Last week Mike the Mad Fermentationist gave me a sample of his spontaneously-fermented DCambic. I had just made some brilliant green agar plates, from BKyeast’s excellent post on media. This media is antibiotic, killing most types of bacteria while allowing yeast to grow. My first thought was to plate some sort of lambic, hopefully being able to isolate the yeasts present. I figured what better lambic than a local one, and Mike was kindly willing to give me a sample.
For this plating I did my first serial dilution. I had previously only done streaking using a needle, whereas this involves placing a small amount of dilute liquid and spreading it around the whole plate. I diluted it 1:1000. I picked that factor somewhat randomly. I was planning on doing at least 10^6 as I had read somewhere, but since I was starting with a clear beer sample and not the dregs, I figured it wouldn’t need anywhere as much dilution. I had previously prepared vials with 9mL of distilled water, so I just had to pick three of these vials. I added a small amount to a brilliant green agar plate and a simple malt extract agar plate, and spread it around with a cell spreader (“hockey stick”). At the same time I also added about 5mL to 50mL of sterile wort to see what would grow in liquid media.
I did this on Saturday, and as of today, Tuesday, there is nothing growing yet. The plates are clean, and the liquid sample is still clear. I am afraid I made a dumb move and tried to do this after the sample had settled in the fridge, without shaking it up. If nothing grows soon I will try plating again. I already added another 5mL of the shaken-up sample to the liquid. Hopefully there will be some growth there soon. In any case, I will definitely post updates as this goes on. Who knows, maybe I’ll be able to isolate an interesting Brett from it. I would also love to isolate the saccharomyces that did most of the fermenting, though there might not be any more in suspension at this point.
DC Yeast Lab 
Hey man! Doing well I see.
Don’t be too disappointed if nothing grows. If you look at my Cantillon yeast isolation, you’ll see that I also didn’t get anything with diluted dregs. Only when I plated undiluted dregs did I get a few colonies (like literally 8 or something like that). So give it a couple more weeks and if nothing appears try plating neat and see what happens.
Hi there,
I am very happy for you mastering the plating technique. Well done! I am very excited what kind of yeasts will appear on the plates. I totally agree on bkyeast’s opinion, give the plates more time. It just takes time. I never plated dregs of a beer directly on a plate. I always did a kind of starter first and then plated the liquids from these starters. And even then, it took nearly three weeks until yeast colonies appeared. Good luck with your yeast hunting
Cheers, Samuel
Thanks guys! I will have to leave it be for a little while, then. Hopefully I will get something growing in the liquid sooner, though, so I can at least have some clue what might be in there. I actually had a chance to taste the beer a few months ago and recall it being very much like the “real deal,” though I don’t remember it being particularly sour. Certainly a good beer, though. I think Mike made the same comment about it.
I’m seriously thinking about getting a microscope this summer. I am moving soon and just have to make sure I will have the room for it. If I get new strains from this experiment I will definitely be itching for a microscope even more so that I can have some idea what bugs I got.
I am very interested in doing something similar to what you are trying so I’ve been following you and the commenters closely. I am trying to source supplies right now and wonder if you would mind sharing how you sourced everything for the brilliant green agar plates?
I bought the brilliant green solution on eBay. Search for “brilliant green” — there’s some listed right now. I use agar flakes from Whole Foods. The other ingredients I used you can get from a homebrew shop — Wyeast nutrient (to replace the yeast extract from boullion cubes), lactose and dextrose, or just use table sugar for the latter. It seems to work, but Saccharomyces grew very slowly. I would imagine that’d have to do with the low amount of sucrose, and hopefully not with the substitution I did. That’s just a guess, though.
Good luck! I like your blog a lot.
Thanks for the quick reply. I saw the “brilliant green” on there, so I will probably order that and I have the rest of the list. I’ll just need to order some plates.
Do you recommend the glass plates so you can reuse them or just buy disposable sterile plastic ones? I’m probably not going to be doing a ton of plating, I just want a few isolated Brett strains in order to try some Brett only fermentations.
Keep up the good work.
Check out the post before this one for some tips. I went with glass plates originally because of the way I was preparing them, but now I am leaning towards using disposable plates. Sometimes I get organisms other than what I wanted, such as mold and in one case a smelly bacterium, so it’d be nice to just be able to toss them completely without having to worry about cleaning them. Also, although you can sterilize the plates at the same time as the media, you get more water that way. Thus I end up sterilizing the plates first and storing them taped shut. It’s just one additional step that I could be avoiding.
Just go with disposable plastic ones. They come gamma-irradiated so you’re sure of their sterility and don’t need to play around with sterilizing plates (which REALLY SUCKS at home setting unless you have a dry autoclave in your house).
Is this the stuff you were talking about on Ebay? – [link removed]
I think it’s a pretty generic product, so that would likely be the same. If you search for “russian anticeptic” [sic] there’s another vendor who ships from the US, though.
Brilliant green is a dye that’s used for some stains, but mainly it’s used as an antiseptic. There is a ton of it all over the place including internet or pharmacies. You can even buy it as a powder so there is no particular brand. It usually comes as a 1% solution (by weight) in 60% ethanol. It’s usually used at something like 0.01-0.03mg per liter which would be something like 1-3mL of that solution per liter of medium.
I’m pretty sure I talked about it in my selective media post, but didn’t fully characterize it at the time of that post. By now I really like it and continue working at tweaking it to give even more specificity (actually poured a few batches yesterday and doing a few more today).
Great info guys.
You are starting to make this previously confusing topic make a lot more sense to me. I’ll be sure to share my results with you, plus any good strains that I hopefully isolate.
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