Freezing yeast...with sucrose?

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RadicalEd

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I was browsing another thread here, which directed me to this page:
http://maltosefalcons.com/tech/MB_Raines_Guide_to_Yeast_Culturing.php

I call you attention to this snippet:
Maltese Falcons said:
Freezing- Yeast can be frozen if a cryoprotectant is added. Glycerin and sucrose are commonly used and should be added to exponentially growing yeast at a final concentration of 5-15% (5-15 g/100 ml).

So then, is simple sucrose an adequate replacement for somewhat expensive and hard-to-find glycerin?

Your thoughts...
 
The addition of glycerol helps stabilize the membrane of the yeast under freezing conditions. What will happen, is that as the water begins to go from liquid to solid state, ice crystals will nucleate on the surface of the cells. As the ice crystals propagate, they effectively act as needles puncturing the cell. Now, adding glycerol prevents this, as it creates a pocket of hydrophobicity on the cell surface, devoid of nucleation sites. It doesn't really work that well, though, unless you're flash-freezing your cells (by immersing in a dry-ice/ethanol bath (-78˚C), for example). Without flash-freezing, the cells will still be "stabilized", but they'll end up being dehydrated in the freezing process.

Sucrose does something different entirely. It works by stabilizing minute pockets (vesicles) inside outer membrane of the yeast...kind of like hardening the shell, so that when it freezes, rather than being squeezed every which way it gets compressed more evenly.

Now, will sucrose be as effective as a glycerol flash freeze? Yes, ceterus peribus. The biggest impact occurs what the osmotic pressure of the media is.

This paper is worth the read if you want more details. It's specific to S. cerevisiae.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1392953

I hope that helps.

EDIT: I feel like I should mention...don't go sticking a chunk of dry ice in a bunch of 151 to get a "flash-freezer". -78˚C is really, really cold, and poses some risks as far as what kind of a containers won't shatter, etc.
 
Haha, yes, I'm very familiar with the effects of dry ice; overclocking is a hobby of mine, so I know the dangers. But a fair warning to others :D.

Thanks for such an informative response! And welcome to HBT, too. I'll definitely read that paper when I get a chance.
 
Third Scientist here. I keep my yeast strains in 30% glycerol in the -80 and grow my starters in the lab at the optimum temps for each strain. Probably over-kill but it's hard to ignore the decade of microbiology education and I get a nice fermentation rolling almost immediately following pitching.
 
biohazzardbrewing said:
Third Scientist here. I keep my yeast strains in 30% glycerol in the -80 and grow my starters in the lab at the optimum temps for each strain. Probably over-kill but it's hard to ignore the decade of microbiology education and I get a nice fermentation rolling almost immediately following pitching.

Cheers to that. Do you add polyvalent metals (particularly ZnCl2) into your brew water?
 
Fourth scientist here. Glycerin is not that hard to find for sale, and actually many drugstores and supermarkets will carry it. You should be able to get 16 oz. for ~ $10. That's good for A LOT of yeast storage. It is also readily available online. You may see it also called glycerol or glycerine - all the same stuff. That may be part of the confusion in finding it.

I store my yeast, autoclave my starters, and innoculate them at work, but typically then take them home to grow them up. I also autoclave my kegs sometimes if I remember to bring them in, and before I kegged I always autoclaved my bottles.

I'm not a microbiologist, but I find myself growing lots of bacteria and yeast in my work studying plants.
 
Fifth scientist here. Any of you use DMSO to freeze your yeast? Works beautifully for mammalian cells.

mrkristofo -- do you have a source for the info on sucrose? That's not correct as I understand it. Sugar or sugar alcohol solutions all act primarly as vitrification agents to the best of my knowledge. To quote from the article you linked, "Most improvements in freezing have resulted from the use of cryoprotective agents (CPAs) (10). The purpose of CPAs is to lower the freezing point of the cellular cytoplasm by increasing the concentration of intracellular solutes. At the same time, addition of CPAs involves osmotic stress, which decreases the cellular volume before freezing (6). By these means, intracellular ice formation effects are reduced (1)." No mention of differing mechanism of action. I'm not trying to be a contrarian -- just interested!

You guys who work with your brewing yeast in your labs ... do your PI's know about that? I'm pretty sure my boss would kill me if he found out I was doing that (which I'm not -- WAAAAAY too damn much work to culture my neurospheres to risk contaminating them with that much yeast, so the primary tissue culture hood is out. The secondary hood is for bacteria work, and it's too much work to get a nice clean batch of yeast to want to risk bacterial contamination in there, and our third hood is for human tissue, so that's out, too. :) )
 
Yeah, as a general rule, bacteria and yeast go nowhere near tissue/cell culture stuff. This is a microbiology lab...yeast and bacteria everywhere. As for the use of a hood, I thought that my methods were over-kill. This is something that people do in there basements and garages by boiling flasks of wort on their stovetops. You definitely dont need a sterile hood to make a starter; the bench top will be fine.
 
DMSO works fine too, but that is harder for the lay-person to find. No tissue culture in our hood, lots of bacateria and yeast. Mostly the wussified general lab strains and the occassional plant pathogen.
 
DMSO can actually be found at most herbal stores. Hippies love the stuff. That said -- I'll probably use glycerol myself, mostly 'cause there's more data on using it to successfully preserve yeast.

