Appendix D: Preparation of Microscopical Material 223 



To Prepare Earthworms for Sectioning, secure good-sized speci- 

 mens, wash them in water and place them in a vessel containing 

 moist filter paper. There must not be sufficient water to drown 

 the worms nor little enough to let the paper dry out. Put only 

 a few worms in each dish and adjust the cover so as to admit a 

 little air. After 12 to 24 hours it is well to remove any dead or 

 injuried specimens and to change the filter paper. The dish should 

 be kept from direct sunlight in a cool place. After two or three 

 days the grit and dirt in the alimentary canal will have been 

 passed out and its place taken by paper which the worms have 

 eaten. They are then ready to kill and preserve or section. 



Place the worms in a flat vessel and pour on sufficient water 

 to cover them. During the next two hours add a little alcohol 

 from time to time until the strength of the liquid is increased to 

 about 8 or 10 per cent. Then wash all mucous from the body of 

 the worms and replace them in 10 per cent, alcohol until they no 

 longer respond to pricking or pinching with forceps. Transfer 

 them to 50 per cent, alcohol for several hours, keeping them 

 straightened out as much as possible; then to 70 per cent, alcohol 

 for 12 hours, followed by 95 per cent, alcohol for 24 hours. 

 Preserve finally in 70 per cent, alcohol. 



For sectioning the preliminary steps are the same but from 

 10 per cent, alcohol the worms should be placed into Zenker's 

 fluid (reagent 6, Appendix B) for 4 to 6 hours. For washing, 

 etc., follow the directions given in the discussion of the reagent. 

 To facilitate penetration of the fluid, it is well to slit open the 

 body cavity of the worm in places that are not to be sectioned. 

 The most instructive sections are cross-sections of the middle of 

 the body, and sagittal sections of the anterior end which include 

 the pharynx. The worms may be stained in bulk (24 to 36 

 hours) in borax-carmine (reagent 32) or hematoxylin (reagent 

 49) before sectioning. 



Entire nephridla together with a small part of the septum 

 which they traverse should be carefully dissected out, stained in 

 borax-carmine (reagent 32), dehydrated, cleared, and mounted 

 in balsam. 



