PEACTICAL PHYSIOLOGY PRACTICAL PHYSIOLOGY EDITED BY M. S. PEMBKEY CONTRIBUTORS A. P. BEDDARD, M.A., M.D. LEONARD HILL, M.B., F.R.S. Assistant Physician, late Demon- Lecturer on Physiology, The strator of Physiology, Guy's Hospital London Hospital J. S. EDKINS, M.A., M.B. Lecturer on Physiology, St. Bartholomew's Hospital J. J. R. MACLEOD, M.B. Professor of Physiology, Western Reserve University, Cleveland, U.S.A. MARTIN FLACK, M.A., M.D. M. S. PEMBREY, M.A., M.D. Demonstrator of Physiology, London Hospital Lecturer on Physiology, Guy's Hospital ILLUSTRATED BY NUMEROUS DIAGRAMS AND TRACINGS THIRD EDITION LONDON EDWARD ARNOLD 41 AND 43 MADDOX STREET, BOND STREET, W. 1910 [All rights reserved] BIOLOGY LIBRARY G PREFACE TO THE FIRST EDITION. PHYSIOLOGY is the basis of medicine, and the further advance of these sciences depends mainly upon the "experimental method." The medical student, the future physician, should undergo a training in practical physiology, for thereby he learns the most important of all lessons ; he learns to observe, to draw conclusions from his observations, and to unravel the causes of his failures. The importance of practical physiology is undoubted, but as to the nature and scope of the experimental work, which is most suitable for the medical student, there is considerable difference of opinion among teachers of physiology. In this country, perhaps, too much stress has been laid upon the physiology of muscle and nerve ; for the hope that a study of the properties of these tissues will unfold the enigma of life is likely ever to remain without consummation. An advance in the knowledge of the living organism as a whole, one organ reacting upon another, has been gained by experiments upon the living animal, treated as a unit and not as a collection of separate organs and tissues. Such practical physiology needs extension in the courses of instruction given to students. It should, as far as possible, have a direct relation to medicine. The methods which are used in the investigation of the respiratory system, the circulation, the body heat, the nervous system and special senses ; the chemistry of the blood, of digestion, and of urine — these are the subjects which are especially required by the clinician. These sub- jects, moreover, afford as excellent a mental training as the study of muscle and nerve. In the present work the authors have attempted to give some exten- sion to practical physiology along the lines just indicated. The book has been divided into an elementary and an advanced por- tion. Part I. treats of elementary experimental physiology (the physiology of muscle and nerve, circulation, respiration, animal heat, vi PBEFACE the central nervous system, and the special senses) ; Part II. of elementary physiological chemistry ; Part III. of advanced experimental physiology ; and Part IV. of advanced physiological chemistry. The experiments upon the physiology of muscle and nerve are based upon the course given at Guy's Hospital — a course modelled on a reduced scale upon the excellent practical courses given at Oxford by Professor Burdon Sanderson and Professor Gotch. The experiments in this section have been limited as far as possible to those which can be conveniently performed with simple apparatus by a large class of students. For this reason the experiments with the galvanometer and capillary electrometer have been restricted to demonstrations, and very few details of such experiments are given. There are some important experiments upon the circulation and respiration, which for various reasons cannot be properly performed by the student ; these have been collected together as demonstrations in Parts I. and III. The subject of vision is so important from a medical as well as a physiological and psychological point of view, that it has here received more extensive treatment than is usually the case in works on practical physiology. In those portions of the book which treat of physiological chemistry, an attempt has been made to demonstrate, step by step, the chemical relationships which exist between the various substances, and to illus- trate, by suitable experiments, the different properties of those bodies. The drawings of crystals were executed by Mr. W. E. M. Turtle, to whom the authors are deeply indebted. Figures have been borrowed from The Physiological Action, of Drugs, by M. S. Pembrey and C. D. F. Phillips. For the loan of numerous blocks illustrating physiological apparatus the authors are indebted to Messrs. Baird & Tatlock, of Hatton Garden, E.G. The sources of other diagrams and tracings, which have been borrowed, are indicated in the description of the figures. The initials of the author, who took the record of the original tracings, are appended to the respective curves. Sept., 1902. PREFACE TO THE SECOND EDITION. IN the present edition considerable changes have been made in those portions of the work which deal with Physiological Chemistry. The new exercises have involved a slight increase in the total number of pages, and several new figures have been added. July, 1905. PREFACE TO THE THIRD EDITION. THE present edition is in many respects a new book, for many parts have been rewritten and the arrangement of the whole has been altered. The book now consists of two parts, Part I., which deals with Experimental Physiology, and Part II., which is devoted entirely to Physiological Chemistry. The experiments upon muscle and nerve have been reduced in number and the observations which can be made upon man have been increased. The Authors wish to thank Drs. H. D. Haskins and J. H. Ryffel for their able assistance in the revision of the chapters on Physiological Chemistry and Dr. Kennaway for many criticisms and suggestions upon the whole of the work. Dr. Hertz has contributed a special chapter upon the " Investigation of the Motor Functions of the Alimentary Canal by means of the X-Rays," and Dr. Ryffel one upon "Lactic Acid, its Estimation and Significance." For this valuable assistance hearty thanks are given. Sept., 1910. CONTENTS. PART I. MUSCLE AND NERVE. CIRCULATION. RESPIRATION. ANIMAL HEAT. CENTRAL NERVOUS SYSTEM AND SPECIAL SENSES. By A. P. BEDDARD, J. S. EDKINS, L. HILL, and M. S. PEMBREY. CHAP. PAGE Introduction, - 1 I. Electrical Apparatus for Physiological Experiments. By A. P. B., 2 II. The Graphic Method. Maximal and Minimal Stimuli. Uni- polar Excitation. By A. P. B., - - - - - - 14 III. A Single Contraction of a Gastrocuemius Muscle. By A. P. B., - 22 IV. The Conditions which affect Single Muscular Contractions. By A. P. B., - .... 29 V. The Conditions which affect Single Muscular Contractions (continued). By A. P. B., - - - 32 VI. The Conditions which affect Single Muscular Contractions (continued). By A. P. B., - - - - - - 35 VII. Two Successive Stimuli. Genesis of Tetanus. Tetanus. By A. P. B., - - 40 VIII. The Properties of Nerve, Minimal and Maximal Stimuli. By M. S. P., - 44 IX. The Relation between Muscle and Nerve. By M. S. P., - 48 X. The Effect of a Constant Current upon Muscle and Nerve. By M. S. P., - - 50 XL The Electromotive Properties of Muscle and Nerve. By M. S. P., - 51 XII. Extensibility and Elasticity of Muscle when at Rest and Contracted. Comparison with Rubber (Advanced). By A. P. B., - - - - - - - - - - - 53, CONTENTS CHAP. XIII. Load and After-load. Work done with Increasing Loads (Advanced). By A. P. B., XIV. Summation of Stimuli (Advanced). By A. P. B., - XV. Effect of Distilled Water arid of Various Salts on Muscle (Advanced). A. P. B., 67 XVI. Fatigue of a Voluntary Movement and of a Muscle-Nerve Preparation with its Circulation intact (Advanced). By A. P. B., \J1& XVII. The Kate of Transmission of a Nervous Impulse (Advanced). By M. S. P., - 76 XVIII. The Polarisation of Electrodes and Unpolarisable Elec- trodes (Advanced). By M. S. P., 78 XIX. Transmission of a Nervous Impulse in both Directions (Advanced). By M. S. P., - 79 XX. The Relation between Muscle and Nerve. The Inde- pendent Excitability of Muscle (Advanced). By M. S. P., 80 XXI. The Effect of a Constant Electrical Current upon the Excitability and Conductivity of Nerve (Advanced) By M. S. P., 81 XXII. The Absence of Fatigue in a Stimulated Nerve (Advanced). By M. S. P., - 87 XXIII. The Electromotive Properties of Muscle and Nerve (Advanced). By M. S. P., - - - 88 XXIV. The Electromotive Properties of Muscle and Nerve (continued). The Galvanometer and the Capillary Electrometer (Advanced). By M. S. P., - - - 90 XXV. The Anatomy of the Frog's Heart and its Contraction. By L. H., 92 XXVI. Methods of Recording the Heart. By L. H., 97 XXVII. The Stannius Heart. By L. H., - - 100 XXVIII. The Cardiac Nerves and Ganglia. By L. H.5 - - 103 XXIX. The Sino-auricular Junction. The Action of Drugs. By L. H., 109 XXX. The Effect of Nicotine, Chloroform, and Ether upon the Heart. By L. H., - 112 XXXI. Dissection of the Heart. The Cardiac Impulse. ByL.H., 115 XXXII. The Pulse. Human Blood Pressure. ByL.H., - - 121 XXXIII. Blood. The Haemoglobinometer and the Haemacyto- meter. By L. H., - 128 CONTENTS xiii CHAP. PACK XXXIY. Circulation of the Blood (Elementary Demonstrations). ByL. H., - 131 XXXV. The Heart (Advanced). By L. H., 149 XXXVI. The Heart (continued). The Action of Drugs. By L. H., 151 XXXVII. Gaskell's Clamp and the Effect of Local Warmth on the Heart (Advanced). By L. H., - - - - 154 XXXVIII. Action of the Cardiac Nerves (Advanced). By L. H., 156 XXXIX. The Pulse (Advanced). By L. H., - 161 XL. Vaso-motor System (Advanced). By L. H., - - 164 XLI. Intracardiac Pressure. Blood Flow (Advanced Demonstra- tions). By L. H., - 166 XLII. Effect of Haemorrhage and Saline Transfusion (Advanced Demonstrations). By L. H., - - - - - 171 XLIII. Respiration. By M. S. P., 174 XLIV. Intrathoracic Pressure. By L. H., - 176 XLV. Ventilation of the Lungs. The Spirometer and the Stethograph. By M. S. P., - 177 XLVI. Chemistry of Respiration. By M. S. P., 180 XLVII. Determination of the Respiratory Exchange in Man. By M. S. P., 184 XLVIII. Respiration Apparatus. By L. H., - 186 XLIX. The Chemistry of Respiration. The Gases of the Blood. By L. H., 187 L. The Oxygen Capacity of Blood. By M. S. P., 190 LI. The Effects of Changes in Atmospheric Pressure. By L. H., - 192 LII. The Influence of Carbon Monoxide. By M. S. P., - 194 LIIT. The Regulation of Respiration. By M. S. P., 195 LIV. Cheyne-Stokes Respiration. By M. S. P., 197 LV. The Influence of the Vagus upon Respiration. By M. S. P., - 199 LVI. Animal Heat. By M. S. P., - 200 LVII. Investigation of the Motor Functions of the Alimentary Canal by means of the X-Rays. By A. F. Hertz, 203 LVIII. Salivary Secretion. By L. H., 210 LIX. The Functions of the Central Nervous System. By M. S. P., 214 LX. Reaction Time. By M. S. P., 216 LXI. The Rate of Discharge of Nervous Impulses from the Central Nervous System. By M. S. P., - 217 xiv CONTENTS CHAP PAGE LXII. The Functions of the Anterior and Posterior Roots of the Spinal Cord. The Bell-Majendie Law. By M. S. P., 220 LXIII. Miiller's Law of the Specific Energy of Nerves. By M. S. P., - - 222 LXIV. Cutaneous Sensations. Sensations derived from Move- ments (Advancd). By J. S. E., - - - - - 223 LXV. The Dissection of the Eye. By J. S. E., ... 226 LXVI. The Eye as an Optical Instrument (Advanced). By J. S. E., - - 228 LXVII. The Refracting Media of the Eye. By J. S. E., - - 232 LXVIII. The Retina, By J. S. E., - - - - - - 240 LXIX. Sensations of Light and Colour. By J. S. E., - - 245 LXX. Binocular Vision. By J. S. E., - - - - - 254 LXXI. The Optical Defects of the Eye. By J. S. E., - - 258 LXXII. The Optical Defects of the Eye (Advanced). By J. S. E., 261 LXXIII. The Instruments used in the Clinical Investigation of the Eye. By J. S. E., - - - - 263 LXXIV. Dissection of the Ear in the Skate. Auditory Sensations. By J. S. E., - 267 PART II. PHYSIOLOGICAL CHEMISTRY. By J. J. R. MACLEOD and M. FLACK. Introduction, 270 I. Carbohydrates. By J. J. R, M., 272 II. Carbohydrates (continued). By J. J. R. M., - ... 279 III. Carbohydrates (continued). By J. J. R. M., - 288 IV. Proteins. By M. F., 297 V. Proteins (continued). By M. F., - - - - - 304 VI. Fats, Fatty Acids, Phosphorised Fats and Cholesterol. By J. J. R. M., - 313 VII. Milk. By M. F., 324 VIII. Blood. By M. F., 331 IX. The Spectroscopic Examination of Haemoglobin and its Derivatives. By J. S. Edkins, - 342 CONTENTS xv CHAP. PAGE X. Muscle. By M. F., - - . . . - 350 XI. Dietetics, Food, Metabolism. By M. F., - - - 359 XII. Digestion in the Mouth. By J. J. R. M., - - - - 371 XIII. Digestion in the Stomach. By J. J. R. M., - - 375 XIV. Digestion in the Intestine. By J. J. KM.,- - - - 387 XV. Bile. Bacterial Digestion. By J. J. R. M., - - - 397 XVI. The Chemistry of Urine. By J. J. R, M., - - - - 407 XVII. Urea. By J. J. R. M., - - 415 XVIII. Uric Acid and other Purine Bodies. By J. J. R. M., - - 422 XIX. The Inorganic Acid Radicles of Urine. Urinary Deposits. By J. J. R. M., - - - - - - - - - 434 XX. Pathological Urine. By J. J. KM.,-- - 442 XXI. Lactic Acid, its Estimation and Significance. By J. H. Ryffel, 454 XXII. Haemolysis and Precipitins. By J. J. R. M., - - 46O XXIII. The Pigments of Urine. By J. S. Edkins, - - 467 Appendix— Analytical Tables. By J. J. R. M., - - 469 Index — Part I., Experimental Physiology, - 474 Index — Part II., Physiological Chemistry, - 478- WEIGHTS AND MEASUKES. LENGTH. Metric or Decimal. 1 Metre (M.) i Decimetre (dm.) 1 Centimetre (cm.) - 1 Millimetre (mm.) - 1 Micro-millimetre (mkm) English. = 39-3701 inches. = 3-9370 = 0-3937 „ = 0-0393 „ = 0-000039 , The unit of the Metric System is the Metre, which represents one ten-millionth part of a quarter of the meridian of the earth. The multiples and subdivisions are obtained by the use of decimals; the former being designated by Greek prefixes, the latter by Latin prefixes. Myriametre(Mm.) = 6 '2137 miles. Kilometre (Km.) - 0*6214 „ Hectometre (Hm.) - - v - =109-361 yards. Dekametre (Dm.) = 32*8084 feet. Metre (M.) = 39 '3701 inches. WEIGHT. Metric or Decimal. 1 Kilogramme (Kgm.) 1 Gramme (Gm.) 1 Decigramme (dgm.) 1 Centigramme (cgm.) 1 Milligramme (mgm.) English. = 2-2046 pounds. = 15 -4323 grains. = 1-5432 „ = 0-1543 „ = 0-0154 The unit is the Gramme which represents the weight of a cubic centimetre of water at 4° C. APOTHECARIES WEIGHT. 437 "5 grains (gr. ) = 1 ounce. 16 ounces (§) =1 pound (lb.). j 60 grains ( 20 grains = 1 drachm (3). = 1 scruple 0). 1 grain = 0*0648 gramme. *Not official. AVOIRDUPOIS WEIGHT. 16 drachms = 1 ounce (oz.). 16 oz. =1 pound (lb.). 281bs. = 1 quarter (qr.). 4 quarters = 1 hundredweight (cwt. ). 20 cwt. = 1 ton. 1 pound =453*592 grammes. 1 ounce = 28 '35 grammes. WEIGHTS AND MEASURES CAPACITY. Metric or Decimal. English. 1 Dekalitre (Di.) - - = 2-1998 Imperial gallons. 1 Litre (L.) - - . =35-196 Imperial fluid ounces. 1 Decilitre (dl.) - 3'5196 ,, 1 Cubic centimetre (c.G.)1 or }• - 0-0352 1 Millilitre(ml.) 60 minims (TTl) • • - =1 fluid drachm (3). 8 fluid drachms • • - =1 fluid ounce (§). 20 fluid ounces - - - =1 pint (0). 8 pints .... =1 gallon (C). 1 cubic centimetre - - =16*9 minims. 1 fluid ounce - - - = 28 '42 cubic centimetres. 1 pint =568-34 cubic centimetres. 1 gallon - - - - =4-54 litres. THERMOMETERS. FAHRENHEIT AND CENTIGRADE SCALES. To convert degrees F. into degrees C., deduct 32, multiply by 5, and divide by 9. To convert degrees C. into degrees F. , multiply by 9, divide by 5, and add 32. F. C. F. C. 212° 100°-0 80* 26-7 112 44-4 70 21-1 106 41-1 60 15-6 104 40-0 50 10-0 102 38-9 41 5-0 101 38-3 32 0-0 100 37-8 23 - 5-0 99 37-2 14 -10-0 98 36-7 5 -15-0 97 36-1 AVERAGE WEIGHTS AND HEIGHTS. Average weight of a healthy male child at birth - - - = 6 '8 Ibs ,, „ ,, six months' old • =12*4 ,, „ ,, twelve ,, - =18-8 „ An adult man (dressed) 5 feet 8 inches in height, should weigh 11 st. 1 Ib. and should have a chest circumference of 38^ inches. PART L MUSCLE AND NERVE. CIRCULATION. RESPIRATION. ANIMAL HEAT. CENTRAL NERVOUS SYSTEM AND SPECIAL SENSES. THE PHYSIOLOGY OF MUSCLE AND NERVE. Introduction. — Physiology, the study of the properties of living organisms, can be properly appreciated and learned only when it is approached from the practical and experimental side. The study of the simplest forms of life, the unicellular organisms, is as yet only in its infancy, and at the present moment experimental physiology deals almost entirely with the functions of the various tissues and organs which together make up a vertebrate animal. The cold-blooded vertebrate, the frog, is the most suitable animal for elementary experiments upon muscle and nerve; it is readily obtained, and its tissues under suitable conditions retain their vitality for many hours after they have been excised and cut oft' from their supply of blood. The muscular, nervous, and vascular systems of the frog are the most important in an experimental course of physiology, for although muscle and nerve are highly differentiated forms of protoplasm with correspondingly characteristic functions, yet they show only in an exaggerated way properties which are common to all living matter. Thus in muscle the power of contraction or movement is highly developed; in nerve the property of excitability or irritability, the response to a stimulus. A CHAPTER I. ELECTRICAL APPARATUS FOR PHYSIOLOGICAL EXPERIMENTS. IN experimental physiology the stimulus most frequently used is an electrical one, for it is convenient, easily graduated, and less injurious to tissues than efficient thermal, chemical, or mechanical stimuli would be. The Daniell Cell, which has an electromotive force (E.M.F.) of 1*1 volts, is the best source of electricity, for it yields an almost constant strength of current. It consists (Fig. 1) of (i) a plate of copper dipping into a solution of copper sulphate which is kept saturated by crystals of the salt, and (ii) a rod of amal- gamated zinc placed in a porous pot filled with a 10 per cent, solution of sulphuric acid ; the porous pot is surrounded by the solution of copper sulphate. The whole is generally placed for convenience in a glazed earthenware pot with a handle. When the copper and zinc elements are connected by a wire the zinc dissolves in the sulphuric acid, forming ZnS04-rH2. The H ions thus liberated become charged with the electricity originally stored in the zinc; they migrate through the porous cell into the copper sulphate and split it up into H2S04 + Cu, and their charge of electricity is transferred to the Cu ions. These in turn deliver up their charge of electricity to the copper plate and, as they discharge, become deposited on the plate as metallic copper. Thus inside the cell electricity passes from the zinc, or positive FIG. 1. — Diagram of a Daniell cell seen in section. ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 3 element, to the copper or negative element; outside the cell the current passes from the copper binding-screw, the positive pole or anode of the battery, to the zinc binding-screw, the negative pole or kathode. If plates of copper and zinc were simply immersed in 10 per cent, sulphuric acid, the chemical action set up would soon cause the copper plate to be covered with bubbles of hydrogen gas. This would cause a resistance to the flow of current inside the cell, and further, hydrogen being electro-positive to zinc, a polarisation current in the opposite direction to the original battery current would be set up in the cell and rapidly reduce its E.M.F. Daniell, by placing the copper plate in a solution of copper sulphate, which the hydrogen splits up, prevented polarisation from taking place within the battery.1 Therefore as long as there is free sulphuric acid present and the copper sulphate is saturated, the current produced by the cell remains constant. Pro- vided too that the porous pot, which is to prevent the deposition of copper on the zinc rod, remains permeable to the H ions. The zinc rod has to be amalgamated because commercial zinc con- tains iron and other metallic impurities ; these in the presence of the sulphuric acid would, with the zinc, constitute a number of minute batteries. By covering the impurities with zinc amalgam their dis- turbing action is removed, and as the zinc is dissolved away, the mercury combines with fresh zinc so that the electromotive properties of the zinc rod remain constant. 1 A more accurate description of the chemistry of a Daniell cell is as follows : The cell consists of two metals, zinc and copper, dipping into an electrolyte containing various ions in solution ; these are H, S04, OH, Cu and S04, of which Cu and H, being positive ions, will work their way towards the negative element, the copper plate and the OH and SO4 being negative ions towards the zinc. When in use chemical changes take place around both metallic plates. The zinc is attacked by the S04 ions discharging, forming ZnS04, and energy is liberated, which is conducted across the electrolyte by the ions in solution. Around the copper plate the copper sulphate is being split up into S04 and Cu ions, in which process energy is stored up. But the energy liberated at the zinc plate is greater than that stored in the neighbourhood of the copper plate, therefore the cell, when working, is always liberating a balance of energy which appears as an electric current. The S04 ions, constantly being liberated in the copper sulphate solution and charged with electricity, migrate through the porous pot towards the zinc, discharge forming ZnS04 and a liberation of energy as explained. Towards the copper plate both H and Cu ions charged with electricity are constantly streaming. That it is the Cu ions and not the H ions which discharge and become precipitated on the plate depends simply upon the fact that it requires a less energy and a lower E.M.F. to separate Cu than H ions. Therefore as long as there are sufficient Cu ions present to conduct the current, Cu ions and not H ions will discharge and be precipitated on the copper plate. PRACTICAL PHYSIOLOGY Keys are instruments for making or breaking electrical circuits and for short-circuiting currents. The Mercury Key consists of a small cup hollowed out of a piece of vulcanite (Fig. 2). From the cup, which is nearly filled with clean mercury, pass in opposite directions two stout copper wires with FIG. 2.— The mercury key. FIG. 3.— The spring key. binding-screws ; one wire and binding-screw are fixed to the vulcanite base, the other wire can be raised out of or lowered into the mercury by an insulated handle. In some forms of mercury key the wires connecting the binding-screws to the mercury cup run through the vulcanite ; the ends of these wires are liable to become oxidised and dirty, and in consequence they make bad contact with the mercury. In order to avoid this it is only necessary to fix the insulated wires from the battery to the binding-screws and to turn the naked ends of these wires over into the mercury. The Spring Key is made of a block of lacquered wood, to one end of which is attached a broad brass spring with a binding-screw, and co the other end a plate of brass with a binding-screw (Fig. 3). When the spring is depressed by the finger its free end touches the brass plate and connects together the two binding-screws. The brass plate carries a clip which can clamp the spring in contact with the plate. The Du Bois Key consists of two metal blocks each carrying two binding-screws and attached to a vulcanite base (Fig. 4). The FIG. 4.— The Du Bois key. ELEMENTARY EXPERIMENTAL PHYSIOLOGY. 5 metal blocks can be connected by a thick brass bar attached to an insulated movable handle. This key, like the mercury and spring key, may be used as a simple make and break key (Fig. 5) ; but its Fia. 5.— Plan of the use of a Du Bois key, as a simple make and break key. Flo. C.— Arranged as a short-circuiting key: key shut. Fio. 7.— Arranged as a stort circuiting key : key open. proper use is as a short-circuiting key (Figs. 6 and 7) ; and when a Du Bois key is directed to be used, it must be inserted into the circuit as a short-circuiting and not as a simple key. Fio. 8A.— The Pohl's reverser. A and B the two side cups ; C, D, B and F the four corner cups ; S the handle made of glass or vulcanite. FIG. 8s.— Universal key (Gotch). The key is used by rotating the arm containing the screws connected with the wires A and B, which come from the battery. In the position shown the current flows from the wire of C to that of D ; if rotated through 45* there is a complete double break of the battery -circuit ; if rotated through 90° then the current is remade and the current flows from the wire of D to that of C. 6 PRACTICAL PHYSIOLOGY The Pohl's Reverser consists of six mercury cups hollowed out in a block of vulcanite, each cup being connected to a binding-screw (Fig. 8A). The four corner cups are connected diagonally by stout copper wires which do not touch each other. The two side cups are joined by stout copper wires to a non-conducting cross-piece, which acts as a handle Each end of the handle also carries a semicircle of copper wire which is connected to the wire going into the side cup, and is of such a length that it will dip into the cup at either end by turning the handle over towards that end. If the handle is in such a position that a current, entering the reverser by one of the side cups, emerges by an end cup of the same side, then, by turning the handle over, the cross-wires come into use, and the current will now emerge by the end cup of the opposite side. The instrument may also be used to send a current into either of two circuits. The cross-wires are removed, the wires from the battery are connected to the two side binding-screws, and to each pair of end cups the wires of the two alternative circuits (Fig. 9). Then by turning the handle over the current may be sent into either of these two circuits. A much more efficient instrument is the universal key (Fig. 8B), which has recently been introduced by Gotch. It can be used as a double break-key, a reverser and a shunt. The term Electrodes is applied to the free ends of the two wires which conduct the current to the tissue to be stimulated. They consist Pio. 9.— Plan of the arrangement of the two alternative circuits. -/Fm^V^r^*" .._ .^— ^ FIGS. 10 AND 11. — Two forms of electrodes. of two insulated wires, the ends of which are clean and free from insulating material, carried in some form of holder ; this is generally made by running the wires through a piece of vulcanite, cork, or model- ELEMENTARY EXPERIMENTAL PHYSIOLOGY 7 ling wax (Figs. 10 and 11). A form of electrode sometimes very useful is made by soldering the free end of each wire to the head of a needle. The Rheochord is used to alter the strength of a constant current to be sent through a muscle or nerve. In its simplest form it consists of Fio. 12. — Simple form of monochord. a single straight or zig-zagged wire with a binding- screw at either end and a movable contact between them (Fig. 12). If a Daniell cell be A + FIG. 13. — To illustrate the principle of the monochord. connected to the two ends of the monochord A and B (Fig. 13), there will be a fall of potential in it from A to B. If from A and the Fio. 14.— The rheochord arranged to vary the strength of a current passing through a nerve. It consists of two parallel wires connected by a movable metal slider 8. By moving the slider S to the right the resistance of the rheochord in circuit and therefore the amount of battery current passing through the nerve would be in- creased. movable contact S two electrodes pass to a nerve, the current from the battery has two circuits open to it and can pass either through the nerve or along the monochord back to the battery. The 8 PEACTICAL PHYSIOLOGY amount of current which will pass through the nerve will be directly proportional to the difference in potential between A and S, i.e. if the fall in potential in the monochord is uniform, proportional to the distance between A and S, being greater as S is moved away from A ; it is also inversely proportional to the resistance of the circuit through the nerve. But the resistance of this circuit may be considered con- stant for all positions of S, since the resistance in the nerve itself is enormously greater than that caused by any change in the length of the monochord wire in the circuit. Although the Daniell cell is the most convenient source of current, and its strength can be regulated by a rheochord, and although the FIG. 15.— The induction-coil. make and break of a constant current do act as a stimulus to muscle and nerve, it is often more convenient to use induced currents. These are obtained by connecting a Daniell cell to an induction coil, and their advantages are: (1) That being of extremely brief duration as com- pared with the make of a constant current, they set up practically no polarisation in the tissues (see page 78). (2) Having a comparatively large E.M.F. and rapid development, as compared with the galvanic current, they constitute a much more effective stimulus. For, the law of excitation states that the effectiveness of a current as a stimulus depends not only upon the total variation in its intensity, but also upon the amount of such variation in the unit of time, i.e. the greater the rapidity of the total variation, the more effective is the current as a stimulus. The Induction-coil (Fig. 15) consists of two coils, of which the primary is made up of a few turns of insulated thick copper wire with only a small resistance. This is wound round a core of iron wire to increase the number of lines of magnetic induction which pass through it. The ends of the wire forming the primary coil are con- nected with the top binding-screws 1 and 2 (Fig. 16). The secondary coil is made up of a large number of turns of insulated ELEMENTARY EXPERIMENTAL PHYSIOLOGY 9 fine copper wire. The large number of turns of wire in the secondary as compared with the primary coil, transforms the low E.M.F. of the current in the primary circuit into a high E.M.F. in the secondary circuit ; for each turn in the primary coil induces an effect in every turn of the secondary coil, so that the sum of all these effects is a single one of greatly increased intensity. The long fine wire of the secondary coil gives it a great resistance, but when the induced currents are passed through the relatively enor- mous resistances of animal tissues this is unimportant.1 The ends of the wire of the secondary coil are connected to the binding-screws 3 and 4 (Fig. 16). The E.M.F. of the induced current varies with the following factors : (1) It varies directly with the intensity of the change of current in the primary circuit, so that if no current or a current of constant strength be running through the primary coil no induction occurs ; but if the strength of the current in the primary circuit does change, whether it be an increase or decrease, the greater the change the stronger will be the induction. (2) It varies directly as the rate of change in the s.c - Fio. 16. — Diagram of an induction-coil and its connections. strength of the inducing current, so that, if the constant current be increased or decreased greatly in strength, but sufficiently gradually, no induction takes place ; on the other hand, for a given change in the constant current the more rapid the change the greater the induction. (3) It varies with the angle between the primary and secondary coils in such a way that when the two coils are accurately at right angles there is no induced current ; but the strength of the induction increases as the angle between the coils is altered until the maximum is reached, when the wires are parallel to each other. If the secondary coil be xThe resistance of a piece of a frog's sciatic nerve 1 cm. long is about 100,000 ohms. 10 PRACTICAL PHYSIOLOGY movable horizontally on a central point, the strength of the induced current can be graduated by altering the angle between the two coils. (4) It varies inversely as the distance between the two coils, being greatest when the secondary is completely over the primary coil, and becomes less and less as the coils are separated. The strength of the induced current is usually regulated by varying the linear distance between the coils, and most induction-coils are graduated by a milli- metre scale fastened to the side of the carrier, so that the pointer in the secondary coil is at the zero of the scale when the one coil is exactly covered by the other. This graduation, however, is purely arbitrary, for the absolute decrease in the strength of the induced current becomes less and less for every centimetre that the coils are separated. An exact graduation can be obtained by a scale corresponding to equal galvanometric deflections. The direction of the induced current in the secondary coil is, at make of the battery-circuit, in the opposite direction, and at break of the battery-circuit, in the same direction as the battery-circuit in the primary coil. Most coils are so wound that when at make the battery current enters the primary coil by one top binding-screw, the induced current leaves the secondary coil by the binding-screw of the opposite side (Fig. 16). The Use of Make- and Break-Induction Shocks as Stimuli. — Two wires are connected with the poles of a Daniell cell ; the free end of one wire is fastened to one binding-screw of a spring-key, and to the other screw of the key is fixed a third wire. The clean free ends of the wires are placed on the tongue, and the key is opened and closed ; no shock is produced, but only a sensation of taste ; the intensity of the current is insufficient to produce a marked excitation. The free ends of the wires are now connected with the screws, or terminals, 1 and 2 of the induction-coil and a Du Bois key is placed in the secondary circuit (Fig. 16). The secondary coil is pushed far apart from the primary, and the Du Bois key is opened; make and break of the primary circuit produces no excitation, for the induction- currents are too weak. The secondary coil is gradually moved towards the primary, and the spring-key is opened and closed from time to time, until a point is reached at which a shock is felt at break, but not at make of the constant current. The position of the secondary coil on the scale is noted. As the secondary coil is moved up further, the break-shock becomes greater, and a slight shock is also felt at make ; in a similar way the two shocks can be further increased, but the break-shock remains greater than the make-shock. It is especially to be noted that there is no induction-shock if the ELEMENTARY EXPERIMENTAL PHYSIOLOGY 11 primary circuit remains closed by the spring-key. An induction shock is produced only at the 'make or the break of the constant current. Closure of the Du Bois key short-circuits the electrodes, and no shock will be felt on make or break of the constant current. By means of this key the make- or break-induction shock, or both, can be shut off from the electrodes. The secondary coil is now removed from the grooves of the carrier, and is placed close to, but at right angles to, the primary coil : no shock is produced when the primary circuit is closed or broken. The secondary coil is gradually turned on its vertical axis, and the spring- key is opened and closed from time to time. A shock will be felt first at break, then at make, and these will increase until the maxima are reached when the secondary coil is parallel to the primary. These simple experiments show that the make and break of a galvanic current can act as weak stimuli; that on connecting the Daniell cell with the induction-coil induced currents are produced in the' secondary coil only at make and break of the battery-current and not when it is running with constant strength through the primary coil ; that the induced currents are very effective stimuli, can be easily graduated in strength and short-circuited by a key. It has further been shown that the break induction-shock is stronger than the make. The cause of this difference lies in the primary coil, and needs explanation. When the battery-current enters the primary coil, it induces a current in it as well as in the secondary coil. This " self-induced " or make extra current, like that induced in the secondary coil, is a momentary current in the opposite direction to the battery -current ; hence it delays the rapidity with which the battery-current reaches its maximal intensity in the primary coil and weakens the effect which change in current in the primary coil will induce in the secondary coil. On the other hand, when the battery-current is broken, the current in the primary coil suddenly runs down to nothing ; and although a "break extra current, running momentarily in the same direction as the battery-current, is induced in the primary coil, it cannot delay the rapidity of the fall of the battery- current, because a primary circuit no longer exists in which the extra current could run. Demonstration of the Break Extra Current. — Connect a cell with "binding-screws 1 and 2 of the induction-coil, placing a spring-key in the circuit. Fasten to the same binding-screws of the primary coil two wires, the free ends of which are placed on the tongue. On closing the spring-key no shock is felt, but, ou opening it, the shock of the break extra current. 12 PRACTICAL PHYSIOLOGY A purely physical proof of the break extra current can be obtained by connecting one pole of a battery to the primary coil, and by touching .with the other wire from the battery the milled head of the other binding-screw of the primary coil. Every time that the battery circuit is broken, the break extra current will pass across from the screw to the wire as a minute spark ; no spark, or a very feeble one, is seen on touching the first terminal, for in this case there is no current in the primary coil. Equalisation of Make and Break Induced Currents. — From what has been said it is clear that, if the break extra current were provided with a circuit to run in, the strength of the current induced in the secondary coil at break would be reduced to that of the current induced at make ; and so they would be equalised. In order to effect this the battery-circuit is not broken, but is nearly completely short-circuited out of the primary coil by a Du Bois key (Fig. 17). Now again test the relative strengths of the make and break induced currents. FIG. 17. — Arrangement of apparatus for equalising the make and break induced currents. They may be approximately equal, but the original difference is not infrequently overcorrected, and now the break-shock is the weaker. This is caused by the make and break extra currents running in circuits of different resistance. At make the extra current runs not only through the primary coil but also through the resistance of the Daniell cell ; but at break the extra current has to run only through the resistance of the primary coil, hence it is the more effective current of the two, and reduces the effect induced in the secondary coil at break more than the make extra current does on closing the primary circuit. Faradic or Tetanising Shocks. — Induction-coils are provided with an automatic arrangement for rapidly making and breaking the primary circuit by means of Wagner's hammer. Connect up the battery to screws 5 and 6 of the coil, interposing a spring-key, and follow out the primary circuit (Fig. 18). The current passes up the pillar A along the spring H to the screw Sj, through the primary ELEMENTARY EXPERIMENTAL PHYSIOLOGY 13 coil to the electro-magnet E, and so to the pillar B. When the circuit is thus made, E becomes an electro-magnet, pulls down the spring H from its contact with Sj and breaks the circuit ; consequently E ceases to be a magnet, the spring flies up into contact with Slf and again J».C FIG. 18. — Diagram to show the action of Wagner's hammer. completes the circuit. The number of times the circuit will be thus made and broken per second depends upon the length of the spring H ; in most coils it is of such a length as to give 50 complete vibrations per second. At each make and break of the circuit a current is induced in the secondary coil, just as when the circuit was broken by hand ; FIQ. 19. — Diagram to show the action of the Helmholtz side-wire. further, the break-shock is stronger than the make-shock, and for the same reason as before. Determine the distance necessary between the two coils for the shocks just to be felt on the tongue. 14 PEACTICAL PHYSIOLOGY Helmholtz showed that it is possible to equalise these Faradic shocks by short-circuiting, instead of completely breaking, the battery-current, and for the reason already explained. For this purpose (Fig. 19) a stout wire, W, connects the binding-screws 7 and 1, Sj_ is screwed up out of reach of the spring, and S2 is screwed up. Follow the circuit of the current which passes from binding-screws 7 to 1 by the side-wire, and so to the primary coil, back to the electro-magnet E, to binding-screw 6 and to the battery. When, however, the current reaches E, it becomes a magnet, and pulls down the spring into contact with S2. This short-circuits the battery-current out of the coil, for the current will now pass from the pillar A, by way of H, to the pillar B, and so back to the battery. There is still left the circuit 7 W, 1, PC, E, H, A, 7, in which the break extra current can run and reduce the strength of the current induced in the secondary coil at break. Determine the distance between the coils at which the shocks are now just felt on the tongue ; it will be found to be reduced, showing that the break-shock which was alone felt before has been reduced down to or even below the strength of the make-shock. CHAPTER II. THE GRAPHIC METHOD. MAXIMAL AND MINIMAL STIMULI. UNIPOLAR EXCITATION. THE graphic method is applied to muscle in order to obtain a permanent magnified record of the change in form of a muscle during contraction, and further, to investigate the time-relations of the con- traction. For this purpose it is necessary to describe the method of preparing the muscle and then three special pieces of apparatus : (1) a magnifying lever, the muscle lever, or myograph, which can write on (2) a surface either stationary or moving at a uniform rate, the drum, and (3) an instrument for recording time on the drum, the chronograph, which will be described in Chapter III. The Muscle- and Nerve-Preparation. — The quickest way to kill a frog is to "pith" it. The articulation between the skull and the vertebral column can be felt with the tip of the finger ; it is severed by a transverse cut with a pair of scissors, and then a probe or blanket- pin is inserted into the skull to destroy the brain. The spinal cord is destroyed in a similar way, and this final stimulation of the nerve-cells ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 15 causes a discharge of motor impulses to the muscles of the body, which give a series of convulsive twitches or contractions. These twitches quickly cease, the body and limbs are in a toneless, relaxed condition, and all reflexes have been abolished. The frog is then placed belly downwards on a frog-board, and the skin at the ankle is divided by a circular incision ; the tendo-Achillis is exposed and a thread passed under the tendon and tied just above the sesamoid bone. In this way a ligature is attached to the muscle with- Fio. 20. FIG. 21. Muscles of the frog's leg. (After Ecker.) FIG. 20.— Dorsal aspect. 1. Trinceps femoris. 2. Biceps femoris. 3. Rectus interims. 4. Semi-membranosus. 6. Gastrocnenoius. 6. Tendo Achillis. FIG. 21.— Ventral aspect. 1. Rectus internus. 2. Gracilis. 3 Adductor lougus. 4 Vastus internus. 5 Sartorius. 6 Adductor brevis. 7 Adductor magnus. 8 Gastrocnemius. 9 Tendo Achillis. out damage to or irritation of its fibres. The tendon is divided below the sesamoid bone, and a pull upwards towards the knee frees the gastrocnemius muscle and the skin from the remaining structures of the leg, which are cut away just below the knee. The gastrocnemius muscle is protected from drying and from contact with foreign sub- stances by drawing down the " trouser " of skin. The sciatic nerve is now dissected in the following way. The skin over the posterior surface of the thigh is divided by a longitudinal incision in the middle line, the biceps and semi-membranosus muscles are separated, and the sciatic nerve is exposed. The nerve must not be pinched with forceps, for it is easily damaged. The muscles on each side of the urostyle and then the urostyle itself are cut away; the three constituent ends of the sciatic nerve are now exposed. The spinal column is divided transversely between the 6th and 7th vertebrae and the 9th, 8th, and 7th vertebrae are bisected. The piece of bone, from which the nerve to be prepared issues, can be grasped with the forceps without damage 16 PRACTICAL PHYSIOLOGY to the nerve, and the sciatic nerve is freed from the surrounding tissues as far as the knee. The thigh is then severed from the body by a FIG. 22. FIG. 23. Diagrams of a muscle- and nerve-preparation. (Pembrey and Phillips.) FIG. 22.— The first stage of dissection. FIG. 23. — The second stage of dissection. The sciatic nerve exposed and the gastroc- neniius muscle covered by skin. transverse cut close to the articulation of the head of the femur (Figs. 22 and 23). In order that the best results may be obtained the muscle- and nerve-preparation should be as fresh and irritable as possible, and in order to obtain this the following precautions should be observed, (a) All apparatus for the experiment should be in working order before the dissection is commenced, (b) The muscle must be prevented from drying by keeping the "trouser" of skin pulled down over it, and since the nerve is even more easily killed by drying, it should, when not required for immediate stimu- lation, be allowed to lie among the muscles of the thigh, the FIG. 24.— The crank-lever, muscle-board and stand. fo lymph of which will keep it moist and irritable. The nerve must not be placed upon the frog's ELEMENTAKY EXPERIMENTAL PHYSIOLOGY 17 skin, the secretions of which quickly injure it. (c) When the nerve is on the electrodes it must be kept moist by normal tap-water saline solution ('70 per cent, sodium chloride in tap-water) upon a piece of filter-paper, but care must be taken that the current from the electrodes is not short-circuited thereby, (d) The nerve itself should not be picked up by forceps, but should be lifted by the pieces of the vertebral column. Consequently the whole length of the nerve should always be dissected out; as a rule it should not be cut across in the thigh nor simply exposed in the thigh and two electrodes pushed under it. The Muscle-lever takes one of two chief forms : (a) The crank-lever (Fig. 24) consists of an L-shaped piece of metal, the horizontal arm of which is long and carries the writing FIG. 25.— The simple lever with after-loading screw. P, clamp ; L, lever ; M, muscle. point, whilst the vertical arm is short and to this the thread round the tendo-Achillis is firmly tied. The muscle rests, in the same straight line as the lever, on the muscle-board, a horizontal piece of wood covered with cork. The whole is carried on a vertical stand (Fig. 24), the arm of which is movable on the base, so that the writing point of the myograph can be swung towards and away from the drum without altering the position of the base of the stand. When the thread has been tied to the lever, a pin is pushed through the lower end of the femur into the cork; this gives the muscle a fixed point from which to pull. It is necessary to see that, when the muscle is at rest, the thread attached to the lever is taut, and that there is no "slack" to be taken in when contraction begins; further, the writing arm should be horizontal. In this form of lever the movement of the writing point is at right 18 PRACTICAL PHYSIOLOGY angles to the movement recorded. The magnification of the movement of the muscle recorded by the lever is calculated by dividing the dis- tance of the writing point from the axis by the distance from the axis of the point of attachment of the thread from the tendon. The nearer to the axis the muscle is attached the greater will be the magnification. It is quite sufficient to magnify the movement of the muscle 5 times. (b) The simple lever (Fig. 25) consists of two parts : a rigid femur- clamp to hold the piece of femur, and a horizontal writing lever below it to which the thread on the tendo-Achillis is tied. Care must be taken that the femur-clamp and lever lie in the same plane, and that the muscle is tied to a point on the lever vertically below the clamp. In this case the movement of the writing point is in the same plane as that of the movement recorded. The magnification, as before, is calculated by dividing the distance of the writing point from the axis by the distance of the point of attachment of the muscle from the axis. The writing lever must be as light as possible (see page 27, Chap. III.), but it must be sufficiently rigid to prevent vibra- tions being set up in it. For this purpose writing levers are generally made of light metal, glass, Japanese cane or straw. The actual writing point is made of thin metal foil or moderately stiff paper bent at its free end slightly over towards Fio. 26.— Kymograph. .. , mi . - . L _ .. the drum. The writing point must lie as nearly as possible parallel to the recording surface, or, in other words, at right angles to a radius of the drum. Further, the bend near its end is necessary ; it acts as a weak spring and keeps the writing point up against the recording surface in different positions of the lever. For the end of the lever describes a curved line, and the more it leaves the horizontal position the greater will be the distance of the end of the straw from the recording surface. The Kymograph or recording drum (Fig. 26) consists essentially of a stout brass cylinder which is made to revolve round a vertical axis by either clockwork or string belting from a motor. It is necessary to have some arrangement by which the speed of revolution can be altered within wide limits ; this is obtained by various mechanical devices in different patterns of drum, one of which is shown in Fig. 26. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 19 The drum is covered with white glazed paper, the surface of which is then blackened by a thin layer of soot, obtained by revolving the drum through either the luminous part of a broad gas flame or the smoke of burning turpentine or camphor. The writing point of the lever, as the drum revolves, rubs off the layer of soot and leaves a white magnified image of the movement of the muscle or heart or whatever change is being recorded. The white paper is of the same width and longer than the surface of the drum, and the under-surface of the overlap is gummed. The paper must be laid evenly and without wrinkles round the drum, the gum is then moistened and the paper fastened. The layer of soot obtained from the gas flame should be dark brown in colour, and care must be taken to revolve the drum sufficiently rapidly through the flame to prevent scorching or burning of the paper. The film of soot from camphor is less firmly attached to the paper, and must not be made too thick, otherwise the writing point does not, without undue friction, rub off enough of it to leave a distinct tracing. In recording it must be so arranged that the tracing does not come at the overlap, for the joint in the paper is liable to make the point of the lever jump. Further, it is very important that the drum should be made to revolve away from and not towards the writing point, in other words, the tracing as it is taken should pass from the writing point, not towards but away from the lever. When the tracing is finished, the paper is cut through at the overlap and the details of the experiment written on it. The tracing is preserved by drawing it once through a varnishing solution l and pinning it up to dry. This graphic method, as we shall see, introduces several errors, but such accuracy as it has must depend upon the drum remaining a true cylinder ; it is therefore very important that a drum should never be dropped or in any way dented. Minimal and Maximal Stimuli. — If the strength of the stimulus applied to a muscle be varied within certain limits, it is found that the muscular response also varies, so that the greater the excita- tion the greater is the shortening of the muscle. In order to demonstrate this, connect up a Daniell cell to an induc- tion coil so as to give single induction shocks, placing a mercury key in the primary circuit and a Du Bois key in the secondary circuit ; cover and smoke a drum. Dissect out a gastrocnemius preparation and attach it to the myograph lever, arrange the electrodes to stimulate the muscle directly; one needle-electrode is used which passes through 1 A rapidly drying varnish is made by dissolving 250 c.c. of the best white hard varnish in a litre of methylated spirits and then adding 10 c.c. of castor oil. 20 PKACTICAL PHYSIOLOGY and fixes the lower end of the femur ; the other wire from the Du Bois key is joined to a piece of capillary copper wire which has been threaded by means of a needle through the tendo-Achillis. In this way the current can be passed through the length of the muscle, and the very fine wire will not cause any obstruction to the free movement of the muscle when it contracts. Bring the writing point on to the surface of the stationary drum. With the secondary coil at 20 cm. and the Du Bois key open, make and break the primary circuit, no contraction will take place. Gradu- ally move up the secondary coil towards the primary, opening and closing the key in the primary, circuit at each new position. With the secondary coil at about 16 cm. the muscle will contract at break but not at make, showing that the break induction shock is stronger than the make-shock. The contraction is recorded on the drum by a nearly vertical line, and shows a minimal contraction in response to a minimal stimulus; the make-induction shock is still a sub-minimal stimulus and no contraction results. Rotate the drum on a short distance by hand, move the secondary coil up 1 cm. and stimulate again. Repeat this process, moving the drum on after each contraction and increasing the strength of the stimulus after each make and break of the primary circuit (Fig. 27). As the strength of the stimulus is increased the contraction at break increases in height rapidly at first and then more FIG. 27.— Heights of contraction of a muscle with different strengths of stimuli. M marks the make and B the break of the primary circuit. The numbers refer to the distances in cms. of the secondary from the primary coil. (A.P.B.) slowly until, with the secondary coil at about 7 cm., a point is reached beyond which the height does not increase. At 7 cm., therefore, the break-shock and the contraction which it causes are maximal. All stimuli intermediate in strength between minimal and maximal are called sub-maximal. At a certain point the make-shock will be found to become an effective stimulus and cause a minimal contraction. As the make-shock is increased in strength, the contraction rapidly increases in height until, with the secondary coil at about 7 cm., ELEMENTAKY EXPEEIMENTAL PHYSIOLOGY 21 it becomes maximal and of about the same height as the break contraction. The higher the contractions become the more obvious is it that the writing point describes on the stationary drum, not a straight line, but an arc of a circle. The shortening of the muscle, after allowing for the magnification by the lever, is measured not by the length of this arc but by a perpendicular line dropped from its highest point on to the base line. It is necessary to point out here that, when the primary circuit is made — and the same is true if it be broken — a momentary induced current is both made and broken through the nerve, and yet there is only one contraction of the muscle. It has been found that in a current of such short duration the break stimulus is ineffective because it falls within the refractory period of the make stimulus (see Chap. VII., p. 40). In both cases, whether the primary circuit is made or broken, the effective stimulus to the nerve is only the make stimulus of the induced current. Unipolar Excitation. — Connect a battery to a coil so as to give tetanising shocks ; connect a wire to one pole of the secondary coil and place its free end on the tongue. If the secondary coil be moved completely over the primary, faint shocks will be felt. The explana- tion of this phenomenon is that the making and breaking of the primary circuit causes free electricity to collect at the end of the wire connected with the secondary coil; when the E.M.F. of this charge is sufficient to overcome the resistance of the tissues of the body, the circuit is completed through the body, the floor and desk, and so back to the other pole of the secondary coil. With the wire still on the tongue, touch the other pole of the secondary coil with a moistened finger; much more powerful shocks are felt because a more direct circuit from one pole to the other of the secondary coil has been provided. Repeat the experiment on a sciatic-gastrocnemius preparation in the following way, with either tetanising or single-induction shocks. Lay the preparation on a perfectly clean and dry glass-plate and place a wire connected with one pole of the secondary coil under the nerve; no contraction of the muscle takes place because the dry plate insulates the preparation and the secondary circuit cannot be completed. Now touch the muscle with a wire, the other end of which rests on a gas or water pipe; the muscle contracts because the circuit is completed through the earth. It is not even necessary that the conductor should touch the preparation, for, if a moistened finger is brought as near the muscle as possible without touching it, the muscle contracts, especially 22 PRACTICAL PHYSIOLOGY if a moistened finger of the other hand touches the other pole of the secondary coil. In this case the human body acts like a condenser charged with electricity, which by its approach can stimulate muscle or nerve. Further, if the nerve be ligatured between the electrode and the muscle, or cut across and the two cut ends laid over each other, which will prevent the passage of a nervous impulse along it, contrac- tion of the muscle is still produced, because the discharge takes place along the whole length of nerve and muscle between the electrode and the point by which the muscle is connected to the earth, so that any irritable tissue in the course taken by the charge is stimulated. If, however, the muscle and nerve preparation is laid on an ordinary moistened muscle-board, the insulation is so slight that one electrode, connecting the nerve and the secondary coil, will by itself cause the muscle to contract. It is in order to guard against accidental stimulation of muscle and nerve by unipolar action that a Du Bois key must always be placed in the secondary circuit, and must always be kept closed except when the tissue is being intentionally stimulated. The brass bridge of the key, which has many thousands of times less resistance than the tissue between the electrodes, affords a perfect closure of the secondary circuit and prevents static electrification of the electrodes. Errors from unipolar action are liable to take place, especially in the study of the electromotive phenomena of muscle and nerve by the electrometer and galvanometer (see Chap. XV III.). CHAPTER III. A SINGLE CONTRACTION OF A GASTROCNEMIUS MUSCLE. IN order to study the contraction given by a muscle in response to a single stimulus, it is not sufficient to inspect the curved line traced by the myograph-lever on a revolving drum. It is also necessary to study the length of time occupied by the whole twitch and the time- relations of different parts of it. For this purpose a time-tracing must be simultaneously recorded by a special apparatus, which generally takes one of two forms. (1) The Tuning Fork; to one prong of this a writing point, similar to that on the myograph-lever, is attached. With the writing point lightly touching the blackened surface of the drum, a sharp tap is given to the fork, and the drum set in motion ; care must be taken that the drum does not make more than one revolution, otherwise the time- ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 23 tracing will run over itself. The number of complete vibrations per second and the time value of each will depend upon the note of the fork. The most useful fork is one that gives 100 complete vibrations per sec. When more rapid vibrations are required the above method is not suitable, because the vibrations of a fork of a higher note cease so soon after a single tap. In order to obtain a time-tracing in Tj^ths or less of a second, it is necessary to use — (2) A Chronograph or time-marker, which records on a drum the number of times per second a current through it is made and broken by FIG. 28.— A time-marker. another special piece of apparatus. The chronograph (Fig. 28) consists essentially of an electro-magnet, which, when the current through it is made, attracts and pulls down a metal lever carrying a writing point. When the current through the electro -magnet is broken, a spring at the other end of the lever raises the writing point. The apparatus used to make and break a current through the chrono- graph at any definite known rate is a tuning-fork of the corresponding note. To one prong of the fork is attached a platinum wire which, Fio. 29.— A tuning-fork with electro-magnet. with each complete vibration of the fork, makes and breaks the chrono- graph circuit by touching and receding from a brass contact or mercury cup (Fig. 29). The tuning-fork, when once started vibrating by a tap, is kept vibrating automatically by an electro-magnet in the same circuit (Fig. 30). Thus, when the platinum wire touches the mercury cup the battery current is made through the chronograph and the writing point is pulled down; at the same time the current is made through the other electro-magnet, which attracts the tuning-fork and pulls the platinum point away from the mercury. Both electro-magnets now cease to act, the writing point of the chronograph is pulled up by the 24 PRACTICAL PHYSIOLOGY spring, and the platinum wire of the tuning-fork again touches the mercury, thereby making the circuit again. To record the contraction of a muscle in response to a single maximal induction- shock, the apparatus is set up in the following way (Fig. 31). Fio. 30.— Diagram of the chronograph circuit. Connect one pole of a Daniell cell to one top binding-screw of the primary coil, and the other binding-screw of the coil to a binding-screw on the base of the stand of the drum. The current passes through the metal work of the stand to a metal striker carried beneath the drum on its axle. As the drum revolves this striker touches a strip of naked wire attached to, but insulated from, the rest of the stand. The binding-screw in connec- tion with this naked wire is connected to the other pole of the battery. It is only when the striker and naked wire are in contact that the primary circuit is completed. A sciatic and gastroc- nemius preparation is made and attached to the myograph-lever, which is weighted near its axis with 10 or 20 grams, and should then be horizontal. The nerve is laid across the electrodes coming from the Du Bois key, and the secondary coil is arranged to give maximal induction-shocks. A tuning-fork giving 100 complete vibrations per second is arranged to write just beneath the myograph lever. Before the two writing points are brought into contact with the smoked surface, the drum should be made to revolve in order to see that it will rotate away from the writing points and at a suffi- Fio. 31. — Diagram of the apparatus for recording a single muscular contraction. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 25 ciently rapid rate ; the rate of rotation should not be less than 20 cm. per sec. Adjust the writing points to touch the smoked paper lightly, and with the Du Bois key open, and the fork vibrating, let the drum make one revolution and no more. The curve of the muscular contraction and the time below it in y^ths of sec. will be recorded (Fig. 32). Close the Du Bois key, remove the tuning-fork, but do not alter the position of the base of the stand carrying the myograph. With the writing point of the lever accurately on the abscissa line of the muscle curve let the drum revolve so as to complete a base line beneath the actual curve corresponding to the muscular contraction. With the writing point still on the base line, rotate the drum by hand until the striker just touches the naked wire. At this position of the FIG. 32.— Single contraction of gastrocnemius in response to a maximal make shock. Muscle loaded with lever and 30 grms. at axis of lever ; actual load on muscle, 6grms. Magnification, 5. Temp., 15° C. Time marker, 100 per sec. (A.P.B.) drum a maximal make induction-shock was sent into the nerve ; with the finger on the lever make the writing point describe a vertical arc, which cuts the time-tracing below and the abscissa line above. In the same way, by rotating the drum by hand, vertical arcs are drawn through the muscle-curve and time-tracing at the three following points : (1) the point at which the curve leaves the base line, (2) the highest point of the curve, and (3) the point at which the curve regains the base line. It will be noted that, during the single revolution of the drum, the primary circuit has not only been made but also been broken again by the striker leaving the naked wire. The nerve has consequently received a maximal make and then a maximal break shock, but has only responded by a contraction to the first; for, owing to the rapid rotation of the drum, the second stimulus has reached the muscle too soon after the first for the muscle to be able to respond (see Re- fractory period of muscle, p. 42). If, however, the drum is revolving but slowly, the second stimulus may follow the first after a sufficient interval of time for the muscle to partly respond to it. This leads 26 PRACTICAL PHYSIOLOGY to a deformation of the curve (Fig, 32), in which the hump near the top of the up stroke of the lever is caused by the muscle re- sponding to the second stimulus (see Effect of two successive stimuli, Chap. VII, p. 40). If with a slowly revolving drum it is desired to send into the nerve a single stimulus, it is only necessary to place the secondary coil at such a distance from the primary that the break but not the make shock is effective. The curve (Fig. 32) occupies about T^ths sec. and can be divided into three parts. (1) The first part extends from the point at which the stimulus reached the nerve to that at which the contracting muscle began to raise the lever. This is the latent period, and is seen to last about yj^th of a sec. During this period several distinct processes take place ; (a) a nervous impulse has to pass down the strip of nerve between the point stimu- lated and the muscle, this will occupy about T ^^ths of a second (see Velocity of nervous impulse). Of the remaining y^^ths (b) the v ' Fto. 33.— Contraction of the same preparation . as in Fig. 32, recorded on a drum revolving at passage of the nerVOUS impulse a slower rate. The hump near the top of the upstroke of the lever represents a second con- aloilg the fine motor traction in response to the break shock. Time ,. marker, 100 per sec. (A.P.B.) endings OCCUplCS about sec., and (c) the latent period of the muscle itself about y^^ths of a sec. This in turn is due to several factors, of which two must be mentioned. When muscle fibres begin to contract a certain time must elapse before the muscle is able to exert a sufficient pull to move the recording lever; in other words, there is instrumental inertia to be overcome. Again, when muscle, which is highly extensible, begins to contract, every part of every fibre does not simultaneously begin to shorten; but the contracted part of a fibre stretches at first the uncontracted part, and is therefore not united to the lever by a rigid connection. It is only when the tension in the stretched part has sufficiently increased, or the fibre as a whole has passed into a state of contraction, that the lever begins to be pulled upon. (2) The second period extends from the point at which the lever begins to rise to the point highest above the base-line. This is the period of active contraction or shortening of the muscle, and occupies about y^ths of a sec. (3) The third portion extends from the highest point of the curve to the point at which the curve rejoins the base-line. This is the period ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 27 of relaxation, and lasts about T£^ths of a sec. Relaxation is a passive process brought about by the falling lever and weight doing the same amount of work on the muscle as the muscle during its period of shortening has done in raising the lever and weight to a certain height. The muscle-curve, although roughly a magnified record of the change in length of the muscle, is deformed by certain errors of instrumental origin, which it is necessary to mention in order to avoid, so far as they are preventible. The most important are the mass and length of the lever and the disposition of the weight along it. They affect all parts of the curve. The weight of the lever tends to prevent the muscle from beginning to raise it (inertia of position) and so lengthens the latent period ; therefore the lever should be as light as possible. During the stage of shortening the lever, when once in motion, tends to be carried on by its own momentum after the muscle has ceased to FIG. 34.— Single contraction of gastrocnemius. Muscle loaded only with a rather heavy lever. Magnification, 5. Temp., 15° C. Time marker, 100 per sec. (A.P.B.) pull on it (inertia of motion), and so makes the muscle appear to have shortened more than it really has. For the same reason, during the period of shortening, the tension on the muscle is not uniform, but becomes less as the lever undergoes acceleration ; during the relaxation exactly the opposite happens, a heavy lever as it falls again undergoes acceleration and increases the tension on the muscle throughout the relaxation and may even stretch it beyond its original resting length (Fig. 34). In order to reduce these errors the lever again should be as light as possible. On the other hand, to attach to the muscle no other weight than that of a very light lever would introduce fallacies. For, unless the muscle is sufficiently weighted to keep it taut, there may be, when the muscle begins to contract, a certain amount of ' slack ' to be taken in which would cause an apparent lengthening of the latent period. 28 PRACTICAL PHYSIOLOGY Again, when the muscle does begin to pull on the lever, it will do so with a sudden jerk, which may cause a light lever to fly up out of control of the contracting muscle ; this, again, makes the muscle appear to have undergone greater shortening than it really has (see, however, Chapter XIII.). Further, the relaxation of a muscle being purely passive, the period of relaxation of an insufficiently weighted muscle is much prolonged, and the writing may fail to reach the base- line again.1 In order to get over these instrumental difficulties, the muscle-lever must be as light as is consistent with rigidity, and the muscle must be suitably loaded, the weight being attached near the axis of the lever for the following reasons : the nearer it is to the axis, the less move- ment will it undergo, and therefore the less will be its inertia of move- ment and the more uniform the tension on the muscle throughout the curve. This disposition of the weight also helps to reduce the after- vibrations or ' shatter '-curves which are frequently seen following the relaxation (Fig. 34). Compare with this Fig. 32 taken from the same muscle ; by hanging a weight of 30 grams near the axis of the lever the shatter curves have been nearly eliminated, and are represented by the slight oscillation between the two vertical lines at the end of the curve. It may be pointed out that in the living body the muscles are always weighted when they contract, and even when relaxed they are under considerable tension ; for they are really shorter than the distance between their points of origin and insertion, and their antagonists are always exerting a certain pull on them, and some muscles, such as the deltoid, are considerably stretched by the weight of a limb. The length of the lever is of some importance ; for, besides the fact that length reduces the rigidity of a light lever, a further deformation of the curve is introduced by increasing the magnification. As the writing point is raised, it tends to leave the drum, and in the course of a much magnified curve is only kept on the drum by the lengthening out of the spring formed by the writing point. Therefore the more the writing point is raised above the horizontal, the more the magnifi- cation is constantly increasing. For this reason the muscular move ment should not be magnified more than is sufficient to make the record of it clear. Although muscle curves, as accurate records of the muscular move- 1 Muscles during the cold of winter, even when properly weighted, frequently show this 'contraction-remainder.' If cold be the cause, turnback the 'trouser* of skin and pour over the muscle some normal tap-water saline heated in a test tube to 25° C. Cf. footnote on p. 33. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 29 ment, have fallacies inseparable from the method of recording them, it is possible to make two rough deductions from them : (1) The amount of actual shortening a muscle undergoes during con- traction can be calculated by measuring the vertical height of the top of the curve above the base line and dividing it by the magnification ; in Pig. 32 the height is 20 mm., and the magnification 5, therefore the muscle became shorter by 4 mm. The length of the resting muscle when loaded by lever and weight was 25 mm., consequently the muscle during contraction became shorter by 4 x ^V, i.e. nearly a sixth of its original length. (2) The amount of work done by the muscle during its contraction is the product of the load and the height to which it was raised, W = L x H. In Fig. 32 the actual load which the muscle raised was not the whole of the 30 grams, hung near the axis of the lever, but a proportion of it, calculated by multiplying by the distance from the axis of the point of the suspension of the weight, and dividing by the distance from the axis of the point of attachment of the muscle ; this fraction was J, and the actual load lifted 6 grams. The height to which it was raised was 4 mm. ; consequently the work performed was 24 gramme millimetres. CHAPTER IV. THE CONDITIONS WHICH AFFECT SINGLE MUSCULAR CONTRACTIONS. (a) Different Muscles, (b) Veratrine. — The curve produced by the contraction of a muscle may be altered not only by such influences as temperature, load, fatigue, and drugs, but also by the differences in structure of various muscles. The muscular fibres of the frog are found to present two varieties, clear and granular, which differ both in structure and in physiological properties. The gastrocnemius may be taken as an example of a muscle whose fibres consist largely of the clear variety, and the hyoglossus of the granular variety, i.e. a muscle in which the majority of muscle-fibres contain more nuclei and are rela- tively richer in undifferentiated living material, the sarcoplasm. The chief physiological difference between granular and clear muscles are, that granular muscles have a slower and more prolonged contraction, are less excitable, more easily tetanised, and less readily fatigued. In mammals the same differences between red and white muscles can be shown to exist. Red muscles, such as the masseter or soleus of the rabbit, differ structurally in having more sarcoplasm and nuclei in their fibres, and are redder in colour owing to a much richer capillary net- 30 PRACTICAL PHYSIOLOGY work between their fibres and to the presence of myohaematin in the fibres themselves ; physiologically they are far less readily fatigued and show a contraction four or more times as long as that of the white gastrocnemius (Fig. 35). For comparison with the single twitch of the gastrocnemius, that given by the hyoglossus may now be studied. This muscle, arising from the anterior edge of the body of the hyoid cartilage, runs forwards into the substance of the tongue. A Hyoglossus Preparation is made by cutting off the whole of the lower jaw, including the tongue and hyoid cartilage. Place it on the myograph board, mucous surface upwards, turn the tongue Fio. 35.— Comparison of contractions of red and white muscle of rabbit, stimulated indirectly. Upper curve is response of the red soleus and lower curve that of the white gastrocnemius. Time marker, 50 per sec. The tracing to be read from right to left. (M.S.P.) forwards, and connect its tip to the lever by a thread. Firmly fix the hyoid cartilage by running a pin through it into the cork. Two needle electrodes transfix the base of the muscle just in front of the hyoid. All the other connections are the same as when studying the single contraction of the gastrocnemius; a weight of 5 or 10 grams is placed near the axis of the lever. Compared with the single twitch of the gastrocnemius, that given by the hyoglossus (Fig. 36) shows the following differences : the whole contraction lasts more than twice as long, the latent period is slightly longer, but it is the period of shortening and still more that of relaxa- tion which is more gradual and prolonged. Action of Veratrine. — A brainless frog is poisoned by injecting into the dorsal lymph sac 5 minims of a saturated (1 in 1000) solution of veratrine in normal tap-water saline. In order that the drug may ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 31 be rapidly absorbed it is important not to ' pith ' the frog, but to destroy its cerebrum with a pair of Spencer- Wells pressure forceps. In about ten minutes it will be observed that the hind legs are very slowly and imperfectly flexed after a jump, and a few minutes later the frog will be seized by a spasm when it jumps. As soon as these FIG. 86.— Contraction of the hyoglossus muscle. Time marker, 100 per second. (A.P.B.) symptoms appear the remaining portions of the central nervous system are destroyed, and a sciatic and gastrocnemius preparation made. In the meantime the action of veratrine may be studied on the hyoglossus preparation used in the previous experiment. Five minims of the veratrine solution are injected into the lymph sac in which the muscle lies. The drum is arranged to revolve at a slow rate of about 2 cm. in 10 sees., and a simple key instead of the " striker " of the drum is placed in the primary circuit. After waiting a few minutes the muscle is stimulated by a single maximal induction- Fio. 37. — Contraction of the gastrocnemius muscle of a frog. The effect of vera- trine. The first two contractions show the characteristic effect of the drug ; further stimulation produced twitches without the prolonged contraction. The curve has been reduced to one-half the actual size. The time is marked in seconds. (Pembrey and Phillips.) shock, and its contraction recorded. The curve shows that the response is a single slow contraction with an enormously prolonged relaxation* Replace the hyoglossus by the gastrocnemius and sciatic preparation and stimulate it in the same way. As soon as the first contraction is over, the muscle is stimulated again, and so on for half a dozen contractions. It will be seen that the first contraction (Fig. 36) con- sists of a smart initial twitch followed by a much longer contraction, and an even more prolonged relaxation. The second contraction shows the same characters to a less extent, and the subsequent con- tractions become of shorter and shorter duration until they reach the 32 PRACTICAL PHYSIOLOGY normal. If the muscle be allowed to rest, the veratrine effect returns again. The absence, in the case of the hyoglossus, of the sharp initial twitch seen in the gastrocnemius contraction, is probably due to more complete poisoning of all the muscle-fibres. The gastroc- nemius is more bulky, some of its fibres remain unpoisoned and respond with a normally rapid contraction, followed by the slower and more prolonged contraction of the poisoned fibres. CHAPTER V. THE CONDITIONS WHICH AFFECT SINGLE MUSCULAR CONTRACTIONS— CONTINUED. (c) Temperature. — Since the shortening of muscle during its contrac- tion is but the outward and visible sign of chemical changes taking place in the muscle, it is not surprising that changes in temperature should greatly affect the single muscle-twitch. In warm-blooded animals whose bodily temperature does not undergo a greater variation than about 2° C., the effect of different temperatures on muscular activity is unimportant. But it is quite otherwise in cold- blooded animals whose range of bodily temperature is that of their external medium. In them, the muscular activity of which they are capable at any moment is determined largely by the temperature of their muscles. Again, the subject becomes important for warm-blooded animals when, from any cause, their bodily temperature is materially altered, as it may be by disease. These abnormal variations in their temperature may be sufficiently great to affect the muscular activity of which the animal is capable. More frequently, however, they are important because of the effect which an abnormally high bodily tem- perature, especially when long continued, may have upon the actual chemical constituents of muscle, and especially upon its proteids. In order to study these effects, the apparatus is arranged to stimulate a muscle with single maximal induction shocks, using the "striker" of the drum, in the primary circuit. Either a hyoglossus or gastroc- nemius preparation may be used ; if the latter, it must be prepared without a covering of skin, in order that its temperature may be more readily altered. Also, the muscle must be stimulated directly and not through its nerve, since changes of temperature affect nerve. It is important to use maximal stimuli, for cold increases the excitability of muscle, and a stimulus which is minimal at 5° C. will be sub-minimal at 25°. The lever should be weighted near its axis and the drum should revolve at a rate of about 20 cm. per sec. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 33 Cold tap-water saline solution, which has been cooled by ice to nearly 0° C., is slowly poured upon the muscle ; the temperature of the solution is noted, the muscle is stimulated, its contraction recorded and the point along the tracing at which the stimulus was sent into the muscle is marked. Swing the writing point off the drum, but do not move the base of the stand carrying the myograph. Take a series of super- imposed curves at temperatures of about 3°, 13°, 23°, and 33° C. (Fig. 38). Sufficient time must be given and fluid used to allow the bulk of the thick gastrocnemius to attain approximately the temperature of the saline solution. In order to get exact results, it would be necessary to FIG. 38. — The effect of temperatu -e upon the contraction of the gastrocnemius muscle. The time is marked in. y^ second. The tracing should be read from right to left. Figures on curve are the temperatures of the salt solution. (Pembrey and Phillips.) suspend the muscle in the solution at a given temperature until its substance had attained that temperature. It will be seen that cold lengthens the whole curve, especially the latent period and the phase of active contraction; the period of relaxation is relatively less affected, but a tendency to incomplete relaxation is often seen.1 As the muscle is warmed, the liberation of energy becomes more and more rapid, consequently the time occupied by the whole twitch decreases progressively, and especially the latent period and period of shortening; the passive stage of relaxation is Pooled excised muscles, even when weighted, are liable to show a 'contrac- tion-remainder,' or incomplete return to their former length after contraction. It is also seen after strong direct stimulation, in poisoning with veratrine, and as fatigue or death come on. C 34 PKACTICAL PHYSIOLOGY relatively less shortened, although muscle does become more extensible as its temperature rises from 0° to 30° C. (Fig. 38). The relation between temperature and the height of the contraction is not quite so simple. Between 0° and about 15° C. the actual height of the contraction may fall slightly, and for two reasons : as the tem- perature increases, the irritability of the muscle decreases ; further, other things being equal, the more slowly a muscle contracts, the more time it has to shorten up as much as it will in response to a given stimulus. From 15° to 25° the height of the curve rapidly increases; this is largely, if not entirely, instrumental in origin, and is- FIG. 39. — Curve of the shortening of the gastrocnemius muscle during heat-riyor. (Pembrey and Phillips.) due to the fact that, as the liberation of energy becomes more rapid> the lever receives a considerable jerk from the rapidly contracting muscle. In other words, the increased height of the contraction is due, not to a greater liberation of energy, but to the greater rate at which the same quantity of energy is liberated. From 25° to 35° C. the irritability of muscle and its height of contraction rapidly fall. Now pour on some solution warmed to 50° C. When the muscle- fibres reach a temperature of about 40° C. they undergo a rapid shortening (Fig. 39), which, as the temperature of the muscle rises,, passes into the permanent shortening of * heat-rigor.' This condition is due to coagulation of some of the muscle proteids, and in consequence the muscle becomes hard, opaqur, inelastic, and has permanently lost its irritability. