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 QP44.P831903 Experiments for stud | 
 
 EXPERIMENTS 
 
 FOR STl'DEKTS IN THE 
 
 HARVARD MEDICAL SCHOOL 
 
 STfu'tti Etrition 
 
 By W. T. PORTER 
 
 :ap 
 
 COLUMBIA UNivPRPtTY 
 
 DEPARTMENT OF PHrSlfHOHY 
 
 College of Physicians and Surgeons 
 437 west fifty ninth stkeet 
 
 NEW YORK 
 
 THE UNIVERSITY PRESS 
 
 Camijrtfigr, fftass. 
 1903 
 
 
Columbia <18ntoer*ftp 
 
 College of $'op£trians anb iburgeons 
 Hibrarp 
 
Digitized by the Internet Archive 
 
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 Columbia University Libraries 
 
 http://www.archive.org/details/experimentsforstOOport 
 
EXPERIMENTS 
 
 FOR STCDEXTS IX THE 
 
 HARVARD MEDICAL SCHOOL 
 
NOTICE 
 
 The experiments performed by Harvard med- 
 ical students are contained in the following 
 publications : 
 
 1. An Introduction to Physiology, Parts I and 
 II, containing experiments in general physiology, in- 
 cluding muscle and nerve, and in the circulation of 
 the blood. 
 
 2. An Introduction to Physiology, Part IV, 
 Physiological Optics. 
 
 3. Experiments for Students in the Harvard 
 Medical School, Third Edition, containing experi- 
 ments upon the central nervous system, skin, general 
 sensations, taste, vision, fermentation, digestion, blood, 
 respiration, and metabolism. 
 
 Additional experiments will be added as rapidly as 
 possible. 
 
EXPERIMENTS 
 
 FOR STUDENTS IN THE 
 
 HARVARD MEDICAL SCHOOL 
 
 QftixXi lErjittcm 
 
 By W. T. PORTER 
 
 THE UNIVERSITY PRESS 
 
 Cambridge, fflass. 
 
 1903 
 
4\ 
 
 no3 
 
 Copyright, 1903 
 By W. T. Porter 
 
cry 
 
 CONTEXTS 
 
 The Central Nervous System 
 
 Simple Reflex Actions 1 
 
 The spinal cord a seat of simple reflexes — Influence of 
 afferent impulses on reflex action — Threshold \alue 
 lower in end organ than in nerve-trunk — Summation 
 of afferent impulses — SegnYental arrangement of rellex 
 apparatus — Reflexes in man. 
 
 Tendon Reflexes 6 
 
 Knee jerk — Ankle jerk — Gower's experiment. 
 Effect of Strychnine on Reflex Action ... 8 
 
 Complex Co-ordinated Reflexes 8 
 
 Removal of cerebral hemispheres — Posture, etc. — Bal- 
 ancing experiment — Retinal retire — Croak reflex. 
 
 Apparent Purpose; in Reflex Action 12 
 
 Reflex and Reaction Time 13 
 
 Reflex time — Reaction time — Reaction time with choice. 
 
 Inhibition of Reflexes 15 
 
 Through peripheral afferent nerves — Through central 
 afferent paths ; the optic lobes. 
 
 The Roots of Spinal Nerves 17 
 
 Lttdwig's demonstration — Localization of movements at 
 different levels of the spinal cord. 
 
VI CONTENTS 
 
 Distribution of Sensory Spinal Nerves .... 19 
 
 Muscular Tonus 20 
 
 Brondgeest's experiment. 
 
 II 
 
 The Skin 
 
 Sensations of Temperature . 21 
 
 Hot and cold spots — Outline — Mechanical stimulation 
 — Chemical stimulation — Electrical stimulation — Tern 
 perature after-sensation — Balance between loss and gaiii 
 of heat — Fatigue — Relation of stimulated area to sen- 
 sation — Perception of difference — Relatively insensitive 
 regions. 
 
 Sensations of Pressure 24 
 
 Pressure spots — Threshold value — Touch discrimina- 
 tion — Weber's law — After-sensation of pressure — 
 Temperature and pressure — Touch illusion ; Aristotle's 
 experiment. 
 
 Ill 
 
 General Sensations 
 
 Tickle 29 
 
 Irradiation — After image — Topography — Summation — 
 Fatigue. 
 
 Pain 30 
 
 Threshold value — Latent period — Summation — Topog- 
 raphy — Individual variation —Temperature stimuli. 
 
 Motor Sensations 31 
 
 Judgment of weight — Sensation of effort — Sensation of 
 motion. 
 
CONTEXTS VU 
 
 IV 
 
 Taste 
 
 Threshold value — Topography — Relation of taste to area 
 
 stimulated ■ — Electrical stimulation 32 
 
 Vision 
 
 Mapping the blind spot — Yellow spot — Field of vision. 
 Color Blindness 35 
 
 Method of examination and diagnosis. 
 
 VI 
 
 Fermentation 
 
 Specific Action 38 
 
 Conversion of starch to sugar by germinating barley — Con- 
 version of starch to sugar by salivary diastase (ptyalin) — 
 Extraction of diastase from germinating barley — Specific 
 action of ferments. 
 
 Proteid Digestion by Pepsin 41 
 
 Gastric digestion of cooked beef and bread — Artificial 
 gastric juice — Digestion with artificial gastric juice — 
 Extraction of pepsin — Change of proteid to peptone by 
 pepsin. 
 
 Splitting of Casein by Renntn 44 
 
 Rennin extract — Separation of rennin — Precipitation of 
 
 casein — Experiments of Arthus and Pages. 
 Precipitation of Fibrin by Fibrin Ferment ... 48 
 Buchanan's experiment — Extraction of fibrin ferment — 
 
 Extraction of fibrinogen — Precipitation of fibrinogen by 
 
 fibrin ferment. 
 
 Ammoniacal Fermentation of Urea by Urease . . 50 
 Extraction of urease. 
 
Vlll CONTENTS 
 
 Splitting and Synthesis of Fats 54 
 
 Chemistry of fats and soaps — Splitting of fats by the pan- 
 creatic juice — Preparation of neutral fat — The emulsion 
 test for fatty acid — Extraction of lipase — Hydrolysis 
 of ethyl butyrate by lipase — Syuthesis of neutral fat 
 by lipase. 
 
 Immunity 67 
 
 Ehrlich's ricin experiments — Ricin antitoxine — Theory 
 of immunity. 
 
 HAEMO LYTIC AND BACTERIOLYTIC FERMENTS .... 76 
 Bordet's experiments. 
 
 Oxidizing Ferments 79 
 
 Schonbein's experiment — Further oxidations by animal 
 tissues — Oxidation by nucleo-proteid — Oxidation about 
 the nucleus — Glycolysis in blood —Oxidation not de- 
 pendent on living cells of blood — Relation of glycolysis 
 to the pancreas and the lymph— Glycolytic ferment of 
 pancreas. 
 
 Alcoholic Fermentation 87 
 
 The yeast plant — Chemical relations of carbohydrates. 
 
 VII 
 
 Blood 
 
 Specific Gravity . 94 
 
 Drawing the blood — Determination of specific gravity- 
 Counting the red corpuscles — Counting the white cor- 
 puscles. 
 
 Estimation of Haemoglobin 100 
 
 Hoppe-Seyler's method (modified). 
 
 Haemorrhage and Regeneration ....... 101 
 
 Alkalinity 102 
 
 Zuntz-Loewy-Engel method. 
 
 Coagulation Time 104 
 
CONTENTS IX 
 
 VIII 
 
 Respiration 
 
 Chemistry of Respiration 104 
 
 Estimation of oxygen, carbon dioxide, and water. 
 
 Mechanics of Respiration 106 
 
 Artificial scheme — Inspiration — Expiration — Normal 
 respiration — Forced respiration — Obstructed air pas- 
 sages — Asphyxia — Coughing : sneezing — Hiccough — 
 Perforation of the pleura. 
 
EXPERIMENTS 
 
 FOR STUDENTS IX THE 
 
 HARVARD MEDICAL SCHOOL 
 
 I 
 THE CENTRAL NERVOUS SYSTEM 
 
 Simple Reflex Actions 
 
 The Spinal Cord a Seat of Simple Reflexes. — 
 1. By means of a hook or thread passed through 
 the lower jaw suspend vertically a frog the brain 
 of which has been destroyed with the seeker ; the 
 legs must not touch the table. Pinch a toe with 
 the forceps. 
 
 The leg will be drawn up. 
 
 A stimulus to the skin has caused the con- 
 traction of muscles. The afferent impulse set 
 going by the sensory stimulus is changed into 
 a motor efferent impulse. This is an example of 
 reflex action. 
 
 2. Destroy the spinal cord with the seeker. 
 Stimulate the skin of the right leg electrically 
 and mechanically. 
 
2 THE CENTRAL NERVOUS SYSTEM 
 
 In no case will the sensory stimulus call forth 
 the reflex contraction of a skeletal muscle. Yet 
 the nerves coming from the skin and going 
 to the muscles are still intact. Only the spinal 
 cord has been destroyed. 
 
 The conversion of sensory into motor impulses 
 for skeletal muscles is a function of the central 
 nervous system. 
 
 Influence of Afferent Impulses on Reflex Action. — 
 Destroy the brain of a strong frog with the seeker. 
 Gently pinch a toe of the right foot. 
 
 Only the right leg will be drawn up. 
 
 Pinch a toe of the left foot. 
 
 Only the left foot will be drawn up. 
 
 Pinch a finger. 
 
 Only the corresponding arm will move. 
 
 Pinch the whole foot sharply. 
 
 More extended movements will be made. 
 
 The character and location of the stimulus 
 affect the resulting contraction. 
 
 Threshold Value Lower in End Organ than in 
 Nerve-Trunk. — 1. Carefully expose the sciatic 
 nerve. Determine the least strength of tetanizing 
 current that will cause a crossed reflex when 
 applied to the skin of the foot. Now apply the 
 same stimulus to the trunk of the nerve. 
 
 As a rule, the intensity required to produce 
 reflex action is less when the stimulus is applied 
 
SIMPLE KEFLEX ACTIONS 6 
 
 to the peripheral endings of the sensory nerves 
 than when the nerve-trunks are stimulated. 
 
 2. Divide the skin over the back in the median 
 line. Raise the skin on one side until the small 
 nerves which pass across the dorsal lymph sac to 
 innervate the skin come into view. Sever from 
 the surrounding skin a piece about one centi- 
 metre square containing the endings of one of 
 the nerves. Let the isolated piece with its nerve 
 endings remain connected with the body only by 
 the trunk of the nerve. As before, determine 
 the least strength of tetanizing current that will 
 cause a reflex movement when applied to the 
 nerve-endings in the skin and to the nerve-trunk 
 respectively. * 
 
 The threshold value for reflex action will again 
 be found lower in the nerve-endings than in the 
 nerve-trunk. 
 
 Summation of Afferent Impulses. — Pass two 
 fine copper wires about the -frog's foot a centi- 
 metre apart and connect them with the secondary 
 coil. Connect the primary coil through a simple 
 key with a dry cell. Stimulate with regularly 
 repeated make induction currents of such strength 
 that single stimuli cause no reflex contraction. 
 
 Summation of the subminimal stimuli will 
 finally cause reflex contraction. 
 
 Determine that the number of stimuli neces- 
 
4 THE CENTRAL NERVOUS SYSTEM 
 
 saryto produce a reflex becomes smaller when (1) 
 the strength of the induction currents is increased, 
 and (2) when the interval between the stimuli is 
 lessened. 
 
 Segmental Arrangement of Reflex Apparatus. — 
 1. Gently pass the seeker over the abdominal 
 walls on one side. 
 
 The muscles in that region only will twitch. 
 
 Eepeat the stimulus, but use a stronger 
 pressure. 
 
 The area contracting will increase in extent 
 approximately in proportion to the increase in 
 the stimulus. The afferent nerves from any one 
 region are more closely related to the efferent 
 nerves of that same region than to those of other 
 regions. The fact that both afferent and efferent 
 fibres spring from the cord at the same level 
 suggests that their nerve C3lls lie also at approxi- 
 mately the same level. On increasing the stim- 
 ulus the afferent impulse spreads from segment 
 to segment of the cord. Further evidence of the 
 segmental arrangement will be gained by the 
 following experiment. 
 
 2. With a clean, sharp knife make transverse 
 sections of the spinal cord, beginning in the cer- 
 vical region. A short time after each section 
 test the reflexes from the hind limb by mechani- 
 cal stimulation. 
 
SIMPLE REFLEX ACTIONS 5 
 
 Note the level below which no section can be 
 made without rendering the reflex impossible. 
 The nerve cells concerned in this reflex lie on 
 the caudal side of this line. 
 
 Now in a second frog make transverse sections, 
 beginning at the caudal end of the cord, and test 
 the reflexes as before, until the level is reached 
 beyond which a section will destroy the reflex. 
 
 Observe that the portion of the cord comprised 
 between the two levels determined forms a seg- 
 ment which contains the central apparatus con- 
 cerned in the reflex studied. 
 
 Reflexes in Man. — 1. From the Skin. — Eub 
 the plantar surface of the foot gently with some 
 hard object. 
 
 The foot will be retracted reflexly. 
 
 Similar results may be obtained by rubbing 
 the skin of the inside of the thigh, which will 
 cause contraction of the cremaster muscles ; or by 
 rubbing the skin of the abdomen, which will be 
 followed by contraction of the abdominal muscles. 
 
 These reflexes are of importance in clinical 
 diagnosis because by means of them the seat of a 
 diseased area in the central nervous system may 
 sometimes be defined, since the reflex depends on 
 the integrity of the corresponding reflex arc. 
 
 2. Cornea Reflex. — Touch the cornea gently 
 with a thread. 
 
6 THE CENTRAL NERVOUS SYSTEM 
 
 The eye will be closed involuntarily. 
 
 3. Throat Reflex. — Touch the posterior wall 
 of the throat. 
 
 The movements of swallowing will usually 
 follow. 
 
 4. Pupil Reflexes ; Light Reflex. — Close one 
 eye for several seconds, then open it quickly. 
 
 Note the contraction of the pupil. 
 
 5. Consensual Reflex. — Close one eye as before, 
 but watch the pupil of the other eye when the 
 first is opened again. 
 
 The pupil will contract. 
 
 6. Accommodation Reflexes. — Look alternately 
 at a near and a far object. The pupil will con- 
 tract when the eye adjusts itself to see the near 
 object. 
 
 Tendon Eeflexes 
 
 Knee Jerk. — Sit in such a position that the 
 knee is bent at a right angle, and the foot hangs 
 free. Let an assistant strike the patellar liga- 
 ment with the side of the hand. 
 
 Note the sudden contraction of the extensors 
 of the thigh, the so-called knee jerk. 
 
 Flex the knee at different angles and deter- 
 mine in which position the resulting contraction 
 is greatest. 
 
 Knee jerk can be obtained only within certain 
 limits of extension. 
 
TENDON REFLEXES 7 
 
 Let the subject immediately before the stimu- 
 lus is applied forcibly contract some other group 
 of muscles ; clench the hand, for example. 
 
 The knee jerk is reinforced. 
 
 Ankle Jerk. — Bend the foot at ri<?ht angles to 
 the leg, and strike the tendo Achillis. The ex- 
 perimenter should hold the end of the foot in his 
 left hand.' 
 
 Contraction of the gastrocnemius muscle will 
 be observed. 
 
 Gower's Experiment. — Strike the side of the 
 tendo Achillis. 
 
 A contraction will result. 
 
 Support the other side of the tendon so that 
 the gastrocnemius muscle will not be stretched 
 by the blow. Eepeat the experiment. 
 
 No contraction follows. The tendon jerk re- 
 quires for its production a rapid stretching of the 
 muscles involved in the contraction. 
 
 Try to obtain tendon jerks from other muscles ; 
 for example, the triceps humeri, flexors of hand, 
 and masseter muscles. 
 
 Normally no response will be obtained. 
 
 The experiments are of value in diagnosis of 
 diseases of the central nervous system. 
 
8 the central nervous system 
 
 Effect of Strychnine on Reflex Action 
 
 Inject with a glass pipette a- few drops of 0.5 
 per cent solution of sulphate of strychnine into 
 the dorsal lymph sac of a frog the brain of which 
 has been destroyed with a seeker. 
 
 After a few minutes, very weak afferent 
 impulses will be sufficient to call forth general 
 spasmodic reflex actions. Note that (1) the 
 strychnine reflexes are paroxysmal, (2) the mus- 
 cles fall into more or less prolonged rigidity (teta- 
 nus), and (3) the extensors overcome the flexors, 
 the limbs being strongly extended. 
 
 The characteristic action of strychnine is evi- 
 dently not dependent on the brain. 
 
 Destroy the spinal cord with a seeker. 
 
 Stimulation of muscles and nerves will not 
 cause spasmodic contractions. 
 
 Strychnine acts on the spinal cord, but not on 
 the muscles or the peripheral nerves. 
 
 Complex Co-ordinated Eeflexes 
 
 Removal of Cerebral Hemispheres. — Place a 
 frog under a glass jar containing a small sponge 
 wet with ether. Be very careful not to kill the 
 frog. When insensibility is complete, place the 
 animal on a frog-board. Cut through the skin in 
 the median line of the skull, from the nose to the 
 
COMPLEX CO-ORDINATED REFLEXES 9 
 
 vertebral column. Connect the front margins of 
 the two tympanic membranes by a transverse in- 
 cision through the skin. This transverse line 
 will pass over the junction of the cerebral lobes 
 with the optic lobes. Strip off the parietal bones 
 with forceps, beginning at the anterior end oppo- 
 site the anterior margin of the orbit. When the 
 cerebral hemispheres are uncovered, they may be 
 removed from before backwards. Avoid injuring 
 the optic lobes. Work rapidly but carefully. If 
 the ether effect diminish before the operation be 
 finished, replace the frog under the glass jar for 
 a few moments. As soon as the hemispheres are- 
 removed, sew up the wounds in the skin. 
 
 Note the signs of profound inhibition. 
 
 If the operation be done carefully, the shock 
 will gradually pass away, and the functions possi- 
 ble in the absence of the cerebrum may then be 
 determined. Put the frog aside, moistening his 
 skin occasionally, but not otherwise disturbing 
 him, and prepare a second frog for the experi- 
 ment upon the "croak reflex" (page 10). When 
 this operation is completed, resume the observa- 
 tions on the first frog, while the second frog re- 
 covers from the shock. 
 
 1. Posture, etc. — Write down the differences 
 between the frog from which only the cerebral 
 hemispheres have been removed and a frog in 
 
10 THE CENTRAL NERVOUS SYSTEM 
 
 which the whole brain has been destroyed with 
 the seeker, in respect to posture, power to regain 
 feet when laid on back, respiratory movements, 
 position of eyelids, leaping and swimming. 
 
 2. Balancing Experiment. — Place the frog on a 
 somewhat roughened board, about 20 inches long, 
 8 inches wide, and 1 inch thick. Tilt the 
 board gradually. 
 
 The frog remains motionless until his centre 
 of gravity is disturbed. He then moves forward 
 in an attempt to reach a stable position. By 
 careful management, he can be made to climb up 
 the inclined board, perch upon the narrow edge, 
 and, the board still turning, descend head-first on 
 the opposite side. 
 
