TECHNIC. 307 



this method the bile capillaries finally become filled with the indigo-car- 

 min by a gradual elimination of the substance from the blood- and lymph- 

 vessels and passage through the cells into the biliary system, while the 

 blood-vessels themselves are distended by the carmin-gelatin. In the 

 frog, the demonstration of the biliary passages is more easily accomplished 

 by injecting 2 c.c. of the indigo-carmin solution into the large lymph- 

 sac and killing it after a few hours. The liver is then fixed in the manner 

 described above and is then ready for further treatment. 



The bile passages may also be injected directly through the 

 hepatic duct or the ductus choledochus. For this purpose it is best to 

 use a concentrated aqueous solution of Berlin blue (Berlin blue that is 

 soluble in water). The results obtained by this method are not, however, 

 always satisfactory, and even in the best of cases only the peripheral por- 

 tions of the liver lobules are successfully injected. 



The bile capillaries may be impregnated with chrome-silver. 

 Fresh pieces of liver tissue are placed for two or three days in a potas- 

 sium bichromate-osmic acid solution (4 vols. of a 3% bichromate of 

 potassium solution and i vol. of i % osmic acid) and then transferred to 

 a 0.75% aqueous solution of silver nitrate. After rinsing in distilled 

 water the specimens are cut with a razor, the sections again washed with 

 distilled water, placed for a short time in absolute alcohol, cleared in 

 xylol, and finally preserved in hard Canada balsam. Both celloidin 

 and paraffin imbedding are admissible, but either process must be hurried, 

 as the preparation always suffers under such treatment. In the finished 

 specimen, the bile capillaries appear black by direct light. 



Another method which brings to view more extensive areas of the 

 bile capillaries is as follows : A piece of liver tissue from a freshly killed 

 animal is fixed in rapidly ascending strengths of potassium bichromate 

 solution (from 2% to 5%). After three weeks the specimen is placed 

 in a 0.75% silver nitrate solution, when after a few days (very marked 

 after eight days) the bile capillaries, if examined in sections, will appear 

 black by direct light (Oppel, 90). 



Sometimes the bile capillaries are brought out in preparations 

 treated by the method of R. Heidenhain, although only small areas are 

 colored and these not constantly. The application of other stains, as for 

 instance the method of M. Heidenhain following the gold chlorid treat- 

 ment, often results in the staining of small areas of bile capillaries. 



In all the methods used for the demonstration of the bile capil- 

 laries, whether physiologic autoinjection, direct injection, or impregna- 

 tion, the secretion vacuoles of the liver-cells are clearly brought to view.. 



By treating pieces of liver tissue according to the method of 

 Kupffer (76) the connective tissue of the liver, especially the reticular 

 structure \Gitterf asern) t is shown. Fresh liver tissue is cut with the 

 double knife and the thinnest sections placed for a short time in a 0.6% 

 sodium chlorid solution or in a 0.05% solution of chromic acid. From 

 this they are transferred to a very dilute solution of gold chlorid (Gerlach) 

 (gold chlorid i gm., hydrochloric acid i c.c., water 10 liters), and kept 

 for one to several days in the dark until they assume a reddish or violet 

 color. If the staining has been satisfactory (which is by no means always the 

 case), the reticular fibers, and occasionally also the stellate cells, are 



