RESUMO
A fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), which had been developed previously for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeasts. Fluorescence intensities of the yeast population were measured by fluorescence spectrophotometry upon exposure to antifungal agents after staining with 2-NBDG and were compared to the number of colony forming units (CFU). A good correlation was obtained between the yeast viability, determined by the CFU, and the accumulation of 2-NBDG by yeast cells (correlation constant: r=0.98). Susceptibility testing of amphotericin B and miconazole against yeast strains by plate count and 2-NBDG fluorescence method yielded corresponding results. In conclusion, we found that staining with 2-NBDG is a rapid and sensitive method for the assessment of yeast cell viability.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Antifúngicos/farmacologia , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismo , Anfotericina B/farmacologia , Contagem de Colônia Microbiana , Corantes Fluorescentes , Glucose/metabolismo , Miconazol/farmacologia , Coloração e Rotulagem , Fatores de Tempo , Leveduras/efeitos dos fármacosRESUMO
Single-cell viability assessment by means of plural dye probes require the spectral and temporal analysis of microscopic images of the test cells. To meet this requirement, we have developed a simple and compact spectro-imaging system using an image slicer and a grism. The image slicer was made of a bundle of 100 optical fibers. The field of view is divided into 10 x 10 sections. The spectral data of each section could be recorded every 5 s in the range from 400 to 800 nm at 5 nm resolution. The viability changes of yeast or tobacco single-cells were measured with this system. Using BY-2 cells, for example, the response to a chemical stress of saponin was measured by means of two fluorescent probes. The spectral-spatial-temporal data of fluorescein and DNA bound ethidium bromide provided us with useful information about the dynamic change of cell membrane permeability from which the cell viability was assessed.