ey Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924031263357 Cornell University Library arV18394 Wi iT 924 031 263 3 olin, are A COURSE OF ELEMENTARY PRACTICAL PHYSIOLOGY AND HISTOLOGY. A COURSE OF ELEMENTARY PRACTICAL PHYSIOLOGY AND HISTOLOGY, aS BY MI’ FOSTER, MD. FRS., PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF CAMBRIDGE. $ 7 - 604—412 Appitions To InpEx . ‘ 7 a 3 , é 413 LESSON I. DISSECTION OF A RABBIT AND OF A DOG. In the following, the descriptions in large type apply more particularly to the rabbit, but the general directions for dissection serve also for the dog: some points in which the two animals differ, and some which are better seen in the dog, are printed in small type. A. 1. Make a median incision through the skin, down the whole length of the front of the body from the neck to the pubis, and reflect the skin as far as possible on both sides. In dissecting a female rabbit note, just under- neath the skin, the thin arborescent mammary glands, one to each mamma. - 2. Observe the thin, pale, abdominal muscles. It is better to dissect out the individual mus- cles in the dog as below, but the dissection may be done on the rabbit. In the dog observe a. The tendinous aponeuroses of the abdominal muscles forming in the middle line the linea alba,- L. 1 ELEMENTARY PHYSIOLOGY, [1 b. The obliquus externus abdominis, a thin muscle, with descending fibres; it arises from the ribs by separate bundles, from the back by a broad tendon and runs to the linea alba and to the pubis. c. The rectz abdominis, one on either side of the middle line, covered by the tendon of the exter- nal oblique. z If & be carefully reflected, there will be seen under- neath ; d. ° The internus obliquus abdominis, with ascending fibres, it arises from the pubis and lumbar fascia and runs to the linea alba and lower ribs; and underneath this e The transversalis abdominis, it arises. from the lower ribs, the dorso-lumbar fascia and the pubis, and runs to the linea alba. Lift up the abdominal wall and cut it through in the middle line from the sternum to the pubis, being careful to avoid puncturing the intestine. From the middle of this cut make transverse cuts nearly as far as the spinal column, Hook or pin back the four flaps. Simply turning the parts.over without cutting or tearing anything, trace out as far as possible the alimentary canal, noting the narrow eso- phagus entering into the stomach about the middle of its concave upper portion, the pyloric end of the stomach placed on the right side and continuous with the small intestine which is not distinctly divisible into duodenum, jejunum and ileum, the large dark thin-walled cecum I] DISSECTION OF A RABBIT AND OFA DOG. 3 having a shallow spiral constriction around it, the rather thick-walled, light coloured appendix proceeding from the end of the caecum, the large intestine of much smaller diameter than the cecum, much puckered in the first part of its course, less puckered in its median portion, and becoming soon quite smooth and passing without change into the rectum. The latter part of the large intestine and the rectum usually contain balls of feeces, 4. Trace out the mesentery which supports the intestine ; observe its continuity with the peri- toneum or membrane lining the abdominal , cavity, note the manner in which the blood- Lf vessels run in it. Observe in the dog the loose fold of mesentery loaded with fat, hanging from the lower border of the stomach and forming the great omentum. Observe the spleen, an elongated dark red body lying near the broad end of the stomach to which it is attached by a mesenteric fold (gastro- splenic omentum). 6. Turn the stomach over to the left’, gently stretch out the duodenum and observe in the mesentery belonging to it, the diffuse, pale-red pancreas ; trace the entrance of the pancreatic duct as a pale thin band into the duodenum: this occurs rather more than a foot below the pylorus, where the duodenum turns back on itself to form a loop. or * Right and left are used throughout for the right and left of the animal, 1-2 4 . | ELEMENTARY PHYSIOLOGY. ~ {. tn the dog the pancreatic duct is close-to the entrance of the bile duct (see § 14). Observe the mesenteric lymphatic glands, small greyish white lumps, more abundant in the duodenal mesentery than elsewhere. 7. Turning the stomach and intestines over to the right side observe the dorsal aorta’ and inferior vena cava lying close together in the median line, trace the aorta upwards to the point where it descends through the diaphragm, tearing through the mesentery as little as is consistent with tracing the aorta. 8. Note the right suprarenal body, small, ovoid and yellowish white, lying close to the aorta, carefully tear away the connective tissue above and medially of this and note the solar plexus consisting of three or more greyish semi-trans- parent ganglia connected by bundles of pale nerve fibres. Into the laterally placed ganglion runs the main branch of the splanchnic nerve, trace this up alongside the aorta as far as the diaphragm. 9. Note the celiac artery given off by the aorta a little below the diaphragm, and the superior mesenteric artery given off somewhat lower down, possibly underneath the suprarenal body, and a, little farther down, the renal artery, run- 1 When an artery and a vein run together, as here, they may be distinguished by the artery having thicker walls and containing less blood than the vein; the artery too has generally a bluish-white tint whilst the vein has generally a dark red tint with a tinge of blue. 1] 10, 11. 12. DISSECTION OF A RABBIT AND OF A DOG. 5 ning to the hilus of the kidney: note the renal vein running parallel to the renal artery into the vena cava. Follow the superior mesenteric artery a short distance and observe the branches given off to the pancreas, these are more easily seen when the intestines are turned to the left. Tearing through the mesentery around the lower part of the cesophagus, observe the right and left pneumogastric nerves (cp. C §§ 17, 24,) dividing into several fibres which spread out over the stomach. One or more branches may be traced to the solar plexus. Observe the number of pale nerves which are given off by the ganglia of the solar plexus; bundles of them may be followed along the celiac, mesenteric and renal arteries. Then turning the stomach and intestines over to the left side, carefully tear away the mesentery over the aorta and note the right splanchnic nerve close beside it, trace the nerve on its course (being careful not to puncture the vena cava) past or underneath the right suprarenal body into a ganglion a little removed from the rest of the solar plexus. Lift up the stomach, and viewing from the right the mesentery below it, note the portal vein, a large vein dividing close to the posterior ‘ surface of the liver and running into it, This vein is formed by the union of the lieno-gastric and mesenteric veins, the former is much the smaller and joins the latter close to the liver; 13. ELEMENTARY PHYSIOLOGY. [. follow for a short distance the course of the mesenteric vein, noting the small numerous branches received by it from the pancreas. Viewing the mesentery from the left side note the juncture of the splenic and gastric veins to form the lieno-gastric. Trace out the branches of the celiac artery; it first gives off the splenic artery which besides giving off a row of smaller arteries to the spleen sends several branches to the greater curvature of the stomach and some small branches to the pancreas, it then gives off at short intervals ‘branches to the lower part of the cesophagus, the stomach and the upper part of the duode- num and a branch, the hepatic artery, which runs to the liver. In the dog, pull the spleen downwards and to the left away from the stomach, a branch of the lieno-gastric artery will be seen sending branches to the spleen and to the greater curvature of the stomach ; the corresponding veins are best seen on turning the spleen over towards the stomach. Double ligature and cut through these vessels, pull. the spleen downwards as before, a-smaller branch of the lieno-gastric artery and vein will be seen; centrally of the lieno-gastric vessels will be seen two or more gastric and pancreatic arteries and veins. Pull the pancreas to the left over the spleen and note the junction of the lieno-gastric and me- senteric veins. Then pull the duodenum over the part of the pancreas previously showing and 1] DISSECTION OF A RABBIT AND OF A DOG. 7 note the fairly large vein from the pancreas and the upper part of the duodenum joining the previously mentioned vein to form the portal vein; note also the branch from the celiac axis dividing into the hepatic artery and an artery supplying the greater part of the ‘pancreas and the upper part of the duodenum ; it then gives off branches to the lower part of the cesophagus and the stomach and finally divides into two branches, one the hepatic artery going to the liver, the other going’ to the lower part of the stomach and the upper part of the duodenum (with branches to the pancreas). 14, Turning the liver up towards the diaphragm, the 16. gall-bladder will be seen in a hollow on the under surface of the posterior right lobe: trace the cystic duct or duct from the gall-bladder to the point where it joins the hepatic duct, proceeding from the liver itself; trace the united duct or common bile duct into the duodenum, close to the pylorus. Ligature the esophagus and the rectum and cut through both, the former above the ligature the latter below it. Turning the intestine to the right, cut through the mesentery close to its abdominal attachment and remove from the abdomen the alimentary canal and its appen- dages except the liver. Observe now the posi- tion and form of the liver, especially i in relation to the diaphragm. Pull the. liver down from the diaphragm, 17. 18. 19. ‘ ELEMENTARY PHYSIOLOGY. ‘ie through the transparent tendon of the diaphragm the lungs will be seen in close contact with it. Puncture the tendon on the right side and note the collapse of the right lung as soon as air ‘enters the pleural cavity. With the liver still pulled down, note the short hepatic veins proceeding from the liver to join the vena cava inferior just below the diaphragm. Cut through the hepatic veins as close to the liver as possible and remove the liver. Cut open one of the hepatic veins and trace it in this way back into the substance of a liver lobe. Observe on its inner surface the opening of numerous smaller veins; cut through the lobe ‘near its base, and try to distinguish the portal veins from the hepatic by the small bile duct and small thick-walled artery running alongside ' the former. Cut away the mesentery from the alimentary canal, and trace out the latter along. its whole length, observing more fully the features men- tioned in § 3, and noting in addition one or more white patches (Peyer’s patches) on the free sur- face of the ileum, due to clumps of lymph-follicles; also note the connection of the cecum with the small and large intestine, the thin walls of the cecum and the thicker spotted walls of its appendix, ‘Note in the dog, the wider cesophagus entering into the stomach nearer the cardiac end than is the case with the rabbit; note also the shorter I] 20. 21, 22. 24, DISSECTION OF A RABBIT AND OF A DOG. 9 ‘length of the intestine, the small cecum, and the less difference between the large and small intestines. The small intestine may be washed out by tying a funnel into the duodenum, and letting water from a tap stream down the funnel. The large intestine may be similarly treated. Cut through the stomach along the lesser curva- ture, throw away its contents and wash the mucous membrane. Note that the mucous membrane of the greater curvature is pale red, that of the pylorus is greyish-white and semi- transparent. The contrast is more marked when the whitish superficial layer of mucous cells is removed. The mucous membrane may be used to prepare a glycerine extract of pepsin (cp. Lesson xvt.), Wash out the duodenum, its inner surface has a velvéty look which is characteristic of the mucous membrane of the small intestine; it is caused by the villi, examine these with a lens. Observe the openings of the biliary and pancrea- tic ducts, and carefully pass bristles through them into the ducts. Cut open a piece of the large intestine, wash it, and with a lens examine its inner surface; it has “no villi. . 28, - Note again the position of the suprarenal bodies. Note the position of the kidneys, the left being much nearer the pelvis than the right; observe on either side the ureter, a pale semi-transparent 10 26. 27. 28. ELEMENTARY PHYSIOLOGY. [r. duct passing downwards from each kidney over the muscles of the back towards the middle line; trace them to their entrance into the urinary bladder. . Trace out the renal artery and vein noted in § 9, follow them into the substance of the kidney. Divide one kidney longitudinally, note the single pyramid opening into the pelvis. In dissecting a female rabbit, observe the uterus, with its two cornua, from each cornu proceeds a Fallopian tube which taking a winding course upwards for some little distance ends in a clump of processes or fimbriz. Near the end of each Fallopian tube a little below the kidney will be seen a small, ovoid spotted body, the ovary. In dissecting a male rabbit, observe in each side of the lower part of the abdominal cavity a white convoluted tube the vas deferens. Cut through the symphysis pubis with bone forceps, stretch the halves apart and cut away as much bone on each side as may be necessary. Trace the vasa deferentia downwards cutting open the scrotal sacs; each vas deferens is continuous with a coiled mass of tubes, the epididymis, attached to one side of the testis. Note that the smooth membrane, tunica vaginalis, lining the scrotal sacs is continuous with the peritoneum. Lay open the bladder, observe its neck ending in the urethra, note the openings of the ureters into the dorsal part of the bladder and in the male the openings of the vasa deferentia near its neck. 1] DISSECTION OF A RABBIT AND OF A DOG. 11 B. 1. Make a median incision over the skull from the nose to behind the level of the ears. Reflect the skin on each side. Cut away the attachment of the muscles of the neck to the occiput until the occipito-atlantoidean membrane between the occiput and the atlas is laid bare. Carefully divide this with scissors and observe the medulla oblongata. With a trephine saw through the roof of the skull in its broadest part, a little behind the orbits, working very carefully when the bone is nearly sawn through. With a lever raise the circular piece of bone and remove it. Then with the bone forceps cut away piecemeal the rest of the roof of the skull. Note the thickish membrane, the dura mater covering but not attached to the brain, it dips down between the cerebral hemispheres as the falx cerebri and between the cerebrum and cerebellum as the tentoriwm; cut away the dura mater and observe the very thin vascular mem- brane, the pia mater, clinging to the surface of the brain. Make a rough sketch of the exposed cerebrum, cerebellum and medulla oblongata for com- parison with the same parts in the dog. Note particularly that in the rabbit the cerebral hemispheres are smooth and that the olfactory lobes are directly in front of the cerebral ‘hemispheres, being separated from them by a constriction only. * ELEMENTARY PHYSIOLOGY. [r. 5. In the dog a, The dura mater is much thicker and the pia mater more obvious. . b. The cerebral hemispheres have deep fissures, c. The pia mater dips down into the ‘fissures, above the pia mater and bridging over the fissures may be observed the thin transparent arachnoid membrane, also distinctly visible as a covering to the pia mater at the base of the brain. In the space between the arachnoid and pia mater is contained the clear watery sub- arachnoid (or cerebro-spinal) fluid. A smaller quantity of fluid also exists between the arachnoid and dura mater. d. Compare the exposed surface with the sketch made of the surface of the brain of the rabbit, noting the relative sizes of the cerebrum and cerebellum in each. With a scalpel divide the front of the cerebral hemispheres from the olfactory lobes. Lift up with the handle of a scalpel the extreme front of the cerebrum, and turning it backwards bring into view the optic nerves. Cut these through with a sharp pair of scissors close to the skull. Still turning the brain back cut through succes- sively all the other cranial nerves. A little behind the optic nerve is the small but evident third nerve (motor oculi), close behind this the considerably smaller fourth nerve (trochlear), farther back in the hollow behind the attach- ment of the tentorium lies the thick fifth nerve, to the median side of which the small sixth 1] DISSECTION OF A RABBIT AND OF A DoG. 13 (abducens) is fairly conspicuous, A little behind and to -the outside of the fifth, in the hard petrous bone are seen together the seventh (facial) and eighth (auditory). Some distance back and nearer the middle line come the ninth (glossopharyngeal), tenth (pneumogastric), and the small eleventh (spinal accessory). Lastly, still farther back is the twelfth (hypoglossal). Cut through the spinal cord below the medulla oblongata, and remove the brain entirely. The outlying lateral portions of the cerebellum will probably be left in the skull. Do not injure the | skull in attempting to get these out’. 7. Cut and scrape away the tissue above the cervical vertebree ; with bone forceps remove the arches of the vertebree and cut them away at the sides piece by piece so that the spinal cord is well exposed. Pull the cord a little to one side and note the nerves running into it, one between each pair of vertebra, Carefully cut through the dura mater and pull it up with forceps, a row of fine nerve fibres will be seen issuing from the spinal cord; they converge and form one bundle the posterior root of the spinal nerve. Cut through these filaments, and pull the dura mater a little farther from the spinal cord; ventrally of the above set of fine nerve fibres will be seen another similar set which unite and form the anterior 1 The brain may be placed in spirit to harden and be dissected later: most of the points of structure of the dog’s brain given in Lesson xxx. can also be made out on the rabbit’s brain. 14 ro ELEMENTARY PHYSIOLOGY. [. root of the spinal nerve. Observe carefully the roots on the outside of the dura mater, they join almost immediately forming the nerve trunk, on the posterior root at or a little before its junction with the anterior root note the swelling caused by the spinal ganglion. Examine again the diaphragm (cp. A § 16). Observe the large central tendon, with the vena cava and esophagus passing through and tightly attached to it. The muscular part of the diaphragm consists of a costal and vertebral portion, The former is attached by short tendons to the ribs and sternum. The latter is attached to the upper lumbar vertebra; it is a somewhat thick mass of muscle divided into right and left portions by the descending aorta, the right is much the larger; the two form the pillars of the diaphragm. Pull down the diaphragm by its pillars, on its unpunctured side the lung will follow it. Observe the pectoral muscle proceeding from nearly the whole length of the sternum to the humerus, cut it through together with the vessels and nerves going to the arm and note its attachments. Several muscles will now be exposed, note the serratus anticus major proceeding from the lower part of the internal border of the scapula to the 3rd to 9th ribs inclusive. Cut it through and reflect the parts. 1] 4, my DISSECTION OF A RABBIT AND OF A DOG. 15 Note the sealenus medius running from the neck to the upper ribs (2nd to 5th); cut this through where it is inserted into the ribs and turn it forward, the scalenus anticus will be seen attached to-the Ist rib at its junction with the costal cartilage. The serratus anticus minor running from the upper part of the internal border of the scapula to the lower cervical vertebra and 1st and 2nd rib. The serratus posticus, a thin inconspicuous muscle proceeding by rather a long broad tendon from the cervical vertebre and dorsal fascia. It is inserted into the 4—12th ribs about the middle part of their course. These muscles having been cut through the small scalenus posticus will be seen running from the neck to the Ist rib laterally of the scalenus anticus. The three scaleni originate from one or more of the transverse processes of the 4th to 7th cervical vertebre. Note the thick muscle the longissimus dorsi covering the ribs dorsally ; cut away this and the adjoining muscles and note the inconspicuous levatores costarum proceeding from the trans- verse processes of the dorsal vertebree to the ribs below. Clear away all muscles and tendons attached to any two of the ribs (say 4th and 5th) except the intercostal muscles joining them. Note the external intercostal muscle, the fibres run 16 10. 