ii^v*.-'.'..-!.':,-;--^^. fytmll Hmmsitg pitmtg BOUGHT WITH THE INCOME FROM THE SAGE ENDOWMENT FUND THE GIFT OF Henirg M. Sage 189X .Au.^.s:jA.r.. ^.;^^/.^.,>.^. 5474 .„__ Cornell Unlveratty Library arV18397 _^ 3 1924 031 274 537 Cornell University Library The original of tliis bool< is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924031274537 A COUESE OF ELEMENTABY PEACTICAL PHYSIOLOGY AND HISTOLOGY A COUESE OP ELEMENTAEY PEACTIOAL PHYSIOLOGY AND HISTOLOaY BY M. FOSTEK AND J. N. LANGLEY. SEVENTH EDITION EDITED BY J. N. LANGLEY, D.Sc, F.KS. and L. E. SHOEE, M.D. Sontion MACMILLAN AND CO., Limited NEW YOEK: THE MACfflOLLAN COMPAJSTST 1899 [All Bights reserved.] First Edition printed 1876. Second, April 1877. Third, December 1877. Fourth, reprinted with alterations, October 1880, November 1880, 1881, 1882, 1883. Fifth, 1884. Reprinted, 1887. Sixth, 1888. Reprinted 1891, 1893, 1894, 1896, 1898. Seventh, 1899. PEEFACE TO THE SEVENTH EDITION. TN this Edition, most of the Lessons have been -^ re-written, and a few have been added. But the general arrangement, planned by Professor — now Sir Michael — Foster in the first edition has been kept. One divergence from the original plan has been made, namely, the omission of the Lesson on the Dissection of the Rabbit and Dog. The specialization of study which has taken place in the last twenty-five years seemed to make this omission inevitable; but the conclusion was reached with regret. Some of the directions are given in small type, they refer on the whole to the less elementary work. But each Teacher will decide for himself, according to the stress of his teaching and the resources of his laboratory, which sections best come in a first year and which best come in a second year course. The portions of the book dealing with chemical physiology, and with the physiology of muscle and VI PREFACE. nerve have been in large part revised or written by Dr Shore, whose name in consequence has been added to the Title-page. The proofs of the sections on chemical physiology have been kindly read by Dr F. Gowland Hopkins, to whom we owe many valuable suggestions. A similar kindness has been rendered us by Dr W. F. R. Rivers in the Lessons on the physiology of the special senses. In the histological part of the book I have had invaluable assistance from Miss Greenwood — now Mrs Bidder — who has given me the benefit of her experience of the difficulties which beset the elementary student, and who has corrected the proofs with a care which I cannot too gratefully acknowledge. I desire also to thank Mr W. B. Hardy and Dr H. K. Anderson for suggestions on various points. J. N. LANGLEY. CONTENTS. LESSON I. structure of Corpuscles of Blood and Lymph PAGE 1-11 II. Measurement of Objects under the Micro- scope. Counting Blood Corpuscles. Some Characters of Blood 12-19 III. Serum. Characteristics of Proteids. Clotting of Blood 20-27 IV. Staining and Mounting Sections . 28-37 V. Section Cutting and Imbedding . 38-47 VI. Hyaline Cartilage 48-52 VII. Connective Tissue 53-61 VIII. Fibro-Cartilage. Elastic Cartilage. Bone. Teeth 62-69 IX. Structure of Contractile Tissues . 70-78 X. Chemistry of Muscle. Derived Albumins. Albuminoids. Egg-White 79-86 XL Physiology of Muscle and Nerve. I. . 87-106 XII. Physiology of Muscle and Nerve. II. 107-113 XIII. Physiology of Muscle and Nerve. III. 114-122 XIV. Nerve Fibres 123-128 XV. Peripheral Ganglia and Nerve Cells 129-135 XVI. Endings of Efferent Nerve Fibres in Muscle 136-139 XVII. Reflex and Automatic Action 140-145 XVIII. Structure of Blood Vessels .... 146-154 Vlll CONTENTS. liESSON XIX. structure and Action of the Heart PAGE . 155-166 XX. Blood Pressure . 167-174 XXI. Salivary Glands. Certain Carbohydrates. Saliva ., 175-186 XXII. Stomach. Gastric Juice. Nucleo-Pro- teids. Milk 187-198 XXIII. Intestine. Bile . 199-207 XXIV. Pancreas. Pancreatic Juice . . 208-211 XXV. The Lymphatic System . 212-221 XXVI. Structure of Liver. Glycogen . 222-226 XXVII. The Lungs. Mechanic^ of Respirat ion 227-231 XXVIII. The Colour df Blood. Eespiration ^ . 232-238 XXIX. Structure of the Kidney . 239-244 XXX. Urine and its Chief Constituents . 245-259 XXXI. The Ductless Glands . 260-262 XXXII. Skin. Touch . . 263-274 XXXIII. Taste and Smell . 275-281 XXXIV. The Eye . . 282-291 XXXV. Vision . 292-307 XXXVI. The Ear . . 308-316 XXXVII. Spinnl Cord . 317-323 XXXVIII. The Brain . 324^341 XXXIX. Dissection of the Larynx . 342-345 XL. Tissues of Eeproduction . 346-353 Appendix 354-378 Chemical Examination of Fluids foe Certain OF THE Commoner Substances of Physio- logical Importance 379-388 Index 389-406 LESSON I. STRUCTURE OP CORPUSCLES OF BLOOD AND OF LYMPH. A. Corpuscles of Frog or Newt. Read the Notes on the Use of the Microscope in the Appendix, p. 354. 1. A frog is given you the brain and spinal cord of which have been destroyed (cp. App. p. 376). Take the frog in one hand, and with a stout pair of scissors cut off a foot at the ankle joint. Touch a clean slide with the cut surface, so as to leave a small drop of blood on the slide. Place on it a cover-slip. Later, drops of fresh blood should be obtained by cutting off the other foot, the leg at the knee, the fore arm at the elbow-joint ; lastly, blood should be obtained direct from the heart ; in order to do this cut through the skin in the median ventral line, cut transversely through the lower part of the sternum just above the epigastric vein, with stout scissors cut through the sternum in the middle line and so expose the heart; cut off the tip of the ventricle, and touch a slide with L. 1 2 ELEMENTARY PHYSIOLOGY. L^ it. For §§ 5, 6, 8, blood which has flowed from one of the cut surfaces will serve. Examine the mounted drop of blood under the microscope with a low magnifying power' and observe the numerous corpuscles floating in the plasma. Examines it with a high magnifying power' and observe the red corpuscles ; if a large drop of blood has been taken the corpuscles will overlap one another, in which case another preparation should be made with a smaller drop. a. The red corpuscles are flattened ellipsoids ; note their spindle shape when seen on edge as they roll over. The great majority are of the same size and tint. h. They appear at first homogeneous, but soon a certain number show a central paler, oval nucleus. 0. A single corpuscle is pale yellow, the colouring substance (haemoglobin) being equally diffused through- out it; when several corpuscles lie over one another they together appear red. 2. Examine the colourless corpuscles in parts of the specimen where the red are not very numerous. a. They are much fewer than the red. They are smaller than the red, but they vary in size. 6. They are usually spherical, when first mounted, but soon many of them put out processes and become irregular in form. 1 For convenience the term ' low power' will be used throughout for a combination of lenses which magnifies 50 to 100 diameters, and the term 'high power' for a combination of lenses which magnifies 250 to 500 diameters. l] STRUCTURE OF CORPUSCLES. 3 c. The majority have no distinct granules (hyaline corpuscles). Some have comparatively large and re- fractive granules; these are the coarsely granular corpuscles. d. The nucleus can seldom be made out, though it may be seen if the corpuscle is very extended. Do not confound a heap of granules or a protuberance with the nucleus. e. Choosing a corpuscle, either elongated or having several processes, watch carefully its amoeboid move- ments; make half-a-dozen drawings of its outline at intervals of about twenty seconds. As a rule the coarsely granular corpuscles put out few and rounded processes, and the finely granular corpuscles put out more numerous and more pointed processes. /. If it is desired to watch the movements for an hour or so the drop should be protected from evaporation, (a) Melt a little glycerine jelly (op. App. p. 372) on a warm bath, keep the cover-slip in place by gently holding a lifter against one edge, and with a small brush, brush a little glycerine jelly over the edges all round. (/3) If the slide is to be warmed, use melted paraffin (of low melting point) instead of glycerine jelly. Before applying the parafSn, dry the slide at the edges of the cover-slip with blotting-paper, and warm the slide. Olive oil may be used instead of glycerine jelly or paraffin. 3. ffanging drop. Cut out a number of pieces of blotting- paper about 3 cm. by 2 cm. Place them together so as to make a pad. With scissors or cork borer cut out from the centre of the pad a hole a little smaller than the cover-sUp to be used. Dip the pad in salt solution 0'6 p.c. and place it on the slide. On the centre of a cover-slip place a small drop of lymph or blood, and lower the cover-slip over the hole in the pad, so that its edges rest on the pad, and the drop of lymph hangs in the centre and does not run oflF to the pad. Thus a small moist chamber is 1—2 4 ELEMENTAET PHYSIOLOGY. [I formed. The pad should be kept wet by adding a little water to one edge from time to time. Examine the movements of the white corpuscles under the microscope. 4. Lymph crowded with white corpuscles, many in active movement, may be obtained in the following way. The brain of a frog is destroyed, and the wound plugged with cotton- wool. A drop of curari is injected under the skin ; this paralyses the lymph hearts, so that the lymph accumulates in the lymph sacs. The frog is kept in a dish containing water about J inch deep, for half-a-day to a day. Lymph may be obtained from any of the lymph sacs, but, if the frog has been placed in a normal position, in especial quantity from the ventral sacs, beneath the skin of the abdomen. Since the lymph clots quickly when withdrawn, not more should be taken up in the pipette than is required at the time. The white corpuscles which have ingested foreign particles will be seen, if a drop of milk or if a drop of water containing hay bacillus, or Indian ink, be injected into a lymph sac about an hour before examining the lymph. The early stages of action may be seen by mixing a little lymph and bacillus culture, and at once examining in a hanging drop. 5. Irrigation. Mount another small drop of blood. Place a small drop of ■! 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 shows some irre- gular granules; a granular nucleus comes into view. As a rule the nucleus is horse-shoe shaped in the coarsely granular cells; and is round or consists of clumps connected by thin threads in the hyaline cells. I] STRUCTURE OF CORPUSCLES. 5 b. In the red corpuscles the nucleus becomes obvious; it is when first seen nearly homogeneous, and oval in outline, later it becomes granular and usually irregularly rod-shaped. c. The red corpuscles swell up owing to absorption of water, most after a time become spherical (if strong acid be used the corpuscles usually preserve their shape). d. They become colourless, the haemoglobin being dissolved from the stroma ; occasionally the haemoglobin is massed round the nucleus before complete solution takes place (effect of water), and occasionally it stains the nucleus yellow (effect of acetic acid). e. Finally the outline of the corpuscles is seen as a faint line at some distance from the nucleus. Observe the not infrequent excentric position of the nucleus. /. Some corpuscles are much more readily acted on than others. 6. Irrigate a drop of blood, first with 30 or 50 p.c. alcohol and then with a strong aqueous or alcoholic solution of Spiller's purple or magenta. The red corpuscle becomes spherical and its peri- pheral rim, the psevdo-memhrane, stains. The nuclei both of the red and of the colourless corpuscles stain deeply. 7. 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 underneath the cover-slip from 6 ELEMENTARY PHYSIOLOGY. [l one side of it to the other (cp. § 5). The first small drops will have partially clotted and will serve as an imperfect barrier to the coi-puscles 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 for a short time largish clumps of colourless corpuscles will be seen. 8. Evaporate to dryness on a slide a drop of a strong solution of urea ; on this place a small drop of blood, mount quickly, and observe the breaking up of the red corpuscles into spheres ; sometimes a corpuscle will put out a varicose filament which breaks up later into spheres. 9. ' (Ecoid' and ' zooid.' Let a little fresh blood of a frog or newt run into about five times its volume of a 2 p.c. aqueous solution of boracio acid, and mount at once a drop of the mixture. The corpuscles, nearly normal in appearance when first mounted, rapidly become altered ; in all or nearly all cases the hsemoglobin leaves the stroma or the outer part of it, and becomes accumu- lated around the nucleus. As this is taking place the hsemoglobin may form a star-shaped figure in the stroma ; from some cor- puscles, but as a rule, from a few only, the mass formed by the nucleus with the hsemoglobin, i.e. the so-called ' zooid,' is extruded from the colourless ' cecoid.' In mammalian corpuscles a 2 p.c. solution of tannic acid causes a similar separation of the hesmoglobin from the stroma. 10. 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. l] STRUCTURE OF CORPUSCLES. 7 11. Irrigate a, drop of blood with 95 p.o. alcohol; a granular precipitate will be formed in the stroma. Irrigate with Spiller's purple, the precipitate will stain deeply. 12. Preparation of a dry film of blood. Make a film preparation in one of the following ways. a. Moisten with blood a small glass rod (1 to 2 mm. in diameter) and rub it over the surface of a clean cover-glass. The film of blood should be so thin that it dries in a few seconds on waving the cover-slip in the air. b. Take two clean cover-slips. Hold one in forceps, and touch a small fresh drop of blood with the centre of the glass; place it on the other cover-slip and at once withdraw it laterally. 13. Staining a film with eosln. See that 3 small wide-mouthed bottles are ready, the first containing a saturated solution of eosin in 75 p.c. alcohol, the other two containing 75 p.c. alcohol. Have ready also a pad of half-a-dozen pieces of smooth filter or blotting-paper, without fluflf. Take up the cover-slip with forceps and hold it for a minute in the eosin solution, let the excess of fluid drain off (5 seconds), hold the cover-slip for 5 seconds in the first bottle of alcohol and for 5 seconds in the second bottle of alcohol. Then place without delay between the pads of filter-paper and lightly press to remove excess of fluid. Wave the cover-slip in the air to dry it. When dry place a small drop of Canada balsam on the centre of the cover-slip, and lower this slowly on a glass slide. Note the deep red stain of the granules of the coarsely granular leucocytes (the oxyphil granules). 8 ELEMENTARY PHYSIOLOGY. [l The red corpuscles should be obvious, and there should not be any precipitate. 14. Staining a film, with eosin and methylene hliie. Add to the bottles used in § 13, two, one containing methylene blue' in 75 p.c. alcohol, the other 75 p.o. alcohol. Proceed as in § 13, but after the cover-slip has been placed in the second alcohol hold it for 10 seconds inHhe methylene blue, dip it for one second in the 75 p.o. alcohol, then without delay press lightly between the blotting pads. The nuclei of the various cells will be stained blue. If ceUs with basophil granules are present, the granules wUl also be stained blue (op. p. 58, § 10). 15. Preservation of hcemoglobin in red corpuscles hy heat. Place a dry film of blood for 5 to 10 min. at a temperature of 120° 0. This may be obtained by placing a Bunsen flame under one end of a long strip of copper ; the part of the strip which is at a temperature of 100° 0. can be determined by noting where a small drop of water boils ; the specimen should be placed a little nearer the flame than this, the filmed side uppermost. (Instead of this, the cover-slip is sometimes passed twice through the flame of a Bunsen burner.) The film may be stained as in § 13 or § 14. The fixation of the hsemoglobin in the corpuscles may be observed later in sections of tissues hardened in osmic acid vapour, osmic acid, mercuric chloride dissolved in salt solution, Miiller's fluid, formol. 16. Platelets. Mount a small drop of blood direct from the blood vessels or from the heart (cp. § 1). Examine at once. Note the elongated pale cells (plate- lets) about \ the size of the red corpuscles. Some are found singly, others sticking together in groups. The nucleus can generally be made out, it is large in com- parison with the amount of cell substance. The nucleus soon becomes round and the cell substance indistinct. 1 A saturated solution of methylene blue in 75 p.c. alcohol, diluted with an equal bulk of 75 p.c. alcohol. l] STRUCTURE OF CORPUSCLES. 9 17. Let a drop of fresh blood of a frog or newt drop into 5 c.c. of a mixture containing 0"6 p.c. NaCl, 0'6 p.c. peptone, 0"02 p.c. methyl- violet ; and stir at once. In this fluid the platelets are preserved for a day or so. The nuclei of all the elements of the blood are stained rather deeply ; the cell substance of the white corpuscles and of the platelets tg,kes a light stain (cp. 3, § 5). 18. The platelets and othei corpuscles of the blood may also be preserved by allowing a drop of blood to run into 5 c.c. of (1) a mixture containing 0*5 p.c. osmic acid, and 0'75 p.c. NaCl., (2) 1 p.c. oxalate of potassium. In the former fluid they do not stick together, and to a slight extent only in the latter. Permanent preparations may be made from the blood mixed with osmic acid. The mixture is allowed to stand for a day; the fluid decanted, water is added to the residue and shaken; when the cor- puscles have settled, the water is poured off ; the process is repeated ; 50 p.c. alcohol is then added, in this the corpuscles can be kept till required ; with a little care the corpuscles can be stained. B. Corpuscles of Man. 1.- With a sharp needle^ prick the finger a few millimetres from the root of the nail ; touch with the centre of a cover-slip the drop of blood which issues ; lower the cover-slip on a slide (cp. A. § 1). Observe the red corpuscles. a. They roll about readily when the cover-slip is lightly touched. Soon after being taken from the body they stick to one another, and, owing to their., shape, usually in rouleaux. ' It is better to use the triangular needle specially made for the purpose. 10 ELEMENTAEY PHYSIOLOGY. [l 6. They are biconcave discs. Note that on focus- sing down on the circular face a darkish centre and a light rim is first seen and then a light centre with a . darkish rim: when viewed in profile and the centre focussed they appear somewhat dumb-bell shaped. c. They appear homogeneous, their colour is like that of the red blood corpuscles of the frog (cp. A. § 1, c). d. Towards the outside of the drop, where evapo- ration is going on, many of the red corpuscles are crenate. e. They are much smaller than the red corpuscles of the frog. 2. Observe the colourless corpuscles. They are larger than the red, they resemble in general appearance the white corpuscles of the frog (A. § 2, c. d. e.) ; to observe their amoeboid movements a drop should be protected from evaporation (A. § %f) and, preferably, warmed to the temperature of the body. In stained specimens there will be seen small cells with a spherical nucleus (lymphocytes), a few hyaline cells, a considerable number of finely granular oxyphil cells, and a few coarsely granular cells. 3. Irrigate with '5 p.c. acetic acid (cp. A. § 5). a. The red corpuscles swell up and become spherical, their haemoglobin is dissolved, leaving the hardly visible stroma. (Effect of water.) 6. No nucleus is brought into view. c. In the white corpuscles the cell-substance be- comes more transparent, and the nucleus comes into view. l] STRUCTURE OF CORPUSCLES. 11 4. Platelets. Kill a rat or mouse with chloroform. Expose the heart, and cut through the pericardium. Cut through the ventricle and let one drop of blood fall into 5 o.c. of (a) methyl- violet solution (A § 11), (b) osmic acid and salt (§ 18), (c) 1 p.c. oxalate of potassium. Stir and examine a drop for platelets. They are small, colourless, oval bodies, i to J the size of the red corpuscles and without nuclei. 5. Wash the tip of the finger, place on it a drop of the methyl-violet solution or of the potassium oxalate sohition. Prick the finger through the drop. As soon as blood comes transfer to a slide and examine. DEMONSTRATIONS. 1. Oirculation of blood in the mesentery of the newt to show the platelets sticking for a variable time to the walls of the vessels (cp. Lesson xviii. § 9, b). 2. Specimens to show amphibian and mammalian platelets. 3. Specimens to show the chief stages of indirect nuclear division in tissue cells of larval salamander (Flemming's fluid'). 4. Hanging drop to show leucocytes with ingested bacilli. 5. Stage for varying temperature of hanging drop, and passing gases over it. ' The fixing agent in which the tissue is placed on being removed from the body is, here and later, given in brackets. The details of treatment are given in the Appendix. LESSON II. MEASUREMENT OF OBJECTS UNDER THE MICROSCOPE. COUNTING BLOOD COR- PUSCLES. SOME CHARACTERS OF BLOOD. 1. Drawing and Measurement of Objects under the Microscope. Place a drawing-board (made so that the drawing surface is at an angle of 15° to 20° with the table), in front of the microscope, and touching its foot, or at an ascertained distance from it. Fix a piece of smooth paper in the middle of the drawing-board. A simple form of camera lucida is shown in Fig. 1. Slip the ring (r) over the tube of the microscope (t) then replace the eye-piece (o). Ai-range the brass rod carrying the prism (A) on the left-hand side and shift the prism till (1) the edge (a) stretches half-way over the lens of the eye-piece, (2) the lower angle of the prism nearly touches the edge of the eye-piece, (3) the anterior exposed surface of the prism is parallel with the drawing-paper. Then turn the prism round the vertical axis so as to uncover the eye-piece. Focus with a low power a stage micrometer, i.e. a n] MEASUREMENT UNDER THE MICROSCOPE. 13 millimetre divided on glass, into tenths, and one of the tenths divided into hundredths. When the lines are Fig. 1. The upper sketch shows the camera from the side, the lower sketch shows it from above. seen distinctly, turn the prism back again into position. On looking down the microscope with the eye close to a, the lines of the stage micrometer and the point of a pencil placed on the drawing-paper should be visible. 14 ELEMENTAKY PHYSIOLOGY. [H If they are not, watch the lines carefully, for the appearance of the pencil point over them as the prism is turned slowly and slightly around the horizontal axis, the point of the pencil at the same time being moved; try also the effect of shading the drawing-paper. Draw the ^th micrometer lines. Then place the drawing alongside a millimetre scale, and note the length in millimetres of the scale drawn. If it mea- sures 80 mm., then the scale drawn, which on the micrometer was one millimetre, has been magnified 80 times. 2. Draw similarly on another piece of paper the lines of the stage micrometer with the high eye- piece, and objective. It will be sufiBcient to draw the lines indicating y^th of a mm. Ascertain the magnifi- cation of the drawing. If the ^th of a mm. drawn measures 38 mm., the magnification will obviously be 380. 3. Substitute now a fresh preparation of human blood for the stage micrometer, and draw some red corpuscles under the high eye-piece and object-glass; taking care that the drawing-board and paper are in the same positions as in § 2. Fold the scale drawn in § 2 at right angles to, and through the lines ; and measure with it the diameter of the corpuscles you have drawn. One division of your scale is ^^ mm. i.e. 10 fi. (fj. = micron, j^ mm.) The red corpuscle drawn should occupy rather less than one division of your scale since the red corpuscle is ordinarily 7 to 8 /t in diameter. 4 Write on each scale drawn, the eye-piece and object-glass with which it was drawn, and the position of the drawing-board. ll] MEASUREMENT UNDEK THE MICROSCOPE. 15 Later, in drawing microscopic objects, draw them in the condition of one of the two scales. Thus the scale will allow the diameters of the object to be at once measured. The phrase magnifying power of the microscope is used for the magnification of the scale, when it is drawn on a level with the stage of the microscope. 5. Measurement of size of objects hy means of an ocular micrometer. The ocular micrometer consists of a disc of glass with a scale engraved on it. A ledge to receive it is placed in the eye-piece on a level with the focus of the upper lens. Take the high eye-piece and objective, place the ocular micrometer face downwards in the ocular. Note the number of divisions of the ocular and of the stage micrometer which corre- spond exactly with one another. Reckon from this how many of the smaller divisions of the stage micrometer,— each of which is 10 /I — one division of the ocular micrometer corresponds to. Thus if 5 ocular divisions cover exactly 4 stage divisions ; 1 ocular division = ^x 10/i=8/i, and a human red blood corpuscle would almost exactly be covered by one division of the ocular micrometer. If the microscope has a draw tube, draw it out to its full length, and redetermine the value of the ocular divisions. Determine similarly the value of one division of the ooiilar micro- meter with the low ocular and objective. 6. Reaction of blood. Frick the finger and touch the drop of blood with litmus paper made of glazed paper and neutral litmus (see App. 374). After 10 — 20 seconds remove the drop of blood with a cloth or with distilled water. The paper is not stained with haemoglobin and a blue patch is seen showing that the blood is alkaline. 7. Determination of the alkalinity of blood. A decinormal solu- tion of tartaric acid {0-75 %) is prepared; 1 c.o. of this should be exactly neutralised by 0-004 gr. NaHO. Dilute this tenfold with 16 ELEMENTARY PHYSIOLOGY. [ll water. Take a series of watch-glasses and into each pat 1 c.c. of the xhi normal acid as follows : — 1. 0-9 0.0. y^ normal tartaric acid+0-1 c.c. NajSOj sol. 2. 0-8C.C +0-2 0.C 3. 0-7 CO. „ „ „ +0-3 0.C and so on. Add to each watch-glass 0-1 c.c of freshly drawn blood. After stirring determine by litmus paper which is neutralised by the blood. The amount of acid which neutralises 0*1 c.c. of the blood is thus found, and since the amount of NaHO which will neutralise this acid is known, the alkalinity of 100 c.c. of blood can be expressed as equivalent to a definite amount of sodium hydrate. The alkalinity of normal human blood is equivalent to '2 to -3 gr. per cent, of NaHO. 8. Determination of the specific gravity of blood. A series of solutions of glycerine and water of specific gravity varying between 1035 and 1068 are prepared. Pour a little of one of them, of sp. gr. 1050 for example, into a narrow but deep glass vessel. Prick the finger and suck up a drop of blood into a fine glass pipette, the point of which is bent at right angles to the main tube. Gently blow the drop of blood out of the pipette so that it issues horizontally into the middle of the glycerine solution. If the blood immediately sinks, take a glycerine solution of higher sp. gr., if it rises, one of lower sp. gr. and make another observation with another drop of blood. After a few trials a solution will be found in which the blood neither sinks nor rises immediately. The blood is of the same sp. gr., as this solution. 9. Enumeration of the red eorpascles. Count the number of red corpuscles with Thoma's hsemacytometer in the following manner. See that the counting cell and the pipette are clean and have ready a large coverslip and a watch-glaes containing the diluting fluid. This may be sodium sulphate solution of sp. gr. 1025, sodium chloride -8 % solution, or Hayem's fluid, which consists of 1 part of sodio chloride, 5 parts of sodic sulphate, -a-parts of corrosive sublimate in 200 parts of water. Prick the finger and when a sufficiently large drop of blood Il] MEASUREMENT UNDER THE MICROSCOPE. 17 is formed, gently suck blood into the pipette as far as the mark 1. With a cloth or the finger remove any blood adhering to the point of the pipette. Wash the blood on into the diluting chamber of the pipette with the diluting fluid selected, gently turning the pipette to assist the mixing and very slowly draw the fluid up exactly to the mark 101. Put the finger on the tip of the pipette, leaving the capillary tube full of the diluting fluid, and by gently shaking thoroughly mix the blood and the fluid, taking care that the contents of the capillary tube are not drawn up into the diluting chamber. The blood is thereby diluted 1 in 100. Allow the contents of the capillary tube and a few drops of the diluted blood to escape, and then at once carefully place a small drop of blood on the centre of the counting chamber. Put on the cover-slip by a sharp lateral thrust ; this assures its close apposition to the wall of the chamber and Newton's colour rings should appear. Allow two or three minutes for the corpuscles to settle, then count the number of corpuscles in at least 10 adjacent squares, including in each square any corpuscles overlapping the upper and the left boundary lines but, by way of compensation, excluding those overlapping the lower and right boundary lines. In order to facilitate the process of counting the squares are divided into groups by lines bisecting every fifth horizontal and vertical column of squares. Determine the average number found for a square. Since each square has an area of jj^ sq. mm., and the depth of the ceU is ^ mm., the cubic contents of the fluid on each square is jj^nj <"i^' iw'"- Hence the average number of corpuscles found multiplied by 4000 gives the number in 1 cub. mm., of the diluted blood, and 100 times this is the number in 1 c.mm. of the blood. 10. Envmeration of the white corpuscles. Dilute the blood 1 in 10 with the mixing pipette provided for the purpose, using the same precautions as before, and taking care that a large drop of blood is collected before the point of the pipette is put into it. The same diluting fluids may be used, but the counting is rendered more easy if '3 % acetic acid is used since this render,? L. 2 18 ELEMENTAEY PHYSIOLOGY. [ll the red corpuscles invisible. Count the number of white cor- puscles lying on the large squares formed by the lines bisecting every fifth horizontal and vertical column of small squares. Count the number on ten of these large squares, using a rather low power unless the microscope is fitted with a mechanical stage. Calculate the average number for a large square. A large square has an area of ^ sq. mm., and a cubic content of yJs cub. mm. Hence the average number found multiplied by 160 gives the number in 1 cub. mm., of the diluted blood, and 10 times this is the number in 1 cmm. of the blood. 11. If Gower's hsemaeytometer is used proceed in the following manner. Pill the larger pipette with sodium sulphate solution of sp. gr. 1025 up to the mark on the stem, it then contains 995 o.mm. ; empty it into the measuring glass. Fill the small pipette with freshly drawn blood up to the line marked 5 cmm. ; 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 ore marked at the bottom of the glass cell. Since the depth of the cell is I nun. and the side of each square is^mm., there is beneath each square ^^ cmm. of the mixture, i.e. tin'oao cmm. of blood, hence the number of corpuscles in 10 squares multiplied by 10,000 gives the number of corpuscles in 1 cmm. blood. 12. Determination of the relative amount of kcemoglobin. Determine the relative amount of haemoglobin in the blood with Gower's hsemoglobinometer in the following manner. Put a few drops of water into the graduated tube. Prick the finger and let a large drop of blood accumulate. By means of the pipette suck up 20 cmm. of blood and eject it into the graduated tube. Draw up distilled water into the pipette and wash it out two or three times into the graduated tube. Thoroughly mix the blood and the water in the graduated tube and then add water drop by drop, mixing after each addition until the tint in the graduated II] MEASUREMENT UNDER THE MICROSCOPE. 19 tube is the same as that of the tube of standard tinted jelly. The reading of the level of the fluid in the graduated tube gives the relative amount of hsamoglobin in the sample of blood in percentage of the normal amount. DEMONSTEATIONS. 1. Determination of the specific gravity of the blood (see §8). 2. Enumeration of the corpuscles (see § 9). LESSON III. SERUM. CHARACTERISTICS OF PROTEIDS. CLOTTING OF BLOOD. 1. Observe the clotting 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, squeezing out more and more serum. 2. With a pipette remove from the clot a quantity of serum. It is colourless in the rabbit, yellow in the dog and horse, reddish yellow in the ox, and yellow in man. Frequently it is coloured by the discharge of hsemoglobin firom the red corpuscles. 3. Determine the specific gravity. This varies somewhat, but is about 1028 in man, 1030 in the ox. 4. Dilute serum tenfold with water^ and with it observe the following general reactions of proteids. ' This will be obtained by the Demonstrator. ' The addition of this amount of water usually makes the fluid slightly cloudy from the precipitation of a small amount of para- globulin. Ill] SERUM. CHARACTERISTICS OF PROTEIDS. 21 a. Xanthoproteic reaction. Take a little of the dilute serum, add a few drops of nitric acid, and boil. 'J'he 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. h. To another small quantity of the serum add a few drops of Millon's re-agent'. A precipitate will be formed which turns 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. 0. Add a drop of cupric sulphate solution to an excess of sodium hydrate. To the blue solution so formed add a little dilute serum, the fluid will become violet (cp. albumose and peptone, Lesson xxil). The violet colour becomes deeper on warming. d. Add strong spirit, a precipitate is formed (peptone in solution is precipitated with diflSculty). e. 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). 5. a. Place a test-tube containing diluted serum in a beaker of water and heat the water not too slowly to about 80° C. By means of a thermometer placed in the test-tube observe the temperature at which the ' See Appendix, p. 374. 22 ELEMENTARY PHYSIOLOGY. [ill solution becomes opalescent, but note that the pre- cipitate does not become particulate nor the fluid clear. b. Just acidulate diluted serum with a drop or two of weak acetic acid and repeat the experiment. The coagulation now becomes complete, the precipitate running together leaving a clear fluid. Note that the coagulation chiefly takes place at 73° C. to 75° C. Coagulation however begins at 70° G, and is not com- plete until 82° C. 6. a. Take some serum in a beaker and saturate it with magnesium sulphate, stirring to assist the solution of the salt. Paraglobulin is precipitated. When the saturation is complete filter, or dilute with an equal volume of saturated MgSOi solution and filter. Wash the paraglobuUn with saturated MgS04 solution on the filter. 6. Dissolve the paraglobulin by adding a little water and so forming, with the salt adhering to the precipitate, a dilute MgS04 solution. 0. Determine the coagulating point of the solution as in § 5. b. Paraglobulin coagulates at 75° 0. d. Dilute serum tenfold with water, and taking a rather large quantity cautiously add a drop or two of acetic acid. At a certain point of acidification a pre- cipitate of paraglobulin occurs. It dissolves if too much acid is added. Let the fluid stand, decant, and filter off the precipitate. Take the precipitate up in distilled water, it does not dissolve. Add a particle of salt (NaCl or MgSOj), the paraglobulin passes into solution. Ill] SEBUM. CHARACTERISTICS OF PEOTEIDS. 23 e. Let a drop of serum fall into a large quantity of distilled water. A faint cloud due to precipitated paraglobulin is seen. 7. Take the filtrate from § 6. a., dilute it with water and heat it. A coagulation of serum albumin takes place. 8. Dilute the filtrate from § 6. a. with five or six volumes of water and determine its coagulating point. Serum albumin in a fluid containing the same percentage of salts as serum coagulates at 73° C. but the presence of a large amount of MgSOi in this fluid lowers the coagulating point. Large dilution with water brings the coagulating point nearer to the normal, but this will not be actually reached unless the salt is largely removed by dialysis. 9. Half saturate serum with ammonium sulphate by adding to it an equal volume of a saturated solution of the salt. Paraglobulin is precipitated. Filter. Add solid ammonium sulphate to the filtrate to saturation. Serum-albumin is precipitated. Filter; apply the xanthoproteic and other proteid tests to the filtrate. No proteids are now present. 