W/9 Cornell University Library QP 519.W19 "'■^ Exercises in practical phys'^^^^^^ CORNELL UNIVERSITY. THE IKosmen p. Wlnm^v Xibrairg THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897 EXERCISES IN PRACTICAL PHYSIOLOGY AUGHSTCS D. WALLER, M.D., F.E.S. PART I. ELEMENTARY PHYSIOLOGICAL CHEMISTRY Bit AUGUSTUS D. WALLER AND W. LEGGE SYMES. LONGMANS, GEBEN, AND Co. LONDON, NEW YOBK, AND BOMBAY 1897 « CORNELL UNIVERSITY. THE THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897 ELEMENTARY PHYSIOLOGICAL CHEMISTRY. Examination op the Mobe Important Peoximate Principles Proteids. Albuminoid. Pats. Carbohydrates. Examination op the More Important Articles op Diet . . Bread. Potato. Butter. Commercial Peptone. Beef Tea. Examination op the More Important Tissues and Fluids op the Body . . . . . . . . . . . . . . 10 Saline Extract of Liver. Mineral Matter of Bone. Saline Extract of Brain. Ethereal Extract of Brain. Blood. Blood Serum. Saline Extract of Muscle. Milk. Urine. Bile. Saliva. Gastric Juice. Pancreatic Juice. Intestinal Juice. General Hints por the Qualitative Examination op Simple Solids and Fluids, containing substances op Physiological Interest . . . . . . . . . . . . . . 21 Quantitative Estimation op the pollowing Bodies in Urine . . 22 Sodium Chloride. Phosphoric Acid. Sugar. Urea. ^ jot GENEEAL DIEECTI0N8. Ascertain the reaction of every fluid that you test, by dipping strips of litmus paper in it. Acid fluids make neutral, or blue litmus paper red. Alkaline fluids make neutral, or red litmus paper blue. To neutralise a fluid, — add a -1 per cent, solution of caustic soda, or a -1 per cent, solution of hydrochloric acid, until it does not change the colour of neutral litmus paper. The specific gravity of a fluid is measured by immersing a hydrometer in it, and noting to what degree the instrument sinks. Unless otherwise directed, you are to use fresh material for each reaction. EXAMINATION OP THE MOEE IMPOETANT PROXIMATE PEINCIPLES. PBOTEIDS. Genebal Reactions. Directions. Result. 1 Buru a little dry proteid with soda-lime in a It becomes charred, and ^P°°'^ smells of ammonia. 2Vje charring proms tlie presence of carbon, the sviell shotvs that nitro- gen is present. '2 Xanthoproteic reaction. Add nitric acid to a proteid solution and "°il ■ ■ • • ■ • . . . . . . A yellow colouration or precipitate appears, then cool, and add ammonia . . . . The colour deepens to orange. 3 Biuret reactimi.^ Add one drop of copper sulphate solution, and one volume- of strong caustic soda solution . . .,. . . . . . . A violet or pink colour results. i Add ,T volume of Millon's re-agent . . A precipitate is formed. then boil the solution The precipitate becomes reddish. 5, Add \ volume of potassium ferrooyatiide solution, and excess (one volume or more) of acetic acid . . . . . . . . A precipitate appears. Peptones and some albumoses do not give this reaction. 6 Saturate" with sulphate of ammonium . . A precipitate forms. Peptmies are not precipitated. 1 Properly speaking, the term "biuret reaction" is applicable only to the pink colour obtained witti digested proteids, and with biuret itself. 2 " One volume " means an equal bulk. 3 You will not really saturate the fluid. To do so would require prolonged agitation. It will be enough for you to shake the solution for five minutes with half its bulk of crystals. You may see the gradual formation of precipitate by pouring fluid on to a layer of crystals and leaving it undisturbed in a test tube. As the salt dissolves a turbid ring forms at the junction of fluid and crystals. This applies also to the globulin test by magnesium sulphate. Special Eeactions. Directions. Mesult. Native albumins {solutions of egg albumin, and of serum albumin free from globulins). 7 Faintly acidify and boil 8 Saturate with sulphate of magnesium . 9 Acidify, add one volume of ether, and shake The albumin is coagulated. The albumin is not pre- cipitated. ,' Egg albumin is usually ] coagulated. I Serum albumin is not \ usually coagulated. Globulins (sohdion of serum globulin, or of muscle globulin). 10 Faintly acidify, and boil . . . . . . The globulin is coagulated. 11 Saturate with sulphate of magnesium . . The globulin is precipi- tated. 12 Let a few drops fall into distilled water . . A cloud, i.e., a faint precipi- tate of globulin appears. Derived albumins (prepared by treating native albumins, or globulins with dilute acid or alkali) . 