COLUMBIA LIBRARIES OFFSITE 
 
 C HEALTH SCIENCES STANDARD 
 
 HX64141713 
 QP51 9 .M31 2 Manual for the physi 
 
 RECAP 
 
 MANUAL 
 
 FOR THE 
 
 PHYSIOLOGICAL CHEMICAL 
 LABORATORY 
 
 JOHN A. MANDEL 
 
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 Columbia tBntoerafap 
 mtljfCttpoflfttigork 
 
 College of $tjpgtctansi anb burgeon* 
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 Gift of 
 Dr. Jerome P Wet s~ber 
 

MANUAL 
 
 FOR THE 
 
 PHYSIOLOGICAL CHEMICAL 
 LABORATORY. 
 
 FOR THE USE OF MEDICAL 
 STUDENTS. 
 
 JOHN A. MANDEL, 
 
 Adjunct Professor of Physiological Chemistry at the 
 Bellevue Hospital Medical College. 
 
 NEW YORK. 
 
 JOS. V. STANDISH. 
 
 1897. 
 
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 Ube 1knicf?ei'bocher press IRew lOorf? 
 
Digitized by the Internet Archive 
 
 in 2010 with funding from 
 Columbia University Libraries 
 
 http://www.archive.org/details/manualforphysiolOOmand 
 
Manual for the Physiological 
 Chemical Laboratory. 
 
 Lesson I. 
 
 CARBOHYDRATES. 
 
 A.compound containing C, H, and O, where the H 
 and O are in the proportion to form water. 
 Three groups. 
 
 MONOSACCHARIDES. DISACCHARIDES. POr.YSACCHARIDKS. 
 
 C 6 H 12 O c . C 1S H SS 1 . 1 . C 6 H 10 O 5 . 
 
 Dextrose, glucose, Cane sugar, Starch, 
 
 Laevulose, Lactose, Dextrin, 
 
 Inosite, Maltose, Glycogen, 
 
 Galactose, Cellulose. 
 
 DEXTROSE, GLUCOSE, GRAPE, SUGAR, ETC. 
 
 The most important member of the first group is 
 dextrose, which occurs in certain fruits and grains, and 
 in the blood, lymph, chyle, diabetic urine, etc. 
 
 Experiment i. Place some in a test tube with water. 
 It dissolves. It has a sweetish taste. 
 
 Experiment 2. Fill a test tube one quarter full with 
 silver nitrate (AgN0 3 ) solution, and add a few drops 
 
 1 
 
2 LESSON I. 
 
 of ammonium hydrate (NH 4 OH) until the precipitate 
 formed just dissolves. Add a few drops of the above 
 dextrose solution to this, and warm in water-bath. A 
 deposit of a metallic silver mirror is obtained, showing 
 that dextrose has a reducing action on certain metallic 
 solutions when in an alkaline condition, and most of 
 the chemical tests for dextrose depend on this prop- 
 erty. 
 
 Experiment j. Trommer s test. Place a few drops 
 copper sulphate (CuS0 4 ) in a test tube, and an equal 
 amount of water, and then caustic soda (NaOH) until 
 a little of the copper hydrate formed remains undis- 
 solved. Add to this a drop or two of the dextrose 
 solution. The copper hydrate dissolves. Apply gentle 
 heat. A yellow reduction of hydrated sub-oxide of 
 copper, and then red sub-oxide of copper (Cu 3 0), sepa- 
 rates even below the boiling point. 
 
 Care must be taken not to have an excess of either 
 copper sulphate or caustic soda in this test, otherwise 
 interferences are liable to occur. 
 
 Experiment 4.. Moore's test. Make some of the dex- 
 trose solution alkaline with caustic soda, and apply 
 heat. The solution turns yellow, red, brown, or black, 
 depending upon the amount of dextrose in the solu- 
 tion. Add a few drops of nitric acid (HNO s ) to the 
 solution. The color disappears, and an odor of caramel 
 or burnt sugar is given off. 
 
 Experiment 5. Fehlings test. Place some copper 
 sulphate in a test tube, and an equal amount of caustic 
 soda solution, then a solution of Rochelle salt (sodium- 
 potassium tartrate NaKC 4 H 4 O e , 4.H 3 0) until the pre- 
 cipitate is dissolved, and a clear, deep blue solution is 
 
CARBOHYDRATES. 3 
 
 obtained. This constitutes Fehling's solution. Heat 
 the upper part of the liquid to the boiling point by 
 holding the test tube at the lower end and applying 
 the heat above. No change, should occur. Add a 
 drop of the dextrose solution. Immediate reduction 
 of the yellow hydrated sub-oxide of copper, and then a 
 red sub-oxide of copper. 
 
 Experiment 6. Barfoed's test. Add some of Bar- 
 foed's solution (a solution of copper acetate with acetic 
 acid) to some of the dextrose solution in a test tube, 
 and apply heat. A reduction of copper sub-oxide is 
 produced. This test distinguishes dextrose from lac- 
 tose and maltose. 
 
 Experiment y. Boettgers test. Fill a test tube one 
 half full of water, add a little of the dextrose solution 
 and some solid bismuth sub-nitrate, and now make al- 
 kaline with sodium carbonate (Na 2 CO s ). Heat to 
 boiling. The white bismuth sub-nitrate will turn gray, 
 brown, or black, depending on the amount of dextrose 
 in the solution, and will collect at the bottom of the 
 test tube. 
 
 Experiment 8. Place some of the dextrose solution 
 in the saccharometer, and add a small piece of yeast 
 thereto. Place in a warm locality at a temperature of 
 about 40 C. Bubbles of gas collect after a time. 
 This is called fermentation, and the gas which collects 
 is carbon dioxide (C0 2 ), while another body, alcohol 
 (C 2 H 3 OH), remains in the liquid. C 6 H 12 6 = 
 2C 2 -H 5 OH + 2C0 2 . 
 
 CANE SUGAR. 
 Occurs in the juices of many plants, in fruits, in 
 flowers, and in honey; does riot occur in the animal 
 
4 LESSON I. 
 
 body, but undergoes transformations when partaken 
 of. 
 
 Experiment p. Place some cane sugar in some water 
 in a test tube. It dissolves readily, forming a solution 
 having a sweetish taste. 
 
 Experiment 10. Repeat Experiment 5, using a solu- 
 tion of cane sugar instead of dextrose. No reduction 
 on the application of heat. 
 
 Experiment 11. Fill a test tube one half full with 
 the cane sugar solution, acidify with a drop or two of 
 sulphuric acid (H 3 S0 4 ), and boil for a few minutes. 
 This operation will convert the cane sugar into a mix- 
 ture of dextrose and laevulose, called invert sugar, 
 C 12 H 33 O n + H 3 = C 6 H 13 6 + C 6 H 13 6 . 
 
 This process is similar to the action of the invertin 
 of the intestinal juice on cane sugar in the intestin. 
 
 Experiment 12. Neutralize this solution with caustic 
 soda (NaOH), and repeat Experiment 5. A reduction 
 of the red sub-oxide is obtained, showing the presence 
 of dextrose and laevulose. 
 
 Cane sugar does not ferment under the action of 
 yeast, but readily undergoes lactic acid fermentation 
 under the action of certain ferments. 
 
 LACTOSE OR MILK SUGAR. 
 
 Important constituent of the milk of mammals and 
 is sometimes found in the urine of women in the early 
 days of lactation, or after weaning. 
 
 Experiment ij. Place some of the lactose in water 
 in a test tube. It does not dissolve very readily and 
 has a feeble sweet taste. 
 
 Experiment 14.. Repeat Experiment 5, using a solu- 
 
CA A' BOH YDKA TES. 5 
 
 tion of lactose. A reduction similar to that obtained 
 with dextrose is the result. 
 
 Experiment ij. Add some Kephir ferment to some 
 lactose solution and keep warm for a short time, the 
 solution turns acid as shown by placing a drop of the 
 solution on a piece of blue litmus paper. This acid is 
 lactic acid C l2 Y[ 22 O lx -f~H 2 = 4C 3 H 6 3 .. 
 
 Lactose. Lactic acid. 
 
 MALTOSE OR MALT SUGAR. 
 
 Formed by the action of malt diastase on starch. Is 
 the chief product of the action of saliva and pancreatic 
 juice on starch during digestion. 
 
 Experiment 16. Dissolve some in water and observe 
 the sweet taste of the solution. 
 
 Experiment ij. Repeat Experiment 5, using the 
 above solution of maltose. A reduction is obtained 
 but not so pronounced as with dextrose. 
 
 Experiment iS. Apply Barfoed's test (see Experi- 
 ment 6) to the maltose solution. No reduction. 
 
 It really undergoes fermentation by yeast, and when 
 heated with very dilute sulphuric acid it yields dex- 
 trose. Diastatic enzymes have a similar action. 
 
 STARCH. 
 
 Found in nearly all plants. Is most abundant in the 
 cereals. Occurs as microscopic granules of various 
 sizes and structure. Does not occur in the animal 
 body except as food. 
 
 Experiment ig. Place some in water. Does not 
 dissolve but disintegrates yielding a white mud. 
 
 Experiment 20. Boil some water in a test tube and 
 
6 LESSON I. 
 
 add some of the above mud thereto. An opalescent 
 solution or jelly-like mass is the result, depending upon 
 the amount of starch added. 
 
 Experiment 21. Place a little starch solution in a 
 test tube and dilute with water. Add a drop of tinct- 
 ure of iodine thereto. Deep blue coloration is obtained, 
 which disappears on heating and reappears on cooling 
 again. 
 
 Experiment 22. Boil some starch solution with some 
 dilute hydrochloric acid (HO) for some time, neutral- 
 ize with caustic soda (NaOH) and repeat Experiment 
 5, using a portion. 
 
 A reduction is obtained showing the presence of 
 dextrose. The action of the acid is expressed by the 
 following equation : 
 3C 6 H 10 O 5 +H 3 O = C 6 H 12 O 6 +C 6 H 10 O 5 +C 6 H 10 O g 
 
 Starch. Dextrose. Achroo-dextrin. Erythro-dextrin. 
 
 Experiment 2j. Moderately heat some of the starch 
 solution with an infusion of malt (containing diastase) 
 for some time. It first becomes clear due to the for- 
 mation of soluble starch. Apply Fehling's test (Ex- 
 periment 5) to the product. A reduction is obtained 
 due to the presence of maltose. The change is ex- 
 pressed by the following equation : 
 ioC 6 H 10 O 5 +4H 3 = 4C 12 H 33 O tl +2 C 6 H 10 O 5 . 
 
 Starch. Maltose. Dextrin. 
 
 The action of the ptyalin of the saliva and amylopsin 
 of the pancreatic juice on starch is also represented by 
 the above change. 
 
 GLYCOGEN OR LIVER STARCH. 
 Occurs chiefly in the liver, muscles, and nearly all 
 tissues of the animal body, and is a regular constituent 
 of all developing animal cells. 
 
CAR BOH YDRA TE S. J 
 
 Experiment 2cf.. Place a little in some water in a test 
 tube. It dissolves yielding an opalescent solution 
 having no marked taste. 
 
 Experiment 25. Add a drop of tincture of iodine to 
 a portion of the above solution. A wine red coloration 
 is obtained, which disappears on heating. 
 
 Experiment 26. Boil the other portion of the gly- 
 cogen solution with some dilute hydrochloric acid 
 (HO), neutralize with caustic soda, and then test for 
 dextrose by repeating Experiment 5. The boiling with 
 the acid has converted the glycogen into dextrose and 
 dextrin, similar to Experiment 22. Certain ferments 
 such as that of the liver change glycogen into dextrose 
 and dextrin. 
 
 DEXTRINS. 
 
 Found in the blood and muscles of animals to a 
 slight extent. They are products of the diastatic fer- 
 ments on starch, etc. Two varieties — Achroo-dextrin 
 and Erythro-dextrin. Common dextrin is chiefly Ery- 
 thro-dextrin. 
 
 Experiment 2j. Place some in water in a test tube. 
 It dissolves and a strong solution forms a gummy and 
 sticky mass. 
 
 Experiment 28. Add some tincture of iodine to 
 the dextrin solution. Red coloration. 
 
