Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5-(and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail. The uptake rate of the prefluorochromes increased in direct proportion to the concentration and was not saturable, which suggests that transport occurs via a passive diffusion process. The permeability coefficient for cFDA was 1.3 ؋ 10 ؊8 m s ؊1 . Once inside the cell, the esters were hydrolyzed by intracellular esterases and their fluorescent products accumulated. FDA hydrolysis (at 40؇C) in cell extracts could be described by first-order reaction kinetics, and a rate constant (K) of 0.33 s ؊1 was calculated. Hydrolysis of cFDA (at 40؇C) in cell extracts was described by Michaelis-Menten kinetics with an apparent V max and K m of 12.3 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 and 0.29 mM, respectively. Accumulation of fluorescein was most likely limited by the esterase activity, since transport of FDA was faster than the hydrolysis rate. In contrast, accumulation of carboxyfluorescein was limited by the much slower transport of cFDA through the cell envelope. A simple mathematical model was developed to describe the fluorescence staining. The implications for optimal staining of yeast cells with FDA and cFDA are discussed.

A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.

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.

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both Rphycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.The free-living amoeba Naegleria fowleri (3, 16), found in diverse freshwater environments, produces a rapidly fatal primary amoebic meningoencephalitis after exposure to contaminated water (7,11,12). N. fowleri infects mostly young and healthy people swimming in contaminated water. Symptoms occur in a few days, followed by a dramatic clinical course and death. Therefore, risk prevention is essential and necessitates environmental monitoring using a rapid and accurate assay to distinguish pathogenic N. fowleri from other free-living amoeba in water samples.Current methods for detection and enumeration of Naegleria species are based on culture techniques (8) followed by identification using monoclonal antibodies (19,21), PCR (10,20), or enzyme electrophoresis (15). Additionally, isolates are tested for pathogenicity in mice. These methods are timeconsuming, and novel methods are being developed to increase the sensitivity and rapidity of detection and thus reduce the amount of time required to obtain results. The main challenges for the development of an assay are to provide tools for the real-time monitoring of the pathogen in the aquatic environment which are highly quantitative and sensitive.Epifluorescence microscopy and flow cytometry are commonly used for the detection and enumeration of cells after fluorescent staining (1, 6). However, none of these techniques can be applied to the detection of low concentrations of pathogens in the aquatic environment because of their low quantitative sensitivity (10). The ChemScan system (Chemunex, Ivry, France) is a recently developed solid-phase cytometer that uses fluorescent labeling of microorganisms after concentration of organisms by filtration on a membrane in combination with an automated detection and counting system (13, 23). Solid-phase cytometry is the only technique that allows the accurate enumeration of rare events (down to one cell on a filtration membrane), providing the same sensitivity as traditional culture methods (10). This sy...

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