately 50/50 dark and clear quotients. I shook and used the combined.

PS I didn't thaw a full 15ml phil, because I was concerned about too much carry over of glycerine into the 125ml starter. The article indicates that this is not necessarily a worry because yeast metabolises glycerine.

My questions now are, slow or flash freeze, and slow, or quick defrost? I'll try as planned, and see how it goes.

 

[mike77]

How old is your sample?

 

[shuggie159]

A week old Wyeast 1335 top cropped.

 

[McMullan]

http://www.homebrewtalk.com/freezing-yeast.html

This post here seems to suggest thawing the sample quickly in warm water. Also suggests lower concentrations of glycerin

Rapid thawing is thought to result in more viable yeast cells in a typical lab situation. Trying this with a 50mL vial, compared to a 1.5mL vial, though, might lead to localised heat shock and partial death? I don’t know, I’m guessing. You’d have to compare rapid vs slow thawing to know for sure.

How much glycerol to use depends on who you ask. 5-60% I think. Outside a lab environment and without -80 storage, I err on using more.

 

[McMullan]

Yup! Just read all that... I've got too high a glycerine ratio, and I flash froze. So by all accounts my chances a iffy.
It's important to learn by mistakes so I'll keep you posted.

I should note that my frozen sample had separated into approximately 50/50 dark and clear quotients. I shook and used the combined.

PS I didn't thaw a full 15ml phil, because I was concerned about too much carry over of glycerine into the 125ml starter. The article indicates that this is not necessarily a worry because yeast metabolises glycerine.

My questions now are, slow or flash freeze, and slow, or quick defrost? I'll try as planned, and see how it goes.

Drop about 1 degree/minute. The cooled vials can be placed in an insulated styrofoam box before going in the freezer. That should slow things down enough.

 

[shuggie159]

Ooh erh.
You guys are worrying me! Technically I seem way out of my depth.

My experiment continues... stage 1

The wort was put on the stir plate, oxygenated, and then gently combined with my sleeping beauties now at room temp. We await ... I don't expect anything for 24hrs.

 

[mike77]

Rapid thawing is thought to result in more viable yeast cells in a typical lab situation. Trying this with a 50mL vial, compared to a 1.5mL vial, though, might lead to localised heat shock and partial death? I don’t know, I’m guessing. You’d have to compare rapid vs slow thawing to know for sure.

How much glycerol to use depends on who you ask. 5-60% I think. Outside a lab environment and without -80 storage, I err on using more.

To be honest I was just planning to defrost it enough to break it loose from the vial and then dump the frozen yeast in a 1l starter like an ice cube and let it thaw and return back to room temp naturally. If there is an easy option I'll give it a go.

Checked my yeast again this evening and it seems 4 froze and 2 didn't. I only checked 1 this morning so that may have always been the case. Suspect I perhaps got a bit more glycerin in the 2 that haven't frozen.