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 35 CHAPTER VI. THE CONDITIONS WHICH AFFECT SINGLE MUSCULAR CONTRACTIONS— CONTINUED. (d) Load. — In order to study the effect of variations in load upon a single muscular contraction, the apparatus is arranged for stimu- lating the muscle by a single maximal induction-shock, the drum being placed as a key in the primary circuit and arranged to rotate at a fast rate. Make a gastrocnemius-sciatic or hyoglossus preparation. FIG. 40. — The effect of load upon the contraction of the gastrocnemius muscle (A P.B.) FIG. 41 is the continuation of the experiment in Fig. 40. Single contractions of the gastrocnemius with different loads. The figures on the curves represent the weights in grms. hung at the axis of the lever ; actual load on muscle was in each case one- fifth. Magnification, 5. Temp., 12' C. (A.P.B.) Record a single contraction of the muscle weighted only by the lever, mark the latent period and draw a base-line. Then hang on to the lever near its axis weights increasing by 20 grams at a time, and for each addition of weight record a contraction. The base of the stand carrying the myograph should not be moved during the experi- ments, but the curves should be superimposed as in Figs. 40 and 41. Each increase of weight stretches the muscle, consequently it is PRACTICAL PHYSIOLOGY necessary to bring back the writing point accurately on to the base-line before each contraction is recorded. The general effects to be noticed are — that, as the load is increased, the latent period becomes slightly longer, the height of the contraction generally becomes less, the rise of the lever during the period of active contraction becomes more gradual, and the period of relaxation, which may be at first much decreased, gradually lengthens out again. If the muscle be fresh and in really good condition, the early effect of increasing the load may be to increase the height of the first few contractions (Fig. 43). This stimulatory effect of initial tension on the power of a muscle to liberate energy during a subsequent contraction, is seen, within certain limits, in all kinds of muscular tissue; and it is of importance. For, in the body, as has been already pointed out, the skeletal muscles are, even when relaxed, under a certain tension produced by the pull of their antagonists and their being really shorter than the distance between their points of origin and insertion. But when we study the work done by the muscle during a series of contractions with increasing loads, and not merely the height of the individual contractions, the stimulating effect of increased load is much more obvious. After the tracing has been varnished and dried, measure off the vertical heights of the curves corresponding to the different loads, and calculate the work done during each contraction (see p. 29). In the following table are given the details of the work done during the contractions recorded in Figs. 40, 41. Number on Contraction. Actual Load in grins. Actual Height of Contraction in mm. Work done in gTDl. Ill 111. 20 4 4 16 50 10 3-8 38 80 16 3-6 57'6 100 20 3-4 68 150 30 3-3 99 200 40 3-2 128 300 60 2-8 168 500 100 2-6 260 It will be seen that, although the height of the contraction decreases as the load increases, the work performed increases throughout. This process of course has limits, which will be dealt with on p. 60. The important deduction to be made from these results is that muscle as ELEMENTARY EXPERIMENTAL PHYSIOLOGY sg fd li |s. I Jjj !*s if 18 I 38 PEACTICAL PHYSIOLOGY a machine for doing work is found to have its output of energy regulated, not merely by the strength of the stimulus reaching it, but also to a large extent by the amount of work it is called upon to do (see p. 60). Effect of Fatigue. — When discussing the fatigue of muscle it is necessary to draw a distinction between the fatigue of a movement produced by the voluntary contractions of the muscle concerned in it (see p. 70), and the fatigue of a muscle caused by the artificial stimula- tion of the muscle itself or of the nerve supplying it (see p. 72). Further, in the second case there is a marked difference in the effect of continued stimulations on a muscle whose circulation is still intact (see p. 74), and on one which has been excised from the body. Here we shall deal only with the simplest case of a muscle excised from the body and stimulated directly and not through its nerve, in order to exclude any possibility of fatigue of nerve or of nerve endings. Prepare either a hyoglossus preparation to be stimulated by two needle electrodes, or a gastrocnemius-sciatic preparation to be stimulated by one needle-electrode and by fine capillary copper wire threaded through the tendo-Achillis, as the other electrode. The drum is placed in the primary circuit, so that each time it revolves the muscle receives a maximal make induction-shock ; it should revolve at such a speed that the muscle will be stimulated once or twice a second. Weight the muscle near the axis of the lever, using 20 grms. for a hyoglossus and 50 grms. for a gastrocnemius preparation. With the Du Bois key closed, describe a base line and mark on it the point at which the stimulus will enter the muscle. Now open the Du Bois key, allow the drum to revolve, and record the first contraction and every tenth or twentieth subsequent contraction. For this purpose, directly the first contraction is over, the writing point is swung away from the drum, which goes on revolving and causing the muscle to contract. The base of the stand carrying the myograph must not be moved so that for each con- traction the point of entrance of the stimulus will be the same. The writing point should be a fine one, otherwise the number of super- imposed curves will to some extent obliterate each other. When a series of curves taken in this way is examined (Fig. 43) it is seen that they show the following changes as fatigue progresses, — the latent period becomes slightly longer, the shortening of the muscle takes place more slowly and reaches its maximum more gradually, but the actual height of the curves does not begin to decrease much until the other features of fatigue are well marked ; the lengthening out of the period of relaxation is the most marked feature, it is evident from the first, and, as it progresses, a ' contraction remainder ' also appears. 40 PRACTICAL PHYSIOLOGY The rate at which fatigue comes on in a muscle under the above conditions is increased by raising the temperature and the load. Another method of studying the effects of fatigue on a hyoglossus or gastrocnemius muscle is as follows. In this case the primary circuit is made and broken by hand, and the contractions are recorded as nearly straight lines on a drum revolving at the slowest possible speed. The secondary coil is moved up to the primary until both make and break shocks are maximal, and the muscle receives a stimulus once every 5 sees. In this way Fig. 45 was produced. It will be seen that the height of the contractions, after remaining fairly constant at the beginning, gradually decreases until, at the end of 15 minutes, the muscle was incapable of lifting the load. Further, it is seen that in the last two-thirds of the tracing the basal points of the twitches gradually fail to reach the base line, thus showing a 'contraction remainder.' If the muscle had been stimulated at shorter intervals, this appearance would have come on earlier ; for, as soon as the period of relaxation began to increase, the next stimulus would have reached the muscle before there had been time for relaxation to be completed. If the muscle be allowed to rest for a few minutes and then the stimulation is continued, it will be found that even excised muscle is capable of slight recovery from fatigue (Fig. 45). One other point shown by Fig. 45 must be referred to ; the -height of the first twenty twitches increases, showing a 'stair-case* effect. This short and temporary improvement in the condition of muscle, brought about by the repetition of a stimulus of constant strength, was at one time thought to be peculiar to cardiac muscle (see Heart) ; but although shown best perhaps by the heart, it is also shown by all forms of muscular tissue. CHAPTER VII. TWO SUCCESSIVE STIMULI. GENESIS OF TETANUS. TETANUS. WHEN a second stimulus reaches a muscle after the contraction caused by the first is over, the muscle responds with a second contraction similar to or perhaps slightly higher than the first (see Fig. 45). When, however, the second stimulus reaches the muscle before the contraction caused by the first is completed, the response given by the muscle to the second stimulus depends upon the exact phase of its twitch, in which it happens to be when the second stimulus reaches it. In order to investigate this point, arrange the drum and circuits as ELEMENTARY EXPERIMENTAL PHYSIOLOGY 41 Fro. 46.— Effect of two successive maximal stimuli, with gradually diminishing intervals, upon the gastrocnemius. To be read from below upwards. B. Time tracing, 50 per sec. In the two upper curves are shown both the con- traction in response to the first stimulus alone and the combined contractions caused by the two successive stimuli. (M.S.P.) A. Time tracing, 100 per sec. Recorded on a drum revolving at a much faster rate. The second stimulus was sent in well within the latent period of the first. (A.P.B.) 42 PRACTICAL PHYSIOLOGY in experiments for recording a single maximal contraction on a rapidly revolving drum (p. 24); it is only necessary in addition to place a second ' striker ' in the primary circuit through the drum. If the rate of revolution of the drum remains constant, then, by simply altering the angular distance between the two ' strikers/ a second stimulus can be sent in at varying intervals after the first. Make a gastrocnemius preparation and stimulate it either directly or through its nerve. Set the drum in motion and, with the Du Bois key open, approximate the 1 strikers' until the muscle clearly to the eye just responds with a complete contraction to each stimulus. Close the Du Bois key, bring the writing point on to the bottom of the drum, describe a base line and mark on it the point at which each stimulus enters the preparation ; then open the key, record both contractions, and close the key again. Now raise the myograph until the writing point will just clear the top of the curves, approximate the strikers a little, and again record the contractions, after marking a base line and the points of entrance of the two stimuli. This process is repeated until the * strikers ' are finally at such a distance apart that the second stimulus falls within the latent period of the first. In this way Figs. 46 A and B were obtained. It shows that when a second maximal stimulus reaches a muscle during any part of its period of relaxation or of shortening, the rest of the contraction due to the first stimulus is omitted and the muscle starts off on a fresh contraction in response to the new stimulus. Since the second contraction may be as high as the first and starts with the writing point above the base line, it follows that the height of the second twitch above the abscissa is greater than and may be nearly double that of a single contraction ; in other words, a summation of contraction has taken place. If, how- ever, the second stimulus falls within the latent period of the first, then the muscle responds by a contraction only to the first stimulus (Fig. 46 A) ; that is, the muscle is refractory to the second stimulus so far as its being able to respond by a second contraction is concerned ; therefore in skeletal muscle the ' refractory ' period corresponds in time to the latent period (cf. the 'refractory' period of cardiac muscle, p. 64). Genesis of Tetanus. — In order to study the response of a muscle to a series of stimuli, it is necessary to have an apparatus which will auto- matically make and break the primary circuit of an induction coil at any desired rate. The vibrating reed is a convenient form and consists of a flat steel spring which can be clamped in various positions along its length ; by -altering the length of spring which is allowed to vibrate, the number of ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 43 vibrations per second can be changed. The spring has numbers stamped on its upper surface, corresponding to the position at which it must be clamped to give that number of complete vibrations per second. The free end of the spring carries a platinum point which makes and breaks contact with a mercury cup in connection with the primary circuit {Fig. 47). In order to maintain the vibrations of the spring it is usual to place above it, and in the same circuit, an electro-magnet, so that, when the spring makes contact with the mercury, it is attracted out of the cup again by the magnet. In performing a complete vibration, the spring will both make and break the primary circuit and, in order that the two stimuli may not cause contractions of unequal height, the secondary coil must be so placed that either the make shock is just Fio. 47.— Diagram of the vibrating reed in circuit. ineffective, in which case the number of effective stimuli per sec. will be the same as the number of complete vibrations of the spring, or the make and break shocks are made equal and maximal, in which case the number of contractions per sec. will be double that of the complete vibrations of the spring. Place the vibrating reed in the primary circuit so as to give 10 effective stimuli per sec. Make a gastrocnemius and sciatic preparation, with the Du Bois key closed, set the spring vibrating and bring the writing point of the myograph on to the surface of the drurn, rotating at a slow rate, about 3 to 4 cm. per sec. ; open the Du Bois key and record the contractions for about 1 sec. Stop the drum, adjust the spring to give 20 effective stimuli per sec., and record the contractions as before. Eepeat again with 30 stimuli per sec. Then remove the vibrating reed from the primary circuit, connect the battery with the coil so as to set the Wagner's hammer vibrating, and record the contraction of the muscle for a few seconds. Since each twitch of a gastrocnemius at 20° C. lasts about T^th sec., a muscle at that temperature could just respond without any summation to 10 stimuli per sec. If, however, the muscle is colder or fatigued, and each contraction therefore lasts longer, with 10 stimuli per sec., some slight summation may be seen, i.e. relaxation is not complete before the next 44 PEACTICAL PHYSIOLOGY contraction begins, and the line joining the apices and basis of the successive contraction ascends slightly. With 20 stimuli per sec. the summation and fusion of each individual contraction is more complete > but the apex of each individual contraction will probably still be seen : the curve is therefore one of incomplete tetanus (Fig. 48). With 30 stimuli per sec. fusion may be complete from the first, i.e. complete tetanus, or if not complete at first, it gradually becomes so. This gradually increasing fusion (Fig. 48) is really due to fatigue : for the period of relaxation of the individual contraction tends to become longer and longer, and therefore the next stimulus reaches the muscle progressively earlier in each individual twitch, until a point is reached in which there is no time for the muscle to begin to relax between the stimuli, and fusion becomes complete. With the Wagner's hammer, which causes the muscle to receive 50 or more stimuli per sec., fusion is com- plete from the first. One other point is to be noted in nearly all these curves : at first the rise in the lever is very rapid, then it suddenly becomes more gradual, but, even when fusion has been complete from the first, the lever may still rise slowly for a short time until the muscle has reached the utmost shortening of which it is capable. If the stimulation is still continued, this height may be maintained for a short time, but sooner or later the lever will begin to drop, showing the onset of marked fatigue. In all cases when the stimulation ceases, the relaxation is at first extremely rapid, then becomes more gradual and a 1 contraction-remainder ' varying in extent according to the degree of fatigue is generally seen. The same experiments may be performed with a hyoglossus prepara- tion. This muscle, however, being of the * granular' variety and having a contraction which lasts twice as long as that of the ' clear gastrocnemius (see p. 30), is sent into complete tetanus with half the number of stimuli, i.e. about 15 per sec. CHAPTER VIII. THE PROPERTIES OF NERVE, MINIMAL AND MAXIMAL STIMULI. A NERVE is not a unit ; it is that branch of a nerve-cell which conducts an impulse to, or from, the periphery. A nerve-cell with its dendrites and axis-cylinder process or axon forms a unit, the neuron. It is con- venient, however, to examine the characteristics of a nerve apart from its nerve-cell. The chief of these are excitability and conductivity. Excitability, or, as it is sometimes called, irritability, is the response to a, ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 45 46 PEACTICAL PHYSIOLOGY stimulus; a nervous impulse, the real nature of which is unknown, is started at the point stimulated, and is transmitted or conducted along the nerve. Nerves can be stimulated by electrical, mechanical, chemical or thermal agents; of these the most important in experimental physiology is the electrical, for it can be finely graduated, is of extremely short duration, and can be applied repeatedly without damage to the nerve. The first experiments will therefore be the electrical stimulation of nerve. The Electrical Stimulation of Nerve. — An induction-apparatus is arranged for single induction-shocks, and a simple pair of electrodes is connected with the secondary coil by means of a Du Bois key. A preparation of the sciatic nerve in its entire length and of the gastroc- nemius muscle of a pithed frog is made, and near the origin of the nerve is applied the pair of electrodes. On the passage of an induction-current through the electrodes the nerve is stimulated, and an impulse is sent down the nerve, reaches the muscle, and causes it to contract. This is indirect stimulation of the muscle, and is, if a weak current be used, not due to an escape of the electric current along the nerve towards the muscle. This is proved by the following experiment. A moistened thread is tightly tied round the nerve at a point between the electrodes and the muscle. The passage of a weak induction-current of the same strength as that previously used will stimulate the upper portion of the nerve, but the nervous impulse will not pass through the block produced by the thread. A breach in the physiological continuity has been produced, and the nervous impulse is not conducted through the ligatured nerve. The moistened thread would not prevent the passage of a purely electric current. The response of the nerve to a stimulus bears within certain limits a relation to the strength of the stimulus. This can be shown by the following experiment. Maximal and Minimal Stimuli. — The muscle of the preparation is attached to a myograph, the"* lever of which is arranged to write upon a drum covered with smoked paper. The electrodes are placed between the muscle and the ligatured portion of the nerve which was used in the previous experiment. The induc- tion shock is made so weak that no response is obtained, and is then gradually increased until a contraction is observed with the break-she ck Contraction does not follow each break-shock ; the stimulus is sub minimal. The contraction is recorded as a vertical line upon the stationary drum, and before each stimulation the drum ELEMENTARY EXPERIMENTAL PHYSIOLOGY 47 is turned by hand about half an inch. The strength of the current is slowly increased until a small contraction follows each break-shock ; this is the minimal stimulus. The distance in centimetres of the secondary from the primary coil is noted upon the drum. The make- shock is weaker than the break, so that it is necessary to use only the one or the other in this experiment. The intensity of the current is still further increased until the most powerful contraction of the muscle, as indicated by the height of the nearly vertical lines upon the drum, is obtained ; the stimulus is now maximal. Any further increase in the strength of the stimulus is not accompanied by a bigger contraction; a supra-maximal stimulus only produces a maximal contraction, and is liable to damage the nerve. It may be, as Gotch has suggested, that the difference between maximal and minimal stimulation depends upon the number of the constituent fibres of the nerve stimulated. A weak electric current may affect only a few fibres, and therefore the result will be only a slight contraction, due to the excitation of those muscle-fibres alone which are supplied by the nerve-fibres. It will be found that the excitability of the nerve changes, so that with the same strength of stimulus there will not be the same minimal point. A loss of excitability readily occurs if the nerve be allowed to dry, but during this process there may be irregular fluctuations in the excitability of the nerve above and below the normal. Mechanical Stimulation of the nerve can be shown by pinching the nerve with a pair of forceps ; the muscle contracts, showing that a nervous impulse was produced. Such a method of stimulation injures the nerve, but by means of simple arrangements a nerve can be stimulated mechanically without damage. A light hammer worked by an electro-magnet may be used to tap the nerve, or small drops of mercury from a funnel may be allowed to fall upon the nerve. Such methods are useful in experiments in which an electrical stimulus might introduce a source of fallacy, but for ordinary experiments they are undesirable, since there is a difficulty in maintaining a constant strength of stimulus, and there is a danger of damage to the nerve. Thermal Stimulation is next shown by the application of a hot wire to the nerve. The muscle contracts. The damaged portion of the nerve is cut away, and to the end of the living nerve is applied a crystal of common salt ; the muscle soon shows irregular twitches due to the chemical stimulation of its nerve. The last form of stimulus is obviously limited to special experiments, for the stimulus is not easily graduated and damages the nerve. 48 PRACTICAL PHYSIOLOGY CHAPTER IX. THE RELATION BETWEEN MUSCLE AND NERVE. THE motor nerves by means of their end-plates are so intimately con- nected with the muscle-fibres that it is impossible to stimulate the muscle-substance alone by the direct application of a pair of electrodes to the intact muscle. The question, therefore, arises whether muscle possesses independent excitability, whether it can respond to a stimulus without the intervention of its nerve. The development of muscle from protoplasm, which is contractile and excitable although possessing no nerves, would suggest that muscle itself is excitable and can respond to a stimulus. This can be shown, for the fully developed muscle, after its nerve has been paralysed by the action of a drug. Curare1 is an alkaloid used as an arrow-poison by some natives of South America. The following experiments show that it paralyses the terminations of the motor nerves, but that the muscle still responds to direct stimulation : (i) Two watch-glasses are almost filled with a 1 per cent, solution of curare in normal tap-water saline. Two muscle and nerve- preparations are made, care being taken to bisect the lower portion of the vertebral column and thus obtain the entire length of the sciatic nerve. The excitability of the nerve and of the muscle in the case of each preparation is tested by the determination of the minimal stimuli. Then the nerve of preparation A is placed in one watch-glass full of the poison, but its muscle is left outside upon a piece of filter-paper moistened with normal tap-water saline. The gastrocnemius muscle of the preparation B is placed in the solution of the drug and its nerve upon the damp filter- paper (Fig. 50). Stimulation of the nerve B will soon produce no contraction, even if the strongest induction-shocks be used ; on the other hand, an examination of the nerve A will show that its excitability has practically undergone no decrease. Stimulation of the muscle B which has been exposed to the action of the drug readily produces a contraction. The poison, therefore, must act upon some portion of the terminations of the nerves, probably upon the end- plates, for both muscle-substance and nerve-trunk retain their excita- bility even after long exposure to the drug. Muscle will contract on direct stimulation even after its nerves have degenerated. This experiment, however, is not suitable for a class, for it would be necessary to keep the animal alive for two or three weeks in order that the nerve-fibres might completely degenerate. JIt is prepared from various plants of the genus Strychnos. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 49 A further experiment with curare can be made, (ii) The cerebral hemispheres of a frog are destroyed, and then the sciatic nerves are carefully exposed in each thigh ; a strong ligature is passed under the sciatic nerve of one side, A, and is tied tightly around all the structures of the thigh except the nerve. The circulation of the blood is thus com- pletely stopped in the structures below the ligature. Stimulation of either sciatic nerve produces a contraction of the muscles of the corre- sponding leg. Under the skin of the back of the frog are injected two or three drops of a 1 per cent, solution of curare. The poison is FIG. 50. — Diagram of the experiment on the action of curare. absorbed by the blood-vessels and is circulated in all parts of the body except those below the ligature. Paralysis is produced, and the frog lies in a toneless condition and does not move if its toes be pinched. Stimulation of the sciatic nerve produces in the case of the ligatured leg, A, a contraction of the muscles, but in the case of the other leg, B, no contraction occurs. The muscles, however, of the leg, B, contract on direct stimulation. Both nerves in their upper portions have been exposed to the poison, the muscles of both legs respond to direct excitation, but the ligatured leg alone to indirect stimulation. The ligature has prevented the poison from reaching the terminations of the nerves inside the muscles. It is upon these terminations that the curare acts. The independent excitability of muscle can also be shown in the case of cardiac muscle. The apex of the ventricle of the frog's heart con- tains no ganglia, but it responds to a stimulus, and under appropriate conditions will even contract rhythmically. Further experiments upon the independent excitability of muscle are given in Chapter XX. 1 This operation should be performed with a pair of Spencer- Wells pressure- forceps in order that no blood may be lost. 50 PRACTICAL PHYSIOLOGY CHAPTER X. THE EFFECT OF A CONSTANT CURRENT UPON MUSCLE AND NERVE. MUSCLE and nerve consist of complex chemical substances, and con- tain about 70 per cent, of water in which various salts are dissolved. Moreover they are bathed in lymph. The passage of a constant current through a liquid produces electro- lysis; thus, in the case of water, oxygen is given off at one plate, hydrogen at the other. Animal tissues, containing, in addition to a large percentage of water, salts and proteins, are also the seat of electro- lysis during the passage of a constant current ; the ions are probably of a complex nature. These changes in nerve and muscle are shown by alterations in excitability and conductivity. These it is necessary to consider in relation to the changes which occur at the anode and kathode during the make and break of the FIG. 51. — Diagram of the frog's heart to show the effects of the make and break of a constant current upon muscle. In A the ventricle is represented as pale and contracted, with a small shaded area to represent the flushed and uncontracted portion of the ventricle ; that is, a local diastole during general systole. This condition can be produced by the make of the anode or the break of the kathode of a constant current. In B the ventricle is dilated and flushed, with a small pale area of contracted muscle ; that is, a local systole during general diastole. This condition can be produced by the make of the kathode or the break of the anode. constant current. The simplest experiment can be made upon the frog's heart. The Effects of Anode and Kathode upon the Frog's Heart. — The brain and spinal cord of a frog are pithed and then the heart is exposed. Care should be taken to avoid the severance of large blood-vessels in order that the vascular system may be well filled with blood. The pericardium is opened and the heart is observed ; the ventricle during systole is pale owing to the contraction of its muscle fibres forcing out the blood from its spongy walls ; during diastole, when the muscle is relaxed the ventricle is flushed owing to its distension with blood. There are no blood-vessels in a frog's cardiac muscle. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 51 The ends of two pieces of ordinary insulated wire are well cleaned and are connected with a Daniell battery ; the clean free ends of the wires are bent back so that there will be smooth surfaces to apply to the heart. The wire connected with the copper of the battery is the anode, that with the zinc is the kathode. In the frog's mouth is placed the kathode, for there good contact is obtained with a moist conductor; the anode is placed upon the ventricle. Now it will be found that during the systole of the ventricle that portion of the muscle which is around the anode will be flushed, uncontracted, and bulging outwards — the anode at the make of the circuit produces a local diastole during general systole (Fig. 51 A). The rhythmic power of the cardiac muscle around the anode is diminished, so that it remains uncontracted. If now the wire be suddenly removed from the heart, the break of the anode causes an increased excitability of the muscle to which it had been applied, there is a local pallor ; the cardiac muscle is here contracted during the general diastole of the heart. The break of the anode produces a local systole during a general diastole. The kathode is now applied to the heart and the anode is placed in the frog's mouth. There is produced a local systole during the general diastole of the heart. The kathode increases the excitability of the cardiac muscle, and thus the fibres affected remain contracted. The end of the wire is kept in contact with the ventricle for about a minute and is then suddenly removed ; a flushed and bulging spot will indicate the region to which the wire had been applied. The break of the kathode produces a local diastole during general systole, for the dis- appearance of the condition of katelectrotonus is accompanied by a fall in excitability. This simple experiment shows that the make of the kathode and the break of the anode excite, that the make of the anode and the break of the kathode depress. This is also true in the case of nerve. CHAPTER XL THE ELECTROMOTIVE PROPERTIES OF MUSCLE AND NERVE. IN uninjured and resting muscle and nerve there is no electric current, but during activity a current, the 'current of action,' is produced. Injury causes local activity around the damaged tissue, and is there- fore accompanied by an electric current, the so-called * demarcation or 52 PKACTICAL PHYSIOLOGY injury-current' This electrical current produced by injury is, as Gotch pointed out, to be considered as a current of action. These facts can be demonstrated by the following experiments. The Rheoscopic Frog. Galvani's Experiment, Contraction without Metals. — A long length of the sciatic nerve is dissected in a pithed frog and the muscles of the thigh are exposed and cut across. The trunk of the sciatic nerve is laid along the longitudinal surface of the muscles of the thigh, and then by raising the end of the nerve by a small glass rod the transverse section of the nerve is allowed to fall upon the cut surface of the muscles (Fig. 52). At this moment a twitch of the muscles of the leg moves the foot or toes. The circuit of the electric current in the muscle has been completed through the nerve. The section of the muscle-fibres has produced a local contrac- tion of the fibres, and this is accompanied by an electrical change which is sufficient to produce excitation when it is passed through an excitable nerve. Secondary Contraction or Secondary Twitch. — Two muscle- and nerve-preparations are made ; the nerve of A is so placed upon the muscle B that the cut surface of the nerve lies upon the tendon and its longitudinal surface upon the muscle-fibres FIG. 52.-Diagram of Gal- (Fig. 53). The nerve of preparation B is stimu- vani's experiment. Con- , , , , . -, . , , , A, traction without metals, lated by a weak induction-shock, and thus its muscle is excited and made to contract ; the muscle A will also contract. The contraction of the muscle B is accompanied by an electrical current, the * current of action,' which passes through the nerve A and thus produces a contraction in the muscle A. This is not due to an escape of electrical current from the electrodes, for a secon- dary twitch can be obtained if A.\ mechanical or thermal stimuli be used to excite the nerve of pre- paration B. Further, ligature of the nerve B with a moist thread will show that there is no escape With a Weak induction-Shock ; the Flo. 53.-Diagram of the experiment on ligature destroys the physiologi- secondary twitch. cal continuity and prevents the passage of the excitatory state but not that of an electrical current. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 53 Secondary Tetanus. — If the nerve be stimulated with a rapid series of induction-shocks the muscle B goes into tetanus and its * currents of action ' stimulate the nerve A, with the result that the tetanus is also observed in the muscle A. This ' secondary tetanus ' can be produced by rapid mechanical stimuli. Further experiments upon the electromotive properties of muscle and nerve are given in Chapter XXIII. CHAPTER XII. (Advanced). EXTENSIBILITY AND ELASTICITY OF MUSCLE WHEN AT REST AND CONTRACTED. COMPARISON WITH RUBBER. MUSCLE is both extensible and elastic, that is, it can be stretched beyond and will return more or less to its original length when the extending force is removed. These are important properties ; for, unless muscle were readily extensible the sudden contraction of one set of muscles would in the body be liable to rupture their antagonists. In the study of these properties a gastrocnemius preparation may be used, but a muscle whose fibres run more nearly parallel to each other is preferable, such as a sartorius preparation from a large frog or better still a gracilis-semimembranosus preparation. A gracilis-semimembranosus preparation consists of the two large internal thigh muscles (Figs. 2Q, 21). The gracilis is a large muscle lying along the inner side of the sartorius ; it arises from the ischial sym- physis and is inserted into the head of the tibia. The semimembranosus is a bulky muscle behind the gracilis on the posterior aspect of the thigh ; it also arises from the ischial symphysis and is inserted into the back of the head of the tibia. To make the preparation, isolate these two muscles from those surrounding them near their points of insertion, cut through the tibia below this point and through the femur just above the knee joint. Holding this piece of bone, separate the two muscles up to the symphysis and remove with them the bone from which they arise. If a larger or longer muscle still is required, a double preparation may be made with the muscles of both thighs and the two hung side by side, or one below the other, united in the middle by the piece of the symphysis. The following experiments should be performed. The bone at the 54 PRACTICAL PHYSIOLOGY upper end of the preparation is rigidly fixed in a clamp and to the lower end is attached by a short thread or pin a brass mm. scale, having its zero at the bottom. The lower end of the scale has a small tray to carry weights or a hole by which weights can be hooked on. A pointer carried by a separate stand is placed opposite the zero of the scale. A weight of 10 grms. is attached to the scale and the amount of extension read off; then another 10 grms. is added and so on until the load is 100 grms. or more. It will be found that the length to which the muscle is extended is not proportional to the weight used, but that, by each increase of weight the muscle is stretched rather less, the greater the previous extension. By removing the weights one by one the elasticity of the muscle is observed ; it is not complete ; for when all the weights have been removed the muscle does not at once return to its original length. An * extension-remainder ' is present, and this is the more marked the more the muscle is fatigued by the degree and duration of the extension. Therefore the observations should be made as rapidly and on as fresh a muscle as possible. It is probable that muscle in the body with its circulation intact is completely elastic. If the muscle is replaced by a suitable piece of rubber band and the same observations are repeated on it, it will be found that the series of elongations are more nearly proportional to the weights used, thus con- forming nearly to Hooke's Law, which states that the successive increments in length produced by equal increments of weight are, in a perfectly elastic body, equal. Also, as the weights are successively removed, it will be found that the elasticity of rubber is more nearly perfect. But, if the extension be great and of long duration, an ' exten- sion-remainder ' does appear and only gradually disappears. Another method of demonstrating the same properties is to fix the upper end of a muscle-preparation in the clamp of a simple myograph and to attach its lower end to the lever by a bent pin. Attached to the lever vertically below the muscle is a scale-pan or hook to which weights can be suspended. The writing point of the lever is brought on to the surface of a stationary smoked drum and a zero line described by rotating the drum by hand. The drum is rotated back so that the point of the lever is 5 mm. from the beginning of the zero line, a weight of 10 grms. is attached to the lever, the muscle will be extended and the writing point will record a new vertical line on the drum. Turn the drum by hand so that the writing point will describe a horizontal line 5 mm. long,1 attach 1 By thrusting the points of a pair of fine forceps through a thin piece of cork a means of measuring off equal distances is obtained ; there is a mm. scale on the induction-coil. ADVANCED EXPERIMENTAL PHYSIOLOGY 55 another 10 grms. and repeat the process until 100 grms. or more are extending the muscle. In the same way reverse the process and remove the weights of 10 grms. one by one. If now the lower ends of the vertical lines drawn by the fall and rise of the lever are joined, a curved line will be formed, showing that the extension of the muscle becomes less and less for each additional weight. Further, when all the FIG. 54. — Curve of extensibility and elasticity of gastrocnemius. The figures on the curve are weights in grms. Temp., 15° C. (A.P.B.) weights have been removed, the writing point will be below the original zero line, showing an * extension-remainder ' (Fig. 54). It will also be seen that the line corresponding to the elasticity of the muscle is a flatter and more gradual curve than that corresponding to the extension ; this is caused by the long continued load impairing the elasticity of the muscle. PIG. 55.— Elasticity curve of quiescent muscle. To be read from right to left. The figures on the curve are for weights in grms. (M.S. P.) If the experiment be repeated on a piece of rubber band, the line join- ing the lower ends of the vertical lines will be nearly straight, and little or no 'extension-remainder' will be seen. Figs. 55, 56 show a com- parison of the lines thus described for a muscle and piece of rubber loaded from 0 to 500 grms. and then gradually unloaded again. A contracted muscle is more extensible than a resting one. This is of importance in the body ; for, otherwise, a sudden and powerful con- traction of a muscle, trying to lift a heavy weighty would be liable to rupture either the muscle itself, or its tendon, or the bones to which it 56 PRACTICAL PHYSIOLOGY is attached. As a matter of fact, of these three structures muscle, owing to its increased extensibility during contraction, is the least often ruptured. In order to demonstrate this properly the muscle-prepara- tion is attached to the clamp and lever, as in the last experiment. Arrange the apparatus for stimulating the muscle directly with single Fio. 56.— Elasticity curve of rubber tubing. The figures represent weights in grms. (M.S.P.) maximal induction-shocks, using a spring-key in the primary circuit. Bring the writing point on to a stationary drum and, with the muscle weighted only by the lever, describe an abscissa line corresponding to the resting muscle. With the writing point again at the beginning of this line, stimulate the muscle once and, from the top of the ordinate so marked, draw another abscissa line corresponding to the muscle when contracted. Rotate the drum by hand, so that the writing point is now 5 mm. along the 'resting' abscissa line; hang 20 grms. on to the lever and stimulate, so as to record a second ordinate 5 mm. from the first. Repeat this process, increasing the weight by an equal amount each time. In this way Fig. 57 was produced. It is clear that the distance of the lowest point of each ordinate below the c resting ' abscissa line represents the extension of the resting muscle by a given weight, and that the distance of the top of the same ordinate below the ' contracted ' abscissa line represents the extension, by the same weight, of the muscle when contracted. If the lowest and then the highest points of the ordinate are joined, two curved lines are produced which represent respectively the curves of extension of resting and contracted muscle (Fig. 57). It will be seen that the extensibility of contracted muscle is absolutely greater, and increases more rapidly, than that of resting muscle. Hence, if the observations were carried ADVANCED EXPERIMENTAL PHYSIOLOGY 57 far enough, the two curve lines would ultimately cross; this means that if a muscle were loaded by a weight greater than it could lift, it FIG. 67. — Comparative extensibility of resting and contracted pastrocnemius. Temp. 12* C. Magnification, 5. Figures represent actual weights in zrms. U is the ' resting ' and C the ' contracted ' abscissa line. (A.P.B.) would during its stimulation actually lengthen (Weber's paradox). If this were not so, we should, when trying to lift a load greater than the muscle could move, run a great risk of rupturing our muscles. CHAPTER XIII. (Advanced}. LOAD AND AFTER-LOAD. WORK DONE WITH INCREASING LOADS. MUSCLES may be loaded in two ways ; the load may be applied before the muscle has begun to contract, or only after it has already begun to contract ; this latter method, in order to distinguish it from the former, is called ' after-loading/ Most of the muscles in the body are both loaded and after-loaded; that is, they are constantly loaded by the pull of their antagonists, and it is only after they have already begun to shorten that the main load — the weight of the limb, etc. — is applied to them. The deltoid, however, is an instance of a muscle constantly loaded by the weight of the arm ; the ventricle of the heart, on the other hand, is a muscle which is only after-loaded. 58 PRACTICAL PHYSIOLOGY The effect of load, and of its method of application on a single muscular contraction, will be studied in the following ways : (a) the contraction given by a muscle loaded and after-loaded with the same weight will be compared; (b) a con- stant load will be thrown on to a muscle as an after-load later and later in its period of shortening, and the effect on the contractions noted ; (c) the muscle being just completely after- loaded, the height of contraction, with increasing loads, will be mea- sured and the work done with each calculated. Comparison of the Contractions of a Loaded and After-loaded Muscle. — Arrange the apparatus for stimulating IJj a muscle with single maximal induc- |~ tion shocks, using the drum as a key 2"! in the primary circuit. Fix a gastro- j| cnemius preparation to a myograph •gg lever, provided with an after-loading j §> screw ; by raising the screw the metal •g* part of the lever can be supported at |? any level (Fig. 25). Hang a weight J "!• of 50 grms. near the axis and raise the J | screw until the whole of the weight | is just after-loaded ; this point can be 5 ascertained by supporting the weight I with the finger, and when the muscle | no longer tends to raise the lever off o the after-loading screw, the muscle is J unstretched by any load. Arrange the | apparatus so that with the screw in | this position the lever is horizontal. jj Record a single contraction of the o- muscle on a rapidly revolving drum, mark the point of stimulation, and draw an abscissa. Then lower the after-loading screw until the muscle is loaded with the whole weight, and super-impose on the same abscissa and with the same point of stimulation a contraction of the loaded muscle (Fig. 58). ADVANCED EXPERIMENTAL PHYSIOLOGY 59 The main differences between these two curves are — in the purely after-loaded muscle there is an appreciable lengthening of the latent period owing to the muscle in its unstretched condition having to take in ' slack ' ; a diminution in the height of the contraction, owing to the absence of tension on the muscle before the contraction began. In other words, moderate initial tension increases the power of a muscle to do work. Progressive After-loading of a Muscle. — With the same arrangement of apparatus as in the preceding experiment, record a single con- Fio. 59.— Effect of progressive after-loading of a gastrocnemius. Actual load on muscle, 4 grms. Magnification, 5. Temp., 10*0. (A.P.B.) traction of the muscle when just after-loaded, draw a base line and mark the point of stimulation. Now raise the after-loading screw until the writing point is on a level with the highest point of the preceding curve; draw a fresh abscissa at this level and record a contraction; the point of stimulation will be the same as before. Repeat this process until the muscle can no longer lift the lever off the after-loading screw (Fig. 59). From this experiment we see that, in a series of contractions each more after-loaded than the last, a muscle is able to undergo a little further shortening each time until it reaches its maximal shortening. Also by measuring the heights of the contractions above their respective abscissae, we learn that the longer after stimulation it is before the muscle meets the resistance of a given weight, the less is the muscle then able to overcome that resistance and raise the weight. In other words, as a muscle contracts its extensibility progressively increases, arid its absolute contractile force decreases, until at the height of its contraction its extensibility is greatest and its absolute contractile force 60 PEACTICAL PHYSIOLOGY nil. Hence a muscle would contract under the most favourable circumstances, if the load, as it was raised, progressively decreased. Relation of Load to Work done during Contraction. — In order to record the height of contraction for a large range of weights, it is more convenient to record on a stationary drum simply the heights of a series of twitches than to super-impose a large number of curves. The apparatus is arranged for stimulating the muscle with a single maximal induction-shock, using a simple key in the primary circuit. A weight is hung near the axis of the lever of such a size that the actual load on the muscle is 50 grms. ; the method of calculating this weight has been already given on p. 29. The muscle is just completely after-loaded throughout the experiment in order to get rid of the effect of alterations in the initial tension. With the lever horizontal, the muscle is stimulated, and the height of its contrac- tion recorded on a stationary drum. The drum is rotated a short distance by hand; an additional load of 50 grms. is hung from the lever, and another contraction recorded. The process is repeated until the muscle is no longer able to raise the load off the after- loading screw. Fig. 60 gives the result of such an experiment; in it the magnification was 5, and the actual load on the muscle halt of the weight hung near the axis of the lever. The following table gives in grm. mm. the work done by the muscle with the various loads. Actual load in grm. Actual lift in mm. Work in grm. mm. 50 4-0 200 100 3'2 320 150 2-2 330 200 1-8 360 250 1-2 300 300 1-0 300 350 -8 280 400 '5 200 450 -4 180 500 -3 150 550 -2 110 600 -1 60 700 0 0 From the last column in this table we see that, although the height of the contractions diminishes continuously, the actual work done by the muscle increases at first rapidly and then more slowly, until it reaches its maximum with a load of 200 grms. After that point the work done begins to decrease slowly, and then more rapidly until at 700 grms. a load is reached which the muscle is unable to ADVANCED EXPEKIMENTAL PHYSIOLOGY 61 lift. This weight represents the * absolute contractile force* of this muscle, that is, the load which, brought to bear on the muscle at the instant of contraction, is just able to prevent it from shortening. Although the muscle is unable to lift this load, and therefore, when stimulated, does no visible mechanical work, it nevertheless liberates energy chiefly as heat. FIG. 60.— Height of contractions of gastrocnemius with increasing load. The number above each contraction is its observed height in mm. Magnification, 5. The number below each contraction is the weight in grm. hung at the axis of the lever ; the actual load on the muscle was half of this number. (A.P.B.) We are now in a position to recapitulate, so far as load is concerned, the conditions necessary to obtain an optimal contraction of a muscle and to see how far they exist in the living body. Initial tension, we have seen, decreases the latent period and increases the power of the muscle to do work. In the body the muscles are constantly loaded to a slight extent, and are thus kept stretched and free from * slack.' In this way movements with a short latent period, and with an absence of jerkiness are obtained; and the muscles by being stretched are kept irritable, awake and fit for sudden work. On the other hand we see that a muscle, when purely after-loaded, is at a disadvantage for doing work; yet in the body the main load is thrown on as an after-load. The advantage of this arrangement depends upon the increased extensibility of contracting muscle; for, in this way liability to rupture is reduced ; further, there is a saving of energy in pulling at a dead weight through an elastic spring, instead of through an inelastic cord, since some of the energy expended would be lost in a sudden jerk, but, in the case of the spring, is stored up in it and given out again as its elastic recoil. Thus smooth- ness is imparted to even the most sudden movements. We have 62 PRACTICAL PHYSIOLOGY also seen that as a muscle shortens its absolute contractile force decreases ; therefore, it is clear that the after-load should be thrown on to the muscle at the instant of contraction, when the contractile force of the muscle is at its maximum, and not later; this is the arrangement in the body. Further, it would be an advantage if the load decreased as the contractile force of the muscle during its con- traction decreased; this is not usually the case in the body, but it does occur in certain movements, as, for instance, in jumping or when, with the upper arm horizontal, a weight in the hand is raised by flexing the forearm on the elbow. CHAPTER XIV. (Advanced). SUMMATION OF STIMULI. IN a previous chapter the subject of summation of contractions has been dealt with. This summation of 'effect' must be distinguished from the summation of stimuli, by which an inadequate stimulus, if repeated sufficiently often, becomes first adequate and then for a time increas- ingly effective. This is a summation of 'cause,' and probably plays an important part in the life of all living matter. In order to demonstrate the summation of stimuli, arrange the apparatus for stimulating a gastrocnemius muscle directly with single induction-shocks, using a simple key in the primary circuit. Place the secondary coil at such a distance from the primary that the break- shocks are just subminimal. Repeat the stimulus every 5 seconds. ADVANCED EXPERIMENTAL PHYSIOLOGY 63 It will be found that sooner or later the summed excitations will cause a contraction, and, if the contractions are recorded on a slowly revolving drum, that a well-marked ' stair-case ' effect is produced (Fig. 61). In dealing with the response of muscle to two successive stimuli, it has been seen that, when the second stimulus falls within the latent period of the first, the muscle is refractory, so far as being able to FIG. 61.— Effect of subminimal stimuli repeated every 5 seconds on gastrocne- mius stimulated directly. The dots mark the points at which stimuli were sent in before they became obviously effective. Time marking in seconds. (A.P.B.) respond with a second contraction is concerned ; but it is not true that a muscle during its refractory period always entirely ignores a second stimulus. In order to investigate this point, the apparatus is arranged as in demonstrating the effect of two successive stimuli (p. 42). The two ' strikers ' are placed at such an angular distance apart that the second stimulus falls well within the latent period of the first ; the muscle is stimulated directly. The secondary coil is placed at such a distance from the primary that when, by rotating the drum by hand, one of the strikers is made to pass over the naked wire, a minimal or submaximal break, but no make contraction is obtained. A tuning fork is arranged to write under the myograph-lever, the drum is allowed to make one revolution at a rapid rate, a base line is drawn, and the points of stimulation corresponding to each * striker' are marked. Swing the lever away from the drum, but do not alter the position of the base of the stand carrying the myograph. The single contraction so recorded is the response of the muscle to two break shocks. In order to determine whether the muscle has been in any way influenced by the second stimulus, raise the second 'striker,' so that it will no longer touch the naked wire, and record the contraction due to the first stimulus alone (Fig. 62). It will be found that the contraction in 64 PKACTICAL PHYSIOLOGY response to the single stimulus is not so great as that due to the two stimuli. In other words, there has been a summation of stimuli during the refractory period. In the same way subminimal stimuli can be summated, but two maximal stimuli are summated only when they follow each other after an interval of less than T J^th second. As has been pointed out on p. 25, when a ' striker ' passes over the naked wire, there is both a make and break of the primary circuit; consequently in these experiments the muscle really receives four induction-shocks, of which, accord- ing to the position of the secondary coil, all four might be individually subminimal, or the two break-shocks might be alone effective, or all four might be effective. In order to deal with the summation of two break-shocks alone, it is usual to perform these experiments with the following special piece of apparatus. The Spring or Trigger Myograph. (Fig. 63). — It consists of a heavy metal base which is clamped to the bench. The essential part of the apparatus is an oblong metal frame carrying a smoked glass plate, the recording surface, which is shot on two horizontal wires past the writing points. In order to prepare the apparatus for use, the frame is pulled to one side by one of the arms attached to it ; this compresses a spring on the other arm, and the frame is held in position by a catch or trigger. When the catch is re- leased the spring gives the frame and ADVANCED EXPERIMENTAL PHYSIOLOGY 65 glass plate a rapid and uniform horizontal motion, and the momentum carries the recording surface across until stopped by the buffers at the opposite side. The frame carries on its under surface two pins which knock over two vertical keys and so breaks two primary circuits (Fig. 64). TTj is fixed, but K2 is movable horizontally, and its position can Via. 63. —The spring myograph. be adjusted so that it will be knocked over at any desired interval after Kr A pointer is attached to K2) and when this is opposite the zero of the scale this key will be knocked over at the same instant as K^ ; there- fore, in order that K2 may be knocked over after and that the second FIG. 64. — Diagram of the spring myograph in circuit. stimulus may still fall within the latent period of the first, it is necessary to move K% a short distance along the scale from Kr Place both keys in the primary circuit of the same coil and arrange the secondary coil at such a distance from the primary as to give sub- maximal break-shocks. With the spring compressed, the catch down and both keys vertical, the writing points of the lever and tuning fork are placed against the recording surface at its spring end in order that the whole contraction may be recorded. Release the catch. The frame is then pulled back to its original position, both keys are made vertical E PRACTICAL PHYSIOLOGY again, and the pins on the frame are slowly brought up against the two keys in turn and the points along the curve marked at which the two stimuli entered the muscle; the second stimulus should have fallen well within the latent period of the first. Reset the apparatus, leaving K2 horizontal, but placing K^ vertical, and record the contraction due to the first stimulus alone. This second contraction will be found to be smaller than that caused by the summation of the two sub- maximal stimuli. Fig. 65 shows the contractions obtained by a Pendulum Myograph which is fundamentally the same as a spring myograph, and differs only in that the smoked plate, instead of being shot horizontally across by a spring, swings across at the end of a long and heavy pendulum and describes an arc of a circle. The glass plate in either case is varnished in the ordinary way, and, when dry the curves are reproduced by exposing to daylight sensitive paper covered by the smoked plate. ADVANCED EXPERIMENTAL PHYSIOLOGY 67 CHAPTER XV. (Advanced). EFFECT OF DISTILLED WATER AND OF VARIOUS SALTS ON MUSCLE. THE various tissues of the body are all bathed in the same fluid, the lymph, which so far as the water and salts it contains are con- cerned, has a uniform composition. The tissues, although immersed in the same fluid, show different and characteristic properties owing to their difference in structure and chemical composition. If, however, the composition of the fluid, in which any given tissue is immersed, be altered, the composition and consequently the properties of its proto plasm must also be altered. The first effect on living matter of such a change is to cause its stimulation, and then if the change be sufficiently profound and long continued to produce its death. Only two changes in the tissue fluids will be considered here, namely — (a) Gross change in the osmotic pressure of the fluid, by using distilled water or a strong saline solution ; and (b) Change in the ions in solution without alteration in the osmotic pressure of the fluid, by using solutions of various salts isotonic with frog's blood -plasma. Effect of Distilled Water. — Dissect out a gastrocnemius muscle and place it, without a 'trouser' of skin, in a watch-glass containing distilled water. For a few minutes the muscle may show irregular contractions, then it becomes opaque, swollen and incapable of re- sponding to a stimulus with a contraction. The muscle is said to have passed into a condition of 'water-rigor.' Test the muscle with induction shocks and demonstrate that it will no longer contract. By placing the muscle into distilled water two effects are produced — the inorganic salts in the muscle diffuse out into the water, and water is attracted by osmosis into the muscle so that each fibre becomes greatly distended with fluid. The first effect of these changes is to produce stimulation, but, as the muscle fibres are distended with fluid, they become incapable of contracting, and finally there are not enough salts left in the muscle to keep the globulins in solution ; hence the muscle becomes gradually opaque and dies. Effect of Strong Saline Solutions. — This effect will be exactly the opposite of that due to distilled water; for water will be abstracted from the tissue, and large quantities of the salt will diffuse into the muscle. The effect on a tissue of mere abstraction of water from it is best seen by allowing a nerve to dry. Majce a gastrocnemius and sciatic 68 PRACTICAL PHYSIOLOGY preparation, keep the muscle and lower half of the nerve just moist with tap-water saline, but allow the upper half of the nerve to dry. As the nerve begins to dry, irregular contractions of the muscle come on which are stopped by moistening the nerve ; showing that loss of water acts as a stimulus to nerve. If the drying is allowed to continue, the dry portion loses its irritability and dies. Now place upon the muscle a few crystals of NaCl; irregular con- tractions will soon appear. These are partly due to the abstraction of water, but also, as we shall see in the next experiments, to the stimulatory effect of NaCl. The above experiments show that, in order to keep muscles and nerves irritable and in good condition, they must be moistened with a fluid which will neither give up nor abstract water from the tissue, i.e. which is isotonic with the animal's lymph. For this purpose a •7 per cent, solution of NaCl in distilled water has frequently been used. This solution, although isotonic with frog's blood, does not contain the calcium and potassium salts found in blood-plasma and lymph; and the question arises whether this alteration of the ions in solution affects in any way the properties of muscle. In order to investigate this point, prepare two sartorius preparations with their bony attachments and without injury to their muscular fibres. Place one muscle in Biedermann's solution (-5 grms. NaCl, •2 grms. Na2HP04, '2-04 grms. Na2C03 in 100 c.c. distilled water), and the other in •? per cent. NaCl in distilled water. The muscle in Biedermann's solution, especially if the solution be cool (3° — 10° C.), will after a shorter or longer interval begin to show fibrillary twitches and may even contract regularly and rhythmically as a whole. As soon as the result has been obtained, transfer the muscle to a solution made by adding to 100 c.c. of *7 per cent. NaCl solution in distilled water, 10 c.c. of a saturated solution of CaS04, or of a 10 per cent, solution of CaCl2 in distilled water. The spontaneous contractions will soon cease. The other muscle placed in the pure NaCl solution may remain quiescent; very often it will show fibrillary twitchings and irregular contractions, which are rapidly stopped by transferring the muscle to the solution containing a calcium salt as well as NaCl. Should the muscle, however, remain perfectly quiescent1 it can still be shown that it is no longer in a perfectly normal condition. After it has remained in the solution for half an hour, remove it and connect it 1 Frog's muscle differs somewhat in its behaviour in any given solution accord- ing to the time of year, there being a marked difference between muscle in the autumn and spring. ADVANCED EXPERIMENTAL PHYSIOLOGY 69 to a myograph lever and stimulate it with a single maximal break shock. The contraction recorded on the drum will be no longer an ordinary single contraction, but a series of tetanic twitches of abnormal height and duration. Now remove the muscle, immerse it for ten minutes in the solution containing the added calcium salt, and again record its response to the same stimulus. A normal single contraction will be obtained. It is clear that sodium salts, when acting alone on skeletal muscle, have a powerful stimulatory effect, and that this can be neutralised by adding a certain proportion of calcium salt. For this reason ' normal ' saline solution is always made with tap-water instead of with distilled water. Some tap-waters, however, do not contain nearly enough calcium to bring about complete neutralisation of the sodium salt. From the above experiments we learn certain facts of considerable practical importance. We see that tissues are greatly affected by changes in the osmotic pressure of the fluid surrounding them. Care must therefore be taken not to expose the tissues of an animal or man to fluids which are not isotonic with the blood-plasma. In man the solution of NaCl isotonic with the blood -plasma is only just under 1 per cent., and therefore differs widely in strength from the solution for a frog ; it is very necessary to bear this in mind when injecting fluid into veins or under the skin, and when irrigating the peritoneal cavity during operations. We further see that, when isotonic solutions of electrolytes are used, the tissues are by no means indifferent to the ions in solution. A really 'normal' saline solution would, therefore, be one which contained the same salts in the same proportion as the animal's own blood-plasma. Ringer's l fluid is an attempt to make such a solu- tion for the frog. Since in man it would often be difficult to obtain such a solution when wanted, it might be preferable, instead of using an imperfectly ' normal ' saline solution, to use an isotonic solution of a non-conductor, such as dextrose. A 5*8 per cent, solution of dextrose is isotonic with human blood-plasma. In all the above experiments it has been found that skeletal muscle responds to the abnormal constant stimulus by an activity which is not constant, but intermittent or rhythmical. This raises the question whether the rhythmical contraction of the heart may not be the normal response of that particular kind of muscle to the constant chemical stimulus of the blood-plasma, and the same might be also partly true of the rhythmical activity of the respiratory and vasomotor centres. 1 A modified Ringer's solution contains NaCl '7 per cent., CaCl, *0026 per cent., and KC1 "035 per cent. 70 PRACTICAL PHYSIOLOGY CHAPTER XVI. (Advanced). FATIGUE OF A VOLUNTARY MOVEMENT AND OF A MUSCLE- NERVE PREPARATION WITH ITS CIRCULATION INTACT. WHEN a voluntary movement is repeated sufficiently often fatigue is produced. The seat of this fatigue has to be investigated ; it might be in some part of a neurone in the central nervous system, or in some part of the peripheral nerve and muscle : in other words, the fatigue might be primarily central or peripheral. As the result of certain ergographic experiments it has been answered that this fatigue is of central origin. The experiments consisted in lifting a heavy weight suspended over a pulley by flexing a finger and registering the height of each successive lift. When the movement had been repeated until the muscle was no longer able to lift the weight at all, it was found that electrical stimulation of either the nerve supplying the muscle or of the muscle itself caused the weight to be again lifted, but to a less height than before. When the electrical stimulation had in turn fatigued the movement it was found that a voluntary contraction of the muscle was again able to lift the weight, owing, it was supposed, to the resting of the cells in the central nervous system. From these experiments it was argued that the fatigue of a voluntary movement is purely central. The methods used in the above experiments are open to grave objections, and it is necessary to touch upon some of these in order to avoid them. The use of a heavy weight is open to the objection that the muscle, when no longer able to lift that weight, is still capable of contracting, and could well lift a lighter weight ; therefore, it is better to make the muscle bend or pull on a spring, which will enable the feeblest as well as the strongest pull exerted by the muscle to be recorded. Again, electrical stimulation of a nerve or a muscle can be a much more powerful stimulus than that resulting from the maximal discharge of a motor nerve-cell ; consequently the fact that peripheral stimulation can make the muscle again lift the weight after voluntary impulses fail, is no proof that the fatigue was central. Further, when a nerve or muscle is stimulated by electrodes placed upon the skin, it is impossible to produce equal stimulation of all fibres ; some muscle-fibres will receive a maximal and others only a sub-maximal or minimal stimulus, and the pull of the muscle as a whole will be equivalent to that of a weaker muscle. When the muscle appears to be fatigued by peripheral stimulation, then a return to volitional stimulation, by pro- ADVANCED EXPERIMENTAL PHYSIOLOGY 71 ducing equal stimulation of every fibre, leads to an apparent recovery of voluntary power. In this way is to be explained the apparent paradox, that a muscle fatigued by either voluntary or peripheral stimulation shows a recovery of power when stimulated in the opposite way. In order to investigate this subject we shall compare the curve of voluntary fatigue taken with a spring ergograph from the human abductor indicis, with the curve obtained from the frog's gastrocnemius, with its circulation intact and stimulated through the sciatic nerve. The Spring Ergograph. — A simple form of this instrument is shown in Fig. 66 to consist of a rigid upright iron bar which is clamped to FIG. 66.— Spring ergograph. (i'orter.) the table. From the upper end of this projects a horizontal straight steel spring, the free end of which carries an ordinary writing point. The spring carries on its under side a short vertical steel arm, the lower end of which fits over the distal end of the second phalanx of the index finger. When the abductor indicis contracts the spring is pushed up ; by sliding the vertical arm along the spring the magnification of the movement and the strength of the spring can be altered. The hand is placed along the vertical side of the wooden support and the three outer fingers tied to it, leaving the thumb and index finger free. The forearm should be fixed to the bench in some form of support, but care must be taken not to tie down the arm sufficiently tightly to interfere with its circulation. The subject of the experiment should sit comfortably and with his eyes shut, should not be spoken to nor in any way have his attention diverted, but should confine himself to giving a maximal contraction of his muscle every time he hears the beat of a metronome, which is set to give a beat every second. The observer takes the time of the experiment in minutes and so calculates the number of contrac- tions recorded, further he has to see that the vertical arm does not slip 72 PKACTICAL PHYSIOLOGY out of position along the finger. In this way take 300 to 600 con- tractions on a drum revolving at an extremely low rate (Fig. 67). At first sight the most striking feature of the curve is the more or less rhythmical waxing and waning in the height of the contractions ; this seems to be purely central in origin and to be due to variations in the strength of the voluntary impulse communicated to the muscle. Practice to a large extent does away with this rhythm. When the height of the contraction is measured it will be found that the average height decreases during the first 180 contractions and then attains a fairly constant level, which represents about 85 per cent, of the height of the original contractions. The initial decrease is better marked in Fig. 68, and here the fatigue-level was only about 45 per cent, of the original height. The characteristics of an ergographic fatigue-curve, therefore, are an initial fall which takes place during a variable number of contractions, and the attainment of a fairly constant level, which represents varying percentages of the height of the original con- tractions. This curve strongly suggests that during a series of con- tractions two processes are at work ; one by which available combustible material is being used up and the products of katabolism are accumu- lating, and the other by which both these defects are made good by the eirculation. During the early part of the curve the first process preponderates over the second and the height of the contraction decreases, but as soon as the two processes exactly balance each other a uniform level is maintained for hundreds of contractions. The probable seat of these processes will be referred to after the next experiment has been performed. In order to obtain a record of the contractions of the gastrocnemius with its circulation intact, arrange the apparatus for stimulating the sciatic nerve with maximal induction shocks, using a simple key in the primary circuit. The cerebrum of the frog must be destroyed and the muscle-nerve preparation made without causing bleeding. The cerebral hemispheres are destroyed by compression, leaving the medulla and spinal cord intact, and the gastrocnemius is prepared in the usual way. A string ligature is placed beneath the gastrocnemius and tied tightly round the upper part of the tibio fibula and the remaining muscles; the leg is then cut through below the ligature. The whole frog is placed belly downwards on the myograph-board, a strong pin is pushed through the lower end of the femur and driven firmly into the cork. A piece of moistened flannel is then pinned down over the trunk to prevent the contractions of the muscles of the trunk from disturbing the lever connected with gastrocnemius. The skin over the middle of the thigh is divided longitudinally for a short distance, the muscles carefully ADVANCED EXPERIMENTAL PHYSIOLOGY 73 74 PRACTICAL PHYSIOLOGY separated and the sciatic nerve exposed ^nd freed ; the nerve is gently raised by slipping a thread beneath it and the electrodes, insulated ADVANCED EXPERIMENTAL PHYSIOLOGY 75 from the underlying muscles by a small piece of cork, are placed beneath the nerve. It is essential that the nerve should not be injured and 31 i! should be kept properly moistened throughout the experiment. The muscle is suitably weighted and just after-loaded. The nerve is stimulated by a maximal shock every 5 sees., and the contractions recorded on a drum revolving at the slowest possible rate (Figs. 69, 70). 76 PRACTICAL PHYSIOLOGY It will be seen that the height of the contractions, although increasing at first, gradually falls off until at the end of about 200 contractions it reaches a uniform level, which represents about 85 per cent, of the original height and was then maintained with scarcely any alteration for three-quarters of an hour. This curve, therefore, is identical in general form with that obtained by the ergograph. Here again we see an initial fall and then a constant level of contraction, representing probably the equilibrium between two opposite processes, which must in this case be affecting some part of the peripheral nerve and muscle. The actual seat of this peripheral change is not absolutely certain (see further Expts. in Chapter XXII. ). Now cut through the leg in the middle of the thigh, so as to destroy the circulation through the gastrocnemius and continue the stimulation (Fig. 71). It will be seen that the height of the contractions rapidly and continuously decreases, and that at the end of about 320 contrac- tions the muscle is no longer able to lift the lever off the after-loading screw. CHAPTER XVII. (Advanced). THE RATE OF TRANSMISSION OF A NERVOUS IMPULSE. THE rate at which an impulse is transmitted along a nerve is important because it throws some light upon the nature of the impulse. It travels much more slowly than an ordinary electric current, and, although it is accompanied by an electric change, it is something more complex. Its rate of propagation is 27 metres per second (88J feet per sec.) in the frog's sciatic nerve, and 60 metres per second (196 feet per sec.) in the motor nerves of man. (a) In the Motor Nerves of the Frog. — The following experiment should be performed for the determination of the velocity of the nervous impulse in the sciatic nerve of a frog : A recording drum is arranged with a ' striker ' for completing the circuit of the primary current of the induction-coil. To the secondary coil are attached two Du Bois keys in the manner shown in the diagram (Fig. 73) ; from these pass two pairs of electrodes, one of which will be applied to the upper portion of the nerve, the other to the lower ADVANCED EXPERIMENTAL PHYSIOLOGY 77 portion. The entire length of the sciatic nerve is dissected out, and the gastrocneraius muscle is connected with the lever of a myograph ; the drum is arranged for rapid revolution, and a maximal shock is to be used for excitation. The latency of the muscular contraction (Chapter III., p. 22) is then determined, first for stimulation by the upper pair of electrodes, the lower pair being short-circuited by closure of its Du Bois FIG. 73. — Diagram of the experiment on the rate of transmission of a nervous impulse. key ; then the experiment is made with the lower pair of electrodes for the exciting point. The time of this latency is determined by recording underneath the curves the vibrations of a tuning fork with 100 vibra- tions per second; the difference in time between the moment of stimulation and the resulting contraction in the two cases represents the time taken for the nervous impulse to pass along the length of nerve between the two pairs of electrodes (Fig. 73). This piece of nerve is measured in millimetres,1 and then the velocity of the transmission of the nervous impulse is calculated. For the accurate determination of the rate of propagation of a nervous impulse a very rapid rate of movement of the recording surface is required ; for this reason the spring-myograph (Fig. 63, p. 65) or the pendulum-myograph may be used with advantage in the place of the drum. (b) In the Motor Nerves of Man. — The velocity of the transmission of a nervous impulse in the motor nerves of man can be determined in the following way : A thick-walled india-rubber ball, similar to that used with a photographic 'shutter,' is connected with a recording tambour. Two clinical electrodes are moistened with strong saline solution in order to improve their conduction and contact with the skin; the large flat electrode is fastened to the leg of the subject, and the small electrode placed above the clavicle will be pressed over the brachiai nerves. These electrodes are connected with the secondary 1 There is a millimetre scale upon the slide of the induction-coiL 78 PEACTICAL PHYSIOLOGY coil of an inductorium, and in the primary circuit is interposed the 1 trigger ' key of the spring-myograph. The india-rubber ball is held between the middle finger and the thumb, and the contraction of the flexor muscles will be recorded by the lever of the tambour, when the nerve is excited. The moment of stimulation is determined in the usual way (p. 25), and then the experi- ment is again performed, but with the small electrode pressed over the median nerve at the bend of the elbow. The moment of stimulation is again determined, in order to show that the resting position of the point of the lever has not been changed. The difference between the latency in the two contractions is measured by a tuning-fork vibrating 100 times per second, and the length of nerve between the two points of stimulation is estimated ; from these data the rate of transmission of the nervous impulse can be calculated. CHAPTER XVIII. (Advanced). THE POLARISATION OF ELECTRODES AND UNPOLARISABLE ELECTRODES. Polarisation of Electrodes.— Ordinary metal electrodes in contact with a muscle or nerve will be surrounded by lymph, and in this fluid electrolysis will take place during the passage of an electric current. The ions resulting from this electrolysis will be positive and negative respectively; if, therefore, the circuit of this seat of chemical and electrical change be suddenly made or broken, a shock will be produced, for the wires of the electrodes surrounded by the electrolysed fluid will form a minute battery. This can be demonstrated by the following experiment : A pair of electrodes, connected with a Du Bois key, is placed under the sciatic nerve, which has been exposed in the thigh of a pithed frog. Making or breaking the circuit causes no contraction. The two wires of a Daniell battery are connected with each side of the Du Bois key, and the current is allowed to pass through the nerve for several seconds. Then these two wires are rapidly disconnected from the battery and key ; the key is closed and opened, and each time a contraction of the muscles of the leg is caused. This make and break can be repeated several times with a similar result, until the polarisation has disappeared. This experiment shows the necessity of unpolarisable electrodes in experiments upon the effects produced in nerve and muscle by the ADVANCED EXPERIMENTAL PHYSIOLOGY 79 passage of a constant electric current, and also the necessity of using a Du Bois key as a bridge to short-circuit the electrodes. Unpolarisable Electrodes. — The preceding experiment has shown that the electrolysis occurring around the ordinary metal electrodes may easily act as an exciting electric current, and thus cause errors in experiments. In order to avoid this unpolarisable electrodes are used. The electric current from the battery is conducted through media which are not liable to polarisation. The structure of Burdon-Sanderson's electrodes is shown in the following diagram (Fig. 74). A smooth amalgamated zinc rod dips into a saturated solution of zinc sulphate, which in turn conducts the current by means of a plug of kaolin or china clay, made into a thick paste with normal saline solu- tion ('75 per cent, sodium chloride). The plug rests upon a small glass tube with a flange; this delays the spread of the zinc sulphate into the kaolin. The nerve or muscle can be placed in contact with the plug of kaolin, or may be connected thereto by threads saturated with normal saline solution and kaolin. The plug must be kept moist with normal saline solution, for the electrodes have a high resistance. The electrodes must be set up with clean hands and material, other- wise polarisation will occur. The solution of zinc sulphate must not be allowed to touch the tissue, for chemical excitation would occur. Kaolin and normal saline solution do not stimulate muscle and nerve. The previous experiment on the polarisation of electrodes should be repeated with the unpolarisable electrodes. The result will be negative if the electrodes have been well and truly made. Fio. 74.— Unpolarisable electrode. Burdon- Sanderson's pattern. CHAPTER XIX. (Advanced). TRANSMISSION OF A NERVOUS IMPULSE IN BOTH DIRECTIONS. THE excitatory state produced by stimulation of a nerve can be transmitted in both directions. This can be shown by the following experiments. Sartorius Experiment. — The sartorius muscle is dissected out and its 80 PRACTICAL PHYSIOLOGY iliac end is divided into two portions (Fig. 75). Stimulation with a weak induction shock at (a) or (a'), when there are no nerve-fibres, will produce a contraction of the one half of the muscle. Excitation, how- ever, at (b) or (V), where there are nerves, will evoke a contraction of both halves. Gracilis Experiment. — The gracilis muscle of the frog is in two por- tions completely separated by a tendinous intersection (Figs. 21, 76). Both halves of the muscle are supplied by a single nerve, the individual FIG. 75. — Diagram of the sartorius experiment to show the transmission of a nervous impulse in both directions. Fio. 76. — Diagram of the gracilis experiment to show the transmission of a nervous impulse in both directions. fibres of which divide and supply both halves of the muscle. Stimula- tion of any kind at (a) or (a') where there are no nerve fibres causes only the corresponding half of the muscle to contract ; but excitation at (b) or (b')t where the nerves lie, will cause both halves to contract. CHAPTER XX. (Advanced). THE RELATION BETWEEN MUSCLE AND NERVE. THE INDEPENDENT EXCITABILITY OF MUSCLE. IN addition to the experiments which have been described in the elementary course (page 48), the following experiment upon the eartorius muscle should be performed. The sartorius muscle lies on the ventral surface of the thigh (Fig. 21), ADVANCED EXPERIMENTAL PHYSIOLOGY 81 and its outlines can be made distinct by sponging it with the frog's heart full of blood. The muscle is carefully dissected out and will contract when its nerve, which passes into the muscle at the middle of its inner border, is cut across by the scissors. If the muscle be placed between two glass- slides and examined under a microscope, the distribu- tion of its nerve can be seen to resemble that shown in the diagram (Fig. 77). The finer branches of the nerves and even the end plates can be more readily seen if the muscle be treated with acetic acid. There are no nerves in the terminal portions of this muscle, which consists of fibres running in a direction parallel with its length. The sartorius muscle is dissected from the other thigh and the nerveless parts are stimulated by a pinch with a pair of forceps or by an electrical shock ; they contract, the muscle possesses independent excitability. The absence of nerves from the terminal portions can also be shown in the following way. The muscle is suspended from its tibial end and is lowered until the cut iliac end touches some strong glycerine contained in a watch-glass ; it does not contract. A thin transverse slice is cut away and the muscle is again lowered into contact with the glycerine; there is still no contraction. This pro- cedure is repeated until the nerves are cut across and on contact with the glycerine are stimulated and make the muscle pass into a contracted condition. FIG. — Dia- gram of the sartorius muscle to show the distribution of its nerves. CHAPTER XXL (Advanced). THE EFFECT OF A CONSTANT ELECTRICAL CURRENT UPON THE EXCITABILITY AND CONDUCTIVITY OF NERVE. THE passage of a constant current produces changes in the excitability of nerve, at the anode there is a condition known as anelectrotonus, the excitability is diminished ; at the kathode there is an increase in excitability, a state of katelectrotonus. The conductivity is also affected, there is a fall in both the anodic and kathodic regions. These effects can be shown by the following experiment, F 82 PEACTICAL PHYSIOLOGY One Daniell battery is connected by two wires with a Pohl's reverser whereby the direction of the current can be changed ; from the reverser the wires pass by means of a Du Bois key to a pair of unpolarisable electrodes. This is the polarising circuit. The stimu- lating circuit is set up separately for the production of single induction- shocks (Fig. 78). A preparation of the sciatic nerve and gastrocnemius muscle is carefully made from a recently pithed frog, and is placed in a moist chamber ; a pin is fixed through the lower extremity of the femur, and the tendo Achillis is connected by a thread with a lever. FIG. 78. — Diagram of the experiment on the effects of a constant electrical current upon the excitability and conductivity of nerve. The sciatic nerve is placed across the kaolin plugs of the unpolarisable electrodes. The drum can be moved by hand. A minimal stimulus for the nerve is obtained, care being taken to use only the break or make-shock. The minimal contraction is recorded on the stationary drum. The current from the polarising circuit is closed in an ascending direction, so that the current enters the nerve on the side near the muscle and immediately above the stimulating electrodes, which are connected with the inductorium. The nerve around the point of entry or anode of the polarising current is depressed in its excitability, and the application of a minimal, or even stronger, stimulus is no longer effective (Fig. 79). The polarising current is short-circuited by the Du Bois key, and by means of the reverser is changed in its direction, so that on opening the Du Bois key the current is descending, and the area of nerve near the stimulating electrodes passes into a condition of ADVANCED EXPEEIMENTAL PHYSIOLOGY 83 katelectrotonns. The minimal stimuli now become maximal, owing to the increase in the excitability of the nerve ut the kathode. II III = 4 gg1| fi.sgglai illl Hill The above experiments can be repeated with a crystal of common salt placed in the position of the stimulating electrodes. The salt causes chemical excitation, and the muscle shows incomplete tetanus, which is quelled by anelectrotonus, and augmented by katelectrotonus (Figs. 81, 82). 