tTT/. QlPA-4 COLUMBIA UNIVERSITY DEPARTMF;Nr OF PHYSIOLOGY THE JOHN G. CURTIS LIBRARY "W VUOIOCV outlines""" PRACTICAL PHYSIOLOGY: /IDanual for tbe pb^^siolooical Xaboratori^ INCLUDING CHEMICAL AND EXPERIMENTAL PHYSIOLOGY, WITH REFERENCE TO PRACTICAL MEDICINE. WILLIAM STIRLING, M.D., Sc.D., PROFESSOR IN THE VICTORIA DKIVERSITT, BRACKENBrRT PROFESSOR OP PHYSIOLOGY AFD HISTOLOGY IN THE OWENS COLLEGE, MANCHESTER, AND EXAMINER IN PHYSIOLOGY IN THE TJNITEBSITY OP OXFORD. With 14*2 Illustrations. CHARLES GRIFFIN & CO., LONDON. PHILADELPHIA: P. BLAKISTON, SON & CO., No. 1012 WALNUT STREET. 1888. (All Rights Reserved.) \\l M Digitized by the Internet Archive in 2010 with funding from Columbia University Libraries http://www.archive.org/details/outlinesofpracti1888stir BcMcateC) MY REVERED AND BELOVED MASTER, CARL LUDWIG. PREFACE. The present work was written to sup])ly 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 ho was str-uck 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 difl'erent 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 Lehrhuch 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-Book of Physiology by Landois and Stirling. WM. STIRLING. The Owens College, Manchester, January, 1888. CONTENTS. LESSON I. u. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. PART I.— CHEMICAL PHYSIOLOGY. PAGES The Proteids and Albumenoids, . . . . . 1-10 The Carbohydrates, Fats, Bone, 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 Hamoglobin, ...... 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, 120-127 Appendix, 127-129 PART II.— EXPERIMENTAL PHY^SIOLOGY. XXII. Galvanic Batteries and Galvanoscope, . . . 130-133 XXIII. Electrical Keys, Rheochord, 133-139 XXIV. Induction Machine, Electrodes, 139-142 XXV. Single Induction Shocks, Interrupted Current, Break Extra-Current, Helmholtz's Modification, . . 142-144 XXVI. Pithing, Ciliary Motion, Nerve-Muscle Preparation, Normal Saline, ....... 145-147 XXVII. Nerve-Muscle Preparation, Stimulation of Nerve, . 148-152 CONTENTS. LHSSOK rAi,B,3 XXVIII. Single and Interrupted Induction Shocks, Tetanus, Constant Current, 152 155 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-lGl XXXI. Graphic Method, Moist- Chamber, Single Contrac- tion, Work Done, 162-168 XXXII. Muscle Curve, Pendulum and Sirring Myographs, Time Marker, Signal, 16S-173 XXXIII. Influence of Temperfa.ture, Load, Veratria on Mus- cular Contraction, ...... 17-1-176 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 Shock.s, Action of Drugs on Excised Muscle, 184-186 XXXVIII. Galvanometer, N.P. Electrodes, Shunt, Muscle Currents, 186-192 XXXIX. Nerve and Heart Currents, Capdlary Electrometer, . 192-194 XL. Galvani's Experiments, Secondary and Paradoxical Contraction, Kiihne's Experiment, . . . 194-197 XLI. Electrotonus, 198-201 XLII. Electrotonus, Pfliiger's Law, Pvitter's Tetanu.s, . . 201-20.3 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. Stamiius'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 Pi-essure, Apex Preparation, . . . 224-226 LL Tonometer, Gaskell's Clamp, 226-229 LII. Valves of Heart, Stethoscope, Cardiograph. ]Meio- cardia, Auxocardia, Retlex Inhil)ition, . . 229-233 LIII. Pulse, Sphygmograph, Sphygmoscope, Plethysmo- graph .' . 233-236 CONTENTS. XI LESSON LIV. LV. LVI. LVII. LVIII. LIX. LX. LXI. LXII. LXIII. LXIV. LXV. Pulse-Wave, Rigid and Elastic Tubes, Schema, Rheometer, ....... Blood- Pressure, Kymograph, Lymph-Hearts, Chest Movements, Elasticity of Lungs, Hydrostatic Test, Vital Capacity, Carbonic Acid Excreted, Heywood's Experiment, Laryngoscope, Vowel Sounds, Reflex Action, Poisons, Knee-Jerk, .... Fmictions of Nerve Roots, Reaction Time, Formation of an Image, Aberration, Accommodation, Scheiner's Experiment, Near and Far Points, Pur- kinje's Images, Phakoscope, Astigmatism, Blind Spot, Direct Vision, Maxwell's Experiment, Phos- phenes, Retinal Shadows, Duration of Impression, Talbot's Law, ........ Perimetry, Irradiation, Imperfect Visual Judgments, Artificial Eye, Colour - Sensations, Colour -Blindness, Contrast, After-images, Haploscope, Ophthalmoscope, ........ Touch, Smell, Taste, Hearing, ..... 237-241 241-246 247-249 249-254 255-258 258-262 2iJ;J-27l 272-277 278-283 284-294 295-297 297-302 LIST OF ILLUSTEATIONS. FlC. PACE 1. Starch, 11 2. Hajmocytometer (Goicers), ....... 28 3. Spectroscoije {Broioning), ........ 30 4. Hamoglobin Spectra, ........ 32 5. Blood Spectra, .......... .35 6. Spectroscope for W.L., ........ 38 7. Ha^min Crystals, 41 8. Hajmoglobiuometer (Gowers), ....... 44 9. Hajmometer [v. Fleischl), ........ 45 10. Kiilme's Dialyser {Krukenberg'), 54 11. Cholesterin, .......... 65 12. Milk iStlrllng), 70 13. Porous Cell (Stir/ing), 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. Urea Oxalate, 92 23. Decomposition of Urea, ........ 93 24. Steele's Apparatus ((?. 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 (Jo/iM-sort), . . . . . . .118 31. Cystin and Oxalates, ........ 122 32. Leucin and Ty rosin, . . . . . . . . .123 33. Daniell's Cell (Stirling), 130 XIV LIST OF ILLUSTRATIONS. 34. Grove's Cell, 35. Bichromate Cell, 36. Detector {Elliott), 37. Du Bois Key, 38. Scheme of 37 {Stirlhig), 39. 40. Plug Key, 41. Morse Key {Stewart and Gee), 42. Spring Key {Elliott), . 43. Bheochord {S/irllng), 44. Rheochord, 45. lleverser {ElJh>tt), 46. Induction Coil, . 47. T)u Bois Electrodes, 48. Break Extra- Current {Stirling), 49. Helmholtz's Modification, 50. Frog's Leg-Muscles {Ecker), 51. Frog's Sciatic Nerve {Ecker), 52. Nerve-Muscle Preparation, 53. Straw-Flag {Stirling), 54. Scheme for Single Induction Shocks {Stirling), 55. Scheme of Constant Current {Stirling), 56. Frog's Sartorius {Ecker), 57. Rheonom {Stirling), 58. Curare Experiment, 59. Pohl's Commutator {Elliott), 60. Revolving Cylinder, 61. Moist Chamber (Stirling), . 62. Crank Myograph (Stirling), 63. Pendulum Myograph, 64. Pendulum Myograph Curve (Stirling), 65. Spring Myograph, 66. Revolving Drum, 67. Time Marker (Stirling), 68. Electric Signal (Stirling), 69. Muscle Curve with Load (Stirling) 70. Veratria Curve (Stirling), 71. Fatigue Curve (iS'/irS«(7), 72. Pfliiger's Myograph, . 73. Scheme for Tetanus (Stirling), 74. Tetanus Curves (Stirling), . 75. Tetanus Interrupter (Stirling), LIST OF ILLUSTRATIONS. XV nc. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 109a 110. 111. 112. 113. 114. 115. 116. Marey's Tambour, Wild's Apparatus (Stirliny), Galvanometer, .... Lamp aucl Scale, N.P. Electrodes, Sliuut, ..... Apparatus for Muscle Current (Sfiii Nerve N.P. Electrodes, Galvani's Experiment {StirUng), Secondary Contraction, Scheme of 85 (Stirlhuj), Paradoxical Contraction {Stlrllnrj), Kiihue's Experiment (StirUng), Electrotonus (Stirling), Electrotonns, Scheme of 90 (StirUng), Pfliiger's Law (Stirling), V'elocity of Nerve Energy, Unequal Excitability of a Nerve (Stir Kiihne's Experiment (Kilhnc), Frog's Heart, Frog's Vagus (Stirling), Vagus Curve (Stirling), Frog's Sympathetic (after Gaslnell), Frog's Heart Support (Stirling), Staircase of the Heart, Double Cannula, Heart Apparatus, Tonometer, .... Gaskell's Clamp (StirUng), Cardiograph, .... Marey's Sphygmograph, Dudgeon's Sphygmograph (Maiv Son <& Thompson), .Ludwig's Sphygmograph (Petzoldt), Sphygmoscope (Bothc) Pvheometer, Kries's Apparatus, . Lymph-Hearts. Stethograph, Miiller's Valves {Stirling), Hey wood's Experiment (Stirliiig), /), ling) PA6B 184 185 187 188 189 189 190 193 194 195 196 196 197 198 200 200 202 204 206 207 209 209 216 217 219 220 221 223 225 226 228 232 234 235 235 236 240 245 245 248 250 251 XVI LIST OF ILLUSTRATIONS. FIG. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 7. View of the Larj^nx, Koenig's Apparatus, . Neuramcebometer (after Obersteiner] Schemer's Experiment, DifiFusion (HebnhoUz), Phakoscope, .... Marriotte's Experiment, . Blind Spot (HelmJioltz), . Bergmann's Experiment (Helmliollz Disc for Talbofs Law (Hdmhollz), Perimeter, Irradiation, [Helmholtz), . Imperfect Visual Judgment, Zbllner's Lines, . Perception of Size [Hel'mlioltz), Spiral Disc for Contrast, (Helmholtz), Kiihne's Eye [Jung), Partly Coloured Disc for Contrast {Helmholtz), Rotatory Disc [Helmholtz), R. Scina's Experiment (Rood), Carriage for Rabbit (Stirling), Aristotle's Experiment, PAGE 253 253 254 260 266 267 269 272 272 273 275 277 278 280 280 280 281 281 282 283 285 288 290 291 297 298 PART L— CHEMICAL PHYSIULUGY. LESSON I. THE PROTEIDS AND ALBUMENOIDS. Tlte White of Egg may be taken as the Type. 1. Preparation of a Solution. — Place the white of an e^o 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 filters 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 difi'uses slowly through animal membranes. (a.) To some of the fluid in a test-tube add strong nitric acid = a precipitate, wdiich on being boiled turns yellow. Allow the liquid to cool, and add strong ammonia = an orange precipitate or colour (Xanthoproteic Reaction). (6.) To another portion add Millon's Reagent = a preci- pitate which Ijecomes 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 ditluse 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 difi'usible.) jV.B. — The reactions d, e, /, and ff are not obtained with peptones. Preparation of Millon's Reagent. — Dissolve mercury in its own weight of strong nitric acid, specific gravity 1 '4, and to the solution thus obtained add two vohimes 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 sohition occurs. Dihite with two volumes of watei', and decant the clear 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 lirst, and then strongly. After cooling, add care- fully 3 cc. of water to the NaCy 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 w^ater 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 " (Ganigee). 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 (O'l 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 C, 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° C. 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 I., 1. In addition, perform the following experiments : — (a.) Evaporate some of the fluid to dryness at 40° C. 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. (6.) 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 O'l 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 pi'ecipitated on saturation with sodic chloride or magnesic sulphate (compare " Globulins "). (2.) Serum- Albumin.— Dilute blood serum until it has the same specitic 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 : — I'ROTEIDS. EGG-ALBUMIN. {a.) Readily precipitated by hydrochloric acid, but the pre- cipitate is not readily soluble in excess. SERUM-ALBUMIN. [a. ) It is also precipitated by hydrochloric acid, but not so readily, while the precipitate is soluble 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. (6.) It is not coagulated by ether. (c.) The corresponding preci- pitate is much more soluble in excess of acid. ((/.) The corresponding preci- pitate is soluble in strong nitric acid. [(e.) When injected under the skin, or introduced in large quantities into the stomach or rectum, it is given off by the urine.] [(e.) "When injected under the 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 (O'l 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. {h.) 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. III. — 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. (6.) 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. (/.) Lieberkilhn'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 stitf 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 mu.scle, 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. (0.) 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. (b.) 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 fil>rin in a test-tube, add some 0"1 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 eflfect of a dilute acid and pepsin (see "Diges- tion "). (f/.) 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. (h.) 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. {(I.) 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 diltite 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). (/.) 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. (/i.) 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. Diifusibility of Peptones. — Place a solution of peptones in a dialyser covered with an animal membrane, as directed in Lesson I., 1, (/i.), 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. (6.) 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). (yi) 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 (OgH^o05)„ 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 concenti'ic markings become more distinct. (&.) At this stage it is advantageous to compare the microscoj^ic characters of other varieties of starch — e.g., rice, arrowroot, etc. (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 held is dark, each granule shows a dark cross on a white refractive ground. {(1.) Squeeze some dry starch powder between the thumb and forelinger, and note the peculiar crepitation sound and feeliner. 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 filtrate with iodine. It gives no blue colour. (b.) 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 (CgHioOg). 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 (h.) 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, GqH.^qO^. 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, CeH^^Og. 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. {b.) 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 Febling'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, OioHoPj^. (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. (6.) Test for a reducing sugar with Fehling's solution or other suitable test. (See also " Salivary digestion.") 8. VI.— Lactose, 0^2^22011 + H^O (see " Milk "). 9. VII.— Cane Sugar, Ci.HooOii. (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 loevulose. 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 a = 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 ; (0)0 = the specific rotation for light corresponding to the light of a sodium flame ; then The sign -t- 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. (6.) Place some common salt (or fused common salt, and sodic 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. (c7.) 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 CARliOHYDUATES. 17 in weight gives the amount of dextrose in 10 cc, ; so that the amount in 1 cc. is got at once = ;;. (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 1 1 '6° ""^ - -11 X 2 - ^"^ ^ 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 - 3.5-5°; serum-albumin - 56°; syn- tonin - 72° ; alkali-albumin prepared from serum-albumin — 86", ■wdien prepared from egg-albumin — 47°. Carbohydrates. — Glucose + 56°; maltose + 150°; lactose + 52-5^ 13. Reactions. NEUTRAL FATS. (a.) Use almond oil or lard, and observe that fat is soluble in ether, chloroform, and hot alcohol. (b). 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. 9 8 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 capaV)le 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 (CO.,) 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 filtrate 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.) Xote 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 filtrate of (a.) by acidulation and heat for albumin. Confirm by other tests. (c.) Test the filtrate of (b.) 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 hlood on a strip of dry, smooth, glazed, neutral litmus paper. Allow it to remain for a short time ; then wash it ofi" 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 0. 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 («.) at printed matter. (6.) To C add a solution of taurocholate of soda. Test its transparency as above. In 2, the htemoglobin is still within the blood corpuscles. In the others — 3 (a.), (h.) — ^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 0, It is opaque, 5. Haemoglobin does not Dialyse, {a.) Place a watery solution of deiibrinated 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 line pores, close them with a little white of egg, and coagulate it with a hot iron, (b.) After several hours observe that no hfemoglobin has passed into the water. (c) Test the difiusate 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 (O'To per cent, salt solution) in a test-tube sur- rounded Avith 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 huffy 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 ofi* 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. (6.) 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.), (a.), (h.), (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 thei'e is precipitated a small quantity of a somewhat slimy white body, fibrinogen. {h.) 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°0. for some hours, when coagulation occurs, a clear pellucid clot of fibrin being obtained. (6.) 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 witli 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 oo°G., 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. LESSOX IV. THE COLOURED BLOOD-CORPUSCLES. SPECTRA OF HEMOGLOBIN AND ITS COMPOUNDS. Enumeration of the Corpuscles. — Several forms of instruments are in use, e.g., those of Malassez, Zeiss, and Gowers. 1. The Hasmocytometer of Gowers' consist of (a.) A small pipette, which, when filled to the mark on its stem, holds 995 c.mm. (Fig. 2, A). (b.) 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 l of a millimetre deep, and with its floor divided into squares y\j mm., in which the blood-corpuscles are counted. (/.) The diluting solution consists of a solution of sodic sulphate in distilled water — specific gravity, 102.5. 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). 28 CHEMICAL PHYSIOLOGY. (b.) 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' Hremocytometer. — A, Pipette foi* measui-ing the diluting solution ; B, for measuring the blood ; C, cell with divisions on the floor, mounted on a slide, to which springs are flxed 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 (0), 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 on its base. (d.) When the corpuscles have subsided, count the number in 10 squares, and this, when multiplied by 10,000, "ives 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 ji^ mm., so that 10 squares have an area of Jjy mm. As the cell is ^ mm. deep, the volume of blood in 10 squares is tV ^ T = :^ c.mm. 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 Ijlood. On multiplying this by ^^5 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. HEMOGLOBIN AND ITS DERIVATIVES. 3. Preparation of Hgemoglobin Crystals. (a.) Rat's Blood.- — Place a drop of the defibrinated 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-htemoglobin, 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 Itlood is rendered lakj/, 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 ci'ystals under the microscope. 4. Ozone Test for Hgemoglobin. — ^Mix some freshly-prepared alcoholic solution of guaiacum with ozonic ether ; the mixture becomes turbid, and on adding even a dilute solution of htemo- 30 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. 8. — 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, h, 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. (&.) 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. {h.) Adjust the spectroscope as follows : — Light a fan-tailed gas-burner, fix the spectroscope in a suitable Iiolder, 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 Jong 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. (c?.) 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 apjyearances seen with each dilution, and indicate opposite each the degree of the latter. {/.) 