The stereoscan electron microscope (Cambridge Instrument Co.) enables the surface of objects to be examined over a wide range of magnifications, but the object must be carefully washed to remove substances which might obscure its outlines, and when the object is biological the substances washed away may be an important part of the surface.
With this limitation in mind, samples of ejaculated bull and rabbit, and testicular ram spermatozoa were prepared in the following way: (1) Seminal plasma (or testicular fluid) was removed from semen by centrifuging at 20,000 g for 20 min. (2) Spermatozoa were resuspended in 40·0 ml phosphate-buffered saline (0·125 m-NaCl, 0·02 m-phosphate buffer, pH 7·4) and centrifuged at 1000 g for 10 min; the supernatant was removed. This was repeated four times. (3) The spermatozoa were fixed by adding 0·1 ml of sperm suspension to 0·9 ml 2·5% glutaraldehyde at pH 7·0 and keeping at 4·0° C for 1 hr. (4) The fixative was removed by centrifuging at 1000 g for 10 min. The spermatozoa were resuspended in distilled water. This process was repeated twice. (5) A drop containing fixed spermatozoa in distilled water was allowed to evaporate on a glass cover slip and examined in vacuo with the stereoscan microscope.
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