OUTLINES OF PRACTICAL PHYSIOLOGY: BEING A /iDanual for tbe pbEsiolocjical OLaborators, INCLUDING CHEMICAL AND EXPERIMENTAL PHYSIOLOGY, WITH REFERENCE TO PRACTICAL MEDICINE. BY WILLIAM STIRLING, M.D., Sc.D., PROPESSOR IN THE VICTORIA UNIVERSITY, BRACKENBURY PROFESSOR OP PHYSIOLOGY AND HISTOLOGY IN THE OWENS COLLEGE, MANCHESTER, AND EXAMINER IN PHYSIOLOGY IN THE UNIVERSITY OP OXFORD. With US Illustrations* CHARLES GRIFFIN & CO., LOND PHILADELPHIA: P. BLAKISTON, SON & CO., No. 1012 WALNUT STREET. 1888. (All Rights Reserved.) BeMcatefc TO MY REVERED AND BELOVED MASTER, CARL LUDWIG. PBEFACE. THE present work was written to supply the wants of the Students attending the course of " Practical Physiology " in The Owens College, but it is hoped that it will be found useful also to students of Medicine and Science in other Colleges and Universities. This course was instituted by my predecessor, Dr. Arthur Gamgee, and extended by him and the late Mr. W. H. Waters, M. A. Mr. Waters himself intended to write a short Manual for the guidance of the members of this class, but he was struck down, to the sincere regret of many of us, before he could accomplish his purpose. The experiments herein described are performed by every member of the class, and they are practically a repetition of some of those which I am in the habit of showing to illustrate my lectures on Physiology. It will be seen that much of the apparatus is of a simple character. Of course, no experiments are given which involve the infliction of pain upon living animals. A considerable portion of the chemical part was in print several years ago, although in a somewhat different form. In the preparation of the experimental part, however, I have had the advantage of knowing the system and methods that were followed by Dr. Gamgee and Mr. Waters. In arranging the experiments, the following works have afforded me much valuable information : — Dr. A. Gamgee's Physiological Chemistry, The Handbook for the Physiological Laboratory, Practical Exercises on Physiology by Professor Burdon-Sanderson, Practical Physiology by Professor Foster and Mr. Langley, Professor Grainger Stewart's Lectures on Vlll PREFACE. Albuminuria, the printed slips of Dr. Sheridan Lea, Gruen- hagen's Lehrbuch der Physiologie, Helmholtz's Physiologische Optik, Krukenberg's Grundriss der Medicinisch-Chemischen Analyse, and Hoppe-Seyler's Handbuch der chemischen Analyse. One feature valuable in any Text-book designed for practical ends, viz., the keeping in view of the fact that " the Student of to-day becomes the Practitioner of to-morrow," has been con- stantly before me. Hence, the aim has been so to arrange the exercises as to give them a bearing on, and to lead gradually up to, the methods used in Practical Medicine. I am indebted to Mr. John T. Millett, B.Sc., and to my demonstrator, Mr. A. F. S. Kent, B.A., for reading the proof- sheets. Many of the illustrations were drawn for me by my pupil, Mr. Philip Worley, and some were photographed from apparatus in the Physiological Laboratory of The Owens College, by Mr. William Charles, Steward of this department. I have also to express my thanks to several scientific instrument-makers, including Messrs. Browning, Elliott, Maw Son & Thompson, Jung of Heidelberg, Rothe of Prague, and Carl Reichert of Vienna. Some of the illustrations are from the Text-Rook of Physiology by Landois and Stirling. WM. STIRLING. •» THE OWENS COLLEGE, MANCHESTER, January, 1888. CONTENTS. i. ii. in. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. PART I.— CHEMICAL PHYSIOLOGY. PAGES The Proteids and Albumenoids, . . . . . 1-10 The Carbohydrates, Fats, Borne, 11-20 The Blood, Coagulation, its Proteids, .... 20-27 The Coloured Blood-Corpuscles, Spectra of Haemo- globin and its Compounds, ..... 27-37 Wave -Lengths, Derivatives of Haemoglobin. Estima- tion of Haemoglobin, ...... 37-46 Salivary Digestion, . . . . . . . 47-51 Gastric Digestion, ....... 51-56 Pancreatic Digestion, ....... 56-61 The Bile, 62-67 Glycogen in the Liver, ...... 67-69 Milk, Flour, Bread, 69-75 Muscle, 75-77 The Urine, 77-83 The Inorganic Constituents of the Urine, . . . 83-90 Organic Constituents of the Urine, .... 90-94 Volumetric Analysis for Urea, ..... 94-99 Uric Acid, Hippuric Acid, Creatinin, . . . 99-107 Abnormal Constituents of the Urine, .... 107-112 Blood, Bile, and Sugar in Urine, 112-116 Quantitative Estimation of Sugar, .... 117-120 Urinary Deposits, Calculi and General Examination of the Urine, ....... Appendix, . . 120-127 127-129 PART II.— EXPERIMENTAL PHYSIOLOGY. Galvanic Batteries and Galvanoscope, . . . 130-133 Electrical Keys, Rheochord, 133-139 Induction Machine, Electrodes, 139-142 Single Induction Shocks, Interrupted Current, Break Extra-Current, Helmholtz's Modification, . . 142-144 Pithing, Ciliary Motion, Nerve-Muscle Preparation, Normal Saline, 145-147 Nerve-Muscle Preparation, Stimulation of Nerve, . 148-152 X CONTENTS. L13SOH PAGES XXVIII. Single and Interrupted Induction Shocks, Tetanus, Constant Current, 152155 XXIX. Rheonom, Telephone, Direct and Indirect Stimu- lation of Muscle, Rupturing Strain, Muscle Sound, Dynamometers, . . . . . 155-157 XXX. Independent Muscular Excitability, Curare, Commu- tator, 157-161 XXXI. Graphic Method, Moist- Chamber, Single Contrac- tion, Work Done, . . . . . . 162-168 XXXII. Muscle Curve, Pendulum and Spring Myographs, Time Marker, Signal, . . . . .168-173 XXXIII. Influence of Temperature, Load, Veratria on Mus- cular Contraction, 174-176 XXXIV. Elasticity and Extensibility of Muscle, . . . 176-177 XXXV. Fatigue of Muscle, 178-181 XXXVI. Tetanus, Metronome, Thickening of Muscle, . . 181-184 XXXVII. Two Successive Shocks, Action of Drugs on Excised Muscle, 184-186 XXXVIII. Galvanometer, N.P. Electrodes, Shunt, Muscle Currents, 186-192 XXXIX. Nerve and Heart Currents, Capillary Electrometer, . 192-194 XL. Galvani's Experiments, Secondary and Paradoxical Contraction, Kiihne's Experiment, . . . 194-197 XLI. Electrotonus, 198-201 XLII. Electrotonus, Pfluger's Law, Ritter's Tetanus, . . 201-203 XLIII. Velocity of Nerve Energy, Double Conduction in Nerve, 204-207 XLIV. The Frog's Heart, the Effects of Heat and Cold, Cutting the Heart, 208-211 XLV. Graphic Record of Frog's Heart, Effect of Tempera- ture, 212-213 XLVI. Stannius's Experiment, Intra-Cardiac Inhibitory and Motor Centres, 214-215 XLVII. The Vagus and Sympathetic in the Frog, . . 215-219 XLVIII. Effect of Drugs, Constant Current, and Destruction of Nervous System on the Heart, . . . 219-222 XLIX. Perfusion of Fluids, Piston Recorder, . . . 222-224 L. Endocardiac Pressure, Apex Preparation, . . . 224-226 LI. Tonometer, Gaskell's Clamp, 226-229 LIL Valves of Heart, Stethoscope, Cardiograph, Meio- cardia, Auxocardia, Reflex Inhibition, . . 229-233 LIII. Pulse, Sphygmograph, Sphygmoscope, Plethysmo- graph, 233-236 CONTENTS. XI LESSON LIV. Pulse-Wave, Rigid and Elastic Tubes, Schema, Rheometer, ....... LV. Blood-Pressure, Kymograph, Lymph-Hearts, LVI. Chest Movements, Elasticity of Lungs, Hydrostatic Test, LVII. Vital Capacity, Carbonic Acid Excreted, Heywood's Experiment, Laryngoscope, Vowel Sounds, LVIII. Reflex Action, Poisons, Knee-Jerk, .... LIX. Functions of Nerve Roots, Reaction Time, LX. Formation of an Image, Aberration, Accommodation, Schemer's Experiment, Near and Far Points, Pur- kinje's Images, Phakoscope, Astigmatism, LXI. Blind Spot, Direct Vision, Maxwell's Experiment, Phos- phenes, Retinal Shadows, Duration of Impression, Talbot's Law, LXII. Perimetry, Irradiation, Imperfect Visual Judgments, LXIII. Artificial Eye, Colour - Sensations, Colour -Blindness, Contrast, After-images, Haploscope, LXIV. Ophthalmoscope, LXV. Touch, Smell, Taste, Hearing, 237-241 241-246 247-249 249-254 255-258 258-262 263-271 272-277 278-283 284-294 295-297 297-302 LIST OF ILLUSTRATIONS. FIG. PAGE L Starch, 11 2. Hsemocytometer (Gowws), ....... 28 3. Spectroscope (Browning), 30 4. Haemoglobin Spectra, ........ 32 5. Blood Spectra, 35 6. Spectroscope for W.L., 38 7. Hsemin Crystals, ......... 41 8. Haemoglobinometer (Gowers), ....... 44 9. Hsemometer (v. Fleischl), ........ 45 10. Kiihiie's Dialyser (Krukeriberg), ...... 54 11. Cholesterin, 65 12. Milk (.Stirling), 70 13. Porous Cell (Stirling), 71 14. Lactoscope, .......... 73 15. Urinometer, 79 16. Urinary Deposit, 82 17. „ 83 18. Triple Phosphate, 87 19. Burette Meniscus, 89 20. Erdmann's Float, 89 21. Urea and Urea Nitrate, .91 22. UreaOxalate, 92 23. Decomposition of Urea, 93 24. Steele's Apparatus (G. Steele), 98 25. Uric Acid, 100 26. 101 27. Urate of Soda, 103 28. Hippuric Acid, 104 29. Creatinin-Zinc-Chloride, 105 30. Picro-Saccharimeter (Johnson), . . . . . . .118 31. Cystin and Oxalates, . . , 122 32. Leucin and Tyrosin, . ..*... . . ' . . 123 33. Daniell's Cell (Stirling), ..•'... . . . 130 xiv LIST OF ILLUSTRATIONS. no. PAGB 34. Grove's Cell, 131 35. Bichromate Cell 132 36. Detector (Elliott), 132 37. Du Bois Key, 133 38. Scheme of 37 (Stirling), . . . .' . . . .133 39. „ „ ........ 134 40. Plug Key, 135 41. Morse Key (Stewart and Gee), 135 42. Spring Key (Elliott) 136 43. Rheochord (Stirling, . . . . . . . .136 44. Rheochord, 137 45. Reverser (Elliott), 139 46. Induction Coil, 140 47. Du Bois Electrodes, 141 48. Break Extra- Current (Stirling), 144 49. Helmholtz's Modification, 144 50. Frog's Leg-Muscles (Ecker), 146 51. Frog's Sciatic Nerve (Ecker), 146 52. Nerve-Muscle Preparation, ....... 149 53. Straw-Flag (Stirling), . 151 54. Scheme for Single Induction Shocks (Stirling), .... 152 55. Scheme of Constant Current (Stirling), 153 56. Frog's Sartorius (Ecker), 154 57. Rheonom (Stirling), . 155 58. Curare Experiment, . . . 160 59. Pohl's Commutator (Elliott), 161 60. Revolving Cylinder, . 162 61. Moist Chamber (Stirling), . . .164 62. Crank Myograph (Stirling), . . ' 167 63. Pendulum Myograph, . . 169 64. Pendulum Myograph Curve (Stirling), 170 65. Spring Myograph, 171 66. Revolving Drum, 172 67. Time Marker (Stirling), 173 68. Electric Signal (Stirling), . . . . . . .173 69. Muscle Curve with Load (Stirling), . . . . . . 175 70. Veratria Curve (Stirling), . . . . . . . .175 71. Fatigue Curve (Stirling), . . . . . . . .178 72. Pfliiger's Myograph, . •••..'• • • • ' '•" . . 179 73. Scheme for Tetanus (Stirling), 182 74. Tetanus Curves (Stirling), . . . . . . . .182 75. Tetanus Interrupter (Stirling), .183 LIST OF ILLUSTRATIONS. XV FIG. PAGE 76. Marey's Tambour, 184 77. Wild's Apparatus (Stirling), 185 78. Galvanometer, 187 79. Lamp aud Scale, • . . 188 80. N.P. Electrodes, 189 81. Shunt, 189 82. Apparatus for Muscle Current (Stirling), . . . . .190 83. Nerve N.P. Electrodes, 193 84. Galvani's Experiment (Stirling), . . . . ' . .194 85. Secondary Contraction, ........ 195 86. Scheme of 85 (Stirling), 196 87. Paradoxical Contraction (Stirling), ...... 196 88. Kiihne's Experiment (Stirling), 197 89. Electrotonus (Stirling), 198 90. Electrotonus, 200 91. Scheme of 90 (Stirling), 200 92. Pfluger's Law (Stirling), 202 93. Velocity of Nerve Energy, 204 94. Unequal Excitability of a Nerve (Stirling), .... 206 95. Kiihne's Experiment (Kilhne}, 207 96. Frog's Heart, 209 97. „ 209 98. Frog's Vagus (Stirling), 216 99. Vagus Curve (Stirling), 217 100. Frog's Sympathetic (after GasJcell), 219 101. Frog's Heart Support (Stirling), 220 102. Staircase of the Heart, 221 103. Double Cannula, 223 104. Heart Apparatus, 225 105. Tonometer, 226 106. Gaskell's Clamp (Stirling), . . . . . . .228 107. Cardiograph, 232 108. Marey's Sphygmograph, ........ 234 109. Dudgeon's Sphygmograph (Maw Son & Thompson), . . . 235 109a.Ludwig's Sphygmograph (Petzoldt), 235 110. Sphygmoscope (Rothe) 236 111. Rheometer, 240 112. Kries's Apparatus, 245 113. Lymph-Hearts. . 245 114. Stethograph, 248 115. Muller's Valves (Stirling), . -" 250 116. Heywood's Experiment (Stirling), .251 XVI LIST OF ILLUSTRATIONS. FIG. PAG« 117. View of the Larynx 253 118. „ . 253 119. Koenig's Apparatus, ......... 254 120. Neuramcebometer (after Obersteiner), 260 121. Scheiner's Experiment, 266 122. Diffusion (Helmlwltz), 267 123. Phakoscope, 269 124. Marriotte's Experiment, ........ 272 125. „ 272 126. Blind Spot (Helmholtz), . 273 127. Bergmann's Experiment (Helmhollz) 275 128. Disc for Talbofs Law (Helmholtz}, 277 129. Perimeter, 278 130. Irradiation, 280 131. ,, (Hzlmholtz), 280 132. „ ,, 280 133. Imperfect Visual Judgment, .281 134. Ziillner's Lines, 281 135. Perception of Size (Helmholtz), 282 136. Spiral Disc for Contrast, (Helmholtz), 283 137. Kiihne's Eye (Jung), 285 138. Partly Coloured Disc for Contrast (Helmholtz), . .288 139. Rotatory Disc (Helmholtz), 290 140. R. Scina's Experiment (Rood), 291 141. Carriage for Rabbit (Stirling), ....... 297 142. Aristotle's Experiment, 298 PAKT L— CHEMICAL PHYSIOLOai. LESSON I. THE PROTEIDS AND ALBUMENOIDS. The White of Egg may be taken as the Type. 1. Preparation of a Solution. — Place the white of an egg in a porcelain capsule (taking care that none of the yolk escapes), and cut it freely many times with scissors to disintegrate the membranes, and thus liberate the albumin. Add twenty volumes of distilled water, and place the mixture in a flask. Shake well until it froths freely. Cork the flask, and invert it, mouth downwards, over a porcelain capsule ; the membranes will rise to the surface, and, after a time, if the cork be gently withdrawn to allow the fluid to escape, a comparatively clear, or slightly opalescent, fluid will be obtained. If the fluid be too opalescent, strain through flannel or several folds of muslin. Such a solution niters slowly, so that it is better to employ several small filters if a clearer solution be required. If the fluid be alkaline, neutralise it. This solution contains about 5 per cent, of albumin, and diffuses slowly through animal membranes. (a.) To some of the fluid in a test-tube add strong nitric acid = a precipitate, which on being boiled turns yellow. Allow the liquid to cool, and add strong ammonia = an orange precipitate or colour (Xanthoproteic Reaction). (b.) To another portion add Millon's Reagent = a preci- pitate which becomes reddish on boiling. A red colour of the fluid is obtained if only a trace of proteid be present. 1 2 CHEMICAL PHYSIOLOGY. (c.) To a third portion add a drop or two of very dilute solution of cupric sulphate, and then a solution of caustic soda (or potash) = a violet colour (Biuret Reaction). (d.) Make a fourth portion strongly acid with acetic acid, and add potassic ferrocyanide = a white precipitate. (e.) Heat a portion of the neutral solution = a coagulum about 70° C. (/".) To a solution of white of egg add glacial acetic acid, and heat to get it in solution ; gradually add concentrated sulphuric acid = a violet colour (The Reaction of Adam- kiewicz). (g.) Wash finely powdered albumin first with alcohol and then with cold ether, and heat the washed residue with con- centrated hydrochloric acid = a deep violet-blue colour. This is best done in a white porcelain capsule, or on a filter-paper in a funnel ; in the latter case, the boiling acid is poured gently down the side of the filter-paper (Liebermann's Re- action). (h.) Non-diffusibility of Proteids.— Place some of the solu- tion either in a dialyser, or in a sausage-paper made of parchment-paper, and suspend the latter by means of a glass rod thrust through the tube just below the two open ends, as in fig. 10, in a tall glass jar or beaker filled with distilled water, so that the two open ends are above the surface of the water. The salts will diffuse readily (test for chlorides by nitrate of silver and nitric acid), but on applying any of the above tests no proteid will be found in the diffusate. (Peptones, however, are very diffusible.) N.B. — The reactions d, e, /, and g are not obtained with peptones. Preparation Of Millpn's Reagent. — Dissolve mercury in its own weight of strong nitric acid, specific gravity 1 '4, and to the solution thus obtained add two volumes of water. Allow it to stand, and afterwards decant the clear fluid ; or take one part of mercury, add two parts nitric acid, specific gravity 1 "4, in the cold, and heat over a water-bath till com- plete solution occurs. Dilute with two volumes of water, and decant the olear fluid after twelve hours. PROTEIDS. 3 2. Presence of Nitrogen and Sulphur in Albumin. (a.) Place some powdered dried albumin in a reduction tube, and into the mouth of the tube insert (1) a piece of red litmus paper, and (2) a lead acetate paper. On heating the tube the former becomes blue from the escape of ammonia, which can also be smelt, and the latter black from the for- mation of lead sulphide. (b.) Heat some dry proteid with excess of soda-lime in a hard dry tube, when vapour of ammonia is evolved. (c.) Place a few grains of the dry proteid, with a small piece of metallic sodium, in a dry hard tube, and heat slowly at first, and then strongly. After cooling, add care- fully 3 cc. of water to the NaOy residue, filter, and to the filtrate add a few drops of ferric chloride and ferrous sul- phate, and then add excess of hydrochloric acid. If nitrogen be present, there is a precipitate of Berlin blue, sometimes only seen after standing for a time. 3. Determination of Temperature of Coagulation. — "A glass beaker containing water is placed within a second larger beaker also containing water, the two being separated by a ring of cork. Into the water contained in the inner beaker there is immersed a test-tube, in which is fixed an accurately graduated thermo- meter, provided with a long narrow bulb. The solution of the proteid, of which the temperature of coagulation is to be deter- mined, is placed in the test-tube, the quantity being just sufficient to cover the thermometer bulb. The whole apparatus is then gradually heated, and the experimenter notes the temperature at which the liquid first shows signs of opalescence " (Gamgee). 4. Circumstances Modifying the Coagulating Temperature. — Place 5 cc. of the solution of albumin in each of three test-tubes, colour them with litmus, and label them A, B, C. To A add a drop of very dilute acetic acid (Ol per cent, acetic acid diluted five or six times) ; to B add a very dilute solution of caustic soda (0-1 per cent, of soda or potash similarly diluted) ; C is neutral for comparison. Place all three tubes in a beaker with water and heat them gradually, noting that coagulation occurs first in A, next in 0, and last of all in B, the alkaline solution. CHEMICAL PHYSIOLOGY. CLASSIFICATION AND PROPERTIES OF THE CHIEF PROTEIDS. 5. I. — Native Albumins are soluble in water, and are not precipitated by alkaline carbonates, sodic chloride, or very dilute acids. They are coagulated by heat at 65° to 73° 0. When dried at 40° C. they yield a clear yellow, amber-coloured, friable mass " soluble albumin," which is soluble in water. (1.) Egg-Albumin. — Prepare a solution as directed in Lesson L, 1. In addition, perform the following experiments : — (a.) Evaporate some of the fluid to dry ness at 40° 0. over a water-bath to obtain " soluble albumin." Study its charac- ters, notably its solubility in water. This solution gives all the tests of egg-albumin. It is more convenient to purchase this substance. (b.) Precipitate portions of the fluid with strong mineral acids, including sulphuric and hydrochloric acids. (c.) Precipitate other portions by each of the following : — Mercuric chloride ; basic lead acetate ; tannic acid ; alcohol ; picric acid (see Lesson XVIII.) (d.) Take 5 cc. of the fluid ; add twice its volume of (H per cent, sulphuric acid, and then add ether. Shake briskly = coagulation after a time, at the line of junction of the fluids. (e.) The solution is not precipitated on saturation with sodic chloride or magnesic sulphate (compare " Globulins "). (2.) Serum- Albumin.— Dilute blood serum until it has the same specific gravity as the egg-albumin solution. Neutralise the solution with very dilute acid until a faint haziness is obtained. Repeat all the tests for egg-albumin, but, in addition, note that the two solutions differ in the following respects : — PROTEIDS. EGG-ALBUMIN. SERUM-ALBUMIN. (a.) Readily precipitated by (a.) It is also precipitated by hydrochloric acid, but the pre- hydrochloric acid, but not so cipitate is not readily soluble readily, while the precipitate is soluble in excess. (6.) It is not coagulated by ether. (c.) The corresponding preci- pitate is much more soluble in excess of acid. (d.) The corresponding preci- pitate is soluble in strong nitric acid. in excess. (6.) A non-alkaline solution is coagulated by ether. (c.) The precipitate with nitric acid is soluble with diffi- culty in excess of the acid. (d.) The precipitate obtained by boiling is but slightly solu- ble in boiling nitric acid. [(e.) When injected under the [(e.) When injected under the skin, or introduced in large quantities into the stomach or rectum, it is given off by the urine.] skin, it does not appear in the urine.] 6. II. — Globulins are insoluble in pure water, but are soluble in weak solutions of neutral salts — e.g., sodic chloride — but insoluble in saturated ones. The solutions in these salts are coagulated by heat. They are soluble in dilute acids and alkalies, yielding acid- and alkali-albumins respectively. Most of them are precipitated from their saline solution by crystals of sodic chloride. (1.) Myosin, see " Muscle." (2.) Serum-Globulin. (a.) Neutralise 5 cc. of blood-serum with a few drops of dilute sulphuric acid (0*1 per cent.), and then add 75 cc. of distilled water, and allow the precipitate to settle. Pour off the fluid and divide the precipitate into two portions, noting that it is insoluble in water, but soluble in excess of acid. (6.) Boil a portion of the neutralised fluid = coagulation. 6 CHEMICAL PHYSIOLOGY. (c.) To 5 cc. of blood-serum in a test-tube, add an excess of crystals of magnesic sulphate, and shake briskly for some time. On standing, a white precipitate of serum-globulin falls. Pour off the supernatant fluid, and observe that the pre- cipitate is redissolved on the addition of water. Filter the supernatant fluid and test it for other proteids, which it still contains — viz., serum-albumin. (d.) Take 5 cc. of blood-serum and pour a saturated solu- tion of magnesic sulphate down the side of the glass to form a layer at the bottom of the tube. Where the two fluids meet, there is a copious white deposit of serum-globulin. (e.) Treat another portion of serum to saturation with crystals of sodic chloride, and observe the same results. (f.) Take another portion of serum, precipitate the serum- globulin with magnesic sulphate, and filter. To the filtrate add powdered sodic sulphate in excess, which gives a further precipitate. The filtrate still gives the reactions for pro- teids. (3.) Fibrinogen, see " Blood." 7. IIL — Derived Albumins are insoluble in pure water and in solutions of sodic chloride, but readily soluble in dilute hydro- chloric acid and dilute alkalies. The solutions are not coagulated by heat. (1. ) Alkali-albumin, or Alkali-albuminate — Preparation of Solu- tion.— Prepare a 5 per cent, solution of egg-albumin, as directed in Lesson I., 1. (a.) To the egg-albumin add a few drops of solution of caustic soda or potash (0-1 per cent.), and heat it gently for a few minutes -- alkali-albumin. Boil the fluid ; it does not coagulate. (b.) Test the reaction, it is alkaline. (c.) Cool some of the alkali-albumin and colour it with litmus solution. Neutralise it carefully with very dilute acid = a precipitate on neutralisation, which is soluble in excess of the acid. PROTEIDS. 7 (d.) Repeat (c.); but, before doing so, add a few drops of sodic phosphate solution (10 per cent.), and note that the alkaline phosphates prevent the precipitation on neutralisa- tion, until at least sufficient acid is added to convert the basic phosphate into acid phosphate. (See " Casein " under " Milk.") (e.) Precipitate it by saturating its solution with crystals of common salt. (/.) Lieberkfthn's Jelly is really a strong solution of alkali- albumin. Place some undiluted white of egg in a test-tube, and add, drop by drop, a strong solution of caustic potash. The whole mass becomes stiff and glue-like, so that the tube can be inverted without the mass falling out. (2.) Acid-Albumin or Syntonin. Preparation. — (A.) To a 5 per cent, solution of egg-albu- min add a few drops of dilute acid (e.g., 0*1 per cent, sulphuric acid, or hydrochloric acid *2 per cent.), and warm gently for several minutes = acid-albumin. (B.) To finely-minced muscle, free from fat, add ten times its volume of dilute hydrochloric acid (4 cc. of acid in 1 litre of water), and allow it to stand for several hours, taking care to stir it frequently ; filter, the filtrate is a solu- tion of syntonin. (C.) Allow concentrated hydrochloric acid to act on fibrin, for a time, and filter. Use the clear filtrate from A or B for testing. (a.) The reaction is acid. (6.) Boil the solution, it does not coagulate. (c.) Neutralise another portion with very dilute potash or soda. A precipitate occurs, which is soluble in excess of the alkali. Employ litmus as on previous occasions. (d.) Repeat (c.), but add sodic phosphate before neutralis- ing ; the syntonin is precipitated as before. 8 CHEMICAL PHYSIOLOGY. (e.) Add strong nitric acid = a precipitate which dis- solves on heating, producing an intense yellow colour. (/) It also gives the biuret test, and that with Millon's reagent (Lesson I., 1). 8. IV. — Fibrin is insoluble in water and in weak solutions of common salt. When prepared from blood and washed, it is a white, fibrous, soft, and very elastic substance, which exhibits fibrillation under a high magnifying power (see " Blood "). (a.) Place some well-washed fibrin in a test-tube, add some O'l per cent, hydrochloric acid, and observe that the fibrin swells up and becomes clear in the cold, but does not dissolve. (b.) Place a test-tube as in (a.) on a water-bath at 60°C. for several hours ; filter, and test the filtrate for acid-albumin by neutralisation with very dilute potash. (c.) For the effect of a dilute acid and pepsin (see "Diges- tion "). (d.~) It decomposes hydric peroxide, and turns freshly- prepared tincture of guaiacum blue (see " Blood "). (e.) Place a very dilute solution of cupric sulphate in a test- tube, add a flake of fibrin. The latter becomes greenish, while the fluid is decolourised. On adding caustic potash, the flake becomes violet. This is merely the biuret reaction common to proteids generally. 9. V. — Coagulated Proteids are insoluble in water, dilute acids, and alkalies, and are dissolved when digested at 35° to 40°C. in gastric juice (acid medium), or pancreatic juice (alkaline medium), forming peptones. They give Millon's reaction. Preparation. — Boil white of egg hard, and chop up the white. (a.) Test its insolubility in water, dilute acids, and alkalies. (b.) It is partially soluble in acids and alkalies, when boiled for some time. PROTEIDS. 9 (c.) Bruise some of the solid boiled white of egg, diffuse it in water, and test it with Millon's reagent. (d.) For the effect of the digestive juices see " Digestion." 10. VI. — Peptones are exceedingly soluble in water. Their solutions are not precipitated by sodic chloride, acids, or alkalies, nor are they coagulated by heat. They are precipitated by tannic acid, and with difficulty by excess of absolute alcohol. Preparation (see "Digestion"). — For applying the tests, dis- solve a small quantity of Darby's Fluid Meat in water, and filter, or dissolve some pure peptone in water. The latter can be bought as a commercial product. (a.) Boil a portion, it is not coagulated. (b.) To another portion add strong nitric acid; and boil = a faint yellowish colour ; allow it to cool, and add strong ammonia = orange colour (Xanthoproteic Reaction). (c.) Acidify a third portion strongly with acetic acid, and add ferrocyanide of potassium = no precipitate. (d.) Test separate portions with tannic acid, mercuric chloride, picric acid, and lead acetate. Each of these causes a precipitate. In the case of picric acid the precipitate disappears on heating, and partly reappears on cooling. (e.) To another portion add a few drops of very dilute solution of cupric -sulphate, and then caustic soda (or potash) =a rose colour ; on adding more cupric sulphate, it changes to a violet (Biuret Reaction). (f.) To another portion add a drop or two of Fehling's solution = a rose colour ; on adding more Fehling's solution it changes to violet (Biuret Reaction). (g.) Neutralise another portion = no precipitate. (h.) To another portion add an excess of absolute alcohol = a precipitate of peptone, but not in a coagulated form. (i.) Precipitate a portion with ferric acetate. 10 CHEMICAL PHYSIOLOGY. 11. Diffusibility of Peptones. — Place a solution of peptones in a dialyser covered with an animal membrane, as directed in Lesson I., 1, (A.), and test the diffusate after some time for peptones. THE ALBUMENOIDS. 12. I. Gelatin is obtained by the prolonged boiling of con- nective tissues, and from the hypothetical substance " Collagen,^ of which fibrous tissue is said to consist. Preparation of a Solution.— Use commercial gelatin. Make a watery solution by allowing it to swell up in water, and then dissolving it with the aid of heat. (a.) It is insoluble, but swells up, in cold water. (b.) After a time heat the gelatin swollen up in water ;. it dissolves. Allow it to cool ; it gelatinises. (c.) Precipitate separate portions by each of the following: Mercuric chloride, tannin, alcohol, and platinic chloride. (d.) It is not precipitated by acids (acetic or hydrochloric), or alkalies, or lead acetate. (e.) It is not precipitated by acetic acid and potassic ferrocyanide (unlike albumin). (f.) It is not coagulated by heat (unlike albumin). (g.) It gives the xanthoproteic and biuret tests, and that with Millon's reagent. It is precipitated by picric acid; the precipitate is dissolved on heating, and reappears on cooling. 13. II. Chondrin is derived by prolonged boiling from the matrix of cartilage, which is supposed to consist of the hypothe- tical substance " Chondrogen." It seems to be really a mixture of mucin and glutin. 14. III. Mucin, see " Bile." THE CARBOHYDRATES. 11 LESSON II. THE CARBOHYDRATES, FATS, BONE. 1. I. Starch (06H10O5)M Preparation. — Wash a potato thoroughly, and grate it on a grater into water in a tall cylindrical glass. Allow the suspended particles to subside, and after a time note the deposit ; the lowest stratum consists of a white powder or starch, and above it lie coarser fragments of cellulose and other matters. Decant off the supernatant fluid. (a.) Microscopical Examination. — Examine the white de- posit of starch, noting that each starch granule shows an eccentric hilum with concentric markings. Add a very dilute solution of iodine. Each granule becomes blue, while the concentric markings become more distinct. (b.) At this stage it is advantageous to compare the microscopic characters of other varieties of starch — e.g., rice, arrowroot, &c. (Fig. 1). Fig. 1. — e, Tahiti arrowroot ; d, Potato starch. (c.) Polariscope. — Examine starch granules with a polari- sation-microscope. With crossed Nicol's, when the field is dark, each granule shows a dark cross on a white refractive ground. (d.) Squeeze some dry starch powder between the thumb and forefinger, and note the peculiar crepitation sound and feeling. 12 CHEMICAL PHYSIOLOGY. 2. Prepare a Solution. — Place 1 grm. of starch in a mortar, rub it up with a little cold water, and then add 50 cc. of boiling water, and rub up the whole together until the starch is apparently dissolved and a somewhat opalescent fluid is obtained. Allow the solution to cool. [In reality, the starch is only imper- fectly dissolved by hot water.] (a.) Add powdered dry starch to cold water. It is in- soluble. Filter, and test the nitrate with iodine. It gives no blue colour. (6.) The above 'method shows that it is imperfectly dis- solved in warm water. If more starch be used, a thick " starch-paste," which sets on cooling, is obtained. (c.) To a portion of the above fluid add a solution of iodine = a blue colour, which disappears on heating and re- appears on cooling — provided it has not been boiled too long. Place the test-tube in cold water to cool it. (d.) Render some of the starch solution alkaline by add- ing caustic soda solution. Add iodine solution. No blue colour is obtained. (e.) Acidify (d.) with dilute sulphuric acid, then add iodine = blue colour is obtained. (/.) To another portion of the solution, add a few drops of dilute cupric sulphate and caustic soda (or Fehling's solution), and boil = no reaction (compare " Grape Sugar"). (g.) Add tannic acid = yellowish precipitate, which dis- solves on heating. 3. Starch is a Colloid. — Place some strong starch solution in a dialyser or parchment-tube, and the latter in distilled water. Allow it to stand for some time, and test the water for starch ; none will be found. 4. II. Dextrin (C6H10O5). Prepare a Solution. — Dissolve some dextrin in boiling water, and observe that the solution is not opalescent. ^ (a.) This proves its solubility in water. THE CARBOHYDRATES. 13 (b.) To a portion of the solution, add a solution of iodine = reddish-brown colour, which disappears on heating and returns on cooling. [The student ought to use two test- tubes, placing the dextrin solution in one, and an equal volume of water in the other. Add to both an equal volume of solution of iodine, and thus compare the difference in colour.] (c.) Precipitate some from its solution by adding alcohol. (d.) Render some of the dextrin solution alkaline by adding caustic soda solution. No red-brown colour is obtained with iodine. Acidify and the reddish-brown colour appears. 5. III. — Glycogen or Animal Starch, 06H10O5. Prepare a Solution (see " Liver "). (a.) Take a portion of the solution ; note its opalescence ; add solution of iodine (made by adding iodine to water in which a crystal of potassic iodide is dissolved) = red-brown or port-wine-red colour. As in the dextrin test, use two test- tubes, one with water and the other with glycogen, to com- pare the difference in colour. The colour disappears on heating, and reappears on cooling. 6. IV.