EMBRYOLOGIOAL METHODS. 297 



Before putting it into the hardening fluid^ the portion of 

 vitelline membrane that covers the blastoderm should be 

 removed with forceps and shaking. 



Fixation in 10 per cent nitric acid has the advantage of 

 greatly facilitating the separation of the blastoderm. The 

 acid should be allowed to act for ten minutes, after which it 

 is well to bring the preparation into 2 per cent, solution of 

 alum [rf. Hofmann, Zeit. wiss. mik., x, 1893, p. 485). Mitko- 

 PHANOW {Anat. Hefte, xii, 1899, p. 200) fixes with nitric acid 

 of 3 per cent., Suschkin {Nour. Mem. Soc. Nat. Moscow, xvi, 

 1899, p. 34) with sublimate ; Fischel {Blurph. Jahrh., xxiv, 

 1896, p. 371) with Rabl's platiuo-sublimate, § 76 (embryos 

 of the duck) ; Patterson (Biol. Bull. Wood's Hole, xiii, 

 1907, p. 252) with picro-sulphuric acid containing 8 per cent, 

 of acetic acid, for an hour (ova of Columba) ; Hoskins 

 {Kansas Univ. Sci. Bull., iv, 1907, p. 176), after removing 

 shell, for 5 to 15 minutes in a mixture of 3 parts of 10 

 per cent, formol with 1 of 10 per cent, nitric acid, and then 

 excises the embryo. 



In order to counteract the turning up of the edges of the 

 blastoderm that generally happens during the process of 

 hardening, it is well to get the blastoderm spread out on the 

 convex surface of a watch-glass, and leave it so during the 

 hardening. 



For hardening Hbnneguy pi-efers the osmic acid and alcohol 

 mixture of Ranvier and Vignal, or Flemming's mixture fol- 

 lowed by successive alcohols. 



Stain and imbed by the usual methods. 



Up to about the fiftieth hour embryos may be mounted 

 entire in glycerin or balsam. 



596. M. Duval's Orientation Method [Ann. 8c. Nut., 1884, 

 p. 3). — In the early stages of the development of the ova of 

 Aves, before the appearance of the primitive streak, it is 

 difficult to obtain a correct orientation of the hardened cica- 

 tricula, so as to be able to make sections in any desired direc- 

 tion. Duval, starting from the fact that during incubation 

 the embryo is almost always found to be lying on the yolk in 

 such a position that the big end of the egg is to the left, and 

 the little end to the right of it, marks the position of the 

 blastoderm in the following way. 



