CASTLE: EMBRYOLOGY OF CIONA INTESTINALIS. 213 
IIL 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 reageuts were employed, viz. Flemming's fluid, Her- 
mann's fluid, picro-nitric, corrosive-acetic, and Perenyi's fluid! 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 tjme 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. 
