Yeast and cell culturing

[dennisdk2000]

After reading Yeast: The Practical Guide to Beer Fermentation I'd like to try my hand at freezing yeast. The protocol involves YPD broth, glycerin and ascorbic acid. I've had a quick look at these - and they are all pretty expensive! So my question is, does anyone have experience with sort of hard-core cell culturing? Does anyone know where to get the where-with-all without paying a fortune? Also, what sort of grade reagents should I be looking for? Solutions etc can be home-sterilized, so that should be okay, but any other requirements?

Dennis

 

[MacKiwi]

Timely thread - I've just started reading this, and suspect I'll be trying my hand at freezing pretty soon too. So can't help, but thought I'd register my interest in any answers people my have!

 

[dennisdk2000]

Time for a BUMP! :grin:

Dennis

 

[Devonhomebrew]

just get 2L of glycerine from ebay i think i paid 15 quid but it does lots of culture

 

[orlando]

I have done quite a bit of yeast culturing and have now settled on slanting as my storage method. Freezing seemed to me to be quite complicated and only properly carried out at temperatures I was unable to achieve. Although it takes a little more effort reslanting a yeast after 3 or 4 months just seems easier than freezing. I can see that if you are keen on rarely released yeasts it can be attractive but for what I want slanting is good enough. Have to say I admire you for trying this method and will be intrigued by the results. Keep posting about the results and method used.

 

[graysalchemy]

Freezing is easy.

You mix glycerine with boiled cooled water in a 50/50 mix then you add this to your yeast slurry in a 50/50 ratio give it a good shake and freeze it in a cold freezer. Simples.

I use 50ml centrifuge tubes. The glycerine you can buy from boots next to the cough mixture here, cheap enough.

Now Evanvine has assured me that he has just been freezing yeast slurry in 250ml PET bottles and doesn't even make a starter though he does acknowledge that fermentation may take 36 hrs to get going.

 

[dennisdk2000]

Thanks for all the replies! :thumb:

I have to say I the freezing process doesn't appear all that complicated to me, but there is so much conflicting information - as usual! I could do with doing side by side comparisons of methods and brew with the resulting yeasts and repeat a few times. :hmm:

I understand that the YPD is for teh yeast to prepare themselves to be frozen and improve viability and vitality, and the ascorbic acid is to protect the yeast cells from oxidation whilst frozen at -20°C. It might well be that this is over-complicating things.

graysalchemy - ever had any problems with viability?

orlando - what is your slanting/re-slanting protocol? I decided to look at freezing because it seems less skill is needed than for slanting!

Dennis

 

[mike77]

I have thought about freezing yeast before but never went anywhere with it. I think I'll give Grays method a go as it sounds pretty straight forward. The practical guide may be best practice based on industry techniques. But it sounds like people are having good results using easier methods. If you make a starter first then you'll know if you are pitching viable yeast or not.

 

[graysalchemy]

Yes there will be loss of viability and no doubt mutations, but I think that for my needs it is acceptable. :thumb:

 

[orlando]

Freezing might appear easy but there are some requirements for it to work well that I decided meant it wasn't worth it.

Yeast has to be in near perfect condition with large reserves of glycogen & trehalose. You really need a non frost-free freezer as they have a warming cycle to help prevent frost build up, something capable of -20 is a good compromise to the usual requirement for highly successful outcome of -80. A really fast freeze will improve viability but most domestic freezers don't really do this. Packing the vials in styrofoam to help keep temperatures stable improves storage life. There is a high risk of killing the yeast when it thaws as the relatively slow freeze is not conducive to yeast surviving it. Of course you will also need the glycerol and all the rigmarole too.

Slanting is a lot simpler and easier to perform with a very high survival rate of the yeast cells. Looking forward to your results.

 

[mike77]

So when I first read up on it a while back I think I came to the same conclusion that it was too complicated and may not work. I saw the post several days ago that Grays referred to, in which one poster (Evanvine I think) just freezes slurry in pet bottles with no problems. So now I'm seeing some evidence that in the real world it can be done without all the complication.

A fast freeze could possibly be achieved using my ice cream maker (which I don't use). Sounds stupid but the only time I used it it froze the ice cream in seconds which was too fast.

I'll try the 50/50 glycerine and I'll make a starter so that I know I have viable yeast first before pitching. If the yeast isn't viable then I've lost nothing and I'll just pitch a packet of dried yeast or something.

 

[orlando]

I saw the post several days ago that Grays referred to, in which one poster (Evanvine I think) just freezes slurry in pet bottles with no problems. So now I'm seeing some evidence that in the real world it can be done without all the complication.

I wonder whether that works because the slurry offers some more protection to the yeast. Chris White has this to say, When freezing as "the medium around the yeast freezes there is less and less liquid water at the cell surface, which creates an osmotic gradient. This pulls water out of the cell by osmosis and kills the cell." The other factor is that freezing that much slurry he is hedging against massive cell death by sheer numbers. There is much more of a problem if you are trying to freeze tiny numbers in a small vial.

 

[dennisdk2000]

Some interesting points to consider.

I saw the post several days ago that Grays referred to, in which one poster (Evanvine I think) just freezes slurry in pet bottles with no problems. So now I'm seeing some evidence that in the real world it can be done without all the complication.

I wonder whether that works because the slurry offers some more protection to the yeast. Chris White has this to say, When freezing as "the medium around the yeast freezes there is less and less liquid water at the cell surface, which creates an osmotic gradient. This pulls water out of the cell by osmosis and kills the cell." The other factor is that freezing that much slurry he is hedging against massive cell death by sheer numbers. There is much more of a problem if you are trying to freeze tiny numbers in a small vial.

