Collection, Preparation, and Preservation of Embryos. 3 



poured from the remaining portion of the shell into normal salt 

 solution. By means of a pair of small forceps and a pipette, as 

 much as possible of the surrounding albumen was removed. An 

 incision was then made into the yolk sac near the edge of the 

 blastoderm. The yolk contents flowed out instantly, but the 

 embryo was preserved from submergence in yolk granules by 

 washing gently with a pipette in a direction contrary to that in 

 which the yolk was streaming. When freed from the mass of yolk, 

 the embryo was floated into a watch crystal, in which the blasto- 

 derm was fixed in Kleinenberg's weaker picrosulphuric solution. 

 After remaining from one to two and a half hours in this solution, 

 the blastoderm was transferred to seventy per cent alcohol and 

 eventually into ninety per cent. 



The only staining reagent used was seventy per cent alcoholic 

 haematoxylin. This stain only was used, since it gave entirely 

 satisfactory histological and differential results. The different 

 germ layers are often made out satisfactorily by the aid of this 

 stain when a separation into definite layers could not otherwise be 

 traced. As a clearing reagent cedar oil was used. In this the 

 embryos could remain without fear of brittleness, and thus an 

 excellent opportunity was afforded for restudying surface views. 

 The blastoderms were embedded in paraffin and cut into sections 

 10 ,ti in thickness. Since all embryos were treated in the same 

 manner, histological differences between the embryos described 

 cannot be due to the effect of different reagents. After a slight 

 staining in haematoxylin in order to render the outlines of the shield 

 more distinct, dorsal and ventral surface views of the embryos 

 were outlined by means of the camera lucida. It seems very proba- 

 ble that the dorsally vaulted appearance of my shields is partially 

 due to my method of hardening, i. e. after removal from the yolk. 

 Mitsukuri ('93, p. 269) has thus accounted for the bulged appear- 

 ance of his own earlier embryos. Unfortunately, I have no embryos 

 hardened upon the yolk to compare with the isolated shields.* 

 This vaulted condition of the shield tends to throw into shadow the 

 notochordal cavity ; hence, when viewed from the ventral side, it is 

 often difficult to make out, on views of the depressed ventral surface, 

 the honeycomb structure described by Mehnert and Mitsukuri. In 



* Since this paper was written, I have had an opportunity to harden shields 

 upon the yolk before removal. The embryos thus killed show little or no ten- 

 dency to bulge. 



