Vol. 103, No. 2 October, 1952 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY STUDIES ON BASOPHILIA OF NUCLEIC ACIDS : THE METHYL GREEN STAINABILITY OF NUCLEIC ACIDS 1 MAX ALFERT Dcpt. of Zoology, University of California, Berkeley 4, Calif. A number of reports in the recent cytochemical literature have dealt with the use of the basic dye methyl green as a specific and quantitative stain of desoxyribo-nucleic acid (DNA). Pollister and Leuchtenberger (1949) first demonstrated by photometric methods the high degree of reproducibility of nuclear methyl green staining in mouse liver and discussed the impairment of stainability caused by pre-treatment of slides with hot water. Kurnick (T950a) concluded from test tube experiments that methyl green combines specifically with highly polymerized DNA only. He used the stain for quantitative determinations of DNA in fixed histo-logical material (1950b; Kurnick and Herskowitz, 1951) and to measure depoly-merase activity of serum (1950c). Vercauteren (1950) and Devreux ct al. (1951) also tested the dye-binding capacity of DNA solutions under various conditions and concur with Kurnick's opinion. The methyl green stainability of some types of degenerating chromatin was investigated by Leuchtenberger (1950), Klemperer ct al. (1950) and Korson (1951), and the influence of x-radiation on stainability of nuclei was tested by Moses ct al. (1951) and Harrington and Koza (1951). Impairment of methyl green stainability as it was found to occur in some in-stances, and as it can be induced by treatment of slides with hot water or mild acid hydrolysis, would, according to Kurnick's view, be due to a depolymerization of the DNA. Pollister and Leuchtenberger (1949) have also used the term "depoly-merization" to designate an unspecified change in the configuration of the DNA molecule without implying that depolymerization in the chemical sense (breakage of internucleotide linkages) was necessarily involved. Although methyl green has long been regarded as an exclusive nuclear stain, there are some indications that it does not possess absolute specificity for DNA, since its ability to distinguish DNA from ribonucleic acid (RNA) depends on the type of fixation used : following fixation in acetic alcohol, staining is entirely re-stricted to nuclei in many types of tissues, while after fixation in formalin, specificity is often lost and cytoplasmic RNA also binds methyl green strongly. Furthermore, in some tissues RNA is methyl green stainable even after acetic alcohol fixation. 1 The work reported here started when the author was a Public Health Research Fellow of the National Institutes of Health. At present the author receives support from the University of California Board of Research and from California Cancer Research Funds. 145
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