QP IC-NRLF C7 372 DIRECTIONS FOR A PRACTICAL COURSE IN CHEMICAL PHYSIOLOGY w. BIOLOGY LIBRARY DIRECTIONS FOR A PRACTICAL COURSE IN CHEMICAL PHYSIOLOGY BY W. CRAMER, PH.D., D.Sc. SECOND EDITION LONGMANS, GREEN, AND CO 39 PATERNOSTER ROW, LONDON NEW YORK, BOMBAY AND CALCUTTA 1915 All rights reserved. PRINTED AT HE DARIEJM J'RE EDINBURGH PREFACE. IN writing this book for the use of medical students the author has departed from the method usually followed. The subject matter is practically limited to experiments and deductions from the experiments, and is not meant to supply the full and ordered information obtainable from a text-book of physiological chemistry. The arrangement of the work differs from that generally followed, in that the student is at the outset provided with substances familiar to him, such as a potato, an egg, lard, butter, etc. From these he prepares, by simple chemical manipulations, the proximate constituents and their decomposition pro- ducts, and studies their chemical reactions and physical properties. In this way he is introduced to the subject without interposing complex chemical conceptions, which the usual arrangement of dividing the subject into the study of carbohydrates, fats, and proteins necessarily involves. In teaching practical physiological chemistry to students, who have had only an elementary training in organic chemistry, it seems on the whole more satisfactory and more scientific to impose no tax on the faith of the pupil, even if the work covered is less comprehensive than it might otherwise be. The knowledge acquired in such a course as outlined in this book is at least real, and questions beyond the scope of personal demonstrations by the student may well be left to treatment in lectures. In order to induce the student to record his own observations, to draw his conclusions from them, and to correlate the facts observed in 343086 IV the laboratory with the theoretical matter taught in the systematic lectures or in the text-books, the text is interspersed with questions which the student is supposed to answer. In describing the experiments special care has been taken to refer to the numerous technical details that must be observed and the fallacies that must be excluded in order to obtain a trustworthy result. PHYSIOLOGY DEPARTMENT, UNIVERSITY OF EDINBURGH, March 1914. PREFACE TO THE SECOND EDITION. IN the present edition the essential features of the first edition, which appear to have found favour with teachers in other universities and colleges, have been retained. The sequence of the experiments in the first part of the book has been altered in order to obtain a more logical arrangement. A few unessential experiments have been replaced by experiments designed to familiarise the student with the use of the polari- meter. The most essential addition consists in directions for some simple experiments on blood coagulation. There is perhaps no chapter in physiology which it is more difficult to teach convincingly, or which puts a greater strain on the faith of the student. The experiments have been selected with the view of demonstrating to the student some -of the most essential facts concerning the coagulation of blood. These essential facts appear to have been somewhat lost sight of, since recent text-books and publications treat almost exclusively only of the last stage of blood coagulation, namely of the nature and mode of the action of the coagulating agent, and omit to deal with the preliminary stages of the process, namely the origin of the coagulating agent and the conditions under which it is produced. The tests for aceto acetic acid and acetone are not those usually given in text-books. They are recommended as the result of personal observation. PHYSIOLOGY DEPARTMENT, UNIVERSITY OF EDINBURGH, December 1914. CONTENTS. PART I. I'AGE ANIMAL AND VEGETABLE TISSUES AND FLUIDS. POTATO i Preparation of Starch from Potato. COMPLETE HYDROLYSIS OF STARCH BY ACIDS. FERMENTATION OF GLUCOSE 3 Preparation of Glucose and Alcohol from Starch. PARTIAL HYDROLYSIS OF STARCH BY DILUTE ACIDS AND BY FERMENTS 8 Preparation of Dextrine and Maltose from Starch. LARD, OLIVE OIL 9 Solubility of Fats. HYDROLYSIS OF FATS BY ALKALI. SAPONIFICATION - 9 Fatty Acids of Lard. Emulsification of Fats. EGG ii Osmosis. Lipoids of Egg Yolk. Proteins of Egg White. General Protein Reactions. MILK - 17 Separation of Constituents. Preparation of Caseinogen and Lactose. Milk Fat. Its P^atty Acids. Comparison of Lactose with Glucose, Maltose, and Cane Sugar. Clotting of Milk by Rennet. FLOUR AND BREAD - 21 Proteins and Carbohydrates of Flour and Bread. MUSCLE AND GLAND 22 Proteins of Muscle and Gland. LIVER - 23 Carbohydrates of Liver. Glycogen. Iron in Liver. NERVOUS TISSUE 25 Separation of Lipoids of Nervous Tissue : Cholesterin, Phosphatides, Cerebrosides and Phospho-Cerebrosides. Histochemistry of Lipoids. VI 11 BLOOD 28 Coagulation of Blood. Haemolysis. Oxidising Ferment of Blood. Oxygen-Capacity of Blood. Chemistry of Blood Pigment. Absorption-Spectra of Haemoglobin and its Derivatives. Chemistry of Blood-Serum. Separation of Globulin and Albumin. Con- stituents other than Proteins. METHOD OF CHEMICAL EXAMINATION OF TISSUES 44 PART II. DIGESTION. SALIVA 46 GASTRIC DIGESTION - 48 Gastric Juice. Gastric Contents I. Acids. 2. Products of Peptic Digestion. PANCREATIC DIGESTION 54 Pancreatic Juice. Products of Tryptic Digestion. METHOD OF TESTING FOR FERMENTS CONTROL EXPERIMENTS 55 BILE 56 Effect of Bile on Emulsification of Fats. Bile Salts. Bile Pigments. PART III. NORMAL METABOLISM. URINE 59 Constituents of Normal Urine. Quantitative Estimation of certain Nitrogenous Constituents : Total Nitro- gen, Urea, Ammonia. Significance of Nitrogen Distribution in Urine. Quantitative Estimation of Chlorides. PATHOLOGICAL METABOLISM. GASTRIC CONTENTS - 79 Quantitative Estimation of Hydrochloric Acid. " Free," " Physiologically Active," and " Combined" Hydrochloric Acid. ABNORMAL CONSTITUENTS OF URINE - 82 Proteins. Reducing Sugars. Estimation of Glucose. Aceto-Acetic Acid and Acetone. Method of Examining the Urine in Cases of Acidosis. Blood Pigment. Bile. Pathological Urinary Deposits. FORM FOR REPORT ON URINE ANALYSIS- . .6 Animal and Vegetable Tissues and Fluids. POTATO. Experiment i. Preparation of Starch from Potato. Scrape a potato as finely as possible with a knife, collect the scrapings in a beaker, stir with about 100 c.c. water, and strain the water which contains a large part of the starch through a piece of unbleached muslin. Mix the residue with more water and strain again. After repeating this with several portions, collect the water in one vessel and allow the starch grains to settle to the bottom. Examine some starch grains microscopically ; observe the effect of adding a drop of very dilute iodine solution, and sketch. Pour off the water, add more, and allow again to settle, repeating till the starch appears white. Experiment 2. Preparation of Starch Solution. Rub i grm. of starch to a thin paste with a little water, and pour the mixture slowly, with constant stirring, into 1 50 c.c. boiling water. Use the solution so obtained for Experiments 3-18. Experiment 3. (a) To some of the starch solution in a test tube add a drop of dilute iodine solution. Note the result, (fr) Heat the tube gradually. What change takes place? (c) Allow the solution to cool, and observe the result, (d) Make alkaline with a few drops of sodium hydroxide. Note the result, (e) Neutralise with a few drops of dilute hydrochloric acid. Explain the result. Experiment 4. Repeat the preceding test, first adding sodium hydroxide solution before testing with iodine. Record and explain the result. Experiment 5. To 5 c.c. (roughly a quarter of a test tube) of the starch solution, add three times its volume of saturated ammonium sulphate solution. Shake, and allow to stand for fifteen to thirty minutes. Filter and test separately the precipitate and the clear filtrate with iodine for starch. Experiment 6 To 5 c.c. of the starch solution add alcohol until a precipitate appears. Apply the iodine test for starch to the precipitate, and to the filtrate separately, and note the result. COMPLETE HYDROLYSIS OF STARCH BY ACIDS. FERMENTATION OF GLUCOSE. PREPARATION OF GLUCOSE AND ALCOHOL FROM STARCH. Experiment 7. To 100 c.c. of starch solution, in a flask, add about 3 c - c< f concentrated hydrochloric acid, and boil continuously over a small of starch, flame. Add occasionally some water, previously heated in a test tube, to replace the water which has evaporated, (a) What change do you observe in the appearance of the starch solution ? Remove every five minutes about 5 c.c. of the solution by means of a pipette, place in a test tube, cool, and test with a drop of dilute iodine solution. () Record the results obtained in each case. Continue boiling the bulk of the solution in the flask until the iodine test is negative, i.e., until a drop of dilute iodine solution, when added to about 5 c.c. of the solution, removed from the flask and cooled, fails to produce a colour. This point will be reached after about thirty minutes ; the time of boiling required varies with the concentration of the starch paste and of the acid. The solution thus obtained is used for Experiments 8- 1 6. (c) What has taken place? parationof Experiment 8. (a) To about 15 c.c. of the glucose solution obtained menfati * n Experiment 7 add dilute sodium hydroxide until the reaction is almost llucose. neutral to litmus, but still slightly acid. Add a small quantity of fresh yeast, and gently shake so as to break up the yeast. With the emulsion of yeast in the glucose solution fill the closed limb of a fermentation tube, so that no air bubble is left, and place the tube "for about an hour in a warm water-bath at 40 C. What takes place ? (b) Put the fermentation tube into the cupboard. After twenty-four hours remove the solution and filter. Examine the filtrate for alcohol by tsfor means of the iodoform test and the bichromate test. [Tests for alcohol (a) lodoform test. To the solution add a solution of iodine in potassium iodide until it is brown, then add just sufficient sodium hydroxide to decolorise the mixture. Warm gently. A yellow crystalline precipitate with the characteristic smell of iodoform is formed, (b) Bichromate test. Dissolve a crystal of potassium bichromate in the solution and add a little dilute sulphuric acid. Heat. The solution turns green, and the characteristic odour of aldehyde will be observed. What reaction has taken place?] Carefully neutralise, with sodium hydroxide, the rest of the solution obtained in Experiment 7, and perform the following tests for glucose. Experiment 9. () To 5 c.c. of sodium hydrate in a test tube add a solution of copper sulphate. A blue flaky precipitate forms which does not dissolve on shaking. What is it? Boil. Describe the result. Explain and compare with the following : (b) Trommer's Test. Add to 5 c.c. of the glucose or sugar solution an equal volume of strong sodium hydrate or potassium hydrate solution, then a dilute solution of copper sulphate, drop by drop, shaking after each addition, as long as the bluish precipitate formed continues to dissolve readily. If an excess of CuSO 4 is added, the precipitate remains, and must be dissolved by adding a few crystals of sodium potassium tartrate (Rochelle salt). The result is a deep blue solution. Why is no precipitate formed in this case ? Heat the solution, and note the result. Explain the chemical changes which have taken place. Experiment 10. Fehling's Test. Prepare Fehling's solution. Dis- solve in 80 c.c. of distilled water 7 grms. of copper sulphate, and label solution "A." Dissolve 34.5 grms. sodium potassium tartrate in 120 c.c. sodium hydroxide 10 per cent, and label solution " B." Mix " A " and " B," and fill the mixture into bottle labelled " Fehling's Solution." Boil 5 c.c. of Fehling's solution in a test tube (it should remain clear), and then add a few drops of the glucose solution. Let it stand a while and note the result. Repeat the test without heating. Experiment n. Caramelisation (Moore's Test). Boil 5 c.c. of the glucose solution with a little caustic soda. The solution turns yellow owing to the formation of caramel. Boiling with alkaline salts, e.g., sodium carbonate, also induces caramelisation. 5 Experiment 12. Bottger's Test. To a quarter of a test tube of the glucose solution add a little solid bismuth subnitrate, and about double the quantity of sodium carbonate. Heat and keep boiling for two minutes. Describe and explain the result. Experiment 13. Nylander's Test. Prepare Nylander's reagent. Dissolve 4 grms. sodium potassium tartrate and 2 grms. bismuth subnitrate in 100 c.c. sodium hydroxide 10 per cent. Pour the reagent into bottle labelled " Nylander's Reagent." Add one part of Nylander's reagent to ten parts of sugar solution, and keep boiling over a small flame for two minutes. Compare with Bottger's test. Repeat tests 8 to 12 with a very*dilute glucose solution in order to test their delicacy. Tests 8 to 12 are reduction tests. Why are they called so, and what is the rationale of these tests ? What happens if these tests are applied to starch solution ? Experiment 14. Osazone Test. To about 10 c.c. of the glucose solution add 10 drops of phenylhydrazine and an equal amount of glacial acetic acid. Shake the test tube and keep in the boiling water-bath for J to I hour. Allow to cool slowly and examine crystals under the micro- scope, and sketch. They consist of glucbsazone. Experiment 15. Molisch's Test. To a few c.c. of the solution add a few drops of an alcoholic solution of ^-naphthol. Incline the test tube and allow about 5 c.c. of concentrated sulphuric acid to flow down the side of the tube so that the acid settles at the bottom of the tube. At the junction of the two liquids a green ring may at first be formed (if traces of nitrates are present in the sulphuric acid), and above this, in a short time, a reddish-violet ring. The green ring, if present, must be disregarded. If the two liquids be mixed and cooled by gently shaking in running water, so that excessive heating is prevented, the mixture assumes a bluish-red or dark blue colour. [All carbohydrates and some proteins give this reaction. The test is used to demonstrate the presence of a carbohydrate group in proteins.] Experiment 16. Optical Activity. Polarimeter. Examine by means of a polarimeter the effect of the glucose solution on polarised light Proceed as follows. 1 Examine the polarimeter. It consists of a hollow brass tube which, at the one end, contains a Nicol prism, the " polariser," together with a double quartz plate. Attached to the brass tube by means of a hinge and a spring catch are at the other (upper) end another Nicol prism, the "analyser," together with a small telescope and a graduated arc divided into whole degrees. The " analyser " can be rotated by means of a milled head. The rotation can be read accurately to a tenth of a degree by means of a vernier. Lock the hinged part and place the instrument so that light falls into the " polariser " from a white surface. Make the following preliminary observations : 1. Rotate the analyser so that the vernier reads zero. On looking through the instrument the field of vision will appear as a circle divided by a straight line into two halves, which have exactly the same violet tint. Move the small telescope so that the dividing line is sharply focussed. 2. By means of the milled head, rotate the analyser slightly to the right and to the left. Note that a very slight rotation of the analyser is sufficient to produce a marked contrast in colour between the two halves of the field, the one half becoming red, the other blue. 3. Rotate the analyser by 90 to the right. Note that both fields are again tinted alike, but the tint is now yellow. Rotate slightly to the right and to the left. Note that now slight rotations of the analyser do not produce a marked contrast in colour between the two halves of the field, i.e., the yellow tint is not so sensitive in indicating rotation as the violet tint. 4. Starting again from zero, rotate the analyser through 360. Note that the two halves of the field are always unequally tinted except at four points of the circle: at o and at 180 the field shows in both halves the 1 The following description applies to a polarimeter of the Soleil type, and more particularly to the hand-polarimeter supplied by Winkel, Gottingen. same sensitive violet tint ; at 90 and at 270 the field shows in both halves the same non-sensitive yellow tint. Note that in order to take a reading the instrument must be set so that 1 . The dividing line betzveen the two halves of the fields is sharply focussed by means of the telescope. 2. The tivo halves of the field show exactly the same sensitive violet tint y the so-called " critical colour? After these preliminary observations insert a tube filled with a solution of glucose (about 5 per cent.). The observation tube must be clean and completely filled with the solution, so that air bubbles are excluded. Release the spring catch, which holds the part containing the analyser in position, insert the observation tube, and lock the instrument. The light, after having passed through the " polariser," and having become polarised, must now traverse the liquid in the observation tube before it reaches the analyser. Place the vernier on zero, and focus the telescope. Note that now the two halves of the field show a marked contrast, one half being blue, the other red. Rotate the analyser slightly until the two halves of the field show exactly the same violet tint (the critical colour). Note the position of the vernier ; it will be to the right of the zero point. The solu- tion of glucose is, therefore, " optically active," and has rotated polarised light to the right : it is " dextro-rotatory." Compare the effect produced by a solution of glucose with that produced by other organic substances, urea for instance. Insert a tube containing a 5 per cent, solution of urea, and place the vernier on zero. Note that the two halves of the field remain equally tinted. The solution of urea is " optically inactive." 8 PARTIAL HYDROLYSIS OF STARCH BY ACIDS. PREPARATION OF DEXTRIN. Reparation of Experiment 17. Proceed as in Experiment 7, but add only ten drops ]d*ffydro- * concentrat ed hydrochloric acid to 100 c.c. of starch paste. Remove every > sis of three minutes about 5 c.c. of the solution, cool, and test with dilute iodine solution. Discontinue the boiling when a purple or red colour is obtained with iodine. The solution then contains erythrodextrin. To the re- mainder of the solution apply Trommer's, Fehling's, and Nylander's tests. Record your results. PARTIAL HYDROLYSIS OF STARCH BY FERMENTS. PREPARATION OF DEXTRIN AND MALTOSE. 'reparation of Experiment l8. Prepare some dilute saliva by rinsing out the Mtosety moutn f r l minute with 5 c.c. warm water. Collect the washings in a tion of beaker. Repeat. Filter the dilute saliva. Place a series of drops of terSL** iodine on a porcelain plate. Put some starch solution (about 5 c.c.) in a test tube and add about half a test tubeful of saliva. Shake the mixture and place in the water-bath at 40 C. Every minute take out a drop with a glass rod and apply it to one of the iodine drops on the porcelain slab. What changes take place in the mixture ? Keep the mixture in the water- bath for J to I hour, until all the starch has been transformed into maltose. This solution is used for Experiment 19. By what constituent of the saliva is starch transformed into maltose ? estsfor Experiment 19. With the solution of maltose obtained in Experiment Wtose. l8 carr y ou t the tests 9 (b) to 14. Note the results. How does maltose differ from glucose, and how can it be distinguished from it ? LARD, OLIVE OIL. olubility of Melt a little lard in a porcelain basin on a boiling water-bath ; with the melted fat carry out the following experiments : Experiment 20. Add a few drops of the lard to each of five test tubes containing (i) acetone, (2) alcohol, (3) ether, (4) chloroform, (5) water, and note the solubility of the fat in these solvents. Experiment 21. Let a drop of the alcoholic or ethereal solution fall on a piece of white paper, and note the grease spot which remains after the solvent has evaporated. Experiment 22. To a test tube containing water add a few drops of the alcoholic solution of the fat. A precipitate appears. Explain. Experiment 23. Repeat Experiments 20 to 22 with olive oil instead of lard. HYDROLYSIS OF LARD BY ALKALI (SAPONIFICATION). PREPARATION OF FATTY ACIDS. 