— Glucose, Dextrose, or Grape-Sugar, C6H12O6. In commerce it occurs in warty uncrystallised masses, of a yellowish or yellowish-brown colour. It is readily soluble in water. Prepare a solution by dissolving a small quantity in water. (a.) To the solution add iodine = no reaction. (6.) To the solution add a trace of a dilute solution of cupric sulphate, and afterwards add caustic soda (or potash) until the precipitate first-formed is redissolved, and a clear blue fluid is obtained. Boil gradually ; if grape-sugar be present, the blue colour disappears, and a yellow precipi- tate of hydrated cuprous oxide is obtained. It is well to boil the surface of the fluid, and when the yellow precipitate occurs, it contrasts sharply with the deep blue-coloured 14 CHEMICAL PHYSIOLOGY. stratum below. If no sugar be present, only a black colour may be obtained (Trommer's Test). (c.) To the solution add Fehling's solution; boil = a yellow or yellowish-red precipitate of hydrated cuprous oxide. [For the precautions to be observed in using Fehling's solution, and for other tests for glucose, see " Urine."] 7. V.— Maltose, 012H22On. (a.) Take 1 grm. of ground malt, mix it with ten times the quantity of water, place the mixture in a beaker, and keep it at 60°C. for half an hour. Then boil and filter ; the filtrate contains maltose and dextrin. (5.) Test for a reducing sugar with Fehling's solution or other suitable test. (See also " Salivary digestion.") 8. VI.— Lactose, C12H22On + H2O (see " Milk »). 9. VII.— Cane Sugar, C12H22On. (a.) Observe its crystalline form and sweet taste. (b.) Its solutions do not reduce Fehling's solutions (many of the commercial sugars, however, contain sufficient grape- sugar to do this). (c.) Place some cane-sugar in a beaker, pour on it strong sulphuric acid, and add a few drops of water ; soon the whole mass is charred. (d.) Inversion of Cane-Sugar.-— Boil a strong solution of cane-sugar in a flask with one-tenth of its volume of strong hydrochloric acid. After prolonged boiling the cane-sugar is " inverted," and the solution contains a mixture of dextrose and laevulose. Test its reducing-power with Fehling's solution. 10. Conversion of Starch into a Reducing Sugar. — Place 50 cc. of starch solution in a flask on wire gauze over a Bunsen burner, add one drop of strong sulphuric acid, and boil for five to ten THE CARBOHYDRATES. 15 minutes, observing the spluttering that occurs, the liquid mean- time becoming limpid. (a.) Test a portion of the liquid for glucose, taking care that sufficient alkali is added to neutralise the surplus acid. (b.) Test it with iodine = a blue colour, showing that some soluble starch (amidulin) still remains unconverted into a reducing sugar. 11. Circumpolarisation. — Certain substances when dissolved possess the power of rotating the plane of polarised light — e.g., the proteids, sugars, &c. The extent of the rotation depends on the amount of the active substance in solution. The direction of rotation — i.e., to the right or the left — is constant for each active substance. Of course, light of the same wave-length must be used. The light obtained from the volatilisation of common salt is used. The term "specific rotatory power," or "specific rotation" of a substance, is used to indicate the amount of rotation expressed in degrees of the plane of polarised light, which is produced by 1 grm. of the substance dissolved in 1 cc. of liquid, when examined in a layer 1 decimetre thick. Those substances which cause specific rotation are spoken of as "optically active ;" those which do not, as "inactive." If n — the observed rotation ; p = the weight in grammes of the active substances con- tained in 1 cc. of liquid ; I = the length of the tube in decimetres ; (a)D = the specific rotation for light corresponding to the light of a sodium flame ; then The sign + or — indicates that the substance is dextro- or laevo-rotatory. Various instruments are employed. Use Laurent's Polarimeter. — This instrument must be used in a dark room. 12. Determination of the Specific Rotatory Power of Dextrose. 16 CHEMICAL PHYSIOLOGY. (a.) Fill one of the decimetre tubes with distilled water, taking care that no air-bubbles get in. Slip on the glass disc horizontally, and screw the brass cap on the tube. Place the tube in the instrument, so that it lies in the course of the rays of polarised light. Jb.) Place some common salt (or fused common salt, and ic carbonate) in the platinum spoon, and light the Bun- sen's lamp, so that the soda is volatilised. If a platinum spoon is not available, tie several platinum wires together, dip them into slightly moistened common salt, and fix them in a suitable holder, so that the salt is volatilised in the outer part of the flame. In the newer form of the instru- ment supplied by Laurent, there are two Bunsen burners, placed the one behind the other, which give very much more light. Every part of the apparatus must be scrupu- lously clean. (c.) Bring the zero of the vernier to coincide with that of the scale. On looking through the eye-piece, and focussing the vertical line dividing the field vertically into two halves, the two halves of the field should have the same intensity when the scale reads zero. If this is not the case, then adjust the prisms until it is so, by means of the milled head placed for that purpose behind the index dial and above the telescope tube. It is well to work with the field not too brightly illuminated. (d.) Remove the water tube, and substitute for it a similar tube containing the solution of the substance to be examined — in this case a perfectly clear solution of pure dextrose. Place the tube in position, and proceed as before. The two halves of the field are now of unequal intensity. Rotate the eye-piece until equality is obtained. (e.) Repeat the process several times, and take the mean of the readings. The difference between this reading and the first at (c.), when the tube was filled with distilled water — i.e., zero — is the rotation due to the dextrose = (a.) (/.) Place 10 cc. of the solution of dextrose in a weighed capsule, evaporate to dryness over a water-bath, let the capsule cool in a desiccator, and weigh again. The increase THE CARBOHYDRATES. 17 in weight gives the amount of dextrose in 10 cc. ; so that the amount in 1 cc. is got at once = p. (g.) Calculate the specific rotatory power by the above formula. It is about + 53°. For practice, begin with a solution of dextrose containing 11 grms. per 100 cc. of water. Make several readings of the amount of rotation, and take the mean. Example.- — In this case, the mean of the readings was 11-6° 11-6° Repeat the process with a 4 and 2 per cent, solution. It is necessary to be able to read to 2 minutes, but considerable prac- tice is required to enable one to detect when the two halves of the field have exactly the same intensity. Test the rotatory power of corresponding solutions of cane- sugar, and any other sugar you please. Test also the rotatory power of a proteid solution. The following indicate the S.R. for yellow light :— Proteids. — Egg-albumin - 35 '5°; serum-albumin - 56°; syn- tonin - 72° ; alkali-albumin prepared from serum-albumin - 86°, when prepared from egg-albumin — 47°. Carbohydrates. — Glucose + 56°; maltose + 150°; lactose + 52-5°. NEUTRAL FATS. 13. Reactions. (a.) Use almond oil or lard, and observe that fat is soluble in ether, chloroform, and hot alcohol. (6). To almond oil add caustic soda, and boil = saponifi- cation. (c.) Shake oil containing a fatty acid — e.g., De Jongh's cod-liver oil, with a few drops of a dilute solution of sodic carbonate. The whole mass becomes white = emulsion. Examine it microscopically, and compare it with milk, which is a typical emulsion. 2 18 CHEMICAL PHYSIOLOGY. (d.) Shake up olive oil with a solution of albumin in a test-tube = an emulsion. Examine it microscopically. (e.) Heat in a porcelain capsule for an hour or more some lard mixed with plumbic oxide and a little water. The fat is split up, yielding glycerin and a lead soap. (f.) Heat some lard and caustic soda solution in a capsule to form a soap ; decompose the latter by heating it with dilute sulphuric acid, and observe the liberated fatty acids floating on the top. BONE. 14. A.— Organic Basis of Bone. (a.) To Decalcify Bone. — Place a small thin dry bone in dilute hydrochloric acid (1 : 8) for a few days. Its mineral matter will be gradually dissolved out, when the bone, although retaining its original form, loses its rigidity, and becomes pliable, elastic, and so soft as to be capable of being cut with a knife. What remains is the organic matrix or ossein. Keep the solution obtained. (b.) Wash the decalcified bone thoroughly with water, in which it is insoluble. Boil it for a long time, and from it gelatin will be obtained. Test the solution for gelatin (Lesson I., 12). B. — Mineral Matter in Bone. (a.) Examine a piece of bone which has been incinerated in a clear fire. At first the bone becomes black from the carbon of its organic matter, but ultimately it becomes white. What remains is calcined bone, having the form of the original bone, but now it is quite brittle. Powder some of the white bone-ash. (b.) Dissolve a little of the powdered bone-ash in hydro- chloric acid, observing that bubbles of gas (CO2) are given off, indicating the presence of a carbonate ; dilute the solu- tion, add excess of ammonia = a white precipitate of phos- phate of lime and phosphate of magnesia. THE CARBOHYDRATES. 19 (c.) Filter, and to the nitrate add ammonium oxalate — a white precipitate of oxalate of lime, showing that there is lime present, but not as a phosphate. (d.) To the solution of mineral matters 14, A. (a.) add acetate of soda until there is free acetic acid present, re- cognised by the smell ; then add ammonium oxalate = a copious white precipitate of lime salts. Exercises on the Foregoing. — The solution may contain one or more proteids or carbohydrates. A. Proteids. (1.) Note the colour, odour, and transparency of the solution. (2.) Test its reaction. Neutralise by dilute sodic carbonate or hydrochloric acid, if necessary. If the precipitate gives the xanthoproteic reaction, it is acid or alkali-albumin. If not, it is earthy phosphates. (3.) Do the xanthoproteic reaction, which shows the presence of a proteid. (4.) Boil. If there is coagulation it is either egg-albumin, serum-albumin, or globulin. (a.) Test if the solution is precipitated by crystals of mag- nesic sulphate = globulin. Filter. (6.) Test the nitrate of (a.) by acidulation and heat for albumin. Confirm by other tests. (c.) Test the filtrate of (6.) for peptones. (5.) Test for gelatin. B. Carbohydrates. (1 .) Remove any proteids present, except peptones, by acidi- fication and boiling, and use this solution for testing. (2.) Add iodine after acidulation if necessary, a blue colour = starch ; a port- wine colour = dextrin or glycogen. Confirm by other tests. 20 CHEMICAL PHYSIOLOGY. (3.) If no peptones are present, test for sugar. (4.) If peptones are present, evaporate to dryness, dissolve the residue in 98 per cent, alcohol, filter. Evaporate the alcohol and redissolve the residue in water, and test for sugar. LESSON III. THE BLOOD — COAGULATION, ITS PROTEIDS. 1. Reaction. — Prick your finger with a needle and place a drop of the freshly shed blood on a strip of dry, smooth, glazed, neutral litmus paper. Allow it to remain for a short time ; then wash it off with a stream of distilled water from a wash-bottle, when a blue spot upon a red or violet ground will be seen, indicating its alkaline reaction. 2. Blood is Opaque. (a). Place a thin layer of defibrinated blood on a micro- scopic slide, and try to read some printed matter through it. This will be found impracticable. 3. To make Blood Transparent or Laky. — Place 10 cc. of the defibrinated blood provided for you in three test-tubes, labelling them A, B, and C. Keep A for comparison. (a.) To B add 5 volumes of water, and warm slightly, noting the change of colour by reflected and transmitted light. When looked at by reflected light, it is much darker in colour, in fact, it looks almost black, but by transmitted light it is transparent. Test this by looking as in 2 (a.) at printed matter. (6.) To C add a solution of taurocholate of soda. Test its transparency as above. In 2, the haemoglobin is still within the blood corpuscles. In the others — 3 (a.), (b.) — it is dis- solved out, and in solution. THE BLOOD. 21 4. Action of Saline Solution. (a.) Take 2 cc. of defibrinated blood in a test-tube, label it D, add 5 volumes of a 10 per cent, solution of sodic chloride. Observe the change of colour. It becomes a very bright florid red, more brick-red than the original blood itself. Compare its colour with that in A, B, and C. It is opaque. 5. Haemoglobin does not Dialyse. (a.) Place a watery solution of defibrinated blood in a dialyser, and suspend it in a large vessel of distilled water. Carefully test the dialyser beforehand to see that there are no holes in it. If there are any fine pores, close them with a little white of egg, and coagulate it with a hot iron. (b.) After several hours observe that no haemoglobin has passed into the water. (c.) Test the diffusate for chlorides. 6. Phenomena of Coagulation. — Place a small porcelain capsule on the table ; decapitate a rat, and allow the blood to flow into the capsule. Within a few minutes the blood congeals, and when the vessel is tilted the blood no longer flows as a fluid, but as a solid. It then coagulates completely. Allow it to stand, and after an hour or so, pale-yellow coloured drops of a fluid — the serum — are seen on the surface, being squeezed out of the red mass, the latter being the clot. 7. Frog's Blood — Coagulation of the Plasma.— Place 5 cc. of normal saline (0'75 per cent, salt solution) in a test-tube sur- rounded with ice. Expose the heart of a pithed frog, and cut into the ventricle, allowing the blood as it escapes to flow into the normal saline. Mix the two, and the corpuscles (owing to their greater specific gravity) after a time subside. After they have subsided remove the supernatant fluid — the plasma mixed with normal saline — by means of a pipette. Place it in a watch- glass, and observe that it coagulates. 8. Mammalian Blood. (A.) Study coagulated blood obtained from the slaughter- house. Run the blood of a sheep or ox into a tall cylindrical 22 CHEMICAL PHYSIOLOGY. vessel, and allow it to coagulate. Set it aside for two days, and then observe the serum and the clot. Pour off the pale, straw-coloured serum, and note the red clot, which has the shape of the vessel, although it is smaller than the latter. (B.) If the blood of a horse can be obtained, study it, noting that the upper layer of the clot is paler in colour ; this is the buffy coat. 9. Circumstances influencing Coagulation. Effect of Cold. — Place a small platinum basin — a brass or glass thimble will do quite well — on a freezing mixture of ice and salt, decapitate a frog or rat, and allow the blood to flow directly into the cooled vessel. At once it becomes solid or con- geals, but it is not coagulated. As soon as the blood becomes solid, remove the thimble and thaw the blood by placing it on the warm palm of the hand, when the blood becomes fluid, so that it can be poured into a watch-glass; if the vessel be once more placed on the freezing mixture, the blood again congeals and solidifies, and on its being removed becomes fluid. Observe at the same time that the colour of the blood changes, becom- ing darker and transparent. This is the laky condition due to the discharge of the haemoglobin from the corpuscles. Place the vessel with the fluid blood on the table, and it clots or forms a firm jelly. 10. Influence of Neutral Salts on Coagulation. — Take to the slaughter-house a vessel capable of holding 500 cc., but previously place in it 170 cc. of a saturated solution of sodic sulphate or magnesic sulphate. Allow enough blood from an animal to run into the saline solution to fill the vessel, and mix them thoroughly. The blood does not clot but remains fluid. Place the vessel aside on ice, and note that the corpuscles subside, leaving a clear yellowish layer on the surface — the plasma mixed with the saline solution, and known as salted plasma. (a.) Pipette off the salted plasma — use 2 cc. — add to it 3 to 5 volumes of water, and observe that it clots after a time. The clotting is hastened by the action of gentle heat. In laboratories where a centrifugal apparatus is in use, the corpuscles can be rapidly separated from the plasma, and enough of the latter obtained for the purposes of a large class of students. THE BLOOD. 23 (b.) Place 15 cc. of the salted plasma — separated by means of the centrifugal apparatus — in a tall, narrow, cylindrical, stoppered glass. Add powdered sodic chloride, and shake the whole vigorously, when a white flocculent precipitate is thrown down. Allow the precipitate to subside. Decant off the supernatant fluid and the salt solution. Filter through a filter, moistened with a saturated solution of sodic chloride, and wash the precipitate on the filter with saturated solution of sodic chloride. This is the plasmine of Denis. Scrape the washed precipitate off the filter by means of a knife. Dissolve it in a small quantity of distilled water, and filter quickly. The filtrate if set aside will clot after a time. It is better to do the several operations rapidly to ensure success, but I have frequently found coagulation occur when the plasmine was not dissolved in water until many hours after it was deposited. 11. Defibrinated Blood. — In the slaughter-house allow blood to run from an animal into a vessel, and with a bundle of twigs beat or whip the blood steadily for some time. Fine white fibres of fibrin collect on the twigs, while the blood remains fluid. This is defibrinated blood, and although set aside for any length of time, it does not coagulate spontaneously. (a.) With a few thin twigs, or the barbed end of a quill, beat some freshly-shed blood, and observe the fibrin sticking to the twigs. Wash it. 12. I. — Fibrin. — Take the twigs coated with fibrin of the pre- vious experiment. Wash away the colouring-matter with a stream of water until the fibrin becomes quite white. (a.) Study its physical properties: it is a white, fibrous, highly-elastic substance. Stretch some fibres to observe their extensibility ; on freeing them, they regain their shape, showing their elasticity. (b.) Place a few fibrils in absolute alcohol to rob them of water, when they become brittle, and lose their elasticity. (c.) Place a flake in a test-tube with some 0-2 per cent, hydrochloric acid in the cold. It swells up and becomes clear and transparent, but does not dissolve. 24 CHEMICAL PHYSIOLOGY. (d.) Repeat (c.), but place the test-tube in a water-bath at 60° C., the fibrin is dissolved forming acid-albumin. Test for the latter (Lesson I., 7, III., 2). (e.) Place a few fibrils in a watch-glass, and pour over them some hydric peroxide; bubbles of oxygen are given off. Immerse a flake in freshly-prepared tincture of guaiacum (5 per cent, solution of the pure resin in alcohol), and then in hydric peroxide, when a blue colour is developed. If the fibrin contain much water, it is preferable to place it first of all for a short time in rectified spirit to remove the water. [Other substances give a blue colour under similar con- ditions.] (/.) Suspend some fibrils of fibrin in water in a test-tube, and observe that they give (1) the Xanthoproteic reaction, and that with Millon's reagent (Lesson I., 1). (g.) Prick a finger with a needle; collect a drop of blood on a microscopic slide, cover, and examine under a microscope ( x 350). After a time observe the formation of threads of fibrin between the rouleaux of coloured blood-corpuscles. 13. II. —Blood-Serum. — By means of a pipette remove the serum from the coagulated blood (Lesson III., 8). If a centri- fugal apparatus is available, any suspended blood-corpuscles can easily be separated by it. Observe its straw-yellow colour. Test its reaction ; it is alkaline. Study its proteids. Test first for the general reactions common to all proteids. (a.) Dilute 1 volume of blood-serum with 50 volumes of water, and use this for testing. (b.) Test separate portions by neutralisation and heat ; nitric acid and the subsequent addition of ammonia ; acetic acid and ferrocyanide of potassium ; Millon's reagent, and the biuret reaction (Lesson I., 1). The solution gives all these reactions. Study its individual proteids. (A.) Preparation of Serum-globulin (Para-globulin or Fibrino- plastin). THE BLOOD. 25 (a.) Take 10 cc. of blood-serum ; add 200 cc. of ice-cold water, and pass a stream of carbon dioxide through it for some time = a white precipitate of serum-globulin. This method does not precipitate it entirely (Schmidt's method). No precipitate is obtained unless the serum be diluted. (b.) Dilute 10 cc. of blood-serum with 150 cc. of water; add 5 drops of a 20 per cent, dilution of acetic acid = a white precipitate of serum-globulin, or as it was called, " serum-casein " (Panum's method). All the serum-globulin is not precipitated. (c.) To 5 cc. of fresh blood-serum in a test-tube, add crystals of magnesium sulphate in large excess, and shake briskly for some time. The excess of crystals falls to the bottom, and on their surface is precipitated a dense white flocculent mass of serum-globulin (Hammarsten's method). Allow the excess of the salt and the precipitate to settle. Decant the bulk of the supernatant fluid, and filter the remainder. Wash the precipitate on the filter with a saturated solution of magnesic sulphate ; add a little dis- tilled water to the precipitate. It is dissolved — i.e., it is a globulin, and is insoluble in excess of a neutral salt, but is dissolved by a weak solution of the same. The solution does not coagulate spontaneously. (d.) The solution obtained in (c.) gives all the reactions for proteids with the special reactions of a globulin. (e.) Allow a few drops of blood-serum to fall into a large quantity of water, and observe the milky precipitate of a globulin = serum-globulin. This is best observed by placing a dead black surface behind the vessel of water. We can then trace the "milky way " of the falling drops of serum as they mix with the water. (B.) Serum-Albumin.— From (A.), («.),-(&.), (c.), filter off the precipitate, and test the filtrate for the usual proteid reactions, so that the filtrate still contains a proteid which is serum-albumin (Lesson I., 5, 2). 14. Precipitation by other Salts. (a.) Precipitate the serum-globulin of blood-serum with magnesic sulphate. Filter, and to the filtrate add sodic 26 CHEMICAL PHYSIOLOGY. sulphate, when serum-albumin is precipitated. Sodic sul- phate, however, gives no precipitate with pure serum. (b). Precipitate blood-serum with potassic phosphate. All the proteids are thrown down after prolonged shaking. (c.) Precipitate blood-serum with magnesic sulphate and sodic sulphate, or the double salt sodio-magnesic sulphate. All the proteids are thrown down. 15. Preparation of Fibrinogen. (a.) Dilute 10 cc. of hydrocele fluid with 150 to 200 cc. of water, and pass through it for a considerable time a stream of carbon dioxide, when there is precipitated a small quantity of a somewhat slimy white body, fibrinogen. (b.) Take 10 cc. of hydrocele fluid and add powdered crystals of common salt to saturation, as for the preparation of paraglobulin (Lesson III., 13, A.) 16. Coagulation Experiments. (a.} Andrew Buchanan's Experiment— Mix 5 cc. fresh blood-serum (preferably from horse's blood) with 5 cc. hydrocele fluid, and keep the mixture at 35°C. for some hours, when coagulation occurs, a clear pellucid clot of fibrin being obtained. (b.) To 5 cc. of hydrocele fluid, add some solution of para- globulin (prepared as in Lesson III., 13, A); coagulation will result after a time. (c.) Modify (a.) in the following manner : — To 2 cc. of fresh blood-serum, add 2 cc. of a solution of fibrinogen (pre- pared as in Lesson IV., 15, b) = coagulation. (d.) To 2 cc. of salted plasma, prepared as in Lesson III., 10 (which is known to clot slowly on the addition of water), add 10 volumes — i.e., 20 cc. of a watery solution of fibrin- ferment, prepared by the demonstrator = coagulation. 17. The Salts present in blood are to be tested for in the usual way. THE COLOURED BLOOD-CORPUSCLES. 27 18. Preparation of Fibrin-ferment. — It must be kept in stock. Precipitate blood-serum with a large excess of alcohol, collect the copious precipitate ; cover it with absolute alcohol, and allow it to stand at least a month — the longer the better. Dry the pre- cipitate at 35°C., and afterwards over sulphuric acid. Keep it as a dry powder in a well-stoppered bottle. When a solution is required, extract some of the dry powder with 100 volumes of water ; filter. The filtrate contains the ferment. LESSON IV. THE COLOURED BLOOD-CORPUSCLES. SPECTRA OF HAEMOGLOBIN AND ITS COMPOUNDS. Enumeration of the Corpuscles. — Several forms of instruments are in use, e.g., those of Malassez, Zeiss, and Gowers. 1. The Haemocytometer of Gowers' consist of (a.) A small pipette, which, when filled to the mark on its stem, holds 995 c.mm. (Fig. 2, A). (6.) A capillary tube to hold 5 c.mm. (B). (c.) A small glass jar in which the blood is diluted (D). (d.) A glass stirring rod (E). (e). Fixed to a brass plate a cell \ of a millimetre deep, and with its floor divided into squares ^ mm., in which the blood-corpuscles are counted. (/.) The diluting solution consists of a solution of sodic sulphate in distilled water — specific gravity, 1025. This instrument can be used with any microscope. 2. Mode of using the Instrument. (a.) By means of the pipette (A) place 995 c.mm. of the diluting solution in the mixing jar (D). CHEMICAL PHYSIOLOGY. (6.) Puncture a finger near the root of the nail with the lancet projecting from (F), and with the pipette (B) suck up 5 c.mm. of the blood, and blow it into the diluting solu- tion, and mix the two with the stirrer (E). Fig. 2.— Gowers' Hsemocytometer. — A, Pipette for measuring the diluting solution ; B, for measuring the blood ; C, cell with divisions on the floor, mounted on a slide, to which springs are fixed to secure the cover glass ; D, vessel in which the solution is made ; E, spud for mixing the blood and solution ; F, guarded spear-pointed needle. (c.) Place a drop of the mixture on the centre of the glass cell (C), see that it exactly fills the cell, and cover it gently with the cover-glass, securing the latter with the two springs. Place the cell with its plate on the stage of a microscope, and focus for the squares ruled 011 its base. (d.) When the corpuscles have subsided, count the number in 10 squares, and this, when multiplied by 10,000, gives the number in a cubic millimetre of blood (e.) Wash the instrument, and in cleaning the cell do this with a stream of distilled water from a wash-bottle. THE COLOURED BLOOD-CORPUSCLES. 29 Take care not to brush the cell with anything rougher than a camel's- hair pencil, to avoid injuring the lines. Each square has an area of yj^ mm., so that 10 squares have an area of y1^ mm. As the cell is 4 mm. deep, the volume of blood in 10 squares is yV x i = "sV c-mi]a' On counting the number of corpuscles in 10 squares, and multiplying by 50, this will give the number in 1 c.mm. of the diluted blood. On multiplying this by -^^j we get the number in 1 c.mm. before dilution. Thus we arrive at the reason why we multiply the number in 10 squares by 10,000 to get the number of cor- puscles in 1 c.mm. of blood. HAEMOGLOBIN AND ITS DERIVATIVES. 3. Preparation of Haemoglobin Crystals. (a.) Rat's Blood. — Place a drop of the defibriiiated rat's blood provided for you on a slide, add three or four drops of water, mix, and cover with a cover-glass. Examine the slide with a high power of the microscope ; after a few minutes, especially at the edges of the preparation, small crystals will begin to form, and gradually grow larger. The crystals are those of oxy-hsemoglobin, and have the form of thin rhombic plates, disposed singly or in groups. (b.) Dog's Blood. — To 15 cc. of the defibrinated dog's blood provided for you, add, drop by drop, 1 cc. or so of ether, shaking the tube after each addition of ether. By this means the blood is rendered laky, a condition which is recognised by inclining the tube, and observing that the film of blood left on it, on bringing the tube to the vertical again, is transparent. Add no more ether, but place the tube in a freezing-mixture of ice and salt ; as the tempera- ture falls, crystals of haemoglobin separate. If the crystals do not separate at once, keep the tube in the freezing- mixture for one or two days. Examine some of the crystals under the microscope. 4. Ozone Test for Haemoglobin. — Mix some freshly-prepared alcoholic solution of guaiacurn with ozonic ether ; the mixture becomes turbid, and on adding even a dilute solution of hsemo- CHEMICAL PHYSIOLOGY. globin, a blue colour results. Or the reaction may be done on filter paper. 5. Spectroscopic Examination of Blood. — Use a small Browning's straight-vision spectroscope (Fig. 3). Fig. 3.— Browning's Straight Vision Spectroscope. Preliminary. — Observe the solar spectrum by placing the spectroscope before the eye, and directing it to the bright day- light. Note the spectrum from the red to the violet end, with the intermediate colours, and particularly the dark Frauenhofer's lines, known as D, E, b, and F, their position and relation to the colours. Make a diagram of the colours, and the dark lines, indicating the latter by their appropriate letters. (a.) Fix the spectroscope in a suitable holder, and direct it to a gas-flame, the edge of the flame being towards the slit in the spectroscope, noting that the spectrum shows no dark lines. (b.) Fuse a piece of platinum wire in a glass tube, and make a loop at the free end of the platinum wire. Dip the platinum wire in water and then into common salt, and burn the salt in the gas-flame, having previously directed the spectroscope towards the gas-flame, and so arranged the latter that it is seen edge-on. Note the position of the bright yellow sodium line D. 6. I. Spectrum of Oxy-hgemoglobin. (a.) Begin with a strong solution, and gradually dilute it. Place some defibrinated blood in a test-tube, and observe its opacity and bright scarlet colour. (b.) Adjust the spectroscope as follows : — Light a fan-tailed gas-burner, fix the spectroscope in a suitable holder, and THE COLOURED BLOOD-CORPUSCLES. 31 between the light and the slit of the spectroscope place a test-tube containing the blood or its solution. Focus the long image of the gas-flame on the slit of the spectroscope. The focal point can be readily ascertained by holding a sheet of white paper behind the test-tube. (c.) Add 10 to 15 volumes of water, and note that only the red part of the spectrum is visible. Make a sketch of what you see, noting the dilution. (d.) Add more water until the green appears, and observe that a single dark absorption band appears between the red and green (Fig. 4, 1). Continue to dilute until this single broad band is resolved into two by the transmission of yellow-green light. Burn a bead of sodic chloride in the gas-flame, to note distinctly the position of the D line, and observe that of the two absorption bands the one nearest D, conveniently designated by the letter a, is more sharply defined and narrower ; while the other, conveniently desig- nated by the letter (3, nearer the violet end, is broader and fainter. At the violet end the spectrum is shortened by absorption (Fig. 4, 2). (e.) Continue to dilute the solution, and note that the band near the violet end is the first to disappear. Sketch the appearances seen with each dilution, and indicate opposite each the degree of the latter. (f.) A very instructive method is to make a pretty strong solution of blood, showing only one undivided band. Place a little of this in a test-tube, and pour in water, so that the water mixes but slightly with the upper strata of the blood. Examine the solution spectroscopically, moving the tube so as to examine first the deeper strata of fluid until the surface is reached. At first a single band is seen ; but as the solution is more dilute above, the two bands begin to appear, and as the solution gets weaker above, the /3-band disappears, until, finally, with a very weak solution, such as is obtained in the upper strata only, the a-band alone is visible. 7. Hsematinometer. — For accurate observation, instead of a test-tube the blood is introduced into a vessel with parallel sides, 32 CHEMICAL PHYSIOLOGY. the glass plates being exactly 1 cm. apart (Fig. 6, D). Study this instrument (Hoppe-Seyler). 8. Hsematoscope. — By means of this instrument the depth of the stratum of fluid to be investigated can be varied, and the variation of the spectrum, with the strength of the solution, or the thickness of the stratum through which the light passes, at once determined (Hermann). Study this instrument. Red. Orange. Yellow. Green. Blue. g. 4— Spectra of Haemoglobin and its compounds.— 1, Oxy-haamoglobin 0-8 per cent.; 2, oxy-haemoglobin, 0'18 per cent.; 3, carbonic oxide haemoglobin ; 4, reduced haemoglobin. 9. II. Reduced Haemoglobin. (a.) To a solution of oxy-hsemoglobin showing two well- defined absorption bands, add a few drops of ammonium sulphide, and warm gently, when the solution becomes purplish or claret-coloured. (b.) Study the spectrum, and note that the two absorption bands of oxy-haemoglobin are replaced by one absorption band between D and E, not so deeply shaded, and with its edges less defined (Fig. 4, 4). By shaking the solution very THE COLOURED BLOOD-CORPUSCLES. 33 vigorously with air, and looking at once, the two bands may be caused to reappear for a short time. Observe the absorption of the light at the red and violet ends of the spectrum according to the strength of the fluid. (c.) Dilute the solution, and observe that the single band is not resolved into two bands, but gradually fades and dis- appears. (d.) To a similar solution of oxy-haemoglobin showing two well-defined bands, add Stokes's fluid, and observe the single absorption band of reduced haemoglobin. Shake the mixture with air and the two bands reappear. (e.) Use a solution of oxy-hsemoglobin where the two bands can just be seen, and reduce it with either ammonium sulphide or Stokes's fluid, and note that, perhaps, no absorp- tion band of reduced haemoglobin is to be seen, or only the faintest shadow of one. (/.) Compare the relative strengths of the solution of oxy- hsemoglobin and reduced haemoglobin. The latter must be considerably stronger to give its characteristic spectrum. Stokes's Fluid. — Make a solution of ferrous sulphate ; to it add ammonia after the previous addition of sufficient tartaric acid to prevent precipitation. It is usual to add about three parts by weight of tartaric acid to two of the iron salt. It should be made fresh when required. 