Yeast freezing question

Hello everyone-

I am very new to Home brewing and have been reading up quite a bit on numerous topics and something that I have really noticed is the cost of yeast. I am interested in freezing yeasts and something that I have not yet read is flash freezing. I am a big foodie and have always been a fan of Alton Brown's "Good Eats" on the Food Network. One one of his episodes, he tackles the issue of freezing strawberries. Freezing conventionally results in mushy fruit when thawed because of ice crystals fracturing cell walls because the ice crystals grow slowly. His solution was to cool the fruit close to freezing temperature and then to use dry ice to immediately flash freeze. The science behind this is that the ice crystals are supposed to be much smaller and cell walls do not get ruptured. I have tried this approach on strawberries and blueberries and it does work. Would this approach help with freezing yeast?

Glycerin is a heck of a lot easier

Why not both to ensure success?

naperboiler said:

Why not both to ensure success?

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Flash freezing is certainly what the pros do in their microbiologiy labs, so it is a theoretically viable practice. But, they've also got freezers that hold a constant -70C. Chris White suggests using a high enough concentration of glycerin to actually prevent freezing in standard home freezers, and this is what I do. So I wouldn't want to flash freeze because I don't want my yeast to freeze at all.

I'd love to see a system that uses something like this, but I'm skeptical that it would actually improve viability. If you get positive data to the contrary, please post it.

How much would the dry ice and ethyl alcohol bath cost you?

As I stated in my original post, I am a novice so I have no idea if this would work, hence the post. As far as cost is concerned, I think the last time I did this with fruit, the local ice cream store sold dry ice for about $6 / lb. Combine that with high proof ethanol (which could be re-used), I am guessing it would cost less than $10 per batch.

I am not really sure about what you mean about the yeast not freezing as I was horrible at chemistry so I probably do not understand the reason for using glycerin in the first place.

naperboiler said:

As I stated in my original post, I am a novice so I have no idea if this would work, hence the post.

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sure thing!

naperboiler said:

I am not really sure about what you mean about the yeast not freezing as I was horrible at chemistry so I probably do not understand the reason for using glycerin in the first place.

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Glycerin has two different effects. The first is to act as a cryoprotectant, which "pads" the yeast cells and ensures that large ice crystals do not form and tear their cell walls. There is an additional second effect of dropping the freezing point of the solution, as described here. If you use the right concentration, the glycerin/yeast will stay cold but will never actually freeze in a home freezer. It is debatable if this is a good thing or not, but Chris White (who wrote the book Yeast, which is really quite good and is targeted at the homebrewer) argues for it.

From the large thread on making a frozen yeast bank, I remember reading that the solution did actually freeze when the op's concentrations were followed. What would happen if the water was replaced with more glycerine? Could propylene glycol be used I wonder?

A home freezer is usually set at about -10 degrees F or around -20 deg C.
Folks seem to use 15% by volume (the Guide to Making a Frozen Yeast Bank thread). The density of glycerine (aka glycerol) is 1.261, so 15% by volume is (15/1,261) ~ 12% by weight. The chart MalFet references would have that freeze at about 27 deg F (by eyeball guesstimate), in other words, frozen in a home freezer.

naperboiler, if you want to save money on yeast, why not consider yeast washing? It looks like a low-tech, inexpensive way of getting more out of your yeast dollar.
(FWIW, I don't do it myself because I don't brew enough and grow my yeast off petri plates streaked from -80 deg C glycerol stocks.)

You need a cryoprotectant which is what the glycerol does, there are others you can use too. It's not as much cell damage thing more so than an osmotic pressure thing. If you freeze all the water up the osmotic pressure will encourage the yeast to spill its guts.

StMarcos said:

From the large thread on making a frozen yeast bank, I remember reading that the solution did actually freeze when the op's concentrations were followed. What would happen if the water was replaced with more glycerine? Could propylene glycol be used I wonder?

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Standard procedure around here, including on the thread you mention, is to use somewhere in the vicinity of 15-30% glycerin. This does indeed freeze solid in domestic freezers. If you add more glycerin, it doesn't freeze. This is actually what Chris White recommends in his yeast protocol, and I mentioned in my last post that this is what I have been trying lately. Still to early to offer results. What would be the advantage of propylene glycol? It has a bad reputation for being nasty to microorganisms.

naperboiler said:

Hello everyone-

I am very new to Home brewing and have been reading up quite a bit on numerous topics and something that I have really noticed is the cost of yeast. I am interested in freezing yeasts and something that I have not yet read is flash freezing. I am a big foodie and have always been a fan of Alton Brown's "Good Eats" on the Food Network. One one of his episodes, he tackles the issue of freezing strawberries. Freezing conventionally results in mushy fruit when thawed because of ice crystals fracturing cell walls because the ice crystals grow slowly. His solution was to cool the fruit close to freezing temperature and then to use dry ice to immediately flash freeze. The science behind this is that the ice crystals are supposed to be much smaller and cell walls do not get ruptured. I have tried this approach on strawberries and blueberries and it does work. Would this approach help with freezing yeast?