10. a. Clotting of blood. With a feather stir slowly about 10 c.c. of freshly shed blood' ; a consider- able 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. h. Eepeat the experiment, but this time stir quickly, filaments of fibrin will be obtained ; note that the fibrin • This will be obtained by the Domonstrator, 24 ELEMENTARY PHYSIOLOGY. [HI is extensible and elastic ; leave the defibrinated blood for a day, no further clot is produced. 11. Apply the xanthoproteic and Millon's test for proteids (cp. § 4) to fibrin chopped up and suspended in water. 12. Take two test-tubes and in each place a few flocks of fibrin. a. Add water and place in a water-bath at about 30° C. for a day; the fibrin does not dissolve (it thus differs from albumin and peptone). h. Treat similarly but with dilute (1 p.c.) solution of sodic chloride ; the fibrin does not dissolve (it thus differs from globulin). 13. 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 ; neutralize the acid with NajCOs, the fibrin shrinks to its original size. If the fibrin is warmed with the acid, solution slowly takes . place, acid-albumin being formed (cp. Less. x. p. 81). 14. Examine the plasma of horse's blood kept, by means of cold, from coagulating^ ' 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 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. Clotting sometimes takes place, but the remaining fluid may still give a clot on appropriate treatment. Ill] SERUM. CHARACTERISTICS OF PROTEIDS. 25 a. Transfer with a pipette 2 or 3 c.c. of the plasma into a small test-tube. Observe the coagulation as the temperature rises. 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 dot has already shrunk away from the sides of the vessel, it may since it is colourless be overlooked unless the fluid be carefully examined. 6. Dilute 1 c.c. of the plasma with 50 c.o. 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. 15. Examine the plasma of blood prevented from coagulating by the presence of neutral salts i. a. Remove 1 or 2 c.c. carefally with a pipette, avoiding blood-corpuscles as much as possible, and dilute five to tenfold with water. The mixture will clot, probably in about halfran-hour, if placed in the warm chamber; more slowly if left at the ordinary temperature. 6. Determine the temperature at which plasma coagulates as in § 5. 6. A coagulation oi fibrinogen occrffs at 56° 0. Filter ofi" the coagulated fibrinogen. Dilute the filtrate as in § 15. a. It does not clot however long it is left. c. Half saturate plasma with NaCl by adding an equal volume of a saturated solution of the salt. Fibrinogen is precipitated. Let the tube stand a few minutes, then filter. Dissolve the precipitate in the dilute saline solution formed by adding to it a very little water. ' 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. If sodio sulphate is used an equal volume of the saturated salt is mixed with the blood. The corpuscles are separated from the blood by oentrifugalisation and the plasma pipetted off. 26 ELEMENTARY PHYSIOLOGY. ^11 d. Set some of the fibrinogen solution prepared in this way in the warm bath. After a short time clotting may take place. 16. Examine the plasma of blood prevented from clotting by the precipitation of the calcium salts i, a. Dilute a little decalcified plasma with two or three vols, of water and add a few drops of 1 "/o calcium chloride solution and place the tube in the warm bath. The plasma clots in a few minutes. 6. Prepare fibrinogen from decalcified plasma by half satura- tion with NaCl as in § 15. c. Dissolve the precipitated fibrinogen by adding a very little water and divide the solution into two parts. To one part add a drop of calcium chloride solution and set the two tubes in the warm bath. The part to which calcium chloride has been added clots, the other does not. 17. a. Take some fibrin washed free from entangled hsemo- globin and let it soak in a 5 "/o solution of NaCl in the warm bath for some hours. Strain away the fluid. This contains fibrin ferment. b. Throw some serum into alcohol and let the precipitated proteids stand in the alcohol for some days. Decant oflf the alcohol and dry the precipitate on a filter-paper in the air. Extract the precipitate with a little water, filter the aqueous extract. It contains fibrin ferment. c. Add a few drops of a fluid containing fibrin ferment to diluted salted plasma and set the tube in the warm bath. Clotting takes place much more quickly than without added fibrin ferment. Salted plasma when diluted usually clots slowly because it contains a little, but only a little, fibrin ferment. 1 The blood is received into a vessel containing a 1 % solution of potassium oxalate, 10 c.c. of the solution to every 100 c.c. of blood to be collected, and mixed by gentle shaking. The plasma is obtained by centrifugalisatiou. IllJ SERUM. CHARACTERISTICS OF PROTEIDS. 27 d. In a similar way show that added fibrin ferment hastens the clotting of fibrinogen solutions. Fibrinogen solutions pre- pared as in § 15. c. may clot because some fibrin ferment may be already present. e. Heat the solution containing fibrin ferment to 65° C. The ferment is destroyed and the solution no longer hastens the clotting of diluted salted plasma. 18. a. Add to hydrocele fluid or to other exudation fluids which have not clotted spontaneously some solution containing fibrin ferment and put it in the warm bath. In a few minutes the fluid clots 1. 6. Prepare fibrinogen from hydrocele or other exudation fluid as in § 15. c. and add to its solution fibrin ferment and put it in the warm bath. In a short time the fibrinogen clots. The clotting of isolated fibrinogen may be most readily obtained by extracting it from exudation fluids. DEMONSTRATIONS. 1. The action of albumoses, when injected into the vascular system, of preventing the clotting of blood. 2. Intravascular clotting caused by the injection of extracts of lymphatic glands and nueleo-proteids. ' Some samples of hydtocele fluid do not clot if fibrin ferment only is added. LESSON IV. STAINING AND MOUNTING SECTIONS. 1. Staining sections and mounting them in Canada balsam. Sections are given you in 75 p.c. alcohoP. Take seven watch-glasses and fill them respectively i full with (1) Delafield's hsematozylin diluted with four volumes of diluting fluids (2) picro- carmine^ (3) water, (4) 30 p.c. alcohol, (5) 50 p.c. alcohol, (6) 75 p.c. alcohol, (7) 95 p.c. alcohol. Pour a few drops of clove oil into another watch-glass. Cover up the 95 p.c. alcohol with a watch-glass to prevent evaporation. Lift up four sections on a glass rod 1 to 2 mm. in diameter' and place them in 50 p.c. alcohol for a minute; transfer them in the same way to 30 p.c. alcohol for a minute ; then transfer two sections to haematoxyUn, and ' Sections of spleen hardened in potassium bichromate may be taken for §§ 1 to 8, and the results compared. ' For the method of preparing staining fluids see Appendix, p. 365. ' Instead of a glass rod, a, glass tube finely drawn out may be used, the end being closed by fusing in a Bunsen flame. IV] STAINING AND MOUNTING SECTIONS. 29 two, first to water, and afterwards to picro-carmine. The sections in hsematoxylin will be stained in about a quarter of an hour, those in picro-carmine in half-an- hour to an hour'. With the fine glass rod, take a section from the hsematoxylin and place it in 30 p.c. alcohol for a minute, gently moving it. Place it for the same time in 50 p.c. and in 75 p.c. alcohol. Leave it for 3 to 5 minutes in 95 p.c. alcohol. Take it up on the glass rod, gently touch the end of the watch-glass with it to remove excess of alcohol, and place it in clove oil, — the clearing agent — for 2 or 3 minutes. In the clove oil the folds in the section will usually disappear. If the section is folded when in 50 p.c. alcohol, it should be transferred from fluid to fluid on a lifter instead of on a glass rod. Arrange the section flat on a lifter, hold it in place by touching it lightly with a needle as the lifter is raised from the fluid ; let excess of fluid drain from the lifter (taking care that the section does not become dry) ; and then lower the lifter gently into the next fluid. Take a section from picro-carmine and move it gently in water, before placing it in 30 p.c. alcohol; after this treat it in the same way as the section from haematoxylin. ^ Delafield'a hsematoxylin nndiluted stains sections in 1 to 2 minutes, Ehrlich's acid heematoxylin, and Mayer's carmalum in ^ to 1 hour. The stain with the acid hsematoxylin will be deeper, if the sections are washed with tap-water instead of distilled water. The rate of staining varies with the agent used to fix the tissue. Tissues fixed with alcohol or mercuric chloride stain more quickly than those fixed in a fluid containing chromic acid or osmic acid. 30 ELEMENTARY PHYSIOLOGY, [iV With a lifter transfer the sections from the clove oil to a slide, and examine them under a low power of the microscope. They should be transparent through- out. If either has any opaque spots, put it on the warm bath for a few minutes. If the opaque spots — which are caused by the presence of water — do not disappear, the section should be thrown away^, and another passed from the staining agent through the alcohols, especial care being taken to remove all excess of 95 p.c. alcohol before it is placed in the clove oil. The complete dehydration of the sections is rendered more certain by placing them in absolute alcohol after 95 p.c. but absolute alcohol is expensive and is not necessary, since clove oil will take up a small quantity of water. When xylol instead of clove oil is used as the clearing agent, greater care must be taken to dehydrate the specimen. When the sections are transparent, tilt the slide slowly and let the clove oil run off, keeping the sections in place with the aid of a needle. Let the slide stand vertically on a piece of blotting-paper for a minute or two to drain: with the clean-cut edge of a piece of blotting-paper remove the clove oil around the specimen. Clean a cover-slip by rubbing it on a smooth, hard surface, with a piece of clean silk or linen. Let a small drop of fluid Canada balsam dissolved in xylol fall on the section. With the aid of a needle gently lower the cover-slip on the balsam. 1 The section may be dehydrated by removing it from the clove oil to 95 p.c. alcohol (or better, absolnte alcohol), for about 10 minutes and then replacing it in clove oil ; but the student is recommended to take a fresh section. IV] STAINING AND MOUNTING SECTIONS. 31 2. Place a section for half-an-hour in each of the following fluids, acid hsematoxylin, alum carmine, carmalum. Mount them (cp. § 1) and compare the staining. 3. Staining with methylene blue and with saffranin. With these — and a number of other reagents — it is generally best to overstain the sections and then to decolorize them to the required extent. Sections are left in a solution of safErauin^ in SO p.c. alcohol or in 1 p.c. aqueous solution of methylene blue for 2 to 24 hours. They are then passed through the series of alcohols. In these the colour is more or less rapidly extracted and the sections must be passed through the fluids the more quickly the more rapidly the extraction is observed to take place. Clove oil dissolves these staining agents, so that cedar wood oil or xylol, in which they are insoluble, should be used as the clearing agent, in place of clove oil. Sections of a young salamander may be stained ; the changes in the nucleus during division will be seen. 4. Staining sections and mounting them in glycerine. Take two sections stained as in § 1, remove them from the staining agent to water, stir the water with a glass rod till no more colouring matter comes from the section (a minute or two); then (a) place one in a watch-glass with a drop of glycerine, and move it gently about till the glycerine has penetrated it ; place a small drop of glycerine on a slide, to this remove the section on a needle, spread out the section, and cover with a cover-glass. Sections stained with any carmine stain may advantageously be mounted in formic glycerine (glycerine containing 1 p.c. of formic acid 116 sp. gr.), b. Treat the other section similarly, but with 1 A saturated solution of safFranin in 50 p.c. alcohol, diluted with an equal volume of 50 p.c. alcohol. 32 ELEMENTARY PHYSIOLOGY. [iV dilute glycerine (equal volumes of glycerine, and water) instead of with strong glycerine; use a lifter to transfer the section to the slide; remove excess of fluid and cover. 5. When it is desired to keep sections mounted in glycerine, they may be treated in the following way. Place the slide and a small bottle of glycerine-jelly on a warm bath. There should be no glycerine beyond the edges of the cover-slip, but if there is, remove it with blotting-paper. With a small brush, brush a little glyceiine-jeUy round the edges of the cover-slip. Put the slide aside for a day or more, then brush gold size (zinc white, Brunswick black, Canada balsam will also serve) over the glycerine-jelly, 6. Double staining with haematozylin and eosin. Arrange watch-glasses as in § 1. Add one con- taining a saturated solution of eosin in 50 p.c. alcohol. Stain rather deeply with hsematoxylin ; after the stained section has been placed in 50 p.c. alcohol, place it in the eosin solution for one minute, then back again in 50 p.c. alcohol. Pass through the stronger alcohols, the clove oil and mount. If the section when in 95 p.c. alcohol has a blue tinge only, it should be placed once more in the eosin and passed more quickly through the alcohols. The nuclei are stained with hsematoxylin, the cell substance and most other tissue with eosin. If the tissue has been hardened in Miiller's fluid, potassium bichromate, or by brief treatment with osmic acid, the haemo- globin of the red corpuscles — ^which in these cases is usually preserved — will be stained orange with eosin. 7. After-staining vnth picrio acid. Picric acid may be used after any other stain. It is perhaps best after carmine or IV] STAINING AND MOUNTING SECTIONS. 33 hsematoxylini. The section is stained and treated in the usual way up to 75 p.c. alcohol. From this it is removed to a watch- glass containing picric acid* dissolved in 95 p.c. alcohol. After staying about two minutes in this, it is placed in 95 p.c. alcohol and moved about till a faint yellow tinge only is left. It is then transferred to clove oil and mounted. Picric acid stains red corpuscles brilliantly when the haemo- globin is preserved, it stains also elastic tissue and the horny layer of the skin ; it stains muscular tissue more readily than white fibrous tissue ; and striated duct cells more readily than the alveolar cells of glands. In slight excess it stains the whole section. 8. Hoematoxylin and rubin S^ with picric acid. Stain with haematoxylin. After the section has been passed through alcohols up to 75 p.c, place it for one minute in the rubin mixtvire. Trans- fer it to 95 p.c. alcohol, and treat in the usual way. If required, a little more picric tint may be given by treating it as in § 7, after it has been stained with the rubin mixture. Bubin stains especially the connective tissue, and is best used when a smaU amount only of this tissue is present. 9. Mounting separately, stained sections im- bedded in paraffin^ a. Place a section in a watch- glass, and pour over it a few drops of turpentine. See that the balsam and a clean cover-slip are ready. With a lifter transfer the section to a slide, tilt the slide and wipe away the excess of turpentine, then add a drop of balsam, and cover with a cover-slip, ' A piece of arytenoid cartilage may be taken. ° A 2 p.c. solution of picric acid in 95 p.c. alcohol. ^ Also called acid-magenta, and acid-fuchsin. For proportion of mixture see Appendix, p. 369. * A compact or fairly compact structure should be taten, as a piece of liver, pancreas, ureter or bladder, hardened in potassium bichromate. The piece should be stained with heemalum for a day before imbedding, for § 9, and left unstained for § 10. L. 3 34 ELEMENTARY PHYSIOLOGY. [iV 6. Mount another section, using xylol instead of turpentine. XyloU is very volatile, and the section after removal of excess of xylol will rapidly dry on the slide and become useless unless this is prevented by the addition of balsam. Xylol is preferable to turpentine for tissues that have been treated with osmic acid, since turpentine is apt to dissolve some of the stained fatty substances. 10. Staining separately sections imbedded in paraffin. Take two or three sections and dissolve the paraffin with turpentine as in § 9. Take up the sections on a thin glass rod, touch blotting-paper with them to remove excess of turpentine and place in 95 p.c. (or absolute) alcohol in a watch-glass. Leave for 5 to 10 minutes, then pass through successively weaker alcohols, stain and mount as in § 1 (cp. App. p. 371, for another method). 11. Mounting serial stained sections. Place a small drop of collodion dissolved in clove oil, on a slide. With a small brush (or with the finger) rub the drop so that it forms a very thin film over a portion of the slide a little larger than a cover-slip. Take the ribbon of nine sections^ given you, and cut it with a scalpel into three series of three sections each. Place these on the film of collodion so that the second series of three is below the first, and the third below the second. Press a small brush lightly on the centre of each section and roll it to right and left, so as to flatten the sections and make them stick to the slide. Place the slide on a warm bath at a temperature just sufficient to melt the paraffin. When the paraffin is melted, dip the slide into a bottle ' Benzol, toluol, and a mixture of four parts of turpentine with one part of creosote, are also used to dissolve paraffin. * A piece of small intestine hardened in mercuric chloride may be taken ; stained with hematoxylin for §§ 11, 12; unstained for § 13. IV] STAINING AND MOUNTING SECTIONS. 35 containing turpentine, for one to two minutes. Then wipe the turpentine from the lower surface of the slide, and stand the slide up to drain. Wipe away the turpentine on either side of the sections. Place a line • of dilute balsam on a cover-slip near one edge ; let this edge first touch the slide, and gently lower the cover- slip so that the balsam flows over the sections. 12. Flattening folded sections, a. Clean the slide by pouring on it a drop or two of strong spirit, and rubbing with a clean cloth. Place a flat basin of distilled water over a small flame, regulating the flame so that the temperature of the water is 35° to 37° 0. Place the sections on the water, they will spread out and become quite flat. Dip the slide in the water obliquely, draw the sections to the slide with a needle, hold the upper edge of the paraffin on the slide with the needle and gently lift the slide out of water, with the sections on it. Stand up the slide to let the water run off, so that the sections are in contact with the glass, place between smooth pads of blotting-paper, and touch lightly to remove most of the remaining water and to make the tissue adhere to the glass. Place on a bath at about 35° C. for half-an-hour or longer to dry. Then place on a warmer bath to melt the paraffin and mount as in §11- b. Clean a slide ; pour water on it, place the sections in the water. Place the slide on a warm bath. As the water becomes warm the sections will flatten. As soon as the sections are flat, remove the slide, let the water run off, and treat as after the corresponding stage in a. With delicate sections it is better not to remove excess of water with blotting-paper, but after 3—2 36 ELEMENTARY PHYSIOLOGY. [iV draining, to let the sectious dry in the warm bath ; in this case they should be left at 35° C. till next day. The drying period may be shortened thus : after the slide has been at 35° C. for half-an-hour (i) dip it in 95 p.c. alcohol, drain, and put on the bath for another half-an-hour or (ii) dip in absolute alcohol, drain, put in turpentine at once. c. Proceed as in 6, but use 50 p.c. spirit instead of water. 13. Staining serial sections on the slide\ a. The several fluids required (turpentine or xylol, alcohols, staining agents) are arranged in a row in bottles without necks, and of such size that the shde just fits into them. Proceed at first as in § 12, either a, h, or c. If the shde taken was clean and the process has been properly carried out, the sections will in most cases adhere to the slide during the subsequent staining and mounting. After the paraffin has been dissolved in turpentine or xylol ; wipe ofi' excess of fluid and place in absolute alcohol for two or three minutes ; transfer for a minute to 95 p.c. alcohol, and so on through the successive alcohols to the staining agent, e.g. Delafield's hsematoxylin. When the section is stained wipe away excess of staining fluid, pass back through the alcohols, removing excess of fluids at each transfer. After absolute alcohol, leave the shde in turpentine or xylol for a couple of minutes ; then stand it up to drain ; mount as in §11- Some sections, especially those of tissues which have been kept long in osmic acid or in chromium compounds, are apt to come away from the slide when treated as abova In such cases a fixative is used as in 5, and o, following. h. If the sections do not require to be flattened, rub the slide with a mixture of egg-albumin and glycerine in the manner in which collodion was used in § 11. ' The sections may be fixed to the cover-slip instead of to the slide. This method does not require so large a quantity of the varions fluids, and there is less chance of their being spoilt. IV] STAINING AND MOUNTING SECTIONS. 37 c. If the sections require flattening, proceed as in § 12, 6, using dilute egg-albumin (2 c.c. filtered white of egg in 100 c.e. water) instead of water. In either case when the paraffin has been dissolved from the sections, leave the slide for 5 to 10 minutes in absolute alcohol, then pass through alcohols and stain as in a. In this process the alcohol fixes the sections to the slide by coagulating the film of egg-albumin. The drawback of the process is that the egg-albumin is stained by most reagents. LESSON V. SECTION CUTTING AND IMBEDDING, 1. Preparation of Hardened Tissue for Freez- ing. If the piece of tissue to be cut has been kept in alcohol, place it in 50 p.c. and then in 30 p.c. alcohol, each for about 10 minutes. Remove the tissue to water for 1 to 3 hours in order to extract the alcohol. The extraction is hastened by placing on the warm bath, at about 38° 0. Place then in dilute gum^ for an hour to a day as convenient. Successful section cutting depends largely upon using a sharp razor, it should not be markedly hollow ground, or it may bend and give sections consisting of bands of unequal thickness. The razor should be placed flat on a microscope stage, and the edge examined with 1 The gum solution is prepared by dissolving gum arable in sufScient warm water to make a freely flowing solution, and filtering through linen. When it is required to keep the piece of tissue in a given position whilst freezing, it may be transferred to a thick solution of gum a few minutes before placing it in the plate of the microtome ; it can then be kept in position by a needle for the short time that elapses before it is frozen. Since gum alone freezes to a hard mass, a little sugar solution may with advantage be added to it. V] SECTION CUTTING AND IMBEDDING. 39 a low power, if there are notches in it it requires re-grinding. 2. Ice and Salt Freezing IVIicrotome. Ice is broken into fragments about the size of walnuts ; salt is sprinkled liberally over the ice ; and the mixture packed in the box of the microtome'. The plate of the microtome remains below freezing-point for 2 to 3 hours. Clean the plate of the microtome and ■ the grooves in it. Place the piece of tissue (given you in gum) in the centre of the plate. If there is much gum around the tissue cut it away after it is frozen. Take the tripod frame which carries the razor, and see that the razor is firmly fixed by the screws. Then adjust the level of the razor by means of the three screws supporting the frame in the following manner : — Screw up the front screw till ^ to | of it is below the frame. Adjust the back right-hand screw so that the right-hand edge of the razor will just cut into the frozen gum over the tissue ; adjust similarly the back left-hand screw; repeat these two adjustments, so that whatever part of the razor is used in cutting, sections of nearly the same thickness will be obtained. Hold the frame firmly, but without unnecessary pressure, and push it straight forward over the tissue, this should cut off a portion of the gum. Lift up the frame — or its anterior end,— carry it back, turn down- ward the front screw ^th of a revolution, push the 1 The freezing mixture made by pounding together equal parts of ice and salt causes too great a lowering of temperature, and the frozen tissue is less easy to cut. 4U ELEMENTARY PHYSIOLOGY. [V frame straight forward again, and so on. In a short time, sections of the tissue will be obtained. With a small brush moistened with water remove each section, as it is obtained, from the razor, and float it off in a dish of water. A number of sections may be cut, and these removed together from the razor, but the sections are apt to become entangled and they are generally less perfect than the sections which are removed sepa- rately as they are cut. After cutting some sections, of a thickness corre- sponding to ^th of a revolution of the front screw, cut others, turning the screw less than ^th between each section and note the smallest angle through which the screw can be turned, complete sections still being ob- tained. Stain and mount the sections. (See Lesson iv.) It must be remembered that the gum unless dissolved out of the sections will form a granular precipitate in them when they are treated with strong alcohol. After the sections are cut, carefully clean the razor and the plate of the microtome. The proper rate to carry the razor through the tissue varies with the temperature of the frozen mass ; when it is a few degrees only below zero, the movement may be the quickest possible ; when it is frozen hard, it should be carried slowly through the tissue, other- wise the sections are apt to curl or break up. Note. In cutting large sections, it is best to place on the razor a number of small drops of water and to cut slowly ; the section folds up on the razor. The razor (with the frame) should then be dipped under water and the section floated off ; it should be taken out on a glass slide and treated on the slide with 30, 50, 76 p.o. alcohol, etc. ; V] SECTION CUTTING AND IMBEDDING. 41 care should be taken to remove as much as possible of the clearing agent, otherwise the Canada balsam in which the section is mounted may remain a long time fluid. 3. Ether Freezing Microtome. The fine nozzle of the ether spray apparatus is easily stopped up by fine particles of dust. To avoid this, see that the bottle to contain the ether is clean, and filter into it the ether to be used. The piece of tissue should not be more than 3 to 4 mm. thick ; for the upper surface of pieces thicker than this freezes slowly. Before beginning the ether spray, adjust the razor (see § 2) so that the edge just touches the top of the tissue as it lies on the plate ; place a brush and dish of water in readiness. Then set the ether spray going by means of the hand bellows, keeping the second bag of the bellows just pressing against the net sur- rounding it. The tissue should be frozen in about a minute, the time varying with the temperature of the room. If the freezing does not begin rapidly look at the spray, if there is not a good jet, the nozzle of the apparatus probably requires cleaning out. As soon as the tissue is frozen, begin cutting sections as with the ice and salt microtome. Immediately the gum on the plate at the edge of the tissue begins to lose its dense white colour, cease cutting sections, and ply the ether spray to re-freeze. Then cut more sections. When the sections are out, clean the razor and the microtome plate. 4. Cutting Fresh Tissiies. A piece of tissue fresh from the body may be placed direct on the microtome plate and frozen. It is best to cut it a few degrees only below freezing-point. The ice rapidly blunts the razor, and ice crystals are apt to distort ■ and tear delicate structures. This can be more or less avoided by placing the piece of tissue for 5 to 15 minutes in dilute gum, this however causes some changes in the tissue cells. a. Cut sections of e.g. a piece of rat's kidney. Transfer the sections to salt solution 0'9 p.c. and with a brush gently unfold them. Dip a slide obliquely under a section, with a needle hold one corner of the section on the slide, lift up the slide, if necessary pull out the edges of the section so that it lies flat and unfolded, 42 ELEMENTARY PHYSIOLOGY. [V drain off the salt solution. Treat another section in the same way. Place a cover-glass on one of the sections and examine it. (A drop of dilute methylene blue or Spiller's purple may be added to another section, in 5 minutes the drop drained away, and a cover-glass put on.) Let two or three drops of 95 p.c. alcohol fall on the oth^. Leave it for a quarter of an hour or longer, then stain e.g. with hsematoxylin and mount in Canada balsam. b. Cut sections of e.g. a small lymphatic gland. Transfer the sections to 30 p.c. alcohol. With a brush move them about in the alcohol to remove some of the leucocytes. Stain and mount. (Sections after unfolding in 30 p.c. alcohol may be transferred flat on a lifter to any of the usual hardening agents.) 5. Imbedding in Paraffin. Take the piece of tissue given you, which has already been stained in bulk'. Place it for \ hour in 95 p.c. alcohol, and for \ hour in absolute alcohol. Transfer it to a little turpentine'' in a watch-glass, cover up and place on a warm bath at about 45° C. for 5 to 10 min. Place on the warm bath a pair of forceps and a piece of blotting- paper. Take up the tissue with the warm forceps, drop it on the blotting-paper, and roll it over, wipe the points of the forceps, take up the tissue and drop it into one of the dishes of melted paraffin in the paraffin bath. The paraffin used should have a melting point of about 45° 0. Leave the tissue in the paraffin for J hour to 3 hours according to its size. ' The piece of tissue should if practicable only be a few millimetres thick. It is stained in bulk by leaving it for 1 to 2 days in acid haematozylin or alum carmine. It is washed in a stream of water for several hoars, and then passed through the alcohols (cp. p. 29). ^ When the student has become accustomed to imbedding, xylol or cedar-wood oil should be used in place of turpentine (cf. Less. iv. § 9, b). V] SECTION CUTTING AND IMBEDDING. 43 Rub the inside of a watch-glass with glycerine, removing excess so that the glycerine only remains as a smear. If in the paraffin dish there is more than about half a watch-glass full of paraffin, pour the excess into another dish. Light a Bunsen burner, and have ready a piece of wire or an old scalpel. Pour the paraffin with the tissue into the watch-glass ; warm the wire in the Bunsen flame, and with it adjust the tissue so that the face to be cut is towards the edge of the watch-glass ; and if air-bubbles are present remove them by means of the heated wire. If the paraffin begins to set round the tissue, when an air-bubble still remains attached to it, heat the wire and melt all the paraffin around the tissue. Carefully take up the watch-glass and place it in a shallow dish of cold water ; when the paraffin has set, immerse the watch-glass cautiously in water; then place the glass surface under a stream of water from a tap. The more quickly the paraffin is cooled, the less chance there is of crystals forming, and the easier it will be to cut. Cut away the paraffin on each side of the tissue, and remove the block with the imbedded tissue. Cut the paraffin into a rectangular block, cutting away the paraffin close up to the tissue on the face to be cut and on the adjoining faces, but leaving 2 to 3 mm. of paraffin on the face opposite to that to be cut. A piece of smooth glass is given you with a central mass of paraffin fixed to it. Heat a scalpel in a Bunsen burner and melt the central part of the paraffin, on this place the paraffin end of the block with the imbedded tissue. Pass the heated scalpel once or twice 44 ELEMENTARY PHYSIOLOGY. [V along the point of junction of the paraflSn mass and the block, to melt it thoroughly and make a good union. With the heated scalpel, heap up a little paraffin about the base of the block. Leave for a minute, then cool under the tap. 6. Cutting sections. Take the tripod razor, adjust as in § 2, and cut sections in a similar manner, carrying the razor rapidly through the tissue, and using the same portion of the razor. The successive sections will stick together and form a ribbon. When a ribbon of half-a-dozen sections has been made, place it aside on a piece of paper, and cut a fresh ribbon. The sections are mounted as in Lesson IV. § 9, 11, or 12. Note. The method given above, viz., of pouring paraffin with the tissue into a watch-glass serves very well for small objects ; but as the surface of the watch-glass is curved, it is better when objects more than half a centimetre in length are to be imbedded, to use instead of the watch-glass, either a small card-board or paper box, or two L-shaped imbedding frames. The latter are made of brass or of lead, with sides about a centimetre high ; they are placed on a glass slide so that the long limb of each is in contact with the short hmb of the other, they thus enclose a space, the size of which can be varied according to the size of the tissue ; into the space paraffin with the tissue are poured, and the tissue is adjusted by a hot scalpel. 7. The Rocking Microtome. Note the mechanism of the microtome (Pig. 2). Turn up and down the large screw at the end of the lever, and note the extent of the movement of the anterior end of the lever which carries the paraffin. Turn similarly the miUed wheel at the base of the screw. PuU the handle backwards and forwards, note that in pulling it forward, a catch engages with the milled wheel and pushes it round, i.e. pushes the paraffin on the lever towards the razor. Move V] SECTION CUTTING AND IMBEDDING. 45 backwards and forwards the guard of the catch, the position of this determines how far the wheel is pushed round by the catch, I.e. the thickness of the section. Pull the handle forward, and then slowly move it back, counting the clicks of the catch as it passes over the teeth of the milling ; adjust the guard so that there are 8 to 10 clicks. Eight teeth give sections approximately 5 /I in thickness. Procedure. Imbed the tissue as in § 5, and shape the paraffin mto a rectangular block. Take the paraffin carrier from the microtome. On this fix the imbedded tissue, thoroughly melting the adjoining surfaces of the paraffin by means of a heated knife. With a sharp scalpel, carefully shape the part of the block sur- rounding the tissue so that the surface to be out is rectangular. Screw the large screw of the microtome downwards as far as it will go. Fix the carrier to the microtome, adjusting it so that the paraffin reaches nearly up to the position to be taken by the razor. Fix the razor firmly in the frame. Leave the right arm on the end of the lever carrying the paraffin so as to bring the paraffin on a level with the edge of a razor. Adjust the paraffin carrier so that the edge of one block to be cut is parallel to the edge of the razor and about half a millimetre from it ; in doing 46 ELEMENTARY PHYSIOLOGY. [V this, do not put the left hand over the razor, but adjust the carrier from the right-hand side. Screw up firmly the carrier. Push the guard of the catch back as far as it will go, and move the handle until the first section is obtained, then adjust the guard so that the catch turns the milled wheel 8 to 10 teeth. Each forward movement of the handle now gives a section which is flat and adheres to. the preceding section. When 3 or 4 are obtained, lift them up with a forceps, so that the ribbon of sections runs free of the razor. The chief causes of bad sections are (1) over-hardness of the tissue ; this was normal to the tissue, or produced by warming in agents such as xylol, or to overheating in the warm bath; (2) imperfect imbedding ; this may be due to a lack of sufficient penetration of the paraffin, to crystals of paraffin having been formed, or to the presence of small bubbles of air. The siuface of the block to be cut should be examined with a lens after it has been shaped, if patches of crystals or bubbles are visible it is best to re-imbed; (3) to bluntness or notches in the razor. 