 Experiment 29. Boil some of the dextrin solution 
 with dilute hydrochloric acid (HC1) for a few minutes. 
 Neutralize with caustic soda (NaOH) and test for dex- 
 trose by Fehling's solution (Experiment 5). The acid 
 has converted the dextrin into dextrose as shown by 
 the reduction. 
 
Lesson II. 
 
 FATS. 
 
 Almost all fats and oils are compound ethers of 
 glyceryl, C 3 H 5 ", or triglycerides. 
 
 The most important fats are tri-stearine C 3 H 5 
 (C 18 H 35 3 ) 3 , tri-palmitine C 3 H 5 (C 16 H 31 3 ) 3 , 
 and tri-olein C 3 H g (C 18 H 33 O s ) 3 . These are com- 
 pound ethers of stearic, palmitic, and oleic acids with 
 glyceryl. 
 
 Experiment i. Place some of the fat in water in a 
 test tube. It does not dissolve but floats on the liquid, 
 hence it is lighter than water. 
 
 Experiment 2. Apply heat to the water and ob- 
 serve that the fat melts much before the water begins 
 to boil. 
 
 Experiment j. Rub a little of the fat on a piece of 
 paper and observe that it makes a transparent spot. 
 
 Experiment ./. Heat a little of the fat on foil with 
 some potassium bisulphate (KHS0 4 ) and note that it 
 melts readily, flowing about the foil and giving off a 
 marked and characteristic irritating odor of acrolein. 
 
 It inflames, burning with a sooty flame. 
 
 Experiment 5. Shake some of the liquid fat given 
 with water in a test tube. The oil globules readily 
 conglomerate and rise to the surface. 
 
 s 
 
FA TS. 9 
 
 Experiment 6. Shake another portion with an albu- 
 min solution. The oil globules do not readily con- 
 glomerate, but forma milky white emulsion. 
 
 Experiment 7. Shake a third portion of the liquid 
 fat with water containing a little soap solution. An 
 immediate and permanent emulsion is obtained on 
 slightly shaking. 
 
 These facts are of the greatest importance in the 
 digestion and assimilation of fats in the intestinal 
 tract. 
 
 Experiment S. Add an alcoholic solution of caustic 
 soda to some of the solid fat and heat on the water- 
 bath for some time. The product, a soap, is character- 
 ized by its forming a lather if shaken with water. This 
 process is called saponification, and may be expressed 
 by the following equation : 
 
 C 3 H s (C 18 H 35 O a ) 3 +3NaOH= 3 NaC 18 H 35 3 + 
 
 Tri-stearin. Caustic soda. Sod. stearate or soap. 
 
 C 3 H 8 3 . 
 
 Glycerin. 
 
 Experiment g. Treat some of the soap solution with 
 some dilute sulphuric acid (H 2 S0 4 ). A decomposi- 
 tion with the precipitation of the insoluble fatty acids 
 is the result. 
 
 2NaC 18 H 35 3 -fH 3 S0 4 = 2C 18 H 36 0, +Na 2 S0 4 . 
 
 Soap. Sulphuric acid. Stearic acid. Sod. sulphate. 
 
 Experiment 10. Treat a small quantity of the liquid 
 fat with some pancreatin and add a small amount of 
 sodium carbonate (Na 2 C0 3 ). Heat in the water-bath 
 at 40 C. for some time. The fat-splitting ferment of 
 the pancreas will decompose a part of the fat into fatty 
 
10 LESSON II. 
 
 acids and glycerine which combine with the sodium 
 carbonate forming soaps which in turn cause the 
 unacted on fats to form an emulsion on shaking. 
 
 C 3 H 5( C i8 H 35 2 N >3+3H 2 = 3C 18 H3 6 2 4-C 3 H 8 C) 3 . 
 
 Tri-stearin Stearic acid. Glycerin. 
 
 Experiment n. Shake some water containing a 
 little oil with ether. The ether dissolves the fat 
 readily. Decant the ethereal solution into a watch 
 glass, and allow the ether to evaporate, leaving the fat. 
 
 Experiment 12. Apply a little of the fat to a piece 
 of blue litmus paper. No reaction. 
 
 Experiment ij. Apply a little of the rancid fat to a 
 piece of blue litmus paper. A marked acid reaction is 
 obtained, showing that the rancidity of fats is due to 
 free fatty acids. 
 
 The free fatty acids form soaps directly, without de- 
 composition, with sodium carbonate (Na 3 C0 3 ) with 
 the disengagement of carbon dioxide (C0 3 )gas. They 
 do not evolve an odor of acrolein when heated with 
 potassium bisulphate. The fatty acids have an acid 
 reaction. These properties distinguish them from the 
 neutral fats. 
 
Lesson III. 
 PROTEIN SUBSTANCES. 
 
 The most important class of substances that occur 
 in artimal or vegetable organisms and necessary for 
 the phenomena of life. They are highly complex in 
 their constitution and contain C,H,0,N,S, and some P. 
 The nitrogen is the most important element. 
 
 The protein substances are divided into various 
 groups having different properties and characteristics. 
 They are as follows: albumins, globulins, nucleo-albu- 
 mins, albuminates, albumoses and peptones, coagulated 
 proteids, haemoglobin, glyco-proteids, nucleo-proteids, 
 keratin, elastin, collagen, etc. 
 
 Experiment i. Place a little of the albumin on foil 
 and apply heat. It blackens, burns with a character- 
 istic odor of burnt hair or horn, and leaves no residue. 
 This odor is characteristic of nitrogenous organic com- 
 pounds. 
 
 Experiment 2. Place some in water in a test tube. 
 It dissolves, but not readily, yielding an opalescent 
 solution. 
 
 Experiment 3. Apply heat to some albumin solution 
 in a test tube. The solution becomes cloudy and fin- 
 ally a coagulum forms. This is called heat coagula- 
 tion. Add a drop of acetic acid to the above and the 
 coagulum will be increased. 
 
12 LESSON III. 
 
 Experiment </. Saturate some albumin solution with 
 ammonium sulphate (NH" 4 ) 3 S0 4 . A precipitate of 
 albumin is produced. All protein substances with the 
 exception of peptones are precipitated by saturation 
 with this salt. This is a means of separating peptones 
 from other protein substances. 
 
 Experiment 5. Place a solution of albumin contain- 
 ing some sodium chloride (NaCl) in a parchment dialy- 
 ser as described, and place it in a vessel of water. It 
 will be found that the water outside will acquire a salty 
 taste after a time, because the salt has passed through 
 the membrane. The presence of the salt may be tested 
 by placing some of the water in a test tube and adding 
 some silver nitrate (AgNO s ), which will give a white 
 precipitate. No albumin will be found in the water on 
 the outside. 
 
 This process is called dialysis or diffusion through 
 membranes. The salts are diffusible and are called 
 crystalloid substances, while the protein bodies, with 
 the exception of peptones, do not, or with difficulty, 
 diffuse, and are called colloid substances. Dialysis is 
 made use of in separating colloid from crystalloid 
 bodies. 
 
 Experiment 6. Color reactions. Add some Millon's 
 reagent to a little of the albumin solution. A precipi- 
 tate is formed, which slowly at the ordinary tempera- 
 ture, but quickly at the boiling point, turns red, 
 depending upon the amount of albumin present. 
 
 The solid proteids give the same reaction. 
 
 Millon's reagent consists of a solution of mercuric 
 nitrate in nitric acid containing some nitrous acid. 
 
 Experiment 7. Xantho-proteic reaction. Add a drop 
 
PROTEIN SUBSTANCES. I 3 
 
 of nitric acid (HN0 3 ) to some solid albumin on 
 a watch glass. It turns yellow and orange-yellow. 
 After a little while add a few drops of ammonium 
 hydrate (NH 4 OH) or caustic soda (NaOH) when the 
 spot becomes red. 
 
 Experiment 8. Biuret reaction. Place one or two 
 drops dilute copper sulphate (CuS0 4 ) in a test tube 
 and add an excess of caustic soda (NaOH). Add some 
 albumin solution to this and apply heat. A violet 
 color is produced which deepens in tint on boiling. A 
 reduction of copper sub-oxide may take place. 
 
 Experiment p. Add two or three drops nitric acid 
 (HNO,) to some dilute albumin solution. A precipi- 
 tate occurs which re-dissolves on adding an excess of 
 the acid. 
 
 Experiment 10. Place some nitric acid (HN0 3 ) in a 
 test tube and allow the filtered albumin solution to 
 flow down gently on its surface. A well-defined ring 
 or zone of precipitated albumin appears at the juncture 
 of the two liquids. Most mineral acids act in the same 
 manner. 
 
 Experiment II. Add some mercuric chloride (HgCl 2 ) 
 to some albumin solution. A precipitate of albumin is 
 obtained. Most metallic solutions have the same action, 
 hence the use of albumin as an antidote in cases of 
 metallic poisoning. 
 
 Experiment 12. Acidify some albumin solution with 
 acetic acid (C 2 H 4 2 ) and add a few drops of potassium 
 ferrocyanide (K 4 Fe(CN) 6 ) solution. A white precipi- 
 tate of albumin is obtained. 
 
 Experime?it ij. Acidify some albumin solution 
 with acetic acid and add a few drops picric acid 
 
14 LESSON III. 
 
 (C 6 H 2 (NO) 3 OH) solution. A white precipitate of 
 albumin is produced. 
 
 Experiment 14. Add some alcohol to some albumin 
 solution in a test tube. The albumin will be precipi- 
 tated. 
 
 Experiment ij. Place a little dilute albumin solution 
 in a test tube, add some pepsin and one drop hydro- 
 chloric acid, and place in the water bath at about 40 
 C. for fifteen minutes. 
 
 The pepsin will convert the albumin into albumoses 
 and peptones, which are the chief products of the pro- 
 teolytic ferments, such as the pepsin of the gastric 
 juice and the trypsin of the pancreatic juice. 
 
 Acidify the solution with a few drops of acetic acid 
 and heat to the boiling point. This precipitates the 
 unconverted albumin, which may be removed by filtra- 
 tion and the presence of albumoses and peptones deter- 
 mined by applying the Biuret test. (See Experiment 
 8, which gives a rose-red color.) 
 
 Experiment 16. Repeat Experiment 15, but instead 
 of using pepsin use pancreatin and show that the pro- 
 ducts are about the same in both cases. 
 
 In this experiment do not make the solution acid 
 with hydrochloric acid, but add one drop of sodium 
 carbonate solution instead. 
 
 Experiment iy. Add some peptone to some water 
 in a test tube. It dissolves readily. 
 
 Experiment 18. Apply heat to a portion of this solu- 
 tion. No coagulation showing a difference from other 
 protein substances. 
 
 Experiment 19. Add some nitric acid to some of the 
 peptone solution. No precipitation. 
 
PROTEIN SUBSTANCES. I 5 
 
 Experiment 20. Apply the Biuret test to some pep- 
 tone solution. A rose-red color is obtained instead of 
 a violet color, as given by other protein substances. 
 
 Experiment 21. Place a peptone solution in a parch- 
 ment dialyser and place it in a vessel of pure water. 
 After a time the peptones will have diffused through 
 the membrane, as shown by applying the Biuret test 
 to the water on the outside. 
 
Lesson IV. 
 
 DIGESTION. 
 
 Is to separate those constituents of the food which 
 serve as the nutriment of the body from those which 
 constitute the waste and to separate each in such a 
 form that it may be easily taken up by the blood from 
 the alimentary canal. 
 
 SALIVARY DIGESTION. 
 
 Experiment i. Collect as much saliva in a test tube 
 as possible. 
 
 Experiment 2. Apply a little to a piece of red litmus 
 paper. It turns blue, showing it to be alkaline. Di- 
 rectly after a meal it may be acid. 
 
 Experiment j. Add a drop of hydrochloric acid 
 (HC1) to some saliva in a test tube and then a few 
 drops of ferric chloride (Fe 2 CL 6 ). A blood-red color- 
 ation denotes the presence of sulphocyanides. The 
 depth of color varies with different saliva, because the 
 amount of sulphocyanides varies very markedly. 
 
 Experiment /f.. Make some dilute starch solution 
 (as described in Lesson I., Experiment 20) and add 
 thereto some fresh saliva and keep in the water-bath 
 at a temperature of 50°-55° C. for some little time. 
 Observe that the starch solution becomes clear. Test 
 a portion of the solution with tincture of iodine when, 
 
 16 
 
DIGESTION. 17 
 
 instead of a blue reaction for starch, a red coloration 
 due to dextrin will be obtained. 
 
