8 ILLINOIS BIOLOGICAL MONOGRAPHS 1112 



The main points of a simplified and rapid technique suitable for the 

 microscopical preparation of the larger free living and parasitic nematodes 

 may be found in an article by the author (Hetherington 1922), the essen- 

 tials of which will be repeated here for convenience, with the addition of a 

 few suggestions and comments. 



The greatest obstacle to successful nematode preparation is the almost 

 impenetrable cuticula in which the animal is encased, as it were, offering a 

 splendid barrier to the entrance of the ordinary fixatives and clearing 

 media, particularly the resinous ones in which one often desires to mount 

 specimens. 



During a series of experiments with various killing and dehydrating 

 fluids it was noticed that little collapse and crumpling took place in fluids 

 containing acids as lactic and acetic. The liquids seemed to diffuse with 

 greater ease through the cuticula if some agent were present which kept it 

 soft and pliable during the stages of dehydration, especially between 85 

 per cent alcohol and the clearing agent where the greatest difficulty was 

 always encountered. In every case of shrinkage and collapse the indica- 

 tions were that diffusion pressures caused the damage and that, were a 

 series of dehydrating and clearing fluids possible which had very similar 

 diffusibilities or penetrabilities, the greatly unbalanced diffusion pressures 

 arising during the passage of the material from one liquid to the next 

 would be eliminated and with them the distortion of the specimens. 



By the use of Carnoy-phenol, itself water free, the killing and dehydra- 

 tion processes can be accomplished at once in the following way: The ma- 

 terial freshly collected and freed from adhering dirt and slime is placed in 



Carnoy-phenol 



Absolute alcohol 20 cc 



Chloroform 15 cc 



Glacial acetic acid 5 cc 



Phenol crystals to raise the volume by 10 cc 



Total 50 cc 



If the fluid is too strong for very delicate worms, it may be weakened 

 by the addition of a very small quantity of water. After killing the 

 worms should be placed in the fluid of full strength before further opera- 

 tions are undertaken. With material so killed only two operations are re- 

 quired to bring the objects into paraffin or balsam, and one to clear them in 

 glycerine; if killed in other media and stored in alcohol, three operations 

 attain the same end. Nematodes may be taken from 70 to 80 per cent 

 alcohol, glycerine, lacto-phenol, or formol in which they have been stored 

 and placed directly in the fluid. Smaller worms are cleared almost in- 

 stantly so that a rapid survey if desired may be made of their internal 



