ffi:'i^:i^:5;i^:':'i:;'i*::";^:^--s ^l^jffl"'""""^'^""" ff Cornell University Library QP 44.F53 1913 Elementary exercises in physiology, 3 1924 000 329 890 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000329890 ELEMENTARY EXERCISES IN PHYSIOLOGY BY PIERRE A. FISH. D.Sc..D.V.M. PROFESSOR OP VETERINARY PHYSIOLOGY NEW YORK STATE VETERINARY COLLEGE CORNELL UNIVERSITY THIRD EDITION REVISED PUBLISHED BY CARPENTER & CO. ITHACA, N. Y. U'Ni Vi UUl'r Y 1^ 1 1-: (l0 Y. /Vo.5l£.34 COPYRIGHT, 1906 BY PIERKE A. FISH y, / T a'^"" f b3 \'li3 PREM OF W. F. HUUPMEY, QENEVA, M. V Preface to the Third Eldition The laboratory covirse is the center around which the other courses in Physiology should be given. The power of observation, cultivation of skill and accuracy and obtaining knowledge at first hand are nqt.availabieto the same extent in the other courses as in the laboratory. Because of this there should be an unusual degree of discrimination shown in the assemblage of the material used. Where professional students are involved such a manual should be constructed not merely from the standpoint of physiology alone (valuable as that may be), but should be adapted as a foundation and introduction to later work in pathology and medi- cine particularly. Although imperfectly carried out this idea has been in the mind of the writer and it is hoped that the student will obtain a more complete perspective of the subject and that the information coming to him first hand will remain with him for a longer period. Acknowledgments are due to my assistants past and present, for suggestions which have been incorporated in the work to a greater or less extent. January, 1913. P. A. F. t z- I f u lu 10 10 — ir CQ f> t ii 1 r. _«Q ' 1 1 K •-I 1 < i^ < w He- If) Ul r I bl J u l. " T ( p j 1 ~i ' ' ■ - 1 — -J ^1 R ^^^ III 1=11 p=>) ^ S~I -i >^ ^ alkali-albimun. Add to it a few drops of a sodium phosphate solution, color with litmus, and neutralize as before. Note that the alkali-albumin now requires more acid for its precipitation than in A, the acid which is first added converting the sodium phosphate into acid, sodium phosphate. 20. Now remove C from the bath. Boil it. Again there is no coagulation, the protein having been converted into acid-albu- minum or syntonin. After cooling, color with litmus and neutralize with 0.1% alkali. At the neutral point a precipitate is formed soluble in excess of acid or alkali. (Acid-albumin is formed more slowly than alkali-albumin, so that it is well to take plenty of time) . 21. Metallic albuminates. Add to separate tubes of albumin solution, a crystal each of copper sulphate, silver nitrate and a small amount of mercuric chloride. In each of the three tubes metalUc albuminates wUl be precipitated. 22. Coagulated proteins are obtained by the action of heat, enzymes, acids, and other reagents on native proteins, by a process of unknown nature, and have been found in the liver and other glands. Fibrin is a coagulated protein formed by the action of the fibrin ferment on the fibrinogen of blood plasma. 23. Proteoses or Albumoses. Proteoses are the products of the hydrolysis of proteins. They are important intermediate products in the digestion of proteins in the animal bod^, are soluble in water, not coagulated by heat, and are precipitated by saturating their solution with ammonium sulphate. 24. Compound proteins on hydrolysis yield as products of the first splitting a simple protein and some non-protein substances. They are subdivided, according to this non-protein result, as hemoglobins, glycoproteins, and nucleoproteins. 25. Hemoglobins on hydrolysis yield a simple protein and hematin. Hemoglobin is the coloring agent of the blood and 24 enters into combination with certain gases — ^for instance, carbon dioxide, nitrogen dioxide, and hydrocyanic acid — ^more readily than with oxygen, and the poisonous properties of these gases are due largely to their power of satisfying the affinities of the hemo- globin, and in this way rendering it incapable of taking up oxygen. Hemoglobin is soluble in water, in dilute solutions of albumin, of the alkalies and their carbonates, and in sodium or ammonium phosphate. It is insoluble in strong alcohol, ether, and in the volatile and fatty oils. With the spectroscope both oxyhemoglobin and reduced hemoglobin show characteristic absorption bands. Hemoglobin crystals may be obtained, which differ in shape and solubility in water according to the species of animal from which the blood is obtained. 26. Glycoproteins }deld a substance capable of reducing an alkaline solution of cupric oxide. They are divided into mucins, mucoids, and chondroproteins. Mucins are secreted by mucous glands and certain mucous membranes. Mucin also occurs in connective tissue and in the umbilical cord. Mucin gives the protein color reactions, and forms a mucilaginous solution with water containing a little alkali. This solution is not coagulated by heat, but forms a precipitate with acetic acid insoluble in an excess of acid. Mucoids include colloid and ovamucoid. They occur in the organism and differ from mucins in physical properties and solu- bility, and are not precipitated by acetic acid. Chondroproteins yield on hydrolysis chondroitin, sulphuric acid, and an ethereal sulphuric acid in combination with a carbo- hydrate. This acid and nucleic acid have the power of forming with proteins a compound precipitated by acetic acid, which is occasionally found in the urine, and is called nucleoalbumin. Important chondroproteins are chondromucoid, found in cartilage, and amyloid, fotind in various organs pathologically. 27. Nucleoproteins, on hydrolysis, yield nucleins. Three varieties are known, differing in hydrolytic products. (i). CeH-Nucleins 3deld a protein, ortho-phosphoric acid, and xanthin bases, and occur chiefly in the nuclei of cells, but also in the protoplasm, and may pass into the animal fluids when the cell is destroyed. 25 (2). Pseudonucleins yield protein and ortho-phosphoric acid, and occur in almost all animals and vegetables. Casein of milk is a nucleoprotein containing a pseudonuclein. (3). Nucleic acid 3rields ortho-phosphoric acid and xanthin bases, and occurs in the nuclei of the spermatozoa alone. AU give the protein color reactions, are soluble in water con- taining a little alkali, and are precipitated from this solution by acetic acid. Nucleins are not decomposed by gastric juice, and are obtained as an insoluble residue after the artificial digestion of nucleoproteins with pepsin. 28. Albtuninoids. Albuminoids are a group of proteins whose general properties suggest them to be anomalous simple proteins. They consist of a number of bodies which, in their general characters and elementary composition resemble proteins, but diflEer from them in many respects. They are amorphous. Some of them contain sulphur, and others do not. The decompo- sition-products resemble the decomposition-products of proteins. The principal albuminoids are keratin, elastin, collagen, reti- culin, and skeletin. Keratin occurs in the horny portions of the skin and its appen- dages. Elastin occurs in connective, especially yellow elastic, tissue. Collagen includes ossein the chief organic constituent of bone; chondrigen of cartilage is a collagen mixed with a small quantity of other material. On boiling with water, more readily with very dilute acid, collagens are converted into gelatin. Gelatin is obtained by the prolonged boiling of connective tissues, for example, tendon, ligaments, bone, as well as from the substance collagen. Gelatin is a colorless or straw-colored solid, usually occurring in flakes or sheets, swells with water, and when heated dissolves, forming a clear solution, with the property of preventing the formation of precipitates by holding them in sus- pension in a finely divided condition; so that they pass through filter paper. 29. Make a watery solution of gelatin (5%) by allowing it first to swell up in the cold water, and then dissolving it with the aid of heat. It is insoluble, but swells up in about six times its volume of cold water. Note which of the following tests differen- tiate the gelatin from albumin. 30. After dissolving with the aid of heat, allow a small portion to cool; it gelatinizes. 31. Apply the xanthoproteic test for proteins to some of the dissolved portion; make notes of any differences as compared with proteins in this and in the following tests: 32. Use Millon ' s reagent . 33 . Try Piowtrowski's reaction. 34. Add some acetic acid and potassium ferrocyanide to another portion. Is there a precipitate? 35. Does it coagiilate by heat? 36. Is it precipitated by saturation with magnesium sulphate? 37. What is the result of the addition of tannic acid? 38. Add picric acid (saturated solution); if a precipitate appears apply heat and note any change that may occur upon cooling. 39. What is the effect of adding alcohol to the gelatin solution? 40. Add a little solution of mercuric chloride to the gelatin solution. 41. Bone. Organic basis obtained by decalcification. Place a small thin dry bone in dilute hydrochloric acid' (i part of the acid to 8 of water) for a few days. Its mineral matter is dissolved out, and the bone, although retaining its original form, loses its rigidity, and becomes pliable, and so soft as to be capable of being cut with a knife. What remains is the organic matrix of ossein. The experi- ment in the succeeding paragraph may be carried out in a subse- quent exercise after the bone has become thoroughly softened. 42. Wash the decalcified bone thoroughly with water, in which it is insoluble, place it in a solution of sodivim carbonate and wash again. Boil it in water, and from it gelatin will be obtained. Neutralize it with sodium carbonate. The solution gelatinizes. Test the solution for gelatin. (30-38). Ill CARBOHYDRATES 43. The term carbohydrates includes an important group of substances, occurring especially in plants. Starch and sugar make up a large proportion of the parts of plants, while celltdose 27 forms the chief material from which many parts of plants are constructed. Carbohydrates also occur to a less extent in animals, where they are represented chiefly by glycogen and some forms of sugars. In elementary composition they are non-nitrogenous and the majority consist of CH and with the H and O in the same pro- portion as in water, that is, 2 atoms of H to i atom of 0. (This proportion is also obtained in other substances not belonging to the carbohydrate group). Carbohydrates are indifferent bodies with a neutral reaction and form only loose combinations with other bodies, especially with bases. Carbohydrates are classified as monosaccharids or glucoses, (simple sugars); disaccharids or saccharoses; polysaccharide or amyloses. The monosaccharids (CgHuOe) include dextrose (glucose or grape sugar), galactose, levidose, glycuronic acid. They cannot be broken down into simpler sugars. The disaccharids (Ci^Hj^Oii) on taking up one molecule of water split and yield two simple sugars. Examples are saccharose (cane sugar), maltose (malt sugar), lactose (mUk sugar). The polysaccharids (C6Hio05)n do not resemble sugars. They have no sweet taste, and form simple sugars only after several reactions. Examples are starch, dextrin, animal gum, glycogen, and cellulose. 44. Starch (C6Hio05)n is one of the most widely distributed substances in plants, and it may occur in all the organs of plants, either (a) as a direct or indirect product of the assimilation of COj in the leaves of the plant, or (b) as reserve material in the roots, seeds or shoots for the later periods of generation or vegeta- tion. 45. Squeeze some dry starch powder between the thumb and forefinger, and note the peculiar crepitation sound and feeUng. 46. Place I gram of starch in a mortar, rub it up with a little cold water, and then add 50 cc. of boiling water. Transfer to an evaporating dish and heat for ten minutes over boiling water. Does the starch go into solution? Filter and test the filtrate with a drop or two of the iodine solution. 47- Add powdered dry starch to cold water. Is it insoluble? Filter and test the filtrate with a solution of iodine. A blue color denotes the presence of iodide of starch. 48. To some of the boiled portion of starch, add solution of iodine. Heat and note any change that occurs. If not boiled too ■ long another change may occur when cooled. 49. Render some of the starch mixture alkaline by adding slight excess of caustic potash. Add iodine solution. What is the result ? 50. Acidify with dilute sulphuric acid, then add iodine. What is the result ? 51. Add some solution of tannic acid. Note result and then heat. 52. Place some strong starch mixture in a dialyzer and the latter in distilled water. Allow it to stand for some time and test the water for starch. 53. Saturate a portion of the starch mixture with crystals of ammonium or magnesium sulphate. Filter. Dilute the filtrate with an equal voltime of water and add a drop or two of the iodine solution. Is the starch precipitated by the salt? 54. Glycogen (CgHmOj)!! is a polysaccharid found exclusively in animals chiefly in the liver, in the leucocytes, in all embryonic tissues, and in muscle. It is also known as animal starch. It forms an opalescent solution in water, gives a reddish-brown color with iodine. On boiling with acids it is converted into dextrin, then maltose and dextrose. The amylolytic enzjmies, by hydrol- ysis, produce similar changes. Basic lead acetate precipitates glycogen. Barfoed's reagent is not reduced. 55. Dextrin. (CsHiqOj) is an intermediate product in the hydration of starch. 56. Dissolve some dextrin, about 2%, in boiling water (100 cc.) and cool. Add iodine solution — a reddish-brown color appears, and disappears on heating and returns on cooling. (The student should take two test tubes placing the dextrin solution in one, and an equal volume of water in the other. Add to both an equal voltime of iodine solution and thus compare the difference in color.) Dextrin is made commercially by heating starch to 200° C. 57. Saturate a solution of dextrin (56) with ammonium sul- phate. Note result. Filter. Dilute with an equal volume of water and test the filtrate for dextrin. 58. Test a solution of dextrin (56) with Barfoed's reagent and heat. 59. Test a solution of dextrin (56) with a few drops of a solution of basic lead acetate. Is there a precipitate? (The lead acetate must be basic. To insure this the solution of lead acetate may be boiled with litharge for ten minutes, the filtrate will be basic lead acetate.) 60. Cellulose. (C6Hio05)n occurs in every tissue of the higher plants, where it forms the walls of cells and the great mass of hard parts of wood. It is also found in the outer investment of the animals known as Tunicates. Purified absorbent cotton and filter paper are good examples of cellulose. Cellulose is insoluble in the ordinary solvents, but can be dissolved in the strong mineral acids, being converted into dextrin. Iodine does not stain the unaltered cellulose, but does so after it has been acted upon by the acid. Cellulose is only slightly attacked, by the digestive ferments of man, though the herbivorous animals digest it to a greater extent. By the continued action of acids it is converted into glucose. 61. Immerse a piece of filter paper or absorbent cotton in a 1% solution of potassium iodide. Let dry. Immerse for an instant in sulphuric acid and then immediately rinse in water. If cellulose is present a blue color will appear. 62. Schidtze's reagent will turn cellulose blue. 63. Immerse a strip of filter paper for a moment in concen- trated sulphuric acid. Then rinse it immediately in plenty of cold water. If the time of immersion has been correct, the paper will be semi-transparent after washing, and as tough as an animal membrane. It is called vegetable parchment and can be stained blue by iodine. IV 64. Dextrose or Glucose (Grape sugar) (CeHuOg) exists in fruits and in small quantities in the blood and other fluids and organs. It is the form of sugar found in diabetic urine. It is readily soluble in water. Use 100 cc. of 2% solution. Dextrose is made commercially by boiUng starch with a dilute acid. 65. To a portion of this solution add a little iodine solution. Compare with starch. 30 66. Heat another portion of the solution with sulphuric acid; — ^it darkens slowly. If not successful add more dextrose and repeat. 67. Trommer's test. To another part of the solution add a few drops of a dilute solution of copper sulphate, and afterwards add potassium hydroxide solution in excess, that is, until the precipitate first formed is re-dissolved and a clear blue fitiid is obtained. The hydrated oxide of copper precipitated from the copper sulphate is held in solution in presence of glucose. Heat slowly, turning the tube in the flame. A Uttle below the boiling point, if glucose be present, the blue color disappears and a yellow (cuprous hydrate) or red (cuprous oxide) precipitate is obtained. If the upper surface of the fluid has been boiled, the yellow precipi- tate, when it occurs, contrasts sharply with the deep blue-colored stratum below. The precipitate is first yellow, then yellowish red, and finally red. It is better seen in reflected than transmitted light. If no sugar be present, only a black color may be obtained. 68. Fehling's solution. Keep the two solutions in separate bottles and mix a few cc. of each (equal parts) when ready to make a test. A deep clear blue fltiid is the result of the mixture, the Rochelle salt holding the cupric hydrate in solution. If kept too long it is apt to decompose. If in doubt as to the efficiency of the solution boil it, and if it remains blue it is good. Add some of the Fehling's solution to a portion of the glucose; boil, a yellowish (cuprous hydrate) or reddish (cuprous oxide) precipitate results. 69. Add to a portion of the glucose solution some strong potas- siimi hydroxide solution and then a very small amount of the sub- nitrate of bismuth. Boil; a black precipitate results which sometimes forms a mirror on the walls of the test tube. This is known as Boettger's test. Albumin because of the sulphur present, gives a similar reaction and if present must be removed if a reliable sugar test is to be obtained. 70. The Phenyl-hydrazine Test. To 5 drops of phenyl- hydrazine and 10 drops of glacial acetic acid in a test tube is added I cc. of a saturated solution of sodium chloride. After shaking the mixture, add 3 cc. of the dextrose solution and boil the contents of the test tube for about two minutes. The fluid is then allowed to cool slowly in order that the crystals may form. The canary 31 yellow precipitate may be examined in from 20 to 60 minutes under the microscope for the characteristic glucosazone crystals. The following test also gives good results, but is longer: The Phenyl- hydrazine Test. To about 10 cc. of the glucose solution in a test tube add 0.2 gram of phenylhydrazine hydrochloride, and 0.3 gram of sodium or potassium acelate. Boil in the water-bath for 20-30 minutes; then cool the test tube by allowing cold water to run upon it and set it aside. A yellow crystalline precip- itate is formed which is known as phenyl-glucosazone. Examine some of this precipitate under a lower power of the microscope and note the needle-like and feathery crystals sometimes arranged in the form of rosettes. Phenyl-glucosa- zone has a melting point of 204°C. 71. Conversion of starch into glucose. Boil some of the starch solution with a few drops of sulphuric acid until the fluid becomes clear and a few drops of it give no blue color with the iodine solution. Neutralize a small portion with sodium car- bonate; test it for glucose. 72. Crush a piece of compressed yeast about the size of a pea. Place it in a test tube and add 10 cc. of the dextrose solution. Agitate thoroughly and transfer the mixture to a saccharometer. Leave in a warm place for 24 hours. If fermentation occurs bubbles of carbon dioxide will be found in the long arm of the saccharometer. 73. Test a portion of the dextrose solution with Barfoed's reagent. Compare with Fehling's. 74. Lactose. Milk Sugar, (Ci^H^^On+H^O). This is a reducing sugar and is found in the milk of all maminals and occasionally, during pregnancy, in the urine. Lactose is less soluble in water than dextrose and is insoluble in alcohol. With pure yeast it does not ferment. By the action of certain other ferments, however, it undergoes alcoholic fermentation, with the production at the same time of lactic acid, forming the drinks known as "koumiss" when made from mare's milk, and "kephyr" when from cow's milk. The ordinary souring of milk is due to the formation of lactic acid from the lactose by micro-organisms. Lactose must be transformed into dextrose before it can be assimi- lated. If injected into the veins it appears in the urine. Use a 2% solution of lactose. 75. Test a portion of the solution with Barfoed's reagent. Compare with the similar test for dextrose. 32 76. Heat a portion of the solution carefully with stilphuric acid, — ^it chars slowly. (See 66). 77. Add to another portion excess of potassium hydroxide and a few drops of copper sulphate solution and heat, — a yellow or red precipitate appears, (Hke glucose). 78. Test another portion with Fehling's solution, — there is a reduction Hke glucose, but its reducing power is not so great as glucose. It requires 10 parts of lactose to reduce the amount of Fehhng's solution that will be reduced by 7 of glucose. 79. Apply the phenylhydrazine test and compare carefully the form of the crystals with those obtained in the dextrose solution. 80. Sucrose. Cane sugar, (Ci^H^jOn). Cane sugar is found in plants, not in the animal kingdom. It has no reducing power, but is decomposed by heating with acid into a molecule of dextrose and one of fructose (fruit sugar). Make a 2% solution of cane sugar. 81. A portion of the solution should not reduce Fehling's solution. (Many of the commercial sugars, however, contain sufficient reducing sugar to do this.) 82 . Trommer's test. Add excess of potassitun hydroxide and a drop of copper stdphate (it gives a clear blue fluid), and heat. With a pure sugar there should be no reduction. 83. Pour strong sulphuric acid on a little dry cane sugar in a test tube. Add a few drops of water with a pipette, the whole mass is quickly charred. 84. Boil a solution of cane sugar with a little sulphuric acid added. Neutralize the solution with a little sodium carbonate and test for dextrose. 85. Apply Barfoed's, Boettger's and the phenyl-hydrazine tests to portions of the cane sugar solution and note if any reduc- tion occurs. 86. Maltose. Malt sugar (Ci^Hj^On+H^O). The reducing power of maltose is one-third less than dextrose. Maltose can be easily transformed into dextrose by acids and ferments, but dex- trose cannot be converted into maltose... Maltose must be trans- formed into dextrose before it can be absorbed into the blood. One molecule of maltose decomposes into two molecules of dex- trose. Use a 2% solution of maltose. 