DS 11.10 



DYE STAINS OF GENERAL APPLICATION 



273 



])ecauso of the very large cliromosonios 

 wliicli it possesses. It is a disadvantage, 

 however, that spermatogenesis in Triturus 

 occurs only in the summer months im- 

 mediately following the breeding cycle. 



The rats selected should bo young 

 males, and are most conveniently killed 

 with chloroform. The scrotal sac is then 

 opened by a median inci.sion, the testes 

 are removed, and the epididymis trimmed 

 away. The testes should be placed on a 

 clean glass plate and slashed with a sharp 

 scalpel or razor about two-thirds of the 

 way thi-ough. The slashes should be spaced 

 a few millimeters apart and should be 

 made before the testes are thrown into the 

 selected fixative solution. Few fixatives for 

 this i)urpose can improve on the fluid 

 originally suggested by Bouin 1897 (Chap- 

 ter 18, F 5000. 1010) ."This fixative today 

 has many uses of doul:)tful validity; but 

 for this, the material which it was origi- 

 nally designed to preserve, it has rarely 

 been surpassed. At least 100 milliliters of 

 fixative should be emploj-ed for a normal 

 size testis, and the bottle containing it 

 should be reversed once or tw'ice during 

 the first few hours to avoid tlie accumula- 

 tion of diluted fluid at the bottom. The 

 length of time allowed for fixation is not 

 of any great importance, but it should, in 

 any case be overnight, and should not in 

 general exceed several weeks. 'After the 

 object is removed from the fixative, it may 

 be washed for about an hour in running 

 water befoie being transferred to 70% al- 

 cohol to complete the removal of the picric 

 acid. It must be emphasized that washing 

 it in water after putting it in picric-acid 

 fixatives results in considerable vacuola- 

 tion of the cytoplasm. This does not in the 

 present instance interfere with the study 

 of the nucleus. After three or four changes 

 of 70% alcohol, leaving the testes in a 

 considerable volume of solution for at 

 least two or three days between changes, 

 the final removal of the picric acid may be 

 completed by adding a small quantity of 

 dry Hthium carbonate to the alcohol used 

 for washing. It will be impossible to re- 

 move the whole of the yellow color, some 

 of which is caused by 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 mate- 

 rial to which objection is raised during the 

 l)assage of the material through paraffin; 

 it is the fact that unless most of the picric- 

 acid be removed, there will l)e a crystal- 

 lization which will damage the tissues. 



Small i)ieccs may now be removed from 

 the testis itself for embedding. It is best 

 to select a jjiece about one millimeter in 

 from the surface and of about a two-milli- 

 meter side. These should be embedded in 

 paraffin and cut about five microns thick 

 in the usual manner. These sections should 

 then be attached to a slide. Particular 

 attention must be paid to the fact that 

 the slides are clean and that not too much 

 of whatever adhesive is employed be al- 

 lowed to remain. 



The dry sections attached to the slide 

 should now be warmed on the underside 

 until the paraffin melts, placed in xylene 

 until the paraffin is completely removed, 

 and then run down in the ordinary way 

 through absolute alcohol and lower-per- 

 centage alcohols to distilled water. Thej' 

 should then be lifted from the distilled 

 water and examined carefully. If there is 

 a tendency for the water to gather in 

 droplets on the slide, or if upon shaking 

 the water from the slide each section ap- 

 pears to retain around itself an adherent 

 coat of water, it is an indication that the 

 wax was not properly removed in the 

 xylene or that the X3'lene itself is so old as 

 to have a wax content too high to be use- 

 ful. Such sHdes must be returned through 

 the alcohols to absolute alcohol and thence 

 to clean x.ylene, in which they should be 

 left for a few minutes before again being 

 brought down to water and reexamined. 

 There is no more common cause of the 

 failure of the stains to take than the im- 

 perfect removal of the wax. 



Only tw^o solutions are required for 

 staining. These are a 2>^% solution of 

 ferric alum and a ^2% solution of hema- 

 toxylin. The onlj' difficulty in making the 

 ferric alum solution is to secure a pure 

 and unoxidized sample of the reagent. 

 Most of the crystals in a new bottle are of 

 a clear violet color, but after it has been 

 opened for some time, particularly if the 

 stopper be loose, most of the crystals be- 



