^JJa&fiSjftSTTY OF CALIFORNIA 
 
 
A MANUAL OF 
 
 PRACTICAL LABORATORY 
 DIAGNOSIS 
 
 BY 
 LEWIS WEBB HILL, M.D. 
 
 Graduate Assistant, Children's Hospital, Boston 
 
 WITH ii FIGURES AND 
 8 PLATES 4 IN COLORS 
 
 BOSTON 
 
 W. M. LEONARD, Publisher 
 1916 
 
 LIBRARY 
 
COPYRIGHT, 1916, BY 
 W. M. LEONARD 
 
PREFACE 
 
 THE author realizes that it may seem superfluous 
 to add another to the very many excellent books of 
 laboratory diagnosis now in use. His purpose is this: 
 
 It has seemed to him for a long time that all the 
 worth-while laboratory tests that a practical medical 
 man needs, could be put together i a small and 
 compact volume. This should be gotten up in such 
 a way that medical students, and house-officers doing 
 their hospital ward and dispensary work can carry it 
 in their pockets for ready reference, without going 
 to the trouble of hunting through all the various al- 
 ternative methods of performing a test given in most 
 of the larger books. Much material usually in- 
 corporated in laboratory manuals has been left out 
 of this one, and purposely, for many methods and 
 tests which are of scientific value and of theoretical 
 interest, are not practical for the average medical 
 man to use. It has been the author's aim to include 
 the information that an every day practitioner wants, 
 but to weed out carefully all tests and methods which 
 are not of ordinary clinical application, or which re- 
 quire for their performance complicated apparatus 
 and trained technicians. Thus, such things as tissue 
 staining and fixing, the Wassermann reaction, the 
 gold chloride test, etc. have been purposely omitted, 
 as these are not ordinarily performed by medical 
 students, house-officers, and general practitioners, for 
 whom this book is intended. 
 
 O A 00-1 
 
IV 
 
 It is a pleasure to thank Dr. John Lovett Morse, 
 Dr. William H. Smith, and Dr. Francis W. Peabody, 
 for their helpful suggestions and criticisms. 
 
 For the plates illustrating the sputum we are in- 
 debted to Dr. W. H. Smith, and to Mr. L. H. Brown, 
 who made the photographs. 
 
 Most of the plates in the chapter on urine were 
 taken from Austin's " Clinical Chemistry." 
 
 BOSTON, May 7, 1916. 
 
CONTENTS AND INDEX 
 
 CHAPTER I 
 
 THE URINE 
 
 PAGE 
 
 COLLECTION OF TWENTY-FOUR HOUR SPECIMEN i 
 
 AMOUNT i 
 
 APPEARANCE i 
 
 COLOR i 
 
 SPECIFIC GRAVITY 3 
 
 REACTION . 3 
 
 ACIDITY 5 
 
 ALBUMIN 5 
 
 SUGAR 9-17 
 
 OTHER REDUCING SUBSTANCES 17 
 
 ACETONE AND DIACETIC ACID 19 
 
 AMMONIA 19 
 
 BILE 21 
 
 UROBILINOGEN. 23 
 
 MELANIN 23 
 
 BLOOD PIGMENTS 23 
 
 FORMALDEHYDE 23 
 
 INDOXYL 25 
 
 METALLIC POISONS LEAD, MERCURY, ARSENIC 25-31 
 
 SEDIMENTS ORGANIZED AND UNORGANIZED 3i~47 
 
 KIDNEY FUNCTION TEST 47 
 
 CHAPTER II 
 THE BLOOD 
 
 HEMOGLOBIN ESTIMATION 51 
 
 COLOR INDEX 51 
 
 COUNTING THE BLOOD CELLS 53 
 
 COUNTING THE BLOOD PLATELETS 55 
 
 v 
 
VI 
 
 PAGE 
 
 EXAMINATION OF THE STAINED SPECIMEN 57 
 
 THE BLOOD CELLS, NORMAL AND ABNORMAL FORMS . . 61-73 
 
 THE BLOOD IN INFANCY 73~75 
 
 MALARIAL PARASITES 75 
 
 DOHLE'S LEUCOCYTIC INCLUSION BODIES 79 
 
 WIDAL REACTION 81 
 
 BLOOD FRAGILITY TEST 81 
 
 HEMOLYSIS TEST 85 
 
 COAGULATION TIME . 87 
 
 OBTAINING BLOOD FOR CULTURES, ETC 89 
 
 CHAPTER III 
 FECES 
 
 COLOR 93 
 
 REACTION 93 
 
 CONSISTENCY AND FORM 93 
 
 MACROSCOPIC EXAMINATION 95 
 
 INTESTINAL PARASITES 97 
 
 CHEMICAL EXAMINATION 99 
 
 MICROSCOPIC EXAMINATION 101 
 
 THE STOOLS IN INFANCY 113 
 
 CHAPTER IV 
 GASTRIC CONTENTS 
 
 EXAMINATION OF THE FASTING CONTENTS: 
 
 AMOUNT 123 
 
 CONSISTENCY 123 
 
 COLOR 123 
 
 FOOD . . : 123 
 
 ODOR 125 
 
 MICROSCOPIC EXAMINATION 125 
 
 CHEMICAL EXAMINATION 127 
 
 EXAMINATION OF THE TEST-MEAL CONTENTS: 
 
 AMOUNT 129 
 
 COLOR 129 
 
 CHEMICAL EXAMINATION 131 
 
Vll 
 
 CHAPTER V 
 
 SPINAL FLUIDS 
 
 PACK 
 
 PRESSURE 137 
 
 APPEARANCE 137 
 
 FIBRIN CLOT 137 
 
 AMOUNT 137 
 
 CHEMICAL EXAMINATION 139 
 
 MICROSCOPIC EXAMINATION 139 
 
 CHARACTERISTICS OF THE SPINAL FLUID IN VARIOUS DIS- 
 EASES 145 
 
 CHAPTER VI 
 PLEURAL AND PERITONEAL FLUIDS 
 
 TRANSUDATES 151 
 
 EXUDATES 151 
 
 EXAMINATION OF EXUDATES 153 
 
 CHAPTER VII 
 SPUTUM 
 
 SOURCE 155 
 
 MACROSCOPIC EXAMINATION ." 155 
 
 MICROSCOPIC EXAMINATION 159 
 
 CHAPTER VIII 
 MISCELLANEOUS 
 
 GRAM'S STAIN FOR GONOCOCCUS 175 
 
 SPIROCHAETA PALLIDA 175 
 
 SCHICK TEST 175 
 
 VON PIRQUET TEST 177 
 
 GRAM-POSITIVE AND GRAM-NEGATIVE ORGANISMS 177 
 
 LEUCOCYTOSIS IN VARIOUS DISEASES 179 
 
CHAPTER I 
 THE URINE 
 
 Collection of Twenty-four Hour Specimen. Empty 
 the bladder at 7 A.M. and throw the urine away. 
 Save all the urine passed up to 7 A.M. the next day, 
 passing it exactly at 7, and adding it to what has 
 already been saved. 
 
 Amount. The normal amount of urine is about 
 1000 to 1500 c.c. in the twenty-four hours. This, of 
 course, is subject to many variations, depending upon 
 the fluid intake, the amount of water lost with the 
 stools, perspiration, etc. 
 
 Appearance. Ordinarily a normal urine is clear, 
 but a cloudy urine is not necessarily pathological. 
 Turbidity may be due to : 
 
 1. Urates or phosphates (usually normal). Urates 
 are particularly likely to be precipitated in a con- 
 centrated urine in a cold room. If due to phosphates, 
 the turbidity clears up on the addition of a little 
 acetic acid ; if due to urates, it clears on heating. 
 
 2. Pus, or a combination of pus, blood, casts and 
 epithelial cells. 
 
 3. Fat (Lipuria). A rare condition. The ad- 
 dition of ether dissolves the fat and renders the urine 
 clear. 
 
 Color. The color depends largely upon the amount 
 or urine passed, and upon the percentage of total 
 solids. The normal color may vary from a light 
 
yellow to a rather deep orange. The main abnormal 
 colors are: 
 
 1. Pink, due to the precipitation of amorphous 
 urates in an excessively acid urine. 
 
 2. Greenish brown, due to bile. 
 
 3. " Smoky " or mahogany colored, due to blood 
 cells and casts or to blood pigment and bloody 
 detritus. 
 
 4. Brown or black. Urine which when passed is 
 light colored, but which turns dark brown or black on 
 standing (oxidation) usually contains either melanin 
 (in subjects with melanotic sarcoma) or alkapton 
 (a metabolic product). 
 
 Specific Gravity. The normal specific gravity 
 varies within rather wide limits 1015 to 1025. The 
 gravity depends, as does the amount, on several fac- 
 tors, such as fluid intake, loss through sweat, etc. 
 In general a high gravity goes with a small amount, 
 and vice versa. The principal diseases in which a low 
 specific gravity is found are diabetes insipidus (1001 
 to 1005) and chronic interstitial nephritis (1008 to 
 1015). The urine in diabetes mellitus is of high 
 gravity (1030 to 1050), due to the presence of 
 sugar. 
 
 Reaction. The normal urine is usually acid, the 
 acid reaction being due to acid phosphates in solution. 
 If a urine is alkaline when passed, the alkalinity is 
 most likely to be due to ammonia formation within 
 the bladder, from bacterial activity (cystitis) or to 
 the presence of large amounts of carbonates of the 
 alkalis, which are derived from the oxidation of the 
 salts of the organic acids of vegetable foods. Nearly 
 all urine becomes alkaline on standing, due to bac- 
 
terial decomposition with ammonia formation from 
 the urea in the urine. 
 
 Quantitation of the Acidity. In certain cases where 
 there is increased frequency or burning micturition, 
 it is of value to know whether the acidity of the 
 urine is abnormally high. 
 
 FOLIN'S METHOD. To 25 c.c. of urine add a drop 
 or two of a I per cent alcoholic solution of phenol- 
 phthalein, and 15 or 20 grams of finely powdered 
 potassium oxalate. Shake thoroughly, and titrate 
 with a decinormal sodium hydroxide solution. The 
 acidity is expressed in terms of grams of hydro- 
 chloric acid. Each cubic centimeter of decinormal 
 sodic hydrate used is equivalent to .00365 gram of 
 hydrochloric acid. The acidity of the normal twenty- 
 four hour amount of urine corresponds to between 
 1.15 and 2.3 grams of hydrochloric acid. 
 
 ALBUMIN 
 
 The ordinary protein occurring in urine is serum 
 albumin. 
 
 Nitric Acid Test. To about 15 c.c. of filtered urine 
 in a small urine glass add about 5 c.c. of concentrated 
 nitric acid, letting it run gently down the sides of the 
 glass. A white, flocculent ring at the zone of contact 
 indicates albumin. This test reacts to all urinary 
 protein except peptone. If there is an excess of 
 uric acid in the urine, a light ring similar to the 
 albumin ring is formed, but it is one or two centi- 
 meters above the zone of contact, and disappears on 
 gently warming or diluting the urine. Bile pigment 
 produces a greenish ring at the zone of contact. 
 Indoxyl produces a violet ring. A rough idea of the 
 
amount of albumin present may be formed by the 
 thickness of. the ring, and may be reported as follows: 
 
 1. Slightest possible trace (S. P. T.). The slight- 
 est haze which can be detected, against a dark back- 
 ground. 
 
 2. Very slight trace (V. S. T.). Slightly more. 
 
 3. Slight trace (S. T.). A fairly thick ring, but 
 one which can be hardly seen from above, when look- 
 ing down through the glass. 
 
 4. Trace (T). Shows a thick, heavy ring, which 
 does not transmit light when looked down upon from 
 above. Not flocculent and not opaque. About .10 
 per cent albumin. 
 
 5. Large trace (L. T.). Anything more than 
 this. 
 
 Heat Test. Fill a test tube one-third full of urine 
 and gently boil the top part of it. If a white precipi- 
 tate appears in the heated area it may be due to 
 albumin or to phosphates. Add a drop or two of 
 acetic acid. If the precipitate is due to phosphates it 
 disappears, if it is due to albumin it remains. 
 
 If a urine contains protein, it is nearly always serum 
 albumin, but such substances as serum globulin, 
 nucleo albumin, mucin and albumose may be present 
 and give the nitric acid or the heat test. For all 
 practical purposes the only one worth considering is 
 albumose. This may be present in the urine of 
 patients with multiple myelomata of the bone mar- 
 row. It reacts to the nitric acid and heat tests, but 
 may be distinguished from other proteins by the 
 fact that on heating gently a flocculent precipitate 
 falls at 60 C., which dissolves when the urine is 
 boiled, and reappears when it is cooled. 
 
The quantity of albumin present may be approxi- 
 mately estimated by the method of Esbach as fol- 
 lows: Fill the Esbach tube with filtered urine to the 
 mark U. Then add Esbach's reagent (10 gm. of 
 picric acid and 20 gm. of citric acid in a liter of water) 
 to the mark R, and invert the tube several times. 
 Let it stand over night, and read off from the gradu- 
 ated tube the height of the precipitate. This reading 
 must be divided by 10 to obtain the percentage of 
 albumin. 
 
 SUGAR 
 
 The only important sugar found in the urine is 
 dextrose (grape sugar). Very small traces of it occur 
 normally, but these small amounts are not recogniz- 
 able by any of the tests ordinarily in use. Many 
 tests are used for dextrose, but only those which have 
 been found most serviceable by the writer in practice 
 will be given. 
 
 In any reducing test for sugar, be sure that no chloro- 
 form or formaldehyde has been added to the urine as a 
 preservative. 
 
 1. Fehling's Test. To 5 c.c. of hot Fehling's solu- 
 tion in a test tube add, a few drops at a time, 5 c.c. of 
 urine, boiling after each addition. If sugar is present, 
 a yellow or a red precipitate of copper oxide is formed. 
 
 2. Benedict's Test. To 5 c.c. of Benedict's 
 reagent add 8 drops of the urine to be examined. 
 The fluid is boiled from i to 2 minutes, and then 
 allowed to cool of itself. If dextrose is present there 
 results a red, yellow or green precipitate, depending 
 upon the amount of sugar present. If no sugar is 
 present the solution may remain perfectly clear or 
 
II 
 
 become slightly turbid, due to precipitated urates. 
 This is a more delicate test than Fehling's. 
 
 Fehling's solution consists of two reagents, which are kept in 
 separate bottles and are mixed in equal volumes when ready for 
 use. 
 
 (a) Copper solution: 34.65 gm. of pure copper sulphate in 
 500 c.c. water. 
 
 (6) Alkaline solution: 173 gm. crystallized potassium and 
 sodium tartrate (Rochelle Salt) and 125 gm. of potassium hydrox- 
 ide dissolved in water and made up to 500 c.c. 
 Benedict's solution has the following composition: 
 
 Copper sulphate * 7 3 gm. 
 
 Sodium citrate J73-O gni. 
 
 Sodium carbonate 100 gm. 
 
 Distilled water to .. 1000 c.c. 
 
 3. Fermentation Test. If Fehling's test is positive 
 and there is any doubt as to whether sugar is present 
 or not, the fermentation test is useful as a confirma- 
 tory test. About 100 c.c. of urine is put into a small 
 flask and a quarter of a yeast cake is crumbled up 
 into it, and the whole left in a warm place for from 
 24 to 48 hours. The reduction test is again tried 
 and if negative this time, proves,, that a fermentable 
 sugar, presumably dextrose, was present in the 
 original sample of urine. 
 
 4. Ozazone Test. A special property of sugars is 
 the formation of ozazones when treated with phenyl- 
 hydrazine. Each sugar has its own ozazone, of 
 definite melting point and crystalline configuration. 
 
 Add I c.c. of phenylhydrazine acetate solution (i 
 part glacial acetic acid, I part water, 2 parts phenyl- 
 hydrazine, by volume) to 5 c.c. of urine in a test tube, 
 and heat on water bath for half an hour. Allow the 
 liquid to cool slowly, and examine the crystals micro- 
 scopically (see plate, p. 31). 
 
13 
 QUANTITATIVE TESTS FOR DEXTROSE 
 
 1. Benedict's. Measure with a pipette 25 c.c. of 
 Benedict's solution into a porcelain dish, add 9 or 
 10 grams (approximately) of solid sodic carbonate, 
 heat to boiling, and while boiling, run in the urine 
 from a burette until a white precipitate forms. Then 
 add the urine more slowly, drop by drop, until the 
 last trace of blue disappears. The urine should be 
 diluted so that not less than 10 c.c. will be required 
 to give the amount of sugar which the 25 c.c. of 
 reagent is capable of oxidizing. A dilution of I to 10 
 usually answers. 
 
 CALCULATION. Five divided by the number of 
 cubic centimeters of undiluted urine run in, equals the 
 per cent of sugar. 
 
 Benedict's quantitative solution is prepared as follows: Dis- 
 solve 9.0 gm. of copper sulphate in 100 c.c. distilled water. 
 (The copper sulphate must be weighed very accurately.) Dis- 
 solve 50 gm. anhydrous sodic carbonate, 100 gm. sodic citrate, 
 and 65 gm. of potassium sulphocyanate in 250 c.c. of distilled 
 water. 
 
 Pour the copper solution slowly into the alkaline citrate 
 solution. Then pour the mixed solutions into the flask without 
 loss, and make up to 500 c.c. 25 c.c. of this solution is reduced 
 by 50 mgm. of dextrose, or 67 mgm. of lactose. 
 
