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Full text of "Practical physiology"

CORNELL 

UNIVERSITY 

LIBRARY 




FROM 

The Estate of S. Simpson 



Cornell University Library 
QP 34.P371 1910 



Practical physiology. 




3 1924 003 128 729 




The original of tiiis book is in 
tine Cornell University Library. 

There are no known copyright restrictions in 
the United States on the use of the text. 



http://www.archive.org/details/cu31924003128729 



PKACTICA.L PHYSIOLOGY 



PRACTICAL 
PHYSIOLOGY 



EDITED BY 

M. S. PEMBEEY 



CONTRIBUTORS 
A. P. BEDDARD, M.A., M.D. LEONARD HILL, M.B., F-KS. 



Assistant Physician, lato Demon- 
strator of Physiology, Guy's Hospital 

J. S. EDKINS, M.A., M.B. 

Lecturer on Physiology, 
St. Bartholomew's Hospital 



Lecturer on Physiology, The 
London Hospital 

J. J. E. 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 Br NUMEROUS BIAORAMS AND TRACINOS 



THIRD EDITION 



NEW YORK 

LONGMANS, GREEN, & CO. 

LONDON: EDWARD ARNOLD 

1910 



PREFACE TO THE FIEST EDITION. 



Physiology is the Ijasis 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 PEEFACE 

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. 



PKEFACE 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 iu 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. Eyffel 
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. Eyffel 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. Phmbret. 

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., ] 4 

III. A Single Contraction of a Gastrocnemius Muscle. By 

A. P. B., 22 

IV. The Conditions which affect Single Muscular Contractions. 

By A. P. B., 29 

V. The Conditions which aifect Single Muscular Contractions 

(continued). By A. P. B., 32 

YI. The Conditions which aflfect Single Muscular Contractions 

(continiied). 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 

XI. The Electromotive Properties of Muscle and Nerve. By 

M. S. P., 51 

XII. Extensibility and Elasticity of Muscle when at Best and 
Contracted. Comparison with Eubber (Advanced). By 
A. P. B., 53 



70 



CONTENTS 

CHAP. J"*"^ 

XIII. Load and After-load. Work done with Increasing 

Loads (Advanced). By A. P. B., 57 

XIV. Summation of Stimuli {Advanced). By A. P. B., 62 

XV. Effect of Distilled Water and of Various Salts on Muscle 

{Advanced). A. P. B., §7 

X VI. Fatigue of a Voluntary Movement and of a Muscle-Nerve 
Preparation with its Circulation intact {Advaiiced). 
By A. P. B., 
XVII. The Bate 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., 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. By L. H., 121 

XXXIII. Blood. The Haemoglobinometer and the Haemacyto- 

meter. By L. H., 128 



CONTENTS xiii 

CHAP. PAGE 

XXXIV. Circulation of the Blood {Elementary Demonstrations). 

By L. 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 Eespiration. By M. S. P., 180 
XL VII. Determination of the Respiratory Exchange in Man. 

By M. S. P., 184 

XL VIII. Eespiration Apparatus. By L. H., 186 
XLIX. TJie 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 

LIII. 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 



V CONTENTS 

CHAF. PAGE 

LXII. The Functions of the Anterior and Posterior Roots of 
the Spinal Cord. The Bell-Majendie Law. By 
M. S. P., - 220 

LXIII. MUUer'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 

LXXIL 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 Sisate. Auditory Sensations. 

By J. S. E., - 267 



PART II. 

PHYSIOLOGICAL CHEMISTEY. 

By J. J. R. MACLEOD and M. Flack. 

Introduction, 270 

I. Carbohydrates. By J. J. R. M., 272 

II. Carbohydrates {continv^d). 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., 35& 

XII. Digestion in the Mouth. By J. J. R. M., 371 

XIII. Digestion in the Stomach. ByJ. J. E. M., 375 

XIV. Digestion in the Intestine. By J. J. R. M., - 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. R. M., 442 
XXI. Lactic Acid, its Estimation and Significance. By J. H. 

Ryffel, 454 

XXII. Haemolysis and Precipitins. By J. J. R. M., 460 

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 ABB MEASUEES. 



Length. 

Metric or Decimal. English. 

I Metre (M.) - =39-3701 inches. 

1 Decimetre (dm.) . . . = 3-9370 

1 Centimetre (cm.) - = 0-3937 

1 Millimetre (mm.) - = 0-0393 

1 Micromillimetre (mkni) - =0-000039 „ 
The nnit of the Metric System is the Metre, which represents one ten-milliontb 

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 Grreek 
prefixes, the latter by Latin prefixes. 

1 Myriametre (Mm.) = 6-2137 miles. 

1 Kilometre (Km.) - - = 0-6214 „ 

1 Hectometre (Hm.) =109-361 yards. 

IDekaraetre (Dm.) - = 32-8084 feet. 

1 Metre (M.) • - - = 39-3701 inches. 



Weight. 
Metric or Decimal. 
1 Kilogramme (Kgm.) 
1 Gramme (Gm.) 
1 Decigramme (dgm.) 
1 Centigramme (cgm.) 
1 Milligramme (mgm.) 
The unit is the Gramme which represents the weight of 
water at 4° C. 



English. 
= 2,-2046 pounds. 
= 15-4323 grains. 
= 1-5432 „ 
= 0-1543 „ 
= 0-0154 „ 

cubic centimetre of 



Apothecaries Weight. 
437 "5 grains (gr. ) =1 ounce. 
16 ounces {§) =1 pound (lb.). 



I 60 grains 
! 20 grains 



= 1 drachm (3). 
= 1 scruple O). 



1 grain =0-0648 gramme. 

*Not official. 



Avoirdupois Weight. 
16 drachms = 1 ounce (oz. ). 
16 oz. =1 pound (lb.). 

28 lbs. =1 quarter (qr.). 

4 quarters = 1 hundredweight (cwt. ) 
20cwt. =lton. 

1 pound =453-592 grammes. 
1 ounce = 28-35 grammes. 



WEIGHTS AND MEASURES 
Capacity. 



Metric or Decimal. 
1 Dekalitre (Dl.) 
1 litre (L.) 
1 Decilitre (dl.) - 
1 Cubic centimetre (c.c.) 

or 
1 MUlilitre (ml.) 



}■ 



English. 
= 2-1998 Imperial gallons. 
= 35-196 Imperial fluid ounoee. 
= 3-5196 „ 

= 0-0352 



60 minims (Tn.) 
8 fluid drachms - 

20 fluid ounces 
8 pints 

1 cubic centimetre 
1 fluid ounce 
1 pint - 
1 gallon 



= 1 fluid drachm (3). 
= 1 fluid ounce (5). 
= lpint(0). 
= lgaUon{C). 



= 16 '9 minims. 
= 28*42 cubic centimetres. 
=568-34 cubic centimetres. 
= 4-54 litres. 



THERMOMETERS. 

Fahrenheit and Cbktigeade 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. 



r. 

212° 

112 

106 

104 

102 

101 

100 

99 

98 

97 



C. 

100°-0 
44-4 
41-1 
400 
38-9 
38-3 
37-8 
37-2 
36-7 
36-1 



F. 


C. 


80- 


26-7 


70 


21-1 


60 


15-6 


50 


10-0 


41 


5-0 


32 


0-0 


23 


- 5-0 


14 


-10-0 


5 


-150 



AVERAGE WEIGHTS AND HEIGHTS. 

Average weight of a healthy male child at birth • - . = 6-8 lbs. 
It .. ,. six months' old ■ =12*4 ,, 

J. » ,, twelve ,, - =18-8 „ 

An adult man (dressed) 5 feet 8 inches in height, should weigh 11 st. 1 lb. and 
should have a chest circumference of 38J inches. 



PAET I. 

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 off 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 ZnSO^ -i- H2, 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 HgSO^ + 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 




' ^BoriAofiMioj'e Pot 
Flo, 1. — Diagram of a Daniell cell seen in 
section. 



ELEMENTARY 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 ilow 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.' 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. 

' 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, SO4, OH, Cu and SO4, 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 ziuo 
is attacked by the SO4 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 SO4 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 SO4 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. 



PEACTICAL 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 




Fio. 2.— The mercury key. 



Fio. 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 

J bad contact with the mercury. In 

I order to avoid this it is only necessary 

L to fix the insulated wires from the 

^ ^fy '^ ^^ii^Tcy to the binding-screws and to 

r' * iiPiTO_jBBiBM turn the naked ends of these wires 

<(i I lOflHRRH 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 




FlQ. 5. — Plan of the use of a Du Boia key, as a simple make and break key. 




Flo. G. — Arranged as a short-circuiting key : key shut. 




Fw. 7. — Arranged as a short 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. 




A + 



Fio. 8A.^The Pohl'a reverser. A and B the two side 
cups ; C, Bf B and P the four corner cups ; S the handle 
made of glass or vulcanite. 

Fig. 8b.— Universal key (Gotch). The key is used by 
rotating the arm containing the screws connected with 
the wires A aud 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 B to that of C. 




6 PRACTICAL PHYSIOLOGY 

The PoM'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 




Fio. 9. — Plau of the arrangement of the two alternative circuits. 




^*jm!lMSlU- 



Pih 






Flos. 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 



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 Bheochord 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 



vmm 



;^V HS — 



■«"Br 






^^ ' J 

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 



4 + 




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 pai-allel wires connected by a movable metal slider S. 
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 PRACTICAL 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 thau 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 b.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.MF. 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.^ 

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 




Flo. 16. — DiagraiD 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 sufficiendy 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 

^The 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 
Hnding-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, on opening it, the shock of the 
break extra current. 



12 PRACTICAL PHYSIOLOGY 

A purely ph3'sical 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. 

EcLualisation 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. 



S.C. 




f xo. 17. — Arransfemeut of apparatus for equalising the make and break iuduced 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 



ELEMENTAEY 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 S,, and again 




Fig. 18. — Diagram to show the actlou 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 ; 




Via. 19. — Diagram to show the action of the Helmholtz side-wire. 

further, the break-shock is stronger than the make-shock, and for the 
sime reason as before. 

Determine the distance necessary between the two coils for the 
shocks just to be felt on the tongue. 



14 PRACTICAL 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 explaiited. 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 Sj 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 £, it becomes a magnet, and pulls down the spring into 
contact with Sj. 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 



ELEMENTARY EXPERIiVIENTAL 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- 





Flo. 20. FlO. 21. 

Muscles of the frog's leg. (After Ecker.) 



Fio. 20. — Dorsal aspect. 

1. Trineeps femorls. 

2. Biceps femoris. 

3. Rectus internus. 

4. Semi-membranosus. 

5. C^strocneiDius. 
& Tendo Achillis. 



Fig. 21. — Ventral aspect. 

1. Rectus Internus. 

2. Gracilis. 

3. Adductor longus. 

4. Vastus internus. 

5. Sartorius. 

6. Adductor brevis. 

7. Adductor magnus. 

8. Gastrocnemiiis. 

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 b}' 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 6 th and 7th vertebrae and the 9 th, 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. 25. 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- 
nemius 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-preparaiion 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. (6) 
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 
lymph of which will keep it 
The nerve must not be placed upon the frog's 




Fig. 24. — The crank-lever, muscle-board and stand, 



moist and irritable. 



ELEMENTARY 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 




«==- 



Fio. 26. —The simple lever with after-loading acrew. F, clamp ; L, lever j 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. 
(6) 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 sti£f paper 
bent at its free end slightly over towards 
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 difierent 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. 




"imiaTAiioeK. 
Fio. 26. — Kymugraph. 



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 ofi' 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 suflSciently 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 ^ 
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 

' 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 PRACTICAL 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. Sotate 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 conttaction 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 uie primary colL (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., 



ELEMENTAEY EXPEKIMENTAL 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 hj 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. 

Eepeat 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 eacjb 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. XVIII.). 



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- 



ELEMENTARY 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 ^^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 




Fio. 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. 





J r'^T'^ 



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). 




Fia. 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 
grams, and should then be 




Fio. 31. — Diagram of the apparatus for recording a 
single muscular contraction. 



weighted near its axis with 10 or 20 

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- 



ELEMENTAEY 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 




Fio. 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, 
6 grms. 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 Ee- 
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 ^nr^^^ ^^c- ^^^ 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 

•j^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 i^nny*^® °^ * second 
(see Velocity of nervous impulse). 
Of the remaining , A ^ ths (6) the 

Fig. 33.— Contraction of the same preparation d iuuu \ / 

as in Fig. S2, recorded on a drum revolving at paSSage of the UerVOUS impulse 

a slower rate. The hump near the top of the i i « 

upstroke of the lever represents a second con- along the fine motor nerVO- 

traction in response to the breai: shock. Time ^. . 

marker, 100 per sec. (A.P.B.) eudlUgS OCCUpieS about looft ths 

sec, and (c) the latent period of the muscle itself about xjnsTr*^^ *'f * 
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 x^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 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 27 

of relaxation, and lasts about xrirths of a sec. Eelaxation 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 




Fio. 34. — Single coiitiaction of gastrocnemius. Muscle loaded only with a I'atLer heavy 
lever. Magnification, 5. Temp., 16° C. Time marker, 100 per sec. (A.F.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 sufiiciently 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 instramental 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- 

' Muscles during the cold of winter, even when properly weighted, frequently 
show this ' contraction-remainder.' If cold be the cause, turn back 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 
rig. 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 4xt^, 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 \, 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. 



CHAPTEE 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 difierences 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 ofiF 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. S5. — Gompaiison 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 



ELEMENTARY 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 latei 
the frog will be seized by a spasm when it jumps. As soon as these 




Fia. 36. — Contraction of the hyoglossus muRcle. 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 
ia few minutes the muscle is stimulated by a single maximal induction- 




FlQ. 37. — ContiactioD of the gastrocnemius muscle of a frog. The etfect of vera- 
trine. The first two contractions show the characteristic effect of the drug ; further 
stimulation produced twitches without the prolonged contraction. Tiie curve has 
f been reduced to one-half the actual size. The time is marked in seconds. (Fembrey 
and Phillips.) 

shock, and its contraction recorded. The curve shows that the response 
is a single slow contraction with an enormously prolonged relaxation^ 
Eeplace the hyoglossus by the gastrocnemius and sciatic preparation 
and stimulate it in the same way. As soon as the iirst 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 eflfect 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. vrill 
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 EXPBEIMENTAL 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 




KiG. jj8. — 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. (Fembrey and 
Phillips.) 

suspehd 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.^ 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 

' Cooled 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 
fatigae or death come on. 

C 



M PRACTICAL 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 




Fio. 39.— Curve of the shortening of the gastroenemius muscle during heat-ri|;ar. 
(Pembi'ey and Fhillips.) 

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 poar 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, opaquo, inelastic, and has permanently lost 
its irritabilitj-. 



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. 




Fio. 40. — The effect of load upon the contraction of the gastrocnemius muscle (A F.B.) 




Fio. n is the continuation of the experiment In Fig. 40. Single contractions of the 
gastrocnemius with different loads. The figures on the curres 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.F.B.) 

Eecord 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 4L 
Each increase of weight stretches the muscle, consequently it is 



36 



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 grms. 


Actual Height 

of Contraction 

in mm. 


Work done in 
grm. mm. 


20 


4 


4 


16 


50 


10 


3 8 


38 


80 


16 


3-6 


57-6 


lOO 


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 increnses 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 



37 



(» o 

as 

aft 



9 9 



S - 
° a 
» o 

« g 



11 

3 a 






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. 






S i3 






SB ^ 

s3 



il 






S'g 



G S 
O CI 




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 tetn|»orary 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 



ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 



41 




Fm. 46.— Effect of two auccegslTO maximal stimuli, with gradually dlminisliing 
Intervals, upon the gastrocnemius. To be read from below upwards. 

S. Time tracing, 60 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 
'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 stijnuli. 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). 

(Jenesis 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 ilat 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 



ELEMENTARY 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 

p.c 




Fig. 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 drum, 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. 
Repeat 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 j^g^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 PRACTICAL 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 
' contraction-remainder ' varying in extent according to the degree of 
fatigue is generally seen. 

The same experiments ma>y 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 STIMULL 

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 amductimty. 
Excitability, or, as it is sometimes called, irritability, is the response to a. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



45 






■a 



0, 
□Q 



a 



S 



.9 

to 



46 PRACTICAL 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 sui ■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 diflference between 
maximal and minimal stimulation depends upon the number of the 
constituent fibres of the nerve stimulated. A weak electric current 
may afiect 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 indejaendent 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. 

Curare^ 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. 

' It is prepared from various plants of the genus Stryohnos. 



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 





Fio. 50.— Biaf^^m of the ezperimeiit 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. 

■■ThiB operation should be performed with a pair of Spencer-Wells pressure- 
forceps in order that no blood may be lost. 

D 



AO PEACTICAL 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 oflF 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 
alterationaJn 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 





Fio. 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 
poi-tion of the ventricle ; that is, a local diastole during general systole. This 
condition can he produced by the make of the anude or the break of the kathode of 
a constant current. In B the veutiide is dilated and flushed, with a small pale 
area of contracted muscle ; that is, a local systole during general diastole. This 
condition can he 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 
amade, 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 heal 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 XI. 

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 



PRACTICAL PHYSIOLOGY 



vnjwry-cwrreni.' 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 Bheoscopic Frog. Oalvani'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 
Pio. 62.— Diagram of Gal- (Fig. 53). The nerve of preparation B is stimu- 
toaction^witiiout 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 'cwrent 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 
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 
ligature destroys the physiologi- 
cal continuity and prevents the passage of the excitatory state but 
not that of an electrical current. 





Fia. ss.- 



-Diagram of the experiment on 
secondary twitch. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 63 

Secondary Tetanus. — If the nerve be stimulated with a rapid series 
of induction-shocks the muscle B goes into tetanus and its ' cvrrertts 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. 



CHAPTEE 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. 20, 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 PEACTICAL 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 
jpointer 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.i attach 

' 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 EXPEEIMENTAL 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 aH the 



Fia. 54..— Curve of extensibility and elasticity of gastrocnemiuB. The figtireB 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. 



Fio. 56. — Elasticity curve of quiescent muscle. To be read from right to left. 
The figures on the curve are for weights in grms. (M.8.F.) 

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 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 weight, would be liable to 
rupture either the muscle itself, or its tendon, or the bones to which it 



56 PEACTICAL 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 







/ 




\ 


5C 


1 1 1 1 
'0 "J-OC 300 200 100 




1 ! 1 1 1 

) 100 200 300 400 500 



Fio. 56. — Elasticity curve of rubber tubing. The figures represent weights in grms. (M.S.F.) 

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. Eotate 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. Eepeat 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 
' 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 




Fia. 67. — Comparative extensibility of resting and contracted gastrocnemius. 
Temp. 12' C. Magnification, 5. Fi|;rures represent actual weights In grms. U is 
the ' resting' and G 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; (J) 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 
eflfect 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 
a muscle with single maximal induc- 
tion shocks, using the drum as a key 
in the primary circuit. Fix a gastro- 
cnemius preparation to a myograph 
lever, provided with an after-loading 
screw ; by raising the screw the metal 
part of the lever can be supported at 
any level (Fig. 25). Hang a weight 
of 60 grms. near the axis and raise the 
screw until the whole of the weight 
is just after-loaded ; this point can be 
ascertained by supporting the weight 
with the finger, and when the muscle 
no longer tends to raise the lever off 
the after-loading screw, the muscle is 
unstretehed by any load. Arrange the 
apparatus so that with the screw in 
this position the lever is horizontal. 
Eecord a single contraction of the 
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 progresBiTe after-loading of a gastrocnemius. Actual load on 
musdO} 4 grms. Magnification, 6. Temp., 10* C (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. 
Eepeat 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, and 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. 
Belation of Load to Work done during Contraction.— Tn 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 hali 
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 









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 EXPERIMENTAL 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. 




FiQ. 60.— Height of coutractiona 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 
suflSciently 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. Eepeat 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 




Fio. 61. — Efifect 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, lime marking in secunds. (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 



PRACTICAL 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 -g^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 eflFective. 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 EXPEETMENTAL 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). Ki is fixed, but K^ is movable horizontally, and its position can 




Fia, 63. —The spring myogrraph. 



be adjusted so that it will be knocked over at any desired interval after 
Ky A pointer is attached to K^, 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 K^ may be knocked over after K^ and that the second 




FlQ. 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 K^ 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 



ee 



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 K^ 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 
sufiioiently 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. Make a gastrocnemius and sciatic 



68 PEACTICAL 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, solutioa 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. NajHPO^, 2-04 grms. NajCOg in 100 c.c. distilled water), 
and the other in -7 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 CaSO^, 
or of a 10 per cent, solution of CaClg 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 quiescent ^ 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 

'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 NaOl 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. Einger's ^ 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. 

'A modified Ringer's solution contains NaCl "7 per cent., CaCL, -0026 percent., 
and KCI '035 per cent. 



70 PEACTICAL 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 successivo 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 EXPERIMEKTAL PHYSIOLOGY 



VI 



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. (Porter.) 



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 musclo every time he hears the beat of a metronome, which 
is set to give a beat eviery 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 PEACTICAL 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 he 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 kataboljsm 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 





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74 



PRACTICAL PHYSIOLOGY 



separated and the sciatic nerve exposed ind freed ; the nerve is gently 
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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 







C 3 




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 6 sees., and the contractions 
recorded on a drum revolving at the slowest possible rate (Figs. 69, 70). 



76 PKACTICAL 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 



CHAPTER XVII. (Advanced). 

THE RATE OF TRANSMISSION OF A NERVOUS IMPULSE. 

1'he 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 ekctric current, and, although it is 
accompanied by an electric change, it is something more complex. Its 
rate of propagation is 27 metres per second (88^ 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 EXPEEIMENTAL PHYSIOLOGY 



77 



portion. The entire length of the sciatic nerve is dissected out, and 
the gastrocnemius 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 




Fio, 73. — Diagram of the experiment on the rate of transmiBsion 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,'^ 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 
brachial nerves. These electrodes are connected with the secondary 

' There is a millimetre scale upon the slide of the induction-coil. 



78 PRACTICAL PHYSIOLOGY 

coil of an inductorium, and in the primary circuit is interposed the 
' 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 t>ining-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. 



CHAPTEE XVIII. {Advanced). 

THE POLARISATION OF ELECTRODES AND UNPOLARISARLE 
ELECTRODES. 

Polarisation of Electrodes. — Ordinary metal electrodes in contact 
with a muscle or nerve will be surrounded by lymphj 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 efiects 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. 




Flo. 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 



PEACTICAL 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 (&'), 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 





Fio. 75, — Diagram of the sartorius 
experiment to show the transmission of 
a nervous impulse in buth directions. 



Fig. 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 {¥), where the nerves lie, will cause both halves to contract. 



CHAPTER XX. (Advanced). 



THE RELATION BETWEEN MUSCLE AND NERVE. 
INDEPENDENT EXCITABILITY OF MUSCLE. 



THE 



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 EXPEEIMENTAL 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. 



Fia. 77. — Bin- 
gram of the Bartorius 
musclo to show the 
distribution of its 
nerves. 



CHAPTEE XXI. (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 



PRACTICAL 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 ciicuit 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 




84 



PRACTICAL PHYSIOLOGY 




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ADVANCED EXPEEIMENTAL PHYSIOLOGY 87 

The effect of the constant current upon the conductivity of the nerve 
is determined upon the same preparation. The stimulating electrodes 
are placed upon the central part of the nerve; a minimal stimulus 
is found, and its effect is recorded upon the stationary drum. The 
polarising circuit is now closed through the nerve in either the 
ascending or descending direction, and then the minimal stimulus is 
again applied. It is no longer effective owing to the decrease in the 
conductivity of the nerve. This change in the conductivity of nerve 
is also shown in the experiment upon the absence of fatigue in a 
stimulated nerve (Chapter XXII ) 



CHAPTER XXII. {Advanced). 

THE ABSENCE OF FATIGUE IN A STIMULATED NERVE. 

Nerves are not subject to fatigue, even if they be repeatedly stimulated 
for long periods of time. The following experiment not only demon- 
strates this fact, but at the same time shows that the passage of a 
constant electrical current through a portion of a nerve blocks the 
transmission of the excitatory state which is produced in the nerve by 
a stimulus applied above the polarising electrodes (page 86). 

An induction coil is arranged for faradic shocks, and a pair of un- 
polarisable electrodes are connected by a Du Bois key with a Daniell 
cell. The two sciatic nerves of a pithed frog are dissected up to their 
points of exit from the vertebral column, which is then cut across above 
the nerves. The thighs are out away above the knee, and the two legs 
with their nerves are placed in a moist chamber, and are fixed by pins 
pushed through the lower extremities of the femora. The stimulating 
electrodes, which are connected with the secondary coil by means of a 



88 PRACTICAL PHYSIOLOGY 

Du Bois key, are placed under both sciatic nerves ; the unpolarisable 
electrodes are placed under one sciatic nerve midway between the 
muscle and the stimulating electrodes. The induction shocks are now 
allowed to pass through both nerves for a few seconds ; the muscles of 
both legs are thrown into tetanus. The stimulation is stopped and 
the polarising current is passed through the one sciatic nerve. The 
faradisation of both nerves is again commenced; the muscle in the 
one case will be sent into tetanus and quickly fatigued, but the other 
muscle shows no contraction, for the polarising current passing 
through its nerve blocks the passage of the nervous impulses evoked 
by the stimulating electrodes. When the first muscle is fatigued the 
polarising current should be broken ; the block is removed from the 
course of the sciatic nerve of the other muscle, which is at once 
tetanised by the stimulation of its nerve. 



CHAPTER XXIII. {Advanced). 
THE ELECTROMOTIVE PROPERTIES OF MUSCLE AND NERVE. 

Three simple experiments upon the electromotive properties of 
muscle have already been described (page 51). The following ex- 
periments require more care and very excitable tissues. 

Secondary Twitch from the Heart.— If a freshly prepared and very 
excitable nerve be laid upon the heart of a frog,i so that the cut end 
of the nerve is on the base and the longitudinal surface upon the 
apex of the ventricle, a twitch of the muscle connected with the nerve 
is observed at each contraction of the ventricle. Each time the 
muscle-fibres of the ventricle contract, a "current of action" is pro- 
duced and stimulates the nerve 

A fine glass rod should be placed under the middle portion of the 

' For these preparations the frogs should have been kept cold for some time 
before the experiment. 



ADVANCED EXPERIMENTAL PHYSIOLOGY 89 

length of nerve, which lies on the ventricle, so that the current may 
not be short circuited. 



Flo, 84. — Diagram of the experiment to show the stimulation of a muscle by the 
"current of action " of another muscle. 



Stimulation of a Muscle by the "Current of Action" of another 
Muscle. — The sartorius muscle is very carefully dissected on each 
side, and then the one muscle is placed overlapping the other; the 
contact of the two muscles is secured by gentle pressure with two 
pieces of cork (Fig. 84). Stimulation of one muscle will produce 
a contraction in both; the "current of action" in the first stimulates 
the second muscle. 




Fio. 85. — Diagram of the experiment to show the stimulatiou of a nerve by its own 
"current of injury," 

Stimulation of a Nerve by its own "Current of Injury." — Two 
plugs of kaolin moistened with normal saline solution are placed upon 
a piece of glass, and the tails of the plugs are made to hang over 
the edge (Fig. 85). The sciatic nerve of a pithed frog^ is carefully 
dissected down to the knee, the thigh is cut across, but the leg and 
foot are left intact. The nerve is so placed that its cut surface is 
upon one plug and its longitudinal surface upon the other plug. A 
watch-glass filled with strong saline solution, which is a good con- 
ductor of electricity, is suddenly brought in contact with the ends of 
the kaolin plugs ; thus the circuit is suddenly made and can be 
suddenly broken by the removal of the watch-glass. If the prepar- 
ation be very excitable, a twitch is observed at each make and 

1 For these preparations the frogs should have been kept cold for some time 
before the experiment. 



90 PEACTICAL PHYSIOLOGY 

break of the circuit : the nerve is stimulated when the circuit of its 



"current of injury" is completed or broken. 



CHAPTER XXIV. {Advanced). 

THE ELECTROMOTIVE PROPERTIES OF MUSCLE AND NERVE— 
Continued. THE GALVANOMETER AND THE CAPILLARY 
ELECTROMETER. 

Demonstrations. — The galvanometer and the capillary electro- 
meter are delicate instruments which are easily damaged; they are 
employed to investigate the electromotive properties of muscle and 
nerve. The essential experiments upon that subject have already been 
performed by means of the so-called " rheoscopic frog." In this course. 




Fig. 86.— Galvanometers. 




therefore, the experiments with the galvanometer and the capillary 
electrometer will be demonstrated to the student and only brief 
details will here be given. 

The Galvanometer employed in these experiments is Kelvin's 
reflecting galvanometer. It consists of a suspended system of magnets 
so arranged as to make the system nearly " astatic " ; the magnets are 



ADVANCED EXPERIMENTAL PHYSIOLOGY 91 

surrounded by coils of many turns of fine insulated wire. The resist- 
ance is high, from 5000 to 20,000 ohms. The movements of the 
mirror attached to the magnets are indicated by a spot of light upon 
the scale. 

The amount of current sent through the galvanometer is regulated 
by means of a shunt, which is a resistance box whereby yV'^j tJi7*^> °^ 
TTTiTS*^ of the total current can be sent through the galvanometer 




Fio. 87. — Scale nnd lamp for tlie reflecting galvanometer. 

The electric current from the muscle or nerve is led off by means of 
unpolarisable electrodes, but before an experiment is performed the 
electrodes are tested, for in most cases they are not perfectly iso- 
electric. Any small deflection of the galvanometer due to this cause is 
compensated by a graduated current from a standard battery sent 
through the galvanometer in the opposite direction. 

Perfectly uninjured muscle and nerve are iso-electric, but they are 
generally slightly damaged during the process of dissection and 
preparation. The deflection due to this current of injury or demarca- 
tion current (wrongly called the current of rest) is measured and is 
then increased by a more pronounced injury caused by touching one 
end of the muscle with a hot wire. The muscle is now stimulated by 
a tetanising current applied to its uninjured end ; the deflection of the 
galvanometer is in the reverse direction, due to the current of action 
(formerly called the negative variation) which is produced when the 
muscle contracts. 

The current of injury is, as Gotch pointed out, to be considered as a 
local current of action ; around the injured portion the tissue is in a 
condition of excitation. 

Similar experiments are demonstrated upon nerve. 

Lippmann's Capillary Electrometer. — This instrument is a delicate 
electrical manometer, and is more suitable than the galvanometer for 



92 



PEACTICAL PHYSIOLOGY 



the investigation of the electromotive properties of the fi-og's heart ; 
it responds to very rapid changes of electrical potential. It consists 
(Fig. 88) of a glass tube drawn out at one end to a fine capillary 
tube; this is filled with mercury and is connected with a pressure 
apparatus by the rubber tubing BT. The capillary tube dips into a 
small trough filled with 10 per cent, sulphuric acid ; the bottom of this 
vessel is covered with mercury M in order to provide good electrical 
conduction with the platinum wire. The movements of the column of 
mercury in the capillary tube are observed by means of a microscope 
fitted with a micrometer scale. 

The passage of an electrical current through the 
capillary tube alters the surface tension, and this 
alteration causes a movement of the mercury in 
the capillary tube. The movement of the column 
of mercury is from positive to negative, and the 
extent of the movement is roughly proportional to 
the difference in electrical potential. Based upon, 
these facts are the determination of the direction 
of, and the measurement of the electromotive force 
of, the current which is under investigation. 

"With the capillary electrometer the electromotive 
properties of the frog's heart are demonstrated. The 
base and the apex of the ventricle are led off by 
unpolarisable electrodes to the electrometer : each 
time the heart contracts there will be a diaphasic 
variation, the contracted portion at first becomes negative and then 
positive to the uncontracted part. 




Fio. SS. — DLigram 
of tlie capillary elec- 
trometer. 



CIRCULATION. 

CHAPTER XXV. 

THE ANATOMY OF THE FROG'S HEART AND ITS CONTRACTION. 

Anatomy of the Frog's Heart. — The cerebrum can be destroyed by 
the application of a strong pair of Spencer-Wells forceps to the skull. 
The medulla oblongata and spinal cord are left intact, so that the vaso- 
motor control continues and the circulation is unimpaired. The frog is 
pinned on the corkplate belly uppermost. The skin over the abdomen 
is pinched up and slit up to the mouth. The abdominal wall is then 
divided slightly to one side of the mid line to avoid cutting the anterior 



EI,EMENTAEY EXPERIMENTAL PHYSIOLOGY 



93 



abdominal vein. By a transverse cut the xiphisternum is divided and 
the junction of the anterior abdominal vein with the heart preserved. 
The pectoral girdle is next divided in the middle line. The inner 
blade of the scissors is kept hard against the sternum to avoid injuring 
tlie heart beneath. The divided halves of the pectoral girdle are pulled 
widely apart. The heart is now seen enclosed in a thin membrane — 
the pericardium. This is picked up with forceps and slit open. A 
slender band of connective tissue — the fraenum — connects the posterior 
surface of the heart with the pericardium. A thread is passed under 
the fraenum with fine pointed forceps and tied. The fraenum is then 
divided on the side of the thread remote from the heart. 



Flo. 




"The frog's heart. 



A, Anterior view ; B, Posterior view ; 
section. (Mudge.) 



C, Lonuitiidinnl 



B3' means of the thread the heart can be lifted up and turned over 
for examination. In the front aspect of the heart a single blunt pointed 
ventricle is seen with the bulbus arteriosus and the two auricles — the 
bulbus ascends over the right auricle from right to left. It separates 
into two aortae. Each aorta is divided by longitudinal septa into three 
channels which soon separate and become the carotid, the aortic, and the 
pnlmono-cutaneous arches.^ The auriculo-ventricular groove separates 
the auricles from the ventricle. On turning the heart over the sinus 
venosus is seen, and the white crescentic line which marks the 



' The frog respires both by Bkin and lungs. 



94 PRACTICAL PHYSIOLOGY 

junction of the sinus with the right auricle. Entering the sinus from 
below is a large vein, the vena cava inferior, into which open the 
hepatic veins. Above there enter the two smaller superior venae 
cavae. These are seen ou gently displacing the auricles. The small 
pulmonary veins enter the left auricle. 

The Contraction of the Heart. — The venae cavae and sinus beat first, 
then the auricles, and lastly the ventricle and bulbus arteriosus. The 
blood is returned from all parts of the body to the sinus venosus, 
whence it passes to the right auricle. From the pulmonary veins the 
blood passes into the left auricle. The two auricles simultaneously 
contract and expel the blood into the ventricle. The two blood streams 
do not readily mix in the ventricle owing to the muscular meshwork 
within its cavity. When the ventricle contracts the venous blood on 
the right side is the first to enter the bulbus arteriosus. It is directed 
by a spiral valve within the bulbus into the pulmono-cutaneous arteries. 
The spiral valve is then driven over and closes the orifice of the 
pulmono-cutaneous arch, and the blood (partly arterial and partly 
venous) now passes into the systemic or carotid arch. The resistance 
is least in the systemic arch, so most of the blood at first takes this 
pathway. Finally, as the pressure increases in the systemic arch, the 
pure blood from the left side of the ventricle is expelled into the 
carotid artery. Between the auricles and ventricle there hangs the 
auriculo-ventricular valve. The bulbus arteriosus contains two sets of 
pocket-shaped valves in addition to the longitudinal spiral valve. 

The ventricle becomes smaller, harder, and pale in colour during 
systole, as the blood is driven out of the muscular sponge-work of 
which it is composed. It reddens in diastole. Count the beats per 
minute. 

The Tissue of the Heart possesses Automatic Rhythmic Power. — 
Excise the heart, cutting widely round the sinus venosus, and place it 
in a watch glass. Note the immediate effect and the after-eifect 
on the rhythm. The beats may at first intermit and then become 
more frequent, but quickly settle down to about the same rate as 
before. 

The Effect of Temperature on the Rhythm.— Pour on the heart some 
normal saline solution which has been cooled in ice. The frequency 
becomes greatly lessened. Replace the cold with warm saline (25° C). 
The heart-beats become frequent as the temperature rises. If heated to 
40°-43° 0. the heart stops still in diastole, but may recover if quickly 
cooled. If not cooled the heart passes into the condition of heat rigor. 

Rhythmic Contraction — the function of the Heart Muscle. — Talcing 
another heart, cut away the sinus at the sino-auricular junction. After 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 95 

a short period of inhibition both parts begin to beat, but with a 
different rhythm. The sinus is the more injured, and beats at a slower 
rate. If the cut be made through the auricles, the sinus beat continues 
and is unaffected by the injury. Cut off the ventricle just above the 
auriculo-ventricular groove. After a period of inhibition both auricles 
and ventricle beat. The auricles recover first. Cut through the ventricle 
below the auriculo-ventricular groove. The apex preparation does not 
beat spontaneously. It responds to a prick by a beat, and may in some 
cases be taught to beat rhythmically by rhythmic stimulation. A 
crystal of common salt placed on the apex or the passage of the 
galvanic current through the apex preparation provokes its rhythmic 
contraction. 

Cut out small pieces of the bulbus arteriosus, and place them under 
the microscope in a watch glass containing Einger's fluid. The pieces 
will beat rhythmically. There are few if any nerve cells in the bulbus, 
and there are certainly none in some of these pieces, so the rhythm is 
probably the function of the heart muscle. In support of this are the 
following facts : 

A frog's heart painted with nicotine (1 per cent, solution) continues 
to beat. Nicotine paralyses nerve cells. 

Isolated portions of the mammalian heart will beat rhythmically 
for hours if fed through their nutrient arteries with oxygenated blood. 

The structural elements of the heart are nucleated, branched, and 
cross-striated cells. The muscle-cells are joined together into net- 
works and bands, so as to form one functional whole, and hence 
excitation of any one part leads to the contraction of the whole. The 
first part to begin to functionate in the embryo is the venous end. In 
the mammalian heart it has been shov.'n that muscle fibres of a 
peculiar type connect the auricles with the ventricles ; they form the 
auriculo ventricular bundle. 

The above experiments suggest that rhythmic contractility is the 
inherent function of the cardiac muscle. The muscle of the sinus and 
auriculo-ventricular junction is more embryonic in structure and possesses 
greater power of initiating rhythm. It is less excitable, and conducts 
a stimulus less rapidly than the muscle of the auricles and ventricle. 
The auricular and ventricular muscle is more differentiated in structure. 
The cross striae are more marked. It does not so easily initiate 
rhythm. Owing to its greater excitability and conductivity it follows 
the lead of the sinus. 

During the period of systole the heart is refractory to artificial 
excitation. The excitability returns with diastole, increasing as 



96 PRACTICAL PHYSIOLOaY 

diastole proceeds. The energy of any cardiac contraction depends on 
the previous activity of the heart, on the pressure of the diastolic 
filling, on the resistance to systolic outflow, temperature, nutrition, 
etc. It is indepefadent of the strength of the stimulus so long as the 
latter is efficient. Owing to the refractory period, the slow rate of 
contraction, and the independence of the amplitude of contraction on 
the strength of stimulus, the heart cannot be tetanised. 

By the study— with the aid of the capillary electrometer or string 
galvanometer — of the electrical current of action which accompanies 
the systole, it has been shown that the contraction of the heart is a 
simple twitch, and not a tetanus. The current of action is triphasic in 
the mammal— (1) base negative, ("i) apex negative, (3) base negative. 
The excitatory wave travels from base to apex and from apex to base, 
following the course of the muscle-bands, which start from the base, 
run to the apex, and, turning in there, ascend on the inner wall 
of the ventricle. The current of action travels at the same rate as the 
excitatory state. 

The power of slow, sustained contraction seems to depend on the 
richness of the heart-muscle in sarcoplasm. The heart-muscle possesses 
tone, and this varies with the temperature and nutrition. Muscarine, 
acids and chloroform weaken, while digitaline, caflfeine, and alkalies 
increase the tone of the heart. The auricular muscle of the toad 
exhibits rhythmic alterations in tone. 

Antiperistalsis is difficult to produce because the excitatory process 
in the ventricle is slow, and does not easily affect the more rapidly 
contracting auricle. The refractory period which persists during 
systole also prevents antiperistalsis. The excitatory wave is delayed in 
passing through the more embryonic type of muscle in the sino-auricular 
and auriculo-ventricular junctions, and therefore the auricle beats in 
sequence to the sinus and the ventricle in sequence to the auricle. By 
cooling the sinus and warming the ventricle the sequence of the heart 
can be reversed, for the excitability of the ventrical is by these means 
raised, while that of the sinus is lowered. 

By gently clamping a strip of tortoise auricle muscle between two 
little bits of. cork an artificial block can be created, and the piece of 
auricle below the clamp then beats in sequence to the piece above the 
clamp. The natural delay in conductivity at the auriculo-ventricular 
junction is thus imitated (Gaskell). The conductivity is decreased by 
the clamp. 

The nerve cells of the heart are placed in the least differentiated 
parts : in the sinus (Eemak's ganglion), in the inter-auricular septum 



ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 



97 



(v. Bezold's ganglion), and in the auriculo-ventricular groove 
(Bidder's ganglion). The nerve cells are the cell stations of the 
vagus nerve. The nervous system regulates, but does not initiate 
either the rhythm or sequence of the heart. The maintenance of the 
rhythm depends on the blood, and there is evidence to show that 
it especially depends on the oxygen, and on the mineral salts which are 
in solution in the blood. 

The chief mineral salts, chlorides and phosphates of sodium, potassium, 
and calcium, are dissolved in the blood in minute traces, and are in a 
state of ionisation. The presence of these ions seems to be absolutely 
necessary for the production of the excitatory state. As the mineral 
salts in the serum, with a due supply of oxygen and water, are 
sufficient to maintain the heart in rhythmic activity for hours, it is 
clear that the heart muscle contains a large supply of contractile 
material in its sarcoplasm. 



CHAPTER XXVI. 

METHODS OF RECORDING THE HEART. 

The Suspension Method of Recording the Heart-beat. — The frog is 
placed on a cork plate which is fixed to the stand beneath the lever. 




Pig. so.— Suspension method of recording the contraction of the frog's heart, with 

use of rubber thread as a spring. 

G 



98 PRACTICAL PHYSIOLOGY 

A fine pin is bent into the shape of a hook and passed through the tip 
of the apex of the ventricle. A thread is attached to this hook and to 
the lever. The tissues round the base of the heart are pinned down to 
the cork plate on which the frog rests. 




Pio. 91.— Contractions of the frog's neart. A=auricular, V=ventricular con. 
traction. The time is marked in seconds. The curve should be read from left 
to right. (L.H.) 




Flo. 92. — Contraction of the frog's heart. The curve should be read from right to 
left. The effect of rendering heart bloodless. Note the plateau on the top of the 
normal ventricular curve, and the pointed top after the blood has escaped at the point 
marked by the star. Time marked in fifths of seconds. (L.H.) 

The lever is provided with a long light straw. A fine wire spring is 
attached to the lever, and the heart pulls slgainst this. Adjust its 
tension so that the lever is horizontal,^ and record the heart-beats on a 

^ With the form of heart-lever (Fig. 90) the contraction is represented by the 
down-stroke ; with the lever (Fig. 93) the contraction is indicated by the up- 
stroke. The curves obtained with the former lever can be best compared with 
those made with the latter by turning the tracing upside down and reading from 
right to left. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



99 



drum (slow rate). Note the auricular and ventricular curves, and the 
rounded top or plateau 
of the ventricular curve. 
Kender the heart blood- 
less by opening an 
auricle. The apex of the 
ventricular curve becomes 
pointed. Internal ten- 
sion excites the muscle 
of the heart to more 
prolonged and sustained 
contractions. 

Another method of re- 
cording the heart is shown 
in Fig 93. A long light 
straw lever is taken, and 
a needle is passed through 
it. The needle plays in 
holes in the brass upright 
as shown. 

Effect of Heat and Cold on the Excised Frog-heart. — Expose the 
heart of a pithed frog. Pass a small hook attached to a thread through 
the tip of the ventricle. Excise the whole heart, cutting widely round 
it, and pin the tissues surrounding the base of the heart to a cork 
which is attached to the bottom of the vertical limb of a T-piece. 
The T-piece is placed beneath the recording lever, and the thread which 




Fia. 93.- 



- Lever, for recording the frog's heart. 
(Pembrey and Phillips.) 



The thread from the heart is attached 




FiQ. 94. — Heart chamber. 

was attached to the ventricle is fastened to the lever (Fig. 94). Take 
a tracing of the heart when immersed in a beaker of Ringer's fluid at 
room temperature (12-15° C). Ringer's solution is made by saturating 
0"65 % NaCl with calcium phosphate and adding to each 100 c.c. of 
this solution 2 c.c. of 1 % KCl. Next fill the beaker with Ringer's 



100 PRACTICAL PHYSIOLOGY 

fluid which has been kept in broken ice, and take another record. 
The cooled heart gives slow and forcible beats. The periods of con- 
traction and relaxation are prolonged, the frequency greatly diminished. 
Now fill the beaker with Einger's fluid at 25° 0. The frequency 
becomes greatly increased, and the period of contraction and relaxation 



Fio, 95. — Contraction of the frog's heart recorded by the suspension method 15" C 
and tiiva immersed in saline at 25** O. The curve should be read from left to right. 
The time is marked in secouds. (L.H.) 

greatly shortened. A temperature of about 35° C. causes diminution 
of the tone of the heart. The ventricle ceases to follow the auricular 
rhythm, although it still responds to excitation. At 38° to 43° C. the 
whole heart ceases to beat, and gradually passes into the condition of 
heat rigor. The heat contraction, when once fully established, is not 
set aside by cooling. 



CHAPTER XXVII. 

THE STANNIUS HEART. 

The Stannius Heart. — The heart of a pithed frog is exposed and a 
thread is tied to the fraenum which is then cut away from the posterior 
surface of the pericardium. Pass a ligature under the two aortae and 
then by means of the thread attached to the fra«num gently pull the 
heart towards the mouth of the frog. The dorsal aspect of the heart is 
now readily seen. Draw the ligature round the white crescentic line 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 101 



riAJUiJVAAJU^ 



./iyvA-Ay>vi/i./iAMjVi 



83P 



43" 



FiQ. 96. — Contraction of the frog- s heart recorded by the suspension method. Efifect 
of pouring over the heart normal saline at the temperatures indicated. The water 
cools rapidly when this method is used, aud the heurt is not heated throughout its 
mass to the temperature indicated. (Pembrey and Phillips.) 



102 



PEACTICAL PHYSIOLOGY 





Pig. 97. — Stanniua heart. The first and second 

ligatures (Hedou). 

1, Auricles ; 2, Sinus ; 3, Ventricle. 



which marks the sino-auricular junction and tie it exactly over this line. 
The sinus continues to beat, while the auricles and ventricle, after giving 
a few rapid beats, stand still. The sinus, with its more embryonic type 

of muscle, possesses the greatest 
power of initiating rhythmic con- 
traction. The more specialised 
muscle of the auricles and ven- 
tricle is more excitable, and 
conducts an excitatory wave 
more rapidly, but is less capable 
of initiating rhythm. The ex- 
citatory wave which is started 
from the sinus is blocked by the ligature; thus the auricles and 
ventricle cease to beat. Prick the ventricle; it will respond by a 
single beat to each stimulus. The Stannius preparation is like a muscle 
preparation, and can be used to record the contraction of the heart and 
the latent period. Tie a second ligature just above the auriculo- 
ventricular groove. Both auricle and ventricle are excited by the 
ligature and start beating. The rhythm is no longer the same in the 
three chambers of the heart. The mere contact of the lever or elec- 
trodes resting on the Stanniused heart sometimes evokes rhythmic 
contractions. The inhibitory effect of the first ligature has been 
attributed by some authors to excitation of the vagus nerve. 

The Heart cannot be thrown into Complete Tetanus. — Set up a 
circuit for giving single induction shocks (see Fig. 16, p. 9). Apply 




Pio. 98.— Contraction o£ the frog's heart recorded by the suBi)ension method. The 
effect of tightening the first Stannius ligature at first gently and then firmly. The 
curve should be read from right to left. The time is marked in seconds. (L.H.) 

the electrodes to the Stanniused heart and record the effect of rapidly 
repeated excitations. The heart gives an incomplete tetanus curve. 
Owing to the refractory period it cannot be completely tetanised. 



ELEMENTARY EXPEEIMENTAL PHYSIOLOGY 103 

The Extra-systole and Compensatory Pause.— Excite with a single 
induction shock a rhythmically beating heart. The heart is recorded 
as in Fig. 90 or 93. An extra contraction excited during the diastolic 




Fig. 99.— Effect of tetanising the Stanniused heart. The curve should be read from 
left to right. The time is marked in seconds. The third line shows the period of 
stimulation. (L.H.) 

period of the rhythmically beating heart is followed by a compensatory 
pause. Note that the heart does not respond when excited during 
systole — the refractory period (Fig. 150). 

This period of inexcitability is seen in skeletal muscle (p. 42), but 
is of much shorter duration than the refractory period of the heart. 
The difference probably depends upon a slower metabolism in the 
cardiac muscle. 