 Test the presence of sugar (maltose) in the liquid by- 
 means of Fehling's or Boettger's test. (See Lesson I.) 
 
 GASTRIC DIGESTION. 
 
 Experiment j. Test the reaction of the artificial 
 gastric juice given by means of a piece of blue litmus 
 paper. It is found to be acid. 
 
 Experiment 6. Add a drop of silver nitrate to a little 
 of the gastric juice. A white precipitate of silver 
 chloride (AgCl) shows the presence of hydrochloric 
 acid (HC1). This precipitate is soluble in ammonium 
 hydrate (NH 4 OH). 
 
 Experiment 7. Place a drop of the gastric juice on a 
 piece of Congo red paper. It turns blue, due to the 
 free hydrochloric acid. 
 
 Experiment 8. Spread a few drops of Gunsburg s 
 solution (consisting of an alcoholic solution of phloro- 
 glucin-vanillin) out in a thin layer upon a watch glass, 
 or porcelain dish, and then gently warm ; then a drop 
 of the gastric juice is allowed to flow across it, or a 
 glass rod dipped in the solution is drawn across the 
 plate. If free hydrochloric acid be present, a deep 
 scarlet-red streak is developed. 
 
 Experiment g. Test for lactic acid. Add a few 
 drops of the gastric juice to some Uffelmann's solu- 
 tion. This solution is made by mixing a few drops 
 neutral ferric chloride (Fe 2 Cl 6 ) solution with 2 drops 
 of pure carbolic acid (C 6 H 5 OH), and adding water 
 until the solution has a beautiful amethyst-blue color. 
 
1 8 LESSON IV. 
 
 In the presence of lactic acid the blue color is changed 
 to yellow instantly. 
 
 Experiment 10. Place a piece of hard-boiled egg in 
 a test tube and add a little of the gastric juice. Keep 
 this at a temperature of about 40 C, and observe the 
 swelling up of the edges, the corrosion and the gradual 
 disappearance or solution of the albumin being con- 
 verted into albumoses and peptones. 
 
 Experiment 11. Test the presence of peptones in 
 the solution by means of the Biuret test. (See Lesson 
 3, Experiment 20.) 
 
 Experiment 12. Warm a little milk to about 40 C. 
 and then add some of the neutral gastric juice. An 
 immediate coagulation of the milk is observed, due to 
 the enzyme called Rennin in the gastric juice. 
 
 PANCREATIC DIGESTION. 
 
 Experiment ij. Apply some of the pancreatic juice 
 to a piece of red litmus paper. It turns it blue, show- 
 ing it to be alkaline. 
 
 Experiment 14.. Make some dilute starch solution 
 and add some pancreatic juice thereto, and keep at a 
 temperature of about 40 C. for some little time. Ob- 
 serve that the starch solution becomes clear and the 
 presence of sugar (maltose) may be shown by applying 
 Fehling's test to the solution. 
 
 The amylopsin of the pancreatic juice converts starch, 
 etc., into maltose and dextrin. 
 
 Experiment 15. Place some liquid fat with some 
 water in a test tube and add a little pancreatic juice ; 
 keep at a temperature of 40 C. for some time. A 
 
DIGESTION. 19 
 
 perfect emulsion of the fat will be obtained if the con- 
 tents of the tube is shaken. (See Experiment 10, 
 Lesson 2.) 
 
 Experiment 16. Place a piece of hard-boiled egg in 
 a test tube and add some of the pancreatic juice and 
 keep at 40 C. for some time. Observe the action on 
 the albumin and the gradual solution of the piece. 
 
 The trypsin converts the albumin into albumoses 
 and peptones which may be tested for by the Biuret 
 test. (See Lesson 3, Experiment 20.) 
 
Lesson V. 
 BLOOD AND BILE. 
 
 BLOOD. 
 
 Observe the obtainment of salt plasma from the 
 blood of a dog. 
 
 Also the collection of blood in a vessel surrounded 
 with an ice mixture. 
 
 Observe the coagulation of the salt plasma on the 
 addition of water. 
 
 Also the coagulation of the cold blood on allowing 
 it to attain the temperature of the room. 
 
 Allow this last to stand quietly, so that the clot may- 
 settle, leaving the clear serum above. 
 
 Experiment i. Place a drop of the serum on a piece 
 of red litmus paper. It turns blue, showing the blood 
 serum to be alkaline. 
 
 Experiment 2. Saturate some of the serum with 
 magnesium sulphate (MgS0 4 ). A precipitate of ser- 
 globulin is produced. Filter this precipitate off, and 
 keep the nitrate. 
 
 Experiment j. Remove some of the substance col- 
 lected on the filter, and place it in a test tube with 
 water. 
 
 It dissolves, due to the salt retained by the precipi- 
 
BLOOD AND BILE. 21 
 
 tate. Pure ser-globulin is insoluble in water, but dis- 
 solves in dilute salt solutions. 
 
 Experiment 7. Heat a portion of the above ser- 
 globulin solution. It coagulates. 
 
 Experiment 3. Apply the Biuret test (see Experi- 
 ment 8, Lesson 3) to another portion. A violet reac- 
 tion will be obtained. 
 
 Experiment 6. Add some sodium sulphate (Na 2 S0 4 ) 
 to the cold filtrate obtained in Experiment 2. A 
 precipitate of ser-albumin occurs. Collect the preci- 
 pitate on a filter. 
 
 Experiment 7. Remove the precipitate from the 
 filter, and place it in a test tube with water. It dis- 
 solves. 
 
 Heat a portion, and observe that it coagulates. 
 
 Apply Experiments 9, 10, 12, and 13, Lesson 3, to 
 some of the solution. The relationship of ser-globulin 
 to ser-albumin in human blood serum is 1:1.5, m oxen, 
 1:0.842, in dogs, 1:1.8. 
 
 Experiment 8. Wash some of the blood clot in 
 water until nearly white and free from blood cor- 
 puscles. 
 
 Observe the thready or fibrous character of the 
 fibrin. 
 
 Experiment <p. Apply Experiments 6 and 7, Lesson 
 3, to some of the fibrin, showing it to be a protein 
 substance. 
 
 Experiment 10. Place a thread of fibrin in some 
 hydrogen peroxide (H 3 2 ). A rapid evolution of oxy- 
 gen gas is the result. The oxygen may be tested for 
 by its relighting a spark on the end of a match. 
 
 Experiment 11. Mix a drop of defibrinated blood of 
 
22 LESSON V. 
 
 a guinea pig on a slide with a drop of water, put on a 
 cover-glass, and in a few minutes the corpuscles are 
 rendered colorless, and then the oxyhemoglobin crys- 
 tallizes out as rhombic tetrahedra (four-sided pyramids). 
 The form of the crystals varies with the blood from 
 different animals. 
 
 Experimetit 12. Treat a little of the haemoglobin 
 given with a drop or two of nitric acid (HNO s ) on a 
 watch glass, and apply gentle heat to dryness. When 
 cold, test the presence of iron in the residue by means of 
 a drop of ammonium sulphocyanide(NH 4 CNS), which 
 gives a blood-red coloration in the presence of iron. 
 
 Experiment ij. Observe the estimation of haemo- 
 globin in a specimen of blood by means of a haemo- 
 globinometer. 
 
 Experiment i/f.. Make a dilute blood solution in a 
 test tube, and place it in front of the slit of the spec- 
 troscope, allowing the light to pass through the solu- 
 tion. 
 
 An absorption spectra will be seen with two (2) 
 absorption bands between the Fraunhofer lines D and 
 E, namely, in the yellow and green parts of the 
 spectrum. 
 
 This absorption is produced by the oxyhaemoglobin. 
 
 Experiment 15. To another portion of dilute blood 
 add some Stoke's reducing solution (an ammoniacal 
 solution of iron tartrate). Observe the change of color 
 of the solution, and place it in front of the spectroscope 
 as before. 
 
 An absorption spectra will be seen with only one (1) 
 broad absorption band between the Fraunhofer lines 
 D and E. 
 
BLOOD AND BILE. 23 
 
 This absorption is produced by haemoglobin or re- 
 duced oxyhemoglobin. 
 
 Experiment 16. Place a drop of blood on a slide, 
 and add a grain of common salt, and cover with a 
 cover-glass. Now add some glacial acetic acid under 
 the cover-glass, and warm gently until bubbles rise. 
 When viewed with the microscope, dark brown, long 
 rhombic crystals of hsemin are seen. If no crystals 
 appear after the first heating, warm again, and, if 
 necessary, add some more acetic acid. These crystals 
 are sometimes called Teichmann's crystals, and are 
 characteristic of blood-coloring matters. 
 
 Experiment ij. Add some tincture of guaiacum to 
 some dilute blood solution in a test tube, and then a 
 drop or two of hydrogen peroxide (H 2 O s ). A bluish- 
 green, and then a blue coloration is obtained. A 
 frothing of the liquid is also observed, due to the evolu- 
 tion of oxygen from the hydrogen peroxide. 
 
 Experiment 18. Apply the same test to a blood 
 stain on a piece of cloth. The same coloration is ob- 
 tained as above. 
 
 BILE. 
 
 Experi?nent ig. Pettenkofer's test for bile acids. 
 Treat a few drops of bile in a porcelain dish with 
 a few drops concentrated sulphuric acid (H 2 S0 4 ) and 
 warm gently. Then add a few drops of a ten-per-cent. 
 solution of cane sugar, continually stirring with a glass 
 rod. A beautiful red liquid, which gradually changes 
 to bluish violet, is obtained. This shows the presence 
 of bile acids (glycocholic and tauracholic acids). 
 
24 LESSON V. 
 
 Experiment 20. Gmelin s test for bile pigments. 
 
 Place some yellow nitric acid (containing nitrous acid) 
 in a test tube and allow some bile to flow down gently 
 on its surface. A series of colored layers are obtained 
 at the juncture of the two liquids in the following 
 order from above downwards: green, blue, violet, red, 
 and reddish-yellow. The green ring must never be 
 absent, otherwise the reaction is not characteristic of 
 bile pigments. 
 
 Experiment 21. H upper? s test for bile pigments. 
 
 Add some calcium chloride (CaCl 2 ) solution to some 
 bile solution in a test tube and precipitate with am- 
 monium hydrate (NH 4 OH). Filter off the precipitate, 
 wash with a little water, transfer while moist to a test 
 tube, and treat with alcohol which has been acidified 
 with sulphuric acid. Now boil for some time, when 
 the solution becomes emerald-green or bluish-green in 
 color. 
 
 Experiment 22. Smith's test for bile pigments. 
 
 Place some bile solution in a test tube and allow some 
 tincture of iodine to flow on its surface. A green ring 
 is seen at the juncture of the two liquids. 
 
 Experiment 23. Cholesterine. 
 
 Treat a crystal on a watch glass with a mixture of 
 5 parts sulphuric acid and 1 part water, when colored 
 rings are obtained, first a bright carmine-red and then 
 violet. 
 
Lesson VI. 
 
 MILK. 
 
 Consists of an emulsion of very finely divided fat 
 globules suspended in a solution of albuminous bodies, 
 milk sugar, and salts in water. 
 
 Experiment I. Test the reaction of the milk given 
 by means of litmus paper. It may be amphoteric, that 
 is, it may turn blue litmus paper red, and at the same 
 time turn red paper blue, showing it to have both an 
 acid and an alkaline reaction to litmus. 
 
 Experiment 2. Take the specific gravity of the milk 
 by means of the hydrometer at a temperature of 15 C. 
 
 Pure milk should have a specific gravity of 1028 to 
 1034. 
 
 Experiment j. Mix a little water with some milk 
 (about £ its volume) and take the specific gravity at 
 the same temperature as before. The specific gravity 
 is diminished depending upon the amount of water 
 added. 
 
 Experiment 4. Boil some of the milk in a test tube. 
 No coagulation or change is observed, with the excep- 
 tion of the formation of a skin on the surface. 
 
 Experiment 5. — Slightly acidify a little of the milk 
 with acetic acid and then warm. An immediate coagu- 
 lation or curdling of the casein is produced. 
 
