Practice question

Viable protoplasts fluoresce bright green when stained with fluorescein diacetate FDA, vital fluorescent dye used routinely for viability assessment before fusion or transformation to ensure batch quality above 80 percent. FDA itself is non fluorescent lipophilic molecule that diffuses across intact plasma membrane permeable only to living cells with intact barrier. Inside cytoplasm active non specific esterases present exclusively in living cells hydrolyze ester bonds releasing fluorescein highly fluorescent polar molecule that accumulates inside membrane intact vesicle because its negative charge prevents efflux, emitting green fluorescence 520 nm upon excitation blue light 490 nm under fluorescence microscope. Dead protoplasts with compromised membranes lack esterase activity and cannot retain fluorescein thus remain non fluorescent and also allow entry of counter stain propidium iodide marking nucleus red. Assay provides rapid quantitative estimation of viability percentage and metabolic vigor as fluorescence intensity correlates with esterase activity and capacity to regenerate wall and divide in culture medium with hormones. Dual staining FDA propidium iodide enables simultaneous live dead discrimination via flow cytometry sorting heterokaryons for somatic hybridization applications.