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GIFT OF 
 
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A LABORATORY MANUAL 
 
 OF 
 
 Physiological Chemistry 
 
 BY 
 
 ELBERT W. ROCKWOOD, M.D., Ph.D. 
 
 PROFESSOR OF CHEMISTRY AND TOXICOLOGY AND HEAD OF THE DEPARTMENT OF CHEMISTRY 
 
 IN THE UNIVERSITY OF IOWA; AUTHOR OF AN INTRODUCTION TO CHEMICAL 
 
 ANALYSIS FOR STUDENTS OF MEDICINE, PHARMACY 
 
 AND DENTISTRY 
 
 SECOND EDITION, REVISED AND ENLARGED 
 
 TKHttb ne Colored plate anfc Ubree plates of 
 Microscopic preparations 
 
 PHILADELPHIA 
 F. A. DAVIS COMPANY, PUBLISHERS 
 
 1907 
 
COPYRIGHT, 1899 
 
 BY 
 
 THE F. A. DAVIS COMPANY 
 
 COPYRIGHT, 1906 
 BY 
 
 F. A. DAVIS COMPANY 
 
 [Registered at Stationers' Hall, London, Eng. 
 
 
 -..... 
 
 " %: 
 
 
 Philadelphia, Pa., U. S. A. 
 
 The Medical Bulletin Printing House 
 
 1914-16 Cherry Street 
 
PEEFACE TO THE SECOND EDITION. 
 
 IN no department of biology have the advances of 
 recent years been more marked than in that which is 
 studied under the name of physiological or biological chem- 
 istry. While the subject has always been regarded as im- 
 portant in the study of the vital processes, its value is 
 being more than ever emphasized as fundamental in a 
 biological or medical education. A revision of this man- 
 ual, with the addition of new material, has therefore seemed 
 advisable. No change has been made in the original plan, 
 but suggestions arising from its use in this university or 
 from the experience of the author's colleagues have been 
 adopted wherever they promise to add to the success of 
 the work. For such suggestions particular thanks are due, 
 among others, to Dr. D. W. Fetterolf, of the University of 
 Pennsylvania, and Dr. Paul Bartsch, of Howard Univer- 
 sity. 
 
 ELBERT W. KOCKWOOD. 
 
 THE UNIVERSITY OF IOWA, 
 1906. 
 
 (iii) 
 
PEBFACE. 
 
 IN view of the results attained from the course given 
 in physiological chemistry in this University, as well as 
 the experience of others, the author is firmly convinced of 
 the superiority of the laboratory method of instruction 
 over the didactic, believing that it is only by practical work 
 that the student can become familiar with the physio- 
 logical changes in progress in the animal body and their 
 products. This book has been prepared with the aim of 
 imparting accurate knowledge through the student's own 
 observation. It has seemed advisable to include with the 
 directions for experimental work a brief explanation of 
 the facts observed, so as to call attention to their meaning; 
 or, at times, to state others which are important, but which 
 could not well be demonstrated in such a course as this. 
 Some acquaintance with general chemistry and with 
 chemical manipulation is presupposed." 
 
 For the purpose of making the course flexible, the 
 less important experiments, or those which are not of 
 general interest, have been printed in smaller type. A 
 few blank pages have been inserted for additional notes 
 by the student. It has been found that the time usually 
 assigned to chemistry in one year of a medical course is 
 sufficient for the performance of most of the experi- 
 mental work. 
 
 As far as possible, the work has been so arranged as 
 to require but a small stock of apparatus and reagents 
 
 (v) 
 
VI PREFACE. 
 
 and such as are readily obtainable. By this means a large 
 class can carry on the work together. Complicated ex- 
 periments have been omitted or put in small type for 
 the use of advanced students or those who choose to 
 spend more time upon the subject. 
 
 The animal substances which are required albumin, 
 blood, bile, and others can be found in the market or 
 obtained from the slaughter-house. If no hospital is 
 near, gastric juice, urine, etc., corresponding to patho- 
 logical specimens can be prepared artificially for testing 
 by the student. The expense of the course is very small. 
 
 ELBERT W. KOCKWOOD. 
 
 UNIVERSITY OF IOWA, 
 JULY 31, 1899. 
 
TABLE OF CONTENTS. 
 
 PAGE 
 
 INTRODUCTION 1 
 
 THE CARBOHYDRATES 1 
 
 Starch 4 
 
 Dextrin 8 
 
 Glycogen 8 
 
 Cellulose 11 
 
 Glucose 12 
 
 Lactose 10 
 
 Sucrose 20 
 
 Maltose 21 
 
 Pentoses 21 
 
 THE FATS 23 
 
 The Lecithins 30 
 
 THE PROTEINS 32 
 
 Albuminous Substances 34 
 
 Albumins 39 
 
 Globulins 44 
 
 The Albuminates 46 
 
 Proteoses and Peptones 48 
 
 Fibrin 51 
 
 Coagulated Albumin 51 
 
 Compound Proteins 51 
 
 THE MUCINS 52 
 
 The Nucleoalbumins 53 
 
 The Nucleins 56 
 
 Haematogen 58 
 
 The Albuminoids 58 
 
 Collagen 58 
 
 Gelatin 59 
 
 Elastin 61 
 
 Keratin 61 
 
 FERMENTATION 62 
 
 THE SALIVA 66 
 
 THE GASTRIC JUICE 71 
 
 Gastric Digestion 75 
 
 Methods of Testing Gastric Juice 81 
 
 (vii) 
 
Vlll CONTENTS. 
 
 PAGE 
 
 THE PANCREATIC JUICE 93 
 
 The Pancreatic Digestion 96 
 
 Leucin and Tyrosin 99 
 
 THE BLOOD 101 
 
 Composition 101 
 
 The Corpuscles 101 
 
 The Serum 106 
 
 The Coloring Matters '. 107 
 
 Haemoglobin 107 
 
 Oxyhsemoglobin Ill 
 
 Methaemoglobin 113 
 
 Hsematin and Hsemin 114 
 
 Carbonic Oxid Haemoglobin 114 
 
 Hsemochromogen 115 
 
 Hsematoporphyrin 115 
 
 Methods of Testing Stains 121 
 
 THE BILE 122 
 
 Biliary Acids 125 
 
 Cholesterin 128 
 
 Biliary Pigments 129 
 
 CONNECTIVE TISSUE 131 
 
 White Fibrous Tissue 131 
 
 Collagen 131 
 
 Cartilage 132 
 
 BONE 132 
 
 MUSCULAR TISSUE 134 
 
 The Plasma 138 
 
 The Extractives 139 
 
 THE BRAIN 141 
 
 MILK 142 
 
 THE URINE 146 
 
 Secretion and Physical Properties 140 
 
 Reaction and Fermentation 150 
 
 Urea 152 
 
 Uric Acids and Urates 161 
 
 Hippuric Acid 165 
 
 Creatinin 167 
 
 Chlorids 167 
 
 Phosphates 169 
 
CONTENTS. IX 
 
 PAGE 
 
 Sulphates, Inorganic and Organic 172 
 
 Albuminuria 178 
 
 Globulinuria 182 
 
 Albumosuria 182 
 
 Peptonuria 182 
 
 Fibrinuria 184 
 
 Glycosuria 184 
 
 Acetonuria 186 
 
 Diaceturia 187 
 
 Laetosuria 188 
 
 Clioluria - 189 
 
 Haemoglobinuria and Hsematuria 193 
 
 Mucinuria 195 
 
 Lipuria, or Chyluria 196 
 
 URINABY SEDIMENTS 197 
 
 Classification . . ' 199 
 
 Pus 200 
 
 Mucus 200 
 
 Epithelium 201 
 
 Blood 202 
 
 Casts 202 
 
 Bacteria 205 
 
 Spermatozoa 206 
 
 Uric Acid and Urates 207 
 
 Calcium Oxalate 208 
 
 Phosphates 209 
 
 Calcium Sulphate 211 
 
 Calcium Carbonate 211 
 
 Tyrosin 211 
 
 Fat 212 
 
 SYSTEMATIC TESTING OF UKINE 212 
 
 UBINABY CALCULI , . 215 
 
 THE METKIC SYSTEM 218 
 
 COMPOSITION OF REAGENTS 220 
 
 INDEX . . 223 
 
PLATE I. 
 
 1. tf, Wheat-starch granules. 
 1), Potato-starch granules. 
 
 2. a, Corn-starch granules. 
 
 b, Buckwheat-starch granules. 
 
 3. Hsemin crystals, color brown. 
 
 4. Cholesterin, colorless, transparent. 
 
 5. Phenyl-glucosazone, yellow. 
 
 6. a, Urea, colorless. 
 
 6, Urea nitrate, colorless. 
 
PLATE I. 
 
PLATE II. 
 
 7. Calcium phosphate, crystallized and amorphous forms, 
 
 both colorless. 
 
 8. Triple phosphate, "coffin-lid" crystals, colorless. 
 Sodium urate, brown spherical masses with spicules. 
 Bacteria. 
 
 9. Ammonium urate, "thorn-apple" forms, color brown. 
 Calcium carbonate, spherules and dumb-bell forms, 
 
 colorless. 
 
 10. Calcium oxalate, "dumb-bell" and "envelope- shape" 
 
 crystals, colorless. 
 
 11. Uric acid crystals, yellow to dark brown. 
 Amorphous urates, brownish. 
 
 12. o, Calcium sulphate crystals, colorless. 
 6, Impure leucin, nearly colorless. 
 
 c, Tyrosin, colorless when pure. 
 
PLATE H. 
 
PLATE III. 
 
 13. o, Normal pus-corpuscles or mucus-corpuscles, gran- 
 
 ular. 
 
 b, Pus-corpuscles swollen with acetic acid, showing 
 
 nuclei. 
 
 c, Pus-corpuscles showing amoeboid movement. . All 
 
 colorless. 
 
 d, Blood-corpuscles, nearly colorless. 
 
 14. Different forms of epithelial cells, colorless. 
 
 15. Granular casts, colorless. 
 
 16. Epithelial casts, colorless. 
 
 17. Hyaline casts. 
 
 a, Broad or waxy, colorless. 
 
 6, Narrow, colorless, and extremely transparent. 
 
 18. a, Fat-casts. 
 
 6, Yeast-fungi in urine. 
 c, Spermatozoa. 
 
PLATE TIT. 
 
PLATE IV. 
 
 ABSORPTION-SPECTRA . 
 
 1. Oxyhsemoglobin. 
 
 2. Haemoglobin. 
 
 3. CO-hsBmoglobin and CO-hsemochromogen 
 
 4. Methsemoglobin, alkaline. 
 
 5. Hoematoporphyrin, acid. 
 
 6. Hsematoporphyrin, alkaline. 
 
 7. Hsemochromogen, alkaline. 
 
 8. Hsematin, acid. 
 
 9. Hsematin, alkaline. 
 
 10. Sulphur metheemoglobin. 
 
 11. Methsemoglobin, neutral or faintly acid. 
 
 12. Pettenkofer's test for biliary acids. 
 
INTRODUCTION. 
 
 THE principal materials which enter into the com- 
 position of the animal body, as well as of the food neces- 
 sary for its support, may be divided into several general 
 
 classes: 
 
 fl. Water. 
 I. Inorganic n , r . , , . 
 
 j 2. Mineral substances. 
 
 ^ 1. Non-nitrogenous 1 (a) Carbohydrates. 
 II Organic J compounds, such as j (5) Fats. 
 
 " I 2. Nitrogenous compounds, such as the 
 Proteins. 
 
 There are a number of nitrogenous compounds in the 
 animal body which cannot be classed under the proteins, 
 and others which contain no nitrogen, but which do not 
 belong to the carbohydrates or fats; nevertheless, these 
 three classes include, by far, the largest part of the organic 
 constituents. 
 
 THE CARBOHYDRATES. 
 
 The carbohydrates are composed of three elements: 
 carbon, hydrogen, and oxygen. The latter two are always 
 present in proportion to form water, and in this the carbo- 
 hydrates differ from the fats, which contain less oxygen. 
 The name of the group is derived from their composition, 
 
THE CAEBOHYDKATES. 
 
 ^ although "tiiey "cannot be made directly from carbon and 
 water. Most of them contain in a molecule six atoms of 
 carbon or some multiple of six. Many organic compounds 
 of carbon, hydrogen, and oxygen, however, have the two 
 latter in the proportion to form water, but do not belong to 
 the carbohydrates. 
 
 Carbohydrates are found in both the animal and vege- 
 table kingdoms, but are most abundant in the latter. 
 
 The different members of the group differ greatly in 
 their properties, such as the power of crystallization, fer- 
 mentation, reducing effect, action on polarized light, taste, 
 etc. 
 
 They may be divided according to their molecular 
 composition into three classes: 
 
 I. Glucoses, or monosaccharids, C 6 H 12 6 , including: 
 
 1. Glucose, or grape-sugar; also called dextrose. 
 
 2. Fructose, or fruit-sugar; also called laevulose. 
 
 3. Less important are galactose, mannose, and sev- 
 
 eral others. 
 
 II. Saccharoses, or disaccharids, C 12 H 22 11 , includ- 
 
 ing: 
 
 1. Sucrose, or cane-sugar. 
 
 2. Lactose, or milk-sugar. 
 
 3. Maltose, or malt-sugar, and some others. 
 
 III. Amyloses, or polysaccharids, (C 6 H 10 5 ) X , includ- 
 ing: 
 
 1. Starch. 
 
 2. Dextrin. 
 
 3. Glycogen. 
 
 4. Cellulose; also a number of gums and others of 
 
 less importance. 
 
THE CARBOHYDRATES. 3 
 
 In addition to the above classes there are a number of 
 compounds containing three, four, five, seven, eight, or 
 nine atoms of carbon in a molecule. Their chemical names 
 are compounded from a prefix which indicates the number 
 of carbon atoms with the suffix ose, thus, triose, tetrose, 
 pentose, etc. As yet they are not known to be of any great 
 importance in physiological chemistry. 
 
 The first class, the monosaccharids, is so named be- 
 cause they contain one of the groups of six carbon atoms. 
 They are mostly crystalline, easily soluble in water, and 
 have a sweet taste. Chemically they are aldehyde or ketone 
 compounds of the hexatomic alcohols. The former are in- 
 dicated by the prefix aldo-, the latter by keto-; thus aldo- 
 pentose, keto-hexose, etc. They have the power of reduc- 
 ing the oxygen compounds of the metals and of forming 
 compounds with phenyl-hydrazin. They will also undergo! 
 fermentation with yeast. 
 
 The disaccharids contain in a molecule twelve atoms of 
 carbon. They may be conceived of as composed of two 
 molecules of a monosaccharid minus one molecule of water: 
 
 C 6 H 12 6 + C G H 12 6 = C 12 H 22 1 + H 2 0. 
 
 By the action of acids or ferments they take up a 
 molecule of water and form two molecules of a mono- 
 saccharid. This formation of one or more simple sugars 
 from a molecule of a disaccharid is called inversion, and 
 the resulting sugar is known as invert-sugar. The di- 
 saccharids do not undergo fermentation with yeast until 
 they have been inverted. They are all soluble in water and 
 have a sweet taste. 
 
 The polysaccharids contain more than two groups of 
 six carbon atoms, though the number is, in most cases, not 
 
4 THE CARBOHYDRATES. 
 
 positively known, and therefore is represented by x. They 
 have probably a much higher molecular weight than the 
 other classes. Their constitution is not known. They are 
 mostly amorphous, insoluble in water, and, consequently, 
 tasteless. Ferments and acids convert them into the mono- 
 saccharids. They do not have reducing power. 
 
 STARCH. 
 
 Starch occurs in the cells of the plant. It is in the 
 form of grains or granules, which vary in size in different 
 plants from about 0.002 of a millimeter to ten times that. 
 The granules are composed of two parts: an inner, soluble 
 one, called granulose, and an outer one, called cellulose. 
 This latter is insoluble in water and protects the starch 
 from the action of many of the weaker ferments. When 
 boiled or acted upon by alkalies it is broken, allowing the 
 granulose to escape and forming starch-paste, or soluble 
 starch. The shape and size of the granules differ so much 
 in the different plants that the source can often be deter- 
 mined by its microscopic appearance. Those of the potato 
 have a shape somewhat similar to that of a clam-shell, those 
 of wheat are round and smaller, and those of buckwheat 
 more irregular. (Plate I, 1 and 2.) 
 
 Starch can be obtained from the parts of the plant 
 where it is stored up, like the tuber of the potato or the 
 kernel of grain, by macerating it, then washing out the 
 starch with cold water. 
 
 Starch is a colloid. A colloid is a substance which 
 when dissolved will not pass through an animal membrane 
 or parchment. They are the opposite of crystalloids, which 
 are usually crystalline and which will diffuse through such 
 membranes. This process of diffusion or separation of 
 colloid from crystalloid substances is called dialysis. As 
 
STAECH. 5 
 
 starch cannot pass through an animal membrane, it must 
 be changed to a diffusible form before it can be absorbed. 
 This is effected by ferments in the saliva and pancreatic 
 fluid. 
 
 By heating to 160 to 200 starch is converted into 
 dextrin. 1 By boiling a solution with a dilute acid it is 
 changed first into dextrin, then into glucose. Ptyalin 
 changes it first into dextrin, finally to maltose. The dias- 
 tase of malt gives the same products. 
 
 Starch gives an intense-blue color with a solution of 
 iodin. This color disappears on heating the liquid; but 
 if it is not heated too long it becomes blue or purple again 
 when it cools. The color will also be destroyed by the ad- 
 dition of anything which will form a compound with the 
 iodin, such as sodium thiosulphate, silver salts, or the alka- 
 line hydrates. 
 
 1. Starch may be prepared from a potato by grating it upon 
 a tin grater, stirring it up with a little water, and squeezing the 
 water, which contains a large part of the starch, through a piece 
 of unbleached muslin. After repeating this with several portions 
 collect the water in one vessel and allow the starch to settle to the 
 bottom. Pour off the water, add more, and allow to settle again, 
 repeating till the starch appears clean and white. Take what is 
 needed for the experiments and" let the rest dry. Enough starch 
 for the microscope examination can be obtained from the scrap- 
 ings from a potato without washing. The cellulose- fibers will then 
 be seen also. 
 
 2. Examine the starch under the microscope. No- -Y 
 tice the shape of the granules. 
 
 3. Place a drop of very dilute iodin solution upon 
 the slide so that it runs under the cover-glass and notice 
 
 1 Unless otherwise stated, all degrees of temperature will be 
 understood as referring to the centigrade scale. 
 
6 THE CARBOHYDRATES. 
 
 the markings which are thus brought out upon the gran- 
 ules which are least colored. 
 
 4. Examine in the same way starch from other 
 sources: corn, wheat, buckwheat, etc. Observe the differ- 
 ence in the size and shape of the granules. Sketch these 
 and hand in the results. 
 
 5. Prove that starch does not dissolve in cold water 
 by filtering after shaking powdered starch in a test-tube of 
 water. lodin gives no color to the filtrate. 
 
 6. Add about a gramme of starch to 100 cubic centi- 
 meters of cold water, mix it thoroughly, and boil. The 
 starch has dissolved, as is shown by filtering and, after 
 cooling, testing a portion of the filtrate with iodin solution. 
 A deep-blue color is produced. It is destroyed by heating, 
 but reappears as purple or blue again upon cooling. 
 
 V 7. Use a piece of parchment dialyzing tube to test 
 diffusibility. First see that this does not leak. It should 
 hold water when suspended by the two ends. Place inside 
 some of the starch solution made in the preceding experi- 
 ment and hang the whole in a small beaker of water, so 
 that the liquids inside and outside are at the same level. 
 ir Instead of the tube a piece of parchment can be placed in 
 a funnel from which the stem has been broken, as if the 
 liquid were to be filtered. Pour the starch solution into 
 this and suspend the whole in a beaker of water. Allow 
 it to stand several hours, then test the water outside with 
 iodin for starch. It does not pass through because it is a 
 colloid. Then put a little glucose in the dialyzer. It dif- 
 fuses out and can be found by Trommer's reaction. 
 
 8. Examine under the microscope the starch-paste 
 which has been made by heating starch in water. The 
 granules have been burst open and destroyed. 
 
STAECH. 7 
 
 9. Prove that starch can be decomposed by acids or 
 ferments by means of the following: 
 
 In about 100 cubic centimeters of water in a porce- 
 lain dish boil enough starch, previously moistened with 
 cold water, to make a thin paste. Add about 10 cubic cen- 
 timeters of dilute sulphuric acid and boil, stirring at first 
 until the liquid becomes thinner. Keep the solution up to 
 its original volume by the addition of water. If this is not 
 done the strong acid will turn the liquid brown or black. 
 From time to time remove a portion, cool, and test with 
 iodin. When the iodin gives a red color the starch has 
 been converted into dextrin. When no color appears on 
 the addition of iodin it has been changed to glucose. Test 
 a portion for glucose by adding an equal volume of sodium 
 hydrate solution, then, drop by drop, cupric sulphate solu- 
 tion till a deep-blue color is produced. Heating this will 
 give a yellow or red precipitate, showing the presence of /? 
 glucose. This is known as Trommels test for glucose. 
 
 10. To a clear solution of starch add a solution of 
 tannic acid. A yellowish precipitate forms which dissolves 
 on heating. 
 
 11. Try Trommer's test with the starch solution. It 
 does not respond. 
 
 * 12. Add gradually to the remainder of the solution 
 which has been boiled with the acid, while it is still hot, 
 powdered calcium or barium carbonate until it is neutral. 
 Filter and evaporate the nitrate to dryness on the steam- 
 bath. 1 Glucose remains: examine its properties and pre- 
 serve it for subsequent tests. 
 
 1 To evaporate a liquid on a steam- or water-bath the 
 evaporating dish in which it is contained can be heated by stand- 
 ing it on a beaker of boiling water. This removes all danger of 
 burning the residue. 
 
THE CARBOHYDRATES. 
 
 DEXTRIN. 
 
 Dextrin is the intermediate product in the change from 
 starch to glucose or maltose. There have been several 
 varieties described: erythrodextrin, which is colored red 
 by iodin; achroodextrin, which is not so colored, etc. 
 
 It is formed from starch by the action of heat, acids, 
 or ferments. It is soluble in water, making a sticky liquid, 
 often used for a mucilage. It is produced when bread is 
 toasted, and is also found in the crust. Toast or bread- 
 crust, then, has its starch partially changed into a more 
 diffusible substance. 
 
 13. Prepare dextrin from starch by heating in a porcelain 
 dish on a sand-bath half a spoonful of powdered starch previously 
 dampened with a few drops of dilute nitric acid (made by adding 
 a few drops of nitric acid to a test-tubef ul of water) . The starch 
 must be stirred with a glass rod until it has turned yellowish or 
 brown, when it has been changed to dextrin. 
 
 14. Dissolve some dextrin in water and test with a 
 drop of iodin solution. A red or brown color is produced, 
 not a blue, if the change has been complete. If commer- 
 cial dextrin is tested it will probably be found to contain 
 undecomposed starch. 
 
 GrLYCOGEN. yit 
 
 Glycogen is found in a few of the lower plants, in some 
 shell-fish, and in many fluids and tissues of the bodies of 
 mammals. It is most abundant in the liver, and next in 
 the muscles. It is also called liver-sugar or liver-starch. 
 In the animal body it is most plentiful when the animal is 
 well nourished, especially after a full meal. At such times 
 it may be in as large an amount as 10 or 12 per cent, of 
 
GLYCOGEN. 9 
 
 the liver, but it is usually not more than 3 or 4 per cent. 
 It disappears completely from the liver after long starva- 
 tion, or more quickly through severe work or great fright. 
 
 It is best obtained from the liver. After boiling to kill 
 the ferments which are always present, dissolving in water, 
 and removing the nitrogenous substances, it can be pre- 
 cipitated by alcohol. 
 
 Glycogen is an amorphous, white, tasteless powder. 
 In water it dissolves to an opalescent solution. With iodin 
 it gives a red color, which disappears on heating. It does 
 not have a reducing action upon cupric hydrate. Boiling 
 with acids converts it into dextrin, then maltose, then glu- 
 cose. The salivary and pancreatic ferments produce the 
 same change. 
 
 The glycogen of the liver seems to be formed mostly 
 from the carbohydrates of the food, but partly, at least, 
 from the nitrogenous compounds. 
 
 It is deposited in the liver as a reserve material, just 
 as the starch is stored for a reserve material in the plants. 
 When it is needed by the body it is converted by a ferment 
 into grape-sugar, and this passes into the circulation. It 
 is probable that it is used to furnish energy for the body. 
 After death the glycogen quickly disappears from the tis- 
 sues of the body, being decomposed by the ferments which 
 are present. If these are destroyed by boiling the tissue 
 for a short time the glycogen is not destroyed, but can be 
 extracted. 
 
 15. PREPARATION OF GLYCOGEN. In a mortar grind with 
 sand or glass about 25 grammes of the adductor muscle of the 
 scallop (pecten irradiens), extract several times with 50 cubic centi- 
 meters of cold water, repeating the operation with hot water. 
 Boil the liquid to coagulate the proteins, filter and concentrate the 
 nitrate to about 50 cubic centimeters, then add alcohol to 70 per 
 cent, in strength. This precipitates the glycogen. Filter this off. 
 
10 THE CARBOHYDRATES. 
 
 If the dry powder is desired, wash with alcohol, then with ether, 
 and dry in a desiccator. 
 
 Prepare glycogen from the liver of a freshly-killed, well-nour- 
 ished animal. The animal is best killed while digestion is in 
 progress. If a rabbit, this may be an hour after introducing 10 to 
 15 grammes of sugar into the stomach through a tube. Remove 
 the liver as soon as posssible, cut it into lumps, and immediately 
 put it into about four times its weight of boiling water. Let it 
 boil half an hour, then rub up the lumps as much as possible in a 
 large mortar, add water, and boil again. Filter through muslin, 
 concentrate upon the water-bath to about one-fourth its volume, 
 and allow the solution to cool. Then precipitate the gelatin and 
 other protein compounds by adding alternately small quantities of 
 hydrochloric acid and potassio-mercuric iodid 1 as long as anything 
 is thrown down. Filter and add to the filtrate twice its volume of 
 alcohol to precipitate the glycogen. Wash with alcohol. To purify 
 the substance it should be dissolved in a little water and precipi- 
 tated again with alcohol. If the anhydrous powder is desired, the 
 water must be removed as far as possible before drying. To accom- 
 plish this wash the precipitate next with absolute alcohol, then 
 with ether to remove the alcohol. Dry in a vacuum-desiccator over 
 sulphuric acid. If the pure substance is not desired, the tests may 
 be made on the solution after the removal of the protein com- 
 pounds. 
 
 1.6. If the dry substance has been obtained, try its 
 taste and its solubility in water. Test the solution with 
 iodin. It gives a red color. 
 
 17. Try Trommer's test (Experiment 9). There is 
 no red color if the glycogen has been purified. If it has 
 not been it contains glucose, which responds to the test. 
 
 18. Convert one portion of the solution into glucose 
 by heating with hydrochloic acid and another by the action 
 of saliva. Test each for the glucose by Trommer's test. 
 
 19. Prove that the glycogen is destroyed (changed 
 
 Prepare by precipitating mercuric chlorid with potassium 
 iodid, washing the precipitate and then adding it to a hot solu- 
 tion of potassium iodid as long as it dissolves. 
 
CELLULOSE. 11 
 
 to a reducing sugar) in the liver after death by the action 
 of a ferment, making the test upon some liver from the 
 market. (Instead of this a part of the liver from Experi- 
 ment 14 can be used. This should be after it has stood 
 several hours in a warm place.) Chop it finely and extract 
 with boiling water. Acidify the solution slightly with 
 acetic acid, add a little sodium chlorid, and boil to pre- 
 cipitate the protein compounds. After filtering, test the 
 filtrate for glycogen by means of iodin and also for sugar 
 by Trommer's test. 
 
 20. Add a little blood to a test-tube of the glycogen 
 solution and after it has stood ten minutes in a beaker of 
 water at body-temperature slightly acidify with acetic acid, 
 boil, and filter to remove the albumin, and test the filtrate 
 for glucose and glycogen. The latter has been converted 
 into glucose by a ferment which is found in the blood. 
 
 CELLULOSE. 
 
 Cellulose forms the membrane of the plant-cells, and 
 is not found as a constituent of the animal body, except in 
 a few of the lower forms. Cotton and filter-paper are two 
 of the most common examples. It is distinguished from 
 the other polysaccharids by its insolubility. It is insoluble 
 in the ordinary solvents, but can be dissolved in the strong 
 mineral acids, being converted into dextrin. It also dis- 
 solves in a solution of cupric hydrate in ammonia. 
 (Schweitzer's reagent), and in a solution of zinc chlorid 
 (Schultze's reagent). Sulphuric acid changes paper into a 
 parchment-like substance by covering the surface with a 
 coating of its decomposition-products and so sticking the 
 fibers together. Iodin does not stain the unaltered cellu- 
 lose, but does so after it has been acted upon by the acid. 
 
12 THE CARBOHYDRATES. 
 
 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 con- 
 verted into glucose. 
 
 21. Show that cellulose is not stained by iodin. Use 
 absorbent cotton or starch-free filter paper. 
 
 _22.-=Try the solubility of cotton or filter-paper in solution of 
 zinc chlorid (Schultze's reagent) and also in a solution of cupric 
 hydrate in ammonium hydrate (Schweitzer^s reagent). It can be 
 precipitated from these solutions by dilution with water. 
 
 23. To one volume of water in a beaker add slowly 
 two volumes of concentrated sulphuric acid, stirring mean- 
 while. Cool the mixture; then immerse in it for a few 
 seconds a piece of heavy filter-paper, plunging it into a 
 large beaker of cold water as soon as it is removed. If the 
 time of immersion has been correct it will be semi-trans- 
 parent after washing, and as tough as an animal mem- 
 brane. It is called vegetable parchment. It can be stained 
 blue by iodin. 
 
 24. Let another piece of paper remain in a small 
 amount of the warm acid until it has entirely disappeared. 
 Then dilute a little of the acid with water and test it for 
 glucose by Trommer's test (Experiment 9), being sure that 
 enough alkali has been added to give it an alkaline reac- 
 tion. 
 
 GLUCOSE (C 6 H 12 6 ). 
 
 Glucose is also called dextrose and grape-sugar. It is 
 found in the vegetable kingdom as well as in the animal. 
 It is normally present in the blood and lymph and in other 
 fluids of the body. Pathologically it is found in consider- 
 able quantities in the urine, sometimes in as large amounts 
 as 10 per cent, or more. The urine may also temporarily 
 
GLUCOSE. 13 
 
 contain grape-sugar after a diet rich in carbohydrates. 
 Whether it may normally occur in very small amounts in 
 the urine is a question which is often discussed, but upon 
 which there is no general agreement. 
 
 Glucose is made commercially by boiling starch with 
 a dilute acid. It can be produced from any of the poly- 
 saccharids or disaccharids in the same manner. They unite 
 with one or more molecules of water, forming glucose: 
 
 (C 6 H 10 5 ) x +x H 2 = x C 6 H 12 6 . 
 C 12 H 2 Ai + H 2 2C 6 H 12 6 . 
 
 Pure glucose can be made from pure cane-sugar by 
 dissolving it in alcohol and adding hydrochloric acid. The 
 glucose crystallizes out on standing. 
 
 Glucose is a crystalline substance, but crystallizes with 
 difficulty from water. It can better be crystallized from 
 methyl alcohol or ethyl alcohol. Its taste is sweet, but less 
 so than that of cane-sugar. It is easily soluble in water or 
 hot alcohol. With yeast, glucose ferments best at about 
 25 C., forming alcohol and carbon dioxid: 
 
 C 6 H 12 6 = 2C 2 H 5 OH + 2C0 2 . 
 
 In the presence of milk or cheese it ferments to lactic 
 acid. Calcium carbonate or oxid of zinc must be added to 
 keep the solution neutral if it is desired that the action go 
 on for a long time, as the presence of the acid kills the 
 ferment: 
 
 C 6 H 12 6 = 2C 3 H 6 3 . 
 
 By the action of another ferment the lactic acid is 
 changed into butjrric acid: 
 
 2C 3 H 6 3 = C 4 H 8 2 + 2C0 2 + 4H. 
 
14 THE CARBOHYDRATES. 
 
 25. Prepare pure glucose from cane-sugar by the following 
 method: 
 
 Acidify 100 cubic centimeters of 90-per-cent. alcohol with 
 4 cubic centimeters of concentrated hydrochloric acid, warm the 
 liquid upon the water-bath to 45, and add gradually 30 grammes 
 of finely-powdered cane-sugar, stirring until it has dissolved. The 
 temperature should not rise above 50. After two hours at 50 
 the sucrose has been inverted. Then let it stand in a cool place. 
 The glucose commences to crystallize out in about a week, but 
 crystallization may be hastened by adding to the cold solution a 
 few crystals of glucose and by frequent stirring. After the glucose 
 has crystallized from the solution filter, best with the aid of a 
 filter-pump; wash free from the acid by 90-per-cent. alcohol, then 
 by absolute alcohol; finally dry the crystals. It may be purified 
 by dissolving in pure methyl alcohol by the aid of heat and 
 allowing it to again crystallize out. 
 
 26. Prove that cupric hydrate (made by the addi- 
 tion of a few drops of copper sulphate to a sodium hydrate 
 solution) is soluble in a solution of glucose, giving a deep- 
 blue liquid. Also show that this blue solution of copper 
 and sugar is decomposed by heating, and yellow or red 
 precipitate of cuprous oxid is produced. This is Trom- 
 mels test for grape-sugar. To perform the test, mix about 
 equal volumes of sodium hydrate or potassium hydrate and 
 the glucose solution, then drop in copper sulphate solution 
 until a permanent precipitate begins to form or until the 
 mixture is deep blue ; finally, heat the solution. 
 
 27. Show that cupric hydrate is also soluble in a 
 solution of Rochelle salt or glycerin in water if an alkaline 
 hydrate is present, but that these solutions are not decom- 
 posed by boiling. Add to each of them a little grape-sugar 
 and heat. Cuprous oxid is formed in both cases. The 
 former is called Fehling's test, the latter Haines's test for 
 glucose. 
 
GLUCOSE TESTS. 15 
 
 28. Show that a glucose solution will reduce copper 
 acetate acidified with acetic acid 1 when heated for some 
 time in a water-bath. This is Barfoed's test. Observe the 
 difference between glucose and lactose with this test. 
 
 29. Prove that when heated alone in water cupric 
 hydrate gives the black cupric oxid and not the red cuprous 
 oxid; also that with an excess of the copper solution the 
 black may hide the red oxid if only a small amount of 
 sugar is present. 
 
 30. Show that glucose will also reduce the sub- 
 nitrate, or basic nitrate, of bismuth if its solution is made 
 alkaline by sodium hydrate or carbonate and boiled with 
 the bismuth compound. The bismuth oxid which is formed' 
 is a black powder, but if mixed with much of the unre- 
 duced bismuth subnitrate it may appear gray. This is 
 Boettger's test. The bismuth subnitrate, like cupric hy- 
 drate, is soluble in an alkaline solution of Eochelle salt, 
 and this solution when heated with glucose gives the black 
 oxid as a precipitate (Nylander's test). 
 
 Heat a little of the bismuth subnitrate in a solution of 
 albumin which has been made strongly alkaline with sodium 
 hydrate, and notice that the sulphid of bismuth, which is formed, 
 has the same appearance as the black oxid which is produced by 
 the glucose; that is, albumin gives a similar result to that obtained 
 with grape-sugar. 
 
 31. Dissolve in a small amount of water as much 
 phenyl-hydrazin hydrochlorid as can be taken up on the 
 point of a knife-blade and twice as much sodium acetate, 
 filtering if it is not clear. Add it to half a test-tubeful of 
 the sugar solution, place the tube in a beaker of boiling 
 
 x The mixture of solution and sugar solution should contain 
 1.0 per cent, of cupric acetate and 1.0 to 1.25 per cent, of acetic acid. 
 
16 THE CABBOHYDRATES. 
 
 water, and heat it for an hour. Then cool it and examine 
 the precipitate with the microscope. It is phenyl-gluco- 
 sazon : bright-yellow, needle-shaped crystals. They may be 
 single, but are more often in clusters (Plate I, 5). They 
 can be distinguished, if necessary, from similar compounds 
 of the other sugars by their melting-point, which is 204 C. 
 If they separate in the amorphous state, they may be crys- 
 tallized, after filtering, by dissolving in a little hot alcohol, 
 then evaporating the alcohol to a small volume, and letting 
 it stand. Make sketches of these : hand them in. 
 
 32. Crush a piece of condensed yeast as large as a 
 pea in a test-tube of water, and wash it two or three times 
 by decantation to remove any fermentable substances which 
 may be present. Fill the tube completely full of a glu- 
 cose solution. Mix and place it, still full of the liquid, 
 with the mouth downward in a beaker which contains a 
 little water, or, better, some of the grape-sugar solution. 
 Let it stand for twenty-four hours in a warm place. The 
 carbon dioxid, which is formed., is found in the test-tube 
 and the alcohol in the liquid. The gas may be proved to 
 be carbon dioxid by shaking it with lime-water, which it 
 turns white. The presence of the alcohol is shown by 
 warming the liquid after the addition of sodium hydrate 
 and a little iodin. lodoform separates out in yellow scales, 
 or, if the amount of alcohol is very small, the odor alone 
 may be perceived. 
 
 A convenient piece of apparatus for carrying on this 
 fermentation is the saccharimeter. This is essentially a 
 graduated tube with bulb to hold the liquid which is forced 
 out by the gas. From its reading the amount of glucose 
 can be learned. 
 

 
 
 
 
GLUCOSE. 17 
 
 QUANTITATIVE TEST FOR GLUCOSE. 
 
 Fehling's Method. The solutions used are: (A) 34.64 
 grammes of cupric sulphate (CuS0 4 , 5H 2 0), dissolved in 
 enough water to make the volume 500 cubic centimeters. 
 The crystals used must be dark blue and not effloresced; 
 (B) 187 grammes of pure Eochelle salt and 68 grammes of 
 sodium hydrate in water enough to make the volume 500 
 cubic centimeters. These solutions must be kept separate. 
 
 33. For each determination mix 5 cubic centimeters 
 of A with 5 cubic centimeters of B, measuring carefully 
 with a pipette. Add about 40 cubic centimeters of water, 
 and heat to boiling in a beaker or porcelain dish. If the 
 solution is good there will be no red precipitate. 
 
 The best results are obtained when the solution con- 
 tains from 0.5 per cent, to 1.0 per cent, of sugar; that is, 
 when from 5 to 10 cubic centimeters are necessary to 
 destroy the blue color of the Fehling solution. If it con- 
 tains more than this, it must be diluted with water to 5 or 
 10 times its volume, measuring accurately the water added 
 and mixing thoroughly. 
 
 The Fehling solution after dilution is heated to boil- 
 ing, and the sugar solution run in from a burette until the 
 blue color has been destroyed, leaving the liquid colorless 
 above the red precipitate. If too much sugar has been 
 added it begins to turn yellowish. The amount of sugar 
 is ascertained most quickly by making two determinations: 
 first, a rough one, then one which is made more carefully. 
 Make the first by running in the sugar solution, 2 or 3 
 cubic centimeters at a time, as long as the blue color is 
 well marked, then 1 cubic centimeter at a time, heating to 
 boiling after each addition. It can be learned by this first 
 test within 1 or 2 cubic centimeters how much will be re- 
 
18 THE CARBOHYDRATES. 
 
 quired. Then rinse out the beaker, take again 10 cubic 
 centimeters of the Fehling solution, diluted as before; heat 
 to boiling and run in at once within 1 cubic centimeter of 
 the necessary amount of the sugar solution. Bring it to a 
 boil. Then add the sugar solution a few drops at a time, 
 heating after each addition, until the blue color has just 
 been decolorized. 
 
 Since 10 cubic centimeters of the Fehling solution is 
 decolorized by 0.05 gramme of glucose, the amount of the 
 sugar solution or urine which has been used from the 
 burette must have contained 0.05 gramme of glucose. Eead 
 the volume which has been poured from the burette, and 
 calculate the percentage of sugar in the original solution. 
 If this has been diluted with water the amount in the dilute 
 solution must be multiplied by the number of times it was 
 diluted. Eemember that however much of the sugar solu- 
 tion may have been used to destroy the blue color, it con- 
 tained 0.05 gramme of sugar. For example, if the amount 
 used was 10 cubic centimeters, there would be 0.005 
 gramme of glucose in 1 cubic centimeter; that is, in 1 
 gramme of solution. In 100 grammes there would be 0.5 
 gramme of glucose, or 0.5 per cent. 
 
 The floating red precipitate of cuprous oxid obscures the encl 
 reaction and makes the titration slow if time is allowed for this 
 to settle. Purdy's modification of Fehling's reagent consists in 
 the addition of a large excess of ammonium hydrate which pre- 
 vents the precipitation of the cuprous oxid. The reagent con- 
 tains: 
 
 Cupric sulphate 4.742 grm. 
 
 Glycerin, pure 38 cc. 
 
 Dissolve in about 200 cc. of water. 
 
 Potassium hydrate 23.5 grm. 
 
 Dissolve in about 200 cc. of water. 
 
LACTOSE. 19 
 
 When these solutions have cooled mix them, add 450 cubic 
 centimeters of concentrated ammonia (sp. gr. 0.9) and dilute to 
 1000 cubic centimeters. 
 
 For the titration use 35 cubic centimeters copper solution 
 diluted with about twice its volume of water. Heat this to boil- 
 ing, then drop in slowly from a burette the urine or glucose solu- 
 tion until the blue has been destroyed. Three to five seconds 
 should elapse between the drops. When this has occurred 0.02 
 gramme of glucose has been added. The solution should then be 
 colorless. If it is yellow an excess of sugar has been added. From 
 the volume of solution used calculate the per cent, of glucose by 
 weight. 
 
 LACTOSE (MILK-SUGAR: C 12 H 22 1;l + H 2 0). 
 
 Lactose is found in the milk of all mammals and occa- 
 sionally during pregnancy in the urine. It can be obtained 
 from the milk by crystallization after the removal of the 
 nitrogenous constituents. 
 
 It is a crystalline substance, soluble in water, with a 
 faint sweetish taste. With pure yeast it does not ferment. 
 By the action of certain other ferments, however, it under- 
 goes alcoholic fermentation, with the production at the 
 same time of lactic acid, forming the drinks known as 
 <r koumiss," when made from mares' milk, and "kephyr" 
 when from cows' milk. The ordinary souring of milk is 
 due to the formation of lactic acid from the lactose by 
 micro-organisms. 
 
 Milk-sugar gives with many reagents the same results 
 as glucose. It can be distinguished from glucose by its not 
 fermenting with yeast and by its having a less strong power 
 of reduction, it is unable to reduce cupric compounds to 
 cuprous in acetic acid solutions (Barfoed's test). 
 
 34. PREPARATION OF MILK-SUGAR. Dilute 200 
 cubic centimeters of milk with 800 cubic centimeters of 
 water, and add very cautiously not more than 0.1 per cent. 
 
20 THE CARBOHYDRATES. 
 
 of acetic acid to precipitate the casein (when enough has 
 been added the liquid is nearly clear). Filter. Boil the 
 nitrate and filter off the .coagulated albumin. Evaporate 
 the filtrate upon a water-bath to a syrup and allow it to 
 stand until the sugar has crystallized out. It may be puri- 
 fied by recrystallizing it. 
 
 35. Test the milk-sugar with Trommels, Fehling's, 
 and the phenyl-hydrazin tests, and notice that the results 
 are similar to those obtained with glucose. 
 
 36. Try Barfoed's and the fermentation tests as 
 made with the glucose, and observe that the results are 
 negative. 
 
 37. Boil the milk-sugar solution with a little hydro- 
 chloric acid, neutralize, and try the fermentation or Bar- 
 foed's test. Glucose has been formed and this will now 
 ferment, forming carbon dioxid and alcohol. 
 
 SUCROSE (CANE-SUGAR: C 12 H 22 11 ). 
 
 Cane-sugar is found in plants, not in the animal king- 
 dom. It has no reducing power and does not respond to 
 the tests where such a reducing action occurs,* such as 
 Trommer's, Fehling% and Boettger's. It is decomposed 
 by heating with acid into a molecule of glucose and one 
 of fructose. 
 
 38. Apply Trommer's or Fehling's test to a solution 
 of pure cane-sugar. It gives no results. 
 
 39. Boil a solution of cane-sugar with a little sul- 
 phuric or hydrochloric acid, neutralize the solution, and 
 prove that it contains glucose. 
 
 40. The "invert sugar" which results from the decomposition 
 of cane-sugar by acids can be separated into glucose and fructose 
 by adding to 10 parts 6 parts of calcium hydrate and 50 parts of 
 water. Both sugars form calcium compounds. That with glucose, 
 
 
MALTOSE. PENTOSES. 21 
 
 being liquid, can be pressed out of the fruit-sugar compound, 
 which can then be decomposed, the fruit-sugar being set free, by 
 the addition of oxalic acid as long as a precipitate is produced. 
 Filter and obtain the fructose by the evaporation of the filtrate. 
 
 MALTOSE (MALT-SUGAE: C^H^O^, H 2 0). 
 
 41. Boil a small lump of starch with 25 cubic centi- 
 meters of water, cool it to nearly body temperature, and 
 add an aqueous extract of ground malt, made at the same 
 temperature. Observe that the mixture becomes thinner 
 and that finally a sample is not turned blue by iodin. Then 
 test for the maltose with phenyl-hydrazin as in the glucose 
 reactions. Try also Trommels test or Fehling's test. It 
 responds to all. 
 
 42. PREPARATION OF PURE MALTOSE. One hundred grammes 
 of starch are to be mixed with 500 cubic centimeters of cold water 
 as thoroughly as possible, then heated on a water-bath until it 
 makes a paste. Make a watery extract of malt at 40 C. from 6 
 or 7 grammes of dry malt. When the starch-paste has cooled 
 down to 60 or 70, add the malt-extract and keep it at this tem- 
 perature for an hour. When the starch has been converted to 
 maltose the liquid becomes thin and watery. Then boil, filter, 
 and evaporate to a syrup upon the water-bath. Dissolve the 
 maltose from the residue with small portions of 90-per-cent. 
 alcohol. Distill the alcohol off from this solution, and evaporate 
 to. a syrup. Let this stand until it crystallizes. This may be 
 hastened by the addition of a little crystallized maltose, which 
 can be prepared by evaporating a few drops of the solution in a 
 thin layer on a piece of glass. It may be purified by recrystal- 
 lizing from methyl alcohol. 
 
 PENTOSES. 
 
 43. Normally but small amounts of the pentoses are found in 
 the urine and these doubtless originate mostly in certain vegetable 
 foods. In pathological conditions they may be more abundant. 
 
THE CARBOHYDRATES. 
 
 Like glucose, they are reducing agents, responding to Trommer's 
 and Fehling's tests. They also form osazones which, however, have 
 different melting points from the glucosazone. They do not fer- 
 ment with yeast, nevertheless they may be mistaken for reducing 
 sugars because of their other reactions. Arabinose, one of the 
 most common, can be obtained by boiling cherry-gum for 10 to 15 
 hours with 4 per cent, sulphuric acid. 
 
 Prepare the arabinose from cherry-gum as indicated above, 
 neutralize and use for testing. 
 
 44. Saturate 5 cubic centimeters of hydrochloric acid (30 per 
 cent.) with phloroglucin (about 25 milligrammes). Add to this 
 the urine or solution which is to be tested for pentoses and heat. 
 The presence of pentoses is indicated by a red color and this shows 
 absorption bands between D and E of the spectrum. Glucuronic 
 acid gives a similar result. 
 
 45. Repeat the test as in the last experiment substituting 
 orcin for phloroglucin. Pentoses produce a green color. Glu- 
 curonic acid does not act in this manner. 
 
 BEACTIONS OF THE CARBOHYDRATES. 
 
 
 d 
 
 o 
 
 ^"^ 
 
 l* 
 
 Phenyl- 
 hydrazin 
 
 Solubility in 
 Water 
 
 Fermentation 
 with Yeast 
 
 a 
 ll 
 
 -2 
 
 s 
 
 a 
 
 Is 
 "02 
 f, 
 
 5 
 
 r 
 
 Starch . . . 
 
 Blue 
 
 
 
 Colloid 
 on 
 
 
 
 
 
 Right 
 
 Dextrins .... 
 
 Red to 
 Yellow 
 
 4 
 
 + 
 
 Heating 
 
 - 
 
 - 
 
 Sweet- 
 ish 
 
 - 
 
 Right 
 
 Glycogen .... 
 
 Red to 
 Violet 
 
 - 
 
 - 
 
 Opal- 
 escent 
 
 - 
 
 - 
 
 - 
 
 - 
 
 Right 
 
 Cellulose. . . . 
 
 
 
 ~ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 . . . 
 
 Saccharose . . 
 
 - 
 
 - 
 
 - 
 
 + 
 
 Only after 
 Inversion 
 
 Slight 
 
 Sweet 
 
 + 
 
 ight 
 
 Maltose 
 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 Slight 
 
 Sweet 
 
 + 
 
 Right 
 
 Lactose 
 
 
 
 + 
 
 + 
 
 + 
 
 
 
 
 
 Sweet 
 
 + 
 
 Kight 
 
 Glucose .... 
 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 Slight 
 
 Sweet 
 
 + 
 
 Right 
 
 Fructose. . . . 
 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 Sweet 
 
 + 
 
 Left 
 
 Arabinose . 
 
 - 
 
 + 
 
 + 
 
 + 
 
 - 
 
 - 
 
 Sweet 
 
 + 
 
 Right 
 
THE FATS. 23 
 
 THE FATS. 
 
 The fats occur in both plants and animals. When pure 
 they are colorless, odorless, and tasteless. They are in- 
 soluble in water and have a lower specific gravity. They 
 dissolve in hot alcohol more easily than in cold, and are 
 easily soluble in ether, gasolin, or benzene. The fats mix 
 with water when the two are shaken violently together, but 
 they soon separate, the fats going to the top. If, however, 
 something like soap or a solution of albumin is added to 
 the mixture which will form a coating around the minute 
 globules of fat, they are prevented from reuniting, and 
 form an emulsion, that is, a mixture of small fat-globules 
 with the liquid, not a solution. It is destroyed by anything 
 which will remove the coating, the fat separating again 
 from the liquid. 
 
 The fats are composed of three elements: carbon, 
 hydrogen, and oxygen. They contain a much smaller per- 
 centage of oxygen than the carbohydrates, the hydrogen 
 and oxygen not being in the proportion to form water. 
 When the fats are kept at the temperature of superheated 
 steam or subjected to the action of the pancreatic ferment, 
 they take up water and are split into two compounds: 
 glycerin, on the one hand, and one or more of the fatty 
 acids, on the other. Thus, stearin gives 
 
 C 3 H 5 (C ]8 H 36 2 ) 3 + 3H 2 = C 3 H 5 (OH) 3 + 3C 17 H 35 C0 2 H. 
 
 stearin -f- water = glycerin + stearic acid 
 
 They may be considered then as made up of glycerin 
 and a fatty acid less water. 
 
 This splitting up of the fat-molecule is called saponifi- 
 cation. It occurs when fats become rancid. It can be also 
 
24 THE PATS. 
 
 effected by boiling the fat with a caustic alkali. Here, in- 
 stead 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: 
 
 C.H B (C 18 H M O a ) + 3KOH = C 3 H 6 (OH) 3 + 3C 17 H 85 C0 2 K. 
 
 stearin + alkali = glycerin + soap 
 
 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. The 
 compounds of the heavy metals with the fatty acids are in- 
 soluble, and can be formed by adding a solution of their 
 b-alts to a soap solution. The lead soap or lead plaster used 
 in medicine is made by heating lead oxid with one of the 
 fats. The soluble soaps can be thrown down from their 
 solutions by saturation with a neutral salt. If a strong 
 acid is added to a soap solution the soap is decomposed, 
 the metal uniting with the strong acid and the fatty acid 
 being set free as an insoluble substance: 
 
 C 17 H 3B C0 2 K + HC1 = KC1 + C 17 H 35 C0 2 H. 
 
 goap acid fatty acid (stearic) 
 
 These fatty acids which enter into the most of the 
 animal and vegetable fats are one of the unsaturated series: 
 oleic acid (C 17 H 33 C0 2 H); and two of the saturated series: 
 stearic acid (C 17 H 35 C0 2 H) and palmitic acid (C 15 H 31 C0 2 H). 
 Besides these acids which constitute, by far, the larger 
 part of those present in fats there are found in some cases 
 certain of the lower members of the saturated series, such 
 as butyric (C 3 H 7 C0 2 H), capronic (C^^GO^), caprylic 
 (C 7 H 15 C0 2 H), and capric (C 9 H 19 C0 2 H), which occur in 
 butter. 
 