'reparation of Experiment 24. Slowly add about 5 c.c. of the melted lard to 50 c.c. ' a *H ^drofsis ^ an a ^ cono ^ c solution of caustic potash, contained in a flask, and kept on a f Fats. boiling water-bath. Mix thoroughly and heat for ten to twenty minutes. Add a few drops of the mixture to some water in a test tube as in Experi- ment 22. No oil globules separate out. Why? (If oil globules are still seen, the reaction is not complete, and heating must be continued with more alcoholic soda.) When the reaction is complete, slowly pour the solution in the flask into a beaker containing 100 c.c. of warm water and mix thoroughly. To the watery solution (which contains soap) add some 25 per cent, sulphuric acid (one part concentrated sulphuric acid slowly added to three parts of water), and heat on the water-bath until the melted fatty acids separate out as an oily layer floating on the top of the liquid. IO Cool. The fatty acids solidify, and can then be removed and freed from adhering sulphuric acid by rinsing with cold water. Experiment 25. Suspend some of the fatty acids in water and add dilute sodium hydroxide. The fatty acids dissolve. Soap is formed again. What is soap ? Use the solution of soap thus obtained to demon- strate the following properties : (a) Shake up some of the soap solution with warm water. A soap lather is produced. (b) To some soap solution add solid sodium chloride until the solution is saturated. The soap is precipitated : " salted out." (c) Add a few drops of calcium chloride solution to some of the soap solution. A precipitate is formed. Of what ? The remainder of the soap solution is used to demonstrate the part played by soap in EMULSIFICATION OF FATS. Experiment 26. Label two test tubes " (a) " and " ()." Place in (a) some water and in (6) some soap solution. Add to each three drops of olive oil and shake. A permanent emulsion is formed in (b) but not in (a). Explain. Experiment 27. The same result is, of course, obtained if neutral fat containing some fatty acid is shaken up with a little dilute alkali solution. For instance, to half a test tubeful of water add one drop of a 10 per cent, solution of sodium hydroxide and 2 c.c. of ordinary olive oil, which always contains some free fatty acid. An emulsion is formed. Explain. A neutral fat, which does not contain any free fatty acids, will not give a permanent emulsion with dilute sodium hydroxide. This reaction may, therefore, be used to test for the presence of free fatty acids in a fat. These are present if a fat is rancid. Another method of detecting whether a fat is rancid is as follows : Experiment 28. To some alcohol in a test tube add a drop of phenolphthalein and one or two drops of very dilute caustic soda, just enough to produce a red colour. Add this fluid to a solution of olive oil in alcohol. If fatty acids are present the red colour disappears. Why? 1 1 EGG. Demonstration of Osmosis. Three eggs, from which the shells have been removed by immersion in hydrochloric acid, are weighed. The one is immersed in distilled water, the other in a 0.9 per cent, salt solution, the third is immersed in glycerin. Note the change in volume and weight. Demonstration of a semi-permeable membrane. Separation of Constituents of Egg. Break a raw egg and collect separately in two beakers the white and the yolk of the egg. The white of the egg consists mainly of proteins, while the yolk, besides other protein substances and some fat, contains lipoids (e.g., lecithin, cholesterin). Experiment 29. Lipoids of Yolk. Preparation of Lecithin and Cholesterin. Mix the yolk thoroughly with about 20 c.c. of ether, pour into a flask, and close flask. Shake vigorously, and allow it to stand for some time. (In the meantime the solution of egg proteins may be prepared as directed below in Exp. 32.) (N.B. In working with ether all gas flames in the neighbourhood must be extinguished.) Pour off from the residue the ethereal, deeply coloured solution into a porcelain capsule, and extract the residue again with 20 c.c. of ether. The ethereal extracts are concentrated to a small volume by placing the capsule in the fume chamber in a previously heated water-bath (use no flame !). Acetone is then added in excess until a distinct precipitate is formed. The precipitate is a crude mixture of phosphorised fats. This mixture is frequently described by the name of its main constituent " lecithin." Remove the precipitate by filtration. The filtrate, which contains choles- terin, is evaporated on the water-bath. Cholesterin separates out, admixed with some lecithin. 12 (a) LECITHIN. Experiment 30. Dissolve in alcohol the precipitate on the filter. The bulk of it dissolves. Drop the alcoholic solution into water and stir. " A white precipitate of " lecithin " is formed. Proceed in the same way, but drop the alcoholic solutions of lecithin into ether. Compare the solubility of lecithin in water, acetone, alcohol, and ether with that of ordinary fats in the same solvents, and record. Boil the watery emulsion with caustic soda. It becomes clear. Notice the smell of trimethylamine. On acidifying, fatty acids separate out. Explain the change which has taken place. For further tests for lecithin (demonstration of phosphoric acid in its molecule), see Experiment 61. (6) CHOLESTERIN. Experiment 31. Remove all the water from a test tube by washing it out with alcohol and ether. Place some chloroform in this dry test tube, and dissolve in it some cholesterin. Add an equal bulk of concentrated sulphuric acid, and shake gently. The chloroform solution which rises to the top turns red, the acid at the bottom of the test tube shows a green fluorescence (Salkowski's test). The necessity for using a dry test tube is shown by the fact that the red colour of the chloroform solution disappears if it is poured into a wet test tube. For further tests for cholesterin (crystalline form), see Experiment 60. Experiment 32. White of Egg. Cut up the egg-white with a pair of scissors. The viscid fluid thus obtained is faintly yellow in colour, alkaline in reaction, and has a specific gravity of 1.045. It contains about 10 per cent, of proteins ; the greater proportion is egg albumin. A portion of the white of egg (about 5 c.c.) is diluted with nineteen times its volume of water. A slight, but well marked, precipitate of a protein appears, but the bulk of the protein remains in solution. The precipitate readily dissolves if the concentration of salts, which by dilution with water has been greatly lowered, is slightly raised again by the 13 addition of a few drops of a saturated solution of sodium chloride. In order to distinguish the proteins which remain in solution when the concentration of salts is lowered from those, otherwise similar, proteins which fall out under these conditions, the former are called albumins, the latter globulins. A more detailed study of their behaviour and solubilities will be made later, when the composition of blood-serum is being examined, because in serum they are present in about equal proportions, while in the white of egg the amount of globulin present is relatively small. The globulins and albumins are the most typical and widely distributed groups of " simple proteins." They show the property of being coagulated by heat. Experiment 33. Heat Coagulation of Albumins and Globulins. (a) Heat 5 c.c. of the diluted egg-white in a test tube to boiling point. The solution becomes opalescent, but there is no definite coagulum. (b) Heat 5 c.c. with I to 2 drops of dilute acetic acid. A coagulum is formed. (c) Heat 5 c.c. with 2 drops of glacial acetic acid. No coagulum occurs. Acid albumin is formed, which does not coagulate on boiling. (d) Heat 5 c.c. with 2 to 3 drops of dilute sodium carbonate solution. There is no coagulation. Alkali albumin is formed, which does not coagulate on boiling. The globulin and albumin of egg-white is thus only coagulated by boiling when the solution is neutral or faintly acid. If the solution be alkaline as in (d), the proteins are acted on by the alkali, as the temperature rises, and converted into alkali-albumin. If strongly acid, as in (V), the proteins are converted into acid- albumin. These compounds, which are grouped together as metaproteins, are not coagulated on heating their solutions. They are precipitated on neutralising their solutions, and dissolve again in an excess of either acid or alkali. With the diluted egg-white, which represents approximately a 0.5 per cent, solution of ovalbumin and ovoglobulin, carry out the following tests, by which proteins can be recognised. H GENERAL PROTEIN REACTIONS. Experiment 34. Colour Tests : (a] Riuret Test. To a portion of the protein solution add sodium hydroxide so that the solution is strongly alkaline, then one or two drops of very dilute copper-sulphate solution. (An excess of copper-sulphate must be avoided.) The solution becomes violet. On what group in the protein molecule does this test depend ? Prepare some biuret by heating a few urea crystals in a dry test tube until the melted mass begins to solidify again. Allow to cool, dissolve in a little water, and apply the biuret test. What is the structural formula of biuret? (b] Milloris Test. To a portion of the protein solution add a few drops of Millon's reagent (solution of mercurous and mercuric nitrates). A precipitate forms, which, on heating, becomes brick-red. The red colour constitutes the essential part of the test. On what group in the protein molecule does this test depend ? Repeat with a dilute solution of phenol. Apply Millon's test to the protein solution after having added some sodium chloride. What takes place ? Explain. Millon's test can be applied to insoluble proteins. (c] Xanthoproteic Test. To a few c.c. of the protein solution add one- third of its volume of strong, pure nitric acid; a white precipitate may or may not be produced (according to the concentration and nature of the protein). Boil. The precipitate or liquid turns yellow. Cool the test tube, and carefully add excess of ammonia, so as to form a layer above the nitric acid. An orange colour is produced at the junction. This constitutes the essential part of the test. Mix the ammonia with the acid by shaking, and note that the yellow colour of the solution deepens. On what group in the protein molecule does this test depend ? Repeat the test with benzine. This test can be applied to insoluble proteins. Experiment 35. Precipitation by Strong Mineral Acids (Nitric Acid or Heller's Test). Place 5 c.c. of the protein solution in a test tube, 15 and by means of a pipette add I or 2 c.c. of strong pure nitric acid very care- fully to the bottom of the solution, so that it forms an under layer. A white ring of coagulated protein appears at the junction of the two fluids. (Allow ten minutes if the reaction is slow in appearing.) Experiment 36. Precipitation by Alkaloidal Reagents. Proteins in acid solution are precipitated by reagents which precipitate alkaloids ; e.g. : (a) Hydroferrocyanic Acid. Make a portion of the protein solution distinctly acid with acetic acid ; then add a few drops of potassium ferro- cyanide solution. A precipitate is formed. Why is it necessary to carry out this test in this way, instead of adding free hydroferrocyanic acid to the protein solution ? (b) Picric Acid. To some protein solution add picric acid drop by drop. Note that a precipitate forms around each drop as it falls in. On shaking, this precipitate dissolves at first. If sufficient picric acid has been added the precipitate remains. (c) Salicylsulphuric Acid. To a portion of the protein solution add a few drops of salicylsulphuric acid solution. A precipitate is formed. A number of other alkaloidal reagents (tannic acid, phosphotungstic acid, etc.) give similar precipitates with protein solutions. What is an alkaloid ? Why do albumins and globulins in acid solution behave like alkaloids towards these reagents ? Experiment 37. Test the delicacy of the protein tests 33 to 36 by applying them to (a) 2 c.c. egg-white diluted with 98 c.c. water (i : 50 dilution), (b) I c.c. egg-white diluted with 99 c.c. water (i : 100 dilution). Record your results. Experiment 38. Precipitation by Alcohol To the protein solution add an excess of alcohol. A precipitate is formed. Allow the precipitate to stand in contact with alcohol for half an hour. Decant the alcohol and add water to the precipitate. It has become insoluble in water, having been coagulated by the alcohol. What is the difference between "pre- cipitation " and " coagulation " ? i6 >sting for Experiment 39. Carbohydrate Material of White of Egg. Egg- ^resenceof wn i te does not contain any polysaccharide of a starch-like nature or a oteins. reducing sugar in the free state. Demonstrate this by applying the iodine test, Fehling's and Nylander's test to the diluted egg-white solution, from which all proteins have previously been removed by heat coagulation. Record. Note that the tests for carbohydrates can only be applied after all proteins have been removed. Test with the biuret test for the absence of proteins before applying the tests for carbohydrates. If, however, Molisch's test (Exp. 15) is applied to the egg-white solution (still containing proteins), a positive result is obtained. This shows that while no free carbohydrate is present in the white of egg, the protein of egg-white contains a carbohydrate group chemically bound in its molecule. MILK. Ik. Experiment 40. Test the fresh milk with litmus paper. The reaction is either faintly alkaline or amphoteric (i.e., turns blue litmus red and red litmus blue). Determine the specific gravity of milk. Record. Why has skimmed or separated milk a higher specific gravity than fresh milk ? Experiment 41. Separation of Constituents. Extract the fat by shaking 5 c.c. of milk in a test tube with twice its volume of ether. Pour off the ether and allow to evaporate until only a few drops are left. Pour these on to a filter paper. A greasy spot indicates the presence of fat. seinogen. Dilute 2O c.c. of milk with 80 c.c. of water. Add carefully dilute acetic acid drop by drop, stirring the fluid after each drop has been added, until a flocculent precipitate is formed, and the solution in which it floats appears clear. An excess of acid must be carefully avoided, as it would dissolve the precipitate. The precipitate consists of caseinogen the main protein of milk with adherent fat. Filter. With the precipitate, carry out the experiments described below under caseinogen (Exp. 43). ctalbumin. The filtrate from the caseinogen, which still gives the tests for proteins, ctoglobuiin. contains (besides small amounts of lactalbumin and lactoglobulin) lactose the carbohydrate material of milk. Remove the albumin and globulin by heat coagulation in the following way : Carefully neutralise the acid filtrate with dilute sodium carbonate solu- tion. Heat the neutral solution to boiling point, and add a few drops of dilute acetic acid. If the heat coagulation is carried out correctly, the lactalbumin and lactoglobulin separate out as a flocculent precipitate, which is removed by filtration, and the filtrate does not give any tests for protein. i8 When all the protein has been removed, the filtrate may be used for the experiments described below under lactose. It contains, besides lactose, inorganic phosphates of calcium, which can be detected by the ordinary test for phosphates. Label the filtrate " Lactose'' For the examination of milk fat butter is given out. Experiment 42. Milk Fat, its Fatty Acids. Different fats differ in the nature of the fatty acids which are combined with glycerine. Liberate the fatty acids of milk fat by saponification (see Exp. 24). Proceed as follows : Heat a little butter with a small quantity of alcoholic soda until a clear yellow solution is obtained. Pour the solution into a beaker containing hot water (no oil drops should be seen) and heat to expel the alcohol. Then acidify with dilute sulphuric acid, warm again, and note the smell of butyric acid and caproic acid. Compare briefly butyric acid and the higher fatty acids obtained from lard with regard to their solubility in water, their volatile nature, and their molecular weight. Experiment 43. Caseinogen. Dissolve the precipitate of caseinogen obtained in Experiment 41 in dilute sodium carbonate solution. The adherent fat remains suspended. Filter through a wet filter paper, and apply to the caseinogen solution the colour tests for proteins. Record. Determine whether caseinogen is a protein coagulable by heat. (See Exp. 33.) Record. Experiment 44. Lactose- Evaporate some of the filtrate obtained in Experiment 41, and labelled "Lactose," to a syrup on the water-bath, and allow to stand in the cold until the sugar has crystallised out. To another portion of the filtrate labelled " Lactose " apply the following tests for sugar : Fermentation test (No. 8). Reduction tests : Trommer or Fehling (Nos. 9 and 10) ; Bottger or Nylander (Nos. 12 and 13). Osazone test (No. 14). Sugar. Experiment 45. Comparison of Lactose with Cane Sugar. Apply reduction tests, fermentation test, and osazone test to a solution of cane sugar which is given out. Record your results. Boil 5 c.c. of cane sugar solution with five drops of concentrated hydro- chloric acid for two minutes. Neutralise with caustic soda, and apply Fehling's and Nylander's tests. Record your results. Experiment 46. Action of Different Disaccharides on Polarised Light before and after Hydrolysis. Examine solutions of lactose, maltose, and cane sugar of equal concentration in a polarimeter. Proceed as described in Experiment 16. Hydrolyse solutions of lactose, maltose, and cane sugar by boiling for five minutes with five drops of concentrated hydrochloric acid, and examine solutions of hydrolysed sugars again. Record your results. Why is hydrolysed cane sugar called "invert-sugar" ? From Experiments 8,9, 10, 12, 13, 14, 16, 19, 44, 45, and 46, construct the following table : tions of Reactions of Sugars. Nature of Sugar. Trommer or Fehling. Nylander. Optical Activity. Fermen- tation. Form of Osazone. Mono- saccharide Glucose Maltose . Disaccharide* Lactose Sucrose (Cane Sugar) Verify the table by applying the various tests to the solutions of the different sugars which are given out. 20 on of Ren- Experiment 47. Clotting of Milk. To 5 c.c. of milk in a test tube 711 Milk. ac [d 1 cc O f a neu tral solution of rennet ferment (prepared by extracting a calf's stomach with glycerine). Place the mixture in a water-bath, kept at 37 to 40. After a few minutes a firm clot forms, from which, on standing, a clear fluid exudes. The clot contains casein and fat. What is casein ? The clear fluid the whey contains all the other constituents of the milk. Demonstrate their presence by suitable tests and record your results. Experiment 48. Prove that the Formation of the Clot Depends upon the Presence of Soluble Calcium Salts. Precipitate all the calcium present in 5 c.c. of milk by adding 2 c.c. potassium oxalate solution (i per cent.). Then add i c.c. of rennet and place in water-bath. No clot forms if all the calcium has been precipitated. Then add to the unclotted milk 2 c.c. CaCl 2 solution (2 per cent.). A flocculent precipitate is produced. Explain the results. 