302 
METHODS IN PLANT HISTOLOGY 
In dealing with the whole anther, it is necessary to select stains 
which will not overstain. Alum cochineal and Mayer’s haem-alum 
might be suggested. It would be worth while to try a combination 
of the two methods. Fix the entire anther in chromo-acetic acid, 
wash in water, and then stain 
in iron-haematoxylin. When 
the last stage in staining is 
reached—the extraction of the 
stain in iron-alum—remove the 
pollen mother-cells and watch 
the differentiation; then wash 
in water and follow the Vene¬ 
tian turpentine method. 
The pollen grain at the time 
of shedding generally consists 
of two cells, the tube cell and 
the generative cell, which 
afterward divides and forms 
two male cells or two male 
nuclei. Lilium and Erythro- 
iiium furnish good illustrations 
of pollen shed in the two-cell 
stage (Fig. 107). In Silphium, 
Sambucus, and Sagittaria the 
generative nucleus divides 
before the pollen is shed. The 
division of the generative cell 
to form the two sperms takes 
place just before fertilization; 
consequently, in forms like 
Silphium, fertilization is likely 
to occur within less than 72 hours after the division of the genera¬ 
tive cell. 
Sections should not be more than 5 ju thick, if they are to show 
a clear differentiation of exine, intine, starch, and other structures. 
If sections have been stained in iron-haematoxylin, staining in safranin 
for from 3 to 7 minutes will give the exine a bright-red color and will 
not obscure the haematoxylin. A rather sharp stain in gentian- 
violet will stain the starch and also the intine. In Asclepias and many 
Fig. 107 .—Erythronium americanum: photomi¬ 
crograph of mature pollen grains; the one at the 
top, which is cut longitudinally, shows both the 
tube nucleus and the conspicuous generative cell; 
the other is cut transversely and shows the genera¬ 
tive cell, but not the tube nucleus; stained in 
safranin and gentian-violet; from a preparation by 
Dr. Lula Pace; Cramer contrast plate; 4-mm. 
objective; ocular X4; yellowish-green filter; bel¬ 
lows, 85 cm.; exposure, 3 minutes. X615. 
