Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re‐hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non‐culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat‐killed mixtures. However, for re‐hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.

[1]  N. Ashbolt,et al.  Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. , 1994, Cytometry.

[2]  P. Hunter,et al.  Cytometric Evaluation of Antifungal Agents , 1993 .

[3]  D. Deere,et al.  Fluorescent probes and flow cytometry: new insights into environmental bacteriology. , 1996, Cytometry.

[4]  D. Lloyd,et al.  Flow cytometric monitoring of rhodamine 123 and a cyanine dye uptake by yeast during cider fermentation , 1996 .

[5]  D. Kell,et al.  Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. , 1996, Microbiological reviews.

[6]  C. Edwards,et al.  Direct measurements of natural planktonic bacterial community viability by flow cytometry , 1995, Applied and environmental microbiology.

[7]  P. Stewart,et al.  Physiological assessment of bacteria using fluorochromes. , 1995, Journal of microbiological methods.

[8]  David Lloyd,et al.  Flow Cytometry in Microbiology , 1993, Springer London.

[9]  P. Costantino,et al.  Enumeration of viable Candida albicans blastospores using tetrabromofluorescein (eosin Y) and flow cytometry. , 1995, Cytometry.

[10]  N. Ashbolt,et al.  Detection of specific microorganisms in environmental samples using flow cytometry. , 1994, Methods in cell biology.

[11]  D. Deere,et al.  Direct analysis of starved Aeromonas salmonicida , 1996 .

[12]  C Laplace-Builhé,et al.  Application of flow cytometry to rapid microbial analysis in food and drinks industries , 1993, Biology of the cell.

[13]  L. Alberghina,et al.  Alteration of cell population structure due to cell lysis in Saccharomyces cerevisiae cells overexpressing the GAL4 gene , 1993, Yeast.

[14]  T. Abee,et al.  Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry , 1994, Applied and environmental microbiology.

[15]  C. Edwards,et al.  Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting , 1993, Applied and environmental microbiology.

[16]  K. Rosén,et al.  Production of baker’s yeast , 1987 .

[17]  G. Pettipher.  Preliminary evaluation of flow cytometry for the detection of yeasts in soft drinks , 1991 .

[18]  Richard P. Haugland,et al.  Handbook of fluorescent probes and research chemicals , 1996 .

[19]  M. Laurent,et al.  Use of flow cytometry in oenology to analyse yeasts , 1994 .

[20]  D. Deere,et al.  Evaluation of the suitability of bis-(1,3-dibutylbarbituric acid) trimethine oxonol, (diBA-C4(3)-), for the flow cytometric assessment of bacterial viability. , 1995, FEMS microbiology letters.

[21]  Zbigniew Darzynkiewicz,et al.  Practical flow cytometry (3rd edn): by Howard M. Shapiro, Wiley-Liss 1995. £49.95 (542 pages) ISBN 0 471 303763 , 1995 .