Wild Yeast Project: Plating Brettanomyces on WLN/WLD Media

About two weeks ago I posted about an interesting experiment involving a new selective growth media that I made for wild yeast. To summarize, I made yeast agar plates that contained cycloheximide (WLD), an inhibitor of protein synthesis. This chemical is interesting because I routinely use it in the lab to study the half life of proteins. This drug kills most mammalian cells, including cancer cells, and I was surprised to learn that Brettanomyces is unaffected by it.

The idea was to plate the dregs from wild commercial bottles and weed out the brewers yeast and isolate the Brettanomyces and other wild flora, including lactobacillus and pediococcus. From here, I can grow up the wild yeast from single colonies and begin test batches of homebrew. Here were the different samples for the experiment:

1. Wyeast 1056 (negative control – should be no growth on WLD)
2. Wyeast Brett B (positive control – should grow on WLN and WLD)
3. Wyeast Brett L (another positive control)
4. Russian River Temptation dregs (experimental sample)

It is important to note that WLN is media without the drug and WLD contains cycloheximide. For each sample, the utmost care in sterility was taken, including bunsen burners, sterile pipettes and tips. For Wyeast 1056 and the Russian River Temptation dregs, I used frozen yeast cells, while the Brett B. and L. came from a Wyeast pack. I dilution streaked each sample onto WLN and WLD plates and grew the yeast for four days at 30°C.

The Results (WLN on left and WLD on right; click on photo for more detail):

Wyeast 1056 (Chico ale). As expected this strain grew fast and strong on the WLN. No growth of the Chico strain on the WLD is consistent with the notion that brewers yeast does not tolerate cycloheximide at all.

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Wyeast Brettanomyces bruxellenis. Excellent growth on both plates consistent with the fact the Brettanomyces can tolerate cycloheximide. The growth of these cells took longer on both plates compared to Wyeast 1056. Interestingly, note the larger colony morphology on the WLN (no drug) plate compared to WLD (with drug).

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 Wyeast Brettanomyces lambicus: Almost the same as Brett B above. Again, notice the larger colonies on the plates without the drug compared to the one with the drug.

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Russian River Temptation Dregs. No growth whatsoever. These plates were left at 30°C for another two weeks, just to make sure I missed any cells.

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Conclusions:

Often in science, when one does not do a properly controlled experiment one cannot interpret the results. If you don’t have a baseline for comparison, the experimental sample is meaningless. That’s not the case here. Both controls worked admirably, 1056 strain died in the presence of drug (brewer’s yeast), and Brett did not (wild yeast). If there were ANY live cells in my temptation dregs they would have grown in both plates. This can only lead me to conclude that freezing down cells from the dregs of this commercial example killed the cells. This is in contrast to my MYPG plating where I saw growth but did not freeze them prior to plating.

Moreover this suggests that freezing wild yeast from dregs (even with glycerol) is bad and when I do isolate yeast it will have to be from a freshly opened bottle. No more freezing and plating of dregs from wild ales, unless my technique is horribly wrong. Now I only need a good opportunity to open some wild ales and plate right away.

Another interesting observation is that on both Brett plates without drug were giant colonies that grew much faster than other colonies surrounding them. These colonies did not grow on WLD plates (with drug) indicating there was brewer’s yeast contaminating the Wyeast sample. I don’t feel that this is a big deal since the number of colonies was very small compared to all the colonies, suggesting trace contamination. This is not surprising since the sheer volume of brewers yeast they produce will cross-contaminate wild yeast preps. However, it is important to realize the Brettanomyces purchased from Wyeast *may* be contaminated with brewers yeast. I emphasize *may* because only hardcore molecular biology, such as polymerase chain reaction (PCR) will cleanly identify the contaminant.

Future:

I learned some important facts here. WLN/WLD media is great for differentiating between wild yeast and brewers yeast. I also learned that my source of wild yeast needs to be as fresh as possible. For my next experiment, I will plate fresh dregs from a wild ale and there are some candidates to select from. Importantly, Chad Yakobson from the Brettanomyces Project and Crooked Stave suggested that I isolate strains from lambics. He mentioned that Russian River wild ales are fermented with commercial strains of wild yeast and the really interesting critters lie at the bottom of Belgian goodness.