MEDICAL R.F. TOMLINSON / 7 fy/fa/wt**^ fisbcd by joiuing gpaaps of Resistance K> obtained by tibe use of Ike ing of cofls of German-sBvcr wire -. , . .... ._ £ --- ^ _. 1 — » - - * i- • - -— --. .,.• mcuiis QE DoctflU •HOBS SHBCKPOBB of the rheocoid, oonsistnig of a kng loop of the same LABORATORY MANUAL OF PHYSIOLOGY. may be short-circuited anywhere along its length by means of a sliding metal rider. 5. Induced Currents. — If a conductor through which a current is flowing is brought near to another and parallel conductor, an in- stantaneous current is in- duced in the latter and op- posite in direction to that of the former. When the first conductor is again removed from the vicinity of the second, a current is again induced in the second, but now in the same direction as that in the first. The same effect is pro- duced by making and break- ing the circuit in the first wire or by alternately bringing a conductor into and away from the vicinity of a magnet. The Inductorium, used in the physiological laboratory, is based on these principles (see Fig. 6). It consists of a primary coil of heavier wire of few windings (P), surrounding a movable core of iron wire, a secondary coil (S) of finer wire and many more wind- ings, not connected with the primary coil, and an automatic inter- rupter which may or may not be placed in circuit with the primary coil. II. PRACTICUH. Take two hollow spools, one considerably larger than the other, winding the small one with heavy insulated copper wire and the large one with fine insulated copper wire. The sma1! coil is the primary and is to be connected with the battery. The large coil is the secondary and is to be connected with the galvanometer. [16] FIG. 6.— Diagrammatic Drawing of In- ductorium. P, Primary coil ; S, second- ary coil ; C, core ; B, battery ; Af, magnet of interrupter ; A, armature of interrupter ; S#, spring of interrupter. MUSCLE-NERVE. 1. Place the primary inside of the secondary. By means of a key interposed in the primary circuit (a) make the current. Is there any movement of the galvanometer needle ? What is the di- rection and degree of this movement ? (b) Break the primary cir- cuit. What is the movement of the galvanometer needle? Does the needle remain in the position which it assumes upon either the make or break of the primary circuit? (c) Repeat (a) and (b), moving the primary farther and farther from the secondary. Is there any difference in the excursion of the galvanometer needle ? Is there any difference in the degree of excursion of the needle at make, as compared with break of the primary circuit ? 2. (a) With the key of the primary circuit closed, suddenly withdraw the primary coil from the secondary. What is the effect on the position of the galvanometer needle? (b) Now suddenly approach the primary toward the secondary and note the effect on the galvanometer. 3. (a) With the primary placed to one side, introduce a per- manent magnet rod into the secondary. What is the effect on the galvanometer? (b) Withdraw the magnet. What is the re- sult? 4. With a simple rheocord in the primary circuit, suddenly in- crease or decrease the resistance by quickly moving the slider back and forth. Is any current induced in the secondary through changing the intensity of the primary current ? 5. Apply the electrodes from the secondary coil of an inducto- rium to the tip of your tongue. Open and close the primary circuit with the primary some distance removed from the secondary. Re- peat, gradually moving the secondary toward the primary until the shocks produced are too strong for comfort. Which shock is first detected by the tongue? Why should the break shock be stronger than the make ? What is the possibility of the production of induced currents in the coils of the primary itself ? What would be their effect on the make as compared with the break currents ? These currents are known as make-extra and break-extra cur- rents. LABORATORY MANUAL OF PHYSIOLOGY. III. INTERRUPTERS. For interrupting the primary circuit, any one of a number of de- vices may be used. Where very rapid succession of shocks is de- sired, as in the production of the so-called tetanizing current, the Neef's hammer is used as shown in Fig. 6. Where known fre- FIG. 7.— Tuning-fork Interrupter. (According to Kronecker.) i, Tuning fork; 2, electro-magnet, alternately made and broken ; 3, battery ; 4, mercury contact ; 5, time marker. quency of interruption is necessary, a Bowditch clock or similar arrangement may be used for low frequencies and metronomes and electrically maintained tuning forks, for higher frequencies (see Fig- 7)- IV. DISSECTION OF THE FROG'S THIGH AND LEG. With a preserved frog or a fresh one that has had brain and cord destroyed, carefully dissect and identify the muscles of the thigh and leg (see Fig. 8). 1. Gastrocnemius-sciatic Preparation. — Pith a frog. Re- move the skin from one thigh. Make a circular incision through the skin at the knee and another at the lower end of the leg. Slide this up as far as the knee. This is to be slipped back, later, over the muscle to keep it from drying. Separate the gastrocnemius [18] MUSCLE-NERVE. muscle from the tibia. Cut the tibia through, just below the knee, being careful to avoid injury to the nerve where it enters the muscle on its upper and dorsal aspect. Tie a thread about the t.a. A. B, FIG. 8.— Muscles of Frog's Thigh and Leg. A, Dorso-lateral view, gl, Gluteus; c.e, coccygeus ; py, pyriformis ; v.e, vastus externus; r.a, rectus anterior; t.f, triceps femoris ; r.i, rectus internus ; s, semimembranosus ; b, biceps ; g, gastrocnemius ; p, peroneus; t.a, tibialis anticus. B, Ventro-lateral view, sar, Sartorius ; ad.l, adductor longus ; ad.b, adductor brevis ; ad.m, adductor magnus ; r.i.mi and r.i.ma, rectus internus minor and major (or gracilis); g, gastrocnemius ; t.a, tibialis anticus; t.p, tibialis posticus. tendo Achillis, just above the sesamoid cartilage. Cut the tendon below the ligature. On the dorsal side of the thigh, carefully separate the gluteus maximus muscle and biceps from the semimembranosus, using the fingers for the purpose. This exposes the sciatic nerve. Carefully separate it from the surrounding muscles, avoiding pulling and stretching as much as possible. It is well to handle the nerve with LABORATORY MANUAL OF PHYSIOLOGY. a glass hook. Cut the thigh muscles near their insertions about the knee, being careful not to cut the nerve. Cut the muscles, also, near the pelvic articulation of the femur. Remove all the abdom- inal viscera, thus exposing the nerve in the lumbo-sacral plexus. Cut through the vertebral column just above the last two verte- brae which join the urostyle. Remove the muscles on each side of the urostyle. Cut through the junction of the last ver- tebra with the urostyle, and, using the two vertebrae as a handle, lift the nerve from its bed from above downward, cutting its branches and carefully free- ing it from the groove over the femoro- pelvic articulation. Cut through the femur just below its articulation with the pelvis and the preparation is com- FIG. 9.- Gastrocnemius- sciatic lt ^ pj x The femur Preparation. be clamped in the muscle clamp and the thread about the tendo Achillis, to the myograph lever. Both nerve and muscle should be kept moist with physiological salt solution. 2. Double Semimembranosus-gracilis Preparation. — Dis- sect out the semimembranosus and gracilis muscles of both sides, using the same precautions as in the previous preparations. Both femurs should be disarticulated at the hips and the pelvis cut through transversely, thus leaving the two muscles joined by a thin piece of bone. This may be secured in the femur clamp. 3. The Sartorius. — This corresponds to the muscle of the same name in human anatomy. It is a long, thin muscle having its ori- gin from the symphysis pubis and its insertion into the capsule of the knee, fascia of the leg, and tibia. This muscle is to be used where parallel fibres are desired. V. ELASTICITY or MUSCLE. Make a gastrocnemius-muscle preparation, clamp the femur in the femur clamp, attach the tendon to the lever of the myograph, [20] MUSCLE-NERVE. and adjust the writing point against the smoked paper of a drum. The lever should be nearly tangent to the surface of the drum and the drum should revolve away from the point of the lever. Ar- range the drum so that it may be revolved by hand. 1. To Show the Elasticity of a Rubber Band. — Attach a rub- ber band to the femur clamp and myograph lever and adjust for writing on the smoked drum. Carefully place a lo-gram weight in the pan attached to the lever. Move the drum slightly to record the amount of stretching. Remove the weight, allow the lever to return through the elasticity of the band and revolve the drum again slightly. Repeat this with 20 grams, with 30 grams, with 40 grams, with 50 grams. Does the lever return each time to its orig- inal position ? 2. To Show the Elasticity of Muscle.— Repeat the above ex- periment, using the muscle already prepared. How does the elas- ticity of the muscle compare with that of the elastic band ? If the elasticity is not perfect for the amount of stretching force em- ployed, are there any factors of error in the apparatus and method that may, in part at least, account for the results ob- tained ? 3. To Show the Tensile Strength of Muscle.— With the same preparation used in the previous experiment, carefully add weights to the pan of the lever until something gives way. Which breaks first, the muscle or the tendon ? Prepare a fresh muscle and re- peat, inserting needle electrodes into the muscle and stimulating with a tetanizing current from an inductorium for each addition of weight. How much will the muscle lift ? 4. Hooke's Law. — This is embodied in the statement that the power of any spring is in direct proportion to the tension under which it is placed. Does the muscle in experiment (b) respond to Hooke's law where small weights are used ? Recent experiments by Professor Haycraft, with improved apparatus from which all sources of error have been eliminated, show that, within physio- logical limits, all the simple tissues of the body follow this law. [21] LABORATORY MANUAL OF PHYSIOLOGY. VI. IRRITABILITY OF NERVE AND MUSCLE TO VARIUS STIMULI. Nerve and muscle tissue have in common the properties of ir- ritability. The muscle has, in addition, the power of contractility. In the following experiments the stimulating agents will be ap- plied to the nerve and the contraction of the muscle will be used as an indicator of the efficacy of the stimulus. The stimulating agents may be grouped as mechanical, thermal, chemical, and electrical. 1. Mechanical Stimuli. — Make a sciatic-gastrocnemius prepa- ration. Place the nerve on a glass plate and keep both muscle and nerve moist with physiological salt solution, (a) Cut the nerve near its origin from the cord with sharp scissors. Does the muscle contract ? Tap the nerve with a scalpel handle. (b) Cover the nerve with moistened filter paper, place a thin sheet of cork over this; place a small beaker very carefully on the cork and slowly pour mercury through a glass tube of small calibre into the beaker. Does the muscle contract? Which is the more efficacious, a stimulating force gradually applied or one suddenly applied ? 2. Thermal Stimuli. — Heat a needle or a copper wire in the flame of a Bunsen burner to a red heat. Touch the nerve with it. Result? Will this piece of nerve respond again to stimulation? Explain. 3. Chemical Stimuli. — Make a fresh muscle-nerve preparation. Cut the nerve high up. Place on a glass plate, allowing the end of the nerve to hang over the edge. Use a watch glass or other small vessel for containing the reagents to be used. This is brought up to the nerve until its end dips into the contained reagent. That portion of the nerve used is usually destroyed by the chemical so that it is necessary to cut off the end of the nerve after each test. In this manner, try the effect of the following reagents. (a) Concentrated solution of sodium chloride. (b) Concentrated solution of sodium or magnesium sulphate. MUSCLE-NERVE. (c) Fiftv-per-cent alcohol. (d) Glacial acetic acid. (e) Five-per-cent sulphuric acid. (/) Ammonia. (g) Zinc chloride. (h) Allow the nerve to dry. 4. Electrical Stimuli. — For this purpose, induced currents from an inductorium will be used. The effect of the constant current will be taken up later. Make a muscle-nerve preparation, arranging for recording upon the smoked drum. Set up the inductorium for single shocks, in- terposing a short-circuiting key in the primary circuit for this pur- pose. Remove the secondary coil, as far as the instrument will allow, from the primary. In those instruments where the secondary is movable to form angles of varying degrees with the primary, the intensity of the secondary currents may be diminished by increas- ing the angle between the primary and secondary wires. Apply the electrodes from the secondary coil to the nerve or to the muscle directly. Close the primary circuit. Result ? Record on drum. Rotate the drum slightly and break the primary circuit. Result ? Move the secondary nearer to the primary and repeat the make and break as before, recording results. Repeat again and again, gradually moving the secondary toward the primary until the muscle ceases to increase in the height of its contraction. Which is the more efficacious, the make or the break shock from the inductorium, and why ? VII. PERIOD OF LATENT STIMULATION AND FORM OF THE SINGLE TWITCH. Arrange a drum to be rapidly spun by hand. With a little prac- tice, this method gives as good results as the pendulum or spring myograph. Arrange a muscle-nerve preparation with point of writing lever touching the smoked surface of the drum. Arrange the writing points of two signal magnets (a) and (ft), exactly under the writing point of the muscle lever. Place signal magnet (a) in LABORATORY MANUAL OF PHYSIOLOGY. circuit with the primary. Place signal magnet (b) in circuit with the tuning fork giving 100 interruptions per second. Introduce a short-circuiting key in each circuit. Place the nerve of the muscle preparation on the electrodes from the secondary coil of the in- ductorium. The three levers, the one attached to the muscle, that of signal magnet (a), which is in circuit with the current stimulating the nerve, and that of signal magnet (b), which is in circuit with the tuning fork, will then begin their tracings directly under each other, so that exact time comparisons may be made. Close the short-circuiting keys in both circuits. Start the drum to spinning rapidly. When the drum has reached the height of its speed, open both keys. Close the tuning fork key immediately. Stop the drum. There will be three tracings to interpret. The signal magnet (a) marks the exact instant that the nerve was stimulated. The muscle lever marks the period of contrac- tion of the muscle. The tuning-fork lever marks the time re- lations. Does the muscle twitch begin exactly at the moment of stimula- tion ? If not, how much time elapses between the application of the stimulus and the onset of contraction ? What is the form of the single-twitch curve ? How does the pe- riod of contraction compare with that of relaxation ? VIII. THE VELOCITY OF THE NERVE IMPULSE. With the same arrangement of apparatus as in the previous ex- periment, ascertain the period of latent stimulation when the stim- ulating electrodes are on the nerve near the muscle; when the elec- trodes are on the nerve some distance from the muscle. Measure the length of nerve between the two points of stimulation. Esti- mate the difference in time of latent stimulation. This difference will be equivalent to the time that it takes the nerve impulse to travel over the length of nerve measured. From this the velocity per second can be easily determined. MUSCLE-NERVE. IX. DIRECT IRRITABILITY OF MUSCLE; ACTION or CURARE. Inject into the dorsal lymph sac of a frog a few drops, about one- half cubic centimetre of a one-per-cent solution of curare. First, however, dissect out one sciatic nerve, passing a ligature under it and tying it tightly about the thigh. All of the frog except that por- tion below the ligature will come under the influence of the drug (see Fig. 10). In fifteen or twen- ty minutes the drug action should be complete. Make two sciatic- gastrocnemius preparations, one of the curarized side and one of the non-curarized side. Set up the inductorium for tetanizing currents. Attach muscles to myograph levers for recording. Stimulate nerve of curarized side. Result? Stimulate the nerve of the non-curarized side. Re- sult ? Stimulate the muscle of the curarized side directly. Result ? What is the action of curare, as deduced from these observations? Is the muscle fibre itself directly irritable, aside from its nerve ? In the experiment under electrical stimulation, it was demon- strated that, up to a certain point, the height of a single muscle twitch is in direct proportion to the strength of the stimulus; i.e., a minimal stimulus is accompanied by a minimal contraction and a maximal stimulus by a maximal contraction. This will be com- pared, later, with the action of heart muscle under similar circum- stances. X. INFLUENCE OF LOAD ON MUSCLE TWITCH. When the weight is continuously supported by the muscle, both when at rest and when contracting, the muscle is said to be loaded. FIG. 10. — Curare Experiment, i, Ligature around thigh ; j, sciatic nerve, not included in ligature. Shaded area, affected by curare; non-shaded area, unaffected by curare. LABORATORY MANUAL OF PHYSIOLOGY. When the weight is supported by the muscle, only during the period of contraction, the muscle is said to be after-loaded. Compare the muscle curves obtained with load and after-load, using first small weights and then heavier and heavier weights. XI. EFFECT OF TEMPERATURE UPON THE MUSCLE CURVE. For the study of the effects of changes of temperature upon the muscle curve, the muscle warmer of Porter is very convenient. Where this is not at hand, the same results may be obtained by the use of a bath of physiological salt solution, which may be cooled or heated to the desired temperature and in which the muscle may be immersed, except for the short period required for stimulating and recording contractions. 1. Place the musde in a small test tube surrounded by an ice pack until the temperature of the interior of the tube has fallen to zero or less, i.e., until the freezing point has been reached. Re- move the muscle and record a single twitch. Label the tracing. 2. Warm the muscle to 5° Centigrade and again record a twitch. 3. Warm the muscle to 10° C. and again record. 4. Warm to 15° C. and record again. 5. Warm to 20° C. and repeat record. 6. Warm to 30° C. and record again. 7. Warm to 40° C. and record again. 8. Bathe the muscle with salt solution heated to 45° C. and note result. The muscle passes into rigor. Compare the curves obtained at the different temperatures and tabulate your results. WTiat is the effect on the height of contrac- tion ? On the time of the contraction phase ? On the relaxation period? XII. INFLUENCE OF FATIGUE ON THE FORM OF THE SINGLE MUSCLE TWITCH. i. Set up the apparatus for automatic stimulation of the muscle or nerve as shown in Fig. n. Make a sciatic-gastrocnemius preparation. Arrange the inductorium for maximal stimulation. [26] MUSCLE-XERVE. Place the drum contact arrangement in circuit with the primary. Adjust the myograph lever to the smoked surface of the drum. Allow the drum to revolve at its greatest speed. According to the arrangement shown in Fig. n, six contractions will occur during Jfl FIG. ii.— Drum Arrangement for Muscle-nerve Stimulation, d, Drum, m, mus- cle ; », nerve ; e, electrodes ; £, battery ; », inductorium ; f, collar with contacts which, as drum revolves, make and break primary circuit with/", metal strip. every revolution of the drum. Ever}* sixth contraction will be re- corded at the same place on the drum. In this way a number of superimposed contractions are recorded. Allow the drum to revolve until the muscle ceases to respond by a contraction. What changes does the contraction curve undergo with the progression of fatigue? Compare the fatigue curve with the temperature curves ob- tained in the previous experiments. 2. Make a fresh muscle-nerve preparation. Let the drum re- LABORATORY MANUAL OF PHYSIOLOGY. volve slowly. Attach the muscle to the myograph lever. After- load with a lo-gram weight. Adjust lever to drum. Stimulate the nerve once a second with a submaximal induction shock. A fatigue record formed of single twitches, written one after the other, will thus be obtained. 3. Repeat the above fatigue experiment with a loaded muscle. XIII. VOLUME OF CONTRACTING MUSCLE. In answer to the question, does the volume of the muscle change during contraction, some such device as that shown in Fig. 12 may be used. The muscle should be put in a container filled with physiological salt solution, the ends of the muscle being attached to elec- trodes from the secondary coil of an inductorium arranged for tetanizing shocks. The mouth of the container is closed with a tightly fitting cork, perforated for the passage of a fine glass tube in which the water from the container rises. Stimulate the muscle with tetaniz- ing induction shocks and observe the level of the fluid in the capillary tube connected with the muscle container. Does the fluid rise or fall ? Does the volume of the muscle change during contraction ? XIV. SUMMATION OF STIMULI. FIG. 12.— To Determine Volume of Contracting Muscle. Af, Mus- cle ; e e, electrodes ; c, capillary Arrange inductorium with second- ary coil removed from primary until single break shocks are just insufficient to cause a muscle twitch. Let the muscle rest for several minutes. Now stimulate the nerve every four or five seconds. Does the muscle finally contract ? Explain. [2&] MUSCLE-NERVE. XV. SUMMATION OF CONTRACTIONS AND GENESIS OF TETANUS. Make a semimembranosus-gracilis muscle preparation. Clamp bony attachment in the muscle clamp. Attach the other end to the muscle lever. Connect muscle up for direct stimulation with the secondary coil of an inductorium, arranged for single shocks. In order that the stimuli may be of equal intensity, it is well to use B FIG. 13.— Cut-out Key. Bt Battery ; P, primary coil of inductorium ; S, secondary coil; a, &, and c, ct, metal strips ; i and 2, metal clip contacts to complete circuit be- tween a, b, and c, d ; 3, crank to revolve clip contacts i and 2. Contacts revolved in direction of arrow. Contact at a, b, is made an instant before that at t, d. The secondary circuit is therefore short-circuited before the primary is made. The secondary is opened before the primary is broken. Hence the make shock is cut out. Connect S with c, d, and P with a, t>, to cut out break shock. some form of cut-out key, so that either the make or break shock may be eliminated (see Fig. 13). 1. Arrange key to give only break stimuli. Stimulate muscle with one break shock. Note form of contraction curve and height of contraction. The drum should be revolving at medium high speed. 2. Allow the muscle to rest for a moment. Stimulate again, and, [29] LABORATORY MANUAL OF PHYSIOLOGY. before the relaxation of the muscle is complete, send in a second stimulus. Repeat, decreasing the interval between stimuli until the two contractions merge to form one. This is known as the sum- mation of contractions. How does the height of the two summed- up contractions compare with that of the single twitch? Where the second contraction begins during the relaxation phase of the first, what determines the height of the second ? 3. Place the primary of the inductorium in circuit with a metro- nome or vibrating rod interrupter. Stimulate the muscle four times per second; five times per second; six times per second; eight times per second; ten times per second; twelve times per second; fifteen times per second ; twenty times per second. Allow the muscle sufficient rest between the series of stimuli in order to avoid fatigue. How do the contraction curves change as the frequency of stim- ulation is increased ? How many stimuli per second are needed to cause the individual curves to merge into one apparently contin- uous curve ? The latter condition is called tetanus. Where the in- dividual twitches are still visible, the condition is one of incomplete tetanus. 4. That this condition of complete contraction is apparent rather than real may be shown by stimulating the nerve of a muscle and connecting the muscle itself with a capillary electrometer which will show a series of action currents corresponding to a series of single twitches. The capillary electrometer and the action cur- rent of muscle will be described later. 5. Influence of Temperature on the Production of Tetanus. — Repeat the previous experiments with a muscle cooled to 5° C.; with a muscle warmed to 35° C. How does the frequency of stim- ulation necessary to produce tetanus in the cold muscle and in the warm muscle compare with that required for the muscle at room temperature ? 6. Influence of Fatigue on the Production of Tetanus.— After the muscle has become tired from repeated stimulation, re- peat the foregoing experiments. Result ? Explain. [30] MTSCLE-KERVE. XVI. To DETERMINE ACTUAL SHORTENING OF A MUSCLE LN CONTRACTION. Divide distance of the writing point of the muscle lever from the axis of the lever by the distance of the muscle attachment from the axis. Then divide the height of the recorded curve by thi> factor. The result is the actual shortening of the muscle during con- traction. XVII . To DETERMINE THE WORK DONE BY THE MUSCLE dur- ing any particular contraction, multiply the actual shortening by the load. Thus, if the actual shortening or height of contraction is 5 millimetres and the load is 10 grams, then the work done would be 50 gram-millimetres, 1. On a drum revolved by hand, record the heights of contrac- tion of a gastrocnemius which is receiving submaximal stimuli. After-load the muscle successively with 10, 20, 30. 40, 50, 70, 100, 150, 200, 250, 300. 350, 400, 450, and 500 grams. 2. Estimate the actual work done according to the formulae given above. Plot a curve, marking, on the abscissa, intervals to represent 50-gram weights; on the ordinates, intervals to repre- sent gram-millimetres. What conclusions can you draw from the data thus plotted ? XVIII. FATIGUE OF HUMAN VOLUNTARY MUSCLE. Ergography. — The contraction of voluntary muscle is normally brought about through nerve impulses originating in nerve cells. The nerve cell, as will be shown later, has a certain rhythmic ac- tivity, sending out from 6 to 10 impulses per second. This seems to be sufficient to keep the muscle in a state of tetanus. The short- est voluntary muscle contraction, then, brought about through the discharge of nerve impulses from nerve cells, is a tetanus. The single twitch occurs only under abnormal circumstances, or through artificial stimulation of the nerve or muscle directly. Any stimu- lation of nerve cells, sufficient to cause a discharge of nerve im- pulses, will produce a tetanus in the muscle receiving the impulses. LABORATORY MANUAL OF PHYSIOLOGY For recording the contractions of human voluntary muscle, either the ergograph of Mosso or that of Porter may be used. With the former, the flexion of the middle finger is recorded ; with the latter the contractions of the abductor indicis. 1 . If the Mosso instrument is used, place the forearm and fingers in the securing attachments of the apparatus and weight the mid- dle finger with one or two kilograms. Contract the muscles, vol- untarily, once every two seconds, keeping time to the beat of a metronome, until you are no longer able to bring about a contrac- tion in this way. The contractions should be recorded upon a slowly revolving drum. Now stimulate the flexor muscles directly with electrodes placed over the forearm, using the same frequency of stimulation as before, one every two seconds. Does the muscle respond to direct stimulation after fatigue has been induced to vo- litional impulses ? 2. Repeat with a new subject, reversing the procedure. In other words, stimulate the muscle artificially until it no longer re- sponds and then attempt to flex the finger voluntarily until com- plete fatigue is obtained. 3. With a fresh subject, induce voluntary fatigue and record the time. Allow the muscles to rest for five minutes and repeat voli- tional contractions until fatigue has again occurred. How does the time of fatigue onset, after the rest, compare with that of the first series of contractions ? 