Class Book Rf ^V? i.^ ]0 GjpiglitlN" CDEffilGHT DEPOSm MANUAL OF LABORATORY DIAGNOSIS Compiled and Elaborated by Herman John Bollinger, S. B., M. D. Assistant in Bacteriology Johns Hopkins University Preface by Sidney R. Miller, S. B., M. D. Associate in Clinical Medicine Johns Hopkins University BALTIMORE. MD. MEDICAL STANDARD BOOK CO, .i'^ ^ iP^\ Copyright 1919 Medical Standard Book Co. Baltimore, Md. mR "5 19 Press of , > ' The Medical Standard Book Co. Baltimore CI.A5L2476 This book is compiled irom lectures given by the following instructors at the Johns Hopkins Medical School. S. R. Miller, M. D., Lectures on Urine, and Blood. Marjorie D. Batchelor, M. D., Lectures on Stomach Analysis. F. A. Evans. M. D., Lectures on Sputum and Stools. W. A. Baetjer, M. D., and S. R. Miller, M. D., Lectures on Blood. C. G. Guthrie, M. D., Lectures on Parasites. PREFACE. Requests have come from time to time for out- lines of the general course in Clinical Microscopy, as given at the Johns Hopkins Medical School, or for summaries of the individual subjects covered. Such outlines have not any particular merit other than that they serve as guides for teaching, or compends, useful for the quick reviewing of any particular topic. It has always seemed a wiser policy to en- courage each student, carefully to take his own notes and prepare for himself such a working out- line as best suited his own needs. Condensed notes and compends in general are too prone to stimulate superficiality to warrant their unqualified recom- njendation. Moreover the practice here has been to have each instructor cover a different subject each year, thereby ^\idening the scope of each man's knowledge and interests; each improving on his predecessor's lectures, if possible. Consequently, no set plan is followed and stereotyped lectures have been consistently avoided. The present summary of the lectures given in the past year, is published chiefly as a result of the en- thusiasm and painstaking work of one of the stu- dents. It is not intended that these notes shall be comprehensive: the interpretation of tests, etc., has not been given much space, and, by reason of the universal "war-time lack of time," careful correction has not been possible. The notes are presented merely as representing the essential subject m^^tter covered in the lectures on urine analysis, exaniina tion of the gastric contents, sputum, feces, parasi- tology and hematology. The publication of these notes had the sanction of the late Dr. Theo. Janeway, whose interest in the application of clinical labora- tory methods to the diagnosis of disease was always keen and enthusiastic. It had been his intention to write this preface as evidence of his approval of the project. B}' reason of their absence in France, Drs. W. A. Baetjer, C. G. Guthrie and Frank Evans have been unable to review the sections previously cov- ered by them. Knowing full well their great interest in the subject, it has seemed wise for me to stand sponsor for them all, and to permit publication, but only with a clear understanding of the limits of the book, the constant changing of methods and inter- pretations, and the lack of balance unavoidable in a work of this kind. Sydney K. Miller^ M.D. The Clinical Laboratory, Johns Hopkins Medical School. ALBUMINURIA Definition: The accumulation of a coagulable protein in the urine. The protein has, in true cases, escaped through the renal parenchyma. Two groups : 1. True condition in which the renal parenchyma is faulty. 2. False. Condition in Avhich the protein has been added to the urine during its passage through the G. U. tract. Origin : 1. Blood protein. 2. Secretion or degeneration of renal cells. 3. Substances added along G. U. tract. No chemical method has been devised to differ- entiate these ditlerent forms. Most of protein is of blood origin all agree. Serum albumin. Greatest in frequency and amount. 5 to 40 grams per day. 20 to 100 mg. oc- curs normally, but is not detectable. Albumin is only clinically present when it can be detected by the ordinary laboratory means. This is set as a standard. Serum albumin is soluble in water, co- agulable by heat, precipitated by alcohol and salts of the heavy metals, and by saturation with magnesium sulphate, and is Levo-rotatory 62 degrees. Serum globulin. This is always present with serum albumin. It represents from 10 to 75 per cent of total protein. It is insoluble in water and dilute acetic acid in the cold. It is precipitated by saturation with magnesium sulphate and half saturation with am- monium sulphate and partially precipitated by so- dium chloride saturation. Pseudo-glohuliUy euglo'dulin^ and fibrinogen. These belong to the globulin group. Fibrinogen is practi- 8 cally never en countered and when it is is perfectly obvious, for the urine undergoes spontaneous clot- ting. Euglobulin and pseudo-globulin are probably the same as nucleo-albumin. They are of no practi- cal importance. jSiucleo-histones are of no practical significance. Bence-Jones protein is protein sui generis. Alhumoscs are characterized by a tendency to be soluble upon heating and insoluble upon cooling. They are increased in acute types of nephritis and in the absorption of exudates. OCCURRENCE OF ALBUMINURIA. 1. With renal lesion. 2. Without renal lesion. Occurrence of albuminuria without definite renal lesion. 1. Severe muscular exercise. 2. Severe emotional upsets. 3. After cold baths. 4. Alimentary ingestion of excessive amounts of proteins. 5. New-born, first 8 to 10 days of life. 6. Pregnancy, toward the end or after parturiton. 7. Adolescence, 12 to 16 years of age. 8. Hypostatic, associated with large spleens, no- ticeable after the patient has lain down for some time. 9. Cyclic, orthostatic, or lordotic. This type oc- curs in young adults, 12 to 20 years of age. Albu- min appears only after the patient has been up and about for some time. Early morning urine is al- bumin free. These patients usually have abnormal lordosis, and w^hen corrected the albuminuria clears up. According to Jehle there is a deficiency of renal function while the patient is standing, which clears up when he lies down. The condition does not predispose one to nephritis, bnt tends to clear up as the x^atient grows older. Salient features of functional disturbance. 1. Albumin slight. 2. Occurs chiefly in young people. 8. Transient or intermittent. 4. Occurs without other evidences of renal disease. 5. No renal disease etiology. 6. Patients tend to be a bit peaked and have a neurotic tendency, with vasomotor instability, der- matographia, etc. Alhurninuria in those with transient renal injury. 1. Fevers (with casts). Disappears with the dis- appearance of the fever and patient does not suffer inconvenience during rest of life. 2. Toxic. Caused by drugs and secondary to other states such as pj'^orrhea, genito-urinar\^ infec- tion, etc. Dental treatment, etc., may clear up con- dition. 3. Hematogenous. Caused by altered condition of blood as in leukaemia, pernicious anaemia, pur- pura, scurvy, beriberi, and jaundice. 4. Traumatic. Caused by injury in the vicinity of the kidney or in remote parts of the body. Occurs in epilepsy, brain tumors, and sometimes after bimanual palpation of the kidney as well as other abdominal manipulations. 5. Intermittent, characterized b}' periodic occur- rence, indicative of: (a) Insidious development of nephritis. (b) Stasis. Summary. Kegard faint traces of albumin in people under 20 years of age in good condition as functional. Ex- 10 amine last voiding at night and iirst in tlie morning to determine whether it is orthostatic, or hypostatic. Use care to exclude extra-renal sources of albumin, bladder infection, vaginal discharge, etc. Regard albuminuria in febrile and toxic conditions as ne- phritis, at that time at least. One cannot judge the extent or the type of nephritis by the frequency of albuminuria. The absence of albumin and casts does not exclude nephritis. Detection of proteins in the urine: Serum albu- min, serum globulin, nucleo-albumin, and albumoses. 1)1 order to detect these substances the iirine must he: 1. Absolutely clear, especially if small traces are to be detected. Make urine clear by filtration or by the us^e of kieselgMhr or animal charcoal. When the urine comes tiirough the filter paper turbid add about 1/10 volume of kieselguhr and refilter. 2. Urine should be faintly acid. ^. Urine should not be too concentrated or too dilute. In cases where it is very dilute 1005 to 1006 specific gravity, it should be made more concen- trated by the addition of about 1/10 volume of so- dium chloride. 4. In doubtful cases don't rely on one test alone. Heat and acetic acid test. After the urine has been made clear by the above methods take a test tube about 2/3 full of the clear urine and heat the upper third in a bunsen flame. If no cloud appears the urine is x)robably normal. If a marked cloud appears it can be : 1. Calcium phosphate. 2. Calcium carbonate. 3. Albumin. In order to differentiate these add 5 per cent acetic 11 acid drop b}' drop. If cloud is due to calcium phos- phate it disappears ; if it is due to calcium carbonate it disappears with the evolution of gas ; if it is due to albumin it persists, Ix-comes greater or becomes slightly less, in accordance Avith the amount of the former substances present. It must be remembered that these protein substances are soluble in an ex- cess of acid or alkali. To detect very faint traces use a dark background. Sources of error: (a) Technique, (b) Other pre- cipitable substances. 1. ^ucleo-albumin. This is precipitated with 5 per cent acetic in the cold. Albumin is only pre- cipitated after heating. 2. Eesinous acids such as cubebs, guiac, etc., give a precipitate with heat and acetic acid. These if shaken with alcohol or ether are dissolved. 3. Albumoses. The precipitate of these comes down after cooling, and reheating gives re-solution. Heat and nitric acid test. Heat urine to boiling and add 20 per cent HNO3 drop by drop. A cloud is fairly indicative of albu- min. It has the same sources of error as the heat and acetic acid test. These two tests are sensitive to .005 grams in 100 cc. Heller's test. Urine and concentrated H]S^Oo are layered in equaJ parts, the urine above and the acid below. A cloud at the line exactly can be : 1. If white, albumin. Its breadth depends upon the quantity of albumin present. xA^fter a time it tends to diffuse upwards. 2. Thymol. This has a grayish or brownish color. There is also a zone of oreenish color below and a 12 reddish one abo\e llic^ line of contnct. ronfirm b}' t^haking with petroleum ether. 3. Albumoses. Tlie cloud given by these is ideii- lical with that of albumin. J>y heating this cioud disappears. 4. Urea nitrate. This cloud is yollowisli brown and is made up of crystals which give a shimnier. A cloud above the line of contact may be: i. A broad, yellowish band about II/2 cm. above line of contact due to urates in concentrated urines. They are dissolved by heating. 2. Xucleo-albumin gives a cloud ^4 to % cm. above the line of contact. It tends to disappear by dilu- tion. By diluting the urine to 1005 to 1008 nucleo- albumin and albumoses can be eliminated. This test is sensitive from .002 to .007 grams per 100 cc. Potassium ferrocyanide and acetic acid test. From 5 to 10 cc. of urine in a test tube to which is added acetic acid. Do not heat. Filter and to the nitrate add 10 per cent potassium ferrocyanide, drop by dro]). Albumin gives a whitish precipitate. Sources of error: 1 . Iron present in large amounts. Color different. 2. Boiling urine in a glass container. 3. Reagent itself. 4. Albumoses which upon heating disappear, 5. ^ucleo-albumin same as albumoses. QUANTITATIVE TESTS FOR ALBUMIN. The most common and easily performed is the em- ployment of the Esbach tube. It differs in accuracy from the Kjeldahl metliod by .3 gm. Urine is added to the mark U and reagent to the mark E. The tube is inverted ten or twelve times to insure thor- ough mixins,- of the contents and is then allowed to 13 stand for about 24 hours and read. The reading below gives the number of grains per liter. Reagents useds 1. Picric acid. This is merely mentioned to be condemned. It not only precipitates albumin but also albumoses, uric acid, creatinin, resinous acids, etc. 2. Tsucliiya'S reagent. Phosphotungstic acid 1.5 grams Concentrated HCl 5.0 cc. 95 per cent alcohol g. s. ad. . . .100.0 cc. The advantage of this reagent is that it eliminates the above error. The disadvantages of the test are that it is inac- curate in urines containing over 4 per cent albumin ; and in some cases the precipitate sticks to the sides, may float or may settle unevenly. OLOBTTLINS: Purdy's test. Globulins are soluble in salt solution, but insolu- ble in distilled water. Dilute the urine and get a cloud, or, better still, drop the urine in a test tube half full of distilled water and watch for a cloud against a black background. Ammomum suliyliata method. Make the urine alkaline with amrnroniura hyd^65^ ide and filter. Layer equal amounts of the filtrate and saturated ammonium sulphate. A cloud at the line of contact is fairty indicative of globulin. For more accurate determination do following test : Urine plus equal volume of saturated ammonium sulphate solution. Let stand an hour and filter. Wash pre- cipitate with half saturated amonium sulj^hate until filtrate is albumin free. To further differentiate it dissolve precipitate in water and heat on Avater 14 bath to coagulate the proteins. Filter and wash the precipitate with water. To the precipitate add 1 per cent solution of sodium carbonate and heat on the water bath. Filter and neutralize with acetic acid. A precipitate signifies globulin (Web- ster). ' - BENCE-JONES PROTEIN. (S. K. Miller and Walter A. Baetjer, J. A. M. A., 1-19-18, vol. 70, pg. 137-139.) Tliis is the most infrequent type of protein found in the urine. In 1816 Bence-Jones described the characteristics of the protein, which were tlie forma- tion of a gelatinous precipitate at a low temperature wliich disappeared upon boiling and reappeared uj)on cooling. In 1876 Rustizky described multiple myeloma. In 1889 Kahler described a case of mul- tiple myeloma with Bence-Jones protein in the urine. The association of multiple myeloma with Bence-Jones protein is almost constant but not spe- cific, for it does occur in other conditions. In no case, however, where Bence-Jones protein has been found has the bone-marrow been found to be normal. Multiple myeloma are bone tumors similar to sar- coma, occurring usually in people beyond forty years of age, and in men more than in women. The condition is one of a deep-seated bone lesion asso- ciated with pain, emaciation, cachexia, etc. ; it is con- fined to the marrow and lives at the expense of the cancellous, tissue; it is limited to the long bones, the sternum and* ribs, rarely invoMng the skull. Bence-Jones protein has /been demonstrated in five types of cases : '"■■■■'■ 1. Multiple myeloma composed of (a) myeloblasts, (b) myelocytes, (c) erythroblasts. . 15 2. Chronic leukaemias (a) lympliatic. (bi niTelo- cytic. 3. Metastasis to bone from cancer elsewhere. 4. Other bone diseases present. 5. Cases without any lesions present. Usually not over two years elapse from the dis- covery of Bence-Jones protein to the death of the individual. In tAvo cases there was reported a high blood pressure. Xature of Bcncc-Joncs protein. It gives rise to amino acid partition and is there- fore a real protein, but it differs from any other known protein and is never found to be a part of the metabolism of the individual. It contains a car- bohydrate radicle, but no phosphorous, and is there- fore not a nucleo-protein. It also contains from 1 to 2 per cent of sulphur. It has been injected subcu- taneously, intravenously and intrarectally, but is al- ways excreted as such. Theories regarding Bence-Jones protein. 1. A substance derived from the secretory activity or degeneration of tumor masses themselves. 2. The tumor itself secretes an enzyme which can influence protein metabolism in such a manner that this protein is formed. 3. Individuals with Bejice-Jones protein have anomalies in urine excretion and metabolism similar to those excreting alkapton and cystin substances. It is probable that these individuals on account of their perverted metabolism are more prone to tumors. Character of the urine. The amount varies from 1200 to 2000 cc. per day with a normal specific gravity. It has a pale smoky color but is clear; it is acid in reaction, rarelv neu- 16 tral or alkaline ; it has a tendency to foam on shak- ing, which foam is more abundant and more per- sistent than normal. There may be as mncb as 70 grams of the protein present in the 24 honi speci- men, and there is no other type of protein nor any casts present unless there is some nephritic condi- tion superimposed. ClwracteriMics of the urine in this disease. 1. Continuous excretion regardless of everything. 2. Intermittent type, in which days or months may elapse without its excretion. 3. The appearance of the Bence-Jones protein sometimes antedates any demonstrable lesion. 4. The largest amounts are excreted in multiple myeloma which may run as high as 50 to 75 gm. per day. In other cases, such as metastatic cancer or sarcoma, from 20 to 35 gm. are excreted. 5. There is also a decreased chloride content. 6. A spontaneous precipitate may occur in the urine after standing for some time or it may occur in the bladder and give rise to a urine resembling chyle. Tests. 1. Heat and acetic acid. The urine clouds at about 55 degrees. It is coagulated and precipitafed from 10 to 20 degrees lower than any blood protein (40 to 60 deg.) It dissolves upon boiling and re- appears upon cooling. Coagulation depends upon the acid and salt concentration of the urine. 2. Urine treated with 25 per cent HXOg in the cold gives a cloud which redissolves at first until excessive acid is added. This precipitate is soluble at 100 deg. and insoluble at 60 deg. Any dilute mineral acid will do the same. 3. Urine plus 2 volumes of saturated ammonium 17 sulphate gives a complete precipitation at room tem- perature. This precipitate is readily soluble in water and does not pass through a dialysing mem- brane, differing from albumoses and pentoses. 4. Urine plus 2 volumes of a saturated solution of sodium chloride gives no precipitate unless the urine is previously acidified with acetic acid. The precipitate is not soluble in water. 5. Urine j)lus 2 volumes of 95 per cent alcohol gives complete precipitation. Immediately after precipitation, the precipitate is soluble in water, but if kept in contact with the alcohol for some time, it becomes insoluble in water, but is soluble in a dilute solution of ammonia. 6. It gives a positi\'e biuret reaction. SEDIMENTS OF THE CRINE. Inorganic. 1. Acid urine a Uric acid. 6 Amorphous urates. c Calcium oxalate and sulphate. d Xanthine. e Cystiu. / Leucin. g Tyrosin. h Hippuric acid. i Bilirubin. / Cholesterin. 2. Alkaline urine. a Amorphous phosphates and carbonates. h Magnesium phosphate, c Triple phosphates. d CaCOg. e Ammonium biurate. One mav find anv kind of acid crvstals in an 18 alkaline urine i'or they may persist after the urine has become alkaline. Uric acid crystals. There are three conditions necessary for their for- mation : 1. Concentrated urine; 2. High acidity; 3. Cold. They form crystals brick red in color and have a tendenc}' to clins to the sides of the container. They mean nothing concerning purine metabolism. They take a variety of shapes, rhomboid, prisms, etc. When made arti-licially they are colorless. They are soluble in HCl and insoluble in acetic acid. Urate sediments. These also occur when the urine is : 1. highly acid ; 2. concentrated, and 3. cold. They take the urinary pigments with them, urochrome and uroerythrin. Their shape is amphorous, sometimes resembling needles. They are soluble by heating to 50 to 60 degrees, and with acetic and mineral acids. Calcium oxalate crystals. These are formed mostly from vegetables such as,.. rhubarb, celery, spinach, peas and beans, and also, from green fruits. A small portion is formed from: the body (nucleins — uric acid — oxalic acid). In ox- aluria as much as 20 to 30 grams may be excreted in a day on a mixed diet. Neurasthenia is often as- sociated with its excretion but not the cause. Its excretion is sometimes increased in jaundice, and hepatic disease, and definitely increased in gout 19 when it is most likely formed from uric acid. In this condition determine whether it is excreted fre- quently and whether it is associated with uric acid ])erversion and calculi formation. They occur as four-sided prisms with a square base and also in dumbbell crystals. They are perfectly white in color, are insoluble in acetic acid, but soluble in HCl. Calcium sulphate. These are perfectly white in color and rare in occurrence. Their shape is long and oblong. XantMn, white and somewhat egg shaped. (After Hawk) Tyrosin, colorless and in needles arranged in sheathes and rosettes. Leucin, never a spontaneous sediment. Cystin, colorless, four-sided or prism shaped crystal?. Insoluble in acetic acid and soluble in HCl. o^ 20 Hippuric acid. Occur rarely. They are irregu- lar, six-sided crystals. C^ Bilirubin crystals, yellow or reddish browu in color, occur rarely in hemorrhagic nephritis, after transfusion, jaundice, and acute yellow atroph}^ Oc- cur in needles and rhomboid shaped crystals. Cholesterin crystals, white, and, in the form of steps, one upon the other. Soluble in chloroform. AmorpTioiis pTiospliates and carbonates. These constitute the chief sediment of alkaline urine. Both are soluble in acetic acid, the car- bonates in addition give off gas. Magnesiutn pliosphate. These are generally amorphous but occasionally resemble triple phosphates. Triple phosphates. (MgNH^PO^) ^^ These are one of the commonest forms of xls found in standing urine, and are characteristic of am- moniacal urine. These are coffin-lid, or when eroded, fern-leaf in appearance. They are colorless and soluble in acetic acid. 21 Calcium carhonate crystals. These may be amorpiious or crystalline and are generally found with amorphous phosphates. In shape they are either dumb-bell or spheroidal with concentric radiations. They are colorless and solu- ble in acetic acid with evolution of gas. Ammonium blur ate crystals. / These occur in the shape of thorn-apples and are dark brown in color. They dissolve in acetic acid and give uric acid. They are of no significance. bCHEME FOR RUNNING DOWN INORGANIC SEDIMENTS. 1. Warm sediment. a Urates readily soluble. 6 Calcium sulphate soluble with difficulty. 2. If not soluble by (1) add 3 to 5 drops of glacial acetic acid. Solution indicates : a Amorphous phosphates and carbonates. h Triple phosphates. c Calcium carbonate. Non-solution indicates : a Uric acid. h Calcium oxalate. c Calcium sulphate. d Organized sediments. e Tyrosin. / Cystin. g Leucin. 3. Add from 3 to 5 drops of concentrated HCl to insoluble sediment. Solution indicates: 22 (/ Calcium sulphate. h Cystin. \ c Tyrocin. ( These are also soluble in d Leucin. ( IS^H^OH. e Xanthin. ) 4. Uric acid is insoluble in acids and ammonia; but is soluble in KOH. 5. Cliolesterin is soluble in chloroform. 6. Hippuric acid is soluble in alcohol. ORGANIZED SEDIMENTS. Mucus threads. Poorly refractile, interlacing, fibres found nor- mally and of no significance. Kesponsible for the nubecula formation. Epithelial cells. These may arise from any part of the genito- urinary tract, viz. : pelvis of kidney, bladder, ure- thra, prostate. Irregular cells occur in the bladder and vaginal tract. Sheets of cells are more fre- quent from the vagina. Kenal cells tend to be romid or cuboidal, a little larger than a pus cell, and i.a\e a large vesicular nucleus. They may occur in av) type of nephritis, but are more apt to occur in larger nimibers in acute nephritis. Similar cells arise from the ureter and prostate, those from the latter being about twice as large. One cannot tell the type, location or extent of the lesion by the type of cell found, although renal epithelium does nr t oc- cur normally. Tailed cells were formerly thoiiglit to arise from the pelvis of the kidney, but they are also found in the deeper layers of the bladder and vagina. Pus cells. A few may be encountered in normal sj)ecimGn,-^., especially in women. Large numbers indicate patho- 23 logical conditions either of tlie G. U. tract or outside. The number of pus cells occurring in diseases of tlie cortex of the kidney is few ; the number occurring in pyelitis, pyelonephritis, and kidney abscess is many. Cystitis is the most frequent cause of pus in the urine. They are better identified in acid urine, so accordingly, if the urine be alkaline as it usually is in cystitis, it will aid in identification by adding a lew drops of acetic acid under the cover slip, which will bring out the nuclei. Tripperfadcn. Are shreadlike bits which float about. Micro- scopically they are masses of pus cells in the meshes of mucus. They occur in chronic urethritis. Red Wood cells. These never occur normally except in women dur- ing menstruation. They disintegrate very rapidly, tend to be slirunken and crenated in concentrated urine, laked in urine of low specific gravity, and are often difficult to recognize. When they arise from the kidney they tend to be mixed with the urine, when from the bladder they tend to occur in clumps. In alkaline urine they tend to go to pieces very quickly. Spermatozoa. Disintegrate very rapidly. Found occasionally in adult males. Tissue jragments. Kenal or bladder new^ growths. Casts. ( Cylindruria ) . Descending tubular elements from the kidney. Theories of origin: 1. Casts represent visible coagulated albumins which have escaped from the kidney. 2. Irritated kidney cells excrete a colloidal sub- stance which is coagulated in the tubes and may en- 24 gulf any cellular substance present there at the time. They indicate a condition of renal irritation and are more indicati\ e of it than albumin. TA'pes of casts. 1. Cellular. 2. Granular. 3. Amorphous. Cellular casts. a Epithelial. & Pus or W B C. c Eed blood cell. Granular casts. a Fine and coarsely granular. & Fatty. G Urate. d Bacterial. Amorphous casts. a Hyaline. 1) Waxy. Theory. Hyaline casts are the fore-runners of all the others, and are the matrix upon which the others are built. They have straight sides, rounded ends, are uniform caliber throughout, narrow, may be short or long, and are poorly refractile. One can build all the other forms around them. Any cast containing even but one cell is called a cellular cast and designated b}^ the type of cell it contains. They are encountered in a number of conditions, acute and chronic nephritis, etc. Cellular casts change into granular casts, going through a stage of fatty degeneration. Coarsely granular casts are yello^^- ish in color and finely granular casts are colorless. Granular casts can finally revert to the hyaline form. Waxy casts differ from hyaline casts in being 25 highly refractile. They seem brittle, have irregiilai' ends, aud may be wavy in outline. They tend to be broader and have transverse lines of fission. They stain with Lugal's solution but are not necessarily limited to amyloid disease. They are a fairly good indication of tubular stasis and probably represent metamorphosed hyaline casts. Occurrence of casts. Casts are occasionally encountered in normal in- dividuals, but whenever they are found they should be regarded as evidence of renal irritation, whether slight, transitory, and of no importance or persist- ent and of serious import. They occur after strenu- ous exercise and in febrile states. Neither the type nor the number allows one to judge the extent of the renal injury, for the more normal the kidney the greater is the response to irritation. Casts tend to appear and run parallel with albuminuria but are more sensitive. The occurrence of albuminuria without casts usually indicates that one has not looked long enough or that the urine has become alkaline and they have disintegrated. Albumin without casts does not predispose to the development of nephritis. It gives some evidence of tuberculosis and other diseases higher up. Albumin with few casts represents in general the same thing as above. There is, however, a higher mortality incidence. Albumin with granular casts indicating cardio- renal disease, has in ten years a much higher mor- tality incidence than the two previous. Casts tend to be more cellular the more acute the condition. Waxy casts are indicative of tubal stasis. It is not so much the type nor the number of casts, but it is their relation to other clinical evi- dence that is of importance. (Read in Osier's book 26 of rcprLiits "On Ike Advantage of a Trace of Albuiuin and a Few Tnbe Casts in the Urine of Certain Men Above Fifty Years of Age.") Cylindroids. These may possess any and all the characteristics of casts. They are usnally straight and hyaline and larely Avaxy. References. Atlas der Clin. Mic. des Harnes, Rieder. Micro- scope der Harnes Sediment, A. Daiber, BACTERIOLOGY OF THE URINE. BaciUuria. Indicates bacilli in the urine irrespective of their sonrce. Types found : 1 . Tubercle bacilli. They may be excreted through the kidney and not represent a lesion of the G. U. tract. When associated Avitli r. b. c. and w. b. c, their local source is more likely. Don't confuse with Smegma bacilli. To obtain a more certain dagnosis inoculate a guinea pig intraperitoneally, keep 3 or 4 weeks, kill, and look for typical findings in the retro-peritoneal lymph glands, spleen or liver. Blad- der tbc. is often associated with an acid urine and sterile pus. 2. Gonococci. Gram negative and intracellular. Difftcult to find them in ^eisser cystitis. 