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. Such a view is further supported by our experience that the success of the double staining with methyl green-pyronin is very much dependent on the proportion of the two dyes which are applied to the slides as a mixture. If pyronin is present in excess, chromatin stains less green and thus takes lavender or even pink-lavender coloration. On the contrary, when the concentration of pyronin is too low as compared with that of methyl green, PNA stains very faint pink and DNA stains bright green. It would be expected that the nature of the differential staining with methyl green-pyronin may further be disclosed by finding out some more or less effective substituents of either methyl green or pyronin in order to obtain a comparable result. Attempts to replace pyronin with other basic dyes of similar coloration have hitherto not succeeded with only one exception, namely with acridine red (Grubler), a basic dye which -was already reported by Ehrich et al. ('49) to be as effec tive as pyronin in this respect. However, from a study of paper chromatography acridine red was found very much alike to ordinary pyronin. Acridine red seems even superior to pyronin by its far more deep staining of nucleic acids. It stains, of course, DNA as well as PNA when applied alone to the tissue section. Other reddish or pink basic dyes were found to be inferior to pyronin in differentiating nucleic acids of the cell, since they stain cytoplasm just as pyronin does, but they compete with methyl green for combining with DNA, chromatin, stains, thus in more or less reddish tinge. An effective substituent for methyl green is iodine green which is

1 Staining tissue sections with the two dyes separately one after another in either order gave less satisfactory differentiation of nucleic acids. 1952 Differential Staining of Nucleic Acids . 319 closely related to methyl green in one respect that two basic dissociating groups are present within the molecule. Malachite green (cf. Ehrich et al., 49, Kurnick, '50a) was found less effective, and Gentian violet by the far inferior to methyl green, although the results obtained by these dyes in combination with pyronin suggested the same trend as methyl green in differentiating nucleic acids of the cell. When applied independently, malachite green stains both DNA and PNA of the cell; it stains PNA less intensely than DNA but does so more prominently than methyl green does. The result with Genitian violet is more advanced towards the same direction, but staining of PNA with this dye is certainly unsatisfactory as compared with thionin or toluidine blue which are recommended as good stains of nucleic acids in general. Malachite green-pyronin stains PNA pink while it stains DNA blue-violet. Gentian violet-pyronin stains PNA pink-lavender and DNA blue-lavender, differentiating effect thus being much inferior to methyl green-pyronin. In both of these cases basophilia of the nucleolus is not satisfactorily differentiated from the remainder of the basophilic structures of the nucleus, or DNA. It should be mentioned that for obtaining such results the proportion of these triphenylmethane dyes to pyronin must be much more lowered than in the case of methyl green-pyronin; otherwise the differentiation of PNA and DNA is highly unsatisfactory, a bluish shade predominating in the cytoplasm. It should be added here that rosaniline, being the simplest basic triphenylmethane dye, stains both DNA and PNA intensely. These findings suggest that staining effect of nucleic acids with basic dyes of triphenylmethane series is somewhat different from that with thiazine and other related groups of basic dyes, PNA staining less prominently with the former groups than the latter. Furthermore, the ability of staining PNA seems to be reduced progressively with the introduction of methyl groups into the amino groups of triphenylmethane: thus, the number of methyl groups per nitrogen atom is in rosaniline 0, in Gentian violet somewhat variable but probably between 1.7-2.0, in malachite green 2.0 and in, methyl green 2.5, and the shifting of absorption maxima of these dyes apparently parallels to this property. Of course a detailed quantitative investigation would be required to establish the presence of such correlation, but attention might be given to the fact that differentiation in staining of PNA and DNA to an appreciable extent is realized by combination of dyes which stain PNA less intensely than DNA with pyronin or acridine red, the most typical example of such peculiar dyes being methyl green. And, although it may be fully fortuitous, in the series of such dyes the less a dye stains PNA when applied independently, the more effectively competes it with pyronin for staining DNA. Accordingly, methyl green is an ideal dye 320 A. SIBATANI Cytoiogia 16

for differentiating PNA (and also dDNA) and pDNA when applied in combination with pyronin. In this connection it should be added that fixation affects the result of methyl green-pyronin stain appreciably. Thus .nuclei stain in purer green with Carnoy than with formalin as the fixative. The. most bril liant results have been obtained with frozen-dried preparations.