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 uj^per 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 [Hoiype-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. A a B ( 4o mImhI 60 n.lnnlu, D 30 E 70 M 1 1 1 u 1 1 1 l\ : b F 80 00 1 1 n n ' M 1 J. M M 100 no III H 2 ill 1 j 1 1 4. nil 1 1 Fig. 4. — Spectra of Hemoglobin and its compounds. — 1, Oxy-hannoglobin 0*8 per cent.; 2, oxy-hannoglobin, 0'18 per cent.; 3, carbonic oxide ha-moglobin ; 4, reduced haemoglobin. 9. II. Reduced Hgemoglobin. («.) 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 2)urplish 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 Ijands, but gradually fades and dis- appears. (d.) To a similar solution of oxy-ha?nioglobin showino- two well-detined 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-hajmoglobin where the two bands can just he seen, and reduce it with either ammonium sulphide or Stokes's fluid, and note that, perhajDS, no absorp- tion band of reduced htemoglobin is to be seen, or only the faintest shadow of one. (/.) Compare the relative strengths of the solution of oxy- hsemoglobin and reduced hjemoglobin. 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-htemoglobin, or detibrinated 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 HbOg to show the aljsorption bands. Two absorption bands nearly in the same position as those of HbO.,, but very slightly nearer the violet end (Fig. 4, 3). Make a map of the spectrum and bands. {b.) The bands are not aflected 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- hiemoglobin 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-hfematin. (b.) 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-Hsematin 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 hsematin is obtained. (b.) 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-hsematin. {a.) Take a solution of acid-hfematin ; neutralise it with caustic soda until there is a precipitate of hjematin ; on THE COLOURED BLOOD-CORPUSCLES. 35 adding more soda, and heating gently, the precipitate is re- dissolved and alkali-ha?matin 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. ini i I nil Mil III im ill ill i|i.ui.i|ii.aiu r.u ulii h i mm iim iii u i jiLiil 4o 50 bo 70 80 qo 100 118 A a B C D E F Fig. 5. — Spectra of Derivatives of Ha?moglobin. — 5, Haematin in alcohol with sulphuric acid ; 6, haematin in an alkaline solution ; 7, reduced hjematin. (b.) Shake (a.) with air to obtain oxy-alkali-hsematin. Observe its spectrum, one absorption band just to the red side of the D line. It is much nearer D than that of acid- htematin (Fig. 5, 6). Much of the blue end of the spectrum is cut off. 14. Reduced Alkali-hgematin or Hsemo-chromogen. (a.) Add to 13, V. (b.) a drop or two of ammonium sulphide and warm gently = reduced alkali-hseraatin, Stokes's reduced hsematin, or haemo-chromogen, and observe its spectrum ; two absorption bands between D and E, as with HbO., 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. Methgemoglobin. (a.) To a medium solution of oxy-htemoglobin, add a few- drops of a 1 per cent, solution of potassic permanganate, warnx 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 pei'manganate until the two bands disappear. Finally, acidify the solution, and with a spectroscope look for the spectrum of metha^moglobin, viz., one absorption band in the red near 0, 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. (6.) 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-h^emogiobin is formed. (e.) To a solution of oxy-ha^moglobin, add a crystal or two of potassic chlorate ; dissolve it with the aid of gentle heat ; after a shoit time the spectrum of methgemoglobin 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 (/. ), the spectrum changes to that described in (a.) Add ammonium sulphide DERIVATIVES OF HAEMOGLOBIN. 37 or Stokes's fluid, the spectrum of reduced hremogloliin appears, and on shaking up with air, the bands of oxy- hsemoglobin appear. 16. VII. Haematoporphyrin. (Iron-free Haematin.) (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 ha?matin 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 htematin solution (in strong sulphuric acid), add a large excess of water, which throws down part of the lipematoporphyrin 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. ((/.) 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 h 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 HEMOGLOBIN— 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-Hseraoglobin. (a.) Arrange the apparatus as shown in Fig. 6. A is the telescope through which the observer looks and sees the Fig. 6. — Arrangement of the spectroscope for determining wave-lengths. — A, Telescope ; B, collimator tube ; C, scale tube ; D, hasmatinometer. spectrum obtained by the light passing through B, and dis- persed by the flint-glass prism in the centre of the apparatus. In 0 is fixed a scale photographed on glass and illuminated by a fan-tailed burner. D is the hiematinometer containing the dilute blood. (h.) 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 n8'9 of the scale, has a wave-length of 589 millionths of a millimetre. The wave-lengths of Frauenhofer's lines are: — A - 760-4, B = 687-4, 0 = 656-7, D = 589-4, 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), till 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 hiematinometer, 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-hiemoglobin 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-hjemo- 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 — noticinsr 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 0-2 per cent. (b.) Observe the single absorption band less deeply shaded, and with less defined edges between D and E, conveniently designated by the letter y. 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-ha^moglobin 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 HaBmoglobin. (a.) Use a dilute solution of carbonic oxide-hremoglobin 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-O^, but both are ve7y slightly more towards the violet end of the spectrum, a extends from about W.L. 587 to 564, and /3 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 hi^matin is obtained. Re-extract the residue several times with boiling alcohol, and mix the alcoholic extracts. The solution is dichroic. (6.) Acidify the alkaline filtrate of (a.) with dilute sul- phuric acid, filter, and keep the filtrate. Observe the spec- trum of acid-hivmatin in the filtrate (Fig. 5, 5). (c.) Add excess of ammonia to the acid filtrate of (5.), and filter ofi" the precipitate, keep the filtrate, and observe that DERIVATIVES OF HAEMOGLOBIN. 41 it is dichroic. Observe the spectrum of alkali-h;i?matin 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 hjvmatin 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-hjematin. 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 l)rown residue is impure hfematin. When dissolved in alcohol and caustic soda it gives the spectrum of alkali-h;vmatin, and on adding ammonium sulphide that of hremochromogen. If it is dissolved in H2SO4, and filtered through asbestos, the red filtrate gives the spectrum of hsematoporphyrin [MacMitnn). 5. Haemin Crystals. — Place some powdered dried blood on a glass slide, add a crystal of sodium chloi'ide, and a few drops of glacial acetic acid. x Cover with a cover-glass, and heat care- ^^ y ^ fully over a flame until bubbles of gas _ \ v. v are given ofi". After cooling, brown ^v ^ ""^ ^ ^ or black rhombic crystals of hsematin ,\f^ ^ ^ are to be seen with a microscope — ^J^^ n" ^ '^ (Fig. 7). :-"•..- - i 6. Detection of Blood Stains. — Use a piece of rag stained with blood. (a.) Moisten a part of the stain » ^ / V x. with glycerin, and after a time Fig. 7.— H;einiu Crystals express the liquor, and observe it preitared from traces microscopically for blood-corpuscles. °^ blood. (6.) 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 PHYSI0L0C4Y. 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 lia^min test, doing the test in a watch-glass (Lesson V., 5). AMOUNT OF HEMOGLOBIN IN BLOOD. 7. Colorimetric Method (Hoppe-Seyler's Method). — A standard solution of pure hamioglobin 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. («.) The demonstrator will prepare a standard solution of haemoglobin of known strength. (h.) Spread a sheet of white paper on a table in a good light opposite a window, and on it place two haematino- 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. (c. ) Take 10 cc. of the standard solution of haemoglobin and dilute it with 50 cc. of water, and place it in one of the haematinometers. {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 haematinometer (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 haemoglobin. Example {Hoppe-Seykr).—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 HEMOGLOBIN. 43 meter, 3S 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 1,5"20 cc, thus — 10 : 400 : : 38 : a: X = ],5"J0 cc. 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 dcgi'ce of dilution as the standard solution. The standard solution on analj'sis was found to contain 0'145 grnis. of haemoglobin in 100 cc, so that the total amount of the ha;moglobin in the diluted blood is found, thus — 100 : 1,920 : : 0145 : x X — 2 '784 grms. Since, however, this amount of haemoglobin was obtained from 20'1S6 grnis. of the original blood, the amount in lUO parts will be found, as follows: — 20-186 : 100 : : 2-784 : x X = 13-79 grms. per cent. 8. The Hsemoglobinometer 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 Avater 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 hfemoglobin 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 I'O c.mm. of blood, in 2 cc. of water (1 in 100). The second tube (C) is graduated, 100° = 2 c. (100 times 20 c.mm.) The 20 c.mm. of blood are measured by a capillary pipette (B). («) Place a few drops of distilled water in the bottom of the graduated tube (0). (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 titlie ; D, tube with standard dilution ; Y, 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 h;i:;moglobin contained in it, compared to the normal. For instance, the 20 c.mm. of blood from a patient with an?emia gave the standard tint of 30° of dilution. Hence it contained only 30 per cent, of the normal quantity of hsemoglolnn. By ascertaining with the hjemacytometer the cor- puscular richness of the blood, we are able to compare the two. A fraction, of which the numerator is the percentage of hpemo- 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 ^ or I 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 HAEMOGLOBIN. 4& the tubes between the eye and the window, or by placing a piece of white paper behind the tubes ; tlie former is perhaps the best. In practice it will be found that, during G or 8 degi'ees 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 standai'd, 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 Hajmometer. 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 th(^ blood to be investigated. A scale (P) on the slot of the instrument enables one to read off directly the percentage of hsemoglobin. (a.) Fill with a pipette the compartment («') 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. (6.) Prick the finger as in 8 with the instrument supplied for the purpose. Fill tlie 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 (6.) 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 hpemoglobin. 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 mouth, 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 the presence of (1) squamous epithelium, (2) salivary corpuscles, (3) 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. (6.) 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 sulplio -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 (HNOg, and AgNOg), 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. («.) Dilute the saliva with 5 volumes of water. Label three test-tubes A, B, and 0. 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 0 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 sacchai'ifying process which follows. 4. Stages between Starch and Maltose. — Mix some starch and saliva in a test-tube as in 3 («.) 0, and place it in a water-bath at 40° C. 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 jDorcelain 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 foi'ming after iodine has ceased to react with the fluid, and its presence is easily ascertained by Fehling's solution. 5. Effect of Dififerent 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 stai'ch mucilage and saliva, to B add a few drops of hydrochloric acid, and to 0 caustic potash. Place all three at 40^ 0. 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 C because strong acids and alkalies arrest the action of ptyalin on starch. (p.) If a test-tube containing starch mucilage and saliva be prepared as in 3 («.) 0, 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° C. the conversion is rapidly efiected. (c.) Mix some raw starch with the saliva and expose it to 40° 0. Test it after half an hour or longer, when no sugar will be found. 6. Starch is a Colloid, but Sugar dialyses. 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 diflTusate 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. (b.) 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 filti'ate. (c.) Place the starch paste of (a.) in a flask, and cool to 60° C., add the extract of (b.), 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 (Oj2H220j^j + H^O). 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. Keutralise 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 Banger'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. («.) 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° 0. 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 G 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^ 0. for an hour, and examine them at intervals of twenty minutes. (6.) After twenty minutes A begins to be turbid, and the fibrin is dissolvins;. 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 C 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 ofi'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 pi'oteids. 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 difierences 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. {/.) Neutralise part of the filtrates of B and 0. They give no precipitate, nor do they give the reactions for pep- tones. In B the ferment pepsin was destroyed by boiling, while in 0 the ferment cannot act in an alkaline medium. ((/.) If to the remainder of C acid be added, and it be placed again at 40° C, 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. ib.) Add 1 or 2 cc. of glyceinn 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 antialbumose 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 Kiihne'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- ing out. By-and-by whey is Fig. 10. 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. (6.) To the test-tube add 5 cc. of O-l 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 Avater-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 compai'ed 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-Tropaeolin dissolved in alcohol. The very dilute mineral acid in A renders it rose-pink, while the far stronger organic acid does not afiect its colour. (6.) 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 CHEIMICAL 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. HCl (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 tine 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. (&.) Make a glycerin extract of the pancreas in the same way as described for the stomach (Lesson VII., 1, h.) 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 ofi" 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° 0. 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° C. (6.) 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 filtrate with very dilute hydrochloric or acetic acid = a precipitate of hemialbumose. Filter off the precipitate, and on testing the filtrate, peptones are found. (d.) Filter B and C, and carefully neutralise the filtrates. They give no precipitate. No peptones are found. (e.) Test the pi'oteolytic power of an extract of Kuhne's "pancreas powder" (Lesson YIII., 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, when leucin 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 suflacient 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 mici"oscopic 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 Tyrosin (Hofmann). — Dissolve some crystals by boiling them in water, add Millon's reagent, and boil, which gives a rosy-red colour. {/.) Test a solution of tyrosin obtained by the prolonged Ijoiling of horn shavings and sulphuric acid, with Millon's reagent, as in (e.) {g.) Indol. — The i-emainder 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 VIIL, 1, e). One must be careful to regulate the strength of the acid. (A.) Acidify strongly with hydrochloric acid a small quan- tity of the highly otfensive 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 (Kiihne). III. The Action on Fats is twofold. 6. A. Emulsification. (ct.) 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 emulsification. Take two samples of cod-liver oil, one perfectli/ 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 ethei', the ethereal extract separated from the insoluble portion, and the ether evaporated over warm water. The oil should now be perfectly neutral (Krickenberg). (b.) Mix the oil with finely-divided, perfectly fresh pan- creas (not a watery extract), and keep it at 40° 0. 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 altogf^ther 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. Gl and keep it at 40° 0. 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. («.) 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-tulje, add a drop or two of jDancreatic extract, or the Liquor Pancreaticus of Benger. Expose on a water-bath at 40^ 0. for half an hour. Note that the casein is first curdled and then dissolved, and as this occurs, the milk changes fi'om a white to a yellowish colour. (6.) 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 watei', and heated to a luke-warm 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. («.) 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 filtrate 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. ((/.) Bile, besides the ordinary salts, contains so much iron as to give the ordinary reactions for iron. To bile add hydrochloric acid and potassic ferrocyanide. A blue colour indicates the presence of iron. It is better to use the filtrate of (d.) free from mucin. If fresh bile is not obtainable, use a watery solution of the " Eel bovinum " of the Pharmacopceia. 2. To Prepare the Bile Salts or Bilin (glycocholate and tauro- cholate of sodium). (rt.) Mix ox bile with animal charcoal in a mortar to form a thick paste. Evaporate to complete dryness over a THE BILE. 63 water-bath. Keep this for preparing an alcoholic solution of the bile salts. (b.) Take some of the dry charcoal-bile mixture, and add five times its volume of absolute alcohol. Cork the flask or test-tube in which the mixture is placed. Shake up the mixture from time to time, and after half an hour, filter. To the filtrate add much ether, which gives a white precipi- tate of the bile salts. If no water be present, sometimes the bile salts are thrown down crystalline ; but not unfrequently they go down merely as a milky opalescence, which quickly forms resinous masses. It is best to allow the mixture to stand for a day or two, to obtain the large glancing needles which constitute Plattner"s Crystallised Bile. 3. Pettenkofer's Test for Bile Acids and Cholic Acid. («.) Place some bile in a test-tube, add a drop or two of syrup of cane sugar, and mix. Pour in concentrated sul- phuric acid, and at the line of junction of the two fluids a purple colour is obtained. The white deposit seen above the line of junction is pre- cipitated bile acids. They are insoluble in water. (b.) Or, after mixing the syrup with the bile, add the strong sulphuric acid drop by drop, mixing it thoroughly. Heat gently, and the fluid becomes a deep purple colour. Take care not to add too much syrup, and not to overheat the tube. If the requisite amount of sulphuric acid be added, the temperature becomes sufiiciently high (70^ C) without requiring to heat the tube. (c.) A better way of doing the test is as follows : — After mixing the bile and syrup, shake the mixture until the upper part of the tube is filled with froth. Pour sulphuric acid down the side, and a purple-red colour is struck in the froth. (d.) It may also be done by mixing a few drops of bile in a porcelain capsule, with a small crystal of cane sugar, and after the sugar is dissolved, adding sulphuric acid drop by di'op. (e.) Strasburg's Modification (e.y., for bile in urine).