33 87- Apply Barfoed's test to a portion of the maltose solution and compare with dextrose. 88. Apply the phenyl-hydrazine test and compare the crystals with those obtained in the dextrose solution. 89. To other portions of the maltose solution apply Trommer's, Fehling's, and Boettger's tests respectively, and compare with dextrose. 90. Fats. The fats occur in both plants and animals. They are insoluble in water and have a lower specific gravity. They dissolve in hot alcohol more easily than in cold, and are easily soluble in either, gasoline, or benzol. Fats are composed of three elements: carbon, hydrogen, and oxygen. They contain a much smaller percentage of oxygen than the carbohydrates, the hydrogen and oxygen not being in the pro- portion to form water. When the fats are kept at the temperature of superheated steam or subjected to the pancreatic enzyme — lipase, they take up water and are split into two compounds: glycerine, on the one hand, and one or more of the fatty acids, on the other. They may be considered, then, as made up of gly- cerine and a fatty acid less water. This splitting up of the fat molecule is called saponification. It occurs when fats become rancid. It can also be effected by boiling the fat with a caustic alkali. Here, instead of the free fatty acid being left, it unites with the alkali to form a salt. These metallic salts of a fatty acid are the soaps. The soaps of the alkalies are soluble in water, the potassium compound being hygroscopic and forming soft soap. The sodium compound forms a hard soap. 91. Neutral fats. The neutral fats of the adipose tissue of the body generally consist of a mixture of the neutral fats, stearin, palmitin, and olein, the two former being solid at ordinary tem- peratures, while olein is fluid, and keeps the other two in solution at the temperature of the body. They are lighter than water: Sp. gr. 0.91-0.94. 34 92. Try the reaction of a fresh fat, like lard or olive oil, with a piece of litmus paper. It is neutral ; but, if the fat has been stand- ing for some time and has become rancid, it may be slightly acid. 93. Test the solubility of a few drops of olive oil in a test tube of water. It mixes when shaken violently, but soon separates at the top on standing. Add now a few drops of a soap solution and shake again. The liquid becomes milky and the fat does not separate. If the oil is not fresh it may be necessary to add a few drops of sodium carbonate to neutralize the free acid. 94. Take a little lard or olive oil, and observe that fat is soluble in ether, also chloroform. Take some of the ethereal solution of lard and let some of it fall upon some paper. The ether soon evaporates but a permanent greasy stain is left. 95. Shake a few drops of cod-liver oil with a small amount of dilute solution of sodium carbonate. The mass should become white — an emulsion. In an emulsion the particles of oil are broken up into innumerable finer particles which remain discrete, that is, do not run together. Milk is a tjrpical emulsion. Examine some of the cod-liver oil emulsion under the microscope. 96. To about 10 grams (11 cc.) of olive oil add 20 cc. of a 10% solution of potassiiun hydroxide. Boil the mixture, gently stirring, meanwhile, until the odor of the oil has largely disappeared and it appears homogeneous and no oil separates when a few drops are poured into water. This may require half an hour. Add water as the solution evaporates, to keep the original volume. The product is a mixture of potassium soap and glycerine. 97. Convert a portion of the above soap into the sodium or hard soap by adding some saturated salt solution and allowing it to stand until cold. It will dissolve on warming. 98. To another portion add some solution of calcium chloride. A calcium soap is formed which is insoluble in water. It is this compound which is produced by the action of soap on "hard water." Many of the heavy metals give similar compounds. Solutions of lead, iron, copper, etc., may be tried. 99. To the remainder of the potassium soap solution add sulphuric acid slowly until it is plainly acid to test paper. The fatty acids are set free as insoluble substances, the glycerine remaining in solution. Filter out the acids by means of a wet filter paper, through which the acids will not pass. The filtrate 35 contains the glycerine, and must undergo still further treatment before the glycerine can be obtained in a pure form. VI EXAMINATION FOR A TEST SOLUTION OF PROTEINS AND CARBOHYDRATES I GO. Note the physical characters of the solution as to color, transparency, odor, and taste. A persistent froth suggests an albimiinous solution. Filter the solution if not clear. Divide it into two portions, and follow the outlines below. A. Proteins. Test reaction to determine if acid or alkaline. Neutralize. If a precipitate forms it is acid or alkali albtimin. If either is present, filter. Pour a few drops of the filtrate into water. A precipi- tate or turbidity shows globulins, if present. Pour remainder of filtrate into excess of water and filter. To this filtrate add acetic acid and sodium stdphate and boil. Albumins, if present, are precipitated. If precipitate is formed, filter. Test filtrate for gelatin. If present, saturate thoroughly with ammonium sulphate (crystals) and filter off precipi- tated gelatin. Test filtrate for peptones. Biuret test (cold). B. Carbohydrates. Test original solution for starch. Then saturate thoroughly with ammonium sulphate (crystals). Starch and glycogen, if present, are precipitated together with proteins. Dextrin, if present, will remain in solution. Filter, and save precipitate (a) and filtrate (6). (a) Wash precipitate on filter with small portions of a saturated solution of ammonium sulphate till portions of washings give no trace of dextrin. (In testing washings for dextrin dilute each time with an equal volume of water). When washings are entirely free from dextrin pass two or three cc. of water (cold) through filter and test for glycogen 36 with a single drop of iodine. A red brown or mahogany- color results if glycogen is present. Basic lead acetate precipitates glycogen but not dextrin. (b) Dilute filtrate (b) with an equal volume of water and test for dextrin. Test original solution for reducing sugars — ^first precipitating out the proteins with acetic acid and sodium sulphate and boiling. VII SALIVARY DIGESTION 1 01. The saliva is a mixtvire of the secretions of the parotid, submaxillary, and sublingual glands with that of the glands of the membrane of the mouth. The reaction of the mixed saliva is usually alkaline but may on fasting, also during the night toward morning, and 2-3 hours after meals, or after much talking, become acid. On standing some hotirs it may become acid and a film of calci-um carbonate form on the surface. The normal mixed saliva contains inorganic constituents consisting of: carbonates, chlorides, sul- phates, and nitrites of magnesium, calciimi, potassium, and sodium, also the sulphocyanide of potassium. The nitrites and sulphocyanide are often absent. The organic constituents are albumin, mucin, and ptyalin. The ptyalin has the power to convert starch into dextrin, maltose, and some dextrose. It is not able to penetrate the granule of unboiled starch, or does so very slowly, differing in this respect from the corresponding enz5mie of the pancreas. It acts best at about the temperature 4o°C. Ptyalin is destroyed by acids — especially, the mineral acids. In the saliva of some animals, as the horse, the enzjmie is not present in the free state but as a zymogen from which it readily forms in mastication. [Novy]. To obtain mixed saliva. Chew a small piece of paraffin or chewing gum, or inhale ether for a short time to stimulate the flow of the secretion. Collect it in a graduate until you have about 50 cc. Note that, in a short time, more or less of a sediment occiirs due to the deposition of epithelial cells, debris of food, bacteria, etc. Numerous air bubbles are usually present upon the surface. 37 Filter. Is it translucent? Is there any great amount of viscidity? What is its reaction to litmus paper? The specific gravity is 1002-1006. Test the specific gravity with an urin- ometer. 102. To a small portion add acetic acid. A precipitate indi- cates mucin. Not soluble in excess. 103. With another -portion test for traces of proteins with the xanthoproteic reaction and MUlon's test. 104. To a few drops of saliva in a porcelain evaporating dish add a few drops of dilute acidulated ferric chloride, — a red colora- tion indicates the presence of sulphocyanide of potassium, the color does not disappear on heating, nor on the addition of an acid, but is discharged by mercuric chloride. Meconic acid gives a similar color, but it is not discharged by mercuric chloride. The sulphocyanide is present only in the secretion from the parotid gland. ' 105. Test for nitrites with a few drops of a starch solution acidified with a little dilute sulphuric acid and containing a small amount of potassium iodide. A nitrite immediately gives a blue color. 106. Test for chlorides by adding to the saliva a few drops of nitric acid followed by a few drops of silver nitrate. A white precipitate indicates the combination of the chloride with the silver to form silver chloride. 107. Test another portion of the saliva with a few drops of barium chloride -for sulphates. 108. Digestive action on starch. Prepare a mixture by placing I gram of starch in a mortar and adding a few cc. of cold water, and mix well with the starch. Add 200 cc. of boiling water, stir- ring all the while. Boil the fluid for a few minutes. This gives a 0.5% mixture. 109. Dilute the saliva with an equal volume of distilled water. Label four test tubes. A, B, C, and D. Into A place some saliva, boil it and later add some starch mucilage. In B and C, place starch mucilage and saliva, to B add a few drops of concentrated hydrochloric acid and to C some 20% potassiurn hydroxide. To D add merely the saliva to the starch mixttire. Place all four in a water bath not exceeding 4o°C., and after a time test a small portion of them for sugar with Fehling's solution. 38 Reserve a small amount of D. Why is no sugar formed in A? In B and C a strong acid and alkali arrest the action of ptyalin. Neutralize a portion of B and C and test again. Is there any result? In D the starch nas been converted by the ptyalin into a reducing sugar. no. Test portions of D with Fehling's and iodine solutions. The absence of any blue color with the iodine indicates that the starch has disappeared, having been converted into a reducing sugar — maltose. Also test the remainder of A, B, and C with the iodine solution. 111. Test another portion of D with phenyl-hydrazin, (70) crystals of phenyl-maltosazone should develop. Examine under the microscope. 112. The intermediate products of salivary digestion may be detected by proceeding as in D (109) and testing a few drops of the mixture every two minutes with a drop of iodine upon a porcelain plate. At first there is a blue color denoting soluble starch ; later there is a reddish violet color indicating the presence oi erythrodextrin; still later there is only a slight yellowish brown color, or no color at all, when the drop of iodine is added, and this indicates achroodextrin — ^the achromic point — when a reducing sugar maltose is also present. At this point the solution should reduce Fehlings. Any undigested starch may be precipitated by alcohol which leaves the maltose in solution. Saturation with ammonium sulphate crystals also precipitates the starch but does not affect the dextrins or maltose. 113. The effect of drugs on salivary action. The following may be used: carbolic acid 2%, saturated aqueous solutions of salicylic, benzoic and boric acids, corrosive sublimate i — 1000, quinine bisulphate 2%, alcohol 50%. Place in each test tube 2 cc. boiled starch 2%, 2cc. sodium carbonate 5%, i cc. saliva, 5 cc. of the given drug. Shake and set in the water bath at 40° C for an h-our. The activity of digestion may be compared by testing a small portion of each tube with the iodine solution, to see if the starch has disappeared; or another small portion with Fehling's to see if maltose has formed. A cruder method is to add to each tube an equal volume of 10% caustic soda or potash with a little dilute copper siilphate solution (Trommer's test). The amount of precipitate or depth of color roughly corresponds to the amount of digestion. 39 114- Bread. Crumble up a small piece of bread in a test tube and add some cold distilled water until it softens and with slight shaking disintegrates. Divide the mixtiire into two por- tions. 115- Apply a drop of iodine solution: blue color indicates starch. 1 1 6. Apply the xanthoproteic reaction to the other portion. Any crumbs that may be in the solution if colored orange would indicate the presence of a protein. The liquid portion may not show as deep a color, indicating a lesser amount in solution. 117. Try the above tests hurriedly by dropping a little iodine solution upon the bread. Similarly with the xanthoproteic test by letting a drop of nitric acid fall upon the bread and then a drop of ammonia upon the spot already covered by the nitric acid. 118. Potato. Boil a small piece of potato in water and let it cool. Divide the liqtiid into two parts. Test one portion for starch with the iodine solution. Without boiling, the starch might give no reaction as the granules are enclosed in a coating of cellulose. 119. Apply the xanthoproteic test. Only a faint orange color appears, indicating that very little protein is present. 120. Small portions of ground oats, com, wheat, or bran may be mixed in separate tubes with saliva and digested. The inter- mediate and end products may be tested for as in 112. VIII GASTRIC DIGESTION 121. The gastric juice, secreted by the glands of the stomach- differs from the other digestive fluids in having an acid reaction. It is a clear thin liquid having a specific gravity of 1 002-1 006, The average composition of man's gastric juice is as follows : Water 99.26 Pepsin, rennin and other organic niatter 0.30 Free hydrochloric acid 0.22 Alkali chlorides 0.20 •Phosphates of alkalies, calcium, magnesium and iron 0.02 40 There is more hydrochloric acid than can unite with the bases, and this must consequently be in the free state. The most important of the organic substances are the two enzymes: pepsin and rennin. Differences exist in different animals, e. g., in cami- vora there is a higher percentage of acid than in others. An artificial digestive fluid giving very good results may be made by dissolving 0.3 gram of commercial pepsin in 1000 cc, of a 0.2% solution of hydrochloric acid. It is more desirable in many ways, however, to prepare an extract from the gastric mucous membrane itself. The writer has found the following method to answer very satisfactorily. To each gram of the mucous membrane add i cc. of 1% solution of acetic acid. Triturate thoroughly in the mortar; then add 10 cc. of chloroform water for each gram of the mucous membrane. This may be kept for some time. When ready for use, filter and add 2 cc. of the extract to 8 cc. of the 0.2% hydrochloric acid, or equal volumes of the extract and the acid may be used. From a comparative standpoint, extracts may be made from each of the three great groups of animals: omnivora (pig), camivora (dog), and herbivora (horse or cow), and differences in the rate of digestion noted. 122. Label 6 test tubes, A, B, C, D, E, F. In A fill the tube half full of distilled water, and add 30 drops, or 2 cc. of the extract. Fill B half full of 0.2% hydrochloric acid. Treat C similarly to B, but add 30 drops of the extract. Fill D half fuU of a 1% solution of sodium carbonate and add 30 drops of the extract. Place 30 drops of the extract in E and add 2 or 3 cc. of distilled water and boil, then add enough 0.2% hydrochloric acid to make the tube half full. In F put 2 cc. of the extract and 2 cc. of bile and fill the tube half full of 0.2% hydrochloric acid. In each of the 6 test tubes put a small thread of weU-washed and boiled fibrin. Place all the tubes in a water bath at 4o°C., and after an hour note any changes that may have occurred in any of the tubes. The rapidity of action will indicate the strength of the ferment. Explain in your notes why no action has occurred in certain of the tubes. Test tube C is to be plugged with cotton and reserved for later examination, in the next exercise. 123. The following tubes are to be prepared exactly as C but omitting the fibrin. In the first tube a very small piece of meat; 41 in the second a cnimb of bread; in the third a bit of boiled potato; . in the fourth a small piece of dried albumin ; in the fifth add a small piece of butter; in the sixth, i cc. of milk diluti|ksvith s cc. of dis- tilled water; in the seventh test tube a small piece of gelatin; in the eighth tube small amounts of all the above substances. These tubes, also, are to remain in the water bath at 40" C, and tested later for intermediate and end products. (See 125, 126, 128). IX 124. The contents of tube C is to be divided into 4 parts, 3 of which are to be used in the following tests, and the other part to be held in reserve, if needed, to correct any of the other tests. 125. Color one portion of the fluid with the litmus solution and neutralize by the contact method with 1% sodium carbonate. (See plate I). At the neutral zone a precipitate will appear indicating acid-albumin (syntonin). The contact test without the litmus is equally delicate. 126. Add to another portion of the solution enough crystals of neutral ammonium sulphate to saturate it. This brings down the proteoses or albumoses in the form of a white precipitate. Pro- teose like peptone is soluble in water, and gives the biuret reaction. Ammonium sulphate precipitates all of the proteins but peptone. Another test for proteose is to add sodium chloride and a few drops of nitric acid. A precipitate should appear which is dissolved on heating, but reappears on cooling, indicating the presence of proteose. 127. Peptones behave differently from the native proteins in the copper sulphate and potassitim hydroxide test, if only a trace of copper sulphate is used. They give a pink instead of a violet color. (Also true of proteoses). The pink color is also given by the substance called biuret, hence the test is often called the biuret reaction. (Biuret is formed by heating urea; ammonia passes off and leaves biuret, thus: 2CON2H4 (urea) — NH3 (ammonia) equals CjOjNjHj (biviret). 128. To the third portion add neutral ammonium sulphate to saturation. This precipitates all of the proteoses and proteins while the peptones remain in solution. Filter and test the filtrate 42 for peptones by the biuret test as follows: Take another test tube and put a few drops of i% solution of copper sulphate in it; empty it out so that the merest trace of the copper sulphate is adherent to the wall of the tube, then add the filtrate and a few drops of strong potassium hydroxide. A pink color (biiiret reac- tion) should be produced. 129. If digestion has been quite long and complete the tests for acid-albumin and proteose may not be very satisfactory as these substances may have been converted into peptones. They are more readily found shortly after digestion has begun. The main fact, however, that an indiffusible protein, before being converted into a diffusible peptone, must pass through intermediate forms — acid albumin and proteose — is important, and must be kept in piind in this and succeeding experiments. 130. After filtering, treat the contents of the tubes containing meat, bread, potato, albumin, butter, milk, gelatin, and mixed silbstances for acid albumin, proteose and peptone. 131. Drugs on gastric digestion. Use the same preparations as in 113. Put in each test tube 4 cc. 0.2% hydrochloric acid, 2 cc. gastric extract and 4 cc. of the given drug. Keep the tubes at 40°C. for a number of hours or over night and test for peptones. 132. Take two pieces of moist fibrin of equal size. Tie one of the pieces in a bunch with thread and place it in a test tube con- taining some gastric extract and 0.2% hydrochloric acid. Tear the other piece of fibrin into small flakes and place it in another test tube with the same amount of extract and acid. Let the two tubes digest at 4o°C. for an equal length of time and note in which most digestion has occurred. In a crude way this experiment shows the effect of mastication upon gastric digestion. Large lumps are acted upon slowly and with difficulty, while an equal amount of material in a state of fine division is readily digested. 133. Rennm or chymosin is the milk-curdling enzyme of the stomach. It is apparently a constant constituent of the gastric juice of vertebrates. It is especially abundant in the mucous membrane of the stomach of the calf (rennet). A solution for experimental purposes may be prepared as in 1 2 1 . Both rennin and pepsin go into solution. The preparation should not stand too long (2 or 3 days), and should be neutralized with 1% sodium carbonate before using. Pepsin digests rennin in an acid 43 medium. Commercial rennin may be used in fluid or tablet form experimentally. 134. To 10 CO. of milk in a test tube add a few drops of the fluid extract of rennin or a i grain tablet of rennin and keep the tube for some minutes at a temperature of about 38°C. After a short time the milk becomes solid, forming a curd, and after a time the curd of casein contracts and squeezes out a fltiid — ^the whey. 135. Repeat the experiment but first boil the rennin. Com- pare and explain the result. 136. Half fill a test tube with 0.2 hydrochloric acid. Put in a little fibrin and add a tablet of rennin. Keep at a temperature of 38° — 4o*'C. for a few hours and test for peptones and intermediate products. 137. To 10 cc. of milk in a test tube add a few flakes of com- mercial pepsin. Keep at a temperature of 38°C. and note if there is any coagulation of the milk. See that the reaction is neutral. Test also for peptones. 