THE PATS. 25 
 
 The acids which enter into the fats resemble the latter 
 in many of their properties. They differ from them in 
 having a slight acid reaction, the fats being neutral. They 
 can also be distinguished by their not giving the irritating 
 odor of acrolein as the fats do when they are heated. This 
 is produced by the decomposition of glycerin, either in a 
 fat or when heated alone. It is most easily obtained by 
 adding before heating some substance which will assist in 
 removing the water. The chemical change is 
 
 C 3 H 5 (OH) 3 2H 2 = C 3 H 4 0. 
 
 The free acids can be neutralized by even weak alkalies, 
 forming the soaps. 
 
 The animal and many of the vegetable oils are true fats, 
 differing only in that they are liquids at ordinary tempera- 
 tures instead of solids. We must, however, distinguish be- 
 tween these and the essential or volatile oils which are 
 found in plants, but which are not fats. The fats produce 
 spots on paper which are not volatile and do not disappear 
 on standing. The essential oils will disappear when left 
 exposed to the air. The mineral oils belong to an entirely- 
 different class of compounds, and do not contain oxygen. 
 
 The fats are named from the acid which they contain. 
 Thus the compound of stearic acid is called stearin; of 
 palmitic acid, palmitin; and of oleic acid, olein. Some- 
 times the prefix tri is used with these names, as tristearin, 
 etc. They differ principally in their melting-points, olein 
 being a liquid at ordinary temperatures; palmitin and 
 stearin, solids, the former melting more easily than the 
 latter. In the animal body these fats are usually mixed, 
 the consistence of the fat varying with the composition. 
 Thus, the fat of the ox, or tallow, is a firmer solid than 
 the fat of the hog, or lard, because it contains less of the 
 
26 THE FATS. 
 
 olein. The animal oils contain more olein than stearin and 
 palmitin, consequently they melt at temperatures below the 
 ordinary ones, and are liquids. Human fat contains 67 
 per cent, to 80 per cent, of olein. 
 
 46. 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 standing some time and has become rancid, it 
 may be slightly acid. 
 
 47. Try 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 be- 
 comes 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. 
 
 48. Examine a drop of the emulsion so formed under 
 the microscope. It will be found to consist of a great 
 number of minute globules the size being smaller the more 
 thoroughly the liquid is shaken. They are kept apart by 
 the thin film of soap which covers each one. 
 
 49. Add a small amount of hydrochloric acid and 
 shake. The soap will be decomposed and the fat will col- 
 lect at the top, as at first. 
 
 50. Try the solubility of a fat in ether, chloroform, 
 or gasolin, avoiding carefully the vicinity of a flame. It 
 is easily soluble. 
 
 51. Try the solubility of tallow or lard, first in cold, then in 
 warm, alcohol. It is readily soluble in the warm alcohol and sepa- 
 rates on cooling in the crystalline form. The crystals can be ex- 
 amined with the microscope. 
 
 52. Show that the fats are non-volatile by -placing 
 a little upon paper and warming it over a flame. It does 
 not disappear. 
 
THE PATS. 27 
 
 53. To about 10 grammes (11 cubic centimeters) of 
 olive-oil add 20 cubic centimeters of 10-per-cent. potassium 
 hydrate solution. Boil the mixture, gently stirring mean- 
 while, 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 fifteen 
 minutes to half an hour. Add water as it evaporates, to 
 keep the original volume. The product is a mixture of 
 potassium soap and glycerin. 
 
 54. Convert a portion of the soap into the sodium 
 or hard soap by adding some saturated salt solution and 
 allowing it to stand until cold. It will dissolve on warm- 
 ing. 
 
 55. To another portion add a calcium solution. 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. Try it with solutions of iron, lead, copper, etc. 
 
 56. 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 glycerin remaining in solution. Filter out the acids 
 by means of a wet filter-paper, through which they will 
 not pass. Save the filtrate for the extraction of the glyc- 
 erin. Wash out the sulphuric acid with distilled water 
 until the wash-water is no longer acid, and try the reaction 
 of the fatty acids with litmus-paper. They are acid to 
 litmus. 
 
 57. Dissolve the fatty acids in hot alcohol, let this 
 cool slowly, observe and sketch the crystals. 
 
 58. Allow the fatty acids to stand until the water has 
 drained off or dry them by the aid of filter-paper. Heat them in 
 
28 THE FATS. 
 
 a dry test-tube until they commence to decompose, and see if they 
 give the irritating odor of acrolein. If the acids were washed 
 clean and if the fat was completely saponified this should not ap- 
 pear. Try the same test on any of the fats, and the odor will be 
 given. It is best produced by adding before heating a little acid 
 potassium sulphate. 
 
 59. Neutralize the first filtrate from the acids, which 
 contains the glycerin, by adding a little powdered chalk, 
 then evaporate the mixture to complete dryness on a water- 
 bath. Extract the glycerin from the powdered mass by 
 alcohol and evaporate this on the water-bath. If the glyc- 
 erin is to be further purified this residue should be ex- 
 tracted with absolute alcohol and the alcoholic filtrate 
 evaporated. The thick liquid is the glycerin, as is shown 
 by the taste. 
 
 60. Mix 2 grammes of lead monoxid (litharge) with 11 cubic 
 centimeters of olive-oil and 75 cubic centimeters of water. Boil 
 until the oil is entirely converted into the lead soap. This is 
 "lead plaster." Filter it off; wash the insoluble soap with water 
 and evaporate the filtrate with the wash-water in an evaporating 
 dish on a waterbath, as long as its volume decreases. The glycer- 
 in remains. It contains lead; if the pure substance is desired 
 this can be removed by diluting with water and passing hydrogen 
 sulphid into< it. 
 
 Glycerin dissolves many metallic oxids, e.g., precipitated, 
 washed cupric oxid dissolves to a blue liquid. Try this. A borax 
 bead when dipped in glycerin gives a green color when heated in 
 the Bunsen flame* 
 
 61. THE PREPARATION OF PURE PALMITIC ACID. Melt in 
 hot water 10 grammes of bayberry wax which is nearly pure 
 palmitin and add 20 cubic centimeters of a 20 per cent, solution of 
 sodium hydrate. Boil it until a drop makes a soapy solution 
 when poured into a test-tube of water. Sodium palmitate has 
 been formed together with glycerin, carbon dioxid and water. 
 (Write the equation for the reaction.) Acidify slightly the soap 
 
THE FATS. 29 
 
 solution with dilute hydrochloric acid, filter out the palmitic acid, 
 wash it with cold alcohol until it is white, dissolve it in the least 
 possible quantity of hot alcohol and let it stand until it is cold. 
 Examine with a microscope the crystals which have separated and 
 make sketches of them. Test the reaction to blue litmus paper. 
 
 62. THE PREPARATION OF PURE SOAP. Melt at a low tem- 
 perature most of the palmitic acid from the last experiment and 
 slowly add sodium hydrate solution until the reaction is alkaline, 
 warming meanwhile. A hard soap is produced. (Write the equa- 
 tion.) If a little alcohol is mixed with this before it cools a 
 transparent soap results. 
 
 63. Hold a piece of small glass tubing in the Bunsen flame 
 until it is soft, turning it continually. Then draw it out into a 
 capillary tube about six inches long. Break this in the middle, 
 forming two open capillaries each with a large end. Melt a fat 
 at low temperature and draw it up into the capillary by exhaust- 
 ing the air, allowing it to cool and solidify there. Seal up the 
 large opening by means of a blast lamp or Bunsen burner. At- 
 tach the tube to the bulb of a thermometer by means of a rubber 
 band cut from a piece of tubing. Hold this in a beaker of water, 
 warming the latter slowly. Make a note of the melting point 
 the temperature at which the fat liquifies. In this manner deter- 
 mine the melting points of a number of fats, like mutton-tallow, 
 beef-tallow, lard, butter, also of palmitic acid and stearic acid, 
 beef tallow, lard, butter, also of palmitic acid and stearic acid. 
 Hand in the results. Place the thermometer with the capillary 
 of melted fat in water a little above its melting point and let it 
 cool slowly, noting the point at which solidification occurs. Notice 
 that this is below the melting temperature and that it varies with 
 the speed of cooling. 
 
 64. Try the solubility of fate, fatty acids and soaps in 
 chloroform, ether, water, hot and cold alcohol. Tabulate the re- 
 sults and determine what methods can be employed for their 
 separation. 
 
 65. Make the acrolein test upon the water-free glycerin by 
 heating in a dry tube with some crystals of potassium bisulphate. 
 The glycerin is decomposed, giving the acrolein odor, and showing 
 that the odor when obtained from fats is due to the glycerin radi- 
 cal, and not to the acid part. 
 
30 THE LECITHINS. 
 
 66. Show that olive-oil is not a simple fat, but a 
 mixture, by cooling it to the freezing-point either by nat- 
 ural cold or by a freezing mixture. It separates into two 
 parts: one crystalline, consisting mostly of palmitin, and 
 the other olein, which remains liquid. 
 
 THE LECITHINS. 
 
 The lecithins are found in nearly all animal and vege- 
 table cells. They are very abundant in the brain and 
 nerves and in the yelk of eggs. They are sometimes called 
 phosphorized fats. The formula of one of those most com- 
 mon in the animal body is.C 44 H 90 KP0 9 , and the composi- 
 tion is probably 
 
 CH0 
 
 It is therefore a fat where for one stearic acid radical 
 has been substituted the group 
 
 -P0 3 OH C 2 H 4 N(CH 3 ) 3 OH. 
 
 In others, instead of the stearic acid radical we may 
 have the oleic or palmitic radical. Like other fats, they 
 can be decomposed or saponified. They then give phos- 
 phoric acid, a fatty acid, and a base, cholin: 
 
 HO C 2 H 4 N(CH 3 ) 3 OH. 
 
 Lecithin is a soft, waxy substance, which swells in 
 water to a pasty mass. This, under the microscope, has the 
 form of oily drops or threads, the so-called "myelin" forms. 
 It resembles nuclein in the readiness with which it unites 
 
THE LECITHINS. 31 
 
 with, albuminous substances. It is found in the yelk of the 
 egg in an unstable union with vitellin. 
 
 67. PREPARATION OF LECITHIN. In the following work the 
 student should remember that ether and petroleum-ether are very 
 inflammable. 
 
 Separate the albumin of an egg as completely as possible from 
 the yelk. Place the yelk in a cylindrical, glass-stoppered bottle, 
 add two or three times its volume of ether, and shake the bottle 
 until they are well mixed. Allow it to stand until the ether above 
 becomes clear, and then decant the latter into a distilling flask. Re- 
 peat this extraction several times, when most of the coloring matter 
 should have been dissolved. Preserve the insoluble for the 
 preparation of hsematogen. Mix the portions of ether and distill 
 off the ether. The residue contains the lecithin mixed with fats, 
 cholesterin, and coloring matters. Dissolve this in petroleum- 
 ether and filter. Pour the filtrate into a separatory funnel, add 
 about one-fourth its volume of 75-per-cent. alcohol, and shake. 
 When the two liquids have separated draw off the alcohol, which 
 contains most of the lecithin. Repeat this extraction with alcohol 
 several times and unite the alcoholic solutions. Distill off the 
 remainder of the petroleum-ether from these, and let the solution 
 stand several days in a cool place. A precipitate of cholesterin 
 and other impurities, will form, from which the solution is to be 
 decanted through a filter. Boil the filtrate with a little animal 
 charcoal to decolorize it, and filter. Evaporate at a temperature 
 of 50 to 60 to a syrupy consistency. Cool this and dissolve in 
 ether. If it does not dissolve completely, filter it. Evaporate the 
 ether, when the lecithin remains nearly pure. If desired it can 
 be purified further by dissolving in as small an amount as possible 
 of warm absolute alcohol and placing this in a freezing mixture 
 of 5 to 15 9 , when the lecithin crystallizes out. It should be 
 filtered in the cold. 
 
 68. Place a little lecithin in water and examine with the mi- 
 croscope. Notice the myelin forms. 
 
 69. Warm this mixture with water and notice that after a 
 time the lecithin turns brown and the reaction becomes acid from 
 decompo sition. 
 
32 THE PBOTEINS. 
 
 70. Mix: a little lecithin with dry, powdered potassium nitrate 
 in a small porcelain crucible, and warm, at first gently, then, after 
 deflagration, until the dark color has disappeared. After cooling 
 dissolve in water and test for phosphoric acid by nitric acid and 
 ammonium molybdate. At once or after warming a yellow pre- 
 cipitate will appear. 
 
 THE PKOTEINS. 
 
 The protein compounds constitute the greater part of 
 the solid matter of the blood, muscles, nerves, and other 
 organs of the animal hody. The urine, tears, and perspira- 
 tion, in a normal condition, never contain more than a 
 trace. The proteins contain carbon, hydrogen, nitrogen, 
 oxygen, and usually sulphur. A few contain phosphorus 
 and a few others iron. When heated they are charred, 
 giving off water, inflammable gases, and ammonia, at the 
 same time emitting a strong odor similar to that of burnt 
 horn or wool. Upon further ignition they leave an ash, 
 though whether this was originally a part of the protein 
 molecule has not been decided. They are often spoken of 
 simply as the nitrogenous constituents of the body or the 
 food, although not all of the nitrogenous compounds found 
 there belong to this class. 
 
 The proteins are very complex substances with a high 
 molecular weight, and it is probably owing to this fact 
 that they are so easily decomposed, as is seen by the putre- 
 faction which sets in soon after life has ceased. To the 
 large molecule, too, is probably due the inability of most 
 of them to pass through a parchment or animal membrane. 
 
 GENERAL PROPERTIES OF THE PROTEINS. 
 
 71. Burn a small piece of dry albumin or other 
 protein compound on a piece of porcelain or platinum foil, 
 
PROPERTIES OF THE PROTEINS. 33 
 
 or on a wire. Notice that it turns black from the presence 
 of carbon. Observe the characteristic odor. On continued 
 heating it will all disappear except the mineral matters, 
 or ash. 
 
 72. Mix a few fragments of dry albumin with an 
 excess of powdered soda-lime and heat the mixture in a 
 dry test-tube. Test the vapors which escape for ammonia, 
 both by the odor and by their action, on a piece of red lit- 
 mus-paper. The ammonia proves that the albumin con- 
 tained nitrogen. 
 
 73. Make a solution of lead hydrate by adding so- 
 dium hydrate to a small amount of lead acetate solution 
 until the precipitate first formed has dissolved. Add to it 
 a protein compound, like albumin, and boil. The presence 
 of sulphur (cystein sulphur) in the protein compound is 
 shown by the dark-colored lead sulphid, which it forms by 
 uniting with the lead. 
 
 74. Very small quantities of sulphur maybe shown by chang- 
 ing it to a sulphid and testing for the latter with sodium nitro- 
 prussid. By this means it can be found in a single hair. Shut off 
 the air from a Bunsen burner and, after turning it low, cover the 
 hair with sodium carbonate and hold it on a wire in the middle of 
 the flame. Allow the substance to fuse, being careful to keep it in 
 the yellow flame to prevent oxidation. Then dissolve the mass in 
 a few drops of water in a porcelain dish. Add to the solution a 
 very small crystal of sodium nitroprussid. The presence of sul- 
 phid is shown by the production of a purple or violet color, which 
 is destroyed by an excess of the nitroprussid. 
 
 75. Test a solution of egg-albumin or any other albuminous 
 substance to see if it will pass through a dialyzer. Only the pep- 
 tones will pass through the membrane. The biuret test can be 
 used to detect them (Experiment 76). 
 
34 
 
 THE PROTEINS. 
 
 CLASSIFICATION OF THE PRINCIPAL PROTEIN COMPOUNDS. 
 
 
 Albumin. 
 
 
 
 Globulin. 
 
 
 Albuminous 
 substances 
 
 Albuminates -j 
 
 Acid albumin. 
 Alkali albumin. 
 
 or simple 
 
 Proteoses. 
 
 
 proteins 
 
 Peptones. 
 
 
 
 Fibrin. 
 
 /"> ,,1J.J !! 
 
 
 Proteins 
 
 Compound 
 proteins 
 
 I Albuminoids - 
 
 Coagulated albumin. 
 
 Mucin. 
 Hemoglobin. 
 Nucleoalbumin. 
 Nuclein. 
 
 f Keratin. 
 
 Elastin. 
 
 Collagen. 
 
 Gelatin. 
 I Etc. 
 
 ALBUMINOUS SUBSTANCES. 
 
 These are sometimes called proteins, though it is 
 more convenient to reserve this name for the whole class, 
 including also the albuminoids. They form the principal 
 part of the protoplasm which is found in animal and plant 
 cells. The constitution of the molecule and even the 
 exact formula of the different members of the group is 
 uncertain. They are known to be very complex, those 
 which have been most studied having several hundred 
 atoms' in a molecule. They differ somewhat from each 
 other in composition, but their constituents usually lie 
 within the following limits; 
 
substance. 
 
 ALBUMINOUS SUBSTANCES. 35 
 
 Average of Most Analyses. Approximation. 
 
 C 50.0 to 55.0 per cent. 52 per cent. 
 
 H 6.5 to 7.3 per cent. 7 per cent. 
 
 20.0 to 23.5 per cent. 23 per cent. 
 
 S 0.3 to 2.2 per cent. 2 per cent. 
 
 N 15.0 to 18.0 per cent. 16 per cent. 
 
 Phosphorus is sometimes found in less amounts than 
 1 per cent. 
 
 A few of the albuminous substances have been ob- 
 tained in a crystalline form, but most of them are amor- 
 phous. They differ in their solubilities and are classified 
 largely upon this basis. The peptones will diffuse through 
 an animal membrane, but they do not pass through rapidly. 
 
 The albuminous substances, like some other organic 
 compounds which do not belong to this class, are thrown 
 out of solution when to the solution certain neutral salts 
 are added until it is saturated. Ammonium sulphate will 
 precipitate all but the peptones and perhaps a few of the 
 albumoses. Magnesium sulphate and sodium chlorid will 
 precipitate many of them. 
 
 When the albuminous substances are heated with 
 water, many of them are coagulated, passing into an in- 
 soluble modification. The temperature at which this takes 
 place is called the coagulation-point. This is a different 
 one for most of the different substances, and may be used 
 in their identification and separation. It may vary, how- 
 ever, from the presence of other substances. It may be 
 raised, prevented, or the coagulation made incomplete by 
 alkalies or by some organic acids, like acetic acid. Coagu- 
 lation is favored and the coagulation-point is lowered in 
 the presence of neutral salts or small amounts of a mineral 
 
36 THE PROTEINS. 
 
 acid. The concentration of the solution also may make 
 it vary. Through coagulation the nature of albuminous 
 substances is altered and they acquire other properties. By 
 the action of alcohol albuminous compounds are precipi- 
 tated, at first in an unaltered form; but if the alcohol is 
 strong and acts for some time they are coagulated, and are 
 then insoluble in water. 
 
 Coagulation, when spoken of with respect to the 
 protein compounds, must be distinguished from precipita- 
 tion, which it resembles. When albumin is coagulated 
 e.g., by boiling, by mineral acids, or by the continued 
 action of strong alcohol it becomes insoluble in water. 
 It may be precipitated by ammonium sulphate without be- 
 ing coagulated or by not too large an amount of alcohol 
 and still retain its original properties, being soluble again 
 upon the addition of water. 
 
 Some of the albuminous compounds are coagulated by 
 the action of ferments; for example, the fibrin, which is 
 so formed from the blood or lymph. 
 
 Albuminous substances are easily decomposed by the 
 action of the putrefactive bacteria, the nitrogen and sul- 
 phur uniting with hydrogen to form hydrogen sulphid, 
 and ammonia, or, these two together, ammonium sulphid. 
 Other nitrogen compounds are also formed, like the amido 
 acids which contain the amido group, KH 2 , such as leucin 
 and tyrosin. Indol is also one of the nitrogenous putre- 
 factive products. 
 
 Many of the albuminous substances are precipitated 
 by the mineral acids, but upon standing with an excess of 
 the acid, or more quickly by heating, they are dissolved, 
 going into acid albumins. Many will also form insoluble 
 compounds with salts of the heavy metals, such as mercury, 
 copper, and lead. With copper in an alkaline solution they 
 
REACTIONS OF ALBUMINOUS SUBSTANCES. 37 
 
 give a blue or purple color and upon boiling with an excess 
 of nitric acid a yellow, which becomes more reddish upon 
 being rendered alkaline. Millon's reagent, which gives a 
 red with all compounds containing a benzene nucleus 
 united with an hydroxyl group, produces the same color 
 with albuminous compounds, whence it is believed that 
 the above complex is contained in albumins. The xan- 
 thoproteic reaction is attributed to the same or the indol 
 group. Similarly each of the other tests appears to be 
 produced by some definite constituent of the protein mole- 
 cule. 
 
 GENERAL REACTIONS OF THE ALBUMINOUS SUBSTANCES. 
 
 The tests may be made upon any albuminous com- 
 pound ; for. example, egg-albumin. 
 
 76. Make the solution alkaline with sodium hydrate 
 and add a few drops of a dilute cupric sulphate solution. 
 A blue or purple color results. An excess of the copper 
 solution must be carefully avoided, as it may produce a 
 blue color when no protein compound is present. (Biuret 
 test.) 
 
 77. Add a small quantity of concentrated nitric acid 
 to the albumin solution and heat to boiling. A yellow color 
 is produced which becomes orange red when the liquid is 
 made alkaline with sodium hydrate or ammonia. (Xantho- 
 proteic reaction.) 
 
 78. Make the solution of albumin acid with acetic 
 acid, then add at least an equal volume of a saturated solu- 
 tion of ammonium sulphate, and heat to boiling. Most 
 albuminous compounds are thrown down as a white pre- 
 cipitate. 
 
38 
 
 THE PROTEINS. 
 
 
 
 
 
 
 
 
 
 
 
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 PS 
 
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 CLASSIFI 
 
 
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ALBUMINS. 39 
 
 79. Acidify the solution with acetic acid and add a 
 few drops of potassium ferrocyanid. A white precipitate is 
 formed. 
 
 The two last reagents fail in case of the peptones. 
 
 80. Add an excess of Millon's reagent and boil. A 
 red color is produced with all albuminous compounds ex- 
 cept antipeptones. 
 
 81. Many of the albuminous compounds are also precipi- 
 tated by, (1) trichloracetic acid; (2) metaphosphoric acid; (3) 
 alcohol or ether; (4) from solutions acidified with HC1 or HNO 3 
 by potassium mercuric iodid or phosphotungstic acid; (5) from 
 acetic acid solutions by picric or tannic acid; (6) from weakly 
 alkaline solutions by salts of mercury and some of the other heavy 
 metals. Try these. 
 
 No reaction is characteristic in itself; therefore in test- 
 ing for these bodies a number of tests should ~be tried. 
 
 ALBUMINS. 
 
 The albumins are soluble in water. They are not pre- 
 cipitated by dilute acids or alkalies. They are precipitated 
 by saturation with ammonium sulphate and by some of the 
 salts of the heavy metals, such as mercuric chlorid and 
 salts of copper, silver, and lead. For this reason albumin 
 is successfully used as an antidote for poisoning by many 
 of these metallic salts. By the action of acids it is con- 
 verted into acid albumin and by the caustic alkalies into 
 alkali albumin. 
 
 There are several different varieties of albumin usually 
 named from their sources, such as the serum-albumin of 
 the blood; egg-albumin; and the albumin of milk, or 
 lactalbumin. We know that these are not the same sub- 
 stances from their physiological action, as well as from 
 some chemical and physical properties. If, for example, 
 
40 THE PROTEINS. 
 
 egg-albumin be injected into the circulation it usually 
 passes in a short time into the urine. Serum-albumin in- 
 jected in the same manner does not pass unaltered through 
 the kidneys. Serum-albumin is more easily dissolved by 
 an excess of mineral acid than egg-albumin. They differ 
 also in their coagulation-temperatures. 
 
 Albumin may be obtained for the experiments by dis- 
 solving the commercial dry egg-albumin or serum-albumin 
 in cold water, filtering if it is not clear. From fresh eggs 
 it can be prepared by separating the albumin from the 
 yelk, being careful not to mix them. Then beat the albu- 
 min, to break up the membranes in it, mix with twice its 
 volume of water, and filter through a piece of unbleached 
 muslin. 
 
 82. Place a moistened piece of parchment or a parch- 
 ment filter in a funnel from which the stem has been 
 broken, and hang the funnel in a beaker of water so that 
 the water rises nearly to the top. Into the parchment pour 
 a solution of egg or serum albumin with a little salt, and 
 let it stand all night. Test the outer liquid by the xantho- 
 proteic and biuret reactions for proteids. None will be 
 found, although it contains a chlorid, as shown by adding 
 silver nitrate with nitric acid, when a white curdy precipi- 
 tate of silver chlorid appears. 
 
 83. Tie a piece of moistened parchment over the 
 mouth of a thistle tube, then fill the bulb with an albumin 
 solution and suspend the tube by a clamp, so that the bulb 
 is covered by the water in a beaker below. The liquid rises 
 in the tube above the level of that outside owing to the 
 inward osmotic pressure of the water being greater than 
 the outward one of the albumin solution. This is the 
 action that occurs when animal cells are surrounded by 
 pure water. 
 
ALBUMIN. 41 
 
 Purified egg-albumin may be prepared by the following 
 method: 
 
 84. Separate the white from the yelk and cut it for some 
 time with scissors or beat it up to a strong foam. Allow this to 
 subside; filter or press it through clean, unsized muslin; mix it 
 with an equal volume of water, and, after the precipitate has sub- 
 sided, filter it. Saturate the filtrate at about 20 with very finely 
 powdered magnesium sulphate, added in small portions with con- 
 tinual stirring. When it is saturated filter off the precipitated 
 globulins and dialyze the filtrate until a portion gives only a faint 
 turbidity with barium chlorid. The solution will increase in vol- 
 ume during the operation. It should be concentrated in flat dishes 
 at 40 to 50, again dialyzed, and finally evaporated to dryness at 
 the above temperature. It still contains 1 to 3 per cent, of ash. 
 
 85. To obtain purified serum-albumin the blood must be 
 drawn into a dry vessel and the fibrin coagulated by beating. 
 Filter this out through muslin and separate the corpuscles from 
 the serum by a centrifugal machine or by allowing it to stand in a 
 tall cylinder until the corpuscles have settled. Horse-blood, if 
 obtainable, is best for this purpose. Then draw off the clear 
 serum with a siphon, dilute it with three volumes of a saturated 
 solution of ammonium sulphate, and add gradually, stirring con- 
 tinually, finely powdered ammonium sulphate until the liquid is 
 saturated. Filter off the precipitate, which contains serum- 
 albumin and paraglobulin, and wash it with a saturated solution 
 of ammonium sulphate. It will be purer if it is dissolved in a 
 small quantity of water and the precipitating and washing are 
 repeated. Dissolve the precipitate while it is still moist in the 
 smallest possible quantity of water and dialyze it. The para- 
 globulin separates out as a white flocculent precipitate, while the 
 albumin remains in solution. Filter out the globulin and wash 
 it with water. It is insoluble in water, but soluble in dilute 
 solutions of neutral salts. It is precipitated by neutral salts like 
 ammonium sulphate, magnesium sulphate, or sodium chlorid. Its 
 sodium chlorid solution coagulates at about 75. 
 
 If the solution of serum-albumin contains no globulin, it will 
 not be made turbid by adding to a small portion magnesium sul- 
 phate to saturation. In that case it is to be carefully neutralized 
 with ammonia, the salts dialyzed out, the contents of the dialyzer 
 
42 THE PROTEINS. 
 
 filtered, if necessary, and evaporated at 40 to a small volume. 
 The solution should be precipitated by alcohol and immediately 
 filtered. Press out the excess of alcohol as much as possible, and 
 wash out the remainder with ether. 1 Allow the ether to escape by 
 stirring the albumin in a mortar. 
 
 86. THE PREPARATION OF A CRYSTALLIZED VEGE- 
 TABLE PROTEIN. The edestin of the hemp seed will serve 
 as an example. Digest about 10 grammes of ground hemp 
 seed for half an hour at 50 to 60 with about 50 cubic 
 centimeters of 10 per cent, sodium chlorid solution; then 
 filter. Divide the nitrate into two parts. Dilute one part 
 with warm water until it begins to be turbid. Let it cool 
 very slowly to nearly the freezing point. Put the second 
 part in a dialyzer and allow the salt to diffuse into the 
 surrounding water. In both cases microscopic crystals 
 will be found,, octahedral or tetrahedral in form. The 
 more slowly they are formed the more perfect they will be. 
 
 87. Show that the crystals are insoluble in water, 
 but soluble in salt solutions. This solution precipitates 
 on dilution with water and coagulates by heating. It also 
 gives the Millon's and the xanthoproteid reactions. The 
 edestin is therefore a globulin. 
 
 88. THE PREPARATION OF CRYSTALLIZED EGG ALBUMIN. 
 To 100 cubic centimeters of egg-white from fresh eggs add an 
 equal volume of a saturated solution of ammonium sulphate. 
 Beat the mixture thoroughly with an egg-beater or a fork to break 
 up the membranes and let it stand in a cool place for several 
 hours, then filter through muslin or paper until the filtrate is clear. 
 Add to the filtrate more saturated ammonium sulphate until a 
 permanent precipitate forms, after which drop in distilled water 
 until this dissolves, avoiding an excess. Now add acetic acid, 
 
 1 This must be done away from the vicinity of any light or 
 fire. 
 
ALBUMIN. 43 
 
 drop by drop, until a slight precipitate appears, and let it stand 
 over night, when there should be a separation of the albumin in 
 rosette-like clusters of needle-shaped crystals. If the eggs are 
 not fresh a little more acid must be used enough to make a fairly 
 bulky precipitate and then the solution must stand over night in 
 a closed flask. To recrystallize, dissolve in cold water, add a few 
 drops of acetic acid, then saturated ammonium sulphate solution 
 as before till the precipitate commences to form and let it stand. 
 The yield should be about 40 per cent. 
 
 89. Try the effect of various reagents upon the co- 
 agulation-temperature of albumin by placing about 10 
 cubic centimeters of an albumin solution in a number of 
 test-tubes, first filtering if necessary to obtain a clear 
 liquid, and adding to each three drops of the following 
 solutions (1) sodium chlorid; (2) concentrated sodium 
 chlorid and acetic acid; (3) dilute hydrochloric acid; 
 (4) sodium hydrate; (5) a solution of sodium carbonate; 
 (6) nothing. Set the test-tubes in a beaker of water and 
 heat to boiling, observing which coagulate first. It will be 
 found that acids and neutral salts favor coagulation and 
 that alkalies hinder it or prevent it altogether. 
 
 90. Eather strongly acidify a concentrated solution 
 of albumin with hydrochloric or sulphuric acid. The albu- 
 min is precipitated. Is it coagulated? Test by diluting 
 with water. 
 
 91. Suspend a small beaker in a larger one, keeping them 
 separate by wedges of cork; fasten by a clamp. Nearly fill both 
 with water and place in the inner one a test-tube with enough 
 solution of filtered egg albumin to stand at the same level as the 
 water. The test-tube should not touch the beaker wall. Heat 
 the water gradually, having a thermometer in the albumin solu- 
 tion. Above 45 the temperature should not be allowed to rise 
 faster than 1 per minute. Make a note of the temperature where 
 the liquid becomes opalescent and where a precipitate forms. 
 
44 THE PHOTEINS. 
 
 92. Put some of the clear albumin solution into three test- 
 tubes. Make one of them faintly acid by a drop of very dilute 
 hydrochloric acid; add to the second enough of a very dilute solu- 
 tion of sodium carbonate to make it faintly alkaline; have the 
 third exactly neutral. Heat them as in the last experiment, ob- 
 serving the coagulation temperature by means of a sensitive ther- 
 mometer. Report results. 
 
 93. Test the solubility in cold water of some egg- 
 albumin which has been dried at a low temperature. It 
 dissolves slowly, showing that coagulation has not occurred. 
 The solution will give the albumin reactions. 
 
 94. Place some of this thoroughly dry egg-albumin 
 in a dry test-tube and let this stand for several minutes 
 in a beaker of boiling water. After removal it will dis- 
 solve in cold water as before. Coagulation has not taken 
 place, since for this the presence of water is necessary. 
 
 95. Test solutions of serum-albumin and egg-albu- 
 min with an excess of nitric acid without heat and also by 
 warming, and notice that the serum-albumin is more 
 easily soluble. Try the same with concentrated hydro- 
 chloric acid. 
 
 96. Show that a solution of egg-albumin forms an 
 insoluble compound when added to solutions of mercury, 
 silver, or copper. 
 
 GLOBULINS. 
 
 The globulins are distinguished from all the other 
 albuminous substances by being soluble in dilute salt solu- 
 tions, but insoluble in water. From this solution they are 
 precipitated by diluting freely with water or by removing 
 the salt by diatysis. They can, in this manner, be sepa- 
 rated from the albumins which remain in solution. The 
 globulins are, as a rule, precipitated by saturating the 
 
GLOBULINS. 45 
 
 solution with neutral salts, like 'sodium chlorid. They 
 are coagulated by heating with water. By the action of 
 dilute acids they are converted into acid albumin. A com- 
 mon example is myosin, which is found in muscle. 
 
 97. Prepare myosin from lean meat by chopping 
 about an ounce finely, then stirring it well with cold water 
 to remove the albumin. Filter through muslin, and repeat 
 the treatment with water until it is white or nearly so. 
 Squeeze out most of the water and treat the residue with 
 a 10-per-cent. solution of ammonium chlorid. For thor- 
 ough extraction it should stand several hours, but enough 
 for testing can be obtained by stirring for five minutes. 
 Filter through muslin, then through paper. The filtrate 
 contains the globulin (myosin). 
 
 98. To a beaker full of pure water add a little of the 
 solution. The myosin is precipitated. 
 
 99. Heat some of the globulin solution. It is coagu- 
 lated. 
 
 100. To 5 cubic centimeters of the myosin solution add as 
 much saturated ammonium sulphate solution. A precipitate of 
 the globulin forms. Is it chemically changed? 
 
 101. Saturate 10 cubic centimeters with magnesium sul- 
 phate. The globulin is precipitated, but dissolves again on dilu- 
 tion with water. Has it been changed chemically? 
 
 102. To the solution of myosin add enough hydrochloric 
 acid to make it contain 0.1 per cent. After it has stood a few 
 hours syntonin, an acid albumin, has been formed. If the change 
 is complete it does not coagulate upon heating. If there is a 
 coagulum it is some of the unchanged myosin. In this case filter 
 it out and test the filtrate for the acid albumin by carefully neu- 
 tralizing it with dilute sodium hydrate. The acid albumin which 
 is formed from the globulin by the dilute acid is precipitated. 
 
 103. Try the general tests for albuminous substances (xan- 
 thoproteic, biuret, Millon's). The myosin responds to all. 
 
46 THE PKOTEINS. 
 
 THE ALBUMINATES. 
 
 These are called also acid and alkali albumins. They 
 are formed from the albuminous substances by the action 
 of acids or alkalies. They are soluble in water which con- 
 tains a small amount of acid or alkali, but are not in neu- 
 tral solution. Consequently they are precipitated when 
 their solution is neutralized. They differ from the glob- 
 ulins by being insoluble in dilute salt solutions. They are 
 not coagulated by boiling. The alkali albumin has the 
 properties of an acid, giving a slight acid reaction. Simi- 
 larly the acid albumin has a slight alkaline reaction. They 
 are named from the manner in which they are produced, 
 and not according to their reaction. 
 
 ACID ALBUMIN. 
 
 104. Prepare by adding dilute HC1 to a solution of 
 egg-albumin till it contains 0.1 per cent, of the acid. 
 Allow it to stand an hour, at about body-temperature, then 
 filter and neutralize with very dilute sodium hydrate, being 
 careful not to add an excess, as this would dissolve the 
 precipitated acid albumin. Wash the precipitate in water. 
 
 105. Notice that the acid albumin is soluble in acids, 
 though insoluble in water. Make a solution in dilute HC1 
 and boil. It is not coagulated. 
 
 106. Make acid albumin by the action of strong 
 HC1, HN"0 3 , or acetic acid on serum-albumin or egg-albu- 
 min, warming if necessary. It is formed very quickly. 
 Neutralize a portion with sodium hydrate. It is precipi- 
 tated. Show that it gives the xanthoproteic reaction and 
 biuret reaction, though it is not precipitated by boiling. 
 
THE ALBUMIN ATES. 47 
 
 ALKALI ALBUMIN. 
 
 107. Prepare from a solution of albumin by warm- 
 ing with alkali, like sodium hydrate. 
 
 108. The solid alkali albumin can be obtained by adding 
 strong sodium hydrate, drop by drop, to the white of an egg, 
 stirring continually. No more must be added after it has become 
 gelatinous, as it will then dissolve. Wash the solid in cold water, 
 in which it is insoluble, though it is soluble without difficulty in 
 warm water. (It is known as Lieberkiihn's jelly.) 
 
 109. Observe that the alkali albumin is soluble in 
 dilute acids or alkalies. If the conversion is incomplete 
 complete solution does not occur. An excess of acid may 
 precipitate it. 
 
 110. Dissolve some of the solid substance in hot 
 water or use the solution obtained from Experiment 107. 
 Add a few drops of phenolphthalein, which gives a red 
 color, showing that the solution is alkaline. Add slowly 
 dilute acid until the red color has just disappeared, when 
 the solution will be neutral. The alkali albumin is pre- 
 cipitated. If, now, more acid is added, the precipitate dis- 
 solves. It is, consequently, like the acid albumin, precipi- 
 tated by neutralizing. 
 
 111. Show that dissolved alkali albumin, like acid 
 albumin, is not coagulated by boiling. 
 
 112. Show there is a cleavage of egg-albumin during 
 the formation of alkali albumin with a splitting off of the 
 loosely combined (cystin) sulphur. 
 
 First heat the albumin solution with sodium hydrate 
 for several minutes. The sulphur which has been split off 
 from the albumin molecule remains in the solution as so- 
 dium sulphid and does not discolor a piece of filter paper 
 
48 THE PROTEINS. 
 
 which is moistened with lead acetate solution and held 
 over the mouth of the test-tube. Upon acidifying the hot 
 solution with hydrochloric acid the sulphur previously 
 liberated is given off as hydrogen sulphid gas, which im- 
 mediately turns the paper brown. 
 
 113. In the same manner as in the previous experi- 
 ment test with lead paper, the gas from a solution of albu- 
 bin when boiled with hydrochloric acid, as in the formation 
 of acid albumin. No discoloration appears; that is, the 
 albumin has not been decomposed to such a degree as 
 in the last experiment. 
 
 114. The thoroughly washed, solid alkali albumin when 
 pressed upon moist litmus-paper reddens it, and its solution in 
 calcium hydrate has an acid reaction, if no excess of the latter is 
 present. To obtain these results, however, great care is necessary 
 to free it from the alkali used in its preparation. 
 
 PROTEOSES AND PEPTONES. 
 
 The peptones are the final products of the digestive 
 action of pepsin upon the albuminous compounds. The 
 albumoses are intermediate products between the albumi- 
 nous compounds and the peptones. They may both be 
 formed by the putrefaction of albuminous substances. 
 
 The proteoses give the general reactions of the al- 
 buminous substances, but they do not, like the former, 
 coagulate on boiling. They are distinguished from the 
 peptones by giving a precipitate with nitric acid or potas- 
 sium ferrocyanid acidified with acetic acid. They are also 
 precipitated by saturating their solution with ammonium 
 sulphate or sodium chlorid, then acidifying. They diffuse 
 with difficulty through an animal membrane. 
 
 The peptones do not give any of these reactions, but 
 respond to the general ones of albuminous compounds, espe- 
 
...... (r 
 
 
 \j t, 
 
 
THE PROTEOSES AND PEPTONES. 49 
 
 cially the biuret test, where the resulting color is a reddish 
 pink. They do not coagulate on boiling, and, unlike the 
 other albuminous substances, will pass through a parch- 
 ment or animal membrane. They cannot be precipitated 
 by ammonium sulphate or with potassium ferrocyanid. 
 Tannin or alcohol precipitates them from their solutions. 
 
 A number of different classes have been described, two 
 of the principal ones being those designated by the prefixes 
 anti, which resist the action of ferments and are not 
 easily decomposed further; and Tiemi, which are more 
 easily decomposed. Thus we have antialbumoses and hemi- 
 albumoses, and antipeptones and hemipeptones. The other 
 classes differ principally in their solubilities. 
 
 In the dry state the proteoses form an amorphous 
 powder. The peptones also have an amorphous form, but 
 are extremely hygroscopic, dissolving, to a resinous mass, 
 in the water which they absorb from the air. Their taste 
 is unpleasant. 
 
 115. PREPARATION or PROTEOSES. Boil for twelve 
 to fifteen hours about 10 grammes of fibrin or coagulated 
 egg-albumin with three to four times its weight of 4 per 
 cent, sulphuric acid. Keep the volume constant by the use 
 of an inverted condenser or by the addition of water. The 
 liquid turns violet, then brownish. If on testing a 
 small sample by neutralizing with sodium hydrate much 
 acid albumin is precipitated, continue the heating until 
 such a precipitate is no longer produced or is but slight. 
 Then filter off any insoluble matter, neutralize with am- 
 monium hydrate, and saturate the liquid with ammonium 
 sulphate. The proteoses are precipitated. Filter and 
 wash them with a saturated solution of the last reagent. 
 Then dissolve the proteoses in a little water, place this 
 solution in a dialyzer, and dialyze until the outer liquid 
 
50 THE PROTEINS. 
 
 gives no reaction for sulphates with barium chlorid. If 
 the water is warm some antiseptic like thymol or chloro- 
 form must be used. The albumose reactions can be made 
 with this solution from the dialyzer. 
 
 If the dry proteoses are desired concentrate the solu- 
 tion to a small bulk and precipitate them with an excess 
 of alcohol, washing with the same after filtration. Dry 
 in a vacuum desiccator over sulphuric acid. 
 
 116. Dissolve some of the proteose in water and show that 
 it is precipitated by potassium ferrocyanid in a solution acidified 
 with acetic acid, avoiding an excess of the former. 
 
 117. Show that a solution of proteose in water is not coagu- 
 lated by neutralizing nor boiling. Nitric acid gives a white pre- 
 cipitate which dissolves on heating and reappears on cooling. (Dis- 
 tinction from albumin and albuminates.) 
 
 118. PREPARATION OF PEPTONE. Digest blood-fibrin in a 
 neutral solution with a watery extract from a chopped pancreatic 
 gland at about body-temperature. If it is allowed to stand many 
 hours, add a few crystals of thymol to prevent putrefaction. 
 Boil after solution has taken place, filter, concentrate by boiling, 
 and saturate while boiling with ammonium sulphate to precipitate 
 the albumoses. Filter these out, first by muslin, then by filter- 
 paper. The solution may be used for testing or the ammonium 
 sulphate may be removed largely by evaporation and crystalliza- 
 tion. The remainder can be removed by adding first barium 
 hydrate, then barium carbonate, and boiling, until a portion of 
 the filtrate gives no precipitate with barium chlorid. 
 
 119. Commercial peptone may be used for testing. 
 Test the solution of peptones by adding first sodium hy- 
 drate, then not more than two or three drops of copper sul- 
 phate (biuret test). A red or pink color is produced, 
 which is characteristic of the peptones. If too much of 
 the copper solution is added, the color is bluish. 
 
 120. Place some of the solution in a dialyzer and 
 leave it an hour, then test the solution outside for the 
 
INSOLUBLE PROTEINS. 51 
 
 presence of peptones. They will be found to have passed 
 through the membrane, although they do not dialyze rap- 
 idly. 
 
 121. Show that tannic acid precipitates peptones in 
 a neutral solution. 
 
 122. Show that the peptones are not precipitated by 
 potassium ferrocyanid acidified with acetic acid, as are 
 the albumoses, if they contain none of the latter. 
 
 FIBRIN. 
 
 Fibrin is formed as a gelatinous mass when fresh 
 blood coagulates. If the blood is beaten during its coag- 
 ulation the fibrin collects together into strings as elastic as 
 caoutchouc, and remains so as long as it is moist. It can 
 be freed from the blood coloring matter by washing with 
 water or a salt solution. It is coagulated by heating with 
 water. 
 
 COAGULATED ALBUMIN. 
 
 The albumins may be converted into the coagulated 
 form by heating with water or by the continued action of 
 strong alcohol. This is insoluble in water, but can be dis- 
 solved by caustic alkalies or by heating with the strong 
 mineral acids, being thereby converted into alkali or acid 
 albumins. 
 
 COMPOUND PROTEINS. 
 
 This class of substances is more complex than the 
 albuminous substances. They can all be decomposed into 
 albuminous substances, on the one hand, and, on the other, 
 bodies which are not albuminous. Thus the albuminous 
 compound is, in hemoglobin, united with the hsematin 
 molecule; in the nucleins, with phosphoric acid, etc. They 
 
52 THE PROTEINS. 
 
 can be considered, then, as unions of an albuminous sub- 
 stance with some other substance. Most of them are coagu- 
 lated by boiling. 
 
 THE MUCINS. 
 
 Mucins are found in some of the secretions of the body, 
 especially in those of the mucous membrane and saliva, 
 and also as a constituent of the tendons and umbilical cord. 
 In their composition they resemble the albuminous sub- 
 stances, but contain less nitrogen. Their characteristic 
 property is that when boiled with a dilute mineral acid they 
 .are decomposed, giving two substances: an albuminous com- 
 pound and a compound containing little or no nitrogen and 
 having the power of reduction, as is shown by its changing 
 cupric hydrate to cuprous oxid in an alkaline solution. 
 By this they can be distinguished from all similar albu- 
 minous compounds. There are several kinds of mucin, 
 although as yet they are not well differentiated from one 
 another. 
 
 The mucins are colloid substances, insoluble in pure 
 water, but soluble in small amounts of dilute alkalies, such 
 as calcium hydrate. They are mucilaginous and can be 
 drawn out into threads. They are precipitated by the 
 addition of acetic acid, if neutral salts are absent. They 
 are not coagulated by boiling, but give many of the reac- 
 tions of the albumins. Like the nucleoalbumins, they are 
 acid in reaction. 
 
 There have been sometimes included with the mucins a class 
 of substances which resemble them in being decomposed by acids 
 into albuminous substances and substances with the power of 
 reduction, but which are not precipitated by acetic acid. They 
 are more properly called mucoids. Such are the pseudomucin, 
 found in ovarial liquids; chondromucoid of cartilage, and a few 
 others. 
 
MUCIN. NUCLEOALBUMINS. 53 
 
 123. PREPARATION OF MUCIN. Mince finely a sub- 
 maxillary gland of an ox and extract it with water. Filter 
 and add to the filtrate strong hydrochloric acid until the 
 liquid contains 0.15 per cent, of acid, avoiding an excess. 
 The mucin is at first precipitated, but dissolves again upon 
 stirring. Then add two or three volumes of water, which 
 will precipitate it. Separate it from the liquid by filtration 
 or decantation and repeat the dissolving and precipitation 
 as before. Wash with water and, if the dry substance is de- 
 sired, with alcohol and ether. 
 
 124. Try the solubility in calcium hydrate, and pre- 
 cipitate from this solution by acetic acid. 
 
 125. Boil mucin for some time with dilute hydro- 
 chloric acid, and, after making the liquid alkaline, show 
 by Fehling's test that there is a reducing body present. 
 
 126. Show that solutions of mucin in an alkali will 
 give the biuret test. 
 
 THE NUCLEOALBUMINS. 
 
 The nucleoalbumins occur widely distributed in the 
 animal and vegetable kingdoms, forming one of the princi- 
 pal constituents of protoplasm. They are found especially 
 in the cell, but sometimes in the secretions, such as the milk, 
 which contains casein. Some authors, however, include 
 in the class of nucleoalbumins only those found in the cell, 
 and exclude such as the casein of milk and the vitellin of 
 eggs. 
 
 Chemically they are composed of the same elements 
 as albumin, but contain, in addition, phosphorus and some- 
 times iron. The}'' resemble in their properties the globu- 
 lins and alkali albumins. They differ, however, in contain- 
 ing phosphorus. They are also insoluble in neutral salt 
 
54 THE NUCLEOALBUMINS. 
 
 solutions, unlike the globulins, and differ from the alkali 
 albumins by being decomposed by the action of gastric juice 
 into an albumin, which is digested, and an indigestible 
 phosphorus compound called nuclein. The nucleoalbumins 
 may be considered, then, as compounds of a nuclein and an 
 albuminous substance. 
 
 The nucleoalbumins are insoluble in water or dilute 
 acids. They can be dissolved in alkaline solutions. They 
 have the properties of dibasic acids, as is shown by the fact 
 that the solution in an alkali, where not too much of the 
 alkali has been used, has a slight acid reaction. The acid 
 reaction is also shown by their setting free the carbonic 
 acid of carbonates. The nucleoalbumins can be precipi- 
 tated from their alkaline solutions by acidifying. The 
 acid removes the alkali with which they are united and sets 
 the nucleoalbumin free as an insoluble substance. This 
 may be shown in the precipitation of the casein in milk. 
 
 The nucleoalbumins are soluble in strong acetic acid 
 or an excess of hydrochloric acid, being at the same time 
 decomposed, nuclein being set free and the albumin being 
 converted into acid albumin. They are also decomposed 
 and coagulated by suspending the free nucleoalbumin in 
 water and boiling. 
 
 In general, being compounds of the albuminous sub- 
 stances, the nucleoalbumins respond to the same tests. 
 
 127. PKEPAKATION OF CASEIN. Dilute about 100 
 cubic centimeters of milk with 400 cubic centimeters of 
 water, and precipitate the casein until the liquid above 
 is nearly clear by adding acetic acid drop by drop, avoiding 
 an excess. Filter and wash with water. If fat-free sub- 
 stance is desired, it must be extracted in an extraction- 
 apparatus with ether. For many tests this is not neces- 
 sary. The casein can be to some degree purified by dis- 
 
CASEIN. 55 
 
 solving in dilute ammonia and reprecipitating with 
 acid. 
 
 128. Test the casein for nitrogen and sulphur in the 
 same manner as albumin was tested (Experiments 72 and 
 73). 
 
 129. Test it also for phosphorus by mixing about 
 a gramme of the dry substance with equal parts of sodium 
 carbonate and potassium nitrate and fusing in a porcelain 
 crucible. After cooling, dissolve the mass in water, acidify 
 strongly with nitric acid, and add ammonium molybdate. 
 A yellow precipitate, at once or after warming, shows the 
 presence of phosphoric acid. 
 
 130. Try the solubility of casein. It is insoluble in 
 water, but soluble in alkalies. Its alkaline solution is not 
 coagulated by heat. With lime water it forms a milky 
 solution. Its solutions give the biuret reaction and Millon's 
 reaction. 
 
 131. Demonstrate the acid nature of freshly precipi- 
 tated casein by dissolving it in a very dilute solution of 
 sodium carbonate. If not quite enough of the latter is used 
 to produce a complete solution and if the mixture is then 
 filtered, the nitrate will have an acid reaction showing 
 that the sodium casein which it contains has some of the 
 properties of an acid salt. Boiling causes no coagulation, 
 but the casein can be set free as a precipitate if hydro- 
 chloric acid is added to strong acid reaction. (Compare 
 with results of acidifying solutions of soap or of soluble 
 carbonates.) 
 
 132. If the rennin ferment is at hand or can be pre- 
 pared, test with it a solution of casein in lime-water. The 
 casein is changed into paracasein calcium (cheese) which 
 is only slightly soluble in water. 
 