— To the urine add a little syrup and mix. Dip filter-paper into 64 CHEMICAL PHYSIOLOGY. the fluid and dry the paper. On placing a drop of sulphuric acid on the latter, after some time, a purple spot which has eaten into the paper is observed. (/.) Repeat any or all of the above processes with a watery solution of the bile salts. 4. Similar colour reactions are obtained with many other sub- stances— e.g., albumin and fats. Albumin and Sulphuric Acid. — To a solution of syntonin and syrup add strong sulphuric acid, and a similar tint is obtained. The spectra, however, are different, the red-purple fluid from bile gives two absorption bands, one between E and F, and another between D and E. In the albuminous solutions, only one absorption band exists between E and F. 5. Action of Bile or Bile-Salts in Precipitating Sulphur. {a.) Take two beakers and in one (A) place diluted bile, and in the other (B) water. Pour flowers of sulphur on the surface of the water of both. The sulphur falls in a copious shower through the fluid of A, while none passes through B. {h) Test to what extent bile may be diluted before it loses this property, which is due to the diminution of the surface tension by the bile-salts (J/. Hay). (c.) Perform the same experiment with a solution of the bile-salts. Bile Pigments. — The chief are bilirubin (red), biliverdin (green), and urobilin. 6. Gmelin's Test for the Bile Pigments. (a.) Place a few drops of bile on a ivldte jDorcelain slab. With a glass rod, place a drop or two of strong nitric acid containing nitro%is acid near the drop of bile, bring the acid and bile into contact, when there is immediately a play of colours, beginning with green and passing into blue, violet, red, and dirty yellow. (6.) Place a little nitric acid in a test-tube. Slant the tube and pour in bile, a similar play of colours occurs — THE TULE. 05 green above, blue, violet, red, and yellow l)elow. It is better to do this reaction after removal of the mucin by acetic acid (Lesson IX., 1, d). Or add the nitric acid, and shake after the addition of every few drops, the successive colours from green to yellow are obtained in great beauty. (c.) For a modification applicable to urine, see " Urine." 7. Cholesterin and Gall Stones. {a.) Preparation. — Powder a gall-stone and extract it with ether. Heat the test-tube with ether in warm water, and see that no gas is burning near it. Allow a drop of the ethereal solution to evaporate on a glass-slide or watch- glass, and examine the crystals under the microscope. They are flat plates, with an oblong piece cut out of one corner (Fig, 11). (h.) Heat a few crystals in a watch- glass with a few drops of moderately strong sulphuric acid, and then add iodine ; a play of colours, passing through violet, blue, green, red, and brown, occurs. (It requires a little practice to get this test always to succeed.) Fig. 11.— Crystals of Cholesterin. (c.) Dissolve some crystals in chloroform, add an equal volume of concentrated sulphuric acid, and shake the mix- ture. When the chloroform solution floats on the top, it becomes blood-i'ed, but changes quickly on exposure to the air, passing through violet and blue to green and yellow. A trace of water decolourises it at once. The layer of sul- phuric acid shows a green fluorescence. [This reaction is not easily performed.] {d.) The crystals when acted on by strong sulphuric acid become red. Do this on a slide under the microscope. (e.) Place a crystal on a piece of porcelain, add a drop of strong nitric acid, evaporate to dryness at a gentle heat, until a yellow residue is obtained. A drop of ammonia 66 CHEMICAL PHYSIOLOGY. strikes a red colour, which is not altered by the addition of caustic soda. (/.) Examine microscopically crystals of cholesterin float- ing in hydrocele fluid. The crystals may not be quite perfect, but their characters are quite distinct. 8. Action of Bile in Digestion. (rt.) Action on Starch. — ^Test if bile converts starch muci- lage into a reducing sugar, as directed for saliva (Lesson VI., 3). (b.) Action on Fats. — Mix 10 cc. of bile with 2 cc. of almond oil or oleic acid. Shake them together, and observe both by the naked eye and the microscope to what extent emulsion occurs, and how long it lasts. Compare a similar mixture of oil and water. In the former case a pretty fair emulsion will be obtained. In the latter the oil and water separate almost immediately. (c.) Mix 10 cc. of bile with 2 cc. of almond oil, to which some oleic acid is added. Shake well, and keep the tube in a water bath at 40° 0. A very good emulsion is obtained. The bile dissolves the fatty acids, and the latter decompose the salts of the bile acids ; the bile acids are liberated, while the fatty acid unites with the alkali of the bile-salts to form a soap. The soap is soluble in the bile, and serves to increase the emulsifying power, as an emulsion once formed lasts much longer in a soapy solution than in water. (d.) Favours Filtration and Absorption. — Place two small funnels exactly the same size in a filter-stand, and under each a beaker. Into both funnels put a filter-paper ; moisten the one with water (A), and the other with bile (B) ; pour into both an equal volume of almond oil ; cover with a slip of glass to prevent evaporation. Set the whole aside for twelve hours, and note that the oil passes through the filter B, but scarcely any through A. (e.) Effect on the Proteid Products of Gastric Digestion.— Digest some fibrin in artificial gastric juice, filter, and to the filtrate add drop by drop some ox bile, or a solution of bile-salts. It causes a white precipitate of peptones and GLYCOGEN IN THE LIVER. 67 parapeptones. The acid of the gastric juice splits up the bile-salts, so that the bile acids are also thrown down. (/.) Action on Syntonin. — Prepare syntonin in solution (Lesson I., 7,/), and add a few drops of bile or bile-salts. It causes a curdling of the whole mass. Be careful not to add too much bile. In (e.) and (/.) it is better to add the bile- salts, l)ecause the free hydrochloric acid gives a precipitate with bile. LESSON X. GLYCOGEN IN THE LIVER. 1. Preparation. (a.) Boil some water slightly acidulated with acetic acid, and keep it boiling. Feed a rat, and three or four hours thereafter decapitate it. Rajndly open the abdomen, remove the liver, cut one half of it in pieces, and throw it into the boiling acidulated water. Lay the other half aside, keeping it moist in a warm place for some hours. After boiling the first poi'tion for a time, pound it in a mortar with sand, and boil again. Filter while hot. The filtrate is milky or opalescent, and is a watery solution of glycogen. The acetic acid coagulates the proteids, while the boiling water destroys a ferment in the liver, which would convert the glycogen into grape-sugar. (b.) Feed a rabbit on carrots, and after from two to three hours decapitate it. Open the abdomen, tear out the liver, cut it rapidly in pieces, and take one half — laying the other half aside as in (a.) — throw it into boiling water, boil it, and afterwards pound it in a mortar and boil again. Filter while hot, and observe the opalescent filtrate, which is a solution of glycogen and proteids. The filtrate should flow into a cooled beaker, placed in a mixture of ice and salt. Precipitate the proteids by adding alternately hydrochloric acid and potassio-mercuric iodide, until all the proteids are precipitated. Filter off the proteids, and the beautiful opal- 68 CHEMICAL PHYSIOLOGY. escent filtrate is an imperfect solution of glycogen (Briicke's method). (c.) Instead of a rat or rabbit's liver, use oysters, and pre- pare a solution of glycogen by methods (a.) or (b.) (d.) Use the other half of the liver of the rat or rabbit that has been kept warm, and make a similar extract of it. 2. Precipitate the Glycogen. — Evaporate the filtrate of (a.) or (6.) to a small bulk, and precipitate the glycogen as a white powder by adding a large amount of alcohol. 3. Preparation of Potassio-mercuric Iodide. — Precipitate a saturated solution of potassic iodide with a similar solution of mercuric chloride ; wash the precipitate, and dissolve it to satura- tion in a hot solution of potassic iodide. 4. Tests. (a.) To the opalescent filtrate add a solution of iodine= a port-wine red colour (like that produced by dextrin). If much glycogen be present the colour disappears, and more iodine has to be added. Heat the red fluid ; the colour dis- appears on heating, but reappears on cooling. JV.B. — In performing this test, make a control-experiment. Take two test-tubes, A and B. To A add solution of glycogen ; to B, an equal volume of water. To both add the same amount of iodine solution. A becomes red, while B is but faint yellow. (b.) Test a portion of the glycogen solution for grape- sugar. There should be none, or only the faintest trace. (c.) To a portion of the glycogen solution add saliva or Liquor Pancreaticus (A), and to another portion add blood (B), and place both on a water-bath at 40° C. After ten minutes test both for sugar. (A) will now be transparent, and give no reaction with iodine. Perhaps both will give the reaction ; but certainly (A) will, if care be taken that the solution is not acid, after adding the saliva. The ptyalin converts the glycogen into a reducing sugar. (d.) Boil some glycogen solution with dilute hydrochloric GLYCOGEN IN THE LIVER. 69 acid in a flask ; neutralise with caustic soda, and test with Fehling's solution for sugar. (e.) To another portion add plumbic acetate = a precipitate (unlike dextrin). {/.) To another portion add plumbic acetate and ammonia = a precipitate (like dextrin). 5. Test the watery extract of the second half of the liver (d). (a.) It will perhaps give no glycogen reaction, or only a slight one. (b.) It contains much reducing sugar. 6. Extract of a Dead Liver. (a.) Mince a piece of liver from an animal {e.g., an ox) which has been dead for twenty-four hours. Place the finely-divided liver in water or in a saturated solution of sodic sulphate, and boil to make a watery extract. Filter, and observe the filtrate ; it is clear and yellowish in tint, but not opalescent. (b.) Test its reaction = acid. (c.) Test with iodine after neutralisation with sodic car- bonate and filtration = no glycogen. (d.) Test for grape-sugar = much sugar. After death the glycogen is rapidly transformed into grape- sutcar. LESSON XL MILK, FLOUR, AND BREAD. 1, Milk. — Use fresh cow's milk. (a.) Examine a drop of milk under the microscope, noting 70 CHEMICAL PHYSIOLOGY. the numerous small, highly-refractive fat globules floating in a fluid (Fig. 12, M). (i.) Add a drop of acetic acid, and observe how the globules run into groups. (ii.) To a fresh drop add osmic acid, and observe how the globules first become brown and then black. (iii.) If a drop of colostrum is ob- tainable, examine for the " colostrum corpuscles " (Fig. 12, C). (b.) Examine the " naked-eye " charac- ters of milk. Fig. 12. — Microscopic appearance of milk. — The upper half, ]\I, is milk; the lower half colostrum, C. (c.) tion Test with its reac- litmus- paper. It is usually alkaline. (d.) Take the specific gravity of perfectly fresh unskimmed milk with a hydrometer or lactometer. It is usually be- tween 1025 - 1030. Take the specific gravity next day after the cream has risen to the surface. The specific gravity is higher. (e.) Dilute milk with ten times its volume of water, and carefully neutralise it with dilute acetic acid, observe that there is no precipitate, as the casein is prevented from being precipitated by the presence of alkaline phosphates (Lesson I., 7). Cautiously add the acetic acid until there is a copious granular-looking precipitate of casein, which as it falls, entangles the greater part of the fat in it. If desired the precipitation is hastened by heating the fluid to 70° C. {/.) Filter (e.) through a moist-plaited filter. Keep the residue on the filter. The filtrate of (/) should be clear. Divide it into two portions. Take one of the portions. MILK, FLOUR, AND liUEAD. 71 divide it into two and boil one = a precipitate of serum- albumin. Filter, and keep the filtrate to test for sugar. To the remainder add potassic ferrocyanide, which also precipitates serum-albumin. {{/•) With the second half of the filtrate test for milk-sugar or lactose with Fehling's solution, or by Trommer's test (Lesson II., 6; IV., b., c.) Instead of proceeding thus, test for the presence of a reducing sugar with the filtrate of {/.) after the separation of the serum-albumin. (h.) Scrape off the residue of casein and fat from the filter (/.) ; wash it with water from a wash bottle, and exhaust the residue with a mixture of ether and alcohol. On placing some of the ethereal solution on a slide, and allowing it to evaporate, a greasy stain of fat is obtained. (i.) To fresh milk add a drop of tincture of guaiacum, which strikes a blue colour ; l)oiled milk does not do so. 2. Separation of the Casein by Salt. — To one volume of milk in a test-tube, add two volumes of a saturated solution of common salt, and then excess of powdered salt, (or magnesic sulphate may be used). Shake the tube vigorously for a time, when the casein and fat separate out, rise to the surface, and leave a clear fluid or whey behind. This fluid contains the lactose, salts, and serum-albumin. 3. Separation of the Casein and Fat by Filtration. — ^Using a Bunsen's pump, filter milk through a porous cell of porcelain. The particulate matters — casein and fat — remain Ijehind, while a clear filtrate containing the other substances in milk passes through. The porous cell is left empty and fitted with a caoutchouc cork with two glass tubes tightly fitted into it. One tube is closed with a clip (Fig. 13), and the other is attached to the pump. Place the porous cell in an outer vessel containing milk. On exhausting the porous cell, a clear watery fluid slowly passes through. Test it for proteids and sugar. Notice the absence of fat and casein. pj„ 13. —Porous Cell for the Fil- 4. Souring of Milk. — Place a small quantity tration of Milk, of milk in a vessel in a warm place for several days, when it turns sour and curdles. It becomes acid — test 72 CHEMICAL PHYSIOLOGY. this [Lesson Til., 8, (e.)] — having Tindergone the lactic acid fermentation, the lactose being split np hj a micro-organism into lactic acid. 5. To Separate the Butter. — Place a little milk in a narro-w, cvlindrical, stoppered bottle : add half its volume of caustic soda and some ether, and shake the mixture. Put the bottle in a ■water-bath at a low temperature ; the milk loses its whit* colour, and" an ethereal solution of the fats floats on the surface. On evaporating the ethereal solution, the butter is left behind. 6. Curdling of Milk. (a.) By an Acid. — Place some milk in a flask ; warm it to 40" C, and add a fe^r drops of acetic acid. The mass clots or curdles, and separates into a solid curd (casein and fat), and a clear fluid, the whey, "which contains the lactose- Filter. (b.) By Rennet-Ferment. — Take 5 cc. of fresh milk in a test-tube, heat it in a water-bath to 40° C, and add to it a small quantity of extract of rennet, or an equal volume of a glycerin extract of the gastric mucous membrane, which has been neutralised with dilute sodic carbonate, and place the tube again in the water-bath at 40^ C. Observe that the whole mass curdles in a few minutes, so that the tube can be invert.ed without the curd falling out. By-and-by the curd shrinks, and squeezes out a clear slightly- yellowish fluid, the whey. Filter. (c.) Using commercial rennet-extract, repeat (b.), but boil the rennet flrst, it no longer efiects the change described above. The rennet-ferment is destroyed by heat. (d.) Boil the milk and allow it to cool ; then add rennet ; in all probability, no coagulation will take place. Boiled milk is far more difficult to coagulate with rennet than unboiled milk. (e.) Take some of the curd of 6 (a.) Dissolve one part in caustic soda and the other in lime-water. Add rennet to both, warm to 40° C. The lime solution coagulates, the soda solution does not. 7. The ordinary Salts are present. MILK, I'LOUK, AND BREAD. 73 (a.) Usin^^ separate. ^ ^-^ 0 /X (6.) Examine them micro- "^.v m /A C ) n scopically (Fig. 22). X^ % I ^ r-i (^•) ^^^"-^ oxalic acid to a ^L\^^ ^ concentrated solution of ♦ ^Jy^ D ^/^ urea = a precipitate of urea ^^ oxalate, which may have „. „_, ^, , , J. 1 . P many forms — rhombic rig. 22. — Lrvstals 01 oxalate or , , -^ . n- i 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(N03)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 : — (ffi.) 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 jnire 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 ofi", consisting of carbon dioxide and nitrocfen. {e.) Put some of the urea nitrate precipitate obtained in 4 {a.) into the test-tube A (Fig. 23), and some lime Fig. 2.3. water in B. Add nitrous acid to A. Cork the tube. The precipitate dissolves. CO., and X are given off, the CO^ makes the lime water in B white. Urea. Nitrous Acid. Carbon Dioxide. Nitrogen. Water. COK,H, + 2(HX0.,) = CO, + N^ + 3H,0 (,/!) 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 otf, 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 Ijlood, 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. (6.) 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 ansemia, 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 urpemia, 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. ih.) 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. (6.) 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 j. This precipitates the phosphates, sulphates, and carbonates. (cl.) 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. JV.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. Run 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 ofl" the number of cc. of the SS. used. ((/.) 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 "Ol, 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. 1 OAA '0^ 10 : 1200 : : "25 : x . -^ — ^7^ — ~ = ^^ 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 niti^ate = precipitate. (6.) 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. Baiyta 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 HNOg, evaporate to a syrup, and then dilute with water to 1 litre. A few drops of HNOg 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 Requked. — Bui-ette fixed in a stand, funnels, VOLUMETRIC ANALYSIS FOR URKA. ^7 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 GOg (which is absorbed by the caustic soda), and N, which is disengaged in bubbles and collected in a suitable apparatus. Sodic Carbon Sodic Urea. Hypobromite. Dioxide. Nitrogen. Water. Bromide. OON.H, + SKaBrO = CO, + N2 + 2Hp + 3NaBr 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, Russel and West, Graham Steele, Simpson, Dupre, Charteris, n.l>y hafiiatim - tion of a solution of h^mo^oliin into a vein ; and after extaiaxve' destruction of the skin by homing. It also oeems in pvrpma, scurvy, often in typhus or aearl^ fovi^ pamiciows maJaria, in " periodic ha&moglobfniiriay'' aod al^er llite inhalation of araen- iuretted hydrogen- [ct.) The urine gives the same reactions as in hemadnaorEa, but no blood-corpuscles are detected by the microscope^ 3. Bile in nrine. — The l^li&ry etaistltuents appear in the urine in cases of jaundiee and in rAw«wTwg with phosplusros^ One may test for the ItUe^igmtadSf or the h^e^teida^ or hoth. A. Bfle-Figmfiots. ('esent. QUANTITATIVE ESTIMATION OF SUGAR. 