Current Microbiology

, Volume 61, Issue 1, pp 57–63 | Cite as

Dual Fluorochrome Flow Cytometric Assessment of Yeast Viability



A novel staining protocol is reported for the assessment of viability in yeast, specifically the biocontrol yeast, Pichia anomala. Employing both the red fluorescent membrane potential sensitive oxonol stain DiBAC4(5) (Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol), a structural analog of the commonly used DiBAC4(3) (Bis-(1,3-dibutylbarbituric acid)trimethine oxonol), with one of the esterase dependent green fluorogenic probes such as CFDA-AM (5-Carboxyfluorescein diacetate, acetoxymethyl ester) or Calcein-AM (Calcein acetoxymethyl ester), a two-color flow cytometric method was developed, which yields rapid quantitative information on the vitality and vigor of yeast cell cultures. The method was validated by cell sorting and analysis of live, heat killed, and UV-treated yeast.


  1. 1.
    Attfield PV, Kletsas S, Veal DA, van Rooijen R, Bell PJL (2000) Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts. J Appl Microbiol 89:207–214CrossRefPubMedGoogle Scholar
  2. 2.
    Bräuner T, Hülser DF, Strasser RJ (1984) Comparative measurement of membrane potentials with microelectrodes and voltage-sensitive dyes. Biochim Biophys Acta 771:208–216CrossRefPubMedGoogle Scholar
  3. 3.
    Deere D, Porter J, Edwards C, Pickup R (1995) Evaluation of the suitability of bis-(1, 3-dibutylbarbituric acid) trimethine oxonol, (diBA-C4(3)-), for the flow cytometric assessment of bacterial viability. FEMS Microbiol Lett 130:165–170PubMedGoogle Scholar
  4. 4.
    Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D (1998) Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast 14:147–160CrossRefPubMedGoogle Scholar
  5. 5.
    Dinsdale MG, Lloyd D, McIntyre P, Jarvis B (1999) Yeast vitality during cider fermentation: assessment by energy metabolism. Yeast 15:285–293CrossRefPubMedGoogle Scholar
  6. 6.
    Epps DE, Wolfe ML, Groppi V (1994) Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(1, 3-dibutylbarbituric acid)trimethine oxonol (Dibac4(3)) in model systems and cells. Chem Phys Lipids 69:137–150CrossRefPubMedGoogle Scholar
  7. 7.
    González JE, Tsien RY (1995) Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys J 69(4):1272–1280CrossRefPubMedGoogle Scholar
  8. 8.
    Hernlem BJ, Srienc F (1989) Intracellular pH in single Saccharomyces cerevisiae cells. Biotechnol Tech 3(2):79–84CrossRefGoogle Scholar
  9. 9.
    Hua ST, Baker JL, Flores-Espiritu M (1999) Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus. Appl Environ Microbiol 65(6):2738–2740PubMedGoogle Scholar
  10. 10.
    Hua ST (2006) Progress in prevention of aflatoxin contamination in food by preharvest application of Pichia anomala WRL-076. In: Mendez-Vilas A (ed) Recent advances in multidisciplinary applied microbiology. Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim, Germany, pp 322–326CrossRefGoogle Scholar
  11. 11.
    Lloyd D (1999) Flow cytometry of yeasts. Curr Protoc Cytom 11.10.1–11.10.8Google Scholar
  12. 12.
    Lloyd D, Harris JC, Biagini GA, Hughes MR, Maroulis S, Bernard C, Wadley RB, Edwards MR (2004) The plasma membrane of microaerophilic protists: oxidative and nitrosative stress. Microbiology 150(5):1183–1190CrossRefPubMedGoogle Scholar
  13. 13.
    Lloyd D, Hayes AJ (1995) Vigour, vitality and viability of microorganisms. FEMS Microbiol Lett 133:1–7CrossRefGoogle Scholar
  14. 14.
    López-Amorós R, Mason DJ, Lloyd D (1995) Use of two oxonols and a fluorescent tetrazolium dye to monitor starvation of Escherichia coli in seawater by flow cytometry. J Microbiol Methods 22:165–176CrossRefGoogle Scholar
  15. 15.
    Melin P, Håkansson S, Eberhard TH, Schnürer J (2006) Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulation at different temperatures, assessed by flow cytometry. J Appl Microbiol 100:264–271CrossRefPubMedGoogle Scholar
  16. 16.
    Novo D, Perlmutter NG, Hunt RH, Shapiro HM (1999) Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry 35:55–63CrossRefPubMedGoogle Scholar
  17. 17.
    Plášek J, Sigler K (1996) Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis. J Photochem Photobiol B 33:101–124CrossRefPubMedGoogle Scholar
  18. 18.
    Shapiro HM (2000) Membrane potential estimation by flow cytometry. Methods 21:271–279CrossRefPubMedGoogle Scholar
  19. 19.
    Suller MTE, Stark JM, Lloyd D (1997) A flow cytometric study of antibiotic-induced damage and evaluation as a rapid antibiotic susceptibility test for methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 40:77–83CrossRefPubMedGoogle Scholar
  20. 20.
    Zhang S, Crow SA Jr (2001) Toxic effects of Ag(I) and Hg(II) on Candida albicans and C. maltosa: a flow cytometric evaluation. Appl Environ Microbiol 67(9):4030–4035CrossRefPubMedGoogle Scholar

Copyright information

© US Government 2010

Authors and Affiliations

  1. 1.Agricultural Research Service, Western Regional Research Center, Foodborne Contaminants Research UnitU.S. Department of AgricultureAlbanyUSA
  2. 2.Agricultural Research Service, Western Regional Research Center, Plant Mycotoxins Research UnitU.S. Department of AgricultureAlbanyUSA

Personalised recommendations