8. Cutting sections in pwraffin meltinff at 58° 0. Paraffin of high melting point allows thinner sections to be cut than paraffin of low melting point. On this account paraffin melting at 58° C. is sometimes used. But sections of the paraffin melting at 58° 0. are apt to curl and do not adhere to a ribbon at ordinary room temperatures. They form a ribbon, however, if the block is coated with paraffin of a low melting point. Imbed a piece of tissue in paraffin melting at 58° 0., shape the block, and fix it in the usual way to the carrier of the microtome. Then dip the block up to the base in melted paraffin of melting point about 40° C, leave it there for 2 to 3 seconds, then lift it out, and hold it vertically for about 10 seconds for the paraffin to set. Put it aside for a minute or two to cool (or cool it in water, removing afterwards the adhering water). With a sharp scalpel cut away the soft paraffin from the surface to be cut, and from the sides of the block, leaving it only on the sides which will be parallel to the razor edge in cutting. Examine the junction of the hard and soft paraffin with a lens. If the two do not adhere, the process of re-coating should be repeated. V] SECTION CUTTING AND IMBEDDING. 47 9. Superficial Imbedding. This can be employed with small firm pieces of tissue. It is often advantageous with portions of the nervous system treated by the Golgi-Cajal method (cp. p. 341). The piece of tissue is placed in 95 p.c. (or absolute) alcohol for 5 to 15 minutes. The imbedding mixture consists of 2 parts of hard paraffin and 1 part of vaseline. Sections of this curl less than sections of hard paraffin ; and fold less than soft paraffin. Take a short oblong or cylindrical block of paraffin-mixture. Scoop out a small hole at an end of it. Take the tissue up with forceps, roll it over on blotting-paper to remove excess of alcohol, but without allowing it to dry, dry the points of the forceps, and place the tissue in the hole in the paraffin. With a warm suction pipette, fill the hole with melted paraffin mixture. Heat a needle or wire in a Bnnsen flame ; and with it remove any bubbles that may be present, being careful not to touch the tissue with the wire when it is over- heated. Let the paraffin cool a little, then cool it further in water. Pare down the paraffin to the surface of the tissue, and level the edges. Blow strong alcohol on the razor from a wash-bottle and cut sections free hand, removing the section to 95 p.c. alcohol ; the tissue must throughout be kept moistened with alcohol. The sections can be at once transferred to clove oil and mounted, or if the paraffin has penetrated into the outer layers, they may be transferred to absolute alcohol, then to xylol and mounted. If an unstained piece of tissue has been taJ^eu the sections may be stained in the usual way. LESSON VI. 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. a. Mount it in '6 p.c. sodium chloride solution" and with a high power note that The matrix is studded at tolerably regular intervals with cartilage cells or corpuscles. 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 trans- parent. Most of the cells entirely fill up the cavities in which they lie. Along the cut edge some cavities from which the cells have fallen out may generally be seen. Except at the free edge, the cells are two or more layers deep. ' Instead of this, sections of the head of the humerus or femur of any young animal may be made. ' See note below. VI] HYALINE CARTILAGE. 49 The matrix is hyaline or faintly granular, and is comparatively (cp. §§ 3, 4) in small quantity. If the newts are not quite young the cartilage will vary from the above in the quantity of matrix, the shape and arrangement of the cells, and in the cells possessing small fat globules and often two nuclei. b. Irrigate the piece with acetic acid 1 p.c. The following changes will be seen as the irrigation pro- ceeds : The nucleus becomes much more granular and distinct. The cell substance becomes granular and more or less hides the nucleus. The cell substance becomes transparent. The cell substance shrinks from the matrix, and presents an irregularly serrated border. Note the space thus formed between the cell and the matrix. Ifote. Examination of fresh tissues. When a piece of tissue is examined fresh, in order to see as nearly as possible its appearance during life, it must on no account be washed or mounted in water, since water causes rapid and extensive alteration. The best method, as a rule, is to remove a small piece of fresh tissue as quickly as possible to a slide, and to put on a cover-slip at once without adding any fluid. Drying may be retarded by putting a little moist blotting-paper round the edges of the cover-slip. When fluid is added to the tissue, it should be one which causes as little change as possible. A "6 p.c. to '75 p.c. solution of sodium chloride in tap water is used for the tissues of the frog and newt, and a "75 p.c. to '9 p.c. L. 4 50 ELEMENTARY PHYSIOLOGY. [VI solution for the tissues of the mammal. These are called normal salt solutions. The tissue should not, if it is avoidable, be soaked in the normal salt solution, but only moistened with it. In most cases, fresh aqueous humour, and fresh blood serum, may with advantage be used, instead of normal saline. 2. Place a small piece of newt's cartilage in gold chloride solution "5 p.c. 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 acidulated with acetic acid ; leave it exposed to the light. When it has become a red-purple colour, mount it in formic glycerine. The reduction takes a few hours only in bright sunlight, and one to two days in cloudy weather. 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. 3. Transverse sections of a cartilaginous rib (picric acid)' of a fully grown, but not old, animal. Stain one section in picrocarmine for J hour to 1 hour, and another in acid hsematoxylin for 15 to 20 minutes. Mount the former in glycerine, and the latter in balsam. 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 carti- lage (capsule) around each cell, sometimes the whole of ' The fixing agent in which the tissue is placed on being removed from the body is here and later given in brackets. The details of treatment are given in the Appendix. VI] HYALINE CARTILAGE. 51 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. 4. Take a piece of costal cartilage from a recently killed oldish animal; cut thin transverse sections either free hand or with a freezing microtome and transfer them with a brush to a watch-glass containing normal salt solution. Mount one section in normal salt solution. Place another in two or three drops of osmic acid "5 p.c. in a watch-glass and cover it up. a. In the former observe under a high power, that The cells frequently show signs of partial degenera- tion, containing fat globules which may be nearly as large as the cell ; these are very highly refractive and so have a very distinct outline. In places the matrix may be fibrillated and towards the centre of the section there may be a nodular semi- opaque deposit of lime salts. 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 connective tissue, cp. Lesson vn. §2). b. Mount in dilute glycerine, and examine the section which has been treated with osmic acid ; the fat globules are stained a deep brown-black, the cell substance, the nuclei and the matrix are but slightly stained. On keeping, the fat globules become black. 4—2 52 ELEMENTARY PHYSIOLOGY. [VI 5. Sections of cartilage from the head of a cuttle- fish (picric acid). Stain a section with acid hssmato- xylin, wash it, and mount in glycerine. If it is too deeply stained, place it in a little 1 p.c. acetic acid and when it is gufificiently decolorised, wash out the acid. Observe a. The groups of cartilage cells. h. The marked processes proceeding from some of the cells. Sometimes the processes of neighbouring groups may be seen to join. 6. Take a small piece of the ear of a freshly killed mouse or young rat, remove the skin and scrape away the tissue smrounding the ear-cartilage. Mount the cartilage in normal saline solution. Note the cartilage cells closely packed together, most have one or more refractive fat globules in them. (The fat increases up to a certain Umit with the age of the animal.) Unmount, stain the specimens with osmic acid. DEMONSTRATION. Transverse section of ear of adult rat. (Alcohol, acid hsematoxylin, or gold chloride and formic acid.) Note the parenchymatous cartilage. The cell-spaces, generally polygonal, are separated by very narrow partitions of matrix. The spaces contain very little cell substance. LESSON VII. CONNECTIVE TISSUE. 1. Elastic Tissue. Tease out in water a morsel of ligamentum nuchse, e.g. of ox. It is almost entirely made up of rather large, branchingj and anastomosing fibres having distinct outlines, and curling at their ends. Irrigate with acetic acid (1 to . 5 p.c.) ; the elastic fibres are unaffected; the small amount of white fibrous tissue present, swells up ; a few nuclei only come into view. 2. Tendon. Cut off the tip of the tail of a mouse or young rat ; cut through the skin for a short distance, break off a tail vertebra, and pull straight outwards. A bundle of small tendons will be obtained. (A similar bundle can be obtained in the same way from each vertebra.) Gently stretch this over a glass slide from edge to edge, and hold it in this position till the edges are dry, touching the mid portion with a brush moistened with normal saline solution ; the fibres are thus kept extended. Cover with a cover-slip. Note the white fibres consisting of parallel bundles 54 ELEMENTARY PHYSIOLOGY. [VII of wavy fibrillse. Slowly irrigate with acetic acid 1 p.c, watching the while under a high power. Between the bundles of fibrillse rows of cells will become visible. Note in each the round or oval nucleus, the rectangular outline of the cell, and the finely granular cell-substance. Indications 'of flange-like lateral processes may be seen as lines running 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 indistinct, and between the swollen bundles scarcely anything is seen but rows of elongated shrunken nuclei. (Cp. Action of acetic acid on cartilage cells. Less. VL § 1 6.) 3. Place a piece of tendon (see § 2) in a saturated aqueous solution of picric acid for a day. Wash with water. Stain deeply with picrocarmine. Wash with water. Take a small piece of the tendon, tease one end carefully, without adding fluid, using very fine needles ; add a drop of water, cover, and examine with a high power. Note the very small fibrillse, now more or less separated from one another. 4. Pull out another bundle of tendons : place them in '2 p.c. nitrate of silver for 5 to 10 minutes. Remove to water, move them to and fro once or twice, and gently separate them, renew the water and expose to light, now and then turning them over. In about half-an-hour pass a tendon through alcohols to clove oil. Place on a slide and examine with a low power. The la^/er of epithelioid cells covering the tendon, will in places be seen (some are often rubbed off in pulling out the tendon from the tail). Out out the successful pieces, mount in balsam, and examine with a high power. 5. Transverse sections of a rather large tendon' such as that of the digastric muscle of a cat or rabbit. ' A tendon may be fixed by placing it fresh in 30 p.c. alcohol for a, few hours, and then in 7S p.c. alcohol; or by placing it in a saturated aqueous solution of corrosive sublimate for a day. VIl] CONNECTIVE TISSUE. 55 Stain and mount in balsam. Note the connective tissue sheath aroutid the tendon, the septa from the sheath dividing the tendon into bundles, the branching tendon-oells between the white fibres of the tendon. 6. Branched connective tissue cells of cor- nea. Take a pithed frog, cut away the nictitating membrane, squeeze the side of the head to make the eye bulge out, then slice boldly at the edge of the cornea; take up its edge with forceps, and with fine scissors cut through the attached part at its junction with the sclerotic; 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. Put it in gold chloride "5 p.c. solution, gently extend it into its normal shape with two finely pointed glass-rods, leave it for thirty to forty minutes, wash well with water, transfer to about 20 c.c. of 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 carefully scrape both surfaces to remove the epithelium, examine it under a low power to see that the epithelium is removed, when it is, mount in formic glycerine and examine it under a high power. If the cornea does not become properly stained in a day, place it in a test- tube with a saturated or nearly saturated solution of tartaric acid, and heat for a few minutes to a tempera- ture at which the test-tube can barely be held in the palm of the hand. This will probably complete the reduction, 1 Probably it will not be stained until the following day. 56 ELEMENTARY PHYSIOLOGY. [VII Note the stained connective tissue corpuscles, with numerous fine branching processes which anastomose with the similar processes of neighbouring cells. This preparation should be preserved for examination of the nerves of the cornea (Lesson XIV. § 11). Branched ccmnective tissue ceUs aie well seen in the tail of the tadpole (Perenyi's fluid) stained with pierocarmine. The surface epithelium is removed by brushing or partial teasing. The prepara- tion may also show a stage in the deyelopment of capillaries. 7. Areolar Tissue. Cut transversely through the skin of the back of a recently killed frog. Turn the lower piece of skin downwards, cutting it through at the sides. Note the connective tissue stretching from the skin to the body at the level of the posterior end of the urostyle. Using fine-pointed scissors and forceps, cut out on one side this piece of connective tissue. Shake it in normal salt solution to wash away blood ; touch blotting- paper with it to remove excess of fluid, and put it on a dry slide. Spread out the piece of tissue with needles ; drawing out gently the edges all round till they adhere to the slide. As soon as it remains extended, put on a cover-slip, and lightly press it down; this must be done before the tissue becomes dry. The tissue is chiefly composed of white fibres having a wavy course, and more or less distinct fibrillation; the bundles are of unequal size and run across one another in all directions. Some small elastic fibres running singly will also be seen, their outlines are more distinct than those of the bundles of white fibres ; they branch and anastomose freely with one another. Where the film is well stretched the elastic fibres run VII] CONNECTIVE TISSUE. 57 for the most part quite straight, elsewhere they are curled at their ends and have a more or less sinuous course. Irrigate with acetic acid, the elastic network then becomes distinct. (The stretching of a piece of tissue on a slide or cover-slip as above, so that it adheres but is not allowed to dry, will be spoken of as making a moist film. It is often simpler to extend the tissue by smoothing it outwards with the fingers, instead of using needles. The film may be fixed by adding a few drops of 95 p.c. alcohol, and leaving it for half-an-hour, covered up. This will be spoken of as fiidng a moist film with alcohol. The film may then be stained.) 8. Inter-musovlar connective tissue of frog. Make a complete transverse incision of the skin of a pithed frog, in the abdominal region. Tear the skin backwards so as to expose the thigh muscles. With forceps lift up the muscles of the ventral surface of the thigh near the knee, and cut away all the muscles over the femur. Place the muscular mass on cork, deep surface uppermost, and gently pull laterally the muscles on either side of the sartorius, a thin layer of connective tissue on either side of the sartorius will come into view. Pin out the muscles so that the connective tissue layer is stretched, wash it with salt solution to remove adhering blood, pour over it 95 p.c. alcohol and leave for half-an-hom". Then cut out the connective tissue and stain it with hsematoxylin and eosin. The connective tissue fibres will be stained with eosin, the nuclei of the cells with hsamatoxylin. A similar preparation may be made of the connective tissue between the muscles of the rat or rabbit. In this case, the piece of tissue between the muscles may be picked up with forceps, cut out, and spread out to make a moist film in the manner given in § 7. When the edges are dry, the tissue is fixed with alcohol. 58 ELEMENTARY PHYSIOLOGY. [VII 9. Dissect back a piece of skin of a recently killed young rat, 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 from the small bullae of tissue so raised. After a few minutes snip off with a sharp pair of scissors one or two pieces as thin as possible, and place them in a watch-glass containing 25 p.c. formic acid^. When the pieces are well coloured, which wUl probably be in two or three hours, shake them gently in a glass containing distilled water. Spread out a piece on a slide till the edges adhere (cp. § 6), put a small drop of formic glycerine in the centre of a cover-slip and lower it on the preparation and press gently. The preparation will improve in a day or two. Observe a. The usually angular 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. 6. Connective tissue corpuscles rather larger than the leuco- cytes, and having processes which, in some cases, are seen to be connected with the processes of other similar cells. They are however swollen and indistinct. c. Larger, more rectangular cells, with coarsely granular cell substance and oval nuclei ; they are often in rows especially by the blood vessels ; on a side view these appear as thin, long cells. These are sometimes called plasma cells. This preparation should be preserved for the observation of fat tissue. 10. Make on a cover-slip a moist film of connective tissue from a rat (cp. § 7). Hold it for a minute in methylene blue dissolved in, 75 p.c. spirit (cp. Less. I., § 14), dip it in 75 p.c. spirit, and at once press lightly between sheets of blotting-paper. Put it aside to dry. When dry, add a drop of balsam and mount. Note that the coarse granules of the cells mentioned in § 8 c. stain deeply with methylene blue. 1 Formic acid 1'06 sp. gr. VIl] CONNECTIVE TISSUE. 59 11. Snip off a small piece of the subcutaneous tissue in a foetal or new-born mammaU. Spread it out on a slide till the edges stick, then (a) -fix with alcohol and stain with hsematoxylin and eosin, or (6) treat '5 p.c. gold chloride on the slide, for 30 to 45 minutes, wash, reduce with 25 p.c. formic acid, or (c) add a drop or two of 1 p.c. aqueous solution of Spiller's purple or methylene blue, leave for a minute or two, wash with water, and mount in water. Note the cells of various shapes, for the most part with faint outlines, but with distinct and rather large nuclei ; irrigate with dilute acetic acid, watching closely the early changes produced. This may also serve to show the development of fat-cells. 12. Note in the section of costal cartilage prepared in Lesson vi. § 6 the connective tissue layer outside and closely attached to it forming the perichondrium; in places sections of tendons running into the cartilage will probably be seen. 13. Pigment cells. Pin out on a frog-board one of the webs between the toes of the frog used in §6, 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. Cut off the foot, and pin out on cork the web between two of the toes. Pour a little 95 p.c. alcohol 1 An animal just lulled serves best, but one preserved In piorio 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 trans- ferred to alcohol stronger than 50"/„. 60 ELEMENTARY PHYSIOLOGY. [VII on the foot. In about 5 minutes cut out the web, and put it in 95 p.c. alcohol in a watch-glass. In a quarter of an hour transfer to fresh 95 p.c. alcohol for another quarter of an hour. Then remove excess of alcohol by pressing the web for a moment with dry blotting-paper, and place in oil of cloves. When the web is transparent, mount in balsam; and examine again the pigment- cells. Pigment-cells will be seen later in preparations of the mesentery, and of the lymphatica cistema magna (Lesson xxv. §§ 1, 2). 14. Fat-cells. 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 groups of highly re- fractive fat-cells. b. Under a high power, the variable size of the fat-cells, the apparent absence of a nucleus, the con- nective tissue passing between and over the cells. 15. Place a similar piece of omentum in osmic acid ■5 p.c. for about half-an-hour, wash with water, cut in half, mount one half at once in dilute glycerine, and the other half, after staining it for thirty minutes in picrocarmine. Observe that the fat-cells are stained a deep brown-black (cp. Less. vi. § 4, 6). If the tissue before being mounted be kept in alcohol for a day the tint of the fat globules will become deep black. 16. Take a small piece of omentum, which has been kept for some time in alcohol, stain it deeply with VIl] CONNECTIVE TISSUE. 61 hajmatoxylin, pass through the alcohols to turpentine or xylol and leave for a quarter of an hour. Mount in balsam. Observe the groups of shrunken cells from which the fat has been thus removed. Note the small amount of cell substance forming the boundary of a cell, and the flattened deeply stained nucleus in the cell substance. 17. In the gold chloride preparation of subcutaneous tissue made above, § 9, observe The network of capillaries in a small collection of fat-cells. In some fat-ceUs a nucleus and a small amount of protoplasm surrounding the fat may be seen. DEMONSTEATIONS. 1. FibrillsB of tendon under a high power, cp. § 3. 2. Epithelioid cells covering tendon, cp. § 4. 3. Gold chloride preparation to show cells of areolar connective tissue of rat, cp. § 9. 4. Film preparation of connective tissue stained with methylene blue, cp. § 10. 5. Section of umbilical cord to show mucous con- nective tissue. LESSON VIII. FIBRO-CARTILAGE. ELASTIC CARTILAGE. BONE. TEETH. 1. Fibro-cartilage. Sections of semi-lunar carti- lage of knee joint of dog (picric acid ; cut frozen) cut parallel with the mass of the fibres. Stain one section with picrocarmine and mount in formic glycerine, stain another in hsematoxylin and mount in balsam. Note The bundles of white fibrous tissue. The rather large cartilage cells lying here and there between the bundles of fibres, and usually in rows; each is surrounded by a capsule consisting of a small amount of hyaline cartilage. 2. Sections of intervertebral disci with adjoining faces of the vertebrae, cut at right angles to the faces of the disc. Stain with hsematoxylin or picrocarmine. Note, The intervertebral disc, consisting partly of fibro-cartilage, partly of ligamentous tissue. The bundles cross, chiefly in two directions at right angles to, and parallel to, the faces of the disc. ' Babbit. Eemove the Bpinal cord. Take several successive vertebrea. Saw through them about 3 mm., from the intervertebral disc. Place the discs with the bone attached, in a hardening and decalcifying fluid (cp. App. p. 364). VIIl] ELASTIC CARTILAGE. BONE. TEETH. 63 The fibro-cartilage passing on the faces of the disc either into a thin layer of hyaline cartilage or directly into the decalcified bone, and on its surface into fibrous connective tissue. A thin layer of spongy bone, the spaces containing red marrow with a considerable number of fat-cells. A little farther from the disc, a layer of cartilage, with cells in rows as if ossifying ; and a little farther still, spongy bone with red marrow and fat-cells. 3. Elastic Cartilage and transition to Hyaline Cartilage. Vertical sections of sheep's arytenoid carti- lage * (picric acid). Stain (a) one section with picro- carmine, and another (6) first with hsematoxylin and then with dilute aqueous picric acid; mount in glycerine. Examine (h. p.), proceeding from the deeper part of the section to the free edge. Observe — In (a) the hyaline cartilage, showing at its upper border, fine granular fibres — the elastic fibres— in the matrix. These rapidly increase, till the matrix consists largely of a close meshwork of fibres, leaving a hyaline portion immediately around the cells. In places, the elastic substance may take the form of thick nodular bars. Externally, areolar tissue ; note the transition of the elastic cartilage into this by the elongation of the cells, the disappearance of the capsules, and the substitution of white fibrous tissue (stained red) for elastic fibres. In (b) the elastic meshwork stained brilliant yellow, and the hyaline substance around the cartilage cells * In the sheep the deeper part of the arytenoid cartilage is hyaline. The cartilage is cut through this, the apex cut off and the mucous membrane removed. The epiglottis may be taken instead of the arytenoid, but it does not contain hyaline cartilage. 64 ELEMENTARY PHYSIOLOGY. [VIII stained blue. The hyaline substance, being stained, appears to be much more abundant than in (a). Here and there a patch may be seen with very little blue stained substance around the cells, this is elastic fibro- cartilage. 4. Structure of Bone. Transverse section through the shaft of a long bone. (The bone is dried; thin pieces are cut with a saw, ground down and mounted in balsam.) a. Examine under a low power. Most of the smaller spaces {i.e. Haversian canals, lacunae, canaUcuU) will be filled with air or debris and will therefore appear dark. Observe The central cavity surrounded by a small amount of spongy bone 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. In the compact bone the Haversian systems, each consisting of a Haversian canal surrounded by con- centric lamellae, which are chiefly marked off from one another by the lacunae which lie between them. The intersystemic lamellas between the Haversian systems ; in the outer part of the bone these run for the most part parallel with the surface (circumferential lamellae). In the spongy bone the lamellae run in the main concentric with the spaces. b. Examine under a high power. Observe The irregularly fusiform lacunae giving off numerous wavy branches, the canaliculi, which run across the lamellae to join the similar branches of neighbouring vm] ELASTIC CARTILAGE. BONE. TEETH. 65 lacunsB. In addition to this connection, canaliculi open into the Haversian canals in the compact bone, into the Haversian spaces and central cavity in the spongy bone. 5. Longitudinal section of the shaft of a long bone. (Prepared as in §4.) a. Examine with a low power. Observe The Haversian canals running in the main parallel with the surface of the bone, they have connecting branches and open here and there on the surface and into the central spaces. The lamellae running for the most part parallel with the Haversian canals. Where a Haversian system is cut obliquely the lamellse are concentric with the canal. h. Examine under a high power. * The individual lacuna are much as in the transverse section, but rather longer; observe the arrangement of the canali- culi (cp. § 4, h). 6. Transverse sections (in paraffin) through the shaft of a decalcified and stained long boneS as femur of rat. Mount in balsam. Note The periosteum formed of connective tissue closely attached to the bone. In the bone, each lacuna is occupied by a cell, the bone corpuscle, probably shrunken from the walls of the lacuna. The canaliculi are scarcely visible. ' For methods of decalcification op. Appendix. The tissue may be stained in bulk with picrocarmine, but it is better to stain the sections on the slide (Less. iv. § l.B). 1. 6 66 ELBMENTAKY PHYSIOLOGY. [VIII The Haversian canals are chiefly occupied by small blood vessels. The white marrow, consisting mainly of fat-cells (cp. Less. VII. § 14). Small clumps of marrow cells are seen, most of them are either leucocytes or resemble leucocytes, and here and there giant cells, large cells with irregularly lobed nucleus. 7. Eemove the periosteum from a decalcified parietal bone (nitric acid) ; 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 projecting from the surface and the apertures through which similar fibres have passed. Examine carefully the thinnest part of the strip (con- taining fewest lameUse) for the fine decussating fiyres of the ' matrix ; add acetic acid, both the perforating and decussating fibres swell up and become indistinct or lost to view. 8. Ossification. Longitudinal sections through the head of the femur of a newly-born (or foetal) cat or rabbit (picric or chromic acid). Stain with eosin or picrocarmine, mount in balsam. Observe, passing from the head to the shaft The normal hyaline cartilage. The longitudinal rows of cartilage cells; many of the cells are triangular in section. The layer of large, probably shrunken, cartilage cells; they are also in rows and have only a small amount of matrix around them. The irregular spaces (secondary areolae) of the primary endochondral bone. The bony trabeculae may show a thin central portion of decalcified cartilage less stained with carmine than the osseous tissue, but the VIIl] ELASTIC CARTILAGE. BONE. TEETH. 67 distinction will probably not be very obvious'. Covering the trabeculse and in the spaces are osteoblasts, cells larger than ordinary leucocytes, with a single ovoid or spherical nucleus, and finely granular cell substance. The spaces also contain blood vessels and some jelly like connective tissue. 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. Note on the outside of the bone below the head, the periosteum, and the thin layer of spongy periosteal bone beneath it. 9. Transverse section of the shaft of the bone used in § 8. Note The spongy periosteal bone beneath the periosteum, the osteoblasts especially numerous in the lower layer of the periosteum, and in the peripheral bone spaces. Many of the osteoblasts are elongated and their smaller ends appear to run into the matrix. The spongy endochondral bone in the centre ; there may be the beginning of a central space but no fat- cells are present. 10. Structure of Teeth. Longitudinal sections of teeth prepared as bone, § 4. a. Examine under a low power. Observe the dentine surrounding the pulp-cavity, the cement or crusta petrosa covering the dentine of the fangs, and the enamel covering the ' The two parts are very distinct in sections stained in the Ehrlich-Biondi mixture. 5—2 68 ELEMENTARY PHYSIOLOQT. [VIII dentine of the crown. Note the general arrangement of the dentinal tubules. b. Examine under a high power, and study in detail The Dentine. In the matrix, apparently homo- geneous, 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 trans- versely. Here the central dark spot indicates the space formerly occupied by the dentinal fibre, the ring round this is the dentinal sheath. The Cement or Crusta Petrosa. This differs little from bone, but Haversian canals are generally absent. The canaliculi of the lacunae next to the inter- 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, consisting of striated fibres or prisms, perpendicular to the surface of the dentine. 11. Sections of lower jaw of a fcetal mammal (chromic acid 0'2 p.c). Stain with picrocarmine. Note the stages of the development of the teeth, and also the osteoblasts and osteoclasts of the young sub-maxillary bone. VIIl] ELASTIC CARTILAGE. BONE. TEETH. 69 DEMONSTRATIONS. 1. Transverse section of rib to show red marrow \ Note the numerous marrow cells, the scattered fat-cells and occasional giant cell. 2. Transverse sections of teeth through the crusta petrosa and through the crown. Compare with the longitudinal sections described in the text. 3. Longitudinal section of decalcified tooth, showing the pulp. 1 Short piece of rib of growing animal, fixed in mercuric chloride ; decalcified; imbedded ; stained on the slide. LESSON IX. STRUCTURE OF CONTRACTILE TISSUES. 1. Ciliated Cells. Cut off the head of a recently pithed frog, and cut away the lower jaw. With fine forceps and scissors, cut out on one side a piece of the mucous membrane of the roof of the mouth and place this on a slide, mucous surface uppermost. Pour 2 or 3 drops of "5 p.c. osmic acid on the remainder of the mucous membrane of the roof, cover it with a watch- glass for 15 to 20 min. (cp. § 1, b). a. To the piece of membrane on the slide add a drop of "6 p.c. sodium chloride ; scrape the epithelium from a portion of the membrane and tease some of the scrapings in the salt solution. Place a fragment of paper at the edge of the drop ; and cover. Note The shimmering appearance caused by the move- ments of the cilia in the larger portions. 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 IX] STRUCTUfiE OF CONTRACTILE TISSUES. 71 (relaxation) ; there is no perceptible pause between the two movements. The results of ciliary action. Granules and blood- corpuscles are driven along ; detached cells may also be seen carried about by the action of their own cilia. The contracted, almost globular form of the isolated cells. b. Remove from osmic acid to water; cut out a piece of the mucous membrane, wash it gently with water, place it in picrocarmine for ^ hour or more, wash, scrape off a little of the epithelium, transfer to a small drop of water faintly coloured with picric acid, tease, cover, and examine (h. p.). Note The patches of epithelium consisting of ciliated cells and of swollen mucous (goblet) cells. Viewed from the side, adjoining ciliated cells are seen nearly to cover in at the surface the mucous cell between them. Viewed from the surface, numerous, closely set, rather faint dots represent the cilia, here and there are seen the small round openings of the mucous cells, at a lower focus the swollen portions of the mucous cells come into view, surrounded by narrow stained bands, formed of the bodies of the ciliated cells. The isolated ciliated cells. (If the isolation is not good, tap the cover-slip.) The cilia are densely studded over the whole surface. Beneath the cilia is a hyaline or faintly striated layer, the hyaline border. A little below this, the cell generally tapers, cp. the shape with that seen in a. The stained nuclei are conspicuous. The preparation may be preserved by adding a drop of dilute glycerine to the edge of the cover-slip so that 72 ELEMENTARY PHYSIOLOGY. [iX it gradually takes the place of the water, but the cilia will then be less obvious. 2. Striated muscle of frog. Cut through the skin of the front of the thigh of the frog used in § 1, note the baiid-like sartorius muscle running somewhat obliquely across the thigh from the pelvis to the knee, seize with forceps the connective tissue lying along each border and tear it away from upper to lower end. Take up with fine forceps a few fibres at one end of the muscle, and gently pull them out to the opposite end. Stretch out the bundle on a dry slide, separate the fibres a little in the centre, place a bristle across them and press gently : remove the bristle, and cover. a. Note (h. p.) the fairly transparent muscular fibres and their varying size. The transverse striation; alternate dim and light bands passing through the whole thickness of the fibre. Longitudinal granular lines or rows of small granules varying greatly in number and distinctness in different fibres. h. Add a drop of normal salt solution. The longi- tudinal granular lines will become more distinct. Some of the fibres may show a distinct longitudinal as well as a transverse striation. Where the bristle was pressed on the fibres, the muscular substance will in some cases be broken, leaving a delicate structureless sheath, the sarcolemma, stretch- ing across the gap ; possibly in other places, as where a fibre is bent, the sarcolemma may be seen as a fine bulging line. IX] STKUCTUEE OF CONTBACTILE TISSUES. 73 c. Irrigate with 1 p.c. acetic acid ; after a time the shrunken elongated nuclei of the fibres come into view scattered throughout the muscle substance. 3. Tear off a very small strip of frog's muscle (extended in alcohol) including one tendinous ending, and tease out as finely as possible in dilute glycerine. Note The transverse striation is more distinct than in § 2. Sometimes a very thin dark line may be seen in the middle of the light one. A longitudinal striation of the fibres is more or less marked, and some of the fibres have been split up into very fine transversely striated threads. At the tendinous end, the abrupt ending of the muscle fibre is seen, and the continuation of the sarco- lemma into connective tissue, cp. with § 4'. 4. 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 muscle, the sterno-cutaneous muscle (m. cutaneus pectoris), will be seen running from the skin downwards towards the lower part of the sternum. Cut through the skin above and below the muscle, and keeping it well stretched, pour a little "5 p.c. osmic acid over it. In a minute or so the muscle will be fixed ; the connective tissue surrounding the muscle may be torn away with ' The fibres are readily isolated and the passage of the saroolemma into the tendon seen in muscle treated as follows. A pithed frog is placed in water at 50° 0. to 55° 0. , allowed to cool, and the muscles preserved in 70 p.e. alcohol. The nuclei may be stained with picro- carmine. 