117 LESSON XX. QUANTITATIVE ESTIMATION OF SUGAR. 1. By the Saccharimeter. Study the use of some form of saccharimeter. The portable form made by Zeiss is very convenient. A coloured urine must tirst be decolourised by acetate of lead [Lesson XVII. , 12 ((?.)]. 2. Volumetric Analysis by Fehling's Solution. — 10 cc. of Fehling's solution = '05 grms. of sugar. (a.) Ascertain the quantity of urine passed in twenty-four hours. (b.) Filter the urine, and remove any albumin present by boiling and filtration. (c.) Dilute 10 cc. of Fehling's solution with about five to ten times its volume of distilled water, and place it in a white porcelain capsule on a wire gauze support under a burette. [It is diluted because any change of colour is more easily observed.] (d.) Take 5 cc. of the diabetic urine, add 95 cc. of distilled water, and place the diluted urine in a burette. (e.) Boil the diluted Fehling's solution, and whilst it is boiling, gradually add the diluted urine from the burette, until all the cuprous oxide is precipitated as a reddish powder, and the supernatant fluid has a straw-yellow colour, not a trace of blue remaining. This is best seen when the capsule is tilted. It is not advisable to spend too much time in determining when the blue colour disappears, as it is apt to return on cooling. {/.) Read off the number of cc. of dilute urine employed. If 36 cc. were used, this, of course, would represent 1-8 cc. of the original urine. {(/. ) Make a second determination, using the data of the tirst, and in this case run in at once a little less of the dilute urine than was required at first. 118 CHEMICAL PHYSIOLOGY, Example. — Suppose the patient passes 8550 cc. of urine, then as 1-8 cc. of urine reduced all the cupric oxide in the 10 cc. of Fehling's solution, it must contain '05 gramme sugar; hence = 237*5 grammes of sugar passed 1-8 :8550 :: -05. 8550 X -05 1- in twenty-four hours. 3. Picro-Saccharimeter of G. Johnson. Solutions Required. (1.) A solution of ferric acetate equal to that yielded by a solu- tion of sugar containing ^ grain per fluid ounce. (2.) Saturated solution of picric acid. (3.) Liquor Potassie B.P.) (a.) Measure 1 fluid drachm of urine into the boiling tube, add 30 minims of liquor potassfe and 80 minims of the saturated solution of picric acid. Make up to the 4-drachm mark on the tube with distilled water. Boil for one minute. (6.) Dip the tube in cold water to cool it. The volume must be exactly 4 drachms. If it is less, add water ; if more, evaporate it. If the colour of the boiled liquid is the same as that of the ferric acetate ^ grain standard, or paler, the urine con- tains 1 grain of sugar per fluid ounce, or less. (c.) Should the colour be darker than the standard, place some of the boiled liquid into the graduated stoppered tube (Fig. 30) to fill 10 divisions of the scale, while the stoppered tube afiixed to the former is filled with the SS. of ferric ace- tate. Fill up the graduated tube with distilled water until the dark red liquid has the same colour as that of the SS. These tints are best compared in the flat- bottomed tubes supplied with the ap- paratus. (d.) Read ofi"the level of the fluid in the Fig. .30— Picro- sacchariineter. QUANTITATIVE ESTIMATION OF SUGAR. 119 saccharimeter, each division above 10 = O'l grain per fluid oz. Thus, 13 divisions = 1-3 grains per fluid oz. (e.) If more than 8 grains per oz. are present, further dilu- tion is required. Full instructions are supplied with the apparatus. 4. Aceto -Acetic Acid is found in certain diabetic urines, but not in all. (a. ) To the urine add ferric chloride ; a red colour is obtained if this acid be present. If there is a deposit of phosphates, filter. The colour disappears on heating. If a diabetic urine containing aceto-acetic acid be distilled, this acid is decomposed, and aceton is obtained. 5. Tests for Aceton. (a.) Lieben's Test. — To a weak, watery solution of aceton add solution of iodine dissolved with the aid of potassic iodide, and then caustic soda. A yellow precipitate of iodoform is obtained. The precipitate is generally described as forming hexagonal plates or radiate stars, but I have generally found it to be amorphous or granular. Other substances give the iodoform reaction. (b.) Smell the peculiar ethereal odour of aceton. (c.) Legal's Test. — Add caustic soda solution, and then a solution of freshly-prepared sodium nitro-prusside, and acetic acid = a red colour. In all cases employ both tests, but they only give a decided reaction in urine when the aceton is in considerable amount. To be quite certain that aceton is present, a considerable amount of the urine must be distilled, and the tests applied to the distillate. 6. Tests for Phenol.— To a watery solution of phenol (a.) Add ferric chloride ~ a bluish-violet colour, (b.) Add bromine water = a yellow (or rather white) precipitate of bromine compounds. 120 CHEMICAL PHYSIOLOGY. (c.) Add Millons' reagent = a beautiful red colour or deposit. This reaction is aided by heat. 7. Pyrocatechin is sometimes found in urine. The method of obtaining it requires too much time to be done in this course. Tests. (a.) To a dilute solution add ferric chloride = a green colour, which becomes violet on the addition of sodic bicar- bonate. (b.) Add ammonia and silver nitrate, which give a black precipitate of reduced silver. LESSON XXI. URINARY DEPOSITS— CALCULI AND GENERAL EXAMINATION OF THE URINE. 1. Mode of Collecting Urinary Deposits. — Place the urine in a conical glass, cover it, and allow it to stand for twelve hours. Note the reaction before and after standing. With a pipette remove some of the deposit and examine it microscopically. There are two classes of deposits, organised and unorganised. ORGANISED DEPOSITS. 1. Pus (p. 120). j 5. Spermatozoa. 2. Blood (p. 112). 6. Micro-organisms. 3. Epithelium. 7. Elements of morbid growths 4. Renal tube casts. ' and entozoa. 2. Pus in Urine (Pyuria) produces a thick creamy yellowish- Avhite sediment after standing, although its appearance varies with the reaction of the urine. If the urine be acid, the precipi- tate is loose, and the pus corpuscles discrete ; if alkaline, and especially from ammonia, it forms a thick, tough, glairy mass. The urine is usually alkaline, and is always albuminous, and rapidly undergoes decomposition. Pus is found in the urine in leucorrhcea in the female, gonorrhcEa, gleet, cystitis, pyelitis, from bursting of an abscess into any part of the urinary tract, &c. URINARY DEPOSITS, 121 {a). Donne's test.— Filter off the fluid, and add to the deposit a small piece of caustic potash, or a few drops of strong solution of caustic potash ; the deposit becomes ropy and gelatinous, and cannot be dropped from one vessel into another — due to the formation of alkali-albumin — the deposit is pus. The same reagent with mucus causes the deposit to become more fluid and limpid, to clear up, and look like unboiled white of egg. (6.) With the microscope numerous pus corpuscles are seen, which when acted on by acetic acid show a bi- or tri- partite nucleus. This test is not absolutely conclusive. (c.) Urine containing pus gives the reactions for albumin, while, if mucus alone be present, it gives only those for mucin. UNORGANISED DEPOSITS. A. Ix Acid Urine. B. In Alkaline Urine. 1. Amorphous. (a.) Urates. — Soluble when heated, redeposited in the cold; when hydrochloric acid is added microscopic crystals of uric acid are formed = urates. (b.) Tribasic Phosphate of Lime. — Not dissolved by heat, but disappears without efferves- cence on adding acetic acid. It is probably tribasic phosphate of lime (Ca32P04). (c.) Oil Globules.— Very small highly refractive globules, soluble in ether (very rare). 2. Crystalline. (a.) Uric Acid.— Recognised hy the shape and colour of the crystals and their solubility in KHO. (b.) Oxalateof Lime.— Octa- hedral crystals, insoluble in acetic acid (Fig. 31, 6, c). 1. Amorphous, (a.) Tribasic Phosphate of Lime dissolves in acids without effervescence. (b.) Carbonate of Lime. (See (f.) below.) 2. Crystalline. {n.) Triple Phosphate.— Shape of the crystals (knife-rest or coffin-lid), soluble in acids. (b.) Acid Ammonium Urate. —Small dark balls, often covered with spines, and also amorphous granules (Fig. 32). 122 CHEMICAL PHYSIOLOGY. (c.) Cystin (very rare), hexa- gonal crystals, soluble in NH4,H0 (Fig. 31, a). (d.) Leucin and Tyrosin (very rare). (Fig. 32.) (e.) Cholesterin (very rare). (Fig. 11.) (c.) Carbonate of Lime. — Small colourless balls, often joined to each other ; efferves- cence on adding acids (micro- scope). (d.) Crystalline Phosphate of Lime. (c.) Leucin and Tyrosin (very rare). Fig. 31. — a, Crystals of cystin ; b, oxalate of lime ; c, hour-glass forms of 6. 3. Urinary Calculi. They are composed of urinary constituents which form urinary deposits, and may consist of one substance or of several, which are usually deposited in layers, in which case the most central part is spoken of as the " mtcleus." The nucleus not unfrequently consists of some colloid substance — mucus, a portion of blood-clot, or some albuminoid matter — in which crystals of oxalate of lime or globular urates become entangled. Layer after layer is then deposited. In certain cases the nucleus may consist of a foreign body introduced from without. Calculi are sometimes classified as primary and seco'ndary ; the former are due to some general URINARY DEPOSITS. 123 alteration in the composition of the urine, whilst the latter are clue to ammoniacal decomposition of the urine, resulting in the precipitation of phosphates on stones already formed. This of course has an important bearing on the treatment of calculous Fig. 32. — « a, Leucin balls; hb, tjrrosin sheaves; c, double balls of amraonium urate. disorders. Calculi occur in acid and alkaline urine. A highly acid urine favours the formation of uric acid calculi, because that substance is most insoluble in very acid urine. A highly alkaliiie urine favours the formation of calculi, consisting of calcium j)hosphate or trlph i^hospliate, as these substances are insoluble in alkaline urine. 4. Method of Examining a Calculus. (a.) Make a section in order to see if it consists of one or more substances ; examine it with the naked eye, and a por- tion microscopically. {b.) Scrape off a little, and lieat it to redness on platinum foil over a Bunsen burner. (A) If it be entirely combustible, or almost so, it may 124 CHEMICAL PHYSIOLOGY. consist of uric acid or urate of ammonia, xanthin, cystin, coagulated fibrin or blood, or ureostealith. (B) If incombustible or if it leaves much ash, it may consist of urates with a fixed base (Na, Mg, Ca), oxalate, carbonate, or phosphate of lime, or triple phosphate. 5. A. Combustible. — Of this group, uric acid and urate of ammonia give the murexide test. (i.) Uric Acid is by far the most common form, and constitutes five-sixths of all renal concretions. Concretions the size of a split-pea, or smaller, may be discharged as gravel. When retained in the bladder they are usually spheroidal, elliptical, and some- what flattened ; are tolerably hard ; the surface may be smooth or studded with fine tubercules ; the colour may be yellowish, reddish, or reddish-brown, very rarely white. When cut and polished, they usually exhibit a concentric arrangement of layers. Not unfrequently a uric acid calculus is covered with a layer of phosphates, and some calculi consist of alternate layers of uric acid and oxalate of lime. Its chemical relations : nearly insoluble in boiling water ; soluble in KHO, from which acetic acid preci- pitates uric acid crystals (microscopic); gives the murexide test (Lesson XVII., 3). (ii.) Urate of Ammonium Calculi are very rare, and occur chiefly in the kidneys of children ; they form small irregular, soft, fawn- coloured masses, easily soluble in hot water. (iii.) If the calculus is combustible and gives no murexide test it may consist of Xanthin, which is very rare, and of no practical importance. (iv.) Cystin is very rare, has a smooth surface, dull yellow colour, which becomes greenish on exposure to the air ; and a glistening fracture with a peculiar soapy feeling to the fingers ; soft, and can be scratched with the nail. It occurs sometimes in several members of the same family. It is soluble in ammonia, and after evaporation it forms regular microscopic hexagonal plates (Fig. 31, a). The other calculi of this group are very rare. 6. (A.) Group. — Apply the Murexide Test. URINARY DEPOSITS. 125 It is J Treat the original ) No odour =: Uric Acid. obtained \ powder with potash. \ Odour of HX3 = Ammonium Urate. The residue is not coloured, but becomes yellowish- | _ \„„i],:„ red on adding caustic potash . . . . S ~" ' The residue is not coloured either by KHO or \ NH4HO ; the original substance is soluble in f _ ^, .. ammonia, and on evaporation yields hexagonal \ ~ crystals On heating, it gives an odour of burned feathers ; ) the substance is soluble in KHO, and is preci- > = Prottid. pitated therefi-om by excess of HNO3 . . ) 7. B. Incombustible. (i. ) Urates (Na, Ca, Mg), are rarely met with as the sole con- stituent. They give the murexide test. (ii.) Oxalate of Lime or Mulherry Calculi, so called because their surface is usually tuberculated or warty ; they are hard, dark bx'own, 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 HCl, it effervesces (COo). Oxalate of lime is not dissolved by acetic acid. (iii.) Cai'bonate of Lime. — Rare in man; when met with they usually occur in large numbers. Dissolve with effervescence in HCl. 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 ivhitish 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 urates. The residue is treated with water. It is soluble, and ( Neutralise ; add platinic chloride, a \ _ p^^^^/j the solution is < yellow precipitate . . . .\ alkaline, . . ( The residue yields a yellow flame . — Sodium. = Calcium, {Ammonium oxalate gives a white \ crystalline precipitate . . _. / Ammonium oxalate gives no precipi-'\ tate, but on adding ammonium I chloride, sodic phosphate, and am- > = Magnesium. 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 HCl, . 'It dissolves with effervescence ""It melts. The origi- There is no j nal stone effervs'ce. I treated Heat in a 1 with KHO. capsule . I It does not melt on heating . Calcic carbonate. Magnesic carbonate. = Calcic oxalate. ^NH ^^ ^ \ ~ ^'''/'^^ phosphate. Evolves no ' NH3 "} 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 1). (v.) Heat. (a.) If a turbid urine becomes clear — urates. (b.) If it becomes turbid = earthy phosjyhates or albumin. Albumin is precipitated before the boiling point is reached (70° 0.), 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 AgNOg (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 hlood 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 XY., 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.) J.f 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 p7'oteids. (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 ^(,rea (Lesson XV., 4, 7). {g.) Ascertain its solubility in warm water, starch (Lesson II., 1), urates (Lesson XYIL, 5), tyrosin (Lesson YIIL, 5). {li.) Test for uric acid (Lesson XVIL, 3). (?'.) 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 J. —EXPERIMENTAL PHYSIOLOGY. Before beginning the experimental part of the course, each student must provide himself with the following : — A large and a small 2Xiir 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'-v:ax ; sealing wax ; two canieVs-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. Darnell'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'sCell. 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 GALVAXOSCOPE. 131 Lift it out and place it on a mercurv on the zinc, and to be amalgamated. Dip the zinc in 10 per cent, sulphuric acid until effervescence commences, shallow porcelain plate. Pour 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 CeU (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 drauglit chamber to prevent the nitrous fumes from affecting the experimenter. 4. Bichromate Cell (Fig. 3.5).— 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 sufl&- 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. (6.) Use a vei'tical 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 tix B. Fisf. 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 lixed 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 tlie Fig. 37. — Du Bois - Reymond'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 token 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. (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 binding screws of the detector. (b.) Observe when the key is depressed or closed, there is no deflection of the needle — i.e., when the current is cut ofi" from the circuit beyond the key or bridge; when the key is raised, the needle is deflected. (a.) As in the scheme Fig. 39. — Scheme of du Bois Key for Short-circuiting. — N, nerve ; M, muscle ; B, 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 ELECTiaCAL KEYS, RHEOCHORD. 135 Fig. 40.— Plug Key. 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 fiidd 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. 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 0 ; 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 C. 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 refei-red to in Lesson XXXII. Fig. 41. — The Morse Key. — The conuection.s are concealed below, but are B to /, 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 boai'd 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, hoAvever, may be connected directly by brass plugs S^ S, ... 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 B h), 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-S^) in their places, thus making the several blocks of brass practically one block. In this posi- tion the resistance oflered by the rheochord circuit is so small as compared with that including the Fig 44.-Eheochorcl 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 S^ 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. (b). 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 S|, 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 XLII., 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 the current is shut otF from the electrodes, while the direction of the current is reversed by raising or lowering the handle. This instrument is used solely for reversing the direction of a current. Fig 45. Thomson's Reverser. LESSON XXIV. INDUCTION MACHINE— ELECTRODES. 1. Induced or Faradic Electricity is most frequently employed for physiological purposes. 