13 Take reaction 14 Boil 15 Cautiously neutralise, shaking after each addition of neutralising re-agent, then add slight excess of the neutralising re-agent Albumoses— 16 Faintly acidify, and boil . . 17 Apply biuret test 18 Saturate with sulphate of magnesium . . 19 Cautiously add nitric acid, or salioyl-sul- phonic acid, and shake . . then heat the test tube and then cool the test tube under the tap Peptones— 20 Faintly acidify, and boil 21 Apply the biuret test 22 Add nitric acid It is acid, or alkaline, not neutral. Derived albumins are not coagulated. Derived albumins are pre- cipitated. The precipitate re-dis- solves. Albumoses are not coagu- lated. A pink colour results. Some albumoses (primary albumoses) are precipita- ted. A precipitate forms. The precipitate dissolves. The precipitate re-appears. Peptones are not coagu- lated. A pink colour results. Peptones are not precipi- tated. COJIPOUND PROTEIDS Directions. Mucin (« compound ofproteid and sugar). 23 Boil 24 Apply xanthoproteic test 25 Add acetic acid Besulf. Mucin is not coagulated. Yellow colour results. IMucin is precipitated, and does not re-dissolve in excess of the acid. Nucleo-albumins {compounds ofproteid with miclein). 26 Apply the xanthoproteic test . . . . An orange colour results. 27 Add acetic acid Nucleo-albumins are pre- , cipitated. 28 Saturate with magnesium sulphate . . Nucleo-albumins are pre- cipitated. Nucleo-albumins react very much like globulins, and like mucin. OxyhSBmOglobin (^^ compound of proteid with hcematin, and oxygen. 29 Boil 30 Dilute and examine with a spectroscope . . 31 Add sulphide of ammonia to remove the oxygen, and then examine as in 30 32 Add tincture of guaiacum, and ozonic ether The proteid is coagulated, and the hasmatin (brown- ish pigmenb) liberated. Two'dark absorption bands are seen between the yellow and green of the spectrum. The two bauds vanish, and a single, broader, fainter band appears in their place. The oxyhasmo- globin is reduced to haemoglobin. A blue colour is developed. ALBUMINOID. Gelatin— 33 Warm the jelly then cool the fluid under the tap . . 34 Apply the biuret test 35 Add nitric acid then complete the xanthoproteic reaction 36 Apply reaction No. 5 It becomes fluid. It gelatinises. A violet colour results. Gelatin is not precipitated. A yellow colour results. Gelatin is not precipitated. If you do not dilute sntliciently, you may see a single very distinct broad absorption band. You must not confuse this with the single faint band of reduced hieinoglobin. SUMMABY OF THE CHIEF REACTIONS OF THE PROTEIDS. Coagulate on_ boiling 'Egg albumin . . . Serum albumin. Globulins /Derived albumins. Albumoses Do not coagu-j late on boiling^ Peptones . Coagulated by ether, not precipitated by sulphate of magnesium. Not coagulated by ether, not precipitated by sulphate of magnesium. Precipitated by saturation with sulphate of magnesium. Violet biuret reaction. Precipitated on neutralisation. Pink biuret reaction. Precipitated by nitric acid and by salicyl-sulphonic acid, the precipitate disappearing with heat, and re-appearing on cooling the solution. Precipitated on saturation with sulphate of ammonium.' Pink biuret re-action in filtrate after saturation with sulphate of ammo- nium. FAT. Directions, 37 Shake up some rancid oil {i.e., liquid fat plus fatty acid) with water then add some alkali and shake again 38 Shake up one volume of oil with four or five volumes of ether then pour a little of the ethereal solution on a piece of paper 39 Take one or two drops of emulsified oil, and boil with alkali Result. The fat is neither dissolved nor emulsified. The fat is not dissolved, but is emulsified. The fat dissolves. The ether evaporates leav- ing a permanent greasy stain. The fat dissolves, yielding glycerine, and forming soap. 1 Subject to tlie qualifications adverted to in Part IT, CARBO-HYDRATES. DirecPkm?. Starch— 40 Acidify and add iodine solution . . then heat the blue fluid and then cool the colourless solution Glycogen— 41 Acidify and add iodine solution . . then heat the brown fluid . . and then cool the colourless solution 42 Add two or three volumes of alcohol Dextrin - 43 Acidify and add iodine solution . . then heat the brown fluid . . and then cool the colourless solution Dextrose— 44 Trommer's test. To some caustic soda in a test tube, add one or two drops of copper sulphate . . then add a little solution of dextrose and then boil . . Result. A blue colour results. The colour disappears. The colour comes back. Reddish brown colour. The colour vanishes. The colour often reappears. Glycogen is precipitated. Reddish brown colour. The colour vanishes. The colour may reappear. 