 3. Retinal Reflex. — Place the frog deprived of 
 cerebral hemispheres in front of a bright light; 
 for example, an incandescent electric lamp. In- 
 terpose some object, such as a small instrument 
 case, between the light and the frog, so that a 
 strong shadow is cast upon the frog's eyes. 
 Stimulate the frog by pinching the skin of the 
 back. 
 
 The frog will jump, but will avoid the object 
 which casts the shadow. 
 
 4. Croak Reflex. — Sever the large hemispheres 
 from the remainder of the brain of another froif 
 
 o 
 
 by passing a knife through the cranium to the 
 
COMPLEX CO-ORDINATED REFLEXES 11 
 
 base of the skull from side to side in a line join- 
 ing the anterior margins of the tympanic mem- 
 brane?. (Where possible, a male frog should be 
 selected for this experiment. Males may be rec- 
 ognized by the cushion-like thickening on the 
 innermost digit of the manus, or hand ; the male 
 Eana esculenta possesses bladder-like, resonating 
 pouches connected on each side with the mouth 
 cavity.) After the immediate shock of the opera- 
 tion has passed, stroke the back over the anterior 
 half of the spinal cord. 
 
 Beflex croaking will be observed. 
 
 The croak reflex can be inhibited by simultane- 
 ous pinching of one of the limbs or other strong 
 stimulation. (Compare*page 15.) 
 
 If the experiments on the frog in which the 
 cerebral hemispheres were extirpated were not 
 satisfactory, repeat them on this frog in which 
 the hemispheres were simply separated from the 
 remainder of the brain. 
 
 These observations teach that very complicated 
 co-ordinated actions are possible in the absence 
 of the large hemispheres. Only simple reflexes 
 are possible when the whole brain is removed. 
 Consequently, the seat of these complicated re- 
 flexes mast lie in the brain between the cord and 
 the cerebral hemispheres. 
 
12 the central nervous system 
 
 Apparent Purpose in Reflex Action 
 
 1. Destroy the brain of a frog with the seeker. 
 Dip small pieces of filter paper in strong acetic 
 acid. Remove the superfluous acid, lay the 
 paper bearing the acid on (1) the frog's thigh, 
 (2) the foot, (3) the back. After each stimulation 
 note the character of the reflex movement, and 
 then carefully wash the acid from the skin. 
 
 The movements are related to the areas stimu- 
 lated in a certain purposeful way. Efforts are 
 made apparently to brush away the acid paper. 
 
 2. Place the acid on the flank of the right leg. 
 Usually the leg stimulated strives to brush away 
 the paper. Hold this leg fast. 
 
 The other leg (the left) will be used to re- 
 move the acid from the opposite limb. (This 
 experiment succeeds best in strong, lively frogs.) 
 
 3. Place an uninjured frog in an evaporating 
 basin containing sufficient water to immerse the 
 frog to the neck and covered with wire gauze 
 to keep him from jumping out. Warm the 
 water. 
 
 As the temperature rises to from 20°-30° C. 
 the frog will attempt to escape. 
 
 Repeat the experiment with the frog the brain 
 of which has been destroyed. 
 
 No movements of escane will be noticed. 
 
KEFLEX AND REACTION TIME 13 
 
 About 35°, muscular twitchings will be seen. 
 At 38°-40° death takes place and the muscles 
 become rigid (heat rigor). 
 
 This observation shows that volition in all 
 probability is absent in the brainless frog. It 
 follows that reflex actions are not volitional; 
 their " purpose " is only apparent. 
 
 Keflex and Eeaction Time 
 
 Reflex Time. — Destroy the brain of a frog with 
 the seeker. Hold one leg of the frog aside with 
 the glass rod. Bring beneath the other a small 
 beaker almost full of dilute sulphuric acid 
 (2:1000). Raise the beaker until the foot is 
 immersed to the ankle.* Count the seconds be- 
 tween the application of the stimulus (sulphuric 
 acid) and the- withdrawal of the foot. 
 
 This interval is the reflex time. 
 ■ Wash the foot carefully in the bowl of water. 
 
 Reaction Time. — Smoke a drum. Raise the 
 drum off its friction bearing by turning the screw 
 at the top of the, shaft. Place the writing point 
 of an electromagnetic signal against the smoked 
 paper. Arrange a tuning fork to write its curve 
 near that of the signal. Connect the signal 
 through two simple keys and a dry cell with the 
 primary coil of an inductorium arranged for 
 maximal single induction currents (posts 1 and 
 
14 THE CENTRAL NERVOUS SYSTEM 
 
 2). Let stimulating electrodes pass from the 
 secondary coil (bridge up) to the tongue of the 
 subject. Let the subject hold one key closed un- 
 til he feels the stimulus on the tongue. 
 
 Direct the subject to shut his eyes. Let the ob- 
 server start the tuning fork, spin the drum, and 
 stimulate the subject by completing the primary 
 circuit. The instant the subject perceives the 
 stimulus, he will break the circuit by releasing 
 his key. By means of the tuning fork curve 
 determine the interval between stimulation and 
 response. This interval is the reaction time plus 
 the errors of observation ; for example, the latent 
 period of the electromagnetic signal. Eepeat the 
 experiment three times and take the mean of the 
 results. 
 
 In the laboratory note-book make a list of the 
 links in the chain between stimulus and re- 
 sponse, and state as far as possible the errors of 
 observation. 
 
 Reaction Time with Choice. — Connect the 
 side cups of a pole-changer (without cross wires) 
 to the posts of the secondary coil. Connect one 
 pair of end cups with the usual stimulating elec- 
 trodes, the other pair with large brass electrodes 
 covered with wet cotton. Let the ordinary elec- 
 trodes touch the forehead, the other pair the hand 
 of the subject. The other connections should re- 
 
INHIBITION OF REFLEXES 15 
 
 main as before. Eepeat the preceding experi- 
 ment but tell the subject to signal only when 
 the tongue (or hand) is stimulated. In order to 
 do this he must add to his former reaction a de- 
 cision as to the part stimulated. 
 
 Eeaction time with choice is longer than sim- 
 ple reaction time. In general, the more compli- 
 cated the mental processes involved, the longer 
 will be the reaction time. 
 
 Inhibition of Eeflexes 
 
 Through Peripheral Afferent Nerves. — Expose 
 the left sciatic nerve for a distance of about 15 
 mm. in a frog the brain of which has been de- 
 stroyed. Tie a thread around the distal end, and 
 sever the nerve at the peripheral side of the liga- 
 ture. Place the central stump of the nerve on 
 the electrodes of the inductorium, the short-cir- 
 cuiting key being closed. Make the primary 
 circuit, and set the hammer vibrating. Xow open 
 the short-circuiting key,, bring the right foot of 
 the frog into the dilute sulphuric acid up to the 
 ankle, and count the seconds from the moment 
 of immersion to the moment of withdrawal, con- 
 tinuing meanwhile the stimulation of the central 
 end of the left sciatic nerve. 
 
 The latent period will be much prolonged. 
 
 Wash off the acid carefully. 
 
16 THE CENTRAL NERVOUS SYSTEM 
 
 Keflex actions may be inhibited by me simul- 
 taneous stimulation of sensory nerves. 
 
 Through Central Afferent Paths ; the Optic 
 Lobes. — 1. Expose the brain according to the 
 directions already given (page 8). Immediately 
 posterior to the cerebral hemispheres lie the optic 
 lobes, two gray spherical bodies. Separate the 
 cerebral hemispheres from the optic lobes by a 
 transverse incision, and carefully remove the 
 hemispheres. Wait until the shock of the opera- 
 tion has passed. Now suspend the frog so that 
 the tips of the toes hang above a shallow dish 
 containing water made stron^lv sour to the taste 
 with dilute sulphuric acid. Determine the reflex 
 time. \Vash off the acid and, after a moment's 
 rest, sprinkle a very little finely powdered com- 
 mon salt on the cut surface of the optic lobes. 
 Again determine the reflex time. 
 
 The reflex time will be found to be markedly 
 increased by the stimulation of the optic lobes. 
 
 2. Prepare a second frog in the same manner. 
 Determine the reflex time. Now instead of stim- 
 ulating the optic lobes, remove them, and again 
 determine the reflex time. 
 
 The removal of the optic lobes shortens the 
 reflex time. 
 
the roots of spinal nerves 17 
 
 The Eoots of Spinal Nerves 
 Destroy the brain of a strong, large frog with 
 a seeker. Divide the skin over the vertebral 
 column from the upper end of the urostyle to 
 the level of the fore limbs. Hook back the 
 flaps of skin. Remove the longitudinal muscles 
 on either side of the spines of the vertebrae, thus 
 exposing the bony arches. Saw through the 
 arches of the 8th, 7th, and 6th vertebra? (there 
 are ten vertebrae in the frog, counting the uro- 
 style) in the order named. Clear away the bone, 
 and the underlying tissues until the last three or 
 four pairs of roots shall be plainly seen. Grasp 
 the filum terminale and cautiously lift the cord 
 until the spinal nerve roots are clearly displayed. 
 The anterior roots are hidden by the large, 
 superficial posterior roots. The conspicuous pos- 
 terior root which seems to be the last is, in real- 
 ity, the 9th, the next to the last ; the last, or 
 10th, is smaller and lies close to the filum termi- 
 nale. Place a silk ligature about the middle 
 of an anterior and a posterior root on the right 
 side. With single induction currents as stimuli 
 observe that (1) the stimulation of only the cen- 
 tral end of the posterior root calls forth a (re- 
 flex) movement, and (2) the stimulation of only 
 the peripheral segment of the anterior root causes 
 movement. o 
 
18 THE CENTRAL -NERVOUS SYSTEM 
 
 On this same side cut all the posterior roots. 
 
 No stimulus applied to the right leg will now 
 discharge a reflex action. But stimuli applied to 
 sensory nerves elsewhere may still cause reflex 
 movements of the right leg. Motor impulses still 
 pass to these muscles. But only the interior 
 roots remain. . 
 
 Hence the anterior roots of spinal nerves trans- 
 mit motor impulses from the spinal cord towards 
 the muscles (efferent impulses) ; the posterior 
 roots transmit sensory impulses from sensory sur- 
 faces towards the spinal cord (afferent impulses.) 
 
 Imdwig's Demonstration. — Destroy the brain 
 of a large frog with the seeker. Kemove the 
 thoracic and abdominal viscera, taking care not 
 to injure the sciatic nerve plexus. Remove the 
 7th and 8th vertebrae, taking the greatest pains 
 not to injure the nerve roots. Divide the body 
 transversely at this level, so that the anterior 
 and posterior halves shall remain connected only 
 by the anterior and posterior sciatic roots. Keep 
 the roots moist with normal saline solution. 
 
 Demonstrate again that the anterior roots 
 transmit efferent, and the posterior roots afferent 
 impulses. 
 
 Localization of Movements at Different Levels of 
 the Spinal Cord. — Separate the three roots which 
 form the sciatic nerve. After tying a thread 
 
DISTRIBUTION OF SENSORY SPINAL NERVES 19 
 
 about each root sever it from the spinal cord by 
 a cut on the proximal side of the thread. Stimu- 
 late each nerve with a very weak tetanizing cur- 
 rent. Note the different results, obtained from 
 nerves arising at different levels of the cord. 
 Stimulation of the most anterior root causes 
 marked flexion of the limb ; stimulation of the 
 middle roots, extension and internal rotation ; 
 and of the most posterior, simple extension. 
 
 In a frog whose nerves have not been cut 
 expose the spinal cord and stimulate it at differ- 
 ent levels in both directions along its length. 
 The various movements of the hind limbs are 
 localized at different levels of the cord. 
 
 Distribution of Sensory Spinal Nerves 
 
 Destroy the brain of a large frog with the 
 seeker. Expose the lower half of the spinal cord 
 by the method already described. On one side 
 cut the dorsal sensory root of the 8th spinal nerve 
 and on the other cut the sensory root of the 7th, 
 9th, and 10th. After the section of each root 
 test the cutaneous sensibility of the limbs by 
 placing upon the skin small pieces of filter paper 
 (two mm. square) moistened, not dripping, with 
 0.2 per cent sulphuric acid. Make a map of the 
 anaesthetic areas in each leg, and note the lack 
 of correspondence. 
 
20 THE CENTRAL NERVOUS SYSTEM 
 
 Many skin areas are supplied by fibres from at 
 least two sensory roots. The fields of distribution 
 overlap. 
 
 Muscular Tonus 
 
 Brondgeest's Experiment. — Fasten a lightly 
 etherized frog back uppermost on the frog-board. 
 
 In a line between the ilium and the coccyx 
 open the pelvic cavity by cautiously dividing the 
 skin, fascia, and muscle. Divide the sciatic nerve 
 roots on the operated side. Pass a hook or thread 
 through the jaw and hang the frog up. 
 
 Observe that the limb the nerves of which 
 have been cut is relaxed, so that the toes hang 
 lower than those of the limb which still retains 
 its connection with the central nervous system. 
 
 Apparatus 
 
 Normal saline. Bowl. Towel. Pipette. Stand. 
 Muscle clamp. Bent hook. Inductorium. Dry cell. 
 Electrodes. Large brass electrodes. Cotton. Key. 
 Frog-board. Fine copper wire. One-half per cent solu- 
 tion of strychnine sulphate. Glass jar with ether and 
 sponge. Balancing board. Strong acetic acid. Filter 
 paper. Evaporating basin. Wire gauze. Bunsen burner. 
 Thermometer. Dilute sulphuric acid (0.2 per cent). 
 Beaker. Kymograph. Electromagnetic signal. Tuning 
 fork. Pole-changer. Vertebral saw. 
 
II 
 
 THE SKIN 
 Sensations of Temperature 
 
 Hot and Cold Spots. — With a lead-pencil point 
 carefully explore an area about an inch square 
 on the back of the wrist or hand. Mark with 
 black ink the places where a distinct sensation of 
 cold is felt, and with red ink those where the 
 -sensation is one of warmth. 
 
 The places indicated are the so-called hot and 
 cold spots. 
 
 Outline. — Attempt to define more exactly the 
 outline of one of the cold spots. 
 
 The spots are of irregular shape, — blotches 
 rather than points. 
 
 Mechanical Stimulation. — 1. Gently tap one 
 end of a small wooden rod the other end of which 
 is placed on a well-defined cold spot. 
 
 The mechanical stimulation of the cold spot 
 will give a sensation of cold. 
 
 2. Stimulate a warm spot mechanically. 
 
 Chemical Stimulation. — Pi lib a menthol pencil 
 over a small area on the back of the hand. 
 
22 THE SKIN 
 
 A sensation of cold will be perceived. This is 
 due to chemical irritation of the cold spots. The 
 temperature of the area does not fall. 
 
 Electrical Stimulation. — It has been found that 
 the stimulation of a well-defined cold or warm 
 spot with moderately strong induced currents 
 causes a sensation of cold or warmth respectively. 
 
 Temperature After-Sensation. — Stimulate a cold 
 spot mechanically with a pencil point. Remove 
 the point. 
 
 The sensation of cold outlasts the stimulus. 
 
 Balance between Loss and Gain of Heat. — Pro- 
 vide three beakers of water. Heat them to 20°, 
 30°, and 40° C, respectively. Place a finger of 
 one hand in the water at 20°, and a ringer of the 
 other hand in the water at 40°. After the re- 
 spective sensations of cold and warmth have 
 disappeared, place both the fingers in the water 
 at 30°. 
 
 The finger from the cold water will seem warm 
 and that from the warm water cold. The tem- 
 perature of the skin equals the balance between 
 its heat loss and heat gain. When this tempera- 
 ture is raised or lowered, the warm spots or cold 
 spots respectively are stimulated. 
 
 Fatigue. — Provide three beakers containing 
 water at 10°, 32°, and 45° C. respectively. Place 
 a finger of one hand in the beaker at 32°, and a 
 
SENSATIONS OF TEMPERATURE 23 
 
 finder of the other hand in the beaker at 45°. 
 After .45 seconds place both fingers in the water 
 at 10°. 
 
 The finger taken from the water at 32° (which 
 is about the normal temperature of the hand) will 
 feel colder than the other finger. Extreme tem- 
 peratures of heat or cold fatigue the temperature 
 spots. 
 
 Relation of Stimulated Area to Sensation. — In- 
 sert a finger of one hand in a beaker of warm or 
 cold water. Note the sensation. Insert a finger 
 of the other hand in the water. 
 
 The intensity of the sensation will increase 
 with the extent of the surface stimulated. 
 
 Perception of Difference. — Provide two beakers 
 of water, one at 30°, the other slightly warmer or 
 colder. By introducing a finger first into the one 
 and then into the other, and varying the tem- 
 perature of the water, ascertain how small a 
 difference in temperature can be detected. 
 
 Usually a difference of 0.5° C. is easily recog- 
 nized. 
 
 Relatively Insensitive Regions. — 1. Compare 
 the temperature sensation perceived on touching 
 with a pencil point the median line of the fore- 
 head, nose, and chin with that perceived on 
 touching the skin on either side of the median 
 line. 
 
24 THE SKIN 
 
 The skin in the median line of. the body is 
 comparatively insensitive to temperature varia- 
 tions. 
 
 2. Similarly compare the mucous membrane 
 with the skin. 
 
 The mucous membranes are much less sensitive 
 than the skin. 
 
 Sensations of Pressure 
 
 Pressure Spots. — Explore the surface of the 
 forearm by bringing the blunted point of a needle 
 gently in touch with the skin. 
 
 At certain spots a distinct sensation of contact 
 will be perceived. Other spots will give only 
 dull sensations. Pressure, like heat and cold, is 
 appreciated by scattered sense-organs in the skin, 
 not by diffuse general sensation. 
 . Note the relation of the pressure points (1) to 
 the hair follicles, and (2) to the warm and cold 
 spots mapped out in previous experiments. 
 
 Threshold Value. — Take from the human head 
 several straight, strong hairs. Cement each to 
 the end of a little stick of soft pine to serve as 
 a handle. Provide a special lever, made as fol- 
 lows : With a hot pin burn a small hole at the 
 middle of a straw about 25 cm. in length. Pass a 
 needle through this hole into a cork held in the 
 muscle clamp. Press the free end of the hairs 
 
SENSATIONS OF PRESSURE 25 
 
 against different parts of the skin of the hand. 
 arm, and face. Select hairs which when pressed 
 against the skin of the respective regions give no 
 sensation of pressure. Shorten the hairs until 
 the pressure is just perceptible. This will be the 
 "pressure threshold." Make a loop in a short 
 silk thread and pass the loop about the lever 
 exactly one millimetre from the axis. Hang on 
 the end of the thread a light bent hook. Coun- 
 terpoise the lever very exactly, so that . the 
 slightest force applied to the end of the straw 
 will raise the lever from the after-loading screw. 
 By counterpoising in this way, the lever becomes 
 a balance. On the bent hook hang a ring of 
 German silver wire weighing one decigram (0.1 
 gram). Find a point on the lever 100 mm. from 
 the axis. The weight of one decigram suspended 
 1 mm. from the axis of the lever will be raised 
 by a force of t ^q of a decigram, equal to one 
 milligram (0.001 gram) applied 100 mm. from 
 the axis. At 50 mm. from the axis, 0.1 gram, 
 suspended 1 mm. from the axis, will be lifted by 
 a force of ^J n gram (0.002 gram). Find the dis- 
 tance from the axis at which each testing-hair, 
 when pressed vertically against the lever, will 
 just fail to lift the lever; in other words, the 
 point at which the pressure will be just sufficient 
 to bend the hair. The number of millimetres 
 
26 THE SKIN 
 
 between this point and the axis of the lever, 
 multiplied by one-tenth, will give the bending 
 pressure of the hair in the fraction of a grain. 
 Make ten observations on each hair and mark the 
 mean bending value on the wooden handle. 
 
