Triple staining of human sperm: technical aspects.

The triple-stain technique according to Talbot and Chacon was adapted for use in routine semen analysis. This staining technique allows evaluation of spermatozoal morphology and determination of the percentage of dead and "live" spermatozoa and of the percentages of spermatozoa with and without intact acrosome. Optimal results were obtained if centrifugation prior to fixation was avoided, the semen was diluted before fixation, and the spermatozoa were fixed in suspension. This modification of the triple-stain technique produces preparations with low background and excellent visibility of all spermatozoal structures. Use of Trypan Blue leads to underestimation of the percentage of dead spermatozoa, but use of other supravital stains was even less satisfactory. Round cells could not be evaluated in the triple-stain technique.