usual methods. The margins of the cells subjected to this treatment will appear black.

Endothelial cells may be demonstrated after the following method : A small mammal (rat, Guinea-pig, rabbit, or cat) is narcotized. Before the heart's action is completely arrested, the thorax is opened and the heart incised. As soon as the blood stops flowing, a cannula is inserted and tied in the thoracic aorta a short distance above the diaphragm, and 50 to 80 c.c. of a 1% aqueous solution of silver nitrate injected through the cannula. About fifteen minutes after the injection of the silver nitrate solution, there is injected through the same cannula 100 to 150 c.c. of a 4% solution of formalin (formalin 10 parts, distilled water 90 parts). The abdominal cavity is then opened, loops of the intestine with the attached mesentery removed and placed in a 4% solution of formalin, in which the tissue is exposed to the sunlight. As soon as the reduction of the silver nitrate has taken place, which is easily recognized by the reddishbrown color assumed by the tissues, the mesentery is divided into small pieces, dehydrated first in 95%, then in absolute alcohol, cleared in oil of bergamot, and mounted in balsam. As a rule, the mesothelial cells covering the two surfaces of the mesentery, and the endothelial cells lining the arteries, veins, and capillaries are clearly outlined by the reduced silver nitrate.

If desired, the tissue may be further stained in hematoxylin (we have used Böhmer's hematoxylin solution) or in a carmin solution after dehydration in 95% alcohol, after which they are dehydrated, cleared, and mounted in balsam. In preparations made after this method the endothelial cells are outlined by fine lines of dark brown or black color.

Silver nitrate may also be dissolved in a 2% to 3% solution of nitric acid, in osmic acid, and various other fluids. Stratified epithelia can also be impregnated with silver nitrate, but only after prolonged immersion. They are exposed to sunlight after sectioning on the freezing microtome, or after hardening and imbedding, followed by sectioning. After the reduction of the silver the sections are dehydrated and mounted in balsam.

131. Kolossow has devised the following excellent method for demonstrating intercellular bridges: Fine membranes, or even minute fragments of previously fixed tissues, are placed for about a quarter of an hour. in a 0.5% to 1% osmic acid (or in a mixture composed of 50 c.c. absolute alcohol, 50 c.c. distilled water, 2 c.c. concentrated nitric acid, and I to 2 gm. osmic acid) and then into a 10% aqueous solution of tannin for five minutes, or into a developer consisting of the following: water, 450 c.c.; 85% alcohol, 100 c.c.; glycerin, 50 c.c.; purified tannin, 30 gm., and pyrogallic acid, 30 gm. In the latter case they are subsequently rinsed in a weak solution of osmic acid, washed with distilled water, and then carried over into alcohol.

132. There are, of course, special methods of fixing and subsequently examining epithelial structures; these, and the methods of examining gland tissue, will be discussed in the chapters devoted to the various organs.

B. THE CONNECTIVE TISSUES.

In the connective tissues, the intercellular substance gives character to the tissue, the cellular elements forming a less conspicuous portion. All the members of this group are developed from the mesenchyme, an embryonic tissue differentiated early in embryonic life from the mesoderm, and consisting of variously branched cells, possessing a small amount of protoplasm and relatively large nuclei. The branches of neighboring cells are united by threads of protoplasm; between the cells is found a homogeneous ground-substance or matrix.

In their fully developed condition some of the members of the connective-tissue group are only slightly altered from embryonic connective tissue. This is the case in mucous connective tissue, which

Cell process.

Nucleus.

Fig. 57.-Mesenchymatous tissue from the subcutis of a duck embryo; X 650. Technic No. 17.

resembles closely mesenchymal tissue. In other members there are developed in the ground-substance, in less or greater number, fibers, known as connective-tissue fibers, thus forming reticular connective tissue and the looser and denser forms of fibrous connective tissue. A more marked condensation of the intercellular substance is observed in cartilage; and in bone and dentin a still greater degree of density is obtained by the deposition of calcareous salts in the intercellular matrix.

The rôle played by the connective tissues in the economy of the body is largely passive, depending on their physical properties. Bone and cartilage serve as supporting tissues; the looser fibrous tissues for binding and holding the organs and parts of organs firmly in place. The denser fibrous connective tissues come into play

where strength and pliability are desired, as in ligaments, or else are used in the transmission of muscular force, as in tendons.

Another important characteristic of connective tissue is that its various members are capable of undergoing transformation into wholly different types; bone, for instance, being developed from fibrous connective tissue and from cartilage. Certain structures are represented by different members of the connective-tissue group in the different classes of vertebrates. In certain fishes the skeleton is cartilaginous, and in certain birds the leg tendons are formed of osseous tissue, etc.

In the different types of connective tissue the cellular elements are morphologically very similar, and do not differ materially from the mesenchymal cells from which they are developed.

The connective tissues receive their nutrition from the lymph. In the denser connective tissues this permeates the tissues through clefts or spaces in the ground-substance, in which the connectivetissue cells are found and which are united by means of fine canals into a canalicular system. In the looser fibrous tissues and in mucous connective tissue the system of lymph-channels is not present; here the lymph seems to pass through the ground-sub

stance.

