26 
METHODS IN PLANT HISTOLOGY 
d) Special Chromo-Acetic-Osmic Solution.— 
Chromic acid. 1 g. 
Glacial acetic acid. 3 c.c. 
1 per cent osmic acid. 1 c.c. 
We have been trying these three ingredients, in various propor¬ 
tions, for many years and have found this combination very good for 
filamentous algae and fungi, even difficult forms like Hydrodictyon, 
Vaucheria, and Saprolegnia showing practically no plasmolvsis. 
The acetic acid may be too strong for some root-tips and probably 
other objects; but the reagent is worth a trial. With 2 c.c. of osmic 
acid, this seems to be as good for mitosis as Flemming’s solutions, 
and it causes less plasmolysis. Besides, in most cases, no bleaching 
is necessary. 
e ) Licent’s Formula.— 
1 per cent chromic acid. 80 c.c. 
Glacial acetic acid. 5c.c. 
Formalin.. 15 c.c. 
This formula has been recommended for coenocytic algae and 
fungi and for embryo sacs. 
/) Flemming’s Fluid (stronger solution).— 
fl per cent chromic acid. 45 c.c. 
[Glacial acetic acid. 3c.c. 
B. 2 per cent osmic acid. 12 c.c. 
Keep the mixture A made up, and add B as the reagent is needed 
for use, since it does not keep well. This fluid is quite expensive on 
account of the osmic acid. For cytological work it has been very 
popular, and it is especially recommended for chromosomes, centro- 
somes, achromatic structures, and mitotic phenomena in general. 
The fluid should be allowed to act for 24 to 48 hours and the washing 
in water must be very thorough. 
Material should be in very small pieces | inch square, or in thin 
slices | inch or less in thickness, for the fluid penetrates poorly. The 
blackening due to the osmic acid may be removed by peroxide of 
hydrogen just before the slide is passed from the alcohol into the 
stain. Harper and Holden, in their work on Coleosporium, recom¬ 
mended 4 hours on the slide in a 3 per cent solution of the peroxide 
of hydrogen. Some prefer a stronger solution of the peroxide of 
