Maintaining A Healthy Yeast Bank Long Term

[rnm410]

Excellent write-up, easy to follow, I will definitely freeze some cultures now, but one question.
Why not just glycerin and freeze straight from the smack pack or WL vial?
Looking at 1 month old liquid yeast you would have 80 billion cells in 125 mL (Wyeast example). You could fill 10 vials at 8 billion cells a piece with 12.5 mL of yeast and 12.5 mL of glycerin.
For the same 1 month old liquid yeast, your method suggest inoculating at a rate of 80 mil/mL yielding a growth rate of about 1.75. You would end up with about 140 billion cells and depending on density about 100 mL of slurry. Divided into 10 individual vials you would have 14 billion cells in 10 mL of slurry, not much different from the above method, or straight from the pack.
Using the above example for 1 month old yeast, either method, 1 vial into 1 L would lead to an inoculation rate of about 10 mil/mL and a resulting growth rate of about 4. Ending cell count would be 40 billion, not enough to brew a typical beer over 1 gallon. It also seems that a multi step starter would be required when growing from 10 billion to 100 to 200+ billion cells.
At growth rates 2-3 i.e. inoculation rates 25-50 mil/mL, pitching 1 thawed frozen vial by the example above would grow from 10 billion to 30 bil. via a 400 mL starter, stepped to 90 bil. via 1500 mL starter and finally 180 bil. via 1800 mL starter. Respective growth rates 3, 3 and 2.
This is my understanding, do you have any input or insight for me/us?

 

[PoppinCaps]

Negative or positive pressure is definitely not needed for doing this work, though access to that would significantly decrease the chance of any contamination. Positive pressure would be pretty easy, just put a high efficiency filter in front of a box fan blowing down into a plexiglass box with one side open to work in. But if you're careful to not keep any liquids uncovered for any long amount of time, that's usually good enough.
Viability is fun to do to get really accurate counts at stages of growth, but as I saw, if you standardize your procedure well, you'll get the same counts time after time, then it's just sort of a waste of time. So I did do it for a while to understand the max densities and growth rates of different strains (which vary widely, take the calculators with a grain of salt). Your terminology is correct though, you only need a hemacytometer and decent 400x total magnificantion. There are a few threads looking at cheap scopes on HBT.
I doubt the home freeze drying machine would work, but if you try it, report back! Active dried yeast is a fairly controlled dehydration process, more than simply freeze drying. Dried yeast are protected somewhat granularly by debris that retains the inner cells...you know, I'll be the first to say I don't know enough about it to even discuss. I will say I traded yeast with some folks on here by taking a drop and letting it dry on paper, wrapping it up and sending it by letter. Viability is shot to hell, but with a plate, you can select strong colonies. So maybe the freeze drier would work....

 

[PoppinCaps]

I think the problem is making the 2L starter from 27B cells only gets 2x more. Let me give an example of what I typically find for cell counts in my starters on a stir plate.
Start with 100-150B cells in a smackpack or vial, add to 1L starter, and you might get 1.5x, maybe less. I find that an average max density of my stirred starters is around 200 million viable cells/ml. So that 1L now has 200B cells. Split that into 10 tubes, 20B each. Take that 20B tube, and add it to a 2L starter and grow. The final density doesn't change with a 1:20 dilution and similar OG, conditions, etc. It will usually still be 200 million/ml. So your 2L starter now has 400B cells. I think in terms of final densities given standard starters, as that is what is important. If you make them happy, they will keep giving you that max density.

 

[PoppinCaps]

Yep, I put it into a cup of slightly warm water, and try to thaw it as fast as possible without a lot of heat. Just swirl it, etc, and try to get it to roughly the same temp as the starter as not to shock it. Yeah, the extra 5 ml is for expansion, or if I didn't eyeball the volume of the decant good enough, and all the tubes have 6ml instead of 5ml, so 12ml total with the glycerin.

 

[PoppinCaps]

1) Totally, you could definitely just freeze the smackpack or WL vial to a final 12% glycerin and be done. The starter just ensures they were viable to begin with.
2) It's totally possible to grow a 10million/ml inoculation rate in a 1L starter into 200B cells. Every strain is different, and has different final density preferences, some 250million/ml and some 90 million/ml, all assuming the same gravity wort, because cell number is determined to a point by grams of extract, not starter size. These are ideal conditions though, with a stirplate and good oxygenation, healthy starter population. But it's typically what I see. Where did you get the growth rate of 4, as a multiple? If the growth rate is a population doubling, then 10B->20B->40B->80B->160B (4 generations). If you use this calculator (http://www.brewersfriend.com/yeast-pitch-rate-and-starter-calculator/), it's pretty close to what I usually see, but more of an average. I see 50% more or less than those final cell counts depending on the strain.
This is very much a YMMV sort of situation, and depends greatly on who is working with the cells, type of extract, temp, stirplate, etc differences. I don't do stepped starters because I saw that I didn't need them, unless I am doing a gigantic high gravity lager. a 1L or 2L is all I need for 5gal batches (disregarding barleywines). I think the key is keeping the inoculation rate above 10M/ml in a standard starter, any lower and they tend to take a hit.

