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Old 10-18-2012, 05:49 PM   #221
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Originally Posted by pvtschultz View Post
This calculator has fallen into favor for me.

http://yeastcalc.com/
I've seen a lot of people mention that here lately. I'll have to check it out.
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Old 10-18-2012, 05:58 PM   #222
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Originally Posted by pvtschultz View Post
This calculator has fallen into favor for me.

http://yeastcalc.com/
Wow, thats quite nice. I like that it shows what growth factor it uses.
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Old 10-22-2012, 01:12 PM   #223
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Great infos!

so here is my experince with freezed yeast.

Up to now I've tried to freeze only 3 strains of yeast:

Wyeast 1099 (15% glycerine)
used only for a test small batch of mint chocolate ale, good fermentation, performed a 200ml starter before pitching in 5L wort (1.040)


Wyeast 1056 (25% glycerine)
On july brewed APA --> 2 steps starter (1st 200ml + 2nd 1,2L )
vigorous fermentation, had to use blow-off tube, lot of yeast came out so I decided to trying top cropping

2 weeks ago brewed an "american bitter" --> 3 steps starter: 200ml + 800ml + 1L (2liters total) too much activity at the 3rd starter, lost a lot of yeast because of krausen our from my flask

Wyeast 13522 (25% glycerine)
on june brewed my belgian IPA recipe, same procedure used for my APA, so same "problems" during fermentation

On setember brewed again the same recipe, this time I've also tried to top crop the yeast, so I've ended up with 4 vials full of just yeast slurry and glycerine (33% of the total volume)


*All starter performed with stirring

So I've brewed 2 belgian ales with wyeast 3522, and two hoppy beers with 1056 reusing freezed yeast.

I've noticed somethings that i want to share with you.
i'm not going in the detail of the freezing process because I'm still trying different glycerine-water-slurry ratio and starters propagation, I'd like to talk about the results in term of final beer quality.

1) Had some problem with my starter procedure.
I've always performed only one starter using wyest pack( max volume 1,75L). As I'm freezing very small amount of yeast I use a 3 steps procedure (200ml+ 800ml + 1L = 2 liters total). As someone mentioned before I've expirenced slow activity with the first one (200ml stirred), but after that the 2nd was fast as usual. Had some problem with the 3rd, always end up with lot of yeast came out of the flask beacuse of high krausen. So I'm planning to perform only 2 starters the next time and to prepare vials with more yest to compensate it.

2)Differences in flavor profile: I've noted different flavor of the final beer in my Belgian IPA. in particular the beer brewed with frozen yeast had higher esters and fenolic profile (less clean final product) but I'm not sure if this is because of the freezing procedure (mutation??) or because of the differences in starter procedure (higher potching rates?).
Want to perform a test: brew again my Belgina IPA, split the wort and ferment one half with new pack of yeast and the other one with frozen yeast

3)Beer stability. I'm very worried about that. All beers brewed with frozen yeast after 4-5 shows a dramatic change. I'm assuming this is due to a more stressed yeast.
On my last belgian IPA I've tried to split the batch and inoculate new yeast at bottling time ( few grams of safale us05 ) only hal to see if there are some differences after 6 months.

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Old 10-22-2012, 07:13 PM   #224
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Originally Posted by SamBrewer View Post
I've noticed somethings that i want to share with you.
i'm not going in the detail of the freezing process because I'm still trying different glycerine-water-slurry ratio and starters propagation, I'd like to talk about the results in term of final beer quality.

1) Had some problem with my starter procedure.
I've always performed only one starter using wyest pack( max volume 1,75L). As I'm freezing very small amount of yeast I use a 3 steps procedure (200ml+ 800ml + 1L = 2 liters total). As someone mentioned before I've expirenced slow activity with the first one (200ml stirred), but after that the 2nd was fast as usual. Had some problem with the 3rd, always end up with lot of yeast came out of the flask beacuse of high krausen. So I'm planning to perform only 2 starters the next time and to prepare vials with more yest to compensate it.

2)Differences in flavor profile: I've noted different flavor of the final beer in my Belgian IPA. in particular the beer brewed with frozen yeast had higher esters and fenolic profile (less clean final product) but I'm not sure if this is because of the freezing procedure (mutation??) or because of the differences in starter procedure (higher potching rates?).
Want to perform a test: brew again my Belgina IPA, split the wort and ferment one half with new pack of yeast and the other one with frozen yeast

3)Beer stability. I'm very worried about that. All beers brewed with frozen yeast after 4-5 shows a dramatic change. I'm assuming this is due to a more stressed yeast.
On my last belgian IPA I've tried to split the batch and inoculate new yeast at bottling time ( few grams of safale us05 ) only hal to see if there are some differences after 6 months.
I don't think the problems you're experiencing are related to frozen yeast.