56 THE NUCLEINS. 
 
 133. Make a gastric juice by adding a few grains of pepsin 
 to 0.2-per-cent. HCI 1 and digest in it for some time at the tempera- 
 ture of the body some casein. The nucleoalbumin is decomposed 
 into two substances: an albumin, which is dissolved, and a 
 nuclein, a compound rich in phosphorus, which remains. Filter out 
 the nuclein, and test the nitrate with the biuret test for the 
 albuminous compound. 
 
 THE NUCLEINS. 
 
 The nucleins occur partly combined with albuminous 
 substances as nucleoalbumins, partly free in the nucleus of 
 the cell. They are composed of phosphoric acid united 
 with an albumin, or sometimes in addition with a nuclein 
 base, such as adenin, guanin, xanthin, or hypoxanthin. 
 Besides the elements found in vhe albumin they contain 
 phosphorus and sometimes iron. The iron in the haamo- 
 globin is probably obtained by the animal organism from 
 such compounds as these. We have no absolute proof that 
 the common salts of iron can be assimilated by the animal 
 organism. One of the nucleins which may furnish iron to 
 the animal body is haamatogen, found in the yelk of eggs. 
 The nucleins may be divided into two classes: 
 1. Those which, when acted on by hot acids or alka- 
 lies, take up water and give, as decomposition-products, 
 phosphoric acid and an albumin. They have been called 
 paranucleins or pseudo-nudeins. Such a one is contained 
 in casein. It can be set free by digesting with gastric juice 
 from the albumin with which it is united. Haamatogen is 
 also of this class, and probably furnishes the iron for the 
 haemoglobin of the young bird. 
 
 1 This strength of acid can be obtained by diluting concentrated 
 hydrochloric acid with 150 times its volume of water. 
 
THE NUCLEINS. 57 
 
 2. The second class is decomposed by dilute acids or 
 alkalies, giving, besides phosphoric acid and an albumin, 
 one or more of the nuclein bases, also called xanthin bases, 
 or purin bases, xanthin, guanin, etc. These nucleins are, 
 then, combinations of albuminous substances with nu- 
 cleinic acids. This class is found principally in the nucleus 
 of the cell and has been called nuclear, or nucleous nu- 
 cleins. 
 
 The nucleins are not attacked by gastric juice, and 
 this is used to separate them from the albuminous com- 
 pounds, which can be digested by it. They are insoluble 
 in water and dilute acids, but soluble in alkalies. They 
 give the biuret and Millon's tests in consequence of the 
 albumin which they contain. 
 
 134. Mix 50 grammes of compressed or brewers' yeast with 
 200 grammes of water, and allow the yeast to subside. Pour 
 off the water and to the residue add O.o-per-cent. potassium 
 hydrate solution; stir, and, after waiting a few minutes to dissolve 
 the nucleins, filter and acidify with hydrochloric acid. Filter out 
 the precipitated nucleins, wash with HC1, then hot alcohol. Dry 
 over sulphuric acid. Try solubility in acids and alkalies, also biuret 
 and Millon's tests. The residue gained by fusion with sodium car- 
 bonate and potassium nitrate gives a yellow precipitate with am- 
 monium molybdate, showing the presence of phosphoric acid. 
 
 A few of the nucleins, both animal and vegetable, contain 
 iron, and these are the principal source of the iron in the animal 
 body, our food-materials containing no inorganic compounds of 
 iron. Hsematogen is an. example of such nucleins. It is found in 
 the yelk of hen's eggs united with an albuminous substance, 
 which can be split off by digestion. 
 
 The iron in such organic combinations as nuclein does not 
 respond to the ordinary chemical tests until the compound has 
 been decomposed by chemical agents or other means. Reagents 
 like ammonium sulphid and potassium ferrocyanid decompose 
 it very slowly, whereas they act immediately upon the inorganic 
 compounds of iron and not at all upon hsematin. The nucleins 
 
58 H^lMATOGEN. THE ALBUMINOIDS. 
 
 which contain iron are important from their being probably the 
 origin of the animal iron compounds. 
 
 H^IMATOGEN. 
 
 135. Prepare from the yelk of an egg. Shake the yelk in 
 a wide-mouth, glass-stoppered bottle with two or three times its 
 volume of alcohol; allow it to stand and when it has settled pour 
 off the alcohol. Repeat this operation twice, then extract in the 
 same manner, or, better, in an extraction apparatus with ether 
 until the residue is white. Digest this in artificial gastric juice 
 (made as in Experiment 133). The nuclein, haematogen, remains. 
 
 136. Dissolve a portion in ammonia. Test for iron by 
 ammonium sulphid. At first there is no color, but after a time 
 the solution turns greenish and, in twenty-four hours, black, as 
 the iron is gradually set free from the compound. In the same 
 manner test with potassium ferrocyanid. The Prussian blue is 
 formed slowly, differing thus from its production with the ordi- 
 nary iron salts. 
 
 137. Add to the nuclein some hydrochloric acid, then, after 
 neutralizing with ammonia, test for iron as before. The acid has 
 decomposed the nuclein, so that the tests are obtained imme- 
 diately. 
 
 THE ALBUMINOIDS. 
 
 The albuminoids are found in the insoluble form, 
 mostly in the bones of the body or the parts which are 
 used for protection. They resemble the albuminous sub- 
 stances, giving many of the same products when they are 
 decomposed, differing, however, in other respects. They 
 are not easily attacked by the reagents which dissolve and 
 decompose albuminous compounds. Only the collagen and 
 its derivative, gelatin, with possibly elastin, are digestible. 
 
 COLLAGEN. 
 
 This substance is found in the animal body in the 
 connective and cartilaginous tissues, tendons, and bones. 
 
GELATIN. 59 
 
 That from the bones was formerly called ossein. It is com- 
 posed of the same chemical elements as the albumins, but 
 contains a little more oxygen. It is probably an oxidation- 
 product of some of the albumins. It is insoluble in water, 
 but by boiling with water it is converted into gelatin or 
 glue. By the action of tannic acid collagen is changed to 
 a form which does not putrefy. This is the action which 
 takes place when leather is tanned. 
 
 GELATIN. 
 
 Gelatin may be considered as the hydrate of collagen, 
 as it is formed by the union of collagen with water. It 
 can be changed back into collagen by heating for some 
 time at 130. It swells up in cold water and dissolves 
 when the water is warmed. On allowing the solution to 
 cool it gelatinizes, or becomes a semi-solid. It acts in this 
 respect oppositely to albumin, which is soluble in the cold, 
 but becomes a solid by the action of heat. 
 
 After it has been boiled a long time with water it is 
 decomposed, and does not gelatinize on cooling, peptone 
 being formed. The sulphur of gelatin is united in the 
 molecule in a different manner from that of the albumin 
 molecule, as is proved by its decomposition-products. 
 
 Gelatin is decomposed by the gastric juice, giving 
 products similar to those from albumin. It has been 
 found, however, that it cannot take the place of the al- 
 buminous materials of food, though it is of value when 
 used with them. 
 
 Gelatin does not give all the reactions of the albumi- 
 nous substances, although it does give the same results with 
 some of them. Like the albuminous compounds, it gives 
 a purple color with the biuret test; it is precipitated by 
 
60 GELATIN. 
 
 picric acid, by mercuric chlorid in the presence of sodium 
 chlorid and hydrochloric acid, by tannic acid in the pres- 
 ence of sodium chlorid, and by saturation with ammonium 
 sulphate. On the other hand, it is not coagulated by boil- 
 ing; it is not precipitated by mineral acids; and it does not 
 give a brown color when warmed with an alkaline solution 
 of lead, as albumin does, the sulphur being apparently too 
 firmly united to be split off and form lead sulphid. It 
 does not give the xanthoproteic reaction when pure. 
 
 138. Prepare collagen from bone by dissolving out 
 the mineral constituents with dilute hydrochloric acid 
 (HC1, 1 part; water, 8 parts) until they are flexible, then 
 wash out the acid. Notice that the collagen is not soluble 
 in dilute acid nor cold water. To remove all the albumins 
 it may be necessary to soak awhile in 5-per-cent. NaOH 
 solution, then wash again. 
 
 139. Convert the collagen into gelatin by boiling it 
 a few minutes with water. Notice that it gelatinizes upon 
 cooling the solution, especially after standing. 
 
 140. Boil a portion of the solution for some time, 
 and notice that it is thus decomposed, so that it will not 
 form a jelly upon cooling. 
 
 141. Test a portion of the gelatin solution with the 
 biuret test. It gives a purple color like albumin. 
 
 142. Show that it is precipitated by tannic acid in 
 the presence of NaCl. 
 
 143. Show that gelatin is not precipitated by nitric or other 
 mineral acids, but is by saturation with ammonium sulphate and 
 also by mercuric chlorid in the presence of HC1 and NaCl. 
 
 144. Show that gelatin contains sulphur by heating with 
 dry sodium carbonate in the reducing flame, then testing with 
 sodium nitroprussid as in the case of albumin (Experiment 74), 
 but that it gives no black sulphid of lead when heated in a solution 
 of lead acetate in an excess of sodium hydrate (Experiment 73). 
 
ELASTIN. KERATIN. 61 
 
 ELASTIC. 
 
 Elastin occurs in the connective tissues, in the cervical liga- 
 ment (ligamentum nuchae) very abundantly. It differs from most 
 of the proteins in containing no sulphur, except possibly some 
 that is loosely combined with the molecule. In the moist state it 
 is very elastic; when dry it is hard and brittle. By the action 
 of the digestive ferments it is decomposed into bodies called elas- 
 toses, similar to the albumoses. It gives the general reactions of 
 the proteins. 
 
 145. Prepare elastin from the cervical ligament of an ox by 
 cutting it into thin slices and boiling it for several days to remove 
 the gelatin. Boil then with 1-per-cent. potassium hydrate for 
 several hours, afterward with water. Repeat the boiling with 10- 
 per-cent. acetic acid; then let it stand twenty-four hours in 5- 
 per-cent. hydrochloric acid. Wash with water, boil with 95-per- 
 cent, alcohol, and extract with ether to remove the fat. For the 
 complete removal of the latter more than a week may be required. 
 
 146. Try the tests used for sulphur in albuminous sub- 
 stances, and see that it is not present. 
 
 KERATIN. 
 
 The keratins are the chief constituent of the horny part of 
 the epidermis, of hair, horns, nails, feathers, etc. They contain 
 a large amount of sulphur, 4 or 5 per cent., a part of which 
 is so loosely united that it is set free by boiling water. It is 
 owing to this sulphur that the salts of lead, silver, and some other 
 metals act as hair-dyes, the sulphur uniting with the metal to 
 form a dark-colored sulphid. The keratin is not at all attacked 
 by the gastric or pancreatic juices. It is decomposed when heated, 
 giving the odor of burnt horn. It is insoluble in water, and gives 
 the xanthoproteic and Millon's reactions. 
 
 147. Prepare keratin by boiling some horn-shavings with 
 water and then digesting them in succession in a dilute solution of 
 pepsin containing 0.2-per-cent. HC1, and a trypsin solution. Wash 
 with water, alcohol, and ether. 
 
62 FERMENTATION. 
 
 148. Boil keratin with sodium hydrate, filter, and test the 
 filtrate for sulphur by lead acetate. It produces a black precipi- 
 tate of lead sulphid. 
 
 149. Show that keratin responds to the xanthoproteic and 
 Millon's tests. 
 
 FEBMENTATIOK 
 
 By fermentation we mean the decomposition of an 
 organic substance into simpler and more stable molecules, 
 the agent which causes the change being itself unaffected. 
 The agents are living organisms or are formed by such 
 organisms. The living ferments such as the yeast-plant 
 or bacteria are often called the organized ferments. 
 They have the power of reproduction and are composed of 
 cells. The non-living ferments are known as the unor- 
 ganized ferments, or enzymes. They may be excreted by 
 the organized ferments or secreted by living cells, which 
 latter is the case with the digestive ferments. They are 
 not reproductive and act outside of the cell where they 
 were formed. 
 
 The enzymes of the animal cell exist in the cells in an 
 inactive condition, called zymogens, but become active 
 after standing exposed to the atmosphere or being brought 
 in contact with certain chemical compounds. The enzymes 
 contain nitrogen and from their properties are apparently 
 identical with the proteins. They are indiffusible and 
 soluble in glycerin and in water. They can be mechanic- 
 ally removed, without decomposition, from their solutions 
 by forming precipitates therein, to which they adhere, also 
 by saturating the solutions with ammonium sulphate. A 
 low temperature stops their action and they are all killed 
 below 100 if moisture is present. Most enzymes act best 
 at about 38 C. The ferment is not destroyed, but its 
 
ORGANIZED FERMENTA. 63 
 
 action is stopped, by a large accumulation of its own 
 products. 
 
 The organized ferments contain albumin, fat, cellu- 
 lose, and some inorganic salts. They survive a high tem- 
 perature better than the enzymes, but are killed at 100 
 except certain spore forms. Moisture is necessary for them 
 to act. 
 
 As is the case with the enzymes, a sufficient amount 
 of their products stops their further action. This is the 
 effect of alcohol upon the yeast-plant. 
 
 150. Add a little yeast to a dilute solution of cane- 
 sugar in water and keep it for some time at the body-tem- 
 perature. 
 
 Test the solution with Trommels test. The copper 
 compound is reduced by the glucose and Ia3vulose which 
 have been formed from the sucrose by the invertin of the 
 yeast. If allowed to stand a long time the glucose is 
 changed by the yeast to alcohol and carbon dioxid. 
 
 151. Stir a little compressed yeast into lukewarm 
 water in a test-tube, and after it has stood a few minutes, 
 add a few drops of chloroform and mix thoroughly by 
 shaking. Fill the rest of the tube now with dilute cane- 
 sugar solution and let it stand inverted for twenty-four 
 hours in a warm place, as in Experiment 31. The alco- 
 holic enzyme (zymase) does not act in presence of chloro- 
 form, so that there is no alcoholic fermentation with a 
 formation of carbon dioxid. 
 
 152. Test the liquid with Trommer's test. It re- 
 sponds to the test, showing that the inverting enzyme is 
 iwt destroyed, but has decomposed the cane-sugar, as be- 
 fore, to glucose and lavulose. 
 
64 FERMENTATION. 
 
 153. Separate the mucous membrane from the muscular 
 coating of a pig's stomach; chop finely and allow to stand several 
 hours with two or three times its weight of dilute phosphoric acid 
 (1 per cent.). Filter and to the filtrate, which contains the pepsin, 
 add lime-water until the reaction is alkaline. The calcium phos- 
 phate which falls carries down the pepsin with it. Filter and dis- 
 solve the precipitate in dilute HC1. Place in a dialyzer, changing 
 the water outside frequently. The acids and salts diffuse out, 
 leaving the pepsin inside the dialyzer, as can be proved by adding 
 it to 0.2-per-cent. HC1 and seeing that it will digest fibrin. 
 
 154. Test a part of the precipitated pepsin which has been 
 obtained with the calcium phosphate for nitrogen. This may be 
 done, after washing with water, by drying the precipitate, still 
 mixed with calcium phosphate, then mixing with twice as much 
 soda-lime and testing in a dry tube. The nitrogen is converted 
 into ammonia, which may be recognized by the odor and by its 
 action on red litmus-paper. 
 
 155. To a part of the dialyzed-pepsin solution obtained in 
 Experiment 153 add finely powdered ammonium sulphate, stirring 
 meanwhile as long as it dissolves. The pepsin is precipitated like 
 the albuminous compounds. Filter, dissolve the precipitate in a 
 0.2-per-cent. HC1 solution, and show that it will digest fibrin. 
 
 156. Collect some saliva in a test-tube, place the 
 latter in a beaker of water, and raise the temperature of 
 the water to 65 or 70 C. Keep it at this point for five 
 minutes. Then let a little of it stand a few minutes with 
 a starch solution, testing afterward with Trommer's test. 
 N~o glucose is produced, the ferment, ptyalin, having been 
 destroyed by the heat. 
 
 157. Collect a considerable quantity of saliva, and 
 put it into two tubes. Quickly cool one nearly to freezing 
 and warm the other to body-temperature. Add to these 
 an equal amount of starch solution. Allow the action to 
 proceed for five minutes, then raise them both to the boil- 
 ing-point to stop fermentation. Determine the amount of 
 
ORGANIZED FERMENTA. 65 
 
 sugar formed by Fehling's quantitative test (Experiment 
 33), or, approximately, by the subnitrate of bismuth test 
 (Experiment 30). There has been little or no fermenta- 
 tion in the cold liquid. 
 
 158. In the same manner cool another portion of 
 the saliva and, after ten minutes, warm to body-tempera- 
 ture and show by its decomposing starch that the ferment 
 is not destroyed by the cold. 
 
 159. PREPARATION OF LACTIC ACID BY FERMENTATION. 
 In 150 cubic centimeters of boiling water dissolve 30 grammes of 
 cane-sugar and add about 30 milligrammes of tartaric acid. Let 
 the solution stand two days, then add 40 cubic centimeters of sour 
 milk and about half a gramme of old cheese. After the addition 
 of 15 grammes of zinc oxid allow the mixture to stand ten days at 
 a temperature of 40 to 50, with repeated stirrings. At the end 
 of that time heat to boiling, filter while hot, and allow to cool. 
 Zinc lactate will crystallize out on cooling if the solution is suffi- 
 ciently concentrated. If it is not it should be allowed to evapo- 
 rate to a smaller volume. It may be purified by recrystallizing. 
 The acid can be obtained by dissolving the lactate in water and 
 decomposing by hydrogen sulphid gas. Filter off the zinc sulphid, 
 evaporate the filtrate to a syrup, and after it is cold extract the 
 acid by dissolving it in ether. 1 When the ether has evaporated 
 the acid will remain. It may be preserved for testing in the gastric 
 juice tests. 
 
 160. PREPARATION OF BUTYRIC ACID. Make a mixture as 
 for the preparation of lactic acid, or use a part of that mixture. 
 Allow the fermentation to go on as before, but for three or four 
 weeks. In that time bubbles of hydrogen and carbon dioxid 
 appear and, after removing the zinc, butyric acid is found in the 
 ether. It can be identified by its acid reaction and characteristic 
 odor. 
 
 1 Remember that the vapors of ether are extremely inflam- 
 mable. 
 
66 THE SALIVA. 
 
 161. PREPARATION OF KEPHYB. The preparation of the 
 ferment requires several days. First stir the kephyr-grains in a 
 little lukewarm water and decant the latter. Then cover them 
 with ten times their weight of milk which has been boiled and 
 cooled. Let them stand for twenty-four hours at about 20 C., 
 then pour off the milk, rinse the grains with a little water, and 
 repeat the digestion with milk for twenty-four hours as before. 
 This should be continued until the milk has an acid reaction and 
 the grains rise to the upper part of the liquid, which may require 
 five or six days. After this the grains may be allowed to stand 
 with a moderate quantity of milk, which becomes filled with the 
 organized ferment. At the end of twenty- four hours this is filtered 
 through muslin, and the filtrate added to ten or fifteen times its 
 volume of previously boiled and cooled milk. The whole is poured 
 into a clean strong bottle, the cork is tied in, and it is allowed to 
 stand two or three days at a temperature below 15 C., when the 
 fermentation is complete. The grains on the muslin can be used 
 an indefinite number of times in the same manner. 
 
 162. Test the acid reaction of the kephyr by litmus-paper 
 (lactic acid) ; also show the presence of alcohol by the iodoform 
 test (Experiment 32). 
 
 THE SALIVA. 
 
 The saliva is a mixture of the secretions of the paro- 
 tid, submaxillary, and sublingual glands with that of the 
 glands of the membrane of the month. Its reaction is 
 normally faintly alkaline. The mixed saliva is a colorless, 
 more or less viscid liquid, often opalescent. On standing 
 it deposits calcium carbonate as a film on the surface. Ex- 
 amined microscopically it is seen to contain epithelial cells 
 from the membrane of the mouth, air-bubbles held by 
 viscid liquid, and salivary corpuscles which resemble the 
 lymph-corpuscles. Bacteria are abundant. 
 
THE SALIVA. 
 
 The normal mixed saliva contains 
 
 I. Inorganic 
 
 II. Organic 
 
 Carbonates ] [ magnesium. 
 Chlorids ! ! calcium. 
 Sulphates. [ | potassium. 
 Nitrites [ sodium. 
 
 Sulphocyanate of potassium. 
 Albumin. 
 Mucin. 
 Ptyalin. 
 
 Nitrites and sulphocyanates (sulphocyanids) are often 
 absent. The latter are most frequently found in the saliva 
 of smokers. The ptyalin has the power to convert boiled 
 starch into dextrin, maltose, and glucose. It is not able 
 to penetrate the granule of the unboiled starch, or does so 
 very slowly, differing in this respect from the correspond- 
 ing ferment of the pancreas. Its presence can be detected 
 by mixing the saliva with about ten times its volume of 
 a solution of boiled starc,h, keeping it awhile at body-tem- 
 perature, and after a few minutes testing for sugar. The 
 ptyalin acts best at about 40, and is, therefore, not the 
 same ferment as the diastase of malt, which decomposes 
 starch most rapidly at a temperature of 55. Ptyalin is 
 destroyed by acids, even as dilute as the 0.2-per-cent. hy- 
 drochloric acid of the gastric juice. It is, however, prob- 
 able that it acts for some time in the stomach before the 
 acid penetrates the mass of food in a large enough quan- 
 tity to stop the fermentation. 
 
 The secretion is influenced by the nervous system. It 
 can be increased by mechanical means, like chewing a 
 pebble or a piece of rounded glass in the mouth; by 
 chemical action, such as touching the tongue with a crys- 
 
68 THE SALIVA. 
 
 tal of tartaric acid or filling it with the vapor of ether or 
 acetic acid ; or by electrical excitation. In collecting saliva 
 for testing it should be accomplished without trying to 
 hasten its flow by suction with the tongue as this increases 
 the amount of secretion from the mucous membrane and 
 so dilutes the secretion of the salivary glands. 
 
 The composition of the saliva is changed by certain 
 pathological conditions. The amount is diminished in all 
 febrile conditions^ also in diabetes and often in nephritis. 
 It is increased by the action of some medicinal substances, 
 like the mercury compounds, pilocarpine, and others ; also 
 by anything which causes irritation or inflammation of the 
 glands. Urea has been found in it abundantly during 
 nephritis. The reaction becomes acid in fevers and in 
 diabetes, and this sometimes happens also after long-con- 
 tinued talking. 
 
 163. Collect for examination some saliva by letting 
 it flow into the mouth without swallowing. Excite the 
 flow by chewing a piece of soft paraffin. Notice the indi- 
 cation of mucin in the viscidity, as well as the lasting foam 
 after beating it with a glass rod. 
 
 164. Let a portion stand exposed to the air, and 
 notice the separation of calcium carbonate as a white film 
 or turbidity. 
 
 165. Test a portion for potassium sulphocyanate by 
 adding to it a very dilute solution of ferric chlorid. A 
 red color indicates the sulphocyanate. This is immediately 
 decolorized by the addition of a few drops of mercuric 
 chlorid solution. 
 
 166. 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 iodid. A nitrite 
 immediately gives a blue color. 
 
SALIVARY TESTS. 69 
 
 167. Test for albuminous substances by the xan- 
 thoproteic reaction. 
 
 168. Test for mucin by adding to the clear saliva 
 acetic acid drop by drop. The mucin separates in white, 
 stringy flakes. They may be washed with water and tested 
 by the mucin reactions. 
 
 169. PREPARATION OF PTYALIN. Collect a large amount of 
 saliva and acidify with phosphoric acid. Then add milk of lime 
 until the liquid has a faint alkaline reaction. The phosphoric acid 
 is precipitated as calcium phosphate and carries the ptyalin down 
 with it. Filter and allow the water to drain off without washing. 
 Place the precipitate in a beaker and add not more water than 
 the original amount of the saliva. Stir it well and filter. This 
 removes the ptyalin from the calcium phosphate, and it goes into 
 the nitrate. Add to the nitrate an excess of alcohol. A white 
 precipitate will separate, which is ptyalin mixed with inorganic 
 salts. To free it from these, dissolve in a little water and pre- 
 cipitate with absolute alcohol. Repeat this operation if necessary. 
 Dry it over sulphuric acid. 
 
 170. Test the aqueous solution of ptyalin. It is not precipi- 
 tated by nitric acid like albumin, nor does it give the xantho- 
 proteic reaction. It can be precipitated after a time by basic lead 
 acetate, the filtrate being without action on starch. 
 
 171. Try the reaction of saliva to litmus and to 
 phenol-phthalein. Normally it is alkaline with the former 
 and neutral with the latter. Since alkaline salts affect 
 litmus while they do not change the color of phenol- 
 phthalein,, it is evident that here no free alkalies are pres- 
 ent, but that the reaction is due to alkaline salts. 
 
 172. In a test-tube treat about 5 cubic centimeters 
 of a 1 per cent, solution of starch (made as in 6) with a 
 few drops of saliva and set it in water of body temperature. 
 From time to time remove a drop and test it with iodin 
 solution. Notice the change of color from the blue first ob- 
 
70 THE SALIVA. 
 
 tained through purple and red to yellow. Then the liquid 
 will reduce Trammer's reagent. Hence the starch has 
 undergone hydrolysis, passing through the dextrins to 
 sugar (maltose). 
 
 173. Try the same experiment with unboiled starch. 
 Glucose is not formed, or, if at all, only after a long time 
 and in small amounts. 
 
 174. Try the effect of dilute acids by diluting 1 
 cubic centimeter of concentrated hydrochloric acid with 
 150 cubic centimeters of water, then adding to the saliva 
 an equal volume of the dilute acid. This makes the acidity 
 of the whole about the same as that of the gastric juice. 
 Let the acidified saliva act on boiled starch as before. No 
 glucose is produced. 
 
 175. Place 2 cubic centimeters of saliva in each of seven test- 
 tubes, adding 2 cubic centimeters of starch solution; both of these 
 are to be measured by a pipette. From a burette containing 0.1- 
 per-cent. HC1 run into the separate tubes 4 cubic centimeters, 2 
 cubic centimeters, 1 cubic centimeter, 0.5 cubic centimeter, 0.2 cubic 
 centimeter, and 0.1 cubic centimeter. The last tube receives no 
 acid. Let them all stand in water at body temperature. Observe 
 the changes in appearance of the solutions. At intervals remove 
 a drop of liquid from each tube by the pipette and test it with a 
 drop of iodin, making notes of the speed of digestion. When 
 a blue color is no longer produced, Trommer's test for maltose 
 can be made. Calculate the strength of acid contained in each 
 tube. 
 
 176. Place about 4 cubic centimeters of saliva in each of 
 four test-tubes. Add from a burette or cylindrical pipette con- 
 taining 1-per-cent. sodium carbonate 1 cubic centimeter, 0.5 cubic 
 centimeter, 0.25 cubic centimeter, and 0.1 cubic centimeter re- 
 spectively, warming with boiled starch in a beaker of water and 
 noting results as before. The alkali retards digestion, but does 
 not prevent it. 
 
 177. Try the effect of free and combined acid on ptyalin as 
 follows: In each of two tubes mix about 4 cubic centimeters of 
 
THE GASTRIC JUICE. 71 
 
 saliva with a few drops of a strong solution of albumin. Use for 
 an indicator two or three drops of tropseolin, which turns red 
 with free acid, but remains yellow if the acid is combined with 
 a proteid. To each tube add very dilute hydrochloric acid, to the 
 first enough to produce a permanent red color, to the second not 
 enough for this. Try their digestive power on starch as before. 
 The trace of free acid has destroyed the enzyme; the acid com- 
 bined with the albumin hinders digestion but does not wholly 
 prevent it. 
 
 178. In each of a series of test-tubes place 2 to 3 cubic centi- 
 meters of saliva, add half as much of the starch solution and a 
 few drops of the following antiseptics: Chloroform, sodium 
 fiuorid, salicylic acid, alcohol, mercuric chlorid, copper sulphate. 
 Let the tubes stand in water at 38 , occasionally removing a drop 
 and testing this for unaltered starch with iodin solution. Since 
 mercury salts form a compound with iodin it will be necessary to 
 use more of the reagent in this instance. When no more remains 
 Trommels test will show the presence of maltose. The antisep- 
 tics do not prevent digestion except the metallic salts which unite 
 with the albuminous substances. 
 
 THE GASTRIC JUICE. 
 
 The gastric juice, secreted by the glands of the stom- 
 ach, differs from the other digestive fluids in having an 
 acid reaction. It is a clear, thin liquid containing, as in- 
 organic constituents, principally the chlorids and phos- 
 phates of the alkalies and of calcium and magnesium. 
 There is more hydrochloric acid than can unite with the 
 bases, and this must consequently he in the free state. The 
 most important of the organic substances are two enzymes, 
 or unorganized ferments: pepsin and rennin. 
 
 The acidity of the gastric juice is caused principally 
 by the free hydrochloric acid, but may be at times due 
 to the acid phosphates, or to the organic acids: lactic, 
 butyric, and acetic. The hydrochloric acid and acid phos- 
 
72 THE GASTRIC JUICE. 
 
 phates are present in the normal juice. The lactic acid 
 may be found in the first stages of digestion, especially 
 when the food contains much of the carbohydrates, but 
 is not normally found after digestion has proceeded more 
 than half an hour. The acetic and butyric acids are not 
 normally present. 
 
 The free hydrochloric acid appears to be formed from 
 the chlorids which are taken with the food. Its forma- 
 tion has been explained as due to the action of disodium 
 phosphate upon calcium chlorid and also to the decomposi- 
 tion of sodium chlorid by a weak acid, like carbonic acid. 
 Neither explanation, however, is perfectly satisfactory. A 
 free acid is necessary for the digestion of the nitrogenous 
 foods by the pepsin, and this is one of the offices of the 
 hydrochloric acid. Recent researches have indicated that 
 one of its most important functions is the prevention of 
 fermentation in the stomach. The mineral acids have 
 antiseptic powers even in such dilution as that of the hy- 
 drochloric acid in the gastric juice; that is, from 0.2 to 
 0.3 per cent. Such a solution will, for several days, pre- 
 vent putrefaction in animal matter, like chopped meat, 
 which would otherwise soon commence to decay. It will 
 also destroy the bacteria of many infectious diseases, 
 though some of these are not affected when in the form 
 of spores. 
 
 The effects of increased fermentation are seen in cer- 
 tain pathological conditions where the secretion of hydro- 
 chloric acid is diminished or stopped. They are especially 
 noticeable in the case of food containing large quantities 
 of carbohydrates. The sugar which is formed by the saliva 
 may be changed by the ferments into different acids: 
 
 C 6 H 12 6 2C 3 H 6 3 . 
 
 cuoel sS lactic acid 
 
GASTRIC DIGESTION. 73 
 
 By further fermentation the lactic acid undergoes this 
 change: 
 
 2C 3 H 6 3 = C 3 H 7 C0 2 H + 2C0 2 + 2H 2 . 
 
 butyric acid 
 
 The glucose may be fermented to alcohol: 
 C 6 H 12 6 = 2C 2 H 5 OH + 2C0 2 
 
 alcohol 
 
 and this be converted to acetic acid: 
 
 C 2 H 5 OH + 2 = CH 3 C0 2 H + H 2 0. 
 
 acetic acid 
 
 The so-called "heart-burn," which is one of the accompanying 
 symptoms of gastric fermentation is caused by the eructations of 
 gas carrying the acids up into the throat, where they cause irrita- 
 tion of the mucous membrane. The methods of treatment based 
 upon administration of hydrochloric acid, creasote, or other anti- 
 septic substances is not for the purpose of removing the acids, but 
 to stop the fermentation. Treatment with alkaline substances like 
 magnesia or sodium bicarbonate neutralizes the acids, but does not 
 prevent fermentation. 
 
 The acid phosphates may be present in the normal 
 gastric juice. When present they increase the acidity of the 
 juice or cause an acid reaction in the absence of free acids. 
 An example is the potassium compound, KH 2 P0 4 , which 
 is found in the stomach after meat has been eaten. 
 
 The pepsin can be obtained from the gastric juice or 
 mucous membrane of the stomach in a number of ways. 
 Like most of the animal ferments, it can be extracted from 
 the membrane by glycerin, and this glycerin solution can 
 be preserved for any length of time. If it is to be used 
 immediately, water containing about 0.2-per-cent. hydro- 
 chloric acid can be employed for extraction. If the dry 
 substance is desired, it may be thrown down with finely- 
 
74 THE GASTRIC JUICE. 
 
 divided precipitates of other substances, as is the case with 
 many of the ferments. 
 
 The pepsin thus obtained is probably not the pure 
 substance. It is a white or yellowish-white, amorphous 
 powder or scale when dried. It is hygroscopic in the air 
 and has a slightly saline or acidulous taste, with no offen- 
 sive odor. It is soluble in about 100 parts of water, but 
 more easily on the addition of hydrochloric acid. Because 
 of its reactions pepsin is generally regarded as a protein. 
 Its molecular composition is unknown. 
 
 Pepsin is inactive in neutral or alkaline liquids, but 
 in slightly acid fluids it dissolves coagulated albuminous 
 compounds, with the formation of proteoses and peptones. 
 It acts most rapidly with hydrochloric acid, though others 
 may be used instead. The best strength of acid for the 
 purpose varies with the kind of material to be digested from 
 0.1 per cent, to 0.3 per cent, of hydrochloric acid. Pepsin 
 from warm-blooded animals digests best at 38. It is 
 destroyed by heating the solution. Its action is hindered 
 by the presence of the products of digestion, but if these 
 are removed as fast as they are formed it will change to 
 the soluble form many thousand times its weight of albu- 
 minous material. 
 
 The second ferment of the gastric juice, rennin, is 
 always present in human gastric juice under normal condi- 
 tions. It exists in the mucous membrane in the form of a 
 zymogen, which is sometimes inactive until it is set free 
 by an acid. Hence if it is extracted by water it may not 
 give its characteristic reaction. This is especially true in 
 the case of birds. or fish. This characteristic reaction is the 
 coagulation of milk or casein in a neutral or faintly-alka- 
 line solution in which calcium salts are present. It does 
 not give the albumin reactions when in a pure state. Ben- 
 
GASTRIC DIGESTION. 75 
 
 nin is more easily destroyed by heating its solution than is 
 pepsin. The methods of obtaining it are similar to those 
 employed with pepsin. 
 
 The gastric juice acts only upon the nitrogenous con- 
 stituents of the food. The albuminous substances are first 
 changed into acid albumin by the free acid; then this is 
 decomposed by the pepsin and hydrochloric acid, forming 
 proteose, which passes into peptone. Since the process is 
 a continuous one, all these products may be found in the 
 stomach at the same time, though in the first stages the 
 acid albumin is in excess and at the last the peptones. 
 The connective tissues are digested by the gastric juice, 
 though the proteolytic ferment of the pancreas does not 
 dissolve them. The membranes which surround the fat- 
 cells are also dissolved, setting the fat free. Thus the food 
 is changed into chyme: a pulpy mass which can be readily 
 penetrated by the intestinal fluids. In the first stages of 
 digestion the saliva may continue to act on the starchy 
 materials, and during the first fifteen to twenty minutes 
 there is a formation of lactic acid, which disappears after 
 this time. Milk is coagulated, partly by the acid, partly 
 by the rennin. The absorption of the peptones commences 
 in the stomach, but the digestion is not completed here, the 
 chyme passing through the pylorus into the intestine. 
 
 179. PREPARATION OF PEPSIN. Separate the mu- 
 cous membrane of a pig's stomach from the muscular tis- 
 sue. After rinsing it with water chop it finely. Make a 
 dilute solution of hydrochloric acid by the addition of 1 
 cubic centimeter of concentrated acid 1 to 150 cubic centi- 
 meters of water. This will contain about 0.2 per cent, 
 of the pure acid. Extract the pepsin from the chopped 
 
 1 The U. S. P. acid containing about 32-per-cent. HC1. 
 
76 THE GASTRIC JUICE. 
 
 membrane by means of this dilute acid. To obtain a strong 
 solution of pepsin it is best to let it stand for twenty-four 
 hours in a cool place, though it will be found in. the solu- 
 tion in a very short time. The rennin, which is dissolved 
 at the same time, is destroyed by the pepsin on standing. 
 Filter the liquid through muslin, then, if necessary, 
 through paper. This solution can be used directly for 
 digestive experiments or for preparing the purified pepsin. 
 
 180. Purified pepsin can be obtained by precipitating it with 
 some substance which is thrown down as a finely-divided precipi- 
 tate. To effect this, neutralize the hydrochloric acid solution of 
 pepsin in the preceding experiment and acidify with phosphoric 
 acid, or extract the pepsin from the membrane with water acidified 
 slightly with phosphoric acid. Precipitate the phosphoric acid 
 solution by adding lime-water or milk of lime until the liquid is 
 alkaline. Filter off the precipitate, which contains the pepsin mixed 
 with calcium phosphate. Dissolve in water with the addition of 
 hydrochloric acid. Remove the salts by dialysis. The pepsin does 
 not pass through the membrane. A purer form is obtained by 
 repeating the solution in acid and precipitation with lime-water 
 or by precipitating with alcohol before dialyzing. If the dry sub- 
 stance is desired, the drying must be at a low temperature. This 
 may be done over sulphuric acid in a vacuum. 
 
 181. PROOF OF THE EXISTENCE OF ZYMOGENS. This is 
 based upon the facts that dilute acids convert the zymogen into 
 the enzyme and that dilute alkalies destroy the latter, but not the 
 former. 
 
 Dilute 5 cubic centimeters of the glycerin extract of the gas- 
 tric mucosa from a recently killed animal in a test-tube (A) with 
 an equal volume of 0.2-per-cent. hydrochloric acid. In another 
 tube (B) dilute the glycerin extract with as much water. Warm 
 both for 15 minutes at 38; then add to each one-half of its volume 
 of 1-per-cent sodium carbonate and warm at 38 for half an hour. 
 Carefully neutralize them and add enough hydrochloric acid to 
 make 0.2 per cent. Test for the presence of pepsin by a shred of 
 fibrin. It is found in B, but not in A. 
 
GASTRIC DIGESTION. 77 
 
 Where the membrane has been for some time exposed to the 
 air before extracting the active enzyme may have been formed 
 and consequently no difference be perceptible. 
 
 182. To make a pepsin solution which may be pre- 
 served, though it is somewhat impure, chop the mucous 
 membrane finely, as in the previous methods, and after 
 squeezing it in a piece of cloth to remove the water as 
 far as possible, cover it with two or three times its volume 
 of glycerin and let it stand for a week. Filter it through 
 a piece of muslin, pressing out the glycerin. This gives a 
 permanent solution which can be used at any time for 
 digestive experiments. 
 
 183. In each of a series of test tubes standing in a beaker of 
 water at 38 place a little pepsin solution and a shred of fibrin. 
 Then add to (a) lactic acid up to 0.8 per cent.; (b) oxalic acid up 
 to 1 per cent.; (c) hydrochloric acid to 10 per cent; (d) hydro- 
 chloric acid to 0.2 per cent. ; but wrap the fibrin tightly with fine 
 thread so that it cannot swell, (e) 0.2-per-cent. hydrochloric acid. 
 Note the speed of digestion. 
 
 184. Make an artificial gastric juice by preparing 
 a 0.2-per-cent. solution of hydrochloric acid as in Experi- 
 ment 179, and adding to this a small quantity of the 
 glycerin solution of pepsin. The solution made directly 
 with the dilute acid can be used if it is fresh. To this add 
 a small handful of washed fibrin. If this is not at hand, 
 boiled egg-albumin may be substituted for it, though in 
 this case the digestion is slower. Warm the mixture care- 
 fully to about body-temperature and keep it at this point 
 until the albuminous substance has dissolved. Notice 
 that the edges first become transparent, then are dissolved. 
 Too high heat will destroy the ferment. The fibrin should 
 be nearly digested in half an hour. It may.be left for 
 
78 THE GASTRIC JUICE. 
 
 several days without danger of putrefaction provided it 
 contains 0.2 per cent, of the free acid. 
 
 When the fibrin has nearly or quite disappeared boil 
 one-half the liquid to coagulate any unchanged albumin- 
 ous compound, and filter. The filtrate contains the prod- 
 ucts of digestion: acid albumin, albumoses, and peptones. 
 The proportion of each varies with the time of digestion. 
 The amount of peptones is usually small until the pepsin 
 has acted for a considerable time. 
 
 185. Precipitate the acid albumin by neutralizing 
 carefully with very dilute sodium hydrate. Filter it out 
 and test as in Experiments 104 and 105. 
 
 186. From the filtrate precipitate the albumoses by 
 saturating the solution with ammonium sulphate by the 
 aid of heat. Filter and test the precipitated albumoses as 
 in Experiments 116 and 117. 
 
 187. The filtrate from the albumoses contains the 
 peptones. Test it according to the tests used in Experi- 
 ments 119, 121, and 122. When the biuret test is made 
 in presence of ammonium salts a large excess of sodium 
 hydrate must be used. Allow the second half of the diges- 
 tion to proceed for a week before testing for peptones, add- 
 ing hydrochloric acid if it is exhausted. 
 
 188. PREPARATION OF RENNIN. Extract the chopped mu- 
 cous membrane with 0.2-per-cent. hydrochloric acid, as in Experi- 
 ment 179. Both rennin and pepsin go into solution. Stir the 
 membrane well in the acidified water, but do not let it stand a 
 very long time, as the rennin is digested in an acid solution by the 
 pepsin. Neutralize carefully, add a small amount 'of magnesium 
 carbonate, and shake well. The pepsin adheres to the carbonate 
 and can be filtered out with it. Shake the filtrate with another 
 portion of magnesium carbonate, filter, and repeat the operation 
 until the pepsin is removed, which can be shown by the failure of 
 an acidified portion of the filtrate to dissolve fibrin. The filtrate 
 containing the rennin should coagulate milk in a few minutes. 
 
RENNIN. 79 
 
 189. In order to purify rennin, precipitate it with basic 
 lead acetate and filter. Wash the precipitate, then suspend it 
 in water, and acidify slightly with sulphuric acid. Filter, and to 
 the filtrate, which contains the rennin, add a solution of stearin 
 soap. The latter is decomposed by the acid, stearic acid being set 
 free as an insoluble precipitate. This carries the rennin with it. 
 Filter and place the precipitate, with a small amount of water, in 
 a glass-stoppered funnel. Add ether, shake, and after the ether 
 has separated above the watery solution draw off the latter. The 
 fatty acids from the decomposed soap have remained in the ether, 
 leaving the rennin dissolved in the water. 
 
 190. Test the solution or a specimen of normal or 
 artificial gastric juice for rennin by exactly neutralizing, 
 then adding it to an equal volume of milk in a test-tube. 
 Place the tube in a beaker of water at body-temperature. 
 If rennin is present the casein will be coagulated in twenty 
 or thirty minutes. 
 
 A solution of casein may be used instead of milk, but 
 in order to make it coagulate a small amount of a calcium 
 salt must be added. 
 
 For demonstrating the properties of rennin the com- 
 mercial product, which is sometimes sold under the name 
 of junket, may be employed. 
 
 191. To 5 cubic centimeters of milk add 1 cubic 
 centimeter of rennin solution and keep at body-tempera- 
 ture. Note the time of coagulating. 
 
 192.- Eepeat the above, adding 2 cubic centimeters 
 of ammonium oxalate solution to the mixture of milk and 
 rennin. This precipitates calcium from its solutions. 
 How does it influence coagulation ? Will it now coagulate 
 if calcium chlorid is added ? 
 
 193. Is the rennin, after it has been boiled, capable 
 of coagulating milk? What does this show as to its 
 nature ? 
 
80 THE GASTRIC JUICE. 
 
 194. VALUATION OF PEPSIN (U. S. P., 1900). Mix 9 cubic 
 centimeters of 10 per cent, hydrochloric acid with 291 cubic centi- 
 meters of distilled water, and in 150 cubic centimeters of this liquid 
 dissolve 0.1 gramme of pepsin. Immerse and keep a hen's egg, 
 which should be fresh, in boiling water during 15 minutes. Then 
 remove it and place in cold water. Separate the coagulated 
 albumin from the pellicle and yelk and rub it through a clean 
 sieve having 40 meshes to the linear inch. Reject the first portion 
 passing through the sieve. Weigh 10 grammes of the second, 
 cleaner portion, place it in a flask of a capacity of about 100 cubic 
 centimeters, then add 20 cubic centimeters of the diluted acid and 
 with a glass rod disintegrate the albumin. Rinse the rod with 15 
 cubic centimeters of the dilute acid, add 5 cubic centimeters of the 
 pepsin solution, cork and shake. Place the flask in a water -bath 
 kept at a temperature of 52 C. for two and one-half hours, shaking 
 every ten minutes, then remove and add 50 cubic centimeters of 
 cold, distilled water. Transfer to a narrow glass cylinder and 
 allow to stand half an hour. The deposit of undissolved albumin 
 should not be more than 1 cubic centimeter. Trustworthy results, 
 particularly in comparative trials will be obtained only if the 
 temperature be strictly maintained and if the contents of the 
 flasks be agitated uniformly and in equal intervals of time. 
 
 The relative proteolytic power of pepsin stronger or weaker 
 than that described above may be determined by ascertaining, 
 through repeated trials, how much of the above pepsin solution 
 will be required exactly to dissolve 10 grammes of coagulated and 
 disintegrated albumin under the conditions given above. Divide 
 15,000 by this quantity expressed in cubic centimeters to ascertain 
 how many parts of egg albumin one part of pepsin will digest. 
 
 In recent years the composition of the gastric juice and 
 its variations in disease are being more and more thoroughly 
 studied and the results of the observations made use of in 
 clinical work. The only obstacle to the general adoption 
 of these tests is probably the difficulty of obtaining the 
 fluid from the stomach. 
 
 With a little experience, the collection of the gastric juice 
 for testing can be easily accomplished. In order to excite the flow 
 
GASTRIC TESTS. 81 
 
 a test-meal should be given. This should be rather simple, and 
 may consist of bread or rolls with weak tea without milk. A 
 large amount of food rich in albuminous materials should be 
 avoided, as the peptones resulting from its digestion interfere with 
 some of the tests. With such a meal digestion is at its height in 
 about an hour, and the collection should be made one to one and 
 a half hours after eating. 
 
 The apparatus for withdrawing the juice is an elastic rubber 
 tube about a yard in length, having a number of small perforations 
 in the end or, if a large opening, it should be on the side, to avoid 
 injury to the mucous membrane by suction. The perforated end 
 is passed slowly down the oesophagus until it reaches the fundus 
 of the stomach, known by the resistance to its further passage. 
 The flow of the juice through the siphon is best started by press- 
 ure upon the stomach, the outer end of the tube being held lower 
 than the stomach and over a collecting vessel. By this means the 
 juice is not diluted. 
 
 Sometimes it is recommended to start the siphon by filling 
 it with water by means of a funnel, then when it is full, pinching 
 the top of the tube to close it and quickly lowering it. The first 
 part of the liquid which runs out is water and should be thrown 
 away, collecting only the last part. Otherwise the amounts of 
 constituents found must be corrected for the water, which dilutes 
 the juice. The addition of water, however, prevents accurate quan- 
 titative tests. 
 
 Before testing the juice it should be filtered through a 
 plaited filter, keeping it covered to avoid loss of water or 
 acid by evaporation. 
 
 The tests which are usually made upon the gastric 
 juice are for: 
 
 1. Eeaction. 
 
 2. Acid phosphates. 
 
 3. Hydrochloric acid. 
 
 (Lactic. 
 Butyric. 
 Acetic. 
 5. Pepsin. 
 
82 THE GASTRIC JUICE. 
 
 Others which are not so important, but the presence 
 of which may sometimes be significant, are for: 
 
 1. Starch. 
 
 2. Albuminous compounds. 
 
 3. Eennin. 
 
 4. Blood-coloring matters. 
 
 Quantative tests are valuable in the cases of: 
 
 1. Total acidity. 
 
 2. Hydrochloric acid, free and combined. 
 
 3. Organic acids. 
 
 The reaction of normal gastric juice is, of course, acid. 
 In some pathological conditions it becomes neutral or alka- 
 line. Litmus-paper can be used for the test. 
 
 To determine the total acidity of the juice filter it, 
 keeping it covered to prevent, as much as possible, evapora- 
 tion; then measure accurately 10 cubic centimeters with a 
 pipette and place it in a beaker. Add to this a few drops 
 of an alcoholic solution of phenol-phthalein, which serves 
 as an indicator to tell whether the liquid is acid or alkaline 
 during the determination, being red with alkalies and color- 
 less with acids. Then add slowly from a burette a solution 
 containing 4 grammes of sodium hydrate to the liter, stir- 
 ring continually, until the liquid is a faint pink color which 
 remains on standing a few minutes. Enough of the stand- 
 ard alkali has then been added to neutralize the acid sub- 
 stances present. Eead off this amount from the burette. 
 If no gastric juice is at hand, a solution for experimental 
 purposes can be made of a mixture of the above acids after 
 greatly diluting them. 
 
 The acid phosphates may be normally present, but 
 cannot perform the functions of the hydrochloric acid in 
 
GASTRIC TESTS. 83 
 
 digestion. Hence it is important to be able to detect them. 
 They can be distinguished from free acids from the fact 
 that they are not neutralized by calcium carbonate in the 
 cold as the free acids are. When, therefore, a gastric juice 
 is neutralized by adding to it finely powdered calcium car- 
 bonate, like precipitated chalk (which must itself be neu- 
 tral), no acid phosphates are present, but the reaction was 
 due to free acids. If the color of the litmus-paper is ob- 
 scured by the excess of calcium carbonate, this may be rinsed 
 off with distilled water. If the reaction remains acid after 
 calcium carbonate has been added, acid phosphates are pres- 
 ent. Their amount can be determined by finding the total 
 acidity of 10 cubic centimeters, then adding calcium car- 
 bonate to 15 cubic centimeters, filtering and determining 
 the acidity of 10 cubic centimeters of the filtrate. This 
 latter is due to the phosphates. The difference between 
 the two is the amount of the free acids. 
 
 195. Test the reaction of the acid phosphates 1 to 
 litmus-paper. 
 
 196. Show that they are not neutralized by calcium 
 carbonate in the cold. 
 
 197. Show that the dilute free acids (both HC1 and 
 lactic) can be so neutralized. 
 
 For a short time after food has been taken hydro- 
 chloric acid may be wanting, or present only in traces in 
 the gastric juice, without any pathological significance, 
 but in one to three hours after a meal it should be found 
 in larger amounts. It is then, under normal conditions, 
 about 0.1 to 0.3 per cent, of the weight of the juice. 
 
 *Acid sodium phosphate, NaH 2 PO 4 , can be prepared by adding 
 carefully orthophosphoric acid to common sodium phosphate until 
 it does not precipitate barium chlorid. An excess of the acid must 
 be avoided. 
 
84 THE GASTRIC JUICE. 
 
 The common tests for the detection of hydrochloric 
 acid cannot he employed in the case of the gastric juice, 
 because the soluble chlorids which are usually present will 
 respond to them. Special tests are used, most of which are 
 based upon the fact that certain organic coloring matters 
 are changed in color by a comparatively strong mineral 
 acid, like hydrochloric, even in the dilute state, but are not 
 affected by the weaker organic acids or acid salts. While 
 these methods are not absolutely accurate, they are suffi- 
 ciently reliable for clinical purposes when carefully per- 
 formed. 
 
 Many methods have been proposed for determining 
 the amount of hydrochloric acid. * Sjoqvist's method gives 
 accurate results. The juice is evaporated with barium car- 
 bonate, which contains no chlorids. The acids unite with 
 the carbonate, forming barium salts. The dry residue is 
 ignited in a crucible, when the salts of the organic acids 
 are destroyed, the barium chlorid remaining unchanged. 
 This is dissolved with hot water and the amount deter- 
 mined by a standard solution of potassium dichromate. 
 This is added until the barium is precipitated and there is 
 a slight excess of the dichromate. The amount of dichro- 
 mate used corresponds to the amount of barium present, 
 and from this the hydrochloric acid is calculated. Toepfer's 
 method is not difficult and the results are good. 
 
