An Eosin-Fast Green-Naphthol Yellow Mixture for Differential Staining of Cytologic Components in Mammalian Permatozoa

Abstract

Air-dried smears of saline suspensions of mammalian spermatozoa were stained for 10-60 min at room temperature in a mixture of eosin Y, fast green FCF, and naphthol yellow S (0.1% w/v, each dye) in 1.0% aqueous acetic acid. They were then blotted, rinsed in 1.0% acetic acid until no more dye was removed (0.5-1.5 min), blotted and allowed to dry completely, rinsed in xylene and mounted in synthetic resin. In all species examined except the rat, acrosomes were stained greenish blue to bluish green or blue depending on their thickness; in the rat, they displayed more affinity for eosin and were reddish. In all species, spermatozoan nuclei were strongly stained by naphthol yellow. In intact sperm heads, postnuclear cap had a yellowish green appearance. Midpieces of rodent spermatozoa, especially those of younger gametes, were stained bright red while those of ejaculated bull and rabbit spermatozoa were stained blue-green. Cytoplasmic droplets associated with rodent spermatozoa were consistently stained a dark green. In all species, the remainder of the flagellum generally was stained bluish to blue-black. Deductions concerning the morphology of spermatozoa derived from the staining experiments were verified by means of scanning electron microscopy. Because it provides reliable information concerning the morphology of the various components of mammalian spermatozoa, this simple staining procedure should prove applicable to a wide variety of studies involving the morphology of intact spermatozoa