138. Digest with the gastric extract in separate tubes small amounts of ground oats, com, wheat and bran, as in sections 123-128. PANCREATIC DIGESTION 139. The pancreatic secretion is a clear thick alkaline fluid- rich in solids, and possesses very active enzyme properties. It contains at least three distinct enzymes, besides albumin, leucin, fats, soaps and salts. These solid constituents make up about 10% of the secretion. The enzymes occur in the gland in the form of inactive zymogens, but are changed to the active form after being discharged into the secretion. The reaction of the juice is alkaline from the presence of sodium carbonate. The extract made from the gland by means of warm water may be acid in reaction from the presence of sarco-lactic acid, especially if the gland is extracted some time after death. The ingestion of food has more or less influence upon the flow of the pancreatic fluid. There is, therefore, no secretion during 44 starvation and it is intermittent in carnivorous animals where some time elapses between meals. On the other hand secretion is going on almost continually in herbivorous animals because digestion is uninterruptedly taking place. The enzymes found in the pancrea,tic juice are : trypsin which digests proteins in an alkaline mediimi, amylopsin which digests starch similarly to ptyalin, lipase which splits up fats into glycerine and fatty acids, and finally there is some evidence of a milk curdling enzyme, although the latter is not universally accepted. A pancreatic extract for digestive purposes with trypsin and amylopsin may be made by running the gland through a food chopper, or triturating it to a pulp in a mortar and adding i cc. of 1% acetic acid for each gram of the pancreas. Then add lo cc. of chloroform water for each gram of the pancreas to extract the enzymes and at the same time, on account of its antiseptic proper- ties, to prevent putrefaction. For use, 2 cc. of this extract may be added to 8 cc. of 1% sodium carbonate, or equal volumes of the two may be employed. Commercial pancreatin 5 grams dissolved in 200 cc. of 1% sodium carbonate will also serve for experimental purposes. 140. Prepare eight test tubes. Each test tube is to be half filled with 1% sodium carbonate and 2 cc. of the pancreatic extract added. To tubes prepared as above add the following: i, a bit of fibrin; 2, a small piece of dried albumin; 3, a piece of meat; 4, a crumb of bread; 5, a bit of cooked potato; 6, a bit of cheese; 7, a small piece of gelatin; 8, a small amount of each of the above substances. Keep these in the water bath at 4o°C. Note par- ticularly any changes that may occur in No. i, and compare with the fibrin digested with the gastric juice. In those tubes which first show signs of digestive action, test the contents for alkali- albtmiin by neutralization. (Similar to the acid-albumin test the only difference being the reaction of the digestive fluid) . Test also for proteoses. Place the tubes in the incubator until the next exercise and, after filtering, again test them for alkali-albumin, proteose and peptone, as with preceding tests. (125-128). Reserve a portion of the contents of tubes No. i and 2 for indol test later. (152). 141. Place a bit of fibrin in two tubes. Half fill the tubes with 1% sodium carbonate. To one tube add 2 cc. of the pancreatic 45 extract. To the other tube add 2 cc. of bile and 2 cc. of the pan- creatic extract. Let the tubes digest at 4o°C. and note in which tube peptone first appears. 142. Take three test tubes, add 5 cc. of boiled starch mixture and 5 cc. of 1% sodium carbonate to each tube. To No. i, add 2 cc. of the pancreatic extract. To No. 2, add 2 cc. of bile. To No. 3, add 2 cc. of bUe and 2 cc. of the pancreatic extract. Place the three test tubes in the water bath at 4o°C. Test a few drops from each tube every minute upon a white plate with a drop or two of iodine and note in which tube the starch first disappears. 143 . Prepare two tubes for starch digestion as follows : In one place s cc. of 2% boiled starch and 5 cc. of 1% soditim carbonate and 2 cc. of pancreatic extract. In the other place i gram of vmboiled starch triturated in 5 cc. of cold water, add 5 cc. of 1% sodium carbonate and 2 cc. of pancreatic extract. Place both tubes in the water bath at 4g°C. and test at intervals as in No. 142. Continue the digestion long enough to find sugar in both tubes by the Fehling's test. XI 144. Prepare four test tubes as follows, labeling them in order, I, 2, 3, 4, and adding a bit of fibrin to each tube. To No. i add 30 drops of the pancreatic extract from the pig, and some distilled water; to No. 2, the same amount of the pig's pancreatic extract and excess of 0.2% hydrochloric acid; to No. 3, some 1% sodium carbonate alone; for No. 4, put 30 drops of the pancreatic extract into a separate test tube, add a little of the 1% sodium carbonate and boil, and then add the fibrin. Put all of the test tubes in the water-bath at 4o''C. After a few hours examine them and explain the result. 145. Emulsion Experiment. Shake up a few drops of olive oil in a test tube with 2 cc. artificial pancreatic juice and 2 cc. 1% sodium carbonate. Place the mixture for a few minutes in the bath at 4o°C., and shake again, compare the results before and after warming. If the oil is neutral, there may be no emulsion or only a poor one. The addition of a few drops of oleic acid will improve it. Why? 46 146. In another tube with a little olive oil add 2 cc. bile and 2 cc. 1% sodium carbonate, shake and place in water-bath at 4o''C. and compare the emulsive effect with 145. Note whether the oil is neutral or not. 147. Action on Fat. For this experiment it is necessary that the fat shovdd be perfectly neutral. Commercial oils usually contain free fatty acids. The following method has been recommended for neutralization by Kruken- berg : Place the oil in a porcelain capsule and mix it with not too much baryta solution, (baryta mixture is prepared by mixing one volume of a solution of barium nitrate and two volumes of barium hydrate, both saturated in the cold) , and boil for some time. Allow it to cool. The unsaponified oil is extracted with ether. The ethereal extract is separated from the insoluble portion and the ether evaporated over warm water. (The flame must not be brought near the ether. Let the water come to a boil, put out the flame and then put the dish containing the ether upon the hot water). The oil should now be neutra- lized. The cream from fresh milk is usually of a neutral reaction and serves very well in the following experiments. 148. Take two test tubes and place in each 2 cc. of cream — neutral fat. Add i cc. blue litmus to color. In the first tube place a small piece of fresh pancreas. Put both tubes in the water bath and observe at intervals. Note if any change of color occurs in the one with the pancreas, due to the formation of fatty acids by the enzyme lipase. A fresh watery extract of- the pancreas also acts favorably. 149. Another form of the experiment is to mix the oil with finely divided perfectly fresh pancreas in a mortar, and keep it for a time at 40°C. It soon becomes acid, owing to the formation of fatty acids. Test with litmus paper. 150. Action on milk. Dilute 2 cc. of cow's milk with 10 cc. of distilled water in a test tube and add 5-6 drops of pancreatic extract. Keep at 4o°C. from }4toi hour. Note any change that has occtirred. 151. Divide the above into two parts. To one part add a little dilute acetic acid; if there is no precipitate it indicates that the caseinogen has been converted into peptones. To the other part apply the biuret reaction for peptones. 152. With the reserved portion from the albumin and fibrin tubes, (140), indol may be found if digestion has continued long 47 enough and if an offensive odor be present. To some of the sus- pected fluid add i cc. of o.oi% solution of sodium or potassium nitrite (fresh) and then a few drops of concentrated sulphuric acid. A pink color indicates the presence of indol. 153. Food test. Use the two digestive extracts furnished. Try the activity of each upon some fibrin and starch solution in an acid (0.2% HCl) medium, and also in an alkaline (1% NajCo,) medium at a temperature of 38 to 40 deg. C. Determine in which medium the best results are obtained and identify the extract. Test with the extracts the foodslufiE provided. Determine if there is pro- teolytic digestion, by testing for acid or alkali albumin, proteose, peptone and indol. Determine if there is amylolytic digestion by testing for dextrin and dex- trose. In addition to the extracts furnished for the experiments, collect your own saliva and try its amylolytic action upon the foodstuflf. 154. Valuation of Commercial Pepsin. Dissolve 67 mg. of the pepsin in 100 cc. of 0.2% hydrochloric acid. Add 5 cc. of the above solution to 95 cc. of 0.2% hydrochloric acid. Place 10 cc of this diluted pepsin sol. in i test tube, 15 cc. in a 2nd, 20 cc. in a 3rd, 25 cc. in a 4th, 30 cc. in a 5th tube. Place all in a water bath at 38-40 degrees C. Place an egg in boiling water and boil for 15 minutes, then place it in cold water. When cold, wipe it dry, remove the coagulated albumin and rub it through a No. 30 sieve. Place I gram of this disintegrated albumin in each of the above test tubes at 38-40 degrees and shake well, to thoroughly mix, being careful not to lose any of the solution or albumin. Keep at 38-40 degrees for six hours, geiitly shaking the tubes every 15 minutes. Note the time at which digestion is completed in each tube. If the pepsin is of U. S. P. strength, this should be at the end of six hours in tube i, only a few thin, insoluble flakes at most being left. From results obtained, determine the relative strength of the pepsin. How many times its own weight of freshly coagulated albumin will it digest in six hours^ 155- Valuation of Commercial Pancreatin. Dissolve .28 gm. pancreatin in loocc. tepid water and add 1.5 gm. sodium bicarbonate. Place 5 cc. of the above solution in one test tube and 10 cc. in a second. Heat some fresh cow's milk on a water bath, to 38-40 degrees, and add 20 cc. of this milk to each test tube. Keep at 38-40 degrees for 30 minutes or longer. Test small portions of each from time to time with Heller's cold nitric acid test. At the end of 30 minutes, no coagulum should be produced in tube one, if the pancreatin is of standard strength. Note the time at which no coagulum is produced in each tube. Repeat the experiment if necessary, using a larger or smaller amount of pancreatin solution as indicated by above tests to digest 20 cc. milk in 30 minutes. From amount of pancreatin solution necessary for this, calculate its relative value. 48 Using a I % starch mixture, determine how many times its weight of starch the pancreatin will digest in 30 minutes. 156. SiKcus Entericus. — Invertase or (invertin) sugar splitting enzyme. This exists in the succus entericus or intestinal juice and the mucous membrane of the small intestine, which splits up cane- sugar, lactose and maltose into dextrose. Lactose is split up into dextrose aiid galactose ; cane sugar into dextrose and levtdose and maltose into dextrose. A molecule of cane-sugar takes up a molecule of .water, and splits into two molecules — one of dextrose, the other of levulose (fructose.) CijHjjO„+H20=C6Hi206+C6Hi206. Cane Sugar Dextrose Levulose. A smiliar enzyme can be extracted from yeast and many plants. An extract of invertase may be made from the small intestine by adding to each gram of the mucous membrane used i cc. of 1% acetic acid, triturating in the mortar and adding 10 cc. of chloro- form for each gram of the mucous membrane. Let the mixture stand for a day or two and before using make slightly alkaline by the addition of 1% sodium carbonate. 157. Place 10 cc. of a 10% solution of cane sugar in a test tube and add 2 cc. of the extract of invertase. Allow the mixture to digest at 4o°C. After a short time test with Fehling's solution for reducing sugar. 158. From the mucosa of the intestine there is also obtained enterokinase an enzyme which activates the inert trypsinogen, converting it into active trypsin. Take two test tubes and place in each the same amount of fibrin. Add the usual amount of pancreatic extract and sodium carbonate solution to both tubes. To one of the tubes add a few drops of the intestinal extract allow- ing the other to remain as it is. Place both tubes in the water bath and observe in which tryptic digestion first occurs by making occa- sional tests for peptones. In the ordinary preparation ^i pancreatic extracts there is. usually more or less contamination with the intestinal mucosa so that in this way there may be introduced into the pan^ degrees of temperature above 60°, add one to the reading of the lactometer; example: Lactometer 114, thermometer 70°, or 10° above standard; add 4 to lactometer reading, making it 118. For each 2}^ degrees below 60°, subtract one from the read- ing of the lactometer; example: Lactometer 114, thermometer SS°, or s° below standard; subtract 2 from the lactometer read- ing, making it 112. In testing milk, the following directions should be observed: Fill the creamometer nearly full with the milk; insert the lacto- meter and note carefully the point on the stem to which it sinks; take the temperature of the rmlk and correct the reading of the lactometer according to the rule given above. If the corrected lactometer reading is less than 106, or if it is suspected that the milk has been skimmed, fill the creamometer to the point marked and place it in a cool place, where it will not be disturbed for twelve hours; in the summer it can be kept in the refrigerator. At the end of that time the cream will have risen and the per- 55 centage can easily be read from the scale on the jar. The lowest safe proportion of cream is 15%, and a percentage lower than that will surely indicate that the milk was poor originally, or has been partly skimmed. 180. Examine a drop of fresh cow's milk under the micro- scope. It consists of a clear fluid containing a large number of highly refractive fat globules. Let a drop of osmic acid solution run under the cover glass; in a short time the globules become stained brown-black. 181. To some milk in a test tube add a few drops of sodium or potassium hydroxide and heat. The liqtiid becomes yellow, then orange, and finally brown. 182. To a 4% solution of lactose add some of the same reagent and heat. The same color is developed as in 181, which is due to the sugar present in the milk. 183. To some fresh milk in a test tube add a few drops of fresh tincture of guaiac (20% solution in alcohol); agitate and add some peroxide of hydrogen (or old turpentine). A blue color develops. A similar result is given by the blood. 184. Repeat the experiment with milk that has been boiled. The blue color is not given — due to changes in the protein. 185. Mix s cc. of fresh milk with 15 drops of neutral artificial gastric juice, and heat in the water bath to 40° C. In a short time the milk curdles so that the tube can be inverted without the curd falling out. By and by the whey is squeezed out of the clot. The curdling of milk by the rennin enzyme present in the gastric juice is quite different from that produced by the "sour- ing of milk," or by the precipitation of caseinogen by acids. Here the casein (carrying with it most of the fats) is precipitated in a neutral fluid. 186. To the same test tube after the above process add 10 cc. of 0.2% hydrochloric acid, and put into the incubator until the next exercise. Note any changes when next examined and test for peptones. 187. Dilute s cc. of milk with 15 cc. of water, add a little dilute acetic acid and warm. A precipitate is formed. Filter and save both precipitate and filtrate. This precipitate is not the- same as that obtained by rennet. The acid precipitate is caseinogen, and is freely soluble in dilute alkali, the rennet clot 56 is "casein," and is much less soluble in dilute alkali. Cheese is made with rennin and cannot be made Tvith acid. i88. The filtrate obtained from 187 is to be divided into two portions. To the first portion apply Trommer's test A red precipitate indicates the presence of a reducing sugar — ^lactose. 189. To the second portion of the filtrate apply the xan- thoproteic reaction. An orange color represents the presence of a protein (lact-albumin). 190. To the precipitate obtained from 187 add a little ether in a test tube and agitate for a few minutes. Pour off the ether upon some paper and note that it leaves a permanent greasy stain indicating the presence of fat. 191. To the residue left in 190 add a little dilute potassium hydroxide (0.1%). A solution is effected. Apply the xan- thoproteic reaction to this fluid. An orange color denotes the presence of a protein — caseinogen. 192. To a test tube half filled with 0.2% hydrochloric acid and 2 cc. of gastric extract add a small piece of cheese. Put the tube in the incubator and examine at the next fexercise for pep- tones and intermediate products. 193. The action of mUk with pancreatic extract is some- what complicated on account of the complexity of mUk itself. The sugar, fat and proteins all undergo some change from the action of the different pancreatic enzjrmes. Perhaps the most interesting of these changes is that produced in the proteins, and is commonly called peptonization. The peptonization, or digestion, of milk is quite often practised in the preparation of food for the sick room, and is illustrated by the following experi- ment. Dilute about 10 cc. of milk with an equal volttme of distilled water and add a half a gram of soditun bicarbonate. Then add a few drops of pancreatic extract, shake the mixture and keep at 40° C. on the water-bath for about a half an hour. Then filter and apply the biuret test for peptones. The pan- creatic extract from beef acts more strongly upon the proteins; that from the pig is very active in converting starch into sugar. 194. Fill a test tube half full of milk and boil it. Add a tablet of rennin. Prepare another test tube in the same way, but use fresh unboiled milk. Place both tubes in the water bath at 38°C. After some minutes compare the tubes. The 57 boiled milk shotild not be coagtdated. The tinboiled milk shotdd be clotted. Leave this tube in the water bath if necessary, until the whey has separated qtiite completely from the curd. Filter and use the filtrate in the following tests. 195. Test one portion of the whey filtrate by adding a few drops of nitric acid and a little ammonium molybdate solution and heat. A yellow precipitate indicates phosphates. Test another portion by adding a little silver nitrate. A white precipi- tate insoluble in nitric acid indicates chlorides (chiefly potassiimi and sodium). To another portion add a little ammonitim oxalate. A precipitate indicates calcium salts. Test the remaining portion for albumin using the xanthoproteic test. 196. To a test tube half full of milk add 3 or 4 drops of a saturated solution of ammonium oxalate; mix, add a tablet of rennin and digest at 38°-4o°C. for at least a half an hour. There should be no coagulum. Then add a few drops of a 2% solution of calcium chloride and digest again. Does the milk coagulate? 197. Separation of caseinogen by salts. To a test tube half full of milk, add crystals of magnesium stdphate or sodium chloride to saturation. The caseinogen and fat separate out, rise to the surface, and leave a clear salted whey beneath. Caseinogen, like globulins, is precipitated by saturation with MgSO^ or NaCl, but it is not coagulated by heat. It was at one time supposed to be an alkali albumin, but the latter is not coagulated by rennin. It appears to be a nucleo-albumin — t. e., a compound of a protein with nuclein, the latter is a body rich in phosphorus. 198. Boil a little milk in a small beaker or evaporating dish. There is no coagulation. A scum forms upon the surface which returns as often as it is removed. This is due chiefly to caseinogen entangled in protein drying on exposure to air. 199. Place a smaU quantity of milk in a warm place for one or two days; then test the reaction, it will be found to be acid; this is due to fermentation, in the process of which the milk sugar is converted into lactic acid. XIV 200. Blood. The blood is a red, thick, opaque fluid. The specific gravity varies from 1.045 to 1.075 with an average for 58 adult human beings of about 1.055; it depends primarily upon the amount of hemoglobin present. For examination, it is convenient to consider the blood as composed of two parts : the corpuscles and the albuminous liquid in which they are suspended — the plasma. The solid blood corpuscles in man may constitute nearly one-half the weight of the blood. In some animals as the ox, they make up but one- third of the weight of the blood. The color of the blood is caused by the red corpuscles. Even comparatively thin layers of the blood are opaque from their presence. The coloring matter (hemoglobin) can be set free from the corpuscles by water or by many chemical reagents. The color then becomes much darker, since the light is no longer reflected from the surface of the corpuscles. The addition of strong neutral salt solutions to blood turns it bright red, because of the increased reflection of light from the shriveled corpuscles. 201. Fresh blood may be obtained and defibrinated at a slaughter house, and a few drops of formalin added to it will prevent putrefaction for some time. It is better, however, when possible, to obtain the blood by bleeding an animal. After the dog, or any other animal of convenient size, has been anesthetized, the carotid or femoral artery is exposed and isolated from sur- rounding parts for an inch or two of its length, and a clamp or ligature applied to the proximal portion of the artery, i. e., as far as possible toward the heart. Apply another clamp about one inch distally and between the clamps make an incision in the artery and insert a glass canula and tie it tightly in place. Remove the clamps and the blood will pass through the caniila, and the animal allowed to bleed to death. -When the animal is apparently dead, an interesting experiment may be performed by injecting into the artery some normal salt solution of the same temperature as the body and note the reviving effect. 202. The blood obtained as above directed is to be caught in four different vessels and each portion is to be treated as follows : One portion of the blood is to be defibrinated by immediate whipping with some broom straws tied in a small bundle and the fibrin as it collects on the straws is to be saved for future use. The defibrinated blood is' also to be reserved for later study. Another portion of the blood is to be collected in a flask and the 59 phenomenon of clotting or coagulation observed. Another portion of the fresh blood is ta be mixed with an equal voltmie of saturated solution of sodium sulphate. And still another portion into a solution of potassitun oxalate in the proportion of I of the oxalate to 4 of the blood. 203. Test the reaction of blood by pricking one of the fingers behind the nail. Put a drop of the blood on a piece of ordinary litmus paper which has been soaked in salt solution. The sub- stances on which the alkaline reaction depend will diffuse out in a ring around the drop, while the hemoglobin remains in its original position. 204. Place a thin layer of defibrinated blood on a glass slide; try to read printed matter through it. The blood is too opaque and the print cannot be read, the light is reflected from the cor- puscles in all directions and but little passes through. 