10. III. Carbonic Oxide-Haemoglobin. — Through a diluted solu- tion of oxy-haemoglobin, or defibrinated blood, pass a stream of carbonic oxide — or coal gas — until no more CO is absorbed. Note the florid cherry-red colour of the blood. (a.) Dilute the solution in a test-tube and observe its spectrum, noting that a stronger solution is required than with HbO2 to show the absorption bands. Two absorption bands nearly in the same position as those of HbO2, but very slightly nearer the violet end (Fig. 4, 3). Make a map of the spectrum and bands. (b.) The bands are not affected by the addition of a re- ducing agent — e.g., ammonium sulphide or Stokes's fluid. 3 34 CHEMICAL PHYSIOLOGY. Add these fluids to two separate test-tubes of the solution of COHb, and observe that the two absorption bands are not affected thereby. There is no difference on shaking the solution with air, as the compound is so very stable. (c.) To a fresh portion of the solution of carbonic oxide haemoglobin add a 10 per cent, solution of caustic soda = cinnabar-red colour. Compare this with a solution of oxy- hremoglobin similarly treated. The latter gives a brownish- red mass. 11. IV. Acid-Hsematin. (a.) To diluted defibrinated blood add water and about 1 cc. of acetic acid, and warm gently, when the mixture be- comes brownish owing to the formation of acid-hsematin. (6.) Observe the spectrum of (a.\ noting one absorption band to the red side of D near 0 (Fig. 5, 5). Observe that there is considerable absorption of the blue end of the spectrum. (c.) The single band is not affected by the usual reducing agents, ammonium sulphide or Stokes's fluid. N.B. — If acetic acid alone be used to effect the change, observe that only one absorption band is seen. 12. Acid-Haematin in Ethereal Solution. (a.) To defibrinated blood add ether and a large quantity of strong acetic acid, which makes the mixture brown. Shake vigorously, and a dark-brown ethereal solution of haematin is obtained. (6.) Observe the spectrum of this solution — -four absorp- tion bands are obtained, one in the red between 0 and D, corresponding to the watery acid-hsematin solution ; a narrow faint one near D, one between D and E, and a fourth be- tween b and F (Fig. 5, 5). The last three bands are seen only in ethereal solutions, and require to be looked for with care. 13. V. Alkali-haematin. (a.) Take a solution of acid-haematin ; neutralise it with caustic soda until there is a precipitate of hsematin; on THE COLOURED BLOOD-CORPUSCLES. 35 adding more soda, and heating gently, the precipitate is re- dissolved and alkali-hsematin is formed. Or to diluted blood add a drop or two of solution of caustic potash, and warm gently. The colour changes, and the solution is dichroic. 90 111 IU jlLII j 100 110 Fig. 5.— Spectra of Derivatives of Haemoglobin. — 5, Hsematin in alcohol with sulphuric acid ; 6, hsematin in an alkaline solution ; 7, reduced heematin. (b.) Shake (a.) with air to obtain oxy-alkali-haematin. Observe its spectrum, one absorption band just to the red side of the D line. It is much nearer D than that of acid- hsematin (Fig. 5, 6). Much of the blue end of the spectrum is cut off. 14. Reduced Alkali-haematin or Haemo-chromogen. (a.) Add to 13, V. (b.) a drop or two of ammonium sulphide and warm gently = reduced alkali-hsematin, Stokes's reduced hsematin, or hgemo-chromogen, and observe its spectrum ; two absorption bands between D and E, as with HbO2 and HbCO, but they are nearer the violet end. The first band to the violet side of the D line is well-defined, while the second band still nearer the violet end (in fact it nearly coincides with the E line) is less defined. They disappear on shaking vigorously with air, and reappear on standing, provided sufficient ammonium sulphide be added. 36 CHEMICAL PHYSIOLOGY. 15. VI. Methsemoglobin. (a.) To a medium solution of oxy-h?emoglobin, add a few drops of a 1 percent, solution of potassic permanganate, warm gently, observe the change of colour, and examine it with a spectroscope. If the two bands of oxy-hsemoglobin are still present, allow it to stand for some time and examine again. If they persist, carefully add more permanganate until the two bands disappear. Finally, acidify the solution, and with a spectroscope look for the spectrum of methremoglobin, viz., one absorption band in the red near C, nearly in the same position, but nearer D than the band of acid-hsematin; the violet end of the spectrum is much shaded. Three other bands are described in the green, especially in dilute solutions. On adding ammonia to render the solution alkaline, the band in the red disappears, and is replaced by a faint band near D. (b.) To an alkaline solution showing the last described spectrum, add ammonium sulphide or Stokes's fluid. This- gives the spectrum of reduced haemoglobin; and on shaking with air, oxy-hremoglobin is formed. (c.) To a solution ofoxy-h?emoglobm, add a crystal or two of potassic chlorate ; dissolve it with the aid of gentle heat ; after a short time the spectrum of methsemoglobin is obtained. (d.) Action of Nitrites.— To diluted defibrinated ox blood, or preferably that of a dog, add a few drops of an alcoholic solution of amyl nitrite. The blood immediately assumes a chocolate colour. (e.) To another portion of diluted blood add a solution of potassic or sodic nitrite. Observe the chocolate colour. (/.) To portions of (d.) and (e.) add ammonia, the chocolate gives place to a red colour. (g.) Observe the spectrum of (d.) and (e.) The band in the red is distinct, and is best seen when the solution is of such a strength that only the red rays are transmitted. On dilution, other bands are seen in the green. Add ammonia, and with the change of colour described in (f.), the spectrum changes to that described in (a.) Add ammonium sulphide DERIVATIVES OF HEMOGLOBIN. 37 or Stokes's fluid, the spectrum of reduced haemoglobin appears, and on shaking up with air, the bands of oxy- hsemoglobin appear. 16. VII. Hsematoporphyrin. (Iron-free Hsematin.) (a.) Place some concentrated sulphuric acid in a test-tube, add some blood, and examine with the spectroscope. Or examine a solution obtained by dissolving some hsematin in concentrated sulphuric acid, and filtering through asbestos — when a clear purple-red solution is obtained. (6.) Observe two absorption bands, one close to and on the red side of D, and a second half-way between D and E. (c.) To some of the hsematin solution (in strong sulphuric acid), add a large excess of water, which throws down part of the hsematoporphyrin in the form of a brown precipitate, which is more copious if the acid be neutralised with an alkali — e.g., caustic soda. Dissolve some of the brown deposit in caustic soda, and examine it spectroscopically. (d.) The spectrum shows four absorption bands; a faint band midway between C and D, another similar one between D and E, but close to D ; a third band near E ; and a fourth one, darkest of all, occupying the greater part of the space between b and F, but nearer the former. In all cases make drawings of what you see, and compare them with the table of characteristic spectra suspended in the Laboratory. LESSON V. WAVE-LENGTHS— DERIVATIVES OF HAEMOGLOBIN— ESTIMATION OF HEMOGLOBIN. Spectroscopic Determination of Wave-Lengths. — Use Zeiss's spectroscope, which is provided with an illuminated scale for this purpose. 38 CHEMICAL PHYSIOLOGY. 1. W.L. of Absorption Bands of Oxy-Haemoglobin. (a.) Arrange the apparatus as shown in Fig. 6. A is the telescope through which the observer looks and sees the Fig. C. — Arrangement of the spectroscope for determining wave-lengths. — A, Telescope ; B, collimator tube ; C, scale tube ; D, hsematinometer. spectrum obtained by the light passing through B, and dis- persed by the flint-glass prism in the centre of the apparatus. In C is fixed a scale photographed on glass and illuminated by a fan-tailed burner. D is the heematinometer containing the dilute blood. (b.) Throw a piece of black velvet cloth over the prism ; light both lamps ; look through B ; adjust the slit in A and the telescope in B, so as to get a good view of the spectrum, and over it the image of the scale. D is supposed not to be in position at first. In a loop of platinum wire burn some common salt in the flame to get the bright yellow sodium line D. Adjust the scale so that this line corresponds to the figures 58-9 on the scale, and fix the spectroscope tubes (A . DERIVATIVES OF HAEMOGLOBIN. 39 and 0) in this position; the scale is now accurately adjusted for all other parts of the spectrum. " The numbers on the scale indicate wave-lengths ex- pressed in one hundred thousandths of a millimetre, and each division indicates a difference in wave-length equal to one hundred thousandth of a millimetre " (Gamgee). Thus, Frauenhofer's line, D, which corresponds to division 58-9 of the scale, has a wave-length of 589 millionths of a millimetre. The wave-lengths of Frauenhofer's lines are: — A = 7604, B = 687-4, 0 = 656-7, D = 5894, E = 527-3, F - 486-5. (c.) Using one of the blank maps provided for you (i.e., the maps supplied with Zeiss's spectroscope — the maps correspond to the scale seen in the spectroscope), fill in, in wave-lengths, the position of Frauenhofer's lines, B toF. (d.) Use a dilute solution of blood or haemoglobin — 1 part in 1000 of water is best — and place it in the heematinometer, D, which is placed in position between the flame and the spectroscope, as shown in Fig. 6. The distance between the parallel faces of D is 1 cm. The spectrum shows the two absorption bands of oxy-hsemoglobin between D and E. The narrower, sharper, and blacker band near D has its centre corresponding with the W.L. 579, and it may con- veniently be expressed by the letter a of the oxy-hsemo- globin spectrum (Gamgee). The other absorption band near E, and conveniently designated (3, is broader, not so dark, and has less sharply defined edges than a. Its centre corresponds to the W.L. 553 -8. Notice that the other parts of the spectrum are seen, there being only slight cutting off of the red, and a slightly greater absorption of the violet end. (e.) Work with a stronger solution of blood, and observe how the two bands become fused into one, while more and more of the red and violet ends of the spectrum are absorbed as the solution is made stronger, until finally only a little red light is transmitted. 2. W.L. of Absorption Band of Reduced Hb. (a.) Adjust the apparatus as before, but reduce the oxy- hsemoglobin solution with Stokes's fluid — noticing the 40 CHEMICAL PHYSIOLOGY. change of the colour to that of purplish or claret — until a solution is obtained, which gives the single characteristic absorption band of reduced Hb. This is usually obtained with a solution of Hb of about O2 per cent. (b.) Observe the single absorption band less deeply shaded, and with less denned edges between D and E, conveniently designated by the letter 7. It extends be- tween W.L. 595 and 538, and is not quite intermediate between D and E ; is blackest opposite W.L. 550, so that it lies nearer D than E (Gamgee), Both ends of the spectrum are more absorbed than with a solution of oxy-hsemoglobin of the same strength. On further dilution of the solution, the band does not resolve itself into two bands, but simply diminishes in width and intensity. 3. W.L. of the Spectrum of Carbonic Oxide Haemoglobin. (a.) Use a dilute solution of carbonic oxide-haemoglobin of such a strength as to give the two characteristic absorp- tion bands. (b.) Observe the two bands, a and /3, like those of Hb-O2, but both are very slightly more towards the violet end of the spectrum, a extends from about W.L. 587 to 564, and j8 from 547 to 529 (Gamgee). (c.) No reduction is obtained by reducing agents. 4. Preparation of Haematin. (a.) Make defibrinated blood into a paste with potassic carbonate. Dry the paste on a water-bath. Place some of the paste in a flask, add 4 volumes of alcohol, and boil on a water-bath. Filter, and an alkaline brown solution of haematin is obtained. Re-extract the residue several times with boiling alcohol, and mix the alcoholic extracts. The solution is dichroic. (6.) Acidify the alkaline nitrate of (a.) with dilute sul- phuric acid, filter, and keep the filtrate. Observe the spec- trum of acid-haematin in the filtrate (Fig. 5, 5). (c.) Add excess of ammonia to the acid filtrate of (6.), and filter off the precipitate, keep the filtrate, and observe that DERIVATIVES OF HEMOGLOBIN. 41 it is dichroic. Observe the spectrum of alkali-hsematin in the filtrate (Fig. 5, 6). (d.) Evaporate the filtrate from (c.) to dryness on a water- bath. Extract the residue with boiling water. The black residue is washed on a filter with distilled water, alcohol, and ether, and dried in a hot chamber at 120° C. This is nearly pure hsematin. (e.) It is convenient to keep in stock hsematin prepared as follows : — Extract defibrinated blood or blood-clot (ox or sheep) with rectified spirit containing pure sulphuric acid (1 : 20). Filter, the solution gives the spectrum of acid-hsematin. Add an equal volume of water and then chloroform. The chloroform becomes brown, and there is a precipitate of proteids. Separate the chloroform extract, wash it with water to remove the acid. Separate the chloro- form, and allow it to evaporate. The dark brown residue is impure hsematin. When dissolved in alcohol and caustic soda it gives the spectrum of alkali-hrematin, and on adding ammonium sulphide that of hsemochromogen. If it is dissolved in H2SO4, and filtered through asbestos, the red filtrate gives the spectrum of hsematoporphyrin (MacMunn). 5. Hsemin Crystals. — Place some powdered dried blood on a glass slide, add a crystal of sodium chloride, and a few drops of glacial acetic acid. x Cover with a cover-glass, and heat care- ** ^~ \ ^ fully over a flame until bubbles of gas ^ % v v are given off. After cooling, brown or black rhombic crystals of hsematin x are to be seen with a microscope " >N 6. Detection of Blood Stains.— Use a * ^ y piece of rag stained with blood. ^ \ v .v **~ (a.) Moisten a part of the stain I ^ ^ \ x. with glycerin, and after a time Fig. 7.— Harniin Crystals express the liquor, and observe it prepared from traces microscopically for blood-corpuscles. °* blood. (b.) Tie a small piece of the stained cloth to a thread, place the cloth in a test-tube with a few drops of distilled water, and leave it until the colouring-matter is extracted. 42 CHEMICAL PHYSIOLOGY. Withdraw the cloth by means of the thread. Observe the coloured fluid spectroscopically. (c.) Boil some of the extract with hydrochloric acid, and add potassic ferrocyanide ; a blue colour indicates the pre- sence of iron. (d.) Use the stain for the hsemin test, doing the test in a, watch-glass (Lesson V., 5). AMOUNT OF HAEMOGLOBIN IN BLOOD. 7. Colorimetric Method (Hoppe-Seyler's Method).— A standard solution of pure haemoglobin diluted to a known strength is used, and with this is compared the tint of the blood diluted with a known volume of distilled water. (a.) The demonstrator will prepare a standard solution of haemoglobin of known strength. (b.) Spread a sheet of white paper on a table in a good light opposite a window, and on it place two hsematino- meters side by side (Fig. 6, D). See that they are water- tight. If not, anoint the edges of the glass plates with vaseline to make them water-tight. J;.) Take 10 cc. of the standard solution of haemoglobin dilute it with 50 cc. of water, and place it in one of the hsematinometer s . (d.) Weigh 5 grammes of the blood to be investigated, and dilute it with water exactly to 100 cc. (e.) Place 10 cc. of this deeper tinted blood (d.) into the second hsematinometer. (/) Fill an accurately graduated burette with distilled water, place it over the second hsernatinometer (e.\ and dilute the blood in it until it has precisely the same tint as the standard solution in the other haematinometer. Note the amount of water added. The two solutions must now contain the same amount of hsemoglobin. Example (Hoppe-Seyler).— 20 '186 grms. of detibrinated blood were diluted with water to 400 cc. To the 10 cc. of this placed in a hsematino- DERIVATIVES OF HAEMOGLOBIN. 43 meter, 38 cc. of water had to be added to obtain the same tint as that of the standard solution, so that the volume of water which would require to be added to dilute the whole 400 cc. would be 1,520 cc., thus— 10 : 400 : : 38 : x x= l,520cc. By adding 1,520 cc. of distilled water to the 400 cc. of blood solution, we get 1,920 cc. of the same tint or degree of dilution as the standard solution. The standard solution on analysis was found to contain 0'145 grms. of haemoglobin in 100 cc., so that the total amount of the haemoglobin in the diluted blood is found, thus — 100 : 1,920 : : 0'145 : x x = 2 '784 grms. Since, however, this amount of haemoglobin was obtained from 20 '186 grms. of the original blood, the amount in 100 parts will be found, as follows: — 20-186 : 100 : : 2'784 : x x — 13 '79 grms. per cent. 8. The Haemoglobinometer of Gowers is used for the clinical estimation of haemoglobin (Fig. 8). The tint of the dilution of a given volume of blood with distilled water is taken as the index of the amount of haemoglobin. The colour of a dilution of aver- age normal blood (one hundred times) is taken as the standard. The quantity of haemoglobin is indicated by the amount of dis- tilled water needed to obtain the tint with the same volume of blood under examination as was taken of the standard. On account of the instability of a standard dilution of blood, tinted glycerin-jelly is employed instead. The apparatus consists of two glass tubes of exactly the same size. One contains (D) a standard of the tint of a dilution of 20 c.mm. of blood, in 2 cc. of water (1 in 100). The second tube (0) is graduated, 100° = 2 c. (100 times 20 c.mm.) The 20 c.mm. of blood are measured by a capillary pipette (B). (a) Place a few drops of distilled water in the bottom of the graduated tube (C). (b.) Puncture the skin at the root of the nail with the shielded lancet (F), and with the pipette (B) suck up 20 c.mm. of the blood, and eject it into the distilled water, and rapidly mix them. (c.) Distilled water is then added drop by drop (from the pipette stopper of a bottle (A) supplied for that purpose) 44 CHEMICAL PHYSIOLOGY. until the tint of the dilution is the same as that of the standard. The amount of water which has been added (i.e., the degree of dilution) indicates the amount of haemoglobin. Fig. 8. — A, Pipette bottle for distilled water ; B, capillary pipette ; C, graduated tube ; D, tube with standard dilution ; F, lancet for pricking the finger. " Since average normal blood yields the tint of the standard at 100° of dilution, the number of degrees of dilution necessary to obtain the same tint with a given specimen of blood is the per- centage proportion of the haemoglobin contained in it, compared to the normal. For instance, the 20 c.mm. of blood from a patient with anaemia gave the standard tint of 30° of dilution. Hence it contained only 30 per cent, of the normal quantity of haemoglobin. By ascertaining with the hsemacytometer the cor- puscular richness of the blood, we are able to compare the two. A fraction, of which the numerator is the percentage of haemo- globin, and the denominator the percentage of corpuscles, gives at once the average value per corpuscle. Thus the blood men- tioned above containing 30 per cent, of haemoglobin, contained 60 per cent, of corpuscles ; hence the average value of each cor- puscle was £g or £ of the normal. Variations in the amount of haemoglobin may be recorded on the same chart as that employed for the corpuscles." " In using the instrument, the tint may be estimated by holding DERIVATIVES OF HEMOGLOBIN. 45 the tubes between the eye and the window, or by placing a piece of white paper behind the tubes ; the former is perhaps the best. In practice it will be found that, during 6 or 8 degrees of dilu- tion, it is difficult to distinguish a difference between the tint of the tubes. It is therefore necessary to note the degree at which the colour of the dilution ceases to be deeper than the standard, and also that at which it is distinctly paler. The degree midway between these two will represent the haemoglobin percentage." 9. Fleischl's Hsemometer. — This apparatus (Fig. 9) consists of Fig. 9. — Fleischl's Hsemometer. a horse-shoe stand with a pillar bearing a reflecting surface (S) and a platform. Under the table or platform is a slot carrying a glass wedge stained red (K), and moved by a wheel (R). On the platform (M) is a small cylindrical vessel divided into two com- partments (a and a') by a vertical septum. In one compart- ment is placed pure water, and in the other the blood to be investigated. A scale (P) on the slot of the instrument enables one to read off directly the percentage of haemoglobin. (a.) Fill with a pipette the compartment (a') over the wedge with distilled water, and see that the surface of the water is 46 CHEMICAL PHYSIOLOGY. quite level with the top of the cylinder. Fill the other compartment (a), that for the blood, about one-quarter with distilled water. (b.) Prick the finger as in 8 with the instrument supplied for the purpose. Fill the short automatic capillary pipette tube with blood. Its capacity is 6-5 c.mm. In filling the tube, hold it horizontally. See that no blood adheres to the surface of the tube. This can be done by having the pipette slightly greasy on the outer surface. (c.) Dissolve the blood obtained in (b.) in the water of the blood-compartment (a'), washing out every trace of blood from the pipette. Mix the blood and water thoroughly. Clean the pipette. Then fill the blood-compartment exactly to the surface with distilled water, seeing that its surface also is perfectly level. (d.) Arrange a lamp in front of the reflector (S) — which is white, and with a smooth matt surface made of plaster-of- Paris — so as to throw a beam of light vertically through both compartments. Look down vertically upon both compart- ments, and move the wedge of glass by the milled head (T) until the colour in the two compartments is identical. Read off the scale, which is so constructed as to give the per- centage of haemoglobin. CHEMISTRY OF DIGESTION. LESSON VI. SALIVARY DIGESTION. 1. To obtain mixed Saliva. — Rinse out the mouth with water. Inhale the vapour of ether, glacial acetic acid, or even cold air through the niouth, which causes a reflex secretion of saliva. In doing so, so curve the tongue and place its tip behind the incisor teeth of the upper jaw. In a test-glass collect the saliva with as few air-bubbles as possible. If it be turbid or contain much froth, filter it through a small filter. 2. I. Microscopic Examination. — With a high power observe e presence of (1) squamous epithelium, (2) salivary corpuscles, ) perhaps debris of food, and (4) possibly air-bubbles. II. Physical and Chemical Characters. (a.) Observe its appearance — either transparent or trans- lucent— and that when poured from one vessel to another it is glairy and more or less sticky. On standing, a white deposit is apt to form. (b.) Test its reaction, neutral or alkaline. (c.) Place a little mixed saliva in a test-tube, add dilute acetic acid = a precipitate of mucin. Filter. (d). With the filtrate from (c.), test for traces of proteids (albumin and globulin) with the xanthoproteic reaction (Lesson I., 1, a), or by the addition of potassium ferrocyanide. (e.) To a few drops of saliva in a porcelain vessel, add a few drops of dilute ferric chloride = a red coloration, due 48 CHEMICAL PHYSIOLOGY. to potassic sulpho-cyanide. The colour is discharged by mercuric chloride. Meconic acid yields a similar colour, but it is not discharged by mercuric chloride. The sulpho- cyanide is present only in parotid saliva, and is generally present in mixed saliva. (/.) Test a very dilute solution of potassic sulpho-cyanide to compare with (e.) (g.) The salts are tested for in the usual way (see " Urine "). Test for chlorides (HISrO3, and AglS"O3), car- bonates (acetic acid), and sulphates (barium nitrate and nitric acid). 3. Digestive Action. Starch Solution. — Place 1 grm. of starch in a mortar, add a few cc. of cold water, and mix well with the starch. Add 200 cc. of boiling water, stirring all the while. Boil the fluid in a flask for a few minutes. This gives a half per cent, solution. Do the tests for starch already described (Lesson II., 2), and especially satisfy yourself that no glucose or reducing sugar is present. Action of Saliva on Starch. (a.) Dilute the saliva with 5 volumes of water. Label three test-tubes A, B, and C. In A place starch mucilage, in B saliva, and in 0 1 volume of saliva and 3 volumes of starch mucilage. Plug all three with cotton-wool, and place them in a water-bath at 40° C., and leave them there for ten minutes. Test for a reducing sugar in portions of all three, by means of Fehling's solution. A and B give no evidence of sugar, while C reduces the Fehling, giving a yellow or red deposit of cuprous oxide. Therefore, starch is converted into a reducing sugar by the saliva. This is done by the ferment ptyalin contained in it. (b.) Test a portion of C with solution of iodine ; no blue colour is obtained, as all the starch has disappeared, being converted into a reducing sugar or maltose. (c.) Make a thick starch mucilage, place some in test- tubes labelled A and B. Keep A for comparison, and to B add saliva, and expose both to 40° 0. Notice that A is un- affected, while B soon becomes fluid — within two minutes — SALIVARY DIGESTION. 49 and loses its opalescence ; this liquefaction is a process quite antecedent to the saccharifying process which follows. 4. Stages between Starch and Maltose. — Mix some starch and saliva in a test-tube as in 3 (a.) C, and place it in a water-bath at 40° 0. At intervals of a minute, test small portions with iodine. Do this by taking out a drop of the liquid by means of a glass rod. Place the drop on a white porcelain plate, and by means of another glass rod add a drop of iodine solution. Note the following stages — At first there is pure blue with iodine, later a deep violet, showing the presence of erythro- dextrin, the violet resulting from a mixture of the red produced by the dextrin and the blue of the starch. Then the blue reaction entirely disappears, and a reddish-brown colour, due to ery thro- dextrin alone is obtained. After this the reaction becomes yellowish-brown, and finally there is no reaction with iodine at all, achroo-dextrin being formed, along with a reducing sugar or maltose. The latter goes on forming after iodine has ceased to react with the fluid, and its presence is easily ascertained by Fehling's solution. 5. Effect of Different Conditions on Salivary Digestion. (a.) Label three test-tubes A, B, and C. Into A place some saliva and boil it, add some starch mucilage. In B and 0 place starch mucilage and saliva, to B add a few drops of hydrochloric acid, and to C caustic potash. Place all three at 40° C. on a water-bath, and after a time test them for sugar by Fehling's solution. No sugar is formed — in A because the ferment was destroyed by boiling, and in B and 0 because strong acids and alkalies arrest the action of ptyalin on starch. (6.) If a test-tube containing starch mucilage and saliva be prepared as in 3 (a.) C, and placed in a freezing-mixture, the conversion of starch into a reducing sugar is arrested ; but the ferment is not killed, for on placing the test-tube at 40° 0. the conversion is rapidly effected. (c.) Mix some raw starch with the saliva and expose it 40° 0. Test it after half an hour or longer, when no su£ will be found. 6. Starch is a Colloid, but Sugar dialyses. to sugar 50 CHEMICAL PHYSIOLOGY. (a.) Place in a short piece of the sausage parchment tube, already referred to (Lesson I. 1, h\ 20 cc. of starch mucilage, label it A, and into another, some starch mucilage with saliva, label it B. Suspend A and B in distilled water in separate vessels. (b.) After some hours test the diffusate in the distilled water. No starch will be found in the diffusate of either A or B, but sugar will be found in that of B, proving that sugar dialyses, while starch does not. Hence the necessity of starch being converted into a readily diffusible body during digestion. 7. Action of Malt-Extract on Starch. (a.) Rub up 10 grms. of starch with 30 cc. of distilled water in a mortar, add 200 cc. of boiling water, and make a strong starch mucilage. (6.) Powder 5 grms. of pale low dried malt, and extract it for half an hour with 30 cc. of distilled water, and filter. Keep the filtrate. (c.) Place the starch paste of (a.) in a flask, and cool to 60° 0., add the extract of (6.), and place the flask in a water- bath at 60° 0. (d.) Observe that the paste soon becomes fluid, owing to the formation of soluble starch, and if it be tested from time to time (as directed in 4), it gives successively the tests for starch and erythro-dextrin. Continue to digest it until no colour is obtained with iodine. (e.) Take a portion and precipitate it with alcohol = achroo- dextrin. (/) Boil the remainder of the fluid, cool, filter, and evaporate the filtrate to 20 cc. Add 6 volumes of 90 per cent, spirit to precipitate the dextrin, boil, filter, and con- centrate to dryness on a water-bath, and dissolve the residue in distilled water. The solution is maltose (Ol2H22On + H2O). If the alcoholic solution be exposed to air, crystals of maltose are formed. GASTRIC DIGESTION. 51 8. Compare the Reducing Power of Maltose and Dextrose. (a.) With Fehling's solution estimate the reducing power of the solution obtained in 7 (/.) (See " Urine.") (b.) Boil in a flask for half an hour 50 cc. of the solution of maltose with 5 cc. of hydrochloric acid. Neutralise with caustic soda, and make up the volume, which has been re- duced by the boiling, to 50 cc., and determine by Fehling's solution the reducing power. The acid has converted the maltose into dextrose, and the ratio of the former estimation (a.) to the present one should be 66 to 100. (c.) A solution of pure dextrose treated as in (b.) is not affected in its reducing power. Saliva has practically the same effect on starch as malt-extract, and may be used instead of the latter. LESSON VII. GASTRIC DIGESTION. 1. Preparation of Artificial Gastric Juice. (a.) Take a part of the cardiac end of the pig's stomach provided for you, which has been previously opened and washed rapidly in cold water. Spread it, mucous sur- face upwards, on the convex surface of an inverted capsule. Scrape the mucous surface firmly with the handle of a scalpel, and rub up the scrapings in a mortar with fine sand. Add water, and rub up the whole vigorously for some time, and filter. The filtrate is an artificial gastric juice. (b.) From another portion of the cardiac end of a pig's stomach detach the mucous membrane in shreds, dry them between folds of blotting-paper, place them in a bottle, and cover them with strong glycerin, letting them stand for eight days. The glycerin dissolves out the pepsin, and on filtering, a glycerin extract with high digestive properties is obtained (v. Wittich's Method). 52 CHEMICAL PHYSIOLOGY. (c.) Instead of (a.) and (b.) it is convenient to use Benger's Liquor pepticus, or the pepsin preparation of Burroughs, Wellcome & Co. All the above artificial juices, when added to hydrochloric acid of the proper strength, have high digestive powers. 2. Both Hydrochloric Acid and Pepsin are required for Gastric Digestion. (a.) Take three beakers or large test-tubes, label them A, B, C. Fill A two-thirds full of hydrochloric acid 0-2 per cent., put into B some water and a few drops of glycerin extract of pepsin, or powdered pepsin, and fill 0 two-thirds full with 0-2 per cent, hydrochloric acid, and a few drops of glycerin extract of pepsin. Put into all three a flake of well-washed fibrin, and place them all in a water-bath at 40° C. for half an hour. (6.) Examine them. In A, the fibrin is swollen up ; in B, unchanged; while in C, it has disappeared, having first become swollen up and clear, and finally completely dis- solved, being converted into peptones. Therefore, both acid and ferment are required for gastric digestion. 3. To Prepare Hydrochloric Acid of 0-2 per cent— Add 6-5 cc. of ordinary commercial hydrochloric acid to 1 litre of distilled water, and shake together. 4. Products of Peptic Digestion and its Conditions. (a.) Take three large test-tubes, labelled A, B, C, and fill each one half full with hydrochloric acid 0-2 per cent. Add to each 10 drops of a glycerin extract of pepsin. Boil B, and make C faintly alkaline with sodic carbonate. The alkalinity may be noted by adding previously some neutral litmus solution. Add to each an equal amount — a few shreds of well- washed fibrin — which has been previously steeped for some time in 0'2 per cent, hydrochloric acid, so that it is swollen up and transparent. Keep the tubes in a water-bath at 40° G. for an hour, and examine them at intervals of twenty minutes. (6.) After twenty minutes A begins to be turbid, and the fibrin is dissolving. In B and C there is no change. After GASTRIC DIGESTION. 53 forty minutes A is turbid, and the fibrin is dissolved. In B and 0 no change. At the end of an hour, filter A and part of B and C. Keep the filtrates. (c.) Carefully neutralise the filtrate of A with dilute •caustic soda. The filtrate becomes turbid and gives a pre- cipitate of parapeptones (antialbumose and hemialbumose). Filter off this precipitate, dissolve it in 0'2 per cent, hydro- chloric acid. It gives proteid reactions. (d.) With a solution of parapeptones (hemialbumose) repeat the ordinary reactions for proteids. Hemialbumose is soluble in water, and gives all the ordinary proteid reactions. It is precipitated by nitric acid in the cold, but the precipitate is redissolved with the aid of heat. (e.) Test the filtrate of (c.) for peptones. Repeat all the tests for peptones (Lesson I., 10, VI.) Note that hemial- bumose gives the ordinary proteid reactions. Note also the differences between peptones and hemialbumose. Hemi- albumose is precipitated by acetic acid and ferrocyanide of potassium ; by acetic acid and a saturated solution of sodic sulphate ; and by metaphosphoric acid : which peptones are not. Like peptones, it is soluble in water. (f.) Neutralise part of the filtrates of B and C. They give no precipitate, nor. do they give the reactions for pep- tones. In B the ferment pepsin was destroyed by boiling, while in C the ferment cannot act in an alkaline medium. (g.) If to the remainder of C acid be added, and it be placed again at 40° 0., digestion takes place, so that neutralisa- tion has not destroyed the activity of the ferment. 