The Limit Between Ectoderm and Entoderm. 43 
of ectoderm and entoderm owing to the persistence of yolk in the 
entoderm cells after it has disappeared from the ectoderm. The 
early development of the mouth has been studied fully in Amblys- 
toma punctatum and the following description applies wholly to this 
form. Necturus and frog embryos have been studied sufticiently 
to control the chief facts. 
While at West Virginia University I made a large collection of 
Amblyostoma embryos and larve from the unsegmented egg to tad- 
poles over an inch in length (about March 20 to July 1). Various 
methods were tried both for fixation and staining. The fixing fluid 
that gave the best results was the formol-sublimate-acetic mixture 
devised by Worcester.? All the embryos used for this study were 
fixed in that fluid. For staining, borax carmine, hemalum, heema- 
toxylin with counter stains have been used. Sections prepared for 
this particular purpose have all been stained by iron hematoxylin 
and counter stained by acid fuchsin. It was found that iron heema- 
toxylin stains yolk granules more intensely than any other element 
in the tissues and remains in the yolk granules in the differentiating 
bath until it is removed from all other structures, even the chromatin. 
By differentiating to the last degree favorable for study of chromatin 
and counter staining with acid fuchsin, clear transparent prepara- 
tions are obtained in which the yolk granules stand out boldly on a 
pink ground, while the nuclei and cell boundaries are lightly stained 
but distinctly seen. 
As is well known, the ectoderm and entoderm differ from the 
earliest stages in the number and size of yolk granules contained 
in the cells. This difference is readily made out in any sections 
of well fixed material by careful study. In preparations stained as 
above described the differences are sharply marked and become more 
prominent and striking as development proceeds. The yolk granules 
in the ectoderm grow smaller and less numerous and disappear at a 
relatively early period. In the brain the yolk persists longer than in 
the ectoderm, but in such small granules that there is no comparison 
with the entoderm. The muscle-forming masses and some parts 
*Formula: take 10 per cent formol, saturate with sublimate, add glacial 
acetic acid to make 10 per cent of the whole. 
