8 ORGANIZATION AND CELL-LINEAGE ОЕ ASCIDIAN EGG. 
in fact, almost every ripe egg of Czoza will develop if fertilized with sperm from 
another individual, but if fertilized with sperm from the same animal the eggs 
rarely if ever develop, as Castle has shown. This is due to the fact that such 
spermatozoa never enter the egg, though they may be quite active. Morgan (1904) 
has recently discussed this interesting fact in a suggestive manner. 
The method which I employed in studying the living eggs of these ascidians 
was very simple; they were placed in several drops of fresh sea water upon a 
glass plate and were covered by a large cover glass, which was supported by 
pieces of thin glass about 200 » thick. In such condition the eggs can be rolled 
over at will by pushing on the cover glass, and, if drops of water are occasionally 
added at the edge of the cover, the eggs will continue to develop normally for 
two hours or more. Inasmuch as the entire development of Cyz/Aza up to the for- 
mation of the free-swimming tadpole normally occupies not more than eight to 
twelve hours, depending upon the temperature, it will be seen that a considerable 
portion of the development can be followed on a single egg. І do not doubt that 
with proper precautions the entire development might be followed on a single egg; 
however, since eggs which have been along time under a cover glass develop slowly 
and may become abnormal, and since there was nothing to be gained for my pur- 
poses by the observation of a single egg through the whole development rather than 
of several eggs through consecutive portions of it, I chose the latter and easier 
method. 
All my studies of the living eggs of these ascidians were made with a dry lens, 
the 4 mm. Apochromat of Zeiss which, with the No. 4 ocular, gives a magnification 
of about 260 diameters. Even with a magnification of 50 diameters or less the 
yellow crescent of the Cyz/Aza egg is plainly visible. In order to see this crescent 
to the best advantage, especially with high powers, it is necessary to use wide angle 
lenses with open diaphragm and clear white light. Тһе fact that Castle studied 
the development of this species but makes no mention of this yellow crescent is 
difficult to explain. І сап only account for it by supposing that he obtained the 
eggs in the evening and studied them by yellow artificial light. 
Preserved material was fixed in various fluids, —Perenyi' s, Kleinenberg's, Picro- 
Acetic, Sublimate and Sublimate-Acetic. For the study of entire eggs and embryos 
Kleinenberg's fluid followed by the Picro-Hematoxylin, which I have used with 
success on molluscan eggs, gave incomparably better results than any other method. 
Eggs so stained were mounted in balsam under thin cover glasses without supports 
of any kind, and were studied with an oil immersion lens, the 3 mm. Apochromat 
of Zeiss. Ву occasionally applying a drop of xylol to the edge of the cover glass 
the balsam remains sufficiently soft so that the eggs can be rolled into any position 
desired. For serial sections, material fixed in Boveri's Picro-Acetic gave the most 
satisfactory results. Such material was stained on the slide in Delafield's Hzema- 
toxylin and Eosin or in Iron Hematoxylin and Bordeau red. 
Castle states that he found it necessary to remove the egg envelopes by 
drawing the egg into a pipette through an opening so small that the egg alone 