 2. Fermentation Test. Take the specific gravity 
 of the 24 urine, put 100 c.c. of it into a flask, and 
 add to it a quarter of a crumbled up yeast cake. 
 Put the flask in a warm place (at about body tem- 
 perature) and allow it to remain over night. The 
 next morning test a sample of the fermented urine 
 for sugar. If no sugar is present make the urine up 
 to 100 c.c. (to allow for the water that has evapo- 
 rated) and take the specific gravity again. Multiply 
 the number of points loss in specific gravity by .23. 
 This gives the percentage of sugar in the urine. 
 
15 
 OTHER SUGARS IN THE URINE 
 
 It is rare to find any sugar but dextrose in the 
 urine, but levulose, maltose, pentose, sucrose, or 
 lactose may occur. 
 
 Levulose. Reduces Fehling's, but more feebly 
 than does dextrose. 
 
 SELIWANOFF'S REACTION. To loc.c. of urine add 
 a small amount of resorcin and 2 c.c. dilute hydro- 
 chloric acid, mix and heat in a test tube. If levulose 
 is present the liquid turns red and precipitates a dark 
 sediment, which is soluble in alcohol, with the forma- 
 tion of a bright red color. 
 
 Maltose. Occurs very rarely in disease of the 
 pancreas, in very small amounts. Of no practical 
 interest. 
 
 Pentose. Pentoses reduce Fehling's solution rather 
 slowly. They do not ferment. They may be de- 
 tected by Bial's orcin test. 
 
 BIAL'S TEST. Five cubic centimeters of Dial's 
 reagent (500 c.c. of 30 per cent hydrochloric acid, 
 i gm. orcin, and 25 drops of 10 per cent ferric chloride 
 solution) are boiled in a test tube and the urine to be 
 tested is added drop by drop. A green color indicates 
 the presence of a pentose. 
 
 Levulose, maltose, and the pentoses occur in urine 
 so rarely as to be of very little practical importance. 
 
 Lactose. Lactose may occur in the urine of preg- 
 nant or nursing women. It reduces Fehling's solu- 
 tion, but does not ferment with pure yeast, and does 
 not give a dextrosazone. Lactose may be detected 
 by Rubner's test. 
 
 RUBNER'S TEST. To 10 c.c. of urine add 3 gm. 
 of lead acetate ; filter off the precipitate and heat the 
 
17 
 
 filtrate in a test tube until a yellowish brown color 
 appears, then add a little ammonia and continue 
 heating. If lactose is present, a brick red color ap- 
 pears and a cherry red precipitate settles at the bot- 
 tom of the test tube, while the liquid above becomes 
 colorless. 
 
 Sucrose. May rarely occur in the urine after eating 
 large amounts of saccharine food. It does not re- 
 duce Fehling's or Benedict's solutions and is of no 
 clinical importance. 
 
 OTHER REDUCING SUBSTANCES IN THE URINE WHICH 
 MAY BE CONFUSED WITH DEXTROSE 
 
 Drugs. Certain drugs, such as chloral, naphthol, 
 antipyrin, aspirin, morphine, camphor and menthol, 
 cause an increase in the conjugate glycuronates 
 (glycuronic acid conjugated with aromatic bodies) of 
 the urine. These substances reduce Fehling's solu- 
 tion, but the reduction is usually of a dirty yellow- 
 green color, and rarely the bright yellow or red of a 
 clean-cut sugar reaction. Conjugated glycuronic acid 
 may be distinguished from sugar by the fact that it 
 does not ferment with yeast. Excessive amounts of 
 creatinine or uric acid may cause a partial reduction 
 of copper solution, and may be distinguished from 
 sugar in the same way that glycuronates are. 
 
 Alkapton, a product of abnormal metabolism, occurs 
 in the urine rarely and reduces Fehling's solution, 
 but does not ferment. Urine containing alkapton 
 turns dark brown or black on standing, or on the 
 addition of an alkali. 
 
 Protein. If a large amount of protein is present in 
 the urine it may cause a reduction of copper solution. 
 
19 
 
 If this is suspected, a few drops of acetic acid should 
 be added to the urine, and it should be boiled a few 
 minutes and filtered to remove the precipitated 
 protein. 
 
 ACETONE AND DIACETIC ACID 
 
 Acetone. To 5 c.c. of urine add a crystal of sodium 
 nitroprusside, acidify with glacial acetic acid, shake 
 a minute, and then make alkaline with ammonium 
 hydrate. A purple color indicates acetone. 
 
 Diacetic Acid. To 5 c.c. of urine add an excess of 
 a 10 per cent solution of ferric chloride. A burgundy 
 red color indicates diacetic acid. After the taking 
 of certain drugs, especially aspirin, a diacetic acid 
 reaction may appear in the urine, which does not dis- 
 appear on heating. The red color if due to diacetic 
 acid, disappears on heating. 
 
 QUANTITATIVE TEST FOR AMMONIA 
 
 To 25 c.c. of urine add 5 c.c. of a saturated solution 
 of potassium oxalate and 2 to 3 drops of a I per cent 
 alcoholic solution of phenolphthalein. Run in from a 
 burette decinormal sodic hydrate, to a faint pink 
 color. Then add 5 c.c. of formalin (40 per cent com- 
 mercial) and again titrate to the same color. Each 
 cubic centimeter of the decinormal alkali used in this 
 last titration equals I c.c. of n/io ammonia, or 
 .0017 gm. of ammonia. Multiply .0017 by the number 
 of cubic centimeters of decinormal alkali used in the 
 titration; this gives the number of grams of ammonia 
 in 25 c.c. of urine. The potassium oxalate and the 
 formalin must both be neutral to phenolphthalein, and 
 the urine must be fresh. 
 
21 
 
 The value of quantitating the ammonia is that it 
 is a rough index to the amount of acidosis in diabetes. 
 The amount of ammonia in the urine varies of course 
 with the amount of protein ingested, but in general 
 an ammonia figure of I to 2 grams per 24 hours may 
 be regarded as normal, anything over this indicates 
 an acidosis, anything over 4.0 gm. a severe acidosis. 
 
 BILE 
 
 The most satisfactory test for bile for general use is 
 the iodine test. To 15 c.c. of urine in a test tube add 
 5 c.c. of a i to 10 dilution of the official tincture of 
 iodine, letting it run gently down the side of the 
 tube. A green ring at the zone of contact of the two 
 liquids indicates bile. 
 
 Gmelin's Test. Filtered urine is allowed to slowly 
 trickle down the side of a test tube containing a few 
 cubic centimeters of concentrated nitric acid. If bile 
 is present several colors (green, blue, violet, yellow) 
 are formed at the line of junction of the two fluids. 
 
 Hammarsten's Test. A mixture of 19 parts of 
 25 per cent hydrochloric acid and I part of 25 per 
 cent nitric acid is allowed to stand at room tem- 
 perature for from several hours to a day, until it has 
 turned slightly yellowish. One part of this acid 
 mixture is added to 5 parts of 95 per cent alcohol. 
 A few drops of urine are added to a few cubic cen- 
 timeters of this acidulated alcohol. If the urine 
 contains bile pigment, a characteristic green color 
 appears almost immediately. This is a delicate test. 
 
 A very simple and fairly satisfactory test is to dip 
 a piece of white cotton cloth into the urine. If bile 
 is present the cloth will be stained yellow. 
 
23 
 UROBILINOGEN 
 
 Half fill a small test tube with urine, and add 3 or 
 4 drops of Ehrlich's reagent (2 per cent solution of 
 dimethyl-paramido-benzaldehyde in 20 per cent hydro- 
 chloric acid) taking care that the reagent remains on 
 top of the urine. A cherry red color, usually appear- 
 ing within an hour, indicates urobilinogen. 
 
 MELANIN 
 
 Urine containing melanin is dark when passed, but 
 turns darker on the addition of an oxidizing agent, 
 such as ferric chloride, potassium dichromate or 
 sulphuric acid. An excess of the oxidizing agent de- 
 colorizes the urine, with the formation of a yellow 
 precipitate. 
 
 Von Jaksch-Pollak Reaction for Melanin. Add a 
 few drops of ferric chloride solution to a third of a 
 test tube of urine, and note the formation of a gray 
 color. Upon the further addition of the chloride a 
 dark precipitate forms consisting of phosphates and 
 adherent melanin. An excess of ferric chloride causes 
 the precipitate to dissolve. 
 
 BLOOD PIGMENTS 
 Guaiac Test (see p. 127). 
 
 FORMALDEHYDE 
 
 Burnham's Test. To 5 c.c. of urine in a test tube 
 add 5 drops of a .5 per cent solution of phenylhydrazine 
 hydrochloride, a crystal of sodium nitroprusside, and 
 shake. Now add a few drops of a concentrated solution 
 of sodic hydrate. If formaldehyde is present a blue 
 color results, slowly passing through blue to brown, 
 
25 
 
 red, and finally yellow. This test is of importance in 
 examining the urine of patients who are being treated 
 for pyelitis with urotropin. If the test for formalin 
 in the urine is negative it shows that the urotropin is 
 not being broken up in the body, and is consequently 
 doing the patient no good. 
 
 INDOXYL 
 
 Obermayer's Test. Add 3 c.c. of a 20 per cent 
 solution of lead subacetate to 15 c.c. of urine, and 
 remove the precipitate by filtering. (This is done to 
 get rid of urobilin and any other coloring matters 
 that may be present, and is not necessary unless the 
 urine is very highly colored.) Then add 15 c.c. of 
 Obermayer's reagent (2 per cent solution of ferric 
 chloride in strong hydrochloric acid) and shake the 
 mixture. After shaking, 3 c.c. of chloroform is added, 
 and the mixture is again shaken. Indoxyl, if present, 
 gives a blue color to the chloroform. 
 
 METALLIC POISONS 
 
 The most common cases of metallic poisoning, that 
 one sees in practice, are those due to lead, mercury or 
 arsenic. Each of these substances appears in the 
 urine, and their quantitative recognition is often a 
 matter of diagnostic or prognostic importance. 
 
 Lead. Two liters of urine is evaporated to a tenth 
 of its volume. An equal volume of 20 per cent 
 hydrochloric acid is added, and 3 gm. of potassium 
 chlorate. The mixture is heated on the water bath 
 to 60 C. As soon as the evolution of chlorine has 
 ceased another portion of 3 gm. of potassium chlorate 
 is added, and the operation repeated until the fluid 
 
27 
 
 no longer gives off the fumes of chlorine on tfye further 
 addition of the chlorate. If the liquid becomes too 
 concentrated, more water is added. The fluid is al- 
 lowed to cool and is filtered after dilution with water. 
 The filtrate is examined for lead with hydrogen sul- 
 phide, sulphuric acid, and potassium bichromate, 
 giving precipitates, if lead is present, of black lead 
 sulphide, white lead sulphate, and yellow lead chro- 
 mate. 
 
 Arsenic. May be tested for by saturating the 
 faintly acid urine with hydrogen sulphide gas, allow- 
 ing to stand from 12 to 24 hours, filtering, washing, 
 treating the precipitate with bromine water, which 
 will dissolve the arsenic sulphide. The solution is 
 placed in a small Erlenmayer flask, to which is added 
 zinc and sulphuric acid, and the stream of hydrogen 
 is conducted into an acid silver nitrate solution 
 (AgNO 3 i to 2 gm., HNO 3 2 gm., water 10 c.c.). If 
 AsH 3 is generated, one gets a blackish brown precipi- 
 tate of metallic arsenic. 
 
 Mercury (method of Vogel and Lee). " 150 c.c. of 
 urine is taken, and in order to break down the organic 
 compound in which the mercury is likely to be pres- 
 ent, it is acidulated with 5 c.c. of concentrated hydro- 
 chloric acid and evaporated until its bulk has been 
 reduced to 25 or 30 c.c. About 2 c.c. of hydrochloric 
 acid is added to replace the loss by evaporation, and 
 enough potassium chlorate to oxidize thoroughly the 
 organic material present. This usually requires 
 about 2 gm. and when it has been oxidized the fluid 
 becomes pale yellow or colorless. It is then "diluted 
 to about 60 c.c. and is boiled vigorously until the 
 chlorine gas previously evolved has been driven off, 
 
Uric Acid Crystals. 
 
 Acid Ammonium Urate. 
 
29 
 
 which is shown by the absence of chlorine odor from 
 the steam. The solution usually darkens again on 
 cooling. A piece of copper wire about 4 cm. in length, 
 bent back on itself twice, and cleaned by boiling in a 
 test tube with dilute hydrochloric acid, is dropped 
 into the solution and allowed to remain an hour or 
 more. If considerable amounts of mercury are present 
 it will be found to be coated with a silvery film of 
 metallic mercury; but this is not sufficient to estab- 
 lish the identity of the metal, and if it exists only in 
 traces the changes in the appearance of the urine may 
 be inconclusive. The wire is accordingly removed 
 from the dish with a glass rod, is washed with a little 
 water, and is gently dried by rolling it on a piece of 
 filter paper, pains being taken to avoid unnecessary 
 handling. 
 
 " It is then put into the bottom of a glass tube from 
 3 to 5 mm. in diameter and 15 cm. long, which is 
 sealed at one end and is followed by a cylindrical plug 
 of gold leaf which is pushed into the tube until it is 
 within 2 cm. of the wire. (Such ^pellets of gold leaf 
 may be obtained from any dentist.) Holding the 
 tube horizontally, the end containing the wire is 
 gradually heated, the part of the tube containing the 
 gold leaf not to be heated. The latter must be ex- 
 amined frequently for any change of color, especially 
 the end of the cylinder toward the wire. If mercury 
 is present it will manifest itself by the appearance 
 of a silver patch of amalgam in this situation. 
 
 " If the amount of the metal is very small there will 
 be simply a pale discoloration of the gold, but if the 
 amount is larger, the deposit on the gold will be very 
 easy to recognize. If further confirmation of the 
 
Glucosazone Crystals. 
 
 Amorphous Urates. 
 
identity of the mercury is required, the gold-foil may 
 be suspended in a tube containing a few crystals of 
 iodine, which are then very gently warmed. The 
 mercury thus becomes converted into red mercuric 
 iodide, or if the amount is considerable the metal may 
 be distilled by heating the gold foil in the tube and 
 looking with the lens for a deposit of very minute 
 droplets of metallic mercury in the cooler parts of the 
 tube." 
 
 SEDIMENTS 
 
 i. UNORGANIZED SEDIMENTS 
 
 Uric Acid. Uric acid occurs in acid urines in a 
 variety of crystalline forms. Some of the more com- 
 mon forms are: " Dumb-bells," " whetstones," hexag- 
 onal plates, rhombic prisms or wedges. The crystals 
 are usually yellowish in color. Uric acid crystals are 
 soluble in alkalis, and hence do not occur as a sedi- 
 ment in alkaline urine. They have little clinical 
 significance, unless associated with blood or other 
 evidence of renal or ureteral irritation, when the 
 possibility of calculus formation should be borne in 
 mind. 
 
 Urates. Urates may occur singly or as a mixture of 
 ammonium, calcium, magnesium, potassium, and 
 sodium urates. Ammonium urates may occur in 
 acid, neutral or alkaline urine, but all the other urates 
 occur as sediment only in acid urine. Calcium, 
 magnesium, and potassium urates are amorphous, 
 ammonium urate is crystalline, and sodium urate may 
 be either amorphous or crystalline. Ammonium 
 urate ordinarily occurs in the form of the so-called 
 " thorn apple," crystals, while sodium urate, when 
 
FIG. I. Calcium Phosphate 
 
 FIG. 2. Ammonio-magnesium Phosphate 
 
33 
 
 crystalline, occurs as fan-shaped clusters or prismatic 
 needles. The urates are all soluble in hydrochloric 
 or acetic acid. The most common urate sediments seen 
 are the " thorn apple " crystals of ammonium urate, 
 and the so-called " brick dust " pinkish precipitate of 
 amorphous urates. Urate sediments have little clinical 
 significance, and are especially seen in rather concen- 
 trated, highly acid urines. 
 
 Phosphates. Phosphates may occur as calcium or 
 magnesium phosphate or as ammonium magnesium 
 phosphate (triple phosphate). Calcium phosphate 
 may occur in crystalline or amorphous form. It 
 appears in the sediment of slightly acid, neutral or 
 faintly alkaline urine. The crystalline form is seen in 
 long glistening prismatic needles, which may be 
 arranged singly or in bundles or rosettes. Amor- 
 phous phosphates (calcium and magnesium) are usually 
 seen in alkaline urines, but may be present if the re- 
 action is slightly acid or neutral. Microscopically 
 they are seen as granular, colorless masses, and are 
 dissolved by acetic acid but not by heat. Magnesium 
 phosphate crystals are not common. They are found 
 rarely in alkaline urines in the form of rhomboid 
 plates, which are soluble in acetic acid. Ammonium 
 magnesium phosphate (triple phosphate) occurs usually 
 in alkaline urines, and is seen in the form of 
 rhomboid prisms of characteristic appearance, the 
 so-called " coffin lid " form. Occasionally they may 
 closely resemble the large envelope forms of calcium 
 oxalate, but may be distinguished from them by 
 their ready solubility in acetic acid. The most com- 
 mon phosphates seen are the amorphous phosphates, and 
 the ammonium magnesium phosphate. These two forms 
 
FIG. 3. Calcium Oxalate Crystals 
 
 FIG. 4. Leucin and Tyrosin 
 I, Leucin and 2, Tyrosin Crystals 
 
35 
 
 commonly occur in urines that have been standing and 
 have become alkaline, and are of little clinical significance. 
 When occurring in freshly passed urine, they indicate 
 ammoniacal fermentation, due possibly to cystitis or 
 to retention of residual urine. 
 
