Do you know how to make a yeast starter? Then why not farm yeast and freeze it?

Milptak, It takes only a small difference in doubling rates of cells for the faster growing yeast to overtake the population when growing from a low concentration to a high density starter. Unless you start with a very high density population that has not gone through too many doublings, or you are not very particular about what you end up with, I would discourage you from trying to propagate blends of yeast.

It wiil be quite difficult to identify the various yeast in a blend unless you know what you are looking for.

By the way, I intend to thaw out the yeast I froze away in October tomorrow and determine viability and recovery rates. It will probably be pretty crude (hey, I'm on holiday ;-) but will tell us whether there are substantial differences in either parameter.

Great thread i was looking for this kind of Info.
Thanks.

I work in a yeast lab and as for percentages of glycerol used to freeze down yeast, standard in our lab is to use 15% glycerol and then pop it into our -80C freezer. There is no need to freeze large cultures, 1 ml will leave you with enough cells to go back to your stock multiple times, take a little scrape with a sterilized toothpick or something and grow it up.

Mcsuck said:

I work in a yeast lab and as for percentages of glycerol used to freeze down yeast, standard in our lab is to use 15% glycerol and then pop it into our -80C freezer. There is no need to freeze large cultures, 1 ml will leave you with enough cells to go back to your stock multiple times, take a little scrape with a sterilized toothpick or something and grow it up.

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That's what a lot of people do, but that is more of a yeast banking approach. What I'm trying to perfect is a more or less "pitchable" frozen yeast reserve similar to that of a store bought vial or smack pack which is something that not very many people seem to have expiremented with. Since most of the strains I use are readily available for purchase, I don't want to invest much time and money in culturing. Once I have some data avialable on viability after the freeze, I should be able to freeze enough cells so that the end result after thawing is roughly equal to a smack pack or vial. This will also reduce lead time when brewing as I won't have to do small step ups to bring the population back up to sufficient numbers.

Ahhh, I see. Personally, I wouldn't recommend that because the long time it would take a large culture to freeze. Also, when you thaw out your yeast reserve completely, you can expect about a 50% loss in viability (i.e. about half the cells won't recover). In our lab, we work a lot with frozen stocks, and this is essentially what we see. This is why it it much more preferential to have a "yeast bank" and make starters from that. So you would need to at the very least have 2x as many cells as a smack pack just to have the same amount of viable cells, not to mention it would be better to have more since your frozen cells are going from dormant to active, so you could expect an extensive lag time before fermentation starts. Another reason why the "yeast banking" is advantageous.

Mcsuck, I have already tested a few things in the lab (I'm also a yeast researcher). I have found, as reported in the literature for yeast and as done routinely with mammalian cells, it is best to freeze slowly and thaw rapidly. We have not routinely done this for our yeast library but about 1 degree C decrease per minute and thawing at 37C is optimal. Recently, I have frozen approximately 2x10e11 packed cells in an equal volume of 15% glycerol and frozen by moving from 4C to -30 to -80C over the course of several hours. Those cells thawed a 37 produced a robust starter culture when inoculated into 3 liters of wort overnight. That would be my preferred approach due to the lag time.

However, I am now testing conditions that are approachable by the home brewer and testing viability and growth rates. Doing a pretty crude set of tests. I should be able to report after the 1st of the month on the effectiveness of freezing for several months at -30C after slow and rapid freezing in 15% or 30% glycerol. I won't have good growth curves, just qualitative assessments and I will have crude viability determinations. Counted and plated those cells today.

I should add that I didn't do a careful determination of lag time or viability of my starters made from -80 since I wasn't pitching directly. However, I'm guessing that they were getting under way pretty well after 4 hours and were at full density within 14 hours. After that I just cold crashed overnight, dumped the spent wort and then pitched. Fermentation was active within 4 hours. My cultures that were innoculated today were all increasing significantly in density after 3 hours suggesting that the lag is not a problem.

Brewitt said:

I should add that I didn't do a careful determination of lag time or viability of my starters made from -80 since I wasn't pitching directly.

Click to expand...

I don't think anybody (including myself) plans to pitch directly from thaw to fermenter (although I have done this for expermentations sake and it did work albeit with less than optimal results). Most people make starters from fresh packs/vials and I plan to always make a starter with the thawed yeast. However, % viability after the thaw is the primary concern so that cell counts can be reasonably estimated before making the right size starter for the target OG. Looking forward to the plate results for the -30 freeze.

orangehero said:

Viability of commercial wine yeasts during freezer storage in glycerol-based media

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There is not really any detail in the paper in how the thaw was performed, but 4-7 orders of magnitude loss in viability is a pretty low number. However they are also using different strains and storage conditions.

