this method the bile capillaries finally become filled with the indigo-carmin by a gradual elimination of the substance from the blood- and lymphvessels 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 lymphsac and killing it after a few hours. The liver is then fixed in the manner described above and is then ready for further treatment.

252. 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 portions of the liver lobules are successfully injected.

253. The bile capillaries may be impregnated with chrome-silver. Fresh pieces of liver tissue are placed for two or three days in a potassium bichromate-osmic acid solution (4 vols. of a 3% bichromate of potassium solution and 1 vol. of 1% 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.

254. 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).

255. Sometimes the bile capillaries are brought out in preparations treated by the method of R. Heidenhain (T. 85), although only small areas are colored and these not constantly. The application of other stains, as for instance the method of M. Heidenhain (T. 65) following the gold chlorid treatment, often results in the staining of small areas of bile capillaries.

256. In all the methods used for the demonstration of the bile capillaries, whether physiologic autoinjection, direct injection, or impregnation, the secretion vacuoles of the liver-cells are clearly brought to view.

From

257. By treating pieces of liver tissue according to the method of Kupffer (76) the connective tissue of the liver, especially the reticular structure (Gitterfasern), 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. this they are transferred to a very dilute solution of gold chlorid (Gerlach) (gold chlorid 1 gm., hydrochloric acid 1 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

seen.

Instead of the double knife the freezing microtome may be used and the method continued as stated (Rothe).

258. The reticular fibers are seen under more favorable conditions by using the following method, recommended by Oppel (91): Fresh pieces of tissue fixed in alcohol are placed for twenty-four hours in a 0.5% aqueous solution of yellow chromate of potassium (larger pieces in stronger solutions up to 5%), then washed with a very dilute solution of nitrate of silver (a few drops of a 0.75% solution to 30 c.c. distilled water), and transferred to a o. 75% solution of silver nitrate. In twenty-four hours the intralobular network surrounding the blood capillaries will have become stained. The best areas lie at the periphery of the specimen, and extend about 1 mm. into the parenchyma. Free-hand sections are made, or the specimens are quickly imbedded in celloidin or paraffin, to be cut afterward by means of the microtome. The same results are obtained by placing small fresh pieces of the tissue for two or three days in a 0.5% chromic acid solution and then one or two days in a 0.5% solution of silver nitrate. The further treatment is as in the preceding method.

259. The method of F. P. Mall (vid. T. 212) is also emploved in the examination of the hepatic connective tissue.

260. The following method is recommended by Berkley for demonstrating the nerves of the liver: Small pieces of liver tissue from 0.5 to 1 mm. in breadth are placed in a half-saturated aqueous solution of picric acid for from fifteen to thirty minutes, and then in 100 c.c. of potassium bichromate solution that has been saturated in the sunlight and to which 16 c.c. of 2% osmic acid has been added. The specimens now remain in this fluid for forty-eight hours in a dark place, and at a temperature of 25° C. After this the tissue is treated with a 0.25% to 0.75% aqueous solution of silver nitrate for five or six days, washed (quick imbedding may be employed), cut, cleared in oil of bergamot, and mounted in xylol-Canada balsam.

261. The cellular elements of the pancreas may be examined without further manipulation in very thin lobules from the rabbit (Kühne and Lea).

262. There are various methods of differentiating the inner and outer zones of the cells. In sections of the tissue fixed in alcohol, carmin stains the outer zone of the cells more intensely than the inner (R. Heidenhain, 83). For the staining of the inner zone, fixation in Flemming's fluid is to be recommended, then staining with safranin, and finally washing in an alcoholic solution of picric acid. The granules of the inner zone (zymogen granules) appear red. These also stain red with the Biondi-Ehrlich mixture (T. 78). The simplest and most precise method of demonstrating the zymogen granules is that of Altmann (T. 124). The secretory and excretory ducts of the pancreas are shown, as in the case of the salivary glands, by the chrome-silver method (compare T. 253).