205. Place I cc. of defibrinated blood in a test tube and add S cc. of distilled water, and warm slightly. Note the change of color by reflected and transmitted light. By reflected light it is much darker — almost black, but by transmitted light it is trans- parent. This constitutes "laky" blood due to the withdrawal of the hemoglobin from the red corpuscles into the water. Test the transparency by looking at some printed matter through this blood as in 204. 206. To 2 cc. of defibrinated blood in a test tube add 5 voltimes of a 10% solution of sodium chloride. It changes to a very bright, florid, brick-red color. Compare its color with No. 205. 207 . Place a watery solution of defibrinated blood in a dialyzer or parchment tube, and suspend in a vessel of distilled water. After several hours note that no hemoglobin has passed into the water. Test the diffusate for chlorides with silver nitrate and nitric acid. Hemoglobin does not dialyze, although it is crystal- lizable. 208. Put a drop of blood on a sHde. Heat it slowly over a flame, so as to evaporate the water. Then add a small crys- tal of common salt and a few drops of glacial acetic acid; put on a cover-glass, and again heat slowly till the liquid just begins to boil. Take the slide away from the flame for a few seconds, then heat it again for a moment, and repeat this process for two or three times. Now let the slide cool and examine with the 60 microscope (high power). The small black or brownish-black crystals of hemin wiU be seen. This test is often important in some medico-legal cases where only a trace of blood is available for examination. If the blood stain be upon a piece of cloth, it may be soaked in a little distilled water and examined by the spectroscope or micro-spectroscope. The liquid may then be evaporated to dryness on the water bath and the hemin test made. Or perform the hemin test directly on the piece of cloth. 209. In the blood saved for clotting, note that in a few minutes the blood congeals, and when the vessel is tilted the blood no longer moves as a fluid, but as a solid. After an hour or so, pale yellow colored drops of fluid — the serum — are seen on the surface, having been squeezed out of the red mass, the latter being the clot and consisting of fibrin. Note in the clot of horse's blood the upper light colored layer of leucocytes — the bufiy coat. Coagulation is slow in this animal and the red and white corpuscles on account of the difference in their specific gravity have time to separate. 210. Salted Plasma. Note that in the flask containing the mixture of blood and sodium sulphate, no coagulation has occurred. Place some of this fluid in the centrifuge to separate the corpuscles and plasma, or let the mixture stand until the corpuscles sink; the plasma mixed with the saline solution is known as the salted plasma. s 211. Oxalate Plasma. Note also that the potassium oxalate blood mixture does not coagulate. Centrifuge the mixture or let stand until the corpuscles fall, to obtain the plasma. The oxalate combines with the calcium which is necessary for coagulation. 212. To a portion of the oxalate plasma add a few drops of a 2% calcium chloride solution. Coagulation results (more quickly at 40°). 213. To another portion of the plasma add a little fibrin- ferment prepared by the demonstrator. The fibrin-ferment is prepared as follows: Take fresh fibrin, wash it under a tap with water (best in a piece of cotton) until perfectly colorless. Squeeze out the water and cover the fibrin with an 8% solution of soditmi chloride. After a few hours, if the solution is filtered it will sho\v the presence of the ferment. Another method is: Precipitate some blood serum with about ten times its 61 volume of alcohol. Let it stand for several weeks, then extract the precipitate with water. The water dissolves out the fibrin-ferment, but not the other coagulated proteins. 214. Add a drop of freshly prepared tincture of guaiacum to a small amoimt of diluted defibrinated blood, and then some hydrogen peroxide or old oil of turpentine. The color changes to blue. This is often used as a test for hemoglobin, but other substances (oxygen carriers) give a blue color under the same conditions. 215. Place some hydrogen peroxide over fresh fibrin in a watch glass; bubbles of oxygen are given off . 216. Immerse a flake of fibrin in freshly prepared tincture of guaiactun, (5% of pure resin in alcohol) and then immerse the flake in hydrogen peroxide. A blue color is developed, due to the ozone liberated by the fibrin and forming a blue color with the resin. Compare 214. 217. The porportion of the corpuscles to the plasma of the blood may be quite readily obtained by the use of the Hematocrit in connection with the centrifuge. The Hematocrit consists of a graduated glass tube 50 mm. in length and 0.5 mm. bore, to receive the blood. The tube is marked by a scale ranging from o to 100, the scale being rendered visible by a lens front (prism form). The outer end of the tube fits into a small cup-like depres- sion at the end of the arm, the bottoms of which are covered with the rubber disks, while the inner extremity is held in position by a spring. To use the Hematocrit in blood examinations proceed as follows : — The rubber tube with mouthpiece at one end is slipped over the end of the Hematocrit, and the latter is filled by suction on the mouthpiece, from a drop of blood obtained by a prick of the finger. The blunt end of the tube is next quickly covered with the finger tip, and the tube is inserted into the arm in the same manner as adjusting the tubes for micro-organisms. The current is next turned on, and the speed increased gradually to 10,000 revolutions per minute, and thus steadily maintained for from two to three minutes. The Hematocrit may next be removed and the percentage of red corpuscles is read off from the scale. In health, the volume of red corpuscles is about 50 per cent. One per cent, by volume represents about 100,000 red blood corpuscles, therefore by adding five ciphers to the percentage of volume, it gives the number of red corpuscles in one cb. nun. of blood. Thus in a given case, if the reading were 25, multiply- that number by 100,000, and the product 2, 500,000 would represent the number of red blood corpuscles in one cb. mm. of blood. The amount of hemoglobin in each corpuscle may be approximately determined, also, by dividing the quantity of hemoglobin ascer- tained by Fleischl's instrument, by the number of corpuscles determined by means of the Hematocrit. The white blood corpuscles or leucoc3rtes will be found to occupy a second but much shorter column immediately above the column of red corpuscles, and if leucocytes be present, even though to a very slight degree, it is easily recognized. XV 218. Protein reactions. Dilute 5 cc. of serum with 35 cc. of water. Add a little litmus solution to color and neutralize with 0.2% hydrochloric acid. Is alkali albumin present? 219. To another portion add a little acetic acid and heat. 220. Apply the xanthoproteic reaction. 221. Acidify another portion strongly with acetic acid and add a few drops of a solution of ferrocyanide of potassium. 222. Apply MiUon's reagent. 223. Apply Piowtrowski's test (6). 224. To another portion add a little alcohol. 225. Saturate another portion with ammonitmi siilphate. This precipitates all of the proteins, globulin and albumin. Filter. The filtrate does not respond to any of the tests for proteins. 226. To another portion of the diluted serum add a little silver nitrate solution. A white, curdy precipitate forms, soluble in ammonia but not in nitric acid. Chlorides are present. 227. Add baritim chloride. A white, heavy precipitate insoluble in nitric acid. Sulphates are present. 228. Add nitric acid and molybdate of ammonia and heat. A yellow precipitate indicates the presence of phosphates. 229. Test with Fehling's solution, and boil. Red, cuprous oxide indicates a reducing sugar — dextrose. 63 230. To a little of the defibrinated blood in a test tube add a few drops of sulphuric acid. Stir up the solution and note the peculiar odor of blood, intensified by the liberation of traces of volatile acids by the sulphuric acid. 231. Detection of paraglobulin (fibrinoplastin, or seruraglobulin). Pass some CO, through a beaker of dilute serum for 20 minutes or more. (The CO, may be generated by the action of dilute hydrochloric acid upon small pieces of marble in a jar and the gas conveyed to the beaker). Let the precipitate settle. It is paraglobulin. Decant and, after washing with water, dissolve some of it in a little dilute saline solution, use Piowtrowski's test and prove it a protein. 232. Take equal quantities of blood and ether in a test tube. Shake thoroughly and let the ether separate. Then pour the ether into a watch-glass or evaporating dish and when evaporated examine for globules of fat. 233. Evaporate a little blood to dryness in a crucible or evaporating dish. Raise the temperature to red heat to convert the blood to ash. When cool add a little nitric acid, heat, dilute with water and filter. Make the following tests with the filtrate: 234. To a small portion of the filtrate add a little sulpho- cyanide of potassium. A red color indicates iron. 235. To another portion add a little ammonium molybdate solution. A yellow precipitate, after allowing the mixture to stand for some time, indicates phosphates. 236. To another portion add a little silver nitrate solution. A white, cloudy precipitate indicates chlorides. 237. Examination of blood with a spectroscope. With a small direct vision spectroscope focus on the sky or bright light until the spectnun shows clearly. Narrow the slit until the spectrum is as distinct as it can be made. Hold the spectroscope so that the red is at the left of the field. Dip a wire into some water, and then into some salt or sodium carbonate, and hold it in a flame of a fish-tail burner. Note the change in the spectrum. 238. Arrange the apparatus with the aid of a demonstrator, so that the spectroscope, gas-flame and substance to be examined, are in their proper relations. Half fill the vial or test-tube with defibrinated blood. Nothing can be seen until the blood- is properly diluted. Continue diluting until two bands of oxyhemo- 64 globin appear in the spectrum. Note their position, and which one disappears first when the solution is diluted far enough. 239. Add a drop or two of ammonium sulphide solution or Stokes' fluid to reduce the oxyhemoglobin. Note the resiilt. 240. Pass some illuminating gas through some blood for a considerable time. Examine with a spectroscope. Add a drop or two of ainmoniimi sulphide or Stokes' Fluid. Compare this with 237. PART II Experimental Physiology 66 XVI Each dissection is to be careftdly demonstrated to one of the instructors before beginning the dissection of a new part. 241. Dissection of Frog's Heart. With a pair of strong scissors and forceps cut through the pectoral girdle. Remove the stemtim and expose the heart, cut carefully through the pericardium and note the division of the heart into ventricle, auricles, and tnmcus arteriosus. (Do not remove the heart until the vagus nerve has been dissected). C«v'iTyo| rijht A uricie; val> Truncus fl.TT«ri»suJ Valve . 'PuitvionAT^ Vfln. — pfnxn^ ok Sinus vena*"*. U^t-ou Title . val ve. .-Ca vi Ty »J Y««Tt1cU, Fig. I Fig. I. Dissection of the frog's heart showing the relationship of the cavities and the principal blood vessels. Raise the apex of the ventricle slightly and note a delicate band of connective tissue binding the ventricle to the body, (the frenum). The ventricle is of a conical shape and is usually of a paler color than the auricles. It has thick walls. The auricles are two in number although externally the division is not easily apparent. The right is larger than the left and both are usually engorged with blood. The walls of the auricles are thin. 67 The trtmcus arteriosus is a cylindrical tube somewhat swollen as it lies upon the auricles. The truncus soon divides into two arches, one passing to the right, the other to the left. Each arch soon splits into three vessels, the carotid, for the head, the pulmo- cutaneous, carrying the venous blood to the lungs and skin to be oxygenated, and the aorta, which curves around to the back to meet its fellow with which it unites to form the descending aorta-v Lift up the ventricle and make out the following structures : The right and the left superior vena cava, bringing back blood from the head and upper extremities; the inferior vena cava, appearing just above the liver; the sinus venosus, (practically a fusion of the venae cavae), the chamber into which the caval veins open. The sinus in turn communicates with the right atiricle. Careftilly slit the heart lengthwise into ventral and dorsal halves. In the ventricle note the comparatively small size of the cavity and the thick walls; note the two openings in the ventricular cavity — one from the auricles, guarded by auriculo- ventricular valves, the other continuous with the truncus arterio- sus; note in the truncus at its base near the ventricle three small semilunar valves, also a longitudinal fold or so-called spiral valve. The swollen portion of the truncus is known as the pylanguim; the distal portion formed by the fusion of the aortic arches is known as the synangitm:!. VAju« nerve. Hy>>»jl««»«l •■g i * 0«s»f>h<^eal I -J r h' fierve. ^>-^'- --.iLr^ ^ '-Tr uncus vaius Fig. 2 Fig. 2. Lateral aspect of the heart, showing its principal parts and the distribution of the branches of the vagus nerve. 242. Dissection of the Vagus Nerve. Introduce a glass rod into the frog's throat to distend the parts. Beginning at the 68 angle of the mouth, remove the skin between it and the arm and tympanum. The projection formed by the articulation of the lower and upper jaws may be cut off. Remove the arm. Dissect away the muscles and expose the scapida; this in turn may be tilted to one side or removed. In front of, and partially under, the scapula nerves may be seen. Two nerves will be found lying close together and accompanied by a blood vessel. The first of these nerves is the glossopharyngeal, the second the vagus with its branches. Follow both nerves toward the cranium and note that both glossophar)mgeal and vagus emerge from the cranitim through the same foramen. Dissect the glossopharyngeal distaUy and note that it supplies the tongue. Dissect the vagus noting that it gives off a branch to the oesophagus, then a smaller one the lar5Tigeal cttrving around the aorta to supply the lar3nix, and finally branches to the limgs and heart. In the ttutle the parts are larger and more satis- factorily demonstrated. Fig. 3 Fig. 3. Ventral aspect of the superficial muscles of the left leg of the frog. 243. Dissection of Muscles of Frog's Thigh and Leg. Ventral Aspect. Remove the skin from the ventral aspect of the leg and expose the superficial muscles. 69 Sartorius, a long narrow muscle crossing the thigh obliquely from the outer to the -inner side. It arises from the Uiac symphysis below the acetabulum and is inserted into the inner side of the head of the tibia. The Adductor Magnus is a large muscle lying along the inner border of the sartorius but passing beneath it at its distal end. Its origin is from the public and ischial symphyses, and the muscle passes under the sartorius to be inserted into the distal third of the femur. The Adductor Longus is a long, thin, narrow muscle lying along the outer side of the adductor magnus and often completely hidden by the sartorius; its origin is from the iliac symphysis beneath the sartorius and unites a little way beyond the middle of the thigh with the adductor magnus. The Rectus Internus Major or Gracilis, is a large muscle lying along the inner side of the adductor magnus and of the sartorius. Its origin is from the ischial S3Tiiphysis and it is inserted into the head of the tibia. The Rectus Internus Minor is a narrow, ribbon-like muscle passing along the inner or flexor margin of the thigh; it arises from a tendinous expansion connected with the ischial symphysis and is inserted into the inner side and just below the head of the tibia. 244. Dorsal Aspect of the Thigh. The Triceps Extensor Femoris is the great extensor muscle of the thigh; it arises by three distinct origins from the ilium and acetabulum and is inserted into the tibia just below its head. The Rectus Anticus Femoris is the middle division of the triceps ; it arises from the ventral border of the posterior third of the ilium in front of the acetabulum; about half way down the thigh it joins the next division. The Vastus Internus is the ventral head of the triceps and lies between the sartorius and the rectus anticus. It arises from the ventral and anterior border of the acetabulum. The Vastus Externus is the dorsal head of the triceps. It arises from the posterior edge of the dorsal crest of the ilium and joins the other two divisions of the triceps at about the junction of the middle and distal thirds of the thigh. 70 The Gluteus lies in the thigh between the rectus anticus and the vastus externus. It arises from the sacrum and is attached to the femtir. The Biceps is a long, slender muscle arising from the crest of the ilium just above the acetabulum. It lies in the thigh along the inner border of the vastus externus and is inserted by a flattened tendinous expansion into the distal end of the femtir and the head of the tibia. U-j. GiuTe u.s_ <^^T». — cloaca, — S'tCtfS, ■R«ctut inlBTnui **• • 1 T. --•Gastrocnemi u4 , V-. Terencus ^■•■Ti'bialij AnTieus Fig. 4 Fig. 4. Dorsal aspect of the superficial muscles of the left leg of the frog. The Semimembranosus is a stout muscle lying along the inner side of the biceps, between it and the rectus internus minor. It arises from the dorsal angle of the ischial symphysis just beneath the opening of the cloaca and is inserted into the back of the head of the tibia. There is an oblique line of tendinous intersection running obliquely through its middle. The Pyriformis is a slender muscle which arises from the tip of the urostyle, passes backwards and outwards between the biceps and the semimembranosus and is inserted into the femur at the junction of its proximal and middle thirds. 245. Ventral Aspects of the Deep Muscles of the Thigh. The Semitendinosus is a long, thin muscle which arises by two 71 heads, an anterior one from the ischitim close to the ventral angle of the ischial symphysis and the acetabulum; and a posterior one from the ischial symphysis. The anterior head passes through a slit in the adductor magnus and unites with the posterior head in the distal third of the thigh. The tendon of insertion is long and thin and joins that of the rectus internus minor to be inserted into the tibia just below its head. The Adductor Brevis is a short wide muscle lying beneath the upper end of the adductor magnus. It arises from the pubic and ischial symphyses and is inserted into the proximal half of the femur. The Pectineus is a smaller muscle Ijdng along the outer (or extensor) side of the adductor brevis. It arises from the anterior half of the pubic sj^nphysis in front of the adductor brevis and is inserted like it into the proximal half of the femur. 246. Dorsal Aspect of the Deep Muscles of the Thigh. The Ilio-psoas arises by a wide origin from the inner surface of the acetabular portion of the ilium; it turns around the anterior border of the ilium and crosses in front of the hip joint, where for a short part of its course it is superficial between the heads of the vastus internus and of the rectus anticus femoris; it then passes down the thigh beneath these muscles and is inserted into the back of the proximal half of the femur. The Quadratus Femoris is a small muscle on the back of the upper part of the thigh ; it arises from the ilium above the aceta- bulum and from the base of the iliac crest; it lies beneath the pyriformis and is inserted into the inner surface of the proximal third of the femur, between the pyriformis and the ilio-psoas. The Obturator is a deeply located muscle which arises from the whole length of the iliac symphysis and the adjacent parts of the iliac and pubic sjTnphyses and is inserted into the head of the femur close to the gluteus. 247. Muscles of the Tibial Portion of the Leg. The Gastrocnemius is the large muscle forming the calf of the leg; it has two heads of origin, the larger of which arises by a strong flattened tendon from the flexor surface of the distal end of the femur; while the smaller head which joins the main muscle about one-fotuth of its length below the knee, arises from the edge of the triceps femoris where it covers the knee. The muscle is thick- 72 est in its upper third and tapering posteriorly ends in the strong Tendon of Achilles, which passes under the ankle joint, being much thickened as it does so and ends in the strong plantar fascia of the foot. The Tibialis Posticus arises from the whole length of the flexor surface of the tibia; it ends in a tendon which passes around the inner malleolus, lying in a groove in the lower end of the tibia and is inserted into the dorsal surface of the astragalus. The Tibialis Anticus lies on the extensor surface of the leg; it arises by a long thin tendon from the lower end of the femur and divides about the middle of the leg into two bellies which are inserted into the proximal ends of the astragalus and calcanetun respectively. The Extensor Cruris lies along and is partly covered by the tibialis anticus. It arises by a long tendon from the condyle of the femur and runs in a groove in the upper end of the tibia and is inserted into the extensor surface of the tibia along nearly its whole length. The Peroneus is a stout muscle which lies between the tibialis anticus and the gastrocnemius. It arises from the distal end of the femur and is inserted into the outer malleolus of the tibia and the proximal end of the calcanevmi. 248. Dissection of the Sciatic Nerve. Expose the muscles of the dorsal aspect of the thigh, careftdly separate the biceps and /imMT^ }/erue muscle prevaration. Fig. 5 73 semimembranosus; closely applied to the deeper margin of the biceps will be found the sciatic nerve accompanied by a blood vessel. Carefully follow the nerve toward the body noting its passage between the pyriformis and the head of the biceps. Fol- low the nerve up to its connection with the lumbar region of the spinal cord. RetvuTi to the middle of the thigh and note that the nerve sends off a branch which passes along the extensor side of the tibia along the peroneus and beyond. Follow the sciatic and note that at the knee joint it again divides, one branch going to the gastrocnemius and the other to the tibialis posticus muscle. The sciatic nerve, gastrocnemius muscle and a portion of the femur comprise a nerve- muscle preparation. XVII 249. Place a frog on its belly and note the movements of the caudal lymph-hearts. They are situated between the hip-joint and the median line in a slight depression. The contractions of these hearts are usually visible through the skin, but are seen more distinctly if the skin is removed without injury to the heart. Later, note that the lymph-hearts cease to beat after the destruction of the caudal portion of the myel (spinal cord). 