aK^ v\\^^ A MAISrUAL EXPERIMENTAL PHYSIOLOGY STUDENTS OF MEDICINE. BY WINFIELD S. HALL, Ph.D., M.D. (Leipsic), PROFESSOR OF PHYSIOLOGY, NORTHWESTERN tTNIVERSITY MEDICAL SCHOOL; PROFESSOR OF PHYSIOLOGY, WESLEY HOSPITAL SCHOOL FOR NURSES; PROFESSOR OF PHYSIOLOGY, MERCY HOSPITAL TRAINING SCHOOL FOR NURSES; LECTURER ON THE PEIYSIOLOGY OF EXERCISE, INSTITUTE AND TRAINING SCHOOL, CHICAGO. WITH 89 ILLUSTRATIONS AND A COLORED PLATE. LEA BROTHERS & CO., IMJJLADELPHIA AND NEW YORK vvi?Trvin Entered according to the Act of Congress, in the year 1904, by LEA BROTHERS & CO., In the OflBce of the Librarian of Congress. All rights reserved. DORNAN, PRINTER. TO KATHAN SMITH DAVIS, M.D., LL.D., RECENTLY DECEASED DEAN EMERITUS OF THE NORTHWESTERN UNIVERSITY MEDICAL SCHOOL, IN HUMBLE RECOGNITION OF THE STIMULUS WHICH HE GAVE TO EXPERIMENTAL MEDICINE IN AMERICA AND IN GRATEFUL REMEMBRANCE OF THE INSPIRATION RECEIVED FROM HIM THIS VOLUME IS RESPECTFULLY DEDICATED BY HIS PUPIL, THE AUTHOR. Digitized by tine Internet Arciiive in 2010 witii funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/manualofexperimeOOhall - PREFACE. This volume represents the accumulated experience of a decade in the presentation of experimental physiology to medical students. The scope of the book has naturally been determined by the needs of medical students who are preparing for the practice of clinical medicine and surgery. The preliminary lessons in Cytology are presented as a feature of the volume. This introductory course has proven to be a most valuable accompaniment to the beginning work in histology, as well as a most substantial foundation to general physiology. The arrangement of the chapters has been determined by two considerations: (1) the degree of difficulty of the technique, and (2) the correlation of other work of the medical course. Cytology — the first chapter — involves the simplest microscopic technique, and the principles of cell life make the foundation of modern medi- cine and surgery. Electro-physiology involves a technique not too difficult for the earlier months of medical study, and, at the same time, it forms a most valuable basis for the experimental work that follows. The order of the chapters on Circulation, Respiration, Hema- tology, and Digestion may easily be changed to suit the curriculum of the institution where the course is given. The exercises have for years been furnished my students in the form of type-written syllibi, undergoing almost annual revision. They represent, therefore, a gradual evolution. At no time during this development of a practical course in experimental physiology has the author lost sight of the fact that his [>iipils were j^reparing for cHnical practice. The experimenls (v) VI PREFACE are carefully chosen and arranged to involve a considerable amount of surgical work and to present to the student those fundamental facts and principles of physiology which form the basis of Internal Medicine. The author takes this opportunity to acknowledge the valuable assistance of his associate, Dr. C. J. Kurtz, who prepared the chapter on Normal Hsematology, and of Professor Charles H. Miller, of the Department of Pharmacology, for his assistance in the preparation of the lessons on the physiological action of drugs. W. S. H. CONTENTS, PAGE IXTRODUCTIOX 17 PART I. EXPERIMENTAL GENERAL PHYSIOLOGY. CHAPTER I. Cytology. I. Algae or Green Plants of Low Order 21 II. The Yeast Plant 26 III. Protozoa or One-celled Animals 28 IV. Normal Ciliary Motion 31 V. Ciliary Motion Modified bj^ the Influence of CO2 and Anaesthetics 33 VI. To Detennine the Amount of Work Done by Cilia 35 CHAPTER II. The General Physiology of Muscle and Nerve Tissue. VII. Electric Apparatus and Units of Measurement 37 VIII. Batteries 41 IX. Methods of "S'arying the Strength of Current 43 X. Muscle-nerve Preparation 46 XI. Electric Stimulation and the Myogram 50 XII. The Typical Mj-ogram, Combined Myograms, and Tetanus 54 XIII. The Work Done by a Muscle . .' 55 XrV. To Send an Electric Current into a Nerve without Response. Fleischl's Rheonom 56 XV. To Determine the Influence of Cathode and .\node Poles .58 XVI. Electrotonus (to Determine the Effect of a Constaiil Current upon the Irritability of a Nerve) 61 XVII. The Law of Contraction 64 XVIII. (a) The Capillary Electrometer. (/>) The M.thod ol (siiig H 66 XIX. Electromotive Phenomena of .Active Muscle 60 viii CONTENTS PART II. SPECIAL PHYSIOLOGY. CHAPTER III. The Circulation op the Blood. PAGE I. The Capillary Circulation and the Movements of the Heart . . 73 11. The Apex Beat and the Heart Sounds 79 III. The Flow of Liquid through Tubes under Constant Pressure . . 81 IV. The Flow of Liquids through Tubes under the Influence of Inter- mittent Pressure 84 V. The Laws of Blood Pressure Determined from an Artificial Cir- culatory System Sft VI. The Radial Pulse and the Sphygmogram 88 VII. To Determine the Arterial Blood Pressure in an Animal .... 91 VIII. The Sphygmomanometer and Pulse Pressure 93 IX. To Determine the Influence of the Vagus Nerve upon the Action of the Heart 95 X. To Determine the Influence of the Cardiac Sympathetic Nerves upon the Action of the Heart 97 XL The Influence of the Vagus and the Cardiac Sympathetic upon the Arterial Blood Pressure 98. XII. The Blood Pressure in the Tissues 99 XIII. The Action of Atropine upon the Heart 101 XIV. The Action of Pilocarpine upon the Heart 102 XV. The Action of Digitalis upon the Heart 103 XVI. The Action of Aconite upon the Circulation 104 XVII. The Action of Adrenalin upon the Circulation 104 CHAPTER IV. ' Respiration. I. Thoracic Movements. Intrathoracic Pressure ...... 106 II. Respiratory Pressure. Elasticity of the Lungs. Pneumatogram . 108 III. To Study the Movements of the Human Thorax 110 IV. Lung Capacity (Chest Measurements, Respiratory Pressure). Recording of Anthropometric Data 113 V. The Evaluation of Anthropometric Data 115 VI. Quantitative Determination of the CO2 and H2O Eliminated from an Animal's Lungs in a Given Time 117 VII. To Determine the Amount of Oxygen Consumed by an Animal in a Given Time 119 VIII. The Respiratory Quotient 121 IX. Respiration under Abnormal Conditions 123 X. To Determine the Influence of the Phrenic Nerve. The Normal Phrenogram 125 CONTENTS j^ CHAPTER V. Xor:mal H.ematology. PAGE Introduction and General Directions 128 I. The Counting of the Blood Corpuscles 13q A. To Count the Red Blood Corpuscles 132 B. To Count the White Blood Corpuscles I35 C. To Count both Red and White Corpuscles at the Same Time . 136 D. Centrifugalization of the Blood. To Detennine the Relative Volume of Red Corpuscles and Plasma. To Estimate the Number of Red Corpuscles from Their Volume .... 137 II. The Estimation of the Percentage of Coloring Matter in the Blood . 138 A. Fleischl's Ha^mometer 130 B. Gowers' Haemoglobinometer 142 C. Dare's Haemoglobinometer I44 D. Tallquist's Hsemoglobinometer I45 E. Estimation of Hjemoglobin by Finding the Specific Gravity . 146 III. Examination of Fresh Blood " 14§ A. Coagulation of Nonnal Blood 148 B. Microscopic Examination of Blood 148 C. Spreading Blood for Staining 150 D. Staining Blood Films 153 E. Differential Counting of the Cells I53 IV. Staining Bone-marrow Igc CHAPTER ^^I. D1GE.ST10N AND Absorption. Digestion l^g I. The Carbohydrates I57 II. Salivary Digestion I59 III. The Proteids lU IV. (a) Diffusibihty of Proteids. (6) Milk ' . 164 V. Gastric Digestion 167 VI. Gastric Digestion (continued) 170 VII. Gastric Digestion (continued) I7I VIII. The Properties of Fats 172 IX. Intestinal Digestion I74 Absorption 170 , CHAPTER VII. Vision. I. DLssection of the Appendages of the Eye 178 II. Dissection of the Eyeball I79 III. Physiological Optics 181 IV. To Determine the Focal Distance of a Lens 183 V. To Locate Experimentally in the Mammalian Eye- the Cardinal Points of the Simple Dioptric System 185 X CONTENTS PAG VI. Accommodation and Convergence 188 VII. Miscellaneous Experiments 192 VIII. Perimetry 193 IX. Determination of Normal Vision 196 X. The Range of Accommodation 200 XI. Normal Ophthalmoscopy (Direct Method) 201 XII. Normal Ophthalmoscopy (Indirect Method) 203 XIII. Skiascopy 204 CHAPTER VIII. The Physiology op the Nervous System. I. ReflexAction 206 II. Reflexes in the Human Subject 208 III. The Action of Strychnine upon the Nervous System .... 210 IV. The Action of Curare upon the Nervous System 212 V. The Action of Veratrin upon the Nervous System 214 VI. Sensation 215 VII. Sensation (continued) 218 VIII. Function of Spinal Nerves 220 CHAPTER IX. The Physiology op the Mtjsculak System. I. Animal Mechanics 222 II. Ergography 226 APPENDIX. 1. Normal Saline Solution . 229 2. Frog Boards 229 3. The Physiological Operating Case 229 4. Galvanic Cells 230 5. Dry CeUs 230 6. To Curarize a Frog 231 7. To Prepare the Kymograph for Work .' . . . 231 8. A Fixing Fluid for Carbon Tracings 231 9. Non-polarizable Electrodes 232 10. The Frog-heart Lever 233 11. The Respiratory Cannula • 234 12. Tambours (Receiving and Recording) . . ' 234 13. The Manometer Talnbour 236 14. Thoracic Cannulse 237 15. The Stethograph 237 16. The Chest Pantagraph 238 17. The Pneomanometer 240 EXPERIMENTAL PHYSIOLOGY. INTRODUCTION. The general method of presenting the subject of physiology is the same as that followed in all of the other experimental sciences, viz., the laboratory method, according to which the student is led to dis- cover for himself certain facts and to formulate from his collected data conclusions which represent fundamental principles of the science. This method of presenting the experimental sciences — chemistry, physics, and the biological sciences, including physiology, psychology, pharmacology, and pathology — is an expensive one, both as to the time and the money involved in it; but from the standpoint of pedagogy it is more economical than the text-book method, because it leads directly and surely to definite results. In answer to the question as to just what these results are which follow the modern laboratory method of instruction, it may be stated first of all that it cultivates in the student the capacity of close and accurate observation; it affords an opportunity for valuable practice in the systematic recording of the observations; it develops the power of logical thought in drawing tenable conclusions from the observed data; and, in the formulation of conclusions, it stimulates the ability to express the thoughts in concise and unambiguous terms. In the second place, practice of this kind makes the student independent and furnishes him with just the mental equipment needed for later life in whatever line his activities may be directed. If such an education is more important for one profession than another, the medical profession is certainly the one in which its importance is greatest. The physics, chemistry, and biology studied in preparation for medicine, and the physiology, pharmacology, and pathology of the medical course should give the student a most admirable equipment for dealing with the complex problems of clinical practice. From what has preceded, it will have been noted that the facts of an experimental science occupy a subordinate position. Facts are only stepping stones leaMre zinc which forms a part of the alloy is presented to the 38 EXPERIMENTAL GENERAL PHYSIOLOGY acid. Chemically pure zinc is acted upon very slowly by 10 per cent, sulphuric acid. Join a wire to the exposed end of each plate; touch the tongue with the free end of each wire separately; touch the tongue with both wires simultaneously. Record results. (3) Place the porous cup with the zinc plate in the receptacle holding CuSO^ with the copper plate. Touch the tongue with one wire, then with the other. Touch the tongue with both at once. Bring the two free ends of the wire into contact with the binding Fig. 13 Fig. 14 The pole changer, or the Pohl commutator. (For description see text.) posts of a galvanoscope. Note results. Touch the ends of the wires together; if the conditions are favorable a minute spark may be seen on touching and on separating the two poles. What conclusions are to be drawn ? (4) Define element or cell as used in this connection. Define plate, pole, electrode. The zinc is arbitrarily taken as the positive plate and the copper as the negative plate. The pole which is attached to the negative plate is the positive pole, and that which is attached to the positive plate is the nega- tive pole. The positive pole or electrode of a galvanic cell or of a battery is called the anode, while the negative pole or electrode of a cell or of a battery is called the cathode. (6) Keys. (1) Study and describe the simple contact key (Fig. 25, K) and the Du Bois-Reymond key (Fig. 13). (2) The two ways of using the Du Bois-Reymond key are shown in the figures : first, as a contact key (Fig. 15); second, as a short-circuiting key (Fig. 16). The Du Bois-Reymond key. GEXEEAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 39 (c) The Pole Changer or Commutator. ]\lost convenient for the physiological laboratory is Pohl's commutator (Fig. 14). This in- strument may be used for the following purposes: Fig. 15 Fig. 16 Fig. 17 Fig. 18 Fig. 19 d) To change the direction of the current. Set up apparatus with cross-bars in place as shown in Fig. 17. Which is the anode when the l)ridge is turned toward a bf Which is the anode when the bridge is turned toward c df 40 EXPERIMENTAL GENERAL PHYSIOLOGY (2) To change the course of the current. Set up apparatus with cross-bars removed as shown in Fig. 18. What course will the current take when the bridge is turned toward a hf What course when the bridge is turned toward c df (3) Pohl's commutator may be used as a simple mercury key (Fig. 19). Is the current open or closed when the commutator bridge is turned toward af How may the current be opened or broken? (d) Work Done by the Electric Cell. The experiments performed show that the galvanic cell may, under proper conditions, liberate energy. This energy is called electricity. But the immediate source of the particular electric energy liberated in the foregoing experiments is the latent chemical energy represented in the plates and liquids of the cell. Under the conditions produced in the working galvanic cell the latent chemical energy is transformed, and at the same time liberated as electric energy. This liberated electric energy may make itself manifest in the contact spark, in moving the galvanoscope needle, or in lifting the armature of a magnet. In the last case mentioned it would not be difficult to determine the amount of work done, though it might be somewhat difficult to determine the amount of work which a cell is capable of performing in a given time. If one were to weigh the copper plate before and after using the cell, one would find that it had increased in weight. This increase in weight is an index of the amount of chemical action in the cell — of the latent chemical energy which has been transformed into electric energy. The amount of electrolysis must be, then, an index of the amount of current that is afforded by a cell or battery. For example, if the negative pole of a cell be attached to a silver or platinum cup containing pure nitrate of silver, and the positive pole be attached to a piece of pure silver which is immersed in the silver nitrate solu- tion, it will be found that one ampere of current will uniformly deposit 0.001118 gm. of. silver upon the cup in one second of time. This brings us to the question of the units of electric measurements. (e) Electric Units. The electric energy available at any point in a circuit — i.e., the current, as it is called — is, according to Ohm's law, equal to the liberated energy — the electromotive force — divided by the total resistance of the circuit. This is expressed in Ohm's » , ^_E.M.F.- E ^ . . lormula, G — — ^ c = ^. It is impossible for the physicist to make any progress in the study of electric energy without arbitrarily assuming units of measurement for current, for electromotive force, and for resistance. (1) Current is measured in amperes. A current of 1 ampere deposits upon the negative electrode of a galvanic cell or battery is 0.001118 gm. of silver per second, or 4.025 gm. per hour. (See above.) GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 41 A concrete idea of the ampere may be gained from the fact that the small-sized Daniell cell produces a current of about \ ampere when the external resistance is reduced to a minimum. (2) Resistance is measured in ohms. An ohm is that amount of resistance opposed to the transmission of electric energy by a column of mercury 1 sq. mm. in cross-section and 106.3 cm. in length. For general purposes an ohm resistance is that of a pure silver wire 1 mm. in diameter and 1 metre in length. (3) Electromotive force is measured in volts. A volt is that amount of electric energy which will produce 1 ampere of current after overcoming 1 ohm of resistance. "The ohm, the ampere, and the volt are thus closely related, and if any two of them be known with reference to any particular electric circuit or portion of a circuit the value of a third may be readily inferred" (Daniell) . For if C = ^, then E=CXR a^nd R=^. The same relations may be expressed thus: 1 ampere current 1 volt E. M. F. ^ ^ 1 volt == — , . , or 1 ampere =_ — - — . 1 ohm resistance 1 ohm Therefore (1 ) volts = amperes X ohms ; (2) amperes = volts -^ ohms ; (3) ohms = volts -7- amperes. The small Daniell cell has about 1 volt E. M. F. and 4 ohms resistance ; the current from such a cell is then equal to approximately J ampere. There are niunerous other units of measurement used by physicists and electricians, but for our purpose it is not necessary to review these more specialized points. VIII. BATTERIES. A Vjattery is a group of two or more elements or cells arranged to produce increased or modified effect. If one wishes to use a stronger current than that afforded by one cell, his first thought is to increase the number of cells, or to procure a larger cell. Experi- mentation will show him that it is not a matter of indifference which of these courses to pursue. In the first place, if he attempts to satisfy the conditions he will find that to increase the size of a cell increases the current only when the external resistance is relatively small, and, furthermore, there are practical limitations to the size of a cell, and these may be much within the requirement which the cell must satisfy. It becomes apparent, then, that he who would use electric energy beyond the most limited field must resort to a battery com- posed of a number of cells. The problem which first confronts him is, Hf>\v shall these cells be arranged? 1. Appliances. Six Daniell cells; wires; galvanoscope (Fig. 24), composed of a simple magnetic needle mounted over a circle divided 42 EXPERIMENTAL OENEBAL PHYSIOLOGY into degrees; rheostat or resistance box, representing at least 100 ohms. 2. Experiments and Observations. (1) (a) Join up apparatus as shown in Fig. 20. With the plugs all fixed in the rheostat — i. e., with no resistance except that of the wires and battery, and the indicator needle at 0° — open the key and then observe the angle at which the needle comes to rest. Fig. 20 (6) Remove from the rheostat the plug which will throw into the circuit an extra resistance of 10 ohms. Allow the needle to come to rest and note angle. (c) Remove from the rheostat plugs which will represent in the aggregate 100 ohms extra resistance. Note angle of indicator as before. (2) Join up two cells in multiple arc, as shown in Fig. 21. That is, join both copper plates to one copper wire and both zinc plates to another. These wires are to be carried to key, rheostat, and galvanoscope, as shown in Fig. 20. (a) Note angle of needle with no extra resistance. (h) Note angle with 10 ohms extra resistance. (c) Note angle with 100 ohms extra resistance. Fig. 21 (3) Join up four cells in multiple arc or "abreast," and repeat the observation of angle at the three resistances as above. (4) Join up six cells in multiple arc and repeat observations with ohm, 10 ohms, and 100 ohms resistance. (5) Join up two cells in series, as shown in Fig. 22. That is, join the copper of the first cell to the zinc of the second. The first cell will have a zinc uncoupled and the second will have a copper plate uncoupled. These two uncoupled terminal plates of the battery are the ones from which to lead off the wires to the GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 43 other apparatus, which should be arranged as shown in Fig, 20. Repeat the observations on the angle of deviation of the needle using the ohm, 10 ohms, and 100 ohms resistance as above. (6) Join up four cells tandem or in series, and repeat the three observations. (7) Join up six cells in series and repeat observations. (8) Tabulate results and draw conclusions: 1. There is a marked difference in the results of the two methods. 2. With low external or circuit resistance the current is indicated by the angle at which the galvanoscope needle stood increased with an increase in the number of cells joined multiple arc or abreast. Fig. 22 3. With high external resistance the strength of the current does not seem to be essentially increased by increasing the number of cells joined up abreast. 4. With low external resistance the strength of the current is not increased by adding cells in series. 5. With high external resistance the strength of current increases with an increase in the number of cells joined up in series or tandem. IX. METHODS OF VARYING THE STRENGTH OF CURRENT. It has already been shown that the strength of current may be varied by increasing the numl^er of cells or by changing their arrange- ment in the battery. This method is indispensable, but it has its limitations. If one has a small cell and wishes to decrease the current, he must have a recourse to another method. E From the formula C= it is evident that one may decrease the R current by increasing the resistance. {a) The Rheostat. 1. Appliances. Resistance l)ox or rheostat; 1 cell; 5 wires; galvanoscope or galvanoinctci'. 2. Experiments and Observations. (I) Set up tiie apparatus as .shown in Fig. 20. 44 EXPERIMENTAL GENERAL PHYSIOLOGY (1) With plugs all fixed in rheostat, needle of galvanoscope at 0°, close key and note angle of deviation. (2) Remove the plug, which will throw into circuit the lowest resistance contained in the rheostat. Note the angle. (3) Add to the above resistance the smallest possible increment and note angle. (4) Proceed in this way, tabulating results. (5) Conclusions. Fig. 23 (II) Another method of using the rheostat. The rheostat may be used in short circuit, as shown in Fig. 23. From this arrange- ment of the apparatus it is apparent that when all the plugs are in place the current will be short-circuited by the rheostat. If the resistance of that part of the circuit leading to the galvanoscope — the long circuit — be considerable, the long-circuit current will Fig. 24 Galvanoscope, composed of a single magnetic needle mounted over a graduated circle. The two heavy copper wires which encircle the compass offer slight resistance to the passage of the electric current. probably not be sufficient to cause any deviation of the galvanoscope needle; for the current varies inversely as the resistance (Coo -i), and if the resistance of the short circuit {R') equals zero, then the current of the long circuit (C) will be incomparably less than the current of the short circuit {C')—i.e., C:C':: i;! , or C:C':: R':R; therefore, if i?' = 0, C must equal 0. GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 45 Suppose that the resistance of the galvanometer circuit {R') be only 10 ohms, and suppose we remove from the rheostat the plug that represented 0.1 ohm resistance, then one-hundredth of the current will pass through the galvanometer. If we make the resist- ance in the short circuit 0.2 ohm, then one-fiftieth of the current will flow through the long circuit. Problem. In this way we may increase the galvanometer current step by step until the maximum is reached. What is the maximum current to be derived when the resistance in the galvanometer circuit (R') equals 10 ohms, the maximum resistance of the rheostat (R) equals 100 ohms, external resistance in circuit between cell and rheostat (r) equals 1 ohm, E. M. F.= 1 volt, and internal resistance of cell 4 ohms? (6) The Simple Rheocord. Besides the methods alreadv used for varving the strength of the current one may use the derived current. The simple rheocord (Fig. 25) may be used for this purpose. Fig. 25 Rheocord with contact key. 1. Appliances. One or more cells; simple rheocord; five wires; galvaiioinctcr. 2. Experiments and Observations. (1) Set up the apparatus as shown in Fig. 25. From the figure we see that from the cell to post A, thence through the German-silver wire to post B and back to tlie cell makes a complete circuit. Having reached the 46 EXPERIMENTAL GENERAL PHYSIOLOGY metallic slider {S) the circuit has two paths presented: 1st, from S direct to B; 2d, from S through G and back to B. The total current is divided into two parts : C, which passes along the wire from S to B, and C , the derived circuit which passes through the galvanometer. Suppose the resistance to the last-named current is R and that to the direct current is R, the relative strength of these two currents is expressed in the following proportion: C':C : : R : R' . But the resistance of the German-silver wire may be conveniently- divided into 100 equal parts (100 r). If the sHder be placed at any position along the wire, say at X cm. from the end, then the formula would be C : C : : xr : R' . Suppose that R=l ohm (r = 0.01 ohm); R' = 2 ohms and a;=0; XT i. e., suppose the slider to hard up to B, then C'= ~C^0. This makes it clear that when the slide is in the zero position there will be no current passing through the galvanoscope. (2) What is the relative strength of the two currents when x=10? (3) What is the relative strength of the two currents when a; =50? (4) What is the relation of C to C when a; =99? (5) What is the relation of C to C when a; =100? From this course of reasoning it is evident that in the simple rheocord we have an instrument with which we can vary a derived current from zero to a maximum. Just what the value of this derived current will be will depend upon the voltage of the cell or battery and the total resistance to be overcome, as well as upon the distribu- tion of that resistance. (6) Verify the theory just developed, making a table of galvano- scope readings. X. MUSCLE-NERVE PREPARATION. (a) The Classic Muscle-nerve Preparation. 1. Appliances. Frog board and pins; operating case; glass nerve hooks, like Fig. 28, A, made as follows: take a 10 cm. piece of glass rod, heat and draw in center to about IJ mm. diameter; cool, cut in two, heat the points to smooth them, and bend the end over to form the hook. Simple myograph or muscle lever (Fig. 26). Watch-glass with salt crystals; 20 cm. of thick copper wire. 2. Preparation. Pith a frog and fix to frog board, with dorsum up. It will be taken for granted that the student is familiar with the anatomy of the frog's leg and thigh. The accompanying cut (Fig.. 27) may serve to refresh the memory. GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 47 Fig. 26 The simple myograph : C, femur clamp ; S, glass slide on which to rest the nerve ; i/, [tendon hook of myograph lever ; T, tracing point of myograph lever. Fig. 27J Sh'jwiriK muKcuIature of the frog'H thigh ami leg: A, ventral aspect; //, tlorsal aspect. 48 EXPERIMENTAL GENERAL PHYSIOLOGY 3. Operation. To make a gastrocnemius "muscle-nerve prep- aration." (1) Make, with scissors, a circular cutaneous incision around the tarsus, corresponding with the lower end of cut B. Make a longi- tudinal cutaneous incision, beginning at the margin of the circular incision where it crosses the external aspect of the tarsus, carry it along the tibia, along the course of the biceps femoris muscle, over the pyriformis to the posterior end of the urostyle, along the whole extent of the urostyle. From the posterior end of the urostyle make an incision posteriorly and ventrally, for 1 cm. or 2 cm. Grasp the free margin of the skin at the point of the circular incision and with a quick traction toward the head of the frog the skin will be removed from the whole field of operation. Fig. 28 A, a glass nerve hook; B, the classic muscle-nerve preparation. (2) Pass a point of the fine scissors under the glistening tendon of the biceps femoris where it is inserted into the tibia, taking care not to injure any of the neighboring tissues. Sever the tendon. Grasp its free end; lift the biceps up, carefully cutting the delicate connective tissue which joins it to neighboring structures; sever its heads. The removal of the biceps and a separation of the cleft which the biceps occupied reveals three bloodvessels and the large trunk of the sciatic nerve. Which of the bloodvessels is the sciatic artery? Which is the sciatic vein? Which is the femoral vein? _ (3) Grasp and lift up the posterior end of the urostyle, sever the iliococcygeal muscles, remove the urostyle. The sciatic plexuses formed by the seventh, eighth, and ninth pairs of spinal nerves will be revealed. (4) Pass a glass nerve hook under the sciatic nerve; gently lift it up, severing, with the scissors, the connective tissue. The pyriformis muscle must also be divided. The whole length of the sciatic nerve GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 49 may thus be readily dissected out. Care should be taken not to stretch, pinch, or cut the nerve during this process. Lay the nerve upon the gastrocnemius muscle. (5) Grasp the triceps femoris muscle, pass a blade of the scissors under its tendon; sever, and remove the whole mass of muscles anterior to the femur. In a similar manner remove the muscles posterior to the femur. (6) Grasp the tendo Achillis, sever it low down where it passes over the calcaneum, lift up the gastrocnemius and sever the tibia and its associated muscles as near the knee-joint as possible. (7) Sever the femur at the juncture of its middle and upper thirds. The finished preparation has the characteristics shown in Fig. 28. A segment of the vertebral column may or may not be left on. (6) The Indirect Stimulation of the Gastrocnemius. 