Flow Cytometry of Yeasts

David Lloyd1

1 University of Wales, Cardiff, United Kingdom
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.10
DOI:  10.1002/0471142956.cy1110s09
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This unit promotes increased interest in the use of flow cytometry in several new environments. The author provides protocols and descriptive detail on measurements of cell cycle, viability, respiratory activity, and β‐galactosidase activity. Many of these assays are described for other biological systems in CPC, but are now provided in detail for yeasts. Despite the increasing usefulness of digital imaging techniques, flow cytometry remains the method of choice for the resolution of population heterogeneities.

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Monitoring the Cell Division Cycle of Yeasts
  • Basic Protocol 2: Determining the Viability of Yeast
  • Basic Protocol 3: Evaluating the Mitochondrial (Respiratory) Function of Yeast
  • Basic Protocol 4: Assaying β‐Galactosidase Activity In Vivo
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Monitoring the Cell Division Cycle of Yeasts

  • Washed yeast suspension
  • 70% (v/v) ethanol
  • Tris⋅Mg2+ buffer (see recipe)
  • 0.5% (w/v) pepsin solution (see recipe)
  • 10 µg/ml RNase A solution (see recipe)
  • 50 µg/ml PI solution (see recipe)

Basic Protocol 2: Determining the Viability of Yeast

  • Yeast suspension
  • Tris⋅Mg2+ buffer (see recipe)
  • 1 µg/ml DiBAC 4(3) solution (see recipe)

Basic Protocol 3: Evaluating the Mitochondrial (Respiratory) Function of Yeast

  • Washed yeast suspension at 1–5 × 106 organisms/ml
  • 10 mM glucose
  • 1 µg/ml rhodamine 123 solution (see recipe)