 Touch Discrimination. — 1. Close the eyes and 
 let an assistant test the different parts of the 
 skin of the hand, arm, and face for discrimina- 
 ting power. For each test separate the points of 
 the aesthesiometer until they can be felt as two 
 (ordinary drawing dividers or compasses can be 
 used for an aesthesiometer). 
 
 Eecord your results in millimetres for finger- 
 tips, palm of hand, back of fingers, back of hand, 
 back of wrist, flexor and extensor surfaces of fore- 
 arm, forehead, cheeks, lips, and tongue. 
 
 2. Separate the points of the aesthesiometer 
 about 20 mm., and draw them gently side by 
 side along the extensor surface of the forearm 
 from the elbow to the wrist. Eepeat the experi- 
 ment on the flexor surface. Try the same for 
 the cheek and lips, beginning near the ear and 
 drawing the points so that one shall go above 
 and the other below the mouth. 
 
 Describe the sensation in each case, and sug- 
 gest an explanation. 
 
 Weber's Law. — Place the hand palm upward 
 in a comfortable position on the table. Close 
 
SENSATIONS OF PRESSURE 27 
 
 the eyes. Let an assistant place on the last 
 phalanx of the middle and index fingers a small 
 round box containing ten small shot. 
 
 When the subject has formed a clear percep- 
 tion of the weight, let an assistant add or 
 subtract shot, and record the number of shot cor- 
 responding . to the smallest difference in weight 
 perceived by the subject (whose eyes of course 
 should be kept closed). Repeat the experiment 
 with 20, 30, 40, and 50 shot in the box respec- 
 tively. Determine in each instance the ratio of 
 the number of shot added or subtracted to the 
 number with which each experiment was begun. 
 
 This ratio will be approximately constant. 
 The degree of stimulation^necessary to cause the 
 perception of difference always bears the same 
 ratio to the degree of stimulation already applied. 
 Weber's law is less true for very small and very 
 large weights than for those of medium value. 
 It is a general law and holds good for visual 
 judgments, etc. 
 
 After-Sensation of Pressure. — Place a rubber 
 band about the head and allow it to remain for 
 several minutes. 
 
 On removing the band, a distinct after-sensa- 
 tion of pressure will be felt. 
 
 Temperature and Pressure. — Place on the back 
 of the hand supported on the table a coin the 
 
28 THE SKIN" 
 
 temperature of which has been made such that 
 it feels neither warm nor cold. Compare the 
 pressure sensation (apparent weight) of this "nor- 
 mal" coin with that of similar coins warmed 
 and cooled. 
 
 The hot or cold coin will seem heavier than 
 the " normal " coin of equal weight. 
 
 Touch Illusion ; Aristotle's Experiment. — Cross 
 the right middle ringer over the right index finger 
 and place them on the palm of the left hand. 
 Place a small shot between the crossed ringers in 
 such a way that it shall touch the ulnar side of 
 the middle finger and the radial side of the index 
 finger. Eoll the shot in the palm of the hand. 
 
 A sensation of two objects will be felt. 
 
 Apparatus 
 
 Black and red ink. Small wooden rod. Menthol pen- 
 cil. Inductorium. Dry cell. Electrodes. Key. Three 
 beakers. Stand. Ring. Wire gauze. Bunsen burner. 
 Thermometer. Needle with blunted point. Muscle lever. 
 Gram and ten-gram weights. German silver ring weigh- 
 ing 0.1 gram. Silk thread. Four small wooden handles 
 for pressure-hairs. Bent hook. Drawing dividers (as 
 sesthesiometer). Small round box containing at least 50 
 shot. Rubber band larce enough to go around the head. 
 
TICKLE 29 
 
 III* 
 
 GENERAL SENSATIONS 
 
 Tickle 
 
 Irradiation. — Gently touch the skin near one 
 nostril with a dry camel' s-hair brush. 
 
 Note (1) the strong sensation produced by 
 the slight stimulus ; (2) the irradiation beyond 
 the spot stimulated. 
 
 After image. — Repeat the stimulus of the pre- 
 ceding experiment. ■ 
 
 Measure in seconds the time during which 
 the sensation outlasts the stimulus (after image). 
 
 Topography. — Test the tickle sensation at vari- 
 ous points on the skin etf the face, hands, and 
 forearms. Determine whether the sensation is 
 greatest about the several openings, where skin 
 joins mucous or serous membranes ; e. g., the 
 nostrils, the conjunctival sac, the auditory canal. 
 Do the results indicate a protective mechanism ? 
 
 Summation. — In one of the sensitive areas 
 found in the preceding experiment determine the 
 difference between the response to a single stim- 
 ulus and to successive stimuli. 
 
 Fatigue. — In any sensitive area determine (1) 
 the quickness with which the apparatus for the 
 sensation of tickle is fatigued ; (2) the duration 
 of fatigue. 
 
30 GENERAL SENSATIONS 
 
 Pain 
 
 Threshold Value. — Arrange an inductorium for 
 tetanizin" currents. Place the electrodes on 
 the tip of the tongue, and move the secondary 
 toward the primary coil until no farther move- 
 ment can be made without causing the stimula- 
 tion to become painful. Determine for this 
 region and for others of the mucous membrane 
 of the mouth and of the skin what distance of 
 the secondary coil from the primary separates the 
 stimulus at which pain is just perceived from 
 that at which the pain is distinct. 
 
 Latent Period. — In several individuals measure 
 approximately the interval between the applica- 
 tion of the stimulus (single break shock) and the 
 resulting painful sensation. 
 
 Summation. — Determine the number of sub- 
 minimal stimuli necessary to produce pain. 
 
 Topography. — Map upon the skin of the face 
 and arm the areas specially sensitive to pain. 
 
 Individual Variation. — Compare the reactions 
 of several individuals, and note the differences in 
 threshold value, latent period, summation, and 
 topography. 
 
 Temperature Stimuli. — Fill two bowls or large 
 beakers with water twenty-five degrees respec- 
 tively, hotter and colder, than the temperature 
 
MOTOR SENSATIONS 31 
 
 of the hand. Determine whether the increase 
 or the corresponding decrease in temperature is 
 the more painful to the immersed hand. 
 
 Motor Sensations 
 
 Judgment of Weight. — Lift the same weight 
 twice, at first very slowly and then quickly. 
 
 The weight will appear lighter when raised 
 quickly. 
 
 Sensation of Effort. — " Hold the finger as if to 
 pull the trigger of a pistol. Think vigorously 
 of bending the finger, but do not bend it. 
 
 " An unmistakable feeling of effort results. 
 
 " Repeat the experiment, and notice that the 
 breath is involuntarily held, and that there are 
 tensions in other muscles than those that would 
 move the finger." (Sanford.) 
 
 Sensation of Motion. — Let the forearm and 
 hand rest upon a table. Bring the four fingers 
 of the hand together, and turn the hand so that 
 it shall rest upright upon the ulnar side of the 
 little finger. Close the eyes. Abduct the first 
 finger. 
 
 The second, third, and fourth finger will seem 
 to move in a direction opposite to the movement 
 of the first. 
 
32 TASTE 
 
 IV 
 TASTE 
 
 Threshold Value. — Prepare solutions of cane 
 sugar of the following strengths : 1 : 1000, 1 : 800, 
 1 : 600, 1 : 400, 1 : 200, 1 : 100. Take half a 
 tea'spoonful of the weakest solution into the 
 mouth, roll it upon the tongue, and swallow 
 it. Note whether a sweet taste can be per- 
 ceived. Rinse the mouth thoroughly. Proceed 
 with solutions of increasing strength until the 
 sweet taste is just perceptible. 
 
 Topography. — 1. Select a solution of sugar 
 slightly more concentrated than that just per- 
 ceived to be sweet. With a small camel's-hair 
 brush apply this solution to the several parts 
 of the tongue and the palate. Determine the 
 regions sensitive to taste. The mouth must be 
 rinsed frequently. 2. Dry the upper surface of 
 the tongue with a handkerchief. With a finely 
 pointed camel's-hair brush apply a twenty per 
 cent sugar solution to the individual fungiform 
 papillae and to the mucous membrane between 
 them. Determine whether only the papillse 
 perceive taste. 
 
 Relation of Taste to Area stimulated. — Swallow 
 a very small quantity of a minimal solution of 
 sugar, as determined in the experiment upon 
 
vision 33 
 
 threshold value. Rinse the mouth," and then 
 swallow a much larger portion of the solution. 
 
 The taste will be perceived more strongly, the 
 larger the area stimulated. 
 
 Electrical Stimulation. — 1. Connect two small 
 zinc electrodes through a simple key to a battery 
 of four dry -cells. Apply one electrode to an in- 
 different region, the other to the tongue. Close 
 the key. 
 
 Note the sour taste at the positive pole and 
 the alkaline taste at the negative. 
 
 VISION 
 
 Mapping the Blind Spot. — Fasten a rod fifteen 
 inches from the table. Beneath the rod place 
 a well-lighted sheet of white paper (a page of 
 the laboratory note-book will serve). Make a 
 small black cross near the left margin. Eest 
 the chin upon the rod in such a way that the 
 right eye shall look directly down at the cross. 
 Place the hand over the other eye. A straw 
 bearing a black pin-head will be drawn by an 
 assistant from the cross along the horizontal 
 meridian toward the temporal side of the eye 
 under observation. The assistant will mark the 
 point where the black object ceases to be visible, 
 and the point at which it -reappears. These are 
 
34 VISION 
 
 the boundaries of the blind spot of the right 
 eye in the horizontal meridian. Determine the 
 boundaries in other meridians. Obtain similarly 
 the outlines of the blind spot of the left eye. 
 
 Yellow Spot. — Close the eyes for half a 
 minute, and then look at the clear sky or a 
 brightly lighted surface through a solution of 
 chrome alum in a glass bottle with parallel 
 sides. The yellow spot will appear rose-colored 
 in the blue-green-red solution. The yellow pig- 
 ment absorbs some of the blue and green rays. 
 The remaining rays form rose color. 
 
 Field of Vision. — Fasten in a vertical position 
 a sheet of white paper about 50 cm. high and 
 60 cm. broad. (It may be pinned to the wooden 
 stand set on edge upon the electrometer box.) 
 About 20 cm. from the left margin and 30 cm. 
 from the lower margin of the paper mark a small 
 cross. Let the subject rest his chin upon a rod 
 clamped to the iron stand in such a way that 
 the right eye shall look directly at the cross. 
 Cement the squares of black, red, green, and 
 blue papers to the ends of separate straws. 
 Carry the black square from without inwards 
 along the horizontal meridian intersecting the 
 cross. Mark the point at which the black 
 object enters the field of vision. 1 This point is 
 the temporal boundary of the visual field in the 
 
vision 35 
 
 horizontal meridian. Determine in the same 
 way the boundary on the nasal side. Kepeat 
 for several other meridians. A line joining the 
 points obtained will bound the visual field. 
 
 Determine the visual field for red, green, and 
 blue. Always pass the test color from without 
 inwards. The subject should be ignorant of 
 the color to be used, and should name the color 
 as soon as it enters his visual field. 
 
 Color Blindness 
 
 The three lame skeins show the test colors. 
 
 1. Light Green. — Palest- (lightest) shade of 
 very pure green, — neither yellow-green nor 
 blue-green to the normal eye. Light green is 
 chosen because, according to the Young-Helm-, 
 holtz theory, it is the whitest of the colors of 
 the spectrum, and, consequently, is most easily 
 confused with gray. Light shades are employed 
 because it is difficult to distinguish between 
 strongly illuminated shades. 
 
 2. Par pie (Rose). — A skein midway between 
 lightest and darkest purple. Chosen because 
 purple combines two fundamental colors which 
 are normally never confounded. 
 
 3. Ilecl. — A vivid, slightly yellowish red. 
 Chosen because it represents the color-group in. 
 
36 vision 
 
 which red (orange) and violet (blue) are com- 
 bined in nearly equal proportions. 
 
 Me':hod of Examination and Diagnosis. — Place 
 the Berlin worsteds on the white cloth in which 
 they are wrapped. They should be well mixed, 
 and not spread out too much. Lay a skein of 
 the first test-color in a well-lighted position two 
 or three feet from the group. Inform the person 
 examined : 
 
 (1) That he must not speak during the test. 
 
 (2) That the skeins are not to be fingered or 
 tossed about. A skein should be touched only 
 after its selection, 
 
 (3) That he must endeavor to pick out skeins 
 resembling the test skein, i. e., a little lighter or 
 darker in shade ; the resemblance cannot be per- 
 fect, as no two shades are exactly alike. 
 
 . Green Test. — The subject must pick out all 
 the other skeins approximately the same shade. 
 The color-blind selects some shade of gray. 
 
 .•Purple {Rose). — The subject should pick out 
 the skeins of the same color, as before. 
 
 . (1) He who is color-blind by the first test, and 
 who, upon the second test, selects only purple 
 skeins, is incompletely par pie-blind. 
 
 (2) He who, in the second test, selects with 
 purple only blue and violet, or one of them, is 
 completely red-blind. 
 
vision 37 
 
 (3) He who, in the second test, selects with 
 purple only green and gray, or one of them, is 
 completely green-blind. 
 
 Remark. — The red-blind never selects the 
 colors taken by the green-blind, and vice versa. 
 Often the green-blind places a violet or blue 
 skein by the side of the green, but only the 
 brightest shades of these colors. This does not 
 influence the diagnosis. 
 
 Red. — This test is applied to those completely 
 color-blind. Continue the test until the person 
 examined has placed beside the specimen all the 
 skeins belonging to this shade, or else, separately, 
 one or more " colors of contusion." 
 
 The red-blind chooses (besides the red, green, 
 and brown) shades which to the normal sense 
 seem darker than red. The green-blind .selects 
 opposite shades, which seem lighter than red. 
 
 Violet Blindness. — Very rare. Recognized by 
 a confusion of purple, red, and orange, in the 
 purple test (see 2). Much care is required to 
 diagnosticate this form. 
 
38 FERMENTATION 
 
 FERMENTATION 
 
 Specific Action 
 
 Conversion of Starch to Sugar by Germinating 
 Barley. — To 5 grams crushed, germinating barley 
 add 10 grams potato starch, and 20 c.c. of cold 
 water. Then add gradually 70 c.c. of hot water 
 with constant stirring. Keep the mixture in a 
 temperature of about 60° C. for one hour. 
 
 The insoluble starch will be converted to a 
 sweet liquid. 1 
 
 Boil 10 c.c. of Fehling's solution, 2 dilute the 
 syrup with water and add it drop by drop to the 
 boiling Fehling's solution. 
 
 1 Kiichoff : Schweigger's Journal fur Chemie und Physik, 
 1815, xiv, p. 3S9. 
 
 2 Fehlirvg 's solution.— In a large watch glass weigh 34.639 gms. 
 pure cupric sulphate (clean crystals). Dissolve the crystals by 
 warming them with about 150 c.c. water in an evaporating dish. 
 Place the solution in a 500-c.c. measuring flask. Wash the 
 remnant from the dish into the flask. Allow the liquid to cool 
 completely. Add water to the mark on the neck of the flask. 
 
 Warm about 173 gms. potassium sodium tartrate in a little 
 water until dissolved. Place the solution in a 500-c.c. measur- 
 ing flask, add 100 c.c. sodium hydroxide, sp. gr. 1.34 (about 
 31 per cent), and, after the mixture has completely cooled, fill 
 the flask to the mark on the neck. 
 
 In use, mix equal volumes of each solution in a dry glass. 
 
 One molecule grape sugar reduces five molecules cupric oxide 
 to cuprous oxide ; 10 c.c. of Fehling's solution equals 0.05 gin. 
 grape sugar. 
 
SPECIFIC ACTION 6\) 
 
 Red cuprous oxide or its yellow hydrate will 
 separate. 
 
 The germinating barlev causes the starch to 
 take up water, thus changing to a reducing sugar. 
 In this instance the agent is a living cell, or 
 some substance or " ferment " secreted by the 
 cell. It is now necessary to inquire whether 
 ferments are separable from living cells. 
 
 Conversion of Starch to Sugar by Salivary Dias- 
 tase (Ptyalin). — To 10 c.c. of starch paste 1 col- 
 ored blue with iodine (blue iodide of starch) add 
 about 2 c.c. of filtered saliva and keep the mixture 
 at 35-40° C. 
 
 The starch paste will liquefy and become sweet. 
 The blue color will become lighter and finally 
 disappear. 
 
 Test with Fehling's solution. Eeduction will 
 take place. 
 
 Saliva hydrolyzes starch to a reducing sugar. 
 
 Saliva is secreted by the cells in the salivary 
 gland, placed some distance from the mouth. 
 The saliva itself contains no secreting cells. 
 There are ferments, then, which act at a distance 
 
 1 Starch paste. — Rub 1 gin. potato starch in a mortar with 
 25 c.c. cold water. Pour the mixture into an evaporating dish. 
 Wash the remnant from the mortar and pestle into the dish with 
 75 c.c. water. Heat the mixture to boiling point with constant 
 stirring. The starch paste will turn blue upon addition of iodine 
 (iodide of starch). 
 
40 FERMENTATION 
 
 from the cells that produce them. There seems 
 thus an important distinction to be made between 
 organized ferments, those acting apparently with- 
 in the living cell, and unorganized ferments, like 
 the salivary diastase, which is secreted by a 
 living cell but remains active after leaving the 
 cell. It will be seen that this distinction cannot 
 be maintained. 
 
 Extraction of Diastase from Germinating Barley. 1 
 — Crush freshly germinating barley in a mortar 
 with about half its weight of water. Keep the 
 mass two hours at 35-40° C. Squeeze out the 
 watery extract in a press, or strain by strong 
 pressure through a linen cloth. Add excess of 
 alcohol. 
 
 Diastase will be precipitated. It may be puri- 
 fied by dissolving it in water and reprecipitating 
 with alcohol. 
 
 Add a little diastase to 10 c.c. starch paste, 
 colored blue with iodine. The starch will be con- 
 verted to sugar. The blue color w T ill disappear. 
 
 It appears, therefore, that ferment action is 
 not dependent on the life of the cell that secretes 
 the ferment. 
 
 Specific Action of Ferments. — The question 
 now arises whether the diastase acts only to 
 
 1 Payen and Persoz : Armales de chimie et de physique, 
 1833, liii, p. 78. 
 
PROTEID DIGESTION BY PEPSIN 41 
 
 change starch to sugar or whether it causes the 
 decomposition of other substances. 
 
 Place a small piece of fibrin in a test-tube and 
 add 2 c.c. filtered saliva. Keep the tube several 
 hours at a temperature of 35-40° C 
 
 The fibrin will not change. 
 
 Place 0.5 c.c. neutral olive oil (page 56) and 2 
 c.c. filtered saliva in a test-tube. 
 
 Noteworthy changes will be absent. 
 