 Calcium Oxalate. Calcium oxalate crystals occur 
 in the urine in two forms, the dumb-bell type and the 
 octahedral type. They are found most frequently in 
 acid urines, but may occur in neutral or alkaline 
 urines as well. They are insoluble in acetic acid, but 
 soluble in hydrochloric. Oxalates are derived from 
 various vegetable foods, particularly spinach and 
 rhubarb, and if present in the urine in large numbers 
 are of some clinical importance, indicating the possible 
 formation of a calculus. 
 
 Calcium Carbonate. Calcium carbonate crystals 
 are not common in human urine. They occur usually 
 in alkaline urine, but may occur in neutral or very 
 slightly acid urines. The crystals are in the form of 
 granules, spherules or dumb-bells. They may be 
 differentiated from calcium oxalate by the fact that 
 they dissolve in acetic acid with the evolution of 
 carbon dioxide gas. They are of no practical im- 
 portance. 
 
 Calcium Sulphate. Calcium sulphate crystals 
 occur rarely in the urine. They are found usually in 
 very strongly acid urines, are of long needle-like 
 shape, and are insoluble in acetic acid. 
 
 Leucin and Tyrosin. Leucin and tyrosin occur 
 rarely in the urine. When they do occur it is usually 
 in the urine of parents with acute yellow atrophy or 
 cirrhosis of the liver, in acute phosphorus poisoning, 
 or in leukemia. They may be in solution, or as a 
 
37 
 
 sediment. Leucin, when crystalline, occurs in spher- 
 ical masses, which show characteristic radial and 
 concentric striations, and are highly refractive. 
 Tyrosin occurs in the characteristic " sheaf " for- 
 mation. 
 
 TESTS FOR LEUCIN AND TYROSIN. As leucin and 
 tyrosin sometimes occur in solution in the urine, if 
 they are suspected, the urine should be evaporated to 
 a small bulk and the following tests applied. 
 
 Leucin. Scherer's Test. To the residue of the 
 evaporated urine add alcohol, which may then be 
 examined for the characteristic crystals. In order 
 to identify the crystals chemically as leucin it is neces- 
 sary to evaporate with concentrated nitric acid on a 
 platinum crucible cover some of the solid residue 
 obtained after adding alcohol to the evaporated 
 urine. With pure leucin the residue remains color- 
 less, but usually a yellowish residue remains. This 
 is heated with a few drops of sodium hydrate solu- 
 tion, when a yellowish or brownish color appears. If 
 it is heated further, the leucin. collects into an oily 
 drop which rolls around on the crucible cover. 
 
 Tyrosin. Evaporate the urine to a small bulk, re- 
 move the fluid and dissolve the residue in water. 
 Morner's test is then used. To this aqueous solution 
 add i c.c. of a reagent composed of I c.c. of formalin, 
 55 c.c. of concentrated sulphuric acid and 45 c.c. water. 
 When the mixture is heated to boiling a green color 
 appears if tyrosin is present. 
 
 Hematoidin. Hematoidin crystals may occur in 
 the urine of persons with various liver diseases. 
 They may be seen as tufts of small needles or as 
 small yellowish red plates. 
 
39 
 
 Fat. Free fat is very rarely seen in the urine. 
 (Lipuria, chyluria.) Before it is decided that the fat 
 droplets seen have actually come from the urinary 
 tract, all sources of contamination must be excluded, 
 such as grease in the vessel in which the urine is 
 obtained, oil on the fingers when manipulating the 
 cover glass, dirt on the slide, etc. Lipuria. is so rare 
 that free fat droplets seen in the urine practically 
 always come from one of these contaminations. Fat 
 droplets may occur quite commonly on fatty casts, 
 however. (See below.) 
 
 Starch Granules. Starch granules may occur in 
 the urine of babies who have had dusting powder ap- 
 plied to their buttocks. They may be easily recog- 
 nized by the fact that they turn blue with iodine. 
 
 2. ORGANIZED SEDIMENTS 
 
 i. Epithelial Cells. As the genito-urinary tract is 
 lined with epithelium and as this is continually 
 desquamating, a few epithelial cells of various sorts 
 are normally found in the urine. If these cells are 
 found in abnormally large numbers their presence 
 indicates irritation or inflammation of the part of the 
 genito-urinary tract from which they come* 
 Epithelial cells may roughly be divided into four 
 classes. 
 
 (a) Small round cells, about the same size as pus 
 cells. They may be differentiated from pus cells by 
 the fact that they are mononuclear. They may 
 come from the renal tubules, or from the urethra or 
 from the pelvis of the kidney. If they adhere to- 
 gether in clumps thay are probably from the renal 
 
pelvis or the urethra; if they are on casts they are 
 from the renal tubules. 
 
 (b) Large round cells two or three times the size 
 of the small ones may come from the neck of the 
 bladder or the membranous or prostatic urethra. 
 
 i- 
 
 3- 
 
 FIG. 5. i. Large round cells. 2. Caudate cells. 3. 
 round cells. 4. Squamous cells. 
 
 (c) Large squamous cells usually come from the 
 the bladder or from the prepuce, or vagina or vulva. 
 
 (d) Caudate cells come from the pelvis of the kidney 
 or from the neck of the bladder or ureter. 
 
 2. Leucocytes (Pus Cells). A few leucocytes may 
 occur normally, in the urine, especially in the urine 
 
FIG. 6. Epithelial Casts 
 
 FIG. 7. Hyaline Casts 
 
43 
 
 of females, who are likely to have leukorrhea. For 
 this reason if there is any question of pus in a female's 
 urine, it is desirable to have a catheter specimen to 
 exclude pus from the uterus, cervix, vagina or ex- 
 ternal genitalia. Be especially careful in young 
 female children and babies, not to make a diagnosis 
 of pyelitis because there are a few pus cells in the 
 urine; these are usually normal. Pus cells may be 
 recognized by their poly nuclear nuclei, and these 
 may be made to show more prominently if a drop of 
 acetic acid is run under the cover glass. 
 
 3. Red Blood Cells. The occurrence of red blood 
 cells in the urine is always abnormal (exclude trauma 
 from the catheter and menstruation). Be careful 
 not to confuse red cells with yeast cells, a great 
 variety of which may appear in stale urine. The 
 yeasts are smaller, are likely to be more irregular or 
 oval in shape and do not show the characteristic 
 refractive outside ring peculiar to red cells, or the flat 
 biscuit shape seen when they are looked at sideways. 
 
 4. Animal Parasites or their Ova. If there is 
 echinococcus disease of the kidney, small cysts or 
 booklets from cysts may be passed in the urine. 
 Other parasites which occur very rarely in the urine 
 are the round worms Ascaris and Filaria, and the 
 embryo of Bilharzia (Egypt). 
 
 5. Yeasts and Bacteria. Normal urine taken by 
 catheter from the bladder is sterile, but on standing 
 many yeasts and bacteria develop, so that if it is de- 
 sired to determine the presence or absence of a bac- 
 teriuria it is necessary to obtain a catheter specimen. 
 The most important bacteria to be looked for in urine 
 are the colon, typhoid, and tubercle bacilli. The 
 
FIG. 8. Fatty Casts 
 
 FIG. 9. Cylindroids 
 
45 
 
 colon and typhoid bacilli may be determined by 
 culture, and roughly, by their characteristic morphol- 
 ogy in the fresh urine specimen examined under the 
 microscope. To recognize tubercle bacilli a smear of 
 the urinary sediment is made and stained (see p. 159, 
 and be sure to exclude smegma bacilli) or a few cubic 
 centimeters of the urine are injected into a guinea 
 pig. The pig is killed in six weeks, and the presence 
 or absence of tuberculosis determined at autopsy. 
 6. Casts. Casts have two particular characteristics : 
 
 1. One end is rounded, sometimes both ends. 
 
 2. Their sides are parallel. (Cabot.) 
 
 They may be divided into six classes: 
 
 (a) Epithelial casts are covered with small round 
 cells from the kidney tubules. 
 
 (b) Bloody casts have red blood cells adherent 
 to them. 
 
 (c) Granular casts are the most common ones 
 seen; they are covered with coarse yellow granules, 
 probably the degenerated remains of epithelial or 
 blood cells. 
 
 (d) Waxy casts are clear, yellowish or bluish, 
 broader than the other varieties, and are usually 
 seen with broken off square ends, as if they were 
 brittle. 
 
 (e) Hyaline casts are pale and watery looking, 
 and cannot be seen well through the microscope unless 
 the light is shut down. 
 
 (/) Fatty casts have small globules of fat ad- 
 herent to them, which may be recognized by staining 
 with a drop of Sudan III solution. Their presence 
 means chronic parenchymatous nephritis. 
 
Waxy Casts. 
 
 Granular Casts. 
 
47 
 
 Cylindroids consist probably of mucus or mucus- 
 like material. They are longer than casts, more 
 irregular in outline, and have sharp, tapering ends. 
 They are of no particular significance. 
 
 SULPHONEPHENOLPHTHALEIN TEST OF KIDNEY 
 FUNCTION. (Rowntree and Geraghty) 
 
 One cubic centimeter of the 'phthalein solution * 
 is injected into the muscles of the back, and all the 
 urine passed in the next two hours is saved. A few 
 cubic centimeters of strong sodic hydrate solution is 
 added to make the urine strongly alkaline, the alkaline 
 urine is made up to 1000 c.c. by the addition of tap 
 water, and the red color is compared with a standard, 
 and the result expressed in terms of per cent of this 
 standard. 
 
 The large Dubosq colorimeter may be used, or a 
 small one made by Hynson, Westcott Co., or a series 
 of standard test tubes made by oneself. 
 
 Directions for Making Standard Tubes. Add a few 
 cubic centimeters of strong sodic hydrate solution to 
 I c.c. of 'phthalein solution in a 1000 c.c.. graduate, 
 and make up to 1000 c.c. with tap water. This is 
 the 100 per cent solution. The other dilutions may be 
 made up from this as follows: To make an 80 per cent 
 solution take 80 c.c. of the first (100 per cent) solution 
 and add 20 c.c. of water; for a 60 per cent solution, 
 60 c.c. of the first solution, and 40 c.c. of water, and 
 
 * .60 gm. of sulphonephenolphthalein and ^.84 c.c. of double 
 normal sodium hydrate solution are diluted to 100 c.c. with .75 
 per cent saline solution, when .15 c.c. more of the double normal 
 sodic hydrate solution is added. The resulting product is filtered, 
 and contains 6 mgm. of sulphonephenolphthalein to the cubic 
 centimeter. This solution may be obtained from Hynson, West- 
 cott Co., Baltimore, put up in very convenient I c.c. ampoules. 
 
49 
 
 so on for all the percentages down to 10. The stand- 
 ard solutions are best kept tightly stoppered in large 
 test tubes in a rack in a dark place, as they deteriorate 
 in the light. 
 
 The normal function in adults varies between some- 
 what wide limits: 50 to 75 per cent. In children it 
 is somewhat higher, from 70 to 90 per cent. 
 
CHAPTER II 
 THE BLOOD 
 
 Hemoglobin Estimation (Tallqvist). A drop of 
 blood is secured from the ear, and is allowed to fall 
 upon a piece of dry white absorbent paper. The 
 blood is allowed to stand a minute until it has lost its 
 glistening appearance, the paper is then folded over 
 so there will be a background of white paper against 
 and underneath the blood drop, and the color of the 
 blood is compared with the graded Tallqvist scale. 
 The hemoglobin is read off directly in per cent. Do 
 not hold your finger as a background against the 
 blood on the paper when you are comparing it with 
 the scale; this makes it look too dark. The normal 
 hemoglobin percentage is 80 to 100. This is the 
 simplest of the hemoglobin tests, but is not accurate. 
 
 Sahli Method. Draw the blood up to the mark in 
 the small pipette. Then blow it out into the gradu- 
 ated tube, and add $ hydrochloric acid up to the 
 mark 10. Mix well by a rotary motion. Then add 
 water drop by drop until the color in the graduated 
 tube matches the color in the standard tube. The 
 height of the column of liquid in the graduated tube 
 indicates the percentage of hemoglobin. 
 
 Color Index. The color index is the percentage of 
 hemoglobin divided by the percentage of the normal 
 number of red corpuscles. The normal color index 
 is of course, I. 
 
 51 
 
Neubauer Ruling. Breuer Ruling. 
 
 FIG. 10. Diagrams showing various hemacytometer rulings. 
 
53 
 
 Counting the Blood Cells. A number of variously 
 ruled counting apparatuses are in use, but the Thoma- 
 Zeiss is the one most commonly used, and is the one 
 referred to here. The counting chamber is .1 mm. 
 deep. The big ruled square is I sq. mm. in area. 
 It contains therefore ^ sq. mm. Each of the small- 
 est squares is -%^-Q sq. mm. in area, and contains 
 
 Wfr <T cu - mm ' 
 
 Count of the Red Cells. The blood is drawn up in 
 
 the red counting pipette to the mark .5, and then 
 the diluting solution* is added to the mark 101, 
 giving a dilution of I to 200. Be sure and shake well, 
 best with a rotary motion, to mix thoroughly the 
 blood and diluting solution. After well mixing, 2 or 
 3 drops are allowed to run out of the pipette and are 
 discarded, after which a small drop is run on to the 
 counting stage and covered with the cover glass, 
 pressing the glass down with the finger, so that the 
 drop is very evenly distributed. The drop should be 
 large enough to cover the stage when the glass is 
 pressed down. Now count the number of cells (the 
 white cells have been dissolved away by the diluting 
 fluid) in four corner blocks of 25 squares each, add 
 the four counts together and multiply the result by 
 8000. This gives the number of red cells per cubic 
 millimeter of blood. It is best to count two drops 
 and take the average. The normal red count is 
 about 5,000,000. 
 
 Count of the White Cells. Draw up the blood to 
 the mark .5 on the white counting pipette, and fol- 
 low with diluting fluid (.5 acetic acid) to the mark n. 
 
 * Gower's Solution = sodium sulphate 7 gm., acetic acid 20 
 gm., water 120 cc. 
 
55 
 
 This gives a dilution of I to 20. Put the drop of 
 diluted blood on the slide as in the red count, and 
 count all the cells in the large ruled square. Multiply 
 this count by 200, which gives the number of white 
 cells per I cu. mm. of blood. If especial accuracy is 
 desired, it is well to count 2 separate drops, add the 
 two counts together, and multiply by 100. 
 
 The normal white count varies from 7000 to 
 10,000. 
 
 COUNTING THE BLOOD PLATELETS 
 
 1. Draw the blood up in the red counter just as in 
 doing a red count, to the mark .5. 
 
 2. Draw up diluting stain * to'the mark 101. 
 
 3. Place a drop of blood on the counting stage 
 just the same as for a red count, put on the cover glass, 
 and let stand for a few moments to allow the platelets 
 to settle. 
 
 4. Count all the platelets in 100 of the smallest 
 squares, 25 at each corner of the large square. 
 
 5. Multiply this count by 8qob. This gives the 
 number of platelets in i cu. mm. of blood. 
 
 The normal platelet count by this method varies 
 from 226,000 to 367,000 (Wright). 
 
 The platelets appear as sharply outlined, oval or 
 
 * The diluting fluid for counting the platelets consists of 3 
 parts of a i to 1400 aqueous solution of potassium cyanide, and 
 2 parts of a i to 300 aqueous solution of brilliant cresyl blue. 
 These two solutions are kept in an ice chest in separate bottles, 
 and are mixed and filtered just before being used. The cresyl 
 blue solution is permanent, but the potassium cyanide should be 
 made up fresh at least every 10 days. 
 
57 
 
 round lilac-colored bodies ; the red cells are decolorized 
 and appear as shadows. 
 
 A common mistake made is to count the yeasts in 
 the staining fluid instead of the platelets. These are 
 much darker and larger than the platelets are, and 
 if the stain is kept on ice there will be no yeasts. 
 The platelets can be seen better if the specially thin 
 cover glass of Zeiss, with a central excavation, is 
 used. 
 
 EXAMINATION OF THE STAINED SPECIMEN 
 
 Technique of Making Smear. The best method of 
 making a blood smear is upon small square cover 
 glasses. It is absolutely essential that these be clean, 
 and free from grease. The best method of cleaning' 
 them is to soak them for a few moments in concen- 
 trated nitric acid, and then to successively wash 
 with water, alcohol, and ether, drying on a soft 
 cloth. 
 
 The ear is punctured and the cover glass touched to 
 the resulting drop of blood. Then the second cover 
 glass is dropped over the first, the drop of blood is 
 allowed to spread, and the cover glasses are quickly 
 drawn apart, with a sideways, but with no up or 
 down motion. The whole success of a blood smear 
 depends on securing a uniform and very thin film of 
 blood. The important points to observe to attain 
 this are: 
 
 1. Do not have too big a drop of blood. 
 
 2. Let the drop of blood spread thoroughly, but 
 do not wait long enough before pulling the glasses 
 apart, for coagulation to have started. 
 
 3. In pulling the cover glasses apart, use only a 
 
59 
 
 sliding motion; do not lift the top cover glass from 
 the bottom one. No fixing, other than drying for a 
 moment, is necessary. The smear is now ready to 
 stain. The most satisfactory stain for general use 
 is Wright's modification of Leishmann's stain. The 
 technique of applying it is as follows (Wright) : 
 
 1. Cover the film with a known quantity of the 
 staining fluid, by means of a medicine dropper. 
 
 2. After one minute add to the staining fluid the 
 same quantity of distilled water, and allow it to re- 
 main two or three minutes, according to the density 
 of the stain desired. A longer period of staining may 
 produce a precipitate. Eosinophilic granules are best 
 brought out by a shorter period of staining. The 
 quantity of diluted fluid on the preparation should 
 not be so large that some of it runs off. 
 