4. Observations. To mount the muscle-nerve preparation in the myograph. Fix the femur in the clamp (Fig. 26, C) ; place a piece of filter paper, wet with normal saline solution, upon the glass nerve- support (S) ; lay the nerve upon the support, make a longitudinal slit in the tendo Achillis, pass the hook of the muscle lever through the slit, and so adjust the height of the clamp as to bring the lever into a horizontal position. (a) Mechanical Stimulation. (1) Snip off with the scissors the central end of the sciatic nerve. If the muscle instantly contracts, thereby lifting the lever, the observer will know that his preparation is successful. If it does not respond to the first stimulation it may to a second or subsequent one. If it responds to later stimuli, but not to the first ones, one may conclude that in making the prepara- tion a portion of the central end of the nerve was killed. (2) What may one conclude if the muscle responds to stimuli applied to a central end of the sciatic nerve, but later fails to respond to stimuli applied farther along the course of the nerve — i. e., nearer the muscle? (b) Thermal Stimulation. Make and mount a fresh preparation. Heat the copper wire in a gas flame and touch the end of the nerve with the hot wire. If the preparation has been successful the muscle will respond by a contraction. If the preparation is a good one, .save at least two-thirds of the nerve for the subsequent experiment. (c) Chemical Stimulation. Cut off the part of the nerve which is (h'iul and lay the central end of the still functional nerve in a saturated .solution of common salt. Await results. Record all results. (d) Electric Stimulation. While in the operation of making a gastrocnemius prej>aration after the sciatic nerve has been freed from the other structures in the thigh, slip the glass nerve hook under it .so that the handle of the nerve hook will hold the nerve away from 4 50 EXPERIMENTAL GENERAL PHYSIOLOGY the other tissues. Press the end of a copper wire against the muscles of the thigh; touch the silver probe to the sciatic nerve, then to the copper wire, first separately then simultaneously. Vary the experiment by using other combinations: silver and steel, copper and steel, etc. Note briefly the original observations of Galvani. Are the observations just made different in any essential respect from the observation which led to the discovery of what we call galvanic electricity? XI. ELECTRIC STIMULATION AND THE MYOGRAM. The simplest work in the field of electro-physiology is that which involves the use of the induction shock as a stimulus and the use of a myograph and kymograph to record the result of the stimulus. The inductorium. 1. Appliances. Inductorium; myograph; kymograph; frog; oper- ating case; glass hook; dry cell; contact key with three wires; shielded electrode with two wires; normal saline solution. 2. Apparatus, (a) The Frog-board Myograph. The frog-board myograph is a new form of myograph, so constructed as to permit all experiments usually performed on the gastrocnemius-sciatic prepara- tion without exposing the active tissues to the atmosphere or dis- turbing the blood supply. The instrument is constructed as follows: An oaken base about one-fourth of an inch in thickness supports a cork plate of equal thickness; the cork plate presents a surface "about 10 cm. by 25 cm. (Fig. 31). The lever holder at the end of the plate is constructed of thin sheet steel and slips from side to side in order to bring it opposite either leg of the frog. The distance from the axis of the elbow lever to the thread-eye is the same as that to the weight; therefore, the weight hfted by the GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 51 muscle is the actual -weight hung upon the weight link. When the lever passes a little below the horizontal position it comes in contact with the rest. This rest can be used in "after-loading" a muscle. For further description of the instrument see Fig. 31 and its legend. Fig. 30 The Neef hammer. In the use of the frog-board myograph one proceeds as follows: The frog is pithed and pinned, dorsum up, on the cork plate, with the feet at the lever end. The tendo Achillis is exposed and loosened from the tarsal ligaments; the tendon hook jV is passed through the tendon and the length of the thread adjusted at C. The skin on the thigh is opened to the extent of 2 cm. and the biceps femoris muscle removed, the sciatic nerve carefully separated from the Fig. 31 Frog-board myograph : S, the shaft which is clamped to the upright stand ; B, the oaken base; C Pt, the cork f>late to which the frog is fixed ; A, the lever axis and slide lever holder; IK, the weight; L, the light lever, about 20 cm. in length ; N, the tendon hook which is joined through the thread T, which passed through the eye and under spring the catch C; R, the lever re»t. sciatic artery and placed on the insulated electrodes. Stimulation may be made from time to time for a period of several hours before the preparation becomes exhausted. (h) The Inductorium. This instrument consists of two spools of wire: a prirnari^ circuit of few turns of coarse wire and a secondary 52 EXPERIMENTAL GENERAL PHYSIOLOGY circuit of many turns of fine wire. It will be assumed that the principle of the inductorium is familiar to the student through his previous work in physics. (See Figs. 29 and 30.) The inductorium used in the physiological laboratory is provided with a vibrating (Neef) hammer which makes and breaks the current with each double vibration of the hammer, the vibration being due to the reciprocal action of an electromagnet and a spring. The instrument must also be provided with a means for either cutting the hammer out of the primary circuit or stopping the vibration of the hammer. The secondary coil or induction circuit must be pro- vided with a short-circuiting key, either as a part of the inductorium or as an extra appliance. Fig. 32 Fig. 33 The kymograph. Drum support for use in smoking the kymograph drums. The secondary coil is movable and may be moved up until it covers the primary coil or moved out along a slide. Some instruments are provided with a long base, permitting the secondary coil to be moved to a considerable distance from the primary coil, while others are provided with a short base and a pivot, allowing the secondary coil to be turned through an angle of 90 degrees after it has been drawn back free from the primary coil. Either arrangement allows one to decrease at will the strength of the induction shocks. (c) The Kymograph (Fig. 32). This instrument is the most important one in any physiological laboratory, because with its help graphic records of all movements of tissues and organs and of all pressure changes may be made. The kymograph or wave-writer is GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 53 thus used in nearly all work in the neuromuscular system, the circulatory system, and the respiratory system. The instrument consists of a cylinder or drum kept in rotation by clock-work. The rate of the rotation is usually governed by fans of varying sizes, also by adjustments of the propelling mechanism. To prepare the kymograph for work, remove the cylinder, stretch a sheet of prepared glazed paper tightly upon the surface and place it upon such a stand as that shown in Fig. 33. Set the drum to rotating and bring a gas flame or preferably a triple flame under the drum. In a few moments it will be evenly covered with a film of carbon, which is as sensitive to touch as a photographer's plate is to light. To fix the carbon tracings and make the record a permanent one see directions in Appendix, 8. 3. Experiments and Observations. Pith a frog; mount it upon the frog-boartl myograph as directed above. Prepare the kymograph for receiving a tracing and adjust it for slowest rotation. Adjust the myograph so that its tracing lever stands horizontal and tangent to the drum with the tracing point lightly touching the side of the drum. Set up the electric apparatus with one dry cell or one Daniell cell so joined in the primary circuit as to avoid the action of the vibrating hammer. Use a contact key in the primary circuit and a short-circuiting key in the secondary circuit. (1) Determine the stimulus of liminal intensity by moving the secondary coil to position of minimum strength; then, while slowly "making and breaking" the current in the primary circuit, move the secondary coil up until the strength of the induction shock is sufficient to cause a contraction of the muscle. The weakest shock which will cause a contraction is the stimulus of liminal in- tensity sought. Note that this occurs on the break of the primary circuit. (2) Determine the stimulus of optimum intensity by starting the kymograph to rotating slowly; meanwhile make and break the primary circuit while continuing to move the secondary coil from the position of liminal intensity toward the position of maximum intensity. The myograph will trace a series of myograms with the rise and fall of the lever, when the muscle contracts and relaxes. The tracings will present a series of sharp-pointed waves varying in height, showing the varying extent of contraction. At first all the contractions occur on break of primary circuit, then on both break and make of the primary circuit. As the secondary coil is moved toward the maximum position the myograms become higher and higher, finally reaching a maximum height which is not exceeded, however strong the stimulus is made. The stimulus of optimum intensity is the weakest stimulus which v:ill produce the maximum contraction. 54 EXPERIMENTAL GENERAL PHYSIOLOGY The increase of the strength of stimulus beyond the optimum will only fatigue the muscle and nerve through overstimulation, without producing greater contractions. 4. Observations. (1) Take tracings of the contractions produced by a series of "make-induction shocks" applied indirectly — i. e., to the nerve. The "make-induction shock" is obtained as follows : (a) With primary circuit not interrupted by the Neef hammer, but closed and opened by the contact key, open the short-circuiting key of the induced circuit. (6) Close the contact key of the primary circuit and make induc- tion shock — i. e., a shock in the induced circuit caused by a closure of the battery circuit will stimulate the preparation. (c) Close the short-circuiting key in the secondary circuit. (d) Open or break the primary circuit. An induced break shock occurs in the secondary circuit, but it is short-circuited by the closed Du Bois-Reymond key. If while the drum rotates one makes, in close succession, the changes above indicated — a-b-c-d, a-b-c-d, etc. — there will be produced a series of contractions, all the result of stimulation by make-induction shocks. (2) Take a tracing of the contractions resulting from a series indirectly applied — break-induction shocks. (3) By leaving the short-circuiting key open one may get a series of contractions due to alternating make-induction shocks and break- induction shocks. Let these be recorded in pairs upon the kymo- graph. XII. THE TYPICAL MYOGRAM, COMBINED MYOGRAMS, AND TETANUS. 1. Appliances. Inductorium; Daniell cell or dry cell; kymograph; myograph; electrodes; keys; wires. 2. Preparation. Pith a frog, make muscle-nerve preparation; mount it on myograph, prepare kymograph for tracing, and adjust for fastest rotation; set up electric apparatus for a series of make- induction shocks or break-induction shocks. 3. Experiments and Observations. (1) Start the kymograph drum to rotating. When it has reached the maximum speed, stimu- late the preparation with a break-induction shock. The lever point should trace upon the drum a typical myogram. Repeat the experi- ment several times with the same preparation. Study the character- istics of the myogram. (2) Trace another myogram while a tuning fork is tracing hun- dredths of seconds upon the drum and while the instant of stimulating the nerve is traced upon the drum, either through the action of an GENERAL PHYSIOLOG Y OF MUSCLE AXD NERVE TISSUE 55 electromagnet and tracing lever or through a tracing lever attached to the key of the primary circuit. There will thus be three tracings upon the drum: (a) the myogram; (b) the time tracing; (c) the stimulus tracing, the latter showing the time when the stimulus is made. Note that the myograph lever does not rise until a certain time after the stimulus is given. This period is the latent period. What is the length of the latent period? (3) Trace another myogram, but as the lever is sinking back toward the abscissa stimulate a second time. Note that the result is a double-crested myogram and that the second is higher than the first. (4) Trace another myogram resulting from a series of stimuli occurring, in rapid succession, if possible about ten times per second. Note that the result is a myogram with a series of crests and that the lever does not fall back to the abscissa between the successive stimuli. Note that the first few crests are progressively higher and higher. This phenomenon is called the "stair-case series of con- tractions," and is usually observed when a muscle is given a series of stimuli after a period of rest. (5) Vary the above experiment by increasing the rapidity of stimuli to 20 per second. This may be done through the use of a toothed wheel as a key in the primary circuit, or through modifica- tion of the Neef hammer, which causes it to vibrate slowly. Use medium speed of kymograph. Note that the result is a myogram with a serrated crest, the serrations indicating the result of the several stimuli. (6) Stimulate with a series of induct shocks caused by the rapid making or breaking of the primary circuit through the vibration of the Neef hammer. Use medium-speed drum. Note that this throws the muscle into a condition of typical tetanus. Trace a series of tetanus curves, each lasting about three or four seconds. XIII. THE WORK DONE BY A MUSCLE. (a) To determine the amount of work done by a single contraction. (h) To determine the total amount of work done by a muscle, (c) Reaction changes in fatigued muscles. 1. Appliances. Same as Lesson XII.; also 50-gram Aveight and 20-gram or .30-gram weight. 2. Preparation. Arrange electric apparatus for a series of break-induction shocks. .J. Operation. Make and mount a gastrocnemius preparation for indirect stimulation. 4. Observations, rjjon a slow di-um record in close order a .series of break contractions. 56 EXPERIMENTAL GENERAL PHYSIOLOGY (a) To determine the amount of work done by a single contraction. (1) What weight is hfted? (2) How high is it raised? (3) What is the ratio between the height of the curve traced by the lever and the height through which the weight was raised? (4) Let ]F=work done. ^= weight hfted. /^= height of curve traced by lever. h= constant of the apparatus, in this case the ratio between the lever arms. Then W=k. g. h. (5) Express the amount of work in ergs. (b) To determine total work done. (6) How many times was the weight lifted before the muscle was fatigued? (7) Through what average height was the weight lifted? (8) Has the value of k or g changed? (9) Give a formula for total height (H^=). (10) Give a formula for total work done (W=). (11) Express in ergs the total work done by the muscle. (12) In the fatigue tracing, did the lever continue throughout the observation to fall back to the original abscissa? If not, describe the general changes in the abscissa. (c) Reaction changes. (13) Apply a piece of neutral litmus paper to the fresh muscle tissue of the frog from which your specimen was taken. Record result. (14) Apply a piece of litmus paper to a fresh-cut surface of the fatigued muscle. Record results. (15) What is the reaction of a muscle of a frog after rigor mortis has been established? (16) What is the reaction of fresh urine? (d) Secondary fatigue (Lagrange, p. 60). (17) Grind a fatigued or exhausted muscle in a mortar and extract with normal saline solution. (18) Inject this extract into subcutaneous lymph spaces of a frog. (19) Observe the effect of this injection upon the second or rested frog. (20) Observe the effect upon the working power of its muscles. XIV. TO SEND AN ELECTRIC CURRENT INTO A NERVE WITH- OUT RESPONSE. FLEISCHL'S RHEONOM. When one is observing the effects of mechanical and thermal stimuli, he finds that he may apply a mechanical stimulus so slowly that the nerve may be severed without calling forth a response; he GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 57 may apply heat to the fresh nerve so gradually that the nerve may l)e actually cooked without causing a contraction of the muscle which it supplies. The proljlem which we have next to solve is to apply an electric stimulus gradually. 1. Appliances. Fleischl's rheonom; one Daniell cell; myograph; contact key; galvanoscope; saturated solution of zinc sulphate; five wires; frog; operating case. The rheonom is constructed as in Fig. 34. Its essential features are: g, the non-conducting base with circular groove; P, the non- conducting rotatable, central standard; the battery binding posts, having zinc connection with the groove; the rotating binding posts, having zinc limbs dipping into the groove. 2. Experiments and Observations. Set up an apparatus as shown in Fig. 34, after amalgamating the zinc tips which dip into zinc sulphate. Fill the groove with zinc sulphate. Fig. 34 (1) Find and mark the zero position for the rotating limbs of the rheonom — i. e., find the position which will give no deviation of the detector needle when the contact key is closed. (2) Find and mark the position which the rotating limbs occupy when the detector needle indicates 10°. (3) Find and mark in succession each higher increment of 10°, until the maximum is reached. (4) Rotate the limbs so gradually as to cause the detector needle to rotate with slow and regular motion from the zero position to the maximum position and back. (5) Make a gastrocnemius muscle-nerve preparation, mount it in the myograph; change the wires from the galvanoscope to the electrodes of the myograph; place the limbs of the rheonom in the maximum position; close the key. With the closing of the key the maximum current is instantly thrown into the nerve and serves as a strong stimulus, in response to which the muscle contracts. ((]) Place the limbs of the rheonom in the minimum position; dose the key. Inasmuch as the muscle-nerve preparation is much more sensitive to electricity than is the low-resistance galvanoscopy, the muscle will probably respond when the conditions are as above iriflifiited. Theoretically, a zero i)oint exists. Practically it may 58 EXPERIMENTAL GENERAL PHYSIOLOGY be difficult to find it for a muscle-nerve preparation. The finding of a position where there is no response on closing the key is, how- ever, not essential in this experiment. (7) Keeping the key closed, slowly rotate the limbs of the rheonom from the minimum position to the maximum position. If the conditions are favorable this can be done without calling forth a response. (8) Without opening the key, slowly rotate the limbs back- ward from the maximum to the minimum position. One may thus send through a nerve a strong current and may withdraw the same without causing a contraction of the muscle. Keep the key closed. (9) Quickly rotate the limbs from minimum to maximum; the muscle responds. Quickly rotate from maximum to minimum; the muscle responds. From the preceding observations one may conclude that response to electric stimulation is elicited not by the simple flow of an elec- tric current through the irritable tissues, but by a more or less sudden change in the strength of the current. The opening and closing of a galvanic current, also its sudden increase or decrease, serves as an efficient stimulus, while the gradual increase or decrease in the strength of the current causes no response. XV. TO DETERMINE THE INFLUENCE OF CATHODE AND ANODE POLES. Many of the phenomena of muscle-nerve physiology were inex- plicable until a difference was noted (von Bezold, 1860) in the influence of the anode and cathode. This difference in the influ- ence of the two poles may be best observed by use of the sartorius muscle of a frog. 1 . Appliances. A double myograph and support ; recording drum ; Daniell cell; Pohl commutator; Du Bois-Reymond key; non-polar- izable electrodes; five wires; electrode clamp and support. 2. Preparation, (a) Set Up a Pair of Non-polarizable Electrodes' (See Appendix, 9.) (b) A Double Myograph. A most efficient as well as convenient and economical double myograph may be arranged for this experi- ment as indicated in Fig. 35. Two common muscle levers, as shown in the figure, are used. These are held in position by common clamps and heavy support. The upper myograph must be reversed and its lever counterpoised by elastic bands. Between the two myographs a small wooden block, with a longitudinal hole for the loop of thread which holds the muscle, is held by a clamp. GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 59 3. Experiment. (1) Ciirarize a frog. (See Appendix, 7.) (2) After the lapse of three hours or more the sartorius muscle may be prepared. (3) Mount the preparation by passing a loop of coarse thread through the hole in the block W, lift the muscle by its tendon of insertion, pass it through the loop, draw the loop gently around the middle of the muscle, and fix by making a single knot around the screw of the clamp. The fine hooks which join the muscles Fig. 35 Doable myograph: CI, femur-clamp holding a wooden wedge (W), through which a loop of thread passes. The sartorius muscle S is held tightly by the loop of thread which encircles its middle. The two tendinous extremities of the sartorius are hoolced to the two levers 1 1. The two levers are pivoted at P and P'. The muscle is put on a stretch by the two rubber bands r and r'. The tracing points Tand 7" are adjusted to a vertical line on the kymograph k. to the levers may now be passed through the tendons, and the proper position of the levers effected In' an adjustment of the clamps. The loop around the sartorius may now be drawn as tightly as possible and not actually .sever the two portiieces of cork and held by an extra support. A "universal" clamp holder is a most desirable acce.s.sory to this apparatus. 60 EXPERIMENTAL GENERAL PHYSIOLOGY The electric apparatus should be set up as shown in Fig. 36. With this arrangement either electrode may be made the anode, the experimenter needing only to reverse the commutator bridge to reverse the position of the anode and cathode. The recording drum or kymograph should rotate rapidly. The recording points of the myograph levers should be adjusted so that the point of the upper one touches the drum vertically over the point of the lower one. Adjust the marker Cr so that it will indicate the time making and breaking the circuit — i. e., so that it will record on the drum the time of making stimulus and the time of breaking stimulus. The recording point of the time marker should, of course, be in the same vertical line with the myograph points. The moist tips of the N. P. electrodes should be so adjusted as to touch the muscle above and below the loop of thread. Fig. 36 (1) Close the key. If the preparation has been successful the half of the muscle in contact with the cathode pole will respond before the other one. (2) Break the current. The anode will respond first. (3) Reverse the direction of the current and repeat (1) and (2). (4) Vary the strength of the current through use of the simple rheocord and determine whether the results are the same for currents of different strength. Law I. The make contraction starts at the cathode and the break contraction starts at the anode. When irritable tissue, muscle or nerve, is subjected to a galvanic current the response to the stimulation begins in the region of the cathode on making the current, and in the region of the anode on breaking the current. Would the foregoing observations justify the following statements ? (1) Cathodic contractions, or make contractions, may be caused by a galvanic current which is too weak to cause anodic contractions, or break contractions. (2) Cathodic contractions, or make contractions, are stronger than anodic contractions, or break contractions. Law II. With a given strength of current the influence of the cathode pole is more irritating than the influence of the anode. GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 61 XVI. ELECTROTONUS fTO DETERMINE THE EFFECT OF A CONSTANT CURRENT UPON THE IRRITABILITY OF A NERVE). At the beginning of the last century Ritter discovered that the vital properties of irritable and contractile tissues were modified when subject to a constant battery current. The modified con- dition was called galvanismus. During the first half of the last century the subject was investigated by Nobili, ^Nlattencci, Valentin, and Du Bois-Reymond ; the last named substituted the word electro- totius for galvanismus and further modified the terminologv. It remained for Pfliiger^ to rework the whole field, to correct, to elabo- rate, and finally to formulate laws. (a) Preliminary Experiment. 1. Appliances. Muscle signal, or myograph; two Du Bois- Reymond keys; two Daniell cells; commutator; eight wires; salt. 2. Preparation. Set up electric apparatus as shown in Fig. 37. 3. Operation. ^lake and mount in the muscle signal a gastroc- nemius preparation. Fig. 37 4. Observations. (1) In which position must the bridge of the commutator stand to give a descending current so that the cathode will be nearer to the muscle than is the anode? Mark the opposite side A. (2) Fig. 37, P, represents the glass plate of the muscle signal. So arrange the triangular platinum electrodes that there shall be a distance of alxjut 3 cm. between the electrodes and both electrodes near that end of the plate farthest from the muscle. Lay the nerve over the electrodes and along the glass plate. The segment of nerve which lies upon the glass plate between the electrodes and the muscle must be subject to various stimuli, mechanical and chemical. At a point about 1 cm. from the electrodes, marked X in the figure, place upon the nerve trunk as many fine crystals of common salt as would l)e taken up on the point of a penknife. Moisten these salt crystals with a drop of water. While the salt ' Untersuchungcii Uber die Physiologic des Electrotonus, Berlin, 1859. 62 EXPERIMENTAL GENERAL PHYSIOLOGY solution is permeating the sheath of the nerve trunk, adjust the commutator for a descending current. When the muscle begins to twitch, note the effect upon the signal. The contractions become more and more tetanic in character. (3) Close the commutator circuit, open the short-circuiting key — i. e., make the "polarizing" current. If the experiment is successful the tetanus is more marked. Which pole is near the point stimulated ? (4) Close the short-circuiting key — i.e., break the "polarizing" current. Reverse the commutator; make the current. The muscle is put completely or almost completely at rest. Which pole is nearer the stimulus? (5) Repeat (3) and (4) several times. It is evident that the irri- tability of the nerve to the salt stimulus is increased in the region of the cathode pole and decreased in the region of the anode pole. This changed condition of the nerve due to the passage of a constant current is called electrotonus. The state of increased irritability in the region of the cathode is called catelectrotonus. The decreased irritability in the region of the anode is called anelectrotonus. (h) Myographic Record of Anelectrotonus and of Catelectrotonus. 1. Appliances. Three or four Daniell cells; three Du Bois- Reymond keys; contact key; two commutators; inductorium; two N. P. electrodes; eighteen wires; kymograph; myograph with moist chamber; two pairs of platinum-wire electrodes to use with induc- tion current. 2. Preparation. Arrange apparatus according to plan as shown in Fig. 38. Fig. 38 3. Operation. Make and mount a gastrocnemius preparation in moist-chamber myograph, or frog-board myograph. Adjust electrodes as shown in diagram. Test apparatus and preparation by sending single make (or break) induction shocks through nerve at M. Let there be a typical response to these stimuli.^ The secondary coil should he removed to a distance that gives the stimulus of liminal intensity. GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 63 To close the constant current ''polarizes" the nerve, or, better, induces electroionus. That segment of the nerve between the anode and cathode is called infrapolar region. Those segments centrally and distally located are called extra- polar. The induced current is called stimulating current. 4. Observations. (1) Adjust for descending, polarizing current. Stimulate in the region of anode. Note extent of muscle contraction. Induce electrotonus; stimulate again in region of anode. If the experiment is successful the contraction will be found to be decreased or absent. The nerve is at this point in a condition of anelectrotonus. (2) Stimulate at M, or in the region of the cathode. Withdraw the polarizing current. After a few minutes stimulate again at M. If the experiment is successful the wave is higher in the former than in the latter case. The stimulation was made in the region of the cathode and the nerve in a condition of catelectrotonus. (3) Adjust for ascending, polarizing current. Stimulate at M — i. e., in the region of the anode. The contrac- tion is weaker than in the normal nerve, or it may be cjuite absent. This region is now in a condition of anelectrotonus. (4) Stimulate in the region of the cathode. The response is probably weak. Withdraw the polarizing current. Stimulate again in the region of the cathode. The response is normal — i. e., it is greater than during the electrotonic condition. But in descending extrapolar catelectrotonus the response was greater than normal. In the experiment just performed we stimulate in the region of ascending extrapolar catelectrotonus. Note that the polarizing current is relatively strong. Co) Remove one cell from the battery and repeat (4). If the response to stimulation is still weaker with than without the polar- izing current, reduce the strength of the polarizing current still farther by the use of the simple rheocord. Finally, with a weak polarizing current the stimulus in the region of extrapolar catelectro- tonus causes a stronger response than normal. The response which the muscle makes must be accepted as a measure of the excitation which it receives from the nerve. But the excitation flelivered by the nerve depends upon two factors — its irritability and its conductivity. When the nerve is stimulated in the region of ascending extrapolar or intrapolar catelectrotonus, its increased irritability is of no avail if there is interposed between that region and the muscle a region of decreased conductivity. With strong polarizing current the region of the anode is not only df-creasefJ in irritability, but in conductivity. 