 198. TESTS FOR FREE HYDROCHLORIC ACID. Make 
 some 0.2-per-cent. hydrochloric solution as in Experiment 
 179, and apply the following tests: 
 
 a. Add a methyl-violet solution. The color changes 
 from violet to blue. 
 
 6. Add a solution of tropaaolin 00. The yellow color 
 is changed to a red. 
 
GASTRIC TESTS. 85 
 
 c. Add Congo red solution. The red changes to blue. 
 Lactic acid may produce a purple color. 
 
 Test-paper can be made from this reagent by dipping 
 a porous paper in the solution and drying. It can be used 
 in testing for HC1 like the solution. 
 
 d. Add to a few drops of liquid an equal volume of an 
 alcoholic solution containing 2 per cent, phloroglucin and 
 1 per cent, vanillin. Evaporate to dryness in a porcelain 
 dish on a water-bath or by carefully warming over a flame. 
 A rose-red color remains. (Gunzburg's test.) 
 
 e. To a few drops of the HC1 solution in a porcelain 
 dish add a little of an alcoholic solution of resorcin and 
 sugar. Evaporate to dryness and a red color appears. 
 (Boas's test.) (See page 221.) 
 
 199. TOEPFER'S METHOD FOR DETERMINING THE 
 FREE AND COMBINED HC1. Into each of three beakers 
 (A., B, and G) measure with a pipette 5 cubic centimeters 
 of gastric juice. Titrate each of them with decinormal 
 ISTaOH (4 grammes to the liter), using, as indicators, in 
 A phenol-phthalein, and adding the alkali until the liquid 
 is a faint red; in B 3 or 4 drops of a 1-per-cent. solution 
 of alizarin sodium sulphonate in water. The NaOH must 
 be added until the liquid is a violet color, not stopping 
 with the reddish shade. The exact shade is very nearly 
 that given by the indicator to a 1-per-cent. solution of the 
 common sodium phosphate. With C the indicator is 3 or 
 4 drops of a 0.5-per-cent. alcoholic solution of dimethyl- 
 amido-azobenzene. HC1 gives a red color with this, and 
 the NaOH is added until this disappears and the color 
 changes to yellow. 
 
 The phenol-phthalein is turned red by all the com- 
 pounds which have an alkaline reaction. A, therefore, rep- 
 resents total acidity. The alizarin sodium sulphonate re- 
 
86 THE GASTRIC JUICE. 
 
 acts with free hydrochloric acid, with organic acids, and 
 with acid phosphates ; that is, with everything except com- 
 bined hydrochloric acid. Consequently A B represents 
 the acidity due to the hydrochloric acid combined in the 
 organic form. Dimethyl-amido-azobenzene is affected only 
 by free hydrochloric acid, and this is shown in C. B G 
 gives the acidity from the acid phosphates, plus the or- 
 ganic acids. 
 
 Calculate from the amounts of sodium hydrate used 
 the percentages of each of these, remembering that one 
 cubic centimeter of the alkali contains 0.004 gramme and 
 neutralizes 0.00364 gramme of HC1. The amounts of acid 
 phosphates and organic acids must be expressed in terms 
 of an equivalent amount of HC1. 
 
 200. DETERMINATION or PERCENTAGE OF FREE HYDRO- 
 CHLORIC ACID (SJOQVIST'S METHOD). To 10 cubic centimeters of 
 filtered gastric juice in a porcelain, nickel, or better a platinum 
 crucible add enough pure barium carbonate to neutralize the free 
 acid. Evaporate with a small name to dryness; cautiously burn 
 the residue and heat it to a low, red heat. After cooling add 
 10 cubic centimeters of water, pulverize the residue, and pour oft' 
 through a filter the water containing the dissolved barium chlorid. 
 Repeat with successive portions of water until the filtrates to- 
 gether amount to about 50 cubic centimeters. Determine in this 
 filtrate the amount of barium chlorid formed from the hydro- 
 chloric acid in the following manner: Make a solution of 10-per- 
 cent, acetic acid and 10-per-cent. sodium acetate in water and add 
 3 to 4 cubic centimeters of this to the filtrate to be tested. Add 
 also to the liquid one-fourth to one-third its volume of alcohol. 
 The latter is to make the barium precipitate more completely, the 
 former solution to prevent the presence of free hydrochloric acid. 
 Add from a burette a solution of potassium dichromate containing 
 7.35 grammes per liter until it is in excess. This is shown by 
 taking out a drop from time to time on a glass rod and putting it 
 on a piece of tetra-methyl-paraphenylene dianiin paper. An ex- 
 cess of the dichromate is denoted by a blue color. When a faint- 
 
GASTEIO TESTS. 87 
 
 blue color is produced, read from the burette the amount of the 
 dichromate solution used. Calculate from this the amount of 
 acid in the gastric juice. The reactions which occur are 
 
 2HC1 + BaCO s BaCl 2 + CO 2 + H 2 
 when the acid is neutralized by the carbonate, and 
 
 2BaCL + K 2 Cr 2 T + H 2 = 2BaCr0 4 + 2KC1 + 2HC1 
 
 when the barium salt is precipitated by the dichromate. 
 
 One molecule of potassium dichromate, weighing 294, pre- 
 cipitates two molecules of barium chlorid containing four atoms 
 of chlorin. These four chlorin atoms were derived from four 
 molecules of hydrochloric acid weighing 4x36.4. Therefore, for 
 every 294 parts by weight of the dichromate used, 145.6 parts by 
 weight of hydrochloric acid were present. One cubic centimeter 
 of the dichromate solution contains 0.00735 gramme, and is, there- 
 fore, equal to 0.00364 gramme of hydrochloric acid. 
 
 The weight in grammes of the hydrochloric acid pres- 
 ent in the gastric juice in the free state or combined with 
 the albuminous compounds is, therefore, obtained by multi- 
 plying the number of cubic centimeters of dichromate used 
 by 0.00364, and, taking the weight of 1 cubic centimeter 
 of gastric juice as 1 gramme, the percentage can be cal- 
 culated. If 10 cubic centimeters of juice were used, it is 
 only necessary to multiply the weight of HC1 in this by 10 
 to give the percentage. 
 
 201. VOLUMETRIC DETERMINATION OF THE FREE ACIDS 
 AND ACID PHOSPHATEIS. To 10 cubic centimeters of filtered 
 gastric juice 1 add 5 cubic centimeters of concentrated calcium 
 chlorid solution and a few drops of phenol-phthalcin as an indi- 
 cator, then Vio normal sodium hydrate until it is neutralized, 
 when the color is a faint pink. The amount of alkali used cor- 
 responds to the total acidity of the juice. Then take 15 cubic centi- 
 
 *A mixture of HC1 and phosphates may be used if juice 
 cannot be obtained. 
 
88 THE GASTRIC JUICE. 
 
 meters more of the filtered gastric juice and add about a gramme 
 of finely-powdered calcium carbonate. Stir well and filter through 
 a dry paper. Measure 10 cubic centimeters of the filtrate into a 
 small flask and by means of a rubber bulb or aspirator (not with 
 the lungs) blow air through it to remove the carbon dioxid. Then 
 add 5 cubic centimeters of the calcium chlorid solution and phenol- 
 phthalein and neutralize with standard sodium hydrate as before. 
 Since the free acids are neutralized by the calcium carbonate, the 
 sodium hydrate used in this second determination corresponds to 
 the acid phosphates, and the difference between the two to the 
 free acid. 
 
 Subtract the number of cubic centimeters of alkali used in the 
 last determination from that used in the first. If lactic and volatile 
 acids are present and have been determined, subtract also the 
 number of cubic centimeters required to neutralize them. The 
 remainder has been used to neutralize the hydrochloric acid. Cal- 
 culate the percentage of the latter, remembering that 1 cubic centi- 
 meter of sodium hydrate equals 0.00364 gramme of hydrochloric 
 acid. 
 
 Lactic acid changes a mixture of gentian violet and 
 ferric chlorid to a green or greenish yellow. None of the 
 other normal or pathological constituents of the gastric 
 juice appear to do the same or to interfere with the use of 
 the above reagents in testing for the lactic acid. 
 
 Lactic acid can also be detected in the gastric juice by 
 the yellow color which it imparts to a solution of ferric 
 chlorid or to the amethyst solution which is produced by 
 adding ferric chlorid to carbolic acid (phenol), although 
 glucose or alcohol gives a similar color. 
 
 It is generally unnecessary to determine the quantity of 
 lactic acid. If this is desired it can be done by measuring off 10 
 cubic centimeters of the juice, diluting to about 100 cubic centi- 
 meters and distilling off the acetic and butyric acids, which can 
 be determined in the distillate. After the volatile acids have been 
 removed by distillation until the liquid remaining is about 10 or 
 20 cubic centimeters, the lactic acid can be dissolved from the 
 residue by shaking it six times with 100 cubic centimeters of 
 
GASTRIC TESTS. 89 
 
 ether, and distilling off the ether. The lactic acid remains. Dis- 
 solve in water, add a few drops of phenolphthalein as an indicator, 
 and see how much decinormal sodium hydrate (4 grammes per 
 liter) is necessary to neutralize it. Each cubic centimeter of the 
 alkali corresponds to 0.009 gramme of lactic acid. 
 
 202. ARNOLD'S TEST FOR LACTIC ACID. Use the two 
 following solutions : 
 
 No. 1. Saturated alcoholic solution of gentian-violet, 
 
 0.2 cubic centimeter. 
 Distilled water, 500.0 cubic centimeters. 
 No. 2. Solution of ferric chlorid (U. S. P.), 5 cubic 
 
 centimeters. 
 Distilled water, 20 cubic centimeters. 
 
 In a porcelain dish place 1 cubic centimeter of No. 1 
 and add No. 2 until a bluish-violet color results. To this 
 add the filtered gastric juice. Lactic acid changes the color 
 to a green or greenish yellow. 
 
 203.* To about 10 cubic centimeters of a 4-per-cent. 
 solution of carbolic acid (phenol) add a few drops of a 
 solution of ferric chlorid. Then dilute with water until 
 the color is amethyst or reddish-violet. Use this as a 
 reagent for the detection of lactic acid. The color is 
 changed to yellow by the lactic acid. 
 
 204. Test 0.2-per-cent. hydrochloric acid in the same 
 way. The solution becomes colorless ; that is, hydrochloric 
 acid gives no color of its own, hence would not conceal the 
 lactic acid if both should be present. Try it. 
 
 205. Test the lactic acid with a solution of ferric 
 chlorid so dilute that it is scarcely colored. The yellow 
 color is made stronger. 
 
 206. Show that glucose or alcohol will give a yellow 
 color with the above reagents. (Experiments 203 and 
 205.) 
 
90 THE GASTRIC JUICE. 
 
 Butyric acid can be distinguished by its odor, which is 
 that of rancid butter. It can be separated from its solution 
 by shaking with ether. The acid is more readily soluble in 
 ether than in water, and hence remains in the ether, and is 
 perceptible when the latter evaporates. It can also be re- 
 moved by distillation, as it passes off with the steam. The 
 acetic acid is also volatile, and if present it distills with the 
 butyric acid. If the steam is condensed the quantity of 
 these volatile acids can be determined by the use of a stand- 
 ard solution of sodium hydrate. Since they both have the 
 same significance, indicating fermentation, it is usually 
 unnecessary to separate them. 
 
 207. Shake 10 cubic centimeters of dilute butyric 
 acid in a test-tube with about 4 cubic centimeters of ether. 
 Pour off the ether and repeat the operation. Allow the 
 ether to evaporate, away from lights and fires, and notice 
 the odor of the acid which remains. 
 
 The presence of pepsin in a solution like the gastric 
 juice is best detected by trying its digestive power on fibrin. 
 Unless the liquid contains a proper amount of acid it must 
 be acidified so as to contain about 0.2 per cent, of hydro- 
 chloric acid. The rapidity of digestion can be best per- 
 ceived by coloring the fibrin dark red with a solution of 
 carmin in ammonia. This coloring matter is insoluble in 
 water, but is set free, coloring the liquid, as the fibrin dis- 
 solves. The depth of color denotes the amount digested. 
 The colored fibrin may be kept on hand for any length of 
 time by pressing out most of the water, then preserving in 
 glycerin or ether. 
 
 208. Stain to a deep red some shreds of washed fibrin 
 with a solution of carmin dissolved in ammonia. After 
 washing with water place these in several test-tubes; add 
 
GASTRIC TESTS. 91 
 
 to each specimens of natural or artificial gastric juice, con- 
 taining different amounts of pepsin, including one with 
 none. If they are not sufficiently acid make them so. Set 
 the tubes in a beaker of water of about body-temperature 
 and notice the setting free of the color as the pepsin acts. 
 
 The rennin is detected by neutralizing and testing 
 with milk for the power of coagulation as in Experiment 
 190. 
 
 The test for starch and its first decomposition-product, 
 dextrin, is iodin dissolved in potassium iodid. According 
 to von Jaksch, neither of these remains in the stomach in 
 normal digestion after the first hour, though they may be 
 present as long as as that when there is an excess of acid or 
 deficiency of ptyalin in the saliva. 
 
 The albuminous substances and digestive products can 
 be detected by the methods given in Experiments 185, 186, 
 and 187, and by the tests described for albuminous com- 
 pounds. 
 
 The gastric juice never normally contains blood, but 
 this is sometimes found in it or in the vomited material in 
 cases of chronic ulceration of the stomach or after poison- 
 ing by the corrosive or strongly irritant poisons. The 
 haemoglobin is usually decomposed and the haamatin which 
 results gives a dark-brown color, to the juice. It is best 
 identified by the hasmin test (Experiment 253). 
 
 It is occasionally desired to test the rapidity of absorption 
 from the stomach. This can be accomplished by determining how 
 long a time is necessary for potassium iodid to pass from the 
 stomach through the circulatory system into the saliva. About 
 5 grains of the iodid is taken in a capsule or in water, in the 
 latter case being careful to thoroughly rinse the mouth afterward, 
 latter case care being taken to thoroughly rinse the mouth after- 
 ward. The saliva is collected and tested every minute after the 
 
92 THE GASTRIC JUICE. 
 
 fifth. Place a little upon a paper dipped in starch-paste and dried, 
 or add it to a few drops of starch solution. Then add a few drops 
 of a solution of calcium hypochlorite (chlorinated lime) to set free 
 the iodin, which colors the starch blue. It should appear in the 
 saliva in from eight to fifteen minutes. If the urine is tested in 
 the same manner there should normally be a positive reaction in 
 15 to 30 minutes. 
 
 The clinical tests for hydrochloric acid given above are open 
 to some objections. Many are interfered with by large amounts 
 of albumin and peptones. If they fail in the tests of a gastric 
 juice the biuret test should be tried, and if any of these substances 
 are present they should be precipitated by a 10-per-eent. solution 
 of tannic acid and, after filtering, the liquid should be again tested 
 for hydrochloric acid. There is also a limit to the delicacy of the 
 tests, so that very minute amounts of free acid may not be de- 
 tected. These are so small when they cannot be thus detected that 
 the condition may be considered pathological. 
 
 Methyl-violet is turned blue by about 1 / 3 of a milligramme 
 of acid. Tropseolin 00 is of about the same delicacy. Congo red 
 is somewhat affected by the organic acids if they are not very 
 dilute. The test-paper made from this has the advantage of easy 
 portability and that it can be preserved and also that something 
 can be judged from the imparted color of the amount of acid 
 present. With a large percentage it becomes a blue black, and 
 with a small amount a lighter blue. Phloroglucin and vanillin 
 make a sensitive reagent, it being possible to detect with it one 
 milligramme of free acid in 10 cubic centimeters of juice. 
 
 The lactic acid test with phenol and ferric chlorid is also 
 in some cases uncertain. When it fails, however, the acid is not 
 present. Some common substances, like sugar and alcohol, give 
 the same results as the acid. WTien it is suspected that this is 
 the case, the liquid should be shaken with ether to dissolve the 
 acid, and the ether, after separation from the water, be evaporated 
 to dryness. Dissolve the residue in a little water and test it for 
 lactic acid. Arnold's test is more reliable. 
 
 The results obtained from chemical testing of gastric 
 juice or vomited material are often of great aid in diagnosis. 
 The presence of the organic acids lactic, butyric, or acetic 
 
GASTRIC TESTS. 93 
 
 more than thirty minutes after taking food indicates 
 fermentative action, usually due to a deficiency of hydro- 
 chloric acid. After the same time a failure or excessive 
 amount of hydrochloric acid can be considered pathological. 
 It may be absent in acute or chronic dyspepsia and in 
 chlorosis. It usually is not found, or is present in only 
 small amount, in carcinoma of the stomach. With dilata- 
 tion of the stomach caused by stenosis of the pylorus there 
 is often an hyperacidity, more than 0.4 per cent, of the 
 acid being present. 
 
 Make analyses of specimens of gastric juice furnished 
 by the instructors, handing in written reports on the pres- 
 ence or absence of: 
 
 1. Free hydrochloric acid. 
 
 2. Acid phosphates. 
 
 3. Lactic acid. 
 
 4. Butyric acid. 
 
 5. Pepsin. 
 
 6. Coagulable protein. 
 
 7. Non-coagulable protein. 
 
 Also quantitative determination of: 
 
 8. Free hydrochloric acid. 
 
 9. Combined hydrochloric acid. 
 
 10. Organic acids and acid phosphates. 
 In each case include in the report a full explanation 
 of the results as showing to what degree the juice is nor- 
 mal or pathological. 
 
 THE PANCREATIC JUICE. 
 
 The fluid secreted by the pancreatic gland contains 
 three ferments which aid in the digestion of the food: tryp- 
 sin, which decomposes the nitrogenous constituents; 
 
94 THE PANCREATIC JUICE. 
 
 steapsin, which acts upon the fats; and amylopsin, which 
 converts starch into glucose. The ferments occur in the 
 gland in the form of inactive zymogens, but are changed 
 to the active form a few hours after death or by the action 
 of water or acids. 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. 
 
 The trypsin dissolves fibrin and other albuminous sub- 
 stances, but differs from pepsin in that it acts in a neutral 
 or weakly alkaline medium. With trypsin the fibrin does 
 not swell and become transparent before dissolving, as is 
 the case with pepsin, nor is acid albumin formed as the first 
 stage of digestion. The principal decomposition products 
 of fibrin by the action of trypsin are, first, a globulin 1 ; then 
 albumose and peptones; then leucin, tyrosin, asparatic 
 acid, and other amido compounds. A substance called 
 tryptophan (skatol-amido-acetic acid), which gives a red- 
 dish-violet precipitate with bromin-water or chlorin, is 
 produced. The antipeptones are not affected by the tryp- 
 sin. The hemipeptones are decomposed. 
 
 The substances produced from albuminous substances 
 by the action of trypsin are: 
 
 1. Globulin. 
 
 I 
 
 2. Albumose. 
 
 I 
 
 3. Peptones (called amphopeptones). 
 
 4. Antipeptoue 5. Hemipeptone. 
 
 (not further changed). 
 
 Leucin, tyrosin, aspartic acid, tryptophan, etc. 
 
 albuminous substances do not give a globulin when 
 acted upon by trypsin; e.g., serum-albumin. 
 
PANCREATIC DIGESTION. 95 
 
 Leucin (amido-caproic acid), 
 
 (CH 3 ) 2 CHCH 2 CH(NH 2 )COOH, 
 
 and tyrosin (para-oxy-phenyl-alpha-amido-propionic acid), 
 C 6 H 4 OHCH 2 CH(KE 2 )COOH, 
 
 are formed in the decomposition of protein substances by 
 putrefaction as well as in the normal processes of digestion, 
 and hence they may be found as a result of pathological 
 processes where there is a destruction of the proteins. 
 Leucin crystallizes in the form of thin plates, which are 
 usually, when impure, grouped together into round knobs 
 or balls. These can be recognized by the aid of the micro- 
 scope. (Plate II, 12, &.) In the impure state tyrosin forms 
 aggregations of crystals which resemble those of leucin, but 
 when purified it appears as fine silky needles often gathered 
 into sheaves or balls. (Plate II, 12, c.) Tyrosin requires 
 for its solution 2454 parts of water at 20. Leucin can be 
 dissolved in 27 parts of cold water. This affords a means 
 of separating them. 
 
 209. In a series of test-tubes place a small amount 
 of pancreatic extract, prepared as in Experiment 210. Boil 
 one (A); acidify the second (B) with hydrochloric acid; 
 make the third (C) slightly alkaline with sodium carbo- 
 nate; make the fourth (D) neutral if it is not already so. 
 Place in each a shred of fibrin and set the tubes in water 
 at 40. Let a fifth tube (E) be made alkaline and stand 
 with fibrin in cold water. Note the change in the appear- 
 ance of the fibrin and the speed of digestion. If the tubes 
 are to be left for a long time putrefaction can only be pre- 
 vented by the use of some antiseptic like chloroform water 
 (chloroform, 5; water, 1000) or thymol. 
 
 Is solution caused by an enzyme? What conditions 
 most favor it? 
 
96 THE PANCREATIC JUICE. 
 
 210. Prepare an artificial pancreatic juice by ex- 
 tracting a finely-chopped pancreatic gland of a hog or ox 
 with lukewarm water. It is better to wait an hour or two 
 after killing the animal, in order to allow of the forma- 
 tion of the active ferment, or to use 0.2 per cent, salicylic 
 acid solution for the same purpose; this also prevents 
 putrefaction. Chloroform water gives an extract with 
 marked proteolytic properties: alcohol gives a strongly 
 lipolytic and amylolytic solution. Filter the extract 
 through cloth and add to the filtrate a little chloroform or 
 thymol to prevent putrefaction, which easily occurs with- 
 out an antiseptic. Place in the liquid 10 to 15 grammes 
 of fibrin, make slightly alkaline with sodium carbonate, 
 and allow it to stand at about body-temperature until it 
 has all dissolved. The products obtained will depend upon 
 the time. If the digestion is stopped too soon they will be 
 largely albumoses and peptones, but later there will be 
 more of the leucin and tyrosin. The best results will be 
 obtained by digesting for several hours, or as much as a 
 day. 
 
 When digestion is well advanced test a small portion 
 of the liquid by adding bromin-water drop by drop. A 
 reddish violet precipitate shows the presence of tryptophan, 
 which only appears after the peptone molecule has com- 
 menced to break down. It is destroyed by an excess of the 
 reagent. 
 
 Filter a second portion and cautiously add to the fil- 
 trate acetic acid until the reaction is neutral. Globulin 
 or alkali albumin is precipitated. This may be coagulated 
 by boiling and removed by filtration, then the filtrate tested 
 for the other products. To this filtrate add a few drops 
 of sulphuric acid, then crystals of ammonium sulphate 
 until it is saturated. The precipitate of albumoses can be 
 
PANCREATIC DIGESTION". 97 
 
 filtered out and the albumose reactions tried. The filtrate 
 from the albumoses contains the peptones. Try the biuret 
 test which should give a pink color. On account of the 
 presence of ammonium sulphate a large excess of the alkali 
 must be employed ; it may be more convenient to add this 
 in the solid form. 
 
 Let the remainder of the original solution digest for 
 a week in the presence of thymol or chloroform as a pre- 
 ventive of putrefaction,, then concentrate the solution to a 
 small bulk and precipitate the albuminous compounds from 
 it by the addition of about twice the volume of alcohol. 
 Filter these off and evaporate the alcohol., or distill it if 
 there is a large quantity. Concentrate the liquid on the 
 water-bath to a thin syrup, then let it stand until the 
 tyrosin crystallizes out. Examine the form of the crystals 
 under the microscope. If there is enough of the liquid to 
 filter, remove the tyrosin and let the leucin crystallize 
 from the filtrate. If there is only a small amount they 
 can both be identified with the microscope. Sketch these 
 and hand in the results. If there is a sufficient quantity 
 they may be tested by the reactions given in Experiments . 
 217-222. Eecrystallization often gives better crystals. 
 
 Steapsin, the second ferment of the pancreatic juice, 
 splits the fats into glycerin and the fatty acid with which 
 it was united. In the process of digestion, as it goes on 
 in the animal body, only a part of the fat is thus decom- 
 posed. The acid which has been set free in this manner 
 unites with the carbonate of sodium which is present in the 
 intestine, forming the sodium salt (a soap), and this serves 
 to emulsify the rest of the fats by surrounding the globules 
 with such a coating that they are not able to unite into a 
 large mass. The sodium carbonate is not able to decompose 
 the fat-molecule or to form a soap with the acid until the 
 
98 THE PANCREATIC JUICE. 
 
 latter has been set free by the ferment, nor will it emulsify 
 the fat if there is no fatty acid present, but a fine emulsion 
 is produced after the decomposition, and the rest of the 
 fat becomes thereby capable of being absorbed through the 
 walls of the intestine. 
 
 The decomposition of the fat-molecule is as follows: 
 
 C 3 H 6 (C 18 H 36 2 ) S + 3H 2 = 80 1T H,.00,H + C 3 H 5 (OH) 3 . 
 
 stearin stearic acid glycerin 
 
 The fat-splitting ferment is easily destroyed by acids; 
 hence it may not be found in a gland which has been kept 
 until it has an acid reaction. 
 
 211. Make a watery infusion of the pancreatic gland, 
 as in the trypsin digestion. If it is not already neutral 
 make it so. Add to it in a test-tube a few drops of some 
 neutral fat, like olive-oil, and let it stand for half an hour 
 in a beaker of water at about 38, shaking occasionally to 
 keep the two liquids well mixed. Then test it with a piece 
 of blue litmus-paper. It will turn the paper red from the 
 fatty acid which has been set free. 
 
 212. Mix 10 cubic centimeters of cream or rich, milk with a 
 little powdered blue litmus, shaking well until an even blue color 
 is obtained. Divide in two test tubes. To one add boiled pan- 
 creatic extract and keep at a temperature of 38; to the other 
 unboiled. The former remains blue, the latter becomes red from 
 the cleavage of the butter-fat into fatty acids and glycerin. 
 
 213. Shake, in a test-tube of water, to which a few drops 
 of sodium carbonate have been added, a few drops of olive-oil 
 which does not contain free acids. (Since the oil easily becomes 
 decomposed on standing, it may be necessary to remove the free 
 acids by first shaking with very dilute sodium hydrate solution 
 and ether. Separate the ether from the water, wash well by 
 shaking it a number of times with pure water, and allow it to 
 evaporate at a gentle heat away from a fire or lamp. The alkali 
 has united with the free acid to form a soap, and this has been 
 
PANCREATIC DIGESTION. 99 
 
 washed out by the water, leaving the fat neutral.) If the oil is 
 neutral it will form no emulsion with the carbonate. Add now 
 a drop of a fatty acid like oleic acid and shake. A fine emulsion is 
 formed immediately. 
 
 214. If a bottle of rancid oil is at hand, shake it with a weak 
 solution of sodium carbonate, and notice that it contains enough 
 of the fre'e acids to form an emulsion at once. 
 
 The third ferment of the pancreas, amylopsin, con- 
 verts starch into maltose as the ptyalin does, except that 
 its action is more energetic. Thus it acts upon raw starch, 
 which the ptyalin will not do, or at most only slowly. 
 
 215. Treat boiled and unboiled starch with a small 
 amount of the watery solution of the pancreatic ferments 
 made as before. Test for maltose by Trommels or Feh- 
 ling's test. Notice that it is found with the boiled starch 
 in a few seconds; after a longer time with the unboiled. 
 
 216. To prepare leucin and tyrosin in larger quantities, take 
 of white horn-shavings 2 parts and boil for twenty-four hours with 
 13 parts water and 5 parts of concentrated sulphuric acid, adding 
 water as it evaporates. Dilute with water, and while warm neu- 
 tralize with milk of lime. Filter, boil the precipitate several times 
 with water, and filter in order to remove all the leucin and tyrosin. 
 Unite these filtrates, and, after concentrating them by boiling, pre- 
 cipitate the calcium by the addition of oxalic acid without using 
 an excess. Filter, extract the precipitate with boiling water, 
 unite the filtrates, and evaporate until it becomes a thin syrup. 
 The last of the evaporation should be performed on a water-bath, 
 to avoid burning. Allow it to stand and crystallize. Tyrosin at 
 first crystallizes out, mixed with a little leucin. Filter, concen- 
 trate the filtrate, and allow the leucin to crystallize. 
 
 Another better way to separate the mixed substances is to 
 dissolve them in a large quantity of boiling water to which am- 
 monia has been added. To the boiling solution add basic lead 
 acetate solution until the precipitate formed is nearly white. 
 Filter, heat the filtrate to boiling, neutralize with sulphuric acid, 
 and filter while hot. After cooling, the tyrosin crystallizes and 
 
100 LEUCIN AND TYROSIN REATIONS. 
 
 can be filtered off, while the leucin remains in solution. Remove 
 the lead from the filtrate which contains the tyrosin by passing 
 hydrogen sulphid through it, filter, and concentrate the filtrate, 
 then boil it with freshly-prepared copper oxyhydrate. (This is 
 made by precipitating a solution of copper sulphate with sodium 
 hydrate and washing by decantation until it no longer has an 
 alkaline reaction.) Part of the leucin is thrown down as the copper 
 salt and a part remains in solution. The precipitate can be re- 
 moved by filtration and the leucin obtained in the pure state by 
 removing the copper by hydrogen sulphid and allowing the leucin 
 to crystallize after concentration. From the deep-blue filtrate the 
 copper salt can be obtained by evaporating it to a small volume 
 and letting it crystallize. It forms sky-blue aggregations of 
 crystals. If desired, the copper can be removed from these also 
 by hydrogen sulphid. The leucin thus obtained is not as pure as 
 the first portion. 
 
 217. Test the tyrosin with Millon's reagent. It gives a red 
 color, showing the presence in the molecule of the group C H 4 OH. 
 (Hoffman's test.) 
 
 218. To a portion of tyrosin crystals as large as half a pea 
 add, in a porcelain dish, a few drops of concentrated sulphuric 
 acid and warm on the water-bath. This forms tyrosin sulphuric 
 acid. This is diluted with water and enough barium carbonate 
 added to neutralize it, then filtered. The filtrate contains the 
 tyrosin compound, which, with a very dilute solution of ferric 
 chlorid, gives a deep-violet solution of tyrosin ferric sulphate. 
 (Piria's test.) 
 
 219. Warm the tyrosin with a mixture of formalin, 1 part; 
 water, 45 parts: concentrated sulphuric acid, 55 parts. An 
 emerald green color results. (Morner's test.) 
 
 220. Evaporate a small portion of the leucin crystals with a 
 drop of nitric acid upon platinum foil. The residue is colorless, 
 but on adding a drop of sodium hydrate becomes yellow to brown, 
 and on gently heating rolls around on the foil in the form of 
 drops. (Scherer's test.) 
 
 221. Place a few crystals of leucin in a dry test-tube and 
 heat gently. They melt with the odor of amylamin and sublime, 
 the leucin appearing on the sides of the tube as wooly flakes. 
 This may not succeed if the leucin is very impure. 
 
THE BLOOET, :r -. ^ ^ ; ; /, 101 
 
 THE BLOOD. 
 
 In the examination of the blood it is convenient to 
 consider it as composed of two parts: the corpuscles and 
 the albuminous liquid in which they are suspended, the 
 plasma. The plasma, on standing, separates into two 
 parts by coagulation, the clot or fibrin and a liquid, 
 the serum. 
 
 The reaction of the blood is alkaline from the pres- 
 ence of the carbonate and phosphate of sodium. The 
 specific gravity varies from 1.045 to 1.075, with an aver- 
 age for adult human beings of about 1.055. 
 
 The color of the blood is caused by the red corpus- 
 cles (erythrocytes). Even comparatively thin layers of 
 the blood are opaque from their presence. The coloring 
 matter (ha3inoglobin) can be set free from the corpuscle 
 by water or by many chemical reagents. The color be- 
 comes then much darker, since the light is no longer re- 
 flected from the surface of the corpuscles. This process 
 is called laking. The addition of strong neutral salt solu- 
 tions to blood turns it bright red, because of the increased 
 reflection of light from the shriveled corpuscles. 
 
 The red corpuscles of the same species of animals 
 have the same shape. The average size of those of one 
 animal of a species will be the same as that of any other, 
 although the size of the individual corpuscle may vary 
 greatly in the same animal. In most mammals they are 
 round, biconcave, non-nucleated disks. In the blood of 
 birds, amphibians, and most fishes they are nucleated and 
 more or less elliptical. A single corpuscle when seen un- 
 der the microscope has a yellowish color, not a red. The 
 size of the red corpuscles can be greatly changed by add- 
 ing water or strong solutions of neutral salts. When 
 
102 
 
 , oV ,, : : TEE 'BLOOD. 
 
 water is added it passes in by diffusion, and the corpus- 
 cle swells and may burst. Likewise by diffusion when 
 they are placed in a liquid and it contains more salts than 
 the blood, water passes out and the corpuscle becomes 
 smaller and shriveled in appearance. In order to dilute 
 the blood without changing the size a solution containing 
 0.5 to 0.6 per cent, of sodium chlorid can be used. Such 
 a solution is called isotonic; that is, its osmotic pressure 
 is the same as that of the fluid within the corpuscles. 
 
 Freshly-drawn blood, when allowed to stand undis- 
 turbed, in a few minutes becomes thickened to a dark- 
 red gelatinous mass. If the coagulation is slow the red 
 corpuscles have time to sink and collect with the fibrin 
 in a mass in the lower part of the vessel. The serum is 
 squeezed out of the mass and surrounds it, above and at 
 the sides. If the blood is beaten during the time of co- 
 agulation the fibrin does not separate as a gelatinous 
 substance, but in stringy masses, which have a high de- 
 gree of elasticity. The coagulation can be prevented or 
 hindered by cold and by the addition of neutral salts, 
 peptones, and some other substances. 
 
 To determine the number of red corpuscles the apparatus of 
 Thoma-Zeiss may be employed. This consists of two pieces: a 
 pipette for measuring and diluting the blood and a cell for count- 
 ing the number with the aid of a microscope. The lower part of 
 the pipette is a graduated capillary tube for measuring the blood. 
 Above is a bulb which, being filled to the mark with the diluting 
 fluid, dilutes 200 times the blood which was measured in the capil- 
 lary tube. The counting-cell when covered with a cover-glass gives 
 a layer of blood 0.1 millimeter in depth. On the bottom of the cell 
 are ruled sixteen squares, each Vwo of a square millimeter in area. 
 They are surrounded by two rows of smaller rectangles. The 
 volume of blood over each of these squares, then, must contain 
 1 /4ooo cubic millimeter. If the number of corpuscles which are con- 
 tained in this V^ cubic millimeter is determined, the number in 
 
THE RED CORPUSCLES. 103 
 
 any volume can be found by multiplication. Several different 
 solutions have been proposed for the dilution of the blood, one of 
 the most convenient being a 3-per-cent. solution of sodium chlorid. 
 For clinical purposes the blood is best obtained from the tip of the 
 finger on the lobe of the ear. For testing the method defibrinated 
 blood from the slaughter-house may be employed. The dilution 
 may be made twice as great by filling the capillary only to the 0.5 
 mark and diluting as before. In this case the number of corpuscles 
 in each square must be multiplied by 8000 to give the number in a 
 cubic millimeter. 
 
 The average number of red corpuscles normally present is 
 5,000,000 per cubic millimeter in the case of a man, and 4,500,000 per 
 cubic millimeter of a woman. This may vary greatly in disease. 
 
 222. DETERMINATION OF THE NUMBER OF RED BLOOD- 
 CORPUSGLES. Fill the capillary tube of the pipette with blood 
 to the mark 1, drawing it in slowly by suction with the mouth 
 and avoiding the presence of air-bubbles in the tube. Quickly 
 wipe dry the end of the pipette with filter-paper and draw in 
 the diluting salt-solution (3 per cent.) to the mark 101. Close 
 the lower end of the pipette witl* the finger, then compress the 
 rubber tube at the upper end of the pipette and shake to thoroughly 
 mix the fluids. The small glass bead in the bulb aids in this mix- 
 ing. Allow the salt solution to flow out of the capillary, place a 
 drop of the diluted blood on the ruled side and cover it with a 
 cover-glass so that air-bubbles are not inclosed. When the cor- 
 puscles have come to rest, count the number in all sixteen squares. 
 Count those upon the upper and left hand line of each square as 
 belonging to the square. Use an objective which will magnify 
 100 to 200 diameters. The squares should be taken in some definite 
 order to avoid counting the corpuscles in the same one more than 
 once. The average number of corpuscles multiplied by 4000 gives 
 the number in a cubic millimeter of the diluted blood. Multiply 
 this by 100 for the original blood. Since a slight error in the 
 average of each square would make a considerable error when 
 multiplied by 4000, it is advisable to repeat the filling of the cell 
 several times before taking the average. After using, the pipette 
 should be rinsed out first with the diluting salt solution, then suc- 
 cessively with water, alcohol, and ether, and finally dried by blow- 
 ing dry air through it. 
 
104 THE BLOOD. 
 
 223. SEPARATION OF THE CORPUSCLES FROM THE 
 SERUM. Add 30 cubic centimeters of a saturated solution 
 of sodium chlorid to 270 cubic centimeters of water, then 
 mix with it 30 cubic centimeters of blood, which has been 
 defibrinated by beating it while freshly drawn. Pour it 
 into a flat-bottomed, shallow dish and allow it to stand 
 until the corpuscles have settled. Decant the serum, and, 
 after mixing the corpuscles with more salt solution as be- 
 fore, allow to settle and decant again. By this means the 
 serum can be entirely removed. Preserve the first portion 
 of the solution for testing in Experiments 234, 236, and 
 237. If any of the corpuscles have been destroyed it may 
 be reddened. 
 
 224. DETERMINATION OF THE SPECIFIC GRAVITY OF 
 BLOOD. Prepare a mixture of benzol and chloroform, of 
 which the specific gravity when tested with a sensitive 
 hydrometer shall be somewhat less than that of blood. 
 Into this mixture allow a drop of blood to fall. Freshly- 
 drawn blood for example, from the end of the finger is 
 best, though the method can be demonstrated by the use 
 of defibrinated blood. When the blood has sunk, add chlo- 
 roform, drop by drop, stirring meanwhile, until the drop 
 floats in the midst of the liquid; that is, it has the same 
 specific gravity. Then filter out the blood, covering the 
 funnel to prevent evaporation, and determine the specific 
 gravity of the mixture by means of a sensitive hydrometer. 
 The mixed liquids can be preserved for future tests. 
 
 225. Wind a string around one of the fingers until it 
 is congested, then prick it at the root of the nail with a 
 needle or knife, sterilized by passing through a flame. 
 Test the reaction of the blood, using a piece of neutral, 
 glazed litmus-paper. After rinsing off the blood with a 
 little distilled water the paper is blue. 
 
THE BLOOD. 105 
 
 226. To a little defibrinated blood in a test-tube 
 add an equal volume of water, and notice the change in 
 color, due to laking. 
 
 227. Show that the blood can be laked by adding 
 a few drops of (a) ether, (b) chloroform, (c) solutions of 
 bile salts, (d) by freezing and thawing, (e) by heating to 
 65. 
 
 228. To another portion add as much saturated salt 
 solution and observe that the color becomes brighter. 
 
 229. Examine each of these under a microscope and 
 compare the appearance of the corpuscles with those of 
 the fresh blood. 
 
 230. Place in a test-tube 5 to 10 cubic centimeters of a cold, 
 saturated solution of sodium sulphate. Open the carotid of a 
 rabbit or other small animal and allow twice as much blood to 
 flow into the tube. Collect as much more in a clean, perfectly dry 
 tube and allow both to stand twenty-four hours. In the first tube 
 there is no coagulation, but the corpuscles settle toward the bot- 
 tom. In the second the corpuscles are mostly held in the mass of 
 coagulated fibrin from which drops of serum are pressed out. 
 
 231. Place in a beaker or flask 3 to 4 cubic centimeters of a 
 4-per-cent. solution of neutral potassium oxalate. Insert a 
 cannula into the carotid artery of a rabbit or cat and let 10 
 times the above volume of blood flow into the solution without 
 first coming into contact with the glass. Let it stand in a cool 
 place or whirl it in a centrifuge until the corpuscles have settled. 
 The clear liquid above is oxalate plasma. Pour it into a test- 
 tube and notice that it does not clot. Add a few drops of calcium 
 chlorid solution when a clot forms. (Compare this with the ac- 
 tion of remain.) 
 
 232. From the oxalate plasma precipitate the fibrinogen 
 with an equal volume of saturated salt solution. Apply to it the 
 protein reactions. 
 
 233. Notice that the serum does not clot with calcium 
 chlorid. Why not? 
 
106 THE BLOOD. 
 
 234. Separate the serum from blood by collecting the 
 freshly-drawn blood in a shallow vessel and letting it stand 
 covered until it has coagulated and the serum is pressed 
 out by the contraction of the coagulated mass. 
 
 235. Show that the serum contains albumins by test- 
 ing a diluted solution by 
 
 1. Heat. 
 
 2. Biuret test. 
 
 3. Xanthoproteic test. 
 
 236. Separate the proteins of the serum by adding 
 to a portion an equal volume of a saturated solution of 
 ammonium sulphate. This precipitates the serum glob- 
 ulin. Filter it out, dissolve it in water, and try the glob- 
 ulin reactions. Into the nitrate stir enough powdered am- 
 monium sulphate to completely saturate it. Serum albu- 
 min falls and can be filtered out, dissolved in water, and 
 tested. Test the filtrate for proteoses with acetic acid and 
 potassium ferrocyanid. They are not present. Peptones 
 are also absent. 
 
 237. Slightly acidify a fresh portion of the serum 
 and boil to coagulate the proteins. Filter and apply Trom- 
 mer's test to the filtrate. Does the serum contain glucose ? 
 
 238. Prepare fibrin by beating freshly-drawn blood 
 with a fork or a bundle of switches. When it has coagu- 
 lated, pour off the liquid and preserve it for further tests. 
 Wash the fibrin, at first in water to which a little salt has 
 been added, then in clear water. Break up the large clots 
 and continue the washing until the coloring matter is re- 
 moved. If it is desired to keep it, it can be preserved in 
 a 1-per-cent. solution of corrosive sublimate. 
 
 239. After noticing the structure and elasticity of 
 fibrin apply the following tests: 
 
HEMOGLOBIN AND ITS DERIVATIVES. 107 
 
 1. Xanthoproteic. 
 
 2. Insolubility in hot and cold water. 
 
 240. Let a shred of washed fibrin stand for an hour 
 or two in a test-tube with 0.1 per cent. HC1. It swells up 
 and becomes transparent, but does not dissolve. Warm 
 at 60 to 70 for several hours; filter and test the filtrate 
 for acid albumin by neutralizing. 
 
 241. Dissolve a little dried blood in nitric acid with 
 the aid of heat. Filter and test the filtrate for iron with 
 potassium ferrocyanid, which produces a blue color. 
 
 HEMOGLOBIN AND ITS DERIVATIVES. 
 
 I. HEMOGLOBIN: Composed of an albuminous substance 
 
 and an iron compound, haemochromogen. 
 
 II. OXYHEMOGLOBIN: A compound of oxygen with haemo- 
 
 globin. 
 
 III. METHEMOGLOBIN: Composition same as oxyhaemo- 
 
 globin. Different arrangement of the atoms. 
 
 IV. HEMATIN: The iron compound united with albu- 
 
 minous substance in oxyhsemoglobin. Haemochro- 
 mogen plus oxygen. 
 
 V. HEMIN: A compound of haematin with HC1, one 
 
 molecule of each. 
 
 VI. HEMOCHROMOGEN: Haemoglobin minus its albumin- 
 
 ous constituent. With oxygen it gives haematin. 
 
 VII. HEMATOPORPHYRIN: Formed by the removal of 
 iron from haematin, haemin, etc. 
 
 HEMOGLOBIN. 
 
 Haemoglobin, sometimes called reduced haemoglobin, 
 is the coloring matter of venous blood. It contains iron, 
 besides the elements which enter into the composition of 
 albuminous substances. The constitution of the mole- 
 
108 THE BLOOD. 
 
 cule has not yet been determined, although the formulae 
 of some varieties are known approximately; but it is very 
 complex, containing a large number of atoms. Like the 
 other members of the protein compound class, it contains 
 an albuminous substance, united in this case with an iron 
 compound. It easily unites with oxygen from the air, tak- 
 ing up one molecule of oxygen for each molecule of haemo- 
 globin and forming the readily-decomposable compound, 
 oxyhaemoglobin. It also forms compounds with carbon 
 monoxid (CO), nitric oxid (NO), and sulphur, all of which 
 are similar to its oxygen compound. 
 
 Haemoglobin can be obtained from oxyhaemoglobin 
 by the removal of the oxygen. This may be effected either 
 by a vacuum, by driving it out by means of a gas which 
 itself does not act on the blood, or by the use of some 
 chemical reducing agent. It is obtained in the crystalline 
 state with more difficulty than its oxygen compound. It 
 is soluble in water, giving a reddish-purple solution. 
 
 The spectrum of haemoglobin is of great value in test- 
 ing for its presence, and the same might be said in the case 
 of the haemoglobin derivatives. When a dilute solution of 
 blood is held before the slit of a spectroscope, the tube being- 
 turned toward a window, the solar spectrum, consisting of 
 the seven primary colors crossed by fine dark lines, is seen, 
 and in addition one or more dark bands, which are due 
 to the coloring matters of the blood. That of haemoglobin 
 has one broad band with rather indistinct edges lying be- 
 tween the D and E lines of the solar spectrum. If the 
 liquid in the tube be shaken with air oxyhaemoglobin is 
 formed, which has two dark bands. For clinical purposes 
 the direct-vision or pocket spectroscope will probably be 
 found to be the most convenient form of instrument. 
 (Figs. 1 and 2 on Plate IV show the spectra.) 
 
HEMOGLOBIN. 109 
 
 A fresh alcoholic solution of guaiacum when oxidized gives 
 u blue color. Many oxidizing agents, like hydrogen peroxid and 
 oil of turpentine which has absorbed oxygen by standing for some 
 time exposed to the air, will not act on the guaiacum alone, but 
 will do so if haemoglobin or its compounds are present to serve as 
 a carrier of oxygen. This is often used as a test for blood, but is 
 only useful in a negative way, for protoplasm will give the same 
 reaction. It can consequently be obtained from any cell, like pus 
 or mucus, or even from such substances as the potato. If the 
 reaction fails, however, there can be no blood present. 
 
 Since haemoglobin is a proteid, containing an albu- 
 minous substance, it will give the general reactions of 
 these compounds. 
 
 The determination of the amount of haemoglobin in 
 the blood is made by comparing the color of the diluted 
 blood with a solution containing a known weight of haemo- 
 globin, or with some other colored liquid or solid. For 
 making a standard solution of haemoglobin the recrystal- 
 lized substance is used (Experiment 257). A solution of 
 this can be preserved in a corked bottle or sealed tube for 
 an indefinite time. The strength is ascertained by evapo- 
 rating to dryness a given volume, and weighing the residue 
 of haemoglobin. This method gives accurate results. 
 
 242. DETERMINATION OF THE AMOUNT OF HEMO- 
 GLOBIN IN BLOOD. Dilute a solution* containing a known 
 weight of haemoglobin with distilled water until it is a very 
 light-red color. Dilute in the same way the blood to be 
 tested until the color is the same. For comparison of 
 colors the two solutions may be placed in flat-bottomed 
 tubes of colorless glass (Nessler tubes) or in small, flat, 
 glass boxes, the breadth of which between the parallel sides 
 is not more than a centimeter. Reckon from the amount 
 of dilution the amount of haemoglobin compared with the 
 standard solution, also the absolute weight and percentage. 
 
110 THE BLOOD. 
 
 For clinical purposes a convenient instrument for the determi- 
 nation of the amount of haemoglobin in the blood is that of Fleischl, 
 called an haemometer. It consists of a short, vertical cylinder for 
 holding the blood, separated by a partition into two compartments; 
 a long movable wedge of ruby glass under one compartment for 
 a standard of color, and a white surface below for reflecting the 
 light up through the wedge and cylinder to the eye of the observer. 
 The amount of haemoglobin is found by filling one compartment 
 of the cylinder with diluted blood, and the other, over the ruby 
 prism, with water. The prism is then moved until the depth of 
 color is exactly the same as that of the blood, when the percentage 
 of haemoglobin compared with the normal amount can be read from 
 the scale. The following is the process: 
 
 Use for a light a lamp or yellow gas-flame, not an incandes- 
 cent light or daylight. Any blood may be used for practice. In 
 clinical cases use that obtained from the tip of the finger by the 
 aid of a lancet. After making the incision force out a drop of 
 blood by gentle pressure. Measure off the required volume of blood 
 (6V 2 cubic millimeters) by filling the small glass tube, open at 
 both ends and mounted on a handle, which accompanies the 
 haemometer. This is accomplished by holding it horizontally and 
 dipping one end into the drop. Wipe carefully all blood from the 
 surface of the tube. This will be more readily done if the surface 
 of the tube is kept slightly greasy by being preserved in an oily 
 piece of chamois. The blood must exactly fill the tube, having 
 neither a convex nor a concave surface at the open ends. 
 
 The compartment over the ruby-glass prism is to be filled 
 with distilled water by means of a pipette and the other one not 
 more than a quarter full. Into the latter the open glass measuring 
 tube is dipped before the blood has coagulated, and the haemoglobin 
 is dissolved by moving the tube back and forth so as to wash out 
 the blood. Then rinse off the tube into the blood solution by the 
 use of a few drops of water from the pipette. Fill the compart- 
 ment from a half to three-fourths full of water and stir well with 
 a wire or with the handle of the measuring tube. Mix the water 
 with the blood until no turbidity is seen and until it is evident 
 that the fluid in the angles is completely incorporated with the rest. 
 Now drop water from the pipette upon the blood solution until it, 
 as well as the water in the other compartment, comes exactly to 
 
OXYH^MOGLOBIN. Ill 
 
 the top of the division between the compartments. If the tip of 
 the pipette is placed slightly below the surface and the water flows 
 slowly it will not mix with the solution of blood below. This is 
 advisable in order to prevent the possibility of any of the haemo- 
 globin's passing over into the other compartment. If both com- 
 partments are filled to the top of the separating partition, so that 
 there is no meniscus at the top of the liquid, they appear, when 
 looked at from above, to be separated only by a narrow black line. 
 A little grease on the top of the partition will help to prevent a 
 mixing of the two liquids. (Some authors recommend that the 
 water be allowed to rise above the partition and a cover glass be 
 then laid on top to prevent currents.) 
 
 Place the instrument with the large screw at the right, turn 
 the reflector so as to illuminate the solution, shade the eye from 
 other light, and move the ruby prism so that the shades of red in 
 the two compartments are the same. The figure in the scale, 
 opposite the middle of the cylinder, gives the percentage of haemo- 
 globin as compared with the average amount found in normal 
 human blood. 
 
 With Dare's hsemoglobinometer but a single drop of blood 
 is necessary. This is drawn by pricking the skin with a needle 
 and allowed to run between two parallel plates of glass fixed at 
 a short distance apart. Comparsion is made with the color on 
 the margin of a disk which can be rotated by means of a milled 
 screw until the same shade is attained. Yellow light is trans- 
 mitted through the blood and colored glass from a candle held 
 opposite; the percentage of normal haemoglobin is read from a 
 scale on the edge of the revolving disk. The operation is simpler 
 and more expeditious than that of Fleischl. 
 
 OXYH^EMOGLOBIN-. 
 
 The crystalline form of oxyhsemoglobin differs when 
 its source is from different species of animal: from human 
 blood being in long prisms; from the squirrel, flat, six- 
 sided plates; and from the guinea-pig, tetrahedra. It 
 can be easily crystallized from the blood of the dog, guinea- 
 
112 THE BLOOD. 
 
 pig, and rat, but with more difficulty from human blood or 
 ox-blood. The color of the crystals is a bright red, and 
 their solution is a much brighter red than that of haemo- 
 globin, which, when pure, approaches a black. 
 