3. Typhoid bacilli. Found during the disease and often for weeks or months after recoverj^ May be due to a bacillaemia or due to local lesion. 4. Colon bacillus. Common invader and cause of pyelitis and cystitis. 5. Paratyphoid. Infrequent cause of pyelitis. C). Cocci often found with Neisser infection. 7. Streptococci often occur from systemic infec- tion, nephritis, etc. o H Q O a. B Improves as Heart May Get Infarction B. P. Nor- mal Oedema (Face 1st) Uraemia Cure Rare Oedema Anasarca Uraemia Cardiac Fail B. P. Incr. Dropsy Rare. Car. Hyper Apo- plexy B. p. Norm or Below Oe- dema Com. Ureamia Rare Associated Willi Fever & Cachexia May Lead Amyloidosis, Septic- aemia or feritoiiltis From Perforation o 03 < CO C V ^ 2 en rs] C en a; en en l2 > o ^^1 c(5 +j _ Xi O w ^ 03 Q < Ph o s ^ S^ ^ IS «J ^ a; ^ v-i ? V = en ■T. 2; < 5 ^ en en en (U -^ < 03 « en rt 2 5 O Ah" ;/5 "to a o 2; 'So o o 03 O 5 ii c o o t-r 8 03 03 ^J3 13 2 D o <1 III 3 -, Vh Z^ (d ^ IS Oh .2 1 ^ ■ ■ <^ in 03 o '5 s 27 8. Any organism in the blood may be swept through the kidney. This may be the cause of much nephritis due to kidney injury. The general methy- lene blue stain detects all the bacteria except two : 1. Acid fast bacilli. Use carbo-fuchsin. 2. Gonococci. Use gram stain. Animal jjarasifes in the urine. 1. Amoeba. 2. Echinococcus (look for hooks and laminated membranes ) . 3. Filarial larvae. 4. Eggs of Schistosoma hematobium (human blood fluke) or Bilharziasis (Egyptian hematuria). 5. Oxyuris (pin worms, occasionally found in young girls. Wanders through urethra into bladder), G.. Trichomonas vaginalis (of no importance). GENERAL CONSIDERATIONS. Urine examined for the folloicing considerations : 1. Renal and G. U. conditions. 2. General metabolism of the body. 3. Food metabolism. Urine examination criteria. 1. Single voidings are of no practical importance. 2. Should be carried out on 24-hour specimens and in some instances the day and night specimens should be kept separate. 3. Fluid intake and general diet should be taken into consideration. 4. Correlate urinary and clinical findings. 5. Abnormalities should be confirmed at subse- quent examinations. Single voidings examined for: 1. Albumin. If but a faint trace is present, ex- amine two specimens, one at night and one in tlie morning, to see whether it is orthostatic. 28 "2. Gross abnormalities, blood, pus, etc. 3. Microscopical elements, such as blood, casts, and various forms of cr3^stals, pathological cells, and parasites. -*. Constituents requiring chemical analysis, such as bile, urobilin, blood, indican, acetone, diacetic acid, sugar, etc. c. Specific gravit}' determination. (). Keaction, acid, alkaline, or amphoteric. Collections of 24:-hour specimens : 1. Establish hours between which the specimen is to run. If the hours determined are between 7 A. M. and 7 P. M., have the patient void at 7 A. M. and throw the specimen aAvay. Collect all voidings, including the one at 7 A. M., the following morning. 1*. Containing vessel should be clean at the start, cool, corked and clearly labeled with: Xame of patient. Date, Hours of collection. Total intake, Preservative used. 3. Day and night specimens should be kept sepa- rate in some cases. 29 Preservatives used for urine : Tj^ie. Amount. Advaiitasro. Disadvantage. Chloroform 2 to 3 drams No Inilk. easily Not good for casts, perlirer. removed, crys- Reducing agent, tals preserved. Formalin. 1/. to 27c by Preserves Adds bulk.reducin-.- volume or 5 casts. agent, error in sugar to S drops test, form^^. crystals per L. with urea, inter- feres with urobilin and indican tests. Thymol. A few crys- Preserves False tests for hile tals. sediments and Heller's albu- min. Toluol A thin layer. No volume. Expensive and in- urinecnnbe flammatory. secured below. Common preservatives in nse are chloroform and tolnol. Physical properties of urine, normal and abnormal. 1. Normally it is a cleav, tranparent shade of yellow. After standing 15 to 45 minutes a "nubec- ula;" or fog, settles to the bottom, Avhich is com- posed of mucus and epithelial cells. 2. Urine cloudy when voided. (a) Frequently voided after heavy meals of vegetable*^. Due to excess of phosphates. Not abnormal. (b) Px'esehce of some kind of organized sedi- ment,' blood," desquamated epithelial cells, pus, bacteria. (c) Cloudy, with the smell of ammonia, indi- cates cystitis. 3. Urine becomes cloudy on standing. (a) Development of bacteria. (b) Precipitates of calcium, phosphorus, and carbonates. Formation of ammonium carbonate bv bacteria. 30 (c) Concentrated nrine, becoming quickly cooled, deposits amorphous urates. Odor of urine. The normal urinary odor is due to aromatic sub- stances. The urinous odor is due to the action of bacteria. A fruity odor occurs in diabetes, due to the excretion of acetone. A foul odor occurs in urines containing large amounts of albumin, as in cancerous and inflammatory conditions in the lower G. U. tract. The ingestion of asparagus giyes a peculiar odor, due to methyl mercapton. Drugs, such as asafedita and yalerian, giye their charac- teristic odor. Turpentine giyes the odor of yiolets; menthol that of pepperment; cubebs, copaiba, tolu and saffran that of spices. Amount of urine excreted depends upon : 1. Indiyidual himself. (a) Sex, males more than females. (b) Children yoid proportionately more, yiz: adults 1 cc per kilo body wt., children 4 cc per kilo. (c) Weight of indiyidual. 2. Fluid intake. 3. Pressure and yelocity of blood current. 4. Condition of renal parenchyma. 5. Loss of fluid by other means: perspiration, res- piration, intestines, formation of transudates and exudates. _ 6. Vasomotor phenomena. The normal excretion yaries bet^yeen 900 and 1200 cc per day. • - Functional capacity is. from 20 -tjo- Bo- liters. Often on forced T^~ater 9 to 10 lifers are ex- creted in typhoid without damage to the kidney. Normally at night less is excreted than during the day, the ratio being 100 parts by day to 60 — 80 parts by night. In some forms of nephritis, hepatic 31 insufficiencT aud cardiac disease, this ratio is re- versed. Nycturia signifies tlie excretion of more urine at night than during the day. It occurs character- istically in chronic diffuse nephritis. Polyuria signifies the excretion of 3000 cc or more urine per day. Causes : 1. Increased fluid intake. 2. Diuretics. 3. Nervous disorders. (a) organic; (b) func tional. 4. Diabetes mellitus and insipidus. 5. Chronic nephritis. 6. Absorption of exudates. 7. Epicritical (end states of acute febrile dis- orders ) . S. Sometimes in ureteral stricture. Oliguria signifies the excretion of 800 cc or less in 24 hours. Causes : 1. Decreased fluid intake. 2. Loss by other means. (a) phj^siological (b) pathological 1. Formation of exudates aiid transu^ dates. 2. Acute febrile states. •3.- Acute nephritis. ^ ..'...' i. End state of chronic nephritis. 5. Chronic diarrhoea. 6. Vomiting. Anuria signifies no urine. Causes : 1. Obstruction to urinary passages. 2. Reflex (functional neuroses^ Dietl's crisis). 32 3. Renal, acute nephritis, or end stage of chronic nephritis. 4. Prerenal conditions. Poisons : bichloride, arsenic, anaesthetics. Occasionally after veronal medication (S. R. Miller). Pollakiuria signifies nnduly frequent passage of urine. Occurrence : 1. Polyuria of nephritis. 2. Prostratic disease. 3. Bladder disease. 4. Ureteral stricture. Specific gravity. Methods of determination: 1. Balance picnometer. 2. Urinometer which is calibrated at 15 deg. C. The reading should be taken at the junction of the lower meniscus and flu^.d. The Sj). gr. depends upon the amount of urine excreted and the amount of solids therein. Sodium chloride and urea are the chief sources of high Sp. gr. in normal urine. Nor- mal urine has a Sp. gr. between 1015 and 1020 for 1200 to 1500 cc per day. Clinical value of specific gravity: 1. Estimation of total solids of urine. Haeser's coefficient equals 2.33 times the last two figures of the reading and gives the weight of solids per 1000 cc. Normally the total solids average from 60 to 70 grams per day on the basis of 1500 cc output. 2. Estimation of urea (Webster). 3. In polyurias, with low specific gravity, it points to chronic diffuse nephritis, and with high specific gravity to diabetes mellitus. 4. Oliguria, with low specific gravity, gives bad prognosis in nephritis. 33 5. Extensive oedema, with low Sp. gr., points to renal trouble. 6. Normally urines collected at intervals during the day show a variation in the Sp. gr. of 10 points. In chronic nephritis there is a tendency to the fixa- tion of the reading at about 1010 (Hyposthenuria). Reaction of the urine. Normal urine is faintly acid, partly from the acid sodium phosphate and partly from free acids, such as sulphuric, oxalic and hippuric, which occur when more animal than vegetable diet is taken. More acid occurs in the morning, and less after heavy meals. Variations in the total acids: 1. Use of drugs, sodium bicarbonate and acid sodium phosphate. 2. Hyperacidity increases; hypoacidity decreases acid. 8. Less after intestinal hemorrhage and after oedema due to nephritis. Classification of reactions : 1. Acid. 2. Neutral. 3. Amphoteric, effecting both red and blue litmus. 4. Alkaline. Alkalinity : 1. Fixed. 2. Volatile. Hold wet litmus in the fumes of the urine and the ammonia tliere present turns litmus blue. This condition leads one to suspect inflam- mation of the bladder. Acidity of urine is due to acid sodium phosphate (NaHoPO,^)* and free organic acids. 34 Quantitative determination of acids : Folin's method : Urine 25 cc. Powdered Potassium oxalate 5 to 20 grams (Prevents dissolution of salts of calcium.) 1% solution plienolphthalein 2 drops Titrate to the end point (a faint pink) with N/10 NaOH. The acidity is expressed in terms of : 1. Total no. of cc. necessary to neutralize total 24-hour output, 617 is about normal. 2. Percentage, number of cc. necessary to neutral ize 100 cc. of urine, 35 to -10 per cent normal. COLORING MATTER OF URINE The normal color of urine is a shade of yellow or amber. Ordinarily the color varies directly with the specific gravity, but here are tAvo exceptions : (1) Diabetes, which is associated with a faint yel- lowish green color and may have a Sp. gr. of 1040 or over. (2) Chlorosis, which may also be asso- ciated with a pale urine and high Sp. gr. Normal pigments of urine. I. Urochrome, which is responsible for most of the yellow color and Avhich is of little importance clinically. Also urocliromogen, precurser of uro- chrome. II. Uroerythrin, which is responsible for the sal- mon-red color. It is a pigment increased on exces- sive meat diet and in fevers. When extracted witji 35 amyl alcohol it gives characteristic spectroscopic bands. III. Urobilin, which is a complicated group of pigments, possessing a pyrrol nucleus. Xormal urine contains 30 to 100 mg. per 24 hours. IV. Urobilinogen, which is unstable and by sun- light is changed into urobilin. Origin of uroljilin : 1. Hematogenous theory, that urobilin may be formed directh' from the blood without the inter- vention of the liver. 2. Hepatic origin. Liver entirely responsible. 3. Nephrogenous theory, that under certain con- ditions the epithelium of the kidney changes bili- rubin into urobilin. 4. Enterogeneous theory, indicated by the dia- gram. Hcmogloltin I Biliruhin (action of the liver) I LrohiIi)i and uroljiUnogeii (action of intestines and bacteria) I I Portal system Part excreted in the feces I Liver I I 'Normal Abnormal I I Bilirubin (Failure to convert) Hemoglobin Urobilin absorbed and excreted by the kidneys. Significance of uro'biVmuria : 1. Bile is entering the intestines. 2. Extensive blood destruction. 3. Hepatic insufficiency. (a) Physiological. (b) Actual hepatic disease. 4. A certain amount of renal efficiency. 5. Absence of urobilin means total obstruction of the bile duct. 36 Occurrence of urobilinuria : 1. Common in hepatic cirrhosis. 2. Chronic passive congestion (prognostic evi- dence). 3. Hemolytic anaemia. 4. Malaria. 5. Pneumonia. Appearing at the time of the crisis is a favorable sign, especiall}^ if the patient is jaundiced before. If in the serum of such a patient the prognosis is usually hopeless. G. Measles and, scarlet fever and all affections which lead to liver damage. Appearance of tlie urine in uroNlinuria. The urine takes on a dark 3^ellowish color. Output is irregular and single void- ings sliOAV great variations in the amount excreted. Tests for urobilin: Schlesslnger's. To about 5 cc. of urine add a few drops of Lugal's solution, 1 to 2 cc. of NH^OH, and an equal volume of 10% alcoholic solution of zinc acetate. Filter and examine filtrate for greenish fluorescence. Spectroscopic examination : Extract with amyl alcohol. Alkaline solution gives broad band between E and F. Acid solution gives intensified bands in same place and the E — b space is filled. Ehrlich's 'benzaldekyde test. Use a 2% solution in concentrated HCl. Use 3 drops of this reagent to 3 to 5 cc. of urine. The pres- ence of a pyrrol derivative gives a cherry-red color. Heating before adding reagent gives intensification of same. 37 V. Iiulican (Iiidoxyl sulphate) 5 to 45 mg. per da 3^ Animal proteins alone give it. _, , ] indol ^ indoxyl Tryptophane= | ^^^^^^ absorbed= j ^^atoxyl These are conjugated with sulphuric acid and ex- creted as sodium or potassium salts. Indicanuria is increased upon a meat diet and is not excreted on a non-protein diet. Urine is generally normal color when voided, but on standing becomes dark. Formation : 1. Extra-intestinal, due to xDrotein decomposition in the body (bronchitis, abscesses and empyemias), 2. People with inborn errors of metabolism. 3. Gastro-intestinal tract diseases or disorders : (a) Transient phenomena, (b) Constant oc- currence, (c) Recurrent type due: 1, to pathological conditions of intestinal tract. 2. Perversion of intestinal tract secretion. Indicanuria is not necessarily associated with constipation, but is due to pathological conditions in the lining of the intestinal tract or of its secre- tions. Subacidity may give it. Tests for indican (Obermayer's). Equal parts of urine and reagent (0.2% solution of ferric chloride in fuming HCl). It is best to filter out bile pigments with PbSO^ before adding reagent. To the urine and reagent add 2 cc of chloroform and shake 12 times. Chloroform ex- tracts indican (blue), which sinks to the bottom of the test tube. KI gives a deep cherry red color with the same test. Albumin^ unless present in large amounts, does not interfere with the reaction. Thymol gives violet red shade, which is obviated with sodium thiosulphate. 38 VT. Hematoporphyriii. Occurs in uriue in such small amounts that it is normally difficult to detect. It is an iron-free derivative of hemoglobin. Increased amounts occur in: 1. Certain diseases, rheumatism, phthisis, Addi- son's disease, paroxysmal hemoglobinuria, exoph- thalmic goiter, lead poisoning, syphilis and other diseases. 2. Use of hypnotics such as trional, veronal, sul- pronal and tetronal. Test for HematoporiDhyrin. Strictly spectoscopic. Take 5 cc of urine and add 10 cc of 10% XaOH. Filter and to the precipitate add 5 to 10 drops of dilute HCl and 15 cc of alcohoL Filter and examine the filtrate spectoscopically. Abnormal pigments of the urine. A. Blood pigments : Hemoglobin, methemoglobin, hematin and hematoporphyrin. Occurrence : I. Hematuria. A condition in which blood as such is present in the urine, and is visible with the naked eye or with the microscope. The urine is turbid, red-tinged, smoky, and sometimes clots are seen. Occurrence of hematuria : 1. General diseases. Yellow fever, typhoid fever, smallpox, leukemia, and purpura. 2. Renal origin: (a) Acute congestion or inflam- mation, (b) acute congestion following poisons, (c) renal infarction, (d) stone in kidney, (e) tbc. of kidney, (f) tumors of kidney, (g) parasites, such as filaria and bilharzia. 8. Genitourinary tract conditions : Passage of stone, Dietl's crisis, stone in bladder, tumors of bladder, and urethral conditions similar. 4. Traumatism, o^Derative or accidental. 5. Renal epistaxis. In this condition sudden 39 unexplained liemorrliages occur with no pain. It occurs in one or the other kidney and does not lead to more serious conditions later on. IT. Hemoglobinuria. In this condition the formed elements of tlie blood are absent and merely the pig- ments are present. The urine is usually clear and dark brown. Occurrence : 1. Toxic conditions, following severe burns, expo- sure to cold, poisons and fevers. 2. Essential or paroxysmal type characterized clinically by: (a) Pronounced hemoglobinuria. (b) Aching in the lumbar region. (c) Chills, fever and headache. This type often follows exposure to cold. In this condition the patient's own blood has amboceptor, Avhich is capable of uniting with patient's own cor- puscles. Cause not known. Majority are spyhilitics, acquired or congenitally so. Tests for hlood and hemoglobinuria: Chemical tests. 1. Heat and acetic acid test. Brown coagulum forms, which tends to float. Decolorized with acid alcohol. 2. Heller's test. Make urine alkaline with sodium or ammonium hydroxide, gently warm. A precipi- tate of phosphates and carbonates forms, which turns brown. 3. Teichmann's hemin test. To urine add XaOH. Filter and wash with water. Dry by pressing be- tween filter papers. Fragments of ppt. are placed upon glass slide, to which is added a crystal of XaCl. Add three drops of glacial acetic acid and place cover slip over mixture. Heat gently, do not 40 boil, and as acid evaporates replace it. As soon as the material becomes brown, allow to cool slowly. A positive test shows, microscopically, crystals rhomboid in shape and in sheathes. These crystals are hyprochlorate of hematin. Errors : 1 . Heating too much. 2. Too rapid cooling. 3. Excess of NaCl. Sensitive in dilution 1 to 100,000 parts. Giiiac Test: For blood — 1. Make fresh tincture of guiac with alcohol; should be shade of light yellow ; mix with equal vol- ume of ozonized oil of turpentine, or HoOg. 2. Four or five cc urine, to to 8 drops glacial acetic acid ; allow to stand 8 minutes ; extract with ether. Pour solution 2 upon 1 in such manner as to form layer ; a deep blue ring will form if blood is present. Benzidine Test for Blood — 1. Two per cent, alcoholic solution is taken and mixed with equal volume ozonized oil of turpentine, or H2O2. 2. Four to five cc urine plus 6 to 8 drops glacial acetic acid; let stand 8 minutes and extract with ether. Stratify 2 upon 1; a greenish ring will form at line of contact if blood is present. III. McthemogJoljin. Occurs in spontaneous de- composition of blood ; also following poisoning by the chlorates, nitrates, arsenic, acetanilid, antipyrene. sulphonal, turpentine. IV. Hematoporpliyrin. See previous discussion. B. Bile pigments. Bilirubin is the most frequent. Sources of choluria (a) Hepatic origin. (b) Hematogenous origin. 41 K- ^- o I ^ ^ ^ ^ I \i- 42 Hepatic origin occurs: 1. Cirrhosis of the liver, 2. Cancer of the liver. 3. Obstruction to the biliary passages. Hematogenous origin occurs : 1. AVhere blood pigments are present in ex- cess of the mobilizing power of the liver, as in pernicious anaemia, ma- laria, pneumonia, yellow fever. (All jaundice is essentially obstructive in origin.) Appearance of the urine in choluria : greenish yellow, brown, pure green. * Tests for l)ilc' : 1. Foam test. Shake a specimen of urine which produces foam. If bile is present the foam x:)ersists and is of yellowish color, ^'ormal urine does not produce much foam, and the foam which is produced is white in color. '2. (xmelin's test. Kegent : Strong HNO3 pl^^s HNO,. This can be made by boiling con. HNO3, in which is placed a match stick, until it takes on a yellowish color. Method : The urine is layered upon the reagent thus made. A positive test shows a green ring at the line of contact, and a yellow ring slightly above. Below the line of contact appear a series of colors ranging from blue to red from above downward. Errors: 1. Too much NHO,. 2. Too much albumin. :]. Urine containing too much indican. 4. Urine too concentrated. 5. Urine containing antipyrene or thymol. These drugs can be ruled out satisfactorily. 43 Rosenhacli's modification of GmelUi's test. Acidif}' urine Avith HCl and filter 4 or 5 times through same filter paper. Let paper dry. Touch a single drop of HNO, plus HNO, solution to the paper. Concentric rings will appear, green on the outside. Smithes test. Make urine acid with 5% acetic acid. Layer upon urine 1% alcoholic solution of iodin. Bile pigments give emerald green at line of contact some time after, which tends to diffuse upward. (Kather indefinite test.) Nakayama^s test. To 5 cc. of urine add from 5 to 10 cc. of 10% solution of barium chloride. Kemove ppt. by decantation. Treat the ppt. with the follow- ing reagent : 95% alcohol 99 cc. Con. HCl 1 cc. Ferric chloride .4 gm. Bring slowly to a boil. Bile pigments give emerald green. If urine changes to green, add HNOg and get red color. (Indefinite test.) C. Melanin. Normal j)ignient of hair and choroid coat of the eye. Pathological increase due to over-activity of the cells which form it. Occurrence: Melanotic sarcoma, Addison's dis- ease, ochronosis and sometimes in malaria. Urine is colorless when excreted, but turns dark either upon standing or the addition of alkaline or oxidizing agent. 44 Tests: All three of the following must be posi- tive : 1. Ferric chloride gives black ppt. 2. Precipitate soluble in sodium carbon- ate (black). 3. Mineral acids plus sodium carbonate solution give black ppt. D. Alkapton hoclies. When in urine designated alkaptonuria. Occurs in people who have an inborn hereditary error in metabolism. It is characterized by the inability of the body to break up the benzene ring. Tyrosin and phenylalanin are changed to uroleucin and homogenstic acid. Urine is nor- mal in color when voided, but becomes dark on standing. Occurs in children Avhose parents are lir.>t cousin?. It is a life-long condition, but not dangerous. Sometimes victims develop ochronosis. E. FJieuoJ do ivatii'cs. These substances consist of sulphuric acid in conjugation with phenol para cresol, pyrocatechin, and hydroquinone. They are excreted mostly in conditions associated with ijutre- faction in the intestinal tract or elscAvhere in the body. Sources: (a) Administration of drugs, (b) Pro- tein metabolism. F. Diazo compounds, due to alloxyproteic acid. Tests : Ehrlicli's Diazo Reaction. Reagent : Solution a. Aqueous sol. sodium nitrite 0.5 % Solution b. Sulphanilic acid 5 grams Concentrated HCl 50 cc. Water q. s. ad 1000 grams 1 part of sol. (a) plus 50 parts of sol. (b) plus equal vol. of urine. Shake quickly. Add .1 volume of 45 NH^OH aud shake quickly. A positive test is a deep- red color throughout the urine and a red foam. Red foam is the most important. After 12 hours a gran- ular greenish precipitate occurs. When doubtful, wait for this test. Xaphthalene, opium, chrysarobin and otlier drugs may give color quite similar, but the foam is not red and no greenish ppt. is obtained. Occurrence: The diazo reaction is never found in health. It does occur in typhoid fever, where it finds its most useful application. It is said to occur in the first or second week of the divsease in 80% of the cases. In case of relapse the test again becomes positive. The reaction also occurs in pneu- monia, scarlet fever, very frequently in measles be- fore the rash, and sometimes in tuberculosis. It never occurs in rheumatic fever or meningitis. If the test is persistently positive in tuberculosis, it indicates a progressive lesion. Ehrlich's egg yellow test : This is due to the pres- ence of urobilinogen. This test is the same as the former, except that after adding the XH^OH the urine takes on a yellow color. Occurrence : Just prior to the crisis in pneumonia. Russo's test: 5 cc. of urine plus 5 drops of 1-1000 methylene blue. Positive reaction gives emerald green color. Blue color is negative. -Occurrence: It is said to be negative in miliary tb'c and positive in typhoid (Dr. Evans did not find it satisfactory). Positive reaction said to occur in measles, small pox and chronic and suppurative tbc. The reaction is said to be negative in varioloid, varicella, scarlet fever, miliary tbc, appendicitis and malaria. G. Medicines. Phenolphthalein, eosin, salol, methylene blue, etc. 46 H. Chijluria, characterized by the excretion of emulsified fat. Uriue varies from cloudy yellow to milky appearance. The surface is covered with a layer of free fat. Causes : 1. Parasitic. Observed in the Far East, due to infection by one form of filaria. Elephantiasis is often as- sociated with it. 2. Non-parasitic. Etiology unknown. Oc- curs spontaneously and intermit- tently in people otherwise well. Sometimes due to rupture or ob- struction of cysterna chyli. I. Lipuria. Characterized by the excretion of fat in microscopical quantities. Causes : 1. Large ingestion of fats. 2. Extensive bone injuries. 3. In diabetes. 4. Frequent in tbc. 5. After catheterization when oil is used. TABLE OF COLOR OF URINE AND ITS CAUSE. Yellow and Orange Pink and Red Normal Hematuria Urobilin Hemoglobinuria Choluria Hematoporphyrin Medicinal #when alkaline Rhubarb # Pyramidon Senna # Trional Santonin # Sulphonal Phenolpbtbalein# Eosin Cbrysopbanic ac# Rosanilic acid Cascara# Brotvn and Black Blue and Green Milky or Whitish Choluria Methylene blue Lipuria Melanuria Urobilin Chyluria Alkaptonuria Phosphates Phenol derv's. Bacterial infect. "Black Water Fever" Dirty container. Indicanuria Medicinal Salicylic ac Salol Resorcin 47 Optical activity of urine: Xorinally levo-rotatorv from .01 to .18 deg. Increased : 1 . In excretion of levnlose. 2. Glycnronic acid excretion. 3. Albnmin (I/2 gni. per 1000 cc). 4. Diabetes (B-Oxybutyric acid). INORGANIC URINARY CONSTITUENTS. The composition of tlie urine depends upon the diet of the individual, both in quality and quantity. Normally 1200 to 1500 cc. are excreted daily with 60 to 70 gms. of solids, organic and inorganic. Inorganic 25 to 30 gms. Organic 35 to 40 gms NaCl 10 to 15 gms. Urea 15 to 40 gms P205 1 to 5 gms. Ammonia .7 to 4.27 gms S03 1 to 3l^ gms. Creatiniii 1.0 to 1.5 gms COS 9 to ? gms. Uric acid .5 to 1.25 gms Na20 4 to 714 gms. Hippuric acid .7 gm. K20 2 to 4 gms. Other CaO .1 to .3 gm. constituent s 1.5 to 2.3 gms MgO * .11/^ to .4 gm. Fe 1 to 11 mg. Chlorides. The excretion of chlorides dep ends upon the intake in health. Source: (a) Vegetable foods in combination with K and Ca. (b) NaCl seasoning most important source. Chlorides are taken in order to prevent the ac- cumlation of K salts in the blood. There is a chloride balance as characteristic as the N bal- ance. Increased ingestion of NaCl is followed by increased excretion. Chloride poor or free diet gives a drop in the excretion to a certain extent, but no further. The proportion in the blood is a constant, as well as that in the tissues. 48 Eetention occurs : 1. Active stage of all fevers. In pneumonia there may be a suppression till the crisis. A chloride crisis also occurs. 2. Formation of transudates and exudates. 3. Profound and continuous vomiting and diarrhoea. 4. Hyperacidity. 5. Oedema (myocardial or nephritic). Theories of retention. 1. Histo-retention theory (Strauss -and Loeb) : Excess of salts in the tissues requires water to maintain osmotic pressure. 2. Colloidal (Fisher) : In cases of oedema the colloids in the tissues have increased hydrating capacity. Salt excretion: 1. Following febrile states. 2. Absorption of transudates and exudates. 3. Polyuria. 4. Pneumonic crisis. 5. FolloAving oedema from salt retention. 6. After chloroform anaesthesia. Quantitative tests: 1. Volhard's method (1874) : Precipitation of chlorides in a known volume of urine by an excess of standardized solution of Agl^Og in HNO3, and then the titration of the excess of AgNOo with NH.SCN, using iron ammonia alum as indicator. (Objection to method is that it requires 4 solutions.) 2. Arnold's method, a modification of the former (1885). Differs from the former in the strength of the standard solution and fil- tration after addition of AgNOg. 