21 FLOUR AND BREAD. Experiment 49. Flour. Knead some flour with a little warm water to form a stiff dough, and allow to stand for about fifteen minutes. Place the dough in a muslin bag and continue kneading in a basin of water ; starch grains pass through. If the water is poured from the basin into a beaker the deposit of starch grains which settle can be easily seen. Prove their chemical nature by examining them microscopically, and by boiling some grains with water and applying the iodine test to the watery solution thus obtained. Remove the grains from the water by filtration, and test the filtrate with iodine for the presence of dextrin (see Exp. 17), and with Fehling's and Nylander's tests for glucose (Tests Nos. 10 and 13). Con- tinue kneading the dough under the tap until all the starch has been removed. A yellowish sticky mass remains. This is gluten, the chief protein of wheat. Prove its protein nature by applying the ordinary colour tests for solid proteins (No. 34, b, c). Experiment 50. Bread. Grate some bread finely, and extract, first with cold water, then with boiling water. Strain the watery extract through muslin. Examine : 1. Cold water solution for glucose, starch grains, dextrin. 2. Hot water solution for glucose, starch, dextrin. In order to demonstrate the presence of dextrin when starch is also present in solution, remove the latter by half saturating the solution with ammonium sulphate. The dextrin passes into the filtrate. 3. The residue is gluten, and can be identified as a protein by the colour tests (No. 34), as in Experiment 49. Record the results. What difference is there between the carbo- hydrates of flour and those of bread ? What changes do the carbohydrates of flour undergo in the process of baking, and how do you account for these changes ? Experiment 51 Extract some crust of bread with water, and test for starch, dextrin, and glucose. Note that in the crust glucose is present in traces only. Why ? 22 MUSCLE AND GLAND. Experiment 52. Proteins of Muscle. In a mortar rub thoroughly 10 grms. of muscle (best from white fish) with 5 grms. of sodium chloride, then to the pulp add 50 c.c. of water, so as to make a 10 per cent, solution of NaCl. Stir the mixture well and strain through muslin. Label the solution, " Salt Extract (A)." Rub the residue with 0.2 per cent. NaOH solution in the mortar and again strain. Label this solution, " Soda Extract (B)." A residue con- taining collagen is left on the muslin. Salt Extract (A). Determine that this extract contains protein by applying the colour tests for protein (Exp. 34). Record the results. Determine that it is a protein coagulable by heat (globulin or albumin), by heating the neutral extract with the addition of a few drops of acetic acid (Exp. 33). (Note the conditions necessary for this test as explained in Exp. 33.) Record the results. koprottin. Soda Extract (B). On careful addition of acetic acid to this extract a precipitate forms which is redissolved in a large excess of the acid. It is the nucleoprotein of muscle. Determine that the nucleoprotein solution gives the colour tests for proteins (No. 34). Experiment 53. Proteins of Glands. Repeat this experiment with glands (pancreas) instead of muscle. Note and record how these two tissues differ with regard to the relative amounts of (i) heat-coagulable protein (albumin and globulin) (salt extract), (2) nucleoprotein (soda extract). LIVER. The student is supplied with A. Small pieces of the liver of an animal which has been fed for some time on a diet rich in carbohydrates (carrots). The liver was placed in alcohol immediately after death. AA. Small pieces of the liver of an animal fed on the same diet, but the liver was placed in alcohol twenty-four hours after death. AAA. Small pieces of the liver of a fasting animal. The liver was placed in alcohol immediately after death. B. Pieces of other glands, e.g., kidney, placed in alcohol immediately after death. With A carry out the following tests : Experiment 54. Glycogen. Cut up a small piece into shreds, place the shreds into a test tube. Add 10 c.c. of water and heat slowly to boiling point. Cool thoroughly. Decant from the shreds and divide the solution into three parts. 1. To one part add two drops of iodine solution. A brown colour results, indicating the presence of glycogen. Care must be taken not to add too much of the iodine solution, since the iodine solution itself has a similar colour. It is useful, therefore, to carry out a control experiment in a test tube which contains the same amount of water as the glycogen solution to be tested. If two drops of iodine solution are added to the water a yellow colour is obtained. The brown colour obtained by adding iodine to the glycogen solution disappears on heating, arid reappears on cooling. It disappears on making the solution alkaline. It reappears on neutralising the alkaline solution. Compare with starch. (Experiment 3.) 2. To another part apply Fehling's test. No reduction results. 24 3- A third portion is boiled with dilute HC1 for five minutes. Neutralise and apply Fehling's test. Reduction occurs. Explain. To which group of carbohydrates does glycogen belong? The presence of large amounts of glycogen in a tissue can be demon- strated rapidly by applying iodine directly to the tissue as follows : Experiment 55. To a piece of liver A apply a drop of dilute iodine solution by means of a glass rod. After a minute remove the iodine solution by rinsing the tissue under the tap. A deep brown colour appears where the iodine has acted. Record your result. Carry out the test with AA, AAA, and B. Record your results. The colour is very faint in AA and AAA, and may even be absent. Why? It is absent in B. Why? Why must the tissues be preserved in alcohol and not in a watery solution (of formalin, for instance), if one wishes to test for glycogen ? Experiment 56. Test for Iron. To a test tube containing 10 c.c. of water add a few drops of ferric chloride. To this ferric chloride solution add a few drops of potassium ferrocyanide. A blue colour results. What has been formed ? Explain the reaction which has taken place in the form of a chemical equation. on in Liver. Experiment 57. Iron in Liver. Keep a piece of liver in a potassium ferrocyanide solution for a few minutes. Then add some dilute HC1 (0.5 per cent.). A faint blue colour appears. Carry out the same experi- ment with a piece of kidney or any other tissue. Record the results. Experiment 58. Apply the same test to a piece of liver from a case . of pernicious anaemia, in which there is an excessive destruction of red blood corpuscles. A distinct blue colour appears. Explain the results. NERVOUS TISSUE. Nervous tissue differs from other tissues in being particularly rich in lipoids, i.e., substances soluble in fat-solvents, such as ether, alcohol, chloroform, and insoluble in water. In order to extract lipoids from tissues it is best to dry the tissues first. This is done by finely mincing the tissue, sprinkling it with formalin in order to avoid putrefaction, and, after removing the bulk of the water, driving off the rest by means of a current of hot air obtained from a fan or by placing it in an incubator at 40. The dried nervous tissue is then treated as follows : Experiment 59. Separation of Lipoids of Nervous Tissue. CHOLESTERIN. ^ration of Place 3-5 grms. of dried tissue in a flask, and extract three times with cold acetone, which dissolves cholesterin. Decant the acetone through a filter, unite all the acetone extracts in a porcelain basin, and evaporate the acetone in a fume cupboard, by placing the basin on a hot- water bath. Scrape off the residue and label " Cholesterin." PHOSPHATIDES (LECITHIN, KEPHALIN). The nervous tissue which has been freed from cholesterin by acetone extraction is now extracted with cold ether in the same way. The ether extracts are united and evaporated in the same way. Label the residue, which contains the ether-soluble phosphatides lecithin and kephalin, " Phosphatides." CEREBROSIDES AND PHOSPHO-CEREBROSIDES (CEREBRON, PROTAGON). The nervous tissue which has thus been freed from cholesterin and phosphatides is now extracted with boiling alcohol or chloroform. These 26 extracts are united and evaporated in the same way. Label residue, ' Cerebrosides and Phospho-cerebrosides." After having in this way separated three groups of lipoids apply the following tests to each of these groups, and verify the presence or absence in each group of the substances tested for. Record your results. Since separation of the various lipoids is not complete, note whether tests are strongly positive or only faintly positive. tsfor Experiment 60. Tests for Cholesterin. (a] Crystalline form. Dissolve lestenn. ^Q solid in very little hot alcohol, filter and place a drop of the alcoholic solution on a slide. Allow the alcohol to evaporate, and examine under the microscope, whether the characteristic plates of cholesterin have been formed. Note that cerebron and protagon also crystallise from alcohol, but that the form of the crystals is quite different. Sketch the crystals obtained. Add a drop of strong sulphuric acid; the edges of the crystals turn red. (b) Salkowski's test. Dissolve some of the solid in chloroform and apply Salkowski's test as described in Experiment 3 1 . t for Experiment 61. Test for Phosphatides. This test depends on the sphatides. p rese nce of a phosphoric acid group in the molecule. Incinerate a little of the material in a crucible. Cool, extract the ash with 2 or 3 c.c. of hot water and filter. To a few drops of the filtrate add about 5 c.c. of ammonium molybdate solution and 3-5 drops of pure nitric acid. Heat gently. A yellow precipitate indicates the presence of phosphatides or phospho-cerebrosides. What is the yellow precipitate ? tsfor Experiment 62. Tests for Cerebrosides and Phospho-Cerebrosides . * b pho*tho These tests depend on the presence of the reducing sugar galactose in the ebrosides. molecule. (a) a-Naphthol test. Boil some of the material with water, cool and apply Molisch's test as described in Experiment 15. If a-Naphthol test is positive verify by (b) Reduction test. Boil some of 27 the material with a little dilute hydrochloric acid, neutralise and apply Fehling's test. (V) Presence of Phosphoric Acid. Test as described in the preceding experiment. A negative result together with a positive test for galactose indicates that cerebrosides only are present. If both the tests for phosphoric acid and for galactose are positive, phospho-cerebrosides are present. Histochemistry of Lipoids (Demonstration). Examine under a polarisation microscope : olein, lecithin, cholesterin, protagon. Note that olein is isotropous, i.e., not double refracting, while lecithin, cholesterin, and protagon are anisotropous, i.e., double refracting. Examine a piece of a sciatic nerve (either fresh or fixed in formalin) under the polarisation microscope. Note that the medullary sheath is double refracting along its entire length. Examine a piece of a degenerated sciatic nerve (about two weeks after the lesion) under the polarisation microscope. Note that the medullary sheath now shows double refracting globules of material alternating with non-double refracting globules. Prepare 2 per cent, chloroform solutions of olein and of the lipoids present in the myelin of nervous tissue : lecithin, cholesterin, protagon. To about i c.c. of each, placed in a small test tube, add one drop of i per cent, osmic acid solution. Note that olein and lecithin blacken rapidly, cholesterin and protagon only slowly. Prepare thin films of these lipoids, and treat them according to Marchi's method for degenerated nerves. Proceed as follows : Allow two drops (not more) of the chloroform solution to fall on a disc of filter paper about 1.5 cm. in diameter. Dry in incubator. Place disc carrying a thin film of lipoid first in a 25 per cent, potassium bichromate solution (Miiller's fluid) for three days at 40, then in a mixture of two parts of Miiller's fluid and one part of i per cent, osmic acid solution, Note that now only olein blackens. BLOOD. Blood is a tissue, the cellular elements of which are suspended in a fluid the blood plasma. COAGULATION OF BLOOD. (Demonstration.) Experiment 63. Formation of Clot and Serum. Blood is allowed to flow from a blood vessel directly into a clean conical glass vessel. A blood clot is formed. Note its soft, spongy character. Allow the clot to stand for several hours without shaking ; the clot contracts and expresses a clear fluid the blood serum. Note that the clot adheres, as a rule, in its upper layers to the walls of the glass vessel. If the process has been carried out carefully, the serum will not show the faintest trace of a red colour. The appearance of such a colour in the serum indicates that haemolysis of the red corpuscles has taken place. (See Exp. 70.) Wash a part of the clot in running water. The red blood corpuscles which are entangled in the clot are broken up, as the deep red colour of the wash-water indicates. If the washing is continued sufficiently long, a yellowish-white stringy mass remains, which gives Millon's test and the Xanthoproteic test for proteins. This is fibrin. Experiment 64. Defibrination of Blood. Preparation of Serum and of a Suspension of Blood Corpuscles. Blood is allowed to flow from a blood vessel into a dish, and stirred there gently but continuously with a wooden stick or any other object presenting a rough surface (bundle of feathers). A stringy mass of fibrin, coloured red by entangled blood corpuscles, gradually collects at the end of the stick. After five to ten minutes' stirring, the stick, with the adherent stringy mass, which is much denser and harder than the clot found in the previous experiment, is withdrawn. The remaining fluid is defibrinated blood. It resembles blood in its external appearance, but differs from it in the fact that all 29 the fibrin, and fibrin-yielding substances, have been removed from it. On standing, or more rapidly on centrifugalisation, defibrinated blood separates into two layers : a layer of red blood corpuscles and a super- natant layer of a clear fluid, the serum, identical in composition with the serum expressed from the clot in the previous experiment. The serum can be removed by means of a pipette. The blood corpuscles are freed from traces of adherent serum by suspending them in 0.9 per cent, saline solution, centrifugalising the solution, and removing the wash-fluid until it ceases to give the biuret test for proteins. 5 c.c. of washed blood corpuscles are then added to 95 c.c. of 0.9 per cent. NaCl solution, and the mixture shaken. It represents a 5 per cent, suspension of red blood corpuscles. Label " 5 per cent, r.b.c." By this method only one constituent of circulating blood can be prepared unchanged the red blood corpuscles. The fluid obtained in this experiment, the serum, differs from the fluid in which the cellular elements of the circulating blood are suspended, the plasma. In order to study the constituents of circulating blood, it is necessary to prevent the coagulation of blood. The following experiment represents the methods most frequently used for that purpose. Experiment 65. Constituents of Circulating Blood (Cellular Elements and Plasma). Methods of Preventing the Coagulation of Blood.- (a) Coat an artery canula and several glass vessels with a mixture of two parts of hard paraffin, two parts of soft paraffin, and one part of stearin. Insert the canula into the artery without touching the tissues surround- ing the artery. Allow blood to flow into the paraffined vessel. The blood does not clot, or if the experiment has not been entirely successful, clots very much more slowly than in Experiment 63. (b) Place in a shallow wide-mouthed bottle 10 c.c. of a I per cent, solution of potassium oxalate in 0.7 per cent. NaCl solution. Allow blood to flow into this solution from a blood vessel, so that the blood does not touch either the tissues of the wound or the walls of the glass vessel. When the mixture of blood and oxalate solution in the bottle has reached 30 a level of 50 c.c., previously marked on the vessel, the bottle is withdrawn, and a fresh one may be substituted. The oxalate, which has then a concentration of 0.2 per cent., has precipitated all the calcium salts of the blood. The blood does not clot. If blood which has been prevented from coagulation by either of these methods is allowed to stand, or, more rapidly, if it is centrifugalised, it separates into three layers : i. A layer of red blood corpuscles at the bottom of the tube. 2. A narrow greyish layer consisting mainly of blood platelets and some leucocytes. 3. A fluid, the blood plasma, which occupies the upper half or two-thirds of the tube. Note that the plasma is fairly clear in its upper layers, but slightly turbid in the lower layer, where it adjoins the layer of platelets. Note further that in standing the platelets agglutinate and form a coherent cake a " white thrombus." Experiment 66. Conditions Inducing the Coagulation of Blood. Receive blood in paraffined vessels as described in the previous experiment. Pour some blood from a paraffined vessel into a glass vessel : coagulation sets in. To some blood contained in a paraffined vessel add a piece of a feather : coagulation sets in. The experiment shows that contact with certain objects (glass and feather) induces coagulation ; contact with other objects, e.g., paraffin, does not do so. Experiment 67. Factors Concerned in the Coagulation of Blood. Separation of Plasma and Blood Platelets. From oxalated blood, ob- tained as described in Experiment 65 (b\ isolate the following constituents. 1. Plasma and Platelets. Centrifugalise oxalated blood ; remove the plasma by means of a pipette. Owing to its contamination with blood platelets the plasma is slightly turbid. Label " Plasma and Platelets." 2. Plasma. Filter some of this mixture of plasma and platelets through a porous porcelain filter (a " Berkefeld " filter), in order to remove all cellular elements. The filtrate is quite clear. Label "Plasma filtered." 3. Extract of Blood Platelets. Remove by means of a pipette parts of the greyish intermediate layer of platelets from centrifugalised oxalate blood. Suspend in 0.9 per cent, saline solution. Centrifugalise until the platelets are deposited at the bottom of the tube. Decant super- natant fluid. Treat the sediment with about 5 c.c. of distilled water, which breaks up the platelets. Label " Platelet Extract." Prepare a I per cent, solution of calcium chloride. Label small test tubes A, B, C, D. Charge with the various solutions as given below, and place in water-bath at 35. Tube. Contents. Result. A i c.c. oxalate blood + 0.2 c.c. CaCl 2 Clot. B i c.c. oxalate plasma and platelets + o. 2 c.c. CaCh> Clot. C i c.c. oxalate plasma filtered + 0.2 c.c. CaCl. 2 No clot. D i c.c. oxalate plasma filtered + 0.2 c.c. CaCl 2 -fo.2 c.c. Clot. platelet extract These results show 1. That calcium salts are necessary for coagulation (tubes A and B). 2. That red blood corpuscles are not necessary (tube B). 3. That platelets are necessary (tubes B and C). 