ORIGIN OF THE PULMONARY VESSELS IN THE CHICK. 15 



To make a suitable glass canula, select a piece of soft glass tubing 12 cm. long 

 and 5 cm. in diameter. Using a Bunsen flame, mold the tubing into the shape 

 of a U . Hold an arm of the U in each hand and draw out quickly until the base 

 measures 5 cm. ; now substitute a small flame (1 cm.) for the Bunsen burner. Gently 

 heat the base of the U near either arm, and as it softens a quick drawing motion com- 

 pletes the canula. A second one can be made from the other arm. Trim the tip 

 to the desired size with a pair of small scissors and the canula is ready for use. 

 Equip with a piece of rubber tubing of convenient length and a glass mouthpiece. 



Injection Mass. It has been found that india ink is more suitable for injection 

 than prussian blue, because of its finer granulation. At first I diluted the ink 

 1 to 1 with water. This was freshly filtered and used at once. These injections, 

 however, were too intense and seemed to embarrass the circulation. Better 

 injections were obtained by diluting the ink 1 to 5 with physiological saline and fil- 

 tering several times through the same paper; the ink is still further diluted in the 

 blood-stream. This gives excellent injections of the capillaries and at the same 

 time renders the large overlying veins transparent, so that they do not obscure the 

 lung- vessels. 



To inject a chick, draw up a small quantity of freshly filtered dilute ink into 

 the canula, followed by a drop of physiological saline to prevent soiling of the field 

 of injection. Prepare a dish of warm Locke's solution, about 37 C, and another 

 dish containing 10 per cent nitric acid for fixation. Place the egg in a shallow 

 glass jar packed with cotton. Remove a sufficient quantity of shell to expose the 

 embryo and permit free access to it. Add a few cubic centimeters of warm Locke's 

 solution to prevent drying. Place the preparation under a low-power binocular 

 microscope and remove the vitelline membrane over the site of the proposed 

 injection. Introduce the tip of the canula into the angle formed by the tributaries 

 of the right vitelline vein and blow the ink into the vein. The heart action com- 

 pletes the injection. In fixing, add the 10 per cent nitric acid first directly to 

 the embryo, then remove the embryo from the shell and place it for 5 minutes in 

 a cover-glass containing the acid solution. The acid fixative makes the tissues 

 more transparent and prevents diffusion of the ink through the vessel-walls. The 

 fixed specimens are washed in several changes of water to remove the excess acid. 

 Some of my specimens were given a light lavender tint with Ehrlich's haematoxylin, 

 but this is not necessary. The embryos are dehydrated with graded alcohols 

 absolute alcohol, absolute alcohol and xylol, xylol then (on an electric stove) 

 through xylol and paraffin, and finally paraffin for embedding and dissection. 

 The larger embryos are not embedded, but are put through benzine into oil of 

 wintergreen for direct dissection. 



Paraffin Dissection. In using whole mounts of injected embryos for the study 

 of the early lung vessels, a difficulty is encountered in the large overlying cardinal 

 veins that obscure the delicate vessels beneath. This difficulty increases with older 

 stages and more complete injections. In efforts to overcome it I have had good 

 results with the following simple method of dissection: The embedded embryo is 

 trimmed into a block so that the broad surface is parallel to the sagittal plane of 



