(d) Very small pieces that contain very little lime-salts, as, for instance, bones in an embryonal condition where calcification has only just begun, can be deprived of their lime-salts by means of acid fixing solutions like Flemming's fluid, chromic acid, picric acid, etc. (e) Bone should be first fixed in some one of the fixing fluids and then decalcified. 158. A method adapted to the study of the hard and soft parts together is that first used by von Koch in studying corals. The specimen is first fixed, and if it be a long bone, the marrow cavity should first be opened to permit the fixing agent to come in contact with all parts of the tissue. After fixing, the bone is stained and then placed in absolute alcohol, and when completely dehydrated the pieces are placed in chloroform, then in a thin solution of Canada balsam in chloroform, and finally put into an oven kept at a temperature of about 50° C. for from three to four months. By this means the pieces are completely penetrated by the Canada balsam, and as the latter becomes very hard on cooling, the sections may be afterward ground without difficulty. Long as this procedure may seem, it is still the one which enables us to see the soft and hard parts of bone in a relationship the least changed by manipu lation. 159. Sections treated by Ranvier's method (vid. T. 154) show the perforating fibers of Sharpey as bright, sharply defined ribbons, appearing as streaks or circles, according to the section made (longitudinal or transverse). If decalcified specimens be first rendered transparent by glacial acetic acid, and then immersed for a minute in a concentrated aqueous solution of indigocarmin, washed with water, and then mounted in glycerin or Canada balsam, the fibers of Sharpey will appear red and the remaining structures blue. Thin sections of bone can be deprived of their organic elements by bringing them for from one-half a minute to a minute into a platinum crucible at a red heat. In such preparations calcified Sharpey's fibers may be seen (Kölliker, 86). 160. Virchow's bone corpuscles may be isolated in the following manner: Very thin fragments or discs of bone are immersed for some hours in concentrated nitric acid. They are then placed on a slide and covered with a cover-glass; pressure with a needle upon the latter will isolate the lacunæ, and occasionally also their numerous processes, the canaliculi. C. MUSCULAR TISSUE. Almost all the muscles of vertebrates have their origin from the middle germinal layer. In the simplest type the protoplasm of the formative cell changes into contractile muscle substance, the cell in the meantime undergoing a change in shape (unstriped muscle-cell). In other cases contractile fibrils are formed which are separated by the remains of the undifferentiated protoplasm (striped muscle-cells). In this case the cells either increase very little in length and possess only a single nucleus (heart muscle), or they grow considerably longer and develop many nuclei (voluntary skeletal and skin muscles). A peculiarity of muscle-substance is that it contracts in only one direction, while undifferentiated protoplasm contracts in all directions. 1. NONSTRIATED MUSCLE-CELLS. The smooth, unstriped, nonstriated or vegetative muscle-cells Fig. 88. Smooth musclecells from the intestine of a cat : In 1, isolated; in 2 and 3, in cross-section; X 300. Technic No. 172. At a the cell is cut in the plane of the nucleus; at c, in the neighborhood of the pointed end. In 3 (from Barfurth) is seen the manner in which neighboring cells are joined to each other by intercellular bridges. belong to involuntary muscle, and are 2. STRIPED MUSCLE-FIBERS. Soon after the segmentation of the mesoderm begins, certain cells of the mesoblastic somites commence the formation of muscle - substance in their interior, a process which is accompanied by increase in the number of nuclei, the formation of a membrane, a lengthening of the cells, and the appearance of fibrils in the peripheral protoplasm of the cells. Voluntary or striated muscle-cells are large, highly differentiated, polynuclear cells, which may attain a length of 12 cm., with a width of 10-100 μ. They are consequently known as muscle-fibers. Their free ends are usually pointed; the ends attached to tendon rounded (Fig. 90). Each striated muscle-fiber consists of a delicate membrane, the sarcolemma, a muscle protoplasm, in which are recognized very fine fibrils and a semifluid interfibrillar substance (the sarcoplasm) and the muscle nuclei. The sarcolemma is a very delicate, transparent, and apparently structureless membrane, which resists strong acetic acid, even after boiling for a long time. If we examine in an indifferent fluid fresh muscle-fibers, the contents of which have been broken without rupturing the sarcolemma, we may see this sheath as a fine glistening line. (Fig. 91.) The fibrils of the muscle-protoplasm constitute the contractile part of the