 25 
 
26 LESSON VI. 
 
 Experiment 6. Warm some milk up to 40 C. and 
 add some rennet powder or rennin. The casein coagu- 
 lates immediately, forming a clot, and a clear liquid 
 called the "whey" separates. Filter the liquid off 
 and retain it for further experiments. 
 
 This process is made use of in the making of cheese. 
 
 Experiment 7. Heat a little of the clot on foil. It 
 burns with an odor of burnt hair, showing it to be 
 nitrogenized. 
 
 Experiment 8. Apply the Biuret test to a portion of 
 the clot. A violet reaction is obtained showing it to 
 be a protein body. 
 
 Experiment p. Shake another portion of the clot in 
 a test tube with some ether. Decant the ether into a 
 watch glass, and let it evaporate. A residue of fats 
 will be found in the watch glass. This shows that the 
 casein in coagulating carries with it most of the fats of 
 the milk. 
 
 Experiment 10. Add some copper sulphate (CuS0 4 ) 
 solution to some milk in a test tube, and then one drop 
 of caustic soda (NaOH) solution. A precipitate of the 
 casein, carrying most of the fats with it, is produced. 
 This is the basis of Ritthausen's method for the esti- 
 mation of casein in milk. 
 
 Experiment 11. Keat some of the " whey " obtained 
 in Experiment 6 with some Fehling's solution. 
 
 A copious reduction of copper sub-oxide shows the 
 presence of milk sugar (lactose). The " whey " should 
 have a sweetish taste. 
 
 Experiment 12. Witness the estimation of fat in a 
 sample of milk by means of the Lactrocrit, and also by 
 Soxhlet's extraction apparatus. 
 
MILK. 27 
 
 Experinunt ij. Place some milk in a narrow test 
 tube, and place it in the centrifugal machine and rotate 
 for a few minutes. The cream, or fat, is made to rise 
 to the surface, and should occupy from ten to fifteen 
 per cent, of the column of the milk. The same result 
 may be obtained by allowing the milk to stand in the 
 test tube for twelve to twenty-four hours. 
 
 Experiment 14. Place a drop of the milk on a slide 
 and cover with a cover-glass. View this under the 
 microscope and observe the number and size of the 
 fat globules. 
 
Lesson VII. 
 URINE. 
 
 i. Collection and measurement of the twenty-four 
 hours' urine. Start at a given time with an empty 
 bladder, and then collect all the urine excreted in the 
 following twenty-four hours. 
 
 Average normal amount for man about 1500 c. c. 
 Women a little less. The quantity of drink greatly in- 
 fluences the quantity of urine voided. 
 
 2. Color, normally amber-yellow. (Refer to Vogel's 
 color chart.) 
 
 3. Transparency, Clear, with the exception of a 
 slight cloud of mucus, which suspends itself midway in 
 the liquid on allowing it to stand. 
 
 4. Consistency. Not ropy. 
 
 5. Odor. Urinous, but sometimes certain foods and 
 medicines give a characteristic odor. 
 
 6. Reaction. Normally slightly acid for the twenty- 
 four hours. Portions voided at different times of the 
 twenty-four hours and after certain food have different 
 reactions. The reaction is determined by placing some 
 of the urine on litmus paper. If it be acid, the blue 
 paper will turn red. If alkaline, the red paper will turn 
 blue. 
 
 The alkalinity may be due to the fixed alkalies, such 
 
 28 
 
URINE. 29 
 
 as sodium or potassium carbonates or phosphates, or 
 to volatile alkali, such as ammonium carbonate. 
 
 If the alkalinity is due to the volatile alkali, on sus- 
 pending the litmus paper, which has turned blue, to 
 the air, the original red will come back again. If the 
 alkalinity be due to the fixed alkalies, the blue color 
 will be permanent. 
 
 Experiment. Test the reaction of the sample of 
 urine given. If alkaline, determine if it is due to fixed 
 or volatile alkalies, as above described. 
 
 Experiment. If the alkalinity is due to volatile al- 
 kali, it will have an odor of ammonia and will give 
 white fumes of ammonium chloride (NH 4 C1) if a glass 
 rod, moistened with hydrochloric acid, is held over the 
 urine. 
 
 7. Specific Gravity. Normally about 1020 at6o° F., 
 or 1 5. 5 C. The specific gravity varies generally with 
 the quantity of urine voided, but not always. 
 
 It is generally determined by means of a urinometer 
 or specific gravity bottle. Always determine the spe- 
 cific gravity at 60 ° F. or 15. 5 C, taking care not to 
 have any air bubbles attach themselves to the instru- 
 ment, and to have the vessel, holding the urine, large 
 enough so that the instrument floats readily. The 
 vessel should not be held by the hand, but should be 
 allowed to stand in a vertical position. It should be 
 filled to the top with urine. 
 
 Read off on the spindle of the instrument where the 
 meniscus of the liquid strikes the graduation. 
 
 Experiment. Determine the specific gravity of the 
 sample of urine given. 
 
 Experiment. Dilute a given volume of this urine 
 
30 LESSON VII. 
 
 with an equal volume of water, and mix thoroughly. 
 Take the specific gravity of the mixture. It will be 
 found to be one half of the original urine. If the spe- 
 cific gravity of the mixture of one volume of urine and 
 two volumes of water is taken, it will be found to be 
 one third of the original urine. 
 
 In cases where too little urine is voided to take the 
 specific gravity, the urine can be diluted with a known 
 volume of water, and then the specific gravity taken, 
 and from this calculate the specific gravity of the origi- 
 nal urine. 
 
 8. Total Solids. The approximate amount of solids 
 may be calculated from the specific gravity by multi- 
 plying the last two figures of the specific gravity when 
 below 1018 by 2 or when above by 2.33. The result is 
 parts of solid in 1000 parts of the urine. 
 
 Experiment. Make the calculation for the urine 
 given, supposing in one case the total quantity of urine 
 voided in the twenty-four hours was 1450 c. c. and in 
 the other case 850 c. c. 
 
 Experiment. Heat some of the evaporated urine 
 given on the foil. It blackens and burns with an odor 
 of burnt hair, due to the nitrogenous organic matter. 
 On continuing the heat all the organic matter burns 
 away, leaving a white residue which consists of the 
 inorganic salts. 
 
 NORMAL PROPORTIONS. 
 
 Total solids... .60 grammes in the 24 hours. 
 
 Organic " 35 " " " " 
 
 Inorganic" ...,25 " " " " 
 
URINE. 31 
 
 Organic Solids. Inorganic Solids. 
 
 Urea 30 grms. Sodium chloride.. 15 grms. 
 
 Uric acid 0.7 " Sulphuric acid. .. .2.5 " 
 
 Creatinin 1.0 " Phosphoric acid. .2.5 " 
 
 Hippuric acid... .0.7 " Potash 3.3 " 
 
 Remaining organ- Ammonia 0.7 " 
 
 ic bodies 2.6 " Magnesia 0.5 " 
 
 Lime 0.3 " 
 
 Remaining inor- 
 ganic bodies. . .0.2 " 
 
 UREA, CH 4 N 2 0. 
 
 Colorless, crystalline needles or prisms. 
 - Very soluble in water or alcohol. It is soluble in 
 its own weight of water and five times its weight of 
 alcohol. 
 
 Experiment. Try the solubility of the urea given in 
 water. Retain this solution for further use. 
 
 Experiment. Try the solubility of the urea in alco- 
 hol. Place a drop of this alcoholic solution on a clean 
 microscope slide and observe the crystallization. 
 
 Urea is a basic compound forming salts with acids, 
 such as urea nitrate CH 4 N 2 0,HN0 3 or urea oxalate, 
 CH 4 N 2 0, C 2 H 2 4 . which are crystalline. 
 
 Experiment. Place a little of the watery solution of 
 urea obtained above in a watch glass and add a few 
 drops of nitric acid thereto. Hexagonal crystalline 
 plates of urea nitrate are formed in the liquid. 
 
 This compound may be employed in the detection of 
 small amounts of urea in liquids. 
 
 Experiment. Treat some of the evaporated urine 
 
32 LESSON VII. 
 
 given with a few drops nitric acid and observe the for- 
 mation of the crystals of urea nitrate. 
 
 Urea readily changes into ammonium carbonate by 
 the action of chemical processes or micro-organisms. 
 (This latter is called alkaline fermentation of the urine.) 
 CH 4 N s O + 2H 2 0=(NH 4 ) 2 C0 3 . 
 
 This change may take place in the body and the 
 urine voided under such circumstances will be alkaline 
 due to volatile alkali, will probably have an odor of 
 ammonia and give white fumes of ammonium chloride 
 with a drop of hydrochloric acid, if held over it on a 
 glass rod. 
 
 Experiment. Heat a little urea in a dry test tube. 
 It melts, decomposes, and gives off ammonia (NH 3 ), 
 and leaves finally a white mass, which if dissolved in 
 water and treated with a drop of copper sulphate 
 (CuS0 4 ) and an excess of caustic soda, gives a beauti- 
 ful reddish-violet liquid. (Biuret reaction.) 
 
 Urea is decomposed by an alkaline solution of sodium 
 hypochlorite, or, better, hypobromite into nitrogen, car- 
 bon dioxide, and water, according to the following 
 equation : 
 
 CH 4 N 2 0+3NaBrO = N 2 +C0 3 +2H 3 0+3NaBr. 
 
 Experiment. Place a little urea solution in a test 
 tube and add some sodium hypobromite. A violent 
 decomposition takes place with the rapid evolution of 
 gas. Let the gas bubbles subside and apply a lighted 
 match to the gas in the test tube. The flame is extin- 
 guished, due to the nitrogen, which is a non-supporter 
 of combustion. The carbon dioxide (C0 2 ) evolved is 
 absorbed by the alkaline solution of sodium hypo- 
 bromite. 
 
URINE. 33 
 
 Experiment. Quantitative Estimation. Fill the nre- 
 ometer with sodium hypobromite (prepared as described 
 below) by holding it in a vertical position and pouring 
 the solution into the bulb, and when it is rather more 
 than one half full, incline the instrument horizontally 
 until the entire tube is filled and a little left in the bulb, 
 say about one third, when in the vertical position. 
 Now fill the nipple pipette up to the I c. c. mark 
 with urine and slozvly inject this amount of urine up 
 under the tube and not in the bulb of the instrument. 
 Allow frothing to cease and let it stand a moment or 
 two, so that the carbon dioxide may be absorbed. 
 Read off on the graduation the weight of urea corres- 
 ponding to the volume of nitrogen collected. The in- 
 struments are graduated to read fractions of a gramme 
 of urea per cubic centimeter of urine. 
 
 The solution of sodium hybromite is prepared as 
 follows : 
 
 Dissolve ioo grammes caustic soda in 250 c. c. water, 
 and adding 25 c. c. bromine to this solution when cold. 
 For use in the ureometer this solution should be diluted 
 with an equal volume of water. 
 
 As this solution does not keep for any length of 
 time, a solution has been suggested by Dr. Rice as 
 follows : 
 
 1. Dissolve 125 grammes bromine, 125 grammes 
 sodium bromide in 1000 c. c. water. 
 
 2. Prepare a solution of caustic soda of a specific 
 gravity of 1.250. 
 
 For use in Doremus's ureometer mix 5 c. c. of each 
 of the two above solutions and dilute with 15 c. c. 
 water. 
 
34 
 
 LESSON VII. 
 
 URIC ACID C 5 H 4 N 4 3 or H 2 C 5 H 2 N 4 3 . 
 
 Is a bibasic acid, thus forming two series of salts. Ex- 
 ample : 
 
 Acid sodium urate, NaHC 5 H 2 N 4 3 . 
 Neutral " " Na 2 C 5 H 2 N 4 3 . 
 
 Soluble with difficulty in cold water (i part in 15,000 
 parts) and more soluble in boiling water (1 part in 1900 
 parts). The acid salts are less soluble than the neutral 
 salts. The alkali salts are more soluble than the 
 earthy, such as calcium or magnesium. Ammonium 
 urate is very insoluble. The uric acid exists in the 
 urine as urates and never free. Sodium phosphate is 
 considered as the solvent for the uric acid in the urine. 
 
 Uric acid may occur in nearly every crystalline form, 
 but generally as small rhombical prisms or plates, dumb- 
 bell shaped crystals, whetstone shaped, etc. When 
 deposited from the urine they are always more or less 
 colored. 
 