Basic Protocol 4: Assaying β‐Galactosidase Activity In Vivo

  • 2% (v/v) Triton X‐100
  • Substrate solution: 2 mg/ml resorufin β‐D‐galactopyranoside (Molecular Probes) in dimethyl sulfoxide
  • PBS ( appendix 2A), prechilled to 0°C
  • 2% (w/v) bovine serum albumin (BSA; Sigma) in PBS (store and use at 0°C)
  • Yeast suspension (106 cells/ml) washed twice in PBS after centrifugation from growth medium and kept at 4°C
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Agar, D.W. and Bailey, J.E. 1982. Cell cycle operation during batch growth of fission yeast populations. Cytometry 3:123‐128.
   Alberghina, L. and Porro, D. 1993. Quantitative flow cytometry: Analysis of protein distributions in budding yeast. A mini review. Yeast 9:815‐823.
   Bell, P.J.L., Deere, D., Shen, J., Chapman, B., Bissinger, P.H., Attfield, P.V., and Veal, D.A. 1998. A flow cytometric method for rapid selection of novel yeast hybrids. Appl. Environ. Microbiol. 64:1669‐1672.
   Brailsford, M. and Gatley, S. 1993. Rapid analysis of microorganisms using flow cytometry. In Flow Cytometry in Microbiology (D. Lloyd, ed.) pp. 171‐180. Springer‐Verlag, London.
   Bruetschy, A., Laurent, M., and Jacquet, R. 1994. Use of flow cytometry in oenology to analyse yeasts. Lett. Appl. Microbiol. 18:343‐345.
   Carlson, C.R., Grallert, B., Bernander, R., Stokke, T., and Boye, E. 1997. Measurement of DNA content in fission yeast. Yeast 13:1329‐1335.
   Carter, E.A., Paul, F.E., and Hunter, P.A. 1993. Cytometric evaluation of antifungal agents. Flow Cytometry in Microbiology (D. Lloyd ed.) pp. 111‐120. Springer‐Verlag, London.
   Costello, G., Rodgers, L., and Beach, D. 1986. Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2. Curr. Genet. 11:119‐125.
   Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogenous microbial populations: The importance of single‐cell analyses. Microbiol. Rev. 60:641‐696.
   Deere, D., Shen, J., Vesey, G., Bell, P., Bissinger, P., and Veal, D. 1998. Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast 14:147‐160.
   Denksteinova, B., Sigler, K., and Plásek, J. 1996. Three fluorescent probes for the flow‐cytometric assessment of membrane potential in Saccharomyces cerevisiae. Folia Microbiol. 41:237‐242.
   Dinsdale, M.G., Lloyd, D., and Jarvis, B. 1995. Yeast vitality during cider fermentation: Two approaches to the measurement of membrane potential. J. Inst. Brew. 101:453‐458.
   Dinsdale, M.G., Lloyd, D., McIntyre, P., and Jarvis, B. 1999. Yeast vitality during cider fermentation: Assessment by energy metabolism. Yeast 15:285‐293.
   Eilam, Y. and Chernichovsky, D. 1988. Low concentrations of trifluoperazine arrest the cell division cycle of Saccharomycescerevisiae at two specific stages. J. Gen. Microbiol. 134:1063‐1069.
   Hayashi, M., Ohkuni, K., and Yamashita, I. 1998. Control of division arrest and entry into mitosis by extracellular alkalisation in Saccharomyces cerevisiae. Yeast 14:905‐913.
   Hutter, K.‐J. and Eipel, H.E. 1978. Flow cytometric determinations of cellular substances in algae, bacteria, molds and yeasts. Antonie van Leeuwenhoek 44:269‐278.
   Lloyd, D. and Hayes, A.J. 1995. Vigour, vitality and viability of microorganisms. FEMS Microbiol. Lett. 133:1‐7.
   Lloyd, D., Moran, C.A., Suller, M.T.E., Hayes, A.J., and Dinsdale, M.G. 1996. Flow cytometric monitoring of rhodamine 123 and a cyanine dye uptake by yeast during cider fermentation. J. Inst. Brew. 102:251‐259.
   López‐Amorós, R., Mason, D.J., and Lloyd, D. 1995. Use of two oxonols and a fluorescent tetrazolium dye to monitor starvation of Escherichia coli.in sea water by flow cytometry. J. Microbiol. Methods 22:165‐176.
   Müller, S., Lösche, A., and Bley, T. 1992. Flow‐cytometric investigation of sterol content and proliferation activity of yeast. Acta Biotechnol. 12:365‐375.
   Ordónez, J.V. and Wehman, N.M. 1995. Amphotericin suseptibility of Candida species assessed by rapid flow cytometric membrane potential assay. Cytometry. 22:154‐157.
   Pettipher, G.L. 1991. Preliminary evaluation of flow cytometry for the detection of yeasts in soft drinks. Lett. Appl. Microbiol. 12:109‐112.
   Plásek, J. and Sigler, K. 1996. Slow fluorescent indicators of membrane potential: A survey of different approaches to probe response analysis. J. Photochem. Photobiol. B. 33:101‐124.
   Porro, D., Sneraldi, C., Martegani, E., Ranzi, B.M., and Alberghina, L. 1994. Flow cytometry for monitoring yeast growth. Biotechnol. Prog. 10:193‐199.
   Pringle, J.R., Preston, R.A., Adams, A.E.M., Stearns, T., Drubin, D.G., Haarer, B.K., and Jones, E.W. 1989. Fluorescence microscopy methods for yeast. Methods Cell Biol. 31:357‐435.
   Rose, A.H. and Harrison, J.S. eds. 1969. The Yeasts, Vol.1: Biology of the Yeasts, Academic Press, New York.
   Sazer, S. and Sherwood, S.W. 1990. Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast. J. Cell Sci. 97:509‐516.
   Seward, R., Willetts, J.C., Dinsdale, M.G., and Lloyd, D. 1996. The effects of ethanol, hexan‐l‐ol and 2 phenylethanol on cider yeast growth, viability and energy status; synergistic inhibition. J. Inst. Brew. 102:439‐443.
   Skarstad, K., Steen, N.B., and Boye, E. 1985. Escherichia coli DNA distributions measured by flow cytometry and compared with computer simulations. J. Bacteriol. 154:656‐662.
   Theorell, B. 1983. Flow‐cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels. Cytometry 4:61‐65.
   Willetts, J.C., Seward, R., Dinsdale, M.G., Suller, M.T.E., Hill, B., and Lloyd, D. 1997. Vitality of cider yeast grown micro‐aerobically with added ethanol, butan‐l‐ol or iso‐butanol. J. Inst. Brew. 103:79‐84.
   Williamson, D.H. and Fennell, D.J. 1979. Mitochondrial DNA. In Methods in Cell Biology, Vol. 12 (D.M. Prescott ed.) pp. 335‐351. Academic Press, New York.
   Wittrup, K.D. and Bailey, J.D. 1988. A single‐cell assay of β‐galactosidase activity in Saccharomyces cerevisiae. Cytometry 9:394‐404.
   Yurkow, E.J. and McKenzie, M.A. 1993. Characterization of hypoxia‐dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: A model for ischaemic tissue destruction. Cytometry 14:287‐293.
PDF or HTML at Wiley Online Library