PMID: 6171060Sep 1, 1981Paper

Factors affecting triple staining of human sperm

Stain Technology
P Talbot, E Dudenhausen

Abstract

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HCl (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjus...Continue Reading

References

Feb 1, 1981·The Journal of Experimental Zoology·P Talbot, R S Chacon
Jan 1, 1981·American Journal of Primatology·Prudence Talbot, Richard S Chacon

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Citations

Oct 1, 1985·International Journal of Andrology·P J MallettL R Fraser
Sep 19, 2002·Molecular Reproduction and Development·Herbert SchuelAlexandros Makriyannis

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