50 CYTOPLASMIC STRUCTURES IN THE SEMINAL EPITHELIUM OF THE OPOSSUM. 



TECHNIQUE. 



Fragments of testicle were fixed in the following fluids: Hermann's, acetic 

 sublimate, Ramon y Cajal's mixture of formalin and uranium nitrate, Benin's, 

 Flemming's, saturated sublimate, Altmann's, Meves's, Benda's, and Regaud's. 

 Fragments of the epididymis were fixed in the latter two reagents. Smears of 

 spermatozoa were fixed either by the action of vapors of 1 per cent osmic acid in a 

 moist chamber for 20 to 30 minutes, or by immersion for 10 to 30 minutes 

 in Regaud's mixture, then for 24 hours in 3 per cent bichromate. Two adults were 

 sacrificed for the purpose of studying the living cells, and were injected with a 

 solution of janus-green dirriny) in 0.85 per cent salt solution. I am greatly indebted 

 to Professor E. V. Cowdry for his help in carrying out these experiments. 



Of the above-mentioned reagents Benda's, Meves's, Regaud's and Altmann's 

 fluids were used for the purpose of fixing the chondriosomes. Perhaps the best 

 preparations were obtained from Regaud's material. Regaud's fluid, however, is 

 an exceedingly one-sided reagent. While it often fixes the chondriosomes perfectly 

 well, other structures, such as centrioles, idiozome, axial filament, or "tingierbare 

 Korner" are scarcely visible, if at all; the cell limits disappear and one no longer 

 wonders at Regaud's peculiar conception, according to which the spermatogonia 

 have no cell-bodies of their own (1908). As to the nucleus, there is no possibility 

 of studying it during the growth or maturation period. In the later stages, however, 

 especially with a good nuclear stain, such as methyl green, the same material proves 

 very valuable. The worst feature in the action of Regaud's fluid is the dislocation it 

 produces in the seminal epithelium, a dislocation due (in part at least) to the fact that 

 the fat is not fixed and is consequently dissolved during subsequent manipulations. 



A number of nuclear stains and (for the study of the chondriosomes) iron 

 hematoxylin, Benda's and Altmann's method, and acid fuchsin-methyl green were 

 used. Excellent preparations were obtained with the latter method, especially 

 after fixation in Regaud's fluid. As it happened that my material would keep too 

 much methyl green, I found it very useful to bring back the slides from the 95 per 

 cent alcohol into distilled water, a procedure which eliminated immediately the 

 superfluous green and improved the preparations greatly, then back to 95 per cent 

 alcohol, and so forth. In order to bring into view the apparatus of Golgi, I used, 

 with the same success as previously (1914), Ramon y Cajal's mixture of formalin 

 and uranium nitrate. The best preparations were obtained by leaving the pieces 

 in the fixative for 9 hours, in the silver nitrate 37 hours, and in the developer 14 

 hours. While undoubtedly this method almost unfailingly impregnates the appara- 

 tus of Golgi, it is nevertheless a capricious one, inasmuch as it is liable to bring into 

 evidence at the same time granulations, most probably mitochondria (fig. 26), 

 fibrils in the connective tissue, cell limits, and eventually other structures difficult 

 to identify. 



Instead of other complicated procedures, at the suggestion of Professor Cowdry 

 I used the following method in the treatment of the sections : The slides were first 



