10 ILLINOIS BIOLOGICAL MONOGRAPHS [114 



Safranin O counterstained with lichtgriin produces a pleasing stain in 

 which, however, the lichtgriin is dominant because of the few nuclei ap- 

 pearing in any one section of material. But the most pleasing stain of all 

 for presenting differentiated pictures is obtained with Mallory's triple 

 stain (Guyer 1917). By this combination of dyes all cuticular parts are 

 colored in shades of blue to purple blue — with an occasional exception 

 where it is orange red. Muscle tissue such as the contractile portions of 

 the muscle cells of the body wall are brilliant red as also are the muscle 

 fibres and bundles of the esophagus and other portions. Protoplasm is 

 pink with a suggestion of a bluish tint; nuclei are darker red with brilliant 

 orange nucleoli. Material fixed in Flemming's reagent and stained with 

 Mallory shows less red with more yellow and purple shades; differentiation 

 being even greater. 



To mount whole preparations in balsam the procedure is the same as 

 for sectioning, including the bringing of the worms into clearing fluid; 

 wintergreen is here to be preferred to the other clearing fluids in general 

 laboratory use because of its rapid penetrating power; xylol shrinks tissues 

 too readily and should be entirely avoided. Now the Syracuse crystal 

 bearing the worms in a small quantity of oil is tipped only slightly and a 

 large drop of pure, unthinned, paper-filtered Canada balsam is placed on 

 the sloping bottom of the dish away from the worms and the whole covered. 

 The resin will flow slowly down and diffuse throughout the oil and speci- 

 mens in the course of 2 or 3 hours. Should the resulting resinous mixture 

 be too thin to dry rapidly upon mounting the objects, more balsam may 

 be added as before. It is important not to rush this process because the 

 thinner medium within the worms will move through to the exterior faster 

 than the balsam can penetrate to the interior with the result that the 

 pressure becomes less within than without and unless the cuticula is thick, 

 collapsing will result; but in all cases the more volatile fluids will vaporize 

 under this reduced pressure and fill the body cavity and interstices between 

 the organs with gas so that the preparations are again valueless, being 

 utterly opaque. If collapsing has not taken place, the difficulty may be 

 remedied by thinning the balsam with chloroform or benzol until the 

 bubbles are gone, then controlling evaporation until the thickness of the 

 fluid is again suitable for mounting. However, should collapsing have 

 occurred, and should the specimens be valuable enough to warrant saving, 

 restoration may be accomplished by running the worms back to Carnoy- 

 phenol and leaving them there until the collapsed portions have filled 

 out. If this does not occur spontaneously, a slight manipulation by rolling 

 the worm gently will usually restore shape but should this not be the case 

 restoration by the lactic acid method may be used (Hetherington 1922). 



Except for low power work with a microscope, toto staining is of little 

 value in examination of relatively large specimens because of the marked 



