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Culturing Yeast

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Asherweef

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Hi all,

I know a lot of members seem to use yeast from previous or commercial beers in their brewing but Im a bit confused how you go about it. Can someone really Janet and John it for me? Do you need a stirring plate? How long does it last? whats the process of keeping and storing?

If you assume I’m a simpleton you wouldn’t be far wrong so really break it down please :laugh8:
 

Galena

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Edit: I didn't read your OP properly and thought this was a general, how to cultivate yeast question, so I ramble on about how to make from a starter, something you probably already know, so please do ignore.

Well, you can cultivate from bottom cropping, top cropping or the way i prefer is to overbuild my starter. So the following is the way I do it.

So, take a pack of liquid yeast like White Labs for instance, this has approximately 100billion cells of yeast.
Then you need a yeast starter calculator, I use Homebrew Dads Yeast Starter calculator that can be downloaded if you google it its an excel spreadsheet.

Supposing you are making a 21 Litre batch of Ale, you input all the details into the calculator and it will tell you what cell count you need for that batch.
So lets say hypothetically you need 157 billion cells, but in your pack there are only 100 billion, what to do?
We make a starter, so in a flask we boil 1 litre of water with 100 grams of DME which sanitises and gives us a S.G. of around 1.040, we add the yeast when cooled and it will build up to around 157 Bn cells which we can then pitch to our beer.

Of course then we have used up all of our yeast and will need to buy a new pack, unless in advance we overbuild our starter.

So what we do is add an overbuild of 100bn cells into our calculator, which means we need 257 Bn cells total.

To do this the calculator tells us to boil 1.5 litres of water and 150gms DME and add the same pack of yeast, a stirrer plate is better but not essential as you can shake it regularly to oxygenate but the calculator will allow for this if you input no stirrer.
You add the same pack of yeast, put on the stirrer and leave for 12-18 hours and then turn off the stirrer, this allows the yeast to prepare glycogen stores as it goes dormant, so I leave it 48 hours in total.
Then after all has gone quiet and the yeast has settled out, shake it all up and decant 0.5 litre into a sanitised Kilkner Jar to the brim and seal, put this in the fridge and it will last a few weeks until you need to make another starter.
The remaining 1 litre in the jar you put in the fridge to let it settle out again, then decant off most of the beer and pitch your yeast.

This video is the method I use to make a starter, then overbuild as above if you want to keep your culture going. Hope that make sense, I like it because it is clean, little risk of contamination if you are hygienic and no messing around washing yeast cropped from the FV, you can keep this going for many generations.

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RichardM

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To re-use yeast from a commercial beer;
Buy a bottle or two of your favourite bottled conditioned beer, for example Proper Job
Let the bottles stand for a day and then pour carefully so as not to disturb the sediment in the bottom. Leave a little beer in the bottom. Swill this around and then pour in to a sanitised jam jar. Repeat with the second bottle.
Now build a one litre starter with the contents of the jar.
 

foxy

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Re using yeast from commercial beer isn't necessarily the fermenting yeast some brewers use a different yeast for bottle conditioning. Article from BYO.

For the benefit of readers who are not up on these methods, I will briefly cover the basics of how a brewer with a basic home microbiology lab can propagate yeast from a bottle of beer. We need an example for this question and I am going to take the easy route and select a bottle of Sierra Nevada Pale Ale.

The first decision that we need to make is do we propagate by simply collecting and growing the yeast solids from the bottom of the bottle, or do we do a little clean up work. I selected a bottle of Sierra Pale Ale because if we really wanted to take the easy route, starting with what is on the bottom of this bottle will most likely work. Why? Because Sierra Nevada Pale Ale is conditioned with the same strain used for fermentation and because their quality control measures are extraordinary. But with all the respect brewers have for Sierra Nevada, simply propping the “dregs” from the bottom of a bottle is pretty crude. The purpose of propagation is to grow cells and whatever ends up in the prop is probably going to grow. So what is a brewer to do? Make some media and streak some plates!