12. ELEMENTARY PHYSIOLOGY, [L downwards and ventrally, and are absent between the costal cartilages, here the internal inter- costal muscle is seén; carefully remove the external intercostal, and so follow the internal intercostal towards the vertebrx; the fibres run downwards and ventrally and near the vertebrae are scanty or absent. Observe more closely the costal cartilages and their connection with the ribs and sternum. 11. The above mentioned muscles, especially the thinner ones, should also be observed in the dog, where they are larger. There are some diiferences in arrangement, The pectoral has an upper portion which: runs not to the scapula but to the humerus. The serratus anticus runs from the whole length of the internal border of the scapula to the lower cervical vertebre and first seven ribs, The origins and insertions of the scalent are somewhat different, The serratus posticus is divided as in man into an upper and a lower portion (s. p. superior and s. p. inferior.) Cut through the costal cartilages on either side close to the sternum, cut through the muscles be- tween the 2nd and 3rd and the 8th and 9th ribs, with bone forceps cut through the 3—8th ribs dorsally and remove them. The pleural cavities will be seen to be separated from one another by the median parietal portions of the pleure, between these is a space, the mediastinum. From the surface of the lungs a shred of a fine 1] 13. 14. 16. DISSECTION OF A RABBIT AND OF A DOG. 17 membrane, the visceral portion of the pleura, may be torn; note that at the base of the lungs this is continuous with the parietal portion of the pleura attached to the walls of the chest and bounding the mediastinum. Note the position of the heart, In the mediastinum attached to the pleura note on either side the phrenic nerve distributed to the muscular fibres of the diaphragm. With fine forceps tear off the membrane over the phrenic nerve in the middle part of its course; another membrane will be seen under- neath, outside of which the phrenic runs, this is the parietal layer of the pericardium ; cut it through, the heart will be seen to lie in a bag formed by it. Remove the middle and posterior portions of the sternum. Trace the connection of the parietal layer of the pericardium with the covering of the heart and of the roots of the great vessels, 15. Turning in the dog the heart and lungs over to the right, pull up the large aortic trunk, and note the almost transparent thoracic duct, lying alongside the esophagus; trace it up. to its termination into the venous system (at the junction of the left jugular and left sub-clavian vein, cp. § 20). With a little care the thoracic duct may also be traced in the rabbit. Prolong the median skin incision to the chin and reflect: the skin as far as possible. Observe on 2 18 17. 18. ELEMENTARY PHYSIOLOGY. [T. . each side the external jugular vein arising an- teriorly from two branches: avoid puncturing it. Cut through in the middle line the thin super- ficial muscle (platysma); draw it to -one side, clearing away the connective tissue. Lying on either side of the muscles immediately surround- ing the trachea will be seen the sterno-mastoid muscle (cp, § 28) diverging from the lower part of the neck. Cut through the connective tissue on the inner side of one sterno-mastoid and draw the muscle outwards; there will be seen the common carotid artery, and, running along the outer side of this, the pneumogastric nerve. Free in one place the carotid, and lift it up with a hook. In the underlying connective tissue will be seen two nerves more or less closely united by tissue ; the larger is the sympathetic, the smaller the superior cardiac (depressor). Clear away the connective tissue from the artery. Draw the larynx from the carotid by means of a hook to which is tied a string having a weight at the end. Passing over the carotid at the level of the larynx will be seen the descendens noni, a branch of the 12th nerve. Cut this through and remove it entirely. Passing under- neath the carotid nearly at the same level is the superior laryngeal branch of the pneumogastric. Trace this with especial care; soon after it leaves the pneumogastric it will be seen to give offa small nerve, the depressor, Follow this down the neck, separating it from the sympathetic. Sometimes 19, 20. 21, 22. DISSECTION OF A RABBIT AND OF A DOG. 19 the depressor receives a branch direct from the pneumogastric; occasionally this is its sole origin. Remove the first rib and the remains of the sternum, avoiding any injury to the tissues below. Observe the thymus, a fatty looking body covering the roots of the great vessels. It may be torn away. Trace out on each side the junction of the external jugular and subclavian veins to form respectively the right and left vene cave superiores: near the junction ends the internal jugular vein, this brings blood from the brain and may be traced from the foramen jugulare (cp. E. § 21) down the neck laterally of the common carotid and vagus. Observe the right vena cava superior passing straight down to join the right auricle; the left vena cava superior passing obliquely downwards underneath the left auricle to jo the night auricle; and the inferior vena cava passing upwards from the diaphragm to join the right auricle. Trace up one phrenic nerve. It makes its way out of the thorax by the side of the superior vena cava, and then passes beneath it. Place a double ligature round the vein and divide be- tween the ligatures. Follow up the phrenic to its origin from the 4th and 5th (and also from the 6th and 7th) cervical nerves. 2—2 20 23. 24. 25. ELEMENTARY PHYSIOLOGY. {i Trace out the arch of the aorta by clearing away the tissue from its upper surface. Take care not to injure the pneumogastric nerves (see next section). Observe on the right side the innominate artery, which gives off first the left common carotid, and then divides into the right subclavian and right common carotid; on the left side the left subclavian. Note the vertebral artery on either side pro- ceeding from the subclavian. On a level with the anterior part of the larynx, note the division of the common carotid into external carotid and internal carotid. The former curls round the angle of the jaw, the latter enters the skull a little in front and to the median side of the tympanic bulla. Trace both pneumogastric nerves downwards, observing the recurrent laryngeal branches passing on the right side round the subclavian artery, and ou the left round the aorta. Place a double ligature round the innominate artery and divide between the ligatures. Trace the recurrent laryngeal nerves along the back of the trachea to the larynx. Pursue the main pneu- mogastric trunks on the cesophagus to the point where they were seen in A, § 10. Trace the sympathetic nerve downwards to the inferior cervical ganglion lying a little above the subclavian artery, and close to the vertebral artery; follow it thence to the first thoracic ganglion. 19 3 DISSECTION OF A RABBIT AND OF A DOG. 21 Observe the branches going from these ganglia towards the heart. Observe also the depressor nerve passing to the heart. From the first thoracic ganglion trace down the thoracic sympathetic nerve trunk lying on the heads of the ribs with the ganglia (twelve in all) and the rami communicantes connecting each ganglion with its corresponding spinal nerve. Trace out the splanchnic nerve on one side; it will be found to separate from the sympathetic at the 8th, 9th, or 10th thoracic ganglion. At first sight it appears to be the continuation of the sympathetic instead of a branch of it; since the sympathetic at its lower part becomes more transparent, and running in a groove between two muscles, is rather easily overlooked. The splanchnic receives branches from each of the thoracic sympathetic ganglia below its origin. Tie a tube in the trachea and distend the lungs, note the appearance of the distended lungs. Cut out the heart* with the lungs attached, and trace the pulmonary arteries and veins. 28. Having reflected on either side the skin of the neck of the dog, and cleared away the fascia of connective tissue, observe the muscles under- neath, a, The sterno-hyoid close to the median line. It runs from the sternum to the hyoid bone. 1 The heart may be dissected in the manner given for the sheep’s heart in Lesson x11, 22 ELEMENTARY PHYSIOLOGY. [I b. The sterno-thyroid lying laterally of (a) and for the greater part of its course close to it, it runs from the sternum to the thyroid cartilage of the larynx. c. The thyro-yoid, a small muscle running from the thyroid cartilage to the hyoid bone, in the upper part of its course it lies lat- erally of (a). d. The sterno-cleido-mastoid lying laterally of (6) and covering it near the hyoid bone, thence it proceeds outwards, and disappears under a white oval mass, the submaxillary glands. These muscles may be dissected in the rab- bit also, the representative of the sterno- cleido-mastoid has however no clavicular attachment and hence is called the sterno- mastoid, it does not come in contact with the submaxillary gland. 29. Carefully separate the sterno-mastoid from the sterno-thyroid; the sympathetic-pneumogastric trunk and the carotid artery will come into view. Observe the following points in which the dog differs from the rabbit : a, There is but one superior vena cava form- ed by the junction of the two innominate veins. (The arrangement of the main arteries is usually that described above for the rabbit, but con- siderable variations occur.) 1] DISSECTION OF A RABBIT AND OF A DOG. 23: B. There is in the neck no separate nerve corre- sponding to the depressor in the rabbit. y. The sympathetic and the vagus run in the neck ina thick sheath common to both. At the lower end of the neck, the sympathetic joins the infe- rior cervical ganglion. From the ganglion run several pale nerves to the heart and lungs, and receives two white ones—the annulus of Vieus- sens—from the first thoracic ganglion, The latter receives rami from the lower cervical and first two dorsal nerves, of these the 2nd dorsal only (the 10th spinal nerve) gives an obvious white as well as a grey ramus to it, 30. Clear away any muscles that may remain around the lower part of the larynx; on either side of it is attached a thin, dark red lobe of the thyroid gland, the lobes run a short way down the trachea, and there join over the ventral surface of the trachea by a very thin connecting piece. D. 1. Cut through the skin in the front of the thigh and turn it back on either side; in the upper median part blood-vessels will be dimly seen through the thin sartorius muscle; cut through this muscle and note the femoral (crural) artery and vein, and the crural nerve run- ning side by side; trace the artery upwards, it unites with other arteries to form the common iliac, which with the common iliac of the other side forms the abdominal aorta; trace similarly the fernoral vein to the common iliac vein and ‘24 ELEMENTARY PHYSIOLOGY. [I. the inferior vena cava. Follow the crural nerve up to the spinal cord, it arises chiefly from the 5th lumbar nerve (receiving branches also from the 6th and 7th). Remove the skin from the back of the thigh, cut through the tendonous line seen over the femur and pull the outside mass of muscle outwards, the large sciatic nerve will be seen, trace this to the top of the thigh, then turn the rabbit over and follow the nerve to its origin from the spinal cord; it arises chiefly from the 7th lumbar and 1st sacral nerve (receiving branches from the 6th lumbar and 2nd and 3rd sacral nerves.) The Student should have a rabbit’s and a dog’s skull before him, and make out the several openings by which the nerves spoken of below issue from the skull. Carry up to the chin the median skin cut and reflect the skin, place the head on one side; just in front and ventrally of the base of the ear will be seen the thin dorsal part of the parotid gland, often much hidden by fat tissue; the gland stretches ventrally a little past the angle of the jaw. From the anterior border of the parotid gland, issues the greater part of the facial nerve (7th) dividing into several branches which run DISSECTION OF A RABBIT AND OF A DOG. 25 forwards across the masseter muscle to their endings in certain muscles of the face. The duct of the parotid (duct of Stenson) runs forward with the facial nerve, from the gland; it is small, thin-walled and inconspicuous, it may sometimes be made evident by. pressing on the gland and so forcing some fluid into it. The branches of the facial should be carefully isolated close to the gland, the connective tissue being cut through with a fine pair of scissors as close as possible to the nerves lest the duct be inadvertently severed; on pulling the nerves to one side the duct will be seen, follow it forwards to the anterior edge of the masseter where it dips down to the mouth; make a small cut in it with scissors and pass a bristle down it. In the dog the duct is much more obvious, the facial nerve does not accompany it. Cutting through the parotid gland, trace the facial nerve to its exit from the skull by the stylo-mastoid foramen; observing the branches going to the muscles of the ear. Behind the parotid gland will be seen a nerve running from the under surface of the sterno- mastoid muscle (cp. C. § 21) dividing into two branches, and passing up the ear. This is the great auricular, which arises from the 8rd cervical nerve, and is the main sensory nerve for the ear. Trace as far as possible its course in the ear. 26 =1 ELEMENTARY PHYSIOLOGY. {i In the dog, reflect the skin of the head, note again the position of the submawillary gland (cp. C. § 28 d.) as seen from the surface ; it lies between two large branches of the jugular vein ; attached to the inner part of the posterior extremity of the lower jaw will be seen the digastric muscle; clear away the connective tissue surrounding it, cut it through, taking care not to injure the parts beneath, and reflect the cut ends; the submawillary duct (duct of Wharton) will be seen running from the gland, trace it forwards, it runs underneath (dorsally of) a muscle with transverse fibres, the mylohyoid, cut through this, turn the lateral part as far back as possible, taking care that the fascia on its lower surface is not attached to it and follow the duct forwards. Attached to the anterior end of the submaxillary gland and stretching for some little way along its duct will be seen the smaller sublingual gland, from this runs the sublingual duct, alongside and laterally of the duct of the submaxillary gland. A short distance from the lower border of the mylohyoid muscle the lingual nerve will be seen crossing the ducts and running on to the tongue: pull the tissues on which the lingual rests well away from the jaw, about three quarters of an inch centrally of the point where the lingual crosses the ducts, it will be seen to give off a small nerve the chorda tympani. This curves towards the ducts and then runs alongside them towards the sublingual and submaxillary glands. 10. 11. DISSECTION OF A RABBIT AND OF A DOG. 27 9. Trace the ducts peripherally, they unite and open underneath the tongue ; trace the lingual nerve peripherally, it supplies chiefly the tip of the tongue. In the rabbit there will be seen lying between the angles of the lower jaw the tolerably com- pact but soft submaxillary glands touching one another in the median line. Each gland is laterally in contact with the ventral lobe of the parotid, its tint is redder than that of the parotid; pull the submaxillary gland laterally and backwards, its small duct will be seen running from it over the muscle attached to the inner surface of lower jaw, and then underneath (dorsally of) the digastric muscle, which here has a conspicuous tendon; cut through the digastric and trace the duct forward underneath the mylohyoid muscle; a short distance from the lower border of the mylohyoid this duct is covered by the lobules of the small sublingual gland, turn this back, the lingual nerve will be seen crossing the duct, with care in dissection fine nerve fibres, chorda tympani fibres, may be seen running from the lingual nerve to the sublingual gland and to the duct of the sub- maxillary gland, the latter fibres are too small to follow towards the gland itself. Now follow up the pneumogastric nerve from the place where it was left in C.§18. A little above the superior laryngeal branch will be seen the pharyngeal nerve, and higher up still a 23 12. 13. 16. ELEMENTARY PHYSIOLOGY. [t. fusiform enlargement, the ganglion of the trunk. Note the hypoglossal, a large nerve running across the pneumogastric a little centrally of its ganglion. Trace it forwards to the muscles of the tongtie. Follow up the sympathetic nerve, it has, at about the level of the ganglion of the pneumogastric, a considerable enlargement, the superior cervi- cal ganglion; observe the fibres which run from this along the carotid artery and its branches. 14. In the dog the sympathetic and pneumogastric nerves which run in a common sheath in the neck (cf. C., § 28 (y)) separate from one another a little distance from their respective ganglia. . Partly saw through the symphysis menti, then use a lever and force the rami asunder, and in the following dissection cut through or remove any muscles necessary. Trace the lingual backwards. It will be found to join the inferior dental (a large nerve entering into the lower jaw), to constitute, with other branches, the inferior maxillary nerve. Trace this back to the front edge of the tympa- nic bulla. 17. Note in the dog the small nerve, chorda tym- pani, which joins the lingual soon after the latter branches off from the inferior dental; trace the chorda tympani centrally, it will be found to make its exit from the tympanic bulla 1] 19. 20. 21. DISSECTION OF A RABBIT AND OF A DOG, 29 close to the Glaserian fissure. Break through the bulla, and observe the chorda running across the tympanic cavity over the handle of the malleus (cp. Lesson xxvii). This course of the chorda tympani may be followed in the rabbit, but the dissection is not easy. 18. Note also in the tympanic cavity the very small nerve running over the promontory, or projec- tion of the cochlea. This is Jacobson’s nerve, a branch of the 9th. Trace up the pneumogastric beyond its ganglion, to its exit from the skull by the foramen jugulare. Note, passing from the skull with the pneumo- gastric, the small spinal accessory nerve behind and the glosso-pharyngeal in front; the communicating branches between these nerves may be neglected. Trace the glosso-pharyngeal forwards to the tongue and pharynx. It runs nearly in the same direction as, but at a higher level than, the hypoglossal, and may be traced to the hinder part and to the sides of the tongue. Cut through the above three nerves, a little distance from the skull, break away with small bone forceps the tympanic bulla, and trace more thoroughly the exit from the skull of these nerves and of the hypoglossal. The latter issues through the condyloid foramen, which is separ- ated by a distinct width of bone from the foramen jugulare, through which the other three issue. 30 ELEMENTARY PHYSIOLOGY. [I. 22. Saw through the base of the skull and the face, from the occiput to the nose, a little on one side of the median line. The nasal: septum will be seen dividing the nasal cavities except posteriorly. Note the anterior and posterior turbinate bones both consisting chiefly of thin folded laminz, pass a bristle through the anterior nasal opening into the nasal cavity, using bone forceps and scissors trace the passage from the nasal cavity through the posterior nasal opening into the pharynx and trachea; note that the posterior turbinate bones are not in the direct course between the anterior nares and the trachea. Cut through the septum nasi dorsally close to the nasal bones, and remove the nasal bones, note that the posterior turbinate bones and the posterior dorsal part, of the septum are covered with a yellowish mucous membrane which is thicker than that lining the rest of the nasal cavities; this is the olfactory part of the mucous mem- brane (Schneiderian membrane). Trace the olfactory nerve forwards from the brain; it divides into a number of fibres which run to the Schneiderian membrane. 