Record for reawakening refridgerated yeast?

[bristela]

When I first started poking around this forum, being a microbiologist, I quickly found the stickied yeast washing protocol:

https://www.homebrewtalk.com/f163/yeast-washing-illustrated-41768/

I've been using this washing for over a year now and haven't had to buy yeast in all that time. Proper thanks go out to Bernie Brewer.

While reading through some of the thread I saw reference to people keeping yeast in the fridge this way for up to a year. I don't remember anybody saying for sure if they had actually done this. There are currently 236 pages of that thread so I haven't been back through to check. Sorry.

I thought I would give this a try. Whenever I wash I always keep two jars worth, one for the next brew and the other for reference in case I need to go back to it. I thought I would try and revive some yeast that I put in the fridge on 12th July 2013, ~13 months ago. You can see it in the first pic with the Wyeast nutrients I use for starter cultures.

I also add honey to my starter cultures before pouring in boiling water so as to discourage growth of other nasties. You can see this covered with foil on my stir plate in the second pic. Once this mixture has reached ambient temperature I pitch in the yeast, after having poured off the supernatant from the canning jar.

On a normal brew day with yeast that has been in the fridge for a few weeks / months, I will let the starter grow for ~4 hours. In this case I saw no sign of activity at that stage. The third pic shows the bubbles I saw just before going to bed, after ~8 hours.

The fourth pics shows growth after ~24 hours at which point I pitched it. I was not prepared to commit to a full brew for this experiment so I thought I could make a gallon batch of cider. This doesn't involve any boiling which is why I call it Lazy Cider.

From past experience I have learned that I need to back-sweeten cider at a ratio of 1/6th the volume. That's what you can see in the fifth pic, just before pitching, with the bottle on the left being kept in the fridge until fermentation has finished. This volume of starter is what I normally use for 5 gallons so I eyeballed ~1/5th the volume when pitching.

The final pic shows the state of it after 3 days in the basement.

Haven't tasted it yet but what's coming out the airlock smells really good. Will update this thread when I do.

If anybody has successfully kept their yeast like this for longer I would love to hear about it.

 

[mb82]

Pesonally for me it was just about the year mark for reawakening a yeast. Actually I did it twice since I had two yeast jars I found in the back of the fridge I had forgotten about.

 

[BullF-16]

I just made a starter from some Pacman yeast i washed and referigerated back in Oct 2010, so nearly 4 years. 1500 ml starter at 1.038 Og. Been on the stirplate for about 12 hrs. Plan to give it 4 or 5 days i guess and see what happens.

 

[bristela]

I just made a starter from some Pacman yeast i washed and referigerated back in Oct 2010, so nearly 4 years. 1500 ml starter at 1.038 Og. Been on the stirplate for about 12 hrs. Plan to give it 4 or 5 days i guess and see what happens.

Four years is pretty impressive. Please keep us updated on that, particularly how much longer the starter takes.

I still have some that are a bit older. Will have to keep them to see if I can try for a longer record

 

[grathan]

I just popped a wyeast 2633 pack dated 4/13. I did a lager a couple months ago with the same date. It took a week to fully swell and with just a 2L starter made an excellent 6 gallon batch from 1.055

 

[jbaysurfer]

Four years is pretty impressive. Please keep us updated on that, particularly how much longer the starter takes.

I still have some that are a bit older. Will have to keep them to see if I can try for a longer record

1500MLs is too much wort for a 4 year old yeast sample IMHO. The n umber of viable cells is exceedingly low. I would've started with 100MLs max and stepped up. If it takes 4-5 days to start up, it's stressed and isn't going to create good beer IME.

PS, this post is for the poster you were responding too, but quick reply doesn't do embedded quotes.

 

[BullF-16]

Four years is pretty impressive. Please keep us updated on that, particularly how much longer the starter takes.

I still have some that are a bit older. Will have to keep them to see if I can try for a longer record

Just got home from work and noticed about a 3/8" layer of foam/kreuzen on top. This morning there was nothing, just a brown liquid stirring on the stir plate. Its been 22 hrs. This is encouraging. It also smelled pretty good.

More to follow.

 

[Hermit]

I just made a starter from some Pacman yeast i washed and referigerated back in Oct 2010, so nearly 4 years. 1500 ml starter at 1.038 Og. Been on the stirplate for about 12 hrs. Plan to give it 4 or 5 days i guess and see what happens.

Wasn't Pacman yeast cultured from slurry someone found in the back of their fridge after 20 years or something? 4? Not such a big deal.

 

[BullF-16]

Just got home from work and noticed about a 3/8" layer of foam/kreuzen on top. This morning there was nothing, just a brown liquid stirring on the stir plate. Its been 22 hrs. This is encouraging. It also smelled pretty good.

More to follow.

Well this morning the kreuzen dropped and there the wort was a nice cream color vs the dark brown color it was before the yeast took off. There was also a nice ring around the flask that was about 3/4 of an inch thick. I will take a gravity reading later today.

I plan to use this in an Orange Cascades Im brewing this Sat. should i decant off as much as i can and do another starter on this same yeast to be sure? Sorry, ive been away from homebrewing for a few years and just starting back up.