 Oxyhaemoglobin is formed by the union of a molecule 
 of oxygen with one of hemoglobin, and it can without 
 difficulty be changed back into haemoglobin. The molecule 
 of oxygen is in this compound very loosely united. Oxy- 
 haemoglobin may be also considered as composed of an iron 
 compound, haematin, with an albuminous substance. It is 
 decomposed into these two substances when its solution is 
 heated, this change being hastened by acids or alkalies. 
 When heated with glacial acetic acid and a little sodium 
 chlorid it is decomposed, the haematin uniting with the nas- 
 cent HC1 formed at the same time and giving haemin, the 
 microscopic crystals of which have a brown color and char- 
 acteristic form. This is one of the best proofs for the 
 presence of blood, although it does not distinguish between 
 the different kinds. No other known substance gives 
 crystals of this color and shape. 
 
 The spectrum of oxyhaemoglobin consists of two dark 
 bands: a narrow one at the right of the D line in the 
 yellow and a broader and less distinct one in the green 
 at the left of the E line of the solar spectrum. They can 
 be made to vary in width as well as distinctness by mak- 
 ing the solution more or less dilute. They can be per- 
 ceived when it contains 1 gramme of oxyhaemoglobin in 
 10 liters of water; that is, 1 part in 10,000. If to this 
 solution is added a few drops of ammonium sulphid, which 
 has a reducing action, the oxygen is removed and in a few 
 minutes the one broad band of haemoglobin is seen in that 
 part of the spectrum between the two oxyhsemoglobin 
 
METH^MOGLOBIN. 113 
 
 lines. It is not so distinct as are the two lines and the 
 solution may have to be strengthened to make it plainly 
 visible. The j;wo lines reappear upon shaking the solu- 
 tion with air, the oxyhaemoglobin being formed again. 
 
 METH^EMOGLOBIN. 
 
 In its percentage composition methaemoglobin differs 
 very little, if any, from oxyhaemoglobin, and probably is 
 formed by a rearrangement of the atoms in the oxyhaemo- 
 globin molecule. It is produced whenever oxyhaemoglobin 
 is dried in the air at ordinary temperatures or when it is 
 acted upon by weak acids. Certain oxidizing agents also 
 will convert oxyhaemoglobin into metha3moglobin. It is 
 also found in transudations and cystic fluids which con- 
 tain blood; moreover in the urine during hgematuria and 
 haemoglobinuria, as well as in the blood itself in certain 
 cases of poisoning or after a large destruction of blood- 
 corpuscles by burns of the skin. 
 
 In the methasmoglobin molecule the oxygen is more 
 firmly attached than in oxyhaemoglobin,, being removable 
 neither by a vacuum nor by another gas. Like oxyhaemo- 
 globin, however, it is changed by weak acids or alkalies 
 into haematin and an albuminous substance. Like oxy- 
 haemoglobin, too, it is converted by reducing agents or by 
 putrefaction, where reducing forces are at work, back into 
 hemoglobin. 
 
 Methasmoglobin crystallizes in brownish-red needles 
 or sometimes in plates. It is easily soluble in water, giv- 
 ing a brown solution, which becomes red on the addition 
 of an alkali. The spectrum of the alkaline methaemoglobin 
 solution has two bands much similar to those of oxyhaemo- 
 globin, one on the D line, the other near the E line. 
 
114 THE BLOOD. 
 
 HEMATIN AND H^EMIN. 
 
 Haematin is the iron compound which is combined 
 with an albuminous substance to form oxyhaemoglobin. 
 It is set free whenever oxyhaemoglobin is decomposed by 
 the action of the gastric or pancreatic juice or by an acid. 
 It is consequently found in the intestine after the eating 
 of meat; also in the stomach after poisoning by a mineral 
 acid. 
 
 The formula is given as: 
 
 C 34 H 35 N 4 Fe0 5 or C 32 H 32 N 4 Fe0 4 . 
 
 It may be obtained from haemin, its compound with hy- 
 drochloric acid. It is an amorphous substance, dark brown 
 or bluish black. 
 
 Hsemin is composed of haamatin and hydrochloric 
 acid, probably one molecule of each. It forms micro- 
 scopic crystals which, in a large amount, have a blue- 
 black color. Under the microscope they are brown, rhom- 
 bic prisms. They are sometimes separate, but two are often 
 crossed or several are collected in clusters or rosettes. 
 (Plate I, 3.) They are insoluble in water, but dissolve 
 in alkalies, the haBmatin being set free. They are often 
 called Teichmann's crystals, and are important in proving 
 the presence of blood. 
 
 CARBONIC OXID HEMOGLOBIN. 
 
 When carbonic oxid, either pure or mixed with other 
 gases, is breathed or passed through blood, it unites with 
 the hemoglobin, forming CO-hsemoglobin: a compound 
 similar to oxyhaemoglobin. It is, however, a more stable 
 compound, the oxygen being unable to drive out the CO 
 
HEMOGLOBIN DERIVATIVES. 115 
 
 and take its place. Consequently the haemoglobin is ren- 
 dered useless as a carrier of oxygen, and cases of poisoning 
 by coal-gas, which contains carbon monoxid, are often fol- 
 lowed by fatal results. The compound has a two-band 
 spectrum much like that of oxyhsemoglobin, but differs in 
 not being changed to hemoglobin by reducing agents. The 
 crystals are similar to those of oxyhaBmoglobin, but more 
 of a bluish red. When mixed with strong sodium hydrate 
 solution blood containing carbonic oxid gives a red mass, 
 while pure blood turns brown, with a greenish cast. 
 
 HEMOCHROMOGEN. 
 
 As the oxyhaemoglobin by the action of acids or alkalies is 
 decomposed into an albuminous substance and hsematin, so by the 
 same agencies haemoglobin gives an albuminous substance and 
 hsemochromogen. The latter in the presence of free oxygen is 
 converted into hsematin. Hence, as we should expect, by the re- 
 moval of oxygen from hsematin by the aid of reducing agents we 
 obtain hsemochromogen. The spectrum of hsemochromogen, in an 
 alkaline solution, has two bands similar to those of oxyheemo- 
 globin, but a little farther toward the blue. The color of the 
 alkaline solution is a cherry red. It is often seen in alcoholic 
 specimens of the liver, muscles, etc., w r hich have stood for a time 
 in alcohol. 
 
 HEMATOPORPHYRIN. 
 
 By the action of acids upon haematin the iron is removed, 
 leaving a violet to red coloring matter hsematoporphyrin. It is 
 found in the contents of the stomach and intestine after poisoning 
 with strong acids. It also is found in some of the dark-colored 
 urines. It is insoluble in water, more soluble in acids, and easily 
 so in alkalies. 
 
 243. Add enough blood to a little water to color it 
 a bright red. Dissolve a small crystal of ferrous sulphate 
 and as much tartaric acid in 10 cubic centimeters of water, 
 
116 , THE BLOOD. 
 
 then enough ammonia to make it faintly alkaline,, and add 
 1 drop to the blood solution. The oxyhaemoglobin gives 
 up its oxygen to the iron compound, becoming changed in 
 a short time to haemoglobin, as is shown by the dark color. 
 An excess of the reducing solution should be avoided. 
 
 244. Shake the dark solution of hemoglobin with 
 air and notice the change in color to a scarlet, showing the 
 formation of oxyhsemoglobin. 
 
 245. Examine a very dilute solution of blood with 
 the spectroscope in the following manner: First examine 
 the solar spectrum by looking through the spectroscope 
 with its slit directed toward a window. Close the slit to 
 a very narrow opening, and focus by sliding the focusing 
 tube until the fine, dark lines are seen clearly. There are 
 hundreds of these so-called Fraunhofer lines in the spec- 
 trum of the sun, but with an ordinary instrument many 
 are indistinct. A few of the most prominent should be 
 noticed and s used to locate the position of the dark bands 
 in the spectra of haemoglobin and its derivatives. The 
 most noticeable are the C line in the red, the D line in 
 the yellow, the E and & not far apart in the green and F 
 in the blue. If there are fine, black lines running length- 
 wise of the spectrum they are caused by dust in the slit. 
 Next make solutions of blood and water of different dilu- 
 tions and examine the spectrum which is given when a 
 test-tubeful of the solution is held before the slit after the 
 solution has been shaken with air to form oxyhaamoglobin. 
 Notice the position of the two bands and observe that this 
 does not change with the different dilutions, although the 
 bands may be wider or more distinct when the solution is 
 concentrated. 
 
 246. To the solutions of oxyhsemoglobin add a few 
 drops of ammonium sulphid, and after they have stood a 
 
OXYH^EMOGLOBIN REACTIONS. 117 
 
 short time examine them again with the spectroscope. No- 
 tice that when the oxygen has been removed by the ammo- 
 nium sulphid the spectrum has changed to that of haemo- 
 globin, which consists of one broad band in the space be- 
 tween that formerly occupied by the two. It is less dis- 
 tinct than the two bands of the oxyhsemoglobin spectrum, 
 but can be rendered darker by adding 3 or 4 drops of a 
 40-per-cent. solution of formaldehyd. 
 
 247. Shake the reduced solutions with air and see 
 that the two bands have returned. If any ammonium sul- 
 phid remains the oxyhaamoglobin will be changed again to 
 haemoglobin on standing. 
 
 248. Through the solution of blood pass a stream of 
 illuminating gas for a few minutes. The carbon monoxid 
 is absorbed, forming carbonic oxid haemoglobin. Examine 
 it with the spectroscope. It gives two dark bands much 
 like those of oxyhaemoglobin, though the one next the green 
 is not as wide as in the oxyhaemoglobin-spectrum. Try 
 now to reduce the compound to haemoglobin by means of 
 ammonium sulphid. The carbonic oxid is not expelled, 
 the two bands remaining unchanged. 
 
 249. Prepare an alcoholic solution of guaiacum by 
 dissolving some of the gum taken from the middle of a 
 lump. No blue color is produced in the solution on the 
 addition of a small amount of old oil of turpentine or 
 hydrogen peroxid, but it is produced upon the further 
 addition of a few drops of blood. 
 
 250. Try the same test on the scrapings from a po- 
 tato. A blue color is produced on standing, without the 
 aid of the turpentine or any similar oxidizing agent. 
 
 251. Add a few drops of glacial acetic acid to diluted 
 defibrinated blood and warm gently. Haematin gives a 
 brown solution. Examine its spectrum. Neutralize with 
 
118 THE BLOOD. 
 
 sodium hydrate, then add enough to dissolve the precipi- 
 tate, warming if necessary. Alkali hsematin is formed. 
 Examine the spectrum. 
 
 252. Acidify slightly a solution of blood, and heat to 
 boiling. Notice the coagulated albuminous substance and 
 the dark-colored haematin coming from the decomposition 
 of the oxyhaemoglobin. 
 
 253. Filter out the hasmatin or use instead a drop 
 of fresh blood and prepare haemin crystals from it by first 
 drying thoroughly on a glass slide, then adding a minute 
 amount of sodium chlorid. Cover with a cover glass ; add 
 a drop of glacial acetic acid, which will flow under the 
 cover. Then heat over a small flame until the acid boils. 
 After cooling examine under a microscope. The crystals 
 sometimes are better if the acid is added and the slide 
 heated two or three times. 
 
 254. To prepare a large amount of hsemin, precipitate the 
 corpuscles from, defibrinated blood by the addition of a large excess 
 of a salt solution which contains 1 volume of a saturated salt 
 solution in 10 to 20 volumes of water. After twenty-four hours 
 pour off the solution and rinse the precipitated corpuscles into a 
 flask by the aid of a small amount of water. Add half its volume 
 of ether, shake, and after pouring off the ether which removes 
 most of the fats allow the solution of blood-coloring matters to 
 evaporate in flat dishes at ordinary temperature to a syrup. Mix 
 this with 10 to 20 volumes of glacial acetic acid and heat in a 
 flask one or two hours on the water-bath. Then pour into a 
 beaker, add several volumes of water, and allow to stand several 
 days. Wash with water and remove the albuminous substances 
 by boiling with acetic acid. 
 
 255. Saturate 2 or 3 cubic centimeters of blood with 
 carbonic oxid by passing illuminating gas through it. Add 
 to it twice its volume of sodium hydrate solution, specific 
 gravity, 1.3, containing about 27 per cent. ISTaOH. Do 
 
CRYSTALLIZED OXYH^MOGLOBIN. 119 
 
 the same with pure blood, and spread the products on a 
 piece of porcelain. Notice that the pure blood gives a 
 brown color, with a shade of green. The carbon monoxid 
 blood has a bright-red color on porcelain. This is a useful 
 test in cases of suspected poisoning by carbon monoxid. 
 
 256. Prepare crystals of oxyhsemoglobin by placing on a 
 microscope-slide a drop of blood (one which crystallizes easily, 
 like that of a dog, rat, or guinea-pig) and cover it with a drop of 
 Canada balsam. Cover the whole with a cover-glass and examine 
 it under the microscope. The crystals will form in a few minutes. 
 They can also be made by mixing the drop of blood with a 
 small drop of water on the slide and allowing it to evaporate 
 until a dry ring has formed around it. Place a cover-glass over it 
 and it will crystallize. It is well to examine several different 
 species of blood if they can be obtained, such as guinea-pig, which 
 gives crystals in the form of tetrahedra (four-sided) ; mouse, giv- 
 ing six-sided plates; cat or dog, giving four-sided needles. 
 
 257. A large quantity of crystallized oxyhsemoglobin can 
 be prepared by the following method: Make a solution of salt 
 containing 1 volume of saturated salt solution to 9 volumes of 
 water. Add 10 volumes of this to 1 of defibrinated blood, and 
 let it stand a day or two in shallow, flat-bottomed vessels until 
 the corpuscles have settled. Pour off the clear liquid, rinse the 
 corpuscles into a separatory funnel with the aid of as small a 
 quantity of water as possible, and add about as much ether. 
 Shake, but not too violently, separate the solution of oxyhsemo- 
 globin from the ether, and filter the former. Cool it to and 
 mix it with one-fourth its volume of alcohol which has been 
 also cooled to 0. Let the mixture stand at a temperature of 
 2 to 10 for several days, until the oxyhsemoglobin has 
 crystallized. This occurs with the blood of dogs and rats almost 
 immediately, but that of the ox crystallizes with much more diffi- 
 culty. After crystallization filter off the crystals in the cold, and 
 dry by pressing between filter-paper. The crystals may be purified 
 by dissolving in a small amount of water, cooling and precipitating 
 in the same manner, repeating several times. The crystals can be 
 preserved for a standard in the determination of the quantity of 
 oxyhsemoglobin in blood. 
 
120 THE BLOOD. 
 
 258. Test a parchment dialyzer or tubing to see that it has 
 no holes, then introduce defibrinated blood diluted with an equal 
 volume of water. Let it stand an hour or more in water. Notice 
 that the large haemoglobin molecules cannot pass out, but the 
 small molecules of chlorids do so, as is shown by adding silver 
 nitrate to the outer liquid. 
 
 259. To a solution of blood in water add a small 
 crystal of potassium chlorate or potassium ferricyanid. 
 Methsemoglobin is formed, the color of the solution chang- 
 ing from a red to a brown. Make it slightly alkaline and 
 it becomes red. Examine the spectrum of the alkaline so- 
 lution. It is much like that of oxyhaBmoglobin, though 
 the first band is broader and extends somewhat farther 
 toward the red. It is reduced to haemoglobin by ammo- 
 nium sulphid, like oxyha3moglobin. 
 
 260. Large amounts of crystalline methaemoglobin can be 
 obtained by adding to a concentrated solution of oxyhaemoglobin 
 enough of a concentrated solution of potassium ferricyanid to give 
 it a deep-brown color. Crystallize, as in the case of oxyhaemo- 
 globin, by cooling to zero and adding one-fourth the volume of 
 cold alcohol. 
 
 261. PREPARATION OF ELEMATOPORPHYRIN. Saturate 75 
 grammes of glacial acetic acid with hydrobromic acid at 10 and 
 add to it in a 300 cubic centimeter flask 5 grammes of dry hsemin 
 crystals. Heat thirty minutes on a water-bath until no more 
 hydrobromic acid fumes escape, then pour into a liter of water. 
 After standing several hours filter and to the red filtrate add 
 sodium hydrate till the liquid is neutral. The coloring matter is 
 precipitated and should be filtered off, washed, and drained upon 
 filter-paper. Digest the moist precipitate with dilute sodium 
 hydrate on the water-bath; filter off the hydrate of iron, which 
 separates, and allow the solution to stand until the sodium 
 compound of hsematoporphyrin has separated in crystalline 
 masses. Dissolve these in water and precipitate the coloring 
 matter by acidifying with acetic acid. Filter, wash with water, 
 and after stirring the precipitate up to a paste with a small 
 
IDENTIFICATION OF BLOOD-STAINS. 121 
 
 quantity of water dissolve by adding hydrochloric acid carefully. 
 Evaporate the dark-red solution in a vacuum over sulphuric acid. 
 The hydrochloric acid compound of haematoporphyrin crystallizes 
 in brownish-red needles. 
 
 262. Examine the spectrum of the hsematoporphyrin. 
 
 263. Dissolve a few haemin crystals in water by the aid of 
 sodium hydrate and examine the spectrum. 
 
 264. Convert the haemin into haemochromogen by removing 
 the oxygen. Use for this purpose a solution of ferrous sulphate 
 with tartaric acid, the whole being made alkaline with ammonia 
 (Experiment 243). Examine the spectrum. 
 
 265. If hsemin crystals are not at hand, prepare the haemo- 
 chromogen from a dilute solution of blood by first making it 
 alkaline with sodium hydrate, then reducing the haematin thus 
 formed by ammonium sulphid or ammoniacal ferrous tartrate. 
 
 TESTING SUSPECTED STAINS FOR BLOOD. 
 
 The following tests can be used, first on a stain made 
 by drying a drop of blood on a piece of cloth, then upon 
 unknown stains. 
 
 1. Soak a small portion of the stain in a few drops 
 of water on a microscopic slide. If no color is imparted 
 to the liquid, blood is not present or the hemoglobin has 
 been so much decomposed that only the hsemin test will 
 show its presence, or, possibly, the albumin and globulin 
 have been coagulated. 
 
 2. If a red color was seen in the water try the spec- 
 troscopic test. By drying, both haemoglobin and oxy- 
 haemoglobin may be changed into methsemoglobin, which 
 gives a somewhat different spectrum. (Plate IV, 11.) 
 
 Ammonium sulphid reduces this to hemoglobin and 
 shaking with air gives the spectrum of oxyhsemoglobin 
 (Experiments 246 and 247). If these are all obtained the 
 Dresence of blood is proved. 
 
122 THE BILE. 
 
 3. To confirm the results, or, if the stain is too small 
 to obtain them, try to obtain the haemin crystals as in 
 Experiment 253. After applying the acid to the dried 
 mass the latter should be broken up with a glass rod to 
 insure thorough mixture. If it is very hard let it soak in 
 the acid for a short time. These haemin crystals are pro- 
 duced only from the coloring matters of the blood. When 
 in doubt as to their identity they should be compared with 
 those obtained from known blood, remembering that dif- 
 ferent specimens of crystals may differ considerably in 
 size. 
 
 4. If there is a sufficient amount of the blood, not 
 too long exposed to the air, or if it is desirable to de- 
 termine the species of animal, it may be soaked in a small 
 amount of 1 / 2 -per-cent.-salt solution and the appearance 
 and size of the corpuscles compared with those of known 
 specimens or measured by a microscope with a micrometer 
 eye-piece. 
 
 THE BILE. 
 
 The bile is normally a brown to greenish, viscid fluid 
 with a bitter taste and a neutral or slightly alkaline re- 
 action. It is a mixture of the secretions of the liver-cells 
 with that from the mucous membrane of the passages, 
 which latter contains a viscous substance similar to the 
 nucleoalbumins. This is usually called biliary mucin, al- 
 though it differs in some respects from true mucin. 
 
 The compounds which make up the larger part of 
 the solid matters of the bile are the sodium salts of gly- 
 cocholic and taurocholic acids. Besides these and the 
 biliary mucin there are present fats, soaps, lecithin, and 
 cholesterin, also a number of inorganic salts of the alka- 
 
THE BILE. 123 
 
 lies, alkaline earths, and iron. The color of the bile is 
 due to the biliary pigments, bilirubin, biliverdin, etc. 
 
 The salts of the biliary acids in the bile of different 
 animals vary in their proportions. In the case of car- 
 nivorous animals only the taurocholic acid is found; in 
 the human bile, as well as that of most cattle, both are 
 present. The biliary acids are both compounds of cholic 
 acid, C 24 H 40 5 . Glycocholic acid, C 26 H 43 N0 6 , is com- 
 posed of cholalic acid united with glycocol, CH 2 ISrH 2 C0 2 H; 
 taurocholic acid, C 26 H 45 ]SrS0 7 , of cholalic acid and taurin, 
 C 2 H 4 NH 2 S0 3 H. They can be decomposed into their con- 
 stituents by the caustic alkalies. With cane-sugar and 
 sulphuric acid the biliary acids give a purple color, and 
 this can be used as a test of their presence. 
 
 This test is an extremely delicate one, and its failure indi- 
 cates that biliary acids are absent. There are, however, other 
 substances like albumin, morphine, and amyl-alcohol which give 
 a similar color. In these cases the spectroscopic test should not be 
 neglected. The biliary acids can be obtained pure by evaporating 
 the solution to dryness, extracting with absolute alcohol, pre- 
 cipitating this solution with ether, and applying the test to the 
 precipitate. The purple solution, when sufficiently diluted with 
 alcohol and examined spectroscopically, gives a dark band between 
 D and E, near to E, and another before F. These are not seen 
 with albumin, etc. 
 
 In concentrated sulphuric acid they give a green color, 
 showing a strong fluorescence. The sodium salts are ob- 
 tainable from the bile by evaporating to dryness and, after 
 dissolving in alcohol, precipitating with ether. 
 
 Cholesterin, C 26 H 43 OH, occurs in most of the fluids 
 of the body, as well as in the bile, and the calculi or 
 concretions of the gall-bladder, of which it forms the 
 principal part. It is not common in the urine, but is a 
 constant ingredient of the faeces. It is insoluble in water, 
 
124 THE BILE. 
 
 but soluble in ether, chloroform, or hot alcohol. It crys- 
 tallizes from ether in fine, silky needles; from alcohol in 
 large plates containing a molecule of water of crystalliza- 
 tion. (Plate I, 4.) In large quantities it has the appear- 
 ance of a mass of white plates with a pearly luster and a 
 greasy feeling. It is distinguished from the fats by its 
 insolubility in the caustic alkalies, even when boiling. It 
 forms compounds with the fatty acids similar to the fats, 
 the cholesterin taking the place of the glycerin. Lanolin, 
 which is found in wool-fat, is an example. These are not 
 easily decomposed by bacteria, hence can be advanta- 
 geously substituted for the animal fats where decomposition 
 is objectionable. The cholesterin as found in the animal 
 body seems rather to be an excrementitious material than 
 to have any function of its own. 
 
 The bile contains at least two well-characterized pig- 
 ments or coloring matters: bilirubin, C 32 H 36 N 4 G ; and 
 biliverdin, C 32 H 36 N 4 8 . The different colors of bile from 
 a brown to a green are due to a preponderance of one or 
 other of these. They seem to be formed from the blood- 
 coloring matters, being found in old blood-extravasations 
 and being increased in amount in the bile when the blood- 
 corpuscles are destroyed, so that the coloring matters are 
 set free in the plasma. 
 
 Bilirubin occurs in many biliary calculi, particularly 
 in and around the nucleus. This is the best source of the 
 pure substance. It is commonly an amorphous powder, 
 orange-red in color. It is insoluble in water, but can be 
 dissolved in chloroform, and crystallizes from the latter in 
 plates and prisms. It unites with strong bases and in cal- 
 culi occurs in union with calcium. By reduction hydro- 
 bilirubin, C 32 H 40 N 4 7 , is formed. This change takes place 
 in the large intestine as a result of putrefactive action, and 
 
BILE PIGMENTS. 125 
 
 the color of the fasces is due principally to the hydrobili- 
 rubin. Bilirubin is acted upon by oxidizing agents, with 
 the formation of biliverdin. This change takes place when 
 an alkaline solution is left exposed to the air. 
 
 Biliverdin is an amorphous, green powder. It differs 
 from bilirubin in being insoluble in chloroform, and the 
 two can consequently be separated by this reagent. 
 
 Both of these biliary pigments when acted upon by 
 yellow nitric acid, such as is formed by allowing the strong 
 acid to stand in a bright light, undergo a change of color 
 through green, blue, violet, and red to yellow. 
 
 Besides these two coloring matters a number of others 
 have been described by different authors. Of them com- 
 paratively little is known. They appear to be derived from 
 biliverdin and bilirubin, and it is to their formation that 
 the play of colors is due when bile is acted upon by oxid- 
 izing agents. Some of these are: 
 
 Biliprasin, greenish black. 
 
 Bilifuscin, brown. 
 
 Bilicyanin, blue. 
 
 Choletelin, yellow to brown. 
 
 Not infrequently there are found in the gall-bladder con- 
 cretions, commonly known as gall-stones. They are sometimes 
 nearly as large as a hen's egg, and may fill the bladder almost com- 
 pletely. They are soft and often worn away from rubbing against 
 one another. If they are cut through the nucleus is generally 
 found to be dark colored and composed of bilirubin- calcium. 
 Around this are concentric layers, usually of eholesterin, but some- 
 times of the bilirubin-calcium. Calcium carbonate is also found in 
 the concretions, as well as others of the biliary pigments in smaller 
 amounts. 
 
 266. SEPARATION or THE SALTS or THE BILIARY 
 ACIDS. The contents of the gall-bladder of an ox should 
 be mixed with washed sand and evaporated to dryness on 
 
126 THE BILE. 
 
 the water-bath. Then pulverize the mass in a mortar, 
 which operation is facilitated by the sand. Dissolve the 
 biliary salts by strong alcohol,, and filter. Evaporate this 
 solution to a small volume on the water-bath, allow it to 
 cool, pour it into a flask, and precipitate with an excess 
 of ether, shaking to mix thoroughly. After standing a few 
 hours the precipitated mass is converted into clusters of 
 silky crystals (sometimes called crystallized bile). These 
 are a mixture of the sodium salts of the taurocholic and 
 glycocholic acids. They can be purified by filtering, wash- 
 ing with water, dissolving in the smallest possible quantity 
 of water, and precipitating again with ether. They crys- 
 tallize then in long, thin, colorless crystals with a silky 
 luster. 
 
 267. PETTENKOFER'S TEST FOR THE BILIARY ACIDS. 
 Use the salts or the ox-bile. Mix in a porcelain dish or 
 test-tube with a small amount of concentrated sulphuric 
 acid, being careful not to let the temperature rise above 
 70 C. It must, however, be above 50. Then add, drop 
 by drop, a 10-per-cent. solution of cane-sugar, stirring with 
 a glass rod. A red color appears. If too much sugar is 
 added or the temperature is too high the sugar is decom- 
 posed by the acid, giving dark-brown products, which con- 
 ceal the red color. 
 
 268. The same result may be obtained by adding the 
 sugar to the liquid to be tested, acidifying with dilute sul- 
 phuric acid, and dipping into it a piece of filter-paper. 
 Allow the paper to dry, or dry it at a moderate heat, to 
 avoid charring. When it is completely dry, the red color 
 appears on the paper. If heated too highly it will be 
 turned black by the acid. 
 
BILIARY ACIDS. 127 
 
 269. Examine the spectrum of the colored liquid obtained in 
 267. There are two absorption-bands: one at F, the other be- 
 tween D and E, near E. 
 
 270. PREPARATION OF THE FREE BILIARY ACIDS. Dissolve 
 in water some of the sodium salts obtained in 266. Add dilute 
 sulphuric acid slowly, until a precipitate commences to form, 
 then add a little ether. After standing in the cold until the acid 
 has separated, filter, wash, and recrystallize from a small amount 
 of hot water. This gives the glycocholic acid. Examine with the 
 microscope and sketch. 
 
 271. PREPARATION OF CHOLALIC ACID. Add to ox-bile 
 one-fifth of its weight of 30-per-cent. sodium hydrate and boil 
 for twenty-four hours, adding more water to replace that which 
 has evaporated. Then saturate the liquid with carbon dioxid, 
 evaporate to dryness, and extract the mass with strong alcohol. 
 The sodium salt of cholalic acid dissolves, as well as some other 
 sodium compounds. Dilute with water until the solution contains 
 no more than 20 per cent, of alcohol, then precipitate with 
 dilute barium chlorid as long as there is a precipitate. Filter 
 and test the filtrate with barium chlorid, which must give no pre- 
 cipitate. Then precipitate the cholalic acid from this filtrate by 
 decomposing its sodium salt by means of hydrochloric acid. Let 
 it stand several hours until it has become crystalline, then re- 
 crystallize from alcohol. Make sketches of the crystals. 
 
 272. Test the cholalic acid thus obtained with concentrated 
 sulphuric acid, and notice that it gives a green fluorescence. Add 
 a few drops of a cane-sugar solution and see that a red color 
 appears, as with the undecomposed biliary acids. 
 
 273. PREPARATION OF TAUROCHOLIC ACID. The bile of 
 dogs is preferable to ox-bile for this purpose. It contains the 
 sodium salt of the acid. Evaporate the bile to dryness on a 
 water-bath, dissolve in alcohol and precipitate with ether as in 
 266. Dissolve the precipitate in water and precipitate the glyco- 
 cholic acid by the repeated additions of small amounts of ferric 
 chlorid, each time nearly neutralizing the acid reaction with 
 sodium hydrate. Filter and precipitate the iron from the filtrate 
 by an excess of sodium carbonate. Nearly neutralize the filtrate 
 and evaporate to dryness. Dissolve in absolute alcohol, evaporate 
 to dryness and dissolve in water. From this precipitate the 
 
128 THE BILE. 
 
 sodium taurocholate by saturating the liquid with sodium chlorid. 
 Filter and add hydrochloric acid until the solution contains 2 
 per cent. If a precipitate of salts appears remove it by nitration 
 and from the filtrate precipitate the taurocholic acid with ether. 
 It should be filtered immediately and can be recrystallized in the 
 same manner, being then obtained in needles or prisms. Its 
 preparation is more difficult than that of the glycocholic acid. 
 
 274. Add the solution of the taurocholic acid acidified with 
 sulphuric acid to solutions of albumin or peptones and observe 
 that they form insoluble compounds. 
 
 275. PBEPAEATION OF TATJKIN. Mix dog-bile with an 
 excess of concentrated hydrochloric acid, and evaporate the liquid 
 to a small volume by boiling. Pour off the solution from the 
 resinous mass of acids which have separated, and evaporate this 
 liquid until the sodium chlorid has, for the most part, crystallized 
 out. Filter, and evaporate the filtrate to dryness. From the 
 residue dissolve the glycocoll with alcohol, then the insoluble 
 taurin in the smallest possible quantity of hot water. 'On cool- 
 ing the taurin crystallizes out in four-sided prisms. 
 
 276. PREPARATION or CHOLESTERIN. Biliary cal- 
 culi or gall-stones are the best source of cholesterin. Pow- 
 der the calculus, remove the bile by boiling water, then 
 dissolve the cholesterin in boiling alcohol, and filter while 
 hot. It separates from the filtrate on cooling. The in- 
 soluble residue, which consists largely of compounds of the 
 biliary coloring matters, can be used for the preparation of 
 these. The cholesterin may be further purified by dis- 
 solving it in an alcoholic solution of potassium hydrate 
 with the aid of heat. After it separates on cooling, wash 
 well with water on the filter, then recrystallize from a 
 mixture of alcohol and ether. 
 
 277. Examine the crystals under the microscope. 
 They are in the form of large rhombic tables or plates. 
 
 278. To a crystal of cholesterin in a test-tube or 
 under the microscope add a drop of concentrated sulphuric 
 
CHOLESTERIN. PIGMENTS. 129 
 
 acid, then a drop of iodin solution. The crystal becomes 
 first violet, then blue, green, and red. 
 
 279. Dissolve a crystal of cholesterin in a few drops 
 of chloroform in a test-tube, then add an equal volume of 
 concentrated sulphuric acid. The chloroform solution be- 
 comes red, then cherry red, and purple. On pouring it 
 into a dish it becomes blue, green, and finally yellow. 
 
 280. Evaporate on a piece of porcelain a small crys- 
 tal of cholesterin with a drop of concentrated nitric acid. 
 A yellow stain remains which, if treated while warm with 
 ammonia, gives a red color. Too high heating prevents the 
 reaction. 
 
 All these reactions can be employed for the identifica- 
 tion of cholesterin. 
 
 281. PREPARATION OF THE BILIARY PIGMENTS. If 
 biliary calculi are not available, bile may be used for ob- 
 taining bilirubin, employing the yellow or brown in pref- 
 erence to the green. Dilute it with a little water and add 
 a small amount of lime-water, avoiding an excess. Mix 
 by shaking. Pass through the liquid a stream of carbon 
 dioxid to convert any excess into calcium carbonate. Filter 
 out the bilirubin, which has been precipitated as the cal- 
 cium compound, and wash it with water. Suspend the 
 precipitate in water, decompose it with a slight excess of 
 hydrochloric acid, and shake it immediately with a small 
 amount of chloroform to take up the free bilirubin, other- 
 wise it will oxidize to biliverdin. Separate the chloroform 
 solution from the water and precipitate the bilirubin from 
 it by alcohol. 
 
 282. If biliary calculi are at hand they may be used 
 instead of the bile. Pulverize the calculi, then dissolve 
 the cholesterin with a mixture of alcohol and ether. The 
 residue from the alcoholic extraction in the preparation of 
 
130 THE BILE. 
 
 cholesterin may be used (Experiment 276). After the 
 cholesterin has been removed decompose with acid and 
 proceed as in the preceding experiment, 281. 
 
 283. Convert a portion of the bilirubin into bili- 
 verdin by dissolving in dilute sodium hydrate and letting 
 the solution stand in an evaporating dish. When it has 
 turned green, precipitate with an excess of hydrochloric 
 acid, filter, and wash. 
 
 284. The biliverdin in an impure state can be obtained from 
 ox-bile by precipitating the mucin with several times its volume 
 of alcohol, then precipitating the biliverdin by barium chlorid. 
 Filter, wash with water, and alcohol, then decompose with hydro- 
 chloric acid. The biliverdin is insoluble in the acid. To remove 
 the fat it must be extracted with ether, then the biliverdin can 
 be dissolved in alcohol, which, after filtering, is left to evaporate. 
 
 285. GMELIIST'S TEST. To a solution of bilirubin in 
 dilute alkali add slightly yellow., concentrated nitric acid, 
 holding the tube in a slanting position and pouring slowly 
 so that the acid flows down under the bilirubin solution. 
 Notice the colored rings : green nearest the top, then blue, 
 violet, red, and yellow next to the acid. The acid must 
 not be too yellow or the pigments quickly oxidize and 
 nothing is seen but a yellow color. 
 
 286. HUPPERT'S TEST. To an alkaline solution of 
 bilirubin add lime-water to precipitate the bilirubin. 
 Filter, wash with water, place in a test-tube half full of 
 alcohol slightly acidified with sulphuric acid, and boil for 
 some time. The bilirubin is oxidized to biliverdin and 
 the alcohol becomes colored green or bluish green. 
 
 287. To a little of the bilirubin solution in a test- 
 tube add a very dilute tincture of iodin so that it floats on 
 top. An emerald-green ring is seen between the liquids. 
 
CONNECTIVE TISSUES.. 131 
 
 CONNECTIVE TISSUES. 
 
 Besides the areolar tissue which may be the white, 
 fibrous, or the yellow, elastic, there are cartilaginous and 
 osseous tissues. 
 
 WHITE FIBROUS TISSUE. 
 
 Nearly pure white fibrous tissue is found in the ten- 
 dons which are made up of fibers, matrix, and cells. The 
 fibers consist of collagen; the matrix contains mucin; the 
 cells contain proteins and salts. 
 
 288. Chop a tendon finely (the tendon of Achilles 
 can be used) and remove the soluble constituents by wash- 
 ing with cold water. Extract the mucin with lime-water 
 and filter. Preserve the insoluble residue and use the solu- 
 tion for the mucin tests. 
 
 289. Show that mucin gives the biuret and Milton's 
 reactions. 
 
 290. It cannot be coagulated by heating nor does it 
 dissolve in dilute acids (distinction from most other pro- 
 teins) . 
 
 291. It does not reduce Fehling's solution unless it 
 has been boiled for an hour or two with an acid like hydro- 
 chloric. A reducing amido compound is thus prod (iced. 
 When the acid solution is neutralized a precipitate of acid 
 albumin falls. Hence the original mucin contained the 
 reducing substance united with a protein. 
 
 COLLAGEN. 
 
 292. For its preparation use the tendon from which 
 the mucin was removed. Heat the tendon with a little 
 water. On cooling it gelatinizes and gives the gelatin re- 
 actions (page 60). 
 
132 CARTILAGE. BONE. 
 
 CARTILAGE. 
 
 293. This contains collagen mixed with chondrogen, 
 chondroitin sulphuric acid and an albuminoid. It can be 
 obtained from pigs' tracheas. Separate the surrounding 
 tissues,, grind the remainder and boil with water. The fil- 
 tered solution yields a jelly when it cools (gelatin from 
 the hydration of the collagen) . 
 
 294. The solution gives no reaction for sulphuric 
 acid with barium chlorid but does so after boiling for some 
 time with hydrochloric acid, showing that it contains an 
 organic sulphate (page 173)., chondroid, in sulphuric acid. 
 
 295. The solution reduces Trommer's or Fehl ing's 
 reagent after boiling, but not before. It gives the protein 
 reactions. 
 
 BONE. 
 
 Bone contains an organic compound, collagen, and a 
 number of inorganic or mineral substances. These latter 
 are the phosphates of calcium and magnesium, mainly the 
 former; also calcium carbonate and small amounts of cal- 
 cium chlorid and fluorid. The inorganic substances can 
 be removed by acids, leaving the bone flexible. If a bone 
 is heated the collagen is decomposed, with an evolution of 
 ammonia, showing that the collagen is a nitrogenous com- 
 pound. Then inflammable gases are set free. If the igni- 
 tion is performed where free access of air is prevented, 
 there remains a black mass known as bone-black or animal 
 charcoal. The black color is due to carbon, which can be 
 removed by burning in the air, leaving the mineral or in- 
 organic constituents only. The bone-black is an extremely 
 porous substance and has the power of absorbing from 
 their solutions many of the vegetable coloring matters and 
 
CONSTITUENTS OF BONE. 133 
 
 also the alkaloids. On this account it is used for decolor- 
 izing liquids as well as for an antidote in cases of poison- 
 ing by strychnine and some other alkaloids. 
 
 296. Fill a dry test-tube one-third full of fragments 
 of dry bone, fasten it horizontally by the upper end in a 
 clamp, and heat, at first gently, then to as high a tem- 
 perature as possible without softening the glass, moving 
 the burner so as not to heat it in one spot. The organic 
 matter is decomposed. First water is given off, then am- 
 monia. Test this with a piece of red litmus-paper. A n 
 oily or tarry mixture distills off with inflammable gases. 
 When the gas has been expelled the mineral matters of the 
 bone remain mixed with carbon. 
 
 297. Take a piece of this and heat it in the air, hold- 
 ing it with the forceps or a piece of wire. The carbon 
 burns away, leaving only the mineral matters as a brittle 
 mass. 
 
 298. Dissolve the bone-ash in dilute nitric acid. 
 Notice that carbonates are present, as shown by the effer- 
 vescence of carbon dioxid gas. 
 
 299. Test a portion of the solution for phosphoric 
 acid, which is present in the phosphates, by making it 
 strongly acid with nitric acid, then adding ammonium 
 molybdate and warming gently. A yellow precipitate 
 shows phosphoric acid. 
 
 300. Test another small portion of the solution for 
 chlorids with silver nitrate. They give a milkiness or a 
 white precipitate, but are present in very small quantities. 
 
 301. Test the remainder of the nitric acid solution 
 for calcium and magnesium after removing the phosphoric 
 acid in the following manner: Add a few drops of ferric 
 chlorid to the solution in a beaker. The iron unites with 
 the phosphoric acid, which was held by the calcium and 
 
134 BONE. 
 
 forms ferric phosphate. Pour a few drops from the beaker 
 into a test-tube and test by adding ammonia to see if all 
 the phosphoric acid has united with the iron. If this is 
 the case, the ammonia gives a yellowish precipitate. If 
 not enough ferric chlorid was added the precipitate is 
 white, and more of the iron solution must be added and 
 the test repeated until all the phosphoric acid has been 
 taken by the iron. Then to the solution add a solution 
 of sodium carbonate until it is nearly neutral; that is, 
 until the precipitate which forms as the sodium carbonate 
 strikes the liquid dissolves slowly on stirring. Then 
 add 1 or 2 grammes of barium carbonate to precipitate 
 the ferric phosphate. Filter after warming. Precipitate 
 the barium from the hot filtrate with dilute sulphuric acid 
 and filter. Having thus removed the phosphoric acid, test 
 the filtrate for calcium by making it alkaline with am- 
 monia and adding ammonium oxalate as long as a pre- 
 cipitate is formed. The calcium is thrown down as white 
 calcium oxalate. Filter and to the filtrate add sodium 
 phosphate. A white crystalline precipitate shows mag- 
 nesium. 
 
 302. Test the absorptive power of bone-black by adding 
 it to a light-blue solution of indigo and warming, then filtering. 
 The coloring matter will have almost or entirely disappeared from 
 the filtrate. 
 
 Directions have been given before (Experiment 138) 
 for the separation of the collagen from the mineral con- 
 stituents of the bone. 
 
 MUSCULAR TISSUE. 
 
 A muscle is made up of fibers or cells, consisting of 
 a sheath (the sarcolemma), composed of a substance similar 
 
MUSCULAR TISSUE. 135 
 
 to elastin, and its contents. The latter are mostly albu- 
 minous matters, alkaline and liquid during life, but be- 
 coming acid and more solid after death. This albuminous 
 liquid, which in many respects corresponds to the plasma 
 of the blood, is called muscle-plasma. It coagulates quickly 
 at the ordinary temperatures, and thus gives rise to the 
 rigor mortis of the muscles after death. The coagulated 
 mass is myosin: a mixture of a number of compounds. 
 The cause of the coagulation of the myosin in this case 
 is probably the formation of lactic acid, which accumulates 
 in the muscle after death. 
 
 If fresh muscular tissue is treated with boiling water, 
 most of the albuminous substances are coagulated and 
 upon filtering remain with the fats in the insoluble residue. 
 The solution contains, besides inorganic matters, a class 
 of organic compounds, sometimes called, from the method 
 of obtaining them, the "extractives." They may be divided 
 into two groups: those which contain no nitrogen and 
 those of which nitrogen is a constituent. Among the non- 
 nitrogenous are lactic acid and its compounds; also gly- 
 cogen and its derivatives: dextrin, maltose, and glucose. 
 The principal ones of the nitrogenous extractives are 
 creatin and creatinin, small quantities of urea and uric 
 acid, and the nuclein bases, such as guanin, xanthin, and 
 hypoxanthin formerly called sarcin. Carnin, which is 
 similar in properties and composition to the nuclein bases, 
 is also found in the watery extract of muscle. 
 
 Creatin: 
 
 H 9 N HOCO 
 
 I I 
 
 I 
 
 I I 
 
 H 3 CN OH, 
 
136 THE UKINE. 
 
 and creatinin: 
 
 HN CO 
 
 H 3 CN CH 2 
 
 are closely related, the latter being derived from the former 
 by taking away one molecule of water, and can be changed 
 back into creatin by adding the water again. 
 
 The nuclein bases, also called purin bases, xanthin 
 bases and alloxuric bases, guanin (C 5 H 5 N 5 0), hypoxanthin 
 (C 5 H 4 N" 4 0), and xanthin (C 5 H 4 N 4 2 ) are similar in their 
 properties and related to uric acid (C 5 H 4 N 4 3 ). They 
 are derivatives of the hypothetical nucleus, purin: 
 
 (6) 
 (1)N= CH 
 
 I (7) 
 (2 ) HC (5) C NH 
 
 II II > CH (8) 
 (3)N C- N 
 
 (4) (9) 
 
 They are formed by the addition to, or substitution in 
 this of oxygen or organic radicals, the position being in- 
 dicated by the figures. Thus guanin is 2-amino-6-oxy- 
 purin. Hypoxanthin is 6-oxypurin; xanthin, 2-6-dioxy- 
 purin and uric acid, 2-6-8-trioxypurin. They occur partly 
 free in the muscular tissue and partly united with phos- 
 phoric acid and albuminous substances in the form of 
 nucleins. All these soluble compounds are found, natur- 
 ally, in the various meat-extracts, which are used in foods. 
 They are probably formed in the body by the decompo- 
 sition of the albuminous materials. When taken as a food, 
 

 PREPARATION OF UREA. 137 
 
 their value is rather in the stimulation of digestion through 
 their agreeable taste than in their absolute nutritive worth. 
 This may be merely because of their increasing the secre- 
 tion of the digestive fluids. 
 
 303. Pith a frog (see Experiment 305) : cut out the 
 gastrocnemius muscles. Let one remain in water at a tem- 
 perature of 50 until rigor caloris has appeared. Put the 
 other into boiling water. Test the reaction of both with 
 litmus paper. The former is acid: the latter is alkaline. 
 
 304. Kill a rabbit or a frog; at once lay bare the 
 muscle and test it with red and blue litmus paper and with 
 lacmoid paper. To the latter it is neutral; the red litmus 
 turns blue and the blue litmus, red that is, the reaction 
 toward litmus is amphoteric. Both free acids and acid 
 salts turn litmus red ; lacmoid becomes red from the action 
 of free acids, not from that of the acid salts. Let the 
 muscle stand and test it as before. It remains neutral to 
 lacmoid, but is acid to litmus. Lactic has been produced, 
 but instead of remaining in the free state it forms lac- 
 tates. The bases are thus removed in part from other salts, 
 leaving them as acid salts (for example, acid phosphates), 
 which latter produce the acid reaction. 
 
 305. Inject a solution of acid fuchsin into the subcutaneous 
 lymph-space of a frog. After it has been absorbed pith the ani- 
 mal; that is, destroy the brain by pushing forward a stout wire 
 inserted through the occipito-atlantoid membrane. This lies in 
 the middle of a line drawn across the back of the head through 
 the posterior margin of the tympanic membranes. Strip the 
 skin from both hind legs and separate the muscles of one thigh 
 until the sciatic nerve is exposed. Hang the frog by a hook 
 through the jaws and repeatedly stimulate the sciatic nerve by 
 electrodes passing under it. The stimulated leg contracts vigor- 
 ously while the other remains passive. In the working muscle 
 lactic acid is formed and this decomposes the colorless, alkaline 
 
138 MUSCULAR TISSUE. 
 
 salt of fuchsin, producing a pink or red color. In the resting leg 
 there is no such acid formation and the color remains pale. 
 
 306. Chop finely 20 to 25 grammes of lean meat and 
 extract it for an hour with three times as much cold water, 
 stirring frequently. Filter through muslin and test the 
 filtrate for myogen, an albumin, sometimes called myo- 
 sinogen. It is coagulable and gives the general reactions 
 for albuminous substances (page 37). It is soluble in 
 distilled water and does not precipitate from this solution 
 on dialysis. 
 
 307. Soak the residue from the last experiment in 
 50 to 75 cubic centimeters of 10 per cent, ammonium 
 chlorid solution, filter through muslin and test the filtrate 
 for myosin, a globulin, sometimes called paramyosinogen 
 or musculin. It responds to the globulin tests (page 45) 
 as well as the general reactions for albuminous substances 
 (page 37) and is coagulated by heat. 
 
 308. Mince finely 10 to 15 grammes of fresh muscular tissue 
 and extract it by stirring with water for a few minutes. Warm 
 the filtered solution in a double beaker (Experiment 91). When 
 coagulation occurs filter and keep the temperature constant until 
 no further change is observed, then increase it. Note the tempera- 
 tures at which the different proteins coagulate and report them. 
 This is the method of separation by fractional coagulation. 
 
 309. PREPARATION OF MUSCLE-PLASMA. Kill a frog and 
 immediately wash the blood from the body by passing in through 
 a cannula inserted in the aorta a cold 0.5-per-cent. sodium chlorid 
 solution. The necessary force can be gained by placing the solu- 
 tion in a doubly-tubulated bottle, which can be raised and lowered, 
 and connecting the lower tubulure with the cannula by a small 
 rubber tube. Cut the muscle up as quickly as possible with a cold 
 knife or pair of scissors and freeze it by stirring in a beaker, pre- 
 viously surrounded by a freezing mixture of ice (3 parts) and salt 
 (1 part). It freezes at about 7 C. Then rub it to as fine a 
 
CONSTITUENTS OF MUSCLE. 139 
 
 powder as possible in a mortar, whir>h, as well as the pestle, has 
 been cooled below this temperature by standing in a freezing 
 mixture. Subject the mass to a strong pressure, which gives a 
 yellowish liquid. Filter this through muslin at a temperature 
 below freezing. The nitrate is the muscle-plasma. Through the 
 whole process care must be taken to preserve a low temperature 
 to prevent coagulation. 
 
 310. Pour a few drops of the plasma into a dish of the 
 ordinary temperature. It coagulates immediately. 
 
 311. Test the reaction of the plasma to litmus-paper. It 
 is alkaline. 
 
 312. Allow the temperature of the rest of the plasma to 
 rise slowly, and notice that it coagulates at a little above freezing. 
 On standing, a yellowish liquid is pressed out of the clot, as in 
 the case of the coagulum of blood-plasma,. This is muscle-serum. 
 
 313. Try the reaction of the muscle-serum to litmus-paper. 
 It is alkaline. 
 
 314. Prove that the coagulated mass is a globulin (Experi- 
 ments, 98, 99, 100, and 101). 
 
 315. Take about 500 grammes of lean beef, and, after re- 
 moving, as completely as possible, the fat and connective tissue, 
 chop it finely. Add an equal weight of water and heat half an 
 hour on a water-bath to 55 or 60. Filter through muslin, press- 
 ing out the water with the hands. Repeat the extraction with 
 half as much water. Unite the nitrates, and boil to precipitate 
 the albuminous compounds. (Instead of the meat a jar of beef- 
 extract can be used after dissolving in water.) Filter and add lead 
 acetate as long as a precipitate forms, avoiding a great excess. 
 Filter and remove the lead by passing hydrogen sulphid gas into 
 the solution. Filter out the lead sulphid and evaporate the nitrate 
 on the water-bath to 5 or 10 cubic centimeters. Allow the yellow- 
 ish syrupy liquid to stand two or three days in a cool place, when 
 the creatin crystals will separate. Filter, and wash with 88-per- 
 cent, alcohol. Unite the nitrate and the washings and remove the 
 alcohol by evaporation on a water-bath. After cooling make alka- 
 line with ammonia and add an ammoniacal solution of silver 
 chlorid. Filter. The precipitate contains the silver compounds of 
 hypoxanthin, xantMn, and guanln. (The nitrate contains lactic 
 acid. Preserve for testing.) Wash with ammonia and dissolve 
 
140 MUSCULAR TISSUE. 
 
 in boiling HNO 8 , sp. gr., 1.1., to which a little pure urea has been 
 added to prevent the decomposition of the bases. While hot, filter 
 from a small amount of AgCl, which may remain, then allow to 
 stand twelve hours. Hypoxanthin-silv&r nitrate separates in small 
 needle-shaped crystals. Filter and wash with water. From the 
 filtrate, by the addition of an excess of ammonia, is obtained a 
 slight precipitate of wantMn-silver oxid. The free xanthin and 
 hypoxanthin may be obtained by suspending their silver com- 
 pounds in water and, after heating and making slightly alkaline 
 with ammonia, adding ammonium sulphid drop by drop until the 
 silver is precipitated, avoiding an excess. On evaporating the 
 filtrate the xanthin and hypoxanthin will be left as microscopic 
 crystals. 
 
 Most of the guanin is left in the precipitate made by the am- 
 monium sulphid. It can be dissolved by boiling with a little very 
 dilute hydrochloric acid. Filter and precipitate it from the filtrate 
 by making it alkaline with ammonia. 
 