23. Looking down into the pharynx, observe the epiglottis and the way in which it when pushed backwards folds over the opening to the larynx. Put the larynx of the dog into weak spirit for dissection later, (Lesson xxx11.) 24, Look at the side of the pharynx for the opening 26. DISSECTION OF A RABBIT AND OF A DOG, 31 of the Eustachian tube, pass a probe up it into the tympanic cavity. Pass another probe down the meatus externus and, rupturing the mem- brana tympani, make sure that the first probe has entered into the tympanic cavity. . Remove one eye from its orbit, cutting through the tissues close to the eye. In the anterior part of the orbit note the white Harderian gland; in the anterior lower part the pale red infra- orbital gland, the duct of which opens into the mouth near the upper molars; and the lachry- mal gland pale-red like the infra-orbital in the posterior part of the orbit. Observe the point of entrance of the optic nerve into the orbit, In the dog the muscles of the globe of the eye may be dissected out, after removing with bone forceps the roof of the orbit. Immediately below, and in front of the eye, the superior maxillary nerve will be found issuing from a foramen in the superior maxillary bone, to supply the skin of the face, &c., with sensory fibres. Cutting away the bone with a small pair of bone forceps, trace this nerve back along the floor of the orbit. 27. In the upper part of the orbit of the dog note the ophthalmic nerve. It passes from the front of the orbit to the forehead. . The superior and the inferior maxillary nerves and the ophthalmic, when traced back, will be found to unite into one large nerve, the fifth or 29. ELEMENTARY PHYSIOLOGY. [1 trigeminal. Observe on the nerve at the junc- tion of the three branches, the swelling of the Gasserian ganglion. Observe also that the nerve in leaving the brain has two roots, a small and a large, that the small root passes beside the ganglion on the large root, without eftering into it, and that the fibres of the small root are, beyond the ganglion, almost entirely confined to the third or inferior maxillary branch. Cut out the tongue taking care to remove the whole of it; on either side of the posterior upper surface, will be seen a small oval patch, the papilla foliata or lateral taste organ; note the parallel ridges running at right angles to the long axis of the papilla. As an introduction to the methods of preserving and hardening tissues, the following should be done by each student. The tissues should be removed from the rabbit as soon as possible after it has been killed, and sections should be cut when the Lessons dealing with the several tissues are being worked through. Cut out from the greater curvature or fundus of the stomach a piece about 1 c.m. square, wash it for a moment in NaCl. ‘6 p.c. to remove any acid or any food substance on the surface of the mucous membrane; with hedgehog quills or small pins fasten it out on a piece of cork with L] DISSECTION. OF A RABBIT AND OF A DOG, 33 the muscular surface downwards, stretching it slightly, and place it in alcohol about 75 p.c. for about an hour, then remove to 95 p.c. alcohol for a fortnight; keep in 75 p.c. alcohol, Cut out of the small intestine a piece about two inches long; tie into each end a short glass tube with lumen about 5 m.m. in diameter, over the free end of each glass tube slip a piece of india- rubber tubing; by means of a syringe wash out the piece of intestine with NaCl‘6 p.c. for about twenty seconds, then inject chromic acid ‘2p.c. When the salt solution has been displaced by chromic acid tie or clamp the peripheral piece of tubing, inject a little more chromic acid to distend slightly the intestine, and tie or clamp the central piece of tubing. Place the distended intestine in ten times its bulk of chromic acid ‘2 p.c. In two to three days cut off both ends of the intestine, cut it open longitudinally, and place it in fresh chromic acid ‘2 p.c.; in about ten days place it in water for some hours, and then in alcohol 30 p.c.; on the next day transfer it to alcohol 50 p.c. renewing the alcohol as long as it becomes coloured and finally place the tissue in strong spirit. Take a piece of costal cartilage about 5 m.m. long and place it in about 10 c.c. of saturated aqueous solution of picric acid; after about ten days wash it well with water and place for a day in 50 p.c. alcohol, then transfer to strong spirit. a 34 ELEMENTARY PHYSIOLOGY. [I. Take of the sciatic or other large nerve a piece about 10 m.m. long and place it in about 10 ¢.c. of ammonium bichromate 2 p.c. In a week renew the ammonium bichromate; in this fluid it may be kept until sections are required, or in a month or more it may be washed with water and placed in spirit as in § 2, LESSON II. STRUCTURE OF BLOOD. A. Buioop or Froa or NEwt. Having destroyed the brain and spinal cord of a frog’, cut through the skin in the median yentral line, cut transversely through the lower part of the sternum just above the epigastric vein, and expose the heart. Cut off the tip of the ventricle; with a glass rod transfer a small drop of blood to a glass slide and place on it a cover- slip. Examine it under the microscope with a low magnifying power” and observe the numerous corpuscles floating in the plasma. Examine it with a high magnifying power’ and observe the red corpuscles; if a large drop of 1 Cp. Appendix. 2 For convenience the term ‘low power’ will be used throughout for a combination of lenses which magnifies less than 100 diameters, and the term ‘high power’ for a combination of lenses which mag- nifies more than 300 diameters. In Zeiss’ microscope, objective A with ocular 2 magnifies 55 diameters, with ocular 3 it magnifies 75 diameters; objective D with ocular 8 magnifies 320 diameters, with ocular 4 it magnifies 440 diameters. If the tube be drawn out the 3—2 36 ELEMENTARY PHYSIOLOGY. [1I. blood has been taken the corpuscles will pro- bably form a continuous layer, in which case a | drop of ‘6 p.c. sodium chloride solution should be made to run under the cover-slip (cp. § 4). a. d. The red corpuscles are flattened ellipsoids; note their spindle shape as they roll over. They appear homogeneous; if however the specimen be not carefully prepared a certain number of the corpuscles will be altered and show a central oval nucleus. A single corpuscle is pale yellow, the colour- ing substance being equally diffused through- out it; when several corpuscles lie over one another they together appear red. The great majority are of the same size and tint. 2. Examine the colourless corpuscles in parts of the specimen where the red are not very nume- rous. a, b. oP They are much fewer than the red. They are smaller than the red, but vary considerably in size. Most have an irregular form, some are spherical. They are colourless and granular; the gra- nules vary greatly in distinctness and size, magnification is of course greater. The $ inch and % inch objectives of English make correspond respectively to the 4 and D objectives of Zeiss, With Hartnack’s microscope the nearly corresponding lenses are oc. 2 or 3, obj. 3 (low power) and oc. 3 or 4, obj. 7 (high power). Ill. ] é. STRUCTURE OF BLOOD. 37 The nucleus can seldom be made out, except when the corpuscle is very extended. Do not confound a heap of granules or a protuberance with the nucleus. Choosing a corpuscle either elongated or having several processes, watch carefully its amosboid movements; make half a dozen drawings of its outline at intervals of about twenty seconds. g. When a drop of blood is first mounted the colourless corpuscles are usually spherical, they soon begin however to put out pro- cesses; if it is desired to watch the move- ments for any length of time a fresh drop should be mounted and protected from eva- “poration in the following manner. With a morsel of blotting-paper dry if necessary the slide at the edges of the cover-slip. Keep the cover-slip in place by gently holding a needle against one edge, and, with a small brush, brush carefully the melted paraffin A.’ (which melts at 39°C.) over the edges all round. The paraffin need not extend more than } or } inch over the cover-slip. With the aid of a camera lucida* make an outline drawing of two or three red corpus- eles; substitute for the specimen a stage micrometer’, and being careful that the mi- croscope and the drawing-pad are in the 1 Op. Appendix, 38 ELEMENTARY PHYSIOLOGY. [II same positions as before, make a drawing of the micrometer lines over the previously made drawing of the corpuscles; then, the real distance between the micrometer lines being known, the diameters of the corpuscles can be at once read off; thus if the micro- meter lines are 4, mm. apart and in the drawing a corpuscle exactly occupies one division its diameter in that direction is evidently ;1, mm. The drawing of the micrometer lines may be kept as a scale, and any object drawn under the same magnifying power and with the pad and microscope in the same relative positions may be directly measured by it. Substitute for the ordinary eye-piece of the microscope one which has a ledge for sup- porting an ocular micrometer’, the values of which have been determined, the size of the corpuscle can then be at once read off. Mount another small drop of blood, place a small drop of ‘1 p.c. acetic acid on the glass slide so that it just touches the edge of the cover-slip; place a piece of blotting-paper on the opposite side just touching the fluid at the edge of the cover-slip, the acetic acid will then run under the cover-slip and mix with the blood. Note the changes which take place. a. In the colourless corpuscle, the cell sub- stance becomes more transparent but shews 1 Cp. Appendix. f STRUCTURE OF BLOOD, 39 several dark granules; a granular nucleus, often irregular or lobed, comes into view, usually more than one nucleus will be seen. In the red corpuscles the nucleus be- comes obvious; it is when first seen nearly homogeneous, and oval in outline, later it becomes granular and usually irregularly rod-shaped. The red corpuscles swell up owing to absorp- tion of water, most after a time become spherical (Gf strong acid be used the cor- puscles usually preserve their.shape). They become colourless, the colouring matter being dissolved; occasionally the colouring matter is massed round the nucleus before complete solution takes place (effect of water) and occasionally the nucleus becomes stained yellow by the colouring matter (effect of acetic acid). Finally the outline of the corpuscles is seen as a faint line at some distance from the nucleus. Observe the not infrequent excen- tric position of the nucleus, Some corpuscles are much more readily acted on than others. Irrigate with a strong aqueous solution of Spiller’s purple or magenta. a. The outline of the red corpuscle becomes distinct, its nucleus stains deeply, around the nucleus a little faintly stained granular 40 ELEMENTARY PHYSIOLOGY. [1. substance is seen which often stretches out to the periphery of the corpuscle in the form of a star. The nuclei of the colourless cor- puscles also stain deeply. Place several very small drops of blood two or three mm. apart on a slide and leave for a few minutes, then cover with a cover-slip, and put under a high power. Take a little blood from a freshly killed frog and establish a current under- neath the cover-slip from one side of it to the other (cp. § 4). The first small drops will have partially clotted and will serve as an imperfect barrier to the corpuscles in the current; in such places note that the shape of the red corpuscles is easily changed and recovered, and that the colourless corpuscles stick to one another and to the glass more than do the red. After the current has passed a short time largish clumps of colourless corpuscles will be seen. Having destroyed the brain and spinal cord of a frog, expose the heart and cut it across, suck up a little blood in a clean pipette and add it to about five times its volume of 2 p.c. boracic acid, stirring gently. Mount a drop of the mixture at once and observe the red corpuscles with a high power. The nuclei scarcely visible at first become in a short time rather deeply stained with hemo- globin; small spheres of hemoglobin appear also in the body of the corpuscle; occasionally the hemoglobin may appear to stretch in rays from I1.] STRUCTURE OF BLOOD. 41 the nucleus through the body (if the rays are not seen irrigate with 2 to 5 p.c. salt solution, but in this case be careful not to mistake foldings of the corpuscle for rays). Later the corpuscle becomes spherical and its body colourless. Whilst the earlier changes are taking place some of the corpuscles may be seen to extrude their nuclei. Dilute a little fresh blood with twice its volume of ‘6 p.c. salt solution; mount a drop of the mixture ; and place it aside for an hour or so to clot; irrigate it with 30 p.c. alcohol and then with Spiller’s purple dissolved in water or in dilute alcohol, Note the deeply stained network of fibrin fibrils and the numerous long threads of fibrin running from the broken-down colourless corpuscles. B. Buioop or Man. With a needle prick the end of a finger, and squeeze out a small drop of blood and mount it (cp. A. § 1). Observe the red corpuscles. a, They roll about readily, when the cover-slip is lightly touched. 'b. Soon after being taken from the body they stick to one another, and, owing to their shape, usually in rouleaux. c. They: are biconcave discs. Note that on fo- cussing down on the circular face a darkish centre and a light rim is first seen and then 42 ELEMENTARY PHYSIOLOGY. {ur a light centre with a darkish rim: when viewed in profile and the centre focussed they appear somewhat dumb-bell shaped. d. They appear homogeneous, their colour is like that of the red blood corpuscles of the frog (cp. A. § 1, ¢). e. Towards the outside of the drop, where evaporation is going on, many of the red corpuscles are crenate. jf. They are much smaller than the red corpus- cles of the frog. Measure them (P. A. § 3). Observe the colourless corpuscles. They are larger than the red, they resemble the white corpuscles of the frog (A. § 2, c. d. e.); to observe their amceboid movements a drop should be protected from evaporation (A. § 2, g) and, pre- _ferably, warmed to the temperature of the body. Irrigate with ‘5 p.c. acetic acid (cp. A. § 4). a. The red corpuscles swell up and become spherical, their hemoglobin is dissolved, leaving the hardly visible stroma, (Effect of water.) b. No nucleus is brought into view. c. The white corpuscles behave like those of the frog (A. § 4, a). Count the red corpuscles with Gower’s hamato- cytometer in the following manner. Fill the larger pipette with sodium sulphate solution of Sp. Gr. 1025 up to the mark on the STRUCTURE OF BLOOD. 43 stem, it then contains 995 c.m.; empty it into the measuring glass. Fill the small pipette with freshly drawn blood up to the line marked 5 c.m.; empty it into the measuring glass, and with the fluid in the measuring glass wash out the blood sticking to the inside of the tube; thoroughly mix the blood and salt solution with the glass spatula, place a small drop of the mixture in the centre of the glass cell and over it lay a cover-slip, arrange the springs on the cover-slip to keep it in position, and under a high power count the number of red corpuscles in ten of the squares which are marked at the bottom of the glass cell, Since the depth of the cell is 1 mm. and the side of each square is j; mm., there is beneath each square 5}, c.m. of the mixture, @.¢. sy¢yqy e.m. of blood, hence the number of corpuscles in 10 squares multiplied by 10,000 gives the number of corpuscles in 1 c. m. blood. DEMONSTRATIONS. The method of using the simple and Stricker's warm stage. The ‘platelets’ of frog’s or newt’s blood (cf. p. 387). Specimens to show the chief stages of indirect nuclear division (cf, p. 395). Norg. If the brain of a frog be destroyed, a drop of curari injected under the skin, and the frog be left for a day in about inch of water, the lymph sacs will become filled with lymph containing numerous white corpuscles, many in a state of active amoeboid movement. LESSON III. COAGULATION OF BLOOD. CHARACTERS OF PROTEIDS: 1. Observe the qoagulation of freshly shed blood’; it is at first fluid but soon passes into a jelly which gradually becomes firm; if then placed aside for some time, drops of clear serum will, by the shrinking of the fibrin, be pressed out on the surface of the clot; later the clot shrinks more or less completely from the vessel squeez- ing out more and more serum. 2, With a feather stir slowly about 10 c.c. of freshly shed blood’; a considerable portion of the blood will form a clot on the feather; squeeze out the clot under a stream of water from a tap; the clot shrinks considerably and a small quantity only of fibrin is obtained. 3. Repeat § 2, but. this time stir quickly, filaments of fibrin will be obtained; note that the fibrin is extensible and elastic; leave the defibrinated blood for a day, no further clot is produced, 1 This will be obtained by the Demonstrator. m1. COAGULATION OF BLOOD, 45 4, Place a small drop of fresh blood on a piece of glazed neutral litmus paper, in about ten seconds wipe off the drop, a blue spot will be left showing that the blood is alkaline. Test also the reaction of serum. 5. Apply the Xanthoproteic and Millon’s test for proteids (cp. § 16) to fibrin chopped up and suspended in water. 6. Take two test-tubes and in each place a few flocks of fibrin. a, Add water and place in water bath at about 39°C. for a day; the fibrin does not dissolve (it thus differs from albumin and peptone). b. Treat similarly but with dilute (1 p. c.) solu- tion of sodic chloride; the fibrin does not dissolve (it thus differs from globulin). 7: Place two or three flocks of fibrin in a test-tube containing a few c.c. of ‘2 p.c. HCl, the fibrin soon swells up and becomes transparent; neu- tralize the acid with Na,CO,, the fibrin shrinks to its original size. If the fibrin is warmed with the acid, solution slowly takes place, acid-albu- min being formed (cp. Lesson IX.) 8. Examine the plasma of horse’s blood kept, by means of cold, from coagulating’. 1 The blood is allowed to run from the animal into a tall narrow vessel contained in a much larger one packed with ice, a little salt may be mixed with the ice, but of course not enough to reduce the temperature so much that the blood is frozen; sometimes also a vessel 46 ELEMENTARY PHYSIOLOGY. [i. a, Transfer with a pipette 2 or 3 cc. of the plasma into a small test-tube. Observe the coagulation and compare it with that of § 1. Avoid shaking. Probably the fibrin will adhere so strongly to the sides of the tube that little contraction will take place. On being freed from the glass it will contract. If the clot has already shrunk away from the sides of the vessel, it may since it is colourless be overlooked unless the fluid be carefully examined. b. Dilute 1 cc. of the plasma with 50 cc. of distilled water or normal saline solution. Carefully avoid shaking and leave it till the next day. Observe the fine delicate fibrils of fibrin which are formed. 9. Examine the plasma of blood prevented from coagulating by the presence of neutral salts*. a. Remove 1 or 2 cc. carefully with a pipette, avoiding blood-corpuscles as much as possi- ble, and dilute five to tenfold with water. filled with ice is placed in the one which receives the blood. Horse’s blood is preferable to bullock’s or dog’s, since it clots less readily and the red corpuscles sink more quickly. 1 In preventing coagulation by neutral salts, blood is collected in a vessel containing a saturated solution of magnesic sulphate; as the blood runs in, it must be mixed well with the salt solution, preferably by stopping the flow of blood now and then and turning the vessel upside down. There should be about 1 vol. of the salt solution to 4 vols. of blood. The vessel may advantageously be surrounded by ice or by ice and salt. On either method (§ 9 or § 10) clotting some- times takes place, but the remaining fluid may still give a clot on appropriate treatment. Im.] COAGULATION OF BLOOD. 