Yes the data is a little confusing and the samples are very small. The study only gives a rough idea, but note the benefits of the addition of ascorbic acid along with the hypothesis that having enough glycerine to prevent freezing at -20C should reduce loss of viablity and mutagenic effect due to the freeze/thaw cycle. Although they were using wine yeast strains, they were all Saccharomyces cerevisiae.

Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae.

Here they emphasize incubation at 4C for 2-3 days to build intracellular trehalose as a cryoprotectant.

Skip to end of post for my recommendation based upon observations and reading to date.

I don't have access to that full article so I don't know what the composition of the medium was and how they did their freezing and thawing. However, if their "orders" refer to "orders of magnitude" rather than "fold", I am amazed at how poorly their yeast did.

I don't have viability counts but from a quick look at the plates it is clear to me that cells settled out and then resuspended in 15% glycerol (at either 70F or 40F) and then allowed to sit at 40F for several hours followed by chilling to -20F (-30C non-frost free freezer, about 15F lower than a standard freezer) for about 2.5 months survived better than cells shifted straight from 70F to -20F after adjusting to 15% glycerol. It also seems that 15% glycerol is better than 30% glycerol (but the effect is not as great). As I stated yesterday, all of the cultures were beginning to grow and divide after 3 hours. All were at full density (although I don't have the numbers yet after 18 hours (I didn't monitor in between). I guessing viability, 50% or better, in the 15% glycerol cultures. I will try to get a more solid number for you.

I did take advantage of all the excess cells from my frozen tubes (about 20 mls of concentrated cells in an equal volume of 15% or 30% glycerol). I inoculated them into 3 liters of fresh wort and let them go overnight with constant shaking. I harvested about 60 mls of packed cells (1/4 cup containing approximately 4x10e11 cells based upon past experience) and resuspended them in an 100 mls (a bit less than half a cup of 15% glycerol, split them into four 40 ml tubes and cooled them stepwise to -112F for later use. These will be each be grown overnight in 2-3 liters of wort for a 5 gal fermentation. Keep in mind that if they retain 50% viability they will only have to go through three doublings (no problem even with 3-4 hours of recovery time) to get to full density.

HERE is my recommendation:

With the caveat that my freezer is not self-defrosting and somewhat colder than yours, I would feel comfortable recommending the following procedure. Keep in mind that I am starting with small volumes of cells from a freezer but could be starting with the yeast from a beer bottle or a small amount of a smack pak, a White Labs vial, or a starter prepared left over after pitching a fermentation. If you start with yeast from a fermenter then you could go straight to step 3 splitting as appropriate for the amount of yeast.

1. Grow yeast with continuous shaking or stirring until full density (after carefully stepping up to the desired volume to avoid contamination) and then move the cells to the refrigerator and allow to settle for 4 hours to overnight. Your cells should be about 1-2x10e11/liter. Grow about a gallon and you will have enough for about six starters.

2. Pour off spent wort retaining as many cells as possible. The bed of cells should be fairly solid, especially after overnight.

3. Add half a cup to a cup of 15% glycerol for cells from a gallon of starter and resuspend the cells by swirling for a bit until the solution of cells is homogeneous.

4. Split the cell slurry into 4 to 6 equal portions and then move to the freezer (surround in ice packs if self-defrosting and seal in bag).

5. When ready to thaw, swirl continuously in water at approximately 100F until completely thawed but not allowed to warm for extended period. This is pitched into 2-3 liters and stirred or shaken overnight to prepare your starter. If you don't have a shaker or stirrer, you will have considerably less yeast (see Mr. Malty or similar for differences in yield).

6. Pitch your starter and enjoy a vigorous fermentation after 3-4 hours. I have cold crashed my starters for several hours to overnight and poured off the spent wort to avoid off flavors but that is a matter of preference. Although not recommended, if you pitch without growing a starter, I would use the frozen yeast from a full gallon of starter culture to account for loss of viability and lag time.

I have done three brews using the procedure posted above (two from yeast stored a month and a half at -20F and another with yeast stored a month at -112F). Both worked great. Hopefully folks using this procedure will report back and tell us the longevity of the cells in standard self-defrosting freezers.

Good luck and tell me where this can be improved upon.

Let me add to the above:

The three brews I have done using this procedure were with two different yeast recovered from California IPAs (maybe the same yeast). I have brewed a high SG Russian Imperial Stout (10% ABV), a 6.5% IPA, and a 6% ginger beer. All had vigorous ferments with trap bubbling within 4 hours and came out very well (well, I'm not so fond of the ginger beer but I think that is simply a matter of taste).

