Will storing yeast this way work?

[solavirtus]

I came up with a plan for storing yeast, and I'd like some other brewers' input on it. I basically wanted to be able to keep yeast around, without having to buy a tube or smack pack every time I brew.

I start with a normal starter in DME on a stir plate, usually between 1 and 2 liters based on MrMalty. Usually 18-36 hours after pitching a White Labs vial or Wyeast smack pack into the starter, I pitch almost all of it into my wort. Sometimes I cool down the starter and decant, if I have enough time. I save about 15-25mL of the yeast starter (or slurry) and mix it 1:1 with a sterilized (boiled) solution of 30% glycerol in water. That makes the final concentration of glycerol 15% and gives me a total of around 50mL, fitting rather nicely in a sterile conical tube.

Then, I put those tubes of yeast in a small styrofoam cooler with a cold pack and that goes in my garage freezer. It's a frost-free freezer, so the styrofoam cooler and cold pack keep the temp inside relatively constant and low enough (I think) through the defrost cycles.

I have only used one of the tubes so far to make a starter, and it seemed to work well. The starter didn't get going quite as fast as a fresh smack pack usually does, when I pitched the starter into the fermenter, it took off as expected. I plan on using this kind of yeast culture and storing only for about 5 generations for a strain, then starting from a fresh smack pack after that. I know mutations and such can accumulate and the yeast get weird an unhappy after long enough.

So my big question is, what are the problems with doing it this way?

 

[jfr1111]

You are basically freezing yeast. I'm not an expert on the matter, but several threads on here talk about the procedure. But it seems to be working for you, so why not ?

 

[Yooper]

I don't do it, but have you considered just using a sterile loop and innoculating a slant that way? That would be less likely to get contaminated, and you're making starters anyway so you'd just build up the slant with a starter. You could take a few samples out of each yeast vial, and have quite a yeast bank, and less risk of contamination.

https://www.homebrewtalk.com/wiki/index.php/Yeast_Slants

https://www.homebrewtalk.com/f163/slanting-yeast-133103/

 

[solavirtus]

I thought about making slants, but the agar and glass tubes seem a little bit of a pain in the neck. If i were planning on keeping the strains around for longer I think that would be my method, but I don't know if it's worth the trouble. Thanks for the links, a little reading up on it should help me out.

 

[MalFet]

Yeast ranching, eh?

Like jfr1111 said, you are using a fairly standard yeast freezing procedure, though with a few modifications. The thing that jumps out at me is that boiling is not generally considered sufficient sterilization for medium or long term yeast storage. There are protocols for sterilizing that don't require pressure, but they are a major PITA. A pressure cooker is pretty much essential equipment for freezing yeast, to my mind. If you think you can get away without one, I would encourage you to buy a few pre-made agar plates sometime and to streak your yeast samples. Speaking from experience (unfortunately), I suspect you'll be unpleasantly surprised by what you see.

If frost-free freezers, Chris White actually recommends using a higher concentration of glycerol to completely prevent the yeast from freezing. I have been doing this lately and have been extremely pleased with the results.

You don't mention your starter process, but start very small and very weak. Gradually build up from there.

My understanding is that home brewers face essentially zero risk of mutation, but depending on your freezing protocol you might be causing unintentional selection pressures that will eventually lead to some weird results. Re-seeding the population after 5 generations is a good, safe plan.

Good luck!

 

[solavirtus]

Thanks for the input MalFet. I think doing a step up starter would be a good idea. Maybe the first step with a 1.020 wort then up to the standard 1.040 or so is what I'll try.

As far as glycerol percentage, do you recall what the honorable Chris White suggests? I have a feeling that too high a percentage of glycerol is bad. Talking to friends that work in laboratories, about 25% glycerol is as high as most people go for storing bacteria, so I would assume yeast are might be even less tolerant, but I'm not expert.

 

[theredben]

As far as glycerol percentage, do you recall what the honorable Chris White suggests? I have a feeling that too high a percentage of glycerol is bad. Talking to friends that work in laboratories, about 25% glycerol is as high as most people go for storing bacteria, so I would assume yeast are might be even less tolerant, but I'm not expert.

They only go to 25% glycerol because they have some very $$$$ -80*C freezers. At home we do not have that luxury. And just to second Malfet's reccomendation about a pressure cooker. Boiling it once is not going to cut it. Google "Tindalization" if you really don't want a pressure cooker taking up space.

 

[MalFet]

Thanks for the input MalFet. I think doing a step up starter would be a good idea. Maybe the first step with a 1.020 wort then up to the standard 1.040 or so is what I'll try.

As far as glycerol percentage, do you recall what the honorable Chris White suggests? I have a feeling that too high a percentage of glycerol is bad. Talking to friends that work in laboratories, about 25% glycerol is as high as most people go for storing bacteria, so I would assume yeast are might be even less tolerant, but I'm not expert.

I've not read anything about damage from too-high glycerol levels, but if you see or have seen anything I would be interested in reading it. Chris White's actual formula is 50% glycerol 50% YPD + 1g/L ascorbic acid, but this is based on the assumption that you are centrifuging your yeast slurry to remove the fluid.

FWIW, my standard procedure is pretty simple. I've got a bottle of glycerol that I dosed at 2g/L with ascorbic acid. I use it to fill six microcentrifuge tubes halfway, close them up and stick them in my pressure cooker along with a graduated syringe. Heat and cool. Open up the smackpack of yeast, load up the syringe and then -- one by one, under the updraft from a flame -- fill up the remaining half of the microcentrifuge tubes of yeast slurry. Stick in the fridge for 24 hours, and then into the freezer.

From there, I plate my samples if I am less than 100% sure of the yeast's viability or my sanitation practices. Otherwise, I just pitch the whole thing right in. The whole thing is mighty quick (<10 min), and the pressure cooker only cost about $25.

 

[Randar]

I've not read anything about damage from too-high glycerol levels, but if you see or have seen anything I would be interested in reading it. Chris White's actual formula is 50% glycerol 50% YPD + 1g/L ascorbic acid, but this is based on the assumption that you are centrifuging your yeast slurry to remove the fluid.

FWIW, my standard procedure is pretty simple. I've got a bottle of glycerol that I dosed at 2g/L with ascorbic acid. I use it to fill six microcentrifuge tubes halfway, close them up and stick them in my pressure cooker along with a graduated syringe. Heat and cool. Open up the smackpack of yeast, load up the syringe and then -- one by one, under the updraft from a flame -- fill up the remaining half of the microcentrifuge tubes of yeast slurry. Stick in the fridge for 24 hours, and then into the freezer.

From there, I plate my samples if I am less than 100% sure of the yeast's viability or my sanitation practices. Otherwise, I just pitch the whole thing right in. The whole thing is mighty quick (<10 min), and the pressure cooker only cost about $25.

Wait wait wait....

This is not entirely correct. The suggested storage media ratios you list are not accurate per White or standard lab practice.

You want a 50% solution of glycerol to be added in equal parts to the YPD. That means the actual glycerol is ~25% of the final volume.

The ascorbic acid can be added to your glycerol solution ahead of time to get to the overall 1g/L concentration as MalFet suggests.


And if the OP is going to do this yeast ranching, you need to get yourself a pressure cooker.

Also, I dose up about 50 vials at a time with glycerol solution and sterilize them all at once. This means I don't have to get out the pressure cooker every time I am going to store yeast, as I usually have a jar of pre-loaded and pre-sterilized tubes and glycerol solution ready for the yeast to be added.

I use disposable plastic pre-sterilized pipettes so I truly only have to get the pressure cooker out every 5 yeast storage days (since I store 10 tubes when I collect yeast)

 

[Randar]

They only go to 25% glycerol because they have some very $$$$ -80*C freezers. At home we do not have that luxury.

It is not related to the -80*C freezer. In the lab, the -80 will freeze solid the 25% glycerol solution with yeast or bacteria and some labs dip in liquid nitrogen as well. The glycerol is there to prevent large ice crystal formation and shearing or rupturing of the cell wall during this freezing process. These -80 stocks will be preserved indefinitely.

In home -20 freezers this concentration will not freeze solid while auto-defrost freezers present the issue of temp cycles. Glycerol in these scenario acts more as an antifreeze and suspension media. You reduce the concentration of glycerol and you will start to lower the freezing temp of the solution and crystal formation and and freeze/thaw will slowly destroy your yeast colony. If done properly, I think you will be able to store yeast in the home freezer for 5+ years with this method. Some have suggest longer, but I have not reached anywhere near that length of time yet to be able to opine from direct experience.

I have seen some homebrewers suggest they will use higher concentrations of glycerol, but I do not see the point, personally.

 

[MalFet]

Wait wait wait....

This is not entirely correct. The suggested storage media ratios you list are not accurate per White or standard lab practice.

You want a 50% solution of glycerol to be added in equal parts to the YPD. That means the actual glycerol is ~25% of the final volume.

Okay, I'm wait wait waiting...now what?

I will have to wait until I get home tonight to double check, but I am relatively sure that Chris White's protocol in Yeast is 50/50 straight glycerol/YPD, not diluted glycerol/YPD. Certainly possible I have been misreading it all this time, though. If anyone has the book handy, it should be easy enough to check.

You and I talked about it in this thread: https://www.homebrewtalk.com/f128/glycerin-water-ratio-freezing-yeast-215319/ Remember?