47 The mixture will clot very speedily if placed in the warm chamber; less speedily if left at the ordinary temperature. . Remove about 10c.c. into a small conical glass. Add powdered sodic chloride to excess, stirring but not more than ts necessary to assist the salt to dissolve: As the point of saturation is reached, a flaky precipitate makes its appearance. If the precipitate be plentiful, remove it with a spatula, put it on a small filter wetted with a saturated solution of sodium chloride and wash with small quan- tities of the same: if the precipitate be small, decant it and the fluid from the undissolved salt; filter, and wash the pre- cipitate on the filter paper with small quan- tities of a saturated sodic chloride solution. Dissolve the substance-so obtained, the plas- mine of Denis, in a small quantity of dis- tilled water, and filter. Probably a portion of it will not dissolve, having already coagu- lated. The clear, colourless fluid filtrate will, if set on one side, clot. Avoid shaking after filtration. If a small quantity only of the fibrin factors be present, the fine threads of fibrin, as they are formed, are loosened by the shaking, and contract; thus the more easily recognized gelatinous stage is lost. This operation is the more successful, the more rapidly it is carried on. 10. To 2c, of hydrocele or other serous fluid which 48 11. 12, 13. 14. ELEMENTARY PHYSIOLOGY, {1i1. has been ascertained not to coagulate, nor to have coagulated spontaneously, add 2c.c. of fresh blood- serum, gently mix, and put on one side. After a while, possibly not until after twenty- four hours, the mixture will have coagulated. The coagulation will be more rapid in the warm chamber. Take 10c.c. of fresh blood-serum and saturate it with magnesic sulphate by adding the salt in powder. Paraglobulin will be precipitated, since like other globulins it is insoluble ina s rated solution of a neutral salt; filter (befor filtering the precipitate may be allowed to settle and most of the fluid removed by decantation), wash on the filter with a saturated solution of the salt, then add 5 c.c, water to the precipitate, the salt solution clinging to the precipitate. will be diluted and the paraglobulin dissolved. It does not coagulate spontaneously. Add a small quantity of paraglobulin solution to hydrocele fluid. Coagulation will result. Treat 10 .c. of hydrocele or pericardial fluid with sodium chloride to saturation, and proceed as in § 11; a precipitate of fibrinogen will be ob- tained, its solution does not coagulate sponta- neously. Add to 1e.c. of a strong solution of fibrinogen an equal volume of blood-serum, and set aside; coagulation will take place. . Take 2c.c. of plasma (§ 9), add to it 16 cc. of IIL] COAGULATION OF BLOOD, 49 water, and determine that the mixture coagu- lates very slowly. Take another 2 c.c. of plasma, and add to it 16 c.c. of an aqueous solution of prepared fibrin fer- ment’; coagulation will quickly take place. 16. Dilute serum ten-fold with water and with it observe the following general reactions of pro- teids. (If sufficient serum is not obtainable, take the white of an egg, cut through the membranes in several places with scissors, add 50 vols, of water, beat up well, filter through flannel and then through filtering paper.) a. Xanthoproteic reaction. Take a little of the 1 The student may prepare fibrin ferment in one of the following ways: a. Let blood run into 10 times its volume of water, tilt it upside down once or twice so that the fluids are well mixed, and let it stand for a day. Filter through muslin and squeeze the excess of fluid out of the clot, chop it up and wash with water until all or nearly all of the colouring substance is removed, place it in 10 times its bulk of 8 p.c. NaCl solution, and warm for one to two days; filter; the filtrate contains fibrin ferment. Add alcohol in abundance to serum until no further precipita- tion takes place, filter, and dry the residue over a water-bath at 35°C.; place the residue in a bottle containing an excess of absolute alcohol, and leave for a month; at the end of this time decant as much alcohol as possible, evaporate the rest at a low temperature (under 40°C.); extract the residue with 200 times its volume of water, and filter. The alcohol will have coagulated the greater part of the paraglobulin and albu- min, etc., and so rendered them insoluble in water, hence the aqueous filtrate will contain little else than fibrin ferment, the more so the longer the alcohol has been acting; the compara- tive absence of proteids should be tested by the reactions given in § 16, L. 4 50 ELEMENTARY PHYSIOLOGY. [rit dilute serum, add a few drops of nitric acid, and boil. The white precipitate of proteid material at first formed becomes yellow and partially dissolves, forming a yellow solution. If the quantity of proteids present is small, the yellow solution only will be obtained. Place the test-tube in a stream of water from a tap to cool and when cold add ammonia; the yellow is turned to orange. . To another small quantity of the serum add a few drops of Millon’s re-agent’. A pre- cipitate will be formed which tarns pinkish on boiling; if the amount of the proteids present be small, no distinct precipitate will be formed but the fluid will turn pink on boiling. Add a drop of cupric sulphate solution to an excess of sodium hydrate. To the blue solu- tion so formed add a little dilute serum, the fluid will become violet (cp. reaction of pep- tone, Lesson xvi). The violet colour be- comes deeper on warming. . Add strong spirit, a precipitate is formed (peptone in solution is precipitated with difficulty). . Add excess of acetic acid and a few drops of | a strong solution of potassium ferrocyanide, a precipitate is formed (peptone is not thus precipitated). 17. With the diluted serum or white of egg observe 1 Cp. Appendix, IL 11] 18. COAGULATION OF BLOOD. 51 in the following manner the coagulation of albumin and globulin by heat. The white of egg contains a small quantity only of globulin. Place a test-tube containing 10 cc. of the fluid in a water-bath at about 50°C. and heat the bath not too slowly up to about 80°C. Note on a thermometer placed in the bath or in the test-tube the temperature at which coagulation begins (fluid slightly milky) and that at which coagulation is. complete (precipitate with clear: fluid).. If the fluid is very slowly heated the coagulate may be small owing to the formation of alkali albuminate (cp. Lesson 1x. § 16); to avoid this the serum should be carefully neutralized with acetic acid before dilution. Add 2¢.c. of serum drop by drop to about 100 cc. of water in a glass beaker. A faint cloudy precipitate of globulin, which is insoluble in water, will be seen (cp. Lesson 1x. § 14). DEMONSTRATIONS. Coagulation of blood over mercury without access of air. Fluidity of blood in living blood-vessels. Coagulation of blood within the vessels during life on foreign bodies or in injured blood-vessels. LESSON IV. HYALINE CARTILAGE. 1. Snip off a piece of the free edge of any of the thin cartilages projecting from the sternum or shoulder-girdle of a freshly killed young newt’. Gently scrape away with a scalpel any tissue attached to it. Mount it in normal saline solu- tion? and with a high power note that a, The matrix is studded at tolerably regular intervals with cartilage cells or corpus- cles. b. Each corpuscle consists of a spherical or ovoid mass of cell-substance, in which lies a relatively large nucleus. Both of these are fairly clear and transparent, though usually showing a variable number of fine granules. c. Most of the cells entirely fill up the cavities in which they lie. 2 Instead of this, sections of the head of the humerus or femur of any young animal may be made. * 2 i.e, -6 p.c. aqueous solution of sodium chloride. In this and the following Lessons whenever a tissue is directed to be mounted in normal saline solution it is to be understood that if either fresh aqueous humour, or fresh blood-serum is obtainable, it is to be preferred. ; IVIV.] HYALINE CARTILAGE, 53 d. Along the cut edge some cavities from which the cells have fallen out may generally be seen. e. Except at the free edge, the cells are two or more layers deep. jf. The matrix is hyaline or faintly granular, and is comparatively (cp. infra §§ 4, 7) in small quantity. If the newts are not quite young the cartilage will vary from the above in the quantity of ma- trix, the shape and arrangement of the cells, and in the cells possessing small fat globules and often two nuclei. Trrigate the piece with acetic acid 1 p.c. The following changes will be seen as the irriga- tion proceeds : a. The nucleus becomes much more granular and distinct. b. Granules appear also in the cell-substance, and more or less hide the nucleus, c. The cell-substance becomes transparent owing to the disappearance of the granules. d. The cell-substance shrinks from the mairix, and presents a granular-looking finely ser- rated border. Note the space thus formed between the cell and the matrix. Place a small piece in gold chloride solution ‘5 p-¢. for about half an hour, (until it is of a light yellow colour,) then wash well with water, and place it in a vessel containing water just acidu- ELEMENTARY PHYSIOLOGY. [tVv. lated with acetic acid; leave it exposed to the light. In one to two days, when it has become a red-purple colour, mount it in glycerine. Observe the cell-substance well coloured, and hardly at all shrunken, the nuclei deeply coloured and having a sharp outline, the matrix coloured very slightly. 4. Take a piece of rib cartilage from a recently killed adult animal and with a razor cut thin transverse sections' and transfer them with a camel-hair brush to a watch-glass containing normal salt solution. Mount one section in normal salt solution and another in osmic acid 1 p.c.; in the former observe under a high power, that a. The cells frequently shew signs of partial degeneration, containing fat globules which may be nearly as large as the cell; these are very highly refractive and so have a very distinct outline. b. In places the matrix may be fibrillated, and in places semi-opaque owing to calcareous deposit; irrigate with 1 p.c. hydrochloric acid, the calcareous deposit is dissolved, but the fibrillation is not affected (it differs thus from the fibrillation of white fibrous connec- tive tissue, cp. Lesson v. § 2b). c. Indications of primary and secondary capsules may be seen (cp. § 6). 1 The sections may be cut with a freezing microtome, ef. Appendix. Iv.] HYALINE CARTILAGE. 55 Examine the section which has been mounted in osinic acid; the fat globules are stained a deep brown-black, the cell-substance, the nuclei and the matrix are but slightly stained. Prepare transverse sections of costal cartilage which has been preserved in picric acid (cp. Lesson 1 F. § 3), the sections may be made as in § 4 or the tissue may be imbedded (ef. Lesson vI. § 1). Stain the sections by placing them in a 2 p.c. solution of picrocarmine for 5 to 10 minutes or in a very dilute solution (e.g. ‘02 p.c.) for a day ; if placed in the strong solution they should be looked at now and then to see that they are not overstained. Wash the sections with water and mount them in glycerine. Observe that the cells are arranged in groups (each group having arisen by division from a single cartilage cell), note the outline of the thin layer of newer cartilage (capsule) around each cell, sometimes the whole of the cells in a group may be seen to be also surrounded by a thin layer just marked off from the rest of the matrix. Towards the outside of the cartilage the cells become flattened in a direction parallel with the surface. This specimen should be preserved’ for examination later (Lesson-v. B. § 7). 1 With a small brush or glass rod spread a fairly fluid solution of Canada balsam over the edges of the cover-slip and the adjoining part of the glass slide; the balsam will dry in a day or so and the specimen can be moved about without fear of the cover-slip being displaced. If the glycerine in which the section is mounted does not stretch to the edges of the cover-slip the balsam will run underneath it and 56 ELEMENTARY PHYSIOLOGY. [Iv. 7. Take a piece of cartilage from the head of a cuttle fish which has been preserved in picric acid and subsequently in alcohol. Cut sections, im- bedding if. necessary (Lesson VI. § 1). Select a thin section and immerse it in hematoxylin for a few minutes watching that it does not stain too deeply, place it in a watch-glass containing spirit to remove the colouring matter simply ad- hering to the tissue (if it is tco deeply stained, place it in a little 1 p.c. acetic acid and when it is sufficiently decolourised, replace it in the spirit), then mount it in glycerine. Observe a. The groups of cartilage cells, b. The marked processes proceeding from the cells of one group, running towards and anastomosing with the processes from the cells of other groups, 8. Take a small piece of the ear of a freshly killed mouse or young rat, remove the skin and scrape away the tissue surrounding the ear-cartilage. Mount the cartilage in normal saline solution. Observe that the cavities of the cells, generally polygonal in form, are separated by very narrow partitions of matrix. From many of the cavities the corpuscles have disappeared altogether, leaving simply a honeycombed matrix. This probably spoil the section; on the other hand if the glycerine spreads beyond the cover-slip the balsam will not stick to the glass, in this case it is generally less trouble to remount the specimen than to wipe away the superfluous glycerine. The Student should make one or two trials to find out the proper quantity of glycerine to take with the cover-slips he uses. HYALINE CARTILAGE. 57 form of cartilage is frequently called “ parenchy- matous.” If it is desired to preserve this specimen or one of newt’s cartilage (§ 1), place it in 75 p.c. alcohol for about half-an-hour, stain with hematoxylin (cp. § 7) and mount in glycerine. LESSON V. CONNECTIVE TISSUE. A. CONNECTIVE-TISSUE FIBRES. 1. 9 ~ Elastic Fibres. a. Tease out in water a morsel of ligamentum nuche. It is almost entirely made up of. rather large branching, and anastomosing fibres having distinct outlines, and curling at their ends. b. Irrigate with acetic acid (1 to 5 p.c.); the fibres are unaffected. White Fibres. a. Place on a slide a small piece of a thin tendon, e.g. from a digit of a frog or from the tail of a mouse (cp. B. § 3) and, keeping it in position at one end with a needle, separate as completely as possible the fibres from one another at the other end by passing a needle through it in the direction of its length. If the unteased end of the tendon is so thick that it would tilt up the cover slip, separate with needles a small outer portion CONNECTIVE TISSUE. 59 and tease this out as above; the tissue should be kept moist with normal salt solution but no more than is necessary should be added. Place a small drop of salt solution on a cover slip and let this fall gently on the tissue. Observe the wavy bundles of fibrille; the outlines of the fibrillee are not very distinct but they may be seen to run parallel to one another; owing to their small size and to the cementing substance between them special preparation is required to isolate the individual fibrillze completely. Irrigate with acetic acid (1 to 5 p.c). The fibrillze disappear, and the whole tissue swells up into a transparent gelatinous mass, in which hardly anything can be distinguished except a few elastic fibres similar in general characters to those of A. 1, but much finer. Pull up with fine forceps the thin connective tissue lying between the muscles of the thigh of a rabbit or frog, cut off with scissors a small piece and tearing it as little as possible spread it out on a slide with the aid of needles, breathing on it if necessary to prevent it from drying. Mount as in § a or press a dry cover- slip gently on the film and allow saline solution to run under the cover-slip; thus the film is kept extended. It is chiefly composed of wavy bundles of fibril- le similar to those seen in (6); the bundles are of various sizes and run across one another 60 ELEMENTARY PHYSIOLOGY. [v. in all directions. Some small elastic fibres running singly will also be seen, their out- lines are more distinct than those of the bun- dles of white fibres; they sometimes branch and sometimes anastomose where they cross one another. Where the film is well stretched the elastic fibres run for the most part quite straight, elsewhere they are curled at their ends and have a more or less sinuous course. If there is any doubt as to which fibres are elastic, irrigate with acetic acid, cp. (6). d. To make evident the corpuscles, take a rather large piece of the film and let the edges partially dry so that it may stick to the glass slide ; add a drop or two of a strong solution of Spiller’s purple in water or in 30 p.c. alcohol, in a few minutes wash away the excess of the purple with water from a pi- pette, and mount in water. B. CONNECTIVE-TISSUE CORPUSCLES. 1. Take a piece of the thin expansion of a tadpole’s tail preserved in chromic acid ‘2 pc. Gently break up a piece in glycerine. Where the hex- agonal cells of the epidermis have broken away, the homogeneous matrix may be seen, imbedded in which are many blood-vessels and a number of stellate cells. The larger dark stellate cells are pigment-cells, the smaller stellate cells are connective-tissue corpuscles, each of which consists of a nucleus and a branched protoplasmic body, Their pro- * v.J CONNECTIVE TISSUE. 61 cesses divide much, but irregularly, and commu- nicate with the processes of other cells. 2. Take a frog, of which the brain and spinal cord have been destroyed’, squeeze the side of the head gently to make the eye bulge out, then slice boldly at the edge of the cornea; if it is not completely separated, take up the edge with forceps, and cut through the cornea with scissors at its junction with the sclerotic; in doing this remember that the success of the preparation greatly depends upon the cornea being placed in gold chloride without having been dragged or pulled about; any blood which may be on the cornea should be removed by placing it in a watch-glass containing normal saline solution and very gently brushing it with a camel-hair brush, Put it in gold chloride °5 p.c. solution for twenty to forty minutes, wash well with water, transfer to water just acidulated with acetic acid, and expose to light When the cornea has become of a red- or blue-violet colour, put it on a slide with glycerine, and brush or scrape both surfaces to remove the epithelium, then mount in glycerine and examine it under a high power. Where the hexagonal surface epithelium has been removed, there will be seen stained connective-tissue corpuscles, with numer- ous fine branching processes which anastomose with the similar processes of neighbouring cells. 1 See Appendix. 2 Probably it will not be stained until the following day. By the tartaric acid method (cp. Appendix) the staining is quicker. 62 ELEMENTARY PHYSIOLOGY. [v. This preparation should be preserved to shew the nerves of the cornea (cp. Less. x. C. § 2). Break off the tip of the tail of a mouse or young rat just killed, and pull out from the stump a bundle of fine tendons. Gently stretch this over a glass. slide from edge to edge, and hold it in this position till the edges are dry, putting a drop of normal saline solution in the middle only; the fibres are thus kept extended. Cover with a cover-slip. On examination the bundles of wavy fibrillze will be seen, but no corpuscles. Slowly irrigate with acetic acid 1 p.c. watching the bundles the while under a high power. Be- tween the bundles of fibrillz will be seen rows of cells. Note in each the round or oval nucleus, the rectangular outline of the cell, and the granular cell-substance. Indications of flange- like lateral processes may be seen as lines run- ning along the cells in the direction of the tendon. After the acetic acid has been added for some little time the cell-substance becomes very indis-. tinct, and between the swollen bundles scarcely anything is seen but rows of elongated irregu- larly shaped nuclei. (cp. Action of acetic acid on cartilage cells, Lesson Iv. § 2.) Cut transverse sections of a tendon which has been placed in 30 p.c. alcohol for a few hours and preserved in 75 p.c. alcohol, imbedding (cp. Less. VI. § 1) if satisfactory sections cannot otherwise be obtained: mount in dilute glycerine. Observe v7] ot CONNECTIVE TISSUE. 