Problem 1 - Yeast Starters

Sounds like you've got healthy yeast if they are blowing off out of the starter vessel. Try a larger vessel, or decant the first two steps worth of wort off before adding the third. I use 1 gallon jugs so I have plenty of head space. I try not to fill my starter vessel more than half way to avoid blow off.

Problem 2 - Different beer flavor - Higher Esters and Phenols

I think this is probably due to pitching rate, not frozen yeast. Your starters are acting normal, so there's no reason to think that the yeast are significantly stessed before you pitch to a batch of beer. It's very possible that you are underpitching. Try pitching more yeast next time and see if that solves the problem.

Problem 3 - Beer Stability.

What kind of changes are you experiencing? What time frame? 4-5 what? Weeks, months? I have some recipes that I've been brewing since well before I started freezing yeast and I can't tell the difference. Some of my kegs sit for weeks or months before I carbonate and drink them and I haven't noticed anything but normal aging.
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Old 10-23-2012, 05:26 PM   #225
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I think that maybe I'm overpitching

Maybe 2 starters are enought to prepare the yeast to do his job, but anyway the split batch using new yeast vs frozen one I'm shure will tell me if there are some differences.
Regarding the beer stability the problems seems to apper 4 months after bottling time

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Old 10-24-2012, 04:12 PM   #226
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Alright, I have some results from my experimentation with freezing conditions. Cells were grown up in a starter until they reached stationary phase. They were then kept in the refrigerator for a few days. I aliquoted 30 billion cells into 9 different 50ml conical tubes, centrifuged them, and removed the supernatant. The cells themselves took up a volume of 15-20ml, so I added 20ml of fresh wort with glycerol added at a concentration of 15%, 30%, or 50%. Since 20ml of wort+glycerol was diluted 2-fold by the volume of the cells, the final concentrations of glycerol were more like 8%, 15%, and 25%. Cells were mixed thoroughly and then frozen at either -20C, -80C, or in liquid nitrogen. For cells frozen at -20C and -80C, the tubes were placed in a styrofoam cooler filled with room temp 70% isopropanol, then put directly into the freezer. The idea was to cool the cells very slowly and protect them being frozen too quickly, which can cause formation of ice crystals that can physically damage and kill the cells. It also prevents osmotic shock during the freezing that can kill the cells. The cells frozen in liquid nitrogen were thrown directly in and were frozen in ~10 seconds. I anticipated that this would result in poor viability. All cells were thawed as quickly as possible in a 37C water bath. Here are the results:

Starting viability before freezing: 78%
-20C 15% glycerol: 80% viability
-20C 30% glycerol: 50% viability
-20C 50% glycerol: 44% viability
-80C 15% glycerol: 70% viability
-80C 30% glycerol: 73% viability
-80C 50% glycerol: 50% viability
Liquid N 15% glycerol: 0% viability
Liquid N 30% glycerol: 0% viability
Liquid N 50% glycerol: 9% viability

Viability was measured 3 different was: 1) manual counting on hemocytometer using trypan blue to identify dead cells, 2) automated counting using Vi-Cell machine and trypan blue, 3) CFU counts on YPD plates. Both the Vi-Cell and the CFU gave pretty similar numbers, but I trust the CFU more. That’s because trypan blue was not a great way to identify dead cells. Dead cells don’t stain dark blue with trypan, but rather just look “less bright”, which is very hard to discriminate between live cells. In fact, by eye I counted ~75% viability in samples that were 0% viable by CFU. The Vi-Cell machine was better at discriminating than my eyes, but since it still uses trypan blue, I doubt it’s 100% accurate.

Conclusions: Freezing at -20C with 15% glycerol gave the best viability. Increasing concentrations of glycerol was actually detrimental at -20. Good viability was also achieved at -80C and the effect of glycerol concentration was less apparent. Flash freezing in liquid nitrogen is bad. CFU is the best way to measure viability.

Future experiments will be needed to measure viability after long-term storage at -20C vs -80C. Future experiments will also be needed to measure viability after freezing in a home freezer. However, I think freezing slowly is essential, and freezing the cells in a cooler filled with 70% isopropanol is something any homebrewer can do. Storing the cells like this should also help protect them from the freeze/thaw of the defrost cycle of home freezers.

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Old 10-24-2012, 05:00 PM   #227
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Any video recommendations for freezing yeast I need some visuals

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Old 10-24-2012, 05:09 PM   #228
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Forkhead,

You rock. Now I need to add a biolab to the Christmas list. Wonder if I can get a gov't grant for one and put it next to the brew room....?

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Old 10-24-2012, 06:41 PM   #229
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Quote:
Originally Posted by Forkhead View Post
Alright, I have some results from my ...