CHAPTER XXVIII. 
THE CARDIAC NERVES AND GANGLIA. 

The Intra-cardiac Ganglia, and Nerves. — The vago-sympathetic 
nerves enter the sinus with the superior venae cavae, and form a plexus 
there which contains many ganglion cells (Eamak's ganglion). The 
nerves pass on to enter the auricular septum, which also contains 
ganglion cells (v. Bezold's ganglion). Leaving the septum the nerves 
enter the auriculo-ventrieular junction, where third groups of ganglion 
■cells lie (Bidder's ganglion). 

To see these structures (Fig. 100), forcibly inject the living heart 
Tvith osmic acid (1 % sol.), passing the needle of the hypodermic syringe 
into the auricle. The osmic acid almost instantaneously fixes the heart 
in distension. Cut out the heart and place it in a watch-glass of 1 % 
osmic. After 5 minutes open the auricles under water, look for the 



104 



PRACTICAL PHYSIOLOGY 



septum and cut it out, including its attachments to the ventricle. 
Mount the septum in glycerine, and examine it microscopically. The 
,nerve fibres and ganglion cells will be apparent in the septum. 

Excitation of the Vagi, without dissection. — Destroy the cerebrum 
and spinal cord of a frog, leaving the spinal bulb uninjured. Stick a 
needle electrode into each tympanic membrane. Record the beat of 
the heart and faradise the spinal bulb ; inhibition is produced. 




Fig. 100. — Inter-auricular aeptum and ventricle showing the vagus nerves and 
ganglia. G. 1 Remak's, G. 2 v. Bezold's, and G. 3 Bidder's. (Hedon.) 

Dissection for Exposing the Vagus in the Frog.^ — Lay the pithed 
frog on its back, and cut through the skin and sternum. Pin out the 
fore-limbs so as to pull the divided halves of the pectoral girdle widely 
apart. Open the pericardium and divide the fraenum. From the 
angle of the jaw on either side trace the thin band-like petro-hyoid 
muscles. These muscles arise from the skull, and circle round to the 
thyroid process of the hyoid. The petro-hyoids are crossed by two 
white nerves, which are clearly visible. One, the glosso-pharyngeal, 
curves round from the angle of the jaw, and disappears among the 
muscles of the floor of the mouth. The other, the hypo-glossal, takes 
the same direction, but lies nearer to the mid-line of the mouth. 

The vagus, dividing into its cardiac and laryngeal branches, lies at 

* See alao another method of dissection, p. 108. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



105 



the lower border of the petro-hyoid muscle. It is a small nerve, and 

not easily seen. Having traced the nerve so far, cut away the lower jaw 

and as much of the larynx as can safely be removed. Next cut away 

the mucous membrane which covers the base of the skull and upper 

vertebrae. You will thus expose on either side a broad muscle, the 

levator scapulae inferior. This 

muscle arises from the skull 

round the jugular foramen, 

and is inserted in the scapula. 

Unpin the frog, and hold the 

skull in the left hand, so that, 

while the skull is horizontal, 

the body hangs vertically. Cut 

through the levator muscle, 

and under the upper part of 

this muscle observe the 

vagus ganglion and the vagus 

and glosso-pharyngeal nerves. 

Trace the sympathetic nerve, 

which is marked by black Fig. I01.-Diagr.m of the origin of the vago..ympa- 

niffment alon? the UDUer 'hetic nerve (V.-S.). L.A.8.=levatoranguli scapulae 

pigmeui,, aioug uie uppei ^^^^i^ Ao.=aorta. 1, 2, 3, 4, =flr8t to fourth spinal 

vertebrae to its iunction with nerves. Sym.= sympathetic nerve. G.P. = Glo8so- 

J pharyngeal nerve. G. =vagus ganglion. (Gaskell.) 

the vagus ganglion. The 

cardiac sympathetic fibres arise from the 3rd spinal nerve, and pro- 
bably have their cell stations in the third sympathetic ganglion, 
a fine thread (by means of a sewing needle) under the sym- 





Fio. 102. — Contraction of the frog's heart. The effect of weak stimulation of the 
vagO'Syiiipathetic nerve. The white line marks the duration of excitation. Note 
the latent time, the acceleration and increased tone and the after-effect. The curve 
should be read from left to right. (Pembrey and Phillips.) 

pathetic at the level of the large brachial (2nd spinal) nerve. Tie 
it, and divide the nerve below the ligature. Pass a thread under 
the glosso-pharyngeal and vagus nerves, but do not tie it. 

Place the frog again on the board, and record the heart by the 
suspension method (slow rate of drum). With the interrupted current 



106 



PEACTICAL PHYSIOLOGY 




ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



107 



stimulate the sympathetic. Use fine electrodes and a strength of 
current just comfortable to the tongue.^ 

The heart-beats are accelerated and augmented after a long latent 
period. This eflFect is prolonged for a considerable time after the 
excitation has ceased. The after efifect is decreSased frequency and 
amplitude. 

Next pass the electrodes under the vago-sympathetic trunk. The 
heart-beats will either be arrested (inhibited) after a brief latent period 
or decreased in frequency and amplitude. There is a short after 
effect; the heart soon escapes, even if the excitation be continued. 
The after effect is usually increased frequency and amplitude. The 
returning beats frequently show the staircase effect. Sometimes the 
sympathetic effect overpowers the influence of the vagus. To stimulate 




Pig. 105 — Excitation of vago-sympathetlc. Note the after eCFect— a staircaBe 
augmentation of the heart-beat. The stars indicate the beginning and end of 
stimulation. The downstroke represents contraction, (See footnote, p. 98,) The 
time is marked in seconds. (L.H.) 

the pure vagus fibres, the cerebrum is destroyed, the cervical cord 
divided, and the spinal bulb excited. During the state of complete 
inhibition the heart may not respond to mechanical excitation. 

There is evidence, in the case of the king crab, that the excitatory 
state is transmitted through the heart and the contraction regulated in 
sequence by the nerve ganglia of the heart. On the other hand, the 
ganglion cells, which wander into the embryonic heart of the chick some 
days after it had started beating, can be removed without disturbing 

' The electrodes may be made of fine covered wires. The ends, for J inch, are 
stuck together by melted para6Sn. The paraffin is grooved with a knife, so as to 
lay bare the wires at a point J inch from their end. The wires are passed 
through slits in a small piece of cork. The cork may then be pinned in any 
suitable position. 




108 PRACTICAL PHYSIOLOGY 

the cardiac rhythm. Extra-cardiac nerve-fibres from the vagus have 
their cell-stations in these ganglia. The sympathetic cardiac fibres 
have their cell-stations in the 3rd sympathetic ganglion in the frog, in 
the stellate or 1st thoracic ganglion in the mammal. Non-medullated 
nerve-fibres spin a fine network through all parts of the cardiac muscle. 
A great many of the cardiac nerve-fibres are centripetal or aflferent, and 
convey impulses up the vagi to the spinal bulb, which reflexly control 
the tonus of the blood-vessels, and possibly the frequency of the heart 
G. c. Vs. Hy. Br. and the respiration. The centri- 
fugal cardiac nerves influence the 
frequency and force of the cardiac 
contraction and the conductivity and 
excitability of the cardiac muscle. 
The inhibitory fibres run in the 
vagus and arise from a centre in the 
spinal bulb which is in tonic action 
Fio. 106.— Diagram of nerrea in the frog's and curbs the heart. The function 

neck. Dissection from behind. (Pembrey 

and PhiUips.) 01 the vagus is to decrease the fre- 

quency, force, conductivity and excitability. The sympathetic fibres, 
which arise in the mammal from the anterior spinal roots in the upper 
thoracic region, antagonise the action of the vagus. The vagus, by 
reducing the heart-beat, causes anabolism, and the sympathetic kata- 
bolism of the cardiac muscle. The after-effect of vagus excitation is 
increased energy of contraction, while that of the sympathetic is exactly 
the opposite. The function of the cardiac nerves is to co-ordinate the 
beat of the heart to the needs of the body, and to co-ordinate the 
functions of the other organs of the body to the needs of the heart. 

Dissection of Vago-Sjrmpathetic Nerve from behind. — The skin in 
the mid-line of the back is divided and the scapula lifted up and cut 
away. The fore-limb is pulled outwards and then removed. A small 
plug of paper is placed in the frog's mouth to put the parts on the 
stretch. In front of the divided brachial plexus (Br., Fig. 106) there 
can be seen (Hy.) a much smaller nerve — the hypoglossal — which is the 
first spinal nerve in the frog and passes down to the floor of the mouth ; 
(VS.) the vago-sympathetic, which can be traced from the skull, and 
runs by the side of the carotid artery (C.) and crosses underneath the 
hypoglossal nerve ; (G.) the glosso-pharyngeal. This nerve issues with 
the vago-sympathetic nerve, but soon turns downwards and forwards 
to the floor of the mouth. The glossopharyngeal and hypoglossal 
nerves are then cut and a small piece of the bone containing the 
foramen from which the vago-sympathetic nerve issues is cut away from 
the skull. By means of this piece of bone the vago sympathetic nerve 
can at any time be lifted up without damage and laid upon electrodes. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 109 

CHAPTEE XXIX. 
THE SINO-AURICULAR JUNCTION. THE ACTION OF DRUGS. 

Inhibition Produced by Excitation of the Sino- Auricular Junction. — 
The heart is recorded by the suspension method. Observe the white 
tendinous line which marks the sino-auricular junction. It is curved with 
its convexity upwards. This is known as the crescent. Pin the cork of the 
:fine wire electrodes to the frog-plate so that the ends of the electrodes touch 
the crescent. The ends must not be more than 2 mm. apart. Start the 
drum (slow rate), record half-a-dozen beats, and then tetanise the 
crescent. The heart, owing to direct excitation of the muscle, at first 




» * 

Fxo. 107. — Contraction of the frog's heart. Excitation of the sino-auricular junction. 
Arres't of auricles and increased rate of ventricle (incomplete tetanus). A pause 
followed the cessation of the excitation. The curve should be read from right to left. 
The stars indicate the beginning and end of stimulation. (L.H.) 

beats faster, and then is arrested in diastole. Sometimes the arrest does 
not take place till the excitation ceases. The heart soon escapes from 
arrest. The arrest is due to excitation of the intra-cardiac branches of 
the vagus. Mechanical stimulation of the ventricle during the arrest 
will cause a reversal of the natural sequence. The sinus continues to 
beat during the period of arrest. The excitatory wave is blocked 
in the auricular muscle. 

Action of Muscarine and Atropine. — Dissect out the vago-sympathetio 
nerve and record the effect of excitation of (1) the vago-sympathetic. 



110 



PRACTICAL PHYSIOLOGY 



(2) the crescent. Next with a glass pipette apply to the heart a few- 
drops of nitrate of musoarine (10% solution). The tone, frequency, and 
amplitude of the heart will decrease until at last the heart becomes 
arrested in diastole. Mechanical excitation may still excite the heart 
to give a single contraction. 

Now apply some drops of a 0*2 — 0*5% solution of atropine sulphate. 
The heart will begin to beat again, at first feebly, and then with 




Fia. 108.— Frog's heart. 1, Normal ; 2, three minutes after one drop of 107, solution 
of museariue ; 3, after the application of a weak solution of atropine sulphate. The 
time is marked in seconds. (Fembrey and Phillips.) 

increasing amplitude. Muscarine abolishes the tone, rhythmic power, 
and conductivity of heart muscle, while atropine has in each respect the 
antagonistic action. This experiment succeeds on any ganglion-free 
strip of tortoise heart. After the application of atropine, excitation, 
either of the vagus or of the crescent, is ineffectual, for atropine 
paralyses the post-ganglionic fibres of this nerve. The effect of atropine 
cannot be antagonised by a further application of muscarine. 

A 1% solution of pilocarpine acts in the same way as muscarine, and 
atropine acts as- its antagonist. 

Muscarine is an alkaloid obtained from the poisonous Fly Agaric — a 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



111 



fungus. It is used as an intoxicant in Siberia. It is excreted, un- 
changed, in the urine, and it is stated that the urine is drunk when the 




supply is short, and thus the intoxicant is handed on from one man 
to another. 

Muscarine nitrate, C5H15NO3, is prepared artificially from cholin, 
CjHjjNOj. Cholin is one of the decomposition products of lecithin. 



112 



PRACTICAL PHYSIOLOGY 



CHAPTER XXX. 

THE EFFECT OF NICOTINE, CHLOROFORM AND ETHER UPON 
THE HEART. 

Action of Nicotine. — Dissect out the vago-sjiupathetic and record 
the beat of the heart by the suspension method. Record the effect of 
excitation of (1) the vago-sympathetie, (2) the crescent. Now apply 
to the heart a few drops of a 1 per cent, solution of nicotine. The 
frequency of the heart is at first lessened and then slightly increased, 
for the nicotine firstly excites and secondly paralyses the synapses of 




Fio, 110 — Contraction of ths frog's heart. I. Kormal neart-beat, II. and III. poisoned 
by nicotine. The downatroke represents contraction. The time is marked in seconds. 
See footnote, p. 98. (I1.H.) 

the vagus fibres with the cardiac ganglia. These ganglia contain the 
cell stations of the vagus fibres. Stimulation of the vago- sympathetic 
trunk no longer produces inhibition, but augmentation and acceleration. 
The cell stations of the sympathetic fibres are in the third sympathetic 
ganglion. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



113 




'S 



*.& 






"sa 






¥ 



114 



PRACTICAL PHYSIOLOGY 




% 



The vagus fibres are meduUated as far as the cardiac ganglia, while 
the sympathetic fibres are non-medullated after leaving the third sym- 
pathetic ganglion (Fig. 113). Stimulation of the crescent still produces 

inhibition, for weak doses of nicotine 
do not paralyse the post-ganglionic 
fibres. Nicotine is similarly em- 
ployed to determine the cell stations 
of all the nerve fibres of the auto- 
nomic system (Langley). Too large 
a dose of nicotine paralyses the 
post-ganglionic fibres, and renders 
the contraction of the muscle slow. 
At this stage stimulation of the 
sinus will cause a series of rapid 
beats due to the excitation of the 
cardiac muscle; this acceleration 
shows as an after-efiect a prolonged 
period of diastole. Nicotine finally 
arrests the heart-beat by poisoning 
the muscle. 

Action of Chloroform and Ether. 
— Excise two frogs' hearts and 
place each in a watch glass con- 
taining 5 c.c. of Einger's fluid. 
To one add one drop of pure 
chloroform and cover with another 
watch glass. The heart will be- 
come feeble, lose tone, and finally 
stop beating. It will take con- 
siderably more ether to produce 
the same efiect on the other heart. 
The causation of death from chloro- 
form is cardiac failure. In the 
mammal the arterial pressure falls, 
and the vagus centre is rendered 
hyperexcitable by too concentrated 
Failure of respiration and syncope result from 



tjlJl 

C S S o a .3 
SS S'S oS 

■i^"« ■" » 2 

1 4i^ ^ 

•ag, ■§'35 




a dose of chloroform. 



inhibition and poisoning of the heart. 



Alcohol, - 

Ether, 

Chloroform, 



Bj Molecules. 
1 

8 

100 



By Weight. 
1 

5 

40 



The relative physiological powers of alcohol, ether and chloroform. 



By Volume. 
1 

5 

75 

(Waller.) 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



115 



CHAPTEE XXXI. 



DISSECTION OF THE HEART. THE CARDIAC IMPULSE. 




The Sheep's Heart. — The heart should, if possible, be obtained with 
the pericardium intact, and the lungs attached to it. Open the 
pericardium and test its strength. It is a strong, inelastic, fibrous 
bag, and prevents the over-distension of the right heart. The parietal 
layer of the pericardium is attached to the roots of the large vessels at 
the base of the heart, it thence 
runs over the surface of the heart, 
forming the visceral layer. The 
pericardium in man is attached 
below to the diaphragm, while 
above it is slung to the spine by 
the cervical fasciae. The heart is 
thereby slung in position, and 
cannot twist over during changes 
of posture. 

The front of the heart is re- 
cognised by a groove filled with 
fat, which runs obliquely down 
the ventricles from left to right. 
The groove starts from about the 
middle of the base of the ven- 
tricles to a point a little below 
the middle of the right margin 
of the heart. Running up the middle of the posterior and flatter 
surface of the heart is a similar shallow groove. The heart is 
divided by these grooves into a right and left side, and each 
of these is again divided by a groove containing much fat, which 
circles round the top of the ventricles. Above this groove lie the 
right and left auricles. Note the musculature of the left ven- 
tricle is thick and firm, that of the right ventricle thinner. Both 
the auricles are thin walled. The appendix of each auricle projects in 
front at the base of the heart, as a flat, crinkled, ear-shaped bag. The 
greater part of the auricles lies at the back and sides of the base of the 
heart, and is concealed by the aorta and pulmonary artery. The 
grooves on the surface of the heart mark the position of the septa, 
which divide the heart into four chambers. Trace the right and left 
coronary arteries, which issue from the right and left sinuses of Valsalva 



Fig. 114. — Diagram of sheep's heart to 
show the course of the blood. S. V.C, superior 
vena cava; I, V.C, inferior vena cava; R.A, 
right auricle ; T. tricuspid valves ; Jt. V. 
right ventricle ; P, pulmonary valves ; PA. 
pulmonary artery ; P. V. pulmonary vein ; 
L.A. left auricle; M. mitral valve; L.V. 
left ventricle ; a. aortic valves ; A. aorta. 
(M.S.P.) 



116 PEACTICAL PHYSIOLOGY 

in the root of the aorta, and run in the auriculo-ventricular groove. The 
left is the more important, and divides into the ramus circumplexus 
and the ramus descendens. Ligation of the left coronary artery causes 
fibrillar contraction, ending in arrest of the heart beat. Ligation of 
either of its chief branches may cause fibrillar contraction. Trace the 
coronary vein in the auriculo-ventricular groove. Li the posterior 
wall of the right auricle find the opening of the vena cava inferior 
below, and that of the superior vena cava above. 

In the posterior wall of the left auricle note the openings of the two 
pulmonary veins — in man there are f(mr. The pulmonary artery arises 
in front of the base of the right ventricle, close to the anterior intrar 
ventricular groove. The aorta arises from the base of the left ventricle 
behind and a little to the right of the pulmonary artery. Tie a glass 
tube V.C. into the vena cava superior, and ligature the vena cava 
inferior and the left vena azygos. Tie a second glass tube P.A. into 
the pulmonary artery. The arterial tube should be two feet, and the 
vena cava tube one foot long. Fill the V.C. tube with water, the water 
runs through the right ventricle into P.A. Rhythmically compress the 
ventricles with your hand. The water sinks in V.C, and rises in P.A. 
Stop the compression. If the pulmonary valve is competent, the fluid 
in P.A. will not sink. Note that the root of the pulmonary artery is 
distended by the pressure of the column of fluid. The same experiment 
can be repeated, using one pulmonary vein and the aorta. Remove the 
tubes, and measure the diameter and compare the sectional areas of the 
two venae cavae, the pulmonary artery and the aorta. Extend the 
aorta and pulmonary artery. Both have extensile and elastic walls, 
and although empty, do not collapse. The pulmonary artery is very 
extensile. The venae cavae and pulmonary veins have thin inextensile 
walls which fall together. 

Pass your finger down the superior vena cava through the right auricle 
into the right ventricle. Feel the size of the auriculo-ventricular orifice. 
Now cut through the pulmonary artery just above its origin, and look 
within. Note the three pulmonary semi-lunar valves. Put the pulmonary 
orifice under the tap. The pocket-shaped valves close and prevent the 
water entering the ventricle. Pass a finger through the pulmonary 
orifice, and another through the superior cava. The two fingers meet 
in the right ventricle. 

Next cut open the right auricle, and observe that it surmounts the 
right auriculo-ventricular orifice like an inverted pocket. 

Note the appendix with its fretwork of muscle — the inter-auricular 
septum with the fossa ovalis ; the Eustachian valve, a membraneous 
fold in low relief, which lies immediately beneath the entrance of 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 117 

the inferior vena cava. It is directed from the posterior wall towards 
the internal wall. 

Note also the size and form of the auriculo-ventrioular orifice. Cut 
away most of the auricle, and put the auriculo-ventrioular orifice for a 
moment under the tap. The valve will float up. The flaps are brought 
into opposition by eddies the moment the ventricular pressure becomes 
greater than the auricular pressure. Note the shape of each flap, and 
the upward convexity of the valve flaps when closed, and the star- 
shaped figure formed by their opposition. Note also the papillary 
muscles and chordae tendineae. A band of muscle — the moderator 
band— crosses the right ventricle of the sheep's heart. 

Next cut through the chordae tendineae, and then place the auriculo- 
ventricular orifice for a moment under the tap. The valve-flaps are now 
driven towards the auricle, and the flap is no longer competent. In- 
troduce a pair of scissors between two of the valve-flaps, and cut down 
to the bottom of the ventricle. Then turn round the scissors and cut up 
close to the septum, towards, but not as far as, the pulmonary artery. 
Observe the colunmae carneae and papillary muscles in the lower part of 
the ventricle. These pack together and obliterate the lower part of 
the ventricle during systole. Acting as elastic cushions, they rebound 
in diastole and produce a momentary negative pressure in the ventricle. 
Note the funnel-shaped, smooth-walled upper part of the ventricle — the 
conus arteriosus — which leads into the pulmonary artery. This part is 
not emptied during systole, and blood thus remains in contact with the 
auriculo-ventricular valve, and ensures its closure. Note the form of the 
flaps of this valve, and their attachment to the auriculo-ventricular ring. 
Some of the chordae tendineae are attached to the edges, and others to 
the under surface of the valves. Owing to the papillary muscles and 
chordae tendineae, the auriculo ventricular valve presses on the blood 
during systole, equally with the rest of the ventricular wall. 

Now lay open the pulmonary orifice and note the shape and attach- 
ment of the semi-lunar valves and the small nodule of tissue in the 
free edge of each flap. Observe also the sinuses of Valsalva. These 
favour the formation of eddies, which bring the valves in opposition the 
moment the intraventricular pressure becomes less than the pulmonary 
arterial pressure. 

Cut open the left auricle in the same manner as the right, and 
observe the two flaps of the left auriculo-ventricular valve, the papillary 
muscles, etc., and thickness of the left ventricular wall. Cut across the 
aorta just above its origin and observe the three aortic semi-lunar 
valves. Insert the nozzle of the tap through this valve into the left 
ventricle and turn on the water. The auriculo-ventricular valve closes 



118 



PRACTICAL PHYSIOLOGY 



and prevents the escape of the water. Lay open the left ventricle in 
the same manner as the right, carrying the first incision down the left 
side of the ventricle. Observe the entrance into the aorta and then lay 
this open. Note the orifices of the coronary arteries from the right 
and left sinuses of Valsalva. 

Scratch away the endocardium and look for the auriculo-ventricular 
bundle along the top of the interventricular septum on each side. The 
right and left septal divisions are clearly seen as whitish bands in the 




Fia. 115.— Right auricle and ventricle of calf. (Keith.) 

I, Central cartilage ; 2, main bundle ; 3. A.V. node ; 4, right septal division ; 

5, moderator band ; 8, orifice of coronary sinus. 



sheep's heart. Twigs from the bundle pass by way of the columnae 
carneae into the musculi papillares and reach all parts of the heart. The 
bundle starts from the A.V. node at the base of the interauricular 
septum on the right side, and is in connection with the sino-auricular 
node which is supposed to initiate the rhythm. The nodes are 
made up of peculiar branched cells ; Purkinje fibres form the bundle. 
Section of the bundle interrupts the normal sequence of the heart 
chambers. 

Demonstration of Action of Valves in the ox or horse's heart. 
(Gad.) Two brass tubes with glass windows are tied one (7 cm. in 



ELEMENTAEY EXPERIMENTAL PHYSIOLOGY 



119 



diameter) into the left auricle, the other (5 cm.) into the aorta. The 
brass tubes are connected by side tubes to the bottom and top respec- 
tively of a reservoir containing water. A small hole is made in the 
apex of the heart, and a glow 
lamp is inserted into the left ven- 
. tricle. The wires of the lamp are 
connected with two Grove cells. 
A tube connected with a rubber 
bag is tied into the apex. The 
bag is full of water. On com- 
pressing the bag the auriculo- 
ventricular valves close, while the 
aortic valves open. On relaxa,- 
tion the aortic valves close, while 
the auriculo-ventricular valves open 
(Fig. 116). 

The Cardiac Impulse. — Observe 
and feel the seat of the cardiac im- 
pulse when the subject is (1) stand- 
ing erect, (2) lying horizontal on 
the left, and (3) on the right side. Fia.ii6.-Gad;s method of showing the 

' ^ ' o action of cardiac valves. (Fredencq.) 

The impulse is felt in the fifth or 

fourth intercostal space about IJ inches below the nipple line, and 

3^ inches from the mid-sternal line. It shifts under the sternum when 





Fig. 117. — Marey's cardiograph. The tube is connected with a recording tambour. 
Pressure is adjusted by the screw and spring. 

the subject lies on the right, and to the nipple line when he lies on the 
left side. Owing to the influence of gravity, a different part of the 



l20 



tEACTlCAL 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 



Fio. 118. — Impulse curve of boy aged 15. Tlie nionients when the beart-sounds were 
beard are marked. Time marked in fifths of a eecund. (L.H.) 

shoulder. Connect the cardiograph by means of a X ^^^^ ^^^^ * 
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 cbanged by altering the position of cardiograph. 
In 3 the chest wall is sucked in during the systolic output. Time marked in filths 
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 PRESSUEB. 

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 
eflfect 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 



PEACTICAL PHYSIOLOGY 




Fia. 120.— Mackenzie's Polygi-aph. 



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'ji 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 




FlQ. 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 



PRACTICAL 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 volv/me 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 efiect 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- -I- 90 mm. Hg. 

Blood Pressure in Man. — The pressure may be measured by the 
Leonard Hill sphygmometer. This consists of a graduated glass tube, 




Fcl. 123. — Ari'aiigement of lovers in 
Dudi^eon's sphygmograph. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 125 




Fio. 124.— Sphygraograph provided with time writer (Jacquet } 



Fiu. 1-J5. — Pulse tracing (sphygmogram) taken by Jacquet s aphygmograph. 

ad=the period of tbe pulse curve, & = the pnmary, c=the dicrotic wave. 

Time marked in fifths of a second. 



126 



PEACTICAL PHYSIOLOGY 



which has a small air-chamber above. Near the open end is a aide 
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 



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Fio. 126. — Effect of abdominal and cheat 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 cuflf 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. J. HICKS SOUMAKER LONDON. PATENT. 
Fig. 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 presswe pulse. 

Bepeat 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 efiect 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 rouud the 
fore-arm, raising the pressure in it, emptying the blood out of a vein 




Flo. 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. 



CHAPTEK 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.i The oxygen capacity of the ox blood from which the 
standard was prepared was 18'5 per cent. This was determined by 
' Coal gas contains carbon monoxide as an impurity. 



ELEMENTAEY 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. — Gower'a haemoglobiiiometer. 



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. Eead 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. 



130 



fBACTlCAL 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 
Hayem's fluid.^ 

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 Hayem's fluid. 
The bulb contains 1 part blood and 99 Hayem's fluid. 



^^'> r. I. T^r[....j-^...tt ^^ 

W r /> 





O.lOOmm. 




C. Zeiss 
Jena. 



Flo. 130.— The Thoma-Zeiss haemacytometer. 

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 j^ sq. mm., and has a volume of 
f^^ c.mm., therefore 1 c.mm. contains 4000 times the average 
number found in a square. The dilution of the blood was 1-100. 
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 

'Sodium chloride, g. 2; sodium sulphate, g.lO; 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. 

%ofHb . . , , , 

iP — r = 'worth 01 corpuscles. 

JNo. m sq. '^ 

The average number of red corpuscles is 5,000,000 per 1 c.mm.; of 
white, 10,000 per 1 c.mm.' 

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. 



CHAPTEE 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. 

' 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 cannula 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 





"""""""""""iillKnt 



Fia. 131.— Simple model to show the effect of gravity on the circulation. 

auricular appendices and 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. 




Fia. 132. — Schema to show the flow in rigid and elastic tubes. (Marey.) 

The Flow in Bigid 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 _L 
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 



PRACTICAL 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 ^-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 



CavOLajrifiS 




To 
Manometer 



CfvpiXLaries 



To 
Manometer 



Flo. 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 



ELEMENTARY 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 




BAIRO S TATLOCK-LONDON. 

Fig. 134. — Another model of artificial schema of circulation. 



the body, forces the blood on past the venous valves and overcomes the 
effect of gravity. Baise 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 beat 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 X. 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 3-14. 

Note the effect on the velocity 
Fio. i36.-The stromuhr. ^f (J) 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 




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 \ seconds measure the time it takes for a red corpuscle to 
move through j^^ 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), and observe that the heart 
fills (a) on compressing the abdomen from below upwards (6) 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 sym pathetic is exposed in the neck, 
where it lies behind the carotid artery, and is traced up to the 
sup erior 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-ilaps 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 _|_ 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 _L 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. 




-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. (Sherrington). 



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 pressura The pulsations 
are distorted by the momentum of the mercury. 

The fnspiratory fall of intra-thoracio pressure aspirates blood into 
the intra-thoracic veins and thin walled auricles, and dilates the 




Fig. 139. — Mercurial manometer fitted with iloat 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, and 
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 




'- BAIRDDeTATLOCKi-DNDON 



Fig. 141.— Bering's apparatus for demonstrating tbe action of the respiratoiy pump. 
A, Glass bell, thorax ; B, air-tight base ; K, diaphragm ; C, trachea leading to lungs ; I, 
manometer ; E, tube opening into A ; P, 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 146 

normal level in the cat. In the hutch rabbit the pressure falls, until 
the medullary centres become paralysed from anaemia. The weight of 




Fia. 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 180 mm., 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 ciu-ve) of a curarised 
animal with artificial respiration. Note tho 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 pressui'e. The time is marked in seconds, 
and the signal line shows the duration of vagus stimulation. (L.H.) 

K 



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. 



A B 

Flo. 144.— Aortic blood pressure. A, Effect of exciting the central cud o£ 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 




_j I 1 I ■ I i__i I I I 



Pig. 14S.— Record of arterial pressure {AP) and plethysniogram of limb (volume 
record LV). Excitation of the depressor nerve at signal J. The limb expanded in 
spite of the tall 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: Eespirations deeper and more ample; heart accelerated and 



ELEMENTARY EXPERIMENTAL PHYSlOLOOY 147 




Fig. 146.— Hill's animal table. The table can be raised or lowered at one end, or be reversed. 



'&i0m^^i0k^^^^^^^ 



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 



'■'''' "'■.■^W?i';vVJ//,,f:/;fl 


' 1' lp'r'''i 


,MV«^*"'**" 


1 .(''I'Vi'i*'' 


■',' ' 




;!((*' 

|.-1,i'' 




-■ 



Fio. 148. — Aortic pressure. Spinal cord divided iu upper dorsal region. Effect of 
placing animal in vertical head up posture. The heart emptied. On the retui'n to the 
horizontal posture the circulation was restored. (L.H.) 



takes place of urine and faeces. 
blood. 



The veins are congested with black 




Fig. 149. — Ai-terial pressure ; effect of asphyxia. Animal anaesthetised and 
curarised. At A the artificial respiration was stopped. The largo oscillations 
are Traubc-Herirtg 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 EXPEEIMENTAL 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 
the ends exactly round the white 
crescentic line which marks the 
sino-auricular junction. Tie the 
ligature. The sinus continues to 
beat, while the auricles and ven- 
tricles stand still. Eecord the 
heart by the suspension method. 

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 
circuiting key is placed in the 
secondary circuit, and the coil is 
arranged to give a break shock 
just perceptible to the tongue. 
Close the short circuiting key, 
and set the drum so that the 
striker is just beyond the trigger 
key. Then close the latter. Place 
the lever at a tangent to the 
drum, and bring the writing 
point lightly in contact. Then 
open the short circuiting key 
and start the drum. Stop the 
drum immediately after record- 
ing the contraction. Close the 
short circuiting key, then close 
the trigger key ; lastly open the 
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 O'l 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, (6) 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 eurarised frogs with intact circulation, and also in the galvanometric 
records of the action current of nerve. Waller attributes the staircase 
effect to the influence of COg formed by the katabolism of the active 
tissue. 




Fig. 152. — Stanniiised 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— Continued. THE ACTION OF DRUGS. 

The Suspension Method of Investigating the Action of Drugs 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 Einger'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 Einger's fluid, and record a series of 
contractions. Now replace the flask of Einger's solution with one 
containing distilled water. 

1. 0-75% NaCl solution in distilled water— followed by a Einger or 
Locke's solutions until the normal beat is recovered. 



j;i52 



PEACTICAL PHYSIOLOGY 




ADVANCED EXPERIMENTAL PHYSIOLOGY 153 

2. 0-75% NaCl solution containing 0-3% KCl (5 c.c. 7% KCl 
solution in 100 c.c. 0-60 NaOl) — followed by Einger or Locke's 
solutions until the normal beat is recovered. 

3. 0*75 % NaCl solution containing a few drops of a 5 % solution 
CaCIo followed by Einger 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. 
Einger's solution contains 0-7 % NaCl, 0-03 % KCl, 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 




Fia. 11 r — ' il 11 liLutiliiiii iiid levtjrs 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 and the other upwards. The heart is held fast by means of a 
screw clamp in the auriculo-ventricular groove.' 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. 

' A screw clip, to the bars of which cork wedges are fastened, will do for the 
clamp. 



ADVANCED EXPERIMENTAL PHYSIOLOGY 155 




Fig. 155.— Record of the contraction of auricle and ventricle (toad) by the use of 
Gaskell's clamp aud levers. The upper tracing is the auricle and here the contraction 
is represented by the down-stroke. The time is marked in seconds. (L. H.) 




Fig. 156.— Record of the contraction of the toad's heart by the suspension method. 
Heat applied by the copper wire method. The signal in tho third line shows the 
period during which the sinus was heated. Acceleration of the whole heart was pro- 
duced. In this curve the down-stroko represents the contraction. The time is 
marked in seconds. (L. H. ) 



ise PEACTICAL PHYSIOLOGY 

The result of warming the sinus is a great increase in the rapidity of 
the beats both of the auricle and ventricle. 




Fio. 157. — Continuntion of Fig. 156. Ventricle heated. "Augmentation of the 
Teutriculai' 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 tbe head of 
the board. Pick up the skin over the neck between the lingers, 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 







Fig. 158.- Pissection of the "vagus, the depressor, and cervical sympathetic nerves 
in the rabbit. (Livon.) 

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 Na2S04. 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. Paradise the 
peripheral end of the vagus and observe the inhibition. Stop the 
artificial respiration for a short time, and observe the effect of asphyxia. 



1S8 



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 and 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. 



CL. 



Pn 




Fig. 159.— Dissection of the stellate ganglion (GB) and cardiac accelei-ators. The 
inferior cervical ganglion (CL) and vago sympathetic (vs) are also shown. (Pn) vagus; 
(ac)caroi;id artery ; (asc) suhclavlon 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 15S 

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, and 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-facial 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. 



3'"'D 



Fio. l^il. — 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 aflferent 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 PEACTICAL 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 aflerent 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 ansesthetised 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. 



CHAPTEE XXXIX. (Jdmnced). 
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- 
miasion 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 notch, indicate the opening and closing of 
the semi-lunar valves. 

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 A'entricle 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 

CHAPTEE XL. (Advanced). 
VASOMOTOR 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 blanketpin 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 siims 
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 Einger's fluid plus 1 in 1000 sodium nitrate; the outflow is 
increased owing to vaso-dilatation. Supra-renal extract produces the 
contrary effect. 



ADVANCED EXPEEIMENTAL PHYSIOLOGY 



165 




a 
s 



I 

M 






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. — HUrthles spring manometer. 
Fig. 166.— Sphygmoscope. 

Htirthle'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. 



/i i»r 




Fio. 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 




Fic 168.— HUrthle'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 




mrnmmMummMmjwi^immmNnNWtMmi 




Fig. 169.— Aortic and venfericular pressure curves taken by Htlrthlc manometcra. (Hiii-tble*). 

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 



I- 




Pin. 170.— Schema to show the velocity and resistance heads. B, Pressure bottle. A, Tube 
with piezometers. B K, Pitot tubes. 

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 
diflerence 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. 



W = QHx 



^^9 




Fio. 171.— Cybulski's photo- 
haematochometer. A cannula 
shaped as shown is introduced 
into the blood-vessel. The os- 
cillations of the mercury-menisci 
are pliotogi-aphed. 



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. 
sol. methylene blue. A side tube is pro- 
vided for the purpose of making this injec- 
tion. Note with a stop-watch, or by an 
electric signal and drum, the time between the injection and the 
appearance of the blue at the beginning of the capillary tube. 

Having obtained V, the output can be reckoned if the sectional 
area (a) of the aorta be obtained and the time {t) of a cardiac 
cycle. Measure the diameter of the artery. Half this and obtain 
the radius. 

Count the number of pulses per minute, and by dividing the number 
found by 60 obtain t. Then (^ = mt. 

Now calculate the work of the pump frbm 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 "mm. Hg, the velocity of flow in the aorta as 320 mm. 
per sec. Mercury 13"5 times heavier than blood 

.-. W = 60xllOxl3-5 + «4^. 

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). 

— AjrCashion, 




Reoarder 
Via. 172.— The Caidiometer. 



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. 1,84). 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 ^^ 
= ^=25c.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. (Admniced 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. 



^^^—"^ 



^3? 



Fit). 173. — Diagram of an oncometer and piston recorder. The i-ubbcr 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, tracheal, 
jugular, and carotid cannulae are introduced. The abdominal cavity 



172 PRACTICAL 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 




Fia. 174. — Arterial pressure (1) 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 blood-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 centi-e the greatest 
rate of blood-flow through the kidney can be produced.) Ligature of the 



ADVANCED EXPEEIMENTAL 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 _L-piece being interposed. Eecord the 
volume curve on a moderately fast drum. The tracing shows pulse 
waves and respiratory oscillations. 




Fio. 175. — Limb plethyamograph. 

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 XLIIl. 
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 not 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 



l'?6 PRACTICAL 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. 

Intrathoracic 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 

„ expiration 
Deep inspiration 

,, expiration 

,, inspiration with air-way closed 

„ expiration „ ., „ 

The intra-tracheal pressure varies from - ] ir.ir. Hg. in quiet inspira- 
tion to + 1 mm. Fg. in expiration. During forced breathing with the 
air-way closed the intra-tiacheal pressure is greater than the intra- 
thoracic pressure by the anrount of the elastic traction exerted by the 
langs. All the structures, e.g. heart and blood-vessels, are affected by 
the respiratory variations of pressure. 



)Oul - 10 


mm. Hg. 


7 




„ - 40 









„ - 100 




„ H- 100 





ELEMENTARY EXPERIMENTAL PHYSIOLOGY 177 

Demonstration. The trachea of a dead rabbit is exposed, and a 
ligature tied round ii. The skin is divided over the thorax on one 
side, and the ribs exposed. The intercostal muscles are carefuUjs 
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 iand causes its collapse. 

Demonstration. In the rabbit anaesthetised with urethane 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 Spiuometee 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 



PEACTICAL 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 
Irom 23 to 10, and the averages for the volume of each breath from 
900 to 250 c.e. 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. -f- complemental air 1 500 
c.c. + supplemental air 1500 c.c. It 
is about 3500 c.c, but too much 




Flo. 176. — Spirometer. T, mouthpiece ; M, 
manometer ; Cp, counterpoise ^ B, scale. 



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 EfiFect 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 resists 
ance to the passage of the air in and out of the chest. Discomfort 



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 appai'atus is low. Connect up two gas meters with a T-piece 




Fig. 177. — A stethograpli employed to record the respiration and cardiac impulse 
of the rabbit or cat. The tambours press on either side of the thorax. The T tube 
leads to a recording tambour. 




Pic. 17S.— Stethograph. A, Metal drum ; B, liooks for tapes which pass round 
neck ; 0, rubber discs ; I), 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 XLVr. 
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,' 
absorbs oxygen. The water in G- 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 gasbui-ette. 

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 T-tube. The tap at P makes it 

'Dissolve 100 grins, of stick caustic potash in 50 c.c. of water. Add 10 grms. 
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 





Fio. 179. — Haldane'6 gas analysis appai-atus. 



Fig. 180. — 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, 003 „ „ 

Nitrogen, 7809 „ „ 

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 EXPERIMENfTAL 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 y^^ x 760= 159'G mm. of mercury or 21 

per cent, of an atmosphere. 

79 
Nitrogen approximately yqqX 760 = 600-4 mm. of mercury or 79 

per cent, of an atmosphere. 



184 



PRACTICAL PHYSIOLOGY 
0-03 



Carbon dioxide approximately j^ 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 

had, therefore, absorbed 21 - 16 = 5 x j^rj:r- = 350 c.c. of oxygen and 

discharged 4 x =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 -j=p = ^^ = ^ = 0-8. 

There is a decrease of about ^ 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 absoiption 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 
^~ 79W "■ 

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 : — 

_ ,. . , . 20-94x80 _, ,„ 

Oxygen of mspired air= — ^ _ — =21-18 c.c. 

Oxygen absorbed =21-18- 16 = 5-18 o.c. 

Respiratory quotient = T=r^=F-ro = 0-77. 
Dg o - J 8 

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 : CgHijOg + 60^ = GCOj + 6H,0. 

C0g_6 
O, 6 '■ 

Albumin (empirical formula) : 

CygHiijNigOjgS + 770, = 63C02 + SSHf) + 9CO(NH2)2 + SO3. 



186 



PRACTICAL PHYSIOLOGY 



? = — = 071 
80 



Olem : C3H5(C,3H3302)3 + 80O, = blCO, + 5mp. 

CO, 
0, 

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-Fembrey 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. 




r^ 



M N A B C D 

Fio. 183. — The Haldaue-Pembrey respirfition 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. 

mu J.- CU„ grms. 32 CO, by volume . ^ ,. ^ 

The ratio .. '' ° x t7= r> t, — i = respiratory quotient. 

Oj grms. 44 Oj 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 OP 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. 




IS next 



12 3 

Pig. 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 i'ubber 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 ijito the eudiometer. The water is returned 
back into F. Several exhaustions are needed to 
extract the gases. The eudiometer tube is filled 
with nrercury and surrounded with a water jacket 
to keep the temperature constant. The eudio 
meter is transferred to a vessel of mercury and 
the volume of gas read, the level of mercury 
inside and outside the eudiometer being the same. 
The temperature of the water in the jacket of the eudiometer is also 




Fio. 186. — C. mercury 
vessel ; t. eudiometer ; 
p. pipotte. 



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 diiference 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: — V' H -/ 

^" 1+i. 0-00367 ■ T60" 
where H = the observed pressure, /the tension of aqueous vapour at 
the observed temperature t. The value of 1+L 0-00367 and of /are 
obtained from tables (cf. Sutton's Vohimetric Analyfiia). 



CHAPTEE 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 Dupr4 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. 088, by diluting with distilled water to -jJiyth. 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 




\^ 



Flo. 187.— Ferrii'yanide method of estimating the oxygen capacity of blood. 



the barometer are taken and the gas is reduced to its volume at 0° 
and 760 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. 

/O o 

Hb/ I + 4Na3(CyeFe) + 4NaHC03 = O^ + Hb<5 

+ 4NajCyeFe) + iCOj + 2H2O. 

/O Q 

In this case Hb(^ | represents oxyhaemoglobin, and Hb^^Q 

methaemoglobin, for it is held that the oxygen atoms are united 
together in oxyhaemoglobin but not in methaemoglobin. 



192 



PEACTICAL PHYSIOLOGY 



CHAPTEK LI. (Demonstmtim). 
THE EFFECTS OF CHANGES IN ATMOSPHERIC PRESSURE. 

Decreased Atmospberic Pressure. — Demonstration. A mouse and 
a frog are placed under the bell glass of the air pump (Fig. 189). A 
side tube is connected with a mercury manometer. The latter must 
be long enough to 'indicate the pressure of the atmosphere. On 
lowering the pressure |-J of the atmospheric pressure the mouse 




Fio, 188. — Geryk air-purnp. The piston is covered with oil and opens a spring 
valve during its aseent. 

becomes unconscious and asphyxiated, while the frog is unaffected. 
The effect of lessening the atmospheric pressure depends entirely 
on the partial pressure of oxygen. The normal pressure of oxygen 
is 20'94 per cent, of an atmosphere. At 10 per cent, of an atmosphere 
there arise restlessness and dyspnoea, and at about 4 per cent , death. 
A partial pressure of oxygen equal to 7 per cent, of an atmosphere cor- 
responds to an altitude of 30 000 feet. Death from want of oxygen is 
common in foul wells, mines, etc.; where "choke-damp'' collects, and has 
occurred in balloon ascents. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



193 



Increased Atmospheric Pressure. — Demonstration. A curarised 
frog, with the brain pithed, is placed in the high-pressure chamber, 
the web of one foot is spread out on a wire ring beneath one of the 
glass observation discs. The apparatus is screwed up and connected 
with an oxygen cylinder. The circulation in the web is observed 
with a microscope using an inch objective.^ The pressure is increased 




Fig, 189. — Receiver used with Geryk 
pump in demonetrating the iufluence 
of lowering the atmospheric pressure 
on animals. The apparatus may also 
he used as a drying chamber. 



Fig. 190.— Hill's apparatus for studying effects of in- 
creased atmospheric pressure. Thick glass discs, provided 
with leather washers, close the ends of the chamber. 



to 20-30 atmospheres. The circulation continues unaffected, for the 
pressure is equally transmitted throughout the fluids of the body. 
After ten minutes the chamber is decompressed. Emboli, formed of 
gas bubbles, may appear in the capillaries, and the circulation ceases. ^ 
Such gas emboli are the cause of the symptoms (paralysis, etc.) 
observed in caisson workers and divers. Every 10 metres in depth of 
water roughly equals one atmosphere. The workers are affected on 
or after decompression. Ee-compression and slow decompression is 
the rational cure for the symptoms when they appear. Compressed 
oxygen is also per se a poison. It lowers metabolism, diminishing 

' The image of the circulation can be projected upon a screen with the aid of an 
are light. 

N 



194 PRACTICAL PHYSIOLOGY 

the output of carbon dioxide and the body temperature. This can 
be observed in mice placed in a high-pressure chamber. A mouse 
is affected with dyspnoea in 10 atmospheres of oxygen, goes into 
convulsions and soon dies. In 50 atmospheres^it is instantly killed. 



CHAPTER LII. 

THE INFLUENCE OF CARBON MONOXIDE. 

Carbon Monoxide is a poisonous gas in virtue of its great affinity for 
haemoglobin ; oxygen is displaced and carboxyhaemoglobin is formed. 
Unconsciousness, convulsions, and death are produced by the lack of 
oxygen which arises when a large portion of the haemoglobin is com- 
bined with carbon monoxide and thus deprived of its power of carrying 
oxygen. 

Carbon monoxide is present as an impurity in coal-gas, and in water-gas, 
which is often used in the adulteration of coal-gas, the percentage is a very 
high one. It is due to this gas that death so often results from coal-gas 
poisoning. In the air of mines after an explosion there is present a 
large quantity of carbon monoxide, due to the incomplete combustion 
of coal dust ; miners overtaken by such a disaster generally die from 
poisoning by this gas. 

Demonstration. A white rat or mouse is selected for the experi- 
ment, for it is easier in such animals to see in the snout and feet the 
change of colour due to the formation of the carboxyhaemoglobin. The 
animal is placed under a glass bell jar and coal-gas is admitted ; it 
becomes restless, unconscious, convulsed, and dies within a few seconds. 
This is one of the quickest methods of killing an animal, and has the 
advantage that it rapidly produces unconsciousness. 

If the animal be removed to free air at the beginning of the stage of 
unconsciousness it may recover. The carboxyhaemoglobin is gradually 
dissociated and oxyhaemoglobin is formed in its place. In rabbits this 
occurs very rapidly ; the animal quickly recovers, passing through a 
stage of incoordination. 

Haldane has shown that the best indicator of the presence of poison- 
ous doses of carbon monoxide is a small warm-blooded animal, such as a 
mouse or bird, which is affected, owing to its rapid respiratory exchange, 
much sooner than a man. This method has been employed with success 
by rescue parties entering a coal mine after an explosion. 

The colour of the snout and feet of the white mouse or rat killed by 
carbon monoxide is pink or cherry red. The blood in the viscera has a 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 195 

similar colour and the contrast between the appearance of an animal 
killed by ordinary asphyxia produced by a blow on the head and one 
killed by lack of oxygen due to carbon monoxide is very striking. 

Perform this simple and practical test for carbon monoxide. Kill two 
animals, one by a blow on the head, the other by coal-gas. Cut open 
their bodies and compare the colours of the viscera. Place a drop of 
blood from each animal in separate test tubes, dilute with distilled water 
and examine in good daylight. The blood containing carboxyhaemo- 
■ globin can be distinguished easily by its cherry red colour ; it is more 
pink and less yellow than the ordinary diluted blood. This test can be 
confirmed by the examination of the two samples of blood with the 
spectroscope (p. 346). 