. . . streaking provides a much better shot at growing what we want versus bringing in a bunch of riff raff into the propagation party.
Yeah, this is sounding pretty advanced but it’s really pretty simple. Making your own media at home is just a few steps away from making Jell-O! Mix up an all-purpose media such as Wallerstein Lab Nutrient (WLN), pressure cook in media bottles for 20 minutes at 15 psig (1 barg) pressure, allow to cool to about 86 ˚F (30 ˚C), and pour the media into pre-sterilized Petri dishes. Once the plates have cooled and solidified they can be used to streak out the dregs from the bottom of the bottle. This is one of the things we do at BYO Boot Camp in a conference room. Fun times!

Why do we want to plate the sediment from the bottom? Because by streaking this stuff onto plates, we can select a single colony-forming unit (CFU in microbiology speak) and propagate that one colony. Each CFU is assumed to represent a single yeast cell and proper dilutions and streaking methods help to produce plates with clear separation among colonies. Suffice to say, streaking plates provides a much better shot at growing what we want versus bringing in a bunch of riff raff into the propagation party.
OK, now what? The textbook methods you reference usually start with transferring a single colony from a plate into about 25 mL of sterilized wort. In about 48 hours, the contents of the propagation flask (usually an Erlenmeyer flask that is about twice the volume of the contents) is increased to 10X. This means that 25 mL of propagating yeast slurry is added to 225 mL of sterile wort in a 500 mL Erlenmeyer flask to yield a 250 mL volume. This method continues until there is enough yeast to pitch into wort. Yes, the propagation volume represents a significant (10%) volume and the color and flavor of the propagation wort is important to your finished beer. But that is really a separate question for another deep dive into what-about-this-wort-ism.

In order to isolate a yeast or bacterial strain, streaking plates is the best way to make sure you are not bringing along unwanted guests. Photo courtesy of Omega Yeast
Now that the basic method has been established, let’s go back and explore some possible shortcuts because this process is somewhat of a pain in the neck (this is when I get to thank the yeast sponsors who generously donate to Team Mr. Wizard . . . thank you yeast sponsors, I am still waiting for those cool rugby shirts I suggested on Facebook). Idea #1 is to skip the intermediate steps and add the starter cells, either a single CFU from a plate or a shot of dregs from a beer bottle, to a volume that is ~1⁄10 the fermenter full volume (remember this is not 10% of the wort volume, but 10% of the wort + yeast volume). The real challenge to this method is transferring the starter cells into the sterile wort without bringing other organisms to the party. The idea with pure culture technique, which is what this is about, is growing cells from a single cell line, and the reality of the situation is that it is nearly impossible to transfer yeast into a sterile environment, i.e., the wort in your Erlenmeyer flask, without introducing other organisms.
So why do the textbook methods suggest incrementally increasing the volume? It’s all about competition, hurdles, and speed. While this may sound like track and field, it’s really about putting yeast into an environment where they will outcompete other critters by quickly reducing pH, gobbling up nutrients, and producing alcohol, which are all hurdles that impede the growth of other organisms that snuck past the bouncer at the door (you). As long as the selected yeast population is the super majority, non-selected bacteria and yeast strains will most likely fare poorly in this environment. “Most likely fare poorly” is not an absolute and yeast labs that do this thing day in and day out have stringent quality methods to ensure that the bouncers were effective.

Circling back on your question, if you put a single CFU or shot of dregs into your 3-L (3-qt.) starter, a lot of time passes before significant pH reduction, nutrient depletion, and alcohol formation occurs. This time represents the period between bar closing and sunrise when all sorts of chaos occurs. Yeast labs do not want this sort of craziness happening . . . ever . . . and have found that incremental increases in propagation volume work pretty darn well.

A corollary to this “hurdles theory” that is so prevalent in the practical application of food microbiology, is selection. Our bottle of Sierra Nevada Pale Ale is probably clean and growing the yeast sediment from one of these bottles will probably turn out just fine. But what if you were going to grow the sediment from a great beer brewed in a garage brewery? The beer is great, we just established that, but great beer can contain bacteria that don’t cause product damage. Let’s call these bottles asymptomatic spreaders. You bring the dregs from an asymptomatic spreader into your propagation flask, encourage cell growth, and what you have is a messed-
up starter culture. This can happen with or without incremental changes in volume, and is much more likely to occur when using dregs as the cell source versus a single CFU.

Back to your original question. Can I just pitch the dregs into a 3-L starter at 1.035–1.040 that is well fed, oxygenated, and on a stir plate? This is a really great question and I sincerely hope that my brief explanation has explained why the answer is “yes, but not recommended.”

