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.
Do you know how to make a yeast starter? Then why not farm yeast and freeze it?
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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)?
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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).
Forkhead
Active Member
That's a beautiful paper that agrees with what we've seen experimentally. Slow freezing, fast thawing is the best for viability.
The difference between w/v and v/v is not very large. I doubt it will make a substantial difference. W/v will always be a higher % glycerol than v/v because glycerol weighs more than water. When mixing liquids in the lab we usually use v/v. When adding dry reagents to liquids we use w/v. If weighing the glycerol is easier for you than measuring the volume, you could correct for the difference by.
It looks like your math is correct. The density of 100% glycerol is 1.25g/ml. So, divide the final desired % v/v glycerol by 1.25 to calculate what it would be in w/v.
In response to diS' question about -80C vs -20C. Yes, I think it is clear that cells frozen at -80 will be stable for longer than those frozen at -20, especially if the -20 is frost free. I have yeast stocks that have been frozen at -80 for 20 years that are still viable. I doubt that would be the case in a -20 frost free freezer, or even a non-frost free one. In addition, Forkhead got numbers that were a bit different in -20 and -80 for optimal glycerol concentration. So there are differences. Nevertheless, it is clear that the yeast strains frozen in glycerol in the range you are suggesting are quite stable for many months and maybe for a year or more.
By the way, that study is the same, or similar, to one I alluded to earlier in an earlier post in this thread. There is another difference with what we are doing besides temperature. Volume is very different and will have different freezing and thawing characteristics. However, I think it is within the error of homebrewing, something important to consider. We could adopt something like the old saying "good enough for government work". Something like "good enough for homebrewers".
By the way, that study is the same, or similar, to one I alluded to earlier in an earlier post in this thread. There is another difference with what we are doing besides temperature. Volume is very different and will have different freezing and thawing characteristics. However, I think it is within the error of homebrewing, something important to consider. We could adopt something like the old saying "good enough for government work". Something like "good enough for homebrewers".
bink00
Well-Known Member
Thanks!!!! I had a fresh harvest of pacman stepped up to a starter size. Some other junk got in the way of the brew I had planned (video games, gun season). So I just stuck the starter in the fridge and ordered some vials off amazon prime and filled up 9 50ml vials tonight. I cant wait to make a starter with one in a few weeks.
Same here. Growing up a starter of SD Super Yeast right now. Grew a double starter. One of them goes in the brew this weekend, if I get to it. The other gets broken up into tubes and goes in the freezer. No washing, no trub, just nice clean yeast. Those will go to join my stocks of 4 other yeasts including a belgian golden ale yeast, a saison yeast, Ballast Point Sculpin (WLP001) and yeast from Racer 5. That way when you want to brew you just run to the freezer and start yourself a starter. Yeast is a renewable resource. Not so for malt extract, grain and hops, unless you are growing those as well.
bink00
Well-Known Member
Hey....I should have thanked you as well for your write up. But anyways. I notice in your picture that your yeast looks nice and evenly distributed in the vials. When I checked mine this morning, a lot of the stuff had settled out. Is this normal?
Thanks
James
The yeast depicted went through a little different process than recommended in the article. I chilled to near freezing temperature and then they were agitated and put in a very cold freezer which caused freezing before the yeast settled. Yours probably had time to settle before freezing. It is just fine. The important thing is that they had time to cool in the presence of glycerine before freezing.
FYI: I just prepared frozen WLP090. I counted them before freezing because there was some question about the numbers of yeast I cited in my article. I found that 20 mls of a thick slurry (a subjective term but lets say easily pourable but noticeably thicker than wort) had 40-50 billion cells (that is about 15 mls of hard packed cells) For my original preparations I used 30 mls of cell slurry and 10 mls of 60% glycerine. That would be 60-75 billion cells. If you are using 50 ml tubes you could easily put 100 billion cells in the tube and adjust to 7.5% glycerol with a concentrated stock. That said, 40-50 billion cells is plenty for an overnight starter.
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BBL_Brewer
Well-Known Member
I just got 27 jars of pacman in the freezer. Won't have to order any of that for a while. I would have had more, but I had to make up some starters for brew day tomorrow. I ended up going with a final concentration of 15% w/v glycerol. ~12% v/v.
I came to a similar conclusion.
I'm using "poor man counting method"- visual estimation of density in transparent vial (Jess from Wyeast advised me and seems that it works) and I usually ends up with 80-90% of solids in vial which corresponds to 2-2.3 B/ml, maybe little less since I don't know how many of cells are vital.
Did you counted cells after freezing (besides those 25% vitality determined before)?
