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

I agree. I was expecting more cells. I weighed out 150 grams of light DME and added 1.5L of water and 1/2 Tsp of yeast nutrient. Boiled for 20 min, cooled to room temp. The original gravity was 1.040 just like I read it should be. I oxygenated using a can of pure oxygen and a diffusing stone for 30 seconds. I warned the yeast for a few hours to room temp, then pitched it. I kept it loosely covered with foil at room temp on a stir plate. I'm pretty sure I did everything correctly but please correct me if I made any mistakes.

Are those yeast calculators based on the assumption that you start with 100 billion cells, or can you enter your starting cell number? I haven't seen any homebrewers validate those calculators with their own real world data.

Forkhead said:

I agree. I was expecting more cells. I weighed out 150 grams of light DME and added 1.5L of water and 1/2 Tsp of yeast nutrient. Boiled for 20 min, cooled to room temp. The original gravity was 1.014 just like I read it should be. I oxygenated using a can of pure oxygen and a diffusing stone for 30 seconds. I warned the yeast for a few hours to room temp, then pitched it. I kept it loosely covered with foil at room temp on a stir plate. I'm pretty sure I did everything correctly but please correct me if I made any mistakes.

Are those yeast calculators based on the assumption that you start with 100 billion cells, or can you enter your starting cell number? I haven't seen any homebrewers validate those calculators with their own real world data.

Click to expand...

Not sure if that was a typo, but was your starter gravity 1.014? That would definitely explain the low cell count. Starter gravity should be between 1.030 and 1.040. I keep mine close to 1.040.

Starting cell count can be manipulated with both calculators. I used a starting cell count of 50 billion.

Oops that was a typo. I corrected the post. The OG was 1.040

Can you give me links to those calculators. The ones I've seen are pitch-rate calculators that tell you how many cells you need depending on the beer your making, but I can't find a calculator that gives you predictions of growth rates and numbers based on starting cell number and volume of culture. Thanks. Sorry if I'm missing something obvious.

Forkhead said:

Can you give me links to those calculators. The ones I've seen are pitch-rate calculators that tell you how many cells you need depending on the beer your making, but I can't find a calculator that gives you predictions of growth rates and numbers based on starting cell number and volume of culture. Thanks. Sorry if I'm missing something obvious.

Click to expand...

Well, actually, the calculators are not designed to be used this way, but it can be done. For Mr. Malty it isn't that bad. I'm pretty sure it assumes 100 billion cells at 100% viability so you have a range of 0-100 billion cells that you can start with by adjusting the % viability. Wyeast on the other hand is a bit more of a pain to use this way. You can start with either an activator pack (100 billion cells) or a propagator pack (25 billion cells). You can use more than 1 pack in the calculation so you can start with an unlimited range of cell counts between 25 billion to infinite, but they have to be in multiples of 25 or 100 billion cells. Here are the links.

http://www.mrmalty.com/calc/calc.html

http://www.wyeastlab.com/hb_pitchrate.cfm

I guess I should explain a little further.

To use Mr. Malty to predict cell counts: Set your % viability to your starting cell count and then adjust the OG of the beer until you get the appoximate starter volume that you plan to use. At this point, the pitching rate represents the predicted cell count.

For Wyeast, it's more strait forward, sort of. Set your starting cell count by selecting the appropriate number and type of packs, then enter your starter volume in gallons. For the second step, enter an arbitrary volume that makes the end volume in the fermenter equal to an even number of gallons. The final pitching rate is reported in million cells/ml so, multiply the end volume by 3785 ml and then multply that by the reported pitching rate to get your final cell count.

Thanks for the detailed explanation. I think I understand where you're getting your numbers from, but just to make sure I'm doing it correctly...

Using the Mr. Malty calculator, if I enter 1.223 for the OG, 5L, and 50% viability, stir plate, it tells me I need a 1.4L starter (which is what I did) and tells me I should have 181 billion cells from 1 vial. So my 115 billion is ~2/3 of 181 billion.

Using the Wyeast calculator, I choose Propagator (which assumes 25 billion @ 100% viability) and 2 packages, stir plate = yes, volume of starter = 0.37 gal (1.4L), it tells me my pitch rate should be 164.8 million/ml, which times 1.4L is 230 billion cells, and my 115 billion is ~1/2 of that.

This is my first starter where I've actually counted cells. Time will tell which one I believe more, my results, or the calculators. I'm sure it can vary depending on conditions between labs and homes. At least I'm not more than 2X off, I think I'm in the right ball park at least.

