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Largely as the result of Brachet's investigations (1), it has become appreciated that the differential staining of tissues with the methyl green-pyronin mixture of Unna and Pappenheim is determined by their nucleic acid components. Thus, the green staining of nuclei was demonstrated to be due to their content of desoxyribonucleic acid, and the red staining of the cytoplasm to be determined by the ribonucleic acid content. It is the purpose of this paper to report some observations on the nature of this phenomenon of differential staining of similar acid substrates by two basic dyes.In tissues, desoxyribonucleic acid apparently occurs as a much higher polymer than ribonucleic acid (2). It appeared to us that this difference in the degree of polymerization might be the determining factor in the differential staining with methyl green-pyronin. It is the purpose of this paper to present observations on the differences in staining between highly polymerized and depolymerized desoxyribonudeic acids. As will be seen, these tend to confirm the hypothesis that the affinity of nucleic acids for methyl green and pyronin is a function of their state of polymerization: methyl green stains preferentially highly polymerized nucleic acid, while pyronin hasa special affinity for low polymers (depolymerized desoxyribonucleic acid and ribonucleic acid). EXPERIMENTAL Nucleic Acids and Nucleoproleins1. Desoxyribonuclcohistone (DNH) was prepared from calf thymus chromosomes by extraction with 1 K NaCI (3). The viscous solution was centrifuged at high speed to remove "residual chromosomes" and nudcoU. The slightly opalescent viscous supemate was stored in the cold with toluene as preservative. The phosphorus analysis of this solution was 0.27 mg./cc.2. Desoxyribonudeic acid (DNA) 1 was prepared from the DNtt solution by repeated mixing ill a Waling blendor with chloroform-octyl alcohol (4) to remove

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We have observed (1) that the selective staining of nucleic acids by methyl green and pyronin is a function of the relative states of polymerization of the nucleic acids. The purpose of this paper is to present studies on the stoichiometry of the reactions, in order to elucidate the mechanism of the selectivity and to explore the possibility of quantitative histochemical application.The studies, as applied to pyronin, met with only very limited success. Unlike methyl green, which appears to form a stable compound with polymerized DNA, pyronin-stained RNA or depolymerized DNA-precipitates lose stain continuously when washed with alcohol or aqueous buffers. The "end point" which is eventually reached, represents the retention of only a few per cent of the original dye bound (cf. Table VI and Table VIII of the preceding paper (1)). Consequently, the possibility of non-specifically adsorbed dye to the pyronin-nucleic acid complex which precipitates on the addition of pyronin to nucleic acid solutions (in high concentrations of dye) cannot be excluded, thus vitiating the significance of the stoichiometric data based upon precipitation experiments in which combined dye was determined by difference between that remaining in the supemate and the original concentration. Likewise, washing probably results in considerable dissociation of the complex so that the "end point" is also not reliable for stoichiometry. However, Table VI (preceding paper (1)) based on precipitation experiments, suggests that the low polymers of NA bind approximately one pyronin molecule for each pair of phosphoric acid groups, indicating that both amino groups are functional in the linkage.Referring again to Table VI (1), a significant difference in staining is noted between DNA and DNH. We doubt that the difference between RNA and RNP is significant, since the preparations are obtained from different sources by unrelated methods. The possibility that the slightly greater staining of RN-P is due to the staining of protein by pyronin is rendered unlikely, but by no means excluded, by the fact that heat-coagulated beef serum fails to stain with pyronin when treated in the same manner as the nucleic acid precipitates.

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