63 a, The fasciculi separated from one another by a small amount of connective tissue. b. In each fasciculus the thin branched tendon cells between the bundles of fibrille of the tendon. The processes of neighbouring cells not infrequently join. Dissect back a piece of skin of a recently killed young and fasting rat or other animal, and into the subcutaneous tissue attached to this inject a ‘5 p.c. solution of gold chloride with a Pravatz syringe, until the fluid begins to: exude. Let this stay for a few minutes, and before the swelling produced subsides snip off with a sharp pair of scissors one or two pieces as thin as possible, and place them in a watch-glass con- taining three parts of water and one part of formic acid 1:06 sp.gr. When the pieces are well coloured, which will probably be in two or three hours, shake them gently in a glass containing distilled water, then mount in the manner given in A. § 2 c, but in acid glycerine’. The pre- paration will improve in a day or two. Observe a. The leucocytes scattered about between the bundle of fibres; the fibrous bundles will be more or less swollen up by the formic acid, and so not offer sharp outlines. b. Connective-tissue corpuscles rather larger than the leucocytes, and having processes 1 Glycerine containing 1 p.c. formic acid, 64 ELEMENTARY PHYSIOLOGY. [v. which, in some cases, are seen to be connected with the processes of other similar cells. (cp. B. §§ 4, 2.) c. Larger, more rectangular cells, generally without evident processes, with oval nuclei, lying over the bundles of fibrous tissue often in rows or patches; on a side view these appear as thin long cells. This preparation should be preserved for the observation of fat tissue (D. § 5). Snip off a small piece of the jelly-like subcuta- neous tissue in a foetal or new-born mamzal’, and mount as in A. § 2, ¢. ; Note the pale granular cells of various shapes, some not unlike those seen in B.§ 1. To make the cells and their nuclei more distinct stain with Spiller’s purple in the manner given in A, § 2 (d), or irrigate with dilute acetic acid watch- ing closely the early changes produced. This may also serve to shew the development of fat-cells (D. § 6). Note in the section of costal cartilage prepared in Lesson Iv. § 6 the connective tissue layer out- side and closely attached to it forming the peri- chondrium ; in places sections of tendons running into the cartilage will probably be seen. 1 An animal just killed serves best, but one preserved in picric acid or Miiller’s fluid will answer the purpose; if it is desired to observe the forming fat-cells the animal must not have been transferred to alcohol stronger than 50 %/). v.] CONNECTIVE TISSUE, 65 C. PigMEentT-CELLS, Pin out on a frog-board one of the webs between the toes of the frog used in B, § 2, and observe first under a low and then under a high power. There will be seen large corpuscles loaded with dark pigment, and possessing numerous branched processes. In some places the pigmented cells will appear as round dots, the processes in this case having been retracted. Every intermediate stage between these two states may be observed by watching from time to time, E. Fat-Ce zs, 1. bo Cut out a small piece of the omentum, from a part containing comparatively little fat. Spread it out on a slide and mount it in normal saline solution. Observe a. Under a low power, the Sree of highly refractive fat-cells, b. Under a high power, the variable size of the fat cells, the apparent absence of a nucleus, the connective tissue passing between and over the cells, Place a similar piece of omentum in osmic acid 1 p.c. for about half-an-hour, wash with water and mount in dilute glycerine. Observe that the fat-cells are stained a deep brown-black (cp. Less. Iv. § 5). If the tissue after washing he placed in alcohol (preferably 75 p.c.) the tint will become much darker. 5 ELEMENTARY PHYSIOLOGY. [v. Take a small piece of omentum, which has been kept for some time in alcohol, and place it in hematoxylin until it has become well stained. Wash it with spirit, place it on a glass slide, and tease it out if it is too thick. Remove as much of the spirit as possible with blotting-paper. Cover the tissue with a mixture of creosote (1 part) and turpentine (4 parts), and let it remain until it is completely transparent, chang- ing the fluid and gently warming if necessary. Remove the excess of turpentine, cover the tissue with a drop of Canada balsam, and put on a cover-slip. Observe the groups of cells from which the fat has been thus removed. Note the shrunken outlines of the cells, the marked membrane, and the presence in each cell of a deeply stained nucleus. A small amount of protoplasm, also stained, may be seen as in § 4. In the gold chloride preparation of sub-cutaneous tissue made above (B. § 5), observe The network of capillaries in a small collection of fat-cells. The large, flat, connective-tissue corpuscles (B. § 5, ce) in greater quantities near the groups. When the fat-cell group is spindle-shaped, these may often be seen proceeding in rows from the ends. In some fat-cells a nucleus and a small amount of protoplasm surrounding the fat may be seen. Do not mistake a deposit of crystals in the fat for a nucleus. CONNECTIVE TISSUE. 67 Take a piece of tissue as in B. § 6 but just outside the border of a small fatty clump visible to the eye. Observe the transitional forms between the granular cells containing a few small fat globules and cells in which hardly anything but fat is to be seen. 5—2 LESSON VI. MODIFICATION OF CONNECTIVE TISSUE AND HYALINE CARTILAGE. A. TRANSITION TO FIBRO-CARTILAGE, 1. Take a piece of intervertebral cartilage’, which has been treated with chromic or picric acid and subsequently with alcohol, and imbed it in the following manner. At one end of a small oblong block of paraffin B® scoop out a small hole; with blotting-paper remove the excess of alcohol from the piece of cartilage and place it in the hole so that the plane of the uppermost surface is at right angles with the plane of the vertebral face. Pour over it a little of the paraffin mixture heated so as to be just melted ; remove any bubbles that may be present and keep the tissue in its proper position by means of a heated needle; when the whole is thoroughly solid pare down the paraffin 1 Cut off from one face of a vertebra of a rabbit a thin slice about 1mm. thick, leaving the intervertebral cartilage in connection with it ; divide the more or less circular piece so obtained into four parts, and treat with chromic or picric acid to dissolve the salts (ep. Appendix). 2 Cp. Appendix. VI] MODIFICATION OF CONNECTIVE TISSUE, ETC. 69 to the surface of the cartilage, at the same time bevelling the edges. In cutting the sections, cover the tissue and the blade of the razor with spirit (for convenience from a wash-bottle) and remove the sections by means of a camel-hair brush from the razor to a watch-glass, With the platinum “lifter” transfer them to hema- toxylin and treat as in § 7, Lesson Iv. It is to be remembered that if the chromic acid’ has not been completely removed from the tissue the staining will be very imperfect; in this case the sections should be placed in a little Na,CO, 1 p.c. for a few minutes and then washed with water before staining. Observe in the sections a. The softened bone passing into b. The thin layer of hyaline cartilage; outside this c. The arrangement of the cartilage cells in rows, and the concomitant appearance of fibres in the matrix, this gradually but rapidly merging into d. Fibro-cartilage consisting of bundles of white fibrous tissue and of ratherlargecellsarranged in rows between the fibrous bands; these cells are like the cells of hyaline cartilage and each has a thin hyaline capsule around it. e. Towards the outer part of the cartilage, the capsules and cells become smaller and more 1 Miiller’s fluid, potassium bichromate and ammonium bichromate, must similarly be completely removed from tissues which are to be stained, ; 70 ELEMENTARY PHYSIOLOGY. [VI. elongated, the capsules disappear, and so the fibro-cartilage merges into f Connective tissue. The fibro-cartilage does not form one continuous band connecting the vertebra, but passes in parallel bundles, between and at right angles to which are tendinous bundles which thus will be seen in cross sections; the cartilage cells may in some bundles also be absent. 2. Prepare through the junction of the round liga- ment and head of the femur in a young animal a section parallel to the direction of the fibres. If the tissue has been treated with picric acid, stain the section in picrocarmine; if in chromic acid, with hematoxylin. Observe as the ten- don approaches the cartilage the transition of the flat rectangular branched cells of the ten- don to oval fuller cells with round nuclei. Note the gradual disappearance of distinct fibres and their replacement by a hyaline matrix, the cells at the same time becoming scattered. Thus the tendon passes into fibro-cartilage, and fibro- cartilage into hyaline-cartilage. B.. TRANSITION To Exasric CARTILAGE. Dissect out the arytenoid cartilage of a sheep, preserved in alcohol, and cut out a small piece of. the tissue immediately above the hyaline cartilage, and together with a little of the hyaline cartilage itself. Imbed, and cut sec- v1] MODIFICATION OF CONNECTIVE TISSUE, ETC. 71 tions, including the hyaline cartilage and the tissue above it. Stain with picrocarmine and mount in glycerine. Observe a, That at the upper surface of the hyaline cartilage, the matrix becomes granular, and the granules are arranged in rows, which then often have less the appearance of rows of granules than of granular fibres; these may be traced into ordinary fine elastic fibres, which increasing in quantity, form b. The elastic cartilage in which the cells stained red with carmine are similar to those of hyaline cartilage; they have thin clear outlines around them, the capsules, and these are surrounded by a meshwork of elastic fibres stained yellow with picric acid lying in a varying quantity of matrix. This meshwork may have not so much the appearance of distinct elastic fibres, as of a system of thick bars, recalling in general feature parenchy- matous cartilage. c. Above (6) ordinary loose connective tissue, ‘note the transition of the elastic cartilage into this by the elongation of the cells and the breaking up of the elastic fibres into loose bundles and the appearance in those of white fibrous tissue staining red. LESSON VII. BONE, OSSIFICATION, TEETH. A. Structure oF Bone. 1. Examine under a low power prepared transverse sections’ through the shaft of a long bone. Most of the smaller spaces (i.e. Haversian canals, lacunz, canaliculi) will be filled with air or debris and will therefore appear dark. Observe a. The central cavity surrounded by a small amount of spongy bone which is arranged in a network with rather large irregular spaces (Haversian spaces); externally the spongy bone passes into the compact bone: note the transition from the Haversian spaces to the Haversian canals. b. In the compact bone the Haversian systems, each consisting of a Haversian canal sur- rounded by concentric lamella which are chiefly marked off from one another by the lacune which lie between them. 7 It is simplest to buy these sections. VIVIL] c. BONE, OSSIFICATION, TEETH. 73 The intersystemic lamelle between the Haversian systems; in the outer part of the bone these run for the most part parallel with the surface. In the spongy bone the lamella run in the main concentric to the spaces. Examine the specimen under a high power. Observe a. The irregularly fusiform lacune giving off numerous wavy branches, the canaliculi, which run across the lamellz to join the similar branches of neighbouring lacune. In the compact hone, canaliculi open into the Haversian canal; in the spongy bone into the Haversian spaces and central cavity. Examine with a low power prepared longitudinal sections of the shaft of a long bone. Observe a. The Haversian canals running in the main parallel with the surface of the bone, they have connecting branches and in places open on the surface and into the central cavity. . The lamelle running for the most part parallel with the Haversian canals. Here and there a Haversian system may be cut obliquely, in such cases the lamellz will be concentric to the canal. Examine the specimen under a high power. The individual lacunz are much as in the trans- verse section, but rather longer; observe the arrangement of the canaliculi (cp. § 2). 74 ELEMENTARY PHYSIOLOGY. [ VII. Prepare transverse sections through the shaft of a long bone which has been decalcified’. Stain with picrocarmine (cp. Lesson Iv. § 6). Wash with water and mount in glycerine. Observe that a. Each lacuna is occupied by a cell or bone corpuscle. b. The canaliculi are scarcely visible. The Haversian canals and spaces are occupied by cells, connective-tissue fibres and blood- vessels (red blood corpuscles if present will be stained yellow). d. If the tissue of the central cavity has not fallen out of the section, it will be seen to consist of fatty tissue. e. The periosteum is closely attached to the out- side of the bone; it consists externally chiefly of white fibres, internally largely of small elastic fibres some of which may be seen to run into the bony matrix. Take a bone (e.g. the parietal) which has been decalcified by nitric or hydrochloric acid, well washed with water and preserved in spirit; remove the periosteum, tear off from the surface thin strips of the bone matrix and mount them with the inner side uppermost in water. Under a high power note the perforating fibres pro- jecting from the surface and the apertures through which similar fibres have passed. 1 Cp. Appendix for methods of decalcification. vit] BONE, OSSIFICATION, TEETH. 75 Examine carefully the thinnest part of the strip (containing fewest lamella) for the fine decus- sating fibres of the matrix; add acetic acid, both the perforating and decussating fibres swell up and become indistinct or lost to view. B. OSSIFICATION. 1, Cut a longitudinal section through the head of the femur of a newly-born rabbit, cat, etc. The femur should have been divided longitudinally into four parts, and treated with picric or chromic acid to harden it and to dissolve the salts. Stain the sections with eosin or picro- carmine, wash with water, place first in dilute and then in strong spirit, clear with creosote and turpentine’ (or turpentine alone) and mount in Canada balsam (cp. Lesson Iv. footnote). Observe a. The normal hyaline cartilage. 6. The cartilage cells arranged in rows. Note that many of the cells in section are tri- angular, the broad side of one lying above the angle of the other; this indicates an 4 The creosote and turpentine is used since it dissolves any paraffin that may be attached to the tissue (from the imbedding), makes the tissue transparent, and mixes well with the Canada balsam; carbolic acid may be used instead of the creosote, it is cheaper but does not dissolve the paraffin mixture quite so readily. Clove oil causes less shrinking in many tissues than creosote and turpentine, but it is much dearer than either of them and does not dissolve the paraffin mixture in the cold, The clearing of the sections takes place more readily and certainly if they are placed for a few minutes in absolute alcohol before they are placed in the clearing agent. 76 ELEMENTARY PHYSIOLOGY. [ VII. origin of two cells from one by oblique division. ce. A layer of cells much larger than the previous ones, with clear cell-substance (in the speci- men probably shrunken) and a distinct nucleus; they are also arranged in rows and have only a small amount of matrix between them. d. The large irregular cavities below this layer surrounded by bone matrix, and containing many osteoblasts, cells very similar in ap- pearance to white blood corpuscles, but larger, most of them are in contact with the bone matrix. In the centre of some of the cavities may also be seen a blood-vessel with surrounding connective tissue. e. Osteoclasts may be seen here and there in contact with the bone matrix; they are large multinuclear cells, and where they touch the bone are often striated at right angles to the surface. 2. Imbed a piece of the shaft of any long bone’ of a foetal mammal that has been preserved in picric or chromic acid, any remaining salts in the bone may be extracted by placing it for a day in spirit containing ‘5 p.c. nitric acid. Cut transverse 1 Instead of these, sections may be made of the lower jaw of a foetal mammal; the developing sub-maxillary bone shews admirably the osteoblasts and osteoclasts but the section is confusing to beginners owing to the number of other tissues present. VII] BONE, OSSIFICATION, TEETH. v7 sections and stain them with picrocarmine, and mount in glycerine or Canada balsam. Observe a. The periosteum consisting externally of coarse connective tissue, internally of fine connective tissue with numerous cells. b. The trabecule of the bone matrix, exter- nally these give off numerous small projec- tions into the inner layer of the periosteum. c. The osteoblasts forming a layer on the outer surface of the bone matrix; some will also be seen on the trabecule throughout the section; note the fine connective tissue and the blood vessels in the trabecular spaces. Many of the osteoblasts especially in the ex- ternal layer are elongated and their smaller ends appear to run into the matrix. d. Some osteoclasts as in § 1, e. may be seen. C. Structure or TEETH. 1. Examine under a low power longitudinal sec- tions of teeth prepared in the same manner as bone in B, Observe the dentine surrounding the pulp cavity, the cement or crusta petrosa covering the dentine of the fangs, and the enamel covering the dentine of the crown. Note the general arrangement of the dentinal tubules. 2, Examine under a high power the same section, and study in detail 78 ELEMENTARY PHYSIOLOGY, (vi. The Dentine. a. In the matrix, apparently homogeneous, are numerous dentinal tubules which run in a wavy course from the pulp-cavity outwards. These, dividing as they go, and giving off many anastomosing lateral branches, finally end either in loops or in small irregular cavities, the so-called interglobular spaces, on the surface of the dentine. In some places the dentinal tubules are cut transversely. Here the central dark spot in- dicates the space formerly occupied by the dentinal fibre, the ring round this is the dentinal sheath. The Cement or Crusta Petrosa. a. B. This differs little from bone, but Haversian canals are generally absent. The canaliculi of the lacune, next to the in- ter-globular spaces, open into these, thus bringing the dentinal fibres into connection with the bone-corpuscles. Where the cement is massive, wavy ‘contour’ lines may be seen, indicating the successive deposits. The Enamel. The striated enamel fibres or prisms are arranged in sets, perpendicular to the sur- face of the dentine. In mounted specimens the indications of the separate fibres are generally not well marked, but are visible at VIVIL.] BONE, OSSIFICATION, TEETH. . 79 places. The line of junction of the enamel and dentine is generally very conspicuous, in consequence of a change of level in the specimen, the dentine having been ground thinner than the harder enamel. The enamel is frequently split or cracked, 3. Examine prepared transverse sections of Teeth, taken at different heights, and compare with the above. LESSON VIII. STRUCTURE OF CONTRACTILE TISSUES. 1. Cut off the head of a frog which has just been killed, remove carefully the lower jaw and upon the mucous membrane of the roof of the mouth pour a little osmic acid 1 p.c. (before doing this a small piece of the membrane may be cut out to serve for § 2). Cover up the preparation and leave it for about half an hour’ then scrape a small area of the mucous membrane and tease out the flakes so obtained in water or in dilute glycerine, putting a small piece of paper under- neath the cover-slip to avoid pressure. Examine under a high power. Groups of ciliated cells intermingled with mucous (goblet) cells will be | seen; neglect the latter, note in the former a. The shape of the cell varies, it often branches irregularly at its attached end, 1 Or the mucous membrane after treatment with osmic acid as above may be gently washed with water to get rid of the fumes of the acid, removed with forceps and scissors, pinned out with hedgehog quills and placed in osmic acid 1 p.c. for 1 to 24 hours as convenient. The oesophagus and the mucous membrane of the floor of the mouth which also have cilia may be similarly treated. VIL] STRUCTURE OF CONTRACTILE TISSUES. 