2. Induction Appai'atus of du Bois-Reymond. — In Fig. 4G the primary coil (R') consists of about 1-30 coils of thick insulated copper wire, the wire being thick to ofler slight resistance to the galvanic current. The secondaiy coil (R") consists of 6000 turns of thin insulated copper wire arranged on a wooden bobbin ; the whole spiral can be moved along the board (B) to which a milli- metre scale (I) is attached, so that the distance of the secondary from the primaiy spiral may be ascertained. At the left end of apparatus is Wagner's hammer as adapted by Neef, wJiich is an automatic arrangement for opening and breaking the primary circuit. When Is eef's hammer is used to obtain what is called an interrupted current, the wires from the battery are connected as in the figure, but when single shocks are required, the wires 140 EXPERIMENTAL PHYSIOLOGY. from the battery are connected with a key, and this again with the two terminals of the primary spiral, S" and S'". J s" Fig. 46. — Induction apparatus of du Bois-Reyraond. — E.', Primary; R", secondary spiral ; B, board on whicli R" moves ; I, scale ; + - , wires from battery ; P', P", pillars ; H, Neef's hammer ; B', electro- magnet; S', binding screw touching the steel spring (H); S" and S"', binding screws to whicli are attached wires wlaen Neef's hammer is not required. 3. Ordinary Hand Electrodes. — Take two pieces of flexible gutta- percha coated wire (No. 20) 60 cm. long, and two pieces of thick glass tubing 8 cm. long, and with a bore sufficient to admit the wire. Push a wire through each tube, and allow the end of the wire to project 3 cm. beyond the tube, scrape the gutta-percha off the free ends of both wires. Fix the wires in the glass tube with sealing-wax, and with a well-waxed thread bind the two tubes together. A very handy holder is made by thrusting two fine insulated wires (No. 36) through the bone handle of a crotchet needle. 4. For some purposes "shielded electrodes" are used, i.e., the platinum terminals are exposed only on one side, the other being sunk in a piece of vulcanite (Fig. 61, E). 5. Du Bois-Reymond's Electrodes (Fig. 47). — The two wires end in triangular pieces of platinum (P), which rest on a glass plate. The whole is supported on a stand (V), and can be moved in any direction by the universal joint (B). INDUCTION MACHINE — ELECTRODES. 141 6. For Non-polarisable Electrodes see Lesson XXXVIII., 2. 7. Polarisation of Electrodes — Apparatus. — Pair of ordinary electrodes, two wires, du Bois key, spring key, Daniell's cell, frog, and instruments. Fig. 47. — Du Bois-Reymond's Platinum Electrodes. The nerve is placed over the two pieces of platinum, P, which rest on glass ; B, universal joint; V, support. (a.) Pith a frog (Lesson XX., 1), lay it belly downwards on a frog plate, and expose one sciatic nerve. (b.) Clamp the du Bois key to the table, place the elec- trodes under the sciatic nerve, and connect their other ends each with the outer binding screw of the brass plates in the du Bois key. Close the key, and observe that no con- traction of the leg muscles occurs. (c.) By two wires connect a Daniell's cell with the du Bois key, introducing a spring key in the circuit. Open the key to allow the constant current to pass through the nerve for three minutes or thereby, and observe that there is no con- traction as long as the constant current is passing. Close the key, i.e., short-circuit the battery, and at once a contrac- tion occurs. Remove the battery, close and open the key. 142 EXPERIMENTAL PHYSIOLOGY. Contractions occur, but they gradually get feebler. The contractions are clue to polarisation of the electrodes. LESSON XXV. SINGLE INDUCTION SHOCKS— INTER- RUPTED CURRENT— BREAK EXTRA- CURRENT— HELMHOLTZ'S MODIFICATION. 1. Single Induction Shocks — Apparatus. — Grove's cell charged, induction machine, live wires, two du Bois keys, and ordinary electrodes. (a.) Connect one wire from the battery to the binding screw, s", of the induction machine (Fig. 46). Join the other wire from the battery to a du Bois key, and use the third wire to connect the key with the binding screw, s"\ The key is used according to the first method, i.e., for make and break, so that the primary current can be made or broken at will. To the binding screws of the secondary coil attach two wires, and connect them to the short-circuiting du Bois key, and to the latter the electrodes as in Fig. 54. (6.) Open the short-circuiting key, push the secondary coil pretty near to the primary, and place the points of the electrodes on the tip of the tongue, or hold them between the forefinger and thumb moistened with water. Close the key in the primary circuit, i.e., make the circuit, and instan- taneously at the moment of making, a shock is induced in the secondary coil, R", and is felt on the tip of the tongue or finger. It is called the closing or make induction shock. All the time the key is closed the galvanic current is cir- culating in the primary spiral, but it is only when the primary current is made or broken that a shock is induced in the secondary spiral. (c.) Break the primary current by raising the key, and THE BREAK EXTRA-CURRENT OF FARADAY. 143 instantaneously a shock is felt as before ; this is the opt-ning or break induction shock. ((/.) Observe that the break is stronger than the make shock. Push the secondary coil a long distance from the primary, and with the electrodes on the tongue, make and break the primary circuit, and gradually move the secondary near the primary coil. Observe that the break shock is felt first, and on pushing the secondary nearer the primary coil both shocks are felt, but the break is stronger than the make shock. 2. Interrupted Current by using Neef s Hammer — Apparatus. — The same as for single shocks. (a.) Connect the battery wires as in Fig. 46, i.e., to the binding screws in the erect pillars, P' ( + ) and P" ( - ). In- troduce a du Bois key as for the make and break arrange- ment. The automatic vibrating spring, or Neef's hammer, is now included in the primary circuit. Set the spring vibrating. Make the current by depressing the handle of the key. The elastic spring, H, is attracted by the temporary magnet, B', thus breaking the contact between the spring, H, and the screw, S', and causing a break shock in the secondary coil. B' is instantly demagnetised, the elastic spring recoils and makes connection with S', and causes a make shock. Thus a series of make and break induction shocks following each other with great rapidity is obtained, but the make and break shocks are in alternately opposite directions. (b.) While Neef's hammer is vibrating, apply the electrodes to the tongue or finger as before, noting the effect produced and how it varies on altering the distance between the secondary and primaiy spirals. 3. The Break Extra- Current of Faraday. — When a galvanic current traversing the primary coil of an induction machine is made or broken, each turn of the wire exerts an inductive in- fluence on the others. When the current is made, the direction of the extra current is against that of the battery current, but at break it is in the same direction as the battery current. — Appara- tus. — Daniell's cell, two du Bois keys, five wires, induction coil, ordinary electrodes (and nerve muscle preparation). (a.) Arrange the apparatus according to the scheme 144 EXPERIMENTAL PHYSIOLOGY, Fig. 4S. — Scheme of the Break Ex- tra-current.—B. Battery ; K and K', keys ; P, primary coil ; N, nerve. (Fig. 48). Notice that lioth keys and the primary coil of the induction machine are in the primary circuit, both keys being so arranged that either the primary coil, P, or the electrodes attached to key K', can be short-cir- cuited. (6.) Test (a) either by elec- trodes applied to the tongue, or (/3) by means of a nerve muscle preparation (/3 to be done after the student has learned how to make a nerve- muscle preparation). (c.) Close the key, K, thus short-circuiting the coil. Open and close key, K'. There is very little efiect. (d.) Open K', the current passes continuously through the primary coil. Open key K, a marked sensation is felt, due to the break J feS extra-current. 4. Helmholtz's Modification. — The break shock is stronger than the make, and to equalise them Helmholtz devised the following modification : — (a.) Connect the battery wires as before to the two pillars (Fig. 46), P' and P", or to a and e (Fig. 49). In Fig. 49 connect a wire from a to J, thus bridging or " short-cir- cuiting" the interrupter. Elevate the screw {/) out of reach of the spring (c), but raise the screw (d) until it touches the spring means the make and break Fig. 49 . — Helmholtz's Modification of Neef's Hammer. — As long as c is not in contact with d, g h remains magnetic; thus c is attracted to d, and a secondary circuit, a, h, c, d, e is foi-med ; c then springs back again, aud thus the process goes on. A new wire is introduced to connect a with /. K, battery. at every vibration. By this shocks are nearly equalised, but both shocks are weaker. CILIARY MOTION. 145 LESSON XXVI. PITHING— CILIARY MOTION— NERVE- MUSCLE PREPARATION- NORMAL SALINE. 1. To Pith a Frog. — Wrap the body, fore and hind legs, in a towel, leaving the head projecting. Grasp the towel enclosing the frog with the little, ring, and middle fingers and thumb of the left hand, leaving the index finger free. With the index finger bend down the frog's head over the inner surface of the second finger until the skin over the back of the neck is put on the stretch. With the nail of the right index finger feel for a depression where the occiput joins the atlas, marking the position of the occipito-atlantoid membrane. With a sharp, narrow knife held in the right hand, divide the skin, membrane, and the medulla oblongata. Withdraw the knife, thrust a " seeker " into the brain cavity through the opening just made, and destroy the brain, To prevent oozing of blood, a piece of a wooden match may be thrust into the brain cavity. If it is desired, destroy also the spinal cord with the seeker or a wire. The knife used must not have too broad a blade, else two large blood-vessels will be injured. The operation should be performed without losing any blood. 2. Ciliaiy Motion. (a.) Pith a frog, destroying the brain and the spinal cord. Place the frog on its back. Divide the lower jaw longitudi- nally, and carry the incision backwards through the pharynx and CESophagus. Pin back the flaps. Moisten the mucous membrane, if necessary, with normal saline. (b.) Make a small cork flag, and rest it on the mucous membrane covering the hard palate between the eyes. It will be rapidly carried backwards by ciliary motion towards the stomach. Repeat the experiment, and determine the time the flag takes to travel a given distance. (c.) Grains of charcoal or Berlin blue are carried back- wards in a similar manner. 10 146 EXPERIMENTAL PHYSIOLOGY, (d.) Apply heat to the preparation, and observe that the cork travels much faster. 3. Anatomy of the Nerve-Muscle Preparation. — Before making Fig. 50. — The Muscles of the left leg Fig. 51. — Distribution of the Sciatic Nerve (I.) of tlie Frog (see also Fig. 50). — s.t., Semi-ten din osus ; ad'", adductor magnus ; (II.) its tibial; and (III. ) peroneal divisions. of a frog from behind. — c.i. , Coccy- geo-iliacus ; gl, gluteus ; p, pjrri- formis ; r.a., rectus anterior ; v.e., vastus externus ; tr, triceps ; r.i.", rect. int. minor ; s.m., semi-mem- branosus ; b, biceps ; g, gastro- cnemius ; La., tibialis anticus ; pe, peroneus. this preparation the student must familiarise himself with the anatomy of the lower limb of the frog. On a dead frog, or on a NORMAL SALINE. 147 'dissection of a frog already prepared, study the arrangement of the muscles, as shown in Fig. 50. The skin of the frog is sup- posed to be removed, and the frog placed on its belly, and the muscles viewed from behind. On the outside of the thigh, the triceps femoris {t.r), composed of the rectus anterior (r.a), the vastus externus (v.e), and the vastus internus, not seen from behind. On the median side, the semi-memhranosus (s.m), and between the two the small narrow biceps (b). Notice, also, the coccygeo-iliacus (c.i), the gluteiis (gl), the 2^y'>'iformis (jj), and the rectus internus minor (r.i). In the leg, the gastrocnemius (g), with] its tendo achillis, the tibialis anticus (t.a), and the peromrus {pe). 4. Make a Dissection. (a.) Remove the skin from the leg of a dead frog ; with a blunt needle, called a " seeker " or a " finder," gently tear through the fascia covering the thigh muscles, and with the blunt point of the finder separate the semi-membranosus from the biceps, and in the interval between them observe the sciatic nerve and the femoral vessels. Carefully isolate both, beginning at the knee, where the nerve divides into two branches — the tibial and peroneal — and work upwards (Fig. 51). (6.) Follow the nerve right upwards to its connection with the vertebral column, and observe that it is necessary to divide the pyriformis {}>) with small scissors, and also the ilio-coccygeal muscle, when the three spinal nerves — the 7th, 8th, and 9th — which form the sciatic nerve, come into view. 5. Normal Saline. — Dissolve 7 '5 grammes of dried sodic chlo- ride in 1000 cc. of distilled water. This is the best fluid to use to moisten tissues. 148 EXPEKIMEXTAL PHYSIOLOGY. LESSON XXYII. NERVE-MUSCLE PREPARATION— STIMU- LATION OF NERVE— MECHANICAL, CHEMICAL, AND THERMAL STIMULI. 1. Prepai'e a Nerve-Muscle Preparation — Apparatus. — A frog, pithing-needle, or " seeker " in liandle, narrow-bladed scalpel, a small and a large pair of scissors, forceps, towel, and a porcelain plate on which to place the frog. (a.) Pith a frog with a pithing-needle, destroying the brain and spinal cord, and place the frog on its belly on a porcelain plate. With the small scissors make an incision through the skin along the back of one thigh — say the left — from the knee to the lower end of the coccyx, and prolong the incision along the back, a little to the left of the uro- style. Reflect the skin, which is readily done, as there are large lymph sacs between it and the muscles, thus exposing the muscles shown in Fig. 50. (b.) Gently separate the semi-membranosus and biceps with the "seeker," and bring into view the sciatic nerve and femoral vessels. Still working with the seeker, and begin- ning near the knee, clear the sciatic nerve of any connective tissue around, but on no account is the nerve to be touched with forceps, nor is it to be scratched or stretched. With the small pair of scissors divide the pyriformis and ilio- coccygeus, and ti'ace the nerve up to the vertebral column. (c.) With a sharp-pointed, large pair of scissors divide the spinal column above the seventh lumbar vertebra; seize the tip of the urostyle with forceps, raise it, and with the strong scissors cut it clear from all its connections as far as the last lumbar vertebra, and then divide the urostyle itself. Divide the left iliac bone above and below, and remove it with the muscles attached to it. The lumbar plexus now comes nerve-:mu.scle preparation. 149 clearly into view. Bisect lengthways the three lower ver- tebrae, and use the quadrilateral piece of bone by which to manipulate the nerve. With the forceps lift the fragment of bone, and with it the sciatic nerve ; trace the latter down- wards to the knee, dividing any connections and branches with fine scissors. If the parts tend to become dry, moisten the whole preparation with normal saline solution. (d.) Divide the skin over the gastro- cnemius, and expose this muscle. Divide the tendo achillis below its libro-cartilage, lift the tendon with a pair of forceps, and detach the gastro- cnemius from its connections as far up as the lower end of the femur. Cut across the knee-joint, and remove the tibia and fibula with their attached muscles. Taking care to preserve the sciatic nerve from injury, clear the muscles away from the lower end of the femur, and then divide, with the large pair of scissors, the femur itself about its middle. This preparation (Fig. 52) consists, therefore, of the gastrocnemius, and the whole length of the sciatic nerve, to which is at- tached a fragment of bone, by which the preparation can be manipulated without injuring the nerve. Fig. 52. — Nerve-muscle Preparation. — S, Sci- atic nerve — the frag- ment of the spinal column is not shown ; F, femur ; and I, tendo achillis. iV^.i)'. — The nerve must not be touched with instruments, neither stretched nor scratched, nor allowed to come into contact with the skin, and it must be kept moist with normal saline. (a.) Another method is sometimes adopted. Pith a frog. With the left hand seize the hind limbs and hold the frog with its belly downwards. With one blade of a sharp- pointed pair of scissors transfix the body immediately behind the shoulder-blades, and divide the spinal column. The head now hangs down, and by its weight it pulls the ventral from the dorsal parts. (6.) With the scissors divide the wall of the abdomen on both sides parallel to the vertebral column, and remove all 150 EXPERIMENTAL PHYSIOLOGY, the abdominal viscera. With the left hand seize the upper end of the divided spinal column, and with the right the skin covering it, and pull. At once the whole lower end of ; the trunk and the lower limbs are denuded of skin. (c.) Take the thigh muscles between the thumb and fore- finger of the left hand, and with the point of one blade of a pair of scissors tear through the fascia between the biceps and semi-membranosus to expose the sciatic nerve, and then proceed as directed in 1. 2. Stimuli may be classified as follows : — (1.) Mechanical — e.g., cutting or pinching a nerve or muscle. (2.) Chemical — e.g., by dipping the end of a nerve in a saturated solution of common salt. (3.) Thermal — e.g., applying the end of a heated wire to the nerve. (4.) Electrical — (a.) Continuous current. (6.) Single induction shocks, (c.) Interrupted current. 3. Stimulation of Muscle and Nerve. — It is convenient to modify somewhat the physiological limb, in order to render the muscular contraction more visible. — Apparatus. — Frog-pithing needle, scalpel, scissors, forceps, straw-flag, pins, muscle-forceps, camel's-hair brush, saturated solution of common salt in a glass thimble, ammonia, copper wire, spirit lamp or gas flame. 4. Mechanical Stimulation. (a.) Pith a frog, destroying its brain and spinal cord. Remove the skin, and proceed as directed by the method (Lesson XXVII., 1) for preparing a nerve-muscle prepara- tion, as far as the isolation of the sciatic nerve, but modify the subsequent details as follows : — [h.) After the nerve is cleared as far as the spine, clear all the muscles away from the femur, and divide the latter CHEMICAL STIMULATION. 151 about its middle. Divide the sciatic nerve as high up as possible. The preparation consists of the leg, the lower end of the femur, and the sciatic nerve, ter- minating in the leg muscles. Pin a straw-flag to the toes by means of two pins. Fix the femur in a clamp or pair of muscle-forceps sup- ported on a stand, and shown in Fig. 53, taking care that the gastro- cnemius is upwards. The nerve hangs down, as shown in the figure, Yi<^. 53.— Straw-flag at- and must always be manipulated tached to a frog's leg with a cam el' s-hair brush dipped in fixed in a clamp. N, normal saline. Nerve; F. flag. (c.) Pinch the free end of the nerve sharply with forceps, the muscles contract and the straw-flag is suddenly raised- Cut ofl" 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. («.) 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 ofl" 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 saHne solution, the nerve floats on the surface, but sufiicient salt difiuses 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 aSected by the salt, and the spasms will cease. 152 EXPERIMENTAL PHYSIOLOGY. (6.) 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 Avhole 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 tlieir 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 lirst 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. («.) 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 Fi" 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- stant current. — B, bat- tei-y ; 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° C. 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 ofi" the long narrow sartorius from the inner side of the thigh. Stretch it on a slip of glass (Fig. 56, s). (b.) 