49 Solutions: 1. AgXOg solution such that 1 cc. equals .01 gm. NaCl or 29.075 gm. AgNOg in 1000 cc. H^O. 2. jN^H^SCX solution such that 2 cc. equals 1 cc. Ag^S^Og sol. 3. Saturated solution of iron ammonia alum. 4. Pure HXOo. Procedure : Uriue 10 ce ^ Pl'^ce in a 100 cc. volumetric flaslv ' for 10 iniimtes. agitate from time to i time. Overcome coloring by 3 to 5 Std. AgXOs 20 cc. I drops of 10% sol. of potassium per- ^ mangnate. Add H,0 up to 100 cc. H>0 50 cc.) ,„^„ flij^^ To 50 cc. of the filtrate add 5 cc. of the iron am- monia alum sol. for indicator andi titrate with NH^SCN to end reaction, which is red-brown color. 3. Lutke Martins method (1S92) : Solution : A. AgXO.. 17.5 gms. 2d% HXO3 000.0 cc. 25% HXO3 equals 225 cc. con. XHO3 in 675 cc. of distilled H.O 10% iron ammonia alum 50.0 cc. Water q. s. ad. 1000.0 cc. B. X/10 XH.SCX Titrate solution A against a X/10 HCl solution. In making solution B dissolve 7.6 gm. of XH^SCN in less than 1000 cc. of water. Add water till 1 cc. of it equals 1 cc. of A, which is determined by titration. Method of performing determination: 10 cc. of urine plus 25 cc. of X/10 AgXOg plus water q. s. ad 100 cc. Add a few drops of potas- sium permangnate to decolorize urine if necessary. Allow to stand and filter or draw otf 50 cc. Titrate the filtrate with X/10 XH^SCX to the end point, using iron ammonia alum as indicator. 50 Calculation: Example 6.2 cc. :NV10 NH.SCN to produce end reaction. 2 times 6.2 equals 12.4 cc. AgNOg are uncom- bined in 10 cc. of urine. 25 minus 12.4. equals 12.6 cc. N/10 AgNOg are combined as AgCl in 10 cc. 1 cc. X/10 AgXOg equals .00585 gm. XaCl. 12.6 times .00585 equals gms. NaCl per 10 cc. (10 to 15 gms. per 21 lirs. normal), rrinciple : When X/10 XH.SCX is added it com- bines with the unused X/10 silver nitrate as long as there is any present. When it is all in combi- nation with NH^SCX it begins to combine with the iron ammonia alum, wliich gives a brown color and is the end point in the reaction. Purdy's centrifuge method : 10 cc. of urine, 1 cc. of NHOg and 4 cc. of a 5% solution of AgXOg are placed in a special centrifuge tube. Agitate and centrifuge at 1200 revolutions for three minutes. Eead the number of grams per 1000 on the scale. Bayne-Jones method : Either use a special flask or an ordinary 25 cc. graduate. In case tlie special flask is used, the urine is added to the mark U. Titrate with N/20 XH^SCN solution to the end point and read grams per liter on the scale (iron ammonia alum indi- cator). In case the 25 cc. graduate is used, add 5 cc. of urine and 10 cc. of X/20 AgXOg. Titrate as before with X/20 XH.SCX, and note the reading. 51 . Calculation : V equals total volume at eud of titratiou. Y-15 equals number ec. of N/20 NH^SCN V-15 equals number cc. N/10 AgNO^ uncombined 2 Y-15 10 equals number ec. N/10 AgNOg combined as AgCl 2 1 cc. N/10 AgCl equals .00585 gm. NaCl V-15 10 X .00585 equals gm. NaCl in 5 cc. urine 2 Y-ls 10 X .00585 X 200 equals gm. NaCl in 1000 cc. 2 20.475 minus .585 Y equals gms. per 1000 cc. (.1 to .3 gm. error) Phosphates. Excreted as sodium, potassium, cal- cium and magnesium salts of HoPO^ as well as with glycerin from the breaking down of lecithin. Source: 1. Food ingested. 2. Breaking down of proteins, ingested or endogenous. Nucleo-proteins most abundant source. Amount : 1 to 5 gm. per day expressed in terms of P2O5 Excretion : Depends upon amount taken. Greater on alkaline diet. Majority of earthy phosphates are excreted in the feces (Ca and Mg). Phosphate metabolism is still in a state of uncertainty, for it is not known where P2O5 is stored. Bone takes up a great amount, but does not account for amount ingested. Eatio between N excretion and PgOg is P :N : :1 :7. Increased : 1. Animal diet. 2. Ph^^sical exercise. 3. Starvation. 4. Conditions where protein disintegration is sroins: on. 52 5. Phospliatic diabetes, where urine lias char- acteristics of diabetes, but no sugar. There is associated with it dryness of the skin and excessive thirst. The c-h metabolism is not abnormal. G. Phosphorous poisoning. 7. Meningitis and tuberculosis. 8. Emotional states. 9. Mental work. Decreased : 1. Vegetable diet. 2. Certain diseases : Addison's disease, Hepatic cirrhosis. Certain types of nephritis. Acute yellow atrophy, Chronic lead poisoning, Certain types of bone disease. 3. Pregnancy. 4. Euns parallel with uric acid excretion in gout. Phosphaturia is characterized by the frequent and constant excretion of cloudy urine at the time of voiding. It is noticed mostly in nervous, sexual neurasthenics of the male sex. Noticed also in cyclic vomiting of children and hypoacidity. This condi- tion represents not a disease, but a condition in which the amount of acidity of urine is diminished, now called alkalinurin. Stilpliates. Excreted in three forms: 1. Preformed or neutral. 2. Conjugated or ethereal. 3. Xeutral, unoxidized or organic. Total excretion about 2l^ gm. in 24 hours on a mixed diet, expressed in terms of H2S0^. Source: Amount in diet relatively small. Most 53 of it arises in the destruction of protein. Amount excreted increases with increase in protein cata- bolism. Ethereal sulphates result from the conjugation of indol, saktol and phenol ^Yith sulphuric acid, and can be taken as a fair estimate of protein decompo- sition. Occurrence : 1. Excessive intestinal putrefaction. 2. Kich protein diet. 3. Hypoacidity. 4. Massi\'e pus formation anywhere in body. Neutral sulphates : increased in jaundice and cys- tinuria. Ckirhonates : Yarj with the amount of carbonate forming material in the food. Vegetables contain organic acids whicJi are easily converted into car- bonates. Generally sedimented as CaCOo. SocUuin and potassium : Excreted as salts, amount determined as XaoO (-1 — Ti/o) and ICO (2 — 4). Sources : 1. NaCl in the food. 2. Potassium in vegetable foods. The excretion is of little significance clinically. Calcium and magnesium: Excreted generally as phosphates (CaO .1— .3) (MgO .U/2— .4). Source : Food, most of it is lost in the feces ; bone destruction, diabetes ; excretion is little understood ; its significance is increasing. Iron : Always present in small amounts. Increased in blood destruction, such as fever, malaria, perni- cious anameia. In diabetes it runs parallel with the sugar output, 2% mg. of iron to 100 grams sugar. Heavy metals : Mercury, lead and arsenic found pathologically. 54 NITROGENOUS BODIES OF URINE. Amount normally excreted varies between 10 and 16 grams a day. Amount excreted depends upon : 1. Amount of food ingested. 2. Tissue metabolism. In health there is a nitrogen equilibrium, i. e., the N. excreted equals the N ingested. In starvation a level is reached on the fourth or fifth day, which will last three or four weeks. When food is again taken the amount of excreted N is less than the ingested N. People feel best on from 4 to 6 grams of N excretion. To determine the N excretion the following factors must be taken into consideration: 1. Total N of food. 2. Character of food in terms of ability to produce alkaline or acid urine. 3. Age of patient. 4. Previous state of nutrition. 5. Previous diet. Previously there should be seven days of diet with regulation of : a. Diet, b. Exercise, c. Amount of food intake. d. The condition of the intestines. iSiitrogen Partition : Mixed D. N-free. Mixed. N-free. Total N 16 gms. 3.6 gms. 100% lOOfo Urea 13.9 2.2 86.87 61.7 NH3 .7 .42 4.37 11.3 Uric Acid 12 .09 .75 2.5 Creatinin 58 ..6 3.63 17.2 Undetermined.. .7 .29 4.37 7.3 Urea N represents great bulk of total N output in 24 hours. In N free diet, urea N decreases; am- monia and creatinin are increased, at expense of urea. 55 ^^itrogcn excretion : Physiological — Increased : 1. On heavy protein intake. 2. Fonr to 6 days after birth. 3. Excessive intake of water. 4. Exercise. Decreased on diets rich in fats and carbohy- drates. Pathological — Increased : 1. In acute febrile conditions with high fevers. There is no relation between the height of temperature and the amount of N excreted. N balance can be maintained by proper diet. 2. Chills. 3. Increased respirations. Urea is relatively decreased, while ammonia salts, organic acids and uric acid are increased. 4. Toxic conditions, such as cancer, leukemia, exophthalmic goiter. 5. Diabetes mellitus and insipidus. 6. Absorption of exudates. 7. JS'ephritis, especially in albuminuria of marked degree. Decreased : 1. Convalescence after fever. 2. Dysentery. 3. Formation of exudates. Methods of N determination. Kjeldahl : The principle of this method is the oxidization of the organic products with the production of sul- phates. XH3 plus HoSO^ equals (NHJ^SO^. The NH3 is liberated by the addition of strong NaOH and is received in a known amount of standard acid. 5^ Procedure : luto a pyrex kjeldahl flask place Urine 10 cc. K2SO4 10 grams or a teaspoonfiil (oxi- dizer). CUSO4 1 medium-size crystal (caty- litic action). Con. H2SO4 15 cc. Heat gradually to the boiling point and continue to boil till the solution takes on an absolutely green color, and boil five minutes longer. Allow the solu- tion to cool and then add the following : Water 250 cc. Talcum 2 tablespoonsful. 40% NaOH 120 cc. This should be added last and in such a manner that it does not mix with the solu- tion. Pour on side of tilted flask and it will go to bottom. Connect the flask with a distilling apparatus and mix the contents by shaking. Either distill 30 min- utes or distill over 150 cc. This distillate is re- ceived in 25 cc. of N/4 H2SO4 and the excess of acid is titrated with N/10 NaOH, using phenolphthalein as an indicator. 1 cc. N/10 H2SO4 equals .0017 grams NH3. 1 cc. N/4 H2SO4 equals .00425 grams NH.. N equals .823 XNH. by weight. .00425 X. 823 equals .003497 grams N for each cc. of N/4 H2SO,. .0017X.823 equals, .0014 grams N for each cc. of N/10 H2SO4. Folin's method: This is a colorimetric test. 1 cc. of urine is put through the oxidizing process and received in 57 N/HCl. 5 cc. of Nessler's reagent: Hgl 10 grams KI 5 NaOn 20 H.O 100 cc. are added. The color of this is compared with a standard color made up of 5 cc. of the reagent plus 1 mg. of N. TJrea. About 80 to 90% of the total N in a mixed diet is excreted in the form of urea. GO to 65% of the total N is excreted in the form of urea in an N-free diet. From 15 to GO grams, or an average of 30 grams, are excreted in a mixed diet per day. Modes of formation : 1. NHo salts. (Protein — hydrolTsis — amino acids — NH3.) Action of bacteria and enzyme action of tissues. 2. Deamidization method. (Protein — amino acid — blood — tissues — protein.) When amino acids are in excess NH3 is split off in the liver and oxy-organic acids are formed. The liver protects the body from NH3 poisoning. 3. Arginin plus arginase gives urea and or- nithin. Variations in excretion. 1, Physiological 1 Increased and decreased same 2. Pathological J as total N. 3. Relation of urea to liver function. 4. Relation of urea to renal function. One can't determine the renal function by the esti- mation of the urea alone, but one must take 58 into consideration the amount of urea in the blood. 5. Kelation of urea to acidosis, formal reac- tion of blood alkaline. The fixed alkalinity of the blood can't be reduced beyond a cer- tain point without certain things happen- ing. As an excess of acids is formed^ an excessive amount of NH3 salts is formed. Urea decreases as output of NHg increases. Qualitative tests for urea. (Not used clinically.) 1. Biuret. Crystals of urea heated to boiling give cyanuric acid and biuret. Add CuSO^ and XH4OH and get blue color. 2. Furlurol. 1 to 2 drops of furfurol plus 1 to 2 drops con. HCl, plus 1 to 2 drops urine. Positive test gives dark blue to black color. (Quantitative estimation of urea : Knop-Hufner method. This test relies upon the decomposition of urea v\irh sodium hypobromide and the measurement of the N evolved. The reaction takes place according to this equation : CO(XH2)2 + SNaOBr = 3NaBr -f 2X + COo -f '2Ii.A\ The COg is absorbed in an ex- ce:^s of alkali and the amount of N gas measured. Doremus Ureometer. The principle of the Knop-Hupfer method is used and a graduated ureometer measures the amount of gas. Solutions : 1. 20% XaOH. 2. Bromine. When ready to use, add 1 cc. of the bromine to 40 cc. of the XaOH solution. Fill the ureometer tube and add by means of a curved pipet 1 cc. of urine to the upright part of the tube. Read grams off on the scale. Method unsatisfactory. 59 PoUn metJiod. The principle of this method is that crystalized magnesium chloride boils in its own water of crjs- talization at 160 degrees centigrade. If urea he present it is decomposed into ammonia and carbon dioxide. If acid be present the ammonia formed will combine with the acid and can be titrated. Urease method (Marshall). The principle of this method is this : The soy bean extract contains an enzyme capable of splitting urea into ammonium carbonate. Characteristics of the enzyme : 1. It is soluble in water. 2. It reacts quantitatively with urea. It will split off a certain amount in a given time and no more. 3. It does not form NHg from any other source than urea, and will form NH3 in the pres- ence of any other substance, except. (5j. 4. Its optimum activity is 55 deg. C. 5. Its activity is destroyed by acids, heavy metals and alcohol above 20%. 6. It has a self-retarding effect. After a cer- tain amount of (^2:4)2003 is formed its action becomes inhibited. Direct method of performing test: Test tube A. 10 cc. of urine. Test tube B. 10 cc. of urine. 1 — 2 urease tablets dissolved in water. Allow these to stand: 1. At room temperature over night, or 2. 45 to 50 deg. C for 1 hour, or 3. 37 deg. C. for 3 hours. Titrate both tubes for alkalinity, using methyl orange as an indicator. 60 Subtract the no. of cc. used in titrating A from the no. used in titrating B. 1 cc. of N/10 HCl equals .003 grams of urea or .0014 grams X. The disadvantage of the test is that the whole solution becomes clouded and the end reaction is an (obscure one. Indirect method. Test tube A. 5 cc. of urine. Aqueous solution of 1 or 2 urease tablets. A layer of kerosene to prevent foaming. Stopper and allow to stand as before. Test tube B. 25 cc. of X/10 HCl. 2 drops of 1% sol. alizarin, A layer of kerosene. After tube A has stood its proper length of time, connect it up to a suction apparatus in such a way that the indrawn air first passes through a solution of HgSOi. Allow air to be drawn through for about a minute in order to remove any free ammonia that may be present. Now, connect tubes A and B in such a way that the air current passes from A into P. When this has been done, add a teaspoonful of dry potassium carbonate to tube A, quickh^ cork, shake and start suction apparatus. Allow air cur- rent to pass from A into B for about 30 minutes. The ammonia in A is freed and caught in the N/10 HCl in B. Titrate the excess of HCl in B with X/10 XaOH. Subtract the number of cc' s necessary to bring about the end reaction from 25 cc, the amount of X/10 HCl originally added. Repeat this process, using 5 cc of urine to which no urease solution is added. This gives the preformed ammonia. This value subtracted from the former gives the amount of XHo formed from urea. Make the calculation of the amount of urea on 6.1 the basis of: 1 cc. of X/10 HCl equals .003 grams of urea or .0014 grams X. ZJiic acid. Amount : .2 to 2.0 grams per day. Mixed diet .37 gm. or .8% total X. N-free diet .09 gm. or 2.5% total X. Origin. Uric acid is not a product of protein decomposi- tion. Xucleo-protein plus pepsin, trypsin, etc., gives nucleic acid and protein. Xucleic acid plus tetranuclease gives purin and ' pyrimidin dinucleotide. Purin nucleotide plus pliosplio-nuclease and purin nuclease give: the former, adenosin and guanosin ; the latter, adenin and guanin. Adenin plus adenase gives hypoxanthin. Guanine plus guanase gives xanthin. Adenosin plus adenosin deaminase and guanosin plus guanosin deaminase give xanthin and inosin, which when the c-h radical is split off give xanthin and hypoxanthin. Xanthin and hypoxanthin plus xanthin oxi- dase (liver) gives uric acid. Sources of uric acid : 1. Exogenous. The greater portion of nucleic acid comes from the diet : sweet bread, liver, caiTein and theobromin. 2. Endogenous. a. Breaking down of tissue nuclei. b. Purin bases free in the body. 3. Synthesis. There is no evidence that this occurs in man, but it does occur in birds. Uric acid destruction in the body. In birds it is .destroyed by ferments and allan- toin is the excretory product. 62 The following facts are known : 1. Uric acid fed can be recovered quantitatively in the urine. 2. Endogenous purine metabolism in persons on an N-free diet is constant. 3. Uric acid can be broken down by other routes than allantoin. a. Glycocoll. b. Oxalic acid-urea. c. Persons fed on purin bases excrete half in the form of urea. Purin bodies can be excreted in forms not going through the uric acid s'tage. 4. When uric acid reaches the system it is ex- creted as such. C/haracteristic properties of uric acid. In the cold it is sparingly soluble in H2O (1 — 40,000). It is fairly soluble in blood serum (1 — 1,000). Its best solvent is a solution of urea. It is insoluble in alcohol, ether, chloroform and acetic acid. It is somewhat soluble in HCl, II2SO4 and weak alkalines. Solutions of uric acid in water are neutral to litmus. Uric acid in sufficient con- centration reduces Fehling's solution, but not Nylander's. Uric acid is kept in solution under the following conditions : 1. With neutral phosphates. 2. When pigments are present. 3. As neutral urates. 4. As monosodium or biurate — Ammonium biurate (urinary type of sedi- ment). Sodium biurate (gout). Excretion of uric acid. 63 Increased : 1. Purin or meat rich diets. 2. Increased protein decomposition. 3. When lencocvtosis is predominant feature in leucaemia, especially when treated with X-ray. From 12 to 15 gms. per day is sometimes excreted. 4. Pernicious anaemia. (In secondary anaemia less than normal.) 5. Articular rheumatism and after medication with salicylates, atophan, colchicum and urotropin. Decreased : 1. Vegetable diet. 2. Certain types of chronic nephritis. 3. Chronic lead poisoning (diagnostic). 4. Quinine and opium. Uric acid in relation to gout : All theories have fared badly. 1. In cases of chronic gout the excretion of uric acid upon a given diet will be the same as in normal individtials on the same diet in most cases. 2. The excretion of uric acid in chronic gouty in- dividuals persists in being lower than normal. 3. Gout}' patients fed with purin bases will show a lower curve of uric acid excretion than normal. 4. On a purin free diet gotity patients will show a lower endogenous excretion than normal. 5. The blood of gouty patients contains more uric acid than normal, 4 to 5 mg. instead of 1 mg. 6. The elimination of uric acid is less than normal from 1 to 3 days preceding an acute gouty attack. 7. With the start of the acute symptoms there is a rise of the excretion above normal which 64 lasts for three or four days, and then is fol- lowed by a much lower output than normal. 8. The relation between an excess of uric acid in the blood and in the urine in acute gouty manifestations is not known. 0. Gouty individuals show utter abnormal condi- tions of metabolism. 10. Probable explanation of gout is that gouty individuals do not possess necessary uric acid elimination and certain tissues seem to pos- sess an aflfinity for uric acid. Factors modifying uric acid excretion. 1. Diet. 2. Tissue metabolism. 3. Indeterminate amount of free purin bases. 4. Amount of conversion of uric acid into urea. 5. Amount of uric acid destruction in the body. 6. How much synthesis occurs. 7. Varyino: degree of blood retention. 8. Kenal capacity of eliminating uric acid. 9. Amount of urinary phosphates. 10. Degree of urinary concentration. 11. Akalinity of urine. Quantitative uric acid determination. Folin method : Urine 300 cc. Folin's reagent To* cc. (gives precititate of phosphates) Folin's reagent : Am. Sulph. 500 gm. Uranium acetate 5 gm. 10% acetic 60 cc. H2O q. s. ad. 1,000 cc. Wait 15 minutes and filter. Place 125 cc. of the filtrate (which equals 100 cc. of urine) in an Ehrle- meyer flask. Add 5 cc. of concentrated XH^OHj let 65 stand 24 hours and filter. Collect the precipitate of ainmoniiTiTi urate, wash it with ammonium sulphate until the filtrate no longer shows a reaction with AgXOo, pierce the filter paper and wash the precipi- tate into a breaker Avitli 100 cc. of water. Add 15 cc. of con. HoSOj, lieat over a fiame, and while still hot titrate witli N/20 potassium xjermanganate (1.5(>7 gn?. in 100 cc. ) to end point, Avhich is a red- dish blush for from 15 to 30 seconds. Calculation : 1 cc. of X/20 potassium permanga- nate equals 3.75 mg. of uric acid. (Uric acid has the proi)erty of reducing i^otassium permanganate.) Correction: ]"or each 100 cc. of urine add 3mg. of uric acid, for a certain amount of urate is soluble and does not change into uric acid. Aiiiinoiiia : De])en upon protein, intake and runs parallel with X excretion. On a mixed diet about .7 gms. are excreted in 24 hours or 4.37% of the total N, and upon an N-free diet about .42 gm. or 11.3% of the total N is excreted. Increased : 1. Decreased oxidation. 2. Acute febrile conditions. 3. Liver diseases. 4. Uraemia. 5. Toxic vomiting of pregnancy. 6. Diabetes, characteristicalh' abnormal. 7. Starvation. 8. Chloroform poisoning. Decreased : 1. Xephritis. 2. Most akaline therapy. 3. Conditions with Ioav HCl. XHo elimination is associated with acidosis. In this condition one finds : 1. Increase of acids with formation of neutral salts. 2. Lowered blood and 66 tissue alkalinity. 3. Carrying capacity of the blood for CO, lowered and CO2 accumulates in the tissues (tissue asphyxia). 4. Increase in the H concentra- tion of the blood stimulates the respiratory center and air-hunger results. Occurrence of acidosis : 1. Diseases with clinical symptonis. Diabetic acidosis. Acute nephritis and acute diseases associated A\ith nephritis. In late stages of primary and secondary con- tracted kindey. Food intoxication of children. Atrophic cirrhosis. 2. Diseases without clinical symptoms. Acute febrile conditions. Advanced cachexia. Severe anaemia. After general anaesthesia. Cardiac conditions. Quantitative ammonia determination. Schlosing method : The objection to this method is that the results are too high. Folin's method : 10 to 20 cc. of urine are introduced into an aerat- ing cylinder and a layer of kerosene added to reduce tlie tendency to foam. The apparatus is connected with a flask containing a knoAvn quantity of X/10 HoSO^ (20 cc.) and sufficient water to well cover tlie absorption tube. About one gram of sodium carbonate is added to the urine and the ammonia liberated is aspirated by means of a suction pump into the known acid solution. The current of air should first pass through a dilute £[280^ solution in order to absorb any ammonia that may be x)resent 67 in the indrawn air. Aerate for one hour and titrate the excess of acid Avith X/10 NaOH, using methyl orange or alizarin as indicator.. 1 cc. of N/10 H.SO, equals .0017 grams of XH.. or .0014 grams of X. Ci'catiu'ui : About .50 gm., or 3.0% of the total X, is excreted upon a mixed diet, and about .0 gm., or 17.2% of the total X, is excreted on an X-free diet. It is an anhydride of creatin, but the relationship between tiie two is not clear. Creatin probably comes from arginin, and creatinin is formed by the action of anliATlrating ferments. There is a remark- ably constant excretion of creatin irrespective of the X intake. Folin considers creatinin a valuable index of protein metabolism, while Shaffer thinks it an index of some special i)rocess of normal meta- bolism occurring mostly in muscles. Exogenous elimination depends upon the ingestion of meats. Endogenous elimination has the following char- acteristics : 1. Constant on X-free diet. 2. Varies with different individuals. o. Apparently independent of total X metabolism - J:. Depends upon weight of individual. A normal individual excretes from 7 to 12 mg. per kilo in 21: hours. Source: Largely due to muscle metabolism of the body. It is, however, formed in other than muscle tissue. There is a diminished excretion in anaemia, marasmus, myositis ossificans, chlorosis, phthisis, chr. diffuse nephritis, progressive muscular atrophy, and pseudohypertrophic paralysis. It is said to be increased in the acute stages of some fevers and exophthalmic goiter. The X excretion runs more or less parallel with protein decomposition, but creati- 6S nin does not. Tlie liver is one main source of forma- tion. Qualitative tests for creatinin. JaftVs test: To urine add picric acid and dilute NaOH (10%), which turns a brilliant sliade of red and which be- comes darker on standing. Upon the addition of acetic acid it changes to an ochre color. Weyl's test : To urine add nitroprussid of soda and a few drops of 10% XaOH. A deep red orange or ruby color appears, which, upon the addition of a little glacial acetic acid, turns to green and upon heating to Ber- lin blue. Quantitative determination. Folin method (colorimetric) : A standard solution is made up by the following method : One gram of pure creatinin is dissolved in 1000 cc. of water. 1 cc. of this plus 20 cc. picric add plus 1.5 cc. of 10% NaOH plus enough- water" to make 100 cc. gives the standard color. One cubic centimeter is treated in the same man- ner, and the resulting color is compared with the standard solution by means of a colorimeter. Undetermined Nitrogen. Mixed diet .7 grams or 4:.31% X-free diet .29 grams or 7.3 % This is determined by subtracting the sum of the other forms of N from the total N. Source : 1. Amino acids which are known to be excreted. Tyrosin, leucin, giycocoll (1^-2 grams per day). In cystinuria there is a perverted metabolism to amino acid; 181 such cases have been reported. These patients do not have the power to reduce the amino acids 69 further than cvstiii. Gystin calculi fre- quently form. A marked hereditary ten- dency prevails in this disease in which males are more frequently effected than females. In diaminouria putrescin and cadaverine are excreted. 2. Hippuric acid. 0.1 to 1 gram per day. Ben- zoic acid foods, such as fruit, berries, prunes, etc., combine with giycocoll. 3. Oxyproteic acid 1 These are responsible for Alloxyproteic acid J Ehrlich's diazo reaction. Their excretion is probably increased in cancer. They contain an S mollecule. 4. Allantoin. This is normally present only in traces. It exists normally and abnormally in conditions little understood. REDUCING BODIES OF THE URINE. Subjects to be considered : 1. General review of carbohydrates. 2. Qualitative determination of glucose. 3. Sugars and reducing bodies other than glucose. 4. Quantitative determination of sugar. 5. Scheme for running down reducing sub- stances in urine. 6. Acid bodies. 7. Diabetes. Carbohydrates. The available carbohydrates of the body exist as glycogen, which is stored in the liver and muscle tissues until called upon b}^ the tissues for energy, heat and tissue formation. Sugar is constantly being converted by the liver into glycogen and back again into sugar in accordance to the body needs. The balance between these two in health is such that the percentage in the blood is from .1 to .15%. 70 Origin of carbohydrates : 1. Sugars and starches. 2. Proteins and amine acid bodies. ?>. Fats. Function of carbohydrates : 1. Energy. 2. Heat. "^ B. Protecting agency for protein. 4. When in excess forms fats. 5. Actual synthesis of tissues (nucleic acid). Kegulation : 1. Supply (glycogenetic function). 2. Storage ( glycogenesis ) . 3. Tissue consumption (glycolysis). When any one of these breaks down glycosuria may result, also in cases of renal permeability for carbohydrates (renal diabetes and phloridzin dia- betes). Tlie adrenal and pancreas are antagonistic in func- tion. In health the two are in perfect equilibrium, but in diabetes the restraint is removed from the liver. The i)ancreas also seems to give off some- thing to the tissues T\iiich enables them to burn carboliydrates to COg and HgO. In diabetes this falls short and sugar is eliminated through the kidneys, due to its accumulation in the blood. A'^ormal urine contains a small amount of glucose (animal gum or isomaltose). It amounts to about .3 to .G grams in 24 hours. Glycuronic acid occurs in amounts of about .004 grams in 100 cc. Traces of pentose occur occasionally after the ingestion of fruits. Lactose and galactose occur frequently in sucklings. 71 Assimulation limit for carbohyrates (two kinds) : .1. Individuals who show sugar after the inges- tion of large amounts of starch (glyco- suria ex amylo). 