Sperm Viability Assay by Eosin-Nigrosin Staining

The viability of sperm was assessed by eosin-nigrosin staining (Merk, Germany). Based on this staining, the head of dead sperms turns red or dark pink, but the head of live sperms remains colorless. At first, the 1:2 ratio (by volume) of sperm suspension and eosin was mixed in the microtube. Following 30 sec, an equal volume of nigrosin solution was added. Then, a thin spread of the sample took place on the slide. After drying the smear, using a light microscope (100x magnification), 200 sperms per sample were counted. Eventually, the percentage of live and dead sperms in the samples was calculated (17).

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Dorikhani A., Omidi A., Movahedin M, & Halvaei I. (2024). Chronic demyelination interferes with normal spermatogenesis in cuprizone-intoxicant C57/BL 6 mice: An experimental study. International Journal of Reproductive Biomedicine, 22(1), 43-54.

Publication 2024

Corresponding Organization :

Other organizations : Tarbiat Modares University

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Variable analysis

independent variables

Ratio of sperm suspension to eosin (1:2 by volume)

dependent variables

Percentage of live and dead sperms

control variables

Nigrosin solution (added in equal volume to the sperm-eosin mixture)
Sample preparation (thin spread on slide and drying)
Microscope magnification (100x)

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