. The treatment of cases of carbon monoxide or coal-gas poisoning is to 
give oxygen to increase the dissociation of carboxyhaemoglobin and to 
keep the patient warm in order that his metabolism and the excitability 
of his nervous system may be raised. 



CHAPTER LIII. 

THE REGULATION OF RESPIRATION. 

The ventilation of the lungs is regulated by a nervous centre in the 
medulla oblongata. This can be proved by a series of experiments, in 
which different portions of the central nervous system are destroyed. 

Demonstkation. The medulla of an anaesthetised animal is 
destroyed in the region of the calamus scriptorius ; respiration ceases 
immediately and the animal dies of asphyxia. 

By experiments upon other animals it can be proved that destruction 
of no other part of the central nervous system will produce this sudden 
cessation of all respiratory movement. If the spinal cord be divided 
close to the medulla the chief respiratory muscles will be paralysed, 
but the movements of the nares will show that the centre is not 
destroyed. 

The respiratory centre is influenced in two ways : (i) by the 
composition of the blood which supplies it, and (ii) by nervous 
impulses which affect its excitability. Experiments upon these points 
can be performed by the student upon himself; he can alter the 
composition of the air in his lungs and thus affect the gaseous 
composition of his blood. 

Influence of breathing air containing carbon dioxide. — The subject 
of the experiment breathes air through a mask and valves and- the 



196 PRACTICAL PHYSIOLOGY 

ventilation of the lungs is determined by a meter. Then, unknown to 
the subject, the air to be breathed is taken from a gas bag containing 
air with 3 or 4 per cent, of carbon dioxide. The breathing is 
increased. Carbon dioxide stimulates the respiratory centre. In 
order to check any effects of change in resistance or of suggestion the 
gas bag should, unknown to the subject, be filled with pure air and the 
experiment repeated. Air containing 8 or 9 per cent, of carbon 
dioxide will produce intolerable discomfort or distress. 

Influence of breathing different percentages of oxygen. — After 
breathing air for some time, the subject breathes pure oxygen from a 
bag: the rate and volume breathed generally show no change, if 
precautions have been taken to avoid the effects of suggestion. If the 
oxygen be moistened with water most men cannot distinguish it from 
air taken from a similar bag. 

Air containing about 15 per cent, of oxygen can be collected free 
from carbon dioxide by breathing slowly through a flask or tin of soda 
lime into a gas bag. Experiments with this gas will show no change 
in the rate or volume of the air breathed. A fall of 5 or 6 per cent, in 
the amount of oxygen in the air is not detected. When the oxygen is 
only 10 per cent, effects are produced; these will be studied in later 
experiments. 

Influence of holding the breath. — Hold the breath to the " breaking 
point " and then collect a sample of alveolar air. The carbon dioxide 
will rise to 7 or 8 per cent. ; the oxygen will fall to about 10 per cent. 

Eepeat the experiment after breathing oxygen for two or three 
minutes. The " breaking point " will not occur so soon, but the rise in 
the carbon dioxide will be the determining factor, for the oxygen in 
the alveolar air may be above 20 per cent, at the end of the 
experiment. The carbon dioxide may rise to 10 per cent. 

Influence of forced breathing. — Take a series of rapid and deep 
breaths for about half a minute, recording the movements by the 
stethograph. Stop breathing when a sensation of giddiness is ex- 
perienced. There will be no inclination to breathe for about a minute. 
The condition is one of apnoea, due to the washing out of carbon 
dioxide from the lungs and blood. The composition of the alveolar air 
will indicate the changes which occurred, as shown by the following 
example. The subject breathed rapidly and deeply, 17 times in 18 
seconds. A sample of alveolar air from the last expiration yielded on 
analysis 2'50 vols, per cent, of carbon dioxide and 19'23 of oxygen. 
Apnoea followed. The sample of the first expiration, when a desire to 
breathe was felt, had the following composition : carbon dioxide 5'59 
vols, per cent., oxygen 12-59 per cent. 



ELEMBNTAEY EXPERIMENTAL PHYSIOLOGY 197 

The experiment should then be repeated with this diflference; 
oxygen instead of air should be breathed. The period of apnoea will 
be much longer, for the subject of the experiment will have more 
oxygen in his lungs and more in his venous blood. 

Forced breathing interferes with the circulation and often produces 
giddiness. An examination of the pulse will show that the systolic 
pressure is diminished by each inspiration. If oxygen is taken in 
during forced breathing there is less discomfort; the brain receives 
more oxygen even if its circulation of blood is disturbed. 

Influence of Muscular Exercise. — The subject of the experiment 
should take vigorous muscular exercise sufficient to produce hyper- 
prioea, but not long enough for the production of " second wind." A 
sample of alveolar air taken immediately after the exercise will show 
in many cases a considerable rise in the percentage of carbon dioxide 
and a small fall in that of oxygen. If the exercise be continued until 
"second wind" has been established, the alveolar air will show less 
carbon dioxide and more oxygen. This accommodation varies in 
different subjects, but the following example may be given. 

Percentage Composition of Alveolar Air. 

COj 
0/ 

0-79 At rest. 
1 '06 After running i mile. 
0'9.3 After running J mile more. 
' ' Second wind. " Sweating. 

" Second wind " appears to be a complex adjustment of the respira- 
tion and circulation to the demands of muscular work. 



CHAPTER LIV. 

CHEYNE-STOKES RESPIRATION. 

In certain cases of heart-disease a well-marked alternation of apnoea 
and hyperpnoea was observed and described by Cheyne and Stokes. 
This phenomenon is characterised by a period of waxing and waning 
respiration followed by a period of apnoea (Fig. 191). 

In some healthy men Haldane and Douglas have shown that this 
type of periodic breathing can be produced in the following way. The 
subject breathes through a small tin of soda lime provided with wire 
gauze to prevent the suction of small pieces of soda lime into the 



Carbon dioxide. 
Vols. 


Oxygen. 
Vols. 


5-27 


14-32 


7-36 


14-03 


5-91 


14-62 



198 



PRACTICAL PHYSIOLOGY 



mouth, and connected at the far end with a piece of tubing about 260 
cm. long and of about 2 cm. bore. The subject thus rebreathes his 
own expired air after it has been deprived of carbon dioxide by the 
soda lime. The percentage of oxygen necessarily falls, the respiratory 
centre becomes excited and hyperpnoea begins. Some fresh air from 
outside the tube will be taken in with each deep breath and the 
percentage of oxygen will rise. The hyperpnoea, however, has washed 




IMIIIIIIIIIIMIIIIIIIIMIIMIMH 



iMIIIIMIinilllMlllllllinillllltMl 



IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIMIIIKII 



Fig. 191. — Oheyne-Stokes respiration. (Pembrey and Allen). 



out a quantity of carbon dioxide from the blood and air in the hings, 
and apnoea results owing to lack of suf5cient carbon dioxide to excite 
the respiratory centre. Thus this alternation of breathing and apnoea 
may continue for several minutes or, it may be, hours. Some healthy 
men exhibit Cheyne-Stokes respiration readily when they perform this 
experiment; others do not. 

Apnoea can be abolished by either (i) air containing 3 or 4 per 
cent, of carbon dioxide, or (ii) pure oxygen, or (iii) air containing a 
deficiency of oxygen, about 1 2 per cent, of oxygen. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 199 

CHAPTER LV. 

THE INFLUENCE OF THE VAGUS UPON RESPIRATION. 

The lungs are supplied with motor and sensory fibres from the vagus 
nerve ; the motor fibres pass to the involuntary muscle fibres of the 
bronchioles and control their contraction ; the sensory fibres carry 
impulses from the lungs to the respiratory centre to co-ordinate the 
respiratory movements. The latter group of fibres can be stimulated 
by the degree of distension of the alveoli, and may be divided into 
expiratory and inspiratory fibres. 

Demonstration. A rabbit is anaesthetised with chloral, urethane, 
or ether. The trachea and vagi nerves are exposed and a cannula is 
placed in the trachea. The respiratory movements can be recorded and 
rendered visible to a large class by fastening one end of a thread to a 
tuft of fur in the epigastric region and the other end to a lever. The 
rate and depth of respiration are observed, then the effect of blowing 
air into the lungs, and, after an interval, the effect of sucking air out 
of the lungs. Positive ventilation produces expiratory apnoea, 
negative ventilation a condition of continued contraction of the 
diaphragm. 

One vagus is now divided ; little or no change will be observed in 
the rate and depth of respiration, but when the other vagus is cut the 
breathing becomes very slow and deep. The experiments of positive 
and negative ventilation are repeated. The effects previously seen are 
absent. It is possible, however, by excessive ventilation of the lungs 
to reduce the pressure of carbon dioxide in the blood and produce 
apnoea. 

The rabbit is now killed, and the tracheal cannula is connected with 
a water manometer. Excitation of the peripheral end of the vagus with 
a faradic current will cause a small rise of pressure in the manometer, 
due to the contraction of the bronchial muscles. 

The rhythm of respiration is maintained by the changes in the 
composition of the blood, not by reflex action due to alterations in the 
distension of the lungs. The vagus, however, influences the respiratory 
centre, thereby co-ordinating the respiratory movements, and, in 
addition, it is probable that it influences at the same time the cardiac 
centre and harmonises the working of the heart and lungs. 

After section of both vagi the animal dies in a day or two from 
septic broncho-pneumonia, due to the passage of food or saliva into the 
trachea and bronchi. The larynx is paralysed by section of the vagi 



200 PEACTICAL PHYSIOLOGY 

high up in the neck, and even if the nerves be cut below the laryngeal 
branches, the paralysis of the oesophagus leads to such an accumulation 
of food therein that particles pass over into the respiratory tract. Dogs 
with a double oesophageal fistula have lived for months after section 
of both vagi ; the fistulae prevent the passage of food or saliva into the 
trachea and lungs. 



CHAPTEE LVI. 
ANIMAL HEAT. 

Difference between Wanu-blooded and Cold-blooded Animals. — 
Warm-blooded animals, such as mammals and birds, regulate their 
bodily heat so that their internal temperature remains constant not- 
withstanding changes in the temperature of their environment ; there 
is little or no difference in the internal temperature of men whether they 
be living in the tropics or in the arctic regions. Cold-blooded animals 
cannot regulate their bodily heat; their internal temperature varies 
with and in the same direction as that of their surroundings. There 
is, however, no hard and fast distinction between the warm-blooded 
and the cold-blooded animals. Hibernating mammals, such as the 
hedgehog, dormouse, and bat, are warm-blooded during the time of 
activity, but become cold-blooded when they hibernate. Young 
mammals and birds in a natural condition of immaturity, when they 
are naked and blind, cannot maintain their temperature at a constant 
level; they need the warmth of the parent's body. A similar con- 
dition is seen in delicate or premature infants. 

The Temperature of Man. — The average temperature of man is 
98°'4 F. (36°-89 C). It is taken by means of a clinical thermometer 
which is either inserted in the rectum, axilla, or mouth, or the subject 
micturates over the bulb of the thermometer. Take the temperature 
of your mouth at each hour of the day. Chart out the results on a 
temperature chart and observe the daily variation (Fig. 192). Take 
the temperature before and immediately after muscular exercise, such 
as a fifteen minutes' run. The temperature may rise to lOC-lOrF. 
(37°'78-38°"33 C.) or even more on a hot day. A rise of temperature 
can be constantly observed if the thermometer be placed in the rectum 
or stream of urine ; the buccal temperature may for the reasons given 
below show a fall in temperature during muscular work. It is impor- 
tant to remember that the daily range in the internal temperature of a 
healthy man may be from 97°-0 F. (36°-l C.) to 99°-6 F. (37°-56 C.) ; 



ELEMENTARY EXPERIMENTAL PHYSIOLOaY 



201 



and that observations taken in the mouth, even when it is firmly closed, 
are liable to be low, owing to the danger of cooling of the tissues of 
the mouth, externally by cold air, internally by the inspired air. 

Heat Begulation. — Take a large frog, and insert a small thermometer 
in the rectum or flex up the thigh, and insert the thermometer 
between it and the abdomen, and record its temperature. Place the 
frog in warm water at 30° C. After 10 minutes observe its tempera- 
ture. It will have reached the same temperature as the water. Cool 
the frog again in cold water and take its temperature again. Then 
place it for 10 minutes in a thermostat heated to 35' C. In the dry 



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Fig. 192. — Daily variation of temperature of mjin. (M, S. P.) 

warm air the frog's temperature will not rise to more than about 
30°-33° C. This is owing to the evaporation of water from the frog's 
skin. Take the temperature of a mouse in the rectum and then place 
it in a dry thermostat at 30° C. for 10 minutes. The temperature of 
the animal will scarcely vary. Note the quickened respiration of the 
animal. This increases the evaporation of water from the lungs. 
Note the way it sprawls out its limbs so as to increase the loss of heat 
by radiation, convection, and conduction. A man cannot bear for 
more than a few minutes immersion in a bath of water at a temperature 
of 44° C, but he can stay for twenty minutes in a dry atmosphere 
heated to 121° C. The body temperature is then regulated by 
sweating. 

Loss of Heat. — An approximate estimation of the amount of 
moisture lost by a man during exercise or exposure to heat can be 
made by weighing him naked before and after the exercise. Moisture 
is lost from the skin and lungs, chiefly from the former. 

The temperature of the skin also influences the loss of heat by 



202 PRACTICAL PHYSIOLOGY 

radiation, convection and conduction. It may be readily taken by a 
mercurial thermometer with a flat bulb. A difference of 10° C. may 
be observed in the temperature of the skin of the hand in summer and 
winter; in warm weather the cutaneous blood-vessels are dilated, in 
cold weather they are contracted. The temperature, however, of those 
parts of the body which are constantly covered with clothing shows 
little change. 

Clothes diminish the loss of heat from the body by enclosing layers 
of stationary air, so that the surface of the trunk and limbs is sur- 
rounded by a layer of air nearly as warm as the skin. 

Experiment. Take the temperature of the skin of the hand and 
compare it with that of the chest or abdomen. Compare also the 
temperatures recorded in the air space between the coat and waistcoat, 
between the waistcoat and shirt, between the shirt and vest, and lastly 
between the vest and skin. In cold weather it will be found that the 
temperature of these strata of air shows a progressive rise, so that the 
air between the vest and the skin is almost as warm as the skin itself. 

The heat lost from the skin depends upon the temperature and 
moisture of the air. The temperature recorded by the wet-bulb 
thermometer is the important factor ; it can be taken by wrapping 
some moist cotton round the bulb of a thermometer and waving it in 
the air, but always keeping it upright, so that no mechanical displace- 
ment of the mercury may occur. 

Sweat. — The discharge of sweat is under the control of the nervous 
system, and a simple experiment will prove the existence of sudorific 
nerves. A cat is killed by an overdose of ether or chloroform. The 
sciatic nerve is exposed and stimulated by a strong faradic current ; 
after a short delay beads of sweat will be seen on the pads of the foot. 
The pad of the opposite leg will serve as a contrast. 

Effect of Anaesthesia on the Temperature of the Body. — Demon- 
stration. A small mammal is anaesthetised with chloral or urethane 
after its rectal temperature has been taken. IF the animal be now laid 
on a table with its limbs spread out, and be exposed to the ordinary 
temperature of a room, its temperature will fall. This is chiefly due to 
the cessation of muscular movement and the paralysis of the central 
nervous system, which regulates the temperature of the body. The 
same effect follows curarisation ; section of the spinal cord in the lower 
cervical region ; and the administration of large doses of alcohol. 

Anaesthetised patients must be protected from cold. Drunkards 
who fall asleep on the roadside on a winter's night are easily " frozen 
to death." 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 203 



CHAPTER LVII. 

INVESTIGATION OF THE MOTOR FUNCTIONS OF THE ALIMENTARY 
CANAL BY MEANS OF THE X-RAYS. 

By A. F. Hertz, M.A., M.D., F.R.C. P., Assistant Physician, late Demonstrator 
of Physiology, Guy's Hospital. 

The soft viscera are transparent and the salts of the heavy metals are 
opaque to the X-rays. When therefore any part of the alimentary 
canal contains food mixed with such a salt, it casts a shadow on the 
florescent screen, when X-rays pass thiough the body. Bismuth salts 
are those generally employed, as they are not absorbed and do not 
irritate the mucous membrane. The oxychloride is the most useful for 
this purpose, as it is unaffected by the hydrochloric acid of the gastric 
juice, and passes through the alimentary canal without influencing its 
motor functions in any way. 

A small breakfast should be taken on the morning of the examination 
in order that the stomach may be as empty as possible when the 
bismuth meal is eaten. Half a pint of bread and milk mixed with two 
ounces of bismuth oxychloride forms the meal. A penny should be 
fixed over the umbilicus by means of strapping, so that the position of 
the stomach and intestine in relation to the umbilicus may be recog- 
nised. It is unnecessary to take photographs, but the outlines of the 
shadows seen on the screen should be marked out with blue chalk on a 
superimposed piece of glass, and subsequently copied on to paper. 

Swallowing. — The examination is begun in the vertical position. 
A large mouthful of the bread and milk is swallowed, and its passage 
through the oesophagus into the stomach is watched. For this purpose 
the rays should pass in an oblique direction through the thorax from 
the front of the right side to the back of the left side in order that 
nothing should interfere with the view of the oesophagus, which 
traverses the clear area between the shadow of the heart in front and 
that of the spine behind. In the vertical position the food passes with 
great rapidity to the back of the pharynx, and thence equally quickly 
down the upper part of the oesophagus. A mouthful of ordinary size 
occupies at any given moment between one and two inches of the 
length of the oesophagus. If several mouthfuls are swallowed in rapid 
succession the whole of the oesophagus becomes visible as a dark 
shadow. 

When the fluid reaches the cardia, its rapid progress is atrested 
owing to the sudden diminution in the lumen of the oesophagus. The 



204 



PEACTICAL PHYSIOLOOy 



lower end of the column of food tapers to a point which represents the 
cardiac orifice of the stomach, the upper limit becoming horizontal. 
At a comparatively slow rate the upper horizontal limit of the shadow 
descends, the lower part remaining unaltered in shape and position 
until the last trace of the shadow has disappeared. This means that 
the fluid runs slowly through the narrow cardia into the stomach after 
having been shot rapidly down the greater part of the oesophagus. 

The time which elapses between the initiation of the deglutition act 
and the disappearance of the last trace of fluid from the oesophagus 
should be measured with a stop-watch. It varies between four and nine 



/<<*a- 




J^M*-3n. " 








L.' S" 6" 

Fig. 193. — Diagrams of position of shadow in oesophagus at intervals of a second 
after swallowing. 



seconds in different individuals. About one-half of the total period is 
required for the food to reach the lower end of the oesophagus, the 
other half being required for its passage through the cardia. 

Fig. 193 represents diagrammatically the shadow as seen at intervals 
of a second. 

In the horizontal position the fluid passes along the oesophagus, 
slightly less rapidly than in the vertical position. A similar but more 
prolonged delay takes place while the food passes through the fcardia, 
the prominent end of the column being in this case rounded. Some- 
times a small quantity of the food follows more slowly, and appears as. 
a thin streak instead of the comparatively broad band seen when the 
oesophagus is filled. 

In the inverted position, with the head directed downwards, tha 



el:ementaey experimental physiology 



205 



food can be seen steadily ascending the oesophagus at about one-third 
the rate it descends in the vertical position. Owing to its slower 
passage, the final delay at the cardia is less obvious. Sometimes a 
little fluid runs back from the stomach into the cardiac end of the 
oesophagus, whence it once again passes into the stomach. 

The Stomach. — The X-ray tube is now lowered so that the rays may 
traverse the abdomen, and the individual faces directly forwards. 
Under the left half of the diaphragm a transparent area is visible, which 




Fig. 194. — Diagram of shadow of stomach. F=fundus ; Pc=pyloric canal ; U=umbilicus. 



represents the gas normally present in the fundus of the empty 
stomach. More of the bread and milk is now swallowed, and it can be 
seen entering the fundus to the right of this clear area ; the shadow of 
the stomach becomes gradually more obvious as more of the food is 
taken. The tone of the stomach diminishes as more food enters, so 
that the intragastric pressure remains constant. Consequently the upper 
and lower limits of the shadow remain almost constant, whatever quantity 
of food is present. When the whole of the meal has been taken, the 
outline of the stomach should be marked on the screen, together with 
the position of the umbilicus. In nearly every case the greater 



206 



PEACTICAL PHYSIOLOGY 



curvature reaches a short distance below the level of the umbilicus. 
The main part of the stomach is almost vertical, and is situated to the 
left of the middle line. The pyloric end, however, passes upwards and 
to ther right across the middle line (Fig. 194). The upper limit of the 
gastric contents is situated about IJ inches below the diaphragm, and 
is bounded by a horizontal line, above which is the gas-containing fundus. 
On voluntarily contracting the abdominal muscles the lower border 
of the stomach is raised several inches, and on relaxing them it 
generally drops an inch or two. 




Fig. 195. — Diagram of shadow of stomach in the horizontal position. 

The peristaltic waves can be seen passing from the centre of the 
greater curvature towards the pylorus. They can, however, be more 
conveniently studied in the horizontal position. 

The examination should be continued in the horizontal position. 
The greater curvature is now seen to have risen above the umbilicus, 
and the clear area in the fundus is no longer visible (Fig. 195), the gas 
having moved to the most superficial part of the stomach, corresponding 
with which a resonant area can be marked out by percussion below and 
to the left of the area of cardiac dulness. 

Peristalsis should now be studied in more detail. The waves start 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



207 




208 



PEACTICAL PHYSIOLOGY 



about midway along the greater curvature. As they pass slowly 
towards the pylorus they become steadily deeper, until about one inch 
from the entrance into the pyloric canal, the extreme pyloric end of 
the stomach is, as a rule, completely separated from the rest of the 
organ (Fig. 197). The part thus cut off gradually diminishes in size 
owing to the further passage of the peristaltic wave and the simul- 
taneous contraction of its longitudinal muscle-fibres. Its contents 
can be seen to pass partly backwards as a reflux stream into the 
stomach and partly through the narrow pyloric canal into the 
duodenum. 






m. 





DT 




vr 



Fig. 197. — Peristalsis in the stomach. 

Intestines. — The shadow of the duodenum cannot, as a rule, be very 
definitely seen, owing to the rapid passage of the bismuth out of it 
and the diminished concentration of the salt due to the large quantities 
of digestive secretions mixed with it. The motor activity of the 
small intestine can therefore only be satisfactorily studied some hours 
later. 

A second examination should be made between four and five hours 
after the bismuth meal. The stomach is then generally empty. The 
shadow of the caecum is seen in the right iliac fossa, and in some 
individuals a small part of the ascending colon is also visible; the 
appendix can only be recognised on rare occasions. This examination 
shows that about four hours are required for the passage of food 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 209 

through the small intestine. A diffuse shadow is generally present at 
this time in the pelvis. It consists of the terminal coils of the small 
intestine, the last few inches of which can often be recognised as they 
join the caecum. With a narrow diaphragm for the X-rays, short lengths 
of intestine can generally be clearly defined and their movements studied. 
A general forward movement of the shadow as a whole, due to 
peristalsis can be recognised. At the same time segmentation is seen 
to occur. The shadow of a short length of intestine, at first of uniform 
thickness, becomes constricted in its centre. The constriction increases 
until the single shadow is more or less completely divided into two. 
Then each half undergoes a similar division, the two central segments 
of the four produced by the second division joining together. The 



y f 

W Q Umbilicus, ^k 



/o 



^ 



Fin. 198. — Segmentatiun of email intestine. 

new central segment then divides again, the segmentation continuing 
at the rate of about seven divisions a minute. The process is shown 
diagrammatically in Fig. 198. 

A further examination should be made on the following day as early 
in the morning as convenient. If possible, the bowels should not be 
opened before this examination. The whole of the large intestine is 
generally visible, and its position should be marked out in the vertical 
and in the horizontal position. In the horizontal position the trans- 
verse colon is approximately on a level with the> umbilicus ; in the 
vertical position it is considerably lower. Both the hepatic and 
splenic flexures are generally acute, especially in the vertical position, 
and the two limbs of the flexures may form a single shadow. The 
effect of straining, as it occurs in defaecation, should be observed : the 

o 



210 



PEACTICAL PHYSIOLOGY 



whole of the colon is greatly depressed, the caecum and ascending colon 
together forming a rounded shadow. Peristalsis is never seen, owing 
to the extremely slow progress of the waves. 

The individual should now retire and open his bowels. On returning 
a tracing of the colon should again be made. The whole of the large 
intestine will be seen to have taken part in the act, even the caecum 
being less full than it was before. In most cases everything beyond 
the splenic flexure is evacuated in defaecation (Fig. 199). 

The above description refers to an average case. Very considerable 
variations occur between diiferent individuals ; sometimes, for example, 
the whole of the bismuth is collected in the pelvic colon at the examina- 
tion on the second morning, the rest of the colon being invisible. 



M 



10.30 a.m-, immediately 
before defaecation. 



10.85 a.m., immediately 
after defcecatioa. 



a 



^ 



12.20 p.m. 



Fia. 199.— Defaecation. 

Bemarkable variations in the shape, size, and position of the different 
parts of the alimentary canal are also observed in perfectly normal 
individuals. 

Care should be taken to expose the body, and especially the hands 
and the testicles, to the rays for as short a time as possible. So long 
as no part of the body is subjected to the direct action of the rays for 
more than ten minutes during the three examinations, it is unnecessary 
to wear any special protective covering. 



CHAPTEE LVIII. 
SALIVARY SECRETION. 

Salivary Secretion. — Demonstration. The submaxillary gland is 
situated within and a little behind the posterior angle of the lower jaw 
bone. 

The animal anaesthetised with ether and chloroform is placed on its 
back, and its head extended. An incision is then made along the 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 



211 



internal border of the jaw bone. The internal border of the digastric 
muscle is thus exposed. This is pulled aside by a hook so as to expose 
the transverse fibres of the mylohyoid muscles. 

The mylohyoid is carefully severed following the line of the digastric 
muscle. The submaxillary and sublingual ducts crossed by the lingual 
nerve are now exposed in the depth of the wound. Wharton's duct is 
the larger and external to the sublingual duct. Just where the lingual 
nerve crosses the ducts it gives off a small branch — the chorda 
tympani. In the angle formed by the origin of the chorda tympani 
from the lingual nerve there lies the sublingual ganglion (it is erroneous 




Hyp M'.h. 



Flo. 200. — Dissection of the submaxillary (G.8.max) and sublingual glands and ducts 
and the lingual nerve L. The chorda tympani leaves the lingual and runs along the 
ducts. J.ext, external jugular vein ; V.G, branch of vein to gland; Hyp, hypoglossal 
nerve ; H.h., mylohyoid ; dig., digastric ; mass., masseter muscle. (Bernard). 



to term this ganglion " submaxillary "). A ligature is placed beneath 
the lingual nerve, central to the origin of the chorda tympani, and the 
lingual nerve is divided central to the ligature. Two ligatures are 
passed under Wharton's duct, and one is tied. The chorda tympani is 
then tetanised and the duct filled with saliva. A V-shaped slit is 
then made into the duct, and a fine glass or silver cannula inserted 
and tied in. 

The sympathetic fibres run into the gland with the arteries. To 
expose these the digastric muscle is divided close to its insertion on the 
jaw bone, and the posterior end of the muscle hooked back. A triangu- 
lar cavity is thus exposed. The carotid artery with the nerves lie in 
the lower part of this, while Wharton's canal and the artery of the 
gland appear in the upper part. The gland itself lies a little more 
to the back. 



212 



PRACTICAL PHYSIOLOGY 



On exciting the cervical sympathetic, or the sympathetic nerve 
filaments which accompany the artery of the gland, the gland will 
pale owing to vaso-constriction. A little thick secretion will at the 
same time appear in the cannula. On exciting the chorda tympani, an 
abundant secretion of thin watery saliva appears. At the same time 
the gland becomes red and turgid. The same effect may be produced 
reflexly by excitation of the central end of the lingual nerve. 

The submaxillary gland is enclosed in a firm capsule. It is fed by a 
branch of the external maxillary artery which enters the hilus of the 
gland. The gland also receives small branches from the great or 




Fia. 201. — Diagram of the submaxillary and sublingual glands and ducts and their 
nerve supply from chorda tympani and sympathetic. F, facial ; Li, lingual ; ct, chorda 
tympani; C.cs., cerrical sympathetic nerve; gl, submaxillary; g^, sublingual 
gland ; cwa, e.c ducts of glandB. (Bernard.) 



posterior auricular artery. The Veins are usually two, but are variable. 
One enters the internal and the other the external maxillary vein close 
to where these veins join to form the external jugular vein. The blood 
coming from the salivary gland can be collected by tying a cannula 
in the external jugular vein and ligating all branches excepting those 
coming from the gland. The exchange of blood-gases in the gland can 
thus be determined. 

Nicotine, 30-40 mgrms. in dog, 10 mgrms. in cat, injected intra- 
venously, paralyses the preganglionic fibres of the chorda tympani for 
about 15 minutes. The ganglion cells of the submaxillary gland are in 
or near the hilus of the gland. 

Atropine sulphate, 10-14 mgrms. in dog, 5-15 mgrms. in cat, injected 
into the blood paralyses the secretory fibres of the chorda tymp?ini, 
while it leaves the vaso-dilator fibres untouched. Pilocarpine nitrate. 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 213 

1-2 mgrms., produces prolonged and plenteous secretion. The anta- 
gonism may be shown by injecting atropine into the blood and then 
injecting a little 2 per cent, solution of pilocarpine into the gland by 
way of the duct cannula. 

If the duct cannula is connected with a mercury manometer and the 
chorda tympani stimulated, the secretory pressure will be observed to 
rise higher than the pressure in the carotid artery. 

The submaxillary gland has been placed in a plethysmograph and its 
volume recorded (Bunch). Stimulation of the cervical sympathetic 
causes very considerable diminution in volume and a scanty secretion. 
Excitation of the chorda tympani is followed by diminution in volume 
in spite of vaso-dilatation. This is due to the copious secretion. 
After injection of atropine the volume is increased by chorda excitation. 

When a cannula was placed in the cervical lymphatic just above where 
it enters the thoracic duct the effect of stimulating the salivary gland on 
the outflow of lymph was observed (Bainbridge). 

Stimulation of the chorda or injection of pilocarpine increases the 
outflow of lymph 2^ times. If Wharton's duct be obstructed the 
lymph flow is not so great. After injection of atropine no such 
increase is found. Stimulation of the sympathetic also increases the 
flow of lymph. 

When a permanent salivary fistula is made, and the duct cannula is 
arranged to empty into a vessel attached to the dog's neck, it is found 
that the character of the secretion varies with the nature of sensory 
excitation (Pawlow). Stones placed in the dog's mouth are rejected 
without flow of saliva. Sand is washed out by watery saliva which 
contains almost no solid or ferment. Food provokes the secretion of 
saliva rich in ferment. The reflex and sub-conscious nervous mechanism 
which controls the secretion of saliva thus carries out actions which are 
similar to voluntary or willed actions. 



NERVOUS SYSTEM. 



CHAPTER LIX. 
THE FUNCTIONS OF THE CENTRAL NERVOUS SYSTEM. 

The Effects of Bemoval of both Cerebral Hemispheres. — In the 
frog the cerebral hemispheres contain only a single layer of nerve- 
cells and have reached only a very low stage of development. If the 
cerebral hemispheres be destroyed by rapidly compressing the anterior 
part of the skull between the blades of a pair of Spencer Wells' forceps 





Fio. 202. —Diagram of the frog'E Fio. 203. —Diagram of a reflex 

brain. 1, Olfactory lobe; 2, arc, 7n='the motor nerve arising 

cerebrum ; 3, pineal gland ; 4, f i ora a nerve-cell in the anterior 

tbalamencephalon ; 5, uptic lube ; horn of the spinal cord and end- 

6, cerebellum; 7, fourth ventricle ing by a motor end plate in a 

and medulla oblongata. muscle ; « = the sensory nerve 

arising from a nerve-cell in the 
posterior root ganglion and form- 
ing a series of dendrites around 
the motor nerve-cell above and 
possessing a sensory nerve end- 
ing in the skin below, 

there will be no loss of blood and the optic thalami will escape injury. 
The first effect of the operation will be a general depression of the 
nervous system, a condition known as shock. This will quickly pass 
off and the brainless frog will show spontaneous movements, will swim 
if placed in water, will turn over if placed upon its back, and will 
behave generally as a normal frog. 

If, however, the corpora striata and optic thalami be destroyed, 
the frog will show no spontaneous movements, will not feed, and will 
soon die unless the evaporation of water from its skin be prevented 
by placing it in a shallow plate filled with water and covered by a 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 215 

bell-jar. The destruction of these portions of the central nervous 
system produces marked shock, but, after this has passed off, the frog 
will still be able to jump, swim, maintain its equilibrium, and perform 
other complicated and co-ordinated movements when it is stimulated 
in the appropriate manner. 

The cerebellum and medulla oblongata are now destroyed by passing 
a blanket-pin through the foramen magnum of the skull, and by 
lateral movements of the pin breaking up the nervous tissue. The 
frog now lies in a limp, toneless condition; shock is well marked, 
and does not pass off quickly. The respiratory movements of the 
nares and of the floor of the mouth cease. The circulation of the blood 
is disordered by the destruction of the vaso-motor centre. 

The "Spinal Animal." — The frog now possesses only its spinal 
cord, but it still shows co-ordinated movements. Its hind legs possess 
tone, and are drawn up against the flanks ; if one leg be pulled away 
from the body, or be stimulated by pinching a toe, it will be with- 
drawn from the source of irritation. The movements are of a reflex 
nature, a response to a stimulus (Fig. 203). 

When placed upon its back such a frog does not right itself, and 
when thrown into water it generally sinks to the bottom, and may or 
may not swim for one or two strokes. 

If such a frog be suspended by the lower jaw, it does not move 
unless stimulated. 

A small piece of filter-paper soaked in strong acetic acid will, if 
placed upon the skin of one flank, act as a 
stimulus, and the leg of the corresponding side 
will be raised to wipe off the offending body. If 
this experiment be repeated five minutes after 
the frog has been dipped in a beaker of water 
to remove the acid, and the leg be held down 
by the hand, then the leg of the opposite side 
will be raised in an apparent endeavour to wipe 
ofl^ the irritating piece of paper. The frog is 
again dipped in the beaker of water to remove 
the acid. 

Ttlrck's experiment upon the time of responsel 
of the spinal animal to a stimulus can now be J?'"- 20*- -a metronome. 
performed. A small beaker is filled with dilute sulphuric acid (1 in 
1000), and is gradually raised until the toes of one of the hind legs 
dip into the acid ; this moment is noted, and then the interval between 
the application of the acid and the withdrawal of the toes is measured 
by a watch or a metronome (Fig. 204). After washing off the acid the 




216 PEACTICAL PHYSIOLOGY 

experiment is repeated with acid of the strength 1 in 600. In each case 
the time of response is much longer than the true time of a reflex action. 

The Action of Strychnine and of Chloroform. — The cerebrum of a 
frog is destroyed by means of Spencer Wells' forceps, and then under 
the skin of the back are injected 10 minims of a saturated solution of 
strychnine (1 in 6700). In two or three minutes it will be noticed 
that the frog cannot readily recover its hind legs after a jump, and 
soon the reflex excitability of the spinal cord is so augmented that a 
slight touch or pufF of wind upon the skin causes a general spasm of the 
muscles. Convulsions quickly follow, and the rigid body of the frog 
rests on the mouth and toes, a position known as emprosthotorms. This 
attitude is due to the different strength of the various muscles ; all are 
thrown into contraction, but the stronger overcome the weaker. The 
muscles are somewhat relaxed after the spasms, but are again sent 
into tetanus by the slightest touch applied to the skin. 

The tonic contractions are followed by prolonged twitches or dmus. 

If during the stage of convulsions a probe be pushed down the 
vertebral canal, and thus the spinal cord be destroyed, the convulsions 
cease at once, showing that the strychnine acts upon the ganglion 
cells and their dendrites in the spinal cord. (See page 220.) 

The action of strychnine should be contrasted with that of chloro- 
form. Under the skin of the back of a frog, whose cerebrum has 
been destroyed by Spencer Wells' forceps, are injected 5 minims of 
chloroform. The first effect is one of stimulation, but this stage of 
excitement is quickly followed by marked inco-ordination and weak- 
ness. In about ten minutes there is marked anaesthesia, paralysis, 
and total absence of reflexes. If the frog be kept moist in a shallow 
plate full of water, and covered by a bell jar, it may recover from 
the efifects of the chloroform in about eight or nine hours. 



CHAPTER LX. 

REACTION TIME. 

The time which elapses between the application of a given stimulus 
and the prearranged response of the subject to that stimulus is known 
as the reaction time. It is obviously more complex than a reflex 
action; this will be readily understood from a consideration of the 
following determination of the reaction time. 

The diagram 205 shows W. G. Smith's reaction time apparatus as 
modified by Colls. The electro-magnetic tuning-fork, T, with 100 



BLEMENTAEY EXPERIMENTAL PHYSIOLOGY 



217 




C3 



Fio. 205. — Diagram of the apparatus for the determination 
of reaction time. 



vibrations per second, is connected with two Daniell cells and with the 
chronograph C. By means of either of the two Du Bois keys, Kj and 
Kj, the chronograph can 
be short circuited. The 
key Kj is closed and Kj 
is open; the tuning-fork 
is set vibrating, but does 
not affect the chrono- 
graph. The subject, 
•whose reaction time is 
to be determined, is told 
to listen for the sound 
of the opening of the 
key Kj and to close the 
key Kg directly he hears the sound. When the key Kj is opened the 
chronograph vibrates in unison with the tuning-fork and the vibrations 
are recorded upon a revolving drum; the closure of the key Kg by 
the subject of the experiment brings the chronograph to rest. The 
number of vibrations recorded upon the drum gives the reaction 
time for sound in j^ths of a second. 

The total reaction time in this experiment is composed of — (1) the 
time taken by the sound to reach the ear ; (2) the time taken for the 
reception of the stimulus by the sensory endings of the auditory nerve 
and the transmission of the nervous impulse to the sensory area ; (3) 
the time for the transmission to the higher centres so that volitional 
impulses may be started in the cerebral motor centres ; (4) the time for 
the propagation of those motor impulses to the nerve cells of the spinal 
cord; (5) the time required for the generation of impulses in these cells 
and their passage down the motor nerves to the muscles of the hand ; 
and (6) the latency of the contraction of those muscles. 

The reaction time for sound is about 0'150 second, for light O'l 95 
Eecond, and for touch about 0'145 second. 



CHAPTER LXI. 

THE RATE OF DISCHARGE OF NERVOUS IMPULSES PROM THE 
CENTRAL NERVOUS SYSTEM. 

The rate at which nervous impulses can be discharged by the central 
nervous system can be investigated in the frog by exciting the nerve 
cells by means of a drug such as strychnine and recording the resulting 
incomplete tetanus ; or in man by the record of the contraction of a 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 219 

muscle thrown into contraction voluntarily, or involuntarily as in 
shivering. 

(a) The Incomplete Tetanus produced by Strychnine. — The cerebral 
hemispheres of a frog are destroyed by com- 
pression with a pair of small pliers or Spencer 
Wells forceps, and then the gastrocnemius 
muscle is prepared with the circulation intact. 
A piece of string is placed under the gas- 
trocnemius muscle and is then tightly tied 
round the upper portion of the tibio-fibula 
and the remaining muscles ; the leg is now 
cut away below the ligature. In this manner 
haemorrhage is prevented, the circulation in 
the muscle is intact, and the muscle is free to ^^H^^^I^H -H 
move with each contraction. A strong pin is ^^H^^^l^^l s 
placed through the lower extremity of the 
femur and is pushed firmly into the cork of 
the myograph ; a piece of moist flannel is 
pinned down over the body of the frog in 
order to prevent the contraction of the 
muscles of the trunk and limbs from disturb- 
ing the lever connected with the gastro- 
cnemius muscle. 

Strychnine is sparingly soluble in water, 
1 in 6700, but a dose of 10-15 minims ^^HI^^H^H g 
(0-592 - 0-888 c.c.) of a saturated solution of 
the drug in normal tap-water saline solution 
will in a frog produce the characteristic con- 
vulsions and death. Such a dose is injected 
under the skin of the frog's back. Twitches 
and convulsions soon begin and the contrac- 
tions of the gastrocnemius muscle are recorded 
simultaneously with the movements of a 

signal marking seconds (Fig. 206). The ^^^|^^|^^| J: 
number of contractions is about 8 or 10 per 
second. This is a measure of the rate of dis- 
charge of the nervous impulses from the 
nerve-cells of the spinal cord. The stage of 
incomplete tetanus is followed by prolonged 
twitches or donus. If the spinal cord be 
destroyed by a probe during the stage of 
tetanus the contractions will cease at once, 



« 



220 



PRACTICAL PHYSIOLOGY 



showing that the convulsions were due to the action of the drug upon 
the nerve-cells and dendrites in the spinal end. 

Becord of a Voluntary Contraction. — If a finger be placed upon a 
muscle voluntarily thrown into contraction, a series of vibrations can be 
felt. These can be recorded and their rate determined in the following 
way. 

A receiving tambour, with a button or a piece of cork fixed upon the 
rubber membrane, is connected with a bellows recorder (Fig. 208), 




Pio. 208. — Brodie*s beUowe recorder. The bellows are made of aluminium plates and 
peritoneal membrane. 

which is arranged to write upon a revolving drum. A chronograph 
is set up for marking the time in seconds. The button of the tambour 
is placed upon the adductor pollicis, or the masseter muscle of the 
subject. When the muscle is voluntarily contracted the lever shows 
a number of vibrations; these are recorded (Fig. 207). The curve 
obtained resembles an incomplete tetanus with 6 or 8 vibrations per 
second. 

CHAPTEE LXIL 



THE FUNCTIONS OF THE ANTERIOE AND POSTERIOR ROOTS OF 
THE SPINAL CORD. THE BELL-MAJENDIE LAW. 

The researches of Bell and of Majendie showed that the anterior 
roots of the spinal cord were motor, and the posterior were sensory ; the 
former nerves are efferent, carrying nervous impulses from the spinal 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 221 

cord to the periphery, the latter are afferent, carrying impulses from 
the periphery to the spinal cord. This law can be proved by experi- 
ments upon a brainless frog, but careful dissection and manipulation 
are necessary. 

The following are the several stages in the experiment. A small 
pair of electrodes is made by passing the bared ends of two pieces of 
fine insulated wire through a piece of cork, and the induction-coil is 
arranged for single shocks. The cerebrum of a large frog is destroyed 
by compression with a pair of Spencer Wells forceps, and then the frog 
is placed belly-downwards upon a cork board, and is confined to this 
position by a piece of wet flannel fastened down tightly by pins. A 
slit is made through the flannel in the line of the vertebral column, and 
the skin is reflected as far as the end of the urostyle. The ilium is 
carefully removed on one side, care being taken to avoid cutting any 
large blood-vessels, for loss of blood would lower the excitability of the 
spinal cord and obscure the dissection. For a similar reason the 
medulla oblongata, which contains the vaso-motor centre, was left 
intact. After the removal of the ilium the nerves of the sacral plexus 
can be easily found and followed up to the spinal cord. Starting from 
the top of the urostyle the laminae of the vertebrae are carefully 
removed by scissors, the points of which should not be plunged deeply 
inwards, otherwise the spinal cord will be injured. After the removal 
of several laminae one of the large nerves of the sacral plexus is 
followed up to its intervertebral foramen, where a black swelling about 
the size of the head of a pin will be seen. This is the posterior root- 
ganglion. It is freed from the foramen by careful dissection, and the 
roots are traced therefrom to the spinal cord. Fine threads are placed 
under the roots, which are then divided in the middle of their length 
by clean sharp scissors. 

Stimulation of the peripheral end of the motor root will cause a con- 
traction of the muscles of the corresponding leg; stimulation of the 
central end with a weak induction shock will cause no movement. On 
the other hand stimulation of the peripheral end of the posterior root 
produces no movement, but a similar stimulus applied to the central 
end sets up a sensory impulse which produces reflex movements. 

The roots of the spinal nerves are longest in the lower segments of 
the spinal cord ; for this reason the experiment is most readily per- 
formed in this region. During development the vertebral column grows 
more quickly than the spinal cord, and thus the lower posterior root- 
ganglia in the intervertebral foramina are separated from the spinal 
cord by a longer length of nerve-roots than in the case of those 
supplying the upper limb. 



222 PBACTICAL PHYSIOLOGY 

CHAPTEE LXIII. 
MiJLLEE'S LAW OF THE SPECIFIC ENERGY OF NERVES. 

The Law of the Specific Energy of Nerves propounded by Johannes 
Miiller states that each sensory nerve gives rise to its own particular 
sensation, whatever may be the means whereby it is excited. Thus the 
retina only gives a sensation of sight, whether it be stimulated by 
light, a blow or an electrical shock. 

This law can be demonstrated by the following experiments. 

Sight. — (i) Two clinical electrodes moistened with strong saline 
solution are connected by means of a key with a Daniell cell; one 
electrode is placed upon the forehead, the other upon the nape of the 
neck. On make or break of the constant current the subject will have 
a sensation of a flash of light. 

(ii) The retina can be stimulated mechanically by pressure on the 
sclerotic. A sensation of light will be experienced. 

Taste. — The end-organs of taste can be stimulated not only by sapid 
substances, but also by mechanical and electrical means, (i) Gentle 
tapping of the front of the tongue gives a sensation of a sweet taste. 

(ii) When the free ends of two wires connected with a Daniell cell 
are placed upon the tongue and the current is opened or closed, a 
sensation of taste is experienced. This experiment can be performed 
with suitable unpolarisable electrodes, so that the objection, that 
electrolysis is produced and the resultant ions are tasted, may be con- 
sidered negatived. Moreover, weak faradising shocks, which would 
cause but little electrolytic action, also give rise to sensations of taste. 

The anode appears to produce an acid taste, the cathode an alkaline 
taste. 

Smell. — The olfactory nerve-endings give rise to a sensation of 
smell when they are stimulated with an electric current. The experi- 
ment can be performed in the following way. The electric current is 
sent through the nose by one electrode connected with the nose by 
filling the nasal cavity with normal saline solution ; the other electrode 
is placed on the forehead. The odour is said to resemble that of 
phosphorus. 

Cutaneous Sensations. — Sensations of touch, cold, warmth, and 
pain can be evoked by gentle application of the point of a metal rod to 
the skin of the hand. The areas or spots which on stimulation give 
rise to the different sensations should be mapped out with ink. 



ADVANCED EXPERIMENTAL PHYSIOLOGY 223 

Mechanical stimulation with a metal rod warmed to the same tempera- 
ture as that of the skin of the hand will give rise to sensations of 
touch, temperature, or pain according to the area stimulated. Miiller's 
law is thus demonstrated in the case of these sensations. 

There is some doubt whether there are specific nerves for painful 
sensations ; it may be that excessive stimulation of any sensory nerve 
causes pain. 



CHAPTER LXIV (Advanced). 

CUTANEOUS SENSATIONS. SENSATIONS DERIVED FROM 
MOVEMENTS. 

The sensations which arise from stimulation of the sensory nerves 
of the skin include four separate qualities. These cutaneous sensations 
may therefore be divided into (a) sensations of pressure, (J) sensations 
of warmth, (c) sensations of cold, (d) sensations of pain. 

On the other hand, surfaces in the interior of the body, such as the 
membranes of the alimentary canal, etc., furnish only sensations of 
pain, which are bound up with sensations referred to the skin, referred 
pain. The defensive mechanism connected with pain has been desig- 
nated protapathk, and is of a lower developmental type than the 
mechanism which furnishes us with the means of making the fine dis- 
criminations of touch and temperature. This more highly specialised 
mechanism has been designated ejpioritie. It has not yet been found 
possible to definitely connect these different forms of sensations with 
diflFerent varieties of sensory nerve endings. 

By simple experiments it is easy to show that the arrangement of 
the machinery which originates these sensations is not regular and 
continuous, an important feature being the pmidiform distribution of 
the cutaneous senses. 

I. Methods Adapted to Ascertaining the Distributions 
OF THE Cutaneous Senses. 

a. The sense of pressure. — It is necessary to distinguish between 
those lighter pressures which aiTect practically only the epidermis and 
the heavier pressures which can excite the subcutaneous tissues. Only 
the first of these can be strictly considered to be concerned in the 
sensation of Unwh. 



224 PEACTICAL PHYSIOLOGY 

Experiment I. Define an area of the skin, for example, on the 
volar surface of the forearm, about 5 cms. square. Carefully work 
over this area with a small camel's hair brush,^ lightly touching 
adjacent small areas and marking with coloured ink the places where 
the contact is felt. The subject of the experiment should close his 
eyes, the observer marking the spots according to the replies of the 
subject. Instead of a camel's hair brush, a fine hair fixed to the end of 
a match may be used. 

b. The sense of temperature. — Bodies at a lower temperature than 
the skin give a sensation of cold, at a higher temperature a sensation of 
warmth. The distribution of these two senses of temperature is not 
identical. 

