INOS i] VARIATIONS IN LIMULUS POLYPHEMUS. 23 
embryos. But in view of the long delay that might attend the 
publication of the first part, and among other reasons, on account 
of the interest now centred in variation and abnormal develop- 
ment, it seems advisable not to delay further the publication 
of the present paper. 
But in order to afford some ready means of comparison with 
normal embryos, prepared by my methods and interpreted on 
the same basis as the abnormal ones, Pl. I has been introduced, 
in which are represented the more important stages in the 
development of normal embryos. 
Methods of Hardening, Staining, and Clearing the Embryo 
for Surface Views and for Sections. 
Surface views of opaque embryos are useful for some pur- 
poses, certain points being brought out with special clearness 
shortly after the eggs are put into the hardening fluid. But 
in order to make out many important details, it is absolutely 
essential to stain the egg, and clear in clove oil, balsam, or oil 
of cedar. The latter often gave the best pictures and the eggs 
could be kept longer in this fluid without discoloring the yolk, 
than in oil of cloves. 
To obtain the best surface views, the embryos should be 
stained and mounted as soon as possible after hardening, or if 
that is not convenient, preserved in perfectly clean alcohol in 
glass-stoppered bottles, as the tannin, or other substances in cork 
stoppers, is dissolved out by the alcohol and discolors the yolk. 
Either picro-nitric or undiluted picro-sulphuric acid or 
Perenyi’s fluid may be used for hardening. The eggs should 
be immersed in ¢he cold solution from ten to twenty-four hours. 
The yolk is thus made quite hard, and the membranes swell so 
that they can be easily removed by fine-pointed forceps. The 
membranes must always be removed before placing in alcohol ; 
otherwise they shrink, and the embryos are injured or dis- 
torted. Moreover, a white albuminoid substance collects under 
the membranes, and is precipitated on the embryo when it is put 
into alcohol, obscuring very much the beauty and clearness of 
the preparations. 