 To obtain the lactic acid from the filtrate from the precipitated 
 hypoxanthin, etc., first precipitate the silver by HjjS and filter. 
 Concentrate the filtrate on the water-bath until most of the am- 
 monia has been expelled. Then cool and acidify strongly with 
 dilute sulphuric acid. The lactic acid is thus set free and can 
 now be separated by shaking gently with about one-fifth of its 
 volume of ether, which dissolves the lactic acid, but not the 
 sulphuric. After shaking in a glass-stoppered funnel, allow it to 
 stand until the ether has all risen to the top of the liquid. Then 
 draw off the water and the ether into separate flasks. Repeat the 
 operation a few times with fresh portions of ether. Mix the differ- 
 ent portions of ether and distill or evaporate it. The residue con- 
 tains the lactic acid mixed with a little sulphuric. Dilute with 
 water and boil a minute with zinc carbonate until it has lost its 
 acid reaction. Filter, and evaporate the filtrate on the water-bath 
 to a small volume. Then let it stand, and the zinc lactate will 
 crystallize in four-sided prisms: (C 8 HA) 2 Zn + 2H 2 O. Filter 
 these from the remaining liquid, and dry on filter-paper. 
 
 To obtain the free acid dissolve some of the crystals in water 
 and precipitate the zinc with hydrogen sulphid gas. Filter, and 
 evaporate the filtrate. The acid will be left as a syrupy liquid. 
 Test it for its acid reaction and sour taste. It differs from fer- 
 
THE BRAIN. 141 
 
 mentation lactic acid in that it rotates the plane of polarized light 
 toward the right. Fermentation lactic acid does not do this. 
 
 316. Convert a part of the creatin into creatinin by boiling 
 fifteen minutes with very dilute sulphuric acid. Neutralize the 
 acid by adding powdered barium, carbonate as long as it effer- 
 vesces. Evaporate to dryness on a water-bath and extract the 
 creatinin from the residue with strong alcohol. Upon evaporat- 
 ing, the creatinin is left in the form of crystals. 
 
 317. Dissolve a little of the creatinin in a small amount of 
 water, add a solution of zinc chlorid, and allow to stand. Charac- 
 teristic crystals in clusters or rosettes appear. They are a double 
 salt of creatinin and zinc chlorid. 
 
 318. To a creatinin solution add a few drops of a freshly- 
 prepared solution of sodium nitroprussid, then, drop by drop, 
 dilute sodium hydrate. The liquid becomes ruby red, soon chang- 
 ing to straw color. If it is now strongly acidified with acetic acid 
 and boiled, it becomes green, then blue. This is WeyPs test. 
 
 319. To a solution of creatinin add a few drops of sodium 
 hydrate, then of picric-acid solution. A red color is obtained. 
 This is Jaffe's test. 
 
 320. With a needle tease out some shreds of muscle 
 from a recently killed frog. Place them on a microscope 
 slide and expose for a few minutes to ammonia gas from a 
 strong solution of the hydrate. When covered with a cover- 
 glass and examined they are seen to contain stellate crys- 
 stals of ammonium magnesium phosphate,, NH 4 MgP0 4 . 
 Explain its production. 
 
 THE BEAIN. 
 
 321. Clean the brain of a sheep, pig or calf, pulp in a mortar, 
 extract several hours with 88-per-cent. alcohol and filter. The 
 lecithin, cholesterin and the substance known as protagon pass 
 into the filtrate. Cool this to 5, when they solidify, then re- 
 move them by filtration. Dissolve the lecithin and cholesterin in 
 cold ether (danger from fire!). The so-called protagon remains. 
 After this has been dried fuse a portion with potassium nitrate 
 
142 MILK. 
 
 in a nickel dish or crucible. Dissolve the residue in water and test 
 for phosphoric acid with magnesia mixture; or test for the same 
 substance after acidifying by nitric acid with ammonium molyb- 
 date. Mix another portion of the dry protagon in a glass tube 
 with soda-lime, hold horizontally by a clamp and heat. Show that 
 it contains nitrogen from the evolution of ammonia as is demon- 
 strated by the action of the gas on red litmus paper. 
 
 322. Pulp another brain and warm it with barium hydrate 
 solution (saponification). Filter. The mass on the filter contains 
 cerebrin, cholesterin, barium soap, and connective tissue. Heat 
 this with alcohol and filter while hot, thus dissolving out the 
 cholesterin and cerebrin. When the liquid cools they separate and 
 are recognizable by their crystalline forms. The cholesterin is in 
 flat thin plates, the cerebrin in clusters of needle-shaped crystals. 
 
 Extract the cholesterin by ether, and recrystallize. Test a 
 part of the cerebrin by heating with soda-lime for nitrogen as in 
 Experiment 72. Boil a part for an hour with dilute sulphuric acid 
 to hydrolize it; then show its reducing power with Trommer's 
 reagent. Observe that before hydrolysis it does not reduce. 
 
 MILK. 
 
 The solids of milk are partly dissolved and partly in 
 suspension in the liquid. Of the dissolved -constituents the 
 most important are milk-sugar, an albumin, a globulin, and 
 some mineral salts. Among the suspended compounds 
 are casein, fat, and calcium phosphate. The average 
 amount of solids in normal cows' or human milk is 12 or 
 13 per cent, by weight. The reaction of fresh cows' or 
 human milk is nearly neutral, or may be amphoteric to 
 litmus; that is, it turns red paper blue and blue paper red. 
 
 The specific gravity should be between 1.029 and 
 1.033 at 15, and of milk which has been skimmed after 
 standing twenty-four hours it should be between 1.0325 
 and 1.0365. Thus the removal of fat raises the specific 
 
MILK. 143 
 
 gravity and the addition of water lowers it. The average 
 percentage composition of milk is given by Konig as fol- 
 lows : 
 
 Cows', water, 87.17; proteins, 3.55; fats, 3.69; lac- 
 tose, 4.88; mineral matter, 0.71. 
 
 Human, water, 87.41; proteins, 2.29; fats, 3.78; lac- 
 tose, 6.21; mineral matter, 0.31. 
 
 Casein, which is a nucleoalbumin, is not in true solu- 
 tion in milk, since it can be filtered out by unglazed por- 
 celain, though not by filter-paper. It is precipitated by 
 weak acids, as is seen when the milk becomes sour, but is 
 not coagulated by boiling. Eennin breaks up the casein 
 into two compounds: an albumose and an insoluble cal- 
 cium compound (paracasein calcium, or cheese). Coagu- 
 lated human casein is not as hard as that of cows. The 
 difference is partly due to its chemical composition, but 
 largely to the fact that cows' milk contains more casein 
 and calcium than human milk. It can be made to form 
 a soft and spongy coagulum similar to the human by dilu- 
 tion or by the removal of the calcium compounds. 
 
 The fats of milk are a mixture of stearin, palmitin, 
 and olein, with a small amount of the glycerids of some 
 lower members of the fatty-acid series, butyric, caproic, 
 caprylic, capric, etc. The fat exists as an emulsion, a coat- 
 ing of albumin keeping the globules separate. They may 
 be made to collect by dissolving this coating by a chemical 
 agent, like sulphuric acid. Babcock's method for deter- 
 mining the percentage of fat in milk is based upon this 
 principle. Here the volume of the fat is measured and 
 this gives the relative amount in the milk. If the fat rises 
 for twenty-four hours without such decomposition it should 
 form a layer 10 or 15 per cent, of the depth of the milk, if 
 the latter is normal. 
 
144 MILK. 
 
 The method of obtaining the milk-sugar has been 
 given. (Experiment 34.) 
 
 323. Test the reaction of fresh milk to red and blue 
 litmus-paper. 
 
 324. Determine the specific gravity with an accurate 
 urinometer. 
 
 325. Eemove the fat from milk by a centrifuge, or 
 after standing, and determine the specific gravity again. 
 
 326. Try the same test after adding from 10 to 25 
 per cent, of water to the milk. 
 
 327. In a weighed porcelain or platinum crucible evaporate 
 10 cubic centimeters of milk to dryness on a water-bath and weigh 
 quickly to find the amount of the total solid matter. The drying 
 will take place much more rapidly if a weighed quantity (about 20 
 grammes) of dried sand is added, but the residue cannot be used 
 for the next experiment. 
 
 328. Heat the dried substance in the crucible, at first gently, 
 then until no black remains. The residue is the mineral matter, 
 or ash. There should not be over 1 per cent, of the weight of the 
 milk. 
 
 329. Compare the action of rennin upon cows' and 
 human milk. Try the rennin in cows' milk to which 50 
 per cent, of water and a few drops of ammonium oxalate 
 have been added to remove the calcium salts. 
 
 330. To separate the nitrogenous constituents of 
 milk first precipitate the casein by saturating the milk 
 (skimmed milk can be used) with sodium chlorid. Filter 
 and to filtrate add powdered magnesium sulphate as long as 
 it dissolves, stirring meanwhile. This precipitate is para- 
 globulin, the same compound that is found in the blood. 
 Filter and apply the globulin tests (Experiments 98 to 
 101). Acidify the filtrate with a few drops of dilute acetic 
 acid and boil. The albumin of milk lactalbumin is co- 
 
MILK. 145 
 
 agulated. To determine the quantity of protein subtract 
 the sum of fats, sugar, and ash from the total solids. 
 
 331. Examine with the microscope a drop of milk 
 under a cover-glass. 
 
 332. Destroy the emulsion by adding 10 cubic centi- 
 meters of concentrated sulphuric acid to an equal volume 
 of milk. Let it stand, and the fat rises to the top in large 
 globules. The separation is complete in a few minutes if 
 a centrifuge is used. The volume of the fat can be better 
 seen by using a narrow-necked flask and, after mixing with 
 the acid, nearly filling with warm water. 
 
 333. Fill a 100-cubic-centimeter graduated cylinder 
 to the upper mark with milk and let it stand twenty-four 
 hours. There should be 10 or 15 cubic centimeters of 
 cream. 
 
 334. To DETERMINE THE PERCENTAGE OF LACTOSE 
 IN MILK. Dilute 20 cubic centimeters of milk to 400 
 cubic centimeters with water. Drop in acetic acid slowly 
 until it coagulates; then pass carbon dioxid gas into the 
 liquid fifteen minutes and let it stand until it settles clear. 
 Filter and wash; coagulate the albumin and globulin in 
 the filtrate by boiling. Filter, wash, and use the filtrate or 
 a part of it for the sugar-determination by Fehling's solu- 
 tion as in the determination of glucose (Experiment 33). 
 For every 10 cubic centimeters of the solution which is 
 decolorized 0.067 gramme of lactose is present. 
 
 335. BABCOCK'S METHOD FOB THE DETERMINATION OF FAT 
 IN MILK OK CREAM. When sufficient milk is available, 17.6 cubic 
 centimeters may be taken for each determination. The bottles in 
 which it is treated have a special scale etched upon the rather long 
 neck. 
 
 Mix the milk thoroughly by pouring it several times from 
 one vessel to another. With a pipette graduated at 17.6 cubic 
 
146 THE URINE. 
 
 centimeters measure the milk and pour it into the graduated flask, 
 adding as much as 90-per-cent. sulphuric acid (sp. gr., 1.82). Mix 
 by gently shaking until the curd has completely dissolved, then 
 revolve in the centrifuge at 600 to 800 revolutions per minute for 
 six or seven minutes. Always make duplicate tests, placing the 
 two bottles opposite each other in the machine. Now carefully 
 fill the bottles about to the highest graduation with hot water, 
 which should have been previously made ready, and whirl again 
 for one or two minutes. Holding the bottle in a perpendicular 
 position, read on the scale the differences between the upper and 
 lower margins of the fat which gives the percentage present in the 
 milk. If the test is successful the fat layer is clear or but 
 slightly cloudy. 
 
 With breast-milk where the available amount is frequently 
 limited smaller flasks and less milk may be employed. Cream 
 must be diluted 5 to 10 times with water. 
 
 THE UEINE. 
 
 The urine is a solution which contains the final prod- 
 ucts from the chemical changes in progress in the animal 
 body. A part of these are excreted in the expired air and 
 from the skin, and a still smaller part through the mucous 
 membrane of the intestine, but, if we omit the carbon di- 
 oxid from the lungs, by far the greater proportion of these 
 final products is found in the urine. A study of its com- 
 position and variation, therefore, is often of great value 
 in judging of changes which are going on in the body. 
 
 Among the most common inorganic constituents 
 normally found are the chlorids, sulphates, and phosphates 
 of sodium, potassium, calcium, and magnesium. Of the 
 normal organic compounds there are urea, uric acid and 
 its salts, creatinin, etc. The following, when found in 
 more than minute amounts, may be regarded as patho- 
 logical: Glucose, albuminous substances, blood, bile, pus, 
 fat, mucin, leucin, and tyrosin. Others which are more 
 
THE URINE. 147 
 
 rare will be spoken of later. All of these either are taken 
 as such into the body with the food or are formed in the 
 body by chemical action. The significance of each may 
 depend upon the amount which is present, as well as upon 
 its mere presence or absence. In interpreting the mean- 
 ing of each of the constituents of the urine its method of 
 formation must be considered, as well as the factors which 
 may cause this to vary. 
 
 Considerable variations are found in the composition 
 of urine which has been collected at different times of the 
 day. That which is passed immediately after rising may 
 differ from that excreted an hour or two after the first 
 meal both in the kind and amount of the dissolved solids. 
 Sugar and albumin are more commonly excreted after a 
 meal, and may be found then, yet not be present in the 
 night's urine. In order to obtain a fair sample for testing, 
 the urine should be collected for twenty-four hours and, 
 after mixing, a part taken for analysis. In all quantitative 
 determinations the volume for twenty-four hours must be 
 measured, and when it has been determined how much of 
 the substance is present in the portion tested, the amount 
 contained in the whole day's urine should be calculated. 
 A statement of the percentage alone has little value if the 
 quantity of the urine is not taken into account. To avoid 
 fermentation the vessels should be clean and the tests 
 should be made as soon as possible. 
 
 The average volume of the urine in twenty-four hours 
 is, for an adult, between 900 cubic centimeters and 1200 
 cubic centimeters (30 and 40 ounces). This, however, is 
 subject to great variations. It is increased by diuretics, by 
 diseases, like diabetes and others; it is diminished in febrile 
 diseases, in acute nephritis, in some other diseases of the 
 kidneys, and usually before the fatal termination of a dis- 
 
148 THE URINE. 
 
 ease. Its variation gives indications of the progress of the 
 disease. The volume will be also affected by the amount 
 of drink or liquid food and, in general, varies inversely 
 with the perspiration. 
 
 From the presence of ferments, the urine begins to 
 undergo a change after it has stood a few hours. The re- 
 action becomes alkaline, owing to the production of am- 
 monium carbonate from the urea, and this precipitates 
 some of the solids, so that the liquid loses its transparency. 
 This and other decompositions produce disagreeable odors. 
 
 The odor of normal urine is characteristic. Certain 
 foods and medicines change this; e.g., oil of turpentine 
 gives an odor of violets. When it putrefies the odor is 
 ammoniacal and offensive. In cystitis it is ammoniacal 
 when passed. In suppurative diseases the odor may be 
 putrid. 
 
 Fresh, normal urine is clear, but after standing a 
 short time a cloud of mucus appears. Pathologically it 
 may be cloudy with matters which settle as a sediment. 
 They will be discussed under that subject. 
 
 The color of urine is normally some shade of yellow, 
 varying from nearly colorless to reddish yellow. The 
 former is true of urines containing much water, and the 
 latter where the urine is concentrated and of high specific 
 gravity. The latter is constant in febrile conditions and 
 their severity can here often be judged from the color. 
 Pathologically the urine assumes many other shades. 
 Presence of blood gives a red or, when methaBmoglobin is 
 present, a brown. Jaundice gives a greenish cast or 
 brownish green; melanotic cancer, almost black; typhus 
 or cholera, sometimes blue, from indigo formed by decom- 
 position. Some medicinal or poisonous substances change 
 the color; thus senna or rhubarb gives a reddish or brown- 
 
SPECIFIC GRAVITY. 149 
 
 ish color, which changes to blood-red on adding an alkali. 
 Santonin gives a yellow; carbolic acid and salol a dark 
 green to black; antipyrin and quinine often darken it. 
 
 The specific gravity of urine varies with the amount 
 of water and dissolved solids. With an increase of the 
 water it approaches 1.000, and becomes greater as the 
 solids increase. Hence it is easy to ascertain the amount 
 of the solids which are present. If the second and third 
 decimal figures of the specific gravity are multiplied by 
 2.33 it will give very nearly the weight of dissolved sub- 
 stances in one thousand parts of urine (grammes per liter). 
 Thus, urine of sp. gr. of 1.021 contains about 49 grammes 
 in a liter. 
 
 The specific gravity varies under normal conditions 
 from 1.002 to 1.030. It is usually between 1.015 and 
 1.025. If sugar is not present the variation in specific 
 gravity is due almost entirely to that of the urea. Clin- 
 ically the specific gravity of urine is determined by an 
 hydrometer, called a urinometer, which consists of a spin- 
 dle weighted so as to float in pure water at the line marked 
 1.000. The specific gravity is indicated by the figures on 
 the spindle at the surface of the liquid. Urinometers 
 should always be tested in pure water and if they are not 
 correct the reading in the urine must be changed to corre- 
 spond with the error. Since the specific gravity varies 
 with the temperature some standard temperature must be 
 adopted. Most instruments are graduated at 60 F. (15.6 
 C.). The urine must be brought to this temperature be- 
 fore testing or, if accuracy is desired, the reading corrected 
 by adding 1 in the fourth decimal place for every degree 
 Fahrenheit above 60 or subtracting 1 for each degree 
 below 60. In order to obtain accurate results the degrees 
 should not be too close together on the spindle. 
 
150 THE URINE. 
 
 The importance of a knowledge of the specific gravity 
 is rather to detect marked changes in the urine from a 
 series of observations than to be able to infer the presence 
 of some abnormal constituent, like glucose, which would 
 certainly be found by the subsequent tests. Thus, in 
 nephritis a decrease in specific gravity without change in 
 the volume indicates that the urea is not being excreted 
 and that uremia may be feared. 
 
 336. Test the accuracy of the urinometer in water, 
 then take the specific gravity of urine. The cylinder must 
 be wide enough for the urinometer to float in it without 
 touching. Foam on the liquid should be removed by a 
 piece of filter-paper. 
 
 337. Test with an accurate urinometer the difference 
 in specific gravities of freshly-passed urine when at a tem- 
 perature of from 95 to 98 F. and that at 60 F. or below. 
 
 The reaction of normal mixed human urine passed 
 during twenty-four hours is acid. Quantitative determina- 
 tions of the salts in the urine show that the bases (kations) 
 are not present in large enough amounts to replace all the 
 hydrogen of the acids. This fact is commonly expressed in 
 the statement that the acid reaction is due to acid salts, 
 principally acid phosphates of sodium and potassium. 
 However, since these as well as the other inorganic com- 
 pounds are more or less dissociated, it is preferable to say 
 that the hydrogen ions (hydrions) cause the acidity. The 
 administration of alkaline drugs is followed by the urine's 
 becoming less acid or even alkaline. The same effect is 
 produced by vegetable foods. These contain the potas- 
 sium salts of organic acids citric, malic, tartaric, and 
 others which are oxidized to potassium carbonate in the 
 system. A similar result is brought about a short time 
 after a hearty meal, when hydrochloric acid is being set 
 
REACTION. 151 
 
 free from its salts in the mucous cells of the stomach. 
 The bases which are freed at the same time remain to in- 
 crease the alkalinity of the blood. Part of them pass into 
 the urine, producing the "alkaline tide," or alkaline reac- 
 tion, which is often noticed at this time. The urine of 
 herbivorous animals is normally alkaline from this cause. 
 On the other hand, an acid food or one from which acids 
 are produced during its decomposition in the body will 
 increase the acidity. Such a one is lean meat, which con- 
 tains acid potassium phosphate, and also sulphur and phos- 
 phorus compounds, which form sulphuric and phosphoric 
 acids by oxidation. Hence the reaction of the urine may 
 be to a considerable extent regulated by the selection of 
 foods. 
 
 Upon standing all urine becomes alkaline by fermenta- 
 tion. This is produced by the action of a number of micro- 
 organisms upon the urea, resulting in the formation of 
 ammonium carbonate: 
 
 CO(NH 2 ) 2 + 2H 2 = (NH 4 ) 2 C0 3 . 
 
 If these ferments are introduced into the bladder by 
 an unclean catheter the same action is often produced 
 there. In chronic inflammation of the urinary tract am- 
 monium carbonate is usually present. The latter alka- 
 linity from ammonium carbonate can be distinguished 
 from that produced by sodium and potassium salts by the 
 litmus-paper's resuming its red color after drying, if am- 
 monia were the alkali, but not otherwise. 
 
 In determining the degree of acidity of the urine by 
 the use of a standard alkaline solution, litmus cannot be 
 used to indicate when the neutralization is complete, on 
 account of the interference of the phosphates. 
 
152 THE URINE. 
 
 Excessive acidity 1 of the urine causes, in time, an irri- 
 tation of the urinary passages, and is favorable to the 
 formation of uric acid concretions. Continued alkalinity 
 makes a sediment in the urine, and tends to produce phos- 
 phatic calculi. It also produces irritation or inflammation 
 of the mucous membrane. 
 
 338. Test the reaction of urine with sensitive litmus- 
 paper, and if alkaline determine whether it is caused by 
 ammonium carbonate by the paper's turning red again 
 after drying, or whether a sodium or potassium compound 
 is the alkali by the paper's remaining blue on drying. 
 
 339. To DETERMINE THE ACIDITY OF URINE. To 50 cubic 
 centimeters in a flask add 25 cubic centimeters of l / 10 normal 
 sodium hydrate, and heat to boiling, then remove the flame. 
 Thereupon add 25 cubic centimeters of barium chlorid solution 
 of about 5 or 10 per cent. Filter through a dry filter, and take 
 50 cubic centimeters of the filtrate, corresponding to 25 cubic 
 centimeters of the urine, for testing. Dilute it to about 250 cubic 
 centimeters with water, add a few drops of phenolphthalein for 
 an indicator, then from a burette, Vio normal sulphuric acid until 
 the red color is just destroyed. Subtracting the number of cubic 
 centimeters of acid used from 12.5, the number of cubic centimeters 
 of standard sodium hydrate in the half of the liquid used, gives 
 the number of cubic centimeters of sodium hydrate neutralized by 
 the acid in 25 cubic centimeters of urine. 
 
 340. Collect the urine of the day in periods of three hours 
 each and determine the variations in its acidity. 
 
 341. Take internally sodium acetate in 2 to 3 
 gramme doses, and note its effect upon the reaction of 
 the urine. 
 
 UKEA. 
 
 About 86 per cent, of the nitrogen in the urine of a 
 healthy man has been found to be in the urea, CO(NH 2 ).,. 
 Under pathological conditions, however, it may vary greatly 
 
UREA. 153 
 
 from this. The absolute weight varies between 20 and 40 
 grammes daily, being somewhat less for a woman than for 
 a man. In round numbers, we can say that it is about one 
 ounce in twenty-four hours for the adult male. 
 
 Urea crystallizes in long, colorless, rhombic prisms. 
 It is easily soluble in alcohol and in water; hence it never 
 forms a sediment. It forms double compounds with acids, 
 some .of which, like the. nitric and oxalic acid compounds 
 are not easily soluble, and are used in separating the urea 
 from urine. It forms similar insoluble compounds with 
 many salts of the heavy metals, mercury, copper, etc. 
 
 When urea is brought into contact with a hypobro- 
 mite or a hypochlorite, it is decomposed into carbon dioxid, 
 nitrogen, and water: 
 
 CO(NH) 2 + 3NaOBr = 3NaBr + C0 2 + N 2 + 2H 2 0. 
 
 This decomposition is made use of to determine the 
 amount of urea in urine by measuring the volume of the 
 nitrogen set free. There are a great number of modifica- 
 tions in form of the apparatus employed, Hiifner's, 
 Doremus's, Squibb's, and many others, all based upon the 
 same principle. They do not give absolutely accurate re- 
 sults, but are sufficiently exact for clinical tests, and have 
 the advantage of requiring but a short time for their exe- 
 cution. Where it is desirable to learn accurately the 
 amount of nitrogenous compounds excreted, it is best to 
 find the total nitrogen by KjeldahPs method. The solu- 
 tion of sodium hypobromite should be freshly prepared 
 from bromin and sodium hydrate, as it decomposes on 
 standing. 
 
 Doremus's ureometer for determining the percentage 
 of urea in urine consists of a short graduated tube closed 
 at the upper end. Below it is bent upward and expands 
 
154 
 
 THE URINE. 
 
 to a bulb. The graduations represent for each division 
 0.001 gramme of urea. That is, 0.001 gramme of urea 
 evolves enough nitrogen to fill one division. Since one 
 cubic centimeter of urine is used, weighing very nearly a 
 gramme, nitrogen to fill one division corresponds very 
 nearly to 0.1 per cent, of urea in the urine. With the tube 
 is furnished a 1-cubic-centimeter dropping pipette. 
 
 D 
 
 Apparatus for Determining Urea in Urine. 
 
 An apparatus can be simply and cheaply made, after 
 the principle of SquibFs, from two 4-ounce wide-mouth 
 bottles (see figure). One of these (A) contains a vial (C), 
 which serves to hold the urine. 
 
 Outside (7, in A, is placed the solution of sodium hy- 
 pobromite. B contains water and is connected with A by 
 a rubber tube. When the rubber stoppers are tightly in- 
 serted the urine is brought into contact with the hypo- 
 bromite by tipping A, the nitrogen of the urea being 
 liberated. This forces from B an equal volume of water. 
 The water is collected from the tube D, and when meas- 
 
SOURCE OF UREA. 155 
 
 ured gives the volume of nitrogen set free. The glass 
 tubes (D, etc.) should be of small diameter. If there is 
 no leak in the apparatus, pressing the stopper into the 
 bottle A will force water from B into the tube D, and it 
 should remain full, without running out, as long as the 
 apparatus is not disturbed. 
 
 Urea is probably formed in the liver. Its source is 
 the nitrogenous compounds of the food and the tissues, 
 including the blood, most of the nitrogen of such com- 
 pounds being excreted from the body in the urea. Hence 
 any increase in the destruction of these substances is ac- 
 companied by an increased formation of urea and vice 
 versa. For this reason the urea is considered as a measure 
 of the decomposition of the proteins in the body. 
 
 Some things which bring about an increased decom- 
 position of proteins are: a large amount of nitrogenous 
 food, like meat; excessive exercise, which causes a de- 
 struction of tissue, though here the urea is not propor- 
 tional to the exertion; fevers and inflammations up to the 
 crisis, owing to the rapid loss of muscular tissue. After 
 the crisis it is diminished. In phosphorus poisoning and 
 diabetes mellitus the urea is excessive for the same reason. 
 A greater excretion of water, either from excessive drink- 
 ing or diuretics, carries with it a larger amount of urea, 
 which seems to be thus washed out of the system. 
 
 On the .other hand, less urea is excreted during hunger 
 and sleep, when the metabolism of the body is lessened. 
 Interference with the excretory power of the kidneys like- 
 wise diminishes the urea. This is seen in acute nephritis 
 and other diseases of the kidneys. In such cases the pro- 
 duction of urea is not stopped, but it accumulates in the 
 system, often being accompanied by urasmic poisoning. 
 Since the urea is formed, at least in part, in the liver, we 
 
156 THE URINE. 
 
 find that less is excreted in carcinoma and cirrhosis of this 
 organ. 
 
 The fermentation of urea to ammonium carbonate, 
 caused by the action of micro-organisms, has been already 
 referred to. 
 
 PREPARATION OF UREA. 
 
 342. PROM URINE. (a) If only a small amount is 
 desired, evaporate half a test-tubeful of urine to dryness 
 on the water-bath. Dissolve the urea from the residue 
 with 95-per-cent. alcohol. Allow a drop of the alcoholic 
 filtrate to evaporate on a microscope-slide without the aid 
 of heat. Examine the crystals under the microscope. If 
 the form is not distinct, dissolve in a drop of water and 
 again observe the crystals after this has evaporated. Add 
 a drop of dilute nitric acid to the slide, let it stand a few 
 minutes, then examine the crystals of urea nitrate. (Plate 
 1,6.) 
 
 (b) A larger quantity can best be obtained by evapo- 
 rating half a liter to a liter of urine to a thin syrup upon 
 the water-bath, then cooling it in ice-water, and adding 
 about three times its volume of nitric acid of a specific 
 gravity of 1.3 which has been boiled to expel the oxids 
 of nitrogen and cooled with ice-water. Filter off the 
 urea nitrate through an asbestos or glass-wool filter, wash- 
 ing with a small quantity of ice-cold concentrated nitric 
 acid. Dissolve the crystals in hot water and decolorize by 
 chlorin-water or a small quantity of potassium chlorate. 
 Add, then, small portions of pure barium carbonate as long 
 as it dissolves and until the liquid is neutral. Evaporate 
 the whole upon the water-bath to dryness. Pulverize the 
 residue and dissolve the urea in absolute alcohol, which 
 does not dissolve the barium nitrate. If the alcoholic solu- 
 
SYNTHESIS OF UREA. 157 
 
 tion is colored it can be decolorized by filtering it through 
 bone-black. Distill off the alcohol or allow it to evaporate 
 to obtain the urea. 
 
 343. SYNTHETICALLY. Coarsely powder 50 grammes 
 of potassium ferrocyanid and heat in an iron dish over a 
 Bunsen flame, stirring continually, until it has become a 
 white powder and the lumps show no yellow color when 
 they are broken. If it turns brown the heat is too high. 
 Pulverize the mass as finely as possible in a mortar., mix it 
 thoroughly with half its weight of finely-powdered man- 
 ganese dioxid, and heat in an iron dish under the hood, 
 stirring meanwhile, until the mass glows and becomes thick 
 and sticky. Heat until a small test dissolved in hydro- 
 chloric acid gives no blue color with ferric chlorid. Then 
 allow it to cool ; dissolve the potassium cyanate, which has 
 thus been formed with cold water. Convert this into am- 
 monium cyanate by the addition of 38 grammes of dry 
 ammonium sulphate. Filter and evaporate upon the water- 
 bath at about 60 to 70, at which temperature ammonium 
 cyanate is converted into urea. The potassium sulphate 
 crystallizes out first, and should be removed from time to 
 time. At last evaporate to dryness and dissolve out the 
 urea with absolute alcohol as before. 
 
 344. Mix a few of the . dry crystals with soda-lime 
 and heat in a dry test-tube. The presence of nitrogen is 
 shown by the evolution of ammonia. 
 
 345. Warm some crystals of pure urea in a dry test- 
 tube. It melts, then decomposes, yielding ammonia, which 
 can be identified by litmus-paper. When the substance has 
 solidified, cool it, dissolve in water, make alkaline with 
 sodium hydrate, and add a few drops of copper sulphate 
 solution. The color is due to the presence of biuret, IS[II 2 - 
 CONHCONH 2 . Write the equation for its formation. 
 
158 THE URINE. 
 
 346. To 5 cubic centimeters NaOH add a drop of 
 bromin, and after this has dissolved a few crystals of urea. 
 Explain the result. 
 
 347. PREPARATION OF SODIUM HYPOBROMITE. In a 
 thin glass flask or beaker containing 20 cubic centimeters 
 of water dissolve 8 grammes of sodium hydrate. Cool, 
 and from a dropping pipette or funnel add slowly 2 cubic 
 centimeters of bromin, stirring or shaking meanwhile. 
 Handle the bromin under a hood or in a draft of air to 
 avoid the vapors, which are especially irritating to the 
 eyes and lungs. As the bromin-gas is heavy, it should be 
 held below the level of the face while pouring, rather than 
 above. 
 
 348. Determine the percentage of urea in urine by 
 the use of Doremus's ureometer. First fill the tube with 
 the hypobromite solution and invert it, having no more 
 of the liquid in the bulb than is necessary to keep the tube 
 full. Fill the pipette exactly to the mark with urine, in- 
 sert the lower end into the ureometer, and slowly and 
 steadily force the urine out by compressing the rubber 
 bulb. The urine, being the lighter liquid, rises in the ureo- 
 meter and the urea is immediately decomposed. The car- 
 bon dioxid is dissolved in the solution and only the nitro- 
 gen is collected. No gas-bubbles should be allowed to 
 escape into the ureometer-bulb or back into the pipette, 
 thereby causing a loss. When the foam has disappeared, 
 read off the quantity of gas and calculate the percentage 
 of urea. Duplicate tests should not differ more than 0.1 
 per cent. If the volume of urine in twenty-four hours is 
 known, calculate the weight of urea excreted in that time. 
 
 349. Determine the amount of urea in urine by ap- 
 paratus, page 154. Fill B nearly full of water. Into C 
 by a pipette put exactly 2 cubic centimeters of urine. Out- 
 
DETERMINATION OF UREA AND NITROGEN. 159 
 
 side of C in A put 20 or 25 cubic centimeters of the hypo- 
 bromite. Insert the stopper (E) tightly, thus filling the 
 tube D with water. If it remains full, showing that the 
 apparatus is tight, place an empty beaker under D and 
 gently mix the urine and hypobromite. Avoid as much 
 as possible raising the temperature by holding the bottle 
 in the hand, as the expansion of the gas causes a consid- 
 erable error. Allow it to stand until no more water passes 
 from D, which must remain full of water during the whole 
 test; then measure the expelled water in a graduated cy- 
 linder. 
 
 One gramme of urea contains 371 cubic centimeters 
 of nitrogen; but, when it is decomposed in this manner, 
 only about 354 cubic centimeters are obtained. For ordi- 
 nary clinical purposes the percentage of urea in urine can 
 be calculated from the following formula: 
 
 100 x number of c. c. of N 
 percentage of urea = " 
 
 350. DETERMINATION OF THE TOTAL NITROGEN IN URINE 
 (KJELDAHL'S METHOD* 1 ). I. Prepare the following solutions: 
 
 1. Standard sulphuric acid containing about 25 grammes per 
 liter, of which the strength has been accurately determined. 
 
 2. Standard ammonia, of which about five volumes are neces- 
 sary to neutralize one of the acid. Determine this accurately and 
 calculate the amount of ammonia by weight in 1 cubic centimeter. 
 
 3. Sodium hydrate free from ammonia, and nitric acid, about 
 270 grammes per liter. 
 
 4. Congo-red, of which the solution contains 0.2 gramme in 
 100 cubic centimeters. This is turned red by alkalies and blue by 
 acids the opposite of litmus. 
 
 1 This method can be used for finding the amount of N in 
 most animal and vegetable compounds. 
 
160 THE URINE. 
 
 Have also at hand: 
 
 1. Sulphuric acid, sp. gr. 1.84, free from compounds of nitro- 
 gen. 
 
 2. Yellow mercuric oxid. 
 
 3. Powdered potassium permanganate. 
 
 4. Crystallized sodium thio-sulphate. 
 
 II. Operation. With a pipette measure accurately 5 cubic 
 centimeters of urine. Place it in a flask holding about 250 cubic 
 centimeters, best of hard (Bohemian) glass. Add 0.4 gramme of 
 mercuric oxid and 10 cubic centimeters of the concentrated sul- 
 phuric acid. Lay the flask in a slanting position on a wire gauze 
 over a flame small enough to just bring it to boiling. Perform this 
 operation under a hood or where there is a good draft to carry away 
 the fumes of the acid. Continue the heating until the liquid is 
 colorless or straw-colored, which may require from thirty minutes 
 to an hour. Then remove the flask from the flame and very 
 slowly add to it a small amount of the powdered permanganate 
 until it is colored reddish or greenish. Allow it to cool and pour 
 it into an 800-cubic-centimeter flask which contains from 200 to 
 300 cubic centimeters of distilled water, rinsing the small flask 
 into the large one. The organic matter has been oxidized in this 
 process, the nitrogen being converted into ammonia, which is con- 
 tained in solution as ammonium sulphate. 
 
 The ammonia is now to be set free by sodium hydrate, then 
 distilled into a known amount of standard acid, and its amount 
 found by ascertaining the loss of strength of the acid through its 
 neutralization by the ammonia. For this purpose a Liebig con- 
 denser is to be arranged so that the flask can be connected with 
 the upper end by means of a bent tube; this should be at least 
 Y 4 inch in diameter inside and a foot long, to prevent small drops 
 of the boiling liquid's being carried over. The insertion of a bulb 
 between the flask and condenser, also having the lower end of the 
 bent tube, in the flask, cut off obliquely, will aid in preventing this. 
 Add, now, to the liquid in the flask a few fragments of granulated 
 zinc to make it boil more quietly, about a gramme of sodium thio- 
 sulphate to precipitate the mercury, and 80 cubic centimeters of 
 the sodium hydrate, or enough to make it alkaline. Connect 
 immediately with the condenser through which a stream of cold 
 water is flowing and distill into a 400 or 500 cubic centimeter 
 
URIC ACID. 161 
 
 conical flask (Erlenmeyer) which contains exactly 10 cubic centi- 
 meters of the standard acid (not the concentrated!). Continue 
 the distillation until at least half has been distilled over and the 
 distillate coming from the condenser no longer turns red litmus- 
 paper blue. 
 
 Then find how much ammonia has been taken up by the 
 standard acid. To do this add a few drops of Congo-red solution to 
 the distillate. It will be colored red, because of the acid reaction. 
 From a burette add the standard ammonia, stirring meanwhile, 
 until the red just changes to a blue, when the liquid is neutral. 
 Subtract the number of cubic centimeters of ammonia used from 
 the number which are required to neutralize 10 cubic centimeters 
 of the standard acid. The difference represents the volume of 
 standard ammonia equal to that which was distilled from the 
 oxidized urine. Calculate the weight of NH 3 in this. Fourteen- 
 seventeenth of the NH 3 is the weight of the nitrogen in 5 cubic 
 centimeters of urine. Calculate the percentage. 
 
 URIC ACID ("LITHIC ACID"). 
 
 Uric acid is normally present in solution in the urine 
 of mammals. With birds and snakes it is the principal 
 nitrogenous excretory product. Its formula is C 5 H 4 N 4 3 
 and the constitution of the molecule is probably 
 
 CO 
 
 It is consequently 2-6-8-trioxypurin. 
 
 The daily amount varies much, but averages from 0.2 
 to 0.8 gramme. Except that it must be formed from the 
 nitrogenous compounds in the body, we know little of its 
 production or of the cause and significance of its variations. 
 
162 THE URINE. 
 
 Uric acid is comparatively insoluble in water or acids, 
 but dissolves readily in the fixed alkalies, forming salts of 
 uric acid, or urates. In the urine the acid exists in the 
 form of these salts or united with some organic base. It 
 is a dibasic acid like sulphuric acid, having two atoms of 
 hydrogen which can be replaced by metals. It can thus 
 have two series of salts, the acid and the normal, corre- 
 sponding to HKS0 4 and K 2 S0 4 . Of these classes the nor- 
 mal salts are quite soluble in water, but the acid salts do 
 not dissolve so easily. The acid can be set free from its 
 salts by the use of a stronger acid. The solubility of the 
 acid salts is much less in cold water than in warm. Con- 
 sequently they frequently separate from urine which was 
 clear when passed but has stood in a cold room, and they 
 can then be redissolved by warming. 
 
 When it is pure, uric acid exists in the form of color- 
 less crystals. As it is found in the urine, it, as well as its 
 salts, is always colored yellow to brown by the coloring 
 matter which has been carried down from the urine. The 
 simplest form of crystals is tabular with curved sides and 
 pointed ends. These are frequently united at right angles, 
 making a star-shaped form, two of the rays often being 
 smaller than the other two. In urinary sediments many 
 crystals may be united, making a rosette-like form. In 
 strongly-acid urine the crystals sometimes have jagged 
 edges like the teeth of a broken comb. Many different 
 forms may be obtained by precipitating with various 
 strengths of acid. (Plate II, 11.) 
 
 Uric acid and its salts have, in some degree, the power 
 of reducing copper compounds in an alkaline solution and 
 thus give with Fehling's or Trommer's test results which 
 are similar to those obtained with glucose. When the dry 
 substance is warmed with nitric acid it is oxidized, and 
 
URIC ACID. 163 
 
 then gives with ammonia a reddish-purple salt, which 
 serves to detect and identify the acid. 
 
 The urates as found in the urine are either in solu- 
 tion or form a sediment. The latter is generally amor- 
 phous and is always colored yellow to brown. Acid sodium 
 urate may occur in spherical aggregations of microscopic 
 acicular crystals. Ammonium urate, formed when urine 
 becomes alkaline by fermentation, may be found as 
 brownish spherules covered with irregular spicules, the 
 so-called "thorn-apple" crystals. (Plate II, 9 and 11.) 
 The amount of uric acid in urine is sometimes found by 
 precipitating from a measured volume of urine by hydro- 
 chloric acid, the albumin having first been removed if it 
 is present. After washing the crystals they are weighed. 
 The results thus obtained are too low, because of the slight 
 solubility of the crystals in water. Volumetric methods 
 may also be employed. These are as accurate and no more 
 difficult. 
 
 351. Prepare uric acid from urine by quite strongly 
 acidifying a beakerful with hydrochloric acid. In twenty- 
 four hours the uric acid will have separated. Examine the 
 crystals under the microscope. It can be purified and 
 gradually freed from the color which it derives from the 
 urine by repeatedly dissolving in concentrated sulphuric 
 acid and reprecipitating by diluting with water. 
 
 352. Precipitate from urine the uric acid with acids 
 of varying concentration, acting for different times. Sketch 
 the principal forms obtained. 
 
 353. Dissolve a few of the crystals of the acid in 
 sodium hydrate and add a few drops of Fehling's solution. 
 Boil and the red cuprous oxid will be formed, best seen 
 by the use of a dark background. 
 
164 THE URINE. 
 
 354. To a small quantity of uric acid in a porcelain 
 dish add a few drops of dilute nitric acid and evaporate 
 to dryness, holding the dish over a small flame with the 
 hand in order to avoid heating too highly. A reddish-yel- 
 low residue is left. Pour into the dish a drop of ammonia 
 without at first letting it come directly into contact with 
 the residue. In a short time the residue becomes colored 
 reddish purple. The ammonia may be added directly to 
 the residue if an excess is not used. An excess destroys 
 the color. The addition of a drop of' sodium hydrate 
 changes the color to a bluish purple, which is destroyed on 
 warming. The test is called the murexid test. 
 
 355. PREPARATION OF AMORPHOUS ACID URATES. Dissolve 
 uric acid in a slight excess of sodium hydrate, and then pass carbon 
 dioxid into the cold solution until it is saturated. Acid sodium 
 urate separates in amorphous masses. 
 
 356. Test the solubility of the acid sodium urate by 
 warming with a small quantity of water. It will dissolve, 
 and, if not too much water has been used, will separate out 
 again when it cools. 
 
 357. Prepare crystallized acid urates by dissolving a 
 little uric acid in a warm solution of sodium phosphate. 
 Filter, if necessary, and allow the filtrate to stand and 
 evaporate. The sodium urate will crystallize as masses of 
 acicular crystals. 
 
 358. FOMN'S METHOD FOR THE QUANTITATIVE DETERMINA- 
 TION OF URIC Acnx Prepare a solution in water, one liter of 
 which shall contain 500 grammes of ammonium sulphate, 5 
 grammes of uranium acetate and 6 cubic centimeters of glacial 
 acetic acid. Of this add 75 cubic centimeters to 300 cubic centi- 
 meters of urine in a 500 cubic centimeter flask, mix, and after five 
 minutes filter through a plaited filter. Take two portions of the 
 
HIPPURIC ACID. 165 
 
 filtrate, 125 cubic centimeters each, pour them into beakers, add 
 5 cubic centimeters of concentrated ammonia and let the precipi- 
 tated urates stand until the next day. Filter and wash the urates, 
 using a 10-per-cent. ammonium sulphate solution for this as well 
 as for transferring to the filter. Then spread out the paper and 
 wash off the precipitates into beakers, using about 100 cubic centi- 
 meters of water for each. Add 15 cubic centimeters of concen- 
 trated sulphuric acid and titrate immediately with one-twentieth 
 normal potassium permanganate, containing 1.57 grammes per 
 liter, stopping when the solution is first pink throughout. For 
 each cubic centimeter of permanganate 0.00375 gramme of uric 
 acid has been oxidized. Calculate the amount in the urine, adding 
 a correction of 0.003 gramme for every 100 cubic centimeters of 
 urine employed because of the solubility of the urates. 
 
 Heintz's Method for the quantitative determination of uric 
 acid is less accurate. It consists in adding to 200 cc. of urine 
 which contains no albumin or sugar 20 cc. of concentrated hydro- 
 chloric acid; the precipitated crystals are collected on a weighed 
 filter, washed with water until the drop of the filtrate shows no 
 chlorin reaction with a drop of silver nitrate and nitric acid, then 
 washed with alcohol. After drying three hours at 110 the uric 
 acid is to be rapidly weighed on the paper. It is customary to 
 add to the weight 0.00038 grm. for each 10 cc. of filtrate and wash 
 water as a correction for the acid which is dissolved. 
 
 HIPPURIC ACID (C 6 H 5 COHNCH 2 C0 2 H). 
 
 This occurs normally in the urine, but in that of 
 human beings only in very small quantities. It is found 
 here in larger amounts after the internal use of benzoic 
 acid. It increases with a vegetable diet and is abundant 
 in the urine of herbivorous animals. It forms translucent, 
 four-sided prisms, somewhat soluble in water. The acid 
 can be made synthetically by heating benzoic anhydrid, 
 (C 6 H B CO) 2 0, with glycocoll, NH 2 CH 2 C0 2 H: 
 
 (C 6 H 5 CO) 2 + 2NH 2 CH 2 C0 2 H = 2C 6 H 5 CONHCH 2 CO 2 H +H 2 O . 
 
166 THE URINE. 
 
 When hippuric acid is heated with mineral acids or alka- 
 lies it decomposes again into glycocoll and benzoic acid. 
 
 359. PREPARATION OF HIPPURIC ACID. Take inter- 
 nally 2 grammes of pure sodium benzoate and collect the 
 urine for the next twenty-four hours. Make it strongly 
 alkaline with milk of lime. Warm, filter, and evaporate 
 the filtrate to a syrup on the water-bath. After it has 
 cooled acidify strongly with concentrated hydrochloric acid. 
 Stir and filter, washing with a little very cold water. Dis- 
 solve the crystals in the smallest possible amount of boiling 
 water. To destroy the coloring matter pass chlorin gas 
 into the hot solution until it is light yellow. Then cool 
 it, filter, and wash the crystals with a very little cold water. 
 If they are still colored they can be still further purified 
 by dissolving in water and boiling with a little animal 
 charcoal. Filter, and let the acid crystallize from the 
 filtrate. Sketch the crystals and hand in sketches. 
 
 360. Heat a few of the dry crystals in a glass tube. 
 They melt and turn red, then give, at first, a hay-like odor, 
 afterward the odor of bitter almonds, from the hydrocyanic 
 acid formed. On the cooler part of the tube is a sublimate 
 of benzoic acid. 
 
 361. On a few crystals in a test-tube pour about a 
 cubic centimeter of concentrated nitric acid, and bring to 
 a boil. Evaporate to dryness in a porcelain dish on a 
 water-bath. The residue, when heated in a dry glass tube, 
 gives the odor of bitter almonds. This test can be used to 
 detect small quantities of hippuric acid. 
 
 362. SEPARATION OF GLYCOCOLL, FROM HIPPURIC ACID. 
 Boil in a flask 1 part of the acid for ten to twelve hours with 4 
 parts of dilute sulphuric acid. Use an inverted condenser to pre- 
 vent evaporation. Let the liquid cool and filter out the benzoic 
 acid. Concentrate the nitrate and mix it in a separatory funnel 
 
CREATININ. CHLORIDS. 167 
 
 with ether, which removes the last traces of benzoic acid. Sepa- 
 rate the aqueous solution of glycocoll from the ether, add to it 
 barium carbonate until the sulphuric acid has been neutralized, 
 filter, wash and evaporate the nitrate until the glycocoll com- 
 mences to crystallize. It has a sweet taste, whence its name. 
 
 CREATININ. 
 
 363. To separate the creatinin from urine add to 100 cubic 
 centimeters of the latter 5 to 6 cubic centimeters of saturated solu- 
 tion of sodium acetate, then 20 to 25 cubic centimeters of a 
 saturated solution of mercuric chlorid. The precipitated sul- 
 phates, phosphates and urates are now to be filtered out and the 
 filtrate allowed to stand for twenty-four hours. There is a 
 separation of the creatinin combined with mercury as transparent 
 globules. Make sketches of its appearance under the microscope. 
 
 CHLORIDS. 
 
 In the urine the excreted chlorin, of which there is 
 normally in a day 6 to 10 grammes, is united principally 
 with sodium. There is a small part with potassium as 
 potassium chlorid. The excretion of chlorids in health is 
 increased with salt food and with large quantities of drink. 
 Chlorids are necessary in the fluids of the body' for the 
 proper performance of their functions. When more chlorin 
 is required by the body the chlorids are held back by the 
 kidneys from the urine. When there is a less demand in 
 the body the kidneys excrete the chlorids. Thus, in pneu- 
 monia and other diseases, where there are serous exuda- 
 tions, the chlorids are withdrawn from the circulation to 
 form the constituents of these fluids, as is shown by their 
 decrease in the urine. When the pathological exudations 
 are absorbed the amount of urinary chlorids increases. In 
 fevers there is a decrease in the chlorids of the urine until 
 
168 THE URINE. 
 
 the crisis, then an increase. In chronic diseases the amount 
 of chlorin gives some indication of the digestive power, 
 6 to 10 grammes per day being normal, and less than 5 
 grammes daily showing weakness of digestion, providing 
 that an excessive amount has not been removed by other 
 means, like serous exudations or diarrhoeic discharges. An 
 excessive excretion of chlorin (15 to 20 grammes daily) 
 is found in diabetes insipidus. In dropsical conditions it 
 is a favorable sign, showing the absorption of the fluid. 
 The quantity of chlorin can be determined by ascer- 
 taining how much silver nitrate is required to precipitate 
 it. 
 
 NaCl + AgN0 3 = AgCl + NaN0 3 . 
 
 58.4 parts 170 parts 
 
 To ascertain when the chlorin has all united with the 
 silver a little yellow potassium chromate is added. The 
 silver forms first a white silver chlorid, and when the chlo- 
 rin has been precipitated it forms the red silver chromate. 
 
 364. Acidify a portion of urine in a test-tube with 
 nitric acid and add a little silver nitrate. A white precipi- 
 tate which turns dark in the sunlight indicates the pres- 
 ence of chlorids. 
 
 365. DETERMINATION OF QUANTITY or CHLORIN IN 
 URINE. For clinical purposes the following method is suf- 
 ficiently accurate : Measure with a pipette 10 cubic centi- 
 meters of urine and dilute with about 100 cubic centi- 
 meters of water. Add a few drops of yellow potassium 
 chromate solution ; then allow to flow into it from a burette 
 a solution which contains 17.000 grammes of fused silver 
 nitrate in a liter. As soon as the color of the precipitate 
 changes from white to reddish, read off the volume of silver 
 solution which has been used. Each cubic centimeter of 
 
PHOSPHATES. 169 
 
 this will precipitate 0.00354 gramme of chlorin, equal to 
 0.00584 gramme of sodium chlorid. Calculate the per- 
 centage of chlorin by weight in the urine. The change of 
 color from white to red can be more plainly seen by yellow 
 light (gaslight) than by daylight. There are present in 
 the urine some other substances which are precipitated by 
 silver nitrate like the chlorin. To make approximate cor- 
 rection for these, 1 cubic centimeter may be subtracted 
 from the number used. 
 
 366. A more accurate result can be obtained if the organic 
 matter is first destroyed. To 10 cubic centimeters of urine in a 
 thin porcelain or platinum dish add about 1 gramme of sodium 
 nitrate and 2 grammes of potassium nitrate, both free from 
 chlorids. Evaporate to dryness and carefully heat to fusion. 
 Cool, dissolve in water, slightly acidify with nitric acid, then make 
 exactly neutral with sodium carbonate and titrate with silver 
 nitrate, calculating the amount of chlorin as in the preceding 
 experiment. 
 
 PHOSPHATES. 
 
