362 
The Ohio Naturalist. 
[Vol. XV, No. 1, 
buds were killed in Schaffner’s weaker ehrom-acetic acid with a 
trace of osmic acid added, being left in this for twenty-four 
hours. After being thoroughly washed in water, the material 
was dehydrated by passing it through the various grades of 
alcohol to 70%, where it was left until September, when it was 
passed through the higher grades into chloroform, from which 
it was gradually passed into pure parafine and imbedded. Sections 
10m to 13 m thick were cut. 
Several methods of staining were used. The first tried 
was analin safranin, which was a fairly good stain, but it did 
not make enough differention between the chromatin material 
and the cytoplasm to be easily studied. Next Heidenhain’s 
iron-alum haemotoxylin was used and found to be very good, 
staining the chromatin material black and the surrounding 
tissues brownish. In using this stain, the slides were passed 
through turpentine, xylol, the different grades of alcohol to 
water, then passed into iron-alum, where they were left for two 
hours; after being well washed in water they were left four hours 
or longer in Heidenhain’s haemotoxylin after which they were 
washed and placed in iron-alum to clear, and after dehydrated 
they were mounted in Canada balsam. The? most satisfactory 
stain was Delafield’s Haemotoxylin. The slides were passed 
through the alcohols to 25%, then into Delafield’s Haemotoxylin 
where they were left for two hours, after which they were washed 
in water and passed up through the alcohols and mounted. 
INVESTIGATION. 
The earliest preparations show the resting cells after the last 
archesporial division, but before the tapetum has become 
differentiated. In the youngest sporocytes the nuclei are small, 
measuring 9 m or 10m, and the cells fit closely together forming a 
compact mass. In many nuclei there are several nucleoli present 
which do not appear spherical, but have one or more finger-like 
projections. In the youngest sporocytes the chromatin material 
seems to be arranged in a loose reticulum (Fig. 1), which is not 
uniformly spaced throughout the nuclear cavity, and is easily 
distinguished in it. Following this reticular stage the chromatin 
material has a tendency to draw together in masses which are 
rather definite in shape, spongy and flaky in appearance, and have 
fine threads radiating in all directions from the central lumps. 
(Fig. 2). 
There is a tendency for these spongy masses to become more 
compact and definite in shape, approximating the reduced number 
of chromosomes, (Fig. 3), and without doubt these are the pro¬ 
tochromosomes described by various authors, and designated 
as “prochromosomes” by Overton and Strasburger. It is prob¬ 
ably at this stage that the univalent chromosomes are paired in 
