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

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Agreed but is there any reason that's not allowed. And, also, does anyone know how to post a formatted MS Word doc that will show up in the thread rather than as a downloadable file? What format?
 
I regularly have to make freezer cultures of bacteria in my lab. I haven't perused the entire thread to see if this has been noted, but you'll get marked viability increases the faster you freeze cells because of both the ice crystals being smaller and the presence of a cryoprotectant (glycerol) preventing the formation of the standard hexagonal crystal lattice.

We use 50% v/v glycerol and liquid nitrogen. Liquid nitrogen is a bit hard to come by for most folks, but dry ice will work to achieve faster freezing.

Also, at least in bacteria, thawing quickly reduces cell viability. When we remove cultures from our -80C freezer, we put them on ice until they thaw fully.
 
Works better opposite for yeast: slow cooling, rapid thaw. Yeast will continue to build up trehalose which also serves cryoprotectant functions.
 
Yes, iit seems to be true that slow cooling and fast thawing works better in terms of maintenance of viability. This is based both on the literature and our own experiments.
 
Brewitt said:
Would anyone be offended if I consolidated the information gleaned from this thread and wrote up a Tutorial to post on the site. I think we have a critical mass of information that should be summarized. Credit where credit is due, of course. One of us should definitely do it.

If you would like too, I can put your write up in the wiki. I have done a few wiki articles myself. Let me know and send me a word doc link pm if you do want it there. This is a wealth of info and should be there where we can link it to farming and washing threads as well. If I do this, you lab guys will need to take some more photos to make the article more like a magazine. I really need to update my pressurized fermentation wiki article as well. Anyways, it would be fun and beneficial to have it done in wiki. Great thread guys.
 
WortMonger said:
If you would like too, I can put your write up in the wiki. I have done a few wiki articles myself.

Thanks. I'm not familiar with the wiki. I submitted it to the technical thread for approval. The directions say that it needs to not be submitted anywhere else. I don't know if that also applies to anywhere on this forum. I also don't know how long it takes to get approved.
 
What are people mixing the glycerol with to freeze? I used fresh wort because I was worried about osmotic stress, but it was a pain because the yeast was making CO2 when they warmed up and made things very messy. I actually had a yeast bomb go off in my apartment!!! What a disaster!

Will putting yeast in straight water be bad for viability? I know with mammalian cells they'll all die instantly in water.
 
What are people mixing the glycerol with to freeze? I used fresh wort because I was worried about osmotic stress, but it was a pain because the yeast was making CO2 when they warmed up and made things very messy. I actually had a yeast bomb go off in my apartment!!! What a disaster!

Will putting yeast in straight water be bad for viability? I know with mammalian cells they'll all die instantly in water.

I decant off most of the spent wort after the starter is done and then add sterile glycerol/water solution. So it's mostly water, glycerol and yeast with a little bit of spent wort. I was wondering how the wort thing would work out for you. Even with my method I still have to do some venting when I pitch a jar. When I swirl to get the yeast in suspension I get a little off gassing.
 
I agree with BBL_Brewer. I have typically made a dense slurry leaving in whatever spent wort is necessary to have a slurry instead of packed cells. I then bring up in an equal volume of glycerol prepared in water. The spent wort (if fully fermented out) should not cause a problem. Washing yeast with water is not particularly detrimental to viability, so the water in the glycerol should not be a big problem. Between the cell wall and effective ion pumps, they do a respectable job of maintaining their osmolarity.
 
OK, thanks for the info! I'm growing up another culture to repeat the freeze/thaw. I'd like to optimize the procedure a little bit. I'll try water+glycerol instead of wort+glycerol. I'm also going to test a few other parameters.
 
You might want to try cold crashing a dense culture overnight and comparing it to spinning down directly from growth conditions. Keep us posted.
 
So thanks to your links I found out those white lab tubes are called baby soda bottles, which opened up a ton of links to buy them.

It looks to me that these are thin walled polycarbonate tubes. That is not really a problem but they certainly won't have the durability of White Labs tubes which are very thick walled. They are about the same diameter and hold only 40 mls. They may have problems during freezing and thawing but that remains to be seen. I believe you would be better off with the centrifuge tubes which are quite durable and close tightly.

Does anyone know differently?
 
It looks to me that these are thin walled polycarbonate tubes. That is not really a problem but they certainly won't have the durability of White Labs tubes which are very thick walled. They are about the same diameter and hold only 40 mls. They may have problems during freezing and thawing but that remains to be seen. I believe you would be better off with the centrifuge tubes which are quite durable and close tightly.

Does anyone know differently?

