DEVELOPMENT OF PRIMITIVE BLOOD-VESSKLS. 67 



and most of the yolk removed. It is more difficult to study the blastoderm with 

 the dorsal surface against the cover-slip than from the ventral aspect, because 

 the ectoderm does not adhere to the glass as well as does the endoderm, and it is 

 necessary to have the embryo very flat in order to see the cells with higher powers. 

 In regard to the blood-vessels, it is of course preferable to study the embryo from 

 the ventral aspect, since the blood-vessels are nearer the endoderm than the 

 ectoderm. In the study of the developing blood-islands it would be very advan- 

 tageous to have the dorsal surface of the embryo against the cover-slip, as the 

 blood-islands are farther dorsal and because the cells of the ectoderm over the 

 area opaqua are much thinner than the cells of the endoderm over the same area. 

 The cells of the endoderm of the area opaqua are so thick and so filled with glob- 

 ules of yolk that one can seldom focus through them in the living embryo. From 

 the ventral aspect there is also an area of the axis of the embryo that it is very 

 difficult to study with high-power lenses, namely, the portion of the axis just caudal 

 to the head-fold, because the heart lifts the embryo from the cover-slip and the 

 cells can then be studied only with dry lenses. 



All of the chicks have been fixed in Bouin's mixture of saturated aqueous 

 picric acid 75 parts, formalin (40 per cent) 20 parts, and glacial acetic acid 5 parts, 

 for about 12 hours. They are then placed directly in 60 per cent alcohol. In the 

 case of the chicks which have been growing on the cover-slip it is very necessary to 

 have the embryo stick to the cover-slip throughout the fixation and dehydration. 

 If the specimens are to be mounted in toto, they are mounted on the same cover-slip 

 on which they were growing. If they are to be embedded and cut they can be 

 removed from the cover-slip after they have been cleared in the oil of wintergreen. 

 The specimens do not become as brittle in the oil as in xylol. In the blastoderms 

 which are kept on the cover-slips it is possible to watch the effects of dehydration 

 much more accurately than with free specimens. The edge of the specimen around 

 the outer margin of the area opaqua clings very closely to the cover-slip; in fact, in 

 mounting the embryo strands of tissue are pulled out which dry slightly and help 

 the specimen to remain fixed. If the specimens are put into alcohol weaker than 

 60 per cent this outer margin will stick to the cover-slip, but the entire area pellu- 

 cida will become free from the cover-slip and swell into a bleb. The space beneath 

 fills in with fluid, and in the subsequent dehydration there is an uneven shrinkage 

 which distorts the tissues. Thus, weak alcohol or water, or a dilute stain, macerates 

 and swells the tissues and the subsequent shrinkage distorts the cells. On the 

 other hand, if the specimens are placed directly in alcohol as strong as 60 per cent, 

 a plexus of cells, which has been studied in the living specimen, can be readily 

 identified in the fixed specimens. If the specimens are to be stained in toto they 

 must be placed in a stain sufficiently strong so that the tissues will not macerate 

 before they react to the stain. From such an experience one should avoid washing 

 embryos in water and should also avoid the use of the lower grades of alcohol. I 

 have used several changes of 60 and 65 per cent alcohol to eliminate the excess of 

 picric acid. The dehydration can be done by changes of 5 per cent. If it is carried 

 out too rapidly the specimens will crack, but the shrinkage with the higher grades 