 3. Wash the preparation in water for thirty 
 seconds, or until the thinner portions of it become 
 yellow or pink in color. 
 
 4. Dry and mount in balsam. 
 
 THE BLOOD CELLS 
 
 The Red Blood Cells. The normal red blood cells- 
 appear as nearly round bodies of a fairly uniform size 
 and color, of about 6 to 9 microns in diameter. 
 
 The Blood Platelets. These are small bodies, stain- 
 ing purple, about one-half to one-third as large as a 
 red cell, which usually occur in groups. They have 
 been variously considered to be disintegration prod- 
 ucts of leucocytes, remnants of primitive erythro- 
 blastic nuclei, and extrusion processes of large bone 
 marrow cells. 
 
.-0. 
 
 
 A , 
 
 
 
 
 
 ' 
 
 10 
 
 11 
 
 12 
 
 :.-W* 
 
 13 
 
 THR Ri.non CF.T.T.S. 
 
 15 
 
 
6i 
 
 THE BLOOD CELLS 
 
 1. Normal red cells. 
 Achromic red cells. 
 Blood platelets. 
 
 2. Macrocyte. 
 Microcytes. 
 
 3. Poikilocytes. 
 
 4. Stippled and basophilic 
 
 cells. 
 
 5. Normoblasts. 
 Megaloblas?. 
 
 6. Cells containing " Howell- 
 
 Jolly " bodies. 
 Reticulated cell. 
 
 7. Tertian malaria. 
 
 8. Quartan malaria. 
 
 9. Estivo-autumnal malaria. 
 
 10. Poly nuclear neutrophile. 
 
 1 1 . Small lymphocytes. 
 Large mononuclear cell. 
 
 12. Mast cell. 
 Eosinophile. 
 
 13. Neutrophilic myelocytes. 
 
 14. Basophilic myelocyte. 
 Eosinophilic myelocyte. 
 
 15. Polynuclear leucocytes 
 
 containing Dohle's in- 
 clusion bodies (stained 
 with methyleneblue). 
 
63 
 
 The most important types of abnormal red cells 
 are the following: 
 
 1. Microcytes. Cells smaller than normal. 
 
 2. Macrocytes. Cells larger than normal. 
 
 3. Poikilocytes. Cells of distorted shape. 
 
 4. Achromic Cells. Pale cells with very little 
 coloring matter in the center of the cell, indicating a 
 deficiency in hemoglobin. 
 
 5. Polychromatophilic Cells. Cells which show a 
 tendency to take the basic portion of the stain, and 
 thus appear of a bluish or a bluish gray color. 
 
 6. Stippled Cells. Cells containing many fine 
 dark staining granules. 
 
 7. Normoblasts. Nucleated cells, the embryonic 
 form of red blood cells. These cells may have round 
 nuclei or may have karyorrhectic or multiple dividing 
 nuclei. They may be basophilic and stippled or 
 may take the stain like a normal red cell. 
 
 Megaloblasts. Cells of somewhat the same ap- 
 pearance as the preceding, only 4 or 5 times as large. 
 
 8. Skeined Forms. Cells which when stained with 
 brilliant cresyl blue, show a skeined or reticulated 
 appearance of the protoplasm. They may occur in 
 any anemia, and a few are present in normal blood, 
 but they are especially numerous in chronic family 
 (hemolytic) jaundice. The technique of staining is 
 as follows : To 5 c.c. of a saturated solution of brilliant 
 cresyl blue in .85 per cent salt solution, add 5 c.c. of 
 .85 per cent salt solution and 2 c.c. of a 2 per cent 
 sodium oxalate solution. Filter. A centrifuge tube 
 is now nearly filled with this mixture, and a few 
 drops of blood obtained from the ear are added. The 
 whole is centrifuged for a few moments, washed once 
 
65 
 
 with .85 per cent salt solution, and a smear is made 
 from the sediment of blood in the bottom of the 
 centrifuge tube. The usual tendency is not to get 
 the saturated solution of cresyl blue strong enough; 
 other than this there are no difficulties in the tech- 
 nique. 
 
 9. Howell- Jolly Body Containing Forms. Cells 
 which contain one or more small black dots, known as 
 " Howell-Jolly " bodies. These bodies are probably 
 portions of the nucleus of the parent normoblast, and 
 occur especially in the blood of pernicious anemia 
 patients after splenectomy has been performed. All 
 of these abnormal forms may occur in severe anemias. 
 
 Secondary anemia is characterized by a low color 
 index, therefore considerable achromia. There may 
 be a good deal of tendency to polychromatophilia 
 and stippling, with moderate variations in size and 
 shape (anisocytosis and poikilocy tosis) . Normo- 
 blasts and skeined forms may occur. It is not 
 characteristic of the blood in secondary anemia to 
 have extreme anisocytosis and poikilocytosis, and it is 
 extremely rare to find megaloblasts. 
 
 Pernicious anemia is characterized by a high color 
 index, therefore there is usually no achromia. Certain 
 special characteristics of the morphology of the red 
 cells in pernicious anemia are: 
 
 1. A tendency to extreme variations in size. 
 Many very small cells will be seen as well as a great 
 many very large ones. The general tendency is to 
 the production of large cells, and the occurrence of 
 many very large cells is almost diagnostic of per- 
 nicious anemia. 
 
 2. A tendency to extreme distortions and varia- 
 
67 
 
 tions in shape. Many elongated and pear-shaped 
 forms are likely to occur. 
 
 3. The occurrence of megaloblasts. It is rare to 
 find megaloblasts in a blood of secondary anemia. 
 
 Chlorosis is characterized by a very low color in- 
 dex, with consequently a great deal of achromia. 
 There is little tendency to anisocytosis and poikilo- 
 cytosis, and nucleated forms are rare. 
 
 THE WHITE BLOOD CORPUSCLES 
 
 The following varieties of white blood corpuscles 
 occur normally. 
 
 1. Polynuclear neutrophiles constitute about 70 
 per cent of the white blood cells. They are two or 
 three times the size of a red blood cell, and have 
 irregularly shaped nuclei, with granular, lilac staining, 
 neutrophilic protoplasm. 
 
 2. Lymphocytes make up about 25 per cent of the 
 white cells. Their usual size is a little larger than 
 that of a red blood cell, although there is considerable 
 variation. Very large forms occur in abnormal 
 bloods, especially in certain cases of acute lymphatic 
 leukemia. The nucleus of a lymphocyte stains 
 dark purple, is usually round or oval, or occasionally 
 indented, and is very clean-cut in its outline. The 
 protoplasm is scanty, showing usually as a narrow 
 ring around the nucleus. It stains light blue, and is 
 usually homogeneous, although sometimes small 
 reddish " azur " granules are seen in it. 
 
 3. Large mononuclear and " transitional " cells 
 make up from 3 to 5 per cent of the white blood cells. 
 It is best to classify them together. Their exact 
 
6 9 
 
 status is not definite, but it is probable, although not 
 certain, that they represent one and the same cell, 
 in different stages of development. The large mono- 
 nuclears are a little larger than the polynuclear 
 neutrophiles, have a single round, oval, or indented 
 nucleus, which takes the stain rather poorly, and* a 
 wide zone of basophilic, non-granular protoplasm. 
 These cells may sometimes be confused with lympho- 
 cytes. The principal points of differentiation are that 
 the lymphocytes are more intensely basophilic and 
 deeply stained than are the large mononuclears, are a 
 good deal more clean-cut and regular in outline, and 
 do not have such a wide zone of protoplasm around 
 the nucleus. The " transitional " cells are like the 
 large mononuclears, except that the nucleus is in- 
 dented or horseshoe shaped, and there may be a few 
 neutrophilic granules in the protoplasm. They are 
 called transitional cells, because they were formerly 
 thought to be an intermediate stage in the develop- 
 ment of the polynuclear leucocyte. The present view 
 is that they have nothing whatever to do with the 
 polynuclear leucocytes. 
 
 4. Eosinophiles are usually about the size of a 
 polynuclear neutrophile and constitute about 2 to 4 
 per cent of the total number of white cells. The 
 nucleus has the same shape as that of the neutrophile, 
 but is likely to appear a little bluer and lighter in 
 color. The protoplasm of these cells is filled with 
 large, round, coarse red granules which take the acid 
 portion of the stain. Sometimes if the diluted stain 
 has been left on too long the granules in the protoplasm 
 of the polynuclear neutrophiles may appear reddish, 
 but they are not nearly so large, red or sharply de- 
 
fined as those in the eosinophiles, and there should be 
 no difficulty in differentiating the two types of cells. 
 
 Mast cells (polynuclear basophiles) are cells the size 
 of the eosinophiles, but they show an especial affinity 
 for the basic part of the stain. The nuclei are like 
 those of the polynuclear neutrophiles, except that they 
 usually stain a little more deeply. The protoplasm 
 is filled with large, dark purplish granules. Mast 
 cells form about .5 to I per cent of the total number of 
 white corpuscles. They have no particular signifi- 
 cance. 
 
 ABNORMAL CELLS 
 
 Myelocytes. The myelocytes are the primitive 
 forms from which the polynuclear neutrophiles, 
 basophiles and eosinophiles are derived. They do 
 not occur in normal adult blood, but may be seen in 
 certain blood diseases, especially myelogenous leu- 
 kemia. They vary a good deal in size, sometimes 
 being a little smaller, sometimes being a little larger 
 than the common potynuclear neutrophile. Three 
 types of myelocytes are recognized : 
 
 1. Neutrophilic Myelocytes. These cells stain 
 rather faintly, are not very sharp in outline, and have 
 a large round or oval or occasionally a kidney-shaped 
 nucleus, and a neutrophilic granular protoplasm. 
 They are differentiated from the other neutrophilic 
 cells by the shape of the nucleus, and from the large 
 mononuclears by their granular neutrophilic proto- 
 plasm. 
 
 2. Eosinophilic Myelocytes are mononuclear cells 
 similar to the preceding, with many eosinophilic gran- 
 ules scattered through the protoplasm. 
 
73 
 
 3- Basophilic Myelocytes are similar to those of 
 the eosinophilic form, except that the granules in the 
 protoplasm are basophilic. 
 
 THE BLOOD IN INFANCY 
 
 The essential fact to remember about the blood in 
 infancy is that it tends more closely to resemble 
 embryonic blood than adult blood does, and that for 
 this reason, certain cells, such as myelocytes and 
 primitive erythrocytes (normoblasts and megalo- 
 blasts) may appear in the blood of an infant without 
 having the same significance that they would have in 
 the blood of an adult. In the anemias of infancy, 
 the blood tends to revert to its primitive form, and 
 comparatively slight causes may produce an enormous 
 change in its constitution, with the appearance of 
 many abnormal forms of cells. 
 
 QUANTITATIVE DIFFERENCES 
 (Morse: Case Histories in Pediatrics) 
 
 Hemoglobin. The hemoglobin varies between 100 
 and 125 per cent during the first 3 or 4 days of life; 
 it then drops to the minimum of 60 per cent at three 
 weeks, after which it gradually rises to 70 per cent 
 at six months. It remains at this point during the 
 rest of the first two years, after which it slowly rises, 
 reaching the adult standard at about 6 years. 
 
 Red Blood Cells. During the first few days of life 
 the red cells number 6,000,000 to 7,500,000 per 
 cubic millimeter, then the count rapidly falls to the 
 normal infantile limit of about 5,500,000 which it 
 reaches at about two weeks. During the rest of in- 
 fancy the blood count remains at about this figure, 
 
75 
 
 and gradually diminishes during early childhood, 
 reaching the adult standard at six years. 
 
 White Blood Cells. During the first few days of 
 life there is a marked increase, sometimes up to 
 36,000. The count rapidly drops to 12,000 or 14,000, 
 where it remains during the first six months. The 
 normal limits during the rest of infancy are between 
 10,000 and 12,000, and the number from this time on 
 is approximately the same as for the adult, 7000 to 
 10,000. 
 
 In young children there is a relatively greater per- 
 centage of lymphocytes and a smaller percentage of 
 polynuclear cells. The following table taken from 
 Chapin and Piseks' " Diseases of Children," shows the 
 percentages for different ages. 
 
 Polynuclears Mononuclears 
 
 Year % % 
 
 i 35 53 
 
 2 38 5i 
 
 3 42 47 
 
 4 47 4i 
 
 5 52 39 
 
 6. 52 37 
 
 7 * 53 35 
 
 8 54 33 
 
 9 55 3i 
 
 10 .' 60 30 
 
 MALARIAL PARASITES 
 
 In the examination for malarial parasites, the 
 blood is stained with Wright's stain, as in making an 
 ordinary blood smear, and if the parasites are present, 
 they may be easily recognized. Sometimes, however, 
 it takes a long and patient search to find them. By 
 beginners, artefacts or blood platelets lying on red 
 blood cells are likely to be mistaken for parasites, but 
 
77 
 
 if there is any doubt whatever about the body seen 
 being a parasite, it is probably not one, for they stain 
 very prettily, and stand out against the reddish back- 
 ground of the red blood cell in a very clean-cut manner. 
 The protoplasm of the malarial parasite stains light 
 blue and always contains one or more red granules. 
 
 Tertian Malaria (Plasmodium Vivax) appears first 
 in the red cell as a small ring-like body, which soon 
 becomes pigmented, and enlarged so greatly that the 
 infected red cell may be swollen to three or four 
 times its normal size, with only a thin ring of proto- 
 plasm showing around the body of the parasite. 
 
 Quartan Malaria (Plasmodium Malaria) starts as a 
 small ring-shaped body, soon becoming larger and 
 pigmented, as does the tertian. In the quartan para- 
 site the pigment granules are more likely to be ar- 
 ranged around the periphery of the organism, whereas 
 in the tertian they are distributed all through it. 
 The principal point of differentiation between the 
 two forms is that red cells infected with the quartan 
 form do not swell to the large size attained by those 
 containing the tertian parasite, usually remaining 
 about the normal size, or even shrinking a little. 
 
 Estivo Autumnal Malaria (Plasmodium Falcipa- 
 rum). The estivo-autumnal form appears in the 
 red cells first as a very small ring-shaped body much 
 like the ring forms of the tertian and quartan, only 
 smaller. The ring is thicker at one side than the 
 other, thus giving it the so-called " signet ring " ap- 
 pearance. The last stage of the estivo-autumnal 
 parasite may occur in the characteristic " crescent " 
 shape, which easily differentiates it from the tertian 
 and quartan forms. 
 
79 
 DOHLE'S LEUCOCYTIC INCLUSION BODIES 
 
 These bodies are small coccus or bacillus-shaped 
 bodies occurring in the polynuclear leucocytes in a 
 number of diseases, especially in scarlet fever, and 
 were first described by him in 1911. They may be 
 stained with the ordinary Loeffler's methylene blue, 
 but the best stain for them is Granger and Pole's 
 modification of Manson's stain, prepared as follows: 
 methylene blue 1.5 grams; absolute alcohol 10 c.c.; 
 5 per cent aqueous solution of phenol 100 cc. Methyl 
 alcohol is used as a fixative, it being sufficient to 
 dip the blood smear into the alcohol, wash with water 
 and immediately stain. With this stain the inclu- 
 sions stain a deep blue, the cell nucleus a deep blue, 
 and the cell protoplasm a pale homogeneous blue. 
 
 The inclusion bodies have the following signifi- 
 cance : 
 
 1 . They are present in a majority of cases of scarlet 
 fever up to the tenth day of the disease; in nearly 
 all cases before the fourth day. 
 
 2. They are not specific for "scarlet fever, being 
 found in a number of the infections, most particularly 
 erysipelas, sepsis, pneumonia and tonsillitis. They 
 are more likely to be found in diseases with which 
 the streptococcus is associated. 
 
 3. They have the following diagnostic value : If they 
 are not found in a doubtful case which has a rash 
 and a marked fever, the case is probably not one of 
 scarlet fever. 
 
 4. They are probably in the nature of a reac- 
 tion of injury of the cell nuclei, caused by bacteria 
 toxins, and are small fragments broken off from the 
 nuclei. 
 
8i 
 
 WIDAL SERUM REACTION FOR TYPHOID 
 
 The ear is punctured and a few drops of blood are 
 allowed to run into a small test tube. This is al- 
 lowed to stand until the clot and the serum have 
 separated. Now place 9 drops (platinum loop) of an 
 active motile twelve to twenty-four hour old culture 
 of the bacillus typhosus on one end of a glass slide, 
 and add one drop of the serum, mixing well with the 
 platinum loop. Place four drops of water at the 
 other end of the slide, and then add to it one drop of 
 the I to 10 dilution which has already been made, 
 making a dilution of I to 50. Cover with cover glasses 
 and let stand one hour. Controls should be made by 
 the same method as given above, except omitting the 
 blood serum. If the reaction must be done with 
 dried blood (as in the board of health outfits) add a 
 drop or two of water to the dried blood and proceed 
 as before. Widal's reaction is positive when there is 
 complete agglutination and loss of motility in an 
 hour, in the I to 50 dilution. . The performance of 
 the test is very easy, but it is sometimes hard to know 
 whether to call a reaction positive or not. Assurance 
 in this can come only after having performed a few. 
 
 A and B para typhoid (done the same as typhoid). 
 
