Example 12 



Demonstration of Spermatogenesis in the Rat Testis, Using the 

 Iron-Hematoxyiin Stain of Heidenhain 



The laboratory white rat is one of the best forms in which to show sperma- 

 togenesis for the reason that it has a continuous breeding period, and all stages 

 are available, therefore, in almost every section examined. 



The rat selected should be a young male. It is killed most conveniently with 

 chloroform. The scrotal sac is opened by a median incision, and the testes are 

 removed, trimming away the epididymis. The testes should be placed on a 

 clean glass plate and slashed with a sharp scalpel or razor about two thirds 

 of the way through by cuts spaced a few millimeters apart before being thrown 

 into the fixative solution selected. Few fixatives for this purpose can surpass 

 Allen's fluid (see p. 11). At least 100 cc. of fixative should be employed for a 

 normal-sized testis, and the bottle containing it should be reversed once or 

 twice during the first few hours to avoid the accumulation of diluted fluid at 

 the bottom. The time of fixation is not of any great importance but, in any 

 case, should be overnight and, in general, should not exceed a few weeks. 

 After the object is removed from the fixative, it may be washed for about an 

 hour in running water before being transferred to 70 per cent alcohol to com- 

 plete the removal of the picric acid. It must be emphasized that washing a 

 specimen in water, after picric acid fixatives, results in a great deal of vacuo- 

 lation of the cytoplasm. Of course in the present instance, this does not inter- 

 fere with the object being studied. After three or four changes of 70 per cent 

 alcohol— the testes remain in a large volume of solution for at least two 

 or three days between changes — the final removal of the picric acid may be 

 accomplished by adding a small quantity of dry lithium carbonate to the 

 alcohol used for washing. It will be impossible to remove all the yellow color, 

 some of which is occasioned by the combination of the picric acid with the 

 albuminoids present, but the last alcohol used for washing should be only 

 very faintly tinted with yellow. It is not the color of the fixed material to which 

 objection is raised during the passage of the material through paraffin; it is 

 the fact that, unless most of the picric acid is removed, there will be crystal- 

 lization with consequent damage to the tissues. 



Small pieces may now be removed for embedding from the testis itself It is 

 best to select pieces about a millimeter in from the surface and of about a 2 mm. 

 side. These should be embedded in paraffin and cut about 5 microns thick in 

 the usual manner. Then the sections should be attached to slides, particular 

 attention being paid to the fact that the slides are clean and that not too much 



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