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Even labs that use liquid nitrogen to flash freeze use glycerol solution but they use a -80C freezer and the sample is frozen rock solid. Freeze fast, freeze slow, won't matter. Either way, if you don't use a cryoprotectant to minimize ice crystal size and density you will cleave the holy hell out of the yeast cells and end up with a much smaller potentially viable number of cells.

Rupturing cell walls in fruit is a lot different than rupturing yeast cell walls considering what you are trying to achieve out of the methods being used.

Also, as previously mentioned, you won't freeze the solution solid with proper glycerol concentration in a home freezer. So if you go with the glycerol solution you skip the flash freeze step since you don't want to force freeze/thaw cycles if you can help it.

I didn't know it was bad for microorganisms. Just thought it was non toxic and had a low freezing point.

StMarcos said:

I didn't know it was bad for microorganisms. Just thought it was non toxic and had a low freezing point.

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All the protocols I've seen call for glycerin, so you may as well stick with that. Glycerin is also non-toxic and has a low freezing point. I seem to remember somewhere reading about why propylene glycol is bad, but I can't remember for the life of me where I read it or why it is a problem.

MalFet said:

All the protocols I've seen call for glycerin, so you may as well stick with that. Glycerin is also non-toxic and has a low freezing point. I seem to remember somewhere reading about why propylene glycol is bad, but I can't remember for the life of me where I read it or why it is a problem.

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Also, you may find some literature for yeast metabolizing glycerol, but Saccharomyces cerevisiae does not, so do not be scared off by such google or other search results.

Randar said:

Also, you may find some literature for yeast metabolizing glycerol, but Saccharomyces cerevisiae does not, so do not be scared off by such google or other search results.

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Actually, yeast can metabolize glycerol during respiration (growth in the presence of O2), not in fermentation.
It's used in YPDG (Yeast extract, Peptone, 0.1% Dextrose, 3% Glycerol) agar to allow the grands (non-petite, wild-types) to grow a LOT bigger, as they have access to more energy & carbon from the glycerol, while the petites have to skimp by on the less-than-usual amount of sugar.
That said, they don't metabolize any during the time it takes to walk to the freezer and put the tubes with the yeast and glycerol into their storage box.

JohnMc said:

Actually, yeast can metabolize glycerol during respiration (growth in the presence of O2), not in fermentation.
It's used in YPDG (Yeast extract, Peptone, 0.1% Dextrose, 3% Glycerol) agar to allow the grands (non-petite, wild-types) to grow a LOT bigger, as they have access to more energy & carbon from the glycerol, while the petites have to skimp by on the less-than-usual amount of sugar.
That said, they don't metabolize any during the time it takes to walk to the freezer and put the tubes with the yeast and glycerol into their storage box.

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Interesting...would you recommend using YPDG for standard propagation agar plates? Right now, I'm streaking plates from microcentrifuge tubes of 50% slurry/50% glycerol solution that I am storing in my home freezer. I'm still trying to get a sense of the mechanics involved, but I haven't been thrilled with the growth I'm getting on the plates.

Basically, I'm getting reasonable colony density on my first pass, a little bit on my second, and virtually nothing on my third and forth. Am I just not getting enough fluid on my inoculation loop, or might the issue be my agar formulation? Right now, I'm using 1.040 wort with a bit of nutrient and agar.

JohnMc said:

Actually, yeast can metabolize glycerol during respiration (growth in the presence of O2), not in fermentation.
It's used in YPDG (Yeast extract, Peptone, 0.1% Dextrose, 3% Glycerol) agar to allow the grands (non-petite, wild-types) to grow a LOT bigger, as they have access to more energy & carbon from the glycerol, while the petites have to skimp by on the less-than-usual amount of sugar.
That said, they don't metabolize any during the time it takes to walk to the freezer and put the tubes with the yeast and glycerol into their storage box.