 The Widal reaction appears usually only after the 
 first week of the disease, and may persist for years in 
 the blood of patients who have had typhoid fever. 
 
 BLOOD FRAGILITY TEST 
 
 A determination of the resistance of the red blood 
 cells to hypotonic salt solution is sometimes of value 
 in the diagnosis of " chronic family jaundice " (hemo- 
 
83 
 
 lytic), and especially in the differential diagnosis of 
 jaundice due to this disease and that due to obstruc- 
 tion of the bile passages, such as occurs in cases 
 of gallstones, cancer -of the pancreas, etc. The red 
 cells in hemolytic jaundice show a lessened resistance 
 to hypotonic salt solution; in obstructive jaundice an 
 increased resistance. The method used by the writer 
 is as follows: 
 
 About 6 c.c. of blood is drawn from the arm with a 
 needle and glass syringe. This is transferred to a 
 test tube half full of 0.5 per cent sodium citrate solu- 
 tion in 0.9 per cent sodium chloride. The tube is 
 then inverted two or three times to insure proper 
 mixing. As soon as possible (certainly within three 
 hours) the blood is centrifuged and the cells washed 
 twice with 0.7 per cent sodium chloride. As much of 
 the supernatant fluid as possible is drawn off with a 
 pipet, and the remaining blood cells are used in the 
 test, without further dilution. The hypotonic sodium 
 chloride solutions are made up from a I per cent solu- 
 tion of chemically pure sodium chloride and distilled 
 water. The solutions run in strength from 0.70 to 
 0.175 P er cent, and are kept in tightly corked 100 c.c. 
 bottles. Exactly I c.c. of each one of these solutions 
 is drawn off in a pipet, and placed in series of small 
 test tubes; 0.05 c.c. of the blood cells is then run into 
 each tube, from a small accurately graded pipet, each 
 tube is inverted twice and allowed to stand two hours 
 at room temperature; at the end of this time the 
 tubes are read. As initial hemolysis take the point 
 at which there is the first tinge of pink in the salt 
 solution; as complete hemolysis the point at which 
 there can no longer be seen any sediment of blood 
 
cells in the bottom of the tube. The average points 
 of hemolysis as obtained by this method are: 
 
 Normal blood. ... .457 (initial) 
 
 Secondary anemia .475 (initial) 
 
 Pernicious anemia . 477 (initial) 
 Chronic family (hem- 
 
 olytic) jaundice .600 (initial) 
 Obstructive jaun- 
 dice 400 (initial) 
 
 3.40 (complete) 
 .322 (complete) 
 .322 (complete) 
 
 .400 (complete) 
 .225 (complete) 
 
 HEMOLYSIS TEST 
 
 (To Determine the Compatibility of Donor's and Recipient's 
 Blood in Transfusion) 
 
 Donor. Secure two samples of blood of 5 c.c. each, 
 from the arm vein. One of these samples is placed 
 in a dry test tube and allowed to clot (to obtain the 
 serum), the other sample is placed in a test tube half 
 full of .5 sodium citrate in 90 per cent sodium chloride 
 (to prevent clotting). 
 
 Recipient. The same is done 'with the recipient's 
 blood. The test is carried out as follows (Lindeman) : 
 ' The red blood cells of recipient and donor are washed 
 three times with normal saline; variable quantities of 
 recipient's serum are placed in three separate small 
 test tubes. To each of these is added 0.25 c.c. of a 
 2%>er cent suspension in normal saline of washed 
 blood cells of the donor. The same is done with the 
 donor's serum and the recipient's cells. Controls are 
 made of donor's serum and donor's cells recipient's 
 serum and recipient's cells. Controls are also made 
 with donor's cells in normal salt solution and recip- 
 
87 
 
 ient's cells in normal salt solution. The total volume 
 in each tube is raised with normal saline to 0.5 c.c. 
 The test tubes are incubated in a water bath for 
 a period of two hours, and readings are made. They 
 are then set in an icebox over night and readings 
 are again made the following morning. When the 
 case is urgent the icebox test is eliminated. The 
 icebox test should be eliminated only when absolutely 
 necessary by the extreme condition of the patient 
 where time is the important factor. When the 
 amount of blood taken from the patient for tests is 
 small, only 0.25 c.c. of serum is used and controls of 
 patient's serum are eliminated." 
 
 COAGULATION TIME 
 (Method of Lee and White) 
 
 This is a very simple and fairly accurate method. 
 It is performed as follows: One cubic centimeter of 
 blood (approximately) is withdrawn from the arm 
 vein by a small needle and syringe, both of which 
 have previously been washed with normal salt solu- 
 tion, and is put into a small tube of about 8 mm. in 
 diameter, which has also been previously washed in 
 normal salt solution. The time of withdrawal is ac- 
 curately noted ; and the tube is inverted gently every 
 30 seconds until the blood will no longer run down 
 the sides of the tube, but remains in the bottom of 
 the tube in a solid clot. This is the end point. By 
 this method the coagulation time for normal blood is 
 from 4 to 8 minutes. 
 
8 9 
 OBTAINING BLOOD FOR CULTURES, ETC. 
 
 At the present day there are so many serological 
 blood tests and cultures being made, that every 
 practitioner should be able to withdraw the necessary 
 amount of blood from the veins with the least pos- 
 sible discomfort to the patient. A few suggestions 
 may be of service. 
 
 Do everything as quickly as you can; it irritates the 
 patient to see you fussing around with your needles 
 and syringes. 
 
 Apply a turniquet of small rubber tubing to the 
 upper arm, tightly enough to make the veins of fhe 
 elbow stand out, but not tightly enough to stop the 
 pulse at the wrist. 
 
 Have the patient's arm resting on a table. Use a 
 5 or 10 c.c. all glass syringe, with a small needle, it is 
 useless to torment the patient with a large one. 
 Wash off the arm at the bend of the elbow gently with 
 a little alcohol, with the fingers of the left hand hold 
 the skin tightly on the stretch, and insert the needle 
 with the right, taking care not to run it in too 
 deeply. 
 
 Now gently and slowly pull the piston back, and 
 if the needle is in the vein and the syringe is tight, 
 there will be no difficulty in getting as much blood 
 as desired. Use a sharp needle, and be sure that it 
 fits the syringe, and that the syringe is tight. If it 
 is not you will be troubled with air bubbles. 
 
 In the case of a very fat patient where the veins 
 cannot be seen, the small needle is inserted in the 
 same way, and gently prodded around in all directions 
 until a vein is struck (most of the pain is in the first 
 skin puncture). 
 
91 
 
 In the case of babies the veins of the scalp or the 
 jugulars may be used. A practiced operator can 
 secure enough blood for a Wassermann in a very few 
 seconds, and can save the patient a great deal of dis- 
 comfort by obtaining the required amount in the 
 shortest possible time. 
 
CHAPTER III 
 FECES 
 
 Color. The most important abnormal colors are : 
 
 1 . Grayish black due to the ingestion of iron or 
 bismuth compounds. 
 
 2. Tarry black, from digested blood (seen in bleed- 
 ing cancer or ulcer of the stomach). 
 
 3. Green from calomel. 
 
 4. Red from blood coming from the rectum or 
 low down in the large intestine. 
 
 5. Clay colored from an excess of fat and an 
 absence of bile (obstructive jaundice). 
 
 Reaction. Normally neutral or slightly acid or 
 alkaline; an excessively acid stool is due to carbo- 
 hydrate fermentation, a strongly alkaline one to pro- 
 tein putrefaction. 
 
 Consistency and Form. The normal may vary a 
 good deal. The longer a stool stays in the colon or 
 rectum, the harder and drier it becomes, owing to 
 the absorption of water from it through the intestinal 
 wall. In abnormal stools all degrees of consistency 
 may be seen, from the very watery thin ones occurring 
 in cholera, to the round, hard, bullet-like ones seen 
 in certain cases of constipation with an impacted 
 rectum. 
 
 93 
 
95 
 
 MACROSCOPIC EXAMINATION 
 
 Spread out a small amount of feces with a little 
 water on a plate. 
 
 1. Abnormal food residue may consist of cellulose 
 or meat or jelly-like starch remains. 
 
 2. Mucus. Any but the smallest amount of mucus 
 is abnormal, indicating catarrh or irritation some- 
 where in the intestinal tract. Mucus from the large 
 intestine is usually present as a thin coating over the 
 outside of the stool that from the small intestine is 
 well mixed with the fecal matter. Do not confuse 
 soft food residues with mucus. 
 
 3. Blood. Blood from the rectum usually appears 
 as bright red streaks on the outside of the stool, that 
 from the rest of the lower intestinal tract is inti- 
 mately mixed with the stool, and gives it a dull brick 
 color or may appear in streaks (as in amebic 
 dysentery) . Blood from the upper intestinal tract or 
 stomach, if present in sufficient amount, gives a dark 
 black color to the stool. 
 
 4. Pus may appear as yellowish masses or streaks. 
 
 5. Shreds of intestinal mucus membrane from 
 ulcerative disease of the intestine. 
 
 6. Gallstones usually may be recognized by their 
 hardness, and characteristic shape, with facets. 
 
 7. Intestinal sand consists of small granules of 
 gray or reddish-brown gritty material occurring rarely 
 in the stools. Its origin is not certain. " False in- 
 testinal sand " usually consists of the seeds of bananas, 
 or the hard portions of fruits, covered with calcium 
 salts. Neither variety has any particular practical 
 significance. 
 
97 
 
 INTESTINAL PARASITES 
 (Adapted partly from Kilgore) 
 
 The most common intestinal parasites are: 
 
 1. Ascaris lumbricoides (the common " round 
 worm ") varies in length from 15 to 40 cm. It 
 occurs usually in small numbers, and there should be 
 no difficulty in recognizing it, as it is much larger 
 than any of the other ordinary round worms seen in 
 the feces. 
 
 2. Oxyurus vermicularis (the ordinary pin-worm) is 
 a small worm 3 to 10 mm. long, and usually occurs in 
 the rectum or colon. 
 
 3. Uncinaria duodenalis (hookworm) is 8 to 1 8 
 mm. long. The buccal cavity has three pairs of 
 inward curving hook-like teeth (uncinaria (necator) 
 Americana has a dorsal and a ventral pair of cutting 
 plates instead of hooks.) 
 
 4. Trichuris trichiura is 4 to 5 cm. long, two- 
 thirds of the length appearing as a whip-like tail. 
 
 5. Taenia saginata (beef tapeworm). The head 
 is 1.5 to 2 mm. in diameter, and has four suckers, but 
 no hooks. The ripe segments are 16 to 20 mm. by 
 5 to 7 mm. The uterus has a multitude of fine 
 branchings. The genital openings are irregularly 
 alternate on the margin. 
 
 6. Taenia solium (pork tapeworm) is very rare in 
 America. The head is .6 to I mm. in diameter, 
 and has four suckers on the sides, and on the end a 
 crown of 22 to 32 hooks. The ripe segments are 9 to 
 10 by 4 to 5 mm. The uterus consists of a large 
 median stem with but 7 to 10 coarse branches, each 
 of which again branches. The genital openings are 
 
99 
 
 at the margin and alternate from side to side with 
 considerable regularity. 
 
 7. Bothriocephalus latus (fish tapeworm). The 
 head is I mm. broad and 2 or 3 mm. long, is flat, 
 almond or spoon shaped, with two deep grooves at 
 its sides. The ripe segments are 10 to 15 by 3 to 4 
 mm. The genital opening is on the side, not the 
 edge, and the uterus is arranged about it. 
 
 8. Hymenolepis nana (Tsenia nana) is most com- 
 mon in Italy and Egypt, but is occasionally seen in 
 the United States. It is a small worm, only 8 to 25 
 mm. long, and .5 mm. broad. The head is round and 
 has 4 suckers and 24 to 28 hooklets. They may 
 occur in the stools in extraordinarily large numbers. 
 
 CHEMICAL EXAMINATION 
 
 Blood. Guaiac test (see gastric contents, p. 127). 
 
 Benzidin T.est. In a test tube place a small pinch 
 of benzidin, 2 c.c. of hydrogen peroxide, and a few 
 drops of glacial acetic acid. Shake well. Rub up 
 with a glass rod a small portion of feces in another 
 test tube in 5 c.c. of water, and heat to boiling. 
 Pour a few drops of mixture No. 2 into mixture No. I. 
 If blood is present, a blue-green color results. This 
 is an extremely delicate test, and is of no value un- 
 less the patient is known to have been on an abso- 
 lutely meat free diet for two or three days. The 
 writer never uses this test, much preferring the 
 guaiac test, which is delicate enough to recognize 
 blood in any amount of practical importance. 
 
 Bile (hydrobilirubin). Rub up a small portion of 
 feces in a mortar or evaporating dish, with a con- 
 
lor : : --*;. ,^ fj , t 
 
 centrated aqueous solution of corrosive sublimate, and 
 let it stand for 3 hours. 
 
 A brick red color indicates the presence of bile. 
 
 MICROSCOPIC EXAMINATION 
 
 For microscopic examination a small portion of 
 feces is rubbed up with a little water on a glass slide, 
 and examined .first with the low and then with the 
 high-power lens. 
 
 i. Food Residue 
 
 (a) Fat. Fat may exist in the stool as fatty acids, 
 neutral fat, or soaps; it occurs most commonly as 
 calcium soap, which usually looks yellow under the 
 microscope. Normally there is a certain amount of 
 soap in the stools, while any but the smallest amount of 
 neutral fat is abnormal. Unstained neutral fat ap- 
 pears as small refractile globules, or sometimes with 
 fats of high melting point, as irregular flakes, the fatty 
 acids as flakes or needle-like or plate-like crystals, 
 and the soaps as yellowish masses or occasionally 
 crystals. Fat is best seen when stained with an 
 alcoholic solution of Sudan III. A drop of the stain 
 is added to the rubbed up feces; neutral fat droplets 
 stain bright red, amorphous fatty acids, if. present, 
 stain a light red crystalline fatty acids and soaps 
 do not stain at all. A little more stain and a drop or 
 two of glacial acetic acid is now added, and the slide 
 is gently heated. Neutral fat stays as it was before, 
 soap is broken down to fatty acid and an alkali, and 
 stains in bright red droplets, fatty acid melts and 
 stains in bright red droplets. 
 
103 
 
 Resume. For neutral fat and amorphous fatty 
 acids, stain with Sudan III alone; for total fat stain 
 with Sudan III + acetic acid and heat. 
 
 (b) Starch. Stain the rubbed up feces with a 
 drop of dilute iodine solution, or with Lugol's solution 
 (IKI). Undigested starch granules stain dark blue, 
 partially digested granules stain a brownish color. 
 The spores of certain fungi may stain blue with 
 iodine, but may be differentiated from starch granules 
 because they lack the characteristic concentric rings, 
 and are smaller and usually oval instead of round. 
 Certain small particles of cellulose may take on a 
 dark color with iodine, but may be differentiated by 
 their irregular shape. 
 
 (c) Meat Residue (muscle fibers). Meat residue 
 occurs in the form of small muscle fibers, which ap- 
 pear yellow. Sometimes these look very much like 
 calcium soaps, but they may be differentiated by the 
 fact that they are striated ; soaps are not. 
 
 (d) Cellulose is the fiber structure surrounding 
 vegetable cells. Normally a certain amount of it 
 appears in the feces, and may be easily recognized by 
 its cellular arrangement. With iodine it may stain 
 a dark purplish black, or a dark brown. 
 
 Be careful not to confuse round or oval cellular 
 remains with parasitic ova; the ova are always 
 more clean-cut and symmetrical than any cellulose 
 cell. 
 
 Unorganized Matter. Various crystals, such as 
 ammonium magnesium phosphate, calcium oxalate, 
 calcium sulphate, cholesterin, etc., occur in the stools, 
 but are of little or no practical importance. After 
 the administration of bismuth salts, irregular crystals 
 
of black bismuth suboxide may be seen. Iron is 
 passed as black amorphous granules. 
 
 2. Cellular Elements 
 
 (a) Epithelial Cells. Normally very few epithelial 
 cells appear in the feces. If present in large numbers 
 they indicate an inflammatory condition. 
 
 (b) Pus. A few leucocytes may be found in a 
 normal stool, but are hard to recognize on account of 
 being partly digested. If present in abnormal num- 
 bers they may come from inflammatory disease of 
 the intestine itself, or may come from a collection 
 of pus in some other organ perforating into the 
 intestine. 
 
 (c) Blood cells are not commonly seen in the feces 
 unless there is fresh blood present from low down 
 in the intestinal tract. If the blood comes from the 
 upper portion of the tract it is practically always 
 digested. 
 
 3. Parasites 
 
 (a) Bacteria. The bacteriology of the stools is an 
 enormous subject and cannot be dealt with in a book 
 of this sort. Two bacteriologic examinations, how- 
 ever, which are of importance and which can be per- 
 formed so simply that they are of value in ordinary 
 clinical routine are those for the tubercle bacillus and 
 for the " gas " bacillus (Bacillus serogenes capsu- 
 latus) . 
 
 Tubercle Bacillus. Mix feces and water together 
 in equal parts and centrifuge. Tubercle bacilli, if 
 
107 
 
 present, will come to the top of the tube, and can be 
 stained in the thin scum there. (See p. 159.) 
 
 Gas Bacillus (Technique used at the Children's 
 Hospital, Boston). 
 
 1. Fill a fermentation tube and a test tube with 
 concentrated nitric acid, let stand 3 minutes, and 
 empty out the nitric acid. 
 
