INDIVIDUALITY OF THE GERM-NUCLEI. 247 



few cases has the individuality of the germ-nuclei during this 

 stage been demonstrated with any considerable success; the 

 observations of Hacker (1892), Riickert (1895) and Conklin 

 (1901) are of most importance in this connection and will be 

 discussed later. 



In the developing egg of the amphibian Cryptobranchus alle- 

 gheniensis I have found material very favorable for the study of 

 this problem. The early cleavage nuclei are invariably double 

 throughout the entire resting stage, each consisting of two 

 separate though closely apposed nuclear vesicles, and the origin 

 of each vesicle from a single germ-nucleus has been clearly 

 traced. Indeed, my observations indicate that typically the 

 germ-nuclei maintain their individuality throughout cleavage, 

 though certain irregularities, real or apparent, occur with in- 

 creasing frequency in the later segmentation stages. 



TECHNIQUE. 



Most of the sections used in making the observations recorded 

 in the present paper were cut from eggs killed with bichromate- 

 acetic-formalin. This mixture, made up in the right propor- 

 tions (Smith, 1912, I.), preserves the form of the egg perfectly 

 in all stages, enables it to be sectioned by the paraffin method, 

 and gives very satisfactory histological and cytological results. 

 For comparison, sections were made of a few eggs killed in 

 Zenker's fluid, also in corrosive-acetic-formalin. These mixtures, 

 particularly Zenker's fluid, gave excellent results in the fertiliza- 

 tion and early cleavage stages, but in the blastula and gastrula 

 stages their use is not advisable since the roof of the blastoccele 

 or gastroccele collapses during the process of infiltration with 

 paraffin. 



Most of the observations were made on sections of eggs stained 

 in toto with Grenacher's borax-carmine, differentiated in acid 

 alcohol, and counterstained on the slide with a mixture of Lyons 

 blue and picric acid. This treatment leaves the chromatin 

 stained red, the yolk granules green, the centrospheres and cyto- 

 plasmic structures blue. The method has the advantage of 

 rapidity of manipulation, an important consideration since a 