84 PKACTICAL PHYSIOLOGY !£! ADVANCED EXPERIMENTAL PHYSIOLOGY 85 Hi •^ ~A ^3 o rt C3 a) ^ ^rrf^i i;ii^sii Ills o EH BiSa PHYSIOLOGY heart comes in contact with the chest wall in each posture. Apply the button of the cardiograph to the seat of the impulse, and fix it with tapes. One tape is fastened round the chest and one over the right Flo. 118.— Impulse curve of boy aged 15. Tlie moments when the heart sounds were heard are marked. Time marked in fifths of a second. (L.H.) shoulder. Connect the cardiograph by means of a J_ ^UDe w^^ a recording tambour, and take records on a moderately fast drum. The J_ tube is used to regulate the pressure in the tambour. A small metal Fio. 119. — Form of impulse curve changed by altering the position of cardiograph. In 3 the chest wall is sucked in during the systolic output. Time marked in fifths of a second. (L.H.) box — the size of a penny and open at the bottom — does very well, if connected to a tambour and pressed over the region of the apex. To interpret the curve, adjust another receiving tambour to the carotid pulse and arrange the writing styles of the receiving tambours to write ELEMENTARY EXPERIMENTAL PHYSIOLOGY 121 in the same perpendicular. Set up a signal, spring key, and battery, in circuit. Listen to the heart sounds and try to mark the first and second sounds beneath the cardiogram (Fig. 118). The signal must write exactly under the writing style of the tambour. The reaction time of a trained observer for making signals in answer to sounds is 0'15 to 0-20 seconds. The curve is only typical when the button of the instrument is exactly applied to the seat of the impulse. Elsewhere the thorax is drawn in, as blood is expelled from the thorax during the period of systolic outflow (Fig. 119). The impulse of the heart occurs where the ventricular wall touches the chest. It is produced by the sudden hardening of the ventricular muscle. During the first part of systole — the period of rising tension — the blood cannot escape from the ventricles. The second tambour can then be placed over the jugular vein and a venous pulse recorded. This will show the time of the auricular con- traction. (See p. 122.) CHAPTER XXXII. THE PULSE. HUMAN BLOOD PRESSURE. Pulse. — Examine the radial pulse with the finger. Note (1) the size of the swelling, composed of the artery and its venae comites, which occupies the radial sulcus ; (2) the tension of the artery, which is estimated by the pressure required to obliterate the artery and stop the pulse ; (3) the condition of the arterial wall, which can be ascer- tained by rolling the vessel upon the bone ; (4) the character of the pulse wave — its frequency, regularity, amplitude, and period of duration. Note also whether the chief secondary or dicrotic wave is perceptible. Compress the brachial artery, and notice that the radial pulse ceases. Compress the upper arm, excluding the brachial artery, and note the effect on the veins and pulse. The pulse may be recorded by a sphygmograph. The principle of this instrument is a button resting on the artery and pressing against a steel spring. The spring in its turn is made to press either against a lever (Fig. 121) or a tambour. The lever is provided with a writing style, while if the tambour be used it is connected with a recording tambour. The Dudgeon sphygmograph 122 PRACTICAL PHYSIOLOGY Flo. 120.— Mackenzie's Polygraph. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 123 is convenient, but Mackenzie's polygraph allows long continuous tracings to be taken in ink on white paper, and is a far more valuable clinical instrument. Apply the sphygmograph to the radial artery, as in Fig. 121. The right position of the button may be found by marking Fio. 121.— Dudgeon's sphygmograph. the position of the pulse with ink. The pressure of the instrument can be varied both by the straps and by the dial which regulates the pressure of the spring. The instrument should be applied with a pressure sufficient to flatten the artery, and then the pressure should Fio. 122.— Marey's sphygmograph. be diminished until the maximal excursion is obtained. We have no means of accurately reading the pressure of the spring or the changes of pressure indicated by the pulse curve. The instrument gives us the form of the pulse curve only. When the smoked paper is in position, and the writing style placed upon it, and the maximum excursion obtained, the clock is started and the record taken. The pulse curve consists of a primary and several secondary waves. The primary wave is the wave of expansion produced by the systolic output of the heart 124 PBACTICAL PHYSIOLOGY and travels down the elastic arteries at the rate of about 5-8 metres a second. The secondary waves are produced by the elastic vibra- tions of the wall of the large arteries which result from their sudden distension. The first secondary, or predicrotic wave is probably pro- duced by the final contraction of the base of the heart, which wrings the blood out of the ventricle. The second or dicrotic wave follows the dicrotic notch. The dicrotic notch is synchronous with the tension of the closed semilunar valves and the second sound of the heart. The dicrotic notch is caused by the back swing of the blood towards the closed aorta valves, and the wave by the rebound. The size of the dicrotic wave depends on its relation to that phase which the primary wave happens to be in, as well as on the con- ditions which increase the back swing and rebound. A forcible beat and an arterial system which can quickly empty itself favour dicrotism. The sphygmograph fixed by a band round the wrist may act like a plethys- mograph, and be affected by changes of volume in the limb. To avoid this, the instrument is suspended, and thus applied to the artery without use of the band. Take another pulse tracing, using this device, and forcibly inspire and expire during the record. A deep intercostal respiration, if not prolonged, yields a fall of pressure, and, conversely, a deep diaphragmatic inspiration yields a rise. But the effect of respiration is very complex, and it is difficult to say what the effect of normal respiration in any individual case will be. The ordinary statement that inspiration raises and expiration lowers blood pressure is not borne out by the records. (T. Lewis.) The pressure falls during forced breathing. The fall occurs with inspiration, and is caused by the violent contraction of the diaphragm obstructing the vena cava inferior. In Valsalva's experiment, a deep expiration with mouth and nose shut, the abdominal and arterial pressure rise. A stiff walled rubber tube inserted into the rectum and connected with a manometer indicates the big changes of abdominal pressure thus produced, e.g. + 20- + 90 mm. Hg. Blood Pressure in Man. — The pressure may be measured by the Leonard Hill sphyginometer. This consists of a graduated glass tube, FGI. 123. — Arrangement of levers in Dudgeon's sphygmograph. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 125 Fio. 124.— Sphygmograph provided with time writer (Jacquet.) Flu. 1-25.— Pulse tracing (sphygmogram) taken by Jacquet s sphygmograph. ad = the period of the pulse curve, 6 = the primary, c=the dicrotic wave. Time marked in fifths'of a second. 126 PRACTICAL PHYSIOLOGY which has a small air-chamber above. Near the open end is a side hole- the zero of the scale. On placing the open end in water, a meniscus rises to the side hole. A short length of rubber tubing, attached to a rubber bag, is slipped over the open end so as to cover the side hole. The bag is half distended with air, and is enclosed in a silk cover. The bag being enclosed by the palm of the hand, and the gauge held erect between FIG. 126.— Effect of abdominal and chest breathing. (Lewis. ) the fingers, the bag is pressed down upon the radial artery of the subject, the thumb exerting counter pressure against the back of the wrist. The index finger of the other hand feels when the pulse is obliterated, while the second finger prevents, if necessary, the pulse getting through from the anastomoses with the ulnar artery. The water used for the meniscus is made alkaline with potash to avoid the effect of grease in the tube. If the meniscus does not rise to the side hole, blow through this and try again. The air-chamber acts as a spring, and the instrument is a spring manometer. The meniscus is set before each reading, so as to avoid errors due to alterations of temperature and barometric pressure. The ELEMENTARY EXPERIMENTAL PHYSIOLOGY 127 instrument is graduated empirically. While taking the reading the hand of the subject must be placed on the same level as the heart, so as to avoid the influence of gravity. Measurement of Systolic Pressure with Hill-Barnard Armlet and Mercurial Manometer. — Strap the armlet lightly around the middle of the upper arm. Palpate the radial artery. By working the pump, raise the pressure in the cuff until the radial pulse is imper- ceptible. Now manipulate the exhaust stop cock so that the pressure in the cuff very gradually falls, and observe carefully the height of the mercury in the manometer. Whenever the radial pulse reappears, note the pressure. Continue the decompression (best by slightly lowering it a few mm. at a time), and note the pulsations of the J.d. HICKS SOLE MAKER LONDON. PATENT. Fi«i. 127. — Leonard Hill sphygmometer. mercury, which became evident at the systolic pressure, get gradually of greater amplitude until, by further lowering of pressure, they become smaller again. The pressure at which the pulsations are of maximal amplitude is the diastolic pressure. The difference between the two pressures is the pressure pulse. Repeat the above observations until constant results are obtained. Then let the observed person run up and down stairs several times, and repeat the observations. Try the effect of putting the hand in very hot water, and palpate the artery at the elbow and wrist. The pulse will disappear earlier at the wrist. A contracted artery conducts the crest of the systolic wave better than a softened artery. The effect of the hot water is more evident after exertion, when the pulse waves are larger. The systolic arterial pressure is 100-110 mm. Hg in healthy young men. It may fall during sleep 10-20 mm. Hg, and rises to 130-140 and to even 200 mm. Hg during mental excitement or severe effort. The arterial pressure is as constant as the body temperature from day to day. In the horizontal posture the arterial pressure will be found to be the same in all the big arteries. In the erect posture the pressure is 128 PEACTICAL PHYSIOLOGY higher in the post-tibial than in the brachial by the height of the column of blood which separates the two arteries. The effect of gravity is compensated. The reverse is the effect in states of debility, and the pulse frequency is then greatly accelerated in the vertical posture. The venous pressure may be obtained by placing the armlet round the fore-arm, raising the pressure in it, emptying the blood out of a vein FIG. 128. — Spring sphygmometer. The leather armlet encloses a rubber bag. The bicycle pump is used to raise the pressure. The spring manometer indicates the maximal pulsation and the pressure. above the armlet by digital pressure, and then diminishing the pressure until the vein suddenly fills. Note the pressure when this happens. Place the other armlet round the upper arm and raise the pressure in it nearly to the systolic pressure, keep it at that, and observe that the venous pressure rises after a minute or so to this pressure. Observe the effect on the veins of holding the arm in the dependent posture and motionless, of contracting the muscles, of raising the arm, etc. CHAPTER XXXIII. BLOOD. THE HAEMOGLOBINOMETER AND THE HAEMACYTOMETER. Gowers-Haldane Haemoglobinometer. — The maximal error of this admirable instrument is not more than 0'8 per cent. The standard solution in tube D is a 1 per cent, solution of ox blood saturated with coal gas.1 The oxygen capacity of the ox blood from which the standard was prepared was 18*5 per cent. This was determined by 1 Coal gas contains carbon monoxide as an impurity. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 129 displacing the oxygen from laked ox blood with ferri-cyanide of potassium, and measuring the amount of gas. The percentage of haemoglobin corresponding to 18'5 per cent, is about 13*8 per cent. The normal human blood when saturated with CO and diluted with water to the mark 100 in tube C corresponds in tint to the standard, and has therefore an oxygen capacity of 18'5 per cent. Add distilled water to tube C up to the mark 20. Take exactly 20 c.mm. of blood in the pipette, and blow it into C. Pass a narrow glass tube connected with a gas burner into the free part of tube C. Turn the gas on and push the glass tube down near to the blood. The gas tube is then withdrawn, and tube C quickly closed with the finger to FIG. 129. — Gowcr's haemoglobinometer. prevent the gas escaping. The tube is then inclined up and down about a dozen times, so that the haemoglobin becomes saturated with CO. Distilled water is then added drop by drop from the dropping pipette A, until the tint appears equal to the standard. After half a minute read the percentage, and then add another drop or drops till the tints appear just unequal. Read the percentage again, and take the mean of the two readings as correct. In comparing the tints hold the tubes against the skylight, and frequently change the tubes from side to side. Many other forms of haemoglobinometer have been con- trived, but in comparison with this instrument none of them are worth notice. The number of Corpuscles in the Blood.— The Thoma-Zeiss Haema- cytometer consists of a counting chamber «and an accurately calibrated pipette. I 130 PRACTICAL PHYSIOLOGY The finger behind the nail is cleaned with alcohol and ether, and a drop of blood is drawn by the stab of a lancet-shaped needle. The finger should not be constricted by a ligature during this operation. The point of the pipette is placed in the drop, and the blood is aspirated as far as the mark 1. The traces of blood on the point of the pipette are then removed, and the pipette is dipped into Hay em's fluid.1 This fluid is sucked up until the diluted blood reaches the mark 101. The tip of the mouth-piece is then closed by the finger, and the pipette shaken. The glass bead in E mixes the blood and Hay em's fluid. The bulb contains 1 part blood and 99 Hay em's fluid. 1 f> FIG. 130.— The Thoma-Zeiss haemaoytometer. Now blow gently into the mouth-piece, reject the first few drops, and then place a drop upon the centre of the counting chamber. The cover-slip is then placed in position, and the counting chamber is placed on the stage of the microscope, and left at rest for a few minutes. When the corpuscles have subsided, count the number in 10 squares, and take the average. Count those corpuscles which happen to lie on the lines on two sides of each square only. Each square covers an area of T^ sq. mm., and has a volume of Y^-Q c.mm., therefore 1 c.mm. contains 4000 times the average number found in a square. The dilution of the blood was I'lOO. Thus the number in a square x 4000 x 100 = number of corpuscles in 1 c.mm. of blood. In counting the white corpuscles it is best to dilute the blood with 1 per cent, acetic acid. This destroys the red corpuscles and brings the white clearly into view. By comparing the number of the red 1 Sodium chloride, g. 2; sodium sulphate, g.10; corrosive sublimate, g. 1; water, g. 400. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 131 corpuscles in a square with the percentage of the haemoglobin, the worth of the corpuscle in haemoglobin is obtained. = ' worth ' of corpuscles. Mo. in sq. The average number of red corpuscles is 5,000,000 per 1 c.mm. ; of white, 10,000 per 1 c.mm.1 Specific Gravity of the Blood. — A number of test tubes are taken and filled with mixtures of glycerine and water, which vary in specific gravity from 1030 to 1075. A pipette is taken with the point bent at a right angle. The skin is pricked behind the finger nail, and a drop of blood is drawn into the pipette. The blood is blown in small droplets into the middle of the solution in several of the test tubes until the solution is found in which the blood neither sinks nor rises. The specific gravity of this solution is determined with the hydrometer. The behaviour of the droplet must be noted at the moment when it enters the solution. The blood quickly alters owing to osmotic change. The specific gravity of the blood is about 1060, of the plasma 1026-29. The specific gravity of fragments of muscle or other tissues may be determined in the same way. The method is thus employed to deter- mine the amount of tissue-lymph in the organs. CHAPTER XXXIV. CIRCULATION OF THE BLOOD (ELEMENTARY DEMONSTRATIONS). Proofs of the Circulation of the Blood. — A mammal is anaesthetised with ether and chloroform. The external jugular vein is exposed and the carotid artery. A clip is placed on the jugular vein. Note the central end of the vein empties, while the peripheral end becomes enlarged. A clip is next placed on the carotid artery, the central end becomes distended and pulsates, while the peripheral end shrinks and ceases to pulsate. The clips are now removed and two ligatures placed in position (but not tied) under each vessel. The vein is pricked, Note the dark blood which flows out from the peripheral end steadily and without force. The vein is then tied above and below the opening. The artery is next pricked. Note the blood spurts out forcibly and in jets from the central end. The artery is then tied above and below the opening. 1 After using, clean the pipettes of these instruments. Suck water, alcohol, and ether up them in turn, and let the liquids run out. Never blow down the pipettes. 132 PRACTICAL PHYSIOLOGY A tracheal cammla is placed in the trachea and connected with the artificial respiration apparatus. The sternum is divided in the mid-line, and the thorax opened, so as to expose the heart. The pericardium is slit open. Observe the systole and diastole of the FIG. 131.— Simple model to show the effect of gravity on the circulation. auricular appendices arid ventricles. Ligatures are now passed under the superior and inferior venae cavae and tightened. The heart quickly empties. On loosening the ligatures observe the immediate filling of the right heart. A ligature is next passed under the aorta and tightened. Observe the engorgement, firstly, of the left, and then the right heart. On loosening the ligature note the effect. A ligature is next passed under the pulmonary artery and tightened. The right heart becomes engorged while the left empties. On loosening the ligature note the result The heart is now excised, the right ventricle quickly opened. The papillary muscles may be observed contracting synchronously with the ventricular wall. FIG. 132.— Schema to show the flow in rigid and elastic tubes. (Marey.) The Flow in Rigid and Elastic Tubes. — Arrange an experiment as shown in figure 132. The two tubes are 1 metre long and of the same bore, but one is a rigid tube and the other elastic. A toy-rubber balloon ELEMENTARY EXPERIMENTAL PHYSIOLOGY 133 inserted in the course of a glass tube will act better than a rubber tube. The middle of a piece of glass tube is drawn out into capillary size and divided, so that outflow orifices are obtained of the same size. Rhythmically open and shut the compressor. The flow from the rigid tube is intermittent, while from the elastic tube it is continuous. The latter delivers more fluid in one minute than the former. Observe that the outflow from the elastic tube becomes intermittent when the outflow orifice is enlarged. The increased and continuous flow from the elastic tube is due to the potential energy stored up in the stretched wall of the tube, which maintains the flow during diastole. The Artificial Schema.— The two ends of a Higginson syringe B are connected with a soft rubber tube about \ inch in diameter and a yard long. The tube divides into two channels ; ( 1 ) a short length of wide glass tube (a lamp chimney with a cork will do) filled with shot repre- senting the capillaries, (2) a rubber tube closed by a screw-clip. The screw-clip represents the muscular wall of the arterioles. These are connected with the inner tube of a bicycle tyre, which represents the capacious venous system. A mercury manometer is connected by a J_ tube with the artery and another with the vein. A loose cotton wad plug is placed in the open end of each manometer to prevent the mercury being accidentally forced out. The system is filled with water, and air removed by tilting the board to which the schema is fixed and working the pump; the air is allowed to escape through a side tube at the top of the schema. The schema is filled only so far that the vein is not distended and there is no positive pressure when the pump is at rest, so as to represent the conditions in the dead body. The bulb of the syringe may be worked by hand. The valves act as the mitral and aortic valves. When the screw clip is widely open, there is little resistance to flow. The outflow from the artery into the vein ceases during the diastole of the syringe. The conditions are the same as if the artery were a rigid tube. The diastolic and systolic variations of pressure are very great, and affect both manometers to a like extent. Screw up the clip. The flow, as the resistance increases, becomes less and less intermittent and finally continuous. The mean pressure rises in the arterial manometer. The systolic and diastolic variations of pressure become greatly reduced. The systolic variation disappears in the venous manometer. When the vascular system is formed of a wide tube free from constrictions, each systolic pulse-wave travels with so great a velocity that the whole system reaches the same pressure before the next systole of the heart occurs. The conditions are otherwise when the clip is screwed up, for the friction of the blood flowing through the narrow channels prevents the blood from passing 134 PBACTICAL PHYSIOLOGY with anything like the velocity of the pulse-wave. In the vascular system the pulse-wave travels in the arteries 8 metres per second, while the blood travels J-metre. The resistance to flow is chiefly situated in the arterioles, where the velocity is high. It is due to the friction of the moving concentric layers of blood against one another, and against the stationary layer .which wets the walls of the blood vessels. It is proportional to the surface area, to the viscosity of the blood — nearly proportional to the square To Manometer To Manometer =1= To Mmometer FIG. 133.— Artificial schema of the circulation. of the velocity of flow, and inversely proportional to the sectional area of the vessel. In the arterioles the velocity is high, the total wall surface wet by the blood great, the sectional area of each arteriole very small. In the schema the resistance is increased by diminishing the sectional area of the arterioles and increasing the velocity of flow. Owing to the resistance to the outflow the arteries are expanded by each systolic output, and the elasticity of their walls comes into play, causing the outflow to continue during the succeeding diastole of the heart. The larger part of the kinetic energy of the systolic outflow is stored up as potential energy by the stretched arteries, and converted into kinetic energy during diastole. Stop the pump, the pressures in the manometers fall to the same level. Start the pump again. The fluid is taken from the vein and ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 135 piled up in the artery, for at each systole a greater quantity of blood is driven into the artery than can escape through the capillaries. With each succeeding systole, therefore, the pressure in the artery rises, and the pressure in the vein falls. Venous pressure cannot sink below the atmospheric pressure, for the flaccid walls of the veins collapse. The venous side is capacious, and possesses little elasticity. Thus the changes of pressure in the venae cavae, when the heart is arrested or starts beating, are insignificant. A slight positive pressure is maintained in the veins by the action of the muscles, which, at every movement of Fio. 134. — Another model of artificial schema of circulation. the body, forces the blood on past the venous valves and overcomes the effect of gravity. Raise the end of the board to which the pump (heart) is attached. The water under the influence of gravity distends the lower part of the vein, the upper part empties, and the circulation is impossible. Compress the lower part of the vein with your hands and the circulation is restored. This shows the effect of relaxation of the muscles in fainting and the method of restoring the subject by compressing the abdomen. The continuous flow of blood established through the capillaries is due to the difference between the pressure in the arteries and veins. This difference depends: on (1) the energy of the heart, (2) the elasticity of the arteries, (3) the peripheral resistance. The energy of the heart is spent in overcoming the resistance, and is dissipated into heat. Vary (1) by lessening the rate of the pump; vary (2) by opening the screw-clip— the difference in pressure diminishes in either case, and the flow becomes intermittent. When the screw-clip is open a 136 PRACTICAL PHYSIOLOGY very frequent heat of the pump is required to make the flow con tinuous, and scarcely any fluid passes through the capillary tube. By means of the vaso-motor nerves the arterioles are similarly dilated or constricted, and the current switched on to or off an organ, according to its functional activity. Velocity of Flow.— (1) Insert the Ludwig stromiihr (Fig. 135) into the artery. It is convenient to fill one side with water, and leave the other full of air. In actual practice one tube is filled with defibrinated blood and the other with oil. Set the pump going, and find the number of times the stromiihr must be turned per minute. Turn rapidly the moment the water reaches the mark A^. Each turn means the flow of the quantity of water con- tained in one half of the stromiihr. Measure the capacity of the stromiihr and the diameter of the artery. The capacity of half the stromiihr multiplied by the number of revolutions gives the volume, and this divided by the time and the sectional area of the artery gives the mean velo- city per second. The sectional area of the artery equals the radius x 3*1 4. Note the effect on the velocity of (1) opening the clip on the rubber tube, (2) of increasing the frequency of the pump. If the energy of the heart is constant, then in proportion as the peripheral resistance increases so the lateral pressure rises and the velocity in the aorta lessens. On the other hand, as the peripheral resistance decreases the pressure falls and the velocity increases. If the peripheral resistance be constant, then as the energy of the heart increases or decreases both the pressure and the velocity in the aorta together become greater or less. By compensatory changes taking place in the heart and the resistance, the velocity may remain constant while the pressure varies, or the pressure may remain constant while the velocity varies. The average velocity at any part of the vascular system is inversely FIG. 135.— The stromUhr. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 137 proportional to the total cross section at that part. If the total cross section of any one part of the circuit be dilated the velocity becomes slower there, while it proportionately increases in the other parts. This must be so if the blood continues to circulate round the whole system in the same time. Vaso-dilatation in one part is normally compensated for by constriction in other parts. Velocity in the Capillaries.— Pith the cerebrum of a frog and plug the hole. Lightly curarise the frog, and spread the web over the hole in the web-board. Examine the circulation under the microscope, and with the aid of an ocular micrometer and a clock beating i seconds measure the time it takes for a red corpuscle to move through ^ mm. Note in an arteriole that the red corpuscles move the fastest in the axial stream, while the white corpuscles roll slowly along the margin. Place on the web a drop of hot water (50°-60° C.). The flow at first is accelerated owing to vaso-dilatation, but soon slackens as the red corpuscles clump together owing to the escape of the plasma through the damaged capillary walls. The Influence of Gravity on the Circulation of the Snake. — Pith the brain of a grass snake or eel. (This experiment can be carried out on the frog, but is less striking.) Fasten the animal on to a board. Expose the heart, which may be seen beating beneath the skin, about 2-3 inches below the mouth. Place the animal head down in the vertical position. Notice the pericardium prevents the over-distension of the heart by the weight of the super-incumbent column of blood. Slit open the pericardium and observe the result. The heart becomes greatly congested. This is especially marked in the eel, when reflexly excited to writhe. Turn the animal head uppermost. The heart gradually empties, and becomes at last pale and bloodless. Slowly tilt the board and observe the blood as it runs up the inferior vena cava and fills the heart Place the animal again in the vertical posture (head up), arid observe that the heart fills (a) on compressing the abdomen from below upwards (b) on sinking the animal in a bath of water up to the level of the heart. In (b) the weight of the water outside tends to balance the weight of the blood within. The vagus nerve may easily be found at the side of the neck in the snake, and the effect of its excitation noted. In the eel reflex inhibition of the heart is very easily brought about by striking the abdomen or gills. Demonstration of Vaso-Motor Nerves.— A white rabbit is chosen, or one with a white ear; the brain of the animal is pithed and 138 PRACTICAL PHYSIOLOGY artificial respiration established at once; a tube is put into trachea and connected with a small hand bellows; the ear is shaven and fixed by threads to a loop of stout wire. This wire is clamped in front of the lantern, so that the blood vessels in the ear can be plainly seen. The cervical sympathetic is exposed in the neck, where it lies behind the carotid artery, and is traced up to the superior cervical sympathetic ganglion. The thread is tied round the nerve, and the latter is cut. Observe that at this moment the blood vessels in the ear dilate and the ear becomes warmer. The palpebral fissure at the same time becomes narrowed. The change will be much more marked had the ear of the rabbit been previously exposed to cold. The cervical sympathetic exercises a tonic action. On exciting the peripheral end of the nerve with the faradic current, the vessels in the ear will be seen to constrict, and this will take place to such a degree that all the smaller vessels will disappear from view." The ear will at the same time become several degrees cooler. Note that the latent time is considerable between the excitation and the effect. Note that the pupil also dilates, the nictitating membrane retracts, and the palpebral tissue is widened. The eyeball at the same time projects forwards. The pupillo dilator fibres arise from the first three thoracic anterior roots, the vaso- constrictor fibres from the second to the fifth, and even to the seventh, in the rabbit. If the superior cervical sympathetic ganglion be painted with nicotine, excitation of the preganglionic fibres will no longer have any effect on the ear, while excitation of the post ganglionic fibres will still be effectual. The sympathetic fibres to the head have their cell-stations in this ganglion. The Circulation Time of the lesser Circulation. — The carotid artery is exposed. A piece of thin rubber membrane is placed beneath it. Between the membrane and the artery a piece of white paper is inserted. The artery is illuminated by a strong light. The external jugular vein is exposed on the other side of the neck, a clip is placed on the vein below and it is tied above, and into its central end a cannula is inserted. The vein cannula is connected with a glass syringe containing a 0*2 per cent, solution of methylene blue in physiological saline at body temperature. Put a screw clip on the piston so that one-third of the contents shall be ejected. The clip is removed from the vein and at a signal from the assistant who times the experiment the syringe is pressed. The stop-watch is stopped by the assistant the moment the blue appears in the artery. The observation is repeated several times with the same amount of injection. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 139 Demonstration of Arterial and Venous Pressure by the Method of Stephen Hales.— An incision is made in the mid-line of the neck, from the larynx to the sternum of the anaesthetised cat. The skin-flaps are pulled apart, and the sterno-mastoid and sterno-thyroid muscles separated, so as to expose the carotid artery. With an aneurism needle the artery is freed from the carotid sheath for the space of about an inch. Two ligatures are then placed beneath the artery, and one is tied at the upper end of the exposed portion. On the lower end an artery clip is placed. With sharp scissors an oblique cut is next made into the artery, and the nozzle of the arterial cannula is inserted, and tied in with the second ligature. Lastly the ends of this ligature are brought round the bulb of the cannula, and tied to make the connection secure. The arterial cannula is J_ shaped and provided with a bulbous enlargement. This shape is chosen both to hinder clotting and to FIG. 136. — Arterial cannula. facilitate the washing out of clots. One limb of the JL is fitted with a short piece of rubber tube, and this is closed by a piece of glass rod or a clip. The other limb is connected by a short length of thick rubber tube (pressure tubing) to a long length of fine bored glass tubing. The latter must be at least 5 feet in length, and is held in the vertical position by a clamp. The glass tube and cannula are filled with 1 per cent, sodium citrate, and this decalcifies the blood and so prevents clotting. The solution is coloured with methylene blue, and a long strip of white paper scaled in centimetres is placed behind the tube. By cutting through the attachment of the sterno-mastoid muscle, the junction of the jugular with the subclavian veins is next exposed. The innominate vein is picked up and cleaned with the aneurism needle. Two ligatures are placed under it, and a clip on the part nearest the 140 PRACTICAL PHYSIOLOGY heart. One of the ligatures is tied round the junction of the jugular and subclavian veins. As the vein is clipped before the ligature is tied, it becomes distended with blood, and this facilitates the introduction of the cannula. The straight vein cannula is connected with a short length (1 foot) of glass tubing. The latter is clamped in the vertical position, and is filled with sodium citrate solution. The cannula is also filled with sodium citrate solution, and to retain the solution a clip is placed on the rubber tube, which connects the cannula with the glass tube. The positive pressure in the glass tube must not be more than 3 to 4 inches of the solution. The innominate vein is now slit and the cannula introduced. Then the clip on the vein is removed, and the cannula is pushed down into the superior vena cava. The clip on the rubber tube is next opened so as to place the venous cannula in connection with the vertical tube. The fluid in this will now oscillate with each respiration at a level of about 2-3 inches. The clip on the artery is next opened. The fluid in the arterial tube will oscillate at a height of about 4 to 5 feet. Notice in each tube the cardiac pulsations and respiratory oscillations. The arterial pressure rises in inspiration — the venous in expiration. 1 . If the abdomen be compressed the pressures will rise in both the artery and vena, but to a greater extent in the former. The heart is better filled in diastole and the peripheral resistance is increased by the compression of the splanchnic vessels. 2. If the thorax be squeezed so as to compress the heart and prevent its filling, the arterial pressure will fall very greatly, while the venous pressure will rise slightly. Record of Arterial Pressure, Effect of Excitation of the Vagus and Depressor Nerves. Effect of Gravity. Effect of Asphyxia.— The artery is now clipped, the cannula washed out, and is connected to a mercurial manometer by a piece of pressure tubing, a J_ piece being interposed. The cannula and tube are filled by means of a pressure bottle or syringe with sodium citrate 1 per cent, solution, and the pressure in the manometer is raised to about the arterial pressure. The vagus nerve is exposed, ligatured in two places, and divided between the ligatures. The depressor nerve is exposed, ligatured, and divided below the ligature. The depressor in the cat runs separately from the vagus on the left side. On the right side it can generally be separated from the rest of the vagus without much difficulty. In the rabbit the depressor runs separately on both sides. In the dog it is bound up in the vago-sympathetic trunk. The trachea is opened and a tracheal cannula inserted. This is con- nected with the anaesthetic bottle and by a side tube with a recording ELEMENTARY EXPERIMENTAL PHYSIOLOGY 141 tambour. The writing styles of the manometer float and of the tambour are brought to write on the kymograph exactly beneath one another. R' -T -F FIG. 137. — Arrangement of cannula, pressure bottle, and mercurial manometer for recording blood pressure. C, cannula ; p, p', clips ; F, float ; S, writing style. A clock marking seconds and an electric signal placed in the primary circuit are also brought to write on the kymograph. The primary circuit is arranged to give tetanising shocks, and shielded electrodes FIG. 138.— Electrodes for exciting vagus and other nerves. (Sbcrriugton). are connected with the secondary coil by means of a Du Bois key, and are placed in position under the peripheral end of the vagus nerve. The clip is then removed from the carotid artery and the kymograph started. Note the height of the arterial pressure, the cardiac pulsations, 142 PRACTICAL PHYSIOLOGY and the respiratory oscillations of arterial pressure. The pulsations are distorted by the momentum of the mercury. The inspiratory fall of intra- thoracic pressure aspirates blood into the intra-thoracic veins and thin walled auricles, and dilates the FIG. 139. — Mercurial manometer fitted with float and writing style. pulmonary vessels. The descent of the diaphragm expresses blood from the liver and abdominal vessels into the right heart in the living animal. Thoracic and abdominal breathing have a contrary effect. Thoracic breathing produces an inspiratory fall of arterial pressure, arid abdominal an inspiratory rise. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 143 ^^ Stimulate the peripheral end of the vagus nerve. The heart is inhibited, and the arterial pressure falls. Complete arrest cannot be obtained in the cat. It is easily obtained in the dog. In the chloro- formed dog with low blood -pressure, vagus excitation, produced by inhalation of concentrated chloroform vapour, may arrest the heart FIG. 140.— The kymograph. for so long a period as to kill the animal. This is one cause of chloro- form syncope in man. The heart soon escapes from vagus arrest if the blood pressure is high. The pressure (after vagus inhibition) for a brief space of time rises to a higher level. The electrodes are now transferred to the central end of the vagus. Excitation produces either a slight rise (pressor effect) or a slight fall (depressor effect) of pressure. The heart rate is reflexedly slowed, and the respiration is stopped with the diaphragm in inspiratory spasm. The electrodes are next transferred to the central end of the 144 PRACTICAL PHYSIOLOGY depressor nerve. On excitation the blood-pressure slowly falls, and remains at a lower level so long as the excitation is maintained. The rhythm of the heart is as a rule unaffected. The second vagus nerve is now exposed and divided. The heart accelerates, and the arterial RDSkTATLOCKl-ONDON FIG. 141. — Bering's apparatus for demonstrating the action of the respiratory pump. A, Glass bell, thorax ; B, air-tight base ; K, diaphragm ; C, trachea leading to lungs ; I, manometer ; B, tube opening into A ; F, heart with valves V. The action of the dia- phragm pumps air in and out of the lungs and water through the heart. The lungs and heart are thin rubber bags. pressure rises. This is very marked in the morphinised dog. The vagus centre tonically controls the rhythm of the heart. The Effect of Posture. — The animal is placed on a swing board, with the arterial cannula in the axis of rotation. A swing board can be improvised thus : through two staples on the under surface of the board on a level with the point of insertion of the carotid cannula, an iron rod is passed and the end of this clamped to two stout retort stands. On dropping the animal into the vertical posture, with the head up, the arterial pressure falls. It may rise again to, or even beyond, the ELEMENTARY EXPERIMENTAL PHYSIOLOGY 145 normal level in the cat. In the hutch rabbit the pressure falls, until the medullary centres become paralysed from anaemia. The weight of FIG. 142.— Record of arterial pressure and respiration (A) before and (B) one minute after dividing the vagi. The upstroke marks inspiration. The arterial pressure rose from 150 to 180mm., the pulse rate from 110 to 260. Respiration fell from 24 to 10. The expirations became strenuous. (Burdon Sanderson.) FIG. 143. — The effect of excitation of the peripheral end of the vagus nerve upon the blood pressure in the aorta (top curve) and the vena cava (second curve) of a curarised animal with artificial respiration. Note the inhibition of the heart ; the great fall of aortic and the insignificant rise of vena cava pressure ; the escape of the heart from the vagus action and the after effect on the aortic pressure. The time is marked in seconds, and the signal line shows the duration of vagus stimulation. (L.H.) 146 PRACTICAL PHYSIOLOGY the blood in the vertical posture is supported by the taut skin, the tone of the skeletal muscles, and the tone of the arterial system. FIG. 144.— Aortic blood pressure. A, Effect of exciting the central end of vagus. The effect was depressor. B, On shifting up the electrodes to a fresh unexposed part of the nerve the effect changed to pressor. The time is marked in seconds. (L.H.) The blood is largely returned to the heart by the action of the skeletal muscles, aided by the valves in the veins, and the respiratory pump. If the spinal cord be divided in the lower cervical region, or the FIG. 145.— Record of arterial pressure (AP) and plethysun _ signal A. The limb expanded in ysmogram of lirnb (volume record L V\ Excitation of the depressor nerve at signal spite of the fall of arterial pressure. The time is marked in seconds. (Bayliss.) administration of chloroform be pushed, these mechanisms are paralysed, and the blood congests in the lower parts, and the heart fails to fill. In such case the circulation is immediately restored by placing the animal in the horizontal posture. Asphyxia. — The trachea is clamped. Note the sequence of events. 1st stage : Respirations deeper and more ample; heart accelerated and ELEMENTARY EXPERIMENTAL PHYSIOLOGY 147 FIG. 146. — Hill's animal table. The table can be raised or lowered at one end, or be reversed. FIG. 147.— Aortic blood pressure. Effect of posture. A, Vertical head up ; B, horizontal ; C, vertical head down ; D, horizontal. (L.H.) 148 PRACTICAL PHYSIOLOGY more forcible. In the normal animal loss of consciousness now occurs and convulsive movements. 2nd stage : Respiration convulsive, less frequent; blood pressure rising; heart slow. At the end of the second stage the pupils dilate and emission FIG. 148.— Aortic pressure. Spinal cord divided in upper dorsal region. Effect of placing animal in vertical head up posture. The heart emptied. On the return to the horizontal posture the circulation was restored. (L.H.) takes place of urine and faeces. The veins are congested with black blood. FIG. 149. — Arterial pressure ; effect of asphyxia. Animal anaesthetised and curarised. At A the artificial respiration was stopped. The large oscillations are Traube-Hering curves. (L.H.) 3rd stage : The inspirations, which have occurred at longer and longer intervals, finally cease. The heart beats slowly and with great force. Finally the heart accelerates, and the blood pressure falls to zero. ADVANCED EXPERIMENTAL PHYSIOLOGY 149 CHAPTER XXXV. (Advanced}. THE HEART. The Contraction Curve and Latent Time of the Stanniused Heart. — Expose the heart of a pithed frog. Pass a ligature under the two aortae, and draw ISBBBSSil^^^BRi H * the ends exactly round the white ' T^SfPr^SBBiSi^BP «J sino-auricular junction. Tie the ligature. The sinus continues to ^^K^S^^BS^^BII -= J§ beat, while the auricles and ven- ^MJ|^^^^|9|jpBlifcj| tricles stand still. Record the ^^^^^_^^^^^^^_ heart by the suspension method. B^fl BU 15 Two needle electrodes passed through a piece of cork are pinned in such a position that one touches either side of the heart. The drum is set at a moderately fast rate, and the trigger key is placed in the primary circuit. A short BSBI Efl circuiting key is placed in the secondary circuit, and the coil is arranged to give a break shock sgiMHHMH^MMWWKBW #A * just perceptible to the tongue. ^SSB&SBRSSSSSlii ^" HH^VraHBi& *™ and set the drum so that the striker is just beyond the trigger key. Then close the latter. Place SJBPSBHBB^BI^BB ^M the lever at a tangent to the drum, and bring the writing point lightly in contact. Then —.•...•• ••.^•••» open the short circuiting key HBJtHBlBB^^BlLBI "2-5J and start the drum. Stop the HBgH^^BH^^Bfll Jl-s drum immediately after record- BRBBBSl^^Efli l'l> il™"iia™ 1^| »-.fS » short circuiting key, then close ^^^^^^^^^^^s^— the trigger key; lastly open the flB^^^^BI^^^BlB *" short circuiting key. Bring the 150 PRACTICAL PHYSIOLOGY drum round slowly by hand until the striker just opens the trigger key. The heart will contract and the lever write a line marking the moment of excitation. Take another curve with the electrodes placed on either side of the base of the ventricle. The latent period will be less. In the first case the excitatory wave was delayed in the auriculo- ventricular groove. With the tuning fork (100 per sec.) take a time tracing just beneath the heart curves, and measure the latent period. It equals about 0*1 sec. The periods of contraction and relaxation will together last 2 sec. The contraction is much slower than that of striated muscle. FIG. 151,-Stanniused heart. Staircase effect produced by excitations at the points marked on the lowest line. The time is marked in seconds. (L.H.) Any Stimulus, if effective, causes a Maximal Contraction. — Place a spring key and an electric signal in the primary circuit. Set the drum at a slow rate and bring the heart lever and signal to write on the drum. Record the effect of excitation at intervals of a minute or more, with varying strengths of current. The heart gives 'all or nothing,' i.e. if excited at all it gives its full contraction. The Refractory Period. — Record the effect of throwing in a second excitation (a) during the systole, (b) during diastole. The heart is refractory during the whole period of systole, i.e. it makes no response to a second stimulus. The excitability returns with diastole, and becomes greater as diastole proceeds. (Fig. 1 50). Staircase Phenomenon. — A Stannius preparation is excited with single induction shocks once in every five seconds. The stationary drum is moved on by 2 mm. between the excitations. The heights of the first four or five contractions form an ascending series. The heart responds to any stimulus which is effective by a maximal con- traction. The height of the contraction depends on the condition of the heart muscle, not on the strength of the stimulus, so long as the latter is effective. For the first few beats each contraction makes the heart more excitable. The same phenomenon is observed in the muscle ADVANCED EXPERIMENTAL PHYSIOLOGY 151 of curarised frogs with intact circulation, and also in the gal vanome trie records of the action current of nerve. Waller attributes the staircase effect to the influence of C0.2 formed 'by the katabolism of the active tissue. FIG. 152. — Stanniused heart. Summation of stimuli. A, ineffective, and B, effective stimuli. The time is marked in seconds. (L.H.) Summation of Stimuli. — Pull out the secondary coil until the break shock is just ineffective, and rhythmically stimulate the Stannius pre- paration with this inadequate stimulus. The heart will respond to the repeated excitation, and the first few beats will show the staircase phenomenon. CHAPTER XXXVI. (Advanced). THE HEART-CoNTiMJED. THE ACTION OF DRUGS. The Suspension Method of Investigating the Action of Druis on the Frog's Heart. — Large frogs and great care are necessary for this experiment. Pass a ligature under the vena cava inferior, where it is joined by the hepatic veins and enters the sinus. Make a V-shaped incision, and tie in a fine glass cannula. The cannula must be provided with a rubber tube ending in a syphon tube. The tube is provided with a clip, and the whole is filled with Ringer's solution, which is contained in a flask. Attach a hook to the ventricle apex, and record the heart by the suspension method. A slit is made into the aorta. Open the clip, circulate the Ringer's fluid, and record a series of contractions. Now replace the flask of Ringer's solution with one containing distilled water. 1. 0*75 % NaCl solution in distilled water — followed by a Ringer or Locke's solutions until the normal beat is recovered. 152 PRACTICAL PHYSIOLOGY ADVANCED EXPERIMENTAL PHYSIOLOGY 153 2. 0-75% NaCl solution containing 0-3% KC1 (5 c.c. 7% KC1 solution in 100 c.c. 0*60 NaCl) — followed by Ringer or Locke's solutions until the normal beat is recovered. 3. 0'75 % NaCl solution containing a few drops of a 5 % solution CaClo followed by Ringer or Locke's solution until normal beat is recovered. 4. Distilled water. Water distilled in glass is less noxious than water distilled in copper or lead. Merely hanging a strip of copper foil in distilled water over- night increases its poisonous properties. It is calculated that there is not more than 1 part of copper in 70 million of the water. The heart is at first stimulated by sodium ions, but after some time becomes weaker, and finally stops in diastole. Tap-water contains traces of calcium salts, which are beneficial. Normal saline should therefore be made with tap-water. The calcium ions present in small quantities in the blood help to maintain contractility and irritability. Excess of calcium throws the heart into a contracted state — calcium rigour. Potassium ions in excess relax the heart and abolish excitability. Ringer's solution contains 0-7 % NaCl, 0-03 % KC1, 0 025 % CaCl, and keeps the heart in good state. A 2 per cent, solution of digitalin causes increased tone of the heart, vigorous systole, and incomplete diastole. The heart finally is arrested in a state of systolic contraction. Caffeine and veratrine also act tonically on the heart. Supra-renal extract, or adrenalin, at first slows and then increases the tone and the frequency of the heart. Adrenalin is the active principle of the medulla of the supra-renal gland. A solution containing 1 part in 10,000 constricts vessels of the conjunctiva. Weak solutions of acid bring the heart into diastolic arrest. Alkalies produce systolic arrest. 154 PRACTICAL PHYSIOLOGY CHAPTER XXXVII. (Advanced). GASKELL'S CLAMP AND THE EFFECT OF LOCAL WARMTH ON THE HEART. Gaskell's Clamp and the Effect of Heat on Sinus and Ventricle.— The heart of a large frog or toad is required. The contraction of the auricle and ventricle are registered by means of two levers which are FiG. 154. — Gaskell's heart clamp and levers for recording the contraction of auricle and ventricle. attached by means of threads to the apex of the ventricle and auricle respectively ; the one lever is pulled downwards against an elastic spring arid the other upwards. The heart is held fast by means of a screw clamp in the auriculo-ventricular groove.1 The clamp is provided with a fine screw, which can easily be adjusted so as to hold the heart firmly without injuring the tissue (Gaskell). In this way the contractions of auricle and ventricle are registered separately. Take a thick copper wire, bent into a hook at one end, and place the hook round the sinus. Warm the other end of the wire in a flame. 1 A screw clip, to the bars of which cork wedges are fastened, will do for the clump. ADVANCED EXPERIMENTAL PHYSIOLOGY 155 FIG. 155.— Record of the contraction of auricle and ventricle (toad) by the use of Gaskell's clamp and levers. The upper tracing is the auricle and here the con! raction is represented by the down-stroke. The time is marked in seconds. (L.H.) Fio. 166. — Record of the contraction of the toad's heart by the suspension method. Heat applied by the copper wire method. The signal in th« third line shows the period during which the sinus was heated. Acceleration of the whole heart was pro- duced. In this curve the down-stroke represents the contraction. The time is marked in seconds. (L.H.) 156 PRACTICAL PHYSIOLOGY The result of warming the sinus is a great increase in the rapidity of the beats both of the auricle and ventricle. Fro. 157.— Continuation of Fig. 156. Ventricle heated. Augmentation of the ventricular contraction, but no change in frequency. (L.H.) Now warm the ventricle in like manner. No alteration of rate of rhythm is produced by heating the ventricle, but each ventricular contraction is augmented. The observation of the local effect of warmth may be carried out equally well on a heart recorded by the ordinary suspension method. CHAPTER XXXVIII. (Advanced). ACTION OF THE CARDIAC NERVES. Dissection of the Cardiac Nerves in the Pithed Cat or Babbit.— The cat has been instantly killed for you by pithing. Quickly tie out the legs of the animal to the nails on the board provided, and tie a string tightly round the lower jaw, and this to the nail at the head of the board. Pick up the skin over the neck between the fingers, and cut through it with scissors. Pick up the trachea between the fingers and incise it, and insert the tracheal tube, which is connected with the bellows which are kept working by the motor. Artificial respira- tion is thus set going. Separate the sterno-laryngeal muscles from the sterno-mastoid along one side of the trachea and expose the carotid sheath. Separate (Ca) the carotid artery : (P.n.) the vagus, ADVANCED EXPERIMENTAL PHYSIOLOGY 157 this is the largest; (Dep) the depressor, a fine nerve which may be traced up to where it arises by two branches, from the superior laryngeal nerve and from the vagus ; (Sy) the cervical sympathetic, a slender thread, which may be traced up to the superior cervical sympathetic ganglion. In the cat vagus and sym- pathetic are one, and the depressor is separate on the left side only as Dig MJv. FIG. 158.- Dissection of the vagus, the depressor, and cervical sympathetic nerves in the rabbit. (Li von.) a rule. Tie threads round these nerves, ready for their excitation. Pass two ligatures under the carotid, and tie the upper one. Put a clip on the artery below. Make a V-shaped incision, and insert and tie in the cannula. Connect the cannula with the mercury manometer by pressure tubing, and with the glass syringe fill the tube and cannula with a sat. sol. of Na.7S04. Close the side tube of the cannula with a piece of rubber tube, in which a solid glass rod is inserted. Carefully raise the pressure in the manometer by means of the syringe to about 100 mm. Hg, and clip off the syringe. Open the clip on the artery, and record the blood pressure on the slow drum. Faradise the peripheral end of the vagus and observe the inhibition. Stop the artificial respiration for a short time, and observe the effect of asphyxia. 158 PRACTICAL PHYSIOLOGY Next divide the skin over the upper part of the sternum and reflect the left skin flap. Pass threads round the sternal ends of the left first and second ribs. Tie these and divide the ribs between the threads arid the sternum. Pull the ribs outwards by means of the threads, separate the inter- costal muscles with the knife, and by cutting through the spinal attachments of these ribs remove them. , GE FIG. 159.— Dissection of the stellate ganglion (GE) and cardiac accelerators. The inferior cervical ganglion (CL) and vago sympathetic (vs) are also shown. (Pn) vagus; (ac) carotid artery ; (asc) subclavion artery. (Dubois.) The stellate or first thoracic ganglion may now be found and cleaned from the surrounding adipose tissue. It lies just in front of the spinal attachment of the first rib. Branches enter the stellate ganglion from the first, second, and third thoracic roots. Below the sympathetic cord is attached to it, and above a nerve passes to it from the 8th cervical root. The ganglion sends off branches, which form the annulus of Vieussens, and pass to the inferior cervical ganglion. From the annulus and from the inferior cervical ganglion branches pass to the cardiac plexus. The stellate ganglion is the cell-station of these accelerator and augmentor fibres. The stellate ganglion is also the cell station of the fibres which pass to the brachial plexus (vasomotor, pilomotor, sudoriferous) and to the vertebral artery. The cervical sympathetic fibres pass through the ganglion, and have their cell-stations in the superior cervical sympathetic ganglion. Excitation of the (1) cervical sympathetic dilates the pupil, retracts ADVANCED EXPERIMENTAL PHYSIOLOGY 159 the nicitating membrane, causes separation of pupils and projects the eye with the axis of the eyeball straight forwards. It constricts the blood-vessels of the skin, glands, arid mucous membrane of the head. FIG. 160. — Arterial pressure. Effect of exciting the stellate ganglion (accelerate nerves). The time marked in seconds. (L.H.) It dilates the vessels in the bucco-f acini region of the dog. It excites secretions of the glands of the head, both salivary and sweat glands. It erects the hairs in the cat and monkey over certain regions of the face and scalp. V/%*^^ 3*-GD FIG. 161. — Record of arterial pressure. Cardiac acceleration produced by excita- tion of the third dorsal root during the time shown by the signal line. (Bradford. ) The time is marked in seconds. (2) The depressor nerve is an afferent nerve which runs from the heart to the spinal bulb, and causes general dilatation of the blood- vessels— especially in the splanchnic region. (See Fig. 145.) It thus lowers the arterial pressure. The depressor is bound up with the vagus in the dog. (3) The vagus is the inhibitory nerve to the heart, the motor nerve to the bronchial muscles. It conveys both inhibitory and augmentary impulses to the alimentary canal. 160 PRACTICAL PHYSIOLOGY It is a secretory nerve to the gastric glands and pancreas. It contains afferent fibres from the heart which provoke reflex movements, pressor or depressor effects, and reflex cardiac inhibition. The afferent fibres of the vagus coming from the lungs regulate the rhythm of respiration. The superior laryngeal branch of the vagus is the motor nerve to the crico-thyroid muscles and the sensory to the larynx. The inferior laryngeal branch is the motor nerve to the intrinsic muscles of the larynx. Spinal Preparation of Mammal. — Sherrington employs the following preparation for studying blood pressure and spinal reflexes, action of vagus on heart, oncometry of kidney, action of nerves of bladder, etc. : The animal (cat) being deeply anaesthetised with chloroform, a cannula is inserted into the trachea. Both common carotids are ligated. A transverse incision through the skin is made over the occiput and extended laterally close behind the pinnae. The skin is retracted backwards so as to expose the neck muscles at the level of the axis vertebra. The ends of the transverse processes of the atlas are then felt for and a deep incision made through the muscu- lature just behind these processes. The large spinous process of the axis is notched with the bone forceps. A strong thick ligature is passed by a sharp-ended aneurism needle close under the body of the axis and is tied tightly in the groove left by the incision behind the transverse processes of the atlas and the notch made in the spinous process of the axis. This compresses the vertebral arteries where they pass from transverse process of axis to transverse process of atlas. A second strong ligature is then looped round the neck at the level of the cricoid and is so passed as to include the whole neck except the trachea. Decapitation is then performed with an ampu- tating knife passed from the ventral aspect of the neck through the occipito-atlantal space, severing the cord just behind its junction with the bulb. The ligature round the neck is drawn tight at the moment of decapitation. The severed head of the deeply narcotised animal is then destroyed. Haemorrhage is extremely slight. If there is oozing from the vertebral canal it is arrested by raising the neck somewhat above the rest of the carcase. The carcase is placed on a small metal-topped table warmed by an electric lamp below. Artificial respiration is employed to ventilate the lungs, the fresh air supplied from the bellows being warmed by passing through a chamber containing a small electric lamp. The skin flaps are stitched together, covering the exposed end of the spinal cord and other structures bared ADVANCED EXPERIMENTAL PHYSIOLOGY 161 by the amputation wound. The carcase will continue for several hours to exhibit good reflexes employing the skeletal muscles, although the arterial blood-pressure is low, often not more than 80 mm. Hg. The scratch and other reflexes may be studied very well on this preparation. CHAPTER XXXIX. (Advamed). THE PULSE. The Velocity of Transmission of the Pulse Wave. — Mackenzie's polygraph is used. The recording tambours write on a roll of paper which is unwound by the clockwork. The writing styles are wet with red ink. A time marker writes seconds. Two tambour sphygmographs are taken, and one is applied to the carotid, and the other to the radial artery. The recording tambours are brought to write exactly beneath one another on a fast drum, and a time tracing is taken with the tuning fork. Mackenzie's polygraph is most convenient for these experiments. The distance between the carotid artery and the radial is measured. The rate of transmission is about 5-8 metres a second. The rate of transmission increases as the coefficient of elasticity of the arterial wall. It is therefore greater with high than with low arterial pressure. The velocity of transmission from carotid to radial may be lessened by placing the arm in water so as to produce vaso-dilatation. The length of the pulse wave is the product of the velocity of transmission by the time occupied by the wave in passing any given point. Calculate this value from the record. It is about 5 metres, so the pulse wave reaches the periphery before it has left the aorta. Jugular and Radial Pulse Curves. — The jugular pulse and the radial venous pulse are simultaneously recorded in man, and by this means time relations of the cardiac cycle are determined. The jugular pulse is recorded by means of a receiving tambour, which is pressed down upon the jugular vein just above the clavicle on the right side. The tambour is not closed by a membrane, but is pressed down on the skin by the fingers until an air-tight junction is made and a good tracing is obtained. The subject must lie down with his head slightly raised by a cushion and bent to the right side. The venous pulse shows three positive waves, A, C, and V. A marks the auricular systole. C is synchronous with and chiefly caused by the output of blood from the ventricles into the arteries. The carotid L 162 PRACTICAL PHYSIOLOGY artery lying close by influences the venous trace. V is due to the filling of the auricle during the ventricular systole. The negative wave Fio. 162. — Tambour-sphygmographs arranged for measuring the velocity of trans- mission of pulse-waves. after V is probably due to the rapid dilatation of the ventricle and entry of blood therein. The venous pulse and radial pulse taken simultaneously demonstrate whether the cardiac rhythm and Fio. 163. — Impulse (I.) and pulse curves (II.) The vertical lines, marking the ascent of the pulse curve and the dicrotic the semi-lunar valves. notch, indicate the opening and closing of sequence are normal. In pathological states extra systoles may occur, or the ventricle beat independently of the auricle with its own rhythm. Alterations in the auricle-ventricular bundle are ADVANCED EXPERIMENTAL PHYSIOLOGY 163 probably the cause of such disturbed action. While the record is being taken place both hands on the subject's belly and compress the abdomen ; both venous and arterial pressures rise. The rise in arterial pressure may be measured with the sphygmometer. One tambour is now placed over the carotid and another over the cardiac impulse. The beginning of the impulse curve marks the beginning of the ventricular contraction. The beginning of the carotid pulse curve marks the beginning of the period of systolic out- put and the opening of the aortic valves. Between these points is the period of rising tension, when the ventricle is raising the blood pressure up to that in the aorta. The beginning of the dicrotic notch cor- responds with the closure of the aortic valves and the end of out- put. The time lost in the transmission of the pulse-wave from the heart to the carotid artery should be deducted in making these time measurements, but it is almost negligible. In a man with a pulse frequency of 70 the duration of systole was 0-379 sec., of diastole 0-483 sec. It is interesting to repeat the observations after the frequency of the heart has been accelerated by running up and down stairs. The diastolic period is shortened much more than the systolic period. When the pulse varied in the proportion 100 : 270 the duration of a systole varied in the proportion 136 : 100. With the two armlets and mercurial manometer and syringe bulb measure the systolic pressure in the arm and leg using the radial and posterior tibial or dorsalis pedis arteries as indices of the obliteration of the pulse. Let the subject be horizontal. Repeat after the subject has run up and down stairs, and again after he has put one hand in hot water for some minutes. The pressures which were equal will now appear unequal for the artery softened by the effect of heat conducts the pulse less well. In cases of aortic regurgitation the leg and arm readings are unequal, the leg being the higher owing to the leg arteries being stiffer and conducting the wave better. Measure the pressure in the leg and arm (1) with the subject lying horizontal, (2) with the legs raised in the L-shaped position, (3) stand- ing erect, the observed leg being kept in an easy relaxed position and the weight thrown on the other leg. In each case measure the difference in height between the upper edges of the armlets with the metre rule. Calculate the difference in terms of mercury by dividing by 13, and compare the difference in the readings found with the calculated differ- ences. The pressure differs by the column of blood separating the two points of measurement. The compensatory vasomotor mechanism keeps the pressure in the aortic arch and its branches about the same in all three postures, while the pressure in the leg arteries varies greatly. 164 PRACTICAL PHYSIOLOGY CHAPTER XL. (Advanced). VASO-MOTOR SYSTEM. Innervation of the Blood-vessels. — Pith the cerebrum of a large frog and plug the hole with a blunt-pointed match to prevent haemo- rrhage. Curarise the frog lightly, place it on the cork board provided for studying the circulation in the web. Tie out the toes so as to spread the web over the hole in the board. Observe the rate of circulation. Next pass a pin through the occipito-vertebral membrane and destroy the spinal bulb. The circulation will become more rapid owing to dilatation of the arteries. Now remove the frog from the board and expose the heart. Suspend the frog in the vertical head-up position. Note that the heart and large vessels are filled with blood. Pass a blanket-pin down the vertebral canal and destroy the spinal cord. The heart and vessels will soon become bloodless owing to the loss of vaso-motor tone. The blood sinks into the dilated abdominal vessels under the influence of gravity. Perfusion of Frog's Blood-vessels. — Destroy the brain and plug the hole in the skull. Expose the heart. Tie one aorta. Place a ligature under the other, snip it with sharp scissors, and allow the blood to escape. Insert a fine-glass cannula into it pointing away from the heart. Fill the cannula with normal saline by means of a capillary pipette. Connect a rubber tube to a glass funnel and clip the tube. Fill the funnel and tube with Ringer's fluid. Connect the tube with the cannula. No air bubbles must be introduced. Snip the sinus venosus and open the clip. Hang the frog in the vertical position. The fluid circulates, runs out of the sinus, and drops from the toes of the frog into a measure glass. Measure the outflow per minute. Circulate Ringer's fluid plus 1 in 1000 sodium nitrate; the outflow is increased owing to vaso-dilatation. Supra-renal extract produces the contrary effect. ADVANCED EXPEEJMENTAL PHYSIOLOGY 165 166 PRACTICAL PHYSIOLOGY CHAPTER XLI. (Advanced Demonstrations). INTRACARDIAC PRESSURE. BLOOD FLOW. Intracardiac Pressure. — Owing to inertia the mercurial manometer is unable to respond to the rapid changes of intracardiac pressure. The pulse curves obtained by the mercurial manometer are also distorted by the swings due to the momentum of the mass. To record the changes of intracardiac pressure an instrument must be contrived which is able to follow a change of pressure equal to 1500 mm. Hg per second. FIG. 165. — Hiirthles spring manometer. FIG. 166.— Sphygmoscope. Hiirthle's spring manometer consists of a small tambour, 5*5 in diameter, covered with rubber membrane. A button attached to the membrane works against a steel spring. The movement of the spring is magnified by a light lever. Inertia is proportional to the mass and the square of the velocity. By making the tambour very small and the lever very light the errors due to the inertia of the fluid and lever are reduced to a minimum. FIG. 167. — Arterial pressure recorded by a spring manometer. Effect of weak excitation of the vagus during the period marked by the signal m. (Dubois). The sphygmoscope is an equally good instrument. One end of a rubber finger-stall is drawn over the end of a rubber cork. The cork is inserted into a short piece of wide tube. A glass tube passes through this cork into the small air-space which is left at the top of the finger- stall. The other end of the wide tube is closed by a rubber cork. A ADVANCED EXPERIMENTAL PHYSIOLOGY 167 glass tube passes through this cork and is connected with a recording tambour. The finger-stall acts as the spring. Connect by side tubes the mercurial manometer and the Hiirthle manometer with the artery in the artificial schema. Take records FIG. 168.— Hlirthle's differential manometer. with each instrument on a moderately fast drum, and compare the results. Connect by side tubes one side of the Hiirthle differential manometer with the chamber of the pump, and the other side with FIG. 169.— Aortic and ventricular pressure curves taken by HUrthle manometers. (Hurthle). 1. Beginning of systole. 2. Opening of semilunar valves. 3. End of rise of ventricular pressure. 4. Dicrotic notch, closure of semilunar valves. the artery close to the valve. Take a record, and observe how the instrument records the moment when the valve opens and shuts. If a time tracing be taken, the time relations of the pump (ventricular contraction) can be exactly determined. The period of ventricular 168 PRACTICAL PHYSIOLOGY systole is divided into three: (1) the period of rising tension, when all the valves are closed; (2) the period of output; (3) the period of relaxation. In simultaneous records of intra ventricular pressure and aortic pressure the beginning of the aortic rise (2) marks the opening of the aortic valve and beginning of output. The end of output occurs when the semilunar valves close at the beginning of the dicrotic notch (4). The period of rising tension lasts from the beginning of systole (1) to the opening of the semilunar valves (2). Velocity of Blood Flow.— Insert the Pitot tubes E and F into a tube A through which water is flowing from a constant head of pressure B (Fig. 170). Note that the water rises to different levels Fir,. 170.— Schema to show the velocity and resistance heads. B, Pressure bottle, with piezometers. B P, Pitot tubes. A, Tube in the tubes. E represents the resistance head plus the velocity head. F records the resistance head minus the velocity head. Measure the outflow per minute from the tube A, and notice the difference between the heights of the menisci in E and F. Lessen the velocity by partly screwing up the clip on the end A. Measure the outflow per minute, and note that the menisci are nearer together. Close the end of A. The flow ceases, and the menisci in the two tubes reach the same level as that of the head of pressure B. Cybulski makes use of this principle in the construction of the photohaematochometer, an instrument by which alterations in velocity can be recorded. Fig. 171. ADVANCED EXPERIMENTAL PHYSIOLOGY 169 The velocity can also be measured in the artificial schema by injecting 1 c.c. of methylene blue sat. sol. into artery, and noting by means of a stop-watch (or electric signal and drum) the moment of injection, and the moment when the blue fluid reaches the capillary tube. The Circulation Time. — In the artificial schema measure the circulation time by injecting methylene blue into the vein V, and noting how long the blue takes to reach the venous end of the capillary tube. The Work of the Heart.— To estimate the work of the heart in the artificial schema the mean pressure H, and velocity in the aorta V, and the volume of the systolic output Q, must be obtained. FIG. 171.— Cybulski's photo- haeinatochometer. A cannula shaped as shown is introduced into the blood-vessel. The os- cillations of the mercury -menisci are photographed. M = the mass of the output in grammes = Q multiplied by the specific gravity of the blood. Close the clip on the arteriole tube and start the pump. Note the mean pressure H indicated by the manometer M. To obtain V inject into the artery, at 1 metre from the capillary tube, 1 c.c. of sat. 2.* Count the number of pulses per minute, and by dividing the number found by 60 obtain t. Then Q = ad. Now calculate the work of the pump from the data obtained. The work spent in maintaining velocity is almost negligible in comparison with that spent in overcoming resistance. In man the output may be taken as 60 grms., the average aortic 170 PRACTICAL PHYSIOLOGY pressure as 1 1 0 mm. Hg, the velocity of flow in the aorta as 320 mm. per sec. Mercury 13*5 times heavier than blood The right heart is considered as doing one-third of the work of the left heart. The total work of the human heart is estimated to be about 12,000 kilogramme-metres per day, or 500 kg.m. per hour. This equals about 28 kilo-calories (425 kg.m. = 1 kilo-calorie). H — Air Cushion, Recorder FIG. 172.— The Cardiometer. In the dog the output can be obtained by estimating the amount of oxygen taken up by the blood from the inspired air in one minute. This can be obtained by Fredericq's or Zuntz's method (see p. 184). At the same time samples of arterial and venous blood are obtained, and the oxygen difference between the two samples estimated by the blood pump or Haldane's ferricyanide method (see p. 190). The number of heart beats per minute is also counted. Suppose 100 c.c. of oxygen are taken up per minute, the arterial blood contains 5 c.c. per cent, more oxygen than the venous blood, and the heart beats 80 times per minute. Then, as every 100 c.c. of blood carries away 5 c.c. O2, 2000 c.c. of blood must have passed through the heart in the minute. Thus the output 2000 80 = 25 c.c. The output in mammals is reckoned to be about *0012 of the body weight per sec. The Cardiometer. — Demonstration of the method of recording the volume of the output of the heart. The Cardiometer is made of ADVANCED EXPERIMENTAL PHYSIOLOGY 171 the end of a large thistle funnel, which is first covered with thin rubber membrane, and then a large round hole is made in the membrane by burning it with a soldering iron. The thorax is opened in the pithed cat after establishing artificial respiration, the pericardium is incised and the heart inserted through the hole in the rubber membrane into the funnel and this connected with a piston recorder. The rubber membrane fits snugly to the base of the ventricles and renders the cardiometer air-tight. The carotid artery is connected as in the figure and the blood returns to the jugular vein, the circulation being confined by ligatures to the heart and lungs. The blood pressure is regulated by sinking the tube more or less deeply in the mercury. CHAPTER XLII. (Advanced Demonstrations). EFFECT OF HAEMORRHAGE AND SALINE TRANSFUSION. IN the anaesthetised and weighed animal a carotid cannula is introduced and another in the jugular vein. A third cannula is placed in the femoral artery. Observe the effect on the blood pressure tracing (1) of bleeding from the femoral artery, (2) running in physiological salt solution into the jugular vein. Note the volume of blood withdrawn and saline introduced. To produce a fall of pressure 25 c.c. per kg. of body weight should be removed. After running in the saline, bleed the animal to death, Fio. 173.