Carbocyanine dye diS-C3(3) was repeatedly employed in monitoring the plasma membrane potential of yeast and other living cells. Four methods of measuring and evaluating probe fluorescence signal were used in different studies, based on following fluorescence parameters: fluorescence intensity emitted within a certain spectral interval, F(580)/F(560) fluorescence emission ratio, wavelength of emission spectrum maximum, and the ratio of respective fluorescence intensities corresponding to the diS-C3(3) bound to cytosolic macromolecules and remaining dissolved in the aqueous cell medium (i.e., unbound, or free). Here we show that data corresponding to the three latter spectral assessments of diS-C3(3) accumulation in cells is mutually convertible, which means that their alternative use cannot lead to ambiguities in the interpretation of the results of biological experiments. On the other hand, experiments based on the effortless measurements of fluorescence intensities should be interpreted cautiously because controversial results can be obtained, depending on the particular choice of cell-to-dye concentration ratio and emission wavelength.

• Keywords
• Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials * MeSH
• Saccharomyces cerevisiae chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

The long-term action of recommended (RC) and near-recommended concentrations of several commercial biocides (Lonzabac 12.100, Genamin CS302D, benzalkonium chloride and 2-phenoxyethanol) on cells of S. cerevisiae wild-type strain DTXII was described using plating tests while short-term effects were determined using the potentiometric fluorescent probe diS-C3(3) that detects both changes in membrane potential and impairment of membrane integrity. A 2-d plating of cells exposed to 0.5xRC of benzalkonium chloride and Genamin CS302D for 15 min showed a complete long-term cell killing, with 2-phenoxyethanol the killing was complete only at 2xRC and Lonzabac caused complete killing at RC but not at 0.5xRC. The diS-C3(3) fluorescence assay performed immediately after a 10-min biocide exposure revealed several concentration-dependent modes of action: Lonzabac at 0.5xRC caused a mere depolarization, higher concentrations causing gradually increasing cell damage; benzalkonium chloride and Genamin CS302D rapidly damaged the membrane of some cells and depolarized the rest whereas 2-phenoxyethanol, which had the lowest effect in the plating test, produced a concentration-dependent fraction of cells with impaired membranes. Cell staining slightly increased during the diS-C3(3) assay; addition of a protonophore showed that part of the remaining undamaged cells retained their membrane potential. Comparison of short-term and long-term data implies that membrane depolarization alone is not sufficient for complete long-term killing of yeast cells under the action of a biocide unless it is accompanied by perceptible impairment of membrane integrity. The results show that the diS-C3(3) fluorescence assay, which reflects the short-term effects of a biocide on cell membranes, can be successfully used to assess the microbicidal efficiency of biocides.

• MeSH
• Anti-Infective Agents pharmacology   MeSH
• Benzalkonium Compounds pharmacology   MeSH
• Cell Membrane drug effects   MeSH
• Ethylene Glycols pharmacology   MeSH
• Fluorescent Dyes metabolism   MeSH
• Spectrometry, Fluorescence MeSH
• Carbocyanines metabolism   MeSH
• Membrane Potentials drug effects   MeSH
• Microbial Sensitivity Tests methods   MeSH
• Cell Membrane Permeability drug effects   MeSH
• Saccharomyces cerevisiae drug effects   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Anti-Infective Agents MeSH
• Benzalkonium Compounds MeSH
• Ethylene Glycols MeSH
• Fluorescent Dyes MeSH
• Carbocyanines MeSH
• phenoxyethanol MeSH Browser