 

[rnm410]

Jamil and Chris Whites yeast book.
Extracted and posted here:
http://beersmith.com/blog/2011/01/10/yeast-starters-for-home-brewing-beer-part-2/

 

[rnm410]

They also suggest inoculation rates of 50-70 mil/mL as ideal. But empirical and "what works for us" are two different subjects.

 

[neovox]

Great read. Thank you for taking the time to write this up.

 

[PoppinCaps]

Agreed, we're not commercial, and it's very hard to standardize one rule of thumb for all strains, for all beers. Even so, I generally go with the standard commercial pitching rule of 0.75M/ml/deg plato for an ale and 1.5M/ml/deg plato for lagers (that's 10-20mil/ml for a 1.050 beer. That would be an inoculation of 9B cells in a 1000L starter for an 1.050 ale or 18B for the same lager (matching inculation rates for the beer, gets them used to that growth cycle).
Again, I wrote this all around doing your own ranching, not using smack packs into a starter, which the BeerSmith link refers to. In those cases, yeah a growth rate of 1-2 is all you'll likely see. The inoculation rates that I use are more like making a batch of beer, which results in a heck of a lot more healthy cells than 4x. My problem with the link is the statement that "No matter how large a single starter is we are not going to get a growth factor greater than 6.0." That's just too general, and misleading. He also at the end suggests that for a 1.050 ale, 80B cells from a smack pack will be needed to add to a 3.5L starter to get 177B cells. Does anyone making a lager use 2 smackpacks in 7L? The method I show here will get you 200B+ cells in a 1L starter, with 9 more just like it from one smackpack. Ridiculously sized starters just empirically are not necessary to get commercial grade pitching rates. Again, this is for ranching your own cultures, not starting from (and paying for) smack packs, and maybe that's the difference that I should have added to the article.
Thanks for the discussion; these are heated topics, and always lead to some disagreement! The important thing is that people, who want to, understand how these numbers are calculated, and some data from homebrewers like us to confirm these calculators should always be part of the discussion.

 

[Tomcat0304]

These look like great instructions. I'm interested in the freezing method and have a few questions:
1. What should I look for in a pressure cooker? I will need to buy one, but I would like to also use it for canning, cooking, and kitchen use. The electric ones look nice, but seem like they are not useful for much else.
2. If I get a larger size for general kitchen use, when making the glycerin solution, can I sterilize it in a smaller flask inside the pressure cooker? I would think it would be less wasteful than making a larger batch of it.
Thanks again for the write up, this method seems practical and achievable now.

 

[sdenue57]

I have been using the "harvesting from the starter" method for the past year or so, thanks brulosophy. I really only use Conan, since I rarely brew anything other than pale ale or IPAs, and I love the character it brings to the brews. I use 4 oz jelly jars, so it's a perfect size. It doesn't use up much of my starter, and when I'm ready to brew again, I just pull out a jar and make a new starter with it. Then I repeat the cycle.

 

[kadozen]

Very nice. Looks like I might start a frozen yeast bank, this write up really demystified it.
But the important question I have from this article: Where do we get a vial/smack pack of that amber bee yeast?

 

[PoppinCaps]

Any size will do, but get one that you can put a few mason jars in. And when I say pressure cook it, I mean in glass jars that you can then seal shut once the cook is done. Sorry if I made it seem like the cooker was full of solution, that's not what you want to do. I use a cheap aluminum one for the stove, my wife has the electric one

 

[PoppinCaps]

Yeah, I don't think this guy is down with spreading the love. From an article on him and Fossil Fuels Brewing Co, "His only worry is that the unfiltered nature of this beer means that some of his yeast will invariably settle to the bottom of the glass or bottle, and an unscrupulous brewer could collect that and use it in another beer. The microbiologist has applied for a patent on his strains and has sequenced the genomes so he can tell if someone else has stolen it. "I am the keeper of the family jewels," Cano says."
Yikes. Here is the patent: http://www.google.com/patents/WO2010042896A1?cl=en

 

[PoppinCaps]

It's all about standardizing your own protocol, and it looks like it works great for you! I've got some Conan in the freezer that I need to bring out for a brew, haven't had a chance to try it. I need to see what everyone is raving about...

