426 EDWARD E. WILDMAN 
of both nuclear and cytoplasmic structures when followed by 
either iron hematoxylin-Bordeaux red, or the Ehrlich-Biondi 
stain. ‘These combinations and those suggested by Benda were 
indeed the only ones that proved to be of value in this study. 
The most instructive reactions obtained were those caused by 
Benda’s fixative and stain. A late spermatocyte with this tech- 
nique shows the numerous refringent bodies stained blue, the 
karyosome or chromatic nucleolus red-brown, and the plastosome 
and certain small granules throughout the cytoplasm (Meves’ 
‘plastochondria’) yellow. While these different colors tell us 
nothing concerning the chemical composition of these structures 
we must conclude that they are not alike chemically. The prime 
value of Benda’s stain for our present purpose, therefore, is that 
it is more selective than any other used. The other combinations 
mentioned will distinguish the plastosome and small granules 
from the karyosome and the refringent vesicles, but the latter 
so closely resemble each other in color that they might easily be 
3 Owing to frequent injury to sections in following Benda’s directions for 
staining, I modified his method slightly. As used by Benda, Meves, Duesberg 
and others, the treatment of material for this stain is as follows: after fixation in 
strong Flemming’s or Hermann’s fluid, the tissue is washed in water, then left 
in a mixture of equal parts of pyroligneous acid and 1 per cent chromic acid to 
mordant. Then for a like period of time, say twenty-four hours, in a 2 per cent so- 
lution of bichromate of potash for the ‘second chromization.’ The material is 
then washed, dehydrated and imbedded as usual. After sectioning, iron alum in 
a 2 or 4 per cent solution is used as a mordant, and also’an aqueous solution of 
sodium sulfalizarine. After washing in water, a solution of ‘krystal violet’ is 
poured over the sections and the slide is held over a flame until steam arises from 
the solution. The stain is then poured off and the sections are washed in water. 
Next they are destained in 30 per cent acetic acid, and after being thoroughly 
rinsed in water they are dried between sheets of blotting paper. The sections 
are then put directly into absolute alcohol for a moment, and are cleared and 
mounted as usual. I found, however, that heating the sections, destaining them 
in 30 per cent acetic and finally drying them between blotting or filter paper ruined 
most of the sections. I got just as clear-cut and permanent stain differentiation 
by using the following method: after washing the excess alizarine solution from 
the sections by carefully dipping the slide into water, they were stained by putting 
the slide into a 3 per cent solution of ‘krystal violet’ (3 cc. aniline stain in 100 ce. 
distilled water) for ten minutes. They were then rinsed in water and put into 
80 per cent alcohol for about five seconds; then into 95 per cent and finally into 
absolute alcohol, where the destaining was watched carefully. Then the sections 
were cleared and mounted as usual. 
