Spectrophotometric characteristics and assay of pure pyronin Y
SummaryThe spectrophotometric characteristics of analytically pure pyronin Y have been investigated. Addition of metal ions (Fe3+, Zn2+, and Mg2+) and of dextrin were shown not to influence the absorption characteristics. The composition of the solvent strongly influenced the value of the extinction coefficient. Aqueous ethanolic solutions with a content of about 50% ethanol gave higher ε-values than those found for more concentrated ethanol solutions. The difference can be explained by the existence of a solvent-solute complex in the less concentrated ethanol solutions. A new spectrophotometric assay is proposed using the ε-value 11.7×104 lmol−1 cm−1 found in aqueous ethanol (52%) as standard.
Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the Methyl Green-Pyronin technique with the Gallocyanin chromalum and Feulgen procedures using image cytometry
SummaryFor simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA.The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen analtion. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm.After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum.We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.
Thin-layer chromatography of some cationic dyes commonly used in histology.
A number of cationic dyes commonly used in histology may be conveniently and effectively analysed using the thin-layer chromatographic system of Marshall and Lewis. Commercial samples of Acridine Orange, Crystal Violet, Janus Green B,Methyl Violet, Neutral Red, Pyronin B, Pyronin Y(G), Safranin, Victoria Blue B and Victoria Blue 4R have been analysed with this system. All have been found to be complex mixtures of coloured components. Chromatographic data on these components are presented.