 

[dfborn]

What size pressure cooker do you recommend for this?

 

[hartlesj]

Do these methods work for Lactobacillius? Also, I'm making a cider with Nottingham. How would that hold up to these methods. Washing Nottingham from a cider then storing it? I know Nottingham is inexpensive, it's more about availability for me. Banking yeast means I don't have to wait, or more importantly pay for overseas shipping rates.

 

[neovox]

What size are the tubes you're showing in the picture?

 

[PoppinCaps]

I use a small one that holds 4 quart size mason jars, so maybe it's 8qt? Plenty big for doing this kind of stuff.

 

[PoppinCaps]

Yep, bacteria will do fine with all of these procedures. Nottingham will work fine as well. If you're banking to save some money on shipping, I would make a master batch right from the package and freeze. After a run through a stressful batch (most ciders/high gravity due to lacking nutrients), the yeast may not be as pure or viable. If you're already got the cider going, take a sample after racking and make a starter, tasting and smelling, making sure they're still healthy.

 

[PoppinCaps]

15ml

 

[wyowolf]

Thank you for this, I found some sterile tubes but wondering is there a certain type of glycerin needed for this? or any will do?
So you make 100 mL of the solution glycerin to water, sterilize it by Pressure cooker, then split into the tubes? 30% gly to 70% water ratio? then add your yeast after its all cooled down?

 

[PoppinCaps]

Any USP glycerin from the drug store will work. I would make a stock of the glycerin/water solution in a mason jar so you always have it on hand, or just put it into the tubes after it's cooled a bit, either works. You can do 30% glycerin or 25% glycerin and water (typo in the protocol), they both will work. Which ever way you do it, you'll want a final glycerin concentration of 12.5-15% in the frozen slurry, which is why you add 5ml each of the glycerin stock and slurry to the tube before freezing.

 

[KnightDesign]

This is a brilliant article! I'm now in the process of upgrading my yeast growing and storage systems. Thanks a bunch!

 

[DICKERBEER]

Nice article! Working with cells I always was curious how it translates to homebrewing. I usually use DMSO as a cyro-preservative in lab instead of glycerol. My question is though that we usually spin down the cells and decant/aspirate the supernatant to remove the cryo-preservative prior to seeding. Obviously centrifuges are not a common household item and would be costly for the average homebrewer. Your instructions just have pitching the thawed tube straight into the starter, my question is if there is any worry in the glycerol affecting the flavor or anything of the beer? I guess for a 5 gal batch you will be at a 1:3800 dilution but just curious

 

[PoppinCaps]

Even though DMSO would work as well, definitely not the right application here, and in case anyone else is reading this, DON'T USE DMSO!
But as far as glycerin, I'm not too concerned. If you take that 5ml of 25% glycerin (1.25ml pure glycerin), and dump it all in the 1000ml starter, you would have a 1:800 dilution. If you then dump that whole starter in the 5 gal beer (19000ml), it would be a 1:15,000 dilution. And if you first decanted the starter for pitching and retained 50ml of slurry, it would be around 1:300,000 dilution. At this dilution, I wouldn't have a problem drinking a FDA appointed GRAS component, and I doubt anyone would detect any flavor addition to the beer.

 

[DICKERBEER]

OK cool I will definitely try this. One more question, what kind of isopropanol box are you using for 15 mL tubes? I only know of the Mr Frosty boxes for the 1-2 mL cryovials

 

[PoppinCaps]

I just use a mason jar and fill the alcohol to the level of the liquid in the tubes. The number of tubes and volume of alcohol don't seem to affect the viability noticeably. Some day I'll actually track the temperature drop in the tube to see how far off I am from the 1degC/min.

 

[wyowolf]

Ok, I looked for some tubes that were sterile, I saw the ones you posted up above... but 300 seems like a lot.. I did find some but they were 50ml I guess thats probably too big?
So you make the 70/30 solution in a mason jar, in pressure cooker, and you seal it and use it as needed? or everytime you make a new set of yeast banks?

 

[PoppinCaps]

Here are some more, smaller pack: http://amzn.com/B00ES3TIRG
I make a 300ml stock solution of 25-30% glycerin in a pint size jar, pressure cook and just use it as needed until empty. It's enough for 6 batches (60 tubes).