4. Now, instead of mere rest, give the forearm five minutes' mas- sage and repeat the ergograph experiment. Is the onset of fatigue delayed as compared with the first series of contractions, or with the second, or with both ? What is the effect of massage ? XIX. INFLUENCE OF TENSION ON THE MUSCLE CONTRAC- TION. ISOMETRIC CONTRACTION. In the preceding experiments, the resistance offered to the muscle during its contraction, as measured by the weight lifted, has been nearly uniform, of course excepting the inertia of the weight at the beginning of the lift. A muscle twitch under these circumstances [33] MUSCLE-NERVE. is called isotonic. When the shortening of the muscle is prevented by a constantly increasing resistance, so that all its power is used in overcoming the resistance, the contraction is called isometric. The resistance is usually obtained by the use of a spring to which the muscle is attached, the energy of the muscle being stored in the spring in the form of tension, to be liberated as heat as soon as the muscle relaxes. 1. Graduation of the Isometric Spring. — In order to estimate the isometric value of a muscle contracting against resistance of- fered by a spring, it is necessary to interpret the spring's resistance in terms of weight. Reverse the spring of the heavy myograph (Porter's), attaching its hook to the scale pan beneath. Bring the writing point of the lever against the smoked paper of a drum ar- ranged to be moved by hand. Revolve the drum a half turn to re- cord a base line. Place a loo-gram weight in the scale pan. The spring will be bent to a certain extent and the lever will mark a descending line on the drum. Move the drum, slightly, to record the lower limit of the spring's bend. Repeat with a 2oo-gram weight, and so on up to 800 grams. 2. Make a gastrocnemius-sciatic preparation. Attach the tendo Achillis to the isometric spring. Adjust the writing point of the lever against the smoked paper of a rapidly revolving drum. Stim- ulate the nerve with a maximal break shock from an inductorium. An isometric curve will thus be obtained. 3. Release the muscle from the spring and attach it to the ordi- nary writing lever weighted with 20 grams. The lever, in this case, should be as long as that used for recording the isometric curve. With the drum revolving at the same rate as before, stimulate the nerve so as to record a twitch, as nearly as possible, under the re- corded isometric curve. 4. Compare the two curves (i) and (2), as to form and as to work done. To find the amount of tension overcome, as indicated by weight, compare the height of the isometric curve with the de- pression of the spring in (i). What influence does tension have on muscle work? How does 3 [33] LABORATORY MANUAL OF PHYSIOLOGY. this compare with the muscle under isotonic conditions? Is any muscle in the body, normally, under isometric conditions to any extent ? XX. ELECTRIC PHENOMENA OF MUSCLE AND NERVE. 1. Galvani's Experiment with Metals. — Pith a frog. Evis- cerate and remove everything above the urostyle and the last two vertebrae. Remove the skin from both legs. Pass a hook, made of clean copper wire, under both lumbar plexuses. Suspend the preparation by the copper wire from a clean iron or steel rod. Swing the preparation until some part of it comes in contact with the rod. Result ? Explain. 2. Contraction without Metals. — Make a muscle-nerve prepa- ration, cutting the nerve high up. Handle the nerve with a glass hook, allowing the nerve to fall across the muscle. There should be a twitch of the muscle every time the nerve comes in contact with it. This twitch may be due to one of two things. If the muscle is uninjured, and the injured portion of the nerve falls across it, the twitch may be due to the completion of a circuit between the injured and uninjured portion of the nerve itself, which are of different electrical potentials. Or it may be due to the completion of a FIG. 14.— Secondary Contraction. slt Sciatic of first preparation ; J2, sciatic of second preparation; w,, muscle of first preparation ; »za, muscle of second prep- aration ; e, electrodes. circuit between injured and un- injured muscle through the nerve. This is known as the current of injury or demarcation current of muscle or nerve. 3. Secondary Contraction. — Make two muscle-nerve prepara- tions. Allow the nerve of preparation 2 (see Fig. 14) to rest on [34] MUSCLE-NERVE. the muscle of preparation i. Stimulate the nerve of preparatu n . with a tetanizing current from an inductorium. Both muscles will be thrown into tetanus. The nerve of the second preparation is stimulated by the action currents of the first preparation. The action current is due to a change of potential in an inactive, as compared with an active muscle fibre. The same change may be demonstrated in a nerve over which an impulse is passing. This may be well shown by means of some form of delicate current detector, such as the galvanometer or capillary electro- meter. 4. The Capillary Electrometer. — This instrument, as commonly em- ployed, consists of a capillary tube containing mercury and dipping into a vessel containing sulphuric acid. The surface tension of the mercury is so great that it does not flow through the fine capillary tube, and its upper and lower meniscus is convex instead of concave as is the case with water. The sulphuric acid is connected with a platinum wire. The mercury in the capillary is also supplied with a plati- num wire for connection with any source of current (see Fig. 15). The upper end of the capillary tube is connected, through a T tube, with a mercury manometer and with a pressure bottle or syringe bulb. By raising the pressure bottle or pressing on the bulb, pressure is exerted upon the mercury in the capillary tube. This pressure is measured by the manometer. By pressing the mercury in the tube downward and then releasing the pressure, some sulphuric acid is drawn up into the tube, into con- FIG. 15.— Capillary Electrome- ter. (Lippmann's.) e e, wires leading to source of current ; Hg, mercury in capillary tube; Hy SO4, sulphuric acid. LABORATORY MANUAL OF PHYSIOLOGY. tact with the mercury. If a current be passed through the mercury and sulphuric acid, the surface tensions of the fluids are so changed that the mercury meniscus will move in the direction of the current. The extent of the excursion of the mercury is in direct proportion to the strength of the current. This may be measured by mercury pressure as determined by the manometer. A fairly exact gradu- ation of the instrument may be made by placing it in circuit with currents of known strengths, and recording in terms of mercury the amount of pressure needed to bring the meniscus back to its original position. This instrument is exceedingly sensitive to very small currents. 5. Current of Injury of Muscle. — Very carefully dissect out the gracilis and semimembranosus muscles, avoiding crushing or tearing as much as possible. Place the muscle on two non- polarizable electrodes, connected by fine wire with the galva- nometer or the capillary electrometer. Test the non-polarizable electrodes first, by bringing them in contact with each other. There should be no deflection of the galvanometer needle or of the mercury meniscus of the capillary electrometer. (a) Interpose a key between the muscle and the current de- tector. Close the key, so that the galvanometer is brought in cir- cuit with the electrodes on the muscle. There should be but little if any deflection of the needle. If the muscle were absolutely free from injury and at rest, there should be no difference of potential between any of its parts. (b) Cut across one end of the muscle with sharp scissors or scal- pel. Place one electrode on the cut surface, the other on the smooth surface. Bring the galvanometer in circuit again. There is now a deflection of the needle. This is an indication of the current of injury or the demarcation current of the muscle. (c) Cut off the nerve near the muscle and repeat (&), using the nerve instead of the muscle. 6. Action Current of Muscle. — Make a careful sciatic-gastroc- nemius preparation. Place the muscle on the non-polarizable elec- trodes, connected with the galvanometer. Place the nerve on elec- [36] MUSCLE-NERVE. trodes from the secondary of an inductorium arranged for single make-and-break stimuli. Close the galvanometer key. There will, probably, be more or less deflection of the needle due to the demar- cation current of the muscle which was injured in preparation. With the galvanometer key closed, stimulate the nerve with a single make or break shock. The muscle will respond with a single twitch. Is there any movement of the galvanometer needle ? If so, how much and in what direction ? 7. Action Current of Frog's Heart. — Pith a frog. Remove the heart, being careful to include the sinus venosus. The heart will, probably, continue its pulsation after its removal from the body of the frog. Place the heart on non- polarizable electrodes, connected with the capillary electrometer. With a low power of the microscope, watch for movements of the menis- cus of the mercury in the capillary tube. How many movements can you make out ? How do they corre- spond with the beating of the heart ? 8. Paradoxical Contraction. — Pith a frog. Make a sciatic-gas- trocnemius preparation, tracing out and cutting the anterior tibial branch of the nerve. Stimulate the cut branch (see Fig. 16). The muscle will contract. 9. Muscle Tone of Rabbit's Gastrocnemius. Demonstra- tion.—Narcotize a fair-sized rabbit with a hypodermic injection of one grain of morphine sulphate. Complete anaesthesia with ether. Tie rabbit, belly down, on the rabbit board, with the hind limbs well stretched out. Make a longitudinal incision through the skin and separate the dorso-lateral thigh muscles. The large shiny white sciatic nerve will be exposed, deep in the wound. Tie a [37] FIG. 16. — Paradoxical Contrac- tion. ' J, Sciatic nerve ; /, branch to peroneus muscle ; e, electrodes ; £•, gastrocnemius muscle. LABORATORY MANUAL OF PHYSIOLOGY. ligature about the nerve as high up as possible. Cut the nerve above the ligature. Place the cut nerve on shielded electrodes, connected with the secondary coil of an inductorium. Place the primary of the inductorium in circuit with a strong constant cur- rent interrupted by the tuning-fork interrupter, vibrating one hundred times per second. Place a short-circuiting key in the secondary circuit. Place the small bell of a stethoscope over the muscle. Open the short-circuiting key. The muscle will be thrown into tetanus and the sound of the vibrating tuning-fork will be heard with the stethoscope. That this reproduction of the tuning-fork tone is really due to the vibration of the muscle fibres to each individual stimulus from the inductorium is shown by the next experiment. 10. Action Currents. Detection of, with the Telephone.— With the same preparation as in the previous experiment, insert needle electrodes into the body and tendinous portion of the gas- trocnemius muscle. Connect these with a telephone receiver and again stimulate the sciatic with one hundred shocks per second. You will now hear the sound of the tuning-fork reproduced in the telephone. This is due to the development of action currents in the muscle corresponding, in frequency, to the number of im- pulses coming to the muscle. This sound is known as the artificial muscle tone, to distinguish it from the muscle sound which occurs when the muscle is con- tracted under the influence of volition and which is called the nat- ural muscle tone. This may be heard by placing the stethoscope on the biceps muscle and strongly flexing the forearm on the arm. XXI. IRRITABILITY AND CONDUCTIVITY OF NERVE AND MUSCLE DURING AND AFTER THE PASSAGE OF A CONSTANT CUR- RENT. ELECTROTONUS. During the passage of a constant current through a nerve, the irritability and conductivity are increased at the kathode, where the current leaves the nerve, and diminished at the anode, where [38] MUSCLE-NERVE. the current enters the nerve. Immediately after the cessation of the current, these conditions are reversed ; the irritability and con- ductivity are increased at the anode and decreased at the kathode. \ FIG. ^.-Arrangement for Studying Effect of Constant Current on Irritability of Nerve. Current running in direction of arrow is a descending current. Inductoria (i and 2) connected with battery through current changer (3) in such a way that cur- rent may be passed through primaries of either i or 2, so as to stimulate nerve (4) m region of anelectrotonus, about anode (6), or in regions of kathelectrotonus, about kathode (8). Nerve impulse indicated by twitch of muscle (5). Through Pohl's commutator (7), with crosspieces in, the constant current maybe reversed and be- come an ascending current, instead of a descending as shown in the figure. The dotted line, running below the nerve at the anode and above at the kathode, repre- sents, respectively, the diminution and increase in irritability of nerve in the anodic and kathodic regions. [39] LABORATORY MANUAL OF PHYSIOLOGY This condition of change in a nerve or a muscle, since the muscle itself reacts in the same manner as the nerve, is known as electro- tonus. The condition of the nerve about the anode is called anelec- trotonus; that about the kathode is called katelectrotonus. The conditions of anelectrotonus and katelectrotonus are most marked in the immediate vicinity of the anode and kathode. From these poles they gradually diminish in the extrapolar and interpolar regions, until, in the latter, a neutral point is reached about midway between the two poles (see Fig. 17). When the kathode is near the muscle and the anode farther from the muscle, the current is said to be descending. When these con- ditions are reversed, that is, when the anode is near the muscle, the current is said to be ascending. In the following experiments the electrotonic conditions will be tested with the nerve, the muscle twitch being used as a convenient indicator. i. Make a sciatic-gastrocnemius preparation. Save the whole length of the nerve. Arrange moist chamber with non-polarizable electrodes placed in circuit with one or more battery cells, rheo- cord, and Pohl's commutator or current-changer. Place the nerve upon the non-polarizable electrodes as shown in Fig. 17. Place a pair of platinum electrodes from the secondary coil of an induc- torium on the nerve at the anode of the constant current, and an- other at the kathode of the constant current. Arrange as in Fig. 17, so that the nerve may be stimulated with the induced current at either pole of the constant current. By means of the Pohl's commutator, the constant current may be reversed in direc- tion; i.e., it may be made either an ascending or a descending current. Attach the tendon of the muscle to the writing lever of a myo- graph. Let this record the contractions of the muscle on a drum arranged to be revolved by hand. Send an ascending current through the nerve. While the constant curfent is passing, stimu- late the nerve in the anodic region with a medium strong single- break shock from the inductorium. Mark result of muscular con- traction, if any, on drum, as well as data needed to identify what [40] MUSCLE-NERVE. you have done. Revolve drum a sufficient distance and repeat, stimulating, this time, in the kathodic region. Record data as before. 2. Now, reverse the constant current so that the anode is away from the muscle and the kathode is near the muscle. Repeat the stimulation with the induced current in the anodic and kathodic regions, as before. Record the muscle response, or the lack of it, on the drum and make careful note of all the data. From these experiments, what conclusions can you draw con- cerning the effect of the passage of a constant current upon the irritability of a nerve ? 3. Repeat the above experiments with ascending and descend- ing currents, stimulating in the anodic and kathodic regions im- mediately after the cessation of the constant current. What is the after-effect of the passage of the constant current upon the irrita- bility of the nerve ? XXII. THE CONSTANT CURRENT AS A STIMULUS. PFLU GER'S LAWS. As we have already seen, a sudden increase in intensity of a stim- ulus which is being applied to a nerve or a muscle is effective in producing an impulse in the same. A gradual increase, on the other hand, is not effective. In the same way a sudden increase in irritability will, of itself, act as a stimulus. Thus, when a constant current of sufficient strength is passed through a nerve, there is a sudden increase in irritability in the region of the kathode and a sudden decrease in irritability at the anode. There will then be a stimulation of the nerve in the kathodic region which will cause an impulse to be transmitted toward the muscle. If the current is an ascending one, however, and the conductivity is sufficiently diminished at the anode, the impulse will be blocked and the muscle will not respond with a contraction. When the constant current ceases to flow, the irritability sud- denly falls at the kathode and rises at the anode. The conductivity [41] LABORATORY MANUAL OF PHYSIOLOGY. changes likewise, but not necessarily to the same degree. If the current is ascending and the increase in irritability is sufficient, the muscle will respond with a contraction. If the current is descend- ing, and the fall in conductivity at the kathode is sufficient, this will act as a block between the anode and the muscle and no con- traction will be obtained. The relative increase and decrease of irritability and conductivity at the anode and kathode vary with the strength of the contsant current employed. Other things being equal, the make of the constant current is more efficacious as a stimulus than the break. These facts have been formulated as Pfluger's laws. Briefly, they are as follows: Strength. Direction of Current. Ascending. Descending. Weak Make. No Yes Yes No Break. No No Yes Yes Make. Yes Yes Yes Yes Break. No No Yes No Medium Medium strong Strong The words "yes" and "no" in the above table indicate the oc- currence or absence of a muscle contraction under the circum- stances noted. The strength of the current is regulated by increas- ing or decreasing the resistance by means of a rheocord or resist- ance box. With a fresh muscle-nerve preparation, make and break a con- stant current through the nerve as indicated by the above table. In the light of the explanations which have been given and the previous experiments performed, how are these results to be ex- plained ? XXIII. STIMULATION OF HUMAN NERVES. In human nerves, in the body during life, it is obviously imprac- ticable to bring the electrodes into direct contact with the nerve. There is more or less insulation from the intervening integument [4*] MUSCLE-NERVE. and fat. In this case the electrode, anode or kathode, is brought into as close relation to the nerve as possible. Only a small por- tion of the current will traverse the nerve, longitudinally. The greater part of the current will traverse the nerve diagonally, form- ing current loops which spread through the tissues, finally concen- trating to pass through the nerve again to the other electrode. Those points at which the current enters the nerve are known as physiological anodes, and those where the current leaves the nerve, as physiological kathodes. Thus, at each pole groups of physio- logical anodes and kathodes are found. The contraction of the muscle which occurs when the current is closed represents irritation at the physiological kathode. That contraction occurring at the break of the current represents irritation at the physiological anode. Since there are both physiological anodes and kathodes at each pole, any one or more of the following results may be obtained through the opening or closing of the constant current: 1. Anodic Closing Contraction. — Contraction following the change developed at the physiological kathode beneath the phys- ical anode. 2. Anodic Opening Contraction. — Contraction following the change produced in the nerve at the physiological anode beneath the physical kathode. 3. Kathodic Closing Contraction. — Contraction following the change produced in the nerve at the physiological kathode beneath the physical kathode. 4. Kathodic Opening Contraction. — Contraction following the change produced in the nerve at the physiological anode be- neath the physical kathode. The following abbreviations for these contractions, from i to 4 respectively, are used: ACC, AOC, KCC, KOC. KCC and ACC are stronger than KOC and AOC. KCC is stronger than ACC, and AOC is stronger than KOC. The effect of change of strength of current is shown in the fol- lowing table: [43] LABORATORY MANUAL OF PHYSIOLOGY. Weak Currents. Medium Currents. Strong Currents. KCC KCC Arc KCC Arr Aor AOP "K"OP With a strong current, tetanus sometimes occurs both at the make and the break. 1. Experiment. — Set up 8 or 10 dry cells in series. Place a commutator and simple key in circuit with the brass electrodes to be used for the stimulation of the human nerve. Place the anode electrode on the back of the neck and the kathode over the ulnar nerve at the elbow. (a) Make and break the circuit with the simple key. If there is no accompanying muscular contraction, add more cells to the circuit until a contraction is obtained. Record results and com- pare with the table given above. (b) Change the direction of the current by means of the com- mutator key so as to make the electrode, over the nerve, the anode. Again make and break the circuit, starting with weak currents and increasing the strength of current as before. Keep a careful record of the various strengths of current and the appearance of the different contraction responses. 2. Reaction of Degeneration. — In a muscle whose nerve has been cut off from its controlling cell, after a time certain definite changes in irritability to the constant and induced currents occur. There is a gradual diminution in excitability to the induced cur- rent and at first an increased excitability to the constant current. Later, this diminishes also. The muscle contraction may also be- come greatly prolonged and a condition called galvanotonus (tonic contraction) may be easily produced. The normal contraction formula is departed from, the most characteristic change being a reversal of the usual appearance of KCC and ACC. Normally, [44] MUSCLE-NERVE. KCC appears before ACC. In well-marked degeneration ACC appears first. Experiment. — Narcotize a rabbit with morphine and ether. Ex- pose and cut one sciatic nerve as high up as possible. Sew up the wound and test the muscle at intervals of two days for the reaction of degeneration. XXIV. ACTION OF CERTAIN DRUGS UPON THE SINGLE MUSCLE TWITCH. 1. Veratrine. — Make a saturated solution of veratrine in 0.6- per-cent sodium-chloride solution. Pith a frog and inject, under the skin, about five drops of the veratrine solution. In a few min- utes (ten to fifteen), if the frog be made to jump, it will be seen that the recovery of the flexed position of the hind limbs is very slowly brought about. A little later, a spasm of both limbs occurs at every attempt to jump. The flexor muscles, being the weaker, are overpowered by the extensors. Make a gastrocnemius-sciatic preparation from the frog. At- tach the tendon to the writing-lever of a myograph. Adjust the point of the lever to the smoked paper of a drum revolving at me- dium speed. Stimulate the nerve with a single maximal break shock from an inductorium. How does the recorded contraction compare with the normal muscle twitch ? 2. Adrenalin (active principle of the suprarenal gland). — In- ject into the dorsal lymph sac of a pithed frog 10 drops of a i to 10,000 solution of adrenalin chloride. After ten minutes, make a muscle preparation and proceed as in i . Is there any departure from the normal muscle twitch ? Prepare the muscle and nerve of the other leg or of another frog. Immerse the muscle in the adrenalin for a few minutes and again record a contraction. XXV. INVOLUNTARY MUSCLE. Synonymes : Plain or smooth, slow, non-striated, i . Remove the stomach from a pithed frog. Make two parallel cuts through the viscus running at right angles to the long axis and about one-half [45] LABORATORY MANUAL OF PHYSIOLOGY. centimetre apart. Pass a bent pin through the ring thus made, and support this in the femur clamp of the myograph. Pass a fine copper wire through the lower portion of the muscle ring and at- tach it to the muscle lever and the binding post of the lever. Con- nect this post and that of the femur clamp with a dry cell, inter- posing a simple key and a signal magnet in the circuit. Adjust the point of the muscle lever, the point of the signal magnet, and the point of a chronograph lever, marking hundredths of a second, in a straight perpendicular line against the smoked paper of a drum arranged to revolve at high speed. Start the drum, place the chro- nograph in circuit with the vibrating tuning-fork, open and close the key in the battery circuit. Compare the length of the latent period and the form of the con- traction curve with that of skeletal muscle. 2. Make a second similar preparation, omitting the electrical apparatus for stimulation, and allow the lever of the myograph to rest against a drum revolving once an hour. Observe tonic- contractions of the muscle. i46] CHAPTER III. NERVOUS SYSTEM. I. REFLEX ACTION. CHLOROFORM a frog. Make a longitudinal incision through the skin in the middle line of the skull. Cross this with another incision from ear drum to ear drum. Turn back the skin flaps and expose the skull. Carefully remove this piecemeal with strong scissors and forceps from before backwards. Expose the brain, noting its relations to the landmarks on the skull. Compare with Fig. 18. 1. Reflex Action with Cerebrum only Removed. — Partly anaesthetize a frog. Cut through the skull with sharp scissors or scalpel, transversely, just in front of the ear drums. This will serve to eliminate the cerebral lobes. Clamp the lower jaw in a femur clamp and suspend the frog from an upright stand. Keep the wound made, moist with physiological salt solution. Allow the frog time to recover from the shock of the operation and try the following experiments: (a) Immerse one foot in a beaker contain- ing a dilute solution of sulphuric acid (i to 10,000). Note the time elapsing between the application of the stimulus and the first muscular contraction. Which muscles contract first? Does the reaction extend to any [47] 9 FIG. 18. — Frog's Brain . i, Olfactory nerves; 2, olfactory lobes ; 3, cerebral lobes ; 4, epiphysis cerebri (pineal body) ; 5, optic thalamus ; 6, optic lobes ; 7, cerebellum ; 8, medulla ; 9, rhomboid fossa (fourth ventricle). LABORATORY MANUAL OF PHYSIOLOGY. other muscles if the application of the stimulus is continued? Explain. (b) Wash the foot thoroughly in plain tap water and dry with filter paper. Repeat experiment (a), using a stronger solution of the acid (i to 1000). Record reflex time as before. (c) Wash the feet again and dry with filter paper. Repeat ob- servations, using a still stronger solution of acid (i to 500), and re- cord results. What is the effect on reflex time of increasing the strength of the stimulus ? (d) Instead of the acid, use medium make-and-break shocks from an inductorium as the stimulating agent. Make and break every three seconds for ten shocks. Is there any reflex response ? (e) Increase the frequency of stimulation to one per second; to two per second. Is there any reflex? If so, how many stimuli must be applied to the skin before the reflex arc is completed? > Where are the main places of resistance in the reflex arc ? i 2. Reflex Action with Optic Lobes also Removed.— Using the same frog as in the previous experiments, make an incision through the skull and brain just behind the tympanic membrane. Allow time for the nervous system to recover from the shock of the operation before proceeding with the next series of observations. Repeat experiments (a) to (e) of series i. How does the reflex time of this set of experiments compare with that of the previous set? What is your conclusion concerning the influence of the optic lobes, in the frog, on cord reflexes ? 3. Reflex Action with Medulla Removed. — Complete the pithing of the frog and repeat the previous series of experiments. Conclusions? J 4. Diffusion of Impulses within the Cord. — Pith another frog. Suspend as before. Apply a strong and continuous stimulus to one foot. Note the successive groups of muscles that become involved in the reflex reaction. Also note the time at which each group becomes involved and the order of response. [48] NERVOUS SYSTEM. 6. Apparent Purposive Character of Reflex Responses. — A pithed frog is prepared as in the previous experiments. Take a small piece of filter paper wet with acetic acid diluted one-half with water, and apply this to the ventral aspect of one thigh. Note the attempt to remove this with the foot of the same side. If this is unsuccessful, or if the leg be held fast, the foot of the opposite side will be brought into play and even the fore limbs, in an attempt to brush off the offending irritant. 