 2. Rinse both tubes with hot tap water until 
 neutral to litmus paper. 
 
 3. Place a small bit of stool, about a gram of 
 dextrimaltose and about 15 c.c. of hot tap water 
 in the test tube, and boil vigorously for half a 
 minute. 
 
 4. Put the contents of the test tube into the fer- 
 mentation tube, taking care that it is filled up to the 
 top, and that no air bubbles remain in it. 
 
 5. Plug the tube with flamed cotton and incubate 
 for 24 hours. 
 
 Gas in the top of the tube indicates that the gas 
 bacillus is present, in greater or lesser numbers, de- 
 pending upon the amount of gasformed. 
 
 (b) Protozoa. In this part of the world the only 
 three protozoans .of importance commonly met with 
 in the feces are the entameba coli, entameba histo- 
 lytica, and entameba tetragenus. Amebse should be 
 examined for in a fresh stool which has been passed 
 into a vessel containing warm water. If examined 
 for in this way, while still warm, they can often be 
 seen moving about and thrusting out their character- 
 istic pseudopodia, which serve to differentiate them 
 absolutely from any other cells or organisms found 
 in the stool. 
 
109 
 
 fl im . i I si sit 
 
 Hlll^i 1 111111 
 
 ^ 8 1^ S 3llll?1 
 
FROM CABOT'S PHYSICAL DIAGNOSIS. 
 
 Heterophyes 
 heterophyes. 
 
 Distomal 
 felineum.j 
 
 Dictocoeliuit! 
 lanceolatum 
 
 Bilharzia Diplogonoporus Bilharzia Dibothrio- BUharzia 
 
 haematobium. grand is. haematobium. cephalus latus. haematobium. 
 
 Ascaris Oxyuris Paragonimus Taenia nana.. Ascaris 
 
 lumbricoides. vermicularis. westermani. lumbricoides. 
 
 Anchylostoma 
 duodenale. 
 
 Necator Strongylus 
 
 americana subtilis. 
 
 Strongyloides 
 stercoralis 
 
 DRAWINGS OF EGGS OF INTESTINAL PARASITES. 
 All are magnified 250. (After Looss.) 
 
Ill 
 
 (c) Ova of various worms. 
 
 Ascaris lumbricoides. These eggs are usually 
 easily recognized by their rough bile-stained envelope. 
 The protoplasm is unsegmented. 
 
 Oxyurus vermicularis. These eggs are small, 
 smooth, oval, and are usually rather light colored. 
 They are more likely to be found on the skin about 
 the anus than in the feces. 
 
 Trichiuris trichiura. May be easily recognized by 
 the characteristic light yellow roundish knobs, one on 
 each pole of the egg. These eggs, occur often in the 
 stools of people who are perfectly well; the parasites 
 may occasionally cause symptoms, however. 
 
 Necator (Uncinaria) americana may usually be 
 recognized by the fact that when seen the protoplasm 
 is likely to be segmented. Often the embryos may 
 be seen curled up within the egg. 
 
 Taenia saginata. Usually round or slightly oval - 
 has an outer and an inner shell, with striations be- 
 tween the two. 
 
 Taenia solium is very similar* to the above; it is 
 very rare in America, however. 
 
 Hymenolepis nana. These ova are a little larger 
 than the preceding. They have a double shell, and 
 the space between the inner and outer shells is filled 
 with a granular substance, and is not striated. 
 
 Bothriocephalus latus. Rather a large egg, larger 
 than the preceding three varieties of tapeworm eggs. 
 They have a lid at one end of the egg, which some- 
 times may be open, and the contents of the egg looks 
 like a bunch of grapes. 
 
H3 
 THE STOOLS IN INFANCY 
 
 The examination of the stools in infancy is of the 
 utmost importance in determining whether the baby 
 is digesting his food well, and if not, to what particu- 
 lar element of the food the indigestion is due. There 
 is no absolute standard of normality for an infant's 
 stool, it varies so much according to the composition 
 of the food, the relation between the different ele- 
 ments of the food, the nature of the intestinal bacteria, 
 the motility of the intestine, and the absorptive and 
 digestive power of the particular baby in question. 
 
 Number. The number of evacuations varies. 
 The normal should not be over four in the twenty- 
 four hours. Breast-fed babies are likely to have 
 more than are bottle-fed babies, owing to the high 
 sugar percentage of breast milk, but, on the other 
 hand, breast-fed babies may sometimes be extremely 
 constipated. Dextrimaltose as a rule is constipating, 
 maltose is laxative and in general the higher the 
 sugar percentage is in the food, the greater will be 
 the number of stools. 
 
 Fermentative and infectious processes in the in- 
 testine give rise to an increased number of stools; in 
 fermentative or infectious diarrhea there may be as 
 many as twenty or thirty movements in the twenty- 
 four hours. Nervous influences may cause an in- 
 creased intestinal peristalsis, and hence an increased 
 number of stools. Sometimes a large amount of in- 
 soluble soaps in the stools causes a severe constipation. 
 
 Form and Consistency. The infant's stool is 
 usually unformed, and of a mushy consistency. 
 Formed stools, however, may be normal. Consti- 
 pated stools due to increased soap content are scyb- 
 
H5 
 
 alous and crumbly. The longer a stool stays in the 
 rectum the harder it is likely to be. The stools of 
 simple indigestion, or of indigestion with fermentation 
 may vary all the way from a consistency that is 
 slightly thinner than normal, to one that is watery. 
 A common type of stool seen is of the consistency of 
 thin scrambled eggs, with many small white fat 
 curds scattered through it. Fat curds are small, soft, 
 soluble in ether, and are of very common occurrence. 
 Casein curds are large, smooth, white masses from 
 the size of a small bean to that of a large one, are in- 
 soluble in ether, and become very hard when put 
 into formalin. They are not of such common occur- 
 rence as the fat curds. A foamy, bubbly stool .is 
 due to fermentation in the intestine, usually of 
 carbohydrate, but sometimes of protein. 
 
 Color. The color varies according to the type of 
 food the baby is being fed on. The typical breast- 
 milk stool is of a golden yellow color. Most stools 
 are usually yellowish, or a dull yellowish brown. 
 Darker brown stools are usually- due to an increased 
 protein percentage in the milk. White stools are 
 due to a high insoluble soap content. A stool which 
 is green when passed is almost always abnormal, and 
 is usually due to a sugar fermentation. The green 
 color is due to the presence of biliverdin. Normally 
 bile is present in the stools as bilirubin, which is 
 colorless, but under the influence of intestinal fer- 
 mentation it is oxidized to the green biliverdin. A 
 stool which turns green on the outside after standing 
 in the air is not abnormal. Black stools are due to 
 the ingestion of bismuth or iron salts, or rarely (in a 
 baby) to digested blood. 
 
Reaction. The determination of the reaction to 
 litmus paper of abnormal stools is of the utmost im- 
 portance in telling to what food component the in- 
 digestion is due. Normal stools may be slightly 
 acid, neutral, or slightly alkaline a strong acidity 
 or alkalinity usually indicates abnormality. Under 
 normal conditions the reaction of the stool depends 
 upon the relation between the fat and the protein, 
 a high fat percentage tending to produce acidity. 
 The stools of normal breast-fed babies are likely to 
 be acid on this account for there is a relatively 
 high fat percentage in breast milk. Under abnormal 
 conditions of fermentation, however, the acid re- 
 action of the stool is due to a high sugar percentage in 
 the milk. 
 
 In the infant's intestine two processes are always 
 working against each other: decomposition of carbo- 
 hydrate food, with acid end products, and decompo- 
 sition of protein food, with alkaline end products. 
 Normally these two processes just about balance each 
 other when one is greatly in preponderance trouble 
 usually results, and a diarrhea ensues, due to the 
 irritating action upon the intestine of the too strongly 
 acid or alkaline end products. Strongly acid stools 
 due to fermentation of carbohydrate are very com- 
 mon, occurring in the common fermentative diarrhea 
 that one sees so often in summer. Strongly alkaline 
 stools from excessive protein decomposition are not 
 so common. 
 
 Odor. The stools of a breast-fed baby usually 
 have a not unpleasant aromatic odor. A high protein 
 percentage in the food causes a rather cheesy and 
 sometimes foul odor. A high sugar percentage, with 
 
119 
 
 fermentation of a part of the sugar, causes an acid 
 odor, sometimes very intense, due to the presence of 
 acetic and butyric acids. The stools in infectious 
 diarrhea may be nearly odorless, or acid, or foul 
 smelling. 
 
 Mucus. Any more than the very slightest amount 
 of mucus is abnormal. Mucus may occur around the 
 outside of a constipated stool, owing to its hard 
 character, and consequent irritation of the rectum. 
 Mucus occurs almost always in large amounts 
 in the stools of fermentative . diarrhea. This is 
 because of the great irritant action of the volatile 
 fatty acids of sugar fermentation upon the intestine. 
 Mucus also always occurs in infectious diarrhea. 
 
 Mucus stools mixed with blood streaks may occur 
 in intussusception. 
 
 Blood. Digested blood is rarely seen in the stools 
 of infants. Blood is usually due to infectious diar- 
 rhea if it occurs streaked with mucus throughout the 
 stool, or it may occur on the outside of the stool if a 
 fissure of the anus or a bleeding rectal polyp is present. 
 
 MICROSCOPIC EXAMINATION 
 
 Fat. Technique is the same as for adult stools. 
 (See p. 101.) 
 
 Neutral fat rarely occurs, except in very small 
 amounts. Most of the fat residue present in infant's 
 stools occurs as calcium or magnesium soap, and nor- 
 mally not an inconsiderable amount may be present, 
 sometimes as high as a fifth of the weight of the dried 
 stool. It is useless to try to tell anybody, except in 
 very general terms, how much fat should be present 
 
121 
 
 
 
 in a normal infant's stool ; every observer has to form 
 a standard for himself by repeated examinations. 
 
 In " soapy " stools, the " seifen stiihle " of the 
 Germans, nearly the whole stool may be made up of 
 soap, and under the microscope the whole field is 
 composed of fat globules when the stool is treated 
 with Sudan III, acetic acid and heat. 
 
 Starch. Technique is the same as for adult stools. 
 (See p. 103.) 
 
 Starch residue is not commonly found in infants' 
 stools except in cases where too much starch is being 
 fed. The occurrence of more than the smallest 
 amount of starch in the stool is an indication for 
 cutting down the starch in the diet. 
 
 Remember that nearly all dusting powders used on 
 babies' buttocks contain starch. 
 
CHAPTER IV 
 GASTRIC CONTENTS 
 
 I 
 EXAMINATION OF THE FASTING CONTENTS 
 
 The fasting contents is obtained through the 
 stomach tube in the morning, when no food or water 
 has been taken into the stomach since the previous 
 night. 
 
 GROSS EXAMINATION 
 
 (a) Amount. Usually the amount is small 10 
 or 15 c.c. Anything over 50 c.c. is distinctly ab- 
 normal, indicating stasis or too profuse gastric 
 secretion. 
 
 (b) Consistency. Normally the 'fasting contents is 
 thin and watery, with a little mucus. An excess of 
 mucus or an increased consistency due to food re- 
 mains is abnormal. 
 
 (c) Color. The normal color is a light yellowish 
 green, or colorless. A darker green color indicates 
 excessive bile (probably from retching due to the 
 passage of the tube). A chocolate or " coffee ground " 
 color usually indicates digested blood. Fresh blood, 
 when present, is in light red streaks. 
 
 (d) Food. Any macroscopic food residue is ab- 
 normal, and indicates stasis, of a lesser or greater 
 degree, depending upon the amount. 
 
 123 
 
125 
 
 (e) Odor. Normally there is very little odor to the 
 fasting contents; if there is fermentation, the char- 
 acteristic odors of acetic and butyric acids may be 
 recognized. 
 
 MICROSCOPIC EXAMINATION 
 
 (a) Food Residue. 
 
 Starch. Stain with dilute iodine solution, or 
 Lugol's solution (IKI). The starch granules stain 
 dark blue. 
 
 Fat. Stain with Sudan III. The fat globules ap- 
 pear red. 
 
 Normally there may be a very few microscopic 
 particles of food residue; any considerable amount of 
 either fat or starch indicates stasis. 
 
 (b) Blood. Red blood cells may be recognized by 
 their characteristic morphology. Do not confuse 
 them with yeasts. 
 
 (c) Pus. Pus is very rarely found in the gastric 
 contents (unless it has been swallowed with the 
 sputum) . Pus cells may be easily" recognized by add- 
 ing a drop of acetic acid to the material to be ex- 
 amined, which makes the characteristic polynuclear 
 nuclei stand out sharply. Pus cells seen in the gastric 
 contents are likely to be partially digested. 
 
 (d) Epithelial Cells. A few epithelial cells may be 
 present in the fasting contents. An excess is abnormal, 
 indicating gastritis. 
 
 0) Tumor Cells. Occasionally, in cases where 
 there is cancer of the stomach, small pieces of cancer 
 tissue or groups of cancer cells may be brought up 
 by the tube, and sometimes are of great assistance in 
 making a correct diagnosis. If the piece of tissue is 
 
127 
 
 large enough it may be " run through " and sectioned 
 by a competent pathologist, and an exact diagnosis 
 of the sort of tumor present made. 
 (/) Organisms. 
 
 1. Sarcinae are occasionally seen. They are small 
 cocci, arranged in squares or tetrahedra, and have a 
 very characteristic appearance. They mean stasis, 
 but are rarely found when there is no free hydro- 
 chloric acid present. 
 
 2. Yeasts. Several forms of yeasts of varying 
 sizes and shapes may occur. These may usually be 
 recognized by their tending to show buds. 
 
 3. Boas-Oppler Bacilli. These are very large, long 
 Gram-positive bacilli, which occur commonly in the 
 gastric contents of patients with cancer of the stomach, 
 especially if there is stasis and lactic acid present. 
 They are rare in non-malignant disease of the stomach. 
 
 CHEMICAL EXAMINATION 
 
 (a) Free HC1. A drop of Top'fer's reagent (a .5 per 
 cent alcoholic solution of dimethyl-amido-azo-benzol) 
 is added to a small portion of the fasting contents. 
 If free hydrochloric acid is present a red color re- 
 sults. A still simpler test is with Congo red paper. 
 If a drop of the fasting contents is put upon the red 
 paper, the presence of free hydrochloric acid is indi- 
 cated by the production of a dark blue color. 
 
 (b) Blood. (Guaiac Test.) To 10 c.c. of gastric 
 contents (or less if 10 c.c. is not available) add a few 
 drops of glacial acetic acid, and 10 c.c. of ether. 
 Shake vigorously. After the ether has separated 
 and has risen to the top, decant. Add this ethereal 
 
I2 9 
 
 extract to a few cubic centimeters of a freshly pre- 
 pared tincture of gum guaiac. Finally add a few 
 drops of hydrogen peroxide; a blue color indicates 
 the presence of blood. Be especially careful that no 
 blood has come from the teeth, or from abrasions in 
 the mouth, and that no meat is in the fasting contents 
 you are testing. Irritation by the stomach tube, with 
 the consequent production of small hemorrhages, is 
 often given as the source of a positive guaiac test. 
 Practically speaking, the writer believes that it is 
 very rare for the stomach tube to produce enough 
 irritation to cause a positive guaiac test, and most of 
 the cases which he has seen with positive tests, 
 supposedly due to trauma from the tube, have after- 
 wards turned out to have had bleeding ulcer or 
 cancer of the stomach. 
 
 II 
 
 EXAMINATION OF THE GASTRIC CONTENTS AFTER A 
 TEST MEAL 
 
 The test meal ordinarily used in Boston is that of 
 Ewald, and consists of a medium-sized slice of bread, 
 and a glass of water. It is given on an empty stomach, 
 and is withdrawn by the stomach tube at the end of 
 an hour. 
 
 Amount. The normal amount is from 50 to 125 c.c. 
 Larger amounts suggest stasis, hypersecretion or 
 hypomotility. 
 
 Color. The normal color of the gastric contents 
 after a test meal is the white color of the bread given. 
 If there is digested blood in the stomach, the color 
 will be brown. 
 
CHEMICAL EXAMINATION 
 
 (a) Blood. It is wise to do guaiac tests on both 
 the fasting and the test meal contents. 
 
 (6) Free HC1. The free HC1 and the total acidity 
 are determined quantitatively. To exactly 10 c.c. of 
 the unfiltered contents add a drop of Topfer's reagent. 
 If free HC1 is present a red color results. Now run 
 in -fo sodic hydrate until the red color changes to yel- 
 low. This end point is not a very sharp one, and care 
 must be taken not to over-titrate. Multiply the num- 
 ber of cubic centimeters of TO sodic hydrate used in 
 this titration by 10. This gives the amount of free 
 HC1 present in 100 c.c. of gastric contents in terms 
 of y\ sodic hydrate. The per cent of hydrochloric 
 acid may be obtained if the above quantity is multi- 
 plied by .00365. The normal quantity of free HC1 
 is equivalent to between 20 to 60 c.c. T\ sodic hydrate 
 per 100 c.c. gastric contents, or from .07 to..i8 per 
 cent. 
 
 Total Acidity. To the same specimen of gastric 
 contents in which the free hydrochloric acid has al- 
 ready been neutralized with sodic hydrate, add a 
 drop or two of a I per cent alcoholic solution of 
 phenolphthalein. Run in the sodic hydrate, drop by 
 drop, until a faint pink color appears. Multiply the 
 number of cubic centimeters of y\ sodic hydrate used 
 in both titrations by 10; this gives the total acidity 
 of 100 c.c. of the gastric contents in terms of fV sodic 
 hydrate. The result may be indicated in per cent of 
 hydrochloric acid by multiplying this result by 
 .00365. The normal quantitative values for total 
 acidity are from .15 to .30 per cent or 40 to 80 c.c. -fV 
 sodic hydrate for 100 c.c. gastric contents. 
 