— Diagram of an oncometer and piston recorder. The rubber bands fasten the glass lid in position. and note the effect on the blood pressure tracing and the quality of the blood compared with that collected before transfusion. Look for fluid in the abdomen and observe the organs after death. PLETHYSMOGRAPHS. Plethysmographs and Oncographs. — In the pithed cat, trachea!, jugular, and carotid cannulae are introduced. The abdominal cavity 172 PKACTICAL PHYSIOLOGY is opened and the greater splanchnic nerve exposed by blunt dissec- tion where it lies just outside the supra-renal capsule. A ligature is tied round the nerve and the peripheral end stimulated. Note that blood pressure rises. A cannula bent at right angles is placed in the bladder, and the left kidney in the oncometer. The kidney is laid on one of the pieces of the oncometer, its vessels being placed in the groove. The india-rubber bag filled with water at 38° C. is placed round it, and the other half of the oncometer put in position and the rubber bands applied. The tube of the rubber bag is connected with a water manometer, the water FIG. 174. — Arterial pressure (I) and oncometer tracing (2) of kidney volume. Be- tween the points starred the 10th dorsal root was excited. The time is marked in seconds. (Bradford.) being coloured with methylene blue. The manometer should show pulsations. Cover the abdomen with warm compresses of wool wrung out from hot 0*75 per cent, saline solution. Measure the outflow of urine for five minutes, and note the effect of stimulating the splanchnic nerve. Note the effect of injecting 30 c.c. physiological salt solution into the jugular vein. So long as the venous pressure is constant any increase in renal volume will denote increased blood-pressure in and increased Wood-flow through the kidney. The secretion of urine varies as the volume of blood passing through the kidney per minute. (By dividing the renal nerves and exciting the spinal cord or vaso-motor centre the greatest rate of blood-flow through the kidney can be produced.) Ligature of the ADVANCED EXPERIMENTAL PHYSIOLOGY 173 renal vein stops the secretion of urine. After a temporary obstruction albuminous urine is secreted. Half a grain of citrate of caffeine injected intravenously will produce a fall of arterial pressure and a preliminary contraction of the kidney, followed by expansion and increased flow of urine. Observe the effect of extract of pituitary gland. This is the most active diuretic known (Schafer). Plethysmography of the Arm. — The arm is placed in the rubber gauntlet of the plethysmograph. The plethysmograph is connected with a recording tambour, a J_-piece being interposed. Record the volume curve on a moderately fast drum. The tracing shows pulse waves and respiratory oscillations. ; ;fl FIG. 175. — Limb plethysmograph. Fasten the armlet of the sphygmometer round the upper arm and record the curve of venous congestion which results from raising the pressure in the armlet. Repeat this ; after exertion the curve rises much more steeply. This is a good comparative method of studying the velocity of blood flow into man. 174 PRACTICAL PHYSIOLOGY CHAPTER XLIII. RESPIRATION. Examination of the Chest of Man. — Much can be learned by simple methods of examination, and it is of the greatest importance that the medical student should rely more upon his sight, hearing and touch, than upon the graphic records obtained with different forms of apparatus. Inspection. — The chest of a man stripped to the waist is examined and the following points are noted : (i) The shape, whether the thorax is strongly built and symmetrical, (ii) its mobility, whether the two sides move equally. The condition of the abdominal wall should then be examined, and attention paid to the development of its muscles and the movements during respiration. The measurement round the chest of an adult man is about 35 inches and can be taken with a tape. The increase in circumference produced by inspiration is about 2 to 3 inches. It is impossible, however, to determine by such measurements whether a man has a good " wind " or not. A well-developed chest generally means that a man has lived an active life and has a good heart and lungs, but great variations are found in the shape of the chest of healthy men. The true test of a man's heart and lungs is whether he can respond to the demands of muscular exercise without undue breathlessness and distress. Even this test must be applied with intelligence, for the man may be under-fed, and may have led a very sedentary life. A graphic record of the shape of the chest in different planes can be obtained with the cyrtometer. This simple instrument consists of two pieces of narrow lead piping hinged by a piece of rubber tubing. The hinge is placed over the vertebral column and the lead tubing is moulded round the sides of the chest in a horizonal plane ; the cyrtometer is then opened, removed from the chest, placed in position on a sheet of paper, and its outline traced with a pencil. The movements of the chest and abdomen should be observed and their relationship to inspiration and expiration determined. Some subjects show marked abdominal or diaphragmatic breathing, others breathe more by the thorax. In women the movement of the upper part of the chest is greater than in men ; the causes of this differ- ence are to be ascribed to the constriction of the abdomen and lower portion of the thorax by corsets and to the greater mobility of the thorax, due to the fact that in civilised countries the women do less ELEMENTARY EXPERIMENTAL PHYSIOLOGY 175 muscular work than the men. If hard work is frequently performed with the arms the upper portion of the thorax becomes more rigid, and this is an advantage, for it gives a better purchase for the contract- ing muscles. There is no sound basis for the dogmatic teaching about thoracic and abdominal breathing of some so-called specialists in physical training. Healthy children do riot need lessons in breathing, but opportunities for muscular exercise, for games in the open air. No reasonable athlete would attempt to improve his "wind" except by training it by progressively graduated runs. A good " wind " is something more complex than a big or mobile chest ; it involves the heart which forces the blood through the lungs. Artificial breathing exercises are unsound ; healthy games and sports train the whole body, the component parts of which are mutually dependent. At rest breathing is performed by healthy subjects with the mouth closed, but during severe work it is opened instinctively and with advantage, for there is then less resistance to the passage of the air in and out of the chest, and the loss of heat is facilitated. The rate of respiration in healthy adult men at rest varies from about 10 to 23 per minute : men who breathe slowly take deep breaths ; those who breathe quickly take shallow breaths. Palpation. — By placing the flat of each hand upon corresponding portions of the chest it is possible to compare the movements of the two sides of the thorax. If the subject be told to speak, to say " ninety-nine," for example, the vibration of the voice, weal fremitus, is propagated through the bronchi to the wall of the chest, and thus to the hands of the examiner. Percussion. — If a tap with the finger be given to the top of a table, the note will be dull over the part directly supported by the leg, but more resonant in the middle of the table. It is also easy for most men to detect a difference in the sense of resistance when the tap is given ; it is greater with the dull note. In a similar manner the level of water in a tub can be determined. Such a method of investi- gation of underlying structures is known as percussion. Firmly place the index finger of the left hand on the chest and tap it with the middle finger of the other hand. Determine the differences in note and resistance over the various parts of the thorax. On the right side the resonance extends from the apex of the lung in the supra-clavicular fossa to the beginning of the dulness produced by the liver under the 6th rib. On the left side it extends to the cardiac dulness which begins at the 4th rib. Make the subject take a deep breath, and then by percussion 176 PEACTICAL PHYSIOLOGY demonstrate that the limit of resonance is increased owing to the expansion of the lungs. Auscultation. — The respiratory and cardiac sounds can be heard by placing the ear against the chest, or by means of a wooden or binaural stethoscope. Over the trachea, or at the level of the 7th cervical spine, the harsh blowing sounds, due to inspiration and expiration, are heard; these "bronchial sounds" are produced by the vibration of the air at the orifices of the vocal cords and divisions of the trachea and bronchi. Another sound, the "vesicular murmur," is heard on listening to parts of the chest wall where the lung is in contact. It is a soft breezy sound which increases during inspiration and dies away during the first third of expiration. There are several views about the causation of this sound ; it may be due to conduction of the bronchial sounds. CHAPTER XLIV. INTRA-THORACIC PRESSURE. Intra-thoracic Pressure. — The thoracic cavity, when opened, is far larger than its contents, for the lungs, owing to their elasticity, collapse as soon as the intra-pulmonary and pleural pressures become equal. The intra-pleural pressure is less than the atmospheric pressure by that amount of the atmospheric pressure which is required to overcome the elasticity of the lungs and distend these organs to the size of the thoracic cavity. The intra-thoracic pressure or elastic traction exerted by the lungs on the thoracic wall varies as follows : — Normal inspiration about - 10 mm. Hg. „ expiration „ - 7 „ Deep inspiration „ - 40 „ „ expiration - ,, 0 „ ,, inspiration with air-way closed ,, —100 „ „ expiration „ „ „ „ +100 „ The intra-tracheal pressure varies from - 1 irm. Hg. in quiet inspira- tion to + 1 mm. Hg. in expiration. During forced breathing with the air-way closed the intra-tracheal pressure is greater than the intra- thoracic pressure by the airount of the elastic traction exerted by the lungs. All the structures, e.g. heart and blood-vessel?, are affected by the respiratory variations of pressure. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 177 DEMONSTRATION. The trachea of a dead rabbit is exposed, and a ligature tied round it. The skin is divided over the thorax on one side, and the ribs exposed. The intercostal muscles are carefully separated between two ribs. Note that the lung is in contact with the thoracic wall. The ligature round the trachea is now divided ; the air escapes, and the lung, owing to its elasticity, will collapse. On opening the pleural cavity the pressure within and without the lung becomes atmospheric. The elasticity of the distended lung then comes into play and causes its collapse. DEMONSTRATION. In the rabbit anaesthetised with ure thane or chloral the skin is divided over an intercostal space. The intercostal muscles are then separated with care, and a piece of rib removed, while the parietal pleura is left quite uninjured. The lung will not collapse so long as the pleural cavity is not opened. On the contrary it will be seen gliding to and fro with each movement of respiration. Note how easily the pleural surface of the lung glides over the parietal pleura. A glass cannula attached to a water manometer is pushed throughout the intercostal muscles until the end comes to lie in the thoracic cavity. Notice the negative pressure indicated in the manometer, which becomes greater in inspiration and less in expira- tion. Note the immediate collapse of the lung on opening the pleural cavity. CHAPTER XLV. VENTILATION OF THE LUNGS. THE SPIROMETER AND THE STETHOGRAPH. THE ventilation of the lungs is determined by a gas-meter through which the subject breathes by means of an anaesthetic mask, provided with inspiratory and expiratory valves. Meters with a very low resistance are more convenient than the special instrument known as the spirometer (Fig. 176), although the latter is very useful for some experiments. The subject of the experiment should breathe through the mask and meter for a minute or two before a record is taken, in order that he may become accustomed to the novel conditions. Then the volume of each breath and the number in periods of consecutive minutes should be determined. A table should be made to show the results obtained with each member of the class, for the differences in the rate and depth of breathing in healthy men are considerable ; some men breathe slowly M 178 PRACTICAL PHYSIOLOGY and deeply, others take rapid and shallow breaths. The volume of air breathed per minute varies from 9 to 5 litres, the number of breaths from 23 to 10, and the averages for the volume of each breath from 900 to 250 c.c. It is important to remember as a general rule that what is lost in frequency is compensated in depth, so that the volume breathed per minute by a man with a frequency of respiration of 10 may be the same as that of a man whose ordinary rate of breathing is 22 per minute. The tidal air is the volume of air breathed at each respiration, and it varies from 900 to 250 c.c. in dif- ferent individuals. After breathing out the tidal air the subject should expire as deeply as possible ; an additional 1500 to 2000 c.c. will be recorded. This is called the supplemental air. Now let the subject take as deep an inspiration as possible; it will be about 1500 to 2000 c.c. above the tidal air. This quantity is known as the complemental air. The so-called vital capacity is the greatest volume of air that can be expired after the deepest possible inspiration ; it is composed of tidal air 500 c.c. + complemental air 1 500 c.c. + supplemental air 1500 c.c. It is about 3500 c.c., but too much importance should not be attached to it, for it depends largely upon practice and control of the inspiratory and expiratory muscles. A broken-winded bandsman, who is accustomed to control the blast of air which he delivers to his instrument, may have a so called vital capacity greater than that of an athlete. The Effect of Muscular Exercise upon the Respiration is very great ; within a few minutes, varying according to the severity of the work and the condition of the subject, the volume of air breathed may be doubled, the number of breaths showing a smaller increase. The breathing is deeper, and the mouth is opened to diminish the resist- ance to the passage of the air in and out of the chest. Discomfort FIG. 176. — Spirometer. T, mouthpiece ; M manometer ; Cp, counterpoise ; R, scale. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 179 or distress is caused by any resistance, and for this reason it is impossible to determine the true volume unless the resistance of the recording apparatus is low. Connect up two gas meters with a T-piece Fio. 177. — A stetbograph employed to record the respiration and cardiac impulse of the rabbit or cat. The tambours press on either side of the thorax. 1 he T tube leads to a recording tambour. FIG. 178.— Stethograph. A, Metal drum ; B, hooks for tapes which pass round neck ; C, rubber discs ; D, hooks for attaching tapes which are tied round thorax ; E, tube leading to the recording tambour. and determine the volume and rate of respiration before and after running down and up a flight of stairs. 180 PRACTICAL PHYSIOLOGY The Graphic Record of the Respiratory Movements. — For this purpose an instrument known as the stethograph is used. There are various forms, two of which are shown in Figs. 177 and 178. A receiving tambour constructed like a drum is fastened to the chest, and is connected with a recording tambour, the lever of which writes on a smoked drum The subject of the experiment should not be allowed to see the movements of the lever, for the respiration is easily affected by nervous impressions. Take a graphic record of the respirations and mark the time relations of inspiration and expiration by means of a chronograph giving seconds. CHAPTER XLVI. CHEMISTRY OF RESPIRATION. The Composition of Inspired Air, Expired Air and Alveolar Air. — For the analysis of these different samples of air the best apparatus is that of Haldane. The gas is measured in the graduated gas-burette A, provided with a three way tap. Surrounding the /gas-burette is a water-jacket. The whole is supported by a clamp and retort stand. The gas-burette is connected by pressure tubing to the levelling tube B, which is held by a spring clamp attached to the retort stand. A and B contain mercury, and by raising or lowering B gas can be expelled from or drawn into A. One of the connections of the three-way tap is used for taking in the sample, the other connects the burette with an absorption apparatus arranged as in the figure. The bulb E, filled with 20 per cent, caustic potash, absorbs carbon dioxide. The bulb F, filled with alkaline pyrogallic acid solution,1 absorbs oxygen. The water in Gr and H protects the pyro. solution from the air. F can be emptied and refilled through K when it is necessary. The tap on the absorption pipette places either E or F in connection with the gas burette. The pressure in the burette is adjusted by using the potash pipette as a pressure gauge and bringing the potash before every reading of the burette to the mark M. In order to make the reading of the burette independent of changes in temperature and barometric pressure during analysis a control tube N is employed. N is connected with the potash solution by means of a 1-tube. The tap at P makes it 1 Dissolve 100 grms. of stick caustic potash in 50 c.c. of water. Add 10 grins, of pyrogallic acid to this solution. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 181 possible to ren ler the pressure in N equal to that of the atmosphere. At the beginning of the experiment the potash is adjusted to the mark R by altering S, P being open. P is then closed, and not opened again B Flo. 179.— Haldane's gas analysis apparatus. FIG. ISO.— Hempel's burette for col- lecting a sample of expired air. till the analyses are complete. The barometer and the temperature of the water-jacket are read. Each time a reading of the burette is made the potash is brought to the mark R by altering S, and to the mark M by means of the levelling tube B. As the control tube and the gas- 182 PRACTICAL PHYSIOLOGY burette are kept moist, variations in the tension of aqueous vapour in the burette are also corrected by the control tube. A sample of expired air is obtained by- breathing through the tube into the burette B (Fig. 180). A and B are filled with acidulated water, and B is controlled by a clip. The portion of B which lies beyond the clip is squeezed empty of air before it is inserted over the entrance tube of the Haldane gas-burette. The sample is then taken over by lowering the levelling tube and opening the clip. Atmospheric Air, measured dry at standard temperature and pressure, 0° and 760 mm., has the following composition : — Oxygen, - 20'94 volumes per cent. Carbon dioxide, - 0'03 „ „ Nitrogen, - . 78-09 „ „ Argon, 0-94 There are also traces of helium, krypton, neon, xenon, and hydrogen. The nitrogen and argon appear to be inert as far as the higher animals are concerned, and in ordinary analyses are given together as nitrogen. The Expired Air varies in composition according to the rate and depth of respiration ; this is shown by the following analyses made by Speck. Volume of air expired Type of per minute. Percentage of Percentage of breathing. c.c. oxygen. carbon dioxide. Normal, - 7,527 16-29 4-21 Very shallow, - 5,833 15'50 4'63 Very deep, - 17,647 18'29 3'17 Stated in whole numbers the composition may be given as follows : — Volumes per cent. Oxygen. Carbon dioxide. Nitrogen. Inspired air, 21 (0'03) 79 Expired air, 16 4 80 There are other differences between inspired and expired air. Under ordinary conditions expired air is warmed nearly to the temperature of the body and is saturated with water vapour ; it has about 6 per cent, of moisture, whereas ordinary atmospheric air has about 1 per cent. The expired air is a mixture of air from the so-called " dead space " of the respiratory tract and of air from the alveoli of the lungs, where the exchange of gases between the blood and the air takes place. The " dead space " extends from the nose to the alveoli and has a capacity of about 150 c.c. in an adult man. In an ordinary expiration the first portion of air to leave the nose or mouth is from this "dead space," then mixed air, and finally air from the alveoli. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 183 The Alveolar Air. — The composition of the alveolar air is deter- mined, according to the method introduced by Haldane and Priestley, by an analysis of the last portion of the air expired in an ordinary expiration. The experiment may be performed in the following way. An anaesthetic mask is connected by a T-piece to a piece of tubing 80 cm. long and 1'8 cm. internal diameter; to the free end of the T- FIG. 181. — Apparatus for collection of a sample of Alveolar Air. piece is connected (Fig. 181) a gas-sampler with a capacity of 50 cubic centimetres. The subject of the experiment fits the mask to his face and makes an ordinary expiration ; as soon as the expiration ceases, the tap of the gas-sampler, the air of which has previously been removed by a vacuum-pump or gas-pump, is opened and a sample of the last portion of the expired air is collected before the mask is removed from the face. The analysis of the air is performed in the manner already described. The percentage composition is about 5*5 carbon dioxide, 14*5 oxygen and 80 nitrogen. It is an advantage to determine the volume of each expiration by a spirometer attached to the end of the tubing, and it is important that the subject of the experiment should by a little practice with the apparatus learn to breathe naturally, otherwise a fair sample will not be obtained. The partial pressure, or, as it is often called, the tension of the component gases is : — Dry atmospheric air : 21 Oxygen approximately ^-^ x 760= 159*6 mm. of mercury or 21 per cent, of an atmosphere. 79 Nitrogen approximately y^ x 760 = 600'4 mm. of mercury or 79 per cent, of an atmosphere. 184 PRACTICAL PHYSIOLOGY Carbon dioxide approximately -^- x 760 = 0'228 mm. of mercury or 0'03 per cent, of an atmosphere. The tensions of the gases of the alveolar air are calculated in a similar way, but the tension of aqueous vapour must be deducted from the pressure of the atmosphere. CHAPTER XLVII. DETERMINATION OF THE RESPIRATORY EXCHANGE IN MAN. AN estimation of the intake of oxygen and output of carbon dioxide can be made by analyses of continuous samples of the air expired FIG. 182. Zuntz respiration apparatus. The subject expires through the meter. The inlet and outlet tubes are controlled by valves D and C, made of pieces of intestine which have been soaked in glycerine. A small sample of the expired air is steadily drawn off into the burette A by the escape of mercury from the tube which is lowered by the revolution of the meter B. The meter gives the total volume of air breathed. The measured sample in the burette is analysed by Haldane's gas apparatus. through a meter. The method introduced by Zuntz for the purpose of collecting such a sample is illustrated in Fig. 182. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 185 It will be sufficient if the student makes the determination in the following way. He should collect a sample of expired air and analyse it; then he should determine the average volume of air which he breathes in a minute. The methods involved have been described in previous chapters. From the data obtained a calculation can be made as follows: — The man breathed 7 litres per minute, and the composition of the expired air was 16 per cent, oxygen and 4 per cent, carbon dioxide; he 7000 had, therefore, absorbed 21 - 16 = 5 x —— = 350 c.c. of oxygen and discharged 4 x -^-TTT- = 280 c.c. of carbon dioxide. His respiratory quotient, the ratio of the volume of carbon dioxide discharged to the volume of oxygen absorbed is -^r-2 = J^ = ^ = 0'8. v/2 OD\) v There is a decrease of about -V in the volume of the expired air as compared with the inspired air, when both are measured at 0° and 760 mm. ; the deficit is due to the absorption of a small quantity of oxygen which does not reappear in combination with carbon as carbon dioxide, but passes out of the body in other products of oxidation. The increased proportion of nitrogen in the expired air must be taken into account when the respiratory quotient is calculated from volumetric analysis ; thus for every 100 c.c. of expired air the slightly larger volume of inspired air contained the following volume of oxygen : — „ _20"94 x Nitrogen of expired air 79-07 The respiratory quotient, therefore, in a case in which the percentages of nitrogen, oxygen and carbon dioxide are 80, 16 and 4, would be correctly calculated as follows : — c • • j • 20-94x80 01 10 Oxygen of inspired air= — — =21 '18 c.c. /"'07 Oxygen absorbed =21 '18 - 16 = 5'18 c.c. Respiratory quotient = TT^-^TTQ^0^"- U._j O'Jo The respiratory quotient varies according to the nature of the food which undergoes oxidation in the body; thus, for carbohydrates it is 1, for protein 0*8, and for fat 0'7. The following formulae represent the oxidation of these different substances : — Dextrose : C6H1206 + 602 = 6C02 + 6H20. C02_6_ 02 ~6~ Albumin (empirical formula) : C72H119N18022S + 7702 = 63C02 + 38H20 + 9CO(NH2)2 + SO3. 186 PRACTICAL PHYSIOLOGY Olein : C3H5(C18H3302)3 + 8002 = 57C02 + 52H20. ^C02_57 ~OT~80~ The effect of muscular exercise upon the respiratory exchange is most marked ; hard work may increase it three or four times. For exact work upon the respiratory exchange of man a respiration chamber is required. Few laboratories possess such an expensive apparatus, but the principles can be studied in the simple form of respiration apparatus for mice. CHAPTER XLVIII. RESPIRATION APPARATUS. The Haldane-Pembrey Respiration Apparatus for the Mouse. — The apparatus is constructed as in Fig. 183. Each double absorption tube is fitted with a wire loop, so that the glass need not be touched with the hand. The animal chamber- a light beaker — is provided with a thermometer and is also fitted with a wire loop. The moisture given off by the animal is absorbed by pumice saturated with sulphuric acid in the tubes AB. The carbon dioxide is removed by soda lime in the tube C, and the water given off by the soda lime is caught by the sul phuric acid tube D. M N A B c D FIG. 183.— The Haldaiie-Pembrey respiration apparatus for the mouse. The animal is weighed in the beaker (with the tubes closed) before and after the experiment. A dummy beaker is placed in the opposite scale pan. The tubes AB and CD are also weighed against a dummy pair of tubes. During the weighings the exit and entrance tubes are left unstoppered. By these means errors due to condensation of moisture and changes of barometric pressure or temperature are avoided, and the weighings can be carried out to less than a milli- gramme. The air entering the chamber is freed from carbon dioxide and water ELEMENTARY EXPERIMENTAL PHYSIOLOGY 187 by soda lime in M and sulphuric acid pumice in N. The amounts of water and carbon dioxide given off in 15 minutes are determined by the increase in weight of AB and CD respectively. The amount of oxygen absorbed is found by subtracting the loss in weight of the animal weighed in the chamber from the total loss of carbon dioxide and water, for the animal absorbs during the experiment oxygen and loses water and carbon dioxide. , CO0 grms. 32 C0« by volume The ratio -7^--— — x — = „ 2, J — } = respiratory quotient. O2 grms. 44 O2 by volume The effect of external temperature upon the respiratory exchange may be studied with this apparatus. EXAMPLE. The beaker containing a full-grown mouse was placed in a water-bath at 9'5° C. ; the mouse gave off from 250-315 decimgrms. of carbon dioxide per 10 minutes, and was active. When the temperature of the bath was 30° C. the mouse gave off 103-116 decimgrms. carbon dioxide per 10 minutes, and was quiet. The rectal temperature of the animal scarcely varied during the experiment. Mammals born in a helpless condition, naked and blind, such as rats and rabbits, behave like cold-blooded animals, and are unable to compensate for low external temperature by increased metabolism ; the output of carbon dioxide sinks as their body temperature falls. CHAPTER XLIX. THE CHEMISTRY OF RESPIRATION. THE GASES OF THE BLOOD. IN a former chapter experiments were given to prove that the air which is taken into the lungs loses a portion of its oxygen and gains carbon dioxide; these changes correspond to differences in the gaseous contents of the blood; the venous blood loses carbon dioxide and gains oxygen in passing through the lungs, and thus becomes arterial. Analysis shows that blood contains about 60 volumes per cent, of gas, thus 100 volumes of arterial blood will yield 20 volumes of oxygen, 40 of carbon dioxide, and about 1 of nitrogen ; 100 volumes of venous blood will yield 12 volumes of oxygen, 48 of carbon dioxide, and 1 of nitrogen. Extraction and Analysis of the Gases of the Blood. — There are many forms of pump for the extraction of the gases of the blood ; the general principle is the exposure of the blood to a barometric vacuum. It will be sufficient for the student to work with the simple form of 188 PRACTICAL PHYSIOLOGY pump introduced by Leonard Hill. For other methods see Barcroft's papers on the subject in the Journal of Physiology or Ergebnisse der Physiologie, 7*" Jahrgang, 1908. The pump consists of a mercury reservoir A, which is connected with a second reservoir B by means of pressure tubing. The con- nection is surrounded by a mercury cup. The upper end of B is closed FIG. 184.— Hill's blood-gas pump. by a three-way tap fitted with mercury cups. By means of this tap B can be put in connection with either the tube E leading to the blood- receiver F, or with the tube C leading to the eudiometer H. The blood-receiver F is constructed of three bulbs, so as to prevent the blood frothing over into B during the extraction of the gases. On the lower end of F is a three-way tap. To the upper end of F is fixed a piece of thick small-bored pressure tubing provided with a clip. The mercury used to fill the pump must be cleaned and the pump ELEMENTARY EXPERIMENTAL PHYSIOLOGY 189 evacuated before use. In using the pump the manipulations are as follow : F is placed in the position indicated by the dotted line. A is raised and B is put in connection with F, and F is filled with mercury. The tap on the rubber tube at the upper end of F is then closed, and A lowered until F is exhausted, except for 2 or 3 c.c. of mercury which are purposely left within. F is next 1 2 3 FIG. 185.— The three-way tap of the mercury pump. The screw-clip on the lower end of F is next closed, and F is then detached from the pump and weighed. A sample of blood is collected in the following way : The arterial or venous cannula is connected by a rubber tube to F, and the tap turned so that the cannula and tube as far as the tap are filled with blood. A sufficient quantity of blood is now withdrawn by turning the tap into connection with F. It is now detached, and the blood is defibrinated by shaking it with the mercury left within F for the purpose. F is then again weighed, and the weight of the sample obtained, affixed to the tube E, and E is exhausted. Finally the screw clip between E and F is opened, and the gases are withdrawn and collected in the eudiometer. To facilitate the escape of the gases F is placed in warm water and shaken. If the blood froths too violently the frothing can be allayed by pouring some warm water on the tube E. The tap is so manipulated that the gases only, and not the water which condenses in B, are driven over into the eudiometer. The water is returned back into F. Several exhaustions are needed to extract the gases. The eudiometer tube is filled with mercury and surrounded with a water jacket to keep the temperature constant. The eudio meter is transferred to a vessel of mercury and , , iiii,. Fl°- 186.— C. mercury the volume of gas read, the level of mercury vessel; t. eudiometer; inside and outside the eudiometer being the same. p' plpctte> The temperature of the water in the jacket of the eudiometer is also 190 PRACTICAL PHYSIOLOGY read and the barometric pressure. Potash solution 20 per cent, is then introduced into the eudiometer by means of a pipette provided with a bent end. The carbon dioxide is thus absorbed and the difference in volume read. Pyrogallic acid is then introduced and the oxygen absorbed. The remainder is nitrogen. The temperature of the water jacket is kept constant by adding cold water during the estimation. To correct the volume of gas to 0° and 760 mm. the following formula is employed : — y H -/ l+t. 0-00367 where H = the observed pressure, /the tension of aqueous vapour at the observed temperature /. The value of 1 +t. 0-00367 and of / are obtained from tables (cf. Button's Volumetric Analysis). CHAPTER L. THE OXYGEN CAPACITY OF BLOOD. The Ferricyanide Method of Determining the Oxygen Capacity of Blood. — Haldane has introduced a simple method of determining the oxygen in combination with the haemoglobin of the blood. It depends upon the fact that the combined oxygen is liberated rapidly and com- pletely on the addition of a solution of potassium ferricyanide to laked blood. The gas can be easily collected and measured with apparatus similar to that of Dupre for the determination of urea in urine. The apparatus used by Haldane is shown in Fig. 187. The process is conducted in the following way : — 20 c.c. of oxalated or defibrinated blood, thoroughly saturated with air, are measured from a pipette into the bottle A. To this are added 30 c.c. of a weak solution of ammonia made from ordinary strong ammonia solution, sp. gr. 0'88, by diluting with distilled water to -^jo^h- The ammonia prevents the evolution of carbon dioxide and the distilled water lakes the corpuscles. The mixture is thoroughly shaken to complete the laking. Into the tube B are placed 4 c.c. of a freshly saturated solution of potassium ferricyanide. The rubber cork is inserted into the bottle A and the water in the burette is brought to a level close to the top by opening the tap and raising the levelling tube. The tap is closed and the reading of the burette taken. The water gauge attached to the temperature and pressure control tube is adjusted by sliding the rubber tubing backwards or forwards on the glass tube D. The bottle A is tilted so that the ferricyanide in B escapes and the mixture is shaken until the evolution of gas has ceased. If the pressure ELEMENTARY EXPERIMENTAL PHYSIOLOGY 191 gauge indicates an alteration in the temperature of the water this is adjusted by the addition of cold or warm water to the bath. After allowing the temperature to become constant and levelling the water in the burette and levelling tube, the amount of gas is read. The temperature of the water surrounding the burette and the height of Pio. 187.^-Ferricyanide method of estimating the oxygen capacity of blood. the barometer are taken and the gas is reduced to its volume at 0° and 700 mm. The chemistry of the process appears to be as follows :— The ferri- cyanide is reduced to ferrocyanide, for if ferricyanide be added to laked blood it will be found that the solution gives with ferric chloride the blue colour which indicates the presence of ferrocyanide. Oxygen is rendered available for the formation of methaemoglobin after the oxygen of the oxyhaemoglobin has been liberated. Hb/ | + 4Na3(Cy6Fe) + 4NaHC03 = 0.2 + Hbphthalmomctcr. This adjustment is carried out by turning the telescope towards a milk glass plate on the left of the subject, and moving the eyepiece till the thread is defined. The telescope is then directed towards the subject's eye, and moved with its stand backwards or forwards towards the observed eye till either of the reflected images of the illuminated areas on the quadrant is clearly defined. In the quadrant is a fixed area opposite a white line corresponding to the number 20 on the scale. Let the quadrant be first placed in a horizontal plane, with the fixed illuminated area to the left. According to the varying position of the right illuminated area two pairs of images will now be seen reflected from the cornea, and attention should be directed to the two middle of these images, which may or may not overlap (Fig. 211). The right moveable area should now be adjusted on the quadrant so that the edge of one image just touches the edge of the other, the 'stepped' image being to the left and the rectangular area to the right. A white line on the back of the right illuminated area will now point to some number on the scale ; when the images are adjusted as above, this number + the 20 corresponding to the position of the left illuminated area, will express numerically the degree of curvature of the cornea. According to the constants of the instrument if the number 337 be divided by the number expressing the curvature of the cornea as above, the quotient represents 232 PRACTICAL PHYSIOLOGY the radius of curvature of the cornea in the horizontal meridian examined. The use of the instrument for measuring astigmatism may here be detailed. EXPERIMENT. Method of Measuring Astigmatism. — By the use of the ophthalmometer represented in Fig. 211 the difference of curvature of different portions of the cornea can be easily ascertained. FIG. 211. —The images in the astigtnometer. The apparatus is adjusted as described above, and the horizontal meridian is first observed. If the curvature in this meridian is regular the four figures will be seen to stand on a level base. If this is not the case, the rotating quadrant must be moved till continuity of base line is obtained. The moveable illuminated area is then adjusted till the four reflected images are as in the figure. The quadrant is then rotated, and as it approaches the vertical the two central images will probably overlap. Note the meridian where the greatest amount of overlap is observed. This will be the most retract- ing meridian. Each tread of the steps in the illuminated area corre- sponds to one dioptre1 of curvature. The excess of curvature of the most refracting meridian may thus be read off at once. CHAPTER LXVII. THE REFRACTING MEDIA OF THE EYE. Ktihne's Artificial Eye. — The nature of the refraction produced by the various media of the eye is conveniently illustrated by means of this instrument (Fig 212). It consists of an oblong box, one of the long vertical sides being generally made of opaque material, the other of glass. The front end of the box is bounded by a curved glass surface, the hinder end is a plane sheet of glass. Various accessories are supplied with the instrument, such as a double convex lens which can be placed in the axis of the box behind the cornea, a frosted glass JA lens in which the focus for parallel rays is at one metre is taken as the standard lens, and its degree of refractive power is represented as one dioptre. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 233 screen which is used as a receiving surface for the refracted rays, and an opaque screen with a central hole. The box is first filled with water, and in order to make rays of light the clearer, a few drops of some fluorescent solution (e.g. eosin) are added to the water. An external luminous object is then arranged. This may be conveniently done by placing a metal plate, in which a vertical arrow has been stencilled out, in front of a good source of light, such as the naked arc light of an electric projecting lantern, with the condenser and focussing lens removed. This stencilled plate is placed four or five feet from the front of the instrument. FIG. 212.— Ktihne's artificial eye. 1. The Action of the Cornea. — If the illuminated arrow be placed approximately in the optic axis of the artificial eye, the rays of light will be seen passing through the box and converging somewhat in their progress. If the frosted glass screen be placed in the box, however far back it be arranged, no image of the arrow will be obtained. If, how- ever, a screen be placed some distance behind the box an image will be formed. We have here illustrated the fact that without some specially strong refracting medium in the eye, external objects would be focussed behind the position of the retina and therefore not clearly visible. This is the case after the operation for cataract in which the crystalline lens is removed. 2. The Action of the Crystalline Lens. —Let the double convex lens supplied be now placed in the box at the front end. This at once 234 PRACTICAL PHYSIOLOGY causes a much greater convergence of the rays, and it will be possible to obtain an image of the arrow upon the frosted glass screen, when this is placed about three inches from the hinder end of the box. This image may be easily seen on looking obliquely through the glass end, or may be projected by a convex lens on a lantern screen sufficiently clear for a number of observers to see. 3. The Action of the Iris. The iris improves the definition of the image by cutting out the more circumferential rays which in consequence of spherical aberration would not be focussed in the same plane as the more central. If the opaque screen having a central hole about an inch and a half in diameter be placed in front of the convex lens the total amount of light passing behind the lens is decreased, but the image is now much more sharply defined. 4. The Position of the Image. — It will be noticed that if the illuminated arrow point upwards the image on the artificial retina will point down- wards. Images on the retina are therefore always inverted, the lower half of the retina corresponding to the upper half of the field of vision and conversely. By experience we always refer images on the retina to their proper position in the field of vision. This rectification corre- sponds to what is done by the second convex lens in projecting the retinal image upon the lantern screen. The etfect of this second lens is to re-invert the image, so that on the lantern screen the image appears in the same position as in the original object. 5. Accommodation. — It is not possible with the artificial eye to mimic the changes that occur in the lens on accommodation. A clear image of objects at different distances can only be obtained by shifting the artificial retina backwards or forwards. ACCOMMODATION. 1. The eye is Able to see objects at varying distances from the eye. It has the power of adapting itself so as to form a clear image on the retina of different objects. Unless the eye had this power images of external objects at different distances would not always be formed at a constant distance behind the crystalline lens, where the retina is situated. EXPERIMENT. Standing about 15 feet from a window and looking towards it, hold up a needle about two feet from the eye. If the needle be seen clearly the window sashes will be blurred, since the image of these will be in front of the retina. If the window sashes be looked at and seen clearly then the needle will be blurred, since the image of this is behind the retina. 2. Range of Accommodation. Determination of Near and Far ELEMENTARY EXPERIMENTAL PHYSIOLOGY 235 Points of Accommodation. Line of Accommodation. — At a certain distance close to the e}re the power of accommodation is lost. EXPERIMENT I. Hold a needle about 2 feet from the eye and gradu- ally bring it nearer ; it is for a certain time possible to obtain a clear image. At a certain distance, in spite of effort, the image begins to get blurred. The least distance at which one obtains a clear vision of the needle corresponds to the near point of accommodation. This is generally about 8 inches. In short-sighted persons a far point of accommodation may also be shown. If the distance between the two objects be not too great, although they are both in the line of sight, they may be seen clearly at one and the same time. That is to say that accommodation of a certain degree will enable the observer to see objects at varying distances from the eye. The maximum distance at which two objects in the line of sight may be separated will vary with the distance of the nearer of them to the eye. As the nearer object recedes from the eye the line of accommodation or the distance between the two objects increases. [ADVANCED EXPERIMENT. Place two pins in the line of sight and note the distance apart at which they are both visible as single objects at the same time. Make observations with the nearer at 20 cm., 50 cm., 2 m. It will be found that the line of accommodation lengthens with a greater distance from the eye.] 3. Formation of Image in Excised Eye. — The excised eye is accom- modated for objects at a distance. EXPERIMENT. Remove the sclerotic and choroid from a fresh sheep eye, and place it, cornea outwards, at the end of a cylinder of brown paper. Direct it towards the window, and on looking down the tube an inverted image of the window will be seen. This experiment can be still more easily performed on the eye of a freshly-killed albino rabbit, which, for convenience of handling, should be fixed in a ring of modelling wax or clay. In this case the sclerotic and choroid are sufficiently thin to obviate the necessity for their removal. 4. Action of Iris in Accommodation, and its Changes with Variations in Amount of Light. — The iris cuts off the more peripheral rays imping- ing on the cornea, otherwise the clearness of the image on the retina would be diminished. This is especially the case when viewing near objects, as here the angle of incidence of the circumferential rays is greater. EXPERIMENT I. In not too bright a light direct the subject's attention from a far to a near object. It would be noticed that the pupil becomes smaller. 236 PEACTICAL PHYSIOLOGY EXPERIMENT IT. Make the subject close one eye and shade the open eye from the direct light. Observe the size of the pupil when the eye is shaded. Then remove the shade ; the pupil will be seen to diminish in size. From this experiment it may be inferred that the amount of light entering the eye is controlled by the iris. [ADVANCED EXPERIMENT. Make a pinhole near the edge of a card, and hold the card about 15 centimetres from the right eye, so that it does not interfere with the field of the light. Let a good source of light be placed about 60 centimetres from the eye, and allow a thin paper-screen to shield the light from the right eye. The left eye, when open, will look directly at the light, the right eye at the pinhole, and the illuminated paper through the hole. Close the left eye, and accommodate as nearly as possible for the distance of the pinhole. Note the size of the hole. Then alter the accommodation by attempting to look far away through the pinhole. The hole will immediately become distinctly larger, though less definite, on account of the blurring of the edges. Keep varying the accommodation, and the edge of the hole will similarly vary. Whilst accommodated for far distance open the left eye. The sudden entry of light in the left eye will cause reflexly a diminution in size of both pupils. The pinhole will now become smaller. Close the left eye again and it enlarges. The size of the blurred image of the pinhole depends upon the size of the pupil, and hence variations in size of the pupil appear as variations in size of the pinhole.] 5. The changes in the Lens during Accommodation. Purkinje Sanson Images. — During accommodation for a near object, the ciliary muscle contracts, with the consequence that the suspensory ligament is slackened. The lens by its natural elasticity becomes more curved in its anterior aspect, and its thickness through the optical axis is increased. This change of curvature can be measured by means of the ophthalmometer. The existence of such a change may be inferred from the following experiments in which observations are made upon the images reflected from the anterior surface of the cornea, the anterior surface of the lens, and the posterior surface of the lens. EXPERIMENT I. (PRELIMINARY). In a dark room place on a table, rather to the right of the observer, a convex lens mounted on a stand. Hold a watch glass a few inches in front of the lens, with the convex surface of the glass forward. Still more to the right let a lighted candle be placed. The candle and the observer's eye should be symmetrically arranged on either side of the optic axis of the lens and watch glass. Observe the images reflected from the surface of (a) the watch glass ; (b) the anterior surface of lens ; (c) the posterior ELEMENTARY EXPERIMENTAL PHYSIOLOGY 237 surface of lens. The images at (a) and (b) are erect ; at (c) is inverted ; the image at (b) appears to be the most deeply situated of the three. EXPERIMENT II. In a darkened room let the observer bring a lighted candle near the eye of the subject, rather to one side of his optic axis. The observer places himself so that his eye is sym- metrical in position to the candle on the other side of the optic axia of the subject. When properly adjusted there should be observed reflected from the eye of the sub- jecc three images the first bright and erect, reflected from the cornea ; a second near the centre of the pupil, but much feebler than the first, and apparently the most deeply situated of all the images, this being reflected from the an- terior surface of the lens ; a third image represented by a mere spot of light differs from the other two 0 . . FIG. 213.— The phakoscopc. in being inverted, ir now the accommodation of the subject be shifted from a far to a near point, the middle image will advance but grow smaller, and will approach the corneal image. The other images do not alter. During varying accommodation it is found that this image is the only one to change, thus indicating that the change is in the anterior surface of the lens. EXPERIMENT III. — The Phakoscope. — This instrument is specially adapted for viewing the reflected images of Experiment II. It is repre- sented in Fig. 213. Fig. 214 represents diagrammatically the arrange- ment and course of the rays of light. It consists of a dark box, roughly triangular in shape, with the angles of the triangle bevelled off, and at S and 0 fitted with windows (Fig. 214). The observer's eye is at 0, the subject's at S. At 0 two prisms are arranged vertically so as to allow two illuminated squares to fall upon the eye at S. The eye at S can either be focussed for the vertical needle at Wt or (since this lies in an opening) for distant objects beyond the opening. With the alteration of the lens corresponding to the change of accommodation, the images from the anterior surface of the lens will vary as in Experiment II. 238 PRACTICAL PHYSIOLOGY 6. Schemer's Experiment. — If the eye be accommodated for an object at any particular distance, the effect of preventing the retina Fio. 214. — Diagram of the course of the rays of light in the phakoscope. receiving all the rays from the object (as by a screen with holes pricked in it and held close to the cornea), is simply to diminish the brightness of the image, on account of the lessening of the amount of light entering the eye. Any object at a distance for which the eye is not accommodated will form a blurred image on the retina, and if rays from the object by this partial screening of the retina have several paths by which to impinge on the retina, there will be formed upon the retina as many blurred images as there are openings in the screen. When, however, the eye is accommodated for this second object, these blurred images fade into one clear image. EXPERIMENT I. To form a screen take a thin piece of cardboard and prick two holes in it, separated by less than the diameter of the pupil. About one-sixteenth of an inch will answer. Place in a strip of wood about a yard long two vertical needles, distant eight and twenty-four inches from the eye. Close one eye and with the other, holding the screen close to cornea, look at one of the needles. The other needle will be also seen, but represented by a double blurred image. If the more distant needle be accommodated for, a double blurred image of the nearer will be obtained. Cover one of the holes in the screen with another card. If the right hole be covered the left blurred image will disappear, and conversely. Let the eye be now accommo- dated for the nearer image. A double blurred image of the more distant needle will be seen. If the right hole of the screen be now covered the right blurred image will disappear, and conversely. EXPERIMENT II. A slight modification of this experiment and the material requisite is provided in the Milton Bradley Pseudoptics, Section I., exp. 4. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 239 EXPERIMENT III. Experiment I. can be most instructively performed with Kiihne's Artificial Eye. A special screen for the experiment is provided in which one hole is covered with red mica. Accommo- dation for the different distances is provided by shifting the retinal screen backwards or forwards, and the illuminated arrow can be used as an external object. It is found that if the screen be shifted forward so as to accommodate for objects beyond the arrow, that two blurred images of the arrow obtain. Covering either hole will block either image. But when the eye is accommodated for a more distant object it will be observed that covering the left hole removes the left retinal image. If the images be projected, as before, on the lantern screen, the opposite image will of course be removed. The apparent contradic- tion between Experiments I. and III. is obviously due to the fact that in I. the imag- s are referred to the field of vision, in III. (without the use of further projection on the lantern screen) they are actually viewed as formed on the retina. EXPERIMENT IV. The near point of accommodation can be conveniently ascertained by noting the least distance at which a single image of a needle can be seen, when using the perforated screen of Scheiner's experiment. EXPERIMENT V. In Experiment II. note that the thread on which the needle hangs remains clear as a single thread for a certain distance on either side of the needle, but that beyond this distance it gradually bifur- cates into a double threa 1. This singleness of the thread corresponds to the length of the line of accommodation. 240 PRACTICAL PHYSIOLOGY CHAPTER LXVIII. THE RETINA. 1. The Blood-vessels of the Retina. — The blood-vessels supplying the retina are distributed to the anterior portion of the retina, the main vessel entering the eyeball at the spot where the optic nerve passes in. These blood-vessels then lie between the vitreous and the sensitive part of the retina, and under certain circumstances they may throw shadows upon this portion of the retina. EXPERIMENT I. Purkinje's Figures. —Make the subject of the experiment turn one eye inwards, and with a lens concentrate a good light upon the exposed sclerotic, focussing the light so as to make a small but strongly-illuminated area. Let the subject look towards a dark wall. Give the lens a gentle rocking or circular movement. The field will appear to the subject as reddish-yellow, and dark figures will be seen by the subject appearing in the field, which branch and have the character of the retinal blood-vessels, of which they are really the shadows. In the direct line of vision a small area will be seen free from these branching shadows. This is the yellow spot. EXPERIMENT II. Through a pinhole in a card held close to the eye, look at a brightly and evenly-illuminated surface, as a white cloud or a sheet of thin white paper held in front of a lamp. Give the card an up-and-down movement, and a number of vessels will be seen running horizontally in general. Move the card from side to side, and vertically-running vessels will be apparent. Give the card a circular movement and the general distribution will be visible. Note that in the direct line of vision is a small area in which no vessels are seen, the macula lutea or yell >w spot. EXPERIMENT III. Remove the objective fr»m a microscope, arrange the mirror for a good light, and move the microscope in the same way as the card was moved in Experiment II. The results will be as in that experiment. In all these experiments the movement of the light or the illuminated field is essential. The retina appreciates these shifting shadows better than if they were continually applied to any fixed point of its surface. Further, a moving object will arouse attention more readily ihan one of constant position, which tends to be neglected. 2. The Circulation in the Blood-vessels of the Retina.— EXPERI- MENT.— Look through a thick piece of blue glass at a white cloud. Many finely-illuminated points will be seen traversing the field. These ELEMENTARY EXPERIMENTAL PHYSIOLOGY 241 again are followed by slight shadows. Fix the gaze and note that these points move in constant directions. They probably represent small local collapsings of fine capillary blood-vessels, caused by tem- porary clogging of the red corpuscles. The re-filling of the vessel brings about the shadow following the bright point. 3. The Blind Spot. — A certain region of the retina, to the inner side and somewhat below the macula lutea, is insensitive to light, inasmuch as the optic nerve here enters the eyeball, and the layer of the retina which reacts to the stimulus of light is here absent. This insensitive region is spoken of as the optic disc or blind spot. Experiments showing the nature of the blind spot may be con- veniently carried out with the material in Section H. of the Milton Bradley Pseudoptics series. EXPERIMENT I. Using cards H.2, or H.3, close the left eye and fix the gaze of the right eye on the cross. At a distance of about eighteen inches the tree in H.2 or the red disc in .H.3 will disappear. EXPERIMENT II. Arrange the cards H.4 and H.5 at such a distance that when the left eye is closed and the right eye gazes at the cross, the house in H.4 or the red spot in H.5 falls on the blind spot. It will be found that similarly, with the right eye closed and the left eye fixating, the cro-s, the church, and the yellow disc will be invisible. Having found the proper distance, open both eyes and place the card H.4x close to the nose and in the plane of the septum of the nose. It will be found that when the gaze is directed to the cross the surface of the cards nppears uniformly white. EXPERIMENT III. If a dot and a cross be drawn about four inches apart, the dot being about half-an-inch above the horizontal level of cross, and if then the left eye be closed and right eye gaze at the dot, at the distance of about a foot, the cross will be invisible, as its image falls on the blind spot. When any image falls upon the blind spot it is invisible. By imagina- tion we Jill in this region of any image falling upon the retina by sensations similar to those in the neighbouring regions. This is well illustrated in the following experiments. EXPERIMENT IV. Using the cards H.6, H.7, H.8, and H.9, and ascertaining the distance at which they should be placed, as in Experiment I., notice that when the coloured discs fall upon the blind spot, the place of the discs is taken by a combination of the background on which the discs lie. In H.9 in particular there seems no break in the chequered pattern forming the background to the red disc. The blind spot may be mapped out with ease in the following manner. Q 242 PRACTICAL PHYSIOLOGY EXPERTMRNT V. Let the head rest in a fixed position, as by placing the chin in a tin mug, and place a sheet of white paper vertically in front of it at a distance of eighteen inches. Put a dot in the centre of the paper Close one eye and with the other fixate the dot. Take a thin strip of white card-board and blacken about two millimetres of the end. Move the blackened end over the region of the field of vision corresponding to the blind spot, and note the points where the black area disappears, marking them on the white paper. A sufficient number of these points can be taken, nnd a curve drawn through them will indicate the margin of the field of the blind spot. 4. The Yellow Spot. — The experiments performed to exhibit the retinal circulation have shown that there is a certain region in the direct line of vision where the retinal blood-vessels are not visible. This region is coloured by a pigment which absorbs the blue and green of the spectrum, and therefore appears of a reddish-yellow colour and is called the yellow spot. EXPERIMENT. Take a flat-sided bottle containing a fairly strong solution of chrome alum, or use a sheet of purple or violet gelatine. Look with one eye closed through the coloured medium at a bright white surface. A rose-coloured oval spot will appear in the centre of the field. The pigment of the yellow spot absorbs the blue and green, and transmits the rest, and hence the predominant red tinge imparted to the area corresponding to the macula hi tea. 5. Acuteness of Vision in different Regions of the Eetina. — In order to differentiate similar objects grouped closely together it is necessary that these should subtend an angle of a certain magnitude with respect to the eye. To be more precise, the angle subtended is at the nodal point of the schematic eye, and this angle again is equal to that sub- tended at the nodal point by the image of the differentiated objects on the retina. In order that objects be differentiated it is apparently necessary that their contiguous margins and the space between should form an image on the retina, which is of certain length. Helmholtz found that a subtended angle of 63 '75", equivalent to a retinal distance of -00463 mm, was necessary for discrimination. As far as this method of investigation is concerned it appears to connect visual acuity with the distribution of the cones. [ADVANCED EXPERIMENT. Set up in a good light the parallel line diagram used in the experiment on chromatic aberration (Experiment III ). Or arrange a series of five black wires, separated by their own diameter, against the sky. Walk backward from either of these objects till they can just be no longer discriminated. Calculate the size of the retinal image. ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 243 The visual acuity diminishes rapidly on the retina as we recede from the fovea. The diminution is more marked in the vertical than in the horizontal meridian.] [ADVANCED EXPERIMENT. Place on a card two dots, each 2 mm. in diameter and separated by a distance of 2 mm. Let the gaze be fixed on a mark on a vertical white sheet of paper, and let the card be moved in a horizontal meridian gradually nearer the mark till the two dots can be discriminated. Compare the vertical and horizontal meridia in this respect.] The acuteness of vision at the fovea is ordinarily tested by noting the distance at which letters, which at a given distance subtend an angle of 5' at the eye, can be read. This method may be applied either to ascertain what error of refraction may exist in the eye, or if this be absent or corrected, what the acuteness of vision in the particular eye is. EXPERIMENT III. Using Snellen's or Jaeger's test types, ascertain whether the letters can be correctly named at the normal distance in a good light. If this distance can be exceeded or if it cannot be reached an expression for the condition of the acuteness of vision may be written as follows : where d = distance of person from the types and D = number of smallest type which a person can read at that distance. 6. Mechanical Stimulation of the Retina. — Phosphenes. — The retina can be stimulated by pressure on the sclerotic. An image will be produced which is referred to the opposite portion of the field of vision. EXPERIMENT I. Close one eye and turn it as far as possible towards the nose. Press with a pencil point on the sclerotic, through the eye- lid, at the edge of the orbit on the outer side. Note the circle of light which appears on the nasal side. The retina is stimulated just beneath the pressure and the image is referred to the nasal side of the field of vision. 7. The apparent Inversion of Shadows thrown upon the Retina. — If a beam of light falling upon the retina be intercepted by some object close to the cornea, an erect shadow of the said object will be thrown upon the retina. This, however, will be projected into the field of vision as an inverted image. EXPERIMENT. The Experiment No. 6, Section I. in Milton Bradley Pseudoptics, illustrates the nature of retinal shadows well. 8. The Perception of Colour in the Peripheral Portion of the Retina. —The sensibility of the retina for colour varies in different zones of the 244 PRACTICAL PHYSIOLOGY retina, and for different colours. Blue and yellow can be recognised at a greater distance from the fovea than red and green. Still more peripherally all colours appear as black, grey, or white. EXPERIMENT I. Milton Bradley Pseudoptics, Section H, Experiment No. 1, conveniently illustrates the variation in the sensibility of the retina for colour. EXPERIMENT II. If a perimeter or campimeter be used the boundaries of the field for the different colours can be defined. (See use of perimeter.) 9. The Perception of Light in different Regions of the Retina. — A faint light is often more easily seen when its image does not fall on the fovea, but a few degrees away from this. The recognition of a light at sea on a dark night is often facilitated by directing the gaze some ten degrees to the right or left of the supposed luminous object. Faint stars again may be seen more readily if not directly gazed at. 10. After-images. — After-images may be of two kinds, those which reproduce the original body in all its brightness, those that are the reverse in brightness to the original body. The first are called positive after-images, the second are negative after-images. Positive after- images may be either of similar colour to the original body or comple- mentary in colour, negative after-images are always complementary. They are due to certain changes taking place in the retina and are best observed in the early morning after waking. EXPERIMENT I. Close the eyes for two minutes to rest them and then for the briefest possible interval look at some bright source of light as the lamp or the window, closing the eyes again. A bright positive after-image of the source of light will be seen. EXPERIMENT II. Look at the incandescent filament through a piece of red glass, as in Experiment I. The positive after-image will appear red. Again look at the filament but for a prolonged period of about half a minute. On closing the eyes the after-image will appear bright but greenish in colour. By an alteration of light and dark backgrounds the after-image may be changed from negative to positive. EXPERIMENT III. Look at an incandescent lamp for half a minute and so get a well marked after-image. If the eyes be directed to a white surface the after-image will be negative, if to a dark surface it will appear positive. [ADVANCED EXPERIMENT. Note the colour of the after-images in Experiment III., and the gradual change in colour which they show. If the after-images tend to fade blink the eyes several times rapidly and they will become more marked. Notice especially the effect of ELEMENTARY EXPERIMENTAL PHYSIOLOGY 245 blinking on the negative afterimage seen on the white surface. It will become during the shutting of the eyes converted into a positive after-image.] FIG. 215.— Disc for the experiment on after-images of motion. [ADVANCED EXPERIMENT. Look at an incandescent lamp with the right eye, the left eye being closed. After the lapse of half a minute, shut the right eye and look with the left at a dot on a white sheet of paper, as far as possible without blinking. After a time the field will gradually darken and a positive after-image will be seen. This is really the after-image seen with the right eye, which is not visible till a certain amount of retinal insensibility has occurred in the left eye.] [ADVANCED EXPERIMENT. After-images of motion may be shown by gazing at the disc in Fig. 215 slowly rotated and then shifting the gaze to some uniformly mottled surface.] CHAPTER LXIX. SENSATIONS OF LIGHT AND COLOUR. MANY theories have been advanced to explain the phenomena con- nected with colour vision. The most important of these theories are those connected with the names of Young-Helmholtz and Hering. The theories are all concerned in referring the multiplicity of colour 246 PRACTICAL PHYSIOLOGY sensations to fusion of certain simpler sensations, which are described as primary colour sensations. In the Young-Helm lioltz theory the primary sensa ions are those corresponding to red, green, and blue- violet; in the tiering theory they are grouped in pairs, which are the red and green sensations, the yellow and blue sensations, and the white and black sensations. It is necessary to assume the existence of certain photo-chemical substances in the retina, which can be acted upon by the light of the primary colours. The light at the ends of the spectrum would, in accordance with the Young Helmholtz theory, act upon either the red visual substance or the violet visual substance, in the intermediate part of the spectrum upon all three visual substances to different extents. If all are affected more or less equally, the com- pound sensation of white is produced. In the Bering theory there would also be assumed to exist three primary visual substances, but according to the chemical changes in any single substance, whether of the constructive or destructive variety, so a sensation corresponding to one of the complementary colours of the different pairs would be brought about. A certain classification of colours is necessary. They may be con- veniently described as varying in hue, tint, or shade. The hue of a colour is its colour tone, corresponding to its wave length. The tint of a colour depends upon its purity, or whether it is admixed with white— in other words, depends upon its saturation. The shade of a colour is an expression of its brightness or intensity, or, what comes to much the same thing, the degree to which it is admixed with black. 1. Colour Tone. — In reviewing the changes of hue that are appreciable in examining the spectrum, it is to be noticed that the changes do not occur at any regular intervals corresponding to wave lengths. Changes of colour tone are most easily appreciated in the yellow, green, and blue green. At the red end and violet ends there appears to be little or no change of hue. The variations in saturation or tint can be seen by using the red and white discs of a colour mixed in varying proportions and noting the corresponding sensations produced. 2. Intensity. — Variations in intensity cause changes in the quality of colours. At their maximum brightness colours tend to give the sensa- tion of white, though they never completely do this. The yellow will the most easily ; the blue and violet approach close to it. The red is most distant in producing the sensation of white. EXPERIMENT I. Take a small square of red paper and a similar piece of blue paper which in a light of moderate brightness appear of approximately equal intensity. Carry these to an almost dark room ELEMENTARY EXPERIMENTAL PHYSIOLOGY 247 and note the dulness or even blackness of the red whilst the blue may still appear bright. 3. The Fusion of Distinct Sensations of Black and White. Flicker. — This fusion depends upon the persistence of the positive after-images each separate stimulus brings about. If separate stimuli follow each other sufficiently rapidly a blending of the different sensations occurs, as is well exemplified in the presentation of the series of rapidly succeeding views in the cinematograph. The phenomena upon which this depends can be shown in a revolving disc divided into rings of sectors of white and black, increasing in number from the centre to the circumference. Such a disc is included in the Petzold series. EXPERIMENT I. Rotate a disc such as (Fig. 216) slowly, and note that at a certain rate the peripheral ring appears as a uniform grey, a flickering sensation is produced on the neighbouring rings, but the central rings show an alternation of white and black. Increase the rate and note that these can also be caused to blend. In general it may be stated that when fusion in any way occurs the resulting sensation of grey is the same as if the light reflected inter- mittently were replaced by the same quantity of light continuously reflected, in other words, as if a uniform grey of a certain shade were substituted for the series of sectors ; moreover, if the rate at which the sectors are successively presented to the retina be increased above that necessary for fusion, the intensity of the resulting sensation is not altered. (Talbot-Plateau Law). 248 PRACTICAL PHYSIOLOGY The rate necessary for the flickering sensation to pass into complete fusion depends upon the intensity of the light. [ADVANCED EXPERIMENT. With a metronome, note the rate of revolu- tion necessary to produce complete fusion in the outer ring. Darken the room and observe whether the rate be altered. It will be found that with diminished light a slower rate of revolution brings about fusion. The converse is true up to a certain limit. The point at which flicker passes into fusion has been used as a means of determining the condition of persistence of visual sensations. It is to be noted that the flicker may be coarse or of a fine tremulous character. The transition of this fine flicker into fusion should be taken as the limiting sensation. The excitability of any portion of the retina is influenced by the stimulation of that portion of the retina (temporal induction) and changes are simultaneously induced in neighbouring regions of the retina (spatial induction). These factors may be of very considerable influence in determining the point at which flicker passes into fusion. A 'physiological' state is brought about by a certain 'physical' stimulus, and thereby the effect of the stimulus may be increased or diminished. If then a succession of stimuli of say blue and black be presented to the retina at a certain rate flicker will pass into fusion. But if the blue be intensified by being placed on a black background this rate will no longer be sufficient. This may be shown in the following manner.] FIG. 217. [ADVANCED EXPERIMENT. Take a disc like that shown in Fig. 217 with black and blue semi-circular rings, and yellow and black back- ELEMENTARY EXPERIMENTAL PHYSIOLOGY 249 grounds On rotating this disc it will be observed that the flicker persists much longer in the outer blue and black ring than in the inner blue and black ring. FIG. 218. Fechner showed that certain colour effects may be produced by slow rotation of discs which consist of black sectors of increasing size on a white ground. They may also be seen in a disc showing black circular lines of different circumferences on a white semicircular area, the other half of the disc being black. Such a disc is shown in Fig. 218. It has been shown that a bright object on a dark background appears, when suddenly exposed, to be surrounded with a red border lasting a fraction of a second. If the illumination be brighter a blue green effect is visible. These facts in part explain the appearance of colours shown when the discs below are rotated.] 4. The Fusion of Colour Sensations. — Several methods have been devised with the object of enabling us to fuse separate colour sensations. These depend either upon separate colours forming images on the retina in such rapid succession as to be inseparable, or else upon separate colours forming images in the same portion of the retina so that the sensations are super imposed. The first method is generally carried out by means of the separate colours being arranged as sectors in a circle, which is rapidly revolved about its centre, the instrument adapted for the purpose being known as a colour-mixer. Discs of different colours, such as the Wundt series, are obtainable, and each disc has a radial slit at one point so that these can be arranged upon a common centre and a circle may be made up of sectors of various discs. It is desirable to have discs of two sizes, one about ten inches across, the other four or five inches. It is to be remembered that these discs are not coloured with pure colours of the 250 PRACTICAL PHYSIOLOGY spectrum, and the results of their mixture yields various colours which are largely mixed with grey. EXPERIMENT I. Take two large discs of red and green and two small of black and yellow. Adjust the proportion of the red and green so that rapid revolution produces a yellow. This will be dark in shade and can be matched by the inner discs of yellow and black. EXPERIMENT II. Take large discs of green and violet and small discs of blue and black. With the large discs a blue can be obtained and matched with the smaller discs. EXPERIMENT III. Take three large discs of red, green, and violet. To bring about a good result the red should correspond to the red in the spectrum at wave-length 6300, the green to wave-length 5150, and the blue to wave-length 4700. Arrange these so that red constitutes about 118°, green about 146°, and blue about 96°. Arrange also two smaller discs of white and black. As the result of revolution the larger discs will give a grey, which can be matched by about 285° black and 75° degrees white of the smaller discs. EXPERIMENT IV. Using the three discs of Experiment III., work gradually through the whole spectrum, using different sized sectors of each for the different regions of the spectrum. The sizes of these sectors will roughly correspond to the different degrees in which the three primary colour sensations according to the Helmlioltz theory are evoked. The best method of fusing the colours sensations is to superimpose the various colours of the spectrum by projection of the same on .a white screen.1 By means of lenses the spectrum can be recomposed as white light. By introducing shutters eliminating certain portions of the spectrum the result of fusion of the remaining colours can be examined. 5. Complementary Colours. — For every colour in any part of the spectrum there is a colour in another part of the spectrum which, when mixed with it, will yield a white or grey. Such colours are said to be complementary. EXPERIMENT I. Take from the series of colour discs one of an orange colour. If no disc can be found which in any proportion with the orange disc will give a white or grey, take the blue and green discs and adjust all three so that a grey is obtainable. (This should be estimated by smaller discs of black and white). A certain proportion will exist between the blue and the green. If now the whole circle be 1 See Abney, Colour Vision, p. 18 et seq. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 251 divided up into blue and green in this proportion, revolution will give the hue of the colour complementary to the orange originally selected. It will be found by such experiments as this that orange is comple- mentary to greenish-blue, red to bluish-green, yellow to blue, yellowish- green to violet, and green to purple. EXPERIMENT II. If a coloured object be viewed on a white surface it may provoke a negative after-image in colour complementary to that of the original object. In illustration of this perform the experiments Nos. III. and IV. of Section E in the Milton Bradley Pseudoptics series. 6. Contrast. — Besides the effect which different colours produce when presented simultaneously, or practically simultaneously, to the retina, as in colour-mixing, other effects also will come about when different colours are presented successively and comparatively slowly to the same portion of the retina, or again, when different colours are pre- sented simultaneously to adjacent areas of the retina. In the first of these two cases we have the conditions of Successive Contrast, in the second we have Simultaneous Contrast. The second experiment in the section on Complementary Colours affords illustration of Successive Contrast. In general the nature of successive contrast may be shown as follows. EXPERIMENT I. Take a number of small squares of various colours each about 1 cm. in size. Arrange also a series of fields of different colours, as well as one of white ; these may be squares of 1 or 2 decimetre side. Taking a small red square, place this in the centre of the large white square and in a good light gnze at it for t*o or three minutes. Blow the small object away and continue the gaze. An after-image of the object will be obtained of a colour complementary to that of the original. Substitute for the large white square squares of different colour and perform the experiment again. It will be found that the after-image varies in colour according to the ground on which it is viewed. If red be the colour of the original small square, the after- image on white will be green or bluish-green. If projected on violet the after-image will be blue and if on orange a dull brown. EXPERIMENT II. By projection successive contrast may be easily demonstrated as follows. Two lantern slide glass plates are taken, and on one is marked out, in centre of plate, two concentric circles of about 1-5 and 3 cm. radius, enclosed by black lines of just perceptible thickness and having a central dot of about the same 2 or 3 mm. diameter. On the second glass plate are fixed rings of coloured gelatine of similar size to the two circular rings, the colours 252 PRACTICAL PHYSIOLOGY chosen being preferably complementary. The two slides are pro- jected simultaneously and the rings are gazed at (the central dot being used as fixation point), for half a minute. The slide with the coloured rings is then suddenly removed, the gaze remaining on the dot, when the two rings will be seen in colours complementary to the original colours. Simultaneous contrast may be shown in the following shadow and mirror experiments. [ADVANCED EXPERIMENT. Arrange two sources of light about six inches apart, and allow each of these to throw a shadow of some opaque object upon a screen held about a yard from the source of light. (8 candle-power and 16 candle-power electric incandescent lamps answer very well for the two sources of light.) Interpose a coloured glass plate in front of the weaker light. The shadow corresponding to this will be the same colour as the plate, the other shadow will become coloured complementarily. Observe the variation in intensity of colour according to the proximity of the two shadows. If the object be moved away from the screen the two shadows will separate and the colours will be dull, if the object approach the screen closely the shadows will almost touch and the colours will be extremely vivid.] [ADVANCED EXPERIMENT. Arrange a mirror horizontally, so as to reflect light from a white surface, e.g. a white lamp shade. Place a coloured glass plate over the mirror. Interpose an opaque object, a pencil or the finger, in the course of the white light incident on the mirror. Observe that two reflected images of this are seen, one from the surface of the coloured glass and of the same colour as the glass, the other reflected from the surface of the mirror and complementary in colour. Gently tilt the coloured glass so as to separate the images. It will be found that they are most brilliantly coloured when slightly overlapping.] EXPERIMENT III. Place the dark grey papers of Experiments III. and IV., Section G, of the Milton Bradley Pseudoptics on the different coloured fields arid cover with tissue paper. Observe the contract colour that appears in the grey paper. EXPERIMENT IV. Arrange on the colour-mixer the discs of Experi- ment V., Section G, of the Milton Bradley Pseudoptics. On rotating these, the black and white rings will assume a colour in contrast with that of the general field. [ADVANCED EXPERIMENT. The Experiments I. and If., of Section G, Milton Bradley Pseudoptics, illustrate the effects of contrast in black and white alone.] ELEMENTARY EXPERIMENTAL PHYSIOLOGY 253 The above experiments on Complementary Colour and Contrast depend upon variations in excitability in the retinal area involved or in adjacent retinal areas. The change in excitability that occurs in any retinal area when affected by incident light is spoken of as caused by temporal induction, and the change that is brought about in adjacent areas as resulting from spatial induction. Successive contrast depends upon temporal induction, simultaneous contrast upon spatial induction. The phenomena connected with the formation of after-images are examples mainly of temporal induction. With regard to the complementary colour of after-images, this is thought by some to be simply the result of fatigue. Others regard the phenomena as due to initiation of processes, the converse of those brought about by the original stimulus. Bering's theory of colour vision involves an explanation of these processes in accordance with the latter view. In this connection it will not be out of place to refer to a pheno- menon known as Irradiation. EXPERIMENT V. Let a black square be inscribed in a white square of three times the size, and conversely, let a white square be inscribed in a black square of three times the size. The side of the inner square will be equal and should be about a centimetre long If the two figures be placed side by side, the inner white square will appear larger than the inner black square. The material for this experiment on a larger scale is also provided in the Milton Bradley Pseudoptics, Section C, Experiment IV. The explanation of this may be due to the dispersive power of the lens, as the appearance is more con- spicuous with a large pupil, or it may be due to the chemical processes of a certain kind (katabolic) in the retina tending to encroach on adjacent fields of the retina, the opposite processes (anabolic) apparently not having that tendency. EXPERIMENT VI. A line passing through the adjacent edges of tv^o rows of black squares, arranged so as to overlap appears oblique. See Milton Bradley Pseudoptics, Section B, Experiment V. 7. Colour Blindness. — The inability to distinguish different hues of colours constitutes the condition of colour blindness. It may vary much as regards the failure shown. A person may be red blind and then only appreciates the colour of red objects as far as they show other con- stituents of white light. Such a person, according to the Helmholtz theory of colour vision, would be entirely lacking in the production of the red sensation. Or a person may lack the green sensation and be green blind, and very rarely violet blindness may exist. 254 PEACTICAL PHYSIOLOGY If a red blind person be examined as to his sensations along the range of the spectrum, he sees nothing at the extreme red end of the spectrum at all. A glimmer of what he calls dark green is seen in the position of the red lithium line, and this green gradually becomes more conspicuous to him through the yellow to the proper green. Passing to the blue green he says the colour is grey, being similar to his idea of white admixed with a certain amount of black. Passing further to the blue end he recognises the blue and speaks of the violet as dark blue. Similarly, a green blind | erson will recognise a grey in the middle of the spectrum, but rather more in the green than the locality thus named by the red blind. Colour blindness can be conveniently tested by the use of a series of coloured wools of great variety of hue and tint. Such a set of wools are spoken of as Holmgren's wools. The method, however, is not a safe test. EXPERIMENT. Spread out the wools on white blotting paper in a good light. Avoid mentioning the names of the colours of any of these wools, but pick < ut a whitish green, and request the subject to collect all those wools which approximate in hue or tint to the colour presented. If any errors are made, proceed to test whether he is red blind, green blind, or violet blind. Give him a skein of a magenta hue. If he is red blind he will pick out blue and violet ; if green blind he will con- fuse green and grey. The matching of colours may be also carried out by rotating the various cards of the colour-mixer, and thus matches of any colour under examination can be obtained. The same result can be obtained by projecting various portions of the spectrum as mentioned in colour mixing. CHAPTER LXX. BINOCULAR VISION. THE images formed on the two retinae of an external object amongst its surroundings will not be identical. The lack of identity enables an observer to form a judgment" as to its position in space. Such a judg- ment is more easily formed when the object is comparatively near than when far off, as in this latter case 'the images are approximately similar. Though the images for objects at a certain distance are not identical, it is necessary that they should be thrown on certain corresponding parts of the retina in order that a single sensation should result. In order that a single image then should result, it is necessary that ELEMENTARY EXPERIMENTAL PHYSIOLOGY 255 various movements of the eyes should occur, so that the two images should fall on corresponding points. With reference to the movements of the eyes, it is customary to regard them as taking place about three axes : (a) the sagittal axis, corresponding nearly to the line of sight; (b) the frontal axis, extending from right to left in each eye ; and (c) the vertical axis. These axes are regarded as intersecting at one point the centre of rotation of the eye. With the head in fixed position the extent of space in which objects can be seen by allowing the maximum of eye movement is called the Field of Regard. If the head and body are erect and the eyes are directed towards the distant horizon, the position assumed is spoken of as the Primary Position. The point upon which the eyes are fixed is called the Principal Point of Regard. A position which the eyes may take up which does not conform to the requirements of the Primary Position is called a Secondary Position. If an observer shift his gaze from the principal point of regard to some other point in the field of regard, he may pass directly to this new position, or may pass over a varied number of different points in the field of regard before reaching this final position. The amount of rotation about the different axes of the eye finally involved in adopting this new position will be the same whether the eye pass to it directly or by a number of varied inter- mediate positions. In other words, only one position is possible when the gaze is shifted to this second point. This is called Donders' law. An extension of the rule is seen in Listing's law, which lays down that in moving from the primary position there is no rotation at all upon the sagittal axis, but merely upon the horizontal and vertical axes. 1. Binocular Direction. — In judging of the position of near objects, they are referred not to either eye separately, but to an ideal eye situated midway between the two actual eyes, the so-called Cyclopean eye of Hering. A line drawn through the object to the centre of such an eye is the Binocular Line of Regard. EXPERIMENT. Make a pinhole in a sheet of paper, and starting with the hole well to the right of the right eye, draw the paper across the eye horizontally, so that the pinhole will pass across each eye succes- sively. First one and then a second image of the pinhole will be. seen as it passes over each eye, but in either case the hole will be referred to the median plane or the Cyclopean eye, and will seem like a succession of two holes over this eye. 2. Single and Double Images. — If the two eyes be directed towards an object about two feet off, and a finger be held up in the binocular 256 PRACTICAL PHYSIOLOGY line of regard about a foot from the eyes, a double image of the finger will be seen. In this case the images of the finger will fall upon non- corresponding parts of the retina, and hence the images will not combine to form a single sensation. EXPERIMENT I. Place a rod vertically about two feet from the eyes. Adjust the vision for a clear image of the rod. Then hold up a finger in the binocular line of regard about twelve inches from the eyes. A double image of the fingers will be seen. Close the left eye, the right image will disappear. Then accommodate for the finger, and a double image of the rod will be seen. Close either eye, and the image on the same side will disappear. This experiment may also be performed with the material in the Milton Bradley Pseudoptic^, Section I., Experiment I. The double images seen, the above experiment may be crossed or uncrossed. If crossed they are spoken of as heteronymous images, if uncrossed, as homonymous images. In general, if the optic axes of the two eyes converge towards a certain point, and a circle be described passing through this point and the two centres of rotation of the eyes, then an object outside the circle will produce homonymous images, and an object inside the circle, heteronymous images. With a definite point of regnrd, then, it will be found that if a circle be described through this point as above, objects lying on this circle will be seen single. Such a circle is called a horopteric circle, and the complete surface (inter- sected as above by a horizontal plane, forming a circle) is referred to as a horopter. Double images of single lines may be shown in performing the Experiments II. and III, Section I., of the Milton Bradley Pseu- doptics. When double images lie, not symmetrically with regard to the line of regard, but both to one side of that line, that nearer the line of regard is the more distinct, and the other is hardly discernible. [ADVANCED EXPERIMENT. Fix the eyes on some remote object, and hold a pencil about six inches from the right eye and about two inches to the right of a line passing from that eye to the remote object. The image falling upon the right retina will alone be appreciated. Close the right eye, and the second image will also be observed. In general the image falling upon the nasal side of one retina will dominate over that falling on the temporal side of the other retina.] 