45 Moore's test. Add one volume of caustic soda solution and boil A bluish precipitate (cupric hydrate) forms. The precipitate dissolves, giving a blue solution. The ouprio hydrate is re- duced giving a, red pre- cipitate of cuprous oxide. A yellow, or brownish col- our (caramel) results, ac- cording to the amount of sugar present. Moore's test is onVy to be used as a rough test for dextrose in the presence of proteids. Lactose— 46 Apply Trommer's test Saccharose— 47 Apply Trommer's test 48 Boil with a few drops of dilute hydrochloric acid, to convert the saccharose into re- ducing sugar, and apply Trommer's test. The cuprio hydrate is reduced as by dextrose. The cuprio hydrate is not reduced, and so no red precipitate is formed. A red precipitate results, as with dextrose. BXAiVlINATION OF THE MOEE IMPOETANT AETICLBS OF DIET. Directions. 49 Pour a drop of iodine solution on bread 50 Apply the xanthopro- teic reaction to another piece BREAD. Result. A blue colour is developed An orange colour results Inference. Starch is present. Proteid is present. POTATO. 51 Boil a bit of potato in A blue colour ap- Starch is present. water, cool, and add pears some iodine solution Ymt could not detect the starch without boiling are enclosed in a coating of cellulose. 52 Apply the xanthopro- Only a faint orange Very little teio test colour appears present. because the granules proteid is 53 Shake up a little butter with some ether. BUTTER. It dissolves, leav- ing little or no residue It is composed almost entirely of fat. COMMERCIAL "PEPTONE.^ 54 To a little solution of commercial peptone, apply the biuret test 55 Saturate some of the solution with am- monia sulphate. 56 Remove the precipi- tated albumose by filtration and apply the biuret test to the filtrate.' Pink colour re- The only proteids that can sults be present are peptones and albumoses. A copious precipi- Much albumose is present. tate falls A faint pink If so a little peptone is colour may de- present. velop T 1 Great excess (4 or 6 volumes or more) of oaust.ic soda is nen.essar)'. BEEP TEA (HOT) MADE BY BOILING MEAT ATSID BONES. Directions. 57 Cool some under the tap 58 Apply the xantho- proteic test 59 Shake a little with ether and pour the ether ou some paper. 60 Add a, little sodium nitro-prusside and caustic soda then boil and while boiling add acetic acid Result. It gelatinises . . A faint yellow colour results, not usually orange A slight greasy stain may re- main A red colour ap- pears The colour fades A blue or green colour results Inference. Gelatin is present. Little, if any, proteid is present. If so a little fat is present. Greatiuin is present. BEEF TEA MADE WITH EXTRACT OP MEAT' ('LIEBIG' 61 Apply the xauthopro- teic test 62 Shake a little with ether, and pour the ether on a filter paper 63 Add some iodine solu- tion 6i To another portion add sodic nitro-prusside and caustic soda then boil and while boiling add acetic acid A faint yellow colour appears No greasy stain results A faint . brown colour may appear A red colour develops The colour fades A blue or green ' colour appears Little, if any, proteid is present. Pat is absent. If so, a little glycogen or dextrin''' is present. Creatinin is present. 1 This fluid has been decolourised by shaking it with animal charcoal and filtering. 3 The body is present in such small quantity that you cannot tell whether it is Rlycogen or dextrin. You would expect to find the former rather than the latter. 10 EXAMINATION OF THE MOEE IMPORTANT TISSUES and" FLUIDS OF THE BODY. SALINE EXTEAOT OF LIVER, i.e.. Directions. 65 Apply the xanthopro- teic test 66 Faintly acidify, and boil 67 Saturate with sulphate of magnesium 68 Filter the mixture ob- tained in test 65 and boil / 69 Dissolve bone ash in dilute nitric acid, warming, to accel- erate solution 70 To the solution add ammonic molybdate, and boil Result. Deep orange colour Coagulation oc- curs Precipitation' oc- curs Little or no co- agulation oc- curs BONE ASH. Effervescence oc- curs, the gas being colourless A copious crystal- line yellow pre- cipitate falls OF EPITHELIAL CELLS. Inference. Proteid is present. This proteid is either na- tive albumin, or globu- lin, or both. Some of the proteid may be globulin. Little or no albumin is present. Some carbonate is present. Much phosphate is present. SALINE EXTRACT OF NERVOUS TISSUE. colour Proteid is present. 