4. That the function of the platelets can be replaced by a watery extract of platelets (tube D). Summary. In the reaction which leads to the formation of a blood clot, the following constituents are concerned: (i) Blood platelets; (2) soluble calcium salts, which are present in circulating plasma; (3) yet another constituent of the plasma. Experiment 68. Separation from Plasma of Mother Substance of Fibrin : " Fibrinogen." Difference in Composition between Plasma and Serum. Prepare "oxalate plasma filtered" as described in the previous experiment. Prepare fresh serum by any of the methods described above. To 10 c.c. of filtered plasma add an equal volume of a saturated NaCl solution, so that the salt concentration of the mixture is that of a half-saturated NaCl solution. A white precipitate appears of one of the protein substances of plasma, namely fibrinogen. Repeat the same experiment with serum. No precipitate appears. (For further investigation of proteins of serums see Experiments 85, 86.) Separate the fibrinogen precipitate from the plasma by centrifugalising, decant the supernatant fluid. Wash the precipitate with a half-saturated solution of NaCl (in which it is insoluble), and dissolve it in 10 c.c. of a 0.9 per cent. NaCl solution. Label " Fibrinogen solution." With a little of the fibrinogen solution perform biuret test and heat coagulation test for proteins. Both are positive. Label lour test tubes E, F, G, H ; charge with the various solutions, and place in water-bath kept at 35. Tube. E F G H Contents of Tubes. I c.c. fibrinogen solution + 0.2 c.c. CaCl 2 I c.c. fibrinogen solution +0.2 c.c. CaCl. 2 + o.2 c c. platelet extract i c.c. fibrinogen solution + 0.2 c.c. CaCl. 2 + o.2 c.c. serum i c.c. fibrinogen solution + 0.2 c.c. CaCl. 2 -fo.2 c.c. plasma, filtered Result. No clot. Clot. Clot. No clot. These experiments show : 1. That the protein precipitated from plasma by half saturation with NaCl is the third constituent concerned in the formation of a blood clot. 2. That this protein when acted upon in the presence of soluble calcium salts by the coagulating agent present in the platelets gives rise to fibrin (tube F). 3. That this protein (fibrinogen) is absent from serum. 4. That the coagulating agent remains active after the clot has been formed, i.e., it is present in serum (tube G). It is absent from plasma (tube H). From these experiments the main facts of the process of blood coagulation can be tabulated as follows : 33 Q O O PQ O ^_Q T 13 | | r. '"S -S;U91U9]9 *, O ^ -f O . ^ < ? ^ $5 o g & ^ ^ 1v> "^ O Q fill ^ 1 1 r '-I ^ ! ^ ^ PC (A) | | i t o ^ :^ i 1 ^ U a & 8 J2 1 C/2 -5 ^ ^ ^ *> ^ 1 ffl * c1& <"^ -^ :s S 1 - 1 ^ lo -" ^L-^iJ^ * ^ P M ^ 1 * ^ '** in ^ ^ cL ^ ^ *** i ^ "^ < A 2j3 ^ |D lab "rf ^^ > .^ S ^ H 3 ||| | III || 34 HAEMOLYSIS. RESPIRATORY FUNCTIONS OF BLOOD. For Experiments 69-83 defibrinated blood is used. Experiment 69. Reaction of Blood. Apply a drop of defibrinated blood to glazed litmus paper. Allow it to remain for a minute and then wash off with water. Why is it necessary to use glazed litmus paper ? Determine in the same way the reaction of freshly drawn blood obtained by pricking the finger. What bearing has the reaction of the blood on its respiratory function ? Experiment 70. Laking of Blood. Haemolysis. Label three test tubes A, B, and C. Place in each I c.c. ( = twenty drops) of defibrinated blood. To B add 5 c.c. of water, to C two or three drops of ether. Then fill all three test tubes with 0.9 per cent. NaCl solution, and compare. In A the solution is opaque, because the blood corpuscles have remained intact ; in B and C it is transparent, because the blood corpuscles have been laked. Explain the results. iac Test Experiment 71. Oxidising ferment of blood " Guaiac test"- lood. Dilute 5 drops of blood with about 10 c.c. of water, add some hydrogen peroxide and float on the surface of the fluid two drops of alcoholic tincture of guaiac resin so that the latter forms a resinous- ring above the fluid. A blue colour gradually develops in the resinous ring. Explain the reaction. The reaction is not specific for blood, but given by many other animal and vegetable tissues and secretions such as milk. Blood differs from the other tissues, however, in so far as the reaction is given by blood even after it has been boiled, i.e., after the ferment has been destroyed. Repeat the experiment with diluted blood which has first been boiled. A positive result is obtained. In blood, therefore, the reaction is due not only to an oxidising ferment but also to some thermostable chemical constituent, probably the blood pigment. Sensitiveness of the Guaiac test. The test is very sensitive for blood. Determine the greatest dilution of blood with which the test can still be obtained. gen Capa- Experiment 72. Determination of Oxygen-Capacity of Blood. of Blood. The oxygen-capacity is the maximum amount of oxygen that can be held by blood. It is dependent on the amounjt of haemoglobin present. 35 The apparatus consists of a burette, which is inverted in a tall jar filled with cold water, and is connected by tubing to a bottle containing a small test tube. The bottle is placed in a vessel (water-bath) filled with cold water. Place some blood in a porcelain dish and stir vigorously with a glass rod, until it becomes completely saturated with oxygen. By means of a pipette run exactly 20 c.c. of blood into the bottle. Add to it 30 c.c. of dilute ammonia (i : 500). The water lakes the blood ; the ammonia is added in order to absorb the carbonic acid formed by the action of potassium ferricyanide on oxyhaemoglobin. In the small test tube place 4 c.c. of saturated potassium ferricyanide solution. Put the test tube into the bottle, taking care that the ferricyanide is not spilt. Close the bottle with the stopper, and put it into cold water. Test whether the apparatus is air-tight by raising the burette ; if there is no leakage the column of water in the burette remains standing at a higher level than the water in the jar. Allow the apparatus to stand for five minutes, so that all parts of it acquire the same temperature. Then open for a few seconds the clip on the tubing, which connects the inside of the bottle to the outer air. The pressure inside the apparatus is now the same as the pressure outside. Close the clip. The level of the water inside the burette is then at the same height as the level of the water in the jar. Read off the height in the burette with the water inside and outside at the same level. Now tilt the bottle so that the ferricyanide solution is upset, and shake gently as long as gas is evolved. When no more gas is evolved replace the bottle in the water and wait five minutes. What reaction has taken place ? Read the burette in the same way as before, />., with the water inside and outside at the same level. (Why is this necessary?) The difference in readings gives the amount of oxygen held by 20 c.c. of defibrinated blood. Record the result. A T .B. In measuring gases, as in this experiment, the temperature must be kept as constant as possible. (Why?) The apparatus should there- fore be touched with the hands as little as possible, and no source of heat (gas flame, bunsen, etc.) must be near it. CHEMISTRY OF BLOOD PIGMENT. ABSORPTION SPECTRA OF HEMOGLOBIN AND ITS DERIVATIVES. Solutions of haemoglobin and its derivatives give characteristic absorp- tion spectra, which can be examined with a spectroscope. What is an absorption-spectrum ? Examine the spectroscopes provided. Note that the width of the slit at one end can be adjusted, 1 and that the spectrum can be focussed by adjusting the eyepiece. The sharpness of the spectrum is dependent upon the width of the slit, and is the sharper the narrower the slit. Experiment 73. Solar Spectrum. Direct the spectroscope to the sky. If the slit and the eyepiece are properly adjusted, fine vertical dark lines can be seen in the spectrum. These are Fraunhofer's lines. How are they produced ? The lines are designated by letters. Line B is in the red, line D in the yellow, line E in the green, line F in the blue. Their position in the spectrum is constant, and they can be used to locate certain parts of the spectrum. Experiment 74. Spectrum of White Light Now direct the spec- troscope to a luminous gas flame (or to any other source of artificial light). The spectrum is still visible, but it does not show Fraunhofer's lines. Explain why Fraunhofer's lines are absent. Experiment 75. Spectrum of Sodium in White Light Introduce into the gas flame an asbestos stick soaked in sodium chloride, so that a sodium flame is produced. A bright yellow band is seen in the yellow part of the spectrum, and if the slit of the spectroscope is sufficiently narrow this yellow band can be seen to consist of two sharply defined 1 In some spectroscopes the width of the slit is fixed. 37 narrow lines. These lines are in the same position as the black line D of the solar spectrum. Most of the absorption spectra of haemoglobin and its derivatives lie in the neighbourhood of the D line, and their position with reference to the D line can be easily recognised by using a sodium flame as described in Experiment 75. NOTE. By convention the spectrum is usually arranged in such a way that the red end is on the observer's left-hand side, In observing the spectrum of any fluid the degree of dilution is of importance. Record diagrammatically the absorption spectra of oxyhaemoglobin, haemoglobin, carboxyhaemoglobin, methaemoglobin, and haemochromogen on charts, using the following chart as a model : Red. D. Violet. Experiment 76. Oxyhaemoglobin. Take some defibrinated blood in a test tube and run in water slowly from the tap, holding the tube obliquely under the end of the pipe ; allow the water to continue running after the tube is full. Thus one obtains a solution of oxyhaemoglobin diluted in such a way that the upper part of the tube contains almost pure water, the lower part a very concentrated solution of the pigment, and the middle part all gradations between the two. The corpuscles are of course laked. Adjust the spectroscope as described above ; place the upper end of the test tube, which must be quite dry, against the slit, holding the tube by the lower end with the left hand. On looking through the spectroscope .probably no bands will be seen. Gradually raise the tube so as to bring a stronger solution of oxyhaemo- globin in front of the slit. Two bands will appear, one narrower than the other and nearer the red end of the spectrum. In stronger and stronger solutions these two bands fuse into one, and broaden out so as to obscure the whole spectrum. Prepare a solution of oxyhaemoglobin so that the two bands can be clearly seen. Direct the spectroscope towards a sodium flame. It will be seen that both bands lie to the right of the D line, the left band lying close to the D line. Experiment 77. Haemoglobin. (Reduced haemoglobin). Reduce the solution of oxyhaemoglobin prepared in Experiment 76, which shows the two bands of oxyhaemoglobin distinctly. Reduction is performed by adding a reducing agent five drops of Stokes' solution, made alkaline with a few drops of ammonia (see below), or five drops of ammonium sulphide. If ammonium sulphide is used for reduction, the tube must be warmed gently to about 50. Note that the scarlet-red colour of oxyhaemo- globin (arterial blood) gives place to the bluish-red colour of haemoglobin (venous blood). Examine spectroscopically. The spectrum now shows a single broad band, which overlaps the space enclosed by the two bands of oxyhaemoglobin, and is fainter than either. Locate its position with reference to the D line. It is immediately to the right of the D line. Preparation of Stoked Solution. Dissolve 2 grms. of tartaric acid in a little water. In another small quantity of water dissolve 2 grms. of ferrous sulphate. Mix the two solutions and make the mixture up to 100 c.c. with water. Fill into bottle labelled "Stokes' solution." Before use a few drops of ammonia are added, so that the solution is just alkaline. Experiment 78. Close with the finger the test tube containing haemo- globin as prepared in Experiment 77, and shake vigorously for two to three minutes. Note that the colour of the solution changes from bluish red to scarlet red. Examine at once with the spectroscope ; the two bands of oxyhaemoglobin have reappeared. (Why?) Allow the test tube to stand for two or three minutes ; reduction takes place again owing to the presence of excess of the reducing agent, and the single band of haemoglobin is again seen in the spectrum. 39 Experiment 79. Carboxyhaemoglobin. Prepare some CO-hsemo- globin by filling a test tube with coal gas, and adding some diluted blood ; close the tube with the thumb, and shake. Notice the pink colour. This pink colour persists even if the pigment is much diluted with water. (Con- trast with oxyhaemoglobin, which becomes yellow if much diluted with water.) Examine CO-hsemoglobin spectroscopically, diluting the solution until two absorption bands are distinctly visible. The two bands are apparently similar to those of oxyhaemoglobin. Verify by locating the position of the bands with reference to the D line, using a sodium flame. The two bands have almost the same position immediately to the right of the D line. Carboxyhceinoglobin is not acted upon by reducing agents: Distinction from oxyhcemoglobin. Add five drops of ammonium sulphide. No change takes place : the bands persist. Add five drops of Stokes' solution, made alkaline with ammonia. Same result. Experiment 80. Methaemoglobin. To 5 c.c. of water add four drops of blood. To the strong solution of oxyhaemoglobin thus prepared add a few drops of potassium ferricyanide. The solution becomes brown, and the spectrum shows a distinct band in the red. Locate by means of the D line, using a sodium flame : the band is to the left of the D line. This is the characteristic band. With this concentrated solution there is marked absorption in the blue end of the spectrum. Dilute with an equal bulk of water ; two faint bands appear in the green. Add to the dilute solution of methaemoglobin a few drops of ammonium sulphide and warm gently. The colour changes to red. Examine immedi- ately with the spectroscope. At first the spectrum of oxyhaemoglobin appears, then that of reduced haemoglobin. What is the difference between methsemoglobin and oxyhaemo- globin ? Experiment 81. Alkaline Haematin. Haemochromogen (or reduced alkaline haematin). Prepare alkaline haematin by warming some diluted blood with caustic soda. \Varm gently at first, then heat to near boiling 40 point, but do not allow to boil. The colour changes to brown. Cool. The spectrum is indistinct. It shows a faint band in the red to the left of the D line. Now add a few drops of ammonium sulphide ; the very distinct spectrum of hsemochromogen appears. It consists of two bands in the green. Locate by means of the D line, using a sodium flame. The two bands are to the right of the D line, as in the case of the spectra of oxyhaemoglobin or CO- haemoglobin, from which the bands of haemochromogen differ by lying further towards the blue end of the spectrum. There is a distinct space between the left band of haemochromogen and the D line, while in the case of oxyhaemoglobin and CO-haemoglobin the left band abuts against the D line. If the solution of haemochromogen is diluted, the left band persists longer, being the stronger band, so that in very weak solutions only this band is seen. Experiment 82. The formation of haemochromogen is a very delicate test for blood. Dilute blood until the spectrum of oxyhaemoglobin cannot be readily seen. Convert this very dilute solution of oxyhaemoglobin into haemochromogen as detailed in Experiment 81. The formation of haemo- chromogen can be detected by the presence of the left band. Experiment 83. Haematoporphyrin. Place 3 c.c. of pure concentrated sulphuric acid in a test tube, and allow one drop of defibrinated blood to fall into it. The fluid shows a purple colour. Examine spectroscopically. Dilute, if necessary, with concentrated sulphuric acid. The spectrum shows two bands, one which is near the red end of the spectrum being much narrower than the other. Locate their position with reference to the D line. The narrower band is immediately to the left of the D line, so that the D line is enclosed by the two bands. Why is concentrated sulphuric acid necessary for the formation of this derivative ? What is the essential difference between the chemical composition of haematoporphyrin and that of the other haemoglobin derivatives ? CHEMISTRY OF BLOOD-SERUM. Experiment 84. Reaction and Specific Gravity of Serum. Test the reaction of serum against litmus. Determine the specific weight by means of a hydrometer. Wipe the instrument clean, and float it in the centre of the cylinder containing the serum, taking care that it does not touch the sides of the vessel. Place the eye level with the true surface of the serum (not the top of the meniscus around the shaft of the hydrometer), and read the division of the scale. Record your result. Experiment 85. Proteins of Serum. Separation of Globulin and Albumin. Apply to serum the protein tests. (Exp. 33, 34.) Note that they are all very strongly positive. Separate the proteins present in the serum by the " salting out " method. Proceed as follows : oteins of T IO c - c - f tne serum add an equal volume of a saturated solution of wd-Serum. ammonium sulphate, thus obtaining a half-saturated solution. Filter off the precipitate of serum-globulin which appears, and remove the filtrate, which contains serum-albumin. Wash the precipitate two or three times with a half-saturated ammonium sulphate solution, and dissolve it, together with the ammonium sulphate which adheres to it, in about 20 c.c. of water. This yields a clear solution of a globulin in a dilute salt (ammonium sulphate) solution. Label it " Serum-globulin" To the filtrate from the serum -globulin add solid ammonium sulphate until the solution is saturated, keeping the solution at a temperature of 20 to 30 C. in order to facilitate the dissolving of the ammonium sulphate. Higher temperatures must be avoided. Why? When the solution is saturated a precipitate of albumin is formed. Note the difference between the dead-white, heavy crystals of ammonium sulphate lying at the bottom of the vessel and the yellowish-white, light, flocculent precipitate of albumin which is suspended in the liquid. Filter. Wash the precipitate on the filter with a saturated solution of ammonium sulphate, and dissolve it in about 20 c.c. water. A clear solution of an albumin is obtained. Label it " Serum-albumin" 42 Experiment 86. Solubility of Albumin and Globulin in Water and in Concentrated Salt Solutions. Add a few drops of the globulin solu- tion to a large volume of distilled water in a beaker. A slight, cloudy precipitate is formed. Do the same with the albumin solution. No precipitate is formed. Saturate some of the globulin solution with solid sodium chloride. The globulin is precipitated. Saturate some of the albumin solution with solid sodium chloride. The albumin is not precipitated. Record in tabular form the solubilities of albumin and globulin according to the following table : Solvent. Albumin. Globulin. Distilled water Dilute NaCl solution - Saturated NaCl Half-saturated (NH 4 ) 2 SO 4 solution Full-saturated (NH 4 ) 2 SO 4 Apply to the albumin and globulin solutions the heat coagulation test. Note that both albumin and globulin are coagulable by heat. Apply the colour tests for proteins. Note that the xanthoprotein test is quite distinct, but that the biuret test may be negative. This is due to the fact that the albumins and globulins have been separated by means of ammonium sulphate, and that ammonium salts interfere with the biuret test. Study the effect of the presence of an ammonium salt on the biuret test in the following way : Apply the test to (a) water ; (b) water to which some ammonium sulphate has been added ; (V) serum ; (d) serum to which 43 some ammonium sulphate has been added. The following colours will be obtained : (a) Water - - faint blue. () Water + (NH 4 ) 2 SO 4 deep blue. (V) Serum - - violet. (d) Serum + (NH 4 ) 2 SO 4 deep blue. Only (c) gives the characteristic violet colour which indicates the presence of proteins. Ammonium salts therefore interfere with the test. This must be borne in mind when the test is applied after having used ammonium salts for the separation of proteins. The difficulty can be overcome by using in such a case a very large amount of caustic soda before adding the copper sulphate. Try this with some serum to which ammonium sulphate has been added. Experiment 87. Serum Constituents other than proteins. Method of removal of heat-coagulable proteins. In order to determine the presence of substances other than proteins in tissue fluids or tissue extracts it is necessary to remove completely the proteins, as their presence interferes with the tests used for other tissue constituents (see also Exp. 39). If the proteins are coagulable by heat, as is the case with serum, the proteins are best removed by heat coagulation. It is important to carry out this process carefully so that all the proteins are completely precipitated. Pro- ceed in the following manner : Dilute 20 c.c. of serum with 100 c.c. of water ; heat the neutral solution to boiling point, stirring constantly. Make faintly acid with a few drops of dilute acetic acid, and filter. If the heat coagulation has been carried out correctly (see Exp. 33), the filtrate will not give the biuret test, i.e., it will be free from proteins. Concentrate the filtrate to about 20 c.c., and apply to it the following tests : (a) Test for chlorides with silver nitrate and nitric acid. () Test for phosphates with ammonium molybdate and nitric acid. (c) Test for sulphates with barium chloride and hydrochloric acid. (d) Test for a reducing sugar with Fehling's or Trommer's test. Record your results. 