3. Binocular Fusion of Dissimilar Images. — If two partially dis- similar images, or at any rate not absolutely identical images, fall upon ELEMENTARY EXPERIMENTAL PHYSIOLOGY 257 corresponding points of the two retinae, the sensations corresponding to a single image result. EXPERIMENT I. Place on a stereoscopic slide, or on a sheet of cardboard, red and green postage stamps at a distance from each other equal to the interocular distance, and similarly arranged. Observe these in the stereoscope, and the sensation of a single image of a black or brown postage stamp will result. EXPERIMENT II. Perform the experiment in the Milton Bradley Pseudoptics, Section K, Experiment III. The fusion of the two retinal images gives the impression that one is looking through a round hole in the hand. 4. Binocular Perception of Relief. — The perception of relief which enables a judgment as to solidity to be formed depends upon the fact that the two pictures presented to the retinae are not identical. The amount of variation in the pictures will depend upon the interocular distance and the propinquity of the objects. The first being con- stant, it follows that a judgment as to solidity is more easily formed in the case of near objects than distant objects. Similarly, a judgment as to the relative distances of an object from the observer depends upon the difference in position of an object with respect to surrounding objects which exists in the two views presented to the two eyes. The difficulty in forming a judgment as to the precise position in space of an object when viewed with only one eye may be shown in the following experiment. EXPERIMENT I. Stick a knife into the wall, and balance on the handle a cork. The height from the ground should be about five or six feet. Close the left eye, and, starting at about ten feet from the wall with the right hand extended forward, walk rapidly to the cork, and by a sweep of the hand attempt to remove the cork. A lack of success will frequently attend the effort. It is seen from this experiment that it is difficult to locate any object precisely in space when a single ocular view is alone obtained. On the other hand, if perfectly flat pictures be taken differing from each other to the same degree as actual pictures presented to the two eyes would differ, and if such flat pictures be combined by some form of stereoscope, or by crossing the eyes, the resulting sensations will correspond to a single picture on which the different objects are differently projected into the space embraced by the picture, in which the quality of depth is added to the flatness shown by each picture separately. R 258 PRACTICAL PHYSIOLOGY These effects can perhaps best be shown by examining the Martius- Matzdorff l series of diagrams with a stereoscope. Visual Illusions. — The study of Visual Illusions is somewhat beyond the scope of the present work, but the student may advantageously perform various of the experiments on the Milton Bradley Pseudoptics, which illustrate many of these illusions. The Sections A, B, C, D, and J are specially recommended in this connection. CHAPTER LXXI. THE OPTICAL DEFECTS OF THE EYE. 1. Spherical Aberration. — This is probably of little consequence in the eye, as the action of the iris eliminates it largely. 2. Chromatic Aberration. — Eays of coloured light are refracted differently according to their position in the spectrum. Those of shorter wave length, as the violet and blue, come to a shorter focus than do those of longer wave length, as the red. EXPERIMENT I. Look through the upper part of a window towards the sky. Pass a card before the eye with the edge parallel to the upper side of the window frame. If the card be passed from below upwards, when it has covered about half the pupil the frame will be seen to have a border of blue. If the card be passed from above downwards, when it covers half the pupil the edge of the frame will be seen to have a reddish-yellow fringe. In the first case the less refracted red constituents of the margin of the white light are cut off by the card, in the second case the more refracted blue rays. EXPERIMENT II. Look at the incandescent filament of an electric lamp. Pass a card across the pupil with the edge parallel to the filament. When the edge of the card is almost covering the filament, the filament is seen to have a red fringe on the side nearer the card, and a blue fringe on that more remote. 3. Astigmatism. — It is frequently the case that the curvature of the cornea, or lens, in the vertical meridian is greater than that in the horizontal meridian. Therefore, accommodation for a horizontal bar at a certain distance means under-accommodation for a vertical bar at the same distance. Persons who have such a spoon-shaped cornea are said to suffer from regular astigmatism. The cornea, or lens, may have irregular curvatures in various meridia, resulting in irregular astigmatism. JThe diagrams can be obtained from Winckelmann und Sohne, Berlin; Petzoldt, Leipsic; or from Messrs. Baird & Tatlock, Cross Street, Hatton Garden, London. From the latter firm can be obtained any of the instruments mentioned above or the Milton Bradley Pseudoptics Series. ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 259 EXPERIMENT I. Adopting the method of ascertaining the near point of accommodation in Schemer's experiment (Experiment IV., page 239), observe the near points of accommodation for a pin held vertically and one held horizontally. Note if the distances are identical, EXPERIMENT II. Draw a rayed figure as follows : First, draw two lines intersecting in the centre at right angles, and each about 5 cm. long. Bisect each right angle by two other lines intersecting at the same point, and each of these smaller angles bisect further by four other lines. Bring this rayed figure to the near point of accom- modation. Observe which of the lines can be distinctly seen, and which are blurred. It will generally be found that the horizontal and those adjacent will be clearly seen, when no effort will bring about definition of the vertical. EXPERIMENT III. Using Kiihne's artificial eye, place in front of the cornea the special glass trough (filled with water) designed for exhibit- ing the nature of astigmatism. This has a plane surface posteriorly. The anterior surface, however, is that of a cylinder, curved in horizontal meridia but not in vertical meridia. Substitute for the arrow used in earlier experiments as a source of light a stencilled cross, each bar being about 5 cm. long and J cm. wide. Before introducing the astigmatic lens, ascertain the position of the retinal screen necessary for definition of the luminous object. Then place the lens in position. The image will become changed. It will be found that the upper and lower edges of the horizontal bar and the ends of the vertical bar are still distinct, but otherwise definition of the vertical bar is absent. In order to obtain definition of the vertical bar it will be necessary to move the screen much closer, when a reversed effect will we seen— viz., definition of the vertical bar, its end, however, blurred ; the end of the horizontal bar clear, but its edges altogether undefined. At no intermediate posi- tion between the two foci can a clear image of the cross be obtained, and it will be necessary in order to compensate for the presence of this lens, convex in horizontal meridia, to introduce a second lens, plane on one surface, and concave in horizontal meridia. This indicates the method of correction of the defect in the human eye. 4. Errors of Refraction.1 — In this division of the optical defects are included the conditions of Myopia or short-sight, Hypermetropia or long-sight, Presbyopia or the sight of old people. The normal eye in which the far point of accommodation is practically infinity and the near point 20 cm. (8 ins.), is spoken of as emme tropic. Presbyopia. — As a result of advancing age the power of accommoda- 1 Properly speaking, astigmatism should be included in this section. We have thought it best, however, to consider it in a separate section. 260 PRACTICAL PHYSIOLOGY tion for near objects may become diminished. Parallel rays are still focussed on the retina, but the ciliary muscle is unable to bring about sufficiently increased curvature of the crystalline lens to accommodate for objects as near as eight inches. It may here be mentioned that in the normal eye continuous exercise of the full power of accommodation rapidly produces fatigue. It is impossible without fatigue to use more than a half to two-thirds of the full accommodation for any protracted period. The normal-sighted person instinctively avoids placing near objects, e.g. a book, closer to the eye than about sixteen inches. Similarly a presbyopic person whose near point is, say, ten inches, will hold a book at about twenty inches distance. Unless the illumination be extremely good the small size of the retinal image causes some diffi- culty to occur in reading. This, however, may easily be corrected by assisting the crystalline lens through using convex glasses, the degree of convexity corresponding to the extent of failure of accommodating power. These are, of course, only necessary when looking at objects close to the eye. It may be mentioned here that the distance of the near point gradually increases from infancy to old age. According to Landolt it is about 3 inches at 10 years of age, 4 inches at 20 years, 5J inches at 30 years, almost 9 inches at 40 years, 16 inches at 50 years, 40 inches at 60 years, at 70 years about 13 feet, and at 75 there is practically no near point, in other words the power of accommodation is generally lost. Ametropia. — This is a term applied to all conditions of the eye in which the retina does not lie at the focus for parallel rays. The retina may lie in front of this focus when we have the condition of hypermetropia, or behind when myopia is the result, or the focus may be a linear one for any distant luminous point when we have the condition of astigmatism. Hypermetropia. — In this condition the antero-posterior axis of the eye is generally too short. By some effort of accommodation, distant objects may form a clear image on the retina, but the individual suffering from this optical defect does not possess sufficient power of accommodation to focus clearly near objects. Though the emmetropic condition with much facility of accommodation is acquired at about the age of eight years, before this stage is reached the eye is naturally hypermetropic. A young child with marked hypermetropia and deficient power of accommodation will in viewing near objects (e.g. reading), make every effort with both eyes to accommodate for such objects. In- cluded in this effort would be an exaggerated action of the muscles pro- ducing convergence of the optic axes of the eyes, leading to squint, but such squint will frequently be removed on correcting the optical defect. ELEMENTARY EXPERIMENTAL PHYSIOLOGY 261 EXPERIMENT. Using Kuhne's artificial eye, place the retinal screen in the position necessary to obtain a clear image of the external luminous arrow. In this position of the retina the condition of the eye may be regarded as emmetropic. Now move the screen about an inch nearer the corneal surface. The image at once becomes blurred. This represents the condition of the hypermetropic eye. Now place in front of the cornea a very weak convex lens. The image will become much clearer, and with little difficulty a lens of sufficient converging power may be chosen which will exactly correct the defect. Myopia. — This defect is usually congenital, the result of the antero posterior diameter of the eye being too long. As a result parallel rays are brought to a focus in front of the retina, and the eye cannot form a clear image of an object beyond a certain distance (far point of accommodation). The most common cause of acquired myopia in children is the reading of books with insufficient light. The child brings the book close to the eye to get a sufficiently large image of the words and this finally leads to a myopic state. EXPERIMENT. Using again Kuhne's artificial eye, which, as in the last experiment, is first adjusted as the emmetropic eye, shift the retinal screen about an inch away from the cornea. The arrow now becomes blurred and the eye resembles the myopic eye. Place in front of the cornea a concave lens. The image will become much clearer if the degree of concavity corresponds to that of the defect. It is neces- sary in this case to use a lens of dispensive power in order that the image may be thrown back on to the retina. CHAPTER LXXII. (Advanced). THE OPTICAL DEFECTS OF THE EYE. 1. Myopia and Hypermetropia. — The condition of the refractive media of the eye when either hypermetropia or myopia are present are conveniently tested by what is known as the shadow test. If one take a concave mirror (such as that of an ophthalmoscope used for the indirect method), and reflects the light of a lamp at the side of the subject into the pupil of the eye, on looking through the aperture in the mirror the back of the eye is seen to be partially illuminated. If the subject be emmetropic the amount of illumination is small, and on tilting the mirror a little to the right or left a scarcely perceptible movement of the light area may be seen in the opposite direction of the tilt. The image of the lamp formed by the concave 262 PRACTICAL PHYSIOLOGY mirror is the direct source of illumination of the subject's eye, and this image moves to the right when the mirror is tilted to the right, and in accordance with the inversion of the image on the retina the illuminated area will seem to pass to the left. The general impres- sion that one obtains of the result of tilting the mirror on the emme- tropic eye is that the illumination suddenly disappears. With the hypermetropic eye the illuminated area is more distinct, as a large part of it can now be seen, and the passing of this area to the right or left inversely to the tilting of the mirror to the left or right is clearly visible. In the case of myopia the observer must be beyond the far point of the eye and then will see an inverted image of the illuminate area. As the result the apparent illuminated area will be an inversion of the actual area. When therefore the mirror is tilted and the image of the lamp passes across from right to left, the apparent movement will be from left to right, so that the movement of the light on the retina appears to be the same as the tilt of mirror. A small amount of myopia cannot be made out by this method. EXPERIMENT. If subjects possessing the defects of myopia and hypermetropia cannot be obtained, using the ophthalmoscopic mirror as directed above, observe the movement of the light on the retinal screen in Kiihne's artificial eye adapted for these defects. Compare the actual movement of the light on the screen with the apparent movement when observing in front of the eye as above. 2. Imperfections of the Refracting Media, Entoptic Phenomena. — (a) Certain bright, cloudy appearances may be seen, which disappear after blinking the eyelids. Wavy lines or speckled patches may appear after rubbing the eyes. These are all due to the condition of the corneal surface, and have been more properly called ' pseudentoptic ' phenomena. (b) Dark specks or irregularly stellate figures may be seen, depending upon imperfections in the lens or its capsule. (c) Muscae Volitantes. These appear as moniliform threads, clusters of bright or dark circles, and are referable to imperfections in the vitreous. EXPERIMENT. Place a card which is pierced by a pinhole a little more than a centimetre from the eye (i.e. in the position of the principal anterior focus of the ' reduced ' eye). Look at an evenly but brightly illuminated surface beyond, as a sheet of thin white paper held in front of a lamp. The rays of light falling on the retina are now approxi- mately parallel, and any shadows that form in consequence of imper- fections in the refracting media are rendered more distinct. Notice any of such shadows that may be received by blinking, due to im- perfections in the cornea or any comparatively fixed figure due to imperfections in the crystalline lens. These may be practically absent. ADVANCED EXPERIMENTAL PHYSIOLOGY 263 No difficulty will be experienced in recognising 'muscae volitantes.' These will appear as small particles or threads which appear to move away rapidly when the gaze is directed at them. When the gaze is fixed, as by a mark on the white paper, they are still seen to move slowly downwards. This implies that actually their shadows are moving slowly upwards, and that the objects themselves are similarly slowly ascending in the vitreous. If, whilst gazing at some distinct cluster of muscae volitantes, the eye move upwards, the cluster will appear to move upwards too. This actually means that the shadow of the cluster is moving downwards on the retina. If the card be moved downwards the same result, as far as the shadows are concerned, will occur. From this it may be inferred that the objects producing the shadow are behind the nodal point (situated in the crystalline lens), and therefore, if the movement of shadow be appreciable, on the vitreous. Objects in front of the nodal point, such as impurities on the cornea, would appear to move upwards when the gaze is directed downwards, and conversely. CHAPTER LXXIIL THE INSTRUMENTS USED IN THE CLINICAL INVESTIGATION OF THE EYE. The Measurement of the Field of Vision.- If the eye be fixedly directed to some particular point it is possible to see objects at some distance from this point. The area in which objects can be seen with the eye thus fixated is spoken of as the field of vision. With the head fixed and the eye allowed to move as far as possible in any direction a much larger area can be viewed. This area is spoken of as the field of regard. Though fairly satisfactory results can be obtained by using a com- paratively simple form of apparatus called a campimeter, it is customary to employ an instrument called a perimeter to obtain accurate details of the extent of the field of vision. The perimeter (see Fig. 219), consists of a quadrant upon which a white spot can be moved, and this quadrant can be revolved about a line continuous with the optic axis. At K is the chin rest, double, so as to enable either eye to be adjusted against 0. The subject having taken 264 PRACTICAL PHYSIOLOGY his position covers one eye and fixes the eye that is to be examined on the mark at /. The quadrant is then placed, say in the vertical meridian, and at the back of the wheel which revolves with the quadrant is inserted in the frame a special chart adapted for recording FIG. 219. —The perimeter. perimetric observations. Starting at the extreme distance the mark Ob is gradually moved along the quadrant and at a certain angle the white spot will be just visible. The angle indicates the limit of vision in this meridian and can be recorded on the chart. Similar observations ELEMENTARY EXPERIMENTAL PHYSIOLOGY 265 are made in other meridia. In this manner the limits of vision in the different meridia of the field of vision can be recorded. It is of course essential that the subject keep his eye fixed on / the whole time the spot is being moved. The area bounded by a line drawn through the limiting points in the different meridia is properly the area of the field of vision. It is, how- ever, often desirable to refer this area to the retina. If the meridia be inverted, the figure traced would then correspond to the sensitive portion of the retina. It will be found that perimeters are generally so constructed that the limiting marks in the different meridia are inverted on the chart, so that the latter becomes a chart of the extent of the sensitiveness of the retina. This is indicated in the figure above. The Ophthalmoscope. — Prior to the invention of the Ophthalmo- scope it was not possible to view the interior of the eye. The reason of this is that when the interior is illuminated an image of the source of illumination is formed in the retina, and the reflected light passing from the illuminated area out again from the eye will be subject to the refracting mechanism of the eye, and form a small image in the line of incidence of the source of light. The Ophthalmoscope(Fig. 220) is really a contrivance to enable an observer to direct his vision along the axis of the pencil of light illuminating the subject's eye, and thereby to enable him to receive light reflected from the retina of the subject, in other words, to actually see the illumi- nated retina. The instrument consists essentially of a mirror, in which is a central aperture. The mirror is arranged so as to reflect light from some source through the pupil into the in- Flo. 220. -Ophthalmoscopes. terior of the eye. The observer, looking through the central aperture, is able to view the illuminated posterior wall of the eye. Two methods are usually adopted of using the ophthalmoscope, one being known as the direct, the other as the indirect. In the first case there is obtained an erect view of a small area of the retina, magnified 266 PRACTICAL PHYSIOLOGY about thirteen times ; in the second case a less magnified and inverted view is obtained of a larger area of the retina. The Direct Method. — The source of light is placed at the side of the head of the subject, so that no light falls directly on the cornea. The mirror, which is somewhat strongly concave, is held a few inches from the subject's eye, and is so tilted that light is directed into the pupil. The observer uses his left eye to examine the subject's left eye, and similarly his right eye for the subject's right eye. By bringing the light very close to the mirror, and this again close to the eye, the subject will not be able to accommodate for the image of the source of light, and consequently a dispersion circle of light will fall upon the retina. If the observer look through the aperture and the subject's eye be emmetropic he will obtain a clear view of the details of the retina. The reflected light from the subject's retina will issue as parallel rays and thus be in an appropriate state to impinge on the observer's cornea without requiring him to make any effort of accommodation. The Indirect Method. — In this case a somewhat larger, but less con cave or a plane mirror is used. The mirror is held at a distance of about eighteen inches, and if the accommodating power of the subject is intact his eye will accommodate for the source of light or its image formed by the mirror. An inverted image of the illuminated area of the retina will be formed at a certain distance behind the mirror. If the rays issuing from the eye be intercepted by a rather strong convex lens held close to the cornea a new image will be formed, smaller and more brilliant but still inverted. The observer then looks through the aperture of the mirror, and holding a lens as above against the cornea obtains a clear view of a considerable portion of the illuminated retina. Ophthalmoscopes are generally supplied with a revolving disc of lens of different strengths, which are used to correct any error of refraction in the subject's or observer's eyes. It is frequently a matter of difficulty to obtain a clear view of the back of the eye or fundus in the subject unless some drug previously has been applied which causes dilation of the pupil. For practice in the use of the ophthalmoscope, an albino rabbit, the eye of which has been treated with atropin, can be advantageously substituted for the human subject ; or artificial eyes, such as Frost's or Perrin's artificial eyes, may be used. In absence of these, the ocular of a microscope furnishes the material for the construction of an artificial eye. If the lower lens of this be removed and a disc painted to represent the fundus be inserted and blocked behind, an artificial eye is obtained which can be used with advantage. HEARING. CHAPTEK LXXIV. DISSECTION OF THE EAR IN THE SKATE. AUDITORY SENSATIONS. Ear of Skate.1—!. Pare away the cartilage between the eyes of a skate. When the brain is reached continue the paring laterally, and in the cartilage at the side of the hinder part of the brain there will eventually be exposed one of the semicircular canals. When this is reached remove the upper wall as far as possible. In the hollow formed by the cartilage will be seen the fine membranous canal, dilating at one end into an ampulla. On continuing the exposure of the mem- branous canal it will be seen to join a rather large membranous sac, the utricle. Separated by a slight constriction is a smaller sac, the saccule, and at the anterior end of this is a small membranous projection which represents the cochlea. 2. Continue the dissection further so as to completely expose the three semicircular canals. Note also a tube leading towards the sur- face from the utricle, representing the recessus vestibuli. 3. Observe that the ampullae are more rigidly adherent to the cartilaginous walls than the length of the membranous canals. Open one such ampulla where comparatively free and note the crista acustica running transversely across the tube for about a third of the circumference. AUDITORY SENSATIONS. Range of Appreciation of Sound. — EXPERIMENT. In a room as free from noise as possible, let the subject sit with eyes closed and one ear plugged with cotton-wool. Let a watch be held in a line joining the 1 A dog-fish can be used for this dissection. 268 PRACTICAL PHYSIOLOGY two ears, and let it be placed opposite the open ear at such a distance that its ticking is just appreciable. In a quiet room this distance may vary from 2*5 to about 5 meters. Repeat the experiment with the other ear. 2. Auditory Fatigue. — The full effect of any sudden sound tends to temporary fatigue, to abolish appreciation of the fainter echoes which succeed it. If the full effect be avoided the fainter echoes may be heard. EXPERIMENT I. Let a sudden intense sound (such as may be made by striking a bench with a hammer) be produced, (a) with the ears open, (b) with the ears closed for about half a second after the impact. In the first case the intense sound will alone be noticed, in the second case fainter echoes will be noticed in opening the ears. EXPERIMENT II. Strike a tuning fork and place it on the crown of the head with gentle pressure. When the sound is no longer heard, remove it for a few seconds and then replace it again when the sound will be again appreciated. EXPERIMENT III. Using a binaural stethoscope, sound a tuning-fork on a stand, and standing symmetrically with respect to the fork let the opening of the stethoscope be directed towards the fork. Then pinch one tube of the stethoscope, and the sound will be located by means of the patent tube only. When the sound has almost died away reopen the pinched tube, and now the sound will appear differently located and more intense to the ear which has not been fatigued. 3. Appreciation of Pitch. — EXPERIMENT. With Galton's whistle or some apparatus which will provide variation in pitch, observe the highest pitch in which tone can still be recognised. Conversely, note the lowest audible pitch in which tone can still be heard. 4. Recognition of Absolute Pitch. — By practice a trained musician can name the pitch of different tones. Education is required more for this probably than in naming fine differences of colour. EXPERIMENT. Sit with the back to a piano and name the notes struck at random by the observers. In many cases this experiment may be impracticable. 5. Beats. — If two tones of different pitches be produced at the same time they mutually interfere and the resultant sensation is marked by a rhythmic variation in intensity, and is described as characterised by beats. EXPERIMENT I. Put two tuning-forks of different pitches into vibration, and frequently the rhythmic beating is easily recognised. ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 269 EXPERIMENT II. Take two tuning-forks which produce beats when simultaneously caused to vibrate. Place one at such a distance from the ear that it can scarcely be heard. Bring the other fork gradually closer to the ear and the beats will be recognised. 6. Compound Tones. — The tones produced by musical instruments are not simple tones, but blended with other so called overtones. The lowest tone of the group gives the fundamental tone. EXPERIMENT. Stretch a violin string between two fixed points. Set this into vibration by pulling it near one end, and immediately touch it in the centre with the finger. The tone will seem to be pitched an octave higher. The fundamental tone of the original group is obliterated, and the lowest tone now is an octave higher, and thus a new fundamental with other less evident overtones give the tone to the group. 7. Location of Tones. — EXPERIMENT I. Sound a large tuning-fork and press it against the vertex. The sound will appear to come from inside the head. Then close one ear, and the sound will seem to be localised in the other ear. EXPERIMENT II. Sound a tuning-fork as above and note the effect of placing it on different parts of the head. EXPERIMENT III. Sound a tuning-fork and let its foot rest upon the teeth. Close one ear and localise the apparent change in position of the sound. PART II. PHYSIOLOGICAL CHEMISTRY. INTRODUCTION. PHYSIOLOGICAL Chemistry, Chemical Physiology, or Bio-chemistry, is the subject which treats of the chemical processes connected with life. It comprises a study of the chemical constitution of the various tissues and of the chemical nature of the interchanges undergone by the food- stuffs in their passage through the organism. The chlorophyll in the leaves of green plants absorbs certain of the spectral rays of sunlight (the red, yellow and orange) and utilizes the absorbed energy to bring about a reduction of carbon dioxide and water. In this process oxygen is evolved and there is formed a carbo- hydrate in which the energy absorbed from the light becomes locked up in potential form, as the carbohydrate can again combine with oxygen with the liberation of energy. A chemical synthesis is said to occur, and although simple carbohydrates are invariably the first products of this synthesis that we can isolate, yet, by further chemical transformations of the same nature, more complex carbohydrates, fats and proteins are evolved. Animals eat the products of plant life and decompose them so as to liberate the potential energy, that is to say, to convert it into kinetic energy, which is then used in the functions of the animal body. The ultimate source of animal energy is, therefore, certain of the sun's rays. In thus decomposing the large molecules supplied them by the plant animals absorb oxygen and evolve carbon dioxide which again the plants absorb and thus complete the cycle. Plants with no chlorophyll — such as the parasites and saprophytes, etc.— cannot perform these syntheses, but like animals they absorb oxygen, decompose complex molecules and liberate carbon dioxide, Even green plants exhibit this latter process, although in day light it is masked by the more active synthetic changes. In the daik, however, green plants behave like chlorophyll-free plants. PHYSIOLOGICAL CHEMISTEY 271 All the food-stuff digested by the animal is not decomposed, a certain amount of it being used in order to build up the tissues themselves (e.g. muscle, glands, etc.), and a certain amount being laid aside as storage material (e.g. fat, glycogen), which the organism can use as food in times of need. The chemical substances which exist in the food-stuffs and tissues may be divided into inorganic and organic, the former include water and the mineral salts, and the latter consist of organic compounds containing the elements carbon, oxygen, hydrogen, and, in some cases, nitrogen. The organic substances are divided into two groups de- pending on whether or not they contain nitrogen. The nitrogenous food-stuffs include protein, which is the most important constituent of the tissues, and without which, as a food-stuff, animal life is im- possible. The non-nitrogenous food-stuffs include the fats and carbo- hydrates, both of which may be regarded as combustion materials; fat, moreover, is the principal storage substance for surplus food-stuff assimilated. The chemical composition of fats and carbohydrates is fairly accu- rately known, but at the present date we are only beginning to understand the structure of the apparently much more complex protein molecule. Much less do we know of the chemical constitu- tion of living protoplasm of which protein is the chief constituent, for living matter cannot be analysed since it is killed by the process of analysis, and the results obtained show only the decomposition products of dead matter. These bodies, fats, proteins, and carbohydrates, really represent the elementary constituents of the organism, and they are frequently called the ' proximate principles.' We shall first of all study the chemical nature of the proximate principles, then the variety and amount of these contained in the various tissues and foods. We shall then be in a position to investi- gate the nature of the chemical interchanges in the organism, and, in order to do this, we shall require to study the chemical compo- sition of the various excretory bodies given off in the urine and other excreta. 272 PRACTICAL PHYSIOLOGY CHAPTER I. CARBOHYDRATES. Chemical Relationships. — These are compounds of carbon, hydrogen, and oxygen, in which the latter two elements usually exist in the same proportion as in water. Their general formula is therefore QA.O.. Carbohydrates are found chiefly in vegetable tissues, but also occur in animal tissues. They form very important food stuffs, for they are easily digested and assimilated, and moreover are much cheaper than proteins and fats. (See Diet.) The simplest form of carbohydrate is called a monosaccharide, and all other carbohydrates can be broken down into two or more monosaccharide molecules by the chemical process of hydrolysis. When, by this process, two monosaccharide molecules are produced, the carbohydrate is called a disaccharide ; when more than two are produced, the carbohydrate is called a polysaccharide. The monosaccharides and disaccharides being sweet to the taste are together spoken of as sugars, I. MONOSACCHARIDES. Chemically considered, monosaccharides are either aldehydes or ketones ; the former are called aldoses, the latter ketoses. The aldoses are classed according to the number of carbon atoms in the molecule, e.g. pentose C5H1005, hexose C6H1206. Aldoses. — An aldehyde is the first oxidation product of a primary alcohol, and it contains the end group - CHO. A primary alcohol is one in which the " OH " or " hydroxyl group " is attached to the last carbon atom of the molecule— as in primary propyl alcohol, CHS-CH2-CH2OH, and it contains the end group - CH2OH. If, on the other hand, the hydroxyl group be attached to a central carbon atom — as in secondary propyl alcohol, ^ _ CHOH _ ^ the alcohol is called secondary, and contains the group - CHOH. Thus, if ethyl alcohol be heated with potassium bichromate and sulphuric acid, it is oxidised and acetic aldehyde is formed : CH3 - CH2OH + O = CH3 - CHO + H20. Ethyl alcohol. Acetic aldehyde. PHYSIOLOGICAL CHEMISTRY 273 This group, - CHO, is, however, not a stable one, but very readily under- goes further oxidation to produce the acid (carboxyl) radicle - COOH, CH3 - CHO + 0 = CH3 - COOH. Acetic aldehyde. Acetic acid. As a consequence of this tendency to absorb oxygen aldehydes are strong reducing agents, and it is this property which constitutes one of their most important group reactions, for the reaction is frequently accompanied by a visible change in the colour of the solution. Their power of reducing cupric hydroxide, which is blue in colour, to cuprous oxide, which is red, and of reducing silver nitrate to metallic silver, is of especial value as a test. Similar reactions are obtained with certain bismuth and mercury salts. In order to produce these reactions, it is necessary that the liquid be alkaline in reaction. EXPERIMENT I. Demonstrate the reducing power of a simple alde- hyde, such as formaldehyde, on cupric salts in alkaline solution. Place one drop of a weak solution of cupric sulphate in a test tube. Add about ten drops of formalin (40% formaldehyde), and then, drop by drop, a strong solution of caustic soda. The first drop or so of the latter produces a precipitate of cupric hydroxide, but this afterwards becomes redissolved, provided there be only a trace of copper present. Now boil and note that a red or yellow precipitate of cuprous oxide is produced. This is called Trommer's test. The chemistry of the reaction is illustrated by the following equations : — 1st Stage. CuSO4 + 2NaOH = Cu(OH)2 + Na2SO4. Cupric sulphate + caustic soda. Cupric hydroxide + sodium sulphate. The cupric hydroxide is kept in solution by the aldehyde to form a clear blue solution. By heating it is believed that a hypothetical cuprous hydroxide is formed, thus : — 2nd Stage. 2Cu(OH)2 + R* . CHO = Cu2(OH)2 + R . COOH + H20. Cupric hydroxide + aldehyde. Cuprous hydroxide + acid. The cuprous hydroxide then loses a molecule of water and changes into the oxide : Cu2(OH)2 - H20 = Cu2O. EXPERIMENT II. Demonstrate the reduction of silver nitrate by a simple aldehyde. Place about 5 c.c. of an ammoniacal solution of silver nitrate (prepared by adding ammonia to a solution of silver nitrate till the precipitate formed just redissolves) in a test tube, and add to it about ten drops of formaldehyde. Boil. Reduction takes place, and the metallic silver is deposited as a mirror on the wall of the test tube : Ag2O + R CHO = 2 Ag + R . COOH. * R stands for the more or less complicated group or radicle to which the - CHO group is attached. In the case of formaldehyde this is H. 8 274 PRACTICAL PHYSIOLOGY Reactions of Monosaccharides depending on the fact that they are aldehydes. /. Their Reducing Power. — Dextrose is the aldehyde corresponding to the hexatomic l alcohol, sorbite. CH2OH - (CHOH)4 - CH2OH, CH2OH - (CHOH)4 - CHO. Sorbite. Dextrose. It, therefore, manifests strong reducing powers on metallic oxides in alkaline solution. EXPERIMENT III. Demonstrate the reducing power of a mono- saccharide, such as dextrose on cupric salts in alkaline reaction. Trammer's Test. — Place a few drops of a weak solution of cupric sulphate in a test tube; add about 5 c.c. of a 1% solution of dextrose, and then, drop by drop, a 20% solution of caustic soda until the precipitate of cupric hydroxide, which at first forms, becomes re- dissolved, and a clear blue solution is obtained. Boil. Reduction is effected, a red precipitate of cuprous oxide resulting. Repeat experiment without the addition of dextrose. A black precipitate of cupric oxide is obtained on boiling with excess of caustic soda. EXPERIMENT IV. Fehling's Test.— This differs from Trommer's test in that tartrate of sodium and potassium (Rochelle salt) is added to the mixture of CuS04 and NaOH.2 Rochelle salt has the property of dissolving cupric hydroxide forming a blue solution, which is unaltered on boiling, and is therefore of especial value when the solution to be tested contains only a small amount of dextrose or other reducing substance. Boil a few c.c. of Fehling's solution in a test tube. Add the dextrose solution drop by drop, with continued boiling, until reduction results, as evidenced by the blue colour being diminished and an orange red precipitate settling down. EXPERIMENT V. Nylander's Test. — To about 5 c.c. of dextrose solution in a test tube add about 1 c.c. of Nylander's reagent (a solution containing 10% caustic soda, 4% Rochelle salt and 2% bismuth subnitrate). Boil for two minutes. A black precipitate of bismuth forms. Some substances (creatinin, uric acid) which reduce Fehling's solution do not give this test. As regards the sugars, however, where Fehling's test is positive this test will also be positive. 1 A hexatomic alcohol is one which contains six OH groups. Glycerine is called tri-atomic, because it contains three such groups. Ethyl alcohol is mon- atomic, because it contains one. 2 For the exact formula for Fehling's solution see p. 450. PHYSIOLOGICAL CHEMISTRY 275 EXPERIMENT VI. Boil some glucose solution with Barfoed's solution (acid cupric acetate). Reduction occurs. This test applies to the monosaccharides only. Disaccharides do not reduce Barfoed's solution. EXPERIMENT VII. Demonstrate that dextrose also reduces ammonia- cal silver nitrate to metallic silver. 77. Monosaccharides form compounds called Osazones, with Phenyl Hydrazine. — The compounds are very useful in identifying the various forms of sugars, as each sugar forms a slightly different compound. They are also of great interest because it was by producing them that Emil Fischer was able to convert one sugar into another and thus to discover the chemical constitution of the sugars. EXPERIMENT VIII. The production of osazones. Add -25 grm. (enough to cover a sixpence) of phenyl-hydrazine hydrochloride and an equal bulk of sodium acetate crystals to about 10 c.c. of a 1% solution of dextrose. Warm gently till everything is dissolved, and then place for "half an hour in a boiling water bath. Allow to cool, when a yellow precipitate of glucosazone will separate out. Examine this under the microscope, and notice that the precipitate is composed of branching needle-shaped crystals arranged in rosettes or sheaves (Fig. 221). The chemical reaction takes place in two stages, the intermediate body being called a hydrazone. The formula for osazone is CH2OH (CHOH)3 C = N-NH-C6H5 C = N-NH-C6H5 H The excess of sodium acetate in the above mixture reacts with the phenyl-hydrazine hydrochloride so as to form the acetate. When it is desired to produce osazones from dilute sugar solutions, a more certain way to proceed is as follows : Mix two drops of phenyl-hydrazine (fluid) with ten drops glacial acetic acid and add to this 5 c.c. of the sugar solution, shake, and place the test tube for one hour in the boiling water bath. After cooling examine under the microscope for the crystals. With stronger sugar solutions this method yields crystals after a few minutes' heating. The advantage of the phenyl-hydrazine hydrochloride is that it does not readily decompose on keeping, whereas the free base does. The osazone crystals are valuable for distinguishing between the different sugars. Besides microscopical examination, a determination of the melting point 276 PRACTICAL PHYSIOLOGY FIG. 221. — Osazone crystals, x 400. A, Phenyl-glucosazone ; £, Phenyl-maltosazone ; C, Phenyl-lactosazone. PHYSIOLOGICAL CHEMISTRY 277 is often of value. For this purpose the crystals of osazone are collected on a filter paper, washed with water acidulated with acetic acid, recrystallised from water, alcohol or acetic acid, and dried by placing in a desiccator over H2S04. They are then placed in a narrow glass tube closed at one end and tied on to the bulb of a thermometer by a fine platinum wire. The thermometer is suspended in a long necked Jena flask in which is concentrated H2S04 (almost saturated with K^SC^ to prevent fuming) and the temperature gradually raised by heating the flask over wire gauze. The bulb of the thermometer should dip into the sulphuric acid. The exact temperature at which the crystals begin to melt and the temperature of complete fusion are noted. For accurate work, a correction is necessary because the mercury thread is cooler than the bulb of the thermometer.1 and 2 The following are the melting points of some of the most important osazones : Dextrosazone,3 - - - - 204-205° C. Lactosazone, .... 200° C. (Begins to melt at this temp.) Maltosazone, .... 206° C. If the crystals are pure, melting occurs at once, but if they are impure there may be a considerable difference in temperature between the points of commencing and complete fusion. If an osazone be hydrolysed by treating with fuming HC1 it breaks up, phenyl-hydrazine being set free, and a body called an osone resulting. This latter has the formula CH2OH - (CHOH)3 - CO - CHO, from which it is seen that it contains both an aldehyde and a ketone group. The former of these groups can be converted into the CH2OH group of sugar by treating with a reducing agent, CH2OH - (CHOH)3 - CO - CHO + H2 = CH2OH - (CHOH)3 - CO - CH2(OH) which is the formula for laevulose (a ketose). The aldoses can thus be changed into the ketoses, and if the aldose obtained by condensation of formaldehyde be used as the starting point an interesting synthesis from a simple aldehyde to a more complex one and then to a ketose is illustrated. It is believed by some that formaldehyde is the chemical substance which, by a process of reduction, chlorophyll-containing plants first form from C02 and H20. By condensation of several (five or six) formaldehyde molecules pentoses or hexoses become formed, and from these the more complex carbohydrates. By other chemical actions fats and proteins can then be produced. ///. The simple sugars can be artificially prepared by careful oxidation of 1 To make the above correction, a second thermometer must be suspended in the flask with its bulb opposite the middle of the column of mercury of the main thermometer, the formula for correction is then ^(^-^(0 '000154) where L = the height of the mercury column of the main thermometer above the sulphuric acid measured in degrees; T7— the reading of the main thermometer; t the reading of the air thermometer. This correction must be added to the reading T of the main thermometer. 2 Too much reliance must not be placed on a determination of the melting points of osazones in identifying unknown sugars, for they vary with the rate of heating and with the method of purification of the osazone. 3 Laevulose forms the same osazone as dextrose. 278 PRACTICAL PHYSIOLOGY the corresponding alcohols or ly reduction of the corresponding acids. — There are three hexatomic alcohols differing from one another in their constitutional formulae. From each of these a different aldose (or ketose) can be produced by oxidation, and the aldoses can be further oxidised to form three different mono-basic acids, or further still, to form three di-basic acids, thus : Alcohol. Aldose. (Ketose). Mono-basic1 Acids. Di-basic 2 Acids. Sorbite Mannite Dulcite Dextrose Mannose Galactose (Laevulose) (Laevulose) 3 Gluconic Mannonic Galac tonic Saccharic Mannosaccharic Mucic Another monobasic acid is glycuronic — CHO. (CHOH4).COOH which differs from gluconic, etc., in the fact that oxidation has occurred at the C atom furthest removed from the aldehyde group. Its physiological importance will be discussed in the chapter on urine. Ketoses. — As mentioned above, some carbohydrates belong to the group of substances called ketones. A ketone is the oxidation pro- duct of a secondary alcohol and it contains the group— CO — which is situated somewhere in the chain between other groups and not at the end of it as in the case of the - CHO group of the aldehydes. The simplest ketone is acetone CH3 - CO - CH3 which may be ob- tained by oxidation of secondary propyl alcohol, CH3 - CHOH - CH3 + 0 = CH3 - CO - CH3 + H20. Secondary propyl alcohol. . acetone. Ketones form compounds with phenyl hydrazine, but only some of them reduce metallic oxides in alkaline solution. Those ketones which belong to the carbohydrates manifest this reducing power. The only well-known ketose is laevulose. There are several reactions characteristic of ketoses, of these the following is important. EXPERIMENT IX. Seliwanofs Test. — Mix a few cubic centimetres of a solution of laevulose with half its volume of concentrated HC1. Add a few crystals of resorcin and heat the mixture. A deep red colour develops and later a brown precipitate. The colour can be extracted by shaking with amyl alcohol. Repeat this experiment with pure dextrose solution instead of laevulose. A slight red colour develops but no precipitate. 1 The formula for these monobasic acids is CH2OH - (CHOH)4 - COOH. 2 The „ „ dibasic „ COOH - (CHOH)4 - COOH. 3 Laevulose when oxidised does not form the same products as mannose or dextrose but breaks down into products with fewer carbon atoms than itself. This is because it is a ketose. PHYSIOLOGICAL CHEMISTRY 279 CHAPTER II. CARBOHYDRATES— CONTINUED. OTHER REACTIONS OF CARBOHYDRATES. THERE are, however, other reactions of carbohydrates which do not depend on their being aldehydes or ketones. The most important of these are : /. Molisch test. — This is an extremely sensitive test, being especially suitable for the detection of minute traces of carbohydrate. For example, most proteins (e.g. egg albumin) give it, on account of the carbohydrate groups which they contain. EXPERIMENT I. To about 2 c.c. of a very dilute sugar solution, or of a strong solution of egg albumin, add a drop of a saturated alcoholic solution of a-naphthol. Then carefully pour about an equal volume of pure concentrated H2S04 down the wall of the test tube so that it forms a layer at the bottom. On standing a minute or so a deep violet ring forms at the line of contact of the two fluids. The greenish colour which also develops is due to the reagents and is no part of the test. //. Fermentation with Yeast. — By allowing yeast to grow on a solution of dextrose, the latter is split up into alcohol and carbon dioxide, C6H1206 = 2C2H5OH + 2C02 Dextrose. Ethyl alcohol + carbon dioxide. All carbohydrates do not give this reaction, so that it is of value as a distinguishing test for the presence of dextrose in the urine. Com- mercially it is the agency employed in the preparation of alcoholic beverages. To ascertain whether the addition of yeast to a solution produces fermentation, the process should be allowed to proceed in an inverted tube over mercury, or in a Southall's ureometer (see Fig. 244), so that any carbon dioxide gas which develops may be collected, and if necessary tested. EXPERIMENT II. Shake up a 1 per cent, solution of dextrose, which has been previously boiled to expel the air and then cooled, with a piece of yeast the size of a split pea. Pour the opalescent solution thus obtained into a Southall's ureometer (p. 421) so that it completely fills the vertical tube. Now place the tube aside in a warm place for some time, when it will be found that a certain amount of gas has collected at the top of the tube. This gas is C02 as can be shown by 280 PEACTICAL PHYSIOLOGY adding some NaOH to the tube by means of a pipette and shaking : the gas disappears. As a control, a tube filled with water and yeast should also be incubated. This should yield no gas. EXPERIMENT. Repeat the above experiment with similar solutions of maltose, lactose and cane sugar, and note that, after 24 hours, lactose has not undergone any fermentation, whereas it is marked in the case of maltose ; cane sugar also shows a certain amount of fermentation. Yeast contains an invertase (maltase) which readily hydrolyses maltose into dextrose, on which the zymase of the yeast then acts, forming alcohol and carbon dioxide. Another invertase in the yeast acts on cane sugar. These invertases have no action on lactose. ///. Rotation of Polarised Light. — All simple carbohydrates rotate the plane of polarisation of polarised light to the right except laevulose, which rotates to the left. This effect is due to the presence in the molecule of asymmetrical carbon atoms. 6 carbon aldose (hexose). 6 carbon ketose. CH2OH CH2OH *CHOH *CHOH *CHOH *CHOH *CHOH *CHOH *CHOH CO CHO CH2OH * Denotes an asymmetrical carbon atom. Examination of the above formulae shows that the aldoses contain four asymmetrical carbon atoms, whilst the corresponding ketoses contain only three. The different arrangements in space of the hexose carbon atoms allow of the existence of sixteen different hexoses, of which twelve have been identified. Only two, however, are of physiological importance, dextrose and galactose. The different spacial arrangement of the atoms in the molecule accounts for the difference in rotatory powers shown by these aldoses and also for slight differences in chemical properties, such as crystalline form and melting point of the osazones. Polarisation Of Light, — When two slices of tourmaline, a semi-transparent mineral, are cut parallel to the axis of the crystal and laid over one another, it will be noticed that the amount of light which passes through the combination varies according to the relative positions of the two slices to one another. If the slices be at right angles to one another no light passes through, and in intermediate positions only a certain amount, so that an opaque combination is obtained. A ray of ordinary light contains vibrations in all planes passing through the ray ; but when the light passes through a tourmaline plate it vibrates in one plane only. Ordinary light may, therefore, be likened to a wheel, the axle representing the ray of light and the spokes the planes along which it vibrates. On passing through the tourmaline plate, however, the PHYSIOLOGICAL CHEMISTRY 281 light is capable of vibrating in one plane only, which would correspond, in our example, to two opposite spokes. The light which vibrates in one plane is called plane-polarised light, and cannot be distinguished by the naked eye from ordinary light. By placing a second, similarly cut, tourmaline plate in its course, however, it can be detected, for it will pass through this only if its axis corresponds to the axis of the first plate. The first plate is called the polariser and the second plate the analyser. The mechanism of this action of the analyser and polariser can be easily illustrated by a piece of string stretched between two posts ; it can vibrate in all planes. If a comb be placed in the course of the string the vibrations can only take place along one plane corre- sponding to the direction of the teeth of the comb. This comb represents the polariser. If now, a second comb be placed along the string it will permit the vibration of the string or stop it, according to the position of its teeth ; if these be in the same direction as those of the first comb the string will go on vibrating, but if they be placed at right angles the string will cease to vibrate. Polarisation of light by tourmaline illustrates the principle of the polarimeter but in this instrument itself it is found more convenient to use a polariser and analyser made of a Nicol's prism. A Nicol's prism consists of a crystal of Iceland spar. Such a crystal has the power of splitting light into two rays, one of which, the ordinary ray, passes through it as it would through glass, and the other one, the extraordinary ray, is more refracted. Consequently, on looking at a dot on a sheet of paper through a piece of Iceland spar laid flat on the paper, a double image of the dot is obtained, and if the crystal be rotated, one of the dots — the extraordinary ray — will be seen to move round the other — the ordinary ray — which remains stationary. Now both these rays are polarised, but in different planes. If the crystal be cut across along a diagonal line and the two surfaces re-cemented by means of Canada balsam, the ordinary ray, when it meets the balsam, will be totally reflected and pass out at the side of the crystal, whereas the extraordinary ray will be trans- mitted through the balsam, and will finally emerge at the end of the prism, parallel to its original direction ; but, of course, plane polarised. To detect the polarisation a similarly constructed prism, or analyser, must be used. Certain other bodies, e.g. a quartz plate, a solution of sugar or albumin, have the power of rotating the plane of polarised light. Thus, supposing that the plane polarised light vibrates along a vertical plane, one of these bodies will cause it to vibrate in an oblique plane. If the analyser be so placed that none of the plane polarised light can pass through it (i.e. the field is black), and if a piece of quartz be inserted between the polariser and analyser, it will be found that now a certain amount of light passes through the analyser (i.e. the field becomes opaque), and, in order to obtain darkness again, it is necessary to rotate the analyser in the direction of the hands of a watch, as seen by the observer. Consequently, rotation has taken place to the right, i.e. dextro rotation is said to have occurred. If a solution of albumin or laevulose be employed the rotation of the analyser must be to the left, i.e. against the hands of the watch. When the plane of white light passes through the quartz plate, however, the various colours of the spectrum are rotated to a different degree, so that, instead of having a mere opacity (as is the case with inter- mediate positions of two ' tourmaline ' plates) different colours are obtained according to the amount of rotation. There are also samples of quartz which rotate the plane of light to the left. 282 PEACTICAL PHYSIOLOGY Dextrose and a quartz plate produce the same amount of rotation, and there- fore it is possible to determine the rotatory power of a solution of the former by compensating its rotation by means of a quartz plate of known rotatory power. We are now in a position to understand the construction of a polaiimeter or sacchaiimeter. It consists of the following parts : (1) A Nicol's prism, called the polariser. This polarises light in a vertical plane. (2) A biquartz, or other device for rotating, in opposite directions, the two halves of a polarised beam. A biquartz consists of a disc of quartz made of two semicircular halves of equal thickness, but of opposite rotatory powers. Fio. 222.— Polarimeter of Mischerlich with Laurent s polariser. P, polariser and device for obtaining half shadow ; jK, fluid container ; T, scale with vernier c attached to pointer ; A, compensator and analyser ; F, lens. Each half is of such a thickness that it rotates the plane polarised light to 90° in opposite directions so that, on emerging from the disc the plane of light is now horizontal. Instead of a biquartz many instruments contain a semi- circular plate of quartz. (3) A tubular liquid holder to hold 10 c.c of the liquid to be examined. If the length of this tube be 188-6 mm. the amount of rotation in angular degrees will correspond to percentage of dextrose in the fluid (e.g. urine) examined. (4) A Compensator. — This shows how much rotation has been produced by the solution. It is connected with a scale representing angular degrees, and the pointer carries a vernier, so that tenths of a degree can be read off. In some instances the compensator consists of two wedge-shaped pieces of quartz, so arranged on one another that the total thickness of quartz inter- PHYSIOLOGICAL CHEMISTRY 283 posed in the path of the polarised beam can be varied by means of a screw. In other instruments the quartz plates are dispensed with, the amount of rotation being measured by rotating the next part of the instrument, namely the (5) Analyser, so as to obtain uniformity of tint in the two halves of field. (6) A Lens. When the tube (3) is filled with water or an optically inactive fluid, and the compensator or analyser rotated until a violet colour of uniform tint fills the field, the indicator will be seen to stand at zero (if not so, the error must be noted). If now, an optically active fluid be placed in the tube the two halves of the field will become of different tints, i.e. rotation of the plane of polarised light has occurred. In order to measure the amount of this rotation, we must move the screw or pointer connected with the compensator or analyser until the uniform tint is again obtained.1 The amount of 'compensation' necessary is read off on the scale and, if the holder be not 188-6 mm. long, the necessary calculation is made in order to ascertain the strength of the solution (for formula see below). Fio. 223.— Diagram of scale and field of vision of polarimeter. Above is represented the scale for measuring the compensation necessary. In the position represented in the diagram the reading is 27 dextro rotation. The lower part of the diagram shows the three appearances of the field of the polarimeter, the central one representing the appearance at zero, i.e. when there is no rotation. To estimate the percentage of sugar in urine the chief precautions are, (1) to see that it is perfectly dear, and (2) to see that it contains no protein. In order to obtain a specific or comparative number (i.e. a result always obtained under the same conditions) it is necessary to adopt a standard. This consists of the rotation, in degrees of a circle, produced by 1 gr. of the substance dissolved in 1 c.c. of fluid and contained in a tube 1 dcm. long. This is called the specific rotatory power and is represented by (a) D.2 It is determined by the following formula : where a = the observed rotation, Z = the length, in decimeters, of the tube in which the solution is placed, p = the weight, in grammes, of the substance contained in 1 c.c. solvent. The rotation produced by a substance depends upon its concentration in a solution ; if, therefore, the index (a) D of any substance be known, and its 1 In the best modern polarimeters the field is divided into three ; when at zero these are of the same tint otherwise the central band takes a different colour. 2 The ' D ' indicates that sodium light is used. 284 PRACTICAL PHYSIOLOGY rotation be ascertained, its percentage P in any fluid can be ascertained by the formula. lOOa " 8l where s = (a)D. For rapidly and accurately determining the percentage of sugar in any fluid (e.g. urine) the polar imeter — and especially that form of it in which the scale reads percentages of sugar — is a very valuable instrument. It is much used for this purpose in the continental clinics. The Specific Rotatory Power * of certain of the sugars is as follows : Monosaccharides : Dextrose : +52 *7°. Galactose : +81°. Laevulose : -93°. Invert sugar : - 20 '2°. Disaccharides. — The (a) D of these carbohydrates changes when they are hydrolysed. Cane sugar : + 66 '5° — after hydrolysis becomes laevorotatory (vide invert sugar). Maltose : + 137° — after hydrolysis becomes much less. Lactose : +52*5° — after hydrolysis becomes slightly more. IV. Mowe's Test. — When heated with caustic soda a dark substance called caramel is produced. This is also produced when sugar is burnt. Caramel contains several chemical bodies, the most important of which is an acid called levulinic acid (CH3 - CO - CH2 - CH2 - COOH). EXPERIMENT III. Mix equal quantities of a 1 % solution of dextrose, and 40 % NaOH in a test tube ; heat. A yellow to brown colouration results, and an odour of burnt sugar (caramel) is produced. This odour becomes very evident if, after qpoling, the solution be acidified with H2S04. The Chief Monosaccharides are dextrose, laevulose and galactose. Dextrose, grape sugar or glucose (C6H1206), is found in many fruits, and is an important food-stuff. In the healthy animal body it occurs in the blood and muscles. In normal human blood the amount of glucose is usually from O'l to O15 %, but in disease it may rise to such a degree that it appears in detectable amount in the urine (see p. 447). Commercially it exists as a syrup much used in making confections. It is easily crystallised. It is soluble in water and in alcohol. It has only a slightly sweet taste. It rotates polarised light to the right ((a)D = +52-7). 1 The rotatory power of a solution of a sugar is frequently different when the solution is freshly made from what it becomes on standing. This pheno- menon is called mutarotation. The figures given are all for solutions which have been kept long enough to be in equilibrium. Temperature also affects the rotatory power of a solution, particularly in the case of laevulose and invert sugar. PHYSIOLOGICAL CHEMISTKY 285 Glucose readily combines with alcohols, acids, phenols, etc. , to form glucosides. These are resolved into their constituent groups by hydrolysis with acid. To understand their structure, the formula for glucose is best written with four of its C atoms in a ring thus : H CH - (CHOH)2 - CH - CHOH - CILjOH. When the H atom of the hydroxyl group of the C atom which exercises aldehydic functions in the open chain formula for dextrose is replaced by some radicle a glucoside results. Since this C atom is assymetric, two stereoisomeric glucosides (a and ft) are possible. These show striking differences from one another in their susceptibility to ferment action. Laevulose (C6H1206) is found along with dextrose in fruits and honey and results from the hydrolysis of cane sugar (see Disaccharides). It is very rarely found in animal tissues. It is crystallisable with great difficulty, being usually obtained as a putty-like mass. It is laevo- rotatory ((«)D= -93°). Galactose (C6H12O6) is a dextro-rotatory sugar produced, along with dextrose, by hydrolysing lactose (see Disaccharides). Protagon, a lipoid substance in brain tissue, yields galactose on hydrolysis. Certain gums also yield it on hydrolysis. It differs but slightly from dextrose in its reactions. Its presence can be detected by the fact that when oxidised, as by boiling with nitric acid, it yields mucic acid (see p. 278) which forms characteristic crystals. EXPERIMENT. Test for galactose. Add 3 c.c. pure HN03(con.) to 10 c.c. of a strong solution of lactose in a small evaporating dish. Boil gently over a free flame for three minutes, and then gently lower the flame and allow to evaporate till the volume is reduced to about 3 c.c. Transfer to a test tube, cool under the tap, add 2 c.c. water, and allow to stand. Crystals of mucic acid separate out. II. DISACCHARIDES. Chemically, each molecule of a disaccharide consists of two molecules of a monosaccharide less one molecule of water, Their structure can be demonstrated by hydrolysing them, ie. by causing them to take up a molecule of water, in consequence of which they split up. In disaccharides the two monosaccharide molecules are linked together in the same manner as glucose and the other con- stituent radicle in glucosides. The chief means of hydrolysing include boiling with dilute acid and the action of certain ferments called invertases, which are contained in 286 PKACTICAL PHYSIOLOGY the succus entericus and in the protoplasm of many cells such as the yeast plant (see p. 280). The members of this class are cane sugar, maltose and lactose, and of these cane sugar does not reduce metallic oxides in alkaline solution, nor does it form an osazone, whereas lactose and maltose give both these reactions. With yeast maltose and cane sugar are first hydro- lysed, and the monosaccharides thus produced then undergo alcoholic fermentation. Cane Sugar (C12H22On) is the common sugar obtained from sugar cane, beet root, etc. It is very soluble in water and has a sweet taste. It does not reduce metallic oxides in alkaline solution. EXPERIMENT IV. Perform Trommer's test with some cane sugar solution. Notice that, although no reduction occurs, the cane sugar, like other sugars, is capable of holding the cupric hydroxide in solution, so that a clear blue colour is produced. - By hydrolysis, reducing sugars (dextrose and laevulose) are developed. EXPERIMENT V. Boil some cane sugar solution with a few drops of 25 % sulphuric acid. Now neutralise the acid and apply Trommer's or Fehling's test and note that reduction occurs. The monosaccharides developed are dextrose and laevulose, the mixture being called invert sugar. It is often better to employ an organic acid such as citric acid to produce the hydrolysis, because the organic acid does not hydrolyse starch or glycogen, whereas mineral acids do. EXPERIMENT VI. Apply Seliwanoff s test for ketose to a solution of cane sugar (Experiment IX. p. 278). The reaction is as marked as for laevulose, owing to hydrolysis of the cane sugar by the hydrochloric acid employed. EXPERIMENT VII. Heat some cane sugar solution with strong hydrochloric acid. Note the reddish colour developed. This reaction is given by other sugars, but not so readily. A solution of cane sugar is dextro-rotatory ((a)D = +66*54), but after hydrolysis it is laevo-rotatory, the laevo-rotatory power of the laevulose being stronger than the dextro-rotatory power of the dextrose formed. On this account the process of hydrolysis is sometimes called inversion, and the hydrolysing ferments in the succus entericus, etc., are often called invertases. EXPERIMENT. Examine a ten per cent, solution of cane sugar with the polari- scope. Note the rotation and calculate (a)D. Place exactly 50 c.c. of a twenty per cent, solution of cane sugar in a 100 c.c. measuring flask ; add 1 gr. citric acid and boil over wire gauze for five minutes. Cool, neutralise with NaOH solution, and fill with distilled water to the 100 c.c. mark. Examine this solution with the polariscope and calculate (a) D. PHYSIOLOGICAL CHEMISTRY 287 Lactose (C12H22On) is the sugar found in milk, and it has been detected in the urine of nursing mothers. It is not very soluble in water, and is quite insoluble in alcohol and ether. It has only a slightly sweet taste. It does not ferment with yeast in 24 hours, but it undergoes a special fermentation with the bacillus acidi lactici which develops in sour milk. This fermentation results in the production of lactic acid. OH. C12H22On + H20 = 4CH3 - OH/ COOH. Lactose. Lactic acid. By hydrolysis it yields dextrose and galactose. It reduces metallic oxides in alkaline solution. It is dextro-rotatory ((a)D = 52'5). By oxidation with nitric acid it yields mucic acid (cf. p. 278). Maltose (C12H22O11) is important physiologically because it is the sugar produced from starch by the action of ptyalin (the ferment of the saliva), of amylopsin (a ferment in the pancreatic juice) and of glycogenase (a ferment in the liver, blood serum, etc.). Maltose is therefore mainly an intermediary substance in the animal body. Maltose is also produced by the action of malt diastase, which is obtained by moistening barley and allowing it to germinate in heaps at a constant temperature. The diastase acts on the starch of the grain and produces maltose The product when dried is called malt. When malt is dissolved in water, and the yeast plant allowed to grow on the solution, malted liquors, such as beer and ale, are obtained. In this process the maltose is first of all inverted into two molecules of dextrose by the invertase contained in the yeast, and the dextrose then undergoes alcoholic fermentation. It reduces metallic oxides in alkaline solution, but is feebler in this regard than dextrose. It rotates the plane of polarised light more strongly than dextrose ( (a)D = -I- 137 -04°). After hydrolysis, therefore, the reducing power shows an increase and the rotatory power a decrease. EXPERIMENT VIII.— Boil lactose or maltose solution with Barfoed's reagent (Experiment VI. p. 275.) There is no reduction. This reagent is not reduced by disaccharides. Isomaltose. — This sugar is closely related to maltose, differing from it in the fact that its osazone melts at a much lower temperature, 158° C. It has been prepared by pure chemical synthesis — e.g. the condensation of dextrose by strong acids. It is of special interest because it is probably the sugar produced as a result of the reversible action of maltase. 288 PRACTICAL PHYSIOLOGY CHAPTER III. CARBOHYDRATES— CONTINUED. III. POLYSACCHARIDES. A POLYS ACCHARIDE is the condensation product of more than two monosaccharide molecules, and has accordingly the general formula, (C6H10O5)n, where n stands for a variable number.1 Polysaccharides can be hydrolysed, in which process they yield, first of all, polysaccharides (dextrines) of lower molecular weight (i.e. with n of less value), then disaccharides and, finally, monosaccharides. Thus, when acted on by diastatic ferments, dextrines (polysacchar- ides of lower molecular weight) arid maltose (disaccharide) are formed. When boiled with acid, on the other hand, the hydrolytic cleavage goes further and, although dextrine and maltose occur as intermediary products, yet the final product is monosaccharide. The most important members of this group are the starches, the dextrines, glycogen, the celluloses, and the gums. They are very widely distributed in vegetables, and constitute a most important class of food-stuffs. General Characters. They do not form crystals, nor, with few exceptions, are they soluble in cold water. Few possess any sweet taste. As a rule they do not diffuse through parchment and are therefore colloids. Their solutions are optically active. They do not reduce metallic oxides in alkaline solution, they do not form osazones and they cannot be fermented with yeast. Like other colloids, they are precipitated when their solutions are saturated with certain neutral salts, such as ammonium sulphate. They may be sub-divided into three sub-groups, the starches, the dextrines and the celluloses. 1. The Starches. These include ordinary starch and glycogen (C6H1006)n. Starch is the most widely distributed carbohydrate in the vegetable kingdom, for it is in this form that plants store up their excess of carbohydrate. Animals store their excess of carbo- hydrate partly as glycogen, but mainly as fat. If the amount of dextrose produced in the leaves be in excess of the present needs of the plant, it is stored up as starch. These starch grains may be seen in various parts of the plant. They show, under the microscope, concentric markings. In its minute structure the starch granule is 1 It is impossible to give a definite value to n because the molecular weight is unknown. The symbol n signifies that the formula within the brackets is to be multiplied an indefinite number of times. PHYSIOLOGICAL CHEMISTRY 289 thought to be composed of a fine interlacement of acicular crystals, the density of the interlacement varying in different portions of the granule, so as to give the appearance of concentric markings. The outside of the granule has densely packed crystals, so that ferments slowly attack it. By boiling, the crystals absorb water and swell up, so that, after cooling, ferments more readily penetrate and digest the granule. The exact shape of starch grains varies according to the plant from which they are obtained. In this connection they may be divided into two groups : (1) a group in which the contour of the grains is even, such as wheat, barley, arrowroot, potato ; (2) a group in which the contour is marked by facets, either completely, as in oats and rice, or only partially, as in tapioca and sago. EXPERIMENT I. Examine some wheat flour, a scraping of potato, and some ground rice under the microscope. To do this, mix the flour, etc., with a drop of water on a slide, and examine under a cover slip. Starch, like most other polysaccharides, is insoluble in cold water, but it swells up in hot water, an opalescent mixture being formed. This is not a true solution, for it does not depress the freezing point of water (e.g. has no osmotic pressure, etc.). It is a colloidal solution. Starch does not pass through a dialyser. EXPERIMENT II. Place some powdered starch in a test tube, and half fill up with cold water— no solution occurs — now boil, when an opalescent mixture will be produced, and, if of sufficient concentration, this will gelatinise on cooling. Try Trommer's test— no reduction occurs. The standard test for starch is with iodine solution. EXPERIMENT III. To an opalescent cold solution of starch add a drop or two of a very dilute solution of iodine in potassium iodide : a blue colour results, which disappears on gradual heating and returns again on cooling. Excessive heat must be avoided, since the iodine is volatile. Starch granules also give this reaction under the microscope. The cut surface of a potato gives it. Hydrolysis can be effected by boiling with a weak acid or by the action of ferments such as ptyalin, amylopsin, and malt diastase. EXPERIMENT IV. Place some starch solution in a flask, add to it a few drops of 25 % sulphuric acid and boil for about a quarter of an hour. Neutralise and apply the iodine test and note that, instead of a blue, a reddish brown colour is produced (due to dextrine). Apply Trommer's or Fehling's test, and note that reduction occurs. 290 PKACTICAL PHYSIOLOGY EXPERIMENT V. Place some of the starch solution in the mouth, and after a minute or so transfer it again to the test tube ; now apply Trommer's or Fehling's test — reduction occurs. Try the same experiment with some unboiled starch, and note that, with Trommer's test, there is no reduction (i.e. the resistant external layers have not been hydrolysed). The sugar produced by hydrolysing with an acid is dextrose, whereas that produced by ferment action is maltose. Glycogen (C6H1005)n. Just as plants store up excess of carbo- hydrate in the form of starch, so do animals store it partly in the form of glycogen. The chief seats of this storage are the liver and muscles. Glycogen forms a non-crystalline, white powder, the particles of which have no characteristic appearance under the microscope. It is soluble in water and the solution is opalescent. Solutions of glyco- gen are dextro-rotatory. EXPERIMENT VI. A simple method for the preparation of glycogen is that introduced by Frankel. It consists in grinding up fresh liver or the common shell-fish, mussel, in a mortar with about three times its volume of a 3 % solution of tri-chloracetic acid. This re- agent coagulates the proteins The glycogen is contained in the extract, and can be precipitated by alcohol.1 After collecting on a filter dissolve some of the glycogen in water and notice that the solution is opalescent. Add to this a drop or two of iodine solution : a port-wine colour results, which disappears on heating, and returns on cooling. EXPERIMENT VII. Place 5 c.c of glycogen solution in a test tube and add ordinary alcohol carefully until a precipitate forms. Note approximately how much alcohol requires to be added to obtain this (about 55 %). EXPERIMENT VIII. Try Trommer's test with the glycogen solution ; no reduction occurs, but the Cu(OH)2 is held in solution. EXPERIMENT IX. To some of the solution add a few drops of 25 % H2S04 and boil for about ten minutes ; dextrose is produced, as can be shown by applying one of the reduction tests. EXPERIMENT X. Mix some glycogen solution with saliva and place the test tube in water at body temperature. After about ten minutes apply one of the reduction tests. It will be found that a reducing sugar has been produced. The Dextrines (C6H1005)n. During the hydrolysis of starch and glycogen dextrines are formed as an intermediate product. British 1 Where not otherwise specified in these experiments, alcohol refers to the commercial product containing from 92-96 % pure alcohol. PHYSIOLOGICAL CHEMISTRY 291 gum is dextrine produced by heating starch to 200* C. This sub- stance is much employed as the adhesive substance on stamps and envelopes. There are several varieties of dextrine, varying from one another in molecular weight. The highest dextrine is called amylo- dextrine, the next erythrodextrine, then achroodextrine. Dextrine is an amorphous powder, soluble in cold water, and forming a clear solution with which the following reactions can be obtained : EXPERIMENT XI. Add some iodine solution ; a brownish red colour, like that obtained with glycogen, results, which disappears on heating and returns on cooling. It is only one form of dextrine — erythrodextrine — which gives the reaction ; achroodextrine gives no reaction with iodine. The bluish tint sometimes obtained is due to the presence of starch. EXPERIMENT XII. Try Trommer's test: no reduction is obtained, but Cu(OH)2 is held in solution. EXPERIMENT XIII. Hydrolyse some dextrine solution and then apply Trommer's test : reduction occurs. The Quantitative Estimation of Glycogen in Animal Tissues. The importance of a rapid and accurate determination of the amount of glycogen in animal tissues has led to the publication of many different methods. To within a few years ago, however, none of these has been of sufficient accuracy, the difficulty being to separate the glycogen from protein without losing any of the glycogen. It is to Pfliiger that we owe a method which is unquestionably far superior to any of its predecessors. This method depends on two facts : firstly, that glycogen is not affected by heating it on a water bath with thirty per cent, potassium hydroxide solution, whereas protein under such conditions is destroyed ; and secondly, that by the addition of an equal volume of water to the above solution (which will bring the percentage of potassium hydroxide to fifteen) and the subsequent addition of two volumes of alcohol (96%) all the glycogen is precipitated, whereas practi- cally all of the degradation products of protein remain jn solution. The method is as follows x : The liver is cut into small pieces and mixed in an Erlenmeyer flask (Bohemian glass) with 100 c.c. 60% KOH.2 1The following description is for 100 gr. liver, but much less than this amount is sufficient for most purposes. Thus, in the case of a dog, fed on the previous day with bread and meat, 20 gr. liver is a suitable amount, and in the case of a rabbit fed with carrots or other carbohydrate-rich food, 10 gr. is sufficient. In the case of muscle, it is best to take 100 gr., as the percentage of glycogen in this tissue is practically never more than one. 2 Pfliiger specifies ' Merck A ' KOH, but for most purposes ' KOH pure by alcohol ' is of sufficient purity. The strength is best adjusted by the use of a hydrometer (alkalimeter), the specific gravity of such a solution being 1-438 at 15° C. or 44 on the Baume scale. 292 PEACTICAL PHYSIOLOGY The flask is closed with a cork, having a wide glass tube about five feet long passing through it to serve as a reflux condenser, and it is then immersed in a boiling water bath and left there for three hours, with occasional shaking. (Less time than this suffices to completely destroy the protein of liver.) On removal from the water bath, the contents of the flask are allowed to cool, and are then thoroughly shaken, with 200 c.c. water (thus bringing the percentage of KOH to fifteen). 