64 EXPERIMENTAL GENERAL PHYSIOLOGY (c) Laws of Electrotonus. (a) The 'passage of a current through a nerve induces a condition of electrotonus marked by increased irritability in the region of the cathode (catelectrotonus) and decreased irritability in the region of the anode (anelectrotonus) . (6) During electrotonus induced by a strong current the conductivity is decreased in the region of the anode. Further — though not derived from the foregoing experiment — "at the instant that the polarizing current is withdrawn the conducting power is suddenly restored in the region of the anode and greatly lessened in the region of the cathode." — Lombard, in American Text-book of Physiology. XVII. THE LAW OF CONTRACTION. 1. Appliances. The simple rheocord; four Daniell cells; frog- board myograph, or myograph with moist chamber, simple key; Du Bois-Reymond key; commutator; two N. P. electrodes. 2. Preparation. Set up apparatus with four cells in series, simple key as closing key. Commutator with cross-bars; short-circuiting key; the two N. P. electrodes clamped in chamber of myograph or mounted above the frog-board myograph (Fig. 39). Fig. 39 3. Operation. Make and mount a gastrocnemius preparation. 4. Observations. (1) Stimulate with make and break of the very weakest descending current. The first response is elicited by the very weak descending current. A slightly stronger current is required to elicit a response with the ascending current. Record results in such a table as suggested under (5). This table shows what response (contraction or rest) the muscle gives on making GEXERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 65 and breaking of the descending current and on making and breaking of the ascending current. It also shows in a marginal column the gradual increase of the strength of the current through gratlual increase of resistance in the rheocord. (2) ]Make and break with weak ascending current. If the con- ditions are typical the muscle will contract on making both ascend- ing and descending current. (3j Increase gradually the strength of the electrode circuit record- ing results. After a longer or shorter transitional period in which the result will be characterized by a contraction on the make of both the ascending and descending current, one comes to a strength of current which causes a contraction on both make and break of both descending and ascending current. This is the medium strength for the preparation and the condition in question. (4) Let the current be increased still farther and by larger incre- ments. After passing another transitional stage one finally reaches a strength of current which causes a contraction on make of descend- ing current and on break of ascending current. This is the "very strong" current for the preparation under observation. It not infrequently happens that through overstimulation and fatigue of muscle the whole experiment cannot be completed upon one preparation except by increasing the current by larger incre- ments. (5) Pfiiiger's law of contraction may be expressed in the follow- in cr table: Descending. Ascending. Strength of current. Make. Break. Make. Break. Very weak c R R R Weak cc R C R Medium C C c Strong CC c C CC Very strong. . ... CC R (or c) R CO (0) But how shall we account for these results? Let us recall .some of the laws which have been demon.strated. Law I. The make contraction starts at the cathode and the break contraction starts at the anode. Lav: II. I'he make or catliodic stimulus of a constant current is more irritating than the break or anodic stimulus. Law III. 'J'lie pa.s.sage of a con.stant current through a nerve iiifhires a condition of electrotonus, marked by an increased irri- 66 EXPERIMENTAL GENERAL PHYSIOLOGY tability in the region of the cathode, and a decreased irritabihty in the region of the anode. Law IV. During electro tonus induced by a strong current the conductivity is decreased in the region of the anode during the passage of the current and in the region of the cathode after removal or breaking of the current. These laws account for all typical phenomena observed above. XVIII. (a) THE CAPILLARY ELECTROMETER, (b) THE METHOD OF USING IT. In those experiments where we have had occasion to measure the strength of an electric current or the difference of potential between two electrodes we have used the tangent galvanometer. But in all these experiments the strength of current or difference of potential has been considerable, amounting in some cases *to that represented by several Daniell cells joined in series with a moderate amount of external resistance. To detect and measure muscle currents it has been necessary to devise a very delicate and sensitive instrument. The Wiedemann galvanometer has been used for this purpose; but the most simple and satisfactory apparatus is the capillary electrometer. (a) The Capillary Electrometer. Take a piece of 6-mm. glass tubing and draw two fine capillary tubes; clamp these in burette holders with the capillaries pointing vertically downward. Into one pour a few drops of water; it will pass through the capillary and leave its point drop by drop. Into the second tube pour some mercury — enough to fill the capillary — and stand 2 cm. or 3 cm. above the capillary in the tube. The mercury will not flow through the capillary. Note that the upper meniscus of the water — in the undrawn part of the tube — is concave, while the upper meniscus of the mercury is convex. The water wets the glass and seems to be drawn up for a short distance on the vertical surface of the glass, while the mercury does not wet the glass — there seems rather to be a repulsion. If one looks at the lower meniscus of the mercury with a low-power microscope, he will find it to be convex downward. Mercury stands up in nearly spherical globules on a glass surface, and water forms nearly spherical globules on an oiled surface. There is no adhesion between the glass and mercury, while there is a strong cohesion between the molecules of the mercury. This accounts for the fact that the mercury forms globules which but for the action of gravitation would be quite spherical. If a drop of liquid be placed GESERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 67 upon a horizontal plane its shape will be modified by three forces: (1) cohesion, (2) adhesion, (3) gravitation. In the case of the globule of water on an oiled surface, or of mercury on a horizontal glass plane, adhesion is practically /n7, thus leaving the two factors, cohesion and gravitation. Cohesion tends to draw all the molecules toward a common center and thus brings the indi\idual molecules of the surface into a con- dition of lateral tension. This condition is tech- nically called surface tension. The greater the ^^^- ^° preponderance of cohesion over the other forces acting upon the liquid the greater the surface tension. It is surface tension which gives to the mercury in the glass tube a convex meniscus, and keeps it from flowing through a fine capillary of glass. It must be evident that the relation between the mercury and the glass (adhesion) does not vary. If the position of the meniscus varies it must be through a change in one or both of the other forces mentioned above. Gravitation measured by the weight of the column of mercury may vary by changing the height of the column of mercury. Through this variation the meniscus may be made to take any flesired position. Experiment has shown that the passage of an electric current through the cohmin of mercury into sulphuric acid modifies the surface tension of the mercury and thus changes its position. As the modification of surface tension varies propor- tionately with the strength of the electric potential, one may measure this strength by noting the distance through which the meniscus moves. The observation of the meniscus must be made with a microscope, using the low power. Note that in the instrument (see Fig. 40) the wooden back that supports the instrument is cut away (at O) near the capillary in order to permit the microscopic observation of the meniscus to be made with transmitted light. Capillary electrometer. (Description in text.) (h) The Method of Using the Capillary Electrometer. 'IV) adjust the instrument for use clean the capillary absolutely clean through tlie use of 20 per cent. H^SC)^ c. p. and (h'stilled water. Pour into the tui^e enough mercury to bring the meni.scus to the middle of the finest portion of the capillary. Adju.st the parts of the electrometer — the pressure bulb /-*, the manometer m, and the 68 EXPERIMENTAL GENERAL PHYSIOLOGY reservoir R. The reservoir is partly filled with mercury, above which 20 per cent. H2SO4 c. p. fills the reservoir to above the capillary meniscus. Note that platinum wires {w w') are fused into the capillary and reservoir passing into the mercury. These wires pass to binding posts and are kept in contact through a short-circuiting key {K). The acid must be in contact with the mercury in the capillary. To effect this press the bulb P until the mercury is forced to the tip of the capillary; relieve the pressure and the meniscus will recede drawing the acid after it. Fig. 41 shows how the electrometer is Fig. 41 Showing method of joining up the capillary electrometer E. Note that the positive plate (zinc) is joined through the rheocord R to the capillary C, while the negative plate (copper) is joined through the rheocord to the reservoir. The battery wires are joined to the zero and 1 meter posts of the rheocord, or to the zero and 10 meter posts. In the former case the sUder Smust be very near, almost touching, the zero post when the first observation of the change of meniscus is made. to be joined up for use. Certain precautions should always be observed in the use of the electrometer. The two poles of the instru- ment — the mercury in the capillary C and the mercury in the reservoir — should be joined through a short-circuiting key, except when one wishes to test difference of electric potential, when the key may be opened for a few moments. The instrument is so sensi- tive that only the weakest currents should be allowed to traverse the acid between the poles. The current from a Daniell cell is GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 69 much too strong to be permitted to traverse the instrument. In testing the instrument with a Daniell or any similar cell use a rheo- cord joined as shown in Fig. 41, and make the first test with the slider almost touching the zero post, and subsequent tests with small increments until the movements of the meniscus are con- siderable in extent, yet not so much as to carry it out of the field of the microscope. If it is desired to make quantitative tests of electromotive force the electrometer may be graduated. To accomplish this it is neces- sary to have a micrometer in the ocular of the microscope, so that the position of the meniscus may be accurately determined. It is also necessary to have some means of measuring the force of dis- placement of the meniscus. This may be done by means of a mercury manometer, shown in Fig. 40 as a part of the electrometer. Pressure exerted on the bulb is measured by the manometer. The amount of pressure required to bring the meniscus back to its original posi- tion after the opening of the key K is proportional to the electro- motive force that displaced the meniscus during the opening of the key. By testing a series of known values and taking the manometer readings one may easily determine the relation between volts and millimetres of mercury pressure. XIX. ELECTROMOTIVE PHENOMENA OF ACTIVE MUSCLE. (A) The physioloc/ical rheoscope, or the rheoscopic frog. (B) Electromotive force detected by the electrometer. In the process of dissecting out a muscle-nerve preparation (the classical form) one is likely to drop the cut-off central end of the sciatic nerve upon the gastrocnemius muscle. Should this occur a contraction of the muscles is almost sure to occur. Galvani made this observation and cited it as a proof that electricity exists in animal tissues. A classical experiment well adapted to demonstrate the difference of electric potential in living tissues is that known as the rheoscopic frog. (A) The Rheoscopic Frog. 1. Appliances. Frog; two glass slides, 1 inch by 3 inches; oper- ating f;i>('. 2. Preparation. Pith the frog. Make two classical muscle-nerve preparations. Place the two glass slides end to end upon the table (as shown in Fig. 42j, with a muscle on each disposed as shown in 70 EXPERIMENTAL GENERAL PHYSIOLOGY the figure. Note that the nerve from muscle II touches muscle I in two places— at the end and middle. Set up the electric apparatus for single-induction shocks and rest the nerve of muscle I upon the electrodes. Fig. 42 "The rheoseopic frog," an experiment to show the presence of the difference of electric potential in different parts of an active muscle. I. The active muscle, stimulated at E by induction shocks. 11. The second preparation, which can be thrown into contraction only through some influence exerted at the points of contact (Xand 1'). Note that the muscles lie upon glass plates (ffand ff'), and that a glass nerve hook rests upon /in order to ensure two separate points of contact of the nerve from II. 3. Observations. (1) Rule a table as follows: strength of stimulus. Very weak . Weak . Medium Strong . Very strong. Tetanizing . Eesponse. Muscle I. Make. Rest. Break. Contract. Muscle II. Make. Rest. Break. Rest. (2) Stimulate muscle I as indicated in the table and record the response in the proper column. (3) What portion of preparation I is traversed by the electric current ? (4) Does any portion of the stimulating current traverse that part of muscle I between the points X and Y. (5) What causes the contractions of muscle II f Preparation II is called a rheoseopic preparation or a physiological rheoscope. If the contractions are caused by electricity one should be able to detect it through the use of the galvanometer or electrometer. GENERAL PHYSIOLOGY OF MUSCLE AND NERVE TISSUE 71 (B) Electromotive Force Detected by the Electrometer. 1. Appliances. A large frog; non-polarizable electrodes; capillary electrometer. 2. Preparation. Pith the frog; prepare electrodes, using kaolin wet with normal saline solution for the tips. Join the electrodes to the binding posts of the electrometer. Make a muscle-nerve preparation, lay it upon a glass plate, and prepare to stimulate with induction shocks as in the case of muscle I above. 3. Observations. (1) Place the electrode which is joined to the capillary upon the tendon of the muscle; the other electrode upon the belly of the muscle. Adjust the meniscus in the middle of the field of the microscope. Open the short-circuiting key of the elec- trometer while watching the meniscus. It will be displaced. Its displacement suggests a difference of electric potential between the tendon and the belly of the muscle. Such a difference of potential is usually to be observed, and it is called the "demarcation current." It is believed to be due to the injury to the muscle tissue incident to its preparation. It is also called the current of injury. (2) After the meniscus has come to rest stimulate the muscle with a single induction shock. The meniscus will move quickly, but in the direction opposite to that of its first motion. That is, its current of action is greater than its current of injury, and in an opposite direction. Describe phenomena in notes. (3) Bring the muscle into action through other stimuli than electricitv and note results. PART II. SPECIAL PHYSIOLOGY. CHAPTER III. THE CIRCULATION OF THE BLOOD. I. THE CAPILLARY CIRCULATION AND THE MOVEMENTS OF THE HEART. A. To Observe the Capillary Circulation. 1. Appliances. Frog; microscope, with low-power and high- power objective; cork board 10 cm. wide by 20 cm. or 30 cm. long and ^ cm. thick; pins; operating case; normal saline solution; watch- glasses; two 100 c.c. beakers. 2. Preparation. Pin the frog out, dorsum up, upon a cork board, and bring one hind foot over a hole 1 cm. in diameter cut in the corner of the board with a cork borer. By tying a thread to the second and third toes the web between these holes may be stretched over the hole in the Ijoard. Care should be taken not to stretch the web too tightly and thus impede the circulation. Fix the cork board with the frog upon the stage of the microscope in such a manner as to bring the stretched web over the middle of the stage. Illuminate the web and focus under a lower power. Keep the web moist. 3. Observations. (1) Observe the movement of corpuscles within bloodvessels of varying size and irregular course. Make a drawing of the field of observation showing the relative size, the course, and anastomoses of the bloodvessels. (2) Observe whether the motion is equally rapid in all vessels; if not, observe whether the slower currents are in the larger or the smaller channels. Determine which of the vessels are arterioles, which capillaries, and which venules. (3) Have you seen evidence of intermittent force acting upon the corpuscles? If so, describe its influence. Determine whether this intern)ittent force makes itself evident in all the ves.sels; if not, in which class of vessels is it present? 74 SPECIAL PHYSIOLOGY (4) Do the corpuscles change shape? If so; under what circum- stances ? (5) Enumerate all the observed structural and functional features which differentiate arterioles from venules. B. To Observe the Action of the Frog's Heart. 1. Preparation. After the capillary circulation has been observed, the frog may be pithed and stretched upon a cork board, ventrum up. Make a median incision through the skin from the pelvis to the mandible; make transverse incisions and pin out the flaps. Raise the posterior cartilaginous tip of the sternum; insert a blade of the fine scissors under it and divide it transversely, about \ cm. anterior to the tip. Raise the anterior segment of the sternum at the point of the transverse incision; insert the blade of the strong scissors under it and divide it longitudinally in the median line. Withdraw from the board the pins which fix the anterior extrem- ities; make gentle lateral traction upon the fore-feet until the slit sternum is sufiiciently separated to afl^ord a convenient working distance and to expose the whole heart. 2. Observations. (1) Note rate of systole. (2) Note sequence of contraction of auricles, ventricles, and bulbus. (3) Note change in shape of different parts. (4) Note the change in color and the position of the different parts of the heart during the cycle of changes that come with each heart beat. (5) Carefully excise the heart, including the sinus venosus and the bases of the posterior and two anterior vense cavse, also the bases of the two aortic trunks. Place the excised heart in a watch- glass. Observe whether the pulsation continues. If so, what is your conclusion regarding the relation of the heart movements to the central nervous system? (6) If the pulsation continues, note whether or not the rate of pulsation has been notably changed by the excision. (7) Bathe the heart with a few drops of normal solution. Note any change in the rate of the beat. (8) Hold the watch-glass in the palm of the hand and note whether there is any change in the rate of the beat. (9) Float the watch-glass on ice-water and note any resulting modification of rate. (10) If the heart seems vigorous (otherwise procure a fresh one), carefully sever the sinus venosus with the fine scissors. Does the sinus continue to beat? Does the heart continue to beat? Inter- pretation. (11) If the heart beats, sever the auricle from the ventricle through the auriculo-ventricular groove. Note results. THE CIRCULATION OF THE BLOOD 75 (12) If the auricles beat, divide them. If they continue to beat, do they follow the same rhythm? (13) If the ventricle becomes quiescent, stimulate it either mechan- ically or with a single induction shock. How does it respond to a single stimulus? Continue to subdivide the heart until the parts refuse to respond to stimuli. (14) Repeat the experiments and see if the results are the same on subsequent trials. Note results and give your interpretation. C. To Make a Graphic Record of the Frog's Heart Beat. 1. Appliances. Large frog; kymograph; heart lever. (For description of heart lever see Appendix, 10.) Frog-board myo- graph or similar apparatus; chronograph with a chronographic system, adjusted to record seconds upon the kymograph; cover- glass; normal saline; operating case. Fig. 43 Frog-heart lever : t, tripod to support lever ; p, the pivot ; c, counterpoise ; h, frog's heart, on which the cork point rests. 2. Preparation. Pith a frog without destroying its spinal cord. Take great care not to cut a vertebral artery during the pithing operation. Should a hemorrhage occur plug the opening with absorbent cotton. Hemorrhage depletes the circulatory system, and the action of the heart is weakened. In the operation to expose the beating heart, take care not to cut any large vessel, for the reason just given. Pin out the frog, ventrum up, upon the frog-board myograph. Expose the heart as described in the previous lesson. CJpen the pericardium carefully, thus expos- ing the heart to direct observation. Place some resistant object — a cover-gla.ss, for example — under the ventricle. So adjust the heart lever that the wedge-sha})('n the groove which marks tiie line of juncture between the aiirifle and the ventricle. (See Fig. 43.) If the weight 76 SPECIAL PHYSIOLOGY of the lever seems to be too great for the heart, move easily; the long arm may be made relatively lighter by adjusting the counter- poise upon the short arm. If the tracing point of the long arm has a sufficient excursion to make a good tracing, bring the kymo- graph to a position where the point will lightly touch the carboned surface of the drum. The lever should be nearly tangent to the surface of the drum, and so arranged that the rotating surface of the drum turns away from the tracing point of the lever rather than toward it. All tracings should be accompanied by a time tracing or chrono- gram. Study the chronographic system and make drawings of the plan, showing all electric connections. Study the chronograph or time marker, and make a diagram showing its construction. 3. Observations. (1) Note whether the curve is a simple one or composed of a major wave, with crests superimposed upon it. In either case closely observe the phases of the heart cycle and determine the relation of each part of the cycle with each part of the tracing. If the tracing has a single crest, more delicately counterpoise the lever and more carefully adjust the narrow foot of the lever to the auriculo- ventricular groove and repeat the experiment. (2) Take tracings of the auricle alone. Compare these with those of the auriculo-ventricular groove and determine the causes of variation. (3) Without altering the counterpoise take a tracing of the ventricle and compare it with the two preceding curves and account for all the differences. (4) By adjusting the foot of the lever between the ventricle and the bulbus it is possible to get a ventriculo-bulbar tracing which differs from the auriculo-ventricular in having the superimposed crest following the ventricular crest, while in the auriculo-ventricular tracing the superimposed crest precedes the ventricular. (5) If the conditions of the experiment are favorable it is possible to get an auriculo-ventriculo-bulbar tracing. To get this the lever foot must be placed in the auriculo-ventricular groove so that it rests upon the auricles, ventricle, and bulbus. A typical tracing may be recognized by the high central ventricular crest flanked by two superimposed crests made by the auricles and bulbus. (6) If a time tracing be added by means of the chronograph one may determine the time relations of the different phases of the heart cycle. D. To Observe the Movements of the Mammalian Heart. 1. Appliances. Dog or rabbit; operating case, supplemented by haemostatic forceps, heavy scissors and scalpels, clippers, heavy linen thread; hand bellows with tube and respiration cannula (see THE CIRCULATION OF THE BLOOD 77 Appendix, 11); animal holder; morphine solution with hypodermic syringe ; chloroform or ether ; tannic acid ; absorbent cotton ; porcelain- lined trays for instruments; cotton, calipers, and rule. 2. Preparation. The dog of medium or large size is to be pre- ferred for class demonstrations, while rabbits or small dogs may be used for laboratory work by students. When rabbits are used anaesthetize with ether. When dogs are used anaesthetize with chloroform after having given ^ to 1 grain of morphine hypodermic- ally fifteen minutes before the use of the chloroform. Make a litre of one-half saturated solution of tannic acid to be used as an hpemostatic. 3. Operations. (1) To Induce Artificial Respiration. The open- ing of the thorax causes the lungs to collapse, and if artificial respi- ration were not instituted the animal would die in convulsions in a few minutes. The successful induction of artificial respiration involves the opening of the trachea, insertion of respiration cannula, and the maintenance of respiratory movements of the lungs through the use of the bellows. Clip the hair from the ventral surface of the neck; make a median cutaneous incision; with forceps and fingers separate subcutaneous tissue, fascia, and muscles over the middle of trachea, and clear one to two inches of the trachea; cut a longitudinal ventral slit into the trachea and insert tracheal end of respiration cannula, ligating it firmly in place. The animal will now breathe through the cannula. When the thorax is opened — but not before — the bellows should be attached to the cannula through the medium of a rubber tube at least one foot in length, and the bellows should then be brought into rhythmical action, causing the lungs to fill eighteen to twenty times per minute in the case of a dog (twice as fast for the rabbit). After the introduction of the cannula and before the bellows is attached apply the anaesthetic to the distal end of the cannula. When the l)ellows is attached the anaesthetic must, of course, be applied to the intake valves of the bellows. (2) To Expose the Heart. After the introduction of the respiration ■cannula, make a median incision over the sternum from anterior tip to posterior end of the xiphoid appendix. Strip the skin back laterally as far as the junction between the ribs and the costal carti- lages. Saturate with tannic acid solution strips of absorbent cotton large enough to cover all cut surfaces. With strong scalpel cut through the thoracic wall at the junction of the first left rib with its cartilage, carrying the incision (piickly back along the thorax parallel to the sternum until all cartilages are cut. The cut-oft' ends of intercostal arteries will bleed freely, but this can be .stopped in a moment by folding a strij) of al)sorbent coiUiW wet with tannic acid over the cut-off ends of the ril)s. A 78 SPECIAL PHYSIOLOGY strip of dry cotton and a towel may be placed outside of the tannic acid cotton. Before proceeding farther, note that the left lung is collapsed. Begin artificial respiration, continuing the rhythm observed in the animal. Carry the incision transversely across the thorax just posterior to the end of the sternum; catch the cut-off internal mammary arteries with haemostatic forceps; carry the incision forward along- the right side to correspond with the incision already made on the left side, and stop the hemorrhage in the same way. The sternum may be covered with absorbent cotton and a towel and tipped forward out of the way. The heart is now clearly exposed within its. pericardium, and its relation to other structures of the thoracic cavity may be carefully noted before the pericardium is removed. 4. Observations. (1) Note position of heart with relation to lungs, spinal column, diaphragm, oesophagus, trachea, and large bronchi. (2) Note character of pericardium and its attachments. Remove the pericardium by making a free longitudinal incision with scissors and slipping the heart through the incision. Note character of inner surface of pericardium; of outer surface of heart; presence of liquid in the pericardium. (3) Note sequence of contraction of the chambers of the heart. (4) Note change of shape of the heart during several phases of a cardiac cycle. (5) Note change of position of the heart apex during phases of a cardiac cycle. (6) Hold the beating heart in the hand and note the change in the tension of the heart muscle during phases of cardiac cycle,, comparing diastole with systole. (7) With calipers and rule measure carefully changes in the diameters of the heart, comparing end of diastole with the end of systole and observing the lateral diameter and the dorsoventral diameter. (8) Is there a change in the anteroposterior diameter — base to apex? If so, when does this change occur? (9) "Push the anaesthetic" to the limit and note that the animal's heart continues to beat. The same amount of ether or chloroform administered under ordinary conditions would cause the death of the animal through the stopping of respiration. But the respiration being carried on artificially, the amount of chloroform which can be taken is much increased. In the case of the dog, it will be hardly possible to kill with chloroform so long as respiration is kept up. If the respiration be stopped the animal will die very soon in con- vulsions. THE CIRCULATION OF THE BLOOD 79 To terminate the experiment open the right ventricle. The thoracic cavity will quickly fill with blood and the animal will die a quick and painless death, free from any convulsions. II. THE APEX BEAT AND THE HEART SOUNDS. 