74 ELEMENTARY PHYSIOLOGY. [iX fine forceps (but in doing this there is considerable danger of injuring the muscle and its nerve) ; cut it out, being careful to cut the upper end as close as possible to the skin ; place it in osmic acid for 15 to 30 minutes, wash with water. Divide the muscle transversely near its upper end. Mount in glycerine, the upper end having the outer surface, and the lower end the inner surface uppermost (the latter will be required in Less. xiv. § 10). Observe at the upper end of the muscle the endings of the muscular fibres, these will probably be covered by a good deal of connective tissue, but the rounded or conical ends of the muscle-substance of the fibres and the continuation of the sarcolemma into the connective tissue (tendon) will be seen. The muscle, after it has been treated with osmic acid and washed, may be stained with picrocarmine ; or the muscle of the opposite side may be fixed with alcohol (Less. vn. § 13) and stained with picrocarmine ; thus the nuclei will be brought out. The nerve fibres stain better if the muscle after osmic acid is placed for a day in 30 p.c. alcohol. 5. Striated muscle of insect. Bemove with as little injury as possible one of the thin muscles fi-om the leg of Hydrophilus', tease it out a little without adding fluid, and cover. The fibres will for a brief period be seen in a normal condition. In many, the alternate dim and bright striae will be very distinct; in others the transverse markings will be more or less obscured by an appearance of longitudinal fibrillation. Occasionally fibres are met with, having only a confased ' If not obtainable, take the large common water-beetle (Dytisous marginaUs), or failing that a cockroach or a fly. IX] STRUCTURE OF CONTRACTILE TISSUES. 75 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 stria- tion, that, The dim band has a faint longitudinal striation, as if made up of small rods. The bright band shows a dotted line running across its centre. The usually round nuclei are imbedded in a granular mass of protoplasm (sarcoplasm) ; the whole forms a band or rod running a variable distance in the substance of the fibre. If the nuclei are not distinct, irrigate with acetic acid. 6. Tease out a little of the wing (thoracic) muscle of an insect, note the sa/rcostyles, and the considerable amount of sarcoplasm. The sarcostyles are small, but much larger than the fibrillsB seen in § 2. Measure one or two. 7. Sections of a piece of wing muscle from an insect (Flemming's fluid). Stain on a cover-slip (cp. Less. iv. § 7). Note the round areas of the sarcostyles imbedded in the granular protoplasm. 8. Unstriated muscle. Remove the stomach of the frog used in § 4. Cut it open, and pin it out. Take up the edge of the mucous membrane with forceps, partly tear and partly cut it away from the muscular coat. Place a piece of the muscular coat about 3 mm. square in a watch-glass with 35 p.c. potassium hydrate'. ' Potassium hydrate readily isolates unstriated muscle cells, but it makes them more homogeneous looking than they are in life. The nuolens and the faintly granular protoplasm around it are better seen 76 ELEMENTARY PHYSIOLOGY. [iX In 20 to 30 minutes, tease the piece in the watch-glass into smallish fragments, remove one or two of these to a slide, — tease further and cover. Note The isolated muscle cells, long cells tapering at the ends, in some the nucleus — here pale and watery looking — will be seen in the mid and broader region. The tapering ends of the cells projecting from the less teased fragments of tissue. 9. Cut out one half of the bladder of a pithed frog ; put it in normal salt solution and cut it open. Make a moist film preparation, mucous membrane uppermost (Less. vii. § 7), rubbing it gently with the finger to remove the epitheKum. Fix with alcohol. Press with blotting-paper, add haematoxyUn at once. When it is rather deeply stained, wash, blot again, and add eosin in 75 p.c. alcohol. In about a minute, pour off the eosin, wash with 95 p.c. alcohol ; blot, at once add clove oil. Mount in balsam. Note the plexus of unstriated muscle cells. 10. Cardiac muscle. Treat a small piece of the ventricle of just killed rat or mouse with 35 p.c. potas- sium hydrate' by the method given in § 8. In the teased specimen, note The isolated muscle cells; they are short, thin columnar cells, many having a short process coming off obliquely from the body of the cell. At about the centre of each cell is a nucleus. The transverse stria- tion is not very distinct. The cells have no sarcolemma. by the following method. A small strip of unstriated muscular tissue is stretched and placed in potassium bichromate '1 p.c, or in 30 p.c. alcohol for 2 to 3 days, washed with water, stained with picrocarmine and mounted in dilute glycerine. ' See Footnote on p. 75. IX] STRUCTURE OF CONTRACTILE TISSUES. 77 11. Isolate the cardiac muscle cells of the frog by the method given in § 8. Note that the cells generally resemble unstriated muscle cells in shape, but are much shorter and are transversely striated. By this method the striation is not marked. Some cells may be seen with branches or with flattened expansions. 12. Esoamination of muscle with polarized light. Place one Nichol's prism under the stage of the microscope in the position of the condenser. Select a specimen of striated muscle, in which the fibres are isolated, and the planes of the discs at right angles to the stage. Using an ordinary ocular, focus the piece of muscle to be observed. Substitute for the ordinary ocular the frame containing an ocular with a Nichol's prism (the analyser) above it. Put the pointer of the analyser at of the scale on the frame. Turn the frame with the analyser round one complete circle. The field dimng the complete revolution becomes dark twice and light twice. Turn the frame so that the field is at its maximum brightness, and clamp the frame. Focus the muscle. Turn the analyser slowly through 90°, note that at 45° the thick discs are light, i.e. they are doubly refractive ; whilst the rest of the fibre more or less completely disappears. Turn the specimen on the stage through an angle of 45° to see if any other part of the fibre becomes visible. (If the princi- pal plane of a doubly refracting substance is parallel to the principal plane of either prism, no light is transmitted.) The disposition and amount of the singly and doubly re- fractive substance vary with the nature of the muscle and with its condition, whether fresh or hardened. DEMONSTRATIONS. 1. Transverse sections of mammalian striated muscle (chromic acid 'o p.c). Observe The connective tissue (perimysium) around the 78 ELEMENTARY PHYSIOLOGY. [iX bundles of fibres and around tbe whole muscle ; from this runs a small amount of connective tissue (endo- mysium) between the muscle fibres. The cut ends of the fibres are finely dotted, corre- sponding with the fibrillae. The nuclei lie just beneath the sarcolemma, few or none being imbedded in the muscular substance of the fibres (cp. § 5, c). 2. Longitudinal section of mammalian striated muscle (alcohol, picrocarmine) to show the nuclei of the fibres. 3. Teased striated muscle fibres (picric acid) to show the splitting of the fibres into discs. 4. Striated muscle fibres under a polarising micro- scope (cp. § 12). 5. Longitudinal section of the intestine of cat or dog at right angles to the surface. (Alcohol or "2 p.c. chromic acid. Hsematoxylin.) In the longitudinal coat, note the sheet of muscle cells with elongated stained nuclei ; in the circular coat, which is cut transversely, note the bundles of muscle cells ; the nuclei are only seen in some of the cells ; they appear as round, deeply stained central spots. 6. Section of ventricle of a small mammal (alcohol ; hsematoxylin). Note the meshwork formed by the junction of the cells, and the hyaline cement substance at the junctions. LESSON X. CHEMISTRY OF MUSCLE. DERIVED ALBUMINS. ALBUMINOIDS. EGG-WHITE. 1. 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 removed. 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 its 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 80 ELEMENTARY PHYSIOLOGY. [X coarse filter paper. A somewhat viscid fluid is ob- tained ; pour this into a tall vessel containing about a litre of water and observe the precipitate of myosin which takes place (or the 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 5 c.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° 0. the fluid becomes milky (or a precipitate is formed) owing to the coagulation of the myosin. b. Add powdered salt, the precipitate soon dis- solves, but when the fluid is saturated with salt is reprecipitated (the precipitate may be in the form of fibrous-looking clumps). c. Dissolve the suspended precipitate in salt solu- tion, and test for proteids, e.g. with xanthoproteic reaction (op. Less. iii. § 4). 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. IIL §§6, 15). 2. JUuscle-plasma. Wash the blood out of the blood vessels of a frog or, better, out of the limbs of a just killed mammal, with cold normal salt solution (5°C.). When the muscles are more or less completely freed from blood, throw them into a freezing X] DERIVED ALBUMINS. 81 mixture. When frozen chop them up. The fluid which can be expressed from the fragments as the temperature rises is muscle- plasma. This may be obtained more easily in a diluted form by extracting the muscle fragments with a little cooled 10 "/o NaCl solution, or half saturated NagSOi solution. a. To dilute salted miisole-plasma add three times its volume of water and set it in the warm bath. In half-an-hour the fluid will usually have clotted, and myosin be formed. b. Determine the coagulating points of the proteids present in dilute salted muscle-plasma. A small coagulation occurs at 47° C. (paramyosinogen), a large coagulation at 56° 0. (myosinogen), and if this is filtered ofii further small coagulations may be ob- tained at 63° (myoglobulin) and at 73° (albumin). Derived albumins. 3. Acid Albumin or Syntonin. Treat a portion of muscle washed as in § 1 with ten to twenty times its bulk of HOI '2 p.c. and place in the warm chamber at about 40° C, frequently shaking. After three or four hours most of the myosin of the muscle will have been converted into acid-albumin and dissolved. Acid albumin 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, a copious bulky precipitate of acid albumin takes place ; if too much alkaline salt is added the acid albumin will be converted into alkali albumin and redissolved (cp. § 4). Filter and wash the precipitate once on the filter with water, then break through the filter paper and wash the pre- cipitate into a beaker with water. The acid albumin is L. 6 82 ELEMENTARY PHYSIOLOGY. [X thus obtained suspended in water. Place a little in each of two test-tubes. a. Add a little HCl '2 p.c, the precipitate at once dissolves and is not precipitated on boiling ; cool under a tap, and ^st for proteids, e.g. with acetic acid and potassium ferrocyanide (cp. Less. iii. § 4). b. Boil, cool under a tap, and add a little HCl •2 p.c. ; the precipitate suspended in the water has been coagulated by boiling and is no longer soluble in dilute acids. 4. Alkali Albumin. Treat a little washed muscle as in § 3 but with NaHO 1 p.c. instead of ynth HCl. On neutralizing a precipitate of alkali albumin will be obtained like that of acid albumin; 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 alkalis. c. Add a 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. X] DERIVED ALBUMINS. 83 It will have been seen that proteids in solution (except peptone) on warming with dilute acids or alkalis give rise to acid albumin or alkali albumin 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. 5. Test the following properties of gelatin, using commercial gelatin. a. Break up the gelatin into small pieces, add cold water, the gelatin does not dissolve; heat, the gelatin passes into solution. b. Cool the tube ; the solution sets into a jelly provided at least l°/o of gelatin is present. Long continued boiling with water lessens the power of gelatinising. c. To a solution of gelatin add sodic hydrate and a drop or two of copper sulphate; a violet colour is obtained. (Resembling proteids.) d. Apply the xanthoproteic test ; a faint reaction only is obtained. e. Add acetic acid; gelatin is not precipitated. (Distinction from mucin.) /. Add acetic acid and potassium ferrocyanide ; gelatin is not precipitated. (Distinction from pro- teids.) g. Add Millon's reagent ; slight red colour on boil- ing. Pure gelatin does not give Millon's reaction. h. Add a few drops of tannic acid ; gelatin is pre- cipitated. 6—2 84 ELEMENTARY PHYSIOLOGY. [X i. Add a few drops of lead acetate ; gelatin is not precipitated. (Distinction from proteids.) 6. Preparation of Mucin. Procure the submaxillary gland of the ox. Mince it and grind it with sand, adding gradually •1 p. o. potassium hydrate solution, shake from time to time. In half-an-hour to an hour strain through linen and filter through coarse filter paper. One Utre of the alkaline solution to 50 grams of moist gland will give a strong solution of mucin. To mucin apply the following tests. a. Add to a solution of mucin acetic acid drop by drop, a stringy precipitate of mucin occurs which is not soluble in excess of the acid. Remove the precipitate on a glass rod and wash it with water, it does not dissolve. Place it in a test-tube and add •! p.c. NaaCOj, the mucin swells up and slowly dissolves. b. Its solution does not coagulate on heating. c. With sodic hydrate and copper sulphate it gives a violet colour. d. It gives Millon's reaction and the xanthoproteic reaction. e. It is not precipitated by potassium ferrocyanide, but if the solution is strong it may become viscid. (Distinction from proteids, except peptone.) f. It is not precipitated by tannic acid. (Dis- tinction from gelatin.) g. It is precipitated by lead acetate. (Distinction from gelatin.) h. On boiling with a few drops of 25% HjSOi X] EGG-WHITE. 85 mucin is decomposed and a reducing sugar formed. Apply Trommer's test (cp. p. 180). Egg-white. 7. Crack the shell of an egg into two equal parts and allow the white to flow away from the yolkj Test the reaction of the egg-white. It is alkaline. 8. Stir up the egg-white with a whisk, thus breaking up the membranes which hold it and strain through muslin. Add four times the volume of dis- tilled water and mix thoroughly. A precipitate is formed of a mucin-like substance (ovomucin). Filter this off and apply to it the tests of mucin (see § 6). 9. a. To the filtrate add acetic acid drop by drop until the fluid is just acid, a precipitate occurs consisting of globulin (with any remaining ovomucin). b. Saturate another portion of the filtrate with MgSOi. A small precipitate consisting chiefly of globulin occurs. 10. The filtrates from § 9, a, and § 9, b, contain egg albumin. Apply to them the general tests for pro- teids. 11. Heat, the solution coagulates. 12. Take the filtrate from § 9, as, or neglect the small globulin precipitate diie to the addition of acetic acid, and proceed to determine the coagulating point of egg albumin as in Less. iii. § 5, 6. Coagidation begins at about 60° C. but is not complete until 74° 0. 86 ELEMENTARY PHYSIOLOGY. [X 13. Take the filtrate from § 9, b, and saturate it with NagSOi so that the solution is saturated with both MgSOi and NaaSO^ a bulky precipitate of egg albumin occurs. 14. A small quantity of a mucoid body (distinct from ovomucin) remains in solution after globulin and albumin have been coagulated by heat and may be precipitated from the filtrate by alcohol It comes down with the proteids on double satura- tion with Na2S04 and MgS04. LESSON XI. PHYSIOLOGY OF MUSCLE AND NERVE. I. A. Apparatus for Stimulation. 1. Unless otherwise mentioned one Daniell's cell will be sufficient to use as a battery. Ordinary electrodes consist of platinum wires mounted in ebonite and exposed at their ends on one side only, or simply of wires projecting about two centimetres from a stout guttapercha covering. 2. a. A key may be used either as a short-circuit or as an in-circuit key. With covered copper wires connect one side of a key with one pole of a Daniell's cell and with one electrode, and the other side with the other pole and other electrode ; it is then arranged as a short-circuit key. When the key is open the current passes into the electrodes ; place the electrodes on the tongue, and note the sensation on opening and closing the key. b. Connect one side of a key with one pole of a Daniell's cell, the other side of the key with one electrode, and the remaining electrode to the other pole 88 ELEMENTARY PHYSIOLOGY. [XI of the Daniell's cell ; it is then arranged as an in- circuit key. When the key is shut the current passes through the electrodes. The arrangement a or & is spoken of as that for a constant or voltaic current. c. The keys used are of two types, one, as the du Bois-Reymond key (Fig. 3), in which complete contact is only gradually though rapidly made ; and the other, as in the Morse key, in which the whole contact is made practically at the same moment. Fig. 8. d. Morse Key (Fig. 5). The following rliagram (Fig. 6) shows the way in which tlic wires of the key arc (u.sually) arranged. The key is arranged as a short-circuiting key by connecting the battery wires to a and b (or the wire.? of the secondary coil), XI] PHYSIOLOGY OF MUSCLE AND NERVE. I. 89 and the derived circuit (the primary coil, or electrodes) with a and 0. The key is arranged as an in-circuit key, by connecting with b and c ; thus if put in the primary circuit of an induction machine the wires from the primary circuit run to 6 and to one pole of the battery, the other pole of the battery is connected with c. Putting down the key makes the current. e. A commutator for changing the direction of a current (cp. Kg. 4) consists of a disc of ebonite carrying six mercury cups to each of which a binding-screw leads. Into the mercury cups to which the screws a: and y, called the central screws, lead, lie the ends of a copper bar e, the two halves of which are insulated from each other by a central mass of ebonite, each half carries a pair of arms, and the bar can be turned so that the arms dip into the cups connected with the screws a and b or into those connected with c and d. These cups are connected diagonally, a to d and b to c, by cross wires not touching each other. The battery is coimected to x and y, and if the bar e makes contact with a and b, a; is connected to a and y to 6 ; if it makes contact with c and c?, a; is connected to b and y to a by means of the cross wires. A current in wires connected with a and b can therefore be changed in direction. 90 ELEMENTARY PHYSIOLOGY. [XI 3. Induction machine. The end of this is shown in Fig. 7. Fig. 7. a. Connect the poles of a Daniell's cell with the screws at the top of the machine interposing a key. An in-circuit key should be used ; it is better, though not necessary for the experiments to be performed in this Lesson, to use a key of the 2nd type mentioned in § 2, c. Connect the electrodes with the screws of the secondary coil, interposing a short-circuit key. Put the index of the secondary coil at 10 cm., open the circuit of the secondary coil, place the electrodes on the tongue. Note the sharp shock caused by the make and break of the primary circuit. Move the secondary coil away from the primary, and determine the maximum distance at which the make and the break shock can be Xl] PHYSIOLOGY OF MUSCLE AND NERVE. I. 91 felt. The make shock will probably cease to be felt before the break shock. The arrangement is spoken of as that for single induction shocks or for induction shocks of slow rhythm. b. The circuit being open, change the battery wires, from the screws at the top of the machine to those at the base of the two pillars. Screw down / (cp. Fig. 7) out of range of the hammer b, screw down c so that it just touches b. Leave the connections of the secondary coil and electrodes as before. Close the primary circuit (if necessary flick the hammer e), the hammer will be set in rapid oscillations, each downward movement of the hammer breaking the primary circuit, each upward movement making it. Put the index of the secondary coil at 35 cm., place the electrodes on the tongue, and gradually push the secondary towards the primary coil ; note the position at which the shocks are first felt. Note also the position at which the shocks can not be borne comfortably. The arrangement is spoken of as that for tetanizing shocks or for induction shocks of .rapid rhythm. The method in which the primary current is made and broken will be easily understood from the figure. When the current is passing in the primary coil, b is in contact with c, but since the current also passes round the coil m the soft iron core of this coil becomes magnetized, and consequently attracts the plate e; as this goes down the contact of b and c is broken, hence the current ceases to pass in the primary coil, the core of m is no longer magnetized, the plate e flies up, b comes again in contact with c and the cycle of events starts again. 92 ELEMENTARY PHYSIOLOGY. [XI c. Arrangement for equalizing the make arid break shocks. Con- nect, by a short etout wiie, the upper of the two screws on the pillar a with the nearer of the two top screws of the primary coiL Baise the screw c ont of range of the hammer i, and raise the screw / until it just touches 6. Close the primary circuit, flick the hammer, and if the screw / is properly adjusted the hammer will continue in oscillation. Each upward movement of the hammer makes the primary circuit, and each downward movement of the hammer making contact with / breaks the primary circuit, the current passing from the pillar a to the pillar d, except for a small portion of the current which passes by the side wire connecting a with the primary coil. Since the current is not completely broken, the shock induced at the break is less than before, and by adjusting the resistance of the side wire may be made equal to the shock induced at the make. B. Direct Observations. 1. Constant Current. Cut off the head of a pithed frog, and squeeze the body upwards to remove most of the blood. (This is done to avoid bleeding, if vessels are cut in dissecting the nerves.) 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 inner side 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 : 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 carefiil not to pinch the nerve with the forceps, nor to put more strain upon it than is absolutely necessary. XI] PHrSIOLOGT OF MUSCLE AND NERVE, I. 93 Place a piece of thin sheet india-rubber, about 1'5 cm. wide under the nerve; arrange for a constant current (A. § 2, 6), close the short-circuit key, place the points of the electrode under the nerve. Open the key so as to throw the current into the nerve; a sing^le con- traction or twitch of the leg is obtained, the make contraction ; leave the key open for 5 to 10 seconds, note that there is no further movement of the leg, i.e. during the passage of the current without variation of intensity the nerve is not stimulated. (In certain circumstances, stimuli may be set up during the passage of the current.) Shut the key; a single contraction of the leg is obtained, the break contraction. 2. Single and slowly repeated induction shocks. Arrange the induction machine for single induction shocks (A § 3, a), with an in-circuit key in the primary circuit, place the index of the secondary coil at 10 cm., open the key of the secondary circuit. Shut the primary key (i.e. make the primary current) ; there is a single contraction or twitch of the leg, the make induced twitch. Open the primary key (i.e. break the primary current) ; there is a single contraction or twitch of the leg, the break induced twitch. Open and shut the primary key at intervals of a second for 10 seconds, each make and break of the primary current causes a twitch of the leg. 3. Rapidly repeated induction shocks. Ar- range the induction machine for tetanizing currents (A. § 3, b). Close the key of the secondary circuit. Make the primary circuit. When the hammer is steadily oscillating open the secondary key ; the leg is immedi- 94 ELEMENTARY PHYSIOLOGY. [XI ately thrust out straight and kept rigid in tetanus. In a few seconds, break the primary current, the limb at once becomes flaccid. If the stimulus is kept on long, or too often repeated, the muscle will be exhausted ; in that case take the nerve and muscle of the opposite side for §§ 4 and 5. 4. Chemical stimulation. Cut through the tissue above the sciatic nerve up to a quarter of an inch above 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 vigour and will extend over the limb until it becomes almost as rigid as in § 3. 5. Mechanical stimulation. Cut off the part of the nerve which has been dipping in the salt solution; the chemical stimulus 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. 6. Reaction of living and dead muscle. Have ready a piece of faintly blue, and a piece of neutral litmus paper. a. Cut out one of the thigh muscles of the opposite XI] PHYSIOLOGY OF MUSCLE AND NERVE. I. 95 side ; cut it across with a clean scalpel, press one surface on the faintly blue, the other on the neutral litmus paper. Both will indicate an alkaline reaction. 6. Stimulate the sciatic (peripherally of the part pinched in § 5) with tetanizing currents, pushing the secondary coil over the primary so that the induction shocks are strong to the tongue. Continue the stimulus as long as any effect can be obtained. Then cut across the gastrocnemius muscle, and test its reaction as in a. It will be found to be faintly acid. c. Take the gastrocnemius of the opposite side ; place it for five minutes in normal salt solution at 45° 0. or at a higher temperature. Observe that it becomes contracted and opaque, i.e. rigor mortis sets in. Test its reaction, it will be distinctly acid. C. Observations by the Graphic Method. 1. Tracing of a single muscular contraction. Tracings are taken on smoked paper covering a re- volving drum, the rate of revolution of which can be varied. First arrange the apparatus for single induction shocks as in A. § 3, a, connecting through the screws s, s' of the moist chamber (Fig. 8) to the electrodes b. Take a pithed frog and cut through the skin, and the skin only, all round the animal at the middle of the trunk, turn the edge of the skin downward, and seizing it mth a cloth, tear the skin from the lower half of the trunk and then completely from one leg. Place the frog on its belly on a glass or porcelain plate. Expose the sciatic nerve in the thigh as in B. § 1, and 96 ELEMENTARY PHYSIOLOGY. [XI trace it up to the level of the tip of the urostyle. lift up the tip of the urostyle with forceps, and with scissors cut through the muscles passing from the urostyle to the pelvic on each side, keeping the scissors close to the urostyle. Free the urostyle in this way right up to its attachment to the end of the vertebral column and then cut it off. The nerves which unite to form the sciatic on each side will be partially brought into view. Place one blade of a stout pair of scissors between the nerves of either side aiid split the end of the verte- bral column in the middle line for about 1'5 cm. With forceps seize the split half on the side selected, turn it outwards, and cut it across transversely 1 cm. from the lower end. Issuing from the piece of verte- bral column thus isolated will be seen the 7th, 8th, 9th, and 10th nerves forming the sciatic plexus. Raise the piece of bone, and without stretching the nerves cut through with sharp scissors the muscular and other tissue attached to it, and to the nerves, freeing the nerves as far as the sciatic trunk. In a similar way, cautiously cut through the tissue sur- rounding the sciatic and so isolate the nerve down to the knee, cutting through its branches on the way. The nerve must not be touched by the forceps; it must be raised only by means of the piece of bone attached to it, and it must not be stretched. Turn the nerve downwards and let it lie on the muscles below the knee ; cut away the muscles attached to the lower half of the femur, taking care not to injure the nerve, and with stout scissors cut through the femur about 1 cm. from its lower end. Divide the lower (Achilles) tendon of the gastrocnemius close to its XI] PHYSIOLOGY OF MUSCLE AND NERVE. 97 attachment at the foot and separate the muscle from below upwards as far as its attachment at the knee, then cut away the tibia and fibula at the knee. The muscle-nerve preparation so obtained consists of the gastrocnemius muscle attached to the lower end of the femur, and the sciatic nerve with a piece of the vertebral column attached to it. Pass a hook through the tendon and fix the clamp {a. Fig. 8) of the moist chamber to Fig. 8. the piece of the femur ; take up the preparation by the clamp and the fragment of vertebral column ; arrange it in the moist chamber so that the muscle is vertically over the hole in the chamber and the nerve lies on the platinum wires of the electrodes 6, the fragment of 7 98 ELEMENTAKY PHYSIOLOGY. [XI vertebral column being supported on the covered part of the electrodes. Put a piece of blotting-paper well wetted with normal salt solution in the chamber and put on the lid. It is well to moisten the nerve from time to time with a camel's hair brush, wet with salt solution. Attach the hook in the tendon to the lever c, load the lever with 10 grams. See that the lever is horizontal and in the same vertical plane as the muscle, and that the hook lies free in the hole in the floor of the chamber. Bring the lever to mark on the revolving cylinder, adjusting it so that it presses very lightly on the smoked paper. Place the index of the secondary coil at 10. Let the drum revolve at its maximum speed so that the lever may trace a base Ilqc, open the key in the secondary circuit, then open the key in the primary circuit, thus sending in a break induced shock. A tracing of the contraction will be obtained. Stop the drum and examine the curve. The curve rises rapidly but steadily to a maximum, and almost immediately falls similarly but rather less rapidly, and later near the base line distinctly slowly. In the same way take a tracing of the contraction caused by a make induced shock, with the key in the secondary circuit open, closing the key in the primary circuit. A similar curve is obtained. Set the cylinder rotating at slower speeds and take further tracings, note the varying form of curve ob- tained. 2. Latent period. Arrange first the apparatus. A drum is used having a knock-down key : see Fig. 9. XI] PHYSIOLOGY OF MUSCLE AND NEEVE. I. 99 As the drum revolves, a horizontal projecting rod a knocks down the vertical rod b and so disconnects Fig. 9. the two binding screws s and s'. Arrange this key as an in-circuit key (A. § 2, b), in the circuit of a Daniell's cell and primary coil. Connect the secondary coil with the electrodes of the moist chamber, introducing a short- circuit key. Shut the key of the secondary coil. Make a muscle-nerve preparation (C. § 1), and arrange it in the moist chamber. Load the lever with 10 grams. 7—2 100 ELEMENTAKY PHYSIOLOGY. [XI Bring the lever to mark on the revolving cylinder, adjusting it so that it presses very lightly on the smoked paper. Place the index of the secondary coil at 10. Let the drum revolve once so that the lever may trace a base line; stop the drum so that the projecting rod is just beyond the primary key (knock- down key). Close the primary key, open the secondary key, start the drum at its maximum speed. Stop the drum when it has knocked down the primary key (thus sending a break induced shock into the nerve). Close the secondary key. The tracing is now obtained ; mark the moment of stimulation on the curve, thus. Close the primary key, open the secondary key, turn the drum round by hand until the projecting rod is near the primary key ; then very cautiously move the drum till it just touches the primary key and breaks circuit ; the muscle will contract and mark on the drum the moment of stimulation. Close the secondary key, and note in the curve The contraction begins a short time after the moment of stimulation, the time is the latent period. 3. Time measv/rement of the parts of tlie owrve. Connect in one circuit two Daniell's cells, a key (in-circuit), a tuning-fork vibrating 100 times a second, and the bobbin of a time-marker, as in the following diagram. The current flows from the battery by the wire/, through the tuning-fork, down the pin connected with the lower prong to the mercury cup Hg and so to the binding screw e. Here the current divides into two circuits, one passes through the coil d lying between the prongs, and the other passes by the wires g and 6 through the coils of a time-marker, and both circuits are brought XI] PHYSIOLOGY OF MUSCLE AND NERVE. L 101 to the binding screw a, from which the current passes to the battery. Pig. 10. The time-marker is shown in the following diagram (a some- what different form is shown in the preceding flgm'e). Fig. 11. The two ends (w, vf) oi the wire wound round the bobbins (6, b') are connected with the binding screws («c, sc') to which the current is conducted. When the circuit is made the current passes round the bobbins, the soft core of the bobbins becomes magnetized and the lever {I) is drawn down ; when the circuit is broken the lever is drawn away from the bobbins by the spring (sp). At the end of the lever is fixed a stiff bristle (to write on smoked paper) or a small bent glass tube filled with magenta dissolved in dilute glycerine (to write on glazed paper). See that the surface of the mercury is quite clean, put on it a few drops of 95 % alcohol, adjust the pin so that it just touches the mercury, flick the tuning-fork, it will continue to vibrate 102 ELEMENTARY PHYSIOLOGY. [XI making and breaking the current at the mercury contact. Each time the current is made the core of the coU d of the tuning-fork becomes magnetic, the prongs are drawn together and the current broken, the core then ceases to be magnetic and the spring of the tuning-fork makes the current again. When the lever of the time-marker is oscillating steadily in unison with the tuning-fork (if it does not adjust the strength of the spring of the time- marker), set the drum running at the same rate as it was when the tracing of the single muscular contraction was taken, and bring the point of the lever to write on the drum, immediately below the tracing, while the drum revolves round once. An undulating line is traced, each undulation represents jj^ of a second. Unhook the muscle from the writing lever, letting the lever rest on the finger, adjust its point accurately to the commence- ment of the contraction, then let it fall and so trace a line downwards to the time tracing. Shift the lever along the curve and drop lines in the same way from the summit of the contrac- tion, the end of the relaxation, and from the line in front of the contraction indicating the latent period. Bead the duration of the latent period, the contraction and the relaxation in yJxfths of a second. 4. Minimal and maximal contractions. Dis- connect the knock-down key, and substitute an in-circuit key in the primary circuit. Extend the scale of the induction machine to its full length by opening out the half of the scale which lies underneath the machine. Place the secondary coil at 40, open the key in the secondary circuit, then open the key in the primary circuit and so stimulate the nerve with a single break induction shock. Record the height of the contraction if any on a stationary drum. If no contraction results push the secondary coil nearer and nearer to the primary, shifting it half a centimetre at the time, and stimulating the nerve again after each change. In this way find XI] PHYSIOLOGY OF MUSCLE AND NERVE. I. 103 the position of the secondary coil which just gives a contraction — the minimal contraction. Now go on pushing up the secondary coil half a centimetre at the time and record the height of each contraction produced, moving on the drum by hand about one centimetre after each contraction. After each contraction close the secondary key before the primary is closed in order to short-circuit the make induction shock and then open the secondary key before the primary is opened again. Observe that the contractions gradually increase up to a certain point — the maximal contraction — and then remain constant although the stimulus is further increased. Shift the secondary coil back to a position which will give a submaximal contraction on the break of the primary circuit, record the contraction and move on the drum ; then leaving the secondary key open, close the primary key and record the contraction due to the make of the primary circuit. This is less than the break contraction, since the induction shock caused by making the current is less than that caused by breaking it. If no contraction is obtained on the make, shift the coil nearer and stimulate by the break and make again. JNo difference between the make and break contractions will be seen if the stimuli are strong enough to give rise to maximal contractions. 5. Fusion of single contractions to form tetanus. Arrange for single induction shocks, intro- ducing into the primary circuit, in place of a key, an oscillating rod and mercury cup. This is a thin band of steel about 35 cm. long and 2 cm. wide ; at one end 104 ELEMENTARY PHYSIOLOGY. [XI at right angles to the band is fixed a pointer 2 to 3 cm. long. The band can be fixed at any part of its length by a wide brass clamp and connected with the clamp is a binding screw. A cup containing mercury is placed underneath the pointer and the clamp is an-anged on ^.the stand at such a height that when the band descends in oscillation the pointer dips into the mercury. Shift the oscillating rod in the clamp sup- porting it, so as to allow its full length to oscillate, and adjust its height so that the pointer just touches the mercury when at rest. Open the key in the secondary circuit, and by moving the oscillating rod in and out of the mercury by hand and shifting the distance of the secondary coil from the primary determine the position of the coil at which contractions are only obtained at break. If such a position cannot be clearly obtained work with a strength of stimulus which is just maximal for make and break so that the height of the con- tractions is the same. Set the drum running at a rather slow velocity (about 6 cm. per sec), put the rod into oscillation, then open the key in the secondary circuit for rather more than one second and close it again. Stop the drum, shorten the rod in its clamp by about 4 centimetres, adjust it to the mercury level, and take another tracing of about, the same duration. In this way take a series of tracings 6 to 8 in all, with the rod shorter and shorter, shortening it less and less each time. Observe the gradual fusion of a series of single contraction curves into the curve of tetanus. Finally remove the oscillating rod and mercury cup and connect the battery wires though an in-circuit key to the lower screws of the primary coil (cp. A. 3 b) XI] PHYSIOLOGY OF MUSCLE AND NERVE. L 105 and take a tracing, stimulating with the automatically vibrating hammer of the induction coil. A complete tetanus is obtained. 6. Connect in one circuit a Daniell's cell, an in-circuit key, a beating metronome, and a time-marker. The metronome consists of an oscillating pendulum which (at each oscillation) makes and breaks an electric current lead to two mercury cups. The period of oscillation can be changed by shifting a weight along the pendulum so as to give oscillations at rates varying from 40 to 200 per minute. Arrange the metronome to beat once a second, and by means of the time-marker record periods of one second duration on the running drum. Instead of the beating metronome a clock may be used to make and break the circuit, or a chronograph, i.e. a small clock with a recording lever, may be brought to bear directly on the running drum. By means of compasses measure the length on the drum of a period of one second, and count the number of oscillations per second of the muscle in the tracing which just fails to be a com- plete tetanus. 7. Fatigue of muscle. Arrange the apparatus as for recording the latent period (C. § 2). Connect a second cell to the lower screws of the primary coil as for a tetanizing current, introducing an (open) in-circuit key. Load the muscle with 30 grammes. With the coil at 15 take a tracing of a single muscular contraction and mark the latent period. With the knock-down key open, and the drum at rest, so that the lever will not mark over the tracing just taken, close the tetaniz- ing circuit for 10 seconds, throwing the muscle into tetanus. Then without shifting the position of the lever, take 106 ELEMENTARY PHYSIOLOGY. [XI another tracing of a single contraction. This second single contraction will probably be recorded immediately above the first, since the relaxation of the muscle after the tetanus may not be quite complete. Then tetanise the muscle for 10 seconds again, and then record another single contraction. Proceed in this way until 8 or 10 contractions have been recorded. Record the latent period of the last of the series. Note that the relaxa- tion of the muscle after each tetanus becomes less and less complete, and the single contractions are recorded immediately over one another. Observe that the second or third contraction is (usually) a little higher than the first, and then the contractions become lower and lower, rising more and more slowly and falling more and more slowly, the prolongation of the relaxation being more marked than that of the contraction, and the latent period is longer. LESSON XII. PHYSIOLOGY OF MUSCLE AND NERVE. II. 1. Eictensibility of muscle. Place a muscle- nerve preparation in the moist chamber and attach the muscle to the lever. Bring the point of the lever to ■write on the drum, and turning the drum by hand trace a base line with the muscle unloaded (except with the weight of the lever). With a pair of compasses mark off on this a number of points "5 cm. from one another. Put on a load of 20 grammes, the muscle stretches, and the lever falls, recording a nearly vertical line. Move on the drum '5 cm. by hand, and put on another 20 grammes, the lever again falls but less than before. In this way record the magnified extension of the muscle for each successive 20 grammes weight until six or eight have been added, moving on the drum '5 cm. after each addition. The extension becomes less and less, and a curved line is formed if the lower extremities of the lines traced by the fall of the lever are drawn through. When the muscle-nerve preparation is no longer wanted for subsequent experiments remove it; substitute for it a strip of sheet india-rubber, and 108 ELEMENTAET PHYSIOLOGY. [XII test the extensibility of the rubber in exactly the same way. Observe that each weight added stretches the rubber an equal amount and the line joining the extremities of the tracings is a straight one. 2. The Tvork done by a contracting muscle. Set up the apparatus for taking a single muscular contraction (Lesson xi. C, 1). Load the muscle with 20 grammes placed vertically underneath the muscle, and leave the lever horizontal. Determine the position of the secondary coil which gives a maximal contraction on break of the primary, without being unnecessarily strong. When once decided upon keep the strength of stimulus constant. Trace a base line by moving the drum on about one centimetre. Stimulate with a single break shock recording the contraction on the stationary drum. Close the secondary key, move the drum on about one centimetre by hand, add another 20 grammes weight, the muscle stretches, set the lever horizontal, trace the new base line, and stimulate again with a single break shock. Proceed in this way for each additional 20 grammes added until the load is about 200 grammes, or until the muscle fails to raise the weight. Mark opposite each contraction the load the muscle is carrying at the time. Measure with a pair of compasses the height of each contraction and express it in millimetres. (There is a millimetre scale on the induction machine.) Multiply the height of each con- traction by the load the muscle was lifting in each case. The figures so obtained give the relative work done by the muscle at each contraction. Observe that the work done increases as the load increases up to a certain XIl] PHYSIOLOGY OF MUSCLE AND NERVE. II. 109 point and then decreases. Measure the length of the lever from the axle to the point, and from the axle to the attachment of the muscle, and calculate the actual work expressed as gramme millimetres done by the muscle when it was doing its maximum work. Repeat the experiments, but instead of stimulating the muscle with single break shocks throw the muscle into tetanus (Lesson XI. A, § 3, b) for not more than one second each time the load is raised. 3. Effect of heat and of cold on muscular contraction. Take a piece of small lead tubing about '5 cm. diameter, and turn it about 5 times round the end of a rod of 1'5 cm. diameter, and so make a coil of tubing about 4 cm. high. Put this in the moist chamber so that the muscle can hang in the coil, and bring the ends of the lead tubing through holes bored in the floor of the chamber. Put two beakers (of about 2 litre capacity), one to contain ice-cold water, the other water at ordinary temperature, at a level higher than that of the moist chamber, and by means of a bent glass tube to dip into one or other of the beakers and a rubber tube connection ; arrange for a flow of water through the coil of lead tubing, controlling the flow by a screw clamp on a rubber tube beyond the coil. Arrange apparatus for taking a tracing showing the latent period (Lesson xi. C, § 2). Place the secondary coil at 15 cm. Fix a muscle-nerve preparation within the coil in the chamber, taking care that neither the muscle nor the nerve touches any part of the coil. Moisten the nerve frequently with normal salt solution. Take a tracing of a single contraction and 110 ELEMENTAEY PHYSIOLOGY. [XII mark the latent period. Let ice-cold water run slowly through the coil, after 3 minutes take another tracing superimposed on the first. Wait 3 minutes more, then take a third tracing. Note cold increases the latent period, prolongs the whole period of contraction, the rise of the lever and the fall, and there is some con- traction-remainder. The lift is usually diminished or unaltered. Shift the bent glass tube to the other beaker and run through the coil water at ordinary temperature (10° — 15° C), after 3 minutes take another tracing im- mediately above or below the series already recorded and mark the latent period. This tracing wUl be more or less of the ordinary form. Raise the temperature of the water to 26°, run it through slowly for 3 miuutes, then take a tracing superimposed on the last. Note diminished latent period, whole period of contraction shorter, sharp rise and sharp fall of the lever ; the lift will probably be the same. Raise the temperature to 35°, run the water through for 3 mia., and take a third tracing superimposed on the last two. Note, the lift is now increased, but the other features of the tracing are much the same as in the last. Raise the tempera- ture to 45°, run the water for 3 minutes, take a tracing, note the lift is now diminished, if it is not, wait a further period of 3 min., and stimulate again, the other features of the tracing are much the same. Mark each tracing to indicate the temperature of the water at the time ; that of the muscle is of course less. Shift the position of the lever on the drum and run water at 55° through for 3 min. Note the effect of super-heating — ^hft may be much increased, sharp rise, long-sustained XIl] PHYSIOLOGY OF MUSCLE AND NERVE. II. Ill irregular contraction, sharp fall. Spontaneous twitching of the muscle will occur as rigor comes on. Let the water continue running and at intervals take 2 or 3 more tracings; note, the lift becomes smaller and the contraction shorter until in 8 to 10 min. no contraction is obtained. 4. Action of veratrin on muscular contraction. Take a frog with the brain only destroyed. Inject 3 or 4 drops of 1 p.c. veratrin solution under the dorsal skin. After 15 to 20 minutes pinch the lower limbs; note that the reflex movements excited are prolonged, the limbs remaining extended for some seconds. Wait until this condition is well marked, then pith the frog and make a muscle-nerve preparation. Load the muscle with 20 grammes, stimulate with a fairly strong single break induction shock, and take a tracing of the con- traction with the drum running at its fastest rate. Note that the rise of the lever does not differ much from the normal, while the fall is greatly prolonged, the lever not reaching the base .line until the drum has revolved several times. Run the drum slowly, about 2 cm. a second, and take several tracings in quick succession; note that after a time a secondary rise appears on the curve and the relaxation becomes less and less prolonged, and later the secondary rise dis- appears and the curve becomes nearly normal. Let the muscle rest for a few minutes, stimulate again, a curve with prolonged relaxation is again obtained. 5. Action of curare on muscle and nerve. Take a frog with the brain only destroyed. Expose the sciatic nerve in the middle of one thigh, e.g. the left. 112 ELEMENTARY PHYSIOLOGY. [XII (cp. Lesson XL B, § 1) taking care not to injure the ac- companying blood vessels. Gently raise the nerve and pass a ligature underneath it, tie the ligature tightly round all the other structures of the thigh including the blood vessels. With a syringe or small glass pipette inject two drops of 1 p.c. solution of curare underneath the skin of the back. In 20 — 30 minutes pinch the left and then the right leg ; the left to which the poison has no access will be drawn up, the right will not. If the right is drawn up wait until the reflex is completely abolished. Since, on pinching, the left Umb is drawn up, the spinal cord and the sensory and motor fibres of the upper part of the sciatic to which the drug has had access are not, at any rate yet, paralysed. Arrange a coil for stimulating with the interrupted current (Lesson xi. A, § 3, 6). Pith the frog, and expose the sciatic nerves of both limbs from the vertebral column to the knee, and lay bare both gastrocnemius muscles. Stimulate the left sciatic close to the vertebral column where the drug has had access to the nerve trunk, contraction of the gastrocnemius nevertheless results. Stimulate the right sciatic, no contraction of the gastrocnemius is produced in whatever part of its course the nerve is stimulated. Apply the electrodes directly to the right gastrocnemius, contraction is produced. The drug has therefore in all probability paralysed the terminations of the motor nerve in the muscle. Divide the Achilles tendon of the left gastro- cnemius and place the muscle in a watch-glass contain- ing curare solution. In 4 or 5 minutes stimulate the left sciatic, no contraction of the gastrocnemius will result if the drug has sufficiently penetrated the XIl] PHYSIOLOGY OF MUSCLE AND NERVE. II. 113 muscle. Stimulate the muscle directly, contraction results. 6. Unipolar stimulation. Set up the apparatus for stimulating with single induced shocks (Lesson xi. A, § 3, a). Expose the sciatic nerve in a pithed frog lying on a cork board, place the electrodes under the nerve, guarding them from touching surrounding tissue by a piece of sheet india-rubber. Determine the position of the secondary coil which just gives a distinct contraction on break of the primary circuit. Disconnect one elec- trode from the secondary circuit key, remove the wire entirely or let it hang over the edge of the table without touching the table. Leave the secondary key open. Break the primary circuit key, no contraction results. Push up the secondary coil several centimetres, and break the primary circuit again until a position is found when a contraction results. Put the cork board with the frog on a dry glass plate, and stimulate again ; no contraction results. (If the induced currents are very strong a contraction may still be produced.) Now touch the frog with the finger and while touching it stimulate again; a contraction results. When the frog is not insulated or is touched with the finger the induced currents pass by one electrode through the frog to earth. Repeat the experiments with tetanic stimulation, the effects are more obvious. LESSON XIIL PHYSIOLOGY OP MUSCLE AND NERVE. HL 1. Jiate of transmission of a nervous impulse. A drum travelling at a rate of at least 100 cm. a sec. is necessary or a rapidly moving pendulum myograph may be used. This con- sists of a pendulum one metre long, carrying a sheet of smoked glass on which the tracing is taken when the pendulum is moving at its maximum velocity. Introduce a knock-down key into the primary circuit, to be opened by the revolving drum or the swinging pendulum (Less. XI. 0, § 2). Connect the secondary circuit to the central screws of a commutator from which the cross wires have been removed, and lead off from each pair of lateral screws of the commutator to a pair of electrodes in the moist chamber. Arrange a muscle- nerve preparation in the chamber with the nerve lying on both pairs of electrodes placed as far apart as possible, one near the muscle and the other near the distal part of the nerve. Arrange the commutator so as to stimulate the distal end of the nerve, take a single contraction and indicate the latent period. Turn over the bar of the commutator, and stimulating the nerve near the muscle take another tracing superimposed on the first. In this the latent period is shorter ; the difference indicates the time required for the passage of the nervous impulse along the length of nerve intervening between the electrodes. Measure this difference of time by means of a vibrating tuning-fork (Lesson xi. C, § 3), measure the length of nerve lying between the electrodes, and calculate the velocity of the nervous impulse. XIIl] PHYSIOLOGY OF MUSCLE AND NERVE. III. 115 2. Stimulation hy the voltaic current. {Law of polar stimu- lation.) Take a piece of sheet cork about 4 cm. square, thrust the bare ends of two battery wires twice through it parallel to each other about 2 cm. apart and press the wires down into the cork, so that they hardly lie above the level of the cork. Connect the wires through a short-circuit key to a Daniell's cell. Take a frog which has been injected with urari (the right leg of the frog used in Less. xn. § 5 will serve) and tie a thread to the lower tendon of the sartorius (cp. p. 72, § 2), then raising the muscle by the thread cut the connective tissue along its borders, separating the muscle right up to the pelvis and cut it across close to the bone. Lay the sartorius on the wires in the cork so that one electrode is near the upper and the other near the lower end of the muscle. Fix both ends of the muscle to the cork with hedgehog quiUs. Watch the muscle carefully, open the key, and so throw in the current; note that the contraction of the muscle remains as a persistent tetanus at the negative pole. In 3 or 4 seconds close the key, cutting out the current ; note the remaining contraction at the negative pole ceases, and the contraction of the whole muscle on the break often persists for a short time at the positive pole. With a camel's hair brush apply to the muscle a few drops of '2 p.c. veratrin in normal salt, solution and stimulate the muscle again. After a few minutes, as the contractions of the muscle become slower and its irritability diminishes, con- traction wiU be observed only at the kathode on making and at the anode on breaking the current. These wiU be more obvious if the muscle is observed through a lens. 3. Yariaiion in the strength and direction of voltaic current. (Law of contraction.) Make a pair of nonrpolarisable electrodes as follows. Take two U-shaped glass tubes to fit the clips (d, Fig. 8) in the moist chamber, with one Umb 4 cm. and the other 3 cm. long, fill the shorter limb with china clay moistened with normal salt solution. Take care that the clay completely fiUs the limb of the tube and that no air bubbles are included in the clay, this is best done by pushing the clay in by the fingers only. Wipe the outside of the 8—2 116 ELEMENTARY PHYSIOLOGY. [XIII tube dry and free from clay ; leave a small wedge-shaped mass of clay projecting from the end of the tube. Put the tubes in the clips in the moist chamber. With a pipette put saturated 2inc sulphate solution into the longer limb of the tubes, taking care not to put in too much and that none runs down the outside of the tubes, and insert into the zinc sulphate solution small amalgamated zinc rods carrying covered wires, connect these with the inner screws of t, i (Fig. 8). Put a piece of moist blotting- paper in the chamber and cover at once. Fig. 12. Connect two Daniell's cells (6, Fig. 12) to the central screws of a commutator c and lead off from one pair of lateral screws of the commutator to the terminals r, / of a simple rheochord, intro- ducing a key for short circuiting the current. The rheochord consists of a board carrying two binding screws, to which the ends of a German-silver wire about 3 metres long of 8 to 10 Ohms resis- tance are connected. The wire passes up and down the board ten times, being carried round ebonite pegs at the top and bottom, and the board is marked transversely by lines, dividing the wires nmning up and down, into tenths, so that the whole wire is divided into hundredths. A moveable block a canying a binding screw can be placed on the wire, to make electrical contact with it, at any division. Connect one non-polarisable Xlll] PHYSIOLOGY OF MUSCLE AND NERVE. III. 117 electrode to one terminal of the rheochord and the other to the moveable block of the rheochord. Fix a muscle-nerve prepara- tion, the nerve of which has been freed from all adherent connec- tive tissue, in the moist chamber, the nerve lying across the projecting wedge-shaped clay of the non-polarisable electrodes placed about one to two centimetres apart. The portion of spinal cohmrn attached to the nerve may be supported on a cork ; neither electrode should be in contact with the end of the nerve nor with a cut branch of the nerve. Arrange the bar of the commutator so that when the current is sent into the nerve it will be descending, i.e. the anode furthest from^ the muscle. Place the moveable block of the rheochord on the wire of the rheochord close to the terminal to which one of the non- polarisable electrodes is connected. Open the key in the circuit, a minimal current is sent into the nerve. Close the key and shift the moveable block along the wire one division at a time, after each shift open the key and after 3 or 4 seconds close it again. At a certain position of the block contractions will begin to occur when the current is sent in. Record the contractions on a stationary drum, moving the drum a centimetre after each contraction. With weak currents no contractions occur on breaking the circuit. Shift the moveable block further and further along the wire, throwing in the current each time ; at a certain position contractions will occur on breaking the current as well as on making it, minimal at first, gradually increasing until the two are equal. Turn over the bar of the commutator so that the current through the nerve is ascending and take another series of contractions, commencing with the weakest currents. If the nerve is in good condition, a contraction on making wUl occur with weaker currents when ascending than when descending, since when ascending the kathode, which is the seat of stimula- tion, is near the upper or more excitable part of the nerve. Except for this, there wiUbe but little difference between the two series. Remove the rheochord, connect the non-polarisable electrodes through the short-circuiting key to the lateral screws of the 118 ELEMENTARY PHYSIOLOGY. [XIII commutator and arrange the commutator so that the current through the nerve will be descendmg. Open the key and send in the whole current from the two cells and after 3 or 4 seconds close the key. Observe a large contraction on making and a small contraction, or none, on breaking the circuit through the nerve. Turn over the bar of the commutator so that the current is ascending and stimulate again, a small contraction occurs at the make, a large contraction at the break. Stimulate with stiU stronger currents, by intro- ducing one or two more cells into the circuit; observe that a. strong descending current gives a contraction at make but not at break, while a strong ascending current gives one at break but not at make. 4. GJumge in irrUdbUity of a nerve produoed hy a constant current. {Eleetrotonus.) Make a pair of non-polarisable electrodes (§ 3) and place them in the moist chamber. Connect them with two Darnell's cells, introducing a rheo- chord, a commutator and a key for short cu-cuiting the current (cp. Fig. 12). A signal may be introduced into the ch-cuit between the key and the rheochord and its writing-point placed imme- diately underneath the point of the muscle-lever, when the current is sent in, the signal will indicate the period of closure. Arrange a coil to give single induction shocks to the nerve by the platinum electrodes of the moist chamber. Lay the nerve of a muscle-nerve preparation on both pairs of electrodes, the platiniun electrodes being nearest the muscle and as close as possible to one non-polarisable electrode, the non-polarisable electrodes being separated from one another about two centimetres. Bun the drimi at its slowest rate about 2 mm. a sec. Arrange the commutator so that the non-polarisable electrode nearest the platinum electrodes is the kathode. Separate the coils of the induction machine so that a single break shock pro- duces a minimal contraction. Kecord three or four contractions, stimulating regularly every two seconds, opening the key with the same rapidity each time. Place the moveable block of the XIIl] PHYSIOLOGY OF MUSCLE AND NERVE. IIL 119 rheoohord on the wire near the terminal to which one of the non-polarisable electrodes is connected and adjust it so that a contraction is produced at the make only of the constant current. Open the key in the constant current circuit. Stimulate with single break induction shocks regularly every two seconds. Larger contractions are now produced (increased irritability). Close the key in the constant current circuit. Go on stimulating regularly six or eight times immediately after the current is cut out. At first no contractions are produced (after-effect of diminished irritability), later minimal contractions are produced as before. Irregularity will probably appear owing to variation in the rate of break of the primary circuit key. Arrange the commutator so that the anode is nearest the platinum electrodes and send in the constant current. Push up the secondary coil slightly and send in single break induction shocks which give small contractions, i.e. sub-maximal con- tractions. Now stimulate with the single induction shocks, smaller contractions or no contractions result (decreased irrita- bility). Break the constant current circuit. Go on stimulating regularly immediately after the current is cut out. At first maximal contractions are produced (after-effect of increased irritability), later submaximal contractions as before. Irregu- larity will probably appear for the reason given above. 5. Change in irritability of a nerve produced hy change of temperature. Take a piece of glass tubing about 3 cm. long and connect its ends to rubber tubing brought through holes bored in the floor of the moist chamber. Support the glass tube on a cork, placing it so that the nerve can lie conveniently across it. Arrange for a flow of cold water (5° C.) and warm water (30° 0.) through the tubing (cp. Less. xil. § 3). Connect two Daniell's cells through a commutator, a key (short-circuit) and a rheochord (cp. Fig. 12) to a pair of non-polarisable electrodes placed in the chamber. Lay the nerve of a muscle-nerve preparation across the glass tube and on the electrodes, so that the tube is nearest the muscle and quite close to one non-polarisable electrode (or place the lower electrode on the part of ihe nerve which lies on the tube). Arrange the commutator so that the cathode is nearest the glass tube. Set the drum running at its slowest rate. Adjust 120 ELEMBNTAKY PHYSIOLOGY. [XIII the moveable block of the rheochord so that minimal contractions are obtained on throwing in the constant current. Take six or eight contractions, stimulating regularly every two seconds. Then run the cold water through the tube, stimulating regularly aJl the time. The period during which the cold water is runniug may be indicated by a signal in an independent circuit. The contractions win rapidly increase and soon become maximal. When there is no further increase, stop the flow of cold water and run through water at 30° C, stimulating regularly all the time. Note the contractions rapidly diminish, until, probably, no contraction is produced. Bun cold water again and note the gradual increase of the contractions to maximal again. Cold increases and heat diminishes the excitability of nerve to the make of the voltaic current (when the current is closed for at least jj^ sec). Arrange the strength of the constant current so that a con- traction is given at make and at break. Observe the efiect of a rise and of a fall of temperature on the make and break contrac- tions with ascending and with descending currents. Evidence will be obtained that the stimulus is set up at the cathode at the make, and at the anode at the break of the current. 6. Demarcation current of muscle and its negative variation. Arrange a Thomson's reflecting galvanometer of high resistance (10,000 to 20,000 Ohms) to reflect a beam of Ught on to a scale. Connect the terminals of the galvanometer to a shunt, i.e. a resistance box, with resistances of such relations to the resistance of the galvanometer that j^, yj^y, or yg^ of a current led to the shunt may, at will, be sent through the galvanometer and ^, -^^ or ^S^ be short-circuited by the shunt. Set up apparatus for stimulating with tetanizing shocks by platinum electrodes (Lesson xi. A, § 3, 6), leading from the secondary coil by long wires to the short-circuiting key, so that the coU may be 3 — 4 yards from the galvanometer. Take a small zinc rod such as is used for non-polarisable electrodes (op. § 3) and a copper battery wire, and dip them into dilute (1 in 20) sulphuric acid in a watch-glass and connect them vdth the terminals of the shunt, having previously inserted a plug between the terminals, so fhat aJl the current is short- circuited from the galvanometer. Insert a plug in the shunt so XIll] PHYSIOLOGY OF MUSCLE AND NERVE. III. 121 that jg^ij of the current will pass through the galvanometer, then momentarily remove the plug from between the terminals, note in which direction the spot of light on the scale moves, and which terminal of the shunt you have connected with the zinc rod, i.e. made the negative pole. In place of the copper wire and izinc rod, connect to the shunt a pair of non-polarisable electrodes made as in § 3, except that a thick worsted thread, moistened with salt solution and covered with clay, is to be inserted with the clay and left projecting from the tube just enough to support and render slightly moveable the clay which projects from the tube. The electrodes may be held in any convenient clamps standing on a sheet of glass or in the clips of the moist chamber, the whole chamber being placed on glass. Bring the projecting clay of the electrodes into contact and test them to see if they are iso-electrio by sending first ^ and then the whole of any current there may be through the galvanometer. If there is much deflection the electrodes are not properly made. Make a fresh pair. If there is but little deflection, note its amount and which electrode is negative. Cover the electrodes with blotting-paper wet with normal salt solution to prevent them getting dry. Make a muscle-nerve preparation, taking care not to injure the muscle. Fix the muscle horizontally by two clamps, one holding the femur and the other the Achilles tendon, so that it cannot shorten on stimulation. Bring the non-polarisable electrodes into contact with it, so that one touches the muscle at its beUy and the other touches it close to the Achilles tendon. Lay the nerve over the platinum electrodes connected with the coil. Eemove the short-circuiting plug of the shunt and send the whole of any current there may be through the galvanometer. A small deflection is usually obtained and the deflection is in the direction which indicates that the tendon region of the muscle is negative to the belly. In reading the deflection make any correction that is necessary, if the electrodes were found to be not iso-electrio. Short circuit the current at the shunt, with scissors snip the muscle close to the tendon without cutting it through and bring the electrode in contact with the injured spot. Test the electrical condition again ; a large deflection is obtained, the spot of Ught probably passes ofi' the scale. If it does, send in ^ only of 122 ELEMENTARY PHYSIOLOGY. [XIII the current. This is the demarcation current or current of injury. Note that the injured part is negative. Probably at first the spot of light will not remain still, but will move slowly towards zero, indicating that the current of injury is diminishing. After a short time this will probably cease and the spot become steady. Whea it is steady stimulate the nerve with tetanising shocks of moderate strength for 2 or 3 sec. Note that the spot of light moves rapidly towards zero and then comes to rest, indicating a negative variation of the demarcation current as long as the stimulation lasts. 7. Stimulation by the demarcation current and hy the current of action. {Rheoscopio frog.) Arrange a coil for single induction shocks. Take a pithed frog, remove the skin from the legs and dissect out both sciatic nerves from the vertebral column to the knee. Suck up with blotting-paper any blood or lymph from the legs and place the frog on a clean dry cork board. With a scalpel make a transverse incision into the extensor muscles of one thigh, e.g. the left, just above the tendon. With a glass rod lift up the left sciatic, from which the piece of vei-tebral column is removed, and let it fall across the thigh muscles so as to come in contact with the surface and the cut end. As the nerve makes contact, a contraction of the muscles supplied by it will occur. The demarcation current of the thigh muscles stimulates the nerve. Divide the Achilles tendon of the right gastrocnemius, fix the tendon by a pin close to the left knee, and, taking care not to injure the right sciatic, fix the right knee with a pin. Raise the left sciatic with a glass rod and place it across the belly of the right gastrocnemius with its cut end lying close to the tendon of the muscle. Stimulate the right sciatic with single induction shocks, each time the right gastrocnemius contracts the left contracts also. The current of action in the miiscle stimulates the nerve. DEMONSTRATIONS. 1. Isometric contraction. 2. Eleotrotonio currents. 3. Current of action shown by the capillary electrometer. 4. Ei^ographio records in man. LESSON XIV. NERVE FIBRES. 1. Spinal Nerves. Cut off ^ to f of a centimetre 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 them out into the shape of a fan ; add a drop of normal saline solution, and cover with the cover-slip. Observe (h. p.) The meduUated nerve fibres of variable size. Measure the diameter of some of these (Less. ii. § 1). In each fibre the double contour, due to the medullary sheath. The primitive sheath or neurilemma; this is seen with difficulty except at points where the me- dullary sheath has been displaced in mounting. The nodes ; these will be seen as short but distinct breaks in the medullary sheath. Drops and fragments of the medullary sheath, extending from the cut ends of the fibres and showing a double contour. 124 ELEMENTAKY PHYSIOLOGY. [XIV 2. Treat a piece of nerve as in § 1, but add a drop of "2 p.c. nitrate of silver instead of salt solution. Note the rapid staining of the cement substance at the nodes. Expose the specimen to light for half-an-hour. Take off the cover-slip. Dip the nerve in distilled water to remove the sUver solution. Mount in dilute glycerine, spreading out the fibres of the teased end. Note the small dark crosses formed by the nodal cement sub- stance and the axis cylinder near the nodes. Probably the axis will also be stained at and near the cut ends of the fibres. 3. Take a short piece of nerve, and tease it into small bundles of fibres in a drop of "75 p.c. sodium sulphate. Run ofif the fluid, keeping the fibres on the slide with a needle. Add another drop of salt solution, and run it ofi'. Bepeat this. Then add a drop or two of -2 p.c. nitrate of silver, and expose to light for about haJf-an-hour, avoiding drying. Wash in distilled water in a watch-glass, and mount in dilute glycerine, so that most of the fibres are parallel to one another. The staining will thus be more localized at and near the nodes than by method, § 2. 4. Tease out a piece of nerve as in §'l. Add chloroform instead of salt solution, adding more chloroform as evaporation goes on. In the middle of the nerve fibre the transparent axis cylinder will be seen running through the swollen medullary sheath. 5. Take another piece of fresh nerve, and place it in a small quantity of osmic acid '5 p.c. for f to ^ an hour, covering it up to prevent evaporation. Wash it in water, place it in dilute glycerine for a minute or two. Remove to a slide and tease. Select a small bundle separated from the connective tissue sheath, and put the rest back in glycerine for use if required. XIV] NERVE FIBRES. 