71 To a saline extract of brain, apply the xanthoproteic test 72 Faintly acidify another portion, and boil Orange results The proteid is The proteid is native albu- coagulated min or globulin or both. The solids of a saline extract of brain are derived chiefly from the grey matter. ETHEREAL EXTRACT OF NERVOUS TISSUE. Fat is present.^ 73 Pour a little of an ethereal extract of brain on afilterpaper 74 To another portion add cautiously some strong sulphuric acid A permanent greasy stain re- sults A cherry red colour results at the junction of the two fluids Cholesterin is present. The solids of an ethereal extract of brain are derived chiefly from the white matter. ' » 5 1 The ijreoipitatiou is not entirely due to globulin, tat in part to nueleo-albumin. ^ Remember tbat this is not wholly onlinavy fat, but largely a nitrogenous, phosphorised fat, — lecithin. * . nii' 11 BLOOD (DEFIBRIBTATED). Directions. Observe the specific gravity of defibriu- ated blood 75 Dip a piece of neutral glazed litmus paper in the blood, and wash away the blood-stain. 76 Dilute freely, add a little freshly made tincture of guaiaoum, and ozonic ether 77 Dilute freely^ and examine with the spectroscope 78 Add sulphide of am- monium (a reducing agent) and warm gently 79 Pour the fluid back- wards and forwards in two test tubes 80 Dilute and examine with the speotro- scope,blood saturated with coal gas 81 Add sulphide of am- monium, and warm gently 82 Dry a little blood on a slide, add glacial acetic acid, cover, heat until bubbles appear, and examine with microscope BesjiU. It is between 1050 and 1060. The paper turned blue Inference. is Blood is alkaline. A blue colour is formed. Two dark absorp- tion bands are seen between the yellow and green of the spectrum The two bauds vanish and a, single, broader, fainter bandap- pears in their place The two bands temporarily re- appear Two bands like those seen in No. 77 appear The spectrum is not altered Nut-brown cry- stals of hsemin Haemoglobin' is present. Oxy-haemoglobin was pre- y sent and has been "deoxygenated. The hsemoglobin has been temporarily re-oxygen- ated. Carboxy-haemoglobin is present. Haemoglobin is present. I This is notia dis^notive test for hssmoglotin. - If you do not dilute suWiciently, you may see a single very distineli broad absorption band. Yuu must not confuse this with the single faint band of reduced haimoglobin. « * 12 Directions. 83 Place blood-ash in a test tube, dissolve it in hydro-ohlorio acid, and add f errooyauide of potassitun BLOOD ASH. Besiilt. A blue colour forms Inference. Iron is present in blood. BLOOD SERUM (DILUTED). 84 Apply the xanthopro- teic test to soma of the fluid 85 Boil another portion . . 86 Saturate with sulphate of magnesium 87 Remove the precipita- ted globulin by filtra- tion, acidify the fil- trate and boil Orange colour re- sults Coagulation takes place , A precipitate falls Coagulation oc- curs Proteid is present. The proteid is native al- bumin, or globulin, or both. Globulin is present (" se- rum globulin"). Native albumin is present (" serum albumin "). SALINE EXTRACT OP MUSCLE. 88 Apply the xanthopro- teic test to one portion 89 Boil another portion . . 90 Saturate with sulphate of magnesium 91 Remove the precipita- ted globulin by fil- tration, acidify the filtrate and boil An orange colour results Coagulation oc- curs A precipitate falls Coagulation oc- curs Proteid is present. The proteid is native albu- min, globulin, or both. Globulin is present (chiefly myosin). A native albumin is pre- sent (muscle-albumin). 13 Directions. 92 Observe specific gravity 93 Dip neutral litmus paper in the milk MILK. Result. It is between 1025 and 1035 It should become blue Inference. Normal milk is alkaline. London Milk is frequently acid. 94 Dilute a little milk 3 or 4 times, add a few drops of dilute acetic aeid, and warm Filter ofi the precipitate and put it aside. 