#4 Agar plates (#3763 Roeselare Blend)

Eureka, today another post about yeast hunting. The other posts of the pentade about agar plates can be found here. This time about plating Wyeast’s #3763 Roeselare Blend isolated from the liquid of my Flanders Red. I took a sample of the fermenter and plated it on a Sabouraud plate. I then picked some colonies from the first plate and transferred them on separate Sabouraud plates. Lets get into the results.

Roeselare the First:

Plated the liquid from the fermenter and incubated it for nearly three days (Fig 1).

Fig 1: #3763 Roeselare Blend on Sabouraud agar after three days of incubation

I could observe at least two different microorganisms: (and three mold colonies as well)

– Off-white color, even, circular, glossy, convex, 2- 4 mm diameter.

– White, wavy, irregular, convex, not glossy, 2- 3 mm diameter.

The white colonies seem to grow on top of the off-white colonies. I guess the off-white colonies are yeasts. The microscopy will tell. And I have no clue about what the other colonies might be. Wyeast states on their page that they blend a Belgian yeast strain with a sherry strain, two Brettanomyces strains, a Lactobacillus and Pediococcus strain. Assumed that the off-white colonies are the Belgian yeasts, then the white colonies could be the sherry strain or some sort of Brettanomyces. I can exclude the Lactobacillus due to the results from the post about Lactobacillus where I concluded that Lactobacillus do not grow on this agar. I exclude Pediococcus as well as Pediococcus do not easily grow in presence of oxygen.

As there were some molds on the agar, I picked one of the colonies each and streaked them on separate plates.

After incubating for nearly four days, the following colonies appeared on the plate where I streaked the off-white colonies (Fig 2):

Fig 2: Off-white colony of Roeselare blend

Off-white color, even, circular, glossy, convex, 2- 4 mm diameter

No surprise. The colonies still have the same morphology.

I picked a colony and prepared it for a microscopy analysis (Fig 3).

Fig 3: Microscopy picture Roeselare blend off-white colony

My guess seemed to be spot on again. The off-white colonies are indeed a sort of yeasts. The fun part: I isolated the yeast strain(s) from the Roeselare blend with this plate. But as already mentioned, Wyeast states, that there are two different yeast strains in the blend: one Belgian Ale and a Sherry strain. So I can’t say whether strain this is or if it is a blend of both of them. Something is a bit unexpected here. The yeasts do not aggregate as top fermenting yeasts do in general. And a Belgian yeast should be a top fermenting one. Maybe just a coincidence. I could not find any picture of a sherry strain. Maybe this is the sherry strain? How could I find out whether this is a Belgian yeast or a sherry strain? And how could I determine if this is a mixture of both? Well, scientists need questions to find an answer. And I got myself a few new ones…

Lets move on to the other colonies. I could observe the following colonies (Fig 4):

Fig 4: White colony of Roeselare blend

Off-white color, even, circular, not glossy, 2- 3 mm diameter.

The morphology is very similar to the one in the first plate. The colonies seemed to be flat, even and circular this time. These colonies look quite different compared to the yeasts in Fig 2.

Nevertheless, I took a sample for a microscopy analysis to get further information about the colonies.

. Microscopy picture Roeselare blend white colony (Take 1)

Fig 5: Microscopy picture Roeselare blend white colony (Take 1)

Lets go step by step. The cells (Fig 5) have the same size as the yeasts above (Fig 3). So these could be yeasts again. The white colonies (Fig 5) are definitely no normal yeast strains as the off-white colony in Fig 3. But what do I got myself here? The only other, different yeasts in the blend are the sherry strain and Brettanomyces.

Brettanomyces Project has some cool pictures of Brettanomyces available to compare with. And my cells look very similar to Brettanomyces. What kind is difficult to say. The only way to be sure about this is to have a look at pure Brettanomyces cultures myself and compare them. I already ordered some Brettanomyces cultures for some experimenting and I will definitely take some pictures of the different strains. And I end this post with another picture of the same cells:

Microscopy picture Roeselare blend white colony (Take 2)

Microscopy picture Roeselare blend white colony (Take 2)

The results are very cool in my understanding. I might have isolated some Brettanomyces out of a bunch of bugs, right? Another cool thing are the plates with the dregs from the first post. They are still incubating and they look very different now. Will post about these plates in another post soon. Could be the sixth post in my pentade…

The end of the pentade about agar plates is near. It ends with the fifth post about cultivating kombucha bugs on Sabouraud plates. I might publish it tomorrow night. But don’t rely on it: I can pick up my case of Westvleteren 12 tomorrow evening…