Differential Staining of Nucleic Acids. I. Methyl Green-Pyronin Owing To

1952 315 Differential Staining of Nucleic Acids. I. Methyl green-Pyronin By Atuhiro Sibatani Medico-Biological Institute, Minophagen Pharmaceutical Co., Tokyo, and Microbial Diseases Research Institute, University of Osaka,' Osaka, Japan Received December 7, 1951 Owing to the growing knowledge about the biological significance of nucleic acids, interests in the mechanism of staining of nucleic acids have revived in recent years. Efforts have been centered to elucidate the mechanism of classic Unna-Pappenheim stain under the modern aspects of nucleic acid chemistry. In the last few years many papers attacking this problem have been made public concurrently (Kurnick, '50a and b; Kurnick and Foster , '50; Kurnick and Mirsky, '50; Pollister and Leuchtenberger, '49; Taft, '51; C. Vendrely, '50; Vercauteren, '50), among which the author's own papers written in Japanese (Sibatani, '48a, '49a and c) may be enumerated . In this communication the author intends to present a brief summary of the results hitherto obtained by him, which are confined to qualitative aspects of the differential staning of nucleic acids with methyl green-pyronin, and which, therefore, are of preliminary nature. Also some discussions as regards the interpretation of the findings will be included in comparison with those of the other investigators. Materials Dyes For testing various dyes as regards possible contamination with colored materials, and also for identifying them, the technique of paper chromatography with butanol or isopropanol mixed with appropriate amount of water as the solvent system was found to be highly utilizable (Sibatani, '49b; Sibatani and Fukuda, '51.). Methyl green:-Various preparations commercially available in this country may be used with satisfactory results. They were always purified by shaking with chloro form repeatedly to remove the violet contaminant. Pyronin:-Various preparations of pyronin (Merck, Grubler, and Muhlheim, Germany; Ishizu, Japan; and National Aniline Corp., U. S.) were always found to be mixtures of several colored materials. Many of them, however, gave satisfactory results without purification. When necessary, some attempts of purification were made using paper chromatography as an indicator. The following procedure may be recommended: shake filtered one per cent aqueous solution of pyronin with equal volume of chloroform; save water layer; shake the latter with equal volume of n-butanol; discard the water layer; add three volumes of ether and half to equal volume of distilled water to the butanol layer and shake vigorously; the bulk of the pigments now goes into the water layer. Aqueous solution thus obtained may be used directly or mixed with purified methyl green solution in an appropriate proportion to make the solution blue to blue violet. 1 The present adress. 22* 316 A. SIBATANI Cytologia 16 Nucleic acids and nucleoproteins Pentosenucleic acid (PNA):-Merck's yeast. ribonucleic acid; sodium ribonucleate extracted from yeast by Clarke-Schryver ('17) method and purified with chloroform (received through the courtesy of Dr. Shimomura, Department of Chemistry, University of Nagoya); and beef liver PNA extracted by the Davidson and Waymouth ('44) method and purified through barium salt. Desoxypentosenucleic acid (DNA):-A typically fibrous material was obtained from beef spleen by the method of Petermann and Lamb ('48), and considered to be highly polymerized or associated (cf. Jungner et al., '49). This will be called henceforth pDNA. A partially degraded sample was obtained from beef spleen by Feulgen ('14) method using hot alkaline extraction. This will be called below dDNA, and the degradation involved in this and comparable treatments will be expressed as disaggregation. Pentosenucleoprotein:-Extracted from beef liver according to Mirsky and Pollister ('46). Desoxypentosenucleohistone:-Extracted from beef spleen nuclei by the Mirsky and Pollister method ('46). Tissue sections Various tissues of adult rats, especially liver, spleen, pancreas and cerebellum, and rat embryos were fixed with 10 per cent formalin, Carnoy or Helly, and paraffin sections of 5ƒÊ thickness were prepared. Basophilia due to PNA was determined with ribonu clease obtained according to Brachet ('40) or McDonald ('48). DNA was identified by Feulgen reaction. Aqueous solutions of methyl green and pyronin were used for tissue staining. In later experiments 0.2M acetate buffer of pH 4.0 was used as the solvent of the dyes according to Kurnick ('50a). Prolonged treatment of the stained slides with differentiating agents (ethyl and isopropyl alcohols) seemed to destain the cell, so that rapid differentiation essentially similar to that indicated by Taft ('51) was employed to remove excess dyes. Results Essentially the same conclusion as that accepted currently (Kurnick, '50a; Pollister and Leuchtenberger '49; C . Vendrely, '50; Vercauteren, '50) has been reached concerning to the nature of differential staining of methyl green and pyronin: this stain does not appear to differen tiate PNA and DNA, but rather the degree of polymerization (or association) of the polynucleotides, discriminating thus pDNA from both dDNA and PNA. Such an interpretation has resulted from experiments in which dilute aqueous solutions of different preparations of sodium nucleates and nucleoproteins (ca. 0.1 per cent) were mixed with varying amounts of solutions of the two basic dyes (usually 0.1-0.3 per cent) on microscopic slides, dried in 37•‹ incubator, washed with water and alcohol, and then examined under the microscope (Sibatani, '48b). On mixing a small volume of 0.1 per cent solutions of DNA or PNA with an excess of solution of a basic dye, precipitate of dye nucleate was seen to be formed (Sibatani, '48a and c). This property is common to all of the basic dyes examined, methyl green being by no means exceptional in this respect. In a preliminary report I have stated that methyl green is an exception among basic dyes through its non-precipitability with PNA and dDNA (Sibatani, '49a), but this observation was incorrect. 1952 Differential Staining of Nucleic Acids . 317 Of course, stoichiometry of dye nucleates and the readiness with which the individual dyes form precipitates with respective types of nucleic acids might be different with different dyes, but only few data indicat ing the presence of such difference are available at the present time (Kurnick, '50a, Kurnick and Mirsky, '50, Mirsky and Ris, '51). It is now a remarkable fact that on application of an excess of a mixture of methyl green and pyronin in an appropriate proportion to solutions of sodium nucleates or nucleoproteins, PNA, dDNA and pento senucleoprotein produced pink precipitates, whereas pDNA and desoxy pentosenucleohistone gave green precipitates (Sibatani, 49a). From such experiments it has become evident that in the presence of both methyl green and pyronin PNA or dDNA produces insoluble dye-nucleic acid complex preferentially with pyronin while pDNA reacts preferentially with methyl green to form green precipiate. It has been considered that such a situation is responsible to the differential staining of methyl green-pyronin on fixed tissues (Sibatani, 49a). Several additional experiments of staining tissue sections appear now to offer some clues for elucidating the nature of such seemingly selective reactions of methyl-pyronin with the two types of nucleic acids. If tissue sections are stained with pyronin alone, not only PNA but also DNA take the stain vigorously and there is no indication that DNA is inferior to PNA in its staining capacity against pyronin. This is compatible with the experiment on the precipitability with pyronin of isolated nucleic acids. On the other hand, when the tissue sections originating from the same series are subjected to the action of methyl green alone, PNA does stain but seems to be much inferior to DNA in its dye-binding capacity and appreciable staining of the cytoplasm is observable only at portions where the concentration of PNA is known to be high (pancreas or nerve cells). However, the behavior against methyl green of the tissue sections subjected preliminarily to the action of 0.1N HCl at 60•‹ for 25 minutes is more remarkable. DNA of the cell may be assumed to disaggregate into smaller molecular units by such a treatment. Indeed, DNA stained in this case exclusively with pyronin when the mixture of methyl green-pyronin was applied to the section. Control slides treated similarly with distilled water showed DNA stained with methyl green. However, when sections subjected to the treatment with dilute HCl were stained with methyl green alone, DNA did take green color intensely, and a reduction in its staining capacity as compared with the control slide was scarcely conceivable by a visual inspection. It may therefore be concluded that PNA in tissue sections is certainly inferior to DNA in its binding capacity with methyl green, what is difficult to be explained on the basis of the simple qualitative observation 318 A. SIBATANI Cytologia 16 of precipitating reactions" of nucleic acids with basic dyes. However, the -presence of a true selectivity in staining reactions of lower and higher molecular aggregates of nucleic acids against pyronin and methyl green respectively would have to be excluded, because pDNA does stain with pyronin and dDNA and, although to a lesser extent, PNA also do stain with methyl green, when respective dyes are applied separately to the fixed tissues. Thus, we are led to a view that the differential staining of the molecular species of nucleic acids as regards the degree of polymerization (or association) of polynucleotides is to be realized only when both methyl green and pyronin are present simultaneously in the staining solution, and hence could hardly be ascribed to some sort of selective properties of the two types of nucleic acids of the cell in their reaction to the respective ones of these two basic dyes.1 This view is fully supported by the precipitating reactions in vitro of nucleic acids and the dyes. Therefore, a hypothesis of preference rather than of true selectivity of the dyes in combining with nucleic acids seems to be justifiable.

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