CLINICAL LABORATORY METHODS Plate I.—Benedict's Test for Sugar. 
1. Green—Showing only a Trace of Sugar. 
2. Red—Showing a Large Amount of Sugar. 
3. Yellow—Showing a Small Amount of Sugar. (From Gradwolil and Blarvas.) clinical 
LABORATORY METHODS 
BY / 
RUSSELL LANDRAM HADEN/M.A., M.D. 
«v 
Associate Professor of Medicine, University of Kansas, School of 
Medicine, Kansas City, Kansas. Formerly Director of 
Laboratories, Henry Ford Hospital, Detroit 
WITH 69 ILLUSTRATIONS AND 
5 COLOR PLATES 
ST. LOUIS 
C. Y. MOSBY COMPANY 
1923 Copyrighted, 1923, By C. V. Mosby Company 
(All rights reserved) 
Printed in U. S. A. 
Press of 
C. V. Mosby Company 
St. Louis. PREFACE 
Every physician interested in the proper utilization of clinical 
laboratory methods must be impressed with the large amount of 
laboratory work which by reason of its inaccuracy is misleading 
rather than helpful. This is true even of such a simple procedure 
as a blood count. 
Accurate results in the laboratory depend first of all on the 
intelligent and conscientious work of the individual making the 
tests. Equally important, however, is the use of correct procedures. 
This little volume is presented to physicians and laboratory 
workers as a series of procedures which have been thoroughly 
tried out and found to give accurate results. It represents the 
outgrowth of notebooks used in the development and standardiza- 
tion of the laboratory of a general hospital. 
In the selection of the methods the first requirement has been 
the correctness of the underlying principle, and next the adapt- 
ability of the procedure to routine use. 
Only one method is given for each quantitative determination, 
and only one for a qualitative test where a single one is adequate. 
An attempt has been made to present each procedure in a simple 
manner. Wherever possible tables have been inserted to aid in 
the understanding of the technic and to assist in the calculations. 
All discussion of the interpretation of results has been inten- 
tionally avoided. The book is in no sense a textbook. However, 
the normal figures for the method are included in each quanti- 
tative test and occasionally types of abnormal findings are given. 
In the preparation of the book free use has been made of 
original articles in the laboratory journals, the various manuals 
of laboratory technic, such as Folin’s Manual of Biochemistry, 
and notes taken by the author of lectures on clinical microscopy 
at the Johns Hopkins Medical School. Credit has been given 
wherever possible. 8 
PREFACE 
I am much indebted to Dr. Ralph H. Major, professor of 
medicine, University of Kansas, School of Medicine, for his 
interest and helpful criticism throughout the work. Mr. Joseph 
Bobbio, a former assistant, has made numerous valuable sug- 
gestions concerning the procedures in blood chemistry. 
Russell L. Haden. 
Kansas City. CONTENTS 
Qualitative Examination of Urine 17 
Routine Examination, 17; Reagents, 18; Qualitative Tests, 19; Mi- 
croscopic Examination of Urinary Sediment, 22; Identification of 
Reducing Substances in Urine, 29. 
CHAPTER I 
CHAPTER II 
Quantitative Chemical Examination of Urine 35 
Acidity by Titration (Folin), 36; Determination of Hydrogen-Ion 
Concentration (True Acidity), 36; Quantitative Estimation of Glu- 
cose, 37; Quantitative Estimation of Albumin, 41; Quantitative 
Estimation of Chlorides, 41; Determination of Total Nitrogen, 44; 
Determination of Urea Nitrogen (Van Slyke and Cullen), 46; Deter- 
mination of Ammonia, 48; Determination of Uric Acid, 49; De- 
termination of Creatinine (Folin), 51; Determination of Creatine 
(Folin), 54; Determination of Sulphates (Folin), 55; Determination 
of Phosphates, 58; Determination of Calcium and Magnesium, 60; 
Determination of Acetone Bodies, 62; Mosenthal Nephritic Test 
Meal, 67; McLean Index of Kidney Function, 69; Phenolsulphone- 
phthalein Test for Kidney Function, 70; Alkali Tolerance Test, 
72; Determination of Urobilin and Urobilinogen, 73. 
CHAPTER III 
Analysis of Gastric Juice 75 
The Test Meal, 75; Routine Examination, 75; Chemical Examina- 
tion of the Gastric Contents, 75; Microscopic Examination of the 
Gastric Contents, 77; Determination of Urobilin and Urobilinogen 
in Duodenal Contents, 77. 
CHAPTER IV 
Examination of Sputum 79 
Routine Examination, 79; Examination of Fresh Sputum, 79; Exam- 
ination for Tubercle Bacilli, 80; Examination for Elastic Tissue, 80. 
CHAPTER V 
Examination of Feces 81 
Routine Examination, 81; Qualitative Tests, 81; Large Parasites, 
Gallstones, and Other Foreign Bodies, 82; Microscopic Examination, 
9 10 
CONTENTS 
82; Determination of Reaction, 84; Examination of Stool for Amebae, 
84; Quantitative Estimation of Urobilin and Urobilinogen in Stools, 
87; Preservation of Intestinal Parasites, 88; Preservation of Stools 
Containing Ova, 89. 
CHAPTER VI 
Qualitative Examination op Blood 90 
Counting the Blood Cells, 90; Procedure in Counting the Red Blood 
Cells, 92; Procedure in Counting the Leucocytes, 95; Procedure in 
Counting the Blood Platelets (Wright and Kinnieutt), 95; Cleaning 
Counting Chamber and Pipettes, 96; Determination of Hemoglobin, 
97; Determination by the Oxygen Capacity Method, 98; Determina- 
tion by Sahli’s method, 98; Determination with the Hellige Colorim- 
eter, 98; Color Index, 103; Volume Index, 104; Saturation Index, 
104; Examination of Fresh Blood, 105; Preparation and Staining of 
Blood Films, 105; Differential Leucocyte Counts, 108; Pathological 
Red Blood Cells and Leucocytes, 112; Platelets, 113; Determination 
of the Fragility of Erythrocytes, 113; Technic for Vital Blood Stain, 
115; Determination of Coagulation Time of Blood, 116; Determina- 
tion of the Bleeding Time, 117; Sehultze’s Oxydase Reaction, 117; 
Examination of Blood for Malarial Organisms, 119; Qualitative 
Tests for Bile Pigments, Bile Salts and Urobilin in Blood Plasma, 122. 
CHAPTER VII 
Quantitative Chemical Examination op Blood 125 
Collection of Blood for Chemical Analysis, 125; Determination of 
the Relative Hydrogen-Ion Concentration, 127; Preparation of Pro- 
tein Free Blood Filtrates, 129; Determination of Nonprotein Nitro- 
gen, 131; Determination of Urea Nitrogen, 133; Determination 
of Uric Acid, 135; Determination of Creatinine, 138; Determina- 
tion of Creatine, 140; Determination of Sugar, 141; Blood Sugar 
Tolerance Test, 144; Determination of Chlorides, 145; Determina- 
tion of Cholesterol, 148; Determination of Phosphates, 151; Deter- 
mination of Calcium, 158; Determination of Acetone Bodies in 
Blood, 162; Determination of the Bicarbonate Content of the Blood 
Plasma Under Constant Carbon Dioxide Tension, 163; Determina- 
tion of the Oxygen Binding Capacity of Blood (Gasometric Hemo- 
globin Estimation), 171. 
CHAPTER VIII 
Serological Technic 175 
Technic of the Wassermann Reaction, General Considerations, 175; 
Preparation of Glassware, 175; Preparation and Standardization of 
Reagents, 176; Daily Titration of Reagents Preliminary to the Diag- CONTENTS 
11 
nostic Test, 181; The Diagnostic Test, 184; Reading the Diagnostic 
Test, 186; Widal Reaction, 187; Determination of Types of Pneumo- 
coccus, 189; Blood Tests Preliminary to Transfusion, 191. 
CHAPTER IX 
Preparation or Bacteriological Solutions, Stains, and Media . . . 194 
Sodium Citrate Solution for Blood Culture, 194; Physiological Salt 
Solution, 194; Stock Staining Solutions (Wood), 194; Carbol-Thionin, 
194; Andrade Indicator, 195; Gram Stain, 195; Neisser’s Stain for 
Diphtheria Bacillus, 196; Zielil-Neelson Stain for Tubercle Bacilli, 
196; Ponder’s Stain, 197; Capsule Stain (Modified Hiss Stain), 197; 
Huntoon Capsule Stain, 198; Stain for Spirocheta Pallida (Medalia), 
198; Sterilization, 199; Titration of Culture Media to Definite 
Hydrogen-Ion Concentration, 200; Meat Infusion Agar, 202; Glu- 
cose Agar, 203; Meat Extract Agar, 203; Red Blood Agar, 203; 
Brown Blood Agar, 204; Meat Infusion Broth, 204; Meat Extract 
Broth, 204; Endo’s Agar, 204; Russell’s Agar, 205; Krumwiede’s 
Brilliant Green Media, 205; Dextrose Brain Broth, 206; Dextrose 
Brain Agar, 206; Potato Media, 206; Dunham’s Peptone Solution, 
207; Litmus Milk, 207; Gelatin, 207; Loeffler ’s Blood Serum, 207; 
Petroff’s Tubercle Bacillus Medium, 208; Sugar-Free Broth, 208; 
Carbohydrate Broth for Fermentation Reactions, 208; Glucose Broth 
for Blood Culture, 208; Glucose Ascitic Fluid Broth, 209; Carbo- 
hydrate Serum Water Fermentation Media (Hiss Media), 209; 
Lactose Bile Medium, 209. 
CHAPTER X 
General Bacteriological Methods 210 
Blood Culture, 210; Sputum Culture, 210; Stool Culture, 211; Urine 
Culture, 212; Nose and Throat Cultures, 212; Eye and Ear Cultures, 
212; Miscellaneous Cultures, 212; Virulence Test for Diphtheria 
Bacillus, 213; Animal Inoculation for Tuberculosis, 213; Examina- 
tion of Stool for the Tubercle Bacillus, 216; Examination of Urine 
for the Tubercle Bacillus, 216; Examination of Smears for the 
Gonococcus, 216; Classification of Streptococci, 217. 
CHAPTER XI 
Miscellaneous Clinical Pathological Examinations 220 
Examination of Cerebrospinal Fluid, 220; Lange’s Colloidal Gold 
Test on Spinal Fluids, 222; Examination of Ascitic and Pleural 
Fluids, 22; Dark Field Examination for Spirocheta Pallida, 228; 
Preparation of Bacterial Vaccines, 230; Wound Bacterial Count, 232; 
Detection of Mercury in Excretions, 232; Colorimetric Determination 
of the Hydrogen-Ion Concentration of Biological Fluids, 234. 12 
CONTENTS 
CHAPTER XII 
Miscellaneous Chemical Procedures and Solutions 243 
The Use of the Colorimeter and Nephelometer, 243; Standardization 
of Blood Chemical Determinations, 249; Preparation of Volumetric 
Solutions, 253; Calibration of Volumetric Apparatus, 258; Use of 
Indicators, 260; Preparation of Dakin’s Solution, 263; Preparation of 
Nessler’s Solution (Folin Method), 266; Preparation of Creatinin, 
267; Method for Purification of Picric Acid, 269; Preparation of 
Solutions for Intravenous Use, 269; Preparation of Sodium Citrate 
Solution for Blood Transfusion, 271; Preparation of Antiformin, 
271; Methyl Violet Shellac (for Marking Glassware), 271; Bichro- 
mate Cleaner for Glassware, 272; Stopcock Grease (Boothby), 272; 
Foam Killer, 272. 
CHAPTER XIII 
Histological Technic 273 
Preparation of Permanent Sections for Histological Examination, 
273; Frozen Sections, 275; Mallory’s Stain for Connective Tissue 
Fibers, 277; Technic for Staining Mitochondria (Bensley), 278; 
Stain for Tubercle Bacilli in Tissues, 279; Gram-Weigert Method 
for Demonstration of Gram-Positive Bacteria in Tissue, 279; Kaiser- 
ling’s Method of Preserving Gross Specimens, 280; Mixture for 
Sealing Museum Jars, 281. 
CHAPTER XIV 
Examination of Milk and Water 282 
Bacteriological Examination of Water, 282; Examination of Milk 
and Crea: i, 282. ILLUSTRATIONS 
FIG. PAGE 
1. Uric acid crystals 23 
2. Calcium oxalate crystals 23 
3. Calcium phospate crystals 24 
4. Calcium sulphate 24 
5. “Triple phosphate’’ 25 
6. Calcium carbonate crystals 25 
7. Acid sodium urate 26 
8. Ammonium urate crystals 26 
9. Epithelial casts 27 
10. Blood and pus casts 27 
31. Fatty casts 28 
12. Granular casts 29 
13. Hyaline casts 30 
14. Cylindroids 30 
15. Fermentation tube 31 
16. Showing Benedict’s method for the quantitative estimation of sugar 38 
17. Esbach albuminometer 42 
18. Types of pipettes used in chemical and serological work .... 45 
19. Kjeldahl apparatus for the determination of total nitrogen in blood 
and urine by the Folin micro-method 46 
20. Van Slyke and Cullen apparatus for the determination of urea nitrogen 48 
21. Fifteen c.c. graduated centrifuge tube 52 
22. Gooch crucible and holder 55 
23. Porcelain mixing plate for use in the determination of phosphates 
and in blood grouping 59 
24. Dunning colorimeter used in phenolsulphonepthalein test of kidney 
function 71 
25. Record syringe for injecting phenolsulphonephthalein solution ... 71 
26A. Spectrum of urobilin and urobilinogen 73 
26B. Ova in human feces 82 
27. Blood counting pipettes for red and white corpuscles 90 
28. Hausser and Levy counting chambers of Burker type with Neubauer 
ruling 91 
29. Showing the Neubauer ruling of counting chamber as it appears under 
the microscope 92 
30. Machine for shaking blood counting pipettes 93 
31. Tray for holding pipettes and materials for blood counting ... 94 
32. Apparatus used in cleaning blood counting pipettes 96 
33. Pipette cleaning apparatus in position for emptying pipettes . . 97 
13 14 
ILLUSTRATIONS 
FIG. PAGE 
34. Hellige colorimeter 99 
35. Scale showing the average amount of hemoglobin in grams per 100 
c.c. blood at different ages 101 
36. Blood film by the cover glass method 106 
37. Convenient stand for staining blood films made on cover glasses . 107 
38. Tallying register used in making differential leucocyte counts . . 108 
39. Dilutions are made of 0.5 per cent sodium chloride solution by adding 
distilled water by the drop method so that each tube contains 
twenty-five drops of hypisotonic solution 114 
40. One drop of whole blood is added to eaeh tube of hypisotonic solution 115 
41. Tube used in the determination of the coagulation time of blood . 117 
42. Blood chemical bottle used for collecting blood 127 
43. Folin and Wu pipette 130 
44. Folin blood sugar tube 142 
45. Types of curves obtained in blood sugar tolerance test 145 
46. Cholesterol extraction apparatus 149 
47. Tube used in collecting blood for the determination of the carbon 
dioxide combining power 164 
48. Separatory funnel used in saturating blood plasma with carbon dioxide 165 
49. Van Slyke carbon dioxide apparatus 166 
50. Showing position of bulb when the gas volume in the pipette of the 
Van Slyke carbon dioxide apparatus is read 167 
51. Interval timer 177 
52. The most desirable type of burette for general laboratory use . . 177 
53. Showing the reaction of corpuscles of various groups with Group 
II and Group III sera 192 
54. Dunham fermentation tube 209 
55. Fuchs-Rosenthal ruling of counting chamber for spinal fluids . . 221 
56. Block tin still used in distilling water for colloidal gold solution . 223 
57. Reaction types of spinal fluid with colloidal gold solution . . . 227 
58. Old style and new style dark-field illuminators 229 
59. Hopkins vaccine tube 231 
60. Showing H-ion comparison standards, comparator, and sodium 
hydroxide bottle with soda lime guard tubes 236 
61. Arrangement of tubes in comparator, in the determination of hydro- 
gen-ion concentration 240 
62. Duboscq colorimeter 244 
63. Bock-Benedict colorimeter 245 
64. The Duboscq colorimeter converted into a nephelometer with the 
necessary extra parts 247 
65. The nephelometer in position in its box, showing its relation to the 
source of light 248 
66. Burette arrangement 254 15 
ILLUSTRATIONS 
FIG. PAGE 
67. Titration curves of hydrochloric acid and acetic acids, showing the 
gradual color change of methyl red and phenolphthalein within 
their PH zones of transformation 260 
68. Apparatus for making Dakin’s solution from liquid chlorine . . . 263 
69. Babcock test bottle for determining the fat in milk 283 
PAGE 
PLATE I. Benedict’s test for sugar Frontispiece 
PLATE II. Fig. 1.—Osozones; Fig. 2.—Uric acid crystals .... 30 
PLATE III. White blood corpuscles with Wright and Ehrlich stains 108 
PLATE IV. Nucleated red blood corpuscles with Wright and Ehrlich 
stains 112 
PLATE V. Citron’s scale for reading the Wassermann Reaction . . 186 
COLOR PLATES CLINICAL 
LABORATORY METHODS 
CHAPTER I 
QUALITATIVE EXAMINATION OF THE URINE 
Routine Examination 
A routine qualitative examination of the urine is made as 
follows: 
(1) Note the patient’s name, case number and the date. 
(2) If single specimen mark “S.S.”; if a total twenty-four 
hour specimen measure the amount in cubic centimeters. 
(3) Determine the reaction with litmus paper. This may he 
conveniently done by dipping a small piece of neutral litmus 
paper into each specimen. 
(4) Note the color and whether turbid or clear. 
(5) Take the specific gravity with the urinometer. Read 
the urinometer at the bottom of the meniscus. If the specimen is 
very small, dilute the urine with an equal volume of distilled 
water, take the specific gravity and multiply the decimal portion 
of the figure obtained by two. 
(6) Test for albumin with heat and acetic acid or with concen- 
trated nitric acid. Make the test for albumin only on clear urine. 
If the specimen is not clear, shake with infusorial earth and filter. 
Make a quantitative determination of the albumin (page 41) 
on all specimens showing as much as three plus or four plus. 
(7) Test for sugar with Benedict’s reagent. If the test is 
positive, identify the reducing substance according to Table I. 
If a total twenty-four hour specimen is available, determine the 
amount of sugar present. 
(8) Centrifuge a specimen and examine the sediment micro- 
scopically. 
Remarks.—All specimens marked “Catheterized” should be 
so noted on the report. 
17 18 
CLINICAL LABORATORY METHODS 
Test for blood, bile, urobilin, acetone, diacetic acid or indican 
if requested, or if indicated. 
Always record relative amounts as “occasional W.B.C.”, 
“many W.B.C.”, etc. 
Reagents 
Benedict’s Solution (Qualitative).— 
Copper sulphate (crystals) 17.3 gm. 
Sodium or potassium citrate 173 gm. 
Sodium carbonate (crystals) 200 gm. 
Distilled water to make 1000 cubic centimeters. 
The copper sulphate dissolved in about 100 c.c. is poured slowly, 
stirring constantly, into the citrate and carbonate, previously 
dissolved in about 700 c.c. of hot water. The mixture is cooled 
and diluted to 1 liter. 
Nylander’s Reagent—Digest two grams of bismuth subnitrate 
and four grams of Rochelle salt in 100 c.c. of a hot 10 per cent 
solution of potassium hydroxide. The reagent should then be 
cooled and filtered. 
Obermeyer’s Reagent.—Add 2-4 grams of ferric chloride to 
a liter of hydrochloric acid (sp. gr. 1.19). 
Scott-Wilson Reagent.—To 10 g. of mercuric cyanide dissolved 
in 600 c.c. of water add a cooled solution of 180 g. of sodium 
hydroxide in 600 c.c. of water. Transfer this mixture to a heavy- 
walled glass jar, and to it add 2.9 g. of silver nitrate dissolved in 
400 c.c. of water. The silver solution should be added in a slow 
stream, and the addition must be accompanied by constant and 
exceedingly vigorous stirring with a heavy glass rod. If prop- 
erly made, the silver dissolves completely, giving a clear solu- 
tion which is at once available for use. If the solution is turbid, 
it should be set aside to settle for three or four days and the 
clear supernatant liquid removed by means of a siphon. 
In the clear reagent a new sediment gradually forms, so that 
the solution deteriorates slowly and after a few months is not 
serviceable for quantitative work, though still good for qualita- 
tive tests. 
Lugol’s Solution.—Dissolve 4 grams of iodine and 6 grams of 
potassium iodide in 100 c.c. of distilled water. QUALITATIVE examination of urine 
19 
Barfoed’s Solution.—Dissolve 9 grams of neutral crystallized 
copper acetate in 100 c.c. water and add 1.2 c.c. 50 per cent acetic 
acid. 
Ehrlich’s Reagent.—Dissolve 4 grams paradimethyl amino ben- 
zaldehyde in 30 c.c. concentrated hydrochloric acid and add 30 
c.c. distilled water. 
Fehling’s Solution.— 
Solution 1 
Copper sulphate crystals 34.65 gm. 
Distilled water to 1000 c.c. 
Solution 2 
Rochelle salt 173.0 gm. 
Sodium hydrate 125.0 gm. 
Distilled water to 1000 e.c. 
Dissolve the Rochelle salt in hot water, cool, add the sodium 
hydrate, and make up to one liter. 
Equal volumes of Solutions 1 and 2 are mixed in a test tube 
and boiled; the deep blue fluid should remain perfectly clear. 
The urine is added to the hot mixture in small amounts. 
Qualitative Tests 
1. Albumin.—Heat and Acetic Acid.—The urine must be 
acid and clear. If alkaline or neutral, make slightly acid by 
adding a few drops of 3 per cent acetic acid. If cloudy, shake 
with infusorial earth, and filter. 
Fill a test tube two-thirds full of the clear acid urine and 
gently heat the upper half of the fluid to boiling. A turbidity 
is due either to phosphates, carbonates or albumin. Acidify the 
urine further by the addition of 3-5 drops of 3 per cent acetic 
acid, adding it drop by drop to the hot solution. If the precipi- 
tate is due to phosphate or carbonate, it will disappear under these 
conditions; if it is due to albumin, it will become more flocculent. 
Very small quantities of albumin may not appear on* heating, but 
will show after the addition of acid. Examine the tube by trans- 
mitted light against a black background. 
Heeler’s Nitric Acid Test.—Place about 2-3 c.c. of con- 20 
CLINICAL LABORATORY METHODS 
centrated nitric acid in the bottom of a test tube and run in 4-5 
c.c. of urine over the acid with a pipette. There should be a dis- 
tinct layering of the acid and urine. A white layer at the junc- 
tion indicates the presence of albumin. 
Record results as follows: 
Negative 0 
Faint trace Ft. + 
Trace + 
Moderate amount + + 
Large amount + + + 
Very large amount + + + + 
Make a quantitative determination of the albumin on all speci- 
mens showing as much as three or four plus. 
2. Sugar.—To 5 c.c. of Benedict’s reagent in a test tube add 
eight (not more) drops of urine. Boil the fluid vigorously for 
from one to two minutes over a free flame or place in boiling 
water-bath for five minutes and allow to cool spontaneously. 
In the presence of glucose the entire body of the solution wall 
be filled with a yellow or red precipitate (Plate 1). If the glucose 
is below 0.3 per cent, the precipitate forms only on cooling. If 
no sugar be present, the solution remains perfectly clear or 
shows a faint turbidity which is blue in color, due to urates. 
Bulk and not color is the basis of the test. 
Concentrated urines must be diluted before adding to the 
reagent. Large amounts of albumin, if present, must be removed 
before making the test. 
If Benedict’s test is positive identify the reducing body (see 
page 33, “Identification of Reducing Substances in Urine”). 
3. Acetone.—Test with Scott-Wilson Reagent.—In a large 
test tube, such as used for the determination of urea (Fig. 
20), place first 5 c.c. of urine and 1-2 drops dilute acid 
(HC1 or H2S04). Then insert the rubber stopper carry- 
ing the absorption tube. Place the test tube in a beaker of 
lukewarm water (35-40° C.) and aspirate the volatile acetone by 
means of a moderately rapid air current into a test tube contain- 
ing 5 c.c. of distilled water and 5 c.c. Scott-Wilson reagent. If 
acetone is present, even if only in minute traces, the solution 
becomes turbid. If the amount of acetone obtained is extremelv QUALITATIVE EXAMINATION OF URINE 
21 
small, turbidity may not appear for 5 to 10 minutes. This test 
is specifie for acetone. 
Sodium Nitro Prusside Test.—To 5 c.c. of urine in a test 
tube add about 1 gm. of ammonium sulphate and a few drops of 
5 per cent sodium nitroprusside. Shake a few times to dissolve 
the salt, then layer with concentrated ammonia. A violet color 
indicates acetone. 
4. Diacetic Acid.—Gerhardt’s Test.—Introduce about 5 c.c. of 
urine into a test tube and add ferric chloride (about 40 per cent) 
drop by drop. In the presence of large amounts of diacetic acid, 
a Bordeaux red color is produced. 
5. Bile.—Foam Test.—Shake vigorously a test tube one-half 
full of urine; if the foam presents a distinct yellow color, bile is 
present. 
Rosenbach Test.—Acidify the urine with hydrochloric acid and 
pass through a small filter several times. Touch the stain on the 
paper with a drop of yellow nitric acid. If bile is present a play 
of colors is seen at the edge of the drop. From within outward 
the colors are yellow, red, violet, blue and green. 
Iodine Test.—Layer a very dilute alcoholic solution of iodine 
over urine. A green ring at the zone of contact shows bile. The 
iodine solution should be of a pale yellow color, (1 part of tinc- 
ture of iodine plus 30 parts of 95 per cent alcohol). 
6. Blood.—Guaiac Test.—To about 4 c.c. of urine add 1 c.c. 
of glacial acetic acid and 2 c.c. of ether; shake gently; pour off 
the ether and add to it a few drops of freshly prepared alcoholic 
solution of gum guaiac, (1 gm. in 60 c.c. 95 per cent alcohol), 
and 1 c.c. of 3 per cent hydrogen peroxide. If blood is present, 
a blue color develops in the ether. 
7. Indican.—Obermeyer’s Test.—Mix equal parts of Obor- 
in eyer’s reagent and urine in a test tube. Add a small amount of 
chloroform and invert several times. If an excess of indican is 
present the chloroform becomes dark blue. 
8. Urobilin.—Schlesinger’s Test.—About 10 c.c. of acid urine 
are treated with 5 or 6 drops Lugol’s solution to convert any uro- 
bilinogen present into urobilin. The urine is now mixed with an 
equal quantity of a freshly made saturated solution of zinc acetate 
in absolute alcohol and filtered. If urobilin is present, the fil- 22 
CLINICAL LABORATORY METHODS 
trate shows a green fluorescence when held against a dark back- 
ground and examined by transmitted light. Place filtrate in spec- 
troscope and note characteristic absorption spectrum, a wide 
band between b & F (Fig. 26-A). 
9. Urobilinogen.—Add 1 c.c. of Ehrlich’s reagent to 10 c.c. of 
urine in a test tube. If an abnormally large amount of urobil- 
inogen is present, a red color develops in the cold. A red color 
may develop in normal urine on heating. 
10. Bile Salts.—Hays’ Sulphur Test.—This test is based upon 
the principle that bile acids have the property of reducing the 
surface tension of fluids in which they are contained. Cool about 
10 c.c. of urine in a test tube to 17° C. or lower and sprinkle a 
little finely pulverized sulphur upon the surface of the fluid. The 
presence of bile acid is indicated if the sulphur sinks to the bottom 
of the liquid • the rapidity with which the sulphur sinks depend- 
ing upon the amount of bile acids in the urine. The test is said 
to react with bile acids when the latter are present in the propor- 
tion of 1:120,000. 
Foam Test.—(V. Udransky). To 5 c.c. of urine in a test tube 
add three or four drops of a very dilute (1:1000) aqueous solu- 
tion of furfurol. Place the thumb over the top of the tube and 
shake the tube until a thin foam is formed. With a small 
pipette add two or three drops of concentrated sulphuric acid 
to the foam and note the dark pink coloration produced in the 
presence of bile salts. 
Microscopic Examination of Urinary Sediment 
The sediment is obtained for microscopic examination by cen- 
trifuging the urine in a conical centrifuge tube. The specimen 
employed for examination should be perfectly fresh. A drop of 
the sediment is removed with a pipette and transferred to a glass 
slide. The preliminary examination is made with low magnifica- 
tion and with the light cut off as much as possible. If necessary 
a cover glass is placed on the drop of sediment, which is then 
examined under higher magnification. 
In the preparation under examination look for: 
(1) Red blood cells. 
(2) White blood cells with polymorphous nuclei, (pus cells). QUALITATIVE EXAMINATION OF URINE 
23 
(3) Epithelial cells: (a) squamous, usually large with small 
round nucleus; (b) round or cuboidal with large vesicular nucleus. 
(4) Amorphous sediment: urates, which are usually pink in 
color and dissolve on heating; phosphates, white in color, which do 
Fig. 1.—Uric acid crystals. (From Gradwohl and Blaivas, after Hawk.) 
Fig. 2.—Calcium oxalate crystals. (From Gradwohl and Blaivas.) 
not dissolve on heating, but are soluble in acids; carbonates which 
are soluble in acid with the evolution of carbon dioxide. 
(5) Crystals: (a) In acid urine: uric acid, which are usually 24 
CLINICAL LABORATORY METHODS 
Fig. 3.—Calcium phosphate crystals. (From Gradwohl and Blaivas.) 
Fig. 4.—Calcium sulphate. (After Hensel and Weil.) 25 
QUALITATIVE EXAMINATION OF URINE 
Fig. 5.—“Triple phosphate.” (After Ogden.) 
Fig. 6.—Calcium carbonate crystals. (After Hawk.) 26 
CLINICAL LABORATORY METHODS 
Pig. 7.—Acid sodium urate. (After Hawk.) 
Fig. 8.—Ammonium urate crystals. (After Peyer.) 
colored reddish or yellowish brown and occur in a variety of forms, 
such as rhombic prisms, wedges, dumb-bells, whetstones, prismatic 
rosettes and irregular or hexagonal plates, (Fig. 1 and Plate II) ; QUALITATIVE EXAMINATION OF URINE 
27 
calcium oxalate, which are formed as highly refractive octohedra 
or as dumb-bell forms (Fig. 2) ; calcium phosphate occurring 
as slender, colorless, rhombic tablets, often grouped together as 
rosettes (Fig. 3) ; calcium sulphate, which crystallizes in the form 
of long thin needles or prisms (Fig. 4). 
Fig. 9.—Epithelial casts. (After Hawk.) 
Fig. 10.—(.a) Blood casts (yellow in color); (b) Pus casts. (After Hawk.) 
(b) In alkaline urine: ammonia magnesium phosphate, 
(“Triple Phosphate”), which occurs in two forms, prisms and the 
feathery type (Fig. 5) ; calcium carbonate, which appears as dumb- 
bells or spheres with radiating lines resembling similar forms of 28 
CLINICAL LABORATORY METHODS 
calcium oxalate (Fig. 6) ; sodium urate, which occurs as groups of 
fan-shaped clusters or prismatic needles (Fig. 7) ; ammonium urate, 
found in the burr-like form of the thorn apple crystal or yellow 
or reddish-brown spheres covered with sharp spicules (Fig. 8). 
(6) Casts.—These are derived from the renal tubules. The 
sides are parallel, the ends are rounded and fairly abrupt. Casts 
are classified as follows: 
(a) Epithelial Casts composed of renal epithelial cells in 
whole or in part (Fig. 9). 
(b) Pus casts which consist of pus cells, characterized by 
their polymorphous nuclei (Fig. 10). 
Fig. 11.-—Fatty casts. (After Peyer.) 
(c) Blood casts containing one or more red blood cells (Fig. 
10). 
(d) Fatty casts which result from the fatty degeneration of 
the cells of epithelial casts (Fig. 11). 
(e) Granular casts which have coarse or fine granules (Fig. 
12). QUALITATIVE EXAMINATION OF'URINE 
29 
(f) Waxy casts which are opaque, very refractive, homo- 
geneous, and white or yellowish in color. 
(g) Hyaline casts which are very pale, have little refrac- 
tivity, and are difficult to see unless the light is cut 
off (Fig. 13). 
(h) Cylindroids are bodies resembling casts, but are dis- 
tinguished by the fact that they have a tapering end, 
often going to a threadlike process (Fig. 14). 
Fig. 12.—Granular casts. (After Peyer.) 
Identification of Reducing Substances in Urine 
If the test with Benedict’s reagent is positive, do the following 
tests on the specimen in the order given. 
1. Nylander’s Test.—To one-half test tube urine add one-tenth 
the volume of Nylander’s solution; boil for 3 to 5 minutes. A 
reducing substance forms a black precipitate. 
2. Phenyl-hydrazine Test.—To 4 c.c. of albumin-free urine add 
y2 c.c. glacial acetic acid and 5 drops of phenyl-hydrazine. Boil 30 
CLINICAL LABORATORY METHODS 
Fig. 13.—Hyaline casts. (After Hawk.) 
Fig. 14.—Cylindroids. (After Peyer.) Fig. 1.—Osozones. (After Hawk.) Upper form, dextrosozone; lower form, maltosozone. 
Fig. 2.—Uric acid crystals. Normal color. (From Hawk, after Peyer.) 
Plate II. QUALITATIVE EXAMINATION OF URINE 
31 
gently for 1 minute; and add 4-5 drops of 20 per cent NaOII. 
Heat for few minutes; let cool at room temperature. Character- 
istic yellow osozone crystals (Plate II) are obtained within 20 
minutes if sugar be present. If the melting point of the crystals 
is to be determined, dissolve crystals in hot 50 per cent alcohol, 
pour into distilled water and evaporate the alcohol. The oso- 
zones crystallize out. 
3. Fermentation Test.—Rub up a small piece of fresh yeast 
with 50 c.c. of boiled urine; fill fermentation tube (Fig. 15) 
and place in incubator. As control, use (a) normal boiled urine 
and yeast and (b) normal urine and yeast plus glucose to prove 
the activity of the yeast. If a fermentable sugar is present, gas 
will form in the fermentation tube. 
Fig. IS.—Fermentation tube. 
4. Polariscopic Test.—Fill tube of polariscope and determine 
whether the substance rotates the plane of light to right or left 
or gives no rotation. (See page 39.) 
Table I shows the reaction of the reducing substances com- 
monly occurring in the urine to the tests given above. 
If necessary for the identification, the following special tests 
for reducing substances may be used. 
(1) Barfoed’s Test.—Place about 5 c.c. of Barfoed’s reagent 
in a test tube and heat to boiling. Add the urine slowly a few 
drops at a time, heating after each addition. Reduction is indi- 
cated by the formation of a red precipitate of cuprous oxide. If 
the precipitate does not form after boiliing % minute, allow the 32 
CLINICAL LABORATORY METHODS 
tube to stand a few minutes and examine. Monosaccharides give 
a positive reaction; disaccharides do not react. 
(2) Levulose.—Seliwanoff’s Test.—Mix 5 to 10 c.c. of urine 
and an equal volume of 25 per cent hydrochloric acid (two parts 
of concentrated hydrochloric acid and one part of water) ; add 
a few grains of resorcin. Boil gently for a few seconds. If 
levulose is present a red color appears, usually followed by a 
brownish precipitate. Cool the fluid, pour into an evaporating 
dish or beaker, and treat with sodium carbonate in substance 
until the reaction is alkaline. Pour the fluid into a test tube and 
shake with ethyl acetate. If levulose is present, the ethyl acetate 
is colored yellow. 
In performing the test prolonged boiling should be avoided. 
(3) Pentose.—Phloroglucin Test.—To about 5 c.c. of urine 
in a test tube add an equal volume of concentrated hydrochloric 
acid and a liberal knife point of phloroglucin. Heat the mixture 
preferably on a water-bath. A red color appears and soon after- 
ward a dark precipitate. Cool the contents of the tube and extract 
with amyl alcohol. Examine the amyl alcohol extract in the 
spectroscope. If pentose is present a band appears midway be- 
tween D and E, a little to the right of the sodium line. 
Glycuronic acid compounds give a positive phloroglucin test as 
well as pentose. 
Orcin Test.—Mix equal parts of urine and hydrochloric 
acid; add a small knife point of orcin and boil gently. If pen- 
tose is present, a dark greenish color soon develops, and finally 
a turbidity, due to a dark green or blue precipitate. Cool the 
contents of the test tube, until they are lukewarm, and then 
extract with amyl alcohol. The extract is a dark olive green 
color, the depth of which is proportional to the concentration 
of pentose in the urine. Spectroscopic examination shows a band 
at D, the sodium line. 
The orcin test also is given by the paired glycuronic acid 
compounds, but the reaction is much less readily obtained than 
with the phloroglucin. Urine should not be filtered through 
paper before the test. 
(4) Paired Glycuronates.—Tollens’ Test.—To 5 c.c. of urine 
in a test tube add a bit of naphthoresorcin about the size of a QUALITATIVE EXAMINATION OF URINE 
33 
Table I 
The Identification of Reducing Substances Occurring in Urine 
benedict's test 
POSITIVE 
NEGATIVE 
Glucose 
Laevulose 
Lactose 
Maltose 
Paired Glycuronates 
Alkaptone Bodies 
Urates (atypical reduction) 
Pentose 
I 
(Excludes all sugars 
but saccharose) 
nylander's TEST 
POSITIVE 
l 
NEGATIVE 
Glucose 
Laevulose 
Lactose 
Paired Glycuronates 
Pentose 
Maltose 
I 
Alkaptone Bodies (urine 
turns black on standing) 
Urates 
phenylhydrazine test 
I 
OSOZONES FORMED 
DIRECTLY 
OSOZONES NOT FORMED 
DIRECTLY 
Glucose (m.p. 200-204) 
Laevulose (m.p. 200-204) 
Maltose (m.p. 190-191) 
Pentose (m.p. 157-160) 
I 
Lactose 
Paired Glycuronates 
I 
fermentation test 
fermentation test 
I 
positive 
negative 
positive 
NEGATIVE 
Glucose 
Laevulose 
Maltose (slowly) 
Pentose 
(optically inactive, 
phloroglucin and orcin 
tests positive) 
Lactose 
Paired glycuronates 
polariscopic test 
I 
polariscopic test 
dextrorotatory 
Glucose (sp. rot. 52.5) 
Maltose (sp. rot. 137) 
Levulose (sp. rot. 92.3) 
(Seliwanoff test positive) 
LEvorotatory 
dextrorotatory 
Lactose (sp. rot. 52.5) 
(Rubner’s test positive) 
Paired glycuro- 
nates in acid urine. 
lEvorotatory 
(Tollens’ test positive) 
barfoed’s TEST 
POSITIVE 
Glucose 
NEGATIVE 
Maltose 
millet seed and 5 c.c. of concentrated hydrochloric acid. Boil 
gently about one minute and set the tube aside for about four 
minutes. Cool the contents of the tube under running water. 34 
CLINICAL LABORATORY METHODS 
Extract with an equal quantity of ether. If glycuronates are 
present in the urine in excess, the ether extract is dark blue to 
violet, while with smaller amounts a faint bluish or reddish violet 
color is obtained. The ether extract when examined immediately 
in the spectroscope shows a single dark band near the sodium 
line. 
The test is sufficiently delicate to detect the small amounts of 
glycuronates normally present in the urine. 
(5) Lactose.—Rubner’s Test.—The urine is boiled with an 
excess of sugar of lead from three to four minutes, when the 
solution becomes yellow or brown. To the hot fluid is then added 
ammonia as long as the precipitate which forms will still dis- 
solve. An intense brick red fluid is obtained which settles later 
as a copper red precipitate with a colorless supernatant fluid. 
If the specific gravity of the urine be over 1.020, it is best to 
dilute one-half. 
Remarks.—A reducing substance giving a positive Benedict’s 
test and Nylander’s test, fermented by yeast, dextrorotatory, and 
giving crystals directly with phenyl hydrazine is glucose or 
maltose. 
A reducing substance giving a positive Benedict’s test and 
Nylander’s test, not fermented by yeast, dextrorotatory and not 
giving crystals directly with phenyl hydrazine is lactose. 
Suspect levulosuria with glycosuria when the quantity of 
glucose found on polariscopic examination falls short of that 
found on titration with Benedict’s copper solution. A positive 
Seliwanoff reaction and the absence of a levorotatory body after 
fermentation practically confirm it. 
Suspect pentosuria wThen the reduction tests are atypical, when 
they persist after attempts at fermentation, when the urine is 
inactive on polariscopic examination. CHAPTER II 
QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
Collection of Urine.—It is very important that all specimens 
of urine for quantitative chemical examination be accurately 
collected on the 24-hour period. 
The container should be kept in a cool place and have added 
to it 10 c.c. of toluol or 5 c.c. of a saturated alcoholic solution 
of thymol for each liter of urine. 
The calculation may ordinarily be simplified by diluting the 
entire specimen to the nearest round number as 1,000 c.c. or 
1,500 c.c. 
Composition of Normal Urine.—Table II shows the average 
Composition of Normal Urine 
Table II. 
Hydrogen-Ion concentration 
Acidity by Titration 
Total nitrogen 
Urea 
Ammonia 
Amino acid nitrogen 
Creatinine 
Creatine 
Uric acid 
Glucose 
Acetone and diacetic acid 
B-Hydroxybutyrie acid 
Indican 
Total sulphates 
Chlorides 
Ethereal sulphates 
Total phosphates 
Calcium 
Magnesium 
4.80 -7.50 (mean 6.00). 
200-500 (c.c. N/10 alkali to 
neutralize 24-hour output). 
12-18 grams in 24 hours. 
30-35 grams (equals 80-90% of 
total nitrogen). 
0.7 gram (equals 2.5-4.5% of total 
nitrogen). 
0.4-1.0 (equals 2-6% of total 
nitrogen). 
1.25 gram. 
Few milligrams (children may 
show 10-50 mgm). 
0.7 gram. 
0 (common qualitative tests). 
3- mgm. (about one-fourth is 
acetone). 
20-30 mgm. 
4- mgm. 
1.5-3.0 gm. (expressed as SO3). 
10-15 gm. (expressed as sodium 
chloride). 
0.1025 gm. (expressed as S03). 
2.5 gm. (expressed as P205). 
0.1-0.4 gram (expressed as CaO). 
0.1-0.3 gram (expressed as MgO). 
35 36 
CLINICAL LABORATORY METHODS 
acidity and the daily excretion of the substances found in normal 
urine. 
Acidity by Titration (Folin) 
Principle.—Potassium oxalate is added to the urine to pre- 
cipitate the calcium present. The urine is then titrated with 
tenth-normal sodium hydroxide solution. 
Procedure.—Place 25 c.c. of urine in a 200 c.c. flask and add 
15 to 20 grams finely powdered potassium oxalate and 1 to 2 
drops 1 per cent phenolphthalein solution. Shake vigorously 
1 to 2 minutes and titrate immediately with N/10 NaOH until 
a faint but unmistakable pink remains permanent on further 
shaking. Express the result in c.c. N/10 NaOII required to 
neutralize the 24-hour specimen. 
Calculation.—The total acidity of the 24-hour specimen ex- 
pressed in cubic centimeters of N/10 sodium hydroxide equals— 
Total vol. of urine ... , n 
■ 25 x c-c- N/10 sodium hydroxide used. 
Remarks.—The normal titration acidity is 200-500 expressed 
in terms of N/10 alkali required to neutralize the 24-hour output. 
Determination of Hydrogen-Ion Concentration (True Acidity) 
Principle.—The urine is treated with a few drops of the proper 
indicator and the color compared with that produced with the 
same amount of indicator and a solution of known hydrogen-ion 
concentration. 
Reagents.—1. Standard Buffer Solutions.—A set covering 
the range PH 5.0 to 7.0 will allow for the extreme variations en- 
countered. For preparation see “Colorimetric Determination of 
the Hydrogen-Ion Concentration of Biological Fluids,” page 234. 
2. Indicators.—Methyl red, brom cresol purple, and phenol 
red are the most useful indicators. See page 239 for preparation 
of the indicator solutions. 
Procedure.—Find the approximate Pn of the urine by adding 
a few drops of indicator beginning with phenol red and com- 
paring with the standard buffer solutions containing indicator. 
Measure 10 c.c. of urine into a test tube of the same bore as 
those containing the standard. Add 10 drops of the indicator QUANTITATIVE CHEMICAL EXAMINATION OP URINE 
37 
showing the sharpest color changes over the range within which 
the solution falls as determined by the preliminary test. 
Compare the urine in a comparator (Fig. 60) with buffer solu- 
tion containing the same indicator. The tubes are arranged as 
illustrated in Fig. 61. The hydrogen-ion concentration .of the 
urine is equal to that of the standard buffer solution whose color 
it matches. 
Remarks.—The normal values lie between 4.8 and 7.5 with a 
mean value almost exactly 6.0. For vegetarians the mean value 
is about 6.6. 
Quantitative Estimation of Glucose 
A. By Titration with Copper Solution (Benedict’s Solution) 
Principle.—The urine is heated with an alkaline copper sul- 
phate solution containing potassium thiocyanate. A white pre- 
cipitate of copper thiocyanate is formed on reduction instead of 
the usual red precipitate of cuprous oxide. 
Reagents.— 
1. Benedict’s sugar reagent: (quantitative) — 
Copper sulphate (crystallized) 18.0 grams' 
Sodium carbonate (crystallized, % the weight of 
the anhydrous salt may be used) 200.0 grams 
Sodium or potassium citrate 200.0 grams 
Potassium thiocyanate 125.0 grams 
Potassium ferrocyanide (5% sol.) 5.0 c.c. 
Distilled water to make a total volume of... .1000.0 c.c. 
With the aid of heat dissolve the carbonate, citrate and thio- 
cyanate in enough water to make about 800 c.c. of the mixture 
and filter if necessary. 
Dissolve the copper sulphate separately in about 100 c.c. of 
water and pour the solution slowly into the other liquid, with 
constant stirring. Add the ferrocyanide solution, cool and dilute 
to exactly 1 liter. Of the various constituents, the copper salt 
only need be weighed with exactness. Twenty-five c.c. of the 
reagent are reduced by 50 mg. of glucose. 
2. Sodium carbonate. 
3. Powdered pumice stone. 38 
. CLINICAL LABORATORY METHODS 
K 
Fig. 16.—Showing Benedict’s method for the quantitative estimation of sugar. 
(From Gradwohl and Blaivas.) QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
39 
Procedure.—The urine, 10 c.c. of which should be diluted with 
water to 100 c.c. (unless the sugar content is believed to be low, 
when it may be used undiluted), is poured into a 50 c.c. burette 
up to the zero mark. Twenty-five c.c. of the reagent are meas- 
ured with a pipette into a porcelain evaporating dish (25-30 cm. 
in diameter), 10 to 20 grams of crystallized sodium carbonate 
(or one-half the weight of the anhydrous salt), are added, to- 
gether with a small quantity of powdered pumice stone or talcum, 
and the mixture heated to boiling over a free flame (Fig. 16) 
until the carbonate has entirely dissolved. The diluted urine 
is now run in from the burette, rather rapidly, until a chalk- 
white precipitate forms and the blue color of the mixture begins 
to lessen perceptibly, after which the solution from the burette 
must be run in a few drops at a time until the disappearance 
of the last trace of blue color, wdiich marks the end point. The 
solution must be kept vigorously boiling throughout the entire 
titration. If the mixture becomes too concentrated during the 
process, water may be added from time to time to replace the 
volume lost by evaporation. 
Calculation.—The 25 c.c. of copper solution are reduced by 
exactly 50 mg. of glucose. Therefore, the volume run out of 
the burette to effect the reduction contained 50 mg. of the sugar. 
When the urine is diluted 1:10, as in the usual titration of diabetic 
urines, the formula for calculating the percentage of the sugar 
is the following: 
x 1000 = percentage in original sample 
%0 
wherein x is the number of cubic centimeters of the diluted urine 
required to reduce 25 c.c. of the copper solution. 
Remarks.—In the use of this method, chloroform must not be 
present during the titration. If used as a preservative in the 
urine, it may be removed by boiling a sample for a few minutes, 
and then diluting to its original volume. Table III shows the 
percentage of glucose corresponding to the different amounts of 
urine required. 
B. By Polariscopic Method 
The urine must be acid, albumin-free, and clear. If cloudy, 
add 2 grams lead acetate, (sugar of lead), to a portion, shake and 40 
CLINICAL LABORATORY METHODS 
Determination of Glucose in Urine 
Take 25 c.c. of Benedict’s reagent and decolorize with urine diluted 1:10, if the sugar content is high, or undiluted 
if low. 
Table shows the percentage of glucose corresponding to the different amounts of diluted or undiluted urine required. 
URINE DILUTED 1:10 
UNDILUTED 
URINE 
C.C. 
URINE 
PER CENT 
GLUCOSE 
C.C. 
URINE 
PER CENT 
GLUCOSE 
C.C. 
URINE 
PER CENT 
GLUCOSE 
C.C. 
URINE 
PER CENT 
GLUCOSE 
C.C. 
URINE 
PER CENT 
GLUCOSE 
C.C. 
URINE 
PER CENT 
GLUCOSE 
5.0 
10.0 
10.0 
5.0 
30 
1.7 
5.0 
1.0 
10.0 
0.50 
30 
0.17 
5.2 
9.6 
10.5 
4.8 
31 
1.7 
5.2 
0.96 
10.5 
0.48 
31 
0.17 
5.4 
9.3 
11.0 
4.6 
32 
1.6 
5.4 
0.93 
11.0 
0.46 
32 
0.16 
5.6 
8.9 
11.5 
4.4 
33 
1.6 
5.6 
0.89 
11.5 
0.44 
33 
0.16 
5.8 
8.6 
12.0 
4.2 
34 
1.5 
5.8 
0.86 
12.0 
0.42 
34 
0.15 
6.0 
8.3 
12.5 
4.0 
35 
1.4 
6.0 
0.83 
12.5 
0.40 
35 
0.14 
6.2 
8.1 
13.0 
3.9 
36 
1.4 
6.2 
0.81 
13.0 
0.39 
36 
0.14 
6.4 
7.8 
13.5 
3.7 
37 
1.4 
6.4 
0.78 
13.5 
0.37 
37 
0.14 
6.6 
7.6 
14.0 
3.6 
38 
1.3 
6.6 
0.76 
14.0 
0.36 
38 
0.13 
6.8 
7.4 
14.5 
3.5 
39 
1.3 
6.8 
0.74 
14.5 
0.35 
39 
0.13 
7.0 
7.2 
15.0 
3.3 
40 
1.3 
7.0 
0.72 
15 
0.33 
40 
0.13 
7.2 
7.0 
16 
3.1 
41 
1.2 
7.2 
0.70 
16 
0.31 
41 
0.12 
7.4 
6.8 
17 
3.0 
42 
1.2 
7.4 
0.68 
17 
0.30 
42 
0.12 
7.6 
6.6 
18 
. 2.8 
43 
1.2 
7.6 
0.66 
18 
0.28 
43 
0.12 
7.8 
6.4 
19 
2.6 
44 
1.1 
7.8 
0.64 
19 
0.26 
44 
0.11 
8.0 
6.3 
20 
2.5 
45 
1.1 
8.0 
0.63 
20 
0.25 
45 
0.11 
8.2 
6.1 
21 
2.4 
46 
1.1 
8.2 
0.61 
21 
0.24 
46 
0.11 
8.4 
6.0 
22 
2.3 
47 
1.1 
8.4 
0.60 
22 
0.22 
47 
0.11 
8.6 
5.8 
23 
2.2 
48 
1.0 
8.6 
0.58 
23 
0.22 
48 
0.10 
8.8 
5.6 
24 
2.1 
49 
1.0 
8.8 
0.56 
24 
0.21 
49 
0.10 
9.0 
5.6 
25 
2.0 
50 
1.0 
9.0 
0.56 
25 
0.20 
50 
0.10 
9.2 
5.4 
26 
1.9 
9.2 
0.54 
26 
0.19 
9.4 
5.3 
27 
1.9 
9.4 
0.53 
27 
0.19 
9.6 
5.2 
28 
1.8 
9.6 
0.52 
28 
0.18 
9.8 
5.1 
29 
1.7 
9.8 
0.51 
29 
0.17 
Table III 41 
QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
filter several times through the same filter until perfectly clear. 
If albumin is present, it must be removed by heat and acid and 
filtration. 
Fill polariscope tube marked 189.4 mm. Place glass disc over 
end of tube and screw down cap. Avoid air bubbles. After 
focusing, readings are made, first without the urine to determine 
whether the zero point is accurate; next, after refocusing, with 
the tube of urine in place. Starting at zero, the handle is rotated 
until the entire field is equally illuminated. The reading gives 
the percentage of sugar directly. If the tube marked 94.7 mm. 
is used, multiply result by 2. 
Remarks.—The polariscope may be used to determine the per- 
centage (P) of any optically active substance in solution from 
the following equation: 
„ , A x 100 
P equals —r-^p:—^ 
(cc) DxL 
where A equals the angle of rotation 
(cc) D equals specific rotation of the substance in question 
L equals length of polariscope tube in decimeters. 
Quantitative Estimation of Albumin 
Preparation of Tsuchiya Reagent.— 
Phosphotungstic acid 15 gm. 
Concentrated hydrochloric acid 50 c.c. 
Ethyl alcohol (95 per cent) ad.q.s 1000 c.c. 
Procedure.—If the urine is alkaline, acidify with acetic acid. 
Fill an Esbach tube (Fig. 17) with urine to the mark “U” 
and then add Tsuchiya’s reagent to mark “R.” The tube is 
corked and inverted twelve times. Do not shake. Place in ver- 
tical position for twenty-four hours at room temperature and 
read the height of the precipitate on the scale marked on the 
tube. The figure obtained gives the albumin in grams per liter. 
Quantitative Estimation of Chlorides 
Principle.—Volumetric silver nitrate solution is added in ex- 
cess to the urine precipitating the chlorides as silver chloride. 42 
CLINICAL LABORATORY METHODS 
Tlie excess of silver nitrate is titrated with a standard ammonium 
thiocyanate solution using iron alum as an indicator. 
Reagents.—1. Silver Nitrate Solution.—Dissolve 29.075 grams 
of silver nitrate in 900 c.c. of 25 per cent nitric acid; add 50 c.c. 
of a 10 per cent solution of ferric ammonium sulphate; make up 
to one liter with distilled water. One c.c. of this solution is 
equivalent to 0.01 gram sodium chloride. 
Standardize against tenth normal hydrochloric acid. Ten c.c. 
of the tenth normal acid should be completely precipitated by 
5.85 c.c. of the silver nitrate solution. 
2. Ammonium Thiocyanate Solution.—This solution is made 
of such a strength that 2 c.c. of it is equal to 1 c.c. of the standard 
Fig. 17.—Fsbaeh albuminometer. 
silver nitrate solution. To prepare the solution dissolve 12.9 
grams of ammonium thiocyanate, NHJ3CN, in a little less than 
two liters of water. In a small flask place 20 c.c. of the stand- 
ard silver nitrate solution, add water to make the total volume 
100 e.c. and thoroughly mix the contents of the flask. Now run 
in the ammonium thiocyanate solution from a burette until a 
permanent red-brown tinge is produced. This is the end-reaction 
and indicates that the last trace of silver nitrate has been pre- 
cipitated. Take the burette reading and calculate the amount of 
water necessary to use in diluting the ammonium thiocyanate in 
order that 20 c.c. of this solution may be exactly equal to 10 c.c. 
of the silver nitrate solution. Make the dilution and titrate 
again to be certain that the solution is of the proper strength. 43 
QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
Procedure.—Mix 5 c.c. of urine and 10 c.c. of volumetric silver 
nitrate solution in a small flask. Add about 35 c.c. of distilled 
water. Run in the standard ammonium thiocyanate solution 
from a burette until a permanent red-brown tinge is produced. 
Calculation.—If x be the number of c.c. of ammonium thiocy- 
anate used in titrating the excess silver nitrate: 20 - x = grams 
per liter of chloride (expressed as sodium chloride). 
Remarks.—When ammonium thiocyanate is added to silver 
chloride, a small part of the chloride is converted into silver 
thiocyanate. This may be avoided by filtering the solution after 
the silver chloride has been precipitated. The error due to this 
side reaction is so small, however, that the method as outlined is 
sufficiently accurate for routine clinical use. Table IV shows the 
number of grams of NaCl corresponding to the amount of 
NH4CNS used to neutralize the excess of AgNOa. 
Table IY 
Take 5 c.c. of urine and 10 c.c. of the standard AgN03 solution. 
Table shows the number of grams of NaCl per liter corresponding to the 
number of cubic centimeters of NH4CNS used to neutralize the excess of 
AgN03. 
