Microspectrophotometric Analysis of Metachromatic Staining of Nucleic Acids

Abstract

Azure B, a metachromatic dye, when used at pH 4, stains nucleic acids and acid polysaccharides specifically in Carnoy-fixed plant and animal tissues. Absorption curves of the dye bound to nucleic acids in tissues were detd. with a micro-spectrophotometer. The orthochromatic absorption peak ([alpha] -peak) at 650 m/x is always present, while a 2d, [beta]-peak at 590 m[mu] and a 3d, [mu]-peak near 545 m[mu], may also occur, the last giving rise to the metachromatic color observed under some staining conditions. The [alpha]_ and [beta]-peaks are identical with those of a concd. aqueous soln. of the dye, while the [mu]-peak is in approx. the same position as the [mu]-peak of the dye combined with certain chromotropes in soln. The repeated occurrence of these 3 peaks supports an extension of the theory of metachromasy based on dye polymerization in soln. to explain metachromasy in tissues. The effect of alterations of the staining conditions appears to be satisfactorily interpreted by this theory. Increasing dye concn. pH, and temp. (up to 60[degree]C) of the staining soln. increases metachromasy of nucleic acid. This may result from the increase in the amt. of dye bound, which presumably would increase the probability of dye polymerization. Within the range of dye concns. of 0.2-0.4 mg./cc. at 40[degree]C, RNA stains metachromatical-ly (purple), while DNA stains relatively orthochromatic ally (blue-green), making this a useful technic for the differential localization of nucleic acids in tissue sections. The implications of this difference in the degree of metachromasy of RNA and DNA in terms of their molecular structure are discussed.