Certain connective-tissue cells have the function of producing fat. In various parts of the body, masses of fat tissue are formed as a protection to various organs and as a reserve material upon which the body can call when necessary. This type can hardly be considered a separate class of connective tissues, as it can be demonstrated that it is merely modified connective tissue, and can occur wherever the latter is found.

Finally, certain elements of the middle germinal layer are capable of producing colored substances known as pigments. To this class belong the pigment cells and the red blood-corpuscles.

From the above account it will be seen that we have to distinguish between the following kinds of connective tissue: (1) mucous connective tissue, (2) reticular connective tissue, (3) fibrous connective tissue, (4) adipose tissue, (5) cartilage, (6) bone.

The fibrous connective tissues are composed of a ground-substance or matrix in which are imbedded the cellular elements and two kinds of connective-tissue fibers, namely, white and elastic fibers. As the character of the fibrous connective tissue depends largely on the arrangement of the fibers and on the relative proportion of the white and elastic fibers, these will be considered prior to a description of the several types of fibrous connective tissue.

White Fibers.-White fibrous connective tissue consists of exceedingly fine homogeneous fibrillæ, cemented by a small amount of an interfibrillar cement substance into bundles varying in size. In the bundles these fibrillæ have a parallel course, although the bundles are often slightly wavy. The fibrilla of white fibrous connective tissue vary in size from 0.25 to 1 μ, and neither branch nor anasto

mose. They become transparent and swollen when treated with acetic acid, are not at all or only very slowly digested by pancreatin, and yield gelatin on boiling.

Elastic Fibers.-These are homogeneous, highly refractive, distinctly contoured fibers, varying in size from I μ to 6 μ, and in some animals are even larger. They branch and anastomose, and are not cemented into bundles. When extended, they appear straight; when relaxed, they show broad, bold curves, or are arranged in the form of a spiral. The broken ends of the fibers are bent in the form of a hook. F. P. Mall has shown that elastic fibers are composed of two distinct substances—an outer delicate sheath which does not stain in magenta, and an interior substance which is intensely colored in this stain. The interior substance is highly refractive. Elastic fibers are not affected by acetic acid, but are readily digested in pancreatin and less readily in pepsin. They yield elastin on boiling.

Our knowledge concerning the development of the connective

a

Fig. 58.-White fibrils and small bundles of white fibrils from teased preparation of a fresh tendon from the tail of a rat.

Fig. 59.-Elastic fibers from the ligamentum nucha of the ox, teased fresh ; X 500. At a the fiber is curved in a characteristic manner.

tissue fibers is not as yet conclusive; two distinct views are held at the present time. One group of observers maintains that the fibers are developed in the cells of the embryonic connective tissue. These cells are thought to change into fibrous connective tissue by the formation in their interior of thread-like structures—the connective-tissue fibrils-a process which is always accompanied by active nuclear division (Flemming,91, II; Lwoff, Reinke). The cells thus become polynuclear and considerably lengthened, and the fibrils gradually increase in number at the expense of the cell-bodies, so that on examining the tissue the fibrils appear to predominate, and give the impression of forming the ground-substance. According to the other view, the fibers are at all times intercellular, developing in the ground-substance. Mall believes their development to be due to a kind of coagulation, certain cells being held

responsible for the formation of special fluids or ferments which bring about this coagulation. The formation of the ground-substance in which the fibers develop is also attributed to the cellular elements. The intercellular mode of formation of connectivetissue fibers would appear to be the more usual, although some of them may have an intracellular origin. We shall now discuss the several types of fibrous connective tissue.

1. MUCOUS CONNECTIVE TISSUE.

Mucous connective tissue is a purely embryonal type, and scarcely represented in the adult human body. It consists of branched, anastomosing cells imbedded in a gelatinous ground-substance, containing here and there white fibers. The latter as well as the mucous matrix are, directly or indirectly, the products of the cells. During the development of the embryo this tissue is found in large quantities in the umbilical cord, and is here known as Wharton's jelly. It also occurs in the embryo in the cutis, in the region of the semicircular canals of the cochlea, in the vitreous humor, etc.

2. RETICULAR CONNECTIVE TISSUE.

Reticular connective tissue is a fibrous connective tissue in which the intercellular substance has disappeared. The tissue is often described as being composed of anastomosing branched cells, arranged in the form of a network with open spaces. The observations of Ranvier and Bizzozero, and more recently those of Mall,' have shown that the framework of reticular tissue is composed of very fine fibrils or bundles of fibrils. These interlace in all planes to form a most intricate network, surrounding spaces of varying size and shape. According to F. P. Mall, the fibrils of reticular tissue differ chemically from both the white and elastic fibers, although their composition has not been fully determined. Like white fibrous tissue, reticular tissue is not digested by pancreatin, but, unlike white fibrous tissue, it does not appear to yield gelatin upon boiling in water.

The cells of reticular connective tissue, which are flattened and often variously branched, lie on the reticular network, being often wrapped about the bundles of fibrils. Unless they are removed, the reticulum has the appearance of a network composed of branched and anastomosing cells.

Reticular connective tissue is found in adenoid tissue and lymphglands, in the spleen, and in the mucous membrane of the intestinal canal, and in these locations the meshes of the reticulum are filled with lymph-cells and other cellular elements, which, unless removed, obscure the reticulum. Connective-tissue fibrils giving the same reaction as those found in the adenoid reticulum are found associated with white and elastic fibers in the liver, kidneys, and