Discussion I am aware that the observations presented above offer by no means conclusive evidence for the mechanism of differential staining of nucleic acids with methyl green-pyronin, especially because they rest upon qualitative experiments only. Several papers have hitherto'been published by Kurnick, (50a and b), Kurnick and Mirsky ('50), Kurnick and Foster ('50) and Pollister and Leuchtenberger ('49), in which quantitative aspects of the interactions of dyes and nucleic acids are discussed on the basis of chemical as well as cytochemical estimations of the reactions involved in tissue staining. However, some conflicting data are found among them. It seems, therefore, to be desirable to discuss on some of such discrepancies of the views represented by different investigators in comparison with implications of my own data, and to stress my feeling that about the underlying mechanism of methyl green-pyronin staining there is much uncertainty at the present time, which should be eliminated in future studies. Contradictory results published by Leuchten berger, ('50), Pollister and Leuchtenberger, ('49) and Taft, ('51) may partially be due to the unequality of staining procedures employed. However, the purity of pyronin must also be questioned seriously, because paper chromatography of pyronin has shown the co-existence of many components exhibiting different Rf values and different colors in every sample hitherto tested (cf. Sibatani and Fukuda, '51). It may be that staining of protein with pyronin as reported by Pollister and Leuchtenburger ('49) may be due to contaminants of pyronin (Sibatani, '49b). Kurnick and Mirsky ('50) and Leuchtenberger ('50) are in agree ment in pointing out that staining results or reaction in vitro of nucleicc acids with pyronin are of poor reproducibility and tissue staining with pyronin has nothing but qualitative significance. But it seems also that experimental results using purified methyl green alone are sometimes contradictory to conclusions obtained from other experimental sources. Pollister and Leuchtenberger ('49) and Kurnick ('S0a) have claimed that out of various types of nucleic acids methyl green combines speci fically with pDNA. However, it has been observed by Taft ('51) and by this author that also PNA of the fixed cell can be stained faintly with methyl green if slides are treated by this dye alone. This may be due to the unequality in differentiation procedure swith alcohols, as 1952 Differential Staining of. Nucleic Acids. 321 the former authors have employed much more exhaustive washing of the stained slides than the latter. Nevertheless, staining reaction against methyl green alone of dDNA produced by the treatment of fixed cells with warm HCl presents another question. While cellular dDNA thus produced stains exclusively with pyronin if both methyl green and pyronin are present in staining solution, no reduction of methyl green-stainability of dDNA could not be detected when slides are sub jected to the addition of purified methyl green alone. This finding is in apparent harmony with. the quantitative data of Di Stefano ('48). Kurnick ('50a) ascribed Di Stefano's observation to the contamination of methyl green with crystal violet, but such an . explanation can not hold in the result of this author, because the purified dye was employed in this case. On the other hand, Kurnick's quantitative deter mination by microspectrophotometry clearly speaks against these data (Kurnick, '50b), This discrepancy may again be due to the difference in the manner of differentiation employed. However, the author's finding that even with the same differentiation procedure dDNA stains with pyronin if this dye together with methyl green is applied but it does stain with methyl green intensely if the latter dye alone is used for staining the slides remains to be explained. Taft ('51) has questioned the specificity of methyl green to pDNA on the basis of an experiment in which exposure of the fixed cells to low and high pH's has failed to change the staining properties of DNA against methyl green-pyronin, despite the pH's employed are known to disaggregate pDNA (Gulland, '47). Taft's contention may be weakened by considering the possible difference in the readiness with which pDNA in non-soluble form and that in dissolved state are disaggregated by pH change. Elevated temperature would be necessary to bring about dis aggregation of pDNA in the fixed tissues by such means.. As regards the stainability of pDNA with pyronin, the situation is much the same as with methyl green. While Kurnick ('50) has observed only faint staining of pDNA with pyronin, experiments of Taft ('51) and myself ('49a) are evidently in disaccord with Kurnick's result. Moreover, Leuchtenberger ('50) has commented that there is no detect able difference between pDNA and dDNA in the stainability to pyronin as measured microspectrophotometrically. Vercauteren's experiments ('50) show, on the other hand, that the reduction of the affinity to methyl green of desoxyribonucleohistone caused by agents which should disaggregate pDNA is accompanied by an unquestionable increase in its binding capacity to pyronin as well as to methylene blue, which is originally in a very low level. However, Vercauteren's experiments must be interpreted with precaution, because the effect of existing protein, histone, could not be neglected (Kurnick and Mirsky, '50; Siba tani, 52). 322 A. SIBATANI Cytologia 16