125 Tease furtlier the small bundle, and arrange the fibres parallel to one another; place a small drop of dilute glycerine on the centre of a cover-slip, and mount. Select a nerve fibre which is isolated for a con- siderable part of its length, and observe The medullary sheath, stained black with osmic acid. The nodes; note that the distance between two successive nodes is greater in large than in small nerve fibres. The numerous oblique breaks in the medulla, dividing it into short overlapping cylinders. The nuclei of the sheath; there is one to each intemode about halfway between the two nodes, it is an inconspicuous transparent elongated body, usually projecting into the medulla. The nuclei of the aheath may be stained by placing a piece of the nerve, after brief treatment with osmic acid, in picrocarmine or in hsematozylin for an hour. In the former preparation, the cells of the fine coimective tissue around the nerve fibres will also be well seen. 6. Transverse sections in paraffin of a large nerve stained with picrocarmine (potassium bichromate 2 p.c). Mount in balsam. Observe The epineurium surrounding the whole nerve and passing between the nerve bundles; its structure resembles that of areolar tissue (p. 56). The perineurium surrounding the nerve bundles, consisting of two or more concentric nucleated mem- branes. The endoneurium between the nerve fibres, con- sisting of a small quantity of fine fibrous tissue. The cut ends of the nerve fibres varying in diameter, 126 ELEMENTARY PHYSIOLOGY, [XIV and in each the section of the stained axis cylinder surrounded by a transparent ring indicating the former position of the medullary sheath, which has been dissolved or made transparent in the process of mount- ing. The primitive sheath as a limiting circle. 7. Cut through the skin of the frog in mid dorsal line. Cut out one of the small dorsal cutaneous nerves issuing from the muscles of the back and running to the skin. Place it in ■2 p.c. nitrate of silver for 5 to 10 minutes. Wash in water and expose to light for about ^ an hour. Dehydrate and mount in balsam. Note the layer of epithelioid cells — shown by the staining of their cement substance — covering the nerve. The nerve will be seen taking a somewhat sinuous course inside the epithelioid sheath. 8. Sympathetic Nerves. Cut out from the fresh spleen of a large animal (e.g. ox) a small piece of one of the large sympathetic 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 constituting the bulk of the nerves. Add acetic acid to bring out more distinctly the elongated nuclei attached to them at short intervals. 9. Tease out in dilute glycerine a small piece of the cervical sympathetic nerve (osmic acid) of a cat or rabbit. Note the numerous small medullated fibres ; some non-medullated fibres may also be seen. Compare the size of the medullated fibres here and in the sciatic nerve (§ 1). XIV] NERVE FIBRES. 127 10. Peripheral Course of Nerves. In the specimen prepared Less. ix. § 4 : 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. 6. 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 medulla extends. 11. Examine the gold chloride preparation (made in Less. vii. § 6) of the cornea. Note a. The small separate bundles of nerve fibres enter- ing the cornea at its periphery ; the medullated fibres on account of their medulla are more deeply stained than the non-medullated fibres. h. Trace as far as possible the course of one of the nerve bundles ; the medulla soon disappears, the fibres, still showing nuclei at intervals, join with the fibres fi-om 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). The coarse plexus is the primary plexus of the cornea. 128 ELEMENTABT PHYSIOLOGY. [XIV 12. Neurokeratin network. Tease out in clove oil a piece of nerve which has been extracted with alcohol and ether \ Mount in balsam. Note the distinct network in the region formerly occupied by the outer part of the myelin sheath. The size of the meshes varies in different fibres. DEMONSTRATIONS. 1. Transverse sections of sciatic, vagus, and cervical sympathetic nerves. (Cat or rabbit; osmic acid.) Note the relative size of the nerve fibres in the three nerves, and the number of non-medullated fibres in the vagus. 2. Transverse section of non-medifllated nerve (splenic nerve of large animal; osmic vapour). Note the oval or circular outlines of the fibres ; they vary very little in size, being generally 2/* in diameter. 3. Teased degenerating nerve fibres (4 days after section; osmic acid). Note the myelin broken up into masses and globules of various size; in places a considerable stretch of the nerve may show very little alteration. 4. Similar nerve fibres in later stage (20 days after section). The myelin has nearly all disappeared, a few fibres still show here and there rows of fat globules. ' Place pieces of nerve in absolute alcohol for a day, boil in the alcohol on a water bath for about 15 minutes and place in ether for a day. Part should be stained with heematoxylin, and part with oBmio acid. The pieces may be kept in clove oil. LESSON XY. PERIPHERAL GANGLIA AND NERVE CELLS. 1. Transverse section of spinal ganglion^ (Osmic acid ; paraffin.) Mount in balsam. a. Note with a low power, the sheath of the ganglion; the numerous nerve cells having between them meduUated nerve fibres, comparatively few nerve fibres being present between the more peripheral cells ; the transversely cut nerve fibres chiefly grouped at one side of the section. (The fibres of the anterior root may form a separate bundle with a distinct . sheath.) b. Note with a high power The nerve cells of various sizes, the small ones as a rule being stained more deeply than the large ones ; the cell substance varying from nearly homogeneous to coarsely granular ; the large spherical nucleus, and large nucleolus or nucleoli. The numerous large nerve fibres ; some fibres will be seen coiling amongst the cells in the plane of the section. ' In §§ 1 to 3, the ganglion of a cat or dog may be taken. Section 1 and section 4 should be mounted on the same slide. 130 ELEMENTAKY PHYSIOLOGY. [XV The sheath of the ganglion consisting internally of several membranes, and externally of loose connective tissue. 2. Sections of a spinal ganglion (potassium bichro- mate; cut frozen) are given you in water. Stain a section in hiEmatoxylin and mount in balsam. Note around each nerve cell the numerous nuclei of the capsule. 3. Sections of spinal ganglion (mercuric chloride) given in paraffin. Take two cover-slips, fix sections to them, and treat them for staining on the cover-slip (Less. rv. § 13) up to the stage of placing them in 50 p.c. alcohol. a. Place one slip in Nissl's methylene blue for about 20 minutes (or warm it with the solution in a watch-glass, till the fluid begins to steam). Blot it between folds of blotting-paper to remove excess of fluid ; place it before it dries in a mixture of 10 parts aniline oil and 90 parts 95 p.c. alcohol. Gently move it about in this, tiU the blue colour of the section is rather faint (the proper tint can only be told by trial) ; then blot again, transfer to absolute alcohol for 30 to 60 seconds. Clear in xylol, mount in balsam, taking care that the balsam is added to the cover-slip before the xylol evaporates as the sections become dry. Note in the nerve cells the basophil masses and granules stained deep blue, they vary in number and size in different cells, and are often absent from the peripheral zone ; they are for the most part arranged concentrically around the nucleus. b. Place the other slip for 1 to 1^ minutes in erythrosin (or eosin), blot, and treat as in a. The erythrosin stains the parts of the sections left unstained by methylene blue. Methylene blue in 75 p.c. alcohol or Loffler's methylene blue may be used instead of Nissl's methylene blue ; 95 p.c. alcohol may be used instead of aniline oil and alcohol ; but this ia apt to produce somewhat diffuse staining. XV] PERIPHERAL GANGLIA AND NERVE CELLS. 131 4. Transverse section of a sympathetic ganglion from the sympathetic chain. (Osmic acid; paraffin.) Under both low and high power compare this section with that of the spinal ganglion in § 1. Note The nerve cells vary in size, but on the whole are not so large as in the spinal ganglia ; they are more irregular in shape, and a considerable number of non- medullated fibres lie between them. The great majority of the medullated fibres are «mall {2/1 to 3ju). 5. Transverse section of lumbar spinal cord of ox, calf, or dog. (Potassium bichromate; cut frozen; picrocarmine.) Note the large multipolar nerve cells of the anterior cornu ; the processes are given off in all directions, some are cut short, others will be seen to divide in the section into finer and finer branches (protoplasmic processes or dendrons) ; here and there a cell may be seen from which a rather large process is given off which runs outwards through the white substance without branching (axis cylinder process or axon). 6. Tease out in formic glycerine a small piece of the spinal ganglion of a skate (ganglion in 30 p.c. alcohol for 3 days, one day in picrocarmine, washed; kept in glycerine). Note the bipolar cells, forming oval to spherical nucleated swellings on the course of a nerve fibre. The connective tissue sheath of the nerve will be seen to run over the cell and form its capsule. 9—2 132 ELEMENTARY PHYSIOLOGY. [XV 7. Spiral nerve cells of the auricular septum of the frog. Take a pithed frog : expose the heart, cut through the peri- cardium, lift up the edges and out it away so far as it is seen. Turn the ventricle forward, out through its ligament, lift up the heart by the ligament and pass two silk threads under the aortie. Draw one backwards, and tie, underneath the ventricle, the veins running to the'heart. With scissors make an incision into the bulbus just before it branches, being careful not to cut it through. Squeeze the heart a little to empty it, wipe away the blood with a piece of .sponge moistened with normal salt solution. Put a fine pointed cannula provided with a short piece of india-rubber tubing into the bulbus,' prop up the tubing so that the cannula will not slip out, tie it in the bulbus. By means of a pipette fill the cannula with salt solution, squeeze the tubing and so force the salt solution into the heart. Remove the fluid from the cannula by means of the pipette, fill with fresh salt solution, and so on till the fluid returning to the cannula from the heart is colourless. Empty the cannula, fill it with "Sp.o. gold chloride, take a glass rod which fits the tubing, and slowly push it in so that the heart is distended with gold chloride. See that the right auricle is distended for a minute or two ; then lift up the heart by means of the cannula and the thread tied round the veins, and cut peripherally of the ligatures. Place the heart in a little gold chloride solution in a watch-glass for half-an-hour. Transfer to water, cut away the bulbus with the cannula, the superfluous tissue round the auricles, and two-thirds of the ventricle. Move about in the water, and place in 25 p.c. formic acid in the dark till next day. Then wash in water, and under a dissecting lens cut through the projecting wall of one auricle, note the position inside of the thin membrane, the septum aurioularum, and cut away from it the auricles and the rest of the ventricle. Mount it in formic glycerine. Note (a) the two main strands of nerve fibres running through it ; the medullated fibres in these will be stained more deeply than the non-meduUated fibre. (6) The septum itself formed of a plexus of cardiac muscle cells in a connective tissue membrane, (c) The numerous nerve cells attached to XV] PERIPHERAL GANGLIA AND NERVE CELLS. 133 the nerve-strands. The nerve cells are generally pear shaped. The spiral process may be seen coiling round the straight pro- cess, but it is not usually distinct. (The heart may of course be injected from the inferior vena cava ; the method described above is recommended because it is perhaps easier to insert a cannula into the bulbus than into the vena cava.) The heart may be injected with alcohol; in this case inject weak before strong alcohol, and colour the strong alcohol with a little eosin, so that the septum may be more easily seen. Pass scissors down the balbus into the ventricle and cut ; cut oS three-fourths of the ventricle, pass scissors from the ventricle into one auricle and cut. The septum can then be readily separated under a dissecting lens, and stained with any reagent desired. 8. Nerve cells of Auerhach's plexus. Cut through and reflect the abdominal walls of a recently killed mouse or rat. Cut out about two inches of the small intestine, tie a cannula in one end, and using a syringe wash out the intestine with normal salt solution. Tie the free end. With a pipetto fill the cannula with 1 p.c. gold chloride, distend the intestine with this, tie just below the cannula, whilst the intestine is still distended. Place it in a watch-glass in gold chloride solution for half-an-hour. Remove to water ; cut open lengthways, and wash well. Place in 25 p.c. formic acid in the dark till next day. Scrape off the mucous membrane, and mount in formic glycerine two pieces of the muscular coat, one with the outer, the other with the inner surface uppermost. Note the wide meshed plexus of Auerbaoh between the muscular coats ; the meshes are rectangular and groups of close-set small cells lie at the nodal points, their nuclei staining less than the cell sub- stauce. The plexus of Meissner may be seen in places in the sub-mucous layer; it is similar to that of Auerbaoh but the meshes are irregular in shape, the strands and groups of nerve cells smaller. 9. Ganglia of the sympathetic chain in the frog. Lay open the abdomen of a recently killed frog, raise the rectum, and 134 BLEMENTABY PHYSIOLOGY. [XV remove the intestine, liver and stomach, cutting through the mesentery just above the kidneys. Lift up the kidneys and cut between them. Lift up each kidney in turn, the peritoneum — separating the abdominal cavity from the lymphatica cistema magna— will be seen stretching flrom the lateral edge of the kidney to the body wall. Remove the kidneys with this part of the peritoneum. Sponge up any blood that may be present. Note on either side of the aorta a longitudinal pigmented strand, the sympathetic chain, and the fine nerves — the rami communicantes — connecting it with the adjacent spinal nerves. With the aid of a lens, the gangha may be seen as spindle shaped swellings on the sympathetic chain. Bemove the chain on one side ; stretch it out on a sUde, cover, and add normal salt solution. Examine with a high power. Note the nearly globular, transparent nerve cells ; much hidden by the pigment cells. The nerve fibres of the rami are chiefly small medullated fibres. By treating the sympathetic chain with dilute methylene blue as directed in Less, xvi., a cell will here and there be seen with deep blue spiral fibre and showing the branchings of the spiral fibre (synapses) on the cell. Teased ganglia, whether they are taken fresh, or after treat- ment with reagents, do not give satisfactory preparations of nerve cells, aa the process or processes are usually broken ofi" short. DEMONSTRATIONS. 1. Longitudinal section of spinal ganglion, to show the anterior and posterior roots attached to it, the anterior root running past the nerve cells and joining the fibres of the posterior root at or near the lower end of the ganglion. 2. Section to show basophil granules and masses of spinal ganglion cells (cp. § 3). XV] PERIPHERAL GANGLIA AND NERVE CELLS. 135 3. Section of spinal cord of mammalian embryo or of chick prepared by Golgi-Oajal method (cp. p. 341), showing multipolar cells with a single axon, and many dendrons. 4. Section of spinal ganglion of chick prepared by Golgi-Cajal method, showing unipolar cells with dividing process. 5. Septum auricularum of frog (cp. § 7). 6. Plexus of Auerbach in intestine of mouse (cp. §8). LESSON XVI. ENDINGS OP EFFERENT NERVE FIBRES IN MUSCLE. 1. The End-Brush. Take a pithed frog, and expose the sartorius muscle (cp. Less. ix. § 2). Free this at its lower end ; raise it by the short tendon and cut through the connective tissue on either side and below it, close to the muscle, being careful not to injure the muscle fibres. Cut through the upper attachment. Place the muscle on a slide, deep side uppermost, add two drops of "05 p.c. methylene blue dissolved in '6 p.c. NaCl. Line a watch-glass with moist blotting-paper, and with it cover the specimen. Leave for 15 to 20 minutes; then drain off the methylene blue. Gently pull the ends with needles to extend the muscle, add one drop of normal salt solution, and examine with low objective and high ocular. Follow the muscle fibres along, until the small rather opaque nerve entering the muscle is seen ; trace the course of the nerve across the muscle fibres. Here and there, one or two nerve fibres will be seen leaving XVl] NERVE ENDINGS IN MUSCLE. 137 the nerve, running to the muscle fibres, and ending on them in blue stained nerve-endings. Selecting a well stained nerve-ending or end-brush, note that in nearly all cases the branches are given off at right angles to the stem, and that they are not very close together. Fine nerve fibrils accompanying the blood vessels will also be seen. When a number of endings have been examined, put on a cover-slip, gently press. With a low power, put a typical ending in the middle of the field, and then examine it with a high power. Soon after the cover-slip is placed on the muscle, the methylene blue begins to be reduced to a colourless compound, and in consequence the nerve fibres become paler till they are no longer seen. Note. The success of this preparation depends largely upon not injuring the muscle fibres. Where they are injured, they stain deeply ; they should have no stain at a time when the nerve-endings are deep blue. 2. Preeervation of nerve-endings stained with methylene blue. When the nerve-endings are distinct under a low power, the muscle is pinned out with hedgehog quills, and placed in a saturated aqueous solution of picrate of ammonia for 10 to 15 minutes. It is then transferred, without washing, to the following solution, Ammonium molybdate 1 gram. Distilled water 20 c.c. Hydrochloric acid 1 drop, and left for | to 1 hour. It is finally washed with water, passed through alcohols, and xylol, and mounted in balsam. 3. Make a similar preparation of the stemo-cutaneous muscle of the frog (Less. ix. § 4). The nerve-endings resemble 138 ELEMENTARY PHYSIOLOGY. [XVI those of the sartorius, but in general are not so large or so regular. 4. Reflect the skin from the lower jaw. Observe with a dissecting lens the mylo-hyoid muscle on either side, attached to the edge of the jaw, and, in the middle line, to its fellow muscle. Carefully out this out, and treat in the same manner as the sartorius (§ 1). Note in it the plexus of fine nerve fibres and fibrillae throughout the muscle, and the small and irregular nerve-endings. 5. Eind plates. Take a piece about an inch and a half long, of a recently pithed snake. Out through the skin in the mid-ventral Une. Gut through on one side the muscle attached to the skin near the mid-ventral line, and pin out the skin. Take the edge of the ribs in forceps, and pull the body gently away from the skin and upwards. A series of small band like muscles wiU be seen passing from the body to the skin. Pin back the body so that these muscles are stretched, carefully tear away the tissue on either side of the muscles, isolating them up to their connections at either end. Cut out the muscles and treat them with dilute methylene blue, in the manner given in §1. Isolate similarly the muscles of the opposite side up to their origin and insertion ; arrange the pins so that they are well stretched ; bi-ush them with 1 p.c. gold chloride. In about 5 minutes, when the muscles have become stiff, cut quickly the attachments, remove with a glass rod to gold chloride for about 25 minutes. During this interval examine the muscles treated with methylene blue, placing on a slide, and moistening them with normal salt solution. Observe in the end plates, the curved branches of the axis cylinder set closely together, the whole end plate forming an oval or round patch. The muscles which were treated with gold chloride may now be removed to water and washed thoroughly. Place in 25 p.c. formic acid in the dark till next day. Wash and place in formic glycerine, put under a dissecting lens, and tease off as much connective tissue as possible. Mount in formic glycerine, press XVl] NERVE ENDINGS IN MUSCLE. 139 the cover-slip to separate the fibres a little, and examine for end plates, using a lamp for illumination. 6. Nerve-plexus in unstriated muscle. Cut out the oesophagus of the frog used in § 1. Cut it open, scrape gently the mucous surface. Piu it out with hedgehog quills over a hole in a piece of cork, muscular coat uppermost. Cut ofif the quills close to the mem- brane. On the muscular coat place a couple of drops of "05 p.c. saline methylene blue. Cover up with a watch-glass lined with wet blotting-paper for 20 to 30 minutes. Then wash off the blue with a drop or two of "6 p.c. salt solution. Place the piece of cork on a slide and examine with low objective and high ocular, and without diaphragm. Note the very close plexus of fine blue-stained nerve fibrils, generally varicose in the muscular coat. Put on a cover-slip and examine with a high power. 7. Make a methylene blue preparation of the nerve plexus in the muscular coats of the rectum of the frog, treating it in the manner given in § 6. DEMONSTRATIONS. Microscopic specimens showing motor nerve-endings in snake's muscle. (Cp. § 5.) LESSON XVII. REFLEX AND AUTOMATIC ACTION. A. Reflex Action. 1. A frog is given you which has been deprived of its brain but not of its spinal cord (cp. App. p. 376). Place it in the sitting posture. Observe that its hind-limbs are drawn up under the body ; but that it differs from the normal frog in the following respects. Its head is depressed, instead of being erect. 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. There are no respiratory movements, either of the nostrils or of the throat. 2. 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 position 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. XVIl] REFLEX AND AUTOMATIC ACTION. 141 3. Gently tickle one flank with a feather or a blunt needle; a contraction of the flank muscles of that side will be observed. 4. 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. 5. 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: if no disturbing circumstances are brought to bear on it, it will remain perfectly motionless. 6. Place the animal on its back ; it will make no effort to regain its normal position, i.e. all sense of equilibrium has been lost. 7. 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. 8. Gently pinch the tip of one of the toes of either leg ; that leg will immediately be drawn up. 9. Fill a beaker with water; and place a little acetic acid "5 to 1 p.c. acid in a watch-glass : let the tip of one of the toes of the frog touch the acid. In a short time the foot will be withdrawn. Dip the foot 142 ELEMENTARY PHYSIOLOGY. [XVII into the water, in order to wash away the acid. Mea- sure, with the aid of a rapidly beating metronome, the time between the moment when the toe comes into contact with the acid and the moment when it is with- drawn. Make, at intervals of two minutes, three such observations (being careful that the toe dips in the acid to exactly the same extent) and take the mean of the three. 10. Cut a small piece of blotting-paper one or two mms. square, moisten it with 1 to 5 p.c. 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. Wash away the acid. 11. Place similar pieces of acid-paper on different parts of the body ; different movements will be wit- nessed in consequence ; all however tending to remove the irritating substance. 12. 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 acci- dentally much distended with air), and no movements of any kind will be witnessed. Observe that all the movements produced in the foregoing observations, although complicated, co-ordinated, and pur- poseful 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. § 1 should be performed here. XVIl] REFLEX AND AUTOMATIC ACTION. 143 13. 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 sulphate 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 observed. 14. With a straight seeker or a piece of stout wire destroy the whole of the spinal cord. The spasms immediately cease. 15. Repeat any of the above observations (§ 2 — 13). No reflex actions will now be produced. B. Automatic Action. 1. The Lymph-Hearts. Placing the animal on its belly watch the movements 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 (A. § 14) the lymph-hearts cease to beat. 3. The Heart. Expose the heart in a pithed frog, it will be seen in the thin membranous peri- cardium beating with considerable regularity and force. Pinching up the pericardium with a fine pair of forceps. 144 ELEMENTARY PHYSIOLOGY. [XVII cut it away from the surface of the heart, then tilt up the apex of the ventricle ; a small thread of connective tissue (the ligament of the ventricle) will he ohserved passing from the posterior surface of the ventricle to the adjoining wall of the pericardium; cut this through near the peiHcardium. Lift up the aorta and cut through the aortic branches, the superior vence cavae, the inferior vena cava, and the surrounding tissue. 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. 4. Lifting up the apex of the ventricle by means of its ligament, 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 auricles and the upper third will continue to beat with regularity. 5. 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. 6. 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 divided, and moisten the mucous membrane, if it is at all dry, with normal saline XVIl] KEFLEX AND AUTOMATIC ACTION. 145 solution. Place on it in a line as high up as practicable three small thin pieces of cork. The pieces of cork will be seen to be driven by ciliary action towards the stomach; probably the middle piece will travel the fastest. DEMONSTRATION. Peristaltic action of the intestine and of the ureters. 10 LESSON XVIII. STRUCTURE OP BLOOD VESSELS. CIRCULATION. INFLAMMATION. 1. Large Arteries and Veins. Transverse sections of thoracic aorta, carotid artery, and jugular vein of dog (potassium bichromate 2 p.c). Stain with hsematoxylin and eosin (Less. iv. § 6), or with hsematoxylin and picric acid. Mount together. a. Note in the aorta the inner coat, consisting of an epithelioid lining shown chiefly by the nuclei, and of a thin layer of elastic tissue with a small amount of white fibrous tissue. The elastic tissue shows chiefly as transversely cut fibres. The thick middle or muscular coat consisting of bundles of smooth muscle and elastic fibres circularly arranged in alternating layers. The outer coat, much thinner than the middle, consisting of white fibrous tissue and of elastic tissue ; most of the fibres of the elastic tissue are cut trans- versely, they diminish in number in passing outwards. XVIIl] STRUCTURE OF BLOOD VESSELS. 147 b. Note in the carotid artery that the elastic part of the inner coat is chiefly represented b/ a refractive, and (probably) wavy lamina; and that thij elastic tissue is comparatively scanty in the middle coat. c. Note in the vein that the whole wall is thinner; the inner coat is inconspicuous, except as regards the nuclei of its endothelium, also the middle coat is much thinner than the outer. 2. Cut open longitudinally the jugular, or other large vein of a freshly killed rabbit. Pin it out with hedgehog quills, stream it with 'To p.c. sodium sulphate for a moment, add "2 p.c. nitrate of silver and leave for 10 minutes, wash in distilled water, expose to light for half-an-hour, pass through alcohols, clove oil, and mount in balsam. Observe the rather jagged dark lines of the cement substance between the cells ; the cells form a continuous layer, are more or less elongated in a longitudinal direction, and are flat ; indications of their nuclei 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 silver nitrate solution, or exposed too long to light, silver will be deposited in the substance of the cells also. 3. Tear off a strip from the inner coat of a medium-sized artery (potassium bichromate "2 p.c. for two to six days; it may be kept in dilute glycerine). 10—2 148 ELEMENTARY PHYSIOLOGY. [XVIII Tease it out in the preserving fluid ; it will be found to consist almost entirely of elastic laminse. Observe the gradations from an almost homo- geneous elastic perforated membrane to a meshwork of elastic fibres. 4. Remove a small portion of pia mater from the brain of a recently killed animal, brush it well in normal salt solution, and wash it in more salt solution, make a moist film of it, fix with alcohol, stain with hsematoxylin (a trace of picric acid after stain is advantageous) and mount in balsam. Note The smallest arteries ; no distinct external coat is seen; the middle coat consists of a single layer of muscle cells wound transversely to the tube, the nuclei are deeply stained, the outlines of the cells will be seen on focussing; the inner coat is represented by the elongated nuclei of the epithelioid lining. The capillaries showing as thin, nucleated, mem- branous tubes. The small veins, of larger calibre than the small arteries, with no muscular coat (this is special to the central nervous system), and in general appearance resembling the capillaries. 5. A pithed frog is given you. Take one or two small pieces of cotton-wool and plug the hole in the vertebral canal. Expose the heart, cutting through the sternum in the middle line, and pinning back the two parts. Cut away the exposed part of the pericardium, pass a thread under the bulbus aortse, with fine-pointed scissors make a cut in the bulbus near the ventricle. With a sponge, moistened with normal salt solution, wipe away the blood which comes, stroking the abdomen gently upwards to remove as much blood as XVIIl] STRUCTURE OF BLOOD VESSELS. 149 possible. Cut across the inferior vena cava, and sponge up the blood. Pick up the cut edge of the aortic bulb with flne-pointed forceps, and put into the bulb and up the left aorta a fine nozzled cannula, provided with an inch or so of india-rubber tubing. Tie in the cannula, fill it with -75 p.c. sodium sulphate and pinch the tubing, in order to drive blood out of the nozzle of the cannula, where it rapidly clots. Fill the cannula again; take a 5 to 10 c.c. injection syringe, the nozzle of which fits the india-rubber tubing, fill it with •75 p.c. sodium sulphate, and push the nozzle into the tubing, pinching the tubing as the nozzle is inserted, so as to avoid air-bubbles. Inject the sodium sulphate, sopping up with a sponge the fluid which comes from the out vessels; and now and then stroking the abdomen upwards. When the fluid is nearly colourless (injection of one syringe-full should be sufficient), inject in the same way a syringe-full of 0-2 p.c. nitrate of silver. Leave for five minutes. Inject a syringe-fuU of distilled water. Then put the frog in distilled water. Expose the viscera freely, and cut out the following tissues and expose to light in distilled water. The bladder: lift it up by the ends, cut through the membrane attached to it ; remove it and cut it open. The intestine and mesentery : lift up the end of the rectum, cut through the mesentery at its dorsal attachment and remove it with the intestine, cut ofi' the mesentery and take the largest pieces ; cut open the intestine, scrape off the mucous membrane, and pin out the muscular coat with quills. (The lungs, pinned out, and the kidneys may also be taken; the latter will show the epithelioid cells of the capsules.) When the tissues show signs of reduction, dehydrate pieces — ^keeping them stretched out — and mount in balsam. Note the outlines of the flat elongated epithelioid cells of the arteries, of the capillaries and of the veins ; the outhnes 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 larger vessels the cement substance between the cells of the 150 ELEMENTARY PHYSIOLOGY. [XVIII muscular coat will also show as black transverse lines, these are more numerous in the arteries than in the veins. 6. Circulation of Bloods Tho frog given you has been deprived of its brain and curarised (cp. Less. XII. § 5). Lay it on its belly on the frog-board, and tie, not too tightly, a piece of soft cotton round the end of the {e.g.) 3rd and 4th 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 with hedgehog quills, and the quills cut short). Surround the web, and cover the leg and body of the frog with moist blotting-paper. Put a piece of cover-slip over the toes, and with a small brush press the web from below against the slip. (If the web is not in close contact with the glass it may be outside the focal distance of the high objective ; in that case, a triangular piece of cover-slip should be placed on the web between the toes, and excess of fluid sopped up, but by this method the front lens of the objective is apt to be smeared by touching the tissue.) Examine, first with a low and then with a high power. Note The course of the blood from the arteries to the veins. A slight pulsation may be observed in the larger arteries and sometimes in the smaller ones. The greater velocity of the blood in the arteries 1 In the spring it is best to take a male frog for the circulation in the mesentery (§ 9). The male may be recognised by the wart-like projection on the ball of the thnmb. XVIIl] CIRCULATION. 151 (owing to their smaller size) than in the veins; pro- bably in neither can the individual corpuscles be made out. The axial and peripheral zones in the arteries and veins; the peripheral zone is small, and under a low power appears free from corpuscles; under a high power one or two white corpuscles may, if the current is not very fast, be seen in the peripheral zone of the arteries ; in that of the veins a few white corpuscles and occasionally a red one will be seen moving along comparatively slowly. The passage of corpuscles usually in single file through the capillaries ; The elasticity of the red corpuscles; observe the way in which they bend and become deformed, and then regain their normal shape. 7. Inflammation. Eemove the cover-slip ; com- pare the circulation in two adjoining webs under a low power. Soak up the fluid on them ; on one place a drop of xylol, and examine it, comparing it with the other. a. The arteries dilate, the veins become larger, and the capillaries much more distinct. 6. At first the circulation is quicker; later it is slower than at first, though the vessels remain dilated. c. The circulation stops after a time (stagnation), the red vessels being distended with blood (if this is not caused by the xylol, add a drop of turpentine). Where the red corpuscles are pressed together, as in the capillaries, their outline is lost. (For other changes in inflammation cp. § 9.) 152 ELEMENTARY PHYSIOLOGY. [XVIII d. The effects are local, they are not seen in the other web. e. If tho' injury to the vessels has been slight, the circulation may be seen to be re-established ia the stagnated spots, the corpuscles gradually recover their outline and are carried off by the current ; this is not seen if stasis has set in, i.e. if the blood has clotted. 8. Observe now the circulation in the tongue ; the frog being on its belly, draw forward the tongue over the hole in the stage and pin out the two cornua ; the tongue at first pale soon becomes flushed and its vessels full of blood. With a low power the peripheral zone in the arteries and veins will probably be seen better than in the web. 9. Eeplace the tongue. On the right side make a cut through the skin and body wall, 1^ to 2 cm. long, midway between the dorsal and ventral surfaces and rather nearer the hind than the fore limbs. Place the frog against the hole in the side of the frog-board, and pull out the coil of the intestine above the rectum, pin it out with hedgehog quills, cut the quills off short, and put small pads of blotting-paper wet with salt solution round the edge of the intestine, cover with a piece of cover-slip, and observe the circulation in the mesentery. The exposure will probably cause some degree of inflammation, and the stages between the early dilation (§ 7, a) and stagnation (§ 7, c) may conveniently be followed. Note under a high power a. The elongated platelets and the spherical white XVIIl] CIECUIATION. 