Determination of Chlorides in Urine 
C.C. OF 
nh4cns 
USED 
0.0 
0.1 
0.2 
0.3 
0.4 
0.5 
0.6 
0.7 
0.8 
0.9 
0 
20.0 
19.9 
19.8 
19.7 
19.6 
19.5 
19.4 
19.3 
19.2 
19.1 
1 
19.0 
18.9 
18.8 
18.7 
18.6 
18.5 
18.4 
18.3 
18.2 
18.1 
2 
18.0 
17.9 
17.8 
17.7 
17.6 
17.5 
17.4 
17.3 
17.2 
17.1 
3 
17.0 
16.9 
16.8 
16.7 
16.6 
16.5 
16.4 
16.3 
16.2 
16.1 
4 
16.0 
15.9 
15.8 
15.7 
15.6 
15.5 
15.4 
15.3 
15.2 
15.1 
5 
15.0 
14.9 
14.8 
14.7 
14.6 
14.5 
14.4 
14.3 
14.2 
14.1 
6 
14.0 
13.9 
13.8 
13.7 
13.6 
13.5 
13.4 
13.3 
13.2 
13.1 
7 
13.0 
12.9 
12.8 
12.7 
12.6 
12.5 
12.4 
12.3 
12.2 
12.1 
8 
12.0 
11.9 
11.8 
11.7 
11.6 
11.5 
11.4 
11.3 
11.2 
11.1 
9 
11.0 
10.9 
10.8 
10.7 
10.6 
10.5 
10.4 
10.3 
10.2 
10.1 
10 
10.0 
9.9 
9.8 
9.7 
9.6 
9.5 
9.4 
9.3 
9.2 
9.1 
11 
9.0 
8.9 
8.8 
8.7 
8.6 
8.5 
8.4 
8.3 
8.2 
8.1 
12 
8.0 
7.9 
7.8 
7.7 
7.6 
7.5 
7.4 
7.3 
7.2 
7.1 
13 
7.0 
6.9 
6.8 
6.7 
6.6 
6.5 
6.4 
6.3 
6.2 
6.1 
14 
6.0 
5.9 
5.8 
5.7 
5.6 
5.5 
5.4 
5.3 
5.2 
5.1 
15 
5.0 
4.9 
4.8 
4.7 
4.6 
4.5 
4.4 
4.3 
4.2 
4.1 
16 
4.0 
3.9 
3.8 
3.7 
3.6 
3.5 
3.4 
3.3 
3.2 
3.1 
17 
3.0 
2.9 
2.8 
2.7 
2.6 
2.5 
2.4 
2.3 
2.2 
2.1 
18 
2.0 
1.9 
1.8 
1.7 
1.6 
1.5 
1.4 
1.3 
1.2 
1.1 
19 
1.0 
0.9 
0.8 
0.7 
0.6 
0.5 
0.4 
0.3 
0.2 
0.1 44 
CLINICAL LABORATORY METHODS 
Determination of Total Nitrogen 
(Folin: Jour. Biol. Chem., 26, 473', 1916) 
Principle.—The diluted urine is first heated with an acid mix- 
ture. By this procedure the nitrogen is converted into ammo- 
nium salts. The solution is then Nesslerized and read against a 
standard ammonium sulphate solution similarly treated. 
Reagents.—1. Nessler’s reagent. (See page 266.) 
2. Standard ammonium sulphate solution: 
Ammonium sulphate C.P. 0.4716 grams 
Concentrated hydrochloric acid 1 c.c. 
Water ad. q.s. 1000 c.c. 
10 c.c. = 1 milligram nitrogen. 
The ammonium sulphate should be dried in 
hot air for % hour at 110° centigrade. 
3. Acid digestion mixture: Mix 300 c.c. of 85 per cent phos- 
phoric acid with 100 c.c. of ammonia free sulphuric acid (concen- 
trated). Transfer to a tall cylinder, cover well to exclude the 
absorption of ammonia and set aside for the sedimentation of 
calcium sulphate. To 100 c.c. of the clear acid add 10 c.c. of 6 per 
cent copper sulphate solution and 100 c.c. of water. 
Procedure.—Dilute 5 c.c. of urine to 100 c.c., mix and with an 
Ostwald pipette (Fig. 18) transfer 1 c.c. of the diluted urine to a 
200 x 20 mm. Pyrex tube graduated at 35 c.c. and 50 c.c. (The 
pipette must be drained for 15 seconds against the wall of 
test tube and then blown clean). With an ordinary pipette 
add 1 c.c. of the acid digestion mixture together with 
a pebble, to prevent bumping. Heat over a micro burner (no 
hood necessary) until the water is driven off and fumes become 
abundant within the tube (Fig. 19). This should take place in 
about two minutes. When filled with fumes, close the mouth of 
the test tube with a watch glass and continue the boiling at such 
a rate that the tube remains filled with fumes yet almost none 
escape. Within two minutes after the mouth of the test tube 
is closed the contents should become clear and bluish or light 
green. Continue the gentle boiling for 30 to 60 seconds longer, 
provided however, that the total boiling period with test tube QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
45 
closed must not be less than two minutes. Remove the flame and 
let cool for a little less than two minutes, then add water to 35 
c.c. mark. 
Transfer 3 c.c. of standard ammonium sulphate solution contain- 
ing 0.3 mg. of nitrogen into a 100 c.c. volumetric flask. Add 
2 c.c. of the acid digestion mixture, to balance the acid in the un- 
Fig. 18.—Types of pipettes used in chemical and serological work.—A, Ostwald pipettes; 
B, volumetric pipette; C, Mohr pipette; D, serological pipette. 
known, and dilute to a volume of 70 e.c. Whirl and add 30 c.c. 
of Nessler’s reagent. To the test solution add 15 e.c. Nessler’s 
reagent. 
If the unknown Nesslerized digestion mixture is turbid, cen- 
trifuge a portion giving a crystal clear fluid above a white sed- 46 
CLINICAL LABORATORY METHODS 
iment (silica). If the sediment is colored, the Nesslerization was 
not successful and the determination must be discarded. 
Calculation.—Set standard at 20. 
If R be the reading of unknown: 
20 
——== grams nitrogen per 100 c.c. urine. 
R 
Table Y shows the percentage of nitrogen corresponding to the 
different colorimeter readings with a plunger type instrument. 
Fig. 19.—Kjeldahl apparatus for the determination of total nitrogen in blood and urine 
by the Folin micro-method. (After Reyner.) 
Determination of Urea Nitrog’en (Van Slyke and Cullen) 
Principle.—The urea in the urine is converted into ammonium 
carbonate by urease. The ammonia is liberated by the addition 
of alkali, removed by the passage of a strong air current and 
collected in N/100 sulphuric acid. The excess of acid is then 
titrated with N/100 alkali. 
Procedure.—Dilute 5 c.c. of urine to 50 c.c. with distilled 
water. Measure 5 c.c. of diluted urine into large test tube (Fig. 
20) and add two pulverized urease tablets. Leave at room tem- 
perature for y2 hour. Add a layer of alcohol and kerosene, or a 
few drops of foam killer and 5 c.c. of saturated potassium car- 
bonate. Connect at once with second tube “B” containing 50 QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
47 
Table Y 
Determination of Total Nitrogen in Urine 
Dilute 5 c.c. or 2 c.c. of urine to 100 c.c. Use 1 c.c. of the diluted urine 
in a final volume of 50 c.c. Compare with standard containing 0.3 mgm. 
nitrogen in 100 c.c. 
Set standard at 20, or fill 20 mm. Bock-Benedict cell. 
Table shows the total nitrogen in per cent corresponding to the different 
colorimeter readings with the two dilutions of urine, using a plunger type 
instrument. 
COLOR- 
IMETER 
5 C.C. URINE DILUTED TO 100 C.C. 
(Total nitrogen in per cent) 
2 C.C. URINE DILUTED TO 100 C.C. 
(Total nitrogen in per cent) 
READING 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
10 
0.60 
0.59 
0.58 
0.56 
0.55 
1.50 
1.47 
1.45 
1.41 
1.39 
11 
0.55 
0.54 
0.53 
0.52 
0.51 
1.37 
1.34 
1.32 
1.30 
1.27 
12 
0.50 
0.49 
0.48 
0.48 
0.48 
1.25 
1.23 
1.21 
1.19 
1.17 
13 
0.46 
0.46 
0.45 
0.44 
0.44 
1.15 
1.14 
1.13 
1.11 
1.09 
14 
0.43 
0.42 
0.41 
0.41 
0.41 
1.07 
1.06 
1.04 
1.03 
1.02 
15 
0.40 
0.40 
0.39 
0.39 
0.38 
1.00 
0.99 
0.98 
0.97 
0.95 
16 
0.38 
0.37 
0.37 
0.36 
0.36 
0.94 
0.93 
0.92 
0.90 
0.90 
17 
0.35 
0.35 
0.35 
0.34 
0.34 
0.89 
0.88 
0.87 
0.86 
0.85 
18 
0.33 
0.33 
0.33 
0.32 
0.32 
0.84 
0.83 
0.82 
0.81 
0.80 
19 
0.32 
0.31 
0.31 
0.31 
0.30 
0.79 
0.78 
0.78 
0.77 
0.76 
20 
0.30 
0.30 
0.29 
0.29 
0.29 
0.75 
0.75 
0.74 
0.73 
0.73 
21 
0.29 
0.28 
0.28 
0.28 
0.28 
0.72 
0.70 
0.70 
0.70 
0.69 
22 
0.27 
0.27 
0.27 
0.27 
0.26 
0.69 
0.68 
0.67 
0.67 
0.66 
23 
0.26 
0.26 
0.26 
0.26 
0.25 
0.66 
0.65 
0.65 
0.64 
0.63 
24 
0.25 
0.25 
0.25 
0.24 
0.24 
0.63 
0.62 
0.62 
0.61 
0.61 
25 
0.24 
0.24 
0.24 
0.23 
0.23 
0.60 
0.60 
0.59 
0.59 
0.59 
26 
0.23 
0.23 
0.23 
0.23 
0.23 
0.58 
0.58 
0.57 
0.57 
0.57 
27 
0.22 
0.22 
0.22 
0.22 
0.22 
0.56 
0.56 
0.55 
0.55 
0.54 
28 
0.21 
0.21 
0.21 
0.21 
0.21 
0.54 
0.54 
0.53 
0.53 
0.52 
29 
0.21 
0.21 
0.20 
0.20 
0.20 
0.52 
0.52 
0.51 
0.51 
0.51 
c.c. N/100 H2S04 and two drops alizarin. Aspirate for one hour 
and titrate excess H2S04 with N/100 NaOH. 
Calculation.—The number of c.c. of N/100 H2S04 neutralized 
multiplied by the factor 0.028 gives the number of grams of urea 
nitrogen plus ammonia nitrogen in 100 c.c. urine. 
Determine the ammonia at the same time by using 5 c.c. of the 
undiluted urine. Layer with kerosene and alcohol or add a few 
drops of foam killer. Add 5 c.c. saturated potassium carbonate 
and aspirate into 50 c.c. N/100 II2S04. The number of cubic 
centimeters of N/100 II2S04 multiplied by the factor 0.0028 gives 
the number of grams of ammonia nitrogen per 100 c.c. urine. 
This subtracted from the result obtained in the first determination 
gives the number of grams of urea nitrogen per 100 c.c. urine. 48 
CLINICAL LABORATORY METHODS 
Remarks.—A slow current of air should be used during the 
first two minutes of aspiration. The column of acid should be at 
least 50 mm. high in the receiving tube. The solution from which 
the ammonia is derived must contain at least one gram of potas- 
sium carbonate for each 2 c.c. of solution. 
A blank determination should be run with each new lot of 
chemicals and the necessary deduction be made if any ammonia 
is found. 
The air used for aerating should be run through a wash bottle 
containing sulphuric acid. 
Tt> SUCTION POCTf* 
SUUPHURIC hC\V> 
~ WMH BOTTUB 
0CIT3 
100 
Fig. 20.—Van Slyke and Cullen apparatus for the determination of urea nitrogen, 
The figure obtained for urea nitrogen may be converted into 
urea by dividing by the factor 0.467. 
Determination of Ammonia 
Principle.—The ammonia is liberated by the addition of an 
alkali, removed from the urine by the passage of a strong current 
of air, and is collected in N/100 sulphuric acid. The excess of 
acid is then titrated with N/100 alkali. 
Procedure.—Use the same apparatus as that employed in the 
determination of urine by the urease method (Fig. 20). In the QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
49 
first tube “A” place 5 cubic centimeters urine, 5 cubic centi- 
meters of saturated potassium carbonate solution (90 grams to 
100 e.c.) and a few drops of foam killer. In the second tube “B” 
place 50 c.c. of N/100 sulphuric acid and 2 drops of alizarin. 
Allow the air current to pass through for about an hour. Titrate 
excess of acid with N/100 NaOII. 
Calculation—Each cubic centimeter of N/100 sulphuric acid 
neutralized by the ammonia liberated corresponds to 0.0028 
grams of ammonia N, or 0.0034 grams of ammonia per 100 c.c. 
urine. 
Remarks.—A slow current of air should be used during the 
first two minutes of aspiration. The column of acid should be at 
least 50 mm. high in the receiving tube. The solution from 
which the ammonia is driven must contain at least 1 gram of 
potassium carbonate for each 2 c.c. of solution. 
Determination of Uric Acid 
(Benedict, S. R., and Franke, Elizabeth: Jour. Biol. Chem., 
1922, lii, 387) 
Principle.—The urine is diluted and a special reagent and so- 
dium cyanide are added. A deep blue develops. This is com- 
pared with a standard solution of uric acid similarly treated. 
Reagents.—1. Benedict’s uric acid reagent. This is prepared 
as follows: 100 gm. of pure sodium tungstate are placed in a liter 
Pyrex flask and dissolved in about 600 c.c. of water; 50 gm. of 
pure arsenic acid (As205) are now added, followed by 25 c.c. of 
85 per cent phosphoric acid and 20 c.c. of concentrated hydro- 
chloric acid. The mixture is boiled for about 20 minutes, cooled 
and diluted to 1 liter. The reagent appears to keep indefinitely. 
2. Sodium cyanide.—A 5 per cent solution of sodium cyanide, 
which should be prepared fresh once in about two months, is used. 
3. Standard uric acid solution.—A standard solution of uric 
acid, acidified with hydrochloric acid, containing 0.2 mg. of uric 
acid in 10 c.c., is employed. A stock solution is prepared as fol- 
lows: Dissolve 9 gm. of pure crystallized clisodium hydrogen 
phosphate together with 1 gm. of monosodium dihydrogen phos- 
phate in 200 to 300 c.c. of hot water. Filter if not perfectly clear 
and make up to about 500 c.c. with hot water. Pour this warm 50 
CLINICAL LABORATORY METHODS 
solution upon exactly 200 mg. of uric acid suspended in a few c.c. 
of distilled water in a liter flask and shake gently until all the 
uric acid passes into solution. Cool the solution, add exactly 
1.4 c.c. of glacial acetic acid, dilute to 1 liter, and mix. Five c.c. 
of chloroform are then added to prevent bacterial growth. Five 
c.c. of this standard solution contain 1 mg. of uric acid. Unless 
kept in an excessively warm place the solution may be relied on 
to keep for two months. 
The standard solution for use in the test is prepared from the 
stock phosphate standard solution. Fifty c.c. (containing 10 mg. 
of uric acid) are measured into a 500 c.c. volumetric flask and 
diluted to about 400 c.c. with distilled water. Twenty-five c.c. of 
dilute hydrochloric acid (made by diluting 1 volume of the con- 
centrated acid with 9 volumes of water) are added, and the solu- 
tion is diluted to 500 c.c. and mixed. This dilute standard solu- 
tion should he prepared fresh from the phosphate standard every 
ten days to two weeks. 
Procedure.—The urine is so diluted that 10 c.c. will contain be- 
tween 0.15 and 0.30 mg. of uric acid. (Usually a dilution of 1 to 
20 is satisfactory.) Ten c.c. of the diluted urine are measured 
into a 50 c.c. volumetric flask, 5 c.c. of the 5 per cent sodium 
cyanide solution are added from a burette, followed by 1 c.c. of 
the arsenophosphotungstic acid reagent. The contents of the 
flask are mixed by gentle shaking, and at the end of 5 minutes 
diluted to the 50 c.c. mark with distilled water and mixed. This 
blue solution is then compared in a colorimeter with a simultane- 
ously prepared solution obtained by treating 10 c.c. of the stand- 
ard uric acid solution (0.2 mg. of uric acid) in a 50 c.c. flask with 
5 c.c. of the sodium cyanide solution, 1 c.c. of the reagent, and 
diluting to the mark at the end of 5 minutes. 
Calculation.—If R be the reading of the unknown: 
20 y20r=mg. uric acid per 100 c.c. of urine when 10 c.c. of urine are 
P diluted to 100 c.c. for the test. 
20 y40=mg. uric acid per 100 c.c. of urine when 5 c.c. of urine ai*e 
E diluted to 100 c.c. 
20 x80=rcig- uric acid per 100 c.c. of urine when 2.5 c.c. of urine are 
E diluted to 100 c.c. 
Remarks.—The proportional depth of color for uric acid con- 
centrations varying between 0.15 and 0.30 mg. is almost abso- 
lutely exact under the conditions indicated, when 0.2 mg. of uric QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
51 
Table YI 
Dilute 2.5, 5 or 10 c.c. urine to 100 c.e. Take 10 e.c. of the diluted urine for the 
test and make up to a final volume of 50 c.c. Compare with standard containing 0.2 
mg. uric acid made up to a similar volume. 
Set standard at 20, or fill 20 mm. Bock-Benedict cell. 
Table shows the uric acid in mgs. per 100 c.c. urine corresponding to the different 
colorimeter readings using a plunger type colorimeter. 
2.5 C.C. URINE 
DILUTED TO 100 C.C. 
5 C.C. URINE 
DILUTED TO 100 
C.C. 
10 C.C. URINE 
DILUTED TO 100 
C.C. 
COLOR- 
IMETER 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
o;o 
0.2 
0.4 
0.6 
0.8 
READING 
12 
133 
131 
129 
127 
125 
67 
65 
64 
63 
62 
33 
33 
32 
32 
31 
13 
123 
121 
119 
118 
116 
61 
61 
60 
59 
58 
31 
30 
30 
30 
29 
14 
114 
113 
111 
109 
108 
57 
56 
56 
55 
54 
29 
28 
28 
27 
27 
15 
107 
105 
104 
102 
10.1 
53 
53 
52 
51 
51 
27 
26 
26 
26 
25 
16 
100 
99 
98 
96 
95 
50 
49 
49 
48 
48 
25 
25 
24 
24 
24 
17 
94 
93 
92 
91 
90 
47 
47 
46 
45 
45 
24 
23 
23 
23 
22 
18 
89 
88 
87 
86 
85 
44 
44 
44 
43 
42 
22 
22 
22 
22 
21 
19 
84 
84 
83 
82 
81 
42 
42 
41 
41 
40 
21 
21 
21 
20 
20 
20 
80 
79 
78 
77 
76 
40 
40 
39 
39 
38 
20 
20 
20 
19 
19 
21 
76 
75 
75 
74 
73 
38 
38 
37 
37 
37 
19 
19 
19 
19 
18 
22 
73 
72 
72 
71 
70 
36 
36 
36 
35 
35 
18 
18 
18 
18 
18 
23 
69 
69 
68 
68 
67 
35 
34 
34 
34 
34 
17 
17 
17 
17 
17 
24 
67 
66 
66 
65 
64 
33 
33 
33 
33 
32 
17 
17 
16 
16 
16 
25 
64 
64 
63 
62 
62 
32 
32 
32 
31 
31 
16 
16 
16 
16 
16 
26 
62 
61 
61 
60 
60 
31 
3 i 
30 
30 
30 
15 
15 
15 
15 
15 
27 
59 
59 
58 
58 
58 
29 
29 
29 
29 
29 
15 
15 
15 
15 
15 
acid is used as standard. Outside of this range the results are 
not quite satisfactory, hence, if the colorimeter reading is below 
14 or above 26, the test should be repeated using urine diluted 
1 to 40, or 1 to 10. 
It is essential that the volume of the unknown and of the stand- 
ard be the same during the period of the reaction. 
If albumin is present in appreciable amounts it is best to re- 
move it by heat coagulation in the presence of a drop of acetic 
acid, and filtration. 
Table YI shows the amount of uric acid corresponding to the 
colorimeter readings, using a plunger type instrument. 
Determination of Creatinine (Folin) 
Principle.—On adding picric acid and sodium hydroxide to a 
solution containing creatinine a deep red color is produced. The 
intensity of this color in a specimen of urine is compared with 
that of a standard solution of creatinine. Sugar and albumin do 52 
CLINICAL LABORATORY METHODS 
not interfere but acetone and diacetic acid, if present, must be 
removed by heating. 
Reagents.—(1) Standard creatinine solution: 1 gram of cre- 
atinine or 1.61 grams creatinine zinc chloride dissolved in 1000 
c.c. of tenth normal hydrochloric acid (for method of preparation 
of creatinine and creatinine zinc chloride see page 267). 
(2) Saturated picric acid solution (about 12 grams per liter). 
The picric acid should be tested for purity as follows: To 20 c.c. 
of a saturated solution of picric acid, add 1 c.c. of 10 per cent 
sodium hydroxide and let it stand for 15 minutes. The color of 
Fig. 21.—Fifteen c.c. graduated centrifuge tube. 
the alkaline picrate solution must not be more than twice as deep 
as the color of the saturated acid solution. If the picric acid is 
unusually pure the color of the picrate solution will not be more 
than one and one-half times as deep as that of a saturated picric 
acid solution. If necessary the picric acid may be purified as 
described on page 269. 
(3) Ten per cent NaOH. 
Procedure.—By means of an Ostwald pipette (Fig. 18) transfer 
1 c.c. or 2 c.c. of urine to a 100 c.c. volumetric flask. To another 
flask transfer 1 c.c. of the standard creatinine solution, 1 c.c. of 
which contains 1 mg. of creatinine. To each flask add 20 53 
QUANTITATIVE CHEMICAL EXAMINATION OP URINE 
Use 0.5, 1 or 2 c.c. of urine in a total volume of 100 c.c. and a standard containing 1 mg. of creatinine in. 100 c.c. 
Set standard at 20, or fill 20 mm. Bock-Benedict cell. 
Table shows the creatinine in mg. per 100 c.c. of urine corresponding to the different colorimeter readings with the 
varying amounts of urine, using a plunger type instrument. 
USING 2 C.C. URINE 
(Creatinine in mg. 
per 100 e.e. urine) 
USING 1 C.C. URINE 
(Creatinine in mg. 
per 100 c.c. urine) 
USING 0.5 C.C. URINE 
(Creatinine in mg. 
per 100 c.c. urine) 
COLOR- 
0.6 
0.8 
IMETER 
READING 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
15 
67 
66 
65 
64 
64 
133 
132 
130 
128 
127 
267 
263 
260 
256 
254 
16 
63 
62 
61 
61 
60 
125 
124 
122 
121 
119 
250 
247 
244 
241 
238 
17 
59 
58 
58 
57 
56 
118 
116 
115 
114 
112 
236 
233 
230 
227 
224 
18 
56 
55 
55 
54 
53 
111 
110 
109 
108 
106 
222 
220 
218 
215 
213 
19 
53 
52 
52 
51 
51 
105 
104 
103 
102 
101 
211 
209 
206 
204 
202 
20 
50 
50 
49 
49 
48 
100 
99 
98 
97 
96 
200 
198 
196 
194 
192 
21 
48 
47 
47 
47 
46 
95 
94 
93 
93 
91 
190 
189 
187 
185 
183 
22 
46 
45 
45 
44 
44 
91 
90 
89 
88 
87 
182 
180 
179 
177 
175 
23 
44 
43 
43 
43 
42 
87 
86 
86 
85 
84 
173 
172 
171 
169 
168 
24 
42 
42 
41 
41 
41 
83 
83 
82 
81 
81 
167 
165 
164 
163 
161 
25 
40 
40 
40 
39 
39 
80 
79 
79 
78 
78 
160 
159 
158 
156 
155 
26 
38 
38 
38 
38 
38 
77 
76 
76 
75 
75 
154 
153 
152 
150 
149 
27 
37 
■ 37 
37 
37 
36 
74 
74 
73 
73 
72 
147 
147 
146 
145 
144 
28 
36 
36 
36 
35 
35 
71 
71 
71 
70 
69 
142 
142 
140 
140 
139 
29 
35 
35 
34 
34 
34 
69 
69 
68 
68 
67 
137 
137 
136 
135 
134 
Determination of Creatinine in Urine 
Table VII 54 
CLINICAL LABORATORY METHODS 
c.c. of saturated picric acid solution, then add from a burette 
1.5 c.c. of 10 per cent sodium hydroxide to each, and let 
stand for ten minutes. At the end of ten minutes dilute to 
mark with water and mix. Compare in colorimeter with standard 
set at 20. 
Calculation.—If R be the reading of the unknown: 
20 
— -= Milligrams of creatinine in the quantity of urine taken. 
Remarks.—The normal excretion of creatinine is about 20 to 
30 mg. per kilo of body weight, fat persons yielding less and thin 
persons more. On an average diet the creatinine nitrogen equals 
about 3 to 5 per cent of the total nitrogen. 
Table VII shows the amount of creatinine corresponding to 
different readings Avith a colorimeter of the plunger type. 
Determination of Creatine (Folin) 
Principle.—On heating creatine with dilute mineral acid it is 
dehydrated and its anhydride creatinine is formed. At a tempera- 
ture of 117° to 120° 0., the conversion is complete in fifteen min- 
utes. This temperature is reached when the pressure is 1 kilo 
per square centimeter, or 14 pounds per square inch. 
Procedure.—Place 1 c.c. of the urine in a 100 c.c. volumetric 
flask and add 20 c.c. of saturated picric acid solution. Heat in 
the autoclave for twenty to thirty minutes at 15 pounds pressure, 
closing the mouth of flask with tin foil. Cool. Add 1.5 c.c. of 
sodium hydroxide. Determine the creatinine by the method de- 
scribed for creatinine. Prom the amount of creatinine so obtained 
deduct the amount of creatinine determined in the unheated urine. 
The difference will be the creatine content of the original urine 
in terms of creatinine. To obtain the amount of creatine multiply 
this figure by the factor 1.16. The dark color produced by the 
heating usually causes no difficulty, owing to the dilution neces- 
sary in making the mixture for the colorimeter. 
Remarks.—Creatine occurs in the urine of normal adults in 
only small amounts. In the urine of children as much as 
10-15 mg. per day may be found. QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
55 
Determination of Sulphates (Folin) 
Principle.—The sulphates in the urine are precipitated by the 
addition of an excess of barium chloride solution. The precipitate 
of barium sulphate is filtered off, washed, dried, ignited and 
weighed. 
Reagents.— 
(1) Dilute IIC1 (1 part concentrated HC1 to 4 parts H20 by 
volume). 
(2) Barium chloride solution, 5 per cent. 
Procedure.—Into an Erlenmeyer flask place about 100 c.c. of 
water, 10 c.c. of dilute hydrochloric acid, and 25 c.c. of urine. If 
(A) Inorganic 
Fig. 22.—Gooch crucible and holder 
the urine is dilute take 50 c.c. instead of 25, and a correspond- 
ingly smaller amount of water. Ten c.c. of barium chloride solu- 
tion is added drop by drop from a pipette having a short piece of 
rubber tubing slipped over its upper end and provided with a 
screw pinchcock. The urine must not be disturbed while the 
barium chloride is being added. At the end of an hour or later 
the mixture is shaken and filtered through a weighed Gooch 
crucible, (Fig. 22), as described below. The precipitate is washed 
with at least 200 c.c. of water. The crucible is then dried, ignited, 
cooled and weighed. Report results in terms of S03. 
Calculation.—One gram BaS04 = 0.3430 gm. S03. 56 
CLINICAL LABORATORY METHODS 
Remarks.—The urine must be free of albumin. 
To make the Gooch crucible filter, pour a suspension of asbes- 
tos fiber in water into the crucible while strong suction is being 
applied, so that a firm feltwork (about 2 mm. thick) is formed. 
The asbestos is prepared by scraping the crude material with a 
knife and adding the fibers to a large bulk of 5 per cent IIC1 in 
a cylinder. Air is blown through to separate the fibers thor- 
oughly, and the mixture is allowed to settle for a few minutes. 
The upper portion of the fluid containing the finer fibers is de- 
canted and kept separate from the lower. In making the filter 
the coarse material is poured on first and a little of the fine after- 
ward. The filter is then washed by drawing distilled water 
through in a slow stream, is dried at 120 degrees C., ignited and 
weighed. 
In igniting the barium sulphate precipitates the flame must not 
be applied directly to the bottom of the crucible, or mechanical 
losses occur. The Gooch is placed inside an ordinary porcelain 
crucible, and the flame of the Bunsen burner is used first gently 
and finally with full force. 
(B) Total Sulphates 
Principle.—The ethereal sulphates are split by boiling with 
HC1, and the total sulphates resulting determined just as above. 
Procedure.—Twenty-five c.c. of urine and 20 e.c. of dilute HC1 
(or 50 c.c. of urine and 4 c.e. of concentrated HC1), are gently 
boiled for twenty to thirty minutes in an Erlenmeyer flask, into 
which a funnel has been placed to reduce the loss of steam. The 
flask is cooled for two or three minutes in running water, and the 
contents are diluted with cold water to about 150 c.c. The sul- 
phate is then precipitated and weighed as directed under deter- 
mination of inorganic sulphates. 
The amount of these may be obtained by subtracting the 
amount of inorganic sulphates from that of the total sulphates. 
(C) Ethereal Sulphates (Folin) 
(D) Neutral Sulphates 
Principle.—All of the sulphur present is oxidized by heating 
with a reagent composed of copper nitrate and potassium chlorate. 57 
QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
The former on heating decomposes into two vigorous oxi- 
dizing agents: nitrogen dioxide and cupric oxide, the latter form- 
ing a stable compound with the oxidized sulphur. This is dis- 
solved in dilute hydrochloric acid, and the sulphur precipitated 
with barium chloride solution. 
Reagents.— 
(1) Benedict’s solution: 
Crystallized copper nitrate 200 grams 
Sodium or potassium chlorate 50 grams 
Distilled water to 1000 c.c. 
(2) Barium chloride solution, 5 per cent. 
(3) Dilute IIC1 (1 part concentrated HC1 to 4 parts wa- 
ter by volume). 
Procedure.—Ten c.c. of urine is measured into a small (7 to 8 
cm.) porcelain evaporating dish, and 5 c.c. of the reagent added. 
The contents of the dish are evaporated over a free flame, which 
is regulated to keep the solution just below the boiling point, so 
that there can be no loss through spattering. When dryness is 
reached, the flame is raised slightly until the entire residue has 
blackened. The flame is then turned up in two stages to the full 
heat of the Bunsen burner and the contents of the dish thus 
heated to redness for ten minutes after the black residue (which 
first fuses) has become dry. This heating is to decompose the 
last traces of nitrate and chlorate. The flame is then removed 
and the dish allowed to cool more or less completely; 10 to 20 c.c. 
of dilute (1 to 4) IIC1 is next added to the residue in the dish, 
which is then warmed gently until the contents have completely 
dissolved and a perfectly clear, sparkling solution is obtained. 
The solution obtained by dissolving the residue in the porce- 
lain dish is washed quantitatively into a small Erlenmeyer flask, 
diluted with cold distilled water to 100 to 150 c.c., 10 c.c. of 
5 per cent barium chloride solution is added drop by drop, and 
the solution is allowed to stand for about an hour. It is then 
shaken up and filtered as usual through a weighed Gooch filter. 
Wash with at least 200 c.c. water; dry. Ignite, cool, and weigh. 
Calculation.—1 gram BaS04 = 0.1374 gram sulphate. 58 
CLINICAL LABORATORY METHODS 
Determination of Phosphates 
(A) Total Phosphates 
Principle.—The urine is first treated with a solution of sodium 
acetate to convert any monacid phosphate into diacid phosphate. 
Standard uranium acetate is run into a measured quantity 
until all of the phosphate has been precipitated as insoluble ura- 
nium phosphate. An excess of uranium is indicated by a reddish 
coloration with potassium ferrocyanide. This method is accurate 
and gives practically the total phosphorus of urine inasmuch as 
the latter exists generally almost entirely as phosphates. 
Reagents.— 
1. Sodium acetate solution prepared by dissolving 100 grams of 
sodium acetate in 800 c.c. of distilled water, adding 100 c.c. of 
30 per cent acetic acid to the solution, and making the volume of 
the mixture up to 1 liter with water. 
2. Ten per cent potassium ferrocyanide. 
3'. Uranium acetate solution: Dissolve about 35.0 grams of 
uranium acetate in 1 liter of water with the aid of heat and 3 to 
4 c.c. of glacial acetic acid. Let stand a few days and filter. 
Standardize against a phosphate solution containing 0.005 grams 
of P205 per cubic centimeter. For this purpose dissolve 14.721 
grains of pure air-dry sodium ammonium phosphate (NaNH4HP04 
+ 4H20) in water to make a liter. To 20 c.c. of this phosphate 
solution in a beaker add 30 c.c. of water and 5 c.c. of sodium 
acetate solution (see above) and titrate with the uranium solu- 
tion to the correct end reaction as indicated in the method above. 
If exactly 20 c.c. of uranium solution are required, 1 c.c. of the 
solution is equivalent to 0.005 gram P,05. If stronger than this, 
dilute accordingly and check again by titration. 
Procedure.—To 50 c.c. of urine in a small beaker or Erlenmeyer 
flask add 5 c.c. of the sodium acetate solution and heat the mix- 
ture to the boiling point. From a burette, run into the hot mix- 
ture, drop by drop, the standard solution of uranium acetate 
until a precipitate ceases to form and a drop of the mixture when 
removed by means of a glass rod and brought into contact with 
a drop of a 10 per cent solution of potassium ferrocyanide on a 
porcelain test-tablet (Fig. 23) produces instantaneously a brown- QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
59 
ish-red coloration. Take the burette reading and calculate the 
P205 content of the urine under examination. 
Calculation.—Multiply the number of cubic centimeters of 
uranium acetate solution used by 0.005 to determine the number 
of grams of P203 in the 50 c.c. of urine used. To express the 
result in percentage of P205 multiply the value just obtained by 
2, e.g., if 50 c.c. of urine contained 0.074 gram of P205 it would 
be equivalent to 0.148 per cent. 
Calculate, in terms of P205 the total phosphate content of the 
24 hour specimen. 
Fig. 23.—iPorcelain mixing plate for use in the determination of phosphates and in 
blood grouping. (From Gradwohl and Blaivas.) 
(B) Earthy Phosphates 
Principle.—The earthy phosphates are precipitated by making 
the urine alkaline. The precipitate is filtered off, dissolved in 
acid, and titrated with uranium acetate. 
Procedure.—To 100 c.c. of urine in a beaker add an excess of 
ammonium hydroxide and allow the mixture to stand 12 to 24 
hours. Under these conditions the phosphoric acid in combina- 
tion with the alkaline earths, calcium and magnesium, is pre- 
cipitated as phosphates of these metals. Collect the precipitate 
on a filter paper and wash it with very dilute ammonium hydrox- 
ide. Pierce the paper, and remove the precipitate by means of 
hot water. Bring the phosphates into solution by adding a small 
amount of dilute acetic acid to the warm solution, and determine 60 
CLINICAL LABORATORY METHODS 
the P205 content of the mixture according to the directions given 
under the Determination of Total Phosphates. 
Calculation.—Multiply the number of cubic centimeters of 
uranium acetate solution used by 0.005 to determine the number 
of grams of P205 in the 100 c.c. of urine used. Since 100 c.c. of 
urine was taken this value also expresses the percentage of P205 
present. 
Calculate the quantity of earthy phosphates, in terms of P205, 
present in the 24 hour urine specimen. 
The quantity of phosphoric acid present in combination with 
the alkali metals may be determined by subtracting the content 
of earthy phosphates from the total phosphates. 
(McCrudden: Jour. Biol. Chem., 1910, vii, 83; 1911, x, 187) 
Determination of Calcium and Magnesium 
Principle.—Calcium is precipitated as the oxalate by the addi- 
tion of ammonium oxalate. The precipitate is either ignited 
and weighed as CaO or determined volumetrically by titration 
with potassium permanganate. By the use of sodium acetate and 
hydrochloric acid such an acidity is attained as will prevent the 
interference with the procedure of magnesium, phosphates and 
iron. 
Magnesium is determined in the filtrate from the calcium deter- 
mination after the destruction of the organic matter. It is pre- 
cipitated as ammonium magnesium phosphate, ignited and 
weighed as the pyrophosphate. 
Reagents Required.— 
(1) 2.5 per cent oxalic acid. 
(2) 20 per cent sodium acetate. 
(3') 0.5 per cent ammonium oxalate. 
(4) N/10 potassium permanganate solution (for volumetric 
procedure). 
(5) Cone, hydrochloric acid (sp.gr. 1.20). 
(6) Alcoholic ammonia solution (1 part alcohol, 1 part dil. 
ammonia, 3 parts water). 
(7) Dilute ammonia (sp.gr. 0.96). 
Procedure for Calcium.—If the urine is alkaline make it neu- 
tral or slightly acid and filter. Take 200 c.c. of the filtered urine QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
61 
for analysis. If it is only sliglitly acid to litmus add ten drops of 
concentrated hydrochloric acid (sp.gr. 1.20). If the urine is 
strongly acid it may be made just alkaline with ammonia and 
then just acid with hydrochloric acid, after which the ten drops 
of hydrochloric acid are added. Then add 10 c.c. of 2.5 per cent 
oxalic acid. Run in slowly with stirring 8 c.c. of 20 per cent 
sodium acetate. Allow to stand overnight at room temperature 
or shake vigorously for ten minutes. Filter off the precipitate 
of calcium oxalate on a small paper and wash free from chlo- 
rides with 0.5 per cent ammonium oxalate solution. The precip- 
itate may then be dried, ignited to constant weight and weighed 
as calcium oxide or titrated volumetrically as described below. 
Volumetric Titration.—If free from uric acid, the calcium oxal- 
ate precipitate may be washed three times with distilled water, 
filling the filter about two-thirds full and allowing it to drain 
completely before adding more. A hole is made in the paper and 
the calcium oxalate washed in the flask. The volume of the fluid 
is brought up to about 50 c.c. and 10 c.c. of concentrated sul- 
phuric acid added. Titrate with N/10 potassium permanganate 
solution to a pink color which persists for at least a minute. 
Calculation.—One c.c. of N/10 potassium permanganate solu- 
tion is equivalent to 2.8 mg. of CaO. 
Procedure for Magnesium.—Transfer the filtrate from the cal- 
cium determination to a porcelain dish, add about 20 c.c. concen- 
trated nitric acid and evaporate to dryness. Heat the residue 
over a free flame until the ammonium salts are destroyed and the 
residue fuses. After cooling take the residue up with water 
and a little hydrochloric acid and filter if nceessary. Dilute to about 
80 c.c., nearly neutralize with ammonia and cool. Add a slight 
excess of sodium acid phosphate and then ammonia drop by drop 
with constant stirring until the solution is alkaline and then add 
enough more slowly with constant stirring to make the solution 
contain one-fourth its bulk of dilute ammonia (sp.gr. 0.96). Al- 
low to stand overnight. Filter and wash free from chlorides 
with alcoholic ammonia solution. The precipitate with filter 
paper is incinerated slowly and carefully with a good supply of 
air to prevent reduction, in the usual manner, and ignited and 
weighed as the pyrophosphate. 62 
CLINICAL LABORATORY METHODS 
Calculation.—One gram magnesium pyrophosphate is equiva- 
lent to 0.3624 grams MgO. 
Remarks.—The average excretion of calcium (as CaO) is 0.1 
to 0.4 gm. per day. The kidneys excrete about the same amount 
of magnesium (as MgO) daily. 
Determination of Acetone Bodies 
(\ an Slyke, D.D.: Jour. Biol. Chem., 1917, xxxii, 455) 
Principle.—The method for the quantitative determination of 
acetone bodies in the urine is based on a combination of 
Shaffer’s oxidation of beta-hydroxybutyric acid to acetone and 
Deniges’ precipitation of acetone as a basic mercuric sulphate 
compound. Oxidation and precipitation are carried out simul- 
taneously in the same solution so that the technic is simplified to 
boiling the mixture for an hour and a half under a reflux con- 
denser and weighing the precipitate which forms. The acetone 
and acetoacetic may be determined either with the beta-hydroxy- 
butyric acid or separately. Neither the size of sample nor mode 
of procedure requires variation for different times. The same 
process may be used for the smallest significant amounts of 
acetone bodies, and likewise for the largest that are encountered. 
The precipitate is crystalline and beautifully adapted to quick 
drying and accurate weighing, but when facilities for weighing 
are absent, the precipitate can be dissolved in dilute hydrochloric 
acid and the mercury titrated with potassium iodide by the 
method of Personue. 
Preservatives other than toluene or copper sulphate should not 
be used. 
Reagents.—(1) Twenty per cent copper sulphate—200 grams 
of CuS04.5H20 dissolved in water and made up to 1 liter. 
(2) Ten per cent mercuric sulphate—73 grams of pure red mer- 
curic oxide dissolved in 1 liter of H2S04 of 4N concentration. 
(3) Fifty volume per cent sulphuric acid—500 c.c. of sulphuric 
acid of 1.835 specific gravity, diluted to 1 liter with water. Con- 
centration of II2S04 must be readjusted if necessary to make it 
17.0 N by titration. 
(4) Ten per cent calcium hydroxide suspension—mix 100 QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
63 
grams of Merck’s fine light “reagent” Ca(OH)2 with 1 liter of 
water. 
(5) Five per cent potassium dichromate—50 grams K2Cr207 
dissolved in water and made up to 1 liter. 
(6) Combined reagents for total acetone body determination— 
1 liter of the above 50 per cent sulphuric acid, 3.5 liters of the 
mercuric sulphate, 10 liters of water. 
Procedure.—Removal of glucose and other interfering sub- 
stances from urine. 
Place 25 c.c. of urine in a 250 c.c. measuring flask. Add 100 
c.c. of water, 50 c.c. of copper sulphate solution and mix. Then 
add 50 c.c. of 10 per cent calcium hydroxide suspension, shake 
and test with litmus. If not alkaline, add more calcium hydrox- 
ide. Dilute to the mark and let stand at least one-half hour for 
glucose to precipitate. Filter through a dry folded filter. This 
procedure will remove up to 8 per cent of glucose. Urine con- 
taining more should be diluted enough to bring the glucose down 
to 8 per cent. The copper treatment is depended upon to remove 
interfering substances other than glucose, and should therefore 
never be omitted, even when glucose is absent. The filtrate may 
be tested for glucose by boiling a little in a test tube. A precipi- 
tate of yellow cuprous oxide will be obtained if the removal has 
not been complete. A slight precipitate of white calcium salts 
always forms, but does not interfere with the detection of the 
yellow cuprous oxide. 
Determination of Total Acetone Bodies (Acetone, Acetoacetic 
Acid, and B-hydroxybutyric Acid).—Place in a 500 c.c. Erlen- 
meyer flask 25 c.c. of urine filtrate. Add 100 c.c. of water, 10 c.c. 
of 50 per cent sulphuric acid, and 35 c.c. of the 10 per cent mer- 
curic sulphate. Or in place of adding the water and reagents 
separately, add 145 c.c. of the “combined reagents.” Connect 
the flask with a reflux condenser having a straight condensing 
tube of 8 or 10 mm. diameter and heat to boiling. After boiling 
has begun, add 5 c.c. of the 5 per cent dichromate through the 
condenser tube. Continue boiling gently 1% hours. The yellow 
precipitate which forms consists of the mercury sulphate-chro- 
mate compound of the preformed acetone, and the acetone which 
has been formed by decomposition of acetoacetic acid and by 64 
CLINICAL LABORATORY METHODS 
oxidation of the B-hydroxybutyric acid. It is collected in a 
Gooch (Fig. 22) or “medium density” alundum crucible, washed 
with 200 c.e. of cold water, and dried for an hour at 110°. The 
crucible is allowed to cool in room air (a desiccator is unneces- 
sary and undesirable) and weighed. Several precipitates may be 
collected, one above the other, without cleaning the crucible. As 
an alternative to weighing, the precipitate may be dissolved and 
titrated as described below. 
Determination of Acetone and Acetoacetic Acid.—The ace- 
tone plus the acetoacetic acid, which completely decomposes into 
acetone and C02 on heating, is determined without the B-hydroxy- 
butyric acid exactly as the total acetone bodies, except that (1) 
no dichromate is added to oxidize the B-hydroxybutyric acid 
and (2) the boiling must continue for not less than 30 nor more 
than 45 minutes. Boiling for more than 45 minutes splits off a 
little acetone from B-hydroxybutyric acid even in the absence of 
chromic acid. 
Determination of B-hydroxybutyric Acid.—The B-hydroxy- 
butyric acid alone is determined exactly as total acetone bodies 
except that the preformed acetone and that from the acetoacetic 
acid are first boiled off. To do this, the 25 c.c. of urine filtrate 
plus 100 c.c. of water are treated with 2 c.c. of the 50 per cent 
sulphuric acid and boiled in the open flask for 10 minutes. The 
volume of solution left in the flask is measured in a cylinder. The 
solution is returned to the flask, and the cylinder washed with 
enough water to replace that boiled off and restore the volume 
of the solution to 127 c.c. Then 8 c.c. of the 50 per cent sul- 
phuric acid and 35 c.c. of mercuric sulphate are added. The 
flask is connected under the condenser and the determination is 
continued as described for total acetone bodies. 
Titration of the Precipitate in the Above Methods.—Instead 
of weighing the precipitate, one may wash the contents of the 
Gooch, including the asbestos, into a small beaker with as little 
water as possible, and add 15 c.c. of normal HC1. The mixture is 
then heated, and the precipitate quickly dissolves. In case an 
alundum crucible is used, it is set into the beaker of acid until 
the precipitate dissolves, and then washed with suction, the 
washings being added to the beaker. In place of using either a QUANTITATIVE CHEMICAL EXAMINATION OF UK INK 
65 
Gooch or alundum crucible, one may, when titration is employed, 
wash the precipitate without suction on a small quantitative fil- 
ter paper, which is transferred with the precipitate to the beaker 
and broken up with a rod in 15 c.c. of normal HC1. 
In order to obtain a good end-point in the subsequent titration, 
it is necessary to reduce the acidity of the solution. For this 
purpose it has been found that the addition of excess sodium 
acetate is the most satisfactory means. Six to 7 c.c. of 3 Tq. 
acetate are added to the cooled solution of redissolved precipitate. 
Then the 0.2 Tq. KI is run in rapidly from a burette, with con- 
stant stirring. If more than a small amount of mercury is pres- 
ent, a red precipitate of Hgl2 at once forms, and redissolves as 
soon as 2 or 3 c.c. of KI in excess of the amount required to form 
the soluble K2HgI4 have been added. If only a few mg. of mer- 
cury are present, the excess of KI may be added before the Hgl, 
has had time to precipitate so that the titrated solution remains 
clear. In this way not less than 5 c.c. of the 0.2 Tq. KI are added, 
as it has been found that the final titration is not satisfactory if 
less is present. The excess of KI is titrated back by adding 
0.05 iq. HgCl2 from another burette until a permanent red pre- 
cipitate forms. Since the reaction utilized is IIgCl2 + 4KI = 
K2HgI4 + KC1, 1 c.c. of 0.05 tq. IIgCl2 is equivalent in the titra- 
tion to 1 c.c. of the 0.2 tq. KI. 
In preparing the two standard solutions the 0.05 tq. HgCl2 is 
standardized by the sulphide method, and the iodine is standard- 
ized by titration against it. A slight error appears to be intro- 
duced if the iodine solution is gravimetrically standardized and 
used for checking the mercury solution, instead of vice versa. 
In standardizing the mercuric chloride the following procedure 
has been found convenient: 25 c.c. of 0.05 Tq. IIgCl2 are meas- 
ured with a calibrated pipette, diluted to about 100 c.c. and H2S 
is run in until the black precipitate flocculates and leaves a clear 
solution. The HgS, collected in a Gooch crucible and dried at 
110°, should weigh 0.2908 gram if the solution is accurate. 
Both by gravimetric analyses of the basic mercuric sulphate- 
acetone precipitate and by titration, the mercury content of the 
precipitate has been found to average 76.9 per cent. On this 66 
CLINICAL LABORATORY METHODS 
basis, each c.c. of 0.2 rrt. KI solution being equivalent to 10.0 mg. 
of Hg. is equivalent to 13.0 mg. of the mercury acetone pre- 
cipitate. 
Titration is not quite so accurate as weighing, but except when 
the amounts determined are very small, the titration is satis- 
factory. 
Calculation.—One mg. of B-hydroxybutyric acid yields 8.45 
mg. of precipitate. One mg. of acetone yields 20.0 mg. of precipi- 
tate. One c.c. of 0.2 Tq,. KI solution is equivalent to 13 mg. of 
precipitate in titration of the latter. 
Special Factors for Calculation of Results When 25 c.c. of Urine 
Filtrate Equivalent to 2.5 c.c. of Urine, are Used 
Table VTII 
for the Determination 
Determination performed. 
Acetone bodies, calculated as gm. acetone 
per liter of urine, indicated by 
1 gm. of pree. 
1 c.c. of 0.2 m. KI sol. 
Total acetone bodies 
24.8 
0.322 
B-hydroxy butyric acid 
26.4 
0.344 
Acetone=acetoacetie acid 
20.0 
0.260 
In order to calculate the acetone bodies as B-hydroxybutyric 
acid rather than acetone, use the above factors multiplied by 
the ratio of the molecular weights == 1.793. 
acetone 58 
In order to calculate the acetone bodies in terms of molecular 
concentration, divide the factors in Table VIII by 58. To cal- 
culate c.c. of 0.1 rrt. acetone bodies per liter of urine use the 
above factors multiplied by —= 172.4. 
Remarks.—The maximum daily excretion of acetone bodies found 
by Van Slyke in normal men, under usual conditions, was 0.42 
grams calculated as acetone, or 0.75 grams calculated as beta- 
bydroxybutyric acid. Normal adults on a mixed diet excrete on an 
average of 3-15 mg. of acetone and diacetic acid bodies per day. 
Usually about one-fourth is in the form of acetone. Beta- 
hydroxybutyric acid may occur in normal urine to the extent of 
20 to 30 mg. per day. QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
67 
Mosenthal Nephritic Test Meal 
(Mosenthal, H. O.: Arch. Int. Med. 1915, lxvi, 733') 
Principle.—The nephritic test meal is a method for measuring 
kidney function by the elimination of fluid, salt and nitrogen, 
and by the specific gravity of the urine. A fixed, weighed, and 
salt-free diet is given for one day. With each meal 2.3 grams of 
salt are given. Urine specimens are collected at 2 hour intervals 
from 8:00 a.m. to 8:00 p.m. and saved separately. The total 
night urine is collected at 8:00 a.m. on the morning following 
the test. Any food refused is weighed and noted on the diet 
chart. The evening meal must be taken at least three hours be- 
fore the collection of the night urine is begun. 
Procedure.—Measure and take the specific gravity of each 
specimen. Mix the seven day specimens and determine the total 
nitrogen and total chlorides. Make similar determinations of the 
total night urine. 
Table IX shows the diet given, the fluid intake, and the nitro- 
gen percentage of each article of food. Calculate the amount 
of nitrogen in any food refused and subtract from the total 
nitrogen of the test meal. Similarly any fluid or salt not taken 
is to be subtracted from the total offered. Calculate the fluid, 
salt and nitrogen balance. 
Record results as follows: 
TIME VOL. SP.GR. 
8-10 
10-12 
12-2 
2-4 
4-6 
6-8 
SODIUM CHLORIDE 
% GMS. 
NITROGEN 
°/o GMS. 
Total Day 
Total Night 8-8 
Total Excretion (24 hours) 
Total Intake 
Balance 
MOSENTHAL TEST 68 
CLINICAL LABORATORY METHODS 
Table IX 
FLUID 
C.C. 
NITROGEN 
FAT 
GRAMS 
PRO- 
TEIN 
CARBO- 
HYDRATE 
CALORIES 
% 
GRAMS 
GRAMS 
GRAMS 
Brealcfast—8 A. M. 
Boiled oatmeal—100 
gm 
0.448 
0.448 
0.5 
2.8 
11.5 
63 
Sugar, 1/2 teaspoon- 
fuls 
5 
20 
Milk, 30 c.c 
30 
0.528 
0.158 
1.2 
1.0 
1.5 
21.6 
Two slices bread (30 
gm. each) 
1.47 
0.880 
0.8 
5.5 
31.9 
160.8 
Butter, 20 gm 
. . • 
0.16 
0.032 
17.0 
0.2 
.... 
159 
Coffee, 160 c.c 
Sugar, 1 teaspoonful 
200 c.c 
160 
5 
20 
Milk, 40 c.c 
40 
0.528 
0.211 
1.6 
1.3 
2 
28.8 
Milk, 200 c.c 
200 
0.528 
1.056 
8.0 
6.6 
10 
144 
Water, 200 c.c 
200 
... 
— 
— 
Dinner—12 noon. 
Meat Soup, 180 c.c. 
180 
0.70 
1.264 
0.7 
7.9 
1.9 
46.8 
Beefsteak, 100 gm. 
. . • 
4.416 
4.416 
7.7 
27.6 
— 
185 
Potato (baked,mashed 
or boiled) 130 gm. 
0.406 
0.528 
0.1 
3.3 
27.2 
126.1 
Green vegetables, as 
desired 
.... 
• ••• 
Two slices bread (30 
gm. each) 
1.47 
0.880 
0.8 
5.5 
31.9 
160.8 
Butter, 20 gm 
. . • 
0.16 
0.032 
17.0 
0.2 
.... 
159 
Tea, 180 c.c 
180 
.... 
Sugar, 1 teaspoonful 
200 c.c 
5 
20 
Milk, 20 c.c 
20 
0.528 
0.105 
0.8 
0.6 
1.0 
14.4 
Water, 250 c.c 
250 
.... 
Pudding (tapioca or 
rice) 110 gm. ... 
0.116 
0.128 
0.3 
0.8 
79 
359 
Supper—5 p. m. 
Two eggs, cooked in 
any style 
2.11 
2.53 
14.4 
15.8 
202.8 
Two slices bread (30 
gm. each) 
1.47 
0.880 
0.8 
5.5 
31.9 
160.8 
Butter, 20 gm 
... 
0.16 
0.032 
17.0 
0.2 
.... 
159 
Tea, 180 c.c 
Sugar, 1 teaspoonful 
200 c.c 
180 
... 
.... 
.... 
.... 
5 
20 
Milk, 20 c.c 
20 
0.528 
0.105 
0.8 
0.6 
1 
14.4 
Fruit stewed or 
fresh—1 portion . 
300 
• • • 
• • • • • 
.... 
10 
40 
Water, 300 c.c 
... 
— 
TOTAL 
1760 
13.736 
89.5 
85.4 
260.8 
2295.3 QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
69 
Remarks.—The following outline shows the normal standard 
for the Mosenthal Nephritic Test Meal. 
Maximum specific gravity 1.020 or plus 
Degrees variation of specific gravity, usually 9 or plus 
Specific gravity of night urine Of no significance 
Volume of night urine 750 c.c. or less 
N and NaCl per cent in night urine, Normal if 1 per cent 
or highest per cent in any specimen. or higher, not necessarily 
abnormal if less. 
Mosenthal has recently suggested (Medical Clinics of North 
America, 4,209, 1920), a modification of this test, in which the 
special diet is eliminated. 
McLean Index of Kidney Function 
(McLean: Jour. Am. Med. Assn., 1916, lxvi, 415) 
Principle.—The McLean Index expresses the relationship be- 
tween the concentration of urea in the blood and the rate of urea 
excretion by the kidney. 
It is based upon the two laws of Ambard that in normal cases: 
(1) when the concentration of the urea in the urine is constant 
the quantity of urea excreted varies proportionately as the square 
of the concentration of the urea in the blood; i.e., if the quantity 
of urea in the blood is doubled, the amount excreted in a given 
time is quadrupled, and (2) when the concentration of the urea 
in the blood remains constant, the quantity excreted in the urine 
varies inversely as the square root of the concentration in the 
urine, i.e., a quadrupling of the concentration results in a halving 
of the rate of the output. 
The ideal normal rate of excretion is taken as 100. The index 
expresses in direct percentage the rate of urea excretion found 
as compared with the rate of excretion of a normal individual 
under the same condition as to concentration in the blood, con- 
centration in the urine and body weight. 
Procedure.—The patient is given a glass of water to insure 
a free flow of urine. The bladder is emptied (by catheter if 
necessary) and the time is noted to within one minute. One hour 
later 5 to 10 c.c. of blood is withdrawn and prevented from 
clotting by the addition of 5 drops of potassium oxalate solution. 
At the end of two hours from the time of voiding, the bladder is 70 
CLINICAL LABORATORY METHODS 
again emptied and the entire second voiding, taking care to avoid 
the least loss, is at once sent to the laboratory, together with the 
specimen of blood. The patient must take no food or drink dur- 
ing the seventy-two minute period. The patient’s weight, taken 
on the day of the test, must be stated on the label of the blood 
specimen. 
Make a quantitative estimation of the urea in the blood and 
the urea in the urine. 
Calculation.—The formula for the index of urea excretion 
follows: 
T, . ,. DyCx 8.96 
Index of urea excretion (1) = TT — 
Wt. x Ur2 
D = Grams of urea excreted per twenty-four hours (calcu- 
lated from the above two hour period). 
C = Grams of urea per liter of urine. 
Ur == Grams of urea per liter of blood. 
Wt = Body weight of individual in kilograms. 
