The correlation between uptake of methyl green and Feulgen staining intensity of cell nuclei. An image analysis study

Paraffin sections of rat tissue fixed in either formaldehyde solution (3.6% w/v) or in Carnoy's fluid were stained using standardized Methyl Green-Pyronin procedures with the dyes used either simultaneously or in sequence. The sections were evaluated for the uptake of the two dyes by cell nuclei, nucleoli and cytoplasm using colour TV-image analysis. The parameters measured were integrated optical density and the surface area of the object. The sections were then destained and a Feulgen reaction was performed. The coordinates of the cells measured after the simultaneous Methyl Green-Pyronin method were stored in the computer, making it possible to measure the same cells in the Feulgen-restained sections. Image analysis gave results which invalidate the sequential methods as opposed to the simultaneous method. Mean optical densities were significantly increased for both dyes with the simultaneous method after formaldehyde fixation as compared to Carnoy fixation. The quantitative correlation of Methyl Green and DNA in the simultaneous technique was found to parallel exactly that of the Feulgen stain. In conclusion, the simultaneous Methyl Green-Pyronin technique is recommended while the sequential methods seem to be of less value.