ZOOLOaY AND BOTANY, MICROSCOPY, ETC. 533 



angles to the longer axis of the egg. If an egg, after one or two 

 days' incubation, is opened, while held in such a position that its 

 large end is turned to the left and its small end to the right of the 

 operator, it will be found that the caudal end of the embryo is directed 

 towards the operator, while the cephalic end is turned in the opposite 

 direction. Out of 166 cases, Duval found only three that could be 

 regarded as exceptions to the rule. Assuming that the orientation is 

 the same before the appearance of the primitive streak, we have then 

 a very reliable means of recognizing, even in the freshly-laid egg, 

 when the blastoderm has a homogeneous aspect, the future anterior 

 and the future posterior region. But this fact alone is not all that is 

 required for complete orientation ; the blastoderm must be hardened, 

 and the means of orientation must be preserved. That portion of the 

 vitelline sphere which bears the blastoderm must be so marked that 

 the anterior and posterior regions of the blastoderm may be recognized 

 after the process of hardening, and after the blastoderm, together 

 with some of the circumjacent yolk, has been cut free from the rest 

 of the egg. This may be done in different ways, according to the 

 method employed in hardening. 



1. Osmic Acid Method. — 1. Make a triangular box without bottom, 

 by folding a strip of paper 5 mm. wide and 50 mm. long. 



2. After opening the egg carefully from the upper side, remove 

 with a pipette the thin layer of albumen which lies above the 

 cicatricula, so far as this can be done with safety. 



3. Place the triangular box over the blastoderm in such a manner 

 that the base corresponds to the future anterior region, and the apex 

 to the future posterior region. While pressing slightly on the box 

 in order to bring it into close contact with the surface of the yolk, 

 fill it by means of a pipette with osmic acid (1/3-1/4: per cent.), and 

 allow the acid to act for some minutes. 



4. As soon as the area inclosed by the box begins to blacken, the 

 whole should be immersed in a vessel of chromic acid, in which the 

 paper box may be detached, and the vitelline sphere freed from 

 the albumen and the shell. 



5. The vitellus may now be transferred, by the aid of a very deep 

 watch-glass, to another vessel of chromic acid, where it is allowed to 

 remain one or two days, until the peripheral layers harden and form 

 a sort of shell around the central portion which is still soft. 



6. A triangular piece of this shell, inclosing the triangular area 

 browned by the osmic acid, is next to be cut out with a pair of sharp 

 scissors. The excised piece is then left a day or more in the chromic 

 solution before treatment with alcohol. 



II. Alcohol Method. — 1, Open the egg as before, and, without 

 attempting to remove the albumen, place the triangular paper box 

 over the blastoderm ; slight pressure causes the box to sink into the 

 albumen till it is brought into contact with the yolk. By the aid of 

 a pipette, fill the box with absolute alcohol ; this coagulates rapidly 

 the inclosed albumen, while the albumen outside the box remains 

 fluid. 



2. After cutting the chalazeee close to the vitellus, the fluid 