250. Pith the frog. This is accomplished by severing the brain from the myel with a thin-bladed knife at the point where the cranium articulates with the atlas. A slight depression will be felt at this point, which will serve as a guide for the operation. The frog may be firmly held if wrapped in one comer of a towel. 251. After pithing, lay the frog on its back and cut through the skin on the mid-line, and from the middle of this cut make lateral incisions through the skin. Raise up the end of the sternum and cut, a little to one side of the mid-line, through such parts as may be necessary to expose the heart. Pin the parts on the side and note the heart beating with some force and regularity. Count the number of heart beats per minute. Pinch up the pericardium with a pair of fine forceps and remove it from the heart. Tilt up the apex of the ventricle and note a small band of connective tissue passing from its dorsal surface to the adjoin- 74 ing wall of the pericardium. Seize this band with the forceps and divide it between the forceps and the pericardial wall. Con- nect the apex of the ventricle with a heart lever and take a tracing of the heart beat upon the kymograph (revolving drum). After immersion in the varnish and drying paste the tracing in your notes. Lift up the apex of the ventricle, by means of the band already described, and with a sharp pair of scissors cut through the right and left aortae, the pre and post caval veins, and the surrounding tissue, taking care not to injure the sinus venosus. Place the heart in a watch-glass, moistening occasionally with physiological saline solution. The beats will not be interrupted at all, or for a very short time only. 252. Watch the beating of the heart. Do the auricles and ventricle contract simultaneously? What are the number of beats per minute? Compare with 2 s i . Then place in cold saline solution, count again. Then gradually heat,- but. not too high, and note the effect upon the heart beats. 253. Lift up the apex of the ventricle, and with the scissors cut off the apex at the upper third of the ventricle. Watch the separated portions. Is there any difference in the beating? 254. With the scissors separate the two auricles from each other, letting the attached portion of the ventricle remain to each auricle. Do they continue to beat? 255. The same frog, if it has been kept in a moist place, may be used for the following cilia experiment: Place the frog upon its back, and cut through the lower jaw, along the midline, con- tinuing the incision down the oesophagus as far as the stomach. Pin the parts back and moisten the mucosa with physiological salt solution, if it is at all dry. Place a small, thin piece of cork upon the mucosa just below the orbits, and note that the cork is carried toward the stomach by the cilia. Warm a little of the physiological salt solution to 30° C, and repeat the experiment. Apply heavier bits of substance to the mucosa, and note if their positions are changed. Apply to the strip of mucosa a few drops of a saturated solution of chloretone and note whether the motion of the cilia is affected or not. With a scalpel scrape some of the mucosa and examine the ciliated cells in the saline solution under the microscope. 75 2s6. If the caudal lymph-hearts are still beating, pass a tracer or piece of wire down the spinal canal to destroy the myel. If thoroughly destroyed the lymph-hearts will cease to beat. XVIII 257. The circulation of blood. This may be shown very nicely in the delicate external gill filaments of the Necturus, or in the tail of a tadpole, or in the web of a frog's foot which does not contain too much pigment. The animals should be injected with a few drops of a 1% solution of curare, in order that they may not move, and arranged upon the stage of the microscope, so that the parts to be examined may come clearly into the field of vision. Precautions should be taken against drying, by keep- ing the animal well surrounded with moist cloth or absorbent cotton. 258. If the frog is more convenient, prepare it by destroying the brain and injecting the curare under the skin of the back. Place the frog on its belly on the frog board and pin out the digits so that the web will be slightly on the stretch. Keep the parts moist. Put a very small drop of water upon the web, and cover it with a triangular piece of cover-glass, being careful that it does not cut the digits and that no fluid flows over its surface. Examine first with a low power, and then, if possible, with a high power. 259. Note the course of the blood from the arteries to the veins. Arteries may be distinguished from veins by the fact that the blood corpuscles scatter to enter the capillaries diverg- ing from- the artery, while in the veins the corpuscles accumulate from the capillaries converging to form the vein. A slight pulsa- tion may sometimes be observed in the smaller arteries. 260. Note the greater velocity of blood in the arteries than in the veins; the individual corpuscles cannot, perhaps, be made out in either. 261. Note the axial and peripheral zones in the arteries and veins; the peripheral zone is small and under a low power appears free from corpuscles; tuider a high power a few leucocjdies may be seen in the peripheral zone, if the current is not too rapid; 76 in that of the veins a few leucocjrtes and occasionally a red cell will be seen moving along comparatively slowly. 262. Note the passage of the corpuscles usually in single file through the capillaries. 263. Note the elasticity of the red corpuscles, observing the way in which they bend and later regain their normal "form. 264. Study of inflammatory conditions. Remove the cover- glass and absorb the fluid on the web; touch the middle of the web with the tip of a glass rod that has been dipped in creosote (Or a 2% solution of croton oil in olive oil) leaving a minute drop on the web. Put on a cover-glass as before and examine with the miscroscope. If not successful with the web, try the tongue or mesentery. 265. Note the dilation of the arteries, the more distinct appearance of the capillaries, and the enlargement of the veins, accompanied by a quickening of the current. 266. Note a little later, the slowing of the current, the vessels remaining dilated. 267. Note that the leucocytes increase in number in the peripheral zone of both arteries and veins; in the latter the leucocytes begin to cling to the sides, temporarily at first and then permanently. In the capillaries the leucocytes and, less frequently, the red corpuscles stick to the capillary walls, partially or completely blockiag the way. Later stagnation may set in and there is then the appearance of the gradual obliteration of the outlines of the corpuscles. 268. Note the migration of the leucocytes from the capil- laries and veins. This occurs when the circulation becomes slow. Watch, at intervals of 10 minutes, some particular leucocyte adhering to the wall of a capillary or vein. 269. Note the diapedesis of the corpuscles from the capillaries, seen to the best advantage in those capillaries in which the current has almost ceased. 270. Note that the above effects are local, are of greatest intensity in the spot touched, that they extend some distance around the spot, but the circulation in the rest of the web is normal. If the injury has not been too severe, the circulation may become re-established in the stagnated spots, and the inflam- matory appearances disappear. 77 Pig. 7 Fig. 6 Fig. 8 Fig. 9 Figs. 6 to 9. Fig. 5. — Leucocytes sending forth processes which penetrate the wall of the vessel. Fig. 7. — Leucocytes partly through vessel wall, show- ing constriction in centre. Fig. 8. — Leucocytes after penetrating wall regain former shape. Fig. 9. — Appearance of vessel and surrounding tissue after diapedisis has gone on for some time. (After Craig) . Blood. Capillary. f 3 Fig. 10. — Diagrammatic Representation of the Manner in which a Leucocyte Traverses the Wall of a Capillary Blood-Vessel. (After Craig), a, Leucocyte before penetrating; 6, leucocyte sending off process and granules beginning to withdraw to farther end of cell; c, leucocyte partly through wall, granules at upper end of cell; d, granules passing through the wall; e, granules arranged in the portion of the leucocyte farthest from vessel; /, leucocyte, after pene- tration, resuming its original condition; g, leucocyte swept from wall, showing retention of the clear penetrating process. 78 271. Pin out the two horns of the tongue and observe that under the microscope. The tongue is at first pale but soon be- comes reddened as the vessels become filled with blood. With a low power the peripheral zone in the arteries and veins may prob- ably be seen better here than in the web. i2 72. Place the frog on its back, cut through the skin and muscles on one side and draw out the mesentery and pin out a loop of it vuider the field of the objective and observe the circula- tion. The inflammatory phenomena can be well seen in this preparation or that of the tongue. (271). XIX 273. Experiments in reflex action. Pith a frog and place it on its belly. Note the position of its fore and hind limbs. Note the position of the head as compared with a normal frog. Are there any respiratory movements at the nostrils or throat? 274. Pull, very gently, one of the hind-limbs into an extended position and then let go. Does it return to its former location? 275. Gently tickle one flank with a feather or blimt needle. Is there any contraction of the muscles? 276. Pinch the same spot sharply with a pair of forceps. Is there any movement of the leg of the same or opposite side? 277. Pinch the skin aroiuid the anus with a pair of forceps. What is the effect upon the legs? 278. Place the frog on its back. Does it make an effort to get into a natural position? Does it show any sense of equilib- rium? 279. Pass a hook through its lower jaw and hang it to the ring of a retort stand. How do the hind-limbs behave? 280. Pinch very gently the tip of one of the toes; what is the effect? 281. Fill two glasses, one with dilute sulphuric acid, the other with water. Raise the glass containing the acid, until the add just touches the tip of the toes. Is the foot withdrawn? If so, raise the second glass and let the foot be immersed in it, to wash off the add. 79 282. Cut a small piece of filter or blotting-paper, moisten it with strong acetic acid and place it on the flank of the animal. What is the effect upon the leg? Put the piece of paper upon the opposite flank and hold the leg so as to prevent it from moving? Is there any action of the opposite leg? 283. Place similar pieces of paper upon different portions of the body. Note any variety of movements and what seems to be their purpose. 284. Remove the frog from the hook and pltmge it in a basin of water. This will wash off the acid. Does the frog make any movements in the water? Does it float? 285. Open the abdomen and draw out a loop of intestine. Expose the heart and while its movements are under observation, strike or pinch the intestine; the heart is temporarily arrested. If you divide the vagi or completely destroy the oblongata and repeat the stimiolus, the arrest does not occur. Or instead of the intestine you may employ strong stimulation of a limb by the sudden tightening of a string around it. The heart will stop, and the body of the frog will become inert and flaccid, and will not respond to cutaneous stimuli. The bulbo-spinal axis is in a state of "shock". [Waller]. (An unpithed frog may be required for the success of this experiment). 286. Inject 3 to s minims of 0.2% strychnine solution under the skin of the frog's back. Let it remain for a few minutes and then note the effect of the slightest stimtilus, such as jarring the table upon which it lies. Then give 10 to 20 minims of 10% chloral hydrate with a pipette. Make sure that the fluid reaches the stomach. Note if there is any effect upon the convulsions. 287. With a tracer or piece of wire destroy the myel, the convulsions cease. Try any of the preceding stimuli upon the frog now and note the result. 288. Make a nerve-muscle preparation of one of the hind Hmbs. Dissect away the skin and muscles upon the dorsal aspect of the leg, until the sciatic nerve is exposed, leaving it connected with the Itimbar plexus. Denude the femur of its muscles, using the greatest care not to injure the sciatic nerve. Keep the nerve moist with normal salt solution. Pass a copper hook under the sciatic nerve and hang to a tripod. Tilt the tripod so that the leg may come in contact with one of the iron supports. 80 If the tripod has been painted, scrape the paint off. What hap- pens when the contact is made? This is known as Galvani's experiment. 289. Make another nerve-muscle preparation of the other hind limb, but cut the sciatic nerve as near to the myel as possible and separate the leg from the body at the femoro-pelvic joint. Remove the skin as far as the foot. With the forceps crush the gastrocnemius muscle near the tendon of Achilles. See that the end of the nerve is cut off squarely. With a small brush or thin glass rod lift the nerve very carefully in such a way that its cross- section may fall upon the injured portion of the muscle. This stimulates the nerve and causes a contraction of the muscle due to the so-called demarcation currents. XX 290. Induction Machine or Inductorium. Principle of action: If portions of the wires forming two separate circuits Fig. II Fig. II, Induction ceil, ab, binding posts for single induced currents; cd, binding posts for interrupted currents; e, post connecting Neef's hammer with the primary coil; /, electro-magnet; p, primary coil; s, secondary coil. 81 be placed parallel to each other, as in the case of the planes of two spirals or coils of the inductorium, the one wire primary (P), being connected with a source of electricity (battery), the other, the secondary (S), being simply a closed circuit; whenever the P circuit is closed (made), or is opened (broken), currents will at those moments be induced in the S circuit. The make induction current flows in the S circuit in a direc- tion opposite to that of the P circuit : whilst the break induction current flows in the same direction as the original battery current. These induction currents are of very short duration. Fig. 12 Fig. 12, showing the direction of the make and break currents^ In the sec- ondary circuit the direction of the break current is shown by the dotted line and arrow, and the make by the continuous line. Place the induction machine lengthwise in front of you on the table with the interrupter turned to the right. In the DuBois Reymond type the wires are wound into two separate coils; the P coil which is supported by a wooden upright attached to the base of the instrument is composed of relatively thick wire, while the S coil mounted upon a sliding foot is composed of very thin wire, in this case invisible, as it has a protective covering of vul- canite. The parallelism of the wire in the two coils is maintained as long as the axes of the coils coincide. The successive turns of the wire in each coil are also practically parallel to each other. The P coil is provided with a core of soft iron wire which magnetizes when a current passes in the surrounding wire, an electro-magnet being thus produced. The electrical field produced by the coil is greatly intensified by this core, and the effect on the S coil is correspondingly in- creased. The nearer the S coil is to the P coil, the more powerfid will be the induction cvurents. The electromotive force of the currents in the secondary bears a direct relationship to the primary. Thus, if there are 200 turns in the P and 6000 in the S, the electromotive force of the induction cturents would be about thirty times as great as the primary, independently of the influence exerted by the iron core. 291. Connect the Secondary Circuit of the Induc- TORiUM. It is well to do this first in all cases. Fasten a key to the table close to the left end of the machine as it now rests upon the table, and connect the binding screws of the S coil with those of the key by means of two wires, so that when the key is closed the S circuit is thereby also closed. This is the short circuiting key in the secondary circuit. Fig. 13 Fig. 14 Fig. 13— Key closed. Fig. 14 — Key open. Now attach the long circuit wires by means of which the con- nection is to be established, with the seat of stimtilation, i. e., attach the electrodes by their metal tags to the other pair of bind- ing screws of the key. 292. Connect the P Coil for Single Induction Cur- rents. Place the battery upon the table near the right hand end of the coil and attach a key to the table close to it; keep the key open. In making the connections always begin at the battery, and follow the direction the current will take. Connect the C (carbon) pole of the cell to the key by a wire, then wire the other side of the key to the top binding screw, a fig. II, of the P coil, wire b to the zinc pole of the cell. Withdraw the S coil to the end of the scale and let one co- worker hold the electrodes to the tip of the tongue, whilst the other makes the trials. 83 Fig. 14 Fig. 15 — a, b, binding posts of primary coil.- Make and Break the P Circuit with the Key i. Fig. 16. Do this smartly once or twice only and after each trial push the S coil I centimeter towards the P coil. Let the co-worker indicate when he feels the "shock" and whether he does so at closure or opening. Note the position of the coil as soon as the minimal break shock is felt; it is perceived first. Proceed with further trials until the make shock is also felt. Read off the position of the S coil. It is considerably nearer to the P coil. The break shock is the stronger of the two. Continue the approximation of the S coil by short distances to the P coil. The shocks will be stronger each time until finally unbearable. The strength of a stimtdus can therefore be varied by changing the relative position of the S coil. It may approximately be assumed to change inversely with the square of the distance be- tween the two coils. SaXte^, Pig. 16 Fig. 16. Apparatus as set up for make and break shocks. 84 Remove the core of soft iron from the P coil. Find the minimal shocks for break and make shock and compare with the readings in the previous experiments. Next take the S coil out of the slide and place it end on and close up to the P coil. Whilst making and breaking the P circuit turn the S coil so that its axis shall be tiltimately set at right angles to that of the P coil. The shocks will rapidly diminish and finally disappear as the position of the S coil is changed. Explanation : When the battery current at the closure of the circuit is rising in strength in the primary, an opposing induction current is thereby generated in the P coil itself, which retards the battery current from attaining its full strength as soon as it otherwise would, and of course the effect upon the S coil is not so sudden a one. On breaking the P circuit an induction current is likewise generated which has the same direction as the disappearing battery current, and consequently it retards change of the electrical condi- tion but does not interfere much with the suddenness of the sub- sequent drop in potential and therefore the effect upon the S coil is greater than at closure. 293. Interrupted Shocks. Detach the wires from a and b and transfer them to the binding screws c and d. Adjust the top contact screw e so that it touches the spring lightly. Fig. 11. Fig. 17 ing Fig. 17. C, binding post; E, primary coil; D, electro-magnet and bind- post. 85 On closing the P circuit this spring oscillates automatically opening and closing the P circuit, and a succession of induction ciurents is generated in the S coil. The rate of their occurrence depends upon the length of the spring. Explanation: The current from the battery flows through the binding screw d, up the pillar through the spring, up through the top contact screw e to the P coil, and thence round the electro- magnet / and back by the base of c to the battery. Fig. 1 1 . When the cttrrent flows round the circuit, / is magnetized and draws down the spring thus breaking the top contact. Upon this the current stops flowing, the magnet ceases to act, the spring is released and again makes contact with e and so the circuit is re-established and the cycle begins anew. As the break shock is always the stronger of the two, it follows that if these shocks are passed through a tissue for some time that polarization effects will be set up. Ordinarily they are em- ployed for a short time only, and this effect can be disregarded. 294. Electrolysis of Potassium Iodide. An interesting example of electrolysis is seen in the decomposition of potassium iodide. Dip a small piece of filter paper in starch paste to which about s% of potassium iodide has been added and lay the paper over the electrodes. Make and break the circuit using the single induced current. Iodine is set free at the anode and turns the starch blue, forming the iodide of starch. This method may be used to determine which is the anode. The direction of the current in the secondary coil of the inductorium may thus be recognized. (Porter.) I I Fig. 18 86 A make contraction starts from the kathode, a break contrac- tion from the anode. This experiment also shows that the current passes in opposite directions in make and break. 295. The Break Extra-Current. When a galvanic current traversing the primary coil of an induction machine is made or broken, each turn of the wire exerts an inductive influence on the others. When the current is made the direction of the extra current is against, or in an opposite direction to that from the battery, but at break it is in the same direction as the battery current. Pig. 19 Fig. 19. The Break-Extra Current. Apparatus: Battery, two keys, wires, primary coil of induc- tion machine. Arrange the apparatus according to the diagram. Fig. 19. Both keys and the coil are in the primary circuit, the keys being so arranged that either the primary coil, P, or the ■electrodes attached to key 2 can be short circuited. Test either by electrodes applied to the tongue or by means of a nerve muscle preparation. Close key i, thus short-circuiting the coil. Open and close key 2. There is very little effect. Open key i, the current passes continuously through the primary coil. Open key 2 ; a marked sensation is felt, due to the break-extra current. 