 From these experiments it is evident that 
 diastase decomposes starches, but does not de- 
 compose proteids and fats. Its ferment action is 
 thus far " specific." The belief that each ferment 
 has its own characteristic product will be in- 
 creased by the study of the following typical 
 ferment actions. 
 
 Peoteid Digestion by Pepsin 
 
 Gastric Digestion of Cooked Beef and Bread. — 
 
 At 7 a.m. feed cooked beef and bread to a cat 
 which has fasted twelve hours. At 11 A. M. kill 
 the cat, expose the stomach, and apply double 
 ligatures about 1 cm. apart to the duodenum at 
 the pylorus and to the oesophagus at the cardiac 
 orifice. Eemove the stomach. Open the stomach 
 very cautiously by drawing a knife along the 
 greater curvature. 
 
 " The stomach is very full, and still contains 
 
42 FERMENTATION 
 
 much meat and bread not wholly softened. The 
 softening is greater in the portal region and in 
 those portions of the food next the mucous mem- 
 brane than in the middle of the stomach contents. 
 The mucus secreted by the gastric mucous 
 membrane-is very abundant and is strongly acid. 
 The stomach contents have a sour odor." 1 
 
 Artificial Gastric Juice. 2 — 1. Strip the milCOUS 
 membrane from the fourth stomach of a calf. 
 Wash the membrane with cold water until the 
 acid reaction disappears. Dry the mucous mem- 
 brane in the air. Divide some of the dried 
 membrane into small pieces and add dilute hy- 
 drochloric acid. 3 
 
 2. Strip the mucous membrane of the pig or 
 rabbit from the deeper layers of the stomach, cut 
 the mucous membrane into the smallest pieces, 
 wash slightly with water, pour off the water with 
 all possible care, and cover the slightly moist 
 residue with glycerine. 4 Before using, add dilute 
 hvdrochloric acid. 
 
 1 Eberle : Physiologie der Verdauung, 1834, p. 100. 
 
 2 Eberle : loc. cit., p. 79. 
 
 3 Dilute hydrochloric acid. — Add to 10 c.c. officinal HC1, 
 sp. gr. 1.124 (about 25 per cent HC1), enough water to make 
 1000 c.c. This solution will contain about 0.281 per cent HC1. 
 (Salkowski's Practicum, 1893, p. 130.) 
 
 4 Von Wittich : Arcbiv fur. die gesammte Physiologie, 1869, 
 ii, p. 194. 
 
PROTEID DIGESTION BY PEPSIN 43 
 
 Digestion -with Artificial Gastric Juice. — Pre- 
 pare three flasks, A, B, and C. In A place 100 c.c. 
 artificial gastric juice; in B, 100 c.c. 0.2 per cent 
 HC1; and in C, a piece of dried gastric membrane 
 and 100 c.c. distilled water. In each of the three 
 flasks place a small piece of cooked meat, and keep 
 the flasks about five hours at 35-40° C. 1 Com- 
 pare the result with that observed in natural 
 digestion. 
 
 The artificial gastric juice will digest the meat 
 as did the natural juice in the stomach, but neither 
 the acid alone, nor the mucous membrane free 
 from acid, will digest. There is a ferment in the 
 mucous membrane, but it will not act except in 
 an acid medium. *' 
 
 Extraction of Pepsin. — Pepsin more or less con- 
 taminated with proteid (pepsin may itself be a proteid) 
 may be precipitated from a glycerine extract by alco- 
 hol. 2 The pepsin may also be carried down mechani- 
 cally by an indifferent precipitate as in Brticke's 
 method, 3 in which the mucous membrane, acidulated 
 with phosphoric acid, is allowed to digest until the 
 proteids are mostly converted into soluble peptone. 
 The mixture is then neutralized with lime water. 
 The insoluble calcium phosphate thus formed falls as 
 
 1 Eherle : loc. cit. 
 
 2 Von Wittich: loc. cit., p. 195. 
 
 3 Briicke : Sitzungsberichte der konigliche Akademie der 
 Wissenschaften zu Wien, 1862, xliii, p. 601. 
 
44 FERMENTATION 
 
 a fine powder carrying the pepsin with it. The precip- 
 itate is dissolved in very dilute hydrochloric acid, and 
 to this solution is added a solution of cholesterin in 
 alcohol and ether. When the two solutions are mixed, 
 the choiesterki separates as an abundant, fine powder 
 bearing the pepsin with it. The cholesterin is removed 
 with ether, leaving the pepsin. 
 
 Ammonium sulphate may also be used as the me- 
 chanical precipitant. 1 
 
 Change of Proteid to Peptone by Pepsin. — 1. 
 
 Place in a test-tube five drops of the glycerine 
 extract of pepsin with 5 c.c. 0.2 per cent hydro- 
 chloric acid and a small piece of fibrin. 2 Keep 
 the mixture at 35-40° C. 
 
 In a short time the fibrin will be dissolved. 
 Appropriate tests will show that it has been con- 
 verted to peptone. 2. Repeat the preceding ex- 
 periment, using commercial pepsin (never very 
 free from proteid). 
 
 Splitting of Casein by Rennin. 
 
 Rennin Extract. — Allow the mucous membrane 
 of the stomach (preferably the fourth stomach of 
 
 1 Kiihne and Chittenden: Zeitschrift fiir Biologie, 1886, 
 xxii, p. 428. 
 
 2 Preparation of fibrin. — With a bundle of smooth rods 
 whip blood as it flows from an artery until the fibrin gathers on 
 the rods. Wash the fibrin in running water until the red cor- 
 puscles are removed and the fibrin shows its natural color. 
 Preserve the fibrin in glycerine. 
 
SPLITTING OF CASEIN BY RENNIN 45 
 
 the suckling calf) to stand" twenty-four hours in 
 150-200 c.c. 0.1-0.2 per cent solution of hydrochlo- 
 ric acid. Then neutralize the acid with great care. 1 
 
 Separation of Reunin. — The extract just prepared 
 contains pepsin as well as rennin. The rennin may 
 be separated as follows. The neutralized extract is 
 repeatedly shaken with fresh amounts of magnesium 
 carbonate. The resulting precipitates carry down 
 almost all the pepsin and very little rennin. The 
 filtrate still rapidly coagulates milk, but contains only 
 traces of pepsin. This filtrate is now precipitated 
 with lead acetate, the precipitate is decomposed with 
 very dilute sulphuric acid, and the mixture filtered. 
 To the filtrate, which contains the rennin, is added a 
 solution of stearin soap in* water. Thereupon the 
 soap is thrown out of solution and falls, carrying the 
 rennin with it. The soap is then removed by shaking 
 with ether, and the rennin remains. 2 
 
 Precipitation of Casein. — Add 1 C C. of the 
 neutral extract to 25 c.c. fresh milk at 36-38° C. 
 (Xormal milk is amphoteric. If the reaction 
 be acid, the acid should be very carefully 
 neutralized.) 
 
 In a few minutes the milk will separate into 
 
 1 Hammarsten : tfpsala Lakateforenings Forhandlingar, 1872, 
 viii, pp. 63-86. Abstract by author in Maly's Jahresberieht 
 iiber die Fortschritte der -Thierchemie, 1872, ii, pp. 118-125. 
 
 2 Hammarsten : Lehrbuch der physiologischen Cnemie, 1S95, 
 p. 241. 
 
46 FERMENTATION 
 
 curd and whey. The curd is casein together 
 with the fat globules carried down as it precipi- 
 tates. The whey is a dilute saline solution of 
 milk-albumin, milk sugar, etc. 
 
 Test the chemical reaction. The mixture is 
 still neutral. Milk may also be curdled by acid, 
 either added artificially or produced in the milk 
 itself by lactic acid fermentation of milk sugar. 
 The absence of an acid reaction in the above exper- 
 iment excludes precipitation through acid fermen- 
 tation of milk sugar. Casein prepared free from 
 milk sugar is also precipitated by rennin. Finally, 
 rennin, extracted by the method given above, does 
 not act upon milk sugar, but rapidly precipitates 
 casein. 
 
 Analogy suggests that the specific action of 
 the rennin may be the splitting of casein and that 
 the precipitation may be a secondary process. 
 The following experiments determine this matter. 
 
 Experiments of Arthus and Pages. 1 — Prepare 
 two solutions, A and B. 
 
 A. 
 Milk 100 c.c. 
 
 Neutral oxalate of 
 
 potassium 1% 5 c.c. 
 
 Rennin 1 to 250 4 c.c. 
 
 B. 
 
 Milk 100 c.c. 
 
 Neutral oxalate of 
 
 potassium 1 % 5 c.c. 
 Water 4 c.c. 
 
 1 Arthus and Pages : Archives de physiologie, 1890, p. 334. 
 
SPLITTING OF CASEIN BY EENNIN 47 
 
 (Rermin, 1 to 250, is a pastille of Hansen dis- 
 solved in 250 c.c. H 2 0.) 
 
 Keep both mixtures at 38° C. during forty 
 minutes. 1. Boil 25 c.c. from each solution. 
 Solution A coagulates, while solution B shows 
 no trace of coagulation. Hence the action of 
 rennin has rendered the casein in A coagulable 
 on boiling. 
 
 2. To 25 c.c. from each solution add 8 c.c. of 
 a solution of calcium chloride capable of pre- 
 cipitating exactly, in equal volumes, the solution 
 of potassium oxalate. By this addition the cal- 
 cium oxalate is removed and the calcium chloride 
 remains in slight excess. 
 
 A will coagulate ; B will not. Hence the casein 
 in solution A has been so changed by rennin that 
 it is precipitated on the addition of a small quan- 
 tity of calcium chloride. Solution A may also 
 be precipitated by restoring its original content 
 of calcium chloride, i.e. by adding 5 c.c. of the 
 above calcium chloride solution, which will exactly 
 combine with the 5. c.c. of potassium oxalate. 
 
 If small quantities of rennin be added to 
 natural milk and equal portions of the milk be 
 tested from time to time by boiling, the amount 
 coagulated will be greater the longer the rennin 
 acts. An amount of calcium chloride too small 
 to produce coagulation in the early stages of 
 
48 FERMENTATION 
 
 leu niii action is sufficient to produce coagulation 
 when added in the later stages. 
 
 Evidently, in the clotting of milk by rennin 
 two separate phenomena must be distinguished : 
 (1) the chemical transformation of casein by 
 rennin, (2) the precipitation of the transformed 
 casein by the calcium chloride. (This salt favors 
 also the splitting of the casein.) Rennin may 
 therefore be classed with pepsin and trypsin. 
 
 According to Hammarsten the casein is split 
 into phosphorus-free albumose and phosphorus- 
 holding paracasein. Heat is set free. It is the 
 paracasein which precipitates. It is less soluble 
 than casein. 
 
 Precipitation of Fibrin by Fibrin Ferment 
 
 Buchanan's Experiment. — Press blood clot 
 through a linen cloth. Add the liquid thus ob- 
 tained to a serous fluid, wfllch does not clot spon- 
 taneously, such as ascitic fluid, pleural effusion, 
 hydrocele fluid. 
 
 After some hours a firm, translucent clot will 
 form. 1 
 
 Extraction of Fibrin Ferment. Schmidt's Method. 
 — Coagulate one part of serum from the blood 
 
 1 Buchanan : London Medical Gazette, 1835, xviii, p. 51; 
 idem, 1845, xxxvi, p. 617. This discovery was first announced 
 in 1831. 
 
PRECIPITATION OF FIBRIN 49 
 
 of ox, dog, or horse, by adding 15-20 parts strong 
 alcohol. After at least fourteen days, filter, dry 
 the moist residue over sulphuric acid, pulverize 
 the dried substance, stir it with water (twice the 
 volume of the serum originally taken) and after 
 allowing sufficient time for solution, filter. The 
 filtrate contains the fibrin ferment. 1 
 
 Gamgee's Method. — Allow freshly prepared fi- 
 brin (obtained by washing a blood clot free from 
 corpuscles) to stand three days in 8.0 per cent 
 solution of sodium chloride. Filter. 2 
 
 The filtrate is rich in fibrin ferment. 
 
 Extraction of Fibrinogen. — Eeceive three 
 volumes of blood directly from an artery into 
 one volume of saturated solution of magnesium 
 sulphate, which will prevent the blood from 
 clotting. Separate the corpuscles from the liquid 
 plasma by the centrifugal machine. Add to the 
 plasma an equal volume of saturated solution of 
 sodium chloride. Flakes of fibrinogen will be 
 precipitated. Filter as quickly as possible, for 
 that purpose dividing the liquid among several 
 funnels each with a folded filter paper. Press 
 the filter papers containing the residue between 
 fresh filter paper, in order to remove the adherent 
 
 1 Schmidt : Archiv fur die gesammte Physiologie, 1872, vi, 
 p. 457. 
 
 2 Gamgee : Journal of physiology, 1S79, ii, p. 151. 
 
 4 
 
50 FERMENTATION 
 
 liquid. Tear the filter containing the fibrinogen 
 into small pieces. Dissolve the fibrinogen which 
 sticks to the filter as a tough, elastic mass, in a 
 quantity of 8 per cent sodium chloride solu- 
 tion equal to about one-third the quantity of the 
 magnesium sulphate solution originally taken. 
 Filter off the fragments of paper. Purify by 
 reprecipitation with an equal volume of saturated 
 solution of sodium chloride. Filter. Dry as 
 before, and add a small quantity of water to the 
 finely divided filter to which the precipitate 
 clings. This water will take a small quantity of 
 salt from the precipitate, and in this dilute saline 
 solution the fibrinogen will dissolve. 1 
 
 Precipitation of Fibrinogen by Fibrin Ferment. — 
 Add to the dilute saline solution of fibrinogen a 
 solution containing fibrin ferment. 
 
 Fibrin will form. 
 
 Ammoniacal Fermentation of Urea by 
 Urease 
 
 1. Place 100 c.c. fresh human urine in each 
 of three clean flasks marked A, B y C. To B and 
 C add 1 c.c. of urine that has become ammoniacal 
 upon standing in the atmospheric air. Add also 
 
 1 Hammarsten : Arehiv fur die gesaramte Physiologie, 1879, 
 xix, p. 563. Also idem, 1880, xxii, p. 431. Hammarsten's first 
 publication was in Nova acta regia societas scientiarum Upsali- 
 eusis, 1873, (3), ix. 
 
FERMENTATION OF UEEA BY UREASE 51 
 
 to C 2 per cent of a saturated solution of carbolic 
 acid in water. Let B and C stand in a warm 
 place sixteen days. 
 
 2. Withdraw 5 c.c. from flask A. Note 
 whether the urine is clear or turbid, and 
 whether it effervesces on the addition of a 
 dilute acid. Withdraw 2 c.c. from flask A and 
 determine its percentage of urea by the hypo- 
 bromite method. 
 
 Centrifugalize a portion of the remaining con- 
 tents of flask A. With a microscope examine 
 the sediment for crystals of ammonio-magnesium 
 phosphate and for micro-organisms, especially the 
 micrococcus urea?, which Occurs in long curved 
 chains of round cells about 1.5 /jl in diameter. 
 
 3. After sixteen days repeat these observa- 
 tions on the urine in flasks B and C. Record 
 the results obtained from all three flasks in the 
 table on page 52. 
 
 The table shows that the hydrolysis of urea 
 into ammonium carbonate still takes place in 
 urine containing enough carbolic acid to destroy 
 the micro-organisms long known to be the cause 
 of the ammoniacal fermentation. 1 It is therefore 
 probably due to a ferment, which escapes from 
 the cells after their death. 
 
 1 Hoppe-Seyler : Medicinisch-chemische Untersuchungen, 
 Berlin, 1866, p. 570. 
 
52 
 
 FERMENTATION 
 
 v3 
 
 £.! 
 
 ej - 
 
 S it 
 O 
 
 
 
 
 06 | « 
 
 
 
 
 Per cent 
 
 of 
 
 Urea. 
 
 
 
 
 Reaction 
 
 to 
 Acids. 
 
 
 
 
 Clear 
 
 or 
 
 Turbid. 
 
 
 
 
 Content 
 
 of 
 Carbolic 
 
 Aeid. 
 
 
 
 
 go 
 at) 
 
 A 
 
 Normal 
 
 B 
 
 Septic 
 
 C 
 
 Aseptic 
 
FERMENTATION OF UREA BY UREASE 53 
 
 Prior to 1860 ammoniacal decomposition of 
 urine was vaguely classed as a fermentation. In 
 that year Miiller 1 suggested that it might be due 
 to a body like beer-yeast. In 1862 Pasteur 2 
 discovered such a yeast, which he called Torula 
 urece. Colin first classed it with the micrococci. 
 It is aerobic. Miguel finds seven species of 
 bacilli, nine micrococci, and one sarcina, that 
 decompose urea. These obtain their nitrogen 
 ordinarily from proteids, but in the absence of 
 proteids may utilize urea. 
 
 Extraction of Urease. — To 10 C.C. of urine 
 undergoing an active ammoniacal fermentation, 
 add 50 c.c. of strong alcohol, and allow the 
 mixture to stand in a well-corked flask. After 
 five days place the precipitate upon a very small 
 filter and wash it with 50 c.c. of fresh alcohol. 
 (Preserve both filtrates for recovery of the alcohol 
 by redistillation.) 
 
 1. Add a very small quantity of this precip- 
 itate to a neutral 2 per cent solution of urea. 
 Test the reaction. Place the mixture in a water 
 bath at 38° C. 
 
 After a few minutes again test the reaction. 
 
 It will be strongly alkaline. 
 
 1 Miiller: Journal fur praktische Chemie, 1860, Ixxxi, p. 467. 
 
 2 Pasteur : Comptes vendus de l'academie des sciences, Paris, 
 1860, 1, p. 869. See also Van Tieghern, idem, 1864, p. 210. 
 
54 FERMENTATION 
 
 After a short time the odor of ammonia will be 
 perceptible. The alcoholic precipitate contains a 
 ferment capable of quickly hydrating urea. 
 
 "The alcoholic precipitate from the unfiltered 
 urine consists chiefly of various salts together 
 with the cells of the Torula, hence when treated 
 with water some of the salts are dissolved and 
 pass with the ferment through the filter. If this 
 first aqueous extract be again precipitated with 
 alcohol, a portion of the salts will be 'again 
 removed, and if this second precipitate be several 
 times redissolved in water and reprecipitated 
 with alcohol, the body with the ferment proper- 
 ties may be ultimately separated — as an amor- 
 phous white powder soluble to a clear solution 
 in distilled water and not characterized by any 
 special chemical reactions." 
 
 The ferment is not secreted by the cells into 
 the surrounding liquid, but is retained within the 
 cell bodies, for the living cells may be filtered off, 
 and the filtrate will not hydrate the urea. 1 
 
 Splitting and Synthesis of Fats 
 
 Chemistry of Fats and Soaps. — When olive oil 
 is saponified, glycerine appears (Scheele, 1779). 
 
 1 Lea : Journal of Pl^siology, 1885, vi, p. 138. See also 
 Musculus : Comptes rendus de l'academie des sciences, Paris, 
 187-1, lxxviii, p. 132 ; idem, 1876, lxxxii, p. 333 ; Archiv fiir 
 die gesammte Physiologie, 1876, xii, p. 214. 
 
SPLITTING AND SYNTHESIS OF FATS 00 
 
 It is related to the alcohols (Chevreul, 1813), 
 being a compound ether or ester, a combination 
 of an alcohol with an acid. Commercially gly- 
 cerin 3 is prepared by exposing neutral fats, such 
 as stearin, to superheated steam, whereby the 
 neutral fat is split into glycerine and fatty acid. 
 
