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When methylene blue is used to stains fixed tissue after acetylating to block competing protein basic groups, the amount of dye bound per cell reaches a limit largely independent of staining time, temperature and dye concentration and to a lesser extent pH. After extracting the nucleic acids, the tissue is unstained except for acid polysaccharide-containing areas. The intensity of staining does not depend upon alcoholic differentiation and the dye is used as a progressive stain. The amount of dye bound is stoichiometric with nucleic acid concentration. This is demonstrated by comparing the average dye binding to the nucleic acid concentrations in gelatin-nucleic acid model slides and for isolated HeLa nuclei. Although methylene blue is a metachromatic dye, analysis of absorption curves show that there is no significant differences in curve shape when the dye is bound either to tissue ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Hence total dye binding of nuclear chromatin may be used as an index of dye bound to both DNA and RNA. RNA may be extracted by a short hydrolysis in 1 N HCl; dye binding after such extraction represents dye bound to DNA. The amount of dye bound to chromosomal RNA can be estimated by the difference between these two figures and DNA/RNA ratios for methylene blue-stained chromatin can be determined in this manner.

A METHOD FOR THE CYTOPHOTOMETRIC ESTIMATION OF NUCLEIC ACIDS USING METHYLENE BLUE