And bhb -- you're right that about the methods probably being overkill, but it's damn hard to escape that cell culture training, eh?
 
Thanks! Very interesting article. We've been experimenting with freezing some tissue for cryosectioning +/- sucrose cryoprotection, so I've been thinking more about that these days. Which, unfortunately, doesn't help me with my thesis work at all, which is in cancer biology. :)
 
Man, the scientists are just crawling out of the woodwork, here!

Good to see another Wolverine here, e lo. Good luck on your thesis, I having trouble just rebounding from Career Fair ;).

For those of us not in the jargon, what is DMSO? Just in case I happen to run across it before glycerin.

EDIT: Ok so after the reading, may I properly take away these tidbits from that first posted article?

1. Higher solute concentrations decreases mortality.
2. If cooling to warmer than -70*C, cooling rate does not matter.
3. If cooling to warmer than -70*C, warming rate does not matter.
4. Sucrose is just as effective as glycerin.

Please do correct me if I'm wrong!

And when I said earlier that glycerin was expensive and hard to find, it was in reference to sucrose. Everybody has a few pounds of sugar available at a cost of less than a dollar a pound. So sucrose would be orders of magnitude less expensive than glycerine. More importantly, there's the time that actively hunting down glycerin takes, which to me is probably the larger expenditure ;). I'll keep an eye open for glycerin, but for now I won't feel bad using sucrose if #4 above can be assumed true.
 
RadicalEd said:
Man, the scientists are just crawling out of the woodwork, here!

Good to see another Wolverine here, e lo. Good luck on your thesis, I having trouble just rebounding from Career Fair ;).

For those of us not in the jargon, what is DMSO? Just in case I happen to run across it before glycerin.

EDIT: Ok so after the reading, may I properly take away these tidbits from that first posted article?

1. Higher solute concentrations decreases mortality.
2. If cooling to warmer than -70*C, cooling rate does not matter.
3. If cooling to warmer than -70*C, warming rate does not matter.
4. Sucrose is just as effective as glycerin.

Please do correct me if I'm wrong!

And when I said earlier that glycerin was expensive and hard to find, it was in reference to sucrose. Everybody has a few pounds of sugar available at a cost of less than a dollar a pound. So sucrose would be orders of magnitude less expensive than glycerine. More importantly, there's the time that actively hunting down glycerin takes, which to me is probably the larger expenditure ;). I'll keep an eye open for glycerin, but for now I won't feel bad using sucrose if #4 above can be assumed true.

DMSO = dimethyl sulfoxide
1. Yes, because it increases the osmotic pressure such that the cells shrivel up due to osmosis. It essentially "sucks the life" out of them.
2. Incorrect. See Figure 1. Up to -70˚C, the faster the rate of cooling, the higher the viabiity. So, a flash-freeze in EtOH/CO2 would take the temp down to -78˚C in about 5 seconds for a 1mL aliquot of cells....not quote 1800˚C/min, but would put you in the 70-90% viability range.
3. I think you could make this presumption.
4. Yes, ceterus peribus.
 
e lo said:
You guys who work with your brewing yeast in your labs ... do your PI's know about that? I'm pretty sure my boss would kill me if he found out I was doing that (which I'm not -- WAAAAAY too damn much work to culture my neurospheres to risk contaminating them with that much yeast, so the primary tissue culture hood is out. The secondary hood is for bacteria work, and it's too much work to get a nice clean batch of yeast to want to risk bacterial contamination in there, and our third hood is for human tissue, so that's out, too. :) )

I though about doing some yeast culture in our hoods but quickly thought better of it. I would hate to try explain why we needed to have someone come in and treat the hood with formaldehyde. But, I can't say that I don't borrow the occasional piece of glassware that can be autoclaved.

:off:
Have you ever gotten DMSO on your skin? He he...that will about ruin your day fast!
 
@RadicalEd -- Hail to the victors valiant! (Although I'm a MI transplant; I grew up in CO and went to Grinnell College in IA for undergrad.) The thesis is coming along -- submitting a paper next week, which is great.

@Beerific -- yeah, I can't imagine trying to explain that to my PI either. He's a cool guy, but ... And pure DMSO on the skin is no fun for sure.

@mrkristofo -- Respectfully disagree with your assesment of Fig 1. If cooling to -20, cooling as slowly as possible is essentially as effective in terms of preserving viability as -1800 C/min (~80% viability vs. ~85% viability, error bars overlap). However, if cooling to -70, -25C/min is significantly better than -1800C/min (~75% vs ~45% viability.) Oh, and I think you mean "ceteris paribus." ;)
 
e lo said:
@mrkristofo -- Respectfully disagree with your assesment of Fig 1. If cooling to -20, cooling as slowly as possible is essentially as effective in terms of preserving viability as -1800 C/min (~80% viability vs. ~85% viability, error bars overlap). However, if cooling to -70, -25C/min is significantly better than -1800C/min (~75% vs ~45% viability.) Oh, and I think you mean "ceteris paribus." ;)

I think you're right, ceterus paribus.
 
We're an E. Coil lab, so I don't really do anything there for fear of contamination. Need to start freezing some yeast, though. We have about 20 gallons of glycerol. Word.
 
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