 CHoO 
 
 1 
 
 •CO(CH 2 ) 16 
 
 •CH 3 
 
 
 H 
 
 •OH 
 
 
 CHO 
 
 •CO(CH 2 ) 16 
 
 •CH 3 
 
 + 
 
 H 
 
 •OH 
 
 = 
 
 CH 2 
 
 •CO(CH 2 ) 16 
 
 ■CH 3 
 
 
 H 
 
 •OH 
 
 
 
 STEARIN 
 
 
 
 WATER 
 
 
 
 
 CH 2 
 
 i 
 
 •OH 
 
 
 CO 
 1 
 
 •OH(CH 2 ) 16 -CH 
 
 
 
 1 
 CH 
 
 i 
 
 •OH 
 
 + 
 
 1 
 
 CO 
 1 
 
 •OH(CH,) 16 'CH 
 
 
 
 CH 2 
 
 •OH 
 
 
 1 
 CO 
 
 •OH(CH 2 ) 16 -CH 
 
 
 
 GLYCERINE 
 
 
 
 STEARIC ACID 
 
 If an alkali be present, it will combine with 
 the fatty acid to form a soap. 
 
 CO 
 
 •OH(CH. 2 ) 16 -CH 3 
 
 Xa • OH 
 
 
 
 CO 
 
 ■OH(CH 2 ) 16 -CH 3 
 
 + Xa • OH = 
 
 
 
 CO 
 
 •OH(CH 2 ) 16 -CH 3 
 
 STEARIC ACID 
 
 Xa • OH 
 
 SODIUM 
 HYDROXIDE 
 
 CO-OXa(CH 2 ) 16 -CH 3 
 
 
 H-OH 
 
 
 
 CO-OXa(CH 2 ) 16 -CH 3 
 
 + 
 
 HOH 
 
 
 
 CO-OXa(CH 2 ) 16 .CH 3 
 
 
 H-OH 
 
 
 
 SODIUM STEAEATE 
 
 
 WATER 
 
 Splitting of Fats by the Pancreatic Juice. Ber- 
 nard's Experiment — Place 2 c.c. neutral olive 
 
56 FERMENTATION 
 
 oil in a test-tube and add a small quantity of 
 pancreatic juice (or a piece of fresh pancreas or 
 extract of pancreas). Test the reaction of the 
 mixture. It is alkaline. Note that a white, 
 creamy liquid forms almost immediately. This 
 " emulsion " is composed of a multitude of small 
 fat globules. 
 
 Test the reaction again. It gradually becomes 
 acid. 
 
 It is evident that under the influence of the 
 pancreatic juice the fatty matter is not simply 
 finely divided and emulsified, but that it has also 
 been modified chemically. 1 
 
 In order to study the splitting of neutral fats 
 by lipase, a ferment found in the pancreatic juice, 
 it is necessary (1) to prepare a perfectly neutral 
 fat, and (2) to recognize the fatty acid as soon as 
 it is set free. 
 
 Preparation of Neutral Fat. — Shake commer- 
 cial olive oil (which always contains fatty acid) 
 for two hours at 95° C. in a separating funnel 
 with a saturated solution of barium hydroxide. 
 Allow the mixture to stand until the oil sepa- 
 rates from the hydroxide. Eemove the hydrox- 
 ide. Filter the oil. 
 
 The Emulsion Test for Fatty Acid. BriicJie's 
 
 1 Bernard : Comptes rendus de l'academie des sciences, Paris, 
 1849, xxviii, p. 250. 
 
SPLITTING AND SYNTHESIS OF FATS 57 
 
 Experiment. — 1. Shake 1 c.c. neutral olive oil 
 in a test-tube with 5 c.c. 0.25 per cent sodium 
 carbonate solution. 
 
 The oil will be broken up into large globules 
 which will speedily reunite, leaving the liquid 
 clear. 
 
 2. Shake 1 c.c. rancid olive' oil (containing 
 about 5.5 per cent fatty acid) with 5 c.c. 0.25 per 
 cent sodium carbonate solution. 
 
 The mixture becomes instantly milky. The 
 oil is divided into globules of microscopic size. 
 The emulsion is permanent. 
 
 3. Shake 1 c.c. neutral olive oil with 5 c.c. water. 
 The water and oil will not mix. 
 
 4. Shake 1 c.c, neutral oil with water con- 
 taining soap. 
 
 The oil will be emulsified. It is probable 
 therefore that soap contributes to the emulsion, 
 perhaps by coating the fine particles of oil with a 
 membrane that prevents their reunion. 1 
 
 Gad's Experiment. — 1. Fill a watch glass 
 about 5 cm. in diameter with 0.25 per cent solu- 
 tion of sodium carbonate. With a glass rod 
 carefully place a large drop of rancid olive oil 
 (containing 5.5 per cent fatty acid) upon the 
 surface of the soda solution. 
 
 1 Briioke : Sitzungsbeiichte der kaiserlichen Akademie der 
 Wissenschaften zu Wien, 1870, lxi, pp. 613-61-4. 
 
58 FERMENTATION 
 
 The drop will come to rest, and for a moment 
 both the drop and the surrounding liquid remain 
 clear. Very soon, however, the oil is covered 
 with a white layer, and through the soda solu- 
 tion spreads a white cloud which becomes denser 
 and denser until the oil drop, steadily diminish- 
 ing in size, floats in a milky white liquid. 
 
 2. Eepeat the experiment, observing the oil 
 drop under a low power of the microscope. 
 
 Note the extraordinary motion in the neigh- 
 borhood of the oil drop, and how the particles of 
 oil are thrown out in strong eddies. 
 
 3. Examine the completed emulsion under a 
 higher power of the microscope. 
 
 There appear exceedingly small fat drops of 
 very uniform size. The milky fluid is the finest 
 and most uniform emulsion. 1 
 
 BachforcFs Experiment. — " Arrange a series of 
 watch glasses containing 0.25 per cent solution 
 sodium carbonate. Place in a test-tube 2 c.c. 
 neutral olive oil and 1 c.c. pancreatic juice (or 
 extract). Shake the tube and allow the juice 
 and oil to separate, then pipette a drop of oil 
 from the surface and place it on the soda solu- 
 tion in watch glass 1. Again shake the tube 
 and allow the oil and juice to separate, then 
 pipette as before, placing a drop of oil in watch 
 
 1 Gad: Archiv fur Physiologie, 1878, p. 183. 
 
SPLITTING AND SYNTHESIS OF FATS 59 
 
 glass 2. Again shake and pipette as before, and 
 repeat this process every three or four minutes 
 until the experiment is completed. The begin- 
 ning of the experiment and the time of each 
 pipetting must be carefully noted. If the pipet- 
 tings are three minutes apart, then the first drop 
 of oil will have been exposed three minutes to 
 the action ' of the pancreatic juice, the second 
 drop six minutes, the third nine minutes, and 
 so on. 1 
 
 The gradual increase in fatty acid will be 
 shown by the gradual increase in the amount 
 of the spontaneous emulsion. 2 
 
 It has just been shown that lipase will hydro- 
 lyze neutral fats into fat^y acid and glycerine. 
 We must now enquire whether this ferment 
 ean effect the synthesis of fats, in other words 
 whether its action is reversible. For this pur- 
 
 1 Rachford: Journal of physiology, 1891, xii, p. SI. Rach- 
 ford used J c.c. fresh pancreatic juice obtained by placing a 
 glass tube in the pancreatic duct of the rabbit (see page 80). 
 
 2 " There is a possible error in this method which had better 
 be spoken of here. It would seem that the alkali of the pan- 
 creatic juice would combine with the fatty acids forming soap, 
 and in this way the oil would soon be emulsified in the juice 
 itself and not separate after shaking. This would indeed be a 
 serious drawback if it actually occurred, but in truth it does not 
 occur until late in the experiment after we have obtained the 
 information we have sought by the spontaneous emulsion 
 method." (Rachford, loc. cit., p. 82). 
 
60 FERMENTATION 
 
 pose an extract of lipase may be used, first, to 
 split a neutral fat (or glycerol ester) into its con- 
 stituent fatty acid and alcohol (glycerine is a 
 trihydric alcohol), and second, to form a neutral 
 fat from fatty acid and alcohol. 
 
 Extraction of Lipase. From Pancreas. — Re- 
 move the pancreas of the pig within thirty min- 
 utes after the death of the animal. Dissect off 
 as much of the fat as possible. Reduce the pan- 
 creas to a fine pulp in a mortar with coarse well- 
 washed white sand. Extract the lipase with a 
 little water or glycerine. 
 
 From Liver. — Remove the liver of the pig 
 within thirty minutes of the death of the animal. 
 Reduce 50 gms. to a fine pulp in a mortar with 
 about 200 c.c. water. Filter. Dilute the watery 
 extract to 500 c.c. 
 
 Hydrolysis of Ethyl Butyrate by Lipase. — 
 Place in each of two test-tubes, A and B, 4 c.c. 
 water, 0.1 c.c. toluene, 1 and 0.26 c.c. ethyl buty- 
 rate. 2 Cork the tubes tightly. Place them in the 
 water bath for five minutes, to bring them to 
 the temperature of the bath, 40° C. Add 1 c.c. of 
 
 1 Toluene is an antiseptic, which prevents the splitting of 
 the neutral fat by bacteria. 
 
 2 Ethyl butyrate hydrolyzes more rapidly than butter fat. 
 It has the further advantage that the amount split by the 
 temperatures employed during the time of the experiment is too 
 small to be measurable. 
 
SPLITTING AND SYNTHESIS OF FATS 61 
 
 the aqueous extract of lipase to each. Boil tube 
 B. Place both tubes at 40° C. for fifteen min- 
 utes. Eemove them from the bath and plunge 
 them into ice-water (to check further ferment 
 action). Titrate with ^ KOH, using neutral lit- 
 mus as the indicator. 1 The initial acidity of the 
 
 1 A normal. solution contains in each litre one equivalent 
 weight of the active substance, i.e. that mass of the active sub. 
 stance which is equivalent to the atomic weight of a univalent 
 element in the reaction for which the normal solution is to be 
 employed. Equal volumes of different normal solutions are 
 equivalent to each other. Thus, 1 c.c. normal alkali solution 
 requires for neutralization exactly 1 c.c. normal acid, no matter 
 what acid is employed to make the normal solution. 
 
 Preparation of Normal Potassium Solution. — The content of 
 KOH in 1 litre is 56.16 grams. Dissolve 60 gms. purest com- 
 mercial KOH (which always contains considerable water) in a 
 graduated cylinder in about 950 c.c. water. Determine the 
 true content of KOH by titration with a normal oxalic acid 
 solution (prepared by dissolving its equivalent weight 63 gms. 
 in 1 litre water) as follows. Thoroughly stir the potassium hy- 
 droxide solution, fill a burette with a portion of the well-mixed 
 solution. Place 10 c.c. normal oxalic acid solution in a beaker 
 and add a few drops of solution of rosolic acid as indicator. 
 Add the alkali from the burette cautiously until the end point 
 of the reaction is reached, i. e. until the indicator gives a red 
 color which does not quickly disappear. As 10 c.c. of acid solu- 
 tion should exactly neutralize 10 c.c. of alkali solution, pro- 
 vided both were normal, it follows that the quantity of KOH 
 solution necessary to neutralize is to 10 c.c. as the total quantity 
 of the original KOH solution is to x. x will be the number of 
 cubic centimetres to which the KOH solution must be diluted 
 in order to make it normal. A portion of the normal solution 
 should then be diluted 1:20, and preserved in an air-tight 
 
62 FEKMENTATION 
 
 enzyme solution, usually 0.1 to 0.2 c.c. ^o KOH, 
 should be deducted from the cubic centimetres 
 KOH required to neutralize the fatty acid 
 formed. 1 
 
 Fatty acid will appear in tube A, but not in 
 tube B, in which the enzyme was destroyed by 
 boiling. 
 
 Synthesis of Neutral Fat by Lipase. — 1. Place 
 5 c.c. T ^ 7 butyric acid, 2 c.c. 13 per cent alcohol, 
 1 c.c. diluted glycerine extract of pig's pancreas 
 (or aqueous extract of liver) in each of two test- 
 tubes, A and B. Boil the contents of test-tube 
 B. Seal both tubes. Keep them thirty-six 
 hours at 48.5° C. 
 
 On opening the tubes, A will give a distinct 
 odor of ethyl butyrate ; none will be found in B, 
 in which the ferment was destroyed by boiling. 2 
 
 2. Place 5 gms. glycerine, 2 gms. isobutyric 
 acid, 125 gms. water, 1 c.c. neutralized blood serum 
 (or aqueous extract of pig's liver) in each of two 
 
 flask. (Compare Miiller and Kiliani: Kurzes Lehrbuch der 
 analytischen Cheinie, 1900, p. 31 and p. 83). 
 
 At 30° (summer temperature) 0.26 c.c. ethyl butyrate weighs 
 0.2300 gram. This quantity, if completely hydrolyzed, would 
 require 39.7 c.c. ^ KOH. 
 
 1 Kastle and Loevenhart : American chemical journal, 1900, 
 xxiv, pp. 491-525. Also Loevenhart : American journal of 
 physiology, 1902, vi, pp. 331-350. 
 
 2 Kastle and Loevenhart : loc. cit., p. 518. 
 
SPLITTING AND SYNTHESIS OF FATS 63 
 
 test-tubes, A and B. Boil the contents of tube 
 B. Place both at 37° C. At intervals of half 
 an hour titrate a portion from each tube with 
 2*0 KOH solution. The acidity will diminish in 
 both, but much more rapidly in the tube contain- 
 ing the active ferment. 
 
 ' The acidity is diminished by the combination 
 of the fatty acid with the glycerine to form a 
 neutral fat. 1 
 
 Fats are hydrolyzed to some extent in the stomach, 2 
 but stomach lipase is active only in neutral solutions. 
 It is inhibited or destroyed by 0.3 per cent hydro- 
 chloric acid. Other ethereal salts besides the fats are 
 hydrolyzed in the intestine, e. g. salol. 3 
 
 The rate of change by lipase increases with the 
 amount of the enzyme present. 4 
 
 Reversible action is seen in ferments other than 
 lipase, as in the following experiments. 
 
 Splitting of Hippuric Acid by Histozyme. — A pig's 
 kidney was perfused four hours with one litre defibri- 
 nated pig's blood to which 0.8 gram hippuric acid 
 
 1 Hanriot : Coniptes rendus de la societe de biologie, 1901, 
 p. 70. 
 
 2 Marcet: Proceedings Royal Society, London, 1858, ix, 
 p. 306. Ogata : Archiv fur Physiologic, 1881, p. 515. Cash : 
 Archiv fiiv Physiologie, 1880, p. 323. 
 
 3 Baas: Zcitschrift fur physiologische Chemie, 1S90, xiv, 
 p. 416. 
 
 4 Kastle and Loevenhart : loc. cit, p. 511. 
 
64 FERMENTATION 
 
 (sodium salt) had been added. The hlood passed 
 through the kidney 9-10 times. 
 
 Upon analysis, there appeared 0.037 gram benzoic 
 acid, produced from 0.1 276 gram hippuric acid. 
 
 Synthesis of Hippuric Acid by Histozyme. — A pig's 
 kidney was perfused three hours with one litre defibri- 
 nated pig's blood containing a neutral solution of 0.5 
 gram benzoic acid and 0.6 gram glycocoll. The blood 
 passed ten times through the kidney. 
 
 Found : 94 mgm. hippuric acid. 1 
 
 These actions depend upon a ferment, histozyme, 
 extracted by Schmiedeberg. 
 
 Some hypothetical considerations will be of value 
 here. Compounds of carbon may be divided into 
 those in which the carbon atoms are arranged in an 
 open chain, for example ethane, C 2 H e 
 
 H II 
 
 I I - 
 H— C— C— H 
 
 I I 
 H H 
 
 and those in which the chain is closed to form a "car- 
 bon ring," for example, benzene, C 6 H 6 , which consists 
 of six carbon atoms, in a closed, ring-shaped chain, the 
 " benzene nucleus," with a hydrogen atom joined 
 to each carbon atom by its fourth affinity (Kekule, 
 1865). 
 
 1 Schmiedeberg : Arehiv fur experimentelle Pathologie und 
 Pharmakologie, 1881, xiv, pp. 382-383. 
 
 'O* 
 
SPLITTING AND SYNTHESIS OF FATS 65 
 
 H H 
 
 \ / \ / 
 
 = C" C = C 
 
 / \ / \ 
 
 C C- H-C C-H 
 
 ^ // % // ■ 
 
 c - c c - c 
 
 BENZENE NUCLETT 
 OR KING 
 
 H H 
 
 BENZENE 
 
 The benzene ring is not easily opened, but deriva- 
 tives of benzene inay be readily obtained by replacing 
 hydrogen atoms. Thus, in aniline or amido-benzene, 
 C 6 H 5 .NH 2 , one hydrogen atom is replaced by amide 
 radical ; in carbolic acid, or phenol. C 6 H 5 .OH, by 
 hydroxyl ; in toluene or methyl benzene, C 6 H 5 .CH 3 , 
 by the radical CH 3 . The usarbon atom in methyl 
 benzene is not a part of the benzene ring, but is 
 chained to the side of the ring. The hydrogen atoms 
 in the side-chain differ in their affinities from those 
 attached to the ring ; the hydrogen in the ring may 
 be replaced by groups («.^N0 2 ) which will not readily 
 replace the hydrogen of the side-chain. This is a 
 matter of special interest in relation to the specific 
 action of poisons, ferments, etc. By substituting 
 hydroxyl for the hydrogen of the side-chain, benzyl 
 alcohol, C 6 H 5 .CH 2 .OH, is formed. By introducing 
 carboxyl, benzoic acid, C 6 H 5 .CO.OH, is obtained. It 
 has been shown above that benzoic acid and glyco- 
 coll are united in the kidney to form hippuric acid. 
 Glycocoll is amido-acetic acid, CH 2 (XH 2 ).CO.OH. It 
 
66 FERMENTATION 
 
 unites with benzoic acid by replacing the hydroxyl in 
 
 the side-chain, thus forming 
 
 C 6 H 5 .CO.NH. 
 
 ;ch 2 
 
 CO.OH 
 
 HIPPUKIC ACID 
 
 Cinnamic acid, toluene, and other aromatic substances 
 are similarly excreted as hippuric acid when taken 
 internally. 
 
 The reversible action of the kidney ferment is im- 
 portant in hastening the establishment of the equi- 
 librium between benzoic acid and glycocoll. If these 
 two bodies pass through the kidney, a certain amount 
 of hippuric acid is formed ; if hippuric acid itself 
 passes through the kidney, a certain quantity is hy- 
 drolyzed. 
 
 Relation of Reversible Action to Absorption of Fat. — 
 "Pancreatic juice is capable of hydrolyzing all the fat 
 of a fatty meal in the period of pancreatic digestion. 
 In the living intestine the hydrolysis should be com- 
 plete, inasmuch as the removal of the products of the 
 hydrolysis by absorption prevents the establishment 
 of equilibrium. On the other hand, the products of 
 the hydrolysis in their transition through the epithelial 
 cells come in contact with a lipolytic enzyme, the pres- 
 ence of which in these cells has been demonstrated in 
 the above. 
 