Yup, you did it right. You took slightly differnt routes than I did, but it makes no difference.

My yeast is growing really slowly. Anyone have any suggestions? I made my starter with 1.5L of bottled spring water (not distilled) + 150g light Munton's light DME from Rebel Brewer. I added 1/2 Tsp of Wyeast yeast nutrient, boiled x 20 min, cooled to room temp, oxygenated using BernzOmatic pure oxygen and a diffusing stone x 30sec. I warmed 1 vial of White Labs WLP001 to room temp for a few hours, then pitched it and kept in on a stir plate at room temp, loosely covered with aluminum foil.

I've been feeding every couple days by putting the starter in the fridge so the yeast will settle out, then decanting the wort and adding fresh wort. The starter always looks really active when I add fresh wort. I see plenty of bubbles and frothing like it's "awake". The specific gravity (the 1 time I measured it) went from 1.040 to 1.010, so the yeast is definitely active.

Is it possible that the yeast are going into fermentation mode instead of reproduction mode? I'm also worried that the quality of the DME isn't good, or my water chemistry is off. I'd really appreciate any advice

I'll be interested to see what folks have to say about this. I'm kind of at a loss as to why your growth rates are low. What temperature are you keeping the starter at? Are you adding oxygen every time you step it up? Are you using the same volume of wort for every step? What's your current cell count and how many times have you stepped it?

I started off keeping it at room temp (~72oF), then I moved it to 30oC (86oF) to speed up the growth. I don't think that matters much because I had poor growth at both temps. I oxygenate every batch of fresh wort before I add it. I've been adding between 1.2-1.5L of wort. I'm up to ~250 billion cells from the ~50 billion I started with, but I've had to feed the starter 3 times already. I'm averaging ~50 billion new cells per liter of wort, which is really low.

I should also mention that counting the cells has had a bit of a learning curve. I wasn't sure whether to count cells that look like they're about to divide as 1 or 2. It's difficult to tell a dividing cell from 2 cells clumped together. I'm also not sure that Trypan blue can clearly distinguish all the non-viable cells. While it's easy to see dead cells, I think the trypan might not really stain dying cells so well. I think I was over-counting on the hemocytometer because the CFU from 3 different plates has consistently given lower #s.

I'm thinking the problem is my DME or water. I'm gonna try feeding tonight with some wort made from dark DME and tap water.

Are you missing any key nutrients? A little yeast energizer/nutrient may be inexpensive insurance. Something with zinc and FAN. Even for starters, I'll use nutrients.

I'm using this yeast nutrient from Wyeast. http://www.rebelbrewer.com/shoppingcart/products/Wyeast-Beer-Nutrient-%2d-1.5oz.html

Their website says that's all that's needed for starters. Do you think I should be using something else?

Forkhead said:

I'm using this yeast nutrient from Wyeast. http://www.rebelbrewer.com/shoppingcart/products/Wyeast-Beer-Nutrient-%2d-1.5oz.html

Their website says that's all that's needed for starters. Do you think I should be using something else?

Click to expand...

No. I think yeast nutrient is pretty much all the same.

I just spoke with the microbiologist from Wyeast on the phone. Very helpful guy. He gave me some tips that I'll try to implement this weekend. He told me their pitch rate calculator is only for very rough estimates, and that actual numbers will vary a lot based on strain and equipment.

I'll post more details about his tips after I try them out.

So my problem was a lack of oxygen in my cultures. I used an aquarium pump to do continuous aeration in addition to infusing pure oxygen at the start, and I got really nice growth.

I started with 5 billion cells in 1L and they grew up to 70 billion in 12 hrs. I then removed the supernatant and added 1.2L fresh wort and they grew up to 200 billion in 12 hrs. I removed the sup. again and added 1.4L fresh wort and they grew up to 300 billion, which is 220 million/ml. I removed the sup. again and added 1.5L fresh wort, but they only grew up to 350 billion (230 million/ml). I'm pretty sure that's the concentration limit.

In doing some research, I found the "Current protocols in Molecular Biology: Yeast" and it says stationary phase occurs around 200e6/ml, which is what I'm seeing. I don't think it's possible to get much higher cell concentration than that. So if you want to pitch at 1 million/ml/degree Plato and your OG is 1.100 (which is on the pretty high end), you'd need 500 billion cells, and a 2.5L culture to get there.

Someone correct me if I'm wrong, but I think those pitch rate calculators are incorrect at pitch rates above 200 million/ml. Also, you need to have continuous aeration if you want to get cell growth, not just fermentation of your starter.