muscle-fiber. They are exceedingly fine and extend the entire length of the muscle-fiber, and consist of a series of minute, rod-shaped segments with attenuated ends (sarcous elements), united by shorter and much thinner thread-like bridges, on each of which there is found a small granule or globule. Their structure may be expressed in the form of a diagram (Fig. 92) giving the view of Rollett, whose account has here been followed. The ultimate fibrils are grouped into small bundles (0.3-0.5 μ Sarcolemma. in diameter), forming the muscle-columns of Kölliker. In the muscle-columns the fibrils are so placed that the larger segments fall respectively in the same plane. (See Fig. 92.) The same disposition of the fibrils prevails in all the numerous muscle-columns forming a muscle-fiber, and all the muscle-columns bear such a relation to each other that the larger segments of the fibrils fall in the same plane. The semifluid, interfibrillar substance, the sarcoplasm, penetrates between the fibrils of the muscle-columns and separates these from each other and from the sarcolemma. In fresh preparations the substance forming the fibrils appears somewhat darker and dimmer, while the sarcoplasm appears clearer. Accordingly, the narrow zone formed by the larger segments of the fibrils appears slightly darker and dimmer than the zones in the regions of the uniting bridges, where the sarcoplasm is more abundant (see Fig. 92), giving these zones a clearer appearance. This gives the striated musclefibers their characteristic cross-striation. The sarcoplasm is found in greater abundance between the muscle-columns than between the fibrils in the columns. The sarcoplasm between the musclecolumns appears in the form of narrower or broader lines, parallel to the long axis of the muscle-fibers, giving the cross-striated muscle-fiber also a longitudinal striation. The sarcoplasm between the muscle-columns is seen to best advantage in cross-sections Sarcoplasm. -Sarcolemma. Fig. 93. Transverse section through striated muscle-fibers of a rabbit. I and 3, from a muscle of the lower extremity; 2, from a lingual muscle; 900. Technic No. 163. In 2, Cohnheim's fields are distinct; in 1, less clearly shown: in 3, the muscle-fibrils are more evenly distributed. of the muscle-fiber. Here it appears in the form of a network inclosing the muscle-columns. Thus, we have in a cross-section slightly darker areas, the cross-sections of the muscle-columns, known as Cohnheim's fields or areas, separated by the network of sarcoplasm. (Fig. 93.) In a striated muscle-fiber the darker and fainter bands (larger segments of the fibrils) are doubly refracting, anisotropic, while the clearer bands (sarcoplasm) are singly refracting, isotropic. The relative grouping of the two unequally refracting substances is, however, somewhat complicated, a condition which has given rise to much discussion as to the finer structure of the muscle-fiber. It should be remembered that the anisotropic and isotropic substances of the fiber are respectively placed in the same plane, so that the cross-striation of the entire fiber is fairly regular. The thickness of the bands varies considerably, often appearing as fine lines. In a definite segment the grouping is very regular, and the structure of such a segment is exactly repeated throughout the entire length of the fiber. A segment of this description contains in its center a broad disc of anisotropic substance--the transverse disc (Q) (Fig. 94); this is divided through its middle by a less refractive (isotropic) narrow band, known as the median disc of Hensen (). Above and below the transverse disc (Q) is a disc of isotropic substance (); these in turn border upon the intermediate discs of Krause (Z). There are consequently in each segment or muscle-casket four discs-the transverse disc (Q) divided into two parts by the median disc of Hensen (h), with above and below the two isotropic discs (J), outside of which lie the intermediate discs of Krause (Z). Fig. 94. Diagrams of the transverse striation in the muscle of an arthropod; to the right with the objective above, to the left with the objective below its normal focal distance (after Rollett, 85): Q, Transverse disc; h, median disc (Hensen); E, terminal disc (Merkel); N, accessory disc (Engelmann); J, isotropic substance. One of the best objects for the study of transverse striation is the muscle of some of the arthropods (beetles). Here it will be noticed that the disc () is separated by an anisotropic disc through its center into three: (1) an isotropic disc (J); (2) an anisotropic disc (NV), the "accessory disc" of Engelmann, Krause's "transverse membrane "; and (3) still another disc of anisotropic substance (E), Merkel's "terminal disc." In other words, the number of discs in a segmental unit is increased to six: Q divided by h, two J's, two E's, and Z. It should be remarked here that all the doubly refracting substances appear as light, |