286 
METHODS IN PLANT HISTOLOGY 
toward the archegonia, and consequently, in some of the material, 
it is better to retain enough of the tissues of the ovule to keep the 
nucellus in place. In later stages, after fertilization has taken place, 
the developing testa should be removed with great care, for a very 
slight pressure is sufficient to injure the delicate parts within. 
With any fixing agent of the chromic-acid series, the free nuclear 
stages of the female gametophyte and, later, the archegonia and 
proembryo stages like those shown in Figure 101, are likely to show 
Fig. 101. Pinus Laricio: A, top of prothallium with an archegonium just before the cutting 
off of the ventral canal cell; fixed in Flemming’s weaker solution and stained in Haidenhain’s iron- 
alum haematoxylin; collected June 18, 1897; B, C, and D, early stages in the development of the 
embryo; fixed in chromo-acetic acid and stained in safranin, gentian-violet, orange- collected 
July 2, 1897. X104. 
plasmolysis. Stages like those shown in Figure 101 B-D, without 
any shrinking, were secured by Miss Ethel Thomas by using hot 
alcoholic corrosive sublimate-acetic acid with formalin (4 g. corrosive 
sublimate, 5 c.c. acetic acid, 5 c.c. formalin, 100 c.c. 70 per cent alcohol). 
Figures like that shown in Figure 101 are better in chromo-acetic- 
osmic acid, but, in general, we should recommend the corrosive 
sublimate solution. 
The period at which the various stages may be found varies 
with the species, the locality, and the season. In Pinus Laricio 