I'll try to estimate number of my frozen cells, but it will be tough to get right number since I can't count dead cells.
theveganbrewer
Well-Known Member
I'm thinking out my process for doing this tomorrow. I have about 600 billion cells in the fridge. Tomorrow I will go with 5 jars. Im not sure how I should go about getting the glycerine mix ready.
Did I read correctly 4:1 water to glycerine ratio? I need double the volume of yeast in glycerine mix, so 50ml of each?
How is everyone sanitizing everything? I do not have a pressure cooker. Should I mix up 250ml of glycerine mix and boil it? Then add to sanitized glass jars, the add slurry?
After that I'm good with the freezing process, in just getting hung up on sanitation and volumes. I worry I'm going to come up short on yeast since the jars don't have measure marks on them. Am I aiming for 100ml of yeast/glycerine mix per jar? Do I need to account for boil off in sanitizing the glycerine mixture?
Did I read correctly 4:1 water to glycerine ratio? I need double the volume of yeast in glycerine mix, so 50ml of each?
How is everyone sanitizing everything? I do not have a pressure cooker. Should I mix up 250ml of glycerine mix and boil it? Then add to sanitized glass jars, the add slurry?
After that I'm good with the freezing process, in just getting hung up on sanitation and volumes. I worry I'm going to come up short on yeast since the jars don't have measure marks on them. Am I aiming for 100ml of yeast/glycerine mix per jar? Do I need to account for boil off in sanitizing the glycerine mixture?
4:1 glyerine/slurry would correspond to 25% v/v solution, you may want to lower glycerine to 10-15%.
That 4:1 is when you mix glycerine and water in 60% solution and then add it to slurry in 4:1 rate (60/4=15).
Boil glycerine shortly and sanitize jars, you can mark jars before to know how much to fill them. Always leave some free space in jars for freezing expansion.
That 4:1 is when you mix glycerine and water in 60% solution and then add it to slurry in 4:1 rate (60/4=15).
Boil glycerine shortly and sanitize jars, you can mark jars before to know how much to fill them. Always leave some free space in jars for freezing expansion.
Forkhead
Active Member
I thawed some yeast that had been frozen in either 7.5%, 15%, or 30% final concentration of glycerol (v/v). 15% had the best viability and 7.5 & 30% had significantly worse viability.
I would strongly recommend everyone to be using 15% v/v final concentration of glycerol.
I would strongly recommend everyone to be using 15% v/v final concentration of glycerol.
Golddiggie
Well-Known Member
How about mixing the slurry and glycerine solution in the flask, then pouring from that (into tubes)??
Golddiggie
Well-Known Member
Just so I'm clear on this... If I have a slurry mix that's 1L in size, I'll add 250ml of 60% glycerine solution to that. Right? To get the 60% solution, just mix 6 parts glycerine to 4 parts water, correct? Looking at v/v mixing here, so that I don't need to use any more containers than necessary. I do have a 100ml graduated cylinder (pyrex) that I can use for the glycerine mixing (should be fairly easy there).
I did a freeze test with the vials I'll be freezing in, so I know how far I can fill one and not have it burst on freezing (had a small amount of a void left in it). I'm planning on making another starter step with the slurry I've reserved, in order to make sure it's still 100% kosher before doing this. So it could be after Turkey Day now.
theveganbrewer
Well-Known Member
Ok, so I need 1 part of 60% glycerine/water and 4 part yeast, I see the math now, not my strong suit. And since I am going with 40ml of yeast, that means 10ml of glycerine solution.
Thanks for the help.
Thanks for the help.
For 40 mls total you would need 10 mls of 60% glycerine and 30 mls of yeast. 50 mls total you need 37.5 mls yeast and 12.5 mls glycerine. That is, you want to dilute 60% glycerine by 4 to give 15%. 3 parts yeast:1 part glycerine gives 15%.
All of that said, if you did 10 mls of glycerine and 40 mls yeast, you would have 60% divided by 5, which is 12.5%. Close enough for home brewing!
All of that said, if you did 10 mls of glycerine and 40 mls yeast, you would have 60% divided by 5, which is 12.5%. Close enough for home brewing!
theveganbrewer
Well-Known Member
Man, I am really bad at simple math. Accounting wasn't my strong point in business school.
Not exactly, you would end with 5:1 ratio or 20% (1 L/1.25L =0.8; 0.25/1.25= 0.2), and you want 25% of glycerine/water in total volume.
Do your math this way for 25% (or 4:1), it is easier:
Slurry Volume= Total Volume x 0.75
Glycerine/Water Volume= Total Volume x 0.25
Total Volume= sum of above volumes
This gives 937 ml of slurry + 313 ml glycerine/water which is 1250 ml. If you want invariable slurry volume (eg. 1000 ml) then you'll need 333 ml of glycerine/water solution. Total volume is as you see 1333 ml.
