Isolating bugs/brett from dregs

[butterpants]

Quick question..... I've got some Avery, Crooked Stave and Jester King sours that I was thinking of trying to isolate the lacto, pedio and brett from for later pitching to a solera I'm doing, for complexity.

The media I have handy are WLN, WLD, UBA and Maconkey.

UBA of course would grow everything but it might be hard to tell what's what. On WLN I could see what was producing lactic acid but there could be a lot of overall colony types because yeast isn't inhibited. WLD would keep the saccharomyces from growing but I have no experience with harvesting off media that contains cyclohexamide, if there are any concerns.... Just wondering the best to use.

0.25mL spreaded per plate or streak with a loop for the inital plating?

 

[trentm]

Quick question..... I've got some Avery, Crooked Stave and Jester King sours that I was thinking of trying to isolate the lacto, pedio and brett from for later pitching to a solera I'm doing, for complexity.

The media I have handy are WLN, WLD, UBA and Maconkey.

UBA of course would grow everything but it might be hard to tell what's what. On WLN I could see what was producing lactic acid but there could be a lot of overall colony types because yeast isn't inhibited. WLD would keep the saccharomyces from growing but I have no experience with harvesting off media that contains cyclohexamide, if there are any concerns.... Just wondering the best to use.

0.25mL spreaded per plate or streak with a loop for the inital plating?

I have a simple method for isolating Brett from Saccharomyces without selective media but don't know about the bacteria. Hope to hear advice from others on isolating bacteria. If the beer contains Saccharomyces (say from bottling or primary) the Sacc will dominate the culture. Try starting a small culture in liquid wort. I use 15 ml centrifuge tubes with ~5 ml wort and 2 ml dregs. Allow the sacc to complete the initial fermentation. Give it at least a week. After completion the sacc will settle to the bottom of the tube and the Brett will now be the dominant organism in the liquid. Now transfer 4 or 5 loop-fulls of the liquid (without disturbing the sacc) to a wort agar plate and dilution streak. There will still be some sacc colonies grow and these will be visible after a day or two but should be few and not dominate the plate. Most Brett strains are slow growers so the Brett will typically be visible after 3-4 days. As soon as the small colonies are large enough to see, select a few individual colonies to transfer to clean plates by dilution streaking to ensure pure colonies. These will likely be Brett but you can proof them by starting a new culture from a pure colony. The aroma, taste and growth characteristics in wort will confirm* you have what you are looking for.

*Confirm may be too strong of a word. We know one of the largest liquid yeast companies was marketing a yeast strain as Brett until molecular analysis proved it was Sacc. However, if it walks like a duck, quacks like a duck and acts like a duck...odds are, it's a duck.

 

[ColoHox]

Trentm's suggestion would be a good approach for general bulk growth. It sounds like you want isolation though, so I would recommend dilutions and isolation streaks.

This could be a big project if you want ID and full isolation. Dilutions will be important to get nicely isolated colonies on plates. Working with the supplies you have on hand:

WLN media might allow you to differentiate between Brett and Sacch, depending on the strain of Sacch present. On the green media, Brett will take up the dye and metabolize it, resulting in clear/creamy white colonies. Sacch (again depending on the strain), cannot metabolize the dye and usually grow green. Bacteria will grow on this media though.

WLD (with cyclo) will inhibit Sacch, but not Brett. So on WLD you will have bacteria and Brett. Based on colony morphology, you could then streak obviously different colonies onto new plates. You will need to incubate a set of plates aerobically and anaerobically (there are ways to do this with a jar and a candle), to promote Lacto and Pedio growth over aerobic species (like Acetobacter and yeast). No issues with subculturing bacteria grown on a cyclo plate.

This project probably means multiple rounds of subculturing, if you are using performance on different media for identification. Do you have access to a microscope? That would improve your chances dramatically. Based on colony morphology alone, yeasts will be larger, dull/cloudy, creamy white. Bacterial colonies will be smaller, more circular, and slimy looking.

An easy way to get some more information from your cultures, once you have good isolation, is with a catalase test. If you get colonies to grow that you suspect could be Lacto or Pedio, make a small smear of the colony on a slide or small piece of glass. Place a drop of hydrogen peroxide on the smear and check for bubbles. In general, aerobic organisms (Brett, Sacch, Acetobacter) will produce bubbles, Lacto and Pedio will not.

Good luck.

 

[trentm]

Trentm's suggestion would be a good approach for general bulk growth.

ColoHox, Perhaps you missed the point of the suggestion described in post #2. This is a simple method to isolate Brettanomyces from a beer that contains both Brett and Saccharomyces without using selective media. It does not allow for any type of ID. However, for brewing purposes, ID in not required (my opinion). It's sort of like planting a seed of unknown origin when upon harvest you have a fruit that has the appearance, aroma and flavor of a tomato. You can safely use that fruit as if it were a tomato.