95 To some of the filtrate obtained in No. 94 apply Trommer's re- action 96 To another portion of the filtrate obtained in No. 94 apply the xanthoproteic re- action 97 Take the precipitate obtained in No. 94 and wash it vrith ether. Pour a little of the ethereal solution on a piece of paper 98 Dissolve the residue left from No. 97, in dilute alkali and apply the xantho- proteic test 99 To some warmed milk add a little rennet and set aside for 5 or 10 minutes A precipitate' falls. A red precipitate A reducing sugar is pre- is formed sent (lactose). Orange colour re- Proteid is present (milk suits albumin). Permanent greasy Fat is present, stain Orange colour re- Proteid is present (casein- suits ogen). The milk clots' and forms " curd and whey " 1 The precipitate obtained by acid and the clot obtained by rennet areditTerent bodies. The acid precipitate is " caseinogeu " and is freely soluble in dilute alkali ; the rennet clot is " casein " and is much less soluble in dilute alkali. Cheese is made with rennet and cannot be made with acid. Caseinogen is a nucleo-albumin. 14 Directions. Observe the specific gravity 100 Dip a neutral litmus paper into some nor- mal urine 101 Do the same with some stale urine 102 Warm the paper used in No. 101 103 Add a few drops of silver nitrate solu- tion and excess of nitric acid 104 Add a few drops of barium chloride so- lution and excess of hydrochloric acid 105 Add nitric acid and boil, then add ammo- nium molybdate so- lution and boil again 106 Add solution of sodium hypobromite 107 Remove the sulphates and phosphates of urine by adding | volume of baryta mixture, and filter- ing. To a portion of the filtrate add some mercuric ni- trate solution 108 To another portion of the filtrate obtained in No. 107, add some sodium chloride and then merouiic ni- trate as before UBINE. Result. It is between 1015 and 1025. It is turned red . . It is turned blue. The blue fades A precipitate forms A precipitate forms A yellow precipi- tate forms Bubbles of gas (nitrogen) are evolved A precipitate forms (a com- pound of urea and mercury) No precipitate forms until ex- cess of mercuric nitrate has been added Inference. Normal urine is acid. Stale urine is alkaline. The alkalinity of stale urine is not due to fixed alkali, but to a volatile alkali (ammonium car- bonate). Chlorides are present. Sulphates are present. Phosphates are present. A nitrogenous body is present. Urea is present. The presence of sodium chloride hinders the pre- cipitation of urea. 15 Directions. 109 Add a little sodium nitro-prusside and caustic soda then boil and -while boiling add acetic acid 110 Murexide test. — Take some deposit ob- tained from a high- ly coloured, acid urine, and evaporate to dryness' with a few drops of yellow nitric acid Then add a little dilute ammonia 111 Schiff'stest. — Let a drop of an allcaliue solu- tion of uric acid fall on a filter paper, and then let a drop of sil- ver nitrate solution fall on the paper near the other, so that they may touch 112 Apply Trommer's test to the alkaline solu- tion of uric acid, not to urine. Result. A red colour de- velops. The colour fades. Colour changes to blue As it dries it be- comes reddish. The colour changes to vio- let A dark line forms at the junction of the drops Inference. Creatinin is present. Uric acid is present. Uric acid is a reducing agent. A red precipitate may be formed Uric acid is a reducing agent. (This is a possi- ble fallacy in testin urine for sugar.) 1 Great care must be taken not to overheat the residue. 16 Directions. Observe specific gravity 113 Dip a neutral litmus paper in bile 114 Add acetic acid 115 Pettenkofer's test. — Sbake up a little bile with a grain^ of cane sugar, and then add some strong sulphu- ric acid or a solution of furfural- may be substituted for the sugar 116 Gmelin's test. — Smear bile evenly over a white tile and let a drop of yellow nitric acid fall into the film 117 To a solution of white gall stones in chloro- form add some strong sulphuric acid BILE. Result. Inference. It is between 1020 and 1030 It becomes blue . . Bile is alkaline. A precipitate falls Mucin' is. present. The froth and Bile-salts are present, fluid below it are coloured purple Concentric rings Bile pigments are present, of colour appear round the drop Cherry red colour Cholesterin is present, appears 1 This is not wholly mucin but in part iiucloo-albumin. '- The purple colour t^in^s to become blaok if too much .sugar or furfui'al arc used. 17 Directions. 118 Take two test tubes A and B, and label them with your name. In A put starch solution and saliva, and in B starch solution and boiled saliva. Place both in the water bath for ten m.inutes. Meanwhile test saliva as fol- lows : - 119 Addexcess of acetic acid 120 Apply the xanthopro- teic test 121 Add silver nitrate and nitric acid 122 Add ferric chloride . . 