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

  • Brian Martyniak
  • Jason Bolton
  • Dmitry Kuksin
  • Suzanne M. Shahin
  • Leo Li-Ying ChanEmail author
Biotechnology Methods - Original Paper


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.


Brettanomyces Budding Cell counting Cellometer Fermentation Image cytometry Propagation Pseudohyphae 


Compliance with ethical standards

Conflict of interest

The authors DK, SMS, and LLC declares competing financial interests. The work performed in this manuscript is for reporting on product performance of Nexcelom Bioscience, LLC. The performed experiments were to demonstrate novel methods for automated Brettanomyces concentration and viability measurement.


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  • Brian Martyniak
    • 1
  • Jason Bolton
    • 1
  • Dmitry Kuksin
    • 2
  • Suzanne M. Shahin
    • 2
  • Leo Li-Ying Chan
    • 2
    Email author
  1. 1.Cooperative Extension and the School of Food and AgricultureUniversity of MaineOronoUSA
  2. 2.Department of Technology R&DNexcelom Bioscience LLCLawrenceUSA

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