Remarks.—Usually the urea index (1) is 100 to 200. Variations 
between 80 and 300 are not infrequently observed in normal in- 
dividuals. An index below 50 indicates a considerable degree 
of impairment of renal function. 
Phenolsulphonephthalein Test for Kidney Function 
Principle.—A dyestuff, phenolsulphonephthalein, which is 
eliminated rapidly by the kidneys, is injected. The urine is col- 
lected over a fixed period of time and the amount of dyestuff it 
contains determined colorimetrically. 
Procedure.—A few minutes before the test is begun the patient 
is given a glass of water to insure a free flow of urine. The 
patient then empties the bladder and is given intravenously, or 
intramuscularly in the deltoid or lumbar muscles, 1 c.c. of sterile 
phenosulphonephthalein solution, containing 6 mg. of the drug. 
The patient voids exactly one hour and ten minutes later and 
again two hours and ten minutes from the time of injection. The 
two specimens are kept separate. 
Determination of Amount of Phthalein Excreted.—Each speci- 
men is measured, rendered alkaline with 40 per cent sodium 
(Rowntree and Geraghty) QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
71 
hydroxide solution and then diluted to 1,000 c.c. with water. The 
amount of dye in the solution so diluted is determined in a Dun- 
ning colorimeter (Fig. 24) by comparison with tubes of known 
concentration, or in a Hellige colorimeter (Fig. 34) using a 
standard solution containing 6 mg. of phthalein per liter. 
If a Hellige colorimeter is used it should he calibrated as de- 
scribed on page 98. 
Remarks.—The normal excretion of phthalein after intramus- 
Fig. 24.—Dunning colorimeter used in phenolsulphonephthalein test of kidney function, 
Fig. 25.—Record syringe for injecting phenolsulphonephthalein solution. 
collar injection is 50 to 70 per cent in two hours; after intra- 
venous injection 60 to 80 per cent is excreted in two hours. 
The results after intramuscular injection are unreliable if 
there he present any condition which will interfere with absorp- 
tion, such as edema. 
It is very necessary that the phthalein injected be accurately 
measured. The injection is best made with a calibrated 2 c.c. 
Record syringe (Fig. 25). 
The following form is convenient in keeping the record of 
the test: 72 
CLINICAL LABORATORY METHODS 
Phenolsulphonephthalein Test 
Be sure the patient empties the bladder. 
Be sure to save the entire amount of urine voided. 
Patient’s Name Case No 
Date 
Bladder emptied at 
Amount of urine obtained c.c. 
1 c.c. phenolsulphonephthalein. injected (intravenously) 
(into buttocks), at 
Note: Amount injected must be accurately measured) 
1 glass of water taken at 
(This should follow the injection immediately) 
Injected by 
1st collection of urine at empty bladder. 
Amount of urine collected c.c. 
(This collection should be made 1 hour and 10 minutes after 
injection of phenolsulphonephthalein.) 
2nd glass of water taken at 
(This should follow first collection) 
2nd collection of urine at empty bladder 
Amount of urine collected c.c. 
KEEP SPECIMENS SEPARATE 
Urine lost 
(Note amount, when, how lost, etc.) 
Collected by 
Alkali Tolerance Test 
(Peabody: Arch. Int. Med., 1915, xvi, 958. Palmer and Van 
Slyke:- Jour. Biol. Chem., 1917, xxxii, 499) 
Principle.—Sodium bicarbonate is administered by moutli in 
small amounts until the reaction of the urine reaches that of 
the blood (PH = 7.4). 
Procedure.—Give two grams of sodium bicarbonate in 100 c.c. 
water every half hour, and at the same time collect a specimen 
of urine. Record the number of grams of sodium bicarbonate 
taken before the urine (examined fresh) shows a H-ion concen- 
tration of 7.4 (page 36). 
Remarks.—The high normal limit of sodium bicarbonate is 10 
grams. If no acidosis is indicated by the test, its absence can 
be accepted; but if acidosis is indicated, the finding must be con- 
firmed by blood analysis as there may be an alkali retention due 
to the inability of the kidney to excrete alkali. 
Instead of determining the liydrogen-ion concentration, litmus 
paper may be used to roughly indicate the reaction of the urine. QUANTITATIVE CHEMICAL EXAMINATION OF URINE 
73 
Determination of Urobilin and Urobilinogen 
(Wilbur, R. L., and Addis, Thos.: Arch. Int. Med. 1913, xiii, 235) 
Collection of Specimen.—Collect total urine for 24 hours in 
dark brown bottle and keep in darkness. Add a few thymol crystals 
as a preservative. 
Procedure.—Measure urine. Mix 10 c.c. of urine with 10 c.c. 
of absolute alcohol and one gram of zinc acetate and filter. Ten 
c.c. of the filtrate are taken and 1 c.c. of Ehrlich’s solution, 
(paradi-inethyl-amido-benzaldehyd, 20 grams; concentrated hy- 
drochloric acid, 150 c.c.; water 150 c.c.), is added. Keep in dark 
for one hour. Transfer filtrate to a spectroscope cell 1 cm. in 
Fig. 26-A.—Spectrum of urobilin and urobilinogen. 
thickness. Examine the solution with the spectroscope for the 
characteristic absorption bands of urobilin and urobilinogen. 
The presence of urobilin is marked by a broad band in the 
blue end of the spectrum. The violet rays are completely ab- 
sorbed, and if there is much urobilin present nearly all of the 
green may be obliterated. Urobilinogen absorbs a narrow por- 
tion of the spectrum in the yellow at the edge of the green (Pig. 
26-A), and if present in large amount the band may be broad 
enough to obliterate the entire yellow portion of the spectrum. 
It is located by its proximity to the “D” line while urobilin 
extends from between “b” and “F” lines to the violet end of 
the visible spectrum. 74 
CLINICAL LABORATORY METHODS 
Dilute the solution with tap water until the absorption dis- 
appears with full light but can be made out faintly when the 
spectroscope slit is narrowed to just half of its former opening. 
The urobilin and urobilinogen bands differ in their intensity, 
consequently the disappearance of the absorption bands will 
occur with different dilutions. 
Calculation.—The number of dilutions required to obliterate 
the urobilinogen band plus the number required to obliterate 
that of urobilin gives the dilution value for 5 c.c. of urine. 
Multiply the sum of these two by the number of 5 c.c. quantities 
in the 24 hour time. This gives the number of dilutions which 
would have been necessary if all the urobilin and urobilinogen 
had been concentrated in a volume of 5 c.c. of urine. 
Remarks.—With this method a positive result in the 24 hour 
specimens of urine indicates an abnormal increase over the 
amount usually present. 
Instead of diluting the specimen until the bands disappear, 
a Hellige colorimeter with spectroscope attachment, may be em- 
ployed. (Boyd, J. D., Jour. Lab. and Clin. Med., 1919. iv. 495.) CHAPTER III 
ANALYSIS OF GASTRIC JUICE 
The Test Meal 
The patient is given a glass of water, and two arrowroot bis- 
cuits or a slice of bread. A small stomach tube is passed and 
specimens of the gastric contents removed by aspiration at thirty 
minute intervals over a period of two hours. 
Routine Examination 
1. Note the number of minutes which elapse between the time 
of ingestion of the test meal and the removal of the specimen. 
2. Measure each specimen. 
3. Note the gross appearance. Look for blood, mucus, bile, 
and retained food such as raisins. 
4. Determine the amount of free and total acid, or acid deficit 
on each specimen, and do guaiac test on pooled specimens. 
5. Examine microscopically the sediment of the pooled speci- 
mens. 
Chemical Examination of the Gastric Contents 
A. Reagents.— 
1. Gunzburg's Reagent.— 
Phloroglucin 2.0 gm. 
Vanillin 1.0 gm. 
Alcohol, absolute 30.0 c.c. 
Dissolve and keep in a brown bottle. The reagent deteriorates 
in a few months. 
2. Topfer's Reagent.—Dissolve 0.5 gm. of dimethylaminoazo- 
benzol in 100 c.c. of 95 per cent alcohol. 
B. Qualitative Test for Free Hydrochloric Acid.—A few drops 
of Gunzburg’s reagent are evaporated to dryness by warming 
gently over a Bnnsen burner. A drop of gastric contents is 
75 76 
clinical laboratory methods 
brought in contact with the yellowisli-brown stain left and is 
evaporated. If free hydrochloric acid is present, an intense red 
color develops where the reagent and gastric juice have mixed. 
Organic acids do not give the test. 
C. Titration of Free Hydrochloric Acid.—Add one drop of 
Topfer’s reagent to 10 c.c. of clear gastric juice placed in a porce- 
lain dish. In the presence of free hydrochloric acid an intense red 
color develops. Run in N/10 sodium hydroxide until the red 
color changes to a pure yellow. 
Calculation.—The number of cubic centimeters of tenth nor- 
mal alkali required to neutralize the acid multiplied by 10 gives 
the “acidity per cent” of free hydrochloric acid. 
D. Titration of Total Acidity.—Add one drop of phenolphtha- 
lein to 10 c.c. of clear gastric contents. Run in N/10 sodium 
hydroxide until the whole mixture takes on a permanent faint 
pink color. 
Calculation.—The number of cubic centimeters of alkali used, 
multiplied by 10, gives the total “acidity per cent.” 
If desired the phenolphthalein may be added to the specimen 
on which the free hydrochloric acid determination was made, 
and N/10 alkali be added until the pink color develops. The 
amount of sodium hydroxide used plus that required for the 
neutralization of the free hydrochloric acid multiplied by 10 
gives the total “acidity per cent.” 
E. Determination of Hydrochloric Acid Deficit.—If the free 
hydrochloric acid is absent as shown by the test with Gunzburg’s 
reagent, determine the amount of hydrochloric acid it is neces- 
sary to add until a reaction for free acid is obtained. 
Add N/10 hydrochloric acid to 10 c.c. of clear gastric con- 
tents until free hydrochloric acid is present as shown by testing 
with Congo-red paper. In the presence of free hydrochloric acid 
the paper turns blue when a small drop of the gastric juice is 
placed on it. 
Calculation.—The number of cubic centimeters of N/10 HC1 
used multiplied by 10 gives the “deficit per cent.” 
F. Qualitative Test for Lactic Acid.—To 5 to 10 c.c. of gastric 
juice in a test tube, add a few drops of dilute hydrochloric acid, 
and about 10 c.c. of ether. Shake carefully and decant ether into ANALYSIS OF GASTRIC JUICE 
77 
another test tube. Add to the ether a small amount of 0.2 per 
cent ferric chloride solution. A yellow color develops in the 
presence of lactic acid. 
G. Guaiac Test for Occult Blood.—Make 5 c.c. of stomach juice 
strongly acid with acetic acid. Layer with a mixture of sat- 
urated alcoholic solution of gum guaiac and 3 per cent hydrogen 
peroxide. A blue color develops at the line of contact in the 
presence of blood. 
In a fractional gastric analysis the guaiac test is done on a 
sample from the pooled specimen. 
Microscopic Examination of the Gastric Contents 
Place a drop of the sediment on a slide, cover with a cover 
glass and examine for: 
(a) Starch granules. These are normally present in moderate 
numbers. They turn blue on the addition of an iodine solution. 
(b) Red blood cells. 
(c) Pus cells. 
(d) Yeast cells, which are oval and may be distinguished by 
the budding which some cells show. 
(e) Sarcinae, which may be recognized by the typical bundle 
arrangement of the cocci. 
(f) Opper-Boas bacilli. These are large, long, gram-positive 
rods occurring in chains. They are found only in the absence of 
free hydrochloric acid. 
In a fractional gastric analysis the microscopic examination 
is made on the sediment from the pooled specimens. 
Determination of Urobilin and Urobilinogen in Duodenal 
Contents 
(Giffen, Sandford and Szlapka: Am. Jour. Med. Sc., 1918, 
civ, 562) 
Collection of Specimen.—Duodenal contents are collected by 
means of an Einhorn duodenal tube. The fluid should be faintly 
alkaline, clear and light yellow to chocolate brown in color. 
Reagents.— 
(1) Schlesinger's Solution—A saturated solution of zinc ace- 
tate in absolute alcohol. 78 
CLINICAL LABORATORY METHODS 
(2) Ehrlich’s Reagent.— 
Paradimethylaminobenzaldehyde, 4 gms. 
Hydrochloric acid 30 c.c. 
Distilled water 30 c.c. 
Procedure.—To 10 c.c. of duodenal contents add an equal 
amount of Schlesinger’s solution. Shake thoroughly. Filter 
through a single layer of coarse filter paper. To 10 c.c. of the 
filtrate add 1 c.c. of Ehrlich’s reagent. Set in dark for fifteen 
minutes. 
Fill the solution into a spectrum cell with parallel sides of 
such dimensions that the path of the rays of light in passing 
through the fluid are exactly 1 cm. Examine in spectroscope. 
The presence of urobilin is marked by a broad band in the 
blue end of the spectrum (Fig. 26-A). The violet rays are com- 
pletely absorbed, and if there is much urobilin present the entire 
blue portion and nearly all the green may be obliterated. Uro- 
bilinogen absorbs a narrow portion of the spectrum in the yellow 
at the edge of the green, and if present in large amounts the 
band may be broad enough to obliterate the entire yellow por- 
tion of the spectrum. It is located by its proximity to the “D” 
Fraunhofer line while urobilin extends from between the “b” 
and “F” lines to the violet end of the visible spectrum. 
Dilute the solution with 60 per cent alcohol until the absorp- 
tion bands disappear but can be made to reappear faintly when 
the slit is narrowed to just half its former opening. The uro- 
bilin and urobilinogen bands will disappear with different 
dilutions. 
The amount of urobilin and urobilinogen is estimated according 
to the Wilbur and Addis method for 1,000 c.c. by multiplying 
the number of dilutions by 200. The number of units of urobilin 
and urobilinogen are added together and the total number of 
units reported. 
Remarks.—The duodenal contents normally contain 500 to 
1000 units of urobilin and urobilinogen. CHAPTER IV 
EXAMINATION OF SPUTUM 
Collection of Specimen.—The patient should be directed to 
cough up sputum from the deeper bronchi, and expectorate this 
into a sterile container. Specimens can usually be best obtained 
on awakening in the morning. Saliva and pharyngeal material 
are worthless for examination. 
Routine Examination 
1. Note gross appearance: (a) mucoid; (b) mucopurulent; 
(c) purulent; (d) serous; (e) bloody. 
2. Examine preparation of fresh sputum. 
3. Wash sputum and make culture on blood agar plate. (See 
page 203.) 
4. Make a smear from a bit of the washed sputum and stain 
by gram. 
5. Make film preparation and stain for tubercle bacilli. 
Transfer a portion of the fresh specimen of sputum to glass 
plate about the size of a microscope stage, and cover with a 
second smaller glass plate. Examine under microscope with the 
low power objective. 
Note in the preparation: 
(a) Elastic fibers, which are usually in the small greyish yel- 
low particles. They are very refractive and have curling ends. 
(b) Moulds. These may be recognized by the branching 
mycelium and the spores. 
(c) Pus cells. 
(d) Eosinophilic leucocytes. These are distinguished by the 
large granules in the protoplasm which stain red with eosin. 
(e) Alveolar epithelial cells, which are 4 to 5 times the size 
of a leucocyte, are oval, their protoplasm coarsely granular, and 
Examination of Fresh Sputum 
79 80 
CLINICAL LABORATORY METHODS 
with one or several large oval, vesicular nuclei. They may con- 
tain large brown granules of blood pigment. 
(f) Charcot-Leyden crystals. These are long, narrow diamond 
shaped crystals which resemble two very sharp pyramids with 
their bases together. They have a slight yellowish refractivity. 
(g) Curschman’s spirals, which are spirally twisted strands of 
mucus enclosing many pus cells and Charcot-Leyden crystals. 
Examination for Tubercle Bacilli 
1. Ziehl-Neelsen Method.—Pour the sputum into a Petri dish 
and select for examination the yellowish particles of pus. Spread a 
small particle in a thin film on a clean glass slide. Dry in air, 
and fix by passing through flame. Cover with carbolfuchsin (see 
page 196) and steam on hot plate for two minutes. Wash under 
tap. Decolorize in acid alcohol (2 per cent hydrochloric acid in 
80 per cent alcohol) until the thicker parts show only a faint pink 
color. Counterstain with Loeffler’s methylene blue. (See page 
196.) 
2. Antiformin Method.—Put total specimen of sputum in 50 
c.c. centrifuge tube, dilute with distilled water, and add anti- 
formin until the preparation contains 20 per cent. Stir well, 
warm over flame, and leave in incubator for one hour. Dilute 
well with distilled water. Centrifuge for thirty minutes. Mix 
sediment with one drop of blood serum, make slide preparation 
and stain as directed above. 
Examination for Elastic Tissue 
Boil the sputum with 20 per cent NaOII solution until it is 
homogeneous, centrifugalize, and examine the sediment pressed 
out on a glass plate as directed in the examination of fresh 
sputum. 
The fibers of elastic tissue are characterized by their intense 
refractivity, wavy outline, sharp edges, uniform diameter and 
curling ends. CHAPTER V 
EXAMINATION OF FECES 
Routine Examination 
1. Note gross appearance; color, presence of blood, or mucus, 
or remnants of food. 
2. Test for occult blood with benzidine. 
3. Make a microscopic examination of a portion of stool emul- 
sified in water. 
Qualitative Tests 
1. Occult Blood 
(a) Benzidine Test.—Take up a bit of powdered benzidine on 
the tip of a toothpick and dissolve in 2 c.c. of glacial acetic acid. 
Add 20 drops of 3 per cent hydrogen peroxide. 
Smear a small portion of the stool on a white card with a 
toothpick; add a drop of the benzidine suspension and mix thor- 
oughly. If blood is present the mixture turns blue. 
Au atypical or doubtful reaction should be checked up with 
the guaiac test. 
(b) Guaiac Test.—Emulsify a portion of the specimen of stool 
in a test tube with glacial acetic acid. Extract with a few cubic 
centimeters of ether. Layer the ether extract in a test tube on a 
mixture of equal parts alcoholic solution of gum guaiac (1 gram 
in 60 c.c. 95 per cent alcohol) and 3 per cent hydrogen peroxide. 
A blue color develops in the ether if blood is present. 
2. Bilirubin" and IIydrobilirijbin 
Schmidt Test.—Emulsify a bit of stool in a porcelain dish with 
a saturated solution of mercuric chloride. Allow to stand for 
24 hours. If hydrobilirubin is present the specimen becomes 
pink. Bilirubin causes a green color. 
81 82 
CLINICAL LABORATORY METHODS 
Examination for Large Parasites, Gallstones, and Other 
Foreign Bodies 
Mix the specimen of stool with a large amount of water, stir 
well, and filter through a fine mesh wire sieve. 
Microscopic Examination 
Place a drop of water on a slide and mix with it bits of the 
stool from different portions of the specimen. Cover with a cover 
Taenia saginata 
Hymenolepis nana 
Ilymonolepls diminuta 
Triehiurns trlchiurn 
Dlbothrioeephalus latus 
Oxyuris vermicnlnris 
Necator americanus 
Ascnri* iumbricoidos 
(unfcltilizrd) 
Asiaris itMubrieoidos 
(containing embryo) 
Fig. 26-B.—Ova in human feces. (After Barker.) EXAMINATION OP FECES 
83 
An operculum: Ova brown 75x45 Dibothricephalus latus 
No operculum 
(Abbreviated from Medical War Manual No. 6.) 
Ova with a 
single envel- 
ope 
Three transparent membranes 68 Hymenolepis nana 
A single membrane 
Classification of Ova of the Commoner Intestinal Parasites 
Wall ornamented . 
Wall 
smooth 
II. Classification of Ova of Nemathelminthes 
Transparent 
Thick 
Thick and transparent 30-40 Dipylidium caninum 
Thick and opaque 
I. Classification of Ova of Cestodes 
Table X 
Mamillated, brown 
Regularly cribbed with 
depressions, yellow 
2 to 4 blastomeres 
Thick embryo, folded 
in two 
2 to 4 blastomeres 
Bulging on one side, 
flat on the other 
Clear plug at each pole 
Ova spherical 
Ova ovoid 
50x23 Oxyuris vermicularis 
55x25 Trichocephalus 
trichiuris 
60x40 Ankylostomum 
duodenale 
70x40 Necator americanus 
54x32 Strongyloides 
intestinalis 
60x44 Ascaris lumbricoides 
75x65 Ascaris canis 
21-56 Tenia solium 
35x25 Tenia saginata 
Dimensions 
m M. 84 
CLINICAL LABORATORY METHODS 
glass and examine under the lower power of the microscope for 
(1) Pus cells; (2) Red blood cells; (3) An excess of undigested 
muscle fibers; (4) Parasites; (5) Ova. 
The ova in the stool, if present, may be concentrated by the 
following procedure (Kofoid and Barber): Emulsify the speci- 
mens with a saturated salt solution. Make a filter % to % inch 
in thickness of No. 0 or No. 1 long fiber steel wool and pour 
the emulsion through it. 
Allow the preparation to stand one hour. Loop off the sur- 
face, film to an ordinary gloss, slide and examine under the low 
power of the microscope. If amebae are suspected, examine as 
indicated under “Examination of Stool for Amebae.” 
Table N summarizes the characteristics of the ova of the com- 
moner intestinal parasites. The ova commonly found in the 
stool are illustrated by Fig. 26B. 
Determination of Reaction 
(Bruce, W. J.: Jour. Lab. and Clin. Med., 1920, v, 61) 
Prepare a 1 per cent aqueous solution of alizarin. Place two 
small drops of the indicator on a white index card ll/> inches 
apart. Dip a toothpick into the liquid part of the specimen (or 
if the feces are formed, merely puncture the mass). Mix thor- 
oughly in one of the drops, using the other drop as a control. 
An alkaline reaction is indicated by a reddish violet color, 
neutral, no change, an acid a light yellow color. The density of 
the colors will depend upon the amount of acid or alkali present. 
Examination of Stool for Amebae 
(From “Parasitic Amebae of Man,” Craig) 
A very small portion of a freshly passed stool should be placed 
upon a microscopic slide and covered with a cover glass, gentle 
pressure being used to spread the specimen. The material 
selected for examination should preferably be a drop of the liquid 
portion of the stool rather than solid particles. It is always well 
to give a saline cathartic before making an examination as this 
tends to wrash the amebae from the intestinal walls. If present, 
a particle of mucus or any blood-stained material should be ex- EXAMINATION OF FECES 
85 
amined as well. Most stools from amebic dysentery cases con- 
tain gelatinous material which frequently contains numerous 
amebae, and such material should always be fully examined. 
The feces should be examined as quickly as possible after 
they have been passed as the amebae are much more easily recog- 
nized when they are motile. No disinfectant should be used in 
stools which are to be examined for amebae, neither should urine 
be mixed with the stools. In temperate regions, especially in 
the winter, the receptacle used in collecting the specimen should 
be warmed, but care should be taken if water is used for this 
purpose, that it be boiled, as otherwise water amebae might be 
mistaken for parasitic amebae, having reached the feces in this 
manner. 
In making fresh preparations it is always well to dilute a loop- 
ful of the stool with normal salt solution or distilled water. One 
of the most frequent mistakes made in examining such prepara- 
tions is the use of too thick a preparation. One should not be 
satisfied with the examination of a single slide, but should thor- 
oughly examine at least six or eight preparations before a nega- 
tive result is reported. 
The hanging drop method is often valuable in the examination 
of these organisms, a small drop of the stool being placed in 
the center of a cover glass which is then inverted upon a hollow 
ground slide and ringed with vaseline. If a film preparation is 
used it should always be ringed with vaseline, for unless this is 
done evaporation occurs and the preparation becomes useless. 
The preparation should be examined with a one-sixth inch lens 
and a one- or two-inch eyepiece. For the finer details regarding 
the structure of the cytoplasm and the nucleus, as well as the 
reproductive changes, it is necessary to use the one-twelfth inch 
oil immersion objective. Table XI summarizes the important 
differential features of Entameba Coli and Entameba Histolytica. 
Neutral Red.—The use of a solution of %o,ooo of neutral red 
is often of great service in those cases in which the amebae 
are few in number and for the study of structural details. This 
solution is very quickly absorbed by the amebae, coloring them 
pink or red, and does not interfere with their movements if it is 
not used in too strong a dilution. It is a most useful method in 86 
CLINICAL LABORATORY METHODS 
Differential Features of Entameba Coli and Entameba Histolytica 
Table XI 
ENTAMEBA COLI-SCHAUDINN, 
1903 
ENTAMEBA HISTOLYTICA- 
SCHAUDINN, 1903 
size : 
15 to 20 microns. Average 
diam. 25-35 microns. Generally 
smaller than entameba histo- 
lytica. 
10 to 70 microns. Generally 
from 15 to 40 microns. 
PSEUDOPODIA: 
Small, blunt and not clearly 
differentiated from rest of par- 
asite. 
Blunt or slender and finger- 
shaped. Very refractive and 
clearly differentiated from rest 
of the parasite. 
MOTILITY: 
Sluggish, less progressive and 
more indefinite in direction as 
compared with entameba his- 
tolytica. 
Active. 
CYTOPLASM: 
Ectoplasm not distinct except 
when moving and then only be- 
cause it is free from granules. 
Is grayish in color and not 
very refractive. Endoplasm is 
gray, finely granular, few non- 
contractile vacuoles. Is not 
generally phagocytic for red 
blood corpuscles but may con- 
tain bacteria and crystals. 
Ectoplasm is very distinct and 
refractive, in some instances 
even when motionless. Glassy 
appearing. Endoplasm is gran- 
ular, contains numerous non- 
eontractile vacuoles and red 
blood corpuscles when latter 
are present in the feces. 
NUCLEUS: 
Distinct, having a well defined 
nuclear membrane, much chro- 
matin and large karyosome. 
In acute dysentery the nucleus 
is usually more or less indis- 
tinct; has no well defined nu- 
clear membrane; contains but 
little chromatin and has a 
minute karyosome. In chronie 
dysentery the nucleus is dis- 
tinct, has thick nuclear mem- 
brane formed by chromatin, 
large karyosome and clear area 
surrounding the ventricle. 
CYST 
FORMATION: 
Present. Cysts have eight nu- 
clei, are 16-25 microns in diam., 
have thick wall, no chromidial 
bodies and are more refractive 
than those of entameba histo- 
lytica. 
Present. Cysts contain four 
nuclei. Measure from 11-14 
microns, are covered with a 
thin membrane, are less refrac- 
tive than those of entameba 
coli and contain chromidial 
bodies. Four amebae develop 
within cyst. 87 
EXAMINATION OF FECES 
Differential Features of Entameba Coli and Entameba Histolytica 
Table XI (Continued) 
ENTAMEBA COLI-SCHAUDINN, 
1903 
ENTAMEBA HISTOLYTICA- 
SCHAUDINN, 1903 
CULTIVATION: 
Doubtful. 
Negative. 
METHODS OF 
REPRODUC- 
TION : 
By simple division, autogam- 
ous sexual reproduction in cyst 
and by schizogony with the 
production of eight daughter 
amebae. Eight amebae are 
produced within the cyst. 
By simple division and by 
autogamous sexual reproduc- 
tion within cyst, four amebae 
being formed. 
PATHO- 
GENESIS : 
Is not pathogenic, occurring in 
a large percentage of healthy 
individuals and in patients 
suffering from diseases other 
than dysentery. 
Is the cause of a form 
amebic dysentery. 
of 
distinguishing between parasitic amebae and leucocytes, as the 
latter do not stain with this substance. In specimens in which 
the amebae are in scant numbers they are easily distinguished by 
the reddish color given them by the neutral red, as other cells 
occurring in feces are not colored distinctly by this dye. The 
dilution should be made with normal salt solution. 
Quantitative Estimation of Urobilin and Urobilinogen in Stools 
(Wilbur, R. L., and Addis, Thos.: Arch. Int. Med., 1914, 
xiii, 235) 
Collection of Specimen.—All the feces passed in 24 hours are 
collected in the same receiver and kept in the dark. 
Procedure.—The total stool is then washed into a large grad- 
uate and thoroughly ground up with water into a homogeneous 
paste and water added to 0.5, 1 or 2 liters, depending upon the 
size of the stool. 
After thoroughly mixing, 25 c.c. are taken and 75 c.c. of acid 
alcohol (95 per cent alcohol, 1600 c.c., concentrated hydrochloric 
acid, 25 c.c. and water 800 c.c.) are added. Put mixture in a 
shaker for about one half an hour. To 10 c.c. add an equal quan- 
tity of absolute alcohol and 1 gram of zinc acetate. Filter. Add 
1 c.c. of Ehrlich’s reagent (page 19) to 10 c.c. of the filtrate and 
put aside in a dark place until the next day. Transfer to a 88 
CLINICAL LABORATORY METHODS 
spectroscope cell 1 cm. in thickness and examine with spectro- 
scope for the characteristic absorption bands of urobilin and 
urobilinogen. 
The presence of urobilin is marked by a broad band (Fig. 
26-A) in the blue end of the spectrum. The violet rays are com- 
pletely absorbed, and if there is much urobilin present the entire 
blue portion and nearly all of the green may be obliterated. Uro- 
bilinogen absorbs a narrow portion of the spectrum in the yellow 
at the edge of the green, and if present in large amounts the 
band may be broad enough to obliterate the entire yellow por- 
tion of the spectrum. It is located by its proximity to the “D” 
line while urobilin extends from between “b” and “F” lines 
to the violet end of the visible spectrum. 
Dilute with 60 per cent alcohol until the absorption bands dis- 
appear with dull light but reappear when the spectroscope slit 
is narrowed to one just half of its former opening. 
The urobilin and urobilinogen bands differ in their intensity, 
consequently the disappearance of the absorption bands will occur 
with different dilutions. 
Calculation—The total amount of urobilin and urobilinogen 
in terms of dilutions equals: 
—^— x 8 x (E plus S) 
where 
V equals the volume to which the stool was diluted; 
R equals dilutions required to obliterate urobilin band in the 
10 c.c. of filtrate, and 
S equals dilutions required to obliterate urobilinogen band. 
Remarks.—Normally the total 24-hour specimen of stool does 
not contain more than 6500 dilutions of urobilin and urobilinogen. 
Preservation of Intestinal Parasites 
Wash carefully in salt solution and place in 2 per cent formalin. 
If it is desired to clear the specimen after fixation in 2 per 
cent formalin for 14 to 16 hours, it is then placed in the following 
solution until clear: EXAMINATION OF FECES 
89 
Glucose 480 grams 
Water 520 c.c. 
Methyl alcohol 200 c.c. 
Glycerine 100 c.c. 
Camphor (q.s. to keep) 
After the specimen is cleared sufficient quantity of glycerine 
jelly is dropped on a slide and the specimen transferred to it. 
Cover with a cover glass and allow the jelly to harden. 
The glycerine jelly is made by melting 14 grams of the best 
Gold Mark gelatine in 120 c.c. of hot water and adding 120 c.c. 
glycerine. This is then cooled to 50° C. 
The carefully separated whites of two eggs are then added and 
the fluid heated gently without stirring. This is filtered, the 
volume made up by adding water to 240 c.c. and 1 c.c. of 
pure carbolic acid is added. This jelly is solid at ordinary tem- 
perature but is easily melted under the hot water tap. 
Preservation of Stools Containing’ Ova 
The stools are diluted to a soup-like consistency, and one-tenth 
volume of formalin is added. CHAPTER VI 
QUALITATIVE EXAMINATION OF BLOOD 
Counting the Blood Cells 
I. Reagents.—(1) Hayem’s Solution.— 
Mercuric chloride 0.25 gram 
Sodium chloride 0.5 gram 
Sodium sulphate 2.5 grams 
Distilled water 100 c.c. 
(2) Turck’s Solution.—One and one-half per cent acetic acid. 
Color with gentian violet. 
(3') Blood Platelet Solutions: 
Solution I: 
“Brilliant cresyl blue” 1.0 gram 
Distilled water 300.0 c.c. 
Dissolve. Keep on ice to prevent the growth of yeast. 
Solution II: 
Potassium cyanide 1.0 gram 
Distilled water 1400.0 c.c. 
This solution should be made up every ten days. 
II. The Diluting Pipettes.—Each pipette (Fig. 27) consists of 
a capillary tube which opens into a bulb containing a glass pearl. 
Fig. 27.—Blood counting pipettes for red and white corpuscles. 
The capillary tube is divided into ten equal parts. In the red 
pipette the bulb when filled to the line on its upper outlet (marked 
90 QUALITATIVE EXAMINATION OF BLOOD 
91 
101) holds one hundred times the contents of the ten divisions of 
the capillary tube. 
The bulb of the white pipette contains ten times that of the 
capillary tube. 
III. The Counting Chamber.—A counting chamber (Fig. 28) of 
the Burker type with double Neubauer ruling is used. On this 
instrument there are two ruled areas three millimeters on a side 
or nine millimeters square. Each area (Fig. 29) is divided into 
nine large squares, each of which is one millimeter on a side 
or one square millimeter. The central square millimeter which 
is used for counting the red blood cells is subdivided into 400 
,Fig. 28.—A.—Hausser counting chamber of Burker type with double Neubauer ruling. 
The glass parts are in one piece which is mounted in a Bakelite holder. This is by far 
the most desirable type of instrument to buy. 
B.—Bevy counting chamber of Burker type with Neubauer ruling. The ruled area 
is cemented on to the slide which is made of glass. 
small squares, each of which is x/2o millimeter on a side, and 
has therefore an area of %0o square millimeter. By means of 
double lines these smallest squares are grouped into blocks of 
twenty-five. Each of the remaining eight large squares (1 square 
millimeter each) is subdivided into sixteen small squares. The 
depth of the chamber is 0.1 millimeter. 
Accurately calibrated pipettes and counting chamber are ab- 
solutely necessary if satisfactory blood counting is to be done. 92 
CLINICAL LABORATORY METHODS 
It is best to use only apparatus certified as correct by the Bureau 
of Standards. 
Fig. 29.—Showing the Neubauer rilling of counting chamber as it appears under the 
microscope. A, unit used in counting white corpuscles; B, unit used in counting red 
corpuscles and platelets. 
Procedure in Counting’ the Red Blood Cells 
The finger or ear is punctured with a blood lancet or with 
a Ilagedorn needle. The blood should flow freely. The first 
drop is wiped away and the second used. The blood is sucked 
cautiously into the capillary tube of the pipette to the line 
marked 0.5. If the blood is accidentally sucked above the line, 
it may be lowered by drawing the finger across the tip of the 
pipette, provided the column of blood has not passed more than 
1 millimeter above the line; if it has extended farther, the blood 
adhering to the wall of the tube will be sufficient to introduce 
a serious error in dilution. The end of the pipette is wiped free 93 
QUALITATIVE EXAMINATION OF BLOOD 
of blood. Before drawing in the diluting fluid care should be 
taken not to expel any of the blood in the capillary tube. 
The pipette is now filled to the line marked 101 with Hayem’s 
fluid. While the dilution fluid is being drawn up, the pipette, 
held between the thumb and fingers, is revolved to keep the 
glass pearl within the bulb in motion. This mixes the blood and 
diluting fluid and also prevents bubbles adhering to the pearl. 
Place pipette in shaking machine (Fig. 30) and shake for three 
minutes, or shake by hand for three minutes. 
Fig. 30.—Machine for shaking blood counting pipettes. 
Clean cover glass and counting chamber carefully. Discard 
the first drop of fluid in the pipette. 
With the cover glass in position, the space between cover 
glass and ruled area is filled by capillary attraction, when the 
tip of the pipette touches the rectangular ruled strip at the edge 
of the cover glass. 
Allow the cells to settle in the chamber. Count the cells in 
the four squares marked “B” on the diagram of the ruled area. 
(Fig. 29.) 
Cells touching the upper and right boundary line are consid- 94 
CLINICAL LABORATORY METHODS 
ered in the square, those touching the left and lower boundary 
as out of the square. The number of cells in each square should 
not vary more than 25 per cent of the average number per square. 
Calculation.—This area counted equals 100 small squares, 
equals 100 x -7~ x 0.1 equals 4-c.mm. 
400 40 
Fig. 31.—Tray for holding pipettes and materials for blood counting. 
The blood was diluted 200 times. Hence the number of cells 
in the 100 squares counted x 40 x 200 equals number of red blood 
cells in 1 c.c. of undiluted blood. 
Remarks.—The red blood cell count in normal men varies from 
4,500,000 to 5,500,000; in women from 4,000,000 to 5,000,000. QUALITATIVE EXAMINATION OF BLOOD 
95 
Procedure in Counting the Leukocytes 
Draw blood up to 0.5 line on white blood cell counting pipette 
and fill to the eleven mark with Turck’s solution. 
Make preparation in counting chamber as directed for red 
blood cells. Allow cells to settle. 
Count all white cells in four squares marked “A” on the 
diagram of ruled area. (Fig. 29.) 
Calculation.—Each square has a cubic content of 0.1 c.mm., 
since the side of the square is 1 mm. and the chamber is 0.1 mm. 
deep. 
The sum of leukocytes in the four large squares divided by 4 
gives the average number of cells in 0.1 c.mm. of diluted blood. 
To obtain the number of cells in 1 c.mm. of undiluted blood, this 
number is multiplied by 10 and by 20, or to put it simply, the 
total number of cells in the four squares counted, multiplied by 
50 gives the number of white blood cells per cubic millimeter 
of undiluted blood. 
Remarks.—The white blood cell count varies normally from 
6,000 to 8,000. A count over 10,000 is a leucocytosis, a count 
under 5,000 is a leucopenia. 
Procedure in Counting the Blood Platelets (Wright and 
Kinnicutt) 
Draw blood up to the 1 mark on a red blood cell pipette and 
fill to the 101 mark with diluting fluid. The diluting fluid is made 
by mixing two parts of blood platelet Solution I with three parts 
of Solution II and filtering. 
Shake the pipette well by hand or in the pipette shaking 
machine. Fill the counting chamber as for a red blood cell count 
and leave at rest for 10-15 minutes in order that the platelets 
may settle to the bottom. 
The platelets appear as sharply outlined, round or oval or 
elongated, lilac-colored bodies, some of which form a part of 
small spheres or globules of hyalin, unstained substance. The 
red cells are decolorized and appear only as shadows. The 
nuclei of the white cells are stained a dark blue, the protoplasm 
light blue. 96 
CLINICAL LABORATORY METHODS 
Count the platelets in 16 large squares marked “B” on dia- 
gram of ruled area, (Fig. 29) using the high power dry objective. 
Calculation.—The total number of platelets in the sixteen 
squares multiplied by 1000 gives the number per cubic millimeter 
of blood. 
Remarks.—The platelet count of normal adults varies between 
225,000 and 350,000 per cubic millimeter, the general average 
being about 300,000. 
Cleaning Counting’ Chamber and Pipettes 
The counting chamber is cleaned with soap and water, using 
a soft cloth. 
Fig. 32.—Apparatus used in cleaning blood counting pipettes. This is made from 3-way 
stopcocks soldered together. 
The pipette is first emptied and then water, alcohol, ether 
and air are aspirated through in the order named (Figs. 32 and 
33). If any albuminous material remains in the bulb, it may be 
removed by filling the pipette with the following alkaline pan- 
ereatin solution and leaving in the incubator overnight. QUALITATIVE EXAMINATION OF BLOOD 
97 
Sodium carbonate 5 grams 
Pancreatin 0.5 gram 
Water 1000 c.c. 
Chloroform a few drops 
Fig. 33.—Pipette cleaning apparatus in position for emptying pipettes. 
Determination of Hemoglobin 
Choice of Method.—The oxygen capacity method with the Van 
Slyke apparatus affords the most accurate means for the estima- 
tion of hemoglobin. This method should be used in all cases in 
which a very exact hemoglobin determination is of value. 
The acid hematin method of Sahli is probably the most valu- 
able for routine clinical use. Good results will be obtained if the 
pipette is correctly graduated, the standard and comparison 98 
CLINICAL LABORATORY METHODS 
tubes are of equal bore, and the standard solution is accurately 
made. 
The acid hematin method as adapted to the Ilellige colorimeter 
affords a procedure which is especially valuable in hospitals or 
wherever it is desirable to collect the specimen for the hemo- 
globin determination and make the reading in the laboratory. 
The blood is diluted and transported in an ordinary red blood 
counting pipette. 
In all the methods given the hemoglobin reading of a normal 
blood containing 5 million red cells per c.mm. is taken as 100 per 
cent. Such a blood contains 15.6 grams of hemoglobin per 100 c.c. 
Determination by the Oxygen Capacity Method 
The procedure for the determination of hemoglobin by this 
method is given in detail on page 171. 
Determination by a Method of Sahli 
Standard Solution.—A stock acid hematin solution is made as 
described below in the method adapted to the Ilellige colorimeter. 
A 1 per cent solution is made from the stock 10 per cent solution 
using equal parts of glycerine and N/10 hydrochloric acid as 
the diluent, and .filled into the standard tube. The standard 
tube as supplied with the instrument is seldom accurate. 
Procedure.—With a dropper run N/10 hydrochloric acid into 
the graduated tube to the mark 10. With the Sahli pipette draw 
up blood to the 20 c.mm. mark. Blow the blood into the acid 
in the graduated tube. The pipette is thoroughly cleaned of 
blood by sucking up and blowing out the acid several times. The 
hydrochloric acid will in a few minutes change the hemoglobin 
to acid hematin. It is then diluted with distilled water until 
its tint corresponds to that of the standard tube. The tubes 
should be compared with the light transmitted through the milk 
glass background. The hemoglobin in per cent is read off di- 
rectly from the graduation on the tube. 
Determination with the Hellige Colorimeter 
Calibration of the Hellige Colorimeter.—The accuracy of the 
readings with the Hellige colorimeter (Fig. 34) will depend upon QUALITATIVE EXAMINATION OF BLOOD 
99 
the care with which the instrument is calibrated. The calibra- 
tion is easily done as follows. With the same solution in both 
cup and wedge read the colorimeter scale when the color of the 
solution in the cup matches that of the wedge. This determines 
the 100 per cent mark when the unknown and standard are 
equally diluted. We may call this point “Y.” Now raise the 
wedge until the bottom is just above the lower level of the 
aperture through which the readings are made. This determines 
the point on the scale equivalent to 0 per cent. This scale read- 
ing may be designated “X.” Now at any point “R” on the scale 
Fig. 34.—Hellige colorimeter. A, Wedge for standard. B, Specimen cup. C, scale. 
F, Ground glass plate. I, D, Rack and pinion. K, Catch for holding ground glass plate. 
L, Back slide. M, Setscrew for holding wedge in position. N, Cup holder. O, Helm- 
holtz double plate. P, Pointer. Q, Front slide. 
at which the colors match, the percentage of a substance in solu- 
tion in the cup is equal to: 
rp 100 A 
(R'X) X (Y-X) X B 
Where R is the reading of the scale. 
A is the concentration of the standard in the wedge. 
B is the concentration of the substance determined. 
In making the hemoglobin estimation the blood is diluted 100 
CLINICAL LABORATORY METHODS 
1:100 to make a 1 per cent solution. It is convenient to so dilute 
the standard as to make the factor x equal a whole 
I -A x> 
number, preferably two. If X equals 5 and Y equals 85, and 
the blood in the standard be in the dilution of 1:62.5, or 1.6 
per cent then the above factor equals x or 2. 
o 0 l.U 
A flat piece of wood about the thickness of a tongue depressor 
should be fitted in the bottom of the cup holder. This raises the 
cup on a level with the bottom of the aperture, thus enabling 
one to make readings with the fluid in even the smallest red cell 
pipette. 
Reagents.— 
1. Tenth normal hydrochloric acid. This may be made suffi- 
ciently accurate by diluting 11.7 c.c. of cone. HC1 with distilled 
water to a volume of 1000 c.c. 
2. Acid hematin standard. This is prepared as follows : With- 
draw by venipuncture 50 c.c. of blood, carefully defibrinate by 
whipping and strain through gauze. Determine the hemoglobin 
of the defibrinated blood in the Van Slyke apparatus by the 
oxygen capacity method. Dilute the blood with N/10 HC1 to 
make a 20 per cent solution of a blood containing 15.6 grams of 
hemoglobin, or an oxygen capacity of 20.9 volumes per cent per 
100 c.c. of blood. Mix well and let stand for 24 hours. Add an 
equal volume of glycerine. Store in a glass stoppered bottle 
preferably in cool spot away from the light. This solution will 
keep for months. The solution represents a 10 per cent solution 
of a blood containing 15.6 grams of hemoglobin per 100 c.c. 
From this standard any desired dilution, usually 1.6 per cent, 
may be made, using equal parts of glycerine and N/10 HC1 as 
the diluting fluid. 
Procedure.—Draw blood accurately up to the 1 mark in a red 
cell counting pipette and fill to 101 with N/10 HC1. This makes 
a 1:100 dilution of the blood. 
Allow the preparation to stand for 10 minutes or more. At 
least 10 minutes is required for approximate complete conversion 
of the hemoglobin into acid hematin. There is very little change QUALITATIVE EXAMINATION OF BLOOD 
101 
after this time so the reading may be made 24, or even 48 hours 
after dilution. 
Blow the contents of the pipette into the cup of the colorimeter 
after discarding the first drop. 
Fig. 35.—Scale showing the average amount of hemoglobin in grams per 100 c.c. blood 
at different ages. (After Williamson.') 
Read the scale at the point at which the color of the unknown 
in the cup matches that of the wedge. 
Calculation.—If the blood in the standard is so diluted as to 
make the factor x equal 2, the hemoglobin corresponding 102 
CLINICAL LABORATORY METHODS 
to any reading “R” on the scale is 2(R-X) per cent or 
15 6 
2(R-X) x grams per 100 c.c. blood. 
Remarks.—The colorimeter cup must be kept clean by rubbing 
the inside surfaces Avith a small moist cotton swab. A small 
amount of acid hematin adhering to the sides will in time make 
a large error in the reading. 
The wedge holding the standard must be shaken daily to keep 
the acid hematin in suspension. 
It is convenient to figure a table, such as Table XII, for each 
colorimeter. The hemoglobin can then be read off directly in 
per cent or in grams per 100 c.c. blood from the scale reading. 
Figure 35 shows the hemoglobin values for different ages as 
given by Williamson. These results are obtained Avitli the spec- 
trophotometer and do not take into account the red cell count. 
They are higher than those obtained Avith the Van Slyke ap- 
paratus. 
Table XII 
The Table shows the hemoglobin in per cent and in grams per 100 c.c. 
of blood with Hellige colorimeter No. 2312. On this instrument a scale 
reading of 87 equals 100, and a reading of 7 equals 0. 
The blood is diluted 1:100, the standard is a 1.6 per cent solution or a 
dilution of 1:62.5 of a blood having a hemoglobin content of 15.6 grams per 
100 c.c. Hence the hemoglobin in per cent corresponding to any point “R” 
on the scale is: 
100 1.6 
X (R-7) x yy or 2(R-7) per cent 
15 6 
or 2(R-7) x y-h- grams per 100 c.c. blood. 
COLORIMETER READING 
PER CENT HEMOGLOBIN 
GRAMS HEMOGLOBIN 
PER 100 C.C. 
67 
120 
18.7 
66 
118 
18.4 
65 
116 
18.1 
64 
114 
17.8 
63 
112 
17.5 
62 
110 
17.2 
61 
108 
16.9 
60 
106 
16.5 
59 
104 
16.2 
58 
102 
15.9 
57 
100 
15.6 
56 
98 
15.3 QUALITATIVE EXAMINATION OF BLOOD 
103 
Table XII 
COLORIMETER READING 
PER CENT HEMOGLOBIN 
GRAMS HEMOGLOBIN 
PER 100 C.C. 
55 
96 
15.0 
54 
94 
14.7 
58 
92 
14.4 
52 
90 
14.1 
51 
88 
13.8 
50 
86 
13.4 
49 
84 
13.1 
48 
82 
12.8 
47 
80 
12.5 
46 
78 
12.2 
45 
76 
11.9 
44 
74 
11.6 
43 
72 
11.3 
42 
70 
11.0 
41 
68 
10.6 
40 
66 
10.3 
39 
64 
10.0 
38 
62 
9.7 
37 
60 
9.4 
36 
58 
9.1 
35 
56 
8.8 
34 
54 
8.4 
33 
52 
8.1 
32 
50 
7.8 
31 
48 
7.5 
30 
46 
7.2 
29 
44 
6.9 
28 
42 
6.6 
27 
40 
6.3 
26 
38 
5.9 
25 
36 
5.6 
24 
34 
5.3 
23 
32 
5.0 
22 
30 
4.7 
20 
28 
4.4 
18 
26 
4.1 
Color Index 
The color index is the quotient obtained by dividing the per- 
centage of hemoglobin by the percentage of red corpuscles, 5,000,- 
000 cells per cubic millimeter being considered as 100 per cent 
of corpuscles. It is calculated as follows: 
„ , T , T, per cent hemoglobin ... , 
Color Index (C I) = — divided by 
number of red cells per cent hemoglobin x 50,000 
5,000,000 number of red cells 104 
CLINICAL LABORATORY METHODS 
In normal blood the color index is about 1. A color index 
above 1 means that the cells are larger than normal. A color 
index below 1 means that the cells contain a smaller amount of 
hemoglobin or are smaller than normal. 
Volume Index 
The term “Volume Index” is an expression used to designate 
the volume of red cells relative to the normal. It is the quotient 
of the percentage volume of the erythrocytes divided by the 
percentage number of cells. It is a measure of the relative size 
of the red cells. 
To determine the volume index 10 c.c. of blood are withdrawn 
by venipuncture into a dry well vaselined syringe. The blood 
is run immediately into a 15 c.c. graduated hematocrit tube 
(Fig. 21) containing 2 c.c. of 1.6 per cent sodium oxalate and 
mixed by inversion. 
The tube is then centrifuged for 30 minutes at 2500 revolu- 
tions per minute. 
With normal blood and a red cell count of 5,000,000, the red 
cells comprise about 46 per cent. This value for a normal blood 
should be determined for each centrifuge however. The value 
so determined represents 100 per cent volume. The erythrocytes 
are counted at the same time that the volume determination is 
made. 
The volume index is equal the volume per cent divided by 
the number per cent of red cells with 5,000,000 corpuscles being 
considered 100 per cent. 
In normal blood the volume index is 1. An index below 1 
shows that the average size of the cells is less than 1 and index 
above 1 indicates that the average size of the cells is greater 
than normal. 
Saturation Index 
The color index expresses the relationship between the number 
of red blood cells and the amount of hemoglobin but does not 
give the true concentration of hemoglobin in the cells. The rela- 
tionship is a function of both the size of the red cell and the 
amount of hemoglobin contained therein. The actual percentage QUALITATIVE EXAMINATION OF BLOOD 
105 
of hemoglobin in the red cells is given by the “saturation index.” 
This is obtained by dividing the hemoglobin in per cent by the 
percentage volume of red cells. 
To calculate the saturation index blood is obtained as for a 
volume index determination, centrifuged, and the volume of red 
cells read off. The per cent of normal volume is determined. A 
hemoglobin estimation on the same specimen of blood is made. The 
hemoglobin in per cent divided by the percentage volume of red 
cells gives the saturation index. For example, 10 c.c. of blood are 
centrifuged and 2.3 c.c. or 23 per cent of packed cells is obtained. 
Normal blood with a red count of 5 million per c.mm. would give 
with the same centrifuge 4.6 c.c., or 46 per cent packed cells. The 
hemoglobin is 50 per cent. The saturation index is 50 divided by 
2.3 
4.6 or 100' 
Examination of Fresh Blood 
The exact size and shape of red corpuscles are best determined 
in preparations of fresh blood. 
The fresh preparations are made by taking a very small drop 
of blood on the center of a cover slip cleaned carefully as for 
a blood smear and laying it on a slide similarly cleaned. The 
blood will spread out leaving the red cells separate. Seal the 
edges of the cover slip to the slide with a small amount of 
vaseline if the preparation is to be kept under observation for 
some time. 
Note in the fresh preparation the size, shape and color of 
the red blood cells; the relative number of white blood cells; 
and the presence of parasites. 
Cleaning the Cover Glasses.— 
(1) Immerse the cover glasses in concentrated sulphuric acid 
for about 24 hours. 
(2) Pour off the acid and wash in running water. 
(3) Drain off the water and cover the glassware with 95 per 
cent alcohol for an hour or longer. 
(4) Wipe dry with towel and place in hot air sterilizer for 
several hours. 
Preparation and Staining of Blood Films 106 
CLINICAL LABORATORY METHODS 
Preparation of Blood Films—The film is made by placing a 
small drop of blood in the center of one cover slip (Fig. 36) 
and quickly placing a second cover over it until there is a suit- 
able spread of the blood. The cover slips are then drawn apart 
with a sliding motion. The films are allowed to dry in the air. 
Staining the Film.— 
1. Wright’s Stain.—(a) Preparation of Stain: To a 0.5 per 
cent aqueous solution of sodium bicarbonate, add methylene bine 
(B.X. or “medicinally pure”) in the proportion of 1 gram of the 
dye to each 100 c.c. of the solution. Heat the mixture in a steam 
Fig. 36.—Blood film by the cover glass method. With thumb and forefinger of the 
right hand firmly grasp the upper cover glass inch square No. 1) at the diagonal 
corners, a and b, and the lower one at the adjacent corners, c and d, and quickly pull 
apart, keeping the two parallel. (After McJunkin.) 
sterilizer at 100° C. for one full hour, counting the time after 
ihe sterilizer has become thoroughly heated. The mixture is to 
be contained in a flask, or flasks, of such size and shape that it 
forms a layer not more than 6 centimeters deep. After heating, 
the mixture is*allowed to cool, placing the flask in cold water if 
desired, and is then filtered to remove the precipitate which has 
formed in it. It should, when cold, have a deep purple red color 
when viewed in a thin layer by transmitted yellowish artificial 
light. It does not show this color while it is warm. 107 
QUALITATIVE EXAMINATION OF BLOOD 
To each 100 c.c. of the filtered mixture add 500 c.c. of a 0.1 per 
cent aqueous solution of “yellowish, water-soluble” eosin and 
mix thoroughly. Collect on a filter the abundant precipitate 
which immediately appears. When the precipitate is dry, dis- 
solve it in methyl alcohol (Merck’s “reagent”) in the proportion 
of 0.1 gram to 60 c.c. of the alcohol. In order to facilitate solu- 
tion the precipitate is to be rubbed with alcohol in a porcelain 
dish or mortar with a spatula or pestle. 
(b) Method of Staining.—Cover the blood film (Fig. 37) with a 
noted quantity of the stain (about 7 drops for a % inch cover 
glass.) After one minute add an equal number of drops of a 
phosphate buffer solution with PH equal to 6.4, and allow to 
remain for 5 to 6 minutes (MeJunkin). Wash quickly, blot, dry, 
and mount in balsam. If the buffer solution is not available, dis- 
tilled water may be used. 
The phosphate buffer solution with PH equal to 6.4 is made as 
follows: 
Recrystallized primary potassium phosphate 6.63 grams. 
Recrystallized secondary sodium phosphate 
(exposed to air for two weeks to lose its 
water of crystallization) 3.20 grams. 
Distilled water, add quantity sufficient.... 1000 c.c. 
2. Ehrlich’s Stain.— 
(a) Preparation of Stain.— 
Saturated aqueous solution of orange G 13.0 c.c. 
Saturated aqueous solution of acid fuchsin. . . . 7.0 c.c. 
Distilled water 15.0 c.c. 
Absolute alcohol 15.0 c.c. 
Saturated aqueous solution of methyl green.. . 17.5 c.c. 
Absolute alcohol 10.0 c.c. 
Glycerine 10.0 c.c. 108 
CLINICAL LABORATORY METHODS 
The fluids are mixed with the same graduated cylinder which 
should not be rinsed. The receiving flask should be shaken vig- 
orously after the addition of each constituent which is added 
in the order given in the formula. It is essential to add the methyl 
green, second portion of alcohol and glycerine slowly, shaking 
Avell after each addition. The mixture is ready for use immedi- 
ately and does not deteriorate with age. 
(b) Method of staining.—Fix the blood film on a copper bar at 
the spheroidal point for 30 to 45 seconds. Cover with Ehrlich’s 
stain, and allow to remain 5 to 10 minutes. Wash quickly in 
water, blot dry, and mount in balsam. 
Differential Leucocyte Counts 
A blood film stained with Wright’s stain is used for a routine 
count. 
Fig. 38.—Tallying register used in making differential leucocyte counts 
Count 250 leucocytes, employing the 4 millimeter objective and 
10 X eyepiece. The counting is facilitated by the use of a tallying 
register (Pig. 38). 
The normal leucocytes are classified as follows: (Plate III) 
1. Polymorphonuclear neutrophilic leucocytes (P.M.N.) are cells 
with polymorphous nuclei, in whose cytoplasm are numerous fine 
neutrophilic granules. These cells are 9 to 12 micromillimeters 
in diameter. 
2. Polymorphonuclear eosinophilic leucocytes (P.M.E.) are sim- 
ilar to the above, except for the presence of coarse eosinophilic 
or acidophilic granules in the protoplasm. They are usually 
larger than the neutrophiles. Wright Stain 
Ehrlich Stain 
Polymorpho-nuclear 
neutrophile 
Poly mtfrpho -nud ear 
eosinophile 
Polymorpho-nuclear 
basophile 
(mast cell) 
Small mononuclear 
(lymphocytes} 
Large mononudear 
Transitional 
mononuclear 
Neutrophilic myelocyte 
Eosinophilic myelocyte 
Basophilic myelocyte 
* 
Myeloblast 
Cells not present in normal blood. 
