Not enough glycerine...frozen yeast bank

[KyleWolf]

Hey everyone,

So I have been working a bit on organizing a small yeast bank. I am storing these in 16mL glass vials at -20 with glycerine. Well apparently i didn't add enough glycerine to keep the solution from freezing.

What does everyone think I could expect as far as viable yeast counts go? Of course I plan on making a rather large starter, but I wanted to know if everyone body thinks I should just ditch em and forget about this batch.

 

[boo boo]

Adding the glycerine will prevent the cell walls from rupturing when it is frozen.
I made a frozen yeast bank a few years ago, and froze solid my yeast. It made
great beer when thawed and a starter was made out of it.

 

[Wellshooter]

Yep, the glycerine and yeast freeze solid. Bring it up to room temp and pitch in a starter and watch it take off. Takes longer than vial starter, usually about 48 hours, but it will build a healthy starter.

 

[peoplesbrewingcoop]

Sometimes you have to watch out how fast you freeze unicellular organisms. I have not froze yeast yet but I am a microbiologist that works on other eukaryotic unicellular organisms.

The idea of storing organisms in a freezer is to slow down the metabolic pathways. The problem with freezing living things is that ice crystals can form and rupture the cell. Things like glycerol and DMSO allow ice crystals not to form as much as only using pure water.

While these help reduce ice crystals, they do not eliminate them. One problem could be the speed of freezing, two, the amount being frozen at a time, and three, the final freezing temperature.

To address the first problem I suggest freezing the vials you are storing very slowly. When I freeze my organisms I put mix the cells in a cryoprotectant and then put them in this container called a Mr. Frosty. It is ridiculously expensive for what it is. Basically it is a plastic container that surrounds the vials to be frozen. Inside the plastic container you put in isopropyl alcohol to allow the box to slowly freeze. This helps reduce the formation of ice crystals. Maybe get some kind of rack to hold the vials and then put them in a container filled with isopropyl alcohol. Let it sit in the freezer for 1-3 hours and then take out to freeze normally.

For the second potential problem, I suggest buying the right kind of cryotubes (not class or weak plastic). Get the ones that are not too large. You want to freeze the smallest volume that is economically feasible. I would freeze in 0.5 to 2-mL cryotubes.

Three: The final temperature you are freezing could also play a role. Usually the lower the better. I will look through some methods of long term yeast storage to find out what is common to use. Things like bacteria are stored at -80 degrees Celsius in a glycerol mixture. Also, if you freezer's temperature is fluctuating as can occur for defrosting home freezers then your yeast may die or be seriously affected. The idea for freezing is to stop metabolic activity in the cells without killing them. If you thaw the vials and then refreeze then it is possible (if you have not killed them ) that you are selecting for strains that may not make the best beer.

 

[KyleWolf]

Peoplesbrewingcoop. Hey there, I am also a microbiologist here in birmingham (go figure), I don't work with many frozen eukaryotes , but those I do work with are normally in liquid nitrogen. And those are cell lines not yeast. However, I do understand the concept. I was just hoping I added enough glycerine to keep the majority of solution from freezing down. I wish I had DMSO on hand, though I don't know how that would affect my starter/pitching/etc.

I have had to deal with something like a Mr. Frosty a few times. neat idea if not a neat price tag I agree. What I did to control the cooling was to put the vials into a Styrofoam box (use to be a dry ice box) and let it sit in the fridge with a hand towel wrapped around them. After approx. 24hrs at 4C, I transferred them to -20 (I say -20 but I mean my freezer). I used an ice pack in the styrofoam box and put the lid on. once the temp settled, it shouldn't change much with the ice pack there.

I am currently using 16mL glass vials, they are rated to work well in anything except liquid nitrogen. They are small enough to be practical and hold about as much yeast as 3 bottle conditioned beers (My efficiency on this will go up as I work out the kinks in the protocol).

My main concern was I know at -20, a majority of my labs cultures do not thoroughly freeze. As I never worked with yeast, I worried over their cell wall integrity. But it sounds as if my worry was unfounded. back to culturing more yeast. I wonder what my girlfriend would think of me wanting to install a culture hood in my house...

 

[BioBeing]

As long as you are above about 10% glycerol then you should be OK. If you want them to remain liquid even when frozen, you'd need to have about 50% glycerol.

 

[peoplesbrewingcoop]

I got into home brewing so I could have fun with microbiology at work and at home. I have already turned my kitchen into a laboratory, a hood sounds like a great idea. haha

 

[JohnMc]

peoplesbrewingcoop,
check out Fred Sherman's "Getting Started with Yeast"
http://dbb.urmc.rochester.edu/labs/sherman_f/StartedYeast.html

I personally use 25% glycerol and store at -80 deg C in cryovials. I've had stuff that's still viable frozen since 1991.