Experiment II. The most convenient way of testing for cold or 
warm spotS is to take several soldering irons, the points of which have 
been filed down to give a surface of about a square millimetre. These 
are kept in water at the desired temperature till required. Or, a 
hollow copper rod, through which a circulation of water at the desired 
temperature is arranged, may be adopted. 

As in Experiment I. explore the defined area of skin for responses to 
the different contacts in respect of heat or cold. The temperature 
should not be extreme, about 10° above or below that of the surface of 
the skin is sufficient. At extreme temperatures (e.g. 70° C.) other 
sensations come into play. Mark out the distribution of heat and cold 
spots in inks of colours different from that used in Experiment I. It 
may be observed that the temperature spots are readily fatigued. 

c. The sense of pain. 

Experiment III. Some form of algometer is required for the 
purpose of stimulation. Bristles, pointed with a razor, fixed in a light 
wooden rod, an ordinary mounting needle, or perhaps best, a needle 
fixed to the end of a bristle, are alternative forms of algometers. The 
amount of pressure used is of consequence, and to keep this constant 
the degree of bending of the bristle should not vary. Mark out the 
pain spots with distinctive ink. 

It will be found in the above experiments that there is a lack of 
identity in the spots, corresponding to the different sensations, but if 
the marks be allowed to remain for some hours and again the sensations 
are tested, there will be found no alteration of position. 

' Such a brush should be '5 cm. in length and forming a bundle not more 
than '05 cm. at the base. 



ADVANCED EXPEEIMENTAL PHYSIOLOGY 225 

II. Method Adopted for Testing Tactile Spatial 
Discrimination. 

If the skin be touched simultaneously by a pair of compasses the 
points of which are separated, the distance that these must be apart in 
order to appreciate the doubleness of the contact varies in different 
parts of the skin. Special instruments possessing two points, the 
distance between which can be adjusted, are called aesthesiometers. 

Experiment IV. Using either a pair of compasses (the points 
of which are guarded with small pieces of cork) or some form of 
aesthesiometer, note the smallest distance apart the two points must be 
in order that the two contacts may be appreciated, in the case of the 
tip of the tongue, tip of the middle finger, the palm of the hand, the 
forehead, the back of the hand, and the back. 

Sensations Derived from the Contractions of Muscle 
AND from the Accessory Structurks of Movement. 

Nerve endings exist in muscles, tendons and joints, and these are 
liable to be affected by the contraction of muscle, and the tension of 
the tissues adjoining thereby nitiated. It is convenient to speak of 
the resulting sensations as brought about by the mediation of a 
"muscular sense." 

Experiment I. Gun cartridges, filled with shot, are made up to 
different weights. Test the power of discriminating a difference in 
two weights when the absolute weights are small and where com- 
paratively great. It will be found that when the weights are low 
the perceptible diflTerences between two weights is much smaller than 
when the weights are great. 

This is in agreement with Weber's law, which lays down that the 
just recognisable difference between two weights is not a constant 
for any person, but a constant fraction of the weight lifted. Eoughly 
speaking, an increase of 10 per cent, on a weight is just recognisable. 



THE PHYSIOLOGY OF VISION. 

CHAPTER LXV. 

THE DISSECTION OF THE EYE. 

This can be conveniently carried out on the fresh eye of an ox or 
sheep. 

1. Notice in the front of the eye the transparent circular area, the 
cornea, continuous with the greyish opaque border, the sclerotic. This 
coat is continued over the sides and back of the eye, but will be found 
covered with fat. The external eye muscles may be traced in the fat, and 
their tendinous insertion seen in the front part of the sclerotic. The 
optic nerve will be seen penetrating posteriorly. The greyish surface of 
the sclerotic in front is covered by a thin membrane, the conjunctiva, 
which is continued as a lining for the eyelids. 

2. Having removed the fat from a portion of the upper surface of the 
eye so as to expose the sclerotic, make a pair of incisions passing along 
the surface from before backwards, and starting a few millimetres 
behind the corneo-sclerotic junction, let these incisons meet posteriorly. 
Then carefully peel up the sclerotic towards the cornea. Observe the 
dark underlining of the sclerotic, the lamina fusca. Note the choroid 
now exposed, and anteriorly observe that it is covered by a number of 
pale fibres passing forward to the corneo-sclerotic junction, forming the 
ciliary muscle. 

3. Eemove carefully the piece of the choroid lying exposed, and note 
a pale membrane lying beneath, the retina. 

4. Place the eye in a glass basin of water, and make an incision right 
round the eye through all the coats, so as to separate the posterior from 
the anterior half. Examine the posterior half in the water. Note the 
thin retina floating away from the choroid, eccentrically in this the 
optic disc where the optic nerve enters the eye, and the blood-vessels 
radiating from this region. The vitreous humour of jelly-like consist- 
ency will remain attached to the anterior half of the eye. Looking 
through this, note the crystalline lens, at the side of this the radial 
folds of the choroid forming the ciliary processes. The thick portion 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 227 

of the retina can be traced as far as these processes, where it terminates 
with a wavy edge, the era serrata. 

5. Eemove carefully the vitreous humour, and note that it adheres 
to the ciliary processes by its outer coat, the hyaloid membrane. On 
removing the vitreous from the more central portion, note that it 
appears adherent to the posterior surface of the lens. The posterior 
layer of the lens capsule is continuous with the hyaloid membrane. If 
necessary, cut away the vitreous humour so as not to dislocate the 
lens. 

6. Make a r.idial incision from the edge of the sclerotic down to the 
edge of the lens. Carefully separate the iris and ciliary region from the 
lens, and the suspensory ligament will be seen passing from the ciliary 
body mainly towards the front surface of the lens. Carefully separate 
thp lens from this, and the suspensory ligament continuous with the 
capsule of the lens will float up away from the iris. 

7. Cut round the upper half of cornea near its junction with the 
sclerotic. The anterior chamber will be exposed containing a clear 
fluid, the aqtueous humour. Note the thickness of the cornea. At the 
back of the anterior chamber is seen the black cuitain of the iris, with 
its central aperture the pupil. 

8. Notice that the fresh vitreous humour and lens when placed in 
water are not easily seen ; they have almost the same refractive index 
as water. After death the lens slowly becomes turbid. 

9. Hold up the lens and look through it towards a lighted match ; 
it will give an inverted image. 

10. Notice the segmentation of the lens; it is peculiar, and may be 
roughly compared to a segmentation similar to that of an orange com- 
bined with the concentric lamination of an onion. 



228 PEACTICAL PHYSIOLOGY 



CHAPTER LXVI (Advanced). 
THE EYE AS AN OPTICAL INSTRUMENT. 

Preliminary Consideration of the Mechanism of the Eye. — In 
order to understand the refraction of the rays proceeding from external 
objects and forming images on the retina, it is necessary, in the first 
place, to briefly consider the nature of such an optical system as 
constitutes the refractive apparatus of the eye. 

The simplest form of an optical system consists of two media of 
different refractive powers separated by a spheiical surface (Fig. 209). 

If dpe be such a surface, separating a less refractive medium IS from a 
more strongly refractive medium B, n is the centre of curvature, and is 
called the " nodal point." If p be the vertex of tlie curved surface, a 
line through p and n will form the optic axis OA. Eays parallel 
to OA proceeding from S will be conveyed to a point F^ on the optic 
axis. This point is called the posterior principal focus. Eays parallel 
to OA proceeding from B will be conveyed to a point F^, the principal 
anterior focus, p is spoken of as the principal point. These two 
foci, the principal point and the nodal point, constitute the cardinal 
points of such a system. 

- In the actual eye the arrangement is not so simple, as there are 
several refractive media, and three separate surfaces — the anterior 
surface of the cornea, the anterior surface of the lens, and the 
posterior surface of the lens. The arrangement of these is, however, 
symmetrical, and permits of the reduction to two ideal surfaces for the 
three actually existing. This brings the number of cardinal pcints to 
six, as each of these surfaces will possess its own nodal point and 
pi'incipal point, though the anterior and posterior foci will be identical. 

But for practical purposes a further simplification is possible. The 
two nodal points are not far separated, and the two principal points are 
similarly very near, being distant only about -4 mm. from each other. 
We therefore take a "mean" nodal point and a "mean" principal 
point and again reduce the optical conditions to those of a simple 
optical system, consisting of one (ideal) refractive surface. In such 
a "reduced eye" the cardinal points are as follows: — 

Principal point. — 2'3448 mm. behind the anterior surface of the 
cornea, in the aqueous humour. 



ADVANCED EXPERIMENTA.L PHYSIOLOGY 



229 



Nodal point. — -4764 mm. in front of the posterior surface of 
the lens. 





Fia. 209.— Diagrams to Illustrate refraction. 

Posterior principal focus. — 22'647 mm. behind anterior surface of 

the cornea. 
Anterior principal focus. — 12'8326 mm. in front of the anterior 

surface of cornea. 
Eadius of curvature of ideal surface, 5 1248 mm. 

With these data we are now able to understand the formation of the 
image on the retina, and are able to calculate the size of the retinal 
image of an object. 

A ray passing through the nodal point K (Fig. 240) will not undergo 
refraction, and therefore will indicate the position of the image of any 
external point upon the retina. It follows also that the size of the actual 
image may be calculated if we know AB (the size of the external 
image), dK its distance from the nodal point. For 

ab _AB 
Kr~ dK' 



230 PJRACTICAL PHYSIOLOGY 

But c?Z'= distance of object from cornea + distance of nodal point 
behind cornea, which latter is equal to 7'44 mm. 
Kr is equal to 15-17 mm. 

, _ size of external object x 15-17 
distance of object from cornea + 7 44 

If the image be near so as to provoke a considerable effort of accommodation, 
this equation will not represent the size of the formed image. In this case the 
anterior surface of the lens will be more curved than in viewing more distant 
objects, and consequently the constants for the "simple reduced eye" will not 
hold good. The "reduced eye" of Listing corresponds, strictly speaking, to the 
lens accommodated for distant objects. 

The Ophthalmometer, — This is an instrument by means of which the 
radius of curvature of the different surfaces of the eye may be 
measured. The degree of curvature of a reflecting surface will affect the 
size of the image formed from some external object. If some device 
be applied for the measurement of the image and the distance of the 
external object from the reflecting surface be known, then the curvature 
of the reflecting surface can be calculated. 

In Helmholtz's original form of the opthalmometer the measurement 
of the image was achieved by causing the rays reflected from the cornea 
to undergo deviation from their direct course by passing through glass 
plates of a definite thickness. By introducing two glass plates, revolving 
in a common vertical axis, two images could be obtained, and the 
degree of overlapping of these images could be adjusted by altering 
the angle which the two plates made with one another. The distance 
between corresponding points in the two images could be expressed in 
terms of the angle representing the degree of tilt of the plates and the 
refractive index of the glass. The greater the obliquity of the plates 
the more considerable would be the displacement of the images. 

Having obtained a value for the size of the reflected image the 
curvature of the cornea could be calculated from the equation, 

the size of a luminous body (L) distance of body from cornea (d) 

size of its reflected imugo (1) J radius of cornea (|/) 

2dxl 
or r = -^ 

A modification of Helmholtz's ophthalmometer was introduced by 
Javal & Schiotz, in which the double glass plate was replaced by a calc- 
spar crystal and a similar double image obtained. This was still 
further improved by Kagenaar, who substituted compound prisms for 
the crystal, and the instrument so cheapened and improved is generally 
spoken of as an astigmometer. This instrument, which is essentially an 
ophthalmometer, is represented in Fig. 210. 



ADVANCED EXPERIMENTAL PHYSIOLOGY 



231 



It consists of a telescope, which is directed towards the subject's eye, 
the head of the subject rests in the frame, opposite the telescope. The 
eyepiece of the telescope is first adjusted by focusing a thread which 
lies in the plane of the image formed by reflection from the cornea. 




Fig. 210. —The .-tphthalmomcter. 

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 cau be easily ascertained. 




r 



Fio. 211. —The iniagea io the astigmometer. 

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 tlie 
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 refract- 
ing meridian. Each tread of the steps in the illuminated area corre- 
sponds to ons dioptre^ 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. 

KUhne'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 

'A 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 li"ht 
the clearer, a few drops of some fluorescent solution {e.g. eosin) are 
added to the water. An external luminous object is then arranged. 
Ihis inay 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. 








Fia. 212.— KUhne's artificial eye. 



1. The Action of the Comea.-If the illuminated arrow be placed 
approximately m 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 win 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 lena 
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 effect of this second lens is 
tore-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. Bauge of Accommodation. Determination of Near and Far 



ELEMENTARY EXPERIMENTAL PHYSIOLOGY 235 

Points of Accommodation. Line of Accommodation. — At a certain 
distance close to the ej'e 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 eifort, 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 paint 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 dtsgree will enable the observer to see 
objects at varying distances from the eye. The maximum dktance 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 
re-cedes 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 tlie 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 PRACTICAL PHYSIOLOGY 

Experiment II. 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 liole. 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 bluning 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. Fnrkinje 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 .aspeut, 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 ; (6) the anterior surface of lens ; (c) the posterior 



ELEMENTARY EXPERIlVfENTAL PHYSIOLOGY 



237 



surface of lens. The images at (a) and (5) are erect; at (c) is inverted ; 
the image at (6) 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 tlie optic axis 
of the subject. When properly 
adjusted there should be observed 
reflected from the eye of the sub- 
ject 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, 
tliis 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 
in being inverted. If 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 tnis image is the 
only one to change, thus indicating that the change is in the anterior 
surface of the lens. 

ExPERiMKNT 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 fitted with windows (Fig. 214). 

The observer's eye is at 0, the subject's at S. At C 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 fF, 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. 




Fio. 213.— The pbakoscopo. 



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 phakoacope. 



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 Kuhne'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 
80 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 imagi 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'). Thi.s 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 Ketina. — 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 sidf, 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 iw spot. 

Experiment III. Eemove the objective from 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 Ketina. — 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 Fseudoptics 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 HA 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 FI.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 appears 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 blinil 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 

manntT, 

Q 



242 PEACTICAL PHYSIOLOGY 

ExPERiMRNT 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, and 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 reddisii-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 lutea. 

5. Acuteness of Vision in different Begions of the Betina. — 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 spaqe 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 appi-ars 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. 



ELEMENTAKY 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. 

ExPKRiMKNT 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 : 

I)- 

where d = distance of person from the types and T) = 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 Eetina. — 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. 

Expkrimj;nt II. If a perimeter or campimeter be used the 
boundaries of the field f(jr the different colours can be defined. (See 
use of perimeter.) 

9. The Perception of Light in different Regions of the Betina. — 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 



ELEMBNTAEY" 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 tho 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 PHVSIOLOGY 

sensations to fusion of certain simpler sensations, which are described 
as primary colour sensations. In the Young-Helmholtz theory the 
primary sensa ions are those corresponding to red, green, and blue- 
violet; in the Bering 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 npon all three visual substances 
to different extents. If all are affected more or less equally, the com- 
pound sensatiou of white is produced. 

In the Hering 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 lengtli. 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 EXVEEIMENTAL 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 WMte. 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. 




Frr. 5r(l. 

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 



PEACfTICAL PHYSIOLOGY 



The rate necessary for the flickering sensation to pass into complete 
fusion depends u])on 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.] 




Pio. 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. 




FlO. 218. 

Fechncr 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 Helmiioltz 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.! 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 thw green. If now the whole circle be 

' 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 fieMs of different 
colours, as well as one of white j 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 giound 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 r5 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. Tiie 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 cxpeiimenls, 

[Advanced Experiment. Arratige 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 snurce 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.] 

EXPEUIMENT III. Place the dark grey papers of Experiments III. 
and IV., Section G, of the Milton Bradley Pseudoptics on the different 
coloured fields and cover with tissue paper. Observe the contract 
colour that appears in the grt-y 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 II., 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 two 
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 difi"erent 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 bliud, and very rarely violet blindness may exist. 



254 PRACTICAL 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 dut 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 au 
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 



ELEMENTAEY 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 flegard. 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 Begard. 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 difi^erent 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 Bonders' 
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 Eegard. 

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 oif, and a finger be held up in the binocular 



256 PEAOTICAL 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 imago 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 eyp, 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 disnppear. 

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 towaids 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 



ELEMENTAEY 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. 



258 PRACTICAL PHYSIOLOGY 

These effects can perhaps best be shown by examining the Martius- 
Matzdorff 1 series of diagrams with a stereoscope. 

Visual niusions. — 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. 

CHAPTEE 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. — ^Rays 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 tlie 
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. 

'The diagrams can be obtained from Winokelmann nnd Sohne, Berlin; 
Petzoldt, Leipsic; or from Messrs. Baird & Tatloek, Cross Street, Hatton 
Garden, London. From the latter firm can be obtained any of the instruments 
jnentioned above or the Milton Bradley Pseudoptics Series. 



ELEMENTARY EXPERIMENTAIi PHYSIOLOGY 259 

Experiment I. Adopting the method of ascertaining the near 
point of accommodation in Scheiner'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.^ — 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 emmetropic. 

Presbyopia, — As a result of advancing age the power of accommoda- 

' Properly speaking, astigmatism should be incladed in this section. We have 
thought it best, however, to consider it in a separate section. 



260 PEACTICAL 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 di£&- 
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. 

Hypenuetropia. — 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 

ExPEEiMENT. Using Kiihne'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 Kiihne'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. (Advcmced). 
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 PEACTICAL 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 Kuhne'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 Befracting 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. 

(jb) Dark specks or irregularly stellate figures may be seen, depending 
upon imperfections in the lens or its capsule. 

(c) Muscae Volitcmtes. 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 LXXIII. 

THE INSTRUMENTS USED IN THE CLINICAL INVESTIGATION OP 

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 ih&fiAd 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 




Fio. 219. —The perimeter. 



perimetric observations. Starting at the extreme distance the mark Oh 
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- 
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 





Fio. 220. — Opllthalmoacopes. 



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 efi'ort 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 diflBculty 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. 

CHAPTEE LXXIV. 

DISSECTION OF THE EAR IN THE SKATK 
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 membianous 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 
' A dog-fish can be used for this dissection. 



268 PEACTICAL PHYSIOLOGY 

iwo 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. Kepeat 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. 



ELEMENTARY EXPEEIMENTAL 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 cliange 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 foi-m, 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 CHEMISTRY 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-stuflfs 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 
C„H2„0„. 

Carbohydrates are found chiefly in vegetable tissues, but also occur 
in animal tissues. They form very important food stuff's, 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. MONOSACOHAKIDES. 

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 C^HjqOj, hexose CgHj^Og. 

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, CHg-CHj-CHaOH, 

and it contains the end group - CHgOH. If, on the other hand, the 
hydroxyl group be attached to a central carbon atom — as in secondary 
propyl alcohol, CH3 - OHOH - CH3, 

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 + = CHs - CHO + HjO. 

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, 

CHg - CHO + = 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. CuSO^-(-2NaOH = Cu(0H)2 -H NajSO^. 

Cupric sulphate + caustic soda. Cupric hydroxide -I- 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-(-E*.CHO = Cu2(OH)2 -1- R . COOH -1- H^O. 
Cupric hydroxide + aldehyde. Cuprous hydroxide + acid. 

The cuprous hydroxide then loses a molecule of water and changes into 
the oxide : Cu2(OH)2 - HjO = CujO. 

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 : 
AgaO + R CHO = 2Ag -H 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. 

S 



274 PRACTICAL PHYSIOLOGY 

Reactions of Monosaccharides depending on the fact that they are 

aldehydes. 

/. Their Beducing Power. — Dextrose is the aldehyde corresponding to 
the hexatomic ^ alcohol, sorbite. 

CHjOH - (CHOH)^ - CH^OH, CH^OH - (CHOH), - 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. 

Trormmr'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. Keduction 
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 CuSO^ and NaOH.^ 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. Nylandei's Test. — To about 5 c.c. of dextrose 
solution in a test tube add about 1 c.c. of Ny lander'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. 

^ A hexatomic alcohol is one which contains six OH groups. Glycerine is called 
tri-atomic, because it contains three such groups. Ethyl alcohol is monatomio, 
because it contains one. 

Tor the exact formula for Fehling's solution see p. 450. 



PHYSIOLOGICAL CHEMISTRY 275^ 

ExPERiMKNT 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. 

//. 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 

CH^OH 

i 
(CH0H)3 

i 

C = N-NH-C6H5 

I 

C = N-NH-C8H5 

I 
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 




Pio. 221.— Osiizoiie crystals, x 400. 
A, Fhenyl-glucosazone ; B, Fhenyl-maltosazone ; C, Phenyl-laotosazono, 



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 H2SO4. 
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 H2SO4 (almost 
saturated with K2SO4 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.' and ^ 

The following are the melting points of some of the most important osazones : 
Dextrosazone,' - - - 204-205° 0. 

Laetosazone, - - - 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 HCl it breaks up, 
phenyl-hydrazine being set free, and a, body called an osone resulting. This 
latter has the formula GHjOH - (CH0H)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, 

OHaOH - (CH0H)3 - CO - CHO + H2 = CHjOH - (CHOH), - CO - CH^fOH) 
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 CO3 and HjO. 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 

' 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 i(7'-<)(0'000154) where L = the 
height of the mercury column of the main thermometer above the sulphuric aoid 
measured in degrees; jr=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. 

^ 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. 

^ Laevulose forms the same osazone as dextrose. 



278 



PEACTICAL PHYSIOLOGY 



the corresponding alcohols or by reduction of the coiresponding 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-bafiic^ Acids. 


Di-basic^ Acids. 


Sorbite 

Mannite 

Duleite 


Dextrose 
Mannose 
Galactose 


(Laevulose) 
(Laevulose) ^ 


Gluconic 

Mannonic 

Galaetonie 


Saccharic 

Mannosaccharic 

Mucio 



Another monobasic acid is glycuronic — CHO. (CHOH^). 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 CHg - CO - CH3 which may be ob- 
tained by oxidation of secondary propyl alcohol, 

CHg - CHOH - CH3 + = CH3 - CO - CH3 + Bfi. 

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. Seliwanoff's Test. — Mix a few cubic centimetres 
of a solution of laevulose with half its volume of concentrated HCl. 
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. 

Eepeat this experiment with pure dextrose solution instead of 
laevulose. A slight red colour develops but no precipitate. 

^ The formula for these monobasic acids is OHjOH - (CH0H)4 - COOH. 

2The „ „ dibasic „ COOH -(CH0H)4- COOH. 

* 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 

CHAPTEE 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 HjSO^ 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, 

CeHiA = 2C,H,OH + 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 COj 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. 

ExPEBiUENi. Bepeat 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. 

III. notation 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 efiect is due to the presence in the molecule of asymmetrical carbon 
atoms. 

6 carbon aldose (hexose). 6 carbon ketose. 

CHjOH CHjOH 

*CHOH *CHOH 

*CHOH *CHOH 

I I 

*CHOH *CHOH 

*CHOH CO 

CHO CHjOH 

* 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 ia 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 possihle 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 polarimeter 
or saccharimeter. 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. 




{{Ml 



Fig 222.-Polai-iini!ter of Mischerlich with Laurent's polariser. P, polaiiser and device 
for obtamingr half shadow; R, 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 e.o 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.' 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). 




Flo. 223. — Diagram of scale and field of vision of polariraeter. Above is represented 
the scale for measuring the compensation necessary. In the position represented in 
the diagram the reading is 2 "7 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.^ It is 
determined by the following formula : 

pxl 
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 

' 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. 

plOOa 

where 8=(a)D. 
For rapidly and accurately determining the percentage of sugar in any fluid 
(e.^. urine) the polarimeter — 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 : 
Monosaccfiarides : Dextrose: +52 "T"- 
Galactose : +81°. 
Laevulose : —93°. 
Invert sugar : - 20*2°. 
Diaaccharides. — The (o) D of these carbohydrates changes when they are 
hydrolysed. 

Cane sugar: + 66 "5° — after hydrolysis becomes laevorotatory (wde invert sugar). 
Maltose : + 137° — after h3'droly8is becomes much leas. 
Lactose : +52 '5° — after hydrolysis becotaes slightly more. 

IV. Moore'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 (CHg - CO - CHj - CH^ - 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 cooling, the solution be 
acidified with HgSO^. 

The Chief Monosaccharides are dextrose, laevulose and galactose. 

Dextrose, grape sugar or glucose (CgHjgOg), 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-I to 0-15 %, 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 CHEMISTRY 285 

Glucose readily combines with alcohols, acids, phenols, etc., to form glucoaides. 
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 
I 
CH - (CHOH)i, - OH - CHOH - CH2OH. 



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 
(o and ;8) are possible. These show striking differences from one another in their 
susceptibility to ferment action. 

Laeviilose (CgHjjOg) 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 ((a)D= -93°). 

Galactose (CjjHjjOg) 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. 

ExPEKiMENT. Test for galactose. Add 3 c.c. pure HNOgCcon.) 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, 

^CfiHjjOg - HjO = CJ2H22O11. 

Their structure can be demonstrated by hydrolysing them, i.e. 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 PRACTICAL 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 (CjjHjaOji) 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 SeliwanofPs 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. 

ExPERiMKNT. Examine a ten per cent, solution of cane sugar with the polari- 
soope. Note the rotation and calculate (a) D. Place exactly 50 c. o. of a twenty 
per cent, solution of cane sugar in a 100 o.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 o.c. mark. Examine this solution with 
the polariscope and calculate (a) D. 



PHYSIOLOGICAL CHEMISTRY 287 

Lactose (C^^^^i^-^^) 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. 

Ci,H2Ai + H20 = 4CH3-Ch/ 

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 (C12H22OU) 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= -|-137'04°). After hydrolysis, therefore, 
the reducing power shows an increase and the rotatory power a 
decrease. 

Experiment VIIL— 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 h, much lower temperature, 158° 0. 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 PEACTICAL PHYSIOLOGY 

CHAPTER III. 

CARBOHYDRATES— Continued. 

III. Polysaccharides. 

A POLYSACCHAKIDE is the Condensation product of more than two 
monosaccharide molecules, and has accordingly the general formula, 
(CgHjgOs),, where n stands for a variable number.^ 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) and 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 
(CgHjg05)„. 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 efiected 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. 

T 



29D PRACTICAL 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 {C^B..^fi^)„. 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.^ 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(0H)2 is held in solution. 

Experiment IX. To some of the solution add a few drops of 
25 % H2SO4 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 (CgHjo05)„. During the hydrolysis of starch and 
glycogen dextrines are formed as an intermediate product. British 

' 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 : 

ExPBRiMENT 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(0H)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 : iirstly, 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 in solution. The 
method is as follows ' : 

The liver is cut into small pieces and mixed in an Erlenmeyer ilask 
(Bohemian glass) with 100 c.c. 60% KOH.^ 

^ The 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. 

^ 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 PRACTICAL 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 sufiices 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. HCl (cone.) {i.e. 5 c.c. HCl to a 100 c.c. of glycogen 
solution), and heated in a flask on the water bath for three hours.' 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 
'If the glycogen be repreoipitated 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 CHEMISTRY 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 phyaiologie, 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 cupric 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.o. 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 PEACTICAL PHYSIOLOGy 

Preparatioa of SoliUions. Solution /.— 12-5 gr. CnSOi (purified as directed in the 
footnote^) are dissolved by heat in 75 o.e. water and the solution cooled to 25° C. 
In a large porcelain basin 250'0 gr. potassium carbonate, 200"0 gr. potassium 
Eulphocyanide 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 mi^ed 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. hydroxy lamin sulphate or 5 '56 gr. hydroxy lamin chloride ^ 
is dissolved in water and the solution added to one of 200 gr. potassium sulpho- 
oyanide 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 aretaken for the estimation; if it 
contain more, then such a number of c.c. must be taken as will yield -■ total 
amount less than 0-06 gr. In all cases the sugar solution must be made up to 
10 C.C.' 

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 untU 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, C^HigOg. 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 

^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. 

' 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- 

lamin 

'solution, 

c.c. 


Dextrose, 
mg. 


Hydroxy- 

lamin 
solution. 

C.C. 


Dextrose. 


Hydroxy- 

lamin 
solution. 

C.C. 


-Dextrose. 
m.g. 


43-85 


5 


29-60 


19 


17-75 


33 


7-65 


47 


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 


13 05 


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 


2100 


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 peiUosanes. Thus, in gum arabio there is a 
pentosane which yields i-arabinose when hydrolysed by heating with mineral 
acid, and in wood or bran another pentosane yields i-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 racemio 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 arable by heating a solution of it in a water 
bath for twenty minutes, with 5 % HCl. Arabinose i? formed. After neutralising, 
apply reduction and yeast fermentation tests to portions of the solution. To 
another portion apply the following characteristic test "for pentoses (ToUens). 
Add phloroglucin (C6H3(0H)3) in small quantities at a time till no more dissolves 
to a solution of about 5 c.c. of equal parts of concentrated HCl. and water. Then 
add tt 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 tlie above spectrum very clearly. ToUens' 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. gii. 
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 pentosecontaining solution^ and 
again heat just to boiling point. A greenish-blue colour rapidly develops: Thip 
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, whe^ 
draotically no colour change will occur. 



296 



PRACTICAL PHYSIOLOGY 





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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-stuflfs animal cells cannot live. At present, too, they are 
bodies of purely biological origin, no efibrt 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, 
Serum albumin, 
Serum globulin, 
Keratin, - 
Elastin, 
Gelatin, 

The nitrogen and the sulphur are usually contained in two formSr 
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 ta 
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. 



52-93 


6-90 


16-66 


22-26 


1-25 


52-08 


7-10 


15-93 


21-96 


1-90 


52-71 


7-01 


15-85 


23-32 


1-11 


50-65 


6-36 


17-14 


20-85 


5-00 


54-32 


6-99 


16-75 


21-94 


— 


49-83 


6-80 


17-97 


25-13 


0-7 



298 PRACTICAL PHYSIOLOGY 

Recent research has shown that nitrogen may be detected in the protein 
molecule after coniplete hydrolysis with 30 % sulphuric acid as : — 

(1) Amide or Ammonia Nitrogen. This is the loosely combined nitrogen 
mentioned above. 

(2) Diamine 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, 


Monamina N. 


Caseinogen, 


15-62 


1-61 


3-49 


10-31 


Edestin (from hemp), 


18-64 


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). 


1613 


2-97 


•49 


12-51 



(T. B. Osborne.) 

The complex constitution has been studied recently in two ways — 
(1) 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 CHEMISTEY 



299 



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. 


min. 


"Serum 
Globu- 
lin. 


Casein- 
ogen 
(cow). 


Fibrin- 
ogen. 


EdcB- 
tin. 


Kera- 
tin. 


Gela- 
tin. 


Bias- 
tin. 


Glyoin, 








3-5 





3 


3-8 


•4 


16-5 


25-8 


Alanin, 


2-7 


21 


2-2 


■9 


3-6 


3-6 


1-2 


•8 


6-6 


Leuein, 


20 


61 


18-7 


10-5 


15 


20-9 


18-3 


21 


21-4 


Aspartic acid, 


31 


1-5 


2-5 


1-2 


20 


4-5 


2-5 


•6 


+ 


Glutamic acid, • 


7-7 


8 


8-5 


110 


8-0 


6-3 


30 


•9 


•8 


Cystin, 


2-3 


•2 


■7 


•1 


— 


•3 


+ 


— 


— 


Histidin, 


— 


— 


— 


2-6 


— 


1-1 


— 


— 


— 


Lysin, 


— 


— 


— 


5-8 


— 


10 


— 


28 


— 


Arginin, 


— 


— 


— 


4-8 


— 


1-7 


2-3 


7-6 


■3 


Phenyl-alanin, 


31 


4-4 


3-8 


3-2 


2-0 


2-4 


30 


•4 


3-9 


Tyrosin, - 


21 


11 


2-5 


4-5 


3-5 


2-1 


4-6 





■3 


Tryptophan, 


+ 


+ 


+ 


1-5 


+ 


+ 








— 


Other amino acids. 


31 


2-3 


2-8 


5-3 


5-4 


41 


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 
<ii-peptide glycyl-glycin with the elimination of water, thus : — 

OH H 
NHjCHjCO NHCHjCOOH = NHaCH^CO . NHCH^COOH + H^O. 

Glycin. Glycin. Glycyl-glycin. 

The addition of another molecule forms a tripeptide, and so on until 
polypeptides {many linkages) are formed. Pentaglycyl-glycin, for 
example, is : — 

NH2CH2CO(NHCH2CO)^NHCH2COOH. 
But not only has glycin been combined to glycin — other end products, 
such as alanin, leuein, phenylalanin, tyrosin, etc., hav« been combined 
together, giving such bodies as leucyl-glycyl-alanin, and so on. For 
example, the polypeptide (do-deka-peptide) leucyldeca-glycyl-glycin 
has the formula i-r- 



C,H9CH(NH2)CO 
Leuoyl. 



(NHCH,CO),o 
Glyoyl. 



NHCHjCOOH. 
Glycin. 



By many such operations, polypeptides have been obtained, which, if 



300 PEACTICAL 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 ^ 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, diifuse 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. 

' Some 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 oif 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 




Fio. 224.— Crystallised albumin. x600. 

have, however, been crystallised. Certain vegetable proteins, e.g. the 
globulin of hemp seed (edestin), 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 PRACTICAL PHYSIOLOGY 

time, the mixture being briskly stirred between each addition. The precipitated 
globulin is filtered oflf, 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 Beactions. 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 cc). 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 cc. 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 such a ring 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 coagulum occurs, which on warming 
changes to a brick-red curd. 

This reaction differs from the xanthoproteic only in depending upon 
the presence of the benzene ring with an hydroxyl group attached to it 
in addition, or, in other words, the phenolic group. 

(d) The Glyoxylic Acid test (Hopkins' modification of Adamkiewiecz's 

Reaction). 

ExPEiiiMENT 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 XI. To some egg-white solution add an equal amount 
of saturated solution of ammonium sulphate = half satwation. 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 AmgSO^ 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 AmjSO^ therefore precipitates 
globulins ; full saturation precipitates albumins. 

(B) Magnesium Sulphate. 

Experiment XII. Fully saturate (i.e. add crystals) the solution of 
egg-white with MgSO^. A precipitate of globulin results. Filter. 
Prove by heat coagulation and by fully saturating with AmjSO^ that 
protein (albumin) is left in the filtrate. Magnesium sulphate in full 
saturation precipitates globulins, but not albumins (see table, p. 312). 

(C) Sodium Chloride, Ammwnium Chloride. These salts resemble 
magnesium sulphate in their "salting out'' properties. 



304 PEACTICAL PHYSIOLOGY 

(D) Sodnim 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 HNO3. 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) Selero-proteins. 
(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 CggHggNigOf; 
salmin (salmon) and clupein (herring) have the formula C3QH5jNj.70g. 
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 glohin 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. 
(h) In presence of salts they are coagulable by heat, 
(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 diifer, 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 PRACTICAL 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 difiFer, 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 Am^SO^ and MgSO^. 
(IV.) Heaf 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 wa.ter bath at 37° G. 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 behayes 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). 

Thk 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 ia 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 AmjSO^ : 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. 
(iii) 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 mitcins. 

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 mucinoide, bodies distinguished 
from mucin in not being viscous in nature and not being so easily precipitated by 



PHYSIOLOGICAL CHEMISTRY 309 

acetic acid, the precipitate wlien 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 chondroitin-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 %. 

/'CH2(0H) 



Formula of 
Glucosamine. ' 



CHOH 

CHOH 

I 
CHOH 

CH(NH2) 

I 
l^CHO. 

The Nucleo-proteins. — These consist of protein in combination with 
nmlein, 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 PEACTICAL 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 



Schema of Relationship of Nuclein, etc. 

Nudeo-protein 
(digested with pepsin) 

Nuclein (precipitated as a brown sediment, Peptone 

I decomposed by acid alcohol) (goes into solution) 



Acid Meta-protein (in solution) Nucleic Acid (white precipitate) 

heated in closed tube with HCl 

.^ \ 1 

Purin bodies Carbohydrate Phosphoric Acid Pyrimidine bases 

(adenin, gnanin) (hexose or pentose) Oytosin 

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 oflf 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 % HCl. 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. Eender 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). 

ExPERiMKNT XV. Use the solution of Witte's peptone provided and 
perform the following tests : 

(a) Biuret reaction is j)mi. {Proteoses axi6. Peptones.) 

(b) On faintly acidifying with acetic acid and boiling — no coagulwm. 

(c) Add a little HNO3 — 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 AmjSO^ (half saturate). 
A white precipitate of the primary proteoses (except hetero- 
proteose) which are salted out. Filter. 

(e) Saturate the filtrate with crystals of AmjSO^. 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 AmjSO^ 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 HNO3 soluble on 

heating. Therefore, in the presence of other proteins, 
precipitated by HNO3, such as albumin and globulin, they 
can be separated by warming the solution and filtering hot. 



312 PRACTICAL PHYSIOLOGY 

The precipitates of albumins and globulins do not dissolve 
on warming. 

(4) Primary proteoses ^ are salted out by half saturation with 

ammonium sulphate. 

(5) Secondary proteoses ^ are salted out by full saturation with 

ammonium suli^ate.- 

(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. 


Diffuai- 

bility. 


Action 
of heat. 


Biuret 

test. 


Salting 

out 
action of 
AmaSOi. 
Satura- 
tion. 


Salting 
out 

action of 
MgSOi 

(full satu- 
ration). 


Action of 

HNO3 or 

salioyl- 

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 ^ 
proteoses. 


Water. 


Small. 


Not 
coagu- 
lated. 


Pink. 


By half. 


+ 


Precipitate 
sc^. on 
heating. 


Precipi- 
tated. 


Secondary'' 
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. 



' Hetero-proteose is an exception. 



^ See fuller table on page 386. 



PHYSIOLOGICAL CHEMISTRY 



31S 



-The 



CHAPTEE 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. 

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 » 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. 



Fio. 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 : 

CH„-0-CO-X 

I 
CH _0-CO-X 



CH2-O-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 C„H2„+i. COOH. Those commonly occurring 
in fats are stearic acid, in which m=17, and palmitic acid, in which 



314 PRACTICAL PHYSIOLOGY 

n=15. Thus the formula for stearic acid is CH3. (CH2)i6. 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 carboxylio 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. (OHa), . CH = CH(CH2)7. COOH, 
and belongs to the series C„H2„_i. 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. 
Kepeat with an alcoholic solution of stearic acid, when the colour of the 
T)romine 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 olein 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 saponification. 

Experiment II. Saponification 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. 

ExPEKiMENT 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 jj^ 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. 
Eepeat 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 ; (h) 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 Expebimest. 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 PRACTICAL 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 acrolein 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,' and heat. A pungent vapour of acrolein ^ 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. 

JEmulsification. — 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 prelimiuary test with 
the crystals alone. The impure salt can be readily purified by crystallisation. 

^Acrolein is the alJehyde of allyl alcohol and has the formula CH2=CH-CH0. 



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 : — 

^CH,-00C(CH,XsCH3l 

I I J- Stearic acid. 

CH-OOC(CHA6CH3J 



Glycerine. 



/OH /CH, 
CH,-0-P4o ^CHg 



\0-N^CH3 
Phosphoric "--^^CHj — CHjOH. 

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 eerebro-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. Glycerine- 
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 




Fio. 226. — Crystals of cholesterol magnified 300 diameters. 



chemical group, namely, that of the terpen es. 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- 
gested fpr it : — 

(0113)2 = CH — CH2 — CH2 — CjfHjg — CH= CBEj 

CHo CH, 

v 

CH(OH). 



PHYSIOLOGICAL CHEMISTEY 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 Expekimest. Preparation of Cholesterol from Gall-Stones. — 

The gall-stones are finely ground and boiled with 95 per cent, alcohol. The 
aluobolic 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-ff. 
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 
ad<led. 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. (Kosenheim.) 

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.) 

Peotagon. 

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 
bases. (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. 

ExpEEiMBNT. 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, but 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 CHEMISTEY 



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322 PRACTICAL PHYSIOLOGY 

EXPEKIMBNT. 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 -r^ 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 

N 
subjoined table the result is calculated as oleic acid on the basis that 1 c.c. j^ 

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. 

ExPBKlMENT. 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 Co. 
alcoholic potash, a sample of which has just previously been titrated against 

^ HCl, 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 j^ 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 



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324 PRACTICAL PHYSIOLOGY 

CHAPTER VIL 

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 I'S 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. Pat. 


Carbohydrate. 


Salts. 


Cow's milk, 87-4 3-4 31 


4-8 


•7 


Human milk, 90-2 1-5 3-1 


5 


■2 


Other milks used for human consumption are :— 


- 




Water. Protein. Pat. 


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 
Tvith, forins 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 he produced in the adult animal by feeding it upon 



326 PEACTICAL 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 ofif 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 grwdty 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. 

EXPEKIMENT II. Estimate by a hydrometer (p. 409) the specific 
gravity (a) in skimmed milk and (6) in fresh milk. In the former it 
is about 1"0345, in the latter 1-028. By adding water to (a) the 
specific gravity obviously falls, and by removing the cream from (6) 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. ^ 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, 

' 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 NajCOgj 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 rermin (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 + CaClj. 
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 6 ; c does not clot, 
because the enzyme has been destroyed ; d clots only after the addition 
of CaClj ; e clots also on the addition of CaClg 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 r^jrangement 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 PRACTICAL PHYSIOLOGY 

The rennin comes from a precursor pro-rennin in the gastric mucous 
membrane. 
The stages can therefore be tabulated as follows : 

Pro-rennin + HCl 

\/ 
Eennin — 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 o.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 ofiF 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 0.0. 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 O. To A are added a few drops 
of rennin ; to B a, few drops of a 0'5 % phosphoric acid and some rennin ; 
to C a few drops of a 0'2 % solution of calcium chloride and some rennin. 

The three test tubes are placed in the water bath at 40° C. , when it will be 
noticed that coagulation occurs only in B and C, in which, besides the ferment, 
soluble calcium salts are present.' In A, although no visible change has taken 
place, the caseinogen has been converted into the so-called soluble casein, and all 
that is necessary for the production of clotting is the presence of calcium in 
solution. That this is so can be demonstrated by boiling the solution A so as to 
destroy the ferment, then cooling and adding a few drops of a 2 % solution of 
calcium chloride, when a clot will at once form. 

The fluid left after the clotting of the caseinogen is known as whey — 
in this case rennet whey. If the caseinogen be got rid of by acid, it 
is known as " acid whey " ; if by " salting out," as " salt whey " ; if by 
alcohol, "alcoholic whey," and so on. These wheys are different in 
composition; for instance, rennet whey and acid whey contain lact- 
albumin, salt whey and alcoholic whey do not. 

Experiment VI. Apply the xanthoproteic reaction to some acid 
whey: a positive result is obtained. Apply also the other protein 
colour tests. Acidify some of the whey with acetic acid and boil ; the 

1 The phosphoric acid added to B brings some of the Ca salts suspended in the 
opalescent fluid into solution. 



PHYSIOLOGICAL CHEMISTRY 329 

protein is coagulated. The proteins are called lact-albumin and lact- 
globulin. 

II. The Carbohydrate Lactose. 

Experiment VII. Boil some rennet whey which has been weakly 
acidified with acetic acid. Filter off the coagulated proteins. To the 
filtrate apply Trommer's or Fehling's test; reduction is efi'ected. 
Barfoed's reagent is not reduced. 

Lactose does not, like dextrose, readily ferment with yeast, but it is 
capable of undergoing a special fermentation, which changes it into 
lactic acid. This is called the lactic acid fermentation. It depends on 
the presence of a microbe, the bacillus acidi lactici. It occurs in two 
stages as follows : — 

C12H22OJ1 + up = 4CH3 - CHOH - COOH. 

Lactose. Lactic acid. 

Some of the lactic acid is then further split up into butyric acid. 

2CH3 - CHOH - COOH = CH3 - CH2 - CHj - COOH + 2C0., + 2H2. 

Butyric acid. 

The presence of these free acids in the milk leads to the precipitation 
of caseinogen, and this explains the production of the cwrd in sour 
milk. It is quite a different thing from the curd which is produced by 
rennin. Thus, it can be dissolved by means of a weak alkali, and if 
rennin be added to the resulting solution true clotting will follow. 

Milk, however, will undergo alcoholic fermentation by a special 
fungus, kiiown as the kephir fungus. From cow's milk the drink 
kephir is formed, from mare's milk the drink koumiss. They contain 
from 1-3 % of alcohol, and when clotted give a fine clot. For this 
reason they have been recommended for invalids. 

EXPKKIMENT VIII. Take some sour whey. Add a few drops of it 
to Uffelmann's reagent,^ when the dark puiple colour of the latter will 
be changed to yellow. Test for lactic acid (see p. 379). 

III. The salts of milk are chiefly phosphates and chlorides of the 
alkalies and alkaline earths. A trace — "00035%— of iron is also 
present. 

Experiment IX. The Detection of Phosphates and Chlokides. 
— Add to 5 c.c. of protein-free whey half its bulk of nitric acid and 
about twice its bulk of a solution of molybdate of ammonia in nitric 
acid. Warm gently on the water bath, and a yellow precipitate of 
phosphate forms. In rennet or acid whey the phosphates may be 
precipitated by ammoniated magnesium citrate. Filter. Dissolve 

' This reagent is made by adding a trace of ferric chloride to a 1 % solution 
of carbolic acid. ' . 



330 PEACTICAL PHYSIOLOGY 

precipitate in nitric acid and heat as before with ammonium molybdate 
Show the presence of chlorides by means of silver nitrate test — a 
white precipitate insoluble in nitric acid, soluble in ammonia. 

Experiment X. The Detection of Calcium Salts. — To some 
whey, freed from protein by boiling, add a few drops of a solution of 
potassium oxalate — a white haze of calcium oxalate results. 

IV. The Fats of Milk.— Examine a thin film of milk under the 
microscope, and note that the fat consists of small spherical bodies, 
which are transparent and do not adhere to one another. 

The fat can be removed by shaking the milk with ether after the 
addition to it of a few drops of weak NaOH solution. 

Experiment XI. To about 5 c.c. of milk in a test tube add two 
drops of caustic soda (20 %), and then about 5 c.c. of ether. Cover the 
top of the tube with the thumb and shake the mixture, occasionally 
lifting the thumb slightly to allow the vapour of ether to escape. The 
ether will dissolve the fat, and the milk will become much less opaque. 
By adding alkali, a certain amount of the caseinogen is changed in its 
physical condition, so that the caseinogen films, which lie between 
and thereby hold apart the fat globules, are diminished, and conse- 
quently the fat globules are dissolved by ether. So long as they are 
surrounded by caseinogen molecules they are not acted on by ether. 
Not only alkalies, but also acids can effect this change. 

When the milk stands for some time, the fats, being specifically 
lighter, rise to the surface to form the cream, and if this be mechanically 
agitated it solidifies to form butter. Analysis of an ethereal extract of 
milk shows that the fats are olein 40 %, palmitin 33 %, stearin 16 %, and 
about 7 % of lower fatty acids, such as butyrin. There are minute 
traces of lecithin and cholesterol. 

Colostrum. — The milk which first appears during lactation is yellower 
in colour and of higher specific gravity than that secreted later. On 
boiling, it yields a distinct coagulum of albumin and globulin, and if 
examined under the microscope it will be found to contain numerous 
cells — colostrum corpuscles — in the protoplasm of which fat globules are 
present. These cells are, in reality, secretory cells of the mammary 
glands which have been extruded in the first portions of milk 

The Quantitative Determination of the various Bodies in Milk.— 

The methods here described can be employed for other fluids besides milk. 

(1) The Percentage of Water. — A weighed quantity of milk is mixed with 
a weighed quantity of fine quartz sand, which has been previously heated to 
redness and then cooled in a desiccator. The weight of the mixture is accurately 
determined, and it is then placed in a hot air bath heated to 100° C. until all the 
water has been driven off and the weight is constant. The amount of weight lost 
corresponds to the amount of water which the sample of milk contains. 



PHYSIOLOGICAL CHEMISTRY 331 

(2) The Percentage of Protein.— Three grs. of milk are diluted with four 
times its volume of distilled water, a few o.o. of a solution of sodium chloride are 
added, and then a solution of tannic acid until all the protein has been precipi- 
tated. The precipitate is filtered off through an ash-free filter paper, and 
thoroughly washed with distilled water. The filter paper with the precipitate 
is removed to a Kjeldahl's combustion flask, and the nitrogen estimated as 
described on p. 410. The result multiplied by 6'37 gives the total amount of 
protein contained in the sample of milk. 

(3) The Percentage of Fat. — The dietetic value of a milk depends to a 
large extent on the amount of fat it contains. There are, therefore, numerous 
methods employed for the quantitative estimation of this, some of which are only 
approximate. The following method (Adam's) will be found very simple and 
sufficiently accurate for most purposes : 

Measure 5 c.c. milk and drop it on to a strip of Adam's fat-free porous paper ; ' 
allow this to dry in the air bath at 60° C, then roll it up and place it in the 
extractor of Soxhlet's apparatus (see p. 313). The weight of the distilling flask is 
ascertained before beginning the extraction, and then again after the extraction 
has been allowed to proceed for about one hour and the ether has been distilled 
off; the increase of weight gives the amount of fat in 5 c.c. of milk. Sufficient 
ether should be used to fill the Soxhlet one and a half times, and it should be 
made to siphon over at least twelve times. 