It is not related to the -80*C freezer. In the lab, the -80 will freeze solid the 25% glycerol solution with yeast or bacteria and some labs dip in liquid nitrogen as well. The glycerol is there to prevent large ice crystal formation and shearing or rupturing of the cell wall during this freezing process. These -80 stocks will be preserved indefinitely.

In home -20 freezers this concentration will not freeze solid while auto-defrost freezers present the issue of temp cycles. Glycerol in these scenario acts more as an antifreeze and suspension media. You reduce the concentration of glycerol and you will start to lower the freezing temp of the solution and crystal formation and and freeze/thaw will slowly destroy your yeast colony. If done properly, I think you will be able to store yeast in the home freezer for 5+ years with this method. Some have suggest longer, but I have not reached anywhere near that length of time yet to be able to opine from direct experience.

I have seen some homebrewers suggest they will use higher concentrations of glycerol, but I do not see the point, personally.

I don't think that theredben was suggesting that 25% glycerol solution doesn't freeze in -80C, but rather that labs with expensive freezers have a different set of goals, abilities, and concerns than do homebrewers with standard freezers.

The "standard" is certainly 25% glycerol, but I've been pretty happy with the results I have been getting using more. I have done a few side-by-side comparisons and the 50% glycerol has given me better growth both in starters and on plates than does the 25% solution. The point is to prevent freezing all together. It makes sense to me that this would help theoretically, and my at present very limited tests are bearing the theory out. This is still pretty much in the realm of anecdotal evidence, but it is suggestive to me. The fact that Chris White suggests (at least by my reading) more-or-less the same thing is what convinced me to do it this way.

I am still curious to know if there are any downsides to using more glycerol, but haven't been able to find suggestion of any yet.

 

[Randar]

White's book refers to the exact same procedure for -80 freezers as for the -20 method except the ascorbic acid bit, at least by my recollection. In my -20 chest freezer I have no freezing and I use a couple ice packs and a medical grade styrofoam pack for further isolation and temp regulation.

 

[MalFet]

White's book refers to the exact same procedure for -80 freezers as for the -20 method except the ascorbic acid bit, at least by my recollection. In my -20 chest freezer I have no freezing and I use a couple ice packs and a medical grade styrofoam pack for further isolation and temp regulation.

Like I said, I'll check when I get home, but I think they are different. I read over the section several times because (like I mentioned in the other thread) I didn't catch it the first time.

If you are keeping your suspension liquid even with 25% glycerol then we're more or less doing the same thing. But, I'm surprised that happens. The Dow Chemicals link you sent me in the other thread suggests that a 25% glycerol solution freezes at about -7C. 25% glycerol freezes solid in my normal kitchen freezer. 50%-60% (I actually use more like 60%) stays liquid.

The ice packs and styrofoam are also certainly essential, but Ilm not sure they're enough. I do a lot of (non-yeast) freezing for another project, and I was told by food chemists in that context that "soft" freezing (i.e., -20C temps) is still hard on cell membranes, even if the temp is held relatively steady. I didn't have the science to completely understand their explanations, but it had something to do with ice crystal activity. I should have paid better attention in all those physical chemistry classes.

 

[solavirtus]

Seeing what you guys are talking about, I think I'll be bumping my freezer stocks up at least 25%. The last few I made at 15% did freeze, though it's not like ice, more like slush. The one starter I made from a frozen stock so far revived fine, but it had only been frozen for a few weeks. I would guess the long term viability of these will drop off pretty quick at only 15% glycerol.

As far as a styrofoam cooler in the frost-free -20C, I measured with a timed thermometer over the course of a week, and inside with ice packs, it stayed below -20C the whole time, so at least with my equipment, that keeps it cold enough.

 

[MalFet]

For anyone still interested, here is the full procedure for -20C storage from p.201 of Yeast. It is an extremely good book, and I would recommend it to anyone who has even the slightest interest in this topic:

Procedure for 20ºC storage
1. Follow steps 1 through 5 from the -80ºC procedure. (note: see below)
2. Prepare a solution of 50 percent glycerol and 50 percent YPD.
3. Add 1 gram per liter ascorbic acid to your solution.
4. Add 1 milliliter of the solution to the tube, and gently re-suspend the yeast with a sterile pipette.
5. Seal tightly, wrap in Parafilm, place the tubes upright inside a small Styrofoam ice chest and then place the ice chest into the freezer.
6. To revive the yeast, either select a portion of the culture with a pipette tip and plate it or warm the entire culture by holding in your gloved hand until it reaches room temperature, then add it to 100 milliliters of liquid growth medium.

(From the -80ºC procedure:
1. Pick a culture and grow it in 10 milliliters of medium for 48 hours. Growth is done before this time, but the yeast build glycogen reserves after growth.
2. Move the 10 milliliters culture to a 40ºF (4ºC) environment, and hold for another 48 hours, to encourage the yeast to build trehalose.
3. Under the hood or near a flame re-suspend the yeast in the 10-milliliter culture and transfer 1 milliliter to a sterile 1.5 ml microcentrifuge tube. Label the tube with the strain name, number, and date.
4. Centrifuge the tubes for 3 to 4 minutes. Remove carefully and place in a rack under the hood.
5. Carefully discard the liquid, saving the yeast pellet at the bottom.)