PS: I never worry about freezing time; I stick 1.5 mL in the cryovial and stick it in the -80

 

[KyleWolf]

JohnMc, did you find a smaller -80 that could be reasonably used in a home? or do you have a -80 in the lab and store your yeast there?

 

[JohnMc]

Kyle,
I have been fortunate enough to have -80 access in the lab. I can't imagine paying for a home -80, both the initial cost of the freezer itself and the electricity.
I streak onto YPD or malt agar plates and bring the little beasts home happily wrapped in Parafilm.

 

[KyleWolf]

very nice. I don't know what my PI would think of that. Of course, I can always bribe her with beer...Her husband use to homebrew so maybe she has a soft spot.

I have to agree the price tag on keeping a -80 at home is high. Didn't know if you knew something i didn't about a miniature -80 lol. but man, that would be tight.

Oh, and everyone looking for cheap kimax and high quality glassware in, or out of the lab... check out www.cynmar.com its on the cheap for good stuff.
*disclaimer, I am in no way affiliated with cynmar. just a happy customer*

 

[JohnMc]

KyleWolf, on a lark, cause by your post, I looked at a used science equipment site. A used 1 cubic foot benchtop -80 was US$5,000, while they had a few normal size ones for about US$3,000
I can only hope the benchtop one uses less electricity
(www.labx.com, btw)

 

[KyleWolf]

wow...you would figure smaller=cheaper...but I guess that only works down to a certain volume (flasks also seem to be in the same boat). The one piece of equipment that always surprises me on their high price tag are stir plates. Microscopes, -80s, all that I can understand...but a stir plate being $200...always gets me.

 

[musick]

I would have never guessed so many biologists were on this forum!

Add another to the mix...nerd alert ahead.....



RE: peoplesbrewingcoop - Great points made! More food for thought - my understanding is that a slow freeze causes larger crystals to grow and rupture walls as compared to a fast freeze. Techniques and data I have picked up in the lab have shown that harvesting yeast mid-log and adding 12-14% glycerol (For homebrewers, anything between 10% and 50% will keep the yeast alive...12-14% is 'optimal' in my lab experience...of course YMMV) before instant freezing in LN2 and storage at -80C results in higher survivability as compared to samples placed directly to -80C from RT.


RE: JohnMc - "Only the Strong Survive"

In my brewing/professional adventures, I have hypothesized that a more minimal media (0.1-0.5X MA or YPD, for example) allows only the most robust yeast cells to first form a colony from a streak plate. I have always used a minimal media to select stout growers from agar and used that to inoculate my starter (5mls to 50mls to 500mls to 1L).

Again, just another viewpoint. Not trying to dismiss others findings/opinions.

 

[JohnMc]

musick,
I don't worry about "strong growers", the only thing I'd worry about is petites, and even then I haven't really done anything about it. If the starter is malt, the yeast that grow will be the ones that can grow on malt, more or less. Also, super minimal media can cause sporulation, which is another way of saying sexual reproduction. While most brewing strains don't sporulate well, they still can and the "kids" will be different (not that there aren't mutants anyway).
BTW, to select against petites (non-function of mitochondria), grow on a non-fermentable carbon source. YEPG works, where the "G" is glycerol.

 

[jah777]

I have been trying to build up a frozen yeast bank but have become a bit discouraged and concerned now that I am trying to actually use my frozen yeast. I have added about 30% glycerol to each vial and shaken to prevent the formation of ice crystals, but from the image of the vial below, it appears that crystals still formed after freezing.

Question 1: Is there hope for this vial or should I toss it?

A couple of days ago, I took one frozen vial out of the freezer and place it in the fridge to begin thawing. After 24 hrs I pulled it out of the fridge and let it sit at room temperature for about 4 hours before adding it to a starter. The other attached image shows the starter on a stir plate about 12 hours after pitching yeast from my frozen bank, and to the right there is another starter I made at the same time with yeast from a fresh vial from the local brew store. The starter with the fresh vial and no stir plate has taken off, whereas the starter with yeast from the frozen bank looks like it isn't doing anything.

Question 2: Does the lack of activity on the left mean that the yeast from my frozen bank is all dead or does it just take longer to get going?

If there is still no activity tonight (after 24 hours on the plate), I will probably pitch a fresh vial into the starter on the stir plate so I can still brew tomorrow night as planned.