Response by Ashton Lewis.
 

BlackIsland

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When I've used commercial yeast from a bottle, I just boil up a 200ml of a 1.030 to 1.040 wort, decant the bottle, add the cooled wort, stuff in an airlock (I used to just use foil, but fruit flies...), and swirl it around every once in a while for a few days. When I want to use it, I pitch it into a 2-3 liter wort, swirl for a couple days, then chill, decant, and pitch.
 

Asherweef

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Some great replies here, thanks guys. I feel like I’m going to be the guy with all the gear and no idea and might pick Up a flask and stirring plate.

I guess doing this can radically cheapen brews?
 

BlackIsland

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Some great replies here, thanks guys. I feel like I’m going to be the guy with all the gear and no idea and might pick Up a flask and stirring plate.

I guess doing this can radically cheapen brews?
Of course! Instead of culturing from bottles, I grow up a liquid pack and take cultures from them, freezing them. As long as I don't accidentally use my last vial, I don't have to buy the strains I keep.
 

Asherweef

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Any rules/advice on freezing? I did look into some of the liquid yeast packs but couldn’t justify the cost but, I guess this is the way around that.
 

foxy

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Any rules/advice on freezing? I did look into some of the liquid yeast packs but couldn’t justify the cost but, I guess this is the way around that.
Just freezing the yeast in distilled water or wort will kill them, there are plenty of articles from home brewers using glycerin to prevent the cell walls from rupturing.
Here is a BYO article.
Yeast Ranching - Brew Your Own
 

JohnB

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If I were to make one suggestion on this point: "Cleanliness is next to Godliness" Sterilise the sh 1 t out of everything you touch! Here is a clean sample from yesterday - I also dumped 5 plates that looked, well, wrong. Each sphere about the size of a pin head has been grown on from a single cell.

yeast-plate-23-09-2020_sm.jpg


I repeat sterilise then sterilise again. Have a quick hunt round on the Internet for using aseptic techniques. You don't need to do plate streaking or owt like that, but if you understand a bit about aseptic working then you can't go far wrong with other stuff you do.

Regarding freezing - don't, simple advice, don't. You can store yeast at room temp in small vials of distilled water if you use sterile techniques - the yeast goes dormant. One of those little balls of yeast up there in 4mL of sterile water in a sealed vial will be quite happy for 6 months and possibly much longer.
 

The-Engineer-That-Brews

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I thought this video about making yeast slants was really good and well worth a watch. I've bought all the stuff to do it but haven't had a go yet.

One of the school mums who's an ex Roche microbiologist watched the vid and shuddered a bit: she echoed the comments above re the need for extreme attention to sterilisation - 'all horrors known to man will grow on agar'

 

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Any rules/advice on freezing? I did look into some of the liquid yeast packs but couldn’t justify the cost but, I guess this is the way around that.
I freeze my cultures in a 24ml vial, made up of 8ml of a 30% sterile glycerine solution, and 8ml of yeast slurry. When I want to use it, I put it into 200ml of 1.030-40 wort for 1-2 days, then step that up to a 2-3 liter starter for 1-2 days, then chill, decant and pitch.
 

JohnB

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@BlackIsland Hi there, David. I'm really interested in freezing yeast, but I had problems when bringing the stuff out of storage. Can you tell me (us), please how you go about this. The reason I suggested above not to freeze is simple, I'll explain: This was washed yeast from a previous pale ale 12.6 Brix brew and viability was about 97% taken from the primary. I took half a dozen vials and mixed distilled water with glycerine at 70%/30% ratio. Sterilised for 15 minutes. Once down to room temp each vial had 4mL of solution and each vial then had 1mL of yeast slurry added. I then chilled this for 24 hours in a domestic fridge about +4 DegC. then placed in a polystyrene box for insulation. This went into a domestic freezer at -11 DegC. After 3 months I took one vial out placed in the refrigerator again to warm up to +4 then after 24 hours up to room temp. I had a viability of 70% which I admit is a workable solution, but still not good. I'm not sure if it was the freezing process, the storage time, or the thawing out, but somewhere I lost viability. I'd hate to store for 6 months (or more) and find dead yeast. Any tips you can pass on, please? This is something that interests me greatly because I'd like to store yeast long term. Currently I use sterile, inert water and store around 80,000 cells/mL in 4mL vials and at 3 months viability is 90%(+-2%). No good for large volumes of cells because I end up with hundreds of tubes!
Regards, John.
 