1. Appliances. A cardiograph, consisting of a receiving tambour and a recording tambour (Fig. 44). The receiving tambour should be about 4 cm. in diameter and not less than 1 cm. deep. The tambour membrane should be of dentists' rubber-dam and should be stretched tightly enough to give it a resistance about equal to that of the relaxed biceps muscle. Upon the middle of the membrane a small cork (1 cm. long) is glued. Fig. 44 The cardiograph : R, receiving tambour provided with a rubber membrane (m) and a cork button (if) to be placed on the apex beat. The receiving tambour is joined through the rubber tube H to the tracing tambour T, whose lever (L) records the movements of the thoracic wall upon the kymograph K. The recording tambour should be 3 cm. to 5 cm. in diameter and not more than 3 mm. in depth. The tracing lever should be at least 20 cm. long and provided with a delicate celluloid or parch- ment tracing point. The recording tambour should be mounted on a light chemical stand and held by a universal clamp holder. The two tambours sliould be joined through a piece of pressure tubing two feet in length. For construction of tambours see Appendix, 12. Besides the cardiograph one will need a chronograph, a kymo- graph, and a stethcscope. 30 SPECIAL PHYSIOLOGY Study the new instruments and make drawings and diagrams showing their construction. 2. Preparation. Let a student remove the clothing from the chest. Find the apex beat. In which intercostal space is it located ? How far is it to the left of the middle of the sternum? Is the loca- tion of the apex beat the same for all members of the class? In recording the location of the apex beat refer to the bony landmarks of the chest rather than to the nipple. To take a cardiogram place the button (cork) of the receiving tambour upon that point of the thorax most affected by heart beat. The movements of the apex of the heart will be transmitted and magnified by the cardiograph. Trace a cardiogram upon the kymograph. 3. Observations. (1) Take several cardiograms from the same individual, being careful so to adjust the apparatus as to gain the maximum excursion of the lever. What features have all of these tracings in common? What features seem to be accidental and non-essential ? What are the causes of the essential features ? What are the sources of the non-essential features? (2) Take cardiograms of several individuals. Do all of them possess the features which seemed essential in the first series, taken from one individual? If not, how would you account for the differ- ence. (3) With a stethoscope, whose construction you have carefully described in your notes, listen to the heart sounds while the cardio- graph is tracing the record of the heart movements. Note that two sounds are audible and that there is a notable pause following the shorter, sharper sound; let us call the sound which succeeds the pause the first sound. (4) With what part of the cardiogram does the first sound seem to correspond ? With what part of the cardiogram does the second sound seem to correspond? Give reasons for this correspondence. (5) As far as the data will admit, enumerate causes for the first sound; for the second sound; for the essential features of the cardio- gram. Can one locate on the cardiogram that crest or feature which corresponds to the auricular systole? The ventricular systole? The recoil of the ventricles? The closure of the semilunar valves? The opening of the semilunar valves? (6) Giving full attention to the auscultation of the cardiac region of the chest with the stethoscope, note carefully: (a) The point where the first sound is most distinctly heard. Locate this point with reference to the thoracic skeleton, (b) The point where the second sound is most distinctly heard. Locate same with reference to skeleton. (7) Compare the two sounds as to duration, intensity, pitch, and quality. THE CIRCULATION OF THE BLOOD 81 III. THE FLOW OF LIQUID THROUGH TUBES UNDER CONSTANT PRESSURE. Fig. 45 The problems presented by the circulation of the blood through the bloodvessels involve some of the general principles of hydraulics. The supply of blood to the various glands and other active tissues of the body is analogous to the supply of water to the buildings of a city. The blood-circulatory system differs from the water-circulatory system in possessing elastic tubes instead of inelastic ones, and an intermittent initial force instead of the constant force furnished by the "head" of water in the reservoir or stand-pipe. It will be profitable for the student to make a few simple experiments in hydraulics in order to make himself familiar with those physical laws which he will apply later. 1. Appliances. Each table is provided with a reservoir (Fig. 45) consisting of a galvanized-iron reservoir about 10 cm. in diameter and 70 cm. in height, with a sup- ply tank above. At the bottom of the reservoir there is a faucet, to which may be screwed a 6-mm. nozzle or a 3-mm. nozzle, thus varying the radius of the outlet stream. The reservoir is supplied with a gauge which indicates the height of the water above the middle of the outlet nozzle. Each table will need besides the reservoir tliree 6-mm. T-tubes and five 6-mm. glass tubes 50 cm. long; also ten rubber connec- tors, a screw clamp, and centimetre rule. Provide a large flask or jar for catching discharge and a .500-c.c. graduated cylinder for measuring the discharge. 2. Preparation. We have thus a means of varying the radius of the outlet and the height of the water above the outlet. These are the two factors upon which the fjuantity of the discharge Reservoir for use in experi- ments in hydraulics, and illus- trating principles underlying circulation of the blood. 82 SPECIAL PHYSIOLOGY an orifice (or nozzle) in a reservoir is equal to the velocity which would be acquired by a body falKng freely through a height equal to the distance between the orifice and the surface of the liquid." Make out a table which will show for each of the first five seconds of a falling body the distance traversed {d); the velocity (v); the total height at the end of each second respectively {h) ; and derive from this table the value of velocity in terms of g ((7 = acceleration of gravitation, 32 + ft. or 981 cm. per second) and of t (^==time in seconds). (1) V = gt. (2) H = f. Eliminate t from these two equations and express the value of velocity (V) in terms of the acceleration of gravitation {g) and the height (h). (3) V = y'2gH = 1/2x981 H=44.3 -j/hT How does the velocity vary in terms of height? The velocity varies as the square root of the height. (4) V x> y^S: Given the height of the water in the reservoir Qi) and the radius of the nozzle (r), to compute the discharge (D). (5) D ^ area X velocity. (6) D = 7rr« X 44.3 "i/h = 44.3 7rr= i/h = 139.2 r« -j/hT The discharge will vary as the product of the square of the radius multiplied by the square root of the height. (7) Door^-j/h. 3. Observations. To test the influence of the radius and the pressure upon the discharge one uses the law (expressed in D oo r^ l/h). given above. Note that the discharge varies with two different factors. It is a fundamental principle governing all experimental work, that, where one is studying a quantity which varies with two or more factors, he makes all but one of the factors constant and allows the quantity in question to be modified by only one variable factor at a time. We will, therefore, make the radius constant by using the small nozzle while we observe the discharge as modified by varying height. (1) Take the discharge in c.c. through 3-mm. nozzle at ^ = 36 cm.; ^=49 cm.; ^ = 64 cm.: D : d : : i/'H : i/hT (2) Take the discharge in c.c. through 6-mm. nozzle at ^ = 36 cm.; ^=49 cm.; A, = 64 cm. In these observations maintain a constant height in the reservoir by letting water flow in from the supply tank. THE CIRCULATION OF THE BLOOD 83 Use a time unit of ten seconds. Repeat each observation at least three times. (3) From the above results compare influence of a varying radius when the height is constant — i. e., discharge at h = 36, through 6-mm. nozzle; through 3-mm. nozzle. D : d : : R* : r2. (4) Having tested the two variables separately, test the two com- bined variables. D : d : : R^ ^ / g . jS -j/t. (5) To determine the relation of discharge to resistance: Attach to the larger nozzle one length of 6-mm. tubing. Note the discharge in, say, ten seconds. Attach a second length of 6-mm. tubing, taking care that the tubing is approximately horizontal. Note the dis- charge in the same length of time. What is your conclusion? Why does the discharge decrease when the length is increased? Fig. 46 '.V I II III 1/ T Reservoir with piezometers. 71 "^^ (6) To measure the pressure at various points along the course of the fli.scharge tube: (a) Insert a 6-mm. T-tube with an upright limb not less than 50 cm. in length between the two 6-mm. discharge tubes. Is the height of the water in the upright (piezometer) as great as in the reservoir? (h) Add another T-tube to the end of the .second 6-mm. discharge tube; how high does the water rise in the second piezometer? Comparing the height of the water in the reserv'oir and the two piezometers, what are your conclusions as to the j)ressure in different parts of the discharge tui)e? (7) By leaving out the 50 cm. tubes and setting the T-tubes end to end thus (J. X J. JL J. J.) a set of piezometers similar to those shown in Fig. 46 can be set up and new observations made. 84 . SPECIAL PHYSIOLOGY IV. THE FLOW OF LIQUIDS THROUGH TUBES UNDER THE INFLUENCE OF INTERMITTENT PRESSURE. A. The Influence of Intermittent Pressure. 1. Appliances. A glass tube of about 6-mm. lumen and about 100 cm. long; a thin-walled elastic tube of about the same lumen as the glass tube and about 100 cm. long; a double-valved, strong rubber bulb (about 7.5 cm. long); very thick-walled rubber tubing for joining up the apparatus; a 2-litre jar and a flask or water recep- tacle; heavy linen thread; a wide capillary and a jfine capillary or a piece of glass tubing 10 cm. long for constructing the same; 500-c.c. graduated cylinder; piece of 8-mm. rubber tubing about 50 cm. long. 2. Preparation. Join the large elastic tube to the entrance valve of the bulb. Couple the glass tube closely to the exit valve of the bulb. Make all joints as close as possible, and tie tightly with thread. Draw a coarse and fine capillary tube from the 10-cm. piece of glass tubing. Fill the jar with water and immerse the tube from the entrance valve in the water. Clasp the bulb in the hand and make rhythmical contractions at the rate of ten to fifteen in ten seconds. This process will, of course, pump water from the jar into the flask held at the distal end of the long glass tube. One person should pump the bulb and the greatest care should be taken to exert the same force and use the same rate in the several observations. 3. Observations, a. Intermittent force and inelastic tubes. (1) Does the stream of water which is ejected from the exit tube flow in a constant or in an intermittent jet? (2) Attach a wide capillary and repeat. What is the character of the stream? Measure the discharge in ten seconds. (3) Attach a fine capillary and repeat. Measure the discharge in ten seconds. b. Intermittent force and elastic tubes. (4) Disjoin the glass tubing from the bulb and join the 100-cm. elastic tube. Work the bulb as directed above and observe the character of the flow. Measure the quantity of discharge. (5) Join on the coarse capillary and repeat, noting the change in the character of the jet and the amount of discharge. (6) Replace the coarse capillary with the fine capillary and repeat. Sum up results and formulate conclusions. B. The Pulse or Impulse Wave. By putting the finger upon the rubber tube while the bulb is in action the pulse may be felt. To trace this upon the kymograph THE CIRCULATION OF THE BLOOD 85 lay the rubber tube across the frog-board myograph and pass a thread from the proximal end of the board around the pulsating tube and thence to the thread-eye of the tracing lever as shown in Fig. 47. A block or cork will hold the tube in place. Pulsations of the tube will be transmitted to the thread and in turn to the lever and may be traced upon the kymograph. Observations. (1) If the finger be held upon this elastic tube while the bulb is being rhythmically squeezed a series of impulses or pulsations will be felt by the finger. Place one finger upon the elastic tube near the bulb; another finger near the capillary. Let the bulb be pumped with sudden but infrequent contractions. May one note the difference in the time of pulsation felt by the two fingers? If so, which is felt first, and why? What is the cause of the pulsation? Fig. 47 Myograph in use as a pulse-writer : K, kymograph ; L, tracing lever ; 5, short arm of elbow lever ; .If, section of frog-board myograph ; T, cross-section of rubber tube ; C, block of cork against which the tube rests ; WT, weight-link ; P, pivot. (2) To get a tracing of this pulse, pass the rubber tube across the cork board as shown in the figure; adjust to kymograph and 'take tracing. Vary the character of the bulb contractions as follows, taking one complete rotation of the drum for each variation: (a) Slow initial contraction of bulb and slow relaxation. (h) Slow initial contraction of })ulb and quick relaxation. (c) Quick initial contraction of bulb and slow relaxation. (d) Quick initial contraction of bulb and quick relaxation. (e) Same as {d) with slow rhythm (1 contraction per second). (/) Same as (d) with rapifl rhythm (3 contractions per second). f3) Make a careful study of these tracings and determine: (a) The characteristic and essential features. (h) The accidental and non-essential features. (c) The cause of the essential features. (d) The cause of the non-essential features. 86 SPECIAL PHYSIOLOGY V. THE LAWS OF BLOOD PRESSURE DETERMINED FROM AN ARTIFICIAL CIRCULATORY SYSTEM. Having tested by experiment some of the laws of governing the flow of liquid through tubes under the influence of intermittent pressure, we come to the point where we may attempt to reproduce experimentally a set of physical conditions so nearly like those which exist in the animal body that we shall be able to draw conclusions from our experiments that shall hold good for the animal circulatory system. The last preceding exercise demonstrated (1) that the contin- uous and even flow of liquid through the capillaries is made possible by the elasticity of the arterial walls; (2) that the pulse is caused by a varying pressure within the elastic artery; (3) that the varying pressure is due to the alteration of systole and diastole of the heart; and (4) that the pressure within the arteries is largely influenced by the size of the capillary through which the fluid must pass — i. e., by the peripheral resistance. Blood pressure is then the product of two factors: Cardiac force X peripheral resistance ( P = H X R); but cardiac force is in turn due to the product of two factors: Rate X strength; (H = r X s); therefore: Pressure is the product of heart rate X heart strength X 'peripheral resistance (P=r X s X R)- We have here to deal with these three variables. Applying a principle set forth in a previous exercise (to the effect that "when a value which is being tested by experiment is affected by two or more variable factors only one of these must be allowed to vary in any one experiment") one will so arrange his experiment that these three factors of pressure will vary one at a time. 1. Appliances. An artificial circulatory system may be con- structed as foUows: A rubber bulb such as used in the preceding exercise, to which is attached a capacious entrance tube. To the exit tube attach the 100-cm. soft-rubber tube used before. This will serve as the main artery, at the end of which a T-tube may be inserted, one limb passing to the arterial manometer. Beyond the T-tube is another rubber tube leading to a Y-tube. From each limb of the Y-tube lead off a smaller elastic tube, one branch being a small, thin- walled tube supplied with a screw clamp, while the other passes to a large calcium chloride tube which has been filled with sponge to represent the capillary system of minute tubes. (See Fig. 48.) After traversing the capillary system the liquid is collected at a Y and returns to the heart through a tube which is nearly twice as large as the artery. In this vena cava is inserted a T-tube, to which is attached the venous manometer. Between the Y-tubes the blood may be opposed by high resist- ance or, with the screw clamp open, by the low resistance. THE CIRCULATION OF THE BLOOD 87 The bulb may be pumped weak or strong, fast or slow, while the peripheral resistance may be high or low. We have, therefore, a contrivance through which we are able to vary one factor at a time. The arterial manometer should have limbs not less than 50 cm. in length, while those of the venous manometer need not be more than one-half that length. These manometers may be held by clamps to chemical stands which are on or beside the table. 2. Preparation. Set up an artificial circulatory system as shown in Fig. 48. Pig. 48 Fig. 49 o'>X Artificial circulatory syBtem, describerl in detail in text. Mercury manometer. After the sy.stem is set up make a study of the mercury manom- eters, the instruments with which the pressure is to be measured. The specific gravity of mercury is approximately 13.(). ^^hat is the gas pressure at n that will cause a rise of 4 cm. of mercury in the distal tube? (See Fig. 49.) What is the water pre.ssure at n that will cause a rise of cm. of mercury in the distal tube? What is the water pressure at n that will cause a rise of m cm. of mercury in the distal tube? After the system has been freed from air and is at rest, do the pro.ximal and distal columns of mercury in the arterial manometer 88 SPECIAL PHYSIOLOGY stand at the same lever? If not, why? What allowance, if any, should be made for this? 3. Observations. (1) By experiment fill out the following table: Observation. Heart a Strength. etivity. Rate. Peripheral resistance. Arterial manometer. "Venous manometer. 1 2 . 3 . 4 . 5 . 6 . 7 8 . Weak Weak Weak Weak Strong Strong Strong Strong Slow Slow Fast Fast Slow Slow Fast Fast Low High Low High Low High Low High mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. mm. (2) Trace the pulse upon the kymograph as indicated in the foregoing lesson. (3) What are the principal factors which control blood pressure? (4) State concisely just what effect these factors have upon blood pressure. (5) What combination of conditions yield the highest arterial pressure ? (6) What set of conditions yield the lowest arterial pressure? The highest venous pressure? The lowest venous pressure? VI. THE RADIAL PULSE AND THE SPHYGMOGRAM. 1. Appliances. A sphygmograph; tracing slips; a fish-tail gas jet or kerosene lamp, and a holder in which to place the slips while they are being smoked (Fig. 50). Fig. 50 Holder for smoking slips for the Dudgeon sphygmograph. 2. Preparation. That the sphygmograph is so little used by the general practitioner may be attributed to the fact that hurry of business or some other cause has hindered him from making himself thoroughly conversant with the adjustment and use of the instrument, with its limitations and with the interpretation of the tracings. THE CIRCULATION OF THE BLOOD 89 To Adjust the Sphygmo graph. (1) Let the observer stand with his right foot on a chair. This brings his thigh into a horizontal position. (2) Let the snbject stand at the right of the observer, resting the dorsal snrface of the left forearm upon the observer's knee. (3) Let the observer with pencil or pen mark the location of the radial artery. (4) Let the observer wind the clockwork which drives the tracing paper; adjust the latter in readiness for tracing; rest the instru- ment upon the subject's arm with its foot on the radial artery and adjust the position, tension, and pressure in such a manner as to obtain the maximum amplitude of swing of the tracing needle. Take the tracing. Fix in damar-benzole solution. 3. Observations, a. The Location, etc., of the Radial Artery. (1) What are the relations of the radial artery at the distal end of the radius ? (2) How may the relations vary? (3) Is there any variation, among the members of the division, in the location of the radial artery? (4) ^lay excessive muscular development affect the ease with which the artery may be located and its pulsations studied? (o) ^lay excessive deposit of adipose tissue hinder the observations of the pulse? (6) May faulty position of subject or of his clothing affect the pulse ? b. The Digital Observation of the Radial Pulse. (7) Feel the pulse with the side or back of the finger, then with volar surface and tip of each finger of each hand, and note the finger or fingers with which the feeling is most acute. It will be wise to always use these fingers in all tactile examinations. Their acuteness of feeling will increase with practice. One may thus acquire the educated touch — tactu-f eruditus. (5) How much may be learned of the pulse by means of the touch alone? Observe and note: (a) rate; (b) rhythm; (c) volume; {d) strength; ie) compressibility; (/) may anything else be determined by this method ? c. The Sphygmogram. (9) Take at least three pulse tracings of each individual in the division, (a) Compare the tracings taken from one individual; if they cHffer, determine the cause of the differ- ence, (b) Compare the tracings of (h'fferent members of the division. Determine, if possible, the cause of the differences. nOj 1 )o variations of the relations of the artery affect the s})hygmo- gram? Does the adjustment affect the sphygmogram? Does the elasticity of the artery affect the tracing? How does strength or rate of heart beat affect it? Make a list of the facts regarding the condition of the circulatory 90 SPECIAL PHYSIOLOGY system which may be determined with the help of the sphygmograph. Make a hst of the precautions to be observed in the use of the sphyg- mograph. The Carotid Pulse. One will frequently experience difficulty in taking the radial pulse; in fact, not more than one person in three or four is a favorable subject for this observation. The reason for this is not far to seek. Anything beyond a moderate development of the musculature of the forearm is accompanied by such development of the tendons and of the styloid process of the radius that the artery is quite in- accessible for the sphygmograph-foot as usually constructed. A moderate deposit of subcutaneous fat also obscures the radial pulse and makes the use of the sphygmograph most unsatisfactory. Fig. 51 Porter's carotid sphygmograph : R, receiving tambour in form of open bell, that is pressed against the throat over the common carotid ; T, tracing tambour with small, thin membrane and light lever. The simple sphygmograph devised by Dr. Porter, of Harvard, enables one to overcome some of the difficulties mentioned above. This instrument consists (1) of a very small and delicately adjusted recording tambour with high magnification and a delicate tracing point; (2) of a receiving tambour not over 2 cm. or 3 cm. in diameter and at least 1 cm. deep, connected with the recording tambour through a 50-cm. piece of pressure tubing, which is provided with a side vent closed by a clamp. For the receiving tambour a small thistle tube may be used. (See Fig. 51.) THE CIRCULATION OF THE BLOOD 91 To trace the carotid pulse, place the open mouth of the receiving tambour, over which no membrane has been stretched, over the course of the carotid arterv beside the hirvnx, taking care that the side vent of the pressure tube is open while the adjustment is being made. Close the vent, and if the adjustment has been successful the lever will show the carotid pulse. Trace it upon the kymograph. Compare the carotid sphygmogram with the radial sphygmogram. Account, if possible, for any essential difference. One may trace a radial sphygmogram with the same instrument by stretching a rubber membrane rather tightly across the mouth of the receiving tambour cementing a bone collar-button to the middle of the membrane then placing the head of the collar-button upon the radial arterv. VII. TO DETERMINE THE ARTERIAL BLOOD PRESSURE IN AN ANIMAL. 1. Appliances. Dog or a large rabbit; mercurial manometer, with manometer tambour. (See Appendix, 13.) In lieu of the manometer tambour (Fig. 52) one may use the ivory float with tracing point in the distal limb of the manometer; kymograph; chronograph; physiological operating case; glass arterial cannulte; half-saturated solution of sodium carbonate, sodium sulphate, or magnesium sulphate; clippers; dog board or rabbit holder; absorb- ent cotton; one-half grain of sulphate of morphine; hypodermic .syringe; chloroform and ether. 2. Preparation. The manometer should be filled with mercury with at least 10 cm. in each limb. Attach to the proximal limb of the manometer a piece of pressure tul)ing G or S inches in length, to which attach one limb of a T-tube. To the opposite limb of the T-tube attach another piece of pressure tubing not less than a foot in length, into the end of which the glass cannula can be placed. To the side limb of the T-tube attach a rubber tube, which may be carried upward to an inverted flask filled with the non-coagulant (XajCO,, XajSO^ or MgSOj. The tube leading from the reservoir shoulfl have a screw clamp near the T-tube. The reservoir may be suj)})orted near the top of the same stand which supports the manometer. If a dog is used for the ex})erinient he should be given from -\ to ^ grain of morphine twenty minutes before the an.esthesia with chloroform. If a large rabbit is used anaesthetize with ether. '.). Operation. After fastening the animal to the holder, clip the throat, make an incision from the uj)per end of the sternum over the right sternothyroid nuiscle to the middle of the neck, cutting 92 SPECIAL PHYSIOLOGY through the skin and subcutaneous tissue to the surface of the muscle. The external jugular vein lies close to the incision externally. Sponge the oozing vessels until hemorrhage is checked, then using forceps and fingers dissect away the fascia until you reach the external margin of the sternothyroid muscle. Separate this muscle to the inside, making an opening down to the carotid artery, where pulsation may be felt near the trachea. Lifting the sheath which contains the carotid artery and the vago-sympathetic nerve, taking care not to- Fig. 52 The manometer tambour : if, manometer ; Tb, tambour ; R, reservoir filled with one-half saturated solution Na.COa; T, T-tube ; Pt, pressure tube: CI, clamp; C, cannula; P, proximal hmb of manometer ; D, distal limb of manometer ; t, tracing point of tambour lever. wound the internal jugular vein, which lies in close relation to these structures, tear open the sheath of the artery and nerve and separate out the carotid artery to the extent of one or two inches. Choose a glass cannula not larger than the carotid; place the large end of the cannula in the pressure tubing attached to the manometer. Open the screw clamp and allow the tubes to fill with the solution clear to the point of the cannula. Close the screw THE CIRCULATION OF THE BLOOD 93 clamp to stop the flow of the sokition from the reservoir and do not open the clamp after this except to clear the cannula of a clot; and this cannot l)e done, of course, while the cannula is in the arterv. Ligate carotid artery at the upper end of the incision. Clamp the lower portion of the carotid with the seraphin forceps, place the finger under the artery, make a longitudinal incision in the middle of the exposed portion of the carotid. Insert the point of the cannula into the lumen of the artery, tie the cannula in place, and remove the seraphin forceps. 