153 corpuscles increase in number in the peripheral zone of the arteries and veins, if the current is quick enough to show a peripheral zone ; the white corpuscles often roll over along the wall. b. In the veins, and to a less extent in the capillaries, the platelets and white corpuscles cling to the walls, at first for a time only, later permanently. Here and there a mass of adhering corpuscles forms which stretches across the tube, sometimes this is torn away by the current, sometimes it blocks the vessel and causes stagnation. Note in the stagnated vessels the gradual obliteration of the outlines of the cor- puscles. c. Focus the side of a small vein in which the circulation is slow, and look amongst the white corpuscles adhering to the wall for one which projects a little externally. Draw this at intervals of 15 minutes to note its rate of migration. 10. Circulation of blood in the newt. A newt is given you without brain and curarised. Mesentery. The hole in the stage should be small, about 1 to 1'5 cm. long, and -5 to '75 cm. broad. The method is the same as in the frog (§ 9). Pancreas. The skin and abdominal wall are cut through on the left side, the cut beginning a little below the fore leg and extending for 1 to 1^ cm. The pylorus and the part of the intestine below it are pinned out. The circulation- in the mesentery is also seen, but less satisfactorily than in the lower loop of the intestine. The same cut allows the lung to be pulled out, and the circulation in it observed. 11. Preservation of inflamed mesentery. When the circulation has been observed, cover up with a funnel lined with wet blotting-paper till next day. Remove the lower part of the intestine with its mesentery, and wash with salt solution. Fix the mesentery in alcohol, cut it into two parts, stain one with 154 ELEMENTARY PHYSIOLOGY. [XVIII Ehrlich-Biondi fluid, and the other with hsematoxylin and eosin. Mount in balsam. Note the numerous leucocytes with irregularly shaped or fragmented nuclei outside the capillary walls. DEMONSTRATIONS. 1. Action of valves. A cannula is tied in the peripheral end of a vein {e.g. the jugular) of a dead animal. On forcing fluid into the vein it will be seen to swell greatly as far as the proximate valve. 2. Vein cut open and pinned out to show the valves. 3. Longitudinal section of aorta of large animal. 4. Specimen of blood vessels, frog, showing epi- thelioid cells (cp. § 5). 5. Circulation in the lung of the frog or toad (cp. p. 130). LESSON XIX. STRUCTUEE AND ACTION OF THE HEART. A. Heart of Sheep^ 1. Observe the attachment of the parietal pericar- dium to the roots of the great vessels. Remember that the parts of the heart which are right and left in the body are called right and left after removal. The front of the heart may be recog- nized by a groove filled with fat, the interventricular sulcus, which runs from about the middle of the base of the ventricles to rather below the middle of the right margin of the heart. The front is also more convex than the back. Holding the heart with the front towards you, note that the right ventricle, which will be on your left hand, is much more yielding than the left ventricle, which will be on your right hand. Note also the pulmonary artery arising nearly in the middle line of the heart at the upper part of the ventricles, and immediately behind this the aorta. ' The heart should be obtained from the butcher with the pericardium; to secure this it is advisable to purchase the 'bag,' i.e. the heart with the lungs still attached to it. 156 ELEMENTARY PHTSIOLOGT. [XIX 2. Tie a short glass tube into the superior vena cava and connect with it a piece of india-rubber tubing. Ligature the inferior vena cava and the left vena azygos which opens close beside it. Tie a glass tube about two feet in length into the pulmonary artery. Fill the india-rubber tubing with water, and squeezing it press the water onwards. The water will mount in the tube connected with the artery, and will only descend a little way on unclasping the india- rubber tubing. Pour water into the long glass tube by means of a funnel, and observe the column of water which the semilunar valves will sustain. Note the distension of the arterial walls and the bulging at the attachment of the valves. When the pressure of the column of fluid is removed the artery by its elasticity returns to its previous dimensions. 3. Repeat the above observation with the pul- monary veins and aorta. 4. Compare the united sectional areas of the superior and inferior vense cavse when distended, with the area of the aorta below the origin of the innominate artery. 5. Having removed the tubes, lay open the superior and inferior venae cavae, and bring the incisions to meet in the front of the auricle. Note The size and form of the auricular cavity. The auricular appendage with its muscular fretwork. The septum auricularum. The fossa ovalis, or expression of the foetal foramen ovale, which is early closed by the growth of the septum auricularum. XIX] STRUCTURE AND ACTION OF THE HEART. 157 The Eustachian valve, a slightly projecting mem- branous fold, immediately beneath the entrance of the inferior vena cava, and again beneath this The opening of the comparatively large left azygos vein. The auriculo-ventricular orifice. 6. Cut open longitudinally the azygos vein, and observe the coronary vein' opening into it a very short distance from the heart. 7. Cut away most of the auricle, and holding the ventricle in the left hand, pour water suddenly into the auriculo-ventricular orifice. The right auriculo- ventricular or tricuspid valve will float up and close the orifice. Note the star-shaped junction of the valve-flaps. 8. Introduce a pair of scissors between two of the valves, and cut through the wall towards the apex. Having arrived at the bottom of the ventricular cavity, turn the scissors sharp round and carry an incision at an acute angle with the previous one, alongside the septum, towards, but not into, the pulmonary artery. Lifting up the flap, note The thickness of the ventricular wall, the pro- jections of its inner surface or columnse carnese, the band of muscle (moderator band) running from wall to wall of the ventricle across its cavity. The ventricular cavity does not extend to the apex. ^ In man, the left azygos vein joins the light, and this runs into the superioi yeua cava ; the coronary vein (coronary sinus) opens direct into the right auricle. 158 ELEMENTARY PHYSIOLOGY. [XIX The tricuspid valve, its form, and attachment to the auriculo-ventiicular ring, the chordae tendineae, and their attachment to the summits of the papillary muscles. 9. Holding the heart vertically, pour water into the pulmonary artery ; observe from below the form of the semilunar valves, and their mode of closing. 10. To observe the valves from above, insert into the pulmonary artery a short wide tube, fill it with water, and cover it with a piece of glass, excluding air-bubbles. 11. Prolong the incision of § 8 so as to lay open the pulmonary artery. Note The form and attachment of the semilunar valves. The small nodule of tissue in the middle of the free edge of each valve, the corpus Arantii. The slight depressions in the arterial walls opposite each valve, the sinuses of Valsalva. 12. Lay open the left auricle in a manner similar to that employed for the right. Note that the left auriculo-ventricular valve, the bicuspid or mitral has but two flaps. Observe its manner of closing (cp. § 7). 13. Lay open the left ventricle in a manner similar to that employed on the right side, carrying the incision at first along the extreme left of the heart. Note the thick walls, the mitral valve, &c. 14. Lay open the aorta, and examine its semilunar valves, corpora Arantii, and the sinuses of Valsalva, XIX] STRUCTURE AND ACTION OF THE HEART. 159 which are here very distinct. Note that the coronary arteries open respectively into two of the sinuses. B. Heart of Frog. 1. Expose the heart of a just-pithed frog. With the pericardium intact, observe the pulsations of the heart, noting the alternate beats of the auricles and the ventricle; and the synchronous beats of the two auricles. Lay open the pericardium and observe (best with the aid of a lens) a. The synchronous contractions of the two auri- cles, followed almost immediately by b. The contraction of the ventricle ; note that the ventricle during its contraction or systole becomes paler and more conical, and that its apex is thrown forwards and upwards ; the obviousness of these changes depends upon the force of the contraction. c. The slight contraction of the bulbus arteriosus immediately succeeding the ventricular systole. d. The pause, or diastole, which follows before the auricles again beat ; if the heart is beating rapidly this may not be obvious to the eye. e. The increased redness and distension of the ventricle after the auricular, and immediately pre- ceding its own systole. 2. Divide the ligament of the ventricle (cp. Less. XVII. B, § 3) ; turn the ventricle forward. Observe a. The junction of the two superior vense cavae with the inferior vena cava to form the sinus venosus. 160 ELEMENTARY PHYSIOLOGY. [XIX b. The whitish line, roughly V- shaped, marking the junction of the sinus venosus with the right auricle. c. The wave of contraction ; it starts in the end- ings of the great veins, and is most easily seen in the superior venae cavse; then follow in quick succession, contraction of the auricles, the ventricle, and the bulbus arteriosus. Note the distension of the bulbus and the rush of blood through it, as the ventricle contracts. 3. Dissection of the vagus. Make a transverse cut through the skin of the frog just below the lower jaw, and carry the cut as far as the vertebral column. Cut away the skin over the lower jaw. Cut through the superficial muscles connecting the shoulder or sternum with the hyoid bone, the jaw and the skull. Near the mid line of the lower jaw will be seen two nerves on each side, the hypoglossal and the glossopharyngeal. Near the symphysis of the jaw the hypoglossal — which is the more superficial of the two — ^lies on the outer side of the glossopharyngeal (cp. Fig. 13). Trace them backwards; the hypo- glossal crosses first the glossopharyngeal and then the branches of the aorta. The glossopharyngeal on coming to the aortic branches runs towards the angle of the jaw, along the upper border of a small band of muscle (part of the petrohyoid). Push a glass tube down the oesophagus into the stomach, and turn the frog on its side. A third nerve, the vagus, will be seen on the lower edge of the slip of the petrohyoid muscle mentioned above, and close to the internal jugular vein ; a small branch, the laryngeal nerve, is given off by the vagus, and runs for a time parallel with it. Trace XIX] STfiUCTURE AND ACTION OF THE HEART. 161 the vagus to the skull. Pass a needle threaded with a silk thread under the vagus near the skull, tie the thread, cut the nerve close to the skull and isolate it for about a centimetre. Do not isolate near the heart; here it divides into delicate branches for the heart, lungs, and stomach, and these are easily injured. Fig. 13. p.h., petrohyoid muscle ; gl., glossopharyngeal nerve ; hy., hypoglossal nerve ; v., vagus ; I., laryngeal branch ; j., internal jugular vein ; hr., brachial nerve. Place a piece of thin india-rubber membrane upon the tissue under the vagus and stimulate the nerve with a tetanizing induced current (cp. Lesson xi. A, § 3, h) for about 15 seconds. There should be contraction in the larynx, but in no other structure. Watch the heart, and note a. The inhibition of the heart-beat. A second or two after the beginning of the stimulation the heart stops beating, all parts of it being flaccid (diastolic stand-still) ; and it remains so during the stimulation L. 11 162 ELEMENTARY PHYSIOLOGY. [XIX and for a short time afterwards. The inhibitory effect varies greatly with the condition of the frog ; a slowing and weakening of the heart-beats may follow instead of cessation ; and sometimes there is no effect. If no effect is obtained with a weak current, repeat, increasing the strength of the current or dissect out and 'stimulate the other vagus, the effect of one nerve is often more marked than that of the other. 3. Turn the ventricle forwards and stimulate the line of junction of the sinus and right auricle ; the heart will stop beating; there may be a preliminary period of quickened beats caused by the direct stimu- lation of the cardiac muscle. 4. Stannius' experiment. Pass a thread under- neath the aortse, draw it backwards, tilting the ventricle forwards, tie it firmly round the junction of the sinus with the auricle. The auricles and ventricle will cease to beat (there may be one or two beats), the sinus will continue as before. Leave for five minutes, then cut through the junction of the auricles and ventricle; the ventricle will soon or at once begin beating with a slow rhythm, the auricles remaining quiescent. 5. Graphic record of the cardiac inhibition. Hold the head of a pithed frog between finger and thumb. Cut away the skin on the back of the head. Pass a scalpel carefully along the posterior edge of the skull, so as to cut through the muscles attached to it. The vagus nerve, the jugular vein, and the petrohyoid muscle will come into view, and on a little further clearing of the muscles, the glossopharyngeal nerve also. Pass a threaded needle under the vagus; put XIX] STRUCTURE AND ACTION OF THE HEART. 163 the frog on its side, tie the nerve and cut it close to the skull. Now expose the heart; cut away the muscles over the vagus, cut off the fore-leg, pin out the frog so that fixed electrodes can be placed under the nerve without touching any other tissue. Obtain a tracing of the movements of the heart in one of three ways. a. Pass a threaded needle through the extreme tip of the ventricle. Knot the threads about an inch from the heart ; hook the thread by a double hook to a lever, 6 to 9 inches long, which has attached to its upper edge a spiral wire spring'. Arrange so that the writing point of the lever just touches the blackened paper of the drum. b. Tie the ligament of the ventricle, and cut it on the pericardium side of the ligature. Then arrange as in (a). By this method the ventricular movements are less fully recorded than in (a) but the ventricle is less injured. c. Leave the pericardium intact. Take a light needle, fixed at the point by wax to a small square of emery paper, and curved at the other. Pass the curved end through a light lever, and let the other end rest on the pericardium over the base of the ventricle. This method causes less injury to the heart than either (a) or (6) but demands a very light lever. ' It is perhaps better to use a lever which projects beyond the fulcrum on the side opposite the writing point, and to attach the thread to the projection ; on the latter should be placed a light weight as a counterbalauce, 11—3 164 ELEMENTARY PHYSIOLOGY. [XIX This method is good when the brain only has been destroyed and the frog CTirarized. If there has been little loss of blood only, the heart fills during inhibition. Note the form of the ventricular curve with the drum moving fairly rapidly (1 cm. a second). The rise increases at first rapidly, then more slowly to a maxi- mum ; the fall is at first slight, then rapid, and finally slow. Clamp the electrodes in a stand, and place the vagus on the points. The nerve should he touched from time to time with a brush wet with salt solution to prevent it drying. Stimulate the vagus, and note the tracing obtained. Stimulate again, keeping the current on ; after a variable time the heart will begin to beat notwith- standing the stimulus (escape of the heart). When the heart begins to beat after vagus stimu- lation it will for a time probably beat more strongly than before the stimulation. By repeated stimulation the beat will (usually) be considerably improved. 6. Measv/rement of latent period. Arrange a time-marker, marking seconds to write under the other levers ; the coils of the time-marker are arranged in circuit with a clock making and breaking contact every second, or with a metronome. a. Place a time-marker in the primary circuit. Arrange the points of the time-marker and of the heart lever in the same vertical line ; with the drum at rest make a vertical stroke with each writing point. Stimulate by closing the key in the primary circuit. 6. Place the time-marker in circuit with a Daniell's cell and a key. Open and close this key simultaneously with the key of the secondary coil and nerve circuit. For this it is best to use Morse keys. XIX] STRUCTURE AND ACTION OF THE HEART. 165 7. Muscarin and atropin. Expose the heart of a pithed frog, cutting through the pericardium. Add one drop of 1 p.c. muscarin, the heart will become slower and stop beating. (If necessary, add a second drop.) Add two drops of 1 p.c. atropin, after a time the heart will recommence beating. 8. Effect of nicotine and atropin. Take a tracing of vagus inhibition of a frog's heart by method § 5, c, but opening the pericardium. Let fall on it two drops of 1 p.c. nicotine. In a few minutes stimulate the vagus, no inhibition will be produced, but there may bo increase in rate and strength of the heart- beats. Detach the heart from the lever, and stimulate the junction of the sinus and auricle, the heart will stop. Let fall on it two drops of 1 p.c. atropin ; and in a few minutes stimulate again, inhibition of the heart will no longer be produced. 9. Efeot of single induction shochs upon the quiescent ventricle. Stop the beats of the heart by a Stannius' ligature. Connect the ventricle by a thread with a lever writing on a drum. Fix the electrodes so that one is touching one side and the other touching the other side of the ventricle at its middle third. Send a single weaK break induced shock into the ventricle, it contracts once. After an interval of a minute stimulate with strong induction shocks, the ventricle contracts once and to the same height as before {law of maximal response). Stimulate with a moderate induction shock every five seconds ; the succes- sive contractions increase in height for a certain time. (Stair- case effect.) Set the drum going at moderate speed, stimulate with a break induction shock, whilst the ventricle is contracting stimulate again, by making the primary circuit, no contraction is produced by the second stimulus {refractor^/ period). 10. Current of action of the heart. Stop the beats of a frog's heart by a Stannius' ligature. Connect the ventricle by a thread to a lever. Make a pair of non-polarisable electrodes (cp. Less. XIII. § 3) but use straight glass tubes about 5 cm. long, filling half the tube with clay and inserting a worsted thread to 166 ELEMENTARY PHYSIOLOGY. [XIX support the projecting clay, which should be moulded on the worsted to a fine point. Put the electrodes in suitable clips and apply one to the base of the ventricle and the other to a point near the apex. Connect the electrodes to a shunt and galva- nometer (cp. Less. xni. § 6). Examine the electrical condition of the quiescent heart by removing the short-circuit plug of the shunt. A deflection will be obtained in the direction which indicates that the apex is negative to the base. The amount of deflection will depend on the extent of injury caused by the thread tied to the apex. If it is small the negativity may be increased by touching the apex with a hot wire. When the spot of light is steady stimulate the ventricle by touching it near the base with a needle. With each beat thus caused a rapid and large movement of the light occurs in the direction indicating that the base becomes negative to the apex. This is the current of action. DEMONSTEATIONS. 1. Efifect of constant current upon the apex of the ventricle. 2. EfiFect of induction shocks upon the apex of the ventricle (ep. § 9). 3. Rhythmical contraction of ventricle apex under pressure. 4. Blocking of contraction waves in the heart of tortoise. 5. Action of the sympathetic on the heart of frog. 6. The beat of the mammalian heart, and inhibition by the vagus. 7. The stethoscope and the sounds of the heart. LESSON XX. BLOOD PRESSTJEE. A. Minor Abteeial Scheme. This consists of an india-rubber bag, or enema syringe, connected by a tube to a vessel of water and furnished with two valves, one on each side of the bag and opening in the same direction, so that when it is alternately compressed and released by hand water is drawn from the vessel and delivered into tubes beyond the bag. The tubes consist of a piece of glass tubing about 5 or 6 feet long and a piece of rubber tubing of similar length and bore and are connected to the syringe by means of a three-way tube. There are clamps upon the long india-rubber tube close to its junction with the three-way tube and upon the small piece of india-rubber which connects the three-way tube with the glass tube, so that the flow of water may be through either the glass or the india- rubber tube. A small piece of india-rubber tubing is also placed on the end of the glass tube, into which a tube finely drawn out can be inserted. 168 ELEMENTARY PHYSIOLOGY. [XX 1. Clamp the india-rubber tube at its proximal end close to the pump, and leave the glass tube open so that all the water flows through the latter. Work the pump with a uniform force at about 30 to 40 strokes a minute. To ensure regularity, the strokes had better be timed with a metronome. The water will flow from the open mouth of the glass tube in jerks, corresponding to the strokes of the pump. At each stroke as much will issue from the distal end as enters at the proximal end. 2. Introduce into the open mouth of the glass tube a fine nozzle, so as to offer considerable resistance to the outflow of fluid. Work the pump with the same force and frequency as before. The outflow will still be intermittent, though less fluid will issue from, and consequently less enter into, the tube at each stroke. 3. Clamp the proximal end of the glass tube and unclamp the elastic tube. Let the distal end of the latter be quite open. Work the pump as before. There being little resistance to the outflow, the elasti- city of the tube is not called into play, and consequently the flow will be, as in the case of the glass tube, intermittent. 4. Working the pump as before, insert the fine nozzle into the open mouth of the tube. Considerable resistance will now be offered to the outflow of fluid, the elasticity of the walls of the tube will be called into play, and the water will issue from the end of the tube in a continuous instead of an intermittent stream. If the tube be suflBciently long and sufficiently elastic in xx] BLOOD PRESSURE. 169 proportion to the force and frequency of the strokes, the flow will be uniform as well as continuous. B. Major Arterial Scheme. This consists of a rubber bag or metal syringe with valves, as in the minor scheme, to deliver water into a rubber tube P (unshaded in fig. 14) about 30 feet long of about "5 inch diameter. This tube is connected near its distal end by means of T pieces and three-way tubes to Fig. 14. two sets of smaller tubes, oo and y, and a tube of equal calibre, c. Each set of smaller tubes consists of a straight tube, c', c", and of two dilated tubes, made by introducing in the middle of the rubber tubing a dilated piece of glass tubing. The dilated glass pieces are 170 ELEMENTARY PHYSIOLOGY. [XX stuffed with sponge. The straight tubes can be clamped at c, c', c". All the tubes into which the main tube divides are united again into the single tube (shaded in fig.) of similar bore to P, and this leads back to the water supply. At A and V side tubes are inserted by which connection can be made to mercury manometers, and at a and v tubes carrying fine nozzles and clamps are introduced. The pump represents the heart ; the small tubes represent the resistance of the small arteries and capil- laries. The tubes on the proximal side of this resistance represent the arteries, those on the distal side the veins. 1. The Mercurial Manometer. The manometer A is connected with the arterial, V with the venous tubes. a. Open the clamps marked c, c' and c", so that as little resistance as possible intervenes between the arterial and venous tubes. Bring the manometers to mark on the revolving cylinder, placing V about an inch under A, in the same vertical line. Work the pump steadily, regulating the time with the metro- nome. In A, the mercury rises at each stroke, and in the interval between each two strokes falls again to its previous level. (The momentum of the mercury fre- quently carries it below this level, and the descent may be followed by one or more oscillations.) In V, a similar rise and fall is observed, of nearly if not quite the same extent. b. Close the clamps c, c' and c", so that the capillary resistance becomes very considerable. In A, the mercury XX] BLdOD PRESSURE. 171 rises rapidly at the first stroke, and at the end of the stroke begins to fall again, but more slowly than was the case in a. It has not fallen far before the second stroke raises it to a higher level than before. On falling still again, it is once more raised to a yet higher level, but the increase is not so great as before. Each succeeding stroke has a similar eifect. Thus at the end of a few strokes, the mean arterial pressure is reached, marked only by comparatively small oscillations corresponding to the strokes of the pump. On the strokes ceasing, the mercury gradually falls until the previous level is reached. In V the mercury rises to a much less extent than was the case in a, a slight mean pressure much less than in A is established, marked either with no oscilla- tions at all or such as are less conspicuous than those of A. Owing to the presence of the resistance, a mean pressure {arterial blood pressure) is established on the proadmal (arterial) side of the resistance. _ This pressure is marked by oscillations synchronous with the strokes of the pump. On the distal {venous) side the msan pressure is much less a/nd the oscillations are either slight or altogether absent. 2. Flow from Arteries and Veins. Remove the clamps from the fine nozzles a and v. Let the clamps c, c' and c" remain closed. Set the pump going. The flow from a on the proximal (arterial) side is in jets ; that from v (venous) side is uninterrupted or nearly so. 3. Sphygmograph. Bring the levers Sa (arterial side) and S.o (venous side) to write on the revolving drum, one under the other. 172 ELEMENTARY PHYSIOLOGY. [XX a. Open the clamps c, c' and c", and set the pump going. The two levers describe two nearly straight lines, a slight rise only being evident (and that to about the same extent in both) at each stroke. When there is little or no resistance in the capillaries, comparatively'little distension qfthe arterial walls is produced at each stroke of the pump. b. Close the clamps c, c' and c". The lever Sa now describes a well-marked curve with each stroke of the pump. Observe the sudden rise to a maximum, the com- mencing fall, the break in the fall, followed by a slight rise (dicrotic wave) and the final descent. The lever 8^ describes now a straight line. TJie rise in pressure at each stroke indicated by the mercurial manometer is accompanied hy a distension of the proximal (arterial) part of the tubing, indicated by the rise of the lever. This is the pulse. On the distal (venous) side of the resistance no pulse is 4. Progression of the Pulse-wave. Place two levers, one 8a, as near as possible to the pump, the other 8' a, as near as possible to the resistance. Bring the two levers to mark on the cylinder the one exactly beneath the other. (The pressure exerted by the two levers must be as nearly equal as possible.) Observe that each rise of 8a begins a little before, and is over a little before that of 8'a. In other words, the pulse of 8'a is a little later than that of Sa- Measure by means of a tuning-fork and time-marker XX] BLOOD PRESSURE. 173 (cp. Lesson xr. C. § 3) this interval of time, and the length of tubing between the two levers being known, calculate the rate of progression of the pulse-wave. 5. While the pump is working, the clamps being closed and the manometers A and V tracing their curves, gradually diminish the resistance by opening slowly first c' and then c". The arterial pressure curve will gradually fall, still marked by the pulse oscillations ; the venous curve will gradually rise. Diminution of capillary resistance lowers arterial, hut increases venous pressu/re. 6. Close the clamps c and c", and take tracings with the manometers, then gradually reduce the strength of the strokes of the pump. Both arterial and venous pressure will diminish. 7. The clamp c being closed, the main arterial trunk of the scheme divides into two chief branches, X and Y, each with its own resistance and venous tube. Leave the clamps c', c" closed, and put clamps on the tubing immediately beyond x and y. a. Work the pump with great regularity, and measure the quantity of fluid which escapes during a given time (say ten seconds) fi:om the venous tube of X, and from that of Y, by the side tubes x and y. b. The clamp c'' of X remaining closed, open c' of Y, and the pump working exactly as before, measure again the outflow during ten seconds. The outflow of Y will be increased. That of X on the other hand will be diminished, though the resistance in X is the same as before. 174 ELEMENTARY PHYSIOLOGY. [XX Tlie flow of blood through an artery is dependent not only on the resistance offered by its own small arteries and capillaries but also on that of other mrteries. DEMONSTRATIONS. 1. The effects in the rabbit on the temperature of the ear, and on the calibre of its blood vessels, following a. Stimulation of the central end of the great auricular nerve. 6. Section of the sympathetic nerve in the neck. c. Stimulation of the peripheral end of the sympa- thetic. 2. Normal kymographic tracings of the blood pressure of a mammal obtained by the use of a mer- curial manometer. 3. The effects on the arterial blood pressure, as indicated by the tracing, produced by a. Inhibition of the heart through stimulation of the peripheral end of the vagus. b. Dilatation of the small blood vessels through stimulation of the central end of the depressor nerve. 4. Methods of measuring the velocity of the blood current in large vessels. 5. Comparison of venous and arterial pressure. 6. Method of using the sphygmograph. 7. Method of using the cardiograph. LESSON XXL SALIVARY GLANDS. CERTAIN CARBO- HYDRATES. SALIVA. A. Salivary Glands. 1. Albuminous glands. Tease out a small fragment of the parotid gland of a recently killed mammal, e.g. of a rat, in a small drop of normal salt solution. Cover, press lightly, and examine under a high power. The outlines of the alveoli and cells are faintly seen ; the nuclei are not seen, except perhaps in some of the peripheral alveoli, altered by the salt solution; note the numerous granules. Fix the cover- slip (first sopping up excess of salt solution, if the cover-slip moves easily when touched), and observe the crowded granules in the fluid. 2. Section of albuminous gland. (Parotid, or sub- maxillary gland of rabbit. Chromic acid and alcohol mixture; picro-carmine.) Observe a. Under a low power. The alveoli, appearing as small roundish, or short tubular bodies, closely aggregated together to form the lobules ; each will be seen to consist of a group of cells 176 ELEMENTARY PHYSIOLOGY. [XXI surrounded by a small amount of connective tissue. The small ducts chiefly in the centre of the lobules, cut obliquely and transversely. b. Under a high power, The alveoli vary in size; externally the basement membrane shows as a rather sharp outline ; the lumina are small and may not be visible, unless kept open by secretion. In the round alveoli — tubes cut transversely — it will be seen that there is one layer of cells only between basement membrane and lumen. The cells are more or less polyhedral ; they have an irregularly granular appearance, but the cell-granules, seen in the fresh gland, have disappeared. The nuclei ai-e spherical (unless shrunken by the treatment), the nucleus of each cell is placed a little nearer the outer than the inner side of the cell. The ducts consist of a single row of slender columnar cells, the inner borders of which form a distinct ring bounding the obvious but small lumen; the outer boundary is not marked by a sharp line. The outer part of the cells is striated (it stains readily with picric acid). The nucleus is ovoid and situated at about the inner third of the cell. 3. Seoiion of lachrymal gland of a rabbit. (Mercuric chloride ; hsematoxylin.) Note that the terminal secreting tubes are obvi- ously tubular, they will probably show distinction between an outer, fairly homogeneous, stained zone, and an inner, irregular granular, unstained zone. (The zones can be readily seen in the fresh gland.) 4. Mucous glands. Tease out in 2 to 5 p.c. NaCl a piece of the orbital or sub-maxillary gland of a XXl] SALIVARY GLANDS. 177 recently killed dog'. Observe the distinct (mucous) granules at the edges of the specimen. Irrigate with HCl 1 p.c. ; the granules swell up and disappear ; here and there a pear-shaped mucous cell will be seen with a nucleus and small amount of protoplasmic cell substance at the narrow end, and a network of cell substance stretching through the swollen, mucous portion. 5. Section of dog's resting sub-maxillary gland (alcohol ; picrocarmine, or better, Ehrlich-Biondi fluid). Note under the low power that the general features are the same as those of albuminous glands § 2 (a). Note under a high power that two kinds of cells are present in the alveoli, the mucous cells and the demilune cells (the latter are not present in all mucous glands). The mucous cells are comparatively large, and have rounded outlines; most have a disc-shaped nucleus close to the basement membrane ; in a few the nucleus is spherical and farther from the basement membrane. Much the greater part of the cell is clear and homogeneous, consisting of mucin (or mucigen) resulting from the running together of the mucous granules (cp. § 4). The mucin is unstained in the carmine specimens". The protoplasmic cell substance is arranged much as in the cell swollen by acid (cp. § 4). ' It is best to take the orbital gland from the body a day after death ; isolated mucons cells more or less columnar and full of granules may then be obtained, and the nuclei and nucleoli may also be visible. " The mucous part of the mucous cells does not stain with hasmatoxylin, unless hardened in acid reagents ; it stains deeply with thionin. L. 12 178 ELEMENTARY PHYSIOLOGY. [XXI The demilune cells occur usually in half-moon shaped groups, at the ends of the alveoli; they are more or less overlapped by the swollen mucous cells. The outlines of the several cells are not very distinct. The nuclei are spherical, the cell substance, which stains equally throughout, resembles that of the cells of the albuminous glands. The ducts are in general features like those of the albuminous glands (cp. § 2). 6. Active mucous gland. Section of dog's sub- maxillary gland after prolonged secretion'. The section should be stained in the same way as that of § 5. Observe, comparing it with the resting gland of § 5 (it is best to mount the two sections under the same cover-slip), the mucous cells are smaller, owing to a diminution in the mucous part of the cell and have less rounded outlines; the mucin which remains still borders the lumen. The protoplasmic cell substance is more abundant. The nuclei are spherical, have conspicuous nucleoli, and are farther from the basement membrane. The alveoli are not all changed to the same extent ; in some the mucin has almost entirely disappeared, and the demilune cells are more polyhedral, so that the two kinds of cells are not very easy to distinguish; in others the only changes observable are that the nuclei of the mucous cells are spherical and the demilunes more conspicuous. 7. Tease in 5 p.o. neutral ammonium chromate a piece of a dog's Bub-maxillary gland which has been kept for 3 to 6 days in ' In a dog under morphia and chloroform, the chorda tympani (or this with the sympathetic) is stimnlatod at short intervals for three to six hours. XXl] SALIVARY GLANDS. 179 the fluid (or for 3 days in 2 p.c. chloral hydrate). Observe the isolated mucous and demilune cells. The general appearance of a mucous cell is like that produced by dilute HCl in § 4, but the basal end of the cell is seen to be prolonged into a process. 8. Sections of dog's orbital gland hardened in osmic acid vapour, given out in parafSn. (a) Stain some sections on a cover-slip with alcoholic methylene blue. Observe the deeply stained mucous granules stretching throughout the cells. The protoplasmic cell substance is stained greenish. (b) Place a section in xylol to dissolve the paraffin, transfer to absolute alcohol, mount in 95 p.c. alcohol. Observe the brownish mucin granules. Irrigate with 50 p.c. alcohol, watching the cells carefully. The granules will be seen to swell up till their outlines are lost, the cells sweU, their outlines become rounded; an intra- cellular network becomes .visible; thus the general appearance of the hardened gland of § 5 is obtained. Transfer to absolute alcohol and the original appearance wiU return. (If the tissue has been too short a time in osmic acid vapour the granules as they swell may stick together, in this case absolute alcohol brings back the original appearance imperfectly or not at all.) B. Reactions of certain Carbohydrates. 