As to the possibility of replacing pyronin with other reddish basic dyes to obtain results similar to that with methyl green-pyronin, the result reported by Pollister and Leuchtenberger ('49) is not reconcilable with that of the author. They state that employment of basic fuchsin along with methyl green to stain fixed tissues causes no detectable decrease in the intensity of methyl green staining of the nuclei. Accord ing to my own experience, however, the introduction of basic fuchsias in place of pyronin considerably affects the selective staining of DNA with methyl green. Such an "impure" staining of DNA can also be encountered even with methyl green-pyronin. C. Vendrely ('50) has shown that this is due to partial disaggregation of pDNA influenced by the fixatives. This has been confirmed by our experiments, and frozed dried preparations have given the most satisfactory staining as regards both intensity and differentiation of the two types of nucleic acids. However, experiments conducted by the author and his associates suggest that the preponderance of pyronin in the staining solution also affects the "selective" staining of DNA with methyl green decidedly, while that of methyl green gives DNA stained in bright green. This result should be only natural if pDNA, as we claim, is to be stained with pyronin as well. Experiments on the staining of nucleic acids in vitro with methyl green and pyronin, or on their reactions in the aqueous solution were conducted by Kurnick ('50a), Kurnick and Foster ('50), Kurnick and Mirsky ('50), C. Vendrely ('50), Taft ('51), and the author ('49a). Reactions of nucleic acids with the individual dyes will be considered here, because with mixtures of the two dyes the results of different authors indicate the same direction. Kurnick ('50a), or Kurnick and Foster ('50), using relatively dilute dye solutions, recognized a remark able difference in the affinity to methyl green between pDNA on the one hand and dDNA and PNA on the other. Furthermore, he was able to show that with pyronin the entire situation is exactly reversed, pDNA showing thus, in contrast to dDNA and PNA, no more than a slight tendency to combine with pyronin. In author's experiment, however, PNA as well as pDNA produced precipitate _of dye nucleate with methyl green and pyronin of moderate concentration (for example, 0.1 per cent aqueous solution). Kurnick's experiments ('50b) are consistent throughout the parts in vitro and in situ, and suggest the presence of a true selectivity in staining reaction of methyl green-pyronin, but observations of Leuchtenberger (50), Sibatani (49a) and Taft ('51) contradict with his results, as discussed above. The results obtained by the author do not favor the hypothesis of true selectivity of this staining reaction. They suggest rather that the differential staining of methyl green-pyronin could be due to preferential 1952 Differential Staining of Nucleic Acids . 323 tendencies of methyl green and pyronin to combine with nucleic acids of higher and lower degree of polymerization (or association) , respec tively. The idea is supported by a series of the observations of our side, but the appearent poor stainability of PNA in the fixed cell against methyl green remains to be explained. From such a view, however, methyl green-pyronin is merely a fortunate double stain, and attributing too much weight on its chemical specificity does not seem justifiable. This view seems further to be ,supported by the evidence that besides methyl green and its allies malachite green and also Gentian violet are able to give similar differential staining when used in conjunction with pyronin, which is, however, less satisfactory than methyl green-pyronin. Malachite green was already indicated by Kurnick ('50a) as a possible substituent of methyl green, although somewhat inferior to the latter. Lastly it should be emphasized that if the results presented here and some of the other investigators which are in apparent accord with them were incorrect, one point would remain to be explored. This is a question of why such discrepancies have emerged from hands of different investigators. Answering to this question and detecting unknown critical factors which should affect the results are urgently necessary, because the presence of such an apparent lack of reproducibility would evidently restrict the possibility of general and successful applications of this tool on various problems of cytochemistry.

Summary

1. The nature of the differential staining of nucleic acids with methyl green-pyronin was investigated by way of comparing tissue staining with methyl green and pyronin, individually and in mixture, with experiments in which aqueous solutions of these dyes, both alone and mixed together, were added with different types of nucleic acids and nucleoproteins and the precipitates formed were observed microscopically. 2. Evidences have been presented which suggest that differential staining with methyl green-pyronin is not due to true selectivity of non-disaggregated DNA to methyl green on the one hand and of dis aggregated DNA and PNA to pyronin on the other hand, but rather to preferential tendencies of these nucleic acids in combining with respective dyes in fixed tissues as well as in aqueous solutions. 3. Some of the discrepancies among the data published by different investigators on the nature of methyl green-pyronin staining are discussed.

Acknowledgements. I would express my hearty thanks to Dr. O. Itikawa, Director of the Medico-Biological Institute, Minophagen Pharma ceutical Co., for his interest in this work and encourragement through out the course of investigation. Thanks are also due to late Miss H. 324 A. SIBATANI Cytologia 16

Kawamata, Miss S. Takeda, Minophagen Pharmaceutical Co., and to Dr. H. Matsuda, Mr. M. Fukuda and Mr. O. Harikane, University of Osaka for their assistance. Further it is my pleasant duty to mention my indebtedness to Prof. S. Hosoya and Dr. T. Homma, Institute for Infec tious Diseases, University of Tokyo, for facilities in utilizing laboratory equipments in the course of extracting pDNA used in this investigation.

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