2. Individuals excreting sugar following sugar intake (glycosuria e saccharo). A normal individual can take as much as 150 to 200 grams of glucose on an empty stomach without showing glucose in the urine. Per- sons with nephritis have a lower sugar threshold. The liver has the greatest toler- ance for glucose and least tolerance for lactose and galactose. Maltose, levulose and saccharose come in the intermediate group. Hamman's method of making threshold test : From 150 to 200 grams of dextrose are dissolved in 150 cc. of watei^ and flavored with lemon or orange juice. The mixture is made ice-cold with cracked ice and the volume made up to 300 cc. This is taken slowly on a fasting stomach and the blood and urine sugar are followed together, the blood being withdrawn at 1, 2, 3, 1 and 5 hours after taking and the urine being examined at the same time. Alimentary i^lycosiiria • Food — Intestines — Liver Hyperglycaemia — Glycosuria Diabetic Glycosuria Glycosuria C. N. S. — Adrenal . ' ' ' Sugar accumu- lation in blood 1 Tissues I ^— Pancreas impairment — -Liver — Epinepliriu I I These condition can occur under nervous excite- ment, fear, anx- iety and diabetes. 72 QuaJitativG test for glucose. Trommer's test: To 2 parts of urine add 1 part of 10% :N'aOH. To this add 10% solution of CuSOl till a bluish precipitate results and remains after shaking (add the CuSO^ drop by drop). Warm the solution (do not boil), and in the presence of sugar a red- dish or yellowish precipitate will form, spreading graduall}' downward and leaving a decolorized su- ]3ernatent liquid. Reaction : H2O + XaOH + 1 drop CuSO, = Cu ( OH) ^ CuSO, + 2NaOH = :\X.SO, + Cu(0H)2 Cu(0H)2 + heat = CuO (black) + H.O 2CuO + sugar + heat — reduction Yellow = CU2OH (alkalinity low) Red = CuoO (alkalinity high) l^he color depends upon the degree of alkalinity. Uric acid, creatinin glycuronic acid, albumin, allan- toin, mucin, lactose, maltose and alkapton have the propert}^ of holding Cu(0H)2 in solution and reduce it to lower salts by heating. A false reduction by these substances can be avoided by warming and not boiling, for these substances reduce only upon boiling, and it tends to occur on cooling. They give a dirty yellowish green color to the previous blue solution. Dilute urine for more accurate de- termination. Requirements necessary for positive reaction: 1. Prompt formation of a precipitate on warm- ing, settling to the bottom and leaving a clear solution above, before cooling. 2. Avoid adding CuSO^ to excess, which gives black j)recipitate and masks reaction. 7^ P*. Any discoloration after prolonged boiling should be neglected. 4. Albumin 'should be removed before perform- ing test if it is present in large amounts. Qualitative tests for sugar. Fehling's test : Solution A. CuSO, 34.65 gm. H2O qs. ad. 1000 cc. Solution B. Rochelle salts 173 gm. NaOH 50 H2O qs. ad. 1000 cc. Equal parts of solution A and B are mixed, which gives a clear blue solution. Boil mixture in a test tube and add urine not over 1/2 by volume. Sugar gives reduction in from 5 to 10 seconds. If no re- duction occurs, bring the solution back to boil. Prolonged boiling gives misleading results. Sensi- tive to .08%. Errors : Normal Constituents — Uric acid, crea- tinin, blood pigments, giycuronic acid and phenol derivatives ; also reduced by chloroform and formal- dehyde. Drugs — Trional, sulphonal, camphor, mor- phia, salicylates, benzoic acid, antipyrene and phe- nacetine. Benedict's test. Benedict's solution : CuSO, Sodium citrate Sodium carbonate HoO qs. ad. 17.3 gm. 173.0 gm. 200.0 gm. (crystals) 1000.0 cc. Method of preparing: Sodium citrate and sodium carbonate crystals are dissolved in 700 cc. of hot Wiiter. Filter while hot and add a proper amount of solution of CuSO^ to make 1000 cc. This solu- tion is permanently stable, lasting 5 to 10 years, 74 and the test is more delicate than Fehling's. Sensi- tive to .02— .05%. Technique of the test : To 5 cc. of the reagent add 5 dro]>s of urine. Boil vigorously for 2 or 3 minutes and let cool spontaneously. A positive reaction is indicated by a reddish or reddisli yellow precipitate. If less than ."*% is present a positive reaction is likely to occur only as the solution cools. In the absence of sugar the solution remains clear or slight- ly turbid. It is not reduced by normal urinary con- stituent?. Haine's test. Same as former, except that sodium carbonate is replaced by glycerin. Xot as sensitive as Benedict's. Xylander's test. Copper is replaced by bismuth subnitrate. Solution : Bismuth subnitrate 2 gm. 10% NaOH 100 cc Rochelle salts 4 gm. Keep solution in a brown bottle and don't expose to light. Technique of test: To 9 parts of urine add 1 part of reagent. Put in water bath for 5 minutes ; boiling is ijermissible. A positive test is indicated by a black ])recipitate of bismuth. In urine with only a small percentage of sugar the precipitate takes on more of a brownish color somewhat darker at the bottom. Advantages of the test : It is not reduced by nor- mal urinary constituents or alkapton. Disadvantages of test: It is reduced by hexoses, pentose, glycuronic acid, marked indicanuria and urobilinuria. It is also reduced by sulphonal, trional, rhubarb, senna, cascara and large doses of urotropin and quinine. 75 Galactose. Conditions which show galactose in the urine: 1. Infants nursing at breast and having gastro- intestinal disturbance. 2. Used in test for hepatic function (formerly). Disaccharides : 1. Lactose, which splits up into I ^, , ,^ J Galactose 2, Maltose, which splits up into I „, 3. Saccharose, which splits up into I Levulose ) Glucose Lactose : Physiologically it occurs during the puerperium and sometimes throughout the period of nursing. It also occurs in persons on an exclusive milk diet. The tolerance for it is low, 80 to 100 grams. It never occurs in diabetes. It does not ferment with yeast unless it is first broken down to glucose and galactose by ferments or bacteria, the former of which is fermentable by yeast. To obtain osazones the urine should be evaporated down in order to make it more concentrated, extracted with an alco- hol, evaporate extract, take up in a small amount of water and apply phenylhydrazine test. Their melting point is 200 degrees. Kubner's test for lactose: To from 5 to 8 cc. of urine add a large excess of basic lead acetate, boil several minutes and filter. To the filtrate add XHIrOII till a precipitate forms, redissolves and a permanent precipitate recurs, which is a brick red color. The filtrate also takes on a reddish color. A red solution with a yellow precipitate indicates glucose. Lactose is to be expected in urines giving slow reduction and D rotatory. 76 JfaUosr. There are ouly a I'eAN' cases ou record of its occur- rence in the urine. It is associated with interstitial lesions in the pancreas. It should be suspected in urine when rotation is greatly in excess of the amount of sugar present, which is determined by titration. To determine its presence, hydrolyze the urine for one hour with dilute acid. Neutralize and test by titration and with polarimeter. Saccharose. This sugar is hot known in pathological condi- tionr*. Chronic maligners often put this sugar in the urine in order to get into a hospital for the winter. The urine usually has a high specific grav- ity, and is of a syrupy nature. Hydrolyze for an hour with ^^eak acid. Neutralize with Xa2C03, titrate and polarize. Fcn)t€)itatio)i : It is said that only those sugars which have 3 or a multiple of o carbon atoms will ferment, which fact does not hold good, only 2 fermentable sugars occurs in urine — glucose most frequent, levulose next. I'cchniquc: Controls, 1. Normal urine -j- yeast = O 2. Normal urine + yeast -\- dextrose = positive 3. Specimen of urine alone = O Test. 4. Specimen of urine -|- yeast = -\- according to presence of sugar. Do not mix by violent shaking Avhen adding yeast, and thus admit small bubble?. No. 3 can be omitted by first boiling No. -1 . Allow to stand for from 3 to I hours at 371/4 degrees. Sensitive from .1 to .05%. Phenylhydrazine test : Given by all hexoses, pentoses, glucose, levulose iiiul iiiaiiuose, but tlie melting point of the ciystals is different. Teclmiqne: l^lienylliydnizine 5 drops Urine 4 cc. Clacial acetic 0.5 cc. Boil and shake 2 minutes, and while still warm add 4 to 6 drops of 20% NaOH. Do not add enough to make alkaline. Boil from 30 seconds to one min- ute longer. Cool spontaneously. If sugar be pres- ent the solution will become turbid and deposit a yelloAv precitate which indicates crj^stals of osazone. This test is extremely sensitive and is the court of last appeal for the determination of a sugar. The crystals have a characteristic appearance arranged in sheathes. To purify the crystals the filter paper is punctured and the precipitate washed into a theater containing 60% alcohol. Heat and dissolve •the crystals; evaporate down and crystals reappear. Repeat several times. The crystals are insoluble in chloroform, water and ether. They are slightly soluble in absolute alcohol, fairly soluble in hot gla- cial acetic acid, but best solvent in 60% alcohol. Uric acid, ghxuronic acid, oxalic acid and acetone may give positive tests. This test should not be done on a urine containing albumin. Dextrosazone 78 Monosaccharides. Glucose, levulose and galactose. Leviilosuria. There are 3 cliuical groups. 1. Alimentary. 100 grams is the normal limit of tolerance. Churchman (See J. H. H. Bui.) found that 26% of normal persons show levulosuria in 100 grams of the sugar, while some patients with actual hepatic disease do not. Churchman's conclusions on the Strause levulose liver test are: 1. The test is modified by extrahepatic fac- tors of sugar metabolism (renal, adrenal and pancreatic complications). 2. There are difficulties in the test (nausea and vomiting ) . 3. The clinical value of the test is insufficient to warrant its continuance. 2. Diabetic. A good many diabetics have a tolerance for levulose. The occurrence of levulosuria constantly with glucose indi cates a rather serious prognosis. It also gives an error in calculating the glucose excretion. 3. Idiopathic. There are only 8 of these cases reported in the literature. Those patients who show continual levulosuria generally have some complication in the glands of internal secretion. Three of the cases re- ported had hypophj^seal trouble and others had trouble with gonads. Seliwanoff's test for levulose : Urine 10 cc. Concentrated HCl 5 cc. Kesorcin few Xls. Bring the solution to a boil or put in boiling watei 79 for .')() secoiul>'. First a reddish blush a])pears, and upon standing and allowed to cool a granular red- dish precipitate forms. Mannose, maltose and glu- cose in large amounts will give the test, also. Pentosuria. Kinds : 1. Alimentary. Follows heavy ingestion of pen- tose containing food, such as apples, plums, cherries, etc. It probably occurs more fre- quently than recognized. It is character- ized by optically active xylose and arabi- nose. 2. Diabetic. Frequent in severe types. It is characterized by L xylose, Avhich probably comes from broken-down pancreatic nucleo- protein. 3. Essential or ideopathic (Dr. Janeway, Am. J. Med, Sci., Sept., 1906). Features : 1. Constant excretion irrespective of the diet. 2. Hereditary tendency. 3. Patients excrete it for long periods without evil effect. 4. Sugar excreted in this disease optically inac^ five. 5. Amount excreted is usually small (.2 to .6%). One case with 1% reported. C). Specific gravity somewhat increased. 7. Amount of urine not excessive. 8. Acidity high. 9. Pentoses fed come through in the urine as such. 10. Pentose may be formed from glucose, but not determined. 11. Glycuronic acid excretion continues as normal. 80 Tests for pentose. Phloroglucin (Tollen's). To urine add a small amount of HCl saturated with phroglucin. Heat in water bath. A deep-red color indicates a positive reaction. Watch for the initial shade of red to appear and then cease heating. AVhen extracted with aniyl alcohol and examined spectroscopically a band is seen between D and E. Errors : Test given also hj lactose, galactose and glycuronic acid. Orcin. To urine add HCl and a few crystals of orcin. Heat in water bath. The development of a green color and the formation of a greenish pre- cipitate indicates a positive test. Extracted with amyl alcohol gives a band betAveen C and D. Bial's method : Solution : 30% HCl 300 cc. 10% Ferric chloride 30 drops Orcin 1 gm. About 4 or 5 cc. of this solution are heated to boiling, and urine is added drop by drop (not over 1 cc). A positive reaction is indicated by a clear emerald green color. This test is best of the three. Glycuronic acid. Glycuronic acid is an oxidation product of sugar metabolism and is not concerned with protein meta- bolism. It is not a forerunner of diabetes and does not represent anything but detoxication. Its osazone has the loAvest melting point of any met with. It has an aldehyde group, which accounts for its reduc- ing properties. Pure glycuronic acid is dextro-rota- tory. Test for gl^^curonic acid (Tollen's) : To 5 cc. of urine add % cc. of 1% alcoholic solu- tion;of naphthoresorcin and 5 cc. of con. HCl. Put in water bath and allow to stand 15 minutes. Allow 81 to cool at room temperature for -L minutes, and then cool under tap. Add ether, which extracts a blue- violet color, indicating a positive test. Examined spectroscopically, an absorption band occurs at b, but is not specific. Clinical significance : 1. Glycuronic acid combines with toxic sub- stances and appears in the urine as glycu- ronates. Glycuronic acid combines readily with the coal tar products, morphin, cocain, etc. ; therefore, after such medica- tion lools: for glycuronates in the urine. 2. Copper and bismuth reduction absent or atypical. 3. No fermentation occurs. 4. Glycuronates in fi'esh urine are levo-rotatory, and osazones are formed with difficultv (m.p = 114 to 115 deg.). 5. They are easily confused with pentoses be cause reactions are same, but can be differ^ entiated by optical activity, glycuronates being levo-rotatory and after hydrolysis become dextro-rotatorv. 82 SCHEME FOR DETECTION OF AN UNKNOWN REDUCING BODY IN THE URINE. I. Feliling's test: A. Negative, no sugar present. B. Positive, proceed with II. Njlander's test: A. Negative, eliminates confusing substances in I. B. Positive, sugar is probably present, so proceed with III. Fermentation test: A. Positive, sugar present may be glucose or levulose. B. Negative, reducing substance may be: Lactose, maltose, saccharose, pentose, glycuronic acid. These become positive with hydrolysis. IV. Polariscopic examination : A. No. I, II and III positive: Urine D-rotatory : indicates glucose. Urine L-rotatory : indicates levulose. B. No. 1, II positive and III negative : Urine D-rotatory indicates: Lactose maltose, saccharose, alimentary f^en- tosuria. Urine inactive indicates pentose. Urine L-rotatory indicates glycuronic acid. V. Phenylhydrazine : A. Osazones easily obtained with dextrose, levulose, pentose, maltose. B. Osazones obtained only by special proce- dure with lactose. C. Osazones obtained after hydrolysis with glycuronic acid or saccharose. 83 Glycuronic acid osazones have melting point of 114 to 115 degrees. Pentose osazones have melting point of 156 to 160 degrees. Eemaining osazones have melting point of 200 to 205 degrees. 84 QUAXTITATIYE DETEKMIXATIOX OF SUGAE. Urinary output and specific gravity. (Xaimyn.) This method is inaccurate, but in the absence of other means gives some information as to the amount present. Amount of Urine. Specific Gravity. % of Sugar. 2000 cc. 102S to 1030 2 to 3% 3000 cc. 1028 to 1032 3 to 5% 5000 cc. 1030 to 1035 5 to 7% G to 10.000 1030 to 1012 G to 10% ^'/K'(:•//fc gravity-icrincntation method. This method is based upon the principle that fermentation by yeast results in lowering the specific gravity. A small amount of albumin can be neglected and the urine should be acid in reaction; if not so, make acid with acetic. Technique: 50 cc. of a 21-hour specimen of urine are taken and the specific gravity is determined. Add 2 cc. of a thick yeast emulsion and allow to stand from 21 to IS hours at 37 deg. C, until the reduction test is negative. Again make specific gravity determination. The ditference between the first and second determination multiplied by 231 coetiicient equals the per cent of glucose. This metliod is accurate to .1%, but sugar present should not be less than .5%. Fermentation metliod. A. Einhorn's method. Mix a sufficient quantity of urine to fill the special fermentation tube with a piece of yeast the size of a pea. Incubate for 21 hours at room temperature or 6 hours at body temperature. Eead the amount of gas given off and its equivalent percentage of sugar on the scale. Eun controls : Activity of yeast, fermentable substance in yeast, and urine alone without yeast. B. Lohnstein's method. This is the most accurate. To % cc. of urine add .2 cc. of yeast solution (1 85 part yeast to 3 parts water) and layer this solution on the mercury in a special fermentation apparatus. Tilt the apparatus until the mercury in the upright column lies even with the zero point on the scale. Turn the cork so as to shut off the air and incubate from 5 to 6 hours and read. Titration metliod. 1. Benedict's method. Advantages : 1. Single solution. 2. Permanent. 3. Sharp end point, degree. Solution : 4. Accurate to a marked Pure CuSO^ NaCOg crystaline Na or K citrate KSCN (accurate) 200 gm. 18 gm. 200 gm. 125 gm. 5. 5% solution KSCN 5 cc. 6. H2O qs. ad. 1000 cc. Dissolve Nos. 2, 3 and 1 in about 800 cc. of boiling HgO and filter. To filtrate add No. 1 dissolved in hot H2O. Allow to cool and add ^o. 5 and Xo. G. Technique : 25 cc. of the reagent are placed in a porcelain dish and 10 to 20 gm. of NaoCOg added to make the end reaction more clear. Run the urine diluted 1 to 10 in from a buret, rapidly at first, Avhich gives a white precipitate, and then 2 or 3 drops at a time, boiling 30 seconds between each addition. Add distilled water if the solution becomes low. The end reaction is the permanent (lisapperance of the blue color. Calculation: 25 cc. of the reagent equals .05 gm. of glucose or .053 gm. of levulose. x cc. = .05 gm., .05 .05 1 cc. = gm. 1500 cc. = gm. X 10 X 1500. X X 2. Bang titration method. Urine is boiled in a know n excess of alkaline cop- Continued on Page 87 H a 0} Seliwanoff's Phenylmethylhy: drazine Phenylhydrazine after concentra- tion and ex- traction o a Ph ^ M. P. & Rot. of Osazone h4 o hi 2000 Inactive 1560 —1600 Inactive 1880-1930 D. 0.480 in Neuberg's pyridin alco- hol mixture § QQ O g (J Q Pheylhy. drazine 1 > Negative In Urine > 1 .2 1 o eg Q OS Q Essential or Ideopathic inact. Al. & Diabetic slight D o 00 O 1 Ph 0) I 1) > a V ^ > Slow Positive ^ ^ «3 Z > 1 Ph Slow Positive Slow Positive 9S 1) Ph > 1 Slow Positive Slow Positive Slow Positive D t-H 6 en 1 v O G Ph Galactose 87 Continued From Page 86 11 Phloroglucin Orclne After Hydrolysis Heat Urine First and It Does Not Reduce O tH S '^• See Glucose & Levulose > > .2 *en S 2 t O > > If 2 5) D. Rot Iv after Hydrolysis 1 Q 1 1) > 1 1) > ID > > I) 1 > 1 == 1 Positive or Negative > > 1 iz; > 1 4J 1 -s i 1 Positive or Negative > 1 o > 11 2 Conjugated Glycuronic acid % o 1 Homogentis- ic acid of Alkapton- uria 6 O 1 88 per, and the copper remaining is titrated with ft standard solution. Solution: A. 1. Potassium bicarbonate 100 gm. 2. Potassium carbonate 500 gm. 3. KSCN 400 gm. 4. CuS04 25 gm. 5. H2O qs. ad. 2000 cc. Dissolve 1, 2 and 3 in 1300 cc. of hot H^O in the order named. Dissolve No. 4 in 200 cc. of H2O separately. Mix while hot. Cool at room tempera- ture, add No. 5. Solution E. KSCN 200 gm. Hydroxylamine sulphate 6.25 gm. (accurate) H2O qs. ad. 2000 cc. Technique: Urine is diluted to not over .6% of sugar. 10 cc. of the diluted urine are placed in an Erlemeyer flask and 50 cc. of solution A are added and the contents boiled 3 minutes by the watch. All the sugar present is used up, but not all the coi^i^er present is reduced. Titrate the cop- per remaining with solution B until colorless. Calculation: 1 cc. of solution B equals 59.4 mg. of olucose. »' Summary of quantitative determination methods : Specific gravity method should be discarded where other instruments are available. Specific gra\ity- fermentation method is not very accurate unless one has a delicate urinometer. Fermentation methods are all right if sufficient controls are run. Lohn- stein method good. Titration method best of all. Estimation of sugar icith the polariscope. Principles : Light rays vibrate in all directions. Some sub- 89 stances have the power of double refraction, Iceland spar for instance. Light passing through this is resolved into two sets of rays, one of which vibrates in all directions, the other vibrates in but one plane. In the polariscope the entering rays are polarized and the ray vibrating in all directions is deflected by means of a Mchol prism. Another Mchol prism is used near the eye-piece as an analyzer. A quartz plate obscures half the visual field. Some substances have the power of turning this polarized ray to the right or left. The substance whose specific rotation is to be determined is put in a tube of a known length, 1 decimeter, and the polarized ray passed through it. After determining the zero point of the instrument the unknown substance is put in the pathway of the polarized light and the analysing prism turned until both sides of the field are of equal illumination. The degree of rotation is read on the scale. Specific rotation. This is the amount of rotation of 1 gram of a substance per cubic centimeter of water in a tube 1 decimeter in length. Formula : AX 100 P=: Sp. rt. X LD A = reading in degrees. Sp. rt. = specific rotation. LD = length of tube in deci- meters. P = percentage. P — A when the tube is of the proper length, viz : 188.6 m m for glucose. Technique : A mixed 24-hour specimen of urine is 90 ina<]e free from albumin and absolutely clear. To do this the following may be used : 1. Heat and acetic acid and filter. 2. Filter after adding kieselguhr. 3. Normal lead acetate, not basic, filter. 4. 25% HCl and animal charcoal, filter. The zero point of the instrument is determined by taking the ayerage of 5 readings, using a sodium flame. This point is indicated by both sides being equally illuminated. Carefully fill the tube so as to leaye no bubble within, place in the instrument and take the ayerage of 5 readings. Where a 188.6 m m tube is used each degree of rotation equals 1% glu- cose. Essential urinary findings in diahetes. There is generally a polyuria caused by the liyper- glycaeiiiia. Sugar in the blood, not in a colloidal state as nornuiUy, acts as a diuretic. The amount of urine excreted depends upon the amount of hypergiycaemia. From 5 to 8 liters are excreted per day. Polyuria with low specific grayity would indicate a possible development of both diabetes mollitus and insipidus. When polyuria of severe cases diminishes on restriction of diet it indicates a good prognosis. When low polyuria and high specific gravity occur together the prognosis is more favorable than great polyuria and low specific grav- ity. Variations in sugar excretion. By securing specimens every 2 hours it is found that the maximum output of sugar occurs at noon and late afternoon. The minimum output is in the early morning. If one should examine a 24-hour mixed specimen, the concentration of sugar may be so low that it may be missed; hence, keep 2-hour specimens separate in doubtful cases and examine. 91 Amount of sugar excreted. The average percentage of sugar in the urine in diabetes is from 2 to 3%. 3% at 3 liters a day would give 90 grams. Maximum output 1500 gm. 6 to 8% is considered high. More is excreted on a hot than a cold day. In febrile conditions there is a tendency for glycosuria to disappear. The amount of sugar is increased on a carbohydrate rich diet. Sometimes lactose or levulose are tolerated well. The amount of sugar excreted usually falls during diabetic coma. Specific gravity. The specific gravity is usually high, varying from 1025 to 1010. 1074 has been reached. In urines with 1060 readings and over, look out for frauds. Sugar may occur in urines with a specific gravity as low as 1007 to 1016. Color of urine. The color is usually a pale greenish yellow. A pale color with a high specific gravity is character- istic. Acidosis. The characteristics of the urine are : 1. Acid bodies are present, B oxybutyric acid acetone and diacetic acid. 2. The reaction is characteristically acid, rarely neutral or alkaline. By the Folin titration method there is increased alkali tolerance. JS^ormally an individual will excrete alka- line urine upon the ingestion of from 5 to 10 grams of sodium bicarbonate; in dia- betes it will require from 100 to 250 grams a day to make the urine alkaline. Nitrogen elimination in dia'betes. Normally 15 gm. are eliminated in 24 hours. In diabetes 20 to 30 grams are excreted. The nitrogen 92 pardtioii is inicliaiiged iiiitir acidosis appears, when the NHg increases. Severe symptoms are indicated Avhen the XH3 ontpnt reaches 2 or more grams per day. Amino acid nitrogen in diahetes. Xormally .1 gm. per day is excreted. In diabetes this is increased to abont .9 gm. Alhiimijiuria and casts in diabetes. Albnmin and casts are seen in patients passed middle life in whom there is arterial or nephritic change. Kolz's sign is showers of hyaline casts preceding acidosis or coma. Acid hodies in tJie iwinc. These inclnde 3 substances : B oxybntyric acid. Diacetic acid. Acetone. (^H, ' CH3 CH3 ( ^H( )II — H. = CO — COo = CO CH. CH2 CH3 (;00H COOH Although these bodies occur in diabetes, they also occur in other conditions. The}^ may arise from car})()hydrates, fats or proteins. From a carbohy- drate source there is no evidence, but there is a pos- sibility of their coming from the tyrosin group of protein. There is no parallelism, however, between their excretion and destruction of body protein as there is between their excretion and fat destruction. Tests for acetone : LegaFs. To about 8 cc. of urine add a few crystals of sodium nitroprussid and a few drops of NaOH or KOH, which gives a red color and which fades to a yellow both in normal and diabetic urine, except tliat the transition is sloAver in the latter. While still red add a few drops of glacial acetic acid, and if the urine changes to a purple or deep red color it denotes the presence of acetone. If it changes 93 to a green color it shows the presence of creatinin. Le XQble'!^\ This test is better than Legal's, for it eliminates aldehyde bodies and creatinin. To about 8 cc. of urine add a few crystals of sodium nitroprussid and a fcAV droj^s of NH^OH. Before the red color fades to a yellow add a few drops of glacial acetic acid as before. A deep red or purple color indicates the presence of acetone. Lieben's. Depends upon the formation of iodoform crystals. To about 5 cc. of urine add a small quan- tity of Lugal's solution or tincture of iodine and a few drops of XaOH. Warm slightly and examine the precipitate for iodoform crystals. Gunning's modification of Lieben\s is a more spe- cific test. To a small quantity of urine add about 5 cc. of either Lugal's solution or tincture of iodine, and then NH^OH till a permanent precipitate forms. Upon standing this turns to a yellow or yellowish brown color, and upon microscopical examinati<-n hexagonal crystals of iodoform are found. It is preferable to set the solution aside for 24 hours be- fore examination. Frommer's. This test is the most specific, being sensitive in dilution 1 — 1,000,000. The reagent used is a 10% alcoholic solution of salicylaldehyde. To a small quantity of urine add a few drops of NaOH or KOH and 10 to 12 drops of the reagent. Keep in water bath at 72 degrees for 3 or 1 minutes. A purple or dark red color indicates a positive reaction. Le Noble's modification of Legal's test and Gun- ning's test are best clinically. Diacetic acid. Gerhardt's test. Reagent: 10% ferric chloride solution. Add reagent to urine in slight excess, whereupon a precipitate of phosphates forms. Either filter at 94 this point or continue adding the reagent without filtering. It is better to filter. Upon the addition of more reagent the previous reddish-brown color is changed to a Burgundy red. This test is also given by salicylates, conjugated glycuronates, after taking phenacetine, antii)yrene, acetates and trionate medi- cation. These can be differentiated, however, by first heating. Diacetic acid is broken up and color dis- appears ; drugs, on the other hand, continue to give the reaction. B oxybutyric acid. There is no satisfactory test for this. The urine shoAvs a greater titration determination than is in- dicated by the polaroscope. It is L-rotatory, specific rotation being — 24.12 degrees. Urine after fermen- tation still L-rotatory also points to B -oxybutyric acid. Autoketo)iogemc junction of carbohydrates. 1. Administration of carbohydrates diminishes ketones. 2. Lipaemia is associated with maximum excre- tion of acetone bodies. 