Viability was measured 3 different was: 1) manual counting on hemocytometer using trypan blue to identify dead cells, 2) automated counting using Vi-Cell machine and trypan blue, 3) CFU counts on YPD plates. Both the Vi-Cell and the CFU gave pretty similar numbers, but I trust the CFU more. That’s because trypan blue was not a great way to identify dead cells. Dead cells don’t stain dark blue with trypan, but rather just look “less bright”, which is very hard to discriminate between live cells. In fact, by eye I counted ~75% viability in samples that were 0% viable by CFU. The Vi-Cell machine was better at discriminating than my eyes, but since it still uses trypan blue, I doubt it’s 100% accurate.

Conclusions: Freezing at -20C with 15% glycerol gave the best viability. Increasing concentrations of glycerol was actually detrimental at -20. Good viability was also achieved at -80C and the effect of glycerol concentration was less apparent. Flash freezing in liquid nitrogen is bad. CFU is the best way to measure viability.

Future experiments will be needed to measure viability after long-term storage at -20C vs -80C. Future experiments will also be needed to measure viability after freezing in a home freezer. However, I think freezing slowly is essential, and freezing the cells in a styrofoam cooler filled with 70% isopropanol is something any homebrewer can do. Storing the cells like this should also help protect them from the freeze/thaw of the defrost cycle of home freezers.
So, by looking to your results the best way to freezing yeast Is a -20°C and using less glycrol.

sorry but I am a bit confused, when you write "15% glycrole solution" you mean 8% total right?
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Old 10-24-2012, 08:53 PM   #230
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Originally Posted by Forkhead View Post
Alright, I have some results from my experimentation with freezing conditions. Cells were grown up in a starter until they reached stationary phase. They were then kept in the refrigerator for a few days. I aliquoted 30 billion cells into 9 different 50ml conical tubes, centrifuged them, and removed the supernatant. The cells themselves took up a volume of 15-20ml, so I added 20ml of fresh wort with glycerol added at a concentration of 15%, 30%, or 50%. Since 20ml of wort+glycerol was diluted 2-fold by the volume of the cells, the final concentrations of glycerol were more like 8%, 15%, and 25%. Cells were mixed thoroughly and then frozen at either -20C, -80C, or in liquid nitrogen. For cells frozen at -20C and -80C, the tubes were placed in a styrofoam cooler filled with room temp 70% isopropanol, then put directly into the freezer. The idea was to cool the cells very slowly and protect them being frozen too quickly, which can cause formation of ice crystals that can physically damage and kill the cells. It also prevents osmotic shock during the freezing that can kill the cells. The cells frozen in liquid nitrogen were thrown directly in and were frozen in ~10 seconds. I anticipated that this would result in poor viability. All cells were thawed as quickly as possible in a 37C water bath. Here are the results:

Starting viability before freezing: 78%
-20C 15% glycerol: 80% viability
-20C 30% glycerol: 50% viability
-20C 50% glycerol: 44% viability
-80C 15% glycerol: 70% viability
-80C 30% glycerol: 73% viability
-80C 50% glycerol: 50% viability
Liquid N 15% glycerol: 0% viability
Liquid N 30% glycerol: 0% viability
Liquid N 50% glycerol: 9% viability

Viability was measured 3 different was: 1) manual counting on hemocytometer using trypan blue to identify dead cells, 2) automated counting using Vi-Cell machine and trypan blue, 3) CFU counts on YPD plates. Both the Vi-Cell and the CFU gave pretty similar numbers, but I trust the CFU more. That’s because trypan blue was not a great way to identify dead cells. Dead cells don’t stain dark blue with trypan, but rather just look “less bright”, which is very hard to discriminate between live cells. In fact, by eye I counted ~75% viability in samples that were 0% viable by CFU. The Vi-Cell machine was better at discriminating than my eyes, but since it still uses trypan blue, I doubt it’s 100% accurate.

Conclusions: Freezing at -20C with 15% glycerol gave the best viability. Increasing concentrations of glycerol was actually detrimental at -20. Good viability was also achieved at -80C and the effect of glycerol concentration was less apparent. Flash freezing in liquid nitrogen is bad. CFU is the best way to measure viability.

Future experiments will be needed to measure viability after long-term storage at -20C vs -80C. Future experiments will also be needed to measure viability after freezing in a home freezer. However, I think freezing slowly is essential, and freezing the cells in a styrofoam cooler filled with 70% isopropanol is something any homebrewer can do. Storing the cells like this should also help protect them from the freeze/thaw of the defrost cycle of home freezers.
Nicely done sir, thanks for this. Just to clarify 15, 30 and 50% of glycerol is the final concentration the cells are frozen in or we have to cut it by 1/2?
And i would bet that for the long term storage -80 viability will be better than -20
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