Plate III. 
White blood corpuscles with Wright and Ithrlich stains. (After Barker.) MICROSCOPIC EXAMINATION OF BLOOD 
109 
CELLS OF 
NORMAL 
BLOOD 
STAIN 
NUCLEUS 
PROTOPLASM 
GRANULES 
SIZE 
IN 
MICRA 
% 
ABS. 
NO. 
ORIGIN 
REMARKS 
# 1 
Wright 
None 
Light buff 
None 
Round or slightly oval. 
or pink. 
w 
Slaty blue color usual- 
Rarely any 
O 
3 
cd 
ly denotes insufficient 
struct ure 
o 
o 
o\ 
g 
washing. 
seen. 
g 
& 
p 
R.B.C. 
Ehrlich 
None 
Buff 
None 
B 
3 
o 
Lemon yellow—over fixed; 
O 
3 
03 
3 
red brown—under fixed. 
# 2 
Wright 
More polymorphous than 
Relatively 
Generally round, become 
polynuclear, chromatin 
abundant 
very large from crush- 
deep purples slightly retie- 
> 
ing. Protoplasm in young 
ular. 
oo 55 
w 
o 
forms often blue. 
P.M.N. 
Ehrlich 
Blue green or robin’s 
Faint pink 
Lilac if 
, p 
i-i <m 
s 
CD 
Black nuclear mass is 
egg color. Little evi- 
fixed O.K. 
to ® 
bi 
<r> 
g 
due to stain precipitate. 
dence of structure. 
True red 
3 S 
o 
P 
-t 
color with 
£ 3 
>-i 
O 
quick fixa- 
P H* 
CS 
3 
tion. 
Stain is 
specific. 
*The absolute numbers and percentages are taken from Miller, S. R.: Bull. Johns Hopkins Hosp., 1914, xxv, 284. 
Table XIII* 110 
CLINICAL LABORATORY METHODS 
CELLS OF 
SIZE 
ABS. 
NO. 
NORMAL 
STAIN 
NUCLEUS 
PROTOPLASM 
GRANULES 
IN 
% 
ORIGIN 
REMARKS 
BLOOD 
MICRA 
# 3 
Wright 
Larger than #2, less 
pyknotic, stains lighter, 
shows fewer lobulations, 
as rule reticulated. 
Scanty,faint 
pink if seen 
at all 
Red 
H- !> 
P < 
CD 
ctq S 
cd 
<-t QTQ 
CD 
3 M 
to 
cc 
to 
h-4 
GO 
o 
p 
CD 
K 
Size and brilliancy of 
the granules stained with 
Wright distinguishes 
these cells from #2 
similarly stained. 
P.M.E. 
Ehrlich 
Light green color 
Generally 
not seen 
Dark red or 
crimson 
=}£ CTq' 
tO ►=£ 
vf 
6 
# 4 
Wright 
Chromatin scanty, stains 
light purple, not very 
polymorphous. 
Faint pink 
Cells are rarely ever in- 
creased in number. 
P.M.B. 
Ehrlich 
Light green reticular. 
None 
Do not 
stain, ap- 
pear as col- 
orless vac- 
uoles 
Generally smaller 
than Nos. 2 or 3 
p 
bi 
to 
Bone Marrow 
Table XIII—Continued. QUALITATIVE EXAMINATION OF BLOOD 
111 
CELLS 01' 
SIZE 
ABS. 
NO. 
NORMAL 
STAIN 
NUCLEUS 
PROTOPLASM 
GRANULES 
IN 
% 
ORIGIN 
REMARKS 
BLOOD 
MICRA 
# 5 
Wright 
Large chromatin, round 
oval or slightly notched, 
deep purple clear outer 
zone due to nuclear con- 
traction. 
h-4 
o 
3 
to 
to 
1725 
Ul 
§ 3 
p Sr1 
|© 
Nucleus central in large 
forms. Easily overlooked 
when stained with Ehr- 
lich. 
S.M. 
Ehrlich 
Light blue green. 
Light pink 
or violet 
often not 
None 
S’ 
p 
Qj pj 
CTQ 
Ul 
g. P 
seen 
TJ1 sP 
# 6 
Wright 
Large oval indented and 
vesicular, chromatin poor, 
light purple or blue, and 
generally eccentric. 
‘ ‘ Azure ’ * 
granules 
occur. 
h-4 
to 
to 
o 
00 
05 
O 
H 
Qj 
O 
C+- 
o 
Group includes cell of 
type #5 larger than an 
average size P.M.N. Ex- 
tremely fine lilac gran- 
ules occasionally seen 
with Ehrlich stain. 
L.M. 
Ehrlich 
Very pale blue or green, 
hard to see. 
None 
o’ 
p 
S’ 
s 
# 7 
Wright 
Horseshoe, kidney shape, 
or irregular plump nu- 
cleus, deeper purple than 
#6- 
Less rela- 
tively than 
in #6. 
May be a 
few like 
those of a 
P. M. N. 
t-* 
p 
*-* 
cTQ 
a> 
ui 
to 
to 
5= > 
3 ° 
ui ►d 
o o 
Cells are not transition 
form of #2. 
Trans. 
Ehrlich 
Light blue easily seen. 
Few occa- 
sionally. 
o 
o 
Ul 
GO 
00 
i L O 
P 
£ 
05 
Are probably senile forms 
of #6. 
seen 
Ul 
a 
o 
Table XIII—Continued. 112 
CLINICAL LABORATORY METHODS 
3. Polymorphonuclear basophilic leucocytes (P.M.B.) show in 
the protoplasm, basophilic granules which are variable in size, the 
majority being about as coarse as the eosinophilic granules. The 
nucleus is usually simply indented or lobulated. 
4. Small mononuclear leucocytes (S.M.) are cells having a single 
round or oval nucleus and a scanty rim of protoplasm. The pro- 
toplasm is nongranular, although about 30 per cent of the leuco- 
cytes of normal blood possess azurophile granules which are 
demonstrable after staining with methylene azure, but not with 
other stains. The granules vary greatly in number and size. 
Classify as small mononuclears all mononuclear cells smaller than 
an average sized polymorphonuclear neutrophiles. The small 
mononuclears are lymphocytes. 
5. Large mononuclear leucocytes (L.M.) are mononuclear cells 
as large or larger than an average sized polymorphonuclear neu- 
trophile. The nucleus is poorer in chromatin and therefore stains 
less intensely. These cells have relatively more protoplasm than 
the small mononuclears. The protoplasm may contain azurophilic 
granules. 
6. Transitional leucocytes (Trans.) differ from the large mono- 
nuclears in the shape of the nucleus, which is horseshoe-shaped, 
lobulated, or deeply indented, and in the constant presence in 
the protoplasm of numerous dust-like, azurophile granules. 
Table XIII summarizes the important features of the normal 
red blood cells and leucocytes with Wright and Ehrlich stains. 
Pathological Red Blood Cells and Leucocytes 
In anemias the following abnormalities of the red blood cells 
should be looked for in fresh preparations and in stained films. 
(Plates III and IV.) 
1. Irregularity in size—anisocytosis (note especially the pres- 
ence of very large cells), and irregularity in shape—poikilocy- 
tosis. These features can be best observed in fresh blood. Cells 
less than 6 micra in diameter are classed as microcytes; those 
with a diameter of 9 to 12 micra are classed as macrocytes. 
2. Nucleated cells: Normoblasts have the diameter of the 
average red cell, the nucleus is round and pyknotic; microblasts 
are abnormally small nucleated red cells; megaloblasts are large Wright Stain 
Ehrlich Stain 
Normoblast 
Normoblasts 
Intermediate s 
Me gal oblasts 
M egalobiast 
try throe yte -showing diffuse and 
punctate basophilia and nuclear particle 
Nucleated red blood corpuscles with Wright and Ehrlich stains. (After Barker.) 
Plate IV. QUALITATIVE EXAMINATION OF BLOOD 
113 
and have a large oval or round nucleus, which may show beau- 
tiful chromatin network. 
3. Basophilia in which the cells take the basic dye and with 
Wright’s stain appear blue. The basophilia may be diffuse in 
which a uniform basic staining occurs or punctate in which the 
cells contain basic staining granules. 
Pathological leucocytes are seen chiefly in leukemia. The most 
important forms are given below: 
1. Myelocytes—neutrophilic, which have a round or oval 
nucleus and show neutrophilic granules in the protoplasm; eosino- 
philic in which the granules are eosinophilic; basophilic in which 
the granules are basophilic. 
2. Myeloblasts which differ from the myelocytes in having no 
granules in the protoplasm. Both the myelocytes and the myelo- 
blasts show a positive oxydase reaction. 
3. Pathological lymphocytes may differ very much from the 
normal. They vary much in size. The nucleus may be indented 
or convoluted forming the so-called Rieder cells. 
Platelets 
Any relative variation from the normal number of blood plate- 
lets in the smear should be noted. 
Determination of the Fragility of Erythrocytes 
(Griffin and Sandford: Jour. Lab. and Clin. Med., 1919, iv, 465) 
Principle.—Fresh blood is mixed with sodium chloride solu- 
tions of varying concentration. The strength of the solutions in 
which hemolysis begins and in which hemolysis is complete is 
noted. 
Reagent.—Five-tenths per cent solution of chemically pure 
sodium chloride. The sodium chloride should be dried in hot 
air oven at 170° C. for two hours, cooled in a desiccator and 
weighed accurately on the chemical balance. The water should 
be measured in a volumetric flask. 
Procedure.—Set up twelve 4 x % inch test tubes in each of two 
racks (Fig. 39). Number the tubes in each rack from left to right 
25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14. 114 
CLINICAL LABORATORY METHODS 
With a capillary pipette put into each tube the number of 
drops of 0.5 per cent sodium chloride indicated by the figure on 
the tube. Add distilled water with the same pipette so that the 
total number of drops in each tube is 25. 
Withdraw blood from a vein with a dry sterile syringe and run 
one drop of blood (Fig. 40) into each tube of one rack. In a 
Fig'. 39.—Dilutions are made of 0.5 per cent sodium chloride solution by adding 
distilled water by the drop method so that each tube contains twenty-five drops of 
hypisotonic solution. (After Giffin and Sanford.) 
similar manner obtain blood from a normal control and put one 
drop into each tube of the second rack. 
Allow the racks to stand at room temperature for one to two 
hours and read. The dilution in which there is just a slight tinge- 
ing of the supernatant fluid due to laking of a few of the least 
resistant corpuscles is noted as the point of initial hemolysis. 
Reading from left to right the first tube, in which there is no QUALITATIVE EXAMINATION OF BLOOD 
115 
corpuscular residue evident by shaking the tube, indicates com- 
plete hemolysis. 
The percentage of salt in any tube can be immediately deter- 
mined by multiplying the number on the tube by 0.02. 
Remarks.—Normal blood shows initial hemolysis in 0.42 to 
0.38 per cent sodium cldoride solution and complete hemolysis in 
0.36 to 0.32 per cent. 
Fig. 40.—One drop of whole blood is added to each tube of hypisotonic solution. 
(After Giffin and Sanford.) 
Technic for Vital Blood Stain 
Reagent.—Mix 5 c.c. of a saturated aqueous solution of bril- 
liant cresyl blue and one cubic centimeter of two per cent sodium 
oxalate solution and filter. Draw up 1 to 2 drops of this mixture 
into each of the several capillary pipettes and place in incubator 
until the stain is evaporated to dryness. These preparations will 
keep indefinitely. 
Procedure.—Puncture the finger so as to get a free flow of; 116 
CLINICAL LABORATORY METHODS 
blood. With a rubber bulb draw up a good sized drop of blood 
into one of the capillary pipettes containing the stain. Mix 
thoroughly. After standing for several minutes make films on 
cover slips. Dry in air and stain with Wright’s stain in the 
usual way. 
In the cells taking the vital stain a granulo-reticulo-filamentous 
substance is seen. This appears in the form of granular par- 
ticles which are sometimes discrete but are more often threads 
which are woven into a network, or wreaths which fill a great 
part of the cell. 
Determine the percentage of reticulated cells using the oil im- 
mersion objective. The counting is facilitated by using a small 
circle cut from cardboard on the diaphragm of the eye piece. 
Remarks.—In the normal blood of adults less than one per 
cent of the red blood cells show the granulo-reticulo-filamentous 
substance. 
If brilliant cresyl blue is not available, Unna’s methylene blue 
may be used. 
Determination of Coagulation Time of Blood 
(Ilowell, W. H., Arch. Int. Med., 1914, xiii, 80) 
A clean syringe is dried, rinsed with albolene and normal salt 
solution. The space in the syringe between the end of the 
plunger and the needle is filled with salt solution so that the 
blood does not come in contact with air. A vial (Fig. 41) about 
21 millimeters in diameter is thoroughly cleaned with bichromate 
cleaning solution, rinsed in distilled wrnter and dried with alcohol 
and ether. 
Blood is withdrawn by venapuncture and 2 c.c. run into the 
vial. The time of withdrawal of the blood is noted accurately. 
The specimen is examined at short intervals for coagulation. The 
test of coagulation is ability to invert the tube slowly without 
dislodging the blood. 
By this technic normal blood at room temperature (20° C.) 
clots in 20 to 40 minutes. For each degree below this tempera- 
ture, about 1 to 2 minutes wull probably be added to the coagula- 
tion time. QUALITATIVE EXAMINATION OF BLOOD 
117 
Decreasing the diameter of the tube shortens the coagulation 
time. Lee and White suggest that a test-tube of 8 mm. bore be 
used. One c.e. of blood is withdrawn with a small syringe and 
run at once into the tube. The same precautions as to prepara- 
tion of the syringe and tube noted above must be observed. The 
normal coagulation time by this method is 5 to 8 minutes. 
A rough idea of the coagulation time may be obtained by fill- 
ing a capillary tube with blood from a puncture of the ear or 
finger. The tube is broken off at intervals of one-half minute. 
Coagulation is easily recognized by the string of fibrin. The 
coagulation time by this method varies from 2 to 6 minutes. 
Fig. 41.—Tube used in the determination of the coagulation time of blood. 
The bleeding time is determined by the method suggested by 
Duke. A small incision is made in the finger or in the lobe of the 
ear and at half-minute intervals the blood is blotted up with 
smooth filter paper. The cut should be such that the diameter of 
the first blot is about 2 cm. without any squeezing. Each blot 
represents a half minute’s flow of blood. The bleeding time is 
the total duration of the hemorrhage. It varies from 1 to 3 min- 
utes in normal individuals. 
Determination of the Bleeding Time 
Schultze’s Oxydase Reaction 
Principle.—Many white blood cells possess an oxidizing fer- 
ment which they disclose by forming synthetically naphthol blue 
(Evans, F. A.: Arch. Int. Med., 1916, xvii, 1) 118 
CLINICAL LABORATORY METHODS 
Tab,le XIV* 
Blood Report 
Name: No: 
Case No: Date: 
Service: Stain used: 
I. Red Blood Cells: 
1. Total number per c.mm. 
2. Size in fresh preparation 
3. Shape in fresh preparation 
4. Color in fresh preparation 
5. Regeneration forms: 
(a) Nucleated R.B.C. - 
(b) Basophilia: punctate or diffuse - 
(c) Nuclear particles - 
(d) Cabot Rings - 
6. Resistance to hypotonic salt solution: maximal %; minimal % 
7. Percentage of reticulated cells in vital stain: 
II. White Blood Cells: 
1. Total number per c.mm. 
2. Differential count: Cells counted: 
PMN — % PME = % PMB — % 
SM = % LM = % TRANS = % 
3. Presence of abnormal forms: 
(a) Myelocytes: type? 
(b) Myeloblasts - 
(c) Irritation forms - 
III. Hemoglobin: % ( Ilemoglobiiiometcr) 
IV. Color Index : 
V. Volume Index: 
VI. Platelets : 
VII. Coagulation Time: ( Method) 
VIII. Parasites : 
IX. Remarks : 
X. Impressions : 
1. Summary of Important Evidence: 
2. Probable Diagnois: 
Name op Examiner. 
*Tliis form is modeled after one in use at the Johns Hopkins Hospital. QUALITATIVE EXAMINATION OF BLOOD 
119 
when they are treated first with a-naphthol and then with di- 
methyl-phenylendiamin. The method is particularly useful for 
differentiating myeloblasts and myelocytes from cells of the 
lymphocyte series. The myelocytes and myeloblasts give a posi- 
tive reaction, while the lymphocytes are negative. 
The granules which exhibit the oxydase reaction are stained 
deep blue. The preparations are not permanent. 
Procedure.—Make smears on cover slips as for differential 
leucocyte count. 
(a) Staining.— 
1. Fix smears eight hours (under bell jar) in formaldehyde 
vapor. 
2. Stain in saturated aqueous safranin for eight minutes. 
3. Wash quickly in water and dry immediately by careful 
blotting. 
(b) Oxydase Reaction: Put on slide. 
1. One drop of 1 per cent aqueous solution of dimethylpara- 
phenylenediamin, and 
2. One drop of freshly prepared 1 per cent solution of alpha 
naphthol in 1 per cent KOH dissolved by heating. 
3. Mount previously stained smear in this and examine at once. 
Examination of Blood for Malarial Organisms 
Make smears as for differential leucocyte count. Stain with 
Wright’s stain. The staining should be done with distilled water 
instead of buffer solution. A preparation of fresh blood should 
also be made by placing a small drop of blood on a clean slide and 
covering with a cover glass. 
Examine both preparations with the oil immersion lens. 
Tables XV and XVI summarize the characteristics of the dif- 
ferent malarial organisms in fresh and stained preparations. 120 
CLINICAL LABORATORY METHODS 
The Appearance op Malarial Plasmodia in Fresh Blood 
Table XV 
pl. vxvax (tertian) 
PL. FALCIPARUM 
(estivoautumnal) 
PL. MALARIA 
(quartan) 
I. HYALINES 
(a) Shape 
Often irregular, oc- 
casionally ring 
forms. 
Usually ring forms, 
occasionally irreg- 
ular. 
Irregular or ring 
forms. 
(b) Refrac- 
tivity 
Difficult to see; 
much like the red 
cell. Ring forms 
more refractive. 
Easily seen; 
refractive. 
Rather easily seen. 
(c) Motility 
Actively ameboid. 
Occasionally active. 
Rings sluggish. 
Sluggish usually. 
(d) Multiple 
infections 
II. PIGMENT- 
ED FORMS 
(a) Shape 
Infrequent. 
Ameboid, very ir- 
regular. 
Three-quarters and 
full grown para- 
sites round. 
Frequent. May be 
six or more hyalins 
in a single cell. 
Usually no pigment- 
ed forms in circulat- 
ing blood. 
Round or oval, when 
seen. 
Infrequent. 
Irregular, soon be- 
coming round or 
oval. 
‘ ‘ Band ’ ’ forms 
not infrequent. 
(b) Refrae- 
tivity 
Young forms dif- 
ficult to see. 
Easily seen; re- 
fractive 
Easily seen. 
(c) Motility 
Young forms ac- 
tively ameboid. 
Sluggish. 
Sluggish. 
(d) Pigment 
Fine brown gran- 
ules scattered 
throughout p a r a- 
site. 
Fine, dark brown 
granules, centrally 
placed. 
Coarse brown gran- 
ules peripherally 
placed. 
(e) Motility 
of pigment 
Very active in 
younger forms. 
Sluggish. 
Very sluggish. 
(f) Meroz- 
oites or 
daughter 
parasites 
12 to 24, usually 
about 16. 
8 to 24, usually 12 
to 16, small. 
6 to 12, often 8. 
III. SEXUAL 
FORMS 
Shape 
Round. 
Usually crescentic, 
at times round or 
Round. 
1 
IV. INFECTED 
RED CELLS 
Swollen and pale. 
oval. 
Often brassy, shrunk- 
en or crenated. 
Often brassy, no 
swelling. QUALITATIVE EXAMINATION OF BLOOD 
121 
Table XVI 
The Appearance of Malarial Plasmodia in Stained Blood Films 
(Wright Stain) 
PL. VTVAX 
(tertian) 
PL. FALCIPAETXM 
(aestivoautumnal) 
PL. MALAEIA 
(quartan) 
I. 
HYALINES 
Ring of blue proto- 
plasm. 
Round chromatin 
mass; red or violet 
with achromatic 
zone. Whole parasite 
encloses unstained 
area of R.B.C. Chro- 
matin located at 
thin area of ring. 
Protoplasm scanty; 
stains faint blue. 
Chromatin in 1/3 
masses of filaments 
often projecting 
beyond the ring. 
Generally the same 
but chromatin is not 
dense but rather an 
irregular clump of 
red violet granules. 
Chromatin is nearer 
the center than in 
tertian. 
Protoplasm stains a 
darker blue. 
Parasite as whole is 
smaller than tertian. 
II. 
PIGMENT- 
ED FORMS 
(a) Pigment 
Fine, irregularly 
scattered, takes a 
greenish brown stain. 
Collected in masses 
at the periphery of 
presegmentingforms. 
Single mass at center 
of segmenters. 
Amount and distri- 
bution as in fresh 
blood. Takes deep 
brown stain. 
Little influenced by 
stain. Size and dis- 
tribution as in fresh 
blood. 
Dark greenish brown 
or blue black in color. 
(b) Chroma- 
tin 
Becomes more irreg- 
ular with growth of 
parasite. 
In full grown para- 
site it breaks up in- 
to 15-20 clusters 
each in achromatic 
zone. Stains a deep 
red violet. 
Often in several 
masses in ring 
forms. 
Chromatin less dense 
in hyalines. In older 
forms is a cluster of 
fine granules with an 
achromatic zone. 
Stains a more in- 
tense red than with 
tertian. 
(e) Proto- 
plasm 
Stains a definite 
blue, thickest oppo- 
site the chromatin 
in hyaline. 
Irregular in outline. 
With growth of par- 
asite tends to stain 
more deeply. 
Protoplasm 
throughout scanti- 
er than in other 
forms. Tends to 
stain a delicate 
blue in hyaline 
form and not very 
dark in mature 
form. 
Protoplasm through- 
out stains more in- 
tensely and is gen- 
erally regular in out- 
line. 
Circular. 122 
CLINICAL LABORATORY METHODS 
Table XVI—Continued. 
The Appearance of Malarial Plasmodia in Stained Blood Films 
(Wrigi.it Stain) 
PL. VIVAX 
(tertian) 
PL. FALCIPARUM 
(aestivoautumnal) 
PL. MALARIA 
(quartan) 
III. SEXUAL 
FORMS 
(a) Female 
Intense blue proto- 
plasm. 
Chromatin is scanty 
and tends to be 
peripheral. 
Stains a brilliant 
red, and is compact. 
Pigment is blue; 
black rods situated 
peripherally or as a 
nidcentral areola. 
Long and slender. 
Chromatin compact 
and central. Pro- 
toplasm deep blue 
with polar intensi- 
fication. Pigment 
is near center in 
masses or in wreath 
about the chroma- 
tin. It is coarse 
and black. 
In general as in ter- 
tian. These forms 
show more intense 
staining reaction, are 
apt to be smaller, 
and show coarser, 
darker pigments as 
in fresh blood. 
(b) 
Male 
Protoplasm stains a 
faint blue. Chroma- 
tin is abundant and 
loose. Stains intense 
red. Central chroma- 
tin has an achromat- 
ic zone. 
Pigment is greenish 
blue, fine and diffuse. 
Chromatin breaks up 
into 4-8 masses just 
prior to flagellation. 
Kidney shaped. 
Protoplasm stains 
much lighter blue 
than female. Chro- 
matin is not so 
brilliant and forms 
loose network 
often scattered 
throughout. Pig- 
ment finer, diffuse, 
and greenish brown 
in color. 
IV. INFECTED 
CELLS 
Enlargement notice- 
able. Often distort- 
ed, stains poorly. 
Red or purple stip- 
pling may occur, 
especially common in 
tertian type and oc- 
curs in cells with 
early forms. 
Small size; darker 
staining. Stippling 
does not occur. 
Normal or smaller— 
takes a dark reddish 
stain corresponding 
to brassy look. 
Stippling very com- 
mon. 
Qualitative Tests for Bile Pigments, Bile Salts and Urobilin in 
Blood Plasma 
(Blankenhorn, M. A.: Arch. Int. Med., 1917, xix, 344) 
I. Bile Pigments—Put nitric acid under the plasma in a test 
tube with a small pipette. A white coagulum is formed at the 
junction, which soon develops into a white layer of a certain 
thickness and remains fairly constant as the acid dissolves the 
coagulum at the lower border and forms it at the upper, thus QUALITATIVE EXAMINATION OF BLOOD 
123 
ascending through the plasma. The blue green color develops in 
a line at the midst of the white zone if bile pigment be present 
and remains in the same relative position. AYhen the bilirubin 
is present in small amounts, the blue color may not appear for 
half an hour. 
The bilirubin content of the blood is expressed by the dilution 
required to diminish the staining to a point where it is just per- 
ceptible in a column one centimeter deep. The plasma must be 
free from hemolysis. In diluting the plasma with distilled water, 
a faint precipitate usually appears in suspension, which can be 
put in solution by a drop of ammonium hydroxide. 
The end point of the dilution, that is where the yellow color is 
just perceptible, is found by comparing the solution in a small 
test tube with a similar column of distilled water. To facilitate 
reading, the tubes are held parallel and observation made down 
the length of the tubes. A device which serves to bring the two 
columns to still more equal terms is to immerse the ends of the 
tubes in an inch or two of water in an evaporating dish. 
II. Bile Salts.—Three c.c. of plasma are dialyzed into 9 c.c. of 
water. The collected dialysate is concentrated and the Pet- 
tenkofer test applied, and the spectroscopic examination made, 
if any color develops. For the test, two cubic centimeters of 
concentrated dialysate are placed in a small flask with two or 
three drops of a 1 to 1000 aqueous solution of furfurol and two 
cubic centimeters of concentrated sulphuric acid are added, drop 
by drop, from a pipette. The contents of the flask is 
kept at 60 degrees Centigrade by immersing the flask in 
a bath and keeping the mixture agitated. When the acid 
is added too rapidly a dark reddish brown color sometimes ap- 
pears from the charring action of the acid on the small amounts 
of protein or sugar that dialyze. If the temperature remains 
much below 60° C., the color fails to develop. The spectrum of 
the Pettenkofer test for bile salts shows a wide absorption band 
in the blue when the test is first made and is of a cherry red color. 
Later as the cherry turns to purple, the band in the blue fades 
and a smaller band develops in the orange (near I, between D and 
C). Small amounts of bile salts which give the Pettenkofer 
test may not give a characteristic spectrum. 124 
CLINICAL LABORATORY METHODS 
Test dialysate for bilirubin also. 
III. Urobilin.—Precipitate the blood protein by adding one to 
two volumes of freshly prepared saturated alcoholic solution of 
zinc acetate and centrifugalize. Add 5 to 10 drops of Ehrlich’s 
aldehyde reagent (page 19) and a color from pink to dark 
red develops, which gives the characteristic spectrum if uro- 
bilin or urobilinogen is present (Fig. 26-A). CHAPTER VII 
QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
Collection of Blood for Chemical Analysis 
Blood for chemical analysis should be collected in the morning 
before the patient has had breakfast. 
All- specimens except those for calcium determination are to 
have oxalate added. The optimum amount is one drop of a satu- 
rated (about 30 per cent) solution of potassium oxalate to each 
5 c.c. of blood. If too much oxalate be added the coagulation of 
the proteins and the uric acid precipitation will be interfered 
with. 
For a calcium determination 10 c.c. of blood is run into a test 
tube containing 0.1 gram of solid sodium citrate and well shaken. 
The following amounts of blood are necessary: 
(1) Complete analysis including, 
Non-protein nitrogen 
Urea nitrogen 
Creatinine 
Uric acid 
Sugar 
Chlorides 
Bicarbonate content 
20 c.c. 
(2) Single determination of any one of the above: 5 e.c. 
The specimen for the determination of the bicarbonate content 
is to be collected in a centrifuge tube containing paraffin oil and 
potassium oxalate. 
It is very important that specimens for sugar determination be 
sent to the laboratory immediately after withdrawal as the sugar 
is quite rapidly destroyed on standing. 
The blood may be preserved if necessary by adding 1 drop 
of commercial formalin for each 5 c.c. of blood. 
125 126 
CLINICAL LABORATORY METHODS 
CHRONIC 
HYPER- 
EARLY 
NORMAL 
TENSIVE 
CHRONIC 
UREMIA 
OR MILD 
SEVERE 
MODERATE 
SEVERE 
GOUT 
ARTHRITIS 
CHOLE- 
VASCULAR 
NEPHRITIS 
DIABETES DIABETES 
ACIDOSIS 
ACIDOSIS 
LITHIASIS 
DISEASE 
Hydrogen Ion 
Concentration 
(Plasma) 
Total Nonprotein 
7.6-7.8 
7.55-7.4 
7.4-7.2 
Nitrogen 
25-35 
25-35 
35-200 
100-375 
25-50 
25-35 
25-100 
Urea Nitrogen 
10-18 
10-20 
25-175 
75-325 
15-30 
Uric Acid 
1-4 
1-4 
1-6 
5-25 
4-10 
4-10 
1-4 
Creatinine 
1-2 
1-2 
1-5 
4-33 
Creatine 
3-7 
3-7 
3-10 
7-18 
Sugar 
Chlorides as NaCl 
70-110 
120-180 
120-230 
120-300 
300-600 
(Plasma) 
550-650 
550-700 
550-750 
450-650 
Cholesterol 
Alkali Reserve 
150-180 
150-180 
170-350 
150-300 
280-950 
(c.c. C02 in 100 
c.c. plasma) 
50-75 
30-40 
Below 30 
Inorganic Phos- 
phorus as H3 
Up to 
P04 (Plasma) 
Lipoid Phosphor- 
7-10 
75 
us as H3P04 
(Plasma) 
20-25 
Calcium as Ca 
(Plasma) 
10 
1 
Compiled from results in the author’s own experience and from numerous other observers. FIGURES IN MILLIGRAM'S PER 
100 C.C. BLOOD OR PLASMA. 
Composition of Normal Blood and Chem'ical Changes in Certain Pathological Conditions! 
Table XVII QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
127 
Fig. 42 shows a convenient bottle to use in obtaining specimens 
of blood for chemical examination. 
Fig. 42.—Blood chemical bottle used for collecting blood. 
Determination of the Relative Hydrogen-Ion Concentration 
(Levy, Rowntree and Marriott: Arch. Int. Med., 1915, xvi, 389) 
Principle.—The blood is dialyzed against normal salt solution 
and the H-ion concentration of the protein-free clialysate is 
determined by the indicator method, using phenolsulphoneph- 
thalein. 
Reagents.—(1) Standard phosphate mixtures prepared accord- 
ing to Sorensen’s directions as follows: 
1/15 mol. acid or primary potassium phosphate. 9.078 grams of 
the pure recrystallized salt (KH2P04) is dissolved in freshly 
distilled water and made up to 1 liter. 
1/15 mol. alkaline or secondary sodium phosphate. The pure 
recrystallized salt (NA.2HP04 12II20) is exposed to the air for 
from ten days to two weeks, protected from dust. Ten mole- 
cules of water of crystallization are given off and a salt of the 
formula Na2HP04 211,0 is obtained. 11.876 grams of this is 
dissolved in freshly distilled water and made up to 1 liter. 
The solution should give a deep rose-red color with phenol- 
phthalein. If only a faint pink color is obtained, the salt is 
not sufficiently pure. 128 
CLINICAL LABORATORY METHODS 
The solutions are mixed in the proportions indicated below to 
obtain the desired PH. 
PH 6.4 6.6 6.8 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 8.0 8.2 8.4 
Primary potassium 
phosphate c.c. 73 63 51 37 32 27 23 19 15.813.211.0 8.8 5.6 3.2 2.0 
Secondary sodium 
phospate c.c. 27 37 49 63 68 73 77 81 84.2 86.8 89.0 91.2 94.4 96.8 98.0 
(2) Eight-tenths per cent sodium chloride solution. Before 
applying the test it is necessary to ascertain whether the solu- 
tion is free from acids other than carbonic. To determine this, 
a few cubic centimeters of the salt solution are placed in a pyrex 
test tube and 1 or 2 drops of the indicator added, whereupon a 
yellow color appears. On boiling, carbon dioxide is expelled, and 
the solution loses its lemon color and takes on a slightly brown- 
ish tint. In the absence of this change other acids are present, 
and the salt solution is therefore not suitable. If, on the other 
hand, on adding the indicator, pink at once appears, the solution 
is alkaline and hence cannot be used. 
(3) Collodion dialyzing sacs, prepared as follows: One ounce 
of celloidin is dissolved in 500 c.c. of a mixture of equal quan- 
tities of ether and ethyl alcohol. The solid swells up and dis- 
solves with occasional gentle shakings, in 48 hours. As a small 
amount of brown sediment separates out at first, the solution 
should stand for at least three or four days, after which the clear 
supernatant solution is ready for use. A small test tube (120 by 
9 mm., inside measurement) is filled with this mixture, inverted, 
and half the contents poured out. The tube is then righted, and 
the collodion allowed to fill the lower half again. A second time 
it is inverted and rotated on its axis, the collodion being drained 
off. Care must be taken to rotate the tube in order to secure a 
uniform thickness throughout. The tube is clamped in the in- 
verted position and allowed to stand for ten minutes, until the 
odor of ether finally disappears. It is filled five or six times with 
cold water, or it is allowed to soak five minutes in cold water. 
A knife blade is run around the upper rim, so as to loosen the 
sac from the rim of the test tube, and a few cubic centimeters of 
water are run down between the sac and the glass tube. By 
gentle pulling the tube is extracted, after which it is preserved 
by complete immersion in water. 
Procedure.—One to 3 c.c. of clear serum or of blood is run, by 129 
QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
means of a blunt-pointed pipette, into a dialyzing sac which has 
been washed outside and inside with salt solution. The sac is 
lowered into a small test tube (100 x 10 mm., inside measurement), 
containing 3 c.c. of salt solution, until the fluid on the outside 
of the sac is as high as on the inside. From 5 to 10 minutes are 
allowed for dialysis. The collodion sac is removed and 5 drops 
of the indicator (0.01 per cent solution of phenolsulphonephthal- 
ein) are thoroughly mixed with the dialysate. The tube is then 
compared with the standards until the corresponding color is 
found, which indicates the hydrogen-ion concentration present in 
the dialysate. Readings should be made immediately against a 
white background. Results are expressed in logarithmic notation. 
Remarks.—Oxalated blood from normal individuals gives a dial- 
ysate with a PH varying from 7.4 to 7.6, while that of serum 
ranges from 7.6 to 7.8. In clinical acidosis figures from 7.55 to 
7.2 have been noted by this method for serum and for oxalated 
blood from 7.3 to 7.1. A rise in the Il-ion concentration of the 
blood is significant because it indicates a failure on the part of 
the protective mechanism of the body to preserve the proper 
reaction. 
Preparation of Protein Free Blood Filtrates 
(Folin and Wu: Jour. Biol. Chem., 1919, xxxviii, 81) 
Collection of Blood Sample.—The blood should be collected 
over finely powdered potassium oxalate, about 20 mg. for 10 c.c. 
of blood. Large amounts of oxalate interfere with the coagula- 
tion of the protein, and may precipitate some of the uric acid. 
Reagents.— 
1. Ten per cent solution of sodium tungstate. 
2. Two-thirds normal sulphuric acid solution. This may be 
prepared by diluting 35 grams of concentrated C.P. sulphuric 
acid up to a volume of 1 liter. Check by titration. One c.c. of 
the acid is neutralized by 6% c.c. of N/10 alkali. 
Procedure.—Transfer a measured amount of oxalated blood, 
preferably 10 c.c., into a flask having a capacity of fifteen to 
twenty times that of the volume taken. Dilute with seven vol- 
umes of water and mix. Add one volume of 10 per cent solution 130 
CLINICAL LABORATORY METHODS 
of sodium tungstate and mix. With another pipette add one vol- 
ume of % normal sulphuric acid. Close the mouth of flask with 
a rubber stopper and give it a few vigorous shakes. Let stand 
for five minutes. The color of the coagulum changes from bright 
red to dark brown. If this change in color does not occur, the 
coagulation is incomplete, usually because too much oxalate has 
been added. The sample may be saved by adding 10 per cent 
sulphuric acid one drop at a time, shaking vigorously after each 
Fig. 43.—Folin and Wu pipette. 
drop and continuing until there is no foaming and until the dark 
brown color has set in. 
Pour the mixture on a filter large enough to hold the entire 
contents of the flask and cover with a watch glass. The filtration 
should be begun by pouring the first few cubic centimeters down 
the double portion of the filter and withholding the remainder 
until the whole filter is wet. 
If the filtrate is to be kept longer than two days, a few drops 
of toluol should be added. 
Remarks.—To test for excess of II2S04 moisten Congo red paper 131 
QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
with the filtrate. It should show negative, or at most, a bare 
trace of acid. An excess of acid will precipitate some of the 
uric acid. 
Some examples of sodium tungstate contain too much sodium 
carbonate. This may be tested as follows: To 10 centimeters of 
a 10 per cent solution of the sodium tungstate add one drop of 
phenolphthalein and titrate with 0.1 N HC1. Each cubic centi- 
meter of IIC1 is equivalent to 1.06 per cent of Na2C03. The 
amount of acid used should not exceed 0.4 centimeters if the spec- 
imen is to be satisfactory for use. 
Folin states that difficulty is sometimes encountered because 
the sodium tungstate is not alkaline enough. This may be cor- 
rected as follows: Prepare 100 c.c. of 10 per cent sodium tung- 
state using heat if necessary. Cool. Titrate 10 c.c. with normal 
NaOH to a permanent pink color using phenolphthalein as the 
indicator. The pink color should persist for 3 minutes after the 
last addition of alkali. Add to the remainder of the tungstate 
and to subsequent dilutions the amount of alkali indicated by the 
titration. 
The preparation of the protein free filtrate is facilitated by the 
use of the special Folin pipette (Fig. 43). 
Determination of Nonprotein Nitrogen 
Principle.—The protein-free blood filtrate is treated with an 
acid mixture, which converts the nitrogen into ammonia. The 
solution is Nesslerized and read against a standard ammonium 
sulphate solution similarly treated. 
Reagents.—(1) Acid digestion mixture: Mix 300 c.c. of phos- 
phoric acid syrup with 100 c.c. of ammonia-free sulphuric acid 
(concentrated). Transfer to a tall cylinder, cover well to ex- 
clude the absorption of ammonia, and set aside for sedimentation 
of calcium sulphate. To 100 c.c. of the clear acid add 10 c.c. of 
6 per cent copper sulphate solution and 100 c.c. of water. 
(2) Nessler’s reagent. (See page 266.) 
(3) Standard ammonium sulphate solution. (See page 44.) 
Procedure.—Introduce 5 c.c. of the protein-free blood filtrate 
(page 129) into a dry 75 c.c. Pyrex test tube (200 x 25 milli- 
(Folin and Wu: Jour. Biol. Chem., 1919, xxxviii, 81) 132 
CLINICAL LABORATORY METHODS 
Use standard containing 0.3 mg. nitrogen made up to a total volume of 100 c.c. 
Set standard at 20 or till 20 mm. Bock-Benedict cell. 
Make total volume of unknown blood up to 50 c.c. 
Table shows the non-protein nitrogen in mg. per 100 c.c. blood corresponding to the different readings, with 5 c.c., 
2 c.c., or 1 c.c. of blood filtrate, using a plunger type colorimeter. 
USING 5 C.C. OF BLOOD FILTRATE 
(Non-protein nitrogen in mg. 
per 100 c.e. blood) 
USING 2 C.C. OF BLOOD FILTRATE 
(Non-protein nitrogen in mg. per 
100 c.c. blood) 
USING 1 C.C. OF BLOOD FILTRATE 
(Non-protein nitrogen in mg. 
per 100 c.c. blood) 
Colorimeter 
reading 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
10 
60.0 
58.8 
57.8 
56.4 
55.4 
150.0 
147.0 
144.5 
141.0 
138.5 
300 
294 
289 
282 
277 
11 
54.6 
53.6 
52.6 
51.8 
50.8 
136.5 
134.0 
131.5 
129.5 
127.0 
273 
268 
263 
259 
254 
12 
50.0 
49.2 
48.2 
47.6 
46.8 
125.0 
123.0 
121.0 
119.0 
117.0 
250 
246 
242 
238 
234 
13 
46.0 
45.6 
45.0 
44.4 
43.6 
115.0 
114.0 
112.5 
111.0 
109.0 
230 
228 
225 
222 
218 
14 
42.8 
42.2 
41.6 
41.0 
40.6 
107.0 
105.5 
104.0 
102.5 
101.5 
214 
211 
208 
205 
203 
15 
40.0 
39.5 ’ 
38.9 
38.5 
38.0 
100.0 
98.8 
97.3 
96.3 
95.0 
200 
198 
195 
193 
190 
16 
37.5 
37.0 
36.6 
36.1 
35.7 
93.8 
92.5 
91.5 
90.3 
89.8 
188 
185 
183 
181 
179 
17 
35.3 
34.9 
34.5 
34.1 
33.7 
88.3 
87.3 
86.3 
85.3 
84.3 
177 
175 
173 
171 
169 
18 
33.3 
33.0 
32.6 
32.3 
31.9 
83.3 
82.5 
81.5 
80.8 
79.8 
167 
165 
163 
162 
159 
19 
31.6 
31.2 
30.9 
30.6 
30.3 
79.0 
78.0 
77.3 
76.5 
75.8 
158 
156 
155 
153 
152 
20 
30.0 
29.7 
29.4 
29.1 
28.9 
75.0 
74.3 
73.5 
72.8 
72.3 
150 
149 
147 
146 
145 
21 
28.5 
28.2 
28.0 
27.7 
27.5 
71.3 
70.5 
70.0 
69.3 
68.8 
143 
141 
140 
139 
138 
22 
27.3 
27.0 
26.8 
26.5 
26.3 
68.3 
67.5 
67.0 
66.3 
65.8 
137 
135 
134 
133 
132 
23 
26.1 
25.9 
25.7 
25.4 
25.2 
65.5 
65.0 
64.5 
63.5 
63.0 
131 
130 
129 
127 
126 
24 
25.0 
24.8 
24.6 
24.4 
24.2 
62.5 
62.0 
61.5 
61.0 
60.5 
125 
124 
123 
122 
121 
25 
24.0 
23.8 
23.6 
23.4 
23.3 
60.0 
59.5 
59.0 
58.5 
58.3 
120 
119 
118 
117 
116 
26 
23.0 
22.9 
22.8 
22.7 
22.5 
57.5 
57.3 
57.0 
56.8 
56.3 
115 
115 
114 
114 
115 
27 
22.4 
22.2 
22.0 
21.8 
21.6 
56.0 
55.5 
55.0 
54.5 
54.0 
111 
111 
110 
109 
108 
28 
21.4 
21.3 
21.1 
21.1 
20.8 
53.5 
53.3 
52.8 
52.5 
52.0 
107 
107 
106 
105 
104 
29 
20.7 
20.5 
20.4 
20.3 
20.2 
51.8 
51.3 
51.0 
50.8 
50.05 
103 
103 
102 
102 
101 
Determination op Non-protein Nitrogen in Blood 
Table XVIII 133 
QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
meters) graduated at 35 c.c. and 50 c.c. Add a quartz pebble and 
1 c.c. of the sulphuric phosphoric acid mixture. Mix well. Boil 
vigorously over a micro burner (Fig. 19) until the characteristic 
dense acid fumes begin to fill up the test tube. Cut down the flame 
so that the contents just boil and close the mouth of the tube with 
a watch glass. Heat for two minutes counting from the time the 
test tube became filled with the fumes. Cool for seventy to 
ninety seconds and add 15 to 25 c.c. of water. Cool further to 
room temperature and add water to the 35 c.c. mark. Add 15 c.c. 
of Nessler solution. If turbid centrifuge. Compare in colorim- 
eter with standard containing 0.3 milligrams nitrogen (as ammo- 
nium sulphate). Add to it 2 c.c. of the sulphuric phosphoric acid 
mixture, about 50 c.c. of water, and 30 c.c. of Nessler Solution. 
Make up to 100 c.c. and mix. 
Nesslerize the unknown and the standard at approximately the 
same time. 
Calculation.— 
Set standard at 20 
If X is the reading of the unknown. 
20 
— y 30 — mg. nonprotein nitrogen per 100 c.c. 
of blood. 
Remarks.—Normal blood gives by this method 25 to 35 mg. 
of nonprotein nitrogen per 100 c.c. 
If the reading is high the determination should be repeated 
employing 2 or 1 c.c. of the filtrate. 
A method for checking total nonprotein nitrogen determination 
is given on page 250. 
Table XVIII gives the amount of nitrogen corresponding to 
the colorimeter readings, with a plunger type instrument. 
Determination of Urea Nitrogen 
(Van Slyke and Cullen: Modification of Marshall Method, 
Jour. Amer. Med. Assn., 1914, Ixii, 1558) 
Principle.—The urea in the blood is converted into ammonium 
carbonate by urease. The ammonia is liberated by a strong 
alkali, removed by the passage of a strong air current, and col- 134 
CLINICAL LABORATORY METHODS 
lected in N/100 sulphuric acid. The excess of acid is then titrated 
with N/100 alkali. 
Reagents.— 
(1) Urease tablets (Hynson, Westcott and Dunning or Squibb). 
(2) N/100 sulphuric acid. 
(3) N/100 sodium hydroxide. 
(4) Saturated solution of potassium carbonate (90 gm. per 
100 c.c.). 
(5) Alizarin (1 per cent sodium-alizarin sulphonate in 50 per 
cent alcohol). 
Procedure.—Run 3 c.c. of oxalated blood into a large test tube 
(Fig. 20) containing about 10 c.c. of ammonia-free distilled water. 
Add 2 urease tablets (powdered). Leave at room temperature for 
30 minutes. Add a teaspoonful of salt, a few drops of foam killer, 
(see page 272) and finally 5 c.c. of a saturated solution of potas- 
sium carbonate. Drive off the ammonia by aspiration into an- 
other tube “B” containing 15 c.c. of hundredth-normal sulphuric 
acid and 1 drop of alizarin for one hour. Titrate the excess of 
acid with hundredth-normal sodium hydroxide. 
Calculation.—Each cubic centimeter of acid neutralized indi- 
cates 10 mg. of urea per 100 c.c. blood, or 4.67 mg. of urea nitro- 
gen per 100 c.c. blood. In case the blood is one of the rare sam- 
ples containing over 150 mg. urea per 100 c.c., all the acid wrill be 
neutralized and it will be necessary to repeat the determination, 
using only 1 c.c. blood. Fresh blood contains so little ammonia 
it may be disregarded. 
Remarks.—The amount of urea nitrogen found in normal blood 
is 10 to 18 mg. per 100 c.c. 
A slow current of air should be used during the first two min- 
utes of aspiration. 
The column of acid in the tube should be at least 50 mm. high. 
The solution from which the ammonia is driven must contain 
at least 1 gram of potassium carbonate for each 2 c.c. of solution. 
A blank determination should be run with each new lot of 
chemicals and the necessary deduction be made if any ammonia 
is found. 
The air used for aerating should be run through a wash bottle 
containing sulphuric acid. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
135 
The figure obtained for urea nitrogen may be converted into 
urea by dividing by the factor 0.467. 
Table XIX shows the blood urea nitrogen values correspond- 
ing to the amount of sodium hydroxide used to titrate the excess 
of sulphuric acid. 
A method for checking urea nitrogen determinations is given 
on page 250. 
Table XIX 
Take 3 c.c. of blood and 15 c.e. of N/100 H,S04. 
Table shows the blood urea nitrogen in milligrams per 100 c.c. of blood 
corresponding to the number of c.c. of N/100 NaOH required to titrate the 
excess of N/100 H,S04. 
Determination of Blood Urea Nitrogen 
c.c. 
NaOH 
REQUIRED 
0.0 
0.1 
0.2 
0.3 
0.4 
0.5 
0.6 
0.7 
0.8 
0.9 
0 
70.05 
69.58 
69.12 
68.65 
68.18 
67.71 
67.25 
66.78 
66.31 
65.85 
1 
65.38 
64.91 
64.45 
63.98 
63.51 
63.05 
62.58 
62.11 
61.64 
61.18 
2 
60.71 
60.24 
59.78 
59.31 
58.84 
58.38 
57.91 
57.44 
56.97 
56.51 
3 
56.04 
55.57 
55.11 
54.64 
54.17 
53.71 
53.24 
52.77 
52.30 
51.83 
4 
51.37 
50.90 
50.44 
49.97 
49.50 
49.04 
48.57 
48.10 
47.63 
47.17 
5 
46.70 
46.23 
45.77 
45.28 
44.83 
44.36 
43.90 
43.43 
42.96 
42.50 
6 
42.03 
41.56 
41.10 
40.63 
40.16 
39.70 
39.23 
38.76 
38.29 
37.83 
7 
37.36 
36.89 
36.43 
35.96 
35.49 
35.03 
34.56 
34.09 
33.62 
33.16 
8 
32.69 
32.22 
31.76 
31.25 
30.82 
30.36 
29.89 
29.42 
28.95 
28.49 
9 
28.02 
27.55 
27.09 
26.62 
26.15 
25.68 
25.22 
24.75 
24.28 
23.82 
10 
23.35 
22.88 
22.42 
21.95 
21.48 
21.01 
20.55 
20.08 
19.61 
19.15 
11 
18.60 
18.21 
17.75 
17.28 
16.81 
16.35 
15.88 
15.41 
14.94 
14.48 
12 
14.01 
13.54 
13.08 
12.61 
12.14 
11.68 
11.21 
10.74 
10.27 
9.81 
13 
9.34 
8.87 
8.41 
7.94 
7.47 
7.00 
6.54 
6.07 
5.60 
5.14 
14 
4.67 
4.20 
3.74 
3.27 
2.80 
2.34 
1.87 
1.40 
0.93 
0.47 
Determination of Uric Acid 
(Benedict, S. R., Jour. Biol. Chem., 1922, li, 187) 
Principle.—The tungstic acid blood filtrate is heated in a 
water-bath with a special uric acid reagent. A deep blue color 
is developed, the intensity of which is compared with that of a 
standard uric acid solution similarly treated. 
Reagents.— 
1. Benedict’s Uric Acid Reagent.—One hundred grams of pure 
sodium tungstate are placed in a liter flask and dissolved in about 
600 c.c. of water. Fifty grams of pure arsenic pentoxide are now 136 
CLINICAL LABORATORY METHODS 
added, followed by 25 c.c. of 85 per cent phosphoric acid and 
20 c.c. of hydrochloric acid. The mixture is boiled for 20 min- 
utes, cooled and diluted to 1 liter. The reagent appears to keep 
indefinitely. 
2. Standard Uric Acid Solution.—Dissolve 9.0 grams of pure 
crystalline hydrogen disodium phosphate and 1.0 gram of dihy- 
drogen sodium phosphate in 200 to 300 c.c. of hot distilled water. 
Pour this warm, clear solution on 200 mg. of pure uric acid sus- 
pended in a few c.c. of water in a 1000 c.c. volumetric flask. Agi- 
tate until completely dissolved. Add at once exactly 1.4 c.c. of 
glacial acetic acid. Make up to 1000 c.c. Mix and add 5 c.c. of 
chloroform. One c.c. of this solution contains 0.2 mg. uric acid. 
This stock solution should be freshly prepared every two months. 
The standard solution for use in the determination of uric acid 
in blood is made from the stock solution by measuring 10 c.c., 
containing 2 mg. of uric acid, into a 500 c.c. volumetric flask half 
filled with distilled water. Twenty-five c.c. of dilute hydrochloric 
acid (1 volume concentrated acid diluted to 10 volumes with 
water) are added and the flask filled to the mark with distilled 
water. This solution contains 0.02 mg. uric acid in 5 c.c. The 
solution should be made fresh once in two weeks. 
3. Five per cent sodium cyanide solution containing 2 c.c. of 
concentrated ammonia per liter. This solution should be made 
up fresh once in 2 months. 
Procedure.—The blood is precipitated with tungstic acid as de- 
scribed under the preparation of protein-free blood filtrates (page 
129). The blood should be allowed to stand at least 10 to 20 
minutes after adding the tungstate and sulphuric acid before fil- 
tration. The use of excess acid in the precipitation is to be 
avoided. Five c.c. of the water clear filtrate are transferred to a 
test tube and 5 c.c. of water added. Five c.c. of the standard 
solution containing 0.02 mg. uric acid are placed in another tube 
and the volume likewise made up to 10 c.c. To both standard 
and unknown are added 4 c.c. of the 5 per cent sodium cyanide 
solution containing 2 c.c. of concentrated ammonia per liter. The 
cyanide should always be measured from a burette. To each tube 
is then added 1 c.c. of the uric acid reagent. The contents of 
the tube should be mixed by one inversion after the addition of 
the reagent and placed immediately in boiling water where the QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
137 
tubes should be left 5 minutes after the inversion of the last tube, 
but the time elapsing between the immersion of the first and last 
tubes should not exceed one minute. Exactly three minutes after 
the last tube is immersed the tubes are removed and placed in a 
large beaker of cold water for three minutes and read in a col- 
orimeter against the standard as soon as may be convenient. It 
is best to read within 5 minutes. 
S 
——x 4 = mg. uric acid per 100 c.c. blood, when S represents 
K 
the height of the standard and R the reading of the unknown 
solution. 
Remarks.—The normal figure by this method is about 3 mg. 
uric acid per 100 c.c. blood. No evidence of definite uric acid 
retention is evident before a figure of 4.0 mg. or over per 100 c.c. 
is reached. 
Table XX 
Determination of Uric Acid in Blood 
Take 5 e.c. or 2.5 c.c. of the protein-free blood filtrate and make up to a 
final volume of 15 e.c. 
Use 5 c.c. of the standard uric acid solution containing 0.02 mg. uric acid 
and make up to a final volume of 15 c.c. 
Set standard at 20, or fill 20 mm. Bock-Benedict cell. 
Table shows the uric acid in milligrams per 100 c.c. blood corresponding 
to the different colorimeter readings using a plunger type instrument. 
Using 5 c.c. op Blood Filtrate 
(Urie acid in mg. per 100 c.c. 
blood) 
Using 2.5 c.c. op Blood Filtrate 
(Uric acid in mg. per 100 c.c. 
blood) 
COLORIM- 
ETER 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
READING 
12 
6.7 
6.5 
6.4 
6.3 
6.2 
13.3 
13.1 
12.9 
12.7 
12.5 
13 
6.1 
6.1 
6.0 
5.9 
5.8 
12.3 
12.1 
11.9 
11.8 
11.6 
14 
5.7 
5.6 
5.6 
5.5 
5.4 
11.4 
11.3 
11.1 
10.9 
10.8 
15 
5.3 
5.3 
5.2 
5.1 
5.1 
10.7 
10.5 
10.4 
10.2 
10.1 
16 
5.0 
4.9 
4.9 
4.8 
4.8 
10.0 
9.9 
9.8 
9.6 
9.5 
17 
4.7 
4.7 
4.6 
4.5 
4.5 
9.4 
9.3 
9.2 
9.1 
9.0 
18 
4.4 
4.4 
4.4 
4.3 
4.2 
8.9 
8.8 
8.7 
8.6 
8.5 
19 
4.2 
4.2 
4.1 
4.1 
4.0 
8.4 
8.4 
8.3 
8.2 
8.1 
20 
4.0 
4.0 
3.9 
3.9 
3.8 
8.0 
7.9 
7.8 
7.7 
7.6 
21 
3.8 
3.8 
3.7 
3.7 
3.7 
7.6 
7.5 
7.5 
7.4 
7.3 
22 
3.6 
3.6 
3.6 
3.5 
3.5 
7.3 
7.2 
7.2 
7.1 
7.0 
23 
3.5 
3.4 
3.4 
3.4 
3.4 
6.9 
6.9 
6.8 
6.8 
6.7 
24 
3.3 
3.3 
3.3 
3.3 
3.3 
6.7 
6.6 
6.6 
6.5 
6.4 
25 
3.2 
3.2 
3.2 
3.1 
3.1 
6.4 
6.4 
6.3 
6.2 
6.2 
26 
3.1 
3.1 
3.0 
3.0 
3.0 
6.2 
6.1 
6.1 
6.0 
6.0 
27 
2.9 
2.9 
2.9 
2.9 
2.9 
5.9 
5.9 
5.8 
5.8 
5.8 138 
CLINICAL LABORATORY METHODS 
If the reading is high it is best to repeat the determination 
using 2.5 c.c. of blood filtrate instead of 5 c.c. 
It is essential that the volume of the unknown and of the 
standard be the same during the period of the reaction. Hence, 
if only 2.5 c.c. of filtrate be used, distilled water must be added 
to bring the volume up to 10 c.c. before the addition of the cya- 
nide and reagent. 
A method for checking uric acid determinations is given on 
page 251. 