I see the point about the osmotic gradient, but with a small volume it might be possible to freeze the yeast quickly enough that this isn't s great problem. Also, if using YPD medium mixed with glycerine, it might provide some protection for the cells...

I still think I'll give it a go, but I need to wait until I have a bit more time.

Dennis

 

[ibrewthereforeiam]

In all my years of brewing, I have never thought about freezing yeast. I know people do it but I'm not sure that is an avenue I would take. To my advantage I brew every 2 weeks, so I just acid wash my yeast. Freezing kinda scares me haha

 

[dennisdk2000]

ibrew - if I brewed frequently enough, I wouldn't bother with freezing (or slanting) either. Unfortunately, I brew infrequently, so I can't keep yeast rolling over. Also, I like many different styles of beer, and as yeast makes a major contribution to the flavour profile, so keeping multiple strains rolling over would be a gigantic challenge!

Yeast has to be in near perfect condition with large reserves of glycogen & trehalose...

This seems to be the reason for culturing the yeast in YPD prior to freezing. It is fairly expensive to buy, though it goes a long way.

Ascorbic acid is used in the protocol for storing at -20°C to minimise the oxidation of the cell membranes, but it isn't used for freezing at -80°C, presuambly because only a negligible amount of oxidation takes place at such low temperatures.

I was considering freezing with glycerine and ascorbic acid - thought the extra protection couldn't hurt - but tempted to try glycerine alone in a parallel experiment... :hmm: I intend to store small vials or centrifuge tubes, so a quick freeze isn't beyond possibilities, but as this limits the number of cells frozen, I need to keep as many cells alive as possible!

For how long have people successfully kept yeast frozen with glycerine alone?

orlando - could you please share the details of your slanting/re-slanting protocol? I decided to look at freezing because it seems less skill is needed than for slanting! However, I might have another look at slanting.

I much appreciate all feed-back! :thumb: It's interesting to see how others do things - as we all have different requirements etc. there is ultimately many ways of doing things, and thus much to be learnt from others!

Dennis

 

[brewtim]

I have just started freezing yeast in a 30/30/30 split glycerin/yeast/beer.

This is what I'm doing:

Create a 2 litre starter for a brewday (as normal). Cool in the fridge overnight to get a compact yeast slurry.
Take 2 x 125ml urine sample pots plastic and starsan sanitise them.
Turkey baste 30ml of slurry into each pot add Glycerine and beer (from starter).
Shake thoroughly and bung in the freezer.
Use remainder of starter in brewday beer.

From the 2 frozen yeast pots I should be able to step up to a 2 litre starter and repeat the process.

I've no idea if this will work but it's worth a try and it's pretty easy to do, I'll report back from future brews.

 

[orlando]

orlando - could you please share the details of your slanting/re-slanting protocol? I decided to look at freezing because it seems less skill is needed than for slanting! However, I might have another look at slanting.

I much appreciate all feed-back! :thumb: It's interesting to see how others do things - as we all have different requirements etc. there is ultimately many ways of doing things, and thus much to be learnt from others!

Dennis

Hi Dennis, if you follow this link a few posts down it goes into what I did. Any other questions fire away.

 

[Lucy]

I used to work as a scientist in cell line development, part of this was laying down cell line banks for long term storage.

One of the chemicals that we used as a cryoprotectant was DMSO, Dimethyl sulfoxide, It prevents the crystalization of water, this is an issue during the freeze thaw process and can lead to permeation of the cell membrane.
I used to use it at 7.5% of the total volume of the freeze mix, also you will want to have a high cell viability on freeze and to have your cells in exponential phase.

It is important to thaw the cells quickly too, a 'waterbath' read: cup of hot water around 37c will be sufficient.

To help with slow freezing we used to have the tubes in a tub with a chamber of isopropanol (not sure where you can get that).

I will have a look online for a yeast freezing protocol, i'm sure something could be adapted for home use if you remove the centrifugation steps.

 

[Lucy]

just quickly googled and found this, I'm guessing nobody had liquid nitrogen or a -80c freezer at home so will have to experiment with a -20 and see how it works.


Freezing yeast
Materials:

yeast culture

DMSO

glycerol

water
Protocol:
1. Take 5 ml yeast cells from the Erlenmeyer of a freshly grown yeast culture, put in a 15
ml tube
2. Spin for 5 minutes at 4600 RPM
3. Discard supernatant without disturbing the pellet
4. Add half-volume of sterile water and resuspend the pellet
5. Spin for 5 minutes at 4600 RPM
6. Discard supernatant without disturbing the pellet
7. Add 450 µl ‘freezing mix’ and resuspend the pellet. (freezing mix: 5% v/v glycerol, 10%
v/v DMSO in water)
8. Freeze slowly, keep in -30 °C for at least 24 hours.
9. Transfer to a -80 °C freezer

 

[orlando]

just quickly googled and found this, I'm guessing nobody had liquid nitrogen or a -80c freezer at home so will have to experiment with a -20 and see how it works.


Freezing yeast
Materials:

yeast culture

DMSO

glycerol

water
Protocol:
1. Take 5 ml yeast cells from the Erlenmeyer of a freshly grown yeast culture, put in a 15
ml tube
2. Spin for 5 minutes at 4600 RPM
3. Discard supernatant without disturbing the pellet
4. Add half-volume of sterile water and resuspend the pellet
5. Spin for 5 minutes at 4600 RPM
6. Discard supernatant without disturbing the pellet
7. Add 450 µl ‘freezing mix’ and resuspend the pellet. (freezing mix: 5% v/v glycerol, 10%
v/v DMSO in water)
8. Freeze slowly, keep in -30 °C for at least 24 hours.
9. Transfer to a -80 °C freezer

Yeah right. "Darling what I really need is a centrifuge and a -80c freezer, would that be OK?" :whistle: :lol: