Yeast Harvesting

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thebull

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Just finished following the instructions I found on this site for washing yeast in three easy steps. It's simple and the five yeasties are now resting in the fridge for later deployment. I have used the full yeast cake with trub for the last two batches without difficulty, but the cleaner yeast will be better, I'm sure.

I will use a starter for the next five batches and continue to use the 3B oxygenation system for the quickest and shortest lag time.
 

Iordz

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I always plan my brews so that I am repitching the yeast, but I've wondered how long can the yeast remain viable if you pour it into bottles, cap them and store them in the fridge.
 

Philip1993

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Add some glycerin (30-33%) and store in the freezer. That's what I'm doing (for the 1st time).
 

BierMuncher

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Iordz said:
I always plan my brews so that I am repitching the yeast, but I've wondered how long can the yeast remain viable if you pour it into bottles, cap them and store them in the fridge.
I did a Kolsch last weekend with a 3-month old bottle. Turned out fine.

I have total confidence in any yeast I have that is less than 6 months old. Haven't gone longer than 6 months yet so can't really say.
 

Philip1993

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Here are some pics from my first venture into yeast freezing:

First yeast harvest using 8oz SunnyD containers. The didn't reseal well, so I bagged and heat sealed 'em.

A Closeup of the first batch showing the yeast settled. It's difficult to make out since the bags fog instantly when removed.

Eight 32ml (20x150mm) vials of yeast. This is from the same primary that yielded the SunnyD bottles. I split the original yield from the yeast washing and put 32oz in the SunnyD bottles, and 32oz in the fridge to floccate and be xferred when the vials arrived (today). Today, I siphoned off all the liquid and re-placed it with a 33% distilled water/glygerin mixture. That's what you see in the vials.

The yeast vials loaded into my "anti-defrost" bag to protect them from the defrost cycles.

Same bag, without the ice pack.

Same bag again, from the outside. I think we paid only $4.99 for the bag before it served as a lunch carrier for a few years. If you don't have one laying aroung, you can find these for next to nothing at the dollar store. Also, I had the blue ice laying around, but I can't see any reason why you couldn't just use a ziplock bag of ice instead of the blue ice.

And for anyone interested, that's 12 vials (8 - 32ml, 4 - 200ml) of Wyeast 1056 waiting to be dumped onto an unsuspecting wort... :)
 

Philip1993

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Another yeast freezing update:

Of the 8 vials, one froze. I'm not sure why as I was careful to get all the glycerin, water, yeast well mixed prior to and during use. Hmmm... glycerin is very heavy and layers sharply when added (pre-mixing). Perhaps there was some invisible layering taking place post mix. Better add glycerin to individual tubes and mix in situ.

Also the yeast has started changing color. The side facing the exterior of the lunchbox (see previous pic) is now a darker color (like trub), while the side facing the interior of the lunchbox (an icepack) is still light beige. I'm thinking excessive cooling (5* vs. 32*) and/or defrost cycle? In any case, this may warrant an ice jacket on the outside and bottom of the lunchbox for protection.

Yeast tube pic #1
Yeast tube pic #2

The pics are of the same tube. At no time was it allowed to lay on it's side and layer in that fashion.
 

chainsawbrewing

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BierMuncher said:
I did a Kolsch last weekend with a 3-month old bottle. Turned out fine.

I have total confidence in any yeast I have that is less than 6 months old. Haven't gone longer than 6 months yet so can't really say.
was that three months in a fridge, or frozen?


brian
 

Philip1993

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Yet another yeast freezing update:

Frozen yeast after 3 weeks

One sour note. After 3 weeks and a low temp of 2*F, 4 of 12 vials have frozen. The first four have shown no signs of freezing. For these, the yeast was added to the vial and topped off with glycerin (yielding an exact mixture). The remaining 8 vial (of which 50% froze), were mixed as a batch and then poured into the vials. Since the yeast/glycerin was well mixed prior to and during use, I still suspect there was some unobserved layering taking place post mix. As such, I strongly recommend that you add yeast to the vials and top them off to ensure proper ratios in all vials. I have also increased my mix to 50/50 glycerin/water to be safe.
 