The tubes I linked to are THICK walled (along the lines of those from White Labs)... These also close VERY tightly. I have a couple of packages of them on hand, so it was easy to check. :D
 
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The tubes I linked to are THICK walled (along the lines of those from White Labs)... These also close VERY tightly. I have a couple of packages of them on hand, so it was easy to check. :D

Point taken. I apologize for misleading and misinterpreting. I saw people saying in reviews that these were like miniature soda bottles. Sounds like I was wrong and that these are like the the clear 40 ml polycarbonate tubes we use in laboratories. Those should be perfect.
 
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Brewitt said:
Point taken. I apologize for misleading and misinterpreting. I saw people saing in reviews that these were like miniature soda bottles. Sound like I was wrong and that these are like the the clear 40 ml polycarbonate tubes we use in laboratories.

They ARE miniature soda bottles, though possibly not in the sense you're thinking. They are called preforms, and are typically used by the bottling facility by expanding them to full size by heating them and blowing air into them (an effect that resembles glass blowing). They are exactly what White Labs uses, and ARE fairly thick-walled because they contain enough material to make a typical 2L bottle... the walls get thinner as air is blown into them to "stretch" to full-size.

They are popular because they are already manufactured on a massive scale for the beverage industry, making them a very cheap (literally pennies when bought in bulk) alternative to purpose-made labware. Notice how the caps are *identical* to soda bottles? It's not a coincidence.
 
They ARE miniature soda bottles, though possibly not in the sense you're thinking. They are called preforms, and are typically used by the bottling facility by expanding them to full size by heating them and blowing air into them (an effect that resembles glass blowing). They are exactly what White Labs uses, and ARE fairly thick-walled because they contain enough material to make a typical 2L bottle... the walls get thinner as air is blown into them to "stretch" to full-size.

They are popular because they are already manufactured on a massive scale for the beverage industry, making them a very cheap (literally pennies when bought in bulk) alternative to purpose-made labware. Notice how the caps are *identical* to soda bottles? It's not a coincidence.

Thanks for the clarification emjay. That is actually pretty interesting. Good multiple use product! Probably should be way cheaper than some of those links on Amazon. You can buy 2 liters of soda for the price of one of those.
 
I wish there was a cheaper way to get those soda bottle types, but I can't find them locally. I just went with 4 oz canning jars I got at Ace for 9.99 for a 12 pack.
 
What's the consensus on the best way scale up cultures? Should I just add fresh wort to the culture, or is it better to let the yeast drop out and dump the supernatant first? I feel like it's better to drop the yeast and dump the supernatant, but if so, is it best to put it in the fridge or leave at room temp? I wish there was a faster way that didn't involve centrifugation.

It seems like you'd definitely want to dump the supernatant from the starter before pitching to make actual beer, especially if the starter volume is quite large.
 
What's the consensus on the best way scale up cultures? Should I just add fresh wort to the culture, or is it better to let the yeast drop out and dump the supernatant first? I feel like it's better to drop the yeast and dump the supernatant, but if so, is it best to put it in the fridge or leave at room temp? I wish there was a faster way that didn't involve centrifugation.

It seems like you'd definitely want to dump the supernatant from the starter before pitching to make actual beer, especially if the starter volume is quite large.

You might get varying opinions on that. If I've got plenty of room in the starter vessel, I just dump in some fresh and don't worry about decanting. If the vessel is at or near capacity, then I'll decant. I normally don't do more than two steps though and the first step is usually much smaller than the second.
 
If starting from a small number of cells, I step up to my 1.5-3 liter starter. However, if you start with a frozen tube containing around 100 billion cells there is no reason to step up. You can go directly into a large starter volume. You are only looking for a couple doublings of the viable cells in the tube. After growing overnight, I usually cold crash and pour off the spent wort. If I have the motivation and the wort, I resuspend in some fresh room temperature wort while I am brewing and then just pitch. Otherwise I just pitch the slurry of cold crashed cells. It doesn't take much wort to wake them up and it gets the fermentation going quicker but it is definitely not necessary.
 
I should say thanks to everyone who has contributed to the thread and whose data or suggestions led to the procedure suggested in the article.
 
My article on Freezing Yeast has been approved and posted:

https://www.homebrewtalk.com/entries/freezing-yeast.html

Hope you like it and, if you have comments, please make them. I presume I can make edits at a later date or at least post changes and updates in the comments.

Very nice write-up. Thanks for spending the time and effort!

I've only done the freezing once, but I was surprised to read that you can get 100 billion cells to fit into a volume of 25ml. When I centrifuged my slurry to pellet the cells, 30 billion took up 15 ml. I would've guessed 100 billion would take up almost the entire 50 ml tube. Please correct me if I'm wrong.
 
Very nice write-up. Thanks for spending the time and effort!

