ACRIDINE-ORANGE STAINING OF DECAPOD CRUSTACEAN CUTICLE

The fluorescent dye acridine orange (C.I. 46005) was found to be useful for differentiating cuticular layers and therefore can aid in determining the molt stage of decapod crustaceans. For Procambarus clarkii and Callinectes sapidus, best results were achieved by staining 10-mu m sections of alcoholic formalin-fixed, paraffin-embedded tissue with a 0.1% aqueous solution at pH 4.0. The sections were viewed with an epifluorescence microscope after being excited with three specific wavelength ranges: blue, violet, and ultraviolet. Sections also were examined with bright-field illumination. The exocuticle, which emitted red to orange metachromatic fluorescence, was easily distinguished from the endocuticle, which emitted green to blue orthochromatic fluorescence.