They used in %: glucose 2, yeast extract 1, peptone from casein 1, and 50 % (W/V) glycerol; with and without 1 g/L Ascorbic acid, 100 mg/L Catechin, or 1 g/L Ascorbic acid and 100 mg/L Catechin.

Based on your observation that leaving the yeast to sit at 40F for a few hours showed an improvement, I would guess this is due to an increase in intracellular trehalose as found in the last study I linked to. Perhaps allowing it to sit for 2-3 days at 40F would be even better as is also recommended. They also mention that at low temperatures cell permeability is very low for the usual external cryoprotectants.

Would you be willing to compare with an even higher concentration of glycerol that doesn't freeze solid?

orangehero, your link is not working, I get a UConn login page. I found the article on the web but my VPN is not giving me access for some reason. I'll check it out when I get access. The issue with the formation of rho- cells is interesting but probably low frequency and not likely to affect our use. I was surprised that the viability of my 30% glycerol was lower than the 15% (not yet confirmed). I probably should have done all this more carefully but I wasn't able to commit the time. I suspect the effect of the 40F incubation was more on the slow cooling rate than the trehalose accumulation but possible that it is both. There is good evidence for trehalose being protective to stresses of various sorts. To be honest, in the interest of time and having achieved the general goal, I am not able to commit a lot more time to this except as it becomes available, which is sporadic. I am curious about a lot of aspects of this issue from both an academic and brewing perspective. Interesting, but not critical for the current use.

orangehero, I had a chance to look at the article you pointed out. I shouldn't be critical since I just did a very quick and dirty experiment (Then again, I'm not trying to publish my results in scientific journals). However, I don't learn much from this article. They have surprisingly low viability and the viability is quite inconsistent from preparation to preparation. Sometimes they lose 90% of their yeast in 3 days and sometimes the same yeast is stable for a long period. They seem to lose more than 70% of their yeast early in most experiments and then they are relatively stable. It seems they are freezing by just throwing in the freezer as opposed to pre-cooling. They are working at much higher glycerol concentrations than I am. It doesn't seem that they are getting much benefit from using glycerol concentrations that prevent freezing. However, since they don't compare to lower concentrations under the same freezing conditions it is very difficult to say. Finally, they don't say how they thaw the yeast.

That said, for the purposes of those on this list, I think we can say that the procedure I posted is practical and gives the desired outcome. The missing piece is how it translates to a self defrosting freezer and how long the yeast remain sufficiently viable. Three months seems fine.

I ran across this thread while researching how to make my yeast library. I am a newbie to homebrewing, but I have been growing microbes in the lab for a very long time and I couldn't resist commenting.

Brewitt has put together a nice recommendation for yeast storage and I thought I could comment on the science behind his findings.

1. Glycerol content.
Glyceral is a cryoprotectant but it is also toxic to the cells. The goal is to add the smallest amount of glycerol possible that will still prevent ice crystal formation, but not so much that it will kill the yeast cells. The industry standard is 15% volume/volume.

Someone mentioned trehalose in a prior post. Trehalose is a sugar that also serves as a cryoprotectant. It can be added to the freezing liquid or the yeast will produce plenty of it themselves if they are healthy. The best way to have plenty of trehalose is to well aerate your culture and make sure they are healthy when you freeze.

2. Slow cooling.
Best viability is obtained by cooling the culture in 15% glycerol for 1 degree per minute until you reach the desired storage temp. The ability of a homebrewer to do controlled rate freezing is not easy but can be obtained by using a Mr. Frosty container that someone linked to earlier or by freezing in a propanol bath (which is basically what Mr. Frosty is). A propanol bath can be made by putting your yeast tubes in a ziplock bag, squeeze out the air and then put your bag into a larger jar containing propanol. Put your jar into the freezer for 24 hours. After 24 hours you can move your tubes to a more permanent storage container. Warning - isopropanol will wash-off permanent marker so use an ethanol-proof marker to label your vials or make sure your ziplock bag is sealed tight.

3. Fast thawing.
Fast thawing is the way to go. Like I said before, glycerol is toxic so when they wake up, you want them out of that glycerol solution as fast as possible. A few minutes in warm water and then into the wort. I think the slow thaw in the fridge and higher glycerol concentrations that people use may be the reason why they are having to store such large volumes to get decent yeast numbers. If I fast thaw, I can pitch at a 200x fold rate and get great numbers of yeast in 24 hours.