296. Rheocord. The rheocord consists of a brass or German silver wire {in this case 20 meters in length), placed along a square board, with its ends connected with binding posts. On the wire is a "slider" which can be pushed along the wire as desired. On account of the difference in potential of the two poles of the cell, 87 the potential through the wire will fall uniformly from the anode to the kathode. The difference of potential between post o and post I will be practically one-tenth the electromotive force of the element. Connect the battery (or two) through a key with the rheocord, at posts o and i. Arrange the electrodes from the "slider" post to come in contact with the muscle. Close the key and note the effect. Move the "slider" along the wires and note the effect. An electric current can be graduated by changing the number, arrangement and size of the cells, or by using a rheocord to divide the current itself, the battery remaining constant. 297. Unipolar Excitation. Set up the battery and induc- torium to give single shocks, and at first attach only one wire to the secondary coil. Prepare a nerve-muscle preparation and place it upon a dry glass plate, putting the single wire from the secondary coil under the nerve. Open and close the key in the primary circuit; no contraction occurs. Insert a second wire in the other binding post of the secondary coil and attach its other end to a gas pipe thus connecting with the earth. A contraction will now occur on opening or closing the primary circuit. In the latter case, the amount of current which passes through the earth and the glass plate is sufficient to stimulate the nerve. The short- circuiting key in the secondary circuit is therefore used in most experiments in order to avoid excitation of the nerve in this way. 298. Polarization of Electrodes. If a pair of clean platinum wires be immersed in water, and a current sent through them for a time, it is found that both of the platinum terminals become covered with bubbles of gas. The one in connection with the negative pole of the battery is covered with hydrogen, and the other with oxygen. Upon the removal of the battery and con- nection of the electrodes with a galvanometer, a current will be demonstrated having a reverse direction to that first induced. This condition at the electrodes is known as polarization of electrodes. If a piece of fresh animal tissue connects the pair of wire electrodes, instead of the solution, the same polarization occurs. Chemical changes occur where the wires touch the tissue which can act in the reverse manner, and set up a small current if the battery be removed and the electrodes connected by a conductor. 88 Fig. 26 Pig. 26. Polarization of electrodes. This acts as a source of fallacy in many experiments and is of much importance when a very excitable tissue, such as a nerve, is dealt with. The following experiment will illustrate polarization. Arrange the apparatus as shown in fig. 26, open the key k2 and close the key, ki. The current is sent through the nerve and will polarize it. There is no contraction of the muscle while the ciurent is passing. After one or two minutes, open ki, and then rapidly close and open k2, when contractions will occur, which are due to the closing and opening of the small current set up by the polarized electrodes. The contractions diminish quickly in amount as the nerve becomes depolarized. In order to avoid polarization effects, special forms of electrodes may be used. These are known as unpolarizable electrodes and usually consist of a glass tube containing a saturated solution of zinc s\ilphate. The electrode end of the tube is filled up with a pad of china clay or camel's hair brush, upon which the nerve is laid ; the other end of the tube is fitted with a binding post attached to a zinc wire which dips into the zinc solution. The electrodes in the moist chamber are an example of impolarizable electrodes. XXI 299. Each student is to have a frog, which is to be pithed and have its brain and myel destroyed by passing a tracer or seeker through the spinal canal. The legs are to be used for nerve muscle preparations. Dissect one leg for the first series 89 of experiments, and reserve the other leg for the second series. Begin the dissection upon the dorsal aspect of the leg, removing the skin and muscles very carefully until the sciatic nerve is exposed. iDissect out the nerve as long as possible, and moisten frequently with the salt solution. Remove all of the muscles as far as the knee, leaving the femur and nerve entirely isolated. Avoid all injury to the nerve during dissection, and apply the normal salt solution every few minutes with a camel's hair brush. Arrange the nerve muscle preparation by placing the iemwr in a clamp and allowing the nerve to hang freely. A small lever may be pinned to the foot to emphasize any movements that may occur. Apply the following stimuli : 300. Mechanical. Pinch the free end of the nerve sharply with a pair of forceps ; the muscles contract and the foot is raised suddenly. Cut off the pinched portion. Contraction again occurs. 301. Thermal. To the same preparation apply at the free end of the nerve a wire or needle heated to a dull heat or a lighted match. Contraction again occurs. Cut off the dead part of the nerve. 302. Chemical. Place some saturated solution of sodium chloride in a watch glass and let the free end of the nerve dip in it. It requires a few moments for the salt to diffuse into the nerve on account of the difference in the specific gravity. Soon the joints of the toes twitch and by-and-by the whole limb is thrown into irregular, flickering spasms, which terminate in a more or less continuous contraction, constituting tetanus. Cut off the part of the nerve affected by the salt; the spasms cease. (a) Finish the experiment by exposing the nerve to the vapor of strong ammonia in a test tube or bottle. The ammonia must not act directly upon the muscle, the tube should be raised slightly above the level of the muscle and the end of the nerve elevated to the mouth of the tube. There should be no contraction if the vapor has not come in contact with the muscle. Apply ammonia to the muscle. It contracts. 303. Electrical. For the following experiments, use the other leg of the frog, taking the same precautions in the dissection and application of the salt solution. 90 (a) Arrange the nerve-muscle preparation with the femur in a clamp and connect with a recording lever. Arrange the battery and DuBois Reymond Key with the induction coil. Connect the battery wires with the primary coil and remove the secondary coil to the lower end of the scale. Arrange the electrodes with a short-circuiting key and a recording drum with smoked paper, conveniently to the preparation. When all is in readiness, close the connection by means of the key by raising or lowering its lever. Or in other words "making" or "breaking" the current. Gradually move the secondary coil along the scale while making and breaking the cturent. The current is made when the connection is complete, and broken when the connection is interrupted. Note at what point on the scale the first result appears and whether it be from "make" or "break." Make a table of your results as follows: In one column indicate the distance of the secondary coil from the primary. In another, Response at Make, and the last column, Response at Break. This is electrical stimulation in the form of single induction shocks. The highest tracings represent maximal and the lowest minimal stimuli. Submaximal stimuli represent any strength between these two extremes. (b) Remove the induction coil and use only the battery with its wires and the key. Make and break the current as in (a). Notice that if the key be so arranged as to permit the current to flow continuously through the nerve, no contraction occurs, provided there be no variation in the intensity of the current. Rapidly make and break the current by opening and closing the key; a more or less perfect tetanus is produced. This is the constant current form of stimulation. (c) Remove the wires from the primary coil and connect them with the sockets leading to the vibrating hammer. On applying the electrodes to the nerve or muscle the latter is at once thrown into a state of rigid spasm or continuous contraction called tetanus. Compare this tracing with that of (a). This form of stimulation is known as the interrupted current or repeated shocks. 304. Electricity itself is not readily conveyed through the nerve, but the irritation caused by it, generates a stimtilus which is transmitted. Ligate the nerve by tying tightly around it a 91 piece of thread. Stimulate the nerve as before; there should be no result, as the ligature has crushed the nerve and blocks the passage of the stimulus. Scratch your name on the above tracings to identify them. The tracings may be made permanent by drawing the paper through a pan of shellac and allowing them to dry. XXII 305. Secondary Contraction. Arrange the induction ap- paratus for single make and break shocks. Pith a frog and use the hind legs for nerve-muscle preparations, and arrange them upon a glass plate. Place the sciatic nerve of the left leg upon the gastrocnemius muscle of the right leg, fig. 20. Place the Seconda rt/ Conir'acitcon' . Fig. 20 Pig. 20, », nerve; m, muscle. sciatic nerve of the right leg over the electrodes and stimulate the nerve with single induction shocks and note that the muscles of both the right and left leg contract. The contraction in the left leg is called a secondary contraction. Repeat the experiment, using the constant current. Note if there is any difference be- tween the make and break shocks. 306. Secondary Tetanus. Prepare the induction apparatus for interrupted shocks, and again stimulate the right sciatic nerve. 92 The right gastrocnemius muscle is thrown into tetanus. The left gastrocnemius is simultaneously tetanized. This is known as a secondary tetanus, and is a proof of the "action current" in muscle. The left sciatic is stimulated by the variation of the muscle current during the contraction of the right gastrocnemius. Ligate the left sciatic near its muscle ; stimulate the right sciatic ; there should be no contraction of the left gastrocnemius. Leaving the left sciatic in position, tie a ligature around the right sciatic near its muscle and stimulate. Is there contraction in either preparation? This experiment also shows that "electricity, as such, is not transmitted through the nerve as the thread of the ligature is a conductor. The electricity serves merely as a stimulus causing an impulse which can traverse the normal nerve but cannot pass beyond a ligature or a crushed portion of the nerve. electrode . Fig. 21 Fig. 21, n, nerve; m, muscle. 307. Secondary Contraction From Nerve. Make a nerve- muscle preparation of the right hind leg of the frog and lay it on a glass plate. Dissect out a long piece of the left sciatic nerve. Remove and arrange it in such a way upon a block of paraffin that one centimeter of it overlaps a corresponding length of the right sciatic, fig. 21. Stimtdate the left nerve with a single induction shock; the muscle contracts. Stimulate with the interrupted current; the muscle is thrown into tetanus. Stimulate also with the constant current and compare effects. If properly conducted this experi- 93 ment will also show that a nerve impulse can pass in both direc- tions. Stimtilate the left muscle directly by applying the electrode to it and note any effect upon the right muscle. Ligate the left sciatic nerve between the electrodes and the right nerve; stimulate again. The muscle does not contract. In the former case, therefore, its contraction was not due to an escape of the stimulating current. 308. Secondary Contraction From the Heart. Excise the heart; lay the nerve of a fresh nerve-muscle preparation upon it as per diagram, fig. 22. The muscle contracts at each beat of the heart, being excited by the electrical current which accom- panies each beat. ^/y, Fig. 22 Fig. 22. Secondary contraction from the heart. Crush the apex of the ventricle with the forceps and arrange the nerve so that its cut end will come in contact with the injured portion of the heart. Note the result. 309. Paradoxical Contraction. Arrange the battery and key for a constant ctirrent. Pith a frog and expose the sciatic nerve down to the knee. Trace out the two branches into which it divides, fig. 23. Cut off one of these branches as near as possible to the knee and stimulate near its cut end. The muscles, supplied by the other branch of the nerve, contract. Try mechanical or chemical stimulation of the same branch. What is the result? The second branch is stimulated by the electrotonic alteration of the first. Electrotonus — ^modification of the vital properties of irritable and contractile tissues when influenced by a constant battery current. (Stirling). 310. Experiment with Ergograph. Adjust the apparatus so that the lever will write upon a drum revolving at its lowest rate of speed. Tie the three fingers of the right hand leaving the 94 Pa. T-cuiox. I ea i. Contract con. . Fig. 23 Fig. 23, n, nerve; m, muscle. index finger free. Adjust this finger to the vertical rod con- nected with the lever. Raise and lower the finger thus causing a simultaneous movement of the lever, which leaves its record upon the smoked paper. Raise and lower the finger at regular intervals (one second). The abductor indicis is the principal muscle involved. Continue until distinct fatigue occurs. Fix and preserve the tracing. XXIII 311. Elasticity and Extensibility of Muscle. Dissect out the gastrocnemius muscle from the frog's leg. Attach the femur firmly in the muscle clamp and the tendon to the writing lever, to which a small scale pan is attached. See that the lever writes horizontally on a stationary drum. The weight of the scale pan may be neglected. Place in the scale pan, successively, different weights; 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 or more. Put in the 10 gram weight and allow to remain 30 seconds. The lever will descend. Remove the weight and the muscle will return to its original position. Replace the weight, the lever will drop along the line it first made, revolve the drum a very short distance horizontally and add 10 grams more; the lever will descend. Revolve the 95 drum again for a short distance equal to the previous horizontal distance, add lo grams more and repeat the above processes until the heavy weights have been used. The "steps" of the "stair- case" wUl become shorter and if the apices of all of the "steps" be joined, the line will form a hyperbola. At the end of the experi- ment remove the weights and note any contractile phenomena. Does the muscle return to its original position? Repeat the experiment with a thin strip of rubber, substituted for the muscle. Join the apices of the "steps" with a line and compare with that of the preceding experiment. Test in the same way the elasticity of a short strip of aorta (cat) provided for the experiment. Test also a short piece of the frog's intestine. 312. Independent Irritability of Muscle. Apparatus: Battery, induction coil, two keys, wires, electrodes, curare, etc. Arrange the battery and induction coil for an interrupted current with one key in the primary circuit and the other key to short-circuit the secondary. Destroy the brain of a frog. Expose the sciatic nerve and the accompanying artery and vein on the left side, being very careful not to injure the blood vessels. Isolate the sciatic nerve and tie a stout ligature around all of the other structures of the leg. Inject s to 10 drops of the curare solution into the abdominal cavity. The poison will be carried to every other part of the body except the leg below the left ligature. The animal is paralyzed in from twenty to thirty minutes. If the non-poisoned left leg is pinched it is drawn up, showing that it has not lost its reflex powers; while the poisoned right leg has lost its reflex. When the frog is thoroughly under the influence of the poison, i. e., when all reflexes cease, expose both sciatic nerves as far up as the vertebral column and as far down as the knee. Stimulate the right sciatic nerve. There is no contraction. Stimulate the right gastrocnemius muscle; it contracts. The poison has therefore not affected the muscle. Stimtilate the left sciatic nerve above the ligature, the left leg contracts. The poison has not affected the nerve trunk. The nerve impulse is blocked by the curare, in all probability by paralysis of the end plates of the motor nerves within the muscle. Apply several drops of a strong solution of curare to the left 96 gastrocnemius, and after a time stimulate the left sciatic nerve; there should be no contraction, but on stimulating the muscle directly, contraction occurs. 313. Bernard's Method. Two nerve-muscle preparations are made. The nerve of one (A) is immersed 20 to 30 minutes in a solution of curare in a watch-glass. The muscle of the other preparation (B) is immersed in the ' curare in another watch-glass for an equal length of time. 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 paralyzed. 314. Relative Excitability of Muscle and of Nerve. Determine the minimal break shock which will cause a muscle twitch through the sciatic nerve, and then apply the same stimulus to the gastrocnemius muscle directly. It will not cause contrac- tion. Slide the S coil nearer to the P coil until the stimulus is strong enough to cause the muscle to contract, and note the difference in strength required. This experiment does not permit of the conclusion that the muscle possesses independent irritability, as the nerve terminations in the muscle are not excluded. (See Curare Experiment.) 315. Changes in the Excitability of a Nerve When Dying. Dissect out the sciatic nerve of a frog, but do not cut it from its connection with the myel. Place under its whole length a strip of thin rubber or a piece of waxed paper and keep the nerve moist with the saline solution. Carefully raise the nerve with the glass rod or camel's hair brush and explore it from one end to the other with minimal single induction break shocks, the effect of which have been tested fir^t at the middle of the nerve, and note if there is a difference in excitability at any point. There is usually one or two such points. Locate them. Determine this by the change produced in the muscular effect, such as an increase, decrease or absence of contraction. Now cut the nerve at its spinal origin, and compare the excita- bility at the cut end with that at a point near the muscle. Repeat this from time to time. The cut end will soon show a greater excitability which will decrease later until it is completely lost. 97 A d5dng nerve first rises and then falls in excitability and finally loses it altogether. A nerve undergoing the process of dying be- comes for a time, more irritable for this reason. 316. Dead Muscle and Nerve. Immerse a nerve-muscle preparation for a few minutes in warm water (40 degrees C). Apply all of the preceding stimtdi and compare results with those obtained from a normal preparation. 317. Kuehne's Sartorius Experiment. Carefully dissect out the sartorius muscle, and to the tendon which attaches it to the tibia tie a fine thread. The upper end of the muscle may be freed from its attachment to the sjonphysis. Suspend the muscle with its upper end hanging downward and bring up under it a little glycerin in a watch-glass until the end of the muscle just touches the glycerin. Observe for a minute or two.. No contrac- tion shotdd result. Cut off the end which has touched the glycerin and note that the muscle contracts as a result of the mechanical excitation. Again touch the cut surface with glycerine and observe. If only about one millimeter has been cut off there is again no contraction. Cut off a fresh millimeter of muscle and repeat as before. It will be found that when about three* or four millimeters of t?-e cephalic end have been cut away, on contact of the freshly exposed end with the glycerin, the muscle shows irregular twitchings and is at last thrown into a state of incomplete tetanus. This experiment should be completed by showing that if a gastrocnemius nerve-muscle preparation be made and the cut end of the nerve dipped into glycerine, the gastrocnemius is thrown into a similar series of irregular twitching. Nerve fiber is there- fore excitable to glycerin. The same experiment may be tried upon a curarized muscle. The experiment on the sartorius muscle confirms the fact, as shown by histological examination, that no nerve fibers are present in the ends of the muscle; for the same experiment shows the same results for two or three millimeters of the distal end of the muscle. Muscle fiber is not excited by glycerin and not until enough of the muscle was cut away to expose the nerve fibers in the muscle did the irregular twitchings occiu:. 98 XXIV 3i8. The Moist Chamber. Muscle and nerve tissue dry shortly after their removal from the body and the usefulness of an experiment is, sometimes, much hampered by this fact. In order to prevent drying, a moist chamber is employed. This apparatus consists of a glass cover fitting tightly over the myo- graph (muscle electrodes). A thread passing through an aperture connects the muscle with the recording lever. A few pieces of blotting paper wet with the saline solution placed in the chamber keeps the air moist. 319. The Muscle Curve. If a stimulus of very short dura- tion be applied to a muscle or its nerve, the muscle responds by giving a contraction of very short duration. This is termed a simple twitch. The curve obtained from a muscle falls naturally into three parts: (i). From the point of stimulation (a) to the point of com- mencing contraction (b). This is Jsnown as the Latent Period. During this time there is no change in the length of the muscle. A muscle does not contract simultaneously all over, but the contraction starts at some one spot and then spreads in a wave- like manner over the rest of the muscle. Following an excitation at one spot, the fibers in that position contract, but do not at first lead to a movement of the recording lever but rather to a stretch- ing of the remainder of the fiber, both above and below the point of contracting. The parts which have to be moved possess some inertia. (2). From the point of commencing contraction (b) to the highest point of the curve (c). This is termed the Period of Contraction. The curve is at first convex to the abscissa, or base line, which means that the rate of contraction is at first very slow as seen by the acute angle which the first part of the curve makes with the abscissa; it then rapidly increases as shown by the increasing inclination to the abscissa, and very soon reaches a maximum rapidity. From this, again, there is another change in rate, this time in the reverse direction, for the curve becomes concave to the abscissa line and gradually shortening becomes slower until at last it ceases, when the tangent to the curve becomes parallel to the abscissa. o- d abscissa. ''— Fig. 24 Fig. 24, ah, latent period; he, period of contraction; ci, period of relaxa-. tion. (3). The third portion of the tracing is from the highest point (c) to the point (d) at which the lever again reaches the abscissa. This part is called the Period of Relaxation. The terminal point (d) is often a difficult one to determine with any accuracy because the lever does not come instantly to rest ; but as it always possesses some inertia, it oscillates for a time about a mean position which it ultimately reaches. 