Determination of Creatinine 
(Folin and Wu: Jour. Biol. Chem., 1919, xxxviii, 81) 
Principle.—On adding picric acid and sodium hydroxide to a 
solution containing creatinine, a deep red color is produced. The 
intensity of this in the protein-free blood filtrate is compared 
with that of a standard creatinine solution similarly treated. 
Reagents.—(1) Alkaline picrate solution made by adding 5 
c.c. of 10 per cent sodium hydroxide to 25 c.c. of saturated picric 
acid solution. The picric acid should be tested as described 
under “Determination of Creatinine in Urine,” page 52. 
(2) Standard creatinine solution prepared as follows: 
Transfer to a liter flask 6 c.c. of a standard creatinin solution 
containing 1 mg. of creatinine per cubic centimeter (see page 52) ; 
add 10 c.c. of normal hydrochloric acid, dilute to mark with 
water and mix. Transfer to a bottle, add 4 to 5 drops xylol. Five 
c.c. of this solution contain 0.03 mg. of creatinine and this amount 
plus 15 c.c. of water represents the standard needed for most 
bloods, as it covers the range of 1 to 2 mg. per 100 c.c. If needed 
10 c.c. of this standard and 10 c.c. of water may be used for 
the range of 2 to 4 mg. per 100 c.c.; 15 c.c. of the standard 
and 5 c.c. of water for the range of 4 to 6 mg. per 100 c.c.; or 
20 c.c. of the standard for the range of 6 to 8 mg. per 100 c.c. 
of blood. 
Procedure.—Transfer 10 c.c. of protein-free blood filtrate (see 
page 129) to a small flask or test tube, transfer 5 centimeters of 
the standard creatinine solution to another flask and dilute the 
standard to 20 c.c. Then add 5 c.c. of the freshly prepared 
alkaline picrate solution to the blood filtrate and 10 c.c. to the QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
139 
USING 5 C. C. STANDARD 
(Creatinine in mg. 
per 100 c. c. blood) 
USING 10 C. C. STANDARD 
(Creatinine in mg. 
per 100 e. c. blood) 
USING 15 C. C. STANDARD 
(Creatinine in mg. 
per 100 c. c. blood) 
USING 20 C. C. STANDARD 
(Creatinine in mg. 
per 100 c. c. blood) 
Colorimeter 
Reading 
0 
0 
0 
2 
0 
4 
0 
6 
0 
8 
0 
0 
0 
2 
0 
4 
0 
6 
0 
8 
0 
0 
0 
2 
0 
4 
0 
6 
0 
8 
0 
0 
0 
2 
0 
4 
0 
6 
0 
8 
10 
3 
0 
2 
9 
2 
9 
2 
8 
2 
8 
6 
0 
5 
9 
5 
8 
5 
6 
5 
5 
9 
0 
8 
9 
8 
7 
8 
5 
8 
3 
12 
0 
11 
8 
11 
6 
11 
3 
11 
1 
11 
2 
7 
2 
7 
2 
6 
2 
6 
2 
5 
5 
5 
5 
4 
5 
3 
5 
2 
5 
1 
8 
2 
8 
0 
7 
9 
7 
8 
7 
6 
10 
9 
10 
7 
10 
5 
10 
4 
10 
2 
12 
2 
5 
2 
5 
2 
4 
2 
4 
2 
3 
5 
0 
4 
9 
4 
8 
4 
8 
4 
7 
7 
5 
7 
4 
7 
3 
7 
1 
7 
0 
10 
0 
9 
8 
9 
7 
9 
5 
9 
4 
13 
2 
3 
2 
3 
2 
3 
2 
2 
2 
2 
4 
6 
4 
6 
4 
5 
4 
4 
4 
4 
6 
9 
6 
8 
6 
8 
6 
7 
6 
5 
9 
2 
9 
1 
9 
0 
8 
9 
8 
7 
14 
2 
1 
2 
1 
2 
1 
2 
1 
2 
0 
4 
3 
4 
2 
4 
2 
4 
1 
4 
1 
6 
4 
6 
3 
6 
2 
6 
2 
6 
1 
8 
6 
8 
5 
8 
3 
8 
2 
8 
1 
15 
2 
0 
2 
0 
2 
0 
1 
9 
1 
9 
4 
0 
3 
9 
3 
9 
3 
9 
3 
8 
6 
0 
5 
9 
5 
9 
5 
8 
5 
7 
8 
0 
7 
9 
7 
8 
7 
7 
7 
6 
16 
1 
9 
1 
9 
1 
8 
1 
8 
1 
8 
3 
8 
3 
7 
3 
7 
3 
6 
3 
6 
5 
6 
5 
6 
5 
5 
5 
4 
5 
4 
7 
5 
7 
4 
7 
3 
7 
2 
7 
1 
17 
1 
8 
1 
8 
1 
7 
1 
7 
1 
7 
3 
5 
3 
5 
3 
5 
3 
4 
3 
4 
5 
3 
5 
3 
5 
2 
5 
1 
5 
1 
7 
1 
7 
0 
6 
9 
6 
8 
6 
7 
18 
1 
7 
1 
7 
1 
6 
1 
6 
1 
6 
3 
3 
3 
3 
3 
3 
3 
2 
3 
2 
5 
0 
5 
0 
4 
9 
4 
9 
4 
8 
6 
7 
6 
6 
6 
5 
6 
5 
6 
4 
19 
1 
6 
1 
6 
1 
6 
1 
5 
1 
5 
3 
2 
3 
1 
3 
1 
3 
1 
3 
0 
4 
7 
4 
7 
4 
7 
4 
6 
4 
6 
6 
3 
6 
2 
6 
2 
6 
1 
6 
1 
20 
1 
5 
1 
5 
1 
5 
1 
5 
1 
5 
3 
0 
3 
0 
2 
9 
2 
9 
2 
9 
4 
5 
4 
5 
4 
4 
4 
4 
4 
4 
6 
0 
5 
9 
5 
9 
5 
8 
5 
8 
21 
1 
4 
1 
4 
1 
4 
1 
4 
1 
4 
2 
9 
2 
8 
2 
8 
2 
8 
2 
8 
4 
3 
4 
2 
4 
2 
4 
2 
4 
1 
5 
7 
5 
6 
5 
6 
5 
5 
5 
5 
22 
1 
4 
1 
4 
1 
3 
1 
3 
1 
3 
2 
7 
2 
7 
2 
7 
2 
7 
2 
6 
4 
1 
4 
1 
4 
0 
4 
0 
4 
0 
5 
5 
5 
4 
5 
4 
5 
3 
5 
3 
23 
1 
3 
1 
3 
1 
3 
1 
3 
1 
3 
2 
6 
2 
6 
2 
6 
2 
5 
2 
5 
3 
9 
3 
9 
3 
9 
3 
8 
3 
8 
5 
2 
5 
2 
5 
1 
5 
1 
5 
0 
24 
1 
3 
1 
2 
1 
2 
1 
2 
1 
2 
2 
5 
2 
5 
2 
5 
2 
4 
2 
4 
3 
8 
3 
7 
3 
7 
3 
7 
3 
6 
5 
0 
5 
0 
4 
9 
4 
9 
4 
8 
25 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
2 
4 
2 
4 
2 
4 
2 
3 
2 
3 
3 
6 
3 
6 
3 
5 
3 
5 
3 
5 
4 
8 
4 
8 
4 
7 
4 
7 
4 
7 
26 
1 
2 
1 
2 
1 
1 
1 
1 
1 
1 
2 
3 
2 
3 
2 
3 
2 
3 
2 
3 
3 
5 
3 
5 
3 
4 
3 
4 
3 
4 
4 
6 
4 
6 
4 
6 
4 
5 
4 
5 
27 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
3 
4 
3 
3 
3 
3 
3 
3 
3 
2 
4 
5 
4 
4 
4 
4 
4 
4 
4 
3 
28 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
3 
2 
3 
2 
3 
2 
3 
2 
3 
1 
4 
3 
4 
3 
4 
2 
4 
2 
4 
2 
29 
1 
0 
1 
0 
1 
0 
1 
0 
1 
0 
2 
1 
2 
1 
2 
0 
2 
0 
2 
0 
3 
1 
3 
1 
3 
1 
3 
1 
3 
0 
4 
1 
4 
1 
4 
1 
4 
1 
4 
0 
Use 10 c.c. of protein-free blood filtrate made up to a final volume of 15 c.c. 
Make final volume of the standard up to 30 c.c. 
Set standard at 20, or fill 20 mm. Bock-Benedict cell. 
The following table shows the creatinine in mg. per 100 c.c. blood corresponding to the different readings with 5, 10, 15 or 20 
c.c., of the standard creatinine solution, using a plunger type colorimeter. 
Determination of Creatinine in Blood 
Table XXI 140 
CLINICAL LABORATORY METHODS 
diluted creatinine solution. Let stand for eight to ten minutes 
and compare in colorimeter. Never omit to first ascertain that 
the two fields of this colorimeter are equal when both cups con- 
tain this standard creatinine picrate solution. The color com- 
parison should be completed within 15 minutes from the time 
the alkaline picrate was added. 
Calculation.—Set standard at 20. If R is the reading of the 
unknown, - = miUigrams of creatinine in 100 c.c. of 
blood provided 5 c.c. of the standard has been used. If 10 c.c. 
of standard be taken multiply by 3 instead of 1.5; if 15 c.c., 
multiply by 4.5; if 20 c.c., multiply by 6. 
Remarks.—The blood of a normal individual contains 1 to 
2 mg. creatinine per 100 c.c. 
If the creatinine content is near normal the colors are so light 
it is difficult to match them well. If the standard solution is 
made up in saturated picric acid solution and the protein-free 
blood filtrate is saturated with picric acid by shaking with a few 
crystals for 5 minutes before adding the other reagents the color 
will be darker and the readings made easier. 
A method for checking creatinine determinations is given on 
page 251. 
If the readings are high the test should be repeated using a 
larger amount of standard. 
Table XXI shows the creatinine corresponding to the different 
readings with a plunger type colorimeter. 
Determination of Creatine 
(Folin and Wu: Jour. Biol. Chem., 1919, xxxviii, 81) 
Principle.—On heating creatine with dilute mineral acids, it 
is dehydrated and its anhydride creatinine formed. The cre- 
atinine is then determined as for preformed creatinine. 
Procedure.—Transfer 5 c.c. of the protein-free blood filtrate 
to a test tube graduated at 25 c.c. Add 1 c.c. of normal hydro- 
chloric acid. Cover the mouth of this test tube with tin foil and 
heat in autoclave to 130° C. for 20 minutes, cool, and add 5 c.c. 
of alkaline picrate solution prepared by adding 5 c.c. of 10 per 
cent sodium hydroxide to 25 c.c. saturated picric acid solution. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
141 
Let stand for 8 to 10 minutes, then dilute to 25 c.c. The stand- 
ard solution required is 30 c.c. of creatine solution made as de- 
scribed under creatinine determination in a 50 c.c. volumetric 
flask. Add to this 2 c.c. of normal acid and 10 c.c. of the al- 
kaline picrate solution, and after 10 minutes’ standing, dilute 
to 50 c.c. 
Calculation.—Set standard at 20. 
If R be the reading of the unknown solution. 
= creatine plus creatinine in milligrams per 100 
R 
c.c. of blood. 
Remarks.—The normal value for total creatinine by this 
method is about 6 mg. per 100 c.c. of blood. 
Determination of Sugar 
(Folin and Wu: Jour. Biol. Chem., 1919, xxxviii, 81; xli, 367) 
Principle.—A weakly alkaline copper tartrate solution on heat- 
ing with the tungstic acid blood filtrate is reduced by the glucose 
contained therein to cuprous oxide. On adding a special molyb- 
date-phosphate reagent an intense blue color is developed by the 
action of the reagent on the cuprous oxide. This is compared 
with a standard solution of glucose similarly treated. 
Reagents.— 
1. Standard sugar solution. Dissolve one gram of pure an- 
hydrous dextrose in water and dilute to a volume of 100 c.c. 
Mix, add a few drops of xylol or toluol, and bottle. Dilute 5 
c.c. of this stock solution to 500 c.c., giving a solution containing 
1 mg. of dextrose per 10 c.c. Similarly dilute 10 c.c. to 500 c.c., 
giving a solution containing 2 mg. of dextrose per 10 c.c. The 
stock solution is permanent; the dilutions from the stock solu- 
tion should be made each month. 
2. Alkaline copper solution. Dissolve 40 grams of anhy- 
drous sodium carbonate in about 400 c.c. of water and transfer 
to a liter flask. Add 7.5 grams of tartaric acid and when the 
latter has dissolved add 4.5 grams of crystallized copper sul- 
phate; mix and make up to a volume of one liter. If the car- 
bonate used is impure, a sediment may be formed in the course 142 
CLINICAL LABORATORY METHODS 
of a week or two. If this happens, decant the clear solution into 
another bottle. Two c.c. of this solution when mixed with 2 e.c. 
of the molybdate phosphate reagent, should show no color. Two 
c.c. should be decolorized by 1.4 c.c. normal acid. 
3'. Molybdate-phosphate solution. Transfer to a liter beaker 
35 grams of molybdic acid and 5 grams of sodium tungstate. 
Add 200 c.c. of 10 per cent sodium hydroxide and 200 c.c. of 
water. Boil vigorously for 20 to 40 minutes so as to remove 
nearly the whole of the ammonia present in the molybdic acid. 
(Test with litmus paper.) Cool, dilute to about 350 c.c. and add 
125 c.c. of concentrated (85 per cent) phosphoric acid. Dilute 
to 500 c.c. 
Fig. 44.—Folin blood sugar tube. 
Procedure.—Transfer 2 c.c. of the tungstic acid blood filtrate 
(see page 129) to a special Folin blood sugar tube (Fig. 44), grad- 
uated to 25 c.c. To two other similar tubes add 2 c.c. of stand- 
ard sugar solution containing 0.2 and 0.4 mg. of dextrose 
respectively. To each tube add 2 c.c. of the alkaline copper tar- 
trate solution. The surface of the solution must now have reached 
the restricted portion of the tube. If the bulb is too large, a little 
but not more than 0.5 c.c. of 1:1 dilution of the copper solution 
may be added. Heat the tubes in boiling water-bath for six 
minutes. Cool in water-bath without shaking for 2 to 3 minutes. 
Add 2 c.c. of the phosphate molybdate solution. After the 
cuprous oxide is dissolved, dilute to 25 c.c. mark. Insert a 143 
QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
rubber stopper in the tube and mix. Compare the unknown solu- 
tion in the colorimeter with the standard set at 20 millimeters. 
Calculation.'— 
If R is reading of unknown: 
20 
x 100 = milligrams of glucose per 100 c.c. of blood 
when the wTeaker standard is used. 
20 
x 200 == milligrams of glucose per 100 c.c. of blood 
when the stronger standard is used. 
Determination of Glucose in Blood 
Table XXII 
IJse 2 c.c. of the protein-free blood filtrate made up to 25 c.c. and 2 
c.c. of the standard made up to a similar volume. 
Set standard at 20 or fill 20 mm. Bock-Benedict ceil. 
Table shows the glucose in mg. per 100 c.c. blood corresponding to 
colorimeter readings with the two different standards, using an instrument 
of the plunger type. 
STANDARD NO. 1 CONTAINING 
1 MG. GLUCOSE IN 10 C.C. 
(Glucose in mg. per 
100 c.c. blood). 
STANDARD NO. 2 CONTAINING 
2 MG, GLUCOSE IN 10 C.C. 
(Glucose in mg. per 
100 c.c. blood). 
COLORIMETER 
READING 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
5 
400 
385 
370 
357 
345 
800 
769 
740 
714 
689 
6 
333 
323 
313 
303 
294 
667 
645 
625 
606 
588 
7 
286 
278 
270 
263 
256 
571 
555 
540 
526 
512 
8 
250 
244 
238 
233 
227 
500 
487 
476 
465 
454 
9 
222 
217 
213 
209 
204 
444 
434 
425 
417 
408 
10 
200 
196 
192 
189 
185 
400 
392 
384 
377 
370 
11 
182 
179 
175 
172 
169 
363 
357 
350 
344 
338 
12 
167 
164 
161 
159 
156 
333 
327 
322 
317 
312 
13 
154 
152 
149 
147 
145 
307 
303 
298 
295 
290 
14 
143 
141 
139 
137 
135 
286 
282 
278 
273 
270 
15 
133 
132 
130 
128 
127 
267 
263 
260 
256 
254 
16 
125 
124 
122 
121 
119 
250 
247 
244 
241 
238 
17 
118 
116 
115 
114 
112 
236 
233 
230 
227 
224 
18 
111 
110 
109 
108 
106 
222 
220 
218 
215 
212 
19 
105 
104 
103 
102 
101 
211 
209 
206 
204 
202 
20 
100 
99 
98 
97 
96 
200 
198 
196 
194 
192 
21 
95 
94 
93 
93 
91 
189 
188 
187 
185 
183 
22 
91 
90 
89 
88 
87 
182 
180 
179 
177 
175 
23 
87 
86 
86 
85 
84 
173 
172 
171 
169 
.168 
24 
83 
83 
82 
81 
81 
167 
165 
164 
163 
161 
25 
80 
79 
79 
78 
78 
160 
159 
158 
156 
155 
26 
77 
76 
76 
75 
75 
154 
153 
152 
150 
149 
27 
74 
74 
73 
73 
72 
147 
147 
146 
145 
144 
28 
71 
71 
71 
70 
69 
142 
142 
140 
140 
139 
29 
69 
69 
68 
68 
67 
137 
137 
136 
135 
134 144 
CLINICAL LABORATORY METHODS 
Remarks.—The blood of normal fasting individuals contains 
80 to 120 mg. glucose per 100 c.c. 
Table XXII shows the blood sugar values corresponding to 
the different colorimeter readings, with a plunger type of instru- 
ment. 
A method for checking blood sugar determinations is given on 
page 250. 
(Janney and Isaacson: Jour. Amer. Med. Assn., 1918, lxx, 1131) 
Principle.—Glucose is taken by mouth and the blood sugar is 
determined at regular intervals. The patient takes no food after 
the evening meal of the day preceding the test. 
Procedure—Five c.c. of blood are obtained by venipuncture 
and run into a test tube, containing a few crystals of potassium 
oxalate. One and five-tenths grams of glucose are given for 
each kilogram of body weight. The glucose is best given in a 
50 per cent solution flavored with lemon. Five c.c. of blood 
are obtained at 30 minute intervals for the next hour, and at 
the end of two hours, three hours and of four hours. A blood 
sugar determination is made on each specimen by the Folin 
method (page 141). A specimen of urine is collected each time 
the blood is taken and tested for sugar by Benedict’s solution. 
The results are recorded as follows: 
Blood Sugar Tolerance Test 
Sugar Tolerance Test ( 
grams glucose given by mouth) 
Blood, sugar per 
100 c.c. 
Urine sugar per cent 
Fasting 
Mgms. 
per cent 
30 Min. 
1 i 
<< 
60 Min. 
C < 
< i 
2 Hrs. 
6 ( 
( t 
3 Hrs. 
i L 
4 Hrs. 
«i 
( i 
Remarks.—The typically normal blood sugar tolerance curve 
shows the following: 
1. Blood sugar before the glucose is taken 70-120 mg. per 
100 c.c. 
2. Blood sugar within an hour after taking the glucose rises 
to 130 to 180 mg. per 100 c.c., this representing the peak of the 
curve. 145 
QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
3. Blood sugar returns to normal fasting level within 2 to 2y2 
hours. 
The urine shows no sugar normally. 
Fig. 45 shows several types of curves obtained in blood sugar 
tolerance tests. 
Blood Sugnr (Mgms. per 100 c.c. blood) 
Time in hours after ingestion of glucose 
Fig. 45.—Types of curves obtained in blood sugar tolerance test, 
Curve of a normal individual. 
Curve of a mild diabetic. 
Curve of a severe diabetic. 
Determination of Chlorides 
(McLean and Van Slyke, Jour. Biol. Chem., 1915, xxi, 361; 
Gettler, A.O., Jour. Am. Med. Assn., 1921, lxxvii, 1652) 
The chlorides may be determined in whole blood or in plasma. 
The chlorides are not uniformly distributed in blood and plasma, 
the whole blood containing a smaller amount. Hence the varia- 
tion in plasma chlorides is more marked. It is more accurate, 
however, to make the determination on whole blood since unless 
the plasma is separated from the red cells almost immediately 
after withdrawal, its chlorides increase at the expense of the 146 
CLINICAL LABORATORY METHODS 
corpuscles due to the passage of carbon dioxide from plasma to 
the corpuscles or its escape into the air. 
Principle.—The proteins of the blood or plasma are precipi- 
tated with sodium tungstate and sulphuric acid as described 
under the preparation of protein-free blood filtrates (page 129). 
The chlorides in the filtrate are precipitated with silver nitrate, 
the silver chloride is filtered off, and the excess silver nitrate 
determined by titration with potassium iodide in the presence of 
nitrous acid and starch. 
Reagents.—(1) AgN03 solution (1 c.c. equivalent to 2 mg. 
NaCl). 
Silver nitrate 5.812 grains 
Nitric acid (sp. gr. 1.42) 250.0 c.c. 
Water to 1000.0 c.c. 
(2) M/117 potassium iodide solution (4 c.c. equivalent to 1 c.c. 
of the silver nitrate). 
Potassium iodide 1.419 grams 
Water to 1000.0 c.c. 
The potassium iodide crystals are dissolved in less than a liter 
of water and standardized by adding 5 c.c. of the starch solution 
to 5 c.c. of the silver solution and titrating with the iodide to the 
first appearance of green color. The solution of potassium iodide 
is then diluted so that 20 c.c. are exactly equivalent to 5 c.c. of 
the silver solution. 
(3) Starch solution: 
Sodium citrate (Na3C6H507 plus 5-J H20) .... 446.0 grams 
Soluble starch 2.5 grams 
Sodium nitrite 20.0 grams 
Water to 1000.0 c.c. 
Dissolve the starch in 500 c.c. of warm water, add the salts, 
heat to the boiling point, and boil vigorously for ten minutes. 
While still hot, filter through cotton, wash the filter with hot 
water, and after cooling, make filtrate up to 1000 c.c. The citrate 
solution becomes cloudy on standing, but keeps indefinitely. It 
is desirable to add a few cubic centimeters of chloroform to pre- 
vent the growth of moulds in the solution. The citrate is neces- 
sary to regulate the acidity for the endpoint, the nitrite to lib- 
erate the iodine from the iodide. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
147 
Procedure.—Pipette 20 c.c. of tlie protein-free filtrate from 
whole blood or plasma, prepared as described on page 129, into 
a dry beaker. If the determination is made on plasma, for each 
volume of plasma eight volumes of water and one-half volume 
each of 10 per cent sodium tungstate and % normal sulphuric acid 
are added; that is, for 4 c.c. of plasma, 32 c.c. of water, and 2 c.c. 
each of the tungstate and acid would be added. Add 10 c.c. of 
the silver nitrate solution. Shake and allow to stand at least ten 
minutes. It is preferable to allow it to stand overnight. Centri- 
fuge or filter through a dry filter paper. Pipette 15 c.c. into a 
casserole or beaker, add 5 c.c. of the starch citrate solution and 
titrate with the M/117 potassium iodide to the first ap- 
pearance of green color. As the endpoint is approached it is 
advisable, in order to avoid overrunning it, to pause after the 
addition of each drop of iodide, because the green color requires 
several seconds to develop. 
Calculation.—The 10 c.c. of filtrate used in the determination 
are equivalent to 1 c.c. of plasma or blood: 
c cKI 
NaCl in mg. per 100 c.c. = (10 —-) 100 
Remarks.—Plasma normally contains 570 to 620 mg. of chlo- 
rides calculated as sodium chloride per 100 c.c.; whole blood con- 
tains 470 to 520 mg. 
In the titration the final acidity is the most important factor. 
The green color is best developed'in slightly acid solution. Too 
much acid abolishes the blue or green color leaving only the 
brown color of the iodine liberated by the nitrous acid. If the 
solution is not acid enough no color at all will be formed because 
it is necessary for nitrous acid to be present to liberate hydrogen 
iodide from the potassium iodide. When a blue color is formed 
after the addition of each drop of iodide during the titration it 
is an indication that the optimum acidity is approximated. 
The sodium tungstate solution used in the determination should 
be checked against contamination by chloride. This is done by 
mixing one volume of the 10 per cent sodium tungstate with two 
volumes of concentrated nitric acid and filtering into a test tube 
containing silver nitrate. There should be no cloud. 
The tungstic acid filtrate should give no precipitate with an 
equal volume of nitric acid. 148 
CLINICAL LABORATORY METHODS 
Table XXIII shows the chloride corresponding to the amount 
of potassium iodide used in titrating the excess of silver nitrate. 
A method for checking chloride determinations is given on 
page 252. 
Table XXIII 
Determination of Chlorides in Blood 
Take 15 c.c. of the filtrate after the addition of silver nitrate. 
Table shows the number of milligrams of NaCl per 100 c.c. of plasma cor- 
responding to the number of c.c. of KI used to titrate the excess of silver 
nitrate. 
M H M m H 
P hrf O 
rfi.COtO|_iO<X>00~qC5Clt|i. 
1 2 P 
o 
OOlOOlOOlOCnOUlO 
ooooooooooo 
botowt£.£*.CNOi®>a5~3~ci 
o 
O^O^Orf^Orf^Ohf'-O 
oioioioioioiuoiaoiw 
taww^^wcics®^^ 
o 
O^O^-O^O^Orf^CD 
oooooooooo o 
f003 05^^0lCl0505^~^ 
o 
OOOJOOOJCOWCDMlXICOOO 
OtUlUimOlOlCriCNWOlCn 
Mww^^woiaoisM 
00 CO GO CO 00 CO 00 CO GO CO 00 
ooooooooooo 
K)03W^^O!ClO50i<JS 
o 
'1 L0 -q N> -q tO -q tO -q tO 
ClUlOlOlUlOlOlOlOlOlOl 
nojwifk^Maaa^s 
o 
S»S»SMSM<|KIN 
ooooooooooo 
toosw^^oioiaa^s 
o 
QMOJMOiMCiHOJHOi 
ox Ol Ol 01 o\ o\ Ol o\ Ol o\ V\ 
bOWOOLli.^010l0505~4-<) 
ooooooooooo 
GO 
tswWrfi^aoiaaMS 
o 
oiboiowowooiooi 
moio\oiuioioia<oioicn 
(Myers and Wardell: Jour. Biol. Chem., 1918, xxxvi, 147) 
Principle.—The blood plasma is mixed with plaster of Paris, 
dried, and extracted with chloroform. The depth of color pro- 
duced on mixing the chloroform extract with acetic anhydride 
and concentrated sulphuric acid is compared with that of a stand- 
ard solution of cholesterol similarly treated. 
Reag’ents.—Standard stock solution of cholesterol made by dis- 
solving 1 gram of chemically pure cholesterol in 1,000 c.c. of an- 
hydrous chloroform. 
Dilute with chloroform 100 c.c. of this stock solution to 1,000 
c.c., giving a solution 5 c.c. of which contains 0.5 mg. cholesterol. 
Procedure.—One c.c. of blood plasma is pipetted into a por- 
celain crucible, or small beaker containing 4 to 5 grams of plaster 
of Paris, stirred, and dried, preferably in a drying oven. It is 
now emptied into a small extraction shell (4 cm. long) and then 
inserted in a short test tube in the bottom and top of which are 
Determination of Cholesterol QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
149 
a number of small holes. This is attached to a large cork 
on a small reflux condenser, and the tube and cork are inserted 
in the neck of a 150 c.c. extraction flask (Fig. 46) containing 
Fig. 46.—Cholesterol extraction apparatus. (After Myers.) 
about 20 to 25 c.c. of chloroform. Extraction is continued for 
30 minutes on an electric hot plate, the chloroform made up to 
some suitable volume such as 15 c.c., filtered if necessary, and 
colorimetric estimation carried out as follows: 5 c.c. of the 150 
CLINICAL LABORATORY METHODS 
chloroform extract are pipetted into a dry test tube and into a 
second tube 5 c.c. of the standard solution containing 0.5 mg. 
of cholesterol. Add to each tube, 2 c.c. of acetic anhydride, and 
0.1 c.c. of anhydrous concentrated sulphuric acid. After thor- 
ough mixing, the solution is placed in the dark for exactly 10 
minutes to allow the green color to develop. Compare the two 
solutions in the colorimeter. 
Calculation.— 
S V 
— x 0.5 x —p~x 100 =mg. cholesterol per 100 c.c. of plasma. 
it 5 
Where S = reading of standard 
R == reading of unknown 
V = volume to which the chloroform extract is made up. 
Determination of Cholesterol in Blood 
Table XXIV 
Use 1 c.c. of blood plasma, extract with chloroform and make up to 15 
c.c. Compare 5 c.c. or 2.5 c.c. of this extract with a standard containing 
0.5 gm. cholesterol in 5 c.c. making the volume in each case up to 7.1 c.c. 
Set standard at 20 or fill 20 mm. Bock-Benedict celL 
Table shows the; cholesterol in mg. per 100 c.c. of plasma corresponding 
to the different colorimeter readings with a plunger type instrument, using 
different amounts of the chloroform extract. 
USING 5 C.C. OF THE 
CHLOROFORM EXTRACT 
(Cholesterol in mg. 
per 100 c.c. plasma). 
USING 2.5 C.C. OF THE 
CHLOROFORM EXTRACT 
(Cholesterol in mg. 
per 100 c.c. plasma). 
COLORIMETER 
READING 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
10 
300 
294 
289 
282 
277 
600 
588 
577 
564 
554’ 
11 
273 
268 
263 
259 
254 
546 
536 
526 
518 
508 
12 
250 
246 
242 
238 
234 
500 
492 
484 
476 
468 
13 
230 
228 
225 
222 
218 
460 
456 
450 
444 
436 
14 
214 
211 
208 
205 
203 
428 
422 
416 
410 
406 
15 
200 
198 
195 
193 
190 
400 
395 
389 
385 
380 
16 
188 
185 
183 
181 
179 
375 
370 
366 
361 
357 
17 
177 
175 
173 
171 
169 
353 
349 
345 
341 
337 
18 
167 
165 
163 
162 
159 
333 
330 
326 
323 
319 
19 
158 
156 
155 
153 
152 
316 
312 
309 
306 
303 
20 
150 
149 
147 
146 
145 
300 
297 
294 
291 
289 
21 
143 
141 
140 
139 
138 
285 
282 
280 
277 
275 
22 
137 
135 
134 
133 
132 
273 
270 
268 
265 
263 
23 
131 
130 
129 
127 
126 
261 
259 
257 
255 
252 
24 
125 
124 
123 
122 
121 
250 
248 
246 
244 
242 
25 
120 
119 
118 
117 
117 
240 
238 
236 
234 
233 
26 
115 
115 
114 
114 
113 
230 
229 
228 
227 
225 
27 
111 
111 
110 
109 
108 
224 
222 
220 
218 
216 
28 
107 
107 
106 
105 
104 
215 
213 
211 
210 
208 
29 
103 
103 
102 
102 
101 
207 
205 
204 
203 
202 QUANTITATIVE CHEMICAL EXAMINATION OP BLOOD 
151 
Remarks.—Normal blood contains 140 to 170 mg. cholesterol 
per 100 c.c. (plasma). 
For the proper color development it is necessary that the acetic 
anhydride, the chloroform, and the sulphuric acid be anhydrous. 
The acetic anhydride may be redistilled using the distillate be- 
tween 134° C. and 140° C. 
Table XXIY shows the cholesterol values corresponding to 
different colorimeter readings, with a plunger type instrument. 
A method for checking cholesterol determinations is given on 
page 252. 
Determination of Phosphates 
(Bloor, W. R.: Jour. Biol. Chem., 1918, xxxvi, 31) 
Principle.—The determination of phosphates is based on the 
precipitation of the phosphoric acid by strychnine molybdate 
as strychnine phosphomolybdate and the measurement of the 
amount of precipitate by comparing it nephrometrically with the 
precipitate produced, under conditions as nearly identical as 
possible, in a standard phosphate solution. 
Reagents.—1. Strychnine Molybdate Solution.—A solution 
of sodium molybdate is first prepared as follows: 72 grams of pure 
molybdic acid are mixed with about 300 c.c. of water and neutral- 
ized with 40 per cent sodium hydroxide (free from all but traces 
of phosphate). Pure acid requires the theoretical amount of 
100 c.c.; impure samples require less. The molybdate, now in 
clear solution, is boiled for one half an hour, adding water to 
beep the volume constant, and alkali if the solution becomes 
turbid. About 1 gram of talcum powder is added, and after a 
further 5 minutes’ boiling, the solution is filtered, and the filter 
washed once with hot water, adding the washings to the main 
filtrate. After cooling, the solution containing approximately 
100 grams of sodium molybdate is ready for use. Sufficient of 
the sodium molybdate solution is taken to contain 30 to 35 gm. 
of sodium molybdate (or this amount of dry sodium molybdate 
dissolved in a small amount of water) is measured into a pre- 
cipitating jar or large beaker (2 liters), and 250 c.c. of a mixture 
of equal parts of concentrated HC1 and water are added with 
stirring. Five hundred c.c. of water are mixed with the solution 152 
CLINICAL LABORATORY METHODS 
and 40 to 50 c.c. of saturated (about 3 per cent) strychnine sul- 
phate solution slowly added with stirring. Two hundred c.c. 
more of the dilute acid and 500 c.c. more water are added, and 
after mixing, the turbid solution is allowed to stand overnight 
or longer if convenient. After the precipitate has settled, most 
of the liquid may be poured off clear. The remainder is filtered 
through a hardened phosphorus-free filter. For use in the deter- 
mination, 25 c.c. of this solution are taken without further ad- 
ditions. 
2. Acid Ammonium Sulphate.—Saturated ammonium sulphate, 
free from all traces of phosphate, to which has been added 15 
c.c. of glacial acetic acid per liter. 
3. Standard Phosphate Solution.— 
(a) Stock standard, containing in 100 c.c. 0.0834 gram of pure 
acid potassium phosphate. 
(b) Standard for use: Dilute 25 c.c. of the above to 500 c.c. 
Each 5 c.c. of this standard contains 0.15 mg. of H3P04. The 
dilute solution deteriorates in hot weather and should be made at 
least once a month. 
4. Sodium Hydroxide (from sodium) 10 to 20 per cent. 
5. Concentrated Sulphuric Acid and Nitric Acid free from all 
but traces of phosphates. 
6. Dilute Sulphuric Acid.—One part of the concentrated acid 
and 3 parts of water. 
Blanks run with the acids, alkalies, and ammonium sulphate 
should show only a slight cloudiness on standing for one hour 
with the reagent. The water should be stored in glass. 
(1) Procedure for Determination of Total Phosphates in 
Plasma.—One half c.c. of plasma is measured with a 0.5 c.c. 
Ostwald pipette into a large (200 x 20 mm.) test tube, four glass 
beads about 3 mm. in diameter, and 1.5 c.c. of a mixture of con- 
centrated sulphuric acid and nitric acid are added, and the whole 
heated with a microburner in the hood. The heating is carried 
out in three stages. In the first stage the mixture is raised to 
boiling and then the flame is turned down until only a slow but 
continuous bubbling takes place. Heating is continued at this 
rate until red fumes cease to come off. This ordinarily does not 
require more than fifteen minutes. In the second stage of heat- QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
153 
mg the flame is increased until the water is driven off and strong 
heating with volatilization of a part of the sulphuric acid is con- 
tinued for 8 to 10 minutes, taking care not to heat so strongly 
that the tube approaches dryness, in which case loss of phosphoric 
acid may occur. The sulphuric acid solution should be now clear 
and colorless. If it is brownish in color a drop of UNO, should 
be added and the heating continued for one minute. In the third 
stage the mixture is allowed to cool somewhat (say for about 2 
minutes) and then one or more drops of one per cent cane sugar 
are added. (The amount added should be enough to produce a 
deep browning of the hot solution and the color should disappear 
when it is boiled. If too much sugar has been added, and the 
brown or yellow color persists after a half minute of cooling, a 
trace of nitric acid should be added and the boiling continued). 
The solution is then boiled until the moisture is gone—about one 
minute—then cooled and about 10 c.c. of water are added, rins- 
ing down the sides of the tube. The solution in the tube is 
neutralized by approximate titration with 10 per cent NaOH 
(from sodium) using one drop of 0.3 per cent phenolphthalein as 
indicator, noting the amount of alkali added, then just made 
acid with a drop or two of dilute sulphuric acid (25 per cent). 
It is then cooled, transferred quantitatively to a 25 c.c. glass- 
stoppered, graduated flask, and the mixture made up to mark 
with water, and the whole mixed. 
Five c.c. of the standard acid potassium phosphate solution 
(containing 0.15 mg. of H3P04) are measured into a 25 c.c. glass- 
stoppered, graduated flask, a drop of phenolphthalein added, and 
the amount of alkali used in neutralizing the digestion mixture 
above run in. The solution is then made just acid with the 25 
per cent 1I2S04, cooled, made up to the mark with water, and 
well mixed. Twenty-five c.c. portions of the strychnine molyb- 
date reagent are measured into each of two 50 c.c. glass-stoppered, 
graduated flasks. Five c.c. of the standard solution are run into 
one of the flasks, and 5 c.c. of the test solution are similarly added 
to the other. When the solutions are well mixed they are al- 
lowed to stand at least three minutes, then filled up to the mark 
with water, and mixed by inverting several times, after which 154 
CLINICAL LABORATORY METHODS 
they are ready to be compared in the nephelometer. (See page 
246.) 
The nephelometer tubes are tilled with the solutions to the 
same height and to the point at which, when the tubes are in 
position in the nephelometer, the meniscus is just out of reach 
of light. 
Calculation.—If R be the reading of the test solution and the 
standard be set at 20: 
20 
—— x 30 = mg. phosphoric acid per 100 c.c. plasma. 
it 
(2) Procedure for the Determination of Lipoid Phosphoric Acid. 
—Two c.c. of plasma are measured into a 50 c.c. volumetric flask 
containing 35 to 40 c.c. of a mixture of 3 parts alcohol and 1 
part ether (both redistilled). The blood is made to enter in a 
slow stream of drops and the liquid in the flask kept rotating 
rather rapidly so as to prevent the formation of large aggregates 
of precipitate which are difficult to extract. The flask and con- 
tents are then immersed in boiling water with frequent and strong 
rotation (to prevent superheating) until the liquid begins to boil, 
cooled to room temperature, made up to volume, mixed and 
filtered. For the determination 15 c.c. (equals 0.6 c.c. plasma) 
are measured into one of the large test tubes, glass beads added, 
and the whole evaporated to dryness in a boiling water-bath. 
The tube should be shaken frequently until boiling begins, after 
which the solution will proceed quietly to dryness. It should 
be left in the bath a few minutes after it is apparently dry to 
remove traces of alcohol which would interfere with the subse- 
quent oxidation. One and one-half c.c. of sulphuric-nitric acid 
mixture are added, distributed by shaking to the material on 
the sides of the tube, and the mixture is digested in the same 
way as directed for total phosphates. 
Calculation.—If R be the reading of the test solution and the 
standard be set at 20: 
20 
x 25 = mg. lipoid phosphoric acid per 100 c.c. plasma. 
R 
(3) Procedure for Inorganic, Acid Soluble, and Other Forms 
of Phosphoric Acid.—Three c.c. of plasma are run slowly into 
20 c.c. of acid ammonium sulphate solution in a 25 c.c. glass- QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
155 
stoppered, graduated flask, the volume made to the mark with 
water, the whole mixed, and let stand with occasional shaking 
for at least 10 minutes. It is then filtered. After the filter has 
drained it should be folded in the funnel and pressed out with 
a clean stirring rod to get as much filtrate as possible. The 
filtrate is clear and colorless and contains no detectable protein. 
With this filtrate determine: 
(1) Inorganic.—(a) Ten c.c. of the filtrate (=1.2 c.c. of plas- 
ma) are measured into one of the 25 c.c. flasks and made to the mark 
with water. A standard is prepared by adding to another flask 
3 c.c. of the standard phosphate (0.09 mg. of H3P04) and acid 
ammonium sulphate equal to that present in the test solution (8 
c.c. is a sufficiently close approximation). The flask is then filled 
to the mark with water. The determination should be made 
promptly after filtering. 
Twenty-five c.c. portions of the strychnine molybdate reagent 
are measured into each of two 50 c.c. glass-stoppered, volumetric 
flasks, and 5 c.c. of the standard solution are run into one of the 
flasks and 5 c.c. of the test solution are similarly added to the 
other. When the solutions are well mixed they are allowed to 
stand at least 3 minutes, then filled to the mark with water, and 
mixed by inverting several times, after which they are ready 
to be compared in the nephelometer. 
Calculation.—If R be the reading of the unknown solution 
and the standard be set at 20: 
R 
—- x 7.5 = mg. inorganic phosphoric acid per 100 c.c. plasma. 
(b) Where many determinations are to be made it is advisable 
to make a special standard—when determinations can be made 
more simply as follows: 
Standard.—Made by measuring 2 c.c. of the strong stock stand- 
ard phosphate solution (= 1.2 mg. of II3P04) into a 100 c.c. 
flask, adding 80 c.c. of the acid ammonium sulphate and making 
up to 100 c.c. with water. Of this standard 5 c.c. contain 0.06 
mg. of H3P04. For the determination 5 c.c. of the filtrate (= 0.6 
c.c. of plasma) are measured directly into 25 c.c. of the strych- 
nine molybdate reagent in one 50 c.c. graduated flask, 5 c.c. of 
the standard solution into another, and after mixing and stand- 156 
CLINICAL LABORATORY METHODS 
ing 3 minutes, the flasks are filled to the mark, the solution mixed, 
and the determination made. 
Calculation.—If R be the reading of the unknown solution 
and the standard be set at 20: 
R 
,r~-x 10 = mg. inorganic phosphoric acid per 100 c.c. plasma. 
<uU 
(2) Acid Soluble.—Ten c.c. of the filtrate are measured into 
one of the large test tubes, glass beads and 1.5 c.c. of the sul- 
phuric-nitric acid mixture are added and the mixture digested. 
The digestion presents some difficulties because of the large 
amount of ammonium sulphate present. The first stage is passed 
over quickly, then in the second stage when the mixture thickens 
and begins to foam, the heat is moderated, and so continued until 
foaming ceases and the salt fuses to a small volume in the tube. 
Heating is then carried on at a rate just sufficient to prevent loss 
of ammonium sulphate from the tube by volatilization for 10 
minutes. The tubes are then cooled, treated with one drop of 
0.3 per cent cane sugar solution, and reheated in the regular 
way. After dissolving in water the solution is titrated with 
alkali, noting the amount used. Transfer to 25 c.c. volumetric 
flask and make up to mark with water. The standard is prepared 
by adding to another 25 c.c. flask, 3 c.c. of the standard phosphate 
solution (= 0.09 mg. H3P04), 8 c.c. of the acid ammonium sul- 
phate solution, and the amount of alkali used to neutralize the test 
solution. The mixture is then neutralized and made up to the mark 
with water. Twenty-five c.c. portions of the strychnine molybdate 
reagent are measured into each of two 50 c.c. glass-stoppered 
flasks. Five c.c. of the standard solution are run into one of the 
flasks and 5 c.c. of the test solution are similarly added to the 
other. When the solutions are well mixed they are allowed to 
stand at least three minutes, then filled to the mark with water, 
and mixed by inverting several times, after which they are com- 
pared in the nephelometer. 
Calculation.—If R be the reading of the unknown and the 
standard be set at 20: 
R 
—x 7.5 = mg. acid soluble phosphoric acid per 100 c.c. plasma. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
157 
TOTAL PHOSPHORIC ACID 
(H3P04 in mg. per 100 c.c. plasma). 
LIPOID PHOSPHORIC ACID 
(H3P04 in mg. per 100 c.c. plasma). 
ACID 
SOLUBLE OR INORGANIC 
PHOSPHORIC ACID 
(HgPO,, in mg. per 
100 c.c. plasma). 
NEPHEL- 
OMETER 
READING 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
15 
40.0 
39.5 
38.9 
38.5 
38.0 
33.3 
32.9 
32.4 
32.1 
31.7 
10.0 
9.9 
9.7 
9.6 
9.5 
16 
37.5 
37.0 
36.6 
36.1 
35.7 
31.3 
30.9 
30.5 
30.1 
29.8 
9.4 
9.3 
9.2 
9.0 
8.9 
17 
35.3 
34.9 
34.5 
34.1 
33.7 
29.4 
29.0 
28.7 
28.4 
28.1 
8.8 
8.7 
8.6 
8.5 
8.4 
18 
33.3 
33.0 
32.6 
32.3 
31.9 
27.8 
27.5 
27.2 
26.9 
26.6 
8.3 
8.2 
8.1 
8.1 
8.0 
19 
31.6 
31.2 
30.9 
30.6 
30.3 
26.3 
26.0 
25.7 
25.5 
25.3 
7.9 
7.8 
7.7 
7.6 
7.6 
20 
30.0 
29.7 
29.4 
29.1 
28.9 
25.0 
24.7 
24.5 
24.3 
24.1 
7.5 
7.4 
7.3 
7.3 
7.2 
21 
28.5 
28.2 
28.0 
27.7 
27.5 
23.7 
23.5 
23.4 
23.1 
22.9 
7.1 
7.0 
7.0 
6.9 
6.8 
22 
27.3 
27.0 
26.8 
26.5 
26.3 
22.8 
22.5 
22.3 
22.1 
21.9 
6.8 
6.7 
6.7 
6.6 
6.6 
23 
26.1 
25.9 
25.7 
25.4 
25.2 
21.7 
21.6 
21.4 
21.1 
20.9 
6.5 
6.5 
6.4 
6.4 
6.3 
24 
25.0 
24.8 
24.6 
24.4 
24.2 
20.8 
20.7 
20.5 
20.3 
20.2 
6.2 
6.2 
6.1 
6.1 
6.0 
25 
24.0 
23.8 
23.6 
23.4 
23.3 
20.0 
19.8 
19.7 
19.5 
19.4 
6.0 
6.0 
5.9 
5.9 
5.8 
Determination of Phosphoric Acid in Blood 
In determining total phosphates use 0.5 c.e. oxalated plasma made up to 25 c.e.; and compare with a standard contain- 
ing 0.15 mg. of phosphoric acid in a similar volume; for lipoid phosphoric acid use the equivalent of 0.6 c.c. plasma 
and compare with a standard containing 0.15 mg. phosphoric acid; for inorganic or acid soluble phosphates employ the 
equivalent of 1.2 c.c. plasma and compare with standard containing 0.09 mg. phosphoric acid. 
In each determination set standard at 20. 
Table shows the amount of different forms of phosphoric acid in mg. per 100 c.c. of plasma corresponding to the dif- 
ferent nephelometer readings. 
Table XXY 158 
CLINICAL LABORATORY METHODS 
(3) Other Forms op Phosphoric Acid.—Obtained by subtract- 
ing inorganic from acid soluble phosphoric acid. 
Remarks.—The average amount of the different forms of phos- 
phoric acid found in normal individuals, per 100 c.c. plasma is 
as follows: (1) Total, 32 mg. (2) Acid soluble, 10.4 mg. (3) 
Inorganic, 8.7 mg. (4) Lipoid, 22.1 mg. (5) Other forms, 1.73 
mg. 
The quantities of plasma taken are based on average values for 
normal human plasma. Variations in amount of blood, or 
strength of standard may be necessary in special cases. 
The readings obtained in the nephelometer are not exactly pro- 
portional to the amount of phosphoric acid in the solution. Solu- 
tions up to about 25 per cent stronger or weaker than the stand- 
ard may be used, however, without correction. 
Table XXV shows the phosphoric acid values corresponding to 
different nephelometer readings. 
Determination of Calcium 
(Lyman, H.: Jour. Biol. Chem., 1917, xxix, 169) 
Principle.—The calcium in the blood plasma is precipitated as 
the oxalate under conditions to minimize the occlusion of mag- 
nesium; the precipitate is centrifuged, washed, and dissolved in 
nitric acid, and treated with a solution of ammonium stearate. 
The resulting cloud of calcium soap is read in a nephelometer 
against a standard solution of calcium oxalate similarly treated. 
Collection op Blood Sample.—Withdraw 10 c.c. of blood by 
venipuncture and run into a tube containing 0.1 gram of solid 
sodium citrate. Centrifuge and use plasma for the test. 
Reagents.—(1) Trichloracetic acid, 6.5 per cent aqueous solu- 
tion. 
(2) Indicator, methyl orange, 0.1 per cent. Dissolve 0.1 gram 
of methyl orange in 10 c.c. of alcohol and make up to 100 c.c. 
with water. 
(3) Oxalic acid, 4 per cent solution. 
(4) Sodium acetate. Dissolve 20 gm. of the crystallized sodium 
acetate in 100 c.c. of water. 
(5) Ammonium oxalate, 0.5 per cent solution. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
159 
(6) Ammonium stearate reagent. Dissolve 4 grams of stearic 
acid and 0.5 c.c. of oleic acid in 400 c.c. of hot alcohol. Add 20 
grams of ammonium carbonate dissolved in 100 c.c. of hot water 
and allow the mixture to boil a few minutes. Cool. Add 400 c.c. 
of alcohol, 100 c.c. of water, and 2 c.c. of ammonium hydroxide 
(sp.gr. 0.9). Filter. This solution should be as clear as freshly 
distilled water and perfectly colorless. If well stoppered it 
keeps indefinitely. Before using for analysis test as follows: 
Into two flasks pipette respectively 5 and 10 c.c. of the calcium 
oxalate standard and to the 5 c.c. add 5 c.c. of nitric acid, 0.05 N. 
Treat both with 25 c.c. of the ammonium stearate reagent and 
read in the nephelometer. If they do not read exactly 2 to 1 
there is some impurity present in the chemicals used. The alco- 
hol, if it has stood in a wooden barrel, will give a yellowish col- 
oration with ammonia and will contain suspended particles which 
reflect light in the nephelometer. It should be redistilled with 
calcium carbonate. Stearic acid may be purified by recrystal- 
lizing from boiling alcohol. Ammonium carbonate may be 
resublimed. 
(7) Calcium oxalate standard.—Dissolve 72.9 mg. of pure cal- 
cium oxalate (CaC20 + 1H20) in 25 c.c. of nitric acid, 2N, and 
make up to 1,000 c.c. with distilled water. 
10 c.c. of this standard contains 0.2 mg. of calcium; 7.5 c.c. con- 
tains 0.15 mg.; 5 c.c. contains 0.10 mg. 
(8) Nitric acid, 0.1 N and 0.05 N. These do not have to be 
exact. They are conveniently made from the 2N acid. 
(9) Ammonium Hydrate, 2N. Thirteen and one-half c.c. of 
ammonium hydrate, sp.gr. 0.9, made up to 100 c.c. of water will 
serve the purpose. 
Use calcium-free filter paper. Baker and Adamson’s paper 
“A” will answer the purpose. For filtering the reagents absorb- 
ent cotton washed first with 10 per cent hydrochloric acid, and 
then with water until the wash water is no longer acid to litmus, 
and finally dried, may be used. 
Procedure.—Run 5 c.c. of blood plasma into a small flask con- 
taining 15 c.c. of trichloracetic acid, 6.5 per cent, while agitat- 
ing the flask. Mix and let stand for a few minutes. Filter 
through a calcium-free filter paper. Pipette 10 c.c. of the filtrate 160 
CLINICAL LABORATORY METHODS 
into an Erlenmeyer flask of about 50 c.c. capacity. Add one drop 
of methyl orange, 0.1 per cent. Add 2 N ammonium hydrate drop 
by drop until just yellow. Add nitric acid, 0.05 per cent, drop- 
wise until just pink, and then 1 c.c. more. Add 1 c.c. of oxalic 
acid, 4 per cent. Add 1 c.c. of sodium acetate, 20 per cent, drop- 
wise. Cool under the water tap until a faint cloud appears. 
Shake ten minutes or let stand overnight, at room temperature, 
as convenient. Rinse the stopper with ammonium oxalate, 0.5 per 
cent. Pour into a centrifuge tube and centrifuge. Pipet off the 
supernatant liquor. Rinse the flask with 5 c.c. of ammonium 
oxalate, 0.5 per cent, pour into a centrifuge tube, stir, rinsing the 
rod with 0.5 per cent ammonium oxalate, and again centrifuge. 
Pipet off the supernatant liquor. Dissolve the precipitate in 
5 c.c. of 0.1 N nitric acid by means of stirring, and pour into the 
original flask. Agitate a moment to dissolve any precipitate ad- 
hering to the walls. Rinse the rod and centrifuge tube with 5 c.c. 
of water, and pour into the flask. 
Prepare three standards in Erlenmeyer flasks of 100 c.c. capac- 
ity as follows: No. 1, 20 c.c. of the standard calcium oxalate 
solution; No. 2, 15 c.c. of the standard and 5 c.c. of distilled 
water; No. 3', 10 c.c. of the standard and 10 c.c. of water. Pipette 
50 c.c. of the ammonium stearate solution into each of three dry 
beakers, and 25 c.c. into a fourth. Pour the three standard solu- 
tions respectively into the beakers containing the 50 c.c. of re- 
agent and the unknown into the 25 c.c. and pour back twice. 
Stopper and let stand ten minutes. Compare the unknown in the 
nephelometer with the standard it most nearly matches. 
Calculation.—Set the standard at 32. If R be the reading of 
32 
the unknown, x 8 equals mg. of calcium (as Ca) per 100 c.c. 
of plasma if standard No. 1 be used. 
32 
p x 6 equals mg. of calcium per 100 c.c. if standard No. 2 be 
used. 
32 
— x 4 equals mg. of calcium per 100 c.c. if standard No. 3 be 
R 
used. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
161 
USING STANDARD NO. 1 
(Calcium as Ca per 100 e.c. plasma) 
USING STANDARD NO. 2 
(Calcium as Ca per 100 c.c. plasma) 
USING STANDARD NO. 3 
(Calcium as Ca 
per 100 c.c. plasma). 
NEPHEL- 
OMETER 
READING 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
0.0 
0.2 
0.4 
0.6 
0.8 
24 
10.6 
10.6 
10.5 
10.4 
10.3 
8.0 
8.0 
7.9 
7.8 
7.8 
5.3 
5.3 
5.3 
5.2 
5.2 
25 
10.2 
10.1 
10.0 
10.0 
9.9 
7.7 
7.6 
7.6 
7.5 
7.5 
5.1 
5.0 
5.0 
5.0 
5.0 
26 
9.8 
9.8 
9.7 
9.7 
9.6 
7.4 
7.4 
7.3 
7.2 
7.2 
4.9 
4.9 
4.8 
4.8 
4.8 
27 
9.5 
9.4 
9.3 
9.3 
9.2 
7.1 
7.1 
7.0 
6.9 
6.9 
4.7 
4.7 
4.7 
4.6 
4.6 
28 
9.0 
9.0 
9.0 
8.9 
8.8 
6.9 
6.8 
6.8 
6.7 
6.6 
4.5 
4.5 
4.5 
4.5 
4.4 
29 
8.8 
8.7 
8.7 
8.6 
8.5 
6.6 
6.6 
6.5 
6.5 
6.4 
4.4 
4.4 
4.4 
4.3 
4.3 
30 
8.5 
8.4 
8.4 
8.3 
8.3 
6.4 
6.3 
6.3 
6.3 
6.2 
4.3 
4.2 
4.2 
4.2 
4.2 
31 
8.3 
8.2 
8.1 
8.1 
8.0 
6.2 
6.2 
6.1 
6.1 
6.0 
4.2 
4.1 
4.1 
4.0 
4.0 
32 
8.0 
7.9 
7.9 
7.8 
7.8 
6.0 
5.9 
5.9 
5.9 
5.8 
4.0 
4.0 
3.9 
3.9 
3.9 
33 
7.8 
7.7 
7.6 
7.6 
7.5 
5.8 
5.8 
5.7 
5.7 
5.7 
3.9 
3.9 
3.8 
3.8 
3.8 
34 
7.5 
7.5 
7.4 
7.4 
7.4 
5.6 
5.6 
5.6 
5.5 
5.5 
3.8 
3.7 
3.7 
3.7 
3.7 
35 
7.3 
7.3 
7.2 
7.2 
7.2 
5.5 
5.5 
5.4 
5.4 
5.4 
3.7 
3.6 
3.6 
3.6 
3.6 
36 
7.1 
7.1 
7.0 
7.0 
6.9 
5.3 
5.3 
5.3 
5.2 
5.2 
3.5 
3.5 
3.5 
3.5 
3.5 
37 
6.9 
6.9 
6.8 
6.8 
6.8 
5.2 
5.2 
5.1 
5.1 
5.1 
3.5 
3.4 
3.4 
3.4 
3.4 
38 
6.7 
6.7 
6.6 
6.6 
6.6 
5.0 
5.0 
5.0 
4.9 
4.9 
3.4 
3.4 
3.3 
3.3 
3.3 
39 
6.5 
6.5 
6.5 
6.5 
6.4 
4.9 
4.9 
4.9 
4.8 
4.8 
3.3 
3.3 
3.2 
3.2 
3.2 
40 
6.4 
6.4 
6.3 
6.3 
6.3 
4.8 
4.8 
4.7 
4.7 
4.7 
3.2 
3.2 
3.2 
3.2 
3.1 
Determination- of Calcium in Blood 
Make volume of the unknown solution up to 35 c.c.; that of the standards up to 70 c.c. 