(4) The Percentage of Sugar.— Ten c.c. milk are mixed with twice that 
amount of alcohol (meth. spt. ) so as to precipitate all the protein, which is then 
■filtered off. The precipitate is thoroughly washed with alcohol, and the washings 
are then placed on the water-bath till all the alcohol has evaporated. The 
contents of the evaporating basin are then carefully washed into a 100 c.c. 
measuring cylinder, and the volume made up to 100 c.c. This is then placed in 
a, burette and titrated with boiling Fehling's solution, as described on p. 45.0 
Ten c.c. Fehling's solution correspond to 0'0676 g. lactose, therefore the number 
of c.c.'s of the diluted extract required contains 0"0676 grm. lactose. In order to 
calculate the percentage it must be remembered that each c.c. of the solution in 
the burette corresponds to 0"1 c.c. of the original milk. 

(5) The Percentage of Ash.— A weighed quantity of milk is evaporated to 
•dryness on a water bath in a weighed crucible. The crucible is carefully heated 
over a free flame until » perfectly dry and black ash has been obtained. The 
flame is now strengthened and the ash is heated until it becomes white. The 
crucible is then allowed to cool in a desiccator, after which it is weighed. 



CHAPTEE VIII, 

BLOOD. 

To the unaided eye, ordinary vertebrate blood appears to be a homo- 
geneous red fluid, but microscopical examination shows that the red 
«olour is really due to certain formed elements, the red corpuscles, 

' The paper can be obtained from any of the dealers. 



332 PRACTICAL PHYSIOLOGY 

suspended in an almost colourless fluid, the plasma. In this fluid, too, 
are certain other corpuscles, which, being colourless, are known as the 
white corpuscles. When blood is shed, it sets at first to a red jelly. 
After a time this jelly contracts and gradually squeezes out a pale 
yellowish fluid, known as the serum. The blood of difi'erent species 
clots at different rates, but the process is essentially the same. 



The Clotting of Blood. 

Demonstration. — Inspect the blood clot in the large vessel placed 
for demonstration purposes. Notice that the shrunken clot floats in 
the serum. If it be a clot of horse's blood, notice the " bufiy coat " at 
the top, due to the fact that the heavier red corpuscles have had time 
to sink before the blood clotted, thereby leaving the white corpuscles 
in abundance at the top. These form " the bufFy coat." 

Experiment I. Carefully sterilise a needle, prick the finger, and 
draw some blood into a fine capillary tube. Place aside and examine 
under the microscope at the end of the lesson. 

In order to study the nature of the processes involved in the 
coagulation of blood, it is essential to stop clotting from taking place. 
This can be done in several ways, such as by receiving blood into 
certain neutral salts (\ volume of magnesium sulphate, equal volume of 
sodium sulphate), or into a soluble citrate, oxalate, or fluoride. How 
these bodies act we shall consider later. Upon standing, the corpuscles 
will gradually sink, and the supernatant plasma can be pipetted ofl^, or, 
what is better, the mixture can be centrifugalised and the plasma 
more quickly obtained. The plasma bears the name indicating its 
method of preparation; we get therefore "salted plasma," with magnesium 
sulphate and sodium sulphate, "oxalate plasma," "fluoride plasma," 
and so on. 

Experiment II. Of the "salted plasma" provided, take about 
5 c.c. in three tubes, a, h, c. Dilute each six times with water. Leave a 
as it is. To b add a few drops of serum. To c add a few drops of 
serum which has been previously heated for several seconds at 60° C. 
Place all three in a water bath at 37° C. Note that a and c clot at 
about the same time, b clots much more quickly. 

From this we learn : — (1) that salted plasma clots on dilution ; (2). 
that the process is quickened by the addition of serum ; (3) tha,t the. 
quickening power of the serum is destroyed by previously heating 
to 60° C. 

Experiment III. Take about 5 c,c. of oxalate plasma in four 
tubes, a, b, c, d. Leave a as it is. To b add a few drops of serum. To c- 



PHYSIOLOGICAL CHEMISTRY 



333 



add a few drops of calcium chloride solution. To d add an equal 
volume of saturated sodium chloride solution. Filteir off the flocculent 
precipitate and keep for the next experiment, which should be per- 
formed as soon as possible. To the filtrate add some calcium chloride 
solution. Place all in a water bath at 37° C. It will be found that a 
has not clotted, that h and c have clotted, that the filtrate d has not 
clotted. 

From these experiments, we gather (1) that blood will not clot when 
the calcium salts have been removed by an oxalate ; (2) that serum can 
clot oxalate blood (that is, blood without the presence of calcium salts), 
because it contains the necessary enzyme already formed in it; (3) that 
oxalate blood will clot when calcium is added to it, because with free 
calcium available the coagulating enzyme is formed ; (4) that the body 




Fig. 227.— Collection of blood. 



coagulated is a protein thrown out of solution by half saturation with 
sodium chloride solution. This body is known as fibrinogen. It is 
insoluble in distilled water and easily thrown out of solution by 
saturation with salts, and, therefore, belongs to the globulin class of 
proteins. 

Experiment IV. Quickly redissolve in water the precipitate 
obtained in Experiment III. d. The salt adhering to the precipitate form s 
a dilute saline solution, in which the precipitate dissolves. Test the 
solution obtained for protein by the colour tests. 

Experiment V. Into the bent capillary tubes provided collect, as 
shown in Fig. 227, some of your own blood, first introducing a small 
quantity of anticoagulant fluid, preferably 10 % sodium citrate, since 
sedimentation is most rapid with this solution ; 1 % potassium oxalate 
or 3 % sodium fluoride may also be used. Having sealed ofi' the ends, 
under the demonstrator's supervision, hang it upon the centrifuge by 
the bent end. With the plasma so obtained, perform experiments such 



334 PEACTICAL PHYSIOLOGY 

as those given in Experiment III. Sodium citrate, it will be found, 
acts like oxalate. This is not because it precipitates the calcium, but 
because it combines with it to form a soluble citrate, a salt which does 
not ionise (dissociate) in solution, and therefore leaves no calcium free 
to aid in the formation of thrombin. If fluoride has been used, it will 
be found that the addition of calcium salts to the plasma does not cause 
a clot to form, showing that the fluoride in some way prevents the 
formation of the enzyme from the pro-enzyme. 

We are now in a position to discuss the chief phenomena concerned 
in the coagulation of blood. We have learnt that a soluble protein, 
fibrinogen, under the action of an enzyme called thrombin, is turned 
into an insoluble protein termed fibrin. In the absence of calcium 
salts blood will not clot, not because this enzyme cannot act, but 
because it cannot be formed from the pro-enzyme thrombogen in the 
absence of such salts. It has recently been shown also that the admix- 
ture of tissue juice plays an important part in the liberation of the 
enzyme from the pro-enzyme. It is well known that jagged wounds 
stop bleeding more easily than clean-cut wounds, as also do wounds 
through a thick piece of skin compared to those through a thin piece. 
Similarly, the coagulation of bird's blood, which normally clots very 
slowly if the blood has not come in contact with foreign tissues, can 
be greatly hastened by the addition of some tissue juice. This body 
existing in the tissue juice is known as thrombokinase. The pro- 
enzyme is present in the plasma, as it is precipitated with fibrinogen 
from bird's plasma on the addition of distilled water. In mammals the 
thrombokinase is also yielded by the disintegration of the white cor- 
puscles and the platelets. ^ We can therefore draw up the following 
table for the coagulation of blood : — 

Thrombokinase + Thrombogen -t- Ca salts. 

(from tissue juice, (plasma) (plasma) 

white corpuscles, 

platelets). 



Thrombin = Fibeinogen (soluble) 
Fibrin (insoluble). 
(Compare this with the clotting of milk (p. 328), noting the diflference 

' Different authorities hold different views as regards blood platelets. Some 
regard them as an actual corpuscle. Others look upon them rather as products 
of asphyxiated blood — coming either from the disintegration of the white cor- 
puscles or the fine coagulation of the blood proteins. 



PHYSIOLOGICAL CHEMISTRY 335 

in the action of the calcium salts and in the part played by the 
enzyme.) 

Conditions which retard Clotting. — (1) CoW— receive the blood into 
a vessel placed in ice (i.e. keep it at a temperature a little above 
freezing point). The enzyme action is inhibited by cold. The blood 
clots on warming. 

(2) Contact with blood-vessel wall. — "The living test tube." This is 
made by ligaturing in two places a vein of a large animal, such as the 
jugular vein of the horse. In the tube thus formed the blood does not 
clot, and if it be hung up the corpuscles gradually sink to the bottom, 
leaving the unclotted plasma above. 

(3) Addition of certain neutral salts. — "Salted plasma" (ef. Experi- 
ment II.). 

(4) Addition of a soluble oxalate. — "Oxalate plasma" (cf. Experi- 
ment III.) 

(5) Addition of a soluble citrate. — " Citrate plasma " (see Experi- 
ment v.). 

(6) Addition of a soluble fluoride. — "Fluoride plasma." This plasma 
will not clot upon the addition of calcium (see Experiment V.). 

(7) Addition of leech extract (Hirudin). — This is a secretion produced 
by the salivary glands of the leech, and which can be obtained by 
extracting the heads with water. It acts because it contains an anti- 
thrombin. 

(8) Contact with oil. — Receive the blood into a smooth vessel smeared 
with oil. 

(9) Intrormtam methods. — These consist in injecting certain substances 
into the blood-vessels of the animal before bleeding it. These sub- 
stances are : — 

(a) Commercial peptone, which consists mainly of proteoses. 

(j8) Soap solution. 

(y) A weak alkaline solution of nucleo-protein injected slowly — the 
so-called " negative phase " of nucleo-protein injection. 

Peptone acts by causing the liver to form a large amount of anti- 
thrombin, which normally keeps blood from clotting inside the vessels. 
The exact action of nucleo-protein is not well understood. 

Conditions which hasten Clotting. — (1) Bodj/ temperatwe. 

(2) The addition of some clotted blood (clot or serum). 

(3) Agitation, e.g. whipping the blood with a bunch of twigs. This 
is a very general method of keeping blood fluid when it is not desired 
to study the phenomena of clotting. ^ 

'It is important to remember that this is no longer normal blood, but de- 
fibrinated blood. 



336 PRACTICAL PHYSIOLOGY 

(4) Contact with a rough surface (cf. effect of receiving into oil). 

(5) Addition of caldvm salts. 

(6) Intra-vitam methods causing blood to clot within the vessels : — 
(a) Injury or death of blood-vessel wall. When an artery is crushed, 

as in a contused or lacerated wound, a clot forms, which acts as a 
natural plug to prevent haemorrhage. When the arterial wall under- 
goes degeneration a clot or thrombus, as it is termed, may form. 
Similarly, when a blood-vessel is ligatured the inner coat is injured, 
and a clot forms for a short distance from the ligature. This clotting 
is due to the liberation of thrombokinase from the injured tissues, 
causing the formation of some thrombin. That the clot does not extend 
indefinitely in the blood is due to two causes : {a) thrombin is adsorbed 
into the fibrin it precipitates ; and (6) the formation of anti-thrombin. 

(6) Rapid injection into a vein of a strong alkaline solution of 
nucleo-protein ; the so-called '• positive phase" of nucleo-protein injection. 

Preparation of Fibrin Ferment. — Blood serum, or some defibrinated blood, 
is mixed with twenty times its bulk of alcohol. A copious white precipitate is 
obtained. Allow this to stand under the alcohol for two months. By this time 
all the other proteins present will be coagulated, except fibrin ferment. The 
fluid is pipetted off, the sediment carefully collected on a filter, and after the 
alcohol has drained off ground up in a mortar with water. This extracts the fibrin 
ferment. Filter, and keep filtrate. 

Blood Serum. 

Proteins. Experiment VI, Divide into three portions — a, b, c. 

(a) Allow a to drop gradually into a beaker filled with distilled water; 
a cloud forms round each drop as it mixes with the water. This is due 
to the precipitation of the globulin present, as there is now too little 
saline present to keep it in solution. 

(J) Saturate b with crystals of magnesium sulphate ; a precipitate of 
globulin occurs. Filter. Show that the filtrate contains albumin (i) 
by faintly acidifying with acetic acid and heating in a water bath — note 
the temperature at which the albumin coagulates (77°-79° C); (ii) fully 
saturating the solution with ammonium sulphate. 

Redissolve the precipitate of globulin in water ; faintly acidify and 
note the temperature of heat coagulation (75° C). 

(c) To c add an equal amount of fully saturated AmjSO^ {half 
saturation). The globulin is precipitated. Filter and fully saturate 
(add solid crystals) with AmjSO^ ; a precipitate of albumin results. 

Salts. 

Experiment VII. Faintly acidify the serum and boil to coagulate 
the proteins. Filter. Test the filtrate for : — 



PHYSIOLOGICAL CHEMISTEY 337 

(a) chlorides by silver nitrate — white precipitate insoluble in 

nitric acid ; 
(6) phosphates — white precipitate on addition of ammoniacai 
magnesium citrate solution. Filter off this precipitate. Dis- 
solve in nitric acid, and heat with nitro-molybdic acid — 
yellow precipitate ; 
(c) sulphates— white precipitate with barium chloride, insoluble in 
hydrochloric acid. 
In all three tests phosphates are precipitated, but in (a) they are 
soluble in nitric acid, in (c) they are dissolved by hydrochloric acid. 
(Of. salts of urine.) 

The amount of sulphate present is usually very small. This filtrate 
may also be tested for sugar by Fehlirig's or Nylander's test. 

The Estimation of Sugar in Blood.— To estimate sugar in blood it is 
necessary that the proteins and haemoglobin be removed. This is most easily 
done by Waymouih Seid's method. Into a beaker of abont 600 c.c. capacity are 
placed 250 c.c. of a 7% solution of phospho-tungstio acid containing 2 % HCl 
and the whole is weighed. The blood is then added, the contents well stirred, 
and the beaker again weighed. The difference in weight gives the amount of 
blood added. The beaker is then heated on a sand bath (or better still an oil 
bath), its contents being meanwhile briskly stirred. The proteins including 
the haemoglobin are, by this treatment, precipitated and form at first a brown 
gummy mass floating in u, clear liquid. After a little the eoagulum becomes 
brittle and sinks to the bottom of the beaker. Great care must now be taken 
that the beaker does not crack. When all the eoagulum has settled to the 
bottom, the beaker is cooled and the supernatant fluid filtered through paper 
into an evaporating dish, the paper well washed into the same dish, the contents 
of the latter nearly neutralised with NaOH, but left faintly acid, and the 
evaporating dish then placed on a boiling water bath. 

While the above fluid is evaporating the brittle protein precipitate is removed 
from the beaker to a mortar, ground up with some water tMl a chooolate-like 
paste is obtained and then washed on to a large suction filter plate and sucked 
dry. It is washed with water three times. The washings are then transferred 
to a 2 litre flask, nearly neutralised and boiled down to a small volume (50 c.c.) 
with the flask on the slant. The evaporated washings are then mixed with the 
contents of the evaporating dish (the evaporated supernatant fluid) and the whole 
brought to a volume of 50c.o. , after which it is cooled, almost neutralised, 
filtered through a small filter paper, the filter washed and the volume of the 
filtrate and washings brought up to 100 c.c. The sugar is then estimated in this 
by one of the methods described on page 450. 

Blood Plasma. — All the above bodies are present in plasma, which 
contains one substance in addition, namely. Fibrinogen. We have 
already shown this (Expt. III. d). Plasma, however, does not contain 
thrombin ; serum does. 

When we remember the function of the blood it is obvious that 
there are many bodies other than the above present in both plasma 

Y 



338 PRACTICAL PHYSIOLOGY 

and serum in small quantities. Thus the blood carries the food 
materials to the tissues, and the products of metabolism away from 
them. We have, therefore, in addition to ammonia, small quantities of 
nitrogenous extractives : — urea, uric acid, creatinin, xanthin, hypo- 
xanthin, etc. ; of non-nitrogenous extractives, fats, cholesterol, lactic 
acid, and other organic acids. 

In the plasma, too, in very minute quantities, are bodies which play 
an important part in regulating bodily functions — namely, the internal 
secretions of such glands as the thyroid, parathyroid, suprarenal, 
pituitary and sexual organs; also such protective bodies as immune 
bodies, antitoxins, haemolysins and precipitins. 

The percentage composition of the plasma varies with different 
animals. The following are two analyses : 

Watei 
SolidS: 



.er. 


90-29\ 
9-7U 


90-80 


is, 


9-16 


-^ ^ fibrin. 


■40 


101 


Proteins < ,, 

^ other, 


7-88 


7-76 


Exlraotives, 


■56 


•52 


Inorganic salts. 


•85 


•81 



GlobnliTi 


Fibrinogen 


28^3 


4-2 


22-6 


6 


30 


4-6 


47^9 


4-5 



It will be seen that the amounts of the different proteins present 
vary in the plasma. This can also be seen from the following table 
showing the amount in 1000 parts of plasma : 

AnimaL Total protein. Albumin. 

Man, - - 72-6 40^1 

Dog, - - - 60 3 31-7 

Sheep, . 72-9 38^3 

Horse, - 80^4 28^0 

When it is remembered that each of these proteins has probably also 
a different composition, it can easily be realised how different in 
composition are the plasmas of different animals (see also chapter on 
Precipitins, etc.). 

The Chemistry of the Leucocytes. 

These are morphologically the same as other cells, and they contain 
the same chemical substances. 'Yh^ protc/plasm consists mainly of water. 
The solids consist of various proteins, which chiefly belong to the 
group of compound proteins (gluco-proteins and nucleo-proteins), and 
there is also a small amount of albumin and globulin. The protoplasm 
may also contain such substances as glycogen, fat, mucin, etc., which 
have either been produced by the activity of the protoplasm, or which 
are simply deposited in the cell for storage purposes. 

The nudeus seems to consist mainly of nucleo-proteins, nuclein and 
nucleic acid. The nucleo-protein of the nucleus is said to contain a 
higher percentage of phosphorus than does that of the protoplasm. 



PHYSIOLOGICAL CHEMISTRY 339 

The Haemocytes or Red Blood Corpuscles. 

Structurally these are said to consist of a stroma containing in its 
meshes a chromo-protein called Haemoglobin. It is, however, impossible 
to demonstrate this stroma histologically, and some authorities believe 
that the haemoglobin is merely contained in a colloidal state in a 
protein envelope. 

Chemically they contain about 60 % of water and nearly 36 % of 
haemoglobin, the remaining 4 % — represented by the so-called stroma — 
consisting of lecithin, cholesterol and nucleo-protein. 

Haemoglobin. — This is a compound protein containing 0"4 % of iron. 
When decomposed by acids or alkalies it splits up into a protein of 
the nature of a histone (see p. 305) called globin and into a pigment 
called haematin, which contains all the iron. A pure solution of 
haemoglobin can be obtained by centrifugalising defibrinated blood, '^ 
removing the serum with a pipette, shaking up the corpuscles with a 
0"85 % sodium chloride solution ^ (which is nearly isotonic for the blood 
of the ox, horse, or man), and again centrifugalising. 

By this means the corpuscles are thoroughly washed free of serum, 
etc. They are then collected and treated with two or three times their 
bulk of distilled water, in this the haemoglobin dissolves, a deep red 
solution resulting. 

Experiment VIII. Heat carefully some haemoglobin solution. It 
decomposes at about 60° C, and the protein coagulates on further 
heating. Also test the solution for protein ; it gives several of the 
ordinary protein reactions, but in each case a splitting into protein 
and haematin simultaneously ensues. 

Besides being dissolved out by distilled water the haemoglobin may 
be set free from the red corpuscles by (i) warming to 50° C; (ii) the 
addition of a little ether, or of dilute ammonia solution; (iii) the 
addition of bile, saponin, or the serum of another species of animal. 
The " laking " of blood can be recognised by the greater translucency 
of the laked blood (see also chapter on Haemolysis). 

Experiment IX. Compare tubes of : — 

(a) whipped blood diluted with distilled water ; 
(6) whipped blood diluted with physiological saline. 

'Horses' blood should be used for this purpose as the oorpusoles sink more 
quickly than do the oorpusoles of any other blood. 

' A salt solution of this strength has the same osmotic pressure as the contents 
of the red blood corpuscle, and consequently no swelling or crenation of the 
corpuscle is produced. 



340 PEACTICAL PHYSIOLOGY 

That haemoglobin contains iron can be shown by the following 
experiment : — 

Experiment X. Dissolve some dried blood by heating with 
strong nitric acid. Evaporate nearly to dryness in a dish. Dissolve 
in water and add potassium sulphocyanide solution. A blood-red 
colour indicates the presence of iron. 

Crystals of haemoglobin. — These are most easily obtained from such 
animals as the rat or guinea-pig ; with more difficulty from man and 
most other mammals. 

Experiment XL Mix a drop of rat's blood with a drop of water 
upon a slide. After several minutes examine under the microscope for 
haemoglobin crystals. 

Haemoglobin, as we have seen, is a compound protein consisting of 
two parts, the iron containing portion " haematin " and the protein 
portion "gloUn." Haematin has the formula C32H32N404re. It itself 
does not crystallise, but a compound of haematin with hydrochloric 
acid and some other body (acetic acid, or an alcohol according to the 
method of preparation) called haemin can be obtained from haemo- 
globin, which crystallises in chocolate-brown rhombic plates. This 
forms one of the chemical tests for blood. 

Experiment XH. Preparation of Haemin Crystals. — Place a drop of 
blood upon a glass slide and warm until dry. Scrape loose the brown 
residue, add a little glacial acetic acid, cover with a cover glass and 
warm very gently until bubbles form. Remove from flame. If 
necessary add a little more acid, and warm again till bubbles form. 
Repeat the operation two or three times. When cold examine with 
microscope for the dark-brown haemin crystals (Fig. 228). 

There is sufficient chloride in blood to give the test without the 
addition of any sodium chloride. If, however, an old blood stain be 
used, it is necessary to add a small crystal of sodium chloride in case 
the chloride of the blood has been washed out. Bromide or iodide may 
be used instead of chloride, yielding a haemin with a corresponding 
change in composition. 

Another chemical test for blood depends upon the fact that the iron 
containing portion of the haemoglobin will, in the presence of such 
oxidising agents as hydrogen peroxide, or old " ozonised " turpentine, 
convert a coloured body like tincture of guiac (red) to another 
coloured derivative (blue). 

Experiment XIII. Boil some diluted blood. Add 2 drops of 
tincture of guiac (or of an alcoholic solution of guaiconic acid), 
then sufficient alcohol to dissolve the precipitate, and lastly a little 
ozonic ether, ozonic alcohol, or old oil of turpentine. A blue colour 



PHYSIOLOGICAL CHEMISTRY 



341 



is formed in the presence of blood. Ascertain in what dilution blood 
gives this test. Ozonic ether and alcohol contain hydrogen peroxide. 
The solution is first boiled to destroy any oxidising enzymes present. 
These bodies can effect the same change, as also can many salts of 
metals, such as copper, iron, gold, cobalt, strong sodium chloride 
solution, and various other fluids such as milk, saliva, mucus, sweat, 




Pio. 228— Haemin. X1600. 

and juices of vegetable origin (extract of pea flour, fruit juices, etc.). 
If, however, the solution be flrst boiled, in the absence of metallic 
salts, the reaction is to be regarded as a reliable one for blood. In 
any case, if the test be negative, most investigators regard it as certain 
that blood is absent. 

Other bodies such as aloin, benzidin, the leuoo-base of malachite green and 
phenol-phthalin can take the place of gniac. 

Aloin is said not to be so sensitive as guiao, benzidin is generally reported to 
be more sensitive, but according to Kastle the most sensitive is phenol-phthaline, 
with which he has detectedl in 8,000,000 of blood. 



342 PRACTICAL PHYSIOLOGY 

ExpESiMBNT XIII. a. To 1 0.0. of blood solution add 2 o.o. of phenol-phthaline 
and hydrogen peroxide reagent.^ Note the purplish-pink colour which develops 
upon standing from 5 minutes to 1 hour. Ascertain in what degree of dilution 
a blood solution will give this test. 

Another test for blood is the biological test (see chapter on Pre- 
cipitins). 

The function of haemoglobin is to carry oxygen to the tissues. 
This power of taking up oxygen can easily be demonstrated by 
shaking up venous blood with air (see under Spectroscope). The 
oxygen carrying capacity of blood can be ascertained as follows : — 

Experiment XIV. In the bottle of a Dupr6 apparatus (see Fig. 242 
in chapter on Urea) take 20 c.c. of oxygenated blood. Cover with 
dilute ammonia. In the small tube take 5 c.c. of fresh saturated 
potassium ferricyanide solution. Having adjusted the water to the 
zero of the apparatus by means of the clip — upset the ferricyanide 
into the blood. Shake well. Eeadjust the level of the water and 
read how much oxygen has been given oflf. 



CHAPTEE IX. 

THE SPECTROSCOPIC EXAMINATION OF HAEMOGLOBIN AND 
ITS DERIVATIVES. 

A SPECTROSCOPE Consists essentially of a screen, in which there is 
a small slit, through which light from any desired source can pass, 
a prism, and a series of lenses forming the telescope, through which 
the observer looks. 

For qualitative work the small direct vision spectroscope (Fig. 229) 
is serviceable. When the position of the bands, however, is required, 
one of the larger compound forms is necessary. 

Adjustment of the Spectroscope. — It is necessary to have an exact 
focus of the image of the slit. In the small direct vision spedvoscope this 

1 This reagent is prepared as follows : — ^Phenol-phthaline is added in excess (over 

N 
•032 grms. ) to 1 o.o. of :fjjNaHO. A little redistilled water is added. Shake well. 

" N 

Filter. To filtrate add 20 o.o. TTjNaHO and make up to exactly 100 o.o. with 

redistilled water. Add "1 o.o. of 3% commercial H^Oj. This solution is best 
kept in the dark. It may go faintly pink but not sufficiently to afiect its value 
in testing for blood. Water redistilled from glass should be used, ordinary 
distilled water being distilled from copper. If this be used, the reagent will not 
keep (Kastle). 



PHYSIOLOGICAL CHEMISTRY 



343 



may be obtained by directing the instrument towards a white cloud, 
and moving the eye-piece till the various Fraunhofer lines are clearly 
defined, or, in absence of daylight, obtaining a clear image of the upper 
and lower edges of the slit, i.e. of the upper and lower edges of 
the spectrum. The slit should not be too widely open. If the source 
of light include a sodium flame, a clear image of the D-line will be 
obtained when the slit is in focus. 




Fig. 229. — Small direct vision spectroscope. 




j.jaj^i,,.j,ii^jia>.y,iiii!iii .iijiiii II 

Pig. 230. —Compound spectroscope. 

In the larger forms of spectroscope, three tubes are generally found radiat- 
ing from the central prism or prisms^ (Fig. 230). One of these has its end 
blocked by a screen, in which there is a slit where width can be varied 
by a small screw. Attached near to the slit there is generally a small 
prism, which can be moved so as to cover half the slit, and affords the 
means of introducing a second source of light into the instrument. The 
tube, at the end of which is the slit, is called the collimator tube, and 
contains a lens so that the image of the slit can be brought to bear 
on one face of the prism. The distance of the slit from the lens 
is variable, and should be adjusted so that the rays issue from tube into 
the prism as parallel rays. After refraction through the prism they are 

^For studying absorption spectra, the two-prism form with its greater 
dispersion of the spectrum is less well adapted. 



344 PRACTICAL PHYSIOLOGY 

collected by a second tube, the telescope, and the eye-piece of this 
should be arranged to receive parallel rays from the surface of the 
prism. The eye-piece is frequently fitted with cross wires, if so, the 
eye-piece should be first adjusted so that they are seen distinctly. 

It will be found that the telescope has some lateral movement 
so that the vertical cross-wire can be made to coincide with any 
required part of the spectrum. 

The third tube will contain a small scale which can be focussed on to 
the surface of the prism and will then become reflected along the axis 
of the telescope. It will be necessary first to adjust the movable 
end of the scale-tube so that the scale is in focus, and it may be 
necessary to move it laterally so that the scale is brought into the field 
of the telescope. 

The scale must remain in a fixed position during any series of 
observations. 

Construction of a chart for determining wave-lengths of bands on the spectrum. 

With the scale in fixed position, notice the position on the scale of the sodium 
line, and the lines observable when the sodium flame is replaced by one coloured 
with a salt of strontium, calcium, lithium, barium, caesium, and potassium. Take 
observations of the positions of about twelve of these lines, the wave-lengths 
of which are known. Obtain the values corresponding to these wave-lengths, and 
on a piece of paper, ruled in sq^uares, plot out their position, regarding the 
abscissae as degrees of the scale and the ordinates as wave-lengths. Draw a curve 
through the several points. The wave-length of any part of the spectrum can 
now be ascertained. Observe where such a part intersects the scale, follow the 
ordinate corresponding to the degree of the scale to the point of intersection with 
the curve, and a line parallel to the abscissae line wiU indicate the wave-length. 

Having arranged the spectroscope so that the scale is illuminated and 
visible through the eye-piece, and the slit is illuminated by a light 
(an argand or incandescent burner) placed about one foot off; notice 
the position of the D-line on the scale. If sodium chloride be sprinkled 
into the illuminating flame, the D-line will be manifest, but a better 
method is to arrange between the illuminating flame and the slit a 
Bunsen flame in which asbestos soaked with a strong solution of 
sodium chloride is placed. 

A piece of glass tubing about two feet long may be taken, which has the lower 
six inches bent back to form an angle of 60° with the main stem. This is filled 
with 6 p.c. solution of sodium chloride. The short arm is then plugged with 
asbestos. At the end of the long arm is a short piece of rubber tubing 
clamped fairly tightly. This tube is held by a burette clamp, and the projecting 
asbestos can be allowed to just touch the Bunsen flame. In this manner a 
constant D-line is furnished. 

1. The visible Spectrum of Oxyhaemoglobin. — Take some defibrin- 
ated blood which has been thoroughly shaken with air, and dilute 





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Fig. 2 J. — Abaorption-Bpectra. 

1, Oxyhaemoglobin (very weak solution) ; 2, Oxyhaemoglobin (weak solution) ; 3, Oxy 
haemoglobin (strong solution): 4, Haemoglobin (reduced haemoglobin); 6, Cai bun- 
monoxide haemoglobin ; 6, Acid haematin ; 7, Acidhaematin (ethereal extract); 8, Alkaline 
haematin; 9, Haomochromogen (reduced haematin); 10, Haematoporphyrin (acid solu- 
tion); 11, Haematoporphyrin (alkaline solution). 



346 PBACTICAL PHYSIOLOGY 

it with about ten times its volume of water. Place some of this behind 
the slit of the spectroscope, preferably in a flat-sided vessel about 1 cm. 
thick, but a test-tube will answer fairly well. It will be noticed that 
the whole of spectrum is blocked out except a portion of the red end. 

Dilute this solution carefully. At a certain stage some of the green 
will be evident (see Spectrum 3 in Chart), there being a wide absorption 
band between the red and green. On diluting still further, this wide 
absorption band will resolve itself into two bands (Spectrum 2). These 
two bands are both on the blue side of the D-line, and their centres 
correspond to A, 579 and X. 543-8. Note carefully the position of these 
centres on the scale and the width of the bands. Observe also the 
limits of the visible spectrum at the red and blue ends. 

On diluting still further it may be possible to cause the band on the 
blue side to disappear, whilst the band on red side is still just 
appreciable (Spectrum 1). 

2. The visible Spectrum of Haemoglobin (reduced Haemoglobin). — 
If some diluted defibrinated blood be left standing undisturbed for 24 
hours, the oxyhaemoglobin will lose its oxygen. This result may be 
arrived at more rapidly by treating some diluted defibrinated blood 
which shows fairly wide oxyhaemoglobin bands with a reducing 
reagent, such as ammonium sulphide or Stokes' reducing fluid.^ If 
ammonium sulphide be used, the mixture should be warmed. It will 
now be noticed that the blood loses its bright scarlet appearance and 
becomes more purple in tint. Examine this by the spectroscope, 
and it will be found that the two bands of oxyhaemoglobin have 
disappeared, and are replaced by one band, the centre of which is 
between the two bands of oxyhaemoglobin. The band is a broad one, 
shading ofi' more gradually on the red side, and the darkest part corre- 
sponds in wave-length to X 550 (Spectrum 4 in Chart). 

3. The visible Spectrum of Carbon-Monoxide Haemoglobin. — If a 
stream of carbon monoxide, or even of coal-gas, be passed through some 
diluted defibrinated blood, the scarlet tint is changed to a carmine 
or cherry colour. The oxygen is replaced by carbon monoxide. 
Examined spectroscopically the blood shows two bands differing from 
those of oxyhaemoglobin in being slightly shifted towards the blue 
end. The two bands have centres corresponding in wave-length to 
A. 575 and A, 540 approximately (Spectrum 5). 

The proportion of red and blue unabsorbed at the ends of the spectrum 
is different in oxyhaemoglobin and CO-haemoglobin, there being more 

• 2 gms. of ferrous sulphate are dissolved with 3 gms. tartaric acid in 100 cc. of 
water. Ammonia is added till the solution is alkaline. Stokes' fluid must be 
freshly prepared. 



PHYSIOLOGICAL CHEMISTRY 347 

blue unabsorbed in CO-haemoglobin than in the former. Hence, com- 
paring dihite solutions of similar strength in test tubes of the same 
diameter, the CO-haemoglobin has a distinct bluish tinge, contrasting 
markedly with the yellowish-red of the oxyhaemoglobin. This dif- 
ference of end-absorption can be best shown as follows : Take a fairly 
dilute solution of oxyhaemoglobin showing the two characteristic bands 
clearly, but not strong enough to produce any intermediate shading 
Note as carefully as possible where the red and blue are first visible. 
Pass a stream of coal gas or carbon monoxide through the solution by 
means of a fine nozzle for two or three minutes. Note the change in 
•colour produced, and again examine the spectrum. It will now be 
found that rather more of the blue is visible, whilst the red is un- 
altered or slightly more absorbed. 

An important difference between oxyhaemoglobin and CO- 
haemoglobin is seen in the effect of reducing reagents. If CO- 
haemoglobin be treated with Stokes' fluid or ammonium sulphide, 
it is unchanged. 

4. The visible Spectrum of Methaemoglobin. — To a solution of oxy- 
haemoglobin, in which the two bands are so wide as to partially 
overlap, add a few drops of a strong solution of potassium ferricyanide. 
The colour changes to a chocolate tint. If this be spectroscopically 
examined, a distinct band is seen on the red side of the D-line, the 
wavelength of its centre being about A 635 (Spectrum 12). On 
diluting the solution down, other bauds may be seen — one just on 
the blue side of the D-line (A 581), another still further towards 
the blue (A, 540), and a fourth may be made out on the bluish-green 
(A 500) (Spectra 13 and 14). The two middle bands are probably 
not due to any traces of oxyhaemoglobin, but are characteristic of 
methaemoglobin. 

If such a solution of methaemoglobin be treated with ammonium 
sulphide, a transient spectrum of oxyhaemoglobin may be seen, suc- 
ceeded by a permanent spectrum of reduced haemoglobin. 

If the solution of methaemoglobin be rendered alkaline with 
ammonia, the colour changes to a more distinct red, and the absorption 
band in the red disappears and is replaced by a band immediately on 
the red side of the D-line (Spectrum 15 in Chart). 

By the action of nitric oxide on oxyhaemoglobin, a product is 
formed called nitric oxide haemoglobin. This is characterised by two 
bands, which are between the D and E-lines: the band on the red side 
is somewhat nearer the red end than the corresponding band of oxy- 
haemoglobin (Spectrum 16). 

If oxyhaemoglobin be treated with a nitrite, as sodium nitrite or 



348 



PEACTIOAL PHYSIOLOGY 



amyl nitrite,* there is formed a certain amount of methaemoglobin 
and a certain amount of nitric oxide haemoglobin. Tlie combination 
of the two gives a spectrum very similar to simple methaemoglobin 
(Spectrum 17 in Chart). 

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12, Methaemoglobin (strong solution) ; 13 and 14, Methaemoglobin (weak solutions) ; 
15, Methaemoglobin (alkaline solution); 16, Nitric oxide haemoglobin; 17} Mixture of 
Methaemoglobin and nitric oxide haemoglobin. 

If the product of the action of nitrites be treated with ammonium 
sulphide, the spectrum passes through a transient oxyhaemoglobin 
stage, succeeded by reduced haemoglobin, and later becomes nitric 
oxide haemoglobin. 

5. The visible Spectrum of Acid-haematin. — If some diluted de- 
fibrinated blood be treated with a little glacial acetic acid and gently 
warmed, it will assume a dark brown colour. If it be diluted suffi- 

' Care must; be taken to avoid excess of amyl nitrite, or so-called photo-met- 
haemoglobin, characterised by one broad band between D and E, wiU be formed. 



PHYSIOLOGICAL CHEMISTRY 349 

ciently, and examined spectroscopically, it will present a spectrum 
characterised by one band on the red, the wave-length corresponding 
approximately to X 645. The blue end of the spectrum will be very 
largely absorbed (Spectrum 6 in Chart). 

There is frequentl}' a considerable amount of general absorption in 
the acid-haematin prepared as above, and the band referred to may 
only be made clear by filtering the solution. More satisfactory results 
are obtained by extracting the colouring matter with ether, or treating 
with chloroform and excess of acetic acid, as follows : — 

(a) Take defibrinated blood, and add about half its volume of glacial 
acetic acid and about an equal quantity of ether. Shake at once. 
The ether will extract the colouring matter, and, on examining the 
same spectroscopically, three distinct bands will be seen — one on the 
red similar to that already described, but apparently shifted nearer the 
D-line (X 640), one on the green (A 550), another on the green but 
nearer the blue (X515). A very indistinct band may be seen on the 
yellow (A, 590) (Spectrum 7 in Chart). 

(6) Take defibrinated blood, warm and add half its volume of glacial 
acetic acid. Cool and add half the volume of chloroform, and more 
acetic acid if any precipitate appears. The solution will become clear 
and give a spectrum similar to that shown in the ethereal extract. 

6. The visible Spectrum of Alkaline Haematin. — Take some diluted 
defibrinated blood, and add a few drops of strong caustic soda, and 
warm. The colour will change to a greenish-brown tint, and when the 
solution is examined spectroscopically, it will show a single band on the 
orange (wave-length, X. 600). A more satisfactory method of preparing 
the alkaline haematin is to form a paste of potassium carbonate and 
defibrinated blood ; dry it over a water-bath and extract with alcohol, 
when a reddish-brown solution is obtained which shows the distinguish- 
ing absorption band clearly (Spectrum 8 in Chart). 

7. The visible Spectrum of Eaemochromogen (reduced Haematin). — 
If a watery solution of alkaline haematin be warmed with a few drops of 
ammonium sulphide, the brownish colour is replaced by a more marked 
red. If the solution be examined spectroscopically, the one band of 
alkaline haematin is found to be replaced by two bands on the green, 
the wave-lengths of their centres being approximately X 557 and X 525 
(Spectrum 9). 

8. The visible Spectrum of Haematoporphyrin. — Take some strong 
sulphuric acid (10 c.c.) in a test tube and add a few drops of blood, and 
shake up the mixture. A purple colour will result, due to the decom- 
position of the haemoglobin and the formation of the iron-free pigment, 
haematoporphyrin. This examined spectroscopically will, in the above 



350 PRACTICAL PHYSIOLOGY 

acid solution, show two bands, the centres being approximately A, 605 
and A, 565 (Spectrum 10 in Chart). 

If a large excess of water is added to the above a precipitate i» 
thrown down. If this be dissolved in a little caustic soda, a solution 
of haematoporphyrin in an alkaline medium is obtained, which shows 
a four-banded spectrum when examined, the positions of the bands 
being A. 630, A. 580, A 550, and A 520 approximately (Spectrum II 
in Chart). 

Haematoporphyrin may be regarded as iron-free haematin, and 
identical in composition with bilirubin. The following equation 
represents the change brought about by sulphuric acid : — 

CgjHjoN.FeOg + 3H,0 = 2(C„Hi8N A -l- Fe). 
Haematin. Haematoporphyriif. 

Solutions of haematoporphyrin exhibit a red fluorescence. This 
pigment must be regarded as normally present in small quantities 
in urine. 



CHAPTER X. 

MUSCLE. 

Muscle forms the most abundant tissue in the body. It is here 
that a great part of the food-stuffs undergo combustion, as a result of 
which energy is liberated and appears either as a muscular movement 
or as heat. The food-stuff's, along with the oxygen necessary for their 
combustion, are carried by the blood to the muscle, and the effete 
products are removed by the blood coming from the muscle. 

Muscle also constitutes one of the commonest food-stuffs, meat being 
the form in which we take much of our protein and a considerable 
amount of our fat (see chapter on Food). 

The Chemical Composition of Muscle. 



Water, 

Proteins, 

Organio Extractives, 

Fat, 

Inorganio Salts, 



75 per cent. 

- 20-21 „ 

• 0-3--4 „ 

■ 2-3 „ 

10-1-3 „ 



The Proteins. — To study these we require a muscle extract. 

Preparation of Muscle Extract. — A rabbit is killed, and a cannula 
tied into its aorta, by which the blood-vessels are washed free of blood. 
The muscles are then removed and quickly passed through the mincing 



PHYSIOLOGICAL CHEMISTRY 351 

machine. The mince is mixed with a 5 % solution of magnesium 
sulphate, the mixture being placed on ice and left standing all night. 

Divide some of the extract provided into two parts, a and h. 

Experiment I. (a) Dilute with four volumes of water, and place in 
the water-bath at body temperature. A clot forms, leaving mmcle 
serwm. 

(b) Add some acetic acid. A precipitate forms. Filter. Neutralise 
the filtrate with NajCOg solution, and dilute it with water. Place it in 
the water-bath at 37° C, and note that no coagulum forms. 

These two experiments show us that the extract contains in solution 
a substance which is precipitated by acetic acid, and which becomes 
transformed into an insoluble clot under suitable conditions. This 
body is protein in nature. Prove this by dissolving the clot in (a) in 
10 % sodium chloride and applying the protein tests. The soluble 
body is called myosinogen, and the clot myosin. 

Besides myosinogen the extract contains, however, other proteins. 

Experiment II. Take some of the muscle serum in (a), or of the 
filtrate in (b), and half saturate with ammonium sulphate. A precipi- 
tate of globulin results. Filter off this globulin and test the filtrate for 
albumin by full saturation with AmgSO^, or by coagulation by heat 
after faintly acidifying. 

Experiment III. Use the saline extract of muscle provided. 
Heat 5 c.c. carefully in a test tube in the water-bath. Note the 
temperature of the first signs of coagulation. Filter off coagulum and 
heat the filtrate, noting the temperature at which the flocculi of a 
second coagulum appear. It will be found that the first protein 
(paramyosinogen, also termed myosin) is coagulated at 4:7°C; the 
second protein (myosinogen, also called myogen) at 56°C. 

Both these bodies serve as the source of the coagulated myosin in 
muscle. In the clotting associated with rigor mortis it is stated that 
paramyosinogen is converted directly into myosin ; whereas myosinogen 
is first converted into a soluble form (soluble myosin), which is then 
turned into insoluble myosin. Soluble myosin can be identified by its 
low-heat coagulation point (40°C). 

The coagulation points of the chief proteins of frog's muscle have 
already been graphically studied (see Heat Rigor, p. 34.) 

Experiment IV. Show that the watery extract- of muscle provided 
contains less protein than the saline extract. It contains albumin, but 
not globulin. Demonstrate this fact. Coagulate the albumin by heat, 
and save the filtrate to test for phosphates later. 

Organic Extractives. These are organic substances which are soluble 
in water, but not protein in nature. They may be divided into two 



352 PRACTICAL PHYSIOLOGY 

classes, (a) nitrogenous, (b) non-nitrogenous. The chief members of the 
first group are creatin (C^HgNjOj), and the purin bodies hypoxanthin 
(CgH^N^O) and xanthin (CjH^N^Og). The most important non- 
nitrogenous extractive is sarcolactic acid (CgH^Og). 

Creatin is the most abundant extractive in muscle, amounting to 
•4 - -45 % in rabbits, -3 % in bullocks, -26 % in frogs, and -2 % in hedge- 
hogs. Chemically it is closely related to urea, and can be changed 
into this body and a substance called sarcosin by boiling with baryta 
water. 

Creatin H- Water = Urea + Sarooaln 

or, C,HgN302-i-H20 = CON2H, + C3H7N02. 

Although this reaction takes place outside the body, there is no 
evidence that such is the fate of creatin in the body, although many 
attempts have been made to prove that such is the case. Injection of 
creatin into the body leads to no increase in the formation of urea of 
the body. 

Another interesting relationship of creatin is to the substance called 
creatinm. If creatin be boiled with dilute mineral acids it loses a 
molecule of water and becomes changed into creatinin. 

CH^COOH \N<CHXco. 

Creatin -Water= Creatinin 

or, C,H9N302-H20 = C,H7N30. 

This reaction is the best means of recognising creatin, since it 
gives no characteristic tests, whereas creatinin does. 

Experiment V. Take about 10 grammes of fresh muscle, grind 
with alcohol, filter, evaporate at about 50° C. Dissolve in water and 
divide into two equal portions, a and b. To a add 15 o.c. of saturated 
picric acid solution and 5 c.c. of 10 % caustic soda. Allow to stand 
5' and dilute to 500 c.c. Note that there is no change in the colour of 
the solution, therefore creatinin is absent. To b add half its volume 
of N . HCl and heat in a flask fitted with a cork and glass tube to act 
as air condenser on water-bath for five hours. Neutralise with caustic 
soda, add picric acid solution and caustic soda, and dilute as before. 



PHYSIOLOGICAL CHEMISTRY 353 

Note red colour, due to picramic acid. Creatinin is now present. (For 
other tests for creatinin see chapter on Urine.) 

The exact relationship of creatin to creatinin in the body is still 
a matter of doubt. It was thought at one time that the creatin of the 
muscles gave rise to the creatinin of the urine. This now appears not 
to be the case, since creatinin is never present in muscle even after very 
prolonged muscular exercise. The amount of creatinin in the urine is 
extremely constant under all conditions, and it is therefore regarded 
by Folin as a measure of the endogenous metabolism of the body. 
Whence it arises is not certain, but the recent work of Mellanby points 
to the fact that creatinin is formed in the liver from unknown pre- 
cursors. From the creatinin thus formed the creatin supply of the 
body is replenished, and the creatinin that is not wanted is excreted by 
the kidney in the urine. We thus see that creatinin probably gives rise 
to the creatin of the body, and not the creatin to the creatinin. 
Further work is required to amplify these facts and also to find out 
the fate of creatin in the body, which is at present unknown. 
Recently Cathcart has pointed out that creatin, which does not 
normally appear in the urine, is present when the carbohydrate 
supply of the body is cut off. (See also small print at end of chapter.) 

Eypoxanthin and Xanthin. These are members of the group of 

bodies known as the purin bodies. They are thus termed because the 

so-called purin ring 

N— C 

I I 

C C— N. 

N— C— N^ 

is the basis of their constitution, purin itself, a synthetic body being 

N=C— H 

I I 

H— C C— NH. 

II II >C-H 
N— C — N'^ 

or more simply CjH^N^ 

Hypoxanthin is monoxypurin, and is represented by the formula 

HN— C=0 

H— C C— NH. 

|_J,_^^C-HorC,HAO 



354 PEACTICAL PHYSIOLOGY 

Xanthin is dioxypurin : 

HN— C=0 

I I 
0=C C-NH. 

I II \C-H or C^H^N^O, 

HN— C — N^ 

Lastly trioxypurin, which occurs in muscle only in traces, is v/ric 
acid, CsH^N^O.,. (See Chapter XVIII. p. 422.) 

Hypoxanthin and xanthin result in part from the breakdown of the 
nuclein present in the muscle, but their amount is normally so large 
compared to the amount of nuclein present that this cannot be their 
sole source ; the other source of supply is at present unknown. It is 
probable that they normally give rise to uric acid, since it is found 
that some time after muscular exercise the uric acid output of the 
urine is considerably increased. (For the isolation of these bodies 
from muscle extract see end of chapter.) 

Lactic Acid (CgHgOg). This variety of lactic acid differs from that 
obtained by the fermentation of lactose, which does not rotate the 
plane of polarised light. The lactic acid of muscle, often termed 
sarcoladic acid, rotates the plane of polarised light to the right. This 
behaviour depends upon a difference in position of the various side 
chains in relation to the central carbon atom. (Of. carbohydrates, p. 280.) 
The amount of lactic acid increases very much during the death of a 
muscle, and also during muscular activity. These points can be shown 
by the following experiments : 

(a) To some of Uffelmann's reagent (a mixture of ferric chloride and 
carbolic acid) add some of the muscle extract provided. This probably 
contains lactic acid from the dying muscle; if it does the violet 
colour of Uffelmann's reagent will be turned to yellow by the lactic 
acid present. 