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Looks like you are correct:

"Utilization of glycerol, a non-fermentable carbon source, by Saccharomyces cerevisiae is repressed by glucose. After the depletion of any faster growth-supporting substrate, the enzymes necessary for the utilization of glycerol are induced, and an exponential growth phase on glycerol will follow a diauxic lag phase. "

I must have been reading about some specific strain or mutation that repressed glycerol metabolism.

MalFet said:

Interesting...would you recommend using YPDG for standard propagation agar plates? Right now, I'm streaking plates from microcentrifuge tubes of 50% slurry/50% glycerol solution that I am storing in my home freezer. I'm still trying to get a sense of the mechanics involved, but I haven't been thrilled with the growth I'm getting on the plates.

Basically, I'm getting reasonable colony density on my first pass, a little bit on my second, and virtually nothing on my third and forth. Am I just not getting enough fluid on my inoculation loop, or might the issue be my agar formulation? Right now, I'm using 1.040 wort with a bit of nutrient and agar.

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The colony density you describe is about right for streaking onto a plate from a frozen stock. The idea with that method is to get single colonies. If you want a large number of colonies then yes, you will have to get a lot more of the fluid from the frozen stock onto the plate.

I have never done it with yeast at home but I have been working in a yeast genetic research lab for 3 years now.

Also, DON'T use YPD or any variant thereof for your plates. You have been doing the right thing by using malt extract. Apparently growing the yeast on YPD before pitching into wort makes for some terrible smells and/or flavors in the resulting beer. I remember reading a thread about on here that a long time ago.

dinertime said:

The colony density you describe is about right for streaking onto a plate from a frozen stock. The idea with that method is to get single colonies. If you want a large number of colonies then yes, you will have to get a lot more of the fluid from the frozen stock onto the plate.

I have never done it with yeast at home but I have been working in a yeast genetic research lab for 3 years now.

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Sounds good. Thanks for the reassurance. I'm getting single colonies, but I'd like to get more of them so I can be a bit choosier when inoculating my first 10mL step. I'll try globbing a bit more up from the snap tube next time to see how it goes.

dinertime said:

Also, DON'T use YPD or any variant thereof for your plates. You have been doing the right thing by using malt extract. Apparently growing the yeast on YPD before pitching into wort makes for some terrible smells and/or flavors in the resulting beer. I remember reading a thread about on here that a long time ago.

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Interesting. I think I found the thread you are talking about here. I guess it makes sense by the same logic that says you don't want to make a starter out of table sugar. I'll stick with the DME agar medium. Thanks for the tips!

Randar, it's the 'petites' that can't use glycerol. They are deficient in respiration (usually missing or messed up mitochondria). Even on normal plates, which gives access to air, the colonies are smaller. This is because there's less energy to be had from sugar if respiration is deficient; less energy = less growth. The beauty of the YPDG is that the differentiation is enhanced between wild-types and petites. If you want to read about it, glycerol is converted to glyceraldehyde phosphate by a couple of steps and enters towards the end of the Embden, Meyerhof, Parnas pathway (glycolylsis), after the energy producing steps. Then a few more steps and it's converted to pyruvate and enters the Krebs cycle, making energy.

MalFet, I wouldn't use YPDG agar for normal stuff, only you're into the science part of things. If you just made YP glycerol (YPG can be either galactose or glycerol, IIRC), leaving out the glucose, petites couldn't grow. The only kind of colony you'd see would be wild-type with regard to respiration. That would be kind of useful, in that beer needs a respiration phase at the beginning.

Also, DON'T use YPD or any variant thereof for your plates. You have been doing the right thing by using malt extract. Apparently growing the yeast on YPD before pitching into wort makes for some terrible smells and/or flavors in the resulting beer. I remember reading a thread about on here that a long time ago.

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I do that all the time. In fact, it's the only plate media I use. I usually even grow my initial 5 ml 'starter starters' in it. The actual starter, however, is malt and yeast nutrient. No off flavors at all.

JohnMc said:

I do that all the time. In fact, it's the only plate media I use. I usually even grow my initial 5 ml 'starter starters' in it. The actual starter, however, is malt and yeast nutrient. No off flavors at all.

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Hm interesting. Like I said I don't propagate this way at home so I have no personal experience. If there is an effect, your full-size starter in malt must give the yeast a chance to re-equilibrate to the new media.

dinertime said:

your full-size starter in malt must give the yeast a chance to re-equilibrate to the new media.

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That's pretty much it. It's mostly to have the maltose digesting enzyme, maltase, pre-made. This is so your yeast get a running start, rather than having to wait for maltase production (hours).