Advertisement

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

Abstract

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.

Keywords

Brettanomyces Budding Cell counting Cellometer Fermentation Image cytometry Propagation Pseudohyphae 

Notes

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.

References

  1. 1.
    American Society of Brewing Chemists (2004) Methods of Analysis, 9th Ed. Yeast-3A Yeast stains, -4 Microscopic yeast cell counting, -6 Yeast viability by slide culture. ASBC, St. PaulGoogle Scholar
  2. 2.
    Chan LL-Y, Kury A, Wilkinson A, Berkes C, Pirani A (2012) Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae. J Ind Microbio Biotech 39:1615–1623CrossRefGoogle Scholar
  3. 3.
    Chan LL-Y, Lyettefi EJ, Pirani A, Smith T, Qiu J, Lin B (2011) Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method. J Ind Microbiol Biotechnol 38:1109–1115CrossRefPubMedGoogle Scholar
  4. 4.
    Ciani M, Maccarelli F, Fatichenti F (2003) Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of Aerobiosis. World J Microbiol Biotechnol 19:419–422CrossRefGoogle Scholar
  5. 5.
    Dickinson JR (1996) ‘Fusel’ alcohols induce hyphal-like extensions and pseudohyphal formation in yeasts. Microbiol. 142:1391–1397CrossRefGoogle Scholar
  6. 6.
    Freer SN, Dien B, Matsuda S (2003) Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH. World J Microbiol Biotechnol 19:101–105CrossRefGoogle Scholar
  7. 7.
    Gilis J-F, Cabri C, Ducournau P (2016) Brettanomyces: Study of red wine contamination by Brettanomyces yeast. http://www.micro-oxigenation.com/gb/brett_intro.asp. Assessed 24 May 2016)
  8. 8.
    Kirsop BE, Kurtzman CP, Nakase T, Yarrow D (1988) Yeasts. Living resources for biotechnology. Cambridge University Press, New YorkGoogle Scholar
  9. 9.
    Kurtzman CP, Fell JW (2010) The yeasts—a taxonomic study. Elsevier, Amsterdam; OxfordGoogle Scholar
  10. 10.
    Laverty DJ, Kury AL, Kuksin D, Pirani A, Flanagan K, Chan LL (2013) Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method. J Ind Microbiol Biotechnol 40:581–588CrossRefPubMedGoogle Scholar
  11. 11.
    Layfield JB, Sheppard JD (2015) What brewers should know about viability, vitality, and overall brewing fitness: a mini-review. MBAA TQ. 52:132–140Google Scholar
  12. 12.
    Oelofse A, Pretorius IS, Du Toit M (2008) Significance of Brettanomyces and Dekkera during winemaking: a synoptic review. S Afr J Enol 29:128–144Google Scholar
  13. 13.
    Piatz S (2005) Learn how to put the “British brewing industry fungus” to work in your home brewery. You don’t need to be a lambic brewer to show an interest in this barrel-loving wild yeast. Plus: two wild Brett clone recipes. In: Brew Your Own. http://byo.com/mead/item/262-brettanomyces
  14. 14.
    Saldi S, Driscoll D, Kuksin D, Chan LL-Y (2014) Image-based cytometric analysis of fluorescent viability and vitality staining methods for ale and lager fermentation yeast. J Am Soc Brew Chem 72:253–260Google Scholar
  15. 15.
    Steensels J, Daenen L, Malcorps P, Derdelinckx G, Verachtert H, Verstrepen KJ (2015) Brettanomyces yeasts—from spoilage organisms to valuable contributors to industrial fermentations. Int J Food Microbiol 206:24–38CrossRefPubMedGoogle Scholar
  16. 16.
    Yakobson C (2010) Pure culture fermentation characteristics of brettanomyces yeast species and their use in the brewing industry. Masters Degree. Heriot-Watt University, EdinburghGoogle Scholar

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

Personalised recommendations