(b) Hopkins' Test for Lactic Acid. Take about 5 c.c. of strong sulphuric 
acid in a dry test tube, add 1 or 2 drops of a solution of muscle 
extract, 2 or 3 drops of saturated solution of copper sulphate. Warm 
in boiling water for about two. minutes ; cool and add a few drops of 
alcoholic thiophene solution (20 minims in 100 c.c. alcohol), and warm 
gently. With lactic acid a cherry red colour develops. 

(c) Take a pithed frog which has been kept on ice for half an hour. 
Quickly cut off the muscles of one hind limb ; cut off the other limb at 
the pelvic girdle, and stimulate electrically until irritability is nearly 
lost. Cut off the muscles. Treat both sets of muscles as follows: 
Grrind with cold absolute alcohol and sand, filter, evaporate the alcohol, 
dissolve in water, heat with a little animal charcoal, filter, evaporate, and 



PHYSIOLOGICAL CHEMISTRY 355 

apply the thiophene test. It will be found that the muscles of the tetanized 
limb give a positive reaction; those of the non-tetanised do not. 
The tetanised limb having no circulation was poorly supplied with 
oxygen. If it had been kept in an atmosphere of oxygen, the lactic 
acid formed on tetanisation would have rapidly disappeared, so that 
fatigue would have developed more slowly, and would have dis- 
appeared rapidly on keeping the limb at rest in the oxygen of the 
ordinary room temperature (Fletcher and Hopkins). The explanation 
is that with plenty of oxygen any lactic acid formed is rapidly 
destroyed with the evolution of carbon dioxide. With an intact circu- 
lation lactic acid is certainly formed in the muscles with even short 
periods of violent exercise, since Rylfel has shown that under these 
conditions lactic acid is present in increased amount in the urine. 
This being the case it is probable that the formation of this acid is one 
of the causes leading to the hyperpnoea which attends hard muscular 
exercise (see page 459). 

Another important nitrogen-free extractive is glycogen {CgH.ifi^)n. 
The relative amount of this is small (0'5 to 1 %), but it varies in 
different animals, and is much diminished after muscular activity. 
Although the percentage is small the total amount contained in all the 
muscles of the body has been found, in the case of the cat at least, to 
be nearly the same as that contained in the liver. 

The less important extractives are : Urea, carnic acid (C10H15N3O5) 
(which exists in muscle combined with phosphoric acid as phospho- 
carnic acid), dextrose (trace), inosite (hexahydroxybenzene), and 
lecittain. 

Carnic Acid. — If » weak solution of ferric chloride be added to a muscle 
extract (from which the proteins have been removed by boiling and the phosphates 
by the addition of calcium chloride and ammonia) a brown precipitate is 
obtained. This is called Camiferrin, and consists of the iron salt of phospho- 
carnic acid. If, further, the phosphoric acid and iron be split oflf from this we 
obtain oamio acid, and this, curiously enough, has the same formula as, and gives 
nearly all the reactions of, one of the varieties of peptone known as antipeptone. 

Inorganic Salts. — These consist of salts of the alkalies and alkaline 
earths. The chief acid radicle present is phosphoric add, and this exists 
in several states — (a) Inorganic phosphates, (J) phosphorus of lecithin, 
(c) phosphorus of nuclein, {d) phosphorus of phospho-carnic acid, (e) 
besides these the watery extract contains another phosphorus-con- 
taining organic compound of unknown composition. 

Phosphorus, therefore, seems to be a very important constituent of 
muscle, and its form of combination changes after muscular work, the 
organically combined phosphorus being split off as inorganic phosphates 



356 



PRACTICAL PHYSIOLOGY 



which are then washed out of the muscle by the blood, and appear in 
the urine. It is on this account that the phosphates in the urine are 
increased after muscular work. 

Experiment VI. The watery extract of muscle has been freed of 
proteins by boiling it. Add to the clear filtrate an ammoniacal solution 
of magnesium citrate. A white precipitate of phosphates results. 




Fio. 233. — Crystals obtained from meat extract, mostly creatln, a few sodium chloride. 

Show that this precipitate consists of phosphates by dissolving it in 
nitric acid and testing with ammonium molybdate. 

ExPEKiMENT VII. As a general revision experiment test the 
solution of commercial extract of meat provided for proteins, glycogen, 
creatin, creatinin, mineral salts, etc. 

Preparation of Extractives of Muscle.— 500 grammes of meat, from which 
as much fat and tendon as possible have been removed, are finely minced ; the 
mince is thoroughly mixed with 500 c.e. of water and heated for half an hour on 
a water-bath at 0. The extract is strained through muslin and the residue 
extracted several times in a similar manner, tha extracts being mixed together. 



PHYSIOLOGICAL CHEMISTflY 357 

The protein in the extract is then coagulated by boiling, and, after cooling, the 
ooagulum removed by filtration. 

A similar extract may be prepared by dissolving some commercial meat extract 
in water. ^ 

To remove the phosphates and the last traces of proteins from this extract a 
saturated solution of subaoetate of lead is added to it until no more precipitate is 
produced. (Care should be taken that an excess of the subacetate solution is not 
added. This may be ascertained by filtering samples of the extract and seeing if 
these yield further precipitates with the subacetate solution. ) The precipitate 
thus obtained is removed by filtration. 

The excess of lead is precipitated from the filtrate by passing a current of 
sulphuretted hydrogen through it. The precipitate of lead sulphide is removed 
by filtration. The filtrate is then evaporated to small bulk (any sulphur which 
may separate out being removed by filtration) and allowed to stand on ice for two 
or three days, when a large number of oblique rhombic crystals of creatin will 
have separated out. (See Fig. 233.) These are collected on "• filter (for which 
purpose a suction pump will be found necessary) and are thoroughly washed with 
alcohol until no more pigment is removed. The filtrate is preserved for the 
isolation of the other extractives. 

Xanthin and Hypozanthin. — The oreatin-free filtrate is made strongly 
alkaline with ammonia, and is then mixed with ammoniacal solution of silver 
nitrate. The purin bodies are thus precipitated. The precipitate is collected on 
a filter paper and thoroughly washed with dilute ammonia, and the hypoxanthin 
and xanthin are separated from it by the following method : the precipitate is 
removed from the filter paper and dissolved in boiling nitric acid (spec. grav. I'l), 
a few crystals of urea being added to the solution so as to destroy any nitrous 
acid which may be present, and which would decompose the purin bodies. When 
all the precipitate has dissolved the solution is quickly filtered hot, and the 
filtrate is allowed to stand over night, when it will be found that a precipitate 
consisting of fine needle-shaped crystals (Fig. 234) has separated out. This 
consists of hypoxanthin silver nitrate combined with nitric acid ; to remove the 
nitric acid wash it with distilled water, transfer it from the filter to a small 
beaker and boil it with ammonia until the crystals break up and become 
amorphous, and then, to remove the silver, pass in HgS, filter off the silveir 
sulphide, and evaporate the filtrate slowly to dryness, when a white chalk-like 
mass of hypoxanthin will be obtained. In order to obtain the xanthin silver salt 
the filtrate from hypoxanthin should be treated with ammonia, when a few 
yellow flakes of the salt will be obtained. To separate the xanthin this pre- 
cipitate is treated in exactly the same way as for hypoxanthin. 

Test for Hypoxanthin. — Place a piece of hypoxanthin in a small evaporating 
dish with a few drops of concentrated pure nitric acid and evaporate slowly to 
dryness : a brilliant yellow residue is obtained. Cool, and then add a drop of 
sodium hydrate solution, when the residue will change to orange. If the residue 
be dissolved in water and the solution again evaporated to dryness the orange 

' The following amounts are suitable for this preparation : Ten grm. bovril are 
dissolved in 200 o.c. water, and to this is slowly added 60 o.c. of a saturated 
solution of subacetate of lead. After the precipitate has settled down a sample 
of the supernatant fluid is removed by a pipette to a test tube and tested with 
the subacetate solution to be certain that no more precipitate is produced. 



358 



PRACTICAL PHYSIOLOGY 



colour persists, thus differing from the murexide stain which, when similarly 
treated, loses Its colour (see p. 425). 

Test for Xanthin. — Repeat the same test as for hypoxanthin and note that 
the sodium hydrate produces in this case a deep red colour, which persists on 
dissolving in water and evaporating. 

Sarcolactic Acid. — The ammoniaoal filtrate, from which the alloxuric bodies 
have been separated, is treated with sulphuretted hydrogen gas so as to remove 
the silver which it contains : the silver sulphide is filtered ofif, and the filtrate 




Fio. 234. — Hypoxanthin silver nitrate. x300. 



-Zinc sarcolactate. x300. 



evaporated till all the ammonia has been expelled. It is then made strongly acid 
with phosphoric acid, and the lactic acid, which is hereby liberated, is dissolved 
out by shaking it in a separating funnel with ether. 

After extracting three or four times, the ethereal extracts are combined and 
the ether evaporated away by placing on a water bath heated to about 60° C, the 
flame underneath which has been extinguished. An acid syrup remains behind ; 
this is impure lactic acid. In order to purify it, dilute three times with water, 
bring the resulting solution to the boil, and then carefully add powdered zinc 
carbonate until the reaction is neutral. Filter. Evaporate the filtrate to small 
bulk, and add an equal bulk of spirit and allow to stand, when zinc earcolactate 



PHYSIOLOGICAL CHEMISTEY 359 

will crystallise out (Fig. 235). The zine salt is filtered o£f, washed several times 
with spirit, dissolved in water,' and the zinc separated by passing a stream of 
sulphuretted hydrogen through the solution. The zinc-free filtrate is then freed 
of water by evaporation, when the lactic acid is obtained as a syrup. 



CHAPTEE XI. 

DIETETICS, FOOD, METABOLISM. 

Food is taken into the body for two purposes — (1) to replace tissue 
waste ; (2) to supply energy for work and for heat. 

It is necessary that such food should be taken in a proper amount, 
in a digestible form, and adapted to the climatic circumstances and 
occupation of the eater. It is also essential that the food should be 
as varied as possible, for experience shows that an unvaried diet is 
not eaten with relish and may impair the general health and resistance 
to disease. The appetite of a healthy man is the best guide for the 
selection of his food ; it is the expression of physiological needs which 
have not been investigated sufficiently to justify dogmatic statements 
as to what a man should and should not eat. 

We may, therefore, divide the proximate principles ^ of food into 
two groups, viz. : 

Tissue formers — Protein, inorganic salts, water. 

CoTnbustion material — Protein, fats, carbohydrates. 

It will be observed that, of the organic food stuffs, protein may 
act in either capacity, and hence, that a diet containing protein and 
salts alone can serve as an efficient food. That this is so is proved 
by the fact that the Indians of the Pampas live entirely on flesh. 
Without protein it is impossible to maintain life; but if more than 
is necessary for the repair of the broken-down tissues be supplied, 
the excess serves as a fuel. 

The most serviceable combustion material seems, however, to be 
carbohydrate ; next comes protein, and the least available is fat, this 
latter being pre-eminently the form in which the excess of food over 

'The watery solution should be evaporated until the crystals of zinc sarco- 
lactate begin to appear, this being ascertained by examining a drop under the 
microscope. 

^ These nutritive constituents are called the proximate principles of food, 
because, consisting as the3' do of carbon, hydrogen, oxygen, and nitrogen 
combined into highly complex bodies, they are really elementary constituents 
of the organism. 



360 PRACTICAL PHYSIOLOGY 

present requirements is laid by for future use. Thus, during summer, 
hibernating animals store up a large quantity of fat, and this is called 
upon during the winter sleep to furnish the energy necessary for life. 

In judging whether any diet he efficient the first thing we must deter- 
mine, therefore, is whether it contains a sufiicient amount and a 
suitable mixture of the nutritive constituents of food. In practice 
it is found that these facts can be determined by estimating the 
amount of carbon and of nitrogen which the diet contains. We can 
find out how much of these two elements is necessary by estimating 
the amount of them contained in the excreta. 

An ordinary man under ordinary circumstances eliminates about 
300 grammes of carbon per diem (chiefly as carbon dioxide), and about 
15 grammes of nitrogen (chiefly as urea, etc., in the urine). Now, the 
only food-stuff which contains both these elements is protein, and to 
supply the required amount of nitrogen it would be necessary to give 
only about 100 grammes of this. Such an amount would, however, 
only furnish about one-sixth of the necessary amount of carbon. This 
difficulty could be surmounted by giving about 600 grammes of protein, 
but then the tissues would be supplied with six times more nitrogen 
than they required. It is advantageous, therefore, to mix with the 
protein some food stuff containing an excess of carbon, but no nitrogen. 
Such a food stuff is fat or carbohydrate. Experience teaches us that of 
these two the more serviceable is carbohydrate, and for two reasons : 
firstly, because it is more easily digested ; and, secondly, because it is 
cheaper. 

When muscular work is performed the excretion of carbon rises, 
whereas that of nitrogen is scarcely affected at all, so that in such cases 
the diet should contain an abundance of carbon. 

Another method of determining how much food will be required, 
is to estimate how much energy must be liberated in order to meet 
the needs of the organism. We can do this by placing the person in 
a respiration-calorimeter in which all the actual heat which leaves the 
body is collected and measured. By adding this result to the thermal 
equivalent of the muscular work which he meanwhile performs, we 
obtain the total amount of energy eliminated. This result is expressed 
in calories, a kilo-calorie being the amount of heat necessary to raise 
the temperature of one kilo of water through one degree centigrade. 
In this way it is found that about 3,500 kilo-calories are necessary 
per diem for an adult doing ordinary work.^ 

1 In physical chemistry the unit of heat chosen is one thousand times smaller 
than the physiological Calorie, it being in this case the amount ol heat necessary 
to raise the temperature of one gramme of water through one degree centigrade. 



PHYSIOLOGICAL CHEMISTEY 



361 



Having determined how much energy will be required, we must now 
find out how much food must be supplied to yield it. We can deter- 
mine the caloric value of the various food-stuffs by burning them in a 
chemical calorimeter. Since the end products of the combustion of 
fats and carbohydrates (viz., COj and HgO) are the same in the body 
as in' vitro, their physical caloric values for 1 grm. of the dry substances 
are the same as their physiological, viz., 4-1 for carbohydrates and 9 3 
for fats. A very important end product of the metabolism of protein 
is, however, urea, which still contains some potential energy, so that 
it has a physical heat value of its own. In order to find the physio- 
logical heat value of protein, therefore, we must deduct from its physical 
value the physical value of the amount of urea arising from it. By 
this means it has been shown that the physiological heat value of 
protein is practically the same as that of carbohydrate, viz. 4 1. 

By an examination of the diets taken by various classes of people, 
averages of the relative amounts of the various classes of food-stuflfe 
have been obtained. Such a table for a man doing an ordinary amount 
of work is the following : 



Protein, 

Carbohydrate, 

Fat, 

This diet yields : 






125 grammes.^ 

500 grammes. 

50 grammes. 




Carbon. 


, Nitrogen. 


Kilo-Calories. 


Protein, 

Carbohydrate, 

Fat, 


62 grm. 

200 grm. 

38 grm. 


20 grm. 


125x4-1= 512-5 C. 

500x4-1 = 2050 C. 

50x9-3= 465 C. 


Total, 


300 grm. 


20 grin. 




30-27 -5 C. 



Such a diet is represented approximately by : \ lb. prime lean meat; 
1^ lbs. bread; 2 oz. butter; J pint milk; 1 lb. potatoes; and \ lb. 
oatmeal. 

Demonstration. — The quantities of foods given above have been 

The small calorie is written with a small "c," the large one with a capital "C." 
'J'he. heat unit can he transformed into units of work by multiplying by 425-5, a unit 
of work being expressed as the amount of force necessary to raise a weight of one 
gramme to a height of one metre —a gramme metre — or of one kilogramme to the 
same height, a kilogramme metre. 
' Dryweight. 



Protein. 
88 


Fat. 
108 


Carbohydrate. 
345 


Calories. 
2,501 


/125 
\116 


137 

158 


476 
538 


3,364 
3,762 


(U5 
\U5 


195 

235 


557 
666 


4,223 
4,954 



362 PEACTICAL PHYSIOLOGY 

weighed out. Carefully gauge the amounts so as to gain an idea of 
the food necessary daily for a person doing light muscular work. 

For persons leading a sedentary life only 2,500 to 2,700 calories are 
required. For moderately hard muscular work it is well to allow 
3,300 — 3,700 C, for hard and very hard muscular work from 4,150 — 
6,000 C. are required. Specimen diets are given in the following table : 

Sedentary, 
Moderate hard work, 

Very hard work, - 

Most investigators have given diets fairly closely resembling these ; 
and although some of them include more fat than the specimen diet we 
have taken, it must be remembered that fat is considerably dearer as 
an article of food. 

Chittenden, as the result of his experiments, believes that the body 
is better with a low protein diet that is considerably less than those 
given above. It has recently been shown, however, that the con- 
stituent parts of the protein vary considerably in their value for the 
maintenance of health. The ringed amino-acids (tyrosin, tryptophan, 
etc.) appear to be the most important. Bodies without these, such as 
gelatine, cannot support life. Yet if these bodies be added to gelatine 
or other such inadequate diet {e.g. the products of digestion not con- 
taining the benzene ring), such addition renders the diet adequate. It 
is worthy of note, also, that the diet supplied to the young animal 
contains much protein (caseinogen), rich in such "ringed" bodies. 
The kind of protein in a diet must, on this account, be taken into 
account. It would seem therefore that it is well, as the analyses of the 
diets of diiferent nations show, to have about 125 grammes of protein in a 
diet in order to ensure an adequate supply of substances from which 
the organism can select what is needed for its nutrition. 

These facts, too, show us how important it is to know the exact 
composition of the various food-stuffs, so that we may be in a position 
to draw up an adequate diet sheet. 

The organic food-stuifs may conveniently be divided into two classes, 
the animal and the vegetable. 

The Animal Food-Stuffs. — One of the most important of these, viz. 
milk, has been discussed in a separate chapter. Meat and eggs form 
another important class of animal foods. (See Table, p. 366.) 

Eggs. — A hen's egg weighs about two ounces or fifty grammes. The 
forms about 12 % of the total weight, and consists mainly of 



PHYSIOLOGICAL CHEMISTRY 363 

carbonate of lime. The white consists of a multitude of very fine 
fibrous envelopes filled with a solution of protein (chiefly egg-albumin, 
but also traces of egg-globulin and egg mucoid), containing traces of 
sugar (0-5 %), fatty substances, and inorganic salts. About 85 % of the 
white is water. We have already studied this. (See Proteins.) The 
yolk contains about 51 % of water, the solids being mainly fats (31-75 %), 
the chief of which is the phosphorised fat lecithin. It also contains 
about 16 % of protein, and this is mainly of the nature of a phospho- 
protein called vitellin. The proteins and fats are intimately united 
with one another in the yolk, the exact nature of the resulting com- 
pounds not being clearly understood. 

The yolk contains about 1 % of inorganic salts, and it is important 
to note that the phosphorus exists mainly in organic combination (partly as 
lecithin and partly as phospho-protein). The same is true of the iron, 
which also exists in organic combination. Both these inorganic bodies 
are much more easily assimilated when presented to the tissues in 
organic combination. 

Meat. — As meat consists mainly of the muscle of certain animals, we 
have already studied its composition in the previous chapter. It con- 
tains beside muscle a small amount of connective tissue and of fat. 
(See Table, p. 366.) Much of this fat is not visible to the naked eye, 
being situated between the muscle fibres. The amount of this fat 
varies in difiierent animals, and in the same animals constitutes one of 
the chief differences between "prime" and "inferior" meat. Owing 
to the large amount of assimilable protein, meat forms one of our 
chief sources of nitrogen. 

Experiment I. Cut very finely the piece of meat provided; 
grind with saline. Filter, and test the filtrate for proteins and salts. 

The Vegetable Food-StufFs. — The most important groups of these 
are the pulses and the cereals. 

Pvlses. — The pulses include such bodies as peas, lentils, beans. They 
contain relatively little water, and are rich in proteins, carbohydrates, 
and salts. (See Table, p. 366.) They are therefore a very valuable 
form of food-stuff, more particularly so owing to their cheapness; 
although it must be remembered that they are not so easily digested as 
meat, and more is found undigested in the faeces. 

ExPEEiMENT II. Extract some of the pea-meal provided with 
saline. Note the precipitate of globulin on adding a drop to water. 
Perform the general protein tests. Also extract some with cold water. 
On heating, a coagulum is obtained showing albumin. Filter. Test 
filtrate for salts. 

Cereals. — These are obtained from the seeds of various artificially 



364 



PRACTICAL PHYSIOLOGY 



cultivated grasses, and they all. contain representatives of the various 
nutritive constituents of foods. The following table gives their general 
average composition : — 



Water, 
Protein, 
Carbohydrate, 
Fat, - 
Mineral matter, 



10-12 per cent. 
10-12 per cent. 
65-75 per cent. 
0-5-8 per cent, 
about 2 per cent. 



The more important varieties have the following composition ; 





Water. 


Protein. 


Fat. 


Carbo- 
hydrate. 


Cellulose. 


Ash. 


Wheat, - 


12-0 


12-11 


1-7 


71-2 


2-2 


1-9 


Oats, 


10 


10-9 


4-5 


59-1 


12-0 


3-5 


Barley, 


12-3 


101 


1-9 


69-5 


3-8 


2-4 


Rice, 


12 


7-2 


2 


76-8 


1-0 


10 



The most important of the cereal foods is wheat, of which it is 
estimated that 6 bushels per head of population are consumed every 
year. It is in the form of flour and bread that wheat is mainly 
consumed. 

FLOUR. 

Ordinary white flour is obtained from the endosperm of wheat grains 
and contains from 70 to 75 % of starch, about 8 % of protein, and about 
1 % of fat. The protein is mainly of the nature of a globulin, and it 
has the property of becoming viscid when mixed with water. This 
viscid body is called gluten, and it is in virtue of this body that dough 
is formed when water is added to flour, as in the manufacture of bread. 

Experiment III. Eoll up some flour loosely in a piece of muslin ; 
place the bag thus formed in a small beaker containing some water and 
knead it. The starch grains pass through the muslin into the water, 
so that this soon becomes opaque and a sample placed in a test tube 
gives a blue colour with iodine. Apply Trommer's test to another 
sample and note that no sugar is present. After kneading for some 
time remove the bag and examine the contents, when it will be found 
that a sticky mass has been produced — gluten. Remove a piece of this 
and suspend it in water in a test tube. Apply Millon's and the 
xanthoproteic tests to it, and note that the suspended pieces of gluten 
react positively to both tests. 

1 Taken from Hutchison's Food and the Principles of Dietetics. 



PHYSIOLOGICAL CHEMISTRY 365 

The gluten is formed from two proteins in the flour, gliadin (a protein 
soluble in 70 to 80 % alcohol) and glutenin (soluble in alkali). It is to 
the gliadin portion that gluten owes its viscidity; grains poor in 
gliadin do not form dough when mixed with water, e.g. rice, oats, etc. 

Good flour does not contain sugar, and if that be present it shows 
that a certain amount of germination has occurred. 

Whole flour is obtained by crushing the entire grain minus the husk 
and outer portion of the bran. It contains somewhat more protein and 
fat than does white flour and is accordingly more nutritious, but on 
account of the admixture of bran which it contains, it is less digestible 
and acts as a mild laxative on the intestine. 

BREAD AND BREAD MAKING. 

The gluten which is formed when water is added to flour cannot be 
directly used as a food, for, on account of its soddenness, it is impervious 
to the digestive juices, and cannot therefore be digested. Before it 
can be digested it must be aerated, i.e. rendered porous, and in this 
state it forms bread. The agency employed to aerate the bread is 
carbon dioxide gas, which is generated in the mass of gluten or " dough" 
by the action of the yeast plant on sugar. 

The following is a, brief account of the process of bread making : — The first 
stage in the process consists in preparing an active culture of the yeast plant. 
This was originally done by allowing dough to stand exposed to the air, when 
some of the yeast cells, which appear to be omnipresent in the atmosphere, 
settled on it, and grew and multiplied there until a fermenting mass or "leaven" 
was obtained. Unfortunately for this process, however, the atmosphere contains 
other bacteria which also settle on the dough, and by their growth lead to the 
production of organic acids, in consequence of which the mass became very sour. 
To make bread a little of the leaven was added to fresh dough, in which it grew 
and multiplied until the whole was leavened, the aerated mass being then heated 
so as to stop the fermentation. Such bread is very sour, and although the 
process is still carried out in some parts of Germany, it is almost obsolete. 

In the modem process the leaven gives way to the so-called ferment, which is 
produced by adding some pure yeast obtained from the brewery to a culture 
medium consisting of a mixture of mashed potatoes and flour, the culture being 
kept in a warm place for about five hours. By this time the mass is swarming 
with young actively-growing yeast cells, and, provided that contamination with 
bacteria has been prevented, none of the sour acids which develop in leaven are 
present. Besides the yeast, an unorganised ferment called diastase, originally 
present in the flour, becomes active and hydrolyses some of the starch of the flour 
into sugar. The yeast cells then act on this to produce alcohol and carbonic acid 
gas, so that a fermenting mass is obtained. More flour is now added to this, and 
the process allowed to proceed five or six hours longer, until the developed gas 
causes the top to burst, after which the remainder of the flour is added. The 
mass is now called dough. It is thoroughly mixed by machinery, and allowed to 



366 



PRACTICAL PHYSIOLOGY 



ferment for another hour, when it is weighed out into loaves, which are then 
placed in pans and heated to about 232° C. in an oven for an hour and a half. 
The heat kills the yeast, but at the same time causes the enclosed bubbles of gas 
to expand, so that the dough becomes filled with little cavities. The heat also 
causes the outer part of the dough to become hardened by coagulating the 
protein, and at the same time it converts the starch into dextrin and soluble 
starch, and so forms the crust. The crust is glazed because of the dextrin, and 
it is coloured and its taste different from the rest of the bread, because of the 
caramel produced by the action of the heat on the sugar which is developed. 

Experiment IV. Shake up some bread thoroughly with cold 
water and filter off the extract. Test the residue for starch by adding 
iodine, and for protein by the colour tests. Test the filtrate for sugar 
by Trommer's test. All the reactions are positive. If a similar 
extract be made of the crust it will be found to give a purplish colour 
with iodine, due to the soluble starch and dextrin which it contains. 

The colour tests for protein and starch can also be well demonstrated 
by pouring the necessary reagents upon different areas of a piece of 
bread. Do this, taking care to use a minimum amount of the reagents. 
To bring out the red colour of Millon's test, it is necessary to toast the 
bread slightly over the Bunsen flame. 



PERCENTAGE AVERAGE COMPOSITION OF SOME OF THE MORE 
IMPORTANT FOOD-STUFFS. 

(Adapted from various sources.) 





Water. 


Proteins. 


Fats. 


Carbo- 
hydrates. 


Salts. 


Beef (best quality). 


72 


21 


6 


— 


1 


Biscuits, 


8 


15 


1-3 


73-4 


1-7 


Bread (wheaten). 


40 


8 


1-5 


49-2 


1-3 


Butter (fresh). 


12 


2 


85 


— 


1 


Cheese, 


41 


28 


23 


1 


7 


Eggs, 


73-5 


13-5 


11-6 


— 


1-4 


Fish (salmon). 


76 


15 


7 


— 


2 


Fish (sole). 


86 


12 


0-5 


— 


1-5 


Flour (fine wheaten), - 


16-5 


13 


1-5 


68-3 


0-7 


Lentils, 


12-5 


24-8 


1-8 


58-4 


2-5 


Milk (cow's), - 


86-9 


4-7 


3-5 


4-2 


0-7 


Mutton, 


76 


18 


5 





1 


Oatmeal, 


15 


13 


6 


63 


3 


Peas, - 


15-6 


22 


2 


58 


2-4 


Potatoes, - 


74 


2 


0-2 


21-8 


1 


Rice, - 


10 


5 


01 


84-4 


0-5 



PHYSIOLOGICAL CHEMISTRY 367 

In the above table note particularly the great food value of such bodies as 
bread, flour, cheese (particularly), lentils, peas, oatmeal, rice ; all comparatively 
cheap articles of diet. 

The Methods for the Estimation of General Metabolism.— Metabolism 
is the subject which treats of the changes undergone by the food-stuffs after they 
are absorbed from the intestine. There are two subdivisions of the subject ; the 
one, called general metabolism, has to do with the building up or breaking down 
of the tissues. It derives its information from a comparison of the amount of the 
various foodstuffs absorbed, with the amount of their excretory products. The 
other, called special metdboliam, has to do with the exact chemical changes which 
absorbed food-stuffs undergo, and the localisation of the actual organ or organs in 
which the various changes are effected. 

Space will only permit us to indicate some of the methods employed in study- 
ing general metabolism, and to describe briefly how the results obtained may be 
interpreted. The actual methods of analysis are fully described in other chapters, 
and in the following description reference will be made to the pages on which the 
most suitable method for each determination can be found. 

General Metabolism. — In order to study this a balance sheet must be drawn 
up, on one side of which is placed the intake (the amount of food and oxygen 
absorbed), and on the other the output (the amount of the various bodies excreted 
in the urine, faeces, breath, and sweat). 

I- The Intake. — The value of a diet can be expressed either as its chemical 
value, or as its physical value. The chemical value means the amount of protein, 
fat, carbohydrate, and salt which it contains. This is determined by referring 
to analytical tables of the various food-stuffs (especially serviceable for this 
purpose are the tables of Atwater). The amount of the various food-stuffs 
administered can then be easily determined by multiplying the percentage given 
on the tables by the amount of food given. When it is desired to be specially 
accurate an actual analysis of the food is necessary, and when the metabolism of 
protein is being specially studied, it is customary to determine the amount of 
nitrogen which the food-stuff contains (Kjeldahl's method, p. 410), and this 
multiplied by 6 "3 gives the amount of protein. ' 

The physiological heat value of a diet means the number of calories which it can 
yield during its metabolism in the body. To find the total heat value of the 
diet, all that is necessary is to multiply the physiological heat values of the 
administered food-stuffs by the amount of each which the diet contains. 

The Form in which the Food-Stuffs are best given for Metabolism 
Experiments. Protein. — This is usually given as meat, from which all the 
visible fat and tendon are, as far as possible, removed. When calculating the 
amount of protein from the nitrogen present, the gelatin and extractives which 
the meat contains may be neglected, for gelatin, in the presence of an excess of 
protein, has almost the same metabolic value as native proteins, and the 
extractives exert no influence on the metabolism since they pass through the 
tissues unchanged. Protein may also be administered as white of egg or as milk. 

Carbohydrate. — This is best given as bread a day old, and always obtained 
from the same source, so that its composition is constant. 

^JThis figure would not be correct if the food contained nitrogenous substances 
other thanjrotein. 



368 PEACTICAL PHYSIOLOGY 

Fat.— About 1 % fat has to be reckoned as contained in the meat prepared as 
above. The rest is given best as butter. 

When the investigation is being carried out on an animal, the whole diet 
should be weighed out in the morning, after collecting the previous day's excreta. 
It is seldom necessary to cook the food, but where there is difficulty in persuading 
the animal to take some unpalatable food substance, this latter may be mixed 
with the soup prepared from the meat. When the experiments are being carried 
out on man, it is of course necessary to cook the meat, and frequently also some 
of the other food-stuflfs. The various constituents must be weighed out before 
cooking, as it is impossible to know, after the food has been prepared, the 
proportion of the substance used in cooking. For observations extending over 
any length of time the diet should be carefully chosen, and exactly the same 
amounts given each day. 

II. The Output, — By referring to the above scheme it will be seen that the 
only food-stuff which contains nitrogen and sulphur is protein. We have, 
therefore, two excretory products from the amount of which we can determine 
protein metabolism. In the case of carbohydrates and fats, on the other hand, 
there is no exclusive end product, so that, in order to estimate the metabolism 
of these two bodies, it is necessary to make a calculation. 

S H2SO, 

Proteins! j,^^^ 1 C}co, |.„rea, etc. 



I Carbohydrates j tj VHjO I 

1. Protein. — The output of this is determined : 

(a) From the Amount of Nitrogen Excreted.— Nearly the whole of this 
occurs in the urine in which it is determined by Kjeldahl's method (p. 410). A 
certain amount, however, appears in the faeces. With an ordinary diet most of 
this latter comes from the unabsorbed protein, and must accordingly be deducted 
from the amount administered in order to ascertain the actual amount absorbed. 
A certain amount of it, however, comes from nitrogenous bodies, which are 
excreted into the intestine from the blood. The actual amount of this excretory 
nitrogen has been determined by feeding an animal with a protein free diet, and 
for man it amounts on an average to 1 grm. per diem. During starvation it only 
amounts to 0"2 grm., so that it is obvious that it comes from the digestive juices 
and excreta poured into the intestine.' 

The amount of nitrogen excreted in the sweat is so small as to be negligible. 
The urinary nitrogen, plus one gramme per diem, as nitrogen excreted into the 
intestine, gives us, therefore, the total amount of nitrogen excreted. Since 
protein contains 16 % of nitrogen, each gramme of nitrogen corresponds to 6'25 grm. 
of protein, and since meat contains on an average 3 '4 % of nitrogen, each gramme 
of the latter will correspond to 30 grm. of muscle. 

(6) From the amount of Sulphur Extracted.— The proteins of food con- 
tain 1 % of sulphur. This is excreted in the urine largely as sulphuric acid, and the 

1 In accurate metabolism determinations it is necessary to collect the faeces for 
each day, to dry them slowly on a sand bath, and then to make the following 
determinations : 

(a) The total amount of nitrogen. 

(ft) The total amount of fat {i.e. extract with Soxhlet's apparatus). 

(c) The total amount of solids. 



PHYSIOLOGICAL CHEMISTRY 369 

amount of this excreted bears a constant relationship to that of nitrogen, viz. 1 of 
sulphuric acid for every 5 "2 gr. of nitrogen. Being less in amount, its deter- 
mination is not so accurate as that of nitrogen, but it affords us a valuable control 
in estimating protein metabolism, and is the only way by which we can estimate 
this when nitrogenous bodies other than protein are contained in the diet. 

2. Fat and Carbohydrate.— The end products of the metabolism of both 
these bodies are water and carbon dioxide gas, and, of these two bodies, 
the only one which it is possible to estimate with ease is the latter. 
Protein, however, also contributes to the excretion of carbon dioxide, so that, 
before we can know how much carbohydrate and fat are being oxidised in the 
body, we must find out what proportion of the total carbon excreted is derived 
from the metabolism of the protein. 

To estimate the total amount of carbon excreted, the expired air must be 
collected, and a determination of the amount of carbon dioxide which it contains 
made by one of the methods described (see p. 184). The obtained result 
multiplied by 0'273 gives the amount of carbon excreted in the breath. A certain 
amount of carbon is aim excreted in the urine. This latter amount could be 
directly determined by making an elementary analysis of the dried urine, but 
such a method would, of course, be too laborious for metabolism work. In order 
to determine this amount of carbon it is sufficiently accurate to multiply the 
nitrogen excreted by 0'67, for it has been determined that, for every gramme of 
nitrogen excreted, there is this amount of carbon, and that this ratio is a 
constant one. 

Having estimated what the total excretion of carbon is, we must now ascertain 
how much of it comes from protein. To do this multiply the total amount of 
nitrogen excreted by 3 '3 (since proteins contain approximately 52-8 of carbon, 
and 16 of nitrogen). If this amount of carbon be deducted from the total amount 
excreted, the remainder corresponds to the carbon derived from the combustion 
of fat and carbohydrate. As to which of these two bodies it is from which the 
carbon really comes, we have no means of telling definitely, except by a 
determination of the respiratory quotient, but since there is very much more 
fat than carbohydrate in the tissues we usually reckon it as fat. Each gramme 
of carbon corresponds to 1"3 grammes of fat (because fat contains 76 "5 grammes 
carbon). 

3. The Amount of Energy given out by the Body.— The energy is 
liberated in the body partly as heat, and partly as muscular work. The amount 
actually lost as heat may be determined by placing the animal in a respiration 
calorimeter, but it is difficult to estimate the amount lost as mechanical work. 

There are, however, certain indirect methods by which the total amount of 
energy liberated may be determined, and these are as follows : (a) By comparing 
the amount of food-stuffs taken in with the amount which reappears in the 
excreta, we can find out how much of each food-stuff has actually undergone 
metabolism in the tissues. It is now quite easy to find how much energy this 
corresponds to, by multiplying the amount of each food-stuff metabolised by its 
caloric value. (Where the diet contains both fat and carbohydrate, and where an 
accurate balance of intake and output of carbon does not exist, we must reckon 
the excess or deficit as fat, since there is much evidence to show that the amount 
of carbohydrate in the body remains pretty constant. ) 

(6) The extent of oxidation in the tissues is determined, not by the amount of 
oxygen inspired, but by the activity of the tissues. We can, therefore, employ 

2a 



370 



PRACTICAL PHYSIOLOGy 



the amount of oxygen absorbed by the tissues as an index of the amount of energy 
liberated in them. In order to do this, however, it is necessary to remember that 
the amount of energy liberated, when dififerent food-stuflFs are burnt, is not the 
same ; thus 100 gr. of oxygen are necessary for the combustion of 35 gr. of fat, 
the amount of energy hereby liberated amounting to 325 calories. The same 
amount of oxygen will burn up 84'4 grm. of carbohydrate, and yield thereby 
346 calories, or 74-4 grm. protein yielding 362 calories. It is therefore necessary, 
before employing the oxygen absorbed as an index of the amount of energy 
liberated, to ascertain that, when the determination is being made, the food-stuffs 
undergoing oxidation are always the same. This can be ascertained by estimating 
the respiratory quotient, the value of this being influenced mainly by the nature 
of the food-stuff undergoing combustion at the time (see p. 185). So long as the 
R.Q. remains constant, any increase or diminution in the amount of oxygen 
absorbed represents more or less energy liberated. In order that we may be able 
to compare the oxygen assimilation of different individuals under the same 
conditions, Zuntz has suggested that the determination should be made the first 
thing in the morning, immediately on awakening, and twelve hours after the last 
diet (which should not contain much carbohydrate) has been taken. The 
estimation should be made by Zuntz respiratory apparatus. The amount of 
oxygen absorbed, and of carbon dioxide exhaled, is then reckoned for each kilo, 
body weight, and for each minute. The normal amounts for man are 3 to 4"5 c.c. 
oxygen, and 2'5 to 3'5 c.c. carbon dioxide. 

Example of a Metabolism Investigation. — It is desired to know whether 
a diet containing 125 grammes protein, 50 grammes fat, and 500 grammes carbo- 
hydrate is sufficient for a man doing a moderate amount of work. 





In 


TAKE. 








Carbon, 




Nitrogen. 


Calories. 


Protein, 


62 grm. 




20 grm. 


512-5 


Carbohydrate, - 


200 




— 


2050-0 


Fat, - 


38 




— 


465-0 


Total, 


300 grm. 


20 grm. 


30-27-5 




Output. 










Carbon. 




Nitrogen. 


In urine. 


- 


11 grm. 


(16-5 X 0-67) 


16-5 grm. 


In faeces. - 




5 




1-0 


In the breath, 




254 




— 



Total, 



270 grm. 



17-5 grm. 



Retained in body = 30 grammes carbon and 2-5 grammes nitrogen. This 
amount of nitrogen represents 2-5x6-25 = 15-6 grammes protein, or 75 grammes 
muscle. Now, this amount of protein will account for 8-25 grammes carbon ; so 
that 30 -8 -25 =21 -75 grammes carbon represent 21-75x1-3 = 28-3 grammes /a<. 
On this diet, therefore, the subject retains in his tissues 15-6 gr. protein and 
28-3 gr. fat per diem. 

To express this result in terms of energy liberated, we know that 3027-5 C. 
were supplied and that all these have been used except 15-6 x 4-1 = 64 retained as 
protein, and 28-3 x 9-3 = 263-2 retained as fat, or in toto 327-2 C. We find, there- 
fore, that 3027-5-327-2=2,7000. have been required. 



PHYSIOLOGICAL CHEMISTRY 371 

CHAPTEE XII. 

DIGESTION IN THE MOUTH. 

The salivary glands — parotid, sublingual, and submaxillary — along 
with the numerous isolated gland acini scattered in the buccal mucosa, 
pour into the mouth a secretion known as saliva. The composition of 
this mixed saliva is as follows : 

Water, 99-42 % 

Organic matter, 0'36 ,, 

Mucus and epithelial cells. Ftyalin and soluble protein. 

Potassium sulphocyanide (KCNS). 

Inorganic matter, 0-22,, 

Chlorides, phosphates, and carbonates of alkalies and alkaline earths. 

It is, therefore, a very dilute secretion (specific gravity about 1005). 

The total secretion during twenty-four hours amounts to about the 
same as that of the urine, i.e. 1500 c.c. 

The saliva secreted by the different glands varies somewhat in com- 
position; that from the parotid contains no mucus, and is consequently 
a thinner fluid than that of the submaxillary, which contains much 
mucus. The sublingual saliva also contains a certain amount of 
mucus. 

Collect some saliva in a test tube,^ and perform the following reactions 
with it : 

I. To identify the various Constituents. 

Experiment I. Place a drop of saliva on red litmus paper ; a blue 
stain results. The reaction may, however, become acid where decom- 
position is taking place in the mouth, as is the case in decaying teeth. 

Experiment II. Place a drop of saliva on a slide, cover and examine 
under the microscope: two kinds of cells will be seen, viz. (1) large, 
flat, squamous cells, which have been desquamated from the surface of 
the stratified epithelium of the mouth; (2) small round cells like 
leucocytes, which come either from the glands themselves or from the 
tonsils. 

Experiment III. Place some saliva in a test tube and dilute it with 
an equal quantity of water; now add a few drops of 10 per cent, acetic 
acid, when a stringy precipitate of mucus will form. Filter off this pre- 
cipitate, and note that the filtrate is watery, showing that the stringy 
character of saliva is due to the mucus which it contains. To the 

1 The secretion of saliva may be stimulated by inhaling acetic acid through the 
mouth, or by chewing rubber. 



372 PRACTICAL PHYSIOLOGY 

filtrate add a few drops of Millon's reagent and boil. The result shows 
the presence of protein. 

Experiment IV. Add to some saliva in a test tube a drop of a 
weak solution of ferric chloride (Liq. Ferri. Perchlor. B.P.) and a drop 
of hydrochloric acid. A red colour is sometimes produced. This is 
due to the production of ferric sulphocyanide by interaction between 
the ferric chloride and a sulphocyanide which is contained in saliva. 
The red colour is discharged by adding a few drops of a solution of 
mercuric chloride (1-1000). A more sensitive way of performing this 
test is to place a drop of saliva at one end of a piece of filter paper, 
and then to allow a drop of ferric chloride solution (acidified with HCl), 
spread to the edge of the saliva drop; a deep red stain will result 
where the two moistened areas meet. 

Experiment V. If some saliva be allowed to stand for an hour or 
so, it becomes milky or a thin surface film forms on it. This is due to 
the precipitation of calcium carbonate, which exists in fresh saliva in a 
soluble state as calcium bicarbonate. On standing exposed to the air, 
however, carbonic acid gas is given off, in consequence of which the 
bicarbonate changes into carbonate, which is insoluble. A similar pre- 
cipitation of calcium carbonate, carrying with it a certain amount of 
calcium phosphate, sometimes occurs in the ducts of the glands and 
leads to the formation of calculi, or it may form on the teeth, where it 
leads to the formation of tartar. 

II. To Study the Action of the Ferment Ptyalin. 

Experiment VI. Place a few cubic centimetres of a 0'5 per cent, solu- 
tion of starch in two test tubes, a and h. To 6 add about an equal amount 
of saliva, and place both a and h in the water-bath heated to body 
temperature. Note that in a very few minutes the solution in h loses 
its opalescence and becomes clear. Bj means of a glass rod transfer 
drops from each solution, about once a minute, to a white slab or dry 
evaporating dish, and add to each drop a little iodine solution. In the 
drops from the test tube h the blue colour becomes at first purplish and 
then reddish brown, and ultimately disappears. When this stage has 
been reached, apply Trommer's or Fehling's test to the contents of the 
test tube, and note that reduction occurs. In the case of a the blue 
colour persists throughout and reduction of cupric salts does not occur. 

What has occurred in h is that the ptyalin has hydrolysed the poly- 
saccharide starch (blue with iodine and no reducing power), first into a 
simpler polysaccharide dextrin (red with iodine, no reducing power), 
and then into the disaccharide maltose (no colour with iodine, reduces 
cupric salts). If left in contact with the maltose for some time the 
saliva can invert this, yielding dextrose. This indicates the presence 



PHYSIOLOGICAL CHEMISTRY 373 

of maltase (see p. 403). There are several varieties of dextrin formed 
during the hydrolysis, one of these gives the iodine reaction described 
above, and is called erythro-dextrin ; another, called achroo-dextrin, 
gives no reaction with iodine. The latter exists as an intermediate 
stage between erythro-dextrin and maltose. 

The very first efiect of ptyalin on starch is to convert it into so-called 
soluble starch (sometimes called amylodextrin). This gives a clear 
solution with water and a blue reaction with iodine. During each step 
in the hydrolytic break down, a certain amount of maltose is set free. 
This is small in amount at first, but becomes progressively more with 
each successive dextrin formed. 

Experiment VII. Place some of 0-5 per cent, solution of starch in the 
mouth, and after about two minutes transfer it to a test tube. Ascer- 
tain if reduction of cupric salts occurs. Eepeat this experiment with 
some unboiled starch, and note the difference in the two cases (see 
Carbohydrates, p. 290). 

The ptyalin will only act in neutral or very faintly alkaline reaction, 
but not in the presence of free acid {e.g. O'OOS % HCl can practically 
stop its action). Stronger alkalinity destroys it. 

Experiment VIII. If Experiment VI. be repeated with the addition 
of a few drops of 0*2 per cent, hydrochloric acid, so that the fluid just 
reacts acid to litmus, it will be noticed that no dextrin is produced. (If 
the acid mixture be heated for a considerable time a trace of reducing 
sugar may appear because of the hydrolysing action of the acid.) 

From the result of Experiment VIII. we may conclude that it would 
be impossible for the action of the ptyalin to proceed in the stomach 
after the gastric contents had become distinctly acid. If the stomach be 
empty at the beginning of the meal, however, the action of ptyalin 
may proceed in this viscus for some considerable time, since the first 
portion of acid which is secreted becomes bound to protein, so that it 
does not exercise its inhibiting influence on the ptyalin which has been 
swallowed. 

A certain amount of the hydrochloric acid secreted by the stomach 
will also combine with the alkalies of saliva to form chlorides. These 
chlorides have a marked accelerating influence on the action of saliva. 
Although, therefore, ptyalin has little chance in the mouth to carry its 
action on starch far, it can, nevertheless, continue acting for some con- 
siderable time in the fundus of the stomach. The extent of this action 
no doubt varies in different cases, being probably more prolonged when 
the food is taken without much liquid. 

One of the chief functions of the saliva is undoubtedly a mechanical 
one, acting as a solvent for certain foods, and assisting in the mastica- 



374 PEACTICAL PHYSIOLOGY 

tion and swallowing of others. A body must be in solution before it can be 
tasted, so that the saliva assists in the appreciation of taste. It is also 
necessary for articulation and for preserving the sensitiveness of the 
nerve endings of taste and common sensation. This explains why a 
fever patient cannot taste things so well as during health. It is inter- 
esting to note that in some animals the saliva contains little or no 
amylolytic ferment {e.g. dog and cat). 

For accurately studying the action of ptyalin (or any other amylolytic ferment) 
on starch one may estimate the reducing power (Bang's method, gravimetric 
method, polarimeter) of the incubated solution after a certain time. Besides being 
tedious, this method is uncertain, because of the different reducing powers of 
maltose and dextrose, both of which sugars frequently result by salivary digestion, 
especially when this is prolonged. 

A simpler and more serviceable method depends on the colour reaction of 
starch with iodine, and is conducted as follows : — 

Prepare a 1 per cent, starch paste solution,^ and place the beaker containing it in 
ice water. Collect some saliva and dilute 1 c.c. of it to lOcc. with distilled water, 
and filter. Take a series of five test tubes labelled A, B, C, etc., and with a 1 c.c. 
pipette graduated in 100 parts deliver into tube .il 1 c.c. of the diluted saliva ; 
into B 0-75 c.c; into C O'S ; into D 0-25 ; and into E 0-1. 