81 b, The free surface is densely studded with cilia. The cilia seen in profile appear to form a row, but when a cell is seen in face, they are foreshortened into mere dots and are seen to be spread over the whole surface. The nucleus is not very obvious but may, with its nucleolus, be made out; the cell- substance is somewhat granular but has a non-granular, hyaline border just below the cilia. Cut out a small piece of the same membrane in a recently killed frog, place it on a slide, from a portion of it scrape off the epithelium and tease out the scrapings in normal salt solution; with this mount the unteased portion of membrane putting a fragment of paper underneath the cover-slip. Note a. In the unteased portion the shimmering ap- pearance caused by the movements of the cilia. The movements of the cilia in the isolated cells or clumps of cells. Observe carefully the cilia which are moving slowly; it may be seen that the down-stroke (contraction) takes place more quickly than the return (relaxa- tion); there is no perceptible pause between the two movements. The results of ciliary action. Granules and blood-corpuscles are driven along ;. detached 6 82 ELEMENTARY PHYSIOLOGY. [vut. cells may also be seen carried about by the action of their own cilia. d. The contracted, almost globular form of the cells which have been set free. . Tear off the smallest possible strip of a frog’s muscle, which has been kept exterided and preserved in alcohol, and tease it out as finely as possible in dilute glycerine. Note a. The varying size of the fibres. b. The striation of the fibres: alternate dim and bright cross-bands passing through the whule thickness of the fibre. c. The breaking up of the fibre into fibrille, the latter also striated. Try to obtain as fine fibrillee as possible. The fibres sometimes split transversely into discs; this is usually the case when the muscle has been preserved In picric acid; the surfaces of the discs ap- pear dotted. Cut through the skin of the front of the thigh of the frog used in § 2, note the sartorius muscle running somewhat obliquely across the thigh from top to bottom, seize with forceps the con- nective tissue lying along its inner border in the upper part of the thigh and tear this back to the knee, remove similarly the connective tissue at its outer border, thus the tissue covering the muscle will also be removed. Take up with fine forceps a few fibres at one end of the muscle, and Vul.] STRUCTURE OF CONTRACTILE TISSUES. 83 gently pull them out to the opposite end. Lay them on a glass slide, and separate them, a little in the centre, place a bristle across them and press gently: remove the bristle, put on a cover- slip and let a drop of normal saline solution run! underneath it (cp. method, Lesson V. A. § 2 a, ¢). Where the bristle was pressed on a fibre the muscular substance will be seen in many cases to be broken across leaving its delicate trans- parent sheath, the sarcolemma, stretching across the gap; an indication of the sarcolemma as a fine line bulging out from the muscle substance may also be seen where a fibre is bent. Irrigate with acetic acid ‘5 p.c. and observe a, The fibres at first become cloudy and semi- opaque. b. The precipitate causing this cloudiness is after a while dissolved, and the fibres be- come more transparent than normally. c. The nuclei of the fibres come into view scat- tered throughout the muscle substance ; they are elongated in the direction of the fibre: lines of granules proceeding from their ends may usually be seen. Cut through the skin in the mid ventral line in a small frog, lift up the flap of skin on one side over the middle of the sternum, a thin band of musele will be seen running from the skin down- wards towards the lower part of the sternum. 6—2 84 ELEMENTARY PHYSIOLOGY. [VIL Cut through the skin above and below the muscle, and tear away the connective tissue around it, then, keeping it stretched, pour a little 1 p.c. osmic acid over it. In a minute or so the muscle will be fixed; with fine forceps tear away carefully from the muscle any connective tissue that can be seen on its surface and cut it out being care- ful to cut the upper end as close as possible to the skin ; place it in osmic acid for a few minutes, wash with water, mount it in either dilute glycerine or after treatment with alcohol etc. in Canada balsam, keeping that surface upper- most which was next the body. (This specimen should be preserved for examination later, Lesson x. C. § 1.) Observe at the upper end of the muscle the endings of the muscular fibres, these will prob- ably be covered by a good deal of connective tissue, but the rounded or conical ends of the muscle-substance of the fibres and the continua- tion of the sarcolemma into the connective tissue (tendon) will be seen. Remove with as little injury as possible one of the thin muscles from the leg of Hydrophilus’, tease it out a little without adding fluid and mount asin§4. The fibres will for a brief period be seen in a normal condition. In many, the alternate dim and bright striz will be very dis- 1 If not obtainable, take the large common water-beetle (Dytiscus marginalis), or failing that a cockroach or a fly. VIII.] STRUCTURE OF CONTRACTILE TISSUES, 85 tinct; in others the transverse markings will be more or less obscured by an appearance of longi- tudinal fibrillation. Occasionally fibres are met with, having only a confused granular aspect. If the preparation has been successfully made, waves of contraction may, at times, be seen to travel along the fibres. Observe in the fibres with distinct transverse striation, that, a, The dim band has a faint longitudinal stria- tion, as if made up of small rods. b. The bright band shews a dotted line running across its centre, thus dividing it into two parts. If the preparation be good, and a high magnifying power be used, this may be seen to consist of two rows of dots. c, The usually round nuclei, imbedded in a granular mass of protoplasm; the whole forms a band or rod running a variable distance in the substance of the fibre (cp. § 5c), Imbed a small piece of mammalian muscle, hardened in chromic acid ‘5 p.c., and cut trans- verse sections, stain with heematoxylin, and mount in glycerine. Observe a. The connective tissue (perimysium) around the bundles of fibres and around the whole muscle, from this runs a small amount of connective tissue (endomysium) between the muscle fibres, 86" bj ELEMENTARY PHYSIOLOGY. [vit The cut ends of the fibres are finely dotted corresponding with the fibrille; sometimes the cut ends are divided into larger polygonal areas (areas of Cohnheim), this appearance is produced by the hardening agent. The nuclei lying just beneath the sarcolemma, few or none being imbedded in the muscular substance of the fibres (cp. § 5 c), In some part of the section, the fibre-frag- ments may be turned over and lie’ length- ways; in these the striation and tendency to break into fibrille may be noticed as in frog’s muscle. Remove the peritoneum from a part of the intestine of a recently killed mammal, and tear ‘off with fine forceps a strip as thin as possible of the longitudinal muscular coat. Place this in potassium bichromate ‘1 p.c. or in 30 p.c. alcohol for two days, wash with water, stain with picro- carmine, wash and tease out in dilute glycerine. Note a The isolated fibre-cells. These are long, and, wher not broken off in the preparation, fusi- form masses of hyaline or faintly granular protoplasm. They appear very much thinner when seen in one direction than in another; that is to say, they are much flattened. In the middle of each fibre an elongated nucleus will be seen stained deeply. In the neigh- bourhood of the nucleus, especially at its VIII] 10. 11. STRUCTURE OF CONTRACTILE TISSUES, 87 ends, granules are more abundant in the protoplasm. b. The arrangement of the fibre-cells in bands - and sheets: the fibres may frequently be seen projecting like a palisade from the torn end _ of a-band.. Take a small strip of unstriated muscle as in § 9 and tease it out in salt solution. The sheets and bands of the tissue are fairly transparent, the individual fibre cells are scarcely visible. Add acetic acid, *1 to ‘5 p.c. the cells become for 2 short time somewhat more obvious, and the nuclei very distinct, often showing one or two nucleoli. Make longitudinal vertical sections of the mus- cular coats of the cardiac end of a cat’s or dog’s stomach. The tissue should have been pinned out, placed in Miiller’s fluid for two to four weeks and then treated with alcohol, Stain with carmine or hematoxylin and mount’, Observe The cells are arranged in bundles, probably this will be distinct in the inner (circular) coat only, the outer (longitudinal) coat being cut parallel to the direction of its bundles, Note the nuclei of the cells in the inner coat; these will be seen in some only of the cells and will appear as largish deeply stained cen- 1 When it is indifferent whether a tissue be mounted in glycerine or in Canada Balsam, neither is mentioned; if balsam is used, of course the tissue must be treated with creosote, ete. (ep. p. 73). 88 ELEMENTARY PHYSIOLOGY. [vut. tral spots. (This section should be preserved for examination later, Less, xvi. A. § 3.) The reduction of nitrate of silver by the cement substance between the muscle cells will be seen later, Less. x11. C. § 4. 12. Tease out thoroughly a smalf portion of cardiac muscle preserved in potassium bichromate’. Note that a. The striation is less distinct than in skeletal muscle. 6. No sarcolemma is present. c. At short intervals along the fibres are not very distinct transverse bands of hyaline cement substance, this joins together the miuscle cells of which the fibres are composed. At about the centre of each muscle cell is a nucleus. (The nuclei will become more evident if the tissue be stained with alum- hzmatoxylin or picrocarmine.) d. The isolated muscle cells may frequently be seen to have a short obliquely running pro- cess ; in the fibres the processes of the cells are joined to other similar processes by a little cement substance, forming thus the anastomoses of the fibres. 1 Good preparations can also be obtained by the following method (Ranvier). Thin sections in the direction of the fibres are cut from a fresh heart, placed in chromic acid -02 p.c. for one day, then washed ’ well with water, placed in picrocarmine for one or more days, washed and mounted in acid glycerine. viii] STRUCTURE OF CONTRACTILE TISSUES. 89 DEMONSTRATIONS. 1. The structure of a living muscular fibre of an Insect as seen under a high power. 2. The appearances of muscular fibre under polarised light. LESSON IX. PROPERTIES OF CONTRACTILE TISSUE. 1, Constant Current. Destroy the brain and spinal cord of a frog and make a longitudinal incision through the skin of the back of the thigh. With fine-pointed scissors cut through the connective tissue between the large semi-membranosus muscle and the small biceps: the former lies on the inside of the thigh, the latter is seen along the outer and lower border of the semi-membranosus. The sciatic nerve and the femoral artery will come into view; at about the upper third of the thigh, the artery will be seen to give off two small transverse branches running, in the position of the frog, over the nerve; from this point down to the knee, isolate the nerve by tearing away with a “seeker” the connective tissue around it; if the tissue is resistant it is better to lift up the nerve with a seeker and to cut through the connective tissue with fine scissors. Be careful not to pinch the nerve with the forceps, nor to put more strain upon it than is absolutely necessary, and further not to puncture the artery. Ix.] PROPERTIES OF CONTRACTILE TISSUE. 91 Take a pair of electrodes which have their pla- tinum points exposed on one side only, and connect them with two Daniell’s cells arranged in series (i.e. with the copper of one joined by means of a wire with the zinc of the other) in- terposing a key’ to short circuit the current. Instead of Daniell’s cells any other cells of cor- responding strength may be used. The key being shut’, place the electrodes under the nerve so that the nerve, and the nerve only, touches the platinum points, then open the key, so as to allow the current to pass into the nerve, and after a few seconds shut it again. A move- ment in the leg caused by contractions in the muscles to which the nerve is distributed, will be seen when the key is either opened or shut, or on both occasions. Save in exceptional cases, there is no movement during the passage of the current through the nerve, but only when the cur- rent is thrown into or shut of from tt (the current being supposed to be tolerably constant). “Do not repeat this experiment more than once or twice on the same nerve, lest it be too much ex- hausted for the succeeding observations. 1 Cp. Appendix for a description of the instruments and for the methods of using them, ' 2 For convenience of description thé terms ‘shut’ and ‘open’ which apply to a Du Bois Reymond key are used, but if the student remembers that to shut the key is to let the current pass through it, and to open the key is to break the current passing through it, he will have no difficulty in using a Morse or other key in the place of that of Du Bois Reymond. 92 2. ELEMENTARY ‘PHYSIOLOGY. [rx. Single Induction Shocks. Now connect a Daniell’s cell with the top screws of the primary coil interposing a key to break the current, connect the electrodes with the secondary coil of a Du Bois Reymond’s in- duction machine’, place the marker of the se- condary coil at 10 on the scale, and, the frog’s leg being at rest, shut and open the key several times. At each shutting and opening of the key, ie. at each momentary induced current, a single sharp movement of the leg will take place. The ‘single induction shock’ causes a single contraction or spasm of the muscles of the leg. If the nerve slips off the electrodes during the movements, be careful not to pinch it in replacing it, If good contractions are not obtained with the secondary coil at 10, push it up gradually over the primary coil, one division at a time. Shut and open the key as rapidly as you can, ‘for afew seconds. The contraction of the muscle taking longer time to develop than the induced current, each contraction will take place before the preceding has disappeared, and the leg will remain rigidly contracted, or nearly so, as long as the breaking and making of the primary cur- rent is kept up. In other words, tetanus is pro- duced. This is however more easily brought about as follows. 1 Cp. Appendix. Ix.] 4, PROPERTIES OF CONTRACTILE TISSUE. 93 Interrupted Current. Introduce into the secondary circuit a key to short circuit the current, and remove the wires of the Daniell’s cell from the screws at the top of the primary coil to those at the base’. The machine should work in such a way that im- mediately the key in the primary circuit is put down the hammer is at once thrown into oscilla- tions (as indicated by its noise). The key in the secondary circuit being open, shut the primary key. Immediately that the sound of the hammer is heard, the leg is thrust out straight by the tetanic contractions of the muscle, and remains so as long as the current continues to be thus made and broken. Open the primary key; at once the limb becomes flaccid and quiet. The ‘interrupted current’ should not be applied for more than a few seconds, Chemical Stimulation. Expose the heart (cp. Less. 11. A. § 1), and bleed the frog by cutting across the aorta. Cut through the tissue above the sciatic nerve up to a quarter of an inch beyond the end of the urostyle, and there cut it across, with scissors and seeker isolate the nerve up to the cut. Let the extreme end of the nerve dip into a saturated solution of sodium chloride; watch the leg. In a variable time twitchings of the toes will be seen, and after a while these will increase in 1 Cp. Appendix. 94 ELEMENTARY PHYSIOLOGY. [rx. vigour and will extend over the limb ‘until it becomes almost as rigid as in § 4. Mechanical Stimulation. Cut off the part of the nerve which has been dipping in the salt solution; the chemical sti- mulus being thus removed, the leg will become flaccid again. Pinch the remaining nerve sharply with a pair of forceps several times. At each pinch the muscles of the leg will contract. The pinching kills or injures the part of the nerve pinched; hence it is necessary to begin at the cut end, and work down towards the muscle. The gastrocnemius muscle of this lex may be used for § 13, b. Curve of Single Muscular Contraction. Place the frog on a glass or porcelain plate; on the left side cut through the skin at the back of the thigh and carry the skin cut half way up the back. Lift up the urostyle and cut through the muscles attached to its lower half, then holding it well up cut through the muscles at- tached to its upper half: it is as well to avoid cutting the 10th nerve which runs along the under surface of the muscles. Note the 7th, 8th, 9th and 10th nerves which unite to form the sciatic; with strong scissors cut away the uro- style and cut through the vertebral column above the 7th nerve: lift up the lower piece of the vertebral column and. without injury to the 1x.] PROPERTIES OF CONTRACTILE TISSUE. 95 nerves of the left side cut away the rest of the tissue attached to it. Then lifting up the piece of bone without stretching the nerves, cut through with scissors the tissue attached to them, follow the nerves on to the sciatic and cut: _ through the tissue above and attached to the sciatic Including the branches given off by the nerve ; isolate thus the sciatic down to the knee. Letting the nerve rest on the muscles of the thigh, cut through the skin of the upper part of the thigh; seize the flap of skin with stout forceps, and tear it away from the leg as far as the foot. Placing the nerve on the muscle below the knee cut away the muscles attached to the lower half of the femur, and with a strong pair of scissors cut through the middle of the femur, Cut through the lower (Achilles) tendon of the gastrocnemius muscle close to its attach- ment to the foot, and seizing the tendon with for- ceps tear the gastrocnemius away from the other muscles up to its attachment with the femur; cut through the tibia and fibula just below the femur. The preparation so obtained is called a nerve-muscle preparation’. Now pass a hook through the Achilles tendon, clamp the femur, and taking up the preparation 1 When two nerve-muscle preparations are to be obtained from one frog, the lower part of the vertebral column should be bisected longitudinally. It is perhaps somewhat better not to bleed the frog before commencing the dissection, but in this case considerable skill and patience are required to avoid cutting small vessels and so covering the nerve with blood. . ; 96 ELEMENTARY PHYSIOLOGY. [rx. by the clamp and by the piece of vertebral column, arrange the preparation in the moist chamber as in Fig. 2 Appendix. Load the lever with 15 or 20 grammes, and bring the lever to mark on the revolving cylinder. Connect the electrodes with the induction ma- chine arranged for single induction shocks as in § 2. Set the cylinder rotating at its swiftest speed and with the marker of the secondary coil at 10 take a tracing of the contraction caused by a break in- duction shock (the make shock is prevented from reaching the nerve by shutting the key in the secondary coil, arranged for short circuiting, before that in the primary is shut; on opening in the reverse order the breaking shock passes into the nerve). Observe the rapid but steady rise to a maximum, passing almost immediately into a similar but rather less rapid fall; the later stages of the fall are distinctly less rapid than the earlier ones. Take another tracing over the previous one with the secondary coil at 5; the height of the con- traction is somewhat greater. Place the secondary coil at 25, the key in the secondary circuit being open, shut that in the primary circuit and after a short interval open it; if no contraction follows shift the secondary coil nearer and nearer the primary, stimulating at each shift. Since the induction shock caused by breaking 1X.] 10. PROPERTIES OF CONTRACTILE TISSUE, 97 the current is stronger than that caused by making it, a contraction will be obtained earlier on opening than on shutting the primary key. Tetanus. Now arrange the induction machine for an inter- rupted current (§ 4), the secondary coil being at 15 or 20 to begin with. With the cylinder moving slowly, take a tracing of a tetanic contraction of a muscle, stimulating for not more than three or four seconds. Observe the rise to a maximum at first rapid but afterwards more and more slow, the mainte- nance of the maximum during the application of the current, and the fall, at first rapid but after- wards more slow, after the removal of the current. Watch during the tetanus the muscle itself as well as the curve. Connect one wire from the Daniell’s cell with one of the top screws of the primary coil, and the other to the binding screw of the oscillating rod (cp. Appendix), connect a wire with the other top screw of the primary coil and bending the wire place its opposite end in the mercury cup. A key for short-circuiting should be kept in the secondary coil. Allow the full length of the rod to oscillate, open the key in the secon- dary circuit and take a tracing of the muscular contractions; then take successive tracings with the rod shorter and shorter. Observe the gradual fusing of a series of single contraction curves into the curve of tetanus. 