 Experiment. Place a little uric acid in a test tube 
 and add some water. Note insolubility. Heat to boil- 
 ing and notice the apparent insolubility. 
 
 Experiment. Add a few drops of sodium carbonate 
 to the above solution. The uric acid dissolves readily 
 with the formation of sodium urate. 
 
 Experiment. Add a few drops of hydrochloric acid 
 to a portion of the above solution of sodium urate. A 
 white precipitate of uric acid is produced. The HC1 
 added combines with the sodium, setting the insoluble 
 uric acid free. 
 
 Experiment. Saturate some of the solution of uric 
 acid in sodium carbonate with ammonium chloride. A 
 
URINE. 35 
 
 white gelatinous precipitate of insoluble ammonium 
 urate is formed. (This is the basis of Hopkin's method 
 of estimating uric acid in urine.) 
 
 Experiment. Murexid test. Place a few grains of 
 the uric acid on a watch glass, moisten with a drop of 
 nitric acid, and gently heat to dryness over flame. A 
 yellowish-red residue is obtained. When cold add a 
 drop of ammonium hydrate. A purple coloration is 
 the result, which becomes darker on the addition of 
 caustic soda. The purple color is ammonium pur- 
 purate. 
 
 Experiment. Schiff's test. Dissolve a little of the 
 uric acid in water, to which some sodium carbonate 
 (Na 2 C0 3 ) has been added. Add a drop of silver ni- 
 trate (AgN0 3 ) to a portion of the solution. A black 
 reduction of silver oxide is obtained. The test may 
 also be performed by applying a drop of the solution 
 of uric acid to a piece of paper previously moistened 
 with silver nitrate (AgN0 3 ). A black stain appears 
 on the paper. 
 
 Experiment. Place a little Fehling's solution in a 
 test tube, and add a little of the uric acid solution. A 
 white precipitate of copper urate is produced, which if 
 heated is reduced to red copper sub-oxide. This should 
 be considered in testing for sugar (dextrose) in the 
 urine. Alkaline solutions of bismuth are not reduced 
 by uric acid. 
 
 Experiment. Place some uric acid on foil and apply 
 heat. It blackens, gives off an odor of burnt hair, and 
 is entirely consumed, showing it to be entirely of an 
 organic nitrogenous nature. 
 
 Experiment. Heat some ammonium urate ( (NH 4 ) 3 
 
36 - LESSON VII. 
 
 C 5 H 3 N 4 3 ) on foil. It blackens, gives off an odor of 
 burnt hair, and entirely disappears. 
 
 Experiment. Heat some potassium urate (K 3 C 5 H 
 N 4 O s ) on the foil. It blackens, gives off an odor of 
 burnt hair, and leaves a residue of potassium carbonate 
 (K 3 C0 3 ), which, if placed on moistened red litmus 
 paper, turns it blue because of its alkalinity. 
 
 Quantitative Estimation. Acidify 200 c.c. of the 
 urine previously filtered and free from albumin, with 
 10 c.c. hydrochloric acid in a beaker, stir and allow this 
 mixture to stand in a cool place for twenty-four to 
 forty-eight hours. Filter off the precipitated uric acid 
 through a previously weighed filter, taking care that 
 all the particles of uric acid are removed from the sides 
 of the glass vessel and transferred to the filter. Wash 
 the precipitate with water until it is free from chlorine, 
 as shown by adding silver nitrate to a portion of the 
 filtrate. Now dry and weigh. 
 
 As some uric acid is dissolved by the wash water, 
 the filtrate and wash water should be measured, and 
 for each 10 c.c. of filtrate and wash water 0.00048 grms. 
 uric acid must be added to the amount found by 
 weighing. 
 
 Hopkins Method. Treat 100 c.c. of the urine in a 
 beaker with 30 grms. powdered ammonium chloride 
 (NH 4 C1), and allow to stand in a cool place for twenty- 
 four hours. Filter the precipitated ammonium urate 
 through a small filter, and wash with a cold saturated 
 solution of ammonium chloride. Puncture the filter 
 and wash the precipitate into a beaker with a small 
 amount of boiling water, and treat with 5 c.c. dilute 
 hydrochloric acid and heat on the water bath. When 
 
URINE. 37 
 
 cold filter the uric acid through a weighed filter and 
 proceed as above described. 
 
 HIPPURIC ACID, C 9 H 9 N0 3 . 
 
 Crystallizes in semi-transparent, milk-white, long, 
 four-sided rhombic prisms or columns, or in needles on 
 rapid crystallization. 
 
 Hippuric acid dissolves in 600 parts cold water and 
 more easily in hot water. It is soluble in alcohol. It 
 is a monobasic acid forming hippurates with bases, 
 which are soluble in water and alcohol. 
 
 Experiment. Boil a watery solution of hippuric 
 acid with a drop or two of hydrochloric acid. It is 
 split into benzoic acid (C 6 H 5 COOH) and glycocoll 
 (C 2 H 5 N0 2 ). This change may also be brought about 
 by certain micro-organisms. 
 
 Experiment. Add some ferric chloride (Fe 2 Cl,.) to 
 some of the above solution. A bluish-red coloration 
 shows the presence of benzoic acid. 
 
 Experiment. Heat some of the hippuric acid (not 
 solution) with nitric acid (HN0 3 ) in a test tube. An 
 odor of oil of bitter almonds (nitro-benzole) is de- 
 veloped. 
 
 Other organic bodies occurring in the urine : 
 
 Creatinin C 4 H,N 3 
 
 Oxalic acid C 2 H 2 4 
 
 Allantoin C 4 H 6 N 4 3 
 
 Xanthin C 5 H 4 N 4 2 
 
 Hypoxanthin C 5 H 4 N 4 
 
 Guanin C 5 H 5 N g O 
 
 Adenin C 5 H 5 N 5 
 
Lesson VIII. 
 
 URINE. 
 
 Occur chiefly as sodium chloride (NaCl) with small 
 amounts of potassium chloride. They are estimated 
 as sodium chloride. Sodium chloride is very soluble 
 in cold and hot water, hence it never occurs in urinary 
 sediments or calculi. 
 
 Experiment. Add some silver nitrate to some dilute 
 hydrochloric acid in a test tube. A white precipitate 
 of silver chloride (AgCl) is produced. Divide into two 
 portions. Add ammonium hydrate (NH 4 OH) to one 
 portion and notice that the precipitate dissolves. Add 
 nitric acid (HN0 3 ) to the other portion. It does not 
 dissolve. * 
 
 Experiment. Place some sodium phosphate (Na 2 - 
 HP0 4 ) in a test tube and add some silver nitrate 
 (AgN0 3 ). A yellow precipitate of silver phosphate is 
 formed. Add nitric acid (HNO s ) to this and observe 
 that the precipitate is dissolved. 
 
 Experiment. To detect chlorides in a sample of 
 urine, first add a few drops nitric acid and then silver 
 nitrate, so as to prevent the phosphates from being 
 precipitated by the silver nitrate, and in the presence 
 of chlorides a white precipitate is obtained. 
 
 Quantitative Estimation. Place 20 c.c. of the urine 
 
 38 
 
URINE. 39 
 
 in a clean porcelain dish and add thereto, drop by 
 drop, a standard solution of silver nitrate (see below) 
 from a burette, constantly stirring with a glass rod. 
 From time to time place a drop of the contents of the 
 dish on a piece of paper previously moistened with a 
 solution of potassium chromate. When a faint red 
 stain is obtained on this paper stop adding the silver 
 nitrate solution. This indicates that enough silver 
 nitrate solution has been added to combine with all 
 the chlorine in the 20 c. c. of urine in the dish. Read 
 off the number of cubic centimetres of silver nitrate 
 solution used. Each c. c. of the standard solution of 
 silver nitrate is equal to 0.01 grammes sodium chloride 
 (NaCl). From the weight of NaCl in the 20 c. c. of 
 urine calculate the total amount in the twenty-four 
 hours. 
 
 The standard solution of silver nitrate is prepared by 
 dissolving 29.075 grammes fused silver nitrate in 1000 
 c. c. of distilled water. Each c. c. = 0.01 grms. NaCl. 
 
 SULPHURIC ACID, S0 3 . 
 
 The sulphuric acid of the urine occurs either in com- 
 bination with bases as potassium (K 3 S0 4 ) and sodium 
 sulphate (Na 3 S0 4 ) or as ethereal sulphates, such as 
 potassium phenol sulphate (C 6 H 5 .O.S0 2 .OK), or 
 potassium indoxyl sulphate (C 8 H 6 N.O.S0 3 .OK). 
 
 Chiefly formed from the sulphur of the protein 
 bodies by oxidation and a small portion from the 
 sulphates of the food. 
 
 Experiment. Place some magnesium sulphate (Mg- 
 S0 4 j in a test tube and add some barium chloride 
 
40 LESSON VIII. 
 
 (BaCl 3 ) thereto. A white precipitate of barium sul- 
 phate (BaS0 4 ) is produced. 
 
 Try the solubility of this precipitate of barium 
 sulphate in nitric acid and in caustic soda. It will be 
 found insoluble in both. 
 
 Experiment. Acidify some urine with acetic acid 
 and add some barium chloride. A white precipitate 
 of barium sulphate is produced. Filter this off and re- 
 tain the filtrate. 
 
 This precipitate represents the sulphuric acid exist- 
 ing as potassium and sodium sulphates. 
 
 Experiment. Add some hydrochloric acid to the 
 above filtrate and boil for a few minutes. This de- 
 composes the ethereal sulphates setting more sul- 
 phuric acid free, and this is then precipitated as white 
 barium sulphate by the barium chloride in the filtrate. 
 
 Quantitative Estimation, ioo c. c. of the filtered 
 urine are acidified with 5 c. c. concentrated hydrochloric 
 acid and boiled for fifteen minutes. While boiling add 
 2 c. c. of a strong solution of barium chloride and warm 
 for a little while until the barium sulphate has com- 
 pletely settled. Filter through a small filter and wash 
 with hot water and then with alcohol (to remove any 
 resinous bodies). Dry the filter, incinerate and weigh 
 according to prescribed methods. From the weight of 
 barium sulphate calculate the amount of sulphuric acid 
 in the urine used. 
 
 PHOSPHORIC ACID, P 3 5 . 
 
 It occurs in urines combined with sodium, potassium, 
 ammonium, calcium, and magnesium, and as phosphoric 
 acid is tribasic, three series of salts occur, namely acid, 
 
URINE. 4 1 
 
 neutral, and basic phosphates. The phosphates of 
 potassium, sodium, and ammonium are called alkali 
 phosphates, and are very soluble in the urine ; while 
 the phosphates of calcium and magnesium are called 
 earthy phosphates and are difficultly soluble in acid 
 urine, but insoluble in alkaline urines. 
 
 The chief quantity of phosphoric acid is derived 
 from the phosphates of the food, but a small amount 
 is derived from the oxidation of organic bodies con- 
 taining phosphorous such as nuclein, lecithin, protagon. 
 
 Experiment. Place some sodium phosphate (Na 2 - 
 HP0 4 ) in a test tube with water and add some magne- 
 sium sulphate (MgS0 4 ) solution, then some ammonium 
 chloride (NH 4 C1) and lastly some ammonium hydrate 
 (NH 4 OH). A copious white precipitate of magnesium- 
 ammonium phosphate (MgNH 4 P0 4 ,6H 2 0, triple phos- 
 phate) is formed. This is a good test for phosphoric 
 acid or phosphates. 
 
 Experiment. Add some caustic soda (NaOH) to 
 some urine in a test tube and warm gently. A flocu- 
 lent precipitate of earthy phosphates is produced. 
 
 Filter this precipitate off and make the filtrate acid 
 with acetic acid, add magnesium sulphate, ammonium 
 chloride, and then ammonium hydrate. A white pre- 
 cipitate of magnesium-ammonium phosphate is formed, 
 derived from the alkali phosphates in the urine. 
 
 Experiment. Add some ammonium hydrate to some 
 urine in a test tube. A white precipitate of calcium 
 phosphate (Ca 3 (P0 4 ) 2 ) and crystalline magnesium- 
 ammonium phosphate (triple phosphate) is formed. 
 This forms whenever urine becomes ammoniacal, by 
 decomposition of urea, on standing. 
 