 The phosphoric acid of the urine is united with two 
 classes of bases: the alkalies, sodium and potassium, 
 and the alkaline earths, calcium and magnesium. The 
 compounds are called, respectively, "alkaline" and "earthy" 
 phosphates. The alkaline phosphates are soluble in water. 
 The earthy phosphates are insoluble in water or alkalies, 
 but are dissolved by acids. They consequently appear in 
 the urine in the insoluble form whenever it becomes alka- 
 line, either by fermentation or by the addition of reagents. 
 They may also be precipitated by boiling. The amorphous 
 white precipitate thus obtained is often mistaken for albu- 
 min. It can be distinguished by being easily soluble in 
 acids, which is not the cr.se with albumin. "When ammonia 
 is present, as in fermentation, the magnesium forms an 
 
170 THE URINE. 
 
 insoluble salt with two bases, NH 4 MgP0 4 . In urinary 
 analysis it is referred to as triple phosphate. It is crystal- 
 line, sometimes in the form of snow-flakes, but more com- 
 monly in prismatic crystals often spoken of as "coffin-lid 
 crystals," from their supposed resemblance to the lid of a 
 coffin. (Plate II, 8.) 
 
 The phosphoric acid of the urine is mainly that taken 
 in the food, but a part conies from the oxidized phos- 
 phorus compounds of the tissues, such as lecithin and the 
 nuclein compounds. The presence of a sediment of the 
 earthy phosphates shows simply that the urine is alkaline, 
 and is no indication that an excessive amount is being ex- 
 creted. Animal foods are richer in phosphoric acid com- 
 pounds than vegetable; hence with these we find more in 
 the urine. 
 
 Experience has shown that there is a diminution of 
 the excreted phosphoric acid in many pathological condi- 
 tions. This is true in most acute infectious diseases, in 
 nephritis, gout, and rheumatism. In diabetes mellitus 
 there is an increase. Still, with the exception of the bones, 
 the tissues of the body contain but comparatively small 
 amounts of phosphorus compounds, and with our present 
 knowledge it is difficult to draw definite conclusions re- 
 garding the decomposition of such tissues from the varia- 
 tions in the eliminated phosphoric acid. 
 
 367. Make a specimen of urine alkaline with sodium 
 hydrate. The earthy phosphates are precipitated in an 
 amorphous form. Examine under the microscope. See 
 that they are dissolved again by acidifying with even a 
 weak acid, like acetic. 
 
 3G8. Filter out the earthy phosphates and test the 
 filtrate for the phosphoric acid of the alkaline phosphates 
 
PHOSPHATES. 171 
 
 by adding magnesia mixture. (This is magnesium sul- 
 phate made alkaline with ammonia and enough ammonium 
 chlorid to dissolve the precipitate first formed.) With 
 phosphoric acid or its salts it gives a white crystalline 
 precipitate. 
 
 369. Form triple phosphate by making urine faintly 
 alkaline with ammonia and allowing it to stand until the 
 precipitate settles. Examine under the microscope for the 
 "coffin-lid" crystals. They can be more abundantly formed 
 for microscopic examination by adding to the urine a little 
 of a solution of magnesium sulphate before making it alka- 
 line with ammonia. 
 
 370. DETERMINATION OF AMOUNT OF PHOSPHORIC ACID. 
 Prepare the following solutions: 
 
 1. Uranium acetate: Dissolve about 34 grammes of crystal- 
 lized uranium acetate in water and dilute to one liter. This solu- 
 tion will be a little too strong. Its exact strength must be found 
 by the method to be described later. 
 
 2. A solution of Na 2 HP0 4 , 12H 2 (crystallized disodium phos- 
 phate), one liter of which shall contain 10.085 grammes of the pure 
 crystallized salt. This salt gives up its water of crystallization 
 when exposed to the air, and cannot then be used. The crystals 
 must be perfectly bright. Fifty cubic centimeters of the solution 
 contain 0.1 gramme of P 2 O B (phosphoric anhydrid). 
 
 3. Solution of sodium acetate of which one liter contains 100 
 cubic centimeters of 30-per-cent. acetic acid and 100 grammes of 
 sodium acetate. 
 
 4. Solution of cochineal made by digesting for some time 1 
 gramme of powdered cochineal in a mixture of 20 volumes of 
 alcohol \vith 60 volumes of water. Filter or decant the liquid. 
 
 Operation. First ascertain the strength of the uranium solu- 
 tion. To accomplish this, measure with a pipette 50 cubic centi- 
 meters of the sodium phosphate solution into a beaker; add 5 
 cubic centimeters of the sodium acetate solution and a few drops 
 of cochineal. Heat to boiling, and then from a burette run in the 
 
172 THE UEINE. 
 
 uranium solution, drop by drop, until a greenish color is produced. 
 The phosphoric acid has then been precipitated. Since 1 cubic 
 centimeter of the uranium solution ought to precipitate 0.005 
 gramme of P 2 6 , exactly 20 cubic centimeters should have been 
 used for the 50 cubic centimeters of sodium phosphate. If this is 
 not the quantity which has been used, first ascertain accu- 
 rately how much is needed and then dilute the uranium solution 
 so that 1 cubic centimeter precipitates 0.005 gramme of P 2 O 6 . If, 
 for instance, 17.5 cubic centimeters have been used instead of 20 
 cubic centimeters there must be added 2.5 cubic centimeters of 
 water for every 17.5 cubic centimeters of the uranium solution. 
 
 The amount of P 2 O 6 in urine can now be determined in the 
 same manner, using urine instead of the sodium phosphate solu- 
 tion. Calculate the percentage of P 2 O 6 present, knowing that there 
 is 0.005 gramme for each cubic centimeter of uranium solution 
 which has been used. 
 
 If it desired to determine the amount of phosphoric acid 
 combined with the alkaline earths (calcium and magnesium), as 
 distinguished from the phosphates of sodium and potassium, the 
 former class can be precipitated from 200 cubic centimeters of 
 urine by ammonium hydrate and, after settling, can be removed 
 by nitration. Wash the precipitate well with ammonium hydrate, 
 then pierce the filter and rinse the precipitate by a jet of water 
 into a beaker. Dissolve it in as small a quantity of acetic acid as 
 possible, add 5 cubic centimeters of the sodium acetate solution, 
 dilute to 50 cubic centimeters and titrate as in the preceding 
 operation. This gives the P 2 O 6 which was united with the calcium 
 and magnesium. If the total has been determined, the difference 
 represents the phosphoric acid originally combined with sodium 
 and potassium. 
 
 SULPHATES. 
 
 The sulphates of the urine are of two classes: (1) 
 those of which the base is a metal, like K 2 S0 4 and Na^SO^ 
 and (2) those in which a part or the whole of the base has 
 been replaced by an organic radical, like KC 6 H 5 S0 4 . 
 
SULPHATES. 173 
 
 Those of the first class are called the inorganic, and the 
 second the organic, or ethereal, sulphates. The latter dif- 
 fer from the inorganic in not forming an insoluble precipi- 
 tate upon the addition of a barium salt as the inorganic 
 do. The two classes can be separated by this means. After 
 the removal of the inorganic sulphuric acid by barium 
 chlorid the organic sulphates can be decomposed by means 
 of boiling hydrochloric acid: 
 
 KC 6 H 5 S0 4 +.H 2 == C 6 H B OH + KHS0 4 . 
 
 The acid will then give the white precipitate of barium 
 sulphate if barium chlorid be added. 
 
 The total amount of combined sulphuric acid excreted 
 by an adult in twenty-four hours is 2 to 3 grammes. It 
 is derived partly from that already formed in the food, 
 which passes without change into the urine, but, for the 
 most part, from the oxidation of sulphur compounds, like 
 albumin, in the body. Variations in the total sulphuric 
 acid in general indicate the rate of oxidation of sulphur 
 compounds. It is increased by taking* such compounds, 
 e.g., by a meat diet. It is decreased by a vegetable diet. 
 
 The organic sulphates normally make up about 
 one-tenth of the total sulphates. The organic bases of 
 these are such compounds as phenol (C 6 H 5 OH), cresol 
 (C 6 H 4 CH 3 OH), indoxyl (C 8 H 6 NOH), etc. These bases are 
 formed by the putrefaction of albuminous substances; con- 
 sequently, when such putrefaction is in progress in the 
 body the organic sulphates increase in the urine. They 
 may be formed in the intestine or absorbed from some 
 other source. ' In the former case they are increased when- 
 ever there is a serious stoppage of the food, as in ileus or 
 in peritonitis with atony of the intestine. In ordinary con- 
 stipation there is no marked increase. In diseases which 
 
174 THE URINE. 
 
 are accompanied by an internal formation of pus there is an 
 increased amount of organic sulphates in the urine, and this 
 fact may be used to judge whether the pus-forming stage 
 has been reached. This is the case in foetid bronchitis, 
 carcinoma of the stomach or intestine, diphtheria, pyaemia, 
 etc. If the formation is from putrefaction in the intestine 
 it will be diminished by taking antiseptic remedies, like 
 calomel, or those which, by their purgative action, remove 
 the contents of the intestine before, this putrefaction has 
 occurred. 
 
 The compound of indol which is found in the urine 
 goes by the name of indican. The indol, 
 
 C 6 H 4 CH 
 
 CH 
 
 formed by the putrefaction of albuminous substances, is 
 oxidized after it has been absorbed from the intestine or 
 elsewhere in the body and becomes indoxyl: 
 
 C 6 H 4 COH 
 
 I II 
 HN CH 
 
 This unites with potassium and sulphuric acid to form 
 indican: 
 
 C 6 H 4 C S0 4 K 
 
 CH 
 
 Indican may be easily oxidized by chlorin or other oxidiz- 
 ing agents, and then forms indigo blue: 
 
 C 6 H 4 CO CO C 6 H 4 
 
 I I I I 
 
 C = C NH 
 
ORGANIC SULPHATES. 175 
 
 Putrefaction of nitrogenous compounds in the small in- 
 testine seems to be more productive of indican than when 
 it goes on in the large intestine. Sometimes the indican is 
 decomposed in the urine, the indigo being set free in the 
 form of blue or red microscopic crystals. It is usually dis- 
 solved as a sulphate, however, until the indigo is formed 
 by an oxidizing agent. It is normally present in large 
 quantities in the urine of the horse, where, because of the 
 long intestine, the residue from the food requires a con- 
 siderable time to pass from the body. 
 
 371. PREPARATION OF POTASSIUM PHENOL SULPHATE 
 (KC 6 H 5 SO 4 ). First prepare, if it is not at hand, potassium pyro- 
 sulphate by mixing 25 grammes of finely-powdered potassium 
 sulphate with 15 grammes of concentrated sulphuric acid, then 
 heating (best in platinum dish). The heating should be done 
 under a hood, to avoid the acid fumes. The heat should be 
 gently applied at first, stirring until all the crystals have dissolved. 
 When it ceases to bubble increase the heat to low redness. Allow 
 it to cool, but before it solidifies it is best to carefully pour it upon 
 a piece of clean sheet iron. Powder finely the potassium pyro- 
 sulphate (K2S 2 O 7 ) thus obtained. 
 
 In a, thin glass flask holding about a liter dissolve 15 grammes 
 of potassium hydrate in 20 or 25 cubic centimeters of water, then 
 add 25 grammes of crystallized phenol (carbolic acid, C fi H 5 OH). 
 When it has dissolved let it cool to 60 or 70 C., and, while stir- 
 ring well, add gradually in small quantities 30 grammes of potas- 
 sium pyrosulphate powdered as finely as possible. Keep it at a 
 temperature of "from 60 to 70 for from eight to ten hours, shak- 
 ing often. Then add about 125 cubic centimeters of boiling 95- 
 per-cent. alcohol, and filter while it is hot. This filtration is best 
 performed in a hot-water funnel that is, one which is surrounded 
 with a hot-water jacket. Otherwise the salt will crystallize out 
 before the liquid has passed through the filter. As soon as the 
 filtrate cools, the potassium phenyl sulphate crystallizes in pearly 
 plates. It should be filtered out and recrystallized from a small 
 quantity of boiling alcohol. 
 
176 THE URINE. 
 
 372. Test a solution of this organic sulphate with 
 barium chlorid. There is no precipitate. Compare the 
 result with that obtained from an inorganic sulphate, like 
 magnesium sulphate, with barium chlorid. 
 
 373. Acidify a solution of an organic sulphate with 
 hydrochloric acid, boil, and add barium chlorid. The acid 
 has decomposed the sulphate; so that a precipitate of ba- 
 rium sulphate is now obtained. 
 
 374. Show that a mixture of the two classes of sul- 
 phates, as in urine, can be detected in this way. First 
 acidify by acetic acid, then, after adding barium chlorid, 
 let the test-tube stand at least half an hour in a beaker of 
 boiling water. The inorganic sulphates are thus precipi- 
 tated as barium sulphate, but not the organic. Filter, and 
 test the nitrate with a drop of barium chlorid. If enough 
 was added at first there will be no precipitate. If there is, 
 more barium chlorid must be used, and the heating re- 
 peated. When the filtrate remains clear, acidify with hy- 
 drochloric acid and boil. The precipitate is from the de- 
 composed organic sulphates united with the barium chlorid 
 previously added. 
 
 375. Insert into a rabbit's stomach a wide, flexible 
 catheter or rubber tubing, passing it through a short piece 
 of glass tubing held between the animal's teeth. Intro- 
 duce by this tube a gramme of ortho-nitro-phenyl-propiolic 
 acid and collect the urine for twenty-four hours. It will 
 contain a large quantity of indican. Eead the literature 
 on the relationship of the above acid to indigo, and explain 
 the formation of indican. 
 
 376. Test urine for indican by adding to half a test- 
 tubeful an equal volume of concentrated HC1. Then add 
 a minute fragment of calcium hypochlorite ("chlorinated 
 lime") and a few drops of chloroform and shake gently. 
 
INDICAN. 177 
 
 Let the chloroform settle to the bottom. If indican is pres- 
 ent in the urine it will be thus oxidized to indigo blue, and 
 this colors the chloroform. A second piece of the hypo- 
 chlorite may be added and the shaking repeated. An ex- 
 cess will destroy the blue color. Instead of calcium hypo- 
 chlorite, a few drops of chlorin water, bromin water, or 
 hydrogen dioxid can be used. This is Jaffe's test. . 
 
 377. Obermayer's reagent for indican contains 2 to 
 4 grammes of ferric chlorid in a liter of concentrated hy- 
 drochloric acid. An excess does not destroy the indigo. 
 Mix equal volumes of the reagent and urine and shake. 
 Indican is oxidized to indigo-blue. This can be taken up 
 by a drop of chloroform, as in the preceding test. 
 
 After the internal use of iodin compounds the iodin is ex- 
 creted largely through the urine. The reagents of either Jaffe's 
 or Obermayer's tests will set it free and it will dissolve in the 
 chloroform with a color which simulates or conceals the indican 
 reaction. A few drops of sodium thio-sulphate solution will, how- 
 ever, destroy the iodin color, but not that of indican. 
 
 378. Add to 10 cubic centimeters of urine a few drops of a 
 solution of potassium iodid and make Jaffa's or Obermayer's test, 
 following this with sodium thio-sulphate. 
 
 379. THE QUANTITATIVE DETERMINATION OP URINARY 
 INDICAN: WANG'S METHOD. This depends upon the conversion 
 of indican to indigo by Obermayer's reagent and, after extracting 
 it with chloroform, determining its amount by titration with 
 standard potassium permanganate. The quantity of urine em- 
 ployed for each test should be modified according to the amount 
 of indican as revealed by a preliminary test, 50 cubic centimeters 
 being enough when much is present and 250 or more when very 
 little is found. 
 
 If the urine is alkaline acidify with acetic acid and precipi- 
 tate with a 20-per-cent. solution of basic lead acetate, avoiding an 
 excess. Find the volume of this mixture, filter, measure off most 
 of the filtrate and to it add as much of Obermayer's reagent. 
 Shake this with renewed portions of chloroform until the latter is 
 
 12 
 
178 THE URINE. 
 
 not colored, separating the two liquids by means of a glass-stop- 
 pered funnel. Distill the chloroform from its solution, dry the 
 residue on a water-bath and wash it with hot water, passing this 
 through a small filter to avoid the loss of indigo. Dry the paper, 
 extract it with warm chloroform, add this to the washed residue 
 and distill off the chloroform. Warm the indigo on a water-bath 
 for five to ten minutes with concentrated sulphuric acid and pour 
 it into .about 100 cubic centimeters of water, rinsing the vessel 
 with a little more water. Filter and titrate with a standard 
 solution of potassium permanganate until the color is colorless or 
 yellowish. 
 
 The permanganate contains about 0.1 gram to the liter. 
 It must be frequently standardized against a solution of oxalic of 
 known strength. The indigo blue corresponding to each cubic 
 centimeter is 1.04 times the weight of the oxalic acid used. From 
 this the weight of indican can be calculated. 
 
 ALBUMINOUS COMPOUNDS OF THE URINE. 
 
 The principal albuminous substance occurring in the 
 urine is serum-albumin. Besides this there may be found 
 there serum-globulin, albumose, fibrin, and possibly pep- 
 tones. The nucleoalbumins also are not uncommon, being 
 often mistaken for mucin. 
 
 ALBUMINURIA. 
 
 Serum-albumin may find its way into the urine either 
 from the kidneys (renal albuminuria) or from serous 
 liquids,- like blood, pus, or lymph, mixing with it at 
 some point in the urinary tract below the kidneys. When 
 it is due to degenerative changes in the kidney it is usually 
 accompanied by epithelium from the tubules, often in the 
 form of cylinders or casts. Changes in the composition 
 of the blood or in the blood-pressure may allow albumin 
 to pass through the kidney. This is seen in anaemic con- 
 
ALBUMINURIA. 179 
 
 ditions, after some poisons, and in some infectious dis- 
 eases, the kidneys in any of these cases not being neces- 
 sarily in a pathological state. Severe muscular labor may 
 cause the temporary appearance of albumin. The quantity 
 present varies greatly under different conditions, and is 
 not necessarily a measure of the severity of the disease. 
 Still comparative tests in the same case will indicate some- 
 thing of its progress. 
 
 The amount of albumin in the urine can be deter- 
 mined accurately by precipitating, drying, and weighing, 
 but the process is a long one for clinical purposes. For a 
 practical test, sufficient to show the variation in amount, 
 Esbach's method can be used. This depends upon precipi- 
 tating the albumin with a solution containing 1-per-cent. 
 picric acid and 2-per-cent. citric acid. The operation is 
 performed in a graduated test-tube, called an albuminom- 
 eter, the height of the precipitate indicating its amount. 
 Variations in temperature greatly affect the height of the 
 precipitate; consequently in comparative determinations 
 the conditions of temperature must be always the same. 
 The results are most accurate when not more than 4 
 grammes of albumin are contained in a liter. 
 
 A more accurate method for the determination of the 
 amount of albumin in urine is to weigh it after coagula- 
 tion. Filter it if it is not clear, then drop in two or three 
 drops of very dilute acetic acid and heat to boiling. Filter 
 on a filter which has been weighed after drying at 100, 
 and wash with warm water first, then with alcohol. Dry 
 at 120 until its weight is constant. For exact results the 
 precipitate and filter must be burned, and the weight of 
 the ash subtracted to get the true weight of the coagulated 
 protein. If globulin is present it will also be found in the 
 precipitate with the albumin. 
 
180 THE URINE. 
 
 TESTS FOR ALBUMIN IN THE URINE. 
 
 If the urine is not clear it must be filtered before 
 testing. 
 
 380. THE HEAT, OR BOILING, TEST. Heat the urine 
 to boiling in a test-tube, then acidify with a few drops of 
 concentrated nitric acid. If albumin is present, a white 
 precipitate remains. The earthy phosphates precipitate on 
 boiling, but are soluble in acids. 
 
 381. HELLER'S TEST. Pour half an inch of concen- 
 trated nitric acid into a small test-tube. From a pipette, 
 the end of which is held just above the surface of the acid, 
 drop the urine slowly or hold the tube in a slanting posi- 
 tion and slowly pour upon the acid an equal volume of 
 urine. If albumin is present a white cloud forms at the 
 point of contact of the two liquids. If the amount is ex- 
 ceedingly small, it may not appear for half an hour. (If 
 biliary pigments are present the ring may be colored. See 
 test for these, Experiment 285.) 
 
 382. Acidify 2 or 3 cubic centimeters of potassium 
 ferrocyanid solution with about 1 cubic centimeter of acetic 
 acid, and fill the test-tube half full of urine. Albumin 
 gives a white, cloudy precipitate. An excess of ferrocyanid 
 interferes with the accuracy of the test. 
 
 383. Add to the urine in a test-tube about one-sixth 
 of its volume of a saturated solution of sodium chlorid, 
 acidify with acetic acid, and boil the upper part of the 
 liquid, holding the tube by the bottom. Albumin gives a 
 white precipitate, which shows plainly above the clear 
 liquid in the lower part of the tube. 
 
 Each of these tests has some objections to it which must be 
 recognized in interpreting the results. By the action of heat and 
 nitric acid some of the albumin is decomposed; hence the first test 
 
ALBUMIN TESTS. 181 
 
 is not as sensitive as some others. This decomposition is greatly 
 increased if the urine is boiled after adding the acid. Besides 
 albumin, there may be precipitated by this test uric acid from the 
 urates in very concentrated normal urine, and also resinous matters 
 after the administration of turpentine or the balsams. The resin- 
 ous compounds are soluble in alcohol, which does not dissolve albu- 
 min. The uric-acid compounds are colored instead of being white, 
 like albumin, and can be filtered out and tested. 
 
 The ring-test with nitric acid is very sensitive. It precipitates 
 other substances than albumin, such as the urates, mucin, and 
 resinous substances. The urates do not form a ring at the plane 
 of contact of the two liquids, but above it; and if the urine is 
 previously diluted with two or three times its volume of water 
 they do not appear. The resinous matters dissolve in alcohol. The 
 mucin precipitate forms a cloud in the upper part of the liquid 
 where the acid is dilute. It dissolves in strong nitric acid. 
 
 Potassium ferrocyanid and acetic acid will detect very small 
 quantities of albumin. Albumose is also precipitated if present. 
 If the acid alone produces a cloudiness it is mucin or resinous com- 
 pounds. These must be removed by filtration before adding the 
 ferrocyanid. 
 
 In the sodium chlorid and acetic acid test the precipitate 
 formed on boiling is acid albumin, which is insoluble in the salt 
 solution. Resinous matters may be precipitated, but not mucin. 
 
 384. DETERMINATION OF AMOUNT OF ALBUMIN IN 
 URINE BY ESBACH'S METHOD. The urine must not have 
 a specific gravity above 1.008, otherwise it must be diluted. 
 If it is not distinctly acid in reaction it must be made so 
 by acetic acid. Fill the albuminometer with urine to the 
 mark U. Add the reagent to the mark R. Close with a 
 cork and mix gently, avoiding hard shaking, which intro- 
 duces air bubbles into the precipitate and thereby prevents 
 its settling. Let it stand at the temperature of the room 
 (60 to 70 F.) for twenty-four hours. The height of 
 the precipitate indicates the number of grammes of albu- 
 min per liter, or parts in a thousand. 
 
182 THE URINE. 
 
 GLOBULIN, ALBUMOSE, AND PEPTONES. 
 
 Globulin is found in the urine only with albumin. 
 It passes into the urine in much the same manner, and 
 has no especial diagnostic value. 
 
 385. To 10 cubic centimeters of the clear urine (fil- 
 tered if necessary) add an equal volume of a saturated 
 solution of ammonium sulphate. The serum globulin will 
 be precipitated, but not the albumin. 
 
 386. Saturate the clear urine with finely powdered 
 magnesium sulphate without warming. Serum globulin 
 is precipitated. This can be removed by filtration and 
 confirmed by the usual tests for globulins. 
 
 Albumose may be formed in urine by bacterial action 
 from albumin. It may easily escape discovery, since it is 
 not coagulated by heat. It is often the precursor of albu- 
 min, and, as such, a knowledge of its presence is impor- 
 tant. Before testing for its presence the other albuminous 
 substances must be removed. After removing albumin by 
 boiling the liquid slightly acidified by acetic acid, albumose 
 can be detected by its giving a precipitate upon saturation 
 with sodium chlorid, which dissolves on heating and re- 
 appears on cooling. 
 
 The results of the latest research have shown that 
 much of what has been regarded as peptone in urine is one 
 of the albumoses which closely resembles it, and it is an 
 open question whether peptones are ever found in this ex- 
 cretion. Nevertheless we may temporarily retain the name 
 peptonuria for the condition, with the understanding that, 
 as our knowledge becomes greater, it may have to be aban- 
 doned. The peptones or albumoses are not normally found 
 in the blood, being converted in the intestinal mucous 
 
PEPTONES. 183 
 
 membrane into another form, probably into an -albumin. 
 When anything interferes with this conversion, or when 
 they are otherwise introduced into the blood, they pass into 
 the urine. Diseases of the intestine, like carcinoma or 
 ulceration, may prevent conversion to albumin, giving rise 
 to enterogenic peptonuria. Peptone and albumose are 
 formed by the decomposition of albuminous substances by 
 other means than by digestion; as, for example, by putre- 
 faction. Diseases which are characterized by a formation 
 of peptones are often accompanied by peptonuria. This is 
 the so-called "pyogenic peptonuria/' It is found when 
 there is much formation of pus in a body-cavity, as in 
 croupous pneumonia and with deep-seated abscesses. 
 
 The following tests can be used with urine containing 
 a considerable peptone : 
 
 387. Heat 50 cubic centimeters of urine to boiling; 
 acidify if necessary with a few drops of acetic acid, filter- 
 ing if it precipitates, and, while hot, add powdered am- 
 monium sulphate to the filtrate as long as it dissolves and 
 until there are some crystals in the bottom. Filter after 
 cooling. This leaves the peptone in solution. To insure 
 complete precipitation of the other proteins the saturation 
 with ammonium sulphate may have to be repeated. When 
 this has been done and no further precipitate results, test 
 portions of the filtrate with (1) tannic acid with twice 
 its volume of water; (2) potassio-mercuric iodid. Each 
 should give a yellowish-white precipitate. The biuret 
 test can be tried, but is not as sensitive as the others. With 
 peptones, if no excess of copper sulphate is used, it should 
 give a pink with no shade of blue. A large amount of 
 sodium hydrate must be present. 
 
184 ' THE URINE. 
 
 FIBRINURIA. 
 
 Through haemorrhage or exudation of serous fluids 
 into the urinary passages the urine sometimes becomes 
 mixed with fibrinogen, and this may form clots or semi- 
 gelatinous masses. It may cover the bottom of the vessel 
 or occasionally cause the whole mass to gelatinize. The 
 fibrin can be filtered from the liquid through muslin and, 
 after washing, can be tested (Experiments 77 and 80) . It is 
 very similar to the deposit of pus from fermenting urine. 
 The pus, however, can be thinned with water. The fibrin 
 is insoluble. 
 
 GLYCOSURIA. 
 
 Glucose is not normally found in large amounts in 
 the urine, although traces are frequently and perhaps 
 always present. More than a slight trace may be re- 
 garded as pathological if it continues for any length of 
 time. A transitory form of glycosuria (alimentary gly- 
 cosuria) is often caused by excessive quantities of carbo- 
 hydrates, especially of sugar in the food. It may be pro- 
 duced by puncture of the fourth ventricle of the brain, 
 by injuries of the pancreas, by a number of medicinal sub- 
 stances which act upon the vasomotor nerves of the liver, 
 such as phloridzin, etc. 
 
 The urine is generally, though not always, of a high 
 specific gravity (1.030 to 1.050), having a light color and 
 a whey-like odor. The daily volume may be increased to 
 ten times the normal, the solids being likewise increased. 
 When poured or shaken it retains the foam for a consid- 
 erable time. 
 
GLUCOSE TESTS. 185 
 
 388. Test diabetic urine with 
 
 1. Trommels test (26). 
 
 2. Fehling's test (27). 
 
 3. Bismuth subnitrate test or Nylandar's test (30). 
 
 4. Phenyl-hydrazin test (31). 
 
 5. Fermentation test (32). 
 
 Notice that the other urinary constituents may mod- 
 ify the results obtained with the solution of pure glucose. 
 
 For the detection of glucose in urine the tests above given 
 may be employed. No one of these, however, is an absolute proof 
 of the presence of glucose. Other constituents of urine have a 
 slight reducing power, and may respond to the tests with alkaline 
 solutions of copper or bismuth, where the action is that of re- 
 duction, for example, uric acid and its salts; creatinin, mucin, and 
 others occurring in smaller amounts have this power of reduction 
 and will reduce Trommer's and Fehling's reagents. The same 
 is true of many medicines which pass into the urine. Trommer's 
 and Fehling's tests are very sensitive under ordinary conditions, 
 but they may fail in some decomposing urines, the ammonia which 
 is present keeping the cuprous oxid in solution. Long boiling will 
 expel the ammonia, and the test may then succeed. Large amounts 
 of uric acid, creatinin, or albumin may act in the same manner, 
 keeping the red oxid from precipitating. It must be borne in mind 
 that the earthy phosphates will always be precipitated when the 
 urine is made alkaline, and consequently, appear in many of the 
 glucose tests. They are always colorless, as can be shown by 
 washing them on a filter, whereas the oxid of copper or bismuth 
 reduced by the sugar are colored. 
 
 In the test with the subnitrate of bismuth the salt is not so 
 easily reduced by other compounds than glucose. Consequently 
 there is not so much danger of mistaking these for sugar. With 
 a very large excess of the alkali this reduction may occur. This 
 is said not to be the case with Nylander's modification of the test 
 (Experiment 30). Albumin is, however, decomposed under such 
 circumstances, giving a black precipitate. It must, therefore, be 
 removed from the solution before the test is made. With this test 
 
186 THE URINE. 
 
 very small quantities of sugar can be detected. Many medicinal 
 substances pass into the urine and react with this test also. 
 
 The phenyl-hydrazin test is not affected by the reducing 
 matters of the urine, but it gives a similar precipitate with milk- 
 sugar. Pure phenyl-hydrazin must be used. If it is the hydro- 
 chlorid, the crystals should be white, not brown. 
 
 The fermentation test is not very sensitive. It may be inter- 
 fered with by the presence of some drugs which stop the action 
 of the yeast. If the urine is not acid, it should be made faintly 
 so with tartaric acid. It can be used to distinguish between glu- 
 cose and lactose. Barfoed's test (Experiment 28) may be employed 
 for the same purpose. 
 
 389. Determine the quantity of sugar in diabetic 
 urine,, using Fehling's solution (Experiment 33). Dilute 
 the urine with a measured volume of water if necessary, 
 and use in the burette^ as was done in the case of the pure 
 glucose solution. 
 
 ACETONE, (CH 3 ) 2 CO. 
 
 Normally acetone is present in the urine only in 
 traces. Pathologically it occurs there in severe diabetes, 
 in fevers, in inanition, and cachectic conditions, as well 
 as in psychoses. In diabetes it often is a precursor of the 
 more dangerous diacetic acid. It appears to be formed by 
 the decomposition of albuminous compounds, and it can 
 be produced in the urine by the use of a diet of such 
 substances. It is a colorless liquid of a fruity odor, which 
 boils at 56.5 C. and which can consequently be readily 
 distilled from the urine. The examination of the urine 
 should be made while it is fresh. 
 
 If a large quantity of acetone is present in urine the 
 latter may be tested directly. For small amounts it is best 
 to distill from about a liter one-fourth of its volume after 
 slightly acidifying with sulphuric acid. Place the distillate 
 
ACETONURIA. DIACETURIA. 187 
 
 in a retort and distill from it about 30 cubic centimeters. 
 This latter portion contains most of the acetone. 
 
 390. LIEBEN'S TEST. To a solution of acetone add 
 a little sodium hydrate, then a solution of iodin in potas- 
 sium iodid and warm. lodoform is produced as a yellow- 
 ish powder having a characteristic odor. After a time it 
 may form six-sided plates, which can be seen with a micro- 
 scope. Notice also the odor. Alcohol gives the same result. 
 
 391. Prepare mercuric oxid by precipitating a little 
 mercuric chlorid with sodium hydrate. Wash by decanta- 
 tion and filter and wash. Add this to some of the acetone 
 solution, shake, and filter. The presence of acetone is 
 shown by its dissolving the oxid. This can be proved by 
 pouring a layer of ammonium sulphid solution on top of 
 the filtrate in a narrow test-tube, when the mercury will 
 be precipitated as a black ring between the two liquids. 
 
 392. LEGAI/S TEST. To the liquid containing ace- 
 tone add a drop of a freshly-prepared solution of sodium 
 nitro-prussid and make alkaline with sodium hydrate. A 
 ruby-red color is produced. In a few minutes it changes 
 to yellow. If it is acidified with acetic acid a carmin or 
 purplish-red color appears when much acetone is present. 
 On long standing (forty-eight hours) this changes to blue. 
 (Compare with the results from WeyPs test for creatinin 
 [Experiment 318], which normally appears in the urine.) 
 In cases of doubt the urine may be distilled and the ace- 
 lone sought in the distillate. Creatinin is non-volatile. 
 
 DIACETURIA. 
 
 Diacetic or aceto-acetic acid (CH 3 COCH 2 C0 2 H) 
 never appears normally in the urine, but is found under 
 the same pathological conditions as acetone. In the fevers 
 
188 THE URINE. 
 
 of childhood it is not so dangerous, but with adults it 
 signals the approach of coma, of which it is, perhaps, the 
 cause, through lowering the alkalinity of the blood. Di- 
 acetic acid is a colorless, strongly-acid liquid, soluble in 
 water and ether. On heating it decomposes below 100 
 to acetone and carbon dioxid: 
 
 CH 3 COCH 2 C0 2 H == CH 3 COCH 3 + C0 2 . 
 
 With ferric chlorid it gives a violet-red solution, which 
 disappears on standing twenty-four hours and more quickly 
 upon boiling. This reaction can be used to detect diacetic 
 acid in the urine. A number of other substances like 
 salicylic and carbolic acids, antipyrin, and the acetates 
 give a somewhat similar reddish color. These are stable 
 at ordinary temperatures, and only that from the acetates 
 is decomposed by boiling. The test should be made upon 
 urine which has been comparatively freshly passed. 
 
 393. Test fresh urine for diacetic acid by adding, 
 drop by drop, a solution of ferric chlorid as long as a 
 precipitate forms. This is ferric phosphate, formed from 
 the phosphates of the urine. Filter, and to the filtrate add 
 a few drops more of ferric chlorid. The diacetic acid gives 
 a violet-red color. Allow it to stand several hours, and 
 notice that it fades and disappears. 
 
 394. If this violet-red color was obtained, boil an- 
 other portion of urine for five to ten minutes, and after 
 cooling repeat the test. If the red color was caused by 
 diacetic acid none will be obtained in this second test, since 
 the acid will have been decomposed by boiling. 
 
 LACTOSURIA. 
 
 Milk-sugar may be found in the urine of women 
 toward the end of pregnancy and a short time after child- 
 
LACTOSURIA. CHOLURIA. 189 
 
 birth. Its presence indicates the absorption of the sugar 
 from the fluid in the mammary gland. It may appear 
 with the interruption of nursing or from stagnation of 
 the milk in the gland. When the gland is well developed 
 and lactose is found in the urine during the period of 
 nursing it shows merely that the secretion of milk is 
 abundant. The chemical reactions of lactose are very 
 similar to those of glucose. The principal differences are 
 that lactose ferments with yeast with difficulty or not at 
 all, and that its power of reduction is less than that of 
 glucose. Still, the distinction between the two as they 
 occur in urine is a matter of some difficulty. 
 
 395. Try the fermentation test with compressed 
 yeast, as in Experiment 32, upon urine containing glucose 
 and that containing lactose, and notice that the former 
 ferments, with the evolution of carbon dioxid, and the 
 latter does not. 
 
 396. Try Barfoed's test (Experiment 28) upon the 
 two kinds of urine, and notice that it responds to glucose, 
 but not to lactose. In this test it must be borne in mind 
 that the other reducing substances of normal urine urates, 
 creatinin., etc. may cause a reduction of the copper salt. 
 
 CHOLURIA. 
 
 In examining the urine for bile two classes of com- 
 pounds are sought for: the biliary acids and the biliary 
 pigments. The biliary acids do not normally occur in 
 urine, except in small amounts. The pigments are more 
 commonly found. In the freshly-passed urine usually 
 only bilirubin is present, but by oxidation it may be 
 changed to biliverdin, etc. Urine which contains bile is 
 generally of a yellowish- to greenish- brown color, and the 
 
190 THE URINE. 
 
 sediment, if it contains epithelial cells, is often colored 
 brown. Upon shaking the urine the foam is yellow or 
 greenish. 
 
 A common cause for the appearance of the biliary 
 constituents in the urine is the obstruction of the bile- 
 duct. This may be either from some abnormal growth 
 or merely from inflammation in the passages. The bile 
 is then absorbed by the lymphatics and excreted through 
 the kidneys. The same result may be produced by any 
 abnormal condition of the liver which interferes with the 
 free passage of the bile. A part of the bile may pass from 
 the blood into the tissues, manifesting itself there by its 
 characteristic color (icterus). 
 
 The biliary coloring matters may be formed in the liver, but 
 they can also be produced by the decomposition of the haemo- 
 globin in the blood and the other tissues of the body, and may 
 pass from here directly into the urine. In this case the urine 
 would contain none of the biliary acids, since they do not appear 
 to be formed outside the liver. A large amount of these acids 
 with the pigments in the urine indicates that the bile comes from 
 the liver (hepatogenous icterus). Some authors have described as 
 a distinct form of icterus that in which the biliary pigments ure de 
 rived from the blood-coloring matters (haematogenous icterus). It 
 seems, however, to be certain that the biliary acids may be absent 
 from the urine even when it contains bile from the liver or gall- 
 bladder. 
 
 397. THE PRODUCTION OF ARTIFICIAL JAUNDICE. Insert a 
 small cannula into the common bile duct of an anaesthetized 
 albino rabbit. Allow a dilute solution of indigo carmine to flow 
 into this from a burette. The conditions are similar to those 
 where the bile is reabsorbed in consequence of some obstruction in 
 the common duct. In a few minutes the mucous membranes show 
 the blue color and it soon is seen under the skin in all parts of the 
 body. Make an autopsy, examining the internal organs to learn 
 how extensive is the diffusion of the color through the tissues. 
 
BILE TESTS. 191 
 
 398. Test biliary urine for the pigments by slowly 
 adding urine from a pipette, to yellow, 1 concentrated 
 nitric acid in a test-tube. The acid remains in the bottom, 
 and between the liquids are seen the colored rings, as in 
 Experiment 285. 
 
 399. To 2 to 3 cubic centimeters of Hammarsten's 
 reagent add a few drops of urine and shake: a green or 
 bluish-green color results if bilirubin is present. With 
 minute amounts of bilirubin or when the urine is dark 
 colored, first precipitate the pigments with a little barium 
 chlorid, allow it to settle, pour off the liquid, and stir the 
 precipitate with 1 cubic centimeter of the reagent. The 
 supernatant liquid is green, converted by increasing 
 amounts of the acid mixture or by yellow nitric acid 
 through blue and violet to red and yellow. 
 
 400. If the urine contains much bilirubin, shake a large 
 test-tubeful or more of urine with half an inch of chloroform; pour 
 off the urine and let the chloroform evaporate on a watch-glass. 
 The bilirubin is left in small, red prisms. It may be purified by 
 dissolving in chloroform, filtering, and again evaporating. These 
 crystals give the play of colors when moistened with nitric acid. 
 They also dissolve in alkalies, and the solution becomes green on 
 standing (biliverdin). 
 
 401. If the urine is dark colored from much urobilin or 
 blood-coloring matters so that the colored rings do not show, test 
 it with Huppert's test. Shake a test-tubeful of the urine with a 
 small amount of milk of lime, then immediately pass into the 
 liquid a stream of carbon dioxid to remove excess of lime. When 
 it is neutral, filter and wash the precipitate, which contains the 
 biliary pigments. Moisten the precipitate on the paper with a 
 drop of moderately-strong, yellow nitric acid and observe the play 
 of colors, from red to green. 
 
 1 The yellow acid can be made by allowing the colorless acid 
 to stand for some time in a strong light. 
 
192 THE URINE. 
 
 402. In urine which is highly colored with other substances 
 the bilirtibin may be identified by Stokvis's test. To 20 or 30 
 cubic centimeters of urine in a test-tube add 5 or 10 cubic centi- 
 meters of a 20-per-cent. solution of zinc acetate. Wash the pre- 
 cipitated bilirubin upon a small filter; then dissolve it by the 
 addition of a few drops of ammonia. The liquid which passes 
 through the filter becomes, after standing, brownish green, and 
 shows the spectrum of bilicyanin: an absorption-band between G 
 and D and one between D and E. If much bile is present the 
 liquid becomes blue upon slightly acidifying. 
 
 403. To the urine add a few drops of very dilute tincture of 
 iodin. A green color results. If the iodin is flowed on to the 
 top of the urine by slanting the tube or by dropping from a 
 pipette a green ring is formed. 
 
 404. Test the biliary urine for biliary acids by dis- 
 solving in it a few crystals of cane-sugar, then dipping in 
 it a strip of filter-paper. Dry the paper and place on it 
 a drop of concentrated sulphuric acid. In a few seconds 
 it becomes violet, best seen by holding it before a window. 
 Too much sugar gives a brown color. 
 
 405. Instead of using concentrated acid make the 
 test with dilute H 2 S0 47 as in Experiment 268. 
 
 It is not advisable to depend upon Pettenkofer's test alone in 
 the urine, as other substances may be present and give reactions 
 similar to those of the bile-acids, although their spectra are differ- 
 ent. The pure bile-acids may, in cases of doubt, be extracted by 
 the following method: 
 
 406. If the urine is highly colored or only a slight amount 
 of bile-acids are present, it may be necessary to extract the latter 
 before testing. Add to the urine lead acetate solution and a few 
 drops of ammonia to make it slightly alkaline. Wash with 
 water the precipitate, which contains the acids, then dry it. Ex- 
 tract it several times with warm alcohol, filtering hot. Make the 
 filtrate alkaline with sodium carbonate, and evaporate to dryness 
 on a water -bath. Dissolve the sodium salts of the bile-acids from 
 the residue with hot, strong alcohol and filter. The bile-salts can 
 
H^IMOGLOBINURIA AND H^MATURIA. 193 
 
 be precipitated by adding ether to the cooled alcohol. They 
 become crystalline on standing, or they can be tested for im- 
 mediately in the alcoholic filtrate with Pettenkofer's or other tests. 
 
 ILEMOGLOBINURIA AND H^MATURIA. 
 
 The haemoglobin is found in the urine in two forms: 
 first, dissolved, no corpuscles being present (haemoglobi- 
 nuria), and, second, in the corpuscles (haematuria). 
 
 The color of urine which contains blood is usually 
 some shade of red, but may be dark brown or even greenish 
 brown when the hemoglobin has been changed to met- 
 hgemoglobin. Very small quantities may not be detected 
 by the eye. The liquid is often more or less cloudy from 
 corpuscles and casts. There may be enough blood present 
 to cause coagulation either in the urinary passages or after 
 the urine is passed. 
 
 The free haemoglobin is produced by the destruction 
 of the corpuscles. This may be due to an injection of 
 substances which dissolve the corpuscles, to the trans- 
 fusion of blood, to the action of some poisons and in cer- 
 tain infectious diseases, like typhus, also after severe burns. 
 In this case the urine should be tested for haemoglobin. 
 If there is a sediment the microscope reveals no corpus- 
 cles. 
 
 Haematuria, where corpuscles are present, is more 
 common. It is due to haemorrhage in some part of the 
 urinary tract. The corpuscles appear as a sediment and 
 are usually not in rolls. They may be shriveled or swollen 
 from standing in the urine. If the hasmorrhage is from 
 the kidney, the blood is usually well mixed with the urine 
 and of a reddish-brown color, the reaction being acid. 
 Blood-casts may be present, and if they are it is a proof 
 
194 THE URINE. 
 
 of a renal haemorrhage. This may occur in Bright's dis- 
 ease, also with malignant renal growths or renal calculi. 
 
 If the haemorrhage is from the bladder the urine is 
 often alkaline, and clots of blood are common. It may be 
 caused by vesical calculi, by cystitis or villous growths, and 
 by carcinoma. 
 
 407. Add a very little blood to highly-colored nor- 
 mal urine, and notice that the bands of oxyhgemoglobin 
 are visible through the spectroscope, although to the eye 
 there may be no indication of its presence. If the urine 
 is too turbid to examine with the spectroscope, it should 
 be filtered, and if the residue is reddish on the paper this 
 should be washed with 5 cubic centimeters of water and 
 the washings examined. 
 
 408. Convert the oxyhasmoglobin into haemoglobin 
 as in Experiment 246, and notice that the two bands 
 change to one. 
 
 409. To urine containing a small amount of blood 
 add enough sodium hydrate to make alkaline, and heat 
 to boiling. The phosphates of the alkaline earths will be 
 precipitated, and the precipitate will be colored reddish by 
 the haematin from the decomposed haemoglobin. If no 
 blood were present the precipitated phosphates would be 
 white. This test will detect very small amounts of blood 
 in urine. If the liquid is very dark colored, it may be 
 necessary to filter and wash the precipitate before its color 
 can be determined. 
 
 410. Examine microscopically the sediment from a urine 
 after recent haemorrhage. Observe the presence of red corpuscles 
 and also the change in their form which takes place after standing. 
 
 The guaiacum-hydrogen peroxid test (249) can also be em- 
 ployed, but its fallacies should not be lost sight of. 
 
MUCINURIA. 195 
 
 MUCINUBIA. 
 
 Both normal and pathological urine often contain a 
 substance which, although similar to true mucin, yet dif- 
 fers from it in many respects. On account of this re- 
 semblance it is often called urinary mucin. The latest in- 
 vestigations indicate that it is a nucleoalbumin. In nor- 
 mal urine it appears after standing as a light, fleecy cloud 
 in the middle of the liquid. Its origin is the mucous mem- 
 brane, principally that of the bladder, ureter, and vagina. 
 In small amounts it has no special significance. In catar- 
 rhal inflammation of the bladder it is abundant. In cys- 
 titis and pyelitis it may give the urine a gelatinous appear- 
 ance. Mucin is also increased in febrile conditions, as well 
 as in nephritis. 
 
 Urinary mucin is precipitated from its solution by 
 alcohol or dilute acetic acid without heating. It may be 
 precipitated by very dilute mineral acids, but dissolves in 
 excess. After precipitation by acids it is soluble in alka- 
 lies. Since nucleoalbumins, like the mucin of urine, are 
 composed of an albuminous substance with a nuclein, they 
 give most of the reactions of the albumins, such as those 
 with potassium ferrocyanid, picric acid, the biuret test, 
 etc. Care is necessary, therefore, to avoid confounding 
 urinary mucin with small quantites of albumin. They 
 can be differentiated by the fact that the mucin is precipi- 
 tated in the cold by acetic acid even after the urine has 
 been diluted with water, while albumin is not. 
 
 411. Dilute normal urine with its own volume of 
 water, acidify a small beakerful with acetic acid and allow 
 to stand until the mucin has separated. Filter and wash 
 with water. 
 
196 THE URINE. 
 
 412. Show that the nmcin dissolves by adding a few 
 drops of an alkali,, like sodium hydrate, and that it is re- 
 precipitated from this solution by acidifying again with 
 acetic acid. 
 
 413. If urine containing much mucin can be ob- 
 tained, apply the general tests for protein and albumin, 
 and notice that it responds to many of them. 
 
 LIPURIA,, OR CHYLURIA. 
 
 An abnormal condition of the urine not uncommon among 
 the inhabitants of the tropics, but more rare among those of cooler 
 climates is the presence of fat. Lipuria, or the appearance of fat 
 in the urine, may be due to an abscess or fatty degeneration of the 
 kidney; to an excessive amount of fat in the blood, as in preg- 
 nancy; or to conditions which produce fatty degeneration of other 
 organs, as the liver, and in phosphorus poisoning, whereby the 
 amount of fat in the blood is abnormally increased. The chyluria 
 of the tropics is due to the action of a parasite, which causes a 
 rupture of the lymph-vessels and allows the lymph to pass into 
 the urinary passages. The urine is often milky, and, on standing, 
 a creamy layer forms. It contains also the other constituents of 
 the lymph, albuminous substances, etc. In cases of lipuria where 
 only a small amount of fat is present it may appear in the form 
 of drops upon the surface, or it may be present in microscopic 
 globules, either free or in the casts or epithelial cells of the sedi- 
 ment. The globules can be perceived with the microscope and 
 separated by ether. 
 
 414. Examine microscopically urine containing fat. 
 
 415. To half a test-tubeful of urine containing fat add one- 
 fifth its volume of ether away from the vicinity of a flame. Mix 
 by shaking carefully. Allow to stand until the ethereal solution 
 of fat rises to the top. Notice that the urine loses its milky ap- 
 pearance. Pour off the ether into an evaporating dish and let it 
 evaporate without heating. Dip a strip of white paper in the 
 residue, and notice that a greasy stain remains after drying. 
 
URINARY SEDIMENTS. 197 
 
 UKINAKY SEDIMENTS. 
 
 Besides the soluble constituents of the urine, there 
 are others which appear as an insoluble deposit upon the 
 bottom of the containing vessel or floating in the liquid. 
 They may be present in the freshly-passed urine or may 
 appear after a time. The former are the more important 
 to the physician, although some conclusions as to the con- 
 dition of the system may be drawn from the latter. 
 
 For the collection of these sediments the best method 
 is by the centrifugal machine, or centrifuge, this requir- 
 ing so little time that the examination can be made before 
 changes have occurred in any of the constituents. The 
 centrifugal machine is essentially an apparatus where 
 tubes or other vessels can be set in rapid rotation. These 
 tubes swing from their upper end, and as the speed is 
 increased assume a horizontal position. The solid con- 
 stituents, being heavier than the liquid, are carried by the 
 centrifugal force to the bottom of the tube. The tubes 
 should contain from 15 to 20 cubic centimeters, and be 
 rotated three to five minutes at a speed of at least 1500 
 revolutions per minute. 
 
 If a centrifugal apparatus is not at hand, the sedi- 
 ment is best collected by allowing the urine to stand in 
 a conical glass vessel, containing 4 to 6 fluidounces, until 
 it has settled. Then decant the supernatant liquid or 
 take, by means of a pipette, a sample of the sediment for 
 testing. 
 
 Urinary sediments can be divided into two groups: 
 the organized or anatomical and the unorganized or 
 chemical sediments. Those of the first group are formed 
 by vital processes, and of the latter by chemical force. 
 
198 URINARY SEDIMENTS. 
 
 Of the unorganized sediments some are soluble in acid 
 and some in alkaline fluids. Their presence depends, 
 therefore, upon the reaction of the urine. They fall 
 naturally into two classes in accordance with their solu- 
 bility, and may be farther subdivided according to their 
 microscopic appearance. The table on the opposite page 
 gives the most common varieties. 
 
 Before examining the sediment, test with litmus-paper 
 the reaction of the urine in which it is found. Then place 
 a drop of urine containing the sediment on a glass slide, 
 cover with a cover-glass, and examine microscopically 
 with a 1 / 2 - or 2 /3-rnch objective. The microscopic ex- 
 amination should be made before the liquid evaporates and 
 leaves on the slide the soluble compounds. A higher power 
 may be used afterward if necessary, but generally the low 
 power is preferable. Chemical reagents may be applied 
 on the slide after removing the excess of urine by a piece 
 of porous paper. Place one drop of the reagent on the 
 slide by the side of the cover-glass. It will flow under 
 the cover-glass, and its action can be observed with the 
 microscope as it comes in contact with the different sedi- 
 ments. Care should be taken not to allow the reagents 
 to touch the microscope-stage. If a low power is used 
 without a cover-glass these tests may be made in a flat 
 watch-crystal. Where large quantities of a reagent are em- 
 ployed, as in testing pus with an alkali, the ordinary chem- 
 ical vessels are to be used. 
 
 Urine containing pus is turbid when freshly passed, 
 and gives the albumin reactions. When much pus is pres- 
 ent it soon falls to the bottom as a thick sediment. Small 
 quantities may remain suspended for a long time. In urine 
 of an acid reaction the pus-corpuscles can be seen. They 
 are circular and colorless, about twice the diameter of the 
 
CLASSIFICATION. 
 