 "The lipase now finds itself in contact with only 
 fatty acid and glycerine, and hence in acting catalyti- 
 cally to bring about the chemical equilibrium, it effects 
 
IMMUNITY 67 
 
 the synthesis of a fat. This would offer a satisfactory 
 
 explanation of the presence of fat granules in these 
 cells. As the fatty acid and glycerine diffuse out of 
 the cells through the basement membrane, the fat 
 in these cells would speedily disappear were it not that 
 these substances were constantly being absorbed from 
 the lumen of the intestine. "When absorption ceases, 
 however, the fat present is at once hydrolyzed by the 
 lipase present. This hydrolysis is in all probability 
 complete for the reason that the products of the 
 hydrolysis, viz., glycerine and fatty acid, are being 
 constantly removed by diffusion. According to this 
 view, therefore, no fat ever enters or leaves the epi- 
 thelial cells as such, but as fatty acid and glycerine. 
 
 '•'These two substances then enter the central 
 lacteal, where equilibrium is again established ami 
 there is a large production of fat." 1 
 
 Immunity 
 
 Ehrlich's Ricin Experiments. 2 — Powder Albert 
 biscuits weighing 6.75 grams. Add to each cake 
 
 1 Kastle and Loevenhart : loc cit., p. 522. 
 
 2 Ehrlieh : Deutsche medicinische "Wochensehrift, 1891, 
 xvii, pp. 976-979. 
 
 Ricin is a toxalbumin extracted from the seeds of the castor 
 oil plant. It is poisonous in the slightest traces. Weight for 
 weight it is a billion times more poisonous than corrosive sub- 
 limate. Intravenous injection of* 0.03 milligram (0.00003 gram) 
 per kilo of body weight is fatal. One gram commercial ricin 
 would kill one and one-half million guinea-pigs. The effect is 
 about one hundred times less when taken by the mouth, yet 
 
68 FERMENTATION 
 
 3.2-3.5 c.c. of water containing ricin. The be- 
 ginning content of ricin should be 0.02 gm. ricin 
 for each cake ; 0.035 gm. is fatal in the course 
 of five or six clays. Mix the biscuit powder and 
 ricin solution to a stiff dough, roll the dough into 
 rods, divide them into equal lengths, and dry 
 the portions quickly on a wire sieve. Determine 
 the effect on white mice of successively increas- 
 ing doses, as follows : 
 
 )AY 
 
 DOSE 
 
 DAY 
 
 DOSE 
 
 1 
 
 0.002 gm. 
 
 9 
 
 0.02 
 
 2 
 
 . . . 
 
 10 
 
 0.03 
 
 3 
 
 0.006 
 
 11 
 
 0.04 
 
 4 
 
 0.008 
 
 12 
 
 0.05 
 
 5 
 
 . . . 
 
 13 
 
 0.06 
 
 6 
 
 0.01 
 
 14 
 
 . . . 
 
 7 
 
 0.0125 
 
 15 
 
 0.08 
 
 8 
 
 0.015 
 
 16 
 
 0.01 
 
 On the 17th day inject subcutaneously a fresh 
 mouse with the fatal dose — 1 c.c. of a ^o^o o"o" so " 
 lution per 20 gm. of mouse. At the same time 
 
 even thus 0.18 gram will kill a full-grown man. The cause of 
 death is agglutination of red blood corpuscles, and hence 
 multiple thrombosis, especially of the abdominal vessels. 
 Clinically, violent diarrhoea and progressive exhaustion are ob- 
 served. The toxicity is greatly dependent on species. Guinea- 
 pigs are far more susceptible than white mice. With white 
 mice the fatal subcutaneous injection is 1 c.c. of a solution con- 
 taining zthjW ricin l )er 20 g rams °f Dod y weight. 
 
IMMUNITY 69 
 
 inject the immunized mice with a dose one 
 hundred times as great. 1 
 
 Observe the non-immune and the immune mice 
 for several days and note the results. 
 
 Ehrlieh continued the above experiment until the 
 immunized mouse received daily 0.5 gm. of the ricin 
 by the mouth. Such animals bore safely subcutaneous 
 injections of -1q and even more. The immunity also 
 appeared in that solutions of 0.5-1.0 per cent applied 
 to the eyes of non-immune mice caused violent pano- 
 phthalmitis, while immune mice bore easily the appli- 
 cation of 10 per cent solutions. 
 
 This absolute local immunity was fully established 
 when the general immunity had attained only a 
 middle grade. Normally the subcutaneous injection 
 of 400V00 ricin solution causes severe local inflamma- 
 tion, but thoroughly immunized animals bear T isW* 
 Quantitative experiments show that the resistance to 
 the poison is not increased during the first four days, 
 and the increase is doubtful on the fifth day, but on 
 the sixth day a relatively high (for example thirteen- 
 fold) general immunity is suddenly established. The 
 sudden fall toward normal temperature observed in 
 diseases with a "crisis," such as pneumonia, may de- 
 pend on the " critical " establishment of immunity. 
 
 Immunity is not increased by continued administra- 
 tion of the same dose, day by day. An equilibrium 
 .appears to be established. 
 
 1 The mice in these experiments must be carefully protected 
 against cold and wettincr. 
 
70 FERMENTATION 
 
 The immunity once established endures a consider- 
 able time ; six months and possibly much longer. 
 
 Ricin Antitoxine. — Defibrinate the blood of the 
 immunized mice. Divide it into two portions. 
 1. To one portion add ricin solution in such a 
 ratio that the mixture shall contain yomIFO' *■ e - 
 twice the fatal amount. 
 
 Inject a fresh mouse subcutaneously with 1 c.c. 
 of this mixture per 20 grams of weight. 
 
 The poison will be borne. It has been neu- 
 tralized by the serum of the immune animal. 
 This result accords with the discovery of Behring 
 and Kitasato that immunity in diphtheria and 
 tetanus depends on the power of the serum to 
 neutralize the poison. 
 
 2. Divide the second portion of the antitoxine 
 blood among six small test-tubes. To the first 
 add a few drops yoo^o'o r ^ cm solution. To the 
 others add amounts increasing in a definite ratio. 
 
 At first there will be no effect (immunity). 
 As the amount of ricin added is increased, a point 
 will be reached at which agglutination of red 
 corpusles will be produced. This is the neutrali- 
 zation point. 
 
 Evidently, there is a definite quantitative 
 chemical relation between the toxine and the 
 antitoxine. 
 
IMMUNITY 71 
 
 Theory of Immunity. 1 — Jenner discovered the 
 protective action of vaccinia against sniall-pox. The 
 small-pox virus when passed through a susceptible 
 animal becomes attenuated. This weakened poison 
 introduced into the circulation in man protects the 
 individual for long periods against the original disease 
 — it establishes an artificial immunity against small- 
 pox. Schwann found that fermentation and putre- 
 faction arose through the agency of micro-organisms 
 coming from without. Pasteur, and Koch demonstrated 
 that the inoculation of animals with pure cultures of 
 certain bacteria produced specific infectious diseases, 
 and that these cultures could be modified at will, 
 either by passing through the animal body, as in 
 Jenner's method, or in artificial culture media. Pas- 
 teur produced artificial immunity by using attenuated 
 virus. Behring discovered ^that the blood-serum of 
 animals immunized against diphtheria contained a sub- 
 stance which would protect other animals against the 
 toxine of diphtheria. So also with tetanus. Ehrlich 
 introduced the quantitative study of toxiues and anti- 
 toxines by means of test-tube experiments, thereby 
 eliminating the uncertain factor of the animal body. 
 Thus it was shown in experiments on tetanus toxine 
 that the action of antitoxines is accelerated by heat, 
 retarded by cold, dependent on concentration — in 
 short, that it is a chemical action. In the above'ex- 
 periments on ricin, it is shown that the relation 
 
 1 Ehrlich : Croonian Lecture, Proceedings of the Royal 
 Society, London, 1901, lxvi, pp. 424-118. 
 
72 FERMENTATION 
 
 between toxine and antitoxine is quantitative. These 
 results, obtained by test-tube experiments, have been 
 confirmed by observations on living animals. Thus it 
 was established that a fixed quantity of toxine is neu- 
 tralized by a fixed quantity of its specific antitoxine. 
 
 Chemical substances atfect only those tissues with 
 which they are able to come . into chemical contact. 
 They must first reach the tissue. This general law is 
 illustrated by the experiments of Douitz with tetanus 
 toxine. 1 When the toxine is injected directly into 
 the circulation and immediately followed by a chemi- 
 cally equivalent amount of antitoxine, the animal is 
 not poisoned ; all the toxine circulating in the blood 
 is neutralized. When the same neutralizing dose is 
 injected eight minutes after the toxine, death occurs 
 from tetanus exactly as if no antitoxine had been used. 
 In these eight minutes a lethal quautity of toxine 
 must have left the blood and entered the tissues. 
 This toxine which has entered the tissues may still 
 for a time be withdrawn by injection of the specific 
 antitoxine in quantities much greater than the simple 
 neutralizing dose. The longer the delay, the larger 
 the saving dose. But after a fixed interval, or "period 
 of incubation," no amount of antitoxine, however 
 large, will prevent tetanus. There must, therefore, 
 be present in the brain or cord (the organ princi- 
 pally affected by tetanus toxine) certain atom groups 
 which, like the antitoxine, have a chemical affinity 
 for the toxine. At the close of the period of incuba- 
 
 1 Donitz : Klinisches Jahrbuch, 1900, vii. 
 
IMMUNITY 73 
 
 tion the chemical union between these atom groups 
 and the toxine is complete and the antitoxine is shut 
 out. Wassermann l found that when tetanus toxine 
 was mixed with fresh brain or cord substance from the 
 guinea-pig, the toxine united chemically with the nerve 
 centres so that neither the surrounding liquid nor the 
 mixture itself was poisonous when injected into an 
 animal. 
 
 The stable benzene ring and the less stable side-chains 
 of the benzene derivatives 2 suggested to Ehrlich that 
 living cells also consist of a stable centre and less stable 
 side-chains. The side-chains enable the cell to form 
 chemical combinations with food stuffs and other bodies 
 that possess atom groups having a chemical affinity 
 with the atom groups in the side-chains. It is in this 
 way that the toxine is bound to"the cell. Experiments 
 have shown that the binding atoms in the toxine 
 molecule are not the poison atoms. If for a portion 
 of fresh toxine there be determined quantitatively (1) 
 the killing power and (2) the amount of antitoxine 
 required to neutralize the toxine, aud if the remainder 
 of the toxine be then allowed to stand for a time, it 
 will be found, on again determining the toxic power 
 and the combining power, that the toxic power has di- 
 minished, while the combining power remains almost 
 the same. Hence, two separate and independent groups 
 exist. Ehrlich terms the combining atoms the hapto- 
 phore group, while the poison atoms are the toxophore 
 
 1 Wassermann : Berliner klinisclie Wochenschrift, 1898. 
 
 2 See page 65. 
 
<4 FERMENTATION 
 
 group. The haptophore atom group (a-n-TU), I cling to) 
 unites with the antitoxins, if there be any present, or 
 with any other atom group for which it has chemical 
 affinity. If this latter atom group be in the side-chain 
 of a living cell, its union with the haptophore atoms 
 of the toxine will necessarily bring the poison atoms of 
 the toxine into intimate chemical relationship with the 
 central atoms of the cell. Poisoning will then take 
 place. If the cells of vital organs have no atom groups 
 with chemical affinity for the haptophore group of a 
 toxine, no union between cell-atom group and hapto- 
 phore takes place, the toxophore is not brought into 
 intimate contact with the cell, and poisoning does 
 not occur. The animal is naturally immune to this 
 particular toxine. Thus a toxine in sausages is exces- 
 sively poisonous to ma*n, the monkey, and the rabbit, 
 while even large amounts are not injurious to the 
 dog. 
 
 The haptophore group of the toxine acts immediately 
 after injection into the organism, while in most or all 
 toxines the toxophore group becomes active only after 
 a longer or shorter incubation period. During this 
 period the animal may often be saved by placing it in 
 conditions in which the toxophores cannot act. Thus 
 frogs kept at less than 20° C. are not poisoned by large 
 doses of tetanus toxine, though much smaller doses are 
 fatal at a higher temperature (Morgenroth). 
 
 The toxophile atom group of the cell was not pre- 
 destined to unite with a remotely possible toxine, — 
 it has a normal function, probably that of attaching 
 food to the cell. When it enters, into its firm and 
 
IMMUNITY 75 
 
 enduring union with the haptophore group of a toxine, 
 this normal function is lost. Such a loss acts as a 
 physiological stimulus. 1 Xew side-chains are produced 
 by the cell, only to unite with fresh toxine. The pro- 
 duction and the loss of side-chains continue until all 
 the toxine in the blood is neutralized. By this time 
 the cell has become habituated to a more than normal 
 production of these special atom groups. The excess 
 is cast off like a secretion and circulates in the blood. 
 These free side-chains, possessing a special affinity for 
 one specific toxine, constitute the antitoxine of that 
 toxine. 
 
 Their continued production after the neutralization 
 of all the toxine protects the animal against fresh 
 toxine, i. e. establishes continued immunity. 
 
 It has already been stated that by special means the 
 toxophore group of a toxine may be weakened or 
 destroyed while its haptophore group is unchanged. 
 Such altered and non-poisonous toxines are termed 
 toxoids. As their affinity for the side-chains of the 
 cells remains unaltered, the toxoids by continuing to 
 unite with the side-chains of the cells may stimulate 
 the production of such side-chains in excess, or, in 
 other words, may assist in making antitoxine and thus 
 establishing immunity. 
 
 1 Weigerr : Deutsche medieinische "Wochenschrift, 1896. 
 
76 FERMENTATION 
 
 Haemolytic and Bacteriolytic Ferments 
 
 Bordet's Experiments. 1 — Inject into the perito- 
 neum of a guinea-pig 10 c.c. defibrinated rabbit 
 blood on five successive days. After two more 
 days bleed the guinea-pig and obtain the serum, 
 by allowing the blood to stand in test-tubes in a 
 cool place until the shrinking clot has pressed 
 out the serum. 
 
 1. Mix a drop of serum from a fresh guinea- 
 pig (one not injected with rabbit blood) with a 
 drop of defibrinated rabbit blood and examine 
 under the microscope. The corpuscles show a 
 very slight agglutination, but are otherwise un- 
 injured. The normal serum of the guinea-pig is 
 almost inactive upon rabbit blood. 
 
 2. A. Mix a drop of the serum from the 
 injected guinea-pig with a drop of defibrinated 
 rabbit blood and examine under the microscope. 
 The corpuscles are strongly agglutinated. 2 
 
 B. Mix 0.5 c.c. of the serum with 1.5 c.c. 
 defibrinated rabbit blood. 
 
 1 Bordet : Amiales de 1'Institut Pasteur, 1898, xii, pp. 692- 
 694. 
 
 2 Agglutinated blood looks granular, especially on gentle 
 shaking ; the massed corpuscles sink rapidly ; they will not pass 
 through filter paper. Agglutination of blood corpuscles is 
 similar to the clumping of the typhoid bacillus in the serum of 
 a typhoid-fever patient. 
 
HAEMOLYTIC, ETC. FERMENTS 77 
 
 The corpuscles are agglutinated and their hae- 
 moglobin is set free. The mixture becomes red, 
 clear and limpid in two or three minutes. With 
 the microscope nothing can be found but the 
 stroma of the corpuscles, more or less deformed, 
 very transparent and scarcely visible. 
 
 The continued presence of blood corpuscles 
 of the rabbit in the blood of the guinea-pig has 
 developed in the latter the power to agglutinate 
 the corpuscles and to set free their haemoglobin. 
 It is thus that the guinea-pig protects itself ; it 
 acquires immunity. 
 
 3. Heat 1 c.c. of serum fco 55° C. for half an 
 hour. Add 0.5 c.c. of this to 1 .5 c.c. defibrinated 
 rabbit blood as in Experiment 3. 
 
 The serum which was heated to od° C. no 
 longer destroys the corpuscles, but still strongly 
 agglutinates them. 1 
 
 Evidently the agglutination of the corpuscles 
 and the setting free of the haemoglobin (termed 
 " hiking ") are effected by different substances. 
 The agglutinating body resists a temperature that 
 destroys the blood-laking body. 
 
 4. To the mixture used in the preceding experi- 
 ment, add 2 c.c. of fresh serum from a normal 
 
 1 A very slow destruction of the red corpuscles may be ob- 
 served. This, however, is due to the fresh serum in the 1.5 c.c. 
 defibrinated rabbit blood, as will be evident from Experiment 4. 
 
78 FERMENTATION 
 
 guinea-pig (one that has not been iujected with 
 rabbit blood). 
 
 In a few minutes the mixture becomes limpid 
 and red. The laking power is restored. 
 
 Obviously, with the fresh serum was added 
 the unstable body destructive to red corpuscles.. 
 Ehrlich and Morgenroth have shown that at low 
 temperatures the stable body unites with the red 
 corpuscles while the unstable body remains in 
 the serum ; in this case the haemoglobin is not 
 set free. At higher temperatures the haemoglo- 
 bin separates and the unstable body is found to 
 have left the serum. It has joined the stable 
 body in the sediment. 
 
 Following the side-chain theory already men- 
 tioned, Ehrlich and Morgenroth assume that the 
 stable substance has two combining powers; 
 on the one hand it unites with the red corpus- 
 cles, on the other with the unstable substance, 
 thus bringing it to the cell which it may then 
 destroy. 
 
 Immunity against toxines and foreign red cor- 
 puscles are only two of the protective actions of 
 the blood. The injection of cells of the most 
 varied kinds is followed by the production of 
 specific protective bodies ; * thus, the injection of 
 
 1 Metchnikoff: Annales de l'lnstitut Pasteur, 1900, xiv, 
 p. 369. 
 
OXIDIZING FERMENTS 79 
 
 bacteria causes the formation of bacteriolysines, 
 which. destroy the injurious organism. 
 
 Many haemolysines and agglutines are found 
 in plants ; others, for example, the tetanus bacil- 
 lus, are bacterial ; still others, such as snake 
 venom, are animal secretions. 
 
 Oxidizing Ferments 
 
 Schonbein's Experiment. 1 — 1. To ten grams 
 hydrogen peroxide add tincture of guiac (freshly 
 prepared by dissolving guiac resin in alcohol) 
 drop by drop until the liquid is milky. Now 
 add from eight to ten drops of a somewhat con- 
 centrated extract of malt, prepared in the cold. 
 
 The guiac will be oxiclfzed and will turn blue. 
 
 2. Eepeat the experiment, adding in place of 
 the malt extract from eight to ten drops of blood. 
 
 The guiac will be oxidized, as before. 
 
 Further Oxidations by Animal Tissues. 2 — 
 1. Soak strips of bibulous paper in a diluted solu- 
 tion made as follows : 
 
 a-naphthol 1 ruol. 
 
 sodium carbonate .... 3 " 
 para-phenylenediamine . . 1 " 
 
 1 Schonbein : Zeitschrift fiir Biologie, 1868, iv, p. 367. 
 
 2 Spitzer : Archiv fiir die gesammte Physiologie, 1895, Ix, 
 pp. 322-323. 
 
80 FERMENTATION 
 
 This solution, left in the atmosphere, oxidizes 
 slowly to indophenol (violet color). 
 