6. To Show the Centres of Reflex Exchange in the De- cerebrized Frog. — Using the frog of the previous experiment, run a long needle through the neural canal to destroy the spinal cord. After a sufficient interval, test the frog as before for reflexes. Result ? What is the function of the cord in relatioli to reflex action? Test the excitability of the muscles and nerves with the induced current. Open the thorax and observe the beating of the heart. The frog is still alive so far as the vegetative functions are con- cerned, but the entire cerebro-spinal axis is destroyed and conse- quently all reflex action is abolished. 7. Action of Strychnine. — Decerebrize a frog. Test the re- flexes as in experiment 2. Now inject under the skin a solution containing one-half milligram of strychnine sulphate. Test re- flexes again, as before, at five-minute intervals. If there is no ap- preciable change after ten minutes, repeat the injection. What is the effect upon reflex time as compared with the normal for the frog used ? Repeat the injection until the frog is thrown into tetanic spasms upon the slightest stimulation. What is the character of these spasms? What is the position of the frog during a convulsion? Explain. Now destroy the cord by passing a needle through the neural canal. What is the effect upon the strychnine spasms ? What is the seat of the strychnine action ? 8. Action of Chloral Hydrate. — Pith another frog. Estab- lish the normal reflex time to some stimulus, taken as a stand- ard. Inject under the skin 10 drops (about 0.6 c.c.) of a 2-per- 4 [49] LABORATORY MANUAL OF PHYSIOLOGY. cent solution of chloral hydrate. Test the reflexes as before and compare with the normal taken as a standard. How does the effect of the chloral compare with that of the strychnine? Repeat the injection of the chloral until a lethal effect is produced. Note all accompanying phenomena. II. REACTION TIME. 1. For Sound. — A tuning fork vibrating one hundred times per second is placed in circuit with a time-marker. Two short-cir- cuiting keys are placed in circuit with the time-marker, so that, by closing either one, the time-marker may be cut out of the circuit. The student experimented upon holds the handle of one key. An- other student holds the handle of the other key. The first key is held open, the second key being closed. Let the subject of the ex- periment close his eyes. He should close his key as soon as he hears the opening click of the other key. The»opening of the one key sets the time-marker to recording. The closure of the other key stops the time record. This should be taken upon a rapidly revolving drum. The interval between the opening of the circuit and its closure is marked in hundredths of a second and represents the time occupied for the passage of the sound wave through the auditory apparatus, the auditory nerve to the auditory centres of consciousness; its transfer to a motor neuron; its passage to the muscles involved and the latent period of these muscles. Repeat the experiment ten times for each individual and try a number of different individuals. Estimate the average reaction time for each individual. 2. For Vision. — For the first key to make the circuit, use a mercury contact. Darken the room, so that the spark made when the key is opened may be distinctly seen. Let the subject of the experiment close his key as soon as he sees the spark made by the opening of the other. The interval marked by the tuning fork gives the reaction time for vision. This experiment should also be repeated ten times and the average taken. NERVOUS SYSTEM. 3. For Tactile Sensation. — The circuit-opening key may be so arranged that, upon opening, it will come into sharp contact with the finger of the subject who is to close his key as soon as he feels this contact. This will give the reaction time for tactile sensation. 4. Influence of Drugs on the Reaction Time. — Repeat experiments i to 3 on one of the subjects whose normal reaction time has already been determined, after drinking 30 c.c. of whiskey diluted with equal parts of water. III. REMOVAL OF CEREBRUM IN THE FROG. Remove the cerebral hemispheres in a frog by making a cut through the skull in front of the tympanic membranes. Compare t 2 FIG. 19.— Pigeon's Brain, i, Skull which has been removed in part to show relations of cerebrum 1^2), cerebellum (3), and medulla (4). the frog so treated with a normal frog. Place both the normal and the decerebrized frogs on their backs. How do they react ? Stim- ulate the decerebrized frog. Is there any change in the co-ordina- tion of movements? Place both frogs in water. Can the decere- brized frog swim in a normal manner? Make cut behind the tympanic ' membranes. Compare this condition with the preceding and with the normal. [51] LABORATORY MANUAL OF PHYSIOLOGY. IV. REMOVAL or THE CEREBRUM IN A PIGEON. Remove the cerebral hemispheres in a pigeon anaesthetized with ether. Sew skin over wound. Allow the pigeon twenty-four hours to recover from the effect of the operation. Compare the pigeon so treated with a normal bird. Is there any disturbance of co-or- dination ? Is the pigeon able to sit on a perch ? Is it able to fly ? V. REMOVAL OF THE CEREBELLUM OF A PIGEON. Etherize another pigeon. Remove the bone of the skull over the cerebellar region, leaving a bony bridge in the middle line. Go in from either side with a blunt instrument. Sew skin over wound and allow twenty-four hours for recovery from the shock of the operation. How does this pigeon compare with the normal and with the one which has had its cerebrum removed? Describe all phenomena and reactions to various stimuli. Are there any disturbances of co- ordination ? If so, what are they ? VI. HEMISECTION OF THE SPINAL CORD. Narcotize a dog or a rabbit with morphine and ether. Cut off hair of back in upper lumbar region. Make a longitudinal incision through the skin and muscles over the spinous processes of the first three vertebra. Cut through the spinous processes with bone-scis- sors. Clean the lamina? of muscle. With a small trephine, care- fully remove a button of bone from the lamina of one side. From this opening remove the rest of the lamina with fine bone-cutting forceps. This will expose the cord in its membranes. Make an incision through the membranes with fine-pointed scissors. With a fine sharp scalpel cut through one-half of the cord. After any en- suing hemorrhage has been controlled, sew up the wound with cat- gut or silk. Allow the animal twenty-four or thirty-six hours to recover from the shock of the operation and then make observa- tions on the affected side compared with the other for changes in voluntary motion and sensation. Test the various reflexes also [52] NERVOUS SYSTEM. and compare with the normal. Observe the animal from day to day and note any change in motion or sensation. Try the muscles for the reaction of degeneration. VII. STIMULATION OF THE MOTOR AREAS OF THE DOG'S BRAIN. DEMONSTRATION. Lightly narcotize a dog ether. Tie on dog-board, out and supported on a block of wood placed be- neath it. With the trephine remove a button of bone from one parietal. This opening may be enlarged by repeating the trephin- ing several times. The re- mainder of the bone may then be removed piecemeal with bone forceps and scis- sors. Expose in this man- ner the whole lateral and dorsal aspect of one cere- bral hemisphere. Identify the fissures and motor points as shown in Fig. 20. With fine platinum elec- trodes, having the two poles but slightly sepa- rated, stimulate at the points indicated in the figure. The current used for this purpose is a tet- anizing current from an inductorium of medium strength. with morphine and anaesthetize with belly down, with head well stretched FIG. 20. — Dog's Brain, Showing Various Motor Areas. P, Frontal fissure, sometimes termed crucial sulcus, corresponding to the fissure of Rolando in man. i, Flexion of head on neck in median line ; 2, flexion of head on neck with rotation towards side of stimulus ; 3, 4, flexion and extension of anterior limb; 5, 6, flexion and extension of posterior limb ; 7, 8, 9, contraction of orbicularis oculi, and facial muscles in general. The unshaded part is that exposed by opening the skull. (Dai- ton.) [53] LABORATORY MANUAL OF PHYSIOLOGY. VIII. DIVISION OF THE SEMICIRCULAR CANALS. A young pigeon serves best for this purpose. It is well to make a dissection on a dead bird first in order to become familiar with the position and relations of the canals. Make a transverse in- cision through the skin of the head. Slip these flaps back so as to expose the bone. Scrape away the insertions of the neck muscles. 3 s». I \ FIG. 2i.— Semicircular Canals (Pigeon). Outer plate of skull and cancellous bone removed to expose the semicircular canals : i, Superior (vertical); 2, posterior (verti- cal) ; and 3, anterior (horizontal) . The planes of i, 2, and 3 cut each other at right angles. Remove the outer table of the skull behind each ear, carefully re- moving with the forceps the cancellous or spongy bone between the two plates until the canals are seen (see Fig. 21). Having made the preliminary dissection on a dead bird, repeat the same process on a live pigeon under the influence of chloro- form. After the canals are exposed, cut through one or two of them, with strong scissors, making a careful record of the canals thus injured. Control the bleeding with suprarenal extract. If the bird recovers from the immediate effects of the operation, carefully observe and note its departure from the normal condi- tion of the pigeon with the semicircular canals intact. Compare the behavior of this pigeon with that of the pigeon which had its cerebellum removed. [54] NERVOUS SYSTEM. IX. To DETERMINE THE NUMBER OF IMPULSES DISCHARGED BY A NERVE CELL IN A GIVEN UNIT OF TIME. In an etherized rabbit, adopting the same methods as were em- ployed in the experiment on hemisection of the cord, expose the cord in the middle lumbar region. Insert needle electrodes in the gastrocnemius muscle of one side. Connect these with the capil- lary electrometer. Insert fine needle electrodes through the cord. Stimulate the cord with medium strong single induction shocks at intervals of one second. Is there any response of the gastrocnemius muscle ? If so, what is the nature of this response ? Increase the frequency of stimulation of the cord and note results. Stimulate the cord ten times per second. Does the muscle go into tetanus ? Does the muscular contraction continue after the stimu- lation of the cord has been stopped ? What changes occur in the capillary electrometer ? Place the tuning-fork interrupter in circuit with the primary coil of the inductorium. Stimulate the cord again. Does the muscle go into tetanus? What is the frequency of vibration of the me- niscus of the capillary electrometer ? Listen with a stethoscope to the muscle during contraction. Is the tone of the tuning-fork reproduced in the muscle? If not, is there any sound heard in connection with the contraction ? Compare these results with those obtained when the sciatic nerve was stimulated in the experiment on muscle tone. What is the rate of discharge from the nerve cells in the cord ? ESS] CHAPTER IV. BLOOD. THE blood may be looked upon as the common carrier of the body. It serves to carry food stuffs to the tissues from the aliment- ary canal where they have been absorbed and O and CO, be- tween the lungs and the tissues. It also carries away from the tis- sues waste products, resulting from their metabolism, to the organs of excretion. It acts as a medium of exchange between the tissues themselves, carrying products of glandular activity from one group of cells to another, as in the internal secretions. It is a prime factor in the regulation of body temperature. It is finally, in part, the re- ceptacle for and, in part, the seat of the formation of protective substances which are manufactured by the body as a result of the introduction of toxins from without. In structure, the blood consists of two main elements, a liquid portion or plasma, and a cellular portion, corpuscles. The latter are divisible into two classes, the colored corpuscles or erythro- cytes and the colorless corpuscles or leucocytes. They are also known respectively as the red and white corpuscles. Their num- bers, varieties, and properties will be considered later. I. COAGULATION OF THE BLOOD. Narcotize and etherize a dog or rabbit. The former will furnish more blood. Expose both carotid arteries. Introduce a cannula into each carotid, securing the arteries on the side near the heart with artery clamps. i. Prepare a series of test tubes, as follows: (a) Clean empty tube for receiving a sample of fresh shed blood; (b) tube half full [56] BLOOD. of distilled water; (c) tube half full of o.8-per-cent NaCl solution; (d) tube half full of saturated NaCl solution; (e) larger tube quarter filled with a saturated solution of MgSO4. Open clamp on one carotid and complete the filling of the test tubes with blood. Set tube (e) to one side for use later. Observe what happens in tube (a), which contains undiluted fresh blood. How long before the blood in tube (a) is completely solidified? Invert test tube. The blood does not run out, but adheres to the sides of the tube as a jelly-like mass of the same volume and color throughout as when first shed. Later, the mass shrinks, the sur- face becoming cup-shaped and, as the shrinking continues, more and more of a clear straw-colored fluid collects upon it. This is the serum which is not subject to further coagulation, except that caused by high temperatures in any albuminous fluid. The solid mass remaining finally floats in the serum as this accumu- lates; it consists of a stringy substance, fibrin, and blood corpuscles entangled in its network-like meshes. If a little fresh blood be allowed to drop on a glass slide; and is then covered with a cover slip and placed in a moist chamber to prevent drying, after fifteen to twenty minutes the fibrin fibrils may be seen with a low power of the microscope. Has the distilled water of tube (b) any effect in hastening or de- laying the coagulation of the blood shed into it ? Has the o.8-per-cent NaCl solution of tube (c) any effect in hast- ening or delaying the coagulation? What is the effect of the saturated salt solution of tube (d) ? 2. Compare the color of the fresh undiluted blood with the sat- urated salt-solution dilution and the distilled-water dilution. Com- pare the different tubes in transmitted and reflected light. To what is the opacity of the fresh blood due ? To what is the trans- parency of the water-diluted blood due? 3. Place a specimen from each tube under the microscope and compare the appearances of the red corpuscles. With distilled water and some other reagents the red blood corpuscles lose their pigment (haemoglobin) which goes into solution in the diluted [57] LABORATORY MANUAL OF PHYSIOLOGY. plasma, giving it a transparent color. This process is known as taking and the blood is said to be laked. The corpuscles will ap- pear faintly outlined as ghost or shadow corpuscles. 4. The magnesium sulphate of tube (e) prevents coagulation. Let the mixture stand in a cool place until the corpuscles have set- tled to the bottom of the tube. The supernatant liquid, the "salted plasma," may then be pipetted or siphoned off. Divide this salted plasma into four portions. To each portion add eight times its volume of water. To portion i add a few drops of a half-per-cent solution of ammonium oxalate. To portion 2 add a little of the clot from tube (a). Portion 3, place in a water bath heated to 38° C. Place portion 4 on a water bath heated to 60° C. and add a few drops of ammonium oxalate. Observe the presence or absence of the phenomena of coagulation in the portions of salted plasma treated as above. Are calcium salts necessary to coagulation ? What is the effect of tempera- ture on coagulation ? Why does coagulation take place in por- tion 2? To tubes i and 4 add a few drops of calcium chlorid. What is the effect as far as coagulation is concerned ? There are various theories to explain the coagulation of the blood. The known facts are as follows: Clotting is produced through the formation of a coagulated substance, fibrin; for the formation of fibrin three things are necessary: a globulin, fibrin- ogen, calcium salts, and an enzyme, fibrin ferment or thrombin. Fibrinogen and soluble calcium salts are normally present in the blood plasma. Thrombin is formed at the time of coagulation. The mooted question is the origin of the thrombin. The thrombin is a nucleo-proteid which seems to be formed through cell disinte- gration and especially through the breaking down of leucocytes. 5. Defibrination of Blood. — To defibrinate blood, collect it from a bleeding artery, in a shallow vessel. As the blood is shed, whip or beat it, vigorously, with a glass rod or a bundle of twigs. The fibrin, as it is formed, separates from the blood and adheres to the whip as a sticky, stringy, almost colorless mass. The blood [58] BLOOD. so treated is then filtered through a fine-mesh cloth. Defibrinated blood will not clot spontaneously. II. THE NUMBER OF RED AND WHITE BLOOD CORPUSCLES. 1. Counting the Erythrocytes or Red Corpuscles. — The Thoma-Zeiss haemocytometer is used for this purpose. This con- FlG. 22. — Thoma-Zeiss Haemocyto meter Counting-chamber, s, Glass slide, upon which is mounted a covered disc m, accurately ruled to present one square millimetre divided into 400 squares. This is surrounded by another annular cell, c, which pro- jects in height exactly one tenth of a millimetre above m. sists of a graduated pipette for accurately diluting a known quan- tity of blood with some fluid having the same osmotic pressure as the blood. One of the most satisfactory diluents is physiological salt solution. For human blood, this consists of a solution of so- dium chlorid, 8.5 grams in 1000 c.c. of water. The capillary stem of the pipette, used for diluting the blood for counting the red corpuscles, has a capacity equalling one-hun- dredth of the hollow ball with which it joins (see Fig. 23). If the blood is drawn up to the line marked i on the pipette stem and then the diluent drawn in until the mixture reaches the line 101 FIG. 23.— Thoma-Zeiss Haemocytometer Pipette. marked just above the ball (see Fig. 23), there will be 101 parts of fluid, of which the blood forms i. The contents of the stem, how- ever, do not have to be considered after the dilution is made, since they can be displaced, unmixed. The dilution of the blood will then be i to 100. As the blood and diluting fluid enter the mixing [59] LABORATORY MANUAL OF PHYSIOLOGY. chamber, this should be constantly rotated between the fingers so as to facilitate the mixture and avoid error through coagulation. The other part of the instrument is the micrometer slide upon which the diluted blood is evenly spread for counting the corpus- cles. This consists of a glass slide (Fig. 22), upon which is mounted a covered disc, m, a square millimetre of which is subdivided by a dividing engine into 400 squares of one-twentieth millimetre each. The micrometer, m, is surrounded by an annular cell, c, the sides of which project one-tenth millimetre above the surface of m. This cell is closed by a thin flat glass cover, so that the cubic space in- cluded between each small square of the micrometer and the cover would be ^nnr °f a cubic millimetre. To find the number of corpuscles in a cubic millimetre of undi- luted blood, multiply 4000 by the dilution and this by the total number of corpuscles counted. This result is then divided by the number of small squares counted. If the blood has been drawn only to the 0.5 mark in the diluting pipette, the blood dilution is i to 200 and this number must be substituted for the factor 100 in the formula given above. With normal blood, the higher dilution is advisable. Procedure. — Thoroughly cleanse the tip of the finger or, prefer- ably, the lobe of the ear, with soap and water. Wipe off with a cloth wet with alcohol. Dry thoroughly. With a sterilized needle, or a sharp pen with one nib broken off, make a quick stab of the ear or the finger. Wipe off the first drop of blood. Blood should ooze freely from the puncture without pressure. Insert the point of the pipette well into the blood drop and carefully draw in blood to the 0.5 mark on the stem of the pipette. With a cotton cloth wipe off all blood adhering to the outside of the pipette. Dip the end of the pipette into the diluting fluid and draw this in through the stem and into the ball until the 101 mark is reached. The pi- pette should be gently rotated while the filling is going on, in order that the mixture of the blood and diluting fluid may be assured through the movements of the glass bead in the ball. Close both ends of the pipette with thumb and forefinger and shake well. [60] SLOOD. This is to obtain a uniform distribution of the corpuscles through- out the mixture. To Fill the Counting Cell. — Blow out three or four drops of the diluted blood from the pipette. Now allow a small drop to flow upon the disc of the counting chamber. Cover quickly, pressing the cover gently down until Newton's rings are seen. These are the spectrum colors due to refraction between the two layers of glass. They do not appear if there is any fluid or dirt between the cover and the cell. If any fluid runs over into the moat between the cell and the micrometer, the slide will have to be cleaned and another drop of the diluted blood taken. Repeat until a satis- factory specimen for counting is obtained. Allow several minutes for the corpuscles to sink to the bottom of the cell upon the ruled squares. It is obvious that the counting cell must ke kept in the horizontal position. Place this upon the stage of a microscope and count the corpuscles in all the squares. For convenience of counting, the micrometer is divided into sixteen large squares by double lines, and these, in their turn, are sub- divided into the small squares already mentioned. Count several specimens, in this way, and take the average. Compare the blood of various students. To Clean the Cell and Pipette.— The cell should be carefully rinsed with distilled water and dried with a soft cloth or absorbent cot- ton. The cover should be treated in the same way. Neither alco- hol nor ether should be used since they will coagulate the albumin of the blood. In cleaning the pipette first blow out any blood mixture remaining. Fill with distilled water several times. If all traces of blood are not removed in this way, rinse with an aqueous solution of hydrogen peroxide and again wash out with distilled water. Now draw alcohol through by suction and follow this with ether, drawing through a stream of air until the pipette is thoroughly dry. This is manifest when the glass bead enclosed in the bulb of the pipette no longer adheres to the sides. Counting the White Corpuscles. — Since there is a much smaller number of leucocytes than of red corpuscles, the dilution required [61] LABORATORY MANUAL OF PHYSIOLOGY. is much less. A special pipette is employed, with which a dilution of i to i o or i to 20 may be obtained. The diluting fluid employed is usually a 0.2 of one per cent acetic acid. This accentuates the f FIG. 24. — Human Blood-corpuscles, a, Red blood-corpuscles for comparison; £, small hyaline cell or small lymphocyte ; c, large hyaline cell or large lymphocyte ; , ducts of submaxillary and sublingual glands (S) ; Dg; digastric muscle ; M, mylo-hyoid muscle. with the artery are filaments of the sympathetic nerves. Entering into and passing out of the hilum of the gland are seen the chorda tympani nerve branch to the gland, the branch of the facial artery to the gland, and the gland duct. Beneath the reflected mylo-hyoid muscle is seen the lingual nerve (Fig. 36, L). Trace this nerve to the ramus of the jaw. At this point a small branch will be exposed which, in close proximity [122] SECRETION— DIGESTION— ABSORPTION. to the duct, runs backward to the gland. This is the chorda tym- pani (Fig. 36, T). Pass a thread under the chorda to use later in handling the nerve for stimulation. Divide the hypoglossal nerve and expose the sympathetic filaments. Pass a thread around these also. Iden- tify the submaxillary duct and introduce and tie a cannula into it. The cannula should end in a small rubber tube. This is closed by an artery clip until it is desired to collect the secretion. The introduction of the cannula may be facilitated by first stimulat- ing the chorda for a short time with a weak tetanizing current, thus distending the duct with secretion. Small graduated glass cylinders are provided for collecting the secretion from the cannula in the gland duct. These may be changed at any desired interval of time, say every five or ten minutes. The rate of flow is determined by the amount of secretion eliminated in a given time period. 1. Observe the rate of flow from the gland before stimulation. Has the anaesthetic any stimulating influence on the salivary flow ? This observation should cover a period of five minutes. 2. Stimulate the chorda with a weak tetanizing current. How is the rate of salivary flow affected ? Compare the appearance of the blood-vessels of the gland during stimulation of the nerve with the vascular condition before stimulation. 3. Allow the preparation to rest for several minutes. Stimulate the sympathetic. Is there any marked effect on the rate of flow ? What is the effect upon the condition of the blood supply to the gland ? 4. Paint the submaxillary ganglion with a o.i-per-cent solution of nicotine. Nicotine, in weak solution, paralyzes nerve cells, but not nerve fibres. Stimulate the chorda again. Is there still an ac- celerator effect on the salivary secretion? Are the fibres of the chorda broken by nerve cells in this ganglion ? Now paint the chorda with nicotine where it enters the hilum of the gland. Stimulate the chorda again. Is there any effect on the flow of the secretion ? Stimulate at the hilum itself. Is there any LABORATORY MANUAL OF PHYSIOLOGY. effect on the flow of secretion ? Does the chorda run directly to the gland cells without interruption? If not, where is the point of transfer ? 5. Wash the gland thoroughly with warm physiological saline. After a time the nicotine effect will wear off. Isolate and intro- duce a cannula into the femoral vein, centrally. Inject into the vein J grain atropine sulphate. Note the effect on the flow of sa- liva. Stimulate the chorda. Is there an increased flow of the secretion? Empty the duct of secretion. With a hypodermic syringe inject into the duct a few drops of a 2-per-cent solution of pilocarpine nitrate. Stimulate the chorda again. Is any effect now obtainable on the salivary flow ? After several minutes stimulate again. The atropine effect has once more asserted itself. Does stimulation of the nerve still cause a dilatation of the gland vessels? Is the effect of the nerve stimulation in causing an in- crease in flow of secretion purely a vaso-dilator effect or is it due to a stimulation of the gland cells themselves ? Explain. II. To SHOW CHANGES IN THE GLAND CELLS FOLLOWING CHORDA STIMULATION. Make another preparation of submaxillary gland, duct, and nerve. Stimulate the nerve with a weak tetanizing current until the flow of the secretion ceases. Allow an interval of five minutes' rest and repeat the stimulation. Continue to complete exhaustion of the gland. Remove both submaxillary glands. Cut out small portions of each and make frozen sections of the fresh glands. Mount in normal salt solution or in glycerin. Examine sections from the two glands under the microscope. Compare the appear- ance of the cells of the stimulated gland with that of the resting gland. Harden the remainder of the two glands in absolute alcohol; embed in paraffin; cut sections; and stain with carmine. Com- pare the stained sections of the two glands with each other and with the frozen sections of the fresh glands. SECRETION— DIGESTION— ABSORPTION. III. SALIVARY DIGESTION. 1. Chemical Constituents of Saliva. — Chew a piece of par- affin gum or inhale a little ether vapor. The flow of saliva is thus stimulated. Collect the secretion in a clean porcelain cap- sule. Filter and divide the nitrate into five portions. (a) Test the first portion for its reaction with litmus paper. Is it alkaline or acid ? Is the reaction very decided in either di- rection ? (b) To the second portion add dilute acetic acid. The pres- ence of mucin is indicated by the formation of a precipitate. (c) To a third portion add a few drops of a silver-nitrate solu- tion. A precipitate of silver chlorid which is soluble in ammonia and insoluble in nitric acid is indicative of the presence of chlorids. (d) To another portion add dilute acetic acid and filter. Test the filtrate with Millon's reagent. The presence of proteids is shown by the production of a red coloration or precipitate. 