 

[broadbill]

When I first started poking around this forum, being a microbiologist, I quickly found the stickied yeast washing protocol:

https://www.homebrewtalk.com/f163/yeast-washing-illustrated-41768/

I've been using this washing for over a year now and haven't had to buy yeast in all that time. Proper thanks go out to Bernie Brewer.

While reading through some of the thread I saw reference to people keeping yeast in the fridge this way for up to a year. I don't remember anybody saying for sure if they had actually done this. There are currently 236 pages of that thread so I haven't been back through to check. Sorry.

I thought I would give this a try. Whenever I wash I always keep two jars worth, one for the next brew and the other for reference in case I need to go back to it. I thought I would try and revive some yeast that I put in the fridge on 12th July 2013, ~13 months ago. You can see it in the first pic with the Wyeast nutrients I use for starter cultures.

I also add honey to my starter cultures before pouring in boiling water so as to discourage growth of other nasties. You can see this covered with foil on my stir plate in the second pic. Once this mixture has reached ambient temperature I pitch in the yeast, after having poured off the supernatant from the canning jar.

On a normal brew day with yeast that has been in the fridge for a few weeks / months, I will let the starter grow for ~4 hours. In this case I saw no sign of activity at that stage. The third pic shows the bubbles I saw just before going to bed, after ~8 hours.

The fourth pics shows growth after ~24 hours at which point I pitched it. I was not prepared to commit to a full brew for this experiment so I thought I could make a gallon batch of cider. This doesn't involve any boiling which is why I call it Lazy Cider.

From past experience I have learned that I need to back-sweeten cider at a ratio of 1/6th the volume. That's what you can see in the fifth pic, just before pitching, with the bottle on the left being kept in the fridge until fermentation has finished. This volume of starter is what I normally use for 5 gallons so I eyeballed ~1/5th the volume when pitching.

The final pic shows the state of it after 3 days in the basement.

Haven't tasted it yet but what's coming out the airlock smells really good. Will update this thread when I do.

If anybody has successfully kept their yeast like this for longer I would love to hear about it.

To answer your question, there is no reason to think why you could step up a very old yeast sample provided your process was solid and appropriate.

I would echo the suggesting that old samples will have poor viability, so you should start with a very small starter and step up to pitching volumes as needed.

Also, I'm finding myself moving away from harvesting yeast from the fermenter. I think taking an aliquot from the starter first made from the smack pack/slant/tube and using that for future propagation is a better method than dealing with the crapped-out yeast in the bottom of a fermenter. It is also an easier process for yeast propagation, in my opinion.

I would be more comfortable stepping up a starter from an old yeast sample cultured directly from the source than an old yeast sample from the bottom of a fermenter.

 

[bristela]

Wasn't Pacman yeast cultured from slurry someone found in the back of their fridge after 20 years or something? 4? Not such a big deal.

I'm intrigued now. Do you have a source for this? I'd love to read some more about it. Did a quick search and nothing obvious came up about its origins.

 

[Hermit]

I'm intrigued now. Do you have a source for this? I'd love to read some more about it. Did a quick search and nothing obvious came up about its origins.

I can't find it either. Just something I read somewhere and it stuck. Is it true? Who knows. If we quote it enough it will become true, at least on the internet. :cross:

 

[Warthaug]

I don't know about packman, but the old-school way of storing yeasts (on a slant, underneath mineral oil) reportedly allowed recovery of yeast that were 20-30+ years old. This wasn't normal, as these cultures were typically expected t last 2-3 years.

Bryan

 

[jabba11]

FWIW I dont "wash" yeast anymore either. I simply make about 400ML more starter than I need each time and place in a jar that has sanitized water in it and seal for the next use..easier and just seems like a more reasonable way to do it. I have used yeast as old as three months and typically reuse in my method 4 to 5 times before I "buy" new yeast.

 

[broadbill]

FWIW I dont "wash" yeast anymore either. I simply make about 400ML more starter than I need each time and place in a jar that has sanitized water in it and seal for the next use..easier and just seems like a more reasonable way to do it. I have used yeast as old as three months and typically reuse in my method 4 to 5 times before I "buy" new yeast.

This is what I do, with the exception of of not using any water. From what I understand the fermented starter wort is just as good a media for the yeast to be stored under.

Just fill up a jar from the extra volume in my starter, label and pop into the fridge.

 

[BullF-16]

What the hell do i know here. My 4 year old pacman is on its second step.

 

[grathan]

You making a beer with it?

 

[bristela]

I don't know about packman, but the old-school way of storing yeasts (on a slant, underneath mineral oil) reportedly allowed recovery of yeast that were 20-30+ years old. This wasn't normal, as these cultures were typically expected t last 2-3 years.

Bryan

You can always store yeast (and other bugs) pretty much indefinitely if you keep them below freezing. Obviously, you need to mix them with a cryo-protectant first. In my experience in the lab with bacteria we always used glycerol and that was considered good for keeping forever when stored at -80C. I have some yeast I've mixed with malt extract in a similar way in my freezer at home. Haven't tried reviving any recently, mainly because this washing procedure works so well.