 Place a drop of a known oxidizer, e.g. ferri- 
 cyanide of potash or potassium chroma te, on the 
 saturated paper. 
 
 The color will change at once, in consequence 
 of immediate oxidation. 
 
 (1) C 6 H 4 (NH 2 ) 2 + C 10 H 7 OH + = 
 
 PARA-PHEXYLENEDIAMINE A-NAPHTHOL C«H.iXFTo 
 
 nh < c :h 6 oh + w> 
 
 & KH /' H ' NH! + O - N ^ C » H * NHa +H O 
 * H< C w H 6 OH + ° - ^<C„H.O +H '° 
 
 INDOPHENOL 
 
 Each of the combining molecules has been acted 
 upon by a different oxygen atom ; hence the oxy- 
 gen molecule must have been split. 
 
 2. Bub the test paper with finely divided tissue 
 from the liver or any other organ. 
 
 Oxidation will occur. B 
 
 A drop of blood placed on the test paper is 
 soon surrounded by a characteristically colored 
 ring. 
 
 Extraction of Nudeo-Proteicl from Liver} — 
 Perfuse a fresh liver (dog) with tap water until 
 the washings are no longer colored bv haemo- 
 
 o 
 
 1 Spitzer : Archiv fur die gesammte Physiologie, 1897, lxvii, 
 p. 616. 
 
OXIDIZING' FERMENTS 81 
 
 globin. Grind the liver to a pulp and press 
 through several thicknesses of gauze. Add five 
 volumes of distilled water. Allow the mixture 
 to stand twenty-four hours at low temperatures. 
 Remove the opalescent watery extract with a 
 pipette and filter through linen. Demonstrate 
 with the microscope that liver cells are absent 
 from the liquid. Add T 7 ^ HC1 drop by drop until 
 there is no further precipitation, and the super- 
 natant fluid is clear. Since the precipitate redis- 
 solves in acid, use lacmoid as an indicator. Cease 
 when the lacmoid shows a trace of excess. De- 
 cant the precipitate, filter, wash the residue with 
 water. 
 
 Oxidation by Nucleo-Proteid. — Place in a wide- 
 necked flask 50 c.c. waSer containing 0.2 gram 
 of the fresh, brown substance and 10 c.c. hydro- 
 gen peroxide in a small glass cup. The hydrogen 
 peroxide must be neutralized with from 1 to 1.5 
 c.c. y ? o XaOH. Connect the flask with the lower 
 end of a eudiometer by means of a bent tube. 
 Shake the flask so that the hydrogen peroxide 
 shall come in contact with the tissue. Oxygen 
 is at once set free. Read in the eudiometer 
 the oxygen ' developed from minute to minute. 
 Spitzer found : 
 
 After minutes . . 12 3 4 5 8 9 16 
 
 C.c. 2 developed 19 28 41 55 69 85 87 95 
 
 6 
 
82 FERMENTATION 
 
 Oxidation about the Nucleus. 1 — Introduce the 
 oxidizable solution of a-naphthol and para-phe- 
 nylenediamine (page 79), beneath the cover 
 glass of a fresh preparation of teased thymus 
 or spleen. 
 
 " Granules of the intense greenish-blue oxi- 
 dation product shortly make their appearance 
 within the leucocytes. Their first appearance is* 
 typically at the boundary between nucleus and 
 cytoplasm ; eventually the latter may become 
 so densely laden as completely to obscure the 
 nucleus. . . . The nucleus is the chief agency in 
 the intracellular activation of oxygen. The ac- 
 tive or atomic oxygen is in general most abun- 
 dantly freed at the surface of contact between 
 nucleus and cytoplasm." 
 
 Glycolysis in Blood. Bernard's Experiment? — - 
 125 c.c. dog's blood were divided into five equal 
 parts. The sugar in each was estimated as 
 follows : 
 
 
 
 
 
 Sugar 
 Grams per 1000 
 
 1. 
 
 Analysis 
 
 made 
 
 at once . . . 
 
 . 1.07 
 
 2. 
 
 a 
 
 a 
 
 after 10 minutes 
 
 . 1.01 
 
 3. 
 
 u 
 
 a 
 
 " 30 " . 
 
 . 0.88 
 
 4. 
 
 a 
 
 a 
 
 " 5 hours . 
 
 . . 0.44 
 
 5. 
 
 a 
 
 u 
 
 " 24 " 
 
 . 0.00 
 
 1 Lillie : American journal of physiology, 1902, vii, p. 420. 
 
 2 Bernard : Comptes rendus de I'acadetnie des sciences, Paris, 
 1876, lxxxii, p. 1406. 
 
OXIDIZING FERMENTS 83 
 
 Sugar disappears from the blood on stand- 
 ing. 
 
 It has been found by Lepine and Barral * that 
 the glycolytic power of the blood increases as the 
 temperature rises to 52.5° C, which is the opti- 
 mum. At 54° the ferment is destroyed. 
 
 Oxidation not Dependent on Living Cells of Blood. 
 — Place the following solutions at 3-4-35° C. for 
 six hours, allowing a stream of air to pass through 
 the liquid. Then estimate the sugar. 2 
 
 A. Calf's blood 100 c.c. 
 
 Water containing 1.14 gram grape sugar 10 c.c. 
 Seegen 3 recovered 1.000 gram. 
 
 1 Lepine and Barral : Comptes rendus de Tacademie des sci- 
 ences, Paris, 1891, cxii, p. 146.' 
 
 2 Test the nitrate by adding a drop of acetic acid and a little 
 ferrocyanide of potassium. 
 
 The absence of a precipitate shows freedom from proteids and 
 ferric salts. Concentrate filtrate to 150-200 c.c. 
 
 Titration of the Sugar Extract. — Make the volume of the 
 solution such that its probable content of sugar shall lie 
 between 0.0004 and 0.0010. Causse (Bulletin de la Societe 
 chimique de Paris, 1, p. 625) recommends that 1750 c.c. of 
 water containing 5 grams of ferrocyanide of potassium be added 
 to each 250 c.c. of Fehling's solution. Boil 10 c.c. of this 
 mixture and add the sugar solution drop by drop until the blue 
 liquid is decolorized (Arthus : Archives de physiologie, 1891, 
 p. 425). 
 
 3 Estimation of Sugar in Blood. Extraction of the Sugar 
 from the Blood. — To 350-400 c.c. boiling water add all at once 
 50 c.c. blood containing 5 c.c. one per cent acetic acid. Let 
 
S-l FERMENTATION 
 
 B. Calf's blood 100 c.c. 
 
 Water containing 1.14 gram grape sugar 10 c.c. 
 
 Chloroform 1 c.c. 
 
 Seegen recovered 0.9G0 grain. 
 
 ■ The chloroform destroys the cells, but fails to 
 check the oxidation. 
 
 Relation of Glycolysis to the Pancreas and the 
 Lymph. 1 — Remove the pancreas aseptically from an 
 anaesthetized dog which has fasted thirty-six hours. 
 Estimate the sugar in the urine at intervals of a few. 
 hours. 
 
 Sugar will be present in large and increasing quanti- 
 ties, 2 rising even to twenty per cent. 
 
 Inject into the jugular vein 15-20 c.c. of lymph 
 from the thoracic duct of a dog fed a few hours before 
 upon one litre of milk. 
 
 the mixture boil for a few minutes. Filter through a small 
 linen cloth. 
 
 Separation of Proteids. — Boil the filtrate. Most of the pro- 
 teids will separate by coagulation. The remainder, if necessary, 
 may be removed by adding to each 300 c.c. of filtrate, 5 c.c. 
 saturated solution of sodium acetate, and a small quantity of a 
 dilute solution of ferric chloride, neutralizing almost completely 
 with dilute soda solution, and boiling. The ferric chloride will 
 precipitate as ferrous chloride and will carry down the last 
 traces of proteid substances. Filter. "Wash with boiling water. 
 (Seegen : Centra lblatt fur Physiol ogie, 1891, v, p. 824.) 
 
 1 Lepine : Comptes rendus de l'academie des sciences, Paris, 
 1890, ex, p. 742. 
 
 2 Von Mering and Minkowski : Archiv fiir experimentelle 
 Pathologie und Pharmakologie, 1890, xxvi, p. 371. 
 
OXIDIZING FERMENTS 85 
 
 The glycosuria will greatly diminish. 
 
 After a few hours, the glycosuria will become once 
 more intense, continuing until death. The quantity of 
 sugar in the blood is also greatly increased. 
 
 Glycolytic Ferment of Pancreas. 1 — Eemove the 
 pancreas aseptically from a dog immediately after 
 death. Crush it at once in 100 c.c. sterile water 
 containing- 0.2 gram sulphuric acid. Allow it to 
 macerate two hours at 38° C. Neutralize the 
 acid with soda, add 0.5 gram pure glucose, and 
 keep the mixture one hour at 38° C. Estimate 
 the sugar. 
 
 The loss will be from ten to fifty per cent. 
 
 When pancreatic extract made without acid is used, 
 the loss of sugar is much Jress. Probably, therefore, 
 the glycolytic ferment is produced from a zymogen by 
 hydration. 
 
 Malt diastase, or salivary diastase, kept three hours 
 at 38° in water containing one tenth per cent sulphuric 
 acid loses the power to change starch to sugar, but ac- 
 quires a glycolytic power. 
 
 If the pancreatic juice which flows upon stimulation 
 of the peripheral end of the vagus (Pawlow) is treated 
 with dilute acid, 1 : 1000, the amylolytic power is lost, 
 but glycolytic power is acquired. During the excita- 
 tion of the nerve — while the juice is flowing — the 
 
 1 Lepine : Comptes rendus de l'academie des sciences, Paris, 
 1895, cxx, p. 139.' 
 
86 FERMENTATION 
 
 blood in the pancreatic vein has almost no glycolytic 
 power; after the jnice ceases to run, the blood has 
 considerable glycolytic power. Here the external is 
 balanced against the internal secretion of the pancreas. 
 
 Oxidative ferments are very widely distributed both 
 in animals and plants. The above experiments show 
 their presence in the blood, pancreas, liver, and lymph. 
 They are present also in the urine. 
 
 The stomach contains a ferment that oxidizes lactone 
 to lactic acid. 1 
 
 Urushi, the milky secretion of Rhus vernicifera, dries 
 in the air to a translucent varnish (Japanese lacquer). 
 It contains urushic acid, which does not dry sponta- 
 neously, and a ferment, the addition of which to 
 urushic acid causes the latter to dry to lacquer. A 
 sample of fresh juice boiled to stop the action of the 
 ferment on urushic acid contained 15.01 per cent 
 oxygen ; lacquer dried in the usual manner contained 
 20.52 per cent oxygen. 
 
 Many oxidations are effected by the tissues without 
 the aid of ferments, so far as is yet known. These be- 
 long properly to metabolism, but in passing, it may be 
 noted that while substances exceedingly resistant to 
 oxidation, for example, proteids, are oxidized in the 
 body, other substances very easily oxidizable may be 
 excreted unchanged ; oxalic acid is one of these. 2 
 
 1 Haramarsten : Maly's Jahresbericht derThierchemie, 1872, 
 ii, p. 118. (Original in Swedish.) 
 
 2 Pohl: Arehiv fur experhnentelle Pathologie und Pharraa- 
 kologie, 1896, xxxvii, p. 413. 
 
ALCOHOLIC FERMENTATION 87 
 
 Hoppe-Seylers Theory. 1 — Living tissues consist of 
 easily combustible reducing substances, which split 
 the oxygen molecules, taking to themselves one atom 
 of and setting the other free in active state to 
 unite with any oxidizable substance present. 
 
 Traube's Theory? — Iu living protoplasm oxygen is 
 rendered active by an oxidizing ferment, which brings 
 the oxygen to bodies ordinarily oxidizable only by such 
 powerful agents as heat and strong alkalies. 
 
 Inorganic bodies, e. g. platinum black, the oxides of 
 copper, silver, mercury, and vanadium, and certain iron 
 salts similarly act as oxygen carriers. Thus 
 
 (1) Pt -f 0, + H, = PtO + H 2 
 
 (2) PtO + H 2 = Pt + H 2 
 
 The oxygen carrier reduces H 2 2 , takes one atom 
 to itself, then gives off this atom in an active or 
 nascent state to oxidize #hy oxidizable compound 
 present ; e. g. guiac Grape sugar takes from 
 indigo-blue, producing thereby indigo-white. The 
 indigo-white oxidizes itself to indigo-blue, then gives 
 up another atom of 0, and so on. 
 
 Alcoholic Fermentation 
 The Yeast Plant. — Observe a solution of sugar 
 undergoing alcoholic fermentation. 3 Xote the 
 bubbles of gas, the scum, the sour odor. 
 
 1 Hoppe-Seyler: Zeitsehrift fur physiologische Chemie, 1879, 
 ii, p. 1. 
 
 2 Traube : Berichte der deutscben chemischen Gesellschaft, 
 1883, xvi, pp. 123, 1201, and earlier papers in volumes x and xv. 
 
 3 The fermentation is assisted by providing the yeast plant 
 
88 FERMENTATION 
 
 Examine some of the mixture under the micro- 
 scope. Note the multitude of globular or slightly 
 ovoid bodies, the largest about T ^ mm. in diame- 
 ter. They are motionless. Many have put forth 
 buds. They seem to be plants in active growth. 1 
 
 1. Place 300 c.c. of the nutrient liquid (Ex- 
 periment 1) in a flask holding 500 c.c. Add a 
 piece of fresh compressed yeast the size of a pea. 
 Place the flask in a temperature of 35° C. 
 
 Note that as fermentation advances the yeast 
 increases in quantity. 
 
 2. Place a small piece of fresh compressed 
 yeast in a test-tube. Fill the tube with nutrient 
 liquid and invert it in a dish of similar liquid. The 
 tube may be kept upright by a clamp. Let the 
 mixture stand twenty -four hours in a warm room. 
 
 with the salts present in the ash of yeast (Pasteur). A useful 
 substitute is 
 
 Potassium phosphate ... 20 gms. 
 
 Calcium phosphate ... 2 
 
 Magnesium sulphate ... 2 
 
 Ammonium tartrate . . . 100 
 
 Cane sugar 1,500 
 
 Water 8,376 
 
 10,000 
 (Practical biology, Huxley and Martin.) 
 1 Cagniard-Latour : L'Institut, 1835, iii, p. 150 ; also Annates 
 de chimie et de physique, 1838, lxviii, p. 206. The yeast plant 
 was first observed microscopically in beer-yeast by Leeuwen- 
 hoek, 1680, but he did not associate fermentation with the 
 growth of the yeast. 
 
ALCOHOLIC FERMENTATION 89 
 
 The tube will fill with gas. "With a bent pipette 
 introduce about 1 c.c. of a solution of sodium 
 hydroxide (sp. gr. 1.12 = 11 per cent). The gas 
 will be absorbed, with formation of sodic carbon- 
 ate, and the liquid will rise in the tube. 
 
 The growth of the yeast plant is accompanied 
 by the production of carbon dioxide. 
 
 3. Return to Experiment 1. After the fer- 
 menting liquid has ceased to give off gas, place a 
 stopper with a. bent tube in the mouth of the 
 flask and -distill the contents of the flask in a 
 water bath. Condense the first fifth of the dis- 
 tillate. Saturate this with sodium carbonate. 
 Redistill, and condense. 
 
 Test for alcohol by warming the distillate with 
 potassium dichromate and dilute sulphuric acid, 
 whereby the alcohol will be oxidized to aldehyde, 
 with characteristic odor. 
 
 Alcohol is present. 
 
 The production of alcohol by the yeast is the work 
 of the ferment zymase. 1 This body is closely bound 
 to the protoplasm of the cell, very easily destroyed, not 
 produced in excess, and not secreted free. Only sugars 
 containing three, six, and nine carbon atoms are at- 
 tacked. The saccharobioses must be " inverted" be- 
 fore they can be fermented. Thus, cane sugar must 
 
 1 Buclmer : Berichte der deutschen chenrisclien Geaellschaft, 
 1897, xxx, pp. 117, 1110, 2668. 
 
90 FERMENTATION 
 
 first be inverted to grape sugar by invertin, 1 and 
 malt sugar by maltase. Lactase is present in some 
 yeasts, enabling them to ferment milk sugar. Diastase 
 is also found. 
 
 The action of these several ferments becomes clear 
 when the chemical nature of the carbohydrates is 
 recalled. * 
 
 Chemical Relations of Carbohydrates. — Carbohy- 
 drates were formerly defined to be compounds con- 
 taining six, or a multiple of six carbon atoms, together 
 with hydrogen and oxygen atoms in the proportion in 
 which they exist in water. The researches of E. 
 Fischer have shown that all aldehydes (bodies which 
 are the first oxidation products of primary alcohols, and 
 which contain the carbouyl group CO) and all ketones 
 (bodies which are the first oxidation products of second- 
 ary alcohols and which likewise contain the carbouyl 
 group CO) contain carbon, hydrogen, and oxygen, there 
 being two atoms of hydrogen to one atom of oxygen, 
 as in water. 
 
 The carbohydrates, therefore, no longer occupy an 
 isolated position, but are to be classed with the fats, 
 being metliane derivatives in which the carbon atoms 
 are arranged in an open chain; thus, grape sugar is an 
 aldehyde alcohol, and fruit sugar a ketone alcohol. 
 
 The carbohydrates are divided, according to the size 
 of their molecule, into monosaccharides, disaccharides, 
 and polysaccharides. The monosaccharides (e. g. grape 
 
 1 For extraction, see Lea : Journal of physiology, 1885, vi, 
 p. 142. 
 
ALCOHOLIC FERMENTATION 
 
 91 
 
 sugar) are the first oxidation products of the hexahy- 
 dric alcohols; the higher carbohydrates are anhydrides 
 of the monosaccharides. Most of the higher carbohy- 
 drates cannot be fermented directly, but must first 
 be hydrolyzed (i. e. take up water). This hydrolysis 
 may be accomplished by the prolonged action of dilute 
 acids at high temperatures, by the action of water at 
 still higher temperatures, or by specific ferments, e. g. 
 diastase, at the relatively low temperature of the body. 
 The polysaccharides, consisting of the starches, the 
 gums (e. g. dextrine or starch gum) and the celluloses 
 (wood fibre) differ greatly from the lower carbohy- 
 drates. The polysaccharides are usually amorphous 
 and are not easily soluble in water. 
 
 Carbohydrates. 1 
 
 Glucoses, Monoses 
 CeH^Oe. 
 
 Saccharobioses 
 
 C 12 H 22°11- 
 
 Polysaccharides 
 (C 6 H 10 O 5 )x. 
 
 Grape sugar — 
 Grape sugar — 
 
 -Malt sugar — 
 
 —Starch 
 
 
 Grape sugar — 
 Fruit sugar — 
 
 -Cane sugar 
 
 
 
 Grape sugar — 
 Galactose — 
 
 -Milk sugar 
 
 
 
 
 
 
 
 
 
 
 
 1 Richter's Organic Chemistry, Third American Edition, i, 
 p. 121. 
 
92 
 
 FERMENTATION 
 
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FERMENTATION 93 
 
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94 BLOOD 
 
 The zymase attacks only those sugars which present 
 a specific stereo-contiguratiun. The position of their 
 atoms in space must fit the position of the atoms of 
 the ferment (the lock and the key). Thus, only the 
 dextro-rotatory forms of the aldehyde sugars (d-glu- 
 cose, d-mannose, d-galactose) are attacked ; the sugars 
 that rotate the plane of polarized light to the left 
 are not attacked. It is probable that the zymase 
 of different species of yeast presents characteristic dif- 
 ferences. It is known that the products formed in the 
 fermentation of sugar by different species of yeasts are 
 to a large degree characteristic. Often these products 
 are injurious. Upon this specific action of ferments 
 testa the work of Hansen, 1 who taught the brewers to 
 make pure cultures of the most favorable species of 
 yeast, and thereby raised the- brewing industry to the 
 level of an applied science. 
 