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. Fig. 56. — Muscles of the left leg of a frog seen from the front. — ij), ileo- psoas; s, sartorius; ad', adductor longus ; vi, vastus intemus (see Kgs. 50 and 51). 6. Unipolar Stimulation— Apparatus. — Daniell's cell, induction machine, du Bois key, muscle-chamber, four 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 FLEISCHLS EHEONOM. 155 electrodes are attached. See that the battery and induction machine are perfectly insulated by supporting them on blocks of paraffin. (6.) Prepare a nerve-muscle preparation, and arrange it in the muscle-chamber in the usual way, laying the nerve over the electrodes. One ot the electrodes will therefore be con- nected with the secondary circuit. (c.) Make and break the primary circuit, there is no con- traction. (c?.) 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. ^ig 57.__Fleischrs Rheonom. 156 EXPERIMENTAL PHYSIOLOGY. (a.) Connect two Daniell'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 XXVIIL, 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. Du'ect and Indirect Stimulation of Muscle. — When the stimulus is applied directly to the muscle itself, we have direct Btimulation ; 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 XXVIIL, 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 MUSCULAH 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. (b.) 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 pan will be a kilo, more or less, according to the size of the frog. (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. {b.) 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 Cui'ai'e. — Curare 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). (b.) 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. Therefore, curara has i^aralysed the voluntary motor nerves, hut not the imtscles. 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, Avhich 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. Rosenthal's 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). (&.) 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 Avires 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 ; clamp ; N P, non -poisoned leg ; poisoned leg ; C, commutator ; key. F, P, K, pohl's commutatok. 161 (i.) Set Neefs hammer going, and turn the handle of the commutator so that the current passes through both nerves; only tlie non-poisoned leg (X 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 muscles. 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 tlie non- jjoisoned 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 dilferent „. ^ -g „ , ,, . q__ wires, the cross-bars are removed and wires niutatoi- with cross- are attached to all six cups, 3 and 4, 5 and 6. bars in. On turnincr 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 desii'es, 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 covered with smoked glazed paper, or other moving surface is re- quired. The velocity of the moving surface is usually determined by recording simultaneously the vibrations of a tuning-fork, of known rate of vi- ' V 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 Lud wig's form (Fig. GO), 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. — Liiclwig's revolving cylinder, E., moved taught at once, one by the clock-work in the box, A, and regulated jnust have recourse to by a Foiieault's regulator on the top of the , ,. box. The disc, D, moved by the clock-work, ^^^ arrangement oi presses u]ion the wheel, n, which can be sliattmg, 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 which cylinder to rotate at different rates. The ^^^^^^i j^ums can be cyhntler itself can be raised by the handle, t. , . , -i r\ On the left side 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 Ijy 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 througii 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, gum juniper may be used instead of mastic. (c.) Dissolve 4 oz. of sandarac in 15 oz. 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 llame, and fix it in position. (6.) 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 til^ia and tibula. 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. ^lanipulate 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 Avhen 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 Lreak the primary circuit by means of the Morse key in it. Oljserve 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 conti'action 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 Ijy 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." (b.) Vaiy 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 JIYOGItAPH. 1G7 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 jNIultiply this by the weight lifted, and the product is the work done expressed in gram-millimetres. 10. The Crank Myograph (Fig. G2) 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- 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. (lu 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, tinder 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 " (C). Test this. (b.) Arrange the induction apparatus for single shocks as in Fig. 63, but shortrcircuit 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 MUSCl'LAR CONTRACTION. 169 (c.) Make a nerve-muscle preparation, tix tlie 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 the writing - style of the lever on the smoked sur- face. Connect the secondary spiral either with t\w ■ 63. — Scheme of the arrangement of the pendulum. — B, Battery ; I., pi'imary, II., secondary spiral of the induction machine; S', tooth ; K', key ; C, C, catches ; K' in the comer, scheme of K' ; K, key in primary circuit. 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. (/i.) Remove the paper, varnish the tracing, hang it up to dry, and next day measure the tracing. Bring ordinates vertical, a', b', 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. 250 DV. Fig. 64. — Pendulum Myograpli Curve. — S, Point of stimulation ; A, latent period ; B, peiiod 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 MYO(iUAl*H. 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 liy means of the catch. Tc.'f^^ 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 (e.) 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 tix 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, 120 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. (a.) 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 vil)ration, and driven by means of a battery (Fig. 68). The three recording levers are all fixed on the same Fig. 66. — Arrangement for Estimat- ing tlie Time-Relations of a Single Muscular Contraction. — B, battery ; K, key in primary circuit ; I., pri- mary, II., secondary spiral, without a short-circuiting key ; I, muscle lever ; e, electro-magnet in primary circuit ; t, electric signal ; St, sup- port ; KC, revolving cylinder. stand, which should prefer- ably be a tangent one — i.e., the rod beai'ing the re- cording styles can by means of a handle be made to rotate so as to luring 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 tlie 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-Kecorder (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. ^^^^-. Fig. ()7. — 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 sianal Fig. 68. — .Sii,'nal and Vibrating Tnning-Fork in an Electric Circuit. — D, Drum ; C, signal ; EM, electric tuning-fork ; Ft, 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 witli a crank-myograpli as in Lesson XXXI., 10, but do not remove the skin of the leg. Take a tracing at the normal temperature. (b.) 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-lam]^. Take a tracing. The contraction is shorter than in 1 (b.) Do not overheat the muscle. {d.) A piece of thin gas piping can be bent, and the muscle laid on it. M^ater 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. (/) 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. (32). INFLUENCE OK VEKATUIA OX 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 orank-my ograph . (b.) Take a tracing with the muscle weighted with the lever only. (r.) Then load the lever successively with ditferent weights 250 DV. Fig. 69. — Peuduhim 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). Xorinal Muscle Curve (Lower). 3. Influence of Veratria on Contraction. {a.) Destroy tht^ brain of a frog, and inject into the ventral lymph sac 10 minims of a freshly-prepared 0*1 per cent, solution of veratria. 176 EXPERIMENTAL PHYSIOLOCtY. {f>.) 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. (6.) 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 weicrht 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. (6.) 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 {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. 12 178 EXPERIMENTAL PHYSIOLOGY, LESSON XXXV. FATIGUE OF MUSCLE. 1. Fatigue of Muscle. («.) 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 j^^^^^ ^^ record stimulated with maximal induction shocks . and every fifteenth contraction recorded. ^^ ^ revolving drum. (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 long as possible (N), clamp the femur (/), attach the tendon {m) 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 paper, 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 cm., and the time required for the impulse to travel from 6 to a to be y|^ sec. Then we have 4 : 100 : -j^ : ^", 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. (&.) Stimulate the nerve at A with a strength of current that gives just a minimal contraction. Reverse the commu- 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 (X) 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) terininates a nerve. The gracilis after excision must be laid on a glass plate with a hlach 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- traction occurs in both L and K. This ^% So.-Kbhne's , , , , • , 1 1 , • Experiment on can be due only to centripetal conduction ^j^g 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 COLD —SECTION OF THE HEART. 1. The Heart of the Frog and how to expose it. (a.) Pith a frog, aiid 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 ofi* 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 Inilbus aorta^ (B), which projects in front of the right auricle, and divides into two aortas — right and left, the left l)eing the larger (Fig. 96). Fig. 96. — Frog's Heart from the front.— V, Single ventricle ; Ad, As, right and left auricles ; 13, bulbus arteriosus ; 1, carotid ; 2, aorta ; 3, pulmocutaneous ar- teries ; C, carotid gland. Fig. 97. — Heart of Frog from be- hmd. — s.v. Sinus venosus opened; c.i, inferior; c.s.d, c.s.s, right and left superior vente cava^ ; v.p, pulmon- arj' vein ; Ad and As, right and left auricles ; Ap, communication between the rioht 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 cavfe (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 " frjenum," containing a small vein, passes from the peri- cardium to the posterior aspect of the ventricle. Divide it with a iine pair of scissors. Count the number of beats per minute. Seize with forceps the pai't of the frasnum attached to the ventricle, and lift up tlie heart therewith ; and with a sharp pair of scissors cut out the heart l)y dividing the inferior vena cava, the two superior vena^ cavte, and the two aort£e. Place the excised heart in a watch-glass, and cover it with another watch-glass. (6.) 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°0. 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. (b.) 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 venti-icle at its upper third just below the auriculo- ventricular groove. Observe that the auricles with the upper thii-d 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. '2\\ 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, Avhich 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 aortte, at the same time becoming pale, while its apex is tilted forwards and upwards. As the auricles continue to till 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 ol)servation 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 aortte immedi- ately following the ventricular systole. (cZ. ) The diastolic phase or pause when the whole heart is at rest before the auricles begin to contract. (e.) Ligature the fra?num 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 aortfe, 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 tilling. 212 EXPERIMENTAL PHYSIOLOGY. LESSON X L Y. 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 tlien 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. (h.) 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 papei', 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 frrenum, 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-l)OX, 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 recoi'd 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. {h.) 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 rHYSIOLOGY. 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 aortas from the auricles, and with an aneurism needle pass a moist stout thread be- tween the two aortse and the superior vense cava?, turn up the apex of the heart, divide the freenum, 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 aftei- it is amputated, the venti"icle begins to beat. 2, Intra-Cardiac Inhibitory Centre. (a.) Expose the heart in a pithed frog, tie a fine silk ligature round the frtenum, 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-sliaped "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 XLVIII., 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 pai^allel cuts into three portions. The middle portion con- tains the auricular septum, in which lie ganglionic 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 216 EXPERIMENTAL PHYSIOLOGY. the sternum, and iniW 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 cesophagus 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 Fig. 98. —Scheme of the Dissection of the Frog\ Vagus. — SM, Submentalis; LU, lung ; V, vagus ; GP, glossopharyngeal ; H, hypoglossal ; L, Laryngeal ; PH, SH, GH, OH, petro-, sterno-, genio-, omo-hyoid ; HB, hyoid ; HG, hyoglossus ; H, heart ; BR, brachial plexus. with a blood-vessel (V), and still further forward is the glosso- pharyngeal (GrP). 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 inhihition, is manifest in the curve by the lever recording merely a straight line. If Heart Beat. M I M I I I I I I I I I I _j Stimulation Fig. 99. — Va^us Curve of Frog's Heart. the lai-yngeal 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 heart-lever. (h.) Arrange an induction machine for an interrupted current, and keep Neef's hammer vibrating. Into the secondar?/ 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 on 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. ((/.) Short-circuit the secondary current again, and observe how the heart gradually resumes its usual rhythm, sinus venosus, auricles, and ventricle. (e.) Repeat (c.) several times, noting that the heart after arrest goes on beating in spite of continued stimulation. 4. Gaskell's Method. — Instead of recording the movements of the heart by means of a lever resting on it, a very convenient method is that of Gaskell. (a.) An ordinary writing-lever is placed above the frog- plate on which the frog rests, and supported in the horizontal position by a thin thread of india-rubber, as in Fig. 106. Expose the heart of a pithed frog, and leave it in situ. Tie a thread to the apex of the ventricle, clamp the aorta to fix the heart in position, and then attach the apex thread to the lever. Every time the heart contracts it pulls down the lever, and the latter is brought into position again, when the heart relaxes, by the piece of elastic. 5. Action of the Sympathetic on the Heart of the Frog. (a.) Pith a frog or preferably a toad, cut away the lower jaw, and continue the slit from the angle of the mouth downwards for a short distance. Turn the parts well aside, and expose the vertebral column where it joins the skull. Remove the mucous membrane covei'ing the roof of the mouth. The sympathetic is easily found before it joins the vagus emerging from the cranium (Fig. 100). Carefully isolate the sympathetic. It lies immediately under the levator anguli scapulpe, which must be carefully removed with fine forceps when the nerve comes into view, usually lying under an artery. Put a ligature round it as far away from the skull as practicable. ACTION ON TIIK HEAUT OF THE FROG. 219 (b.) Expose the heart and attach its apex to a lever sup- ported by an elastic thread as in GaskelFs method. Record several contractions, and then stimulate the sympathetic with interrupted shocks by means of fine electrodes. The heart beats quicker. If the heart is beating quickly, reduce the number of beats by cooling it. (c.) If desired, the vagus may be isolated and stimulated, and the effects of the two nerves compared (although the vagus outside the skull is really the vago-sympathetic). Fig. 100. — Scheme of the Frog's kSympathetic. — LAS, Levator anguli scapulpe ; vSym, sympathetic ; GP, glossopharyngeal ; V-S, vago-sym- pathetic ; G, ganglion of the vagus ; Ao, aorta ; SA, subclavian artery (Gadell). Stimulation of the intra-cranial vagus — i.e., before it is joined by the sympathetic — is somewhat too difficult for the average student, and is therefore omitted here. LESSON XLVIII. ACTION OF DRUGS AND THE CONSTANT CURRENT ON THE HEART— DESTRUCTION OF THE CENTRAL NERVOUS SYSTEM. 1. Action of Drugs on the Heart— Muscarin and Atropin. — Either the excised heart, placed in a watch-glass, or the heart, in sittc, may be used. 220 EXPERIMENTAL PHYSIOLOGY. (a.) Pith a frog, expose its heart, and with a fine pipette apply a drop of serum or normal saline containing a trace of muscarin, which rapidly arrests the rhythmical action of the heart, the ventricle being relaxed — i.e., in diastole — and dis- tended with blood. (b.) After a few minutes, with another pipette apply a few drops of a 0"2 per cent, solution of sulphate of atropia in normal saline, the heart gradually again begins to beat rhythmically. (c.) Ligature and divide the fra?num, raise the heai't by the ligature, and faradiso the crescent or inhibitory centre ; the heart is no longer arrested, because the atropin has paralysed the intracardiac inhibitory mechanism. (d.) In another frog, arrest the action of the heart with pilocarpin, and then apply atropin to antagonise it, observing that the heart beats again after the action of atropin. 2. Effect of a Constant Current on the Heart. («.) Expose a pithed frog's heart. Cut out the heart, dividing it below the auriculo-ventricular groove, thus obtaining an "apex" prepar- ation which does not beat H /-b; spontaneously. (6.) By means of sealing- wax, fix a cork to a lead base 0 cm. square, cover the upper end of the cork with sealing- wax, and thrust through it two wires to serve as elec- trodes, about 4 mm. apart (Fig. 101). Cover the whole with a beaker lined with moist blotting-paper. Place the heart-apex with its base against one electrode, and its apex against the other. Fig. 101. ^Support for Fro Heart. — E, Electrodes; heart. H, (c.) Arrange two Daniell's cells in circuit, connect them with a key, and to the latter attach the electrodes. Pass a continuous current in the direction of the apex. The heart THE STAIRCASE. 221 resumes its rhythmical beating, and continues to do so as long as the constant current passes through the living pre- paration. 