66 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 



Direct injections into the aorta can be made in the following manner: When 

 the embryo is placed in a strong light the myotomes are of course very plainly 

 visible, and along their lateral border can be seen a faint opaque streak, which is 

 the intermediate cell-mass or nephrotome. Between the nephrotome and the 

 lateral border of the myotomes is a thinner line which is more transparent. The 

 canula is then introduced between the lateral border of the myotomes and the 

 intermediate cell-mass, with the point toward the head of the embryo. If the 

 canula enters the aorta, and only a slight pressure is used, there need be no 

 extravasation at the point of puncture. After the embryo has been injected it is 

 fixed by dropping Bouin's mixture on the specimen while it is still on the yolk. 

 It is kept flooded with the fixing agent, and is not removed from the yolk until it 

 is well hardened. 



In regard to the injection of young mammalian embryos there are a few 

 special points in technique which are of interest. In order to identify the young 

 embryo pig the mucosa is examined carefully for long strings of chorion, which 

 are so inconspicuous that they are more readily found by running the finger over 

 the mucosa than by sight. These strings of chorion are then very carefully coiled 

 on a glass slide or piece of filter paper until the embryo is found. In the case of 

 the pig, I have never succeeded in puncturing the veins on the yolk-sac or the 

 umbilical vein. The latter is so large that it might be punctured if it contained 

 enough corpuscles to render it visible. The aorta, on the other hand, is readily 

 punctured opposite the mid-body region. Here in early stages the two aortse seem 

 slightly dilated, or later are fused into a single vessel. The needle is introduced 

 ventral to the myotomes. The injection mass in every case was india ink. Silver 

 nitrate seems to damage the tissue much more markedly in very young embryos 

 than in later stages. After injection the pigs were fixed in Carnoy's mixture of 

 absolute alcohol 6 parts, chloroform 3 parts, and glacial acetic acid 1 part. They 

 were then placed in 80 per cent alcohol, dehydrated in graded alcohols, and cleared 

 by the Spalteholz method of benzine followed by oil of wintergreen. 



The study of the blastoderm of the living chick embryo in a hanging-drop 

 preparation depends on the methods originated by Harrison and developed by a 

 large group of workers Burrows, Carrel, M. R. Lewis and W. H. Lewis, and others. 

 In 1912 McWhorter and Whipple applied the method to the study of the growing 

 blastoderm of the chick, which was its first application to the entire embryo, so 

 far as I am aware. These investigators mounted the blastoderm in clotted plasma 

 and used the method to test the question as to whether blood-vessels arise from 

 fusion of isolated vesicles. In 1913 Brachet published a study on the growth of 

 a mammalian embryo in a hanging-drop preparation. 



In studying the blastoderm of the chick by the method of the hanging-drop 

 I have followed the technique of Margaret Reed Lewis, of growing the embryo 

 in Locke's solution. In this way the embryo can be kept growing for several 

 hours, and the cells which are nearest the cover-slip can be seen with great clear- 

 ness and followed with an oil-immersion lens. The embryo is removed from the 

 yolk and placed in a dish of warm Locke's solution and the vitelline membrane 



