SPERMATOPHYTES—GYMNOSPERMS 
273 
The development of the pollen tube and its structures must be 
studied in sections of the nucellus. As soon as the integument is 
removed the nucellus is exposed and the position of the pollen tubes 
is easily determined, since the haustorial portions of the tubes form 
brownish lines radiating from the nucellar beak. Having learned 
the location of the pollen tubes, it is better not to remove the integu¬ 
ment, but to remove the female gametophyte; then cut from the 
underside of the nucellus against the hard, stony layer of the integu¬ 
ment so as to remove a small piece of the nucellus 5 to 7 mm. square, 
according to the species. Fix in chromo-acetic-osmic acid (1 g. 
chromic acid, 2 c.c. acetic acid, 1 c.c. 1 per cent osmic acid to 100 c.c. 
water). Nothing surpasses iron-alum haematoxylin for all the 
stages in the development of the male gametophyte (Fig. 94 A-C). 
By using a 2 per cent iron-alum for 5 or 6 hours and staining over 
night, or even 24 hours, the stain can be drawn so precisely that the 
portion of the cilia between the blepharoplast and the surface can be 
differentiated from the free portion (Fig. 95). 
The pollen tubes, with their sperms, make instructive prepara¬ 
tions when mounted whole. Fix the nucellus, with its pollen tubes, 
as if for paraffin sections. About 6 per cent formalin in water has 
proved successful. Wash in water for half an hour and stain in 
aqeous safranin, 1 or 2 hours. Extract the stain until it is satis¬ 
factory, and then transfer to 10 per cent glycerin and follow the 
Venetian turpentine method. When the turpentine becomes thick 
enough for mounting, tease the pollen tubes from the nucellus and 
mount with pieces of cover-glass under the cover to prevent crushing. 
Iron-alum haematoxylin, which is so satisfactory for staining sections 
of sperms, is the most unsatisfactory stain we have tried for staining 
the cycad pollen-tube structures whole. 
The immense sperms of the cycads, more than 100 ix in diameter, 
can be seen with the naked eye. If the tip of the nucellus with the 
pollen chamber be removed when the sperms are mature, the large 
pollen tubes, 300 to 500 m in diameter, are very conspicuous. Even 
with the naked eye, the movements of the sperms can be seen; and 
with a pocket lens one can see some details. Place the piece of 
nucellus on a slide in a drop of water and examine with a 16 mm. 
objective. The amoeboid movements of the sperm and also a quick 
movement reminding one of the sudden jerky movement of Vorti- 
cella, are easily seen; and, by closing the diaphragm a little, some 