133 
 
 Lactic Acid. Lactic acid is rarely present when free 
 hydrochloric acid is. Its presence indicates the lactic 
 acid fermentation of carbohydrate food, and probably 
 stasis. It may be tested for as follows: 
 
 Half fill two test tubes with a very dilute ferric 
 chloride solution. Add a few drops of the gastric 
 contents to one of the test tubes, and compare the 
 color with that of the control. An intensification 
 of the yellow color indicates that lactic acid is 
 present. 
 
 Pepsin and Rennin. Pepsin is always present 
 when free hydrochloric acid is. If free hydrochloric 
 acid is absent it may be tested for as follows: To a 
 small portion of the gastric contents add enough 
 hydrochloric acid to make it give the test for free 
 hydrochloric acid, then add a small open tube of 
 coagulated egg albumin to the contents, and leave it 
 in a warm place for 12 hours. If pepsin is present 
 the egg albumin will show signs of digestion. 
 
 Rennin also is always present when free hydro- 
 chloric acid is. It may be tested for by neutralizing 
 5 c.c. of gastric contents, adding it to 5 c.c. of milk, 
 and incubating the mixture. If a normal amount is 
 present, the milk will coagulate within 15 minutes. 
 
 Mercury. In testing stomach contents or feces the 
 same method is employed as is used for urine (see p. 
 27), although the quantity of material taken is 
 usually smaller. The material must be well mixed to 
 insure getting a uniform sample of the specimen, 
 especially in stomach contents if egg albumin has been 
 given as an antidote, as the mercury is then in the 
 form of albuminate. The oxidation also takes longer, 
 and larger quantities of potassium chlorate are re- 
 
135 
 
 quired. Before the wire is placed in the solution, the 
 latter should be filtered in order to remove any fatty 
 substances, carbon or other insoluble materials. 
 This test recognizes mercury in a I to 100,000 
 dilution. 
 
CHAPTER V 
 SPINAL FLUIDS 
 
 Pressure. The normal spinal fluid is under very 
 little pressure. Pathological fluids are usually under 
 pressure, the amount depending upon the amount of 
 the fluid. 
 
 Appearance. Normal spinal fluid is perfectly clear ; 
 pathological may be opalescent or purulent. 
 
 Blood may appear in the fluid in cases where there 
 has been a fractured skull or a ruptured blood vessel 
 from other cause; or it may be due to trauma of 
 small blood vessels caused by inserting the needle. 
 Hemorrhage during lumbar puncture is sometimes 
 alarming, but it almost always stops soon if the needle 
 is withdrawn and the patient kept quiet. 
 
 Fibrin Clot. In normal fluid* no fibrin clot forms 
 on standing; in pathological fluids a fibrin clot is 
 likely to form, the time of its formation depending 
 upon the intensity of the inflammation and the 
 amount of fibrin present. 
 
 Amount. The normal amount obtained by lumbar 
 puncture may vary a good deal, but in general it is 
 not over 20 c.c. In pathological fluids the amount 
 is increased. 
 
 In certain cases of meningitis, however, a very 
 small amount, or no fluid at all may be obtained, 
 owing to the formation of adhesions, which prevent 
 the circulation of the fluid. 
 
 137 
 
139 
 CHEMICAL TESTS 
 
 Pathological spinal fluid usually contains an excess 
 of globulin, the amount depending upon the activity 
 of the inflammation present. This may be tested for 
 as follows: 
 
 Globulin 
 
 Noguchi's Test. To one part of spinal fluid add four 
 parts of 10 per cent butyric acid in normal salt 
 solution. Boil, and then add as much normal sodic 
 hydrate as there was spinal fluid to begin with, and 
 boil again. Normal fluids may give a faint opal- 
 escence, pathological ones are likely to give a flocculent 
 precipitate, indicating an excess of globulin. 
 
 Nonne's Test. To about 2 c.c. of spinal fluid in a 
 small test tube, add an equal amount of a saturated 
 solution of magnesium sulphate, and compare this 
 tube with a tube containing spinal fluid alone; a 
 white precipitate indicates an excess of globulin. 
 
 Sugar. Normal spinal fluid contains sugar, and 
 gives a prompt reduction of Benedict's reagent. 
 Most pathological fluids do not. In general, the less 
 reduction there is obtained, the more severe is the 
 inflammation. Sugar may be tested for with Bene- 
 dict's or Fehling's reagent. 
 
 MICROSCOPICAL EXAMINATION 
 
 i. Cell Count. The normal cell count varies. 
 Most normal fluids contain only 3 or 4 cells per 
 cubic millimeter. Authorities differ somewhat as to 
 what is the upper limit of normality in the cell count. 
 A count of over 8 may usually safely be regarded as 
 abnormal. 
 
As the number of cells in a spinal fluid may vary 
 from I to 10,000 or more per cubic millimeter, the 
 technique of counting must necessarily be varied 
 somewhat according to the nature of the fluid. 
 
 (a) Purulent fluids are best counted with exactly 
 the same technique used for the white blood cells 
 (see p. 53). 
 
 (b) Clear or Slightly Turbid Fluids. The Zappert 
 modification of the Thoma-Zeiss ruling is the best rul- 
 ing for the counting slide for clear spinal fluids. In 
 this ruling, besides the large central square, which is 
 the same size as in the ordinary Thoma-Zeiss ruling, 
 there are eight other large squares of equal size around 
 the central square. (See p. 52.) 
 
 Technique of Count. Rinse the white blood count- 
 ing pipette out with glacial acetic acid (to make the 
 cell nuclei stand out), and draw up spinal fluid into it, 
 using no diluting agent. Put a drop of the undiluted 
 spinal fluid on the counting stage the same as for a 
 blood count, and count 5 of the large squares. Do 
 the same with a second drop. 
 
 The sum of the two counts equals the number of 
 cells per cubic millimeter. 
 
 If a counting chamber with the modified ruling is 
 not available, the ordinary Thoma-Zeiss ruling may 
 be used, as follows: Count all the cells in the large 
 central square in 5 different drops of spinal fluid, and 
 multiply the sum of the counts by 2. 
 
 Differential Count. A differential count of the 
 cells may be made at the same time, in the counting 
 chamber, as the acetic acid with which the pipette 
 has been rinsed makes the nuclei stand out very 
 clearly. Normally all the cells are mononuclears. 
 
H3 
 
 2. Smear. Smears for the microscopic study of 
 the spinal fluid are made from the centrifuged sedi- 
 ment. It is rather hard to prepare these satisfactorily, 
 but it will be found that if the smear is allowed to 
 dry of itself, without heat, the best preparations will 
 be obtained. The best staining is done with methyl- 
 ene blue. In the stained specimen the following may 
 be seen : 
 
 (a) Lymphocytes the normal cell of cerebro- 
 spinal fluid. 
 
 (b) Pus cells indicating a purulent inflammation. 
 
 (c) Large endothelial cells especially likely to 
 be seen in anterior poliomyelitis or cerebrospinal 
 syphilis. 
 
 (d) Bacteria. 
 
 1 . Meningococci. These organisms appear as small, 
 biscuit-shaped diplococci both within and without the 
 leucocytes. They may be present in small or in fairly 
 large numbers. 
 
 2. Pneumococci. Small or large cocci growing in 
 pairs or in short chains, sometimes showing the 
 characteristic surrounding capsule. When pneumo- 
 cocci are present they usually appear in extraordi- 
 narily large numbers, the field under the microscope 
 often resembling a smear from a pure culture of the 
 pneumococcus. 
 
 3. Streptococci and staphylococci may be easily 
 recognized by their characteristic morphology. 
 
 4. Influenza bacilli appear as small, thin, rather 
 faintly staining rods, both within and without the 
 leucocytes, in large numbers. 
 
 5. Tubercle bacilli are present in the spinal fluid 
 in all cases of tubercular meningitis. There are 
 
H5 
 
 certain experts who can find them nearly every time, 
 but the average laboratory worker will miss them 
 much more often than he finds them. 
 
 The two best methods of looking for them are : 
 
 1. Make a smear from the fibrin clot that has 
 formed after the spinal fluid has stood for some time, 
 and stain it for the bacilli. (See p. 161.) 
 
 2. Centrifuge the spinal fluid at a high speed for 
 one hour, and make a thick smear from the sediment, 
 letting it dry of itself in the air. 
 
 CHARACTERISTICS OF THE SPINAL FLUID IN 
 VARIOUS DISEASES 
 
 The spinal fluid may vary considerably in different 
 examples of the same disease, or at different periods 
 during the course of the disease. The data given 
 below refer to the average. 
 
 1. Tubercular Meningitis. Almost always slightly 
 opalescent rarely clear as compared with water. 
 Under slight or considerably increased pressure. May 
 vary in amount from 20 to 125 c.r., and in cell count 
 from 15 or 20 to 1000 cells per cubic millimeter. 
 The cell count is practically never below 10, and the 
 most common count that one sees is somewhere in 
 the neighborhood of 100. Nearly all the cells are 
 mononuclear. The globulin test may be positive or 
 negative, more often positive. A fibrin clot usually 
 forms. Sugar is absent in about 25 per cent of the 
 cases. 
 
 2. Meningococcus Meningitis. Almost always 
 cloudy, sometimes very thick. The amount is from 
 5 to 125 c.c., and it may be under slight or consid- 
 erable pressure. The cell count is high, varying from 
 
H7 
 
 2OO to 10,000 cells per cubic millimeter. The cells 
 are mostly polynuclear, and the characteristic biscuit- 
 shaped diplococci may be seen both within and with- 
 out the leucocytes. The globulin test is positive, 
 and a fibrin clot forms if the fluid is not too purulent. 
 Fehling's solution is usually' not reduced. 
 
 3. Pneumococcus, streptococcus and staphylococcus 
 meningitis have much the same sort of fluid that 
 epidemic meningitis does, except that the etiologic 
 organism is likely to be present in greater numbers 
 than is the diplococcus intracellularis in epidemic 
 meningitis. 
 
 4. Influenza Meningitis. A cloudy fluid, under 
 moderately increased pressure, 25 to 100 c.c. in 
 amount. The cell count varies; in three cases re- 
 ported by Brown of Toronto, from 1600 to 3600. 
 The cells are nearly all polynuclears, and the in- 
 fluenza bacilli are seen in large numbers both within 
 and without the leucocytes. The globulin is in- 
 creased, and the sugar decreased or absent. 
 
 5. Poliomyelitis. Usually under moderately in- 
 creased pressure, 20 to 100 c.c. in amount. Usually 
 clear. The cell count is moderately increased, from 
 20 to 50 per cubic millimeter. In the incubation stage, 
 most of the cells are polynuclear, later on in the course 
 of the disease they are mononuclear, up to 95 per 
 cent. Endothelial cells are especially likely to occur. 
 There is usually a slightly increased globulin content, 
 and there may or may not be a fibrin clot. Sugar is 
 usually present. 
 
 6. Encephalitis is now thought to be the same 
 disease as poliomyelitis, but is confined to the brain. 
 The fluid is the same as that of poliomyelitis. 
 
149 
 
 7. Serous Meningitis (Meningismus). Under 
 slightly increased pressure. The amount may vary 
 between 10 and 100 c.c. The fluid is usually clear, 
 has a normal cell" count, and reduces Benedict's solu- 
 tion. It may occasionally have a slightly increased 
 globulin content. 
 
 8. Hydrocephalus. A greatly increased amount, 
 under great pressure. Cell count and globulin 
 normal. Reduces Benedict's. Wassermann may be 
 positive. 
 
 9. Tabes Dorsalis. A clear fluid under slightly in- 
 creased pressure. The amount may vary from 10 to 
 30 c.c. The cell count may vary a good deal, in 
 certain cases being almost normal, and in others con- 
 siderably increased. The usual count is from 25 to 
 75. The Wassermann and the globulin tests may be 
 either positive or negative, but are more likely to be 
 negative. Sugar is usually present. 
 
 10. General Paresis. The amount and macro- 
 scopic character of the fluid is about the same as it is 
 in tabes. The cell count is usually a little lower, 
 running from 15 to 50. 
 
 The Wassermann and Globulin tests are usually 
 positive. Sugar is present. 
 
 11. Cerebrospinal Syphilis. There may be a good 
 deal of pressure as well as a considerably increased 
 amount, with a clear or slightly opalescent fluid, de- 
 pending upon the acuteness and severity of the 
 syphilitic process. 
 
 The Wassermann and Globulin tests are positive, 
 the cell count is from 100 to 1500, with a mono- 
 nuclear formula. Sugar may or may not be 
 present. 
 
CHAPTER VI 
 PLEURAL AND PERITONEAL FLUIDS 
 
 A transudate is an accumulation of non-inflamma- 
 tory fluid in one of the body cavities, due not to in- 
 fection, but usually to some mechanical disturbance of 
 circulation or to impaired kidney function. 
 
 An exudate is an inflammatory fluid due to irri- 
 tation of the serous lining membrane of one of the 
 body cavities by some microorganism. 
 
 Transudates. Transudates are usually colorless, 
 or pale straw-colored, of a thin consistency, with a 
 specific gravity of usually under 1018, and an albumin 
 content of below 2 per cent. The sediment shows a 
 few epithelial and endothelial cells, and a very few 
 leucocytes. Transudates very rarely clot. 
 
 Exudates. Exudates may vary from a thin con- 
 sistency like that of a transudate, to a thick, creamy 
 consistency, due to the presence of pus. The color 
 varies from light to dark straw color. The specific 
 gravity is usually over 1018, and the albumin content 
 over 2 per cent. Transudates usually clot spon- 
 taneously. 
 
 The sediment contains many more cells than does 
 that of transudates, the cells being either lympho- 
 cytes or polynuclear cells according to the nature of 
 the infection. The causative microorganism may 
 sometimes be seen in the stained specimen, often in 
 large numbers. 
 
153 
 
 It is often of importance to distinguish between 
 tubercular and other fluids. As a rule, tubercular 
 fluids are clear, and pale, and may be very close to a 
 transudate in some of their properties. There may 
 be few cells in the sediment, or a good many, and 
 these are nearly all lymphocytes. Any pleural or* 
 peritoneal exudate which shows a predominance of 
 lymphocytes is presumably a tubercular fluid. As 
 it is not at all easy to demonstrate the tubercle 
 bacillus in tubercular exudates, it is best to inject a 
 few cubic centimeters into a guinea pig, kill the pig 
 after six weeks, and determine whether or not he has 
 developed tuberculosis. 
 
 EXAMINATION OF EXUDATES 
 
 Albumin. May be determined by the Esbach 
 tube, as used for urine (see p. 9). As exudates 
 usually have a much higher albumin percentage 'than 
 any urine that is ever examined, it is best to dilute 
 I to 10. 
 
 Staining. The staining is best done immediately 
 after the fluid is withdrawn. If it is desired to prevent 
 clotting a few cubic centimeters of a 2 per cent solu- 
 tion of sodium citrate may be added. The fluid is 
 centrifuged and a smear made from the sediment, 
 drying in the air with no heat, and using methyl 
 alcohol as a fixative if one is desired. The best stain 
 is methylene blue, and the important things to ob- 
 serve in the stained specimen are the differential count 
 of leucocytes and the number and variety of any 
 bacteria which may be present. 
 
 Purulent fluids may be stained with Smith's stain 
 (see p. 163). 
 
CHAPTER VII 
 SPUTUM 
 
 Source. The sputum may be composed of ma- 
 terial from the mouth, nose, naso-pharynx, trachea 
 and larynx, bronchi or lungs. Also, material from 
 diseased conditions in other organs may find its way 
 into the sputum. Examples of this are amebic liver 
 abscesses or pyogenic sub-diaphragmatic abscesses 
 perforating into the lung, or pus from an empyema 
 perforating into a large bronchus. 
 
 MACROSCOPIC EXAMINATION 
 
 Amount. The amount varies greatly. Especially 
 large amounts are likely to be seen in perforating 
 empyema, pulmonary gangrene or abscess, bronchiec- 
 tasis, and certain cases of bronchitis. The amount 
 may sometimes reach a liter or more in the twenty- 
 four hours. 
 
 Consistency and Appearance. May be very thin 
 or very thick and tenacious. On the basis of the 
 character of the sputum the following classification is 
 usually made. 
 
 1. Frothy seen especially in acute edema of the 
 lungs. 
 
 2. Mucoid especially likely to be seen in certain 
 cases of bronchitis and in edema of the lungs due to 
 chronic cardiac conditions. 
 
 155 
 
157 
 
 3. Muco-purulent common in many diseases: 
 pneumonia, bronchitis, tuberculosis, etc. 
 
 4. Purulent seen in any inflammatory or de- 
 structive process within the respiratory tract, where 
 there is pus present (tuberculosis, bronchiectasis, 
 bronchitis, abscess, perforating empyema, etc.). 
 Purulent sputum may be homogeneous, or the pus 
 may be present in small masses, in which case it is 
 said to be nummular. 
 