42 LESSON VIII. 
 
 Experiment. Add some uranium acetate solution to 
 some sodium phosphate in a test tube. A white pre- 
 cipitate of uranium phosphate is obtained. This is one 
 of the best tests for phosphoric acid or phosphates. 
 
 Quantitative Estimation. Place 50 c. c. of the filtered 
 urine in a clean porcelain dish and add a few drops 
 acetic acid or better a solution of sodium acetate. 
 Warm over water the bath and add a standard solution 
 of uranium acetate from a burette drop by drop, stirring 
 all the while, until a drop taken out and placed on a 
 piece of paper moistened with potassium ferrocyanide 
 (K 4 Fe(CN) 6 ) gives a faint chocolate brown stain. 
 Place it on the water bath again and warm and apply 
 a drop to the potassium ferrocyanide paper. If the 
 stain is permanent stop adding the uranium acetate 
 solution, otherwise go on until a permanent stain is ob- 
 tained. Each c. c. of the standard solution of uranium 
 acetate corresponds to 0.005 grammes phosphoric acid 
 (P s 6 ). From the above result calculate the total 
 amount of phosphoric acid in the twenty-four hours 
 urine. 
 
 Experiment. To determine the P 2 5 in combination 
 as alkali or earthy phosphates separately, first precipi- 
 tate the earthy phosphates from 100 c. c. of the urine 
 by the addition of a very small amount of ammonium 
 hydrate. Filter this precipitate off and collect 50 c. c. 
 of the filtrate. Acidify this with acetic acid and deter- 
 mine the quantity of P 2 O g therein by means of the 
 standard solution of uranium acetate as above directed. 
 This gives the amount of P 2 O g combined with sodium 
 and potassium or alkali phosphates. On subtracting 
 this result from the total amount of P 3 O g in the urine 
 
URINE. 43 
 
 we get the amount of P 2 O g existing in combination 
 with calcium and magnesium, or earthy phosphates. 
 
 ABNORMAL CONSTITUENTS.— PROTEIN SUBSTANCES. 
 Abnormal urine may contain ser-albumin, ser-globu- 
 lin, peptones, mucin, fibrin, etc., but the most common 
 substance is ser-albumin. 
 
 SER-ALBUMIN. 
 
 In testing the presence of albumin in a sample 
 of urine it should be perfectly clear, and if not, it 
 must first be clarified by filtration through one or 
 more folds of filter paper. If it cannot be clarified by 
 simple filtration (due to the presence of micro-organ- 
 isms), add some magnesium sulphate (MgS0 4 ), ammo- 
 nium chloride (NH 4 C1), and then some ammonium 
 hydrate (NH 4 OH), forming a precipitate of triple 
 phosphate, which retains the bacteria, and which may 
 be removed by filtration. The filtrate must be made 
 acid with acetic acid before applying the tests for 
 albumin. 
 
 Before applying the tests to a sample of urine first 
 test the reaction of the urine, and if alkaline make it 
 neutral or slightly acid with acetic acid. 
 
 Experiment. Fill a test tube two thirds full with 
 some of the clear urine and apply heat to the upper 
 layer of the liquid, holding the test tube at the bottom. 
 A precipitation or coagulation may occur, but it may 
 be due to earthy phosphates or albumin, or both, be- 
 cause they are precipitated by heat alone. 
 
 Now add three (3) drops of nitric acid to the liquid. 
 If the precipitate dissolves completely on the addition 
 of the acid it is due to earthy phosphates, but if it does 
 
44 LESSON FIJI. 
 
 not, and the urine remains opalescent and cloudy, it 
 contains albumin. Boil again and observe if the pre- 
 cipitate increases. 
 
 Experiment. Heller s test. Place about one half an 
 inch of nitric acid in a test tube and allow the urine to 
 flow gently on the surface of the acid without mixing 
 with it. This may be done by allowing the urine to 
 flow from a filter, holding the point of the funnel on 
 the side of the test tube, inclining it slightly. The 
 urine may also be floated on the surface of the acid by 
 means of a nipple pipette, holding the test tube in an 
 inclining position. 
 
 If albumin is present a sharply-defined opalescent 
 ring or zone will be observed at the point of contact 
 between the two liquids. In the presence of traces of 
 albumin some time is required for the development of 
 the zone. With a urine containing an excess of uric 
 acid a zone is obtained, but it is higher up in the liquid 
 and not sharply defined as the albumin zone. 
 
 The ring due to coloring matters should not be con- 
 founded for the albumin zone. 
 
 Experiment. Fill a test tube one half full of clear 
 urine and add some clear potassium ferrocyanide 
 (K 4 Fe(CN) 6 ) solution and mix thoroughly. Now add 
 ten drops acetic acid, and if albumin is present a white 
 cloudiness or precipitate is obtained. The obtainment 
 of a cloudiness with this test as above directed shows 
 the presence of albumin and nothing but albumin. 
 
 It is advisable in all these tests, in the presence of 
 faint traces of albumin, to have a second test tube con- 
 taining the same amount of clear urine, without any 
 reagents, and hold the two test tubes side by side and 
 
URINE. 45 
 
 look through the liquid at a dark background. By 
 this means the faintest cloudiness may be detected in 
 the test tube to which the reagents have been added 
 by difference in appearance. 
 
 Experiment. Tanret's test. Acidify some of the 
 urine in a test tube with a few drops of acetic acid, and 
 add a few drops of a solution of double iodide of mer- 
 cury and potassium. A white cloudiness or precipitate 
 is obtained in the presence of albumin. This test may 
 also be applied by the contact method ; the reagent is 
 placed first in the test tube, and then the urine allowed 
 to float on top. A white ring or zone at the point of 
 contact of the two liquids is the result in the presence 
 of albumin. 
 
 The reagent is prepared by adding potassium iodide 
 to some mercuric chloride solution in a test tube and 
 just re-dissolving the red precipitate of mercuric iodide 
 formed by a slight excess of potassium iodide solution. 
 
 This test is very delicate, but peptones, mucin, vege- 
 table alkaloids also respond to it. The precipitate 
 obtained with peptones and alkaloids is dissolved by 
 the application of a gentle heat and reappears on cool- 
 ing again. 
 
 Experiment. Robert's Nitric-Magnesium test. Float 
 the urine on the surface of a solution of magnesium 
 sulphate containing nitric acid. In the presence of 
 albumin a sharply-defined zone or ring is seen between 
 the two liquids. 
 
 The solution is made by adding one ounce of strong 
 nitric acid to five ounces of a saturated solution of mag- 
 nesium sulpate. This modification is said to be more 
 delicate than Heller's test. 
 
46 LESSON VIII. 
 
 Experiment. Acidify some of the clear urine with 
 acetic acid and add a few drops of a picric acid solution. 
 A turbidity or cloudiness is seen as the picric acid dif- 
 fuses through the liquid. 
 
 Peptones, alkaloids, such as quinine, morphine, etc., 
 are precipitated by this reagent, but the precipitate 
 disappears on the application of heat and reappears on 
 cooling again. 
 
 Experiment. Make some of the urine acid with 
 acetic acid, and add a few drops of a solution of sodi- 
 um tungstate. In the presence of albumin a cloudi- 
 ness or precipitate is observed. In addition to albumin 
 this reagent precipitates peptones and mucin but not 
 the alkaloids. The precipitate obtained with peptones 
 is dissolved by gentle heat. 
 
 Quantitative Estimation. Heat 50 c.c. water contain- 
 ing 10 c.c. acetic acid to boiling, and add gradually, 
 while boiling, 100 c.c. of the clear urine, and boil for a 
 few minutes to insure complete precipitation. Filter 
 through a weighed filter, wash with hot water until free 
 from acid ; dry, and weigh. 
 
 Esbacli s Method. Fill the albuminometer tube to 
 the letter U with the clear urine, and then to the letter 
 R with the test solution. (See below.) Close the end of 
 the tube with a rubber stopper, invert several times, 
 and allow the mixture to stand twenty-four hours. 
 When the precipitate has completely settled, read off 
 on the graduation on the tube the number of grammes 
 of ser-albumin per litre of urine. The percentage is 
 derived from this by moving the decimal point one 
 figure to the left ; thus 4 grammes per litre would be 
 0.4 per cent, of albumin. If the urine contains con- 
 
URINE. 47 
 
 siderable albumin, the urine must first be diluted with 
 one or two volumes of water before testing, and the 
 result multiplied by two or three, as the volume is 
 doubled or trebled. 
 
 The test solution is prepared by dissolving 10 grammes 
 picric acid and 20 grammes citric acid in 1000 c.c. dis- 
 tilled water. In order to save time the tube may be 
 placed in a centrifugal machine, and the albumin pre- 
 cipitated in three or four minutes. 
 
 MUCIN. 
 
 Mix some saliva with some normal urine and apply 
 the following tests : 
 
 Experiment. Dilute some of this urine in a test tube 
 with water, and acidify with an excess of acetic acid. 
 A precipitate of mucin is formed. This precipitate 
 dissolves on the addition of caustic soda, and is again 
 precipitated on the addition of an excess of acetic 
 acid. 
 
 Experiment. To another portion add some acetic 
 acid, and then some potassium ferrocyanide. No 
 cloudiness or precipitate. 
 
 Experiment. Repeat Heller's test, with some of the 
 urine containing mucin. A zone or ring is obtained, 
 but it is not so sharply defined, and is not at the point 
 of contact of the two liquids. 
 
 Experiment. Treat the urine with an equal volume 
 of a saturated solution of common salt, and then add 
 a solution of tannic acid, prepared by dissolving 5 
 grammes tannic acid in a mixture of 10 c.c. 25 per cent, 
 acetic acid and 240 c.c. 40-50 per cent, alcohol. 
 
48 LESSON VIII. 
 
 In the presence of the smallest amounts of mucin a 
 precipitate is formed. 
 
 PEPTONES. 
 
 Add a little peptone to some normal jirine and use 
 this for the following test : 
 
 Experiment. Saturate some of the urine with am- 
 monium sulphate after having slightly acidified it with 
 acetic acid. This precipitates the albumin, globulin, 
 and albumoses, leaving the peptones in solution. Filter 
 and test the filtrate for peptones by the Biuret test, 
 Tanret's test, and with picric acid. 
 
Lesson IX. 
 
 CARBOHYDRATES. 
 
 Dextrose. Urine containing sugar as a rule has a high 
 specific gravity, a light color, and is large in amount, 
 sometimes three or four times the normal amount. 
 
 Before testing the urine for sugar see that it does 
 not contain any protein substances, otherwise they 
 must first be removed by coagulation with heat and 
 acetic acic and filtration. Albumin interferes with the 
 tests for sugar. 
 
 Experiment. Moore s test. Add some caustic soda 
 to some urine in a test tube, and apply heat to the 
 upper part of the liquid. In the presence of sugar the 
 urine becomes yellowish-brown in color, as seen by 
 observing the difference in the color between the 
 heated and the unheated portions. The depth of color 
 depends upon the quantity of sugar present. 
 
 The color disappears on the addition of a few drops 
 of nitric acid, and develops an odor of burnt sugar 
 (caramel). 
 
 This test is best applied to decolorized urines. 
 
 Bile pigments produce a brown color with caustic 
 soda without the application of heat. 
 
 Experiment. Trommer s test. Treat some of the urine 
 with about one fourth its volume of caustic soda, and 
 
 49 
 
50 LESSON IX. 
 
 then, while shaking, add a 10 per cent, copper sulphate 
 solution until the precipitate formed just redissolves, 
 and a deep blue solution is the result. Apply a gentle 
 heat to the upper part of the liquid and a reduction of 
 yellow hydrated cuprous oxide, and then red cuprous 
 oxide is obtained. It should not be boiled too long, 
 neither should the precipitate of earthy phosphates be 
 mistaken for a reduction of cuprous oxide, which settles 
 quickly to the bottom of the test tube. 
 
 Experiment. Fehling's test. The solution as sug- 
 gested by Fehling does not keep, hence it is advisable 
 to make two solutions and keep them separated. I. 
 Dissolve 34.65 grammes pure copper sulphate in 1000 
 c. c. water. 2. Dissolve 173 grammes Rochelle salt in 
 350 c. c. water, adding 600 c. c. of a caustic soda solu- 
 tion of a specific gravity of 1. 12 and dilute to 1000 c. c. 
 with water. For use mix equal parts of the above so- 
 lutions and dilute with an equal volume of water. 
 
