DEVELOPMENT OP PLUSTRELLA HISPIDA. 439 



complete penetration. As soon as possible after fixation the 

 colonies were removed from the Facus and after washing 

 transferred to 70 per cent, alcohol. Chromo-acetic acid and 

 corrosive acetic have given the best results when dealing 

 with material fixed in bulk. 



Isolated Larvae. — The removal of larvae from the colonies 

 is best effected by slicing off the front wall of the colony with 

 a sharp razor ; the larvae lie immediately below this wall, 

 enclosed in the tentacle-sheath of the polypides, and they can 

 then be readily removed by means of a scalpel. Before 

 attempting to preserve the larVcB a considerable amount of 

 washing is necessary in order to free them from a mucus-like 

 substance in which they lie imbedded. 



The best fixing reagents for the isolated larvae appear to 

 be corrosive acetic, and acetic alcohol saturated with sub- 

 limate ; chromo-acetic acid sometimes gives good results ; and 

 the fixing reagents containing osmic acid have proved useful, 

 especially in the study of entire eggs and larvae before 

 clearing. Material preserved in chromo-acetic requires 

 very prolonged washing and frequently proves difficult to 

 stain. 



Entire Eggs and Larvee. — The external characters of 

 isolated eggs and larvae of all stages have been studied 

 during life. After fixation the larvae were again examined 

 unstained in 70 per cent, alcohol, and were then stained either 

 in borax carmine followed by acid alcohol, or in safranin, 

 and re-examined. After clearing with cedar- wood oil or clove 

 oil — both of which reagents gave good results — the larvae 

 were either mounted entire in Canada balsam or imbedded in 

 paraffin for sectioning. Staining the larva with borax carmine 

 after acetic alcohol and corrosive sublimate or corrosive 

 acetic brings out the nuclear spindles and also the yolk 

 nucleus very clearly, and so greatly assists in the interpreta- 

 tion of the external appearance of a segmenting egg. In a 

 few cases the embryo was removed from Canada balsam 

 after having been examined and drawn, and was imbedded 

 in paraffin for sectioning. 