44 METHOD OF CHEMICAL EXAMINATION OF TISSUES. From the foregoing experiments it follows that the three main groups of organic substances which may be present in tissues and tissue fluids namely, (i) proteins, (2) carbohydrates, and (3) fats and lipoids, can be separated from each other by extraction with different solvents. The various substances of which each group is composed can then be recognised by their specific reactions and properties. The following general rules for the chemical examination of tissues and tissue fluids can be deduced from the work hitherto performed : The organs must be minced. I. Proteins. (a) Extraction with dilute salt solution dissolves 1. Simple Proteins {Albumins, Globulins]. They give the tests for proteins. They are coagulated by heat. In order to ascertain whether an albumin or a globulin is present, determine the solubility in distilled water, in half-saturated and in fully-saturated ammonium sulphate solution. If both are present they can be separated by their different solubility in ammonium sulphate solution. 2. Mucin. It gives all the tests for proteins. It is precipitated by- acetic acid, and is insoluble in excess of this acid (see Exp. 88). On boiling with dilute HC1 it yields a reducing substance, glucosamin. This last test can be carried out only if sufficient material is obtained. The saline extract will also contain carbohydrates and extractives (see below). (b] Extraction with I per cent, sodium carbonate or sodium hydrate solution dissolves Nucleoprotein. It gives all the protein tests. It is precipitated by dilute acetic acid, but soluble in excess of this acid. This alkaline extract will also contain Mucin (see above). 45 II. Carbohydrates. Extraction with hot water will dissolve all the carbohydrates : Starch, glycogen, dextrin, sugars. These may be recognised by their tests. The extract may contain a small amount of protein sub- stances (test by means of biuret test) ; these must be removed by precipitation or heat coagulation before the tests for carbohydrates can be applied. The watery extracts also contain inorganic salts and organic extractives, such as urea, creatin, purin-bases, etc. The commercial meat extracts consist mainly of such organic extractives. III. Fats and Lipoids. The tissue is dried and extracted successively (a) With acetone, which extracts mainly fats and cholesterin. (b) With ether, which extracts mainly phosphatides. (c) With chloroform (or hot alcohol), which extracts lastly cerebrosides, phospho-cerebrosides, cholesterin esters. If one wishes to remove all fats and lipoids without separating them, extraction with chloroform, which dissolves all lipoids, is applied at once. The method is essentially the same if tissue fluids, exudates, etc., are to be examined, except that the tests for proteins are applied directly to the fluid. If any proteins are present, they must be removed before examining for carbohydrates. Fat is extracted by shaking the fluid with ether, which, after allowing the two fluids to separate, is removed by means of a pipette, or by decantation. If contents of the alimentary canal are to be examined, products of protein digestion may be present, and must be tested for by the method described under digestion (see below, Exp. 109). Digestion. SALIVA. Experiment 88. Collect about 10 c.c. of saliva in a beaker. Test its reaction to litmus. Record. ;/. Add dilute acetic acid to some saliva in a test tube. A stringy precipi- tate of mucin is formed. It is insoluble in excess of acetic acid. Prepare some dilute saliva as described in Experiment 18. With this dilute saliva carry out the following experiment : stion by Experiment 89. Prove that Saliva contains an Amylolytic (Diastatic) ' a - Ferment: Ptyalin. To 5 c.c. of starch solution (i per cent.) in a test tube add 5 c.c. of dilute saliva. Mix by shaking and place in a water-bath kept at 37 to 40. Place a series of drops of dilute iodine solution on a porcelain slab. Every half minute remove a drop of the salivary digest by means of a glass rod, and apply it to one of the iodine drops. At first a blue colour will be produced, then a reddish-violet colour, then a light brown colour, finally no colour will appear. (State what chemical changes are indicated by' the changes in colour.) The point when first a colour fails to appear is called the llc - to dryness on the water-bath. Cool, and extract the residue with ether. Decant the ethereal solution into a beaker and again extract with ether. Decant again, collect the ethereal extracts, and allow the ether to evaporate. (All gas flames must be extinguished?) When the ether has been evapor- ated a residue remains, which should be dissolved in a few drops of warm alcohol. Examine microscopically the crystals which separate out on cooling, and apply the tests for cholesterin. Under certain pathological conditions cholesterin separates out from bile. What is the result? Normal Metabolism. URINE. CONSTITUENTS OF NORMAL URINE. Each student is expected to provide for these experiments about 200 c.c. of his own urine. Colour The colour is of a transparent yellow. The froth which forms on shaking soon disappears. Normal urine contains no sediment when passed. Deposits may form later (see below). Experiment 126. Reaction. The urine is acid to litmus. Test. It may react acid to blue litmus, and alkaline to red litmus : " amphoteric reaction." Two or three hours after a meal it may have an alkaline reaction. Why ? On standing urine becomes alkaline unless kept sterile. This is due to " ammoniacal fermentation," produced by the Micrococcus tirece which converts urea into ammonium carbonate. Experiment 127. Specific Gravity. Total Solids. Determine the specific gravity by means of a urinometer, as described in Experiment 84. The urinometers are graduated for a temperature of 15 C. The specific gravity of normal urine varies between 1.015 and 1.025, and gives a rough indication of the amount of solids present in the urine. The last two figures of the specific gravity multiplied by 2.33 give approxi- mately the solids in grammes per litre. Example: specific gravity 1.020. 20 : 2.33 = 46.6 grammes of solids per litre of urine. Volume. The volume varies. The average volume is 1,200 to 1,400 c.c. in twenty-four hours. 59 6o CHLORIDES. Experiment 128. To 1 5 c.c. of urine add a drop or two of concen- trated nitric acid (if the urine is alkaline add nitric acid till reaction is acid) and five drops of silver nitrate solution (2 per cent). A coherent clump of silver chloride is precipitated if the chlorides are present in normal quantity. If the chlorides are much diminished, as in febrile conditions, the precipitate is more or less diffuse, according to the diminu- tion. Why is nitric acid added ? Add ammonia ; the precipitate dissolves. What is the average quantity of chlorides in normal urine ? What is the source of the chlorides in urine ? SULPHATES. Experiment 129. (a) Inorganic Sulphates. To 10 c.c. of urine add about 3 c.c. of barium chloride solution. A thick precipitate forms, which consists of the phosphate and sulphate of barium. Acidify with a few drops of concentrated HC1. The barium phosphate dissolves and an opaque milkiness remains, indicating the presence of inorganic sulphates. If the precipitate which remains is thick the inorganic sulphates are in excess. Experiment 130. (/;) Ethereal Sulphates. To 10 c.c. of urine add barium chloride as long as a precipitate continues to form. Make alkaline with a few drops of sodium carbonate solution, and filter. Acidify the filtrate, which is now free from inorganic sulphates, with concentrated HC1, and boil for three minutes. A faint cloud of barium sulphate is formed on standing, indicating the presence of ethereal sulphates. If ethereal sulphates are present in excess, a distinct precipitate is formed, which settles to the bottom. What is the average daily quantity of sulphates in urine ? What is the source of the sulphates in urine? What does an excess of ethereal sulphates indicate ? Experiment 131. Prepare an ethereal sulphate as follows : Warm ten drops of absolute alcohol, with five drops of concentrated sulphuric acid. After cooling, make alkaline with 10 per cent, sodium 6i hydrate ; add barium chloride as long as a precipitate continues to be formed ; then heat to boiling point, and filter. The filtrate contains barium ethyl-sulphate. To it add half its volume of concentrated hydro- chloric acid, and boil. A precipitate of barium sulphate forms. Compare the solubility in water of barium sulphate and barium ethyl-sulphate. PHOSPHATES. Experiment 132. To a test tube full of urine add a little strong ammonia and heat. A white precipitate of the phosphates of calcium and magnesium " earthy phosphates " forms. (Such a precipitate is often found in alkaline urine as a crystalline deposit. See below, under Deposits.) Filter. The filtrate contains the phosphates of sodium and potassium " alkaline phosphates." A. Precipitate. " Earthy PJwsphates" Place the precipitate in a test tube with some water, and add a few drops of dilute acetic acid. It dis- solves. The presence of .phosphoric acid in this solution can be verified by the ordinary tests ; for instance, by the formation of ammonium phospho- molybdate. To the acid solution add some nitric acid and about 5 c.c. of ammonium molybdate. Heat gently over small flame to about 6o f . The solution turns yellow, and a yellow crystalline precipitate is formed. What is the yellow precipitate ? B. Filtrate. "Alkaline Phosphates" To the filtrate add a little magnesia mixture and warm gently. A white precipitate is formed, indicating the presence of phosphates in the filtrate. What is this pre- cipitate ? The presence of phosphates may also be determined by testing with ammonium molybdate as detailed above. Note the difference in the bulk of this precipitate and that of " earthy phosphates " obtained on adding ammonia to the urine. Are the " earthy " or the " alkaline phosphates" present in larger amount in urine? Experiment 133. Formation of Deposit of Ammonium Mag- nesium Phosphate. ("Triple Phosphate"). --Set some urine aside in a beaker for two or three days. Ammoniacal fermentation occurs (the reaction of the urine becomes alkaline ; test with litmus), and a crystalline deposit of ammonium - magnesium phosphate settles out. 62 Examine microscopically. Sketch. Crystals of calcium phosphate may also be present, and may be recognised by the stellar arrangement of the crystals. Experiment 134. On heating neutral urine a precipitate of earthy phosphates is often formed. Try this. It dissolves on the addition of a few drops of acetic acid. This is important in testing for albumin by the heat test. The explanation for the appearance of this precipitate is given by the following experiment : Experiment 135. Treat a solution of calcium chloride with sodium phosphate, and then with excess of sodium carbonate. Calcium phosphate is precipitated. Add acetic acid, drop by drop, till the precipitate just dissolves : acid calcium phosphate is formed. Heat : calcium phosphate is precipitated again owing to the alteration of the reaction as the carbon dioxide is evolved. The precipitate dissolves on adding a drop or two of dilute acid. Experiment 136. Precipitation of Phosphates by Uranium Nitrate. To a little urine add a few drops of acetic acid and some sodium acetate, and then uranium nitrate. A precipitate forms. In order to complete precipitation the urine must be heated to boiling point. This reaction is used for the quantitative estimation of phosphates. What is the average daily quantity of phosphates in the urine ? What is the source of phosphoric acid in the urine ? By which other channels are calcium and magnesium excreted ? DEPOSITS OF PHOSPHATES IN URINE. From alkaline urine deposits of phosphates may separate out. All these deposits are easily soluble in acetic acid. Earthy Phosphates. Amorphous granules of Ca 3 (PO 4 ) 2 and Mg 3 (PO 4 ) 2 . Ammonium- Magnesium Phosphate^ MgNH 4 PO 4 . (Triple phosphate.) Large colourless prisms in the shape of'" knife-rests" or " coffin lids," or in the shape of feathery crystals. 63 Calcium-Hydrogen Phosphate, CaHPO 4 . Prismatic crystals arranged in rosettes. These may also occur in acid urines. DEPOSIT OF OTHER CALCIUM SALTS IN URINE. From acid urines : Urinary Calcium oxalate, either in the form of highly refractive octahedra, ^aicnnn Salts " enve ^P e " sna P e > or in the form of ovoid bodies, " dumb-bell " shape. They are insoluble in acetic acid ; soluble in hydrochloric acid. A con- siderable sediment of calcium oxalate is pathological. From alkaline urine : Calcium carbonate. Spherical or ovoid crystals with concentric striation. Readily soluble in acetic acid with effervescence. Common deposit in the urine of herbivorous animals. AMMONIA. Ammonia. Experiment 137. Make urine alkaline with sodium carbonate solu- tion, and warm. Note the smell of ammonia. A moist piece of red litmus paper held over the mouth of the tube turns blue. Within what limits does the average amount of ammonia excreted by a normal person in twenty-four hours vary ? How can it be increased ? How can it be diminished ? PREPARATION OF UREA FROM URINE, Preparation of Experiment 138. Place about 100 c.c. of urine in a porcelain capsule and evaporate in a water-bath to about 20 c.c., so that the urine is now concentrated to a syrup. Cool by floating the capsule on cold water. Filter into a beaker, and place beaker in cold water. Slowly add while stirring an equal volume of cold 50 per cent, nitric acid (pure). (Why can fuming yellow nitric acid not be used ?) The mixture must be kept cold. Crystals of urea nitrate separate out. Filter off the crystals ; dry them by pressing between successive sheets of filter paper. When the crystals are dry, mix with excess of barium carbonate, and add a little alcohol to form a paste. The urea nitrate is decomposed, giving barium nitrate, CO 2 , and urea. Dry the mass on the water-bath, extract with alcohol, and filter. Concentrate on 64 the water-bath the alcoholic filtrate to a small volume. Cool. Urea crystallises out in long needles. Examine microscopically. Sketch the crystals. If no immediate crystallisation occurs, allow the alcoholic solution to stand overnight. Is urea or urea-nitrate more soluble in water? Within what limits does the amount of urea excreted by a normal person in twenty-four hours vary ? How can it be increased ? How can it be diminished ? PREPARATION OF URIC ACID FROM URINE. Preparation of Experiment 139. To 25 c.c. of urine in a beaker add 5 c.c. of strong *and of C Urates HC1, and allow to stand for twenty-four hours. Examine microscopically the crystals of uric acid which separate out. Sketch the crystals. Note that the crystals are deeply pigmented with urinary pigment. To some of the crystals add caustic soda. The crystals dissolve. To the alkaline solution add excess of strong HC1. The crystals are again formed after some time. The crystals give the murexide test (see below, Exp. 146). PREPARATION OF AMMONIUM URATE FROM URINE. Experiment 140. To 25 c.c. of urine add two drops of ammonia and solid ammonium chloride, stirring all the time until the solution is saturated. Avoid an excess of the salt. A gelatinous, amphorous precipitate of ammonium urate is formed. Examine microscopically. All the uric acid present in the urine is completely precipitated as ammonium urate. This reaction is therefore used for the quantitative estimation of uric acid. The deposit gives the murexide test (see below). What is the source of the uric acid excreted in urine ? Within what limits does the quantity of uric acid excreted in twenty- four hours by a normal person vary? How can it be increased ? How can it be diminished ? UREA. Urea. What is the structural formula for urea ? Prepare biuret from urea (see Exp. 34*2). 65 Experiment 141. Solubility. Determine the solubility of urea in (a water ; (b) cold alcohol ; (c) cold ether. Record the results. Prepare a dilute watery solution of urea for the following experiments. Experiment 142. To 5 c.c. of the urea solution add some sodiun hypobromite. Bubbles of nitrogen are evolved. State in the form of an equation the process which takes place. Thi reaction is used for the quantitative estimation of urea. It is, howevei given, not only by urea, but by all substances having amido groups and b; ammonium salts. Repeat the test, using a dilute solution of ammonium sulphate instea< of the urea solution. Record the result. Experiment 143. To some urea solution add some yellow nitric acic which contains nitrous acid. Bubbles of nitrogen and of CO 2 are evolved. This reaction is also given by all substances having an amido group. State in the form of an equation the process which takes place. Experiment 144. Boil some urea solution with caustic soda. Notio the smell of ammonia which is evolved. State the reaction which takes place. URIC ACID. What is the structural formula for uric acid ? Experiment 145. Solubility. Determine the solubility of uric aci( in (a) water, cold and hot ; (b) alcohol, cold and hot ; (c) caustic soda o sodium carbonate, cold and hot ; (d) dilute HC1, cold and hot. Dissolve some uric acid in warm caustic soda, acidify with dilute HC1 and allow to cool slowly. Uric acid crystallises out. Examine micro scopically. Sketch the crystals. What is formed when uric acid is dissolved in caustic soda? Experiment 146. Murexide Test. To a small crystal of uric acic on a chip of thin porcelain or a crucible lid, add two drops of strong nitri< acid. Evaporate over a small flame, in a fume chamber, to complete dry ness. A red deposit remains. Add with a glass rod a drop of very dilute ammonia. The residue turns to a violet colour. 