123 Divide the contents of test tube A into two parts, acidify one part and add iodine solution 124 To the other part apply Trommer's test 125 Test some of the con- tents of tube B as in No. 123 126 To the remainder apply Trommer's test SALIVA. Result. Inference. A precipitate falls An orange colour appears A precipitate falls A red colour ap- pears A somewhat pur- ple colour ap- pears Or a reddish brown colour develops Or the solution remains colour- less A red precipitate falls A blue colour ap- pears No red precipitate' falls Mucin is present. Proteid is present. Chlorides' are present. A thiocyanate is present.' Starch and dextrin are present (reddish brown, plus blue = purple). In this case erythrodextrin and no starch is present. In this case neither ery- throdextrin nor starch is present. Sugar is present (chiefly maltose). Starch is unaltered boiled saliva by 1 Silver nitrate precipitates both mucin and albumin, aud so these bodies ought, properly speaking, to be removed before testing for chlorides, but the precipitate here is too copious to be entirely due to mucin and albumin. 2 Assuming the absence of acetate and meconate. 3 A small precipitate may fall owing to presence of sugar in your saliva. 18 GASTRIC JUICE Vessels are placed on the tables labelled products of gastric digestion. Directions. 127 Take two test tubes G and D and place apel- let of fibrin in each. To C add dilute gas- tric glycerin plus an equal volume of '3 per cent, hydrochloric acid, and to D add only -15 per cent, hydrochloric acid. Put them in the water bath and leave for 30 minutes Meanwhile examine the fluids X and Y. In X albumin has been di- gested with gastric juice, for a short time, in Y for a longer time. 128 Neutralise a little of the fluid X X and Y and containing Inference. 129 Apply the biuret test to a little of the fluid Y 130 Neutralise,and saturate another portion with ammonic sulphate 131 Eemove the precipitat- ed albumose by fil- tration and apply biuret test to the filtrate ' 132 Examine the tubes C and D . The fibrin in both is swollen. 133 Filter their contents and apply biuret test to both filtrates A precipitate falls on neutralisa- tion A pink colour results A precipitate falls Acid albumin is present. The only proteids present are albumose and pep- tone. Albumose is present. A pink results colour Peptone is present. A pink results a violet. colour in C; inD The fibrin in C is digested, that in D is not digested. 1 In applying the biuret test to a filtrate after saturating with ainmonic sulphate it is neces- sary to add great excess (three or four volumes or more) of caustic soda. This dilutes the peptone considerably and so the pink will be much less distinct than in No. 129. For this reason you would probably fail to find peptone in the fluid X. 19 PANCREATIC JUICE. Vessels labelled Z are placed on the tables containing products of the pan- oreatic digestion of proteids. Directions. Result. 134 In a test tube E place a pellet of fibrin with, a little dilute pancreatic glycerin, and an equal bulk of a 2 per cent, solution of sodic carbonate. Place in the water bath for 30 minutes. 135 In another test tube F place some starch mucilage with pan- creatic glycerin and sodic carbonate as above. Put it in the water bath for 10 minutes. 136 In other test tubes G and H place some oil and add some blue litmus. To G add a small frag- ment of pancreas and place both in the water bath for 30 minutes. Whilst these " digests " are progressing, exam- ine the fluid placed on the table ; notice its smeU. 137 Neutralise some of it, and saturate it with sulphate of ammo- nium 138 Remove this precipi- A pink tate, if necessary, by results filtration and apply biuret test Inference. Very little pre- cipitate falls, if any Digestion is practically complete. colour Peptone is present. 20 Directions . 139 Dip the eudof a wooden matoh into hydro- chloric acid, and then into the fluid liO Boil a little of the fliii:! with an equal volume of Millon's re-agent 141 Examine the test tube E. The fibrin in it is eroded, not swollen as it was in No. 132 142 Filter the contents of E and apply the biu- ret test to the filtrate 143 To the contents of E apply Moore's test 144 Compare the contents of 'g and H Besttlt. The match may be stained red A port wine col- our may appear Inference. If so indol is present. If so, tyrosin is present A pink colour Theflbrinhas been digested, results A yellow or brown colour results In G the litmus has become red The starch has been di- Acid has been liberated by the pancreas. ACTION OP INTESTINAL JUICE. 