4. Storage volume.
I know you guys really want to make a "pitchable" product, but your best bet is going to be freezing in small volumes and pitching a starter. The extra step is the price you pay for being able to store your yeast in the freezer in large quantities for longe periods of time.
The Pros of a small frozen volume is:

• space - you can store many more tubes of a smaller volume. I can store 100 x 1ml tubes in the same space you can put 2 x 50ml tubes.
• washing - pitching the starter allows you to dilute the glycerol and wash it away by the time you pitch your homebrew. Glycerol tastes like soap and I am sure it's not a flavor you want to add to your beer. Also - like I said before . . . it is toxic to the cells so the more you can get out of the culture before you add to your fermenter, the healthier your brew will be.
• viability is less important - because you are pitching a starter, you don't have to worry about yeast counts and post-thaw viability. You only need a little to live to get the starter going and grow until you get the right amount of yeast. I know everyone struggles to quantify how much yeast you have, but you can go by optical density. You can literally tell how much yeast you have by how opaque it is. Once you get used to culturing yeast, you can tell just by glancing at it.

5. Storage temp.
Yeast is viable in a non-defrosting freezer at -20C for about 9 months to a year, at -80C for many years and in liquid nitrogen forever. This is because the yeast are still slightly metabolically active at -20C and the cryoprotectant you use to prevent ice crystals will slowly kill the yeast over time. They are less metabolically active at -80C and completely inactive in liquid nitrogen.

6. Testing viability.
Because most homebrewers are going to be using a -20C (standard freezer temp) then to get a good measure of your freezing technique, you should test your yeast viability within a couple of days of freezing it. If you wait longer, you are actually testing your freezing technique AND your freezer's ability to maintain a stable low temp and it is impossible to separate the 2 factors.

All that being said, I am about to start making my own yeast libraries and have access to all my lab equipment of you guys would like to put some real numbers to the protocol parameters. I know this is what Brewitt proposed, but it sounds like he has been swamped at work. What do you think? Tell me what you want to test and I will generate the data.

Brewitt said:

I guessing viability, 50% or better, in the 15% glycerol cultures. I will try to get a more solid number for you.

Click to expand...

Well, my guess was somewhat off. After 10 weeks in the freezer the viability was ~25% in the culture which went into the cold overnight and then was adjusted to cold 15% glycerol, allowed to sit and then moved to the freezer. The cells that were allowed to settle at room temperature, adjusted to room temperature 15% glycerol, allowed to sit and then moved to the freezer retained >10% viability (about 1/3 of the other). The one that was treated the same as the first except adjusted to 30% glycerol fared somewhat worse. Keep in mind that these are rough numbers.

So, my recommendation stands. Even with 25% viability, you should have an expansion of 8 fold or greater overnight yielding a sufficient starter for pitching. The dead cells will make a negligible contribution.

I'm trying two such starters this weekend and will report back.

katy_bug said:

I ran across this thread while researching how to make my yeast library. I am a newbie to homebrewing, but I have been growing microbes in the lab for a very long time and I couldn't resist commenting.

Click to expand...

Katy_bug,

Very nice writeup that covers lots of ground. I like the propanol bath which I was not familiar with. I also wasn't aware that 30% glycerol as a cryoprotectant was toxic. The only thing I will reiterate from an earlier post is that the goal is to have a pitchable culture after overnight rather than stepping up from a small innoculum. Since we work in a formal laboratory environment, we have the advantage of being able to autoclave large volumes of wort (at least that is what I do) and using ultracold freezers, so some of the issues are moot but I am trying to adapt this to home use. Of course I am storing 1 ml samples of my yeast. But my goal was to store samples of about 40 mls containing on the order of 10e10 to 10e11 cells. I can pitch one of those into 1-2 liters (a large dilution of glycerol) at night and have 2-4x10e11 cells by morning for pitching 5 gal of wort. I pour off the spent wort after cold crashing so put less than 2 mls of glycerol into 5 gal (about 2000 fold dilution). [EDIT: I forgot to say that a home brewer has concerns about sterility leading to the recommendation to step up in 10X steps. Since the sterility of their wort and environment are not great, that is a good recommendation. The approach I suggest creates a culture which is at the last step before pitching.]

I did this last night and pitched cells cold crashed from 1.5 liter stirred starter into 6 gal of a black imperial IPA and it was bubbling actively within 1 hour. That is the fastest fermentation start I have gotten to date (only 12 brews under my belt). I have to admit, although I am meticulous about generation of research data, for my brewing I am settling with "the proof is in the pudding". The procedure I recommend is working and I think is adaptable to home conditions.

However, if you have the time and patience to do the exercise that you propose, my scientific side would love to know that answer. I don't know what your constraints are but I really don't think this issue has been adequately addressed in the literature. The small sample freezing data from at least one study that I referred to in an earlier post looks reasonably good. However, since it has relatively little use in the laboratory environment, freezing large samples has not been carefully looked at. I have no idea whether it is important to you but perhaps a well controlled study could be publishable in a brewing industry or technical journal (just a thought in terms of making your effort have some academic value).