66 Experiment 147. Uric Acid reduces Fehling's Solution. Dilute some Fehling's solution and heat to boiling point. To the boiling solution add repeatedly a few drops of a solution of uric acid in caustic soda, heat- ing after each addition. Red cuprous oxide separates out. If the urate is present in excess a white precipitate of cuprous urate will be formed at the same time. The presence of uric acid may therefore be a fallacy in examining urines for sugar by means of Fehling's test, especially if the boiling is prolonged. This can be shown to be the case as follows : Experiment 148. Heat simultaneously in two test tubes equal volumes of normal urine and of half-diluted Fehling's solution to boiling point. When boiling mix the contents. Pour one-half of this mixture of urine and Fehling's solution into a test tube and allow to cool. This will remain blue if the urine is normal. Boil the other half for about three minutes : if sufficient urates are present the solution may become decolor- ised and acquire a brown colour, owing to the reduction of the cupric salt by urates. Experiment 149. Boil 5 c.c. solution of uric acid in caustic soda with i c.c. of Nylander's reagent. No reduction takes place. Experiment 150. The presence of uric acid is therefore no fallacy if Nylander's reagent is used. Demonstrate this by adding to some urine (5 c.c.) one-tenth of its volume (J c.c. = 10 drops) of Nylander's reagent ; boil over a small flame for three minutes. No reduction to metallic bismuth occurs. DEPOSITS OF URIC ACID AND URATES. From acid urines a crystalline deposit of uric acid may separate out. It has a sandy red colour, and is therefore called " cayenne pepper deposit." This deposit corresponds to the one obtained experimentally in Experi- ment 139. It is recognised by its crystalline form and by the murexide test. From acid urines a deposit of urates (mainly sodium urates) may separate out. It maybe amorphous or crystalline. It has a pinkish-red colour, and is therefore called " brick dust deposit." This deposit is found 67 frequently in concentrated urines on cooling. It dissolves on heating the urine, and thus differs from deposits of phosphates. The deposit is soluble in hot water or hot acids. On adding hydro- chloric acid to the watery solution, and allowing to cool, crystals of uric acid separate out. The deposit gives the murexide test. It is not readily soluble in cold acetic acid. From alkaline urines a deposit of ammonium urate may separate out in the form of yellow or brownish spheres, with or without projecting spicules " hedgehog crystals." They dissolve in hydrochloric acid ; on standing uric acid crystallises out of the acid solution. The deposit gives the murexide test. A considerable sediment of uric acid or urates does not necessarily indicate a high uric acid content. CREATININE. What is its structural formula ? Experiment 151. Weyl's Test. To 5 c.c. of urine add a few drops of a freshly prepared solution of sodium nitroprusside. Then render alkaline with caustic soda. A red colour results. Strongly acidify with acetic acid. The solution is decolorised. Experiment 152. Jaffe's Test To 5 c.c. of urine add picric acid. Then render alkaline with caustic soda. The solution becomes red in colour. What is the average amount of creatinine excreted by a normal person in twenty-four hours ? Creatinine, like uric acid, is capable of slightly reducing Fehling's solution. It has at the same time the power to keep in solution a small amount of cuprous oxide. If, therefore, a dilute glucose solution contain- ing creatinine is tested with Fehling's solution, a brown clear solution instead of a red or yellow deposit may be obtained. This may also occur if a urine of normal concentration containing only a small amount of sugar is examined by Fehling's test. 68 INDICAN. Experiment 153. To about 10 c.c. of urine add an equal volume of strong fuming HC1, one or two drops (not more !) of a 5 per cent, solution of calcium hypochlorite, and 3 c.c. chloroform. Close the mouth of the tube with the thumb, cautiously invert a few times, and allow the mixture to stand for a few minutes. The chloroform becomes blue. Note the change in the colour of the urine. The amount of indican present is proportional to the depth of colour of the chloroform extract. A rough estimate of the amount of indican present can be obtained by comparing the depth of the blue colour with a blue standard solution, e.g., Fehling's solution. From what substance is the indican formed ? What relation does the amount of indican present in the urine bear to intestinal putrefaction ? Repeat the test, using about five to ten drops of ferric chloride solution, instead of the calcium hypochlorite. The same result is obtained. Repeat the test, with one or two drops of calcium hypochlorite, as detailed above. After the chloroform has become blue add a few more drops of calcium hypochlorite, and invert the tube repeatedly. What takes place ? Why is the addition of calcium hypochlorite necessary for this reaction, and by what substances can it be replaced ? Why must an excess of calcium hypochlorite be avoided ? Is the addition of chloroform necessary for the formation of indican ? QUANTITATIVE ESTIMATION OF CERTAIN NITROGENOUS CONSTITUENTS OF THE URINE. Each student is expected to collect accurately a twenty-four hour specimen of his own urine, and to measure and record its volume. The urine should be protected against ammoniacal fermentation by the addition of 2 c.c. of a 5 per cent, solution of thymol in chloroform, which is supplied. A sample (about 150 c.c.) of the total twenty-four hours urine should be kept on hand for the experiments. Decinormal solutions of acid and alkali are supplied. NORMAL SOLUTIONS. A normal solution is one which contains the equivalent weight in grammes of a given substance dissolved in 1,000 c.c. of water. What is meant by the term " equivalent weight " ? As an example take hydrochloric acid. The equivalent weight of = 35.5, H=i, therefore HC1 = 36.5. Therefore a normal HC1 solution contains 36.5 grms. HC1 in 1,000 c.c. of water, a decinormal solution con- tains 3.65 grms. HC1 in 1,000 c.c. of water, and I c.c. of a decinormal HC1 solution contains 3.65 mgs. HC1. Normal solution is written n/i, decinormal n/io, and so on. The same holds good for, say, NaOH, or for H 2 SO 4 . Note that for a dibasic acid, such as H 2 SO 4 , the equivalent weight is half the molecular weight. What is the equivalent weights for NaOH ? for H 2 SO 4 ? for NH 3 ? Since normal solutions contain equivalent weights dissolved in equal volumes, it follows that each c.c. of any n/io acid will require for neutralisa- tion exactly i c.c. of any n/io base. Try this as follows : Experiment 154 Run from a burette into a beaker exactly 10 c.c. of n/io acid. Dilute with about 20 c.c. of distilled water, add two drops of 70 an indicator (phenolphthalein). Then add slowly from a burette ;//io alkali, until the addition of one drop will just produce a purple colour. Record your result. Experiment 155- Repeat the titration, using rosolic acid as an indicator instead of phenolphthalein. Experiment 156. Estimation of the Total Nitrogen by Kjeldahl's Method. Principle. First Stage. Acid Incineration. By boiling with concen- trated sulphuric acid all organic nitrogenous compounds are converted into ammonium sulphate. This reaction is accelerated by the addition of a small amount of copper sulphate, which acts as a catalyst. (What is a catalyst?) Second Stage. Distillation. The ammonium sulphate formed in the first stage of the process is decomposed by the addition of an excess of caustic soda. The ammonia, which is set free, is distilled into a measured amount of standard n/io sulphuric or hydrochloric acid. The amount of the acid that has been neutralised by the ammonia is found by subsequent titration with standard n/io sodium hydrate. Process. First Stage. From the twenty-four hours' urine take exactly 5 c.c. by means of a pipette and place in a Kjeldahl flask in such a way that the urine does not touch the sides of the neck of the flask. Add about 10 c.c. concentrated sulphuric acid (measured with a measuring cylinder, not with a pipette], and a small crystal of copper sulphate. Place the Kjeldahl fla.sk in the fume-chamber and heat. After all the water is driven off a more or less violent reaction will take place. Continue the boiling until a clear, almost colourless, solution is obtained (30 to 45 minutes in the case of urine). In the meantime arrange the apparatus for distillation by connecting the upper end of a condenser with a spurting bulb, while the lower end is connected with an " adapter," a glass tube, which passes down into a receiving flask. Second Stage. Distillation. Allow the incinerated urine to cool. In the meantime place in receiving flask from a burette an accurately measured quantity (between 25 c.c. and 50 c.c.) of /io acid, e.g., 30 c.c. Note the amount of acid placed in the receiving flask. When the Kjeldahl flask has cooled add distilled water to the contents, so that about a third oi the flask is filled. (The first addition of water must be done carefully because the Kjeldahl flask contains concentrated sulphuric acid.) Add two drops of an indicator, e.g., rosolic acid (which gives a red colour when alkaline), to the contents both of the Kjeldahl flask and of the receiving flask. Now add a pinch of talc to the contents of the Kjeldahl flask (in order to prevent bumping), then make the contents strongly alkaline by the addition of strong caustic soda, and at once close the Kjeldahl flask by inserting the spurting bulb. (Note. The addition of soda is best carried out through a funnel, so that the mouth of the flask remains dry, otherwise it will be difficult to keep the spurting bulb in position.) See that the tube connected with the lower end of the condenser dips into the acid. Now heat, at first carefully, then with a full flame. Continue boiling until all the ammonia is distilled over. This will last from 30 to 45 minutes. The distillation is complete when a piece of red litmus held against the mouth of the adapter no longer turns blue. Note I. In distilling great care must be taken to prevent a " sucking back " of the contents of the adapter into the distilling flask. This will occur as soon as the pressure in the distilling flask is allowed to fall by removing the flame or by careless heating. If there is any danger of "" sucking back," the receiving flask should be lowered, so that the adapter -does not dip into the acid. Note 2. If during distillation the contents of the receiving flask turn pink, indicating that the reaction has become alkaline, a measured amount of standard acid must be added at once. Otherwise ammonia will be lost. When distillation is complete, remove first the receiving flask, then turn out the gas. Titrate the acidity of the contents of the receiving flask, and from that calculate the total amount of nitrogen excreted in twenty- four hours. Calculation. Example : Total volume of twenty-four hours' urine = 1,550 c.c. Amount taken for estimation = 5 c.c. Amount of n/io acid placed in receiving flask = 30 c.c. Amount of n/io alkali used in titration= 17.8 c.c. Therefore amount of n/io acid neutralised by ammonia = 12.2 c.c. Now i c.c. n/io acid= I c.c. n/io ammonia. Since 1,000 c.c. njio ammonia contain 1.7 grms. NH 3 , I c.c. n/io ammonia contains 1.7 mg. NH. 5 . Therefore I c.c. n/\o acid= 1.7 mg. NH 3 . Further, 1.7 mg. NH 3 contains 1.4 mg. N, because the atomic weights are N=i4, H 3 = 3. Therefore I c.c. ;//io acid ndicates 1.4 mg. N. Therefore the amount of N in milligrammes is obtained if the number of c.c. ofn/io acid, neutralised by ammonia, is multiplied by 1.4. In this case 5 c.c. of urine contain 12.2 x 1.4 mg. N= 17.08 mg. N. From this the total amount of nitrogen excreted in twenty-four hours can be calculated (in this case =5. 29 grms.). Experiment 157. Estimation of Acidity and of Ammonia. This method is based on the fact that ammonium salts (and also other sub- stances containing an amido group) react with formaldehyde in such a way that the ammonia and formaldehyde form a complex organic com- pound (hexamethylentetramin or urotropin), while the acid which was combined with the ammonia is liberated. The amount of acid set free is determined by titration, and is a measure of the ammonia present. In carrying out this estimation, the urine is at first neutralised by adding from a burette n/io alkali. The amount of n/io alkali added is a measure of the acidity of the urine. Then neutral formaldehyde is added. Owing to the liberation of acid which takes place when the formaldehyde has combined with the ammonia, the urine acquires again an acid reaction. This second acidity is titrated again with /io alkali, and this second titration is a measure of the amount of ammonia present. At least two estimations should be carried out. 73 Process. Dilute 25 c.c. of urine with an equal volume of water, add 15 grms. of finely powdered neutral potassium oxalate in order to pre- cipitate all the calcium salts, and four or five drops of phenolphthalein. Shake thoroughly for one or two minutes, and, whilst the solution is still cold from the effect of the oxalate, titrate with TZ/IO NaOH until a per- manent pink tint remains. Record the number of cubic centimetres added. This is a measure of the acidity. Now dilute 10 c.c. of formalin with two volumes of water. This mixture will be slightly acid owing to the presence of some formic acid in the formaldehyde. It must be made neutral to phenolphthalein by adding TZ/IO NaOH until a faint permanent pink colour appears. Add this neutral formaldehyde to the urine. The urine becomes acid again and the colour disappears. Run the n/io NaOH into the mixture until a permanent pink tint is again obtained. Record the number of cubic centimetres^ added in this second titration. This is a measure of the ammonia. Calculate from your results : 1. The acidity of the urine expressed in terms of njio acid (a) as per- centage ; (&) for the total twenty-four hours' quantity. 2. The ammonia in grammes excreted in twenty-four hours. 3. The fraction of total nitrogen which is excreted as ammonia. Express the fractions in terms of percentage of total nitrogen. Calculation. Example : Total volume of urine = 1,550 c.c. Total N excreted in twenty-four hours = 5. 29 grms. Amount taken for estimation = 25 c.c. First titration, 2.2 c.c. n/io alkali. Second titration, 7.7 c.c. n/io alkali. i. Acidity. Since 2.2 n/io alkali neutralises 25 c.c. of urine, its acidity is 2.2 c.c. n/io acid. The acidity of 100 c.c. of urine is therefore 8.8. c.c. n/io acid. The acidity of the total urine is ~ X 1 >$5 c.c. 136.5 c.c. n/io acid. 74 2. Total NH y Since i c.c. n/io alkali=i c.c. /io NH 3 , 25 c.c. urine contains 7.7 c.c. n/io NH 3 . i c.c. n/io NH 3 = 1.7 mg. NH 3 . Therefore 7.7 c.c. njio NH 3 = 77x 1.7 mg. NH 3 = 13.09 mg. NH 3 . This is the amount present in 25 c.c. urine. The amount excreted in twenty-four hours is therefore 1,550x13.09 grm 3. Ammonia N. Now 17 grms. NH 3 contain 14 grms. N. Therefore 0.830 grm. NH 8 contain HX 0.830 grmg N=o683 grm N Of 5.29 grms. of total N, 0.683 g rm - N are excreted in form of NH 3 . Of 100 grms. of total N, grrns . are excreted in form of NH 3 = 12.9 grms. N. 1 2. Q per cent, of total N is excreted in the form of ammonia. Experiment 158. Estimation of Urea. In the following method urea is estimated by measuring the amount of nitrogen liberated from the urine by sodium hypobromite. From i grm. urea 354 c.c. of nitrogen are evolved. As stated in Experiment 142 some other substances also liberate nitrogen if acted upon by hypobromite, so that the method gives only approximate results. Apparatus. Connect a bottle containing a short test tube, and closed with a rubber stopper, with an inverted burette standing in a tall glass cylinder filled with water. The apparatus is the same as the one used for the estimation of the oxygen capacity of the blood (Exp. 72). The bottle is standing in a bath filled with water. The water in the jar and in the bath should have room temperature. Method. Place 20 c.c. of hypobromite solution in the bottle, without letting it touch the mouth of the flask. Run, with a pipette, 5 c.c. of urine accurately measured into a small test tube, and place the test tube into the bottle, taking care not to upset any of the urine into the hypobromite. Put in stopper tightly, place the bottle in the water-bath, connect one tube 75 with the burette, leaving the second tube open. After five minutes close the second tube with a clip. Read the burette with water-level outside and inside equal. Take the bottle out of the water-bath, and tilt, so that the urine mixes with the hypobromite. Gently shake bottle from side to side, holding it upright so that the froth does not enter the tube. Tilt again and shake again. Place the bottle back in water-bath. After five minutes read the burette again with water-level outside and inside equal. The difference in the readings gives the amount of N evolved. Record your results and calculate from them: (i) Amount of urea excreted in twenty-four hours; (2) that fraction of the total N which is excreted as urea. Calculation. Example : Amount of urine used, 5 c.c. ; N liberated, 8.7 c.c. Volume of urine excreted in twenty-four hours, 1,550 c.c. Amount of N excreted in twenty-four hours, 5.29 grms. 1. Total Urea. Since 354 c.c. N are liberated by i grm. urea, 8.7 c.c. N Q f. are liberated by grms. urea = 0.0245 grm. urea. Since 5 c.c. of urine contain 0.0245 grm. urea, 1,550 c.c. urine contain 0.0245x1,550 ^ 1^! grms. urea = 7.0 1 grms. urea. 2. Urea N in Percentage of Total N. Urea (molecular weight = 60) contains N 2 (molecular weight = 28). Since, therefore, 60 grms. urea contain 28 grms. nitrogen, 7.61 grms. L . 7.61 X28 urea contain ^ grms. N = 3 % 55 grms. nitrogen. This amount of N excreted in twenty-four hours in the form of urea represents a fraction of the total N excreted in twenty-four hours. This 3 r r ^ IOO fraction, expressed in terms of percentage of total N, is *= =67.10 per cent. Result. The amount of urea excreted in twenty-four hours is 7.61 grms. The urea nitrogen represents 67.10 per cent, of the total nitrogen. Collect the results from Experiments 156 to 158 in tabular form as under : NITROGEN DISTRIBUTION IN TWENTY-FOUR HOURS' URINE. Date. Volume. Total N. Urea. Ammonia. Absolute Amount. N. Per Cent. Absolute Amount. N. Per Cent. ON THE PRACTICAL SIGNIFICANCE OF THE NITROGEN DISTRIBUTION IN THE URINE. The quantitative estimation of one single nitrogenous constituent- urea or ammonia of the urine, which, in the case of urea, has long been the routine in clinical work, is of little practical use. In the case of both these substances, more especially in the case of urea, the absolute amounts excreted rise and fall with a rise and fall of the nitrogen intake, so that no definite conclusions can be drawn from a quantitative estimation of urea alone or ammonia alone. In order to be able to draw conclusions it is necessary to determine also the amount of total nitrogen excreted, and to calculate therefrom the fraction excreted in the form of urea and of ammonia respectively. If with a fairly constant excretion of total nitrogen the amount excreted in the form of urea diminishes, while that excreted in the form of ammonia increases, a severe disturbance of metabolism is indicated, namely, a condition of acidosis. If an estimation of total nitrogen is .impracticable, estimation of urea and ammonia may give useful information. Normally the absolute 77 amounts excreted rise and fall together, the rise being always more marked in the case of urea than in the case of ammonia. A fall in the urea excretion, accompanied by a rise in the ammonia excretion, indicates an acidosis. It must be remembered that a high percentage of ammonia nitrogen a so-called high "ammonia coefficient" is not in itself evidence of a true acidosis. Such high ammonia coefficients always occur when the protein intake is greatly reduced, i.e., with a low total nitrogen excretion. QUANTITATIVE ESTIMATION OF CHLORIDES IN URINE. Experiment 159. Estimation of Chlorides (Volhard's Method). The principle of this method consists in precipitating all the chlorides with an excess of a standard silver nitrate solution. The excess of silver nitrate used is determined by adding a standard solution of ammonium sulphocyanate in the presence of a ferric salt. The sulphocyanate solution precipitates the soluble silver nitrate as silver sulphocyanate. As soon as all the silver nitrate is precipitated the addition of another drop of sulpho- cyanate solution will produce a red colour, since the ammonium sulpho- cyanate will now react with the ferric salt and form red ferric sulphocyanate The standard sulphocyanate solution is made up in such a way that i c.c. of it will just completely precipitate I c.c. of the standard silver nitrate solution (13 grms. NH 4 CNS in 1,000 c.c.). The standard silver solution is made up in such a way that I c.c. will completely precipitate 10 mg. NaCl (29.04 grms. AgNO 3 in 1,000 c.c.). Method. With a pipette place 10 c.c. of urine in a beaker, dilute with about 90 c.c. of distilled water, add 5 c.c. of a five per cent, solution of