 Glycerine may be used instead of the Rochelle salt. 
 
 In applying this solution to the urine first fill a test 
 tube one half full of the solution and boil for a few 
 seconds. No change or reduction should be observed 
 in the liquid. Now add the urine drop by drop and if 
 sugar is present a reduction of yellow hydrated cuprous 
 oxide and then a red cuprous oxide is seen. The test 
 should not be boiled after the addition of the urine 
 but only warmed. 
 
 If a reduction occurs in the test solution alone on 
 boiling it must be discarded. 
 
 Urines containing large amounts of uric acid, cre- 
 atinin, hippuric acid, hypoxanthin, allantoin, alkaloids, 
 glycuronic acid, etc., may give a marked reduction with 
 
URINE. 5 1 
 
 these preceding tests and may lead to error. After 
 the administration of certain drugs such as benzoic 
 acid, turpentine, chloral, glycerin, salicylic acid, etc., a 
 reducing substance called glycuronic acid is eliminated 
 in the urine, which may mislead the experimenter. 
 Urines containing an excess of ammonia, should be 
 boiled, to drive off the ammonia, before applying the 
 above tests as the ammonia has a tendency to dissolve 
 any cuprous oxide precipitated. 
 
 Experiment. Boettger s test. Make some of the 
 urine alkaline with sodium carbonate and add a little 
 solid bismuth sub-nitrate. Heat to boiling and in the 
 presence of sugar the white bismuth sub-nitrate will 
 turn gray, brown or black, depending upon the amount 
 of sugar present. The sugar reduces the bismuth sub- 
 nitrate into bismuth oxide and metalic bismuth (?). 
 
 Experiment. Nylander s modification of Boettger's 
 test. Dissolve 4 grammes Rochelle salt in a solution 
 of 10.33 grammes caustic soda in 100 c. c. water. Add 
 to this 2 grammes bismuth sub-nitrate and digest on 
 the water bath until as much of the bismuth salt has 
 dissolved as possible. 
 
 Heat 10 vols, of the urine with 1 vol. of the above 
 solution for a few minutes when a black precipitate or 
 a dark coloration is the result. 
 
 Before applying either of the two preceding tests 
 any albumin present must first be removed, otherwise 
 the sulphur of the albumin will turn the bismuth salt 
 black. 
 
 Bismuth sub-nitrate is not reduced by the substances 
 mentioned in connection with Trommer's or Fehling's 
 test. 
 
52 LESSON IX. 
 
 Experiment, v. Jakscti s test. Place 25 c. c. of the 
 urine in a small flask and add I gramme (15 grains) 
 phenyl-hydrazine hydrochloride and 0.75 grammes (10 
 grains) sodium acetate. Warm on the water bath for 
 at least one hour. Allow to cool and the presence of 
 sugar is shown by the presence of a yellowish deposit, 
 which may appear amorphous to the naked eye but 
 which, when examined under the microscope, is seen to 
 contain fine, bright yellow needle-like crystals, either 
 single or in stars. These crystals of phenyl-glucosazone 
 melt at 204 C, and are highly characteristic and cannot 
 be mistaken for any other substance. 
 
 This test does not give any reaction with uric acid, 
 creatinin hippuric acid, or any of the other reducing 
 substances mentioned above. It is one of the most 
 delicate and most reliable of all known tests for sugar 
 in the urine. 
 
 Experiment. Make some of the urine alkaline with 
 caustic soda, add some picric acid solution and heat 
 the upper part of the liquid. In the presence of sugar 
 a dark mahogany-red color will be produced. This test 
 has its interferences, namely creatinin gives a red 
 coloration with this test. 
 
 Experiment. Fermentation test. Shake a little piece 
 of yeast with some of the urine and fill this into an 
 Einhorn Saccharometer and place this in a warm lo- 
 cality (40 C.) for 12 hours. The dextrose will fer- 
 ment with the evolution of carbon dioxide, which will 
 collect in the tube of the instrument. 
 
 This test may also be performed by simply shaking 
 some yeast with four ounces of the urine in a bottle 
 which is tightly corked and allow it to stand in a warm 
 
URINE. 53 
 
 place for 12 hours. If sugar is present, on removing 
 the cork partly, it will be forcibly ejected and the car- 
 bon dioxide will escape as a froth. If much sugar is 
 present the bottle may burst. 
 
 The fermentation test is the most reliable test for 
 dextrose in the urine and is readily performed. If a 
 urine responds to the reduction tests and does not fer- 
 ment with yeast, it is safe to conclude that no dextrose 
 is present, but contains other substances which have a 
 reducing action. (Lactose gives the reduction tests but 
 does not ferment.) 
 
 Quantitative Estimation. Experiment. Robert's diff- 
 erential desity method. Take the specific gravity of 
 the urine at a known temperature, and then add a little 
 yeast and allow it to ferment in a warm place for 24 
 hours. Take the specific gravity again at the same 
 temperature as before. Each degree of specific gravity 
 lost represents 1 grain of dextrose per fluid ounce of 
 the urine, or the percentage may be ascertained by 
 multiplying each degree of specific gravity lost by 0.23. 
 In taking the specific gravity, be careful not to have 
 any gas-bubbles attach themselves to the urinometer ; 
 otherwise an incorrect reading will be taken. 
 
 Einhorns Saccharometer. Fill one of the instruments 
 with some of the urine to be tested containing a little 
 yeast, and the second instrument with normal urine 
 containing the same amount of yeast. Place the two 
 side by side in a warm place for twelve hours. Read 
 off on the instrument the percentage of sugar from the 
 volume of gas collected. The object of the second 
 instrument is to see if the yeast itself gives off any gas. 
 These results are only approximate, as no account is 
 taken of the gas dissolved by the urine itself. 
 
54 LESSON IX. 
 
 Fehlings Titration Method. Place 20 c. c. of the 
 standard Fehling's solution (prepared as above de- 
 scribed) in a flask, and dilute with two volumes water. 
 Heat to boiling and add small amounts of the urine 
 from a burette until the blue color of the solution has en- 
 tirely disappeared. After each addition of urine from 
 the burette it is advisable to boil the contents of the 
 flask and allow it to stand a few seconds so that the 
 reduced cuprous oxide may settle, and so the observer 
 may see if the blue color has entirely disappeared. 
 When the blue color has disappeared, read off the 
 number of cubic centimeters of urine used from the 
 burette. Since it requires 0.05 grammes dextrose to 
 remove the blue color (namely all the copper) from 20 
 c. c. of Fehling's solution, therefore the quantity of 
 urine used must have contained 0.05 grammes dextrose. 
 From this calculate the percentage of dextrose in the 
 urine. 
 
 This operation should be repeated to get exact re- 
 sults. If the original urine contains a large amount of 
 dextrose, then dilute it first with a known volume of 
 water. 
 
 Polariscope. Dextrose has a right-handed or dextro- 
 rotatory action on polarized light. Its specific rotatory 
 power is (a)D = + 52.6. In using this instrument for the 
 determination of the amount of sugar in a urine, first 
 decolorize the urine by shaking it with freshly burnt 
 animal charcoal, filter and place the perfectly decolor- 
 ized and clear urine in the glass tube provided for the 
 purpose. Place this later between the polarizer and 
 the analyser, and rotate the analyser until both halves 
 of the field of view are equally illuminated. Read off 
 
URINE. 5 5 
 
 on the scale by means of the vernir the percentage of 
 dextrose in the urine. 
 
 Urines may also contain : 
 
 Lactose, in the urine of women when nursing. 
 
 Inosite. 
 
 Levulose, occasionally in diabetic urines. 
 
 Maltose. 
 
 BILE. 
 
 Urine containing bile pigments has a color varying 
 from bright yellow to a greenish-brown, and when 
 shaken, readily gives a foam having a yellow color. 
 
 It is generally ropy, with a high specific gravity. 
 
 Experiment. Apply Gmelin's test to the urine as 
 described in Lesson 5, Experiment 20. The test should 
 be allowed to stand some little time so as to allow the 
 characteristic rings of color to develop. 
 
 Experiment. Apply Huppert's test, as described in 
 Lesson 5, Experiment 21, to the urine. 
 
 Experiment. Repeat Smith's test as described in 
 Lesson 5, Experiment 22. 
 
 The bile acids can only be tested for in a urine with 
 great difficulty. 
 
 BLOOD. 
 
 Blood occurs in urines in cases of hematuria where 
 we have the presence of blood corpuscles, and in cases 
 of hemoglobinuria where we find the blood pigments 
 with very few corpuscles. The presence of blood in a 
 sample of urine may be seen by its characteristic color 
 and its smoky and dichroic appearance. The presence 
 of blood corpuscles is determined by microscopic 
 examination. 
 
$6 LESSON IX. 
 
 Experiment. Test the presence of albumin in the 
 urine containing blood. 
 
 Experiment. Make some of the urine alkaline with 
 caustic soda and boil so as to precipitate the earthy- 
 phosphates. They will be colored bright red, due to 
 their retaining blood pigments. Collect this precipi- 
 tate on a filter and use it for the obtainment of Haemin 
 or Teichmann's crystals, as described in Lesson 5, 
 Experiment 16. In this case there is no need of add- 
 ing any common salt. 
 
 Experiment. Repeat Experiment 17, Lesson 5, with 
 the urine, but instead of using hydrogen peroxide, use 
 ether which has been ozonized (by shaking it with 
 hydrogen peroxide). As the ether floats on the mix- 
 ture of urine and tincture of guaiacum it forms a blue 
 ring in the presence of blood. 
 
 Experiment. Slightly acidify the urine with acetic 
 acid, filter and place it in front of the slit of the spectro- 
 scope. Two absorption bands will be seen, as described 
 in Lesson 5, Experiment 14. 
 
 PUS. 
 
 Urine containing pus corpuscles has a cloudy appear- 
 ance and is clarified by filtration with difficulty. 
 
 Microscopical examination reveals the presence of 
 pus corpuscles. 
 
 Experiment. Test for albumin in the urine contain- 
 ing pus. It will be present in large amounts. 
 
 Experiment. Allow the corpuscles to settle or facili- 
 tate by means of a centrifugal machine and treat them, 
 after decanting the urine from them, with a small piece 
 of caustic soda and stir. After a moment or two the 
 
URINE. 57 
 
 corpuscles are converted into a ropy and gelatinous 
 mass. 
 
 Experiment. Acidify the urine with acetic acid, filter 
 and treat a portion of the contents of the filter with a 
 few drops of tincture of guaiacum (which has been kept 
 in the dark), when in the presence of pus the filter 
 paper is colored a deep blue. 
 
 Experiment. Place some of the contents of the filter 
 obtained above on a slide and treat it with a drop of a 
 solution of iodine in water containing some potassium 
 iodide. In the presence of pus a dark, mahogany- 
 brown color, due to the glycogen contained therein, is 
 the result. Epithelium cells do not give this color. 
 
 UROBILIN. 
 
 This is the normal coloring matter of the urine. It 
 is markedly increased in fevers. 
 
 Experiment. Make some of the urine alkaline with am- 
 monium hydrate, and filter off the precipitate of earthy 
 phosphates. To the filtrate add a few drops of a solu- 
 tion of zinc chloride, which produces a beautiful green- 
 ish fluorescence by reflected light. The above solution 
 exhibits, with the spectroscope, a dark absorption band 
 between the Fraunhofer's lines b and F. 
 
 INDICAN. 
 
 This substance is derived from the indol, produced 
 in the intestine by putrefactive processes, which is 
 oxidized into indoxyl in the blood ; this, entering in 
 combination with sulphuric acid, is eliminated in the 
 urine as sodium or potassium indoxyl sulphate or indi- 
 can, (KO.C 8 H 8 N.O.S0 8 ). 
 
58 LESSON IX. 
 
 Experiment. Treat a small amount of the urine in a 
 test tube with an equal volume of hydrochloric acid 
 and two or three drops sodium hypochlorite solution, 
 and then a small amount of chloroform. Shake and 
 put aside for a little while. The indigo set free from 
 the indican is taken up by the chloroform, coloring it 
 blue to a greater or less extent, depending upon the 
 amount of indican present in the urine. 
 