3. Administration of fatty acids is followed by excretion of acetone bodies in animals. This occurs also in people on an insufficient diet or who have an error in metabolism where carbohydrates do not spare fats. This condition is characterized by the following : A. 1. Large amounts of acetone bodies 2. Increased NHg. 3. No characteristic anatomical lesion This condition is found in diabetes, starvation and cachexia. 95 2. Excess of XH.. o. Large amounts of uuoxidized X and lactic acid This condition is found in plios. poisoning, cliloro- form poisoning, toxaemia or pregnancy and cyclic vomiting. Coma supervenes when acetone bodies are in the greatest concentration, in both the blood and the urine. Coma does not occur in their absence. Acidosis is further proved to be the cause of coma : 1. There is decreased alkali in the blood. 2. Concentration of CO^ in the blood reduced from 36 m m to as low as 3.2 m m tension. 3. There is a greatly increased alkali tolerance. 4. Administration of alkali results in improve- ment. Allen's theory of diabetes. 1. Pancreas is the seat of the trouble. 2. Diabetes is a specific disease. 3. It is explained by a lack of amboceptor, which is necessary to produce blood colloidal sugar. Sugar free in the blood and not in a colloidal state acts as a diuretic. Treatment. 1. SuiDply deficient amboceptor. 2. Protection. Restrict carbohydrates up to a point of no sugar in the urine and blood sugar not greater than .17% and no ace- tone bodies in the urine. By restricting the carbohydrates you attack acidosis. There is no explanation for this except that by starvation you make the body learn to metabolize sugar properly. Dia'betes insipklus. This is a condition characterized by the excretion of large amounts of urine with a low specific gravity 96 without any demonstrable kidney lesion. The con- dition frequently shows a hereditary disposition. Symptomatic group. 1. Polydypsia (excessive fluid intake). 2. Central nervous system injuries. Lues r^ften found at autopsy. Ideopathic group. Lately some internal glandular pathology has been determined. Sometimes the posterior lobe of the hypophysis has been found diseased, especially in distrophia adiposa genetalia. The inability of the kidney to concentrate urine is the chief symp- tom. When salt is given to a normal individual he excretes it with a rise in the specific gravity of the urine. In this condition the concentration in the urine remains unchanged, but a greater quantity of fluid is excreted. The total solids are the same, but more fluids are required in the latter condition. Characteristics of the disease. 1. Polyuria with 5 to 49 liters a day. 2. Excessive thirst. 3. Urine: a. Low specific gravity. b. Practically colorless. . c. Weakly acid. d. Hypotonic (bl6od cells rapidly disin- tegrate). e. Polyuria greater at night. f . Free from albumin and sugar. Ferments in the urine. Pepsin. Normal in small amounts and easily de- tected. It is absent in gastric carcinoma, subacidity and occasionally in achylia gastrica. It is increased in pneumonia and is of some prognostic value. Test for pepsin. Fibrin is washed and then soaked in the urine 97 for from 5 to 6 hours. It is then placed in a weak solution of HCl and incubated. If pepsin is present digestion will take place. Lipase. NormallT present, but in smaller amounts than pepsin. It is increased in jaundice, diabetes mellitus and hemorrhagic pancreatitis. Test for lipase (Castle and Loevenhart). Flash A Flash B Flash C Urine 5 cc. Boiled urine 5 c c. Urine 5 cc. Phenolphthalein x cc. N'/IO XaOH x cc. y/iO NaOH N710 NaOH to a .25 cc. ethylbutyrate .2." cc. ethyllnityrate pink color — x cc. 1 cc. toluol 1 cc. toluol Incubate flasks B and C for 21 hours at 37% degrees. At the end of this time add % cc. more of X/10 HCl than X/10 XaOH, extract with 25 cc. of alcohol and 50 cc. of ether. Titrate to end point, using phenolphthalein as indicator. 1 cc N/10 XaOH equals .0088 grams butyric acid. Diastase. Origin not known. It may have its origin from polvmorphoneutrophils of the blood, the pancreas, or the liver. It is formed from great meas- ure from the pancreas, for removal of this organ causes a great diminution in the amount of diastase. Wohlgemuth's test. Keagent : 1% starch solution free from precipitate. Dilute 2 cc. of urine with 6 cc. of distilled water. Into each of 9 test tubes add 5 cc. of the 1% starch solution and then the following amounts of the diluted urine : Tube 1, .88 cc. ; tube 2, .72 cc. ; tube 3, .50 cc. ; tube I, .10 cc. ; tube, 5, .32 cc. ; tube 6, .24 cc. ; tube 7, .16 cc. ; tube 8, .08 cc, and tube 9, .00 cc. x\dd sufficient distilled water to each ttibe to make the volimie up to 6 cc, and a small amount of toluol to prevent bacterial action. Cork tubes and incubate at 37I/2 degrees for 21 hours. At the end of this time check action of ferments by chilling in ice Avater. Fill tubes nearly to top with cold water, 98 add 3 drops of X/10 iodine solution and shake. Determine the highest dilution which shows no blue color, and the amounts of starch and urine therein. For instance, if tube 5, with .32 cc. of diluted urine, should be the first tube not to show the reaction, it would mean that .08 cc. of undiluted urine were capable of completely si)litting 5 cc. of 1% starch. Formula : .08 : 5 : : 1.0 : x or x = 62.5 99 STOMACH ANALYSIS. The study of gastric analysis divides itself into four subdivisions, dependent upon the four chief functions of the stomach: 1. That of a mixing chamber in which the food is prepared for the later intestinal digestion — hence a study of Secretion. 2. That of digestion — hence a study of Ferments. 3. That of a reservoir from which food passes fur ther along, requiring a study of Motility. 4. That of a place of absorption, requiring a study of Absorption. AVe shall limit ourselves to the knowledge obtain- able from a study of the gastric contents and leave to other departments the additional important in- formation to be gained by direct abdominal examin- ation and X-ray. The condition of the gastric contents may be studied from the vomitus or by emptying the stom- ach by means of the stomach tube. By this means we may study the activity of the stomach — as to its powers of secretion, ferment formation, motility, and absorption. I. Secretions. A. Vomitus. Vomitus consists of stomach con- tents plus mucus and saliva. It represents no defi- nite phase in gastric digestion; it is, therefore, of much less value along certain important lines than the gastric contents artificially removed. Much val- uable information can be gained from its study, how- ever, especially in the following particulars : 1' Time of vomiting. 2. Amount. 3. Keaction. 4. Odor. 5. Color. 6. Macroscopical contents. 7. Microscop- ical examination. . 100 Types of Tomiting. 1. Cerebral. a. Organic brain changes (no nausea, projec- tile). b. Drugs. Apomorphine. 2. Systematic. a. Local. Drugs, emetics, acute gastritis. b. Psychic — unpleasant sights or smell. c. Toxic. Uraemia, pregnancy, ethyl, alcohol. d. Keflex. Gastric crises, acute inflammation of gastrointestinal tract, renal colic. e. Obstructions. Points of special interest. 1. Time. Morning (alcoholism, pregnancy, hypersecre- tion). After meals (ulcer, esophageal obstruction, pyloric obstruction). Definite time (neurotic). 2. Amount (relation to food and water intake). Large (stasis-atony or obstruction — shows food remains). Hypersecretion has little or no food present. 3. Reaction. The reaction is acid, with the fol- ing exceptions : 1. Esor>hageal obstruction. 2. Presence of intestinal contents (bad retching). 3. Occasionally in achylia. 4. Alkali poisoning, or therapy. 4. Odor. 1. Fecal (obstruction, paralytic ileus, perito- nitis with complete motor insufficiency). 2. Almonds (hj^drocA^anic poisoning). 3. Garlic (arsenated hydrogen). 4. Sour (uraemic, acetone), etc.). 101 5. Color. Varies Avith presence of: 1, food; 2, bile; 3, blood; 4, poisous; 5, fungi. 6. Macroscopic appearance. 1. Bile (retching). 2. Food (retention). 3. Blood (dark red or coffee ground). 4. Fecal. 5. Cancer tissue. 6. Mucus. 7. Microscopical examination, (see under Fast- ing stomach), 8. Chemical analysis (see under Test meals). B. Stomach tube. It is better taken if ice cold and lubricated with oil. Some cases warrant cocain- izing the throat. 1. Factors regarding patient. He should be sit- ting upright or lying on side. A rubber apron should be tied around neck so as to prevent soiling clothes. False teeth should be removed and other foreign bodies. He should be instructed to breath normally. 2. Passage of tube. Hold as a pen, avoid pressing on tongue, and push back to posterior wall and then down. A swallowing movement at this point helps. Kesistance is encountered on the floor of the stom- ach, 40 cm. or 18 in. in adults, 9 in. in child). Re- move tube if there is great pallor or cyanosis. 3. Emptying stomach. Have patient cough, or bear down with abdominal muscles or use Politzer or Boas bags. Indications for the passage of the stomach tube. 1. Emptying the stomach in poisoning, disten- tion or stasis. 2. Lavage in stasis, fermentation, mucus, hic- coughs, post-operative vomiting or chronic ulcer with high acidity. A pint of warm water is usually safe for an adult with 102 sodium bicarbonate or HCl, as the case demands. Beware of habit formation. 3. Gavage. Coma, forced feeding, local mouth conditions, pyloric stenosis (infancy). 4. Medication. Castor oil, salts, emetics, silver nitrate, etc. Contraindications for the passage of the stomach tube. 1. All general conditions in which excitement and retching are associated with harmful results to the patient. 2. Local conditions in which trauma associated with its passage might be dangerous as aneurism, esophageal vacicosities, bleeding ulcer, corrosive poisons, etc. C. Fasting stomach examination. The patient should be instructed to eat a supper in which rice or some other easily recognized food is included, such as spinach, raisins, prunes, etc. After this meal nothing should be eaten until after the passage of the stomach tube the following morn- ing. 1. Normal findings. a. Macroscopic. 10 — 100 cc. fluid, often bile tinged from retching. There may be a few streaks of blood from injury to the mucus membrane. No food remains should be present. b. Microscopic. An occasional starch granule and a few bacteria. No yeasts, no Oppler- Boas bacilli, no sarcinae, no food remains. c. Chemical. The gastric contents should be slightly acid (usually test for free HCl). No lactic acid should be present and is not tested for a^ a routine unless suspected. Guiac test should be negative and is not 103 tested for as a routine. Hoiiseiiiann's test should be negative. This test is used to determine retention and is carried out in the following manner : Allow the gastric contents to stand in a conical container for about 30 minutes; pour off the supernatent fluid, add 10 — 15 drops of Lugal's solution or iodine to the sediment and water till transparent. Starch granules show up blue and are in- dicative of retention. 2. Abnormal findings. a. Macroscopic. Food remains, large amount of fluid (hypersecretion), body tissues (cancer fragments, blood, pus, mucus), foreign bodies (rarely), parasites. b. Microscopic. Oppler-Boas bacilli, B. Bul- garicus, sarcinae, yeasts, infusoria, pro- tozoa (amoeba, flagellates), eggs and worms. Food remains (meat fibers, starch, fats). Crystals, fatty acid, triple phos- phates, calcium oxalate, bile crystals (leu- cin, tyrosin, cholesterin). Body cells r.b.c, w.b.c, cancer cells). D. Test meals. The advantage of giving test meals and then obtaining the gastric contents is that a study can be made of the effects of various meals on normal individuals and a standard can be obtained by which we can judge deviations in the abnormal conditions. Test meals should be of such a character as to (1) produce a normal, psychic and chemical flow of gastric juice; (2) be easily taken by even ill people; (3) be easily recovered (not ob- structing stomach tube). 104 Ewald test meal. 40 grams of bread (without crust because of color) well masticated. 400 cc. of water. (Tea as a substitute not ad- visable on account of interference with guiac test.) Remove in one hour. ]^ormal findings : 30 — 50 cc. fluid with residual bread. Free HCl 20—40%. Total acidity 40— G0%. Disadvantages : a. Little psychical stimulation. b. Some lactic acid introduced. Dock modification. 1 shreaded wheat biscuit in place of bread. (More palatable.) Boas test meal. Oatmeal (1 liter of water, 1 tablespoonful oat- meal boiled to 500 cc.) This modification eliminates the introduction of lactic acid. Remove in one hour. !Xormal findings : Ver}^ little residual material, acidity? Riegel test meal. Mid-day heav}' meal : 400 cc. soup, 200 gm. beefsteak, 150 gm. potato, 1 glass of water. Remove in three to four hours. Advantages : a. Good psychicial stimulation of gastric juice. b. Longer period before removal, hence more information. 105 Disadvantages : a. Too heavy for many conditions. b. Blocks tube. 4. Fisher test meal. Bread and tea (modified Ewald). % pound chopped, seasoned, lean beef. Remove in 3 hours. Advantages same as Riegel. Disadvantages : Too heavy for many cases. 5. Salzer test meal. Double meal. Breakfast: Meat, milk, rice and egg. Four hours later : Stale bread and water. Remove one hour after second meal. Normal findings : No remains of first meal. Advantages : Especially adapted for study of motility. G. Sahli test meal. A fat savory soup of which 250 cc. are given and 50 cc. kept for comparison. Remove gastric contents 1 hour after taking in 2 portions : a. As much as can be obtained directly. b. With a known amount of water wash out the remaining. By reckoning the per cent of fat content (method of Mathieu and Remond- Webster, pg. 58) of the various specimens it becomes possible to determine quantitatively the amount of gastric contents in the stomach at the time of its removal; then by reckoning the acidity per cent it becomes possible to tell the actual amount of acid in the stomach and not merely its per cent. 106 E. Examination of recovered gastric contents. 1. Amount. Ewald 30 — 50 cc. ^Vlore in stasis or livper .secretion. 2. Reaction. Acid, rarely alkaline. Duodenal re- gurgitation and a few acliylias may produce an alka- line reaction. Generally su^cient organic acid is present to cause acidity. 3. Odor, Same as for vomitu?. (Uraemic, rancid, acetone, fecal, etc.) 4. Color. Limited also by previous emptying to bile and blood, fresh or coffee-ground. 5. Macroscopic. Layer forination. Xormal. Fluid and food residue. Abnormal. Retained food (not removed by pre- vious emptying). Blood, fresh or old. Con- sider possibility of trauma from stomach tube, also rule out blood from nose, mouth, throat and lungs. Pus. L'lcerated carci- noma, ruptured abscesses, pyorrhea. Tissue. Especially in carcinoma. Mucus. Gastritis. (I. Microscopic, (See Fasting stomach.) 7. Cliemical analysis. a. Routine. 1. Presence or absence of HCl. 2. Amount of HCl present. 3. Amount of combined acid. 4. Organic acids, especially lactic. 5. Occult blood. b. When indicated. 1. Ferments. 2. Special tests (especially for cancer, which are taken up under that subject). For the acid determination the necessity of devis- ing methods for the differentiation of organic and inorganic acids is obvious. When we speak of a test as specific for HCl it really means for inorganic acids in general, but acids other than HCl are elimi- 107 Hated from consideration. Most of the tests we use as specific for HCl Avill, unfortunately, give suspici- ously positive results with organic acids if they are in high enough concentration. 1. Qualitative tests for HCl. 1. Litmus. Turns red from any acid. 2. Congo red paper (filter paper dipped in acids it gives an orange to a pinkish Congo red, alcohol solution, and dried). It is supposed to differentiate organic from inorganic acids ; deep blue with HCl , bluish red with organic acids. AVith suffi- cient concentration organic acids will also give a definite blue. 3. Topfer's (dimethyl amido azo benzol, 5% alcoholic solution). This is the most gen- erally used reagent. It gives a yellow color in alkaline solutions, and with HCl it gives a deep red to a pinkish orange, depending upon the concentration. With organic acids it gives an orange to a deep pinkish orange, depending upon the concentration. It is sensitive to .02 parts of HCl in 1000 cc. It gives suggestive positive reactions with organic acids in concentrations above 5%. 4. Gunzberg's (2 gm. phloroglucin, 1 gm. vanil- lin in 30 cc. of absolute alcohol or in 100 cc. 80%). Keep in dark bottle. Ivoact,^ with no organic acids nor acid salts re- gardless of concentration. Sensitive to .05 parts of HCl in 1000 cc. Method of performing test : 1 drop of reagent is allowed to dry in a porcelain dish. (Heating is permissible without burning.) 1 drop of gastric contents is placed over the drop of the dried reagent and also dried with great care. lo: HCl gives a purple red color. ( Miutz mod- ilication of Gunzberg. 5. Boas. 5 gm. resorcin, :j gill, caiie sugar, 100 cc. 95% alcohol. More stable tban Gimzberg's, with similar delicacy and same color reactions. 0. Tropeolin. Saturated alcoholic solution Tropeolin. Use as Gunzberg's. Less delicate and color ditference between organic and inorganic is too close to be of practical value. II. Quantitative Tests for Free HCl. 1. 10 cc. of gastric contents well mixed and un- filtered. (Filtering causes some H2O loss and, therefore, HCl concentration.) If much mucus is present, filtering will be necessary regardless of slight error associated with it. Avoid including food in the 10 cc. Technique of perfonning test. To 10 cc. of gastric juice add a drop of Topfer's reagent. If a deep pink color results, proceed with quantitative determination. If a i)inkish orange color results, stop at once and do a Gunzberg test to determine the presence of any HCl. A. Should the resulting color be a deep pink or red color, add from a buret, drop by drop, with con- stant stirring, enough ^syi^ XaOH to change the color to a definite Jeinon color. Calculation: Xo. cc X/10 XaOH X 10 = acidity per cent. B. Should The resulting color with the Topfer's be a pink orange and although only one drop were used, a fresh 10 cc. of gastric contents will be neces- sary, for the presence of Topfer's reagent gives a false positive witli Gunzberg's reagent. If tlie qual- 109 itative test with Gunzberg's shows no HCl present, it will be unwise and impossible to carry ont the test. If the fresh gastric contents gives a positive (hmzberg test, then from a buret add, very care- fully, VIO XaOH to 10 cc. of the gastric contents and test with Gunzberg's reagent after the addition of each 1 — 2 drops, for there is not much free HCl present and the end j^oint must not be passed for accurate determination. Calculation : Same as with Topfer's. Note: Normal acidity is .'2 to .0%, probably as high as .5%, when just out of the gland, but quickly falls to .2 to .8% from contact with (1) mucus of gastric glands, (2) protein combination, (3) alka- line duodenal regurgitation. Expression of acidity: 1. In terms of acidity per cent, which is the no. of cc. of N/IO XaOH necessary to neu- tralize 100 cc. of gastric contents. 2. Actual amount of HCl present based upon the fact that 1 cc. of N/IO NaOH is equal to .00365 gm. of free HCl. Determine 100 cc. .2 to .3% equals 20 to 10 acidity per cent. Definition of terms : Euchlorhydria, when free HCl is .2 to .3% or 20 — 10 acidity pex* cent. Hypochlorhydria, when free HCl is decreased .1%. [Chronic gastritis ( subacute), incipient cancer, fe^'ers, severe anaemias, many mental diseases, c.p.c. chronic nephritis, etc.] Anachlorhydria, absence of free HCl. (Cancer, pernicious anaemia, neurasthenia, etc.) Aclijlia, absence of HCl and ferments. Hyperchlorhydria, free HCl from .2 to .9%. (Ulcers, chlorosis, reflex, migraine.) (Surmont- Dahon, defective XaCl output, compensation.) 110 E-,timation of HCl deficit. To 10 cc. of gastric contents add 1 drop of Topfer's reagent, Tvliicli should not give positive reaction. Confirm absence of free HCl with Gnnzberg's test. Into the 10 cc, with Topfer's reagent as an indica- tor, add, drop by drop, X/10 HCl, stirring con- stantly, nil til a definite positive reaction is obtained. For more accurate determination control end point with Gnnzberg's reagent. Calculation: Xo. cc. of X/10 HCl required X 10 =: acidity per cent. III. Combined Acid. In order to calculate the entire HCl acidity per cent, one must determine, in addition to the free HCl, the HCl combined with foods, especially the proteins. The amount of combined HCl can be de- termined by one of two methods : 1. Einhorn method. This method is simple, quick and sufficiently accurate for clinical work. Method of performing test: To 10 cc. of gastric contents add a drop of Top- fer's reagent. Titrate with X/10 XaOH and de- termine the free HCl. When the end point of the above determination is reached add a drop of phe- nolphthalein and continue to add X/10 XaOH until a definite pink color is obtained lasting at least 30 seconds. Calculation: Xo. cc. XVIO XaOH required to bring about reaction after the free HCl has been neutral- ized X 10 =: acidity per cent for combined acid. In cases where no free HCl exists, i. e., negative Topfer's and Gnnzberg's reaction, the HCl deficit and combined HCl should be determined, which will require two 10 cc. portions. Determine the HCl deficit as above. The combined HCl is determined by adding the i)henolphthalein at once and titrating with the X/10 XaQH, The fallacies of this method Ill are obvious, for such a determiuation of conibiued acids includes: 1, combined HCl ; 2, acid salts; 3, organic acids. When the free HCl is high and there is no evidence of stasis, the bulk of combined acid determined will be represented by combined HCl, but where the free HCl is low or absent, especially when associated with stasis, acid salts and organic acids will often comprise the greater part of this acid calculation. 2. Topfer's method. This method is of value in differentiating between the constituents above enu- merated. Procedure : A. Determine total acidity, including free, com- bined, and salts, using phenolphthalein as indicator. (10 cc. gastric contents plus 1 drop of indicator plus ^NyiO XaOH till end point). Calculate acidity per cent. B. Determine free acid, both HCl and organic, and salts as follows : To 10 cc. of gastric contents add 2 — 3 drops of a 1% aqueous solution of alizarin monosulphonate of sodium, which turns yelloAv with acid and violet with alkali. This indicator reacts with free acids, organic and HCl, and salts, but does not react with organically bound HCl. Titrate with N/10 XaOH till a pure violet color not growing darker with the addition of more alkaU. Cal-culate acidity per cent. C. Determine free HCl with Topfer's or Gunz- berg's reagent. Calculate acidity per cent. Explanatory example: A = 80 acidity % (includes all acids, free, salts, and combined). B = 22 acidity % (includes all acids and salts not organically bound). C = 20 acidity % (includes only free HCl). A — B = 58 acidity % = combined HCl (also com- bined organic acids if HCl is insufficient for lactic 112 acid being weaker will not combine as long as HCl is present to do so). B — C = 2 acidity % ^ organic acids and salts. (A— B) plus C = total HCl, or 78 acidity %. In cases where there is a free HCl deficit and organic acids are present, the combined acids may be part HCl and part organic, or all organic. AVhere delicate discrimination is needed and it is necessary to know if any HCl is excreted by the gastric glands, the folloAving method should be adopted: 3. Incineration with BaCOg. If any HCl is present it will combine with BaCOo and form the soluble BaCls- The addition of HgSO^ will precipitate insoluble BaSO^. The appearance of a precipitate after this procedure signifies the presence of HCl. Filtration, incineration, weigh- ing and calculation will give amount of HCl. IV. Organic Acids. Practically all foods contain some fatty acids. Where rancid butter, vinegar, sour milk, etc., are included in the diet, the amount may be consider- able. Carbohydrates, contained in milk, bread, meat, etc., always contain some lactic acid, and the find- ing of an excess amount in the gastric contents after the ingestion of such foods points to a fermentative process at wqx% in the stomach, due possibly to a combination of decreased HCl and decreased motil- ity. Boas thinks that with a normal diet there should be no appreciable formation of organic acids in the stomach during normal digestion. Bacterial decomposition, in the absence of HCl, plan's an im- portant source of their production. The presence of a fat-splitting ferment would also produce them. In fermentation processes lactic acid, but^^ric acid and acetic acid are generalh' found. Lactic acid is used as an index of organic acid production, be- cause (1) it is produced in greater amounts than 113 the others and is thus more easily detected; (2) qualitative tests for it are quick and simple. Quantitative tests for organic acids are not used clinically. Xo clinical information is obtained by quantitative data because there exists no correlation between the amount of organic acid present and the severity of the lesion. A. Qualitative consideration of lactic acid. With the Ewald meal in normal digestion, as a rule, no lactic acid is found in the gastric contents ; but there is a slight abnormality, even within nor- mal limits, in which it may be found. In such bor- derline cases the use of the Boas meal is advisable. Normally the small amount of lactic acid in the EAvald meal is either (1) absorbed or (2) cannot be detected on account of the presence of the normal amount of HCl, which interferes with its tests. Pathologically, lactic acid appears in conditions of stagnation and low HCl output. Such conditions are, found in (1) carcinoma, with obstructive fea- tures ; (2 ) hypochlorhydria or achlorhydria, . with sluggish motility ; (3 ) benign stenosis, with low HCl output. .^ - - :. . . . ; Tests for lactic acid : ■ " .' .....,• .■ ■•• ■ 1^ Uffelman's. -■ - :.: ;. _ Reagent: 1% phenol, 20 cc; dil. ferric chloride,.! drop; dist. water q.s. to mal^e a delicate . amethyst' color. . . Procedure : Divide amount of reagent between two test tubes, and into one add 5 — 8 drops of clear gas- tric juice (filtered, if necessary), and into the other add 5 — 8 drops of distilled water for a control. A positive test is indicated by the formation of a canary yellow color in the tube containing the gas- tric juice. If the reagent is not sufficiently diluted and is of too dark a color, a small amount of lactic acid will not give the characteristic lemon color, but 114 Avill simply decrease the intensity of the purple color. Sources of error: 1, yellow gastric juice; 2, acid sodium phosphate: 3, cane sugar; 4, glucose; 5, alcohol: 6, other organic acids. Modification : If doubt exists, make an ether ex- tract of the gastric juice, evaporate, take up the residue in distilled water and apply the test. 2. Kelling modification of UJielmann's test. Reagent: Distilled water, 20 cc. : 10% ferric chlo- ride, 1 or 2 drops. Procedure: Divide between two test tubes. Into one put a few drops of clear gastric, juice, and into the other a fcAv drops of distilled water for a control. A j)ositive test, as before, is the formation of a canary yellow color. Sources of error : Same as for Uffelmann's, except that acetic acid will not give yellow color. 3. Strauss test. This is the best clinical test, for it detects lactic acid when in pathological amounts. Procedure : Into a Strauss funnel put 5 cc. of gas- tric juice and a drop of HCl to free any organically- bound lactic acid. Upon this pour 20 cc. of alcohol- free ether. Invert several times, avoiding the forma- tion of an emulsion. Allow the gastric juice to es- cape through the stop-cock below and replace it with an equal quantity of distilled water. Shake several times in order that the lactic acid extracted with the ether can be taken up by the water and add a few drops of a 10% solution of ferric chloride. An intense green color will appear if more than .1% lactic acid is present, paler shades of green if less is present. B. Qualitative consideration of butyric acid. Physiologically, none is present in the stomach during digestion unless (1) hea^^^ carbohydrate meal has been taken, (2) it is taken directly in the 115 form of rancid butter, etc., (3) it finds it way into the gastric juice from the mouth. Fugge has shown that it may be formed from lactic acid. Method of detection : 1. Odor specific. 