62 - 
While the following formula, 70 cc. one per cent, chromic acid, }4 cc: 
glacial acetic acid, 30 cc. H 2 O, gave better results with the different 
stages of the sporogonia. Carnoy’s fixing fluid was used twice in 
order to save time, and although the material fixed in this solution 
took a very brilliant stain with the saffranin gentianviolet combination, 
more or less shrinkage resulted. This, however, might have been 
prevented if the material had not been hurried too rapidly through 
the infiltrating and imbedding process. 
The plants were either put into the fixing solution in the field or 
soon after carrying them home. When such a delay was necessary 
the material was kept in a tightly covered tin box until placed in the 
chromacetic solution. In the earlier stages after removing the dirt 
from the rhizoids with needles, entire plants may be placed in the 
fixing solution. But after July, when the young sporogonia begin to 
develop, the thallus should be trimmed down nearly to the sporogo- 
nium. The capsules should be pricked and the surrounding membrane 
removed, otherwise bubbles will form making it impossible for the 
fixing solution to penetrate, and later the paraffine, thus making the 
infiltration process a failure. 
After fixing for about 24 hours, the material was washed in 
running water for about 12 hours and then was carried through the 
usual solutions of alcohol, viz.: 15 per cent., 35 per cent., 50 per 
cent., 70 per cent., comparatively short periods ( 3 hours ) being 
sufficient for the weaker alcohols. In 70 per cent, alcohol much of 
the material had to remain for a long time, from several months even 
to a year. 
When ready for imbedding the material was carried from 70 per 
cent, through 85 per cent., 95 per cent., two changes of absolute 
alcohol and through the three solutions of absolute alcohol and xylol 
into pure xylol and was finally imbedded in paraffine melting at 54° 
C. The sections were cut with a Minot rotary microtome, being 
three, four or five microns in thickness. 
In the earlier stages great trouble was experienced from shrinkage 
of the tissues. After the loss of much time and good material it 
became evident that the difficulty, though caused partly by insuffi- 
cient washing, was chiefly due to allowing for too short periods in 
the absolute alcohol and xylol solutions and also to hastening the 
process of infiltration. Moreover, when all had gone well up to the 
last solution in xylol everything was sometimes ruined by adding the 
paraffine in too large pieces. Even in the most refractory material, 
shrinkage was always avoided by letting it remain a long time ( 24 hrs. 
or longer) in the absolute alcohol-xylol solutions and then putting only 
the smallest, thinnest shavings of paraffine into the pure xylol, 
letting each dissolve before adding the next. 
