110 A CYTOLOGICAL STUDY OF THE KIDNEY CELL 



TECHNIQUE 



Owing to the fact that the kidneys had to be weighed for reasons 

 explained in the above life history, fixation by arterial injection could not 

 be practiced. This would in any case have been impracticable since it 

 would not have permitted vital studies. 



As fixing agents Bensley's(4) formalin-zenker, Regaud's(S) neutral 

 formalin-bichromate, and Kolster's(6) chromalum and fluorchrome mix- 

 tures were used. Controls were also fixed in chemically pure, freshly neu- 

 tralized 10 per cent formaldehyde. It was impossible to obtain osmic acid 

 at the time of this investigation (1918). Tissues in pieces of 2 mm. thick- 

 ness were fixed in these agents from one to three days and then chromi- 

 cized from three to nine days. A temperature of about body heat was 

 maintained throughout the period of fixation. The fluids were changed 

 daily. The fixed material was cleared with either cedar oil, bergamot oil, 

 or xylol. I have found practically no difference in the shrinkage of the 

 tissues with either of the three, and therefore have used xylol in prefer- 

 ence as it is the fastest and least expensive of all clearing agents. Paraf- 

 fin, 60, was used as the embedding medium. Sections were cut serially 

 and about 5 \i thick. Altmann's(7) anilin-fuchsin picric acid method in 

 its original form and as modified by Cowdry(8) and Galeotti(9), Bens- 

 ley's (4) anilin-acid-fuchsin methyl green, Bensley's(4) copper-chrome- 

 hematoxylin and Heidenhain's iron-hematoxylin were used. Simple con- 

 ventional stains were employed as a check on the general picture. Ap- 

 parently it is not necessary to use osmic acid to obtain good fuchsin pic- 

 tures. Such simple bichromate mixtures as mentioned above will pre- 

 serve mitochondria excellently and allow good staining results. 



Among the fixatives the formalin-zenker and the neutral formalin 

 bichromate mixtures with a period of chromization have given by far the 

 best results. Of the staining methods the anilin-acid-fuchsin methods 

 are quicker but otherwise in no way superior to the other methods 

 mentioned. 



Fresh tissues were studied in a normal saline solution of 1 : 10,000 

 Janus green |3. Arnold's (10) neutral red and methylene blue were used 

 in several control animals. Janus green studies of renal tissues are very 

 difficult. First of all, the dye seems to penetrate very slowly, and one is 

 therefore not certain whether the mitochondria have not changed in ap- 

 pearance by the time they stand out plainly; secondly, mitochondria are 

 usually massed so densely in the convoluted tubules that a clear picture is 

 very rarely obtained ; thirdly, in order to obtain thin enough pieces of 

 tissue one must either tease them out or cut them with the freezing micro- 

 tome. Either method furnishes ground for controversy. It seemed to 

 me, however, that sections cut with a good- freezing outfit are preferable 

 to teased-out tissues. 