3. The Staircase. (a.) To a glass slide used for microscopic purposes (3 x 1) fix with sealing-wax two copper wires in the long axis of the slide, and let their two free ends be about 3 milli- metres apart. They will act as electrodes. Connect the other ends of the wires to a du Bois key introduced into the secondary circuit of an induction machine. Arrange the primary coil for single induction shocks, introducing a Morse key in the circuit. (b.) Expose the heart of a frog, make an " apex prepara- tion," and place it on the electrodes on the glass slide. Rest on the heart a heart-lever properly balanced and arranged to record its movements on a slow-moving drum (5 mm. per second). The preparation does not contract spontaneously, but responds to mechanical or electrical stimulation. (c.) Stimulate the apex preparation with single break induction-shocks at intervals of about ten seconds. To do this un-short-circuit the secondary circuit, depress the Morse key, short-circuit the secondary circuit, and close the Morse key again. Repeat this several times every ten seconds, and note that the amplitude of the second contrac- tion is greater than the first, that of the third than the second, the third than the fourth, and then the successive beats have the same amplitude (Fig. 102). Allow the heart- apex to rest for a few minutes, and repeat the stimulation. Always the same result is obtained. From the graduated rise of the first three or four beats after a period of rest, the pheno- ^ ^_ menon is known as the " staircase." The „. ^^.^ c<. . ^ . , , . , . Fig. 102.— Stan-case increment is not equal m each successive chai-acterof Heart beat, but diminishes from the beginning Beat, to the end of the series. ((7.) If, while the apex is relaxing, it be stimulated by a 222 EXPERIMENTAL PHYSIOLOGY. closing shock, it contracts again, so that the lever does not immediately come to the abscissa. (e.) If the Morse key be rapidly tapped to interrupt the primary current, the contractions become more or less fused, and the lever remains above the abscissa writing a sinuous line. 4. Effect of Destruction of the Nervous System on the Heart and Vascular Tonus. (a.) Destroy the brain of a frog, and expose its heart in the usual way, taking care to lose no blood; note how red and full the heart is with blood. (b.) Suspend the frog, or leave it on its back, introduce a stout pin into the spinal canal, destroy the spinal cord, and leave the pin in the canal to prevent bleeding. Observe that the heart still continues to beat, but it is pale and collapsed, and apparently em/jtij, it no longer fills with blood. The blood remains in the greatly dilated abdominal blood- vessels, and does not return to the arterial system, so that the heart remains without blood. If the belly be opened, the abdominal veins are seen to be filled with blood. (c.) Amputate one limb, perhaps not more than one or two drops of blood will be shed, while in a frog with its spinal cord still intact, blood flows freely after amputation of a limb. LESSON XLIX. PERFUSION OF FLUIDS THROUGH THE HEART— PISTON RECORDER. 1. Perfusion of Fluids through the Heart. The Fluid. — (a.) Take two volumes of normal saline, add one volume of defibrinated sheep's blood, mix, and filter. See that the blood is thoroughly shaken up with air before mixing it. This is the best fluid to use. PREPARATION OF THE HEART. i>2:3 (&.) Rub u}) in a mortal- 4 grammes of dried ox blood (this can be purchased) witli GO cc. of normal saline. Allow it to stand some time, add 40 cc. of water, and filter. (c.) Ringer's Fluid.— Take 90 cc. of -6 per cent. NaCl solution, saturate it with calcic phosphate, and add 10 cc. of a 1 per cent, solution of potassic chloride. 2. Preparation of the Heart. («.) Pith a frog, expose its heart, ligature and divide the frtenum behind the ligature. (/;.) Take a two-wayed cannula (Fig. 103), attach india- rubber tubing to each tube, and till the tubes and cannuhe with the fluid to be perfused. Pinch the india-rubber tubes with fine bull- dog forceps to prevent the escape of the fluid. (c.) Tie a tine thread to the apex of the ventricle. To this thread a writing- lever is to be attached. (d.) By means of the frsenum ligature raise the heart, with a pair of fine scissors make a cut into the sinus, and through the opening introduce the double cannula passed through a cork, until its end is well within the ven- tricle. Tie it in with a ligature, the ligature constricting the auricles above the auriculo-ventricu- lar groove, thus making what is known as a "heart-prepara- tion." Out out the heart with its cannula. (e.) In a filter-stand arrange a glass funnel, with an india- rubber tube attached, at a convenient height, fill it with the perfusion fluid, clamp the tube. Attach this tube to one of the tubes — the inflow — connected with one stem of the cannula, taking care that no air-bubbles enter the tube. Adjust the height of the reservoir so that the fluid can flow freely through the heart, and pass out by the other tube of the cannula. Place a vessel to receive the outflow fluid. After a short time the heart will begin to beat. Fig. 103. — Cannula for Frog's Heart. 224 EXPERIMENTAL PHYSIOLOGY. {,/'.) Place the heart in a cylindrical glass tube, fixed on a stand, and arranged so that the cork in which the cannula is fixed fits into the mouth of the tube. A short test-tube does perfectly well. The lower end of the glass tube has a small aperture in it through which the thread (c) is passed, and attached to a writing-lever arranged on the same stand as the glass vessel. See that the lever is hori- zontal, and writes freely on a slow-moving recording drum. Every time the heart contracts it raises the lever, and during diastole the lever falls. In this way it is possible to use various fluids for perfusion. The fluids may be placed in separate reservoirs, each communi- cating with the inlet tube, and capable of being shut ofl:" or opened by clamps, as required. Further, by poisoning the supply fluid with atropin, muscarin, spartein, or other drug, one can readily ascertain the eflect of these drugs on the heart, or the antagonism of one drug to another. Instead of a glass funnel as a reservoir for the fluid, one may use a Marriotte's flask (Fig. 104), the advantage being that the pressure of the fluid in the inflow tube is constant. Another simple arrangement is to have a bird's water-bottle, with a curved tube leading from it to the inflow tube of the cannula. 3. Piston-Recorder (of Schafer). The heart is tied to a two-way cannula as before, and is introduced into a horizontal tube with a dilatation on it. The tube of the recorder is filled with oil, and as the heart dilates it forces the oil along the tube and moves a light piston resting on it. When systole takes place, the oil recedes, and with it the piston. The piston records on a slow-moving drum placed horizontally. LESSON L. ENDOCARDIAL PRESSURE— APEX PREPARATION. 1. Endocardial Pressure in the Heart of a Frog. {a.) Proceed as in the previous experiment (a.), (6.), (omit APEX PREPARATION. 225 104. (b.) Arrange a frog's mercury manometer provided with a writing-style as in Fig. 104. Attach the inlet tube of the cannula to the Marriotte's flasks {a, b), and connect the outflow with the tube of the mercury manometer. It is well to have a T-tube between the heart and the manometer, but in the heart apparatus, as shown and used, the exit tube is preferable. See that no air-bubbles are present in the system. Every time the heart contracts the mercury is displaced and the writing-style is raised, and records its movements on a slow-moving drum. (c.) Take a tracing with the outflow tube and Marriotte's flask shut otf", so that the whole effect of the contraction of the heart is exerted upon the mercury in the manometer. Take another tracing when the fluid is allowed to flow continuously through the heart. The second Marriotte's flask shown in the figure, is for the pei'fusion of fluid of a different nature, and by means of the stop-cock (*■) one can pass either the one fluid or the other through the heart. The little cup ((/) under the heart can be raised or lowered, and filled with the nutrient fluid, and in it the heart is bathed. 2. Apex Preparation. — In this preparation of the heart only the apex of the heart is used. As a rule, it does not beat spontane- ously until sufiicient pressure is applied to its inner surface by the fluid circulating through the heart. (a.) Proceed as in Lesson XLIX., 2, (a.), (6.) (omitc. ), (d.), with this difference, that in (. Varnish, 164. Vascular tonus, 222. Veratria, 175. Vision — Physiology of, 261. Visual judgments, 281. Vital capacity, 249. Vogel's lactoscope, 73. Volumetric processes, 87. „ ,, DuprG'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 soimds, 254. Wave-lengths, 37. Weyl's test, 106. Whistle— Galton's, 302. Wliite of egg, 1. Wild's apparatus, 185. Xanthin, 124. Xanthoproteic reaction, 1. ZoUner's lines, 281. A Selection from Charles Giiffin & Compamfs Catalogue. Professors LANDOIS and STIRLING. HUMAN PHYSIOLOGY (A TEXT- BOOK OF): INCLUDING HISTOLOGY AND MICROSCOPICAL ANATOMY; WITH SPECIAL REFERENCE TO PRACTICAL MEDICINE. By Dr. L. LANDOIS, PROF. OP rHTSIOlOOY, UNIVERSITY OP GKEIFSWALD. XTranelatcft from tbc ffiftb fficrman lEbition, wltb Bnnotations an6 Bfefcitione, By WM. STIRLING, M.D., Sc.D., BRACKENBURY 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., RoyalSvo, Handsome Cloth. Price 42s. Part I.— Physiology of the Blood, Circulation, Ilespiration, Digestion, Absorption, Animal Heat, Metabolic Phenomena of the Body. 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PAGES 1 PAGES The Quiz-Compends, 2 , 3. 4, 5 Pathology and Histology, . ii Anatomy, . 6 Physical Diagnosis, .11 Chemistry, . 6 Physiology, . . . .11 Children's Diseases, . 7 Practice of Medicine, . . 12 Dentistry, 8 Prescription Books, . . 12 Dictionaries, 8 Skin Diseases, . . .13 Eye Diseases, 8 Surgery, . . .13 Electricity, . 9 Throat 14 Gynsecology, 10 Urine and Urinary Organs, 14 Hygiene, 9 Venereal Diseases, . . 14 Medical Jurisprudence, 9 Medical Briefs. A New Miscellaneous, 10 Series 15 Obstetrics. . . 10 New Students' Manuals, . 16 PU «LIS1 iEU BY P. BLAKISTON, SON & CO., Medical Booksellers, Importers and Publishers. LARGE STOCK OF ALL STUDENTS' BOOKS, AT THE LOWEST PRICES. 1012 Walnut Street, Philadelphia. *si,* For sale by all Booksellers, or any book will be sent by mail, postpaid, upon receipt of price. Catalogues of books on all branches of Medicine, Dentistry, Pharmacy, etc., supplied upon application. ?gUIZ-COMPENDS? A JNEW SERIES OF COMPENDS FOR STUDENTS. For Use in the Quiz Class and when Preparing for Examinations. Price of Each, Bound in Cloth, $1.00. Interleaved, $1.25. Based on the most popular text-books, and on the lec- tures of prominent professors, they form a most complete, practical and exhaustive set of manuals, containing infor- mation nowhere else collected in such a condensed, practical shape. Thoroughly up to the times in every respect, containing many new prescriptions and formula", and over two hundred and thirty illustrations, many of which have been drawn and engraved specially for this series. The authors have had large experience as quiz- masters and attaches of colleges, with exceptional oppor- tunities for noting the most recent advances and method?. The arrangement of the subjects, illustrations, types, etc., are all of the most improved form, and the size of the books is such that they may be easily carried in the pocket. They are constantly being revised, so as to include the latest and best teachings, and can be used by students of any college of medicine, dentistry or pharmacy. No. 1. ANATOMY. 117 Illustrations. FOURTH EDITION, ENLARGED, WITH INDEX. A Compend of Human Anatomy, (including Visceral Anatomy, formerly published separately as No. 8 of this series). By Saml. O. L. Potter, m.a., m.d., Late A. A. Surg. U. S. Army; Professor of Practice, Cooper Medical College, San Francisco. 117 lUus. " The work is reliable and complete, and just what the student needs in reviewing the subject for his examinations." — The Physi- cian and Surgeon' s Investigator , Buffalo, N. Y. " The arrangement is well calculated to facilitate accurate memo- rizing, and the illustrations are clear and good." — North Carolina Medical yournal. Nos. 2 and 3. PRACTICE. NEW REVISED EDITIONS. A Compend of the Practice of Medicine, especially adapted to the use of Students. By Dan'l E. Hughes, M.n., Demonstrator of Clinical Medicine in Jefferson Price of each Book. Cloth, $1.00. Interleaved for Notes, $1.25. THE r QUIZ-COMPENDS ?. Medical College, Philadelphia. Second Edition. En- larged and thoroughly Revised. In two parts. Part I. — Continued, Eruptive, and Periodical Fevers, Diseases of the Mouth, Stomach, Intestines, Peritoneum, Biliary Passages, Liver, Kidneys, Intestinal Parasites, etc., and General Diseases. Part II. — Diseases of the Respiratory System, Circu- latory System and Blood, Nervous System, etc. *^* These little books can be regarded as a full set of notes upon the Practice of Medicine, containing the Synonyms, Definitions, Causes, Symptoms, Prognosis, Diagnosis, Treatment, etc., of each disease, and includ- ing a number of new prescriptions. They have been, compiled from the lectures of prominent Professors, and reference has been made to the latest writings of Pro- fessors Flint, Da Costa, Bartholow, Roberts, etc. " It is brief and concise, and at the same time possesses an accu- racy not generally found in compends." — yas. ]\I. French, M.D., Ass't to the Prof, of Practice , Medical College of Ohio, Cincinnati. " The book seems very concise, yet verj' comprehensive. . . . An unusually superior Ijook." — Dr. E. T. Bruen, Demonstrator of Clinical Medicine , Uniz'ersity of Pennsylvania. " I have used it considerably in connection with my branches iu the Quiz-class of the University of La." — y. H. Betniss. "Dr. Hughes has prepared a very useful little book, and I shall take pleasure in advising my cla.ss to use it." — Dr. George W. Hall, Prof, of Practice, St. Louis College of Phys. and Surgeons. No. 4. PHYSIOLOGY. Illustrated. fourth revised edition, with index. A Compend of Human Pliysiology. By Albert P. Brukaker, M.D., Demonstrator of Physiology in Jef- ferson Medical College. Philadelphia. Professor of Physiology, Pennsylvania College of Dental Surgery. Third Edition. Enlarged and Revised. " Dr. Brubaker deserves the hearty thanks of medical students for his Co}7!j>end of Physiology . He has arranged the fundamental and practical principles of the science in a peculiarly inviting and accessible manner. I have already introduced the work to my class." — Maurice N. Miller, M.D., Instructor in Histology , for-^ tnerly Demonstrator of Physiology, University City of New York. " ' Quiz-Compend' No. 4 is fully up to the high standard estab- lished by its predecessors of the same series." — Medical Bulletin^ Philadelphia. " I can recommend it as a valuable aid to the student." — C. N. Ellinivood, M.D., Professor 0/ Physiology, Cooper Medical Col- lege, San Francisco. " This is a well written little book." — London Lancet. Price of each Book. Cloth, $1.00. Interleaved for Notes. SI. 25. THE ? QUIZ-COMPENDS?. No. 5. OBSTETRICS. Third Edition. A Compend of Obstetrics. For Physicians and Students. By Henry G. Landis, m.d., Professor of Obstetrics and Diseases of Women, in Starling Medical College, Columbus. Third Revised Ed. New Illustrations. " We have no doubt that many students will find in it a most valuable aid in preparing for examination." — The American your- nal of Obstetrics. " It is complete, accurate and scientific. The very best book of its kind I have seen." — y. S. Knox, M.D., Lecturer on Obstetrics , Rush Medical College, Chicago. No. 6. MATERIA MEDIOA, THERAPEU- TICS AND PRESCRIPTION WRITING. Fourth Edition, -with Index. A Compend on Materia Medica, Therapeutics and Prescription Writing, with especial reference to the Physiological Actions of Drugs. By Saml. O. L. Potter, m.a., m.d.. Professor of Practice, Cooper Medical College, San Francisco, Late Surgeon U. S. Army. " I have examined the little volume carefully, and find it just such a book as I require in my private Quiz, and shall certainly re- commend it to my classes. Your Compends are all popular here in Washington." — John E. Brackett, M.D., Professor of Materia Medica and Therapeutics, Howard IMedical College, Washington. " Part of a series of small but valuable text-books. . . . While the work is, owing to its therapeutic contents, more useful to the medical student, the pharmaceutical student may derive much use- ful information from it." — TV. V. Pharmaceutical Record. No. 7. GYNECOLOGY. A Compend of Diseases of Women. By Henry Morris, m.d.. Demonstrator of Obstetrics, Jefferson Medical College, Philadelphia. In P>'ess. No. 8. DISEASES OF THE EYE AND REFRACTION, WITH ILLUSTRATIONS AND INDEX. Compend on Diseases of the Eye and Refraction, in- cluding Treatment and Surgery. By L. Webster Fox, M.D., Chief Clinical Assistant, Ophthalmological Department, Jefferson Medical College Hospital ; Ophthalmic Surgeon, Germantown Hospital, Phila- delphia ; late Clinical Assistant at Moorfields, London, England, etc., and Geo. M. Gould, a.b. 6o IUus. Price of each Book, Cloth, $1.00. Interleaved for Notes, $1.25. THE ?QUIZ-COMPENDS?. No. 9. SURGERY. THIRD REVISED EDITION. 77 FORMULA. 91 ILLUSTRATIONS. A Compend of Surgery; including F'ractures, Wounds, Dislocations, Sprains, Amputations and other opera- tions, Inflammation, Suppuration, Ulcers, Syphilis, Tumors, Shock, etc. Diseases of the Spine, Ear, Eye, Bladder, Testicles, Anus, and other Surgical Diseases. By Orville Horwitz, a.m., m.d., Demonstrator of Anatomy, Jefferson Medical College, Philadelphia. Third Revised Edition. 91 Illustrations. 77 Formulae. *:^*This compend has been prepared with great care, from the standard authorities on Surgery and from notes taken by the author during attendance on lectures by prominent professors. "Ail the essential facts of surgery are presented in a well- arranged and condensed manner." — Medical Brie/. "Useful to the student in fixing the essentiab firmly in his mind." — Pro/. G. F. Shears, Chicago. No. 10. ORGANIC CHEMISTRY. A Compend of Organic Chemistry, including Medical Chemistry, Urine Analysis, and the Analysis of Water and Food, etc. By Henry Leffmann, m.d., Pro- fessor of Clinical Chemistry and Hygiene in the Phila- delphia Polyclinic ; Professor of Chemistry, Penn- sylvania College of Dental Surgery. " Compact, substantial and exact; well suited as a remembrancer to students." — Paci/c Medical and Surgical Journal. " It contains, in compact form, the most of modern organic and medical chemistry essential to the student of medicine, and will be of great value in bringing this subject within his grasp." — C. C. //award, Pro/. 0/ Chemistry, Starling Med. College, Columbus. " It has the decided merit of being written in a clear and under- standable langu.ige." — Dr. y . Sickels, /nsiructor in Chemistry , University Medical College, Neiv York. No. 11. PHARMACY. Second Ed. A Compend of Pharmacy. Based upon " Remington's Te.\t-Book of Pharmacy." By F. E. Stewart, m.d., PH.G., Quiz Master in Chemistry and Theoretical Pharmacy, Philadelphia College of Pharmacy ; De- monstrator and Lecturer in Pharmacology, Medico- Chirurgical College, and Woman's Medical College; 2d Edition, thoroughly revised. "Will be most useful to the student. Its questions and answers are very compact and concise, presenting all important facts with- out unnecessary verbiage."^ TXe" Druggist's Circular. 4®" The ? Quiz-Compends ? contain the latest and best infor- mation, in such a shape that it can be easily memorized. Price of each Book, Cloth, $1.00. Interleaved for Notes, $1.26. STUDENTS' TEXT-BOOKS AND MANUALS. ANATOMY, Holden's Anatomy. A manual of Dissection of the Human Body. Fifth Edition. Enlarged, with Marginal References and over 200 Illustrations. Octavo. Cloth, 5.00; Leather, 6.00 Bound in Oilcloth, for the Dissecting Room, $4.50. " No student of Anatomy can take up this hook without being pleased and instructed. Its Diagrams are original, striking and suggestive, giving more at a glance than pages of text description. * * * The text matches the illustrations in directness of prac- tical application and clearness of detail." — Neiv York Medical Record. Holden's Human Osteology. Comprising a Description of the Bones, with Colored Delineations of the Attachments of the Muscles. The General and Microscopical Structure of Hone and its Development. With Lithographic Plates and Numerous Illus- trations. Sixth Edition. 