SPERMATOPHYTES—GYMNOSPERMS 
271 
were then followed and sections were cut with a sliding microtome, 
with the knife placed obliquely, as in cutting celloidin. 
The course of the vascular bundles, as they pass to the cones, is 
quite peculiar. Instructive preparations may be made by cutting 
longitudinal sections, about 3 mm. thick, through the apex of the 
stem and, without staining, clearing thoroughly and mounting in 
balsam. In this way we have mounted sections 5 cm. long, 15 cm. 
wide, and 3 mm. thick. 
The course of the bundles in the xylem zone and in the cortex may 
be traced by clearing the cubes in xylol and then transferring to 
equal parts of xylol and carbon disulphide. Placed on a glass plate 
with an electric light bulb beneath, the bundles are quite distinct. 
The root .—Small roots, up to a centimeter in diameter, are easily cut 
freehand. The tender root-tips and also the peculiar “ root-tubercles” 
should be fixed in chromo-acetic acid and imbedded in paraffin. 
The leaves .—The young tender leaves should be fixed in formalin 
alcohol and imbedded in paraffin. The adult leaves are rigid and 
cut well freehand. It is a good plan to tie several leaflets together 
with a string and then cut across, about a centimeter beyond the 
tied portion, so that the whole will be like a leaflet 5 or 6 mm. thick. 
Dip the whole thing in paraffin two or three times. Of course, there 
is no infiltration but the paraffin holds the leaflets in place. Cut in 
a sliding microtome with an oblique stroke. The sections fall out 
from the paraffin, which is easily skimmed away. Fix the sections 
for an hour in 95 per cent alcohol, stain in safranin and light green, 
clear in clove oil, transfer to xylol, and mount in balsam. 
Spermatogenesis. —Except in the earliest stages, the staminate 
cones are too large to be cut whole. The individual sporophylls, 
with their sporangia, cut easily up to the formation of microspores; 
then the sporangium wall hardens rapidly and cutting becomes 
difficult. Up to the young microspore stage, fix in chromo-acetic- 
osmic-acid solution (1 g. chromic acid, 1 c.c. acetic acid, 1 c.c. 1 per cent 
osmic acid to 100 c.c. water). With a larger proportion of acetic 
acid, our results have not been satisfactory. Fix later stages 
in formalin-acetic acid-alcohol. Transverse sections are more 
instructive and are more easily cut, since the peripheral end of the 
sporophyll can be cut only in younger stages. In all the genera of 
cycads, the microspore germinates while still within the sporangium, 
the pollen grain at the time of shedding consisting of a prothallial 
