218 ORIGIN OF BLOOD-VESSELS IN BLASTODERM OF CHICK. 



unincubated hen-egg contains 1.07 per cent of sodium chloride, and that the 

 amount decreases as the blastoderm develops, I used 1.07, 1.06 and 1.05 per cent. 



With these higher percentages of sodium chloride it was evident at once, not 

 only that the hemoglobin was not laked out of the red blood-cells, but that the solu- 

 tion was a more favorable one for all of the cells of the young blastoderm. To deter- 

 mine the best solution for each of the early stages of the chick would necessitate a 

 much longer series of experiments than I have carried through. So far my best 

 results have been with solutions containing 1.06 or 1.05 per cent of sodium chloride 

 in specimens of less than 20 somites. In specimens having between 20 and 30 

 somites a solution containing 1.07 or 1.06 per cent of sodium chloride will show a 

 considerable amount of crenation of the free red blood-corpuscles, and hence is too 

 strong. It is interesting to note that the young red cells still attached to the islands, 

 or just freed from them, do not tend to crenate as much as the older cells, indicating 

 that there is a slight difference between them. The concentration of the sodium 

 chloride is a very important point for the successful study of the young chick- 

 embryos because all of the cells are very sensitive to it. 



The technique of preparing the specimens is as follows: The blastoderm is 

 cut out in a warm box under sterile precautions, leaving a narrow rim around the 

 area opaca; it is then placed in a dish of warm, sterile solution and freed from the 

 yolk and the vitelline membrane, after which it is transferred to a dish of fresh 

 solution and floated on a sterile coverslip. This must be free from grease, as for 

 blood-smears. The coverslip is then placed on a piece of blotting paper and the rim 

 wiped perfectly dry. I mount the blastoderms, for the most part, with the endoderm 

 against the coverslip. This has a twofold advantage: in the first place, the great 

 majority of the vessels develop ventral to the mesoderm and hence are nearer to 

 the endoderm than to the ectoderm ; in the second place, the cut edges of the blas- 

 toderm always roll clorsalward and hence it is easier to get flat mounts on the ventral 

 surface. Moreover, the endoderm sticks to the glass much more closely than the 

 ectoderm, a matter of great importance in using a thin medium like the Locke- 

 Lewis solution. If the specimen sags away from the glass it interferes very much 

 with the development of the blastoderm. It must therefore be spread out carefully 

 and the edges pulled out to the dry rim of the covershp, leaving room enough for 

 the rim of vaseline which seals the mount but which must not touch the specimen 

 at any point. If in the mounting of the specimen a little of the blastoderm comes 

 in contact with the vaseline it should be remounted, or else a symmetrical point of 

 the specimen be brought in contact with the vaseline. Otherwise surface tension 

 will greatly distort or even destroy the embryo. The covers are mounted on a 

 hanging-drop slide and the amount of fluid which remains on the blastoderm is 

 sufficient to keep the embryo alive. 



Inasmuch as the endoderm of the area opaca is many times as thick as that of 

 the area pellucida, it is clear that there must be a rim of tissue just at the border 

 between the two which will not be in exact contact with the glass. To make this 

 rim as narrow as possible I stretch the specimen on the glass to a considerable extent. 

 If one has a large number of specimens it will become clear that there is a very great 