2. Shake material with ether, evaporate ether ex- tract, take up with water and a small amount of CaCU, and upon Avarming butyric acid w^ill settle out, recognized by (1) droplets, (2) odor of rancid butter. C. Qualitative consideration of acetic acid. Acetic acid is frequently found in large amounts when taken in the form of vinegar. Pathologically, it is found in conditions of stasis with yeasts and fungi. The stomach in this condition is usually dilated and atonic. Yeast action on carbohydrates yields alcohol, which, in turn, when acted upon by yeasts and bacteria, yields acetic acid. Method of detection : Shake the material with ether and extract with water. Neutralize with NaCOo, carefully. Acid prevents the necessary re- action and alkali causes formation of ferric hydrox- ide when ferric chloride is added. Add a few drops of ferric chloride and look for appearance of deep ved color. Upon boiling a reddish precipitate of basic ferric acetate will also form when acetic 'acid is present. D. Detection of blood. The guiac and benzidine tests are carried out in the same manner as for similar tests in the urine. II. Consideration op Ferments. A. Pepsin is the chief ferment and is secreted by the peptic glands. B. Lipase, which is slight in amount and of ques- tionable origin.. Possible origin: 116 1. Regurgitated duodenal contents, pancreatic origin. 2. Derived from secretion of Brunner's glands in intestine. 3. Dual origin from both. C. Diastase, a small amount of Avliich is of gas- tric origin, but the greater amount of which is due to swallowed saliva. A. Pepsin. This ferment is secreted as propepsin, is activated by HCl, and destroyed by alkali. Other acids will activate it, but in higher concentration, viz: HCl, .2— .4%, lactic 1—1 plus ?c: The action of pepsin is continuous, and a great amount of work is accomplished by a small amount of pepsin, pro- vided that the products of its digestion are removed. Stasis hinders its action. Decrease in amount of pepsin. Due to disease of peptic glands. Pepsin is generally present when the HCl is high, low or even absent. Pepsin may be absent in the following conditions : 1 . Carcinoma, especially the liniis plastica type. 2.~ Atrophic gas- tritis. 3. Occasionally in pernicious anaemia. The output of pepsin lias no relation to the amount of HCl present. . ' . . ' - ■' Qualitative tests jor pepsin: --■"'.'.:■. These are- based upon the digestion of protMii-in- the form Of il. Fibrrn, which is obtained by whip- ping ox blood and is then washed, kept in alcohol for two or three days, then carmine for two or three days, and finally preserved in glycerin till used. 2. Albumin, which is made by cutting the whites of hard-boiled eggs into small squares and preserved in glycerin. Procedure : 1 . Into 25 cc. of gastric juice with free HCl place either a number of pieces of fibrin or albumin j^repared as above. Incubate for 15 min- utes to an hour. The fibrin swells up in 15 to 30 117 minutes and liberates the carmine. It is all gone normally in 1 to IV2 hours. The albumin swells up in 30 to 60 minutes and is well gone in three hours. Quantitative tests for pepsin. 1. Hammerschlag's. 1% solution of egg albumin in 0.4% HCl {egg albumin 1 part, 0.4% HCl 13 parts j 10 cc. Gastric juice 5 cc. Incubate at 37 deg. for one hour and run a con- trol tube with 5 cc. of distilled water instead of gastric juice. At the end of an hour run an Esbach determination on both the tubes, also on the original 1% solution. Xormallv 90% of the albumin is digested in the hour. Sources of error: Albuminoses are thrown down with albumin. 2. Mett's method. (The one of choice.) Preparation of tubes : Take several whites of eggs, cut them, filter through gauze and use the liquid portion to fill capillary tubes 10 to 30 cm. long by 1 to 2 mm. bore. Fill tubes by suction, seal with bread crumbs, drop into boiling water (95 to 100 (leg.) for 5 minutes, and then seal ends with paraffin to prevent drying. AVhen read}" to use cut tubes into 2 cm. lengths. Sources of error to be avoided in preparation of gastric juice: 1. If too concentrated, especially after meals, there may be present substances which inhabit di- gestion, i. e., XaCl and carbohj'drates in solution. 2. Schultz's law holds with digestion up to 3.6 mm. The length of the cylinder of albumin digested by any gastric juice is proportional to the duration of digestion and is independent of the diameter of the capillary tube, provided that the length of the ii3 digested column does not exceed 7 mm. For this reason the gastric juice is diluted. 8. Kesult of peptic digestion hinders further di- gestion. 4. Pepsin is activated by HCl and its presence is essential. Procedure: Into a small dish place Filtered gastric juice 1 cc. .18% HCl 16 cc. 2 cm. Mett's tubes 2 Incubate 24 hours. Kead four ends, take aver- age and square. Note: It is often desirable to set up two addi- tional dilutions of gastric juice, using 32 cc. and 64 cc. of .18% HCl to 1 cc. of gastric juice. Unit of digestion equals the amount of pepsin necessary to digest 1 mm. length of albumin in 24 hours in the presence of .18% HCl. Calculation. This is based upon Schultz's law, which may be stated as follows : Relative amounts of pepsin, in constant acid solutions and time of action, are proportional to the square of the length of the column of digested albumin, or the square of the length of the digested cylinder of albumin is proportional to the pepsin concentration, pro- vided the length of the digested column is less than 3.6 mm. Example : Average length 3 mm. 3- X 16 (dilution) = 144 units. Significance: to 70 units — low. 70 to 150 units — average. 150 to 250 units— high. B. Rennin (chymosin). Pepsin and rennin run parallel in most cases, but marked variations may occur. One or the other may be suppressed or follow different curves of secretion. Some have tried to show that pepsin 5 cc. 10 cc. Coagulation shows the 119 and rennin are identical, but this is probably not true, for there are some differences between these two ferments, viz: pepsin acts onh^ in acid media, while rennin acts in acid, neutral, or slightly alka- line media. Its action is to make insoluble casein from caseinogen. Qualitative test for rennin: Neutral gastric juice Fresh milk 5 to Incubate for 15 minutes presence of rennin. Quantitative test (Boas). In a rack place 6 test tubes. In the first tube place 1 cc. of gastric juice and 9 cc. of distilled vv^ater, which gives a dilution of 1 to 10. Into the remaining tubes place 5 cc. of distilled water. With a 5 cc. pipette mix the contents of the first tube by drawing the solution up and down three or four times, then transfer 5 cc. of it to the second tube, mix as before, transfer 5 cc. from the second to the third tube and so on till the last tube, when 5 cc. are discarded after mixing. The dilutions are as follows : 1. 1—10. 2. 1—20. 3. 1—10. 4. 1—80. • 5. 1—160. 6. 1—320. After these dilutions have been made add to each tube 5 cc. of fresh or, preferably, boiled milk, and 21/2 cc. of 1% CaC12. '^ ' Place in a thermostat for 30 minutes and read. In abnormal conditions the coagulation is low, if only in the first tube, interference due probably to acid salts. Normally, there should be coagulation up to 1—160. C. Lipase. This ferment does action is very slight. occasionally occur, but its Gastric digestion is not con- 120 cerned with the splitting of fats. In testing for this ferment be snre to nse a fat-free meal. Qualitative test for lipase : 1. Wash stomach out thoroughly. 2. Give fat-free test meal. Kemove. 8. Incubate, adding some neutral butter. Positive results show presence of fatty acids. Examination of the duodenal contents for fer- ments. Method of obtaining duodenal contents : Give pt. 200 c. of olive oil by mouth. Half hour later pass stomach tube and obtain regurgitated duodenal contents. Set up for examination of duodenal contents for ferments. 50 ^ Q O 9 W ^ > 9p to CD ^2. ahlbaum solution 1% in t ^2 w c-t- a> S3 5' 1- W2. i-5- ^^ p I . . . . . 2 cc 2 cc 2 cc 2 cc II . 1 cc 1 cc 2 cc 2 cc 2 cc III . 0.7 cc 1.3 cc 2 cc 2 cc 2 cc IV , 0.4 cc 1.6 cc 2 cc 2 cc 2 cc V . 0.2 cc 1.8 cc 2 cc 2 cc 2 cc VI . , . 0.1 cc 1.9 cc 2 cc 2 cc 2 cc VII . 0.0 cc Control 2.0 cc 2 cc 2 cc 2 cc Incubate tubes at 38 deg. C for 30 min. in water bath, cool rapidly and read at once. Titrate tubes A with a saturated solution of NaCl, which gives white ppt. Add Lugal's solution to tubes B and obtain blue color if starch is pres- ent. Titrate tube C with N/10 XaOH, using phenol- phthalein as indicator. Method of preparing casein solution: X/10 XaCl 5 cc. Casein 0.1 gm. Dist. water 45 cc. 121 Boil, with constant watching and stirring, for from 3 to 5 minutes. Allow to cool, make neutral with HCl or N/10 NaOH ( phenolphthalein as in- dicator) and bring volume up to 100 cc. with dist. water. Method of preparing starch solution: Starch 1 gm. Dist. water 100 cc. Boil from 3 to 5 minutes, allow to cool, and make up volume to 100 cc. Method of preparing Monobutyrin solution: Monobut^^rin 1 cc. Dist. water 99 cc. Normally, tube No. VI. shows digestion of casein and starch. The lipase test is practically never done because of numerous sources of error and uncertain end point. In diastase test the last positive tube is the tube just before the one which shows blue, probably brownish, due to presence of erythrodextrin. Gastric digestion. A. Protein. Pepsin and HCl change albumin into acid albumin, then into albumoses (four in num- ber), then into peptones. Gastric digestion is not carried beyond the peptones or the slightly lower polypeptid stage. When lower products of digestion are found their presence is due to the action of other ferments, either intestinal or those excreted by malignant growths. B. Carbohydrates. Some inversion of sugar oc- curs in stomach, due to t]ie acid there present. By the action of the salivary ferment starch is changed into soluble starch, erythrodextrin, achrodextrin and, finall}', into maltose. Ptyalin does not act in the presence of free HCl, but its action is so rapid that from 50 to 75% of starch is converted into the 122 soluble forn:. If the HCl is kept low, starch diges- tion will continue much farther than when it is present in higher concentrations. C. Fat. Practically none is digested normally as lipase is present only in a trace. Motility. Disturbance in tliis function of the stomach is of more imj^ortance than its secretory disorders. Food can pass into the intestine and be digested there irrespectiye of gastric secretion. If motility is im- paired, howeyer, food stays and stagnates in the stomach, causing more or less disturbance. Motor disturbances : 1. ^'omiting, already cousidered, important be- cause of loss of food. 2. Hypermotility, in which condition food is rushed through, and often associated with hyperacidity. Consider spasm also in these conditions.) .']. Hypomotility, which is important and seri- ous. Results of motor disturbances: 1. Disorders of secretiou. :?. Disorders of digestion. 3. Disorders of absorption. Causes of stasis : 1. Cancer with obstruction, associated general]}' with hypochlorhydria, achlorhydria or achylia. 2. Fleer with obstruction, associated generally with hyperchlorhy dria . ?. Spasm, generally associated Ayith hyperchlo- rhydria. 4. Atony, witli or without hypersecretion. 5. I'tosis, which interferes Ayith mechanical passage of food. Study of Motility. 1. Xot by acid content. 123 2. Not bj the size of the stomach, for a large stomach may have normal motility. Terms : Ectasia (or ectasis) signifies dilation with motor insufiiciency. Atonic gastric ectasia signifies weakness of muscles of stomach. Hypertonic gas- tric ectasia signifies pyloric stenosis. Food should be out of the stomach in from 7 to 8 hours, no matter how heavy or large the meal. Tests which give information of gastric motility. a. Leube. A Riegel meal is washed out in 6 hours with 1 liter of water. Normal equals slight trace of food. b. Boas. A simple evening meal of meat, bread and butter is washed out the next day. If any food remains there is considerable motor insufficiency. c. Ewald-Siever's method : 1 gram of salol is given after a meal and the urine is collected every 15 min- utes for 2 hours. Test with ferric chloride for phenol, which gives violet color. Salol is broken down in the alkaline media of the small intestine into phenol and salicylates and is excreted in the urine. Difficulties of test: 1. The gastric juice acts slightly upon salol in about 15 minutes, breaking it up. 2. Small amounts of salicylic acid and phenol may be absorbed from the stomach. Normally, phenol appears in the urine in from 45 to 75 minutes and is all excreted in 24 to 27 hours. Abnormally, if not detected before 75 minutes in- dicates motor insufficiency; if not detected before 24 hours indicates stenosis. If detected in 15 min- utes an error is indicated. d. Winternitz. The method is the same as the preceding, except that iodipin is used in place of salol. Iodipin is not touched by the gastric juice, but requires pancreatic secretions and bile to free 124 iodiii. Tlie snliva is tested for iodin with si arch paste. Xormallv, it appears in from 15 to 45 min- utes. e. Sahli test meal (fat soup), (see text books for detail.) The method is incorrect if lipase is pres- ent in the stomach. f. An evening meal of substances easily recog- nized, such as raisins, rice, spinach, etc. Recover}^ of any of the meal (macroscopically or microscopi- cally) the next morning shows impaired motility of a degree depending upon the findings. g. Direct fluoroscopic examination with barium, wliich is the best method when available. Consideration oi absorption. Not of much im- poitance clinically. Penzoldt-Faber test : Two to o grains of KI are given in a capsule after a meal, which is rapidly absorbed by the mucus membrane and appears in the saliva, normally, in oO vo 40 minutes. Test the saliva every few minutes ^^'itll a fcAV drops of HX03 and a small amount of starch paste, when a positive reaction is indicated by a blue color. Indirect examinations. Where it is not possible to remove contents of stonuich, indirect methods may be used. They are not very accurate, but may give much help at times. a. (iunzberg's method: 0.2 gm. of KI are placed in the thinnest possible strongly vulcanized rubber tubing about 2.5 cm. long, which is then tied wath three threads of fibrin hardened in alcohol. Test by placing in water for 1 hour to be sure of no leak. The patient is allowed to swallow the bag three-quarters of an hour after an Ewald meal. Test saliva as in Penzoldt test. The HCl and pepsin digest the fibrin threads and free the KI which is 125 absorbed. It should appear in the saliva in three- quarters of an hour. b. Sahli's Desmoid bag. A bag of ordinary rub- ber-dam, containing a 0.05 gm. pill of methylene blue and 0.1 gm. of iodoform, tied with dried but chemi- cally-untreated catgut, which is digested by the gas- tric ferments, but not the pancreatic. The bag is swallowed after the noon meal. The methylene blue appears in the urine, normally, in 6 hours, coloring it green ; iodin in the saliva in 2 hours. Special test for carcinoma. Wolff Junghan's test. This is a soluble albumin test for carcinoma, very delicate, and applicable only to stomach contents. There should be no free HCl present and no occult blood for the successful application of the test, and it should be read within half hour after it has been set up. Positive results occurring after that time should be ignored. Reagent : Phosphotungstic acid 0.3 gm. HCl (concentrated) 1.0 cc. 96% alcohol 20.0 cc. Distilled water qs. ad. 200.0 cc. Procedure : Six carefully cleaned test tubes (cleaned with soap and water, alcohol and ether) are put in a rack and to them are added the fol- lowing : Tubes I II III IV V VI Filtered gastric contents. Ice O.o cc 0.2 cc 0.1 cc 0.05 cc 0.025 cc Dist. water 9 cc 9.5 cc 9.8 cc 9.9 cc 9.95 cc 9.975 cc Dilution equals ..1-10 1-20 1-50 1-100 1-200 1-400 After these dilutions have been made apply a clean rubber stopper to each tube and invert several times to mix. Do not use fingers as stoppers on account of errors in test. 1 cc. of the I'eagent is now carefully layered on 126 each of the tubes, which layering must be perfect. The presence of a white ring in the first three tubes is normal. A ring in the fourth is non-com- mittal. A ring in the fifth or sixth speaks for car- cinoma and is considered a positive test. Several points of clinical value : 1. High free HCl with low combined suggests liyperchlorhydria with hypermotility. 2. High (or normal) free HCl with high combined suggests hyperchlorhydria with stasis, probably spasm or stenosis. 3. Low free HCl with moderate combined sug- gests hypomotility with perhaps normal acid output. 1. HCl deficit with high combined suggests stasis with organic acid production. 5. HCl and mucus vary inversely as to their amounts. Mucus is never found with a high HCl. In gastritis, where mucus is characteristically pres- ent, HCl is low or absent. 6. Organic acids do not form if HCl is present in normal or increased amounts. 127 SPUTUM. Deriiiition : Sputum mcliides all the secretions which come from the respiratory passages. Collection of sputum. Avoid coutaminatioii from the mouth especially when making cultures. In chil- dren this can he done by stretching a piece of gauze over the finger and tickling the tliroat. The child AviJl cough and raise sputum, Avhicli will adhere to the gauze. When a quantitative examination is to be made, collect sputum in conical glass containers and look for stratification. Sometimes the gastric contents are examined in patients who swallow their sputum. Color of sputum: Gray — mucus. YelloAA' — pus ( purulent ) . Yellowish-gray — pus and mucus (muco-puru- lent) . Red — blood usually. When the lung is the source, it is frothy; when from the mouth, it its not frothy; when from the stomach, it is dark red or coffee-ground in appearance, and is never frothy. Occjurrence of hlood in the sputum {hemoptysis), 1. Pneumonia. 2. Infraction of lung. ?). Weeping aneurism. 4. Tuberculosis. 5. Tumor metastasis to lung. G. Blastomycosis. 7. Echinococcus cysts. 8. Paragonimus westermani (lung fluke). Green — resolving pneumonia, pj^ocyaneus infec- tion, rupture of subdiaphragmatic abscess. Black — coal and iron workers. White — starch granules in sputum of bakers. Various colors — dve workers. 12B An} Oil lit of spuiuni. This varies within a wide range. Large amounts are met with in : J. Branchial affections with much secretion. 1\ Bronchiectatic and tubercular cavities. 3. Pulmonary edema. 4. Perforating empyemas. Odor of sputum. Normally the sputum should have no odor. Sweetish odor — pulmonary abscess, occasionally in tbc. and oral sepsis. Foul — putrid bronchitis, gangrene, bronchiec- tasis. Cheese-like — perforating empyema. Putrefactive — stagnation in lungs or receiving cup. Macroscopic appearance. Layer formation. Top, frothy; bottom, granu- lar ; serous fluid between ; used to be consid- ered diagnostic of bronchiestasis, but it may occur whenever there are large amounts of sputum. Curshmann's spirals. These consist of a central core around which are wound strands of threads. The core is highly retractile and may be fibrin. Microscopical examination is necessary in order to make out the structure Charcot-Leyden crystals and eosinophiles are usually found with them. Dittrich's plugs. These are cheese-like masses usually about the size of a mustard seed, yel- lowish-Avhite to gray in color, sometimes form- ing casts of the bronchi or bronchioles from which they come. They are also of frequent occurrence in the crypts of tonsils of other- wise normal individuals. Upon crushing them they give a disagreeable odor. Microscopi- cally, they show large numbei^s of bacteria, 129 fatty acid crystals, fat globules and cellular detritus. Cheesy masses (rice bodies). These are small, yellowish masses varyinci- in size from that of a pin-]wint to a pea. They may be pigmented by decomposition products of hemoglobin. Upon crushing tliey do not give a disagreeable odor. They occur most frequently in tuber- culosis, also in abscess and gangrene. Fibrinous casts. In as much as not all the material making up these casts is fibrin, they are more properly called "bronchial casts.'' Many are branching and the size of the bron- chus from whicli they came. When composed of fibrin, they stain beautifully with Weigert's fibrin stain. With acetic acid they are not precipitated, and are friable in consistency. They occur in pneumonia and fibrinous bron- chitis. Bronchioliths. Tliese are calcified collections of debris and secretions. Pneumoliths. The majority of these are tuber- cttlous in origin, formed by the calcification of caseotis areas. They have a chalky or cal- carious consistency. Elastic tissue. This appears as grayish, opaqtie flakes, which are most readily recognized by ptitting the sptitum between two glass plates and looking toward the light. The other struc- tures can be dissolved away by boiling the sputum in 10% XaOH, eqtial parts of each, after which the sputum is diltited and the sediment examined. The fibrils of elastic tis- sue are not as wavy as those of fibrous tissue and are not dissolved by XaOH. Origin of elastic tissue: 1. Walls of arteries. Appear in sheets. 130 -. Bronchi. Have branches. 3. Alveoli. Appear in squares. When elastic tissue is found it means lung destruction. Echinococcus membranes. These ma}'^ be ex- pectorated in rather large masses, which are tough, thick and porcelaindike in color. 3Jicroscopically, they show laminated struc- ture. Microscopical exu in i nation. White blood cells. The ordinary ])olymorpho nuclear neutrophil is of little significance and is almost invariably found. The eosinophiles are found in asthma associated Avith Charcot- Leyden crystals. Red blood cells. These, also, are of little im- portance. Epithelial cells. J. Pavement ejnthelium coming from mouthy pharynx and upper larynx. 2. Cylindrical e]nthelium coming from nose and bronchi may be ciliated. '.\. Alveolar epithelium coming from alveoli often contain coal pigment ' (phthisis melanoticaj and are found in normal sputum, especially in the morning. They are increased in any irritation of the respiratory tract. In chronic passive congestion of the lungs they often con- tain hemotoidin or hemosiderin granules, and are then known as ''heart-failure cells." iUifSitals. Patty-acid. These needle-shaped crystals occur singly or in group-. Heat changes them into fat droplets. They are soluble in ether and alkali. They occur in gangrene, putrid bron- chitis, and chronic tuberculosis. 131 Cliolcsteriii. Tliese crystals resemble steps superimposed upon one another. They occur frequently in association with fatty-acid crys- tals in empyema, chronic lung abcesses and chronic tuberculosis. Hematoidin. These crystals are rhomboid or needle-shaped, and ruby-red in color. They occur rarely in lung abscesses, empyenui, per- forating liver abscesses and old hemorrhages. Tyrosin and leucin. These crystals are of rare occurrence and are formed only by the decom- position of protein material. They occur in empyema, lung abscess or perforating liver abscess. Triple phosphates. These occur in the same conditions as leucin and tyrosin. Calcium oxalate. These occur in conditions associated A^■itll decomposition. Charcot-Leyden. These are found in association Avith eosinophiles and are probably derived from them. They occur most frequently in asthma, and less frequently in fibrinous bron- chitis and hav fever. 1 niriial pcn'osites. 1. Amoeba buccalis (A. gingivalis and A. den- talis probably the same). 2. Amoeba histolytica. This parasite ma}' occur in rupture of amoebic abscess through dia- phragm. See section on parasites. .*). Echinococcus granulosus and cysticus. The booklets, scolices, or portions of the cyst wall will diagnosticate the condition. These should be looked for in unexi^lained hemor- rhages from the lungs. 132 4. Pargonimus westermani (lung fluke). Look for the eggs of this parasite, Avhich are operculated on blunt end. They measure .1 mm. X .05 mm. This parasite is a com- mon cause of heilioptysis in Japan. Fungi. 1. Streptothrix actinomyces. The sputum with this infection is glairy and mucilaginous or purulent and contains yellowish gran- ules about the size of a pin head, the so- called ''sulphur granules." If one of these granules is placed upon a slide and crushed with a coyer-slip and examined, threads are found centrally and club-shaped proc- esses are seen peripherally. The}^ can be stained with Lugal's solution. 2. Aspergillus. Look for doubly-contoured threads with brownish pigmented spores. They are best seen after the sputum has been treated with 10% KOH. They occur occasionally in bronchiectasis and tuber- culosis. 3. Blastomyces. Treat the sputum with dilute KOH and look for yeast-like, doubly-con- toured, retractile bodies. They occur occa- sionally in systematic blastomycosis. Bacteria. I. Tubercle bacillus. Pick out grayish opaque flakes and stain with Ziehl-jN^eelsen stain: 5% carbolic acid water 90 parts Concentrated alcoholic fuchsin 10 parts Stain for three minutes, during Avhich time gently heat oyer a bunsen flame and replace the stain as it eyaporates. AYash the excess of stain off with water and decolorize witli a 2% solution of HCl in 80% alcohol. When no more stain comes off', counter- stain Ayith 1% methylene blue. The tubercle bacilli. 133 I'etain tlie red fuslisiii stain wliile tlie other elements; are decolorized and take the bine stain. It is of importance that not all acid-fast organisms are alcohol acid-fast as is the tnbercle bacillus. Allien a more careful search, is desired, the anti- formin method should be used. A good modification is that of Loeffler's : 10 to 20 cc. of sputum are mixed with an equal quantity of Sodium hypochlorite 10 grams Sodium hydroxide 10 grams Water 100 cc. Boil for not longer than 15 minutes and centri- fugal ize at high speed for from 1 to 2 hours. Make smears of the sediment and stain as above. Guinea pig inoculation should also be resorted to when in doubt. The sputum is injected intraperi- toneally. If the animal is first given a strong radia- tion with the X-ray it will succumb much more quickly than othermse. Cultivator of the tubercle baoillus. Petroff egg media: Meat infusion glycerin (500 grams of meat infused with 500 cc. of a 15% solution of glycerin in water for 21 hours) 1 part Beaten eggs (sterilize shells by pour- ing boiling water over them and break into sterile beaker and beat thoroughly. Filter through sterile gauze into sterile graduate) 2 parts Ifo alcoholic solution of gentian vio- let to make a dilution of 1-10,000 Tube in sterile test-tubes and inspissate for three consecutive days, the first at 85 deg. C till solidified and on the two remaining at 75 deg. C for one hour. An equal quantity of sputum and 134 mixed and incubated for 30 minutes to digest the former. Neutralize ^^'itll N/HCl and centrifugalize at liigli speed for 10 minutes. Inoculate the sedi- ment on to a few Petrolf tubes and seal with par- affin. Seven to ten days are required for growth. The gentian violet inhibits the growth of other organisms. ( Method of isolating the organism from the blood. Dr. Mildred C. Clough, J. H. H. Bui., Dec, 1917, page 303.) 10 to 20 cc. of the patient's blood are hemolysed in sterile distilled water and centrifugalized at high speed for 1% hours. Three-fourths of the sediment is planted upon a blood agar slant, which is then sealed with paraffin to prevent drying. A growtli appears after incubation of 7 to 20 days. The other fourtli is inoculated into a guinea pig. II. Pneumococcus. Pneumococci are divided into four groups, in accordance with their serological reactions. See Monograph of the Kockefeller Insti- tute for Medical Research No. 7, October 16, 1917. Tyi)e I. Causes about 33.3 %_of the cases of lobar pneumonia. The disease runs a severe source. The organism is practically never found in normal sputum. Type II. Causes about 13% of the cases of lobar pneumonia. The disease runs a severe course. The organism is practically never found in normal sputum. Tj^pe III. Causes about 13% of the cases of lobar pneumonia. The disease runs a most severe course. The organism is found in about 28.1% of normal persons. Type IV. Causes about 20.3% of the cases of lobar pneumonia. The disease usually runs a mild course. The organisms are found in about 18.2% of noa'mal persons. 135 Types 11-a, lib and li-x are found in about 1S.27(; of normal persons. Isolation of the pncumococcus and determination of group. 1. Mouse inoculation. Obtain a specimen of sputum as free from saliva as possible; wash in six changes of sterile salt; grind a piece the size of a bean in a sterile mortar, add sterile salt solution drop by drop till a homogeneous solution is formed that will readily pass through the needle of a small syringe. Inject 0.5 to 1.0 cc. of this emulsion intra- peritoneally into a mouse. Either wait until the mouse is dead or test by peritoneal puncture for the presence of pus, and kill if pus is found. In either case wash the peritoneal ca\'ity out with 4 to 5 cc. of sterile salt solution after making pre- liminary smears and cultures on blood agar plates. Make a homogeneous suspension of the peritoneal wasliings and set up tubes as follows for agglutina- tion : TuLe 1 2 3 4 5 Put 0.5 cc of suspension into c \ach of^ tubes, O Q Q Q if o 1 , No. 2 serum undiluted 0.5 cc , No. 2 serum 1-20, 0.5 cc 'a Ivpe 1 . . . . +--1- — — — Di s solved Tvpe II . . . lia, III) IIx + ++ — Dissolved Dissolved Tvpe III . . — — + Dissolved Type IV... — — — Di s solved ( Incubate ' for 1 hour at 37 deg. C.) 136 Precipitin ieM. For this test tlie peritoneal wash- ings are centrifngalized until the superuatent fluid is water clear. The clear tluid is pippetted off and the following set-up made: Tube 12 3 4 Put 0.5 cc of supernatent fluid in each tube. Tvpe I — Tvpe II . . . ++ lia, lib IIx — Tvpe III.. — Tvpe IV... — u: zn Ul CD o o Si i-s 1— 1 i-S 1— 1 i-j gM ^ ^^^ S3 ^^3 "n? on ? o? P- to o to Ci Jj3 ++ ++ + (The precipitin reaction usually occurs at once. If it does not, incubate.) 