Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry

“…In fact, changes in mitochondrial Rh123 accumulation can be independent of ∆Ψm and reflect changes in either mitochondrial mass or the cytoplasmic concentration of the probe. An example of the former situation has been reported for an ADP\ATP translocator mutant (Petit et al, 1996). Such a mutant has a nearly normal ∆Ψm ; however, the decrease observed in ∆Ψm by Rh123 staining is associated with a decrease in mitochondrial mass, assessed by cardiolipin staining (by nonyl acridine orange dye).…”

Assessment of viability of microorganisms employing fluorescence techniques

“…The properties of the ideal membrane potential probe are discussed by Lolkema (66) and summarized here: (i) the probe should pass rapidly the membrane, (ii) it should not bind to the membrane or other constituents, (iii) it should be detectable at very low concentrations, and (iv) it should be biologically inert. Distributional fluorescent probes applied in microbiology are Rhodamine 123, positively charged carbocyanines such as 3,3-dihexyloxacarbocyanine iodide (DiOC 6 (3)), 3,3-diethyloxacarbocyanine iodide (DiOC 2 (3)), and 3,3'-dipropylthiadicarbocyanine iodide (DiS 3 (5), and the negatively charged bis-(l,3-dibutylbarbituric acid)trimethine oxonol (DiBAC 4 (3)) (23,51,55,71,72,73,83,94,97,142,145,154). Rhodamine 123, DiOC 6 (3) and DiBAC 4 (3) can all be excited by the 488 nm line and emit green fluorescence.…”

“…Significantly, in eucaryotic cells rhodamine 123 accumulates preferentially in the mitochondria, due to the high membrane potential present in this organelle (26,113). Less hydrophobic cyanines are also expected to accumu-General introduction late into mitochondria (94,97). However, a high intramitochondrial carboxycyanine concentration may well be toxic to the cells, and the fluorescence is likely to be quenched (94,125).…”

Ectopic Potassium Uptake in trk1 trk2 Mutants ofSaccharomyces cerevisiae Correlates with a Highly Hyperpolarized Membrane Potential

“…For propidium iodide, a 590-nm dichroic filter and a 610-nm LP absorbance filter were added. Because the cellular fluorescence was size dependent, we normalized all the results measuring the fluorescence of single small cells (25). In all experiments, a control sample of wild-type cells (W303.1A strain) grown in the arginine medium at 0.5 mM K ϩ was analyzed in parallel with the other samples, and the fluorescence values given by the flow cytometer were always referred to the fluorescence of the control cells and expressed as a percentage.…”