Langdon, Sense-organs of Nereis virens. 5 



of the animals under as nearly as possible normal conditions for a time 

 long enough to allow the stain to be carried all over the body by the 

 blood-vessels. 



The same worm can be used for study for from three to five hours 

 after removal from the dark, probably even longer. When a part of 

 the body was mounted in sea-water and covered with a cover-glass, the 

 stain quickly faded from all parts except the nerve-elements, which kept 

 the stain from one half to three quarters of an hour. 



Parts of the worm which were to be preserved were dropped into 

 Bethe's fixing fluid ( invertebrate formula) which had been previously 

 cooled. The tissues were kept on ice in this fluid for from 4 to 6 hours 

 — or even over night if convenient. They were then washed in a large 

 quantity of distilled water for from 10 to 12 hours, and passed quickly, 

 but by gradual steps, through the various grades of alcohol. The spec- 

 imens were not only kept on ice, but each grade of alcohol was cooled 

 before using. It was not only found that the warm alcohol removes 

 the stain, as stated by Bethe, but also that warm water does the same. 

 Several times tissues which a microscopical examination showed to be 

 richly stained up to the time of warming for the paraffin bath, were at 

 once ruined by this process and permanent mounts of such tissues 

 showed that they had not been thoroughly dehydrated. 



Pure xylol proved to be the best clearing fluid ; the transfer from 

 the absolute alcohol to the xylol was made gradually and the xylol kept 

 on ice until all trace of the alcohol was removed. The tissues were 

 warmed gradually to the melting point of the paraffin used and kept 

 for two or three hours in the paraffin which was changed once or twice 

 before embedding. When embedded in paraffin, even it the paraffin 

 is allowed to cool before all the xylol is removed, the tissues retain the 

 stain well. Material which had been kept in a block of soft paraffin 

 for six months before cutting was in excellent condition and was unin- 

 jured by re-embedding. 



The sections were cut from from 20 to 45 /< thick, and fixed to 

 the slide by means of albumen fixative — the warm water method prov- 

 ing unsatisfactory in this case since it seemed to injure the stain. May- 

 er's alcoholic cochineal, which stains in 10 min., proved to be the best 

 secondary stain. While in the grades of alcohol and in the stain itself 

 the sections were kept cold ; they were cleared in xylol and mounted 

 in xylol balsam. Sections thus prepared show no sign of losing their 

 blue after an interval of three years. The only epidermal structures 

 not well preserved in these preparations are the spiral organs. Lewis 

 ('98) states that the cuticula in Axiothea and Clymene is badly pre- 



