Guide to Making a Frozen Yeast Bank - Page 3

andyp · 10-02-2007, 05:45 PM

Hey FlyGuy.

I've never cultured yeast, but I have cultured many other cell types. In every case I have always encountered slow freezing and fast thaw to minimize cell damage. I would revise your thaw steps such that you thaw in room temp water and immediately pitch into your starter at the same temp.

However, maybe I should look into yeast culturing and see if this is the case. Let me check on it and if I find a discrepancy I'll edit this post. Thanks for the methodology. Very solid home cell banking!

Edit:
Ok. I've looked at a couple of papers. For home freezer temps it doesn't look like thawing rates has much of an impact. Also, one paper basically proves the importance of high levels of osmotic pressure (more glycerol) to help preserve cell viability in the cooling, and optimizing (essentially faster) warming rates to prevent death in the warming. So, basically, slow cool with lots of glycerol and fast warming (even 37C or higher to thaw).

http://aem.asm.org/cgi/reprint/72/2/1330.pdf

"Thus, we found that conservation of cell viability following freezing could be improved by increasing the osmotic pressure of the medium and the warming rate should be optimized."

http://aem.asm.org/cgi/reprint/63/10/3818.pdf

l3lackEyedAngels · 10-03-2007, 03:59 PM

I think I've mentioned this in other posts, but in freezing my yeast, I omitted the glycerine. My girlfriend centrifuged the vials and poured off the water. I've used two of the vials thus far, the last one having been in the freezer for about nine months. Not everyone has access to a centrifuge, but I thought I would point out a slightly different procedure. Excellent write-up, btw.

**fifelee** · 10-03-2007, 04:19 PM

Quote:

Originally Posted by l3lackEyedAngels

I think I've mentioned this in other posts, but in freezing my yeast, I omitted the glycerine. My girlfriend centrifuged the vials and poured off the water. I've used two of the vials thus far, the last one having been in the freezer for about nine months. Not everyone has access to a centrifuge, but I thought I would point out a slightly different procedure. Excellent write-up, btw.

Could you explain that a little more? Do you just spin it, pour off the water, then freeze? Any other steps? Is the theory that the yeast cells are almost dry and therefore there is no ice crystals to damage the cells? Seem that even if spun there would be enough water remaining in the cells to still cause damage.

Nonetheless, a centrifuge seem like a great hombrew project.

**TheJadedDog** · 10-03-2007, 06:03 PM

In case anyone is interested, I've posted this information on the Wiki here:

http://www.homebrewtalk.com/wiki/index.php/Yeast_bank

**bigjohnmilford** · 10-03-2007, 06:09 PM

I agree nice presentation

l3lackEyedAngels · 10-03-2007, 06:44 PM

Quote:

Originally Posted by fifelee
Could you explain that a little more? Do you just spin it, pour off the water, then freeze? Any other steps? Is the theory that the yeast cells are almost dry and therefore there is no ice crystals to damage the cells? Seem that even if spun there would be enough water remaining in the cells to still cause damage.

Nonetheless, a centrifuge seem like a great hombrew project.

I forget the size of the vials, but they were plastic, four inches long, and about one inch in diameter with conical bottoms. I think they were 50mL vials. Anyway, they were filled with yeast slurry, capped, and then spun down. I forget the size of the rotor or the RPMs, but duration couldn't have been more than 10 minutes. After being centrifuged, the yeast formed what my girlfriend called a pellet on the bottom one sixth of the vials with almost crystal clear water on top. I uncapped the vials, poured off the liquid, flamed the caps and rims, and recapped. Then I brought them home and put them in the freezer. I don't know what the theory is. As for water content, when I thawed the yeast out, it wasn't dry. It was a much thicker slurry than what I started with. So, there was some water present. Damage to the yeast may have occurred, for all I know, but the beer they made tasted fine.

Yes, a homemade centrifuge would be sweet, but hilarity/carnage could ensue.

andyp · 10-03-2007, 08:43 PM

From a technical aspect you're probably losing a lot of yeast by freezing them down without any cryopreservative. However, as long as you're pitching enough viable yeast, which would be greatly improved by preparing a starter, it's probably fine.

I don't know if the amount of lysed yeast would be detectable by taste...probably not that much, but that's just another thing to consider.

Writeup looks great on the wiki.

I would amend the part about the slow thaw though...if we're trying to stack the deck in our favour, every little bit helps.

Cheesefood · 10-03-2007, 08:52 PM

Quote:

Originally Posted by rdwj

Ah, good! I'm only a few pieces of equipment away from doing exactly what you're showing here and I've been wanting to set up a steam injection system anyway. Now I have one more reason to get started.

At first, I was all excited thinking "Snizzle Yeast Farm!" but then I remembered that with gas prices today, it's cheaper to buy yeast than to drive to your place to pick up some, even if it was free.

Still, this is a great idea. I've got some 1056 currently fermenting my AIPA. I might pull the sludge from the secondary and see if I can revive it to the point of being able to freeze some for the future.

**FlyGuy** · 10-04-2007, 02:16 AM

Quote:

Originally Posted by andyp

Edit:
Ok. I've looked at a couple of papers. For home freezer temps it doesn't look like thawing rates has much of an impact. Also, one paper basically proves the importance of high levels of osmotic pressure (more glycerol) to help preserve cell viability in the cooling, and optimizing (essentially faster) warming rates to prevent death in the warming. So, basically, slow cool with lots of glycerol and fast warming (even 37C or higher to thaw).

http://aem.asm.org/cgi/reprint/72/2/1330.pdf

"Thus, we found that conservation of cell viability following freezing could be improved by increasing the osmotic pressure of the medium and the warming rate should be optimized."

http://aem.asm.org/cgi/reprint/63/10/3818.pdf

Thanks for the good info, Andy. Much appreciated.

I already modified my original post some time ago to recommend a 48 cooling period prior to freezing. This isn't quite the same as the slow freezing method, but perhaps more practical for homebrewers. The method has been researched by Tom Schmidlin and presented at the most recent NHC. Essentially, the theory is that cooling the yeast in the fridge prior to freezing stimulates a stationary phase where yeast accumulate glucose and trehalose, which appears to promote higher viability during freeze-thaw cycles (see also Kandror et al., 2004). I presume that the cooling period prior to freezing could produce similar results to the slow freezing method you were advocating. Interestingly, Schmidlin also reports that a study by Park et al. (1997) found little difference in viability between frozen yeast warmed rapidly at room temperature vs. those warmed at temperatures just above freezing.

Regardless, I have now seen two suggestions now that faster warming is beneficial, so I will definitely try that. Thanks for the tip. I'll make sure to modify my thread/wiki article too (probably in a couple weeks when I am back from work travel).

Cheers!

andyp · 10-04-2007, 01:51 PM

Great. Yeah, there's probably a benefit to putting them in the fridge before the freezer. Often, when freezing bacteria, I would stick them straight into the -20C freezer first for 4h to overnight, then to -80C. As long as you don't leave them lying around at room temp in the glycerol for too long...4C is likely helpful. Sounds like you've nailed down a good system.

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