Place the tubes in a beaker containing ice water,, sind then deliver into each 5 c.c. 
of 1 per cent, cooled starch solution. The cold prevents any ferment action until 
all are ready. Now remove the tubes to another beaker containing water at 
40° C. , and gently shake them so that the contents become thoroughly mixed. Note 
the exact time at which the tubes are placed in the warm water. At the end of 
half an hour remove the tubes simultaneously to ice water, and shake them gently 
so as to ensure thorough cooling. Fill each tube to within half an inch of the top 

with distilled water and add a few drops of iodine solution ( tt; )." Close each 

tube with the finger and invert so as to mix. It will be seen that there is a grada- 
tion of colours in the different tubes from blue through violet and brown to yellow. 
Note the tube which just shows a bluish tint. The next one higher up in the 
series is taken as that in whicli all starch has just disappeared. From the amount 
of diluted solution added to this, calculate the amount of undiluted saliva required to 
convert 100 c. c. of 1 per cent, starch solution into dextrines in half an hour at 40° C. 
Thus, suppose that the tube containing 0'2d c.c. diluted saliva is found to be that 
which just shows a bluish tint. In the next (viz. containing 0'5 o.c. saliva) all 

0*5 

the starch has disappeared, therefore -y^= 05 c.c. saliva can hydrolyse 5 c.c. 

Ipercent. starch,orlc.c. can invert 100 c.c. Ipercent. starch. The diastatic action 

^ Weigh 1 or 2 gm. of pulverized " soluble starch," and stir it up in a beaker 
with an amount of distilled water suflScient to make a 1 per cent, solution. Place 
on a boiling water bath and continue stirring until a clear opalescent solution is 
obtained. Cool before using. 

2 Care must be taken that sufficient iodine solution is added to give the 
maximal reaction, but an excess must be avoided. The iodine solution is made 
by dissolving 12'7 gr. iodine in water containing 25 gr. potassium iodide, and 
then diluting to 1000 o.c. 



PHYSIOLOGICAL CHEMISTRY 375 

of pancreatic juice, of liver extract, of blood serum or of malt diastase may be 

measured in the same way, but dififerent amounts of the ferment solution must be 

employed. 1 (Thus for blood serum and liver extract it is unnecessary to dilute 

40° 
the solution.) The results may be expressed by the formula'' ^nj; , iu which the 

temperature and the length of time of incubation are shown. In the above 
example U;^=l. 

To study the influence of weak acids, etc. , on the action of ptyalin the above 
method is very satisfactory, i. e. by adding some acid solution to one or more of 
the tubes. In some cases it is desirable to prolong the incubation for twenty-four 
hours, in which case some chloroform or toluol or thymol should be added to 
retard the development of micro-organisms. If very close results are desired, the 
observation should be performed with amounts of ferment solution which vary 
from one another by smaller amounts, or a second observation should be made 
taking amounts of ferment solution lying between the faintest blue and the next 
tube. 

It is of interest to compare by the above method the comparative diastatic 
powers of the various commercial preparations of diastase, taking human saliva 
as the standard. 



CHAPTER XIII. 

DIGESTION IN THE STOMACH. 

The food, after being masticated in the mouth, is passed down the 
oesophagus into the stomach, where it is acted on by the gastric juice, 
and is gradually forced out through the pylorus. Normally the 
stomach is again empty in about five hours after a meal. The food 
collects at first in the fundus of the stomach, which becomes dilated to 
receive it. By gradually contracting the fundus forces the food in 
small quantities at a time into the pyloric region, in which there are 
frequent peristaltic movements which cause the food to be moved 
about, and thus more intimately mixed with the digestive juices. 
When properly disseminated and distinctly acid in reaction this food 
causes the pyloric sphincter to relax, so that it is passed into the 
duodenum ; but only small quantities at a time are allowed to pass, the 
sphincter closing between each portion. While lying in the fundus, 
very considerable digestion of starch by swallowed ptyalin is taking 
place. On entering the stomach the food is very little changed, except 

' Care must be taken when using organ extracts, such as those of liver, that the 
reaction of the incubation mixture is kept constant. This is best accomplished 
by adding a few drops of a saturated solution of Na2HP04 to the solutions. 

''D = diastatic power. 



376 PRACTICAL PHYSIOLOGY 

that it has been masticated. On leaving it, however, its appearance is 
quite altered, being now a thick, more or less coloured, fluid called 
chyme. 

Various methods have been adopted for studying gastric digestion — 
e.g. observing the process through a gastric fistula, and removing 
samples of the gastric contents by means of a stomach tube. In order 
to obtain pure gastric juice the most reliable method is that introduced 
by Pawlow, which consists in resecting a portion of the fundus of the 
stomach, and sewing it up so as to form a bag, which is then sutured 
to an abdominal fistula. This isolated sac of stomach secretes pure 
gastric juice, which may be collected from the fistula. The juice 
secreted by this miniature stomach has been shown to be identical in 
amount and strength with that secreted in the main stomach. 

The Composition of Gastric Juice. — Pure gastric juice obtained by 
Pawlow's method is a clear, colourless fluid, with a specific gravity of 
1003-1006, and of acid reaction. 

Its percentage composition varies in different animals, that of the 
dog and of man being as follows : 

Man. Dog. 

Water, - 9944 973 

Organic matter, chiefly pepsin, 0'32 1"71 

Inorganic matter — 

(a) free hydrochloric acid, 0-2-0-3 0-3 ^ 

(6) salts, 0-1-0-2 0-66 

The most important features to be considered in connection with 
this table are : (1) the presence of free hydrochloric acid, and (2) the 
nature of the organic matter. 

The Acidity of the Gastric Juice. 

In marked contrast to most of the other fluids of the animal body, 
the gastric juice has a strong acid reaction towards all indicators. This 
acidity performs a very important r61e in the process of digestion in 
the stomach, which makes it of interest and importance to study 
carefully. The urine also reacts acid towards certain indicators, but 
the acidity in this case can readily be shown to be due to quite another 
cause from that of the acidity of the gastric juice. In certain diseased 
conditions, alterations take place in the degree and nature of the 
acidity of gastric juice, and these alterations are sometimes of value in 
assisting in the diagnosis of the pathological condition. 

From a chemical standpoint acidity is invariably due to the presence 
of excess of hydrogen ions in the solution. For the presence of these 

^Pawlow always found more than 0'3 per cent. — namely, 0'5-0'6 per cent. 



PHYSIOLOGICAL CHEMISTRY 377 

hydrogen ions, one or other of three general causes may be responsible,. 
viz. the presence of free mineral acid, free organic acid, and acid salt. 
The acidity in each case is in direct proportion to the dissociation of 
the acid in watery solution, being greatest for mineral acid. One of 
the first questions, therefore, which confronts us in connection with the 
acidity of gastric juice is : to which of the above causes is the presence 
of hydrogen ions due t The question is most simply answered by the 
use of indicators, for it has been found that the behaviour of these 
varies with the nature and cause of the acidity. 

Let us see, first of all, whether the acidity is due to a free acid or to 
an acid salt. Congo red is the most useful indicator for this purpose. 

Experiment I. To a 0-2 per cent. HCl solution add a few drops of 
congo red solution -.i the red turns to blue. Eepeat with a dilute 
solution of acid sodium (NaHjPO^) phosphate — no blue colour develops. 
Show that the latter solution reacts acid towards litmus or phenol- 
phthalein. Eepeat this experiment, using, instead of a congo red 
solution, pieces of congo red paper prepared by dipping filter paper 
in a congo red solution and drying. 

The result with congo red indicates that the acidity is due to free 
acid, but it does not tell us whether this is a mineral or an organic 
acid, for both of these turn it blue, although the mineral acid does 
so much more readily, i.e. in much greater dilution than the organic. 
(Indeed, if a solution of the acidity of gastric juice — as determined 
by the method described on p. 381 — gives the reaction with congo 
red distinctly, especially when congo red papers are used, it is almost 
certain evidence that free mineral acid, and not organic acid, is the cause 
of the acidity.) 

To further trace the cause of the acidity, use is made of several 

indicators whose behaviour towards dilute organic and combined 

mineral acids is quite different from that occurring in the presence of free 

mineral acid. The most important of these indicators are employed in 

the following experiments which should be performed with 0-2 per cent. 

N 
hydrochloric acid solution, j^ hydrochloric acid solution (0-0365 per 

N . . N 

cent.), YR lactic acid solution (0-9 per cent.) and ytjtt lactic acid solution 

(0-09 per cent.). 

Experiment II. Giinzberg's Test. Place a few drops of the reagent 
(a solution of 2 parts phloroglucin and 1 part vanillin in 30 parts 
95 per cent, alcohol) in an evaporating basin, and add a few drops of 

^ Congo red solution — dissolve 0'5 gra. of congo red in 100 c.c. of 10 per cent, 
alcohol. 



378 PRACTICAL PHYSIOLOGY 

the liquid to be tested. Slowly evaporate to dryness. With dilute 
hydrochloric acid a red colour develops, with lactic acid no red colour. 

Experiment III. The Tropaeolin Test. Place a drop of a saturated 
solution of Tropaeolin-00 ^ in 95 per cent, alcohol in a dry evaporating 
dish, and dry it with moderate heat. To the yellow stain which 
results apply a drop of 0-2 per cent, hydrochloric acid. A purple colour 
is produced. Repeat with the other acid solutions provided. 

Experiment IV. Di-methyl-amino-azo-benzol 

N(CH3),-C,H,-N = N-CeH, 

(Topfer's Test).^ Add 1-2 drops of the reagent to some of the solution 
to be tested. If this contain free mineral acid a pinkish red colour 
develops. Organic acids, even when quite dilute, will also give a faint 
red colour with this reagent. 

It will be found, as a result of these experiments, that the reactions 
obtained with the hydrochloric acid solutions are more or less simulated 
by those of the stronger lactic acid solution, except in the case of 
Giinzberg's reaction. On the other hand, this reagent gives a positive 
result with hydrochloric acid diluted to 1 in 10,000 parts. The 
tropaeolin and the azo-benzol reactions with 0'2 HCl are also quite 
distinguishable from those given by lactic acid solutions of the above 
strengths, but in greater dilutions of HCl the distinction is by no 
means so definite. 

If the contents of the stomach (removed by a stomach tube or 
through a fistula) be tested with any of the above reagents some three 
hours after an ordinary meal, results like those obtained with the HCl 
solutions will be observed. This is taken as evidence of the presence 
of free hydrochloric acid. Absolute proof that it is this, and not some 
other mineral acid, that is the cause of the positive result has been 
furnished by comparing the total amount of bases with the total 
amount of acids in the ash of gastric juice (see p. 381): it has been 
found that there is an excess of chlorine over that required to combine 
with the bases to form chlorides. This chlorine must exist in the juice 
as hydrochloric acid. 

The Functions of the Acid. — These are generally stated as being 
two in number. 

1. To assist in the action of the proteolytic ferment, pepsin. Evi- 
dence of this will be furnished when we study the action of pepsin. 
For the present it is important to note that the acid combines with 
the protein, and that the amount of acid which thus combines increases 

' 00 represents a trade brand. 

^Dissolve O'o gr. di-methyl-amino-azo-benzol in 100 c.c. 95 per cent, n'.cohol. 



PHYSIOLOGICAL CHEMISTRY 379 

as digestion proceeds, because the combining power of the proteoses 
and peptones for hydrochloric acid is greater than that of native 
protein. In consequence of this increasing absorption of hydrochloric 
acid during peptic digestion, the acid becomes combined as soon as it is 
secreted for some considerable time after the start of digestion. After 
a test meal of soup, meat (200 gr.) and bread (50 gr.) it takes from 
three to four hours before there comes to be any free hydrochloric acid 
detectable in the gastric contents, although during all this time the 
acid is being actively secreted. In cancer and in catarrhal conditions 
of the gastric mucosa, and in fever, the secretion of hydrochloric acid is 
depressed so that it may all become combined with protein and never 
appear in the free state, i.e. never come to react with the above 
indicators. 

2. To prevent putrefaction in the stomach. Hydrochloric acid in 
the free state, like any other mineral acid, is a strong antiseptic. The 
micro-organisms with which our food is contaminated are therefore 
destroyed before they have had time to multiply to any degree in the 
stomach. If it were not for this, the stomach contents would serve as 
a most favourable place for the growth of these micro-organisms, and 
putrefaction and fermentation would become excessive in them. This 
is actually what occurs when the secretion of hydrochloric acid is 
■deficient, as in cancer. If, in such cases, the reaction of the stomach 
contents be tested, it will nevertheless be found strongly acid to litmus, 
and if, furthermore, the degree of this acidity be estimated (by the 
method described on page 381), it may be found even higher than that 
•of normal gastric contents. By the application of the indicator tests 
•described above (especially Giinzberg's), it can readily be shown that 
the acidity is not due to free hydrochloric acid. This leaves, as its 
possible causes, hydrochloric acid combined with protein, acid salts, and 
organic acids. Since it is known that micro-organismal growth is apt 
to produce organic acids, especially lactic acid, we next proceed to test 
for the presence of this. 

Experiment V. The test for lactic acid is performed with an ethereal 
extract of the gastric contents, which is made by mixing 5 c.c. of the 
gastric contents with 30 c.c. ether in a separating funnel, then gently 
shaking the two with a rotatory motion (avoid violent shaking, else an 
emulsion is apt to occur), and, after settling, pouring off the ether. 
The ethereal extract is then evaporated to dryness,^ the residue dis- 
.solved in a little water, and the resulting solution tested with Uflfel- 

^In evaporating ether, he, very carefvl that no flame is near. The evaporation 
should be performed on a water batli that has been heated, and the flame then 
.extinguished. 



380 PRACTICAL PHYSIOLOGY 

mann's reagent (30 c.c. 1 % phenol solution and a few drops of ferric 
chloride solution). The blue violet colour of the reagent changes to a 
characteristic yellow when lactic acid is present.^ 

A much more sensitive and characteristic test for lactic acid is 
Hopkms', which is applied as follows : 

Experiment VI. Mix some of the dry ethereal extract of stomach 
contents with 5 c.c. concentrated sulphuric acid, and transfer to a dry 
test tube. Add 3 drops of a saturated solution of copper sulphate, 
mix, heat the mixture in .a boiling water bath for 2 minutes. Cool 
under the tap, and add 2 drops of a 0-2 per cent, alcoholic solution of thio- 
phene and shake. Eeplace the tube in the boiling water bath, and 
examine it frequently, when a cherry red colour will be found to 
develop if lactic acid is present. Prolonged heating causes the solution 
to become very dark. 

The lactic acid is produced by the action of the bacillus acidi lactici 
and other organisms on sugars (see Milk, p. 329). • 

Lactose. Lactic acid. 

The fermentative process seldom stops at the production of lactic 
acid. Other bacteria act on the lactic acid and produce butyric acid> 
carbon dioxide gas, and hydrogen. 

iC,E,0, = 2C,H302 + 4Co2 + m. 
Lactic acid. Butyric acid. 

These gases accumulate in the stomach, causing flatulence. The 
presence of butyric acid usually reveals itself by the odour of the 
gastric contents. When its presence is doubtful, boil a portion of 
the fluid and hold a strip of blue litmus paper in the steam. If this, 
turns red, it indicates a volatile acid (butyric or acetic). Butyric acid 
has a characteristic odour. 

Acid phosphates (NaHjPo^) when present in gastric contents are 
demonstrated by mixing calcium carbonate with a portion of the fluid. 
If an acid reaction still remains towards litmus paper, acid phosphates, 
must be present, since the calcium carbonate will have combined with 
the free acids. 

In the clinical examination of the stomach contents numerous methods have been- 
introduced for the purpose of estimating the total acidity, the total amount of 
hydrochloric acid, and the amount of free (uncomhined) hydrochloric acid contained 
therein. It would be out of place to go exhaustively into these methods here,, 
but a brief outline of the most trustworthy may be of value, 

^ It is indispensable to make an ethereal extract for this test, because alcohol,, 
sugar and phosphates give the reaction, and one or other of these is always, 
present in the gastric contents. 



PHYSIOLOGICAL CHEMISTRY 381 

1. Total Acidity. — A measured quantity (10 c.o.) of filtered gastric contents 

is mixed in an Erlemueyer flask with ten times its bulk of distilled water. Two 

or three drops of a solution of phenol-phthalein are added, and the solution is 

N 
titrated with jr: caustic soda solution until a faint pink colour is just obtained. 

The number of c.c. of alkali required is read off, and the result expressed as the 

N 
amount of ^ alkali required to neutralise the acids in 100 c.c. filtered gastric 

N 
contents. Thus, an acidity of 40 would mean that 40 c.c. of ^ alkali had been 

required to neutralise the acids of 100 c.c. gastric contents. The result may also 

be expressed in terms of HCl, and this is the method most useful in physiology. 

N 
1 c.c. jt: alkali equals 0'00365 gr. HCl. If, for example, 100 c.c. of gastric juice 

N 
require 50 c.c. -^ alkali to neutralise it, the acidity in terms of HCl will be 

0'1825. In other words, the percentage of acid will be equivalent to 0'1825 HOI. 

2. Total Hydrochloric Acid (i.e. the free HCl + the HCl combined with 

protein). — 10 c.c. of filtered gastric contents are placed in a platinum dish and 

evaporated to dryness on the water bath. The dish is then heated to a low red 

heat, so that charring is complete, but the resulting carbonaceous material is not 

burnt up. The mineral chlorides alone now remain in the dish. The contents of 

the dish are rinsed with hot distilled water through a funnel into a 100 c.c. 

N 
measuring flask. The flask is cooled, 5 c.c. nitric acid and 20 c.c. y^r silver nitrate 

solution are added, and the contents made up to 100 c.c. The amount of silver 
nitrate used in precipitating the chloride present is then determined by Volhard's 
method (see p. 434). A similar experiment is performed with the same volume 
of the gastric contents, to which slight excess of sodium carbonate solution is 
added before evaporation, and again the amount of silver nitrate used in pre- 
cipitating the chloride determined. The first experiment gives the mineral 

N 
chloride present, equivalent, say, to 5 c.c. ttt silver nitrate. The second experi- 

N 
ment gives the total chloride, equivalent, say, to 10 c.c. tt; silver nitrate. The 

difference gives the volatile chloride, that is, the hydrochloric acid free and com- 

N 
bined with protein. In the hypothetical case this is 10-5=5 c.c. r^ silver nitrate. 

0*365 x5 
The gastric contents, therefore, contain r-- — per cent, total hydrochloric acid. 

Normal human gastric contents obtained after a meal containing very little 
protein usually contain about 0"2 per cent, total hydrochloric acid. This hydro- 
chloric acid determination is of value, as it is the best measure of the secretory 
activity of the gastric mucous membrane in pathological conditions. 

3. Free Hydrochloric Acid.' — This can be approximately determined by 

N 
titrating 10 c.c. gastric juice with y^r caustic soda, using Giinzberg's reagent as 

' In testing for free HCl in solutions containing protein, it is important to bear 
in mind that the HCl will gradually become combined with the protein. The 
solutions should therefore not be allowed to stand for long before testing them. 



382 PEACTICAL PHYSIOLOGY 

indicator. In doing this, the gastric contents are mixed with the standard alkali 
in a small evaporating dish, samples of the mixture being removed with a glass 
rod for testing with the reagent. 

It is from the glands of the cardiac end of the stomach that the 
hydrochloric acid is secreted. These glands differ from those of the 
pyloric end in having parietal as well as central cells, and there is much 
evidence to show that it is the special function of the former to 
separate the hydrochloric acid from the blood. After the injection of 
solutions of neutral salts of iron into the blood, the parietal cells, when 
treated with potassium ferro-cyanide solution, turn blue, because of the 
formation of Prussian blue in them. No other cells in the body show 
this reaction, because they do not contain the necessary acid. 

The Organic Matter. — If pure gastric juice be cooled to 0°C., a 
precipitate falls down. On analysis, this precipitate is found to have 
nearly the same percentage composition as protein ; and on testing its 
action on a solution of protein, it is found to be pepsin. Pepsin of 
similar composition can also be prepared by saturating gastric juice 
with ammonium sulphate, which precipitates it. Whether the actual 
ferment pepsin is what we obtain by these methods is uncertain. The 
methods employed for obtaining ferment from the gastric mucosa after 
death yield a still more impure product, on account of the ferment 
adhering to the proteoses, etc., which are always present in the final 
precipitate. 

To Prepare an Extract of Gastric Mucosa containing large CLuantities 
of Pepsin, the thoroughly washed stomach of the pig is taken, and the mucosa 
is scraped o£f with u, knife. The scrapings are mixed with a large excess (100 
times their bulk) of 0'4 per cent, hydrochloric acid, and the mixture is digested for 
several hours in the incubator. The extract is then filtered through muslin, and 
may be employed for general work without further purification. In order to 
separate the pepsin from the excess of proteoses which this extract contains, the 
digestion should be allowed to proceed for several days Idnger, so that the 
proteoses may become changed into peptones. The product is then saturated 
with ammonium sulphate crystals ; the resulting precipitate of proteoses, which 
carries down the pepsin with it, is pressed free of fluid, and again incubated for 
a few days with several volumes of 0'5 per cent, hydrochloric acid, after which the 
digest is again saturated with ammonium sulphate. This final precipitate is 
approximately pure pepsin. The ammonium sulphate can be removed from the 
preparation by dialysis through parchment. 

The scrapings of mucosa, after being treated with weak acid to convert the 
pepsinogen into pepsin, can also be extracted with glycerin. This is the method 
which is most used commercially. The various commercial preparations of 
pepsin are very suitable for the experiments about to be described. 

Prior to its secretion, pepsin exists in an inactive form as granules in 



PHYSIOLOGICAL CHEMISTRY 383 

the gland cells of the stomach mucosa. This precursor or zymogen 
(see p. 388) is called pepsinogen. It differs from pepsin in that alkali 
does not destroy it, whereas alkali destroys pepsin. 

Advanced Experiment. Scrape off the mucosa from about three square 
inches of the stomach, grind it with some sand in a, mortar, and gradually add 
about 20 c.c. of 1 per cent, sodium carbonate solution. Filter. When about 
10 c.c. of filtrate has collected — which will take some time on account of the 
mucilaginous nature of the extract — place a piece of washed fibrin in the filtrate 
and incubate at body temperature. No digestion occurs. In half an hour add 
3 per cent. HCl, drop by drop, to the solution until it reacts faintly acid towards 
litmus and again incubate. The fibrin soon digests. The acidity has converted 
pepsinogen into pepsin. Divide the resulting solution into two parts. To one 
of these add 1 per cent, sodium carbonate solution until faintly alkaline, and set 
aside at warm temperature for 15 minutes, after which again render it faintly 
acid with 3 per cent. HCl. To both test tubes now add similar pieces of fibrin 
and warm to body temperature. It will be found that the fibrin becomes quickly 
digested in the tube, which has been kept at acid reaction, but not in the other 
tube, because of the alkalinity of the solution having destroyed the pepsin. 
Pepsinogen, therefore, withstands an alkaline reaction, but pepsin is destroyed. 

The most favourable conditions for the action of pepsin may be studied 
in the test tube as described in the following experiments : 

The Action of the Gastric Juice. — The most convenient protein for 
studying the action of pepsin is blood fibrin which has been very 
thoroughly washed with boiling acidulated water so as to remove all 
impurities. Cubes of coagulated egg white may also be employed, but 
they digest more slowly than fibrin. 

Experiment VII. Label six test tubes A, B, C, D, E, F, and place 
a small piece of fibrin in each. Half fill A with water, B with 0'2 per 
cent. HCl, C with water and a few drops of peptic extract, D with 0'2 
per cent. HCl and a few drops of peptic extract,^ E same as D, but boil 
the mixture, and F with 1 per cent, sodium carbonate solution and a 
few drops of the peptic extract. 

Place all these in a water bath kept constantly at body temperature 
(37-38°). Observe that in A the piece of fibrin remains unchanged, 
whereas in B, D, and E, which all contain 0"2 per cent. HCl, it becomes 
swollen and transparent. In F, which contains alkali, it does not swell. 

Experiment VIII. After about half an hour, remove a sample of 
the contents of any of the tubes containing acid, colour it faintly with 
a drop or two of litmus solution, and then carefully neutralise with 
weak sodium carbonate solution (1 part 1 per cent, sodium carbonate + 
2 parts of water). A precipitate of acid meta protein or syntonin is 
usually produced (for reactions, see proteins, p. 310). 

^ Use larger quantities of fibrin and fluid in this test tube, because the products 
of digestion will be required for succeeding experiments. 



384 PRACTICAL PHYSIOLOGY 

The first stage in gastric digestion of proteins consists, therefore, in 
the production of acid meta-protein by the weak HCl. As we shall see 
later, this preliminary change is necessary before pepsin can further 
hydrolyse the protein. 

EXPKRIMENT IX. Eemove a sample of the contents of D and apply 
the following tests: — (a) The Biuret reaction — rose-pink colour; (6) Add 
nitric acid (cone.)— white precipitate, which clears up on heating and 
returns on cooling; (c) Add a few drops of a saturated solution of 
salioyl sulphonic acid. A white precipitate results which disappears on 
heating and returns on cooling. These results show us that proteoses 
have been produced (see p. 310). 

The production of proteoses constitutes the second stage of peptic 
digestion, and it is the pepsin which produces the change. If samples 
of any of the other test tubes than D be examined, no proteose will 
be found, either because no pepsin has been present (as in A and B), 
or because, though present, its action has been destroyed by heat (as 
in E), or there has been no acid present to produce syntonin and help 
its action (as in C and F). 

There are two principal varieties of proteoses developed, namely 
*' primary " and " secondary " ; the former are precipitated by half 
saturation with ammonium sulphate, the latter begin to be precipitated 
at two-thirds saturation with this salt. 

Experiment X. Take a sample of a peptic digest of two days' 
duration. Heat this to near boiling point, and add ammonium sulphate 
crystals till no more will dissolve. Now change the reaction of the 
fluid to alkaline and allow to cool.^ Filter and test the filtrate for 
Peptone. 

1. By Biuret reaction — (remember to add a large excess of caustic 
alkali, so that more than is sufficient to decompose the ammonium 
sulphate may be present in the fluid) — rose-pink. 

2. By nitric acid or salicyl sulphonic acid tests — no precipitate. 

In peptic digestion some of the protein is always further broken 
down to amino acids. 

Method of Separation of Products of Gastric Digestion.— Fibrin is 

boiled first with tap water, and then with O'l per cent, hydrochloric acid to 
purify it. It is placed for 1-2 hours in an incubator along with five times its 
bulk of 0'2 per cent, hydrochloric acid and a sixth its bulk of commercial peptic 
extract. (A solution of Witte's peptone may also be employed). 

The products of digestion can be separated from this digest by the following 
process : — 

^ It is only by thus saturating in the heat, both in acid and alkaline reaction, 
that all traces of secondary proteoses are precipitated. 



PHYSIOLOGICAL CHEMISTRY 385 

(1) Boil the solution in a, beaker or evaporating dish, cool and separate any 
coagulated native protein by filtration. 

(2) Carefully neutralise the filtrate with I per cent, sodium carbonate solution ; 
if a precipitate of metaprotein (s3Titonin) falls down, separate it by filtration. 

(3) The resulting filtrate is now shaken vigorously with an equal volume of a 
saturated solution of ammonium sulphate. A precipitate of primary proteoses 
forms since these are insoluble in half saturated ammonium sulphate solution. 
Collect the precipitate on a filter paper and preserve the filtrate. The further 
investigations of the precipitate and filtrate are conducted simultaneously. The 
precipitate is washed at least once with a half-saturated solution of ammonium 
sulphate, the filter paper floated on to wat«r in an evaporating dish stirred up 
with a glass rod, warmed, and portions of the resulting solution of primary 
proteoses (containing ammonium sulphate) employed for the following tests : — 

1. Add a few drops of a saturated solution of salicyl sulphonic acid — a white 
precipitate forms which dissolves on heating and reappears on cooling. 

2. Add a few drops of pure concentrated nitric acid. The same result is 
obtained as in 1. 

3. Apply the Biuret test using a sufficiency of 20 per cent, caustic soda solution 
to permit of some excess after all the ammonium sulphate has been decomposed 
(see p. 31 

4. Apply the various protein tests described on pp. 302, 303.' 

The filtrate still contains the secondary proteoses. These are precipitated by 
adding sufficient sulphuric acid to the filtrate to render it distinctly acid towards 
litmus, saturating while boiling with ammonium sulphate crystals, adding 
ammonia till just alkaline and allowing to cool. The precipitate is then filtered 
off, dissolved in water, and the tests described under primary proteoses applied 
to the solution. The precipitates with nitric acid and salicyl sulphonic acid will 
be much less marked than with primary proteoses. 

The filtrate after removal of the last traces of proteose is tested for peptones 
by applying the Biuret test with excess of caustic soda, and other general protein 
tests. 

For convenience in testing the large amount of ammonium sulphate present 
may be removed by boiling the solution, adding barium carbonate as long as 
ammonia continues to be evolved, and filtering. 

The taWe on page 386 shows the main reactions of the intermediate 
products of peptic digestion (as occurring in Witte's Peptone) com- 
pared with those of native protein. (See also p. 312.) 

The action of gastric juice in curdling milk is described in the section 
on Milk (see p. 327). This action is usually attributed to the ferment 
rennin, but it is probable that rennin and pepsin are identical, as proteo- 
lytic ferments always have a rennin action, and the proteolytic activity 
of a given ferment is proportional to its rennin activity. 

The gastric juice scarcely affects other foodstuffs. In the case of fat, 
however, it dissolves the protein envelope of the fat cell, and liberates 
the contents, which now float in the chyme as oil globules. On 
emulsified fats the gastric juice can effect hydrolysis into fatty acid 

' The alkaloidal reagents give precipitates with proteoses. 
2b 



386 



PEACTICAL PHYSIOLOGY 



Name of 
protein. 


Precipitatioti 
limits with 
(NH4)2S04 
expressed in 
percentage 
saturations. 


SolubiUty in 
alcohol. 


Salicyl sulphonic 

acid and 

HNO, tests. 


Biuret 

test. 


MiUon's 
reaction. 


Native 
protein. 


Globulins 
(isat.). 

Albumins 
(sat.). 


Relatively in- 
soluble. 


Ppte., becom- 
ing ooagnlum 
on boiling. 


Violet. 


Positive. 


Acid meta 
protein. 


— 


— 


Do. 


Do. 


Do. 


Primary 
proteose. 


24-42 per cent, 
(isat.). 


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 
ptato- 
proteose. 


Secondary 

proteose 

A 


54 -62 per cent. 

(8 sat.). 


Partly insol- 
uble in 70 
per cent. 


Do. 


Do. 


Positive. 


B^ 


70-95 per cent, 
(practically 
sat.). 


Part insoluble 
in 35 percent.; 
part soluble in 
80 per cent. 


Do. 


Do. 


Positive. 1 





100 per cent. 
pliis acid. 


Soluble in 68-80 
per cent. 


Do. 


Do. 


Absent. 


Peptones. 


Not precipi- 
table. 


A insoluble in 
96 per cent. 


Negative. 


Do. 


Absent. 




B soluble in 96 
per cent. 


Negative. 


Do. 


Present. 



and glycerine in virtue of a lipase which is secreted by the gastric 
glands. 

Method of Estimating Activity of Pepsin Solutions, (l) Grutzner's 
Method (Approzimate). — Fibrin, purified as above described, is stained with 
carmine solution, and washed free of adherent stain. Equal weighed quantities 
are then placed in two test tubes, and 10 c.c. of 0-2 % hydrochloric acid are added 
to each. Equal quantities of the pepsin solutions which it is desired to test are 
added, and the tubes placed in the incubator. As the fibrin becomes digested the 
carmine is liberated, and stains the solution. The more deeply stained solution, 
therefore, contains the stronger ferment. The exact amount of carmine liberated 
may be determined by comparing the digests with an artificial scale consisting of 
ten solutions of carmine of different known strengths. A control of 0-2 % HCl 
and carmine fibrin ought also to be studied. 

(2) Mett's Method. — A narrow glass tube 1 to 2 mm. in diameter, and drawn 

^ Proteose B can be fractionated into various sub-varieties which differ from 
one another in their elementary composition and somewhat in their reactions. 
There is one variety of proteose B which gives no reaction with MiUon's reagent. 



PHYSIOLOGICAL CHEMISTRY 387 

to a fine point at both ends, is filled with egg white, the ends closed in the 
flame, and the tubes then heated so that a column of coagulated albumin is 
obtained. It is then cut into segments of equal length, and two of these are 
placed in a test tube which contains the pepsin solution acidified with 0'2% 
hydrochloric acid. Two similar tubes are placed in another test tube with the 
other pepsin solution. Both are placed in the incubator for several (10) hours. 
The length of dissolved protein column is then measured in both cases, and the 
desired result is obtained by squaring this distance. 

Thus if in one test tube the length were 2, and in the other 3, the strength of 
the two pepsin solutions has the ratio of 4 to 9. 

This method is only accurate when weak pepsin solutions are used. If more 
than 4 mm. of protein are digested, the estimation must be repeated with diluted 
solutions. 



CHAPTER XIV. 

DIGESTION IN THE INTESTINE. 

In about half an hour after the food enters the stomach, small portions 
of it begin to pass through the pyloric sphincter into the duodenum. 
These have undergone gastric digestion and constitute chyme. This 
leakage goes on until the stomach has completely emptied itself, the 
length of time necessary for this (3-10 hours) varying with the quantity 
and quality of the food, and with the activity of the gastric juice. 

The chyme, as it leaves the stomach, is strongly acid in reaction to 
all indicators. In the duodenum, it becomes mixed with the secretions 
of Brunner's glands and with those of the pancreas and liver, which 
latter are poured into that portion of the intestine by one common duct, 
and, as it travels on to the jejunum, it also becomes gradually mixed 
with the intestinal juice, secreted from Lieberkiihn's follicles. These 
various secretions are alkaline in reaction, in consequence of which the 
acid of the chyme becomes neutralised, so that the contents of the 
lower portion of the duodenum and of the upper portion of the jejunum 
become alkaline in reaction to litmus. Although the acidity of the 
gastric juice prevents the growths of organisms in it, it does not kill 
their spores, and these are carried into the intestine along with the 
chyme. When this latter becomes alkaline, however, the conditions 
are very favourable for organismal growth, and the spores become 
transformed into the active organisms which multiply quickly, mean- 
while receiving their nourishment from the half-digested foodstuffs. In 
this way the organisms assist the digestive juices in decomposing the 
foodstuffs. Among the products of this organismal growth are several 
organic acids, so that the food, before it has gone far along the intestine, 



388 PEACTICAL PHYSIOLOGY 

again becomes acid in reaction towards litmus. The mucosa of the 
large intestine does not secrete any digestive juices, its sole function 
being one of absorption. In its passage along it the fluid of the 
intestinal contents becomes gradually absorbed, and the unabsorbed 
residue forms the faeces. 

It will be seen, therefore, that there are four distinct digestive 
agencies at work in the intestine, and we will now study the action of 
ea^h of these separately. 

The Pancreatic Juice. Composition. — This can be collected by pro- 
ducing a fistula of the pancreatic duct. The juice is strongly alkaline 
in reaction, gives a coagulum of protein on boiling, and contains, besides 
protein, a considerable amount of organic matter. 

Its percentage composition varies very much with the method 
adopted for collecting it, that obtained immediately after the establish- 
ment of the fistula being very much richer in solids than that secreted 
a few days later. 

Directly after Permanent 
operation. fistula. 

Water, 90-08 97-68 

Total solids, 992 2-32 

Organic, 9-04 1-64 

Inorganic, 0-88 0'68 

In studying its digestive action we may employ, as in the case of 
gastric digestion, an extract of the gland. This extract may be made 
with glycerine, after treating the minced gland with weak acid, or 
allowing it to stand some time, so as to convert the zymogens into the 
active ferments. G-lycerine does not extract all the ferments, however, 
so that it is more usual to employ the minced gland itself, or a watery 
extract of it. 

The secretion of pancreatic juice is stimulated by the presence in the 
blood of a substance called secretin. Secretin is produced in the epi- 
thelial lining of the small intestine by the action of weak acids on it. 
Thus, if some of the inner lining of the small intestine be scraped ofif 
and ground in a mortar with fine sand and 0-4 % HCl, and the resulting- 
mixture boiled, neutralised and filtered, a solution is obtained which, 
when injected intravenously into an anaesthetised animal with a pan^ 
creatic fistula, causes an immediate and abundant secretion of pancreatic 
juice. The secretin does not exist preformed in the intestinal epi- 
thelium, for a saline extract of this, when similarly injected, has no 
eifect on pancreatic secretion. 

The pancreatic juice thus secreted differs in its digestive properties 
from an extract of pancreas. The chief difi"erence lies in the fact that 



PHYSIOLOGICAL CHEMISTRY 389 

it can digest proteins only feebly, because it does not contain active 
trypsin but only its zymogen trypsinogen. To convert the trypsinogen 
into trypsin, mixture with succus entericus is necessary. The succus 
entericus contains a substance called enterokinase which activates the 
trypsinogen. 

There are three active ferments in pancreatic juice, one proteolytic — 
trypsin; one amylolytic — amylopsin or amylase; one steatolytic — 
steapsin or lipase. 

I. Trypsin. —Like pepsin, this ferment hydrolyses protein, and leads 
to the production of proteoses and peptones. In this case, however, 
digestion is more complete. Under suitable conditions the proteoses 
and peptones can disappear entirely, polypeptids, amino acids and 
hexone bases resulting; the ultimate decomposition products are, in 
fact, almost the same as when a strong acid is used as the hydrolysing 
agent (see Proteins, p. 299). 

Experiment I. — A solution of pancreatic extract in 1 % sodium 
carbonate is prepared (Liq. Pancreaticus (Benger), diluted thirty times 
with 1 % sodium carbonate solution). In order to study the action of 
this on proteins, add to it a piece of fibrin which has been soaked over 
night in 1 % sodium carbonate solution, and place on a water-bath at 
body temperature. 

The following points of difierence may be noted between this and 
the peptic digestion of fibrin: (1) The reaction is alkaline; (2) there 
is no preliminary swelling of the fibrin ; it is gradually eaten away 
(erosion) ; (3) when the piece of fibrin has nearly disappeared remove 
a sample of the digest, and neutralise with weak acetic acid. A pre- 
cipitate of alkali metaprotein results (for Reactions, see p. 310). 

Apply to another sample the tests for proteoses and peptones, and 
note that they are positive.^ 

Experiment II. — If the pancreatic extract in Experiment I. be 
boiled before the fibrin is added, no digestion will result. The digestive 
agent is, therefore, a ferment which is destroyed by heat. 

Experiment III. — Repeat Experiment I., making the reaction acid 
by means of hydrochloric acid. Note that, although the fibrin becomes 
swollen up — as this depends on the acid, not on the ferment — no forma- 
tion of proteoses or peptone occurs. The trypsin cannot act in acid 
medium, being destroyed in this reaction. 

(4) Trypsin can carry digestion further than pepsin. 

Leucin, Tyrosin and the other Products of Tryptic Digestion. — 
During digestion of protein by trypsin a number of amino acids are 

' No primary proteose is formed by tryptio digestion ; there is, however, a, 
considerable amount of secondary proteose (see p. 386). 



390 PEACTICAL PHYSIOLOGY 

produced, of which leuein and tyrosin are examples. An amino acid is 
derived from an organic acid (containing therefore the - COOH group) 
by the substitution of one of the hydrogen atoms attached to a carbon 
atom of the chain (e.g. CHj - , = CH - ) by the amino group (NHg). 

Thus acetic acid has the formula CHgCOOH. 

If one of the " H's " of the CHg group be displaced by NH2, the 
result is NHj. CHg. COOH, which is amino acetic acid, also called 
glycin and glyooooll. 

Glycin is formed during the digestion of gelatine and globulin, but 
not of albumin. It also exists in the bile, where it enters into the 
formation of one of the bile salts (e.g. glycocholate of soda is glycin 
+ cholalic acid). It likewise occurs in combination with benzoic acid, 
as hippuric acid, in the urine of herbivorous animals, and to a less 
extent in the urine of man. 

The amino acid corresponding to the next acid of the acetic acid 
series, viz. propionic acid CHg . CHg . COOH is amino- qtt 
propionic acid, or alanin, CHg . CHNH^ . COOH. In 1 
the free state it is only produced from a few proteins,^ /\ 
and is unimportant, but it is frequently combined I I 
with phenol, the resulting compound being tyrosin. I I ^„ 
If in the formula of phenol the H atom in the para | / ^ 

position to the OH group be replaced by amino QH CH 

propionic acid, para-hydroxyphenylamino-propionic \ 

acid, or tyrosin results. It, therefore, belongs to the COOH. 

aromatic group of organic bodies, and because it iyrosin. 

contains hydroxyphenyl (the radicle of phenol) it reacts red with 
Millon's reagent (see Proteins, p. 302). 

Experiment IV. Add Millon's reagent to some pancreatic digest ; 
a white coagulum of proteins results. Filter. Boil the filtrate. It 
turns red, because it contains tyrosin. 

EXPEKIMENT V. Examine the crystals of tyrosin supplied you 
under the microscope, and note that they consist of fine needles 
grouped into star-shaped masses (Fig. 236).^ 

There are no other important amino acids till we come to the 
member of the series which contains six carbon atoms, viz., caproic 

^ By the hydrolysis of haemoglobin, however, alanin is a very abundant decom- 
position product. 

^ A body very closely related to tyrosin in its chemical structure has recently 
been described amongst the products of hydrolysis of proteins by acids. This is 
phenylalanin, differing from tyrosin only in that it does not contain the phenolic 
OH group. It has also been discovered in the products of the prolonged action 
of pepsin on proteins. 



PHYSIOLOGICAL CHEMISTRY 



391 



acid. The amino acid of this is closely related to kiicin. It has 
recently been shown, however, that the true constitution of leucin 
is not amino-caproic acid but rather amino-isobutylacetic acid (0113)2 : 
CH.CH2.CHNH2.COOH. 

Experiment VI. Examine crystals of leucin under the micro- 
scope and note that they consist of round balls not unlike oil globules 
yellowish in colour, and usually having concentric markings (Fig. 236). 




Pio. 236. — Crystals of leucin and tyi-osin. 



Leucin and tyrosin were among the firstKiiscovered composition 
products of proteins, and, on account of the ease with which they 
are isolated, they have been detected in nearly every organ and 
tissue of the body, being probably produced, however, by the chemical 
agencies employed in examining these, and not existing as such in 
the living tissue. They also occur, along with excess of ammonium 
salts, in the urine of patients suffering from severe disease of the liver. 

Not only are amino derivatives of mono-basic acids produced during 
protein decomposition, but we may also have similar derivatives of 



392 PRACTICAL PHYSIOLOGY 

di-basic acids. One of the simplest of these latter is succinic acid, 
COOH.CH^.CHj.COOH. 

If an " H " atom attached to a carbon atom of the chain be replaced 
by the amido group (NHj), asjnrtic acid results, COOH . CHNHg . CHj . 
COOH. Besides being produced in the intestine by the action of 
trypsin on protein, it also occurs plentifully in plants.^ 

Another important di-basio amino acid, which is also a common 
decomposition product of protein, is glutaminic acid. It contains one 
more C atom than aspartic acid, and has the formula: COOH . CHNHj . 
CHj.CHj.COOH. 

All these amino acids retain to a certain extent their acid properties. 
Thus they can combine with bases to form salts. On the other hand, 
on account of the NHj group which they contain, they also show faint 
basic properties, in that they can unite with metallic salts, forming 
double compounds, which are very useful in preparing the pure amino 
acid. Their ethereal salts shew marked basic properties. 

Besides these mono-amino acids, there are also produced bodies in 
which more than one amino group exists. These have a distinctly 
basic reaction, and combine with weak acids, such as phosphotungstic* 
They also form double salts with silver nitrate. These two reactions 
are taken advantage of in separating these bases from the mono-amido 
acids. Since these bases contain six carbon atoms, they are called 
hexone bases, and the most important are lysine (C5Hg(NH2)2COOH) 
and arginine (CgHj^N^Oj). 

Lysine is o-e-di-amino-caproic acid, being therefore somewhat related 
to leucine. Its structural formula is : 

NHj . CH . CH2 . CH2 . CH2 . CHNH2 . COOH. 

Arginine, the most frequently occurring decomposition product of 

protein, is chemically 8-guanidin a-amino-valerianic acid. This is a 

peculiarly interesting decomposition product of protein because on 

hydrolysis it is split into urea and di-amino-valerianic acid or ornithin. 

The urea comes from the guanidin radicle thus : 

H H H NH„ 

I I I I 
NH-C-C-C-C-COOH + H„0 = 

I I I I I 
NH= C H H H H 

I a-amino-valerianic 

JSlHj acid. 

guanidin 
radicle. 

^ If the OH group of the COOH radicle of aspartic acid be further replaced by 
NHj we have asparagin. 
2 This complex acid has the formula H3PO4 . IIWO3 . I2H3O. 



PHYSIOLOGICAL CHEMISTRY 39S 





NHj 




H 

I 


H 

1 


H NHj 

1 1 





1 
= + 

1 


NHj 




-I 
1 


-C-C-COOH 

1 1 




NHj 




1 

H 


1 
H 


i i 




Urea. 


a. 


S. di- 


amino- valerianic 



acid, or omithin. 

Histidine (CgHgNgOj) differs essentially from the above so-called 
hexone bases in containing a ring formation of atoms in the molecule. 
It is believed to be a-amino-;8-imidazol propionic acid : 

/NH-CH NH, 

CH/ II I 

^N - C -CHj-CH-COOH. 

A product of some interest is cystin: — COOH.GHNHg. CHj. S. S. 
CHj. CHNHg. COOH. It is converted into cystein by reduction. 
Cystein is a-amino-/8-thiolactie acid :—SH. CHj. CHNHj. COOH. It 
is, therefore, closely related to alanin (CHj. CHNHj.COOH) or amino- 
propionic acid. Cystin is probably the source of the taurin of bile, 
and is the chief sulphur-containing constituent of protein. 

By reference to the table on p. 299 it will be seen that trypsin 
produces much the same decomposition products as strong acid. 
Certain parts of the protein molecule, however, resist the action of 
trypsin for a long time, such, for example, as those which contain 
the pyrrolidin-carboxylic acid and phenylalanin groups. Between the 
peptones which give the Biuret test and the amino bodies are several 
lower peptones or polypeptids which do not give this test. 

To isolate and identify the various products of tryptic digestion of protein 
would consume far too much time and require too great a bulk of material to make 
it practicable as a class exercise. Two of the commonest degradation products, 
viz. leucin and tyrosin, can, however, be isolated with comparative ease, although 
even for this purpose a considerable bulk of material is required. A method for 
the isolation of tryptophane is also described.'' 

Advanced Experiment. — Mince up a pig's pancreas thoroughly, and shake 
it in a, flask with 500 c.c. of water containing 3 c.c. of a, saturated solution of 
sodium carbonate, and 3 c.c. of chloroform. Add also about 200 grams of blood 
fibrin, which has previously been soaked in 1 % sodium carbonate solution. Place 
the flask in an incubator at body temperature, and after three days test the 
reaction of the digest towards litmus. If acid, add more sodium carbonate till 
distinctly alkaline. Also remove about 10 c.c. and filter into a test tube. To 
this sample carefully add a few drops of bromine water. A violet colour results, 
the intensity of which should be carefully noted. This colour reaction is due to 
tryptophane, an aromatic amino acid which is liberated by the action of trypsin 
(see p. 395). 

' These exercises are introduced here for the benefit of the more advanced 
students. They should, however, be read by the junior student as well. 



394 PRACTICAL PHYSIOLOGY 

Test the reaction towards litmus and the intensity of the tryptophane reaction 
on each succeeding day. When the tryptophane reaction becomes very intense (in 
about five days) proceed to isolate leucin and tyrosin in the following manner : — 

The digest is rendered faintly acid with acetic acid, boiled and filtered hot. A 
sample of the filtrate is removed and tested for proteose. A negative result is 
usually obtained. 

11. Separation of Tyrosin. — The remainder is evaporated on the water-bath 
to a thin syrup. This ia allowed to stand on ice or in a cold place for several 
days. White flocculi of tyrosin separate out. These are filtered through fine 
muslin, and removed to a beaker by means of a jet of cold distilled water and 
washed several times with distilled water by decantation. They are then dis- 
solved by boiling with water made alkaline by the addition of a. few drops of 
ammonia, and the resulting solution is quickly filtered hot. The filtrate is heated 
till all the ammonia is expelled ; it is then cooled, when the tyrosin separates out 
as a white precipitate. This is collected on a filter paper, washed, and dried. 
The following reactions may be applied to the resulting powder : — 

(1) Tyrosin is insoluble in cold water, slightly soluble in hot water, and very 
soluble in dilute alkali. 

(2) A solution in hot water gives a red colour on the addition of Millon's 
reagent. This is because tyrosin contains an aromatic radicle (p. 390). 

(3) Piria's Test. — Place some of the powder in a dried test tube, add about 2 c.c. 
concentrated sulphuric acid, and place the test tube in a boiling water-bath for 
half an hour. Now cool and dilute with water, transfer to an evaporating basin, 
and remove the sulphuric acid by adding powdered barium carbonate ; filter off 
the barium sulphate, evaporate the filtrate to small bulk, and add a drop or two 
of very weak ferric chloride solution. A violet colour results. This reaction is 
due to the formation of tyrosin-sulphuric acid. 