Set standard at 32. 
Table shows the calcium as Ca in milligrams per 100 c.c. plasma, corresponding to the different nephelometer readings. 
Table XXVI 162 
CLINICAL LABORATORY METHODS 
Remarks.—Using this method the average normal calcium per 
100 c.c. of plasma is 10 mg. 
Table XXVI shows the calcium values corresponding to the 
different nephelometer readings. 
Determination of Acetone Bodies in Blood 
(Van Slyke, D.D., and Fitz, R.: Jour. Biol. Chem., 1917, xxxii, 
495) 
The same procedure and reagents used in the determination of 
acetone bodies in urine (see page 62) may be applied to blood, 
after the proteins of the blood have been removed. 
Removal of Proteins.—1. Whole Blood.—Dilute 10 c.c. of whole 
blood with 100 c.c. of water in a 250 c.c. flask, and add 10 c.c. of 
the 10 per cent mercuric sulphate. Shake for a moment until 
the protein coagulates, and fill to the mark. After 15 minutes or 
longer filter through a dry folded filter. 
2. Plasma or Serum.—Dilute 8 c.c. of plasma or serum in a 
20 c.c. flask with 30 to 40 c.c. water and add 15 c.c. of the mer- 
curic sulphate. Shake gently until the precipitate coagulates in 
floccules and then fill to mark with water. After standing 15 
minutes or longer the solution is filtered through a dry folded 
filter. 
Determination.—For the determination of acetone plus aceto- 
acetic acid, of beta-hydroxybutyric acid, or of the total acetone 
bodies together, 125 c.c. of the filtrate corresponding to 5 c.c. of 
blood may be treated exactly as the 25 c.c. of urine filtrate plus 
100 c.c. water in urine analysis. If it is desired to determine 
separately in a single sample of blood both the acetone plus 
acetoacetic acid, and the beta-hydroxybutyric acid the preformed 
acetone and that from the acetoacetic acid are precipitated as 
described for urine and the beta-hydroxybutyric acid determined 
in the filtrate. 
The filtrate from the mercury-acetone precipitate is received 
in a dry flask. One hundred sixty c.c., equivalent to 16%7o x 5 c.c. 
blood of the filtrate is measured into an Erlenmeyer flask and 
10 c.c. water added. The mixture is heated to boiling under a 
reflux condenser, 5 c.c. of the diehromate solution added, and the QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
163 
determination continued as described for total acetone bodies in 
urine. 
The procedure for beta-hydroxybutyric acid followed in urine 
cannot be used in blood because the excess mercury used in re- 
moving the proteins previous to boiling off the acetone, would 
partly precipitate the latter before it escaped. 
Factors for Calculating Results when Filtrate Equivalent to 
5 c.c. of Blood Is Used for Determination , 
Table XXVII 
DETERMINATION PERFORMED 
■ ACETONE BODIES CALCULATED AS GRAM OF 
ACETONE PER LITER OF BLOOD, INDICATED BY: 
1 gm. of precipitate 
1 c.c. of 0.2 KI Sol. 
Total acetone bodies 
12.8 
0.161 
Beta-hydroxybutyrie acid 
13.2 
0.72 
Acetone plus acetoacetic acid 
10.0 
0.130 
Remarks.—Normal blood when analyzed for total acetone bod- 
ies yields by this method 13 to 26 mg. acetone bodies calculated 
as acetone. 
Determination of the Bicarbonate Content of the Blood Plasma 
Under Constant Carbon Dioxide Tension 
(Van Slyke and Cullen: Jour. Biol. Chem., 1917, xxx, 289; 
Van Slyke: Jour. Biol. Chem., 1917, xxx, 347) 
Principle.—A given volume of blood plasma is saturated with 
carbon dioxide under normal tension by shaking it in a separa- 
tory funnel filled with air whose carbon dioxide tension is approx- 
imately equal to that of normal arterial blood. The plasma is 
then run into a special apparatus, acid added, and carbon dioxide 
liberated by the production of a Torricellian vacuum. The car- 
bon dioxide is measured at atmospheric pressure, and the volume 
corresponding to 100 c.c. of plasma calculated. 
Drawing Blood Sample.—For at least an hour before the blood 
is drawn the subject should avoid vigorous muscular exertion, 
as this, presumably because of the lactic acid formed, lowers the 
bicarbonate of the blood. The blood is drawn from the arm vein 
directly into a centrifuge tube containing enough potassium ox- 
alate to make about 0.5 per cent of the weight of the blood. 164 
CLINICAL LABORATORY METHODS 
It is essential that the blood be collected with minimum gain 
or loss of carbon dioxide, as IIC1 is transferred from plasma to 
cells by increase of free C02 in the former, and vice versa, with 
resultant change of not only free carbon dioxide, but also of 
bicarbonate in the plasma. Consequently, overaccumulation of 
carbon dioxide in the venous blood is avoided by using as little 
stasis as possible. When stasis is necessary the ligature is re- 
leased as soon as the vein is entered and a few seconds allowed 
for the stagnant blood to flow out before the main sample is 
33AR/5FFLN Oir 
POTASSIUM oxalate:- 
H SW. 
Fig. 47.—Tube used in collecting blood for the determination of the carbon dioxide com- 
bining power. (After Van Slyke and Cullen.) 
drawn. It is equally necessary to avoid loss of carbon dioxide 
while the plasma is still in contact with the corpuscles in vitro. 
In order to prevent such loss, the blood may be drawn into a tube 
arranged for this purpose (Fig. 47). After the sample has been 
drawn the stopper is loosened and the blood stirred with the 
inlet tube in order to assure distribution of the oxalate. The 
tube should not be shaken or inverted. The blood is centrifuged 
in it within a half-hour. In place of the tube a syringe may be 
used if the blood is drawn with minimum suction, free air space QUANTITATIVE CHEMICAL EXAMINATION OP BLOOD 
165 
in the barrel is avoided, and the transfer to a centrifuge tube 
made with minimum exposure to air. 
The clear plasma, being pipetted off, should, in case it is not 
convenient to determine its C02 capacity at once, be transferred 
to a paraffin-lined tube, where it will keep unchanged for a week 
if placed on ice. 
Saturation with Carbon Dioxide at Natural Tension.—In order 
to correct error from loss of C02 and consequent reversion of 
NaTICOg to Na2C03 after centrifugation, the plasma is resaturated 
with C02 at alveolar tension immediately before analysis. The 
plasma (3 c.c. or more if there is plenty of material), which 
should be at room temperature, is placed in a separatory funnel 
(Fig. 48) of about 300 c.c. capacity and the funnel is filled with 
Fig. 48.—Separatory funnel, used in saturating blood plasma with carbon dioxide 
{After Van Slyke and Cullen.) 
alveolar air from the lungs of the operator. The air is passed 
through a bottle full of glass beads before it enters the funnel, in 
order to bring the moisture content down to saturation at room 
temperature. If one blows directly into the separatory funnel, 
enough moisture condenses on the walls to dilute the plasma ap- 
preciably. The lungs are completely emptied through the funnel 
by a quick, forced expiration. The stopper is inserted just before 
the stream of breath stops. The funnel is then rotated for two 
minutes in such a manner that the plasma is distributed as com- 
pletely as possible about the walls, forming a thin layer, which 
quickly approaches equilibrium with the CO, in the air. 
Analysis—The determination of the carbon dioxide content 
of the saturated plasma is performed as follows: The C02 appa- 166 
CLINICAL LABORATORY METHODS 
ratus (Fig. 49) held in a strong clamp on a ringstand is com- 
pletely filled with mercury, which should fill both capillaries 
above the upper stopcock “e.” The mercury leveling bulb is 
placed about on a level with the lower cock. The cup “b” at the 
top of the apparatus is washed out thoroughly with dilute ammo- 
nia, followed by water, medicine droppers being convenient for 
Position 1 
Position 3 ' 
IS 80 CM BEtiOW 
POSITION 2. 
Fig. 49.—Van Slyke carbon dioxide apparatus. (After Van Slyke.) 
this purpose. One cubic centimeter of the saturated plasma is 
introduced into the cup and allowed to flow down into the upper 
stem of the apparatus. The cup is now washed with two portions 
of about 0.5 c.c. each of water, care being taken that no air enters 
the apparatus with the liquid. One small drop of octyl alcohol, to 
prevent foaming, is now admitted into the capillary connecting 
the cup with the upper end of the apparatus and about 1 c.c. of QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
167 
5 per cent sulphuric acid is poured into the cup. Enough of the 
acid is admitted into the 50 c.c. chamber, carrying the octyl alco- 
hol along with it, so that the total volume of water in the appara- 
tus is exactly 2.5 c.c. A drop of mercury is now placed in the cup 
and allowed to flow down to the upper stopcock in order to seal 
the same and make it capable of holding an absolute vacuum. 
Volume of Gas Measured 
Column of Wafer Solution 
. 'LevjeLof Mercury Surface 
'<s in Leveling Bulb 
'' -LaveL of Hercur/ 
flenibcuB in.Pi.pette, 
Fig. SO.—Showing position of bulb when the gas volume in the pipette of the Van Slyke 
carbon dioxide apparatus is read. 
JiE.t'J. 
The leveling bulb (the lower cock having remained open from the 
beginning of operations) is lowered to such a point that the sur- 
face of the mercury in it is about 800 mm. below the lower stop- 
cock “i,” and the mercury in the apparatus is allowed to fall 
until the meniscus of the mercury has dropped to the 50 c.c. mark 
on the apparatus. As the latter is evacuated, bubbles of C02 are 
seen escaping from the water mixture into the vacuum. 168 
CLINICAL LABORATORY METHODS 
In order to extract the carbon dioxide completely the apparatus 
is removed from the clamp and shaken by turning it upside down 
about a dozen times. It is then replaced, the mercury leveling 
bulb still being at the low level, and the water solution is allowed 
to flow completely into the small bulb “d” below the lower stop- 
cock. The water solution is drained out of the portion of the 
apparatus above the stopcock as completely as possible, but with- 
out removing any of the gas. The mercury bulb is now raised in 
the left hand and the lower stopcock is turned with the right hand 
so that mercury is admitted to the apparatus through the left-hand 
entrance, “c,” of the 3-way cock without readmitting the water 
solution. The leveling bulb is held beside the apparatus so that 
the mercury level in it is even with that in the apparatus (Fig. 50) 
and the gas in the latter is under atmospheric pressure. A few 
hundredths of 1 c.c. of water will float on the mercury in the 
apparatus, but this may be disregarded in leveling. The volume 
of gas above the short column of water referred to is at once 
read off. 
Calculation.—The calculation of the C02 combining power is 
made from the volume of gas as read off from the burette with 
the aid of the two tables given by Van Slyke and Cullen. The 
volume of gas observed is multiplied by the factor The 
barometric pressure is read and this factor ascertained from 
Table XXVIII. For example, if the barometer reading were 744, 
B 
the factor is seen to be 0.979. The volume of gas observed 
7b0 
is multiplied by this and the resultant figure is located in the 
first column of Table XXIX, the column headed “Observed vol- 
g 
ume of gas x 700 *?> ie ne carr^ed across horizontally to 
the column which is headed by the temperature at which the 
reading was made, and the result read off. This is expressed in 
terms of c.c. of carbon dioxide bound by 100 c.c. of plasma, re- 
duced to 0 degrees, and a barometric pressure of 760 mm. 
Plasma of normal adults yields 0.65 to 0.9 c.c. of gas, indicating 
53 to 77 volume per cent of C02 chemically bound by the plasma. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
169 
Figures lower than 50 per cent in adults indicate acidosis. If the 
figure goes below 30 the symptoms of acid intoxication usually 
appear, and with further fall, rapidly intensify. The normal fig- 
ures for infants appear to be 40 to 55 per cent,—much lower than 
for adults. 
Caution in Setting up Apparatus.—The jaws of the clamp in 
which the apparatus is held should be lined with thick soft rub- 
ber. The apparatus has to be clamped very tightly because of 
the weight of the mercury. 
In order to prevent the apparatus from slipping out of the 
clamp an iron rod should be so arranged as to project under the 
lower stopcock, so that it will support the apparatus from this 
point in case it should at any time slip down from the clamp. 
Table XXIX facilitates calculation of the results. It con- 
tains corrections for the air (about 0.05 c.c.) dissolved by the 
2.5 c.c. of water introduced into the apparatus, for an approxi- 
mately equal volume of C02 physically dissolved by the 1 c.c. of 
plasma in addition to that chemically bound as bicarbonate, also 
corrections for temperature, pressure, and vapor tension neces- 
sary to reduce the gas volume to standard conditions; viz., tem- 
perature of 0° C. and pressure of 760 mm. 
Showing Barometer Coefficients 
Table XXVIII 
B 
Values are given below for the ratio - over the range usually 
encountered. 
BAROMETER 
B 
BAROMETER 
B 
760 
760 
732 
0.963 
756 
0.995 
734 
0.966 
758 
0.997 
736 
0.968 
760 
1.000 
738 
0.971 
762 
1.003 
740 
0.974 
964 
1.006 
742 
0.976 
766 
1.008 
744 
0.979 
768 
1.011 
746 
0.981 
770 
1.013 
748 
0.984 
772 
1.016 
750 
0.987 
774 
1.018 
752 
0.989 
776 
1.021 
754 
0.992 
778 
1.024 170 
CLINICAL LABORATORY METHODS 
Table XXIX 
For Calculation of Carbon Dioxide Combining Power of Plasma 
Observed 
vcl. gas 
x B 
760 
C.c. of C02 reduced 
760 mm. bound as 
bonate by 100 c.c. 
plasma 
to 0° 
biear- 
, of 
Observed 
vol. gas 
x B 
760 
C.c. of C02 reduced to 0° 
760 mm. bound as bicar- 
bonate by 100 c.c. of 
plasma 
15° 
20° 
25° 
30° 
15° 
20° 
25° 
30° 
0.20 
9.1 
9.9 
10.7 
11.8 
0.60 
47.7 
48.1 
48.5 
48.6 
1 
10.1 
10.9 
11.7 
12.6 
1 
48.7 
49.0 
49.4 
49.5 
2 
11.0 
11.8 
12.6 
13.5 
2 
49.7 
50.0 
50.4 
50.4 
3 
12.0 
12.8 
13.6 
14.3 
3 
50.7 
51.0 
51.3 
41.4 
4 
13.0 
13.7 
14.5 
15.2 
4 
51.6 
51.9 
52.2 
52.3 
5 
13.9 
14.7 
15.5 
16.1 
5 
52.6 
52.8 
53.2 
53.2 
6 
14.9 
15.7 
16.4 
17.0 
6 
53.6 
53.8 
54.1 
54.1 
7 
15.9 
16.6 
17.4 
18.0 
7 
54.5 
54.8 
55.1 
55.1 
8 
16.8 
17.6 
18.3 
18.9 
8 
55.5 
55.7 
56.0 
56.0 
9 
17.8 
18.5 
19.2 
19.8 
9 
56.5 
56.7 
57.0 
56.9 
0.30 
18.8 
19.5 
20.2 
20.8 
0.70 
57.4 
57.6 
57.9 
57.9 
1 
19.7 
20.4 
21.1 
21.7 
1 
58.4 
58.6 
58.9 
58.8 
2 
20.7 
21.4 
22.1 
22.6 
2 
59.4 
59.5 
59.8 
59.7 
3 
21.7 
22.3 
23.0 
23.5 
3 
60.3 
60.5 
60.7 
60.6 
4 
22.6 
23.2 
24.0 
24.5 
4 
61.3 
61.4 
61.7 
61.6 
5 
23.6 
24.2 
24.9 
25.4 
5 
62.3 
62.4 
62.6 
62.5 
6 
24.6 
25.2 
25.8 
26.3 
6 
63.2 
63.3 
63.6 
63.4 
7 
25.5 
26.2 
26.8 
27.3 
7 
64.2 
64.3 
64.5 
64.3 
8 
26.5 
27.1 
27.7 
28.2 
8 
65.2 
65.3 
65.5 
65.3 
9 
27.5 
28.1 
28.7 
29.1 
9 
66.1 
66.2 
66.4 
66.2 
0.40 
28.4 
29.0 
29.6 
30.0 
0.80 
67.1 
67.2 
67.3 
67.1 
1 
29.4 
30.0 
30.5 
31.0 
1 
68.1 
68.1 
68.3 
68.0 
2 
30.3 
30.9 
31.5 
31.9 
2 
69.0 
69.1 
69.2 
69.0 
3 
31.3 
31.9 
32.4 
32.8 
3 
70.0 
70.0 
70.2 
69.9 
4 
32.3 
32.8 
33.4 
33.8 
4 
71.0 
71.0 
71.1 
70.8 
5 
33.2 
33.8 
34.3 
34.7 
5 
71.9 
72.0 
72.1 
71.8 
6 
34.2 
34.7 
35.3 
35.6 
6 
72.9 
72.9 
73.0 
72.7 
7 
35.2 
35.7 
36.2 
36.5 
7 
73.9 
73.9 
74.0 
73.6 
8 
36.1 
36.6 
37.2 
37.4 
8 
74.8 
74.8 
74.9 
74.5 
9 
37.1 
37.6 
38.1 
38.4 
9 
75.8 
75.8 
75.8 
75.4 
0.50 
38.1 
38.5 
39.0 
39.3 
0.90 
76.8 
76.7 
76.8 
76.4 
1 
39.1 
39.5 
40.0 
40.3 
1 
77.8 
77.7 
77.7 
77.3 
2 
40.0 
40.4 
40.9 
41.2 
2 
78.7 
78.8 
78.7 
78.2 
3 
41.0 
41.4 
41.9 
42.1 
3 
79.7 
79.6 
79.6 
79.2 
4 
42.0 
42.4 
42.8 
43.0 
4 
80.7 
80.5 
80.6 
80.1 
5 
42.9 
43.3 
43.8 
43.9 
5 
81.6 
81.5 
81.5 
81.0 
6 
43.9 
44.3 
44.7 
44.9 
6 
82.6 
82.5 
82.4 
82.0 
7 
44.9 
45.3 
45.7 
45.8 
7 
83.6 
83.4 
83.4 
82.9 
8 
45.8 
46.2 
46.6 
46.7 
8 
84.5 
84.4 
84.3 
83.8 
9 
46.8 
47.1 
47.5 
47.6 
9 
85.5 
85.3 
85.2 
84.8 
0.60 
47.7 
48.1 
48.5 
48.6 
1.00 
86.5 
86.2 
86.2 
85.7 QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
171 
Determination of the Oxygen Binding Capacity of Blood 
(Gasometric Hemoglobin Estimation) 
(Van Slyke, D. D., Jour. Biol. Chem., 1918, xxxvi, 127; Van Slyke, 
D. D., and Stadie, W. C., Jour. Biol. Chem., 1921, 1, 49) 
Principle.—The Van Slyke apparatus (Fig. 49) used for deter- 
mining the carbonic acid binding power of the blood plasma may 
be used with equal facility for determining the oxygen binding 
power of blood. The oxygen in a measured amount of blood is 
set free from combination with the oxyhemoglobin by the addi- 
tion of potassium ferricyanide. The oxygen is then extracted in 
the Van Slyke apparatus and measured at atmospheric pressure. 
Reagents.—(1) 1 per cent saponin (Merck) solution. 
(2) 20 per cent potassium ferricyanide. The ferricyanide solu- 
tion is made air-free by boiling or by shaking in an evacuated 
flask and is kept in a bottle or burette under a layer of paraffin 
oil 2 or 3’ cm. thick to exclude air. 
(3) 0.5 N sodium hydroxide. 
Procedure.—Five to 10 c.c. of blood is introduced into a sepa- 
ratory funnel or bottle and distributed in a thin layer about the 
inner wall, so that maximum contact with the air is assured. 
The vessel is rotated for three or four minutes so that the blood 
is kept in a thin layer, or it may be shaken five or more minutes 
on a mechanical shaker. The blood is then transferred to a cyl- 
inder or heavy walled tube. The blood gas apparatus is washed 
twice with distilled water before each analysis in order to remove 
the alkali used to absorb C02 in any former analysis. For 2 c.c. 
of blood 6 c.c. of water 0.3 c.c. of 1 per cent saponin (Merck), and 
2 to 3 drops of caprylic alcohol are introduced into the cup “b” 
of the apparatus and the apparatus is evacuated by lowering 
the leveling bulb until the level of the mercury in the apparatus 
is just above the lower stopcock. The air is extracted by shak- 
ing for about 15 seconds. The extracted air is expelled and the 
process repeated to make sure that no air is left in the solution. 
Nearly the entire 6 c.c. is then forced up into the cup of the 
apparatus, the blood is stirred to assure even distribution of the 
corpuscles and drawn into a pipette calibrated to deliver 2 c.c. 
between two marks of which the lower is 3 to 4 cm. from the tip. 172 
CLINICAL LABORATORY METHODS 
The pipette is introduced under the water solution in the cup so 
that the tip rests on the bottom near the capillary. As the blood 
flows out of the pipette held in the left hand, the upper stopcock 
is partially opened with the right, so that the blood accompanied 
by some of the water flows at once into the chamber of the appa- 
ratus. The layer of blood need never rise more than 1 to 2 mm. 
above the bottom of the cup and the slight amount adhering is 
washed completely into the chamber by the water which follows 
after all the blood has been delivered. 
Before the last c.c. of water is readmitted, 0.10 to 0.12 c.c. of a 
solution of potassium ferricyanide containing 20 grams per 100 
c.c. is added and thereby introduced into the chamber after the 
blood. A mercury seal is made by placing a drop of mercury in 
the cup and allowing it to run down to the upper stopcock. The 
blood and water in the apparatus are thoroughly mixed and al- 
lowed to stand until the blood is completely laked. This requires 
from 30 seconds to a minute. The apparatus is now evacuated 
until only a few drops of mercury remain above the upper stop- 
cock, and is shaken preferably with a rotatory motion to whirl 
the blood in a thin layer about the wall of the chamber. The 
shaking should be continued for three minutes. Usually two 
minutes, frequently less, are sufficient for the complete extrac- 
tion but three minutes are sometimes required before the last 
traces are extracted. The extracted solution is trapped in the 
bulb below the lower stopcock as in the determination of C02 
and the vacuum is released; 0.5 c.c. of 0.5 N. sodium hydroxide 
solution previously saturated with air or oxygen is admitted 
from the cup of the apparatus and allowed to trickle slowly 
down the inner wall to absorb the C02 from the gas mixture. If 
any of the alkali solution remains just below the stopcock it may 
be dislodged by running in a little mercury from the cup above. 
After the absorption of C02 is complete two minutes must be 
allowed for drainage of the alkali solution before the reading is 
taken. 
Calculation.—The volume of oxygen obtained as read off from 
the burette of the apparatus is reduced to 0° C. and 760 mm. 
pressure by multiplying by the proper factor as indicated in 
Table XXX. QUANTITATIVE CHEMICAL EXAMINATION OF BLOOD 
173 
Factor by which gas measured 
Temperature (t°) 
moist at t°C. and B mm. pressure is 
reduced to 0°C. and 760 mm. 
0°C. 
B 
760 
15 
0.932 
X 
16 
0.928 
X 
( ( 
17 
0.924 
X 
i i 
18 
0.919 
X 
i 6 
19 
0.915 
X 
C ( 
20 
0.910 
X 
i ( 
21 
0.906 
X 
( i 
22 
0.901 
X 
( i 
23 
0.897 
X 
< i 
24 
0.892 
X 
i 4 
25 
' 0.888 
X 
4 i 
26 
0.883 
X 
( ( 
27 
0.878 
X 
l ( 
28 
0.873 
X 
< i 
29 
0.868 
X 
< i 
30 
0.863 
X 
it 
Table XXX 
The result obtained is then multiplied by 50 (when 2 c.c. of 
blood are used in the determination) to bring to a volume per 
cent basis. 
The gas as measured in the burette includes the nitrogen and 
oxygen which is in physical solution in the blood as well as the 
oxygen in chemical combination with the hemoglobin. In order 
to find the oxygen bound by hemoglobin in blood saturated in 
air the nitrogen and dissolved oxygen are subtracted as indi- 
cated below: 
15° C. 2.14 volume per cent 
20° C. 2.10 
25° C. 2.06 “ “ “ 
30° C. 2.02 “ “ “ 
The figure obtained gives the oxygen binding capacity of the 
blood per 100 c.c. This figure may be converted into grams of 
hemoglobin per 100 c.c. by multiplying by the factor 0.746 since 
1 gram of hemoglobin combines with 1.34 c.c. of oxygen. 
Example.—Blood sample 2.00 c.c. 174 
CLINICAL LABORATORY METHODS 
02 and N2 measured = 0.495 c.e. at 20° C., 767 mm. 0.495 x 
0.910 x^=02 and N2 = 0.455 c.e. at 0° C., 760 mm. 50 x 
0.455 = 02 and N2 per 100 c.e. blood = 22.75 c.e. at 0° C. and 
760 mm. Physically dissolved 02 and N2 per 100 c.c. blood = 
2.10 c.c. at 0° C. and 760 mm. 22.75 - 2.10 = combined 02 per 
100 c.c. = 20.65 cc. at 0° C. and 760 mm. 20.65 x 0.746 = hemo- 
15.40 
globin per 100 c.c. = 15.40 grams. x 100 = per cent of 
normal standard = 99 per cent. CHAPTER VIII 
SEROLOGICAL TECHNIC 
Technic of the Wassermann Reaction 
General Considerations 
The essential features of the technic of the Wassermann reac- 
tion as outlined on the following pages may be summarized as 
follows: 
The test is based on the “quarter unit” amount, the total vol- 
ume of the reagents in each tube being 1.25 c.c., one-fourth of 
that originally suggested by Wassermann. 
An antisheep hemolytic system is employed. 
The dose of amboceptor is two units, the unit being determined 
daily with 0.25 c.c. complement diluted 1:10 in a water-bath at 
37° C. for one hour. 
The complement dose is two units, the unit being determined 
daily by titration with two units of amboceptor. 
The antigen dose is not greater than one-fourth of the anti- 
complementary dose. Two doses are used in the antigen control 
with the same preliminary incubation as in the tests. 
The patient’s serum is diluted 1:5. 0.25 c.c. and 0.125 of the 
diluted serum are used in the test, and 0.5 c.c. in the serum con- 
trol tube. 
The preliminary incubation is four hours in the ice box at 8° C. 
with plain antigen and % hour in the water-bath at 37° C. for 
the cholesterin fortified antigen. 
The quantitative readings are made on Citron’s scale. 
Natural hemolysin is disregarded. The pooled guinea pig 
serum is tested for hemolysin and rejected if any is present. 
Preparation of Glassware 
All glassware used in the test must be chemically clean and 
free of all traces of acid or alkali. Acidity or alkalinity may 
give rise to false reactions. The tubes, pipettes and flasks are 
175 176 
CLINICAL LABORATORY METHODS 
thoroughly washed in soapy water; well washed in running tap 
water; dried in metal baskets and sterilized in the hot air oven. 
Very dirty glassware may be cleaned by boiling in dilute acid. 
The test tubes should be of uniform bore. The most desirable 
size is 100 x 12 millimeters. The pipettes and flasks should be 
accurately calibrated. 
Preparation and Standardization of Reagents 
The reagents used in the complement-fixation test for syphilis 
are: 
1. Physiological salt solution. 
2. Sheep’s red blood cells. 
3. Amboceptor (antisheep immune rabbit serum). 
4. Complement (guinea pig serum). 
5. Patient’s serum. 
6. Antigen (alcoholic extract of heart muscle). 
Physiological Salt Solution.—Physiological salt solution is used 
in performing the test, in diluting the reagents and in washing 
the sheep’s blood. It is prepared by dissolving 8.5 grams of 
chemically pure sodium chloride in a liter of distilled water and 
autoclaving the solution for fifteen minutes at fifteen pounds 
pressure. 
The Sheep’s Blood.—Sheep’s blood may be obtained from a 
slaughter house or from a sheep kept for the purpose. The blood 
is collected directly from the sheep into a sterile bottle contain- 
ing glass beads and immediately defibrinated by shaking or al- 
lowed to run into a wide-mouth bottle half full of a 1.5 per cent 
solution of sodium citrate in physiological salt solution. 
The sheep’s cells are washed and prepared in the following 
manner: 
A centrifuge tube is filled one-fourth with defibrinated or eit- 
rated blood, thoroughly mixed with physiological salt solution 
and centrifuged rapidly for a sufficient time to throw the cells to 
the bottom of the tube. The supernatant fluid is decanted; the 
sedimented cells again mixed with salt solution and centrifuged. 
This process is repeated until the cells have been washed four 
times. After the final washing the level of the cells is noted, the SEROLOGICAL TECHNIC 
177 
supernatant fluid discarded and the packed cells diluted with 
salt solution to twenty times their volume. This gives the 5 per 
cent suspension of sheep’s cells as used in the test. The speed 
and duration of centrifugation should always be the same for 
the final washing in order that the packing of the cells may be 
uniform from day to day. 
Fig. 51. 
Fig. 52. 
Fig. 51.—Interval timer. This is a valuable aid in chemical and serological work. 
Fig. 52.-—The most desirable type of burette for general laboratory use. 
Amboceptor.—The amboceptor is prepared by injecting in- 
travenously a full grown healthy rabbit with washed sheep’s 
red blood cells at five day intervals. The corpuscles must 
be freshly obtained and well washed. A 50 per cent sus- 
pension is used for the injection, 4 c.c. being given the first 
time; 6 c.c. the second and 8 c.c. the third. The second and third 178 
CLINICAL LABORATORY METHODS 
injections must be made very slowly to prevent sudden death 
from agglutination of the corpuscles. 
The rabbit is bled from the ear vein seven to nine days after 
the last injection, and the serum after inactivating is titrated for 
hemolytic power. The serum is satisfactory to use if 0.25 c.c. of 
a 1:2000 dilution hemolyzes completely 0.25 c.c. of a 5 per cent 
suspension of sheep’s corpuscles in the presence of 0.25 c.c. of 
guinea pig serum diluted 1:10 after incubation in the water-bath 
at 37° C. for one hour. 
The animal is then bled either from the carotid artery or by 
puncturing the heart directly. The blood is allowed to clot. The 
clear serum is removed with a sterile pipette and heated in the 
water-bath at 55° C. for % hour. 
This heated immune rabbit serum constitutes the amboceptor. 
It is mixed with an equal quantity of glycerine and distributed in 
2 c.c. quantities into small sterile ampoules or test tubes. 
The stock amboceptor keeps indefinitely in the refrigerator. 
The unit of amboceptor is defined as the smallest amount which 
will give complete hemolysis of 0.25 c.c. of a 5 per cent suspen- 
sion of sheep’s corpuscles in the presence of an excess of com- 
plement after an incubation of one hour in the water-bath at 37° 
C. The unit is roughly determined for each new batch of ambo- 
ceptor as follows: Into each of seven test tubes pipet 0.25 c.c. 
physiological salt solution. To the first add 0.25 c.c. of the ambo- 
ceptor diluted 1:100. Mix and transfer 0.25 c.c. to the second 
tube. Mix and transfer 0.25 c.c. to the third tube. Proceed in 
this manner up to and including the seventh tube. Discard the 
last 0.25 c.c. A series of dilutions of the amboceptor ranging from 
1:200 to 1:12800 is thus obtained. Add to each tube 0.25 c.c. of 
guinea pig serum diluted 1:10, 0.25 c.c. 5 per cent suspension of 
sheep’s corpuscles, and 0.5 c.c. salt solution to bring the volume 
to 1.25 c.c. Incubate in water-bath at 37° C. for one hour. Table 
XXXI shows the scheme for this titration. 
After the incubation the titration is read to determine the high- 
est dilution in which hemolysis is complete. This shows the dilu- 
tion in which a unit of amboceptor is contained in a volume of 
0.25 c.c. The amboceptor is used in the dilution so determined in 179 
SEROLOGICAL TECHNIC 
Preliminary Amboceptor Titration 
number 
CORPUSCLE 
COMPLEMENT 
SALT 
OF TUBES 
AMBOCEPTOR 
SUSPENSION 
1:10 
SOLUTION 
C.C. 
Dilution 
C.C. 
C.C. 
C.C. 
1 
0.25 
1:200 
0.25 
0.25 
0.5 
2 
0.25 
1:400 
0.25 
0.25 
0.5 
3 
0.25 
1:800 
0.25 
0.25 
0.5 
Incubate one 
4 
0.25 
1:1600 
0.25 
0.25 
0.5 
hour in water- 
5 
0.25 
1:3200 
0.25 
0.25 
0.5 
bath at 37° C. 
6 
0.25 
1:6400 
0.25 
0.25 
0.5 
7 
0.25 
1:12800 
0.25 
0.25 
0.5 
Table XXXI 
the daily titration of the amboceptor preliminary to the Wasser- 
mann test. 
Complement.—Complement is obtained from guinea pig’s blood. 
Large male pigs furnish the most active complement. The blood 
is aspirated from the heart with a Luer syringe. The chest wall 
should be punctured close to the sternum through the second or 
third right interspace. The needle is directed downward and 
backward for 1 to 2 centimeters. A large pig will withstand the 
loss of 7 to 8 c.c. of blood. The interval of bleedings should not 
be less than three weeks. 
It is preferable to bleed at least three pigs for making any 
test or titration and to pool the blood obtained. The blood is 
allowed to clot at room temperature, placed in ice box overnight 
and is then centrifuged. The clear serum is pipetted off and 
diluted as indicated by the preliminary complement titration. 
The complement should be kept in the ice box as it deteriorates 
rapidly at room temperature. 
The Patient’s Serum.—The patient’s serum is obtained from 
blood withdrawn by venipuncture. The blood is delivered into a 
small test tube and allowed to stand at room temperature until 
the clot retracts. For use in the test the clear serum is removed 
with a pipette, diluted with four parts of physiological salt solu- 
tion and heated in a water-bath at 55° C. for 15 minutes to 
inactivate. 
Spinal fluids are not diluted or inactivated. 
Antigen.—Antigen is prepared from fresh human or beef heart 
muscle. The fat and connective tissue are carefully removed 
with scissors. The muscle is then finely ground in a meat chop- 180 
CLINICAL LABORATORY METHODS 
per. Five hundred c.c. of absolute alcohol are added to 100 grams 
of the ground heart. The suspension is shaken for twenty-four 
hours in a shaking machine or placed in the incubator at 37° C. 
and left from 10 to 14 days, shaking by hand daily. The mixture 
is allowed to sediment at room temperature. The supernatant 
fluid is plain antigen. Guinea pig heart makes a very satis- 
factory antigen, when extracted with alcohol for four months 
in the ice box. The alcohol is added in the proportion of 
25 c.c. to one heart. To make cholesterinized antigen add 0.2 
gram of cholesterin to 100 c.c. of the plain antigen and place in 
incubator at 37° C. until completely dissolved. The stock anti- 
gens will keep almost indefinitely at room temperature. 
In diluting the stock antigens for use in the test, the antigen is 
transferred to a dry graduate with a dry pipette. Physiological 
salt solution is added drop by drop shaking well after the dilution 
of each drop. 
There are two requirements for a good antigen—(1) a long 
range and (2) specificity. The range is the difference between 
the anticomplementary dose, (the smallest dose of antigen that 
is in itself inhibitory), and the minimum fixing dose, the antigen 
unit. 
The antigen is titrated for anticomplementary property by 
incubating varying amounts of antigen with two units of com- 
plement, and adding amboceptor and corpuscle suspension. This 
titration is illustrated in the protocol in Table XXXII. 
Titration of Antigen for Anticomplementary Property 
Table XXXII 
NUMBER 
OF TUBE 
ANTIGEN 
DILUTED 
1:5 
COM- 
PLEMENT 
2 UNITS 
SALT 
SOLUTION 
m O 
u 0 
lJ 00 
AMBO- 
CEPTOR 
2 UNITS 
CORPUSCLE 
SUSPEN- 
SION 
e.c. 
e.c. 
C.C. 
* n 
C.C. 
C.C. 
Incubate 
1 
0.5 
0.25 
0 
X 
0.25 
0.25 
in water- 
2 
0.4 
0.25 
0.1 
O 
0.25 
0.25 
bath at 
3 
0.3 
0.25 
0.2 
J2 § 
0.25 
0.25 
37° C. 
4 
0.2 
0.25 
0.3 
2 .a 
o 
0.25 
0.25 
for % hr. 
5 
0.1 
0.25 
0.4 
M 
0.25 
0.25 
The tube containing the largest amount of antigen in which 
hemolysis is complete shows the largest amount of antigen which 
is not anticomplementary. SEROLOGICAL TECHNIC 
181 
The natural hemolytic property of the antigen is determined 
by incubating varying dilutions of antigen with complement and 
corpuscle suspension as outlined in Table XXXIII. 
Table XXXIII 
NUMBER 
OP TUBE 
ANTIGEN 
DILUTED 1 :5 
SALT 
SOLUTION 
COMPLEMENT 
2 UNITS 
CORPUSCLE 
SUSPENSION 
c.c. 
C.C. 
C.C. 
C.C. 
1 
0.5 
0 
0.25 
0.25 
Incubate 
2 
0.4 
0.1 
0.25 
0.25 
in water- 
3 
0.3 
0.2 
0.25 
0.25 
bath at 
4 
0.2 
0.3 
0.25 
0.25 
37° O. 
5 
0.1 
0.4 
0.25 
0.25 
for % hour. 
Titration op Antigen for Hemolytic Property 
Note the tube containing the largest amount of antigen causing 
no hemolysis. There should be little if any hemolysis even in the 
tubes containing the largest amount of antigen. 
The best way to standardize an antigen either qualitatively or 
quantitatively is by testing its complement-fixing power against 
a large number of known positive and known negative specimens 
of blood serum and spinal fluids. The unknown antigen should 
be run in parallel with a known satisfactory antigen for comparison. 
Some idea of the inhibitory properties of an antigen may be 
obtained by running a regular Wassermann test using serum 
pooled from at least five positive specimens of blood and employ- 
ing successive dilutions of the antigen. 
Daily complete titrations of the antigen are unnecessary. How- 
ever, antigen control tubes are included in each series to show 
that the antigen is not anticomplementary in double the dose used 
in the test and to show that the antigen fixes complement com- 
pletely in the presence of a positive serum. 
The largest amount of antigen which may be safely employed 
in the diagnostic test is one-fourth of the largest dose which is 
not anticomplementary but should be 8 to 10 times as great as 
the smallest dose which gives complete fixation with a pooled 
positive serum. 
Daily Titration of Reagents Preliminary to the Diagnostic Test 
The correct adjustment of the hemolytic system is the crucial 
factor in the technic of the Wassermann reaction. The most 182 
CLINICAL LABORATORY METHODS 
variable factor is the strength of the complement as obtained 
from different guinea pigs. Each day the hemolytic system is 
standardized by titrating the amboceptor in the presence of fixed 
amounts of complement; and by titrating the complement in the 
presence of two units of amboceptor. 
1. Titration of Amboceptor.—A dilution of the amboceptor is 
prepared in which the unit of amboceptor is contained in a vol- 
ume of 0.25 c.c. as determined roughly in the preliminary titra- 
tion of the amboceptor. Varying amounts of this dilution are 
then incubated with complement and corpuscle suspension for 
one hour in the water-bath at 37° C. 
Table XXXIV shows the scheme for the amboceptor titration. 
Table XXXIV 
Amboceptor Titration 
NUMBER 
OF TUBE 
COMPLE- 
MENT 
1:10 
CORPUS- 
CLE 
SUSPEN- 
SION 
SALT 
SOLUTION 
HEMOLYTIC SERUM 
IN DILUTION TO BE 
TESTED FOR UNIT 
c.c. 
C.C. 
C.C. 
C.C. 
.s 
1 
0.25 
0.25 
0.25 
0.50 
S © 
2 
0.25 
0.25 
0.30 
0.45 
2 ?- 
To deter- 
3 
0.25 
0.25 
0.35 
0.40 
« CO 
mine the 
4 
0.25 
0.25 
0.40 
0.35 
§ OS 
unit of am- 
5 
0.25 
0.25 
0.45 
0.30 
boceptor 
6 
0.25 
0.25 
0.50 
0.25 
7 
0.25 
0.25 
0.55 
0.20 
® Fh 
8 
0.25 
0.25 
0.60 
0.15 
"5 .2 
9 
0.25 
0.25 
0.65 
0.10 
03 
g £ 
10 
0.25 
0.25 
0.70 
0.05 
& 
M 
To show that 
11 
0 
0.25 
0.50 
0.50 
each reagent 
12 
0.50 
0.25 
0.50 
0 
has no hemo- 
lyzing effect 
13 
0 
0.25 
1.00 
0 
The reagents are added in the order given. 
The tubes are shaken thoroughly and incubated one hour in 
the water-bath at 37° C. 
The tube containing the smallest amount of amboceptor in 
which hemolysis is complete contains the unit. For instance if 
in titrating a dilution of 1:1000, tubes 1 to 8 show complete 
hemolysis, then 0.15 is a unit. From this we may calculate that 
dilution in which 0.25 c.c. is a unit as follows: SEROLOGICAL TECHNIC 
183 
X : 0.25 :: 1000 : 0.15 
25000 
X = 1600 (about) 
If a dilution of 1:1600 is now made and titrated, it will be 
found that 0.25 c.e. contains one amboceptor unit. In the test 
two units are employed and the dilution is so made that 0.25 c.c. 
contains two units. In the illustration given this would be a 
1:800 dilution. 
Tubes are included in the amboceptor titration to show that 
each reagent has no hemolyzing effect. 
2. Titration of Complement.—A 1:20 dilution of the guinea pig 
serum to be used in the diagnostic test is prepared. Varying 
amounts of the diluted serum are incubated for % hour in the 
water-bath at 37° C. with 0.25 c.c. of the corpuscle suspension and 
0.25 c.c. of the amboceptor containing two units as determined in 
the preliminary amboceptor titration. 
The unit of complement is defined as the smallest amount in 
the presence of which two units of amboceptor will completely 
hemolyze 0.25 c.c. of a 5 per cent suspension of sheep’s cor- 
puscles. 
Table XXXY 
COMPLE- 
NUMBER 
MENT 
SALT 
CORPUSCLE 
AMBOCEPTOR 
OF TUBE 
1:20 
SOLUTION 
SUSPENSION 
2 UNITS 
c.c. 
C.C. 
C.C. 
C.C. 
1 
0.50 
0.25 
0.25 
0.25 
£ 
2 
0.45 
0.30 
0.25 
0.25 
r? O 
3 
0.40 
0.35 
0.25 
0.25 
To determine 
4 
0.35 
0.40 
0.25 
0.25 
■s 
the unit of 
5 
0.30 
0.45 
0.25 
0.25 
* * 
complement 
6 
0.25 
0.50 
0.25 
0.25 
«H 
7 
0.20 
0.55 
0.25 
0 25 
% d 
OS o 
8 
0.15 
0.60 
0.25 
0.25 
9 
0.10 
0.65 
0.25 
0.25 
rO t- 
0 CO 
10 
0.05 
0.70 
0.25 
0.25 
o 
a -g 
M 0 
To show that 
11 
0.0 
0.50 
0.25 
0.25 
each reagent 
12 
0.50 
0.50 
0.25 
0.25 
has no hemo- 
lyzing effect- 
13 
0.0 
1.00 
0.25 
0.25 
Titration of Complement 184 
CLINICAL LABORATORY METHODS 
Table XXXV shows the arrangement of the tubes for the com- 
plement titration, and the order in which the reagents are to 
be added. 
The tubes are thoroughly shaken and incubated in the water- 
bath at 37° C. for % hour. The tube containing the smallest 
amount of complement in which hemolysis is complete contains 
one unit. For instance if Tubes 1 to 7 show complete hemolysis 
then 0.20 c.c. of the 1:20 dilution is a unit. From this we may 
calculate that dilution in which 0.25 c.c. is a unit as follows: 
X : 0.25 :: 20 : 0.20 
5.0 
0.20 
X = 25 
If a dilution of 1:25 is now made and titrated it will be found 
that 0.25 c.c. contains one unit. In the diagnostic test two units 
are employed and the dilution is so made that 0.25 c.c. contains 
two units. In the illustration given two units would be con- 
tained in 1:12.5 dilution. 
The Diagnostic Test 
Reagents.—(1) Patient’s serum diluted with four parts of salt 
solution and heated in the water-bath at 55° C. for 15 minutes. 
(2) Complement—guinea pig serum free from natural hemol- 
ysin and so diluted that 0.25 c.c. contains 2 units. 
(3) Antigen—alcoholic extract of heart muscle which is not 
hemolytic and is so diluted that four times the amount used (0.25 
c.c.) is not anticomplementary. 
(4) Amboceptor—immune rabbit serum so diluted that 0.25 c.c. 
contains two units. 
(5) Sheep’s corpuscles—5 per cent suspension in salt solution. 
Procedure.—Into the first of three tubes pipette 0.50 c.c. of the 
diluted patient’s serum; into the second 0.25 c.c. and into the 
third 0.125 c.c. Add to the second and third tubes 0.25 c.c. 
of the antigen. Finally add to each of the three tubes 0.25 c.c. 
of complement. 
Fix for 45 minutes in the water-bath at 37° C. if cholesterin- SEROLOGICAL TECHNIC 
185 
i—1 
o ® 00 S ® Ol W K H 
NUMBER 
OP TUBE 
ooooooopop 
m to a? • 
to o\ o P 
o\ 
KNOWN 
NEGATIVE 
SERUM 
OOOOppOOOOo 
i—1 to bi b 
to at o 
Ul 
KNOWN 
POSITIVE 
SERUM 
oppooooooo 
H b Ul 
to cn o 
Ol 
PATIENT’S 
SERUM 
PPOOOOOOOOQ 
bi to bo bo to to bo h 
o oi oi oi oi oi cn 
ANTIGEN IN 
STANDARD 
DILUTION 
to to to to to to to to to to b 
UlOtCTlCHOlCHOlOlOlOt* 
COMPLE- 
MENT 
2 UNITS 
Incubate 45 min. in water-bath 
at 37° C. if cholesterinized 
antigen is used; or 4 hrs. in 
ice box at 8° 0. if plain 
antigen is used. 
opppppppppo 
bo bo bo bo to bo bo to to to b 
OlOlOlOlOlOlOlOlOlOl- 
AMBO- 
CEPTOR 
2 UNITS 
ppooppppppa 
tobotobotobobobobobob 
OlOlOlOlOlOlOlOlOlOl' 
1 5 PER CENT 
SUSPENSION 
SHEEP’S CELLS 
Incubate in water-bath at 37° 
C. until serum control tubes 
(Nos. 1, 4, and 7) and antigen 
control tube (No. 10) show 
complete hemolysis. 
Test of Serum for Diagnosis. 
Table XXXVI 186 
CLINICAL LABORATORY METHODS 
ized antigen is used, or in ice box at 8° C. for four hours if plain 
antigen is used. 
Remove from the water-bath or ice box and add to each tube 
0.25 c.c. of a 5 per cent suspension of sheep’s cells and 0.25 c.c. 
of amboceptor. It is convenient to mix equal parts of corpuscles 
and amboceptor just before using and to add 0.50 c.c. of the 
mixture. 
Incubate in water-bath at 37° C. until hemolysis is complete in 
the serum control tube. A test is also made with a known posi- 
tive and a known negative serum as control of the fixability and 
specificity of the antigen. A tube containing a double dose of 
antigen is included in the series to show that the antigen is not 
anticomplementary. 
Tables XXXYI and XXXYII show the arrangement of the 
tubes in the diagnostic test: 
Table XXXVII 
Test of Spinal Fluid fob Diagnosis 
NUMBER 
OF TUBE 
PATIENT ’S 
SPINAL 
FLUID 
UNDILUTED 
ANTIGEN IN 
STANDARD 
DILUTION 
COMPLE- 
MENT 
2 UNITS 
i ice box 
O 
c3 
pT-? 
*3 S3 
CO 
O 
O 
<d> 
£ 
Q 
hJD 
AMBO- 
CEPTOR 
2 UNITS 
5 PER CENT 
SUSPENSION 
SHEEP CELLS 
in water- 
° 0. until 
3 complete 
C.C. 
e.c. 
e.c. 
•H 
o 
'H 
"3 
C.C. 
C.C. 
1 
2 
1.0 
1.0 
0 
0.25 . 
0.25 
0.25 
GO 
** a 
d-rH 
d 
0.25 
0.25 
0.25 
0.25 
CO 
3 
4 
0.50 
0.25 
0.25 
0.25 
0.25 
0.25 
.a 
U 
00 
CO 
N 
3 
*n 
0.25 
0.25 
0.25 
0.25 
§ £ g -2 
5 
0.125 
0.25 
0.25 
Ph 
s 
o> 
4-> 
0.25 
0.25 
Reading the Diagnostic Test 
Citron’s standard is used in reading all tests on serum after 
the final incubation. (Plate Y.) 
Complete absence of hemolysis in tubes 2 and 3 equals posi- 
tive 4 plus or very strongly positive. 
Complete absence of hemolysis in tube 2 and faint hemolysis in 
tube 3 equals positive 3 plus or strongly positive. 
Complete absence of hemolysis in tube 2 and complete or nearly 
complete hemolysis in tube 3 equals positive 2 plus or positive. 
Partial hemolysis in tube 2, complete or nearly complete hemol- 
ysis in tube 3 equals positive one plus or suggestive. Tube 1 
Tube 2. 
Tube 3 
Positive + + + + 
Wassermann Reaction 
Positive + + 
Wassermann Reaction 
Positive + 
Wassermann Reaction 
Negative 
Wassermann Reaction 
Plate V. 
Citron’s scale for reading the Wassermann Reaction. SEROLOGICAL TECHNIC 
187 
Complete hemolysis in all tubes equals negative. 
This standard cannot be used in reading the tests on spinal 
fluids. Complete fixation in any amount of spinal fluid as used 
in the test is considered a positive test. A report is made of 
the degree of fixation with the varying amounts of spinal fluid 
used. 
Widal Reaction 
(Modified Dreyer Technic) 
Principle.—The Widal reaction is a procedure by which the 
agglutinating power of a serum against any organism may be 
tested. Serum in varying dilutions is mixed with a standard- 
ized bacterial suspension. The tubes containing the mixtures 
are incubated for 2 hours at 55° C., and examined for ag- 
glutination. 
The most essential part of the procedure is the use of a stand- 
ardized bacterial suspension. Having this, quantitative fluctua- 
tions in the agglutinin content of the blood can be accurately 
determined. 
Preparation of Bacterial Suspension.—In making a bacterial 
suspension, the organism is subcultured daily in broth for about 
ten days to increase its agglutinability and to diminish any 
tendency to spontaneous agglutination. It is then planted on 
agar slants in Kolle flasks or in large bottles and incubated over- 
night. The growth is washed off with 0.85 per cent salt solu- 
tion to which 0.1 per cent formalin has been added. It is then 
placed in an ice box for 4-5 days and shaken repeatedly. It is 
tested for sterility. The suspension is diluted with salt solution 
to make a suspension containing 2,000 million organisms per c.c. 
This may be done by counting, using Wright’s method or by 
contrifuging and diluting according to the table of Hopkins 
(see page 230). 
The suspension is then ready to be standardized for agglutin- 
ability. A rabbit immune serum of known agglutinin unit con- 
tent is set up in two parallel series in varying dilutions, the 
variations not to be excessive. To one series a standardized sus- 
pension is added, to the other the suspension to be standardized. 
The tubes are then shaken, incubated at 55° C. for two hours 188 
CLINICAL LABORATORY METHODS 
and read for the highest dilutions showing agglutination visible 
to the naked eye. 
That dilution of the standardized suspension is to the dilution 
of the new suspension as the known factor of the standardized 
suspension is to x (the factor of the new suspension). 
Example.—If the dilution in which the standardized suspen- 
sion is agglutinated is 1 to 6400 while that of the new suspension 
is 1 to 3200 and the factor of the standardized suspension is 
1.0, then— 
6400 1 3200 1 
3200 " x or X _ 6400 = 2 
The factor of the new bacterial suspension is therefore one-half. 
The suspension should be bottled in small sterile bottles and 
kept in an ice box. 
Procedure.—Blood is withdrawn by venipuncture, allowed to 
clot, and the serum separated off. Select ten small test tubes 
about 1 cm. in diameter and of uniform bore. In the first tube 
place 1.8 c.c. 0.85 per cent salt solution and in each of the others 
1 c.c. Transfer 0.2 c.c. of blood serum to the first tube, mix 
thoroughly and carry forward 1 c.c. to the next tube. This 
procedure is carried out through the series of ten tubes thus 
making serum dilutions ranging from 1 to 10, to 1 to 5120. 
To each tube in the series 1.5 c.c. of the standardized bacterial 
suspension is added. The tubes are next thoroughly shaken. 
The tubes are then placed in a water-bath at from 50° to 
55° C. for two hours, are removed and cooled for fifteen min- 
utes at room temperature, and read. The highest dilution 
showing agglutination without sedimentation visible to the naked 
eye gives the reading. The dilution of the serum in this tube, 
divided by the factor of agglutinability gives a final reading ex- 
pressed in the number of standard agglutinin units per cubic 
centimeter of serum. It must be kept in mind that the addition 
of the suspension has increased the dilution of the serum one 
and a half times, thus the first tube represents a dilution of 1 to 
25, the last a dilution of 1 to 12,800. 
Remarks.—The Widal reaction is applied principally in diag- 
nosing the existence of enteric fevers. In noninoculated persons SEROLOGICAL TECHNIC 
189 
who have not had typhoid or paratyphoid fever agglutination in 
a dilution of 1 in 25 justifies a strong suspicion of typhoid or 
paratyphoid infection. Marked agglutination in 1 to 50 or more 
is nearly always diagnostic of active typhoid or paratyphoid 
infection. 
In inoculated persons the test is done at intervals of one week, 
and if an active enteric infection exists, there will be a definite 
rise in agglutinin titer. 
The agglutinin titer in active typhoid increases for the first 
three weeks after which it falls, at first rapidly and then slowly. 
Determination of Types of Pneumococcus 
(Blake, F. S.: Jour. Exp. Med., 1917, xxxvi, 67) 
1. Mouse Method.—If mice are available, proceed as follows: 
A small portion of the sputum is selected and washed through 
three or four changes of sterile salt solution in sterile Petri 
dishes. The washed sputum is then transferred to a sterile mor- 
tar, ground up and emulsified with about 1 c.c. of sterile bouillon, 
added drop by drop, until a homogeneous emulsion is obtained 
that will readily pass through the needle of a small syringe. 
One-half to 1 c.c. of this emulsion is inoculated intraperitoneally 
into a white mouse with a sterile syringe. As soon as the injected 
mouse appears sick a drop of peritoneal exudate is removed by 
means of peritoneal puncture with a sterile capillary pipette, and 
examined microscopically for pneumococcus. If there is an abun- 
dant growth of pneumococcus the mouse is killed and the deter- 
mination of type proceeded with. If the growth is only moderate, 
or other organisms are present in any quantity, further time must 
be allowed until subsequent examination of the peritoneal exu- 
date shows an abundant growth of pneumococcus. 
As soon as the mouse is killed the peritoneal cavity is opened 
with sterile precautions, and cultures are made on plain broth 
and on one-half of a blood agar plate. Smears are made and 
stained by gram and for capsules. 
The peritoneal exudate is washed out by means of a sterile 
glass pipette with 4 to 5 c.c. of sterile salt solution, the washings 
being placed in a centrifuge tube. Cultures are then made from 190 
CLINICAL LABORATORY METHODS 
the heart’s blood in plain broth and on the other side of the 
blood agar plate. 
The peritoneal washings are centrifugalized at low speed for 
a few moments until the cells and fibrin are thrown down. 
Decant the supernatant bacterial suspension with a centrifuge 
tube and centrifuge at high speed. The bacterial sediment is 
taken up in sufficient sterile salt solution to make a moderately 
heavy suspension. 
The suspension is then mixed with diluted immune serum of 
the three fixed types, and with undiluted Type II serum, in 
equal quantities of 0.5 c.c. each. 
Table XXXVIII shows the possible reactions after incubation 
in a water-bath at 37° C. for 1 hour. 
Table XXXVIII 
Determination of Pneumococcus Types by Agglutination 
PNEUMOCOCCUS 
SUSPENSION, 0.5 C.C. 
SERUM I 
(1 TO 20) 
0.5 C.C. 
SERUM II 
(undiluted) 
0.5 C.C. 
SERUM II 
(1 TO 20) 
0.5 C.C. 
SERUM III 
(1 TO 5) 
0.5 c.c. 
Type I 
- 
- 
- 
Type II 
+ + 
+ + 
- 
Subgroups II, A, B, X. 
. . - 
+ 
- 
- 
Type III 
- 
- 
+ + 
Group IV 
- 
- 
- 
A fifth tube should be added containing 0.1 c.c. sterile bile and 
0.3 to 0.5 c.c. of bacterial suspension to determine the bile solu- 
bility of the stain for differentiation from the streptococcus. 
Gentle agitation of the tubes will hasten clumping. An organism 
which agglutinates in undiluted Type II serum alone should be 
incubated with undiluted Type I and Type II serum also to 
rule out a Group IV stain that shows cross agglutination in all 
three types of immune serum. If no agglutination occurs in 
any tube and the organism is bile soluble it is reported as pneu- 
mococcus Group IV. 