4. Observations. As soon as the seraphin forceps have been removed the blood will rush into the cannula and tube for a dis- tance of 4 to 8 cm., the mercury will rise in the distal limb of the manometer to a corresponding degree. (1) Measure this rise in the distal limb of the manometer. What is the blood pressure in centimetres of mercury per unit area? (2) Note that the mercury rises and falls in the manometer with a rh\i:hmical motion. Attach the manometer tambour or adjust the float and watch the movements of the tracing point. Feel the pulse of the animal and note whether the movements of the tracing point correspond to the heart beats. (3) Bring the kymograph into position, adjust the tracing point of the blood-pressure apparatus, also the chronograph, and take a tracing. What is the rate of heart beat? (4) Are the respiratory movements evident in the tracing? If so, what is the influence of inspiration upon blood pressure? What is the influence of expiration? Account for the influence of respira- tory movements upon blood pressure. (5) What causes the blood pressure to rise during inspiration? Modification in blood pressure must be due either to the rate or strength of the heart beat or to the condition of peripheral resistance. (6) If a line were drawn through the lowest point of the individual cardiac waves, this waving line would represent the influence of respiratory movements upon blood pressure. If the lowest point of these respiratory waves were joined by a line, would this line be a straight one or would it be a long, undulating curve? If such a curve is observed, it may be recognized as the Traube-Hering curve. This curve represents a gradual rise and fall of the l^lood pressure under the influence of changing peripheral resistance, which in turn is controlled bv the vasomotor nerve centres. VIII. THE SPHYGMOMANOMETER AND PULSE PRESSURE. \'arious clinical instruments have been devised for the purpose of determining blood pressure in the human subject in health and in disease. The most satisfactorv of these devices involves the use 94 SPECIAL PHYSIOLOGY of the mercury manometer in measuring the pressure in a pneumatic arm-girdle so adjusted as to suppress or to modify the pulse. An accurate determination of blood pressure is occasionally of very great importance, and it goes without saying that methods used on the lower animals are not applicable in the case of man because they involve the opening of an artery. The only appliafice needed is the sphygmomanometer (Fig. 53) and the only preparation is for a member of the class to remove clothing from one arm. Observations. (1) Let the subject lie upon his back on the table in an easy and comfortable position, and absolutely relaxed and quiet for five minutes. During this period the girdle may be Fig. 53 The sphygmomanometer : G, arm-girdle with inflatable rubber tube (t) within and sole-leather belt (6) without ; P, pressure bulbs ; m, mercury manometer. fastened about the right arm. While one observer is counting the pulse at the left wrist, another may feel the right pulse. A third observer may watch the manometer while he gradually pumps air into the girdle until the pulse is shut off at the wrist. Read the manometer, relax the girdle pressure until the pulse reappears. Read the manometer. The mean between the two readings as thus made is taken to represent the pulse pressure. Record the pulse rate as counted on the left pulse. Record the pulse pressure as determined by the sphygmomanometer. (2) Let the subject lie on his right side. Take observations as outlined above and record pulse rate and pulse pressure. THE CIRCULATION OF THE BLOOD 95 (3) Let the subject lie on his left side. Record results. (4) Let the subject sit. Record results. (5) Let the subject stand. Record results. (6) Let the subject take vigorous exercise for five minutes. Take observations of pulse rate and pulse pressure while the subject stands. (7) From the tabulated results of the above observations write in a list the postures and conditions which give an increasing series of pulse pressures. (S) Prepare a similar list of the postures and conditions which give an increasing series of pulse rates. (9) Are these two series alike in the order in which the conditions are named? — i. e., do the conditions which give high rate give also high pressure? Account for what you discover. (10) If pulse rate increases, under what conditions could pulse pressure fall {P=Hr X Hs X R)"^ IX. TO DETERMINE THE INFLUENCE OF THE VAGUS NERVE UPON THE ACTION OF THE HEART. 1. Appliances. Operating case; a pair of curved, blunt-pointed shears, or, better, a pair of barber's clippers; a rabbit board; a large sheet of heavy paper; cotton; ether; thread; one dry cell; induc- torium; shielded electrode (Fig. 54); seven wires; stethoscope; a rabbit; contact key; short-circuiting key. Fig. 54 A shielded electrode of hard rubber, bearing copper or platinum wires. 2. Preparation. Let six or eight students be divided into three or four groups of two each. Let the group "a," be responsible for the anaesthesia. Use the sheet of heavy paper to make a conical hood, whose spiral turns may be held in place with sealing-wax or pins. Place a wad of cotton loosely in the mouth of the cone. Let the group "b" perform the operation. Tie the rabbit back downward upon the holder; fix the nose in special holder; with the barber's cjij>pers remove the hair from the ventral side of the thorax and neck; make hanfls and instruments clean; place instruments in shallow basin of warm water; cut two or three ligatures of thread anri place them in the in.strument basin. Let the group "c" arrange the electric apparatus for stimulation 96 SPECIAL PHYSIOL OGY of the nerves. Fill the cell; join up with contact key in the primary circuit, and a short-circuiting key in the secondary circuit. Test the apparatus to see if everything is in order. Group "d" should keep all the records of pulse or other observations. 3. Operation. Group "a." (1) Pour 1 c.c. or 2 c.c. of sulphuric ether upon the cotton in the cone; place the cone over the rabbit's nose; observe and note carefully the three stages of anaesthesia. (2) Carefully note the rate of the heart before beginning anaes- thesia, and the influence of anaesthesia upon rate and strength of heart and respiration. (3) Keep the cotton moist with ether; watch the respiration and pulse, and be careful not to give the animal too much and thus interrupt the experiment. Group "b." Wash the clipped surface of the throat. After the rabbit is completely anaesthetized, make with scissors a median incision through the skin, beginning at the anterior end of the sternum and cutting anteriorly for about 5 or 6 cm. ; divide the subcutaneous connective tissue over the middle of the trachea. Carefully separate from the median line on either side laterally the subcutaneous con- nective tissue with the associated adipose tissue. How many pairs of muscles come to view? What two muscles approach the median line to form the apex of a triangle at the anterior end of the sternum? Observe a pair of thin muscles lying dorsal to the muscles just mentioned, and joining them in the median line to form a thin muscle sheet covering the trachea on its ventral side. What muscles are these? Carefully lift up the median edge of the sternomastoid muscle and separate with the handle of a scalpel or a seeker the delicate intermuscular connective tissue. A bloodvessel and several nerves come into view. Is the bloodvessel an artery or a vein? How many large nerves accompany the bloodvessel? Take hold of the sheath of the vessel; lift it up and note in the connective tissue accompanying the bloodvessels two nerves, one large and one small. When the artery is in its normal position, what relation do these two nerves sustain to it ? Which of the two nerves is external and which is dorsal to the bloodvessel? Which is in close relationship with the artery? The larger of the two nerves is the vagus or pneumogastric. In preparing the nerve for stimulation one should neither grasp it with the forceps nor with the fingers. It may be separated from the delicate connective tissue in which it lies by use of a blunt seeker. Far better than any metallic instrument is a small glass rod drawn to a point, curved and rounded in the Bunsen lamp. Prevent the tissues drying up by occasionally pressing them lightly with pledgets of cotton moistened with normal salt solution. Adjust the electrode THE CIRCULATION OF THE BLOOD 97 carefully upon the vagus and see that no unnecessary tension is allowed to be exerted upon the nerve. It is usually necessary to hold the electrode in place during the observations. 4. Observations, a. Anaesthesia. (Observations of Group "a.") (1) Are you able to make out the different stages of anaesthesia ? (2) How many stages did your animal manifest? (3) Give the characteristics of each stage? (4) AVhat effect did the ether have upon the rate of heart beat? (5) What effect did ether have upon respiration? b. The Stimulation of the Vagus. (Observations of Groups "c" and "d.") (6) Stimulate moderately one vagus. Note with a stethoscope any change in the rate of the heart. (7) Cut both vagi high up in the neck. Note the rate of heart beat at intervals of five minutes for thirty minutes, allowing the rabbit to partially recover from the anaesthesia. (8) Stimulate one vagus. Compare the result with that obtained under experiment (6). (9) Will very strong stimulation bring the heart to a standstill? (10) If the heart were brought to a complete standstill by the stimulation, will it start up spontaneously when the stimulus is removed? Will the rate be the degree of acceleration observed in experiment (7)? (11) Sum up the observations into a concise statement as to the influence of the vagus upon the heart. Note. Dispatch the rabbit with chloroform. X. TO DETERMINE THE INFLUENCE OF THE CARDIAC SYMPATHETIC NERVES UPON THE ACTION OF THE HEART. The appliances should be the same as for the preceding exercise. Let the students who work at one table continue the same grouping that was arranged in the preceding exercise, but rotating in the work: Group "a" to operate; group "b" to arrange electric apparatus and stimulate nerve; group "c" to note pulse rate and keep records; group "d" to give anaesthetic. The operation should be similar to that of the vagus experi- ment. Find the cardiac branch of the cervical sympathetic in the lower part of the neck, where it is in close relation with the carotid artery and the internal jugular vein. The most certain way to recognize it is through its function. Carefully separate out the nerve trunks in the region described; with the glass nerve hook lift up any nerve except the vagus and stimulate nuxlerately. 7 98 SPECIAL PHYSIOLOGY Stimulation of the cardiac sympathetic distinctly increases the pulse rate. Find the corresponding nerve of the opposite side, verifying your choice by observing the effect of stimulation. Cut both cardiac sympathetic nerves and observe the rate of the heart beat at intervals of five minutes through a period of thirty minutes. XI. THE INFLUENCE OF THE VAGUS AND THE CARDIAC SYMPATHETIC UPON THE ARTERIAL BLOOD PRESSURE. 1. Appliances. Dog or large rabbit; animal holder; mercury manometer, with float or tambour and with flushing flask of non- coagulant; with tubing and cannulse, as described in Appendix A, 12; a kymograph, inductorium; Daniell cell; two Du Bois-Reymond keys; operating case; chloroform, ether, morphine; hypodermic syringe. 2. Preparation. Let eight students in four groups of two each have charge of (a) anaesthesia, (6) operations, (c) electric apparatus and stimulation, {d) pressure tracings. 3. Operations, (a) Aneesthetize the animal in accordance with directions given in previous exercises for the dog and rabbit, respec- tively. (h) Remove the hair from the throat; make a cutaneous incision in the median ventral line from the anterior end of the sternum to the anterior end of the larynx. Remove the subcutaneous tissue and expose the sternomastoid and sternothyroid muscles. Let one operator expose the carotid artery of one side while the other operator exposes the vagus and sympathetic of the other side. (c) Adjust the shielded electrode for stimulation. Insert the cannula into the artery. {d) Make the tracing of arterial blood pressure in accordance with directions given in a previous exercise. While the tracing is in progress stimulate the vagus with a moderate tetanizing current for a period of two to five seconds. Repeat the stimulation at intervals of ten to twenty seconds for ten minutes. Adjust the electrode for stimulation of the cardiac sympathetic. Stimulate. 4. Observations. (1) What is the average blood pressure meas- ured in centimetres of mercury in the animal under observation before stimulation of a nerve? (2) What influence does stimulation of a vagus nerve have upon the arterial blood pressure? (3) Is the effect clearly marked on the pressure tracing? (4) What influence does stimulation of the cardiac sympathetic have upon arterial blood pressure? THE CIRCULATION OF THE BLOOD 99 (5) Is the effect clearly marked on the pressure tracing? (6) In the case of which nerve is the influence of stimulation the more pronounced? (7) In one animal cut both vagi nerves and note the influence on blood pressure for a period of one hour after the section, (8) In another animal cut both cardiac s^Tnpathetic nerves and note the influence on blood pressure. XII. THE BLOOD PRESSURE IN THE TISSUES. Fig. 55 A. To Determine Capillary Blood Pressure. 1. Appliances. A set of metric weights from 1 to 100 grams; a common-sized watch-crystal; a |-inch round cover-glass, No. 3; sealing-wax; linen thread; dividers; millimetre scale. 2. Preparation. To make an apparatus for determining capillary pressure mark upon the edges of the watch-crystal and cover-glass, points distant from each other 120° of arc, cut three equal pieces of thread from 10 to 12 cm. in length; fasten the ends to the points marked in the circumference of the glasses with melted sealing-wax. If the threads are of equal length, and if the cover-glass is held in a horizontal plane, the watch-crystal suspended by the threads should be parallel to the cover-glass, and, therefore, in an horizontal plane. If the cover-glass is given a half-turn to right or left, the three threads will cross as seen in Fig. 5.5. A thread should be tied around where this cross occurs and the knot se- cured with sealing-wax. Weigh this ap- paratus and mark upon the watch-glass its weight in grams. Hold the left hand with palm upward, fingers slightly flexed. Hold the apparatus with the cover-glass horizontal and place the middle of the cover-glass on the tip of the ring finger, the watch-crystal hanging below. The weight suspended on the finger woukl be simj)ly the weight of the apparatus. 3. Observations. Place sufficient weight upon the watch-glass scale-pan to nearly exclude capillary circulation from the flattened circuhir area where the cover-glass presses upon the finger. If the capillary circulation is completely excluded from this area the skin Apparatus for determining the capillary pressure. 100 SPECIAL PHYSIOLOGY will look quite white. A sufficient weight should be put on to make the area distinctly paler, but not white. (1) W\x2it is the diameter of the area from which the capillary circulation is excluded? (2) What is the area expressed in square millimetres? (3) What weight was added to the apparatus? (4) What is the total weight resting on the computed area? (5) What is the weight in milligrams resting upon each square millimetre of surface? (6) How high would a column of water be in milligrams that would represent this same pressure per square millimetre? (7) How high would a column of mercury be that would represent this same pressure? (8) What is the capillary pressure in the volar surface of the ring finger in the different members of the class? (9) Is the capillary pressure modified by a variation of the position of the arm? (10) Is the capillary pressure modified by variation in the posture of the subject: lying, sitting, standing? B. The Plethy sinograph. This instrument is designed to determine the tissue pressure in contradistinction to the arterial pressure in larger arterial trunks. When an arm, leg, or finger is thrust into a case just large enough to accommodate the member, any change in the volume of the tissues will change the amount of space between the limb and the case, and this change in volume may be easily traced with a record- ing tambour. Such a case is really a modified receiving tambour and is called a plethysmograph. One of these adapted to the finger is shown in Appendix, 12. 1. Appliances. Plethysmograph; recording tambour, smallest size for finger, medium size for arm; kymograph. 2. Preparation. Pass the finger or the bare arm through the rubber collar of the receiver. The collar shoidd fit the arm above the elbow, or the index finger around the first phalanx tightly enough to prevent any escape of air between the tissue and collar, but not tightly enough to prevent ready return of venous blood. The tube leading from the plethysmograph to the recording tambour should have a side vent, which should be left open while the adjustment of the apparatus is in progress. Closing the vent, one should find that the tracing lever of the recording tambour rises and falls rhythmically, showing a rhythmic change in the size of the limb. THE CIRCULATION OF THE BLOOD 101 3. Observations. Trace a plethysmogram while holding the limb as still as possible. Breathe regularly and deeply. (1) Are the cardiac contractions visible in the tracings; and, if so, does the part get larger or smaller in cardiac systole? (2) Are the respiratory movements evident; and, if so, does the part get larger or smaller during inspiration. Account for results. (3) Wliile the arm is enclosed in the plethysmograph, slowly con- tract the flexor muscles of the forearm. Does the vohmie increase or diminish on contraction? Account for results. XIII. THE ACTION OF ATROPINE UPON THE HEART. 1. Material. Two dogs; atropine sulphate; morphine sulphate; chloroform (or ether); mask. 2. Preparation. Make up following solutions: a strong solution of atropine, 0.4 grm. to 10 c.c; morphine, 0.6 grm. to 10 c.c. Simply restrain dog "a." Fasten dog "6" to board. Give hypodermically 0.03 grm. of morphine to dog "6," then anaesthetize him. Set up inductorium so as to obtain tetanizing current. 3. Experiments and Observations. (1) Expose the vagus of dog "6." Stimulate it with weak induced current, using shielded electrode. (2) Count the pulse; then give 5 mg. atropine hypodermically. (a) Count the pulse at short intervals after the injection of atropine for at least thirty minutes, or until its rate is markedly affected. (6) What is the effect of atropine on the rate of the pulse? Could atropine produce this effect by acting on the vagus centre ? On the vagus fibres? On the heart muscle direct? (3j After the pulse rate has been markedly affected by atropine, stimulate vagus as before, using shielded electrodes. (a) \Yhat is the effect on the rate of the heart's action? ih) Compare this result with that obtained in experiment (2). (c) Had atropine acted solely by depressing the vagus centre, would we have found a difference in results in stimulating the vagus nerve before and after its exhibition? (d) Had atropine acted on the accelerator apparatus, would there be a difference in such results? (e) If now, on stimulating the heart muscle directly, you obtained a normal physiological effect, to what elements have you limited the possible action of atropine? ij) Basing your opinion on the experiments you have performed, to what elements have you limited the possible action of atropine? (4) Further general observations. {a) Note condition of visible mucous membranes with regard to their secretions. ih) If dog can be kept until next day, note size of pupils. 102 SPECIAL PHYSIOLOGY XIV. THE ACTION OF PILOCARPINE UPON THE HEART. 1. Material. One rabbit; one dog; hydrochlorate of pilocarpine; sulphate of morphine; sulphate of atropine; chloroform. 2. Preparation. Make solution of pilocarpine, 50 grm. to 10 c.c. ; atropine, 0.02 grm. to 10 c.c; morphine, 0.6 to 10 c.c. Do not fasten the rabbit to the holder. Fasten the dog to the dog board, after giving preliminary hypodermic injection of 0.03 grm. of morphine. 3. Experiments and Observations. (1) Give, hypodermically, 5 mg. per kg. pilocarpine to rabbit. Record weight, pulse, and tem- perature. Note secretions and size of pupils. (a) Record symptoms as they arise, especially as regards: (I) Secretions. (II) Pulse rate. (III) Size of pupil. (IV) Temperature. (V) Weight. (6) Formulate the total effect of pilocarpine upon the animal. (2) After morphinizing the dog, fasten it firmly to the dog board and lightly anaesthetize; expose both vagi. Count the pulse. Give a subcutaneous injection of 0.03 grm. pilocarpine. After salivation has become profuse count the pulse again. How does pilocarpine affect the pulse rate? (3) Now sever the vagi. (a) How does the severing of the vagi affect the normal animal? (6) How does it affect an animal poisoned by pilocarpine? (c) Could pilocarpine alter the effect produced by severing vagi if it acted on the proximal side of the point at which the vagi were cut? On a point beyond that at which they were cut? {d) Could the pilocarpine alter the effect normally produced by severing the vagi, by acting on the cardiac sympathetic? (e) Enumerate the possible points at which pilocarpine may act to produce the effects observed. (4) Give the same dog 5 mg. atropine, hypodermically. (a) Is the rate of heart beat altered? (6) Where does atropine act to produce alteration in rate of heart beat? (c) Does atropine antagonize the action of pilocarpine in this experiment ? {d) To what elements have you limited the probable action of pilocarpine ? (5) General observations. (a) Compare the action of pilocarpine with that of atropine throughout the range of action observed. (6) Is atropine a physiological antagonist of pilocarpine? THE CIRCULATION OF THE BLOOD 103 XV. THE ACTION OF DIGITALIS UPON THE HEART. 1. Material. Tincture digitalis; sulphate of morphine; sodic chloride; chloroform; two dogs; one frog; sodic carbonate (one-half saturated solution). 2. Preparation. Make solution of morphine, 0.6 grm. to 10 c.c. Pith frog. Morphinize dogs, using 0.03 grm., and chloroform them pre\'ious to operation. Set up induction coil so as to obtain tetanizing current, having contact key in primary circuit. Prepare kymograph for tracing. 3. Experiments and Observations. (1) Fasten a dog firmly to the dog board and lightly anaesthetize. Expose the vagus. Count the pulse. Using shielded electrodes and separating secondary from primary coil, find a current just weak enough not to affect heart when applied to vagus. Now inject subcutaneously 0.3 c.c. tincture digitalis per kilo animal. After waiting at least twenty minutes, in the mean time using no anaesthetic except a repetition of the morphine if necessary, and keeping the wound closed after moistening with saline solution, stimulate the vagus with same current that before the exhibition of digitalis was unable to affect the heart. (a) What is the function of the cardiac fibres of the vagus? (6) What result is produced by the stimulation of these fibres in the normal animal? (c) Does digitalis increase or decrease the influence of the vagus? (Maximum effect occurs after two hours.) {d) With the stimulus applied to the vagus fibres, the cardiac fibres carrying impulses centrifugally, could this altered excitability be due to central action of the digitalis? (2) After morphinizing dog, fasten firmly to dog board and Hghtly anaesthetize; expose carotid artery. Insert the cannula of the manometer tambour apparatus into the artery. There must be no air-bubbles in the apparatus at any point. The anaesthetic should be discontinued as soon as the cannula is in.serted into the artery. Take normal tracing and read pressure as indicated in the manometer. Now give the dog, hypodermically, 0.3 c.c. tincture digitalis for each kilo of weight. (a) Watch effect on elevation of mercury meniscus, making tracings at short intervals. (b) What factors enter into arterial pressure? {(:) How does a "high-pressure" tracing differ from a "low- pressure" tracing? id) What effect has digitahs on arterial pressure? ('3j Having firmly fastened a pithed frog to frog board with web stretchcfl over a hole in the board, focus the microscope upon a certain arteriole in the field, and measure its fliameter with an eye- 104 SPECIAL PHYSIOLOGY piece micrometer. Now inject into dorsal lymph spaces 0.3 c.c. tincture digitalis and measure same arteriole at intervals of ten minutes. Keep the web moist with normal saline solution. (a) What change occurs in the diameter of the arteriole? (6) What effect would you expect this to have on arterial pressure ? (c) Would its action on the arterioles help to account for its effect on arterial pressure? (4) Comparisons. Compare digitalis and atropine with regard to (a) their effects on the rate of the heart beat; (6) their effects on the irritability of the vagus. XVI. THE ACTION OF ACONITE UPON THE CIRCULATION. 1. Material. Tincture aconite; sulphate of atropine; one dog; one frog; sphygmograph. 2. Preparation. Make solution of atropine, 0,02 grm. to 10 c.c. Pith frog. Do not fasten the dog to dog board. 3. Experiments and Observations. (1) Give 1 c.c. tincture aconite hypodermically to the dog. Record symptoms as they arise. (1 c.c. often not fatal.) (2) Fasten the pithed frog on its back to the board. Count the heart beats, exposing heart if necessary. Now give two drops tincture aconite subcutaneously. What effect has aconite on the pulse rate? (To obtain satisfactory results, observations must be made at short intervals, for from thirty to sixty minutes.) (3) Take a sphygmographic tracing of the radial pulse of a student. Note the pulse rate. Administer by mouth 0.2 c.c. tincture aconite and 0.06 c.c. every ten minutes until action on pulse is noticeable. Repeat tracing and counting of pulse at short intervals. (a) How does aconite affect blood pressure? (6) How is the rate of the heart's action affected? (c) What subjective sensations are produced? (4) Comparisons. Compare aconite and pilocarpine with regard to their action on the gastrointestinal system. XVII. THE ACTION OF ADRENALIN UPON THE CIRCULATION. 1. Materials. White rabbit; adrenahn. 2. Preparation. Make solution of adrenalin 0.01 grm. to 10 c.c. of normal saHne solution. Weigh the rabbit and fasten it to the holder, and with probang made of absorbent cotton on probe apply solution to cornea; note local effect on peripheral circulation. 3. Operation. Anaesthetize rabbit, expose carotid, and insert cannula into artery and take blood pressure. Ligate external carotid; THE CIRCULATION OF THE BLOOD 105 inject toward heart enough of the sohition to make 2 grm. per kilo animal; ligate below needle point and withdraw needle. 4. Observations. (1) What is the effect of adrenalin applied locally ? (2) What is the effect upon the peripheral circulation of adrenalin injected intravenously ? (3) Is the rate or apparent force of the heart modified by adrenaUn? (4) Is the blood pressure modified; if so, how may the change be accounted for? CHAPTER IV. EESPIRATION. I. THORACIC MOVEMENTS. INTRATHORACIC PRESSURE. 1. Appliances necessary for these exercises are: Kymograph; physiological operating case; clippers; stethograph; thoracic cannula. (See Appendix, 12.) The stethograph consists of two tambours; the recording tambour is the same as used in other analogous experiments (Fig. 56), while the receiving tambour, joined to the recording tambour through a ^-inetre length of No. ^ pressure tubing, is provided with a cork button which may be placed upon the rabbit's thorax and receive and communicate its movements to the air in the tambour system. Fig. 56 Recording tambour. (Described in Appendix, 12.) 2. To Study the Movements of the Rabbit's Thorax. The problem is to take a graphic tracing or stethogram of the movements of the thoracic walls, and from this tracing to determine the rate and the character of the movements, particularly the latter. To record a stethogram, fasten the rabbit upon its board and hold the button of the receiving tambour upon the thorax, tracing the movement of the lever upon the kymograph. Study the characteristics of this curve. Anaesthetize the rabbit with ether. How does the stethogram vary as the anaesthesia progresses? Is the stethogram of full anaesthesia different in any essential feature from the normal one? RESPIRA TION \ 07 3. To Study the Intrathoracic Pressure. Locate an intercostal space to the right of the sternum and opposite its middle point. ]\Iake an incision 1 cm. long, parallel with the intercostal space and 1 cm. from the sternum. Dissect through the intercostal muscles, taking care not to cut the pleura. Insert into the wound the point of the glass cannula, previously provided with a rubber tube which is clamped, and press it carefully through the pleura into the right pleural cavity. Join the rubber tube to a recording tambour and unclamp. Slowly and gently manipulate the cannula until there is evident communica- tion through the lumen of the cannula and tube from the pleural cavity to the tambour. So adjust the cannula that the recording lever makes the maximum excursion. Bring the levers into such a relation to the kymograph that the tracing point of the stethograph lever shall be vertically over that of the lever which is to record intrathoracic pressure, and 2 cm. to 3 cm. from it. Trace upon the drum a stethogram and chronogram as well as an intrathoracic pressure record, taking care that the tracing points of the recording tambours are in a vertical line. (1) Does the rhythm of varying pressure correspond to the rhythm of the respiratory movements? (2) If so, does that necessarily establish between them the relation of cause and effect? (3) What change of pressure is indicated by the rise of the pressure lever? (4) What movement of the pressure lever corresponds to a rise of the stethograph lever? (5) What is the condition of intrathoracic pressure during inspira- tion? During expiration? (6) Stop the entrance of the air into the respiratory passages by closing the rabbit's nostrils. What effect does this have upon the respiratory movements ? (7j Is the intrathoracic pressure affected by the experiment? If so, explain the effect. (8j If two phenomena correspond perfectly in their cycles, and if a variation of one is always accompanied by a variation in the other, can there be any reasonable doubt that they sustain to each other the relation of cause and effect? (9) Is one of the phenomena in question the cause of the other? If so, state which is the cause and estaljlish your position. (10 J Clamp the rubber tube of the pressure apparatus. Replace the recording tambour with a water manometer. Unclamp. Is the pressure during inspiration positive or negative, and how much? (11) Is the pressure during expiration positive or negative, and how mnch? 108 SPECIAL PHYSIOLOGY (12) If the whole apparatus were filled with water instead of air and water, would it make any essential difference in the result? What effect do the variations of the intrathoracic pressure have upon the circulation? II. RESPIRATORY PRESSURE. ELASTICITY OF THE LUNGS. PNEUMATOGRAM. A. Respiratory Pressure. 1. Appliances. Operating case; clippers; rabbit board; ether; ether cone; absorbent cotton; rabbit stethograph; kymograph; a small mercury manometer, to the proximal limb of which is attached a thick-walled rubber tube, a piece of glass tubing for a mouth-piece, a screw clamp; chronograph; two recording tambours; rabbit. 2. Preparation. Fix the rabbit to the operating board and anaes- thetize; clip the ventral surface of the neck. Join up the manometer as shown below. Fig. 57 Tracheal cannula, with manometer attached. 3. Operation. Make a longitudinal incision over the trachea, through skin and connective tissue. Part the sternothyroid muscles in the median line and expose the trachea. Separate the trachea from the oesophagus and other surrounding tissue's for 3 cm. below the larynx. Carefully pass a strong linen ligature under the trachea. Make a median ventral slit in the trachea anterior to the ligature. Pass through the slit the limb of the Y-tube marked / (Fig. 57). Ligate. 