2. Separation of Leucin.— The tyrosin-free filtrate is evaporated till a skin 
of leucin forms on the surface. It is then mixed while still warm with several 
times its bulk of alcohol, whereby a precipitate (previously known as antipeptone) 
separates out, which after cooling can be removed by filtration. This precipitate 
consists of a mixture of several bodies, including lysine, histidine, and arginine. 
The filtrate is evaporated on the water-bath until all the alcohol has been driven 
off. It is then boiled with lead carbonate and filtered. The lead is removed from 
the filtrate by means of HjS, the PbS separated by filtration, and the final filtrate 
accurately neutralised with weak NaOH. By now concentrating by evaporation 
on the water-bath and cooling leucin will separate out. 

Reactions of Leucin. — (1) It is much more soluble in water than is tyrosin ; it 
is soluble also in alcohol. 

(2) When heated in a piece of dry glass tubing, a sublimate forms on the cool 
parts of the tube. 

(3) Like other amino acids, it gives off ammonia gas when heated in a test tube 
with a piece of solid caustic potash and a few drops of water. If the melt be 
cooled, dissolved in water, and then acidified with sulphuric acid, it gives a smell 
of valerianic acid on heating. 

(4) Scherer's Test. — Ilea.t some leucin with a. drop of nitric acid on a piece of 
platinum foil, add to the dry residue some caustic potash, when a yellow stain 
results. Heat still further, and the stain rises up into a globule which runs off 
the platinum. 

(5) Examine a solution of leucin with the polariscope (p. 282). It is levo- 



PHYSIOLOGICAL CHEMISTRY 395 

orotatory (a)u— in aqueous solntion= - 10'8°. The leucin which is obtained by 
boiling protein with baryta, or that obtained synthetically (by the action of 
ammonia on a-bromocaproic acid) is opticaMy inactive, and the dextrorotatory form 
may be obtained from this by allowing penicillium glancum (a fungus) to grow on 
a solution of it. The fungus destroys the levo-rotatory part, but leaves the 
dextro-rotatory, untouched. Moulds, yeasts and ferments act much more ener- 
getically on naturally occurring than on synthetic isomers. 

Tryptophane.^ — If bromine water be cautiously added to a tryptic digest of 
several days' standing a deep violet-red colour will result, and if the mixture be 
shaken with amyl alcohol, this latter will take up the colour. The glyoxylic 
reaction (see p. 303) will also be very distinct in the digest even after the Biuret 
reaction has disappeared (i. e. after the protein molecule has been quite destroyed). 
Both these reactions are due to tryptophane, which is closely related in its 
chemical structure to certain of the aromatic substances that are produced by 
the bacterial digestion of protein. 

Separation of Tryptophane. — A large amount (500 gr.) of commercial 
casein (plasmon or protene) is mixed with liq. pancreaticus (200 c.c. Benger) and 
0'8 % NajjCOg, and placed in an incubator for about a week. The ferment should 
be added, half at the beginning and the remainder three or four days later. Anti- 
septics should be added. 

Digestion is allowed to proceed until the bromine water reaction is maximal. 
The digest is then boiled, cooled and filtered, and H2SO4 added to the filtrate, so 
as to bring the amount of H2SO4 in the latter to 5-6 %. If any precipitate is 
hereby formed it should be filtered off. The clear filtrate is then mixed with an 
excess of an acid solution of mercuric sulphate (10 % mercuric sulphate dissolved 
in 10 % H2SO4) and filtered. This reagent may precipitate, besides tryptophane, 
some tyrosin and cystin. 

From tyrosin the precipitate is freed by washing it with 5-6% H2SO4, the 
mercury compound of tyrosin being very soluble in this. From cystin (which is 
scanty in a digest of casein) the tryptophane is separated by reprecipitation. For 
this purpose the washed mercury precipitate is suspended in water and decom- 
posed with H2S gas. To complete this reaction the suspension must be saturated 
with the gas, then warmed and saturated again. The HgS precipitate is filtered 
ofl, the filtrate warmed to rid it of H2S, then acidified to 5-6 % H2SO4, and the 
mercuric sulphate reagent added to it until a small permanent precipitate is pro- 
duced. This is mainly cystin, and is filtered off. The tryptophane in the filtrate 
is then completely precipitated by mercuric sulphate, and the resulting precipitate 
treated exactly like the first one. 

In this way a solution of tryptophane in 5-6 % H2SO4 is obtained. The H2SO4 
is now precipitated by adding Ba(0H)2 water in the heat and filtering, tireat 
care should be taken that the filtrate contains no excess either of H2SO4 or of 
Ba(0H)2. The watery solution of tryptophane is then mixed with half its bulk 
of alcohol and evaporated on a water bath. During evaporation small quantities 
•of alcohol are added from time to time to prevent the browning which occurs if 
watery solutions of tryptophane are heated alone. Evaporation proceeds till 

'A single digestion mixture may be employed for the separation of leucin, 
■tyrosin and tryptophane, but in such a case both fibrin and casein ought to be 
added, since casein is the only common protein which yields any large amount of 
tryptophane. It also contains a considerable amount of tyrosin. 



396 PRACTICAL PHYSIOLOGY 

crystallisation commeiicea, when the basin is removed and allowed to stand. 
The crystals (glistening plates) are collected on a filter, and, to purify them, may 
be recrystallised. 

A solution of the crystals gives the bromine and the glyoxylic reactions very 
distinctly, and if the crystals be heated in a test tube indol and skatol (see p. 406) 
are evolved. 

EUinger has shown the constitution of tryptophane to be indolamino-propionic 
acid ; its formula is probably : — 

-CH2-CH(NHj)-C00H 
C'6H4<^ ^CH (o. 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 fafc 
acid and glycerine (see Fats, p. 314). 

Experiment VIII. Some minced pancreas is shaken with water "^ 
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 

' Glycerin does not dissolve steapsin, so that a glycerine extract of pancreas is 
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 nuoleo-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 Hwman 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. 



100 
Water, 


parts 


contain — 


86 




97 


Solids, 






14 




3 


Fiz. organic salts, 






9 




0-9-1 -8 


Mucin and V>ile pigment. 






3 




0-5 


Cholesterol, 






0-2 




0-06-0-16 


Lecithin and fat, 






0-5- 


1-0 


0-02-0-09 


Inorganic salts, 






0-8 




0-7-0-8 



398 PEACTIOAL PHYSIOLOGY 

Besides these, bile also contains traces of soaps, fats and urea. 
Compounds of glycuronic 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 Fettenkofer'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 CjsH^gNOg and taurocholic 
CjgH^gNSOf) 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 — 
COOH . CHNHj . CHa . SO3H. By the loss of a molecule of COj, this 
becomes taurin : NHj . CH^ . CHj . SO3H. \Ye 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 grni. pure animal charcoal with 200 c.o. 
of ox-bile in an evaporating disli, 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.^ 

Experiment V. Dilute some ox bile with an equal amount of 

'This 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 fcili-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 CgjHggN^Og. If we compare this with the formula of haematin — 
CgjHgjN^O^Fe — 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 (C^^HgoNPOg) and Cholesterol {C^B^fiR) (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-stufts, 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 invertinff 
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 PHYSIOLOaY 

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 r61e in the 
metabolism of proteins. 

Advanced Experiment, To Demonstrate the Ereptic 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 NagCoj. 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'S % 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 CHEMISTEY 405 

necessary for bacterial growth are very favourable in the intestine. 
As a result of their gi-owth, 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 

CH 

phenol CgHjOH and its methyl derivative hresol G^^<Cn^- These 

are produced from tyrosin, which, it will be remembered, has the 

OTT 
formula CgH^<^pTT ptt^NH ^ rOOH When this changes into kresol 

and phenol, the amino-propionic acid side-chain loses, first its amino 
group as ammonia, and then its carboxyl and methyl group are 
oxidised and given off as carbonic acid and water. 

The other group is more complex, and contains indol G^^-h^-^t^^CQ. 
and its methyl derivative shatol 

/CH3 

CeH,<NH>CH. 

These are derived from tryptophane, a product of the tryptic digestion 
of certain proteins. Its chemical reactions and constitution are described 
on p. 395. 

Anaerobic bacteria first of all act on the tyrosin and tryptophane, 
and split off from them the amino (NHj) groups as NH3. After this 
has been accomplished, aerobic organisms act on the remaining side 
chain's yielding carbon dioxide and water. 

Certain of these aromatic bodies — especially skatol — have a strong 
faeculent odour which they impart to the faeces. Considerable pro- 
portions of them are, however, absorbed into the blood and reappear 
in the urine as indoxyl and skatoxyl in combination with sulphuric 
acid and alkalies as aromatic sulphates (see p. 435). 

These above products also result when proteins undergo putrefaction 
in vitro, but in this latter case other bodies called ptomaines are also 
produced. These are powerful poisons and it is on account of their 
presence that it is dangerous to eat putrid flesh. 



406 PRACTICAL PHYSIOLOGY 

Experiment X. Preparation and reactions of Indol, Skatol and 
Phenol. Prepare an artificial digestion mixture with pancreatic 
extract, or minced pancreas, and allow it to incubate without the 
addition of an antiseptic, until it has an intense and disagreeable 
odour. The digest is then acidified with acetic acid and placed in 
a large flask connected with a Liebig condenser. Distillation is 
continued as long as the distillate has a marked odour. (Indol 
distils over much more quickly than skatol). The following tests 
are then applied to portions of the distillate : — 

Indol. — 1. Legal's Test. — To a few c.c. of the solution in a test 
tube add a few drops of sodium nitro-prusside solution and then 
ammonia till alkaline. A deep reddish violet colour results, which 
changes to blue on acidifying with acetic acid. 2. Add to a few 
c.c. of the liquid about 2 c.c. of each of the following solutions : 
(i) Para-dimethyl-amino-benzaldehyde 4 parts, 95 per cent, alcohol 
380 parts, hydrochloric acid (cone.) 80 parts, (ii) Potassium persulphate 
2 grams in 100 c.c. water; a reddish pink colour results. 

Skatol. — Warm some of the solution with an equal volume of 
strong sulphuric acid. A red colour results. 

Phenol. — Boil some of the solution with Millon's reagent. A red 
colour, but no precipitate results. 

The action of bacteria on carbohydrates is even more energetic than 
it is on proteins. They can do all that ptyalin and amylopsin can do, 
but besides this they can decompose the monosaccharides into simpler 
bodies such as ethyl alcohol, lactic and butyric acids. They have 
also the power of digesting cellulose whereby methane (CHJ is pro- 
duced as one of the products. 

On fats they act like steapsin, but here also they can carry the 
process a stage farther in that they transform the fat acid, which 
is first of all liberated, into members lower in the fat acid series. 
They decompose lecithin, and prevent the poisonous action of the 
liberated cholin by further breaking it up into carbon dioxide, 
methane and ammonia. 

The meconium of the foetus i is sterile, but very shortly after birth 
micro-organisms gain admission to the intestine with the food. The 
exact varieties of organisms present in the intestine depends mainly 
on the nature of the food and on the presence of oxygen. The 
anaerobic bacillus putrificus is the most important proteoclastic 
organism, its action being supplemented by the aerobic B. coli and 
B. lactis aerogenes, i.e. the latter organisms act on the products 

■* Meconium is a substance which collects in the intestines during intra-uterine 
life. 



PHYSIOLOGICAL CHEMISTRY 407 

produced by the former. The intestinal bacteria are no doubt very 
important as digestive agents. Thus, without their aid, cellulose 
cannot be digested, so that, for herbivora, bacteria are indispensable, 
at least after they have ceased to live on the mother's milk. 
Carnivorous animals could probably live without them. It has, for 
example, been shown that if guinea pig foetuses be excised from 
the uterus just before full term under antiseptic precautions, . and 
kept in a chamber aspirated with sterile air, and fed on sterile milk, 
they thrive, and if after some time the intestinal contents be examined, 
the latter will be found free of bacteria. A repetition of this experi- 
ment on chickens (fed on grain) has given quite a different result, 
for although they took abundance of sterile food, yet they died as 
soon as if no food had been taken. 

The bacteria live in symbiosis with the host in whose intestine 
they grow — that is to say, both they and their host are benefited by 
their presence in the intestine. 

The Faeces. — These are composed of the following substances : — 

1. Substances which have escaped digestion, e.g. pieces of vegetables 
(cellulose, etc.), muscle fibres, elastic tissue, casein, fat, nuclein, haematin, 
etc. 

2. Remains of juices secreted into the intestines, e.g. mucin, traces 
of bile salts and pigments, inorganic salts (alkaline earths), epithelial 
cells, and cholesterol. 

3. Products of digestion, e.g. aromatic bodies (indol and skatol), 
fat acids, methane, ammonia, etc. 

4. Micro-organisms. The faeces contain a certain amount of 
nitrogen, which probably comes from the various secretions rather 
than from undigested foods (about 1 grm. N. per diem on ordinary 
diet). The amount of faeces varies very much with the nature of 
the diet, being about 170 gms. in 24 hours on a mixed diet and 
400-500 gms. on a vegetable diet. 



CHAPTER XVI. 

THE CHEMISTRY OF URINE. 

No portion of Biochemistry is of .so much practical importance to the 
medical practitioner as the chemistry of the urine. It is in the urine 
that the waste products of protein metabolism are chiefly excreted. 
Urea, uric acid, and creatinin are almost entirely derived from protein, 
and an estimation of these products in the urine yields valuable 



408 PRACTICAL PHYSIOLOGY 

information regarding the breakdown of protein in the body. The 
main function of the kidneys is to serve as a regulator of the composi- 
tion of the blood, removing from it any excess of its normal constituents, 
water, dextrose, sodium chloride, etc., and also injurious waste pro- 
ducts, such as urea, uric acid, etc. The capacity of the kidneys for 
their work can be determined most readily by the examination of the 
urine. When the kidneys, or other parts of the urinary tract, are 
diseased, abnormal substances, such as proteins, blood, pus cells, etc., 
are mixed with the urine and can be detected in it in a more or less 
changed state, according to the site of the lesion. 

In studying the chemistry of the urine, therefore, we must ascertain, 
firstly, the nature of its various constituents and of their precursors in 
the blood and tissues ; secondly, the total amount of those excretory 
products which contain the nitrogen of the decomposed proteins ; and 
thirdly, we must look for unusual products, indicating improper 
composition of the blood or organic disease of the urinary tract. 

We must remember that the quantity and the composition of the 
urine Vary considerably within the limits of health, and in order to 
form reliable conclusions we must collect the total urine for a period of 
twenty-four hours. Even with a fair sample thus obtained, we must 
consider the intake and loss of water ; copious drinking will increase 
the quantity and lower the speciiic gravity of the urine ; on the other 
hand, profuse sweating or diarrhoea will have the opposite effect. The 
nature of the diet in relation to the reaction of the urine and the 
quantity of urea must also be considered. 

General Characters of Ukine. 

Quantity. — A healthy man of average weight (65-70 kg.) and height, 
and living on an ordinary mixed diet, excretes about 1500 c.c. per 
24 hours. If we wish to ascertain whether any one of its constituents 
is being excreted in normal amount, a knowledge of the total daily 
excretion of urine is indispensable, a mere determination of the 
percentage in an isolated sample being of very slight value. For 
accurate work {e.g. in making observations in metabolism) the method 
employed is to collect the total urine for the 24 hours in a suitable 
vessel, and then to remove from this a measured sample for analysis.^ 

^ In doing this, the bladder is emptied at some chosen hour (best in the morn- 
ing), and this urine thrown away ; all urine passed subsequently to this is 
collected in a sterile flask or bottle containing a few c.c. of chloroform, and at 
the same hour next day the bladder is again emptied and the urine added to the 
twenty-four hour specimen. When the observation is being conducted on the 
lower animals, it is usually necessary to employ the catheter. 



PHYSIOLOGICAL CHEMISTRY 



409 



The amount of urine is increased by the imbibition of large quantities 
of liquid and by certain drugs called diuretics; it is diminished by 
excessive sweating or diarrhoea, and by failure of the heart's action. 

Specific Gravity. — This is determined by a special form of hydro- 
meter — a urinometer — graduated so that the zero mark — 1000 — cor- 
responds to distilled water (Fig. 237). 

Experiment I. Fill a urine testing glass with urine cooled to 
room temperature, place the urinometer in it, and 
read off the graduation which is on a level with the 
surface of the urine. Be careful that the urinometer 
does not stick to the sides of the vessel. 

The average density varies between 1015 and 1025, 
but a highly concentrated urine, e.g. after severe 
sweating, may reach 1035, or a very dilute one, 
e.g. after huge potations, 1002, and still be healthy. 
A specific gravity over 1030, however, usually indi- 
cates the presence of sugar or the existence of high 
fever, and one much below 1010 should raise sus- 
picions of renal trouble. 

Reaction. — Healthy urine usually reacts acid to 
litmus. This acidity is due to sodium dihydrogen 
phosphate, NaH2P04, not to free acid. 

Experiment II. Test the reaction of urine with 
blue litmus paper and congo red paper. The litmus 
is turned red, but the congo red is not altered, as it 
is not affected by the acid salts of any but the 
strongest acids (see Digestion, p. 376). 

The alkaline phosphate, NajHPO^, may be present 
in urine. It is detected by the addition of calcium 
chloride to the urine, when a precipitate of calcium 
phosphate forms if the alkaline phosphate is present, 
but not if the acid phosphate alone is present. The 
amount of alkaline phosphate may be sufficient to 
cause the urine to have an amphoteric reaction, turn- 
ing red litmus blue and blue litmus red, or even to 
have a definite alkaline reaction. This is often the 
case during the stage of digestion, when hydrochloric 
acid is being poured into the stomach, as the removal of hydrochloric 
acid from the blood leaves an excess of bases. 

Besides the alkaline phosphate, alkaline bicarbonates may be present 
in urine, causing an efiervescence on the addition of acid. This is the 
case when salts of oxidisable acids (e.g. citric, tartaric, etc.) are being 




Fio. 237.— The urino- 
meter. 



410 PRACTICAL PHYSIOLOGY 

taken by the mouth, and when the diet is an exclusively vegetable one. 
Lastly, an alkaline reaction may be due to ammonia, which is produced 
by microbal hydrolysis of urea (see Urea, p. 417). For this reason 
stale urine always reacts alkaline. If the alkaline reaction of freshly 
passed urine is due to ammonia, decomposition must be taking place in 
the bladder. 

Colour. — The straw colour of healthy urine is due to Urochrome, the 
origin of which is uncertain. Another pigment. Urobilin, is normally 
present in traces in the urine. In fever, and when there is liver disease 
or rapid destruction of haemoglobin, the amount of urobilin is often 
much increased, imparting a reddish tint to the urine. Urobilin is 
identical with stercobilin, the pigment of faeces, which is formed from 
bilirubin in the intestine by bacterial action, so that its presence in 
urine is presumably due to absorption from the intestine. Urobilin 
can also exist in the urine as a colourless precursor, or chromogen, which 
yields the pigment, when the urine is acidified with sulphuric acid and 
allowed to stand. 

Experiment III. Examine an acid solution of urobilin or sterco- 
bilin with the spectroscope. A diffuse absorption band is seen between 
the green and the violet of the spectrum. 

A third pigment is woerythrin, the colouring matter of pink urate 
deposits. It appears to be related to skatoxyl sulphuric acid. 
Haematoporphyrin (see Haemoglobin, p. 350) may also occur. 

Indican, or indoxylsulphuric acid (see Ethereal Sulphates, p. 437), is 
the cause of the blue tint sometimes acquired by urine on standing, as 
it is oxidised to indigo blue. Normally a small quantity is present. 
This is increased when there is excessive putrefaction in the small 
intestine, or in an abscess. 

Experiment IV. Test urine for indican. Mix about 5 c.c. of 
urine in a test tube with an equal volume of strong hydrochloric acid 
containing 0*4 per cent, ferric chloride. Add about 3 c.c. of chloroform 
and shake the tube. The chloroform on settling will be tinged with 
blue (indigo) if the urine contains indican. 

The Nitrogenous Constituents. — Over 90 per cent, of the nitrogen 
in combination excreted by the body is present in the urine, the 
remainder occurring in the faeces (about 1 gramme per diem), and as 
urea in the sweat. A determination of the total nitrogen of the urine is, 
therefore, of great importance. The method employed is that of 
Kjeldahl. 

Experiment V. Measure 5 c.c. of urine with a pipette into a Jena 
flask. (This flask should be of at least 400 c.c. capacity. It saves 
time and chance of error to use the flask of 750-1000 c.c. capacity, 



PHYSIOLOGICAL CHEMISTRY 411 

from which ultimately distillation of the ammonia is to be carried out.) 
A.dd approximately 0*5 grm. copper sulphate and 2'0 grm. potassium 
sulphate, and then 10 o.c. of pure concentrated sulphuric acid. Put 
the flask on a rack (Fig. 238), so constructed that the neck of the flask 
lies in a slanting position with the belly of the flask, fitting into the 
depression made to receive it in the asbestos plate or sand-bath. Start 
the heating with a low flame. After 10-15 minutes, if there is no 
excessive foaming, turn up the flame and heat the mixture strongly 




Fig. 238. — Apparatus for determination of total nitrogen. 

until it is a clear greenish blue. Now shake the flask with a rotary 
motion so as to wash down from the wall any carbonaceous particles 
which may be sticking to it. Continue heating until the liquid is 
perfectly clear again. The stage of incineration is now complete. {1st 
stage.) 

The chemical reaction which takes place is that the sulphuric acid 
decomposes the organic matter, the carbon being oxidised to carbon 
dioxide, and the nitrogen changed into ammonia, which immediately 
on its formation combines with the excess of sulphuric acid present to 
form ammonium hydrogen sulphate. The first effect of adding the 
acid is to produce charring {i.e. the mixture becomes black), and the 
reaction is complete whenever all this liberated carbon has been 
oxidised. 



412 PRACTICAL PHYSIOLOGY 

The sulphates of copper and potassium accelerate the process very 
greatly, the former by acting as a carrier of oxygen, the latter by 
raising the boiling point of the mixture. 

Distillation {2nd stage). — When the acid mixture has cooled, add 
100 c.c. of distilled water (free from ammonia if obtainable), and mix. 
If the incineration was performed in a small flask, it will be necessary 
to transfer its contents to a large Jena flask for the distillation,^ and 
rinse with portions of about 100 c.c. water, until the distilling flask is 
nearly half full. If the mixture is already in the distilling flask the 
requisite amount of water is added. Add a little powdered pumice or 
mica to prevent bumping. Then, holding the flask in a slanting 
position, pour strong caustic soda (40 to 50 per cent.) solution, enough 
to render the liquid alkaline, down the neck and wall of the flask so as 
to form a bottom layer of alkali. By this means any ammonia set free 
by the alkali will be caught by the overlying acid mixture. The 
amount of alkali required to render the mixture alkaline must be 
previously determined ; 40 c.c. is usually an excess. 

Attach the flask to the distilling apparatus (see Fig. 239). Place 50 

c.c. YjT sulphuric acid, about an equal volume of distilled water and a 

few drops of methyl orange solution, or other suitable indicator, in 
the receiving flask. Adjust the delivery tube of the apparatus so that 
it just dips below the surface of the liquid in the receiver. Mix the 
contents of the distilling flask. (If enough alkali has been added a 
blue colour will develop owing to the liberation of cuprous hydroxide.) 
Light the burner, and continue the distillation for at least 15 minutes, 
taking care to prevent the sucking back of the contents of the receiver 
into the distilling flask. ^ 

Lower the receiver so that the delivery tube is above the liquid, and 
continue the distillation for a few minutes so as to wash out the inside 
of the tube. Finally wash the outside of the tube with a jet of water 
from the wash bottle, so as to remove adhering acid, and remove the 
receiver for the titration. Should the contents of the receiver become 
neutralised during the distillation, as shown by the indicator changing 

colour, a further measured quantity of ^ts acid must be added .^ 

'750-1000 o.c. capacity. 

2 A vertical condenser, replacing the descending portion of the delivery tube, 
reduces the risk of sucking back and prevents the heating of the liquid in 
the receiver, and is, therefore, desirable in accurate work. 

^It is often necessary to continue the distillation for considerably more than 
15 minutes. When it is doubtful whether all of the ammonia has distilled over 
in this time, distillation should be continued into a second flask containing a 
few o.c. decinormal acid and distilled water. 



PHYSIOLOGICAL CHEMISTRY 



413 



The chemical reaction involved in this stage of the method is the 
liberation of ammonia from the ammonium hydrogen sulphate (pro- 
duced in the incineration stage) by the excess of alkali added to the 
contents of the distilling flask. This ammonia is completely carried 
over into the sulphuric acid of the receiver during the distillation, and 
thus quantitatively neutralises some of the acid. 

Titration (Srd siage).— Cool the receiver under the tap. Eun in ^n 

caustic soda carefully from a burette, until its contents are neutral. 




Fig. 239. — Apparatus for determination of total nitrogen. 

as shown by the change in colour of the indicator employed. Subtract 
the volume in c.c. of jn ^o^a required for neutralisation from the 

volume of j^ acid originally added to the receiver. The remainder 

represents the volume in c.c. of -r^ ^'Cid neutralised by ammonia during 

the distillation. Let this remainder be n c.c. Then, as the ammonia 
distilled quantitatively represents the total nitrogen present in the 
5 c.c. of urine taken, this 5 c.c. of urine contains »x0'0014 grm. 

nitrogen, and, therefore 100 c.c. of the urme contain = 



grm. nitrogen. 

To take an example, suppose that 50 c.c. j^ acid were placed in the 



receiver, and that after the distillation 19-4 c.c. y^ alkali were required 

for neutralisation. Then 5 c.c. urine contain 

(50 - 19-4) X 0-0014 grm. = 0-0428 grm. nitrogen. 
Therefore 100 c.c. urine contain 0-856 grm. nitrogen. 



414 PRACTICAL PHYSIOLOGY 

Experiment VI. Determine by the above method the amount of 
nitrogen contained in an acid solution of ammonium sulphate.^ 

Measure out 5 c.c. of the solution with a pipette, place it in the 
distilling flask A, dilute to 200 c.c. with water. Now measure 

accurately 20 c.c. yn ^"i^ ^^^ place in receiving flask B, adjust 

distilling tube C, add excess of caustic soda, and distil as above. 

The total amount of nitrogen excreted by the urine per diem in the 
case of a man living on an ordinary diet varies between 15 grammes 
and 20 grammes, and, if the total amount of nitrogen taken in the food 
be ascertained, it will be found to nearly correspond to this. By 
special precautions it can be made to accurately correspond when 
the person is said to be in nitrogenous equilibriwn.^ The nitrogenous 
constituents of the urine, which collectively make up this total amount 
of nitrogen, vary in their relative amounts according to the amount of 
nitrogen which the diet contains. This fact has been clearly shown by 
Folin, who, for the purpose of demonstrating it, first of all elaborated 
rapid and accurate methods for estimating these nitrogenous bodies. 
By the use of the older methods, the chemical estimations were too 
laborious and too complicated to permit of a suificient number of 
analyses being made in daily urine. These methods will be described 
in their proper places, but in the meanwhile it may be well to briefly 
consider the main results which have been obtained. 

The chief nitrogenous bodies which occur in the urine are urea 
(CO(NH2)2), uric acid (C5H4N4O3), ammonia (NH3) and creatinin 
(C4H7N3O). There are several otber nitrogenous substances which are 
present only in small amounts and are estimated together as un- 
determined nitrogen. In the following table, the first column gives 
the relative amounts of these bodies in the urine when a diet rich 
in nitrogen was taken, and the second column the corresponding 
data when the diet contained very little nitrogen. The diet rich in 
hitrogen was made up of whole milk (500 c.c), cream (300 c.c), 
eggs (450 grm.), Horlick's malted milk (200 grm.), sugar (20 grm.), 
salt (6 grm.), and water sufficient to make up to 2000 c.c. Besides 
this, 900 cc water were allowed. The diet contained about 19 grm. 
of nitrogen. The diet poor in nitrogen consisted of 400 grm. arrowroot 
starch made into a paste with 1500 c.c. water, then partly digested for 

^A suitable solution for the purpose is made by dissolving 1'32 grammes of 
ammonium sulphate crystals in 100 c.c. of I per cent, sulphuric acid ; 5 c.c. 
of this solution contains '014 gramme N. 

^Allowance must &lso be made for the nitrogen in the faeces. 



PHYSIOLOGICAL CHEMISTRY 415 

half-an-hour with diastase (2 grm.) and taken with 300 c.c. cream. 
It contained only about 1 grm. of nitrogen and could be taken without 
great discomfort to the person for several days. It might have been 
better had it contained twice as much cream, for then the breakdown of 
tissue protein which occurred would have been diminished. 

Nitrogeu— rich diet. Nitrogen— poor diet. 

Volume of urine 1170 c.c. 385 e.c. 

Total nitrogen - 16'8 grm. 3'60 grm. 

Urea-nitrogen 14-7 grm. =87 '5% of total N. 2 -20 grm. = 61 7% of total N, 

Ammonia nitrogen 0-49grm.= 3-0% „ 0-42 grm. = 11 -3% 

Uric acid nitrogen 0-18grm.= 1-1% „ 0-09grm.= 2-5% 

Creatinin nitrogen 0-58grm.= 3-6% „ 0-60 grm. =17-2% 

Undetermined nitrogen 0-85 grm. = 4-9% „ 0-27 grm. = 7-3% 

When the intake of nitrogen by the diet is much reduced, the 
percentage of the total nitrogen excreted as urea markedly falls, whilst 
that of the other nitrogenous bodies rises. It will further be noted, 
however, that the absolute amounts of all these bodies falls, except in 
the case of creatinin, which remains unchanged. 

The total sulphate excretion is made up of inorganic sulphates, 
ethereal sulphates, and neutral sulphur, and the relative amounts of 
these excreted on a rich and a poor diet show striking resemblances to 
the nitrogenous bodies, as will be discussed later. 



CHAPTER XVII. 

UREA. 

Urea is the diamide of carbonic acid. 

Carbonic acid. Urea. 

CO<gH C0<NH, 

In common with other acid amides, urea has weak basic properties, 
forming unstable salts with nitric and oxalic acids. 

Experiment I. To some urine, which has been evaporated to 
small bulk on a water-bath, add some pure, colourless (not fuming) 



416 



PRACTICAL PHYSIOLOGY 



nitric acid, and cool the mixture by holding the test tube under the 
tap. Crystals of urea nitrate separate out. Examine these with 
the microscope, and note that they are either rhombic tables or six- 
sided plates, which overlap each other like the tiles of a roof (see 
Fig. 240). 

Experiment IL Repeat experiment with a saturated alcoholic 
solution of oxalic acid, and note that the crystals are not unlike those 
of the nitrate, being elongated plates with bevelled pointed ends 
(Fig. 241). 




Fio. 240.— Urea nitrate. 



Pig. 241.— TJrea oxalate. 



Urea is decomposed by nitrous acid — HNOj — carbonic acid gas and 
nitrogen being evolved : 

co<Ni:+§:N:§i=co^+2N,+3H,o. 

Experiment III. Add some fuming nitric acid (i.e. containing 
nitrous acid) to urine, and note the effervescence which results. That 
one of the gases evolved is carbon dioxide can be proved by holding 
the mouth of the test tube over another one containing lime water or 
baryta water, when, on shaking, the latter will turn milky. 

A very similar reaction is obtained by adding a hypobromite or 
hypochlorite to urine. 

CO<J5g2 + SNaBrO = CO;, + N^ + BH^O + 3NaBr. 

The carbon dioxide formed combines with excess of caustic soda 



PHYSIOLOGICAL CHEMISTRY 417 

present in the hypobromite. This reaction is employed in the quanti- 
tative estimation of urea (see below, p. 419). 

There are several reactions which are peculiarly interesting, since 
they demonstrate the chemical relationships of urea to its probable 
precursors in the tissues (see below). Thus, if urea be hydrolysed {i.e. 
be caused to take up water) it forms ammonium carbonate : 

PO/NH, HOH _ po<^ONH, 
^°<NH, + HOH - ^"<ONH*. 

(Urea) (Water) (Ammonium 
carbonate) 

This process occurs in urine which has stood for some time, the 
hydrolysis being effected by several kinds of microbes. It may also be 
produced by boiling urea with strong acids or alkalies ; in both cases 
the ammonium carbonate is further decomposed, liberating, in the case 
of alkalies, ammonia gas (the carbon dioxide being absorbed by the 
alkali present), and in the case of acids, carbon dioxide gas (the 
ammonia being absorbed by the acid present). 

Experiment IV. Prepare a solution of pure urea, and divide it 
into two portions, A and B. To A add about 10 drops of sulphuric 
acid and boil, meanwhile collecting the vapour which comes off in a 
second test tube containing lime or baryta water. By this becoming 
milky, the presence of carbon dioxide gas is demonstrated. To B add 
about 5 drops saturated caustic potash and boil. Ammonia gas is 
evolved, so that a moistened strip of red litmus paper is turned blue if 
held in the fumes, which smell strongly of ammonia. 

A substance intermediate between urea and ammonium carbonate, 

and having therefore the formula CO<^t^tt *, 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 (HCNO3), which 
is isomeric with cyanic acid, HONO. 

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 (NH^CNO) to dryness. This 

2d 



418 PEACTICAL PHYSIOLOGY 

salt has the same empirical formula as urea, but its structural formula 
is diflferent : 

/ONH, /NHj 

^N \NH2 

(Ammonium oyanate) (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 o.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 o.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.' 

2. Separation of Urea from Blood, Serous Fluids or Watery Extracts 
of Tissues. — About 100 o.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 ia 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 nf H^S gas through it. 

Far qvantitatively estimating wrea 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 
0"1 grm. urea contained in urine yields 37'1 c.c. moist nitrogen at 
15° C. and 760 mm. pressure. O'l 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 oif 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 Dupr6 (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 



PEAGTICAL 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). 




Pig. 242.— Dupr4's urea appamtus. 




Fio. 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. — Ureometor. 



graduation corresponds to grammes of urea in the quantity of urine 
used. 

IL Momer-Folin It/Lethod.— 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 veith 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° 0.^ When the liquid has been reduced to a few c.c, 
25 c.c. water and » 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. 

EXPEBIMENT. — Place 5 c.c. ui-ine, 5 c.c. HCl (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 HCl 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 HjS04 (con.) is dropped into it). Now lower the flame and connect the 

^To 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 rrTrH2804 as described in connection with Ejeldahl'ls 

method. The distillation requires much longer than in Ejeldahl's process 
(1} hours). 

From the number of o.o. of yrrNHj found must be deducted : — 

(1) The CO. tjjNHj present as such in the 5 o.o. of urine used (determined by 
Folin's or Shaflfer's method, p. 433). 

(2) The c.c. ^NHg present in the reagents for MgCl^ always contains traces of 
NHo. 



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— C« 



20 60— 7N. 

SN_40_N9/ 
Purine nucleus. 



€«; 



HN— CO 

CO C— NH 

\ II >CH; 

HN— C— N 
Xanthine 
(2, 6, dioxypurine). 



HN— CO 

CO C— NH. 

\ II >C0; 

HN— C— NH/ 

Uric acid 
(2, 6, 8, trioxjrpurine). 

HN— CO 
CH C— NH 

^N— C — N 

Hypoxanthine 
(6, oxypurine). 



NH— CO 

C— NH 
\ II >CH; 
NH— C— N^ 


HN— C=NH 
CH C— NH 


Guanine. 


Adenine. 



PHYSIOLOGICAL CHEMISTEY 423 



HN=C 

>CH. 



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 abundantly 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, CsH^N^. 

(Hypoxanthine, CjH^N^O. 

Xanthine, C^H^N^Og. 

Adenine, C5H5N5. 

Guanine, C5H5N5O. 

Uric Acid, C^B.^Nfis. 

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 PBACTICAL PHYSIOLOGY 

synthesised in the liver from oxidation products of lactic acid. 
Whether such a synthesis takes place also in matiimals is at present 
unknown. 

On oxidation with potassium permanganate uric acid yields allanfoin, 
which is present in the urine of the dog and cat, and occasionally in 
that of man. The formula for allantoin is : — 

/NH— CH— NH. 

C0< I >C0. 

^NH— 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 : — 

co<gizS>co. 

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 hypoxanthine in meat, caifeine, 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 
grm., 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 CHEMISTEY 425 

Prepaeation and Eeactions of Uric Acid. 

Experiment I. To 100 c.c. urine add 5 c.c. HCl (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 HCl. 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 HCl. 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 add. 

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 AgNOg. A black stain of reduced silver results. This is 
called Schifi's reaction. In the presence of neutral salts, and more 
especially of magnesium mixture (MgClg, NH^Cl, NHj), 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 




Fio. 245.— Crystals of uric acid. 



PHYSIOLOGICAL CHEMISTRY 427 

Experiment V. Tests for Uric Acid in Urine.— Apply Sdiifs 
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 mwrexide 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 oS, dissolved in a few c.c. of water, and employed 
for the murexide test. 

Estimation of Ukic Acid. 

The most rapid and accurate method ia 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 n/20 potassium 
permanganate. 

The method is carried out as follows : — 

To 300 c.c. urine in n 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 fiuid should have collected in the beaker. Then 15 c.c. H2SO4 (cone.) is 
added to the fluid, and while still hot from the mixing of acid and water, it is 
titrated with 72/20 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.' 

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 

' 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 
Inagnesiiim 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 cm. 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 oft' 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 CgH^. CO. NH. 
CHj. 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 
tbese 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 Eeaction. — 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 





E 




^^e. 


H 




? 


L _ 


E 



for five minutes. Fill up the flask to the 500 c.e. 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- 
tfnin 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. K. HCl. Heat theflask 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 hy the acid into creatinin. The difference hetween 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 j^ 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 1 5 grm. powdered potassium oxalate (neutral 
to phenol phthalein), place in a flask, and add from a pipette 25 c.c. urine 
and an equal voluine of water. Add about 10 drops 1 per cent, alcoholic 
phenol phthalein. Mix and allow to stand for about a minute. Now 

run in ttt caustic soda from a burette until the contents of the flask 

assume a slight pink tint. Eead 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. Eun in 

jjr 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- 

appears. Run in j^ caustic soda until the colour returns, and take 

the reading. 

The first reading of the burette gives the total acidity of 25 cc 

N ■ • ■• 

urine in terms of rrjr soda. Potassium oxalate is added to precipitate 

the calcium in thei 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 
m terms of j^ soda. To calculate the amount of nitrogen in grammes 

present as ammonia in the volume of urine taken multiply the reading 
of this titration in cc. by 0-0014. 

The method of determining ammonia is of suflScient 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 aifords 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 cc. 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 com. 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) 5 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 cc. N/10 H2SO4-1-5 cc 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.o. J\^/10 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 attacbed to a 




r=^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 Erlenraeyer flask and titrated with NllO alkali. For titrating, Folin 



S> Suction/J^unp 




Fio. 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 PRACTICAL PHYSIOLOGY 

Place 50 o.c. urine in a round bottom i litre flask A (Fig. 248), add 20 grm. 
sodium chloride to prevent decomposition and 50 c.o. methyl alcohol to reduce 
the boiling point of the mixture. In flask B place 50 o.c. or less NjlQ acid and 
in O 10 c.c. NjlO acid, diluted in both oases 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 off 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 ilask measure with a pipette 10 

c.c. urine free from albumin. Add 5 c.c. pure nitric acid and 

N 
30 c.c. YjT 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 j^ potassium sulphocyanide 



PHYSIOLOGICAL CHEMISTRY 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 ( - CI.) 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. 

ExPEEiMENT 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 
O'OOS grm. phosphoric acid (V^O^). 

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.). 

2. Ethereal sulphates (phenyl sulphate of potassium) S02'\qq tt 

(indoxyl sulphate of potassium) S02<^qq tt -xj 
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 cc. 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 HCl (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).' The flask must not be shaken 
until after the end of an hour,^ 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 

'More rapid addition of the reagent causes the results to be too high, i.e. 
produces an impure precipitate. 

^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 dilating 125 c.c. of urine with 75 c.c. water, adding 
30 CO. 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 Goooh 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 cc, 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 daya,^ after which the precipitate is 
collected and weighed as above described. 

ExPBEiMBNT 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 difierence. 

These values can be calculated as S or SO3 according to the following ratios :^ 

BaSOi : S = l : 0-1374; BaSOi : S03=l :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 

^It takes this length of time to ensure complete precipitation. 



438 PEACTICAL PHYSIOLOGY 

the sulphur is an integral part of the molecule. Cystin, when present, 
belongs to this group. When the relative amounts of SO3 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 0.0. .385 e.o. 

Total nitrogen, 16'8 grm. 3'60 grm. 

Total SO3, 3-64 grm. 0-76 grm. 

Inorganic SO3, 3-27 grm. =90-0% of total S. 0-46 grm. =60-5% of total S. 

Ethereal SO2, 0-19 grm. = 5-2% „ O'lO grm. = 13-2% „ 

Neutral SO3, 0-18grm.= 4-8% „ 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 /rom a healthy person may deposit thefollomng: — 
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 



439 




Fio. 249,— Sodium 
urate. X 350. 



Fio. 260.— Cystin. 
X S60. 



Fio. 261.— Calcium 
carbonate (from 
human urine). 
X 400. 



44U 



jriiii.<jinjii.ij ru.xai\jiJUKjri 



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 




Fia. 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 CHEMISTEY 



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). 

Add 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 PEACTICAL PHYSIOLOGY 

tion of benzoic 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 usuall)' 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 



44d 




Fia. 264.— 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 : — 

L 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 concentratedl 
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 the 
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-Snlphonic 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 (Mac William). 

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 B. Close the 
tube with a tightly-fitting cork and invert several times, so as to mix 

'These corrections should be made before the urine is measured into the 
Esbach's tube. 



t 

s~ 

— s 

— t 
— s 

I 

Hi -1 



Fio. 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, Soherer's method is employed. 

Advanced Experiment. Place 50-100 o.o. 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 aah-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 /i 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 ImemogloUnwia and meihaemoglohinwia. 



PHYSIOLOGICAL CHEMISTRY 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 oxy haemoglobin. 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 
and 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 pundv/re of the floor 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 PEACTICAL 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 proteia 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 Fhloridzin. — 
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.^ 

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. 
^ 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 Eochelle 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. 
Nylander's reagent.^ 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 L c.c. 
copper solution and 2-5 c.c. alkaline Eochelle 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 

' Nylander's solution : dissolve 4 grm. Rochelle salt in 100 grm. of a caustic 
soda sorution of 1'12 sp. gr. ; add 2 grm. Bismuth subnitrate and heat on water 
bath until it is dissolved. 

''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, ms 
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. 

I/actose 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. 27.5. 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. glyjcuronic 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 difiiculty 
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 th§ 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 3464 grm. pure 



PHYSIOLOGICAL CHEMISTRY 451 

crystallised copper sulphate, 180 grm. Eochelle 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 5i> 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 O'0O5 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 PEACTICAl, 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 uroehrome. 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 uroehrome as 
well as a considerable proportion (75 per cent.) of the uric acid and creatinin, 
Bang and Bohmanneson use blood charcoal and hydrochloric acid. We have 
obtained more constant results, however, by using acetone' 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 «, dry filter paper into 
a test tube. Of the filtrate (which is always perfectly colourless) 5 c.c.'' 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 laemlose is revealed by Seliwanoff's test (p. 278). 

The Acetone Bodies in Urine. — These substances are: — 

(1) iS-oxybutyric acid, CHg . CHOH, . CH^ . COOH. 

(2) Aceto-acetic acid, CH3.CO.CH2.COOH. 

(3) Acetone, CHg . CO. CH3 . 

Aceto-acetic acid is an oxidation product of ^-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 

' The acetone must be chemically pure, otherwise it may contain reducing 
substance. 

'This amount of the filtrate is for a urine containing not more than 2 per 
cent, dextrose. If it contain less than O'S per cent, dextrose 10 c.o. 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 CHEMISTRY 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 sodui^ 
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). 

Kothera's Test. — Add a few drops of sodium nitrojffusside 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 1.5 minutes. This test is more sensitive 
and distinctive than Legal's. 

Iodoform 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. Iodoform is precipitated, and is detected by the charac- 
teristic smell. 

Experiment XI. 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 
Scha£fer'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 constemt) 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 i.s then redistilled, aud the second 
distillate tested for acetone by Bothera's test and by the iodoform test. 

/OH 
Homogentisic Acid is di-oxyphenyl-acetic acid CgHgv— OH 

^CH^COOH. 

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 



464 PEACTICAL 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 — 
CHgOH — group has become oxidised to form COOH, or carboxyl. It 
has, accordingly, the formula COOH— (CH.OH)^— 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 admiin'stration 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. Ryffbl, 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]^ (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 urine. 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 TMopliene Test for lactic acid introduced by Hopkins has already 
l)eeu 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 : 

CHj . CHOH . COOH = CHg . 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 CO., 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 o.c. flask may be employed, the quantities 
given in what follows being halved.) 

To the second distillate are added 0'5 o.c. SohifF's reagent (see later) and 
water to bring the volume to 100 o.c. The flask is stoppered, inverted a few 
times to mix its contents, placed in a glass vessel containing water at 15° C, 
and left for .30 minutes in diffuse daylight. The SchifiCs 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 Pormaldebyde 3 c.c. 

Readings of equal depth of colour, 2-42 cm. 2 cm. 1'46 cm. 

10 -r readings, 4-13 5 6 85 

Then a is equivalent to 3 c.c. +;7-=^ — -r-r^ 

b'oo — 4*lo 

=3 '32 c.c. standard formaldehyde solution. 

The amount of lactic acid in the liquid originally employed = 

3-32 X 3-435 xn 

0^4 "S- 

where 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. 

V'Ae Formaldehyde Standards. — A series of four stoppered flasks is prepared 
containing 0-5 c.c. Schiflf's reagent and 1-5 c.c, 2 o.c, 3 c.c, 5 o.c. respectively 
of dilute standard formaldehyde solution, made up to 100 c.c with water. 
These are placed In » 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 cc 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 

iV 
measured into a, stoppered bottle, 25 cc. jrr 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 jn 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 =& c.c. Then the formaldehyde in 

1 -49 X 6 
mg. present in 1 cc. of the solution=re= — jjt 

The value of n should be nearly 0"4 mg. 



PHYSIOLOGICAL CHEMISTRY 457 

Schifs Reagent. — 1 grm. finely powdered rosaniUne 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 l^ept 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 rag. 
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. 

rrrj; potassium permanganate. 

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. 

Glyouronio 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 rag. at night when determined directly, or to 
about half this quantity when determined after lead acetate treatment. 



458 PEA.CTICAL PHYSIOLOGY 

Lactic acid is, therefore, present in relatively greater amount in the 
blood than irt 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 tliat 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. 

ExPEiiiMENT 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 cleai'ly 
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 lesult 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 und 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 PEACTICAL 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 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 BED 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 
salts. 

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. 

ExFEBiMENT 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. 

ExPEMMBNT II. Determine what 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., 0-70 per cent.^ 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 hypotonioity 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. 

ExPBRiMEKT 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'd5 per cent, solution of sodium 
chloride. 

We can determine the osmotic pressure of a saline solution indirectly by 
observing what efiect it has on the volume of red blood corpuscles. When 
DO 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 

^ Thesfe 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 PRACTICAL PHYSIOLOGY 

make a mixing chamber. By attaching a piece of indiarnbber 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 tht same level as 
before, if it be hypotonic they will stand at a higher level, it 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 
chemiccd, such as the addition of ether and other fat solvents, saponin, bile 
salts, acids and alkalies ; and others as bio-chemiccU, 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. Expbeimbnt V. l. 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 efiect on the corpuscular envelope. 
This question can be s^tt^ed 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.' 

5. Determine the minimum amount of a 0"3 per cent, saponin solution necessary 
to produce laMng of washed dog's corpuscles in Jive minutes at room temperature. 

Label four test tubes A,B,0, 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 3 per cent. 
solation of saponin in 0-9 per cent. NaCl solution, add to A 0-05 c.c. ; to B 
0-1 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 1/lOOth of a o.o. 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 10, then in this experiment use 
0.11 c.c. saponin in A ; 0']2 c.c. in B, and so on). 

6. Dem^mstrate 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 exauiiiied 
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 stroma 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 

• To wash blood corpuscles free of adherent serum a, good centrifuge is 
necessary. The defibrinated blood is first of all oentrifuged 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 PRACTICAL PHYSIOLOGY 

developed in the tissue fluids of an animal when the deflbrinated 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. Haemolysins 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, it the dog be previously inoculated 
with deflbrinated rabbit's blood. ^ Besides causing haemolysis, the serum usually 
causes the corpuscles to run together into clumps. This process is called 
aprjliitination, and when it occurs in blood the corpuscles settle down and can be 
filtered off. 

ExPEBiMENT 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 tabes, mix : 

A. 1 c.c. suspension and 0'5 o.c. 0'9% NaCl (control). 

B. Ice. „ 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 thermostable body is speciflc 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 

^ One intraperitoneal injection of 50 c.c. of rabbit's deflbrinated 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.o. 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 immnne body acts on the corpuscle as o, 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 u 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 obtaiu 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 erythrocytes 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 oentrifugaliae. 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 u, 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 auiount 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 u, 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 prec