The determination of type on the peritoneal washings should 
be confirmed by agglutination tests with a pure bouillon culture 
of the pneumococcus obtained from culture of the heart’s blood 
of the mouse. 
Bouillon cultures from blood, spinal fluid or empyema fluid SEROLOGICAL TECHNIC 
191 
showing a pneumococcus may be centrifuged, the sediment mixed 
with salt solution, and the type determined by the method out- 
lined above. 
2. Avery Cultural Method.—If mice are not available the typ- 
ing of pneumoeoccus may be done by the cultural method of 
Avery. Wash sputum as indicated under mouse method. Grind 
in sterile mortar with 9 c.c. of meat infusion broth with an H-ion 
concentration of 7.6, adding the broth drop by drop. Pour in 
centrifuge tube, add 0.5 c.c. each of sterile 20 per cent dextrose 
and blood. 
Incubate at 37° C. for 5 to 6 hours. ' The tube is now centri- 
fuged at low speed long enough to throw down the red cells but 
not enough to bring down the bacteria. A smear is then made, 
stained by gram, and a blood agar plate is inoculated. 
The. supernatant suspension is used for agglutination tests 
as indicated under mouse method. 
Blood Tests Preliminary to Transfusion 
Before a blood transfusion is done, it is necessary to show that 
the serum of the donor does not agglutinate or hemolyze the 
cells of the recipient, or vice versa. 
Since hemolysis never occurs when agglutination is absent, the 
selection of a proper donor from the point of view of agglutina- 
tion determines the other also. Two tests should be done. First, 
the blood group of both the donor and the recipient hre deter- 
mined. If these are found to be the same, the corpuscles of the 
recipient are matched against the serum of the donor and the 
serum of the recipient against the corpuscles of the donor. The 
two tests act as a check on each other. 
1. Determination of Blood Group.— (A) Preparation of Diag- 
nostic Sera.—Blood is withdrawn by venipuncture from a person 
whose group is known to be II. The serum is separated off and 
put in small sterile tubes or ampoules in 1 c.c. amounts. Similarly 
serum is procured from a person who belongs to Group III. It is 
important that the sera selected be high in agglutinin content. 
(B) Procedure.—Several drops of blood from the person to be 
grouped are collected in a small test tube containing 3 to 5 c.c. 
134 per cent of sodium citrate in physiological salt solution. 192 
CLINICAL LABORATORY METHODS 
With a capillary pipette one drop of the corpuscle suspension 
and one drop of Group II serum are placed on a porcelain color 
mixing plate (Fig. 23) and thoroughly mixed. 
A similar preparation is made with the corpuscle suspension 
and Group III serum. 
Fig. S3.—Showing the reaction of corpuscles of various groups with Group II and 
Group III sera. 
Cover the mixing plate with a moist blotter and examine the 
preparations at intervals. If agglutination takes place the mix- 
ture of serum and corpuscles takes on a “brick-dust” appearance 
(Fig. 53). 
The following diagram shows the agglutination reactions be- 
tween the corpuscles and serum of different groups (Moss classi- 
fication). SEROLOGICAL TECHNIC 
193 
CORPUSCLE GROUP 
SERUM GROUP 
Table XXXIX gives all the possible combinations of reac- 
tions which can be obtained by mixing the corpuscles of an un- 
known group with the serum of Group II and Group III and 
indicates the group to which the unknown blood belongs (Moss 
classification): 
Table XXXIX 
THE UNKNOWN BLOOD 
BELONGS TO GROUP 
X Corpuscles plus Group II Serum equals 0 ) 
X Corpuscles plus Group III Serum equals 0 | 
IV 
X Corpuscles plus Group II Serum equals plus ) 
X Corpuscles plus Group III Serum equals 0 ( 
III 
X Corpuscles plus Group III Serum equals plus ) 
X Corpuscles plus Group II Serum equals 0 J 
II 
X Corpuscles plus Group II Serum equals plus 1 
X Corpuscles plus Group III Serum equals plus } 
I 
Report results as follows: 
Determination of Blood Group 
The blood belongs to Group.. 
2. Transfusion Test.—Having determined that the donor and 
recipient belong to same group, make a preparation as described 
above using the suspension of the donor’s corpuscles, and the 
recipient’s serum, and another from the donor’s serum and the 
recipient’s corpuscles. There should be no agglutination or 
hemolysis. 
Report as follows: Donor 
Transfusion Test No agglutination or hemolysis. CHAPTER IX 
PREPARATION OF BACTERIOLOGICAL SOLUTIONS, 
STAINS, AND MEDIA 
Sodium Citrate Solution for Blood Culture 
Sodium chloride 8.5 grams 
Sodium citrate 15.0 grams 
Water (distilled) 1000 c.c. 
Fill in 60 c.c. Erlenmeyer flasks, in 20 c.c. amounts. Sterilize 
in autoclave. 
Physiological Salt Solution 
Sodium chloride, chemically pure 8.5 grams 
Water 1000 c.c. 
Stock Staining Solutions (Wood) 
Saturation Strengths 
Basic fuchsin in alcohol 3.0 per cent 
Gentian violet in water 1.5 per cent 
Gentian violet in alcohol 4.8 per cent 
Methylene blue in water 6.7 per cent 
Methylene blue in alcohol 7.0 per cent 
Safranin in water 4.0 per cent 
The saturated alcoholic solutions can be kept and aqueous 
staining solutions made from them by adding 5 per cent of the 
saturated alcoholic solution to water. 
Carbol-Thionin 
Saturated solution of thionin in 50% alcohol. ... 10 c.c. 
2% phenol 100 c.c. 
Stain two minutes. 
194 SOLUTIONS, STAINS AND MEDIA 
195 
Andrade Indicator 
0.5% solution of acid fuchsin 100 c.c 
Normal sodium hydroxide 16 c.e 
Gram Stain 
Reagents.— 
1. Sterling’s Gentian Violet Solution.— 
Gentian violet 5.0 gm. 
Anilin oil 2 c.e. 
95% alcohol 10 c.c. 
Water 88 c.c. 
Mix the anilin oil and the 95 per cent alcohol. Shake and add 
the distilled water. Add the anilin solution to the gentian violet 
while grinding in a mortar. Filter through paper. 
This solution has intense staining power and will keep indef- 
initely. 
2. Gram’s Solution.— 
Iodine 1 gm. 
Potassium iodide 2 gm. 
Water 300 c.c. 
3. Counter Stain.— 
Use either Bismarck brown 0.5 per cent aqueous solution; or 
safranin, 0.1 per cent aqueous solution. 
Procedure.—1. Stain the fixed smear in the Sterling’s gentian 
violet for a minute. 
2. Wash in water. 
3. Cover with Gram’s solution for one minute. 
4. Decolorize in 95 per cent alcohol until the smear has a gray- 
ish color. 
5. Wash in water. 
6. Counterstain one minute with the 0.5 per cent Bismarck 
brown, or the 0.1 per cent safranin. 
7. Wash, dry and examine. 
The gram positive organisms are stained purple; the gram 
negative ones are brown if Bismarck brown be used as counter 
stain, or red if safranin be used. 196 
CLINICAL LABORATORY METHODS 
Neisser’s Stain for Diphtheria Bacillus 
Reagents.— 
1. Neisser Stain Number 1 (Methylene Blue Solution).— 
Methylene blue 1 gram 
Alcohol—95% 20 c.c. 
Dissolve and add 
Glacial acetic acid 30 c.c. 
Distilled water 950 c.c. 
Filter. 
2. Neisser Stain Number 2 (Bismarck Brown. Solution).— 
Bismarck brown 2 grams 
Boiling distilled water 1000 c.c. 
Dissolve—filter after the solution cools. 
Procedure.— 
1. Stain in solution Number 1—one to three seconds. 
2. Wash quickly in water. 
3. Stain in solution Number 2—three to five seconds. 
4. Wash quickly, dry and examine. 
The diphtheria bacilli appear a light brown, rods containing 
one to three dark blue granules (polar bodies). 
Ziehl-Neelson Stain for Tubercle Bacilli 
Reagents.— 
1. Carbol-Fuchsin.— 
Basie fuchsin, saturated alcoholic solution 90 c.c. 
5 per cent carbolic acid to 100 c.c. 
2. Acid Alcohol.— 
Hydrochloric acid, cone 20 c.c. 
80 per cent alcohol to 1000 c.c. 
3. Loeffler’s Methylene Blue.— 
Methylene blue, saturated alcoholic solution 30 c.c. 
Potassium hydroxide, 0.01 per cent solution 100 c.c. 
Procedure.—1. Fix the smear by passing it through the flame 
of a Bunsen burner. SOLUTIONS, STAINS AND MEDIA 
197 
2. Cover the smear with carbol-fuchsin and warm it till the 
stain steams. Maintain this temperature for 3’ to 5 minutes. 
3. Wash in running water. 
4. Decolorize in acid alcohol until only the thickest part of 
the smear remains a faint pink. 
5. Wash in water. 
6. Stain in Loeffler’s methylene blue, 30 seconds to one minute. 
7. Wash dry and examine under the oil immersion objective. 
The tubercle bacilli appear red; all else is blue. 
Ponder’s Stain 
Prepare as follows: 
Toluidin blue 0.02 gram 
Glacial acetic acid 1 centimeter 
Absolute alcohol 2 centimeters 
Distilled water 100 centimeters 
The film is made on a cover glass and fixed in the usual way. 
A small quantity of the stain is spread on the film and the cover 
glass turned over and mounted as a hanging drop preparation. 
The metachromatic granules of the diphtheria bacilli stain with 
striking intensity. With diphtheroids the more intense stain- 
ing sharply differentiates from ordinary cocci and bacilli, which 
show in the preparation only as faint light blue bodies. 
Capsule Stain (Modified Hiss Stain) 
Smear the organisms in a drop of animal serum, preferably 
beef blood serum, and make film preparation on a glass slide. 
Dry in air and fix by heat. 
Stain for a few seconds with Sterling’s gentian violet. The 
preparation is flooded with the dye and held for a second over a 
free flame until it steams. 
Wash off stain with 20 per cent copper sulphate solution. 
Decolorize quickly with 95 per cent alcohol. 
Wash again with the copper sulphate solution. 
Dry and mount. 198 
CLINICAL LABORATORY METHODS 
(Huntoon, F. M.: Jour. Bact., 1917, ii, 241) 
Huntoon uapsuie stain 
Solutions.— 
1. Nutrose Diluent Solution.— 
Sift three grams of nutrose into 100 c.c. of distilled water and 
heat to 100° C. in the Arnold sterilizer for one hour. Add 5 c.c. 
of 2 per cent aqueous solution of carbolic acid to serve as a pre- 
servative. Decant into test tubes and allow to settle. Employ 
the supernatant fluid as the diluent. Since the supernatant fluid 
tends to become thinner by constant precipitation of the nutrose 
the solution should be occasionally reboiled. 
2. Fixing and Staining Solution.— 
2 per cent aqueous solution of carbolic acid.. 100 c.c. 
Lactic acid 75 per cent 0.25-0.5 c.c. 
1 per cent acetic acid 1 c.c. 
Saturated alcoholic solution of basic fuchsin. . 1 c.c. 
Carbol-fuchsin (old) 1 c.c. 
Technic.—1. Employ the nutrose solution as a diluent, emul- 
sifying the bacteria in one or two loopfuls and then spreading in 
as thin a film as possible with the loop. 
2. Allow to dry in the air. 
3. Cover the film with the fixative and staining solution and 
allow to act for thirty to forty-five seconds. 
4. Wash quickly in water, dry and examine. 
Remarks.—If nutrose is not available, the following solution 
may be used as a substitute for the 3' per cent nutrose solution: 
Render milk as nearly fat free as possible by means of centrifu- 
gation, add 1 per cent of 2 N sodium hydrate and bring to a boil. 
After cooling add ether and shake. After a few minutes decant 
off the ether. The remaining opalescent fluid may be used in 
place of the nutrose solution. 
Stain for Spirocheta Pallida (Medalia) 
(Medalia: Jour. Am. Med. Assn., 1918, lxx, 914) 
The surface of the lesion is cleaned with a wad of sterile ab- 
sorbent cotton, soaked in sterile saline solution, and all the dried SOLUTIONS, STAINS AND MEDIA 
199 
exudate is removed. The patient is then told to squeeze the 
lesion between the thumb and forefinger until serous exudate 
appears. The appearance of the exudate may be hastened by 
scraping the deeper parts or the ragged edges of the lesion with 
a broken wooden applicator, or with a stiff platinum wire. The 
exudate thus obtained from the deeper parts of the lesion should 
be mostly a serous exudate; real bleeding should be avoided. A 
good smear is one in which only an occasional red blood cell is 
found. The smears may be made on clean slides by placing a 
drop of deep exudate obtained with a broken applicator, a sterile 
toothpick, or a platinum loop, on one slide and spreading it out 
thinly with the other slide. The slides should not be pulled out 
but slid apart. 
The smears are then dried in the air and stained with Wright’s 
blood stain in the ordinary way, except that instead of plain dis- 
tilled water to dilute the stain, one per cent solution of sodium 
carbonate in distilled water is used. The film is covered with 
Wright’s stain, to fix the smear, a sufficient quantity being used, 
a little in excess of what is used in staining blood smears. At the 
end of from one to two minutes, to the staining fluid are added 
from 45 to 50 drops of one per cent sodium carbonate solution in 
distilled water to a slide, or as much as the film will hold without 
running over. The diluted stain is allowed to remain on the film 
for fifteen to twenty minutes, and is gently steamed all the while 
with the flame of an alcohol lamp, as in the staining of sputum 
for tubercle bacilli by carbol fuchsin. The slide is then lightly 
washed with water, dried between filter paper and examined with 
the oil immersion lens. 
The spirochetes appear intensely violet on a pale blue back- 
ground and are easily found. 
Sterilization 
All dry glassware should be sterilized in a hot air sterilizer 
for one hour at 150° C. Petri dishes and pipettes are placed in 
copper boxes or wrapped in paper. Tubes and flasks are plugged 
with absorbent cotton. 
Media is sterilized by steam, using either an autoclave or Ar- 
nold sterilizer. Media containing sugar is heated in the Arnold 200 
CLINICAL LABORATORY METHODS 
on three successive days for thirty minutes. The temperature 
cannot rise above 100° C. since no pressure is used. All other 
media is sterilized by heating in the autoclave at 15-20 pounds 
pressure for 15-30 minutes. Fifteen pounds of pressure is equiva- 
lent to a temperature of 121.3° C., 30 pounds is equivalent to 
134.6° C. 
Titration of Culture Media to Definite Hydrogen-ion 
Concentration 
The reaction of culture media should be accurately adjusted 
to a definite hydrogen-ion concentration. This may be quickly 
and simply done by the colorimetric method. 
Reagents.—(1) Phenolsulphonephthalein (phenol red), 0.02 per 
cent water solution. 
(2) N/10 NaOII and N/l NaOH. 
(3) Standard phosphate mixtures of varying hydrogen-ion 
concentration prepared according to Sorensen’s directions as 
follows: 
One-fifteenth mol. acid or primary potassium phosphate. 9.078 
grams of the pure recrystallized salt (KH2P04) is dissolved in 
freshly distilled water and made up to 1 liter. 
One-fifteenth mol. alkaline or secondary sodium phosphate. 
The pure recrystallized salt (Na2HP0412H20) is exposed to the 
air for from ten days to two weeks, protected from dust. Ten 
molecules of water of crystallization are given off and a salt of 
the formula Na2IIP042H20 is obtained. 11.876 grams of this is 
dissolved in freshly distilled water and made up to 1 liter. The 
solution should give a deep rose-red color with phenolphthalein. 
If only a faint pink color is obtained, the salt is not sufficiently 
pure. 
The solutions are mixed in the proportions indicated in Table 
XL to obtain the desired PH. 
Procedure.—Measure 10 c.c. of the media to be titrated into a 
test tube that has been rinsed with a portion of the media. Add 
0.5 c.c. of the 0.02 per cent phenolsulphonephthalein. Run in 
from a burette a measured quantity of N/10 NaOH until the color 
matches that of the standard chosen. 
To make the color standard measure 10 c.c. of the phosphate SOLUTIONS, STAINS AND MEDIA 
201 
6.4 
6.6 
6.8 
7.0 
7.1 
7.2 
7.3 
7.4 
7.5 
7.6 
7.7 
7.8 
8.0 
8.2 
8.4 
Primary potassium 
phosphate c.e. 
73 
63 
51 
37 
32 
27 
23 
19 
15.8 
13.2 
11.0 
8.8 
5.6 
3.2 
2.0 
Secondary sodium 
phosphate c.c. 
27 
37 
49 
63 
68 
73 
77 
81 
84.2 
86.8 
89.0 
91.2 
94.4 
96.8 
98.0 
Table XL 202 
CLINICAL LABORATORY METHODS 
mixture corresponding to the hydrogen-ion concentration to 
which the media is to be adjusted and add 0.5 c.c. of the 0.02 per 
cent solution of phenol red. Compare in color comparator, hold- 
ing four tubes. Place behind the media to be tested a tube of 
distilled water and behind the color standard a tube of the media 
containing no indicator (Fig. 61). In this way the native color 
of the media is compensated for. 
Calculate the amount of normal alkali or acid to be added to 
the medium to give the proper reaction. For example, if it 
requires 2 c.c. of N/10 NaOH to bring 10 c.c. of the medium to 
PH 7-6 then to bring 1 liter of the medium to the same reaction 
will require the addition of 
x 2 = 200 c.c. N/10 NaOII or 20 c.c. N/l NaOH. 
Media is made more acid by the process of autoclaving. The 
change is small and is much less with meat infusion than with 
meat extract media. 
Table XLI shows the optimum reaction for the growth of vari- 
ous organisms and for the preparation of special media. 
Table XLI 
Optimum H-Ion Concentration of Media for Cultivation of Various 
Organisms, and for Preparation of Special Media 
(Fennel and Fisher: Jour. Infect. Dis., 1919, xxv, 418.) 
B. Typhosus 
6.2 - 
7.2 
B. Paratyphosus 
6.2 - 
7.2 
Pneumococcus 
7.8 
B. Influenzae 
7.8 
Streptococcus Yiridans 
7.6 - 
7.8 
Streptococcus Hemolyticus 
7.6 - 
7.8 
Meningococcus 
7.6 
Gonococcus 
7.4 - 
7.6 
B. Dysenteriae, Shiga, Flexner, and “ Y” 
6.2 - 
6.8 
B. Diphtheriae for toxin production 
8.0 - 
8.2 
Endo Agar 
7.4 - 
7.8 
Bussell Agar 
7.4 - 
7.6 
Brilliant Green Agar 
7.0 - 
7.2 
Meat Infusion Agar 
I. Infuse 500 grams of lean meat twelve hours in 500 c.c. of 
distilled water in ice box. 
Strain through wet cotton flannel or wet cheesecloth. SOLUTIONS, STAINS AND MEDIA 
203 
II. Add 15 grams of thread agar, 10 grams peptone, and 5 
grams sodium chloride to 500 c.c. water and dissolve in autoclave. 
Cool to about 65° C. 
III. Then to Solution I of meat infusion add Solution II of 
agar (at 65° C.). After the two are mixed, stir thoroughly, take 
out a specimen for titration, and lose no time in getting the mix- 
ture into the Arnold or water-bath to keep it from cooling below 
the congealing temperature of the agar. 
Before doing this, quickly measure volume. 
Titrate the specimen removed, calculate the amount necessary 
to bring the entire volume to PH 7.4, add normal sodium hydrate 
solution as required and place the mixture in the Arnold steril- 
izer for one-half hour. 
Filter through gauze and absorbent cotton. Sterilize in the 
autoclave for 15 minutes at 15 pounds pressure. 
Glucose Agar 
Dissolve 10 grams of glucose in one liter of stock agar. Tube 
and autoclave at 15 pounds pressure for 15 minutes. 
Meat Extract Agar 
To 1 liter of tap water add: 
Peptone 10 grams 
Beef extract 3 grams 
Sodium chloride 5 grams 
Agar 15 grams 
Dissolve all ingredients except agar first; add agar and auto- 
clave for one hour at fifteen pounds pressure. Cool to 50° C. 
Add white of an egg and steam in Arnold for thirty minutes. 
Adjust reaction to PH 7.4. Steam for a few minutes. Filter 
through cotton, and autoclave for fifteen minutes at fifteen 
pounds pressure. 
Red Blood Agar 
To 100 c.c. of melted agar at a temperature of 50° C. add 5 c.e. 
of defibrinated blood. Pour plates using 12 to 15 c.c. of agar for 
each 100 mm. plate. 204 
CLINICAL LABORATORY METHODS 
Brown Blood Agar 
To 100 c.c. of melted agar at a temperature of 90° C., add 5 c.c. 
of defibrinated blood. Allow the mixture to stand until it as- 
sumes a chocolate brown color. 
Pour into Petri dishes using 12-15 c.c. of blood for a 100 mm. 
plate. 
Meat Infusion Broth 
One pound of ground beef in one liter of water is heated to 
55° C. for one hour. Filter through cotton. Add peptone, one 
per cent and sodium chloride, 0.5 per cent, and bring to boiling 
to dissolve the peptone. Adjust reaction to PH 7.8-8.0. Filter 
through paper and autoclave in liter quantities. 
For pneumococcus cultures add 0.5 per cent glucose. 
Meat Extract Broth 
Peptone 10 grams 
Meat extract 5 grams 
Salt 5 grams 
Water 1000 c.c. 
Dissolve by heat. Adjust reaction to PH 7.4. Heat for a few 
minutes. 
Filter through paper. Autoclave under 15 pounds pressure for 
15 minutes. 
Endo’s Agar 
To 1000 c.c. of water add: 
Peptone 10 grams 
Beef extract 3 grams 
Sodium chloride 5 grams 
Agar 15 grams 
Dissolve all ingredients except agar first; add agar and auto- 
clave for one hour at 15 pounds pressure. Cool to 50 degrees. 
Add the white of one egg and steam in autoclave for thirty min- 
utes. 
Adjust reaction to PH 7.4. Boil over free flame for 5 to 6 min- 
utes. Filter. Fill in flasks in 100 c.c. amounts and autoclave. SOLUTIONS, STAINS AND MEDIA 
205 
Before pouring the Endo plates the reaction is adjusted to 7.6 
to 7.8. It usually takes 0.9 c.c. N NaOH for 100 c.c. agar. The 
amount required must be determined for each batch. 
Add to each 100 c.c. flask one gram of chemically pure lactose, 
0.5 c.c. of a saturated alcoholic solution of basic fuchsin and 1 c.c. 
of a 10 per cent solution of anhydrous sodium bisulphite. 
Colonies of lactose fermenting bacteria when cultured on this 
media are red; others are colorless. 
Russell’s Agar 
Make meat infusion agar as described on page 202 and adjust 
reaction to PH 7.4. 
Add 10 c.c. of Andrade indicator per liter, and tube in 5 c.c. 
amounts using Wassermann tubes (4 x % in.). Autoclave under 
15 pounds pressure for 15 minutes. While still hot add to each 
tube 0.25 c.c. of a solution containing 20 per cent lactose and 2 
per cent glucose. Cool in such a position that the slant will be 
iy2 inches long, and the butt y2 to % inches in depth. 
In inoculating the tube the needle is drawn along the slant and 
then stabbed through to the bottom of the tube. 
The following table shows the appearance of the media after 
incubation with the common gram negative bacilli. 
B. Typhosus 
B. Paratyphosus “A” 
B. Paratyphosus “B” 
B. Dysenteriae 
B. Coli and members of the 
colon group 
Butt Slant 
Acid Unchanged 
Acid and gas Unchanged 
Acid and gas Unchanged 
Acid Unchanged 
Acid and gas Acid 
Krumwiede’s Brilliant Green Media 
Make stock agar as for Endo medium. Adjust reaction to 7.0- 
7.2. To each 100 c.c. of melted stock agar add 0.25 c.c. of a one 
per cent solution of neutral red, one per cent lactose and 0.1 per 
cent glucose. The optimum concentration of brilliant green must 
be determined for each hatch of agar as follows: 
Pour plates using three dilutions of the dye; namely, 1 to 
200,000, 1 to 330,000, and 1 to 500,000, or expressed in terms 206 
CLINICAL LABORATORY METHODS 
of 0.1 per cent dye solution per 100 c.c. agar the amounts are 0.5, 
0.3', and 0.2 c.c. Add a loopful of a twenty-four hour broth cul- 
ture to 10 c.c. of a stool suspension of moderate density, and 
inoculate the plates containing the different dilutions of brilliant 
green, and a plate of Endo medium as a control. Incubate 18 to 
24 hours. Determine: (1) The concentration of dye which has 
little effect on the number and size of the typhoid colonies but 
which shows a marked restraint of the fecal flora; and (2) the 
concentration which shows a moderate reduction in number and 
size of the typhoid colonies and a still greater reduction of the 
fecal flora. The two optimum dilutions average 0.2 and 0.3 
respectively of a 0.1 per cent solution of the dye to 100 c.c. of 
agar. 
(Rosenow Formula) 
Dehydrated Bacto nutrient broth 8 gm. 
(Digestive Ferments Co.) 
Sodium chloride 8 gm. 
Dextrose C. P 2 gm. 
Andrade indicator 10 c.c. 
Distilled water 1000 c.c. 
Dextrose Brain Broth 
Dissolve the broth and salt by heating. When cool add the 
indicator and dextrose. Tube in large tubes (8 x % in.). Add 
three pieces of calf brain, about 1 cm. square, also 2 or 3 pieces 
of calcium carbonate, preferably as crushed marble, to each tube. 
Sterilize for 20 minutes in the autoclave at 20 pounds pressure. 
If the broth is to be used for blood cultures, 5 gm. of sodium 
citrate is added to each liter, to prevent coagulation of the blood. 
Dextrose Brain Agar 
Dissolve 7 gm. of powdered agar in 1 liter of dextrose broth 
prepared as indicated above. Add the calf brain and calcium 
carbonate, tube and sterilize. 
Potato Media 
Select large potatoes. Remove skin and cut cylinders with an 
apple corer. Wedge-shaped pieces are obtained from the cyl- 
inders by an oblique cut. SOLUTIONS, STAINS AND MEDIA 
207 
Place in test tubes, add a little water and sterilize in autoclave 
under 20 pounds pressure for 15 minutes. 
Dunham’s Peptone Solution 
Dissolve 10 grams peptone and 5 grams sodium chloride in 
1,000 c.c. water. 
Filter through paper until clear. Tube, and sterilize by auto- 
claving for 15 minutes at 15 pounds pressure. 
Litmus Milk 
Place perfectly fresh milk in ice box overnight. Syphon off 
the milk leaving the cream in the bottle. Add litmus solution 
until the milk is a light blue color. Tube, and sterilize for 20 
minutes on three successive days in the Arnold sterilizer. 
Gelatin 
To 1 liter of distilled water add: 
Meat extract 5.0 grams 
Peptone 10.0 grams 
NaCl 5.0 grams 
Finest French sheet gelatin. . 120.0 grams 
Weigh with vessel and dissolve by warming. Bring back to 
original weight, determine volume, titrate, and adjust. Cool to 
60° C., add whites of two eggs and stir. Heat for half an hour. 
Stir and heat again 15 minutes. Adjust weight, filter through 
cotton, and sterilize by fractional sterilization. Gelatin should 
not be subjected to prolonged heating. 
Loeffler’s Blood Serum 
Allow the serum to separate from clotted sheep or ox blood. 
Mix three parts of clear serum with one part of meat infusion 
broth containing 1 per cent dextrose. Fill into small culture 
tubes. Put tubes in slanting position in Koch serum inspissator 
and heat gradually until temperature reaches 90° Centigrade. 
Hold temperature at this point until medium is completely solid- 
ified. Sterilize on the two successive days by placing in Arnold 
sterilizer for 20 minutes. Raise temperature very slowly. 208 
CLINICAL LABORATORY METHODS 
Petroff’s Tubercle Bacillus Medium 
Treat 500 grams of chopped-up meat with 500 c.e. of 15 per 
cent glycerine solution. Keep in ice chest for 24 hours and filter 
through gauze. Sterilize the shells of eggs by immersion in 70 per 
cent alcohol for ten minutes or by dipping them in boiling water 
for five seconds or so. Mix white and yolk of these eggs in a 
sterile mortar and add an equal volume of the glycerine 
meat infusion which should have had added to it before mixing 
1 c.c. of 1 per cent alcoholic solution of gentian violet to each 100 
c.c. of the glycerine meat infusion. 
Put 3 to 4 c.c. of this medium in test tubes and inspissate as 
slants at 85° C. until the medium is solidified. Subject these 
slants to temperature of 75° C. on the second and third days for 
one hour. 
In culturing sputum mix with an equal amount of 3 per cent 
sodium hydroxide and leave in incubator for one to two hours. 
Neutralize with normal hydrochloric acid and centrifugalize. 
Take up sediment and smear out on slants. 
Sugar-Free Broth 
Inoculate a liter of meat infusion broth with Bacillus coli. In- 
cubate for two days. Heat in Arnold sterilizer for 20 minutes. 
Adjust reaction and heat thoroughly again. Put about 5 grams 
pure talc in a large mortar. Add the dead colon culture, stirring 
constantly. Filter through filter paper until perfectly clear. This 
is used as stock broth from which the carbohydrate media is 
made. 
Carbohydrate Broth for Fermentation Reactions 
Add sugar in proportion of one per cent to sugar free broth. 
Put in Dunham fermentation tubes (Fig. 54) and heat in Arnold 
sterilizer for 20 minutes on three successive days. 
Glucose Broth for Blood Culture 
Add 10 grams dextrose to 1,000 c.c. of meat infusion broth. 
Autoclave under 15 pounds pressure for 15 minutes. SOLUTIONS, STAINS AND MEDIA 
209 
Glucose Ascitic Fluid Broth 
Add one part of sterile ascitic fluid to three parts of sterile 
glucose broth. 
Carbohydrate Serum Water Fermentation Media (Hiss Media) 
Collect sheep or ox blood in sterile wide-mouth bottles. Sepa- 
rate clot and place in ice box for 24 hours. Pipette off clear serum 
and add three volumes of distilled water. 
Heat for 15 minutes in the Arnold Sterilizer. 
Add 1 per cent Andrade indicator and the desired sugar in 
Fig. 54.—Dunham fermentation tube. 
proportion, 1 gram to 100 c.c. Dissolve, tube, and sterilize in 
Arnold sterilizer on three successive days. 
In making inulin medium it is advisable to first dissolve the 
inulin in water and sterilize for 15 minutes in the autoclave be- 
fore adding it to the diluted serum. 
Lactose Bile Medium 
To 1,000 c.c. of fresh ox bile add 10 grams peptone and 10 
grams lactose. Dissolve and fdter. 
Place in bottles in 50 c.c. amounts. Autoclave 15 minutes under 
15 pounds pressure. CHAPTER X 
GENERAL BACTERIOLOGICAL METHODS 
Blood Culture 
Before the venipuncture is done, clean up the antecubital space 
very thoroughly with tincture of iodine and alcohol. Withdraw 
blood with sterile syringe. Place 10 c.c. of blood in citrate solu- 
tion (1.5 per cent sodium citrate in physiological salt solution) 
provided in 50 cubic centimeter Erlenmeyer flasks. Pour half 
of citrated blood into a bottle containing 50 c.c. of glucose meat 
infusion broth and incubate. Melt two tubes each of plain and 
of glucose agar, cool to 42°, and add some citrated blood. Pour 
into petri plates. If a typhoid infection is suspected, add some 
citrated blood to a bottle containing lactose bile medium. 
At intervals place drops of broth and of the sediment at the 
bottom of the bottle on a slide, dry, stain with methylene blue 
and examine for organisms. 
If bile cultures are made, transfer a loopful to a Russell agar 
slant on the second, third, and fifth days. If the typhoid bacillus 
is present the butt of this Russell tube will become red and the 
slant remain colorless if Andrade indicator is used, or remain 
blue, if litmus is used as indicator. 
Sputum Culture 
A small portion of sputum is selected and washed through three 
or four changes of sterile salt solution in sterile petri dishes. 
The washed sputum is then thoroughly emulsified in a small 
amount of sterile salt solution or broth in a sterile test tube. 
Smears are made from the washed sputum and stained by 
gram. A drop of the emulsion is transferred to a blood agar plate 
and spread over the surface of the plate with a glass rod. 
Incubate for twenty-four hours. If the plate shows a pure 
culture of pneumococcus the growth may be washed off with 
sterile salt solution and the type identified as indicated under 
210 GENERAL BACTERIOLOGICAL METHODS 
211 
the determination of types of pneumococcus. Single colonies may 
be picked and grown in broth for type determination. Hemolytic 
streptococcus colonies may be identified directly by the clear zone 
of hemolysis and by the Gram stain. 
For the influenza bacillus the plate should be incubated forty- 
eight hours. 
Influenza colonies are minute, colorless, translucent, and dis- 
crete. The influenza bacillus when stained is small, shows polar 
staining and is Gram negative. 
Other organisms present may be identified by smear and fur- 
ther cultures. 
Stool Culture 
If typhoid or paratyphoid bacilli are to be searched for, use 
Endo and Krumwiede’s brilliant green agar; for dysentery bacilli 
use freshly prepared Endo agar. 
The stool should be fresh. Dilute fluid feces or emulsify solid 
feces in peptone water or broth to a density corresponding to 
one part of solid feces to fifteen of diluent. Allow the suspen- 
sions to stand fifteen to thirty minutes for sedimentation of the 
particles and then inoculate plates from the surface of the 
suspension. 
The plates are conveniently inoculated by dipping a bent glass 
rod into the fecal suspension and passing it over three plates in 
succession using a clockwise motion. 
In the case of dysentery, select fragments of bloody mucus, 
rinse free of fecal material and inoculate on Endo plate. Spread 
with a bent glass rod passing the rod over several plates in 
succession. 
Incubate 18 to 24 hours and fish colonies having the appear- 
ance of the typhoid, paratyphoid, dysentery group to Russell 
medium. After incubation a tentative decision as to type can 
be made from the appearance of the tubes (see Russell medium, 
page 205). 
The final identification is made by macroscopic agglutination 
with the appropriate specific immune serum depending upon the 
reaction obtained with the Russell tube, and by further fermenta- 
tion reactions (Table XLII). 212 
CLINICAL LABORATORY METHODS 
Urine Culture 
If the urine is clear, pour a small amount on a plain agar plate, 
rotate or tilt plate so that the specimen is well spread over the 
surface, and drain off excess. 
If the specimen is cloudy use only a few drops and spread over 
surface of plate with a sterile rod. Also pour several drops on 
a Russell agar slant, spread over surface and stab through to 
bottom of tube with platinum wire. 
If the plate shows a growth after incubation, make smear and 
stain by Gram method. Identify a Gram negative bacillus as 
indicated under stool culture. 
Nose and Throat Cultures 
All diagnostic cultures are to be made on both Loeffler’s blood 
serum and blood agar. Streak surface of a blood agar plate and 
a blood serum slant with the same swab. 
The following morning, make smear from the Loeffler tube and 
stain with Neisser. Examine under oil immersion lens for diph- 
theria bacilli. 
Examine the blood agar for colonies of B. influenzae, hem- 
olytic streptococcus or other predominant organisms. 
Eye and Ear Cultures 
Make culture with swab directly on blood agar plate. Incu- 
bate 48 hours and identify organisms present. 
After culture is made, smear swab over glass slide and stain 
with Gram’s stain. Note types of organisms present. 
Report findings of both smear and culture. 
Miscellaneous Cultures 
The most satisfactory medium for routine use is Loeffler’s 
blood serum. Material obtained at surgical operations or from 
other sources in which the use of a special medium is not indi- 
cated are cultured directly on slants of the blood serum. If the 
presence of a hemolytic streptococcus is suspected a culture 
should be made on blood agar also. GENERAL BACTERIOLOGICAL METHODS 
213 
Virulence Test for Diphtheria Bacillus 
Streak the surface of a number of ascitic infusion agar plates 
with the growth from the Loeffler tube. Incubate for 16 hours at 
37 degrees C. and examine the growth along the line of streak. 
Diphtheria colonies are most apt to be found at the edges of the 
streak. Fish suspicious colonies to ascitic infusion broth or Loef- 
fler’s medium. If very few bacilli are present it is well to inoc- 
ulate several tubes of ascitic broth. As the diphtheria bacillus 
will grow mostly on the surface of the broth, this portion can be 
used with success when direct plates fail. 
After the isolation of the bacillus in pure culture, inoculate 
a tube of ascitic fluid broth of PH equal 8.0-8.2. 
One cubic centimeter of the ascitic fluid broth culture (incu- 
bated 48 hours) is injected subcutaneously into a guinea pig 
weighing 250 grams. A second guinea pig is injected with the 
same amount of culture to which has been added 50-100 units of 
diphtheria antitoxin. If the first pig dies within two or three 
days and on postmortem examination shows typical engorgement 
of the adrenals, and the second pig lives, the bacillus is a true 
toxin-producing diphtheria bacillus. Otherwise, it is not. 
Animal Inoculation for Tuberculosis 
The centrifugalized sediment from urine, spinal fluid, etc., or 
the sediment from antiforminized material is emulsified in ster- 
ile 0.85 per cent salt solution and injected subcutaneously into the 
groin of a guinea pig. 
The inoculation should be made into the loose tissue of the 
groin well out and away from the midline, avoiding the mammary 
gland. It is well to always inoculate two pigs. 
A lesion developing in 3 to 4 days is always nontuberculous 
and usually subsides. 
If the suspected material contains tubercle bacilli, the inguinal 
glands begin to swell in from twelve to twenty days; rarely at 
eight to twelve. 
The guinea pig is killed after the inguinal glands show definite 
involvement, and autopsied. Smears are made from the glands 
and stained for tubercle bacilli by the Ziehl-Neelson method. 214 
CLINICAL LABORATORY METHODS 
DEX- 
LAC- 
SACCHA- 
MAL- 
MAN- 
LEVU- 
GALAC- 
DEX- 
LITMUS 
INDOL 
MOTIL- 
TROSE 
TOSE 
ROSE 
TOSE 
NITE 
LOSE 
TOSE 
TRIN 
MILK 
FORMATION 
ITY 
REMARKS 
B. Typhosus 
4* 
0 
0 
+ 
4 
4 
4 
4 
+ 
0 
4 
B'. Paratyphosus A 
++ 
0 
0 
++ 
4-4 
44 
44 
0 
4 
0 
4 
B-. Paratyphosus B 
B. Dysenteriae 
++ 
0 
0 
44 
44 
44 
44 
0 
+ 
0 
4 
(Shiga) 
4 
0 
0 
0 
0 
0 
0 
0 
+ 
0 
0 
B. Dysenteriae 
(Flexner) 
4 
0 
+ 
4 
4 
4 
4 
4 
+ 
4 
0 
B. Dysenteriae 
(Hiss-Russell) 
+ 
0 
0 
0 
4 
4 
4 
0 
+ 
4 
0 
B. Dysenteriae 
(Rosen) 
4 
+ 
0 
4 
4 
4 
4 
4 
4 
4 
4 
B'. Enteritidis 
++ 
0 
0 
4+ 
44 
44 
44 
44 
0 
0 
4 
B. Coli Communis 
++ 
44 
0 
4+ 
4-1- 
44 
44 
44 
4 
4 
4 
Milk coagulated 
B. Coli Communior 
B. Paracolon 
-H- 
++ 
44 
44 
44 
44 
44 
44 
4 
4 
4 
Milk coagulated 
(Day) 
++ 
0 
++ 
++ 
44 
44 
44 
44 
+ 
? 
4 
B. Lactis 
Aerogenes 
44 
44 
-H- 
++ 
44 
44 
44 
44 
4 
4 
0 
Milk coagulated 
B. Acidi Lactici 
44 
44 
0 
4+ 
44 
44 
44 
44 
4 
4 
0 
Milk coagulated 
B. Cloacae 
++ 
++ 
++ 
44- 
44 
44 
44 
44 
4 
0 
4 
Milk coagulated 
B. Fecalis 
and peptonized 
Alkaligenes 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
4 
B. Mucosus 
Capsulatus 
44 
++ 
44 
44- 
44 
44 
44 
44 
4 
4 
0 
Milk coagulated 
B. Proteus 
44 
0 
++ 
0 
44 
44 
44 
? 
4 
4 
4 
Milk coagulated 
and peptonized 
Fermentation Beactions op the Commoner Gram-Negative Bacilli 
Table XLII 
*0 —No fermentation 
+ —Acid formation 
++—Acid and gas formation 
+—Primary acidity with terminal alkalinity. GENERAL BACTERIOLOGICAL METHODS 
215 
DEXTROSE 
MALTOSE 
LEVULOSE 
SACCHAROSE 
LACTOSE 
GALACTOSE 
DEXTRIN 
MANNITE 
DtTLCITE 
INTTLIN 
Meningococcus 
Pseudomenin- 
+* 
+ 
0 
0 
0 
0 
0 
0 
0 
0 
gococcus 
+ 
4- 
0 
0 
0 
0 
0 
0 
0 
0 
Gonococcus 
Micrococcus 
+ 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Catarrhalis 
Micrococcus 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Pharyngis 
Ghromogenic 
Siccus + 
4- 
4- 
4- 
0 
0 
0 
0 
0 
0 
Group I 
CThromogenic 
+ 
4- 
4- 
4- 
0 
0 
0 
0 
0 
0 
Group II 
Chromogenic 
4 
4- 
4- 
0 
0 
0 
0 
0 
0 
0 
Group III 
4- 
4- 
0 
0 
0 
0 
0 
0 
0 
0 
Fermentation Reactions of Gram-Negative Groups of Cocci on Carbohydrate Serum-Water Media 
(Elser, Wm. J., and Huntoon, Frank, M.: Jour. Med. Res., 1909, xx, 427.) 
Table XLIII 
*+ Acid formation. 
0 No fermentation. 216 
CLINICAL LABORATORY METHODS 
Sections are made from the spleen and glands for histological 
examination. 
If the pig shows no signs of tuberculosis, it should be killed 
at the end of six weeks and autopsied. 
Examination of Stool for the Tubercle Bacillus 
Select purulent or mucous particles and make film preparation. 
If no tubercle bacilli are found, dilute feces with three volumes 
of water, mix thoroughly and allow to stand one-half hour. Sat- 
urate the filtrate with dry sodium chloride and allow to stand 
one-half hour. Skim off the film from the surface, dilute mate- 
rial with distilled water and add antiformin to 20 per cent con- 
centration. Treat sediment as in the case of sputum. (Page 80.) 
All cases showing positive findings in stool should he controlled 
with a sputum examination. 
Examination of Urine for the Tubercle Bacillus 
Collection of Specimen.—(a) Men: Patient is cleaned up as 
for a catheterization and then voids into a sterile vessel. The 
first few drops voided are discarded, (b) Women: A specimen 
is collected by catheterization, using routine sterile technic. As 
much urine as possible should be obtained. 
Procedure.—The total specimen is centrifuged at high speed 
for at least one-half an hour. Place a drop of blood serum on a 
slide, mix the sediment with it and spread. 
Dry the smear in the air, fix in flame, and stain by the Ziehl- 
Neelson method. 
If there is a large amount of pus present, mix the sediment with 
distilled water, add antiformin to 20 per cent concentration, place 
in incubator for several hours, centrifuge again and make 
preparation on slide as above. 
Remarks.—In a properly collected specimen all acid-alcohol- 
fast bacilli can be safely considered as tubercle bacilli. Absolute 
differentiation can be made, however, only by guinea pig inocula- 
tion. 
Examination of Smears for the Gonococcus 
A satisfactory examination for the gonococcus can be made 
only on well prepared specimens. In men a preparation is made GENERAL BACTERIOLOGICAL METHODS 
217 
by carefully spreading secretion from the urethra on a glass 
slide. Norris and Mickelberg suggest that specimens be obtained 
in women with a medicine dropper which has been drawn out in 
the flame to 6-8 cm. in length and the thickness of a coarse capil- 
lary tube. The drop of material desired for examination is 
drawn up into the pipette and spread evenly on a slide in weak 
mercury bichloride solution. In female children a soft eye 
syringe is used. Weak bichloride of mercury solution is sucked 
in and out of the vagina several times. The washings are cen- 
trifugalized and the sediment examined for organisms. In mak- 
ing slide preparations all pressure must be avoided since the 
pus cells are easily crushed. 
The smear on the slide is stained by Gram’s method. The 
gonococci are gram negative and appear as small biscuit-shaped 
cocci in pairs. In carefully made preparations they are always 
found largely in pus cells. In a positive case one nearly always 
finds a few pus cells containing a very large number of cocci. 
In urethral smears one may be reasonably sure that diplococci 
morphologically similar to gonococci occurring outside pus cells 
are specific. In the vulvovaginal tract one may find other dip- 
lococci which stain as gonococci hence one cannot be sure here 
that organisms occurring outside the pus cells are really gono- 
cocci. Only those smears showing leucocytes filled with morpho- 
logically typical gonococci are reported positive. 
Classification of Streptococci 
(Holman, W. L.: Jour. Med. Research, 1916, xxxiv, 377) 
Principle.—The streptococci are cultured on red blood agar 
and separated into hemolytic and non-hemolytic types. The or- 
ganisms are further subdivided by their reaction in lactose, man- 
nite and salicin carbohydrate broth. The streptococci are differ- 
entiated from pneumococci by their insolubility in bile and their 
failure to ferment inulin. 
Media.—(1) Blood Agar.—Meat infusion agar with a PH equal 
to 7.4 is sterilized in 100 c.c. amounts in flasks. A flask is heated 
in the autoclave, cooled to 50° C. and 5 cubic centimeters of defi- 
brinated human blood added. The mixture is poured into petri 
dishes to make a layer 2 to 3 mm. thick. 218 
CLINICAL LABORATORY METHODS 
Salicin 
Str. Ignavus 
Str. Equinus - 
I 
Mannite 
l . 
Salicin 
Str. Non-Hemolyticus III 
Str. Non-Hemolyticus II 
Lactose 
(Gram-positive cocci in chains, no capsules, insoluble in bile) 
I 
Salicin 
Str. Salivanus - 
Str. Mitis 
i 
Mannite 
Classification of Streptococci 
Salicin 
Str. Non-IIemolyticus I - 
Table XLIV 
Hemolysis 
Str. Fecalis — 
Str. Subacidieus 
i 
Salicin 
Str. Equi 
Mannite 
I 
Salicm 
Str. Hemolyticus III 
Str. Hemolyticus II 
i 
Lactose 
I 
Salicin 
Str. Angiosus 
I 
Mannite 
Str. Pyogenes - 
I 
Salicin 
Str. Hemolyticus I- 
Str. Infrequens- GENERAL BACTERIOLOGICAL METHODS 
219 
(2) Carbohydrate Media.—Take 200 e.e. of double strength 
meat infusion broth with PH equal 7.4, add to this 100 c.c. of 
water, 4 grams of the test substance and 4 c.c. of Andrade’s indi- 
cator. This is sterilized in a large flask on three successive days 
in an Arnold sterilizer. Beef serum diluted one half with water 
is slowly filtered through a Berkefeld filter and 200 c.c. added to 
the above. The whole is tubed through a sterile funnel into ster- 
ile test tubes and the tubes incubated for two days to eliminate 
chance contaminations. 
Procedure.—The blood agar plate is streaked with a loopful of 
the streptococcus culture. The surface is covered with as many 
parallel lines as it is possible to make in order to thoroughly dis- 
tribute the material. 
After growing the streptococcus on blood agar for 24 hours 
transfer to inulin, lactose, mannite and salicin serum broth tubes 
and incubate for at least a week at 37° C. 
Colonies of hemolytic streptococcus on the blood agar plate 
will show well defined colorless zones of hemolysis. 
There should be no pigmented corpuscles visible under the low 
power of the microscope remaining next to or under the colony. 
If there is any question as to the hemolytic property it is well 
to mix 0.5 c.c. of a 24 hour bouillon culture with 0.5 c.c. of a 
5 per cent suspension of washed human red blood cells in physio- 
logic salt solution and incubate in water-bath at 37° C. for two 
hours. True hemolytic streptococci produce laking of blood un- 
der these conditions. 
Pneumococci may be differentiated by solubility in bile when 
1 c.c. of a bouillon culture is incubated at 37° C. for one hour 
with 0.2 c.c. of sterile ox bile. 
Table XLIV shows the classification of the streptococci using 
the above procedure. CHAPTER XI 
MISCELLANEOUS CLINICAL PATHOLOGICAL 
EXAMINATIONS 
Reagents.— 
(1) Pandy’s solution— 
Saturated aqueous solution of phenol. 
(2) Ross-Jones solution— 
Saturated (by heat) aqueous solution of ammonium sul- 
phate. 
(3) Fuehs-Rosenthal solution— 
Glacial acetic acid 2 c.c. 
Methyl violet 0.1 gram. 
Water 50 c.c. 
(4) Tsuchiya reagent (see page 41). 
Procedure.— 
1. Test for Excess Globulin.— 
Qualitative.—The Pandy Test. To 1 c.c. of Pandy’s reagent 
add one drop of spinal fluid. If an excess of globulin is present 
a white cloud will develop immediately. 
Record results as follows: 
Negative 0 
Borderline ± 
Positive + 
Strongly positive + + 
Ross-Jones Test.—Layer the spinal fluid on a saturated solu- 
tion of ammonium sulphate. If an excess of globulin is present 
a white ring develops at the line of contact. 
Quantitative.—Fill a Felton precipitometer to line marked 
“Spinal Fluid” and add Tsuchiya solution to line marked “Re- 
Examination of Cerebrospinal Fluid 
220 PATHOLOGICAL EXAMINATIONS 
221 
agent.” Invert several times and let tube stand overnight. Read 
off directly in grams per liter. 
2. Cell Count.—This should be done as soon after the with- 
drawal of the fluid as possible since the cells settle out rapidly. 
If the fluid is clear draw up the Fuchs-Rosenthal reagent to 
1 mark on white blood cell counting pipet and fill to the 11 mark 
with spinal fluid. Allow pipette to stand for a few minutes, shake 
and fill a Fuchs-Rosenthal counting chamber (Fig. 55). The 
white cells are stained violet; red cells if present are only faintly 
stained. The number of white cells in the entire ruled area 
divided by 3 gives the number per cubic centimeter. 
If a counting chamber with Fuchs-Rosenthal ruling is not avail- 
able, an ordinary blood cell counting chamber is employed. The 
Fig. 55.—Fuchs-Rosenthal ruling of counting chamber for spinal fluids 
total number of cells in the ruled area (Neubauer ruling) multi- 
plied by the factor gives the number of cells per cubic 
01 
millimeter. 
A cloudy fluid should be diluted as for a white blood count 
and counted in an ordinary counting chamber. 
3'. Wassermann Test.—(See page 175.) 
4. Colloidal Gold Test.—(See page 222.) 
5. A Culture should be made if the fluid is cloudy or if other- 
wise indicated. This is best done by centrifuging the fluid in a 
sterile tube and culturing the sediment on a blood agar plate and 
in dextrose bouillon. 
6. Smears.—A cloudy fluid should be centrifuged, smears made 
from the sediment, and stained with gram and with methylene 222 
CLINICAL LABORATORY METHODS 
blue. Make a different count of the white cells and look for 
bacteria. 
7. Examination for Tubercle Bacilli.—If tuberculosis is sus- 
pected, allow tube to stand overnight in ice box. Float the film 
which forms onto a slide and stain for tubercle bacilli by Ziehl- 
Neelson method. Another good way of getting a film preparation 
is to place a cover glass in a conical test glass, add the spinal 
fluid and allow to stand overnight. The film will collect on the 
cover glass. The fluid is then pipetted off, the cover glass 
removed and the film stained by the Ziehl-Neelson Method. 
Lange’s Colloidal Gold Test on Spinal Fluids 
(Miller, Brush, Hammers, and Felton: Bull. Johns Hopkins 
Hospital, 1915, xxvi, 391) 
A. Preparation of the Colloidal Gold Solution — 
(a) Reagents.— 
1. Merck’s gold chloride (sealed in 
brown glass ampoules) 1 gram. 
Water triply distilled up to 100 c.c. 
2. Chemically pure potassium car- 
bonate (desiccated) 2 grams. 
Water triply distilled up to 100 c.c. 
3. Chemically pure formaldehyde 
(40 per cent solution) 1 c.c. 
Water triply distilled up to 40 c.c. 
4. Chemically pure crystalline ox- 
alic acid 1 gram. 
Water triply distilled up to 100 c.c. 
The reagents need not be made up fresh each day but may be 
kept as stock solutions. 
(b) Cleaning the Glassware.—The beakers and pipettes are 
thoroughly brushed under hot tap water with ivory soap solution. 
They are then rinsed in running water, tilled with bichromate 
cleaner, and left standing for at least one-half hour. When 
needed, the beakers are emptied, washed again in running water 
for five minutes, rinsed with ordinary distilled water, and finally 
with triply distilled water. The pipettes, flasks and graduates PATHOLOGICAL EXAMINATIONS 
223 
are cleaned in the same manner. The glassware should not be 
allowed to dry in the air before use. 
(e) Distillation of Water.—The first distillate is taken from 
an ordinary Stoke’s still, and immediately poured into clean 
Pyrex distilling flasks. Five c.c. of a saturated solution of barium 
Fig. 56.—Block tin still used in distilling water for colloidal gold solution. 
hydroxide for each liter of water is added. The water is then 
distilled twice, using a still (Fig. 56) with either Pyrex glass or 
block tin condensing tubes. Each distilling flask is washed out 
with some of the new lot of water that is to be put into it. The 
water should be used as soon as possible after its last distillation. 224 
CLINICAL LABORATORY METHODS 
There should be no rubber connections in the distilling appara- 
tus. If the tap water contains chlorine it is better to start with 
spring water instead of the distillate from the Stoke’s still. 
(d) Technic for the Preparation of the Solution.—Rinse 
out a beaker with a portion of the triply distilled water and fill 
to the liter mark. Raise the temperature gradually to about 
50° C., and then turn the gas on full. At 60° C. add 10 c.e. of the 
1 per cent gold solution, and 7 c.e. of the 2 per cent potassium 
bicarbonate solution. At 80° C., while stirring with a thermom- 
eter, add slowly 10 drops of oxalic acid. The solution should 
remain colorless and clear. When the temperature has reached 
90° C., the flame is withdrawn and while stirring, 5 c.e. of the 
1 per cent formaldehyde, or enough to produce an initial pink 
color, is slowly added a drop at a time. If a pink color makes 
its appearance before all the reducing agent has been added, 
stop at once. The final end point is marked by the production of 
a beautiful brilliant clear orange-red solution. The solution 
should be kept in a dark place. 
(e) Testing the Solution for.—1. Reaction: To 5 c.c. of the 
solution in a test tube are added 2 drops of 1 per cent alizarin 
red in 50 per cent alcohol. With an alkaline solution a purplish 
red color is produced; the neutral point is of a brownish red 
tinge; an acid solution gives a lemon yellow color. 
Ten test tubes are set up in a rack and 1 c.c. of freshly distilled 
water put into each. Depending upon whether the solution is 
acid or alkaline as shown by the preliminary test, add 1 c.c. of 
N/50 NaOII or N/50 IICL to the first tube. Mix well and trans- 
fer 1 c.c. to the second tube. Again mix and transfer 1 c.c. from 
the second to the third tube. Proceed in this manner up to and 
including the ninth tube. The tenth tube serves as a control. 
Now add to each tube two drops of the alizarin red indicator and 
5 c.c. of the gold solution to be tested. The amount of acid or 
alkali in each successive tube will be as follows: 0.5 c.c., 0.25 
c.c., 0.125 c.c., 0.0625 c.c., etc., each tube having exactly one-half 
of the preceding one. The tube showing the most typical brown- 
ish red is selected as neutral; the amount of acid or alkali in that 
tube is divided by 5 to determine the amount necessary to neu- 
tralize 1 c.c.; this is multiplied by the number of cubic centi- PATHOLOGICAL EXAMINATIONS 
225 
meters of solution to be corrected and the resulting amount of 
fiftieth normal hydrochloric acid or sodium hydroxide added to 
the solution, which is then ready for use. It should be noted that 
a deep dark red with a purplish tinge in the upper layer when 
shaken, as well as a deep purple, is a definite sign of an alkaline 
solution, and that no red without a definite brownish tinge should 
be regarded as neutral. 
2. Protection.—Add 1.7 c.c. of one per cent salt solution to 
5 c.c. of the colloidal gold solution. The gold should be com- 
pletely precipitated in one hour’s time. 
3. Reaction Curves with Known Normal and Abnormal Fluids.— 
Test the fluid with a known paretic cerebrospinal fluid, and four 
known normal fluids. 
(f) Requirements for a Satisfactory Colloidal Gold Solu- 
tion.—1. It must be clear to both direct and transmitted light 
and preferably of a brilliant red orange or salmon red color. 
2. Five c.c. of the solution must be completely precipitated 
by 1.7 c.c. of a one per cent sodium chloride solution in the time 
interval of one hour. 
3. The solution must be neutral in reaction on the day on which 
it is used. 
4. It must give a typical reaction curve with a known paretic 
cerebrospinal fluid. 
5. It must produce no reaction greater than a number one with 
four normal cerebrospinal fluids. 