4. Observations. Respiratory Pressure. The Pneumatogram. (1) After the ligature is tied how does the rabbit breathe? Are the RESPIRATION 109 thoracic and abdominal movements of respiration accompanied by other respiratory movements ? (2) With tube N (Fig. 57) open, is there any variation of the mercury during respiration? (3) With a screw clamp slowly close tube N. As the resistance to the flow of air increases, what change is noted in the manometer? (4) Quickly clamp tube N at end of expiration and carefully note the manometer reading. Is it positive or negative? (5) Clamp tube N at the end of inspiration. Is the pressure posi- tive or negative? (6) You have been determining certain facts regarding respiratory pressure. Are the causes of the changes of respiratory pressure the same as the causes of the changes of intrathoracic pressure? (7) In what way does respiratory pressure differ from intra- thoracic pressure? (8) Disjoin the manometer and join its tube to a recording tambour and trace a pneumatogram, with stethogram and chronogram. (9) Compare the pneumatogram with the tracing of intrathoracic pressure. Account for all differences. (10) While dispatching the rabbit with chloroform trace a pneu- matogram of chloroform narcosis. Describe its characteristics. Does the heart continue to beat after the respiration has ceased? B. Elasticity of the Rabbit's Lungs. (1) After the death of the rabbit open the thorax freely, taking care not to wound the visceral pleura. The lungs will collapse. Why ? (2) Replace the manometer; gently blow into the mouth-piece until the lungs have been inflated to their normal size. Measure carefully the rise of mercury in the distal column. What degree of positive respiratory pressure will the elasticity of the lungs alone cause ? (3) What is the significance of the elasticity of the lungs in respi- ration ? C. The Cardio-pneumatogram. Remove the tube A^ from the Y-tube; join it to a recording tambour. (Ij Let a member of a division sit in perfect repose, and, while the drum of the kymograph rotates very slowly, hold the mouth- piece between the lips. Hold the nose and suspend all respiratory movements for a period. Let some member of the division count the pulse of the experimenter. Trace the cardio-pneuinatograni. (2) Is there a relation between the rhythm of tiie pulse and the waves of the tracing? If so, account for this relation. (3j Account U)V the essential features of the cardi()-f)n('nmatogram. 110 SPECIAL PHYSIOL OG Y III. TO STUDY THE MOVEMENTS OF THE HUMAN THORAX. 1. Appliances. Stethograph (see Appendix, 14); chest pantagraph (see Appendix, 15) ; chronograph and kymograph. 2. Observations. With the stethograph (Fig. 58). (1) How much may be learned of man's respiratory movements by simple inspection? Make a careful enumeration and record. Fig. 58 The human stethograph : St, stand with heavy base, supporting a thoracic frame constructed of gas-pipes and clamps ; a and a', horizontal parallel arms, to he adjusted on either side of the thorax ; a', to touch the thoracic wall ; RT, receiving tambour, constructed as described in the Appendix ; the movements of the cork c, which touches the thoracic wall, are transmitted to the recording tambour rt, thence traced on the kymograph K. (2) Take a stethogram of the lateral diameter in the nipple plane. (3) Take a stethogram of the dorsoventral diameter of the thorax over the middle of the sternum in the nipple plane. Compare. (4) Adjust the stethograph and make a record (a stethogram) of the changes of the lateral diameter of the thorax at the ninth rib. RESPIRATIOX 111 (5) Take a lateral ninth-rib stethogram while the subject reads a paragraph, sighs, coughs, and laughs. Account for the peculiarities. (6) Take a lateral ninth-rib stethogram after the subject has taken vigorous exercise. What changes are to be noted? (7) Compare the stethogram from several individuals. Determine the essential features and give causes of these. (8) Seek the causes of the differences which exist between stetho- grams of different individuals. INIay they be accounted for by stature, condition, occupation, or habit? 3. Observations. With the chest pantagraph. The purpose of this instrument is to record the outline of any horizontal section of the thorax, though it could be used as well for tracing the periphera Fig. 59 The chest pantagraph. For measuring and recording chest contours. The instrument is constructed of brass or of wood with brass or steel semicircle. The joints a, b, x, and y move easily in the plane of the instrument. The semicircle, forty inches in diameter, rotates at x around the diameter t x. The point/is fixed to a table. With/ a fixed point all movements of t, the tracing point, are accompanied by corresponding movements of r, the recording point. The triangle/ r b and ft a are similar triangles in all positions of the instrument fb:/a : : f r : f t ; but f- = .'< therefore the distance/ r is always ' the distance/^. of the abdomen, of the head, or of the limb. To use the pantagraph for the purpose here intended, let the subject sit beside a table adjust- able as to height. Make such adjustment as to bring the circum- ference of the thorax to be observed even with the upper surface of the table. Fix the point / of the instrument to the table. Let the ob- .server locate, with pen or pencil, upon the side of the subject distal from the table, a point which shall serve as a starting point. (See Fig. 59.) When the point (/) of the instrument rests upon this point of the subject's thorax the instrument should be well extended, somewhat more than represented in the figure. Fix a sheet of paper to the table under the recording pencil at r. To take a graphic record of the contf)ur of the thorax proceed as follows: 112 SPECIAL PHYSIOLOGY (a) Let the observer place the tracing point {t) upon the "starting point" on the distal side of the thoracic perimeter. (h) Sweep the tracing point quickly around one-half the perimeter to a point approximately opposite to the starting point. (c) Rotate the curved arm of the instrument upon its axis {t x) through 180 degrees. Fig. 60 Fig. 61 Fig. 60.— The water spirometer. The outer receptacle contains water. The inner inverted reservoir receives the air through the mouth tube, at the right, and is raised. Fig. 61. — Pneomanometer. {d) Sweep the tracing point around the other one-half of the perimeter to the starting point. The movements of the tracing point {i) in the horizontal plane have been faithfully recorded upon the sheet of paper by the recording pencil at r. It is hardly necessary to remind the student that the subject must remain motionless during the observation. I R ESP IE A TION 113 d) Take a thoracic perimeter with the chest in repose. Measure different diameters of the tracing and muhiply bv five to reduce to actual measurements. (2) Take a tracing at end of forced expiration; at end of forced inspiration. Compare diameters. (3) Make a series of these . tracings for different individuals. Compare. (4) Do different individuals of the class represent different types of contour, as broad, medium, and deep? (5) Which t^'pe of chest is capable of adding the greatest area of contour by expansion? IV. LUNG CAPACITY i CHEST MEASUREMENTS, RESPIRATORY PRESSURE . RECORDING OF ANTHROPOMETRIC DATA. 1. Instnunents. Spirometer (Fig. 60); pneoraanometer (Fig. 61); meter tape; steel calipers; standard, with horizontal arm for meas- uring height; scales for taking weight. 2. Observations. (1) Test with spirometer the lung capacity of each member of the division. May differences in lung capacity be accounted for by difference in stature, condition, occupation, or habit? (2) Take with the meter tape the girth of chest over the nipples in a plane at right angles with the axis of the thorax. (a) AVith chest in normal repose. (b) At the end of forced expiration. (c) At the end of forced inspiration. (3j Take the girth of chest over the juncture of the ninth rib with its cartilage, holding the tape in a plane at right angles with the axis of the thorax. (a) With the chest in repose. (6) At the end of forced expiration, (c) At the end of forced inspiration. (4) With the calipers measure the dorsoventral diameter at the level of the nipple, holding the calipers in a plane perpendicular to the axis of the thorax. (a) Normal ; (b) after forced expiration ; (c) after forced in.spiration. (5) Take the lateral diameter in the nipple plane. (a) Normal; (b) after forced expiration; (c) after forced inspiration, (fj) Take the lateral diameter at the ninth ril). (a) Normal; (b) after forced expiration; (cj after forced inspiration. (7) Test with pneomanometer the force of inspiration and expira- tion. IvCt each member of the division test with the pneomanometer the maximum positive pressure which he is able to produce in the respiratory passages during expiration. 8 114 SPECIAL PHYSIOLOGY (8) Test with the same instrument the maximum negative pressure which each individual can produce during inspiration. (9) Does the face become red in either of these tests? If such is uniformly observed, account for it. (10) The preservation of data. Experience has shown that when data are to be preserved for subsequent use in comparison of one class of individuals or cases with another, it is very much more economical in time to record the data upon cards. For the above data one may use such a card as is appended below. In addition to the measurements above given record upon the cards the weight, the height, the bodily condition of the individual, and especially whether the individual has lived in a hilly or in a flat country, and whether he has been active or inactive. Name ... Address Place of residence : level, hilly, or mountainous altitude Previous occupation Habits: Exercise, sports, character, amount f Father's weight height Parents -{ [ Mother's weight height Which parent do you resemble physically ? Which parent do you resemble temperamentally ? . . . . Age Weight .... Emaciated, thin, spare, stout, obese- Height .... Dwarfish, short, medium, tall, very tall, r Inspiration Lung capacity .... Respiratory pressure ^ [Expiration Girth of Chest, Nipple Plane: 1. Repose ... 2. Inspiration . . . .3. Expiration 4. Expansion Per cent Diameter of Chest, Dorsoventral : 1. Repose ... 2. Inspiration . . . .3. Expiration 4. Expansion Per cent Diameter of Chest, Lateral: 1- Repose ... 2. Inspiration .... 3. Expiration 4. Expansion Per cent Examiner Date EESFIEATION II5 V. THE EVALUATION OF ANTHROPOMETRIC DATA. A large proportion of the problems that the medical man has to solve involves the finding of averages of a large number of observa- tions. This is sure to be true in all anthropometric problems. In the course of the preceding lesson valuable anthropometric data were collected and recorded upon cards. The value of this material is purely potential. Before the data will furnish a basis for drawing conclusions it is necessary to subject them to a process of evaluation. This process consists, first, in grouping; second, in getting the average or the median value for each measurement; and, third, in graphically representing the averages. In the previous lesson the observer noted upon each card whether the subject had lived in a hilly or flat country; further, whether he had lived a physically active or inactive life. This gives one an opportunity for four groups when the cards for the whole class are collected. Group I. Active men from a hilly country. Group II. Active men from a flat country. Group III. Inactive men from a hilly country. Group IV. Inactive men from a flat country. Until recently it has been customary simply to write the data for any group in columns and ''strike an average" of each column. If there are only 10 to 20 or 30 individuals in each group this method does not entail the unnecessary expenditure of much energy, but it is not reliable, for one "giant" or "dwarf" in any group would vitiate the whole result. If there are 100 or 1000 individuals in a group, then the use of the old method of finding the arithmetical average is exceedingly wasteful of both time and energy. It must be arlded, however, that when the number of observations is large the chances are that there will be as many dwarfs as giants, thus making the average approximate closely the median value. It is the latter we are seeking, viz., the median value; this may be defined as that value which is so located in the whole series of observations in a single measurement of any group, that there are as many below it as above it — i. e., that the number of values which it exceeds equals the number of values which exceed it. Let us take a concrete case. In a group of 316 seventeen-year-old boys certain physical measurements were recorded upon individual canis. Let us take, for example, the girth of the head recorded in centimetres and tenths. Instead of writing in a column the 316 head-girths, each expressed in three figures, adchng and averaging, let us adoj^t the new method, first suggested by the Belgian astronomer and anthropologist, Quetelet, and later elaborated by Galten, the London anthnjpologist. Arrange the cards in piles, placing in one pile all (;f the cards having girth of head 51 cm., in another pile all 116 SPECIAL PHYSIOLOGY having 52 cm., and so on. In the case in question it was found that the 316 cards were quickly distributed, falhng into the following groups : Girth of head . 51 52 53 54 55 56 57 58 59 60 No. of observations . (i. e., of cards.) 1 7 17 41 70 74 60 29 10 7 The problem is to find the value of the median measurement or the median value. There are 158 values below the median value and as many above it. 1. To Locate the Median Observation. This is equivalent to saying — find in the lower series of numbers (1-7-17, etc.) the 158th observa- tion from either end. It must be located in the pile of cards which number 74. This group may be called the median group. But where in this group is the median observation located ? In order to deter- mine this, add the groups to the left of the median group, these may be called the minus groups, the values which they represent being less than that of the median group: 1, 7, 17, 41, 70 = 136. To this sum we must add 22 observations from the median group to make 158. The median observation is then located in the median group, 22 points from the left. 2. To evaluate the median observation we must take it for granted that the 74 observations of the median group are evenly distributed over the distance between 56 cm. and 57 cm. That being the case, 22 the median value would be 56 and jtt cm. 74 If it is desired to reduce this simple process to a mathematical formula, that can readily be done: Let w=the total number of observations (316). m=the number of observations in the median group (74), Z=the sum of the minus groups at the left (136). r = the sum of the plus groups to the right (106). a=the minimum value of the median group (56 cm.). d=ihe arithmetical difference in the minimum values of the groups (1 cm.). M=the median value to be determined. Then M = a-i- Kl-') or M- /316 56+^\ 2 - — ISe) -„22 74 56.3 cm. After one has found the median value for each measurement in each group, these may be tabulated and the values compared. When the table of median values is large it is almost necessary to carry the work of reduction a step farther and represent these values graphic- RESPIRATION 117 ally in a chart. Another opportunity will be used for giving the methods used in the graphic representation of statistical tables. The table which results from the data collected in connection with the previous lesson is not so large but that the observer can practi- cally comprehend the whole at a glance. Our grouping enables us to answer the following questions : 1. Has general physical activity any essential influence in the development of the respiratory organs and function? 2. Is the climbing of hills in early life a factor in the development of the respiratory organs and function? If both of these questions may be answered affirmatively, then one would expect to find that the median values of Group I. (active individuals from a hilly country) uniformly exceed the values of Group II., and that those of Group III. uniformly exceed those of Group IV., but that the median lines of Group II. may or may not exceed those of Group III. VI. QUANTITATIVE DETERMINATION OF THE COj AND H^O ELIMINATED FROM AN ANIMAL'S LUNGS IN A GIVEN TIME. 1. Appliances. A 4-ounce Woulffe bottle with three necks and with delivery tubes and stopper ground in the necks (Fig. 62, a); Fig. 62 KOH Ba((JlI)-,, CaClq Aninml CaCk CaCk cage Apparatus for the estimatif)n of COj and H2O in exhaled air. three .'j-incli calcium chloride tubes, with side tubes and perforated gla.ss .stoppers, opening and closing the flow of gas (Fig. 62, c, e, /); 118 SPECIAL PHYSIOLOGY Geissler's potash bulbs, with CaCl2 tube ground on (g) ; two small flasks (b, h) with rubber stoppers, double bored, with delivery tubes fitted as shown in figure; a 1 -litre or 2-litre bottle with very wide mouth to use as animal cage, fitted with delivery tubes and with a cork impregnated with paraffin; siphon apparatus, as figured, consist- ing of two 8-litre bottles with paraffined corks and tubes; analytical balances; laboratory balances (correct to 0.01 grm.); drying oven; chemicals, KOH, Ba(OH)2, CaCl2; any small animal whose weight in grams does not exceed one-fifth the volume of the animal cage expressed in cubic centimetres. 2. Preparation. (1) Fill the calcium chloride tubes; put them into the drying oven, where they are to be kept at a temperature of 100° to 120° C. for several hours; cool in a desiccator and weigh upon the analytical balances the tubes e and /, recording the weight in milligrams. (2) Fill the Woulffe bottle and the Geissler's bulbs with a strong solution (50 per cent, or more) of KOH. Fix into position, upon the Geissler bulb, its filled and desiccated CaClj attachment, and fit to each end a rubber connecting tube; clamp with strong serre-fine forceps and weigh upon the analytical balances. (3) Fill the flasks b and h with a strong solution of Ba(OH)2. These flasks serve simply to show whether or not the COg gas has all been absorbed by the KOH through which it has just passed. (4) Pieces e, f, and g should be fixed to a light wooden rack, by which they may be moved; if this is not convenient clamp them to supports. (5) Join up the apparatus a, b, and c. (6) Fill siphon apparatus. (7) Weigh the animal cage without the animal. 3. Operation. (1) Put the animal into the cage; fasten the stopper in so that it will not leak air. (2) Join the animal cage with c and with siphon apparatus at i, leaving out for this preliminary operation the apparatus e, f, g, and h. Start the siphon and note the rate of flow per minute. The level of the water in the lower bottle should be probably 1 metre below that in the upper bottle. Notice whether the animal seems to be respiring normally. If so, it may be taken for granted, after ten minutes, that the ventilation is sufficient. If it seems insufficient, one has only to increase the difference of level in the two siphon bottles. (3) Disjoin the animal cage and weigh the cage with the contained animal upon the laboratory balances. Note the time; join the animal cage in circuit again, attaching it to e, and attaching h to the siphon apparatus at i. Start the siphon. The greater resistance to be overcome will necessitate a greater difference in the level of the two bottles in order to ventilate at the same rate as before. To RESPIRATION ]19 test joints place the finger over the distal tube of the Woulffe bottle (a); if the joints are all right the siphon stream will stop after a few moments. When the water in the upper bottle is lowered nearly to the end of the siphon, clamp the tube joining h to i, set the empty bottle upon the floor and the full bottle on the higher level, join the tube at k, and unclamp. This whole change need only occupy a few seconds. If it is desired to make a determination of the amount of oxygen which the animal consumes in a given time, the air that passes out of the ventilating apparatus after the second change may be caught and tested. (4) It is evident that in the afferent apparatus (a, h, and c) one has a means of robbing the air of COj and H2O, thus furnishing the animal with pure dry air. It is further evident that in the afferent apparatus one has a means of collecting absolutely all of the COj and H2O given off from the animal during the experiment. Further, the weights before and after will show just how much of these excreta have been passed into the collecting apparatus. (5) Note the time (one hour or more); damp siphon tube; turn the stoppers off e and /, clamp x and y; disjoin d and weigh it. (6) Weigh e, weigh /, weigh g. 4. Observations. (1) How much has the animal lost in weight during the period of observation? (2) How much water left the animal cage during the period of observation ? (3) What was the source of this water? (4) Did the animal micturate or defecate during the time of the experiment? If so, is this to be looked upon as a source of error in the experiment? Would such an occurrence tend to increase or decrease the amount of water caught in the CaClj tubes e and jf Would it interfere in any way with the experiment? If so, how may such a source of error be avoided or corrected? (5) How much COj left the animal cage during observation? (6) What is the total amount of COj and HjO collected? (7) Does the amount of these excreta collected equal the loss in weight of the animal ? What should the relation of these two quanti- ties be? Explain in full. (8) What is the respiratory quotient? (9) Formulate several problems which may be solved with this method. VII. TO DETERMINE THE AMOUNT OF OXYGEN CONSUMED BY AN ANIMAL IN A GIVEN TIME. 1. Preparation. The oxygen is determined by a volumetric method, using two or more gas burettes and a solution of potassium pyrogallate. 120 SPEC I A L PH YSIOL OGY The solution of potassium pyrogallate is prepared by mixing two parts of 25 per cent, aqueous solution of KOH and one part of 5 per cent, aqueous solution of pyrogallic acid. Comparison must be made between the oxygen content of the expired air and that of the atmosphere at the time of the experiment. If it is desired to calculate the respiratory quotient it will be necessary to make the oxygen analysis from the air that traversed the animal cage in the previous experiment when the CO2 was being determined. If it is not desired to compute the respiratory quotient it will be necessary only to have it traverse the animal cage, drawn through by the ventilating apparatus. The air should come into the cage from out of doors (brought in through glass or rubber tubes from the window). Fig. 63 % n Position 1. Position 2. Gas burettes to determine oxygen. Position 3 2. Operation. These two constituents of the pyrogallate should be mixed in the pressure tube of the gas apparatus just before the analysis is made. To collect samples of air for analysis, one fills the gas burette (Fig. 63, A) with water by suction. Connection is then made between the exit tube at k, of the respiration apparatus used in the previous experiment (see Fig. 62), and the upper end of the gas burette as shown in Fig. 63, position 1 ; the respired air flows in, displacing the water. The stopcocks are now turned so that no air can escape from the burette. The rubber tube of the pressure tube B, which has been filled with the potassium pyrogallate, is now RESPIBA TION 121 connected to the lower end of the gas burette. After all the air has been expelled from the connections, turn the three-way stopcock in such a position as to permit the pyrogallate to flow up into the gas burette, coming in contact with the air to be analyzed. The pressure tube should now be elevated as high as the connecting rubber will permit and the potassium pyrogallate solution allowed to run into the burette A. The clamp on the connecting tube should now be applied to it close to the lower end of the burette. This operation made positive pressure in the burette, thereby causing a more rapid absorption of the oxygen. The burette should now be taken by the experimenter and its ends alternately raised and lowered. At frequent intervals he should loosen the clamp on the connecting rubber tube and raise the pressure tube, thus permit- ting potassium pyrogallate solution to take the place of the oxygen as it is absorbed. This procedure should continue ten minutes, after which the clamp on the connecting rubber tube should be loosened. The burette and its pressure tube should be allowed to remain ten minutes longer, at the end of which time the solution in the burette should be brought to a level with the solution in the pressure tube by elevating or lowering the tube. This causes the air in the burette to be under the atmospheric pressure existing at that time. The reading for the amount of oxygen is now taken. To calculate the amount of oxygen consumed by the animal, one subtracts the amount of oxygen found in the respired air from that found in the normal air. At least one sample should be analyzed from each 10 litres of respired air, the average being used to obtain the result. VIII. THE RESPIRATORY QUOTIENT. The respiratory quotient being the ratio between the volume of car- bon dioxide exhaled and that of oxygen consumed (R.Q.= — r^ — ^^ ) , it may be'computed from data given in Exercises VI. and VII., or it may be directly determined in the following manner: 1. Appliances. Ventilating apparatus (Fig. 64); animal cage; CaClj tube; Geissler bull)s; two barium hydrate flasks; 25 percent, solution of KC)II; 5 per cent, solution of pyrogallic acid; two gas burettes with pressure tubes; guinea-pig or small rabbit. 2. Preparation. Pass one end of the glass tube out through hole in wiiiflow sash; to inner end attach a rubber tube to whose other end is joined a V>. Operation. I. Prehminary. (a) Put animal "a" into the small jar "a;" count resj)i rations; close the jar. (h) Put animal "b" into jar" b." Before closingcount respirations; close air-tight. {(■) Fill jar "c" one-third full oi water and displace the water with 124 SPECIAL PH YSIOL OGY COj. Put animal "c" into the jar, taking care to allow as little loss of CO3 as possible; close; count respirations. (d) Fill jar "d" full of water and displace with CO2. Put animal "d" into jar, taking care to allow as little loss of COg as possible; close jar and count respirations. (e) Put an animal into a jar; cover the mouth of the jar with a towel; insert into the jar the end of a rubber tube through which illuminating gas (a mixture of CO with various other gases) may be let into the jar. Let the gas in in little momentary puffs every five minutes, noting the effect upon the animal. II. Post-mortem Examination. After an animal dies fix it to the dissecting board and open the abdominal and thoracic cavities; take great care not to cut a large bloodvessel; pin the flaps out so that all of the organs will be exposed in place. 4. Observations, (a) Respiration in Small Closed Space. (1) Make a careful record of number of respirations and general condition of animal "a" in the normal state, and at the end of every five minutes after the closure of the jar. ^Miat changes in rate or depth of respiration have been noted? (2) Note all abnormal signs and symptoms. (3) On post-mortem examination record the condition of heart, large bloodvessels, lungs, liver, kidneys, and the general appearance of the tissues. (4) Compare the conditions with those found in a normal animal, prepared by the demonstrator. (b) Respiration in a Larger Closed Space. (5) Note all symptoms of animal "b" every five minutes after confinement in the jar. (6) Make a post-mortem examination; record in detail the con- dition of the organs as in the case of animal "a." (7) Compare animal "b" with normal animal. (8) Compare animal "b" with animal "a." (c) Respiration in an Atmosphere of One-third COg. (9) Note all symptoms at intervals of five minutes. (10) Compare these observations with corresponding ones from animal "a" and "b." What are your conclusions? (11) Make a post-mortem examination; make a record as before. (12) Compare appearances in animal "e" with those in the normal animal; with those of animal "a"; with those of animal "b." (13) iNIake a generalized statement of the facts discovered in the experiments. (14) What is the cause of death when an animal is enclosed in a small space? (15) What is the cause of death when an animal is enclosed in a large space? (16) Have the relations which you have discovered any bearing upon the future development of animal life upon the earth? RES PIE A TIOX 125 {d) Respiration in COj ("Choke-damp"). (17) Lower a lio-hted candle into a jar of CO,. Record results. (18) What happened to the animal when it was lowered into an atmosphere of COj? (19) Record post-mortem appearances. (e) Respiration in an Atmosphere of One -third Illuminating Gas (00 -f). Record all symptoms. Record post-mortem appearances. How does death in an atmosphere of CO compare, as to symptoms, with death in an atmosphere of COj. Compare it in turn with other forms of death as induced in this and the previous chapter. Compare the post-mortem appearances in this case with those in preceding cases. X. TO DETERMINE THE INFLUENCE OF THE PHRENIC NERVE. THE NORMAL PHRENOGRAM. 1. Appliances. Operating case; chppers; rabbit board or dog board; rabbit or dog; ether or chloroform ; anaesthesia cone; tambours, arranged as used to record the rabbit stethogram ; beaker with warm water; inductorium; one dry cell; two keys; vagus electrode; seven wires; a piece of glass rod 10 cm. which has been rounded at one end and sharpened at the other. 2. Preparation. Fix the animal to the board; anaesthetize; clip the anterior median region of abdomen. Set up electric apparatus with short-circuiting key in secondary coil and with Xeef hammer in primary circuit. 