Further observations on the chemistry of Pararosaniline-Feulgen staining

Summary

Pararosaniline-Feulgen staining of cells in suspension produces nucleus- and chromatin-specific fluorescence as well as color. Experiments were designed to test postulated reaction mechanisms responsible for the fluorescent staining with the nonfluorescent pararosaniline. The reduction in fluorescent-staining intensity by pretreatment of cells with 2.2×10⁻²M K₂S₂O₅ tends to rule out the alkysulfonic acid pathway; conditions favoring the formation of this intermediate reduce staining intensity. The fluorescence enhancement, observed when cells stained in pararosaniline without K₂S₂O₅ are post-treated with K₂S₂O₅, suggests that there is an initial Schiff-base linkage between pararosaniline and an aldehyde of hydrolyzed DNA, and that this linkage is stabilized in the presence of K₂S₂O₅. Microspectrofluorometer measurements of cells stained at various pararosaniline concentrations in 2.2×10⁻²M K₂S₂O₅, show that the fluorescence emission maximum ranges from about 627 nm at 3.1×10⁻³M pararosaniline to about 604 nm at 3.1×10⁻⁵M. All of the employed staining protocols appear to produce the same fluorescent product, perhaps a heterocyclic pyronin analog formed from pararosaniline. Flow microfluorometric analysis of cells stained in suspension verified that the relative fluorescence intensity represents relative DNA content. Staining at reduced pararosaniline concentration (3.1×10⁻⁴M) reduces the coefficient of variation of the flow microfluorometric histograms, showing that maximum quantitation does not necessarily correlate with maximum staining intensity.