A glimpse into copper sulfate agar

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).

IMG_1892

Fig 1: Saccharomyces yeasts on Sabouraud agar (1056 = Wyeast American Ale, 1084 = Wyeast Irish Ale, PtPtince = EBY049, Y05 = EBY050)

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.

IMG_1893

Fig 2: Saccharomyces yeasts on CuSo4-Sabouraud agar (1056 = Wyeast American Ale, 1084 = Wyeast Irish Ale, PtPtince = EBY049, Y05 = EBY050)

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.

IMG_1895

Fig 3: Different yeasts on CuSO4-Sabouraud agar (B04 = EBY004 Brettanomyces, B04green = EBY041 Saccharomyces, B05 = EBY005 Brettanomyces, B02 = EBY002 Brettanomyces)

IMG_1896

Fig 4: Different yeasts/bacteria on CuSO4-Sabouraud agar (B01 = EBY001 Brettanomyces, I10 = EBY024 Bacteria, I05 = EBY009 Brettanomyces, I11 = EBY013 Brettanomyces)

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.

References:

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7 thoughts on “A glimpse into copper sulfate agar

  1. Thanks for this post. I had been curious about this medium since CuSO4 is somewhat readily available online. But it sounds like it might not be worth the cost, though I understand this is just one experiment.

  2. Sam, you may want to try reducing the amount of yeast used in the initial plating of the yeast. It is not uncommon to see little to no growth defect on selective media when larger numbers of organisms are plated down, while strong effects can be seen when organisms are plated at low concentrations. This occurs because at higher concentrations, a portion of the yeast can take up the selective agent, thus reducing it to non-lethal concentrations which then allow the growth of the remaining yeast.

    A standard way of doing this (in the lab) is to do a dilution series – you do a 1:10 serial dilution of your yeast (i.e. 1x 1/10x, 1/100x, 1/1000x, etc), and plate a single drop of each dilution (usually in a row) onto the selective media, with space between each drop. The toxicity of the agent is the reflected by which dilutions give you colonies and those which do not. You can find more info on this method by googling for ‘yeast spotting assay’ or ‘dilution plating’.

    In terms of use for isolating yeasts, the principal is the same – you need to plate out your source yeast at a low enough concentration that you don’t end up with yeasts consuming the selective agent, thus allowing susceptible strains to grow.

    Bryan

    • Thank you very much Bryan for your explanations. I haven’t considered any of the effects you described. I will redo the experiment with a couple of dilutions.

      I currently use normal Sabouraud agar for all my isolation processes. The most striking evidence for Brettanomyces colonies is the time they need to appear. That’s how I get/got most of my current Brettanomyces strains. Sure I do some downstream analysis such as microscopy, sometimes some bromocresol green platings followed by small scale fermentations (20 mL cultures). If I detect Brett specific compounds or even a pellicle and all the others analysis point to Bretts as well, I dare to say that the strain(s) I isolated are Brettanomyces sp. I therefore don’t even use a selection agent (with the exception of the agar media itself) for my isolation processes.

      Thanks again for your explanations, always appreciated. Cheers, Sam

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