@solavirtus - it is worth looking at a glycerol datasheet to find the right concentration. I am still very curious to hear if there are any potential harms to the yeast from too much glycerol, but I haven't heard anything along those lines yet. People have been getting good results with 25% solutions, but that is not to say they wouldn't be getting better results with higher. My hunch is that the 25% thing is a hold-over from lab technique that doesn't necessarily apply in the same way to homebrewers, like HSA or any of the other conventional wisdoms that turned out to be wrong. It's interesting stuff. I'll keep poking at it as my samples age and report back if I get any sudden drops.

 

[Randar]

LOL. I just talked it over again with the SWMBO yeast geneticist and she originally told me that she interpreted that as the way she does it in lab for her -80 and as 50% dilution of glycerol with 50% YPD. WTF... When I just had her re-read it she admitted to misleading me.

Good lord.

SWMBO says spinning 1ml of dense yeast generally gives 0.2-0.3 ml of pellet (of course depending on how dense the sample is). So if you do the math on White's method of adding 1ml of solution to this pellet, that would give ~38-42% glycerol in the final solution of 1.2-1.3 final ml.

So it looks like maybe we're bracketing the actual solution concentrations White is suggesting.

Looks like I will be stepping up my future glycerol concentrations!

 

[MalFet]

LOL. I just talked it over again with the SWMBO yeast geneticist and she originally told me that she interpreted that as the way she does it in lab for her -80 and as 50% dilution of glycerol with 50% YPD. WTF... When I just had her re-read it she admitted to misleading me.

Good lord.

SWMBO says spinning 1ml of dense yeast generally gives 0.2-0.3 ml of pellet (of course depending on how dense the sample is). So if you do the math on White's method of adding 1ml of solution to this pellet, that would give ~38-42% glycerol in the final solution of 1.2-1.3 final ml.

So it looks like maybe we're bracketing the actual solution concentrations White is suggesting.

Looks like I will be stepping up my future glycerol concentrations!

Don't be too hard on her. Make her steal a centrifuge from work for you.

 

[solavirtus]

Yeah, I really should get that book, everybody has good things to say about it. I'm just a little concerned it will convince me to spend some more $$$ on equipment.

From what MAlFet posted here, it looks like White is describing a system where only a small of yeast solution is created and stored, like 1.2mL. The way I've been doing it is have about 40mL total. I just figured that there will be some loss of viability regardless, so why not have a bit more viable yeast (in absolute numbers) to start with when it gets thrown into the starter? This way in a pinch, I could just throw the whole tube of 40mL into a single starter. I'm sure it's not ideal, but it has worked (once so far).

 

[Randar]

Yeah, I really should get that book, everybody has good things to say about it. I'm just a little concerned it will convince me to spend some more $$$ on equipment.

From what MAlFet posted here, it looks like White is describing a system where only a small of yeast solution is created and stored, like 1.2mL. The way I've been doing it is have about 40mL total. I just figured that there will be some loss of viability regardless, so why not have a bit more viable yeast (in absolute numbers) to start with when it gets thrown into the starter? This way in a pinch, I could just throw the whole tube of 40mL into a single starter. I'm sure it's not ideal, but it has worked (once so far).

I think storage size is really the biggest reason to keep the tubes small. If you have space for a couple hundred 40ml tubes and whatnot, why not? I don't, so keeping it in 2 ml tubes is much better. I do a full 2 ml final volume in a 2 ml tube, so I don't stick to the 1.2-1.3 ml size White discusses. I think that is more due to the 1.5ml microfuge tubes being more common in general than the full 2 ml tubes.

 

[MalFet]

Yeah, I really should get that book, everybody has good things to say about it. I'm just a little concerned it will convince me to spend some more $$$ on equipment.

From what MAlFet posted here, it looks like White is describing a system where only a small of yeast solution is created and stored, like 1.2mL. The way I've been doing it is have about 40mL total. I just figured that there will be some loss of viability regardless, so why not have a bit more viable yeast (in absolute numbers) to start with when it gets thrown into the starter? This way in a pinch, I could just throw the whole tube of 40mL into a single starter. I'm sure it's not ideal, but it has worked (once so far).

Like Randar says, the volume you end up saving isn't really critical. If you are pitching the frozen contents right into a starter, bigger will save you a step or two. If you are plating first, all you really need is ten cells so volume doesn't matter. I've got more than a hundred samples stored in a very small insulated box in the back of my kitchen freezer, so the tiny tubes are essential.