Tree Bark Yeast, Part 2

Introduction
This is part 2 of a hopefully 3 part series of catching, isolating, and using a yeast caught from a tree's bark. In part 1, I attempted to catch a yeast from an oak, apple, and pine tree. The pine tree yeast never panned out, but I got possible catches on the apple and oak tree bark.

Objective
To determine what species of yeast I caught on the apple and oak tree bark.

Prediction
The yeast caught on the apple tree is a Brettanomyces strain. The yeast caught on the oak tree is a Saccharomyces strain, possibly S. Paradoxus.

Materials Needed
DME
Agar Agar
DI Water
Yeast Nutrient
Bromocresal Green
Cycloheximide
Pietri Dishes
Alcohol Burner
Loop
Santization Liquid

Procedure
Plating
I used agar DME pietri dish plates, which I found a good recipe HERE, that uses 35g DME + 2.5 agar per 400ml of DI. I added a pinch of yeast nutrient. I also added the Bromocrescol Green. Bromocresol Green is a bacteria inhibitor. This should allow only yeast strains to grow and eliminate the growth of bacteria.

Brett should be able to metabolize the bromocresol while the saccharomyces cannot. By metabolyzing, that means if a strain cannot metabolize bromocresol, the colony will remain green. If it can metabolize, the colony will grow white. Some strains of sacchromyces can metabolize bromocresol, so it is possible to get a false negative or a false positive. My research has shown that this metabolizing factor is not a very solid determination of a Brett or Sacch strain. This part of the experiment isn't that crucial if I don't get a mix of green and white colonies. Bonus points if it happens.

Once enough colonies had formed for both the apple and oak plates, I selected 3 good looking colonies from each and replated those colonies onto new Bromocresol Green plates. This allowed a backup of the colony, as well as allowing me to select even newer formed colonies to go onto the final plates to confirm or deny that these strains are Saccharomyces or Brettanyomyces.

These final plates contain cycloheximide. Cycloheximide inhibits saccharomyces growth, yet allows brett growth, thus this will be my final determination of Brett or Sacch strains.

Here is a flow chart of what I am attempting:

Analysis
Here are the final images of the above's worflow diagram, but in picture form of each plate. Some of the plates are a little old when I took the picture, but they worked flawlessly when they were at their peak.

Apple Tree	Oak Tree

Conclusion
My prediction was mostly correct! The apple tree did in fact have a brett strain in it, as well as two sacch strains. The oak tree was 100% sacch.

What kind of brett and sacch strains did I catch? There is where I start to tread into unknown territory. Unless I sent out a sample to be DNA fingerprinted, the only other option is to do my own PCR and electrophoresis (which I have experience doing on human hair) to fingerprint its ribosomal structure, but I don't exactly have easy access to that kind of equipment nor primers. Future goal?

As for the bromocresol indicator to determine brett vs sacch? Not a worthy tool, BUT! totally worth using to eliminate bacteria. It feels really good to know that there is no bacteria growing on your plates and you only have yeast growing.

Plating "Control"
Because this is my first time doing all of the above, I also did 2 "control" plates. One was all filled with the regular agar, the other with the bromocresol agar. I then looped a commercially bought pure strain of each of sacch, brett, and lacto into each third on each type of plate. This was to see if the bromocresol does in fact inhibit the bacterial growth (it does) and to see what brett and sacch should look like.

Continuing onwards....
For now, its time to move back towards the sensory analysis side of the experiment. I have created 6 75 ml starters, one for each 1/3 of the plate for the apple and oak isolation plates. Now that I have pure strains, hopefully I can get some better ferments than the initial ferments in part 1 of this experiment, where there is no bacteria competing with my live strains to form and off aromas and pellicles.