5. To prepare Hemialbumose and Gastric Peptones in Quantity. (a.) Place 10 grms. of fresh, well-washed, expressed fibrin in a porcelain capsule, cover it with 300 cc. of 0-2 per cent, hydrochloric acid, and keep the whole at 40° C. in a water-bath until the whole of the fibrin is so swollen up as to become converted into a perfectly clear, jelly-like mass, and it becomes so thick that a glass rod is supported erect in it. (b.) Add 1 or 2 cc. of glycerin pepsin extract, and stir 54 CHEMICAL PHYSIOLOGY. the mass, fluid. Within a few minutes the whole becomes (c.) After a short time— fifteen to twenty minutes— before the peptonisation is complete, filter and exactly neutralise the filtrate with ammonia, which precipitates the antialbumpse and hemialbumose. Dissolve these in a 5 per cent, solution of sodic chloride. To isolate the hemialbumose, precipitate it with nitric acid, or dialyse the salt solution of it in a parch- ment-paper tube arranged in Kuhne's dialyser (Fig. 10). The greater part of the hemialbumose is thrown down in flocculi on the parchment tube. (d.) The filtrate after neutral- isation is evaporated, and yields peptones, which can be precipi- tated by alcohol. 6. Action of Gastric Juice on Milk. (a.) Place 5 cc. of fresh milk in a test-tube, and add to it a few drops of a neutral artificial gastric juice. Mix and keep at 40° C. In a short time the milk curdles, so that the tube can be inverted without the curd fall- Fig- 10. ing out. By-and-by whey is Kiihne's Dialyser. squeezed out of the clot. The curdling of milk by the rennet ferment present in the gastric juice is quite different from that produced by the "souring of milk," or by the precipi- tation of casein by acids. Here the casein (carrying with it most of the fats) is precipitated in a neutral fluid. (b.) To the test-tube add 5 cc. of 0-4 per cent, hydrochloric acid, and keep at 40° C. for two hours. The pepsin in the presence of the acid digests the casein, gradually dissolving it, forming a straw-yellow coloured fluid containing peptones. The " peptonised milk " has a peculiar odour and bitter taste. GASTRIC DIGESTION. 55 (c.) To 5 cc. of milk in a test-tube add a few drops of Benger's liquor pepticus, and place in a water-bath. Observe how the casein first clots, and is then partially dissolved to form a yellowish coloured fluid, with a bitter taste and peculiar odour. There generally remains a very con- siderable clot of casein ; and, in fact, the gastric digestion of milk is slow, especially if compared with its tryptic digestion (Lesson VIII., 9). Test the fluid for peptones with the biuret test, and observe the beautiful light pink colour obtained. The bitter taste renders milk "peptonised" by gastric juice unsuitable for feeding purposes. 7. Action of Rennet on Milk. (a.) Place some milk in a test-tube, label it A, add a drop or two of rennet, shake it up, and place the tube in a water-bath at 40° 0. Observe that the milk becomes solid in a few minutes, forming a curd, and by-and-by the curd of casein contracts and squeezes out a fluid — the whey. (b.) Repeat the same experiment, but previously boil the rennet. No such result is obtained as in (a.) 8. Comparison of Mineral and Organic Acids. (a.) Take two test-tubes, A and B. Place in A 10 cc. of a 0*2 per cent, solution of hydrochloric acid, and in B 10 cc. of a 5 per cent, solution of acetic acid. To both add a few drops of o-o-Tropseolin dissolved in alcohol. The very dilute mineral acid in A renders it rose-pink, while the far stronger organic acid does not affect its colour. (b.) Repeat (a.), but add to the acids a dilute solution of methyl-violet, and note the change of colour produced by the mineral acid. It becomes blue. (c.) Repeat (a.) with the same acids, but use threads stained with congO-red, and observe the change of colour to blue produced by the hydrochloric acid. (d.) Instead of threads stained with congo-red, use papers similarly stained, or a watery solution of congo-red. (e.) For lactic acid. Prepare a fresh solution by mixing 10 cc. of a 4 per cent, solution of carbolic acid, with 20 cc. 56 CHEMICAL PHYSIOLOGY. of distilled water, and 1 drop of liquor ferri perchloridi. The blue solution thus obtained is changed to yellow by lactic acid, while it is not affected by 0*2 per cent. HC1 (Uffelmann's Reaction). These reactions for a mineral acid are specially to be noted, as they are sometimes used clinically for ascertaining the presence or absence of hydrochloric acid — e.g., in a vomit. This acid is almost invariably absent from the gastric juice in cancer of the stomach. It is to be noted, however, that the presence of pep- tones interferes with the delicacy of these reactions. LESSON VIII. PANCREATIC DIGESTION. 1. Preparation of Artificial Pancreatic Juice. (a.) Use part of the pancreas of an ox twenty-four hours after the animal was killed. Mince a portion of the pancreas, rub it up with well-washed fine sand in a mortar, and digest it with cold water, stirring vigorously. After a time strain through muslin, and then filter through paper. The filtrate has digestive properties chiefly upon starch. Instead of water a more potent solution is obtained by digesting the pancreas at 40° C. for some hours with a 2 per cent, solution of sodic carbonate. To prevent the putre- factive changes which are so apt to occur in all pancreatic fluids, add a little 10 per cent, alcoholic solution of thymol. (b.) Make a glycerin extract of the pancreas in the same way as described for the stomach (Lesson VII., 1, b.) Before putting it in glycerin it is well to place it for two days in absolute alcohol to remove all the water. This extract acts on starch and proteids. (c.) For most experiments it is more convenient to use the excellent pancreatic extracts now supplied by Mr. Benger, of Manchester, as "Liquor Pancreaticus," or those of Messrs. Savory & Moore, or Burroughs, Wellcome & Co. PANCREATIC DIGESTION. 57 (d.) Weigh the pancreas taken from a dog just killed, rub it up with sand in a mortar, and add 1 cc. of a 1 per cent, solution of acetic acid for every gramme of pancreas. Mix thoroughly, and after a quarter of an hour add 10 cc. of glycerin for every gramme of pancreas. After five days filter off the glycerin extract. The acetic acid is added to convert the unconverted " zymogen " into trypsin. (e.) Kiihne's Dry Pancreas Powder. — This is obtained by thoroughly extracting a pancreas with alcohol and ether, and drying the residue. It is better to purchase the pre- paration. Extract the dry pancreas powder with five parts of a 1 per cent, solution of salicylic acid, and keep it about 40° C. for four or five hours. Filter, and use the filtrate as a glycerin or other extract would be used. It has only proteolytic properties. I find this extract acts much more energetically than those prepared in other ways. 2. I. Action on Starch. (a.) Take thick starch mucilage in a test-tube or beaker, add glycerin extract of pancreas or liquor pancreaticus, and place it in a water-bath at 40° 0. Almost immediately the starch paste becomes fluid, loses its opalescence, and be- comes clear. Within a few minutes much of the starch is converted into a reducing sugar or maltose. (b.) Test for sugar (Lesson II., 6, IV., b., c.) 3. The same conditions obtain as for saliva (Lesson VI., 5). 4. II. Proteolytic Action due to Trypsin, and its Conditions. (a.) Take three test-tubes, labelled A, B, and C, fill each half full with 1 per cent, solution of sodic carbonate, and place 5 drops of glycerin pancreatic extract, or liquor pan- 'creaticus in each. Boil B, and make C acid with dilute hydrochloric acid. Place in each tube an equal amount of well-washed fibrin, plug the tubes with cotton-wool, and place all in a water-bath at 40° 0. (b.) Examine them from time to time. At the end of one hour or so, there is no change in B and C, while in A the fibrin is gradually being eroded, and finally disappears, but it does not swell up, the solution at the same time 58 CHEMICAL PHYSIOLOGY. becoming slightly turbid. After two hours, still no change is observable in B and 0. (c.) Filter A, and carefully neutralise the nitrate with very dilute hydrochloric or acetic acid = a precipitate of hemialbumose. Filter off the precipitate, and on testing the nitrate, peptones are found. (d.) Filter B and C, and carefully neutralise the nitrates. They give no precipitate. No peptones are found. (e.) Test the proteolytic power of an extract of Kiihne's "pancreas powder" (Lesson VIII., 1, e.) 5. Products other than Peptones. Leucin and Tyrosin (Indol). (a.) Place 300 cc. of a 1 per cent, solution of sodic carbonate in a flask, add 5 grammes of boiled fibrin, 5 cc. of glycerin extract of pancreas, and a few drops of an alcoholic solution of thymol. Keep all at 38° C. on a water-bath for six to ten hours. (b.) After six hours take a portion of the mixture, filter, and to the filtrate cautiously add dilute acetic acid to preci- pitate any hemialbumose that may be present in it. Filter and evaporate the filtrate to a small bulk, and precipitate the peptones by a considerable volume of alcohol. Filter to remove the peptones, and evaporate the alcoholic filtrate to a small bulk, and set it aside, whenleucin separates first, and crystals of tyrosin afterwards. Keep them for microscopic examination. (c.) A much better method of obtaining leucin and tyrosin is to digest, at 40° C., for five or six hours, equal parts of fresh moist fibrin and ox-pancreas with a sufficient quantity of thymolised water. Boil part of the liquid, and evaporate a small quantity of it, or merely place a drop on a glass slide and allow it to evaporate, when beautiful microscopic crystals of leucin and tyrosin are obtained. Continue the digestive process of the remainder of the liquid for a few hours, until the mixture emits a very disagreeable odour. This fluid gives the chlorine and indol reaction splendidly. (d.) Examine the crystals of leucin and tyrosin microscopi- cally. The former occurs as brown balls, often with radiat- ing lines, not unlike fat, but much less refractive, and the PANCREATIC DIGESTION. 59 latter consists of long white shining needles arranged in a stellate manner, or somewhat felted (see " Urine," Fig. 32). (e.) Test for Tyro sin (Hofmann). — Dissolve some crystals by boiling them in water, add Millon's reagent, and boil, which gives a rosy-red colour. (f.) Test a solution of ty rosin obtained by the prolonged boiling of horn shavings and sulphuric acid, with Millon's reagent, as in (e.) (g.) Indol. — The remainder of the original digestive fluid after digestion for ten hours or longer, emits an intensely disagreeable odour, due to indol, whose presence is ascer- tained by warming the liquid, and adding first a drop or two of dilute sulphuric acid to some of the filtered liquid, and then a very dilute nitrite solution. A red colour indicates the presence of indol. This test is very readily obtained with the products of digestion by Kiihne's dry pancreas (Lesson VIII., 1, e). One must be careful to regulate the strength of the acid. (h.) Acidify strongly with hydrochloric acid a small quan- tity of the highly offensive fluid, and place in it a shaving of wood or a wooden match with its head removed and soaked in strong hydrochloric acid. The match is coloured a beautiful red, sometimes even an intense red. The match can be dried, and it keeps its colour for a long time, although the colour darkens and becomes somewhat duskier on drying. (i.) Chlorine Reaction. — Add to some of the digestive fluid (g, or preferably c), drop by drop, chlorine water, it strikes a beautiful rosy-red tint. Or add very dilute bromine water (1 to 2 drops to 60 cc. water), the fluid first becomes pale red, then violet, and ultimately deep violet (Kuhne). III. The Action on Fats is twofold. 6. A. Emulsification. (a.) Rub up in a mortar which has been warmed in warm water, a little olive oil or melted lard, and some pieces of fresh pancreas. A creamy, persistent emulsion is formed. Examine the emulsion under the microscope. Or use a 60 CHEMICAL PHYSIOLOGY. watery extract of the fresh pancreas, and do likewise ; but in this case the result will not be nearly so satisfactory. (b.) Rub up oil as in (a.); but this time use an extract of the fresh pancreas made with 1 per cent, sodic carbonate. A very perfect emulsion is obtained, even if the sodic carbonate extract is boiled beforehand. This shows that its emulsifying power does not depend on a ferment. (c.) The presence of a little free, fatty acid greatly favours emulsitication. Take two samples of cod-liver oil, one perfectly neutral (by no means easily procured), and an ordinary brown oil — e.g., De Jongh's. The latter contains much free fatty acid. Place 5 cc. of each in two test-tubes, and pour on them a little solution of sodic carbonate (1 per cent.) The neutral oil is not emulsified, while the rancid one is at once, and remains so. Many oils that do not taste rancid contain free fatty acids, and only some of them give up their acid to water, just according as the fatty acid is soluble in water, or not. 7. B. The Fat-Splitting Action of Pancreatic Juice. (a.) Prepare a perfectly neutral oil. — A perfectly neutral oil is required, and as all commercial oils contain free fatty acids, they must not be used. Place olive or almond oil in a porcelain capsule, mix it with not too much baryta solution, and boil for some time. Allow it to cool. The unsaponified oil is extracted with ether, the ethereal extract separated from the insoluble portion, and the ether evaporated over warm water. The oil should now be perfectly neutral (Krukeriberg). (b.) Mix the oil with finely-divided, perfectly fresh pan- creas (not a watery extract), and keep it at 40° C. After a time its reaction becomes acid, owing to the formation of a fatty acid. This experiment is by no means easy to perform, and some observers deny altogether the existence of a fat- splitting ferment. The free fatty acids thus liberated unite with the alkaline bases of bile, and form soaps. 8. IV.— Milk- Curdling Ferment. (a. ) Add a drop or two of the brine extract of the pan- creas prepared for you, to 5 cc. of warm milk in a test-tube, PANCREATIC DIGESTION. 61 and keep it at 40° C. Within a few minutes a solid coagulum forms, and thereafter the whey begins to separate. (b.) Repeat (a.), but add a grain or less of bicarbonate of soda to the milk. Coagulation occurs just as before, so that this ferment is active in an alkaline medium. (c.) Boil the ferment first. Its power is instantly destroyed. 9. Action on Milk. (a.) Dilute cow's milk with 5 volumes of water. Test a portion, and note that acetic acid throws down a flocculent precipitate of casein. Place some of the diluted milk in a test-tube, add a drop or two of pancreatic extract, or the Liquor Pancreaticus of Benger. Expose on a water-bath at 40° C. for half an hour. Note that the casein is first curdled and then dissolved, and as this occurs, the milk changes from a white to a yellowish colour. (b.) Divide the fluid of (a.) into two portions, A and B. To A add dilute acetic acid, there is no precipitation of casein, which has been converted into peptones. To B add caustic soda and dilute copper sulphate, which give a rose colour, proving the presence of peptones. 10. To Peptonise Milk. — A pint of milk is diluted with a quarter of a pint of water, and heated to a luke-warin tempera- ture, about 140° F. (or the diluted milk may be divided into two equal portions, one of which may be heated to the boiling point and then added to the cold portion, the mixture will then be of the required temperature). Two tea-spoonfuls of Liquor Pan- creaticus, together with about fifteen grains or half a level tea- spoonful of bicarbonate of soda are then mixed therewith. The mixture is next poured into a jug, covered, and placed in a warm situation to keep up the heat. In a few minutes a considerable change will have taken place in the milk, but in most cases it is best to allow the digestive process to go on for ten or twenty minutes. The gradually-increasing bitterness of the digested milk is unobjectionable to many palates; a few trials will, how- ever, indicate the limit most acceptable to the individual patient ; as soon as this point is reached, the milk should be either used or boiled to prevent further change. From ten minutes to half an hour is the time generally found sufficient. It can then be used like ordinary milk. 62 CHEMICAL PHYSIOLOGY. LESSON IX. THE BILE. 1. Use ox bile obtained from the butcher. (a.) Observe the colour of bile of man and that of the ox, the former is a brownish-yellow, the latter greenish, but often it is reddish-brown when it stands for a short time. (b.) Dilute bile and test its reaction = alkaline or neutral. (c.) Pour some ox bile from one vessel to another, and note that it is sticky, strings of mucin connecting the one vessel with the other. (d.) Dilute bile with 5 volumes of water, add dilute acetic acid, which precipitates the mucin coloured with the pigments. Or, dilute bile with its own volume of water, and precipitate the mucin with alcohol. Filter, and observe that the nitrate is no longer sticky, but flows like a watery non-viscid fluid. The mucin remaining on the filter may be washed with dilute spirit and dissolved in lime water. (e.) Bile gives no reactions for albumin. (/.) Fresh human bile gives no spectrum, although the bile of the ox, mouse, and some other animals does. (.) Dry some of the gluten, and heat it strongly in a test-tube ; an arnmoniacal odour similar to that of burned feathers is evolved. Water, which is alkaline (due to ammonia), condenses in the upper part of the tube. (c.) Extract 10 grms. of wheaten flour with 50 cc. of water in a large flask. Shake it from time to time, and allow it to stand for several hours. Filter. If the nitrate is not quite clear, filter again. Heat a part of the clear filtrate, and observe the coagulation of vegetable albumin. (d.} Test another portion of the nitrate from (c.) for the xantho-proteic reaction. (e.) Another portion of (c.) is to be precipitated by acetic acid and ferrocyanide of potassium. (/) Test a third portion of (c.) for the biuret reaction. This is best seen on slightly heating. Take care not to boil the liquid, or the reaction for sugar will be got instead. (g.) Extract some wheaten flour with a 10 per cent, solu- tion of common salt for twelve hours. Filter, and drop some of the clear filtrate into a large vessel of water ; a milky precipitate of a globulin is obtained. (h.) On saturating some of the filtered saline extract of (g.) with powdered NaCl or MgSO4, a precipitate of a globulin is thrown down. (i.) Fats. — Shake up some wheaten flour with ether in a cylindrical stoppered vessel or test-tube, with a tight-fitting cork. Allow the mixture to stand for an hour, shaking it from time to time. Filter off the ether; place some of it on a perfectly clean watch-glass, and allow it to evaporate spon- taneously, when a greasy stain will be left. MUSCLE. 75 (j.) The chief mineral matter, or salts, consists of potas- sium and phosphoric acid. The watery extract gives a yellow precipitate with platinic chloride, showing the pre- sence of potassium ; while heating it with molybdate of ammonium and nitric acid gives a canary -yellow precipitate, proving the presence of phosphates. 11. Pea Meal. (a.) Make corresponding watery and saline extracts, and perform the same experiments with them as in Lesson XI., 10, (C.), (d.), (6.), (/.), (0.), (h.) (b,) Observe the copious precipitate on boiling the watery extract. (c.) Note specially the copious deposit of globulin on adding the saline extract to water. 12. Bread. (a.) Make a watery extract with warm water, filter, and test the nitrate. Its reaction is alkaline. (b.) Test for starch and sugar. LESSON XII. MUSCLE. 1. The Reaction. (a.) Arrange two strips of glazed litmus-paper, one red and one blue, side by side. Pith a frog; cut out the gastro- cnemius, remove as much blood as possible, divide the muscle transversely, and press the cut ends on the litmus- paper; a faint blue patch is produced on the red paper, showing that the muscle is alkaline during life. The blue paper is not affected. (b.) Have some water at 50° C. Dip into it the other gastrocnemius until rigor mortis sets in. Test its reaction ; now it is acid. 76 CHEMICAL PHYSIOLOGY. (c.) Boil some water, and plunge into it any other muscle of the same frog ; it is alkaline. (d.) Test the reaction of a piece of butcher's-meat. It is intensely acid. (e.) Tetanise a muscle for a long time. Its reaction be- comes acid. 2. Watery and Saline Extracts. (a.) Mince some perfectly fresh muscles from a rabbit or dog. Extract with water, stirring from time to time. After half an hour, pour off, and filter the watery extract. Re-extract the remainder with water until the extract gives no proteid reactions. For the purposes of this exercise, half an hour is sufficient. Keep the filtrate, which contains the substances soluble in water. (b.) Take some perfectly fresh muscle from a rabbit, rub it up with sand in a mortar, and extract it with a large volume of 10 per cent, solution of ammonium chloride, NaCl, or 5 per cent. MgSO4. Stir occasionally, and allow it to extract for an hour. A stronger extract is obtained if it be left until next day. Pour off the fluid, keep it as it con- tains the substances soluble in saline solutions, the globulins. 3. With the filtrate of 2 (a.) (a.) Test for proteids — e.g., serum-albumin. (b.) Test the coagulating point of the proteids it contains (45° and 75° C.) (c.) Add crystals of ammonium sulphate to saturation, which precipitates all the proteids. 4. With the filtrate of 2 (b.) (a.) Pour a few drops into a large quantity of water, observe the milky deposit of myosin. The precipitate is redissolved by adding a strong solution of common salt. (b.) Test the coagulating point. Four proteids are coa- gulated by heat at 47°, 56°, 63°, and 73° C., an albumose being left in solution. The fluid is acid in reaction. THE URINE. 77 (c.) Saturate the filtrate with crystals of sodic chloride or ammonium chloride. The myosin is precipitated. (d.) Collect some of the precipitate of 4 (c.), dissolve it with a weak solution of common salt, and test for proteid reactions (Lesson I., 1). Repeat 3 (c.) 5. The Extractives of Muscle. Prepare Creatin, omitting the others. (a.) Make a strong watery solution of Liebig's extract of meat. Cautiously add lead acetate until precipitation ceases, avoiding excess of the lead. Filter, pass sulphuretted hydrogen through the filtrate to get rid of the lead. A pellicle is very apt to form on the surface. Filter, and evaporate the filtrate to a syrup on a water-bath, and set it aside in a cool place to crystallise. Crystals of creatin separate out. (6.) After several days, when the creatin has separated, pour off the mother liquor, add to it 5 volumes of 90 per cent, alcohol to precipitate more creatin. Filter, wash the crystals with alcohol, redissolve them in a boiling < water, allow them to recrystallise, and examine them with the microscope. Sarkin and xanthin may be prepared from the alcoholic filtrate of (6.) LESSON XIII. THE URINE. 1. The Urine is a transparent light straw or amber-coloured watery secretion derived from the kidneys, containing nitro- genous matters, salts, and gases : it has a peculiar odour, bitter saltish taste, and acid reaction. (a.) Evaporate a drop of urine on platinum foil. Do this over the flame of a Bunsen burner, taking care not to burn it. A brownish-yellow stain, with an ammoniacal odour, is obtained. 78 CHEMICAL PHYSIOLOGY. (6.) Evaporate a drop of distilled water to compare with this — no residue. (c.) Burn the stain of 1 (a.) in the flame : it chars, indicating the presence of organic matter, while a faint trace of ash or inorganic matter is left. 2. The Quantity. — Normal. — About 2| pints (50 ounces), or 1500 cc., in twenty-four hours, although there may be a consider- able variation even in health, the quantity being regulated by the amount of fluid taken, and controlled by the state of the tissues, the pulmonary and cutaneous excretions. Increased by drinking water ( Urina potus) or diuretics ; when the skin is cool, its blood-vessels are contracted, and the cutaneous secretion is less active ; after a paroxysm of hysteria, and some convulsive nervous diseases; in Diabetes insipidus and D. mellitus; some cases of hypertrophy of the left ventricle, and some kidney diseases. The increase may be temporary or persistent, the former as the effect of cold, diuretics, or nervous excitement ; the latter in diabetes and certain forms of kidney disease. Diminished after profuse sweating, diarrhoea ; early stage of acute Bright's disease ; some forms of Bright's disease ; the last stages of all forms of Bright's disease ; in general dropsies ; in acute febrile and inflammatory diseases. 3. The Colour. — Normal. — Light straw to amber-coloured. The colour varies greatly even in health, and is due to the presence of pigments, probably largely derived from the decomposition of haemoglobin. The colour largely depends on the degree of dilu- tion of the urine pigments. Pale after copious drinking, in diabetes, anaemia, and chlorosis ; after paroxysmal nervous attacks (hysteria). N.B. — Pale urines indicate the absence of fever. High-coloured after severe sweating, violent muscular exercise, diarrhea, or during febrile conditions. Pathological pigments, purpurine or uro-erythrine in febrile disorders ; bile pigments ; blood. Medicinal Substances. — Creasote and carbolic acid make urine nearly black. This is due not to carbolic acid, but to hydro- chinon. Sometimes these urines become almost black on standing exposed to the air. Rhubarb (gamboge-yellow) ; senna (brownish). 4. The Specific Gravity.— Normal.— Specific gravity 1020 (1018-1025). THE URINE. 79 To take the Specific Gravity. — This is usually done by means of the urine-meter (Fig. 15). The instrument ought to be tested by placing it in a cylindrical vessel filled with distilled water to ascertain that its zero is cor- rect. .1010 _!030 .1040 (a.) Fill a tall cylindrical vessel with urine, and place the urinometer in it. Bring the vessel to the level of the eye, and as soon as the instrument comes to rest, read off" the mark on its stem opposite the lower surface of the meniscus against a bright background. Precautions. — 1. The vessel must be so wide that the urinometer can float freely and not touch the sides. 2. The instrument must be dry before being placed in the fluid. 3. The urine itself must be clear, and free from air-bubbles on the surface; the latter can be readily removed by means of a fold of blotting-paper. N.B. — It is always necessary to take the specific gravity of the "mixed" urine of twenty-four hours. Low Specific Gravity. — All causes which increase the water of the urine only — e.g., drinking on an empty stomach ; after hysteria ; in Diabetes insipidus or Polydipsia. N.B. — If continually below 1015, suspect Diabetes insipidus or chronic Bright' s disease. High Specific Gravity. — When the urine is concentrated; Diabetes mellitus, due to a large amount of grape sugar; first stages of acute fevers ; rapid wasting of the tissues, especially if associated with sweating or diarrhoea. It is highest normally three to four hours after a meal ; and as it varies during the day, it is necessary to mix the urine of the twenty-four hours, and test the specific gravity of a sample of the " mixed urine." N.B. — If above 1025 and the urine be pale, suspect saccharine diabetes. 5. Determination of the amount of Solids from the Specific Gravity. — By Christisoris formula (" Haser-Trapp's coefficient") " multiply the last two figures of a specific gravity expressed in four figures by 2-33. This gives the quantity of solid matter in Fig. 15. Urinometer. 80 CHEMICAL PHYSIOLOGY. every 1000 parts" — i.e., the number of grammes in 1000 cc. (331 oz.) Example. — Suppose a patient to pass 1200 cc. of urine in twenty-four hours, and the specific gravity to be 1022, then 22 x 2-33 = 51-26 grms. in 1000 cc. To ascertain the amount in 1200 cc. 1000 : 1200 : : 51-26 : a: = — ^ = 61-51 grms. This formula is purely empirical, and is not applicable where the variations are very marked, as in saccharine diabetes and some cases of Bright's disease, where there is a great diminution of urea. The normal quantity of solids, or the total solids — sometimes spoken of as " solid urine" — is about 70 grms. in twenty-four hours — i.e., 1000 to 1050 grains. Parkes gives an average of 945 grains per day for an average adult male between twenty and forty years of age. The latter estimate gives about 20 grains of solids per fluid ounce of urine, or about 4 per cent, of solids. 6. The Odour is "peculiar" and "characteristic," somewhat aromatic in health. Certain medicinal and other substances influence it — turpentine (violets); cubebs, copaiba, and sandal wood oil give a characteristic odour, and so do asparagus, valerian, assafsetida, garlic, &c. In disease, note the ammoniacal odour of putrid urine and the so-called " sweet " odour in saccha- rine diabetes. 7. The Reaction.— Normal.— Slightly acid, it turns blue litmus- paper slightly red, and does not affect red litmus-paper. The acidity is chiefly due to acid sodium phosphate (NaH2 PO4), acid urates, and very slightly to free acids — lactic, acetic, oxalic, &c. A neutral urine does not alter either blue or red litmus paper. A very acid urine turns blue litmus paper very red. Sometimes violet litmus paper is used; it becomes red in acid urine and blue in alkaline. (a.) Take two pieces of neutral litmus-paper, put a drop of water on the one, and a drop of urine on the other, and observe the effects. THE URINE. 81 (b.) Test with appropriate litmus-paper a normal, very acid, neutral, and alkaline urine. (c.) Test also with violet litmus-paper. 8. Variations during the Day. — Two or three hours after a meal the urine becomes neutral or alkaline. (See that the bladder be emptied before beginning the experiment.) The cause of the alkalinity is a fixed alkali, probably derived from the basic alkaline phosphates taken with the food (Roberts). Nature of the Food. — With a vegetable diet, the excess of alkali causes an alkaline urine. In herbivora it is alkaline, in carnivora very acid. Herbivora (rabbits) whilst fasting have a clear acid urine, because they are practically living on their own tissues. Perhaps this is one of the reasons why the urine is so acid in fevers. Inanition renders the urine very acid (Chossat). Medicines. — Acids slightly increase the acidity. Alkalies and their carbonates are more powerful than acids, and soon cause alkalinity ; alkalies, e.g., the alkaline salts of citric, tartaric, malic, acetic, and lactic acids, appear as carbonates (Wb'hler). 9. The Alkalinity may be due to the presence of a Fixed or a Volatile Alkali. — In the former case, the blue colour of the litmus- paper does not disappear on heating • in the latter it does, and the paper assumes its original red colour. (a.) Test with two pieces of red litmus-paper two samples of urine, one alkaline from a fixed alkali, and the other from a volatile one. Both papers become blue. (6.) Place both side by side on a glass slide, heat them carefully, and note that the blue colour of the one disappears (volatile alkali), the red being restored, while the blue of the other remains (fixed alkali). The alkalinity may be caused by the presence of ammonium carbonate (volatile), derived from the decomposition of urea ; the urine may be ammoniacal when passed, in which case there is always disease of the urinary mucous membrane ; or it may become so on standing — from putrefaction — when it is always turbid, and contains a sediment consisting of amorphous phos- phate of lime and triple-phosphate, and sometimes urate of ammonium ; it has an offensive ammoniacal odour, and is very irritating to the mucous membrane. 6 82 CHEMICAL PHYSIOLOGY. The acidity is increased during the resolution of febrile diseases; is excessive in gout and acute rheumatism, and whenever much uric acid is given off (uric acid diathesis); in saccharine diabetes, when certain acids are taken with the food (CO2, benzoic). The amount of the acidity may be determined by using a standard solution of caustic soda. 10. Fermentation of the Urine. — When urine is freely exposed to the air it undergoes two fermentations— (1) the add; (2) the alkaline. The urine at first becomes slightly more acid, from the formation of lactic and acetic acids (although this is denied by some observers), then it gradually becomes tieutral, and finally alkaliiie from putrefaction. It becomes lighter in colour, turbid, and a whitish heavy precipitate occurs ; a pellicle forms on the surface, it swarms with bacteria, and it has an ammoniacal odour, which is due to the splitting up of the urea, thus — CON2H4 + 2H20 = (NH4)2 C03 The carbonate of ammonia makes the urine alkaline, and the earthy phosphates are precipitated because they are insoluble in an alkaline urine. The phosphate of lime is precipitated as such, while the phosphate of mag- nesia unites with the ammonia and is precipitated as am- j I rtr\ i- ^ ••*•• JT ~'-v_ ^ ~ •• F——^ 1 -w ff? (Qxs 4*—^. 1 • Fig. 16. — Deposit in "acid fermentation" of urine.— a. Fungus ; b, amorphous sodium urate ; c, uric acid ; d, calcium oxalate. urine may be kept " sweet phosphate or triple phosphate (Mg NH4P04 + 6H20). It is not known what ferment causes this reaction — whether mucus, bacteria-like or- ganisms, or some amorphous fer- ment. N.B.— Although for a long time in perfectly clean THE INORGANIC CONSTITUENTS OF URINE. 83 vessels, still when mixed with decomposing matters it has a marked tendency to putrefy. Insist that all urinary vessels be scrupulously clean ; and that all instruments introduced into the bladder be pro- perly purified by car- bolic acid or other germicide. (a.) Place some normal urine a- side for some days, preferably in a warm place. Ob- serve it from day to day, noting its reaction, change of colour, trans- parency, odour, and any deposit that may form in it. Examine the deposit microsco- pically (Figs. 16, 17). u Fi .g. 17.— Deposit in ammoniacal urine (alka- line fermentation). — a, Ammonio-magnesium phosphate ; d, acid ammonium urate ; c, bac- terium urea3. Fermentation is hastened by a high temperature, and especially if the urine be passed into a contaminated vessel, or the urine itself contain blood, much mucus or pus. It is retarded in a very acid and concentrated urine. LESSON XIY. THE INORGANIC CONSTITUENTS OF URINE. The ratio of inorganic to organic constituents is 1 to 1*2 — 1'7. 