You are right about mixing glycerine and water, simplest way to calculate it is to multiply desired total glycerine/water volume with 0.6, you will get required glycerine volume and then top it up with water to desired total volume..
eg. for 500 ml total you need 300 ml (500 x 0.6) of glycerine and rest of it (200 ml) water.
Golddiggie
Well-Known Member
God I hate math... lol
Might be easier to just aim for 1L total volume. IF I'm following you, that would be [simply] 750ml slurry and 250ml glycerine solution. Now, to figure out the glycerine mixture to get that... Looks like 150ml of glycerine and 100ml of water... Right?? It might be easier to make sure the slurry is no more than 750ml, add the 250ml of glycerine solution, and the 'top off' with sanitized water to get the 1L total volume... Good?
theveganbrewer
Well-Known Member
How great would an online calculator for this be? Integrated into a yeastcalc.com type thing.
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BBL_Brewer
Well-Known Member
It doesn't have to be this difficult. What I reccomend is that you put all of your yeast slurry into a large container with volume markings. Add your glycerine solution and any top of water if needed, then split it up into whatever you're going to freeze in. Unless you're using a centrifuge, there's no reason not to. I have marks on my starter vessels so I just do it in there. I know what I want my final vomue to be (500 ml) so I use the following equation: Final Volume x Desired Glycerine Concentration (%) / Concentration of Glycerine Solution
So for example Let's say 500 ml final volume, 15 % v/v final concentration, 60% glycerine solution. (500 x 0.15) / 0.6 = 125 ml of 60% glycerine solution required.
Add your 125 ml, top off with sanitized water.
So for example Let's say 500 ml final volume, 15 % v/v final concentration, 60% glycerine solution. (500 x 0.15) / 0.6 = 125 ml of 60% glycerine solution required.
Add your 125 ml, top off with sanitized water.
God I hate math... lol
Might be easier to just aim for 1L total volume. IF I'm following you, that would be [simply] 750ml slurry and 250ml glycerine solution. Now, to figure out the glycerine mixture to get that... Looks like 150ml of glycerine and 100ml of water... Right?? It might be easier to make sure the slurry is no more than 750ml, add the 250ml of glycerine solution, and the 'top off' with sanitized water to get the 1L total volume... Good?
That's it.
shogun0660
Member
Just wondering... so is this different than the instructions on the front page of this thread?? Or is it the same/similar since one is vol/vol and one is weight/vol? Just a little confused. Thanks!
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BBL_Brewer
Well-Known Member
It's different. With my original method, I ended up with a final concentration of glycerine of about 20% v/v. I was using 35% w/v glycerine, but I hadn't accounted for the dilution once I mixed it with my yeast slurry. Hope that answers your question.
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BBL_Brewer
Well-Known Member
Somehow I missed this post. So, these results contradict your initial treatment? After a little time had passed the 15% (ie, 30% diluted two fold) had the best viabiltiy?
shogun0660
Member
This certainly calls for a revision of my protocol. 15% is the concentration that worked for me originally at -80C. Now it seems that it is the optimal concentration at -20 as well.
I have added a comment to that writeup but I should probably do a revision.
I've tried to keep up with this thread, but not being a science and math guy, my head is spinning. I hope I'm not out of line in asking for a little clarification/confirmation of my process.
I will be freezing a total volume of 20ml of yeast/glycerine solution. (I know more would most likely be better, but I already bought 24ml glass vials, so I need to use them.)
In order to finish with the proper mixture of glycerine and yeast, I plan on creating a 30% glycerine solution (by combining 30ml of glycerine with 70ml of water). I will combine 10ml of this solution with 10ml of a slurry of yeast (thick but pourable) in each vial. This should give me the desired 15% solution of glycerine to yeast in each vial, correct?
I apologize for my confusion and I truly appreciate all of the work that was put into this thread and the article. There's a lot of new data since I first subscribed to this thread and I've thoroughly confused myself trying to adapt my proposed process.
I will be freezing a total volume of 20ml of yeast/glycerine solution. (I know more would most likely be better, but I already bought 24ml glass vials, so I need to use them.)
In order to finish with the proper mixture of glycerine and yeast, I plan on creating a 30% glycerine solution (by combining 30ml of glycerine with 70ml of water). I will combine 10ml of this solution with 10ml of a slurry of yeast (thick but pourable) in each vial. This should give me the desired 15% solution of glycerine to yeast in each vial, correct?
I apologize for my confusion and I truly appreciate all of the work that was put into this thread and the article. There's a lot of new data since I first subscribed to this thread and I've thoroughly confused myself trying to adapt my proposed process.
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