2. Action on Starches. — (a) To some boiled starch paste add a few drops of iodine. A blue coloration will occur. To some powdered starch add a few drops of iodine. A blue color test will also be obtained. Both cooked and raw starch respond to the iodine test. (b) To a test-tube partly filled with a dilute Fehling's solution add a little of the starch paste. Boil the mixture. There should be no reduction of the copper sulphate of the Fehling's solution. The copper salt is reduced by any of the reducing sugars. (c) To another portion of Fehling's solution add a few drops of a dilute solution of dextrose. Heat to boiling and note the for- mation of a copious precipitate, first yellow, and, as the heating is continued, changing to a reddish color. This is the cuprous and later cupric oxide formed by the reduction of the copper salt in the test solution. (d) Repeat the test with maltose instead of dextrose. Is re- duction obtained? LABORATORY MANUAL OF PHYSIOLOGY. (e) To another portion of the test solution add lactose. Is the copper salt reduced ? (/) Test a solution of cane sugar for reduction. Result ? (g) Partly fill three test-tubes with starch paste. Mark them I, II, and III. To I add some of the filtered saliva. To II add some saliva that has been heated to boiling. To III add some saliva that has been neutralized with HC1 and has had enough acid added to bring the acidity up to i per cent HC1. Place the three tubes in a water-bath or in the incubator kept at a tempera- ture of 38° C. In five minutes remove the tubes and test the three for reducing sugar with Fehling's solution. Is there any reducing sugar in I ? in II ? in HI ? Explain. (//) Make a solution of dextrin. To this solution add a few drops of iodine. Note the wine-color reaction. (t) To a few cubic centimetres of the dextrin solution add an equal quantity of saliva. Place in the water-bath or incubator for one hour at a temperature of 38° C. At the end of this time test the solution with iodine for the red dextrin reaction. Is there any color reaction? Test for reducing sugar. (f) Mix equal quantities of boiled starch and saliva in a test- tube. Place in the warm water-bath. Place several drops of dilute iodine solution on a white porcelain slab. At five-minute intervals, by means of a glass stirring-rod, add a drop of the digesting starch to a drop of the iodine on the slab. Continue this until a color re- action is no longer obtained. \Yhat changes occur in the color reaction as digestion progresses ? (k) Take some boiled starch into the mouth and go through the movements of mastication. At the end of one minute spit this out into some boiling water in a beaker. Test a portion of the mixture for sugar, and another portion for starch . Repeat with another por- tion of starch, keeping it in the mouth two minutes. Chew another portion for five minutes and test again for starch and sugar. Does the lest for starch diminish in intensity, and the test for sugar increase ? (/) Mix a portion of fibrin in a test-tube with some saliva. [126] SECRETION— DIGESTION— ABSORPTION. Place in the incubator for twenty minutes: At the end of this time remove and test the mixture for peptones. Has any of the proteid been digested ? (;;z) Add some saliva to a starch solution in a dialyzer tube of parchment paper for twenty-four hours in the incubator. At the end of this time test the water surrounding the dialyzer for re- ducing sugar and for starch. IV. MECHANISM OF SWALLOWING. 1. Inject a solution of 0.03 gram morphine sulphate under the skin of a medium-sized rabbit. Anaesthetize lightly with ether. Tie the rabbit, back down, upon the rabbit-board, with the neck well stretched out. Clip and shave the hair in the neck region and also over the epigastrium and zvphoid appendix. 2. Make a median incision through the skin and fascia of the neck. Separate the sterno-hyoid muscles and expose the trachea. Carefully separate the trachea from the oesophagus, which lies be- hind it, avoiding injury to the nerves and vessels running beside and between the two. 3. On either side of the trachea and between it and the oesopha- gus, a fine nerve filament will be seen. These are the recurrent laryngeal nerves. Pass a loop of thread around each recurrent laryngeal nerve, but do not tie. Isolate the vagus nerve of each side and secure with untied threads. Follow the vagus on one side as far as the level of the lower part of the thyroid cartilage. At this point it is joined by the superior laryngeal nerve. Isolate and pass a loop of thread around this nerve. 4. Pass a heavy ligature around the trachea as low in the neck as practicable. Cut between the rings of the trachea just above this ligature. Introduce a tracheal cannula and tie it in with the ligature. Excise the piece of trachea between the cannula and the cricoid cartilage. This brings the oesophagus w^ell into view. Pass two threads around the oesophagus, to be tied later. 5. Now make a median incision through the abdominal wall in the median line and expose the stomach. By pulling the stomach LABORATORY MANUAL OF PHYSIOLOGY. down with one hand, and the liver and lower ribs up with the other, the junction of the oesophagus and stomach may be seen. 6. Arrange an inductorium for weak tetanizing current. Place the electrodes from the secondary coil under the superior laryngeal nerve. Arrange a metronome or chronograph marking seconds. This is to assist in taking the time of the swallowing move- ments. The work of the experiment may be divided among four stu- dents as follows : Let one student manage the stimulation of the superior laryn- geal nerve or other nerves that it may be desired to stimulate dur- ing the course of the experiment ; let another make the time ob- servations of the swallowing movements; let a third manipulate the stomach for observation of the lower end of the oesophagus; and let a fourth make careful notes of the observations. 7. Stimulate the superior laryngeal nerve with the weak tetan- izing current until the rabbit swallows. Stimulation of this afferent nerve brings about, among other things, a reflex swallow. Note and time the beginning of the swallowing movement. Note the passage of the peristaltic wave along the cervical portion of the oesophagus, and the end of the peristaltic movement at the stomach. How much time has elapsed between the beginning of the swallow and the ending of the peristalsis at the stomach ? Repeat the ob- servation a number of times. What is the average time occupied by the passage of a peristaltic wave over the length of the oesopha- gus in your rabbit ? 8. Determine whether the mechanism of cesophageal peristalsis is a nervous reflex one or due to muscular conduction of the con- traction wave from one segment of the oesophagus to another. Tie two ligatures around the oesophagus, in the cervical region, and cut completely through the gullet between the ligatures. Mus- cular continuity is thus absolutely severed. While making observations, as before, produce a swallow by stimulating the superior laryngeal. Does the peristaltic wave still pass over the lower segment of the oesophagus to the stomach ? If SECRETION— DIGESTION— ABSORPTION. so, how does the time occupied in the passage of the wave com- pare with the time before the gullet was divided ? If the peristaltic wave ceases to pass below the cut, what con- clusion might you draw concerning the method of conduction of the contraction wave ? If the wave continues to pass after the sev- erance of muscular continuity, what conclusion might you draw ? 9. Secure the vagus of one side with two ligatures and cut be- tween. Again induce a swallow as before. Does the contraction wave still pass over the oesophagus ? Explain. 10. Now cut the other vagus also. Both vagus nerves are now cut. Induce a swallow. Does the peristaltic wave continue to pass over the oesophagus ? From the above observations what conclusions can you draw con- cerning the mechanism of cesophageal peristalsis and the function of the vagi in this connection ? 11. The Vagus as a Motor Nerve to the Stomach. — Enlarge the abdominal incision so as to expose the whole of the stomach including the beginning of the duodenum. Place the peripheral ends of both vagi upon electrodes from an inductorium arranged for medium strong tetanizing currents. Stimulate both vagi contin- uously and note the strong contraction rings which pass over the stomach from the fundus toward the pylorus. Note the opening of the pyloric sphincter and the expulsion of a small quantity of stom- ach content into the duodenum. Note the movements of the duo- denum during and after the entrance of food from the stomach. Keep the stomach covered with a pad of absorbent cotton moist- ened with warm physiological saline, between observations. Over what part of the stomach wall are the contraction rings most distinct and strongest? Where do they begin and in what directions do they pass ? 12. Stimulate both inferior laryngeal nerves. Note the effect upon the upper segment of the oesophagus. What is the nature of the musculature of the first part of the oesophagus ? 13. Free the small piece of trachea connected with the cricoid cartilage from blood and note the position of the vocal bands. 9 [I29l LABORATORY MANUAL OF PHYSIOLOGY. Note the slight opening and closing of the glottis with inspiration and expiration. While observing the movements of the vocal bands, stimulate one inferior laryngeal nerve. What is the effect on the vocal bands? Stimulate both nerves at the same time. What is the effect on the movements of the vocal bands ? 14. Stimulate the superior laryngeal nerves in the same way and note the effect, if any, on the vocal bands. 15. To determine the time occupied for the passage of a liquid from the mouth into the stomach, in man, proceed as follows: Arrange a drum with smoked paper for medium slow revolution. Set up a chronograph to mark seconds on the drum. Place a short- circuiting key in circuit with the time marker. When the key is closed, the lever of the time marker will write a straight line. When the key is opened, a time tracing, in seconds, will be recorded. Take a fellow-student into a quiet room and listen with a stetho- scope over the end of the sternum. Start the drum. Let the sub- ject of the experiment take one swallow of water. You will hear two sounds, one when the liquid is shot into the oesophagus, the other when the liquid enters the stomach. When the first sound is heard, open the short-circuiting key. When the second sound is heard, close the key. How many seconds have elapsed between the two sounds ? V. GASTRIC DIGESTION. 1. Tests for Proteids. — (a) Coagulation by Heat. — Prepare solutions of the following proteids: A, egg albumin, dilute; B, acid albumin in acid solution. This is obtained by subjecting some dilute egg albumin to the action of o.2-per-cent HC1 for sev- eral hours at body temperature. Neutralizing with an alkali will precipitate the acid albumin from its solution. C, myosin, dissolved in a lo-per-cent NaCl solution. This may be prepared by mincing lean meat, freeing from blood by repeated washings, and extracting the myosin by an ammonium-chlorid solution. The salt may be removed by dialysis, leaving the myosin as a gelatinous mass, or it may be precipitated by diluting the solu- SECRETION— DIGESTION— ABSORPTION. tion with distilled water. This precipitate is redissolved in the sodium-chlorid solution as given above. D, proteose. This may be prepared by digesting a small quan- tity of fibrin with o.2-per-cent HC1 and a little commercial pep- sin, at 38° C., just to the point of solution of the fibrin and no more. Neutralize carefully with dilute NaOH, heat to boiling, and filter. Witte's peptone may be used, since it consists chiefly of albumoses. E, peptone. Savory & Moore's preparation is used. Place a small quantity of each of the above solutions in test- tubes and immerse in a water-bath heated to 65° C. Gradually raise the temperature of the bath to 100° C., noting observations at every 5° rise in temperature. Are all of these solutions coagulated by heat ? In those in which coagulation does occur, is the coagulating point the same ? (b) Nitric-acid Ring Test.— Place a small quantity of HNO3 in a test-tube. With a glass tube of small calibre draw up some of solution A into the tube, and with the finger firmly pressed over the other end introduce the end containing the solution into the acid. Remove the finger. If the acid level is slightly higher than the level of the liquid in the tube, some of the acid will be drawn up into the glass tube containing the solution to be tested. Is there any ring of precipitation formed where the two liquids come in contact ? Repeat this test for the other proteid solutions and re- cord results. (c) Xanthoproteic Reaction. — Add an excess of concentrated nitric acid to a little of each of the above tested solutions and heat to boiling. A yellow color is produced. Neutralize and make the solutions alkaline with sodium hydrate or ammonia. The color changes to an orange red. (d) Biuret Test. — Make the solutions of the proteids to be tested alkaline with sodium hydrate. Add a few drops of a dilute cupric- sulphate solution. Be careful not to add an excess of the copper solution, since this may give a test in the absence of proteid. A blue-purple or violet color results. (e) Millon's Test. — Test each of the solutions with Millon's LABORATORY MANUAL OF PHYSIOLOGY. reagent, which is already made up. This is a solution of mercu- rous nitrate together with some free nitrous acid. When mixed with proteid a yellow precipitate is formed which becomes red on heating. 2. To Differentiate Albumins, Proteoses, and Peptones.— (a) What reactions have they in common ? Are the albumins co- agulable by heat? Are the proteoses coagulated D> neat? Are the peptones coagulated by heat ? (b) Do the proteoses give a precipitate with nitric acid? Do the peptones? (c) To a proteose solution add some potassium ferrocyanid acid- ified with acetic acid. Is there any precipitate? Repeat with a pure peptone solution. Is there any precipitate ? (d) To a solution of proteoses add sodium chlorid to satura- tion. Is there any precipitate ? Repeat with pure peptone solution. (e) To a peptone solution add alcohol. Is a precipitate formed ? Repeat using a saturated solution of tannic acid instead of the alcohol. Result ? 3. Artificial Gastric Juice. — Scrape off the mucous membrane of the fresh stomach of a pig. Grind this thoroughly with clean sand in a mortar. Add ten times the volume of a o.2-per-cent so- lution of hydrochloric acid and place in the incubator at blood temperature for twenty-four hours. Grind up another portion of mucous membrane of the pig's stomach with glycerin. Let this stand for several days before using. (a) To a little fibrin in a test-tube add some o.2-per-cent HC1. (b) To another portion of fibrin add some of the glycerin ex- tract of the pig's stomach. (c) To another portion of fibrin add some of the acidulated aqueous extract of pig's stomach. (d) To another portion add some of the acidulated extract which has been neutralized and made slightly alkaline with so- dium carbonate. (e) To another portion add some glycerin extract plus enough SECRETION— DIGESTION— ABSORPTION. o.2-per-cent HC1 to make the acidity of the mixture equal to about o.i per cent HC1. (/) To another portion add glycerin extract plus o.4-per-cent acetic acid. (g) To another portion add glycerin extract plus o.4-per-cent lactic acid. Make the fibrin portion in each tube as near the same quantity as possible and use the same amount of the extract each time. Place all the tubes, properly labelled, in a water-bath kept at a temperature of 38° C. Examine the specimens every three to five minutes, and note the extent of the solution of the fibrin in each tube. Continue the observations for a half-hour. Solution of the fibrin is not an index of complete digestion, but is a sufficient index for rough comparison of the digestive activity of the mixtures in the various tubes. Note results and record observations. The glycerin extract contains pepsin, but no acid. What is the digestive activity of pep- sin in the absence of acid ? What is the digestivity of HC1 alone, in the absence of pepsin ? Is there any digestion with the alkaline mixture of pepsin? Does digestion occur when other acids are substituted for HC1 ? (h) Boil some of the acidulated glycerin extract. Add this to fibrin and note results. Does heat destroy the enzyme ? 4. Filter the contents of one or more of the tubes in which the fibrin has been dissolved. (a) To a portion of this filtrate add dilute NaOH, carefully, until the acid is neutralized. Is there any precipitate ? If so, the presence of what proteid is indicated ? Filter. (b) To a part of this filtrate add an excess of the alkali and then a drop or two of very dilute CuSO4 solution. How is the pres- ence of proteoses and peptones indicated ? What is the necessity for care in adding the copper-salt solution? (c) Heat another part of the filtrate from (a) to 70° C. Is there any coagulation ? (d) Take another portion of the filtrate from (a) and saturate [133] LABORATORY MANUAL OF PHYSIOLOGY. the solution with ammonium sulphate. How is the presence of proteoses indicated ? How are they differentiated from peptones ? (e) Filter portion (d) and test the nitrate for the biuret reaction. If present, what does it indicate ? (/) Take a portion of the fibrin which has been acted upon by the acidulated glycerin extract, neutralize the mixture with sodium carbonate, and place in a dialyzer over night. The next day test the dialyzant for albumin, proteoses, and peptones. Test the fluid remaining in the dialyzer in the same way. Re- sults ? Conclusions ? 5. Place some fresh milk in a test-tube, in a water-bath at 38° C., and add a few drops of rennet extract. What is the action of the rennin ? Remove the fluid part of the milk so far as possible, and add some artificial gastric juice. Is the casein dissolved ? VI. INTESTINAL DIGESTION. 1. Emulsification. — (a) Shake up 5 c.c. of olive oil in a test- tube with an equal quantity of water. The mixture will become milky white because of the distribution of oil globules through it. Do these oil globules remain in suspension ? (b) Repeat the procedure in (a), adding to the mixture, before shaking, 5 c.c. of strained egg albumin. Do the oil globules remain in suspension ? Set this to one side and observe again, some hours later. Has any of the oil begun to separate out ? (c) Repeat (6), substituting gum acacia for the egg albumin. Set aside and observe later for the separation of oil from the emulsion. 2. Saponification. — To 5 c.c. of olive oil or cottonseed oil add twice the volume of a 20-per-cent solution of NaOH or KOH. Shake well. Continue to agitate the mixture at frequent intervals for fifteen to twenty minutes. Now add an excess of water. Has the oil been dissolved ? What chemical reaction has taken place between the oil and the alkali ? 3. Saponification as an Aid in Emulsification. — (a) Mix a small quantity of oil which has become slightly rancid, with a SECRETION— DIGESTION— ABSORPTION. strong solution of sodium carbonate. Shake the mixture vigor- ously. Set the emulsion thus formed aside and compare it later with the emulsions made with egg albumin and gum acacia. (b) Repeat (a), but do not shake the mixture. Is an emulsion produced ? Place some of this mixture under the microscope and observe the disintegration of the oil-drops into small globules. How does this compare with the emulsification of fats in the intes- tine? 4. A pancreatic extract may be made in the following way. Take a pig's pancreas or a dog's pancreas which has lain for twenty-four hours at the room temperature. Cut into small pieces or run through a meat grinder. Crush in a mortar with twice its volume of glycerin. Place the mixture in a bottle and allow to stand for several days before using. For use, strain the mixture through a fine cloth and dilute as needed with four or five times its volume of water containing so- dium carbonate to make the mixture distinctly alkaline. Prepare a series of test-tubes for digestion experiments as follows : A . Place a small quantity of fibrin in a test-tube, adding the alkaline diluted glycerin extract of pancreas until the tube is half full. B. To another tube containing fibrin add the glycerin extract diluted with water, alone, and without the addition of the alkali. C. To another portion of fibrin add glycerin extract of pan- creas and dilute with o.i-per-cent HC1. D. To another portion of fibrin add some glycerin extract which has been boiled, and dilute with o.i-per-cent sodium car- bonate. E. To another fibrin portion add o.i-per-cent sodium carbon- ate, alone. Prepare a second series of tubes in the same way, substituting commercial pancreatin for the glycerin extract. Place all these tubes in a water-bath at a temperature of 38° C. From time to time make an observation of the changes which may be going on in the various tubes. After a half -hour, in which tubes LABORATORY MANUAL OF PHYSIOLOGY. has solution of the fibrin taken place ? At the end of an hour, what is the condition of the fibrin in the various tubes ? In those tubes in which digestion of the fibrin has taken place, what difference can be made out, by direct observation, between the action of pancreatic and gastric juice in their attack upon pro- teids? Filter the contents of those tubes which have shown digestive change and test for albumins, proteoses, and peptones. Place some of the filtrate in a dialyzer, and the following day test the fluid surrounding the dialyzer for proteoses and peptones. 5. Amylolytic Action of Pancreatic Juice. — (a) Make up some starch paste. Test it with Fehling's solution to make sure of the absence of a reducing sugar. Mix some of this paste with dilute pancreatic extract of neutral reaction. Label this tube A. In a second tube of starch paste, B, place pancreas extract of al- kaline reaction. To a third tube of starch paste add pancreas extract made acid with o.i-per-cent HC1. Label this tube C. To a fourth portion of starch paste add o.i-per-cent HC1 alone. Label D. To a fifth portion add strong hydrochloric acid. Label E. To a sixth portion add o.i-per-cent sodium-carbonate solution. Label F. Place all these tubes in the water-bath at 38° C. In ten or fifteen minutes test for reducing sugar in all the tubes, first carefully neu- tralizing those of an acid reaction. Record results. In which tubes has starch digestion taken place ? What is the starch-digest- ing enzyme of the pancreatic secretion ? What is the optimum re- action of the digesting medium ? (b) Mix some starch paste with pancreatic extract which has been previously boiled. Place in the warm water-bath and note results. W7hat is the effect of high temperature on the enzyme ? Is this effect common to all enzymes ? 6. Lipolytic Action of Pancreatic Juice. — Mix a small quan- tity of fresh butter in a test-tube with glycerin extract of pancreas SECRETION— DIGESTION— ABSORPTION. and o.i-per-cent sodium carbonate. Place the mixture in the warm water-bath. What change occurs in the butter ? After a time can you detect the odor of butyric acid ? If so, what is its significance ? 7. Action of Bile. — You will be supplied with ox-bile. What is its reaction ? (a) Test for bile pigments as follows (Gmelin's reaction) : To a little bile on white porcelain add a few drops of fuming yellow nitric acid. Note the changes in color from green to blue, yellow, and brown-yellow. The test may also be done by placing a drop of bile on white filter paper and bringing a drop of the acid in contact with it. Color rings will be formed at the junction of the bile and the acid. (b) Pettenkojer's Test for Bile Acids. — Mix some ox-bile in a test- tube with a small amount of strong sulphuric acid. Test the tem- perature of the mixture with a thermometer, adding the acid slowly. The temperature should not be higher than 70° C. or lower than 50° C. Now add a ic-per-cent solution of cane sugar, slowly, drop by drop, stirring with a glass rod. A red coloration indicates the presence of bile acids. This reaction is masked by using an excess of sugar or too high a temperature, since the sugar is decomposed and colors the mixture a dark brown. (c) Mix some fresh butter in a test-tube with a few cubic centi- metres of ox-bile. Mix a portion of butter in another tube with bile and pancreatic extract. Place in the warm water-bath and note results. 8. Absorption of Fat. — Starve a cat for twenty-four hours and kill. Open the abdomen and note the condition of the mesenteric lymphatics, the lacteals. Open a loop of small intestine. Scrape off some of the mucous membrane. Tease some of the scrapings, on a slide, with glycerin, after previous immersion in osmic acid J per cent for twenty-four hours. Remove the excess of glycerin with filter paper, and, covering the preparation with a cover slip, ex- amine under the microscope. If fat-droplets are present in the epithelium, they will be stained black or dark brown by the osmic acid. Note results. LABORATORY MANUAL OF PHYSIOLOGY. Feed another cat, after a starvation period of twelve hours, with fatty meat or milk and cream. Kill in ten hours after the meal. Prepare the intestinal mucous membrane, as before, with osmic acid. Also note the appearance of the lacteals. Can you demon- strate the presence of fat in the intestinal epithelium ? VII. MECHANISM OF PANCREATIC SECRETION. (DEMONSTRATION.) i. Narcotize a medium-sized dog with morphine sulphate and anaesthetize with ether. Expose and introduce a cannula into the trachea. Expose, isolate, and secure both vagus nerves with un- tied ligatures. Connect the tracheal cannula with the artificial re- spiratory apparatus and anaesthetic flask. Through a median ab- dominal incision expose the duodenum and head of the pancreas. Slit the duodenum longitudinally for an inch or more in the neigh- borhood of the head of the pancreas. Place a cannula in the larger pancreatic duct through its duodenal opening, on a level with the lower border of the pancreas. Connect this with a long glass tube, bent so as to pass over the edge of the abdominal wound. Fill this tube with physiological salt solution. To the rubber membrane of a transmitting tambour cement a light aluminum shovel-shaped lever. Arrange the transmitting tambour in connection with a recording and a slow drum. Bring the end of the pancreas-cannula tube over the shovel lever of the tambour. Each drop from the end of the cannula will then depress the lever of the transmitting tambour, and this will be transmitted to the recording tambour and a record will be written on the paper of the revolving drum. During the continuation of the experiment the exposed abdom- inal viscera should be protected by cotton pads soaked in warm o.8-per-cent NaCl solution. After completion of the operative procedures, note the rate of flow of pancreatic juice for a period of ten or fifteen minutes. Now inject into the duodenum or jejunum 30 c.c. of a o.4-per-cent HC1 SECRETION— DIGESTION— ABSORPTION. solution. Note the rate of pancreatic secretion for another period of fifteen minutes. Allow ten minutes' rest. Then divide both vagus nerves and stimulate with a weak tetanizing current. Note any change in the rate of pancreatic flow. Repeat the injection of the acid. Is the same effect produced that occurred after the first injection ? 