7 98 11. 13. ELEMENTARY PHYSIOLOGY. [rx. Load the muscle with 50 grammes, allowing the lever to write on the stationary cylinder. Note the elasticity of the muscle. The weight when applied stretches the muscle to a certain extent, but when raised so as not to drag on the muscle, the latter returns, at once to almost, and even- tually to quite, its former length. Stimulate with an interrupted current (coil say at 20). Note the amount of contraction. Repeat this with 100, and then with 200 grammes, using in each case the same strength of current for the same time, moving the cylinder with the hand so as to expose a fresh black surface for each observation. Observe that with medium weights the work done (the product of the weight into the height) is greater than with very light or very heavy weights. Observe also that the muscle gradually becomes exhausted, ie. the contractions caused by the same stimulus gradually become less and less. . Probably the preparation will be nearly exhaust- ed; take as in § 7 a tracing of a single muscular contraction, and note the difference in the form of the curve; the rise is less rapid, the fall more gradual, the muscle may take some time to regain its original length, Reaction of Muscle. a. Remove the muscle from the moist chamber, place it on a clean glass slide, and apply the electrodes, connected with the induction IXx.] PROPERTIES OF CONTRACTILE TISSUE, 99 machine arranged for an interrupted current, directly to the muscle, Observe that the current applied directly to the muscle produces the same effect as when applied indirectly through the nerve. To completely eliminate the effect of stimu- lating the nerves urari should be given to a frog the brain of which has been destroyed. Tetanize the muscle with currents of increas- ing strength until it is completely exhausted, pushing the secondary coil right up over the primary. Cut the muscle across in half, with a quite clean scalpel, and press on one section a piece of faintly blue, on the other a piece of neutral litmus paper. Both will be turned red where they come in contact with the muscle, b, Dissect out the gastrocnemius from the leg used in §§ 1—6, and place it for five minutes in normal saline solution heated to 50° C. Observe that it has become contracted and opaque, Le. rigor mortis has set in. Cut the muscle across, and test with litmus paper asina. The muscle will be distinctly acid, even more so than the tetanized muscle in a. ce. Take a fresh, living gastrocnemius from a frog just killed, cut it across as in a and 6b, and test the sections with neutral litmus paper. The muscle will be found neutral or faintly alkaline, 7—2 100 ELEMENTARY PHYSIOLOGY. [rx. 14. Myosin. Strip off the muscles from a rabbit or other animal and chop them up as finely as possible, place them in a large jar filled with water and stir; in about a quarter of an hour place a piece of muslin over the top of the jar and pour off the water ; fill up the jar again with water and let it stand for an hour, then pour off the water and refill the jar as before. When this has been repeated once or twice the greater part of the substances soluble in water will have been re- moved ; itis best however to let the muscle stay in water for a day adding a little thymol or -salicylic acid to prevent putrefaction. The filtrate from a small quantity of muscle which has been in an equal bulk of water for an hour should give no proteid reaction if the muscle has previously been thoroughly washed. Collect the muscle on linen, squeeze out the water, grind it up with clean sand and add 5 times tts bulk of 10 p.c. ammonium or sodium chloride, and stirring occasionally place it aside for an hour or so (naturally if it is left longer more myosin will be obtained). Filter through muslin,* through linen, and then through coarse filter paper. A somewhat viscid finid is ob- tained; pour this into a tall vessel containing about a litre of water and observe the pre- cipitate of myosin which takes place (or the 1 The residue may be again treated with 10 p.c. ammonium chloride. IX.] PROPERTIES OF CONTRACTILE TISSUE, 101 fluid as it filters may be allowed to drop into water). After a short time decant or draw off with a pipette as much fluid as possible, shake up the rest and pour about 5c.c. with the myosin suspended in it into each of three test-tubes. a. Add drop by drop a strong (e.g. 20 p.c.) salt solution, the precipitate soon dissolves ; then place it in a water bath at about 50°C. with a thermometer and heat; at about 57°C. the fluid becomes milky (or a precipitate is formed) owing to the coagulation of the myosin, b. Add powdered salt, the precipitate soon dissolves, but when the fluid is saturated with salt is reprecipitated (the precipitate may bein the form of fibrous-looking clumps). ce. Dissolve the suspended precipitate in salt solution, and test for proteids, e.g. with Xan- thoproteic reaction (cp. Less. 111. § 16 (a)). Myosin belongs to the class of globulins; it will have been seen that it is insoluble in water and in saturated neutral salt solutions, soluble in not too dilute solution of neutral salts, its solution coagulating on boiling (cp. also Less. ur. §§ 11, 13, 17, 18). Syntonin or Acid Albumin. ‘ Treat a portion of muscle washed as in § 14 with ten to twenty times its bulk of HCl ‘1 p.c. and place in the warm chamber at about 40°C, 102 16. ELEMENTARY PHYSIOLOGY. [rx. frequently shaking. After 3 or 4 hours most of the myosin of the muscle will have been con- verted into syntonin or acid-albumin, and dis- solved. Syntonin may be prepared in a similar way from white of egg or from serum. Filter and carefully neutralize the filtrate with a weak solution of sodium carbonate (1 c.c. of HCl ‘Lp.c. requires about 1°5 c.c. of Na, CO, ‘I p.c. to neutralize it), a copious bulky precipitate of syntonin takes place; if too much alkaline salt is added the syntonin will be converted into alkali albuminate and redissolved (cp. § 16). Filter and wash the precipitate once on the filter with water, then break through the filter paper and wash the precipitate into a beaker with water. The syntonin is thus obtained suspended in water. Place 2 c.c. in each of 2 test-tubes, a. Add a little HCl ‘1 p.c, the precipitate at once dissolves and is not precipitated on boiling; cool under a tap, and test for pro- teids e.g. with acetic acid and potassium ferrocyanide (cp. Less. 111. § 16). 6. Boil, cool under a tap, and add a little HCl ‘1 p.c.; the precipitate suspended in the water has been coagulated by boiling and is no longer soluble in dilute acids. Alkali Albuminate, Treat a little washed muscle as in § 15 but with NaHO ‘1 p.c. instead-of with HCl On neutral- izing a precipitate of alkali albuminate will be 1x.} PROPERTIES OF CONTRACTILE TISSUE. 103 obtained like that of syntonin; if too much acid is added the precipitate will be converted into acid-albumin and re-dissolved. Take in separate test-tubes a, little of the precipitate suspended in water. a. Add a little NaHO ‘1 p.c. the precipitate at once dissolves and is not precipitated by boiling ; with the proteid tests a reaction is obtained. b. Boil, cool under a tap and add a little NaHO ‘1 p.c., the precipitate suspended in water has been coagulated by boiling and is no longer soluble in dilute alkalies. c. Adda little NaHO ‘1 p.c. and a little sodium phosphate, with a drop of litmus solution; add drop by drop dilute HCl: on neutralizing no precipitate takes place, add a little more acid a precipitate is obtained, if still more is added the precipitate is re-dissolved. It will have been seen that proteids in solution (except peptone) on warming with dilute acids or alkalies give rise to acid albumin or alkali albuminate respectively, these substances do not coagulate on boiling and so differ from albumin and globulin, and are insoluble in water and so differ from peptone (cp. Less. xvi. C. § 5). DEMONSTRATIONS. The electric currents of resting muscle removed from the body (demarcation currents) 104 ELEMENTARY PHYSIOLOGY, [ax The negative variation of the currents of the muscle during a contraction (action currents) The rheoscopic frog Action of heat and cold on the single muscular contraction Action of veratrin on the single muscular con- traction Curve of elasticity of muscle Measurement of latent period of contraction of muscle by means of pendulum myograph, LESSON X. STRUCTURE OF NERVOUS TISSUES. A. SprnaL NERVEs. 1. Cut off about one-third of an inch of a small perfectly fresh nerve (e. g. a branch of the sciatic of a frog), and place it on a glass slide without any fluid. Fixing one end by pressing on it with the blunt end of a scalpel, pass a needle through the other end in the direction of the nerve fibres, and so spread it out into the shape of a fan; add a drop of normal saline solution, and cover with the cover-slip. Observe a. The medullated nerve-fibres of variable size. b. In each fibre the double contour, due to the medullary sheath or white substance of Schwann. c. The primitive sheath; this is seen with difficulty except at points where the me- dullary sheath has been displaced in mount- ing. d. The connective tissue (endoneurium) running amongst and round the nerve-fibres. 106 ELEMENTARY PHYSIOLOGY. (x. e. Drops and fragments of the medullary sheath, extending from the cut ends of the fibres and shewing a double contour. Some non-medullated nerve-fibres will also be seen (cp. B). Cut off a similar piece of nerve and treat it in the same way, but instead of adding a normal solution, add a drop of chloroform, and irrigate with chloroform as evaporation goes on. In the middle of the nerve-fibre the pale granu- lar axis cylinder will be seen running through the swollen and partially dissolved medullary sheath. Take another piece of fresh nerve, and place it in a small quantity of osmic acid 1 pe. for ten minutes, covering it up to prevent evaporation. Remove it to a slide, and separate a small bundle of fibres, putting the rest back in osmic acid;- isolate the fibres as much as possible, add a drop of water, and put on a cover-slip. If it is desired to preserve the specimen the nerve should be washed with water before teasing and mounted in dilute glycerine. Select a nerve fibre which is isolated for a con- siderable part of its length, and observe a. The medullary sheath, stained black with osmic acid. b. The nodes of Ranvier. These will be seen as short but distinct breaks in the me- dullary sheath ; with Zeiss obj. D. oc. 2 they x.]x.] STRUCTURE OF NERVOUS TISSUES, 107 occur usually at intervals of about twice the diameter of the field, varying considerably, however, in different nerves. The axis cy- linder may be seen. continuing across the nodes. c. The nuclei of the primitive sheath; one to each internodal nerve portion situated about halfway between two nodes, and appearing as a small transparent elongated body pro- jecting usually into the medulla. The nuclei of the primitive sheath may be stained by placing a piece of the nerve, after treatment with osmic acid, in Frey’s carmine for two or more days. In such a preparation, the cells of the fine connective tissue around the nerve fibres will also be well seen. Stain with carmine a short piece of a rather thick nerve which has been preserved in ammo- nium bichromate 2 p.c. (cp. Less. 1. F. § 4 and footnote p. 69). Imbed, and cut thin transverse sections, treat these with creosote and turpentine (cp. p. 75 footnote) and mount in Canada balsam. Observe a. The cut ends of the nerve-fibres varying in. diameter, and in each b. The section of the stained axis cylinder sur- rounded by c. A transparent ring indicating the former position of the medullary sheath, which has been dissolved or made transparent in the 108. ELEMENTARY PHYSIOLOGY. fx. process of mounting. If the nerve has been kept long in ammonium bichromate the medulla will shew an indistinct network stained yellow. d. The primitive sheath as a limiting circle. e. The arrangement of the fibres in bundles. The perineurium around each bundle con- tinuous with the endoneurium between the fibres. B. SYMPATHETIC NERVES. 1. Cut out from the fresh spleen of a large animal (e.g. ox) a small piece of one of the large sympa- thetic nerves running alongside the blood-vessels. Remove the connective-tissue sheath, and tease out the nerve carefully in normal saline solution. Note a. The scanty medullated nerve fibres, b. The non-medullated nerve fibres consti- tuting the bulk of the nerves: they are fairly transparent, and often fibrillated, some give off fibrillae. Add acetic acid to bring out more distinctly the small elongated nuclei which lie on them at short intervals. Take a small piece of the sympathetic nerve from the neck of a freshly-killed rabbit, place it in osmic acid 1 p.c. for five or ten minutes, wash and stain with strong Frey’s carmine or with picrocarmine. Tease out in dilute glycerine. x.] STRUCTURE OF NERVOUS TISSUES. 109 Numerous small medullated fibres will be seen mingled with the non-medullated fibres. Compare the size of the medullated fibres here and in the sciatic nerve (A, § 1). C. PERIPHERAL CoursE oF NERVES. 1. In the specimen prepared Less. VIII. § 6: observe a. Under a low power, the nerve running across the lower part of the muscle and sending off fibres or bundles of fibres at intervals and so spreading out over it. b. Under a high power, that where the lateral bundles (especially the smaller ones) are given off, one or more of the nerve fibres divide into two fibres, the division taking place at a node. Trace a small bundle of nerve fibres, the nodes are very close together. Each nerve fibre apparently ends abruptly over a muscle fibre; in this specimen it can only be traced as far as the blackened me- dulla extends. 2. Make a gold chloride preparation of the cornea’ 1 The mesentery of the frog may be taken instead of the cornea, but it is more difficult to obtain a good preparation. The mesentery of the lower coil of the small intestine is pinned out with the intestine, and placed in gold chloride -5 p.c. for about half-an-hour, it is then well washed, the intestine cut away, and the mesentery exposed to light in acidulated water. The larger nerve bundles, containing a few medullated fibres, accompany the larger arteries and form coarse and fine plexuses of non-medullated fibres as in the cornea, but there are many more nuclei, even very small fibres having nuclei on their course ; varicose fibrils like those of the cornea will probably not be seen. 110 ELEMENTARY PHYSIOLOGY. [x. of a frog as in Less. v. B. § 2 and mount in glycerine a sector of it, including its junction with the sclerotic. Note a. The small separate bundles of nerve fibres entering the cornea at its periphery; the medullated fibres on account of their me- dulla are more deeply stained than the non- medullated. b. Trace as far as possible the course of one of the nerve bundles; the medulla soon dis- appears, the fibres, still showing nuclei at intervals, join with the fibres from other bundles to form a coarse plexus; from this proceeds a plexus of smaller bands which have few nuclei; finally from this plexus run very fine varicose non-nucleated nerve-fibrils in straight lines across the cornea (these fine fibrils may also be seen forming part of the finer bands of the plexus). D. Sprnat GANGLIA. 1. Take a mammal’s spinal ganglion (e.g. a dorsal spinal ganglion of a cat or dog) which has been preserved in ammonium bichromate 2 p.c. about three weeks, and subsequently in spirit. Prepare sections passing through the trunk of the nerve, the ganglion, and the anterior and posterior roots of the nerve. Stain with hematoxylin or carmine, clear (cp. p. 75) and mount in Canada balsam. x. x] STRUCTURE OF NERVOUS TISSUES. 111 Observe with a low power that a. The fibres of the anterior root mix with those of the posterior root below the gan- glion, Most of the ganglion cells lie outside the fibres of the posterior root; some lie in rows between the fibres. Observe with a high power a, In the ganglion cells the rather granular cell-substance, containing a large spherical nucleus which often has one or more distinct nucleoli, The many nuclei of the capsule of each ganglion cell, The stained axis cylinders of the nerve fibres. (The medulla may show an irregular network of substance hardened by the ammonium bichromate.) The nerve fibres coiled amongst the cells, and fibres running more or less transversely from the lateral mass of cells to the central bundle of nerve fibres. Destroy the brain and spinal cord of a frog and cut off its head. Remove the lower jaw and divide the skull in two down the middle line. Tear away the mucous membrane covering the roof of the mouth; the inner and lower part of the eye will be laid bare. Just behind the eye is a depression ; from this two or three small fila- ments, the branches of the fifth nerve, will be 112 ELEMENTARY PHYSIOLOGY. [= seen issuing. Cut through the bone from the middle line to the place where the nerves issue, turn the piece of bone forward, and gently scrape out the brain. A filament (the fifth nerve) will then be seen going towards the branches spoken of above. Between the two is a slight enlargement, the Gasserian ganglion, surrounded by tough con- _ nective tissue and by connective tissue attached to the bone. Cut away this tissue and excise the ganglion. Tease it out a little in 25 p.c. osmic acid’, leave it-in the acid for about half-an-hour, then wash, and place it in strong Frey’s carmine for a day; wash, and tease out in glycerine, during the teasing examine from time to time under a low power and throw away the fragments which consist of connective tissue only. Instead of the Gasserian ganglion a spinal gan- glion from a frog or mouse may be taken. In the frog the spinal ganglia are surrounded by a chalky mass; when this is removed the ganglion can be seen as a greyish semi-transparent bulg- ing on the nerve, the 7th, 8th, 9th are easiest to obtain. Examine carefully the more isolated cells; here and there one will be seen with its cell substance prolonged into a process; from most of the cells the process will probably have been torn away in teasing. 1 Or it may be placed in potassium bichromate ‘2 p.c. for two or three days and then teased out. x.] STRUCTURE OF NERVOUS TISSUES. 113 [E. SYMPATHETIC GANGLIA. Place a sympathetic ganglion e.g. the superior cervical sympathetic ganglion of a rabbit in 1 p.c. osmic acid for a day. Wash it with water and leave it in water for some hours. Transfer to 50 p.c. spirit for an hour, then to strong spirit for a week. Cut longitudinal sections and observe a. The large number of nerve-cells of various sizes. b. Thesmall number of medullated nerves com- pared with the spinal ganglion (D § 2 a). c. The small size of nearly all the medullated nerves (cp. D § 2d), when these are in bundles they are usually at the periphery of the ganglion. d. The bundles of non-medullated nerve-fibres at the ends of the ganglion; the distinctness with which these are seen will of course vary in different sections. Dissection of Sympathetic Ganglia in the Frog. Lay open the abdomen of a recently killed frog, pick up the intestine and cut through the mesentery just above the kidneys; pulling up one kidney, cut through the peritoneum along its edge, and turn over the kidney to the opposite side of the body. Gently stretch the spinal nerves away from the spinal column, a row of small-nerve fibres will be seen running transversely. from the spinal nerves (one from each nerve) to the chain of pigmented, semi-transparent sympathetic ganglia which will be seen lying over the spinal column. L. 8 114 ELEMENTARY PHYSIOLOGY. [x. F. CELLS oF SPINAL CorRD. Cut transverse sections of the spinal cord of a large mammal (e. g. calf or ox) which has been hardened in ammonium bichromate 2 p. c. and subsequently treated with alcohol. Stain with carmine or dilute picrocarmine (it is better still to stain the small piece of cord before cutting sections by placing it in strong Frey’s carmine for several days); clear and mount in balsam. Note a. In the white substance the cut ends of the nerve-fibres differing very greatly in size (cp. with A § 4). The small amount of connective tissue (neu- roglia) between the nerve fibres; this is finely dotted (transverse sections of semi-elastic fibrils), and is more abundant near the grey substance. In the grey substance of the anterior cornu, the large multipolar nerve-cells each with a large nucleus and distinct nucleolus. Select a conspicuous cell and note that the processes run off in all directions; many of them will be seen to branch and these branches to branch again and so on (protoplasmic processes) ; from some of the cells a rather large process which does not branch may be seen to run outwards through the white substance (axis cylinder process). d. In the grey substance of the posterior cornu, the much smaller usually fusiform nerve-cells with branching processes at either end. x.] STRUCTURE OF NERVOUS TISSUES. 