MYPG Plating of Sanctification and Temptation

About a week ago I made some MYPG plates with and without Bromocresol Green. According to the literature, brewer’s yeast are unable to metabolize the dye and thus will appear green when plated on agar. However, wild yeast such as Brettanomyces can metabolize the dye and should be a shade lighter or white. There are some exceptions to this as Chad Yakobson found that B. claussenii (WLP645) is unable to metabolize the green dye.

I decided to plate yeast isolated from the dregs of two Russian River bottles, Sanctification and Temptation, and to find what species of yeast can grow on MYPG + Bromocresol green media. It’s important to note that I plated the yeast from the top portion of a centrifuged slurry. The centrifugation process was low enough (4000 rpm for 5 minutes) to only pellet large cells and debris. This upper fraction contained more Brett cells when I checked it under a microscope. I have yet to plate the pellet and both the upper fraction and pellet are stored at -80°C.

Every experiment needs controls for accurate comparison. In this case I also plated two strains with Wyeast 1056 (Chico) serving as a negative control for Brett species, and Wyeast 9097 (Old Ale Blend) as a positive control. The Old Ale Blend is a mix of an attenuative Sacc and a Brett species so some colonies should be able to metabolize the dye.

Samples:

• 1056 MYPG
• 1056 MYPG + Bromocresol Green
• 9097 MYPG
• 9097 MYPG + Bromocresol Green
• Sanct. MYPG
• Sanct. MYPG + Bromocresol Green
• Temp. MYPG
• Temp. MYPG + Bromocresol Green

Protocol:

1. Centrifuged dregs for 5 minutes at 4000 rpm in a 15 ml sterile conical.
2. Took 20 μls from each centrifuged sample and added directly to plate.
3. Under sterile conditions, dilution streaked each sample.
4. For 1056 and 9097 I used 30-50 μls of frozen glycerol stocks.
5. Grew yeast at 30°C for 4 days (until colonies appeared).

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