145 The vessel K contains No red precipitate The sugar present is not a reducing sugar. The saccharose has been converted into a reduc- ing sugar. saccharose. Apply Trommer's test to some of its contents 146 The vessel L contained saccharose. But it has been acted on by suocus entericus. To some of it apply Trommer's re-action falls A red precipitate falls 21 GENBEAL HINTS FOE THE QUALITATIVE EXAM- INATION OP SIMPLE FLUIDS CONTAINING SUB- STANCES OF PHYSIOLOGICAL INTEEEST. Take the reaction of the fluid. If it is coloured, examine it with the spectroscope, before and after the addition of ammouic' sulphide. Then decolourise with animal charcoal, and test the fluid for proteids. If proteids are present try and find out which proteids they are. Test also for the album.inoids and ferments. Whether proteids are present or not, test the original fluid for — Starch \ Dextrin |-acidifying if necessary. Glycogen J Bile pigments Uric acid ' using Schifi's test if the solution is alkaline, and the \ murexide in any case. Digestive ferments (proteolytic alone, and with amylolytio ferments) Sulphates, phosphates, chlorides. If proteids are absent test also for Urea Sugars Bile salts But if proteids are present take 25-50 cc. of the fluid, evaporate almost to dryness, extract residue with alcohol, filter, evaporate alcohol from filtrate, dissolve this residue in water, and then test for urea, sugars, and bile salts. GENBEAL HINTS FOE THE QUALITATIVE EXAM- INATION OF SIMPLE SOLIDS CONTAINING SUB- STANCES OF PHYSIOLOGICAL INTEEEST. Examine microscopically, looking especially for— Blood corpuscles. Starch' granules. Crystals. Make extracts with — 1 Distilled water ,) and examine them 2 Normal salme, and a 10 per cent, solution of I ^^^ directed o .sodic chloride [ ^^ove. 3 Alcohol I i Ether, and look for fat. 5 Dilute acid, and look for phosphates. If any insoluble residue remain it may be Coagulated proteid, Elastic tissue. 1 Aminonie sulphide must not be added to an acid solution. It necessary therefore neutralise before reducing. 22 ESTIMATION OF SODIUM CHLORIDE IN UBINE. Mohr's Method— You are given a burette containing a standard solution of silver nitrate, and a small flask. Place 10 cc. of urine in the flask with 100 cc. (half a flask) , of distilled water, and 10 or 12 drops of potassium chromate solution. Let the silver solution run gradually into the urine, shaking the latter frequently until a permanent orange tinge appears in the precipitate. Before calculating the amount of sodium chloride present, you must deduct 1 00. from the amount of the standard solution used, because some of the silver is precipitated by the phosphates of the urine.' Then calculate as follows : — Suppose that you required 10 cc. from the burette. 1 cc. of the standard solution precipitates -01 gram of sodio chloride. -.9 CO. „ „ „ -09 •09 gram of sodic chloride are present in 10 cc. of urine. .-. -9 „ „ „ „ 100 cc. „ ESTIMATION OP THE PHOSPHORIC ACID IN URINE. You are given a burette containing a standard solution of uranium acetate, a white basin, a white tile, and a vessel of potassium ferrocyanide solution. Place a few drops of the ferrocyanide solution on different points of the tile. Place 50 cc. of urine iu the basin, under the burette, and boil the urine ; when it boils run iu the standard solution gradually, stopping frequently, and testing some of the urine by dipping a glass rod in it, and then touching one of the ferrocyanide drops on the tile. When the ferro- cyanide is tinged brown by the drop on the rod, the precipitation of the phos- phoric acid is complete, and an excess of the uranium salt has been added. Suppose that you use 15 cc. from the burette, calculate as follows: — 1 cc. of standard solution = -005 gram of P^Oj. .-. 15 CO. „ =-005 X 15 gram of P^Oj. . ■ . -005 X 15 grams of P^O^ are present in 50 cc. of urine. Multiply this amount by 2 and you get the percentage of P.O iu the urine. Remember that Ffi^ does not exist free in the urine, but iu combina- tion with bases. 1 In qualitative testing for the presence of clilorides, you prevent the formation of silver phosphate by adding nitric acid to the urine. You cannot add nitric acid here, because it would prevent the appearance of the orange colour, and you would not Itnow when the pre- cipitation was complete. 