8vo. Cloth, 6.00 Heath's Practical Anatomy. Sixth London Edition. 24 Col- ored Plates, and nearly 300 other Illustrations. Cloth, 5.00 CHEMISTRY. Hartley's Medical Chemistry. A text-book prepared specially for Medical, Pharmaceutical and Dental Students. With 40 Illustrations, Plate of Absorption Spectra and Glossary of Chemi- cal Terms. Cloth, 2.50 *if* This book has been written especially for students and phy- sicians. It is practical and concise, dealing only with those parts of chemistry pertaining to medicine ; no time being wasted in long ■descriptions of substances and theories of interest only to the advanced chemical student. Bloxam's Chemistry, Inorganic and Organic, with Experiments. Sixth Edition, nearly 300 Illustrations. Cloth, 3.75; Leather, 4.75 Richter's Inorganic Chemistry. A text-book for Students. Third American, from Fifth German Edition. Translated by Prof. Edgar F. Smith, ph.d. 89 Wood Engravings and Colored Plate of Spectra. Cloth, 2.00 Richter's Organic Chemistry, or Chemistry of the Carbon Compounds. Translated by Prof Edgar F. Smith, ph.d. Illustrated. Cloth, 3.00; Leather, 3.50 Trimble. Practical and Analytical Chemistry. A Course in Chemical Analysis, by Henry Trimble, Prof, of Analytical Chem- istry in the Phila. College of Pharmacy. Illustrated. Second Edition. Bvo. Cloth, 1.50 49" See j>ages 2 to s for list 0/ ? Quiz- Compends f S'lUDENTS' TEXT-BOOKS AND MANUALS. 7 Chemistry : — Continued. Muter. Practical and Analytical Chemistry. Second Edi- tion, Revised and Illustrated, Cloth, 2.00 Holland. The Urine, Chemical and Microscopical. For Luburatory Use. Illustrated. Cloth, .50 Wolff's Applied Medical Chemistry. By Lawrence Wolff, M.D., Demonstrator of Chemistry in Jefferson Medical College, •Philadelphia. Cloth, 1.50 CHILDREN. Goodhart and Starr. The Diseases of Children. A Manual for Students, and Physicians. By J. F. Goodhart, m.d.. Physi- cian to tlie Evelina Hospital for Children ; Assistant Physician to Guy's Hospital, London. American Edition, Revised and Edited by Louis Starr, m.d., Clinical Professor of Diseases of Children in the Hospital of the University of Pennsylvania; Physician to the Children's Hospital, Philadelphia. Containing many new Prescriptions, a List of over 50 Formulse, conforming to the U. S. Pharmacopoeia, and Directions for making Arti- ficial Human Milk, for the Artificial Digestion of Milk, etc. Just Ready. Demi-Octavo. 738 Pages. Cloth, 3.00; Leather, 3.50 The New York Medical Record says : — " As it is said of some men, so it might be said of some books, that they are ' born to greatness.' This new volume has, we believe, a mission, particu- larly in the hands of the ymuiger members of the profession. In these days of prolixity in medical literature, it is refreshing to meet with an author who knows both what to say, and when he has said it. The work of Dr. Goodhart (admirably conformed, by Dr. Starr, 10 meet American requirements) is the nearest approach to clinical teaching, without the actual presence of clinical material, that we have yet seen. The details of management so gratefidly read by the young practitioner are fully elucidated. .Altogether, the book is one of as great practical working value as we have seen for many months." Day. On Children. A Practical and Systematic Treatise. Second Edition. 8vo. 752 pages. Cloth, 3.00; Leather, 4.00 Meigs and Pepper. The Diseases of Children. Seventh Edition. 8vo. Cloth, 5.00; Leather, 6.00 Starr. Diseases of the Digestive Organs in Infancy and Childhood. With chapters on the Investigation of Disease, and on the General Management of Children. By Louis Starr, M.D., Prof, of Diseases of Children, Hospital of the University of Pennsylvania. IIlus. Cloth, 2.50 4S°* See pages 2 to 5 for list 0/ ? Quiz- Contends > 8 STUDENTS' TEXT-BOOKS AND MANUALS. DENTISTRY. Flagg's Plastics and Plastic Filling. 2d Ed. Cloth, 4.00 Gorgas. Dental Medicine. A Manual of Materia Medica and Therapeutics, by Professor F. J. S. Gorgas, m.d., d.d.s , Pro- fessor of the Principles and Practice of Dental Science, in Den- tal Dept., University of Maryland. Second Edition. Cloth, 3.25 Harris Principles and Practice of Dentistry. Including Anatomy, Physiology, Pathology, Therapeutics, Dental Surgery and Mechanism. Eleventh Edition. Revised and enlarged by Professor Gorgas. 744 Illustrations. Cloth, 6.50 ; Leather, 7.50 Richardson's Mechanical Dentistry. Fourth Edition. 458 Illustrations. 710 pages. 8vo. Cloth, 4.50; Leather, 5.50 Stocken's Dental Materia Medica. Third Edition. Cloth, 2.50 Taft's Operative Dentistry. Dental Students and Practitioners. Fourth Edition. 100 Illustrations. Cloth, 4.25 ; Leather, 5.00 Tomes' Dental Anatomy, Human and Comparative. Third Edition. 191 Illustrations. Preparing. Tomes' Dental Surgery. Third Edition. Revised. 292 Illustrations. 772 Pages. Cloth, 5.00 DICTIONARIES. Cleaveland's Pocket Medical Lexicon. Thirty-first Edition. Giving correct Pronunciation and Definition of Terms used in Medicine and the Collateral Sciences. Very small pocket size, red edges. Cloth, .75 ; pocket-book style, i.oo Longley 's Pocket Dictionary. The Student's Medical Lexicon, giving Definition and Pronunciation of all Terms used in Medi- cine, with an Appendix giving Poisons and Their Antidotes, Abbreviations used in Prescriptions, Metric Scale of Doses, etc. 24mo. Cloth, I.oo; pocket-book style, 1.25 EYE. Arlt. Diseases of the Eye. Including those of the Conjunc- tiva, Cornea, Sclerotic, Iris and Ciliary Body. By Professor Fred. Ritter von Arlt. Translated by Dr. Lyman Ware. Illus- trated. 8vo. Cloth, 2.50 Macnamara. On Diseases of the Eye, Fourth Edition, revised, with Marginal References, numerous Colored Plates and Diagrams, Wood Cuts and Test Types. Cloth, 4.00 Meyer. Diseases of the Eye. A complete Manual for Stu- dents and Physicians. 270 Illustrations and two Colored Plates. 8vo. Just Ready. Cloth, 4.50; Leather, 5.50 Morton. Refraction of the Eye. Third Ed. Illus. Cloth, 1.00 MS' See pages 2 to S /or list 0/ > Quiz-Cotnpends f STUDENTS' TEXT-BOOKS AND MANUALS. 9 ELECTRICITY. Mason's Compend of Medical and Surgical Electricity. With numerous llluslrations. i2mo. See page ij. Cloth, i.oo HYGIENE. Parke's Practical Hygiene. Seventh Edition, enlarged. Illus- trated. 8vo. Cloth, 4 CO Wilson's Handbook of Hygiene and Sanitary Science. Sixth Edition. Revised and Illustrated. Cloth, 2.75 MATERIA MEDICA AND THERAPEUTICS. Biddle's Materia Medica. Tenth Edition. For the use of Students and Physicians. By the late Prof. John B. Biddle, M. D., Professor of Materia Medica in Jefferson Medical College, Phila- delphia. The Tenth Edition, thoroughly revised, and in many parts rewritten, by his son, Clement Biddle, m.d , Past Assistant Surgeon, U. S. Navy, assisted by Henry Morris, m.d.. Demon- strator of Obstetrics in Jefferson Medical College. 8vo., illus- trated. Cloth, 4.00 ; Leather, 4.75 " The larger works usually recommended as text-books in our medical schools are too voluminous for convenient use. This work will be found to contain in a condensed form all that is most valuable, and will supply students with a reliable guide." — Chicago Med. yi. Potter, Materia Medica, Pharmacy and Therapeutics. Including Action of Medicines, Special Therapeutics, Pharma- cology, etc. See last page. Cloth, 3.00; Leather, 3.50 The most complete compendium of its subjects published, con- taining information not hitherto collected in one volume. Roberts' Compend of Materia Medica and Pharmacy. By the author of " Roberts' Practice." Cloth, 2.00 Headland's Action of Medicines. 9th Ed. 8vo. Cloth, 3.00 Waring. Therapeutics. With an Index of Diseases and an Index of Remedies. A Practical Manual. Fourth Edition. Revised and Enlarged. Cloth, 3.00; Leather, 3.50 MEDICAL JURISPRUDENCE. Reese. A Text-book of Medical Jurisprudence and Toxi- cology. By John J. Reese, m.d., Professor of Medical Juris- prudence and Toxicology in the Medical and Law Departments of the University of Pennsylvania ; Vice-President of the Med- ical Jurisprudence Society of Philadelphia ; Physician to St. Joseph's Hospital ; Corresponding Member of The New York Medico-legal Society. Cloth, 3.00; Leather, 3.50 " We might call these the essentials for the study of medical juris- prudence. The subject is skeletonized, condensed, and made 4^ See pages 2 to s /or list 0/ ? Quiz- Compends ? 10 STUDENTS' TEXT-BOOKS AND MANUALS. Medical Jurisprudence : — Continued. thoroughly up to the wants of the general medical practitioner, and the requirements of prosecuting and defending attorneys. If any section deserves more distinction than any other, as to intrinsic excellence, it is that on toxicology. This part of the book comprises the best outline of the subject in a given space that can be found anywhere. As a whole, the work is everything it promises, and more, and considering its size, condensation, and practical character, it is by far the most useful one for ready refer- ence, that we have met with. It is well printed and neatly bound." — Ne~M I 'ork Medical Record. Abercrombie's Students' Guide to Medical Jurisprudence, izmo. Cloth, 2.50 Mann's Manual of Psychological Medicine, and Allied Ner- vous Diseases. Their Diagnosis, Pathology and Treatment, and their Medico-Legal .\spects. Illus. Cloth, 5.00; Leather, 6.00 Woodman and Tidy's Medical Jurisprudence and Toxi- cology. Chromo-Lithographic Plates and 116 Wood engravings. Cloth, 7.50; Leather, 8.50 MISCELLANEOUS. Beale. Slight Ailments. Their Nature and Treatment. Illus- trated. 8vo. Paper cover, .75 ; Cloth, 1.25 Dulles. Surgical and other Emergencies. Illustrated. Sec- ond Edition. 12010. Cloth, .75 Fothergill. Diseases of the Heart and Their Treatment. Second Edition. 8vo. Cloth, 3.50 Tanner. Memoranda of Poisons. Their Antidotes and Tests. Sixth Edition. i2mo. In Press. Allingham. Diseases of the Rectum. Fourth Edition. Illus- trated. 8vo. Paper covers, .75; Cloth, 1.25 OBSTETRICS AND GYNAECOLOGY. Parvin's Winckel's Diseases of Women. Edited by Prof. Theophilus Parvin, Jefferson Medical College, Philadelphia. 117 Illustrations. Cloth, 3.00; Leather, 3.50 Galabin's Midwifery. A New Manual for Students. By A. Lewis Galabin, m.d., f.r.c.p.. Obstetric Physician to Guy's Hospital, London, and Professor of Obstetrics in the same Insti- tution. 227 Illustrations Cloth, 3.00; Leather, 3.50 " The illustrations are mostly new and well executed, and we heartily commend this book as far superior to any manual upon this subject." — Archives 0/ Gyncecology, New York. Glisan's Modern Midwifery. 2d Edition. Cloth, 3 00 Rigby's Obstetric Memoranda. By Alfred Meadows, m.d. 4th Edition. Cloth, .50 9ff See pages 2 to S /"r list 0/ ? Quiz-Co)nJ>ends f STUDENTS' TEXT-BOOKS AND MANUALS 11 Obstetrics and Gyncecology : — Continued. Meadows' Manual of Midwifery. Including the Signs and Symptoms of Pregnancy, Obstetric Operations, Diseases of the Puerperal State, etc. 145 Illustrations. 494 pages. Cloth, 2.00 Swayne's Obstetric Aphorisms. For the use of Students commencing Midwifery Practice. 8th Ed. i2mo. Cloth, 1.25 PATHOLOGY AND HISTOLOGY. Bowlby. Surgical Pathology and Morbid Anatomy, for Students. 135 Illustratiuns. i2mo. Cloth, ;J2. 00 Rindfleisch's General Pathology. By Tyson. For Students and Physicians. By Prof. Edward Rindfleisch, of WUrzburg. Translated by Wm. H. Mercur, m.d., of Pittsburg, Pa., Edited by James Tyson, m.d.. Professor of Pathology and Morbid Anatomy in the University of Pennsylvania. i2mo. Cloth, 2.00 Gilliam's Essentials of Pathology. A Handbook for Students. 47 Illustrations. i2mo. Cloth, 2.00 *;!:* The object of this book is to unfold to the beginner the funda- mentals of pathology in a plain, practical way, and by bringing them within easy comprehension to increase his interest in the study of the subject. Gibbes' Practical Histology and Pathology. Third Edition. Enlarged. i2mo. Cloth, 1.75 PHYSICAL DIAGNOSIS. Bruen's Physical Diagnosis of the Heart and Lungs. By Dr. Edward T. Bruen, Assistant Professor of Clinical Medicine in the University of Pennsylvania. Second Edition, revised. With new Illustrations. i2mo. Cloth, 1.50 *:i;*The subject is treated in a plain, practical manner, avoiding questions of historical or theoretical interest, and without laying special claim to originality of matter, the author has made a book that presents to the student the somewhat difficult points of Physi- cal Diagnosis clearly and distinctly. PHYSIOLOGY. Yeo's Physiology. Second Edition. The most Popular Stu- dents' Book. By Gerald F. Yeo, m.d., f.r.c.s.. Professor of Physiology in King's College, London. Small Octavo. 750 pages. Over 300 carefully printed Illustrations. With a Full Glossary and Index. Cloth, 3.00; Leather, 3.50 " The work will take a high rank among the smaller text-books of Physiology." — Pro/. H. P. Bowditc/t, Harvard Med. School, Boston. " The brief examination I have given it was so favorable that I placed it in the list of text-books recommended in the circular of the University Medical College." — Pro/. Lewis A. Stivtpson, M. D., j>7 East 33d Street, New York. J^f" Seepages 2 to j/or list 0/ ? Quiz-Coiiipends ? 12 STUDENTS' TEXT-BOOKS AND MANUALS. Physiology : — Continued. Kirke's Physiology. iithEd. Illus. Cloth, 4.00; Leather, 5.00 Landois' Human Physiology, Including Histology and Micro- scopical Anatomy, and with special reference to Practical Medi- cine. Second Edition. Translated and Edited by Prof. Stirling. 583 Illustrations. Cloth, 6.50; Leather, 7.50 " So great are the advantages offered by Prof. Landois' Text- book, from the e.xhaustive and eminently practical manner in which the subject is treated, that, notwithstanding it is one of the largest works on Physiology, it has yet passed through four large editions in the same number of years. Dr. Stirling's annotations have materially added to the value of the work. . . . Admirably adapted for the practitioner. . . . With this Te.\t-book at his command, no student could fail in his e.xamination." — -Lancet. Sanderson's Physiological Laboratory. Being Practical Ex- ercises for the Student. 350 Illustrations. 8vo. Cloth, 5.00 Tyson's Cell Doctrine. Its History and Present State. Illus- trated. Second Edition. Cloth, 2.00 PRACTICE. Roberts' Practice. New Revised Edition. A Handbook of the Theory and Practice of Medicine. By Frederick T. Roberts, m.d. ; m.r.c.p.. Professor of Clinical Medicine and Therapeutics in University College Hospital, London. Seventh Edition. Octavo. In Press. " I have become thoroughly convinced of its great value, and have cordially recommended it to my class in Vale 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, o/the Practice 0/ Medicine, University 0/ 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." — N. Y. Medical Record. PRESCRIPTION BOOKS. ^Vythe'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. 32mo. Cloth, i.oo; Pocket-book style, 1.25 Pereira's Physician's Prescription Book. Containing Lists of Terms, Phrases, Contractions and Abbreviations used in Prescriptions, Explanatory Notes, Grammatical Construction of Prescriptions, etc., etc. By Professor Jonathan Pereira, m.d. Sixteenth Edition. 32mo. Cloth, i. 00; Pocket-book style, 1.25 t^'See pages 2 to j /or list 0/ ? Quiz- Compends ? STUDENTS' TEXT-BOOKS AND MANUALS. 13 SKIN DISEASES. Anderson, (McCall) Skin Diseases. A complete Text Book, with Colored Plates and numerous Wood Engravings. 8vo. Just Ready. Cloth, 4.50; Leather, 5.50 " We welcome Dr. Anderson's work not only as a friend, but as a benefactor to the profession, because the author has stricken off mediaeval shackles of insuperable nomenclature and made crooked ways straight in the diagnosis and treatment of this hitherto but little understood class of diseases. The chapter on Eczema is alone worth the price of the book." — Nashville Medical News. " Worthy its distinguished author in everj' respect ; a work whose practical value commends it not only to the practitioner and stu- dent of medicine, but also to the dermatologist." — Jinies Nevens Hyde, M.D., Prof. 0/ 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, izmo. 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 iheN. V. Hospital. lUus. 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. A Manual of Surgical Manipula- tions, Minor Surgery, Bandaging, Dressing, etc., etc. With special chapters on Aural Surgerj-, Extraction of Teeth, Anaes- thetics, etc. 208 Illustrations. 8vo. Cloth, 5.00 Swain Surgical Emergencies. New Edition. 1.50 Walsham. Manual of Practical Surgery. For Students and Physicians. By W.\i. J. Walsham, m.d., f.r c s., Asst. Surg, to, and Dem. of Practical Surg, in, St. Bartholomew's Hospital, Surgeon to Metropolitan Free Hospital, London. With 236 Engravings. Cloth, ^3 00; Leather, {3.50 Watson on Amputation of the Extremities, and their Compli- cations. 2 colored plates and 250 wood cuts. 8vo. Cloth, 5.50 4®~ See pages 2 to S for list 0/ f Quiz- Commends ? 14 STUDENTS' TEXT-BOOKS AND MANUALS. THROAT. Mackenzie on the Throat and Nose. New Edition. By Morell Mackenzie, m.d., Senior Physician to the Hospital for Diseases of the Che^t and Throat; Lecturer on Diseases of the Throat at the London Hospital, etc. Revised and Edited by D. Brysan Delavan, m.d., Prof, of Laryngology and Rhinology in the N. Y. Polyclinic; Chief of Clinic, Department of Diseases of the Throat, College of Physicians and Surgeons, N. Y. ; Sec'y of the Amer. Laryngological Assoc, etc. Complete in one vol- ume, over 200 Illustrations, and many formulae. Octavo. Diseases of the CEsophagus, Nose and Naso-Pharynx, with Formulse and 93 Illustrations. Cloth, 3 00 ; Leather, 4.00 " It is both practical and learned ; abundantly and well illustrated ; its descriptions of disease are graphic and the diagnosis the best we have anywhere seen." — Philadelphia Medical Times. Cohen. The Throat and Voice. Illustrated. Cloth, .50 James. Sore Throat. Its Nature, Varieties and Treatment. i2mo. Illustrated. Paper cover, .75 ; Cloth, 1.25 URINE AND URINARY ORGANS. Acton. The Reproductive Organs. In Childhood, Youth, .■\dult Life and Old Age. Si.vth Edition. Cloth, 2.00 Beale. Urinary and Renal Diseases and Calculous Disorders. 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Cloth, 3,50 4®^ See pages 2 to $ for list 0/ ? Quiz-Compends ? MEDICAL BRIEFS. A new series of short, concise conipends for the Med- ical Student and Practitioner. i2mo. Cloth. Price of Each Book, $i.oo. No. I. POST-MORTEM EXAMINATIONS. With Especial Reference to Medico-I.egal Practice. By Prof. Rudolph Vikchow, of Berlin Charite Hos- pital, author of Cellular Patholofjy ; 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. " \Ve 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 e.xamined 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, ^\.v>.,Professaro/Skin and Venereal Diseases, Rush Medical College, thicago. No. 3. MEDICAL ELECTRICITY. A Com- pend of Electricity and its Medical and Surgical Uses. By Ch.\s. F. M.^soN, m.d., Ass't Surg. U. S. Army ; with an introduction by Charles H. M.w, 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 covipact and practical series of text- - books pttblished. 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. 2d Edition, revised. 301.IIIUS. 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. MID^WIFERY. ByA. L. Galabin, m.d.. Lecturer on Midwifery and Dis. of Women, Guy's Hospital, London. 227 Illustrations. CHILDREN. By J. F. 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