Long term yeast storage: agar vs. water

[spareparts]

Thanks for the mineral oil suggestion. I can see how that would help and I never heard of it before (or never paid attention for whatever reason)

Does mineral oil need to be sterilized?

 

[JohnMc]

Does mineral oil need to be sterilized?

Good question, yes, it does. You can buy it sterile or sterilize it in the oven. It can be sterilized by dry heat, 1-2 hours at 350 deg F in your oven.

 

[chicagobrew]

You're best bet for long term storage is freezing with a cell permeable freezing agent, such as glycerol (15%). You can also use a non-permeable agent such as 1M sorbitol for shorter periods. Ideally you want to store at -80C, but -20C in a non-defrost freezer is sufficient for many years.

I have no doubt you may get viable yeast after many years in distilled water, but odds are you are bottlenecking for a population of yeast that does not resemble your original population.

Professor,

I'll concede that -80C storage is the best option here for long-term storage if you have access to the equipment. I don't want to argue this point. But, I don't have access to this equipment, so I'm considering sterile water storage.

I'm familiar with the concept of bottlenecking and the affects it can have on populations (B.S. in microbiology), but would appreciate a little more explanation on your thinking.

Consider this situation: you get a yeast strain and prepare a master sterile water sample that goes into long-term, archival storage. You also prepare a working sterile water sample(s) from which you culture and prepare starters. Then, every year or so you use the master sample to rebuild the working sample(s).

I realize that the viability of the master sample will degrade over time and there may be occasions (I'm not exactly sure how long the master will stay viable, but let's say 5 years) that you have to create a culture from the master and then rebuild a new master. This could create a bottleneck that could alter the yeast and some testing would be in order, but I wouldn't expect to see a huge drift in the strain between these, essentially, two generations. I guess what I'm getting at is, assuming you rebuild the master every 5 years, over a 20 year brewing career your final yeast strain would only be the 4th generation and any genetic drift would be minimal.

Any thoughts?

Aaron

 

[Wolfy]

Long term storage of yeast needs to not only keep the yeast viable (which is what most information here and elsewhere focuses on) but also ensure the yeast does not mutate and change. Colder temperatures mean less cell mutation, change and genetic drift, if you don't have -80C, then a standard -20C freezer is the next best thing. Using a 50% glycerin mix means that the samples stay liquid and do not actually freeze solid at -20, so it should be easier to maintain them even with freeze/thaw cycles in a normal freezer.

 

[JohnMc]

I guess what I'm getting at is, assuming you rebuild the master every 5 years, over a 20 year brewing career your final yeast strain would only be the 4th generation and any genetic drift would be minimal.

Plus, if one kept good notes and made a few submasters each time, tested them, and discarded any off types and kept a good one, it would probably be even more minimal. Unless one selected for drift by taste preference.

Using a 50% glycerin mix means that the samples stay liquid and do not actually freeze solid at -20, so it should be easier to maintain them even with freeze/thaw cycles in a normal freezer.

that jives with my vague memory on -20C storage, though I've this little feeling there's a caveat that I can't remember. Oh well, I'm on the down side of middle age, so maybe I have an excuse.

 

[COLObrewer]

. . . . .that jives with my vague memory on -20C storage, though I've this little feeling there's a caveat that I can't remember. Oh well, I'm on the down side of middle age, so maybe I have an excuse.

I've read somewhere that glycerin is toxic to yeast but I think it's minimized as long as the freeze/thaw times are kept short and dilution is sought imediately after thaw?

But then the reason to use glycerin is so that the solution is kept in a "liquid" state, so I'm confused on that point if glycerin is toxic to yeast.

But then there are those that say to refrigerate the yeast/water/glycerin samples for two days prior to freezing to stimulate the production of trehalose in the yeast cells (which protects them from the cold better)? So I'm confused on the quick freeze time, etc . . . . . .

I'm just a big ball of confusion and am leaning toward sterile water storage in the fridge? . . . . Because I'm lazy. Does sterile mineral oil keep yeast from sporulating?

 

[chicagobrew]

Wolfy,

You make an important distinction between viability and mutation. I think we've been muddling the waters by mixing these two in the discussion. Let's concede that freezing yields the longest anticipated viability, and that sterile water probably yields viability for as long as we need as homebrewers. I mean, we're not preserving strains for decades here. We're just trying to save yeast at home so we can reduce the cost of brewing.

If we, temporarily at least, leave the viability discussion let's talk about mutation. Are there any microbiologists in the house that can provide some background on the topic? How do yeast mutate and how do you think the different storage methods would affect the mutation rate? What sort of rate would we expect to see?

It's been a long time since I took microbiology, so my understanding of the topic is poor at best, but I always understood mutation to occur during reproduction. In other words, we don't observe mutation until genes are passed to successive generations. If we store a sample, either frozen or under sterile water, we are attempting to prevent reproduction and would not expect to see mutation? How far off base am I here? Can we see mutation within the same generation?