3. Operation. F'rom the posterior extremity of the xiphoid appendix make a median incision through the abdominal walls. The incision should be just large enough to admit the glass rod, and should be located in the rabbit 1 cm. from the tip of the xiphoid and in the dog 3 cm. from the xiphoid. Clamp with the serre-fines any small vessels which may be oozing. The rounded end of the glass rod is passed through the abdom- inal wall and held against the diaphragm A. The point is inserted into the cork button of the receiving tambour. (See Fig. 65.) Any contraction of the diaphragm presses the round end V>ackward and the rod is forced posteriorly, slipping back and forth through the liole in the body wall, the point is pressed back, and the lever of the recording tambour rises. Trace a phrenogram. In the mean time let another member of the division dissect out the left phrenic nerve. This operation to expose the phrenic nerve is the most difficult 126 SPECIAL PHYSIOLOGY operation yet attempted in any of these exercises. The nerve is very small and lies deeply buried in the neck not far from the spinal column and in close relation to other nerves, making it difficult so to describe its relations that the operator will be certain when he has found it. The only sure course is to test it by stimulation, and if it causes a contraction of the corresponding side of the diaphragm the operator may be certain he has found either the main trunk or one of its three roots. The cutaneous incision should be on the course of the sterno- mastoid muscle, just dorsal to the course of the external jugular vein. The cutaneous incision should be ample, extending to the clavicle at least 5 cm. long anteriorly in the rabbit and correspondingly long if the dog is used. Fig. 65 Phrenograph for taking tracings of ttie movements of the diaphragm : T, tambour joined to recording tambour and fitted with a cork button (C); a glass rod passes through a slit in the abdominal wall at i^'and rests against the diaphragm at A. Dissect through the subcutaneous tissues and separate the skin flaps widely, pressing the external jugular toward the median line. The superficial layer of muscles consists of the sternomastoid on the median side and the cleidomastoid laterally in the rabbit (the cephalohumeral in the dog). Divide the connective tissue that separates these two muscles and pass to the deeper layer. On the median side one sees the carotid, the internal jugular, and the nerves that lie in close relation to them; drawing the cleidomastoid outward one exposes the roots of the brachial plexus, emerging from between the deep muscles of the neck and passing downward and backward toward the axilla. Very careful dissection of the delicate connective tissue which lies over the roots of the brachial plexus will reveal a fine nerve thread crossing these roots very near to the line where they first come into view, and passing posteriorly it gradually draws nearer to the median line as it passes under the clavicle (under the subclavian artery in the dog). This nerve is the phrenic. Carefully dissect it out as near the clavicle as possible, lift it gently on the nerve hook, and place it RESPIRATION 127 in the groove of a shielded electrode. Stimulate gently. If you have dissected out only the phrenic and posterior to its three tributaries, the stimulation will be followed by a tetanic contraction of the corresponding side of the diaphragm. If, however, one has taken up with the phrenic a communicating thread, passing from one root of the brachial plexus to another, the stimulation will be followed by a tetanic contraction, not only of the diaphragm, but also of some of the muscles of the front leg of the side operated upon. As this will disturb the result the error must be corrected. The nearer the clavicle one can get the nerve the more unlikely he is to get nerve fibres belonging to the brachial plexus. 4. Observations, (a) Tactile Observation of the Diaphragm. (1) In what condition is the diaphragm during inspiration? Expiration? (2) In what position is the diaphragm during these two phases of respiration? (3) What parts of the diaphragm make the greatest change of position during inspiration? (4) What causes the diaphragm to arch anteriorly during normal expiration? Are the conditions changed during the present observa- tions ? (5) Are the diaphragmatic movements synchronous with the costal movements ? (h) The Normal Phrenogram. (6) Take a phrenogram. What may be learned from it? (I) Without varying the adjustment of the phrenograph take a tracing while repeatedly interrupting the respiration by hold- ing the nostrils. What does the phrenogram show? What is the interpretation ? What effect upon intrathoracic pressure would holding the nostrils have ? (c) The Phrenic Nerve and its Function. (8) Describe minutely the relations of the nervus phrenicus in the neck. (9) Cut the nerve while tracing a phrenogram from the left side of the diaphragm. Note the result. (10) Take a phrenogram from the right side of the diaphragm. Does it differ essentially from the normal? (II) While taking the left phrenogram stimulate the distal end of the left phrenic nerve. Interpret the result. (12j While taking a right phrenogram stimulate the distal end of the left phrenic nerve. Interpret the result. (13j Dissect out and cut the right phrenic nerve. Does the diaphragm cease to move? If it moves, is its movement active or passive? Does it move backward (hiring inspiration anfl forward during expiration? If so, what causes it to make these movements? If the movements are reversed, what has caused the change? Account for the phenomena. Kill the animal with chloroform. CHAPTER V. NORMAL HEMATOLOGY. INTRODUCTION. The examination of the blood, like that of the urine, gives a posi- tive diagnosis in a number of diseases. It assists the diagnosis in many diseases and is often of much value negatively. It is important, then, to be familiar with the characteristics of normal blood. The examination of the normal blood consists of an actual study of the blood by use of the microscope and the determination of many of its properties by the use of various instruments, which will be described in the text. The accurate use of the instruments can be learned only by experience. While the instruments are delicate and easily broken, yet the technique of their use is easily mastered by the student if he is careful, accurate, and persevering. The technique once acquired can be quickly regained in later years, although it may apparently be forgotten for the time being. Speed in the tests can be obtained only by continuous practice. Theoretically all these instruments are accurate, but because of the minute quantity of blood used, slight inaccuracies will be multiplied in the final results and may be large or small according to the experience and careful- ness of the observer. By knowing where these errors are possible and avoiding them by the best-known methods, and by adopting a definite method of use of each instrument, these inaccuracies can be largely eliminated and good comparative results obtained. In the use of blood instruments the observer must constantly avoid manufacturing results. There is always the tendency to read into the test a preconceived result. This is best governed by control tests and by repeated tests. When one can repeat a test three or four times with the same individual's blood and obtain approximately the same results he is quite proficient. Reference Books. — Clinical Examination of the Blood, by Cabot. Clinical Pathology of the Blood, by Ewing. Clinical Haematology, by Da Costa. Histology of the Blood, by Ehrlich and Lazarus. Text-book on Physiology, by Hall. Works on Histology and Physiology. GENERAL DIRECTIONS. All blood instruments must be perfectly clean and dry if the best results are to be obtained. The various pipettes are cleaned by the use of hydrogen peroxide and distilled water; they are then dried NORMAL H^MA TOL OGY 129 by the use of alcohol to remove the water, followed by ether, which will evaporate quickly aud remove the alcohol. Hydrogen peroxide oxidizes organic matter; alcohol and ether coagulates. The cleaning fluids (hydrogen peroxide and water) are used by filling the pipette and rolling it for a few minutes between the thumb and fingers and then blowing or drawing the fluid out. In the use of the drying fluids (alcohol and ether) do not blow the fluids out of the pipettes, as the moisture of the breath will defeat the object which one is seeking. Having filled the pipette with alcohol or ether, draw it into the rubber tube, remove the rubber tube from the pipette, blow the fluid out of the rubber tube, replace it upon the pipette, then draw air through the pipette. After using alcohol in this way, followed by ether, one may be assured that the pipette is absolutely dry. For the student's work, secure the blood from the lobe of the ear or the side of the tip of the third finger. The ear is better, as it contains fewer nerves, gives more blood, and will continue to bleed for a longer time. The ear or finger should be lightly washed with a towel moistened with distilled water, then dried with the towel to remove any dirt or loose epithelial cells. The needle used should be a fair-sized glover's needle. It is a three-sided needle, the sides of which are so ground that each has a fine saw-edge and will cut and not crush the tissues as a saddler's needle will. The needle should be kept clean with distilled water and hydrogen peroxide, and sterilized with alcohol. The puncture should be made by holding the lobe of the ear between the thumb and finger and pricking lengthwise of the ear in its lowest part. The needle should enter about one-quarter of an inch, and should be thrust in quickly while the thumb and finger hold the ear, and when withdrawn it should be given a half-turn and be quickly removed. The first drop of blood should always be wiped away to moisten the skin with blood, and also because it clots quicker than the following drops. The blood should gradually ooze out of itself. It should never be forcibly squeezed out by pinching, as that will give an abnormal specimen; but the ear may he gently pressed an inch or so above the puncture, to make the blood flow more freely. To fill a pipette by suction, take the lobe of the ear between the thumb and finger of the left hand, standing l)ehind and to the right when using the right ear and in front and to the left when using the left ear. Place the tip of the pipette upon the thumb that is behind the ear hold the pipette with the right hand near its upper extremity, with the marks showing in front; then, by turning the thumb, insert the capillary point into the drop of blood and do not allow it to touch the skin of the ear; the column of blood drawn into 9 130 SPECIAL PHYSIOLOGY the capillary must be accurate and complete. It must not remain short of or go beyond the mark desired, and air must not be allowed to enter the pipette. If any of these errors take place the pipette must be recleaned, dried, and filled again. When properly filled, any blood adhering to its outer surface must be completely removed before proceeding farther. It is better to have a large drop of blood at first than to use two or three small drops, as there is less liability of getting air into the capillary and of the blood clotting. Fig. 66 I. THE COUNTING OF THE BLOOD CORPUSCLES. Introductory. In health the number of red cells in the blood is quite constant. The variations that occur are quite small and are due to normal processes. In the male there are about 5,000,000 red cells in each cubic millimetre. In the female there are about 4,500,000 red cells. Any deviation from normal health quickly causes a diminution in the number of red cells. In fact, simple unhygienic surroundings or habits are sufiicient to speedily reduce the number of red cells without other demonstrable pathological conditions. The life of the red cell is probably of about two weeks' duration. There are approximately in the normal male's blood 200,000,000,000 red cells. Then accord- ing to the length of the lifetime of the cells about 14,000,000,000 red cells die and must be disposed of each day. A cor- responding number must be manufactured each day in order to keep the number It will be readily seen that such an im- mense process, which depends upon perfect elimination as well as assimilation, can be disturbed very easily. It is important that this fact about the blood be thoroughly understood. Even though the physician may not estimate the number of red cells in every case, yet he must recognize the fact that every disturbing element in the normal body must disturb the number of red cells contained therein. There are, then, two objects to be gained by actually counting the The Thoma-Zeiss blood-counter. The pipette for use in counting the red corpuscles. within its normal limits. NORMAL HEMATOLOGY 131 red cells and estimating their number. First, to gain a clear idea and understanding of the number of red cells in normal blood, and, second, to be able readily and accurately to estimate the number of red cells per cubic millimetre in any given clinical case. Fig. 67 The haematocrit. The attachment at the ur)per end of the vertical shaft is made to rotate at a speed of 7000 to 10,000 per minute by means of the eear-work of the body of the instrument. Each arm of the rotating attachment is provided with a capillary tube which is graduated into 100 divisions. If the tube be filled with blood and rotated for two or three minutes at the speed abfn-e mentioned the corpuscles will be thrown to the outer end and the volume per cent, may be read off on the tube. B, an enlarged view of tube with centrifugalized blood. There are three methods of estimating the number of red cells per cubic millimetre. 1. The Thoma Haemacytometer. An instrument by which, with accurate dilutifHi, the corpuscles may be actually seen and counted in a known space (Fig. 66). 132 SPECIAL PHYSIOLOGY 2. The Oliver Haemacytometer. This instrument depends upon the transmission of a transverse hne of hght from a candle through a flat glass tube. The blood stops this light until a certain dilution is obtained. The tube is graduated to read in the number of cells per cubic millimetre of the blood used according to the dilution. 3. The Haematocrit. By this instrument is obtained the volume of the corpuscular elements in the blood by centrifugation. From this the number of red cells per cubic millimetre may be estimated except in some special cases (Fig. 67). A. To Count the Red Blood Corpuscles. Appliances. Microscope with one-fifth-inch objective and me- chanical stage; Thoma corpuscle counter, consisting of the ruled counting slide and the diluting pipettes; glover's needle; three small beakers and as many open dishes. Preparation. Wash the counting slide with water or soap and water only when it needs it; the less it is handled the better. Usually rinsing it in clean water and drying with a cloth is sufiicient. Prepare small beakers of distilled water and the diluting solution. Clean the pipette as usual. Technique. Having prepared the apparatus and solutions, make the puncture and fill the pipette by gently sucking a continuous column of blood up to the mark "0.5" or "1" on the pipette, which is near the bulb. Wipe the end of the pipette free from blood with a clean cloth, but do not allow any blood to be drawn out by the capillary attraction of the cloth. As soon as possible now insert the point of the pipette into the diluting solution and suck up a con- tinuous stream of solution until the mark "101" above the bulb is reached. Roll the bulb between the thumb and finger as the blood enters the bulb. If there is not blood enough to reach the mark "1," draw it only to mark "0.5" and proceed in the same manner. Now hold the pipette in the horizontal position with ends free and roll it back and forth for three minutes to thoroughly mix the blood and solu- tion. When thoroughly mixed blow out the contents of the cap- illary below the bulb and then place a small drop on the marked plate of the counting slide, putting just enough of the mixture on it to fill the space between the marked plate and cover-glass, and being careful not to allow any of the mixture to get into the moat. Adjust the cover-glass over the drop quickly and carefully by placing one edge of cover-glass in contact with the slide and letting the opposite edge down gently with a needle. Place the counting slide when properly filled under the microscope and find the upper left-hand corner of the marked area. Wait until the corpuscles come to rest upon the surface of the marked plate, NORMAL HEMATOLOGY 133 then begin the actual estimation by counting all the corpuscles in the first marked space, including those that are on the upper and left-hand lines of the space. Then count those in the space to the right, including the corpuscles on the upper and left-hand lines as before. Continue counting each space to the right until six spaces are counted; then drop down to the next space below and count [ 3 Fig. o ' o 68 o fo o ( B ) ffl A o o ° o o o C c ' o " o n o O ( ) o o o o o o O O o o o O < o o o ' o n o o o O ° o o ( o o o o O o ' o o o o o° o o O o o o ( o ' o o o o O g o ° <"' o o o o c o O o o o o o o o O O o o o o o o o o o ° o ° o o o O n o o o o f c > n o c o O o o O o o o ° o o o o o '^ O O ( o o > °0 o 0° o o o o o ° O o o o o o o c c o O o o o o o °o o n- r ) E 3 Appearance of slide under about 500 diameters magnification. One counts all corpuscles which lie upon the upper and left boundaries of each square. each space to the left until six spaces in the second row are counted. Then drop down to the next row of spaces and continue counting back and forth in the same manner until six rows of six spaces each are counted, as shown in Fig. 68. Place the results of counting on paper in the same relation to each other as the spaces illustrated, as follows: 5 6 5 4 7 5 9 6 C, 6 6 7 4 5 5 5 8 7 7 5 8 6 4 7 6 6 7 7 9 7 6 6 6 6 5 7 37 + 34 + 37 + 34 + 39 + 40 221 -- 36 = 6/y. 221 Having made the count, the slide and cover-glass should be cleaned a.s previously described. The pipette, which has been left in a hori- 134 SPECIAL PHYSIOLOGY zontal position in a safe place, should be rolled again for three minutes. Fill the counter and adjust the cover-glass carefully as before; count another group of thirty-six spaces and record the results obtained. If the averages of the two counts differ more than one, the same procedure must be carried out the third time, and the average of the tvv'o fields nearest alike taken and the estimate made. To compute the number of corpuscles per cubic millimetre, find the average number of cells for each space and multiply this by 4000, as each space is 2^ mm. X xo" t^^^- X iV ^J"^- This Tvill give the actual number of cells per cubic millimetre in the diluted blood. Then make the correction for the dilution of the blood by multiplying by 100 or 200, and the result "^dll be the number of red cells per cubic milHmetre in the specimen of blood taken. In the example given above it would work out as follows: First 36 spaces, 63^; second 36 spaces, 6|-|. Average of the two groups, 6^. 6iX 4000 - 2.5,000 X 200 = 5,000,000,'' the number of red cells per cubic millimetre. Precautions. The cement used on the counting slide is dissolved by alcohol or ether; so these licpids should not be used on the plate. Roll the filled pipette between the thumb and finger, and do not shake the pipette, as some of the solution is sure to be lost. A com- mon source of error that can easily be detected, but which is often overlooked, is the unequal distribution of cells on the marked plate. As soon as the drop is placed on the marked plate the cells begin to settle, and, of course, most of them settle where the drop is thickest, that is, in the center. This can be avoided by getting the cover- glass in place quickly and making the whole drop of an even thickness. Each specimen, before being coimted, should be tested to see that the corpuscles are evenly distributed over the whole drop. For the same reason the filled coimting slide should be kept in a horizontal position. Theoretically, counting the cells in one small space should be suflBcient, and it would be if the measurement and dilution of the blood and distribution of the cells were all perfectly accurate. This is impossible, and the errors are mostly eliminated by the methods given. It is best for beginners always to make three coimts of 36 spaces each from the same pipette and take the average. Questions. 1. ^Miy is alcohol used to dry and not to clean the pipette ? 2. ^Vhy should the marked plate be dried without friction? 3. TMiat does hydrogen peroxide do to clean the pipette? 4. Why rotate the needle while withdrawing it from the ear? 5. Why wipe away the first drop of blood? 6. Why wipe the end of the pipette before piuting it into the diluting solution? 7. "Why roll the pipette as the blood enters the bulb? 8. Why blow out a few drops before putting a drop on the slide ? NORMAL HEMATOLOGY 135 9. Why draw air through the pipette after the ether is drawn out? 10. What kind of a sohition should be used to dihite the blood; that is, what properties should it have? 11. Why are there 101 parts in the pipette instead of 100? 12. Is there any appreciable variation in the number of red cells in normal individuals? 13. If there is a variation, give some of the reasons. 14. Account for the variations observed in members of your section. B. To Count the White Blood Corpuscles. In counting the number of white cells per cubic millimetre in the blood, the principle of diluting and counting is exactly the same as in counting the red cells. There are some differences in the details, since we must first get rid of the red cells so that the white cells can be seen, and because of the small number we must dilute less and count larger areas. Appliances. The instruments are the same, with two exceptions or modifications. The diluting pipette is just like the red-cell diluting pipette, except that it is larger in the capillary and smaller in the bulb, so it can make dilutions of ten to one hundred instead of one to one hundred. The counting plate should have the modified mark- ings as shown in Fig. 68. One can use the lower power (f ) objective of the microscope just as well and save time by it, but it is not necessary. Reagents. The same as when counting the red cells except that a different diluting solution must be used. A \ per cent, solution of acetic acid in distilled water will destroy the red cells and render them invisible, while it will not destroy the white cells, but make them show more plainly. White-cei>l Diluting Solution. Acetic acid 0.5 c.c. Distilled water . . . . . q. s. ad 100.0 c.c. Technique. The technique of obtaining the blood and filling the pipette is the same as with the red cells, except that the capillary tube is so large that we must have more blood. For this reason for l)egin- ners we recommend that only a half-part of blood be taken at first. More accurate results can be obtained by using one part, but much time and practice is necessary to fill the tube easily. The capillary is .so large that .solutions will not stay in it, but run out quickly when the tube is out of the horizontal position. First, then, we must have more blood; usually two or three good-sized drops are sufficient. Scc(jnd, the tube must be held horizontal or the blood and solution will run out. As the capillary tube is large it is very easily cleaned and dried. 136 SPECIAL PHYSIOLOGY Roll the pipette as before when filled and in a few moments the mixture will turn quite dark; when it no longer changes color it is ready to be counted. Allow a few drops to flow out of the tube as in the case of the red-cell pipette, then place a small drop from the end of the pipette on the ruled plate. It is not necessary to blow the fluid out; it will run out. Take the same precautions in filling the counter and adjusting the cover-glass as before, except that there is no need of haste in placing the cover-glass, because the white cells are lighter. Here, because we have a clear field with little in it, and the cells are quite large, we can use a lower power of the microscope and see a whole square millimetre at once. Begin at the upper left-hand corner and count the cells in each space 1 mm. square, and observe the same method in keeping the record as when counting the red cells. Clean the counting slide, roll the pipette for a moment, and refill the marked plate and count the nine spaces again, keeping the records as before. Do this at least three times, so that the area counted will be 27 spaces, each 1 mm. square. The more cells counted the more accurate the results should be, but the three fields should be sufficient. To estimate the number of cells per cubic millimetre in the blood specimen used, add together the number of cells and divide by the number of millimetre spaces counted. Each space is -^q mm.X 1 mm. X 1 mm. or y q- c.mm. Now multiply the average number of cells in each space by 10 to find the number of cells in the diluted blood, and then by 10 or 20 according as the blood was diluted, and that will give the number of white cells per cubic millimetre in the blood specimen, as follows: 33 45 56 47 39 57 51 43 49 48 57 39 55 45 61 37 61 53 61 53 59 43 51 41 57 39 40 142 155 154 145 135 159 145 143 142 = 1320 1320 ^ 27 = 48|X 10X20 = 9777^ white cells per cubic millimetre of the blood examined. Questions. 1. What is the number of white cells per cubic millimetre in the blood in the normal individual? 2. What is the normal variation? 3. What are some of the causes of the variations? C. To Count both Red and White Cells at the Same Time. In general the whole technique is followed out and the same instruments used as when counting the red cells alone. The method consists of using a diluting solution containing a stain that will stain the white cells only, and then counting the red and white cells separately. NORMAL H.EMATOLOGY 137 Colored Diluting Solution. Methyl violet 0.025 gram. Sodium chloride 1.000 gram. Distilled water 100.000 c.c. Count the red cells in a group of thirty-six spaces first, and keep the record as before. Next count the white cells in all of the nine square-millimetre spaces and keep the record as before. This should be repeated until at least two groups of red cells and three or four groups of white cells are counted from different specimens on the counter, and each record should be kept so that the average may be taken and the number per cubic millimetre be estimated in each case. Estimate the number of cells by taking the average and estimating the number just as when counting the red cells alone. Estimate the number of white cells just the same as before by taking the average for each y^o c.mm., but multiply that by 100 in this case instead of 10 or 20, as the blood in this specimen was diluted 100 times. D. Centrifugalization of the Blood. To Determine the Relative Volume of Red Corpuscles and Plasma. To Estimate the Number of Red Corpuscles from Their Volume. Appliances. Electric or hand hsematocrit (Fig. 67) ; small rubber tubing to fit capillary tube; glover's needle; white paper; fine wire for cleaning tubes. Reagents. Di.stilled water, hydrogen peroxide, alcohol, and ether. Preparation. Adjust rubber to capillary tube. Put empty tube in one arm of cross-piece to preserve balance. Use fine wire to remove blood from the capillary tube, then clean and dry as other tubes. Technique. Obtain blood from the lol)e of the ear as heretofore described. Draw capillary tube full of blood. Remove the rubber tube by pushing it off and not by pulling. Remove any blood from the outside of the capillary, and make a record of the amount of bloofl in the capillary. Place the tul)e in the cross-piece of the instrument as quickly as possible and centrifugalize at least three minutes at the rate of 7000 to 10,000 rotations per minute. Take out the tube and lay on a piece of white paper to read the divisions. J)ach degree of the scale is estimated to contain about 100,000 cells; hence, a tube in which the red column stands at .'jO would indicate about .5,000,000 red corpuscles per cubic millimetre. The use of this instrument is designed, however, chiefly to show the volume of red rorpn-srlrs rather than the numher. Precautions. Do not displace the rubl)er i)ads in the outer ends of the rotating arm, as the blood will be thrown out of the tube and 138 SPECIAL PHYSIOLOGY necessitate the repetition of the test. Before starting each test see that the pads are in place. If the tube is not adjusted in the apparatus and set to rotating within a few seconds after the blood is drawn, coagulation will set in and hinder the complete separation of the corpuscles from the plasma. Should separation not be complete in three minutes the test should be repeated. The instrument should be started and stopped gradually, as the sudden starting and stopping injures it. Questions. 1. Determine the volume percentage of red blood corpuscles in a number of normal individuals. 2. Do apparently normal individuals have the same or approx- imately the same volume percentage of red blood corpuscles ? If not, seek for causes of the variations in different individuals. 3. Does the same individual have the same volume percentage of red blood corpuscles all the while? (a) If there is a variation, is there any periodicity to be observed? (6) Seek for causes of any variation in the same apparently normal individual. 4. The volume percentage as recorded by the hsematocrit varies with the product of two factors: the average volume of the individual corpuscles by the number of corpuscles per unit volume. (F^vXn). (a) Is the average volume of the individual corpuscles (v) neces- sarily constant? (6) If it is not constant, would one be justified in drawing con- clusions regarding the number of corpuscle per unit volume (n) after observing the volume percentage (F) with the hsematocrit? 5. What variation of the observation as above made would enable one to determine with reasonable accuracy the number of corpuscles per cubic millimetre? 