94 BERTRAM G. SMITH 



with very uniform success, whether fixed before or after the 

 removal of their envelopes. In particular, stages after the clo- 

 sure of the neural folds give a sharpness of detail in the surface 

 features rarely found in Cryptobranchus; these stages of Nec- 

 turus are very favorable objects for photography. 



C. SECTIONING AND STAINING 



Kerr ('01), in describing the technique employed in studying 

 the egg of Lepidosiren, has well said: ''The investigation of a 

 holoblastic egg 7 mm. in diameter and packed with yolk involves 

 great technical difficulties, for the whole of each egg has to be 

 converted into thin sections. The full extent of these difficulties 

 will only be appreciated by embryologists who have essayed a 

 similar task." In sectioning the heavily yolk-laden stages, Kerr 

 used the celloidin method, and a combination of the celloidin 

 and paraffin methods. DeBussy ('04) used the celloidin method 

 in studying the cleavage stages of Cryptobranchus japonicus. 



For sectioning the embryological material of Cryptobranchus 

 allegheniensis and Necturus I have used the paraffin method 

 exclusively; success with this method was found to be entirely 

 a matter of careful attention to technique. The most important 

 considerations are proper fixation and washing, and thorough 

 infiltration with paraffin. In handling serial sections of large 

 numbers of these eggs the advantage of the paraffin method is 

 obvious. 



With regard to staining, for general purposes the best results 

 were obtained by staining in toto with Grenadier's borax carmine, 

 and counterstaining on the slide with Lyons blue in absolute 

 alcohol ; to the Lyons blue solution sufficient picric acid was added 

 to turn it green. By this method the effect of a triple stain, 

 with excellent differentiation, is obtained. The chromatin is 

 stained red, cell walls and cytoplasm blue ; the yolk is first stained 

 red by the borax carmine, but turns green in the counterstain. 

 It is usually best to cut short the action of the counterstain at 

 a time when the smaller yolk particles are stained green, while 

 the larger ones are left red. The method has the advantage of 