I now have enough yeast to test different freezing conditions, which I'll do this weekend. I'll post my results next week sometime.

Intersting. I like the continous aeration thing. Did you still use a stir plate, or did the air pump keep it agitated enough? I assume you used an air filter? What kind of stone? I think you're wrong about the calculators. They don't calculate anything over recommended pitching rates. Where are you getting 200 million cells per ml for a pitching rate? I just went to Mr. Malty, and to grow 500 billion cells with a stir plate, it recommends a 6.3 liter starter. That assumes that you are starting with 96 billion viable cells. That comes out to 80 million cells per ml if you hit your target cell count. That's no where close to maximum density. The pitching rates that it uses to determine how many cells you need are .75 million/ml/degrees plate for ales and 1.5 million for lagers.

The continuous aeration idea came from the guy at Wyeast that I spoke with. I used an aquarium pump with an inline 0.2 micron filter and no diffusing stone, just blowing bubbles into the wort. I don't know if an air stone would help or not, probably not if you're using a stir plate. I did use a stir plate, and I don't think the bubbles alone would've been enough to keep the cells agitated or promote good gas exchange. The theory behind using continuous aeration goes like this...

Oxygen is required for the cells to synthesize new cell wall components during division. Thus, no oxygen, no division. You'd think the stir plate would help get new oxygen dissolved in the culture, but since the cells are making so much CO2, you get positive pressure within the culture flask that doesn't allow atmospheric gas to get into the flask. So, the only way to get oxygen in there is to pump it in and drive out the CO2. The stir plate on its own is good for driving the CO2 out of solution and keeping the cells in suspension so they don't get too crowded. When you have air being pumped in, the stir plate will also facilitate good gas exchange and keep the culture oxygenated.

I haven't explored the Mr. Malty calculator much, I was basing all of my comments on the Wyeast calculator. For example, their calculator says if you start with 3 activator packs (300 billion cells) in 1L, you'll get 483 billion total cells. I don't think there's any way you'll get 300 billion cells per L (or 300 million/ml) to expand at all let alone generate 183 billion new cells. I think it's safe to assume you'll never get more than 200-250 million cells/ml for any culture under ideal conditions. A 6.3L culture to generat 500 billion cells seems like overkill to me, but might be necessary if you don't have a stir plate or continuous aeration. If you have ideal conditions and can get to 200 million/ml, you should only need a 2.5L culture. I'll be testing that hypothesis out in the future though.

I'm using a pitching rate of 1 million/ml/degree plato for ales, not 200 million. Sorry if that wasn't clear in my earlier post.

In my experience the maximal density you are suggesting is a good guideline for a vigorously shaken culture growing in a nutrient rich medium.
With vigorous shaking or aeration. I also think the pitching rates you are suggesting are in the correct ballpark.

I see. I've always wondered about the Wyeast calculator actually. It is a quite a bit different than Mr. Malty. It's easier to use when determining step ups, which is why I've preferred it. That and I figured they knew what they're doing. I guess if you want something done right you gotta do it yourself.

This calculator has fallen into favor for me.

http://yeastcalc.com/

pvtschultz said:

This calculator has fallen into favor for me.

http://yeastcalc.com/

Click to expand...

I've seen a lot of people mention that here lately. I'll have to check it out.

pvtschultz said:

This calculator has fallen into favor for me.

http://yeastcalc.com/

Click to expand...

Wow, thats quite nice. I like that it shows what growth factor it uses.

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.

SamBrewer said:

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.

Click to expand...

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.

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

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.

Any video recommendations for freezing yeast I need some visuals

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....?

Forkhead said:

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.

Click to expand...

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?

Forkhead said:

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.

Click to expand...

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

Sorry for the confusion, 15%, 30%, and 50% glycerol is the concentration before adding it to the cells. Once added to the cells it will get diluted by the volume of the cells. For me, it was 2-fold, so the final glycerol concentrations ended up being 7.5%, 15%, and 25%, respectively. Hope that makes sense.

I agree that storage at -80 will probably be better for viability than -20. I intend to test that ultimately though.

Forkhead said:

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.

Click to expand...

