8 ORGANIZATION AND CELL-LINEAGE OF ASCIDIAN EGG. 



in fact, almost every ripe egg of Ciona will develop if fertilized with sperm from 

 another individual, hut 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) 

 lias 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 n 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 Cynthia 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. I 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 Cyiitliia 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. The fact that Castle studied 

 the development of this species but makes no mention of this yellow crescent is 

 difficult" to explain. I can 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-Ha?matoxylin, 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. Pyy 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 iixed in Boveri's Picro-Acetic gave the most 

 satisfactory results. Such material was stained on the slide in Delafield's Hema- 

 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 



