Ascidia (Phallusia) , after from three to six hours in 

 chloral hydrate of 0.1 of 1 per cent, is hardened for half an 

 hour in chromic acid of 1 per cent, washed, and transferred to 

 alcohol. 



Ciona intestinalis is killed slowly by putting into the 

 water in which the animals have expanded a few drops of the chrom- 

 acetic mixture No. 2. When the animal has died, which happens 

 in about a half hour, it is to be taken by the anterior orifice, 

 to avoid the discharge of the water within, and put into chromic 

 acid of 1 per cent, making an injection of the same into the body 

 cavity. The transfer to the alcohol series should be made in 

 the same way. 



Certain ascidians (Ascidia and Rhopalea) are killed in 

 the following manner, so as to keep the orifices open: They 

 should be placed in beakers with from 4 to 5 cm. (about 2 inches) 

 of sea water above them. Then slowly drop in 1 per cent chromic 

 acid in such a manner as to form a stratum on the top of the 

 water. Little by little the chromic acid will diffuse through 

 the water, usually killing the animals in from twelve to twenty- 

 four hours. If the animals are not dead by that time, add a 

 little more chromic acid. Harden in chromic acid of 1 per cent, 

 wash in fresh water, and put into alcohol. The animals should 

 not rest against the sides of the beaker during narcotization. 

 If the animal has not a good base upon which to stand, some 

 clean sand may be placed in the bottom of the glass, in which 

 it can be arranged in the desired position. 



Molgula, Polycarpa, Rhopalea, and Chevreulius (Rhodosoma) 

 must remain for twelve hours in chloral hydrate of 0.1 of 1 per 

 cent. Then kill them in chrom acetic No. 2 and transfer at once 

 to 1 per cent chromic acid for a little time to harden. 



Cynthia and Styela are narcotized in 0.2 of 1 per cent 

 chloral hydrate for twenty-four hours and then treated like the 

 last-mentioned genera. Cynthia papillosa, however, sometimes 

 contracts greatly when immersed in chloral hydrate of 0.2 of 1 

 per cent. When it does, put it back into running sea water to 

 expand again, and then try treating it with 0.1 of 1 per cent 

 chloral hydrate. 



Ascidiae compositae. — The gelatinous forms — for example, 

 the Botryllidae, Polycyclus, Circinalium, and Fragarium — are 

 allowed to lie in chloral hydrate of 0.1 of 1 per cent for a few 

 days and then are killed by pouring hot saturated sublimate over 

 them. Immediately afterwards they are transferred to chromic 

 acid of one-half of 1 per cent, where they are left for a half 

 hour before they are washed and transferred to alcohol. 



Distaplia, after it has been narcotized with 0.1 of 1 per 

 cent chloral, is killed with chrom-acetic No. 2, washed, and put 

 directly into weak alcohol. 



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