 BLOOD 
 Specific Gravity 
 
 Drawing the Blood. — Wash the lobe of the ear 
 with a bit of absorbent cotton dipped in clean 
 water. 2 Bub the lobe dry with another piece 
 of cotton. Pass a three-sided surgical needle 
 
 1 Hansen: Untersuelmngen an der Praxis der Gahrungs- 
 Industrie, 1895. 
 
 2 Subjects who are "bleeders" are not to be used for this 
 observation. 
 
SPECIFIC GRAVITY 95 
 
 through a Bunsen flame. (Do not heat the 
 needle red or the temper will be drawn and the 
 sharpness lost.) Stretch the skin of the lobe 
 between the fingers of the left hand. Make a 
 quick puncture one-eighth inch deep in the edge 
 of the lobe. Press gently to start the flaw; 
 The blood must now flow freely. On no account 
 use blood squeezed out. 
 
 Determination of Specific Gravity. 1 — Fill a 
 small beaker half full of a mixture of benzol and 
 chloroform of a specific gravity of about 1059. 
 Let a drop of the blood fall into this mixture. 
 The drop will remain spherical, for blood does 
 not mix with benzol and chloroform. If the 
 drop sinks, add chloroform drop by drop, mean- 
 while stirring the mixture with a glass rod, until 
 the drop neither rises to the surface nor sinks 
 to the bottom but swims with the mixture. If 
 the drop rests upon the surface, add benzol in 
 a similar manner. When the drop neither sinks 
 nor floats, its specific gravity must be that of the 
 benzol-chloroform mixture. Pour the mixture 
 into a glass cylinder, through a piece of linen to 
 hold back the blood-drop, and take the specific 
 gravity of the^benzol-chloroform with an areoni- 
 
 1 Roy : Journal of physiology, 1884, v, p. ix. 
 Hammerschlag, A. : Wiener klinische Wochenschrift, 1890, 
 iii, p. 1018. 
 
96 BLOOD 
 
 eter. The result is also the specific gravity of 
 the blood. 
 
 The values obtained are slightly too low. The 
 error is one unit in the third decimal place. 
 
 Determine the specific gravity of the blood 
 under the following conditions. Record the re- 
 sults in the laboratory note-book. Hand to the 
 instructor a copy of your observations written in 
 ink upon a laboratory blank. The material col- 
 lected by the class will be analyzed statistically 
 by a committee and a report made. 
 
 1. The specific gravity of the blood in a healthy 
 man. 
 
 2. In the same man half an hour after drink- 
 ing 750 c.c. of water. 
 
 3. In the same man one hour after drinking 
 750 c.c. of water. 
 
 4. In the same man after profuse sweating. 
 Note any feeling of thirst. 
 
 5. In a healthy woman. 
 
 Hammerschlag found the specific gravity in 
 chlorosis and nephritis diminished as the haemo- 
 globin diminished. Xo relation was observed 
 between the appearance of oedema and a reduc- 
 tion in the specific gravity. 
 
 Counting the Red Corpuscles. — See that the 
 pipettes of the Thoma-Zeiss apparatus are per- 
 fectly clean and dry. Open the bottle contain- 
 
SPECIFIC GRAVITY 97 
 
 ing Gower's solution (sodium sulphate, 7.3 grams ; 
 acetic acid, 20 c.c; water, 125 c.c). Prick the 
 ear as directed on page 94. In a large drop 
 which has collected without pressure put the 
 point of the smaller Thoma-Zeiss pipette ("red 
 counter"). Fill the pipette to the mark 0.5 by 
 careful suction. Should the mark be passed, 
 lower the column to the mark by touching the 
 point of the pipette to filter paper. When the 
 mark is reached, clean the outside of the pipette, 
 dip the end in Gower's diluent solution, and 
 draw the liquid very carefully up to the mark 
 101. (Should the liquid pass the mark, the 
 pipette must be cleaned and dried and the whole 
 process repeated.) Close, the ends of the pipette 
 with the ringers, and shake it gently for one 
 minute in order to mix the blood thoroughly 
 with the diluent. The blood will now be diluted 
 200 times its volume. 
 
 Remove the rubber tube from the pipette. 
 Blow out the unmixed solution in the capillary 
 tube, between the point and the bulb, and several 
 drops of the mixture in the bulb. Wipe off the 
 end of the pipette. Touch it to the ruled disc. 
 Let a very small drop flow out. Place the cover- 
 glass on the drop. The flattened drop should 
 almost cover the glass. If it spread into the 
 moat, clean the disc and use a second, smaller 
 7 
 
98 BLOOD 
 
 drop. Tf Newton's color-rings cannot be seen 
 between the coverglass and the disc by placing 
 the eyes near the level of the coverglass, another 
 preparation must be made, with cleaner disc and 
 coverglass. 
 
 Use Leitz No. 5 or Zeiss D objective. Bring 
 the drop into focus and then, using the microm- 
 eter screw, find the ruled field. 
 
 On the central portion of the disc 1 square 
 millimetre has been ruled into 400 squares, each 
 square having therefore an area of ^^ square 
 millimetre. Each 16 small squares are sur- 
 rounded by double lines, thus forming a " large 
 square." In the Zappert-Ewing slide, the cen- 
 tral square of 1 mm. is surrounded by eight other 
 squares of 1 mm. each, and the central ruling is 
 extended through the surrounding squares, which 
 are intersected by lines ^ mm. apart. Count the 
 number of corpuscles, square by square, in 200 
 small squares. Corpuscles touching the north 
 and south lines of each area are to be counted 
 in, those touching the east and west lines are to 
 be omitted from the count. 
 
 Each square has an area of ^J^ square milli- 
 metre. The thickness of the layer of blood, i. e. 
 the distance from the ruled disc to the cover- 
 glass, is 0.1 mm. The volume of the space above 
 each square, therefore, is ^J^ cubic millimetre. 
 
SPECIFIC GRAVITY 99 
 
 As the blood is diluted 200 times its volume, and 
 the number of squares counted is 200, the total 
 number of corpuscles in a cubic millimetre is 
 
 x x 200 x 4000 
 200 
 
 x being the total number of corpuscles counted. 
 In short, to obtain the number of corpuscles in a 
 cubic millimetre, multiply by 4000 the number 
 counted in 200 squares. Clean the pipette as 
 soon as the counting is done. 
 
 Cleaning the Pipette. — Draw clean Gower's 
 solution through the pipette, then alcohol, and 
 finally ether. Dry the pipette by sucking (not 
 blowing) air through \tM 
 
 Control Counting. — Count the red corpuscles 
 in a second drop. If the result differ greatly 
 from that of the first count, the corpuscles in a 
 third drop must be counted. 
 
 Counting the White Corpuscles. — Have ready 
 a diluting solution of glacial acetic acid (one- 
 third of one per cent). This solution will make 
 the red cells invisible. Obtain a very large drop 
 of blood. Fill the large Thoma-Zeiss pipette 
 (white counter) by very gentle suction. Keep 
 
 1 Pipettes left dirty will be cleaned at the student's ex- 
 pense, or, where necessary, a new one purchased. The cost is 
 considerable. 
 
100 BLCOD 
 
 the pipette nearly horizontal, both in obtain- 
 ing the drop and in drawing in the diluting solu- 
 tion ; the bottle should be tilted. Count the 
 white corpuscles in the entire ruled disc. Eepeat 
 with a second, drop. 
 
 Estimation of Haemoglobin 
 
 Hoppe-Seyler's Method 1 (modified). — "Weigh 
 about five grams of crystallized haemoglobin. 
 Make a concentrated solution in a measured 
 quantity of distilled water. Saturate with car- 
 bonic oxide. Preserve in glass tubes containing 
 about 6 c.c., the ends drawn out and closed in 
 the Bunsen flame. Before using, dilute with 
 distilled water to 0.2 per cent CO-Haemoglobin 
 and saturate with CO. Place in one compart- 
 ment of the double container with parallel glass 
 sides. In the other compartment place about 
 2 c.c. of 0.1 per cent sodium hydrate solution. 
 Prom a drop of blood obtained from the ear as 
 directed on page 94 fill the pipette to the mark 
 (3 cubic millimetres; by careful suction. Should 
 the mark be passed, lower the column to it by 
 touching the point of the pipette to filter paper. 
 When the mark is reached, clean the outside of the 
 pipette, and then blow the contents of the pipette 
 
 1 Hoppe-Seyler : Handbuch der physiologisch- una patholo- 
 gisch-chemischen Analyse, 1893, p. 412. 
 
HAEMORRHAGE AND REGENERATION 101 
 
 into the 0.1 per cent sodium hydrate solution. 
 Eemove all the blood by drawing the sodium 
 hydrate solution in and out of the pipette. Look 
 at the parallel columns of haemoglobin solution 
 and blood solution through the blackened tube 
 against an evenly illuminated sheet of white 
 paper plaeed ten inches away. Dilute the blood 
 with 0.1 per cent XaOH solution until its color 
 is precisely that of the haemoglobin solution. 
 
 Measure the volume of the two solutions in 
 c.c. Then the volume of the haemoglobin («) 
 solution is to the known quantity of haemo- 
 globin which it contains («') as the volume 
 of the blood solution (b) is to the desired weight 
 of haemoglobin (x) it contains, or the weight of 
 haemoglobin in three cubic millimetres of the 
 blood. 
 
 Haemorrhage and Regeneration 
 
 Determine the specific gravity, number of red 
 and white corpuscles per millimetre, and per- 
 centage of haemoglobin in the same animal under 
 the following conditions : Xormal ; two hours 
 after a profuse haemorrhage ; one day, three days, 
 and five days after the haemorrhage. Plot all 
 three curves upon one co-ordinate system. 
 
102 BLOOD 
 
 Alkalinity 
 
 Zuntz-Loewy 1 -Eiigel 2 Method. — Place j^ tar- 
 taric acid 3 in a finely graduated pipette (1 c.c. 
 in 20) with glass stopcock. 
 
 Place in a small beaker 5 c.c. of water dis- 
 tilled in glass and proved neutral in reaction. 
 Select the larger pipette of the Thoma-Zeiss 
 haemocytometer. Note the cubic millimetres 
 contained in the pipette 4 up to the mark 0.5. 
 Remove this quantity from the water. Dry the 
 pipette with alcohol (page 99). Fill the pipette 
 to the mark 0.5 with blood from the ear. Re- 
 move any blood from the outside of the tube. 
 Place the end of the tube in the neutral water 
 and gently expel the blood. Wash out the last 
 traces of blood by gently drawing the water in 
 and out of the tube. If this be done with care, 
 the mixture will now measure practically 5.0 
 c.c. including the blood. Titrate the mixture 
 with the Y5 tartaric acid solution drop by drop. 
 
 1 Loewy : Archiv fiir die gesammte Physiologie, 1894, lviii, 
 p. 462. 
 
 2 Engel : Berliner klinische Wochenschrift, 1898, xxxv, 
 p. 308. 
 
 3 A normal solution of tartaric acid (C 4 H c 6 ) contains 
 
 -^J — 75 g. in 1 litre. A ^ tartaric acid solution 
 
 contains ff '= 1 g. in 1 litre. 
 
 4 This must be determined for each pipette. 
 
ALKALINITY 103 
 
 Before and after the addition of each drop of 
 the acid let a drop of the mixture fall gently 
 on a piece of light-colored glazed litmus paper 
 previously impregnated with saturated neutral 
 sodium chloride solution. In such paper the 
 salts on which the alkaline reaction of the 
 blood largely depends diffuse more rapidly than 
 the haemoglobin. The latter forms a yellow spot 
 which is surrounded by a clear solution of the 
 blood salts. When the titration acid has com- 
 bined with all the alkali in the blood a sharp 
 red ring will appear around the yellow spot of 
 haemoglobin. This ring is clearer when the 
 blood is wiped off after having rested a few 
 moments on the paper.**" 
 
 Normally, the alkalinity of 0.05 c.c. blood is 
 satisfied by about 5 c.c. y\ tartaric acid. Then 
 100 c.c. blood would require 0.5x20x100 = 
 1000 c.c. y \ tartaric acid. 1000 c.c. = l litre 
 normal tartaric acid saturates 40 g. XaOH. 1 
 litre y \ tartaric acid = -f £ =533 mg. NaOH. The 
 alkalinity of 100 c.c. blood = 533 mg. XaOH. 2 
 Hence 0.05 c.c. blood = 0.25 mg. XaOH. 
 
 1 The same tint of paper should he used in 'comparative 
 experiments. Light colored papers should be observed by di- 
 rect light ; darker papers by transmitted light. Litmus may 
 be replaced by lacmoid (for directions, see Cohnstein : Virchow's 
 Archiv fur allgemeine Pathologie, cxxx, and Bockmann : Che- 
 misch-technische L'ntersuchungs-Methoden). 
 
 2 The average given by Engel is 426.4 — 533.0 mg. XaOH. 
 
104 kespiration 
 
 Coagulation Time. 
 
 Fasten on a glass slide a metal ring pierced 
 with a small hole. Place a little vaseline on 
 the upper surface of the ring. Upon this lay a 
 perfectly clean cover glass upon the middle of 
 the under surface of which has been placed a 
 large drop of blood drawn from the ear with the 
 precautions mentioned on page 94 As soon as 
 the drop is in place within the cell, make it ro- 
 tate by blowing gently against it by means of a 
 pointed glass tube applied to the hole in the metal 
 ring. 
 
 The drop will cease to move when coagulation 
 sets in. 
 
 Note the interval between the drawing of the 
 blood and the onset of coagulation. 
 
 The method is rough, and a fairly correct re- 
 sult requires much care and a number of obser- 
 vations, but even thus it reveals the important 
 diminution in coagulability in certain diseases, 
 e. g. jaundice. 
 
 RESPIRATION 
 
 Chemistry of Eespiration 
 
 Estimation of Oxygen, Carbon Dioxide, and 
 Water. 1 — Weigh bottles 3, 4, and 5 (4 and 5 
 
 1 Apparatus. — Two aspirator bottles, with box. A wooden 
 tray, containing a jar for the guinea-pig, and six bottles, viz. : 
 
CHEMISTRY OF RESPIRATION 
 
 105 
 
 together). Place the guinea-pig in the jar and 
 weigh. During one hour draw air through bot- 
 tles 1 to 6 by placing an aspirator bottle on its 
 box and allowing the water to flow from this 
 bottle to the one remaining on the desk. The 
 rubber connecting tube must be changed when 
 the aspirator bottles are changed. After one 
 hour weigh bottle 3, and bottles -i and 5. 
 Tabulate results as follows : 
 
 Grams. 
 
 Weight of jar and guinea-pig at beginning 
 " " " end . 
 Loss 
 
 Wi. of bottle 3 (sulph. acid) at beginning 
 " t end . 
 Gain (= water absorbed) . . . 
 
 Weight of bottles 4 and 5 at beginning 
 " " " end . . 
 
 Gain (= carbon dioxide absorbed) 
 
 Total water and carbon dioxide absorbed 
 Loss in weight of jar and guinea-pig . 
 Difference (= oxygen absorbed) 
 Respiratory quotient 
 
 ^Tos. 1 and 4, filled with soda-lime, to absorb carbonic acid ; 
 Kos. 2, 3, and 5, filled with pumice stone soaked in sulphuric 
 acid, to absorb moisture ; Xo. 6, a Miiller valve, to prevent air 
 being forced back through the series of bottles by a wrong 
 coupling of the aspirator tubes. 
 
106 respiration 
 
 Mechanics of Eespiration 
 
 Artificial Scheme. — Eaise the left glass" rod 
 above the opening in the rubber tubing. Hold 
 the lower end of the free cylinder even with the 
 rubber balloon, and pour in water till the level 
 just reaches the balloon. Lower the left glass 
 rod to cover the opening. 
 
 The surface of the water in the attached 
 cylinder represents the diaphragm and movable 
 chest-walls; the interior of the cylinder above 
 the water, the thoracic cavity ; and the rubber 
 balloon, the lungs. The left manometer shows 
 the intra-thoracic pressure ; the right manometer 
 shows the intra-pulmonary pressure. The left 
 glass rod closes the entrance to the cylinder, 
 i. e. makes the thoracic cavity a closed cavity, 
 as is normal ; the right glass rod, with its lower 
 end partly covering the opening in the rubber 
 tubing, controls the entrance to the balloon (the 
 respiratory passages). 
 
 Inspiration. — Nearly close the respiratory pas- 
 sage. Lower the water level to the base of the 
 thoracic cylinder. 
 
 Note the change in the size of the lung, and 
 in the pressure in the lung and in the thorax. 
 Give reasons for these changes. 
 
 Expiration. — Widen the respiratory passage 
 
MECHANICS OF RESPIRATION 107 
 
 slightly. Eaise the water level slowly till the 
 lung is slightly but evenly distended. 
 
 Note the pressure in the pleural cavity. Is it 
 positive or negative ? Why ? 
 
 Normal Respiration. — Slowly and rhythmi- 
 cally raise and lower the diaphragm (water level) 
 between the inspiratory and expiratory level, 
 taking care that the lung never becomes even 
 slightly collapsed at the end of expiration. 
 
 Give reasons for the changes in the intra- 
 pulmonary pressure. 
 
 Forced Respiration. — Eaise and lower the 
 diaphragm more quickly. 
 
 Observe that the differences in pressure are 
 increased. *- 
 
 Obstructed Air Passages. — Diminish the inlet 
 in the respiratory tube by moving the glass plug. 
 Eaise and lower the diaphragm. 
 
 The differences of pressure will be increased. 
 
 Asphyxia. — Close the entrance to the lungs 
 entirely. 
 
 Note the effect of movements of the diaphragm 
 upon the intra-thoracic and intra-pulmonary 
 pressures. 
 
 Coughing : Sneezing. — Eemove the glass rod 
 from the respiratory passage. Bring the lung to 
 full inspiration. Close the respiratory opening 
 with the moistened thumb. Eaise the diaphragm 
 
108 RESPIRATION 
 
 half-way toward expiration. Suddenly open the 
 respiratory passage. 
 
 Air is quickly and forcibly expelled from the 
 lung (cough, sneeze). 
 
 Hiccough. — Lower the diaphragm quickly 
 toward full inspiration, and while the lung is 
 expanding close the respiratory opening with 
 the moistened thumb (hiccough). 
 
 Note the sudden changes of pressure in the 
 two cavities. 
 
 Perforation of the Pleura. — Open the inlet to 
 the pleura. 
 
 Note the effect of the opening into the pleural 
 cavity upon the lung and upon the intra-pulmo- 
 nary and intra-thoracic pressure. 
 
 Observe the result of movements of the 
 diaphragm. 
 
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 Experiments for studerrtsj^ 
 
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