FlyGuy

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pldoolittle said:
Yet another yeast freezing update:

Frozen yeast after 3 weeks

One sour note. After 3 weeks and a low temp of 2*F, 4 of 12 vials have frozen. The first four have shown no signs of freezing. For these, the yeast was added to the vial and topped off with glycerin (yielding an exact mixture). The remaining 8 vial (of which 50% froze), were mixed as a batch and then poured into the vials. Since the yeast/glycerin was well mixed prior to and during use, I still suspect there was some unobserved layering taking place post mix. As such, I strongly recommend that you add yeast to the vials and top them off to ensure proper ratios in all vials. I have also increased my mix to 50/50 glycerin/water to be safe.
I suspect you are using too MUCH glycerine. Everything I have read says to use about 10 - 20% glycerine and mix it well. I think if you use too much of it, the resulting solution is too viscous to mix properly. You really don't need much glycerine to prevent crystalization.

Also, I can't remember from your OP whether you used a cooler to store your yeast inside the freezer, but it is necessary to ensure that the yeast vials stay frozen at a constant temperature. Otherwise, the changing temps will quickly kill all the yeast. I suspect that is what you are seeing in the darker colour yeast vials.
 

Philip1993

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FlyGuy said:
I suspect you are using too MUCH glycerine. Everything I have read says to use about 10 - 20% glycerine and mix it well. I think if you use too much of it, the resulting solution is too viscous to mix properly. You really don't need much glycerine to prevent crystalization.
15-25% is what is generally recommended by most yeast freezers here on HBT, but all the scientific/medical sites I found on freezing live samples seemed to indicate that best the highest post freezing viability was obtained at about 33%. I also thought that I read 1/3 glycerin in your thread as well. In any case, I don't think the ratio itself was a problem, but we do agree on the assessment of the cause. The solution was certainly very well mixed at the start, but despite working quickly and periodically re-mixing, I believe it stratified while standing, yielding different ratios in each vial.

Next time I will add the glycerin to the vials directly and top off with slurry to ensure a consistent ration of glycerin/slurry.

FlyGuy said:
Also, I can't remember from your OP whether you used a cooler to store your yeast inside the freezer, but it is necessary to ensure that the yeast vials stay frozen at a constant temperature. Otherwise, the changing temps will quickly kill all the yeast. I suspect that is what you are seeing in the darker colour yeast vials.
Yes, I am using a cooler and an rather large internal ice pack to stabilize temps inside the cooler.


One last note: Many thanks to FlyGuy for the great how-to on making a yeast bank. It was my inspiration for this project. I hope that reading about my experiments, successes, and failures will help others as well.
 

e lo

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For what it's worth, the American Type Culture Collection, the gold standard for obtaining yeast, bacterial and mammalian cell clones for laboratory cell culture work, uses a 10% glycerol (aka glycerine) solution for freezing:

http://www.atcc.org/common/technicalInfo/faqYeastGeneticStock.cfm

However, they also have liquid nitrogen deep freeze (-180 degrees C or so), which most of us don't have access to. I do have access to one, but I don't think my boss would take too kindly to my storing brewing yeast next to our mouse cell lines... :)

*Edit -- sorry, got the name wrong -- It's "ATypeCC," not "ATissueCC." Sheesh.
 

Philip1993

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Good info. I'll drop my next batch back to 15%. Since last update, I added another vial (#9) from the secondary. Also, #5 of the 8 vials has frozen and the vial broke.

I pulled out 4 of the frozen ones and within 30 seconds they were slush. 3 minutes and they would have been 100% liquid. That and the fact that only one of 5 vials (99.99% full) broke, indicates that only partial freezing has occurred.

Having looked at this problem for some time now, I suspect that the glycerin/water/yeast may be separating/layering in the freezer because of the tall, narrow vials. I can't find any other plausible explanation for a vial being at 5* for a month and then freezing. To test this, I am no longer storing them vertically.

In any case, I took the broken vial and made a starter to see if it is still viable and how viable it is.
 

Philip1993

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That starter from the frozen vial worked. It took a long time to start (about 32ml into 375ml), but it kicked off and I upped it to 750ml the next day. I let it ferment and now I'm chilling it to harvest the created yeast.

Interestingly, the vial I captured from the secondary is still in suspension (failed to clear) with minor settling despite over a week at 5*.
 

