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Hum Reprod. 2003 Apr;18(4):813-6.

Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment.

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  • 1Reproductive Biology and Genetics Group, ACU Birmingham Women's Hospital, Department of Medicine, University of Birmingham, B15 2TG, UK.



The one-step eosin-nigrosin staining technique for assessment of sperm vitality was developed in the 1950s for various mammalian species. Although commonly used on human sperm in semen, a validation for this use has not previously been published.


The technique was evaluated on 1235 consecutive semen samples.


The one-step eosin-nigrosin staining technique gave valid results when evaluated with sperm motility data obtained according to World Health Organization standard (1992, 1999). The mean for the sums of stained (i.e. supposedly dead) and motile sperm using the one-step eosin-nigrosin technique was 91% (SD +/- 10%). The distribution of sums for percentage stained and percentage motile sperm was similar, regardless of whether the samples had many or few dead sperm.


Standardization and quality control of basic semen analysis demands robust, reliable and simple techniques that are easy to learn, and easy to continue to perform in the same way. The one-step eosin-nigrosin technique does not need negative phase contrast optics but can be run with ordinary bright-field microscopy. Since it also includes fewer methodological steps to control, it seems preferable in terms of standardization and quality control management. It should therefore be recommended in the basic semen analysis when sperm vitality is to be assessed.

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