1. Water is derived from the food and drink (normal quantity 1500 cc., or about 50 oz.) 84 CHEMICAL PHYSIOLOGY. 2. Chlorides are chiefly those of sodium with a little potassium and ammonium, derived chiefly from the food, and amount to 10 to 15 grammes (150 to 230 grs.) (a.) Test with a few drops of AgNO3 (1 pt. to 8 distilled water) = white, cheesy, or curdy precipitate in lumps in- soluble in HNO3. The phosphate of silver is also thrown down, but it is soluble in HNO3. Variations, increased in amount when the urine is secreted in excess, although the NaCl usually remains very constant (| per cent.); lessened in febrile affections, and where a large amount of exudation has taken place, as in acute pneumonia, when chlorides may be absent from the urine. The reappearance of chlorides in the urine is a good symptom, and indicates an improvement in the condition of the lung. N.B. — The urine ought to be tested daily for chlorides in cases of pneumonia. (6.) Test urine from a case of pneumonia, and compare the amount of the precipitate with that of a normal urine. Estimation. — A rough estimate may be formed of the amount by allowing the precipitate to subside, and comparing its bulk from day to day. 3. Sulphates are those of soda and potash. Quantity 3 to 4 grammes (46 to 61 grs.) They have no clinical significance. (a.) Test with a soluble salt of barium (the nitrate or chloride) = white heavy precipitate of barium sulphate,, insoluble in HNO:J. 4. The Phosphates consist partly of alkaline and partly of earthy salts in the proportion of 2 to 1. The latter are insoluble in an alkaline medium, and are precipitated when the urine becomes alkaline. They are insoluble in water, but soluble in acids ; in urine they are held in solution by free CO2. The alkaline phosphates are very soluble in water, and they never form urinary deposits. 5. The Earthy Phosphates are phosphates of lime and mag- nesia. Quantity 1 to 1-5 grammes (15 to 23 grs.) They are precipi- tated when the urine is alkaline, although not in the form in which they occur in the urine (Lesson XIII., 10). They are THE INORGANIC CONSTITUENTS OF URINE. 85 insoluble in water, readily soluble in acetic and carbonic acid, and are precipitated by ammonia. (a.) To clear filtered urine add nitric acid, boil, and add baric chloride, and boil again = a precipitate of baric sul- phate. Filter, and to the cool nitrate add ammonia = a precipitate of baric phosphate. Clinical Significance. — They are increased in osteomalacia and rickets, in chronic rheumatoid arthritis, after prolonged mental fatigue, and by food and drink, and diminished in renal diseases and phthisis. 6. The Alkaline Phosphates are chiefly acid sodium phosphate, with perhaps traces of potassium phosphate ; they are soluble in water and not precipitated by alkalies, and never occur as urinary deposits. The quantity is 2 to 4 grammes (30 to 60 grs.) They are chiefly derived from the food, and perhaps a small amount from the oxidation of the phosphorus of nerve tissues. (a.) To fresh, clear filtered urine, add ammonia, caustic soda, or potash, and heat gently until the phosphates begin to separate ; let it stand for some time = a white precipitate of the earthy phosphates. Allow it to stand, and estimate approximately the proportion of the deposit. [If a high- coloured urine be used, the phosphates may go down coloured.] (b.) To urine add about half its volume of nitric acid, and then add solution of ammonium molybdate and boil = a canary-yellow precipitate of ammonium phospho-molybdate. N.B. — The molybdate is apt to decompose on keeping. (c.) To urine add half its volume of ammonia, and allow it to stand = a white precipitate of earthy phosphates. Filter and test the filtrate as in 6 (b.) (d.) It gives the reaction for phosphates. This method separates the alkaline from the earthy phosphates. (e.) To urine add half its volume of baryta mixture [Lesson XVI., 2 (c.)] = a copious white precipitate. Filter and test the filtrate as in 6 (c.) It gives no reaction for phos- phoric acid, showing that all the phosphates are precipitated. 86 CHEMICAL PHYSIOLOGY. (/.) To urine add excess of ammonium chloride, and ammonia = a white precipitate of earthy phosphates and oxalate of lime. Filter, and to the nitrate add a solution of magnesic sulphate = a precipitate of the alkaline phosphates as triple phosphate. If the filtrate be tested for phosphoric- acid by 6 (c.) no precipitate will be found. (g.) Instead of 6 (/) use magnesia mixture, composed of magnesic sulphate and ammonium chloride, each 1 part, distilled water 8 parts, and liquor ammonise 1 part. It gives the same result as in 6 (f.) (h.) To urine add a few drops of acetic acid, and then uranium acetate or nitrate - bright yellow or lemon- coloured precipitate of uranium and ammonium double phosphate. 2(Ur2O3)NH4PO4. This reaction forms the basis of the process for the volumetric estimation of the phosphoric acid. 7. The other fact connected with the volumetric estimation of phosphoric acid is, that when a uranic salt is added to a solution of potassium ferrocyanide, a reddish-brown colour is obtained. (a.) To a very dilute solution of uranium acetate add potassium ferrocyanide = a brown colour. 8. In some pathological urines the phosphates are deposited on boiling. (a.) Boil such a urine = a precipitate. It may be phos- phates or albumin. An albuminous precipitate falls before the boiling point is reached, and phosphates when the fluid is boiled. Add a drop or two of nitric or acetic acid. If it is phosphates, the precipitate is dissolved; if albumin, it is unchanged. 9. Microscopic Examination.— As the alkaline phosphates are all freely soluble in water, they do not occur as a urinary deposit. The earthy phosphates, however, may be deposited. (a.) Examine a preparation or a deposit of calcic phos- phate, which may exist either in the amorphous form, THE INORGANIC CONSTITUENTS OF URINE. 87 or the crystalline condition, when it is known as "stellar phosphate" (b.) Prepare " stellar phosphate " crystals by adding some calcium chloride to normal urine, and then nearly neutralising. On standing, crystals exactly like the rare clinical form of stellar phosphate are obtained. (c.) Triple phosphate or ammonio-magnesic phosphate never occurs in normal urine, and when it does occur, indi- cates the decomposition of urea to give the ammonia necessary to combine with magnesic phosphate to form this compound. It forms large, clear "knife-rest" crystals (Fig. 18.) (d.) If ammonia be added to urine, the ammonio-mag- nesic phosphate is thrown down in a feathery form, which is very rarely met with in the investigation of human urine clinically. 10. General Rules for all Volu- metric Processes. (a.) The burette must be carefully washed out with the titrating solution, and must be fixed vertically in a suitable holder. Fig. 18. — Various forms of triple phosphate. (6.) All air-bubbles must be removed from the burette as well as from the outflow tube. The latter must be quite filled with the titrating solution (c.) Fill the burette with the solution up to zero, and always remove the funnel with which it is filled. (d.) Read off the burette always in the same manner, and always allow a short time to elapse before doing so, in order to allow the fluid to run down the sides of the tube. (e.) The titrating fluid and the fluid being titrated must always be thoroughly well mixed. 88 CHEMICAL PHYSIOLOGY. 11. Volumetric Process for Phosphoric acid, with Ferrocyanide of Potassium as the Indicator. 1 cc. of the SS. (Uranium acetate) = -005 gramme of phos- phoric acid. (a.) Collect and carefully measure the urine passed during 24 hours. (b.) Place 50 cc. of the mixed and filtered urine in a beaker. Do this with a pipette. Place the beaker under a burette. (c.) To the urine add 5 cc. of the solution of sodium acetate ; mix thoroughly. (d.) Fill a Mohr's burette with the SS. of uranium acetate up to zero, or to any mark on the burette. See that the Mohr's clip is tight, and that the outflow tube is filled with the SS. Note the height of the fluid in the burette. Heat the urine solution in the beaker to about 8u° C. Drop in the SS. (" Standard Solution ") of uranium acetate from the burette. Mix thoroughly. Test a drop of the mixture from time to time, until a drop gives a faint brown colour when mixed with a drop of potassium ferrocyanide. Do this on a white plate. (e.) Boil the mixture, and test again. If necessary, add a few more drops of the SS., until the brown colour reappears on testing with the indicator. [Paper may be dipped in the indicator solution and tested with a drop of the mixture.] Read off the number of cc. used. Example. — Suppose 17 cc. of the SS. are required to precipitate the phosphates in 50 cc. of urine; as 1 cc. of SS. = -005 gramme of phosphoric acid, then -005 x 17 = -085 gramme of phosphoric acid in 50 cc. of urine. Suppose the patient passed 1250 cc. of urine in 24 hours, then 50 : 1250 : : -085 : x 125° * '°85 = 2-12 50 grammes of phosphoric acid in 24 hours. 12. Solutions Required. Sodium Acetate Solution.— Dissolve 100 grammes of sodium THE INORGANIC CONSTITUENTS OF URINE. 89 acetate in 100 cc. pure acetic acid, and dilute the mixture with distilled water to 1000 cc. Potassium Ferrocyanide Solution. — Dissolve 1 part of the salt in 20 parts of water. Uranium Acetate Solution (1 cc. = -005 gramme H3PO4).— Dissolve 20*3 grammes of pure uranium oxide in strong acetic acid ; dilute with distilled water to a litre. The strength of this solution must be ascer- tained by means of a standard solution of sodium phosphate. Or, dissolve 20*3 grammes uranium nitrate in strong acetic acid, and dilute the solution to 1 litre. Apparatus Required. — Mohr's burette, fitted in a stand, and provided with a Mohr's clip; piece of white porcelain: tripod B O A Fig. 19.— Burette Meniscus. Fig. 20. — Erdmann's Float. stand and wire-gauze ; small beaker ; two pipettes, one to deliver 50 cc. and the other for 5 cc. ; glass rod. 13. Reading off the Burette.— In the case of the burette being filled with a watery fluid, note that the upper surface of the water is concave. Always bring the eye to the level of the same hori- zontal plane as the bottom of the meniscus curve. Fig. 19 shows how different readings may be obtained if the eye is placed at different levels, A, B, C. 90 CHEMICAL PHYSIOLOGY. 14. Erdmann's Float consists of a glass vessel loaded with mercury, so that it will float vertically. It is used to facilitate the reading off of the burette. It has a horizontal line engraved round its middle, and must be of such a width as to allow it just to float freely in the burette. Read off the mark on the burette which coincides with the ring on the float. (Fig. 20.) 15. The Lime, Magnesia, Iron, and other inorganic urinary constituents are comparatively unimportant, and have 110 known clinical significance. LESSON XV. ORGANIC CONSTITUENTS OF THE URINE. 1. Urea (CON2 H4) is the most important organic constituent in urine, and is the chief end-product of the oxidation of the nitrogenous constituents of the tissues and food. It crystallises in silken four-sided prisms, with obliquely cut ends (rhombic system), and when rapidly crystallised, in delicate white needles. It has no effect on litmus ; odourless, weak cool-bitter taste, like saltpetre. It is very soluble in water, alcohol, and almost insoluble in ether. It is an isomer of ammonium cyanate. It {-VTTT NE Urea represents the final stage of the metamorphosis of albu- minous substances within the body. More than nine-tenths of all the N. taken in is excreted in the form of urea. 2. Preparation.— Take 20 cc. of fresh filtered human urine, add 20 cc. of baryta mixture — Lesson XVI., 2 (c.) — to precipitate the phosphates. Filter, evaporate the filtrate to dryness in an evaporating chamber, and extract the residue with boiling alcohol. Filter off the alcoholic solution, place some of it on a slide, and allow the crystals of urea, usually long, fine, transparent needles, to separate out. Examine them micro- scopically. 3. Combinations. — Urea combines with acids, bases, and salts. Evaporate human urine to one-sixth its bulk, and divide the ORGANIC CONSTITUENTS OF THE URINE. 91 residue into two portions, using one for the preparation 01 nitrate, and the other for oxalate of urea. 4. Urea Nitrate, CH4N2O, HNO3. (a.) To the concentrated urine add strong, pure nitric acid = a precipitate of glancing scales of urea nitrate, which, being almost insoluble in HNO3, separate out in rhombic plates or six-sided tables, with a mother-of-pearl lustre, and often imbricate arrangement. (b.) Examine the crystals microscopically (Fig. 21). -)/- b Fig. 21. — a, Urea; b, hexagonal plates; and c, smaller scales, or rhombic plates of urea nitrate. (c.) If only traces of urea are present, concentrate the fluid supposed to contain the urea, place a drop on a slide, put into the drop one end of a thread, apply a cover-glass, and put a drop of pure nitric acid on the free end of the thread. The acid will pass into the fluid, and microscopic crystals of urea nitrate will be formed on the thread. After a time examine the preparation microscopically. 92 CHEMICAL PHYSIOLOGY. 5. Urea Oxalate, (CH4N2O)2 C2H204 + H2O. (a.) To the other half of the concentrated urine, add a concentrated solution of oxalic acid. After a time crystals of oxalate of urea separate. (6.) Examine them micro- scopically (Fig. 22). (c*) Add oxalic acid to a concentrated solution of urea = a precipitate of urea oxalate, which may have cv rtrt - many forms — rhombic Fig. 22.— Crystals of oxalate of , , J . ,,. urea from urine. plates, crystalline scales, easily soluble in water. (d.) Do the same test as described for urea nitrate (4, c.), but substitute oxalic for the nitric acid. 6. Urea and Mercuric Nitrate (2U + Hg(NO3)2 + 3HgO). (a.) To urine or urea solution add mercuric nitrate = a white, cheesy precipitate, a compound of urea and mercuric nitrate. Liebig's method for the estimation of urea is founded on this reaction. 7. Other Reactions of Urea. Make a strong watery solution of urea, and with it perform the following tests : — (a.) Allow a drop to evaporate on a slide, and examine the crystals which form (Fig. 21, a). (b.) Repeat, if you please, the exercises under Lesson XV., 4. (c.) To a strong solution of urea add pure nitric acid = a precipitate of urea nitrate (Fig. 21, b). ORGANIC CONSTITUENTS OF THE URINE. 93 (d.) To a strong solution of urea add ordinary nitric acid tinged yellow with nitrous acid, or add nitrous acid itself; bubbles of gas are given off, consisting of carbon dioxide and nitrogen. (e.) Put some of the urea nitrate precipitate obtained in 4 («.) into the test-tube A (Fig. 23), and some lime Fig. 23. water in B. Add nitrous acid to A. Cork the tube. The precipitate dissolves. CO2 and K" are given off, the C02 makes the lime water in B white. Urea. Nitrous Acid. Carbon Dioxide. Nitrogen. Water. CON2H4 + 2(HNO.2) = CO2 + N4 + 3H2O (f.) Mercuric nitrate gives a greyish-white, cheesy pre- cipitate. (g.) Add caustic potash, and heat. The urea is decom- posed, and ammonia is evolved. 8. With Crystals of Urea perform the following experiments : — (a.) Biuret Reaction. — Heat a crystal in a hard tube ; the crystal melts, ammonia is given off, and is recognised by its smell and its action on litmus, while a white sublimate of cyanuric acid is deposited on the upper cool part of the tube. Heat the tube until there is no longer an odour of am- monia. Allow the tube to cool, add a drop or two of water to dissolve the residue, and a few drops of caustic soda or potash, and a little very dilute solution of cupric sulphate = a violet colour (biuret reaction). (b.) Place a large crystal of urea in a watch-glass, cover it with a saturated watery solution of furfurol, and at once add a drop of strong hydrochloric acid, when there occurs a rapid play of colours, beginning with yellow and passing through green, purple, to violet or brown. This test requires care in its performance. 9. Occurrence. — Urea occurs in the blood, lymph, chyle, liver, lymph glands, spleen, lungs, brain, saliva, amniotic fluid. The chief seat of its formation is very probably the liver. It also 94 CHEMICAL PHYSIOLOGY. occurs in the urine of birds, reptiles, and mammalia, but it is most abundant in that of carnivora. 10. Quantity. — An adult excretes 30 to 40 grammes (450 to 600 grs.) daily ; a woman less, and children relatively more. It varies, however, with — (a.) Nature of the Food. — It increases when the nitrogenous matters are increased in the food, and is diminished by veget- able diet. It is increased by copious draughts of water, salts. It is still excreted during starvation. Muscular exercise has little effect on the amount. (b.) In Disease. — In the acute stage of fevers and inflam- mations there is an increased formation and discharge, also in saccharine diabetes (from the large quantities of food con- sumed). It is diminished in anaemia, cholera, by the use of morphia, in acute and chronic Bright's disease. If it is re- tained within the body, it gives rise to uraemia, when it may be excreted by the skin, or be given off by the bowel. LESSON XVI. VOLUMETRIC ANALYSIS FOR UREA. 1. Before performing the volumetric analysis for urea, do the following reactions, which form the basis of this process : — (a.) To a solution of sodic carbonate, add mercuric nitrate = a yellow precipitate of mercuric hydrate. (b.) To urine, add sodic carbonate, and then mercuric nitrate = first of all a white cheesy precipitate, on adding more mercuric nitrate, a yellow is obtained — i.e., no yellow is obtained until the mercuric nitrate has combined with . the urea, and there is an excess of the mercuric salt. (c.) To urine add hypobromite of soda. At once the urea is decomposed and bubbles of gas — N — are given off. 2. Liebig's Volumetric Process for Urea with Sodic Carbonate as VOLUMETRIC ANALYSIS FOR UREA. 95 the Indicator. — 1 cc. of the SS. (Mercuric Nitrate) = -01 gramme urea. (a.) Collect the urine of the twenty -four hours, and measure the quantity. (b.) If albumin be present separate it by acidification, boiling, and filtration. (c.) Mix 40 cc. of urine with 20 cc. — i.e., half its volume — of a solution of barium nitrate, and barium hydrate (com- posed of one volume of solution of barium nitrate and two volumes of barium hydrate both saturated in the cold). This precipitates the phosphates, sulphates, and carbonates. (d.) Filter through a dry filter to get rid of the above salts. While filtration is going on, fill the burette with the standard solution (SS.) of mercuric nitrate up to the mark 0 on the burette. See that there are no air-bubbles, and that the outflow tube is also filled. (e.} With a pipette take 15 cc. of the clear filtrate and place it in a beaker. N.B. — This corresponds to 10 cc. of urine. Place a few drops of the sodic carbonate solution (the indicator) on a piece of glass resting on a black back- ground. (/.) Note the height of the fluid in the burette. Bun in the SS. of mercuric nitrate from the burette into the 15 cc. of the mixture, in small quantities at a time, until the preci- pitate ceases. Stir and mix thoroughly with a glass rod. After each addition, with the glass rod lift out a drop of the mixture and place it on one of the drops of sodic carbonate until a pale yellow colour is obtained. This indicates that all the urea has been precipitated, and that there is an excess of mercuric nitrate. Read off the number of cc. of the SS. used. (g.) Repeat the experiment with a fresh 15 cc. of the filtrate, but run in the greater part of the requisite SS. at once before testing with sodic carbonate. Read off the number of cc. of the SS. used, and deduct 2 cc.; multiply by *01, which gives the amount (in grammes) of urea in 10 cc. of urine. 96 CHEMICAL PHYSIOLOGY. Example. — Suppose 25 cc. of the SS. were used, and the patient passed 1200 cc. of urine in 24 hours : then, 25 x -01 = -25 gramme urea in 10 cc. 10 : 1200 : : -25 : a . - =30 grammes of urea in 24 hours. This method yields approximately accurate results only when the amount of urea is about 2 per cent. With a greater or less percentage of urea, certain modifications have to be made. 3. Correction for Sodic Chloride. — Two cc. were deducted in the above process. Why ? On adding mercuric nitrate to a solu- tion containing sodic chloride, the mercuric nitrate is decomposed and mercuric chloride formed, and as long as any sodic chloride is present, there is no free mercuric nitrate to combine with the urea. Proofs of this : (a.) To a solution of sodic chloride (normal saline), add mercuric nitrate = precipitate. (b.) To a solution of sodic chloride (normal saline) add a few crystals of urea, then add mercuric nitrate. At first there is no precipitate, or, if there is, it is redissolved ; but by-and-by a white precipitate is obtained. (c.) To a solution of urea (acid) add mercuric chloride = no precipitate. 4. Solutions Required. Baryta Mixture. — Prepared as in Lesson XVI., 2, (c.) Mercuric Nitrate Solution (1 cc. = -01 grm. urea). Dis- solve with the aid of gentle heat 77 -2 grammes of pure dry oxide of mercury in as small a quantity as possible of HNO3, evaporate to a syrup, and then dilute with water to 1 litre. A few drops of HNO3 will dissolve any of the basic salt left undissolved. N.B. — The exact strength of this solution must be estimated by titrating it with a standard 2 per cent, solution of urea. Sodic Carbonate Solution.— 20 grains to the ounce of water. 5. Apparatus Required.— Burette fixed in a stand, funnels, VOLUMETRIC ANALYSIS FOR UREA. 97 beakers, filter paper, glass rod, plate of glass, and three pipettes, 10, 15, and 20 cc. 6. Estimation of Urea by the Hypobromite Method. The principle of this method rests on the fact that urea is decomposed by alkaline solution of sodic hypobromite. The urea yields CO2 (which is absorbed by the caustic soda), and N, which is disengaged in bubbles and collected in a suitable apparatus. Sodic Carbon Sodic Urea. flypobromite. Dioxide. Nitrogen. Water. Bromide. CON2H4 + SNaBrO = CO2 + N2 + 2H2O + SNaBr Every 0*1 gramme of urea yields at the ordinary temperature and pressure 3 7 '3 cc. of nitrogen ; the calculation, therefore, is simple. Many different forms of apparatus have been devised, including those of Knop and Hiifner, E/ussel and West, Graham Steele, Simpson, Dupr4, Charteris, &c. (a.) Study these forms of apparatus, but make the experi- ment with the apparatus of Dupre or Steele. 7. Dupre's Apparatus. — In this apparatus the graduation on the collecting tube represents the percentage of urea, and not cc. of N. The collecting tube, which is clamped above, is placed in a tall vessel containing water, and connected with a small glass flask containing a short test-tube. (a.) Remove the short test-tube from the flask, and in 'the latter, place 25 cc. of the hypobromite solution. (6.) With a pipette measure off 5 cc. of the clear filtered urine, and place it in the short test-tube. With a pair of forceps carefully introduce the tube with the urine into the flask, and place the caoutchouc stopper in the latter. (c.) Test to see if all the connections are tight. Open the clamp at the upper end of the collecting tube, depress the tube in the water until the water inside and outside the tube is at zero of the graduation. Close the clamp, and raise the collecting tube. If the apparatus be tight, no air will pass in, and on lowering the collecting tube the water will stand at zero inside and outside the tube. 98 CHEMICAL PHYSIOLOGY. (d.) Mix the urine gradually with the hypobromite solu- tion by gently tilting over the flask, and ultimately move the flask so as to wash out the test-tube with the hypo- bromite solution. Gas is rapidly given off, the CO2 is absorbed by the caustic soda, while the N is collected in the graduated measuring tube. (e.) Place the flask in a jar of water at the same tempera- ture as that in the tall jar, and slightly lower the measuring tube. After all effervescence has ceased, and when the N collected in the collecting tube has cooled to the temperature of the room — i.e., in three to five minutes — raise the collecting tube until the fluid in- side and outside stands at the same level. Read off the graduated tube ; this gives the percentage of urea. It is to be re- membered that other bodies in the urine, such as uric acid (urates) and creatinin — bu* not KPP«™ acid — also vieid nitrogen by this process ; further, Fig. 24.— G. Steele's apparatus for urea.— A, C, burette ; D, vessel with water ; E, vessel with water to cool A. , that only about 92 per cent, of the N of the urea is given off in the above processes. These sources of fallacy are, however, taken into account in graduating the apparatus. 8. Steele's Apparatus. — This is practically the same apparatus, URIC ACID, URATES, ETC. 99 but a graduated burette is substituted for the graduated collect- ing tube. (a. ) Use this apparatus in a similar manner. (6.) Read off the number of cc. of N evolved, and from this calculate the amount of urea. Every 37 '3 cc. N = 0'l gramme urea. 9. Solutions required. A. FOR DUPRE'S APPARATUS. Hypobromite Solution. — Dissolve 5 cc. of bromine in 45 cc. of a 40 per cent, solution of caustic soda. N.B. — This solution does not keep, and must be freshly pre- pared. B. FOR STEELE'S APPARATUS. Hypobromite Solution. — 20 grammes of caustic soda are dis- solved in water, and the solution is diluted to 250 cc. ; after cooling, add 5 cc. of bromine, and mix. Keep in a stoppered bottle in the dark; as it soon decomposes, it should be made fresh. 10. Use also Charteris's apparatus. The bromine and caustic soda are mixed in a marked measure, so that the hypobromite is always fresh, while the collecting tube for the N is so graduated as to indicate a certain percentage of urea. 11. Study Squibb's apparatus. In all these cases directions are supplied with the apparatus. LESSON XVII. URIC ACID, URATES, HIPPURIG ACID, GREATININ, = Proteid. pitated therefrom by excess of HN03 . . ) 7. B. Incombustible. (i. ) Urates (Na, Oa, Mg), are rarely met with as the sole con- stituent. They give the murexide test. (ii.) Oxalate of Lime or Mulberry Calculi, so called because their surface is usually tuberculated or warty ; they are hard, dark brown, or black. These calculi, from their shape, cause great irritation of the urinary mucous membrane. When in the form of gravel, the concretions are usually smooth, variable in size, pale grey in colour. Layers of oxalate of lime frequently alternate with uric acid. When heated it blackens, but does not fuse, and then becomes white, being converted into the carbonate and oxide. The white mass is alkaline to test-paper, and when treated with HOI, it effervesces (C(X). Oxalate of lime is not dissolved by acetic acid. (iii.) Carbonate of Lime. — Rare in man; when met with they usually occur in large numbers. Dissolve with effervescence in HC1. Sometimes crystals occur as a deposit. They are common in the horse's urine. (iv.) Basic Phosphate of Lime Calculi are very rare, and are white and chalky. (v.) Mixed Phosphates (Fusible Calculus) consist of triple- phosphate and basic phosphate of lime. They indicate that the urine has been ammoniacal for some time, owing to decomposi- tion of the urea. They are usually of considerable size, and whitish ; the consistence varies. When triple-phosphate is most abundant, they are soft and porous, but when the phosphate of lime is in excess, they are harder. A whitish deposit of phos- 126 CHEMICAL PHYSIOLOGY. phates is frequently found coating other calculi. This occurs when the urine becomes ammoniacal, hence in such cases regard must always be had to the condition of the urinary mucous membrane. Such calculi are incombustible, but, when exposed to a strong heat, fuse into a white enamel-like mass, hence the name, fusible calculi. 8. (B.) Group. (i.) The substance gives the murexide reaction, indicates urate.-s. The residue is treated with water. It is soluble, and { Neutralise ; add platinic chloride, a \ _ p j the solution is < _ yellow precipitate . . . . \ ~ . f" alkaline, The residue yields a yellow flame = Sodium. /Ammonium oxalate gives a white \ Scarcely soluble ; I crystalline precipitate . . . / the solution is | Ammonium oxalate gives no precipi-\ i = Calcium. ammonum scarcely alka-< tate, but on adding line ; soluble in I chloride, sodic phosphate, and am- > = Magnesium. acetic acid, . I monia there is a crystalline precipi- 1 \ tate of triple-phosphate . . . / (ii.) The original substance does not give the murexide test. Treat the original substance with hydrochloric acid. It dissolves with effervescence It dissolves without ef- fervescence. Heat the, original sub- stance, and treat it with HC1, . [It dissolves There is no effervs'ce.^ Heat in a* capsule . with effervescence . f^heT^K-lvesl t?.:S3f*s»~«( withKHO./ IM3 -) It does not } melt on > \ heating . ) ( Calcic carbonate. \ Magnesic carbonate. = Calcic oxalate. j- = Triple phosphate. = Neut. calc. phosp. = Acid calc. phosp. 9. General Examination of the Urine. (i.) Quantity in twenty-four hours (normal 50 oz., or 1,500 cc.) (ii.) Colour, Odour, and Transparency (if bile or blood be sus- pected, test for them). URINARY DEPOSITS. 127 (iii.) Specific Gravity of the mixed urine (if above 1030, test for sugar). (iv.) Reaction (normally slightly acid ; if alkaline, is the alkali volatile or fixed ?). (v.) Heat. (a.) If a turbid urine becomes clear = urates. (b.) If it becomes turbid = earthy phosphates or albumin. Albumin is precipitated before the boiling point is reached (70° C.), whilst phosphates are thrown down about the boiling point. It is necessary, however, to add HNO3, which will dissolve the phosphates, but not the albumin. A case may occur where both urates and albumin are pre- sent ; on carefully heating, the urine will first become clear (urates), and then turbid, which turbidity will not disappear on adding HNO3 (albumin). Estimate approximately the amount of albumin present. (vi.) Test for Chlorides, with HNO3 and AgNO3 (if albumin be present, it must be removed by boiling and filtration). (vii.) If sugar be suspected, test for sugar (Moore's, Trommer's, or Fehling's test), and if albumin be present, remove it. (viii.) Make naked-eye, microscopic, and chemical examinations of the sediment. APPENDIX. Exercises on the foregoing. A. The student must practise the analysis of fluids containing one or more of the substances referred to in the foregoing Lessons. Suppose the solution contains one or more of the following — Blood, bile, urea, uric acid, or ferments, proceed as follows : — 128 CHEMICAL PHYSIOLOGY. (a.) If blood is suspected, use the spectroscope if the colour appears to indicate the presence of blood. (6.) If the colour is such as to suggest the presence of bile, concentrate the fluid on a water-bath, and apply Gmelin's test for the bile-pigments (Lesson IX., 6). If proteids are absent, apply Pettenkofer's test for the bile-acids (Lesson IX., 3). If proteids are present, proceed as in Lesson I., p. 19. (c.) In testing for urea, proceed as in Lesson XV., 2, by precipitating with baryta mixture. Filter, evaporate the filtrate to dryness, redissolve the residue with absolute alcohol, and allow some of the alcoholic extract to evaporate on a slide, and use the microscope for the detection of crystals of urea. Apply the other reactions for urea in Lesson XV., 4. (d.) For uric acid, or its salts, add hydrochloric acid to precipitate the uric acid in crystals [Lesson XV. 2, (a.)], and to the latter apply the murexide test. (e.) If ferments are suspected, their action must be tested on fibrin or starch mucilage, as the case may be, the reaction of the fluid being adapted to the ferment tested for. B. If a powder or solid substance be given to you, (a.) Examine it with the naked eye and microscopically, whether it be amorphous or crystalline. (6.) Burn some in a tube ; smell it to detect any odour. Observe if it leaves an ash. (c.) Examine its solubility in water, caustic soda, salt solutions, alcohol, and ether. (d.) Apply tests in Lesson I., 1, for proteids. (e.) If it contain a proteid, test its solubility in water. Native albumins, peptones, and gelatin are soluble; the others are insoluble. Confirm by other tests in Lesson I. (/) If it be not a proteid, or if proteids be present, remove them. Test if it be soluble in cold water, and to the URINARY DEPOSITS. 129 solution apply the tests for dextrin and glycogen (Lesson II., 4, 5), reducing sugars, e.g., grape or milk sugars (Lesson II., 6). The former, in a concentrated solution, is precipitated by absolute alcohol, the latter is not. If cane-sugar be suspected, invert it (Lesson II., 9, d.), and test for a reducing sugar. Test also for urea (Lesson XV., 4, 7). (g.) Ascertain its solubility in warm water, starch (Lesson II., 1), urates (Lesson XVII. , 5), tyrosin (Lesson VIII., 5). (h.) Test for uric acid (Lesson XVII., 3). (i.) Cholesterin is insoluble in cold water and alcohol, but soluble in ether. On evaporation of the ether, the character- istic crystals are obtained (Lesson IX., 7). (j.) Fats melt on heating and are soluble in ether, leaving a greasy stain (Lesson IV., 13). C. Analysis of Urine — The student must also practise the analysis of urines containing one or more abnormal constituents, and he must also practise the estimation of the quantity of the more important substances present. Both sets of processes must be done over and over again, in order that he may perfect himself in the methods in common use. PART I).— EXPERIMENTAL PHYSIOLOGY. Before beginning the experimental part of the course, each student must provide himself with the following : — A large and a small pair of scissors ; a large and a fine pointed pair of forceps ; a scalpel; a blunt needle or "seeker" in a handle; pins ; fine silk thread ; bees' -wax ; sealing wax ; two camel' s-hair brushes of medium size. It is convenient to have them all arranged in a small case. PHYSIOLOGY OF MUSCLE AND NERVE. LESSON XXII. GALVANIC BATTERIES GALVANOSCOPE. AND 1. Daniell's Cell consists of a glazed earthenware pot with a handle (Fig. 33) and containing a saturated solution of cupric sulphate. Some crystals of cupric sulphate are placed in it to keep the solution saturated. The pot itself is about 18 cm. high, and 9 cm. in diameter. In the copper solution is placed a roll of sheet-copper, provided with a tongue, to which a binding screw is attached. Within is a porous unglazed cylindrical cell containing 10 per cent, solution of sulphuric acid. A well amal- gamated rod of zinc provided at its free end with a binding screw is immersed in the acid. The zinc is the negative ( - ), Fig. 33.