 Experiment. Boil equal volumes of the urine and 
 hydrochloric acid, containing a few drops nitric acid ; 
 allow to cool and shake with a little chloroform. The 
 chloroform is colored violet, due to the setting free of 
 indigo blue and indigo red from the indican. 
 
Lesson X. 
 
 SEDIMENTS (Chemical or unorganized). 
 
 From acid urines. From neutral or slightly alkaline 
 
 (fixed) urines. 
 Uric acid. Sodium urate. 
 
 Sodium urate. Calcium oxalate. 
 
 Calcium oxalate. Calcium carbonate. 
 
 Acid calcium carbonate. Di-calcium phosphate. 
 
 Tri-calcium phosphate. 
 Ammonium-magnesium phos- 
 phate. 
 From ammoniacal urines. 
 Ammonium urate. 
 Ammonium-magnesium phosphate. 
 Tri-calcium phosphate. 
 Calcium carbonate. 
 
 In testing the sediments the urine should be allowed 
 to stand in a conical vessel in a cool place until all the 
 sediment has settled to the bottom. It may be col- 
 lected by decantation, or better, by means of a pipette. 
 Hold the first finger over the end of the pipette and 
 introduce it to the bottom of the vessel into the sedi- 
 ment. Now remove the finger slowly and the sediment 
 will rise in the tube. Close the end of the tube tightly 
 again with the finger and remove it from the vessel. 
 Allow the contents of the pipette to flow on a few 
 
 59 
 
60 LESSON X. 
 
 pieces of filter paper, which absorbs the urine, leaving 
 the sediment on the paper, which may be removed by 
 the aid of a knife or spatula, and tested as described 
 below. 
 
 The sediment may also be obtained readily by means 
 of a centrifugal machine. 
 
 URIC ACID, C 5 H 4 N 4 3 . 
 
 Generally colored red. 
 
 Experiment. Heat some on a piece of platinum foil. 
 It blackens and burns up entirely, leaving no residue. 
 
 Experiment. Place a little in water in a test tube. 
 It does not dissolve. When a drop or two of sodium 
 carbonate is added it dissolves readily. 
 
 Experiment. Apply the Murexid test to a portion 
 of the sediment. A purple coloration shows the pres- 
 ence of uric acid. (See uric acid.) 
 
 Experiment. Repeat Schiff's test with the solution 
 obtained above. A black reduction denotes the pres- 
 ence of uric acid. (See uric acid.) 
 
 SODIUM URATE, Na 3 C 5 H 3 N 4 3 . 
 
 Experiment. Heat a little on the foil. It blackens 
 and burns with a yellow color to the flame, if looked at 
 through a piece of blue glass. It does not burn up en- 
 tirely, but leaves a residue, which if placed on a piece 
 of moistened red litmus paper turns it blue, due to the 
 sodium carbonate in the residue. 
 
 Experiment. Place a little in water. It does not 
 dissolve, but dissolves readily when a drop of sodium 
 carbonate solution is added. 
 
 Experiment. Repeat the Murexid test with some of 
 the sediment. 
 
SEDIMENTS. 6 1 
 
 Experiment. Repeat Schiff' s test with the solution 
 of the sediment obtained above. A black reduction is 
 the result. 
 
 CALCIUM OXALATE, CaC 2 4 . 
 
 Heat some on the foil. It blackens slightly but 
 does not burn or disappear. Place this residue in a 
 watch glass and add a little water and a drop of acetic 
 acid. It effervesces, due to the discharge of carbon 
 dioxide, and dissolves. Retain this solution. 
 
 The application of heat to the sediment has con- 
 verted the calcium oxalate into calcium carbonate, 
 which effervesces with an acid. CaC 3 4 = CaC0 3 + CO. 
 
 Experiment. Heat another portion of the sediment 
 for a few minutes in the flame. Place a particle on a 
 piece of moistened red litmus paper. It turns it blue, 
 showing that calcium oxide has been formed by the 
 protracted heating of the calcium oxalate. CaC 2 4 = 
 CaO + CO + C0 3 . 
 
 Place the remainder of the residue in some water in 
 a test tube or watch glass. It dissolves, especially on 
 the addition of a drop of acetic acid. 
 
 Experiment. Add some ammonium oxalate to some 
 of the solution obtained above. A white precipitate 
 of calcium oxalate shows the presence of calcium. 
 
 Calcium oxalate does not give the Murexid test. 
 
 ACID CALCIUM PHOSPHATE, Ca (H 2 P0 4 ) 3 . 
 
 Experiment. Heat some on the foil. It does not 
 blacken nor does it burn up. No apparent change. 
 
 Experiment. Place some in a test tube with water. 
 Add some acetic acid and apply heat if necessary. It 
 
62 LESSON X. 
 
 dissolves. Divide the solution in two portions. To 
 one portion add a solution of ammonium oxalate. A 
 white precipitate of calcium oxalate shows the presence 
 of calcium. 
 
 To the other portion add a solution of uranium 
 acetate. A white precipitate of uranium phosphate 
 denotes the presence of phosphoric acid. 
 
 Acid calcium phosphate does not give the Murexid 
 test. 
 
 Di-calcium phosphate, CaHP0 4 . 
 
 Tri-calcium phosphate, Ca 3 (P0 4 ) 3 . 
 
 Occurring in neutral or alkaline urines are tested for 
 as described under acid calcium phosphate. Their mi- 
 croscopic appearances are different. 
 
 CALCIUM CARBONATE, CaC0 3 . 
 
 Experiment. Heat some on the foil. It does not 
 blacken nor burn up. No apparent change. Place a 
 particle of the heated residue on a piece of moistened 
 red litmus paper. It turns blue, showing the presence 
 of calcium oxide, formed by heating the carbonate. 
 CaCO s = CaO + CO s . 
 
 Experiment. Place some of the sediment in a test 
 tube with water. It does not dissolve, but readily dis- 
 solves on the addition of a drop or two of acetic acid 
 with an effervescence showing that it is a carbonate by 
 the escape of carbon dioxide gas. 
 
 Experiment. Add some ammonium oxalate to the 
 solution obtained in the previous experiment. A white 
 precipitate of calcium oxalate shows the presence of 
 calcium. 
 
 This sediment does not give the Murexid test. 
 
SEDIMENTS. 63 
 
 AMMONIUM-MAGNESIUM PHOSPHATE OR TRIPLE PHOS- 
 PHATE, NH 4 , MG, P0 4 . 
 
 Heat on the piece of foil. It does not blacken, but 
 gives off an odor of ammonia, which gives dense white 
 fumes with a drop of hydrochloric acid on a glass rod. 
 
 A residue of magnesium pyro-phosphate is left on 
 the foil. 
 
 Experiment. Place some of the sediment in a test 
 tube and add some caustic soda. A marked odor of 
 ammonia is evolved, and a piece of moistened red lit- 
 mus paper is turned blue by the vapors, if held at the 
 mouth of the test tube. 
 
 Experiment. Place some of the sediment with water 
 in a test tube and add a few drops acetic acid. It dis- 
 solves without effervescence. 
 
 Experiment. Add some uranium acetate to some of 
 the above solution. A white precipitate of uranium 
 phosphate denotes the presence of phosphoric acid. 
 
 It does not respond to the Murexid test. 
 
 AMMONIUM URATE, (N 2 H 4 )C 5 H 3 N 4 3 . 
 
 Experiment. Heat some on the foil. It blackens 
 and burns up, leaving no residue. 
 
 Experiment. Place some of the sediment on a watch 
 glass and add a few drops caustic soda. Apply a 
 gentle heat to the contents of the watch glass, and 
 hold a piece of moistened red litmus directly over the 
 watch glass. It turns blue, showing that ammonia has 
 been evolved by the action of the caustic soda on the 
 ammonium urate. 
 
 Experiment. Place some of the sediment in water in 
 
64 LESSON X. 
 
 a test tube. It does not dissolve, but readily dissolves 
 on the addition of a few drops sodium carbonate. 
 
 Experiment. Apply Schiff's test to the above solu- 
 tion. A black reduction shows the presence of uric 
 acid. 
 
 Experiment. Apply the Murexid test to some of the 
 sediment on a watch glass. A purple coloration de- 
 notes the presence of uric acid. 
 
 Cystin, leucin, tyrosin, and xanthin have been ob- 
 served in sediments in very rare cases. 
 
 CALCULI. 
 
 Calculi may consist of only one ingredient, but more 
 frequently two or more deposits occur in separate 
 layers. Most calculi have a nucleus generally of uric 
 acid. 
 
 In examining a calculus, it should be wrapped up in 
 a piece of writing paper and cut through the centre by 
 means of a fine saw. Each layer should be examined 
 separately by the following scheme : 
 
ON HEATING THE POWDER ON PLATINUM FOIL. 
 
 Does not burn. 
 
 The powder when treated 
 with H CI. 
 
 Does burn. 
 
 Does not effervesce. 
 
 The gently heated 
 powder with H CI. 
 
 The powder when £ 
 
 moistened with a a 
 
 little NaOH. < 
 
 -. > 
 
 = 3 
 :-- E 
 
 p ■ 
 n 
 
 s - 
 
 p ? Z 
 
 - (I z 
 
 -2 - Z 
 
 -: -: 
 
 "I 
 
 .— p 
 
 o p 
 
 po n 
 
 5 » 
 
 n 
 
 - z 
 
 With flame. 
 
 Without flame. 
 
 X -r 
 
 — c — 
 
 S 3 
 
 — a 
 
 — = 
 
 M 9 Z 
 
 ?r ft 
 
 U r- ;■ 
 
 
 
 
 z2 
 
 r~ 
 
 p' 
 
 
 
 
 ri 
 
 Q 
 
 
 "- 1 r* 
 
 
 
 
 
 5, p_ 
 
 fl — 
 
 g 3'^ 
 
 1 = § 
 
 2 E r 
 
 p — 
 EL S 
 
 ° 
 
 p — 
 
 3* £. 
 
 — » o 
 
 p CL 
 
 a z. 
 
 
 — ZZ. < 
 
 1 1 o 
 
 
 g "o 
 
 
 2nd 
 
 
 a 2 M 
 
 
 3 o B 
 
 
 tfq rt o 
 
 
 
 
 W 
 
 
 
 
 
 
 ~ re 
 
 
 o 
 
 
 
 
 
 
 
 
 ~ re 
 
 
 
 n -> 
 
 
 
 
 
 
 
 
 
 
 Is 
 
 
 
 
 c — 
 
 
 
 
 ft 
 
 
 
 
 nj r* 
 
 
 i. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 . c 
 
 
 
 
 
 
 
 
 
 
 
 
 a < 
 
 
 a a. 
 
 
 S a- 
 
 
 fi 5)' 
 
 
 a c/: 
 
 
 tn c 
 
 
 « 5- 
 
 
 «i ft 
 
 
 < X 
 
 
 
 
 
 
 ~~ 
 
 
 EL~ 
 
 
 ?r —' 
 
 
 ELZ 
 
 
 
 
 " O 
 
 
 
 
 a - 
 
 
 95 <? 
 c 3 
 
 
 
 
 
 
 ^ a - 
 
 
 
 
 
 
 
 
 a - 
 
 
 
 
 
 
 ~ 35 
 
 
 o 2 
 
 
 
 
 ~ ' 
 
 
 The powder 
 gives the Mu- 
 rexid test. 
 
 The powder, 
 when treated 
 with NaOH, 
 gives 
 
 'f o 
 
 =•5 S - 
 
 ^ 3 °*» 
 
 — ~ ' p 3 a 
 
 - 
 
 n 
 
 T, 
 
 £ 
 
 r> 
 
 X 
 
 E£L 
 
 p 
 
 C 
 
 c 
 en 
 
 •«< 
 
 » 
 
 o 
 
 
 ^3 
 
 
 3- 
 
 = 
 
 c 
 
 3 
 
 
 a 
 
 =r 
 
 a 1 
 
 3 
 
 
 p_ 
 
 
 3 
 
 
 
 
 
 
 
 
 - 
 
 
 
 
 
 P 
 
 - 
 
 
 
 
 
 - 
 
 
 
 
 
 
 
 s 
 
 a 
 
 p 
 
 5 
 
 
 
 
 
QP519 M3 i2 
 
 Man del 
 
 Manual for the physiological 
 
 chemical laboratory