Brewitt said:

However, if you have the time and patience to do the exercise that you propose, my scientific side would love to know that answer. I don't know what your constraints are but I really don't think this issue has been adequately addressed in the literature.

Click to expand...

I also have access to autoclaves, shakers, stirplates and -80 and -20 freezers so I would love to evaluate some of these parameters and I can do it in a controlled setting and then try to mimic a homebrew setting.

Since I am still a newbie to homebrewing, help me understand exactly what you want to learn. From the conversation in this thread, this is what I have extracted:

Ultimate Goal - An aliquot of yeast stored in the freezer that can be pitched into a starter and be ready to add to a brew in 24 hours.

Parameters to evaluate -
Ideal % of glycerol as a cryoprotectant
Ideal frozen aliquot size
Freezing protocol that maximizes viability
-Flash freeze
-Cold crash
-Controlled rate freeze
-Sequential freeze (room temp, fridge for a while, freezer)
Ideal freezing protocol
-fast thaw at 37C
-slow thaw in the fridge

How to measure effectiveness -
I have the capability to do live/dead cell counts so I can sample and measure viability immediately post thaw and then 24 hours later.

Any requests? Thoughts? Snide comments?

Katy_bug

Let me start by saying welcome to homebrewing and the forum community

As for your observations, you've pretty much got it covered. The ultimate goal is to have a frozen aliquot which upon thawing has a viable cell count similar to that of a store bought vial or smack pack (roughly 100 billion viable cells). However, if vialbility after thaw is really low (like the 25% in brewitts initial findings, then that may not be practical.

If you have the time and are willing to do some experiments, we welcome your input. The more data the merrier. With that said, I think a lot of us would like to see more data at the home level. Since the vast majority of us do not have access to a lab or -80 freezers, etc, evaluating those parameters won't really help us any. If you could conduct your experiments with an ordinary home deep freeze and self-defrosting freezer, that would be ideal. For any self-defrosting freezer experiments, putting the aliquots in a small cooler with some ice packs is the accepted practice for homebrewers that do not have a deep freeze.

Here are my thoughts on your parameters to evaluate.

Ideal % of glycerol as a cryoprotectant - Definitely
Ideal frozen aliquot size - Definitely. It would be intersting to see if the viablity upon thaw is dramatically affected by aliqot size. For instance, a lot of people yeast bank by freezing small amounts of yeast in vials. Do smaller samples fair better than larger ones?

Freezing protocol that maximizes viability
-Flash freeze - yes, or at least at the home level, putting the aliquot strait into the freezer without a refridgeration step.
-Cold crash - yes, and also optimal cold crash time needed. It seems that cold crashing the yeast helps build trehalose reserves but how much time is really needed to accomplish this effectively?
-Controlled rate freeze - not really of interest since it cannot be acurately controlled at the home level. EDIT: I somehow missed your post above about freezing in propanol. Could this be accomplished with OTC isopropyl alcahol? If so, then that would be a parameter of interest
-Sequential freeze (room temp, fridge for a while, freezer) - I believe this was a parameter that brewitt threw in on the mix. He was experimenting with what temperature to acclimate the yeast to the glycerine solution. I think the idea here is: Is it better to mix cold crashed yeast with cold glycerine solution or is it acceptable to mix the yeast and glycerine solution at room temperature and then refridgerate. This is actually of interest to me as well as some of the strains I use settle great at room temp and some I have to cold crash to settle out, so I actually do both. If I misinterpreted your goal here brewitt, please chime in.

Ideal freezing protocol - I think you meant to say "Thawing Protocol"
-fast thaw at 37C - yes, ideal fast thaw temp
-slow thaw in the fridge - yes

Thank you for your interest, and we look forward to hearing any results that you can contribute.

Cheers

BBL_Brewer said:

The ultimate goal is to have a frozen aliquot which upon thawing has a viable cell count similar to that of a store bought vial or smack pack (roughly 100 billion viable cells). However, if vialbility after thaw is really low (like the 25% in brewitts initial findings, then that may not be practical.

Click to expand...

My impression is that you will not achieve a pitchable population of yeast without some period of recovery and, probably, growth in wort prior to pitching. I do think that you could pitch a large quantity of glycerol frozen cells but the time for strong fermentation would be relatively long (guess: 12 hours). That said, 8 hours of pregrowth may be sufficient.

BBL_Brewer said:

Definitely. It would be intersting to see if the viablity upon thaw is dramatically affected by aliqot size. For instance, a lot of people yeast bank by freezing small amounts of yeast in vials. Do smaller samples fair better than larger ones?

