LYMPH-FLOW AND LYMPHATICS, CHICK EMBRYOS 403 



warm Ringer's solution was dropped on the embryo and yolk 

 sac to prevent drying. The experiments were carried on under 

 the high power of the binocular microscope and, for the injection 

 tests, glass cannulae measuring from 15 to 20yu at the tip were 

 used. The bright illumination necessary for the observations 

 was obtained from direct sunlight or from a desk arc-light. The 

 temperature of the warm chamber should remain between 37 and 

 40°C. for the success of the experiments, and all injury to the 

 embryo, and especially to the blood circulation, must be avoided. 



In chicks of 5 to 6^ days more or less blood is always present 

 in the superficial lymphatics and it is therefore not difficult to 

 inject a few granules of ink directly into a lymphatic capillary. 

 In older embryos, the lymphatics cannot be seen without in- 

 jection and the direct puncture of a vessel is a matter of greater 

 uncertainty. However, with practice and increased familiarity 

 with the position of the lymphatics at various stages it became 

 possible to pierce a lymphatic at practically every attempt, 

 although small extravasations in the surrounding tissue pro- 

 duced no effect on the circulation of granules. It is important 

 to insert the needle directly into the lumen of a lymphatic vessel, 

 and not to plunge it through the opposite wall, to deposit only 

 a very few granules, in order not to plug the vessel, and, finally, 

 to withdraw the needle carefully so as to avoid leakage. 



After testing the direction and character of the lymph-flow 

 by observing the course of the injected granules, the superficial 

 lymphatics were injected. The embryo was then fixed in 

 Carnoy's fluid, dehydrated in absolute alcohol, and cleared by 

 the Spalteholz method in benzol and oil of wintergreen.^ In 

 the cleared specimens, the pattern and position of the superficial 

 lymphatics could be studied in comparison with the records of 

 the lymph-flow in the same embryos. Injections of the deep 

 lymphatics were also obtained at all of the stages, in order to 

 determine their relation to the superficial vessels. For show- 

 ing the relationship of the lymphatics to the venous system, 

 cleared specimens were obtained in which, in addition to the 



* I am indebted to Miss Sabin for this method of fixing and clearing injected 

 specimens. 