2. Action of Secretin (Bayliss and Starling). — That the chief stimulus to the elimination of pancreatic secretion is a chemical one was demonstrated by Bayliss and Starling. They have found that there is a substance present, in the mucous membrane of the upper part of the small intestine chiefly, which they have termed prosecretin. This, in the presence of hydrochloric acid, or other acids to a less extent, is changed to another substance, se- cretin, which is absorbed, passes to the pancreas, and there acts as a stimulus to the elimination of pancreatic juice. An active secretin extract is prepared as follows: Cut out a dog's duodenum and jejunum. Slit open the bowel. Wash thoroughly with water. Scrape off the mucous membrane. Grind this with sand and a little o.4-per-cent HC1 in a mortar. Add three times its volume of o.4-per-cent HC1 and allow to stand for fifteen to twenty minutes. Bring to a boil in a porcelain capsule, and while boiling neutralize and render slightly alkaline with strong NaOH. Acidify slightly with acetic acid, strain through muslin, and filter. Isolate and introduce a cannula into the central end of the fem- oral vein of the dog of experiment i. While a record of flow of the pancreatic juice is being taken, introduce into the vein 5 c.c. of the secretin extract prepared as described above. Note the re- sult on the rate of elimination of the pancreatic secretion. Repeat the injection several times, allowing a period of rest be- tween injections. CHAPTER VII. INTERNAL SECRETIONS. I. LIVER, GLYCOGEN. i. SELECT a well-nourished rabbit. Kill quickly by a sudden blow upon the back of the neck in the region of the medulla. As speedily as possible, open the abdomen and remove a portion of the liver. Cut this into small pieces. Place some of these pieces in boil- ing water; and place one piece immediately on the holder of a freezing microtome and freeze the piece. (a) Make a number of sections with the freezing microtome, and as soon as cut place in Lugol's solution containing iodine and po- tassium iodide. Allow the sections to remain in the staining solu- tion for two or three minutes. Wash in water to remove excess of the iodine solution. Mount in glycerin and examine under the microscope. The glycogen present in the liver cells will be stained a mahog- any red by the iodine. Compare this reaction with that given by dextrin with iodine. (b) Allow some of the rabbit's liver to remain in the incubator at body temperature for an hour or longer. Cut frozen sections of a piece of this and treat with iodine as before. Is there any gly- cogen reaction ? (c) Grind some of the incubated liver with sand in a mortar. Add two or three volumes of water. Allow to stand for several minutes. Strain through muslin and filter through paper, and test for reducing sugar with Fehling's solution. (d) To prepare glycogen, take the pieces of liver which have been immersed in the boiling water, grind them in a mortar with [140] INTERNAL SECRETIONS. fine sand, return to the capsule, and boil again for a short time to make certain that the diastatic power of the liver has been de- stroyed. Add ten volumes of water slightly acidulated with acetic acid. Strain through muslin. To remove the proteids, concen- trate the liquid to a third its volume and add alternate drops of HCl and potassium mercuric iodid until precipitation ceases. Filter off a little of the liquid and test the filtrate for proteids. If there are none present, strain all of the liquid through muslin and filter through paper. To the filtrate add two volumes of alcohol, stirring well. Allow the precipitate, glycogen, to settle, decant off the supernatant liquid, filter the residue, and wash with dilute al- cohol. Transfer the residue to a beaker, cover with absolute alco- hol, and set aside for an hour. Remove the alcohol and dry the residue between folds of filter paper. To some of the glycogen thus prepared add 25 c.c. of water and warm gently. A solution is formed. Compare the appearance of this solution with that of soluble starch. Test a little of the glycogen solution with iodine and KI. Note the color reaction. Does it disappear upon heating ? If so, does it reappear upon cooling ? Test some of the solution of glycogen with Fehling's reagent. Is there any reduction of the copper salt brought about ? To a little of the glycogen solution add some saliva. After a few minutes test with Fehling's reagent for reducing sugar. II. PANCREATIC DIABETES. Effect of Removal of the Pancreas on Carbohydrate Metabolism. — Collect the urine of a medium-sized dog and test for reducing sugar with Fehling's solution. In all probability none will be found. Starve the dog for twelve hours. Inject subcutanrously 0.12 gram of morphine sulphate. Place dog, back down, on the operating- board and continue the anaesthesia with ether. Prepare sterile medium and heavy silk suture material. Sterilize a number of absorbent cotton pads in physiological salt solution. Sterilize instruments by steam or by boiling. LABORATORY MANUAL OF PHYSIOLOGY. Clip and shave the hair over the * midabdominal region and wash the skin with corrosive sublimate, i to 1000, followed by al- cohol. Clean the hands by thorough scrubbing and immersion in the corrosive-sublimate solution. Open the abdomen by an incision in the median line. Locate the head of the pancreas at the duodenum and the tail at the spleen. Carefully separate the pancreas from the omentum and mesentery and from the duodenum, tying the pancreatic ducts and all ar- teries and veins with double ligatures and cutting between. Having controlled hemorrhage in this way, remove the pancreas in tola. Bring the peritoneum and abdominal muscles together with one row of interrupted sutures and close the skin wound with a second row of sutures. Dry the wound with alcohol and ether and paint with collodion. After the animal has recovered from the anaesthetic and the im- mediate shock of the operation, collect the urine every six hours and make quantitative estimations of the sugar by Fehling's method. Also, carefully note the condition of the dog, as to loss of weight, temperature, appetite, weakness, convulsions, etc. The diet may be the same as that given previous to the operation. Make an es- timate of the carbohydrates, proteids, and fat of the diet. If the dog survives more than forty-eight hours after the oper- ation, the diet may be changed to pure proteid and a known amount of raw pancreas given with the food. Immediately after the death of the animal, the liver should be removed and tested for glycogen as described for experiment I. III. THYROID. 1. Ablation of the Thyroid in Dogs.— This is best done in two operations. At the first operation one lobe of the thyroid is removed, and at the second the other lobe. Select a young dog. Record weight. Place under morphine narcosis. Shave and scrub skin of the neck and wash with a bi- chloride solution, i to icoo. The operation should, of course, be INTERNAL SECRETIONS. done with strict asepsis. The instruments should be boiled, liga- tures sterilized, and hands cleansed and soaked in the bichloride solution. Expose the trachea through a median cervical incision, carrying the incision as far as the thyroid cartilage. Pull aside and sepa- rate, by blunt dissection, the longitudinal neck muscles from the thyroid lobe of one side. This is seen as an oval, reddish mass. With blunt hooks and scalpel handle separate it from its attach- ments. Tie all blood-vessels with two ligatures and cut between. The thyroid branch of the carotid artery should not be tied too near its origin, since there is danger of the ligature slipping later, and secondary hemorrhage. If an isthmus is present connecting the two lateral lobes, this also should be removed. The wound is now cleansed with sterile o.8-per-cent NaCl solu- tion, dried, and closed by two rows of interrupted silk sutures, one to draw the muscles together over the trachea, the other to approx- imate the skin. Cover the wound with a thin layer of sterile ab- sorbent cotton and paint with collodion. Keep the animal under careful observation for ten days or two weeks, recording the weight daily. At the end of this time, repeat the operative procedure and remove the remaining thyroid lobe. Keep under careful observation, recording the weight daily, taking the temperature per rectum, and counting the red and white cor- puscles of the blood. Note all symptoms and keep a careful record until death occurs. If the animal does not die or show the charac- teristic symptoms of thyroid removal, all the thyroid has not been removed or there are accessory thyroids present. This may be de- termined at autopsy later. At autopsy the condition of all the organs should be determined and the field of the operation examined to see if the thyroid re- moval was complete and if accessory bodies are present. These are sometimes found in the neck region near the thyroid lobes proper, or in the mediastinum. If found, they should be hardened, embedded, sections cut, stained, and examined for thyroid struct- ure LABORATORY MANUAL OF PHYSIOLOGY. 2. Thyroid Feeding after Thyroid Removal. — Remove the thyroid of another dog in the same way as in experiment i , and at about the same time, so that the symptoms of the two animals may be compared. The second dog should be fed, after the com- plete removal of the gland, with fresh sheep's thyroids, or thyroid extracts may be mixed with the food. What are the symptoms of thyroid removal in the dog ? How d< they compare with those stated to occur in similar conditions in man ? How do they compare with the symptoms of thyroid dis- ease in man? Has thyroid feeding any effect in alleviating the symptoms following thyroid removal in the dog ? If the dog fed with thyroids survives, it should be killed later and careful search made for accessory bodies. IV. SUPRARENAL GLANDS. 1. Ablation of the Suprarenal in the Rabbit. — Demonstra- tion.— Select a large, well-nourished rabbit. The Belgian hare serves well for the purpose. Starve for twenty-four hours. The operation is done in two stages, one suprarenal being removed at the first operation, and the other two weeks later. The left suprarenal is removed most readily by the abdominal route. The right suprarenal is reached best by the dorsal route without enter- ing the peritoneal cavity. Inject under the skin 9 cgm. of morphine sulphate. Strap the rabbit, back down, upon the rabbit-board. Clip and shave the hair from the midabdominal region. Wash with bichlorid. Sterilize instruments by boiling. Sterilize absorbent cotton pads in o.8-per- cent NaCl solution. Beginning just below the xyphoid appendix of the sternum, make an incision in the midabdominal line, through the skin, fascia, and peritoneum. The length of the incision should be about three inches. Cover the intestines and hold them back with absorbent cotton pads wrung out in hot salt solution. Let an as- sistant hold back the edges of the abdominal wound with retrac- tors. Locate the left kidney. Follow the renal vein to its junction INTERNAL SECRETIONS. with the inferior vena cava. A little above the angle formed by these two veins and closely hugging the inferior vena cava, the left suprarenal capsule will be seen. This is a yellowish-white oval body, lying behind the perito- neum. Its blood supply is variable. There are generally one large artery and vein entering the hilum of the gland, and these must be tied before the gland is excised. With small blunt hooks and a blunt-pointed seeker tear through the overlying peritoneum. Separate the gland from its surround- ings slowly and carefully, being especially careful to avoid injury to the inferior vena cava. Remove all cotton pads from the abdominal cavity. Bring the muscles and peritoneum together with one row of silk sutures, and the skin with another row of sutures. Cover the wound with some antiseptic powder and apply a gauze bandage. Place the rabbit in its cage and observe it carefully twice a day for the first two days and then make daily observations. At the end of two weeks the remaining gland may be removed. In the mean time make an ex- tract of the gland as described below. 2. Weigh the suprarenal which you have removed. Grind it in a mortar with fine clean sand and a little o.y-per-cent NaCl solu- tion. Add of the salt solution enough to equal ten times the weight of the gland. Transfer to a tightly stoppered bottle and place in the incubator at 30° C. for fifty-six hours. Strain through muslin and filter through paper. One part of this extract will be equivalent to y1^ of the fresh gland. 3. (a) Prepare another rabbit for a blood-pressure experiment. Isolate both vagi and secure them with untied ligatures. Prepare one jugular vein for injection. Take a normal blood-pressure tracing. While the tracing is being recorded, inject into the jug- ular enough of the suprarenal extract to be equivalent to -f^ of the fresh gland. Note the effect on the heart-beat and on blood pressure. What is the duration of the effect of the in- jection ? (b) Repeat the injection, and immediately after cut both vagus 10 [ US ] LABORATORY MANUAL OF PHYSIOLOGY. nerves. Compare the effect of the second injection upon heart- beat and blood pressure with the result of the first injection, when the vagus nerves were intact. (c) Repeat, using in place of the extract of the rabbit's suprarenal, i c.c. of a i to 10,000 solution of adrenalin chlorid. The effect of the suprarenal extract upon skeletal muscle has already been shown under Muscle-nerve Physiology. 4. Remove the second suprarenal from rabbit i as follows: Place the animal under morphine and ether. Tie, belly down, upon the operating-board, placing a pad under the abdomen for the purpose of bringing the viscera into the field of the operation. Shave and cleanse the skin of the right flank. Beginning at the lower border of the ribs, make a longitudinal incision through the skin and fascia of the flank about three inches long and about two inches from the spinous processes of the vertebrae. Cut through the lumbar aponeurosis of the abdominal muscles, and separate these from the heavy spinous muscles. Locate the kidney. Let an assistant, standing on the right side of the operating-table, hold the kidney down and to the right with one finger, and with two fin- gers of the other hand pull the lateral margin of the wound up and out. The operator should stand to the left of the subject. Follow up the renal vein as before, until the right suprarenal body is seen. Separate this from its attachments in the same way as was done in removing the left suprarenal. Remove the gland en- tire, or, if this cannot be done with safety, remove as much as pos- sible and crush the remainder with forceps. Sew up the wound with two rows of sutures and return the rabbit to its cage. Since death frequently occurs within the first twelve hours following com- plete removal of the suprarenals, the operation is preferably done in the morning, so that the animal may be under observation all day. If the operation has been successfully performed with the minimum of shock and hemorrhage, the animal should regain con- sciousness. Make careful note of all symptoms from the time of the operation until death occurs. INTERNAL SECRETIONS. At the autopsy examine all the organs and make careful search for accessory suprarenals and for any stump remaining after re- moval of the glands. Compare the symptomatology of the rabbit minus suprarenals with the symptomatology of Addison's disease in man. CHAPTER VIII. RESPIRATION. I. RESPIRATORY MOVEMENTS IN MAN. 1. Direct Observation. — Let one of the students of a labora- tory group, preferably one of slender build, strip to the waist. Let him sit straight on a stool with the arms and hands symmetri- cally placed at the sides, and both sides of the chest evenly illumi- nated. (a) Observe first whether or no the two sides of the chest are of equal size and the respiratory movements equally prominent on the two sides. Note the increase in the lateral and antero-posterior diameters of the thorax in inspiration. Note the movements of the abdominal wall in inspiration and in expiration. Note the changes in the intercostal spaces during the respiratory movements. What muscles seem to be involved in quiet inspiration ? in quiet expiration ? In which part of the thorax is the lateral diame- ter enlarged most, during quiet inspiration ? What are the move- ments of the ribs during expiration and inspiration ? Demonstrate these movements on the skeleton thorax. Remembering the attachments of the external and internal in- tercostal muscles, demonstrate their action on the skeleton thorax by the use of heavy elastic bands attached to the ribs in the same way as the intercostals are attached. (b) Now let the subject of the observation make a forced in- spiration, followed by a forced expiration. Compare this with the movements of quiet respiration. What additional muscles are in- volved in inspiration ? in expiration ? (c) By means of some form of pneumograph or stethograph record the respiratory movements. This may be conveniently [148] RESPIRATION. done by strapping to the chest a rubber bulb connected with a re- cording tambour whose lever is allowed to write on a medium fast drum. A time tracing in seconds should also be taken. First record the respiratory movements while the subject is in the recumbent position. Take the pulse rate at the same time. Allow the subject to sit, and record the respiratory movements again. Repeat the record with the subject standing. Compare the rate and character of breathing, together with the pulse rate, in the three postures. Compare the inspiratory phase with the expiratory phase. What is the ratio between the two ? Under what pathological conditions may this ratio be disturbed and in what way ? What is the rela- tion between rate of heart-beat and respiratory rate ? Under what pathological conditions is this ratio disturbed ? (d) Let the subject take some form of exercise for a few min- utes, such as running up a flight of stairs. Record respiratory movements again. Compare with other tracings. Observe rate of heart-beat. (e) While a tracing of the respiratory movements is being taken let the subject take several swallows of water in rapid succession. While the swallowing is going on, what is the effect on the respira- tory movements ? To what is this effect due ? 2. Respiratory Sounds. Auscultation. — In order to distin- guish abnormal respiratory sounds in pathologic pulmonary con- ditions it is necessary to be familiar with the normal. There are normal individual variations which must also be taken into ac- count. It is therefore well to examine a number of normal chests. (a) Vesicular Breathing. — With a stethoscope, first listen, dur- ing quiet respiration, at about the fifth or sixth right intercostal space. A sound more distinct and of longer duration on inspira- tion will be heard. The character and quality of this sound is hard to describe. It is best compared, perhaps, to the sound made when the leaves of a tree are stirred by a light breeze. What is the ratio of inspiratory sound to expiratory sound? Compare this with the ratio of inspiration to expiration. [J49] LABORATORY MANUAL OF PHYSIOLOGY. (b) Bronchial Breathing. — With a stethoscope listen to the breathing over the trachea and over the second costal cartilage on the right side. The latter position is at the bifurcation of the right bronchus. The sounds heard here are due to the passage of air through a tube and in part over the mouth of a tube. This is a fair example of normal bronchial breathing. Compare with ve- sicular breathing. Should bronchial breathing be heard where you listened for vesicular breathing ? What change in the lung might give rise to bronchial breathing ? (c) Listen over the right lung during forced respiration. How do the respiratory sounds differ from those of quiet respiration ? (d) Now go over the chest systematically, first one side and then the other, comparing the two. Start at the supraclavicular space of one side, then listen in the same region on the other side, and so on over all of the anterior, lateral, and posterior aspects of the thorax. Note any differences in the sounds in the different regions of the chest either in intensity, or duration, or character. Explain. 3. Palpation. Vocal Fremitus. — Place the same hand suc- cessively over the various regions of the chest both in front and behind, alternating sides, so as to compare one region with its fel- low of the opposite side. While the hand is applied to the chest wall let the subject say " twenty-one " or some other number. The vibrations of the chest wall during phonation are distinctly felt by the hand. There are pathologic variations of this, either in an ac- centuation of the fremitus on one side as compared with the other, or in a diminution of the fremitus. Listen again over the various regions of the chest while the sub- ject counts "one, two, three." The sound will be transmitted to the ear, but more as a murmur, and not as distinctly enunciated syllables. In certain pathologic conditions the spoken or whis- pered word is very distinctly heard, as if it were spoken directly into the end of the stethoscope. What physical changes in the lung might cause this condition ? 4. Percussion. — Laying the middle finger of the left hand be- tween the ribs, in the intercostal spaces, and using the bent mid- RESPIRATION. die finder of the right hand as a hammer, percuss the entire chest wall. Explain the variations in the percussion note over various regions. Map out the heart and the liver. 6. Chest Measurements. — (a) Let a student strip to the waist. Measure the chest circumference at the axillae at the end of quiet inspiration and at the end of quiet expiration. Repeat this measurement at the level of the end of the sternum. Re- peat both measurements, in forced inspiration and expiration. Record results. (b) In the same individual measure the antero-posterior tho- racic diameters at the junction of the first and second pieces of the sternum and at the end of the sternum, during inspiration and ex- piration, both quiet and forced. Measure the changes in the lat- eral diameters in the same way. A pair of long, graduated calipers serves for the purpose of making the measurements. Record results. For purposes of comparison, measure the length of the trunk from the "vertebra prominens" to the level of the chair upon which the subject is sitting. Repeat these measurements on other students and keep a record for each individual. 6. Respiratory Capacity. — This is usually determined by some form of water spirometer. A long, narrow cylinder, gradu- ated in cubic centimetres, is filled with water and inverted over water in another cylinder. An air tube passes through the second cylinder to the top of the first. The first cylinder is counterpoised by weights and pulleys, so that when air is forced into it the water is displaced, the cylinder rises, and the amount of water displaced by air can be read off on the attached scale. (a) Calibration of Spirometer. — The air cylinder of the spirom- eter should be calibrated before using. This may conveniently be done in the following way: Fill the cylinder with air; note the position of the pointer on the scale; place the outlet air tube under a graduated 1000 c.c. cylinder, filled with water and inverted over water, in a large pan or tub ; slowly depress the spirometer cylin- der until the pointer has passed five or ten spaces of the scale, and read off the amount of water displaced from the graduated cylin- LABORATORY MANUAL OF PHYSIOLOGY. der in the tub", record and repeat the operation until all the spirom- eter scale is estimated in terms of cubic centimetres of the testing graduated cylinder. (b) Tidal Air. — With the pointer of the spirometer at the zero mark of the scale, expire into the spirometer cylinder after an or- dinary inspiration. The amount of expired air recorded is an ap- proximate indication of the tidal air, i.e., the amount that passes in and out of the lungs during quiet respiration. (c) Supplemental Air. — Repeat the spirometer record, taking a normal quiet inspiration, and then forcing as much air out of the lungs as possible. Read the record on the spirometer scale. Sub- tract the reading of (b) from' the latter reading. The difference is the so-called supplemental or reserve air. This is the amount of air which remains in the lung after a quiet expiration and which may be expelled by a forced expiration. After this is expelled, air still remains in the lung which cannot be forced out. This is the residual air. The air which can be inspired in addition to the or- dinary inspiration is known as the complemental air. (d) Vital Capacity. — Take the deepest possible inspiration, and empty the lungs as completely as possible into the spirometer cylinder by a forced expiration. The record obtained indicates the full pulmonary capacity minus the residual air, and is equal to the sum of the tidal air, complemental air, and supplemental air. This is known as the vital capacity. Record the vital capacity of each member of your group, and compare with the various chest measurements already taken. 7. Cardio-pneumatic Movements. — The changes in volume of the heart during systole and diastole cause corresponding changes in the capacity of the thorax and consequently of the lungs. The inspiratory and expiratory movements caused by the heart-beat may be demonstrated in the following manner: Bend one end of a medium large piece of glass tubing into the form of a U. Fill the bend of the U with a little water colored with eosin. Place the end of the horizontal limb of this tube in one nostril. Close the other nostril with the finger and keep the mouth closed. Hold the RESPIRATION. breath. The fluid in the U will move synchronously with the heart-beat. At each systole there will be an inspiratory move- ment, and at each diastole an expiratory movement. II. PULMONARY PRESSURE. Place a rabbit under morphine narcosis. Anaesthetize lightly with ether. Place, back down, on rabbit-board. Expose the trachea through a median cervical incision. Introduce a tracheal cannula. Connect this, through a T-tube, with the proximal end of a mercury manometer. Place a clip on the rubber tubing, leading from the T to the manometer, so that it can be either opened or closed. At the beginning of an inspiration, open the manometer clip and close the air-inlet limb of the T. At the height of inspira- tion, close the clip on the manometer tube and open the air-inlet tube. The inspiratory negative pressure may then be read off from the manometer scale. A tracing of the respiratory movements may be obtained in this way by partially occluding the air-inlet tube. Close the air-inlet tube during several respiratory cycles. Note the positive pressure of the expiratory phase and the negative pressure of the inspiratory phase. After a few respiratory efforts the animal will begin to struggle because of asphyxia. When this occurs, note the change in the character of the respirations and the great difference between inspiratory and expiratory pressure. In quiet respiration, is the positive pressure in expiration much above the atmospheric pressure ? III. INTRATHORACIC PRESSURE. Using the same rabbit as in the previous experiment, connect the proximal limb of a manometer, through a piece of pressure tubing, with a small glass tube. Make a small incision through the skin over the fourth intercostal space on the right side. Make a very small nick through the intercostal muscles and force the glass tube through this opening. The tube should fit so tightly that there will be no leakage of air around it. Note the movements of the mercury in the manometer with inspiration and expiration. Com- LABORATORY MANUAL OF PHYSIOLOGY. pare them with the changes of intrapulmonary pressure. In quiet breathing, what is the constant condition of pressure in the thorax ? How does this pressure change with inspiration and expiration? Explain. Now close the tracheal cannula and induce asphyxia. Note the variations in intrathoracic pressure during the violent respiratory efforts which occur in this condition. For the influence of the respiratory movements on the blood pressure and heart-beat, refer back to your blood-pressure tracings, and study again the respiratory waves of the tracings. When is the intrathoracic pressure lowest? when highest? What effect would changes of pressure within the thorax have upon the pressure of blood in the large arteries and veins and in the heart itself dur- ing diastole ? IV. THE VAGUS NERVE IN RESPIRATION. The same rabbit that was used in the previous experiments may be used for this. Continue the dissection of the neck region very carefully, isolating both vagus nerves, and both superior laryn- FIG. 37.