216 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY. 



great service in sectioning. When this was desired, the egg, previously 

 studied as a whole object, was returned to xylol. The transfer was accom- 

 plished by placing the slide on which it was mounted in a shallow por- 

 celain dish containing a little xylol. Tins soon dissolved away the 

 balsam, and left the egg free and clearly visible against the white back- 

 ground. The egg was next removed to a shallow watch-glass with a 

 perfectly flat bottom, which was previously smeared with a thin layer 

 of glycerine. Any superfluous xylol was removed from about the egg 

 with Alter paper, and a small amount of melted paraffine poured over it, 

 enough to fill the watch-glass to a depth of 3 to 5 mm. The whole was 

 then set over the paraffine bath for fifteen or twenty minutes, when it 

 was placed floating on a dish of water to cool. This being accom- 

 plished, the paraffine block was removed from the watch-glass, and the 

 egg, which of course had settled to the bottom and lay with its long axis 

 parallel to the surface of the block, was oriented under the compound 

 microscope in any manner desired. The thinness of the block generally 

 allowed plenty of light to pass through it for this purpose, and it was 

 usually not difficult, owing to the shape of the embryo, to determine its 

 axes. Sections were usually cut 6|- jx in thickness. 



The staining which was found most advantageous for the study of the 

 egg as a whole object was altogether too faint for sections. These were 

 accordingly given a further staining after fixation to the slide. Ehrlich's 

 hematoxylin was employed, diluted one half with water. After immer- 

 sion in the stain for from twenty minutes to an hour, the sections were 

 washed in water to remove the superfluous stain, then to decolorize were 

 placed in 35% alcohol containing 0.1% hydrochloric acid. Here they were 

 allowed to remain until quite pale in color, usually for about five min- 

 utes. They were then rinsed in 35% alcohol and held for an instant over 

 the unstoppered mouth of an ammonia bottle, a treatment which gave 

 the hematoxylin remaining in the sections a deep blue color, and in- 

 sured the permanency of the stain. The sections were then passed 

 through the grades of alcohol, cleared in xylol, and mounted in balsam. 

 This process, when properly conducted, resulted in a beautiful and sharply 

 differential double stain. The nuclei retained the light rose tint given 

 them by the carminate of lithium, for the superadded hematoxylin stain 

 had been entirely removed from them, except in the chromatic elements, 

 which possessed a deep black color. Cell boundaries, attraction spheres, 

 and other cytoplasmic structures, were clearly brought out, and the fun- 

 daments of various organs, as, for example, chorda, mesoderm, and defini- 

 tive endoderm, were distinguished one from another with great sharpness 