 

[ColoHox]

ColoHox, Perhaps you missed the point of the suggestion described in post #2. This is a simple method to isolate Brettanomyces from a beer that contains both Brett and Saccharomyces without using selective media. It does not allow for any type of ID. However, for brewing purposes, ID in not required (my opinion). It's sort of like planting a seed of unknown origin when upon harvest you have a fruit that has the appearance, aroma and flavor of a tomato. You can safely use that fruit as if it were a tomato.

Thanks for clarifying. Nice analogy, but what if from the beginning you wanted a zucchini and not a tomato? Despite your opinion, ID of the bugs one is using is a big deal, and relatively easy (with the right approach).

The point of the OP was to isolate and identify the bugs in the mixed culture using selective media.

 

[butterpants]

I do have a stereo and compound light microscopes. I can gram and acid fast stain. I can test for catalase.

I do think some type of rudimentary isolation and identification is necessary for my purposes. Being able to slant and catalog would be great for repeatable results later on if the solera beer comes out as planned.

I realize that true identification is beyond the scope of my capabilities but the genus difference between Sac, Brett, Pedio, Lacto and **** bugs not going in my beer would be nice.

 

[ColoHox]

I do have a stereo and compound light microscopes. I can gram and acid fast stain. I can test for catalase.

I do think some type of rudimentary isolation and identification is necessary for my purposes. Being able to slant and catalog would be great for repeatable results later on if the solera beer comes out as planned.

I realize that true identification is beyond the scope of my capabilities but the genus difference between Sac, Brett, Pedio, Lacto and **** bugs not going in my beer would be nice.

You're in business then. Without molecular ID, the use of differential media morphology, some biochemistry (catalase), and microscopy is as good as you can get.

Here is a flowchart for ID of bacteria in the brewery.

 

[butterpants]

OH nice. Thanks for that

 

[butterpants]

What are you guys using for a candle jar? Everything I look at doesn't have a mouth big enough for a 100mm plate to fit in...

You don't think the residual 8% or so O2 in acandle jar will be an issue? Those gas packs are expensive....

 

[spenghali]

You might be able to use a desiccator with a candle, though I am not sure if the seal would be air tight. They are usually used to remove moisture from a system using silica. They have large openings.

https://www.google.com/search?q=des...=dessicator&tbm=shop&spd=16104620673133430293

 

[spenghali]

Also, it might be possible to obtain isolation from streaking on WLD, then separating based on acid production, as LAB will have much higher acid production. Or mounting individual colonies on slides for morphology/size comparison. Brett will obviously be much bigger than bacteria.

 

[butterpants]

You might be able to use a desiccator with a candle, though I am not sure if the seal would be air tight. They are usually used to remove moisture from a system using silica. They have large openings.

https://www.google.com/search?q=des...=dessicator&tbm=shop&spd=16104620673133430293

You'd think they would be air tight...

Those are good ideas man, thank you.

 

[spenghali]

Yea they should be nearly airtight. Whenever I open one it takes some effort and has a good vacuum just from having some silica in the bottom.

 

[butterpants]

WLD mixed (unknown) culture 1:100 dilution 100ul spread plated. Incubated 7 days @ 37C aerobically.

White, Creamy, Dull = Brett
Green, Large =??

Shouldn't be sacc right cuz this has cyclohexamide but it seems too large to be bacteria. All seem to be making acid, hard to tell.

I have yet to subculture or view under the stereo microscope.

View attachment 1432423529694.jpg

 

[butterpants]

According to Yacobsons Brett Project a limited number of strains will not metabolize the bromo green.

 

[butterpants]

Subculture round 1

View attachment 1433217633368.jpg

 

[dantheman13]

Not sure if any of this is useful for you (Bryan Heit's identification video might be): http://www.milkthefunk.com/wiki/Wild_Yeast_Isolation

 

[ColoHox]

Hey @butterpants, how did this work out? Any update?

 

[butterpants]

Thanks for checking in on me.

I recently obtained some vessels to do anaerobic/microaerophilic growth in so I'm most likely going to start with another round of subculture and utilize them. I've got a solid amount of Brett isolated. The LAB, well not so much.

What's the general consensus on brom green inhibiting gram positive bacteria growth? I've come across this in some literature and seeing that I'm using WLD to isolate lacto/pedio it concerns me.

I'm following some of my beers to NHC this week so and need to pour a few dozen plates when I get back so the project is on hold for the moment.

 

[ColoHox]

Brom green is often incorporated into commercially prepared media for LAB.

I know that it can inhibit some mammalian enzymes, but I haven't seen anything about it being inhibitory to LAB.

 

[butterpants]

Hmm ok. Wish I remembered where that comment was but it definitely was a brewing related article.

I have a few plates where the morphology doesn't scream brett but I haven't had a chance to stain them to look for sure. Maybe I did grab some