 5. Bloody. Blood may be seen in the sputum in 
 nearly any inflammatory or destructive process of the 
 respiratory tract. It may exist as bright blood, or 
 dark blood, clotted or unclotted, or it may be present 
 as changed blood pigment. Blood is especially likely 
 to be found in the sputum in pneumonia, where it 
 is very intimately mixed with the thick tenacious 
 sputum, giving it a " rusty " or " prune juice " color, 
 in hemorrhagic infarction of the lung, where the 
 blood is intimately mixed with the sputum, but does 
 not have the rusty color of the pneumonic sputum, or 
 in any destructive process of the lung in which ulcer- 
 ation has occurred (tuberculosis, new growth, gan- 
 grene). Blood is also likely to be present in the frothy 
 sputum of acute pulmonary edema, or it may come 
 from ulcerative processes in the throat or esophagus. 
 
 Odor. Most sputum is odorless or has a mildly 
 offensive odor. In abscess or gangrene of the lung 
 it may be extremely foul. 
 
 Color. Most sputa are light yellow, from mixed 
 pus and mucus. Purulent sputa are a darker yellow, 
 or a rather light greenish yellow. The sputum may 
 be green in jaundice, or in chloroma of the lung. A 
 rusty red or " prune juice " color is characteristic of 
 
159 
 
 lobar pneumonia. The sputum of amebic liver 
 abscess rupturing into the lung has the characteristic 
 brick red " anchovy sauce " color. Gray or black 
 sputum is usually due to inhalation of carbon. A 
 chocolate color may be due to gangrene of the lung. 
 
 Casts of the bronchi may be seen in sputum from 
 fibrinous bronchitis, or in cases of diphtheria where 
 the membrane has extended down into the bronchi. 
 
 Lung Tissue. In destructive processes of the lung, 
 small bits of lung tissue may appear in the sputum, 
 as dark threads or small masses. 
 
 Dittrich's plugs are small, light-colored particles 
 occurring occasionally in the sputum, usually coming 
 from the terminal portions of the small bronchi, or 
 from the tonsillar crypts. They are composed of 
 fatty acids, epithelium and detritus, and have a very 
 offensive odor when crushed. 
 
 " Lung stones " usually are small masses of cal- 
 cined tubercular material, and may occur in con- 
 siderable numbers in the sputum from certain cases 
 of tuberculosis. 
 
 Echinococcus Cysts. Portions of echinococcus 
 cysts are sometimes coughed up in the sputum when 
 there is echinococcus disease of the lung. 
 
 Food remains of various sorts may be present in 
 the sputum, and should not be confused with any of 
 the pathological constituents. 
 
 MICROSCOPIC EXAMINATION 
 
 Bacteria 
 
 Tubercle Bacillus. Select a thick portion of the 
 sputum, and make a smear from it on a cover glass, 
 drying gently with heat. 
 
1. Cover with Ziehl's carbol fuchsin, and steam 
 for I minute, or stain without heat for 5 minutes. 
 
 2. Wash in water. 
 
 3. Decolorize 20 seconds in Czaplewski's solution 
 (3 per cent strong HC1 in 95 per cent alcohol) or in 
 20 per cent sulphuric acid. 
 
 4. Wash in water. 
 
 5. Stain with Loeffler's methylene blue 30 seconds. 
 
 6. Wash in water, dry, and mount in balsam. The 
 cells, etc., of the sputum stain a light blue, and the 
 tubercle bacilli a bright red. They show an especial 
 tendency to occur in small bundles. 
 
 Antiformin Method of Staining. If the bacilli are 
 present in very small numbers they are more likely 
 to be found by the antiformin method, as follows: 
 
 To 10 or 20 c.c. of sputum add an equal volume of 
 antiformin (antiformin is a 10 per cent solution of 
 sodium hypochlorite, containing 5 to 10 per cent 
 sodium hydroxide), and mix thoroughly, until the 
 mixture has become homogeneous. All but the acid- 
 fast group of organisms are destroyed. Centrifuge, 
 and stain the sediment for tubercle bacilli. 
 
 OTHER ORGANISMS 
 
 The sputum contains an extremely mixed flora, 
 and, as many organisms which may be pathogenic are 
 normally present in it, it is unwise to draw too hasty 
 conclusions as to the etiology of a disease from the 
 bacteriology of the sputum. In general, if one 
 organism is present in very large numbers, to the ex- 
 clusion of the other organisms, it is likely to be the 
 etiologic factor of the disease. Thus, one would pay 
 but little attention to a few influenza bacilli in the 
 
CAST OF A BRONCHUS. 
 
 PNEUMOCOCCI. 
 
163 
 
 sputum, but if they were present in large numbers, in 
 nearly pure culture, as they sometimes are, one would 
 expect that they were important in the etiology of the 
 disease. 
 
 A very satisfactory stain for all the bacteria in 
 the sputum except the tubercle bacillus, is that of 
 W. H. Smith. In the routine examination of a 
 specimen of sputum, two preparations should always 
 be made; one stained for the tubercle bacillus, and 
 one stained with Smith's stain, as follows: 
 
 1. Make a thin cover glass smear of the sputum. 
 
 2. Stain with aniline oil gentian violet long enough 
 to run through the flame once or twice, steaming 
 gently. 
 
 3. Stain with Gram's iodine solution, steaming 
 gently for a moment, as before. 
 
 4. Wash with 95 per cent alcohol until no more 
 color comes out. 
 
 5. Wash in water. 
 
 6. Stain with a I per cent aqueous eosin solution 
 for 15 seconds, steaming gently." 
 
 7. Wash in water. 
 
 8. Stain with Loeffler's methylene blue for about 
 30 seconds. 
 
 9. Wash in absolute alcohol, followed by xylol, 
 and mount in balsam. 
 
 ; With this stain the Gram-positive organisms ap- 
 pear dark purple, the Gram-negative light blue, and 
 the protoplasm of the cells pink. Capsules stain pink, 
 and eosinophile cells are easily recognized. 
 
 Pneumococcus occurs as a small, Gram-positive 
 encapsulated diplococcus, but may grow in chains, 
 which (if the capsule does not show up well) are some- 
 
STREPTOCOCCUS Mucosus CAPSULATUS. 
 (Pseudopneumococcus.) 
 
 -~:>T*K '-Ty 
 ZffiifiUm 
 
 ;< ,'-ti ' .* ;'. 
 
 INFLUENZA BACILLI. 
 
165 
 
 times difficult to distinguish from streptococcus 
 chains. The various groups of pneumococci must be 
 differentiated by their agglutination reactions. 
 Pneumococci are present in nearly every sputum in 
 small numbers, and cannot be regarded as the etiologic 
 organism of the disease unless they are numerous. 
 
 Streptococcus is a small Gram-positive organism 
 occurring in chains, long or short. It is seen in many 
 sputa as a secondary infection, but is sometimes the 
 etiologic organism of bronchitis or pneumonia. 
 
 Streptococcus mucosus capsulatus (pseudopneumo- 
 coccus) is a very large, Gram-positive encapsulated 
 diplococcus, appearing usually in large chains. It 
 may be recognized by its large size and distinct 
 capsule. It is a very virulent organism, and if it is 
 the etiologic organism in a case of pneumonia, the 
 prognosis is bad. It is said by some to be present 
 normally in small numbers around the teeth. 
 
 Staphylococcus is a small, Gram-positive coccus, 
 appearing in bunches. It occurs to a greater or lesser 
 extent in nearly every sputum, and is sometimes seen 
 in large numbers as a secondary infective agent in 
 the sputum from cases of tuberculosis, lung abscess, 
 bronchiectasis, etc. 
 
 Influenza Bacillus. This organism is of consider- 
 able importance as an etiologic agent in various 
 respiratory infections, especially in chronic bron- 
 chiectasis and sometimes in acute bronchitis or 
 bronchopneumonia. It is a very small, short bacillus, 
 and when it is the cause of a disease it usually ap- 
 pears in very large numbers in the sputum, both within 
 and without the leucocytes. It does not grow on 
 ordinary media, but must be grown on blood agar. 
 
i6 7 
 
 This is prepared by smearing a drop of blood from the 
 finger (taken under sterile precautions) over the sur- 
 face of the agar in an ordinary culture tube. A loop- 
 ful of the sputum is planted on this. The influenza 
 colonies appear as very small, clear, " dew drop " like 
 dots, which can best be seen with a hand lens. It is 
 Gram-negative. 
 
 Pneumobacillus (Friedlander) occurs as plump rods 
 with rounded ends, varying in size and shape, and 
 often much resembling diplococci. It is Gram-nega- 
 tive, often occurs in pairs, and may have a capsule. 
 
 If this bacillus is found in large numbers in the 
 sputum of a case of pneumonia, the patient is very 
 likely to die, as the mortality is always very high in 
 pneumonia of this type. 
 
 Micrococcus tetragenus is a small coccus in tetrad 
 form, within a capsule, which is likely to appear in 
 mixed infections. It is Gram-positive. It is not of 
 much practical importance. 
 
 Micrococcus catarrhalis is a diplococcus or some- 
 times a tetracoccus, but does not form chains. It 
 looks very much like a gonococcus, but is larger. It 
 is Gram-negative. At one time this organism was 
 thought to be of a good deal of importance in causing 
 various respiratory infections, especially in children, 
 but is not now considered very important. It may 
 occur normally in the sputum. 
 
 MOLDS, YEASTS AND FUNGI 
 
 Actinomycosis of the lung is a rare condition. 
 Macroscopically the actinomyces occur as small 
 " sulphur granule " particles in the sputum. When 
 one of these is crushed on a glass slide, and examined 
 
169 
 
 under the microscope, it is seen to consist of numerous 
 rather fine threads, which radiate from a center in a 
 fan-like manner. The extremities of these threads 
 may be club-shaped. 
 
 Yeasts may be present in the sputum. They may 
 closely resemble fat droplets, but may be distinguished 
 by their property of budding. They are usually not 
 pathogenic. 
 
 Blastomycetes belong to the same general plant 
 group as yeasts. They may cause blastomycosis of 
 the lung, a very rare condition. 
 
 They are oval or round in shape, and have a granular 
 protoplasm with a double capsule. This is separated 
 from the protoplasm by a clear zone. Reproduction 
 in the lung tissue is by budding. They are usually 
 found in the sputum in large numbers when the 
 disease is present. 
 
 Molds. Are not commonly seen in the sputum, 
 but occur occasionally, as the air is full of them. 
 There are over a hundred varieties. They occur 
 usually as a secondary infection in tubercular or other 
 destructive processes, and are characterized especially 
 by their spores, which are likely to be black, and by 
 their filamentous mycelia. 
 
 OTHER BODIES OCCURRING IN THE SPUTUM 
 Epithelial Cells. As the entire respiratory tract, 
 from mouth and nose to lung alveoli, is lined with 
 epithelium, epithelial cells of various sorts may be 
 found in the sputum. They have little practical 
 importance. 
 
 Elastic Tissue. Elastic tissue may be found in 
 the sputum. When found it is an absolute indication 
 
that a destructive process of the lung is going on. 
 It must not be confused with threads of cotton or 
 silk, mycelia of various molds, or elongated fatty acid 
 crystals. True elastic fibers are waxy, extremely 
 
 Elastic Fibers. 
 
 Elastic Fibers in alveolar arrangement. 
 
 refractive, usually rather clean-cut in outline, with 
 rounded ends. They may sometimes occur in alveolar 
 arrangement. The best way to look for elastic tissue 
 is to boil the sputum with an equal volume of 10 per 
 cent sodic hydrate until the mixture is homogeneous, 
 dilute with four times its volume of water, centrifuge, 
 and examine the sediment with the low power of the 
 microscope. 
 
 Charcot-Leyden crystals, eosinophiles, and Cursch- 
 mann's spirals may be considered together, as they 
 usually occur together in cases of bronchial asthma, 
 
CURSCHMANN'S SPIRAL. 
 
 CHARCOT-LEYDEN CRYSTALS. 
 
173 
 
 although they may occasionally be seen in the sputum 
 from other diseases. 
 
 Charcot-Leyden crystals are seen in nearly every 
 case of bronchial asthma. They are rather long and 
 slender, straight, hexagonal double pyramids, are 
 colorless, and may vary a good deal in size. They 
 are probably derived from disintegrated matter of the 
 eosinophile cells. 
 
 Eosinophile cells in the sputum are especially 
 characteristic of bronchial asthma. They are usually 
 mononuclear, not very clean-cut in appearance, and 
 resemble the eosinophilic myelocytes of the blood. 
 
 Curschmann's spirals appear as fine collections of 
 threads twisted spirally, usually containing eosino- 
 philes and Charcot-Leyden crystals in the meshes of 
 threads. They are nearly visible to the naked eye, 
 and hence should be looked for with the low power 
 of the microscope. They usually stand out quite 
 sharply against the structureless background of the 
 sputum. 
 
 Hematoidin crystals may occur in the sputum 
 either in needles or rhombic form. They are es- 
 pecially likely to be seen in sputum coming from 
 closed cavities containing old pus mixed with blood, 
 such as occur in empyema, lung abscess, amebic liver 
 abscess, etc. 
 
 Their color is a bright brownish yellow. 
 
 Cholesterin crystals may be seen under the same 
 conditions. 
 
CHAPTER VIII 
 MISCELLANEOUS 
 
 Gram's Stain for Gonococcus. 
 
 1. Make a thin smear of the suspected pus on a 
 cover glass. 
 
 2. Stain i minute with aniline oil gentian violet. 
 
 3. Wash in water. 
 
 4. Stain 2 minutes with Gram's iodine solution 
 (IKI). 
 
 5. Wash in water. 
 
 6. Decolorize in 95 per cent alcohol until no more 
 color comes out. 
 
 7. Wash in water. 
 
 8. Counterstain I minute with Bismarck brown. 
 
 9. Dry and mount. 
 
 The gonococci appear as rather large, biscuit- 
 shaped diplococci, both within and without the pus 
 cells. 
 
 Spirochaeta Pallida (in primary lesions). Best 
 stained for with Wright's blood stain in the same 
 manner as staining a blood film. 
 
 Schick Test. The value of this test is that it 
 shows whether or not a person possesses a natural 
 immunity to diphtheria. One-fiftieth of the mini- 
 mal lethal dose (guinea pig) of diphtheria toxin, 
 diluted to .1 c.c. with salt solution, is injected 
 intmcutaneously. 
 
 The reaction appears in from 24 to 48 hours after 
 i75 
 
177 
 
 the injection, and consists of an area of erythema, 
 with a brownish tinge, measuring .5 to 2 cm. It 
 usually reaches its height in 48 to 72 hours, and then 
 fades. 
 
 If the reaction does not occur it shows that the 
 patient probably possesses sufficient natural immunity 
 to diphtheria to ward off the infection if exposure 
 should occur. 
 
 Von Pirquet (skin) Tuberculin Test. With a small 
 awl-like implement the skin of the forearm is scarified 
 in three small places about two centimeters apart. 
 Especial care should be taken not to scarify deeply 
 enough to draw the blood. A drop of old tuberculin 
 is applied to the outside scarifications, the inner one 
 being left as a control, and is allowed to dry a moment, 
 and a light gauze dressing is applied. 
 
 The reaction, if -positive, usually appears within 
 24 hours, but should not be called negative until 48 
 hours have elapsed. 
 
 The reaction consists of small, raised, reddened and 
 indurated areas about the points where the tuberculin 
 has been applied. 
 
 GRAM-POSITIVE AND GRAM-NEGATIVE ORGANISMS 
 
 (Mallory and Wright) 
 Gram + Gram 
 
 Staphylococcus pyogenes Gonococcus. 
 
 aureus. Diplococcus intracellularis 
 
 Staphylococcus pyogenes meningiditis. 
 
 albus. Typhoid bacillus. 
 
 Streptococcus pyogenes. Bacillus coli communis. 
 
 Streptococcus capsulatus. Spirillum of Asiatic cholera. 
 
 Pneumococcus. Bacillus pyocyaneus. 
 
 Micrococcus tetragenus. Bacillus of influenza. 
 
 Bacillus diphtherise. Bacillus of glanders. 
 
179 
 
 Gram + 
 
 Bacillus tuberculosis. 
 Bacillus of anthrax. 
 Bacillus of tetanus. 
 Bacillus aerogenes capsulatus. 
 Bacillus of malignant edema. 
 
 Gram 
 
 Bacillus proteus. 
 Bacillus mucosus capsulatus. 
 Bacillus of dysentery. 
 Bacillus of bubonic plague. 
 Bacillus of chancroid. 
 
 DISEASES IN WHICH 
 
 THERE IS A LEUCOCY- 
 
 TOSIS. 
 
 Actinomycosis. 
 Amebiasis (not always). 
 Appendicitis. 
 Bronchitis. 
 Bubonic plague. 
 Diphtheria. 
 Endocarditis (acute). 
 Erysipelas. 
 
 Gonorrhea (complicated). 
 Hydatid disease. 
 Meningitis (cerebrospinal). 
 Meningitis (tubercular). 
 Mumps (lymphocytosis). 
 Osteomyelitis. 
 Pancreatitis (acute). 
 Pneumonia. 
 Purpura. 
 Pyelitis. 
 
 Rheumatism (acute articular). 
 Rickets (in severe cases). 
 Scarlet fever. 
 Scurvy. 
 Tonsillitis. 
 Typhus fever. 
 
 Whooping cough (lymphocy- 
 tosis). 
 
 DISEASES IN WHICH 
 
 THERE IS NO LEUCOCY- 
 
 TOSIS. 
 
 Influenza. 
 
 Malaria (usually leukopenia). 
 Measles (usually leukopenia). 
 Pernicious anemia (leukope- 
 nia). 
 Syphilis. 
 Tuberculosis. 
 
 Typhoid fever (leukopenia). 
 Variola. 
 
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 A manual of practical 
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