— To Record Movements of Diaphragm. JR, Recording tambour ; B, rubber bulb, connected with recording tambour ; D, diaphragm ; L, liver. geals. Make an incision through the abdominal wall below the xyphoid appendix of the sternum, just large enough for the intro- duction of a catheter over the end of which a collapsed rubber bal- loon is tied. Pass this up between the diaphragm and the liver, on RESPIRATION. the right side. Connect the catheter with the recording tambour (see Fig. 37), and the movements of the diaphragm will be recorded upon a drum revolving at medium speed. (a) First take a tracing of the normal movements of the dia- phragm. Take a time tracing in conjunction with the respiratory tracing. Note the rate of the respirations in the rabbit. These may be influenced by the morphine which has been previously given the rabbit. (b) While the tracing is being taken, tickle the rabbit's nose with a feather. Is there any effect on the respiratory movements ? What nerve has been stimulated ? Note the movements of the rabbit's nostrils with inspiration and expiration. These movements continue, even though breathing is no longer taking place through the nose, but through the tracheal cannula. Other associated movements you have already noted, e.g., the opening and closing of the glottis. (c) Pinch the skin or pour a little ether on the shaved abdom- inal surface. What effect has cutaneous stimulation on respiratory movements ? (d) Now tie one vagus with two ligatures and cut between. Mark on the tracing the time at which the vagus was tied and cut. Note any change in the respiratory movements occurring at the time of tying and cutting the nerve or following this operation. If the respiratory rhythm has been disturbed, does it return to nor- mal, after a time ? (e) Allow "five or ten minutes to elapse and then stimulate the central end of the cut vagus with a weak tetanizing current. What is the effect on the respiratory movements ? (/) Stimulate the central end of the cut vagus with a medium strong tetanizing current. Compare this effect with that obtained with the weak stimulation. (g) Apply to the central end of the cut nerve a few crystals of NaCl. Note the change in the respiratory movements. After the effect of this stimulus is sufficiently evident, cut off the small piece of nerve to which the salt has been applied. LABORATORY MANUAL OF PHYSIOLOGY. (h) Stimulate one superior laryngeal nerve with a weak tetaniz- ing current. Note the effect on the respiratory movements. Stim- ulate with a stronger current and note the effect. (i) Now tie and cut the other vagus nerve. Both vagi are now severed. What is the effect on the rhythm, rate, and depth of the respiratory movements ? (y) Stimulate both central ends of the cut nerves with a weak tetanizing current. Note the effect on the rate, rhythm, and char- acter of the respirations. Stimulate with a stronger current. Result? (k) Stimulate both vagi with a weak tetanizing current, and, when the effect of such stimulation begins to show, stimulate both superior laryngeal nerves with a medium strong current. Result ? In all of the above experiments, marks should be made upon the tracings to indicate the operative procedures and their time relation to the tracing; the nerves stimulated and the strength and nature of the stimulus employed. (/) Stimulate the peripheral ends of the divided vagus nerves. Is there any effect on the respiratory movements ? From the above experiments what conclusions can you draw concerning the function of the vagus nerve in relation to respira- tion ? Is the vagus chiefly an afferent or an efferent nerve in re- lation to the lungs? The vagus has now been studied, in part, in connection with the circulation, digestion, and respiration. Compare its functions in relation to the three systems. It contains both afferent and effer- ent fibres for circulation, digestive tract, and respiratory tract. Summarize your knowledge on the subject, secured in part through your own experiments in the laboratory. V. INNERVATION OF THE DIAPHRAGM. i. Still using the same rabbit that was used in the previous ex- periments, expose the phrenic nerve of the right side. This may be done in the following manner: Enlarge the cervical incision to RESPIRATION. the upper end of the sternum. Pull the sterno-mastoid and other longitudinal neck muscles toward the median line. Pull the skin and other muscles to one side and expose the cervical spinal nerves and -the beginning of the brachial plexus. The carotid artery, vagus nerve, and jugular vein should also be pulled toward the median line. Determine the position of the fourth, fifth, sixth, and seventh cervical nerves. Arising by filaments from the pos- terior divisions of these nerves, a fine nerve fibre will be seen run- ning over the heavy spinous muscles, parallel with the spinal col- umn and disappearing under the clavicle. To make sure that you have found the phrenic nerve, place fine platinum electrodes under this nerve filament and stimulate with medium strong single-in- duction shocks. The diaphragm recorder which records the move- ments of the right side of the diaphragm will move with each stimulus. 2. Pass a thread around the upper origin of the nerve. Tie the nerve and sever all connection with the cervical nerves. Note the diminution or complete loss of recorded movement of the dia- phragm. The right side of the diaphragm is paralyzed and moves only as it is pulled upon by the side which is still active. Compare the thoracic breathing after the section of one phrenic with the thoracic breathing before the section of the nerve. Do the two sides of the chest move equally, or is there a differ- ence between the right and the left side ? 3. Stimulate the nerve with single shocks from an inductorium, and take a tracing on the drum. 4. Enlarge the abdominal opening and pull down the abdom- inal viscera so that the movements of the diaphragm may be ob- served directly. Note the movement of the left side, whose nerve supply is still intact. Note the lack of motion on the right side and the position of this enervated side of the diaphragm during in- spiration and expiration. 5. Expose and cut the left phrenic nerve. Both sides of the diaphragm are now paralzyed. Note the change in the respira- tory movements. If the rabbit is young there will be great difn- [157] LABORATORY MANUAL OF PHYSIOLOGY. culty in breathing, amounting to distinct dyspnoea. Note the in- crease in thoracic breathing. Explain the difficulty in breathing following the paralysis of the diaphragm. Is it due simply to the loss of the active movements of the diaphragm in enlarging the vertical diameter of the thorax? 6. Place both phrenic nerves on electrodes from the inducto- rium. Stimulate with a medium strong tetanizing current, inter- rupted about thirty times per minute. Is the dyspncea relieved ? Does the rabbit cease attempts at thoracic breathing ? 7. Close the trachea by means of an artery clamp and kill the rabbit by asphyxiation. Note all phenomena connected with death from this cause. After breathing has ceased, open the thorax and observe the heart. Is this still beating? Note the color of the blood. Note difference between the two sides of the heart. Excise the heart. Does the heart-beat recover for a short time after ex- cision ? VI. EFFECT OF BLOOD TEMPERATURE ON RESPIRATION. 1. Heat. — Narcotize a rabbit. Place, back down, on the rabbit- board. Through a median cervical incision expose both carotid arteries and isolate the vagus nerves. Arrange the apparatus for recording the movements of the diaphragm. Isolate as much of each carotid as possible. Separate the artery from the nerves running with it, by several layers of paper. Tie each carotid, gently, to a small tubing running parallel to the artery. One end of this tubing is connected to a rubber outlet tube. The other end is connected to a rubber inlet tube. The inlet tube leads from a bottle filled with water kept at a temperature of 40 C., by immer- sion in a water-bath. This bottle is elevated sufficiently to give a constant flow of warm water through the tubes in contact with the arteries. The blood passing through the carotids is therefore warmed two or three degrees above the normal. Set up the arrangement as above described. First take a normal respiratory tracing. Then, while a tracing is being taken, let the warm water run through the tubing and note the effect upon res- RESPIRATION. piration. Change in the pulse rate may also be observed by watch- ing the pulsation of the carotid arteries or by feeling the cardiac impulse on the chest wall. How are the respirations affected when the blood going to the brain is warmed above the normal? How is the heart-beat affected? What is the relation between heart- beat, respiratory rate, and rise of temperature ? 2. Cold. — Disconnect the warm-water bottle from the tubes in contact with the carotids, and substitute a receptacle containing ice water. First, let the respiratory rate return to normal. Then while a tracing is being taken, allow the cold water to flow through the tubing in contact with the arteries. Note the effect upon the respiration and the heart-beat. Compare the tracing obtained through the cold application with that obtained when heat was applied. What is the effect of warmed blood upon the respiratory centre ? of cold blood upon the respiratory centre ? VII. EFFECT OF ANEMIA UPON THE RESPIRATORY CENTRE. 1. Using the same animal as in the previous experiments, clamp both carotids with artery clips. Is there any change in respiration following the occlusion of both carotids ? If so, does this changed respiration continue or does it soon return to the normal? Ex- plain. Does occlusion of the carotids materially or permanently diminish the blood supply to the brain? What is the collateral circulation ? What would be the only way completely to cut off the blood supply to the brain ? 2. Tie both carotids as high up as practicable. Introduce a glass cannula into each artery. Take a normal respiratory trac- ing. While this is being taken, open the clips on both arteries and allow the animal to bleed to death. Collect the blood in a grad- uated cylinder and record the phenomena observed after the loss of each additional 10 c.c. of blood. How much blood is lost before the respirations are affected? In what way are the respirations affected as hemorrhage continues ? [159] LABORATORY MANUAL OF PHYSIOLOGY. Note also every few seconds the condition of the various re- flexes. Among these try the reaction of the pupil to light, the con- junctival reflex, the reflex upon tickling the nares, and cutaneous reflexes. Note the time at which each one of these reflexes disap- pears. Note time when struggling begins. How do these respira- tory phenomena differ from, and resemble, those of asphyxia ? VIII. RESPIRATORY CENTRE. Narcotize a rabbit lightly with morphine. Anaesthetize with ether. Expose the trachea and both carotid arteries through a median cervical incision. Introduce a cannula into the trachea and tie both carotids. Change the position of the animal so that it lies on the rabbit- board belly down. Make an incision in the median line through the integument of the skull from the root of the nose to the occiput. Pull the skin flaps to one side, exposing the parietal bones of the skull. Make two trephine openings, one through the parietal bone of each side, enlarging the openings with cutting forceps, until the entire skull cap is removed. Be careful in crossing the median line not to injure the longitudinal venous sinus. 1 . Open and lay back the dura on each side, thus exposing both cerebral hemispheres. With a blunt spade or scalpel handle crush both cerebral lobes. Control the hemorrhage by packing with cotton moistened with adrenalin i to 10,000, or use the actual cautery. Observe the respiratory movements before, during, and after this operation. Do respirations continue after the removal of the cerebrum? Is the controlling respiratory centre located, there- fore, in the excised portion of brain ? 2. Continue the median dorsal incision until all the cervical ver- tebrae are exposed. Continue the removal of the skull cap until the cerebellar hemispheres are exposed. Remove the cerebellar hemispheres in the same way that the cerebral lobes were re- moved. Do the respiratory movements cease in the absence of the cere- [160] RESPIRATION. bellum? Is the controlling respiratory centre, therefore, located in the cerebellum ? 3. Divide the upper part of the medulla by an incision between the atlas and skull. Do the respiratory movements continue ? 4. Divide the cord on a level with the origin of the seventh cer- vical nerve. In the rabbit the respirations are altered but little, since in this animal breathing is chiefly diaphragmatic. By this section the thoracic muscles are cut off from the respiratory centre, but the innervation of the diaphragm is still intact. Impulses are still carried to the centre from the periphery by the intact vagus nerves. Other afferent impulses are in the main cut off from the centre by its isolation from the brain above and the cord below. 5. Expose and cut both vagus nerves in the neck region. Note the change in the character of the respiratory movements and the disturbance of the respiratory rhythm. Are the respiratory move- ments which still continue sufficient to sustain the life of the animal ? Allow the first stages of asphyxia to occur. Then revive the an- imal with artificial respiration continued for a short time. 6. Place the central ends of the divided vagi on electrodes from an inductorium set up for medium strong tetanizing currents. Stimulate the vagi with this current, interrupted about thirty times per minute. Stop the artificial respiration. Is the respiratory rhythm re-established ? Compare the results obtained from this experiment with those obtained through section and stimulation of the vagi in former experiments. What conclusions can you draw concerning the location of the respiratory centre and the regulation of its rhythmic activity ? IX. CONDITION OF LUNG FOLLOWING SECTION OF BOTH VAGI. Narcotize and anaesthetize a rabbit. Under aseptic precautions expose and cut both vagus nerves in the neck region. Sew up the wound and return the animal to its cage. Make careful observa- tions of the subject until death occurs, which will generally be within forty-eight hours. Determine the cause of death at au- ii [161] LABORATORY MANUAL OF PHYSIOLOGY. topsy. Observe particularly the condition of the lungs. Save pieces of the lung tissue for hardening, embedding, and sectioning. Stain sections with haematoxylin and eosin and study with the microscope. What pulmonary condition follows section of the vagus nerves and how is it brought about ? Save these sections for comparison with sections of human lung showing areas of lobar and lobular pneumonia. X. ARTIFICIAL RESPIRATION. The student should be familiar with at least one good nethod of artificial respiration for use in emergencies. One of the best methods for this purpose is the so-called Sylvester's method. It consists in imitating, so far as possible, the normal respiratory movements. In applying this method the operator should assure himself that the respiratory passages of the subject are free. The subject is placed upon his back, the shoulders being elevated by some support placed beneath them. The head should be on a lower level than the feet. The operator should stand at the head of the subject and, grasp- ing the wrists, flex the forearm upon the arm and press both arms firmly against the sides of the chest, pressing down and in on the chest at the same time. This motion forces air out of the lungs. When the pressure upon the chest is released, the thorax through its own elasticity rebounds to its original capacity, and air, by this motion alone, is drawn into the lungs. The thoracic diameters are still further increased by the second part of the operation. This consists of extending the arms and pulling them above the head, giving an extra tug when the position of full extension has been reached. The accessory respiratory muscles, mainly the pectorals, are thus put on the stretch and in their turn pull up and out on the upper part of the thorax. After this has been accomplished, the first position is again as- sumed and expiration is brought about. This alternate forced' ex- piration and inspiration are continued at the rate of fifteen to twenty RESPIRATION. per minute, until the subject begins to make respiratory efforts of his own or no doubt remains that life is extinct. XI. ESTIMATION OF CO2 AND H2O EXPIRED IN A GIVEN TIME. The estimation of carbon dioxid and water expired by a small animal in a given time may be conveniently done by each group of students. Absorption tubes, made for the purpose, may be FIG. 38.— Apparatus for Estimating the Water and Carbon Dioxid Eliminated from the Lungs of a Small Animal. Described in text. bought at a moderate price, as well as the respiratory chamber with air inlet and outlet and tightly fitting stopper. A schematic sketch of the apparatus used is given in Fig. 38. The chamber C is intended for holding some small animal, such as a rat or a mouse. This is tightly stoppered, the stopper being perforated for the passage of two tubes — (a) the air-intake tube, which passes nearly to the bottom of the chamber, and (b) the air- outlet tube, which begins near the upper part of the chamber. The air-intake tube is connected with two absorption tubes — i , contain- ing pumice stone soaked in sulphuric acid, for absorbing the moist- ure from the air which passes to the respiratory chamber, and 2, containing soda lime for absorbing the carbon dioxid of the in- spired air. The air outlet tube is also connected with two absorption tubes — 3, containing sulphuric acid for absorbing the water of the expired air, and 4, containing a strong solution of sodium hydrate for ab- LABORATORY MANUAL OF PHYSIOLOGY. sorbing the carbon dioxid of the expired air. This tube also has a bulb containing CaCl2. Tube 4 is connected with the water pump P. Air is drawn through the system of tubes and respiratory chamber in the di- rection of the arrow. 1. Set up the apparatus as described, and allow the air to be drawn through for fifteen or twenty minutes or until the respira- tory chamber has been freed from moisture and carbon dioxid. Now place the mouse in the chamber, stopper quickly, and weigh. Also quickly weigh tubes 3 and 4. Make a record of the weights for use later. Connect up the absorption tubes with the chamber and air pump, and allow a current of air to pass through the system for twenty minutes to one-half hour. Then disconnect and weigh the chamber and absorption tubes again. The difference between the two weighings of the respiratory chamber indicate the loss, in part, of the animal, in water and CO2. The difference between the weighings of the absorption tubes in- dicates their gain in water and CO2, respectively, and the actual output of the animal in carbon dioxide and water, during the time of the experiment. The difference between the two weigh- ings of the respiratory chamber and the difference between the two weighings of the absorption tubes do not correspond, since what the animal has lost in CO2 it has partly regained in oxygen. The gain in oxygen may be roughly determined by subtracting the difference between weighings of chamber C from the difference between weighings of tube 4. The ratio between the oxygen ab- sorbed and the carbon dioxid expired is known as the respiratory quotient. What is the respiratory quotient for the mouse experimented upon? 2. Remove the mouse from the respiratory chamber. Ventilate the chamber again. Prepare and weigh a new set of absorption tubes. Give the mouse some form of exercise, such as moving the treadwheel of a squirrel cage. Place in the chamber and quickly [164] RESPIRATION. weigh. Determine the CO2 elimination for another period equal to the first period in time. How does the CO2 elimination of the second period compare with that of the first? What is the difference in the respiratory quotient ? Repeat these experiments, using a cold-blooded animal instead of the mouse. The frog will serve as a type. The mouse is an animal of high respiratory activity. The frog is an animal of low respiratory activity. What is the reason for a higher rate of gas exchange in the mouse than in the frog ? Study the nature of the respiratory movements hi the frog. How do they differ in mechanism from those of mammalia? How is respiration carried on in fishes and in gilled amphibia ? What points in common have these three orders of animals as far as respiration is concerned ? What points of difference ? A fair idea of the function of the circulation in respiration may be obtained from a study of the circulation through the gill of Necturus. CHAPTER IX. EXCRETION. IT is assumed that the chemical examination of the urine, both for normal and abnormal constituents, has already been done by the student under the direction of the department of chemistry. The chemistry of the urine, therefore, will not be taken up here. This chapter is limited to an outline of a few experiments deal- ing with the method of urine secretion and excretion. 1. Movements of the Ureter and Bladder. — Narcotize a rab- bit, lightly, with morphine. Anaesthetize with ether, just suffi- ciently to keep the animal quiet. Prepare absorbent-cotton pads soaked in hot physiological salt solution for protecting the abdominal viscera after the abdomen has been opened. Open the abdomen in the median line. Con- tinue the incision to the symphysis pubis, so as to expose the bladder. Note the form of this organ and its relation to the surrounding viscera. If the bladder is full, stimulate it by mechanical irrita- tion or by the application of a tetanizing induced current. Note the character of its contraction. Does it continue to contract and empty itself after the original stimulus has ceased to act ? Collect the urine and save for examination and comparison in color, clearness, specific gravity, reaction, and constituents, with the normal urine of man. Open the bladder and locate the entrances of the two ureters. Observe these, for a time, for the passage of urine into the bladder. Trace the left ureter to the kidney. Dissect this out from its bed, so that the kidney, ureter, and bladder are easily observable. Observe the movements of the ureter. What is their nature? How do they compare with the movements of the intestines? [«S6] EXCRETION. What is the normal direction of the ureter movements ? Can the movements be induced in response to a mechanical or electrical stimulus ? Are the movements rhythmical or irregular ? 2. Urine Flow. Kidney Volume. — Introduce a fine glass can- nula into the ureter, near the bladder or through the ureteral opening into the bladder. The speed of urine flow may be re- corded by allowing the drops from the end of the cannula to fall upon a lever, made for the purpose and connected with a tambour membrane. This tam- bour is connected, through rubber tubing, with a second tambour whose lever is arranged to write upon the smoked paper of a slowly revolving drum. The changes in volume of the kidney may be determined by means of a plethysmograph arrangement known as an oncometer. The oncometer, as generally used, consists of a metal jacket lined with some membrane for enclosing the kidney. There is an open- ing for the passage in and out of the kidney vessels — artery, vein, and ureter. The space between the membrane and the jacket is filled with oil. This space is connected through tubing with a piston recorder whose lever is arranged to write upon the smoked paper of a revolving drum. In this way a curve of kidney volume is written. A simple form of air oncometer is shown in Fig. 39. The kidney is partly encased in a rubber balloon inflated with air. The changes in pressure in the balloon are transmitted, through rubber tubing, to a recording tambour or bellows re- corder. The bellows recorder devised by Brodie is far preferable to the tambour as a recorder of volume changes. It can be easily made hi the laboratory and consists of two rectangles, hinged with thin FIG. 39.— Oncometer, Simple Form. B, Metal jacket ; O, opening for kid- ney vessels; C, rubber balloon, in- flated with air, partly surrounding the kidney and connected through T with a recording tambour. LABORATORY MANUAL OF PHYSIOLOGY. leather, the base being made of vulcanite or wood and perforated for the entrance of the inlet tube. The top consists of a light aluminum frame covered with paper. The sides are made of peritoneal membrane, varnished with a dilute solution of boiled linseed oil to make them airtight.1 Place the kidney in the oncometer, cover the exposed abdominal viscera with the warm cotton pads moistened with physiological salt solution, and arrange the recording apparatus for writing on a medium slow drum. Arrange the recorder for urine flow under the kidney-volume recorder. The urine should also be collected for examination, later. Be careful that the ureter, the renal artery, and renal vein are not obstructed by kinks. Keep the ureter from drying by moistening, from time to time, with physiological salt solution. Observe and record the changes in kidney volume and urine flow for a period of twenty minutes or one-half hour. Is the rate of urine flow constant during this time? Are there any changes in the volume of the kidney? How do urine flow and kidney volume correspond? 3. Blood Pressure and Kidney Volume. — Expose the carotid artery, vagus nerve, depressor nerve, and jugular vein. Introduce cannulae into the artery and vein. Pass thread loops around the nerves for convenience in handling. Connect the artery with the mercury manometer. Record blood pressure on the same drum used for recording kidney volume and urine flow. Note the cor- respondence between the changes in blood pressure and changes in kidney volume. (a) Divide one vagus nerve. Stimulate the peripheral end with a tetanizing current sufficiently strong to cause inhibition of the heart-beat. Note the effect upon the volume of the kidney. (b) Allow the blood pressure to recover from the effect of the vagus stimulation. Now stimulate the depressor nerve with a medium strong tetanizing current until a marked depressor effect is obtained. Note the effect on kidney volume and urine flow. 1 For further details see Journal of Physiology, vol. xxvii., p. 473. [168] ' EXCRETION. (c) Allow the blood pressure to return to normal. Allow the animal to inhale a few. whiffs of amyl nitrite. Note the effect upon blood pressure, kidney volume, and urine flow. (d) After the blood pressure has again returned to normal, in- ject into the jugular vein one cubic centimetre of a i to 10,000 solution of adrenalin chlorid. What is the effect upon the blood pressure, kidney volume, and urine flow ? (e) After the blood pressure has returned to normal, connect the vein cannula with a burette containing warm physiological salt solution. Being careful that there are no air bubbles in the con- necting tubes, allow the solution, under low pressure, to run slowly into the vein. Run in fifty cubic centimetres of the solution. Is there any noticeable rise of blood pressure ? Explain. Is there any change in kidney volume or urine flow ? (/) Run into the vein fifty cubic centimetres more of the solu- tion. Note any effect upon blood pressure, kidney volume, or urine flow. (g) Repeat the perfusion, using fifteen cubic centimetres of a i-per-cent urea solution. 4. Intravenous Injection of Dextrose. — Using the same rab- bit as in the previous experiment, or a fresh animal if necessary, prepare a i -per- cent dextrose solution. Warm this to body temperature and slowly inject twenty cubic centimetres of this solution into a vein. Collect the urine eliminated before and after the sugar injection. Test both with Fehling's solution for reducing substances. Collect samples of the urine every ten minutes after the beginning of the sugar injection. When does the sugar first appear in the urine ? When does it cease to ap- pear in the urine ? 5. Intravenous Injection of Albumin. — Test the urine for al- bumin. If there is none present, inject, into a vein, ten to fifteen cubic centimetres of a i-per-cent solution of egg albumin in physio- logical salt solution. Examine the urine for albumin at intervals of ten minutes. [169] LABORATORY MANUAL OF PHYSIOLOGY. 6. Intravenous Injection of Peptone. — After the disappear- ance of the albumin from the urine, prepare a 2-per-cent peptone solution. Inject 10 c.c. of this solution into the vein. Collect the urine for ten minutes or until enough has been eliminated for testing. Saturate the urine with ammonium sulphate. This precipitates mucin, albumin, and urates. Filter and test the nitrate for pep- tones by means of the biuret reaction. Do peptones occur, normally, in the blood stream ? If not, what becomes of the peptones that are absorbed from the gastric and intestinal mucous membranes ? Is peptone ever found as an ab- normal constituent of the urine ? Under what pathological con- ditions may peptonuria occur? 