Click to expand...

At small vial size, viability is not a real issue (even several logs of killing is OK) since the goal is to be able to recover enough live cells to step up to large starters, but not to generate and quick starter. This is the way we preserve cells for banking and it works just fine.

BBL_Brewer said:

-Sequential freeze (room temp, fridge for a while, freezer): Is it better to mix cold crashed yeast with cold glycerine solution or is it acceptable to mix the yeast and glycerine solution at room temperature and then refridgerate. This is actually of interest to me as well as some of the strains I use settle great at room temp and some I have to cold crash to settle out, so I actually do both. If I misinterpreted your goal here brewitt, please chime in.

Click to expand...

It is correct that there are two components to moving to the cold before adding cold glycerine; first, cold crashing concentrates the cells, second, the slow cooling from room temp to freezing seems to be advantageous (whether due to trehalose or some other form of protection). I would like to see more data but this clearly seems like the way to go.

Based on my own experience and the data I have seen in research papers on the topic, I believe slow cooling and quick thawing are optimal. As you say, the home freezer is the wildcard.

Brewitt said:

My impression is that you will not achieve a pitchable population of yeast without some period of recovery and, probably, growth in wort prior to pitching. I do think that you could pitch a large quantity of glycerol frozen cells but the time for strong fermentation would be relatively long (guess: 12 hours). That said, 8 hours of pregrowth may be sufficient.



At small vial size, viability is not a real issue (even several logs of killing is OK) since the goal is to be able to recover enough live cells to step up to large starters, but not to generate and quick starter. This is the way we preserve cells for banking and it works just fine.



It is correct that there are two components to moving to the cold before adding cold glycerine; first, cold crashing concentrates the cells, second, the slow cooling from room temp to freezing seems to be advantageous (whether due to trehalose or some other form of protection). I would like to see more data but this clearly seems like the way to go.

Based on my own experience and the data I have seen in research papers on the topic, I believe slow cooling and quick thawing are optimal. As you say, the home freezer is the wildcard.

Click to expand...

Ok, maybe I should refrain from using the word "pitchable." The only reason I used that is because that is how commercial yeast companies advertise thier product. However, my goal is not to pitch strait from thaw to fermenter, it is just to have a viable cell count similar to commercial packs or vials so that a large starter can be made from the onset without small steps.

As for the cold crashing, I'm just curious as to whether it makes a difference if you cold crash first then add glycerine vs mixing settled yeast at room temp with glycerine and then refridgerating. I realize that the refridgeration step seems to help viability, but like you said, cold crashing and refridgeration are two different things. Cold crashing generally means chilling the starter with the goal of settling out yeast. Refridgerating before freezing for a period of time is done to build trehalose. The strain I use the most settles very well at room temp. So I have been decanting and adding the glycerine solution then refridgerating for a couple days before freezing. So basically, the question is should you refridgerate to build trehalose before or after adding the glycerine or does it even make a difference?

I realize with small vials viability is less of issue because enough cells will survive that a new population can be built back up. I'm just curious as to whether viability is affected by aliquot size. If I freeze a vial with 1 ml of slurry vs a jar with 20-30 ml of slurry is the survival rate the same?

BBL Brewer, I did the comparison of settling at room temperature, pouring off wort, adding room temperature glycerol, then moving to the cold, as compared to settling in the cold and then adding cold glycerol. The cold cells fared better than the room temperature ones. However, it is true that the cold cells had been crashed overnight and then after glycerol addition left for the same amount of time that the room temperature cells were chilled for. Therefore, the cold crashed cells got more time in the cold. Whether that is the difference or the addition of cold glycerol is the difference, or both, I can't say. I will guess both contribute.

With regard to freezing small versus large volumes, I haven't tested that but the large volumes did "well enough" for the application you are interested in. If I was publishing a paper on this I would have to do it more carefully and better controlled but since we are looking for "useful" outcomes, I think we know enough. That said, I would be happy to know the answer.

I've been trying to search for the question I have, but can't find the answer. Is there any advantage to having both the glycerine frozen yeast as well as some slants in the fridge, or should I just stick with one or the other?
Tom

TomRep said:

I've been trying to search for the question I have, but can't find the answer. Is there any advantage to having both the glycerine frozen yeast as well as some slants in the fridge, or should I just stick with one or the other?
Tom

Click to expand...