ammonia iron alum and 5 c.c. of dilute pure nitric acid. Then add with a pipette a measured excess of standard silver nitrate solution (20 c.c. of standard AgNO 3 will, as a rule, be sufficient). Now run in at once from a burette the standard sulphocyanate solution, stirring all the time, until the reddish tint of the ferric sulphocyanate first extends through the whole liquid. This can be observed most readily by comparing the urine to be titrated with another urine in which the end point has not yet been reached. Note the number of c.c. of sulphocyanate solution used, and 78 calculate in terms of sodium chloride (i) the amount of chlorides in 100 c.c. urine ; (2) the amount of chlorides excreted in twenty-four hours. Calculation. The amount of ammonium sulphocyanate solution used gives at once the excess of silver nitrate used beyond the quantity required to precipitate all the chlorides in 10 c.c. urine. If this excess is deducted from the total amount of silver nitrate added, one obtains the number of c.c. of standard silver nitrate solution necessary to precipitate all the chlorides. Each c.c. of the standard silver solution equals 10 mg. NaCl. Example : Urine used, 10 c.c. ; total volume, 1,550 ; Standard AgNO 8 added, 20 c.c. ; Sulphocyanate solution used, 7.4 c.c. ; therefore excess of AgNO 3 solution = 7.4 c.c. AgNO 3 solution necessary to precipitate all chlorides = 20 c.c. 7.4 c.c. = 12.6 C.C. Since I c.c. AgNO 3 = 10 mg. NaCl, 12.6 c.c. AgNO 3 = 126 mg. NaCl. Since 10 c.c. of urine contain 0.126 grm. NaCl, 100 c.c. of urine contain 1.26 grm. NaCl. Result. The total amount of chlorides excreted in twenty -four hours is in terms of NaCl : Pathological Metabolism. GASTRIC CONTENTS. ESTIMATION OF HYDROCHLORIC ACID SECRETION In certain pathological conditions the gastric cells secrete more hydro- chloric acid than they do normally, while in certain other conditions the amount of hydrochloric acid secreted by these cells is below the normal. The existence of such abnormal conditions can be recognised by a quanti- tative chemical examination of the gastric contents collected, by means of the stomach tube, an hour after a test meal of a fixed composition (for instance, dry toast and a large cup of tea without milk or sugar) has been given. The chemical examination consists in estimating quantitatively : 1. The total amount of hydrochloric acid secreted. Hydrochloric acid may exist in the gastric contents in three forms (a) free HC1 ; (&) HC1 combined with proteins and organic bases ; (c) HC1 combined with inorganic bases, as NaCl. 2. The " total acidity," which is a measure of the " physiologically active HC1," provided that no other acids are present. The physiologically active HC1 comprises free HC1 and HC1 combined with proteins, etc. 3. The " free acidity," which is a measure of the " free HC1." Why are butter, milk, and sugar excluded from the test meal ? ^nation of Experiment 160. Estimation of Total Acidity. " Physiologically vsioiogic- Active HC1." Place into a beaker by means of a pipette exactly 10 c.c. V. ' of the filtered gastric contents. Add two drops of phenolphthalein. 79 8o Titrate with n/io sodium hydrate until a purple colour is just produced. Take the burette reading. Record your result. Calculation. From your result calculate the number of c.c. of n/io alkali necessary to neutralise 100 c.c. of gastric contents. From this cal- culate the weight in grammes of HC1 which the total acidity represents. Example. 6.5 c.c. n/io alkali neutralise 10 c.c. gastric contents. Therefore 100 c.c. gastric contents are neutralised by 6.5 x 10 = 65 c.c. n/io alkali. Now i c.c. n/io alkali = i c.c. n/io HC1 ; i c.c. n/io HC1 contains 3.65 mg. HC1. Therefore 100 c.c. gastric contents contain 65x3.65 mg. HC1 237.25 mg. HC1 = 0.237 grm. HC1. Result. The total acidity of the gastric contents is represented by 0.237 per cent. HC1. Experiment 161. Estimation of Free Acidity. 10 c.c. of filtered gastric contents are placed in a beaker by means of pipette. Add two or three drops of Topfer's indicator. The solution turns red. Titrate with n/io alkali until the red colour is replaced by a lemon -yellow colour. (If the lemon-yellow colour appears at once when the indicator is added, free acid is absent.) Take burette reading. Record the result. Since Topfer's indicator reacts only to free acid which is not combined with protein, the result indicates the amount necessary to neutralise the free acidity of 10 c.c. of the gastric contents. Calculation is carried out as above. Explain the results obtained. Refer to Experiment 100. Experiment 162. Estimation of Total Amount of Hydrochloric ^ C ^ Secreted. To 20 c.c. of the filtered gastric contents apply Volhard's method for the estimation of chlorides as given in Experiment 159, and express the result in terms of HCI. 8i By deducting the " free acidity" from the " total acidity " the amount of" HC1 combined with proteins and organic bases" is arrived at. By deducting the " total acidity" from the total HC1 secreted the amount of" HC1 combined with inorganic bases" is arrived at. Collect the results obtained from Experiments 160, 161, and 162 in tabular form as under : Total HC1 secreted by gastric cells : Free HC1 : HC1 combined with proteins and organic bases : Physiologically active HC1 : HC1 combined with inorganic bases : 82 ABNORMAL CONSTITUENTS OF URINE. Of the substances which may appear in the urine in pathological con- ditions, proteins, blood pigments, bile pigments and bile salts, glucose, acetone^ aceto-acetic acid, occur most frequently. The fact that these substances are dissolved in urine, a fluid having such a complex composition, introduces certain fallacies in the application of the reactions by which the substances mentioned can be recognised if present alone in a simple watery solution. In the case of proteins, for instance, the fact that the urine has a colour of its own excludes the use of the colour reactions (biuret, xanthoproteic, etc.) as tests for the presence of proteins in urine. The guaiac test for blood is given also by pus, and a variety of other substances which may occur in urine. In the case of sugar it has been pointed out already that Fehling's solution may be reduced by other substances than glucose or lactose. In order to avoid these fallacies, as many different tests as possible should be carried out before deciding whether a certain substance is or is not present in the urine. Some drugs are excreted in the urine, and may introduce further fallacies. The previous treatment of the patient must therefore also be taken into- consideration. The following tests for abnormal constituents should be carried out at first on normal urines, then on normal urines to which a very small amount of the abnormal constituents has been added; lastly, on pathological urines obtained from the infirmary. PROTEINS. The proteins of the blood-plasma (globulin, albumin, fibrinogen) are the proteins most frequently met with in the urine. Clinically no distinction is drawn between these different proteins, and the presence of any of them is spoken of clinically as " albuminuria." as/or Pro- Experiment 163. Tests for Protein in Urine. For all the following 1 their Fal tests the urine must be quite clear. If not clear, filter. fes - (a) Heal Test. The urine must be neutral or very faintly acid. Heat to boiling point. A precipitate may appear which may consist of either earthy phosphates or protein. Add three drops of dilute acetic acid to the hot solution and heat again to boil. If the precipitate be one of earthy phosphates it will dissolve. If a flocculent precipitate remains after the addition of the acid, the presence of protein is indicated. Fallacies. If the urine is alkaline when heated, or if too much acid is added, alkali-albumin or acid-albumin is formed, which is not coagulated by boiling and remains in solution. A small amount of protein will there- fore not be indicated by this test, unless it is carried out carefully. (b) Precipitation by Strong Mineral Acids. Hellers Test. Place 2 or 3 c.c. of pure nitric acid in a test tube. Incline the test tube and from a pipette allow the urine to flow slowly down the side, so that it forms a layer above the nitric acid. If albumin is present a white opaque ring appears in the urine at the junction of the two fluids. The test is very delicate, and is given by a dilution of i : 50,000. If only traces of albumin are present, the ring may appear only after a minute. Fallacies. If concentrated urines are examined by this test, a white ring, usually less defined, may be formed, which may be due to the pre- cipitation of uric acid or urea nitrate. If that is suspected, dilute the urine with three times its volume of water, and repeat the test with the diluted urine. If the ring was due to uric acid or urea nitrate the ring will not appear with the diluted urine. After the administration of drugs containing resins a white ring may appear owing to the precipitation of the resins by the acids. This ring will disappear on the addition of alcohol to the urine, while a ring due to protein will persist Note. A coloured ring may also appear at the junction of the acid and urine. This is due to the formation of indigo red in urine rich in indican. It has no relation to the presence of albumin. (c] Precipitation by Alkaloidal Reagents : Precipitation by Hydroferrocyanic Acid. Render 5 c.c. of urine distinctly acid with i to 2 c.c. of acetic acid. Add drop by drop potassium ferrocyanide. If the urine remains clear.no albumin is present. If a pre- cipitate forms protein is present. 8 4 Precipitation by Salicylsnlfonic Acid. To 5 c.c. of urine rendered slightly acid add drop by drop salicylsulfonic acid. If protein is present a pre- cipitate forms. Precipitation by Picric Acid. Place in a test tube 5 c.c. of picric acid solution. Allow a drop of urine to fall from a pipette into the picric acid. If protein is present, a cloud will form round the drop. Fallacies. If alkaloids are administered in large doses, for instance quinine, they may be excreted in the urine and form a precipitate with these reagents. Albumoses. In some pathological conditions it will be found that the heat test is negative, while the other tests give a positive result. Then albumoses are present in the urine. It will be found then that the white ring formed in Heller's test dissolves on heating, and reappears on cooling. This condition of albumosuria is rare. REDUCING SUGARS. The sugar which is found most frequently is glucose. This occurs in diabetes mellitus. If proteins are present they must be removed by heat coagulation before testing for reducing sugars. Experiment 164. Tests for Glucose in Urine. [a] Troinmers Test. To 5 c.c. of urine add i c.c. of caustic soda. Then add, drop by drop, cupric sulphate, shaking after the addition of each drop. On the addition of each drop a flaky precipitate of cupric hydrate forms at first, which, if sugar is present, dissolves on shaking, giving a deep blue solution. Con- tinue to add cupric sulphate, drop by drop, until a little cupric hydrate remains undissolved. Heat the upper part of the solution. A yellow precipitate forms if a reducing sugar is present. (b} Fehling's Test. Heat in two Jest tubes equal volumes of urine and of Fehling's solution to boiling point. The Periling solution must remain clear on boiling. Otherwise it has become decomposed, and a fresh solution must be prepared. When both solutions are boiling, remove the test tubes from the flame and pour the one into the other without further heating. Allow the mixture to stand. If much sugar is present yellow 85 cuprous oxide separates out at once. With less sugar present the colour gradually changes. This is followed by the appearance of a yellow pre- cipitate, which may not begin to appear until the tube has cooled. If little sugar is present repeat the test with Feh ling's solution half diluted with water. Note. Although the principle of Trommer's and Fehling's test is the same, it is best to use both in testing for reducing sugars, as there are certain advantages and disadvantages connected with either of them. Fallacies. The fallacies due to the presence of uric acid and creatinine have already been discussed (see Exps. 147, 152). Further: Glycuronic acid reduces cupric sulphate. This acid may occur in the urine as the result of administration of certain drugs, such as chloral, morphia, camphor^ chloroform, antipyrin, antifebrin, or it may occur as the result of excessive intestinal putrefaction. (Explain its appearance in both these conditions.) If the test for indican shows that a large amount of indican is present, it points to the latter possibility. Glycuronic acid (what is its structural formula?) can be distinguished from glucose by its failure to ferment with yeast. In connection with the use of chloroform as a preservative for urines, it must also be borne in mind that chloroform reduces cupric salts, the mixture turning first red and then dark brown, owing to the reduction of cupric salts to colloidal cuprous oxide (red) and colloidal copper (black). (c] Nylanders Test. To some urine in a test tube (wide tubes are most convenient) add one-tenth of its volume of Nylander's reagent. Boil over a small flame for three minutes and allow to cool. If reducing sugar is present the urine darkens to a deep brown, and eventually a fine black precipitate settles out. Normal urine shows only a slight darkening. Fallacies. The same as in Trommer's or Fehling's test, except that uric acid and creatinine do not reduce Nylander's reagent. On the whole this test is more reliable than Fehling's or Trommer's test. (d] Fermentation. For this test the urine must be clear and as fresh as possible. It must not have entered into ammoniacal fermentation. Test reaction. If alkaline, make faintly acid with a few drops of very dilute acid (tartaric acid). Boil the urine for two minutes and cool 86 under the tap. Place a small piece (size of a pea) of yeast in the cooled urine in a test tube and shake up gently, so that an emulsion of yeast in the urine is formed. Fill the fermentation tube with this emulsion, so that the closed limb contains no air bubbles. Allow the tube to stand in a warm place ( best at 37), and examine after one hour, and again after twenty-four hours. The appearance of a bubble (of CO 2 ) in the closed limb indicates the presence of glucose. Fallacies. This is the most reliable and important test for glucose in the urine, since glucose is the only fermentable substance which may occur in the urine. The only possible fallacies arise from possible irregularities in the behaviour of the yeast. Firstly, the yeast may be inactive, so that no fermentation takes place although glucose is present. Secondly, the yeast may undergo self-fermentation, that is, it may give off gas, even in the absence of glucose. These two fallacies can be excluded by the following two control experiments, carried out at the same time with the same yeast. One tube contains an emulsion of yeast in a dilute glucose solution. Absence of fermentation in this tube would indicate that the yeast is inactive, and therefore useless. The other tube contains an emulsion of yeast in normal urine. If more than a very small air bubble is found in the closed limb of this tube the yeast undergoes self-fermentation, and the results obtained are not reliable. (e) Phenylhydrazine Test. To 10 c.c. of urine add ten drops of phenylhydrazine and equal amount of glacial acetic acid. Shake the test tube to ensure a thorough mixing, and keep in the boiling water-bath for one hour. Allow to cool slowly. If crystals separate out, examine microscopically. If glucose is present the characteristic crystals of the glucosazone should be seen. If only small amounts of glucose are present this test may be negative. Note. The value of this test is that it enables one to distinguish glucose from other reducing sugars, especially lactose, which may be present. The other sugars also form osazones which, however, have a different crystalline form. 8? (/) Polarimetric Examination. If a polarimeter is available the urine should be examined with regard to its action on polarised light. If glucose is present, the urine will produce dextrorotation. If the urine is too deeply coloured it should be decolorised by shaking with solid lead acetate and filtering until the filtrate is clear. tost, its Lactose is sometimes present in the urine. It gives the same tests ttnctwn as gl UC ose, but can be distinguished from it by the form of the osazone i Glucose. s , b 7 crystals and by its inability to ferment. Method of Testing Urine for Reducing Sugars. Proceed as follows: Test first with Trommer's and with Fehling's test. If both are undoubtedly negative no further tests are necessary. If they are positive Nylander's test, Phenylhydrazin test, and fermentation must be carried out before any reliable conclusion can be arrived at. A polarimetric examination is desirable. mitative Experiment 165. Estimation of Glucose by Fehling's Method. 'motion of This titration is based on the principle that a definite amount of copper salt, ation. m this case the amount contained in 10 c.c. of Fehling's solution, is reduced to the red cuprous oxide by a constant amount of glucose (0.05 grm. glucose). When all the blue copper sulphate has been reduced to red cuprous oxide, which falls out, the end-point of the reaction is reached, and the blue colour of the supernatant fluid, which is due to the presence of copper sulphate, totally disappears. The volume of urine used is then known to contain 0.05 grm. glucose. Method. Run exactly 10 c.c. of Fehling's solution from a burette into a porcelain basin, dilute with about 40 c.c. of water, and heat to boiling. While the Fehling's solution is kept boiling gently, run in the urine from a burette. Add \ c.c. of the urine at a time in short intervals. A red pre- cipitate appears suspended in a fluid which is, at first, deep blue. As more and more of the urine is added the blue colour of the fluid in which the precipitate is. suspended becomes fainter and fainter. Finally the red pre- cipitate will be seen suspended in a fluid which is colourless (or slightly yellow). The point when the fluid, in which the precipitate is suspended, first becomes colourless indicates the end -point of the titration. 88 The first titration gives only a rough estimate of the amount of urine necessary to reduce 10 c.c. of Fehling's solution. If the urine contains so much glucose that less than 5 c.c. of urine have been used in this first titra- tion, the urine must be diluted until about 6 to 12 c.c. of urine are required to arrive at the end-point. (If, for instance, 2.8 c.c. of urine have been used in the first titration, the urine should be diluted three times, i.e., 20 c.c. of urine with 40 c.c. of water.) Empty the burette containing the original urine, wash out with the diluted urine, and then fill with the diluted urine. In order to determine the end-point accurately, subsequent titrations are carried out in such a way that instead of adding the urine in small portions, almost all the urine necessary to produce complete reduction is added in bulk to the Fehling's solution. Continue boiling after the addition, and complete the reaction until the supernatant fluid is colourless by adding the urine in small quantities (three drops at a time). The end- point is best seen if, after allowing the red precipitate to settle, the basin is slightly tilted so that the supernatant fluid appears against the white background of the porcelain. Carry out one preliminary and two subsequent titrations. If the two subsequent titrations agree to within 0.5 c.c., take their average, and calculate from it the amount of sugar present in 100 c.c. of-urine. Note. The fallacy to guard against in this titration is the oxidation by air. If, for instance, a titration has been completed, and the red precipitate with the supernatant colourless fluid is allowed to stand in contact with the air, a blue colour will again appear in the supernatant fluid owing to oxidation and solution of the cuprous oxide. If, therefore, the end-point of the reaction has once been reached, subsequent appearance of a blue colour must be neglected. For the same reason the whole titration should be carried out as quickly as possible. Calculation. Example : Preliminary titration. 