800 c.c. of ordinary (96%) alcohol are then added to the solution, the mixture shaken and allowed to stand for several hours (preferably overnight). The more or less white precipitate of glycogen will by this time have settled down, so that the supernatant reddish fluid can with care be poured off into a beaker, after which it is filtered through a filter paper of suitable size, so as to collect on the filter any particles of glycogen which the decanted fluid may contain. The precipitate of glycogen is now thoroughly shaken with about ten times its volume of 66% alcohol (about 700 c.c. alcohol and 300 c.c. water) containing 1 c.c. per litre of a saturated solution of NaCl. This washing fluid removes many of the impurities which adhere to the glycogen. After settling, the wash fluid is decanted into the same beaker as was employed for receiving the original supernatant fluid, and filtered through the same filter. This process is repeated at least once again, after which the precipitate is shaken with ordinary alcohol (about 10 times its volume), and the suspension thrown on to the same filter paper as used above. When the alcohol has all drained off, the precipitate is washed on the filter paper with ether. All the washed glycogen has thus been collected on the filter paper and must now be dissolved, for which purpose the filter is filled up with boiling water, and the solution of glycogen allowed to filter through into a clean Erlenmeyer flask. When the first added water has completely drained through the filter, the filter is filled up with boiling water a second and a third time. It is essential to allow the filter to drain completely before adding more water. To be certain that all the glycogen has been dissolved, some of the final filtrate should be tested with alcohol for glycogen. The resulting opalescent solution can now be employed either for the preparation of pure glycogen or for its quantitative estimation. For the former purpose the glycogen is precipitated by alcohol ; for the latter purpose the glycogen solution is made up to a litre in volume, and of this 200 c.c. are taken, mixed with 10 c.c. HC1 (cone.) (i.e. 5 c.c. HC1 to a 100 c.c. of glycogen solution), and heated in a flask on the water bath for three hours.1 Complete hydrolysis of the glycogen is certain within this time, although the resulting solution often contains a flocculent precipitate which is probably of some protein body. The solution, after cooling, is neutralised with 20% KOH and filtered into a 250 c.c. measuring flask through a small filter' (10 cm.) paper. The flask used for inversion is rinsed three times with distilled water, the washings being each time poured on to the filter and added to the contents of the measuring flask. In this way the volume of the dextrose solution is brought exactly to 250 c.c. Where only 10 or 20 gr. of liver were originally employed, the above measurements must of course be altered, it being usually best to take all of 1 If the glycogen be reprecipitated and redissolved in a known volume of water the resulting solution can be examined in the polarimeter and its glycogen con- tent calculated according to the formula on p. 284. (a) D = 196 '63. PHYSIOLOGICAL CHEMISTKY 293 the glycogen solution for inversion and bring it to a definite volume after neutralising. For the estimation of the dextrose formed Pfliiger uses a special gravimetric method (see Dictionnaire de physiologie, par C. Eichet, t. vii.), but Bang's method, described in the following section, is of sufficient accuracy for most purposes. Quantitative Estimation of Sugars. All the methods employed for this purpose consist in determining the reduction brought about by a measured amount of sugar solution in a known alkaline solution of cupric salts. They may be divided into two classes : (i) Methods in which sugar solution is added to a measured volume of the cupric salt employed, until the reduction to cuprous salt or oxide is complete. To this class belong the methods of Fehling, Pavy and Gerrard, which are described in Chapter XX. For the estimation of sugar in urine they are usually of sufficient accuracy, and are widely employed for this purpose. Their great advantage is simplicity. (ii) Methods in which excess of cupric salt is employed. In Pfliiger's method the precipitated cuprous oxide is weighed. In Bang's method the excess of cupric salt is determined by titration. In both these methods it is possible to keep the conditions of different titrations exactly similar except as to the excess of cupric salt employed, which will naturally depend on the amount of cupric salt reduced by the sugar in the determination. Now it is found that the larger the excess of cnpric salt present the greater is the reduction caused by a given amount of sugar. The reduction is therefore not proportional to the amount of sugar employed in the determination, and it is necessary to construct a table, representing the amount of reduction caused by different known amounts of sugar, from which the results of any given determination can be calculated. These methods are more accurate than those of the first class. EXPERIMENT. Bang's Method. Principle. — In the presence of carbonates and sulphocyanides cuprous oxide forms cuprous sulphocyanide, which becomes dissolved to form a colourless solution in the presence of excess of potassium sulphocyanide. A solution of cupric carbonates containing potassium sulphocyanide is boiled for exactly three minutes with an amount of the sugar solution, which is insufficient to reduce all the cupric salt ; 'after rapidly cooling, the amount of cupric salt which has not been reduced by the sugar is then determined by titration in the cold with a standard solution of hydroxylamin (sulphate). This, like sugar, reduces cupric salts to the cuprous state to form, as above explained, a colourless solution. The hydroxylamin solution must not be run in too quickly, else a precipitate forms and the results are vitiated. Shake sufficiently to prevent the precipitate forming. At least 30 seconds should be taken in titrating. The hydroxylamin solution is of such a strength that 1 c.c. of it exactly decolourises 1 c.c. of the copper solution. From the amount of hydroxylamin solution required to reach the decolourisation point, the sugar content is obtained by using tables which have been constructed for this purpose. 294 PRACTICAL PHYSIOLOGY Preparation of Solutions. Solution I. — 12'5 gr. CuSO4 (purified as directed in the footnote1) are dissolved by heat in 75 c.c. water and the solution cooled to 25° C. In a large porcelain basin 250*0 gr. potassium carbonate, 200 '0 gr. potassium sulphocyanide and 50-0 gr. potassium bicarbonate are dissolved by stirring in 600 c.c. water. During the process of solution of these salts the temperature at first rises and then falls. If the potassium bicarbonate does not become dissolved the basin must be placed on a water bath and the temperature raised to 40° C. (but no higher). The solution is then cooled to 15° C. and the copper solution mixed with it in small quantities at a time with frequent shaking, so as to prevent any large amount of precipitate forming. The solution is then made up to 1 litre in volume. Solution II. — 6 "55 gr. hydroxylamin sulphate or 5*56 gr. hydroxylamin chloride2 is dissolved in water and the solution added to one of 200 gr. potassium sulpho- cyanide in 1500 c.c. water. The volume is then brought up at 2000 c.c. Titration.— The amount of sugar added must be less than 0'06 gr. (to which limit the table has been constructed). If, therefore, the solution to be examined contain less than 0-6 per cent., 10 c.c. of it are taken for the estimation ; if it contain more, then such a number of c.c. must be taken as will yield a total amount less than 0-06 gr. In all cases the sugar solution must be made up to 10 c.c.3 Mix the 10 c.c. sugar solution with 50 c.c. of the copper solution in an Erlenmeyer flask. Place on wire gauze over a Bunsen burner and bring to the boil. Maintain the boiling for exactly three minutes. Cool the solution quickly by holding the flask under the cold water tap. Place under a burette containing the hydroxylamin solution, and add this, with constant shaking of the flask until the blue colour is just discharged. The weight of dextrose corresponding to the amount of hydroxylamin solution used is then read off on the following table. (See p. 295.) Pentoses. Besides the hexoses, animal tissues also contain small amounts of pentoses, that is, sugars containing five carbon atoms, C5H1005. Being aldehydic in nature, they possess reducing powers and form osazone crystals. They do not ferment with pure yeast, but they all rotate the plane of polarised light. In the animal tissues pentoses do not exist in a free state, being, as far as is known, bound to guanylic acid (see p. 310). They are very plentiful in plants, where they 1 Filter a hot saturated solution of copper sulphate into a large evaporating dish, and after covering with a sheet of filtered paper allow to stand for some hours. Collect the crystals which separate out on a filter, and after all the mother liquor has drained, open up the filter and spread out the crystals between several folds of filter paper. Press then between the folds of filter paper, removing them to fresh paper, until perfectly dry. This is ascertained by placing a glass rod on the crystals and then tapping it. If dry no crystals will stick to the rod. 2 The sulphate is recommended by Bang, but we have found the chloride quite as suitable. 3 Where there is no previous knowledge as to the strength of the sugar solution a preliminary titration should be made by boiling 10 c.c. of the solution with 50 c.c. of the copper solution for three minutes. If the blue colour disappears, repeat with 5 c.c., and so on until the amount is found which does not discharge the blue. PHYSIOLOGICAL CHEMISTRY 295 TABLE FOR CALCULATION OF AMOUNT OF DEXTROSE FROM HYDROXYLAMIN SOLUTION USED IN BANG'S METHOD. Hydroxy' lamin solution, c.c. Dextrose, mg. Hydroxy- lainin solution, c.c. Dextrose. mg. Hydroxy- lamin solution, c.c. Dextrose, mg. Hydroxy- lamin solution, c.c. Dextrose, m.g. 47 43-85 5 29-60 19 17-75 33 7'65 42-75 6 28-65 20 16-95 34 7-05 48 41-65 7 27-75 21 16-15 35 6-50 49 40-60 8 26-85 22 15-35 36 5-90 50 39-50 9 26-00 23 14-60 37 5-35 51 38-40 10 25-10 24 13-80 38 4-75 52 37-40 11 24-20 25 1305 39 4-20 53 36-40 12 23-40 26 12-30 40 3-60 54 35-40 13 22-60 27 11-60 41 3-05 55 34-40 14 21-75 28 10-90 42 2-60 56 33-40 15 21-00 29 10-20 43 2-15 57 32-45 16 20-15 30 9-50 44 1-65 58 31-50 17 19-35 31 8-80 45 1-20 59 30-55 18 18-55 32 8-20 46 0-75 60 exist as polysaccharides called pentosanes. Thus, in gum arabic there is a pentosane which yields £-arabinose when hydrolysed by heating with- mineral acid, and in wood or bran another pentosane yields Z-xylose on similar treatment, which is the variety of pentose present in the nucleic acid of animal cells. Pentose sometimes occurs in the urine — the condition being called pentosuria — the variety being racemic arabinose (inactive optically). From what source this is derived is difficult to determine, for it is independent of the pentoses in the food, and its structure is different from that found present in the tissues. It is mostly combined with urea. EXPERIMENT. Hydrolyse gum arabic by heating a solution of it in a water bath for twenty minutes with 5 % HC1. Arabinose is formed. After neutralising, apply reduction and yeast fermentation tests to portions of the solution. To another portion apply the following characteristic test for pentoses (Tollens). Add phloroglucin (C6H3(OH)3) in small quantities at a time till no more dissolves to a solution of about 5 c. c. of equal parts of concentrated HC1. and water. Then add a few drops of the arabinose solution and warm until a red colour develops. Examine with the direct vision spectroscope when an absorption band will be seen between D. and E. lines. By further heating, a precipitate forms which becomes dissolved in amyl alcohol when this is shaken with the solution. The amyl alcoholic solution shows the above spectrum very clearly. Tollens' test can be applied to urine. Repeat this test, using dextrose solution. EXPERIMENT. Heat about 5 c.c. of Bial's reagent (500 c.c. HCL, sp. gr. 1*151, 1 grm. orcinol, 25 drops 10% ferric chloride solution) to boiling in a test tube, and run in not more than 1 c.c. of the pentose- containing solution, and again heat just to boiling point. A greenish-blue colour rapidly develops. This colour can be extracted with amyl alcohol, when it shows an absorption band in the red. Repeat this test, using a dilute solution of dextrose instead of pentose, when dractically no colour change will occur. 296 PEACTICAL PHYSIOLOGY SK • ' ill nr ~§ II 'i •-S I! 0 il 1 03 X S 03 rft3 a I 2° te ta b S .-^ o .&•§ o o o 1 1 o n ,-H Xn „ t- PHYSIOLOGICAL CHEMISTRY 297 CHAPTER IV. THE PROTEINS. THIS group of bodies, as their name signifies (protos = first) are bodies, of great importance. They occur in all animal and vegetable cells; indeed they are intimately connected with the life of the cell. Without them as food-stuffs animal cells cannot live. At present, top, they are bodies of purely biological origin, no effort to make them in the laboratory having as yet been successful. Most of the members of the group are amorphous bodies of high molecular weight. The molecule is made up of the elements carbon, hydrogen, nitrogen, oxygen, and sulphur. The amounts of the elements vary considerably in different proteins, as can be seen from the following table : — Protein. C. H. N. O. S. Fibrinogen, - - 52 -93 6 '90 16 '66 22-26 1'25 Serum albumin, - 52*08 7'10 15*93 21'96 1'90 Serum globulin, - 52'7l 7'01 15-85 23 -32 I'll Keratin, - - - 50'65 6'36 17'14 20'85 5'00 Elastin, - - - 54-32 6 "99 1675 21-94 Gelatin, - - - 49'83 6'80 17'97 25*13 07 The nitrogen and the sulphur are usually contained in two forms, loosely combined and firmly combined. The loosely combined portions can be split off from the molecule by boiling with a caustic alkali (caustic soda, caustic potash, or soda lime). All proteins when heated alone give a smell of burnt feathers, due to the evolution of ammonia, pyridine, etc. EXPERIMENT I. Evaporate to dryness some of the protein solution provided. Heat strongly. Notice the charring and smell of burnt feathers. EXPERIMENT II. The loosely combined Nitrogen. — To about five c.c. of diluted egg-white add a few drops of 20 % caustic soda ; warm slowly, and hold a piece of moistened red litmus paper over the mouth of the test tube. The litmus turns blue, showing that ammonia gas is being evolved. The ammonia may also be detected by its smell, or by holding the stopper of the concentrated hydrochloric acid bottle over the mouth of the test tube when fumes of ammonium chloride are formed. EXPERIMENT III. The loosely combined Sulphur (lead sulphide test). — To about five c.c. of 20 % caustic soda add two drops of lead acetate solution and some solution of egg-white. Boil. A brown to- black colour is developed, due to the lead sulphide which is formed. 298 PRACTICAL PHYSIOLOGY Recent research has shown that nitrogen may be detected in the protein molecule after complete hydrolysis with 30 % sulphuric acid as :— (1) Amide or Ammonia Nitrogen. This is the loosely combined nitrogen mentioned above. (2) Diamino Nitrogen, precipitated by phosphotungstic acid after the amide N has been removed. (3) Monamino Nitrogen estimated in the residuum when the above two have been removed. By this means the difference in composition of proteins is further brought out : — Total N. Amide N. Diamino N. Monamino N. Caseinogen, - - - 15*62 1-61 3'49 10'31 Edestin (from hemp), - IS'64 1'88 5 '91 10'78 Gliadin (from wheat), - 17 '66 4-20 '98 12'41 Glutenin (from wheat), 17 '49 3'30 2'05 11 '95 Zein (from maize), - 16 '13 2 -97 '49 12-51 (T. B. Osborne.) The complex constitution has been studied recently in two ways — (I) by working out the products of hydrolysis (breaking down) of the different proteins; (2) by endeavouring to link together simple cleavage products, and thereby produce some form of protein. As the result of the first method of procedure, we now know that the proteins of the various parts of the body differ greatly in composition ; for example, the protein of the spleen is different from that of the thymus or of the pancreas. Further, the protein of the same tissue differs in animals of different species, e.g. the serum albumin of the blood of one animal has a different constitution to the serum albumin of an animal of another species; likewise, the chief protein (caseinogen) of milk. We can understand, therefore, why it is that the proteins of the food have to be broken down into such numerous end products in the digestive tract. From these end products chiefly those are required which are of value in building up the animal's own particular forms of protein, forms, as we have seen, differing in various parts of the body, and wholly different from the protein ingested. It is only, therefore, by very complete hydrolysis, that these valuable end products can be obtained free from products of lesser value. (See also under Digestion.) In the following table will be seen the varying yields of the different amino acids obtained from 100 parts of various proteins, after complete hydrolysis with hydrochloric or sulphuric acid. Tyrosin and cystin are separated by crystallisation, after neutralising and concentrating the liquid. The diamino acids — arginin, histidin, lysin — are separated from the rest of the products by being precipitated by phosphotungstic acid in acid solution. Tryptophan is separated by precipitation with mercuric sulphate in the presence of 5 % sulphuric acid after tryptic digestion. The other amino acids are separated (after hydrolysis of PHYSIOLOGICAL CHEMISTRY the protein with hydrochloric acid) by fractional distillation of their ethereal salts under greatly reduced pressure. It will be noticed that the figures given for any one protein do not by any means add up to 100 %. This is due to the occurrence of some inevitable loss in the method of separation, and to the fact that doubtless all the components of protein have not yet been isolated. End products. Serum Albu- min. Egg Albu- min. Serum Globu- lin. Caseiu- ogen (cow). Fibriu- ogen. Edes- tin. Kera- tin. Gela- tin. Bias- tin. Glycin, 0 0 3-5 0 3-0 3'8 •4 16-5 25-8 Alanin, 2-7 2-1 2-2 •9 3-6 3-6 1-2 •8 6-6 Leucin, 20-0 6-1 18-7 10-5 15-0 20-9 18-3 2-1 21-4 Aspartic acid, - 3-1 1-5 2-5 1-2 2-0 4-5 2-5 •6 + Glutamic acid, - 7-7 8-0 8-5 11-0 8-0 6-3 3-0 •9 •8 Cystin, 2-3 •2 •7 •1 — •3 + — — Histidin, - — — — 2-6 — 1-1 — — — Lysin, — — — 5-8 — 1-0 — 28 — Arginin, - — — — 4-8 — 1-7 2-3 7-6 •3 Phenyl-alanin, 3-1 4-4 3-8 3-2 2-0 2-4 3-0 •4 3-9 Tyrosin, - 2-1 1-1 2-5 4-5 3-5 2-1 4-6 0 •3 Tryptophan, + + + 1-5 + + 0 0 — Other amino acids, 3-1 2-3 2-8 5-3 5-4 4-1 10 9-6 2-7 The results of the synthetic method of procedure have also been highly interesting. This has been mainly the work of Emil Fischer and his pupils. Starting with a simple end product, such as glycin, monamino-acetic acid, two of these are combined together, forming a da-peptide glycyl-glycin with the elimination of water, thus : — OH NH2CH2CO H NHCH2COOH = NH2CH2CO.NHGH2COOH + H20. Glycin. Glycin. Glycyl-glycin. The addition of another molecule forms a tripeptide, and so on until potypeptides (many linkages) are formed. Pentaglycyl-glycin, for example, is : — NH2CH2CO(NHCH2CO)4NHCH2COOH. But not only has glycin been combined to glycin— other end products, such as alanin, leucin, phenylalanin, tyrosin, etc., have been combined together, giving such bodies as leucyl-glycyl-alanin, and so on. For example, the polypeptide (do-deka-peptide) leucyl-deca-glycyl-glycin has the formula : — C4H9CH(NH2)CO Leucyl. (NHCH2CO)10 Glycyl. NHCH2COOH. Glycin. By many such operations, polypeptides have been obtained, which, if 300 PRACTICAL PHYSIOLOGY not actually having the same composition as any known peptone (see later), have many resemblances to peptones. The proteins have for the most part many physical and chemical reactions in common. THE PHYSICAL AND CHEMICAL PROPERTIES OF PROTEINS. I. Solubility. — All proteins 1 are insoluble in alcohol and ether. They vary as to their solubility in water, the more common proteins (albumins and globulins) being soluble in water (albumins) or in weak saline solutions (globulins). Some, however, are not soluble, even in concentrated saline solutions. EXPERIMENT IV. From the undiluted egg-white provided prepare a solution of egg albumin by adding 10 volumes of distilled water and mixing thoroughly in a flask. An opalescent solution is thus obtained, the opalescence being partly due to the colloidal nature of the solution, although in part to some other protein (ovo-mucin), which has not gone into solution. This can be removed by filtering through fine muslin. Note that this solution, like all colloidal solutions, gives a persistent froth on shaking. The solution prepared above can be used in the subsequent experi- ments, unless otherwise stated. II. Diffusibility. — As the proteins give only colloidal solutions, these solutions will not dialyse, that is, diffuse through animal membranes or parchment paper. In this they are unlike crystalloids, such as inorganic salts, which readily diffuse through such membranes. Of the various forms of dialyser, a tube of parchment is the simplest. EXPERIMENT V. Place a mixture of diluted blood and of a 10 % sodium chloride solution in the dialyser provided. Test a sample of distilled water with silver nitrate, and note that no haze of silver chloride occurs. Place the dialyser in a beaker of this water and allow dialysis to proceed for a day. On now testing the water in the beaker for chlorides with silver nitrate, it will be found that a white precipitate of silver chloride occurs, showing that the chlorides have diffused through the parchment. It can be shown, however, that no protein has dialysed through, by the absence of pigment and by applying the tests for protein given below. III. Heat Coagulation. — Most of the so-called native proteins (albu- mins and globulins) coagulate when their solutions are heated. Dif- ferent proteins coagulate at different temperatures, varying usually from 56°C.-78°C. A faint degree of acidity and the presence of much neutral salt greatly favour heat coagulation. vegetable proteins are soluble in alcohol. PHYSIOLOGICAL CHEMISTRY 301 EXPERIMENT VI. Fill a narrow glass tube with some egg-white solution, faintly acidulated with acetic acid, and fasten off one end. Now fix this to the lower end of the thermometer by means of small elastic bands. Gradually heat in a test tube placed in a water bath and observe the temperature at which the albumin becomes opaque and set. IV. Crystallisation. — Most proteins crystallise with difficulty; the blood pigment of certain animals, however, crystallises readily. (See later under Blood, Chapter VIII.) Egg albumin and serum albumin FIG. 224.— Crystallised albumin. X600. have, however, been crystallised. Certain vegetable proteins, e.g. the globulin of hemp seed (edesbin), crystallise more easily. DEMONSTRATION. Some hemp seed has been thoroughly pounded, extracted with warm 5 % sodium chloride (50°C.) and placed in a dialyser overnight. As the result of dialysis, crystals of edestin have become deposited in the tube. Examine those placed under the microscope. Crystals of edestin may also be obtained, on standing, by cooling with ice the extract of hemp seed. To obtain crystals of egg albumin the whites of several eggs are mixed with an exactly equal amount of a fully saturated solution of ammonium sulphate. This precipitates the globulins. The ammonium sulphate solution must be exactly neutral in reaction and should be added to the egg-white in small quantities at a 302 PKACTICAL PHYSIOLOGY time, the mixture being briskly stirred between each addition. The precipitated globulin is filtered off, and the filtrate, which reacts alkaline to litmus, is treated with ammonium sulphate, drop by drop, until a faint haze of precipitated albumin is obtained. A drop of water is added, so that the haze just disappears. The solution is now treated with 10 % acetic acid, drop by drop, until a pre- cipitate of albumin just forms. The flask is set aside ; in about twenty hours it will .be found that a large number of needle-shaped crystals have become deposited (see Fig. 224). V. Rotation of Light. — All proteins are laevo - rotatory. Some combined proteins, such as haemoglobin and nucleo-protein, are dextro- rotatory, but their protein portion is laevo-rotatory. VI. Colour Reactions. This group of reactions is very important, as each reaction yields information as to the constitution of the protein molecule. The meaning of each test should therefore be carefully noted. (a) The Biuret Reaction (Piotrowski's test). EXPERIMENT VII. Pour a drop of weak copper sulphate into a test tube. Now add some 20% caustic soda until a pale blue colour is obtained (about 15 c.c.). Divide this into three portions, A, B, C. Keep A as control colour. To B add a few drops of diluted egg-white. To C add the same number of drops of the commercial peptone provided. Note the violet colour with albumin, the pink colour with the peptone solution. It is important to keep control tube A, since in using very weak solutions a slight change in colour can be detected by comparison with the control. All proteins give either a purple or pink colour with this test. It shows that the protein contains two or more CO - NH - groups linked together. The same reaction is given by the body biuret formed when urea is heated, hence the name. (b) Xanthoproteic Reaction. EXPERIMENT VIII. To about 5 c.c. of the solution of egg-white add a few drops of strong nitric acid; a white precipitate results. Warm this and the precipitate changes to a yellow curd. Cool under the tap Add a few drops of strong ammonia ; the yellow colour changes to a brilliant orange. The name xanthoproteic (yellow protein) will help the student to remember the colour of the curd obtained. __ This test, shows the presence of the benzene ring in the protein molecule ; hence only proteins containing axSSRVFrng give this test. (c) Millon's Reaction. EXPERIMENT IX. Add a few drops of Millon's reagent (which consists of a solution of mercurous and mercuric nitrates) to some of PHYSIOLOGICAL CHEMISTRY 303 the egg-white solution. A white coagulura occurs, which on warming changes to a brick-red curd. This reaction differs from the xanthoproteic only in depending uponc/ I the presence of the benzene ring with an hydroxyl group attached to it r\fa* in addition, or, in other words, the phenolic ffroup. _ (d) The Glyoxylic Acid test (Hopkins' modification of Adamkiewiecz's Reaction). EXPERIMENT X. To some egg-white solution in a test tube add about 1 c.c. of glyoxylic acid solution, and run in carefully without mixing ordinary strong sulphuric acid. A violet ring is obtained at the junction of the fluid, which extends into the supernatant egg-white solution when the tube is gently agitated. This test depends upon the presence of tryptophan (indol amino- propionic acid) in the protein molecule, and is only given by proteins containing such a grouping. (e) The a-Naphthol test (Molisch's test). This has already been given under carbohydrates (q.v). Proteins containing a carbohydrate moiety yield this test. The purple colour should be very pro- nounced before the test is deemed positive. The green colour obtained plays no part in the reaction. The test is not very reliable. VII. Precipitation by Neutral Salts (" Salting out "). (A) Ammonium Sulphate. EXPERIMENT XL To some egg-white solution add an equal amount of saturated solution of ammonium sulphate = half saturation. A white precipitate of globulin is produced. Filter ; keep the filtrate. After washing the residue with saturated ammonium sulphate dissolve it in a little water and boil. Note that the protein is coagulated in fine flakes. Divide the filtrate : (a) Add crystals of Am2SO4 in excess (full saturation). The albumin is now salted out. (b) Boil ; flakes of coagulated protein show the presence of coagulable protein (albumin). Half saturation with Am2S04 therefore precipitates globulins ; full saturation precipitates albumins. (B) Magnesium Sulphate. EXPERIMENT XII. Fully saturate (i.e. add crystals) the solution of egg-white with MgS04. A precipitate of globulin results. Filter. Prove by heat coagulation and by fully saturating with Am2S04 that protein (albumin) is left in the filtrate. Magnesium sulphate in full saturation precipitates globulins, but not albumins (see table, p. 312). (C) Sodium Chloi'ide, Ammonium Chloride. These salts resemble magnesium sulphate in their " salting out " properties. 304 PEACTICAL PHYSIOLOGY (D) Sodium Sulphate possesses at 30° C. the same protein precipitating powers as ammonium sulphate. It is of great advantage when it is desired to estimate the amount of protein in any fluid. By precipitat- ing with sodium sulphate and determining the total nitrogen in the precipitate by Kjeldahl's method (see Urine) the amount of protein is found by multiplying by 6*25. (E) Zinc Sulphate has also been used for salting-out purposes. VIII. Coagulants of Proteins. — A coagulum differs from a precipitate in that it is no longer soluble in its original solvent ; in other words, its physical or chemical nature has undergone some change. Such is the case in the coagulation of protein by heat. Other coagulants of protein are :— mechanical agitation, mineral acids and salts, and other acids such as tannic, picric, etc. EXPERIMENT XIII. Violently shake some egg-white solution with sand. Strings of coagulated protein are deposited. EXPERIMENT XIV. To some egg-white solution add gently some strong HN03. A white precipitate appears, which is insoluble on heating (cf. Proteoses). EXPERIMENT XV. Acidulate some egg-white solution strongly with acetic acid, then add strong potassium ferrocyanide — a whitish yellow precipitate. EXPERIMENT XVI. Add picric acid — a white precipitate. Many other acids, such as phosphomolybdic, phosphotungstic, trichloracetic and salicyl sulphonic are used to precipitate proteins. Alcohol precipitates all proteins. At first it forms a precipitate; but if the action be prolonged this changes to a coagulum. Peptone . and fibrin ferment (thrombin) take longer to undergo this change ; advan- tage is taken of this to separate these bodies from other proteins (see Preparation of Fibrin Ferment, 336). CHAPTER V. PROTEINS— CONTINUED. THE CLASSIFICATION OF PROTEINS. THE following classification has now been adopted for proteins. It is based partly upon the results of chemical investigation, partly upon .such properties as solubility, " salting out," etc. It cannot, therefore, be regarded as complete. PHYSIOLOGICAL CHEMISTRY 305 (1) Protamines. (2) Histones. (3) Albumins. (4) Globulins. (5) Phospho-proteins. (6) Sclero-protems. (7) Compound proteins. The Protamines. — These substances are the simplest proteins known (Kossel). They occur combined with nucleic acid in the sper- matozoa of certain fishes, such as the salmon, sturgeon, mackerel and herring. Sturin from the sturgeon has the formula C36H6SN1907; salmin (salmon) and dupein (herring) have the formula C30H57N1706. They are difficult to obtain in a state of purity, and the technique is complicated. Upon hydrolysis they yield large amounts of the hexone bases arginin, lysin, histidin, especially arginin. Monamino acids are combined into the protamines, but only one cyclopterin has so far yielded a ringed amino acid grouping. The protein nature of the protamines is shown by the fact that they yield the Biuret test. The Histones occur mainly in combination. Perhaps the best known example is the globin portion of haemoglobin. They also occur com- bined in the nuclei of blood corpuscles, e.g. in the red corpuscles of the goose and in the white corpuscles of the thymus gland. The histones are somewhat more complicated than the protamines. Bang gives the following characteristic reactions : — (a) They are precipitated from watery solution by ammonia — the precipitate being soluble in excess. (b) In presence of salts they are coagulable by hea't. (c) They give a precipitate with nitric acid in the cold, soluble on heating, reappearing on cooling (cf. Proteoses and Peptones). THE ALBUMINS AND GLOBULINS. These two groups have been already studied in the preceding experi- ments with the egg-white solution. The main difference between the albumins and the globulins is that of solubility. It has also been shown chemically that the products of hydrolysis differ, the albumins yielding no glycin. Upon hydrolysis all yield members of the chief amino acid groups (see table, p. 299). Albumins are soluble in distilled water and in saturated solutions of all neutral salts except ammonium sulphate and anhydrous sodium sulphate, in which they are insoluble. They are, however, soluble in half-saturated solutions of these salts (see table, p. 312). Globulins are insoluble in distilled water and in saturated solutions of all neutral salts. They are, moreover, insoluble in half-saturated U 306 PKACTICAL PHYSIOLOGY solutions of ammonium sulphate and anhydrous sodium sulphate. They are soluble in weak saline solutions (see table, p. 312). The chief kinds of albumins are egg albumin, serum albumin (see Blood), and lactalbumin (see Milk). The most important globulins are egg globulin, serum globulin, fibrinogen (for both see Blood), and myosinogen (see Muscle). THE PHOSPHOPROTEINS. The chief members of this group are the caseinogens of milk and the vitellins from egg-yolk. They derive their name from the large amount of phosphorus contained in their molecule. They differ, how- ever, from nucleoproteins in containing no purin bases. Dissolve some commercial caseinogen in 2 % caustic soda, and perform the following EXPERIMENTS : — (I.) Note that it is precipitated with 1 % acetic acid, the precipitate being soluble in excess of acid. (II.) Perform the colour tests for protein, and record your results. (III.) Perform the " salting out " tests with Am2SO4 and MgS04. (IV.) Heat the solution. With the solid substance perform the following experiments : — (V.) Heat some solid caseinogen upon a piece of broken porcelain with " combustion mixture " (a mixture of sodium carbonate and potassium nitrate). When cool, extract with nitric acid, filter, add ammonium molybdate in nitric acid, and heat. The canary yellow precipitate denotes phosphates. (VI.) Heat a little caseinogen with 1 % NaOH in the incubator or on a water bath at 37° C. for twenty -four hours. Phosphoric acid is broken off. Precipitate the phosphoric acid, after acidifying with acetic acid and filtering, by the addition of ammoniacal magnesium citrate. Filter. Dissolve the pre- cipitate in nitric acid, and test with molybdate as above. (VII.) Make a solution of caseinogen in lime water. Show that it is clotted by rennet. In connection with the above experiments it will be found that case- inogen yields all the colour tests except Molisch. It therefore contains no carbohydrate group (see p. 279). The xanthoproteic, Millon's, and the glyoxylic tests will be very well marked, showing that caseinogen is rich in tyrosin and tryptophane. In "salting out" caseinogen behaves like a globulin, being pre- PHYSIOLOGICAL CHEMISTRY 307 cipitated by full saturation with magnesium sulphate and half saturation of ammonium sulphate. Caseinogen is not coagulated by heat (see table, p. 312). THE SCLERO-PROTEINS. This comprises the group of proteins formerly termed albuminoids. They are obtained mainly from " the hard " or supporting structures of the body. Collagen, the precursor of gelatin, forms the chief constituent of white fibrous tissue and of the organic substance of bone. It also exists in cartilage, where, however, it is mixed with several other bodies (see under mucinoids, p. 308). Preparation of Collagen. — A piece of tendon is macerated overnight in 1 % caustic alkali to remove other proteins, and then washed with water till alkali free. The resulting mass is collagen. Place a piece of this in a flask and boil it for ten minutes with water which is rendered faintly acid with acetic acid. By this treatment, the collagen is trans- formed into gelatin and, on cooling the solution, it gelatinises. Gelatin.— This is really the hydride of collagen, the boiling with acidulated water in the above experiment having caused the collagen to take up a molecule of water. Conversely, the gelatin can be recon- verted into collagen by heating it to 130° C., whereby it loses water. EXPERIMENT VIII. Divide a solution of gelatin in lukewarm water into five portions, to which apply the following tests: (1) the Biuret reaction : a violet colour is produced. (2), the xantho-proteic reaction : only a slight colouration is produced. (3) the Millon's test : only a slight reddening of the precipitate occurs on boiling. (4) the glyoxylic test : absent or very faint. (5) half saturation with Am2S04 : salted out. The reason why the second, third and fourth tests are not very distinct, is because gelatin does not yield aromatic bodies on decomposi- tion, and both these tests depend on the presence of aromatic bodies. Some varieties of gelatin give these reactions more distinctly than others, but absolutely pure gelatin is said not to give them at all, so that their presence is held to depend on native protein in the gelatin. The other sclero-proteins are unimportant. They are Keratin, which occurs in the skin and its appendages and in the medullary sheaths of nerves ; it is remarkable for the large percentage of sulphur which it contains ; Elastin, which is found in elastic fibres, and contains a very small percentage of sulphur, but a considerable amount of aromatic bodies. All these sclero-proteins except keratin yield glycin as their chief 308 PRACTICAL PHYSIOLOGY decomposition product. They also yield the hexone bases, since protamin forms part of their molecule (see table, p. 299). EXPERIMENT IX. With pieces of finger-nail show that keratin gives xanthoproteic, Millon's, and lead sulphide test. THE COMPOUND PROTEINS. In this group we have proteins to which groups other than protein are united to form a complex molecule. The chief groups are : — (i) The chromo-proteins. (ii) The gluco-proteins. (Hi) The nucleo-proteins. The Chromo-proteins. — As the name signifies these are " coloured " proteins, and its members are pigments, of which the chief is haemo- globin. (See chapter on Blood.) The Gluco-proteins are compounds of protein with a large carbo- hydrate group. Many proteins not contained in this class, such as egg albumin and nucleo-proteins, contain carbohydrate, but not in such large amounts. The chief members of the class are the mucins. EXPERIMENT X. Collect some saliva in a test tube, note its viscidity; add to it a few drops of 1 % acetic acid; a stringy precipitate of mucin results. It is insoluble in excess of acetic acid. Filter. To residue add a few drops of weak sodium carbonate solution, when the precipitate will dissolve. Test this with protein colour tests, including Molisch. EXPERIMENT XI. Mucin has been prepared from connective tissue where it is very abundant, by extracting the latter with a weak alkali (lime water). The mucin has been precipitated by a weak acid. The resulting precipitate has then been boiled for about ten minutes with hydrochloric acid (1 part concentrated acid + 3 parts water), and the resulting solution cooled and neutralised. Examine portions of the resulting solutions. Divide the solution into portions, a and b. To (a) apply the Biuret reaction — a violet or pink colour is produced, showing the presence of the protein moiety. To (b) add a drop of copper sulphate solution, and, if necessary, some caustic alkali till a blue solution is obtained. Now boil, when reduction to cuprous oxide will occur, demonstrating the presence of the carbo- hydrate moiety. Besides forming the ground substance of the connective tissues, mucin is also secreted on to the surface of all mucous membranes, where it acts as a lubricant. Besides the mucins, this group also contains the mucinoids, bodies distinguished from mucin in not being viscous in nature and not being so easily precipitated by PHYSIOLOGICAL CHEMISTEY 309 acetic acid, the precipitate when formed being soluble in excess. They are represented by the mucoid of ovarian cysts, the ovo-mucoid of white of egg, and bodies (sometimes termed chondro-proteids) which occur along with collagen in cartilage. These last on decomposition with an acid yield protein and a reducing body called chondroi tin -sulphuric acid, which can further be decomposed to yield chondrosin, a body containing nitrogen, but more strongly reducing than dextrose. This body can still further be decomposed to glucosamine, the usual carbohydrate moiety of the mucins. Of this the mucins contain about 30 %. fCH2(OH) CHOH Formula of I (JHOH Glucosamine. l^jj CH(NH2) J3 The Nucleo-proteins. — These consist of protein in combination with nuclein, and form the chief constituent of the nuclei of cells. Nuclein itself is a compound of protein, with an organic acid known as nucleic acid. PREPARATION OF NUCLEO-PROTEIN. METHOD I. A cellular organ, such as the thymus or pancreas, is minced and macerated overnight with water made faintly alkaline with caustic soda or ammonia. The extract is then strained through muslin, litmus added, and then weak acetic acid. When the reaction becomes faintly acid, a copious precipitate of nucleo-protein occurs. The nucleo- protein is filtered off and dissolved in weak alkali (1 % sodium carbonate). METHOD II. Demonstration. — The minced organ is pounded in a mortar, with an equal amount of solid NaCl. Upon throwing the mass into excess of water in a tall cylinder, the nucleo-protein rises as a stringy mass to the top of the water. This is collected and dissolved in 1% sodium carbonate. EXPERIMENT XII. Some of this alkaline solution is supplied— (1) Add acetic acid — white precipitate soluble with difficulty in excess. (Cf. mucin, which is insoluble, and caseinogen, which is readily soluble.) (2) Perform the protein colour tests. (3) Ascertain how it is " salted out." Demonstration. — The precipitate of nucleo-protein has been digested with pepsin hydrochloric acid for twenty-four hours. The protein has become converted into peptone — the liberated nuclein has fallen down as a brown sediment. 310 PRACTICAL PHYSIOLOGY This brown sediment can be further decomposed into nucleic acid and protein by dissolving it in alkali and adding 0*3 % hydrochloric acid in alcohol. This precipitates the nucleic acid. If this be collected and heated in a sealed tube with hydrochloric acid, it splits into a number of simpler bodies. Guanylic acid, the simplest nucleic acid found in the pancreas, yields phosphoric acid, guanine, and pentose. Other nucleic acids which occur with the guanylic acid yield phosphoric acid, guanine, and adenine, laevulinic acid (probably from decomposition of a hexose), the pyrimidine base cytosine, and probably other pyrimidine bases. SCHEMA OF RELATIONSHIP OF NUCLEIN, ETC. Nucleo-protein (digested with pepsin) Nuclein (precipitated as a brown sediment, Peptone decomposed l>y acid alcohol) (goes into solution) Acid Meta-protein (in solution) Nucleic Acid (white precipitate) heated in closed tube with HC1 ! Purin bodies Carbohydrate Phosphoric Acid Pyrimidine bases (adenin, guanin) (hexose or pentose) Cytosin The presence of phosphorus in nucleo-protein may be demonstrated by heating it with combustion mixture (cf. phospho-protein). Its phos- phoric acid, however, is not split off by incubation with 1 % NaOH at 37°C. for 24 hours; nor are nucleo-proteins clotted by the rennet enzyme. We come lastly to the products of protein hydrolysis, which will be dealt with more fully under Digestion. When albumin is subjected to the action of a weak acid or weak alkali it is transformed into a derived protein or meta-protein. These closely resemble the globulins. Acid and Alkali Meta-protein. EXPERIMENT XIII. To some diluted egg-white add two or three drops of 10 % HC1. Place in water bath at body temperature for five minutes. Acid meta-protein is formed. Note. — (a) That no coagulum now appears on heating. (b) It is precipitated by making the solution neutral or very faintly alkaline. (c) It is salted out by half saturation with ammonium sulphate (like a globulin). (d) If neutralised and suspended in water it is coagulated on boiling. PHYSIOLOGICAL CHEMISTRY 311 EXPERIMENT XIV. Render dilute egg-white alkaline, and treat as above. Alkali meta-protein is prepared. It gives similar reactions to acid meta-protein. As, however, in making alkali meta-protein some of the loosely combined nitrogen and sulphur are split off, it cannot be changed into acid meta-protein. The reverse change is, however, possible. Acid meta-protein when prepared from muscle (myosin) is often called syntonin. Alkali meta-protein when prepared by the action of strong alkalis on protein is termed " Lieberkiihn's Jelly.' Acid meta-protein is one of the first products of peptic digestion of protein, alkali meta-protein of tryptic digestion. Proteoses and Peptones. — These occur as the first stages of protein cleavage under the action of such agents as mineral acids, superheated steam, the proteolytic enzymes. They will be more fully studied under Digestion. (See p. 384). EXPERIMENT XV. Use the solution of Witte's peptone provided and perform the following tests : (a) Biuret reaction is pink. (Proteoses and Peptones.) (b) On faintly acidifying with acetic acid and boiling — -no coagulum. (c) Add a little HN03 — a white ring. This dissolves on heating and reappears on cooling. Salicyl-sulphonic acid produces the same effect, but the reaction is more delicate. (d) To the solution add an equal amount of Am2SO4 (half saturate). A white precipitate of the primary proteoses (except hetero- proteose) which are salted out Filter. (e) Saturate the filtrate with crystals of Am2SO4. The secondary proteoses are salted out. ' Filter. (/) With the filtrate perform Biuret and xanthoproteic tests To obtain the Biuret test in the presence of a large quantity of Am2S04 a large excess of caustic soda is required. As peptones are not precipitated by HNO3 the xanthoproteic test manifests itself by a yellow colour on heating the solution, turning orange with ammonia. The positive results show the presence of peptones. From these experiments we learn : (1) That proteoses and peptones give a pink Biuret. (2) That they are not coagulable by heat. (3) That proteoses give a precipitate with HN03 soluble on heating. Therefore, in the presence of other proteins, precipitated by HN03, such as albumin and globulin, they can be separated by warming the solution and filtering hot. 312 PEACTICAL PHYSIOLOGY The precipitates of albumins and globulins do not dissolve on warming. (4) Primary proteoses1 are salted out by half saturation with ammonium sulphate. (5) Secondary proteoses2 are salted out by full saturation with ammonium sulphate. (6) All proteins but peptones are salted out by full saturation with ammonium sulphate (see table below). (The other products of protein hydrolysis are fully dealt with under Digestion). Protein. Solu- bility. Diffusi- bility. Action of heat. Biuret test. Salting out action of Am2SO4. Satura- tion. Salting out action of MgS04 (full satu- ration). Action of HN03 or salicyl- sulpbonic acid. Action of alcohol. Globulin. Saline but not pure water. Nil. Coagu- lated. Violet. By half. + Precipitate insol. on heating. Precipi- tated then coagu- lated. Albumin. Water. Nil. Coagu- lated. Violet. By full. Precipitate insol. on heating. Precipi- tated then coagu- lated. Primary l proteoses. Water. Small. Not coagu- lated. Pink. By half. + Precipitate sol. on heating. Precipi- tated. Secondary2 proteoses. Water. Small. Not coagu- lated. Pink. By full. Little or no precipitate sol. on heating. Precipi- tated. Peptones. Water. Great. Not coagu- lated. Pink. Not. — No precipi- tate. Precipi- tated. Casein- ogen. Weak alkali. Nil. Not coagu- lated. Violet. By half. + — Precipi- tated. 1 Hetero-proteose is an exception. 2 See fuller table on page 386. PHYSIOLOGICAL CHEMISTRY 313 CHAPTER VI. FATS, FATTY ACIDS, PHOSPHORISED FATS AND CHOLESTEROL. THESE bodies are classified together because they are soluble in the same liquids. After extracting an organ or tissue with alcohol, ether or chloroform, and evaporating off the solvent, a more or less syrupy mass is left behind consisting of a varying mixture of the above men- tioned substances. They are often called collectively the lipoids. Method for the Extraction of an Organ or Tissue with Ether.— The simplest method is by means of Soxhlet's apparatus (fig. 225). This consists of an extracting chamber into which opens, near the top, a side tube, connected below with a flask, in which is placed the ether ; above it is connected with a condenser. The flask is placed on a water-bath, and the ether passes into the chamber, and then into the Liebig's condenser, where it is condensed and trickles back into the extracting chamber. The ether thus gradually accumulates in the extraction chamber until it reaches the level of the bend in another side tube opening near the bottom of the extracting chamber, when syphon action is established, and the whole of the ether drains back into the distilling flask. The dried tissue or organ to be extracted is finely ground and placed in a cartridge of porous paper, which is inserted in the extracting chamber. The warm condensed ether as it accumulates in the chamber dissolves out the fat, and carries it into the distilling flask. The process should be allowed to proceed for several hours. The contents of the distilling flask are then removed to a flat dish, and the ether allowed to evaporate. The residue contains the lipoid substances. FIG. 225.— Soxhlet's. apparatus. FATS AND FATTY ACIDS. Neutral fats are the ethereal salts of the fatty acids with the tri- atomic alcohol glycerine, and have therefore the general formula : CH9-0-CO-X C'H _ O - CO - X CH2-0-CO-X. They are named according to the fatty acid they contain, thus : stearin, olein. The fatty acids are monobasic organic acids, containing one car- boxylic group (COOH) attached to a hydrocarbon radicle. They belong to two classes, the saturated and the unsaturated. The saturated acids have the general formula CnH2n+1. COOH. Those commonly occurring in fats are stearic acid, in which ?i = 17, and palmitic acid, in which 314 PRACTICAL PHYSIOLOGY 7i=15. Thus the formula for stearic acid is CH3. (CH2)16. COOH. The first member of this series is acetic acid, CH3. COOH. The unsaturated acids contain relatively less hydrogen in the hydro- carbon chain attached to the carboxylic group. This is due to the fact that there are one or more double bonds (unsaturated) between the carbon atoms of the chain. Thus oleic acid, the commonly occurring unsatur- ated acid of fats, has the formula : CH3. (CH2)7. CH = CH(CH2)7. COOH, and belongs to the series CnH2n_!. COOH. Other unsaturated acids, containing two, or even more, double bonds occur in the fat of the liver, heart and kidney, and in drying oils, such as linseed oil. The unsaturated nature of these acids is shown by their combining directly with chlorine or bromine, thus becoming saturated. EXPERIMENT I. Shake up some oleic acid or its alcoholic solution with dilute bromine water. The colour of the bromine disappears. Repeat with an alcoholic solution of stearic acid, when the colour of the bromine persists. Under suitable conditions unsaturated fatty acids and fats will also combine with iodine. The proportion of iodine with which a given mixed fat will combine therefore represents the amount of unsaturated acid present. This is called the Iodine Number of the mixed fat. (See p. 322). Common fats are made up almost entirely of varying pro- portions of stearin, palmitin (which are solid at ordinary temperatures) and oleiri which is liquid. The more olein a fat contains, therefore, the lower will be its melting point and the higher its iodine number. All the fatty acids possess one property in common, viz. that they form salts. These salts are called soaps. By boiling neutral fat with caustic alkali, it is split up (by a process of hydrolysis) into its con- stituents, the glycerine being set free and the fatty acid uniting with the alkali to form a soap. This process is called saponificatim. EXPERIMENT II. Saponificatwn of Neutral Fat. — Place about 50 c.c. of strong caustic soda in a dish, and add about 10 grammes of fat. Heat to near the boiling point and stir the mixture frequently. When all the fat has disappeared allow the mixture to cool. The soap forms a jelly or cake, and can be washed in cold water to remove any excess of caustic soda. A hard soap is formed if caustic soda is used; but with caustic potash a soft soap is obtained. EXPERIMENT III. Separation of Fatty Acid from Soap. — Place about 40 c.c. of 20 per cent, sulphuric acid in a small beaker, and heat it nearly to boiling point ; drop into this pieces of the washed soap, stirring with a glass rod between each addition, The acid displaces the alkali from its combination with the fatty acid, and the latter separates out on the surface of the water as an oily layer. PHYSIOLOGICAL CHEMISTRY 315 EXPERIMENTS IV. Reactions of Fatty Acids. — Remove some of the fatty acid with a clean glass rod, and place it on a piece of ordinary paper ; a greasy stain will result. In order to purify the fatty acid allow the contents of the beaker to cool, when the fatty acid will solidify and can be easily removed with a penknife, and transferred to distilled water in a small beaker. This removes a great part of the adherent sulphuric acid. But to free it completely it is necessary to dissolve the fatty acid in alcohol, and pour the resulting solution into excess of cold distilled water. The fatty acid which separates is filtered off and washed with distilled water. Use the purified fatty acids for the following reactions :— A. Demonstrate that fatty acid is acid in reaction. For this purpose place some alcohol in a test tube, add a few drops of an alcoholic solution of phenolphthalein (an indicator which turns red with alkali, N but is colourless with acids), and then a few drops of weak ^. caustic soda. Warm the resulting red solution on the water-bath, and drop into it small pieces of fatty acid. The red colour will disappear. Repeat the experiment with a piece of neutral fat ; the result is negative. B. Add a piece of fatty acid to some half saturated solution of sodium carbonate, and warm ; the fatty acid dissolves, carbon dioxide is liberated, and a solution of soap is obtained. Neutral fat is insoluble in cold sodium carbonate solution. C. Press out some fatty acid between filter paper until it is dry, and apply the acrolein test as described in Experiment V. (p. 316). The result is negative. D. To a solution of soap add : (a) a few drops of a solution of calcium chloride — a white precipitate of a calcium soap falls down ; (b) some lead acetate solution — a white precipitate of lead soap falls down (lead plaster). The fatty acids prepared by the above method mainly consist of a mixture of palmitic, stearic and oleic. To separate these from one another, advantage is taken of the fact that they differ in the readiness with which they form salts (soaps) with lead acetate. ADVANCED EXPERIMENT. To separate the Solid from the Fluid Fatty Acids. — Melt the fatty acids in a beaker, and add to the resulting fluid about four times its bulk of 70 per cent, alcohol. Place the beaker on the boiling- water bath for a few minutes, and then filter quickly through a folded filter. Allow the filtrate to cool, when the solid acids will separate out as a crystalline mass, whereas the oleic acid will remain in solution. The two can then be separated by filtration. The further separation of stearic from palmitic acid is a laborious process, and consists of the addition of an alcoholic solution of lead acetate in small quantities 316 PEACTICAL PHYSIOLOGY at a time to a solution of the acids in alcohol. Each addition produces a pre- cipitate, which is filtered off and treated with dilute hydrochloric acid and ether. The hydrochloric acid decomposes the lead salt, and the liberated fatty acid goes into solution in the ether. This process is called fractional precipitation, and the higher the melting point of the acid the more easily is it precipitated by the lead acetate. Besides these reactions of the fatty acid produced from it, neutral fat gives an important reaction, depending on the glycerine which it contains. This is called the acrokin reaction. EXPERIMENT V. Place a small piece of fat in a thoroughly dried test tube, add to it three or four times its bulk of acid potassium sulphate,1 and heat. A pungent vapour of acrolein 2 is given off, which blackens a piece of filter paper which has been dipped in ammoniacal silver nitrate solution. This reaction demonstrates that the vapour acts as a reducing agent. Emidsificatim. — When oil is mixed with water it floats to the surface, but when a soap is present in solution in the water the oil globules remain suspended, and an emulsion results. This is more permanent if some suspending medium such as mucilage be added. EXPERIMENT VI. In one test tube (a) place some soap solution ; in another (6), some water. To each add some neutral olive oil and shake. Allow to stand, and note that a remains emulsified, b does not. EXPERIMENT VII. Place some rancid oil (i.e. containing free fatty acid) in a test tube, add some weak caustic potash solution and shake; an emulsion forms, soap being formed by the alkali combining with the fatty acid. EXPERIMENT VIII. Divide the emulsion produced in Experiment VII. into two parts ; to one of these add a little mucilage or egg-albumin and shake, and note that the emulsion " stands " much longer than that to which no suspending medium has been added. LECITHINS. Soluble in acetone, and otherwise very closely related to the fats, is a group of bodies called lecithins. The lecithins are present in greater or less amount in all the cells of the body. Most plentifully, they occur in the envelope and stroma of the red-blood corpuscles, in nervous tissues and in bile. They are also found in plants. Chemically these bodies consist of a glycerine molecule, two of the hydroxyl groups of which are combined with fatty acid and the remaining one with Commercial acid potassium sulphate is often impure and gives a pungent reducing vapour by itself. It is well, therefore, to make a preliminary test with the crystals alone. The impure salt can be readily purified by crystallisation. 2 Acrolein is the aldehyde of allyl alcohol and has the formula CH2 = CH-CHO. PHYSIOLOGICAL CHEMISTRY 317 phosphoric acid, which, on the other hand, has attached to it an ammonium base, cholin. The two molecules of fatty acid are usually of the same kind, but they may be different. The structural formula for a typical lecithin is therefore : — fCH2-OOC(CH2)16CH3^ V Stearic acid. H- CH-OOC(CH2)16CH3 Glycerine. Phosphoric • — ^CH2 — CH2OH. acid- Cholin. Cholin is closely related chemically to certain basic bodies occurring in plants, one of which is muscarin, an alkaloid with a strong pharma- cological action on the heart and glands. Cholin itself has a pro- nounced pharmacological action, thus, it produces a marked fall in blood pressure. In the free state cholin is not present in the blood, for it appears that the cholin liberated in the intestine by the break- down of lecithin is destroyed before absorption. During degeneration of nervous tissue, of which lecithins are important constituents, cholin may appear in a free state in the blood; in such cases it can be recognised by adding platinic chloride to an alcoholic extract of blood, or cerebro-spinal fluid, when crystals of choline platinochloride separate out. These are yellow in colour and octahedral or prismatic in shape. Similar crystals are produced by adding platinic chloride to ammonium or potassium chloride, but the cholin crystals can be distinguished from those by adding a strong solution of iodine in potassium iodide, when the choline crystals become changed into dark brown plates, which afterwards change into oily droplets. From their chemical structure, we see that the lecithins, besides being closely related to fats, bear some relationship to the nucleins; both contain phosphorus, and it has been suggested that the phosphorus of nuclein is derived from the phosphorus of lecithin. The lecithins can also form various combinations with proteins. These are some- times called lecithides. The lecithins are split up by lipase and possibly reconstructed in the various tissues in which they are found present. Further indication of their importance in the animal economy is found in the fact that they can act on the so-called complement in the laking of red blood corpuscles by such substances as snake venom. They are important constituents of the cell wall, and have therefore to do with the process of absorption into the cell. The lecithins can be recognised chemically by their decomposition 318 PRACTICAL PHYSIOLOGY products. For example, when they are saponified, as above described, they yield fatty acid, glycerinphosphoric acid and cholin. G-lycerine- phosphoric acid is readily formed by bringing together glycerine and phosphoric acid. CHOLESTEROL. Although soluble in the same solvents as fats and the lecithins, cholesterol is not a fat, but belongs to an entirely different FIG. 22(5.— Crystals of cholesterol magnified 300 diameters. chemical group, namely, that of the terpenes. The terpenes are common in plants, examples of them being camphor and turpentine. By its reactions, cholesterol can be shown to contain a double linking and an alcohol-hydroxyl group. The following formula has been sug- for it : — (CH3)2 = CH - CH2 - CH2 - C17H26 - CH = CH2 CH2 CH2 CH(OH). PHYSIOLOGICAL CHEMISTKY 319 Like the lecithins it is very widely distributed in the animal body. In the free state, it is present in the envelope and stroma of the red blood corpuscles ; as an ester-, it is present in the blood serum. It is also present in bile, and it may separate out from this to form calculi, following a catarrhal state of the mucosa of the bile ducts. A variety of cholesterol, called isocholesterol, is found in lanolin (purified wool fat). Although we do not know much about its functions in the animal body, yet there are indications that these must be important. Thus, if added along with lecithin to a suspension of red blood cor- puscles, it prevents haemolysis. It also antidotes the haemolytic action of saponin. (See p. 462.) ADVANCED EXPERIMENT. Preparation of Cholesterol from Gall-Stones.— The gall-stones are finely ground and boiled with 95 per cent, alcohol. The alcoholic extract is filtered hot and allowed to cool, when crystals of cholesterol separate out and can be filtered off, preferably with suction, using a perforated porcelain plate fitted in a glass funnel and covered with a disc of filter paper. The crystals are washed with a little cold alcohol, and may be re-crystallised from hot alcohol. ADVANCED EXPERIMENT. Preparation of Cholesterol from Tissues, e.g. Brain. — The tissue is minced and then ground in a mortar with sand and about three times its weight of plaster of Paris. After standing for some hours the mass, which has now set hard, is ground in a mortar and cold acetone gradually added. This extracts the cholesterol alone. The acetone is filtered and the extraction repeated three times. On evaporation of the extract almost pure choles- terol is obtained. This may be re-crystallised from hot alcohol. (Rosenheim.) Cholesterol is recognised by a number of colour reactions, of which the most important are described in the following experiments : — EXPERIMENT IX. Place some cholesterol crystals on a microscopic slide and distribute them with a glass rod, and examine under the microscope ; or better, dissolve some in absolute alcohol, place a drop of the solution, on a slide, and allow it to evaporate. The crystals are colourless, glancing rhombic plates having usually a square piece removed from one corner. (Fig. 226.) The crystals give distinctive colour reactions. Place some cholesterol crystals under a cover slip on a microscopic slide, and allow a drop or so of a mixture of 5 parts sulphuric acid (cone.) and 1 part water to run under the cover slip. Note that the edges of the crystals become red. Now run in a drop of iodine solution, when it will be noted that a play of colours results (brown, violet, blue, etc.). Other colour reactions can be obtained with solutions of cholesterol. EXPERIMENT X. Dissolve some cholesterol crystals in a few c.c. of chloroform, and add an equal volume of sulphuric acid (cone.). 320 PRACTICAL PHYSIOLOGY Shake gently. On settling, it will be seen that the chloroformic solution becomes coloured blood red and afterwards purple, and the sulphuric acid shows a green fluorescence. If the chloroformic solution be moistened with water, as by pouring it into a moistened test tube the colour disappears. (Salkowski's reaction.) EXPERIMENT XI. Dissolve some cholesterol in acetic anhydride, and, after cooling, add some sulphuric acid (cone.). A play of colours results. (Liebermann's reaction.) PROTAGON. This name is given to a crystalline substance containing phos- phorous and nitrogen. It can be prepared from brain tissue by various methods, but perhaps most simply by extraction, by means of hot acetone, of a mixture of gypsum and brain tissue (see p. 319), from which the cholesterol has been previously removed by treatment with cold acetone. The hot extract, after filtration, deposits crystals of so-called protagon on cooling. By fractional precipitation, or by treatment with different solvents, protagon can be shown to be a mixture of different lipoids, some of which contain large amounts of phosphorus (sphingomyelin), whilst others are phosphorus free (phreno- sin). Hydrolysis of protagon yields galactose, choline, and other Imses. (See table, p. 321.) FAT VALUES. For many reasons it is important that the physiologist and hygienist should be acquainted with the chemical methods used for distinguishing the various fats. It is by an application of such methods that the physiologist has been able to show, among other things, that forced feeding with a fat- rich diet (after previous starvation) leads to the deposition in the tissues of fats very similar to those contained in the food. By such methods, also, the hygienist is able to tell when butter, for example, is of proper composition, for it is easy for the merchant to substitute other fats (oleomargarine) for it. The following are the most important of these methods : — I. Melting Point. — The method for estimating this has been given on p. 277. The absorbability of a fat from the intestine varies inversely with its melting point, e.g. mutton fat with a melting point of 44°-51°C. is absorbed much more slowly than is pig fat with a melting point of 36°-46°C. II. Specific Gravity. EXPERIMENT. Melt pieces of butter and of oleomargarine in two small evapora- ting dishes, and drop the melted fats into alcohol at room temperature (15° C.). The butter will sink, hut the oleomargarine will float, since it is composed of fats of lower specific gravity than those of butter. III. Acid Value. — This refers to the amount of free fatty acid which the specimen of fat contains. When fats become rancid, the acid value rises con- siderably. PHYSIOLOGICAL CHEMISTRY 321 1 1 P Hi o TS w b-^ * | A 1 *-^l '— • 2 t2 03 ^* O BJ °s le 3 g 0 IS 'So o ECITHIK 1 1 II O >_ W (H 1 »I| |:p| Hi 0) <§ So . "^ "o H o 8 ^0 «8 j§ d B -tJ -^ «i •1 fl 1 **> fc>^ Ji *c3 '^ — H r-H ^4 H g s W g 2 • 1^ i^ fl 'o • 3 i. ^ c O O • H O -tn "P« 31 •d o p^'C -5 -d g ll $ >> 5 o .5 .§3 1 W AJ '^'S GD *>S Q'd 5§ 5^ £ 5 00 P |l 5 s •T* - o5 a S ^ a? §3 II 1 ^ 3 'S 1 o o 1"! & S^ o a 1 Sg 08 6 d OT ^ OJ D „ . a5 oJ <» d CQ 11 _> s "5 1 ^ ^g 1 1 1 ! O •^ "o-H-o tf iiijl 1 1 1 a §•§ a p,^ ^s s 1 i • tf^- fe •5 ^ 9 lid d 3 d 1 4 § ^ d US | 58- d 1 I-H 3 -1 O2 S3 1 HH 1 h- 1 o S a P 'o .2 • ^H a i O "5 !H~ ° 1 1 ll| J2 ^ EH -scg 1 3 S^ 1 2 -d i 1 1 _g 1 3 +3 •^ 1 r J u 322 PRACTICAL PHYSIOLOGY EXPERIMENT. Dissolve 1 grm. of fat (butter) in as little alcohol as possible (with the addition of ether, if necessary), and, after adding a few drops of phenolphthalein titrate with ^ KOH. The result is expressed as the number of milligrams of KOH required to neutralise the fatty acid of 1 grm. of fat. In the subjoined table the result is calculated as oleic acid on the basis that 1 c.c. y^ KOH equals 0'0282 grm. oleic acid. IV. The Saponification Value. — This is a measure of the total amount of fatty acid (both free and combined) contained in the fat. The fat is saponified with a known amount of caustic potash which is in excess of that required to produce complete saponification, and the caustic polish which is not neutralised in the process is ascertained by titration against standard acid. EXPERIMENT. Weigh a dry, clean, wide-mouthed Erlenmeyer flask, and add to it 2 grm. of melted and filtered fat. By means of a pipette add exactly 25 c.c. alcoholic potash, a sample of which has just previously been titrated against - HC1, using phenolphthalein as an indicator. Close the flask with a cork having a wide glass tube passing through it. This serves as a reflux condenser. Place the flask on a boiling water bath for half an hour, and shake frequently. Then remove the flask, add 1 c.c. phenolphthalein solution and titrate against ^ HCL The difference between the amount of acid now required and the amount of acid corresponding to 25 c.c. of the alcoholic potash, as determined by the previous titration, corresponds to the amount of fatty acids. The result is usually calculated in terms of the number of milligrams of KOH required to saponify 1 grm. fat. 1 c.c. | KOH contains 0'028 gr. KOH. V. The Ester (ether) value represents the amount of fatty acid which is combined with glycerine. It is obtained by deducting the acid value (III. ) from the saponification value (IV.). VI. The Iodine value is the percentage amount of iodine which a weighed quantity of fat can absorb. This is proportional to the amount of unsaturated fatty acid (oleic, etc.) in the fat (see p. 314). The iodine value is of great im- portance in physiological investigations, since by it we can form an estimate of the relative amount of unsaturated fatty acids in fats. Its determination involves the use of carefully standardised solutions, and is too complicated for description here. VII. The Reichert-Meissl value indicates the amount of volatile soluble fatty acids present. It is of great value in testing the purity of butter, because this contains a considerable proportion of such acids, whereas the cheaper fats, which are sometimes used as substitutes for butter, do not contain much of them. EXPERIMENT. 5 grm. melted fat is saponified with alcoholic potash, the alcohol evaporated, and the resulting soap dissolved in water acidified with sulphuric acid, and distilled. The distillate, which contains the volatile acids, is collected in a flask and titrated with y^- NaOH, the result being expressed as the number of c.c. of decinormal acid contained in the distillate from five grammes of fatty substance. PHYSIOLOGICAL CHEMISTEY 323 jf 'o '53 I c -i I i o 00 55 ^ H -8 i S 6 'T5 p oo CO g « •^ OS os t^, os OS o s g-g i 1 s 2 i oo i i 1 111! TjH CO C* OS •* (N 00 1^ o S 9 7 2 ti. t- i— I OO ! p I 5 ^ A'Q 0 0 0 ^H 0 0 0 gq ® g 1 CO <-* O O (N 05 to oo ie CO 0 222 s "f CO 3 *« ^5 | — . O^ . 1 1 1 1 65, *? 'T' cq w *? o o 3 "Sis OS § 6 6 as S 9 co £ 0 0 o o O o £ • • , . , . - o 1 *"i M % 1 S I s do s i 324 PRACTICAL PHYSIOLOGY CHAPTER VII. MILK. MILK contains proteins, fats, carbohydrate, salts and water. The fat is suspended in the form of a fine emulsion. The proportion of these bodies varies in the milks of different animals. Naturally that pro- vided by the animal is the best for its own species. The more quickly a young animal grows the greater is the percentage of protein and salt, in the mother's milk. Thus, a puppy doubles its weight in eight days; its mother's milk contains 7'1 per cent, of protein and 1-3 per cent, of ash. On the other hand, a child takes half a year to double its weight; human milk contains only 1-5 per cent, protein and 0'2 per cent. ash. The mother's milk forms a perfect food for the young growing animal — but it is deficient in iron. It has been shown that the young animal has sufficient iron stored within itself until it can begin to get its own further supply of iron. Thus the young rabbit contains within itself a sufficient supply until it is able to eat green food. This is important from the medical point of view, and shows the necessity of weaning a child at the proper time in order that a proper supply of iron may be obtained in the food. In everyday life the two kinds of milk of the greatest importance are cow's milk and human milk. As hinted above, the two milks vary in composition. Water. Protein. Fat. Carbohydrate. Salts. Cow's milk, - 87'4 3'4 37 4-8 '7. Human milk, - 90*2 1-5 3'1 5'0 '2 Other milks used for human consumption are : — Water. Protein. Fat. Sugar. Salts. Goat's, - - 87-3 3 "5 3 '9 4-4 -8 Ass's, - - 92-5 1-7 '4 5-0 '4 It will be seen that, as regards cow's and human milk, the amount of fat and of carbohydrate is nearly the same in both, the amount of protein and salts is more in cow's than in human milk. To make cow's milk, therefore, of approximately the same percentage composition as human milk, it is usual to dilute it with an equal bulk of water and to add fat and carbohydrate in proportion. This fat is usually in the form of cream, but, in the case of the very poor, cotton-seed oil will serve a similar purpose. Similarly with the carbohydrate — that of milk is milk-sugar or lactose and is expensive — but the addition of cane sugar serves well and if anything renders the milk more palatable. In passing, it may also be noted that a good non-skimmed PHYSIOLOGICAL CHEMISTRY 325 condensed milk diluted in suitable proportions, such a 1 in 12 to begin with, forms a good inexpensive substitute for human milk. But when these alterations have been made in cow's milk it has only the same percentage composition — and certain well-marked differences must be pointed out. Such differences are : — (1) The proportion between the amount of the different kinds of protein in cow's and human milk. Cow's milk, 3*02 per cent, caseinogen, '53 per cent, lactalbumin. Human „ 1-00 „ „ '50 „ It will be seen that human milk contains relatively far more lact- albumin than does cow's milk, so that even when cow's milk is diluted there is the discrepancy between the relative amount of the proteins to be taken into account. It is stated that if lactalbumin be added to diluted cow's milk it causes it to yield the same light form of clot as does human milk. It is also interesting to note that the first secreted milk, colostrum, is very rich in lactalbumin. (2) The caseinogens of the two milks are not of the same composition either in percentage or actual composition. It has recently been shown that a carbohydrate group is attached to the caseinogen of human milk, such a group is absent from that of cow's milk. (3) The percentage of the salts present differs in the two milks — cow's milk contains more calcium, iron and phosphoric acid; human milk more potassium and chlorine. (4) There is a provision in the mother's milk of protective bodies for her offspring. Perhaps the strongest argument for the human mother to nurse her child wherever it is possible, is shown by the "changeling experiments " of Ehrlich. This eminent researcher performed the fol- lowing remarkable experiments. He immunised a male and a female mouse to the poison abrin before they were allowed to reproduce. He then crossed the immunised male with a non-immunised female, and the immunised female with a non-immunised male. Upon the birth of the young, it was found that those of the immunised female possessed an immunity to the poison, which increased after birth, and was therefore not derived from the placenta alone ; whereas the young of the non-immunised female — that is, of the immunised male — possessed no immunity towards the poison. The families were now changed over, the immunised mother suckling the non-immune young, and vice versa. It was then found that the once non-immune young acquired an immunity, whereas the immune-born young quickly lost theirs. These results are all the more remarkable, because a similar immunity cannot be produced in the adult animal by feeding it upon 326 PRACTICAL PHYSIOLOGY an immunised cadaver, yet the young mouse can be immunised through its food. This points either to a special condition of the antitoxins in the milk of the mother or to a special condition of digestion in the young. In order to study the chemistry of milk, we usually employ cow's milk, because it is easily obtainable. Cow's Milk. — This is an opalescent solution, possessing a character- istic taste, and of amphoteric reaction. EXPERIMENT I. Place a drop of fresh milk on a piece of red litmus paper, and wash it off with distilled water ; a blue stain is left : if the drop be placed on blue litmus, a red stain is left. This peculiar reaction is due to the fact that milk contains a mixture of acid and alkaline salts. By ascertaining how much decinormal acid or alkali are required to produce neutralisation with the aid of different indicators the amount of each of these kinds of salt can be determined. (See Titration Methods.) The specific gravity of fresh milk varies between 1*028 and 1*0345. The more fat (i.e. cream) the milk contains the lower is the specific gravity. EXPERIMENT II. Estimate by a hydrometer (p. 409) the specific gravity (a) in skimmed milk and (b) in fresh milk. In the former it is about T0345, in the latter T028. By adding water to (a) the specific gravity obviously falls, and by removing the cream from (b) it rises. In dairy hygiene, a rough estimate of the richness of milk in cream is obtained by ascertaining its specific gravity, but it is clear from the above experiment that some of the cream can be removed and the consequent rise in specific gravity masked by the addition of water. This fraudulent trick of some dairymen must, therefore, be borne in mind before giving an opinion of the quality of the milk. Fresh milk does not coagulate on boiling, but a skin forms on its surface. A similar skin is produced when any emulsion containing protein is boiled, and in the case of milk it is composed chiefly of caseinogen entangling some fat globules.1 Its formation is due to drying of the protein at the surface of the milk. THE CHEMICAL CONSTITUENTS OF MILK. I. Proteins. — The chief protein of milk is a phospho-protein called Caseinogen. This can be precipitated by adding to the diluted milk a 1 An emulsion of cod-liver oil in diluted blood-serum is given round ; warm it to about 50° C., and a skin will form on the surface. Be careful not to heat above 50° C. , as then coagulation of the proteins will be produced. PHYSIOLOGICAL CHEMISTRY 327 weak acid, or by saturating it with a neutral salt. (See Phospho- proteins, p. 306.) EXPERIMENT III. Place about 5 c.c. of milk in a test tube, and dilute with an equal bulk of water. To this diluted milk add, drop by drop, a weak solution of acetic acid ; a precipitate of caseinogen, entangling fat, falls down. Filter off this precipitate and wash it with water. Now add to it a weak solution of Na2CO3; the precipitate dissolves, and an opalescent solution of caseinogen, still, however, containing some fat, passes through the filter. By repeated reprecipi- tation and filtration comparatively pure caseinogen can be obtained, from which the last traces of fat can be removed by treating with ether. The chief property of caseinogen is its power to clot when treated with rennin (a ferment contained in gastric juice) in the presence of soluble calcium salts. EXPERIMENT IV. Take a pure solution of caseinogen. Divide it into two portions, a and b. To both add about ten drops of rennin ferment. To b add also a few drops of a 5 % solution of calcium chloride. Place both in the water bath at 40° C. ; after about five minutes examine to see if clotting has occurred. It will be found that clotting has occurred in b where both rennin and soluble Ca salts were present. EXPERIMENT V. Make similar experiments with milk, taking five tubes, a, b, c, d, e. a = milk + rennin only. b = milk + rennin + CaCl2. c = milk + rennin (heated). d = milk + rennin + potassium oxalate solution. e = milk + rennin + potassium oxalate solution (heated after 10 minutes). It will be found that a clots, but not so quickly as b ; c does not clot, because the enzyme has been destroyed ; d clots only after the addition of CaCl2 ; e clots also on the addition of CaCl2 any time even after the rennin has been destroyed. From these experiments we learn that the clotting of milk takes place in two stages. (1) The rearrangement of the soluble caseinogen into the form of soluble casein by the enzyme rennin. (2) The combination of the soluble casein thus formed with calcium salts to form insoluble casein or clot. Note that the enzyme rennin does not clot the protein; it merely rearranges it in such a form that it can be clotted. 328 PEACTICAL PHYSIOLOGY The rennin comes from a precursor pro-rennin in the gastric mucous membrane. The stages can therefore be tabulated as follows : Pro-rennin + HC1 Rennin — Caseinogen (soluble) Sol. Casein + Ca Insol. Casein or Clot. (Cf. Blood.) In order to study the conditions necessary for the clotting of milk a solution of caseinogen may be prepared by the following method (Ringer's) : 300 c.c. of milk are mixed with an equal bulk of water, and 10% acetic acid is added till all the caseinogen has been precipitated. The precipitate is filtered off and thoroughly washed with distilled water until the washings are no longer acid in reaction. It is then removed from the filter paper, and ground up in a mortar with solid calcium carbonate. The resulting paste is thrown into 500 c.c. of water placed in a tall vessel, and the solution is allowed to stand for several hours. The fat, which was contained in the precipitate, rises to the surface, the calcium carbonate sinks to the bottom, and the intervening fluid contains the caseinogen in combination with calcium as calcium caseinogenate, which is soluble in water (Osborne). Three samples of the opalescent solution are removed by means of a pipette, and placed in three test tubes labelled A, B, and 2S04 expressed in percentage saturations. Solubility in alcohol. Salicyl sulphonic acid and HNO3 tests. Biuret test. Millon's reaction. Native protein. Globulins (isat.). Albumins (sat.). Relatively in- soluble. Ppte., becom- ing coagulum on boiling. Violet. Positive. Acid meta protein. — — Do. Do. Do. Primary proteose. 