CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 213 



III. METHODS. 



1. Killing, Preservation. 



Whenever it was desired to kill a lot of eggs, a sufficient quantity of 

 them was collected in a pipette from the bottom of an aquarium and 

 transferred to a watch-glass, or directly to a small vial of two drams' ca- 

 pacity, in which the eggs were ultimately stored. After the eggs had 

 settled to the bottom of the dish, the water was carefully removed and 

 the killing reagent applied. 



The eggs were ultimately preserved in 90% alcohol, and the vials 

 tightly corked, or preferably stoppered with cotton plugs and stored in 

 tightly sealing glass jars. When the latter method is employed, the jars 

 must be kept right side up in transportation, otherwise the small eggs 

 will settle into the cotton plugs and be lost. However, the extra trouble 

 which this method necessitates is well worth taking, for it entirely avoids 

 the injurious effects on preserved material sometimes caused by the 

 tannin which alcohol will extract from corks, if they are used. 



Several killing reagents were employed, viz. Flemming's fluid, Her- 

 mann's fluid, picro-nitric, corrosive-acetic, and Perenyi's flu id. ^ The 

 blackening effects of the first two reagents made material killed in them 

 unfit for use in the study of eggs as whole objects. Likewise in the case 

 of sections the results from them were disappointing. The only real 

 service rendered by either of these two reagents was in demonstrating in 

 the egg by their blackening effects the character and distribution of the 

 fatty yolk granules. Most serviceable of all the reagents employed on 

 the eggs and embryos up to the period of hatching was Perenyi's fluid. 

 It renders the abundant yolk clear and transparent, and preserves all 

 structures perfectly, without distortion by either swelling or shrinking. 

 Its use does not in my experience interfere in the least with sharp differ- 

 ential staining. The fluid was allowed to act for about twenty minutes, 

 then followed by 70% alcohol, which, to insure removal of every trace of 

 the killing reagent, was changed once or twice in the course of the next 

 twenty-four hours, and replaced at the end of that time with 90% alco- 

 hol. A longer treatment with the killing reagent, extending to three or 

 four hours, seemed to give no added advantage, but to interfere slightly 

 with subsequent staining. 



Picro-nitric also gave good results, but for the pre-larval stages not so 

 good as Perenyi's fluid, its clearing effects being less. It seems, however, 



1 For the composition of the killing reagents and stains mentioned in this paper, 

 see Lee's "The Microtomist's Vade Mecum," 3d edition, London, 1893. 