IV. ORGANS OF RESPIRATION.

A. THE LARYNX.

THE greater portion of the laryngeal mucous membrane is covered by a stratified columnar ciliated epithelium containing goblet cells, and resting on a thick basement membrane. The epithelium covering the free margin of the epiglottis, the true vocal cords, and

Fig. 231.-Vertical section through the mucous membrane of the human larynx; X5.

part of the arytenoid cartilage as far as the cavity between these cartilages, is of the stratified squamous variety, and is provided with connective-tissue papillæ. The mucosa contains many elastic fibers, and is rather firmly connected with the structures underneath it, but is somewhat more loosely connected in the regions supplied with squamous epithelium. In it are found branched tubulo-acinal glands, which may be single or arranged in groups. These are found at the free posterior portion of the epiglottis, in the region of the latter's point of attachment—i. e., in the so-called cushion of the epiglottis. Larger collections of glands are found in the false vocal cords, and on the cartilages of Wrisberg (cuneiform cartilages), which appear almost imbedded in the glandular tissue. In the remaining parts of the larynx glands are found only at isolated points. The true vocal cords have no glands.

The cartilages of the larynx are of the hyaline variety, with the exception of the epiglottis, the cartilages of Santorini (the latter are derivatives of the epiglottis, Göppert), the cuneiform cartilages, the processus vocalis, and a small portion of the thyroid at the points of attachment of the vocal cords, which consist of elastic cartilage.

The vascular supply of the larynx is arranged in three superimposed networks of blood-vessels. The capillaries are very fine, and lie directly beneath the epithelium. The lymphatic network is arranged in two layers, the superficial being very fine and directly beneath the network of blood capillaries.

The nerves of the laryngeal mucous membrane will be described in connection with those found in the trachea.

B. THE TRACHEA.

The trachea is lined by a stratified ciliated columnar epithelium containing goblet cells and resting on a well-developed basement membrane. The mucosa is rich in elastic tissue. In the superficial portion of the mucosa the elastic fibers form dense strands, which usually take a longitudinal direction. The deeper layer of the mucosa is more loosely constructed, and passes over into the perichondrium of the semilunar cartilages of the trachea without any sharp line of demarcation. Numerous leucocytes are scattered throughout the mucosa, and are also frequently found in the epithelium. Connecting the free ends of the semilunar cartilages, which are of the hyaline variety, are found bundles of nonstriated muscle tissue, the direction of which is nearly transverse.

The trachea contains numerous branched tubulo-acinal glands of the mucous variety containing here and there crescents of Gianuzzi. The glands are especially numerous where the tracheal wall is devoid of cartilage.

The larynx and trachea receive their nerve supply from sensory

nerve-fibers and sympathetic neurones. These have been described by Ploschko (97) working in Arnstein's laboratory. According to this observer, the sensory fibers divide in the mucosa, forming subepithelial plexuses from which fibrils are given off which enter the epithelium of the larynx and trachea and, after further division, end on the epithelial cells in small nodules, or small clusters of nodules. In the trachea of the dog, such fibrils were traced to the ciliary border of the columnar ciliated cells before terminating. Numerous sympathetic ganglia are found in the larynx and trachea. latter they are especially numerous in the posterior wall. The neuraxes of the sympathetic neurones forming these ganglia were traced to the nonstriated muscular tissue of the trachea. The cellbodies of these sympathetic neurones are surrounded by end-baskets of small medullated fibers terminating in the ganglia. Medullated

nerve-fibers, ending in the musculature of the trachea in peculiar end-brushes, were also described by Ploschko.

C. THE BRONCHI, THEIR BRANCHES, AND THE BRONCHIOLES.

The primary bronchi and their branches show the same general structure as the trachea. The epithelium of the bronchi of medium size (up to 0.5 mm. in diameter) consists of a ciliated epithelium having three strata of nuclei. Kölliker (81) distinguishes a deep layer of basilar cells, a middle layer of replacing cells, and a superficial zone consisting of ciliate and goblet cells. The number of the last varies greatly. Glands are found only in bronchial twigs that are not less than 1 mm. in diameter; as in the trachea, they are branched tubulo-acinous glands of the mucous variety.