— Daniell's Cell. and the copper the positive ( + ) pole. 2. Amalgamation of the Zinc.— The zinc should always be well amalgamated. When a cell hisses, the zinc requires GALVANIC BATTERIES AND GALVANOSCOPE. 131 to be amalgamated. Dip the zinc in 10 per cent, sulphuric acid until effervescence commences. Lift it out and place it on a shallow porcelain plate. Pour some mercury on the zinc, and with a piece of cloth rub the mercury well over the zinc. Dip the zinc in the acid again, and then scrub the surface with a rag under a stream of water from the tap. Collect all the surplus mercury and place it in the bottle labelled "Amalgamation Mixture." Take care that none of the mercury gets into the soil pipe. A very convenient method is to dip the zinc into a glass tube with thick walls containing mercury and sul- phuric acid. For convenience the tube is fixed in a block of wood. 3. Grove's Cell- (Fig 34) consists of an outer glazed earthenware glass or ebonite vessel containing a roll of amalgamated zinc and dilute (10 per cent.) sulphuric acid. In the inner porous cell is placed platinum foil with strong nitric acid. The platinum and zinc are provided with binding screws. The platinum is the + positive pole or anode, the zinc the - negative pole or cathode. For physiological purposes, the small Grove's cells about 7 cm. in diameter and 5 cm. in height, are very convenient. When in use the battery ought to be placed in a draught chamber to prevent the nitrous fumes from affecting the experimenter. 4. Bichromate Cell (Fig. 35). — This consists of a glass bottle containing one zinc and two carbon plates immersed in the fol- lowing mixture : — Dissolve 1 part of potassic bichromate in 8 parts of water and add 1 part of sulphuric acid. The zinc is attached to a rod, which can be raised when it is desired to stop the action of the battery. This cell is convenient enough when it is not necessary to use a current of perfectly constant intensity. Fig. 34.— Large Grove's Element. 132 EXPERIMENTAL PHYSIOLOGY. Other forms of batteries are used, but the foregoing are suffi- cient for the purposes of these exercises. 5. The Galvanoscope or Detector. (a.) Charge a Daniell's cell and attach a copper wire to the negative pole (zinc), and another to the positive pole (copper). On bringing the free ends of the two wires together, the circuit is made, and a current of continuous, galvanic, or voltaic electricity circulates outside the battery from the + to the - pole. The existence of this electrical current may be proved in many ways — e.g., by the effect of the current on a magnetic needle. (b.) Use a vertical gal- Fig. 35.— Bichromate Cell. -A, the glass vessel ; K, K, carbon ; Z, zinc ; D, E, binding screws for the wires; B, rod to raise or depress the zinc in the fluid ; C, screw to fix B. Fig. 36.— Detector. vanoscope, or as it is called by telegraphists, a detector (Fig. 36), in which the magnetic needle is so loaded as to rest in a vertical position. A needle attached to this moves over a semi-circle graduated into degrees. Connect the wires from the + and - poles of the Daniell's battery with the binding screws of this instrument, and note that when the circuit is made, the needle is deflected from its vertical ELECTRICAL KEYS, RHEOCHORD. 133 into a more or less horizontal position, but the angle of deflection is not directly proportional to the current passing in the instrument. Break the circuit by removing one wire, and notice that the needle travels to zero and resumes its vertical position. The detector made by Stohrer, of Leipzig, is a convenient form. LESSON XXIII. ELECTRICAL KEYS, RHEOCHORD. It is convenient to make or break — i.e., close or open — a cur- rent by means of keys of which there are various forms. 1. Du Bois Key (Fig. 37). — It consists of a square plate of vulcanite, attached to a wooden or metallic framework which can be screwed to a table. Two oblong brass bars (II. and III.), each provided with two binding screws, are fixed to the ebonite, while a movable brass bar (IV.) with an ebonite handle is fixed to one of the bars, and can be depressed so as to touch the other brass bar. Two Ways of Using the du Bois Key. 2. (1) When the key is closed the Fig. 37. — Du Bois - Raymond's Friction Key. Fig. 38.— Scheme of du Bois Key.— B, battery; K, key; N, nerve; M, muscle. 134 EXPERIMENTAL PHYSIOLOGY. current is made, and when it is opened the current is broken (Fig. 38). Apparatus. — Use a charged Daniell's cell and detector as before, three wires, and a du Bois key screwed to a table. (a.) As in the scheme (Fig. 38) connect one wire from the battery to one brass bar of the key. Connect the other brass bar with one binding screw of the detector. Connect by means of the third wire the other binding screw of the detector with the - pole of the cell. (b.) Observe on depressing the key (i.e., making the circuit) the needle is deflected, on raising it (i.e., breaking the circuit) the needle passes to zero. This method of using the key we may call that for " making and breaking a current." This method is never used for an interrupted current applied to a nerve or muscle. 3. (2) When the key is closed the current is said to be " short- circuited." Apparatus. — Daniell's cell, detector, four wires, and a du Bois key screwed to the table. (a.) As in the scheme (Fig. 39) connect the wire from the positive pole of the battery to the outer binding screw of one brass bar of the key, and the other battery wire to the outer binding screw of the other brass bar of the key. Then connect the inner binding screws of both brass bars with the screws of the detector. binding (b.) Observe when the key is depressed or closed, there is no deflection of the needle — i.e., when the current is cut off from the circuit beyond the key or Fi£ 39.-Scheme of du Bois .bridge; when the key is raised, K^^slTBT the Seedle is defl^d battery ; K', key. When the key is depressed, the current is said to be " short-circuited," for the key acts like a bridge, and so a large part of the current passes through it back to the battery, while only an excessively feeble current passes ELECTRICAL KEYS, RHEOCHORD. 135 through the wires beyond the key, so feeble is it that it does not affect a nerve. On raising the key, the whole of the current passes through the detector or nerve, as the case may be. This method of using the key we may call the method of " short- circuiting." N.B. — In using the key to apply an induction current to excite a nerve or muscle, always use this key by the second method — i.e., always short-circuit an induction current. 4. Mercurial Key. — Where a fluid contact is required the wires dip into mercury. Study the use of this key. It is used in the same way as a du Bois key. 5. Plug Key (Fig. 40).— Two brass bars are fixed to a piece of vulcanite. The circuit is made or broken by insert- ing a brass plug between the bars. Each brass bar is provided with two bind- ing screws, to which one or two wires may be attached, so that it can be used like a du Bois key, either by the first or second method. Fig. 40.— Plug Key. 6. Morse Key (Fig. 41). — If it is desired to make or break a current rapidly this key is very convenient. If this key be used to make and break the primary circuit, connect the wires to B and G ; when the style of the lever, I, is in contact with c, the current does not pass in the primary cir- cuit. On depressing the handle, K, the primary circuit is made. If, however, the wires be connected to A and B the current passes and is broken on depressing K. To use this key as a short-circuiting key, connect the wires from the battery to A and B, and those of the electrodes to B and 0. The current is short-circuited until K is depressed, when the current passes from 0 to B through the electrode wires. 7. The " Trigger or Turn- over Key " is referred to in Lesson XXXII. Fig. 41. — The Morse Key. — The connections are concealed below, but are B to I, A to c, C to c'. 136 EXPERIMENTAL PHYSIOLOGY. Fig. 42.— Spring Key. 8. The Contact or Spring Key (Fig. 42) is also very useful for rapidly making and breaking a circuit. The current can only pass between the binding screws when the metallic spring is pressed down. The left end of the spring is in metallic contact with the upper binding screw, while the second binding screw is similarly connected with the little metallic peg at the right-hand end of the fig. 9. Means of Graduating a Galvanic Current . — Besides altering the number, arrangement, or size of the cells themselves, we can use an arrangement to divide the current itself, the battery remaining constant. This is effected by the simple rheocord. The Simple Rheochord consists of a brass or German-silver wire about 1 metre in length, stretched longitudinally along a board, and with its ends connected to binding screws and insulated (Fig. 43). On the wire there is a " slider " which can be pushed along as desired. Apparatus. — Simple rheochord, Daniell's cell, detec- tor, du Bois key, five wires. (a.) Arrange the experiment as in Fig. 43. When the slider S is hard up to W, practically all the electricity passes along the wire (W, R), back to the battery. (6.) Pull the slider away from W, and in doing so, more resistance is thrown into the battery circuit, and some of the electricity passes along the detector circuit and deflects the needle. The deflection is greater— but not proportionally so — the further the slider is moved from W. 10. Use in the same way a rheochord with two platinum wires, which are connected by an ebonite cup filled with mercury, which Fig. 43.— Scheme of Simple Rheochord.— B, Bat- tery ; K, key ; W, R, wire ; S, slider ; D, detector. ELECTRICAL KEYS, RHEOCHORD. 137 slides along on the two wires. Connect the battery — a key being interposed in one wire — with the binding screws on one end of the rheocord, and to the same binding screws connect two wires to the detector. Observe as the mercury cup is pulled away from the binding screws, there is a greater deflection of the needle, but the deflection is not in proportion to the distance of the cup. 11. The Rheochord of du Bois-Reymond is used to vary the amount of a constant current applied to a muscle or nerve. It consists of a long board or box, with German-silver wire — of varying length and whose resistance is accurately graduated — stretched upon it. At one end are a series of brass blocks dis- connected with each other above, but connected below by a German-silver wire passing round a pin. These blocks, however, may be connected directly by brass plugs Sx S2 ... S5. From the blocks 1 and 2, two platinum wires pass from A to the opposite end of the box (Y), where they are insu- lated. Between the wires is a " slider " (L), consisting of two cups of mercury, which slide along the the wires. In using the instrument, take a Daniell's battery and connect its wires to the binding screws at A and B, and to the same screws attach the wires of the electrodes over which the nerve (c d) of the muscle (F) is laid. We have two circuits (a c d b and a A B b}, the wires of the rheochord introduced into the latter. Push up the slider with its cups (L) until it touches the two brass plates 1 and 2, and insert all the plugs (Sj-S5) in their places, thus making the several blocks of brass practically one block. In this posi- tion the resistance offered by the rheochord circuit is so small as compared with that including the Fig 44.— Rheochord of du Bois- nerve, that practically all the Reymond. 138 EXPERIMENTAL PHYSIOLOGY. electricity passes through the former and none through the latter. Move the slider away from A, when a certain resistance is thrown into the rheochord circuit, according to the length of the platinum wires thus introduced into it, and so a certain fraction of the current is sent through the electrode circuit. If the plug Sj be taken out more resistance is introduced, that due to the German-silver wire (I b), and, therefore, a certain amount of the current is made to pass through the electrode circuit. By taking out plug after plug more and more resistance is thrown into the rheocord circuit. The plugs are numbered, and the diameter and length of the German-silver wires are so selected in making the instrument, that the resistances represented by the several plugs when removed, are all multiples of the resist- ance in the platinum wires on which the slider moves. The instrument is described here for convenience, but its use will be practised later. (a). Connect a Daniell's cell or a small Grove with the binding screws at A and B, introducing a du Bois key in the circuit. To A and B attach two other wires, and connect them to a du Bois key, and to the key attach electrodes, thus short-circuiting the electrode circuit. (6). Prepare a nerve muscle preparation, lay the muscle on glass, and place the nerve over the electrodes. (c), Put in all the plugs and push up the slider close to the blocks. Open the short-circuiting key. Make the battery circuit, perhaps a contraction may be obtained. Pull the slider away from the blocks, and on making the current contraction will occur, and perhaps also on breaking it. Take out plug Slf and pull the bridge still further away, and very probably there will be contraction both at make and break. Proceed taking out plug after plug, and note the result. The result, and explanation thereof will be referred to in Lesson XLIL, 2. 12. Pohl's Commutator.— Sometimes it is desired to send a cur- rent through either of two pairs of wires. This is done by means of Pohl's commutator without the cross-bars (Lesson XXX., INDUCTION MACHINE — ELECTRODES. 139 Fig. 59). At other times it is desired to reverse the direction of a current. This is done by Pohl's commutator with cross-bars, or by means of Thomson's reverser. 13. Thomson's Reverser (Fig. 45) may be used to reverse the direction of a constant current. The wires from the battery are connected to the two lower, and those from the electrodes to the upper binding screws. The binding screws are four in number, and placed behind the circular disc seen in the figure. When the handle is horizontal m, ,g TiP • T F» flaS' (c.) Pinch the free end of the nerve sharply with forceps, the muscles contract and the straw-flag is suddenly raised- Out off the killed part of the nerve, and observe that con- traction also occurs. (d.) Prick the muscle with a needle, it contracts. 5. Thermal Stimulation. (a.) To the same preparation apply gently, either to muscle or nerve, a hot copper wire or needle heated to a dull heat, a contraction results in either case. Cut off the dead part of the nerve. 6. Chemical Stimulation. (a.) Place some saturated solution of common salt in a small glass thimble, or place a drop on a perfectly clean glass slide, and allow the free end of the nerve to dip into it. Owing to the high specific gravity of the saline solution, the nerve floats on the surface, but sufficient salt diffuses into the nerve to stimulate it. After a few moments, the individual joints of the toes begin to twitch, and by-and- by the whole limb is thrown into irregular spasms, ulti- mately terminating in a powerful, more or less continuous, contraction or spasm of the whole musculature, constituting tetanus. Out off the part of the nerve affected by the salt, and the spasms will cease. 152 EXPERIMENTAL PHYSIOLOGY. (b.) Use the same preparation, cover the leg with the skin of the frog, or wrap it in blotting-paper saturated with nor- mal saline. Expose the fresh cut end of the nerve to the vapour of strong ammonia; there is no contraction of the muscle, but the ammonia kills the nerve. Instead of doing this, the whole leg may be laid on a card, covered with blotting-paper moistened with normal saline, with a hole in it just sufficient to allow the sciatic nerve to pass through it. The card is placed over a test-tube containing a drop of ammonia ; the nerve hanging in the vapour of the latter is speedily killed, but there is no contraction of the muscle. Apply the ammonia to the muscle, it will contract. LESSON XXVIII. SINGLE AND INTERRUPTED INDUCTION SHOCKS— TETANUS— CONSTANT CURRENT. 1. Electrical Stimulation. Single Induction Shocks. — Apparatus. — Frog, Daniell's cell, induction machine, two du Bois keys, five wires, flexible electrodes. (a.) Arrange a cell and induction machine, for single in- duction shocks according to the scheme, Fig. 54. Flexible Fig. 54.— Scheme for Single Induction Shocks. — B, Battery; K, K', keys ; P, primary, and S, secondary coil of the induction machine ; N, nerve; M, muscle. electrodes are fixed to the short-circuiting key (K') in the secondary circuit, and over them the nerve is to be placed. (b.) Expose the sciatic nerve in a pithed frog, place the electrodes — preferably a pair fixed in ebonite, and so shielded CONSTANT CURRENT. 153 that only one surface of their platinum terminals is ex- posed under it. Pull the secondary coil (S) far away from the primary (P), raise the short-circuiting key (K'), make and break the primary circuit by means of the key (K). At first there may be no contraction, but on approximating the secondary to the primary coil a single muscular contraction will be obtained, first with the break shock, and on ap- proaching the secondary nearer to the primary coil, also with the make. The one is called a make and the other a break contraction. Record the results obtained. 2. Interrupted Current. (a.) Arrange the induction machine so as to cause Neef's hammer to vibrate as directed in Lesson XXV, 2. On applying the electrodes to the sciatic nerve or gastrocnemius muscle, at once the muscle is thrown into a state of rigid spasm or continuous contraction, called tetanus, this condi- tion lasting as long as the nerve or muscle is stimulated, or until exhaustion occurs. 3. Constant Current — Apparatus. — One, two, or three Daniell's cells, du Bois key, four wires and pair of electrodes, forceps, and nerve-muscle preparation. (a.) Use two Daniell's cells. If two or more Daniell's cells be used, always connect them in series — i.e., the positive pole of one cell with the ne- gative pole of the next one. Connect two wires, as in Fig. 55, to the free + and - poles of the battery (B), and introduce a du Bois key (K'), so as to short-circuit the battery circuit. Fix two shielded electrodes in the other binding-screws of the du Bois key, and having prepared a fresh nerve- muscle preparation, lay the divided sciatic nerve (N) across them, as shown in Fig 55. (6.) Make and break the current, and a single muscular contraction or twitch is obtained either at making or breaking, or both at making and breaking. Fig. 55. — Scheme of con-t stant current. — B, bat- tery ; K', short-cir- cuiting key; N, nerve; M, muscle. Notice that 154 EXPERIMENTAL PHYSIOLOGY. if the key be raised to allow the current to flow continuously through the nerve, no contraction occurs, provided there be no variation in the intensity of the current. The electrodes may also be applied to the muscle directly. (c.) Rapidly make and break the current by opening and closing the key, a more or less perfect tetanus is produced. 4. Dead Muscle and Nerve. — Immerse a nerve-preparation for a few minutes in water at 40° 0. Both are killed, and none of the above stimuli cause contraction. 5. The Sartorius. — The student gets a clear idea of the short- ening and thickening which occur when a muscle contracts by working with the sartorius muscle, because its fibres are arranged in a parallel manner. (a.) Pith a frog, skin it, lay it on its back, and dissect off the long narrow sartorius from the inner side of the thigh. Stretch it on a slip of glass (Fig. 56, *). (6.) Stimulate the muscle first at its ends and afterwards at its centre or equator, as in Lesson XXVIII., 1,2, with (i.) a single induction shock, and (ii.) afterwards with an inter- rupted current. Observe the shortening and thickening, which are much greater in (ii.) than (i.) The muscle may be extended again, and stimulated as frequently as de- sired if it be kept moist. 6. Unipolar Stimulation— Apparatus. — machine, du Bois key, muscle-chamber, ad'. Fig. 56.— Muscles of the left leg of a frog seen from the front.— ip, ileo- psoas; 8, sartorius; ad', adductor longus ; tn, vastus interims (see Figs. 50 and 51). Daniell's cell, induction /our wires. (a.) Connect the Daniell to the primary coil of the induc- tion machine either for single shocks or tetanus, introducing a du Bois key in the circuit. Connect one wire with the secondary coil, and attach it to one of the binding screws on the platform of the muscle-chamber, to which the nerve FLEISCHL'S RHEONOM. 155 electrodes are attached. See that the battery and induction machine are perfectly insulated by supporting them on blocks of paraffin. (b.) Prepare a nerve-muscle preparation, and arrange it in the muscle-chamber in the usual way, laying the nerve over the electrodes. One ol the electrodes will therefore be con- nected with the secondary circuit. (c.) Make and break the primary circuit, there is no con- traction. (d.) Destroy the insulation of the preparation by touching the muscle, or what does better, allow the brass support of the muscle to touch a piece of moist blotting-paper on the inner surface of the glass shade of the chamber. Every time the brass-binding of the shade is touched, or the brass support itself, the muscle contracts. Touch the secondary coil and contraction results. LESSON XXIX. RHEONOM —TELEPHONE EXPERIMENT- DIRECT AND INDIRECT STIMULATION OF MUSCLE— RUPTURING STRAIN OF TENDON— MUSCLE SOUND —DYNAMOMETERS. 1. Fleischl's Rheonom.— This instrument (Fig. 57) is very use- ful for showing du Bois-Reymond's law, that it is variations in the density of a galvanic current which excite a motor nerve. It consists of a square ebonite base, with a grooved circular channel in it, and two binding screws, with zinc attached, and bent over so as to dip into the groove, which is filled with a saturated solution of zinc sulphate. A vertical arm, with binding screws attached to two bent strips of zinc, moves on a vertical support. Fig> 57._Fieischl's Rheonom. 156 EXPERIMENTAL PHYSIOLOGY. (a.) Connect two Darnell's cells with the binding screws, A and B, introducing a du Bois key in one wire. Attach the electrodes, introducing a du Bois key to short-circuit them, to the binding screws, 0 and D. Fill the groove with a saturated solution of zinc sulphate. (b.) Arrange the nerve of a nerve-muscle preparation in the usual way over the electrodes (Lesson XX VIII. , 4). Pass a constant current through the nerve, observing the usual effects, viz., contraction at make or break, or both, but none when the current is passing. Then suddenly rotate the handle with its two zinc arms ; this is equivalent to a sudden variation of the intensity of the current; the current, of course, continuing to pass all the time. The muscle sud- denly contracts. 2. Telephone Experiment. (a.) Arrange a nerve-muscle preparation with its nerve over a pair of electrodes. Connect the latter with a short- circuiting du Bois key. To the key attach the two wires from a telephone. (b.) Open the short-circuiting key ; shout into the tele- phone, and observe that on doing so the muscle contracts vigorously. 3. Direct and Indirect Stimulation of Muscle. — When the stimulus is applied directly to the muscle itself, we have direct stimulation ; but when it is applied to the nerve, and the muscle contracts, this is indirect stimulation of the muscle. (a.) Arrange a nerve-muscle preparation, and an induc- tion machine for single or interrupted shocks (Lesson XXVIII., 1). (b.) Test first the strength of current— as measured by the distance between the secondary and primary coils — which causes the muscle to contract when the stimulus is applied to the nerve — i.e., for indirect-stimulation. (c.) Then with the secondary still at the same distance from the primary, try if a contraction is obtained on stimulating the muscle directly. It will not contract, but make the current stronger, and it will do so. INDEPENDENT MUSCULAR EXCITABILITY. 157 4. Rupturing Strain of Muscle and Tendon. (a.) Dissect out the femur and gastrocnemius with the tendo achillis of a frog. Fix the femur in a strong clamp on a stand, preferably one with a heavy base. To the tendo achillis tie a stout thread, and hang a scale pan on to it. (6.) Into the scale pan place weights, and observe the weight required to rupture the tendon or muscle. Usually the muscle is broken first, and the weight added to the scale will be a kilo, more or less, according to the size of the (c.) Compare the rupturing strain of a frog's gastrocnemius which has been dead for twenty-four hours. A much less weight is required. 5. Muscle Sound. (a.) Insert the tips of the index fingers into the auditory meatuses, forcibly contract the biceps muscles. A low rumbling sound is heard. (6.) When all is still at night, firmly close the jaws, and especially if the ears be stopped, the sound is heard. 6, Dynamometers. (a.) Hand. — Test the force exerted first by the right hand and then by the left, by means of Salter's dynamometer. (b.) Arm. — Using one of Salter's dynamometers, test the strength of the arm when exerted in pulling, as an archer does when drawing a bow. LESSON XXX. INDEPENDENT MUSCULAR EXCITABILITY —ACTION OF CURARE— ROSENTHAL'S MODIFICATION— POHL'S COMMUTATOR. 1. Independent Muscular Excitability and the Action of Curare. — Ourare paralyses the intra-muscular terminations of the motor 158 EXPERIMENTAL PHYSIOLOGY. nerves. — Apparatus. — Daniell's cell, induction machine, two keys, five wires, shielded electrodes, scissors, fine-pointed forceps, fine aneurism needle, or fine sewing needle fixed in a handle, with the eye free to serve as an aneurism needle, fine threads, pithing needle, 1 per cent, watery solution of curara in a glass-stoppered bottle, fine hypodermic syringe or glass pipette, frog. (a.) Arrange the battery and induction machine for an interrupted current with a key in the primary circuit, and a du Bois key to short-circuit the secondary as in Lesson XXVIII., 2). (6.) Pith a frog, destroying only its brain, and inject into the ventral or dorsal lymph sac one or two drops of a 1 per cent, watery solution of curara. The poison is rapidly absorbed. At first the frog draws up its legs, in a few minutes it ceases to do so, and will lie in any position in which it is put, while the legs are not drawn up on being pinched, and the animal lies flaccid and apparently para- lysed. (c.) Place the frog on its back. Expose the heart, and observe that it is still beating. Take care to lose no blood. (d.) Expose the sciatic nerve on one or both sides. (i.) Apply the shielded electrodes under them, and stimulate the nerves with tetanising shocks. There is no contraction. (ii.) Apply the electrodes to the muscles, they contract. TJierefore, curara has paralysed Hie voluntary motor nerves, but not tlie muscles. 2. On what part of the Nerve does the Curare act ? (a.) Keep the induction apparatus as in the previous ex- periment. (6.) Pith a frog, destroying only its brain. Carefully expose the sciatic nerve and the accompanying artery and vein on one side, e.g., the left, taking great care not to injure the blood-vessels, which are to be carefully isolated for a short distance with a finder. Thread a fine aneurism needle with ACTION OF CURARE. 159 a fine silk thread. Moisten the thread with salt solution, and gently pass it under the sciatic artery. Withdraw the needle and ligature the artery. Instead of ligaturing merely the artery, it is better to isolate the sciatic nerve, and then to tie a stout ligature round all the other structures of the thigh. In this way none of the poison can pass by a col- lateral circulation into the parts below the ligature. (c.) Inject a few drops of a 1 per cent, solution of curara into the ventral lymph sac, either by means of a hypodermic syringe or a fine pipette. In a short time the poison will be carried to every part of the body except the left leg below the ligature. Observe that the animal is rapidly paralysed, but if the non-poisoned leg (left) is pinched, it is drawn up, while the poisoned leg (right) is not. (d.) Wait until the animal is thoroughly under the influence of the poison, and then expose both sciatic nerves as far up as the vertebral column and as far down as the knee. (i.) Stimulate the right sciatic nerve. There is no con- traction. Therefore the poison has acted either on nerve or muscle. (ii.) Stimulate the right gastrocnemius muscle, it con- tracts. Therefore the poison has acted on some part of the nervous path, but not on the muscle. (iii.) Stimulate the left sciatic above the ligature, the left leg contracts. Observe that the part of the nerve above the ligature was supplied with poisoned blood, and has been under the influence of the poison, so that the nerve-trunk itself is not paralysed, as may be proved by stimulating any part of the left sciatic as far down as its entrance into the gastrocnemius. Stimulating any part of the left nerve causes contraction. Therefore, neither nerve- trunk nor muscle is affected. The nerve impulse is blocked somewhere, in all probability by paralysis of the terminations of the motor nerves within the muscle. (e.) Apply several drops of a strong solution of curare to the left gastrocnemius, and after a time, stimulate the left 160 EXPERIMENTAL PHYSIOLOGY. sciatic nerve, there is no contraction, but on stimulating the muscle itself contraction takes place. 3. RosenthaTs Modification. (a.) Prepare a frog as in the previous experiment, ligature the left leg — all except the sciatic nerve — and inject curare as before. After complete paralyses occurs, dissect out both legs with the nerves attached, but retain the legs, as in Fig. 58. Attach straw flags (N P and P) of different colours to the toes of both legs by pins, and fix both femora in muscle- forceps (F) with the gas- trocnemii uppermost. Place the nerves (N) on the platinum points of du Bois-Reymond's elec- trodes (Fig. 47). (b.) Arrange the induc- tion apparatus as in Fig. 58. The primary coil is as before, but the ter- minals of the secondary coil are connected by two wires with the piers of a Pohl's commutator (Fig. 58) without cross-bars (H). Two other wires pass from two other bind- ing screws of the commu- tator to the electrodes (N), while two thin wires pass from the other two binding screws (C) and their other ends are pushed through the gas- trocnemii muscles. Place the commutator on a meat plate and fill its holes with mercury. The commutator enables the tetanising currents to be passed either through both nerves or both muscles. It is more convenient if the secondary circuit have a key, so that it may be short-circuited when desired. Fig. 58. — Scheme of the Curare Experi- ment.—B, Battery ; I., primary, II. , secondary spiral; N, nerves; F, clamp ; N P, non-poisoned leg ; P, poisoned leg; C, commutator; K, key. POHL'S COMMUTATOR. 161 (i.) Set Neefs hammer going, and turn the handle of the commutator so that the current passes through both nerves; only the non-poisoned leg (N P) contracts. (ii.) Reverse the handle and pass the current through both muscles, both contract. (iii.) Push the secondary spiral far away from the primary, and pass the current through both inuscles. At first, if the spirals be sufficiently far apart, there is no contraction in either muscle. Gradually push up the secondary spiral, and notice on doing so that the non- poisoned limb contracts first, and that on continuing to push up the secondary spiral, both muscles ultimately contract (Rosenthal's Modification). 4. Pohl's Commutator (Fig. 59) is used for sending a current along two different pairs of wires, or for reversing the direction of the current in a pair of wires. It con- sists of a round or square wooden or ebonite block with six cups, each in connection with a binding screw. Between two of these stretches a bridge insulated in the middle. The battery wires are always attached to the cups connected with this (1 and 2). When it is used to pass a current through different F- 59 _Pohl»s Com. wires, the cross-bars are removed and wires Siutator with cross- are attached to all six cups, 3 and 4, 5 and 6. bars in. On turning the bridge to one side or other the current is sent through one or other pair of wires. To reverse the direction of a current, only one pair of wires, beside the battery wires, is attached to the mercury cups — e.g., to 3 and 4, or 5 and 6, the cross-bars remaining in. 5. The student, if he desires, can prepare two nerve-muscle preparations, and dip the nerve of one (A) and the muscle of the other (B) into a solution of curara in two watch-glasses. On stimulating the nerve of A its muscle contracts ; on stimulating the nerve of B its muscle does not contract, but the muscle contracts when it is stimulated directly. In A, although the poison is applied directly to the nerve trunk, the nerve is not paralysed. 11 162 EXPERIMENTAL PHYSIOLOGY. LESSON XXXI. THE GRAPHIC METHOD— MOIST CHAMBER —SINGLE CONTRACTION— WORK DONE. 1. Recording Apparatus. — For this purpose a revolving cylinder vrered with smoked glazed paper, or other moving surface is re- 1. covered quired. The velocity of the moving surface is usually determined by recording simultaneously the vibrations of a tuning-fork, of known rate of vi- bration, or an electro- magnetic time-marker. It does not matter par- ticularly what form of recording drum is used, provided it moves smoothly and evenly, and is capable of being made to move at dif- ferent rates as required. In Hawksley's form this is accomplished by placing the drum on different axles, moving at different velocities. In Ludwig's form (Fig. 60), this is done by moving a small wheel, n, on a large brass disc, D. Where a num- ber of men have to be Fig. 60.— Ludwig's revolving cylinder, R, moved taught at once, one by the_clock-work in the box, A, and regulated mu*t have recou;se to by a Foucault's regulator on the top box. The disc, D, moved by the clock- of the work, an arrangement of presses ui>on the wheel, n, which can be shafting, moved, say raised or lowered by the screw, L, thus alter- by a water-motor or ing the position of n on D, so as to cause the turbine and from whiVh cylinder to rotate at different rates. The >me^nd cylinder itself can be raised by the handle, t. seTeral drums can be On the left aide of the figure is a mercurial driven by cords. Or manometer. one may use a small gas engine as the mo- tive power, and cords passing over pulleys to move the drums. MOIST CHAMBER. 