I guess when I started this thread I was mostly interested in how long they can be kept in the fridge. Always welcome other suggestions though. Best way to learn about different stuff that way.

 

[Warthaug]

You can always store yeast (and other bugs) pretty much indefinitely if you keep them below freezing

Indefinite storage requires -80C or liquid nitrogen storage - at conventional freezer temps (-15C to -20C) frozen stock are stable for ~2 years (the exact time depends on how stable your freezer is, and how many times you remove the tube from the freezer).

Freezing is easy - a low gravity wort (1.020 or less) provides some protection in the form of extracellular sugars, while the addition of 20% glycerol or 20% DMSO provides intacellular cyroprotection.

Bryan

 

[bristela]

Indefinite storage requires -80C or liquid nitrogen storage - at conventional freezer temps (-15C to -20C) frozen stock are stable for ~2 years (the exact time depends on how stable your freezer is, and how many times you remove the tube from the freezer).

Freezing is easy - a low gravity wort (1.020 or less) provides some protection in the form of extracellular sugars, while the addition of 20% glycerol or 20% DMSO provides intacellular cyroprotection.

Bryan

I always thought the -20C recommendation was a very conservative estimate so that nobody in the lab ever lost any cultures, which would definitely be bad. I suspect it's possible to recover things after a longer period of time, it's just your percentage chance of success will decrease. I certainly don't remember anybody that had actually tested this, it was just part of lab lore. I have some Eppendorfs in my -20C with a mixture of yeast slurry and malt extract. I've had them for nearly two years now, perhaps I'll redo this experiment with them sometime next year

I've found that recipes for -80C freezing vary from lab to lab too. One boss I had insisted that it had to be done with MSG, which may have worked but was really irritating as the vial contents completely solidified. My preferred method is the one I learnt first which was to mix the cells 1:1 with 100% glycerol (final concentration of 50%). This meant that it was easy to scrape some off the top of the vial, so that it was out of the freezer for a very short period of time. I've also known people that insisted on flash freezing cells with liquid nitrogen. Never did see that this helped particularly. As with so many protocols, people tend to inherit them from whatever lab they've worked in and don't always question why they are the way they are.

Cheers

 

[Warthaug]

I always thought the -20C recommendation was a very conservative estimate so that nobody in the lab ever lost any cultures, which would definitely be bad. I suspect it's possible to recover things after a longer period of time, it's just your percentage chance of success will decrease. I certainly don't remember anybody that had actually tested this, it was just part of lab lore. I have some Eppendorfs in my -20C with a mixture of yeast slurry and malt extract. I've had them for nearly two years now, perhaps I'll redo this experiment with them sometime next year

I maintain a large yeast bank, with my day-to-day vials in a -20C and my backups in a -80C. In my experience with this bank, 2 years is actually a pretty accurate estimate of how long the stocks last. According to my notes, I've never had a sub-culture from the -20C tubes fail in anything less than 18 months old. I've had 1 failure (in ~300 "withdrawals") that fell within the 18-24 month window. After 24 months of storage the failure rate starts climbing fast - in vials 3 years old about 1 in 4 "withdrawals" fails.

I'm starting to think that I should do the analysis and do a blog post about it...

I've found that recipes for -80C freezing vary from lab to lab too. One boss I had insisted that it had to be done with MSG, which may have worked but was really irritating as the vial contents completely solidified. My preferred method is the one I learnt first which was to mix the cells 1:1 with 100% glycerol (final concentration of 50%). This meant that it was easy to scrape some off the top of the vial, so that it was out of the freezer for a very short period of time. I've also known people that insisted on flash freezing cells with liquid nitrogen. Never did see that this helped particularly. As with so many protocols, people tend to inherit them from whatever lab they've worked in and don't always question why they are the way they are.

There are about as many ways to freeze bugs as there are labs freezing bugs. I'm not sure why - a lot of research was done on this back in the 1970's so its not like we know what works and what doesn't. 20% glycerol or 20% DMSO work well, and additional adulterants do not appear to improve recovery. A lot of the additional stuff is required for highly advanced methods (e.g. high-pressure cryofreezing), but don't do much when added to conventional freezing media stock.

Bryan

 

[jbaysurfer]

Warthaug:

That's quite a bank you've got there. I may have a couple of strains to trade but I've got to get my home-lab set back up.

Out of curiosity, given your 18-24 month window, does that mean as part of your SOPs you re-streak every yeast at least once during that window? Given the number of yeasts you're banking it must get laborious....

 

[Warthaug]

In a word No. I do not actively maintain my stocks in the sense of scheduled re-streaks/cultures.

I "withdraw" under the assumption it will always work. If it doesn't grow then I start a liquid culture from my -80c stocks that I then re-freeze as my -20c "working" stock. That is the advantage of having -80c capacity - I have "permenent" stock of stable cultures. I don't think I could manage the sized bank I have without the -80c.

I'm always open to trades, just follow the instructions on my blog to get past the spam-trap when you are ready.

Bryan