 

[DrawTap88]

Science Daily

The article linked to above makes it seem that the mutuation takes place in the DNA of the cells when they split. It also provides a very key piece of information as to why the mutations cause bad beer: "the weak survive." So, if we take that at face value, we still have a good deal of perfect yeast. They're just mixed in with just as much bad (mutated) yeast. At least that's how I deciphered it.

<--not a microbiologist (but I did stay at a Holiday Inn last night).

 

[ni*]

Most mutations don't happen at any one particular point, although certain sorts (ie, non-homologous recombinations) only occur doing division.

That experiment was designed to create an environment free of selection so that all mutations would be visible. This allowed "weak" (whatever that is supposed to mean) yeast to live. Agar slants, etc, are definitely not absent selection pressure, although it's almost entirely irrelevant to the question at hand.

I have big doubts that mutation actually plays a serious role in yeast strain deterioration as seen by most homebrewerers. Genetic drift might play a bigger role, but in homogenous populations (as nearly any yeast strain will be) it, too, is likely pretty minor.

In cases where strain deterioration occurs (and while it undoubtedly does, I think it is talked about far more often than it is observed) it is, I am fairly certain, almost solely because of contamination.

"Mutation" is treated by homebrewers as a sort of boogeyman, in a way no one in biology would take seriously. It really probably isn't a problem.

 

[Randar]

Not sure who is still left reading this thread but let me go down the line..

I've read somewhere that glycerin is toxic to yeast but I think it's minimized as long as the freeze/thaw times are kept short and dilution is sought imediately after thaw?

I think the definition of toxic is required here. Concentration is always important in this discussion and the extremely wide usage of glycerin in storage at either -20 or -80 is a viable data point that contradicts the assertion of being toxic. These methods generally result in glycerin being 15-25% concentration of the final solution.

But then the reason to use glycerin is so that the solution is kept in a "liquid" state, so I'm confused on that point if glycerin is toxic to yeast.

Not totally true. In -20 storage it will not freeze, but at -80 it does in fact freeze. The glycerin is meant to prevent the yeast cells themselves from rupturing due to the expansion of the water molecules (remember that water expands when frozen, glycerol does not). So even though the -80 stocks are "frozen", the 25% concentration of glycerin prevents enough of the cells from being compromised that the original culture is recoverable.

But then there are those that say to refrigerate the yeast/water/glycerin samples for two days prior to freezing to stimulate the production of trehalose in the yeast cells (which protects them from the cold better)? So I'm confused on the quick freeze time, etc . . . . . .

You can do this before you collect the yeast into the storage tubes. Jamil/White mention to put yeast in fridge for 48 hrs prior to storing. Lab protocol is to freeze in liquid nitrogen and/or dump straight into the -80. There is no tempering of the solution. Again, if you have a sterile storage of the yeast (a WL vial or WYeast smack pack that has been smacked, grown, then chilled for 48 hrs) there is no need for resting it in the fridge after creating the storage solution.

I'm just a big ball of confusion and am leaning toward sterile water storage in the fridge? . . . . Because I'm lazy. Does sterile mineral oil keep yeast from sporulating?

Storing at -20 is REALLY REALLY easy. Can you get yourself all confused? Sure, but when it comes down to it, there are only a couple of basic steps and a much lower chance of sanitation being compromised compared to liquid/fridge storage techniques (never mind all the other drawbacks to storing at room temp or fridge temps)

 

[JohnMc]

In cases where strain deterioration occurs (and while it undoubtedly does, I think it is talked about far more often than it is observed) it is, I am fairly certain, almost solely because of contamination.

10-4 on that.
I will say I *think* I've seen some strains throw petites (something wrong with the mitochondria), but I've been too lazy to check them on the right media (0.1% glucose and 2% glycerol, IIRC).
Basically, RDWHAHB.

 

[ni*]

Mitochondrial ejection, specifically. But yes, a petite phenotype is normally induced by dodgy media.

 

[bobz]

How long does a homebrewer need to store yeast. Sterile distilled water will get you < 2 years depending on your procedure. I pressure cook distilled water in 8ml vials and pipette 2-3 ml in each vial every time I smack a pack or open a liquid culture. stores at room temp. 400x is the recommended microscope power.
I do 6 vials every time. -------bobz

 

[JohnMc]

Mitochondrial ejection, specifically. But yes, a petite phenotype is normally induced by dodgy media.

Actually, petites can simply be respiration deficient and the mutations can be nuclear or mitochondrial mutations; the mitochondria can still be present, though generally funked up.

How long does a homebrewer need to store yeast.

That depends. If it's to save money and master the process, not long really. If it's to really preserve strains, then longer. I've had MeV Stout in storage since '92 or '93. MeV went out of business in either the late 80's or early 90's. It's not really needful to have it, good stouts can be made with other yeast. It is nice to have, though, because it's very distinct. All of the Brewtek strains would also be nice to have. I think they went under in 2004 or so.
You can get most of them from the ATCC, but they're $275 each, IIRC. I didn't order them at that price...