2. Sputum cultures. Obtain a specimen of sputum as free from saliva as possible and wash through six solutions of sterile salt solution. A blood-tinged flake is the one of choice. After washing the flake place a small portion of it upon a blood agar plate and break it up Avith a platinum wire bent at a right angle. Streak three agar plates with the wire without reinoculation. Incubate for 24 hours and then look for small colonies with a green zone about them. Pick colonies, inoculate broth tubes, and carry out agglutination test as given. 3. Precipitin test in the urine. This test is often 137 positive within 12 lioiirs after the initial chill and remains positive for some days. When it is posi- tive it furnishes a rapid and accurate method of determining the group of the organism. For this test 0.5 cc. of urine are placed in each of three test tubes. To the lirst tube is added 0.5 cc. of Group No. 1 serum undiluted; to the second, 0.5 cc. of Group Xo. 2 serum undiluted, and to the third, 0.5 cc. of Group Xo. 3 serum undiluted. If a precipi- tate does not come down immediately, incubate at 37 deg. C. for one hour. The presence of the pre- cipitin reaction in the urine indicates a severe in- fection and is of bad omen, especially if it increases from day to day. 4. Avery's method. (Jour. A. M. A., Vol. 70, Xo. 1, Jan. 5, 1918, page 17.) This method is based upon the fact that carbohydrates and blood proteins when added to suitable media accelerates the growth of the pneumococcus, and also that bile dissolves the organism. Special media for the test: Meat infusion broth, 0.3 to 0.5 acid to phenolphthalein (sterilized in arnold sterilizer on three consecutive days) . 100 cc. 20% glucose (sterile) 5 cc. Defibrinated rabbit's blood (sterile) 5 cc. Tube media in small tubes about 4 cc. to the tubo. A kernel of sputum the size of a bean is selected and washed in six changes of sterile salt solution, emulsified in broth and inoculated into one of the tubes of special media. Incubate at 37 deg. C. for five hours and streak a blood-agar jDlate for the isolation of the organism and subse<|uent confirma- tion of type. Remove the blood cells from the spe- cial media by slow centrifugalization. Remove 3 cc. of the bacterial emulsion to another centrifuge tube and add to it 1 cc.of sterile ox bile. Incubate till 138 the solution of the piieuiiiococci has taken place and perform the precipitiu test, using 0.5 cc. of serum and bile solution. If ox bile is not obtainable, per- form the agglutination test after the cells have been removed. 5. Krumwiede and Valentine's method. (Jour. A. M. A., Vol. 70, No. 8, Feb. 23, 1918, page 513.) This method is based upon the fact that many sputums are comparatively rich in soluble antigens, and these antigens are not destroyed by heating to boiling. From 3 to 10 cc. of sputum are placed into a test tube, Avhich is then placed into, boiling water till the albumins are coagulated ; that is, if the specimen is a suitable one. Break up the coagulum with a platinum Avire and add just enough ]N^/]S"aCl to carry out the test after it has been centrifuged. In some instances no saline is necessary, for suffi- cient fluid separates to carry out the test. After the coagulum has been broken up agitate it in the saline and place again into boiling water for a few minutes to extract the antigen, shaking gently. Cen- trifugalize and use the supernatent fluid for the test. Place 0.2 cc. of the undiluted anti-pneumococcus serum in narrow tubes and upon each layer an equal quantity of the supernatent fluid. Place in a water batli at 55 to GO deg. C. and observe in several min- ute^\ If a great quantity of the antigen is present in the sputum, a ring y\Hl be observed in a short time, but if snmller quantities are present, longer incubation will be necessary. The advantage of this test and of the other tests is to determine the group of the organism and the employment of serum in tlie treatment should it be Group No. 1. HI. Meningococcus. See monograph by Simon Flexner on ''Mode of Infection, Means of Prevei;- 139 tion, and Specific Treatment of Epidemic Menin- gitis," the Rocli:efeller Institute for Medical Re- search, 1917. ''The meningococcus enters and leaves the body bv way of tlie nasopharyngeal membrane." Mode of idcntificatiou. The West tube is used to swab tJie nasoi)harynx. Tliis tube consists of a glass tube about 7 mm. inside diameter and bent at nearly a right angle at one end. A copper wire carrying a cotton swab at one end and a loop on the other is inserted into the tube, which is then plugged at both ends and sterilized. The nasopharynx is swabbed by inserting the glass tube up behind the soft palate, then pushing the cotton swab out against the pharyngeal wall and swabbing it, and finally pulling the cotton into the tube again and with- drawing the whole tiling. The object is to prevent saliva from getting on the swab, which is destructive to the organism. The inoculated swab is then run over a series of three plates of sheep serum agar, which should be made as follows : Melted meat infusion agar with a plus 0.4 to phenolphthalein and cooled to 50 deg. C. 100 cc. Dextrose 1 % Sheep serum 1 part, distilled water 3 parts (sterilized at 15 lbs. for 10 minutes) 20 cc. Avoid chilling the ])lates and incubate at ">7 deg. C. for from IG to 20 hours. A medium more favorable for growth is made as follows : Nutrient agar melted and cooled to 50 deg. C. 100 cc. Sterile rabbit's blood 5cc laked in sterile distilled water 40 cc. 20 cc. The colonies of the meningococcus are small, deli- cate and their outlines fade away into the medium. Make smears and stains of susi)icious colonies 140 and transfer to laked-blood or sheep sernra agal' slants. The meningococcus together Avith M. catar- rhalis, flavns and phar^ngis-sicctis are gram nega- tive. Incubate the slant inoculations for 24 hours, and then emulsify in salt solution and subject to the following agglutination : Polyvalent 1-50 1-100 1-200 1-500 1-1000 1-2000 Serum 0.8 cc. 0.8 cc. 0.8 cc. 0.8 cc. 0.8 cc. 0.8 cc. Emulsion 0.2 cc. 0.2 cc. 0.2 cc. 0.2 cc. 0.2 cc. 0.2 cc. Incubate at 55 deg. C. for 16 hotirs and read. Any culture which is agglutinated in a dilution of 1-200 and ferments glucose "and maltose with acid production, but does not ferment saccharose, is considered provisionally as meningococcus and the person from whom it Avas isolated is regarded as a carrier. Some differential points between meningococcus and other gram negative organisms of throat: Micrococcus flavus : Colonies velloAv and opaque ; indiscriminate agglutination, agglutinated by normal horse serum in a dilution of 1-50 and by polyvalent serum in a dilution of 1-100 or slightly higher. Micrococcus caturrhalis : Exerts no action on glu- cose, and no agglutination in higher dilutions of specific sera. Other indefinite gram negative micrococci: Fer- ment saccharose. Olitsky method for the identification and isola- tion of the meningococcus. (Jour. A. M. A., Jan, 19, 1918, A^ol. 70, Xo. 3, page 153.) Tins method takes adA^antage of a fluid medium Avhich serAes to eliminate other organisms resem- bling the meningococcus and reduces the time nec- essary for tlie identification to about 12 hours. 141 Medium: Glucose broth, 1% (made from veal infusion and having acidity of from I)lus 0.5 to 0.7 phenolphthalein ) • 100 cc. Sterile, clear, unheated, normal horse serum 5 cc. Tube this medium in 8 or 10 mm. tubes, about 1 cc. to the tube. Suspicious colonies are fished from a plate inoculated with the nasopharyngeal secretion of a suspected carrier and are seeded into these tubes, a colony to a tube. The tubes are then incubated 12 hours, and at the end of this time a great many negatives can be eliminated. Organisms which must be eliminated : 1. Micrococcus flavus, crassus, pharyngis-siccus and unclassified gram positive bacilli will show firm agglutination below and slight turbidity above. 2. Bacillus influenzae will not grow for the want of hemoglobin. 3. Micrococcus catarrhalis grows with a dense turbidity, and often shows a pellicle on the surface. 4. The gram positive staphylococci grow with dense turbidity, show agglutinated masses in the sediment, and often have a pellicle. 5. Streptococci grow with a clear or turbid supernatent fluid, but show an agglutinated sediment. The meningococci give a faint turbidity and a slight sediment forms which emulsifies uniformly when the tube is shaken. The suggestive positive tubes are separated from the definitely negative ones, and to the former is added 0.1 cc. of a 1-10 dilution in 0.85 saline of a high titer polyvalent antimeningococcus serum. Incubate the tubes at 37 deg. C. for two hours in the water-bath and the tubes containing meningococci will show definite agglutination, and those which do not will remain 142 luicljaiiged. From the positive tubes cultures can be obtained for fiirtlier identification. \y. Diphtheria bacillus. It is important to re- meiiiber tliat tliere are organisms in the thvoat of h-altliy persons wliich have the morphological char- acteristics of tlie diphtheria bacillus, but which pro- (luci: no toxin, tlie>e being the diphtheroids; also, that one examination of a diphtheretic throat may give negative results: hence, upon the ability to produce toxin should rest the final diagnosis of the organism and one examination should not suffice in sus])icious cases. P /< Diphtheroid The throat is swabbed Avith a small piece of cot- ton and the surface of a serum agar plate is streaked with it. Incubate from six to eight hours when the diphtheria colonies will be visible and contami- nating organisms will not. Make smears from colo- nies and stain w^ith Loffler's methylene blue. Stained specimens of the cultivated bacillus show the typical beaded appearance Avith clubbed ends. For more careful study use Neisser's stain. V. Bacillus mucosus capsulatus. This gram neg- ative non motile bacillus varies from coccoid forms to longer bacilli. They are surrounded by a large capsule which is easily demonstrable. They groAv easily on plain agar, which growth is mucoid and sticky. They often cause a A^ry fatal broncho or lobar pneumonia J the sputum of Avliich is slimy and stickA\ 143 ^^T. Tiitiueiiza bacillus. This is a very short, mod- erately thick, gram negative bacillus which grows singly in pairs or sometimes forms threads. It has 2 polar bodies which make it resemble a gram neg. diploccns. It is a frequent secondary invader in respiratory and pulmonary infections, such as chronic bronchitis, bronchiectasis and tuberculosis. The orbanisms grow best on blood agai*, upon which the colonies appear as small, colorless dewdrops. It has not yet been proved to be the cause of influenza epidemics. Avery's influenza .media : Delibrinated blood, centrifugalized and cells resuspended in amount of broth equal to original volume of blood 1 cc. Agar (sterile) 95 cc. Sodium oleate 2% (neutralized and auto- claved) 5 cc. This media is said to inhibit other organisms of the throat and to enhance the growth of the influ- enza bacillus. VII. Spirochaeta pallida. This organism is some- times found in the mouth in association with syph- ilitic lesions there. It must be differentiated from S. dentalis, S. buccalis and S. refringens. In gen- eral it may be said that under the dark Held illumi- nator these other spirochaetes have a much livelier movement than the pallida, which hardly moves out of the field. It has a lashing motion, a spinning motion about its long axis and a slight backward and forward motion. It is usually easy to identify the pallida from these characteristics. YIII. Spironema vincenti. This organism is found in Vincent's angina in association with a cigar- shaped bacillus. They are both readily stained with dilute carbo-fuchsin and gentian violet. 144 Sputum in various diseases. 1 . Lobar pneumonia due to the pneumoeoccus. The course of the disease can be followed by the type of sputum. It is blood tinged for the first three or four days and mucoid in consistency. After this blood cells disappear and it assumes a rusty appear- ance from altered blood pigments. This lasts until after the crisis, when it becomes muco-purulent, and later serous. Occasionally one sees x3neunionia without sputum at all. In some cases a green color is noted in the later stages of the disease, due to altered blood pigments. In pneumonia due to the B. mucosus capsulatus the sputum is slimy and sticky and very tenacious. 2. Tuberculosis. This disease may have almost any kind of sputum. As a rule, in the early stages there ma^^ be little or no sputum; later it may be- come mucoid or mucopurulent and blood streaked. On microscopical examination one may find the acid- fast organisms and elastic tissue. In chronic tuberculosis the amount of sputum varies from little to tremendous amounts. It may be bloody, mucopurulent, or purulent. In ulcerative tuberculosis one finds sputum of a feweetish odor, blood clots, a great deal of elastic tissue^ caseous lumps which do not give a bad odor on ci'ushing, and on microscopical examination one mav find all kinds of contaminating organisms. Tn fibroid tuberculosis one may have no sputum, '^ it may be mucoid or mucopurulent. '■, Ab^cesse^. The sputum is abundant, cheesy, u'^c^'ou . and masses of blood may be present. )cca ionally one finds masses of lung tissue and elastic tissue. 4. Gangrene. The sputum is the same as abscess, except that the odor is characteristically very foul and penetrating. 145 5. Infarction. Immediately after the accident the sputum is stringy and mucoid and blood streaked, or there may be a marked hemoptysis. The sputum soon becomes rusty and "prune juice'- in character, which change comes on sooner than in pneumonia. C). Chronic passive congestion. In this condition tlie sputum is thin and abundant. It may be slightly rust;\'. On microscopical examination one finds "heart-failure cells," i. e., alveolar epithelium con- taining blood pigments. 7. Asthma. There is no sputum in the early stages of the attack, but when the attack breaks pearls of Lannec (mucoid globules) make their ap- pearance. On careful examination one finds, also, Curslimann's spirals, Cliarcot-Leyden crystals and eosinophiles. 8. Bronchitis. There may be no sputum or there may be present pearls of Lannec and Charcot-Leyden crystals. Fihri)ioifs bronchitis. In this type of bronchitis there may be fibrin casts of the bronchi. Purulent hionclntis. The sputum has a mucoid base with a yellowish appearance from the presence of i)us cells. riccrativc hronchitis. Epithelial cells remain un- changed. Goblet and ciliated cells appear occa- sionally. Tissue fragments and blood may be pres- ent. Chroidc hroucliitis. The sputum is usually thin, may be tinged Avith blood, and may contain Dit- trich's plugs. In the later stages of the disease the sputum may become foul, abundant and muco-puru- lent. D. Bronchiectasis. The sputum usually occurs periodically in large amounts and has a fetid odor. In tlie early stages it is thin and watery, but later (Continued on Page 146) 146 it may resemble the sputum iu abscess, except that there is not so much pus. Cartilage, elastic tissue, clots of blood and tissue masses may be present. Its separation into three layers, viz., top, brownish froth ; middle, clear and mucoid, and bottom, gran- ular ; is not characteristic for this disease, but occurs whenever there are large amounts of sputum. 10. Pulmonary oedema. This is usually a termi- nal event, but frequently occurs after too vigorous thoracentesis. It may start during the tapping and may last from 5 minutes to 24 hours. Huge amounts of fluid are given otf. It is a safe rule not to draw off more than 1500 cc. at one tapping. STOOLS. Constituents of stools. I. Food remnants. These are undigestible or un- absorbed. Xormally, there are some and it is diffi- cult to draw a line between the pathological and the normal under various conditions. When an excess of meat fibres occur, the condition is called creator- rliea ; and when an excess of fat occurs, the condi- tion is called steatorrhea. It is important to be- come familiar with vegetable cells in order not to confuse them with animal parasites or their ova. Fat. Fat occurs as soaps and fatty acids and sometimes as neutral fat. If it occurs as neutral fat, it is yellow and clear ; while as fatty acid, it is wiiite and glistening. Soaps usually occur as the insoluble calcium and magnesium soaps, but in rapid peristalsis the soluble sodium and ]30tassium soaps nuiy occur. Neutral fats indicate the absence of lipase. Fatty acid increase indicates an excess of fat in the diet over that Avhich can be absorbed or hyperperistalsis. In the decreased secretion of bile an excess of fats occur also. 147 DIFFEREXTrATTOX OF FATS, FATTY ACIDS & SOAPS. Test. Heat Ether Xeutral fat. Melted Dissolved Fatty acid. Melted Dissolved Soap. Sudan III Stained Crystals Globules + Osmic acid Scharlack R. H,0 1 Stained Stained 1 Xa. & IC. dis- solved Ca. & :M.g-. un- ' dissolved KOH Carbol fuchsin + + Soaps can be broken np with acetic acid and tbcn stained with Sndan I IT. Some fatty acids with a h)w nieltino- ])oint appear in globnbir form and stain with Sudan ITT. Sugar and starch. These are not nsnal. Occa- sionally tliey come thronj^h in a cellubir envelope. Test with iodin Avhich turns starch granules blue, and Benedict's solution which is reduced by sugar. Tf. Tnte^stinal tract secretions. Bile. Occult bile never seen in the adult. To de- termine the presence of bilirubin or hydrobilirubin emulsify about 2 or :> cc. of the stool in a concen- trated solution of mercuric chloride. Allow to stand 24 hours and examine microscopically. Green sig- nifies the presence of bilirubin ; red indicates hydro- bilirubin. UrobUui. This is normal in certain amounts, but large amounts are pathological. To determine its presence use Schlessinger's test as follows : Make an acid-alcohol extract of the stool, add a few drops of Lu gal's solution, 1 or 2 cc. of XH^OH, and an equal volume of an alcoholic solution of zinc acetate. Filter and examine the filtrate for a greenish fluo- rescence. Spectroscopic examination of the intesti- nal fluid normally is positive in dilutions up to 1 to 9,000. In higher dilutions than this it is patho- logical. 148 Mucus. When mucus is abundant it means catar- rhal inflammation. In a disease resembling asthma one may get complete casts of the colon, which are usually passed between stools. To test for mucus is usually unnecessary. Ferments. These are usually tested for in order to determine whether the pancreas is functioning normally or whether the pancreatic secretion is en- tering the intestine. Since trypsin and lipase are more easily destroyed by bacteria and each requires actiyation to show its maximum efliciency, and also since proteolytic action of the intestinal bacteria may complicate the tindings, the study of the dias- tase of the stools is probably the best means of obtaining this information. Method of performing test: 1. The patient is given a light meal and a high enema the night before. 2. At 7 A. M. the next day, 750 cc. of milk are given. 3. At 7.30 and 8.00, half an ounce of Epsom salts. 4. At 8.30, a glass of Avater containing a tea- spoonful of bicarbonate of soda. Save all stools up to 2 P. M. in a vessel contain- ing 2 ounces of toluol and keep in the ice-chest or cool room. If less than 400 grams of cc. are ob- tained, an enema of a pint of water is given. From 400 to 1,100 cc. are to be expected. Titration of the diastase: Dilute the stool with enough water to make 3,000 cc. Dilute 2 cc. of this Avith 48 cc. of isotonic salt, which gives a dilution of 1 to 25, and then centrifugalize to clear. Set up tubes as folloAvs : 1 2 3 4 5 6 1 cc stool 1 cc stool 1 cc stool 1 cc stool 1 cc stool 1 cc stool 1-25 1-50 1-100 1-200 1-400 1-800 Add 2 cc of 0.1% starch solution, after dilutions 149 are made, to each. Incubate at 37 cleg. C. for half hour and add a few drops of a 1% solution of iodin to each tube. Blue shows the presence of undi- gested starch. Calculation : % cc. of 1-25 diluted stool digests 2 cc. of 0.1% starch. 1 cc. of 1-25 diluted stool digests 4 cc. of 0.1% starch, or 0.4 cc. of a 1.0% starch solution. 1 cc. of undiluted stool digests 10 cc. of a 1% starch solution. Tlie unit is the digestion of 1 cc. of a 1% starch solution by 1 cc. of undiluted stool. The minimum of normal is 600 units. (See article by Dr. Brown in Boston Med. and Surg. Jour., :N"ov. 30, 1916, Vol. CLXXV, pages 775 to 784.) Schmidt and Strasburger diet for pancreatic effi- ciency : 1. Morning: 500 cc. of milk and 50 grams of zweibach. 2. Forenoon: 500 cc. of oatmeal gruel com- posed of: Oatmeal 40 gm. Butter 10 gm. Milk 200 cc. Water 300 cc. 3. Noon: 125 gm. (raw weight) of chopped beef, broiled rare and 20 gm, of butter. 250 gm. of potato broth composed of : Mashed potato 190 gm. Milk 100 cc. Butter 10 gm. 4. Afternoon : Give same as for forenoon. 5. Evening : Give same as for dinner. This diet should be given three days or longer in order to obtain stools in which it is present. In 150 pathological conditions there will be food remnants of all kinds. ITT. Decomposition products. Gasses: H,S, CH^, ^\ H, and CO,, which are the products of fermentation and putrefaction. Crystalline bodies : Tndol and skatol. These give the feces their characteristic odor. IV. Bacteria. These make up about one-third by weight of the stool. A. Those ^^'hich normally occur: The majority of these belong to the colon group, among which the more common ones are: B. coli, including B. fecalis alkaligenes, and B. acidi lactici ; B. lactis aerogenes, B. subtilis, B. proteus vulgaris, etc. A few strep- tococci and staphylococci are sometimes found. B. Those Avhich occur under j)athological condi- tions. 1. Bacillus typhosus. This organism occurs in the stools of typhoid patients and of typhoid car- riers. One of the best ditferential culture media for identification is Endo's media, which is made, ac- cording to Robinson and Rettger's modification, as follows : Agar 25 grams . Meat extract 5 grams Pepton 10 grams Water 1000 cc. Dissolve the agar, pepton and meat extract, and autoclave at 15 pounds pressure for 30 minutes. Filter through cheese cloth and cotton, add 10 cc. of 10% sodium carbonate, adjust reaction to # plus 0.1 phenoli)hthalein, put in 100 cc. containers and autoclave at 10 pounds for 10 minutes. 151 When ready to use, add to each 100 cc. of agar: 20% lactose solution (Arnoldized) 5 cc. 10% freshly prepared anhydrous sodium sulphite sol. (sterile) 1 cc. Saturated alcoholic fuchsin (basic) 0.5 cc. Pour plates and allo\y to harden without the covers upon them. # Plus 0.1 phenolphthalein means the addition of 0.1 cc. of N/HCl to 1000 cc. of the medium after the neutral point of the solution has been determined with phenolphthalein as indicator. The basic fuchsin is colorless in an alkaline me- dium, but turns red in acid solution. Since the typhoid bacillus does not ferment lactose, the typhoid colonies will remain colorless, while the colon colonies will turn red because the colon bacillus ferments lactose. An emulsion of the stool is made in isotonic salt solution and a loopful of the emulsion is transferred to an Endo plate. With a bent wire the surface of the plate is streaked, and, without flaming, a second plate is treated likewise. Incubate 24 hours and pick colonies. In order to be relatiyely sure that a colony picked is a typhoid one, microscopic aggluti- nation can be done in the following way: A low dilution of anti-typhoid serum is made (1-100), and one drop of this is placed upon a clean slide. A drop of salt solution is placed upon the same slide near the drop of serum, and in it an emulsion of one of the suspicious colonies is made. The two drops are now coalesced, and within 2 or 3 minutes, if the colony picked is one of typhoid, deflnite clumps can be made out. One must be sure in this pro- cedure to haye the drops large enough to prevent drying, which would give false readings. If the colony picked gives agglutination, the remainder is inoculated upon agar for further identification. If 152 one should titrate specific sera with typhoid, para- typhoid (a) and paratyphoid (b), one could deter- mine the dilution of each, wliich Avould agglutinate the organism for Avhich it was specific and not those which it agglutinates in lower dilutions. By this rapid slide agglutination one can, by properly dilut- ing the sera, sometimes determine which of the typhoid group of organisms he is dealing with. 2. Dysentery bacilli. These are a group of bacilli which are resj)onsible for some forms of dysentery and which are differentiated by sugar reactions and agglutination tests. Their isolation can be accom- plished by the use of Endo's media or brilliant green media, for which see Park and Williams Bacteri- ology. These are short gram negative rods, with little or no motility. The colonies are not as large nor as opaque as the typhoid or colon and most of the groups are colorless on Endo's media. They also appear later than the foregoing. By use of the polyvalent serum they can be detected by macro- scopic slide agglutination, and then transferred to other media for more careful study. In making cultures it is desirable to pick the bloody flakes of mucus, 3. Tubercle bacillus. These are occasionally swal- lowed and must be excluded. The smegma bacillus must also be differentiated, which is . not alcohol acid-fast. Particles of blood-stained mucus are most likely to show the organisms. The antiformin method is not very satisfactory unless the organisms are present in large numbers. 4:. Cholera spirillum. This organism can usually be diagnosed from morphology and serum reactions. It can grow in a media too alkaline for the growth of other organisms. In fluid media it seeks the surface where oxygen is most abundant. It liquifies gelatin slowly, unlike S. metchnikovi. 153 Y. Animal i:>arasites. See section on parasitology. VI. Foreign bodies. 1. Gall stones. Tliese may be large or small. Stones as large as the gall bladder itself may ulcer- ate into the intestine and cause obstruction. Smaller stones must be differentiated from cartilage, insolu- ble soaps, enteroliths and fecal masses, >Yhich can be done by crushing, dissolving in ether and allow- ing to crystallize out. In the case of gall stones, one will find cholesterin crystals. 2. Intestinal sand. This is mucus impregnated with calcium salts. These smaller particles may become matted together into larger enteroliths. 3. Objects sTvallowed. Coins, buttons, safety pins, hair, etc. 4. Objects left in the abdominal cavity during operation. Clamps, sponges, knives, etc., may ulcer- ate through the bowel and be passed per rectum. A'll. Tumor fragments. If these come from any distance they are digested beyond recognition. Oc- casionally they aid in the diagnosis of tumor. VIII. Pus cells. A few occur normally. When in very great numbers they may arise from ruptured abscess. When seen in clumps they may indicate some ulcerative process, such as dysenter}', ulcera- tive colitis, syphilis, carcinoma, tuberculosis or typhoid. IX. Blood. That coming from the mouth must be excluded. If the blood is at the bottom of the stool its source must be low down in the intestinal tract; if on top, its source is higher up. If blood comes from high up in the intestinal canal, it will be dark and give the stool a characteristic tarry appearance, although its source may be high up and appear dark red if there be rapid peristalsis. Blood from the stomach gives a tarry black appearance. Blood occurs in any ulcerative condition of tlie 154 iutestinal tract, such as typhoid, ch^sentery, carci- noma, ulcer, etc. It also occurs as a result of rup- ture of blood vessels into the stomach or intestine, as in Banti's disease, aneurism or hemorrhoids. Tests for Mood. The best is the guiac test. Make a watery emulsion of the stool, add a few drops of glacial acetic acid, extract T\dth ether and perform in the same manner as with the urine. The benzi- dine test can be carried out in the same manner. A good preliminary test is the following : Make a smear of the stool upon a glass slide, and before it dries add a solution of benzidine in glacial acetic acid plus an equal volume of hydrogen-perox- ide. The absence of a green color shows that no blood is present. If the material turns a green color, a more accurate test, such as the above, should be performed, X. Crystals. 