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CHAPTER 1.1
staining procedures should be followed exactly, i.e. the semen, stain and slides should be warm (37°C)
and the preparation should be thin allowing the smear to dry rapidly (Fig. 3). For preparing an eosin-
nigrosin stain, the following procedure is recommended (Bart and Oko, 1989):
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a. Place a drop of warm stain near the frosted end of a warm microscope slide.
b. Place a drop of warm semen near the stain and mix the two on the slide (the ratio stain to
semen depends on the concentration of the semen sample).
c. To make a smear, a second slide held at a 30-40° angle, is pushed against the drop of
stained semen and pulled back slowly as shown in Fig. 3.
d. Dry the smear quickly by blowing air across it.
Fig. 3. Method for making a sperm smear using eosin-nigrosin stained sperm.
Sperm morphology is classified in different categories based on the origin of the abnormality
or based on the specific morphological defects. Based on origin, morphological defects are classified
as primary, secondary or tertiary abnormalities. Primary defects, of testicular origin, originate during
spermatogenesis. Secondary abnormalities are associated with the excurrent duct system
(epididymal origin) while tertiary defects are considered artefactual defects caused during sperm
collection and preparation. On the other hand, the classification by the specific morphological
appearance might be preferable since it provides information about the actual defect instead of the
presumptive origin. After all, a specific defect might have different causes. Therefore, sperm can be
classified as either normal or with abnormalities i.e. in the head, neck and midpiece, or tail, and
sperm with excess residual cytoplasm. Within any category, subdivisions are made. In Fig. 4, an