WBC

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I have found a neat and sanitary way to recover yeast in a sterile condition. I use a "used" 2.5 gallon distilled water container (sanitized). I believe they are made of polypropolene plastic. I cut 2 holes in one side and use some beer bucket drains. Now you connect 1/2 inch tubing to the spiggots and hook them inline from your fermenter to the water bucket you use for blow off tubes, thus (closed yeast recovery container). The yeast will collect in the sanitized distilled water container and the blowoff will go from the second spiggot to the blow off bucket. If the "In" side of the recovery vessel has a tube fitted that goes to the bottom, the foam will go back into a liquid if the tube is under some of the yeast liquid. At first some foam escapes and comes through the out spiggot but after some of the yeast turns back into liquid this stops. I came up with this after I saw how much yeast came off my blowoff tubes and thought "How can I save that yeast?". I have a Blichmann 27 gallon fermentor and it can spew a lot of yeast out the blow off tube if the frementer is full. Of course I can recover yeast from the bottom of the Blichman fermenter but it always has some trub in it too. I wanted something for carboys and buckets for yeast recovery. Life is good!!! :mug:
 

Ender

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pldoolittle,

Question about your yeast (great discription and pic btw)

I'm a bit confused about your report. Is the testube, vial, whatever with the yeast inside supposed to freeze solid? I was under the impression it was. I have 5 vials of Wyeast 1469 (Tim Taylor) I mixed w/ glycerin and froze. They are solid. Hop they're going to still be viable
 

Philip1993

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No. The purpose of the glycerin is to prevent freezing solid. Yeast can tolerate cold, but when ice crystals form they burst the cell walls. You and I know that process as "frostbite". Tell me more about your mix ratios, container size, process, etc. Perhaps we can assist.

That said, They may be viable. I pitched one of my frozen vials and it worked, but it took 18-24 hours to start. That's very slow for a 350ml starter w/ yeast nutrient.

FWIW, I've just cultured 4 vials of Northwestern/1332 from a starter that's going now. I mixed glycerin/slurry in vial at a ratio of 5:32, and it's refrigerating now. I'll keep everyone posted on how those proceed.
 

e lo

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Actually, pldoolittle, that's not quite correct.

The purpose of the glycerol/glycerin is to prevent crystals from forming if/when the water does freeze (called vitrification.) A 15% solution of glycerol/H20 should freeze at about -4 C, and your standard freezer is likely at about -20. (http://en.wikipedia.org/wiki/Glycerol_(data_page))

We commonly freeze down cells (including mammalian cells) at -80 for a few days, then transfer to liquid nitrogen. The medium in which they are suspended is frozen absolutely solid. However, because of the addition of a cryoprotectant, crystals don't form, and the cells are viable for many years (I once thawed and successfully grew out a vial of human tumor cells that had been in the freezer for well over 10 years.)
 

Philip1993

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Now you have me perplexed. I wasn't aware that water could freeze without forming a crystalline structure. Can you shoot me a link to some more info?

As for my samples, they were clearly crystalline formations as one would expect with standard H2O freezing.

One last Q: Adding glycerin doesn't alter the physical properties of water. How does one freeze in glass (or any closed vessel) without a significant risk of container rupture?
 

e lo

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http://en.wikipedia.org/wiki/Vitrification has some good info. I'm no physicist by any means, but as I understand it (and as confirmed by that wikipedia article,) you can prevent crystal formation either by very careful controlling of the temp drop or, in the case of using cryoprotectants, adding additional colligative elements that disrupt potential crystal formation. As an interesting sidenote, sugars work very well as cryoprotectant (glycerol, for example, is a sugar alcohol.) For freezing tissues prior to cutting thin sections for microscopy, we soak them in a 30% sucrose solution overnight.

The crystals in your frozen tube suggest that maybe the solution wasn't well mixed. Alternately, the glycerol might not prevent ALL crystal formation, but rather prevent enough of it so that a reasonable percentage of the yeasties survive the freeze-thaw process.

To freeze w/o rupturing, simply use a vessel that can withstand the increased pressure. In lab, we freeze in (sealed) polypropylene tubes every day with no issues.
 

l3lackEyedAngels

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My girlfriend centrifuged some 1065 yeast that I recovered two batches ago. Then I just poured off the water and put the vials in the freezer, no glycerol. I've made one batch so far with the harvested yeast and it came out great.
 

Philip1993

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I've always wondered about the viability of centrifuging. Do the trub/yeast solids layer as cleanly as it does when allowed to fall unassisted?

Yeast fallout raises another issue. When manually seperating (and in yeast washing), the erlenmeyer has to be the worst possible flask. A large graduated cylinder would make thick layers that could likely be seperated manually with no decanting, centrifuge, etc.
 

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