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.
Do you know how to make a yeast starter? Then why not farm yeast and freeze it?
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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.
theveganbrewer
Well-Known Member
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.
Forkhead
Active Member
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.
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.
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BBL_Brewer
Well-Known Member
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.
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.
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.
Forkhead
Active Member
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.
Forkhead
Active Member
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.
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 
Forkhead
Active Member
I think that's a safe assumption as long as you're starting your culture at a concentration higher than 20e6/ml, as illustrated when you express the data as below.
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BBL_Brewer
Well-Known Member
I finally got a chance to look at the article. Good summary. Nice job Brewitt.
BBL_Brewer said:
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 ;-)
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BBL_Brewer
Well-Known Member
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.
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BBL_Brewer
Well-Known Member
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.
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.
Forkhead
Active Member
BBL_Brewer said:
I've been using v/v
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BBL_Brewer
Well-Known Member
Thanks forkhead. Brewitt suggests mixing glycerine 4:1 in his article, so I'm pretty sure he's using v/v as well.
Matthewjscott
Well-Known Member
How do I sanitize the glycerine solution without a pressure cooker
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BBL_Brewer
Well-Known Member
Boil it like you would starter wort.
I would probably put it in a canning jar and boil the jar in water. Boiling 100 % glycerol is probably not a good idea and boiling diluted glycerol will make it hard to know its final concentration. This will be more controllable in a canning jar.
Golddiggie
Well-Known Member
What if you boil the water, then mix that (cooled enough to work with) with the glycerine?
I really didn't want to pick up a pressure cooker JUST for this.
I really didn't want to pick up a pressure cooker JUST for this.
I'm buying glycerol in drugstore and it is sterile, if you're not sure just boil it for few minutes with foil on top (mixed w/water).
What is about v/v vs. w/v dilutions?
I've been using w/v but now I'm not sure about it.. on other hand it depends on what % of gycerin/water solution we use.
I did a little math and it seems that 12% v/v dilution equals to 15% w/v.
When searching about this topic I found article where 10% v/v (12% w/v) is used, samples are frozen to -80°C:
http://nbimcc.org/JCC/2002/JCC312.pdf
Do you think there should be difference in mixing percentage depending on storage temperature (eg -20 vs -80 °C)?
What is about v/v vs. w/v dilutions?
I've been using w/v but now I'm not sure about it.. on other hand it depends on what % of gycerin/water solution we use.
I did a little math and it seems that 12% v/v dilution equals to 15% w/v.
When searching about this topic I found article where 10% v/v (12% w/v) is used, samples are frozen to -80°C:
http://nbimcc.org/JCC/2002/JCC312.pdf
Do you think there should be difference in mixing percentage depending on storage temperature (eg -20 vs -80 °C)?
sub
strongale28
New Member
Would this glycerine be good for freezing yeast? not sure after reading the material safety sheet. The price is pretty good.
http://www.newdirectionsaromatics.ca/glycerine-vegetable-usp-995-p-540.html
http://www.newdirectionsaromatics.ca/glycerine-vegetable-usp-995-p-540.html
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BBL_Brewer
Well-Known Member
Just depends on whether you trust the source or not. The certificate of analysis is certainly vague. At least you know it tastes sweet.
Forkhead
Active Member
Apparently styrofoam coolers are NOT a good choice for holding isopropyl alcohol. I had a styrofoam cooler filled with isopropanol in the freezer since the weekend and when I came back to it today all the isopropanol had leaked out and made a huge mess. I had a somewhat similar thing happen the first time I froze cells but I thought my cooler just had a hole in it. Now I'm pretty sure the isopropanol breaks down the styrofoam and leaks out.
For freezing cells, use a PLASTIC cooler filled with isopropanol to slow down the cooling rate. DO NOT USE STYROFOAM!!!
For freezing cells, use a PLASTIC cooler filled with isopropanol to slow down the cooling rate. DO NOT USE STYROFOAM!!!
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BBL_Brewer
Well-Known Member
LOL. Man, you seem to be hitting a few hurdles there Forkhead. Exploding vials and now melting coolers. At least you're getting those mistakes out of the way for everybody else.
Should be downhill from here right?Forkhead
Active Member
Haha, yeah it's all part of the process. Smooth sailing from here on out (fingers crossed).
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