A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

New advances on the Brettanomyces bruxellensis biofilm mode of life.

The wine spoilage yeast Brettanomyces bruxellensis can be found at several steps in the winemaking process due to its resistance to multiple stress conditions. The ability to form biofilm is a potential resistance strategy, although it has been given little attention so far for this yeast. In this work, the capacity to form biofilm and its structure were explored in YPD medium and in wine. Using microsatellite analysis, 65 isolates were discriminated into 5 different genetic groups from which 12 strains were selected. All 12 strains were able to form biofilm in YPD medium on a polystyrene surface. The presence of microcolonies, filamentous cells and extracellular polymeric substances, constituting the structure of the biofilm despite a small thickness, were highlighted using confocal and electronic microscopy. Moreover, different cell morphologies according to genetic groups were highlighted. The capacity to form biofilm in wine was also revealed for two selected strains. The impact of wine on biofilms was demonstrated with firstly considerable biofilm cell release and secondly growth of these released biofilm cells, both in a strain dependent manner. Finally, B. bruxellensis has been newly described as a producer of chlamydospore-like structures in wine, for both planktonic and biofilm lifestyles.