Well, that kind of depends on your situation and how often you want to reculture. You don't have to freeze yeast to maintain it long term. You can use slants or agar plates as well. If you plan to work only from your own stock and stop buying yeast altogether, then you should do a streak plate to isolate a pure culture before making a new slant. You can maintain a yeast strain indefinitely this way. The draw back is that the yeast will lose viability more quickly storing at fridge temps and you will have to reculture more often. At the same time, freezing some vials for yeast banking purposes would also be a good idea in case your yeast mutates on you or you somehow lose your unfrozen cultures. If you're not planning on long term maintenance and are still going to buy yeast once in a while, then I would stick with freezing. This way you can grow up what you need from a fresh sample and put it strait into long term storage.

A few comments. First off very cool thread! I have some vials at work I was just going to throw away. Quick stop at the store tonight to pickup some glycerine and I'll be in business. I hope to freeze some of the Munich Lager yeast I have going, and will also freeze some of the Chimay I have in a starter (if it takes off). My wife was starting to complain about all of the yeast jars in the fridge, so this should help!

Second, nice avatar BBL_Brewer. And lastly, nice quote you have there. I got problems.

solbes said:

A few comments. First off very cool thread! I have some vials at work I was just going to throw away. Quick stop at the store tonight to pickup some glycerine and I'll be in business. I hope to freeze some of the Munich Lager yeast I have going, and will also freeze some of the Chimay I have in a starter (if it takes off). My wife was starting to complain about all of the yeast jars in the fridge, so this should help!

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Now she'll be complaining about all the jars in the freezer

solbes said:

Second, nice avatar BBL_Brewer

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Thanks

solbes said:

And lastly, nice quote you have there. I got problems.

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I'm right there with ya man

Love the thread.

I've been farming for a little over two years. I'm glad to see some guys that know what they are doing generating some metrics on this. I've spent some time changing my variables around like you guys (slurry%, glycerin%, temps) and also have to mention a variable that I have encountered as well. As much as I hate to admit it, some yeast just don't freeze well. I've had issues with several strains, but on the other had have strains that I am still using from an initial freeze 2 years ago. Go figure.

Do love the thread, though. Just be prepared guys. Every other week or so, I get somebody from the brew club asking if I have some Wyeast/Whitelabs <<insert random strain here>>. But on the plus side, I haven't paid for yeast (with the exception of adding to the Zoo) in almost 18 months!

IsItBeerYet said:

As much as I hate to admit it, some yeast just don't freeze well. I've had issues with several strains, but on the other had have strains that I am still using from an initial freeze 2 years ago. Go figure.

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If you think you can make that claim and not name names, think again.

Would you mind elaborating, perhaps making an ordered list?

IsItBeerYet said:

Love the thread.

I've been farming for a little over two years. I'm glad to see some guys that know what they are doing generating some metrics on this. I've spent some time changing my variables around like you guys (slurry%, glycerin%, temps) and also have to mention a variable that I have encountered as well. As much as I hate to admit it, some yeast just don't freeze well. I've had issues with several strains, but on the other had have strains that I am still using from an initial freeze 2 years ago. Go figure.

Do love the thread, though. Just be prepared guys. Every other week or so, I get somebody from the brew club asking if I have some Wyeast/Whitelabs <<insert random strain here>>. But on the plus side, I haven't paid for yeast (with the exception of adding to the Zoo) in almost 18 months!

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Glad you liked it. There's so much varied info out there on the subject that I thought I'd try to pin down a good all around standard method with some solid data to back it up. As soon as I get the funds, I think I'm going to spring for some plates, stains, a microscope and a hemocytometer. I'm such a data junkie. Plus I have a pretty good spread of older and newer yeast with varied glyercerine percentages now so it will be very interesting to start counting and staining some cells.

So what strains are you having trouble with? That would be good information to keep in mind. Also, what is your current method just out of curiosity and or what method have you had the best results with?

Thanks BBL, this is q great thread. I think I have found my happy medium between slants and washing.

subscribed. nice write up

@ Hermit - I will "attempt" to put up a list of what I current have in the zoo. Unfortunately, my list doesn't log failures and much empirical data. Its pretty much just a list of what I have "on tap", ages, and which generation it is.

I can tell you that there are a few that are particular standouts. English Cider yeast is on its second generation with me. I used all of the first generation last summer and it was approximately 1.5 years old when I cracked the last specimen tube. Belgians/French yeasts are very hardy as well. I cracked a vial of Belgian Saison at almost 12 months and it was probably the most vigorous ferment I've had "from the freezer". I haven't had a success yet (but I keep trying ) with the wheat yeasts. My best success story is WY1968 (WLP002), but the other English yeasts are hit or miss. I keep trying London Ale (1028), but can't say I've had much success. On the other hand, I can't kill 1968. Even the last tube I brought out to build up seemed to laugh at me and was actively fermenting 5 HOURS after I retrieved it from the deep freeze. Hmmmm. Good thing I'm a sucker for an ESB, eh?

