solochrome-cyanine-r and ferric-chloride

Chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) is a valuable biological stain. The dyestuff is supplied as a powder containing a little less than 50% by weight of the monosodium salt of the dye, mixed with colourless crystalline and amorphous fillers. The tetrabasic colour acid was prepared and purified for study of the chemical and spectral properties of the dye. Chromoxane cyanine R is an acid-base indicator, with five different colours corresponding to the colour acid and the four anions. The most conspicuous colour change, from yellow to blue, occurs with ionization of the phenolic hydroxyl group at pH 11-12. The dye is assayed by measuring the absorbance of a strongly alkaline solution at 585 nm, with reference to a standard solution prepared from the purified colour acid. Spectrophotometric evidence has been found for the existence of three dye-metal complexes in solutions of the dye containing added ferric chloride at pH 1.5 (the pH of iron-dye solutions most useful in histological staining). These have the postulated compositions [ Fe2H (dye)]- (red), [ FeH2 (dye)]- (red), and [Fe2(dye)]2- (blue). The first two are probably simple carboxylate complexes of low stability. Increase in pH or molar iron: dye ratio promotes formation of the more stable blue complex, which is a metal chelate. Other blue complexes have been described by other investigators in solutions less acid than those that are useful in microtechnique. The production of blue and various shades of red in tissues stained by solutions containing iron (III) and chromoxane cyanine R probably involves reactions of both the red and the blue complexes of the dye.

Topics: Benzenesulfonates; Chemical Phenomena; Chemistry; Chlorides; Ferric Compounds; Hydrogen-Ion Concentration; Iron; Spectrophotometry; Staining and Labeling

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron:dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe:dye was high), or both red and blue (when Fe:dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron:dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron:dye ratio is due to a simple carboxylate complex, [ Fe2H (dye)]-. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe2(dye)]2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.

Topics: Animals; Benzenesulfonates; Cell Nucleus; Chlorides; Connective Tissue; Cytoplasm; Erythrocytes; Ferric Compounds; Hydrogen-Ion Concentration; Iron; Mice; Myelin Sheath; Rats; Staining and Labeling