Eureka, I would like to publish some preliminary results from my latest plating experiments. I am still interested in isolating Brettanomyces from different sources and still play around with different agar media to see what their impact is on the entire isolation process. The latest experiment I performed was a large scale bromocresol screening on different Saccharomyces yeasts to see whether bromocresol can be used to differentiate between Saccharomyces and Brettanoymces. My insight from this experiment: bromocresol green as a tool to differentiate between Brettanomyces (known to grow as white colonies) and Saccharomyces might only work within a small time frame. In addition, some Saccharomyces strains grew as white colonies in presence of bromocresol green (possible false positive strains).
Yet another approach is to add copper sulfate to the agar media to inhibit the growth of domesticated yeasts [Yakobson, 2010, Taylor et al, 1984]. Wild yeasts therefore should be able to grow in presence of copper sulfate. I wanted to give this agar a go to see if it can be used to differentiate between domesticated Saccharomyces strains and wild yeasts (Brettanoymces in my case). I started by adding 0.6 g copper sulfate to 1 L of Sabouraud agar and streaked some strains on the plates. As controls, plain Sabouraud agar plates were used to test the viability of the strains (not all plates shown).
The four domesticated Saccharomyces strains plated on plain Sabouraud agar showed a nice growth phenotype (Fig 1). Streaking the same strains on copper sulfate containing Sabouraud agar revealed that only one strain (WY1084 Irish Ale) was impaired in its growth (Fig 2). All the remaining Saccharomyces strains grew as normal. From this observation one can already conclude that the addition of copper sulfate to the agar media impaired only 25% of the domesticated Saccharomyces strains tested.
Plating Brettanomyces and isolated Saccharomyces strains on copper agar media revealed a growth phenotype for all tested Brettanomyces strains (Fig 3, 4). Only the Saccharomyces isolate (B04 green in Fig 3) and the bacteria strain (I10 in Fig 4) did not grow on copper sulfate agar. Since B04 green was isolated from a Gueuze, it can be argued that this particular strain might be a non-domesticated Saccharomyces strain. On the other hand, it might be a domesticated yeast strain concluding from the lacking growth on copper sulfate. Including the previous observation that only a small part of domesticated Saccharomyces strains were impaired in their growth makes it even harder to allocate the isolated yeast strain to domesticated or non-domesticated Saccharomyces.
This small-scale experiment revealed that a copper sulfate addition to Sabouraud agar media does not impair most of the domesticated Saccharomyces strains tested. All the Brettanomyces strains tested in this experiment grew in presence of copper sulfate.
It seems to me that copper sulfate used at a concentration of 0.6 g per liter of Sabouraud agar media was not useful to differentiate non-domesticated from domesticated Saccharomyces yeasts. Simply because it could not inhibit the growth of most of the domesticated yeasts tested. As an outlook, one might increase the concentration of copper sulfate to levels where it impairs most of the domesticated Saccharomyces strains. Then test the Brettanomyces under the same conditions and see if they still grow or not. Maybe even change the Sabouraud agar to MYGP like published by Taylor et al. It is not clear to me yet if I even further investigate the use of copper sulfate.
- Yakobson C. (2010) Brettanomyces project, http://www.brettanomycesproject.com/dissertation/analysis-of-culturability-on-various-media-agar/brettanomyces-identification-methods/
- Taylor GT, Marsh AS (1984) MYGP+Copper, A medium that detects both Saccharomyces and non-Saccharomyces wild yeasts in the presence of culture yeast, J. Inst. Brew., Vol 90, p 134-145