23 ESTIMATION OF DEXTROSE IN URINE. Pehling's Method— You are given a bottle of Pehling's solution, a white evaporating dish, and a burette containing a diabetic urine diluted ten times. Place the dish on a tripod under the burette, add 30 cc. of distilled water to it, and apply heat with a Bunsen's burner. Note the level at which the solution in the burette stands, aud when the Pehling boils run in the dilute urine, a few cc. at a time boiling after each addition, until the blue colour has nearly gone. Then work more cautiously, adding a few drops at a time until the Pehling is quite colourless. When all the blue is destroyed, note how much dilute urine you have used, and you will have a rough idea of the quantity required to decolour- ise 10 cc. of Pehling's solution. Now wash out the white dish and repeat the process adding, when the Pehling first boils, nearly enough dilute urine to de- colourise it, and working gradually until you obtain exact decolourisation . To he accurate you ought to make a third determination, but for rough work two are enough.' Then find the average amount of solution used in your two or three determinations, and calculate the amount of sugar present in the urine as follows. Suppose that you used 12 cc. from the burette. 10 cc. of Pehling are decolourised by -05 gram dextrose 12 cc. ot diluted VLrme cout&in .. '05 ,, ,, 1 cc. ,, ,, ,, ■.lOOcc. . 100 cc. iindilnted ,, ,, In estimating sugar observe the following cautions — 1. — Do not allow the Pehling to cool before looking to see whether it is colour- less, because as it cools, it gets blue again. 2. — Do not trust a determination in which you require less than 10 co. from the burette. If less than 10 co. of the fluid decolourise your Pehling, the urine must be still further diluted, and of course this extra dilution must be allowed for in the calculation. 3. — Do not try to be too accurate in the determination of the end. •05 12 n " ■05 12 X 100 grams dextrose •05 12 X 100 X 10 grams dextrose 1 If you have to do -ivitli a fluid, in which the percentage of .sugar is entirehj unltnowu to you you may advantageously commence by estimating rouglily and raindly, applying the test to 1 cc. of Pehling diluted three times in a test-tube. H this is deoolouriseci by less than 1 cc. of urine the amount of sugar is above b per 1,000, and the urine must be diluted. If for example, you find with urine diluted ten times, that -i cc. do not, and that 4 cc. do com- pletely reduce 1 cc. of Fehling, you know that the sugar percentage in the diluted urine is between 2-b and f25 per 1,000, and can then proceed to a nmre accurate determination within these limits. 24 ESTIMATION OP UREA IN URINE. Hypobromite Method— You are given a gag burette, standing in a tall jar of water, and connected by an india-rubber tube with a bottle, which has a small test tube in it, graduated to hold 5 cc. Take out the graduated tube, and place 25 cc. of hypobromite of soda in the bottle. Pour urine into the graduated tube, up to the 5 cc. mark, and lower the tube carefully into the bottle containing the hypobromite solution. Then cork the bottle. This will force the level of the water inside the gas burette, below that outside it. Open the brass clip and the water in the burette will again rise to the proper level. Then close the clip and note the level of the water in the burette. Now tilt the bottle gently so as to mix the urine and hypobromite solution, and the urea will be decomposed into nitrogen, water, and carbonic acid gas. The carbonic acid will be absorbed by the free alkali in the hypobromite, and the nitrogen will pass over into the burette, and depress the water inside it. Allow the bottle to cool and then read the amount of nitrogen evolved. This is done by lifting the burette until the water inside it is at the same level as the water outside it, and then noting the level of the water. Then, knowing the level at which the water stood before the experiment, you can ascertain the quantity of nitrogen evolved, and calculate the amount of urea present, as follows. Suppose that you obtain 30 cc. of nitrogen, 35-5 cc. of nitrogen is yielded by . . . . -l 1 cc. of nitrogen ,, . . . . L 35-5 .•. 30 cc. of nitrogen ,, .. .. 35'5 gram of urea 30 35-5 •1 35-5 -j-g X 30 gram of urea is present in 5 co. of urine. 30 X 20 grams of urea are present in 100 cc. of the urine, i.e., the percentage of urea in the urine is 1-69.