Journal of Applied Bacteriology zyxwvutsrq 1995, 78. 3-15 The ability of membrane potential dyes and calcafluor white to distinguish between viable and non-viable bacteria D.J. Mason, R. Lopez-Amoros, R. Allman, J.M. Stark and D. Lloyd School of Pure and Applied Biology, University of Wales College of Cardiff,Cardiff, UK 4934/05/94: received 25 May 1994, revised 2 November 1994 and accepted zyxwv 4 November 1994 D.J. MASON, R. LOPEZ-AMOR~S, R. ALLMAN, J.M. STARK AND D. LLOYD. iw5. zyxw Various dyes were assessed for their ability to discriminate between viable and non-viable bacteria. Two methods of killing were employed zyxwvut : by heat treatment or by gramicidin treatment. Staining was carried out in two ways; by staining directly in the medium or by washing cells prior to staining in buffer. Carbocyanine and rhodamine 123 dyes only exhibited small changes in fluorescence between viable and non-viable populations of bacteria. Both oxonol dye (bis 1,3- dibutylbarbituric acid trimethine oxonol) and calcafluor white proved much more useful. INTRODUCTION The use of fluorescent dyes as indicators of cell viability is widespread. Dyes such as ethidium bromide, acridine orange, propidium iodide and fluorescein diacetate have all been used successfully for this both in fluorescence micros- copy and flow cytometry. Membrane potential sensitive dyes have also been used as indicators of bacterial cell viability, the fluorescent response of these dyes varies with the magnitude of the membrane potential (Mason et al. 1993). Rhodamine 123 (rh123) is one such dye; a cationic lipophilic dye (accumulated cytosolically by cells with an inside negative transmembrane electrochemical potential), it has been used extensively to study mitochondria in eukaryotic cells (Ronot et al. 1986; Farkas et al. 1989; Skowronek et al. 1990; Rhan et al. 1991) as well as bacterial viability (Matsuyama 1984; Kaprelyants and Kell 1992; Diaper et al. 1992; Davey et al. 1993). The carbocyanine dyes are also a family of membrane potential sensitive lipophilic cations. These have been used to determine membrane potential in a diversity of cells and vesicles including mouse ascites tumour cells (Eddy 1989), cultured mammalian cells (Hargittai et al. 1991), lympho- cytes (Wilson et al. 1985), red blood cells (Sims et al. 1974), yeast (Pena et al. 1984) and bacteria (Zaritsky et zyxwvutsr al. 1984; Mason et al. 1993). Another well used group of membrane potential sensitive dyes are those of the oxonol family; these are lipophilic Correspondence to : Professor David Lloyd, School zyxwvutsr of Pure and Applied Biology, University of Wales College of CardtB; PO Box 915, Cardcff CFI 3TL, UK. anions and thus unlike the carbocyanines and rh123 are not extensively accumulated cytosolically by organisms with an inside negative transmembrane electrochemical potential. Therefore, the fluorescent response is opposite to that of cationic dyes, i.e. the fluorescent response decreases with increase in potential. These dyes have been used in the study of mouse tumour cells (Brauner et al. 1984; Oyama 1991), lymphocytes (Wilson and Chused 1985), mouse thymus cells (Lakos et al. 1990) and to estimate the effects of antibiotics and antihngal agents on microbial popu- lations (Carter et al. 1993; Ordofiez and Wehman 1993). Calcatluor white (CFW) is the disodium salt of 4,4’-bis(4 anilino-bis-diethyl amino-s-mazin-2-ylamino)-2,2’-stilbene disulphonic acid); it is one of a family of compounds used as ‘fluorescence brighteners’ in the dye industry. These compounds are highly fluorescent when excited by U.V. light. Absorption and transport of these dyes by micro- organisms has been investigated (Darken 1962). CFW binds to the chitin of the fungal cell wall (Streiblova 1984). It has also been used to estimate cell viability in rodent cell lines (Berglund et al. 1987). Viable cells are able to exclude this dye whereas non-viable cells appear brightly fluores- cent although the mode of binding is unknown. Measure- ments of cell viability, staining with CFW have been found to correlate well with propidium iodide (PI) and fluorescein diacetate fluorescence, a correlation coefficient of 0.9886 was recorded with PI (Berglund et al. 1987). In this paper flow cytometry has shown the superiority of the oxonol dye bis-( 1,3dibutylbarbituric acid) tri- methine oxonol (DiBAC4(3)) and CFW as indicators of cell vitality over the more commonly used carbocyanine dyes and rh123.