7. Effect of Peptone on the Coagulation of the Blood. — Iso- late and introduce a cannula into one carotid artery. Open the clamp on the artery and collect 10 or 15 c.c. of blood in a small test tube. Note the time taken for solidification of the shed blood. Now inject 15 to 20 c.c. of the peptone solution into the vein. In three or four minutes open the artery clamp again ; allow 2 or 3 c.c. to escape, and then collect in a small test tube 10 to 15 c.c. of blood. Compare the coagulability of this second sample with that of the first portion of blood shed. What is the effect of pep- tone upon the coagulability of the blood? CHAPTER X. SENSATION. AN organism is brought into relation with its environment through its irritability to external stimuli. This property, of irri- tability, is common to all protoplasm. As the organism increases in complexity from single-celled individuals to individuals con- sisting of groups of cells, this property of irritability or sensa- tion becomes differentiated into a variety of sensations, depending upon the part of the external surface or special end-organ stimu- lated and the nature of the stimulus. Conscious sensation first occurs, so far as we know, in those animals provided with a nervous system and brain. The sensory impulse is conducted over nerve pathways to the sensory portions of the cerebral cortex, and there interpreted in terms of sensation and corresponding judgments formed. All sensations occur as a result of some form of stimulus applied to the outer body envelope and its connection through afferent nerves with the centres of consciousness in the brain. For the re- ception and transmission of certain stimuli, the outer envelope has become markedly modified, as, for example, the receiving appara- tus for audition and vision. The localities for the reception of certain sensory impressions are limited to certain sharply defined areas. These include the end-organs of taste, smell, sight, and hearing. Others have a wide distribution over the entire cutaneous surface and, to a lesser degree, over the mucous surfaces. Such are the tactile sense, the sense of temperature, the pain sense, and the pressure sense. The so-called muscular sense also has a wide distribution. All parts of the body are brought into relation with the central nervous system through afferent or centripetal nerves. Only part LABORATORY MANUAL OF PHYSIOLOGY. of these sensations, however, are commonly brought into the realm of consciousness. The majority of such impulses, from the viscera, for example, are either lost, through diffusion in the subsidiary parts of the nervous system, or are transferred, as corresponding efferent impulses, to complete the formation of reflex arcs. When such impulses become abnormally intense, so as to over- come the resistance in the longer nerve pathways sufficiently to reach the realm of cerebral consciousness, the subjective sensations are either vague and indefinable, other than as a feeling of discom- fort or pain somewhere in the region involved, or they are referred, as pain, to some part of the cutaneous surface whose afferent nerve distribution corresponds to the same cord segment as the efferent nerve distribution of the visceral area involved. Such reference of a sensory impression occurs, probably, for the reason that the sen- sorium is in the habit of receiving impulses from the skin area and not from the visceral area ; and where the same terminal neurons transmit the impressions from the two sources, the sensation is referred to the area from which the impulses more usually come. The nature of the conscious impression depends, not so much upon the character of the stimulus applied, as upon the peripheral area stimulated, the afferent nerve involved, and the brain area to which the impulse goes. Thus, a stimulation of the optic nerve, whether it be mechanical, electrical, or through the impact of light waves upon the retina, causes a sensation of light; stimulation of the olfactory nerves gives a sensation of smell; and of the taste nerves, of taste. The cutaneous surface itself has been mapped out into areas or spots which are irritable to stimuli of various kinds. Thus, there are spots which respond to stimuli by a tactile sensation, others which are irritable to heat, others to cold, and others to stimuli which give a sensation of pain, independent of temperature or tactile sensation. Quantitative Relation between Stimulus and Sensation. — In order that a stimulus may be effective in producing a sensation, its intensity must exceed a certain minimum value. This minimum SENSATION. is sometimes spoken of as the threshold value of the stimulus. This threshold value is a variable quantity, varying for different individuals and for the same individual at different times. It de- pends partly upon the condition of the end-organ and the over- lying integument, and partly upon the receptivity of the sensory cerebral area involved. If the intensity of the stimulus is increased progressively above the threshold value, the intensity of the sensation increases also, up to a certain maximum, beyond which an increase in the strength of the stimulus produces no further increase in the intensity of the sensation. This maximum occurs with comparatively weak stimuli. The range of sensory variation is, therefore, not large. Between the maximum and minimum a variation in stimulus is accompanied by a variation in sensation. This variation cannot be measured by the subject of the sensation. He can tell that one stimulus is stronger or weaker than another, but not how much stronger or how much weaker. An increase of the stimulus above the maximum of sensory interpretation very rapidly fatigues the sense organ. Even with weak stimuli, the sensory apparatus rapidly tires. Weber's Law. — E. H. Weber, the first to make systematic obser- vations along these lines (1831), formulated the following conclu- sion, which has since been known as Weber's law: "An increase in a stimulus sufficient to call forth a conscious increase in the sensa- tion must always bear the same ratio to the original strength of stimulus to which it is added." For example, if to a weight of i it is necessary to add a weight J in order that the subject of the experiment may detect a differ- ence, then, if a weight of 10 is used, the added increment necessary to produce an increase of sensation will be 10 divided by 3. I. CUTANEOUS SENSATION. 1. Tactile Sense. — To map out the touch-spots in a certain region of skin, some form of instrument, known as an aesthesiom- eter, is used. A simple form of lesthesiometer is made by fasten- LABORATORY MANUAL OF PHYSIOLOGY. ing a hair at right angles to the end of a wooden handle by means of a bit of sealing-wax. If this hair is pressed, perpendicularly, against the skin, it will exert a certain pressure, depending upon the thickness and character of the hair. This pressure can be determined, for any hair, by pressing it against one scale pan of a balance and finding the largest weight that can be lifted in this way. (a) Prepare a number of hair aesthesiometers, using hairs of different lengths and thicknesses and estimating their pressure values. (b) Gently touch the end of a hair on the back of the hand. Note the sensitiveness of the hair as a touch organ. The end organs of touch are arranged in radiating lines about the roots of the hairs. The hairs act as levers, the long arm projecting above the skin surface and the short arm making pressure against the nerve endings. In this connection consider the so-called touch hairs of the cat and other animals. (c) Shave the skin of the back of the hand, and, starting at the hair follicle, map out the touch-spots in an area of 2 sq. cm. Start with a test hair of least pressure and increase the pressure until sensation is produced. The subject of the experiment should be blindfolded and instructed to say yes immediately upon feeling the application of the test object. Record the threshold value of the stimulus needed to produce sensation in this region. Record, also, the number of touch-spots present in the area of skin tested. What is the arrangement of the touch-spots in relation to the hair follicle? (d) Shave the skin of the back of the leg and map out the touch- spots and determine the threshold value of the stimulus, in the same way as was done for the skin of the back of the hand. (e) Repeat the experiment for the skin of the abdomen, near the median line and some distance from the median line. (/) Test the touch sensation of the skin of the back, over the shoulder. [174] SENSATION. (g) Map out the touch-spots of the cheek, starting near the lobe of the ear and proceeding to the angle of the mouth. In what part of this area are the touch-spots most numerous ? (k) Test the mucous membrane of the upper and lower lips in the same way. (i) Test the palmar surfaces of the finger tips; of the hand. How do the threshold values of the efficient stimuli, for the various regions tested, compare? Is there any relation between the number of touch-spots and the mobility of the regions tested ? (j) Repeat experiments (a) to (i), using, instead of the test hairs, a series of weights of the same surface area (about 4 sq. mm.) . Begin with a weight of 0.0005 gm. and increase until the sensation is obtained. How do the threshold values compare with those ob- tained with the test hair ? In the first series of experiments, single touch-spots were stimulated. In the second series, a number of touch-spots were stimulated simultaneously, the number varying with the region of skin to which the stimulus was applied. The efficacy of any particular stimulus will depend, to a large extent, upon the number of touch-spots in the area stimulated, the proximity of the end organs to the skin surface, and the deforma- tion of the skin effected by the stimulus. The temperature of the weights employed should be approximately that of the skin. (k) Take a number of pieces of flat cork, cut into strips about 3 cm. long, i cm. wide, and i cm. thick, and pass blunted needles through the ends of the strips so that the distance between the needle-points of the different pairs varies from i to 25 mm. With a blindfolded subject, test the ability of the skin of various regions to detect the application of the different pairs of needles as two separate stimuli. The subject should answer, immediately upon the application of the stimulus, one or two, as the sensation is that of one point or two points. In this way test the sensitiveness of the skin of the finger-tips, the back of the hand, the shoulder-blade, the forehead, the cheek, the lips, the tip of the tongue, the skin of the thigh in its long axis, the skin of the thigh in its short axis. LABORATORY MANUAL OF PHYSIOLOGY. In which of these areas are the two points of the test needles distinguished as separate, when brought nearest together ? Why ? In which axis of a limb are the two points of the test object most readily distinguished? Explain. Is there any after-sensation in any of the skin areas stimulated ? (i) Stimulation of the touch-spots, through pressure, occurs as a consequence of deformation of the skin. If a constant pressure is applied to all parts of a skin surface equally, there is no sensa- tion of touch. This may be accomplished by immersing the hand in a vessel of water at the same temperature as that of the skin. So long as the hand remains quiet and the water is still, there is no sensation of touch, except at the junction of air and water at the surface of the liquid. 2. Temperature Sense. — Temperature sensation is of two kinds, sensation of cold and sensation of heat. There are, ap- parently, separate sets of nerve fibres and endings for these two sensations, since certain skin areas are irritable to cold objects but not to warm, and others are irritable to objects warmer than the skin but not to cold stimuli. (a) These areas may be mapped out as so-called warm and cold spots by a method similar to that used in mapping out the touch- spots. Take a metal cannula, drawn to a fine point — a small ar- tery cannula may be used — and run hot water through it for a time. Choose an area of skin, about 4 sq. cm. on the back of the hand, for testing. Bring the tip of the cannula sufficiently near the skin to obtain the sensation of heat without mixing this with the touch sense. Mark those points where the heat sensation occurs, with a fine-pointed colored pencil. (b) Go over the same area of skin as in (#), running cold water through the cannula instead of hot as before. Mark the cold spots with a fine pencil of another color than that employed in marking the warm spots. (c) A rough estimate of the temperature sense, in various skin areas of the body, may be made by filling two test tubes of small calibre with hot and cold water, respectively. These are applied, SENSATION. alternately, to the same skin areas, and the sensation experienced is recorded and compared with that obtained in other skin areas. (d) With a pair of blunt-pointed dividers, determine the nearest distance between the points at which they are detected as two. Now warm the points and repeat the experiment. Is the distance increased or decreased at which the points are separately felt by the skin? 12 [177] CHAPTER XL VISION. I. DISSECTION OF THE EYE. 1. Appendages. — (a) Examine the specimen before you, tracing out the ocular and palpebral conjunctiva, noting the plica semi- lunaris and the caruncula. How do the latter compare in relative size with the human structures ? Locate and describe the puncta lachrymalia and the openings of the lachrymal ducts. How many are there? Is your specimen a right or left eye? (b) Observe carefully the appendages, locating the tarsal carti- lages, Meibomian, sebaceous, and lachrymal glands. Observe the recti and oblique muscles and their actions on the eyeball. Ob- serve the entrance of the optic nerve. 2. By pinning down the flaps of the conjunctiva, fix the eyeball to the board, the cornea downward. Then dissect out the four recti and two oblique muscles, observing the capsule of Tenon. Without injuring important vessels and nerves, remove the heavy retractor muscle. Locate and describe the vence vorticosce. How many are there? Find the anterior ciliary arteries. How many are there ? What structures do they supply ? Find the two long ciliary arteries, the short posterior ciliary arteries, and the ciliary nerves. 3. Eyeball. — (a) Fix the eyeball to the board, cornea up, pin- ning down the dissected muscles as guys. After having observed the cornea remove it with heavy scissors, near the corneo-scleral margin. (fy Through the opening thus made, examine the iris. Where is the posterior chamber ? (r) Holding the margin of the cornea with strong forceps, dissect VISION. the sclerotic coat free from the choroid, for about 3 mm. posterior to the angle of the anterior chamber. Between the insertions of the recti muscles, locate four points on the margin from which incisions may be made antero-posteriorly. From these points, make the incision posteriorly as far as the equator of the eyeball. Dissect each flap free from the underlying choroid. After having removed the pins fixing the recti muscles, draw the flaps back and fix. Observe the iridal and ciliary portions of the choroid. (d) With a fine forceps, grasp the margin of the iris and with small scissors cut out a sector with the ciliary body as a base. Study the posterior chamber, suspensory ligament, and the anterior surface of the ciliary body. (e) Make a circular incision with small scissors, severing choroid and retina at about the line of the ora serrata. Lift off from the vitreous humor the whole ciliary apparatus, placing it upon a plate, anterior surface downward. Observe the posterior aspect of the ciliary body. Describe the lens carefully, making a cross- section. Can you discern its capsule? (I) Observe the retina, as seen through the vitreous, locating the entrance of the optic nerve. Can you locate the f ovea centralis ? II. PHYSIOLOGICAL OPTICS. Light is propagated from a luminous point in every plane and in every direction, in straight lines. These lines of direction are called rays. Rays travel with the same rapidity so long as they remain in the same medium; the denser the medium the slower the passage of light through it. The divergence of the rays of light is proportionate to the distance from which they come. Rays of light proceeding from infinity are parallel. In dealing with rays of light which enter the eye, it will be sufficiently accurate to consider them parallel when they proceed from a point more than six metres distant. A ray of light, meeting with a body, may be absorbed, reflected, or, if the body is transparent, refracted. In dealing with the eye, it is necessary to consider only the latter. LABORATORY MANUAL OF PHYSIOLOGY. 1. Refraction. — A ray of light passing through one transparent medium into another of different density is bent or refracted, unless the ray falls perpendicular to the surface of the denser medium. The ray is spoken of as incident before entering the second medium, and emergent after leaving it. Upon entering a denser medium, the ray is refracted toward the perpendicular and from the perpendicular upon entering a rarer one. Reflection accompanies refraction, the ray dividing at the point of incidence. THE INDEX OF REFRACTION is the relative resistance of a sub- stance to the passage of light. Air is taken as a standard and is called i. The index of refraction of water is 1.3, of glass, 1.5. The diamond has the highest refractive index, which is 2.4. LENSES. — A lens is a transparent substance, usually glass, bound- ed by two curved surfaces, or by one plane and one curved surface. $ FIG. 40.— «, Object ; /', image ; n, nodal point ; L, lens ; F, F/, and/,/, conjugate foci. It may be regarded as a series of prisms. In a convex lens the bases are directed toward the centre, and in a concave lens the bases are directed away from the centre. Rays of light passing through a convex lens are made to converge. Those passing through a concave lens are made to diverge. The point to which rays converge, after passing through a con- vex lens, is its focus. The principal focus of a convex lens is its focus for parallel rays. When rays of light diverge from any point nearer than infinity, they are brought to a focus at a point beyond the principal focus. [180] VISION. The point from which they diverge and the point to which they converge are called conjugate foci. As one approaches the lens, the other recedes, and vice versa. By means of a candle, determine the principal focus of the con- vex lens before you. The foci of concave lenses for parallel or divergent rays are FIG. 41.- Described in text. virtual or negative. They are the points from which rays seem to diverge after passing through the lens (see Fig. 41, F). Parallel rays of light, falling upon a concave lens, are diverged. If these rays were traced backward, they would seem to diverge from a point nearer the lens (see Fig. 41, F). The conjugate foci of concave lenses are also virtual and found in a similar manner. Find them. Formation of images. The image of an object is the collection of the foci of its principal points. FIG. 42.— Described in text. SIMPLE DIOPTRIC SYSTEM. — The simplest form of a dioptric apparatus consists of two media of different refractive indices, [181] LABORATORY MANUAL OF PHYSIOLOGY. separated by a spherical surface. In such an apparatus, the optical properties depend upon the curvature of the surface and the refractive power of the media. Such an apparatus is shown in the accompanying figure (Fig. 42). The line pr represents a curved surface separating media of different refractive power, the lens being on the left The line oa, falling perpendicularly upon the surface at 3, passes through the centre of the sphere, 6. The line o-a is the optical axis. All the lines that cut the surface normally, such as o-dj c-y, and u-i, undergo no refraction and, continuing in straight lines, cross at 6, which is the nodal point. All of the rays are refracted. All rays, parallel to the optical axis, passing through the lens will be bent so as to meet at m, which is the posterior principal focus. The anterior principal focus is at b, in the first medium and in front of the lens. Rays of light, such as b-i-t, passing from it, are so refracted that they become parallel to the optic axis. The principal point is the point where the optic axis cuts the surface. The posterior, anterior, nodal, and principal points are the cardinal points of an optical system. THE EYE AS AN OPTICAL INSTRUMENT. — Having reviewed the general optical principles concerning the refraction of light and the formation of images by convex lenses, we now come to the eye as an optical instrument. Rays of light, as they enter the eye encounter not one refracting medium as in the simple dioptric system, but five, namely: Tears, Cornea, Aqueous humor, Lens, Vitreous humor. The indices of refraction of these various media are such that parallel rays of light, entering a normal eye, are brought to a focus upon the retina. For the sake of simplicity, they may be looked upon as equal to a convex lens of about twenty-three millimetres focus. However, a ray of light falling upon the cornea does not follow the same simple direction it would, were it to pass through a VISION. single medium. Instead, the eye must be regarded as a compound refracting system, composed of a spherical surface and a biconvex lens. The cardinal points are Two. principal points, Two nodal points, Two principal foci. In the diagram, Fig. 43, the cardinal points are shown all upon the optic axis, f-a. At 6, two principal points, situated so FIG. 43.— Described in text. close together in the anterior chamber that they may be regarded as one. At / is the first principal focus, and at a the second. The nodal points correspond nearly to the optical centre of the refractive system. Rays passing through these points are not refracted. They are situated about 7 mm. behind the cornea. THE FORMATION OF RETINAL IMAGES. A luminous point placed above the principal axis has its image formed upon the retina below this axis, and vice versa. Replace these points by an object and the same thing occurs. The retinal image is, as it were, a mosaic, composed of innumerable foci of the object. Construct a simple diagram of the human eye, showing the formation of an image, say an arrow or a candle, upon the retina. Is the image erect or inverted ? If inverted, why do we see it erect ? The human eye has aptly been compared to a camera, the refracting media representing the camera lenses, and the retina its sensitive plate. [183] LABORATORY MANUAL OF PHYSIOLOGY. ADAPTATION OF THE EYE FOR DISTANCE. In the camera, however, it is necessary to adjust the instrument by backward and forward movements of the lenses. In the eye, this adjustment is brought about by changes in convexity of the lens, or accommodation. Accommodation is, therefore, the func- tional adaptation of the eye to distance. If the entire optical apparatus of the eye were rigid and fixed, how could objects at various distances be seen clearly ? Explain what takes place when the eye accommodates. Take 'a sharp-pointed pencil in each hand. With one eye closed, hold the points in a direct line of vision before the other eye — one, about twenty centimetres distant, and the other a full arm's length. Focus on the nearer pencil. Is the image of the distant one clear ? Focus upon the farther pencil. Is the image of the nearer one clear? The near point, or punctum proximum, is the nearest point to the eye to which objects may be brought and still be seen clearly. It averages about 1 2 cm. At this point the accommodation is most active. Determine your own near point. The far point, or punctum remotum, is the farthest point at which objects may be seen clearly by the normal eye. The range of accommodation is the difference between the punc- tum proximum and the punctum remorum. Determine your own range of accommodation. ADAPTATION OF THE EYE FOR DIRECTION. As the eye can functionally adjust itself to distance, it can also change the direction of its visual axis from one object to another, or can follow objects moving within its field of vision. Two students may work together, one as observer and the other as subject. (a) MONOCULAR FIXATION. — The observer and subject being seated opposite each other, let the subject close or screen one eye. VISION. (i) Hold any small object directly in front of the subject and have him fix his eye upon it constantly. Move the object quickly toward the subject's left and notice the immediate fixation of the object in its new position. What muscles are brought into action, in this movement? (2) Move the object quickly to the right, upward, downward, and diagonally, noticing the immediate fixa- tion in all the fields. What muscles are brought into action in each position, and are all movements equally rapid ? (3) Bring the object exactly in front about one metre distance and note the range of lateral movement without causing any appreciable change in the visual axis. (4) Bring the object to the central position and move it very slowly outward in various directions and observe whether the changes of d, direction of the visual axes are equally slow and regular. (b) BINOCULAR FIXA- TION.— Convergence. — It was probably noticed dur- ing the above exercises that, though one eye was screened, it shared in all movements with its fel- low. With both eyes open, let the subject fix a small object, held about one metre distant. Let the observer move the object slowly in all fields, downward, upward, laterally, and around, observing the perfect continuous fixation with both eyes. What muscles or pairs of muscles are involved in the movements in the different directions ? If any variations are noticed in the subjects examined, describe them. STEREOSCOPIC VISION. — Binocular Single Vision. — By this is FlG. 44.- a, 6, Two objects, the images of which (a,, bl and a3, *,) fall on corresponding parts of both retinae, K and /?,. LABORATORY MANUAL OF PHYSIOLOGY. meant the union, in one single impression, of images received simultaneously on both retinae. The external ocular muscles maintain the visual axes parallel, so that impressions of an object fall on correspondingly identical points of both retinae (see Fig. 44)- What happens when paralysis of one ocular muscle destroys balance ? Show by diagram. CONVERGENCE. — Let the subject fix his vision upon an object within an arm's length, and then upon some object more than six metres distant but in the same line of vision. What change takes place in relation of the visual axes to each other? Hold an object one metre distant, directly in front of the subject. Move it directly toward the subject's eyes. Note the convergence of the visual axes. What change takes place in the pupil? What muscles are in- volved in the act? Recall the innervation of the pupil and the internal recti. OPTICAL DEFECTS. The eye is not a perfect optical instrument, since it is not exactly centred and possesses in small degree chromatic and spherical aberration. By chromatic aberration is meant that different rays of the spectrum are bent to different degrees. For instance, violet rays, which are more refrangible than red, have their focus near to the lens. In the manufacture of optical instruments, this is overcome by combining a convex with a plano-concave lens. Practically the same arrangement exists in the eye, and this, combined with the rapid accommodative ability of the lens, makes chromatic aberra- tion a negligible quantity. By spherical aberration is meant that rays of light which traverse the periphery of a lens are brought to a focus sooner than those which pass nearer the centre. The iris corrects this defect by acting as a diaphragm, shutting off the peripheral rays. [186] VISION. MISCELLANEOUS EXPERIMENTS. Blind Spot. — On a white card make a small black cross, and about 8 cm. away, in a horizontal direction, draw a black dot. Looking intently at the dot, one eye having been screened, hold the card about 25 cm. from the eye and then move it slowly toward the eye. At what distance does the cross disappear? Why? Imperfect Visual Judgments. — Draw one line (horizontal), 6 cm. long, and one perpendicular to the same length but not joining. Which line appears the longer and why ? Make three black dots on the same imaginary horizontal line and equidistant from each other. Mark them x, y, and z. Con- nect x and y with a series of equidistant dots of the same size. Which appear farther apart, x and y or y and z ? Draw two horizontal lines 5 cm. long. On the upper one make arrow-heads pointing toward each other. On the lower make arrow-heads pointing away from each other. Which line appears the longer? Draw three parallel lines. On the upper one draw a series of short, parallel intersecting lines, cutting the longer line at an angle. Do the same with the middle line, except to make the angle of intersection equal and opposite to that of the first series. Prepare the lower line the same as the upper line. Do the lines still appear parallel ? Sanson-Purkinje Images. — Darken the room. Hold a lighted candle a little to one side and in front of the subject's eye. The observer, looking at the eye from the other side, sees three images of the flame. The first and brightest is a small, erect image formed by the anterior convex surface of the cornea. The sec- ond, larger and less distinct, is formed by the anterior convex sur- face of the lens. The third, smaller, inverted, and indistinct, is formed by the posterior surface of the lens. Let the subject ac- commodate for a near object. Describe the change in relation that takes place in the size and clearness of the second image and its proximity to the first. LABORATORY MANUAL OF PHYSIOLOGY. Duration of Impressions. — On a circular white disc, mid- way between the periphery and centre, fix a small black ob- long disc. Rotate it rapidly. Note that a ring of gray appears on the black, showing that retinal impressions are of a certain duration. Inversion of Shadows. — Make three pinholes in a card, close together and arranged in a small triangle. Hold the card about 12 to 15 cm. from the right eye. Look through the holes at a bright light. Close the left eye and hold a pin in front of the right eye, so that it just touches the lashes. Note that an inverted image of the pin will be seen in each hole. Retinal images are inverted. Shadows are erect. Therefore the latter, upon being projected outward into space, are seen inverted. NORMAL VISION. Examination of Distant