163 This is the arrangement adopted in the Physiological Depart- ment of Owens College, so that a large number of men can work at the same time, each being provided with recording apparatus for himself. 2. Cover the Cylinder with Paper. — The paper is glazed on one surface, and is cut to the necessary size to suit the drum. The drum can be removed from the clock-work or other motor which moves it, and is then covered with a strip of paper, the latter being laid on evenly to avoid folds. One edge of the paper is gummed, and slightly overlaps the other edge. Leave it for a few minutes until the gum dries. The paper has then to be blackened, by holding the drum and keeping it moving over a fan-tailed gas burner, or paraffin lamp — the former is preferable. Take care that the soot from the flame is deposited evenly and lightly, and see that it is not burned into the paper. The drum is then placed in position in connection with its motor. 3. General Rules to be observed with every Graphic or other Experiment. (1.) In every experiment arrange the apparatus completely, cover the drum with paper, and smoke it, before beginning the dissection of the frog. (2.) See that the secondary circuit is " short-circuited." (3.) Test all the connections stage by stage as they are made. (4.) Each tracing is to be inscribed with the name of the individual who made it, the date, and what it is intended to show, and any other particulars it is desired to record. It is then to be varnished, and the varnish allowed to dry. 4. Myographs. — Various forms are in use, but most of them consist of a lever which is raised by the contracting muscle, and so arranged as to record its movement on a smoked surface of paper or glass. 5. Moist Chamber (Fig. 61). — To prevent a nerve-muscle pre- paration from getting dry, it must be enclosed in a moist chamber, which is merely a glass shade placed over the preparation, while to keep the air and the preparation moist, the sides of the shade are covered with blotting-paper moistened with water. 164 EXPERIMENTAL PHYSIOLOGY. 6. Varnish for Tracings.— The tracing is simply drawn through the varnish and then hung up to dry. (a.) A very good varnish consists of gum mastic dissolved to saturation in methylated spirit. Fig. 61.— Moist Chamber.— N, Glass shade; E, electrodes; L, lever; W, weight ; TM, time-marker ; other letters as in previous figures. (b.) Where a large quantity is used, and economy is an object, guin juniper may be used instead of mastic. (c.) Dissolve 4 oz. of sandarac in 15 o/>. of alcohol, and add half an oz. of chloroform. 7. Single Contraction or Twitch — Apparatus. — Recording drum, Daniell's cell, Morse key, induction machine, du Bois key, wires, electrodes, moist chamber and lever, moist blotting- paper, stout ligatures, hook, pins, lead-weight (20 grammes),, frog, and the necessary instruments. (a.) Arrange the recording apparatus and the drum to move slowly. Cover the drum with glazed paper, and after- wards smoke it over a gas flame, and fix it in position. (b.) Arrange the apparatus as follows : — One Daniell's cell and a Morse key in the primary circuit, the secondary SINGLE CONTRACTION OR TWITCH. 165 circuit of the induction machine short-circuited, and with wires to go to the binding screws on the platform of the moist chamber on the myograph (Fig. 62). [The muscle may be caused to contract either by stimulating it directly — in which case the electrodes are made of thin wires and merely pushed through the two ends of the gastrocnemius — or indirectly through the nerve. It is convenient to use the latter method (Lesson XXIX., 3).] (c.) Make a nerve-muscle preparation, leaving the lower end of the femur in connection with the gastrocnemius, and cut away the tibia and fibula. With the point of a sharp pair of small scissors make a small hole in the tendo Achillis, and insert in it a hook like the letter S, made by bending a pin. Manipulate the nerve with a camel's-hair pencil. Arrange the preparation in the moist chamber by fixing the femur in the muscle clamp, and by means of a stout ligature thread attach the hook in the tendo Achillis to the writing-lever under the ebonite or wooden stage of the moist chamber. See that the muscle or ligature goes clear through the hole in the stage, and that the hook does not catch on anything. Adjust the height of the muscle clamp, so that the writing-lever is horizontal. Place the nerve over the electrodes, and cover the whole preparation with the glass-shade lined on three sides with moist blotting-paper. Load the lever near where the muscle is attached to it by a weight of 20 grammes or thereby, and make the lever itself horizontal. Arrange the point of the lever so that it writes on the cylinder. The writing-style on the tip of the lever may be made of very thin copper foil or parchment paper, fastened on to the lever with sealing-wax or telegraph composition. 8. According as the recording surface is stationary, or moving when the muscle contracts and raises the lever, either a vertical line or a curve will be made upon the paper. In the latter case the form of the curve will vary with the velocity of the drum. Arrange experiments for both. A. Begin with the recording cylinder stationary. (a.) Push the secondary coil far away from the primary, open the key in the secondary circuit, and make and break the primary circuit. There may be no contraction. Close the secondary circuit key. 166 EXPERIMENTAL PHYSIOLOGY. (b.) Gradually approximate the secondary coil, open the short-circuiting key, and break the primary circuit by means of the Morse key in it. Observe when the first feeble con- traction is obtained = minimal contraction. Make the primary circuit, there is no contraction. The break shock is, there- fore, stronger than the make. Record under each contraction whether it is a make (M) or break (B) shock, and the dis- tance in centimetres of the secondary from the primary coil. Move the drum a short distance with the hand, the lever will inscribe a horizontal or base line, the abscissa. (c.) Gradually approximate the secondary spiral, and from time to time test the effect of the make and break shocks, after each test moving the cylinder with the hand, and recording the result as to M or B, and the distance in centi- meters of the secondary from the primary coil. After a time a make contraction appears, and on pushing up the secondary coil the make contraction becomes as high as the break. (d.) Increase the stimulus by bringing the secondary nearer the primary coil, and notice that the contractions do not become higher = maximal contraction. In each case keep the make and break contractions, obtained with each strength of current, close together. Their relative heights can then be readily compared. B. Arrange the experiment as in A, but allow the cylinder to revolve at a moderate speed, 25-30 centimetres per second ; the writing-style records an even horizontal line = the abscissa. (a.) Select a strength of stimulus which is known to cause a contraction, and while the cylinder is revolving, cause the muscle to contract either by a make or break shock. Study the characters of the " muscle -curve." (6.) Vary the velocity of movement of the cylinder, and observe how the form of the curve varies with the variation in velocity of the cylinder. (c.) Remove the tracings in A and B, and varnish them. In this case the moment of stimulation is not recorded. THE CRANK MYOGRAPH. 167 9. The work done. — After the tracing of B is dry, from the abscissa draw vertical lines, or ordinates, and measure their height in millimetres. Measure the length of the lever, and from this calculate the actual amount of shortening of the muscle itself Multiply this by the weight lifted, and the product is the work done expressed in gram-millimetres. 10. The Crank Myograph (Fig. 62) is fixed on a suitable sup- port, so that it can be adjusted to any height desired. The experiment is arranged in exactly the same way as for 7. (a.) Use one hind-limb of a pithed frog; pin the femur firmly to the cork plate of the myograph covered with blotting-paper moistened by normal saline, the tibia being in line with the writing-lever. Or take the pithed frog, lay it on the frog-plate of the myograph, expose the gastro- cnemius, and proceed as above. Detach the tendo Achillis, tie a stout ligature to its sesamoid bone, and fix the liga- 1 Fig. 62.— Crank Myograph.— W,W, Block of wood; M, muscle; F, femur; P, pin to fix F ; L, lever ; WT, weight ; a, screw for after-load ; C, cork ; B,B, brass box. (In this figure the fulcrum should be at the angle of the crank.) ture to the short arm of the lever, add a weight of 20 grammes to the lever, and see that the lever itself is hori- zontal. Thrust two fine wires — the electrodes — from the du Bois key in the secondary coil, through the upper and lower end of the gastrocnemius muscle; (6.) Arrange the style of the lever so that it writes on the cylinder, and repeat the experiments of either A or B, or both. 168 EXPERIMENTAL PHYSIOLOGY. (c.) Use different weights — 5 — 20 — 50 grammes — and observe how the form of the curve varies on increasing the weight attached to the lever. 11. After-load. — In the crank-myograph, under the lever is a screw on which the horizontal arm of the bell-crank rests (Fig. 62, a), so that the muscle is loaded only during its con- traction. 12. Interrupted Current. — If instead of single shocks, the induc- tion apparatus be so arranged that Neef's hammer is in action, then on stimulating the muscle or nerve, tetanus is obtained (Lesson XXVIII, 3), and the curve of a tetanised muscle re- corded. LESSON XXXII. ANALYSIS OF A MUSCULAR CONTRACTION -PENDULUM MYOGRAPH— SPRING MYOGRAPH— TIME-MARKER- DEPREZ' SIGNAL. 1. Muscle Curve by the Pendulum Myograph. (a.) Cover the oblong glass plate with glazed paper, smoke its surface, and fix it to the pendulum. The plate must be so adjusted that the pendulum on being set free from the "detent" (Fig. 63, C) shall be held by the « catch " (0). Test this. (b.) Arrange the induction apparatus for single shocks as in Fig. 63, but shor^circuit the secondary circuit, inter- posing in the primary circuit the trigger-key or knock -over key of the pendulum myograph (Fig. 63, K'). ANALYSIS OF A MUSCULAR CONTRACTION. 169 (c.) Make a nerve-muscle preparation, fix the femur in the clamp, attach the tendo Achillis to the writing-lever (S), and place the nerve over the electrodes in the ordinary muscle moist chamber. Adjust the moist chamber on its stand and raise it to a suitable height. Load the lever with 20 grammes, and direct its point to the side to which the pendulum swings. Fix the pendulum with the detent, and adjust B Fig. 63. — Scheme of the arrangement of the pendulum.— B, Battery ; I., primary, II., secondary spiral of the induction machine; S', tooth ; K', key ; C, C, catches ; K' in the corner, scheme of K' ; K, key in primary circuit. the writing - style of the lever on the smoked sur- face. Connect the secondary spiral either with the muscle directly, or preferably, with the electrodes on which its nerve rests, introducing a short-circuiting key. In Fig. 63 this is omitted, and the wires of the secondary circuit go direct to the muscle. After un-short-circuiting, with the hand break the primary circuit to make certain that a contraction occurs on breaking the primary circuit. (d.) See that the trigger-key (K') of the pendulum (in the primary circuit) is closed, and the key in the secondary circuit open. Allow the pendulum to swing • as it does so, it knocks open the key in the primary circuit and breaks the current, thus inducing a shock in the secondary circuit, whereby the muscle is stimulated and caused to record its contraction or muscle-curve on the smoked surface. (e.) Take the abscissa— i.e., the base line. Rotate the stand on which the moist chamber is supported, so as to withdraw 170 EXPERIMENTAL PHYSIOLOGY. the writing point of the lever from the recording surface. Bring the pendulum back to the detent, adjust the writing- style, close the trigger-key, and keep the secondary circuit short-circuited by closing the du Bois key. Allow the pendulum to swing. This records the abscissa or base line. (/) Ascertain the latent period as follows : — Bring the pendulum to the detent, short-circuit the secondary current, and withdraw the writing-style as in (e.) Close the trigger- key of the pendulum, and with a finger of the left hand keep it closed. Allow the lever to touch the glass plate in its original position, and with the right hand bring the knife-edge of the pendulum in contact with the trigger-key so as just to open it. Thus a vertical line is inscribed on the stationary plate, which indicates the moment of stimulation. (g.) Remove the muscle lever, place the pendulum in the detent, close the trigger-key, take a tuning-fork vibrating, say, 120 or 250 double vibrations per second, and adjust its writing-style in the position formerly occupied by the style of the muscle lever. Set the fork vibrating, either electrically or by striking it. Allow the pendulum to swing, when the vibrating tuning-fork will record the time curve under the muscle curve (Fig. 64, 250 DV). All the conditions must be exactly the same as when the muscle curve was taken. (h.) Remove the paper, varnish the tracing, hang it up to dry, and next day measure the tracing. Bring ordinates vertical, a', 6', c', to the abscissa, and measure the " latent period" (Fig. 64, A), the duration of the shortening (B), the phase of relaxation (C), and the contraction remainder. Fig. 64.— Pendulum Myograph Curve.— S, Point of stimulation ; A, latent period ; B, period of shortening, and C, of relaxation. 2. Spring Myograph (Fig. 65). — The arrangements are exactly the same as for the pendulum myograph, the trigger-key of the myograph being placed in the primary circuit. The instrument must be raised on blocks. SPRING MYOGRAPH. 171 (a.) Cover the slip of glass with glazed paper, smoke it, and fix it in the frame. Push the plate to one side by means of the rod attached to it, and fix it by means of the catch. Fig. 65. — Spring Myograph. Close the trigger-key (h), and introduce it into the primary circuit of the induction machine. (b.) Make a nerve-muscle preparation, and arrange it to write on the glass plates as directed in 1 (c.) Remember to un-short-circuit the secondary circuit. (c.) Press on the thumb-plate (a), thus liberating the spring, when the glass plate moves swiftly to the other side, and in doing so the tooth (d) on its under surface breaks the primary circuit, and the muscle -curve is recorded. (d.) Short-circuit the secondary circuit, push back the glass plate, and fix it with the catch ; close the trigger-key, and shoot the glass plate again. This records the abscissa, or horizontal line. (e.) Remove the moist chamber, and take the time curve. Push the glass plate back again, and secure it by the catch ; close the trigger-key — in order that the conditions may be exactly the same as before — strike a tuning-fork, vibrating, say, 1 20 double vibrations per second, and when it is vibrat- 172 EXPERIMENTAL PHYSIOLOGY. ing adjust its writing-style under the abscissa. Shoot the glass plate again, and the time curve will be recorded. (/.) Remove the tracing, fix it and measure it out, deter- mining the length of the latent period and the duration of the contraction, and of its several parts. 3. Study the improved form of this instrument recently intro- duced by du Bois, in which the glass plate is set free, and the tuning-fork vibrations are recorded simultaneously when a handle is pressed. It is a most elegant piece of apparatus, and has a beautiful mechanism for adjusting the writing-styles for the muscle and abscissa. 4. On a Revolving Cylinder. («.) Arrange the drum to move at the fastest speed. (b.) Arrange an induction machine for single shocks, the secondary circuit to be short-circuited, and arranged to stimulate a nerve attached to a muscle placed in a moist chamber, as directed for the foregoing experi- ments. Into the primary circuit introduce besides the Morse key, an electro-mag- net with a marking lever (Figs. 61, 66, e),and cause its point to write exactly under the muscle lever. Arrange, with its point ex- actly under the other two, a Deprez' chronograph or signal, in circuit with a tuning-fork of known rate of vibration, and driven by means of abattery (Fig. 68). Muscular Contraction.— B, battery; I he three recording levers K, key in primary circuit; I., pri- are all fixed on the same marv, II., secondary spiral, without stand, which should prefer- a short-circuiting key ; /, muscle ably be a tangent one— i.e., lever ; e, electro-magnet in primary . i J i i & • ,1 circuit; t, electric signal; & sup- ^e.rod bearing the re- port ; RC, revolving cylinder. cording styles can by means of a handle be made to rotate so as to bring the writing-styles in contact with TIME-RECORDER. 173 the recording surface. This avoids the overlapping of the time curve which otherwise happens. On un-short-circuiting the secondary circuit, and breaking the primary one, the muscle contracts, and at the same time the style of the electro-magnet is attracted and records the exact moment of stimulation (Fig. 61). 5. Time-Recorder (Fig. 67). — This is merely an electro-magnet introduced into an electric circuit, and the magnet, B, is so arranged as to attract a writing-style, L. vv vv Fig. 67. — Time-Marker. — L, Lever ; B, electro-magnet bobbins; S, support; W,W, wires. 6. Deprez' Signal (Fig. 68). — This small electro-magnet has so little inertia that, if it be introduced into an electric circuit, its armature, which is provided with a very light writing point, vibrates simultaneously with the vibrations of an electric tuning- fork introduced into the same circuit. Arrange the signal EM Pt Fig. 68.— Signal and Vibrating Tuning-Fork in an Electric Circuit.— D, Drum ; C, signal ; EM, electric tuning-fork ; Pt, platinum contact. and tuning-fork as in Fig. 68. The drum must move more rapidly, the more rapid the vibrations of the tuning-fork used. 174 EXPERIMENTAL PHYSIOLOGY. LESSON XXXIII. INFLUENCE OF TEMPERATURE, LOAD, VERATRIA ON MUSCULAR CONTRACTION. 1. Influence of Temperature on Muscular Contraction. (a.) Arrange the experiment with a crank-myograph as in Lesson XXXI., 10, but do not remove the skin of the leg. Take a tracing at the normal temperature. (6.) Alter the height of the drum or that of the myograph. Place ice upon the skin over the gastrocnemius for some time, and then take another tracing, noting the differences in the result, the contraction being much longer. (c.) Adjust a piece of wire gauze over the leg, and allow it to project beyond the end of the plate of the myograph. Heat the gauze with a spirit-lamp. Take a tracing. The contraction is shorter than in 1 (6.) Do not overheat the muscle. (d.) A piece of thin gas piping can be bent, and the muscle laid on it. Water of various temperatures can then be passed through it. (e.) The muscle may be attached to an ordinary horizontal writing-lever. Surround the muscle with a double-walled box, with an inflow and outflow tube, through which water at different temperatures can be passed. A delicate thermometer is placed in the chamber with the muscle. (f.) Perhaps for the purpose of the student the most convenient method is to allow the muscle to rest on a small circular brass box, fitted into the wooden plate of the crank- myograph. The box ( B, B) is provided with an inflow and an outflow tube, through which water of the desired tem- perature can be passed (Fig. 62). INFLUENCE OF VER ATRIA ON CONTRACTION. 175 2. Influence of the Load on the Form of the Curve. (a.) Arrange an experiment with the pendulum myograph as in Lesson XXXII., 1, using either a muscle-lever or a crank-my ograph . (b.) Take a tracing with the muscle weighted with the lever only. (c.) Then load the lever successively with different weights 250 DV. Fig. 69. — Pendulum Myograph Curves showing the Influence of the Load on the Form of the Curve. (5 ... 20 ... 50 ... 70 ... 100 grammes), and in each case record a curve and observe how the form of the curve varies (Fig. 69). (d.) In each case record the abscissa and time curve with the usual precautions. Fig. 70. — Veratria Curve (Upper). Normal Muscle Curve (Lower). 3. Influence of Veratria on Contraction. (a.) Destroy the brain of a frog, and inject into the ventral lymph sac 10 minims of a freshly-prepared O'l per cent, solution of veratria. 176 EXPERIMENTAL PHYSIOLOGY. (6.) Arrange the induction machine for single shocks. (c.) Make a nerve-muscle preparation, fix it in a moist chamber, and arrange the muscle lever to record its move- ments on a slowly revolving drum. Take a tracing, observ- ing the long drawn-out form of the curve, and how long the muscle takes to relax. (d.) The direct action of veratria on muscular tissue may also be studied by the apparatus described in Lesson XXXVII., and by this method it is easy to compare the form of the curve before and after the action of the poison (Fig. 70). LESSON XXXIV. ELASTICITY AND EXTENSIBILITY OF MUSCLE. 1. Elasticity of Muscle. (a.) Dissect out the gastrocnemius of a frog with the femur attached, clamp the femur, attach the tendon to the light writing muscle lever of the moist chamber, and fix a scale pan to the lever. Neglect the weight of the pan, and see that the lever writes horizontally on a stationary drum. (b.) Place in the scale pan successively different weights (10, 20, 30, 40 ... 100 grammes). On placing in 10 grammes the lever will descend, remove the weight and the lever will ascend. Move the drum a certain distance, and add 20 grammes to the scale pan. This time the vertical line drawn is longer, indicating greater extension of the muscle by a greater weight, but nevertheless the muscle lever will rise to its original height on removing the weight. Repeat this with other weights. With the heavier weights care must be taken that everything is securely clamped. If the apices of all the lines obtained be joined, they form a hyperbola. The muscle therefore has not a large amount of elasticity, i.e., it is easily extended by light weights, and on removal of the weight it regains its original ELASTICITY OF MUSCLE. 177 length, so that its elasticity is said to be very perfect. The hyperbolic curve obtained shows further that the increase in length is not directly proportional to the weight; but it diminishes as the weights increase. (c.) Repeat the same observation with a thin strip of india-rubber. In this case equal increments of weight give an equal elongation, so that a line joining the apices of the vertical lines drawn after each weight is a straight line. 2. The Extensibility of Muscle is Increased during Contraction, its Elasticity is Diminished. (a.) Arrange a muscle in a moist chamber, connecting it to a lever to record on a drum, and adjust an interrupted current to stimulate the muscle, either directly or indirectly. (b.) Load the lever with 50 grammes, and in doing so allow the drum to move slowly. Remove the load and observe the curve obtained. (c.) Tetanise the muscle, and while it is contracted to its greatest extent, again load the lever with 50 grammes while the drum is in motion, and remove the load. Observe the curve. (d.) Compare the two curves. The second curve will, of course, begin higher, but notice that its absolute descent is greater than the first curve, and that it does not rise to the horizontal again. (e.) It is better to begin the experiment with the drum stationary, and then to record the tracing with the drum in motion. (/.) A better curve is obtained by using a long counter- poised lever attached to the muscle, which writes on a very slow-moving drum. A weight is made to travel along the lever by means of two pulleys with an endless string. 178 EXPERIMENTAL PHYSIOLOGY. LESSON XXXV. FATIGUE OF MUSCLE. 1. Fatigue of Muscle. (a.) Arrange an induction apparatus for single shocks, but introduce into the primary circuit in addition to the du Bois key a trigger-key, the latter fixed to a stand, and so placed that a tooth on the edge of the drum can knock it over, and thus break the primary current as required. Or attach to the edge of the under surface of the drum a short style ; a strong pair of bull-dog forceps clamped on to it does perfectly well. (6). Make a nerve-muscle preparation, clamp the femur, and adjust the preparation in the moist cham- ber, or a crank- myograph — the whole to be sup- ported on a tan- gent stand. At- tach the muscle to a writing- Fig. 71.-Fatigue Curve.-The sciatic nerve : was leyer to record a revolving drum. stimulated with maximal induction shocks and every fifteenth contraction recorded. (c.) Close the trigger-key, and on allowing the cylinder to revolve, the style knocks it over, breaks the primary circuit, and induces a shock in the secondary. Immediately short-circuit the secondary circuit, close the trigger-key and unshort-circuit the secondary circuit, and allow the drum to revolve. Repeat this until the muscle is fatigued. Record only every fifteenth contraction. In this way the muscle is always stimulated at the same moment, and the various curves are superposed, and can be readily com- pared (Fig. 71). FATIGUE OF MUSCLE. 179 (d.) The best plan is to fix a platinum style on the spindle of the drum, and as it revolves it comes in contact with another piece of platinum introduced into the primary circuit, and fixed to the base of the drum-support, so that a break shock is obtained each time the drum revolves. (e.) Observe how rapidly the height of the curves falls, while their duration is longer. In nearly every case fatigue curves from muscle show a "stair-case" character (Fig. 71), the second curve being higher than the first one, and the third than the second. 2. Instead of recording on a moving surface, a stationary one may be used (or a very slow-moving drum, 1 mm. per sec.), either a drum or a flat glass surface, while the muscle may be attached to a crank-myograph or to Pfliiger's myograph circuit the battery. Fill the cups with mercury, and place the commutator in a 200 EXPERIMENTAL PHYSIOLOGY. small tray to avoid spilling the mercury. From two of the binding screws connect wires with two N.P. electrodes Fig. 90.— Scheme of Electrotonic Variation of Excitability in a Nerve. — K, Cathode; A, anode; N n, nerve. The curve above the line indi- cates increase, and that below the line decrease, of excitability. or the platinum electrodes of du Bois, introducing a short- circuiting key in the electrode circuit (Fig. 91). Fig. 91. — Scheme of Electrotonic Variation of Excitability. — D, Drop of strong solution of salt on the nerve, N ; F, flag on the muscle. (6.) Make a nerve-muscle preparation, attach a straw flag to the foot, as directed in Lesson XXVII., 4, and clamp the femur in a clamp on a stand, as in Fig. 53. Lay the nerve over the electrodes. Trace the direction of the current, and make a mark to guide you as to when the current in the nerve is descending or ascending — i.e., whether the negative or positive pole is next the muscle. (c.) Place a drop of a saturated solution of common salt on the nerve between the electrodes and the muscle. In a minute or less the toes begin to twitch, and by-and-by all the muscles of the leg become tetanic, so that the flag is raised and kept in the horizontal position. PFLUGER'S LAW OF CONTRACTION. 201 (d.) Turn the commutator so that the positive pole is next the muscle, at once the straw falls — i.e, the excitability of the nerve in the region of the positive pole is so diminished as to "block " the impulse passing to the muscle, showing that the positive pole lowers the excitability. (e.) Reverse the commutator so that the negative pole is next the muscle. At once the limb becomes tetanic, the negative pole (kathelectrotonic area) increases the excita- bility. LESSON XLII. ELECTROTONIC VARIATION OF THE ELEC- TROMOTIVITY— PFLUGER'S LAW OF CONTRACTION— RITTER'S TETANUS. 1. The Electrotonic Variation of the Electromotivity. (a.) Arrange a long nerve on the N.P. electrodes, as for determining its demarcation current. Place the free end of the nerve on a pair of N.P. electrodes — the polarising current — arranged as in Lesson XLL, 2, A., so that the current can be made ascending or descending. (b.) Take the deflection of the galvanometer*, needle or demarcation current when the polarising current is shut off. Throw in a descending polarising current, and observe that the spot of light travels towards zero. Reverse the commu- tator, and throw in an ascending current, the spot of light shows a greater positive variation than before, From this we conclude that kathelectrotonus diminishes the electromotivity, while anelectrotonus increases it. 2. Pflttger's Law of Contraction — Apparatus. — Several small Grove's cells, commutator with cross-bars, du Bois and Morse key, rheochord, N.P. electrodes, moist chamber, wires, frog, recording apparatus, and usual instruments. 202 EXPERIMENTAL PHYSIOLOGY. (a.) Arrange the apparatus as in the scheme (Fig. 92). Take two Daniell or small Grove cells, connect them to a Pohl's commutator with cross-bars, and introduce a Morse or mercury key (K) into the circuit; connect the commutator with the rheochord (R). Connect the rheochord with a pair Fig. 92.— Scheme for Pfluger's Law.— R, Rheochord. of N.P. electrodes with a short-circuiting key introduced. Fix to a lever a nerve-muscle preparation — with a long nerve — in the moist chamber, and lay the nerve over the electrodes. (6.) Begin with all the plugs in position in the rheochord and the slider hard up to the brass blocks. Place the com- mutator to give an ascending current, make and break the current — gradually adjusting the slider — until a contraction occurs at make and none at break. Reverse the commutator to get a descending current, make and break, observing again a contraction at make and none at break. This represents the effect of a weak current. (c.) Pull the slider further away and remove one or more plugs, until contraction is obtained at make and break, both with an ascending and descending current. This represents the effect of a medium current. (d.) Use six small Grove's cells, take out all the plugs from the rheochord, and with the current ascending, contraction occurs at break only — while with a descending current, con- traction occurs only at make. This represents the effect of a strong current. Tabulate the results in each case. For this experiment very fresh and strong frogs are necessary, and several preparations will be required to work out all the HITTERS TETANUS. 203 details of the law. Instead of reversing the commutator after testing the effect of an alteration of the direction of the current, the student may use one preparation to test at intervals the effect of weak, medium, and strong currents, when the current is ascending, and a second preparation to test the results with currents of varying intensity when the current is descending. The results may be tabulated as follows : — R, = rest ; C = con- traction. ASCENDING. DESCENDING. C rs. C* On Making. On Breaking. On Making. On Breaking. Weak, . C R C R Medium, C r> C C Strong, . R C C R 3. Ritter's Tetanus. (a.) Connect three Daniell's cells with non-polarisable electrodes short-circuiting with a du Bois key. Prepare a nerve-muscle preparation, and apply the electrode to the nerve so that the + pole is next the muscle — i.e., the current is ascending in the nerve. Allow the current to circulate in the nerve for some time (usually about five minutes is sufficient), no contraction takes place. Short- circuit, and observe that the muscle becomes tetanic. (b.) Divide the nerve between the electrodes, and the tetanus does not cease ; but on dividing it between the + pole and the muscle, the tetanus ceases. Therefore the tetanus is due to some condition at the positive pole. -204 EXPERIMENTAL PHYSIOLOGY. LESSON XLIII. VELOCITY OF NERVE ENERGY IN A MOTOR NERVE— DOUBLE CONDUCTION IN NERVE — KUHNE'S GRACILIS EXPERIMENT. 1. Velocity of Nerve Energy in a Motor Nerve. The rate of propagation of a nerve impulse may be estimated by either the pendulum or spring myograph. With slight modifications the two processes are identical, only in using the spring myograph it is necessary to use such a coiled spring, as will cause the glass plate to move with sufficient rapidity, to give an interval long enough for the easy estimation of the latent period. (a.) Use the spring myograph and arrange the experiment according to the scheme (Fig. 93) — i.e., an induction coil for n Fig. 93.— Scheme for Estimating the Velocity of Nerve Energy. single shocks with the trigger-key of the myograph (1, 2) arranged in the primary circuit; in the secondary circuit (which should be short-circuited, not represented in the diagram) place a Pohl's commutator ivithout cross-bars (C). VELOCITY OF NERVE ENERGY. 205 Two pairs of wires from the commutator pass to two pairs of electrodes (a, b), movable on a bar within the moist chamber. Measure the distance between the electrodes. (b.) Make a nerve-muscle preparation with the nerve as long as possible (N), clamp the femur (/), attach the tendon (ra) to a writing-lever, and lay the nerve over the electrodes^ the distance between them being known. (c.) Arrange the glass plate covered with smoked paperr adjust the lever to mark on the glass, close the trigger-key in the primary circuit, and un-short-circuit the secondary. Turn the bridge of the commutator so that the stimulus will be sent through the electrodes next the muscle (a). Press the thumb-plate, and shoot the glass plate. The tooth (3) breaks the primary circuit, and a curve is inscribed on the plate. (d.) Short-circuit again, replace the glass plate, close the trigger-key, reverse the commutator. This time the stimulus will pass through the electrodes away from the muscle (b). Un-short-circuit the secondary circuit, and shoot the glass plate. Again another curve will be inscribed, this time a little later than the first one. (e.) Replace the glass plate, close the trigger-key, short- circuit the secondary circuit, and shoot the plate. This makes the abscissa. (/.) Replace the glass plate, close the trigger-key, and bring the tooth of the glass plate (3) just to touch the trigger-key ; raise the writing-lever to make a vertical mark. This indicates the moment when the stimulus was thrown into both points of the nerve. (g.) Remove the moist chamber, push up the glass plate, close the trigger-key, and arrange a tuning-fork vibrating 250 D.Y. per second to write under the abscissa. Shoot the plate again and the time-curve will be obtained. Fix the tracing, draw ordinates from the beginning of the curves obtained by the stimulation of a and b respectively, measure the time between them from the time-curve (this gives the time the impulse took to travel from b to a), and calculate the velocity from the data obtained. 