1899] DEVELOPMENT OF THE MICROSPORANGIUM 329 
The Flemming chrom-osmo-acetic acid solution has of late 
years proved of such value that it must now be regarded as the 
very best fixing agent for cytological work. For the following 
studies material from no other fixing agent was used, although 
_ Some was put up in alcohol, sublimate, and picricacid.. None of 
the latter gave satisfactory results. The secret, if there is any, 
in the use of the Flemming solution seems to lie in obtaining 
pid penetration. To accomplish this the material cannot be 
subdivided too much, and when possible even the anthers them- 
selves should be cut open. From six to twelve hours is sufficient 
for complete fixation. The black discoloration caused by the 
_ tsmic acid was removed by the aid of hydrogen peroxid either 
pon the sections themselves or preferably upon the material 
Mm toto, 
For clearing, cedar-wood oil gave the best results. It was 
ways added with great care in order to avoid too rapid change 
of density, otherwise collapse of the cells often occurred. The 
Mrafin used possessed a melting point of 54°. This also was 
added with the same degree of care. The sections ranged in 
thickness from four to six and two thirds microns, and were all 
ton a Minot-Zimmerman revolving microtome. 
: Considerable experimentation was necessary before a suitable 
4 “aining method could be devised. Among others, Rosens’ 
‘chsin-methy lene blue method,! iron-hamatoxylin and the Flem- 
mg ‘afranin-gentian-violet-orange combination were the most 
: “portant. The latter was at length almost exclusively employed, 
; “thy found very satisfactory. Asa general nnclear re 
| *t, better results were obtained by the omission of the 
“nin, and at the same time a large amount of time was saved. 
~ Mange G was always used in a very dilute solution, and 
f i.” Short time, from 15 sec. to 2 min. giving a 
Wace a ora chromosome stain the gentian-violet was 
a short time only, but for differentiating the sp 
r results were 
he stock 
- obta; md Kinoplasmic radiations, much bette 
tained 
‘3 With a very weak solution (2-3 drops of t 
ttrage zur Kenntniss der Pflanzenzellen. Cohn’s Beitrage 5+ 443: 1892. 
