208 Proceedings of the Royal Physical Society. 
cysts definite numbers were taken and fixed on different dates. If more 
cysts were afterwards found in the “stock” which had been set apart, they 
too were isolated. By this means a great number of “stock” Petri-dishes 
and watch-glasses containing the isolated cysts were soon collected and 
experiments tried for initial encystment in the case of the amoebe from the 
“stocks,” and for continuance of development in the encysting and encysted 
amoebze, with varying success. 
FIXATION AND STAINING. 
In 1912 the cysts were fixed in corrosive acetic 10-20 minutes, in 1913 
in corrosive alcohol from 45-60 minutes. The shrinkage seems greater in the 
latter, the outer membranous wall collapsing more readily. 
After fixation they were passed through the alcohols 30 %-90 % + eosin 
(to render them conspicuous after clearing in clove oil); absolute alcohol, 
24 hours in each; clove oil, 24 hours; clove oil and celloidin, 8 days; 
chloroform, 3 hours; paraffin (52° C.), 15 minutes. All the sections were cut 
5 p» in thickness. 
The sections were stained in Ehrlich’s hematoxylin, but after examination 
this stain was replaced by iron hematoxylin except in the case of the sections 
shown in Pl. VII. Figs. 3 and 4. 
As the nucleus breaks down a change seems to occur in the constitution 
of the protoplasm of the amoeba-body. A similar change has been observed . 
by Popoff1in his study of A. minuta. This protoplasm now becomes strongly 
stainable. When the stain employed is borax carmine, or Ehrlich’s haema- 
toxylin, the nucleus still holds its ascendancy, though not great (Fig. 3); but if 
iron hematoxylin has been used then the nucleus parts with the stain almost 
immediately when placed in iron-alum solution while its surroundings are 
still quite black. 
In one case a nucleus similar in condition to that shown in Fig, 4 (N) was 
left in_this iron hematoxylin stain for 36 hours, but on being placed in 
the alum solution parted with the stain in less than 5 minutes. Apparently 
the property gained by the protoplasm is lost by the nucleus. 
The plastin, or whatever complex of plastin and chromatin it may be 
which is passed from the nucleus (Pl. VII, Figs. 4, 5, 6), is most tenacious of 
all stains—a brilliant red in borax carmine, a deep blue in Ehrlich, and black, 
after a very lengthy destaining process, in iron hematoxylin. 
The nuclear membrane can still be seen during the breaking down of the 
nucleus. It finally disappears with the rest of the degenerated portion. 
1 Popoff, “A. minuta,”? Archiv fiir Protistenkunde, Ba, 22, p, 202, 1911. 
