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Stain:

Toluidine Blue

Description:

Section of loose CT stained with toluidine blue and demonstrating both orthochromasia (bright blue color) and metachromasia (dark purple color). Metachromasia, often described as a color change exhibited by certain basic aniline dyes in aqueous medium, is a phenomenon that is characterized by a shift in light absorption to shorter wave lengths. Referring either to a change in the color of the dye itself or to the cells and tissues that are stained in this manner, metachromasia is typically brought about by an increase in dye concentration that results in dye polymerization. These increases can be achieved experimentally by simply adding more dye molecules to monomeric (dilute) solutions or biologically by staining tissues that contain highly electronegative polymers. Examples of these tissue polyanions are glycosaminoglycans (GAGs) and proteoglycans, which essentially concentrate the dye molecules in situ by binding them in close proximate array. In histology, an important dye with metachromatic properties is toluidine blue. In dilute staining solution, its natural color is blue; therefore, those cells and tissues that stain this color are described as orthochromatic. When tissues that contain highly acidic polyanions are stained with a dilute solution of toluidine blue, the resulting close proximity of bound toluidine blue molecules causes the color shift to a metachromatic reddish-purple. Other basic dyes that also exhibit this property are methylene blue, thionin, and the azures. Metachromatic staining with the acid dye Biebrich scarlet has also been reported. In this micrograph, which shows some unmyelinated nerve fascicles (top, middle and lower right) and a small ganglion (lower left) surrounded by loose CT, the CT and nervous tissue nuclei are mainly staining the blue color of toluidine blue (orthochromasia). In contrast, scattered throughout the center of the field, nine intensely stained mast cells are exhibiting the dark purple color of toluidine blue (metachromasia). These CT cells possess numerous cytoplasmic granules that contain proteoglycans and the GAG heparin--polyanions that are responsible for the color shift. Due to the thickness of the section and the intensity of staining, the metachromatic mast cells appear almost black in this preparation. (ag00)

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