MICROSPECTROPHOTOMETRIC ANALYSIS OF ACCUMULATION OF THE FLUORONES K-FLUORESCEIN, ROSE-BENGAL AND PHLOXINE RED IN LIVING PLANT-CELLS

Spectrophotometric investigations of dye solutions in different media and of living stained cells from the upper epidermis of the scaleleaf of Allium cepa were carried out with the dyes K-fluorescein, rose Beng al and phloxine red to elucidate the mechanism of the accumulation of these dyes in the cytoplasm, the nucleus and the cell sap. Thin layer chromatography and paper electrophoresis indicate that the K-fluoresce in used here contains no detectable contaminants, Besides the main com ponent, rose Bengal contains two components in small quantities with R f values of 0.64 and 0.57, plus three more components in traces. Besid es the two main components (Rf values of 0.83 and 0.73), phloxine red also contains five more components in traces. Electrophoretic investig ations reveal that in aqueous solution the fluorones rose Bengal and p hloxine red from pH 2.0-11 show a migration toward the anode. K-fluore scein from pH 2.9-10.4 shows a migration toward the anode, but at pH 1 .9 a migration toward the cathode. By shaking aqueous solutions of K-f luorescein, rose Bengal and phloxine red at different pH values with d ifferent organic solvents, the above used stainings show different spe ctral absorption curves according to the polarity of the solvent. The position of the absorption maxima acid the shape of the absorption cur ves of these three anionic dyes lead to the conclusion that the staini ng of the living cytoplasm and nucleus is due to ion accumulation by m eans of the ''ion trap mechanism'' within the aqueous, phase of the cy toplasm (cytosol) and the nucleus. Adsorption of dye particles in the protein phase of the cytoplasm cannot be excluded. There seems to be a fundamental difference in the vital staining of the protoplasm by ani onic and cationic dyes, the latter apparently accumulating as neutral dye molecules in the lipid phase of the protoplasm. The concentration of the dyes used in the living cytoplasm (cytosol) is approximately 0. 2-0.05%. During natural and artificial displacement of K-fluorescein f rom the cytoplasm to the vacuole, it appears that accumulation of the dye within the vacuole is performed through an ion trap mechanism In t he form of bivalent ions. Along with natural displacement, it is possi ble that ion accumulation also occurs in metabolic products.