1. Fatty acid. Long or short needles singly or in groups. 2. Calcium phosphate. Wedge shaped in rosettes, or singly. 3. Magnesium phosphate. Rhombic plates. 4. Triple phosphates. Coffin-lid crj^stals. 5. Calcium carbonate, sulphate and oxalate. (See urine.) G. Calcium lactate. Radiating needles in sheet-like masses. Found in children on milk diet. 7. Cholesterin. Usually occur as steps, one superimposed upon the other. In doubt- ful cases test with concentrated sulphuric acid. The color changes from yellow to blood red, violet, green and, finally, blue. 8. Charcot-Leyden crystals. These are color- less, double-pyramid crystals. 155 9. Hematoidin crystals. These are reddish yel- low, rhombic plates, groups of needles or amorphous masses. Color of stool. The normal color is brownish, due to h^Tlrobilirubin. Bilirubin occurs in children, but never in adults. The color also depends upon exposure to the air and the character of the diet as well as medication. Milk gives a light-brown color; meat, dark brown; bismuth, black; senna and rhubarb, yellow, and calomel, a green, which is due to bilirubin not changing into hydrobilirubin. Blood gives all shades from bright red to black. Clay- colored stools occur in pancreatic disease due to excessive amount of fat, the action of bacteria re- ducing bile pigments, or the absence of bile due to the occlusion of the bile duct. Yellowish-green stools often occur in hypermotility of the intestine. Odor of stool, formally it depends upon the presence of indol, skatol, methane and hydrogen sulphide gas. The odor is most marked on a heavy meat diet and less marked on a vegetable or milk diet. It is almost lost during fasting. An excess of fermentation over putrefaction will give a sour odor. In some cases of severe dysentery- and carci- noma of the large bowel the stool has an intensel}^ disagreeable odor. Number and amount. There are great individual variations. There may be a normal number of stools and yet be constipation. Xormally from li*0 to 250 "vams are excreted a da v. 156 STOOLS IN DISEASE. There are no characteristic stools in disease, but the stools of some diseases are of some diagnostic help. 1. Obstructive jaundice. (Due to stone in the common or hepatic duct, carcinoma at the head of the pancreas, or duodenitis with temporary closure of the common duct.) The stool is strictly acholic and clay-colored if the obstruction is complete and fat globules and crystals are seen. Clay-colored stools resembling this are sometimes found where no obstruction to the bile excretion exists, but where bilirubin appears in the form of a leukobase and can be demonstrated by the bilirubin test. Clay- colored stools associated with nausea and vomiting and jaundice give a complete picture. If there is a history of colics, search, at least for three days, for stone. If one is found, confirm by crushing, extract with ether, evaporate and examine for cholesterin crystals. 2. I*ancreatic disease. (May be associated with stone shutting off the pancreatic duct.) The stools are ver}^ large and have an abundance of neutral fat. They may resemble vaseline or freshly-fallen snow. They may be odorless or sour. Besides the great quantity of neutral fat, fatty acids are found and great quantities of undigested meat fibres. 3. Carcinoma of the stomach. There is usually associated hyperperistalsis, but occasionally anti- peristalsis. In the cases with hyperperistalsis there occurs a copious diarrhea. If no blood is present, they are pale or yellow in color, but if blood is present, they are brown to black. The odor is usually foul. On microscopical examination one finds undigested meat fibres more frequently in this disease than any other. 4. Carcinoma of the rectum. The stool may be 157 normal in size, shape and consistenc^N', or, if the growth has caused a stricture, they may be ribbon- like. They may show a coating of blood if ulcera- tion has taken place, and mucits. Occasionally tis- sue fragments are found. 5. Annular carcinoma of the sigmoid. The groAvth may constrict the lumen and cause obstruction. There is usually constipation, distension, much gas, and there may be hyperperistalsis. Metastasis oc- curs late and the prognosis is usually good. 6. Amyloid disease. Abundant and frequent stools with little odor, ]!^o tenesmus, pain, blood nor mucus. Diarrhea continuous. 7. Hypoacidity. Foul-smelling, abundant diarrhea, with meat particles, much mucus, and blood if asso- ciated with carcinoma. Often a little hydrochloric acid will relieve condition. 8. Mucus colitis. The cause may be due to a for- eign protein, much like asthma. Charcot-Leyden crystals and eosinophiles are found. 9. Sprue. There is irritation of the whole gastro- intestinal tract. There is stubborn diarrhea and pain. 10. Metallic poisoning. (Arsenic and mercury.) These drugs cause a gaj^tro-enteritis. Mercury is excreted in the colon and causes an intense irrita- tion and watery stool. n, StricLiire of the rectum. This may be due to any of a number of causes: syphilis, carcinoma, and sometimes a congenital malformation. Kibbon stools are characteristic. If there is ulceration, as in carcinoma and lues, there will be an excess of mucus and pus. Eibbon stools always indicate rectal ex- amination. J 2. Constipation. There is a diminution in the number of stools, large appetite, a feeling of depres- sion, and usually headache. This is more common 1<8 and cliionic in women. A decrease in the nnniber c. stools may be due to impaction of fecal masses in tlie rectum. In many cases these can reached Avith the linger and dislodged or broken up. 1.'). ^[egalo-cohni. (Hirschsprung's disease.) There is a congenital weakness of the wall of the colon. The bowels may move once a week, once a uiontli, and one case is reported of once a year. The-c patients present all the signs of constipation. 14. Ty|>hoid. This is associated frequently with con ti])ation, especially in the early stages. During ilu stages of ulceration profuse watery and foul- siiulling stools occur. They may be yellowish-green in coh)r and give rise to the term "pea soup stools." Hemorrhages are frequent which are usually pre- ceded by oozing and the appearance of occult blood. Clinically, this indicates the cessation of hydro- therapy and feeding. Typhoid cultures are easily obtained. 15. Cholera. The stools are usually profuse with an abundant watery discharge, although moderate cases show only a moderate amount of diarrhea. Flakes in mucus, rice-like in appearance, give the term "rice-water stools." 16. Amoebic dysentery. This sometimes runs an intermittent course. There are periods of intense diarrhea, followed by periods of normal stools and even constipation. Amoebae are foUnd in the patches of bloody mucus. 17. Tuberculosis. There is a great variation in the stools, which depends upcn the site of the lesion. In tabes mesenterica there is an interference with absorption, and one finds an excess of fatty acids and soaps in the stools. This is unlike pancreatic disease, where an excess of neutral fat and fatty acid occur. Dietary indiscretions lead to great diarrhea. J 159 A. OUTLIXE OF ROUTINE FOR MAKING QUAL- ITATIVE EXAMINATIONS OF BLOOD. I. Cleaning glass ware (slides and cover slips), a. Gj-oss cleaning — several methods. (1) Wash with soap and water, then with much clean, hot water (never allow the water to cool till all the soap is re- moved), followed by distilled water, finally put in 95% alcohol (may be put into ether or chloroform after alcohol). (2) Soak in acid mixture (cone, nitric acid or a mixture of sulphuric acid and po- tassium bichromate) for 6 to 24 hours, then wash thoroughly in clean water, then distilled water. Finally run through two thorough washings with 95% alcohol and put in ether or chloro- form (ether may be omitted — keep in alcohol — closed vessel) . b. Fine cleaning. (1) Keep in alcohol (ether or chloroform), and when needed polish with a clean cloth (old linen preferable) absolutely free from grease and dust. Ex. unfold an old but clean and ironed handker- chief — use the inside surface which no linger has touched. (2) Take from alcohol (ether or chloroform) and polish, keeping them in a clean, dry, well-closed receptacle until used. Ex. A closed glass dish. (Avoid Avooden or pasteboard boxes, as fragments fall from such materials.) (3) Never hold with fingers, always use clean forceps to handle glass ware. (4) Dust off with clean camel's hair brush before us^ing. 160 (5) It is oCteii advantageous to blow across glass ware just before placing blood on it — care must be exercised that no parti- cles of saliva fall on glass. 1 1, flaking smears. a. Preliminary details. (1 ) I^e glass Avare cleaned in manner sug- gested under T. Cover glasses must be square ( round will not do) ; cover glasses must not be too thick, as oil immersion cannot be focused upon preparation ; size 22 mm. preferable, thickness Xo. 1 essen- tial. (2) Glass Avare must be flat — avoid concave surfaces — blood will not spread. ( .") ) Dip blood sticker in alcohol before using (never flame sticker — dulls point). (1) Wash skin with alcohol and dry before sticking. Alcohol frees skin from grease and is antiseptic ; drying prevents blood drop from spreading over skin. (5) Avoid CA'anosed areas and aA^oid cyano- sis by squeezing area Avhich is to be stuck, in order to make the blood floAA\ Blood examinations from such areas are useless for many purposes. (()) Stick deep enough to bring from 2 to 4 drops of blood— preferably without the assistance of any pressure. While any pressure is undesirable, a slight amount is sometimes necessary. (7) Wipe off the first drop — use the second, Avhich is more representative of actual blood picture. b. Actual smearing. (1) CoA^er slip method. (a) With instruments. 161 1 . Plokl cover slip Xo. 1 in cross bill forceps. 2. Hold cover slip Xo. 2 in clean, plain forceps. .*). Brush the cover slip Avith a clean camel's hair brush to remove any lint or dust. 4. Place drop on cover slip Xo. 2. Drop should be size of small black-headed ])in. Let it reach the cover slip by capillary attraction. Xevei' touch skin with cover slip. 5. With drop on cover slip Xo. 2, j)lace cover slip Xo. 2 on cover slip Xo. 1 in such a maner that tl'.e dro]) is sj)read without caus- ing bubbles. (Ex. Let cover slip X'^o. 2 come down so that cover slip Xo. 1 touches edge of drop first and gradually touches whole drop — or let the drop reach the first cover slip by capillary at- traction). 0. Let drop spread until it has almost stopped spreading — expe- rience is needed for this. 7. Pull cover slip in an absolutely horizontal numner. Any tendency to a vertical pull will ruin the preparation. (Holes in the smear will result.) 8. Place preparations (smear side up) on a clean paper and allow them to dry in the air. 9. Protect smear from insects, ants and flies especially, as they will ' quickly ruin smear. 162 (b) Without instruments. Cover slips may be bandied by the lingers instead of Avith forceps in making smears provided tbe following precautions are ob- served : 1. Use forceps to take cover slips from the clean container. 2. Hold cover slip between fingers so that only the edges are touched and never the surface of the glass. 3. Cover slip No. 1 is held in left hand, usually between thumb and forefinger. Place drop on cover slip Xo. 2, Avhich is held in right hand, and then proceed to make the smear as directed above, with the exception that the pulling is done by holding the cover slip with the fingers rather than with forceps. (2) Slide method (use of slides instead of cover slips). A drop, larger than the one used for cover slip method, is placed on a slide and drawn across it so as to make a smear. Various methods for spreading are suggested. (a) By means of a second slide, prefer- ably with a beveled edge and of a width less than the slide on which the smear is to be made (i. e., break off a corner of a slide to make it nar- rower)., (b) A small glass rod (w^idth less than slide), to which another glass rod has been fused, to serve as handle, in such a way that the combination 163 looks like a T. The cross-beam of the T is used as the spreader. (CI (Mgarette paper, hat pius, long needles, or plain glass rods may also be used as the spreading agent. (d) The spreader (whichever method suggested being used) may be placed to the left of the drop, and the blood in this way pulled across to the right, or fe) The spreader may be made to touch the drop from the right and the drop pulled across the slide to the right. (This method is to be pre- ferred.) (f ) Tlie thickness of the spread can be varied by changing the degree of angulation of the slide and spreader. (X.B. — The drop should be placed well to the left of the slide, so that a long spread may be pulled to the right). c. Criteria for good smears. (1) Smooth, even spreads, with R. B. C. lying flat (never in rouleaux)). Their edges may touch, but there must be no overlapping. (2) At least 8 such areas (low power) with- out holes and without streaks are re- quired to constitute the minimal re- quirement for a good smear. d. Relative merits of cover slip and slide methods. ( 1 ) The cover slip method gives a much bet- ter distribution of W. B. C. and is the method necessary to use in making dif- ferential W. B. C. counts. 164 (2) The slide method gives a good distribu- tion of K. B. C. The large W. B. C. (polymorphonuclear and large mono- nuclear) are pushed to the edges and the lyphocytes remain scattered through the spread. This makes satisfactory dif- ferential counting impossible. When E. B. C. are to be examined for parasites, the slide method is superior to the cover slip method, inasmucli as more fields are available for study and the E. B. C. dis- tribution is as good as Avith the cover- slip method. III. Making stained preparations, a. With Wilsoji stain. (1) The stain. A modified Eomanowski stain — a polychrome methylene blue- eosin stain. (For details of making it see Emerson, Webster, etc.) (2) Technique of applying stain. (a) Use freshly made smear for stain- ing; smears 2 to 4 days old do not stain so Avell as those stained very soon after making. (b I Place preparation with smeared sur- face up (smeared surface has not the gloss of the clean glass surface) in staining forceps on the edge of a table or on a cork for support in such a manner that it may easily be taken up Avith a pair of forceps. (c) With a dropper drop 6 to 8 drops of Wilson stain on the smear, being careful to avoid shaking the bottle of stain before using it in order to keep stain free from any precipitate which may be present. Let the stain 165 remain on the smear 60 sec. (using second hand of watch for accurate calculation) ; then (d) Add the same number of drops of distilled water, and let the prepara- tion stand an addition 4 min. (by the watch) ; then (e) Taking the cover slip with forceps (preferably staining forceps to pre- vent possibility of dropping cover glass) and holding it carefully in a perfectly horizontal manner so that the scum which has formed on the surface may not touch the glass, float off rather than wash off the scum with a stream of water (pref- erably distilled), which is at first run very slowly, and then more briskly so as to free the smear from all traces of excess stain. All this time the cover slip is held abso- lutely horizontally. Washing should take from 5 to 8 seconds. (X. B. — 1. For staining, do not hold smear with any forceps used for liulling smears — forceps used for pulling smears must be kept abso- lutel}^ clean. 2. Each fresh supply of stain re- quires a new formula for stain and water combination. In general equal numbers of drops of stain and water are used.) (f ) After washing is completed, the cover slip, still held in the staining forceps, is tilted and the lower edge touched to a blotter, so that the 166 excess of water is quickly drained off. (N. B. — If the cover slip be held with any forceps other than stain- ing forceps, great care must be ex- ercised to j)revent fluid collected at the forceps' tip from running over the preparation and streaking it. If any such forceps be used, it is best to tilt the cover slip immediately after washing, in such a manner that the excess of water on the cover slip runs toicard the forceps. Remove the cover slip from the for- ceps and holding it in the hand, drain off the excess of water by touching an edge to a blotter. (Pref- erably that edge held previously by the forceps.) (g) After washing, the smears are dried in one of the following ways : 1. By air drying : a. Generally by placing the smear against some sup- port and letting one edge rest on a blotter, b. By waving it gently in the air, holding it with either for- ceps or finger (touching only the edge of the cover slip if the finger method is employed). 2. By blotting : Place preparation between layers of fine blotting paper (it must be absolutely free from dust). Apply light pressure to the upper layers of the blotting paper to facilitate drying. Then pick up the preparation and remove it to a dry place, and again apply light 167 pressure. Be careful not to push the smear along the blotter nor to press too heavily, for holes and streaks in the preparation will be the result. (N. B. — The staining is thought to be better if the blotting method is employed, but many good smears have been ruined by slight negli- gence in the manner of blotting.) (h) After drying the preparation (if it be a cover slip), mount it in Can- ada balsam {acid free) in one of the following ways : 1. Preferably smear side down as the preparation is then protected from insects and any acid or alkali in the air. 2. Smear side up if the cover slip is too thick to permit focusing the oil immersion when mounted smear side down. (N. B. — If slides are used instead of cover slips, the technique is iden- tical except that the preparation is not mounted, and for examination oil immersion is applied directly to the surface of the smear.) (l\) Criteria for a good stain. R. B. C. — Are of a buff color, neither lemon nor red. Platelets — Are well stained — nuclear purple blue stain, with the architecture plainly visible. W. B. C. — Are stained as follows : Polymorphonuclears. P. M. N. — Nucleus — Deep purple, retic- 168 ular, chromatin pronounced, more poly- morplious than |)olynuclear. Grannies — May or may not be seen ; vary in size ; pink or yiolet. rrotoplasm— Faint pink. r. M. E. — Nucleus — Larger than P. M. X., fewer lobes; takes lighter purple color ; reticulated. Granules — Large, round or oyal; bright red; tend not to oyerlie nucleus. Protoi)lasm — Faint pink. P. M. B. — Nucleus — Chromatin scanty, stains light purple. Granules — Large, yary in size; jiurple to black; generally some oyerlying nucleus. Protoplasm — Faint pink. Lymphocytes. — Nucleus — Large, round or oval ; slightly notched, chromatin pro- nounced, deep purple ; clear zone outside. Granules — Normally none; old cells (?) sho\v azure granules ; red yiolet ; yary in size ; few to a cell. Protoplasm — Scanty, crescentic ring; homogeneous sky to deei) blue; slightly reticulated. Large Mononuclears. — Nucleus — Large, oyal, indented, horse shoe, kidney or yery irregular shape ; chromatin poor ; light blue or purple color; generally ec- centric. ProtoiDlasni — Abundant, often irregular, clear, reticulated, pale blue (reticulated nodosities giye granular ap- pearance). Granules — None (azurophil- ogranules frequent). Malarial parasites are beautifully stained with Wilson stain. The areas between the cells must be clear and free from all suggestion of stain. The cells must stand out with distinctness T\ith jio suggestion of hazy edges. There must 169 . be no precipitate present. (4) Common pitfalls in staining with Wil- son stain. (a) Precipitate on the preparation. 1. Due to: a. Faulty washing, l)y not holding preparation horizontal and floating otf scum, thus permit- ting the scum, which ahvavs forms, to touch the smear, b. Permitting dust to settle on the smear. 2. Prevented bv: a. Holding the preparation horizontal all during the washing and learning to play the stream of water in washing to the best advantage, b. Keeping the smears clean during tlie interval which elapses between pulling and staining. (b) An indefinite serum-like stain be- tween the cells, due to insuflicient washing. (c) Tearing preparation by improper blotting. (d) Deterioration of stain supply due to: 1. Acids kept in the same locker. 2, Water from mixing pipettes or putting stain in bottle waslied witli water. b. With Jrinio' stain. (1 I The stain. A simple methylene blue- eosin, alcoholic staiu. (For details see Emerson, Webster, etc. i (2) Technique of a])plying stain, (a) Freshly made smears stain better than those 24 to 48 hours old. (b) Staining, washing, blotting and 170 mounting are carried out in a man- ner like tliat described under Wilson stain with the following differences : 1. Place 6 to 8 drops of stain on the smear and leave for 2 min. (use watch for timing) ^ then add the same number of drops of water and leave for an additional 2 min. (use watch), wash and dry. (N. B. — With each fresh supply of stain a new formula for the stain and water combination, as well as for the time relation, is necessary). (3) Criteria for a good stain. E. B. C. — Are of a darker color than the K. B. C. with Wilson stain, pink rather than buff, although it is possible and advisable to have them look as much like the R. B. C. of the Wilson stain as possible. Platelets — Stain rather jDOorly — a pale blue. W. B. C. are stained as follows : The nuclei are not so well stained as with Wilson stain, but granules stand out well. Polymorphonuclear — Nuclei blue, but distinctly paler than with the Wilson stain. Neutrophile granules a deep pink. Eosinophile granules a very deep pink. Basophile gran- ules purple blue. Lymphocytes — Nuclei a moderate- ly pale blue, protoplasm tinged with blue. Mononuclears — Nuclei paler blue than those of the lymphocytes, proto- plasm a faint blue* Azurophilic 171 granules show less proiiiineiitly than with the Wilson stain. ^Malarial parasites are not well stained. The intercellular areas are clear and shoAV no stain. (4) Pitfalls in using Jenner stain. These are almost absent. The chief difficulty is in giving the nuclei a deep enough blue stain. Improper stains show the nucleus to have a faded-out bluish tinge. By changing the water and stain combination and by washing more quick- ly the nuclei can be made to take a bet- ter stain. c. With EhrUch stain. (1) The stain — a complex triacid aqueous- alcohol stain. Do not shake the bottle before using. (2) Preliminary details : (a) Old smears stain better than fresh. Ex. smears 5 to 10 days old are bet- ter than tliose 5 to 10 hours old. It is advisable to have 15 to 25 smears of the same age when an Ehrlich is to be stained. (b) Before staining, smears must be fixed by heat in the following man- ner : 1. Heat a copper bar for 30 to 10 min. in a place free from draughts, so that the flame will not be shift- ing. (X. B. — Place flame under point of bar.) 2. At the end of that time, with a dropper having a small bored point, drop a few small drops of 172 water (3 to 4) on the bar in an etfort to find the spheroidal i)oint. (Spheroidal point equals point on bar where water rolls off in glob- ules.) Let at least 5 min. elaj^se be- tween trials, so that the heat lost by the application of the water will be surely regained. 3. AYhen the spheroidal point has been determined by repeated tests, draw a chalk line across the bar at that place. ( N. B. — Inaccurate determination of the spheroidal point is to blame for many poor Ehrlichs.) 4. Place preparation, smear side up, outside the spheroidal point, with the edge closest to the flame exactly on the line. The method is carried out as follows : Fix a smear 20 sec, another one 30 sec, still another 40 sec, on up at intervals of 10 sec, until 90 to 150 sec (Devise a method for care- fully differentiating the variously heated smears so that in staining no confusion will arise as to the lixa- tion time of any particular smear, for ex. 20 sec. or 90 sec.) 5. In general, the older the smears the shorter the required time of heating. Ex. smear 10 da^^s old may be well fixed at 30 sec. ; smear 1 day old may be well fixed at 150 sec. or more. (3) Actual staining. (a) Place the stain on the smear and let it remain 5 min. At the end of 173 that time pour the stain hack into the t)ottle, and wash off the adher- ing ainonnt of stain in the sink. Dry and blot as directed under the other staining methods. (Ehrlich stains are not easily ruined in the staining and washing, but are generally spoiled by poor fixing.) (4) Criteria for a good Ehrlich. (a) The cells should stand out clearly with no intercellular stain. (b) The K. B. C. should be buff, without the slightest suggestion of any red color, and without being a lemon yellow. (N. B. — Smears Avhich are under- fixed or markedly over-fixed have a reddish color in the K. B. C. Smears which are moderately over-fixed have lemon color in the K. B. C.) (c) The W. B. C. stain as follows : P. M. X. — Nucleus — Blue green or robin's egg blue; no structure visi- ble. Protoplasm — Faint pink. Gran- ules — Lilac, if correctly fixed ; red from short fixation. P. M. E. — Nucleus — Light green color. Protoplasm — Generally none seen. Granules — Dark red or crim- son. P. M. B. — Nucleus — Light green, reticular. Protoplasm — None seen. Granules — No stain, often seen as colorless vacuoles. Lymphocytes. — Nucleus — Light blue green. Protoplasm — ^Light pink 174 or violet, often very faint. Grannies — None seen. Lartie Mononnclears. — Xnclens— r\iint bine or green. Protoplasm — Fink or light violet. Grannies — None seen, (di Platelets are not stained. ( e ) Malarial parasites are not stained. (."")) Creneral tecliniqne nsed in staining with Ehrlich. ( a I From 15 to 25 smears ; select 3 or 4 of the best and lav them aside. (b) Take the less good smears and rnn through the various times for fixing a Irea d v su gges ted . (c) Stain these, and after mounting ex- amine them carefnlly with oil im- . mersion, and note the following points : 1. Under-fixed smears Avill have reddish staining K. B. C, often with stained intercellular spaces. The W. B. C. will have a dirty appear- ance with granules not standing out distinctly. 2. Well fixed smears will have the R. B. C. of a buff color (neither yellow nor red). The W. B. C. will stand out distinctly with neutroph- ilic and eosinophilic granules well stained and standing out sharply against a clear unstained proto- plasm. 8. Over-fixed smears will have the R. B. C. lemon color. The granules in the polymorphonuclear white cells Ayill not be clear cut, and if '175 stained will be seen lying in a stained protoplasm. There is a smudgy look to the polymorpho- nuclear cells. The mononuclears are often so faintly stained as to be missed. 4. If still further over-fixed, the K. B. C. lose their lemon color, and again take on a reddish hue. (d) Having discovered a well-stained smear, note the heating time for it, and then fix any number of good smears (of the same age) for a sim- ilar time. (N. B. — The need of again deter- mining accurately the spheroidal point is evident since by placing these goods smears at a point which is no longer the spheroidal point, they will be ruined.) d. General points with regard to stained blood preparations. (1) Be careful to mount cover slips in a neufral halsam, if any acidity is pres- ent (as frequently occurs) the color will fade at a rate proportional to the degree of the acidity, the nuclear ele- ment, particularly, being attacked. (2) Keep the preparations away from bright sunlight, from acid or alkaline fumes. (3) After using oil, remove it with xylol. (N. B. — Never attempt this if the smear has been freshly mounted.) lY. Differential Counting. The cover slip method must be used for this as follows : . a. With a mechanical stage count across and 176 up and down over the good areas of the spread. Be careful to use such a technique that the same areas will not be counted twice. b. .'iOO W. K. C. must be counted. In order to get the true proportion it is necessary to include in this nund^er brolvcn cells and undertermined cells as well as those which can be classified. c. A W. P. (\ count should always be nuide at the time a ditferential is counted. It is only by this means that relative or abso- lute changes in the number of cells is de- termined. (1. The classiticati(m of cells is as follows: (1) Polymorphonuclear neutrophiles (P. M. X.). The nuclei — irregular, 2 to ^) lobes, often appearing actually polynuclear. The granules — pink or violet (AVilson). pinker (Jenneri, lilac (Ehrlich). (X. B. — With Wilson the granules may not stand out distinctly an