CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 215 



them to alcohol of a low grade, or to water, the egg envelopes would 

 again stand out clear of the ovum, as in the living egg. By then suck- 

 ing tlie eggs one at a time into a glass tube of the proper calibre, the 

 entire envelope, consisting of follicle cells, test cells, and chorion, could 

 be removed with considerable facility, and in the majority of cases with- 

 out injury to the egg itself. Eggs thus decorticated and then mounted 

 afforded excellent surface views. 



The eggs are rather opaque, on account of the large amount of yolk 

 which they contain, so that any stain except a veiy faint one is an ob- 

 stacle in the study of whole preparations. Excellent results were obtained 

 by mounting in balsam, without any staining whatever, eggs which had 

 been killed in Perenyi's fluid and decorticated.^ But for the 64-cell and 

 later stages staining was found desirable. Many carmine and ha;matoxy- 

 lin stains were tried ; the one which gave by far the best results being 

 Orth's picro-carminate of lithium. The eggs were treated with a small 

 amount of tliis stain in a watch-glass for from six to twenty-four hours, 

 then washed thoroughly in water. By this method resting nuclei are 

 stained bright rose-color, while all other structures take only a faint 

 yellow color from the picric acid, and give up even this if the washing is 

 sufficiently prolonged. But the carmine stain in the nuclei is extremely 

 tenacious, and does not fade in the least upon prolonged washing in water 

 or preservation for months in strong alcohol. After the eggs had been 

 stained and decorticated, they were dehydrated, cleared in xylol or cedar 

 oil, then mounted in balsam, the cover glass being supported with small 

 glass rollers made from fine capillary tubes. These served the double pur- 

 pose of preventing the crushing of the egg and allowing it to be rolled 

 into any desired position by movement of the cover glass. Changing the 

 position of the egg, however, is not often necessary, for at an early stage 

 it takes on a flattened form, which causes it to come to rest with the dorsal 

 or the ventral surface uppermost. This is the case at all periods between 

 the 24-cell stage and that at which the neural tube begins to close, ex- 

 cept for a brief period, when the embryo consists of from forty-six to 

 sixty-four cells, and the vertical axis becomes equal to or even greater 

 than the longitudinal axis. Then there is no single position of stable 

 repose for the embryo, and rolling is often necessary to bring it into the 

 positions desired. 



The self-orientation of the egg during most of the early stages was of 



1 I find that Lillie ('95) has obtained good results in the case of the eggs of the 

 mollusk Unio by mounting, without staining, material killed in Perenyi's fluid. 

 He, however, used glycerine instead of balsam as a mounting medium. 



