Nov 012012

I have been contemplating taming wild yeast. What I mean is I want to isolate clonal strains of wild yeast, be it S. cerevisiae or Brettanomyces species. By far the latter is more intriguing, as there are hundreds of S. cerevisiae strains available. To begin taming Brett stains I need two things: a source and a medium to specifically select Brett versus S. cerevisiae. Well, maybe a third thing – patience, as this is going to get tedious.

In regards to the source, I’ll figure that out later. More importantly, I need a selective medium to isolate non-S. cerevisiae yeast.  Scouring the internet, I found several selective and differential media. The differential media use chemicals to either enhance the barnyard/funk smell of Brett (p-coumaric acid being converted to 4-ethyl-phenol) or change the color of the colony (metabolism of bromocresol green). I’m not interested in differentiating yeast, so I narrowed my options to selective media. There are three major selective media: copper sulfate, lysine, and cycloheximide. I opted for copper sulfate because it’s super cheap, and you don’t need a lot of it.
Base Medium
For my base agar medium, I will be using a modified MYPG. It’s “modified” due to my limited access to yeast extract. Basically I’m using expired YPD agar from the lab, cutting the quantity by three-quarters. This yields 0.25% yeast extract, 0.5% dextrose, and 0.5% agar. I then add dextrose to yield 1%, agar to yield 1.5%, and malt extract to yield 0.3% Here’s a comparison of MYPG and my modified medium:
0.3% malt extract
0.3% malt extract
0.3% yeast extract
0.25% yeast extract
1% dextrose
1% dextrose
0.5% peptone
0.5% peptone
1.5% agar
1.5% agar
Copper Sulfate
There are some discrepancies between various individuals who have reported using copper sulfate as the selective agent when growing Brett. The amount used by van der Aa Kuhle and Jespersen (Int J Food Microbiol 43.
205-213, 1998) was 195 ppm. Chad Yakobson at used 1200 ppm, and Jason at used 312 ppm. I decided to try two concentrations to see what grows – 195 ppm and 390 ppm. My stock solution of copper sulfate was 1.95% w/v CuSO4·5H2­O dissolved in water, so I added 0 μl, 250μl (195 ppm), or 500 μl (390 ppm) of copper sulfate solution to 25 ml of melted agar per plate.

My control for S. cerevisiae inhibition was Wyeast 3522, and my test yeast mixture was the dregs of Jolly Pumpkin Bam Biere.  I chose Wyeast 3522 (WLP550) because that is the primary yeast strain used to ferment Bam Biere.

The Test
I scraped my glycerol stock of Wyeast 3522 and resuspended in 50μl water. I added 10μl to plates and streaked for isolation. Similarly, I added 10μl of Jolly Pumpkin Bam Biere to the plates and streaked for isolation.  These were incubated at 30°C for several days to a week.
The Wyeast 3522 and the S. cerevisiae in the Jolly Pumpkin dregs grew within 2 days.  At that point I saw a lot of colonies on the MYPG plates, with less on the 195 ppm plate.

3522 0 ppm CuSO4

3522 195 ppm CuSO4

3522 390 ppm CuSO4

Jolly Pumpkin 0 ppm CuSO4

Jolly Pumpkin 195 ppm CuSO4

Jolly Pumpkin 390 ppm CuSO4

There were no colonies on the 390 ppm plates.  I kept the latter plates at 30°C for several more days, and finally something (s) grew on the Jolly Pumpkin plate, but not the Wyeast 3522 plate.  I was concerned that what grew out was bacterial in origin, as there is no antibiotic on the plate to suppress bacteria.  I picked a couple Jolly Pumpkin colonies, and analyzed them under the microscope, and they were indeed yeast, not bacteria (not shown).

So what’s next?  Now I’ve got to figure out what source I’m going to use to isolate these wild yeast strains, and then get some time to start taming them.

  2 Responses to “Yeast Isolation Medium”

  1. So are you going with the 390 ppm CuSO4 and have you done any yeast wrangling yet?

    • I have done any further work with Brett isolation – I’m been busy with my eubayanus work. However, when I do get back into wild Brett culturing, I will definitely use 390 ppm. Actually will probably just round up to 400 ppm for simplicities sake.

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