The-Engineer-That-Brews

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I don't know if this is relevant, but I keep sourdough yeast in the freezer; and although it definitely looses viability, the extent doesn't seem to depend on how long it's been in the freezer,,,
 

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I have cultured yeast from Westmalle, St.-Bernardus and Rochefort. You do not really need a stir plate (it might work better), I work from the bottle first with 1 ml of wort, then 10 ml if it stops fermenting, then up to 100 ml. Then I cold crash, remove the liquid and scale up to about 1l, from which I always try to make a couple of bottles of beer. And the next step is to scale up to brewing 5 litre of light beer, in order to get a big scale up. The yeast from that I collect, and then scale bits of it to brew bigger beers (in volume and/or alcohol). I don't reuse the yeast from that then.

It is not fast, but I can do it with simple things: test tubes, jars, simple glass work.
 

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@BlackIsland Hi there, David. I'm really interested in freezing yeast, but I had problems when bringing the stuff out of storage. Can you tell me (us), please how you go about this.
You bet! When I want to save a strain, I start by overbuilding a starter. I grab my already prepared and sterilized (I use a pressure canner) 24ml vials filled to the 1/3 mark with a 30% glycerin solution. When I'm ready to harvest yeast, I decant the spent wort off the starter so that I have as high of a concentration of yeast in the slurry as I can get. I lay out a sheet of aluminum foil, put a lit alcohol lamp in the middle, and use a sanitized glass wine thief to dip out the slurry and fill each vial to the 2/3 mark. I cap them, put parafilm around the neck, and put them in the fridge for 24 hours. Then I shake them up and put them into a styrofoam case, along with some ice packs. I don't know how cold the freezer is, but the styrofoam and ice packs protects against the freezer's frost free cycles.

When I'm ready to use them, I sanitize my flask, add 200ish ml of sterile 1.030-040 starter wort I've canned, add the barely defrosted vial to it, and put it on the stir plate for 24-48 hours. Then I add 2-3 liters of sterile canned starter wort and give it another 24-48 hours.

All of these amounts and times are variable, because I don't check for viability or cell count, I just use visual cues to tell when it's done. For low gravity ales I use 2 liter starters, for lagers and higher gravity ales I use three. The only times this has failed for me is when I've tried to rush the process and pitch the yeast before it was ready. And although I haven't had a problem yet, I expect I may run into problems after multiple generations of doing this, especially on yeast blends.

I suspect genuine yeast experts will cringe at my process, but I get the results I'm happy with, have picked up a few medals along the way, like the beer, and, since I'm often skint, save money!
 

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Thanks for all this info, I'm going to give this a go. Problem with the way I do it is the step-up, remember these are vials of just a few mL of sterile water with tiny amounts of yeast and they take ages to get up to pitching quantities and I ain't always got the time to do 4 or 5 or even 6 steps.
I suspect genuine yeast experts will cringe at my process, but I get the results I'm happy with
If it works why try to fix it? I was struggling with this method and you have solved it. Straight forward instructions, David, thanks. I'll give this a go with the next batch of yeast I'll be separating out in about 3 days time. athumb..
 

peebee

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Good grief. I bet @Asherweef wasn't expecting all this when he posed his question?

Well, I really need to add my OTT response to the thread (I'm feeling left out). You also need one of these? (Not really).
Yeast counting for the bored.
I'd actually been considering one along with one of these kiddie toys:
Kiddies microscope.
At the moment sanity has the better of me and I won't be getting one. But for how long? (Me and sanity don't stay together very well).
 

BlackIsland

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If it works why try to fix it? I was struggling with this method and you have solved it. Straight forward instructions, David, thanks. I'll give this a go with the next batch of yeast I'll be separating out in about 3 days time. athumb..
This week I found it worked better than I expected. I was making a starter for a Sunday brew day, and I was distracted, and instead of pitching my vial into the 200ml starter, I dumped it into the 2L starter, skipping the intermediate step. After 48 hours I have just as much yeast slurry as I would get normally. I can't speak to the vitality yet, though.
 
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