50 DE\ELOPME.\T OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 



METHODS. 



Tlu> injection method has lieen principally used, hut it has been siip])lemented 

 and supported by evidence from both single and serial sections. The collection of 

 pig embryo of the Anatomical Laboratory has been at my disposal, and I have also 

 studied a nimiber of especially prepared scries. Many of the series have been of 

 embryos in which the blood-vessels have been injected, and this has materially aided 

 in their interpretation; in fact, in all the especially i)repared series the blood-vessels 

 were injected. All these embryos were fixed in Carnoj^'s fixing fluid, consisting of 6 

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



The method of fixation is as follows: Place the embrj^o immediateh' in the 

 fluid and allow it to remain there G to 8 hours; thcni transfer directly to 70 per cent 

 alcohol; dehj'drate by ascending grades of alcohol with 2 per cent difference until 

 95 per cent is reached; then change to absolute. This gives excellent fixation with 

 very httle shrinkage. The stains used were Ehrlich's hematoxyUn and a mixture of 

 eosin, aurantia, and orange G. 



Thehijection masses used were india ink, a saturated solution of prussian blue, 

 a 5 per cent aqueous solution of silver nitrate, and an acjueous suspension of lamp- 

 black. The india ink and prussian blue give about the same results, except that 

 the specimens injected with prussian blue are more easily studied after clearing, as 

 the ink renders them more opaque. The india ink, however, flows more easily and 

 hence the injections are more nearlj' complete. The silver-nitrate injections are 

 easiest to analyze and give beautiful preparations, but its caustic action prevents the 

 finer vessels from filling, so that only the larger trunks are injected; however, it 

 furnishes an extremely valuable method of following the principal drainage-lines at 

 different stages. The lampblack is the mass which gives the most nearly complete 

 injections, but unfortunately it i^recipitates in fine flakes and gives a feathery 

 appearance to the specimen, thus rendering it difficult to use for illustrating. 



It will be necessary to review the methods used in injecting the various stages, 

 as they differ considerably and are of especial importance in interpreting the results. 

 The earhest injections were made bj^ filling the jugular sacs from the superficial 

 plexuses and then gently moving the embryo. I have succeeded in injecting the 

 early vessels to the trachea and the lungs in only a few pigs less than 3 cm. long, 

 because the injection mass usually follows the path of least resistance, which is into 

 the jugular vein. 



In injecting embrj'os between 3 and 6 cm. in length, three general methods have 

 been employed: 



(1) The best and by far the easiest method of obtaining good preparations of 

 the left part of the tracheal plexus is to inject through the retroiieritoneal sac in the 

 manner described by Heuer (1909) ; but this seldom gives good preparations of any 

 of the vessels of the lung except those of the lower lobe. However, this method has 

 been of particular importance in following the Ij^mphatics up from the retroperi- 

 toneal sac to the posterior poles of the lower lobes. 



(2) One may inject the tracheal plexus, especially the left part, by plunging the 

 needle deep behind the aorta and injecting cerebral wards; the right plexus is some- 

 times filled also, and often the vessels of the left lobe of the lung. 