206 EXPERIMENTAL PHYSIOLOGY. Example. — Suppose the length of nerve to be 4 cni., and the time required for the impulse to travel from b to a to be T1TT sec. Then we have 4 : 100 : ^ : ^", or 30 metres (about 90 feet) per second, as the velocity of nerve energy along a nerve. 2. Repeat the observation with the pendulum myograph. Practically the same arrangements are necessary. If it be desired to test the effect of heat or cold on the rapidity of propagation, the nerve must be laid on ebonite electrodes, made in the form of a chamber, and covered with a lacquered copper plate on which the nerve rests. Through the chamber water at different temperatures can be passed, and the effect on the rate of propagation observed. 3. Unequal Excitability of a Nerve— Apparatus.— Battery, two keys, wires, commutator, induction machine, two pairs of electrodes. Fig. 94.— Scheme for the Unequal Excitability of a Nerve. (a.) Arrange the apparatus as in Fig. 94, introducing a Morse key in the primary circuit. Dissect out the whole length of the sciatic nerve with the foot attached. Lay the nerve on two pairs of electrodes, A and B, one near the muscle, and the other away from it, and as far apart as possible. Two pairs of wires thrust through a cork or piece of caoutchouc will do quite well. (b.) Stimulate the nerve at A with a strength of current that gives just a minimal contraction. Reverse the conimu- DOUBLE CONDUCTION IN NERVE. 207 tator, and on stimulating at B a much stronger contraction is obtained, because the excitability of a nerve is greater further from a muscle. 4. Double Conduction in Nerve — Kiihne's Experiment on the Gracilis. — The gracilis is divided into a larger and smaller portion (L) by a tendinous inscription (K) running across it (Fig. 95). The nerve (N) enters at the hilum in the larger half, and bifurcates, giving a branch (k) to the smaller portion, and another to the larger portion of the muscle. (a.) Excise the gracilis from a large frog, and cut it as shown in Fig. 96, avoiding injury to the nerves, so that only the nerve twig (k) connects the larger and smaller halves, and in one tongue (Z) terminates a nerve. The gracilis after excision must be laid on a glass plate with a black background, else one does not see clearly the inscription and the course of the nerves. Both are easily seen on the black surface. (6.) Stimulate the tongue (Z) with fine electrodes about 1 mm. apart, and con- trnction occurs in both L and K This can be due only to centripetal conduction the Gracilis. in a motor nerve, and this experiment is adduced by Kiihne as the best proof of double conduction in nerve fibres. PHYSIOLOGY OF THE CIRCULATION. LESSON XLIV. THE FROG'S HEART— BEATING OF THE HEART— EFFECT OF HEAT AND GOLD —SECTION OF THE HEART. 1. The Heart of the Frog and how to expose it. (a.) Pith a frog, and lay it on its back on a frog-plate. Make a median incision through the skin over the sternum, and from the middle of this make transverse incisions. (b.) Reflect the four flaps of skin, raise the lower end of the sternum with a pair of forceps, and cut through the sternal cartilage just above its lower end to avoid wounding the epigastric vein. With a strong pair of scissors cut along the margins of the sternum, and divide it above transversely to remove the anterior wall of the thorax. This exposes the heart, still enclosed within its pericardium, where it can be seen beating. (c.) With a fine pair of forceps carefully lift up the thin transparent pericardium, cut it open, thus exposing the heart. 2. Study the General Arrangement of the Frog's Heart. (a.) Observe its shape, noting the two auricles above (Ad, As), and the conical apex of the single ventricle below (v), the auricles being mapped off from the ventricle by a groove -or furrow which runs obliquely across its THE HEART BEATS AFTER IT IS EXCISED. 209 anterior aspect. The ventricle is continuous anteriorly with the bulbus aortse (B), which projects in front of the right auricle, and divides into two aortse — right and left, the left being the larger (Fig. 96). Fig. 97.— Heart of Frog from be Fig. 96. — Frog's Heart from the hind. — s.v, Sinus venosus opened; front. — V, Single ventricle; Ad, As, right and left auricles; B, bulbus arteriosus ; 1, carotid ; 2, aorta; 3, pulmocutaneous ar- teries ; C, carotid gland. c.i, inferior; c.s.d, c.s.s, right and left superior vense cavse; v.p, pulmon- ary vein ; Ad and As, right and left auricles ; Ap, communication between the right and left auricle. (b.) Tilt up the ventricle and observe the sinus venosus (Fig. 97, s.v), continuous with the right auricle, and formed by the junction of the large inferior vena cava (c.i), and the two (smaller) superior vense cavse (c.s.s, c.s.d). 3. The Heart beats after it is excised. (a.) With a seeker tilt up the apex of the ventricle, and observe that a thin thread of connective-tissue, called the " frsenum," containing a small vein, passes from the peri- cardium to the posterior aspect of the ventricle. Divide it with a fine pair of scissors. Count the number of beats per minute. Seize with forceps the part of the frsenum attached to the ventricle, and lift up the heart therewith ; and with a sharp pair of scissors cut out the heart by dividing the inferior vena cava, the two superior vense cavse, and the two aortse. Place the excised heart in a watch-glass, and cover it with another watch-glass. (b.) The heart goes on beating. Count the number of beats per minute. Therefore its beat is automatic, and the heart contains within itself the mechanism for originating 14 210 EXPERIMENTAL PHYSIOLOGY. and keeping up its rhythmical beats. If the heart tends to become dry, moisten it with normal saline solution, although normal saline containing a little blood is better. (c.) Observe also how during diastole the heart is soft and flaccid, and takes the shape of any surface it may rest on, while during systole when it contracts, it becomes harder, while the apex is raised and erected. 4. Effect of Heat and Cold on the Excised Heart. (a.) Place the watch-glass containing the beating heart on the palm of the hand, and the heart will beat faster ; or place it over a beaker containing warm water, which must not be above 40°C. Observe that as the temperature rises, the heart beats faster — i.e., there are more beats per minute, also that each single beat is faster. (t>. ) Remove the watch-glass from the palm, place it over a beaker containing cold water or ice, when the number of beats will diminish, each beat being executed more slowly and sluggishly. 5. Section of the Heart. (a.) With a sharp pair of scissors divide the ventricle at its upper third just below the auriculo- ventricular groove. Observe that the auricles with the upper third of the ventricle attached to them continue to beat spontaneously, while the lower two-thirds of the ventricle no longer beat spontaneously. If it be pricked with a needle, however, it contracts just as often as it is stimulated mechanically. It responds by a single contraction to a single stimulus, but a single stimulus does not excite a series of rhythmical con- tractions. (b.) With a sharp pair of scissors divide the auricles with the attached portion of the ventricle longitudinally. Each half continues to contract spontaneously, although it is possible that the rhythm may not be the same in both. 6. Movements of the Heart. — Expose the heart of a freshly pithed frog as directed in Lesson XLIV., 1, or better still, MOVEMENTS OF THE HEART. 211 destroy only the brain and then curarise the frog. After dividing the pericardium and exposing the heart, observe (a.) That the two auricles contract synchronously and force their blood into the ventricle, which from being pale and flaccid becomes red, turgid, and distended with blood. (b.) That immediately the ventricle suddenly contracts, and forces the blood into the bulbus aortas, at the same time becoming pale, while its apex is tilted forwards and upwards. As the auricles continue to fill during the systole of the ventricle, on superficial observation it might seem as if the blood were driven to and fro between the auricles and ventricle, but careful observation will soon satisfy one that this is not the case. Observe very carefully how the position of the auriculo- ventricular groove varies during the several phases of cardiac activity. (c.) The slight contraction of the bulbus aortre immedi- ately following the ventricular systole. (d.} The diastolic phase or pause when the whole heart is at rest before the auricles begin to contract. (e.) Ligature the frsenum and divide it, tilt up the ven- tricle by the ligature attached to the frsenum, and observe the sinus venosus. The peristaltic wave, or wave of con- traction, begins at the upper end of the vena cava inferior and sinus venosus; it extends to the auricles, which contract, then follows the ventricular systole and that of the bulbus aortse, and finally, the pause, when the whole sequence of events begins again with the systole of the sinus. (/) Before the ventricular systole is complete the sinus is full, while the auricles are filling. 212 EXPERIMENTAL PHYSIOLOGY. LESSON XLV. GRAPHIC RECORD OF THE FROG'S HEART — EFFECT OF TEMPERATURE. 1. Graphic Record of the Contracting Frog's Heart. (a.) Pith a frog, or destroy its brain, and then curarise it. Expose the heart, still within its pericardium, and arrange a heart-lever, so that it rests lightly on the pericardium over the beating heart. Adjust the lever to write on a revolving cylinder, moving at a suitable rate (5-6 cm. per second). Take a tracing of the beating of the heart. (b.) A suitable heart-lever is easily made with a straw about 12 inches long, or a thin strip of wood about the same length. Thrust a needle transversely either through the straw or wood, or through a piece of cork slipped over the straw about 2 inches from one end of the lever. The needle forms the fulcrum of the lever, and works in bearings, whose height can be adjusted. To the end of the lever nearest this is attached at right angles a needle with a small piece of cork on its free end. The lever is so adjusted that the cork on the needle rests on the heart. The long arm of the lever is provided with a writing-style of copperfoil, or a writing point made of parchment paper, fixed to it with sealing-wax. By using a long lever a sufficient excursion is obtained. (c.) Open the pericardium, expose the heart, and adjust the cork on the lever. To obtain a good tracing, it is well to put some resistant body behind the heart. Raise up the ventricle, ligature the frsenum, and divide the latter outside the ligature, and behind the heart place a pad of blotting- paper moistened with normal saline, or a thin cover slip. Adjust the lever, with its cork pad, on the junction of the auricles and ventricle, to write on the cylinder, moving at a slow rate (5-6 cm. per second), and take a tracing, noting the rise and fall of the lever. EFFECT OF TEMPERATURES ON THE EXCISED HEART. 213 (d.) Fix the tracings, and observe in the tracing a first ascent due to the auricular contraction, and succeeding this a second ascent due to the contraction of the ventricle, fol- lowed by a slow subsidence due to the continuation of the ventricular systole, and then a sudden descent due to the diastolic relaxation of the heart. 2. Auricular Contraction. — Adjust the lever again so that it rests on the auricles alone, and take a tracing. Note the smaller excursion of the lever. In this case the cork resting on the auricles must be small. 3. Ventricular Contraction. — Adjust the lever so as to obtain a tracing of the ventricular movements only. 4. In all the above experiments arrange an electro-magnetic time-marker (Fig. 67) under the recording lever, so that the points of the recording lever and time-marker write exactly in the same vertical line with each other. In this way one can calculate the time-relations of any part of the curve. The time- marker is arranged to record seconds, and is driven by an electric clock. 5. Effect of Varying Temperatures on the Excised Heart. (a.) Excise the heart of a pithed frog, lay it on a cylindrical brass cooling-box, three inches long and one broad, fixed to a support, and fitted with an inlet and outlet tube, like that in Fig. 62. Fix india-rubber tubes to the inlet and outlet tubes of the cooling-box, the inlet tube passing from a funnel fixed in a stand above the box, and the outlet tube discharging into a vessel below it. Adjust the heart lever to record the movements of the contracting ventricle on a slowly revolving drum. If the heart tends to become dry, moisten it with normal saline mixed with blood. Adjust a time-marker, as indicated for other experiments. Take a tracing. (6.) Pass water from 10° to 20° 0. through the cooling-box, noting the effect on the number of the contractions, and the duration, height, and form of each single beat. 214 EXPERIMENTAL PHYSIOLOGY. LESSON XLVI. STANNIUS'S EXPERIMENT AND INTRA- CARDIAC INHIBITORY MOTOR CENTRE. 1. Stannius's Experiment. — Pith a frog, and expose its heart in the usual way. (a.) With a seeker clear the two aortue from the auricles, and with an aneurism needle pass a moist stout thread be- tween the two aortae and the superior vense cavse, turn up the apex of the heart, divide the frsenum, and raise up the whole heart to expose its posterior surface, and the crescent or line of junction of the sinus venosus and the right auricle. Bring the two ends of the thread ligature round the heart — call this for convenience No. 1 ligature — tie them, and tighten the ligature just over the "crescent," so as to constrict the line of junction of the sinus venosus with the right auricle. Before tightening the ligature, observe that the heart is beating freely. On tightening the ligature, the auricles and ventricle cease to beat, and remain in a state of relaxation, while the sinus venosus continues to beat at the same rate as before. After a time, if left to itself, the ven- tricle begins to beat, but with an altered rhythm. If the relaxed ventricle be pricked, it executes a single contraction. (b.) When the heart is still relaxed, take a second ligature (No. 2), and preferably of a different colour to distinguish it from No. 1, place it round the heart, and tighten it over the auriculo-ventricular groove, so as to separate the ventricle from the auricles. Immediately the ventricle begins to beat again, while the auricles remain relaxed or in diastole. (c.) Instead of applying No. 2 ligature, the ventricle may be cut off from the auricles by means of a pair of scissors. Immediately after it is amputated, the ventricle begins to beat. 2. Intra- Cardiac Inhibitory Centre. ^ (a.) Expose the heart in a pithed frog, tie a fine silk ligature round the framum, and divide the latter between CARDIAC VAGUS OF THE FROG. 215 the ligatured spot and the pericardium. Gently raise the whole heart upwards to expose the somewhat whitish V-shaped "crescent" between the sinus venosus and the right auricle. (b.) Prepare previously an induction machine arranged to give an interrupted current. Place the electrodes — which must be fine, and their points not too far apart (2 milli- metres)— upon the crescent, and faradise it for a second ; if the current be sufficiently strong, the auricles and ventricle cease to beat for a time, but they begin to beat even in spite of continued stimulation. (c.) Stimulate the auricles, there is no inhibition or arrest. (d.) If a drop of solution of sulphate of atropia (Lesson XL VIII. , 1) be applied to the heart, stimulation of the crescent no longer arrests the action of the heart, for the atropine paralyses the inhibitory fibres of the vagus. 3. Seat of the Motor Centres. (a.) Expose a pithed frog's heart, cut out the ventricle with the auricles attached to it, and observe that the heart continues to beat. Divide the ventricle vertically by two parallel cuts into three portions. The middle portion con- tains the auricular septum, in which lie ganglioiiic cells. It continues to beat while the right and left lateral parts do not beat spontaneously, but respond by means of a single contraction if they are stimulated. LESSON XLVII. CARDIAC VAGUS AND SYMPATHETIC OF THE FROG AND THEIR STIMULATION. 1. Cardiac Vagus of the Frog — How to Expose it. — In this case a preliminary dissection must be made before the student attempts to stimulate the vagus. (a.) Pith a frog, or destroy its brain and curarise it. Lay it on its back on a frog-plate. Expose the heart, remove EXPERIMENTAL PHYSIOLOGY. the sternum, and pull the fore-legs well apart. Introduce a small test-tube, or stick of sealing-wax into the oesophagus, to distend it; the nerves leaving the cranium are better seen winding round from behind when the oesophagus is dis- tended. Remove the muscles covering the petrohyoid muscles which reach from the petrous bone to the posterior horn of the hyoid bone (Fig. 98). Three nerves are seen coursing round the pharynx parallel to these muscles. The lowest is the hypoglossal (H), easily recognised by tracing it forward to the tongue, above it is the vagus in close relation sai GP--- Fig. 98. —Scheme of LU, lung; Laryngeal HB, hyoid ; HG, hyoglossus ; H, heart ; BR, brachial plexus. with a blood-vessel (V), and still further forward is the glosso- pharyngeal (GP). Observe the laryngeal branch of the vagus (L). The vagus, as here exposed outside the cranium, is really the vago-sympathetic. The glossopharyngeal and vagus leave the cranium through the same foramen in the ex-occipital bone, and through the same foramen the sympa- thetic enters the skull. STIMULATION OF THE CARDIAC VAGUS. '217 2. Stimulation of the Cardiac Vagus. (a.) Adjust a heart-lever so as to record the contractions of the heart on a revolving drum moving at a very slow rate. (b.) Place well-insulated electrodes under the trunk of the vagus, stimulate it with an interrupted current, and observe that the whole of the heart is arrested in diastole. Although the faradisation is continued the heart recom- mences beating. The arrest, or period of inhibition, is manifest in the curve by the lever recording merely a straight line. If Heart Beat. Time in Sees. Stimulation Fig. 99. — Vagus Curve of Frog's Heart. the laryngeal muscles contract, and thereby affect the posi- tion of the heart, divide the laryngeal branch of the vagus. (c.) Note that when the heart begins to beat again, the beats are small at first and gradually rise to normal. In some instances, however, they are more vigorous and quicker (Fig. 99). 3. Determine the Latent Period. — For this purpose a time- marker and an arrangement to indicate when the stimulus is thrown into the nerve are required. (a.) Arrange the heart-lever as before, and adjust a time- marker to write exactly under the hearfc-lever. (b.) Arrange an induction machine for an interrupted current, and keep Neefs hammer vibrating. Into the secondary circuit introduce an electro-magnet with a writing- lever attached to it; so adjust the electro-magnet that its writing-style writes exactly under the heart-lever, and arrange that when the writing-style on the electro-magnet is depressed — e.g., by means of a weight — the secondary circuit is short-circuited, so that no stimulus is sent along the electrodes under the trunk of the vagus. 218 EXPERIMENTAL PHYSIOLOGY. (c.) When all is ready lift the weight off the electro- magnet, whereby the secondary circuit is un-short-circuited, the electro-magnet lever rises up, records its movement 011 the cylinder, and at the same moment the induction shocks are sent through the vagus. Observe that the heart is not arrested immediately, but a certain time elapses — the latent period — usually about one beat of the heart (0-15 sec.) before the heart is arrested. ( perfusion, 220. record of, 212. reflex inhibition, 233. section of, 210. sounds of, 231. staircase of, 221. Stannius's experiment, 214. effect of swallowing on, 233. sympathetic on, 218. effect of temperature on, 210, 213. tonometer, 226. vagus on, 215. valves of, 229. Heat— Effect on cilia, 146. „ ,, muscle, 174. „ heart, 210. Heller's test, 109. Helmholtz's modification, 144. Hemialbumose, 53, 58. Heywood's experiment, 250. Hippuric acid, 104. 20 306 INDEX. Holmgren's worsted, 237. Hydrocele fluid, 26. Hydrochinon, 78. Hydrostatic test, 249. Hypobromite method, 97. Illusions, 300. . Image— Formation of, 263. Indican, 106. Indigo carmine, 1 1 6. Indol, 58. Induced electricity, 139. Induction machine, 139. Inhibition, 262. „ reflex, 233. Interrupted shocks, 143. Intrathoracic pressure, 248. Inversion of sugar, 14. Irradiation, 27t). Judgment of distance, 281. Key — Du Bois-Reymond's, 133. mercury, 135. Morse, 135. plug, 135. spring or contact, 136. trigger, 135, 168. Knee-jerk, 258. Kcenig's flames, 254. Kiihne's experiments, 197, 207. „ eye, 284. ,, pancreas powder, 57. Kymograph, 241. Lactic acid, 55. Lactoscope, 73. Lactose, 14, 129. Lambert's method, 286. Laryngoscope, 251. Laurent's polarimeter, 15. Legal's test, 1 19. Leucin, 58, 122. Lieben's test, 119. Lieberkiihn's jelly, 7. Liebermann's reaction, 2. Liquor pancreaticus, 56. ,," pepticus, 55. Liver, 67. „ dead, 69. Locality, sense of, 297. Lymph-hearts, 246. Malt extract, 50. Maltose, 14, 49, 50. Marriotte's experiment, 272. Maximal contraction, 166. Maxwell's experiment, 275. Mechanical stimulation, 160. Meiocardia, 232. Mercurial key, 135. Metaphosphoric acid, 110. Methsemoglobin, 36. Methyl violet, 55. Metronome, 183, 256. Milk, 54, 69, 71, 72. ,, curdling ferment, 60. ,, digestion of, 54. ,, to peptouise, 61. ,, rennet on, 55. Millon's reagent, 2. Minimal contraction, 166. Moist chamber, 163. Moore's test, 115. Morse key, 135. Mucin, 10, 62. Mulberry calculus, 125. Mailer's valves, 250. Murexide test, 101. Muscae volitantes, 276. Muscarin, 219. Muscle — Action current of, 192. action of heat, 174, ,, veratria, 175. curve of, 168. demarcation current, 1-89. direct stimulation of, 156. effect of load, 175. ,, two shocks, 184. elasticity, 176, electrical stimulation, 152. exhaustion of, 180. extensibility of, 177. extractives of, 77. extracts of, 76. fatigue, 178. independent excitability of, 157. indirect stimulation of, 157. reaction of, 75. rupturing strain, 157. single contraction, 164. sound, 157. stimulation of, 150. tetanus, 181. thickening of, 183. work of, 167. INDEX. 307 Muscular sense, 300. Myographs — Crank, 167. ,, pendulum, 168. ,, Pfluger's, 179. ,, spring, 170. Myosin, 76. Native albumins, 4. Near point, 267. Negative variation, 192. Nerves — Action current, 193. demarcation current, 192. double conduction, 207. exhaustion of, 180. roots of, 258. unequal excitability, 206. velocity of energy, 204. Nerve-muscle preparation, 146. Neuramoebometer, 260. Nitrites, 36. Non-polarisable electrodes, 188. Normal saline, 147. Oils, chemistry of, 17. Ophthalmoscope, 295. Organic acids, 55. Organic substances — Examination of, 128. Ossein, 17. Oxalate of lime, 125. Oxy-hsemoglobin, 30. ,, crystals of, 29. Pancreatic juice, 56. Paradoxical contraction, 196. Paraglobulin, 24. Parapeptones, 53. Pea meal, 75. Pendulum myograph, 168. Peripheral projection, 299. Perrin's eye, 297. Pepsin, 52. Peptic digestion, 52. Peptones, 9, 53. Peptonuria, 110. Perception of size, 283. Perimetry, 278. Pettenkofer's test, 63, 114. Pfluger's law, 201. Phakoscope, 269. Phenol, 119. Phosphates, 19. ,, alkaline, 85. Phosphates, in milk, 72. ,, in urine, 84. ,, volumetric process for, 88. Phosphenes, 275. Phosphoric acid, 88. Picric acid, 109, 116. Picro-saccharimeter, 118. Pithing, 145. Piston-recorder, 224. Plasma of blood, 21, 22. Plethysmograph, 236. Plug key, 135. Pohl's commutator, 138, 161. Poisons on heart, 215. ,, on muscle, 186. ,, on spinal cord, 257. Polar imeter, 16. Polarisation of electrodes, 141. Polariscope, 11. Potassic chloride, 258. Potassic sulphocyanide, 48. Potassio-mercuric iodide, 68. Pressure, sense of, 299. Proteids— Coagulated, 8. ,, exercises on, 19. Psychodometer, 260. Ptyalin, 48. Purkinje's figures, 276. Purkinje-Sanson's images, 268. Pulse, 233. Pulse-wave, 237. Pus, 120. Pyuria, 120. Pyrocatechin, 120. Quantitative estimation of phos- phates, 88. „ sugar, 117, 118. „ urea, 94, 97. Radial movement, 283. Ragona Scina, 291. Reaction — Adamkiewicz, 2. Biuret, 2, 93. ,, Liebermann, 2. ,, Uffelmann's, 56. ,, Xanthoproteic, 1. Reaction time, 260. Recording apparatus, 162. Reflex action, 255. Rennet, 55. ,, ferment, 72. 308 INDEX. Respiratory movements, 247. Retinal shadows, 276. Reverser, 139. Revolving cylinder, 172. Rheochord, 136. Rheometer, 240. Rheonom, 155. Rheoscopic frog, 195. Rigor mortis, 76. Ringer's fluid, 223. Ritter's tetanus, 203. RosenthaTs modification, 160. Roy's tonometer, 226. Rupturing strain, 157. Saccharimeter, 117. .Saliva, 47. Saponification, 17. Sartorius, 154. Schafer's piston-recorder, 224. Schemer's experiment, 266, 285. Schin"s test, 102. Secondary contraction, 195, 196. ,, tetanus, 195. Serum, 24. Serum-albumin, 4, 25. ,, globulin, 5, 25. Shadows on retina, 277. ,, coloured, 291. Shunt, 189. Single contraction, 164. Single induction shocks, 142. Smell, 301. Soap, 18. Specific rotation, 15. Spectroscope, 30. Sphygmograph, 233, 234. Sphygmoscope, 236. Spinal nerve roots, 258. Spirometer, 249. Spring key, 136. ,, interrupter, 183. ,, myograph, 170. Squibb's apparatus, 99. Staircase, 221. Stannius's experiment, 214. Starch, 11. Stereoscope, 294. Stethograph, 247. Stethometer, 247. Stethoscope, 231. Stimuli, 150. Stokes's fluid, 33. Strasburg's test, 63, 114. Straw flag, 151. Strobic discs, 283. Struggle of fields of vision, 294. Strychnia, 257. Successive light induction, 287. Sulphocyanide, 48. Sulphur test for bile, 64. Superposition of shocks, 184. Swallowing, 233. Sympathetic in the frog, 218. Syntonin, 7, 64. Talbot's law, 277. Tambour, 184. Taste, 301. Taurocholic acid, 62, 114. Telephone, 156, 302. Temperature, sense of, 298. Tendon to rupture, 157. Test-types, 271. Tetanus, 181. Thermal stimulation, 161. Thomson's reverser, 139. Time measurements of muscle, 170. Time-recorder, 173. Tonometer, 226. Touch, 297. ,, illusions of, 298. Trigger-key, 135. Triple phosphate, 87. Trommer's test, 14, 115. Tropasolm, 55. Trypsin, 57. Tyrosin, 58, 59. Tubes, rigid and elastic, 238. Turck's method, 257. Twitch, 164. Uffelmann's reaction, 56. Unipolar stimulation, 154. Urates, 102. Urea, 90. ,, nitrate, 91. ,, oxalate, 92. ,, volumetric analysis, 94. Uric acid, 99. Urinary calculi, 122. „ deposits, 120. Urine, 77. „ albumin in, 108. ,, bile in, 113. ,, blood in, 112. INDEX. 309 Urine, chlorides, 84. colour, 78. colouring-matters, 10G. fermentations of, 82. general examination of, 126. inorganic bodies, 83. mucus in, 107. odour, 80. organic bodies, 90. phosphates in, 84. pus in, 120. quantity, 78. reaction, 80. specific gravity, 78. sulphates in, 84. urea in, 90. uric acid in, 99. Urinometer, 79. Urobilin, 106. Urochrom, 106. VagUS in frog, 215, 217. ,, in rabbit, 243. Varnish, 164. Vascular tonus, 222. Veratria, 175. Vision — Physiology of, 261. Visual judgments, 281. Vital capacity, 249. Vogel's lactoscope, 73. Volumetric processes, 87. „ ,, Dupre's apparatus, 97. ,, ,, hypobromite me- thod, 97. ,, ,, for phosphoric acid, 88. ,, ,, Steele's apparatus, 98. ,, ,, for sugar, 117. ,, ,, for urea, 94. Von Wittich's method, 51. Vowel sounds, 254. Wave-lengths, 37. Weyl's test, 106. Whistle— Galton's, 302. White of egg, 1. Wild's apparatus, 185. Xanthin, 124. Xanthoproteic reaction, 1. Zollner's lines, 281. A Selection from Charles Griffin & Company's Catalogue. Professors LANDOIS and STIRLING. HUMAN PH YS I O LOGY (A TEXT-BOOK OF): INCLUDING HISTOLOGY AND MICROSCOPICAL ANATOMY; WITH SPECIAL REFERENCE TO PRACTICAL MEDICINE, BY DR. L. LANDOIS, PROP. OP PHYSIOLOGY, UNIVERSITY OF. GREIFSWALD. ^Translate* from tbe ffiftb German EMtion, witb annotations ant> Btoitfons, BY WM. STIRLING, M.D., Sc.D., BRACKENBCRY PROFESSOR OF PHYSIOLOGY IN OWENS COLLEGE AND VICTORIA UNIVERSITY, MANCHESTER; EXAMINER IN THE HONOURS SCHOOL OF SCIENCE, OXFORD. With very numerous Illustrations. Second English Edition. In Two Vols., Royal 8vo, Handsome Cloth. Price 42s. PART I.— Physiology of the Blood, Circulation, Respiration, Digestion, Absorption, Animal Heat, Metabolic Phenomena of the Body. 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Seventh Edition. Octavo. In Press. " I have become thoroughly convinced of its great value, and have cordially recommended it to my class in Yale College." — Prof. David P. Smith. " I have examined it with some care, and think it a good book, and shall take pleasure in mentioning it among the works which may properly be put in the hands of students." — A. B. Palmer, Prof, of the Practice of Medicine, University of Michigan. Aitken's Practice of Medicine. Seventh Edition. 196 Illus- trations. 2 vols. Cloth, 12.00; Leather, 14.00 Tanner's Index of Diseases, and Their Treatment. Cloth, 3.00 "This work has won for itself a reputation. ... It is, in truth, what its Title indicates."— ./V. y. Medical Record. PRESCRIPTION BOOKS. Wythe's Dose and Symptom Book. Containing the Doses and Uses of all the principal Articles of the Materia Medica, etc. Seventeenth Edition. Completely Revised and Rewritten. Just Ready. 321110. 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" Worthy its distinguished author in every respect ; a work whose practical value commends it not only to the practitioner and stu- dent of medicine, but also to the dermatologist." — -J'lines Nevens Hyde, M.D., Prof, of Skin and Venereal Diseases, Rush Medical College, Chicago. Van Harlingen on Skin Diseases. A Handbook of the Dis- eases of the Skin, their Diagnosis and Treatment. By Arthur Van Harlingen, M.D., Prof, of Diseases of the Skin in the Phila- delphia Polyclinic; Consulting Physician to the Dispensary for Skin Diseases, etc. With colored plates. i2mo. Cloth, 1.75 *** This is a complete epitome of skin diseases, arranged in alphabetical order, giving the diagnosis and treatment in a concise, practical way. Bulkley. The Skin in Health and Disease. By L. Duncan Bulkley, Physician to the N. Y. Hospital. Illus. Cloth, .50 SURGERY. Heath's Minor Surgery, and Bandaging. Eighth Edition. 142 Illustrations. 60 Formulae and Diet Lists. Cloth, 2.00 Pye's Surgical Handicraft. 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Cloth, 1.75 Holland. The Urine. Chemical and Microscopical, for Labo- ratory Use. Illustrated. Cloth, .50 Ralfe. Kidney Diseases and Urinary Derangements. 42 Illus- trations. i2mo. 572 pages. Cloth, 2.75 Legg. On the Urine. A Practical Guide. 6th Ed. Cloth, .75 Marshall and Smith. On the Urine. The Chemical Analysis of the Urine. By John Marshall, M.D., Chemical Laboratory,Univ. of Penna ; and Prof. E. F. Smith, PH.D. Col. Plates. Cloth, i.oo Thompson. Diseases of the Urinary Organs. Seventh - Edition. Illustrated. Cloth, 1.25 Tyson. On the Urine. A Practical Guide to the Examination of Urine. By James Tyson, M.D., Professor of Pathology and Morbid Anatomy, University of Penn'a. With Colored Plates and Wood Engravings, sth Ed. Enlarged. 12010. Cloth, 1.50 VENEREAL DISEASES. Hill and Cooper. Student's Manual of Venereal Diseases, with Formulae. Fourth Edition. i2mo. Cloth, i.oo Durkee. On Gonorrhoea and Syphilis. Illus. Cloth, 3.50 ee pages 2 to 5 for list of ? Quiz- Compends ? MEDICAL BRIEFS. A new series of short, concise compends for the Med- ical Student and Practitioner. tamo. Cloth. Price of Each Book, $1.00. No. i. POST-MORTEM EXAMINATIONS. With Especial Reference to Medico- Legal Practice. By Prof. RUDOLPH VIRCHOW, of Berlin Charite Hos- pital, author of Cellular Pathology ; Translated by T. P. SMITH, M.D., Member of the Royal College of Sur- geons of England. 2d American, from the 4th German Edition. With new Plates. Illustrated by Four Lith- ographs. " We are informed in precise and exact terms how a post-mortem examination should be made, both with regard to the plan to be pursued, and the manner of making the several cuts into the various organs and tissues. 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" I have examined it with care, and find it to be a practical and useful compendium of knowledge on the subjects discussed, well adapted to the use of medical students and those physicians in general practice who have occasional need to consult a work of this kind."— James Neven Hyde, M.D., Professor of Skin and Venereal Diseases, Rush Medical College, Chicago. No. 3. MEDICAL ELECTRICITY. A Com- pend of Electricity and its Medical and Surgical Uses. By CHAS. F. MASON, M.D., Ass't Surg. U. S. Army ; with an introduction by CHARLES H. MAY, M.D., Instructor in Ophthalmology, New York Polyclinic. Illustrated. Just Ready. OTHER VOLUMES IN PREPARATION. Price of Each Book, bound in Cloth, $1.00. The latest, cheapest, most compact and practical series of text- books published. May be used by students of any College. A New Series of Manuals FOR Medical Students, Price of each Book, Cloth, $3.00 ; Leather, $3.50. PRACTICAL SURGERY. By WM. J. WALSHAM, M.D., Asst. Surg. to, and Dem. of Surgery in, St. Bartholomew's Hospital. 256 Illustrations. DISEASES OF 'WOMEN. By Dr. F. WINCKEL, Prof. Royal University of Munich. The Translation Edited by THEOPHI- LUS PARVIN, M.D., Prof, of Obstetrics and Dis. of Women and Children, Jefferson Medical College, Phila. 117 Engravings. PHYSIOLOGY. By GERALD F. YEO, M.D. Prof, of Physiology King's College, London, ad Edition, revised. 301 Illus. MATERIA MEDICA, PHARMACY AND THERAPEU- TICS, including the Physiological Action of Drugs, Special Thera., Official and Extemporaneous Phar., with Tables, For- mulae, Notes on Temperature, Clinical Thermometer, Poisons, Urinary Exam, and Patent Meds. Over 600 prescriptions and formulae. By S. O. L. POTTER, M.D., Prof, of Practice of Medi- cine, Cooper Coll., San Francisco, late A. A. Surg. U. S. A. MIDWIFERY. By A. L. GALABIN, M.D., Lecturer on Midwifery and Dis. of Women, Guy's Hospital. T.ondon. 227 Illustrations. CHILDREN. By J. F. GOODHART, M.D.. 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