115 DEMONSTRATIONS. 1. Preparations to shew the method of ending of motor nerves in muscle. 2. Preparation to shew the nerve-cells of the plexus of Auerbach in the intestine. 3. Mounted transverse section of a nerve which has been treated with osmic acid. Nore to D § 3. Isolation of nerve cells. The ganglion may be placed in osmic acid 1 p.c. for half an hour, washed in water, placed in a mixture of equal parts of glycerine and strong acetic acid for three or four days, and then teased out. LESSON XI. GENERAL PROPERTIES OF NERVOUS TISSUE. AUTOMATIC ACTIONS. A, ReFLex ACTION. 1, Place on its belly a frog, which has been pre- viously deprived of its brain* by the demonstrator. Observe that its hind-limbs are drawn up under the body; but that it differs from the normal frog in the following respects. a. Its head is depressed, instead of being erect. b. Its fore-limbs are spread out, or flexed, instead of being held nearly vertical; thus the angle which the body makes with the table is diminished. 1 This is done by cutting across the spinal cord just below the skull and thoroughly destroying the brain with a seeker; in order to ensure the complete removal of the medulla oblongata, it is advisable to destroy the parts a short distance down the spinal canal. The frog should be left in a moist place for a day. Instead of this the frog may be chloroformed the skull cut open and the brain- re- moved; or the upper jaw with the anterior part of the skull may be cut off with strong scissors and the medulla removed with forceps, with this method the reflex action is often weak owing to the considerable loss of blood. XI] GENERAL PROPERTIES OF NERVOUS TISSUE. 117 c. There are no respiratory movements, either of the nostrils, or of the throat. Gently pull out one of the hind-limbs, until it becomes quite straight, and then let it go. It will be immediately drawn up into its old posi- tion under the body. If this experiment be made soon after the operation of removing the brain, or if much blood has been lost, the leg may be drawn up slowly instead of sharply. Gently tickle one flank with a feather or a blunt needle; a contraction of the flank muscles of that side will be observed. Pinch the same spot rather sharply with a pair of forceps ; the leg of the same side will be first extended, and then drawn up and swept over the | flank, the movement tending to thrust away the points of the forceps. Pinch with the forceps the skin round the anus; both legs will be drawn up and thrust out again; the movement tending as before to sweep away the points of the forceps. Leave the animal alone for five minutes and watch it carefully: ifno disturbing circumstances are brought to bear on it, it will remain perfectly motionless. Place the animal on its back; it will make no effort to regain its normal position, 7.e. all sense of equilibrium has been lost. 118 10. II. ELEMENTARY PHYSIOLOGY. [x1 Pass a hook through the lower jaw, and fasten it to the cross-bar of a stand so that the body can be raised up and down. The hind-limbs, after a few movements of flexion and extension, will remain pendant and motionless, Gently pinch the tip of one of the toes of either leg; that leg will immediately be drawn up. _ Take two glasses, fill one with dilute sulphuric acid (‘1 p.c.) and the other with water: lower the frog until the tip of one of the toes just ‘touches the dilute sulphuric acid. In a short time the foot will be withdrawn. At once let the foot dip into the second glass containing water, in order to wash away the acid. Measure with a rapidly beating metronome, the time between the moment when the toe comes into contact with the acid and the moment when it is withdrawn. Make, at intervals of a few minutes, three such observations, and take the mean of the three. Cut a small piece of blotting-paper one or two mms. square, moisten it with strong acetic acid, and place it on the flank of the animal. The leg of the same side will be speedily drawn up and swept over the flank as if to remove the piece of paper. Place similar pieces of paper dipped in acetic acid on different parts of the body; different movements will be witnessed in consequence; XI.] GENERAL. PROPERTIES OF NERVOUS TISSUE. 119 13. 14. all however tending to remove the irritating substance, Wash off all the acid from the frog, and when it has become perfectly quiet, place it in a basin of water; it will sink to the bottom (unless the lungs be accidentally much distended with air), and no movements of any: kind will be witnessed. Observe that all the movements produced in the fore- going observations, although complicated, co-ordinated, and purposeful in character, are partial, and only by accident bring about locomotion. However stimulated, the animal never springs or leaps forward. In order that the same frog may serve for observations on the. lymph-hearts, B. I. § 1 should be performed here. Make a small cut through the skin of the back and with a fine glass tube inject one drop of a 1 p.c. solution of strychnia. In a few minutes the slightest stimulus applied to any part of the animal will produce violent tetanic spasms of the whole body. A preliminary stage of increased reflex action may also be Gbeorvell, With a straight seeker or a piece of stout wire destroy the whole of the spinal cord. Observe that the spasms immediately cease. 1 Immediately underneath the skin of the back of the frog is the dorsal lymphatic sac, and any fluid placed in this rapidly makes its way into the blood. ° 120 ELEMENTARY PHYSIOLOGY. [XI 15. Repeat any of the above observations (§ 2—13). No reflex actions will now be produced. B, Avromatic Action. I. The Lymph-Hearts. 1. Placing the animal on its belly watch the move- ments of the posterior lymph-hearts. They may be seen beating on either side of the extremity of the urostyle, in a depression between that bone and the hip-joint. The contractions are generally visible through the skin, but become more evident if the skin be removed, care being taken not to injure the lymph-hearts themselves. 2. Observe that after destruction of the posterior part of the spinal cord the lymph-hearts cease to beat. IJ. The Heart. 1. Lay the frog on its back, make a median incision through the skin, and from the middle of this make transverse incisions. Raising up the end of the sternum with a pair of forceps, cut it through a little above the end so as to avoid cutting the epigastric vein. Then again lifting up the sternum cut it through longitudinally with a strong pair of scissors; pin back each part: of the sternum, cutting through such muscles as may be necessary; the heart will XL] GENERAL PROPERTIES OF NERVOUS TISSUE, 121 i) be seen in the thin membranous pericardium beating with considerable regularity and force. Pinching up the pericardium with a fine pair of forceps, cut it away from the surface of the heart then tilt up the apex of the ventricle; a small band of connective tissue will be observed passing from the posterior surface of the ventricle to the adjoining wall of the pericardium. Seizing this band with the forceps, divide it between the forceps and the pericardial wall. Lift up the apex of the ventricle, by means of this band, and with a sharp pair of scissors cut through the aorte:, the superior venz cave, the inferior vena cava, and the surrounding tissue. Take care not to injure the sinus venosus. Place the heart in a watch-glass, moistening it when necessary with normal saline solution. The beats will either not be interrupted at all or for a very short time. In cold weather the heart may stop on being removed from the body, but if the heart be warmed by putting the watch-glass containing it in the palm of the hand, the beats will be resumed. Lifting up the apex of the ventricle by means of the same band as before, cut through the ventricle with a sharp pair of scissors at its upper third. The lower two-thirds of the ventricle will remain motionless without any spontaneous. beat; the upper third and the auricles will continue to beat with regularity. ad III. to ELEMENTARY PHYSIOLOGY. [XL By means of a longitudinal incision divide the auricles with the attached portions of ventricle into two lateral halves. Each half will continue to beat. Cilia. Placing the frog on its back, cut through the lower jaw, in the middle line, and carry the incision down the oesophagus as far as the stomach. Pin back the parts and moisten the mucous membrane, if it is at all dry, with normal saline solution. Place on it, a little below the orbits, a small thin piece of cork. The cork will be seen to be driven by ciliary action down towards the stomach. DEMONSTRATIONS. The electric currents of nerves removed from the body (demarcation currents). The negative variation attending a nervous impulse (action current), The effect of a constant current on demarcation currents (electrotonus). The effect of a constant current on the irritability of nerves (electrotonus as affecting irritability). The law of contraction. XI] GENERAL PROPERTIES OF NERVOUS TISSUE. 123 6. The phenomena of urari poisoning. 7. The function of the anterior and yomeron roots of the spinal nerves. 8. The peristaltic movements of the intestines and of the ureters LESSON XII. STRUCTURE AND PROPERTIES OF BLOOD- VESSELS. A. Tse Larcer ARTERIES. 1. Cut transverse and longitudinal sections of a small piece of the aorta or carotid artery of a dog (or other animal), which has been preserved in potassium bichromate 1 p.c.; stain with picro- carmine. Note a. The thin inner coat thrown into longitudinal folds by the contraction of b. The much thicker middle coat, consisting of alternating layers of elastic and circularly arranged muscular tissue. Both of these are stained of a yellower tint than the white fibrous tissue. The disposition and relative amount of the elastic and muscular tissue will be found to vary much in different arteries. c. The external coat (tunica adventitia), con- sisting mainly of white fibrous connective ‘ XL] STRUCTURE OF BLOOD-VESSELS. .125 tissue, with some elastic tissue not infre- quently arranged in layers, especially at the inner part of the coat. Note the nuclei of the muscle cells (ep. Lesson vit. § 11). As a rule in the more peripheral arteries, the middle coat is formed of bundles of muscular fibres separated from one another by a small amount only of connective tissue with elastic fibres. Take the jugular vein from a freshly-killed rab- bit, cut it open longitudinally, and pin it out with hedgehog quills with the inner surface. uppermost on a thin piece of cork; stream water gently over it for a moment, then pour over it a ‘2 p.c. solution of nitrate of silver and leave it for five minutes, wash well with distilled water, and expose to light, also in distilled water, until it becomes of a brownish colour: spread it out on a slide with the inner surface uppermost, treat it successively with 50 p.c, 75 p.c, 95 p.c. alcohol, clear and mount it in Canada balsam. Observe the rather jagged dark lines of the cement substance between the cells; the cells form a continuous layer, are more or less elon- gated in a longitudinal direction, and are flat; indications of the nuclei of the cells may be seen, and in some places indications also of the fibres of the: muscular coat, as transverse or longitudinal markings caused by a deposition of silver in the cement substance between the muscle cells, If the vein has been left too long in the 126 ELEMENTARY PHYSIOLOGY. [ XII. silver nitrate solution, or exposed too long to light, silver will be deposited in the substance of the cells also. A vein is here taken as being a somewhat easier preparation for a student than an artery; the main features in either case are the same. Cut open longitudinally a large artery, e.g. carotid of sheep, from a freshly killed animal, and very gently scrape the internal surface. Tease out the material so obtained in normal saline solu- tion. Observe in surface and profile the cells of the lining epithelium, with their elongated nuclei. Probably there will also be seen fragments of the elastic fenestrated membrane. Add acetic acid 1 p.c. to distinguish more clearly the elastic from the white fibrous tissue. (The perforations in the fenestrated membrane are rendered more distinct by staining.) Tear off a strip from the inner coat of a medium- sized artery which has been in potassium bichro- mate ‘2 p.c. for two to six days; tease it out in the fluid; it will be found to consist almost en- tirely of elastic lamine. Observe the gradations from an almost homo- geneous elastic perforated membrane to a mesh- work of elastic fibres. XII] STRUCTURE OF BLOOD-VESSELS, 127 B. Tse Larcer VEINS. 1. Cut transverse sections of a fairly large vein, as in A. §1. Relatively the muscular element will be found to be less (though varying considerably in different veins); the elastic element also to be less, and the white fibrous connective tissue more plentiful. Attempt to make fluid pass along a vein of some _ large animal in the wrong direction, in order to shew the action of the valves. Cut open the vein and examine the structure and position of the valves. bo C. CAPILLARIES AND SMALL BLOOD-VESSELS. 1. Place on a slide with a drop of glycerine the tail of a tadpole which has been hardened in Muller's fluid or in chromic acid ‘2 p.c. and subsequently treated with alcohol. Brush off the surface epi- thelium or if this is unsuccessful break it up into not too small fragments with needles (in doing so try to scrape off the epithelium). The tissue may be previously stained by placing it for a day in dilute picrocarmine. Note a. The network of capillaries; in some, blood- corpuscles will probably be seen. b. The nuclei in the walls of the capillaries, es- pecially at the points of bifurcation. c. The branched connective-tissue corpuscles, in places partially surrounding the capillaries, 128 -ELEMENTARY PHYSIOLOGY. [XIL and constituting the simplest form of a tunica adventitia. The connection of some of the processes of the connective-tissue corpuscles with the ca- pillary walls. i Remove a small portion of pia mater from a per- fectly fresh brain of a chloroformed cat or dog allowed slowly to bleed to death, and spread it out carefully in normal saline solution, washing it gently with the same, and mount. Note a. The capillaries, with their nuclei bulging into the channels. The small arteries, their scanty external con- nective-tissue coat or adventitia, and the indications of their muscular coat composed of a single layer of fibres wrapped round the intima. The small veins generally filled with blood- corpuscles, with their muscular coat absent or developed only to a slight degree. Gently irrigate with acetic acid 1 p.c. Note a In the capillaries, the more distinct nuclei. -b. In the small arteries, (a) The fusiform cells and nuclei of the adventitia, arranged longitudinally, with elastic fibres. (8) The nuclei of the muscular coat ar- ranged transversely, By watching the x11] STRUCTURE OF BLOOD-VESSELS. 129 earlier stages of the action of acetic acid, the outlines of the muscle fibres, as well as of their nuclei, will be dis- tinctly seen. (y) The thin inner elastic layer, internal to the muscular fibres, seen as wrinkled longitudinal markings. (6) The oval nuclei of the lining epithelium arranged longitudinally. c. In the small veins, appearances similar to ), consistent with the less development of mus- cular and elastic tissue. A piece of fresh pia mater may be stained with aqueous hematoxylin to bring out more clearly some points of the above ; the following will, however, be found a better method. Place a fresh brain, with the dura mater at least in part removed, in potas- sium bichromate 1 p.c. for two or three days (or longer), then wash it with water, and place it in 75 p.c. spirit for half an hour to twenty-four as convenient, cut off then a small piece of pia mater, stain with hematoxylin, or picrocarmine, and mount. Care should be taken to drag the membrane as little as possible, and to avoid creases. Destroy the brain and spinal cord of a frog, lay bare the heart, cut through the whole length of the muscles of the abdomen a little on one side of the epigastric vein. Cut off the ventricle and cut across the venous sinus (cp. Less. XI B. 11), sop up with a sponge the blood which 9 130 ELEMENTARY PHYSIOLOGY. [xu. flows out, when the bleeding has ceased take a small glass cannula having a piece of india- rubber tubing over one end and filled with normal salt solution and insert it into the aortic bulb, tie it near or at the branching of the aorte with a silk thread. Then take a syringe full of normal salt solution, and the tubing being also full of salt solution gently force the nozzle of the syringe into the tubing, being careful that no air-bubbles get in at the same time. Then slowly inject the salt solution: this.should be repeated until the fluid issuing from the venous sinus is perfectly clear; sop up the fluid and inject with water, then inject once or twice a ‘2 p.c. solution of nitrate of silver, sopping up the fluid which issues from the sinus; inject water to wash out the nitrate of silver, place the frog in a basin of water and cut out the intes- tine; cut through the mesentery at its junction with the intestine and lay open the intestine, brush away its epithelial coat and expose it to light in water or in 30 p.c. alcohol. The out- lines of numerous vessels will soon be visible; then cut out a piece, spread it out on a slide with the inner surface uppermost, wash with alcohol of increasing strength, clear and mount in Canada balsam’. ° 1 If the tissue be left too long before it is mounted the cells as well as the cement substance will become black. From the frog injected as above a piece of the mesentery, or of the lungs, or of the bladder, may be mounted. X11] STRUCTURE OF BLOOD-VESSELS. 131 Note with a low power the small arteries branch- ing into capillaries and the uniting of the capil- laries to form veins which again unite to form larger veins. Note under a high power the outlines of the flat elongated epithelium cells of the small arteries, of the capillaries and of the small veins; the outlines in the capillaries are more irregular than in the arteries or veins ; in the veins the cells are rather broader than in the arteries. In the small arteries the cementing substance between the cells of the muscular coat (here usually a single layer) will also shew as black transverse lines, the small veins are without these transverse markings. D. CIRCULATION OF BLoop. INFLAMMATION. Take a frog with a pale web which a day or two previously has been deprived o1 its brain by the Demonstrator (cp. p. 116, footnote) and kept in a moist place. With scissors make a small cut through the skin of the back, with a fine pipette introduce underneath the skin one drop of a1 p.c. solution of urari, leave the frog under a glass jar until it does not move on being pinched ; this should be the case in about three-quarters of an hour, if it occurs earlier too much urari has been given and the blood-vessels will be abnor- mally dilated. Lay the frog on its belly on the frog-board, and tie, not too tightly, a piece of soft cotton round the end of the (¢.g.) 3rd and 4th 9—2 132 ELEMENTARY PHYSIOLOGY. [XII. digits. Stick two pins into the board a little distance from the hole; by twining the cotton round them the web may be stretched out level above the hole (or the toes may simply be pinned out). Surround the web, and cover the leg and body of the frog, with moist blotting-paper. Put a small drop of water upon the web, and cover it with a triangular piece of cover-slip, taking care that the glass does not cut into the digits and that no fluid flows over it. Examine, first with a low and then with a high power. Note a. The course of the blood from the arteries to the veins.