24 -42 per cent. (I sat.). Hetero - insol- uble in 32 per cent. ; proto- soluble in 80 per cent. Ppte. , clearing up on boiling and returning on cooling. Rose pink. Feeble with hetero- ; strong with proto- proteose. Secondary proteose CH (a. amino-propionic acid) NH (Indol group.) Tryptophane is the mother substance of indol, which, along with its methyl derivative skatol, is largely responsible for the faecal colour. These bodies are produced from tryptophane by bacterial growth (see p. 405). Preparations of trypsin have a rennin-like action on milk if sufficient calcium be added (see p. 327). II. Amylopsin, Pancreatic Amylase. — This ferment acts on starch in exactly the same way as ptyalin does — i.e. it converts it into maltose and achroodextrin. Unlike ptyalin, it is capable of acting on unboiled starch. EXPERIMENT VII. Add some glycerine extract of pancreas to some powdered starch. Shake, and place in the water-bath at 37°. Eemove drops every half minute, and mix on a slab with a drop of iodine solution. Note the appearance of the dextrine reaction. When this disappears, apply Trommer's test, or one of its modifications, to a, sample of the digest; note the reduction due to maltose. III. Steapsin or Lipase. — This decomposes neutral fat into fat acid and glycerine (see Fats, p. 314). EXPERIMENT VIII. Some minced pancreas is shaken with water1 strained through muslin, and the resultant extract divided into two parts. One of these is boiled to destroy the ferment, and is then cooled. To both portions (about 10 c.c. each) are added five drops of melted and filtered butter fat, a few drops of an alcoholic solution of phenolphthalein, and then N/10 caustic soda, until a deep red colour is obtained. After vigorous shaking, so as to obtain a partial emulsion, the test tubes are placed in the incubator, and examined after about half an hour. The lipase-containing fluid will be decolourised (the fatty acid having bleached the phenolphthalein), and, to regain the original red colour, a certain number of c.c. N/10 caustic soda must be added to it. In this way, an approximate estimate can be obtained of 1 Glycerin does not dissolve steapsin, so that a glycerine extract of pancreas ia not suitable for this experiment. PHYSIOLOGICAL CHEMISTRY 397 the fat-splitting power of the extract. Pancreatic lipase is very readily destroyed in acid reaction, thus making it necessary to employ an absolutely fresh gland from which to prepare the extract. The liberated fatty acid is held in solution by the bile, and so is absorbed into the epithelial cells of the villi, wherein it recombines with glycerine to form neutral fat. CHAPTER XV. THE BILE. BACTERIAL DIGESTION. THIS is perhaps the most puzzling secretion in the whole of the physiological chemistry. Its digestive action is very slight, so that it would almost appear, at first sight, to be an excretion of effete products rather than a useful secretion. Against such an idea, however, stands the fact that it is poured into the beginning of the intestinal tract, and not into the end of it, as we should expect were it an excre- tion. Further, some of its constituents are reabsorbed into the portal blood and carried back to the liver, to be re-excreted in the bile. In other words, there exists a circulation of certain biliary constituents, from liver to intestine by the bile, and from intestine back to liver by the portal blood. The absence of this reabsorption of bile constituents into the blood when there is a biliary fistula (produced by attaching the central end of the bile duct to a wound in the abdominal wall) accounts for the bile in such cases containing less solids than the bile obtained from the gall bladder after death. Another reason for this difference in composition is that reabsorption of water occurs in the gall bladder, and that mucin or nucleo-protein are secreted by its mucosa. In the case of a fistula of the bile duct the bile does not collect in the gall bladder. Composition of Human Bile. — In I. the bile was obtained from the gall bladder of persons who had been accidentally killed, while in good health : in II. the bile was obtained from a fistula during life. i. ii. 100 parts contain— Water, 86 97 Solids, 14 3 Viz. organic salts, - 9 G'9-1'8 Mucin and bile pigment, ... - 3 0'5 Cholesterol, 0'2 0'06-0-16 Lecithin and fat, O'5-l -0 0'02-0'09 Inorganic salts, 0'8 07-0-8 398 PEACTICAL PHYSIOLOGY Besides these, bile also contains traces of soaps, fats and urea. Compounds of glycuronie acid have also been found in bile. The daily secretion amounts to about 750 c.c. To study the chemistry of bile we employ that of the ox, since this is easily procurable. EXPERIMENT I. Examine some ox bile. Note that it has a greenish colour, a peculiar musk-like odour, a bitter-sweet taste, a faint alkaline reaction to litmus paper, and that it is of a slimy consistency. EXPERIMENT II. If a few drops of weak acetic acid be added to a few cubic centimetres of bile, a stringy precipitate is produced. This consists, in certain animals (ox) of nucleo-protein, in others (man) of mucin. Filter off this precipitate, and note that the filtrate has lost its slimy character. Boil the filtrate ; no coagulum is produced, there- fore bile contains no native protein. The above proteins are not produced by the hepatic cells, but are added to the bile in its passage along the bile ducts, being secreted by the mucous lining of these. So far as can at present be ascertained, the amounts of pigment and of bile salts do not bear a quantitative relationship to one another, so that it is improbable that they are both derived from the same source. Quantitative estimations of these two bodies in bile, obtained from a biliary fistula, are, however, far from numerous, partly on account of the rarity of suitable cases, and partly because there is no accurate method for quantitatively determining the pigment. EXPERIMENT III. Test another portion of the bile for bile salts by PettenJcofer's reaction. To do this place a drop of bile in a small evaporating dish, and move this about so that a thin film of the bile is produced. Now add to the film a very small drop of a concentrated watery solution of cane sugar, and then a few drops of concentrated sulphuric acid. A purple colour is produced, which can be intensified by warming. This pigment shows absorption bands in the spectrum. The chemistry of this reaction is that the sulphuric acid acts on the cane sugar to produce a body called furfuraldehyde, which then reacts with the cholalic acid of the bile salts to produce the pigment. Where only traces of bile salts are present, the test may be made more delicate by using a solution of furfuraldehyde (1 in 1000) instead of cane sugar. EXPERIMENT IV. Matthew Hay's Sulphur Test. — If a small pinch of powdered sulphur be sprinkled on the surface of bile, or of a solution containing bile salts, it will sink to the bottom of the vessel ; whereas with most other fluids it remains floating on the surface. This reaction depends on the fact that bile salts lower the surface tension of fluids in which they are dissolved. For comparison repeat this test with water. PHYSIOLOGICAL CHEMISTRY 399 The bile salts are two in number, glycocholate and taurocholate of sodium. The two acids (glycocholic C26H43N06 and taurocholic C26H45NS07) are very closely related to one another, for they both yield, on boiling with stronger acids, a common non-nitrogenous body called cholalic acid, and a nitrogenous body of the nature of an amino acid. The amino acid, which is obtained from glycocholic acid, is glycin. The amino acid in taurocholic acid is taurin, which is peculiar in that it contains sulphur which can be demonstrated by fusing some taurin (prepared as described below) on a piece of platinum foil with sodium carbonate, dissolving in water, acidifying and heating the resultant solution : sulphuretted hydrogen is given off, which can be detected by holding a piece of filter paper soaked in lead acetate solu- tion over the mouth of the test tube. Taurin is undoubtedly derived from cystin (see p. 393), which when oxidised yields cysteinic acid — COOK". CHNH2 . CH2 . S03H. By the loss of a molecule of CO^ this becomes taurin : NH2 . CH2 . CH2 . S03H. We see, therefore, that both glycin and taurin are derived from protein disintegration, the latter body being one of the forms in which the sulphur of protein is excreted. Cholalic acid is believed to be related to cholesterol. It has recently been shown to contain two primary alcoholic and one secondary alcoholic groups. There are probably several varieties of it. The relative amount of these two acids in the bile varies in different animals. In the bile of the herbivora, glycocholic acid is much in excess, whereas in that of many carnivora the only acid is taurocholic. In omnivora (e.g. man, etc.) a variable mixture of the two is present. The bile salts are decomposed into their constituents by the action of the bacteria in the intestine. If we examine the faeces, however, no glycin and only a trace of cholalic acid can be detected. The fate of taurin has not been accurately determined. ADVANCED EXPERIMENT. Separation of Bile Salts. — To Separate the Bile Salts as a Whole. Thoroughly mix 50 grm. pure animal charcoal with 200 c.c. of ox-bile in an evaporating dish, and evaporate the mixture to dryness on a water bath. During the drying the mixture should be frequently stirred. The black powder thus obtained can be kept a considerable time. To extract the bile salts from it, mix it with absolute alcohol in a flask and place the flask on the boiling water bath for about a quarter of an hour, cool, filter into a dry beaker, and add ether to the filtrate till a permanent haze is produced. Now cover the beaker with a ground glass plate, and allow it to stand in a cool place till next day, when it will be found that a crystalline mass of bile salts has separated out (Plattner's Crystalline Bile). The crystals can now be collected on a filter paper and allowed to dry in the air. A 1 % solution of the crystals should now be made, and Pettenkofer's reaction (see p. 398) applied to it by the following method : Dissolve a few grains of cane sugar in the solution, and run concentrated 400 PRACTICAL PHYSIOLOGY sulphuric acid down the side of the tube so as to form a layer underneath the watery solution. A violet ring is formed where the two fluids meet. Now place the test tube in a beaker of cold water, and shake gently so as to mix the two fluids. A violet solution is thus obtained. (By cooling the test tube in water too great a rise of temperature is avoided). Divide the violet solution into two parts, A and B. Add A to some ether and examine by means of the spectroscope — a distinct band is seen in the green. Add B to some absolute alcohol and note that, although the spectrum is at first the same as in A, a band gradually develops in the blue, and that, along with the development of this, the tint of the solution changes from violet to brown. ADVANCED EXPERIMENT. To Prepare Pure Glycocholic Acid.— In certain districts of Germany and America it has been observed that the glycocholic acid can be separated from the bile by a very simple process, and, so far as it has as yet been tried, the bile obtained from oxen reared in this country appears to be suitable for the process. The method is as follows : Some ox bile is placed in a stoppered cylindrical vessel, and mixed with ether and hydrochloric acid in the proportion of ten parts of the former and four parts of the latter, for every hundred parts of bile. A few crystals of glycocholic acid are added to the mixture so as to start the crystallisation, the vessel is stoppered, vigorously shaken, and then allowed to stand in a cool place. After some time the mass will be found to be " solid " with crystals. These are collected in a filter paper, and washed with cold distilled water till no more pigment can be removed. They are then removed to a flask and dissolved in boiling water ; the solution is filtered hot, and the filtrate, on cooling, deposits numerous acicular crystals of the acid. These may now be collected, washed with distilled water, and dried (for Chemistry and Reactions, see p. 398). Preparation Of Taurin.— Bile from carnivorous animals— cat or dog— is heated on a sand-bath with one-third its bulk of concentrated hydrochloric acid until a resinous-like mass of the anhydride of cholalic acid (called Dyslysin) has formed. This can be drawn out into brittle threads by means of a glass rod. The dyslysin is filtered off, and the filtrate is evaporated to a small bulk, the sodium chloride, which crystallises out during the evaporation, being removed by filtration. The thin syrup is then poured into fifteen times its bulk of alcohol, and left standing twenty-four hours, when the taurin will have crystallised out. It can be purified by collecting the crystals on a filter paper, and washing with cold water. The Bile Pigments. — These are bilirubin and biliverdin. The former occurs most plentifully in the bile of carnivorous, the latter in that of herbivorous animals. Their presence can be detected by oxidising a mixture containing them with nitrous acid, when a play of colours — green, blue, purple, and then yellow — is produced. This is called Gmelin's test.1 EXPERIMENT V. Dilute some ox bile with an equal amount of aThis test depends on the various colours of the oxidation products of bili- rubin. The first oxidation product is bili-verdin, which is green ; the next is bili-cyanin, which is blue ; the next is bili-purpurin, which is purple ; and the last is choletelin, which is yellow. PHYSIOLOGICAL CHEMISTRY 401 water. Hold the test tube as nearly horizontal as possible, and allow some fuming nitric acid to run down it, so that this forms a layer under the bile. Where the two fluids are in contact, a play of colours is produced. This test can be rendered still more delicate by filtering a little diluted bile through white filter paper, then removing and opening out the filter paper and placing a drop of fuming nitric acid on it. Bilirubin is the least oxidised bile-pigment, and its empirical formula is C32H3GN4O6. If we compare this with the formula of haematin — C32H32N404Fe — we see that it must be from this body that it is derived, the change being the abstraction of iron and the addition of two molecules of water. This is also the formula of iron-free haematin or haematoporphyrin, and of haematoidin, a pigment which crystallises out in old blood clots in the tissues. Although the same empirically, these bodies vary somewhat in their physical behaviour, and neither of them gives Gmelin's test, so that we may assume that they have different constitutional formulae. When it reaches the intestine, the bile pigment is converted by bacteria to another pigment called stercobilin. Some of this pigment is absorbed into the portal blood along with the bile salts. This ^reabsorbed stercobilin is mainly re-excreted in the bile, but a small quantity is excreted in the urine, where it goes by the name of urobilin (see Urine). Stercobilin forms the principal colouring matter of the faeces. EXPERIMENT VI. Bilirubin can be extracted from pigmented gall- stones. The gall-stones are ground to a rough powder and extracted by heating with 95 % alcohol, to which a few drops of strong hydro- chloric acid have been added. (The acid is necessary to decompose the compound of bile pigment with calcium present in the stones.) The coloured extract is then cooled. The crystals of cholesterol, which separate, are filtered off, washed with alcohol and examined. (See p. 318.) The filtered extract is placed in a dish, and pure nitric acid run in, drop by drop, when a brilliant Gmelin's test is obtained. Lecithin (C44H90NP09) and Cholesterol (C27H45OH) (see Chapter VI.). — These two bodies are kept in solution in the bile by means of the bile salts. For their separation, see p. 316. EXPERIMENT VII. Place some bile in a test tube, and add one or two crystals of cholesterol to it and gently warm. The cholesterol dissolves. Before doing this show that the crystals will not dissolve in water. Both lecithin and cholesterol are excretory products. The tissues which contain the highest percentage of them are the nervous, so that 2c 402 PRACTICAL PHYSIOLOGY the bile functionates as the channel by which the products of nervous metabolism are removed. Inorganic Salts. — These are chiefly sodium carbonate and disodium hydrogen phosphate. The Uses of the Bile in Intestinal Digestion. — (1) It is an alkaline fluid, containing a viscid substance (mucin, etc.); consequently, it assists in the emulsification of fats. EXPERIMENT VIII. Shake up some rancid oil with bile in a test tube. Notice that a very stable emulsion is formed. (See Fats, p. 316.) (2) It causes a precipitate when added to an artificial peptic digest. EXPERIMENT IX. Add some bile to a sample of a 24 hours' peptic digestion of egg-white. A precipitate of proteins is produced. It is claimed that by this precipitation the fluid chyme becomes much thicker, and its condition, therefore, rendered more favourable for being further digested in the intestine, since it will adhere to the intestinal wall. (3) It dissolves the free fatty acid produced in the intestine. On account of this latter action, and, to a certain extent, on account of its emulsifying powers, bile assists materially in the absorption of fat. Where bile is not excreted into the intestine (as in Jaundice), the faeces become rich in fatty acid, in consequence of which they appear greasy and pale in colour. The presence of excess of fatty material in the intestinal contents also hinders protein digestion to a certain extent, by coating the particles of food and preventing the juices getting at them. In consequence of this, bacterial growth becomes excessive when there is suppression in the secretion of bile. It is by this means that bile diminishes putrefaction in the intestine, and not on account of any antiseptic properties it possesses, for bile itself quickly becomes putrid on standing. Many other properties have been ascribed to bile, e.g. that it assists the absorption of oil globules and that it acts as a laxative, but these are not of much importance. It may be men- tioned that in some animals bile contains a diastatic ferment. The secretion of bile by the liver is stimulated by the intravenous injection of secretin. It also activates, to a certain extent at least, the lipolytic ferment of pancreatic juice. To sum up, we may state that, although bile contains no ferment by which a chemical change can be produced on any of the food-stufls, it is nevertheless of great value as a digestive fluid, in that it assists the pancreatic juice: (1) by neutralising the chyme; (2) by activating pancreatic lipase ; (3) by dissolving the fatty acid produced by the action of lipase ; (4) by assisting in the emulsification of neutral fat ; (5) by assisting the absorption of fat; and consequently (6) of allowing PHYSIOLOGICAL CHEMISTRY 403 protein to be attacked by trypsin, thereby diminishing bacterial growth and consequent putrefaction ; (7), and lastly, by precipitating the half-digested products of chyme, so that the trypsin may the better act on them. Intestinal Juice. Succus Entericus. — This is secreted by Lieberkuhn's follicles. It may be obtained pure by isolating a piece of intestine and collecting the juice secreted by it. This may be accomplished by cutting out a piece of intestine and stitching both ends to abdominal fistulae (Vella's method), the severed ends of the intestine being sutured together. Or one end of the isolated piece may be sutured, the other being attached to a fistula (Thiry's method). In both these cases the mesentery of the isolated portion is left intact, and the juice can be removed from the loop and its action studied in vitro, or food may be placed in the loop, and afterwards removed and examined. Extracts of the mucous membrane of the intestine, prepared by scraping this off and grinding it with sand and water and then filtering through muslin, usually contain large amounts of ferments. This extract will contain both exoenzymes and endoenzymes. Succus entericus seems to contain three ferments or ferment-like bodies. One of these has been known for long, and is called inverting ferment, because it "inverts" (see p. 285) disaccharides. There are several varieties of inverting ferment depending on the exact nature of the disaccharide on which they act ; e.g. one acting on maltose (maltase), one on lactose (lactase), and one on cane sugar (invertase). Lactase is present in extracts of the intestinal mucosa only when the food contains lactose. It is therefore absent in the intestine of herbivorous adult animals (guinea pig), but is present for some time after birth, i.e. when the animal is living on milk. By feeding milk to adult animals lactose does not reappear in the intestine. Extracts of intestinal mucosa of omnivorous animals (cat and pig) contain lactase through- out life. It cannot be found in the succus entericus, and is therefore an endoenzyme. Invertase is also stronger in extracts of the intestinal mucosa than in succus entericus. Maltase is not confined to the intestine, being present in large amount in blood serum, and in most of the organs and tissues of the animal body. The other two ferments act on proteins. One of them, erepsin by name, hydrolyses casein, proteoses and peptones into simple nitrogenous crystalline products. It cannot, however, act on all native proteins. It differs from trypsin in the fact that it can hydrolyse certain poly- peptides, such as glycyl-glycin ; d. I. leucylglycin, etc., on which trypsin 404 PRACTICAL PHYSIOLOGY has no action, and that it can carry hydrolysis to a further stage than trypsin. Like trypsin, it acts best in alkaline reaction. It is more plentiful in extracts of intestinal mucous membrane than in succus entericus. It is probably, therefore, an intracellular ferment — endoen- zyme — some of it leaking out of the cells into the succus entericus. Since the proteins (i.e. peptones) have to pass through these cells during absorption, they will come under the influence of erepsin. Erepsin is not confined to the intestine, but is present in large amount in other parts of the animal body. Next to the intestine, the largest amount has been found (by Vernon) in the kidney, then, in order, the spleen, pancreas, liver, cardiac muscle, brain, skeletal muscle, serum. These endo-erepsins of the tissues probably play an important role in the metabolism of proteins. ADVANCED EXPERIMENT. To Demonstrate the Breptic Power of Tissues. — Take 20 grm. minced liver, and 20 grm. mucous membrane of the intestine (scraped off with a scalpel). Grind each in a mortar with fine quartz sand and 20 c.c. of a 0'2 % solution of Na2Co3. Filter the extracts through muslin. Divide each extract into two equal parts, A and B. To A of each extract add 1 c.c. of a 2'5 % solution of Witte's peptone, and to B a similar amount of a 2'5 % solution of egg-white. Remove a few drops of the contents of each of the four test tubes, and apply the Biuret test, noting the results. Place the tubes in the incubator at body temperature, and at the end of an hour again remove a little of the contents of each tube, and apply the Biuret test. It will be found that there is no change in the tube (B) containing egg-white, but that in the tube (A), containing the intestinal extract, the test has become very feeble or disappeared entirely. By longer incubation, the Biuret secretion will also disappear from the tube (A) containing liver. By thus ascertaining the time required to split up a standard solution of peptone, so that the Biuret test is no longer given, a comparative estimate may be made of the ereptic power of different extracts. Another ferment-like body in succus entericus is enterokinase. Alone, it has no action on any food- stuff, but when mixed with trypsinogen it converts it into trypsin. On a flesh-free diet, the pancreatic juice, as secreted from the duct of Wirsung, contains very little trypsin, and digests coagulated egg-white only slightly even after several hours. If to this inactive pancreatic juice a few drops of succus entericus be added, digestion of the egg-white proceeds actively. Trypsinogen, which is the form in which the proteolytic ferment is secreted on a flesh-free diet, remains inactive until it gets to the intestine, where it is converted into trypsin by the entero-kinase. Enterokinase is not secreted unless it is required, i.e. if the intestinal mucosa be mechanically stimulated, a juice will be secreted which contains no entero-kinase. Bacterial Digestion. — As has been explained above, the conditions PHYSIOLOGICAL CHEMISTRY 405 necessary for bacterial growth are very favourable in the intestine. As a result of their growth, bacteria decompose the food-stuffs and lead to the production of products in many cases the same as those of the digestive juices, in other cases of a different nature. In the small intestine the bacteria which are most active are those acting on carbo- hydrates, whereas in the large intestine these are largely replaced by bacteria acting on protein. Their action on proteins leads to the production of proteoses, peptones, and amino acids, etc. So far their action corresponds to that of trypsin, but they digest farther and produce a multitude of simple degradation products, such as ammonia, fat acids, carbonic acid, etc., as well as a group of substances belonging to the aromatic series. The aromatic bodies are arranged in two groups. The one contains PTT phenol C6H5OH and its methyl derivative kresol C6H4 can be formed by allowing dry carbon dioxide gas to act on dry ammonia. This is called ammonium carbamate. If heated to 135° C. it splits up into urea and water. A certain amount of ammonium carbamate is always present in watery solutions of ammonium carbonate. Dry heat splits urea into ammonia gas and a body called Biuret. By further heating, Biuret changes into cyanuric acid (HCN03), which is isomeric with cyanic acid, HCNO. EXPERIMENT V. Heat some urea crystals in a dry test tube. Note that they melt and give off ammonia. Continue heating for a few minutes, then cool the test tube and dissolve the residue in water, and to this solution apply the Biuret test. A rose pink colour results (see Peptone, p. 302). Conversely, we can change cyanic acid into urea by evaporating an aqueous solution of ammonium cyanate (NH4CNO) to dryness. This 2D 418 PKACTICAL PHYSIOLOGY salt has the same empirical formula as urea, but its structural formula is different : (Ammonium cyanate) (Urea) It was by this means that Wohler first showed that organic bodies of animal origin could be formed from inorganic substances. PREPARATION OF UREA. 1. From Urine-— To about 400 c.c. urine add barium mixture (1 vol. saturated barium nitrate solution mixed with 2 vol. baryta water) until there is no further precipitate of sulphates and phosphates. Filter and evaporate the filtrate — at first over a free flame, afterwards on a water-bath — to a thin syrup. Now mix this syrup with about 100 c.c. methylated spirit, and, after allowing the mixture to stand for about half an hour so that the precipitate of inorganic salts may settle, filter the alcoholic extract into an evaporating dish and evaporate it nearly to dryness on a water-bath. Allow the residue to cool, and then add to it about double its volume of concentrated pure nitric acid, meanwhile placing the basin in a dish of cold water, and stirring the contents with a glass rod so as to accelerate the formation of the urea nitrate. After about half an hour the crystals of urea nitrate are filtered off by means of a suction filter, sucked as dry as possible, and then placed between several thicknesses of filter paper, between which they are pressed so as to dry them. In order to convert the nitrate into urea, the crystals are placed in an evaporating dish and dissolved in as little water as possible ; the basin is then placed on a heated water-bath, and powdered barium carbonate added with a penknife in small quantities until the fluid reacts neutral. By this treatment the urea nitrate is decomposed, the nitric acid combining with barium to form barium nitrate, and the urea being thereby liberated. The mixture is now filtered, the filtrate evaporated to dryness and the urea taken up from the residue by extracting with absolute alcohol, which does not dissolve the barium nitrate. The alcoholic solution of urea is now evaporated to dryness, when a mass of urea crystals is obtained. The above process may be considerably curtailed by omitting the preliminary precipitation of phosphates, etc., with barium mixture, the evaporated urine being simply mixed in a test tube with nitric acid, which is kept cool by immersing it in a beaker of water. The crystals of urea nitrate are then filtered off, dried between filter paper and treated with barium carbonate as above described.1 2. Separation of Urea from Blood, Serous Fluids or Watery Extracts of Tissues. — About 100 c.c. of the fluid are mixed with four times its volume of methylated spirit, vigorously shaken and allowed to stand over night. By this treatment the proteins are coagulated, whereas the spirit dissolves the urea. The coagulum is now filtered off, washed with spirit, and the washings are combined with the filtrate, the whole being then evaporated to dryness on a water-bath. The residue is extracted with absolute alcohol, the extract filtered, again evapo- rated to dryness and re-extracted with absolute alcohol, this process being 1 By adding powdered animal charcoal after barium carbonate, and boiling and filtering, the final product will be rendered colourless. PHYSIOLOGICAL CHEMISTRY 419 repeated until the evaporated residue is entirely dissolved in the alcohol. The purified residue is now cooled by placing the dish containing it on ice, and is mixed with one or two drops of pure nitric acid, the mixture being allowed to stand on ice till next day, when it is examined for crystals of urea nitrate. The alcoholic extracts usually contain a considerable amount of fatty acid which may mask the separation of urea nitrate. To remove this, the first alcoholic extract should be mixed with a few drops of a solution of basic lead acetate till no more precipitate is produced, after which a few drops of a solution of ammonium carbonate are added to cause the suspended precipitate of lead soaps to settle down. The solution is then filtered, and the lead removed from the filtrate by passing a stream of H2S gas through it. For quantitatively estimating urea the following methods may be employed : — I. By decomposing urea with sodium hypobromite in the presence of free caustic alkali. The alkali absorbs the liberated carbonic acid and the nitrogen is collected in a graduated tube. From the amount of nitrogen evolved the urea can be calculated by remembering that O'l grm. urea contained in urine yields 37*1 c.c. moist nitrogen at 15° C. and 760 mm. pressure. Ol grm. of pure urea should theoretically liberate 39 -76 c.c. nitrogen under the above conditions, but only about 92 per cent, of the urea nitrogen is liberated by the hypobromite. This deficit is, in urine, however, partly compensated by a certain amount of nitrogen being simultaneously split off from the other nitro- genous bodies present. The method is therefore only approximate. There are various forms of apparatus used for collecting the liberated nitrogen. That of Dupre* (Fig. 242) consists of an inverted burette (a) placed in a cylinder of water, and to the neck of which is connected a T-piece (/). With the side tube of this the generating bottle is connected by india-rubber tubing, and the other tube is closed with a piece of tubing and a clip. To make the estimation* 25 c.c. of the alkaline solution of sodium hypobromite are placed in the generating bottle (o) and 5 c.c. urine in a small tube, which is then carefully placed in the generating bottle without allowing the two fluids to mix. The cork of the generating bottle is then inserted, and the meniscus of the water both inside and outside the burette brought to the same level at the zero mark, the clip on the T-piece being open meanwhile, and water being added to, or removed from, the outer vessel if necessary. The clip is now applied, and the burette raised to ascertain that no leakage exists. The two menisci are then readjusted, and the contents in the generating bottle mixed. The evolved N displaces the water in the burette. After the reaction is complete, the generating flask is immersed in a basin of water, so as to bring the temperature of 420 PEACTICAL PHYSIOLOGY the gas contained in it to the same as that of the gas in the burette. After waiting two minutes the two menisci are again brought to the same level, and the number of c.c. of N read off. Another form of apparatus is that of Gerrard (Fig. 243). Fio. 242. — Dupr^'s urea apparatus. FIG. 243. — Gerrard's urea apparatus. For rapid clinical purposes quite satisfactory results may be obtained by using the Doremus ureometer (Fig. 240), with side tube for the urine. In using this 2 c.c. of urine are placed in the small side tube and the main tube is filled with the hypobromite solution. By turning the stopcock the urine is then allowed to run very slowly into the hypobromite, when the nitrogen rises to and collects at the top of the tube. When all effervescence has ceased the apparatus is allowed to stand until it is cooled to room temperature, when the graduation at which the meniscus stands is noted. This PHYSIOLOGICAL CHEMISTRY 421 FIG. 244. — Ureometer. graduation corresponds to grammes of urea in the quantity of urine used. II. MSmer-Folin Method.— Principle.— By the addition of certain reagents to a measured quantity of urine, the greater proportion of the nitrogenous bodies, except urea and ammonia, are precipitated. The precipitate is removed by filtration, and, after expelling the ammonia by heat, the nitrogen of the filtrate is determined. This, multiplied by 2-143, gives the amount of urea present. Solutions necessary. — 1. Powdered barium hydroxide (baryta). 2. A mixture of 1 vol. ether and 2 vol. absolute alcohol. 3. Apparatus, etc., for Kjeldahl's nitrogen deter- mination. Determination. — 5 c.c. urine are shaken in a small stop- pered flask with 1'5 grm. barium hydroxide until no more will dissolve : 100 c.c. alcohol-ether mixture are then added, whereon a copious precipitate falls down. The flask is corked and left standing over night. The contents are then filtered through a small filter paper (10 cm. in diameter), the filtrate being collected in a Kjeldahl combustion flask of 500 c.c. capacity. When all the solution has passed through, the precipitate is washed at least three times with alcohol-ether mixture, and then the flask is connected with a suction pump and placed on the water-bath heated to 55° C.1 When the liquid has been reduced to a few c.c., 25 c.c. water and a pinch of magnesium oxide are added to the contents and the evaporation continued so as to drive off the last traces of ammonia from the solution. When the volume of fluid in the flask has reached about 10 c.c. the urea is determined by Folin's method. Folin's Method. — Unless reducing substances such as sugar are present this method is usually applied directly to the urine without a preliminary treatment by the Morner and Folin method. The principle of this method depends on the fact that urea becomes completely hydrolysed into carbon dioxide and ammonia, when solutions of it are heated for about an hour to a temperature of 150°-160° C. The hydrolysis must be performed in acid reaction so that no ammonia can escape. The above temperature may be obtained, either by heating under pressure (in an autoclave) or, as recom- mended by Folin, by heating the urine with magnesium chloride which, after the excess of water has evaporated, gives a solution boiling at 160° C. EXPERIMENT.— Place 5 c.c. urine, 5 c.c. HC1 (con.) and 20 grm. magnesium chloride in a 200 c.c. Jena flask. Connect the neck of the flask with a glass trap or a wide tube, and heat fairly strongly so as to drive off the water from the contents of the flask. The trap prevents too much HC1 from escaping. When all the water has been boiled off (10-15 minutes) the contents of the flask will change in their manner of boiling (they will behave as warm water does when H2SO4 (con.) is dropped into it). Now lower the flame and connect the xTo employ the suction pump in this way to accelerate evaporation at low temperature the flask is closed by a doubly-bored cork; through one hole the pump is connected, through the other passes a tube ending below the surface of liquid in the flask in a fine capillary point. 422 PRACTICAL PHYSIOLOGY flask with a reflux condenser, keeping its contents just boiling for 1£ hours. After this time, and without permitting the flask to become cold, add distilled water cautiously and transfer the contents to a Kjeldahl distilling flask ; bring the volume of fluid up to 600 c.c. and allow 10 c.c. of 40 per cent. NaOH solution to run down the side of the flask so as to form a layer under the watery solution. Add some powdered pumice and distil the contents into a measured quantity of ^H2SO4 as described in connection with Kjeldahl's method. The distillation requires much longer than in Kjeldahl's process (li hours). From the number of c.c. of y^NHo found must be deducted : — (1) The c.c. TjrNH3 present as such in the 5 c.c. of urine used (determined by Folin's or Shaffer's method, p. 433). (2) The c.c. TjrNHg present in the reagents for MgCla always contains traces of NH3. CHAPTER XVIII. URIC ACID AND OTHER PURINE BODIES. IT will be remembered, from the description of the chemical structure of nuclein (p. 310), that there exist among its decomposition products several bodies belonging to the so-called purine group of chemical sub- stances. Uric acid is also a member of this group. The group receives its name because all the members of it contain, as their nucleus of construction, a body called purine, which exists as a double ring of carbon and nitrogen atoms. The various members of the group differ from one another according to the nature and position of the atoms or groups of atoms which are tacked on to this purine ring. In order to make the relationships clear the structural formulae of the various members should be studied side by side, thus : — iN— C6 HN— CO 2C 5C_7M CO C— NR \ II >CO; — — / HN— C— NH Purine nucleus. Uric acid (2, 6, 8, trioxypurine). HN— CO HN— CO CO C— NH CH C— NH \ || >CH; X l! HN— C— N^ ^N— C — Xanthine Hypoxanthiue (2, 6, dioxypurine). (6, oxypurine). PHYSIOLOGICAL CHEMISTRY 423 NH— CO HN— C=NH HN=C C— NH CH C— NH \ || >CH; X II >CH. NH— C— N" ^N— C — N^ Guanine. Adenine. The atoms in purine are numbered so as to facilitate the description of the location of the side groups. The lowest oxidation product of purine is hypoxanthine (6 oxypurine). It occurs abundant^ in muscle extract (p. 356) and in the extracts of other tissues, and also in the urine. It always exists along with xanthine, which is 2, 6 di-oxypurine. If the oxygen in hypoxanthine be replaced by an imino group ( = NH), the result is adenine, which occurs in nucleic acids. A similar derivative of xanthine is called guanine. It is the only purine found in the variety of nucleic acid called guanylic acid, and exists in certain pigments of insects and fishes. It occurs abundantly in guano. If three oxygen atoms be present we have uric acid (2, 6, 8 tri- oxypurine), and this is the form in which nearly all the " tissue purines " are excreted in the urine. The empirical formulae for these bodies are therefore : — Purine, C5H4N4. f Hypoxanthine, C5H4N40. Purine I Xanthine, C5H4N402. Bases j Adenine, C5H5N5. I Guanine, C5H5N5O. Uric Acid, C5H4N403. Of these, the uric acid is by far the most abundant in urine, whereas the purine bases are most abundant in the tissues. In metabolism the latter form the precursors of the former. The alkaloids of tea and coffee are methyl derivatives of xanthine. Thus, caffeine and theine are 1, 3, 7 trimethyl, 2, 6 dioxypurine, and theobromine (the alkaloid in cocoa) is 3, 7 dimethyl, 2, 6 dioxypurine. The constitutional formula of uric acid given above indicates that it is a diureide, containing two urea groups in the molecule. This fact is demonstrated by the syntheses of uric acid and by the nature of its oxidation products. The simplest synthesis of uric acid, brought about by heating urea with a derivative of lactic acid, trichlor lactamide, is of some physio- logical importance, as there is experimental evidence to show that in birds uric acid, which forms their principal nitrogenous excretive, is 424 PRACTICAL PHYSIOLOGY synthesised in the liver from oxidation products of lactic acid. Whether such a synthesis takes place also in mammals is at present unknown. On oxidation with potassium permanganate uric acid yields allantoin, which is present in the urine of the dog and cat, and occasionally in that of man. The formula for allantoin is : — /NH— CH— NHV C0< | >CO. XNH— CO NH/ On oxidation with nitric acid, uric acid yields alloxan, carbon dioxide, and nitrogen. An intermediate oxidation product, alloxantin, is formed at the same time, which with ammonia forms a red dye, murexide. This reaction is used as a test for uric acid. Alloxan is the ureide of mesoxalic acid : — Further oxidation yields the ureide of oxalic acid. Ordinarily uric acid behaves as a monobasic acid, being soluble in alkalies (caustic soda, ammonia, and boiling solution of sodium carbonate) with the formation of the corresponding salts, which are more soluble in water than the free acid. The dibasic salts can, however, be obtained by the use of excess of concentrated alkali, so that it is the custom to call uric acid a dibasic acid and its ordinary salts acid salts, although their solutions are alkaline, not acid. Strictly speaking, uric acid is a tetrabasic acid, as all four hydrogen atoms in the molecule have latent acid properties. Uric acid is the principal nitrogenous excretive of birds and reptiles. Together with other purine bodies it is always present in the urine of man, having a twofold origin, exogenous and endogenous. The exo- genous purines come from purine bodies in the food (nucleo-proteins in cellular structures, xanthine and hypoxan thine in meat, caffeine, etc.). The endogenous output of purines is fairly constant for a given indi- vidual under ordinary conditions, and is to be traced, partly at any rate, to the breakdown of nucleo-proteins in the body. Burian and Schur found the daily purine excretion of a normal individual on an ordinary diet to be about 1 grm. On a purine free diet this was reduced to 6'0 grin., and was practically independent of the amount of nitrogen in the food. Violent muscular exercise and pyrexia both increase the output of purine bases and uric acid on a purine free diet. This effect is pre- sumably connected with the xanthine and hypoxanthine of muscle (see p. 354). PHYSIOLOGICAL CHEMISTRY 425 PREPARATION AND REACTIONS or URIC ACID. EXPERIMENT I. To 100 c.c. urine add 5 c.c. HC1 (cone.), and allow the mixture to stand overnight. It will then be found that a dark-brown sediment, like cayenne pepper, has settled down, and pro- bably also that a brown scum has formed on the surface. Filter and examine the sediment under the microscope. It consists of large dark- brown clumps of crystals, whetstone or barrel-shaped (Fig. 245). These are crystals of uric acid admixed with pigment. They can be purified by solution in 5 per cent. KOH and reprecipitation by HC1. Preserve the crystals for further use. EXPERIMENT II. Pure crystals can be obtained from the solid urine of a snake or bird. This urine, which consists of sodium urate, is dissolved in caustic potash and acidified with HC1. Pure uric acid separates out. From these two experiments we learn that uric acid exists in urine as a salt. If this salt be decomposed by a mineral acid the liberated uric acid, being very insoluble, is precipitated. The following are the most important reactions of uric acid. EXPERIMENT III. The Murexide Test.— Place some uric acid or bird's urine in a capsule, add a few drops of dilute nitric acid, evaporate slowly to dryness on a water-bath. A yellow residue is obtained. Add a drop of ammonia, a crimson colour results, which is changed to purple by adding caustic soda. If overheated, the residue will turn crimson without the addition of ammonia. EXPERIMENT IV. Uric acid has the power of reducing metallic oxides in alkaline solution. This may be demonstrated by the following tests. Some uric acid is dissolved in weak sodium carbonate solution, which is then poured on to a piece of filter paper moistened with a solution of AgN03. A black stain of reduced silver results. This is called Schiffs reaction. In the presence of neutral salts, and more especially of magnesium mixture (MgCl2, NH4C1, NH3), the uric acid and other purine bodies unite with the silver to form a double salt. This salt separates out as a gelatinous precipitate, and is employed for quantitatively estimating the purine bodies (Salkowski's method). Uric acid can also exercise its reducing powers on cupric salts in alkaline solution. By applying Trommer's test, or one of its modifications, to an alkaline solution of uric acid, it will be noticed that reduction ensues. The reduction precipitate is, however, of a dull brown colour instead of being yellowish red, as it usually is. This is because a certain amount of the cuprous oxide unites with some of the uric acid to form a brown compound. 426 PRACTICAL PHYSIOLOGY FIG. 245.— Crystals of uric acid. PHYSIOLOGICAL CHEMISTEY 427 EXPERIMENT V. Tests for Uric Acid in Urine.— Apply Schifs test to urine. The result is positive, but does not necessarily show the presence of uric acid, as other reducing bodies are present in the urine. The murexide test cannot be applied directly to urine, as urine yields a red pigment on heating with nitric acid. In order to apply this test take about 100 c.c. of urine, add ammonia until it is alkaline, and saturate with ammonium chloride. A precipitate of ammonium urate forms. This is filtered off, dissolved in a few c.c. of water, and employed for the murexide test. ESTIMATION OF URIC ACID. The most rapid and accurate method is that of Hopkins as modified by Folin. In this method the mucoid substances and some of the phosphates of urine are first of all precipitated by a strong solution of ammonium sulphate con- taining uranium acetate and acetic acid, and the filtrate is then rendered alkaline with ammonia ; on standing ammonium urate separates out. This is collected on a filter, washed, and suspended in water and titrated with vi/20 potassium permanganate. The method is carried out as follows : — To 300 c.c. urine in a flask 75 c.c. of the uranium ammonium sulphate re- agent is added (500g. ammon. sulph., 5gr. uranium acetate, 60 c.c. 10 per cent, acetic acid, 650 c.c. water), and in five minutes the solution is filtered through a dry thick filter paper into a dry 250 c.c. measuring cylinder, or into a dry beaker. Two portions of the filtrate of 125 c.c. each are transferred to beakers and 5 c.c. concentrated ammonia added to each. The beakers are then set aside for twenty-four hours, at the end of which time the precipitate of ammonium urate will be found on the bottom of the beaker. The clear supernatant fluid is carefully poured through a hardened filter, after which the sediment is shaken with a 10 per cent, solution of ammonium sulphate and care- fully collected on the same filter and washed once or twice with the 10 per cent, ammonium sulphate solution. It is unnecessary to transfer every trace of pre- cipitate to the filter, and the washing with ammonium sulphate solution does not require to be prolonged. The filter is then opened up and the precipitate washed into the beaker in which the original precipitation was made by means of a spray of distilled water from a wash bottle. As a result of this process about 100 c.c. of fluid should have collected in the beaker. Then 15 c.c. H2S04 (cone.) is added to the fluid, and while still hot from the mixing of acid and water, it is titrated with w/20 potassium permanganate until a faint pink colour remains for five seconds after mixing. The reading obtained, multiplied by 0*00375, gives the grammes of uric acid in 100 c.c. of urine.1 Estimation of the Total Purine Bodies. Modified Camerer's Method. — Principle. — Ammoniacal silver nitrate, in the presence of neutral salts, or, better, of magnesium mixture, combines with all the purine bodies to form an insoluble salt of definite composition (see p. 425). The nitrogen in this can be estimated by Kjeldahl's method, and the result expressed as total purine nitrogen. This result is exceedingly useful in studying the metabolism of purine bodies. If 1 On account of partial solubility of ammonium urate in water, it is necessary to add 3 mg. uric acid for every 100 c.c. of urine. 428 PEACTICAL PHYSIOLOGY it be desired to determine the uric acid and the bases separately, a slight modifi- cation of the process is necessary. Solutions necessary. — 1. Magnesia mixture. This consists of 1 part crystallised magnesium chloride, 2 parts chloride of ammonium, dissolved in 8 parts of water and made strongly alkaline with 4 parts of ammonia. If the mixture be not quite clear (from the presence of magnesium hydrate) more ammonium chloride should be added. 2. Ammoniacal silver nitrate. Dissolve 26 gr. silver nitrate in about 300 c.c. water, add ammonia to this until the precipitate of silver oxide, which first forms, redissolves. Dilute the solution to one litre. 3. Kjeldahl's apparatus and solutions (see. p. 410). Determination. — 240 c.c. protein free urine are mixed with 30 c.c. magnesia mixture, and the solution is made up to 300 c.c. by the addition of a 20 per cent, ammonia solution. This process is best done in a measuring cylinder. After the precipitate has settled, which it does in a few minutes, it is filtered through a dry folded filter and two portions of the filtrate are taken amounting to 125 c.c. each. Each of these corresponds to 100 c.c. of the original urine. They are both treated in exactly the same way, and should yield similar results. Each is mixed with 10 c.c. ammoniacal silver nitrate, and the mixture, after the precipitate has settled somewhat, filtered through an ash-free filter paper (of 10 c.m. diameter). The last traces of the precipitate are removed from the beaker by means of weak ammonia water. The next stage consists in washing the precipitate with distilled water until it is free from ammonia, as the presence of this would vitiate the determination of the nitrogen. In order to do this, the precipitate should be allowed to stand exposed to the air over night so that it may become partially dried, in which state the washing with water 'is much easier than when the precipitate is moist, for then it forms a gummy mass. The washing must be continued until the washings no longer react alkaline to litmus. In order to remove the last traces of ammonia, the filter paper, with the precipitate on it, is carefully removed to a Kjeldahl's combustion flask ; about 50 c.c. of water are added, and then a little magnesium oxide. The mixture is then boiled, whereon the magnesia expels the ammonia. The boiling is continued until only about 10 c.c. of fluid remain, and then sulphuric acid, etc., are added, and the nitrogen determined. To Determine the Bases and Acid separately.— Various methods are recommended. The simplest is probably to precipitate the bases by the Camerer method in the filtrate from which uric acid has been removed, as ammonium urate> as described under Folin's method. Hippuric Acid. — In herbivorous animals a large amount of hippuric acid is excreted, but in man on an ordinary diet and in the carnivora only a small quantity. Hippuric acid may readily be obtained from the urine of a herbivorous animal by the following procedure. The urine is boiled for a few minutes with excess of milk of lime, filtered hot, concentrated on the water-bath, cooled and acidified with hydro- chloric acid. Crystals of hippuric acid separate on standing, which are filtered off and dried. They may be freed from benzoic acid by extrac- tion with petroleum ether, in which hippuric acid is insoluble, and recrystallised from hot water, using animal charcoal to decolourise if PHYSIOLOGICAL CHEMISTRY 429 necessary. Chemically hippuric acid is benzoyl glycine C6H5. CO. NH. CH2. COOH. It may be synthesised by the action of benzoyl chloride on glycine, and decomposes to benzoic acid and glycine on heating with strong hydrochloric acid. The presence of hippuric acid in urine is due to aromatic substances in the food, which are oxidised to benzoic acid in the body and excreted in combination with glycine. A vegetable diet is particularly rich in these aromatic substances. Hence the large quantity of hippuric acid in the urine of herbivorous animals. Creatinin. — The chemical relationships of this body have already been discussed (see Muscle, p. 352). Urine always contains creatinin. On a creatinin-free diet the amount excreted per diem is remarkably constant for a given individual. So much so that a determination of creatinin in a urine of twenty-four hours from a person whose creatinin output is known affords a test as to whether the urine has been properly collected. Creatin is not normally present in urine unless large quan- tities of creatin are taken in the diet. It, however, makes its appear- ance in inanition and in carcinoma of the liver. Tests for Creatinin. EXPERIMENT VI. Weyl's Reaction. — To five c.c. of urine are added four or five drops of a very dilute solution of sodium nitro-prusside, so that the original colour of the urine remains unchanged. If a weak solution of caustic soda be now added drop by drop a ruby-red colour results, quickly changing to yellow. If an excess of acetic acid be added and the solution boiled, a greenish blue colour results, and after standing some time a blue sediment (Prussian blue) settles to the bottom of the tube. Acetone gives a similar colour with the nitro-prusside and alkali, but it does not change to yellow on standing, and turns reddish purple on the addition of acetic acid. Creatinin possesses, to a certain extent, the power of reducing metallic oxides in alkaline solution, and this must be remembered as a possible source of fallacy in testing for dextrose. EXPERIMENT VII. Jaffa's Test. — Add a few drops of a saturated solution of picric acid in water and a few drops of caustic soda 10 per cent, solution to about 5 c.c. urine. A red colour is produced owing to the formation of picramic acid. Estimation of Creatinin. Folin's Method.— For this purpose the urine must be free from aceto-acetic acid and hydrogen sulphide, and must contain not more than traces of acetone. Measure 10 c.c. urine with a pipette into a 500 c.c. graduated flask. Add 15 c.c. saturated aqueous picric acid solution (about 1*2 per cent.) and 5 c.c. 10 per cent, caustic soda solution. Mix and allow to stand 430 PBACTICAL PHYSIOLOGY for five minutes. Fill up the flask to the 500 c.c. mark, and mix well. By means of a Dubosq or other suitable colorimeter determine the depth of liquid required to give in daylight an intensity of colour exactly equal to that given by a depth of 8 mm. of a solu- tion containing 24 '55 grm. pure potassium bichromate per litre. The readings of the colorimeter, of which several should be taken, should be completed within twenty minutes of the dilution, as the reaction liquid fre- quently fades. The zero of both sides of the colorimeter should be tested, and it is as well to test the use of the colori- meter by employing the bichromate solution on both sides before determining the creatinin. The readings in the crea- tinin determination should not differ by more than 0*3 mm. If the average read- ing is less than 5 mm. , the urine should be carefully diluted and another determination made; if above 13 mm., 20 c.c. urine instead of 10 c.c. should be employed. The result of the determination is calculated from the formula : — 10x8-1 FIG. 246.— The Dubosq Colorimeter. Where x is the quantity of creatinin in milligrammes in the volume of urine employed, and a is the colorimeter reading in millimetres. The amount of creatinin is inversely proportional to the colorimeter reading. The formula depends on the fact that, when 10 mg. of pure creatinin was employed for a determination, the colorimeter reading, against 8 mm. of standard bichromate, was 8'1 mm. Estimation of Creatin. — Place in a flask, fitted with a cork and glass tube to serve as condenser, 10 c.c. urine and 5 c.c. N. HC1. Heat the flask in a boiling water bath for five hours. Cool to room temperature. Add sufficient caustic soda to neutralise the acid added, 15 c.c. picric acid solution and 5 c.c. 10 per cent, caustic soda. Allow to stand for five minutes. Wash the contents of the flask into a 500 c.c. flask, make up to 500 c.c., and proceed as for creatinin. PHYSIOLOGICAL CHEMISTRY 431 This determination gives the creatin and creatinin together, as creatin is con- verted by the acid into creatinin. The difference between this result and that for creatinin previously determined represents the amount of creatin present. Ammonia. — In metabolism ammonia is the precursor of urea, being transformed to urea by the liver. The transformation to urea is never absolutely complete, so that urine always contains ammonia. In man under ordinary conditions the output of nitrogen as ammonia varies between 3 and 5 per cent, of the total nitrogen. When measured in N terms of the equivalent amount of ^K alkali, the ammonia is roughly equal to, or rather less than, the total acidity of the urine. When the nitrogen of the diet is low, the ammonia of the urine is decreased in absolute amount, but forms a greater percentage of the total nitrogen (see page 415). The principal cause of a rise in the ammonia of the urine is the presence of acids, which require neutralisation, and so prevent the con- version of some ammonia into urea, the amount of ammonia rising more rapidly than the total acidity. From this cause the ammonia nitrogen may rise in pathological cases till it forms 40 per cent, of the total nitrogen. A similar effect is produced in dogs (carnivora) by the administration of hydrochloric acid. On the other hand, hydrochloric acid given to rabbits (herbivora) causes only a slight rise in the ammonia excretion. These animals rely on mineral bases to neutralise the acid, with the result that they are much more easily poisoned with mineral acid than dogs are. Similarly, ammonium chloride given to man and to dogs is mainly excreted unchanged, because the hydrochloric acid of the salt prevents the conversion of ammonia to urea, which takes place when ammonium carbonate is given ; on the other hand, a large pro- portion of the ammonia of ammonium chloride given to rabbits is converted to urea. EXPERIMENT VIII. Estimation of Total Acidity and Ammonia in Urine. — Weigh out roughly 15 grm. powdered potassium oxalate (neutral to phenol phthalein), place in a flask, and add from a pipette 25 c.c. urine and an equal volume of water. Add about 10 drops 1 per cent, alcoholic phenol phthalein. Mix and allow to stand for about a minute. Now run in y^ caustic soda from a burette until the contents of the flask assume a slight pink tint. Read the burette. Measure into a small beaker 5 c.c. formalin (40 per cent, formaldehyde) and roughly 5 c.c. water, and add a few drops of phenol phthalein solution. Run in caustic soda till a slight pink colour is attained. Add this mixture 432 PRACTICAL PHYSIOLOGY to the flask containing the neutralised urine. The pink colour dis- N appears. Kun in y^ caustic soda until the colour returns, and take the reading. The first reading of the burette gives the total acidity of 25 c.c. N urine in terms of y^ soda. Potassium oxalate is added to precipitate the calcium in the urine as calcium oxalate, as the formation of calcium phosphate would otherwise interfere with the end-point. On the addition of neutral formaldehyde the ammonia in the urine combines with the formaldehyde forming a neutral compound, urotro- pine, thus liberating the acid which it previously neutralised. The second titration, therefore, determines the amount of ammonia present N in terms of y^ soda. To calculate the amount of nitrogen in grammes present as ammonia in the volume of urine taken multiply the reading of this titrabion in c.c. by O0014. The method of determining ammonia is of sufficient accuracy for clinical purposes. The amount of ammonia is always higher by this method than by the more accurate methods which follow. This is due to the fact that formalin combines with amino acids, which are normally present in urine in minute traces, and thus renders them acid to phenol phthalein. This source of error is small, unless amino acids are present in excessive amount, as in cystinuria. The difference between the result of this method and that of one of the methods which follow affords a measure of the amount of amino acids present in the urine. Estimation Of Ammonia. Folin's Method. — Ammonia is liberated from its salts when a solution of the latter is made alkaline with a soluble carbonate, which, unlike a hydroxide, does not decompose the other nitrogenous bodies. By bubbling a fast current of air through such a mixture the ammonia is carried away and may be collected and measured by passing this air through standard acid. Excess of sodium chloride in the mixture not only preserves it against putrefaction, but encourages the evolution of the ammonia. The technique of the method is as follows : — 25 c.c. urine are placed in a large test tube a (Fig. 247) 2£-3 cm. diam. and 20-30 cm. long), and mixed with 8-10 grms. sodium chloride and 5-10 c.cm. petroleum (to prevent excessive frothing), and lastly with 1 grm. sodium carbonate. The test tube is closed by an india- rubber stopper through which pass two tubes, the one for the air inlet passing to the bottom of the test tube, the other connecting the top of the test tube with a wide tube (U tube) 6 containing a loosely packed cotton-wool plug (to catch any particles of fixed alkali which might be sucked over with the air current). This safety tube is connected with a second test tube c (of the same size as the first) containing 15 c.c. N/10 H2S04 + 5 c.c. water, the tubing being so arranged that the air bubbles through the acid. A third tube or bottle d, arranged in the PHYSIOLOGICAL CHEMISTRY 433 same way as the second and containing 10 c.c. NjlQ acid and some ammonia free water, follows this, otherwise all the ammonia would not be caught by the acid. The tubing connected with this tube goes to a Bunsen's air-pump e attached to a a FIG. 247.— Folin's apparatus for estimating ammonia. tap /. A quick stream of air (600-700 litres per hour) is made to pass through the apparatus for 1£ hours. The acid in the two last test tubes is then washed into an Erlenmeyer flask and titrated with jV/10 alkali. For titrating, Folin FIG. 248.— Shaffer's method of estimating ammonia in urine. recommends 2 drops of a 1 per cent, solution of Alizarin red (for 200-300 c.c. fluid), the titration being carried on till a pink (not a violet) colour just appears. This indicator gives a better end-point than methyl orange. Shaffer's vacuum distillation method of estimating ammonia in urine is as accurate as Folin's and much more rapid. 2E 434 PEACTICAL PHYSIOLOGY Place 50 c.c. urine in a round bottom £ litre flask A (Fig. 248), add 20 grm. sodium chloride to prevent decomposition and 50 c.c. methyl alcohol to reduce the boiling point of the mixture. In flask B place 50 c.c. or less JV/IO acid and in C 10 c.c. N/10 acid, diluted in both cases with a little water. The flasks may be tilted obliquely, and should be large enough to prevent loss of acid by spraying during the violent commotion which is set up by the rapid passage of steam. If such loss should occur, the acid may be recovered by rinsing out the flask D. When the apparatus is ready, 1 grm. of dry sodium carbonate is added to the liquid in the flask A, the stopper is rapidly inserted and the suction started. The pump will quickly reduce the pressure to about 30 mm., and the liquid in A, which is warmed up to about 40° C. in a water-bath, will begin to boil. The temperature of the bath must be maintained and should not be allowed to rise above 50° C. for fear of decomposing urea. When the boiling has continued for fifteen minutes, all the ammonia will have been given oft7 and the operation is stopped by slowly letting in air by the stop-cock a. The acid in B and C is titrated, after a few drops of a 1 per cent, solution of Alizarin red have been added as the indicator. CHAPTER XIX. THE INORGANIC ACID RADICLES OF URINE. URINARY DEPOSITS. Chloride. EXPERIMENT I. Add to urine a few drops of nitric acid, and then silver nitrate solution. A white precipitate of silver chloride forms, which is soluble in ammonia. The nitric acid prevents the precipitation of other silver salts such as phosphate. Estimation of Chloride. Volhard's Method. — The chloride is precipitated by the addition of a known excess of silver nitrate in the presence of nitric acid, and the excess of silver nitrate deter- mined by titrating a known part of the filtered solution with potassium sulphocyanide solution, which precipitates the silver, using ferric alum to indicate when the sulphocyanide has been added in slight excess. Into a 100 c.c. graduated flask measure with a pipette 10 c.c. urine free from albumin. Add 5 c.c. pure nitric acid and N 30 c.c. y~r silver nitrate solution (17 grm. per litre) measured with a pipette. Add distilled water up to the 100 c.c. mark and rinse thoroughly. Filter the liquid through a dry chloride free filter into a dry clean beaker. Measure 50 c.c. of the filtrate with a pipette into an evaporating dish. Add 10 to 20 c.c. 10 per cent, ferric alum N solution, and run in from a burette potassium sulphocyanide PHYSIOLOGICAL CHEMISTKY 435 solution (9-73 grm. per litre), until on stirring the liquid assumes a permanent slight red colour. Take the reading on the burette. Let this be a c.c. Then the amount of chloride ( - Cl.) in the 10 c.c. urine employed is equal to (30 - a) 0-00355 grm. The sulphocyanide solution must be exactly equivalent to, or standardised in terms of, the silver nitrate solution. Phosphate. EXPERIMENT II. Boil some solution of ammonium molybdate in nitric acid in a test tube, and add drop by drop boiling urine acidified with nitric acid. A yellow precipitate indicates the presence of phosphate. EXPERIMENT III. Make urine alkaline by the addition of ammonia. A white precipitate of calcium and magnesium phosphate forms. Filter off this precipitate and prove that it contains phosphate by the molybdate test. Estimation of Phosphate. — Standard uranium nitrate solution is added to urine in the presence of sodium acetate and acetic acid until all the phosphate is precipitated, as shown by the presence of slight excess of uranium in the solution. Sodium acetate is added so as to prevent the liberation of nitric acid, in which uranium phosphate is soluble. The acetic acid serves to prevent the pre- cipitation of other uranium compounds than the phosphate and to dissolve any calcium phosphate present. Place 50 c.c. urine in a porcelain basin. Add 5 c.c. of a solution containing 100 grm. sodium acetate and 100 c.c. glacial acetic per litre. Heat the basin and run in from a burette a standard solution of uranium nitrate, until a drop of the solution, placed on a small heap of powdered potassium ferrocyanide on a white tile gives an immediate brown colour, due to the formation of uranium ferro- cyanide. The most convenient standard solution of uranium nitrate contains 35 '5 grm. per litre. 1 c.c. of this solution is equivalent to 0'005 grm. phosphoric acid (P205). Sulphates and Neutral Sulphur. — There are two varieties of sulphates in the urine, each of which has a special significance of its own. These varieties are : 1. Inorganic sulphates (sodium sulphate, potassium sulphate, etc.). OK 2. Ethereal sulphates (phenyl sulphate of potassium) S02<^QQ g OK (indoxyl sulphate of potassium) SO2<^Qp TT -^ Under ordinary conditions the ethereal sulphates constitute only 436 PEACTICAL PHYSIOLOGY about 10 per cent, of the total sulphate excretion. The neutral sulphur is present in organic compounds, such as cystin. EXPERIMENT IV. Place about 10 c.c. of urine in a test tube and dilute with water to about 20 c.c. Add about 2 c.c. of hydrochloric acid (1 part HCl(conc.) + 4 parts water), and then drop by drop about 2 c.c. of 5 per cent, solution of barium chloride. A white precipitate of barium sulphate forms, due to the presence of inorganic sulphates. After allowing the test tube to stand for a few minutes, filter and boil the clear filtrate with a few drops more of hydrochloric acid and barium chloride solution. There is another precipitation of barium sulphate due to ethereal sulphates which have been decomposed by boiling with hydrochloric acid. Quantitative determination. — There is no volumetric method, that is even approximately accurate, available for this purpose, so that it is necessary that a good analytical balance be at command. For accurate work it is further necessary that great care be exercised in carrying out the processes, for the final precipitates are otherwise apt to be quite impure and the estimation inaccurate. The following processes have been worked out by Folin, and the directions must be implicitly followed. Protein must be removed if present. This is best done by boiling the urine acidified with acetic acid in a flask, cooling and filtering. EXPERIMENT V. Inorganic Sulphates. — 25 c.c. of urine are diluted with 100 c.c. of water in an Erlenmeyer flask (of 250 c.c. capacity) and 10 c.c. of dilute hydrochloric acid (1 part HC1 (con.) to 4 parts water) added. A burette contain- ing a 5 per cent, solution of barium chloride is then placed over the mouth of the flask and 10 c.c. of the reagent allowed to drop into the contents of the flask at a slow rate (not quicker than 5 c.c. per minute).1 The flask must not be shaken until after the end of an hour,2 when it is shaken and the precipitate collected on an asbestos mat in a Gooch crucible, washed with about 250 c.c. cold water, dried and ignited. In doing this, the flame must not be applied directly to the per- forated bottom of the crucible, but the crucible must be laid on a crucible lid or specially fitting platinum bottom. The crucible must also be covered with a lid during the ignition. Ten minutes' ignition is sufficient. EXPERIMENT VI. Total Sulphates (Inorganic and Ethereal).— By boiling the urine with acid, nearly all of the ethereal sulphates are decomposed. 25 c.c. urine are mixed with 20 c.c. of dilute hydrochloric acid (1 : 4) in an Erlenmeyer flask of about 250 c.c. capacity, and, after covering the mouth of the flask with a watch-glass, gently boiled for 20 to 30 minutes. The flask is then cooled in running water, its contents diluted with distilled water to about 150 c.c. and 10 c.c. of 5 per cent, solution of barium chloride added, and the further procedure followed as above described. EXPERIMENT VII. Ethereal Sulphates. — It is sufficiently accurate to calcu- late this as the difference between I. and II. If a direct estimation is desired as a JMore rapid addition of the reagent causes the results to be too high, i.e. produces an impure precipitate. 2 Shaking the solution too soon will lower the result, because the precipitate will lose sulphuric acid during ignition. PHYSIOLOGICAL CHEMISTRY 437 check, it may be made by diluting 125 c.c. of urine with 75 c.c. water, adding 30 c.c. dilute hydrochloric acid (1:4) and precipitating the inorganic sulphates with 20 c.c. of 5 per cent, solution of barium chloride, as above described. After standing for one hour the mixture is filtered through a dry filter and 125 c.c. of the clear filtrate, corresponding to 62'5 c.c. original urine, boiled for not less than 30 minutes, whereby a precipitate of sulphate forms on account of the decomposi- tion of the ethereal sulphates. After allowing the solution to cool, this precipitate is collected on a Gooch crucible, washed, ignited, and weighed. EXPERIMENT VIII. Total Sulphur.— 25 c.c. urine (or 50 c.c. if very dilute) is mixed in a large nickel crucible (of 200 to 250 c.c. capacity) with 3 grams of sodium peroxide. The crucible is then carefully heated until its contents solidify (about 15 minutes), after which it is cooled, the fused mass moistened with 1-2 c.c. of water about 7 grms. of sodium peroxide sprinkled over it, and again heated to cause its contents to become completely fused; the mass is kept fused for ten minutes, then allowed to partially cool, 100 c.c. water added and the contents boiled for half an hour. This dissolves the alkali and decomposes all the sodium peroxide. The contents of the crucible are transferred to an Erlenmeyer flask (400 — 450 c.c. capacity) and the crucible washed into the flask with hot water, the volume of the resulting solution made up to about 250 c.c., and then brought almost to the boil ; concentrated hydrochloric acid is now slowly added until the nickelic oxide just dissolves (about 18 c.c.) and the mixture is boiled until it becomes clear. (If it does not become clear by this treatment, it must be cooled and filtered.) 5 c.c. of dilute alcohol (1 : 4 ) is then added and the boiling continued for a few minutes, so as to remove traces of chlorine. The solution is now ready for precipitation of the sulphates, into which, by the above procedure, all the sulphur has been converted. To do this 10 c.c. of 10 per cent, solution of barium chloride is added drop by drop, as above described, and the mixture allowed to stand for two days,1 after which the precipitate is collected and weighed as above described. EXPERIMENT IX. Neutral Sulphur.— This is obtained by subtracting the total sulphates (No. 2) from the total sulphur. It will be seen that at least three separate estimations must be carried through to obtain all the desired data — (1) inorganic sulphates, (2) total sulphates, and (3) total sulphur. The ethereal sulphates and the neutral sulphur can be calculated by difference. These values can be calculated as S or SO3 according to the following ratios :— BaS04 : S = 1 : 0'1374 ; BaS04 : S03= 1 : 0'3429. Metabolism. — One of the results of Folin's investigations on metabolism has been to show the significance of determinations of sulphates in the urine. The total sulphur in the urine is, like the nitrogen, distributed among several substances, which are divided into three groups — the inorganic sulphates, the ethereal sulphates, and the neutral sulphur compounds. The inorganic sulphates are mainly those of sodium; the ethereal sulphates are compounds of phenol, cresol skatoxyl, and indoxyl, with sulphuric acid and potassium (see p. 435), and the neutral sulphur compounds are organic compounds in which 1 It takes this length of time to ensure complete precipitation. 438 PRACTICAL PHYSIOLOGY the sulphur is an integral part of the molecule. Cystin, when present, belongs to this group. When the relative amounts of S03 excreted in the above-mentioned three forms are calculated as percentages of the total SO3 excretion, it is found that the inorganic sulphates on a protein poor diet behave like urea-nitrogen, i.e. become less both in absolute amount and in relative percentage ; that the neutral sulphur under the same conditions behaves like creatinin-nitrogen, i.e. remains constant in absolute amount, whereas the percentage rises and that the ethereal sulphate excretion behaves like that of ammonia-nitrogen, i.e. becomes somewhat less in absolute amount, but that the percentage rises. These facts are clearly shown in the following table, which is an extension of that on page 415. N-rich diet. N-poor diet. Volume of urine, 1170 c.c. 385 c.c. Total nitrogen, - 16 '8 grm. 3 '60 grm. Total S03, - - 3-64 grm. 0'76 grm. Inorganic S03, - 3 "27 grm. =90'0 % of total S. 0'46 grm. =60 '5 % of total S. Ethereal S03, - 0'19grm.= 5'2% „ O'lO grm. = 13*2% „ Neutral S03, - 048 grm. = 4'8% „ 0 "20 grm. =26 '3% The ethereal sulphates cannot, as has been supposed, derive their source entirely from the aromatic bodies formed in the intestine by micro-organismal growth. When this is excessive, or when there is obstruction in the small intestine so that an excessive amount of these aromatic bodies is absorbed, an increase no doubt occurs in the ethereal sulphate excretion, but this increase can be no reliable index of intes- tinal putrefaction, since the relative ethereal sulphate excretion becomes greater when the diet contains little or no protein. Practically the only source of sulphur intake by the food is in proteins. Sulphates are not taken unless for medicinal purposes, because of their disagreeable taste. The sulphur excretion by the urine is therefore a measure of protein katabolism in the organism. URINARY DEPOSITS. Normal urine is quite clear when it is passed, but, on standing some time, a sediment usually separates out, and this varies somewhat under different conditions. Acid Urine from a healthy person may deposit the following: — 1. Urates (see p. 424). — The sediment has a chalky appearance and is usually tinged reddish by uroerythrin. It disappears on warming the urine. Examined microscopically, it is generally amorphous, but may show a crystalline structure, usually as needles, or as balls with spines projecting from them (Fig. 249). It is composed mainly of sodium urate. PHYSIOLOGICAL CHEMISTRY FIG. 249.— Sodium urate. x 350. Fio. 250.— Cystin. X 350. PIG. 251.— Calcium carbonate (front, human urine). X400. 440 PRACTICAL PHYSIOLOGY 2. Uric Acid. — This may be split off from the urates as described on p. 424. It appears as a cayenne pepper-like sediment, and has a definite crystalline appearance under the microscope (Fig. 245). The crystals may vary much in shape, but are always large and tinged a reddish colour. The most usual shapes for the crystals to assume are " sheaves," "whetstones," "rhombic tables," and sometimes "dumb-bells." The presence of the crystals does not necessarily indicate an increased FIG. 252.— Calcium oxalate. x 500. excretion of uric acid, but depends on the concentration and acidity of the urine. 3. Calcium Oxalate. — This is usually a scanty deposit, adhering to irregularities on the surface of the glass of the urine jar, or forming a glistening layer on the top of the mucous deposit that settles at the bottom. The crystals are insoluble in acetic acid. This reaction distinguishes them from phosphates or carbonates. They are also insoluble in ammonia, and are thus distinguished from urates. PHYSIOLOGICAL CHEMISTRY 441 Microscopically they are seen to be very small octahedra, often flattened along one axis, so that they appear like squares with diagonal lines (hence called "envelope" crystals, Fig. 252). Acid urine from a person suffering from disease, or during the administra- tion of certain drugs, may deposit : — 1. Cystin. — This forms a deposit somewhat like that of urates in appearance. Fio. 253. — Stellar phosphate of calcium, x 500. Microscopically, however, it shows a distinct crystalline structure consisting of hexagonal colourless plates or slabs (Fig. 250). When the crystals are present the condition is called cystinuria (see p. 438). 2. Leucin and Tyrosin. — Though very rarely, these two bodies sometimes occur in urine (e.g. in severe hepatic disease), where their appearance is similar to that in a pancreatic digest (see Fig. 236). 3. Hippuric Acid. — This may appear in urine during the administra- 442 PRACTICAL PHYSIOLOGY tion of benzole acid. It crystallises in four-sided prisms. It is quite common in the urine of herbivora. In Alkaline Urine the following may occur : — 1. Phosphates. — Of these there are two kinds, viz. phosphate of calcium and ammonium-magnesium phosphate. (a) Phosphate of Calcium. — The sediment is chalky and never pigmented ; it clears up on adding a few drops of nitric acid ; it is increased by boiling. Microscopically it is usually amorphous, but may exist as long prismatic crystals arranged in star-shaped clusters, hence called Stellar Phosphates (Fig. 253). The crystalline form may also occur in faintly acid urines. (b) Ammonium-magnesium Phosphate, Triple Phosphate. — When urine gets stale and ammonia develops in it, a white sediment and a white surface film form. Under the microscope these are seen to be made up of large clear crystals like "knife rests" or, if excess of ammonia be present, they may look like "feathery stars" This latter type can be easily obtained by adding ammonia to normal urine (Fig. 254). 2. Urate of Ammonia. — This looks like the urate of soda crystals, but is associated with crystals of phosphates, and occurs in an alkaline urine. 3. Carbonates. — In the urine of vegetarians these are not uncommon. The urine effervesces on adding acetic acid. Microscopically the sedi- ment is usually amorphous, but may exist as biscuit-shaped crystals or as dumb-bells (Fig. 251). CHAPTER XX. PATHOLOGICAL URINE. I. Proteins in the Urine — Albuminuria. — Traces of mucin or nucleo- protein may be added to the urine in its passage along the urinary tract, but otherwise healthy urine does not contain any protein. When the kidneys or urinary passages are diseased, however, a certain amount of the plasma proteins leak into the urine, where they can be recog- nised by certain tests, the condition being called Albuminuria. Also when proteins other than serum albumin and globulin gain access to the blood, they are at once excreted in the urine. It is on this account that albuminuria results after the consumption of a large number of raw eggs (egg flip), because the intestinal epithelium allows a certain amount of the unchanged albumin to pass into the blood, PHYSIOLOGICAL CHEMISTRY 443 „, FIG. 254.— Triple phosphates, x 400. 444 PRACTICAL PHYSIOLOGY where it is foreign (in being egg- and not serum-albumin), and is con- sequently immediately excreted by the kidneys. In disease of the red- bone marrow, a body somewhat similar in its reactions to a proteose is added to the blood and is excreted by the urine (Bence Jones' proteose- uria). Although globulin may occur along with albumin in the urine, or even in some cases independent of it, it is of no practical importance to distinguish between them, so that the tests about to be described include both bodies. The tests employed depend on certain of the reactions described under proteins. It is obvious that the colour reactions will not be applicable to the urine; those employed depend on the production of coagula. The most important of these are : — 1. Heat Coagulation. — EXPERIMENT I. Place some clear urine in a test tube, and boil. A white turbidity or coagulum indicates the presence of either albumin or phosphates (earthy phosphates are pre- cipitated by boiling). To the boiling solution, whether it show a turbidity or not, add 3-4 drops of concentrated nitric acid. If due to phosphates, the turbidity will disappear, but will remain if due to protein. In nitric acid any acid- or alkali-albumin which the urine may contain is insoluble. Where there is doubt as to the occurrence of a haze, the test tube should be about three-quarters filled, and only the upper layer should be boiled, the test tube being meanwhile held low down. By holding it against a dark background the slightest haze becomes very evident by this method, on account of contrast with the unboiled layer beneath. 2. Heller's Test. — EXPERIMENT II. Place some clear urine in a test tube. Hold the test tube in a slanting position, and allow concentrated pure nitric acid to run very slowly down the side, so that it forms a layer underneath the urine. Where the two meet, a sharp white ring (of coagulated acid albumin) is formed. The test may also be done by placing the nitric acid first in the test tube, and covering this with th& urine slowly delivered from a pipette. The ring does not disappear on warming. A similar ring may be obtained when proteoses are present,, but in this case the ring clears up on gently warming the test tube, and reappears on cooling. In warming, very great care must be taken that no mixing of the two layers occurs. When mucin is present in excess a diffuse haze may be produced in the portion of urine next the acid. Certain resin acids which may appear in the urine after the administration of such drugs as copaiba also give a haze by Heller's test. Also when the urine is very concentrated, acid urates or urea nitrate crystals may develop and simulate the reaction. In these cases,. PHYSIOLOGICAL CHEMISTRY 445 the urine should be diluted with two or three times its bulk of water, and the test reapplied, when very little doubt will remain as to the reaction. 3. Salicyl-Sulphonic Acid Test. — This is perhaps the most delicate of all the tests. EXPERIMENT III. Add to about 10 c.c. of urine a drop or two of a saturated solution of pure salicyl-sulphonic acid. A white precipitate results, which on boiling changes into a number of coagula. This reaction occurs in a dilution of 1-230,000 albumin. The only other body with which this reagent produces a precipitate is proteose, in which case, however, the precipitate disappears on warming. The reagent, if pure, keeps indefinitely. If impure, however, it turns red on keeping. It has the great advantage over nitric acid in being non-corrosive, and therefore easily carried about (MacWilliam). There are numerous other tests, but their application is superfluous if the above be properly applied. Proteoses are detected by the precipitates produced by nitric and salicyl-sulphonic acids clearing up on heating the urine, and returning when it is cooled. The so-called " proteose " in Bence Jones' proteosuria is coagulated by moderate heat, but redissolves on boiling the urine. Proteose can best be separated from albumin by adding salicyl- sulphonic acid, boiling and filtering. The coagulated albumin remains on the filter paper, and the pro- teose is gradually precipitated in the filtrate as it cools. Quantitative Estimation of Albumin. — For clinical purposes this is done by means of Esbach's albumino- meter (Fig. 255). The determination is made by mea- suring the depth of the coagulum produced by adding to the urine Esbach's reagent (a mixture of 10 grms. picric acid and 20 grms. citric acid in 1000 c.c. dis- tilled water). EXPERIMENT IV. Place clear urine, filtered if necessary, in an Esbach's tube up to the mark U. If the reaction be alkaline, render slightly acid by the addition of acetic acid ; and if the specific gravity be above 1008 dilute it with water till this density, or something below it, is obtained.1 Now add the reagent up to the mark R. Close the tube with a tightly-fitting cork and invert several times, so as to mix 1 These corrections should be made before the urine is measured into the Esbach's tube. 5 FIG. 255.— Esbach's albuminometer. 446 PRACTICAL PHYSIOLOGY the fluids thoroughly. Allow to stand in an upright position for twenty- four hours, and then read off the graduation corresponding to the top of the precipitate. This gives the number of grammes of dried albumin per litre of urine used. If the urine has been diluted the necessary calculation must be made in order to obtain the percentage in the original urine. For more accurate estimation of albumin, Scherer's method is employed. ADVANCED EXPERIMENT. Place 50-100 c.c. urine (according to amount of albumin in it) in a large-sized evaporating dish, and, while stirring, bring to the boil. Carefully add a few drops of dilute acetic acid, and allow the coagulum to settle down. If the supernatant fluid is opalescent, add a little more acetic acid, and bring again to the boil. (It is very important to use as little acetic acid as possible, so that acid albumin may not be formed.) The coagulum must then be collected on a small ash-free filter paper which has been dried between watch-glasses at 120° C. After being collected on the filter, wash the coagulum with boiling water, followed by alcohol and ether, and dry it at 120° C. until the weight is constant. Since the coagulum contains considerable ash, the filter and coagulum must now be transferred to a weighed crucible, incinerated, and the weight of ash deducted from the weight of dried coagulum. Mucus, Pus, and Casts in Urine. — When the kidneys or urinary passages are diseased, besides protein there may be a considerable deposit of mucus in the urine. This body has the general properties and solubilities of mucin (see p. 308), but may consist largely of nucleo- protein. Casts also occur in the deposit from the urine. When these come from the urinary passages, they consist of groups of flattened epithelial cells. When they come from the kidney tubules, they are tubular and consist of polyhedral cells, showing various stages of degeneration. When the kidneys or urinary passages are infected by micro-organisms, pus cells occur in the urine and form a deposit. Strong potash dissolves the pus, forming a viscid solution. Pus also gives a guiac test as for blood, but much more slowly and not after boiling, which destroys the oxidases to which the test is due. The only certain test for pus, however, is to examine the urine or deposit with the microscope. The pus cells appear as colourless, spherical, highly refractive granular bodies, about 9 /x in diameter, the nuclei of which can be stained by adding dilute methylene blue. The urine is usually acid when the pus comes from the kidney, and alkaline when the pus comes from the bladder, due to the decomposition of urea and ammonium carbonate. Haemoglobin in Urine. — This may be due to bleeding from the kidneys or urinary passages, when it is called haematuria, or to excre- tion of haemoglobin or methaemoglobin from the blood plasma by the kidneys, called respectively haemoglobinuria and methaemoglobinuria. PHYSIOLOGICAL CHEMISTEY 447 In any case the tests for haemoglobin can be applied. The guiac test, which is very sensitive, should be applied after boiling the urine to destroy oxidases. The spectroscopic examination is also very sensi- tive when an adequate depth of urine is employed (see p. 344). Haematuria is distinguished by the smoky appearance of the urine and by examination of the urine, or deposit on centrifugalising, when red blood corpuscles are seen. The spectroscope nearly always shows the presence of oxyhaemoglobin. Blood from the kidney is mixed with the urine. That from the bladder is often present as a clot. If the red corpuscles have disintegrated, the urine will present the appear- ance of haemoglobinuria. If the urine is stale, methaemoglobin may be present. In Haemoglobinuria and Methaemoglobinuria red blood corpuscles are not seen, and the urine is clear, not smoky. The two conditions are distinguished by the colour of the urine and by the spectroscope. EXPERIMENT V. Test the urine supplied for blood and haemoglobin. Bile in Urine. — When the bile duct is blocked by a calculus, or its mucous membrane is swollen from catarrh, the bile, which accumulates in the bile channels, is reabsorbed into the blood-vessels and carried to the tissues, which become stained with bile pigment. Under these con- ditions the urine contains bile constituents, the most easily recognised of which are the bile pigments. EXPERIMENT VI. Apply Gmelin's test (see p. 400) to the urine of a jaundiced patient. Where only a small quantity of bile pigment is present it is better to concentrate the pigment by proceeding as follows : — Add calcium chloride solution to the urine, and then sodium carbonate solution, so as to form a precipitate of calcium carbonate and phosphate, which carries down the pigment; filter off the precipitate atfd dissolve it in as small a volume of hot dilute hydrochloric acid as possible ; apply Gmelin's test to this solution. Also apply Matthew Hay's sulphur test for bile salts (see p. 398). II. Sugars in the Urine. — In the disease known as diabetes mellitus, the most important symptom is the presence of dextrose (or glucose) in the urine, or, in other words, glycosuria. This condition can also be produced experimentally: (1) By puncture of the flow of the fourth ventricle. The cause of the glycosuria in this condition is an excessive conver- sion of glycogen to glucose in the liver, whereby the percentage of dextrose in the blood rises above the normal, the excess being excreted by the kidneys. The glycosuria ceases when all the liver glycogen has been used up, and it cannot be produced by a similar experiment in animals which have been previously starved to remove the glycogen from the liver. 