320. Arrange a nerve-muscle preparation in the moist chamber so as to record its contraction upon the drum. Connect the battery with the induction coil introducing into the primary circuit a make and break key and an electro-magnet. Use also a short-circuiting key in the secondary circuit. Arrange the writing tip of the lever from the electro-magnet, so that it will write just below and on the same vertical plane as the muscle lever. Spin the drum at a fairly rapid rate by hand and use single induction shocks by breaking the primary circuit. The lever of the electro-magnet will indicate the instant the current is sent into the nerve-muscle preparation. (The lever rises and falls alternately as the current is made or broken). The muscle lever will rise just after that of the magnet. The tips of the two levers having started side by side from the same vertical plane, the difference on the two abscissas between the rising point of the lever from the magnet and the rising point of the muscle lever, will be the approximate latent period. Verticals drawn through the two abscissas at the rising points of the two levers will be of use in determining more accurately the extent of the latent period. Verify as far as possible on the tracing, the preceding state- ments. Also obtain a curve from the make shock alone (short circuit the secondary coil when the break shock shotild occur). 100 Vary the position of the secondary coil and compare the ciirves. Get a tracing of the contraction of plain muscle, by using a piece of the intestine or stomach of the frog. Compare. 321. Amplification or Magnification. The amplitude of the tracing is measured by the ordinates; it is the distance which separates each point of the tracing from the line of the abscissa. When the primitive length of the muscle does not change as in the period of latent excitation, this distance equals o, and the tracing is confounded with the line of the abscissa. When the muscle shortens, the tracing is raised above this line to a height relative to the degree of shortening. When the muscle elongates the tracing falls below this line to a certain extent. But as the muscle acts on a long lever, the changes in the length of the muscle are amplified on the tracing in a noticeable way. If, for example, the lever has a total length of 150 millimeters and the tendon of the muscle is attached to a point 15 millimeters on the axis of rotation of the lever; each millimeter of muscle shortening will be produced on the tracing by a height (amplitude) of ten millimeters (i centimeter). It is not difficult when one knows the length of the lever and the distance from the point of attachment to the axis, to calculate the actual degree of muscular shortening. Amplitude depends upon the length of muscular fiber; the longer the fibers of the muscle the longer the ctu^e of amplitude. In general the amplitude increases with the intensity of the excita- tion, but there is a limit. Determine the amplitude in your tracing by measuring the length of the lever in millimeters; then measure from the point of attachment of the muscle to the fulcrum and divide the totar length by this and the result will give the degree of magnification of the shortening of the muscle. 322. Work Done During a Single Contraction. Arrange a gastrocnemius to record on a cylinder, but record only the "lift," the cylinder being stationary, move the cylinder by the hand as reqmred. On the lever imder the muscle attachment place weights of known value. With each twitch the muscle lifts the weight, and thus does a certain amount of work which is easily calculated. 101 (a). Measure the height of the tracing from the base line or abscissa. This is conveniently done by a millimeter scale. The work which is done (w) is equal to the load (1) multiplied by the height (h) to which it is lifted; w=lh. But, of course, a long lever being used the tracing is much higher than the actual shorten- ing of the muscle. (b). To determine the exact amount of the lift, one must know the length of the lever and the ratio between its arms. Suppose the one to be ten times as much as the other, then the total work in gram-millimeters must be divided by ten. Try different weights always using the same stimulus. It will be found that at a certain point there will be a maximum contrac- tion after which the contractions will become weaker, because of the greater load and fatigue. Calculate the amount of work done at the maximum contraction. 323. Record of the Thickening of a Muscle. Prepare a nerve-muscle preparation and lay it on a glass plate, keeping it well moistened with the saline solution. Arrange the battery and induction coil as before. Adjust the heart lever (such as used in recording the beat of the frog's heart) so that the vertical portion of the lever rests upon the belly of the muscle. Use the break and then the make shocks as before. Compare these curves with the others. The drum should revolve at its fastest rate. XXV 324. Influence op Veratrine on the Contraction of Muscle. Destroy the brain of a frog, and inject hypodermically four or five drops of a 1% solution of sulphate of Veratria or a drop or two directly in the muscle. When the frog is under the influence of the poison, cause a reflex act by mechanically stimulating the skin of the leg. The limbs are extended, and remain so for several seconds, due to the prolonged contraction of the extensors over- coming the flexors and thus causing extension of the legs. Arrange the induction machine for single shocks and make and break the primary circuit by means of the key. Short-circuit the secondary. Do not stimulate the muscle often as the veratria effect diminishes with the activity of the muscles. 102 Make a nerve-muscle preparation; on cutting the nerve notice the prolonged extension of the legs. Arrange the muscle lever to record its movements on a slow moving drum. Take a tracing. Note that the muscle contracts quickly enough, but the contraction is very high compared with that of a non-poisoned muscle, while the muscle relaxes very slowly indeed. The relaxation phase may last several seconds. The tracing may show an uneven curve due to irregular spasms of the muscular fibers. Take another tracing with a quick revolving drum, and a curve reaching the whole circtraiference of the dnom may be obtained, or the drum may go arQU,nd several times before the relaxation is complete. Note that if the "veratrized" muscle be made to contract several times the effect passes off — only a simple twitch being obtained — ^but is re-established after rest. A high temperature also causes it to disappear. 325. Fatigue of Muscle. Arrange an induction coil for break shocks, i. e., adjust the strength of the stimulus so that only one, the break and not the make, will appear. Prepare a nerve-muscle preparation; use a long lever and a weight of 50 grams. Use a slow revolving drum on which to record the muscle tracings, so slow that the ascent and descent of the lever form merely one line. Break the primary current at regular intervals. Note the "staircase" character of the record, i. e., the second contraction is higher than the first, the third than the second and so on for a certain number of contractions. After that the height of the contraction falls steadily so that a line uniting the apices of all of the contractions forms a straight line approximately. Note later that in the phase of relaxation the lever does not reach the abscissa (contracture). If the march of events be arrested, and time given for repose, then, on stimulating, the lift increases, but the effect lasts only for a short time. After the gastrocnemius muscle is thoroughly fatigued, cut across the middle of the muscle with a scalpel and test with litmus paper the area of the cross section thus exposed. Test in the same way a cross section of the sartorius or some other muscle which has not been fatigued. 103 326. Tetanus. Prepare a nerve-musde preparation. Ar- range to record on drum with the smallest fan. Place a key in the primary circuit, also one in the secondary and wire for interrupted ctirrent. Adjust the special wire and weight in the vibrating hammer, so that it swings at its lowest and gentlest rate. Open the short-circuiting key in the secondary circuit. Make the current for very short intervals in the primary. Adjust the weight so as to get faster vibrations and compare. Fig. 25 Fig. 25. Curve of tetanus. At the beginning ad, the individual contrac- tions are somewhat discernible; these disappear and the general level of the curve rises to e. At this point the stimulus was removed and the curve dropped quickly toward the base line. Study the tracings. The first are indented but gradually there is more and more fusion of the teeth until a curve unbroken by depressions is obtained. In the curve of complete tetanus the ascent is at first steep then slightly more gradual, speedily reaching a maximum, when the lever practically records a horizon- tal line parallel to the abscissa. When the current is shut off the descent is very steep at first, and towards the end very slow. The number of shocks required to produce tetanus depends on the animal, the muscle, and the condition of the latter; the more fatigued a muscle is the more slowly it contracts, and there- fore, the more readily does fusion of contractions take place. A fresh frog's gastrocnemius requires about 27 to 39 shocks per second to produce tetanus. Replace the key in the primary circviit by a metronome; con- nect the wires with the primary coil. Vary the rate of vibration of the metronome and observe the effect on the muscle curve. Compare with the previous tracings. 104 XXVI 327. Influence of Temperature upon the Contraction OF Muscle. Prepare a gastrocnemius muscle, leaving it attached to the femur. The sciatic nerve may be disregarded. Fasten the femur in the clamp on the under side of the cover of the "muscle warmer." Tie the end of a fine copper wire around the tendon of Achilles. Bring the other end of the wire through the opening in the bottom of the muscle warmer and bend the wire around the muscle lever, making stire that the wire connect- ing the tendon with the muscle lever is vertical. Connect the end of the fine copper wire with one of the binding posts of the secondary coil. Cormect the other post of this coil with the bind- ing post on top of the cover of the muscle warmer. Connect the battery with a make and break key to the binding posts of the primary coil of the inductorium for single induced shocks. Fill the outer chamber of the muscle warmer with crushed ice. Bring the writing point of the muscle lever against a smoked drum and let the drum revolve at a fairly rapid speed. Insert a thermoraeter in the top of the muscle warmer and stimulate the cooling muscle at intervals of s degrees with a maximal break current. As the temperature falls, the contraction ctirve becomes longer and the muscle shows a tendency to contracture. Indicate the tempera- ture upon each curve made. Place a fresh paper on the drum and let it revolve slowly. Adjust a flame under the arm of the muscle warmer and stimidate the muscle with a maximal break current at intervals of 5°. Indi- cate the temperattire upon the curves as before. "The height of contraction is least at the freezing point of the muscle ( — 5 degrees). It rises from the freezing point to zero; falls from zero to 19 degrees; increases to 30 degrees, which is the maximum; from 30 to 45 degrees diminishes again, and at 45 degrees the frog's muscle usually enters into a state called rigor caloris; the muscle becomes opaque, inelastic, resistant to the touch, shortens very considerably and undergoes chemical changes of great importance. The duration of contraction lessens with the rising temperature, being least at .30 degrees. Above 30 degrees the duration remains practically unchanged. The latent period is increased at low temperatvires, diminished at high. 105 Above 30 degrees the excitability to electrical stimuli diminishes steadily; it disappears almost entirely before rigor is reached." — Porter. 328. The Influence of Load Upon the Contraction OF Muscle. A muscle to which a load is suspended is said to be "loaded." If a muscle contracts against a small and constant resistance, so as to be extended by a constant force during its contraction, the curve described by a light lever attached to it is termed ' 'isotonic. " If a muscle contracts against a large resistance, e. g., a strong spring, so that it can shorten very little, the curve described by a lever attached to it is termed "isometric." The latter is flat-topped, i. e., it shows a period of maintenance at maximum contraction. The muscle reaches its maximtun tension sooner than its maximum shortening and maintains the maximum tension longer than the maximum shortening. As ordinarily employed, the myograph gives an isotonic curve. Arrange the apparatus as for a simple twitch. Arrange a nerve-muscle preparation and attach to a lever weighted only with the scale pan. Obtain a tracing as thus arranged. Increase the load by adding a 10 gram weight and get tracing. Increase the load still further by adding 30, 50 and 100 grams respectively and compare the tracings. It will be noticed that the latent period increases as the load increases; the period of contraction also tends to increase; the period of relaxation is at first decreased but with heavier loads is increased; the heights of the contraction diminish progressively as the load increases. 329. After Load. In the ordinary study of muscle twitch, the contractions have occurred whilst the load on the muscle was as nearly as possible constant. There is, however, a method which is exemplified in many bodily movements, in which the muscle is under a low tension until it commences to contract, and then, only, experiences a rise of tension. This is called the method of after loading. Arrange the apparatus for taking a simple twitch with a nerve-muscle preparation. Load the muscle with a weight of 20 grams attached to the pully of the muscle lever. Have the screw of the muscle lever adjusted so that the muscle itself bears no weight. Now lower the screw so that the whole load is carried by the muscle and bring the writing tip of the lever 106 so that it will write horizontally upon the drum. Get a tracing of the muscle curve, using the break shock only. Now raise the screw to support the writing lever, so that the writing point is placed at the level of the apex of the curve just taken. Record from this position another curve. It will be found that the muscle still raises the lever. Raise the screw again until the level of the writing point is at the summit of this second curve, and again get tracing. Repeat this process until no further result is obtained. The important and characteristic feature of these curves is that though the weight is supportfed at a level which it just reached at the height of a previous contraction, it is still further raised when the muscle is again stimulated. Under such conditions, the muscle contracts to a greater degree than when freely loaded. The latent period is increased as the height of support of the weight is increased. This is accounted for by the fact that the muscle is taking in any "slack" there may be; also that it is gradually increasing its tension until it is able to lift the load, The first portion of such ■ a twitch is isometric ; but beyond a certain point it suddenly becomes isotonic, and its shortening is then registered. 330. iNDUCTroN IN Nerves. Remove the secondary coil and with the aid of a glass plate, lay the nerve of a well moistened nerve-muscle preparation vipon the primary coil in such a way that the free end of the nerve touches the nerve near the muscle or touches the muscle itself, so as to form a closed circuit. Make and break the primary circuit. Make and break currents will be induced in the nerve and the muscle will contract. 331. Telephone Experiment. Arrange a nerve-muscle prep- aration with its nerves over a pair of electrodes. Connect the latter with a short-circuiting key. Attach the key to the tele- phone by means_ of two wires. Open the short-circuiting key; shout or blow into the telephone, and note that the muscle con- tracts vigorously. Remove the electrodes from the key and connect directly with the telephone. What is the result? 107 XXVII 332. Cardic Vagus of the Frog or Turtle. Refer to the previous dissection of the vagus. Pith the frog. Lay it on its back on a frog board. Expose the heart, remove the sternum and pull the fore legs well apart. Distend the oesophagus by intro- ducing a glass rod or tube; the nerves leaving the cranium are better seen winding from behind when the oesophagus is distended. Remove such muscles as may be necessary. Three nerves are seen coursing round the pharynx. The lowest is the hypoglossal, easily recognized by tracing it forward to the tongue, next is the vagus in close relationship with a blood vessel, and finally the glosso-pharyngeal. Observe the laryngeal branch of the vagus. The vagus as here exposed, outside the cranium, is really the vago-S3mipathetic as it contains fibers from the sjmipathetic system. The glosso-pharyngeal and vagus leave the cranium through the same foramen, in the exoccipital bone, and through the same foramen the sympathetic enters the skull. Stimulation of the vagus. Adjust a heart lever so as to record the contractions of the heart upon a drum moving at a medium Vagrit Pig. 26 Fig. 26. Cardiac Nerves of the Prog. (Foster) . rate of speed. Place well-insulated electrodes under the trunk of the vagus, stimulate it with an interrupted current, and observe that the whold heart is arrested in diastole. 108 If the stimtdation is kept up the heart will finally "escape" from the influence of the stimulus and will recommence beating. Note if the auricles appear to be more inhibited than the ventricle. There is often a difference in effect between the two vagi. Sometimes one vagus is found not to possess any inhibitory fibers, in which case the opposite vagus is usually found especially active. It is generally found that the effect is not identical on the two sides, one usually being more powerful than the other. ■The arrest, or period of inhibition, is manifest in the curve by the lever recording merely a straight line. If the laryngeal muscles contract, and thereby affect the position of the heart, divide the lar3mgeal branches of the vagus. There is an appreciable time or latent period, before the heart reacts to the stimulus and likewise when the stimulus is removed the heart does not at once regain its normal movement. Note that when the heart begins to beat again the beats are at first small and gradually rise to normal. In some instances, however, they are more vigorous and quicker. Cut both vagus nerves and compare the tracing with those just obtained. 333. Action OF Drugs ON Heart. The experiments may be performed upon a heart which has been removed and placed in a watch-glass or preferably upon the heart in its natural position. Muscarine. Pith a frog, expose its heart and with a fine pipette apply a drop of serum or saline solution containing a trace of muscarine, which rapidly arrests the rhythmical action of the heart, the ventricle being relaxed, i. e., diastole, and distended with blood. Get a tracing from the heart while under the influence of muscarine. Atropine. Remove the solution of muscarine as much as possible by absorbing it with filter paper and after a few minutes, with another pipette, apply a few drops of a 0.5% solution of atropine sulphate in saline solution, the heart gradually again begins to beat rhythmically. Get a tracing. Pilocarpine. In another frog arrest the action of the heart by appl3dng a few drops of a 0.5% solution of pilocarpine, and then apply atropine to antagonize it, and observe that the heart beats again after the action of the atropine. Nicotine. Apply a drop or two of a 0.2% solution of nicotine. Stimulate the vagus and note that it no longer arrests the heart's 109 action, but stifflulation of the sinus venosus does; so that nicotine paralyzes the fibers of the vagus and leaves the intracardiac inhibi- tory mechanism intact. XXVIII In this exercise four styles of sphygmographs are available. Each student should obtain a tracing of his ptdse from these instruments; fix and preserve the records for comparison. 334. Ludwig's Sphygmograph. With a soft pencil make a mark upon the skin of the wrist at the point where the radial pulse is most distinctly felt. Apply the instrument so that the button rests upon the pulse. Use the arm rest so that the parts will remain steady. Adjust the instrument so that the writing lever will move freely and give as large a curve as possible. Take tracings on the revolving drum. Suspend respiration for a few seconds and notice whether there is any effect upon the pulse. 335- Von Frey's Sphymograph. Adjust in the same manner as for Ludwig's. See that the clockwork runs properly and take tracing on the small drum. 336. Richardson's Sphymograph. Adjust the pressure of the button upon the artery until a maximum excursion of the marker is obtained. Wind up the clockwork, insert a strip of smoked paper between the guide wheels, and let the paper travel past the recording point as soon as the latter moves regularly. 337. Teske's Sphymograph. Apply this instrument to the radial artery so that the amplitude of the pulse is at its great- est. When all is in readiness, set the smoked paper in motion by pressing the lever. Care must be taken not to move the instru- ment nor the arm of the person while the pulse is being registered. This instrument magnifies the movements of the artery fifty times. The cloclcwork is regulated so that the smoked paper shall pass through in ten seconds. Six times the ntimber of pulsations traced on the paper will give the number per minute. The clockwork will not pass more than two lengths of the paper at the same rate. It is best to wind it up anew after two lengths have passed. 