 199 
 
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 g 
 
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 ^J O PH 
 
 
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200 URINARY SEDIMENTS. 
 
 red blood-corpuscles. They appear granular, but when 
 brought in contact with acetic acid the granulation dis- 
 appears and the nuclei, of which there are two or three, 
 become visible. (Plate III, 13.) When the urine becomes 
 alkaline, either by fermentation or by the addition of a 
 fixed alkali, the corpuscles disappear and the mass be- 
 comes very sticky and gelatinous, so that it can be drawn 
 out by a glass rod into long threads. The turbidity of 
 urine which contains pus resembles that from urates or 
 from the earthy phosphates. It does not disappear, how- 
 ever, like the former, by warming, nor, like the latter, upon 
 the addition of acids. 
 
 The source of the pus in the urine may be anywhere 
 in the urinary tract. When it is from the kidney the urine 
 is apt to be acid in reaction, and round-celled epithelium 
 or casts may be present. When it is from the bladder the 
 urine is usually alkaline. It may be due to simple inflam- 
 mation or to some deep-seated affection of the tissues. 
 
 416. Examine microscopically urine containing pus. 
 Remove the excess of liquid around the cover-glass by 
 means of a piece of filter-paper. Put a drop of acetic acid 
 on the slide and let it run under the cover-glass. Notice 
 the change in the appearance of the corpuscles. 
 
 417. Show that the turbidity does not disappear 
 upon warming or upon acidifying. 
 
 418. Make Donne's test for pus by allowing it to 
 settle, then, after decanting off the urine, making it alka- 
 line with sodium hydrate. The mass becomes extremely 
 viscid, as is shown by stirring or pouring. 
 
 419. Show that the pus responds to the albumin re- 
 actions. 
 
 Mucus as a sediment is in the form of a slimy, viscid 
 liquid, sometimes showing the mucous corpuscles. Its sig- 
 
EPITHELIUM. 201 
 
 nificance lias been explained before. It can be made more 
 visible by adding a little tincture of iodin, which colors it 
 brownish. The addition of acetic acid to the urine precipi- 
 tates nrncin as a fibrous mass. 
 
 The epithelial cells, being continually thrown off 
 from mucous surfaces, are normally present in small num- 
 bers in the urine. In such cases they are usually from the 
 bladder and urethra, and, in women, from the vagina. A 
 large increase, however, is indicative of a diseased condi- 
 tion of some part of the urinary system. The cells from 
 different parts of the system are not all of the same shape. 
 (Plate III, 14.) They may be considered as belonging to 
 three classes: the squamous,, or pavement-epithelium; the 
 round celled; and the long, or spindle-celled, epithelium. 
 The squamous epithelium is composed of large, flat, some- 
 what irregular cells with a distinct nucleus. They may be 
 found singly or united, like the stones of a pavement. 
 They occur chiefly in the outer layers of the mucous mem- 
 brane of the vagina and bladder. The round-celled epi- 
 thelium has smaller cells with a nucleus and nucleolus 
 and are found especially in the tubules of the kidneys. 
 They are also found in the deeper layers of the mucous 
 membrane of other tissues, such as the bladder, urethra, 
 and pelvis of the kidney. They are somewhat larger than 
 the pus-corpuscles, and the nucleus can be seen without 
 clearing by acetic acid. The long-celled epithelium is nar- 
 row and somewhat irregular, with a nucleus visible with- 
 out staining. They are found in the outer layer of the 
 membrane of the renal pelvis or in the deep layers of the 
 bladder, ureters, and urethra. 
 
 Although the presence of a single kind of epithelial 
 cells in the urine may give an indication of their origin, 
 still their occurrence in different tissues often renders this 
 
202 URINARY SEDIMENTS. 
 
 a matter of doubt. The condition of the cells, however, 
 may furnish information of the pathological changes which 
 have taken place. If they appear disintegrated or contain 
 fat-globules, their origin is from the locality of some de- 
 generative process, often of a chronic nature. 
 
 Blood-corpuscles are not normal in urine. In freshly- 
 voided urine they may retain their normal shape, that of 
 a biconcave disk. (Plate III, 13, d.) In acid urine, espe- 
 cially where the specific gravity is high, they shrivel after 
 a time, the margins becoming irregular. In dilute urine 
 and where the reaction is alkaline the corpuscles swell, 
 and become biconvex or spherical. If there is much blood 
 the liquid is reddish, but a slight amount may escape de- 
 tection by the unaided eye. When it is present the albu- 
 min reactions can always be obtained. 
 
 By urinary cast is meant an irregularly-cylindrical 
 mass, composed of various materials, which have been 
 formed in the tubules of the kidney, and hence are of 
 about the same size as the tubules. Opinions vary as to the 
 cause of their formation, but most casts appear to be due 
 to the coagulation of the serum which passes into the 
 renal vessels owing to some pathological condition. The 
 presence of anatomical elements such as epithelium, pus, 
 blood, and fat or their decomposition products in the 
 coagulated mass gives the different varieties of casts. 
 
 Epithelial casts are not very common. They consist 
 of cylindrical-shaped masses of round epithelial cells which 
 are thrown off from the tubules by some pathological proc- 
 ess. The cells may appear normal or they may be more 
 or less decomposed and of a granular appearance, or they 
 may contain minute fat-globules. The cells sometimes 
 seem to compose the whole cast and sometimes to be scat- 
 tered over its surface. (Plate III, 16.) When present, 
 
CASTS. 203 
 
 they indicate inflammation of the kidney. When the cells 
 are degenerated, the indications are that the condition is 
 chronic or has existed for some time. 
 
 Blood-casts consist of coagulated blood often contain- 
 ing so many red corpuscles that they are dark and non- 
 transparent. They may be formed whenever haemorrhage 
 occurs in the urinary tubules, and are the best evidence of 
 this. They are quite rare, and may be obscured under the 
 microscope by the free blood-corpuscles. 
 
 Casts of pus are also very rare, but pus-corpuscles 
 are not infrequently seen in other varieties of casts. 
 
 By the decomposition and metamorphosis of epithe- 
 lium, blood- or pus- cells in casts, the so-called granular 
 casts have their origin. They vary greatly in size, shape, 
 color, and in fineness of granulation. (Plate III, 15.) 
 The finely granular cannot be easily seen except with a 
 high power of the microscope, although the coarsely granu- 
 lar may be observed with a low degree of magnification. 
 They often contain unaltered epithelium, leucocytes, and 
 fat-globules. Granular casts indicate degeneration or a 
 long-continued pathological condition of the kidney. 
 
 Occasionally casts of fat-globules are observed. They 
 result from farther metamorphosis of the granular casts. 
 (Plate III, 18, a.) 
 
 In diseases of the kidney, like interstitial suppurative 
 nephritis, where bacteria are abundant, casts composed of 
 these organisms are often seen. They resemble granular 
 casts, but are not destroyed by mineral acids and caustic 
 alkalies, as are the granular casts. High powers of the 
 microscope should be used in their examination. 
 
 Hyaline casts are almost transparent or at most show 
 only a very fine granulation. On account of their great 
 transparency they are extremely difficult to perceive. 
 
204 URINARY SEDIMENTS. 
 
 They may be colored yellow by adding a solution of iodin. 
 In shape they are usually long and narrow. Besides these 
 narrow hyaline casts, which probably are formed in the 
 smaller tubules, there is sometimes found a broader vari- 
 ety. (Plate III, 17.) These have an indented edge and, 
 in consequence of being more highly refractive, can be 
 seen more easily than the narrow ones. They are called 
 waxy . casts. They often give the amyloid reaction, a 
 brown color with iodin, turning blue to violet upon 
 acidifying with sulphuric acid. They are doubtless formed 
 in the renal pyramids. The narrow casts dissolve readily 
 in acetic acid, but the waxy casts remain in it for some 
 time. Hyaline casts not infrequently have anatomical ele- 
 ments blood- and pus- corpuscles, epithelium, etc. 
 clinging to the surface or included within the mass. 
 
 The origin of the hyaline casts seems to be due to 
 the coagulable elements of the blood. It is doubtful if 
 they are ever present in urine which has not been albu- 
 minous. Their presence, consequently, is indicative of 
 the existence of albuminuria. They may be the best evi- 
 dence of such a condition as interstitial nephritis, where 
 the amount of albumin is small. 
 
 Whatever variety of cast may be present in urine, it 
 shows, without any doubt, that there is a pathological con- 
 dition of the kidney and that the accompanying albumin 
 is of renal origin. 
 
 Besides these cylindrical casts there sometimes appear 
 in the urine the so-called cylindroids. These are flat or 
 ribbon-shaped, rather than cylindrical. They are usually 
 about the diameter of casts, but longer, and resemble in 
 their transparency and solubility the hyaline casts, their 
 composition being probably the same. They are found in 
 nephritis and congestion of the kidneys, also in cystitis. 
 
BACTERIA. 205 
 
 They do not seem to be characteristic of any pathological 
 condition of the kidneys, but rather of some irritation of 
 the lower urinary tract which has extended to the kidneys. 
 
 All casts are decomposed by bacterial action. The 
 examination should, therefore, be made as soon as possible 
 after the urine is passed and the casts have settled. This 
 time may be shortened to five minutes by the use of the 
 centrifuge. Without this it will be necessary to let the 
 urine stand several hours or over night. 
 
 420. To examine urine for casts a few drops from 
 the sediment obtained from standing in a conical glass 
 or from the centrifuge is placed upon the microscope- 
 slide; one with a shallow cell on top is best. Cover it 
 with a cover-glass and remove liquid outside by filter- 
 paper. Focus on the sediment, using a l / 5 -ijich objective, 
 then cut off nearly all light from below. When trans- 
 parent or hyaline casts are sought for swing the mirror to 
 one side and upward and throw the illumination upon the 
 slide obliquely or use a small diaphragm. They will be 
 more plainly visible by this means than with a strong illu- 
 mination. After the casts have been detected their cylin- 
 drical shape can be shown by inclining the stage of the 
 microscope so that they roll in the liquid. 
 
 BACTERIA. 
 
 The freshly-voided normal urine contains no bacteria. 
 They may be present, however, under abnormal condi- 
 tions, and will soon appear in normal urine .upon its stand- 
 ing exposed to the air. On account of the large amounts 
 of organic matter dissolved in the urine, it furnishes a 
 medium in which micro-organisms readily grow. This 
 occurs even in the bladder if they are introduced from 
 
206 URINARY SEDIMENTS. 
 
 the outside, as, for example, by means of an unclean cath- 
 eter. Urine containing bacteria is cloudy and is not cleared 
 by filtration. 
 
 The non-pathogenic organisms are found in putrefy- 
 ing or decomposing urine. This is usually not acid and 
 often is strongly ammoniacal. They may be found thus 
 in the urine of cystitis where ammoniacal fermentation is 
 excessive. Some of these are of large size and can be ob- 
 served with a Y 5 -inch objective without staining. (Plate 
 II, 8.) The pathogenic organisms are such as the pus- 
 organisms, the diplococcus of gonorrhoea, and also the 
 bacillus of tuberculosis and the organisms of infectious 
 diseases. They can be examined and isolated by the com- 
 mon bacteriological methods. 
 
 SPERMATOZOA. 
 
 These may be found in the urine of males after coitus 
 or pollution. They may be present in some diseases, like 
 typhoid, and are constantly found in spermatorrhoea. By 
 straining during defecation there may be a slight emis- 
 sion of semen, and consequently the spermatozoa be mixed 
 with the urine. They are readily recognized by their 
 characteristic shape under the microscope, a flattened oval 
 head united with a long thread-like body and tail. (Plate 
 III, 18, c.) They are most abundant in the first and last 
 portions of the urine. 
 
 In freshly voided urine they may have some motion, 
 but this soon ceases. Acids and alkalies, as well as pure 
 water, stop it immediately. Spermatozoa resist putrefac- 
 tion and the action of chemical reagents, even that of 
 strong acids or alkalies. 
 
URIC ACID AND URATES. 207 
 
 URIC ACID AND URATES. 
 
 The properties of these compounds have been given 
 before. As a sediment, the free acid and its salts differ 
 from all others in being colored yellow to brown. They 
 are not abnormal in urine unless they are present as solids 
 when the urine is passed, or are deposited within a few 
 hours, since normal urine throws down uric acid on fer- 
 mentation. The precipitation of these compounds is 
 largely effected by a concentration or an increase in the 
 acidity of the urine. The normal or dibasic urates are 
 readily soluble in water, and do not occur in sediments. 
 When the acidity of the liquid is increased, either by 
 fermentation or by the addition of an acid, half the base 
 is taken from these salts, leaving the monobasic or acid 
 urates, which are soluble with much more difficulty. If the 
 acidity becomes still greater, all the base is removed, leaving 
 the free acid, which is only very slightly soluble in water. 
 Of course, a decrease in the volume of water would be ac- 
 companied by a corresponding increase in precipitated 
 uric acid and its compounds. Hence a sediment of these 
 may appear in the urine without signifying that an in- 
 creased quantity has been formed in the body. Thus, they 
 are common in fevers, when the urine is of small volume 
 and concentrated. Less uric acid is formed in the body 
 with a vegetable diet than with one of meat. 
 
 Uric acid and urates as sediments occur mostly in 
 acid urine and can be usually identified microscopically. 
 (Plate II, 11.) The color is characteristic. The acid is 
 always crystallized, commonly oval or diamond shaped, 
 sometimes visible to the naked eye, often in clusters or 
 rosettes. The urates are commonly salts of sodium, potas- 
 sium, or ammonium. They may be amorphous when ex- 
 
208 URINARY SEDIMENTS. 
 
 amined with high powers. The so-called "brick-dust" 
 sediment is a mixture of the sodium and potassium urates. 
 Sodium urate is also found in fan-shaped clusters or irregu- 
 lar groups of fine crystals, and sometimes in granules. 
 (Plate II, 8.) Ammonium urate makes up the " thorn- 
 apple" crystals: brown, spherical masses covered with 
 curved spicules. (Plate II, 9.) The urates can be differ- 
 entiated from other sediments by being soluble on gently 
 warming the liquid, as well as in alkalies. The urates, as 
 well as the free acid, give the murexid test (Experiment 
 354). Uric acid is especially important when found as a 
 sediment, from its tendency to form calculi. The same is 
 true, to a less extent, of the urates. 
 
 CALCIUM OXALATE. 
 
 This salt is most frequent in acid urine. It may exist 
 in two forms: the crystalline, or "envelope shaped/' and 
 the "dumb-bell shaped." Its appearance under the micro- 
 scope affords the best method of identification. (Plate II, 
 10.) The crystalline form consists of octahedral crystals. 
 They are never large, often being smaller than a red blood- 
 corpuscle. When sufficiently magnified, they have some- 
 what the appearance of the back of a square envelope, the 
 crossed lines being formed by the angles of the crystal. 
 In the shape of the crystals they resemble some forms of 
 triple phosphate, from which they can be distinguished by 
 their insolubility in acetic acid and by their smaller size. 
 The amorphous form of calcium oxalate is disk shaped, 
 with a contraction on opposite sides, so that it somewhat 
 resembles a dumb-bell. Calcium carbonate has much the 
 same form, but dissolves in acids with effervescence. 
 Calcium oxalate is insoluble in acetic, but soluble in hydro- 
 
PHOSPHATES. 209 
 
 chloric acid. The dumb-bell form gives rise to calculi of 
 the bladder. 
 
 Oxalic acid and its salts are found in many fruits and 
 vegetables, like tomatoes, celery, rhubarb, etc., and 
 when these are eaten it appears as the calcium salt in 
 the urine. It is also produced in the body from certain 
 foods, as from large quantities of nitrogenous foods or 
 from the carbohydrates, where the oxidation is not com- 
 plete. A small amount, then, may be normal, and if it is 
 transitory is of no great consequence. If the excretion is 
 continual it is due probably to some constitutional weak- 
 ness. 
 
 PHOSPHATES. 
 
 The phosphates of the alkalies, being readily soluble 
 in water, do not appear as urinary sediments. The phos- 
 phates of calcium and magnesium are insoluble in water 
 or alkalies, although they dissolve in acids. They, conse- 
 quently, appear as sediments whenever the urine becomes 
 alkaline, but are not found in acid urine unless the acid 
 reaction is very faint. They can be distinguished from 
 other urinary sediments by dissolving in acetic acid with- 
 out effervescence. 
 
 Triple phosphate, NH 4 MgP0 4 , is a salt of phosphoric 
 acid having two bases, ammonium and magnesium. When 
 it is made by precipitating a phosphate by ammonia and 
 magnesium sulphate the crystals are usually stellate or 
 snow-flake formed. As it is made in the urine, however, 
 they are more commonly in the form of rhombic prisms. 
 The terminations of the prisms are commonly truncated; 
 so that the crystals have a shape which approaches that 
 of the end of a coffin, and this gives rise to the common 
 appellation: "coffin-lid crystals." (Plate II, 8.) The 
 
 14 
 
210 URINARY SEDIMENTS. 
 
 angles may not be so truncated and the long axis of the 
 crystal may be so much shortened that it assumes the form 
 of an octahedron, like the calcium oxalate. Unlike the 
 latter, it is soluble in acetic acid. Calcium phosphate in 
 the urine is usually amorphous, and always colorless. It 
 is formed when the urine becomes alkaline in the absence 
 of ammonia. To the unaided eye it resembles pus, but 
 differs from it in its solubility in acids. In acid urine the 
 acid phosphate, CaHP0 4 , may crystallize in long prisms, 
 usually in clusters. Tribasic calcium phosphate, Ca 3 - 
 (P0 4 ) 2 , is colorless and amorphous. (Plate II, 7.) 
 
 The presence of phosphates may be due1;o an excessive 
 formation in the body, and they are then usually accom- 
 panied by systemic disturbances. Alkalinity of the urine 
 causes their appearance when there is no excess. This may 
 be from the food or medicine, from an increase in the alka- 
 linity of the blood, or from fermentation. Excessive men- 
 tal work is often accompanied by phosphatic sediments. 
 Their long-continued presence may excite fear of the 
 formation of calculi. Their temporary appearance is a 
 matter of no grave significance. In urine which has stood 
 for a time after its passage they are the most common of 
 the sediments. 
 
 421. Drop ammonia into normal urine until it is 
 slightly turbid, and after it has settled examine the sedi- 
 ment with the microscope. It is a mixture of the amor- 
 phous calcium phosphate and crystalline triple phosphate. 
 To obtain a larger amount of the latter add to the urine 
 a little magnesium sulphate before it is made alkaline. 
 
 422. Precipitate sodium phosphate with magnesium 
 sulphate after making alkaline by ammonia. Notice the 
 difference in the shape of these stellate crystals under the 
 microscope and those usually formed in the urine. Try 
 the solubility of both forms in acetic acid. 
 
CALCIUM SALTS. TYROSEN. 211 
 
 423. Make normal urine alkaline with sodium hy- 
 drate and examine the precipitated calcium and magne- 
 sium phosphates with the microscope. Try their solubility. 
 
 CALCIUM SULPHATE. 
 
 This does not often occur as a sediment. It may be 
 found in acid urines as long prisms united in clusters. 
 (Plate II, 12, a.) 
 
 424. Prepare crystals of calcium sulphate by pre- 
 cipitating a rather dilute solution of calcium chlorid with 
 a few drops of sulphuric acid. Dissolve the precipitate in 
 boiling water, filtering hot if all does not dissolve. It will 
 reprecipitate upon cooling. Examine with the microscope. 
 
 CALCIUM CARBONATE. 
 
 This compound is often found in alkaline urine with 
 calcium phosphate. It appears as a sandy powder which, 
 when examined microscopically, is seen to consist of 
 spherical bodies formed of concentric layers or to have 
 the dumb-bell shape of calcium oxalate. (Plate II, 9.) 
 It dissolves readily in acetic or other acids, with the evolu- 
 tion of carbon dioxid gas. 
 
 TYROSIN. 
 
 Tyrosin is not often found as a sediment because of 
 its solubility in water, but it sometimes appears as such, 
 though never in a normal condition of the system. It 
 crystallizes in minute needle-shaped crystals, which are 
 usually aggregated into clusters or sheaves. (Plate II, 
 12, c.) Its microscopic appearance is the best means of 
 identifying it. The chemical tests have been given. 
 
212 SYSTEMATIC TESTING OF URINE. 
 
 Tyrosin in the urine has the same source as in diges- 
 tion the decomposition of protein compounds. It is im- 
 probable that it comes from the intestine, but from other 
 parts of the system. It is indicative of retrograde met- 
 amorphosis of the nitrogenous tissues. Thus, it is present 
 in acute atrophy of the liver, in suppurative processes, and 
 in phosphorus poisoning, which is accompanied by degen- 
 eration of the liver. Leucin is often found at the same 
 time. (Plate II, 12, ft.) 
 
 FAT. 
 
 The appearance and significance of fat in the urine 
 (lipuria) has already been discussed. 
 
 * 
 
 SYSTEMATIC TESTING OF TJKINE. 
 
 In the systematic testing of urine the course is often 
 varied, as the symptoms may point to the likelihood of 
 the presence or absence of certain substances. The quan- 
 titative tests may be made use of or not according to 
 circumstances. The following are the determinations 
 which are most important, with the tests which may be 
 employed : 
 
 1. Amount passed in twenty-four hours. 
 
 2. Color ^ XT . 
 
 f Normal or abnormal. 
 
 QCy (If the latter, what is the cause? 
 
 4. Odor J 
 
 5. Chemical reaction. 
 
 If alkaline, is it from NH 3 or fixed alkalies? 
 (Experiment 338). 
 
 6. Specific gravity at 60 F. (15.5 C.). 
 
 7. Urea: percentage and amount in twenty-four 
 
 hours (Experiment 348 or 349). 
 
SYSTEMATIC TESTING OF URINE. 213 
 
 8. Glucose. 
 
 General test, Trommels or Fehling's (Experi- 
 ments 26 and 27). 
 
 Confirmatory tests (Experiments 29, 30, and 31). 
 Quantitative test, Fehling's (Experiment 33). 
 
 9. Acetone. 
 
 Experiments 390, 391, and 392. 
 
 10. Diacetic acid. 
 Experiments 393 and 394. 
 
 11. Albumin. 
 
 General test, heat and HN0 8 (Experiment 380). 
 Confirmatory tests (Experiments 381, 382, and 
 
 383). 
 Quantitative test, Esbach's (Experiment 384), 
 
 or weighing. 
 
 12. Blood. 
 
 General tests, spectroscope (Experiments 245, 
 246, and 247) ; also corpuscles in sediment. 
 Confirmatory test, guaiacum test (Experi- 
 ment 249; also Experiment 409). 
 
 13. Bile-pigments. 
 
 General test, colors with yellow HN0 3 (Experi- 
 ment 398). 
 
 Confirmatory tests (Experiments 401, 402, and 
 403). 
 
 14. Bile-acids. 
 
 Experiments 404, 405, and 406. 
 
 15. Peptone. 
 Experiment 387. 
 
 16. Organic sulphates. 
 Experiment 374. 
 
 17. Indican. 
 Experiments 376 and 377. 
 
214 SYSTEMATIC TESTING OF URINE. 
 
 18. Uric acid: amount. 
 Experiment 358. 
 
 19. Total nitrogen : percentage and amount in twenty- 
 
 four hours. 
 Experiment 350. 
 
 20. Chlorin: amount. 
 Experiments 365 or 366. 
 
 21. Phosphoric acid: amount. 
 Experiment 370. 
 
 22. Identification of sediments if present. (Page 
 
 199.) 
 
 I. UNORGANIZED. 
 
 (A) Crystalline. 
 Uric acid. 
 Calcium oxalate. 
 Calcium phosphate. 
 Triple phosphate. 
 Other rarer compounds. 
 
 (B) Amorphous. 
 Urates. 
 Phosphates,, etc. 
 
 II. ORGANIZED. 
 
 Pus. 
 
 Mucus. 
 
 Blood-corpuscles. 
 
 Bacteria. 
 
 Spermatozoa. 
 
 Epithelium: kind and probable source. 
 
 Casts: kind and probable source. 
 The proof of the presence of any abnormal constit- 
 uent should not be allowed to rest upon one test, but sev- 
 eral should be tried. 
 
VARIATIONS WITH FOOD. CALCULI. 215 
 
 425. THE EFFECT OF FOOD ON THE COMPOSITION OF THE 
 URINE. Let a number of subjects each select food of a different 
 class and eat only this for twenty-four hours, collecting all the 
 urine for the period. The following dietaries will give a variety: 
 
 1. Largely animal. 
 
 2. Vegetable. 
 
 3. Rich in purins, sweetbreads, etc. 
 
 4. Purin free milk, eggs, wheat bread, butter, cheese. 
 
 5. Low in nitrogen. 
 
 6. No food. 
 
 Determine volume, specific gravity, color, reaction; amounts 
 of nitrogen, iiric acid, phosphoric acid, urea. Report results. 
 
 URINARY CALCULI. 
 
 The constituents of calculi are the same as those of 
 the chemical sediments, and the causes which give rise to 
 the formation of the latter will also favor the production 
 of calculi in the bladder. To these various names are 
 applied, according to their size: sand, gravel, stone, and 
 calculi, or concretions. They vary from the microscopic 
 to aggregations as large as an orange. They are generally 
 not composed of a single material, but have at the centre a 
 nucleus, and this is surrounded by layers, often of two or 
 more compounds in alternation. The nucleus may be a 
 mass of foreign matter, or it may be a clot of blood or a 
 particle of one of the sediments around which material, 
 perhaps of a different kind, can be deposited. Uric acid 
 concretions are the most common. They are brown in 
 color, rough of surface, and brittle. The form of the 
 crystals cannot be seen, but they give the murexid test. 
 They dissolve in sodium or potassium hydrate, from which 
 solutions the uric acid may be precipitated in the crystal- 
 line form by the addition of a mineral acid. Uric acid 
 calculi are formed only in an acid urine. 
 
216 URINARY CALCULI. 
 
 The urates are often found mixed with the uric acid 
 deposits or with those of calcium oxalate. The ammonium 
 salt is the most abundant. They are generally small, 
 grayish, and rather soft. They give the murexid test. 
 They are deposited from acid urine, except the am- 
 monium urate, which is formed in an alkaline solution. 
 
 Calcium oxalate concretions are commonly of large 
 size and are very hard. The surface is rough and warty. 
 They are called "mulberry calculi" from the resemblance 
 of the surface to that of the fruit. The urine is generally 
 acid, unless where the presence of the stone has produced 
 cystitis. They are often dark in color from the blood which 
 has been incorporated with them. 
 
 The phosphates can only be present in calculi when 
 the urine is alkaline. They are generally rather soft and 
 easily broken. Calcium phosphate has a chalky appear- 
 ance. Triple phosphate, NH 4 MgP0 4 , is found with other 
 substances. It is more commonly on the outside of the 
 stone, being precipitated by the alkaline reaction produced 
 by the presence of the concretion in the bladder. A mix- 
 ture of the triple phosphate and calcium phosphate is fusi- 
 ble with the blow-pipe and is known as the "fusible cal- 
 culus/' 
 
 Calcium carbonate is not common, although found 
 occasionally. 
 
 The analysis of calculi is made by the use of chemical 
 methods. The stone should be broken or, better, if it is 
 large enough, sawed through the middle. This shows 
 the layers of which it is composed and the nucleus. If 
 there appears to be any difference in the layers, they should 
 be tested separately. Heat a piece upon platinum foil and 
 notice whether it fuses and whether it is combustible or 
 not. If it fuses it is an indication of a phosphate of cal- 
 
ANALYSIS. 217 
 
 cium and triple phosphate. If it is combustible it consists 
 of organic compounds. 
 
 Blackening when ignited is evidence of organic mat- 
 ter, but if slight it may be merely mucus arising from 
 irritation of the bladder, and not an essential part of the 
 calculus. Ignition on the foil will divide the constituents 
 into two classes, although both may be present. 
 
 COMBUSTIBLE, OK INCOMBUSTIBLE, OR 
 
 ORGANIC. INORGANIC. 
 
 1. Uric acid. 1. Calcium phosphate. 
 
 2. Ammonium urate. 2. Calcium oxalate. 
 
 3. Calcium carbonate. 
 
 4. Triple phosphate. 
 
 5. Urates of K, Na, and Ca. 
 
 If it is composed largely or entirely of organic matter 
 try the murexid test (Experiment 354) for uric acid and 
 urates. If inorganic compounds are present, powder a piece 
 and treat in a test-tube with 2 or 3 cubic centimeters of 
 dilute hydrochloric acid. Carbonates dissolve with effer- 
 vescence of carbon dioxid gas, the others without. Warm, 
 if necessary. Filter, if it does not give a clear solution. 
 To one-fourth of the filtrate in a test-tube add sodium 
 hydrate until it is alkaline, and test for ammonia by hang- 
 ing in the tube a strip of moist red litmus-paper, being 
 careful that it does not touch the side of the tube which 
 is wet with the sodium hydrate. The tube can be allowed 
 to stand corked over night or the ammonia-gas can be ex- 
 pelled from the liquid by boiling. If present it will turn 
 the paper blue. 
 
 To the remainder of the solution in hydrochloric acid 
 add ammonia until it is alkaline, acidify with acetic acid, 
 and boil. If there is a precipitate, filter. 
 
218 
 
 ANALYSIS OF CALCULI. 
 
 Precipitate is cal- 
 cium oxalate. Test 
 after washing and 
 drying by heating 
 to a bright-red heat 
 on platinum foil. 
 After cooling it 
 should turn moist 
 red litmus - paper 
 blue. 
 
 To the filtrate add ammonium oxalate, 
 boil, and, if there is a precipitate, filter Avhile 
 hot. 
 
 A white precipi- 
 tate shows cal- 
 cium, probably 
 originally present 
 as phosphate or 
 carbonate. 
 
 The filtrate is to be 
 tested for magnesium 
 and plwsphoric acid. For 
 Mg make one-half alka- 
 line with ammonia and 
 if the liquid remains 
 clear, add sodium phos- 
 phate. A fine, white 
 crystalline precipitate 
 with either reagent in- 
 dicates Mg. For phos- 
 phoric acid make re- 
 mainder acid with strong 
 HNO 3 and add ammo- 
 nium molybdate. A yel- 
 low precipitate appears. 
 
 Urates of K, Na, and Ca can be found by boiling the 
 powdered calculus in water, filtering, and testing the fil- 
 trate by the murexid test. Or if it is evaporated to dryness 
 and the residue is ignited on platinum the sodium and 
 potassium will remain as carbonates, giving an alkaline re- 
 action to litmus-paper. 
 
 THE METEIC SYSTEM. 
 
 In all work in modern chemistry the metric system 
 of weights and measures is employed. The unit of length 
 is the meter (39.37 inches); of weight is the gramme (or 
 gram), which is the weight of 1 cubic centimeter of water 
 at 4; and, of capacity, the liter, which has the volume of 
 1 cubic decimeter. 
 
METRIC SYSTEM. 219 
 
 MEASURES OP LENGTH. 
 
 10 millimeters = 1 centimeter. 
 
 10 centimeters = 1 decimeter. 
 
 10 decimeters = 1 meter. 
 
 10 meters = 1 decameter. 
 
 10 decameters = 1 hectometer. 
 
 10 hectometers = 1 kilometer. 
 
 MEASURES OP WEIGHT. 
 
 10 milligrammes = 1 centigramme. 
 10 centigrammes = 1 decigramme. 
 10 decigrammes = 1 gramme. 
 10 grammes = 1 decagramme. 
 10 decagrammes = 1 hectogramme. 
 10 hectogrammes = 1 kilogramme. 
 
 MEASURES OF VOLUME. 
 
 10 milliliters = 1 centiliter. 
 
 10 centiliters = 1 deciliter. 
 
 10 deciliters = 1 liter. 
 
 10 liters = 1 decaliter. 
 
 10 decaliters = 1 hectoliter. 
 
 10 hectoliters = 1 kiloliter. 
 
 The following are especially to be remembered: 
 
 One gramme is the weight of 1 cubic centimeter of 
 water measured at 4 C. 
 
 A liter contains 1000 cubic centimeters and a liter of 
 water weighs, therefore, 1000 grammes. 
 
220 WEIGHTS AND MEASURES. 
 
 The following are convenient in the conversion of the 
 weights and measures of one system into another: 
 
 1 meter =39.37 inches. 
 
 1 foot =0.304 meter. 
 
 1 liter = 61.03 cubic inches = 1.06 U. S. qts. 
 
 1 liter =33.81 U. S. fluidounces. 
 
 1 gramme = 15.43 grains. 
 
 1 grain = 0.0648 gramme. 
 
 1 ounce (apoth.) =31.1 grammes. 
 
 1 ounce (avoirdupois) =28.35 grammes. 
 
 1 pound (apoth.) =373.2 grammes. 
 
 1 pound (avoirdupois) = 453.6 grammes. 
 
 EEAGENTS. 
 
 FEHLING'S SOLUTION. Make up and preserve in two 
 parts: A and B. 
 
 (A) Dissolve 34.64 grammes of crystallized, non- 
 effloresced copper sulphate (CuS0 4 , 5H 2 0) in water and 
 make up the volume to 500 cubic centimeters. 
 
 (B) Dissolve 173 grammes of pure, crystallized Eo- 
 chelle salt (sodium and potassium tartrate) and 50 grammes 
 of sodium hydrate in water, and bring the volume to 500 
 cubic centimeters. 
 
 Before using mix equal volumes of A and B. 
 
 NYLANDER'S EE AGENT. Dissolve in 100 cubic centi- 
 meters of water 2 grammes of subnitrate of bismuth, 
 4 grammes of Eochelle salt, and 10 grammes of NaOH. 
 
 ESBACH'S REAGENT contains, in a liter, 10 grammes 
 of picric acid and 20 grammes of citric acid. 
 
REAGENTS. 221 
 
 OBERMAYER'S REAGENT FOR INDICAN contains 2 to 4 
 grammes of ferric chlorid in a liter of concentrated hydro- 
 chloric acid. 
 
 MILLON'S EEAGENT. Dissolve 1 part of mercury in 
 2 parts of nitric acid (sp. gr., 1.42), first at ordinary tem- 
 perature, then with the aid of heat. When it has dis- 
 solved add twice its volume of water, and after several 
 hours decant the reagent from any sediment that may be 
 present. 
 
 HAMMERSTEN'S REAGENT FOR BILE PIGMENTS consists 
 of 1 volume of nitric acid and 19 volumes of hydrochloric 
 acid, each 25 per cent. When it has become yellow by 
 standing, dilute with four times its volume of alcohol. 
 
 BARFOED'S REAGENT contains acetic acid and cupric 
 acetate of such a strength that there shall be about 1 per 
 cent, of each when mixed with the sugar solution to be 
 tested. 
 
 GUNZBURG'S EEAGENT FOR HC1. Dissolve 2 grammes 
 of phloroglucin and 1 gramme of vanillin in 100 cubic 
 centimeters of alcohol. 
 
 BOAS'S EEAGENT FOR HC1. Dissolve 5 grammes of 
 resorcin and 3 grammes of cane-sugar in 100 cubic centi- 
 meters of dilute alcohol. 
 
 Methyl-violet ^ 
 
 A solution in water containing about 1 
 Trorjseolin 00 
 
 per cent, of the coloring matter. 
 Alizarin sodium 
 
 sulphonate t 
 
 Phenolphthalein, a 1-per-cent. alcoholic solution. 
 Dimethyl-amido-azobenzene, a 0.5 per cent, alcoholic 
 solution. 
 
222 
 
 REAGENTS. 
 
 lodin, a 1-per-cent. solution of potassium iodid in 
 water with a few crystals of iodin. 
 
 Of the common reagents, the following strengths may 
 be conveniently used: 
 
 Barium chlorid ^j 
 
 Ammonium hydrate 
 
 A . y, . , > 10 per cent, in water. 
 
 Ammonium chlorid { 
 
 Tannic acid J 
 
 Ammonium oxalate 
 Ammonium molybdate 
 Potassium ferrocyanid 
 Potassium ferricyanid 
 Lead acetate 
 Sodium phosphate 
 Ferric chlorid 
 Cupric sulphate 
 Mercuric chlorid 
 Sodium hydrate 
 Silver nitrate 
 
 5 per cent, in water. 
 
 Picric acid 
 
 A saturated solution in 
 
 Lime-water, or calcium hydrate J water. 
 
 Sulphuric acid, 10 per cent.; pour 1 volume into 18 
 volumes of water. 
 
 Nitric acid, 10 per cent.; 1 volume of acid and 6 of 
 water. 
 
 Hydrochloric acid, 5 per cent.; 1 volume of acid and 
 6 of water. 
 
 Acetic acid, 6 per cent. 
 
INDEX. 
 
 Absorption from stomach, test, 
 
 91. 
 
 Acetic acid in stomach, 73. 
 Aceto-acetic acid, 187. 
 Acetone in urine, 186. 
 Acid albumin, 46. 
 
 from digestion, 78 
 Acid phosphates in gastric juice, 
 
 73, 82. 
 tests for, 83, 87. 
 
 in urine, 170. 
 
 Acidity of gastric juice, deter- 
 mination, 85. 
 
 urine, determination, 152. 
 Acrolein, 28. 
 Albuminates, 46. 
 Albuminoids, 58. 
 Albuminous substances, 34. 
 
 classification, 38. 
 
 in urine, 178. 
 Albumins, 39. 
 
 in urine, 178. 
 
 limitations of tests, 
 
 94 
 
 Albuminuria, 178. 
 Albumose from digestion, 78, 
 99. 
 
 in urine, 182. 
 
 Alcohol, production, 13, 16. 
 Alkali albumin, 47. 
 Alkaline phosphates, 169. 
 Alloxuric bases, 136. 
 Ammonium urate, 163. 
 Amphoteric reaction, 142. 
 Amylopsin, 99. 
 
 Amyloses, 2. 
 
 Animal body, composition, 1. 
 
 charcoal, 132. 
 | Anti compounds, 49. 
 
 Babcock's test for fat in milk, 
 
 145. 
 Bacteria as ferments, 62. 
 
 in urine, 205. 
 Bacterial casts, 203. 
 | Barfoed's test, 15. 
 Bile, 122. 
 
 -acids, 123. 
 
 in urine, 189, 192. 
 preparation, 127. 
 salts, preparation, 125. 
 tests for, 130, 191. 
 -pigments, 124. 
 in urine, 191. 
 preparation, 129. 
 tests for, 130, 191 
 Biliary calculi, 128. 
 
 mucin, 122. 
 Bilifuscin, 125. 
 Biliprasin, 125. 
 Bilirubin, 124. 
 Biliverdin, 124. 
 Biuret test, 37. 
 Blood, 101. 
 -casts, 203. 
 
 coagulation, 102, 105. 
 -corpuscles, 101. 
 
 determination of num- 
 ber, 102. 
 isolation, 104. 
 
 (223) 
 
224 
 
 INDEX. 
 
 Blood-fibrin, 106. 
 
 in urine, 193, 202. 
 
 -plasma, 101. 
 
 -reaction, 101. 
 
 -serum, 101, 106. 
 
 specific gravity of, 101. 
 determination, 104. 
 
 -stains, tests for, 121. 
 Boettger's test, 15. 
 Bone, 132. 
 Bone-black, 132. 
 Brain, 141. 
 Butter-fats, 143. 
 Butyric acid, 90. 
 
 in stomach, 73. 
 
 preparation, 65. 
 
 tests, 90. 
 
 Calcium carbonate in urine, 211. 
 
 oxalate in urine, 208. 
 
 sulphate in urine, 211. 
 Calculi of bladder, 215. 
 
 analysis, 216. 
 Cane-sugar, 20. 
 Carbohydrates, 1. 
 
 classification, 2. 
 
 reactions, 22. 
 Carbonic oxid haemoglobin, 114, 
 
 117, 118. 
 Carnin, 135. 
 Cartilage, 132. 
 Casein, 55, 143. 
 
 preparation, 54. 
 Casts in urine, 202. 
 
 bacterial, 203. 
 
 blood, 203. 
 
 epithelial, 202. 
 
 fatty, 203. 
 
 granular, 203. 
 
 Casts in urine, hyaline, 203. 
 
 pus, 203. 
 
 waxy, 204. 
 Cellulose, 11. 
 Cerebrin, 142. 
 Cheese, 55, 143. 
 Chlorin in urine, 167. 
 Cholalic acid, 123, 127. 
 Cholesterin, 123. 
 
 preparation, 128, 142. 
 Choluria, 189. 
 Chyluria, 196. 
 Chyme, 75. 
 
 Coagulated albumin, 51. 
 Coagulation, 36, 43. 
 Coffin-lid crystals, 171. 
 Collagen, 58, 131. 
 Colloid, 4. 
 Connective tissues, 131. 
 
 digestion of, 75. 
 Creatin, 135, 141. 
 Creatinin, 135, 141. 
 
 separation from urine, 167. 
 Crystalloid, 4. 
 Cylindroids in urine, 204. 
 Cystin, 47. 
 
 Dextrin, 8. 
 Dextrose, 12. 
 Diacetic acid, 187. 
 Diaceturia, 187. 
 Dialysis, 4. 
 Digestion, gastric, 75. 
 
 pancreatic, 94, 96. 
 
 salivary, 67. 
 Disaccharids, 2. 
 
 Earthy phosphates, 169. 
 
 in urinary sediments, 209. 
 
INDEX. 
 
 225 
 
 Egg-albumin, crystallization, 42. 
 
 purification, 41. 
 Elastin, 61. 
 Emulsion, 23. 
 
 Enterogenic peptonuria, 182. 
 Enzymes, 62. 
 Epithelial casts in urine, 202. 
 
 cells in urine, 201. 
 Essential oils, 25. 
 Extractives, 135, 139. 
 Fat in urine, 196. 
 
 in milk, determination, 143, 
 
 145. 
 Fats, 23. 
 
 digestion of, 97. 
 Fatty acids, 24. 
 
 casts, 203. 
 
 Fehling's test, 14, 17. 
 Fermentation, 62. 
 
 in stomach, 72. 
 
 in urine, 148, 151. 
 
 of glucose, 16. 
 Fibrin, 51, 89. 
 
 in urine, 184. 
 
 preparation, 102. 
 Fibrinuria, 184. 
 Foods, composition of, 1. 
 
 Gall-stones, 125. 
 Gastric digestion, 75, 77. 
 juice, 71. 
 
 collection, 81. 
 preparation of artifi- 
 cial, 77. 
 testing, 81. 
 tests, interpretation, 92. 
 
 limitations of, 92. 
 Gelatin, 59. 
 Globulins, 44. 
 
 Globulins in urine, 182. 
 Glucose, 12. 
 
 in urine, 184. 
 
 preparation of pure, 14. 
 
 tests, 14, 15, 16. 
 in urine, 185. 
 
 limitations, 185. 
 Glycerin, formation, 23. 
 Glycocholic acid, 123. 
 Glycocoll, 123, 166. 
 Glycogen, 8 . 
 Glycosuria, 184. 
 Glycuronic acid, 22. 
 Gmelin's test, 130. 
 Granular casts, 203. 
 Granulose, 4. 
 Grape-sugar, 12. 
 Guaiacum test for haemoglobin. 
 
 117. 
 Guanin, 135, 136. 
 
 Hsematin, 107, 114. 
 Hsematogen, 56, 58. 
 Hsematogenous icterus, 190. 
 Haematoporphyrin, 107, 115. 
 
 preparation, 120. 
 Hsematuria, 193, 202. 
 Haemin, 107, 114, 118. 
 Haemochromogen, 107, 115. 
 
 preparation, 121. 
 Haemoglobin, 107. 
 
 derivatives, 107. 
 
 quantitative determination, 
 109. 
 
 spectrum, 102, 108, 116. 
 Haemoglobinuria, 193. 
 Haines's test, 14. 
 Heart-burn, 73. 
 Hemi compounds, 49. 
 
226 
 
 INDEX. 
 
 Hepatogenous icterus, 190. 
 Hippuric acid, 165. 
 Huppert's test, 130. 
 Hyalin casts, 203. 
 Hydrochloric acid of gastric 
 
 juice, 72. 
 tests, 84, 85. 
 Hypoxanthin, 135, 136. 
 
 Icterus, 190. 
 
 Indican, 174, 176. 
 
 Indigo, 174. 
 
 Indol, 174. 
 
 Indoxyl, 174. 
 
 Inorganic sulphates, 173. 
 
 Inversion, 3. 
 
 Invertin, 63. 
 
 Invert-sugar, 20. 
 
 Iron of animal body, source, 57. 
 
 Isotonic solution, 102. 
 
 Kephyr, 19. 
 
 preparation, 66. 
 Keratin, 61. 
 KjehldahPs determination of 
 
 nitrogen, 159. 
 Koumiss, 19. 
 
 Lactalbumin, 144. 
 Lactic acid, fermentation, prep- 
 aration, 65. 
 in stomach, 72. 
 tests, 88. 
 sarco-lactic, 137. 
 Lactose, 19. 
 
 in urine, 188. 
 preparation, 19. 
 quantitative test, 145. 
 tests, 20. 189. 
 
 Lacosturia, 188. 
 
 Laking, 101. 
 
 Lanolin, 124. 
 
 Lead plaster, 24, 28. 
 
 Lecithin, 30. 
 
 Leucin, from digestion. 95, 97. 
 
 in urine, 212. 
 
 preparation, 99. 
 
 tests, 100. 
 Lipuria, 196. 
 Lithic acid, 161. 
 Liver-starch, 8. 
 Liver-sugar, 8. 
 
 Maltose, 21. 
 Malt-sugar, 21. 
 Methsemoglobin, 107, 113. 
 
 preparation, 120. 
 Metric system of weights and 
 
 measures, 218. 
 Milk, 142. 
 Milk-sugar, 19. 
 
 in urine, 188. 
 Millon's test, 39. 
 Monosaccharids, 2. 
 Mucin, 52. 
 
 in urine, 195, 200. 
 Mucinuria, 195. 
 Mucoids, 52. 
 Murexid test, 164. 
 Muscular -tissues, 134. 
 Muscle plasma, 135. 
 
 preparation, 138. 
 
 -serum, 139. 
 Myelin, 30. 
 Myogen, 138. 
 Myosin, 45, 138. 
 
 Nitrogen, quantitative determi- 
 nation, 159. 
 
INDEX. 
 
 227 
 
 Nuclein bases, 56, 136. 
 Nucleins, 56. 
 Nucleoalbumins, 53. 
 Nylander's test, 15. 
 
 Oils, 25. 
 
 Olein, 25. 
 
 Olive-oil, 30. 
 
 Organic sulphates, 173. 
 
 preparation, 175. 
 Organized ferments, 62. 
 Ossein, 59. 
 Oxyhaemoglobin, 107, 111. 
 
 preparation, 119. 
 
 spectrum, 112, 116. 
 
 Palmitic acid, preparation, 28. 
 
 Palmitin, 25. 
 
 Pancreatic, digestion, 94, 96. 
 
 juice, 93. 
 Paranucleins, 56. 
 Pentoses, 21. 
 Peptone, 48. 
 
 in urine, 181. 
 Pepsin, 73, 77. 
 
 preparation, 64, 75. 
 
 test, 90. 
 
 valuation, 80. 
 Pettenkofei-'s test, 126. 
 Phenyl-hydrazin test, 15. 
 Phosphates in calculi, 216. 
 
 gastric juice, 73, 83. 
 
 urine, 169, 209. 
 
 Phosphoric acid, determination, 
 171. 
 
 source, 170. 
 Phosphorized fats, 30. 
 Plasma, blood-, 101. 
 
 muscle-, 135. 
 Polysaccharids, 2. 
 
 Protagon, 141. 
 Proteins, 32. 
 
 classification, 34. 
 
 compound, 51. 
 
 crystallization, 42. 
 Proteoses, 48. 
 Ptyalin, 69. 
 
 preparation, 69. 
 Purdy's test, 18. 
 Purin, 136. 
 
 bases, 136. 
 Pus-casts, 203. 
 
 in urine, 198. 
 Pyogenic peptonuria, 183. 
 
 Reagents, 220. 
 Rennin, 74. 
 
 preparation, 78. 
 
 test, 79. 
 Rigor mortis, 135, 137. 
 
 Saliva, 66. 
 
 pathological, 68. 
 Saponification, 23. 
 
 in digestion, 97. 
 Sarcin, 135. 
 
 Sediments in urine, 197. 
 Sjoqvist's quantitative test for 
 
 HC1, 86. 
 Soaps, 24, 27. 
 
 Sodium hypobromite, prepara- 
 tion, 158. 
 
 urate, 163, 208. 
 Spermatozoa, 206. 
 Starch, 4. 
 
 tests, 5, 6. 
 Steapsin, 97. 
 Stearin, 25. 
 Sucrose, 20. 
 Sulphates in urine, 172. 
 
228 
 
 INDEX. 
 
 Sulphocyanates, in saliva, 67. 
 Syntonin, 45. 
 
 Tanning, 59. 
 Taurin, 123. 
 
 preparation, 128. 
 Taurocholic acid, 123. 
 preparation, 127. 
 Test-meal, 81. 
 Tests, Arnold's for lactic acid, 
 
 89. 
 Babcock's for fats in milk, 
 
 145. 
 
 Barfoed's for sugar, 15, 19. 
 Biuret for albumins, etc., 
 
 37. 
 
 Boettger's for sugar, 15. 
 Esbach's for albumin, 179, 
 
 181. 
 
 Fehling*s for sugar, 14, 17. 
 Gmelin's for bilirubin, 130. 
 Guaiacum for blood, 117. 
 Haines's for glucose, 14. 
 Heller's for albumin, 181. 
 Huppert's for bilirubin, 130. 
 Jaffa's for indican, 177. 
 KjehldahTs for nitrogen, 
 
 159. 
 
 Legal's for acetone, 187. 
 Lieben's for acetone, 187. 
 Millon's for albumins, 39. 
 Murexid for uric acid, 164. 
 Nylander's for sugar, 15. 
 Obermayer's for indican, 
 
 177. 
 Pettenkofer's for bile acids, 
 
 126. 
 Phenyl-hydrazin for sugar, 
 
 15. 
 Purdy*s for sugar, 18. 
 
 Tests, Sjoqvist's for acidity of 
 gastric juice, 86. 
 
 Toepfer's for acidity of gas- 
 tric juice, 85. 
 
 Trommer's for sugar, 14. 
 
 Xanthroproteic for albumi- 
 nous substances, 37. 
 Toepfer's test, 85. 
 Triple phosphate, 171. 
 
 in sediments, 209. 
 Trommers' test, 14. 
 Trypsin, 94, 95. 
 Tryptophan, 94, 96. 
 Tyrosin, from digestion, 95, 97. 
 
 in urine, 211. 
 
 preparation, 99. 
 
 tests, 100. 
 
 Unorganized ferments, 62. 
 
 urinary sediments, 199. 
 Urates, 163. 
 
 in calculi, 216. 
 in sediments, 207. 
 Urea, 152. 
 
 preparation, from urine, 
 
 156. 
 
 synthetic, 157. 
 quantitative determination, 
 
 153, 158. 
 
 Uric acid, 136, 161. 
 in calculi, 215. 
 in sediments, 207. 
 quantitative determination, 
 
 164. 
 Urinary calculi, 215. 
 
 analysis, 216. 
 casts, 202. 
 mucin, 195, 200. 
 sediments, 197. 
 
INDEX. 
 
 229 
 
 Urine, amount, 147. 
 color, 148. 
 constituents, 146. 
 daily variations, 147. 
 fermentation, 151. 
 odor, 148. 
 reaction, 150. 
 specific gravity, 149. 
 systematic testing, 212. 
 
 Vegetable parchment, 12. 
 
 Waxy casts, 204. 
 
 White fibrous tissue, 131. 
 
 Xanthin, 135, 136. 
 Xanthin bases, 136. 
 Xanthoproteic reaction, 37. 
 
 Zymase, 63. 
 Zymogens, 62, 76. 
 
DAY AND 1 
 OVERDUE. 
 
 _ -r- "^ 
 
 LD 2l-50m-8,'33 
 
UNIVERSITY OF CALIFORNIA LIBRARY