448 PRACTICAL PHYSIOLOGY (2) By extirpation of the Pancreas. — If the whole of the pancreas be removed in dogs, glycosuria is at once established, and the blood will be found to contain an excess of dextrose. So far, then, the cause of the glycosuria is the same as in the previous condition, viz. an excess of sugar in the blood. If, after the condition has existed several days, the liver be examined it will be found to be glycogen-free, but, unlike the previous condition, the glycosuria still continues, and in a few days it will be noticed that the animal has become markedly emaciated. The cause of the emaciation is that the protein tissues are undergoing dissolution. That such is actually the case is proved by a determination of the nitrogenous excretion, which will be found to be enormously increased in amount. In the course of a few weeks the animal dies of emaciation. These results show us that the pancreas must possess, besides its digestive function, some controlling influence on the metabolism of carbohydrates. (3) The administration of certain drugs, more especially of Phloridzin. — The administration of this drug is immediately followed by glycosuria, which, however, ceases after a few hours. If the liver be examined at this stage it will be found that a large proportion of its glycogen has disappeared. If a second dose be administered the glycosuria will reappear, and will persist so long as the drug is administered, and even after all glycogen has been used up. After some time, however, the animal becomes very emaciated, this being accompanied by an excessive excretion of nitrogen. The percentage of sugar in the blood is normal, or even sub-normal. On this account, it is supposed that phloridzin produces glycosuria by disturbing the controlling mechanism of the kidney, whereby the latter allows too much dextrose to escape into the urine, in consequence of which the percentage tends to become sub-normal in the blood. Increased demands are therefore made on the stored-up glycogen, which at last becomes used up, and then the supply has to be furnished by the proteins, and these break down. In both pancreatic and phloridzin diabetes, therefore, protein is an important source of the excess of dextrose. It has been conclusively shown now that it is from the amino acids, etc., that some of the dextrose is derived.1 The other sugars which the urine may contain are lactose and pentose. The former of these is sometimes found in the urine of nursing mothers, and the latter appears in the urine whenever pentoses (Wood Sugars, p. 294) are given in the food. 1 The conversion of fat into glucose is also possible. PHYSIOLOGICAL CHEMISTRY 449 Tests for Dextrose in the Urine. —The tests for dextrose*, as described, can, with slight modifications, be applied to its detection in urine. The most important of these are : — EXPERIMENT VII. Fehling's Test.— Boil 5 c.c. of Fehling's solution in order to ascertain that the Rochelle salt which it contains has not decomposed into reducing bodies. If no reduction occur, add a drop of the suspected urine and boil again. If no result, go on adding small quantities, boiling between each addition, till 5 c.c. have been added. Fehling's test is quite satisfactory, when sugar is present in considerable quantity. When the amount of reduction is small, however, it may be due to the presence of other substances than sugar. In such cases the following tests should be applied, as they are positive for sugars only. EXPERIMENT VIII. Boettger's Test.— To 10 c.c. urine add 1 c.c. Ny lander's reagent.1 Heat for five minutes on the water bath. If sugar is present to the extent of at least 0*08 per cent., a black precipitate of bismuth forms. EXPERIMENT.— Worm Miiller Test.2— In one test tube mix 1 c.c. copper solution and 2 '5 c.c. alkaline Rochelle salt solution. Place 2-5 c.c. urine in another test tube. Bring the contents of both test tubes simultaneously to the boil. After allowing to cool for 25-30 sec. pour the blue reagent into the urine and without shaking place the test tube on the rack. In the presence of a pathological amount of dextrose, a finely suspended yellow precipitate will develop within 10 minutes. If the solution should turn red but no precipitate develop the result is negative ; in such a case repeat the test, using 3 c.c. of copper solution. Though not so delicate, the following tests are valuable, in that they indicate the nature of the sugar : — 1. The Fermentation Test. — EXPERIMENT IX. Place some diabetic urine in a small beaker, and boil it on a sand bath for ten minutes, to expel any air it may contain. Cool the urine and test its reaction ; if alkaline, render faintly acid with a weak solution of tartaric acid. (This precaution is necessary in order to prevent putrefaction, which would lead to the evolution of ammonia.) Add a small piece (about 1 Nylander's solution : dissolve 4 grm. Rochelle salt in 100 grm. of a caustic soda solution of T12 sp. gr. ; add 2 grm. Bismuth subnitrate and heat on water batli until it is dissolved. 2 Worm Miiller solutions: 1, 2'5 % solution of copper sulphate; 2, Dissolve 10 grm. of Rochelle salt in 100 c.c. of a 4 % solution of caustic soda. 2F 450 PRACTICAL PHYSIOLOGY the size of a, split pea) of German yeast, and stir it in the urine until a milky solution is obtained. Now transfer the fluid to a Doremus ureometer (Fig. 244) so that the upright limb is completely filled with fluid. Place this in an incubator, or in a warm place, as on the mantelpiece, over night when it will be found that gas — carbon dioxide — has collected in the upper portion of the vertical limb. Two control tubes — one with a weak solution of dextrose and yeast, the other with normal urine and yeast — should be arranged so as to prevent any fallacy due to inactive or impure yeast. Instead of using a Doremus ureometer a test tube inverted in a trough of mercury may be employed. Lactose and pentose do not give a positive result by this test. 2. The Phenyl Hydrazine Test. — The method of employing this is described on p. 275. The obtaining of characteristic dextrosozone crystals is positive evidence of the presence of dextrose; glycuronic acid (p. 454) also gives crystals, but less readily. Estimation of Dextrose in Urine. — The polarimeter (see p. 282) may be employed for the estimation of dextrose in urine. The main objection to its use is that other optically active bodies besides dextrose, e.g. glycuronic acid and oxybutyric acid, which are laevo- rotatory, occur in diabetic urine, and therefore to a certain extent vitiate the result. The other method is to determine the reducing power of the urine. For clinical purposes the methods of Fehling, Gerrard, and Pavy are employed. In Fehling's method the amount of urine necessary to decolourise a measured quantity of standard Fehling's solution is determined. It is, however, difficult to observe when the blue colour of the Fehling's solution has disappeared owing to the precipitation of red cuprous oxide. To obviate this difficulty Gerrard prevents the precipitation of cuprous oxide by the addition of potassium cyanide. The solution when reduced has a light yellow tint, so that the end-point is a little troublesome, but for the use of students this is probably the best method. In Pavy's method ammonia is added to the Fehling's solution. There is then no precipitation of cuprous oxide until the ammonia has been boiled off. The reaction liquid is perfectly colourless when reduced, so that the end-point is a good one, but the ammoniacal solution absorbs oxygen from the air, so that the liquid has to be kept boiling in a flask with a small opening. Moreover, owing to the escape of ammonia there is only a limited time in which to perform the titration before cuprous oxide begins to be precipitated. This method, therefore, although very rapid, requires considerable practice. Fehling's Method. — The standard solution contains 34 '64 grm. pure PHYSIOLOGICAL CHEMISTRY 451 crystallised copper sulphate, 180 grm. Rochelle salt and 70 grm. caustic soda per litre. 10 c.c. of this solution are equivalent to 0'05 grm. dextrose. The urine is diluted exactly 10 or 20 times according to the amount of sugar present and placed in a burette. 10 c.c. of the standard solution are measured with a pipette into a porcelain basin, diluted with 40 to 50 c.c. water and heated to boiling. The solution is kept just boiling, and the diluted urine run in carefully with stirring, until the blue colour of the solution has just disappeared. From the volume of diluted urine required in the titration the amount of dextrose in grammes present in 100 c.c. of the original urine is calculated. Several determinations must be made. A flask heated on a water bath may be substituted for the basin in order to minimise the risk of oxidation of cuprous oxide. Gerrard's Method. — The cyanide solution is prepared by adding cautiously an approximately 5 per cent, solution of potassium cyanide to 100 c.c. Fehling's solution diluted with 300 c.c. water, which is kept boiling, until the blue colour of the solution has just disappeared. The resulting liquid is cooled, diluted to exactly 500 c.c. and kept in a stoppered bottle. For the titration 50 c.c. of the cyanide solution and 10 c.c. of standard Fehling's solution are measured into a basin or flask, and the diluted urine run in from a burette in exactly the same manner as in Fehling's method. The urine required in the titration contains 0*05 grm. dextrose. Pavy's Method. — The standard solution contains 120 c.c. Fehling's solution and 300 c.c. strong ammonia per litre. 10 c.c. of this solution are equivalent to 0*005 grm. dextrose. The nozzle of a burette is fitted to a small flask by means of a cork, through which is also passed a short bent tube to allow of the escape of steam and ammonia, when the flask is boiled. The urine is diluted exactly 10 to 50 times according to the amount of sugar present. The burette is filled with this diluted urine, care being taken that there are no bubbles in the nozzle. 10 c.c. Pavy's solution and about an equal volume of water are placed in the flask. The flask is now heated till it boils. The heating is continued and the urine allowed to drop in from the burette at such a rate that ebullition does not cease. When the colour in the flask is perceptibly less, the rate of addition of drops is much reduced, but is continued until all blue colour has disappeared. The first reading will be almost certainly too great, so that other determinations must be made. In the later deter- minations it is a good plan to run in fairly rapidly a quantity of urine, which will be almost but not quite sufficient, to wait till the colour is constant and en very carefully and slowly to add drops from the burette till the blue colour has quite disappeared. The amount of diluted urine employed should not be less than 2 c.c. or more than 5 c.c. 452 PRACTICAL PHYSIOLOGY Normal human urine has an average reducing power equivalent to about 0-2 per cent, dextrose. Of this reducing power 18 per cent, is due to dextrose, 8 per cent, to uric acid (see p. 425), and 25 per cent, to creatinin (see p. 429), the remaining 50 per cent, being probably due to urochrome. Furthermore, the colour of urine renders the end-point of the titration much more uncertain than when a watery solution of dextrose is employed When great accuracy is required, in order to remove all the urochrome as well as a considerable proportion (75 per cent.) of the uric acid and creatinin, Bang and Bohmannsson use blood charcoal and hydrochloric acid. We have obtained more constant results, however, by using acetone1 and blood charcoal according to the following method : — 20 c.c. urine are mixed in a flask with 5 c.c. acetone and 2 gr. charcoal from blood (a teaspoonful). The flask is then shaken occasionally during five minutes, after which its contents are filtered through a dry filter paper into a test tube. Of the filtrate (which is always perfectly colourless) 5 c.c.2 are used for Bang's titration, as described on page 293. Sometimes the urine contains pentose (i-arabinose). In such cases it reduces but does not ferment with yeast ; it gives Bial's test (p. 295). The presence of laevulose is revealed by Seliwanoff's test (p. 278). The Acetone Bodies in Urine. — These substances are: — (1) /3-oxybutyric acid, CH3 . CHOH, . CH2 . COOH. (2) Aceto-acetic acid, CH3. CO.CH2. COOH. (3) Acetone, CH3 . CO. CH3 . Aceto-acetic acid is an oxidation product of /3-oxybutyric acid. Acetone is formed from aceto-acetic acid by the loss of carbon dioxide. A solution of aceto-acetic acid partially decomposes to acetone at ordinary temperatures. On boiling the decomposition becomes complete. Acetone is present in minute traces in normal urine. All three bodies make their appearance in human urine when fat is being metabolised at an unusually rapid rate. They are present therefore in the urine of severe cases of diabetes, in the urine of starvation, and in the urine of many people when the carbohydrate of the diet 1 The acetone must be chemically pure, otherwise it may contain reducing substance. 2 This amount of the filtrate is for a urine containing not more than 2 per cent, dextrose. If it contain less than 0*5 per cent, dextrose 10 c.c. of the filtrate should be taken. If it contain more than 2 per cent. 2-3 c.c. When, however, more than 2 or 3 per cent, dextrose is present the polariscopic method, or Bang's titration, without the addition of charcoal, will probably give close enough results for most purposes. PHYSIOLOGICAL CHEMISTKY 453 is reduced below 70 grm. per diem. Under these conditions the amount of the acetone bodies is increased by exercise. EXPERIMENT X. Tests for Acetone. Legal's Test.— Add to the urine in a test tube a few drops of a fresh solution of sodium nitroprusside and then caustic soda solution till definitely alkaline. A permanent red colour develops, which becomes deeper and assumes a purplish tint on acidifying with strong acetic acid. (Compare with test for creatinin). Bothera's Test. — Add a few drops of sodium nitroprusside solution, ammonia till alkaline, and saturate the liquid with ammonium sulphate crystals. A deep colour similar to that of permanganate develops and reaches its maximum in 15 minutes. This test is more sensitive and distinctive than Legal's. lodoform Test. — Distil a few c.c. of the urine with a few drops of dilute sulphuric acid. To the distillate add a few drops of iodine in potassium iodide solution and caustic soda till the iodine colour disappears. lodoform is precipitated, and is detected by the charac- teristic smell. EXPERIMENT XL Test for Aceto-acetic Acid.— To the urine add ferric chloride solution in excess of that required to precipitate the phosphate present. A deep red colour in the solution indicates the presence of aceto-acetic acid. (Salicylic acid in the urine gives a very similar colour.) There is no simple test for oxybutyric acid- The best procedure is based on Schaffer's method for estimating oxybutyric acid, in which this substance is oxidised to acetone. 50 to 100 c. c. urine which contains acetone is diluted with twice its volume of water, treated with basic lead acetate and ammonia (to remove possible glycuronic acid) and filtered. The filtrate is acidified with 10 c.c. sulphuric acid (cone.) and boiled for half an hour, with the addition of water to keep the volume constant ; this removes the acetone present. It is then distilled (potassium bichromate 0*5 per cent, solution being added from a dropping funnel so as to keep the volume constant) as long as reduction occurs as shown by the colour. To the distillate are added a few c.c. of hydrogen peroxide (free from alcohol) and caustic soda till alkaline. It is then redistilled, and the second distillate tested for acetone by Rothera's test and by the iodoform test. /OH Homogentisic Acid is di-oxyphenyl-acetic acid C6H3^-OH CH2COOH. It reduces Fehling's solution. When present in the urine it causes the latter to become of a dark-brown colour on standing, or this change in colour may be hastened by adding some alkali. It is present in the urine throughout life, and it has been noticed that persons who exhibit it are almost invariably the children of first cousins. It can be easily 454 PRACTICAL PHYSIOLOGY separated from the urine by adding a solution of lead acetate, filtering off the precipitate of inorganic salts which at first forms and allowing the filtrate to stand, when large needle-shaped glancing crystals of the lead salt separate out. If these be collected and treated with sulphuretted hydrogen, so as to remove the lead, the acid is obtained in a pure state. Glycuronic Acid. — Chemically this is dextrose in which the end — CH2OH — group has become oxidised to form COOH, or carboxyl. It has, accordingly, the formula COOH— (CH.OH)4— CHO. It is an intermediate body in the metabolism of dextrose, and usually becomes further decomposed in the organism, to yield carbon dioxide and water. Sometimes, however, it unites with the aromatic bodies (plenol, skatol, etc.) absorbed from the intestine to form a salt. In this combination it takes the place of sulphuric acid (see p. 435). In very small amount, it seems to be always present in the urine, but under certain conditions (as after the administration of certain drugs) it becomes increased to such an extent as to impart to the urine a very considerable power of reducing metallic oxides in alkaline solution. When this is the case it is apt to be confused with dextrose. The only absolute test whereby it may be distinguished from dextrose is that it does not ferment with yeast. It gives the pentose reactions. CHAPTER XXI. LACTIC ACID, ITS ESTIMATION AND SIGNIFICANCE. By J. H. RYFFEL, M.A., B.Sc., Demonstrator of Physiology, Guy's Hospital. THE lactic acid of the body, called sarcolactic acid, is the dextrorotatory variety, the free acid rotating the plane of polarisation of polarised light to the right, while its metallic salts rotate to the left. The specific rotatory power of the zinc salt [a]B (see p. 354) is - 7'5° to - 6 5° according to the strength of the solution. The zinc salt contains two molecules of water of crystallisation, whilst that of the inactive or fermentation lactic acid contains three molecules. Identification and Estimation of Lactic Acid. Ether Extraction Method, — If the material is blood or tissue, it is extracted with alcohol or with boiling water; if urine, it is concentrated and extracted with alcohol. PHYSIOLOGICAL CHEMISTRY 455 The extract is evaporated to small bulk, acidified with phosphoric acid, and extracted repeatedly by shaking in a funnel with many times its volume of ether. The residue after the evaporation of the ether extract is dissolved in water, boiled with zinc carbonate, filtered and evaporated. Crystals of the zinc lactate are thus obtained, which are weighed either after drying in air, when they contain their water of crystallisation, or after complete drying at 110° C. The product is liable to be impure. This is partly obviated by washing the crystals with alcohol. Frequently the aqueous solution obtained after ether extraction is treated with lead carbonate, filtered, treated with hydrogen sulphide to remove lead, and again extracted, but this procedure involves loss. In any case the percentage of zinc in the zinc salt must be determined in order to prove its identity as zinc lactate. The method, though cumbrous, is sufficiently satisfactory when relatively large amounts of lactic acid are present, but, when the amount of lactic acid is small, the zinc lactate is almost certain to be impure, especially if the determination is made on uriiie. Moreover, in the extraction of lactic acid from a watery solution by means of ether there is always some loss, due to incomplete extraction and to oxidation of lactic acid by impurities in the ether. When the amount of lactic acid is small, this loss is relatively very considerable. The Thiophene Test for lactic acid introduced by Hopkins has already been described (see p. 354). It is very sensitive and is distinctive for a hydroxy acids, but cannot be used quantitatively and is difficult to apply to urine. The Distillation Method.— This method depends on the fact that lactic acid, when heated above 140° C. with sulphuric acid, yields acetaldehyde quantitatively according to the following equation : CH3 . CHOH . COOH = CH3 . CHO + H . COOH. 40 c.c. of the liquid, which must be free from sugar and nearly free from protein, are placed in a 500 c.c. Jena distillation flask. 45 c.c. pure sulphuric acid are rapidly added from a dropping funnel, the flask being shaken and cooled under the tap. The flask is then fitted with a rubber cork carrying an inlet tube for steam and a thermometer, so arranged that both dip well below the surface of the liquid. It is then placed in a slanting position on wire gauze on a retort stand and attached to a good vertical condenser. (For this purpose the exit tube of the flask must be bent at a suitable angle.) A flask of about 300 c.c. capacity, immersed in cold water, is placed as the receiver of the condenser with its mouth just touching the jacket of the condenser, so as to prevent loss of aldehyde by evaporation. A gentle current of steam from an ordinary steam generator is then passed into the distillation flask, which is vigorously heated with a Bunsen burner. Distillation will generally begin at about 140° C., but the heating is continued till the temperature reaches 155° C., when the current of steam is increased, and the heat applied to the flask adjusted so that the temperature is kept between 153° and 157° C. When about 100 c.c. have collected in the receiver, or the distillation has lasted nearly 30 minutes, the decomposition is complete. The contents of the receiver are rendered just permanently alkaline by the addition of 2 per cent, caustic soda solution and a little litmus solution, diluted to about 150 c.c., and redistilled into a flask with a 100 c.c. mark in the neck, using the same precautions to prevent loss as before, until about 50 c.c. have 456 PRACTICAL PHYSIOLOGY been collected. (When the amount of lactic acid is excessively small, as is the case in normal urine, a 50 c.c. flask may be employed, the quantities given in what follows being halved.) To the second distillate are added 0'5 c.c. Schiff's reagent (see later) and water to bring the volume to 100 c.c. The flask is stoppered, inverted a ftw times to mix its contents, placed in a glass vessel containing water at 15° C., and left for 30 minutes in diffuse daylight. The Schiff's reagent reacts with the aldehyde present, giving a red colour, which reaches a maximum in 30 minutes and then slowly fades. This reaction may be used qualitatively as a test for lactic acid. For quantitative estimation the coloured liquid (a) is transferred at the end of 30 minutes to one tube of a colorimeter. A convenient depth of liquid is selected. The two formaldehyde standards (see later) are selected which are nearest to a in colour, and the depth of each determined which gives the same intensity of colour as the selected depth of a. The calculation is best described by an example. Formaldehyde 4 c.c. a Formaldehyde 3 c.c. Readings of equal depth of colour, 2*42 cm. 2 cm. 1*48 cm. 10 -r readings, 4 '13 5 6 "85 £ A ,| O Then a is equivalent to 3 c.c. +^ ' O'oO — 4*lo = 3 '32 c.c. standard formaldehyde solution. The amount of lactic acid in the liquid originally employed = 3-32x3-435xw -01 --- mg' whero n is the standard value of the formaldehyde. If the colour of a is much greater than that of any of the standards, another determination must be made, using a more dilute solution of lactic acid. The Formaldehyde Standards. — A series of four stoppered flasks is prepared containing 0'5 c.c. Schiff's reagent and 1'5 c.c., 2 c.c., 3 c.c., 5 c.c. respectively of dilute standard formaldehyde solution, made up to 100 c.c. with water. These are placed in a dark cupboard till required. The colour develops very slowly, and is fairly permanent, so that the standards may be used any time within three days after the first twelve hours. The dilute standard Formaldehyde Solution.— 10 c.c. commercial formalin (40 per cent, formaldehyde) are diluted to 100 c.c. This solution will keep practically indefinitely. To make the dilute standard solution 5 c.c. of this solution are diluted to 500 c.c. This dilute solution will keep practically unaltered for a week if well stoppered. It is standardised, unless made from an already standardised formaldehyde solution, by the following method : 40 c.c. are measured into a stoppered bottle, 25 c.c. j^ iodine solution are added, and then 10 per cent, caustic soda, till the liquid assumes a light yellow colour. The mixture after standing for 10 minutes is acidified with dilute hydrochloric N acid and titrated with j^ sodium thiosulphate solution, until the colour of the iodine just disappears. The volume in c.c. of thiosulphate solution required is subtracted from 25 c.c. Let the remainder = 6 c.c. Then the formaldehyde in 1 -49 x b mg. present in 1 c.c. of the solution = n = — — -- The value of n should be nearly 0'4 mg. PHYSIOLOGICAL CHEMISTRY 457 Schiff's Reagent. — 1 grm. finely powdered rosaniline hydrochloride and 100 c.c. water are placed in a small bottle with a closely fitting stopper. Sulphur dioxide is passed in from a syphon, till the dye just dissolves to a yellow solution, when the liquid is very nearly saturated with the gas. The reagent loses sulphur dioxide rather readily, so that it must be kept closely stoppered, and must be resaturated occasionally with sulphur dioxide. The formaldehyde standard with 0*5 c.c. of the reagent and 5 c.c. dilute formaldehyde solution (2 mg. formaldehyde) made up to 100 c.c. with water should be of such a depth of colour, that by the colorimeter 1'3 to 1'7 cm. is equivalent in colour to 0*7 cm. yrrf: potassium permanganate. J.UU The method can be applied to urine either directly, or after rendering alkaline with sodium carbonate and evaporating on the water bath, but not more than 40 c.c. of urine of specific gravity 1020 should be employed for one distillation in either case, as with more urine frothing is liable to occur. Glycuronic acid forms a source of error, but may be removed by means of basic lead acetate. For this purpose 25 to 200 c.c. urine are measured into a 500 c.c. graduated flask. Slight excess of basic lead acetate solution, 10 c.c. strong ammonia and water to make 500 c.c. are added. The contents of the flask are well mixed, allowed to stand for a short time and filtered through a dry filter into a dry flask. A measured volume of the filtrate (350 c.c. or less) is evaporated in a dish on the water bath, sodium carbonate solution being added to keep the liquid alkaline. The residue in the dish is then washed into the distillation flask with 40 c.c. water and 45 c.c. sulphuric acid and treated as above. This treatment causes a small loss of lactic acid, so that only about 50 per cent, of minute quantities of lactic acid added to urine are recovered. When the quantity of lactic acid is consider- able, however, the loss is negligible. In order to apply the method to blood the following preliminary procedure is necessary. The blood, of which 20 c.c. is usually quite sufficient, is diluted about five times, heated to boiling in order to coagulate the proteins, and filtered. The coagulum is very thoroughly washed with boiling, faintly acidulated water. The total liquid thus obtained is rendered alkaline with sodium carbonate, evaporated and employed for the determination. Lactic acid has been found in all tissues, but the amount present depends on the condition of the tissue and on the method employed for killing it. Muscle, for instance, forms lactic acid during the onset of rigor. Lactic acid appears to be a normal constituent of blood, but its amount is variable. In the venous blood of man at rest its amount varies between 10 mg. and 20 mg. per 100 c.c., but the arterial blood of animals, particularly rabbits, shows higher values up to 100 mg. per 100 c.c. By the ether extraction method lactic acid has never been found in normal urine, but this does not preclude its presence in small quantity. By the distillation method, however, lactic acid is always present in urine, amounting in man on an ordinary diet to about 4 mg. per hour during the day and 2 mg. at night when determined directly, or to about half this quantity when determined after lead acetate treatment. 458 PRACTICAL PHYSIOLOGY Lactic acid is, therefore, present in relatively greater amount in the blood than in the urine under ordinary conditions, being to a consider- able extent kept back by the kidneys. When, however, lactic acid in the blood rises above a limiting value, which in man is less than 70 mg. per 100 c.c , it is actively excreted by the kidneys, so that the urine may often contain 500 mg. per 100 c.c. even when fairly dilute. This result is in man easily brought about by violent exercise such as running. EXPERIMENT I. Collect the urine of a man at rest, or engaged in quiet work, during half an hour, and determine the lactic acid in the urine by the distillation method. Then let him run for at least two minutes at a pace sufficient to produce marked dyspnoea. Collect and measure the urine after half an hour. Determine the lactic acid in 20 c.c. of this urine. A qualitative determination is sufficient, as the reaction obtained with the urine passed at rest is very small. The urine passed at rest will not give the thiophene test. That passed after the exercise will give the test after the following preliminary treatment. The urine is rendered alkaline with sodium carbonate, evaporated and extracted with alcohol. The alcohol is evaporated and the residue dis- solved in a little water, strongly acidified with phosphoric acid and extracted with many times its volume of washed ether in a separating funnel. The ether is separated and shaken up with dilute sodium car- bonate solution, again separated and used for extracting the acid liquid a second time. This process is repeated two or three times. The alkaline liquid so obtained is boiled with animal charcoal till colourless, filtered, and evaporated to dryness. The residue is then dissolved in 5 to 10 c.c. pure sulphuric acid and the thiophene test applied. In spite of boiling with charcoal a reddish brown colour is usually obtained on adding the sulphuric acid, which masks the reaction to a certain extent. Violent exercise also increases the acidity of the urine (see p. 431) and the proportion of ammonia nitrogen to the total nitrogen. The amount of lactic acid in the blood is increased, and the alkalinity of the blood reduced at the end of a short period of violent exercise, but both have returned practically to normal at the end of half an hour. When more moderate exercise is taken, such as walking, there is no increase in the lactic acid of the urine, even if the exercise be continued for many hours, so that the production of lactic acid in exercise clearly depends on the intensity of the muscular activity. There is no •evidence that the blood is less well supplied with oxygen than usual during a short period of violent exercise, so that the muscles are presumably not short of oxygen. The production of an increased PHYSIOLOGICAL CHEMISTRY 459 quantity of lactic by the muscles, which thus passes into the blood, is, therefore, a direct result of great muscular activity. This increased production of lactic acid serves a very useful purpose in making the blood more acid, and so stimulating the respiratory centre, and probably the heart also. The increase of pulse rate, blood pressure and respiration produced by violent exercise are matters of common observation. Although the output of carbon dioxide is much increased, the alveolar carbon dioxide (see p. 197) is not high, except for a short time after the start, and falls markedly below the normal level for about half an hour after the period of violent exercise, so that the increased respiration cannot be due to carbon dioxide alone. If, how- ever, we suppose that the activity of the respiratory centre is always regulated by the acidity of the blood, which depends on the amounts of carbonic and other acids present, we see how lactic acid may take the place of carbon dioxide in increasing the activity of the respiratory centre. Experimentally in animals the urinary output of lactic acid may be greatly increased by reducing the supply of oxygen, or by carbon monoxide poisoning. The experiment has to be continued for several hours with the animal in the collapse stage of asphyxia. In man increased lactic acid excretion has occurred from long-continued acci- dental carbon monoxide poisoning, but not as yet from voluntary shortage of oxygen. Shortage of oxygen is not, therefore, as efficient a cause of increased production of lactic acid as muscular activity, although respiration is increased and the alveolar carbon dioxide much reduced even in short experiments on man. It may be that in this case the respiratory centre is the first to feel the lack of oxygen, and is stimulated by lactic acid, which it itself produces, instead of by the acidity of the general blood stream. The results of the experiments of Fletcher and Hopkins on surviving frogs' muscles are in general agreement with the above results from man. These observers showed that the muscles of frogs at rest contain very little lactic acid, provided the muscles are killed with a minimum amount of stimulation. For this purpose they cooled the hind limbs with ice, then rapidly separated the muscles and ground them up with ice-cold alcohol and sand. The lactic acid was isolated as anhydrous zinc lactate after ether extraction. Resting fresh muscle gave 0*03 to 0*045 per cent, zinc lactate. When the muscle was tetanised the lactic acid rose, until when irritability was lost by fatigue the yield of zinc lactate was about 0-2 per cent. If the muscle was then kept in oxygen, the irritability returned and the zinc lactate fell to about 0*1 per cent. Oxygen inhibited, but did not entirely prevent, the onset of 460 PKACTICAL PHYSIOLOGY fatigue and production of lactic acid, and enabled the muscle at rest to remove preformed lactic acid. Irritation or injury of the muscle in any way increased the lactic acid in it. The production of heat rigor in the muscle gave the maximal yield of lactic acid, from 0'4 to 0*5 per cent, zinc lactate according to the time of year. This yield of lactic acid was independent of the previous manipulation, tetanisation, etc., to which the muscle had been subjected. CHAPTER XXII. HAEMOLYSIS AND PRECIPITINS. A RED blood corpuscle or erythrocyte is usually regarded as consisting of an envelope enclosing haemoglobin and salts of various inorganic bases, of which potassium is predominant in some animals, sodium in others. The interior of the corpuscle is also believed by some to contain a meshwork connected with the envelope and of similar structure. The envelope and meshwork are composed chemically of lipoid substance and protein, and behave physically as a semi- permeable membrane, readily allowing small molecules (such as those of water) to diffuse through but not so readily larger ones, such as those of many inorganic Within the envelope the haemoglobin cannot be present in ordinary solution for its concentration is greater than that of a saturated solution of haemoglobin in water or saline solution. The red corpuscle is developed from a cell, but in its metamorphosis most of the cellular properties become lost, the greater part of the protein constituents of the cell changing into haemoglobin. In the following experiments some evidence will be obtained to show:— 1. That the envelope is semi-permeable; 2. That it possesses certain qualities common to it and other cells ; 3. That lipoid substance is an important con- stituent of the envelope. It is comparatively easy to study alterations in the permeability of the corpuscular envelope, because, when haemoglobin leaves the corpuscle and passes into the fluid surrounding the corpuscle, this fluid becomes tinged with red : laking or haemolysis is said to have occurred. EXPERIMENTS DEPENDING ON THE FACT THAT THE CORPUSCULAR ENVELOPE is SEMI-PERMEABLE IN NATURE. EXPERIMENT I. Examine some blood under the microscope (frog's blood is very suitable because of the large size of the corpuscles). Allow some water to run under the cover slip, and at the interface between blood and water note the rapid swelling of the corpuscles followed by their rupture. To another preparation add a 2 per cent, solution of sodium chloride and note that the corpuscles shrink and become irregular in shape. The explanation PHYSIOLOGICAL CHEMISTRY 461 of these results is that the envelope readily allows water to pass through it but not inorganic salts. The water tends to pass into the corpuscle in the first part of the experiment because the osmotic pressure inside the corpuscle is higher than that outside. To equalise this difference of osmotic pressure water passes in but salts cannot pass out because the membrane is impermeable towards them. The shrinkage of the corpuscle in the second part of the experi- ment bears out this explanation. EXPERIMENT II. Determine, ichat strength of NaCl solution just prevents haemolysis. Into each of a series of test tubes place 20 c.c. of sodium chloride solutions of gradually increasing concentration, e.g. 0*5 per cent., 0-55 per cent, 0'60 per cent., 0'65 per cent., 070 per cent.1 To each tube add five drops of fresh defibrinated (ox or dog) blood ; mix by inverting the tube and allow to stand for some minutes. It will be noted that the super- natant fluid in the case of the stronger solutions is colourless, but that in the tube with 0'55 per cent. NaCl it is slightly tinted red, indicating that the corpuscular envelope has ruptured and the haemoglobin has escaped. The saline solution which just prevents haemolysis stands somewhere between 0'55 and 0'6 per cent, in strength. By estimating the osmotic pressure of blood serum and of the above saline solutions, either by means of the depression of freezing point method or by the microscope or haematocrit (see Exp. IV.), it will be found, however, that a 0'55 per cent, saline solution has a much lower osmotic pressure than that of blood serum (which equals a 0'9 per cent. NaCl solution). The results of the above experiment therefore show us that the corpuscular envelope can withstand a certain amount of hypotonicity before it ruptures. If the experiment be repeated with other salts than sodium chloride it will be found that the strength of solution which just fails to show haemolysis bears a close relationship to the molecular weight of the salt used, i.e. the corpuscular envelope gives way at corresponding osmotic pressures. There are certain salts, however, for which this is not true; the most important of these are the ammonium salts and organic substances containing an ammonium residue, e.g. urea, others are sodium carbonate, glycerine, etc. EXPERIMENT III. Mix 5 drops of ox blood with 20 c.c. of a 0'7 per cent, solution of ammonium chloride. Laking will occur although this strength of solution has a higher osmotic pressure than a 0'55 per cent, solution of sodium chloride. We can determine the osmotic pressure of a saline solution indirectly by observing what effect it has on the volume of red blood corpuscles. When no change in volume occurs with a given solution this must be isotonic with the blood serum ; if it causes swelling it is hypotonic, if shrinkage, hypertonic. Besides employing the microscope to detect these changes in volume, we may •employ an instrument called a haematocrit. EXPERIMENT IV. The haematocrit consists of two capillary glass tubes, graduated in one hundred equal parts ; the upper ends are widened so as to 1 These solutions of NaCl are best prepared in the following manner : Fill one burette with a 1 per cent. NaCl solution and another with distilled water. Into test tube 1 place 5 c.c. of the NaCl solution and 5 c.c. of water ( = '5 per cent.); into test tube 2 place 5'5 c.c. NaCl solution and 4 '5 c.c. water < = 0'55 per cent.), and so on for the other tubes. 462 PEACTICAL PHYSIOLOGY make a mixing chamber. By attaching a piece of indiarubber tubing to the tube, blood is sucked up to the mark 100, clotting being retarded by first of all drawing some cedar oil through the tubes. The tubes are then placed in a holder, the ends being closed by small metal plates held in position by a spring, and rotated on a centrifuge until the corpuscles have been thrown down to a certain mark, which is then noted. After cleaning, blood from the same source is again sucked up to the mark 100 and then an equal volume of some solution. The two are thoroughly mixed in the mixing chamber by means of a fine wire and the instrument again rotated. If the solution is isotonic with the blood serum the corpuscles will stand at the same level as before, if it be hypotonic they will stand at a higher level, if hypertonic, at a lower. It will be seen that by this method we can readily ascertain, in a series of solutions of unknown osmotic pressures, whether one of them is isotonic with the blood serum. The haemoglobin may also be caused to leave the corpuscle by bringing about an alteration in the permeability of the envelope. Such an alteration may be brought about in a variety of ways, some of which may be styled physical, such as heating, freezing or shaking the blood ; others, as purely chemical, such as the addition of ether and other fat solvents, saponin, bile salts, acids and alkalies ; and others as bio-chemical, such as the addition of immune serum. In the present state of our knowledge it is impossible to say in every case definitely what the alteration in permeability is due to, but some very important facts are known. Physical Laking. EXPERIMENT V. 1. Place some blood in a test tube and keep at 60° C. for a few minutes. The blood becomes laked. 2. Repeatedly freeze and thaw some blood and then dilute with some isotonic saline. Note the evidence of laking. Chemical Laking. 3. Place some blood in four test tubes ; to one add some ether, to another some 3 per cent, saponin solution, to a third a solution of sodium taurocholate in isotonic saline, and to the fourth some acetic acid. Laking occurs in all cases. 4. To another test tube containing 1 c.c. of blood add 3 c.c. of a 2 per cent, solution of urea. Laking soon occurs. Since this strength of urea solution has practically the same osmotic pressure as '9 per cent. NaCl, it is of further interest to see whether its laking influence is due to its having, like the above-mentioned chemicals, a damaging effect on the corpuscular envelope. This question can be settled by repeating the above experiment with a solution of 0'9 per cent. NaCl containing 2 per cent, of urea. It will be found that no laking occurs thus showing that, unlike bile salts and saponin, the urea does not damage the envelope. The reason why laking occurs with the pure urea solution must therefore be that the urea molecule penetrates the corpus- cular envelope very readily, perhaps as readily as water itself does. A considerable amount of work has been done on saponin laking, the most important outcome of which has been to show that the haemolytic effect of this drug can be antidoted by something (probably lecithin or cholesterol) contained in normal blood serum. To demonstrate this effect it is of course PHYSIOLOGICAL CHEMISTRY 463 first of all necessary to take blood corpuscles that have been washed free of adherent serum.1 5. Determine the minimum amount of a 0*3 per cent, saponin solution necessary to produce laking of icashed dog's corpuscles in five minutes at room temperature. Label four test tubes A, B, C, D, and place in each 2 c.c. of 10 per cent, dilution of dog's washed blood corpuscles in 0'9 per cent. NaCl solution. Of a 0'3 per cent, solution of saponin in 0'9 per cent. NaCl solution, add to A 0*05 c.c. ; to B O'l c.c. ; to C 0-15 c.c. ; to D 0'2 c.c. Immediately fill each test tube with 0'9 NaCl solution and without shaking allow to stand for five minutes. Then note the tube which just shows complete laking. Repeat the observation taking amounts of saponin in l/100th of a c.c. between the amounts in the previous trial, which did and which did not show laking (i.e. suppose trial gave laking with 0*15 c.c. but not with O'lO, then in this experiment use 0.11 c.c. saponin in A ; 0'12 c.c. in Bt and so on). 6. Demonstrate the antidotal effect of blood serum on saponin laking. Place 2 c.c. of a 10 per cent, suspension of dog's washed blood corpuscles in four marked test tubes: add to each 2 c.c. of dog's blood serum and mix. Then add minimal laking amount of saponin solution (determined as in Experiment 5) to A and to the others amounts increasing by 0'05 c.c. Fill up the test tubes with 0-9 per cent, sodium chloride solution and, after standing for five minutes, see in which tube laking has occurred. The antidotal effect of the blood serum will be clearly shown. There are some very peculiar differences between the action of laking agents. Thus saponin evidently acts on some constituent of the envelope which is closely related to cholesterol (or lecithin), for if a saponin solution be shaken with cholesterol its haemolysing effect will become greatly reduced. In its action on the corpuscular envelope the saponin either dissolves the cholesterol or forms a compound with it which is soluble in the surrounding fluid, and in this way makes the envelope so permeable that the haemoglobin escapes. It is of interest, in this connection, to note that if the mixture of blood and saponin be examined under the microscope, the corpuscle will be seen to swell somewhat before haemolysis occurs, showing that its permeability towards water is first of all lowered. The electrical conductivity increases somewhat during laking by a minimal dose of saponin, probably due to the escape of haemoglobin, but if at this stage more saponin be added, the electrical conductivity still further increases, showing probably that some compound of strorna and inorganic salts has been broken down (Stewart). With bile salts, on the other hand, there is no preliminary swelling of the corpuscles ; their haemoglobin contents are set free immediately. Ether does not merely act by dissolving out lipoid, for ether saturated with cholesterol or with the lipoid substances of corpuscles themselves still causes laking. Bio-chemical Laking, Perhaps the most interesting haemolytic bodies are those which become xTo wash blood corpuscles free of adherent serum a good centrifuge is necessary. The defibrinated blood is first of all centrifuged and the serum removed with a pipette. The centrifuge tube is then filled up with 0'9 per cent, sodium chloride solution and, after shaking, placed in the centrifuge. This process is repeated at least three times and, for more particular work, even more. 464 PKACTICAL PHYSIOLOGY developed in the tissue fluids of an animal when the defibrinated blood of an animal of another species is repeatedly inoculated into it. These haemolysins belong to the general group of anti-bodies in which are included the antitoxins, precipitins, agglutinins, bacteriolysins, etc. Haemolysius also sometimes occur as normal constituents of tissue fluids and secretions; for example, they are present in snake venom. A particular study of these anti-bodies is more properly a bacteriological than a bio-chemical one, but a study of a few of their essential properties must also be included in the course in bio-chemistry, since many ferments seem to act according to the same laws as those which govern the action of haemolysins. Haemolysins. — The washed erythrocytes of the rabbit are laked when mixed with the blood serum of the dog or ox. This haemolytic effect of dog's blood serum becomes much more marked, however, if the dog be previously inoculated with defibrinated rabbit's blood.1 Besides causing haemolysis, the serum usually causes the corpuscles to run together into clumps. This process is called agglutination, and when it occurs in blood the corpuscles settle down and can be filtered off. EXPERIMENT VI. (1) Examine microscopically (under cover slip) the effect of dog's serum on rabbit's erythrocytes. (Agglutination, then haemolysis.) (2) With a 5 per cent, suspension of washed rabbit's erythrocytes in physiological saline perform the following experiments, ascertaining the degree of haemolysis by allowing the corpuscles to settle, and noting the intensity of colour of the supernatant fluid. Mote also any agglutination. In small short test tubes, mix : A. 1 c.c. suspension and 0'5 c.c. 0'9% NaCl (control). B. 1 c.c. . ,, 0*2 c.c. immunised dog's serum. C. 1 c.c. ,, 0*5 c.c. ,, ,, Place the tubes in the water-bath at 40° C., and observe after fifteen minutes. The haemolysin thus produced by inoculation of foreign blood can be shown to consist of two parts, one of which is destroyed by moderate heat (the thermo- labile body), the other being much more resistant in this particular (the thermo- stable body). The thermo-labile body is not specific in its nature, but is a normal constituent of blood serum; it is usually called the complement or alexine. By specific in this connection is meant that the substance is not confined in its action to one kind of corpuscle. The thermo-stable body is specific in nature, being produced as a result of the inoculation of foreign blood and acting only when brought in contact with blood corpuscles which are of the same kind or are very closely related to those of the blood used for inoculation. On account of analogy between the thermo-stable body and anti-bodies in general it is often called the immune body. EXPERIMENT VII. Place 3 c.c. of immunised dog's serum in the water-bath at exactly 50° C. for ten minutes. Allow to cool. Repeat the above experiments, A, B, and C, using the heated serum: no haemolysis will occur, the heating 1 One intraperitoneal injection of 50 c. c. of rabbit's defibrinated blood will render the dog's serum strongly haemolytic in about four days after the injection. If the injection be repeated once a week for three or four weeks the haemolytic power will be very strong. When an animal is inoculated in this way it is said to be immunised. An equally suitable pair of animals is the rabbit and guinea pig, but the amount of serum obtainable is small. PHYSIOLOGICAL CHEMISTRY 465 having destroyed something necessary for haemolysis. Is there any agglutination ? Now add 1 c.c. rabbit's serum to some of the mixture of previously heated serum and blood corpuscles ; haemolysis will occur. To another part add some serum from an uninoculated (normal) dog. The same result will be obtained. The destruction of the complement by heating is called inactivation. That inactivation destroys something which, though necessary for haemolysis, is yet a normal constituent of serum, is shown in the above experiment by the fact that reactivation of the haemolysin can be effected by addition of rabbit's serum ; i.e. of the same serum that the erythrocytes are normally accustomed to. The complement, whatever its chemical nature may be, is a constant con- stituent of serum, but under normal conditions it does not as a rule act on the erythrocytes and cause haemolysis because of the absence of the immune body. To produce the immune body, inoculation with foreign blood is usually necessary. There are in general two views as to the mechanism by which these two con- stituents of serum act in bringing about haemolysis. The French school believe (with Bordet) that the immune body acts on the corpuscle as a mordant acts on cotton in dyeing ; in other words, it sensitises the corpuscle towards the com- plement (thus, the French call it the substance sensibilatrice). The German school (headed by Ehrlich), on the other hand, believe that the immune body first of all combines with the corpuscle, and that the complement then combines with the attached immune body and acts through it on the corpuscle. It would be out of place here to give a detailed account of the experiments that have been brought forward in support of these hypotheses, but one important fact can be easily demonstrated, viz. that the immune body readily unites with the corpuscle in the absence of the complement, and that the subsequent addition of complement then causes haemolysis. EXPERIMENT VIII. Centrifugalise 5 c.c. of a suspension of washed rabbit's erythrocytes, and then carefully remove as much of the supernatant fluid as possible. This yields, as sediment, erythrocytes that are free of complement. Now add to the erythrocytes 2 c.c. of inactivated serum from an immunised dog; shake the tube to obtain complete mixing, and allow to stand for some minutes. No laking occurs. Then fill up the tube with 0*9 per cent, sodium chloride and centrifugalise. Pipette off the supernatant fluid and repeat the washing three times, so as to remove all traces of the inactivated serum, which may be merely adherent to but not combined with the corpuscles. Now add some normal serum to the tube and warm to 38° C. : haemolysis occurs, the complement in the normal serum having acted on the erythrocyte-immune body compound. The immune body acts, therefore, as a link uniting corpuscles with complement. It may be considered to possess two combining groups, and is hence called an amboceptor. One of these groups— called the cytophilic group — first of all unites with a side chain of the erythrocyte, and then the other group — called the complementophilic — unites with the complement, resulting in destruction of the erythrocyte. That the reaction takes place in this order is demonstrated in the following experiment : EXPERIMENT IX. Centrifugalise 5 c.c. of the above suspension of rabbits' erythrocytes, and with a pipette remove as much of the supernatant fluid as possible. Place the sediment in a test tube, and cool in crushed ice to zero. In another test tube likewise cool 0'5 c.c. of immunised dog serum. Mix the cooled corpuscles and serum, and keep them at 0° for one hour, after which transfer the mixture to a cooled centrifuge tube, and immediately centrifugalise. Then remove 2G 466 PEACTICAL PHYSIOLOGY the supernatant fluid carefully, and test it and the sediment for the presence of amboceptor and complement in the following manner : a. Add 0'2 c.c. of the supernatant fluid to 0'5 c.c. of the 5 per cent, suspension of the washed erythrocy tes of the rabbit, and incubate at 40° for ten minutes. No laking will probably be noted. Now add a few drops of inactivated immunised dog's serum (see Experiment VII.), and incubate; laking will probably occur. The conclusion is that the supernatant fluid contained complement, but no amboceptor. If the experiment is unsuccessful it must be repeated again, using a smaller amount of the supernatant fluid. b. Shake the sediment with a few c.c. of ice cold 0*9 per cent, sodium chloride solution, and centrifugalise. Remove the supernatant fluid with a pipette and place some of the washed sediment to a small test tube, and incubate ; only slight laking, if any, will be noted in ten minutes. Now add a few drops of rabbit serum or some of the supernatant fluid used in a, when laking will become marked. The conclusion is that the amboceptors had combined with the erythrocytes, but not with the complement. Precipitins. — Not only cells and toxins, but proteins also, react in the manner above described. The reaction manifests itself in the formation of a precipitate. For example, the serum of one animal can cause the serum protein of another animal to become precipitated, indicating that it contains some body which changes the nature of the protein so as to render it insoluble. It is called a precipitin, and, like anti-bodies in general, e.g. haemolysins, it seems greatly increased in amount by inoculation. Thus, if rabbit serum be mixed with ox serum the mixture remains clear, or only becomes slightly cloudy. If, however, ox serum be injected intraperitoneally or intravenously into a rabbit, and the inoculation repeated every three or four days for some weeks, then the rabbit serum will, when mixed with ox serum, cause a marked precipitation of the proteins. A precipitin has become developed by inoculation. Precipitins are remarkably specific in nature, so that if a series of rabbits be inoculated as above described, each with the blood serum of some different animal, there will be pro- duced in the serum of each rabbit a precipitin which reacts only with the serum of the same kind of animal whose blood was used for inoculation, or with that of some closely allied species. In this way a variety of sera can be prepared (and preserved in sealed tubes), which are peculiarly delicate and specific reagents for the detection of the proteins used to produce precipitins in them. By an application of these facts it is possible to distinguish from what animal a given blood serum (or blood solution) has been obtained. Thus, if it be desired to ascertain whether a blood stain is of human blood, it is dissolved in water and filtered till clear, and then mixed in a small test tube with a few drops of the serum of a rabbit that has been inoculated for some time with human blood. If a precipitate occurs the inference is that the stain was of human blood. It is impossible to cause precipitin formation by inoculation with blood from another animal of the same kind or from one that is closely related. Thus, no precipitin will be developed in a rabbit serum by inoculating the rabbit with guinea pig's serum. For the same reason, the bloods of closely related animals can form precipitins that are common to them all. Thus, if the blood of certain monkeys be inoculated in rabbits, the serum of the rabbit will give the precipitin reaction not only with monkey's blood, but also with that of man. CHAPTER XXIII. THE PIGMENTS OF URINE. WHEN fresh normal urine is examined by means of the spectroscope it usually presents no absorption bands, a diffuse absorption of the violet end being alone conspicuous. The yellow colour of the urine is to be regarded as due almost entirely to the presence of a preformed pigment, urochrome. If this pigment be removed from the urine, the colour of the urine is largely lost. It may be separated from urine by saturating urine with am- monium sulphate and filtering. The filtrate which contains the pigment is shaken with alcohol, and by such repeated extractions from the saline solution practically all the pigment may be removed. The urochrome may now be precipitated by adding an excess of ether. The substance is readily soluble in water and when examined by the spectroscope shows no absorption bands. Urobilin is present in very small quantities in normal urine and the amount normally present is generally in the condition of a chromogen. In abnormal conditions the urine may tend to have a brownish tint added to the ordinary rich orange colour and such urine frequently contains urobilin. A solution of urobilin or urine rich in urobilin will present the spectrum shown in Fig. 256, 1. If a concentrated solution of urobilin in sodium hydrate be taken and hydrochloric be added till the mixture is slightly acid, a turbid condition of the liquid results owing to imperfect re-solution of the pigment in the acid. Examined spectroscopically a band is seen in the position of the E-line, in addition to the normal band at the junction of the green and blue (Fig. 256, 2). If the liquid be filtered the E-band will be no longer seen. As regards the connection of urobilin and urochrome, it is important to remember that when urochrome is acted upon by aldehyde a urobilin- like substance is produced, and if urobilin be oxidised with potassium permanganate a substance similar to urochrome is formed. 468 PRACTICAL PHYSIOLOGY The pink colour possessed by a deposit of urates is due to another pigment uroerythrin. This pigment is never excreted in large amount, but it possesses in high degree a colouring power. If a pink urate deposit be dissolved in warm water the urates may be precipitated by saturation with ammonium chloride carrying down the pigment. This may now be extracted with alcohol, and on shaking the alcoholic solution with chloroform to which one drop of acetic acid has been Fro. 256. 1. Acid urobilin In strong solution. 2. Urobilin precipitated by acid from its alkaline solution and partially redissolved. so-called E-band spectrum. 3. Uroerythrin. 4. Uroerythrin in pink urate sediments. The added the pigment passes into the chloroform. It now gives the spectrum seen in Fig. 256, 3. If the pink urate deposit be simply dissolved in warm water the spectroscopic appearance is different, as represented by Fig. 256, 4. Haematoporphyrin is normally present in very small amount in urine. After certain drugs it may be present in comparatively large amounts. Even in acid urine it is present in the condition in which it shows the so-called alkaline spectrum (p. 345, Fig. 231, 11). APPENDIX. ANALYTICAL TABLES. (OUTLINE OF METHOD FOB DETECTION OF VARIOUS PHYSIOLOGICAL CHEMICAL SUBSTANCES IN A MIXTURE.) The following Physical Properties should be noted : I. Appearance. A. Powder. — Dust some on to a slide and examine under the microscope for starch grains and crystals. Dissolve some in a suitable solvent. B. Solution. 1. Opaque — may be due to : (a) suspended fat globules — clear up with ether ; (b) certain inorganic salts — clear up with mineral acid ; (c) certain proteids. 2. Opalescent — may be due to : (a) glycogen or starch — iodine reaction ; (b) certain proteids. 3. Deeply coloured — suspect blood. II. Eeaction. A. Acid — may be due to : (a) free acid If due to free acid, ascertain whether this be 1. a mineral acid or \apply Gunsberg's and the tropaeolin 2. an organic acid / test. If due to an organic acid, apply Uffelmann's test for lactic acid. B. Alkaline test for carbonic acid (effervescence with mineral acid)> ammonia (smell, etc.), caustic alkali. 470 PRACTICAL PHYSIOLOGY The following Chemical Tests should now be applied to Suitable Quantities of the Solution. I. For Carbohydrates. 1. Apply Trommer's test. A. Positive — indicates monosaccharides, lactose, or maltose. B. Negative, but complete solution of cupric hydrate obtained on adding caustic alkalie, indicates cane sugar. Confirm for this by boiling some of the solution with a mineral acid for a minute or so, and applying Trommer's test to the product — reduction indicates cane sugar. The original solution will also taste sweet. C. Negative, and no solution of cupric hydrate. Absence of monosaccharides and disaccharides. 2. Add Iodine Solution. (a) a blue colour which disappears on heating, and returns on cooling indicates starch. (b) a port-wine colour which disappears on heating, and returns on cooling indicates dextrin or glycogen. Confirm for polysaccharides by heating some of the original fluid for about fifteen minutes with a mineral acid, and testing for sugar in the hydrolysed fluid. To distinguish between Starch, Glycogen, and Dextrin. — Shake up some of the original powder with cold water and filter. By this treatment glycogen and dextrin will dissolve, starch will not. Wash the filter paper thoroughly with water, then add a drop of Iodine solution — a blue stain indicates starch. Add Iodine solution to the filtrate — a red colour indicates dextrin or glycogen ; if the former body be present the filtrate is clear, opalescent if the latter. To distinguish between Dextrose, Maltose, and Lactose. (1) Prepare osazone crystals and examine under the micro- scope— dextrosazone gives long thin needles ; maltosazone, short thick needles ; lactosazone, needles of varying length and thickness. (2) Barfoed's reaction may also be tried. Dextrose reduces this with ease ; lactose and maltose not so readily. II. For Proteins. 1. Apply the Biuret reaction — (a) A violet colour indicates native proteins or albuminoids; (b) a rose pink colour, proteose or peptone. APPENDIX 471 2. Apply Millon's and the Xantho-proteic tests. (a) A well-marked reaction indicates proteins of Kossel's 3rd and 4th groups, (b) A faint reaction (combined with a distinct Biuret, and the absence of coagulation on boiling) points to gelatine ('2nd group). (Confirm by seeing if the solution gelatinises on cooling). If the Biuret Test gives a Violet Colouration, A. Add a drop or so of dilute acetic acid and boil. A coagulum points to native proteins To ascertain which of these is present (i.e. albumin or globulin), half saturate some of the solution with (NH4)2S04. A precipitate indicates globulin ; filter ; if the nitrate still gives a coagulum on boiling, albumin is present. B. Carefully neutralise some of the solution. A precipitate may be : 1. Alkali Meta-protein — original^ the precipitate re- fluid alkaline ! dissolves on adding 2. Acid Meta-protein — original f excess of acid or fluid acid J alkali. 3. Nucleo-protein — original fluids precipitate does not alkaline I disappear on adding 4. Mucin — original fluid alka- 1 a moderate excess line J of acid. To distinguish between Nucleo-protein and Mucin.— This is possible only when a large amount of these bodies is present. The acetic acid precipitate is collected on a filter paper, washed with acidulated water, and divided into two portions a and b. (a) Boil with 20 per cent. HC1 for 10 minutes; cool; neutralise ; apply Trommer's test. A positive reaction points to mucin. (b) Melt in a crucible with fusion mixture; after the ash cools, dissolve it in nitric acid and add molybdate of ammonia solution. A yellow precipitate on warming indicates Nuclein. If the Biuret Test gives a Rose Pink Colouration, add a few drops of concentrated pure nitric acid. A. A white precipitate, which clears up on warming and returns on cooling, points to Proteose. Confirm by the salicyl sulphonic acid test. If proteose be present, saturate some of the original fluid, from which native proteins have been separated by boiling, with sodium chloride. A precipitate indicates 472 PEACTICAL PHYSIOLOGY primary proteoses. Filter and add a drop of acetic acid ; a precipitate points to secondary proteoses. B. No precipitate with nitric acid, but a distinct pink Biuret reaction points to Peptone. Confirm by saturating the original fluid with ammonium sulphate, filtering arid applying the Biuret test to the nitrate. When two or more Proteins are present, the following method will be found very useful. Add a few drops of salicyl sulphonic acid to several c.c. of the original fluid. A white precipitate may indicate native protein or proteoses. Boil. The proteoses dissolve, whereas the native protein becomes coagulated. Filter hot. If a precipitate forms in the filtrate on cooling, it indicates Proteoses. Filter off this precipitate and apply the Biuret test to the filtrate. A rose pink colouration indicates Peptone. III. For Fats. — In watery solution fat may be dissolved as a soap. The presence of this can be detected by pouring some of the original fluid into about 20 c.c. of 20 per cent. H2S04 contained in a small beaker, and heated to near boiling point. If soap be present a film of fatty acid will form on the surface of the fluid. IV. The following substances should also be tested for. I. Bile salts — Pettenkofer's reaction ; II. Bile Pigments — Gmelin's test. V. Urea (1). — Add some fuming nitric to some of the original fluid. Effervescence points to urea. (2) Repeat with hypobromite solution. (3) If 1 and 2 be positive, confirm by obtaining urea nitrate crystals. To do this evaporate about 30 c.c. of the original fluid to small bulk, extract residue with six times its bulk of methylated spirit, evaporate this extract to dry ness, dissolve residue in 3-4 c.c. distilled water, and add to the resulting fluid a few c.c. of pure nitric acid, meanwhile keeping the test-tube cool by holding it under the tap. Crystals of urea nitrate separate out if urea is present. Examine under micro- scope. VI. Uric Acid. — Apply Murexide test. VII. Blood Pigment. — (1) Examine by means of the spectroscope. A, the original fluid; B, the same after reduction; C, the same after the addition of caustic alkali and heating. By this latter method alkali haematin is formed. This itself does not give a very distinct absorption band, but if a reducing agent (NH4HS) be added to it haemochromogen is formed, which has two very distinctly marked bands in about the same position as those of oxyhaemoglob. in APPENDIX 473- (2) Apply the guaiac and ozonic ether test. When it is desired to ascertain whether Ferments be present it is necessary to add a piece of coagulated egg white, or of washed fibrin to the original fluid, and to place the mixture on a water bath heated to body temperature. If, after an hour, the digest gives a distinct proteose reaction, and this was not obtained in the original fluid, the presence of a proteolytic ferment may be assumed; pepsin, if the original fluid react acid, and trypsin, if it react alkaline. If proteoses are present in the fluid itself, Mett's method must be employed to identify the ferment. For the detection of Amylolytic and Steatolytic ferments, the methods described in the text must be employed. For the detection of the various substances which may occur in the urine, the test and reactions described in the text must be applied. INDEX. PART I.— EXPERIMENTAL PHYSIOLOGY. Aberration, chromatic, 258. spherical, 258. Accommodation of eye, 234. After-images, 244. Air, alveolar, 183, 197. complemental, 178. expired, 182. inspired, 180. supplemental, 178. tidal, 178. Alimentary canal, movements of, 203. Ametropia, 260. Anelectrotonus, 50, 83, 8$. Anode, 3. Apnoea, 196, 199. Asphyxia, 146. Astigmatism, 232, 258. Atropine, effect of, upon frog's heart, 110, 111. upon salivary glands, 212. Auscultation, 176. Bell's law, 220. Biedermann's solution, 68. Blind spot, 241. Blood, circulation of, 131. corpuscles, number of, 128. gases, 187. pressure, 121, 139, 140, 144, 165. oxygen capacity of, 191. specific gravity of, 131. Brain, frog's, 214. Break extra current, 11. Cannula, arterial, 139. Capacity, vital, 178. Carbon dioxide, influence of, 195. monoxide, influence of, 194. Cardiograph, 119. Cardiometer, 170. Cerebellum, effects of removal of, 215. Cerebrum, effects of removal of, 214. Chest, auscultation of, 176. examination of, 174. inspection of, 174. palpation of, 175. percussion of, 175. Cheyne- Stokes respiration, 197. Chloroform, effect of, on frog's heart, 112. effect on frog, 216. Chronograph, 23. Circulation of blood, artificial schema of, 134, 135. influence of gravity on, 132, 137, 147, 148. proofs of, 131. time, 138, 169. velocity of, 136. Clonus, 219. Colour blindness, 253. complementary, 250. sensations of, 245. Contraction, remainder, 38, 39, 44. secondary, 52. single, 22, 25, 40. voluntary, 220. Cord, spinal, effects of removal of, 215. roots of, 220. Cornea, action of, 233. Corpora striata, effects of removal of, 214. Curare, 48. Current action, 51, 91. demarcation, 51, 91. injury, 52, 91. Cyrtometer, 174. Daniell cell, 2. Defaecation, 210. Dioptre, 232. INDEX 475 Drum, recording, 18. Du Bois-Reymond's key, 4. Ear, dissection of, 267. Elasticity of muscle, 53. Electrodes, 6. polarisation of, 78. unpolarisable, 79. Electrometer, capillary, 92. Electrotonus, 50, 82, 86. Emmetropia, 259. Emprosthotonus, 216. Endiometer, 188, 189. Ergograph, 71. Ether, effect of, on frog's heart, 114. Exchange, respiratory, 184, 186. Extensibility of muscle, 53. Eye, accommodation of, 234. artificial, 233. dissection of, 226. mechanism of, 228. media of, 232. optical defects of, 258. "reduced," 228. Faradic shocks, 12. Fatigue, 38, 39, 70. absence of, in nerve, 87. effect of circulation of blood on, 72. Flicker, 247. Fremitus, vocal, 175. Galvani's experiment, 52. Galvanometer, 90. Gas, analysis of, 181. of blood, 187. pump, 188. tension of, 183. Gaskell's clamp, 154. Gracilis experiment, 80. Gravity, influence of, on circulation, 132, 137, 147, 148. Haemacytometer, 130. Haemoglobinometer, 129. Hearing, 267.' Heart, anatomy of frog's, 92. anatomy of sheep's, 115. apex preparation of, 95. beat, methods of recording, 97. contraction of, 94, 149. effect of temperature on, 94, 99, 101, 154. effect of distilled water on, 152. effect of drugs, 70, 109, 112, 151. ganglia of, 96, 103. latent period of, 149. nerves of, 103, 156. output of, 170. refractory period of, 103, 150. rhythm of, 94. sounds of, 121. Heart, Stannius, 100. valves of, 115, 119. work of, 169. Heat, loss of, 201. regulation of, 200. Helmholtz, arrangement for equalisa- tion of make and break induced currents, 13. Hooke's law, 54. Hyoglossus muscle, 30. Hypermetropia, 259. Images, Purkinje-Sanson, 336. Impulse, cardiac, 119. nervous, rate of transmission of, 76. nervous, rate of discharge of, 217. nervous, transmission in both direc- tions, 79. Induced currents, equalisation of make and break, 12, 13. Induction coil, 8. Induction shocks, 10. Inhibition of frog's heart, 105, 106. Inspection of thorax, 174. Intestine, movements of, 208. Iris, action of, 234. Irradiation, 253. Isotonic solutions, 69. Katelectrotonus, 51, 81. Kathode, 3. Keys, 4, 5. Kidney, volume of, 172. Kymograph * 18, 143. Latent period of muscular contrac- tion, 26. Law of specific energy, Miiller, 222. Bell and Majendie, 220. Talbot and Plateau, 247. Hooke, 54. Lens crystalline, action of, 233. changes in, during accommodation, Levers for muscle, 17. Lippmann's capillary electrometer, 92. Load, effect upon muscular contrac- tion, 35, 57. Manometer, 127, 142, 166, 167. Medulla oblongata, effects of removal of, 215. Metronome, 215. Miiller's Law, 222. Murmur, vesicular, 176. Muscarine effect on heart, 109. Muscle, independent excitability of, 80. electromotive properties of, 89, 90. elasticity of, 53. action of veratrine on, 29. 476 INDEX Muscle, action of distilled water on, 67. action of salts on, 67. Muscle and nerve, preparation, 14, 16. Muscle-lever, 17. Muscles of the frog's leg, 15. Muscular contraction, 22. shortening during, 29. work during, 29, 57. conditions affecting, 29, 32, 35. red and white muscles, 29, 30. effect of temperature on, 32, 33. effect of load on, 35, 37. effect of fatigue on, 38, 39. Myograph, 16, 17. spring, 64. pendulum, 66. Myopia, 261. Nerve, properties of, 44. electrical stimulation of, 46. mechanical stimulation of, 47. thermal stimulation of, 47. che/nical stimulation of, 47. relation between muscle and, 80. electromotive properties of, 51, 88. vago-sympathetic, 104, 108. impulse, rate of transmission of, 76. depressor, 140, 146. chorda tympani, 211. vagus, dissection of, 157. effect of constant electrical current on, 81. cervical sympathetic, 158. specific energy of, 222. splanchnic, 172. roots, functions of, 220. vaso-motor, 137. sweat, 202. Nervous system, central, 214. Nicotine, 112, 212. Oncometer, 171. Ophthalmometer, 230. Ophthalmoscope, 265. Oxygen, effect of, 196. Pain, sense of, 224. Palpation, 175. Percussion, 175. Perfusion of blood-vessels, 164. Pericardium, 115. Perimeter, 263. Phakoscope, 237. Phosphenes, 243. Photohaematochometer, 169. Piezometers, 168. Pilocarpine, 213. Plethysmograph, 171, 173. Pohl's reverser, 5. Presbyopia, 259. Pressure, blood, in man, 127. blood, 140, 157. Pressure, intracardiac, 166. intra-thoracic, 176. atmospheric, effects of changes in,, 192, 193. Pulse, 121, 161. Pump, blood-gas, 188. air, 192. Purkinje-Sanson images, 236. Purkinje's figures, 240. Reaction time, 216. Recorder, bellows, 220. Reed vibrating, 42, 43. Reflex, 214. Refractory period, voluntary muscle.. 42. of heart, 103, 150. Refraction, errors of, 258. Respiration, movements of, 180. chemistry of, 180, 187. regulation of, 195, 199. centre for, 195. apparatus, 184, 186. in tissues, 402. Respiratory exchange, 184, 186. quotient, 185. Retina, 240. Reverser, Pohl's, 6. Rheochord, 7. Rheoscopic frog, 52. Rigor, heat, 34. Ringer's solution, 69. Rubber, elasticity of, 56. Saline solution, normal, 69. Salivary glands, dissection of, 211,. 212. Salts, action of, on muscle, 67. Sanson-Purkinje images, 236. Sartorius experiment, 79. Schemer's experiment, 238. Secretion, salivary, 210. Sensations, 222, 223. Shatter curves, 28. Sino-auricular junction of frog's heart, stimulation of, 109. Skin, sensibility of, 222, 223. Smell, 222. Sounds, bronchial, 176. cardiac, 121. Specific gravity of blood, 131. Sphygmograph, 123. Sphygmogram, 125. Sphygmometer, 127. Sphygmoscope, 166. Spinal animal, 160, 215. Spirometer, 178. Stannius experiment on heart, 100. " Stair-case " effect in voluntary muscle, 40. cardiac, 150. Stethograph, 179. Stethoscope, 176. INDEX 477 Stimuli, minimal and maximal, 19, 46. successive, 40, 41. summation of, 62. Stomach, movements of, 205. Stromiihr, 136. Strychnine, action of, 216, 219. Submaxillary gland, 211, 212. Summation of contraction, 41, 42. of stimuli, 62. "Suprarenal extract, 164, 165. Swallowing, 203. Sweat, 202. Talbot-Plateau law, 247r Taste, 222. Temperature, effect of muscular exercise on, 200. effect on heart, 94, 101, 154. of body, 201. effect of anaesthesia on, 202. regulation of, 201. sense of, 224. Tetanus, genesis of, 40. incomplete, 44, 45, 219. complete, 44, 45. secondary, 53. Thalami, optic, effects of removal of, 214. Thermometry, 200. Thorax, inspection of, 174. Tuning-fork, 22, 23. Tiirck's experiment, 215. Unipolar excitation, 21. Vagus (vago-sympathetic), dissection of, in frog, 104, 108. stimulation of, effect on heart of frog, 105, 106. influence on respiration, 145, 199. influence on arterial pressure, 145. Varnish, for tracings, 19. Vaso-motor system, 137, 164. Veratrine action upon muscle, 29. Vision, 226. field of, 263. binocular, 254. Water, distilled, action on muscle, 67. action on heart, 151. Weber's paradox, 57. Wind, second, 197. Work done by muscle during con- traction, 35, 57. by heart during contraction, 169. Yellow spot, 242. INDEX. PART II.— PHYSIOLOGICAL CHEMISTRY. Acetone in urine, 452. Achroodextrin, 291. Acid, aceto-acetic, 453. aspartic, 299, 392. carnic, 355. cholalic, 399. cyanuric, 417. fatty, 313. glycocholic, 399. glycuronic, 454. hippuric, 428, 441. homogentisic, 453. hydrochloric, tests for, 377. lactic, 354, 454. nucleic, 310. oleic, 314. oxybutyric, 453. palmitic, 313. sarcolactic, 358. stearic, 313. taurocholic, 399. Acrolein, 316. Adamkiewicz reaction, 303. Adenine, 423. Agglutination, 464. Albumin, 305. crystals, 301. quantitative estimation, 445. Albuminuria, 442. Aldoses, 272. Alexine, 464. Alloxan, 424. Alloxuric bodies, 422. Amboceptor, 465. Amido acids, 389. Ammonia in urine, 431, 433. Amylopsin, 396. Arginine, 392. Bang's method, 293, 295. Barfoed's reagent, 275. Bence Jones' albumosuria, 445. Bial's reagent, 295. Bile, composition of, 397. Bile salts, 399. pigments, 400. Bilirubin, 400. Biliverdin, 400. Biuret, 417. Biuret reaction, 302. Blood, 331. clotting of, 332. Boettgers test, 449. Bread, 365. making of, 365. Calorie, 360. Camerer's method, 427. Carbohydrates, 272-296. Carnic acid, 355. Caseinogen and casein, 298, 3S Cholesterol, 318, 401. Cholin, 317. Chromo-proteins, 308. Collagen, 307. Colorimeter, 430. Colostrum, 330. Complement, 464. Congo red, 377. Creatin, 352, 356. Creatinin, 352, 429. Cystin, 393, 441. Dextrin, 290. Dextrose, 284, 450. Diet, 359. Digestion, 371. bacterial, 404. in intestine, 387. in mouth, 371. in stomach, 375. Disaccharides, 285. INDEX Dubosq colorimeter, 430. Dupre's urea apparatus, 420. Edestin, 298. Eggs, composition of, 362. Elastin, 307. Emulsification, 316. Erepsin, 403. Erythro-dextrin, 291. Esbach's albuminometer, 445. Ester value, 322. Faeces, 407. Fats, 313. Fatty acids, 313. Fehling's test, 274. Fermentation test for sugar, 279, 449. Fibrin, 334. ferment, 336. Fibrinogen, 334. Flour, 364. Folin's method, for ammonia, 432. for urea, 421. for creatinin, 429. Foods, 359, 366. Galactose, 285. Gall-stones, 319. Gelatin, 307. Gerrard's method, for sugar, 451. Gerrard's urea apparatus, 420. Gliadin, 298. Globulins, 305. Gluco-proteins, 308. Glucose, 284, 450. Glutenin, 298. Glycin, 390. Glycogen, 290. Glycosuria, 447, 450. Glycuronic acid, 454. Glyoxylic acid test, 303. Gmelin's test, 400. Grutzner's method, 386. Guanine, 423. Guiac test, 340. Giinsberg's reagent, 377. Haematin, 339. Haematocrit, 461. Haematoporphyrin, 349. Haematuria, 447. Haemin, 341. Haemochromogen, 349. Haemocytes, 329. Haemoglobin, 339. and derivatives, 344. Haemoglobinuria, 447. Haemolysin, 464. Haemolysis, 460. Hay's test, 398. Heller's test, 444. Hexone bases, 392. Hippuric acid, 428. Hirudin, 335. Histones, 305. Hopkins' test, 303, 380. Hypoxanthin, 353, 357, 423. Inactivation, 465. Indican, 410. Indol, 405. Inosite, 355. Inversion, 403. Invertase, 403. Iodine number of fats, 314, 323. Isomaltose, 287. Jaffe's test, 429. Juice, gastric, 376. intestinal, 403. pancreatic, 388. Keratin, 307. Ketoses, 278. KjeldahPs method, 410. Kresol, 405. Lactic acid, 354. Lactose, 287, 329, 448. Laevulose, 285. Lecithin, 316, 401. Legal's test, 406. Leucin, 389, 391, 441. Leucocytes, chemistry of, 338. Lieberkuhn's jelly, 311. Lipase, 396. Lysine, 392. Maltose, 287. Meat, 350. Meat extracts, 350. Metabolism, 359, 367, 437. Meta-protein, 310. Methaemoglobin, 347. Mett's method, 386. Milk, 324. fats of, 330. proteins of, 326. salts of, 329. sugar of, 329. Millon's reaction, 302. Molisch test, 279, 303. Monosaccharides, 272. Moore's test, 284. Morner-Folin method, 421. Mucin, 308. Mucus, 446. Murexide test, 425. Muscle, 350. extractives of, 350. Myosin, 351. Nitrogen, estimation of, 410. Nucleic acid, 310. Nucleins, 310. -480 INDEX Nucleo-proteins, 309. Nylander's test, 274. Olein, 314. Ornithin, 393. Osazone, 275. Palmitic acid, 313. Pancreatic digestion, 388. Parry's method, for sugar, 451. Pentoses, 294, 448. Pepsin, 382. Peptone, 311. Pettenkofer's reaction, 398. Phenol, 405. Phenyl hydrazine, 275. Phosphoproteins, 306. Plasma, 337. Polarimeter, 282. Polarisation of light, 280. Polypeptides, 299. Polysaccharides, 288. Precipitins, 460. Protagon, 320. Protamines, 305. Proteins, 297. classification of, 304. tests for, 300. Proteose, 311. Ptyalin, 372. Purin bodies, 422. Pus, 446. Reichert-Meissl value, 322. Rennin, 327. Rothera's test, 453. Saccharimeter, 282. Salicyl-sulphonic acid test for albu- min , 445. Saliva, 371. Saponification, 314. Saponin, 462. Sarcolactic acid, 358. Schiff's test, 427. Sclero-proteins, 307. Seliwanoff's test, 278. Serum, 336. Shaffer's method, 433. Skatol, 405. Soap, 314. Soxhlet's apparatus, 313. Spectra, 345, 348. Spectroscope, 343. Starch, 288. Steapsin, 396. Stercobilin, 407. •Succus entericus, 403. Sugar, cane, 286. Sugar, estimation, 293. grape, 284, 450. milk, 287. Syntonin, 311. Taurin, 400. Thiophene test, 455. Thrombin, 334. Thrombogen, 334. Thrombokinase, 334. Trommer's test, 274. Tropaeolin test, 378. Trypsin, 389. Tryptophane, 395. Tyrosin, 389, 391, 441. Uffelmann's reagent, 354. Urates, 438. Urea, 415. nitrate, 416. oxalate, 416. quantitative estimation of, 419. Ureometer, 421. Uric acid, 422, 426. quantitative estimation of, 427. Urine, acetone in, 452. ammonia in, 431. bile in, 447. blood in, 446. chlorides in, 434. colour of, 410. composition of, 410. deposits in, 438. inorganic salts of, 434. pathological, 442. pigments of, 467. phosphates in, 435. quantity of, 408. reaction of, 409. specific gravity of, 409. sugar in, 447, 450. sulphates in, 435. total nitrogen of, 410. Urobilin, 467. Urochrome, 467. Uroerythin, 468. Volhard's method for chloride, 434. Weyl's reaction, 429. Wheat, 364. Worm, Muller test, 449. Xanthin, 353, 357, 423. Xanthroproteic reaction, 302. Yeast, 279. Zein, 298. GLASGOW : PRINTED AT THE UNIVERSITY PRKSS BY ROBERT MACl.EHOSE AND CO. LTD. From Mr. Edward Arnold's List of Medical Books. TEXT-BOOK OF GENERAL PATHOLOGY. By the following contributors : A. P. BEDDARD, M.A., M.D., London; A. E. BOYCOTT, M.A., M.D., Manchester; C. H. 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