1. To a few c..c. of starch mucilage^ 1 p.c. add a drop or two of a moderately strong solution of iodine ; an indigo-blue colour will be produced ; if the colour is very dark fill up the test-tube with water. 1 To prepare the starch mucilage take 1 gram of starch and rub it into a thin paste with cold water. Pour it into a beaker containing one hundred o.c. of boiling water, boil for a few minutes and place it aside to cooL It should have no lumps in it and should be thin enough to be measured out readily with a pipette. 12—2 180 ELEMENTARY PHYSIOLOGY. [XXI 2. To a 2 p.c. aqueous solution of dextrin^ add a strong solution of iodine, drop by drop. A deep brown-red colour will be produced. Warm ; the brown- red colour will rapidly disappear, a light brownish-yellow tint due to the iodine remaining; on cooling, the dextrin colour returns. Now add water ; as the dextrin solution becomes more dilute, the red tint becomes less obvious, the fluid appears yellow-brown. That this colour is due to the dextrin can be seen by warming the fluid, and noting its change of tint. 3. To 5 c.c. of a ('1 p.c. solution) of dextrose (grape- sugar) add an excess of a solution of sodium hydrate (5 p.c.) and a couple of drops of a 1 p.c. solution of cupric sulphate; the precipitate of hydrated cupric oxide at first formed will dissolve' giving a blue solution. Boil ; the cupric oxide will be reduced and a yellow or red precipitate of cuprous oxide will be produced (Trommer's test). When a very small quantity of sugar is present no distinct precipitate is obtained, but the fluid is decolorized or turns faintly yellow. Sepeat this, adding half-a-dozen drops of a strong solution of cupric sulphate ; the reaction will be much less obvious, partly owing to the blue colour of the dissolved hy- drated cupric oxide and partly to the brown-black precipitate of anhydrous cupric oxide. 4. Add to a solution of dextrose some strong NaHO and boil ; the solution turns yellow, yellowish-brown, or nearly black according to the amount of sugar present (Moore's test). ' This may be bought or it may be prepared by boiling a little starch with sulphniic acid about 3 p.c, until a drop of the fluid gives a red-brown colour with a drop of iodine. XXl] CERTAIN CAKBOHYDEATES. 181 5. Add to a solution of dextrose some sulphindigotate of soda solution and some NajOOg and boil ; the blue colour turns green, reddish purple, red and yellow. When shaken with air the blue reappears (Mulder's test). 6. Add to a solution of dextrose an equal vol. of saturated aqueous solution of picric acid, and some caustic soda ; boil. The solution becomes very dark red. 7. Add to a solution of dextrose half its vol. of acetic acid and 2 or 3 drops of phenylhydrazin ; place the test-tube in a beaker of water and boil for ^-hour. On cooling, if not before, yellow crystals of phenyl glucosozone separate out. Examine the crystals microscopically. They are acicular and occur singly, in bundles and in star-like clusters. 8. Cane sugar. To a 1 p.c. sol. of cane sugar apply Trommer's test ; no reduction occurs. Add strong HCl to the cane sugar solution, 1 c.c. for each 10 c.c. of sol. taken, and boil gently for 10 min. The cane sugar is converted into laevulose and dextrose. Cool the solution, neutralize it, and apply Trommer's test. 9. Quantitative estimation of dextrose. Pre- liminary estimation. Put the solution of dextrose in a burette, let a few drops escape, so that the nozzle is filled and read the level of the solution. Into a small porcelain dish measure accurately 10 c.c. of Fehling's fluid (see App. p. 374), add about 10 c.c. of sodium hydrate and about 30 c.c. of water ; boil ; as soon as it boils run in the sugar solution from the burette in such amount that the fluid does not cease to boil, and stir continuously. When the fluid undergoes a distinct change of colour add the solution more slowly, and as soon as the fluid turns bright yellow or brick red remove the flame for a few moments and allow the 182 ELEMENTARY PHYSIOLOGY. [XXI precipitate partially to settle. Gently tilt the dish and observe if the fluid is still distinctly blue. If it is add a few more drops of the sugar solution and boil again. Proceed in this way until the fluid is no longer distinctly blue. Note the amount of sugar solution run in. Since 10 c.c. of Fehling is reduced by '05 gram of sugar, the percentage of sugar will be z , where b is the number of c.c. run in. There are certain sources of error in the method given above. (1) The cuprous oxide takes up oxygen from the air and a cupric salt is formed, giving a blue tinge to the solution. The re- oxidation of the precipitated oxide may be lessened by boiling in a small glass flask instead of in an open porcelain dish, but since the former is more easily cracked during boiling, and in it the colour of the fluid is less readily seen, it is not recommended for the beginner. In any case the estimation should be made as quickly as possible. (2) When the solution contains more than 1 p.c. or less than '5 p.c. of sugar, the method is inaccurate ; since 5 c.c. of a 1 p.c. solution of sugar, or 10 c.c. of a "5 p.c. solution contain '05 gram of sugar, i.e. will reduce 10 c.c. of Fehling's fluid, the solution, for an accurate estimation, must be diluted to such an extent that not more than 10 and not less than 5 c.c. of the diluted solution are required to reduce 10 c.c, of Fehling's fluid. The Fehling's fluid is diluted 5 times because with either a greater or a less dilution the results are inaccurate. Second estimation. If the solution is found to contain more than 1 p.c. of sugar, dilute it accurately with a certain number of volumes of water, to bring its percentage amount within the limits given above, and calculate how much of the diluted solution will probably be required to reduce 10 c.c. of Fehling. Even if no dilution is required make a second estimation, working quickly. Boil 10 c.c. of Fehling diluted a.s before and XXl] SALIVA. 183 run in all at once about 1 c.c. less than the amount of sugar solution which will be required. Boil, then remove the burner and observe the colour of the fluid, and finish the estimation by adding the solution a few drops at a time. C. Saliva. 1. Look at a little fresh saliva under the micro- scope (h. p.). A few swollen spherical leucocytes, their granules showing Brownian movement, and some flat epithelium cells from the mucous membrane of the mouth will probably be seen. 2. Test with neutral litmus paper the reaction of a drop of saliva, it will be found to be alkaline. 3. Induce the secretion of saliva by chewing a small piece of india-rubber tubing, or by rubbing the tongue with a crystal of tartaric acid. If time allows let the saliva stand until the turbi- dity has settled down into a sediment. To a few c.c. of the fluid add strong acetic acid ; mucin will separate out as a stringy mass, which does not dissolve in excess of acid (cp. Less. x. § 6, a). Shake gently, or stir it with a glass rod, the mucin will form a clump ; remove it, and if the fluid is cloudy, filter. 4. To the clear fluid add a drop or two of a strong solution of potassium ferrocyanide. The slight pre- cipitate which results indicates the total quantity of proteids present (cp. Lesson iii. e). If the reaction is not obvious, test another small portion with Millon's reagent. 184 ELEMENTARY PHYSIOLOGY. [XXl 6. In this and the following experiments the saliva^ used should be diluted 5 to 10 times. Mix equal quantities (say 5 c.c.) of starch and saliva in a test-tube and place in a water bath at about 37° 0. At short intervals (1 to 3 minutes) take a drop of the mixture and add it to a drop of iodine on a porcelain plate. The blue colour produced at first will later become a blue-violet, a red-violet, a red-brown, and a light-brown yellow, according to the relative amounts of starch and dextrin present, finally there will be no coloration, no more starch or dextrin (ery- throdextrin) being left. Then divide the fluid into two parts. a. Add iodine; no coloration is produced (there may be a little tint from dextrin, since in mixing the drops a faint colour may escape notice which in a larger quantity of fluid is obvious). b. Add an excess of sodium hydrate and a drop of 1 p.c. cupric sulphate and boil ; the fluid turns yellow and a yellow or red precipitate will be formed, showing the presence of sugar. 6. Boil a little saliva, add it to starch in a test-tube and warm. In half-an-hour divide into two parts and test as in § 5, a, b. The blue colour from starch will be as distinct as at first, no trace of sugar will be found ; 1 An aqueous extract of a ptyalin-eontaining gland may be used instead of saliva. To prepare the extract take [e.g.) the parotid glands of a rabbit and having removed the connective tissue around them chop them up 'well and place the pieces in about 200 c.c. of water ; leave in the warm for an hour or two and filter. The aqueous extract thus prepared contains much proteid material, and this obscures the reducing action of sugar on cupric hydrate in Trommer's test when a small quantity only of sugar is present. XXI] SALIVA. 185 hence boiling destroys the ferment (ptyalin) which converts starch into sugar. 7. If the saliva used in § 5 converts starch into sugar very rapidly, dilute it still further for the following experiment. Into each of three test-tuhes pour equal quantities of saliva and starch. Place ^ in a water bath at about 37° C.^ leave B at the temperature of the room (noting it), and place in a vessel with ice (it is best to cool the starch and saliva before mixing them). At short intervals take with a glass rod drops from each and add them to drops of iodine on a porcelain plate and so compare the rate of disappearance of starch (cp. § 5) in the three mixtures. It will disappear much more quickly in A than in B; in G there will be very little change. When no starch is left in A, remove G from the ice and place it in the warm chamber and test at intervals as before, the starch soon disappears. Hence a tem- perature of 0° C. arrests the action of saliva hut does not destroy it. 8. Neutralize a small quantity of saliva ; to 5 c.c. of this add 5 c.c. of HOI '2" p.c, the mixture thus contains "1 p.c. HCl. Place at 37° C. for ten minutes, add 3"5 c.c. Na^COs "4 p.c. and complete the neutraliza- tion with a more dilute solution. Add a few c.c. of starch and place at 37° C. In half-an-hour test for starch and sugar; starch will be found but no sugar, hence the acid has destroyed the ptyalin. ^ In these and in similar experiments 37° 0. is taken, since that is very nearly the normal body temperature of man, but a rather higher or a rather lower temperature will serve equally well. ^ Pure strong commercial hydrochloric acid contains about 33 p.c. HCl. 186 ELEMENTARY PHYSIOLOGY. [XXI 9. Place in one dialyser' {A) 15 c.c. of starch and in another (B) 10 c.c. of starch with a little saliva. Test from time to time the external water in each. That from (A) will give no trace of starch or sugar. That from (£) will contain sugar but no starch. Sugar dialyses, but Btarch does not. 10. Add a little raw starch (or arrow-root) to saliva and place in the warm chamber, shaking fre- quently. The raw starch is converted into sugar very slowly, it may be an hour or more before any sugar can be detected. 11. Apply a drop of saliva to filter-paper which has been dipped into a weak solution of ferric chloride acidified with hydrochloric acid and allowed to dry. A brownish-red stain on the paper will probably be produced — with human saUva — indicating the presence of a sulphocyanate. DEMONSTRATIONS. 1. Transverse section of the hilus of the sub- maxillary gland of a cat or dog. Note ; the branching duct with large lumen and one or two layers of cubical or flattened epithelium cells ; close to the duct groups of nerve-cells forming part of the submaxillary gan- glion; the gland artery, and probably a vein. 2. Section of dog's orbital gland to show the mucous granules (cp. § 8, a). 3. Section of dog's active submaxillary gland hardened in osmic acid vapour to show the inner granular and outer protoplasmic zones. 1 A very convenient dialyser may be made from a short length of parchment paper tubing (Papier-Darme) sold by Carl Brandegger, EUwangen, Wiirtemberg. LESSON XXII. STOMACH. GASTRIC JUICE. NUCLEO- PROTEIDS. MILK. A. (Esophagus and Stomach. 1. Transverse vertical section of the cardiac end of the stomach' (alcohol or chromic acid "2 p.c). Stain with hsematoxylin and eosin. a. Observe under a low power, Externally, the thin connective-tissue layer of the peritoneum. The muscular coat, consisting of an outer longi- tudinal and an inner circular coat of unstriped muscle, the former will appear as a cross section of a number of bundles with connective tissue running in between them from the peritoneum ; the latter as a continuous layer. On the inner side of the circular coat some small oblique muscular bundles may perhaps be present. The submucous coat of connective tissue. If the mucous membrane is in folds the submucous but not the muscular coat will be seen to run up in the folds. ' Stomach of rabbit, oat, or dog. The muscular layers are thinner in the rabbit than in the cat or dog. 188 ELEMENTARY PHYSIOLOGY. [XXII The muscularis mucoss, or thin stratum of unstriated muscle fibres a little external to the glands, this is divided more or less distinctly into an outer longitudinal and an inner circular layer. The mucous coat. Note in this the oxyntic gastric glands with their openings and the ridges between the openings. The bifurcation of some of the glands will probably be made out. b. Observe under a high power, The columnar mucous cells, liniag the mouths of the glands and covering the free surface of the mucous membrane between them; they are long, slender cells, becoming shorter in passing down the mouths of the glands; the upper third of the cell (containing mucin) is much more transparent than the remaining portion ; the nucleus lies at about the lower third. (The stomach must be hardened very soon after death, or these cells will be detached.) An oblique section of the mouths of the glands will cut through two or more of the columnar mucous cells on each side of the lumen, thus a number of small polygonal areas may be seen in the gland mouth, sometimes apparently blocking it up. The large deeply stained ovoid or oxyntic cells with ovoid nuclei, and the short columnar or polyhedral central or chief cells with spherical nuclei. At the base of the glands the central cells are usually most numerous, the ovoid cells being placed between them and the basement membrane ; towards the neck of the glands the ovoid cells increase in number ; in the neck, the majority of the cells are ovoid, and abut on the lumen. The ovoid cells usually cause a bulging out- XXll] STOMACH. 189 wards of the basement membrane, this is especially the case if the animal has been killed soon after it has fed. The connective tissue immediately internal to the muscularis mucosse; it surrounds the bases of the glands, and sends up processes between them. It runs between the glands, — generally with a few muscle-cells from the muscularis mucosse ; — and is seen as thin bands between the bodies of the glands ; the bands spread out near the surface. Leucocytes are present, but not in great numbers. 2. Section of the fundus region of the gastric mucous membrane, through the bodies of the glands parallel to the surface. Observe The central cells forming a tube with very small lumen. The comparatively rare ovoid cells on the outer side of the central cells. 3. Cut with scissors a thin strip of the fundus mucous membrane of a recently killed guinea-pig or rabbit. Mount it without fluid and gently press the cover-slip. Note in the deeper parts of the glands the central mass of granules (granules of the central cells), and the projecting ovoid cells without distinct granules. Irrigate with salt solution, the granules of the central cells in time disappear, and the ovoid cells become irregularly granular. (The central cell granules are not preserved by most hardening agents, cp. § 1.) 4. Vertical section of the pyloric end of the stomach. (Alcohol; hsematoxylin.) Compare these with the sections made of the cardiac end. Note The greater thickness of the muscular layers. 190 ELEMENTARY PHYSIOLOGY. [XXII The wider and longer mouths to the pyloric gastric glands, their more frequent branching, the absence of ovoid cells (if the section passes through the upper part of the pyloric region a few ovoid cells may be seen), the short columnar cells of the bases of the glands and the usually distinct lumina. 5. Examine fresh pyloric glands (cp. § 3). The cells are devoid of the distinct granules seen in the central cells of the fundus. 6. Section of cardiac region of stomach of frog (osmic acid). Observe that the surface cells are in general features like those of the mammalian stomach ; the necks of the glands have swollen mucous cells ; the bodies of the glands have one kind of cell only, in shape somewhat resembling the ovoid cells, but containing distinct granules. (In lower vertebrates the granules of the fresh gland are preserved by osmic acid.) 7. Transverse vertical section of the lower third of a rabbit's oesophagus (potassium bichromate 1 p.c). Stain with hajmatoxylin (or Ehrlich-Biondi fluid) and compare it with the corresponding sections of the stomach. Note the following points of contrast: The muscular coat contains striped as well as unstriped muscular fibres ; sections from the upper part of the oesophagus show no unstriped fibres. The submucous tissue contains small albuminous and mucous glands (cp. Lesson xxi.). Each of these consists of a duct, dividing and ending in dilatations, the alveoli. Traces of the muscularis mucosae internal to the alveoli of the glands. The papillae of the mucous membrane. XXIl] GASTRIC JUICE. 191 The epithelium formiug a layer several cells deep, the deeper being columnar or spheroidal, the superficial cells flattened (cp. Epidermis, Lesson xxxii). B. Gastric Juice. 1. Artificial Gastric Juice, a. Tear off the miicoxis mem- brane from the stomach of a mammal, cutting away the pyloric region (the stomach of a pig obtained from the butcher's will serve). Mince it finely. Put it in a flask with two hundred times its bulk of hydrochloric acid -2 p.c, and place the flask in a water bath at about 40° C. After some horn's a considerable part will be dissolved. Decant, and filter the decanted fluid. A solution of pepsin in hydrochloric acid will be obtained ; it will, however, contain a considerable quantity of peptone. b. Mince another gastric mucous membrane ; remove with blotting-paper the excess of fluid, add five times its bulk of glycerine and place aside, stirring occasionally. It is best to leave the mixture for some days before use, it may be kept almost indefinitely. When required for use filter through miisUn, add to the fluid ten to twenty times its volume of HCl "2 p.c. and filter. 2. Action of Gastric Juice. Use a peptic ex- tract prepared as above or take 5 grams of commercial pepsin to 1000 c.c. of HCl '2 p.c. and dissolve by rubbing up the pepsin with a few drops of the acid in a mortar, then add the rest of the acid and if necessary filter. a. Take four test-tubes. In A place 5 c.c. of hydrochloric acid "2 p.c. In 5 5 c.c. of the peptic extract. In C 3 c.c. of the same fluid, carefully neutra- lized with dilute NajCOj. In D 5 c.c. of the same fluid thoroughly boiled. Add the same quantity of 192 ELEMENTARY PHYSIOLOGY. [XXII fibrin' to each, and place in a water bath at about Zl'C Examine from time to time: A, the fibrin will swell up and become transparent, but will not be dissolved; on neutralization it will appear unaltered. B, the fibrin will be digested. G, the fibrin will be unaltered. D, the fibrin will be like that in A. These experiments show that acid alone (A) and pepsin alone (C) will not digest fibrin, and that pepsin loses its power on being heated to boiling point (D). Now add acid again to C, and place it in the warm chamber. Digestion will take place. The neutraliza- tion has only suspended, not destroyed, the action of the pepsin. b. Take two test-tubes, with 5 c.c. of peptic extract and a morsel of fibrin in each. Place A in the warm. Surround B with ice, or put it in a cold spot. The fibrin in A will be digested rapidly ; that in B very little or not at alL 3. Take 5 c.c. of peptic extract which has been found to digest fibrin rapidly, neutralize it, filter and add an equal bulk of NaaCOj 2 p.c, thus obtaining pepsin in the presence of a small quantity of an alkaline ' Baw fibrin digests more easily than that which has been boiled or kept in alcohol, it often however contains traces of pepsin so that a slow digestion may take place when acid only is added to it. When it is required to measure accurately the amount of fibrin added, raw fibrin finely chopped up should be placed in dUute HCl until it is well swollen, the excess of acid poured off and the fibrin measured in small tubes containing {e.g.) 2 c.c. XXIl] GASTRIC JUICE. 193 salt. Place at about 40° 0. for half-an-hour to an hour. Then add HCl until the mixture is distinctly acid (or neutralize and add an equal volume of HCl '4 p.c). Add a flock or two of fibrin and warm. Little or no digestion will take place. The pepsin has been de- stroyed by the alkaline salt. 4. Preparation of peptone. Place 50 c.c. of peptic extract together with some fibrin or other proteid in a beaker and leave in the warm until a small part only of the proteid remains undissolved. Filter and neutralize carefully, a precipitate of acid albumin (parapeptone) will be obtained (cp. p. 81). Filter off the acid albumin, the filtrate contains pep- tones. Since all samples of commercial pepsin contain peptone, the detection of peptone after a digestion experiment does not indicate that peptone has been formed. 5. Determine the following characters of pep- tones with the solution obtained in § 4 ; or with a 1 p.c. sol. of commercial peptone'. a. Apply the tests for proteids (cp. p. 20), Millon's and the xanthoproteic reaction are obtained, but no precipitate is produced with acetic acid and potassium ferrocyanide. b. Add excess of sodium hydrate and a drop of dilute cupric sulphate, a rose colour is produced (biuret reaction) ; a slight excess of cupric sulphate obscures 1 Commercial "peptone" consists largely of albnmoses. L. 13 194 ELEMENTARY PHYSIOLOGY. [XXII the rose colour, turning the tint to violet or mauve. (Cp. Lesson iii. § 4, c.) c. Boil ; it does not coagulate. d. Pour into one dialyser (A) a, solution of peptone and into another (B) diluted serum or white of egg, having only a small quantity of water outside the dialyser. Leave for an hour or longer, then apply the xanthoproteic test to the fluid outside the dialyser, a reaction will be obtained from {A) only, i.e. the peptone has dialysed, the albumin has not. 6. Preparation, of albtmwse (hemialbumose). Take about ten grams of fibrin, chop it finely, warm it in a beaker with three or four volumes of HCl '2 p.c. till it is swollen up. Pour off excess of fluid. Add to it about ^ of its volume of a strong peptic extract and place in the warm. In about 30 minutes the fibrin should be almost completely dissolved. Then add dilute sodium carbonate until the reaction is neutral or faintly alkaline. A bulky precipitate, chiefly of acid albumin, is formed. Filter. The filtrate contains hemialbumose and but little peptone. Apply to it the following tests *. a. Cool the test-tube containing it, then add a few drops of nitric acid, a precipitate is formed which disappears on warming and reappears on cooling. 6. Add a few drops of acetic acid and a drop of potassium ferrocyanide^ a precipitate is formed, which disappears on warming and reappears on cooling. e. Acidulate with acetic acid, and add NaOI to saturation, hemialbumose is precipitated. d. Saturate with ammonium sulphate, the albumose is precipitated. ^ The hemialbumose reactions may be obtained with a solution of Witte's peptone. XXll] NUCLEO-PROTEIDS. 195 e. Filter off the precipitate from c or d, dissolve it in a little water. Apply the biuret test, a rose colour is obtained as with peptone § 5, 5. f. Take the filtrate from d and apply to it the biuret test, adding excess of NaHO, i.e. twice its volume of 40 p.c. NaHO, a rose colour indicates peptone. 0. NUCLEO-PBOTEIDS. 1. Preparation, (a) Take lymphatic glands of the ox, free them as much as possible from fat, mince, grind up with sand and extract for 1 to 2 hrs., with 10 — 20 times the volume of water, shaking frequently, strain and centrifugaJise. To the fluid add acetic acid drop by drop imtU distinctly acid, a bulky precipitate of nuoleo-proteid is formed. Let it stand for a few minutes, best in the warm, then collect the precipitate by centri- fugalisation, dissolve it in 1 p.c. NajCOj. Purify by repreoipi- tating it with acetic acid. (6) Grind up minced lymphatic glands with an equal volume of solid NaCl. Throw the material into a large volume of water, nucleo-proteid rises to the surface in stringy masses, collect it. 2. Properties of nucleo-proteid of lymphatic glands, a. It dissolves in dilute alkalis and alkaline salts, forming a slightly opalescent solution. Make a solution in 1 p.c. NaaCOj. b. Boil the alkaline solution; no coagulation occurs. Boil nucleo-proteid in suspension in water or in slightly acid fluid, it coagulates and is then comparatively insoluble iu dilute alkali. c. Apply to a solution of nucleo-proteid the general proteid reaction ; all reactions are obtained. d. Add magnesium sulphate or ammonium sulphate to saturation ; the nucleo-proteid is precipitated. 13—2 196 ELEMENTARY PHYSIOLOGY. [XXII e. Add acetic acid drop by drop, nucleo-proteid is precipitated when distinctly acid and is not readily soluble in excess of the acid. /. Add precipitated nucleo-proteid to a pepsin solution in "2 HCl, and place in the warm for 3 — 4) hrs. An insoluble residue of nuclein remains. Filter or centrifugalise off the residue. Boil it in strong nitric acid and add a few drops of ammonium molybdate, a canary-yellow colour, indicating phosphorus, is ob- tained. D. Milk. 1. Examine a drop of fresh cow's milk under the microscope with a high power. It consists of a clear fluid containing a large number of highly refractive fat globules of varying size. 2. Test the reaction of fresh cow's milk with litmus paper. It will be found to be alkaline : occasionally it is acid, owing to the presence of free lactic acid. 3. Dilute a little milk five to ten times with water ; neutralise it cautiously with strong acetic acid, no precipitate will fall. Continue to add the acetic acid drop by drop, a copious precipitate of caseinogen will occur can-ying down with it nearly all the fat. When there is a distinct flocky precipitate no more acid should be added, as caseinogen is soluble though not very readily in excess. To precipitate the whole of the caseinogen the milk must be much diluted. XXIl] MILK. 197 4. Filter ofif the precipitate. The filtrate should be clear; if it is not, either too little or too much acetic acid has been added ; in this case add either a little more acetic acid or a little dilute sodium carbonate and filter again. Boil a portion of the filtrate; a precipitate of albumin (with a little globulin) takes place. Filter, and to the filtrate Apply Trommer's test (p. 180), a yellow or red precipitate will be obtained, showing the presence of a reducing sugar, lactose. 5. Carefully neutralise a portion of the filtrate fi-om § 3 with sodium carbonate. A precipitate forms which does not dissolve in excess of alkali. This is calcium phosphate. Apply the nitric acid and am- monium molybdate test. 6. Boil some of the caseinogen precipitate § 3 with strong NaHO, cool, and shake with a little ether. Allow a drop of the ethereal solution to evaporate on a slide or on blotting-paper. A greasy spot remains. 7. Action of Gastric Juice on Milk. Neutralize with dilute NaaCOs a little artificial gastric juice, filter and add 5 c.c. of the filtrate to 5 c.c. of fresh milk, place in the warm. Observe at short intervals the condition of the milk ; it will soon form a firm clot so that the test-tube can with safety be held upside down, later the clot shrinks and presses out a nearly clear fluid ; the clot continues to shrink for some time. The rennet-ferment in the extract has clotted the 198 ELEMENTARY PHYSIOLOGY. [XXII milk, converting the caseinogen into casein, and this has carried with it the greater number of the fat globules. If the amount of rennet-ferment contained in the extract is large the clotting may be almost instanta- neous; in this case the experiment should be repeated, taking a smaller quantity of the extract and without warming'. The extract is neutralised since (cp. § 3) excess of acid of itself precipitates caseinogen. 8. To the milk clotted by rennet-ferment add 5 c.c. HCl "4 p.c. and warm for an hour or so, the casein will be converted into peptone by the pepsin of the extract in the presence of acid. ' Some samples of commercial pepsin do not contain rennin. Commercial rennet may be ased. LESSON XXIII INTESTINE. BILE. A. Structure of Intestine. 1. Vertical sections of a cat's or dog's small intestine given out in paraflSn (chromic acid "2 p.c. ; stained in bulk with h^ematoxylin)^ The outer coats of the intestine have the same general characters as those of the stomach (Lesson xxii. A. § 1), except that there are no oblique muscular bundles. Observe in the mucous coat, a. The projections of the mucous membrane, or villi, either extended and long, or contracted and short, with the surface thrown into folds. Note in the villi, The epithelium, consisting of rather long columnar cells, each with a hyaline border more or less distinctly striated with vertical lines (the balsam is apt to render this indistinct), rather granular cell substance, and oval nucleus placed at about the lower third of the cell; the hyaline borders of the cells frequently appear to have coalesced into a narrow 1 Or Flemming's fluid, cut in paraffin, stained on a cover-slip with Ehrlich-Biondi fluid. 200 ELEMENTARY PHYSIOLOGY. [XXIII highly refractive hand, which inay be traced over the whole villus. The mucous or goblet cells, irregularly scattered among the former, sometimes abundant, sometimes scanty or absent; they have an upper ovoid portion which is transparent but has sharp outlines, and a lower basal granular portion containing the nucleus (cp. with the mucous cells Lesson xxi. § 4, and Lesson ix. § 1, 6). The connective tissue, forming the substance of the villus: this consists of a mesh work of fibres and membranous cells, for the most part hidden by the numerous leucocytes. The ' lacteal radicle ' may be visible in some of the villi as a central space bounded by a fine line formed by the epithelioid cells. Unstriped muscular fibres as narrow bands running up the villus from the muscularis mucosas. b. The rather deep depressions of the mucous membrane, the intestinal glands or glands of Xiieberkiihn. Note that The epithelium consists chiefly of cubical or short columnar cells; observe their gradation into the cells covering the villi, usually they have a small hyaline border similar to that of the columnar cells of the villi. Some goblet cells will be seen ; (the number and appearance of the mucous cells vary in dififerent animals). There is usually a distinct basement membrane immediately beneath the epithelium, formed of con- nective-tissue cells very much flattened; the outlines of the cells are not seen in the section, but the nuclei are fairly conspicuous. XXlll] INTESTINE. BILE. 201 The lumina of the glands are small but usually distinct. c. The adenoid tissue around the bases of the glands of Lieberkiihn and between them and the muscularis mucosae. This, unlike the corresponding tissue in the stomach (Lesson xxii. A. § 1, b), has a large number of leucocytes in its meshes. d. The lymph follicles; they are round or oval masses of adenoid tissue crowded with leucocytes, lying immediately beneath the surface epithelium and stretch- ing down into the submucous tissue. Villi are absent over them, and glands of Lieberkiihn only occur over their peripheral portions. (The lymph follicles do not occur in every section.) 2. Transverse sections of the villi of the small intestine of dog. (Flemming's fluid, stained in bulk in hsematoxylin.) Note, comparing with § 1, the cells with hyaline border and the goblet cells, the basement membrane, the connective tissue network with contained leucocytes, the capillaries a little below the basement membrane (these will not be obvious, if they are col- lapsed), the bundles of muscle-cells. In some sections the central Ijnmphatic space will be seen, bounded by a sharp line probably showing one or more nuclei of its constituent cells. 3. Snip oflf a few villi from a fresh intestine, and tease in salt solutipn. Note especially the hyaline border of the columnar cells and its striation. 4. Vertical transverse sections of the large in- testine. Observe The longitudinal and circular muscular coats. 202 ELEMENTARY PHYSIOLOGY. [XXIII The mucous membrane, probably thrown into longi- tudinal ridges, the submucous tissue running up into the ridges. The absence of villi. The intestinal glands (glands of Lieberktihn) ; they are broader than in the small intestine, and have more connective tissue (chiefly adenoid) between them. The epithelium covering the free intestinal surface or the ridges between the glands consists of long columnar cells, in the glands the cells are shorter. The cells have usually a thin hyaline border. In some animals {e.g. dog) there are many distinct mucous cells. 5. Examine sections of a small intestine in which the blood vessels have been injected, and note the capillary network round the glands of Lieberktihn, and the small artery running up each villus and dividing into a capillary network just below the epithelium. 6. Section of frog's intestine after feeding with fat (osmic acid). The columnar cells contain numerous fat globules of various size, the hyaline border is free from fat. 7. Feed a frog with a small piece of bacon ; on the next day'- kill the frog, remove the stomach and intestine, pin the tube out on cork, cut it open, and gently wash with salt solution. Note that the mucous membrane of the stomach has a yellowish semi-transparent look, whilst the mucous membrane 1 The difference in the tint of the stomach and intestine is still more obvious if the frog be fed again after two days and killed on the subsequent day. The frog is fed by placing the piece of fat in the upper part of the oesophagus, the fat is then usually swallowed at once. XXIIl] INTESTINE. BILE. 203 of the intestine is of an opaque white, this is more marked in the upper than in the lower part of the intestine ; the reotmn is greyish and semi-transparent. Tease out a small piece of the opsique white mucous membrane in normal salt solution; the epithelium cells are crowded with fat globules, scarcely anything but these being visible. Fat is absorbed by the cells of the small intestine, and is absorbed little or not at aU by the cells of the stomach. 8. Pin out pieces of the. intestine ; place some in 75 p.c. alcohol for an hour, and then in strong spirit ; place others in osmic acid 1 p.c. for half-an-hour, wash and place in 75 p.c. alcohol. In sections of these pieces note that there are no villi and no proper glands of LieberkUhn. The mucous membrane is however thrown up into considerable folds. In the osmic acid specimens, the cells wiU probably be so full of deeply stained fat globules that httle structure can be seen in them except the hyaline free border ; in the submucous connective tissue few or no fat globules are seen. In the alcohol specimens the cell substance wiU be seen as a distinct sponge-work or network, the fat globules having been dissolved. B, Bile. 1. Test the reaction of bile' with litmus paper. If fresh it is slightly alkaline or neutral. 2. To a small quantity add strong acetic acid drop by drop. A curdy precipitate of a mucoid body (mucin or nucleo-proteid or both), coloured with the bile- pigment will be thrown down (op. Less. x. 6 ; xxii. C). Since this body is not formed in the liver but in the glands and cells of the gall-bladder and duct, the longer ^ Ox-gall or sheep's gall may be obtained from a butcher's. 204 ELEMENTAKY PHYSIOLOGY. [XXIII the bile has been in the gall-bladder the greater the precipitate which will be obtained. For the following tests (§§ 4, 5) it is best, although not necessary, to precipitate the mucoid body with acetic acid, to filter and use the filtrate ; before filter- ing, the bile^