338. Sphygmometer. The sphygmometer is an apparatus designed for the purpose of obtaining the blood pressure. It is a 110 matter of some importance to know even approximately the arterial pressure and its variations under normal and abnormal conditions. The first practical method for determining this point upon man was suggested by von Basch (1887) who devised an instrument known as the sphygmomanometer. Since then a number of different instrtiments have been devised for the pur- pose. The brachial and radial arteries .are the vessels ordinarily used, depending upon the kind of apparatus. The general princi- ple in the use of these instruments is to determine the amount of pressure necessary to obliterate the pulse and this can be observed from a registering apparatus which is a constituent of the instrument. The diastolic as well as the systolic presstires may be determined. The Riva-Rocci, Janeway, Stein and other forms of modem apparatus are available and give as up to date results as possible. Directions usually accompany the different makes and should be followed rather strictly to obtain accurate results. 339. The Pneumograph. Apply this apparatus to the thorax. Connect with a tambour and take a tracing on a revolv- ing drum. Study the curves. Does expiration occupy a longer time than inspiration ? Compare with other members of the group. Do not look at a tracing while it is being made, a person un- consciously attempts to regulate the breathing so that a perfect curve is made. Note the effect upon the curve of the following phenomena: squeeze suddenly a rubber bulb held in the right hand; swallow a few mouthfuls of water; hold the breath while you count ten; speak a few words ; laugh. Have your co-worker indicate on the drum when each of these acts are performed. Use a time marker marking half seconds. XXIX 340. The Scheme of the Circulation. The circulatory system may be considered as consisting of a contractile organ — the heart, connected on one side with a system of elastic vessels — the arteries, which expand into a very large area of thin-walled capillaries. The capillaries converge to form the venous system which connects with the opposite side of the heart. The arteries Ill possess thicker walls than the other vessels and if a cut be made into an artery, especially a large one, the blood issues in spurts and with considerable force. If the bleeding is to be checked, pressure must be made between the cut and the heart. The veins are thinner walled and are usually more or less collapsed. If a cut be made into a vein the blood may issue in considerable amount in a constant stream but without much force. If the flow is to be checked pressure should be applied between the cut and the capillaries. If the flow of a fluid through a tube be examined it will be found that the longer the tube the greater is the surface for friction and that the resistance is increasingly greater as the Fig. 27. Fig. 27 Scheme of the circulatory system. diameter of the tube diminishes, so that in the actual circulation the resistance in the very small arteries and capillaries is very great and this peripheral resistance in connection with the action of the heart must be considered a very important factor in regulat- ing the flow of blood through the total circulation. If a fluid be passed through a tube with rigid walls and uniform diameter, it will be found that exactly as much fluid passes out as comes in 112 and if the fluid be added intermittently the outflow will also be intermittent. This will also be true of a rubber tube if the diameter is uniform and its length not too great. If, however, the end of the rubber tube is constricted or resistance be introduced, then the flow will lose its intermittent character and become more or less continuous. This is because a smaller volume can now escape in a given time and because the elastic nature of the walls permit them to expand to accommodate the extra fluid and as the recoil of the walls upon the fluid occurs immediately after the expansion the fluid is therefore forced steadily but continuously through the constricted orifice until the walls have returned to their original size. Many of the important phenomena of the circulation may for all practical purposes be worked out nearly as well upon an artificial scheme consisting of a bulb syringe and rubber tubing, as upon the circulation itself. In the accompanying diagram, H represents the left ventricle of the heart with the mitral and semi lunar valves. A, the arterial system with A M, a mercury manometer to show arterial pressure. C, the capillary system represented by a larger glass tube filled with small pieces of sponge or glass wool to imitate peripheral resistance by retarding the passage of the fluid. C T is a connecting tube between the arterial and venous systems to simulate the arteries and capillaries in a dilated condition. CI, clamp. When this is closed the fluid must pass through the peripheral resistance in the capillaries C. V, venous system with V M its mercurial manometer for recording venous pressure, r is a connection which joins the venous system with that portion (auricle) which conveys the fluid to the ventricle. 342. Disconnect the apparatus at r and fill the system with fluid by allowing the disconnected portions to drop in a basin of water and pumping the syringe. Continue until all of the air is displaced. To insure this it may be necessary to raise the end of the venous tubing so that the air bubbles may escape. Support the end of the venous tube upon the edge of the basin so that the character of the outflow may be observed. See that the clamp on C T is open. Compress the bulb at slow intervals and note that the outflow is intermittent and occurs with each contraction of the bulb also that the mercury in the venous manometer oscillates with each contraction quite as freely as that in the 113 arterial except for the slight friction and expansion of the walls, the conditions are very similar to those of rigid tubes. 343 . Screw up the clamp on C T thus forcing the fluid through the peripheral resistance of the capillaries. When the bulb contracts note that there is a sudden and marked rise of the mercury in the arterial manometer while that of the venous manometer does not rise but may fall a little, indicating a high arterial but low venous pressure. Note the pulsation in the arterial manometer and tubing and the complete or almost com- plete absence of it on the venous side. Note also that the outflow has lost its intermittent character and streams continuously between the heart beats. If no more contractions of the bulb occur the mercury of the arterial manometer sinks gradually to its former level and the pressure becomes equalized. 344. In the body the circulation is a closed system of tubes. Connect the apparatus at r, seeing that the system is full of fluid and carefully exclude air bubbles. There should be a sUght positive pressure and the mercury of the two manometeSfe should be at about the same height. Compress the syringe as before with the clamp closed so that the fltiid must pass through the capillary system. There will be a rise in the arterial and a fall in the venous manometer. If there is but one contraction of the syringe, the pressure will gradually become uniformly distributed and the mercury of the two manometers arrive at the same level. 345. If the contraction of the bulb be carried on at a definite rate the mercury will continue to rise in the arterial manometer until a point is reached when it will rise no higher but merely oscillates with each contraction and expansion of the ventricle. This is known as the mean pressure. The maintenance of pressure at a mean height means that this pressure including the action of the heart, is just sufficient to force out through the peripheral resistance during the time of one complete cycle, exactly the same amount of fluid as comes into the arteries with each contraction of the bulb. If the venous manometer be observed it will be found that the mercury in the free arm falls with the expansion of the bulb as it returns to its original form. Note that the height of the arterial pressure can be changed: (i) by changing the rate or strength of the heart beat, (2) by changing the peripheral resistance, i. e., by opening or closing the clamp cl. 114 346. Effect of gravity. Let the heart H with its tubes hang at a lower level than the rest of the apparatus. There will be a fall in arterial and venous pressures, the vein v becoming more distended under the influence of gravity, by the pressure exerted by the column of fluid between it and the highest part of the appara- tus. If the heart is not working the pressures in the two mano- meters may become negative. Thus, if the heart ceases to beat with an animal in the upright position, the blood of the body will tend to drain into the dependent parts of the venous system. 347. Pulse. With the heart beating rhythmically, so as to main- tain an average pressure on the arterial side, press the finger upon the rubber tubing representing the arteries and note the pulse; tracings may be taken by placing a lever upon the tubing and recording the effect upon a kymograph or by the use of a sphygmograph. The beat of the heart should be maintained as regularly as possible. Tracings may be taken with the clamp cl, closed ; with the clamp slightly opened and, with the clamp ftdly opened. Tracings may thus be obtained corresponding to the high medium and low tension piilse. Fig. 28 Fig. 28. Scheme of the lymph and circulatoiy systems. 348. Scheme of the combined blood and l5niaph systems: As arranged the circulatory portion is essentially the same as in 115 the preceding scheme. The capillaries are represented as dilated. A clamp on V may be adjusted to regulate the pressure in the capillaries. The scheme endeavors to represent the tissue or l3rmph spaces of the body and their relation to the capillary and Ijmiph vessels ; how the lymph may be produced and how the lymph again reaches the blood. The small jar represented by Is is filled moderately full of small pieces of sponge to represent the tissues, while the spaces between represent the tissue or lymph spaces Is. The vertical tubing 1 emerging from the middle of the jar is a lymph, vessel which has its origin in the tissue spaces of the body. Vessels convey the lymph from the tissues to the thoracic duct through which it passes to enter the venous system. 349. To work the apparatus, disconnect the venous system at r and let that portion attached to the heart be submerged in a beaker of water. Compress the b\ilb until the circulatory appara- tus is filled and the air displaced. The small pieces of sponge in the jar should have been previously dampened. Depending upon the force and frequency of the heart beat and the amount of peripheral resistance at cl the amoimt of pressure in Is will vary. If the jars Is and re are thoroughly tight, then when increased pressure occurs in the capillaries some of the fluid will pass out through the capillary walls to the tissue spaces; the pressure in the latter will increase with that of the former. So that if the jar is perfectly tight and the pressure sufficient some of the fluid in Is will be forced through the openings of the beginning lymph vessel 1 and finally reach re the receptaculum chyli of the thoracic duct. If perfectly tight the pressure in re also rises and the fluid is passed on through td, the thoracic duct to enter the venous system. Sometimes if the air has not all passed out or the pres- sure not quite strong enough the fluid hesitates about passing through bl. In this case if bl be disconnected near re and a little suction applied the fluid will usually pass over quite readily. Reconnect and if a similar trouble occur between re and the venous systems try the same plan. Increasing or changing the pressure by regulating the clamp cl will oftentimes be sufficient. 350. After the apparatus has been practised upon, the venous system may be connected at r, being careful to exclude all air. 116 This would simtdate very closely the actual conditions as they exist in the animal body. By the use of manometers as in the previous experiments the differences in pressure may be observed in the arterial, venous and lymph systems. The pulse may be shown to be absent from the lymph as well as the venous system by the use of sphygmographs or writing levers. XXX 351. Blood Pressure in the Frog. Curarize a frog lightly, and expose the heart with the aortae leading off from it. Get ready a fine cannula with a short piece, of rubber tubing attached. Fill the tubing and cannula with a i per cent solution of sodium carbonate and close the end of the tube with a clamp. Dissect out one of the aortae and tie a ligattire around it as far as possible from the heart. Pass a second ligature around the same aorta, without tying, nearer to the heart. Lift the aorta with the second ligature and with a pair of sharp pointed scissors make a slight incision in the vessel and introduce the cannual into this incision and tie it with the second ligature. Fill the proximal end of the manometer with a i per cent solution of sodium carbonate seeing that all air is excluded, so that when the tubing is attached to the manometer, there will be a continuous volume of the soditmi carbonate solution from the cannula to the mercury of the mano- meter. Before attaching the tubing to the manometer, clamp the aorta or have your co-worker compress it carefully with a pair of forceps. Place the frog-board on a wooden stand, so as to bring the heart to a slightly higher level than the level of the mercury in the manometer. Bring the writing point of the lever of the manometer against a smoked drum and revolve the drum once so as to record a line of atmospheric pressure. After the cannula in the aorta, with its tube has been attached to the manometer, remove the clamp or forceps from the aorta and allow the blood from the heart to pump against the soditun carbonate and mercury in the manometer. The columns of mercury in the proximal and distal tubes will be no longer at approximately the same level. The mercury in the proximal tube will fall slightly and will rise correspondingly in the distal 117 tube. Note that with each beat of the ventricle the column rises a short distance above the mean levels and sinks again. Get a tracing of this blood pressure curve upon a very slowly revolving drum. The actual pressure, in millimeters of mercury is ob- tained by multiplying the mean height of the curve, above the atmospheric line, by two. Similar or more satisfactory results may be obtained from the carotid artery of the dog or cat, using a half saturated solution of magnesium sulphate instead of the sodium carbonate. 352. Stannius's Experiments on the Frog's Heart. Some of the early and important experiments relating to the beat of the frog's heart were performed by Stannius, and bear his name. If the sinus venosus is separated from the rest of the heart by a ligature of thread passed under the aorta and drawn tightly around the sinus at its junction with the auricle, the sinus venosus continues to pulsate, but the auricles and ventricle are quiescent. If the auricles are now separated from the ventricle by a thread ligature tied around the auriculo-ventricular groove, the auricles remain motionless, but the ventricle begins to beat, so that the sinus venosus and ventricle are pulsating, but with a different rhythm, while the auricles are at rest. The rate of the ventricular beat is usually much slower than that of the sinus. Fig. 29 Fig. 29. Aur, auricle; V, ventricle; S V, Sinus Venosus. The figure to the left shows the application of the ligature between thesinus and the auricle. In the figure to the right there is shown the second ligature between the auricle and ventricle. The quiescence of the auricles and ventricle, in the first case, has been supposed to show that the motor centers for the entire heart reside in the sinus, and that from them the motor impulses originate which keep up the rhythmical pulsations of the organ. 118 But the fact that the veratricle begins to pulsate on its own account, as in the second case, when separated by another ligature from the auricles, seems to show that it also contains motor centers. The hypothesis has been advanced that both sinus venosus and ventricle contain motor centers, while the auricles contain inhibi- tory centers. So long as the auricles are in connection,- both with the sinus venosus and the ventricle, the motor centers in the latter two parts are supposed to be sufficiently powerful to overcome the resistance offered by the inhibitory centers, and thus the cardiac rh3rthm is maintained. When the motor centers of the sinus are removed, the inhibitory centers of the auricle are supposed to be so power- ful as to keep both it and the ventricle in a state of rest. 3 S3 . Cardiac Delay or Latent Period of Cardiac Muscle. In the case of skeletal muscle, the muscle is at rest and a stimtilus excites it to contraction ; cardiac muscle has the power of contract- ing rhythmically; it will, therefore, be necessary to stop the heart- beat by the application of a "Stannius" ligature. Arrange the apparatus for single induced shocks, and include in the primary circuit a single magnet to mark the exact time the stimulus is applied. Use also a time marker recording in half seconds. After exposing the heart apply the "Stannius ligature" to stop the beat. Attach the apex of the ventricle to the heart lever. Arrange the three levers, heart, signal magnet and time marker so that their writing points will all be exactly in the same vertical line. Let the drum revolve. Stimulate the ventricle with a single induced shock. When the circuit is made or broken the lever of the signal magnet will immediately respond and shortly after the heart lever will also respond. The interval represents the "latent period" and may be about half a second, depending upon temperature and other conditions. Stimulate an auricle in the same way and note the longer "delay;" the wave of contraction travelling slowly and delaying at the groove. Compare the cardiac latent period with the latent period of skeletal muscle. 354. Maximum Contractions Only. Find the weakest stim- ulus that will cause contraction of the vftitricle. Increase the 119 strength of the stimulus but do not stimulate more than once in ten seconds, otherwise "staircase" contractions may result. The force of the ventricular contraction will remain the same in spite of the stronger stimulus. If the heart is capable of responding at all it will, in each case, give a maximum cbntraction. Stimulate either auricle in the same way and note the result. 355. Staircase Contractions of the Heart. Apply the first Stannius ligature over the sino-auricular groove. Connect the apex of the heart with the heart lever. Record on a slowly moving drum. Stimulate the quiescent heart with single induction shocks at intervals of five seconds. Notice that the second beat is higher than the first, the third than the second and so on until a maximum beat is obtained. This is the "staircase" of Bowditch. 356. Location of Motor Centers in the Frog's Heart. Dissect out the entire heart of a frog and note that it continues to beat. Cut the heart vertically into three pieces, so that the middle portion will contain the auricular Septum, in which lie the ganglionic cells. . This portion continues to beat while the right and left lateral parts do not beat spontaneously, but will respond with a single contraction if stimulated. 357. Effect of Temperature Upon the Heart Beat. Leaving the heart in its usual position insert a glass tube into the oesophagus and allow it to project through the stomach. Pass water at different temperatures through the tube and note the number of beats in each case. 358. Bernstein's Experiment. Isolated Apex. — Tie a ligature around the ventricle about half way between its apex and base, or compress it with a clamp or pair of forceps, the object being to destroy physiological continuity but preserving anatomical connection. Remove the ligature. The physiologically isolated apex does not contract. This would seem to indicate that the adult heart muscle is incapable of spontaneous rhythmical con- traction. If the bulbus arteriosus is compressed, the pressure of blood in the ventricle rises and is usually sufficient to stimulate the apex strongly enough to start it beating again. Remove the ligature and apply the pressure to the bulbus and note the effects. 359. Intracardiac Inhibitory Center in the Frog. Expose the^heart of a frog, divide the frenum and tilt the heart upward to expose the whitish V-shaped crescent between the sinus 120 venosus and right auricle. Stimulate the crescent, using fine electrodes, with an interrupted current ; if the current is sufficiently strong, the auricles and ventricle, after a brief delay, will cease to beat for a time, but they begin beating again even in spite of continued stimiilation. Stimulate the auricles; there is no inhibition. Connect the apex of the ventricle with the heart lever. Use a signal magnet marking seconds, in the primary circuit. Its lever will vibrate when the circuit is closed. Arrange so that its writing point will write immediately under and in the same vertical plane as the writing point of the heart lever. Get a tracing of the normal beat, then stimulate the crescent for one or two seconds as- before. Inhibition results. After a pause the beat begins again, the contraction passing as a wave from the sinus, through the auricles to the ventricle. Stimulate the auricles. Note any effect upon the tracing. (During inhibition the sinus beats, but the auricles and ventricle do not, because the excitability is so lowered that they do not propagate the excitatory process). Stimulate the ventricle mechanically, the heart beats in the reverse order from ventricle through auricles to sinus. Apply a few drops of atropine solution to the heart and again stimtdate the crescent. There is no inhibitory effect as the atropine paralyzes the inhibitory fibers. 360. Form and Volume of a Contracting Muscle. Dis- sect out the gastrocnemius muscle of a frog. Connect the hooked electrodes at each end of the volume tube with the muscle. The tube is to be filled with saline solution which has been boiled and allowed to cool down to the temperature of the room. Replace the stopper in the tube in such a way that all air bubbles shall be excluded. The height of the water in the capillary tube may be adjusted to the proper level by moving the glass rod in the stop- per in or out. Connect the electrodes of the volume tube with the secondary coil and, using a single induction current, send a ■maximal break shock into the muscle. Note very carefully the level of the water in the capillary tube before, during and after the contraction of the limb. Does the level of the water in the capillary tube change? SHORT TITLE CATALOGUE OF VETERINARY PUBLICATIONS Veterinary Obstetrics. Williams $8.00 Surgical and Obstetrical Operations. Williams 2.50 Veterinarian's Handbook of Materia Medica and Ther- apeutics. Udall 1.50 General Surgery. Frbhner. Translated by Udall 3.00 Veterinary Doses and Prescription Writing. Fish 1.50 Examination of the Urine of the Horse and Man. Fish. ... 1.50 Elementary Exercises in Physiology. (Laboratory Manual.) 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