39 BOTANICAL GAZETTE {JULY 
very mild one, and germination proceeded with only occasional 
interruption throughout the entire season. Material brought 
into the laboratory at any time after the middle of November 
developed much more rapidly than in the open, and would shed 
the spores within a week or ten days. 
Before placing the material in the fixing agents, the calyptra 
was dissected away and about one-third of the sporogonium cut 
off with a razor, thus freely exposing the spores. In a few cases 
the mass of spores, held together only by the elaters, was 
removed from the sporogonium, but while not nearly so many 
spores were lost as might be anticipated, this tedious method was 
found to be unnecessary, since the other process readily yielded 
smooth sections as thin as 2 or 3p. 
Several fixing agents were used, but only two gave thoroughly 
satisfactory results. These were chrom-acetic acid (0.8% 
chromic acid, 0.5 glacial acetic acid, 100° water) and a modifi- 
cation of Flemming’s solution (0.58™ chromic acid, 0.5 8™ glacial 
acetic acid, I per cent. osmic acid 10°, water 100%). While 
achromatic structures stain more readily after solutions contain- 
ing some osmic acid, equally good preparations were often 
obtained from material fixed in the former solution. 
Most of the sections were cut at 2 or 3m, but sections 5 4, and 
even IO or 15 in thickness, were used in determining the num- 
ber of asters and in counting chromosomes. 
Haidenhain’s iron alum haematoxylin, with or without a slight 
tinge of erythrosin, Congo red, or orange G, gave fairly satis- 
factory preparations, but gentian violet proved to be so much 
superior in differentiating kinoplasmic structures that safranin 
and gentian violet, sometimes with the addition of orange G, 
were used in most of the work. Sections were stained, usually 
over night, in safranin (18™ safranin in 100° of 50 per cent. 
alcohol), then washed in 50 per cent. alcohol until all red color 
was removed from the achromatic structures, and then stained 
for one or two hours in gentian violet (saturated aqueous solu- 
tion). From the gentian violet the preparations were trans- 
formed directly to absolute alcohol, where they were quickly 
dehydrated, the process being hastened by moving the slide to 
Pate.  «S Vi 