Nice treatment Forkhead. It seems that your results align pretty closely with mine although you did more conditions. I'm pleased to see that 15% final glycerol concentration was optimal at -80 by your assessment as well. I didn't go below that. I think the effect of lower glycerol at -20 is likely a consequence of the failure to freeze completely. I have been using the 15% concentration with slow freezing and rapid thawing for some time now and have found the viability to be good after many months (and probably years) at -80 C. Again, I haven't done long term tests at the higher temperatures. I always do a starter and have a rapid recovery and vigorous overnight cultures. I like your solution for slow freezing and may use it in the future since it is quite a bit easier than my stepwise approach. Finally, I am a bit surprised by the very low viability of the LN2 frozen cultures. We have used that approach at times for various stocks and have no problem growing them up on plates. Of course, we use a toothpick full and just expect to a fairly dense growth on plates which requires relatively few viable cells. But still, yours looks unexpectedly low.

Good work, useful information. I'll look forward to future reports.

Good to see there's some consensus Brewitt. I have much more experience with mammalian cells over yeast, but I anticipated that freezing extremely quickly in liquid nitrogen would be bad for the cells. We only do that with mammalian cells when we are going to purify DNA, RNA, or proteins from the cells so viability is not an issue. Anyway, I'm just glad we have a method that works well. Now on to troubleshooting other things.

Forkhead said:

Good to see there's some consensus Brewitt. I have much more experience with mammalian cells over yeast, but I anticipated that freezing extremely quickly in liquid nitrogen would be bad for the cells. We only do that with mammalian cells when we are going to purify DNA, RNA, or proteins from the cells so viability is not an issue. Anyway, I'm just glad we have a method that works well. Now on to troubleshooting other things.

Click to expand...

Agreed, fine to store mammalian cells in LN2 but not freeze them that way. As you say, I would expect quick freezing to be worse but didn't think it would be that bad. For regular yeast stocks we just freeze small aliquots at -80 directly and have no problem but we aren't looking for the level of viability that we are seeing in the slow freeze samples. Those cells that are viable remain so for decades. I presume the same is true for the large samples we are dealing with here. Mine are on the order of 10e11 cells which gives me enough for growth of as much as a gallon of overnight starter. In fact, I have a couple saison strains ready to aliquot and freeze away. Cheers.

Brewitt said:

Mine are on the order of 10e11 cells which gives me enough for growth of as much as a gallon of overnight starter. In fact, I have a couple saison strains ready to aliquot and freeze away. Cheers.

Click to expand...

What kind of tubes do you freeze that many cells in, and what volume are the aliquots?

Forkhead said:

What kind of tubes do you freeze that many cells in, and what volume are the aliquots?

Click to expand...

50 ml plastic screw top tubes. About 50% yeast slurry to 30% glycerol stock.

Sorry, but just so I'm clear. Do I understand you correctly that each 50ml tube is 1/2 full with yeast slurry, then you fill it up with 30% glycerol? So the final concentration of glycerol is actually 15%?

Interested in knowing what the optimal glycerine concentration would be too. I have some on hand (now) and have the yeast starter cold crashing (made from the part I reserved from a starter I pitched into a brew on Sunday). I'm hoping to fill at least 2-4 50ml tubes for freezing/storage. I might keep one in the fridge for use in another 2-4 weeks. The others will get frozen where my hops are kept. The freezer there is kept at its maximum cool setting and very rarely opened.

Golddiggie said:

Interested in knowing what the optimal glycerine concentration would be too. I have some on hand (now) and have the yeast starter cold crashing (made from the part I reserved from a starter I pitched into a brew on Sunday). I'm hoping to fill at least 2-4 50ml tubes for freezing/storage. I might keep one in the fridge for use in another 2-4 weeks. The others will get frozen where my hops are kept. The freezer there is kept at its maximum cool setting and very rarely opened.

Click to expand...

Well, I got the best results by adding a solution of 15% glycerol 1:1 with the yeast slurry so that the final conc. was 7.5% glycerol. Try to freeze the cells as slowly as possible. For example, put them in a plastic cooler filled with 70% isopropanol. Also, thaw them as rapidly as possible, preferably in a 37C (98.6F) water bath.

Forkhead said:

Well, I got the best results by adding a solution of 15% glycerol 1:1 with the yeast slurry so that the final conc. was 7.5% glycerol. Try to freeze the cells as slowly as possible. For example, put them in a styrofoam cooler filled with 70% isopropanol. Also, thaw them as rapidly as possible, preferably in a 37C (98.6F) water bath.

Click to expand...

No room in the freezer for a cooler, nor do I have a styrofoam cooler to use. The freezer is almost 100% full with bags of hops (it's my 10 cubic foot brew fridge, the freezer is on top)...

I do have a 100ml graduated cylinder. SO, I should be able to mark the 50% line (on the tubes the yeast will go into) and fill it with slurry to that level, then the rest with the 15% solution.