I've only done the freezing once, but I was surprised to read that you can get 100 billion cells to fit into a volume of 25ml. When I centrifuged my slurry to pellet the cells, 30 billion took up 15 ml. I would've guessed 100 billion would take up almost the entire 50 ml tube. Please correct me if I'm wrong.

Thanks. I have to admit, I only counted the first time I did it and I was dealing with the equivalent of WLP001. That was approximately what I found. Since I am normally dealing with haploid yeast which are much smaller I was not surprised by that. However, I have not counted a range of yeast. I will check it out again.

That all said, if one uses the cells to inoculate a starter, I think it is a moot point. An overnight starter should give you about a 10X increase in cell number which way exceeds the necessary cell number for a 5-10 gallon batch.
 
That all said, if one uses the cells to inoculate a starter, I think it is a moot point. An overnight starter should give you about a 10X increase in cell number which way exceeds the necessary cell number for a 5-10 gallon batch.

I agree that if you're doing a starter, it shouldn't matter much. However, I've been doing some playing around with the Wyeast calculator and have generated the following graph on the top showing the 'Expected Fold Expansion' vs. 'Starting Cell Concentration'. I think this is the most useful way to represent the data because it can be used for any culture as long as you know your volume and cell #. From it, you can estimate the expected final cell #. The second graph, titled 'Actual Fold Expansion' is data generated from my own cultures. It comes very close to the Wyeast data.

I'd like to point out a few interesting things. 1st, you get more expansion if you start at a lower conc. than at higher. For example, if you made a 1L starter with 100 billion cells, that'd be 100e6/ml, and you could expect the cells to expand 2 fold for a final # of 200 billion. If you made the same 1L starter with only 10 billion cells, i.e. 10e6/ml, you can expect a 7 fold expansion for a final # of 70 billion. This is definitely something to keep in mind when planning your step-ups.

My data pretty much confirms the Wyeast data, except I've been getting better expansion at low concentrations, and worse expansions at high concentrations. The Wyeast data also predicts cultures higher than 200e6/ml to roughly double, whereas I am unable to get hardly any expansion above that concentration, which is why I cut the graph off at that point. I'm pretty sure the Wyeast Pitch Rate Calculator is based on data from cultures with starting concentrations near the middle of the graph, and that the numbers it gives you for higher and lower concentrations are extrapolations, which are not accurate. I should also remind those who haven't been following along that I start all my cultures by oxygenating with pure oxygen and a diffusing stone, and then use a stirplate along with an air pump to provide continuous aeration.

Wyeast.jpg


Forkhead.jpg
 
I would agree with you. I think 2x10e8/ml is about the highest density you can achieve with optimal conditions. To be honest, I just assume about 1-2x10e8/ml as max density when brewing. After all, I'm not publishing my results, I'm drinking them :mug:
 
I finally got a chance to look at the article. Good summary. Nice job Brewitt.
 
BBL_Brewer said:
I finally got a chance to look at the article. Good summary. Nice job Brewitt.

Thanks. I appreciate the feedback. Especially from the originator of the thread. By the way, I did get promoted to Premium but what do I do with it. I'm not too interested in arguing with folks in the debate forums ;-)
 
Thanks. I appreciate the feedback. Especially from the originator of the thread. By the way, I did get promoted to Premium but what do I do with it. I'm not too interested in arguing with folks in the debate forums ;-)

LoL. Well, you should at least browse the boneyard for a minute :)

Yeah, I liked how you pointed out that some of the methods needed further testing. I had planned on doing something similar by revising this thread but I just didn't feel like a major re-write was warranted until more data came in. You left the door open for improvement and hopefully you can edit if and when the process evolves. Plus, now the information is readily available independent of the search function and hopefully folks will benefit from it. :mug:
 
Brewitt / Forkhead,

How are you mixing your glycerine solution? % v/v or w/v. The reason I ask is becasue v/v is a higher concentration than w/v. I'm ready to freeze up a bunch of jars of pacman and was contemplating how much glycerine to use. I already have a couple sterile jars of 60% and 50% w/v solution. I'm going to dilute them and was wondering what you guys have been doing.
 
BBL_Brewer said:
Brewitt / Forkhead,

How are you mixing your glycerine solution? % v/v or w/v. The reason I ask is becasue v/v is a higher concentration than w/v. I'm ready to freeze up a bunch of jars of pacman and was contemplating how much glycerine to use. I already have a couple sterile jars of 60% and 50% w/v solution. I'm going to dilute them and was wondering what you guys have been doing.

I've been using v/v
 
Thanks forkhead. Brewitt suggests mixing glycerine 4:1 in his article, so I'm pretty sure he's using v/v as well.
 
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