Vision. — The sense of sight consists of (i) form sense (acuity), (2) light sense, (3) color sense. The acuteness of direct vision is measured by means of letters, sized to certain definite standards. Those devised by Snellen are in most common use. Snellen determined the normal acuteness of vision to be the power of distinguishing letters subtending the visual angle of 5'. The letters are formed of strokes whose width is one-fifth the size of each letter, hence they are 'seen under an angle of i'. The openings in the letters, and the spaces between the contiguous strokes, are made to conform, as nearly as possible, to the same angle. The relation of the size of the letter to the distance at which it should be discerned by the normal eye is expressed by twice the tangent of half the angle of 5', or, 0.001425. The size of the letter, the perception of which constitutes normal vision at a given dis- tance, may be obtained by multiplying the distance by 0.001425. On this, the standard letters of measuring visual acuity have been built up. Practical experience, however, has shown that letters constructed under the angle of 5' do not always give the best visual acuity of [188] VISION. which the subject is capable; so that figures constructed on the 4' basis are gradually coming into use. For recording visual acuity, the formula V = — is used, V standing for vision; d, for the distance of the subject from the test type; D, the distance from which it should be read. In practice, the acuteness of vision is found by determining the smallest type the subject can read at six metres. Normal vision is represented by the symbol f . That is to say, that at six metres the subject reads the test line marked 6. If, at this distance, the subject can only read the line marked 12, his visual record would be T6^, and so on. In instances where the vision of the subject is lowered to the extent that he cannot see even the largest test types, vision is tested by the ability to count fingers at varying distances. For example, V — fingers, 3 m. If vision is still lower, V = shadows or light. Examination of Near Vision. — This includes the ability of the subject to read print. That is the condition of accommodation. The test types are those of von Jaeger and Snellen. Exercise. — Test the visual acuity of each other, recording your findings, both for distant and near. Light sense is the power possessed by the retina of appreciating variations in the intensity of light. It is measured by the photom- eter, which consists, essentially, of an apparatus by which the in- tensity of two sources of light may be compared. Color Sense. — This is the power the retina has of distinguishing or perceiving colors, or the impression resulting from the impact of light rays having different refrangibilities. Holmgren's test consists of testing the power of a subject to match various colored yarns. Three large test skeins, namely, (i) light, pure green, (2) rose-purple, (3) red, are given the patient, and also smaller skeins, comprising various shades and tints of each color. He is requested to pick out the colors similar to his original three skeins. If, for example, he is red-blind, he will not LABORATORY MANUAL OF PHYSIOLOGY. * red m the purple or related colors, but will dassify these te reds wul be confused with the greens. the ere, in a state of rest, „*• »- •» ^ , , , »• .,,_,,__ +» - - _ . . • .. • : . - ; t condition of (tar si-htedness) is that form of eye, when at rest, focusses parallel rays of It is corrected bj placing before the eye . Why? Make a diagram. arH9gfatedness) is that fonn of ametropia in which at T1p^tJ, fc* imy^ pnolifi ravs of i^ht in front of the It is Maintrtl bj pbring before flic eye a concave fens. Why? Make a dbgram. of ametropia in which the eye, when rajs of fight upon any one spot, rays of fight, coming through different to 2 mOOQS ffl CnKKflnCOK DHUICS* 'DCfDCD' depending upon the in which the rays of light, are equally refracted in all parts of Ac •rriifa». bat the refraction of at least two meridians is in which the various parts is that form in winch the rays are brought to a focus on through the otiier principal meridian a point back of the retina. Cempemd kyptropu astigmatism is that form in which the VISION. rays from both meridians are focussed back of the retina, but at two different points. (e) Simple myopic astigmatism is that form in which one merid- ian refracts rays to a point in front of the retina, and the other principal meridian fooisses rays upon the retina. (/) Compound hyperopic astigmatism is that form in which rays passing through both principal meridians are brought to a focus at different points in front of the retina. (g) Mixed astigmatism is that form in which one meridian fo- FIG. 4$.— Described in text cusses rays in front of the retina, and the other focusses them back of the retina. In simple hyperopic astigmatism the rays are focussed at 2 and 3 (see Fig. 45). In compound hyperopic astigmatism the rays are focussed at i and 2. In simple myopic astigmatism the rays are focussed at 3 and 4. In compound myopic astigmatism the rays are focussed at 4 and 5. In mixed astigmatism the rays are focussed at 2 and 4. (/;) Presbyopia is loss of accommodative power due to sclerosing of the crystalline lens. Although the process commences during the first year of life, the lens does not lose enough of its elasticity to interfere with near vision until about the age of fort}'. It is cor- rected by placing before the eye a convex lens. Why ? Make a diagram. CORRECTION OF REFRACTIVE DEFECTS. The Numbering of Lenses. — Lenses are measured according to their refractive power. A lens whose focal distance is one metre LABORATORY MANUAL OF PHYSIOLOGY. is taken as the unit of measure. It is numbered i, and is called one diopter (D). The refractive power of a lens is the inverse of its focal distance. Hence a lens of 2 diopters has a focal distance of 0.50. What is the focal distance of a lens of 4 D? Given a lens whose focal distance is 2 metres, what is its number ? Parallel rays of light, passing through a convex lens, are made to converge. Parallel rays of light, passing through a concave lens, are made to diverge. A cylindric lens is a lens, one or both surfaces of which are seg- ments of a cylinder. Rays of light, passing through it in a plane parallel to its axis, are not bent. Rays passing in a plane perpen- dicular to its axis, converge or diverge, according as to whether the cylinder is concave or convex. Lenses are designated plus (+) if they are convex, and minus ( — ) if concave. What forms of ametropia would cylinders correct ? OPHTHALMOSCOPY. The ophthalmoscope is, in its simplest form, a mirror with a hole in it. The first instrument of Helmholtz, in fact, consisted of three thin plates of glass, fastened together and mounted in a frame, at an angle of 56°. The whole object of the instrument is to il- luminate the ocular fundus by reflected rays of light and permit the observer to inspect the illuminated area. All patterns are useful. The patterns of Loring and Morton are most popular. Use. — Not all the light entering the pupils is absorbed by the pigmentary layer of the choroid. A certain amount returns from the eye. If, therefore, the observer's eye is placed in the same position as the source of illumination, or directly behind it, the interior of the eye becomes visible. This is the principle of the ophthalmoscope. The mirror, which gathers rays of light from a luminous point, becomes a secondary source of light which is pro- jected into the pupil. Methods.— There are two, direct and indirect. In the direct, the examiner places his eye close to that of the patient and looks [192] VISION. FIG. 46. — Use of Ophthalmoscope. Direct method. S, eye of subject ; £, eye of observer; M, mirror ; L, source of light. directly upon the enlarged and upright details of the fundus. In the indirect, the subject is at an arm's length, and a convex lens is placed between the sub- ject's eye and the exam- iner's mirror. The image, as obtained, is inverted and aerial. The two methods differ, practi- cally, in that the direct image is larger and erect, in a small field, while the indirect image is smaller and inverted, but in a larger field. The two methods are explained in the accompanying figures (46 and 47). Examine the fundus, both by the direct and indirect method, and make an outline drawing showing the disc and retinal vessels in each case. PERIMETRY. In contradistinction to visual acuity which is limited to the macula, the function of sight performed by the other parts of the retina is called indirect vision. The limits of the field of vision are best obtained by an instru- ment, the perimeter, but a fairly accurate map of a field, not larger than 45°, may be obtained on a blackboard. Exercise. — In the centre of the blackboard, in the line of direct vision, locate a dot, the point of fixation. Draw from the dot, as a centre, a series of circles whose distance from each other shall represent an angular distance of 10°. Now draw the meridians which will divide each quadrant into at least three subdivisions. A wooden guide, twenty centimetres long, should be provided LABORATORY MANUAL OF PHYSIOLOGY. to regulate the distance of the subject's eyes from the point of fixa- tion. The subject, with one eye screened, places himself directly in front of the point of fixation and twenty centimetres from it. Make a test object out of a piece of white paper, one centimetre FIG. 47.— Use of Ophthalmoscope. Indirect method. S, eye of subject; £, eye of observer; M, mirror; L, source of light; C, convex lens; /, image of fundus. square, and affix it to a black handle. Let the operator move the test object along one meridian, say the horizontal, from the pe- riphery toward the point of fixation. As soon as the subject sees the test object, make a chalk mark on the meridian, denoting the place where it is first seen. In like manner go over at least eight of the meridians. Join the points so obtained with a line, and the result is the approximate field for white. In the same manner map out the field for blue and red. Which field is the largest, that for white, for blue, or for red? Which is the smallest? DRUGS ACTING LOCALLY ON THE EYE. Those acting directly upon the eye are divided into (i) mydri- atics (dilators of the pupil), such as atropine, homatropine, cocaine, scopolamine, etc. (2) Myotics (pupil contractors), such as eserine and pilo- carpine. [194] VISION. (3) Cycloplegics (paralysants of the ciliary muscles), such as atropine, homatropine, scopolamine, etc. (4) Anaesthetics (local), such as cocaine, eucaine, holocaine. Of the mydriatics, the first four are in most common use. They differ from one another in intensity and duration of effect, in the order named. The mydriatics, as a rule, increase the intraocular tension. Exercise. — Instil a drop of homatropine into a subject's eye. What effect does it have on the pupil ? Upon accommodation ? Explain the action. Instil a drop of eserine in the same eye. What effect is pro- duced ? Instil a drop of cocaine into another subject's eye. Brush the cornea, lightly, with a wisp of cotton. Note the anaesthesia and the effect on the pupil. Which produces the more complete mydriasis, cocaine or homatropine? [195] INDEX. Aberration, chromatic, 186 spherical, 186 Absorption, 121 of fat, 137 Acceleration, heart-beat, 87 Accommodation, visual, 184 Action current of muscle, 35, 36 detection of, with tel- ephone, 38 of frog's heart, 137 reflex, 47 Adaptive sera, 78 Adrenalin, action on blood-vessels, H7> J45 on heart-beat, 92 on muscle twitch, 45 on shock, 117 on urine flow, 169 ^Esthesiometer, 173 After -load of muscle, 26 Albumin, intravenous injection of, 169 Albumins, differentiation of, 132 Albuminuria, 169 Algae, i Ametropia, 190 Amoeba, 5 influence of temperature on, 6 ingestion of foreign bodies by, 6 locomotion of, 6 structure of, 5, 6 Ampere, 14 Amyl nitrite, effect on blood pressure and kidney volume, 169 Amylolytic action of pancreatic juice, 136 Anelectrotonus, 40 Anode, 14 Anodic closing contraction, 43 opening contraction, 43 Apex beat of heart, 109 Apparatus, arrangement of, for clcc- trotonus, 39, 40 for study of ciliary mo- tion, 9 to show action of heart valves, 10 1 electrical, 13 Artificial respiration, 162 Ascending currents, 40 Asphyxia, 158 Astigmatism, 190 Atropine, action on heart, 88 on secretion of saliva, 124 Auricles, record of pulsation, 84 Auscultation, 149 Battery cells, arrangement of, 15 Bayliss and Starling on pancreatic secretion, 139 Bellows recorder, Brodie, 167 Bile acids, test for, 137 action of, 137 pigments, test for, 137 Binocular fixation, visual, 185 Biological introduction, i Biuret test, 131 Bladder, movements of, 166 INDEX. Blind spot, eye, 187 Blood, circulation of, 80 coagulation of, 56 action of peptone on, 170 action of reagents on, 57 calcium salts in, 57, 58 fibrin in, 56, 58 fibrinogen in, 58 corpuscles of, 59 defibrination of, 58 fresh, examination of, 63 haemoglobin of, 73 laking of, 58 microscopic examination of, 63 plasma of, 56 pressure, 105 and kidney volume, 168 effect of depressor-nerve stimulation on, 115 effect of hemorrhage on 116, effect of shock on, 117 effect of vagus - nerve stimulation on, 114 estimation of human, 118 record of in rabbit, 113 serum, 57 smears, 64 specific gravity of, 71 Brain, frog's, 47 pigeon's, 52 motor areas of, 53 Breathing, bronchial, 149 vesicular, 149 Brodie, bellows recorder, 167 Bulbus arteriosus, 85 Calcium salts, action on heart mus- cle, 94 in coagulation of blood, 57, 58 Canals, semicircular, 54 Capillary electrometer, 35 Carbon dioxide, influence on plant cells, 2, 3 influence on animal cells, 8 monoxide, haemoglobin, spec- trum of, 76 Cardiac nerves, extrinsic, 87, 98 dissection of, 87, 98 stimulation of, 88, 100 Cardiogram, 109 Cardiograph, 84, 109 Cardio-pneumatic movements, 152 Caruncula, 178 Cells, blood, 59 difference between animal and plant, 5 nerve, discharge of impulses from, 55 Centres, reflex, 49 vasomotor, 112 Cerebellum, removal of, 52 Cerebrum, motor areas of, 53 removal of, effect on reflexes, 48 in frog, 51 in pigeon, 52 Cervical sympathetic, section of, 115 Chemical stimuli of muscle and nerve, 22 Chest measurements, 151 Chloral hydrate, influence on re- flexes, 49 Chloroform, action on heart, 91, 94 Chlorophyll, i Chorda-tympani nerve, 123, 124 Chromatophores, i, 3 Cilia, work done by, 10, n Ciliary arteries, 178 motion, effect of CO2 on, n effect of other gases on, 12 effect of temperature on, 10 rate of, 9 98] INDEX. Ciliated epithelium, g, 10, n, 12 Circulation of blood, 80 artificial schema of, 102 in mesentery of frog, 81 in respiration, 165 in web of frog, 80 mechanics of, 102 Coagulation of blood, 56 Cocaine, action on heart, 89 Commutator, Pohl's, 39, 40 Compensatory pause of heart, 97 Complementary air, 152 Conjugation in plants, 3 Conjunctiva, 178 Contractions, muscle, Galvani's ex- periments, 34 paradoxical, 37 secondary, 34 summation of, 29 Convergence, visual, 186 Cord, spinal, diffusion of impulses in, 48 hemisection of, 52 reflex centres, 48 Corpuscles, blood, 56 action of reagents on, 62 classification of, 66 differential count of, 64 enumeration of, 59 frog's and mammalian, com- pared, 64 iodine reaction in, 65 staining of, 64 Curare, action of, 25 Current, ascending, 40 constant, as a stimulus, 38, 41 demarcation, 34 descending, 40 effect on nerve irritability, 38 galvanic, 13 of action, 35, 36, 38 of injury, 34, 36 Current, Pfliiger's laws, 41, 42 Currents, electrical, regulation of, 15 induced, 16 Cytolysis, 77 Dare, haemoglobino meter of, 68 Defibrination of blood, 58 Degeneration, reaction of, 44 Deglutition, mechanism of, 127 Demarcation current, 34 Depressor nerve, 100 effect of stimulation of, on kidney volume, 168 Descending current, 40 Dextrin, 126 Dextrose, 125 intravenous injection of, 169 Diabetes, pancreatic, 141 Dialysis, 136 Diaphragm, innervation of, 156 Digestion, 121 gastric, 130 intestinal, 134 salivary, 125 Digitalin, action on heart, 90 Diopter, 192 Dioptric system, 181 Dissection, frog's leg, 18 heart, 82, 97 Diuresis, 168, 169 Drum, arrangement of, for superim- posed twitches, 27 Drugs, action on heart, 88 action on muscle twitch, 45 Duct, lachrymal, 178 sub maxillary, 123 Elasticity of muscle, 21 Electrical stimuli, 23 Electrolytes, 14 Electrometer, capillary, 35 Electromotive force, 14 199 INDEX. Electrotonus, 38 Emmetropia, 190 Emulsification, 134 Epithelium, ciliated, 9 Ergography, 31 Erythrocytes, enumeration of, 59 Ether, action on heart, 91, 93 Euglena viridis, 6 Excretion, 166 Extrinsic cardiac nerves, 87, 88, 98, 100 Eye, adaptation for direction, 184 for distance, 184 appendages of, 178 as an optical instrument, 182 cardinal points of, 183 coats of, 179 dissection of, 178 drugs acting locally on, 194 humors of, 179 lens of, 179 posterior chamber of, 179 suspensory ligament of, 179 Eyeball, 178 Fatigue, effect on muscle twitch, 26 on tetanus, 36 of human skeletal muscle, 31 record of curve, 27, 28 Fats, absorption of, 137 digestion of, 134 Fibrin ferment, 58 formation of, 57 Fibrinogen, 58 Films, blood, staining of, 64 Flagellae, 6 Fleischl, v., hae mo meter, 69 Flow of current, 14 of liquids, 104 Foci, conjugate, 181 Fovea centralis, 179 Fraunhofer lines, 75 Fremitus, vocal, 150 Fungi, 4 Galvani, experiment with metals, 34 without metals, 13, 34 Galvanic cell, 13 current, 13 Gastric digestion, 130 juice, 132 Gastrocnemius-sciatic preparation, 18 Glands, lachrymal, 178 Meibomian, 178 salivary, changes in, following chorda stimulation, 124 dissection of, 122 sebaceous, 178 Glycogen, preparation of, 140 Glycosuria, 169 Gmelin test for bile pigments, 137 Haematin, 73 spectrum of, 77 Haematoporphyrin, 74 spectrum of, 77 Haemin, 73 Haemocytometer, Thoma-Zeiss, 59, 60 Haemoglobin, crystals of, 73 derivatives of, 73, 74, 75 spectra of, 75 estimation of, 68 reduced, 76 Haemoglobinometer, Dare's, 68 Talqvist's, 68 Haemolysis, 77 Haemometer, v. FleischFs, 69 Hammerschlag, estimation of spe- cific gravity of the blood, 71 table of comparison between specific gravity and haemo- globin, 71 [200] INDEX. Heart, action current of, 37 action of frog's, 82, 83 of rabbit's, 98 dissection of frog's, 82 of rabbit's, 97 excision of, 83 extrinsic nerves of, 86, 87 sounds, 109 valves, action of, 101 Heart-beat, action of drugs on, 88 direct observation of, 82, 98 graphic record of, 84 influence of temperature on, 83, 85, 118 nerve action on, 87, 100 reflex inhibition of, 88 Stannius' experiment, 95 Heart muscle, action of salts on, 94 compensatory pause, 97 maximal response of, 96 refractory period, 97 Helmholtz, ophthalmoscope of, 192 Hemisection of spinal cord, 52 Hemorrhage, effect on blood press- ure, 116 Holmgren, test for color vision, 189 Hooke's law, 21 Hyperopia, 190 Hypoglossal nerve, 122 Images, retinal, 183 Sanson's, 187 Impressions, visual, duration of, 188 Impulse, nerve, velocity of, 24 Induced currents, study of, 16, 17 Inductorium, 16 Infusoria, 6 Inhibition of heart-beat, 87, 88 Internal secretion, 140 Interrupters, 18 Intestines, digestion in, 134 Involuntary muscle, 45 Iodine, test for dextrin, 126 for glycogen, 140 for starch, 125 lodophilia, 65 Irritability of nerve and muscle, 22 and effect of constant cur- rent on, 38 Isometric contraction of muscle, 32 spring, graduation of, 33 Isotonic contraction of muscle, 33 Judgments, visual, 187 Kanthack and Hardy, classification of leucocytes, 66 Kathelectrotonus, 40 Kathode, 14 Kathodic opening contraction, 43 closing contraction, 43 Kidney volume, 167 volume and blood pressure, 168 volume and depressor stimula- tion, 1 68 volume, effect of vagus stimula- tion on, 1 68 Kronecker, apparatus for ciliary mo- tion, 9 interrupter, 18 perfusion of frog's heart, 93 water pen, 108 Lachrymal ducts, 178 glands, 178 Laking of blood, 58 Latent period of stimulation, 123 Lenses, 179 numbering of, 191 Leucocytes, classification of, 66 differential count of, 68 enumeration of, 61 migration of, 81 of frog's blood, 6 [201] INDEX. Leucocytes, of human blood, 64 staining of, 64 varieties of, 67 Light, influence on plant cells, 2, 4 refraction, 179 Lipolytic action of pancreatic juice, 136 Liquids, swallowing of, 130 Liver, formation of dextrose in, 141 glycogen of, 140 Load, muscle, 25 LugoPs solution, 140 Macrospores, i Manometer, mercury, 113 Maximum response of heart mus- cle, 96 Mechanism of circulation, 102 Medulla, destruction of, in frog, 48 Meibomian glands, 178 Metabolism, carbohydrate, after pan- creas excision, 141 effect of sunlight on plant, 3, 4,5 MethEemoglobin, spectrum of, 76 Microspores, i, 2 Migration of leucocytes, 81 Milk, digestion of, 134 Millon, reagent of, 131 Monocular fixation, 184 Mosso, ergograph of, 32 plethysmograph of, 108 Motor areas, stimulation of, 53 Muscarin, action of, on heart, 90 Muscle, action current of, 36 action of curare on, 25 actual shortening of, 31 demarcation current of, 36 elasticity of, 20 electric phenomena of, 34 fatigue of frog's, 26 fatigue of human, 31 Muscle, Hooke's law, 21 involuntary, 45 irritability of, to stimuli, 22, 23 isometric contraction of, 33 isotonic contraction of, 33 reaction of degeneration in, 44 secondary contraction of, 34 single twitch, action of drugs on, 45 form of, 23 influence of fatigue on, 26 influence of load on, 25 influence of temperature on, 26 summation of contractions, 29 of stimuli, 28 tensile strength of, 21 tension, influence on contrac- tion, 32 tetanus of, 29 tone, 37 volume of contracting, 28 work doneduring contraction, 31 Muscle-nerve physiology, 13 Muscles, dissection of frog's leg, 18, iQ Myopia, 190 Nerve cell, number of impulses dis- charged in given time, 31, 55 conductivity, influence of con- stant current on, 38, 40 electric phenomena of, 34 impulse, velocity of, 24 diffusion of, in cord, 48 irritability, to stimuli, 22, 23 influence of constant cur- rent on, 38, 40 stimulation of human, 42, 44 Nerves, cardiac, frog, 87 rabbit, 100 chorda tympani, 122, 123 [202] INDEX. Nerves, hypoglossal, 122 laryngeal, inferior, 128, 129 superior, 128 lingual, 122 optic, 179 vagus, 87, 100, 114, 128, 129 Nervous system, physiology of, 47 Newton's rings, 61 Nicotine, action on heart, 90 on nerve cells, 124 Nitric-acid test for proteids, 131 Ohm, 15 Ohm's law, 14 Oncometer, 167 Ophthalmoscopy, 192 Optical defects, 186 Optic lobes, removal of, in frog, 48 nerve, 179 Optics, physiology of, 179 Oxy-haemoglobin, spectrum of, 75 Pancreas, excision of, 141 Pancreatic diabetes, 141 juice, 135 action on fats, 136 action on proteids, 135 action on starches, 136 secretion, mechanism of, 138 Palpation, 150 Paramcecium, 7 Pasteur fluid for yeast, 4 Pepsin, action of, 133 Peptone, effect on blood coagulation, 170 Peptones, 132 Peptonuria, 170 Percussion, 150 Perfusion of frog's heart, 92 of rabbit's heart, 117 Perimetry, 193 Peristalsis, cesophageal, 127 Pettenkofer test for bile acids, 137 Pfliiger, laws of constant currents, 41, 42 Phrenic nerve, 156 Physiological salt solution, diuretic action of, 169 Pilocarpine, action on heart, 89 action on salivary secretion, 124 Pithing, frog, 10 Plasma, blood, 56 salted, 58 Plethysrriograph, 108 Plica semilunaris, 178 Porter, ergograph of, 32 plethysmograph of, 108 Potassium salts, action on heart muscle, 95 Presbyopia, 191 Pressure, pulmonary, 153 intrathoracic, 153 Proteids, tests for, 130 differentiation of, 132 Proteoses, 132 Protococcus, i, 2 Protozoa, 5, 6, 7 effect of gases on, 8, 9 Pseudopodia of amoeba, 6 Pulse, record of, 105, 106 volume, 107 Puncta lachrymalia, 178 Quotient, respiratory, 164 Rabbit, blood pressure of, 27 Reaction, iodine, for blood, 65 of degeneration, 44 time, for sound, 50 for touch, 51 for vision, 50 xantho-proteic, 131 Reflex action, in frog, 47, 48 centres, 49 203] INDEX. Reflex inhibition of heart, 88 responses, purposive character of, 49 time, 48 Reflexes, action of drugs on, 49 diffusion of impulses within the cord, 48 influence of chloral hydrate on, 49 influence of strychnine on, 49 inhibitory, 88 vasomotor, 112 Refraction, index of, 179 Refractive defects, correction of, 191 Refractory period of heart muscle, 97 Rennin, action of, 134 Reproduction, 3 Resistance in circulation of blood, 106 to electric currents, 14 Respiration, 148 artificial, 162 diaphragm in, 157 estimation of CO2and H2O in, 163 vagus nerve in, 154 Respiratory capacity, 151 centre, 159, 160 movements, 148, 154 effect of anaemia on, 159 effect of temperature on, 158 quotient, 164 sounds, 149 Retina, 179 formation of images on, 183 Rheocord, 15 Rheostat, 15 Ring test for proteids, 132 Riva-Rocci sphygmomanometer, 119 Saccharomyces cerevisiffi, 4 Saliva, action on starch, 125 Saliva, chemical constituents of, 125 secretion of, 121 Saponification, 134 Sartorius-muscle preparation, 20 Schema of circulation, 102 Sebaceous glands, 178 Secretin, action of, in pancreatic se- cretion, 139 Secretion, gastric, 132 pancreatic, 138 salivary, 121 collection of, 123 digestion by, 125 effect of atropine on, 124 effect of nicotine on, 123 effect of pilocarpine on, 124 nervous mechanism of, 121 Secretions, internal, 140 Semicircular canals, 54 Semimembranosus-muscle prepara- tion, 20 Sensation, 171 cutaneous, 173 tactile, 173 tests of, 173, 174, 175 threshold value of, 173 Sense of sight, 178 of temperature, 176 Serum, adaptation of, 78 globulicidal action of, 77 Shadows, inversion of, 188 Shock, action of adrenalin in, 117 blood pressure in, 117 Sight, sense of, 178 Smear, blood, method of prepara- tion, 64 Snellen, test letters of, 188 Sodium salts, action on heart mus- cle, 93 Specific gravity of blood, 71 Spectrum, absorption, 75 blood pigments, 75 [204] INDEX. Spectrum, CO2 haemoglobin, 76 Fraunhofer lines of, 75 haematin, 77 haematoporphyrin, 77 haemoglobin, reduced, 76 methsemoglobin, 76 oxy-haemoglobin, 75 sodium, 75 solar, 75 Sphygmomanometer, 119 Spinal cord as a reflex centre, 48 hemisection of, 52 diffusion of impulses in, 48 Spirogyra, 2, 3 Spirometer, calibration of, 151 Stannius' experiment, 95 Starch, digestion of, 125 formation of, in plant cell, 3, 4 Stimulation, latent period of, 23 Stimuli, chemical, electrical, mechan- ical, 22, 23 summation of, 28 Stimulus and sensation, relation be- tween, 172 Stomach, vagus nerve in, 129 Strychnine, influence on reflexes, 49 Sugars, test for, 125 Summation of contractions, 29 of stimuli, 28 Sunlight, effect on organisms, 2 effect on plant metabolism, 4, 5 Supplemental air, 152 Suprarenal glands, 144 extract of, 145 effect on blood pressure, 145 effect on heart-beat, 92 effect on muscle twitch, 45 removal of, in rabbit, 144 Suspension method of recording heart-beat, 84 Swallowing, mechanism of, 127 relation of vagus nerve to, 128 Sylvester, method of artificial respira- tion, 162 Sympathetic nerve, cervical, section of, 115 Tactile sense, 173 Talqvist, hoemoglobinometer of, 68 Tarsal cartilages, eye, 178 Telephone, detection of action cur- rent with, 23 Temperature, influence of, on heart- beat, 83, 85, 118 on muscle twitch, 26 on plant growth, 4 on tetanus, 30 sense, 176 Tenon, capsule of, 178 Tensile strength of muscle, 21 Tension, influence of, on muscle twitch, 33 Tetanus, effect of fatigue on, 30 effect of temperature on, 30 genesis of, 29 incomplete, 30 Thermal stimuli of muscle and nerve, 23 Thigh, frog's, dissection of, 18 Thoma-Zeiss haemocytometer, 59 Thrombin, 58 Thyroid feeding, 142 removal, 142 Tidal air, 152 Tone, muscle, artificial, 37 natural, 38 Torula, 4 Touch-spots, 174 Trypanosoma, 6, 7 Tuning-fork interrupter, 18 Twitch, muscle, effect of fatigue on, 26 effect of temperature on, 26 form of, 23 INDEX. Urea, diuretic action of, 169 Ureter, movements of, 166 Urine flow, 167 Vagus nerve in relation to blood pressure, 114 in relation to heart-beat, 87, IOC in relation to respiration, 154, 161 in relation to stomach move- ments, 129 in relation to swallowing movements, 128 section of, 161 Valves, heart, action of, 25 Vasomotor centres in cord, 112 in medulla, no, in outside of cord, 112 mechanism, no nerves, 115 reflexes, 112 Velocity of nerve impulse, 24 Venae vorticosae, 178 Ventricle, heart, record of contrac- tion, 84 Veratrine, action on muscle twitch, 45 Vision, 178 abnormal, 190 Vision, color sense, 189 distant, 188 light sense, 189 miscellaneous experiments, 187 near, 189 normal, 188 stereoscopic, 185 Visual judgments, 187 Vital capacity, 152 Vocal bands, movement of, 130 Volt, 15 Volta, 13 Volume of contracting muscle, 2:, Vorticella, 7 Water, distilled, effect on plant life, 3 Weber's law, 173 Wright stain for blood corpuscles, 64 Xantho-proteic reaction, 131 Yeast plant, 4 growth of, 4 influence of light on, 5 products of growth, 5 Zoospores, i, 2 Zygospores, 3 [206] MEDICAL SCHOOL LIBRARY THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW Books not returned on time are subject to a fine of 50c per volume after the third day overdue, increasing to $1.00 per volume after the sixth day. Books not in de- mand may be renewed if application is made before expi- ration of loan period. 2wi-4,'36 39169 ZJ. Of