ORIGIN OF BLOOD-VESSELS IN BLASTODERM OF CHICK. 219 



variation in the width of the area pellucida, and since it is only this area that can 

 be seen distinctly in the living specimen, there is considerable variation in the value 

 of different specimens. The stretching of the specimen flattens the embryo some- 

 what, so that the circulation, if begun, is often not reestablished immediately on 

 the coverslip. In this connection it is interesting to note that the circulation is more 

 often impeded in the left side than in the right, since the heart curves to the right, 

 and hence the left vitelline vein is the longer and becomes compressed across the 

 body of the embryo. In spite of this mechanical disadvantage a good circulation 

 is often (indeed usually) reestablished and maintained for 3 or 4 hours. The slide 

 must, of course, be kept at a temperature of 39 C., either in a stage incubator or 

 in a warm box. 



The average life of the specimen is about 5 hours. The heart can often be made 

 to beat, after it has stopped, by a bath in fresh solution, but I have never seen it 

 beat for more than an hour after this procedure. Cell division does not cease when 

 the heart stops, but I have made no tests to determine the actual length of life of 

 cells in these preparations. The most striking sign of the death of the cell in these 

 specimens is that the resting nuclei, which have been practically invisible in the 

 total mounts, become almost as plain as if they had been treated with an acid. 



The specimens are fixed by floating the cover-slip on Bouin's fluid of 75 parts 

 of saturated aqueous picric acid, 20 parts of formol and 5 parts of glacial acetic 

 acid. The picric acid is removed by repeated changes of 70 per cent alcohol without 

 the use of any of the lower grades of alcohol or of water. If placed in water the 

 specimens swell and sag away from the glass. I change the alcohol several times the 

 first day, then keep the specimens in 70 per cent alcohol until white, when they are 

 placed in 80 per cent. I have used absolute alcohol, methyl alcohol, and Kelly's 

 fluid as fixatives, but with less success. 



For stains I have had the best results with hematoxylin alone, or with hema- 

 toxylin and a counterstain of eosin (6 parts), orange G (4 parts), and aurantia 

 (1 part). In staining with hematoxylin the specimens must be hurried through 

 water and not left too long in the dilute stain or they will swell, as in water. The 

 young blastoderms react intensely to hematoxylin and nothing in the tissues 

 reacts to an acid stain except the globules of yolk. A blastoderm, therefore, with- 

 out any counterstain can be analyzed in a total preparation to a considerable extent 

 over the area opaca, while it is seldom possible to focus sharply enough through its 

 thick endoderm in the living specimen. In specimens which have hemoglobin in 

 the cells a counterstain helps to bring out the contrast between angioblasts without 

 any hemoglobin and the true blood-islands. I have used iron hematoxylin, but with 

 less success, for the total mounts. Eosin-azur brings out the basophilic granules of 

 the angioblasts and of the young blood-islands better than hematoxylin, but it is 

 difficult to get good, permanent preparations of total mounts with this stain, since 

 it is impossible to differentiate accurately for a specimen which varies so greatly 

 in thickness. Alum-carmine is also an excellent stain for these specimens. 



If it is desired to mount a specimen with the ectoderm against the cover-slip, 

 a method which offers advantages for studying the area opaca, or if the specimen 