IsItBeerYet said:

@ Hermit - I will "attempt" to put up a list of what I current have in the zoo. Unfortunately, my list doesn't log failures and much empirical data. Its pretty much just a list of what I have "on tap", ages, and which generation it is.

I can tell you that there are a few that are particular standouts. English Cider yeast is on its second generation with me. I used all of the first generation last summer and it was approximately 1.5 years old when I cracked the last specimen tube. Belgians/French yeasts are very hardy as well. I cracked a vial of Belgian Saison at almost 12 months and it was probably the most vigorous ferment I've had "from the freezer". I haven't had a success yet (but I keep trying ) with the wheat yeasts. My best success story is WY1968 (WLP002), but the other English yeasts are hit or miss. I keep trying London Ale (1028), but can't say I've had much success. On the other hand, I can't kill 1968. Even the last tube I brought out to build up seemed to laugh at me and was actively fermenting 5 HOURS after I retrieved it from the deep freeze. Hmmmm. Good thing I'm a sucker for an ESB, eh?

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Hmm, I've sucessfully frozen 1028, in fact I still have one or two jars of it. I stopped using it because I didn't particulary care for it, but I brewed 4 or 5 batches with it out of the freezer.

IsItBeerYet said:

@ Hermit - I will "attempt" to put up a list of what I current have in the zoo. Unfortunately, my list doesn't log failures and much empirical data. Its pretty much just a list of what I have "on tap", ages, and which generation it is.

I can tell you that there are a few that are particular standouts. English Cider yeast is on its second generation with me. I used all of the first generation last summer and it was approximately 1.5 years old when I cracked the last specimen tube. Belgians/French yeasts are very hardy as well. I cracked a vial of Belgian Saison at almost 12 months and it was probably the most vigorous ferment I've had "from the freezer". I haven't had a success yet (but I keep trying ) with the wheat yeasts. My best success story is WY1968 (WLP002), but the other English yeasts are hit or miss. I keep trying London Ale (1028), but can't say I've had much success. On the other hand, I can't kill 1968. Even the last tube I brought out to build up seemed to laugh at me and was actively fermenting 5 HOURS after I retrieved it from the deep freeze. Hmmmm. Good thing I'm a sucker for an ESB, eh?

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Thanks. We might want to start logging this kind of info somewhere. I know the site has a WIKI which would probably be the best bet.

Well, I'm not above mentioning that it may well we an ID-10-T error on my part. The last time I tried 1028 was about 12 months ago. I also haven't had much success with Scottish ale yeasts. But I have sharpened my mad yeast-freezing skills, so I may have better success now.

Well,

After a failed attempt or two to get the yeast list into HTML format, guess I'll just have to attach it as a screenshot. These are my current working strains.

I do remember that I've tried 1028, 1728, and Brett with no or little success.

@BBL - the older strains aren't as large and contain more glycerin (I started at around a 50/50 mix by volume, and have since gone to a 1/3 glycerin by volume mix with water. As a matter of fact, I have some older tubes that are still liquid in my -20f freezer!) When I started, I got a lot of trub in there as well, but since, I have started washing and now, I put almost 100ml of dense yeast slurry in each 125ml tube. One thing that improved my viability (perceived, I have not tested with empirical tests) is a "cold rest". I let my last yeast step-up sit until fermentation is complete and all the yeast has settled, then put it in the refridgerator for a few days. At this point, I will decant as much wort as I can, mix the yeast slurry and remaing wort, and decant into sterilized tubes. I put the tubes into the fridge to settle, and once the slurry had settled and compacted, I decant the last of the wort and replace it with the glycerin/water mix.

A quick shake to mix the yeast and glycerin mix, then off to the freezer.

I have read some things in "the Yeast book" by Jamil and Chris White that talked about mixing up some fresh wort with the glycerin along with a touch of citric acid and using that as a storage medium, but haven't tried it.

Yeah, it looks like the cold rest is vital to increasing viability. I've been doing the quick thaw method with good results as well. Don't know if it is helping viability, but it does cut down on lead time and doesn't seem to hurt anything. I'm also curious about glycerine percentage now since it was mentioned previously that it is cytotoxic and excess amounts could lead to low viability. This is why I just have to get some gear and start running tests myself.

I believe that I read somewhere that a good "cool" storage alerts the yeast that "hard times are a'comin!" and triggers a glycogen uptake, which would increase long-term viability. Althought I am no bio-chemist. Don't even play one on TV.

I've found that a quick reheat is pretty vital to recoverability of the strain as well. I'll usually give a vial to my wife straight from the freezer, who will warm it up in her pocket for 30 minutes or so. Her contribution to my brewing.....