A triple-stain technique for evaluating normal acrosome reactions of human sperm

A triple-stain technique has been developed to score normal acrosome-reacted human sperm in fixed smears. Live and dead sperm are first differentiated using the vital stain trypan blue. Sperm are then fixed in glutaraldehyde, dried onto slides, and the postacrosomal region and acrosome are differentiated using Bismark brown and Rose Bengal. Slides are examined at 1,000 X with a bright-field microscope and assessed for 1) the percentage of sperm that were alive at the time of fixation and 2) the percentage of sperm that had undergone normal acrosome reactions. Experiments are included that show that trypan blue is a reliable stain for dead sperm and that Rose Bengal stains only sperm having intact acrosomes. This technique may have applications in experimental and clinical studies on sperm capacitation, acrosome reactions, and fertilization in laboratory and domestic animals as well as in man.