Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry

Abstract

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H+-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40°C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efilux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability.

Current and future applications of flow cytometry in aquatic microbiology

“…A number of esterase substrates and their applicability to several organisms have been compared [96]. Apart from di¤culties with dye loading, staining problems arise from dye extrusion [18,19,97] as mentioned above or from the pH dependence of £uorescent signals. The use of dichlorocarboxy-£uorescein diacetate [98] or calcein-acetomethyl ester [28] may overcome this drawback.…”

Intracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica

“…The type of carrier implicated here could be, most likely, a transport system for the extrusion of more or less toxic anionic compounds from yeast cells, as hypothe- [7] for the e¥ux of £uorescein in S. cerevisiae. The use of pyranine introduced in yeast cells by electroporation appears to be a convenient method for pH i determination.…”

“…The probe leakage can also be a limitation to the £uorescence use in pH i determination as reported in yeast, for example with the dyes C.SNARF-1 and £uorescein [5]. The probe e¥ux has also been favor-ably used to assess yeast membrane integrity [2,6] and cell viability [7].…”

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

“…Several publications describe yeast viability determination by measuring the fluorescent properties of cells stained with dyes that discriminate cell types according to activity and structural integrity. Examples include oxonol (Ox) and propidium iodide (PI; Carter et al, 1993), rhodamine 123 (Rh123; Lloyd et al, 1996), ChemChrome Y (CY; Bruetschy et al, 1994;Carter et al, 1993), carboxyfluorescein di-acetate (CFDA; Breeuwer et al, 1994;Haugland, 1996), ethidium bromide (EB; Martegani et al, 1993), Fungolight (Haugland, 1996) and eosin Y (EY; Costantino et al, 1994).…”

“…For measurement of membrane integrity, PI was chosen in favour of EB because the former has one more positive charge then the latter and is, therefore, thought to give better live/dead discrimination (Shapiro, 1995). For measurement of intracellular enzyme activity and membrane integrity, CY was chosen over CFDA since the cleavage product of CFDA can be actively extruded from Saccharomyces cerevisiae (Breeuwer et al, 1994). For measurement of membrane potential, two dyes, both Rh123 and Ox, were chosen as they stain in the opposite sense and may, therefore, have potential as a mutually confirmatory dye pair.…”