10 c.c. Periling are reduced by 3.3 c.c. of urine. Dilute 20 c.c. of urine with 40 c.c. of water. Fill burette with urine thus diluted, I : 3. First titration. 9.1 c.c. urine. 8 9 Second titration. 8.8 c.c. urine. Average, 8.95 c.c. urine. 8.95 c.c. diluted urine contain 0.05 grm. glucose. 100 c.c. diluted urine contain grms. glucose = 0.56 grm. glucose. 0-95 Result. The original urine was three times stronger, and contained therefore in 100 c.c. urine 1.68 grms. glucose. From this the amount of glucose excreted in twenty-four hours can, if necessary, be calculated. Experiment 166. Quantitative Estimation of Glucose by Polari- meter. The method of using the polarimeter has already been described (see Exp. 16). Method of Preparing the Urine for Examination by the Polarimeter. The urine must be free from proteins, since these substances are themselves optically active and laevorotatory. If proteins are present, they must be removed by heat coagulation. If the urine is cloudy, it must be filtered until it is clear. Diabetic urine is, as a rule, only lightly coloured, and can frequently be used without removing the colouring matter. If the urine is coloured to such an extent that, after inserting the polarimeter tube filled with urine, the dividing line between the two halves of the field cannot be seen clearly, the urine must be decolorised. This is done by boiling the urine with some animal charcoal or by shaking it with about one-tenth its bulk of lead acetate and filtering. The urine is then cooled to room temperature, and the polarimeter tube filled with the urine in such a way that no air bubbles are present. The tube is then placed in position in the apparatus, and the rotation is read to within one-tenth of a degree. Calculation. The calculation depends on the fact that every optically active substance has a definite rotatory power. The actual rotation produced by a solution of an optically active substance depends on (i) this "specific rotatory power," (2) the wave-length of the light used, (3) the concentration of the solution, and (4) the length of the column of fluid which the polarised light traverses. In the case of glucose, and with the light used in an apparatus of the Soleil pattern, a glucose solution which contains I grm. of glucose in 100 c.c. of water rotates polarised light through 0.59, 90 when the light traverses a column of glucose solution 100 mm. long. If the column of glucose solution is twice as long 200 mm. the rotation is twice as great, namely, 1.18. In all quantitative polarimetric estimation the length of the tube which contains the glucose solution must therefore be known. If a 200 mm. polarimeter tube is used the percentage/ of the glucose solution is found by dividing the actual rotation a, which has been observed, by 1.18. a '-Elf- This formula applies to a tube 200 mm. long. With a tube half that length the result has to be doubled. 2a a P= 1. 1 8 "6.59 Note. Since with a 200 mm. tube a I per cent, glucose solution produces a rotation of 1.18, each degree of rotation corresponds to 0.84 per cent, of glucose. By using a slightly shorter tube, 168 mm. long, it is possible, therefore, to obtain readings so that i of rotation corresponds to I per cent, of glucose. In other words, the degree of rotation indicates directly the percentage of glucose. Instruments for clinical use are fre- quently arranged in this way. ACETO-ACETIC ACID AND ACETONE. These two substances always occur together, since acetone is formed by decomposition from aceto-acetic acid. What are the structural formulae for these two substances ? The appearance of these two substances indicates a severe metabolic disturbance to which the name acidosis has been given. It is frequently associated with other pathological conditions, such as diabetes mellitus, chloroform-poisoning, starvation, withdrawal of carbohydrates, etc. ACETO-ACETIC ACID. Note. In testing for aceto-acetic acid the urine must be quite fresh, as aceto-acetic acid is easily decomposed on standing into acetone. Note the odour. Experiment 167. Nitroprusside-Ammon. -Sulphate Test (Rotheras Modification}. Take 10 c.c. of urine in a test tube and saturate it with ammonium sulphate by shaking it with an excess of the solid salt. Then add two or three drops of a freshly prepared solution (5 per cent.) of sodium nitroprusside. Add 2 or 3 c.c. of strong ammonia. Mix by inverting the test tube once or twice. Allow to stand undisturbed for twenty minutes. If aceto-acetic acid (or acetone) are present a deep permanganate colour appears. The rapidity with which the colour develops and the depth of the colour give a rough measure of the amount of aceto-acetic acid (and acetone) present. The test is very sensitive, and is given by aceto-acetic acid even in con- centrations of i : 200,000 and less. Repeat the test with normal urine. Note that a faint reddish colour is obtained. Repeat the test with urines containing decreasing amounts of aceto- acetic acid. These can be prepared by mixing a urine from a case of acidosis with normal urine in the proportions i : 2, I : 5, I : 10, I : 100. Experiment 168. Ferric Chloride Test. (Gerhard's Test.} To 5 c.c. of urine add, drop by drop, ferric chloride as long as a precipitate of ferric phosphate continues to form. A claret-red colour is produced if sufficient diacetic acid is present. If, owing to the presence of the ferric phosphate precipitate, the colour is difficult to recognise, filter. This test is much less sensitive than the nitroprusside-ammon. -sulphate test, and is given only when the concentration of aceto-acetic acids is i : 1,000 or higher. Fallacies. Carbolic acid, salicylates, antipyrin, etc., give a similar colour. They can be distinguished from diacetic acid, because the colour produced by the latter disappears on boiling, while the colour produced by the drugs named persists. Why is the colour produced by diacetic acid destroyed by boiling? Diacetic acid may be extracted from urine acidified with hydro- chloric or sulphuric acid, by shaking with ether. Remove the ether with a pipette, and add to the ether ferric chloride. A claret-red colour appears if diacetic acid is present. Note the odour. 92 ACETONE. specific Experiment 169. Nitroprusside-Ammon. -Sulphate Test. Carry out ^ the test as described in Experiment 167. The same result is obtained. The test is given both by aceto-acetic acid and by acetone, but is not so sensitive for acetone as for aceto-acetic acid. It is given by acetone only when the concentration is i : 20,000 or higher. METHOD! OF EXAMINING THE URINE IN CASES OF ACIDOSIS. Much significance was attached formerly to the question whether acetone alone was present, or whether both acetone and aceto-acetic acid were being excreted. It was believed that the latter condition represented a more severe disturbance of metabolism than the excretion of acetone alone. This view is, however, erroneous. It probably arose from the fact that the only test formerly available for aceto-acetic acid, the ferric-chloride test, is not so sensitive as the tests formerly used for acetone. An indication of the severity of the disturbance is given by the amounts of abnormal acids formed. This can be determined directly by estimating by means of special methods the amount of acetone and diacetic acid. It can be determined indirectly by determining the "ammonia coefficient" (see page 76), since the organism responds to an increased formation of acid substance by an increased excretion of ammonia and a diminished excretion of urea. So far as the qualitative examination of the urine is concerned, no great significance attaches to the question whether acetone alone or aceto-acetic acid alone are present. It is therefore sufficient to apply first the nitro- prusside-ammon. -sulphate test. If this test is negative, no further tests are necessary. If the test is positive, some indication of the amounts present is given by (i) the intensity of the colour and (2) the rate at which it develops. Some further indication of the degree of acidosis may be obtained by applying the ferric chloride test, which is positive only with a concentra- tion of aceto-acetic acid exceeding i : 1,000. Note that this test must be applied to freshly voided urine. 93 It is not of great importance to apply separate tests for acetone. If aceto-acetic acid is present, acetone will always be present too, especially if the urine has been allowed to stand, since aceto-acetic acid is readily decomposed with the formation of acetone. It may be of interest to determine whether in freshly voided urine acetone is absent or present. In that case the following test, which is very sensitive, may be applied. To 5 c.c. of urine add i c.c. of a 10 per cent solution of salicylaldehyde in alcohol. Mix by shaking gently. Add a piece of a solid caustic soda stick (or caustic potash stick) about one inch long. Allow to stand undisturbed. If acetone is present a deep brownish-red colour develops at the point of contact. BLOOD PIGMENT. Experiment 170. (a) Colour. If a relatively large amount of blood is present the urine has a red colour. Smaller quantities give to the urine an opaque reddish-brown appearance, " smoky urine." Examine microscopically the deposit, if any, for the presence of red blood corpuscles. () Colour of Earthy Phosphate Precipitate. Render 5 c.c. of urine strongly alkaline with caustic soda, and boil. A precipitate of earthy phosphates will separate out on standing. This precipitate is normally greyish white. If blood is present it is brownish red in colour from the haematin which is carried down with it. Fallacies. Cascara sagrada, rhubarb, senna, give to the precipitate a similar coloration. (c] Guaiac Test, To 5 c.c. of urine add i c.c. of hydrogen peroxide and shake. Then add two drops of tincture of guaiac, so that the resin floats on the urine. A blue colour appears at the junction of urine and resin if blood is present. This test is a very sensitive one. Fallacies. Iodides and pus also give a blue colour. The presence of pus can be excluded by previously boiling the urine. If the guaiac is then applied while the urine is still hot, the test becomes even more sensitive. 94 (d) Spectroscopic Examination. Examine urine spectroscopically as described in Experiments 76 to 83. If blood is present the spectrum of methaemoglobin is usually seen. This may have been formed from haemoglobin on standing after the urine has been passed, or it may have been passed as such. If the absorption spectrum is not distinct, prepare hsemochromogen by boiling with caustic soda and reducing with ammonium sulphide. If haemoglobin or methaemoglobin are present the characteristic spectrum of haemochromogen will be seen. Spectroscopic examination is free from fallacies, but it does not indicate traces of blood as the guaiac test does. It is therefore possible that the guaiac test is positive, while Spectroscopic examination is negative. BILE. Experiment 171. Bile Pigments. (a) Colour. If bile pigments are present the urine has a brownish or greenish colour. The latter colour is present especially if the urine has been standing for some time, so that the red bilirubin has become oxidised to the green biliverdin. (U) Gmeliris Test. The following modification is best adapted for the examination of urine. Filter urine repeatedly through the same paper. Unfold the paper and put a drop of yellow nitric acid on it. A ring of colours appears if bile pigments are present. This test is not delicate. (c] Hupperfs Test. Render 10 c.c. of urine alkaline with caustic soda or sodium carbonate solution, and add barium chloride or calcium chloride solution drop by drop under shaking. A precipitate forms, which carries down with it bile pigment. Continue Adding calcium chloride until the fluid in which the precipitate is suspended has the colour of normal urine. Collect the precipitate on a filter ; wash with water. Place the filter paper with the precipitate in a porcelain basin, add 10 c.c. of alcohol, and while stirring add five to ten drops of strong hydrochloric acid, until the precipitate is dissolved. Pour the yellowish solution into a test tube ; add two drops of ferric chloride, and heat. A green colour changing to blue appears if bile pigments are present This is the most reliable test for bile pigments. 95 Experiment 172. Bile Salts. Pettenkofers test for bile salts, if applied to urine, is best carried out as follows (see Exp. 122): Dissolve in the urine a small fragment of a crystal of cane sugar. Filter the urine thus prepared through filter paper. Dry the paper. Allow a drop of concen- trated sulphuric acid to fall on the paper. After half a minute the paper shows in transmitted light a violet colour if bile salts are present. Normal urine gives a reddish or brownish colour. SOME PATHOLOGICAL DEPOSITS. Morphological elements, such as pus cells, epithelial cells, casts, etc., are not dealt with here. Cystin. Hexagonal plates, insoluble in water and acetic acid, soluble in hydrochloric acid and in ammonia. Leucin and Tyrosin. Usually occur together, especially in diseases of the liver. Leucin is deposited in spheres having a radial and concentric striation. It is slightly 'soluble in water. Soluble in acids and alkalis. Tyrosin is deposited in sheaves of fine white needles. The appearance of the crystals is very much like that of glucosazone, except that the latter is yellow. Tyrosin is very slightly soluble in water, soluble in acids and alkalis. It gives a red colour with Millon's test. What are the structural formulae for cystin, leucin, and tyrosin ? What are they formed from ? Calcium Oxalate. See under normal urines (page 63). Is pathological only if present in excess. Experiment 173- Examine urines of cases from the infirmary for pathological constituents, and report on the form given out. Experiment 174. Examine urines of cases from the infirmary for total N, urea, ammonia, acidity, chlorides, sulphates, indican, and report on the form given out. FORM FOR REPORT Name of Patient Diet and Treatment (Drugs) during previous twenty-four hours Total Volume of Urine excreted in twenty-four hours - Colour Deposit, if any Specific Gravity - Reaction Total N excreted in twenty-four hours - Urea. Amount excreted in twenty-four hours ,, Percentage of urea N to total N Ammonia. Amount excreted in twenty-four hours , , Percentage of ammonia N to total N - Acidity of total Urine - Chlorides. Amount excreted in twenty-four hours Sulphates l Ethereal sulphates l Indican ^ --------- I c.c. acid- 1.7 mg. NH 3 =i.4mo;. N; i c.c. standard AgNO 3 =iomg. NaC' ; 10 c.c. 10 1 State by + or - whether test is positive or negative. A doubtful result may be indicated 2 State here your general conclusion; for instance: "Glucose absent, although Fehling's 97 ON URINE ANALYSIS. ABNORMAL CONSTITUENTS. Test. Result. 1 Conclusion.' 2 ,Heat ---- I Nitric Acid Albumin. / Picric Acid ) Hydroferrocyanic Acid \SalicylsulfonicAcid - /-Trommer - j Fehling Reducing f Fermentation Sugar. ~\ Nylander - J Osazone ^Quantitative Estimation f Guaiac - Blood, -j Earthy Phosphates { S pec t rose opic Examination Aceto- acetic Acid. Nitroprusside-Ammon. -Sulph. Ferric Chloride Acetone. Nitroprusside-Ammon. -Sulph. fGmelin - Bile. j Huppert (Pettenkofer - standard Fehling's solution = 5O mg. glucose; i grm. urea liberates 354 c.c. N. by + ?, a strong positive result by + + , and so on. and Trommer's slightly positive." NDEX. ABSORPTION spectra, 36 Aceto-acetic acid, 90 Acetone, 92 Achromic point, 46 Acids in gastric contents, 50 Acidity, estimation of, 69 in gastric contents, 79 in urine, 72 Acidosis, 76, 92 Albumin, in egg white, 13 in milk, 17 in blood serum, 41 in urine, 82 heat, coagulation of, 1 3 properties of 'table;, 53 removal of, 43 solubilities of (table), 42 Albumoses, detection of, 52 formation of, 48, 55 in urine, 84 properties of 'table), 53 Alcohol, 3 Ammonia in urine, 63 estimation of, 72 Amylopsin, 54 BILE, 56 in urine, 94 Biuret test for proteins, 14 Blood, coagulation of, 28-33 laking of, 34 oxygen, capacity of, 34 oxidising ferment of, 34 pigments, 36-40 plasma, 30-33 serum, 41-43 platelets, 30-33 in urine, 93 Bread, 21 CALCIUM SALTS, in clotting of blood, 31 in clotting of milk, 20 in urine, 61, 95 in urinary sediments, 63 Cane sugar, 19 Carbohydrates, in blood, 43 in bread, 21 in flour, 21 100 Carbohydrates, in liver, 23 in milk, 18 in potato, I digestion of, 8, 46 Carboxyh hemoglobin, 39 Caseinogen, 18 Cerebrosides, 26 Chlorides in urine, 60 estimation of, 77 Cholesterin, in bile, 58 in egg yolk, 12 in nervous tissue, 25 Chromic period, 46 Clotting, see Coagulation Coagulation, of blood (table), 33 of milk, 20 of proteins, 13, 15 Creatinine, 67 Cystin, 95 DEXTRIN, 8 Dextrose, see Glucose Dialysis, 53 Digestion, of carbohydrates, 8, 46 of fats, 54 of proteins, 52, 55 salivary, 46 gastric, 48-53 pancreatic, 54 EGG, ii Emulsification of fats, 10 effect of bile on, 56 Enzymes, see Ferments Ethereal sulphates, 60 FAT, digestion of, 54 emulsification of, 10 of milk, 1 8 phosphorised, 11 saponification of, 9 Fatty acids of lard, 9 of butter, 18 Fehling's solution, preparation of, 4 Fermentation, 3 Ferments, acting on carbohydrates, 3, 8, 46, 54 acting on fats, 54 acting on proteins, 48, 54 method of testing for, 55 oxidising, 34 Fibrin, 28 Fibrinogen, 31 Flour, 21 GALACTOSE, 26 Gastric contents, 50 Gastric juice, 48, 79 Glands, proteins of, 22 Globulin, in egg white, 13 in milk, 17 in serum, 41 heat coagulation of, 1 3 properties of (table\ 53 removal of, 43 solubilities of (table), 42 Glucose, preparation of, 3 estimation of, 87, 89 in urine, 84 tests for, 4 Gluten, 21 Glycogen, 23 Glycuronic acid, 85 101 H.EMATIN, 39 Haematoporphyrin, 40 Haemochromogen, 39 Haemoglobin, 38 Haemolysis, 34 Heat coagulation, 13 Hydrochloric acid in gastric juice, 48 estimation of, 79 INDICAN in urine, 68 Inversion, 19 Iron in liver, 24 NITROGEN in urine, estimation of, 69 Nitrogen distribution in urine, 76 Normal solutions, 69 Nucleoproteins, 22, 44 Nylander's reagent, preparation of, 5 OPTICAL activity of sugars, 7, 19 Osazones, preparation of, 5 Osmosis, ii Oxalates in urinary sediments, 63, 95 Oxidising ferment of blood, 34 Oxygen capacity of blood, 34 Oxyhaemoglobin, 37 KJELDAHL'S method for estimating nitrogen, 69 LACTIC acid in gastric juice, 50 Lactose, in milk, 18 in urine, 87 Laking of blood, see Haemolysis Lecithin, 12, 25 Leucin, 95 Lipoids, separation of, 25, 45 histochemistry of, 27 Lipolytic ferment, 54 MALTOSE, 8 Metaproteins, 13 Methaemoglobin, 39 Milk, 17 Mucin, 44, 46 Muscle, proteins of, 22 PANCREATIC digestion, 54 Pepsine, 48 Peptic digestion, 52 Peptones, 52 Phosphates in urine, 61 Phosphatides, 26 Phospho-cerebrosides, 26 Phosphorus in lipoids, detection of, 26 Polarimeter, 6, 89 Potato, i Protagon, 25 Proteins, of bile, 56 of blood plasma, 3 1 of blood serum, 41 of egg white, 12 of glands, 22 of milk, 17 of muscle, 22 in urine, 82-84 digestion of, 52, 55 reactions of, 14 properties of (table), 53 Ptyalin, 46 102 RENNET, 20, 49 SALIVA, 46 Saponification, 9 Soaps, 10 Spectroscope, 36 Starch, digestion of, 8, 46 hydrolysis of, 8 reactions of, i Steapsin, 54 Stokes' fluid, 38 Sucrose, see Cane sugar Sugars, reactions of (table), 19 in urine, 84 Sulphates in urine, 60 TISSUES, chemical examination of, 44 Trypsin, 54 Tyrosin, 95 U RATES, 60 Urea, 63 estimation of, 74 Uric acid in urine, 64 in urinary sediments, 66 Urine, acidity of, 72 deposits from, 62, 63, 66, 95 normal constituents of, 59-68 abnormal constituents o , 82-95 nitrogen, distribution of, 76 Printed at THE PAKIEN PRESS, Edinburgh. 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