6. If the tube were only partly filled at first, could one make an accurate test? If so, tell how to proceed. II. THE ESTIMATION OF THE PERCENTAGE OF COLORING MATTER IN THE BLOOD. The estimation of the coloring matter in the blood is based on the supposed fact that a normal individual under normal surroundings has a normal amount of coloring matter, and that is called 100 per cent. The instruments that have been devised for making the estimation are numerous, and all, while theoretically correct, practically are liable to a greater or less error according to the experience and carefulness of the observer. They are, however, in a skilful and conscientious operator's hands, quite accurate, and are especially so when used to compare the tests of the same patient's blood, week by week. NORMAL H^MA TOL OGY 139 The haemoglobin contains practically all the coloring matter, and it constitutes 90 per cent, of the red cell. The haemoglobin consists of 96 per cent, globulin and 4 per cent, hsematin. In the hsematin is the iron of the corpuscles; the coloring matter of the blood varies as does the amount of iron. Theoretically, the most accurate way to test the haemoglobin would be to measure the amount of iron in a certain amount of blood. But the chemical extraction and weighing of so small an amount of iron is too difficult and tedious. Because of this, other tests have been devised, which depend upon the observer's eye to detect the likeness of shades of red as repre- sented by the blood and colored glass, solutions or paper. Again, the specific gravity of the blood except in rare cases depends upon the amount of iron in the red cells, and varies as the iron does. Then we can estimate the percentage of haemoglobin by finding the specific gravity of the blood. The principal tests may be classified as follows: 1. Estimation of iron in the blood. JoUes' ferrometer. 2. Estimation of percentage of coloring matter by color tests. A. Fleischl's haemometer. B. Gowers' haemoglobinometer. C. Dare's haemoglobinometer. D. Tallquist's haemoglobinometer. 3. Obtaining the specific gravity of the blood by Hammerschlag's method. A. Fleischl's Haemometer. Appliances. Fleischl's haemometer; glover's needle; pasteboard tube two inches in diameter; artificial light; small beaker; a dark room or cupboard. Fleischl's haemometer consists of a sliding colored-glass wedge which moves in a standard underneath a cylindrical metallic cup, and a capillary tube. This cup is divided into two equal com- partments and has a glass bottom and a detached glass top. The capillary tube is very small and is held by a small metallic band on a handle. The glass wedge and the capillary tube are the important parts of the instrument and are made to be used together. There is a number on the handle of the capillary tube, indicating its capacity, and this same number is stamped on the top of the standard; also a number is placed on the end of the sliding frame that holds the glass wedge, and the same number appears on the base of the standard of the instrument to which it belongs (Fig. 69). Reagents. Distilled water and hydrogen peroxide. Preparation. Clean metallic cell or well with water and dry with a cloth only when it needs it. The capillary tube should be cleaned with water and hydrogen peroxide, and then with water again, by 140 SPECIAL PHYSIOLOGY waving it back and forth in the sokitions for a moment or two. Then carefully dry the tube by blowing air through it, holding the tube about two inches from the mouth so as to avoid the moisture of the exhaled air. Fill each side of the metallic cup about three- fourths full of distilled water. Prepare the needle and the ear or finger as in other tests. Technique. Obtain the blood in the usual manner. Hold the lobe of the ear with the thumb and finger. Use the second drop. Hold the capillary tube horizontally and carefully touch the drop of blood with the end of the tube onlv. If the tube is clean it will fill rapidly Fig. 69 Fleischl's hsemometer. by capillary attraction. If there is any blood on the outside of the tube or air-bubbles inside, it must be cleaned, dried, and refilled properly. If the capillary is overfull, remove the excess by touching the tip to a cloth or filter paper. Then quickly put the capillary tube into the water in one compartment of the metallic cell and wave it back and forth or up and down, and the blood, if fresh enough, will readily mix with the water; then allow a few drops of water from the medicine dropper to flow through the capillary into the same com- partment to wash the blood that sticks to the tube. Now fill each compartment almost full with distilled water, taking care that the contents of either compartment does not flow into the other. Take NORMAL HEMATOLOGY 141 the handle of the capillary and stir the one that contains the blood so as to make the mixture complete. Now carefullv sUde the thick cover-glass over the compartments and gradually fill each cell with water as the cover-glass is put on until there is no air left in either cell. Exclude dayhght by use of a dark room or a cupboard, and adjust the reflector so that the artificial yellow light is thrown up through the diluted blood and water from the side of the instrument, thus placing both cells in same relation to the reflector and the light. While making the test alwavs shade the eves from the light bv placinfir some thick paper or a pasteboard tube, that reaches from the instru- ment to the forehead, before the eyes. It is better to use only one eye at a time, and look only for a few seconds at each time, gi^"ing the eve a rest and a chance to regain the abilitv to distinguish tints. Stand at one side of the instrument or turn the instrument so as to face the light and to bring the two cells into similar relations with the eve. Begin with a glass of a lighter color than the blood, and move the colored-glass shde by quick turns about one-fourth of an inch each time until the color or tint of the diluted blood appears to be the same as that of the colored slide; then make the reading. Next turn the colored glass on imtil it is darker than the diluted blood and do the same as before, except in the opposite direction, turning the slide until the color of the glass and blood are the same, and then make the reading. Usually the first reading will be too low and the second too high. The dift'erence will usually be about 10 per cent. The correct result will be between these two readings, which can now be obtained bv carefullv moving the glass back and forth or by taking the middle point between ihe two readings. It is almost impossible to make the reading accurately and honestly unless great care is taken, and the writer has found the method given to produce the best results by far. This method should be practised again and again and done with care. A hasty reading is rarely correct. Repeat the whole test until you can obtain the same result each time with the same individual's blood. Precautions. If the capillary tube is not perfectly clean it will not take blood by capillary attraction. While cleaning the tube always test it by touching a drop of water, when it should fill imme- diately. This will save time and ensure quick work. The amount of blood taken is so small and this is diluted so much that the least error is multiplied many times. We can expect accurate results only when every known chance of error is safely guardetl. If the capillary has moisture or foreign matter in it, the tube will not hold the right amount of blootical conclnHionH. 204 SPECIAL PHYSIOLOGY (c) Observation of the Myopic Eye. Adjust the model to represent 3 D. of myopia. (5) Does the increase of the distance of the lens from the eye cause the image of the papilla to become altered in size or reversed in posi- tion? Account for all phenomena. (6) If the position of the + 12 D. lens, which the observer holds, remains the same — 8 cm. from cornea — will there be any variation in the distance from the cornea of the retinal image for the hyperopic eye and myopic eye? Will the distance of the hyperopic eye be greater or less than for the emmetropic eye? Why? (d) Observation of the Human Eye. At this point of the student's work, let him practice the direct and indirect method of ophthalmos- copy upon his comrades; after two or three days of practice he may pass to the following exercise. XIII. SKIASCOPY. Gould defines skiascopy as "a method of estimating the refraction of the eye by observation, through ophthalmoscopic mirror, of the movements of the retinal images and shadows." Synonyms: Fundus reflex test; umbrascopy; pupiloscopy; koroscopy; kertoscopy; retin- oscopy, etc. 1. Appliances. A simple retinoscope or an ophthalmoscope with a plane mirror; Thorington's skiascopic eye or an equivalent; dark-room lamp, etc. 2. Operation. The observed eye and lamp are to have the same relative position as in ophthalmoscopy. Let the observer sit directly in front with the eye in the same horizontal plane with the lamp and observed eye, and somewhat more than 1 m. distant from the observed eye. Throw the light reflected by the mirror into the observed eye; rotate the mirror slowly and a shadow will be seen in the pupil of the observed eye. 3. Observations, (a) Observation of the Emmetropic Eye. Adjust the model to represent emmetropia. (1) Does the shadow move in the same direction as the mirror rotates or in the opposite direction — i. e., does the shadow move with the mirror or opposite f (2) Is the movement of the shadow quick or slow? (b) Observation of the Myopic Eye. (I) Adjust the model to represent less than 1 D. of myopia. (3) Note that the shadow movement is with the direction of the mirror rotation, and that it is relatively quick. (II) Adjust the model to represent a myopia of more than 1 D. (4) Note that the shadow movement is opposite the direction of the mirror rotation, and that it is quick when the myopia is of low degree; slow when of high degree. VISION 205 (5) Observe alternately the three conditions indicated above until their differences are so familiar that any one of the conditions may be readily and unerringly detected by the observer when they are arranged for him ))y the instructor. (c) Observation of the Hyperopic Eye. Adjust the model to repre- sent any degree of hyperopia. (6) Note that for a low degree of hyperopia the shadow movement is ivith the mirror rotation and quick. (7) Note that for higher degrees of the condition the shadow move- ment is with the mirror and slow. (S) How may one differentiate a high degree of myopia from a high degree of hyperopia? (9) Is there any difference in the size, shape, distance, or position of the shadows in these two conditions? (d) Observation of the Human Eye. Let the student practice upon his comrades.^ 1 Observation of the astigmatic eye is intentionally omitted here. It belongs more espe- cially to the clinical phase of the subject. CHAPTER VIII. THE PHYSIOLOGY OF THE NERVOUS SYSTEM. I. REFLEX ACTION. 1. Material and Appliances. Three large, vigorous frogs; operat- ing case; sulphuric acid, 0.5 per cent.; acetic acid, 50 per cent.; dis- tilled water; cork board, 10 cm. square; hand basin; filter paper; six watch-glasses. 2. Preparation. Pith two of the frogs, taking care to sever the medulla completely; destroy the brain but leave the spinal cord intact. If there is hemorrhage plug the puncture with absorbent cotton. Lay the pithed frogs ventrum down, with legs extended, upon a moist paper. Note that if the toes be pinched the leg will not be flexed. There is no reflex response. The animal is under the in- fluence of shock. This condition will probably last for half an hour or more. Recovery will be indicated by the drawing up of the legs, first one leg and then the other being flexed. 3. Observations, a. Modifications of General Functions by Pithing. (1) The pithed frogs lie upon the ventrum with legs flexed. The position simulates the normal. Make a detailed comparison of the posture of the pithed frogs with that of the normal frog under the bell-jar. (2) Compare pithed frogs with normal as to the appearance of the eyes. (3) Study the respiratory function of the pithed frogs. (4) Is the heart, as observed through the body wall, acting with usual rate and force in the pithed frogs? (5) Gently lower a pithed frog into a basin of cold water. Is there an adaptation to the conditions? Does the frog swim? Vary the experiment by dropping the frog from a height of six inches. Take a yard of cord and tie one end around the brachium of the normal frog. Repeat the experiments with the normal frog and note the character of response. (6) Place a pithed frog upon a cork board; gently tip the board in any direction, noting whether there is adaptation in equilibration. Repeat the experiment with the normal frog. Describe differences. (7) Lay a pithed frog on its back on the table; will it right itself? Try a normal frog. THE PHYSIOLOGY OF THE NERVOUS SYSTEM 207 h. Reflex Response to Various Stimuli. Suspend a pithed frog by a hook through its mancHble. The body and legs should hang freely, and should be several inches from the table. (8) ^Mechanical Stimuli. With forceps pinch one of the toes (not the longest) of the hind leg. Pinch the skin of the flank. Pinch the folds of skin about the anus. Note response when stimulus is varied in strength and applied to either side. (9) Thermal Stimuli. With a hot wire touch the skin at several points, noting response. (10) Electric Stimuli. With single-induction shocks stimulate skin of legs, thighs, or flank, using fine platinum- wire electrodes, and touching the moist skin. If single shocks elicit no response, use a rapid succession of shocks produced by bringing the Neef hammer into the primary circuit. (11) Chemical Stimuli. Cut some pieces of filter paper not over 2 mm square. Dip a piece into 50 per cent, acetic acid, taking care that the paper is saturated and that there is no excess of the acid. Apply the acid paper in turn to the web of the foot; to the flank; to the ventrum; median line; to the anus. Note the character of the response, as to extent, single-sided or double-sided. After each application of acid to the skin of the frog, the acid should be thor- oughly rinsed or swabbed away. c. The Characteristics of Reflex Response. (12) Purposive Char- acter OF Response. In the responses already studied the observer could easily note that the movements possessed the manifest pur- pose of removing the offending object. In many cases the move- ments involved several sets of muscles, but in every case all the muscles involved in the response acted with perfect co-ordination, and the movement was well directed toward the removal of the irritation. This is what is meant by the purposive character. In order further to illustrate this characteristic of response, repeat Foster's instructive experiment: "Choosing a strong frog in which reflex action has been found to be highly developed; suspend it; hold the right leg firmly down, and apply a square of acid paper to the right flank. Twitchings and convulsive movements of the right leg are at first witnessed; then the left leg is brought up to rub the right flank." (Handbook for the Phjj.sioloc/ical Lahoraiory, p. 409.) (13) The Latent Period of Keflex Response. The observer will remember that in most of the above experiments the responses did not follow instantaneously upon the application of the stimulus; this was especially i)oticeable in the case of one of the weaker stimuli. In order further to illustrate this characteristic, prepare five dilu- tions of sulphuric acid in as many watch-glasses; hold a glass so that the tip of the longest toe ju.st dips into the acid. Note the time rerjuired to elicit a resi)onse. After each response rinse off the j)art .stimulated and allow the animal to rest several minutes, testing 208 SPECIAL PHYSIOLOGY other pithed frogs in the mean time. The number of seconds re- quired for response may be counted from a metronome or from a watch. Latent periods in seconds. Strength of stinaulus. , * , Frog A. Frog B. H2S04 0.05 per cent. . H2S0, 0.1 li (( H2S0, 0.2 It u Hi,S04 0.3 <( (( H2SO, 0.4 (( (. (14) The Irradiation of Reflex Action. In the above- described experiment it will probably have been noted that the stronger the stimulus the more extended the response — i. e., the greater the number of muscles brought into action. When only the tip of the toe is touched to very weak acid the response will be a simple flexion of the tarsus, and this only after several seconds. When stronger acid is applied to the toe or web the crus and the femur may both be flexed, and the action is some- times repeated. Repeat some of the experiments, paying special attention to the variation of extent of response, with varying strength of stimulus. Note that in some cases the response involves the opposite side, as well as higher and lower levels of the cord. (15) Location of Reflex Centers. Take one of the pithed frogs that has been responding typically. Run a pithing probe down the spinal canal, thus functionally destroying the spinal cord. Apply any of the stimuli mentioned above. Note results, and account for same. II. REFLEXES IN THE HUMAN SUBJECT. Until one's attention is called to it, he is likely to overlook the great importance of reflex action and the reflexes in the mainte- nance of the human body. The eyes are protected from dust and other irritable substances, the lungs from dust and irrespirable gases, through the intervention of reflex action. The food is moist- ened and the digestive juices started through reflex response to the stimulating influence of food in the mouth and digestive canal. The respiration, circulation, heat regulation, excretion, and various other vital processes are controlled through reflexes. The paramount im- portance of reflex action thus becomes apparent. A disturbance of certain reflexes becomes a clinical symptom of considerable importance. It is proposed here to study briefly a few of these reflexes. Their detailed discussion is left to the text-books. THE PH rsiOL OGY OF THE NER VO US S YSTEM 209 1. Appliances. Glass rod, 20 cm. long, with rounded ends; 3 per cent, carbolic acid; beaker of water; towel; bristle mounted in handle. 2. Observations, (a) Respiratory Reflexes. (1) Make a bristle aseptic. Let one member of the group act as subject. Let the sub- ject close his eyes. The observer should introduce the bristle very gently through the nostril, and, as far as possible, up the nasal passage. The response will probably be in the form of a sneeze. Accidental introduction of irritating substances into the respiratory passages below the glottis causes coughing. (2) Make a bristle aseptic and bend it into a semicircle. Let the subject open the mouth wide, depress the tongue, and say "Ah!" prolonging the sound several seconds. Introduce the bristle into the mouth; pass it over the tongue without touching the latter; turn the point downward back of the tongue, and tickle the glottis. A convulsive, reflex movement of the larynx, sometimes accompanied by a cough, will result. (6) Circulatory Reflexes. (3) Posture influences the circulation reflexly. Let the subject remain sitting quietly while the pulse is counted through a minute. Note the number. Let the subject lie on the back upon the table. After he has rested quietly three to five minutes, take the pulse rate again. Let the subject stand and observe the rate after three to five minutes. (4) Respiration influences the circulation reflexly. Let the sub- ject sit breathing at the rate of thirty respirations per minute for two minutes. Count the pulse during the second minute. Let the sub- ject then drop to ten respirations per minute for three minutes, and then slower, if possible, during the fourth minute, when the pulse is to be counted. (5) Exercise influences the circulation reflexly. This has already been demonstrated. (See Circulation.) (c) Secretory Reflexes. (6) The chewing of anything like paraflSn or rubber incites reflexly the free flow of saliva. In a similar way the presence of food in the stomach and intestines stimulates the secre- tory activity of the digestive glands. (d) Reflexes of Deglutition. (7) Let the subject open the mouth. Introduce the aseptic glass rod into the mouth without touching tongue or cheeks. Gently touch the uvula; it wifl probably rise. Touch the fauces, and observe the convulsive swallowing move- ment. Sometimes this merges into a gaggitig movement. The raising of the uvula is part of a normal swallowing act. The resfjonse of the fauces may be a part of an act of swallowing, or of a protective act (gagging), according to the conditions of the stimu- lation. (e) Visual Reflexes. (8) Let the subject direct his vision toward some distant object. Make a sudflen movement with hand or a book 14 210 SPECIAL PHYSIOLOGY • as if to strike the subject in the face. Observe the winking of the eyeHds — another protective reflex. (9) Let the subject again direct his vision toward a distant object; gently touch the conjunctiva of the eyeball with the sterilized round end of the glass rod. The convulsive winking is a protective reflex. (10) Let the subject sit near a window, and, looking through the window, direct his vision toward some object not more than twenty feet away. Suddenly shade the eyes of the subject for a few moments; then remove the shade and observe the change in the size of the pupil. During the experiment let the subject maintain the same state of accommodation, if possible. (/) Cutaneous Reflexes. (11) Tickle the base sole of the foot. The foot will be involuntarily withdrawn — the plantar reflex. (12) Pinch skin of neck. The pupil will dilate — the ciliospinal reflex. There are various other cutaneous reflexes, such as the cremas- teric, abdominal, epigastric, scapular, and gluteal. (g) "Tendon Reflexes." These phenomena are not really reflexes, though they have been called that for a very long time. They may be studied in this connection. Let the student give reasons why the responses to the stimuli are not necessarily reflexes. (13) Ankle Clonus. Let the subject's leg be supported as by resting it across a chair, the subject being seated. Let the observer place the hand upon the ball of the foot and press suddenly, so as to put the tendo Achillis upon the stretch. There results a series of clonic contractions. This phenomenon is not observed in a healthy subject. (14) "Patellar Reflex" or Knee-jerk. Let the subject cross the legs in a posture frequently assumed when sitting. Tap the tendon below the patella with the edge of the hand, with the back of a thin book, or with a percussion hammer. The quadriceps extensor muscle will be suddenly stretched and will respond with a quick contrac- tion, which will throw the foot forward in a kicking motion. This phenomenon is present in health, and it may be modified in disease. III. THE ACTION OF STRYCHNINE UPON THE NERVOUS SYSTEM. 1. Material. One dog; two frogs; sulphate of strychnine. 2. Preparation. Make a solution of sulphate of strychnine, 0.01 grm. to 10 c.c; also concentrated solution, 0.2 grm. to 10 c.c. pithed frogs. Do not fasten the dog to the dog board. Set up electric apparatus to obtain tetanizing current. 3. Experiments and Observations. (1) Hypodermic injection of 2 mg. strychnine per kg. of the dog. This dose is invariably lethal,, even with early antidotal treatment. THE PH YSIOL OGY OF THE NEB VO US S YSTEM 211 (a) Record the condition before and S}Tnptoms as they arise after exhibition of the drug, especially with reference to: (I) Muscular activity. Describe convulsions. (II) Respiration. How affected by reflexes. (III) Circulation. Rapidity and rhythm of heart. (IV) If death occurs, which stops sooner, the circulation or respiration ? (6) Formulate results. (2) Ligate thigh of frog, except sciatic nerve, at junction with body. Sever all structures, except nerve and femur, just below ligature. Separate cut surfaces with rubber tissue to prevent diffu- sion of the drug. Turn the frog over and make a median abdominal incision. Pressing viscera aside, pick up the sacral plexus of nerves going to the uninjured leg. The sacral plexuses may be readily recog- nized, lying on each side of the median line. Pass a thread loosely around the nerves, so as to quickly find them when wanted. Inject into dorsal lymph space 0.0001 grm. strychnine. (a) ^Yhat part of the frog is reached by poison? What part is protected from it? Illustrate by diagram. (b) Were strychnine a convulsant through its action on the sen- sorium, would the legs be equally convulsed? If it acted on the spinal cord? If it acted on the motor nerves? If it acted on the muscles directly? (c) Are both legs convulsed? (d) To what parts in the reflex arc have you limited the action of the strychnine? (3) Using as a guide the thread formerly passed around it, pick up the sacral plexus and sever it high up. (a) Does this strychnine reach the motor nerve and the muscles of the uninjured leg? (b) If strychnine were a convulsant through its action on either the motor nerves or the muscles, or both, would the uninjured leg still participate in the convulsions? (c) Demonstrate that muscles, sciatic nerve, and sacral plexus below the point at which it was severed are still intact by stimulating distal portions of the latter. (d) To what elements of the reflex arc have you limited the pos- sible action of «;trychnine? ("4) Expose the heart of a frog and ligate the aortte at the base. Operation as follows: P>eely expose sternum by + shaped incision and laying back of flaps. Remove lower half of sternum with scissors, taking care not to injure vessel in abdominal wall, which comes just to the tip of sternum. Freely incise exposed pericardium, l)ringirig heart into view. Grasp apex of heart with forceps, taking care not to use force enough to cut through ventricular wall, and draw heart down and 212 SPECIAL PHYSIOLOGY forward. This gives ready access to bulbus arteriosus and aortse. With an aneurysm needle pass fine thread around latter, taking care iiot to injure auricle, and ligate. With scalpel cut through occipito-atlantoid membrane from side to side, and bend head forward. Remove posterior wall of upper end of spinal canal by inserting smaller blade of strong scissors into spinal canal and cutting, taking care not to injure cord. Allow a drop of the concentrated solution of strychnine to fall directly upon cord; or with fine hypodermic needle inserted 1.5 cm. into the arach- noid space inject two drops of the solution. {a) What effect has ligation of the aortse on the circulation? (h) Would stoppage of the circulation prevent the drug from reaching the peripheral terminations or trunks of the sensory nerves ? Motor nerves? Muscles? (c) Where, then, must strychnine act to produce the observed symp- toms? {d) Would cessation of the circulation delay the action of strychnine on the cord by slowing the rate of its absorption by the latter? (5) After observing results in experiment (4), destroy first the upper, then the lower, portion of the cord by passing a wire down the spinal cord. (a) How does destruction of the upper part of the cord affect the convulsions ? (h) What is the result of the destruction of the entire cord? (c) Do the results agree with those of previous experiments? Note. Destruction of the upper part of the cord during the preparation of the animal may take place; if so, the upper limbs will not take part in the convulsions. (6) Further Observations and Comparisons, (a) Compare the gen- eral effects of strychnine and curare in the dog. (&) Compare results obtained in experiments consisting of ligating the thigh of a frog, except the sciatic nerve, and injecting, in the one case strychnine, in the other curare. IV. THE ACTION OF CURARE UPON THE NERVOUS SYSTEM. 1. Materials. One dog; two frogs; normal saline solution; curare; dry cells; inductorium; hand electrodes. 2. Preparation. Prepare the following solution: sodic chloride, curare, 0.2 grm. to 10 c.c, in acidulated 20 per cent, alcohol. Pith frogs. Do not fasten the dog to the board, but simply restrain him. Set up electric apparatus so as to obtain single induction shocks. 3. Experiments and Observations. (1) Give a hypodermic injec- tion of 0.01 grm. per kg. curare to the dog. THE PHYSIOLOGY OF THE NERVOUS SYSTEM 213 (a) Record the condition of the dog^ just before and every ten minutes after injections of curare, with special reference to: (I) ^Muscular activity. (II) Respiration, number and depth. (III) Circulation, rate and rhythm of heart beat. (IV) Which stops sooner, respiration or circulation? (b) Formulate the total effect of curare upon the animal. (2) Ligate the thigh of a frog, except the sciatic nerve near the knee-joint. Inject into the dorsal lymph space 0.002 grm. curare. (a) What elements enter into the formation of a reflex arc f (b) What motor phenomena would result from increased irrita- bility of any part of the reflex arc? (c) What motor phenomena would result from lessened irritability or destruction of any element in the reflex arc? (d) What effect has the ligature of the thigh on the distribution of the curare? (e) How do the reflex arcs, of which the gastrocnemii are the motor ends, differ with regard to the distribution of the curare? What part of the reflex arc is protected from curare in the ligatured limb ? (/) Describe the relative reaction of the gastrocnemii to stimuli (chemical, mechanical, electric) applied to the various parts of the body and limbs. (g) Is the sensorium intact? Is it reached by the curare? (h) Is the cord intact? Is it reached by curare? (3) Expose the sciatic nerves, near the body, in the frog, used in the experiment. Stimulate them. (a) What elements in the reflex arc enter into consideration in this experiment? (b) Which of these elements are exposed to, which protected from, the poison? (c) Are both sciatics reached by curare? (d) Is there a difference in the reaction of the gastrocnemii to the stimuli applied to the sciatic nerves? (e) To what elements of the reflex arc have you limited the possible action of the curare? (/; Have you proven that curare does not affect the nerve trunks? (4) Expose gastrocnemii by cutaneous incision. Stimulate the muscles directly. (a) Is there a difference in reaction to stimuli? 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