B. Procedure in Making the Test.—Into the first of 10 clean, 
dry test tubes reserved especially for the purpose put 1.8 c.c. of 
fresh sterile 0.4 per cent NaCl solution made from a stock 4 per 
cent NaCl solution. Into each of the remaining nine tubes put 
1 c.c. of salt solution of the same strength. Now add to the first 
tube by means of a clean, dry, certified 1 c.c. pipette, 0.2 c.c. of 
the spinal fluid to be tested. Mix well. Transfer 1 c.c. of the 
resultant 1 to 10 solution of spinal fluid to the second tube and 
again mix thoroughly and transfer 1 c.c. of the solution to the 
third tube. Proceed in this manner up to and including the 
tenth tube. By this method a series of dilutions of the spinal 
fluid is secured in geometrical progression ranging from 1 to 
10 to 1 to 5120. Now add to each tube 5 c.c. of the colloidal gold 226 
CLINICAL LABORATORY METHODS 
solution. Shake each tube thoroughly. Read the tubes after 
standing overnight at room temperature. All readings must be 
done with direct daylight, holding the tubes up against the sky. 
In recording the results of the test, numbers are used to de- 
note the various color changes, as follows: 
Unchanged 0 
Bluish red 1 
Reddish blue 2 
Deep blue 3 
Gray blue 4 
Colorless 5 
Remarks.—The reaction types are classified as follows: 
1. Normal in which there is no reaction or a maximum color 
change of 1. 
2. Zone I (so-called paretic zone) : The greatest reaction is in 
the first three to six tubes and is of the 5 type. The color values 
rapidly fall in the succeeding two or three tubes to 0. 
3. Zone II (so-called luetic type) : The greatest color change is 
in the fourth and fifth tubes but is seldom greater than 4 or 
less than 3. 
4. Zone III (so-called meningitic type) : The maximum color 
change occurs in the higher dilutions beginning with the sixth tube. 
The reaction types are illustrated in Fig. 57. 
Satisfactory solutions cannot be uniformly made with the quan- 
tities of reagents directed. If trouble is experienced, it is best 
to vary the amounts until a successful combination is found. 
If the solutions are turbid, the oxalic acid should be omitted. 
Examination of Ascitic and Pleural Fluids 
Collection of Specimen.—Collect specimen in sterile container, 
diluting the fluid with an equal volume of sterile 1.5 per cent 
sodium citrate in normal salt solution to prevent clotting. 
Frankly purulent fluids need not he diluted. 
Routine Examination.— 
1. Specific Gravity.—Take specific gravity with urinometer. 
2. Quantitative Albumin Determination.—Dilute one to 
twenty with distilled water and make quantitative albumin deter- PATHOLOGICAL EXAMINATIONS 
227 
Number used 
to designate 
color 
Bril .1 i ant 
Red-Orange 
Red-Blue 
Lilac or 
Purp] e 
Blue 
Pale Blue 
Colorless 
Color of 
solution 
No reaction—normal spinal fluid. 
Zone I reaction (paretic type). 
Fig. 57.—Reaction types of spinal fluid with colloidal gold solution. 
Dilutions of Spinal Fluid with 0.4$ Nad 
Zone II reaction (luetic type). 
Zone III reaction (meningitic type). 228 
CLINICAL LABORATORY METHODS 
mination. Fill Esbach tube (Fig. 17) to point marked “W” with 
diluted fluid and to “R” with Tsuchiya reagent. Stopper, invert 
ten to twelve times, and allow to stand overnight. The amount 
of sediment as read on the scale multiplied by twenty gives the 
number of grams of albumin per liter. 
3. Cell Count.—Dilute with 1 per cent acetic acid as for white 
blood cell count and determine the number of white cells in count- 
ing chamber. Centrifuge, make smear of sediment and stain with 
Loeffler’s methylene blue. Make differential count of cells and look 
for bacteria. 
4. Culture.—Make culture on blood agar plate and in tube of 
glucose bouillon. 
5. Guinea Pig Inoculation.—If indicated inject guinea pig sub- 
cutaneously in the groin with centrifugalized sediment from 10 
to 50 c.c. of fluid. 
Report as follows if complete examination is made: 
Pleural 
: Fluid Sp.Gr. 
Ascitic Albumin gms. per liter 
WBC per cmm. 
Differential - PMM 
SM 
Smear shows 
Culture 
If the specimen is purulent only make culture and smear. 
Report the examination of a purulent fluid as follows: 
Pleural fluid 
Smear shows 
Culture shows 
Dark-Field Examination for Spirocheta Pallida 
The simplest and most successful method of finding the spiro- 
cheta pallida in luetic lesions is by use of the dark-field. 
The apparatus consists of an ordinary microscope from which 
the substage condensor is removed and a dark-field illuminator 
(Fig. 58) attached instead. A funnel stop is inserted in the oil 
immersion objective. A strong light, such as that given by an 
arc lamp or a 100 Watt nitrogen bulb, is necessary. The dark- 
field substage must be centered by means of the lateral adjust- 229 
PATHOLOGICAL EXAMINATIONS 
rnent screws. This is accomplished by bringing the rings on the 
apex of the condensor to the center of the field as viewed through 
the low power lens of the microscope. 
Recently an illuminator has been devised in which the light is 
placed in the substage (Fig. 56). 
In obtaining the material for dark-field examination, the lesion 
should be superficially cleansed in order to remove the many con- 
taminating organisms on the surface. In taking a specimen 
from a primary lesion, it is well to cleanse rather roughly, remove 
most of the surface layer, and to squeeze out a little serum from 
the deeper tissues. A drop of the secretion is caught in the 
Fig. 58.—A.—Old style dark-field illuminator. An external source of light is em 
ployed. 
B.—New type dark-field illuminator. The light is an integral part of the instrument 
center or the side of a cover glass, which is then placed on a 
thin slide. Secretion from a gland can be obtained by inserting 
a needle into the gland and massaging the latter for a few 
minutes. A few drops of secretion may he obtained by aspiration. 
A sufficient quantity of immersion oil is placed on the cover 
glass, also on the apex of the dark-field substage. The prepara- 
tion is placed on the condensor gently so that the drop of oil 
on the condensor spreads evenly without bubbles. 
If the condensor is properly centered the correct focus of light 
can be obtained by moving the condensor up and down until 
the center ring of light becomes a point of light in the middle of 230 
CLINICAL LABORATORY METHODS 
the preparation. This adjustment is necessary because of varia- 
tions in the thickness of slides. 
With the proper adjustment the silvery appearance of the 
illuminated organic structures will stand out conspicuously in 
contrast with the background which should be of an intense 
brown color. 
The Spirocheta pallida will appear as a silvery, very slender, 
highly flexible structure, which is spirally wound, having any- 
where from ten to twenty turns. Its length is about twice the 
diameter of a red blood cell. The turns are closely set and of 
more or less equal size and shape. The organism will move 
slowly across the field, the movements being corkscrew-like. 
The Spirocheta pallida must be differentiated from other spiro- 
chetes, of which Spirocheta refringens is the most important. 
Table XLV given by Dourmashkin is of value in differentiating 
the two organisms. 
Table XLY 
SPIROCHETA PALLIDA 
SPIROCHETA F.EFRINGENS 
1. 
Body very slender. 
1. 
Body much coarser. 
2. 
Has from ten to twenty turns. 
2. 
The turns are much fewer, any- 
where from five to eight. 
3. 
The movements are 
regular. 
3. 
Extremely irregular. 
4. 
Moves slowly. 
4. 
Movements very rapid; may be 
compared to lightning; difficult 
to follow it, as the organism 
dashes across the field. 
5. 
Retains its turns. 
5. 
Does not retain its turns. 
6. 
Does not take up 
blue stain. 
methylene 
6. 
Takes up the stain. 
Preparation of Bacterial Vaccines 
(Hopkins: Jour. Am. Med. Assn., 1913', lx, 1615) 
Each organism to be used must be isolated in pure culture. 
Plain agar slants may be employed for the colon, typhoid and 
staphylococcus groups and dextrose broth for the pneumococcus, 
streptococcus and M. catarrhalis groups. It will be necessary to 
use blood agar slants or plates for B. influenzae and dextrose 
ascitic agar slants or plates for the gonococcus. 
Inoculate medium and incubate for 24 hours. Wash the organ- 
isms off solid medium with sterile physiological salt solution and PATHOLOGICAL EXAMINATIONS 
231 
place the suspension in a Hopkins vaccine centrifuge tube (Fig. 
59). If the organisms have been grown in a fluid medium the 
material from the bottom of container is placed in the vaccine 
centrifuge tube. Fasten a sterile cotton plug in centrifuge tube 
with adhesive. Centrifuge for 30 minutes at 2800 revolutions 
per minute. Remove supernatant fluid with sterile pipette and 
add sterile physiological salt solution to make a 1 per cent sus- 
pension. Mix and transfer to sterile tube containing some beads. 
Stopper with sterile rubber stopper and shake until a homo- 
geneous suspension is obtained. 
Fig. 59.—Hopkins vaccine tube. 
Heat in water-bath at 60° C. for one hour. The level of the 
water in the tube must be well above the level of the vaccine. 
Culture by adding one drop of the suspension to a blood agar 
plate and one drop to a tube of plain broth with a sterile pipette. 
Incubate for 72 hours. After culturing add 0.25 c.c. of 10 per 
cent tricresol mixture, or 0.5 c.c. of 10 per cent phenol, to each 
10 c.c. of vaccine and shake thoroughly. 
Below is shown the number of organisms per cubic centimeter 
in a 1 per cent suspension, as given by Hopkins: 
Streptococcus hemolyticus—8 billions per cubic centimeter. 
Staphylococcus aureus and albus—10 billions per cubic centi- 
meter. 
Gonococcus—8 billions per cubic centimeter. 232 
CLINICAL LABORATORY METHODS 
Pneumococcus—2.5 billions per cubic centimeter. 
B. Typhosus—8 billions per cubic centimeter. 
B. Coli—4 billions per cubic centimeter. 
Label with name, date, type of organism and number of or- 
ganisms per cubic centimeter. The suspension may be diluted 
as desired. Keep in ice box. 
Wound Bacterial Count 
Principle.—In order to intelligently control the treatment of 
infected wounds with Dakin’s solution it is necessary to deter- 
mine frequently the number of bacteria in the exudate. 
Procedure.—A smear is made from the infected area on a slide 
and spread uniformly with a platinum wire. The smear is stained 
two minutes with carbol-thionin, washed under the tap, and dried. 
Using the Bausch and Lomb No. 10 eyepiece and the 1.90 mm. 
objective, a standard field is selected in which the leucocytes 
just touch but do not overlap. 
The bacteria in the standard field are counted and if over 60, 
are recorded as infinity. If the number falls between 10 and 60, 
five fields are counted and the average taken; with counts be- 
tween 2 and 10, the average of ten fields is taken, and with those 
below 2, the average of 20 fields. 
In examining the smear the predominant cell type—mononu- 
clear or polymorphonuclear—and the predominant type of or- 
ganism—bacillus, coccus, or streptococcus—are noted. 
The findings are reported as follows: 
WOUND Bacterial count 
Predominant cell type 
BACTERIAL COUNT Predominant organism type 
(Vogel and Lee: Jour. Am. Med. Assn., 1914, lxii, 532) 
Detection of Mercury in Excretions 
Principle.—The organic combination in which the mercury 
exists in the excretions is broken down by oxidation with nascent 
chlorine. Metallic copper is added to the solution containing the 
mercury. By electrolytic double decomposition the mercury is 
deposited on the copper and from this it is distilled on to a piece PATHOLOGICAL EXAMINATIONS 
233 
of dentists’ gold foil. The mercury is easily recognized as glob- 
ules or as a silvery patch of discoloration on the gold. 
Procedure.—As large a volume as possible of the material, 
(urine, gastric lavage, fluid, colon irrigation), is acidulated 
with 10 to 20 c.c. of concentrated hydrochloric acid; a few grams 
of potassium chlorate are added; and the whole is heated in a 
large porcelain evaporating dish. The fluid becomes pale yellow 
or colorless Avhen the organic material is completely oxidized. 
It is not advisable to use more acid or chlorate than is required 
for complete decolorization. Specimens containing a large 
amount of protein, such as stools, vomitus and blood, are first 
diluted with several volumes of water, and require more of the 
oxidizing materials. The excess of acid and chlorine is elimi- 
nated by evaporation and the solution concentrated to about 25 
c.c. The solution is filtered to remove any solid matter. A short 
piece of clean copper wire, a number of small copper discs, or a 
small amount of copper dust, is dropped into the solution and 
allowed to remain for several hours, or overnight, preferably in 
a warm place. The copper is washed with distilled water, dried 
with alcohol and ether, and placed in the bottom of a small glass 
tube. It is then followed by a cylinder of gold foil pushed to 
within 2 centimeters of the copper. 
The tube is held horizontally, and its closed end is carefully 
treated over a microburner almost to the softening point, care 
being taken to avoid heating the part of the tube containing the 
gold foil. The gold foil is examined for any trace of silvery dis- 
coloration, signifying the presence of mercury. A hand lens is 
useful in recognizing small amounts of the metal. If chlorine 
is still present in the concentrated oxidized solution the wire may 
be completely dissolved, in which case the solution should be di- 
luted, again concentrated by boiling, and another wire dropped in. 
If further confirmation of the identity of the mercury is re- 
quired, the gold foil may be suspended in a tube containing a 
few crystals of iodine, which are then gently warmed. The 
mercury thus becomes converted into red mercuric iodide. 
By this method 1 mg. of mercury in 100 c.c. can be detected in 
urine, stomach contents, and stools; the copper being allowed to 
remain in the oxidized mixture 2 hours. 234 
CLINICAL LABORATORY METHODS 
Colorimetric Determination of the Hydrogen-Ion Concentration 
of Biological Fluids 
(Clark and Lubs: Jour. Bact., 1917, ii, 1, 101, 191) 
Principle.—The colorimetric method of determining hydrogen- 
ion concentration is based upon the fact that each indicator has 
a characteristic zone of hydrogen-ion concentration within which 
its color changes occur. The unknown solution is treated with 
a few drops of indicator and the color obtained compared with 
that produced with the same amount of indicator and a solution 
of known hydrogen-ion concentration. 
The essentials are: First, a set of indicators which (1) cover 
the ranges of PH to be studied; (2) are little affected by the pres- 
ence of protein and neutral salts and, (3) do not fade rapidly; 
and second, a set of buffer solutions of known hydrogen-ion con- 
centration. 
Reagents.—1. Standard Buffer Solutions.—The various mix- 
tures are made from the following stock solutions: M/5 potas- 
sium chloride (KC1), M/5 acid potassium phosphate (KII2P04), 
M/5 acid potassium phthalate (KIIC8H404), M/5 boric acid with 
M/5 potassium chloride (H3B03, KC1), M/5 sodium hydroxide 
(NaOH), and M/5 hydrochloric acid (HC1). 
Th*e water used in the crystallization of the salts and in the 
preparation of the stock solutions and mixtures should be redis- 
tilled and preferably “conductivity” water. 
M/5 Potassium Chloride Solution.— (This solution will not be 
necessary except in the preparation of the most acid series of 
mixtures.) The salt should be recrystallized three or four times 
and dried in an oven at about 120° C. for two days. The fifth 
molecular solution contains 14.912 grams in 1 liter. 
M/5 Acid Potassium Phthalate Solution.—Acid potassium 
phthalate may be prepared by the method of Dodge (1915) modi- 
fied as follows: Make up a concentrated potassium hydroxid solu- 
tion by dissolving about 60 grams of a high grade sample in about 
400 c.c. of water. To this add 50 grams of the commercial resub- 
limed anhydrid of orthophthalic acid. Test a cool portion of the 
solution with phenolphthalein. If the solution is still alkaline, 
add more phthalic anhydrid; if acid, add more KOH. When 235 
PATHOLOGICAL EXAMINATIONS 
roughly adjusted to a slight pink with phenolphthalein add as 
much more phthalie anhydrid as the solution contains and heat 
until all is dissolved. Filter while hot, and allow the crystal- 
lization to take place slowly. The crystals should be drained 
with suction and recrystallized at least twice from distilled 
water. Dry the salt at 110° C. to 115° C. to constant weight. 
A fifth molecular solution contains 40.828 grams of the salt 
in 1 liter of the solution. 
M/5 Acid Potassium Phosphate Solution.—A high grade com- 
mercial sample of the salt is recrystallized at least three times 
from distilled water and dried to constant weight at 110° to 
115° C. A fifth molecular solution should contain in 1 liter 
27.232 grams. The solution should be distinctly red with methyl 
red and distinctly blue with brom phenol blue. 
M/5 Boric Acid M/5 Potassium Chloride.—Boric acid should be 
recrystallized several times from distilled water. It should be 
air dried in thin layers between filter paper and the constancy 
of weight established by drying small samples in thin layers 
in a desiccator over CaCl2. Purification of KC1 has already 
been noted. One liter of the solution should contain 12.4048 
grams of boric acid and 14.912 grams of potassium chloride. 
M/5 Sodium Hydroxide Solution.—This solution is the most 
difficult to prepare, since it should be as free as possible from 
carbonate. A solution of sufficient purity for the present pur- 
poses may be prepared from a high grade sample of the hydroxide 
in the following manner. Dissolve 100 grams NaOH in 100 c.c. 
distilled water in a Jena or Pyrex glass Erlenmeyer flask. Cover 
the mouth of the flask with tin foil and allow the solution to 
stand overnight till the carbonate has mostly settled. Then 
prepare a filter as follows. Cut a ‘‘hardened” filter paper to fit a 
Buchner funnel. Treat it with warm strong (1 : 1) NaOII solu- 
tion. After a few minutes decant the sodium hydroxide and 
wash the paper first with absolute alcohol, then with dilute al- 
cohol, and finally with large quantities of distilled water. Place 
the paper on the Buchner funnel and apply gentle suction until 
the greater part of the water has evaporated but do not dry 
so that the paper curls. Now pour the concentrated alkali upon 
the middle of the paper, spread it with a glass rod making sure 236 
CLINICAL LABORATORY METHODS 
that the paper, under gentle suction, adheres well to the funnel, 
and draw the solution through with suction. The clear filtrate 
is now diluted quickly, after rough calculation, to a solution 
somewhat more concentrated than N/l. Withdraw 10 c.c. of 
this dilution and standardize roughly with an acid solution of 
known strength, or with a sample of acid potassium phthalate. 
From this approximate standardization calculate the dilution 
Fig. 60.—Showing H-ion comparison standards, comparator, and sodium hydroxide 
bottle with soda lime guard tubes. 
required to furnish an M/5 solution. Make the required dilution 
with the least possible exposure, and pour the solution into a 
paraffined bottle to which a calibrated 50 c.c. burette and soda-lime 
guard tubes have been attached (Fig. 60). The solution should 
now be most carefully standardized. One of the simplest methods 
of doing this, and one which should always be used in this in- 
stance, is the method of Dodge (1915) in which use is made of PATHOLOGICAL EXAMINATIONS 
237 
the acid potassium phthalate purified as already described. 
Weigh out accurately on a chemical balance with standardized 
weights several portions of the salt of about 1.6 grams each. 
Dissolve in about 20 c.c. distilled water and add 4 drops phenol- 
phthalein. Pass a stream of C02—free air through the solution 
and titrate with the alkali until a faint but distinct and per- 
manent pink is developed. It is preferable to use a factor with 
the solution rather than attempt adjustment to an exact M/5 
solution. 
M/5 Hydrochloric Acid Solution.—Dilute a high grade of hydro- 
chloric acid solution to about 20 per cent and distill. Dilute the 
distillate to approximately M/5 and standardize with the sodium 
hydroxide solution previously described. 
The only solution which it is absolutely necessary to protect 
from the C02 of the atmosphere is the sodium hydroxide solu- 
tion. Therefore all but this solution may be stored in ordinary 
bottles of resistant glass. The salt solutions, if adjusted to 
exactly M/5, may be measured from clean calibrated pipettes. 
Table XLVI shows the proportions in which the stock solu- 
tions are to be mixed to make buffer solutions of varying hydro- 
gen concentration. 
Table XLVI 
Compositions of Mixtures Giving Ph Values at 20° C. at Intervals 
of 0.2 
KOI - HC1 Mixtures 
PH Composition 
1.0 50 c.c. M/5 KC1 97.0 c.c. M/5 HC1 Dilute to 200 c.c. 
1.2 50 c.c. M/5 KC1 64.5 c.c. M/5 HC1 Dilute to 200 c.c. 
1.4 50 c.c. M/5 KC1 41.5 c.c. M/5 HC1 Dilute to 200 c.c. 
1.6 50 c.c. M/5 KC1 26.3 c.c. M/5 HC1 Dilute to 200 c.c. 
1.8 50 c.c. M/5 KC1 16.6 c.c. M/5 HC1 Dilute to 200 c.c. 
2.0 50 c.c. M/5 KC1 10.6 c.c. M/5 HG1 Dilute to 200 c.c. 
2.2 50 c.c. M/5 KC1 6.7 c.c. M/5 HC1 Dilute to 200 c.c. 
2.2 50 c.c. M/5 KHPhthalate 46.70 e.e. M/5 HC1 Dilute to 200 c.c. 
2.4 50 c.c. M/5 KHPhthalate 39.60 c.c. M/5 HC1 Dilute to 200 c.c. 
2.6 50 c.c. M/5 KHPhthalate 32.95 c.c. M/5 HC1 Dilute to 200 c.c. 
2.8 50 c.c. M/5 KHPhthalate 26.42 c.c. M/5 HC1 Dilute to 200 c.c. 
3.0 50 c.c. M/5 KHPhthalate 20.32 c.c. M/5 HC1 Dilute to 200 c.c. 
3.2 50 c.c. M/5 KHPhthalate 14.70 c.c. M/5 HC1 Dilute to 200 c.c. 
3.4 50 c.c. M/5 KHPhthalate 9.90 c.c. M/5 HC1 Dilute to 200 c.c. 
3.6 50 c.c. M/5 KHPhthalate 5.97 c.c. M/5 HC1 Dilute to 200 c.c. 
3.8 50 c.c. M/5 KHPhthalate 2.63 c.c. M/5 HC1 Dilute to 200 c.c. 
Phthalate - HC1 Mixtures 238 
CLINICAL LABORATORY METHODS 
Table XLVI—Cont T) 
Phthalate - NaOH Mixtures 
4.0 50 ec. M/5 KHPlithalate 0.40 c.c. M/5 NaOH Dilute to 200 c.c. 
4.2 50 cc. M/5 KHPlithalate 3.70 c.c. M/5 NaOH Dilute to 200 c.c. 
4.4 50 cc. M/5 KHPlithalate 7.50 c.c. M/5 NaOH Dilute to 200 c.c. 
4.6 50 ee. M/5 KHPhthalate 12.15 c.c. M/5 NaOH Dilute to 200 c.c. 
4.8 50 cc. M/5 KHPhthalate 17.70 c.c. M/5 NaOH Dilute to 200 c.c. 
5.0 50 cc. M/5 KHPhthalate 23.85 c.c. M/5 NaOH Dilute to 200 c.c. 
5.2 50 cc. M/5 KHPhthalate 29.95 c.c. M/5 NaOH Dilute to 200 c.c. 
5.4 50 cc. M/5 KHPhthalate 35.45 c.c. M/5 NaOH Dilute to 200 c.c. 
5.6 50 cc. M/5 KHPhthalate 39.85 c.c. M/5 NaOH Dilute to 200 c.c. 
5.8 50 cc. M/5 KHPhthalate 43.00 c.c. M/5 NaOH Dilute to 200 c.c. 
6.0 50 ec. M/5 KHPhthalate 45.45 c.c. M/5 NaOH Dilute to 200 c.c. 
6.2 50 cc. M/5 KHPhthalate 47.00 c.c. M/5 NaOH Dilute to 200 c.c. 
KH2P04 - NaOH Mixtures 
5.8 50 c.c. M/5 KH3P04 3.72 c.c. M/5 NaOH Dilute to 200 c.c. 
6.0 50 c.c. M/5 KH2P04 5.70 c.c. M/5 NaOH Dilute to 200 c.c. 
6.2 50 c.c. M/5 KH2P04 8.60 c.c. M/5 NaOH Dilute to 200 c.c. 
6.4 50 c.c. M/5 IvH2P04 12.60 c.c. M/5 NaOH Dilute to 200 c.c. 
6.6 50 c.c. M/5 KH„P04 17.80 c.c. M/5 NaOH Dilute to 200 c.c. 
6.8 50 c.c. M/5 KH2P04 23.65 c.c. M/5 NaOH Dilute to 200 c.c. 
7.0 50 c.c. M/5 KH,P04 29.63 c.c. M/5 NaOH Dilute to 200 c.c. 
7.2 50 c.e. M/5 KH2P04 35.00 c.c. M/5 NaOH Dilute to 200 c.e. 
7.4 50 c.c. M/5 KH2P04 39.50 c,c. M/5 NaOH Dilute to 200 c.c. 
7.6 50 c.c. M/5 KH,P04 42.80 c.c. M/5 NaOH Dilute to 200 c.c. 
7.8 50 c.c. M/5 KH2P04 45.20 c.c. M/5 NaOH Dilute to 200 c.c. 
8.0 50 c.c. M/5 KH2P04 46.80 c.c. M/5 NaOH Dilute to 200 c.c. 
Boric Acid, KC1 - NaOH Mixtures 
7.8 50 c.c. M/5 H3B03 M/5 KOI 2.61 c.c. M/5 NaOH Dilute to 200 c.c. 
8.0 50 c.e. M/5 H3BOs M/5 KC1 3.97 c.c. M/5 NaOH Dilute to 200 c.c. 
8.2 50 c.c. M/5 H3B03 M/5 KOI 5.90 c.c. M/5 NaOH Dilute to 200 c.c. 
8.4 50 c.c. M/5 H3B03 M/5 KC1 8.50 c.c. M/5 NaOH Dilute to 200 c.c. 
8.6 50 c.e. M/5 H3B03 M/5 KC1 12.00 c.c. M/5 NaOH Dilute to 200 c.c. 
8.8 50 c.c. M/5 H3B03 M/5 KOI 16.30 c.c. M/5 NaOH Dilute to 200 c.c. 
9.0 50 c.c. M/5 H3B03 M/5 KOI 21.30 c.c. M/5 NaOH Dilute to 200 c.c. 
9.2 50 c.c. M/5 H3B03 M/5 KCl 26.70 c.c. M/5 NaOH Dilute to 200 c.c. 
9.4 50 c.e. M/5 H3B03 M/5 IvCl 32.00 c.c. M/5 NaOH Dilute to 200 c.c. 
9.6 50 c.c. M/5 H3B03 M/5 KCl 36.85 c.c. M/5 NaOH Dilute to 200 c.c. 
9.8 50 c.c. M/5 H3B03 M/5 KCl 40.80 c.c. M/5 NaOH Dilute to 200 c.c. 
10.0 50 c.c. M/5 H3BQ3 M/5 KCl 43.90 c.c. M/5 NaOH Dilute to 200 c.c. 
The mixtures should be made up freshly once a week. They 
are conveniently kept in 200 c.c. bottles, each of which is provided 
with a 10 c.c. pipette. The bottles used for the alkaline borate 
mixtures should be paraffined. 
2. Indicators.—Table XLYII of indicators shows the concen- 
tration of the solution used to test gross color changes and the 
range in which each indicator is useful. PATHOLOGICAL EXAMINATIONS 
239 
Table XLYII 
List of Indicators 
CHEMICAL NAME 
COMMON NAME 
CONCEN- 
COLOR 
RANGE 
TRATION CHANGE 
Thymol sulphone phthalein 
(acid range) 
Thymol blue 
0.04 
Bed-Yellow 
1.2-2.8 
Tetra bromo phenol sulphone 
phthalein 
Ortho carboxy benzene azo 
.Brom phenol blue 
0.04 
Yellow-blue 
3.0-4.G 
dimethyl aniline 
Di bromo ortho cresol sul- 
Methyl red 
0.02 
Bed-yellow 
4.4-6.0 
phone phthalein 
Brom cresol purple 0.04 
Yellow-purple 
5.2-6.S 
Di bromo thymol sulphone 
phthalein 
Brom thymol blue 
0.04 
Yellow-blue 
6.0-7.6 
Phenol sulphone phthalein.. 
Ortho cresol sulphone phthal- 
Phenol red 
0.02 
Yellow-red 
6.8-B.4 
ein 
.Cresol red 
0.02 
Yellow-red 
7.2-8.8 
Thymol sulphone phthalein 
(see above) 
.Thymol blue 
0.02 
Yellow-blue 
8.0-9.6 
Ortho cresol phthalein 
.Cresol phthalein 
0.02 
Colorless-red 
8.2-9.8 
The indicators are supplied in dry form from which stock 
solutions are made as follows: 0.1 gram of dry powder is ground 
in an agate mortar with the following quantities of N/20 NaOH. 
Indicator 
Phenol red 
Brom phenol blue 
Cresol red 
Thymol blue 
Brom thymol blue 
Brom cresol purple 
N/20 NaOII per 0.1 gm. 
5.7 c.c. 
3.0 c.c. 
5.3 c.c. 
4.3 c.c. 
3.2 c.e. 
3.7 c.e. 
When solution is complete dilute to 25 c.c. 
This gives a 0.4 per cent solution. For use the stock solution 
of thymol blue, brom thymol blue, brom phenol blue and brom 
cresol purple are diluted with water to make 0.04 per cent 
solutions, those of phenol red and cresol red to make 0.02 per 
cent solutions. 
Methyl red is made by dissolving 0.1 gram in 300 c.c. of alcohol 
and diluting to 500 c.c. Orthocresol phthalein is used in 0.02 
per cent solution in 95 per cent alcohol. 
3. Comparison Color Standards.—Select test tubes of the same 
bore. Fill into them 10 c.c. of the standard buffer solutions to 
make a set of standards whose PH range from 1.2 to 9.6 with 240 
CLINICAL LABORATORY METHODS 
increments of 0.2 PH allowing extra tubes for the overlapping 
of indicators. 
Add to each 5 drops of indicator solution as given below: 
PR of Buffer Solution 
1.2 - 2.8 
3.0-4.6 
4.4-6.0 
5.2-6.8 
6.0 - 7.6 
7.0-8.4 
7.8 - 8.6 
8.8-9.6 
Indicator 
Thymol blue 
Brom phenol blue 
Methyl red 
Brom cresol purple 
Brom thymol blue 
Phenol red 
Cresol red 
Cresol phthalein 
If corked and sealed with paraffin, these standards are fairly 
permanent except those containing methyl red as the indicator. 
Procedure.—If the approximate PH of the solution is not 
known, find the range within which it falls by adding to a portion 
LIGHT 
ELY El 
Fig. 61.—Arrangement of tubes in comparator in the determination of hydrogen-ion 
concentration. 
the sulplionephthalein series of indicators, beginning with thy- 
mol blue. 
Measure 10 c.c. of the solution to be examined into a test tube 
of the same bore as those containing the standard solutions. 
Add 5 drops of the indicator showing the sharpest color changes 
over the range within which the solution falls, as indicated by the 
preliminary test. 
Compare this solution with those of the comparison color stand- PATHOLOGICAL EXAMINATIONS 
241 
PH 
H-ION CONC. 
(gms. of hydro- 
gen per li.) 
PH 
H-ION CONC. 
(gms. of hydro- 
gen per li.) 
1 
PH 
H-ION CONC. 
(gms. of hydro- 
gen per li.) 
PH 
H-ION CONC. 
(gms. of hydro- 
gen per li.) 
PH 
H-ION CONC. 
(gms. of hydro- 
gen per li.) 
1.0 
1.0 x 10-i 
3.0 
1.0x10-3 
5.0 
1.0 x 10-5 
7.0 
1.0 x 10-7 
9.0 
1.0x10-9 
1.1 
7.9x10-2 
3.1 
7.9 x 10-4 
5.1 
7.9 x 10-6 
7.1 
7.9 x 10-8 
9.1 
7.9 x lO-io 
1.2 
6.3x10-2 
3.2 
6.3 x 10-4 
5.2 
6.3 x 10-6 
7.2 
6.3 x 10-8 
9.2 
6.3 x lO'io 
1.3 
5.0x10-2 
3.3 
5.0 x 10"4 
5.3 
5.0 x 10"6 
7.3 
5.0 x 10*8 
9.3 
5.0 x 10"io 
1.4 
4.0 x 10-2 
3.4 
4.0 x 10‘4 
5.4 
4.0 x 10"6 
7.4 
4.0x10-8 
9.4 
4.0 x 10-io 
1.5 
3.2 x 10-2 
3.5 
3.2 x 10-4 
5.5 
3.2 x 10-6 
7.5 
3.2x10-8 
9.5 
3.2xl0-io 
1.6 
2.5x10-2 
3.6 
2.5 x 10-4 
5.6 
2.5 x 10‘6 
7.6 
2.5 x 10-s 
9.6 
2.5 x lO-io 
1.7 
2.0 x 10-2 
3.7 
2.0 x 10"4 
5.7 
2.0 x 10-6 
7.7 
2.0 x 10-s 
9.7 
2.0 x lO'io 
1.8 
1.6x10-2 
3.8 
1.6 x 10-4 
5.8 
1.6 x 10-6 
7.8 
1.6 x 10-s 
9.8 
1.6 x lO'io 
1.9 
1.2x10-2 
3.9 
1.2x10-4 
5.9 
1.2x10-6 
7.9 
1.2 x 10-s 
9.9 
1.2xl0-io 
2.0 
1.0 x 10-2 
4.0 
1.0x10-4 
6.0 
1.0x10-6 
8.0 
1.0 x 10-s 
10.0 
1.0 x lO'io 
2.1 
7.9 x 10-3 
4.1 
7.9 x 10-5 
6.1 
7.9 x 10-7 
8.1 
7.9 x 10-9 
10.1 
7.9 x 10-n 
2.2 
6.3x10-3 
4.2 
6.3 x 10-5 
6.2 
6.3 x 10-7 
8.2 
6.3 x 10-9 
10.2 
6.3 x 10-ii 
2.3 
5.0 x 10-3 
4.3 
5.0 x 10"5 
6.3 
5.0x10-7 
8.3 
5.0x10-9 
10.3 
5.0 x lO'H 
2.4 
4.0 x 10-3 
4.4 
4.0 x 10-5 
6.4 
4.0 x 10-7 
8.4 
4.0 x 10-9 
10.4 
4.0 x 10-ii 
2.5 
3.2x10-3 
4.5 
3.2 x 10-5 
6.5 
3.2 x 10-7 
8.5 
3.2x10-9 
10.5 
3.2 x 10-n 
2.6 
2.5 x 10-3 
4.6 
2.5 x 10"5 
6.6 
2.5x10-7 
8.6 
2.5 x 10-9 
10.6 
2.5x10-11 
2.7 
2.0 x 10*3 
4.7 
2.0 x 10‘5 
6.7 
2.0 x 10-7 
8.7 
2.0 x 10-9 
10.7 
2.0xl0-ii 
2.8 
1.6 x 10-3 
4.8 
1.6x10-5 
6.8 
1.6 x 10-7 
8.8 
1.6x10-9 
10.8 
1.6 x 10-11 
2.9 
1.2 x 10-3 
4.9 
1.2 x 10-5 
6.9 
1.2 x 10-7 
8.9 
1.2x10-9 
10.9 
1.2x10-11 
True H-Ion Concentration Corresponding to Logarithmic Figures 
Table XLVIII 242 
CLINICAL LABORATORY METHODS 
ards containing the same indicator. The PH of the unknown solu- 
tion is the same as that of the standard buffer solution the color 
of which it most nearly matches. 
If the unknown solution is colored, compare in a comparator 
holding six tubes (Fig. 60). Arrange the tubes as indicated in 
Fig. 61. 
If the native color of the solution still interferes, the solution 
may be diluted up to 1.5 without appreciably changing the hy- 
drogen-ion concentration. 
Remarks.—Table XLVIII shows the true hydrogen-ion con- 
centration corresponding to the logarithmic figures (1.0-10.9) . 
The hydrogen-ion concentration of the body fluids is given in 
Table XLIX. (Clark.) 
Table XLIX 
FLUID 
PH 
FLUID 
PH 
Blood 
7.4 
Muscle juice (fresh) 
6.8 
Urine 
6.0 
Muscle juice (autolyzed) 
Variable 
Saliva 
6.9 
Pancreas extract 
5.6 
Gastric juice (adult) 
0.9-1.6 
Peritoneal fluid 
7.4 
Gastric juice (infant) 
5.0 
Pericardial fluid 
7.4 
Pancreatic juice (dog) 
8.3 
Aqueous humor 
7.1 
Small intestinal contents 
8.3 
Vitreous humor 
7.0 
Small intestinal contents, 
(infant) 
3.1 
Cerebrospinal fluid 
7.5-7.7 
Bile from liver 
7.8 
Amniotic fluid 
7.1 
Bile from gall bladder 
Perspiration 
Tears 
5.3-7.4 
4.5 
7.2 
Milk (human) 
7.0-7.2 CHAPTER XII 
MISCELLANEOUS CHEMICAL PROCEDURES AND 
SOLUTIONS 
The Use of the Colorimeter and Nephelometer 
The Colorimeter.—The quantitative determination of sub- 
stances which are present in only minute amounts but which 
form colored compounds is made with a colorimeter. 
Three types of colorimeter are in common use: (1) plunger; 
(2) wedge and (3) dilution type. With the plunger type, (Fig. 
62), the intensity of the color of either the standard or unknown 
is varied by changing the depth of solution through which the 
light passes with the aid of a plunger. The two halves of the 
field of view are illuminated by the light passing through the 
standard and unknown solutions simultaneously. With the wedge 
type, (Fig. 34) the standard solution is placed in a wedge and 
the unknown in a small cup with sides parallel to the wedge. 
The wedge is moved up and down until the intensity of the color 
is the same as the unknown. With the dilution type the standard 
and unknown are placed in small test tubes of equal diameter. 
The unknown is diluted until it has identically the same color as 
the standard. 
Each type of instrument has its advantages and disadvantages. 
The wedge type is quite satisfactory where determinations need 
to be made quickly and only relatively accurate results are 
necessary. The wedge must be carefully calibrated, however, 
as described on page 98. For the most exact work an instru- 
ment of the plunger type such as the Duboscq (Fig. 62) or 
Bock-Benedict (Fig. 63) is necessary. 
In using a colorimeter of the plunger type, the instrument and 
the mirror should be adjusted to the source of light, by placing a 
colored solution in both cups, setting them at the same point on 
the scale and noting the two halves of the field. If they are not 
alike, the zero point or the optics of the instrument must be 
243 244 
CLINICAL LABORATORY METHODS 
wrong. Suitable allowance must be made in the readings after 
the degree of variation has been calculated. After having deter- 
mined the point at which the two halves of the field are alike, 
change the height of the plungers and then make a color com- 
parison of the color standard against itself, readjusting the moved 
plunger until the fields look alike again. The error should not 
exceed 0.2 mm. 
Fig. 62.—Duboscq colorimeter. (Bausch and Lornb.) 
Haying adjusted the instrument, the solutions are poured into 
the cups. The cup containing the standard solution is then 
lowered to a definite thickness, usually 20 mm. of the standard 
solution between the bottom of the cup and the end of the 
plunger. The cup containing the unknown solution is now moved 
until the two halves of the field are brought to the same intensity 
of color, after which the height at which the two liquid columns CHEMICAL PROCEDURES AND SOLUTIONS 
245 
display this equal absorptive power is read by means of the scale. 
Since the proportion of coloring matter in two solutions is in- 
versely proportional to the heights of the two columns necessary 
to obtain the same intensity of illumination, if the standard tube 
is set at 20 mm. and the solution under examination is the same 
intensity of color at 10 mm. the latter is just twice the concentra- 
tion of the standard. This is expressed by the formula: 
Color of Test Solution Height of Standard Solution 
Color of Standard Solution Height of solution to be tested 
Key to Figure 
A ens 
E Eye-piece 
HM Half size mir- 
ror 
FM Full size mir- 
ror 
S Cell for stand- 
ard solution 
P Plunger 
C Cup for un- 
known solu- 
tion 
R Large reflector 
ST Stage for hold- 
ing cup 
T Thumb screw 
moving stage 
up or down 
SC Set Screw 
Fig. 63.—Bock-Benedict colorimeter. 
If, therefore, the scale reading is 20 mm. for the standard 
and 15 mm. for the solution to be tested, the formula reads: 
20 
= 1.33. If for example the standard solution contains 4 mg. 
of coloring matter in 100 c.c., the solution under test will be 
found to contain 4 x 1.33 = 5.32 mg. in 100 c.c. 
The color comparisons should be made rapidly as the eye 
fatigues quickly. The eye should be closed often while making 
the readings. The optical parts of the instrument must be kept 
free from dust, and cups, plungers and mirror must be kept clean. 246 
CLINICAL LABORATORY METHODS 
Alkaline solutions such as those containing Nessler’s reagent 
must not be spilled on the mirror. The spilling of solutions is 
usually due to tilling the cups too full. 
The focus should be adjusted with the draw tube so the line 
dividing the fields is distinct. The readings are best made in a 
dark-room, both eyes being kept open. 
The more nearly the composition and concentration of the test 
solution equal that of the standard the more accurate will be the 
determination. Hence it is best to vary the strength of the test 
solution or standard until the two nearly match. 
The tables given under the colorimetric determinations in blood 
and urine can be used with a Duboscq, Kober or Bock-Benedict, 
(20 mm. cell) instrument. 
The Nephelometer.—Minute amounts of substances which do 
not form colored solutions may be determined in the nephelom- 
eter. This is an instrument for the determination of the quantity 
of a substance by measurement of the density of a precipitate 
which it produces with a reagent. It differs from the colorimeter 
in that the light which reaches the eye is not transmitted light 
but is light reflected from the particles of the suspension. The 
brightness of the two fields is compared instead of their color. 
The Duboscq colorimeter with movable cups may be easily 
adapted for nephelometric purposes, as suggested by Bloor, (Jour. 
Biol. Chem., 1915, xxii, 145). The method of transformation may 
be seen from the diagrams (Figs. 64 and 65). 
The brass plate carrying the colorimeter plungers is replaced 
by the plate A, with two slots in which are supported the nephel- 
ometer tubes B, with their flanges resting on the edges of the 
slots. The slots are so cut that the center lines of the tubes are 
exactly in line with the centers of the lower opening of the prism 
case E. If desired they may be countersunk to receive the flanges. 
The colorimeter cups are replaced by the jackets F and are 
supported on them by the collars D. They move when the cup 
supports move. The mirror is turned to the horizontal position, 
so that it reflects no light; the light in the nephelometer comes 
from in front and not from below (Fig. 64). 
The nephelometer tubes are small test tubes, 100 x 15 mm., CHEMICAL PROCEDURES AND SOLUTIONS 
247 
preferably made from the same sample of colorless glass tubing 
so that they are of exactly the same bore. The flanges at the 
top should be well made so that the tubes rest firmly and evenly 
in the slots. The glass should be as free as possible from imper- 
fections or striations. After the tubes are made and fitted into 
place the jackets are moved up on each tube by means of the 
Fig. 64.—The Duboscq colorimeter converted into a nephelometer with the necessary 
extra parts. (After Bloor.) 
rack and pinion, until the indicator on the scale is exactly at 
zero. Marks are made on each tube at the point reached by the 
top of the jacket and the portion above that point is made opaque 
by a ring of black paper or paint. Tubes and jackets are then 
marked “right” and “left” and always used on the same side. 
Since it is rare to find two tubes, which when filled with the 248 
CLINICAL LABORATORY METHODS 
same solution, give exactly the same readings, it is necessary to 
take this fact into account and correct accordingly. 
The jackets C are made of tubing—metal or glass—a little 
larger than the tubes and about the same length, (they should 
just clear the mirror when it is turned horizontal), closed at the 
bottom and made light tight by black paint or paper. The col- 
lars D, supporting the jackets may be made of cork or more per- 
manently of metal. A little cotton wool in the bottom of the 
jackets will prevent breakage if the tubes should fall into the 
jackets. 
Artificial light is necessary and the lamp should be enclosed in 
a tight box, into one end of which the nephelometer fits snugly. 
Fig. 65.—The nephelometer in position in its box, showing its relation to the source of 
light. (After Bloor.) 
A partition extending part way up the box, as shown on the dia- 
gram (Fig. 65), serves the double purpose of shutting off the 
light from the lower part of the instrument and of providing a 
stop against which the instrument is pushed, so that its distance 
from the light is kept constant. The box is conveniently made 
without a bottom and the end closed with a dark curtain after the 
nephelometer is pushed into place. The inside of the box should 
be painted black. A dark room is desirable but not necessary, 
as the instrument may be used satisfactorily in a room darkened 
by a dark shade, or even in a dark corner of the laboratory. 
The relations of the nephelometer and light source may be seen 
in the diagram (Fig. 65). The lamp used is an ordinary 50 Watt 
tungsten, supported by a bracket at 30 cm. from the nephelometer CHEMICAL PROCEDURES AND SOLUTIONS 
249 
and at the height of the nephelometer tubes. The extra parts 
necessary for the conversion of the colorimeter into a nephelom- 
eter may be obtained from the International Instrument Com- 
pany of Cambridge, Massachusetts. 
The above description applies only to the type of instrument 
with movable cups. 
The total light reflected from a given depth of suspension de- 
pends not only on the number of reflecting particles but also on 
the condition of the precipitate, such as size and density. Hence 
it is very important that the condition of the particles in the 
standard and in the test solution be the same. It is thus neces- 
sary to make the physical and chemical conditions as regards 
salts, reaction, temperature and volume be as nearly alike in the 
two solutions as possible. 
The readings obtained with the different suspensions in the 
nephelometer are not exactly proportional to the amount of sub- 
stance present, and the difference between the observed and theo- 
retical readings increases as the substance increases in amount. 
Kober has proposed a formula to take care of such corrections. 
But practically where the reading of the test solution does not 
differ from the standard by more than 25 per cent the correction 
falls within the limits of error of the determination and no cor- 
rection need be made. 
The nephelometer tubes should be so filled that the meniscus 
comes just above the bottom of the black collar of the tube B, 
(Fig. 64). 
Standardization of Blood Chemical Determinations 
The technic and reagents employed in blood chemical deter- 
mination should be checked by making quantitative analyses on 
specimens of blood to which known amounts of the substance to 
be estimated have been added. 
The procedure to be followed in the check determination is 
given below. The protocols show the amount to be added in 
each test, and the calculated amount per 100 c.c. of blood with 
which results obtained are to be compared. Blank determinations 
in which no blood is used should also be run with each new lot 
of chemicals. 250 
CLINICAL LABORATORY METHODS 
Blood Urea Nitrogen.—Pipette 3 c.c. of whole blood into each of 
seven urea tubes. Add urea to each tube as indicated in the 
protocol below. Make a urea determination on each specimen in 
the usual way (page 133). Compare the results obtained with the 
calculated figures (Table L). 
Table L 
TUBE NO. 
UREA ADDITION 
calculated 
UREA 
NITROGEN 
1 
0.0 
X 
mg. 
per 
100 
c.c. 
2 
0.3 mg. 
urea= 1 c.c.Si 
X +4.67 
C l 
( C 
( ( 
< ( 
3 
0.6 “ 
“ =2 c.c.S1 
X +9.34 
( c 
( ( 
( ( 
( ( 
4 
0.9 “ 
“ =3 c.c.Si 
X +13.00 
(( 
( ( 
( ( 
C ( 
5 
1.2 “ 
“ = 4 c.c.Si 
X +17.67 
(( 
C ( 
(c 
( i 
6 
1.5 “ 
“ =5 c.c.Si 
X +22.34 
(( 
(( 
(( 
( ( 
7 
1.8 “ 
“ =6 c.c.Si 
X +26.00 
( c 
C l 
(( 
i ( 
Where 
water. 
Si = a solution containing 300 mg. 
of urea dissolved in 
1000 c.c. 
Total Non-protein Nitrogen.—Into each of seven Erlenmeyer 
flasks pipette 2 c.c. of oxalated blood. Add nitrogen and water as 
indicated in the protocol below. Finally add 2 c.c. each of 10 
per cent sodium tungstate and % normal sulphuric acid. Deter- 
mine the non-protein nitrogen on the filtrate in the usual way 
(page 131). Compare the results obtained with the calculated 
figures (Table LI). 
Table LI 
FLASK NO. 
NITROGEN ADDITION 
WATER 
CALCULATED TOTAL 
NON-PROTEIN NITROGEN 
1 
0.0 
14 c.c. 
X mg. per 
100 
c.c. 
blood 
2 
0.2 mg. 
— 2 c.c.Sj 
12 c.c. 
X +10 “ 
£ c 
( i 
£ £ 
£ £ 
3 
0.4 mg. 
= 4 e.c.S-t 
10 c.c. 
X +20 “ 
( < 
t ( 
£ £ 
i £ 
4 
0.6 mg. 
= 6 e.e.St 
8 c.c. 
X +30 “ 
( C 
( C 
£ £ 
£ £ 
5 
0.8 mg. 
= 8 c.e.St 
6 c.c. 
X +40 “ 
( ( 
t £ 
£ £ 
£ £ 
G 
1.0 mg. 
—10 c-c.Si 
4 c.c. 
X +50 “ 
( ( 
£ £ 
£ £ 
£ £ 
7 
1.2 mg. 
=12 c.c.Sj 
2 c.c. 
X +60 “ 
( ( 
f £ 
£ £ 
£ £ 
Where 
ammonium 
St = a solution containing 0.1 mg. 
sulphate per liter). 
nitrogen per 
c.c. (0.4716 gms. 
Blood Sugar.—Pipette 2 c.e. of oxalated blood into each of nine 
Erlenmeyer flasks. Add glucose and water as indicated in the 
protocol below. Finally add 2 c.c. each of 10 per cent sodium 
tungstate and % normal sulphuric acid. Determine the sugar on 251 
CHEMICAL PROCEDURES AND SOLUTIONS 
the filtrate in the usual way (page 141). Compare the results 
obtained with the calculated figures (Table LII). 
FLASK 
NO. 
GLUCOSE ADDITION 
WATER 
CALCULATED ! 
SUGAR 
1 
0.0 
14.0 c.c. 
X 
mg. 
per 
100 
c.c. 
2 
0.5 mg. 
= 5.0 
c.c.Sj 
9.0 c.c. 
X +25 
£ £ 
i £ 
£ C 
£ £ 
3 
1.0 mg. 
==10.0 
c.c.St 
4.0 c.c. 
X +50 
£ £ 
£ £ 
£ £ 
£ £ 
4 
1.5 mg. 
= 7.5 
e.c.S2 
6.5 c.c. 
X +75 
£ £ 
£ £ 
i £ 
• • 
5 
2.0 mg. 
=10.0 
c.c.S2 
4.0 c.c. 
X +100 
( 6 
£ £ 
£ £ 
£ £ 
6 
2.5 mg. 
=12.5 
c.c.S2 
1.5 c.c. 
X +125 
( i 
£ £ 
£ £ 
£ £ 
7 
3.0 mg. 
= 3.75 
c.c.S3 
10.25 c.c. 
X +150 
C 6 
£ ( 
£ £ 
£ £ 
8 
3.5 mg. 
= 4.375 
c.c.S3 
9.625 c.c. 
X +175 
( i 
£ ( 
£ £ 
£ £ 
9 
4.0 mg. 
= 5.0 
c.c.S3 
9.0 c.c. 
X +200 
( 6 
£ £ 
£ £ 
£ £ 
Where S, = 
= standard containing 10 mg. glucose per 
100 
c.c. 
S2 = 
= standard containing 20 mg. glucose per 100 
c.c. 
S3 3 
= standard containing 40 mg. glucose per 100 
c.c. 
Table LII 
Blood Uric Acid.—Pipette 2 c.c. of oxalated blood into each of 
nine Erlenmeyer flasks. Add uric acid and water as indicated in 
the protocol below. Finally add 2 c.c. each of 10 per cent sodium 
tungstate and % normal sulphuric acid. Determine the uric 
acid on the filtrate from each flask in the usual manner, (page 
135.) Compare the results obtained with the calculated figures 
(Table LIII). 
Table LIII 
FLASK 
NO. 
URIC ACID 
ADDITION 
WATER 
CALCULATED 
URIC ACID 
1 
0.0 
14 c.c. 
X 
mg. 
per 
100 
c.c. 
blood 
2 
0.01 
mg. = 1 c.c. St 
13 c.c. 
X +0.50 
£ £ 
( ( 
( ( 
£ £ 
£ £ 
3 
0.02 
“= 2 c.c. St 
12 c.c. 
X +1.00 
£ £ 
i ( 
( ( 
£ £ 
£ £ 
4 
0.03 
“ == 3 c.c. S, 
11 c.c. 
X +1.50 
( ( 
( i 
i ( 
£ £ 
£ £ 
5 
0.04 
“ — 4 c.c. Sj 
10 c.c. 
X +2.00 
i ( 
< ( 
£ £ 
£ £ 
£ £ 
6 
0.05 
“ = 5 c.c. Sj 
9 c.c. 
X +2.50 
“ 
( t 
£ £ 
£ £ 
‘ ‘ 
7 
0.06 
“ = 6 c.c. S, 
8 c.c. 
X +3.00 
( ( 
< £ 
£ £ 
£ £ 
£ £ 
8 
0.07 
“ = 7 c.c. St 
7 c.c. 
X +3.50 
( ( 
( l 
£ £ 
£ £ 
£ £ 
9 . 
0.08 
“ = 8 c.c. 
6 c.c. 
X +4.00 
( ( 
< ( 
£ £ 
£ £ 
‘ ‘ 
Where Sj = a solution containing 10 mg. uric acid (50 
phosphate solution) in one liter of distilled water. 
c.c. of 
the 
stock 
Blood Creatinine.—Into each of eight Erlenmeyer flasks pipette 
2 c.c. of oxalated blood. Add creatinine and water as indicated 
in the protocol below. Finally add to each flask 2 c.c. each of 
10 per cent sodium tungstate and % normal sulphuric acid. De- 252 
CLINICAL LABORATORY METHODS 
termine the creatinine on the filtrate in the usual manner (page 
138). Compare the results obtained with the calculated figures 
(Table LIV). 
Table LIV 
FLASK NO. 
CREATININE ADDITION 
WATER 
CALCULATED CREATININE 
1 
0.0 
14 c.c. 
X 
mg. per 100 cc. 
blood 
2 
0.012 
mg. — 2 c.c.Sj 
12 c.c. 
X 
+0.6 
£ £ 
£ £ 
£ £ £ £ 
£ £ 
3 
0.024 
“ = 4 c.c.Sj 
10 c.c. 
X 
+1.2 
£ £ 
£ £ 
£ £ £ £ 
£ £ 
4 
0.036 
“ =6 c.c.Sj 
8 c.c. 
X 
+1.8 
£ C 
£ £ 
£ £ £ £ 
£ £ 
5 
0.048 
“ = 8 c.c.Sj 
6 c.c. 
X 
+2.4 
(e 
£ £ 
£ £ £ £ 
£ £ 
6 
0.060 
“ =10 c.c.Sj 
4 c.c. 
X 
+3.6 
( c 
£ £ 
£ £ £ £ 
£ £ 
7 
0.072 
“ =12 c.c.Sj 
2 c.c 
X 
+3.0 
( £ 
£ £ 
£ £ £ £ 
£ £ 
8 
0.084 
“ =14 c.c.Sj 
0 
X 
+4.2 
£ £ 
£ £ 
£ £ £ £ 
£ £ 
Where S, 
water. 
= a 
solution containing 6 mg. 
creatinine in 
one 
liter 
of distilled 
Blood Cholesterol.—Introduce into each of six beakers, 1 c.c. 
of blood plasma, 4 or 5 grams of plaster of Paris, and choles- 
terol as indicated in the protocol below. Determine the choles- 
terol in the usual way (page 148). Compare the results obtained 
with the calculated figures (Table LY). 
Table LY 
BEAKER NO. 
CHOLESTEROL ADDITION 
CALCULATED CHOLESTEROL 
1 
0.0 
X 
mg. per 100 cc. plasma 
2 
0-2 mg. 
= 2 c.c. 
X 
+20 “ “ 
c c (< a 
3 
0-4 “ 
= 4 c.c. Sj 
X 
+40 “ “ 
a c c (c 
4 
0.6 “ 
= 6 c.c. S, 
X 
+60 “ “ 
c c c i c e 
5 
0.8 “ 
: - 8 C.C. St 
X 
+80 “ “ 
a a a 
6 
1.0 “ 
=10 c.c. S. 
X 
+100 “ “ 
(c a a 
Where 
chloroform. 
= a solution 
containing 
10 mg. of cholesterol 
in 100 c.c. 
Blood Chloride.—Pipette 4 c.c. of blood into each of five Erlen- 
meyer flasks. Add water and sodium chloride as indicated in 
the protocol below. To each flask then add 4 c.c. each of ten 
per cent sodium tungstate and % normal sulphuric acid. Shake 
and filter. To 20 c.c. of the filtrate of each flask add 10 c.c. of 
silver nitrate solution (page 146). Filter off the silver chloride 
and determine the excess of silver nitrate in the filtrate as de- 
scribed on page 145. Compare the results with the calculated 
figures (Table LVI). CHEMICAL PROCEDURES AND SOLUTIONS 
253 
Table LYI 
FLASK NO. 
SODIUM CHLORIDE 
ADDITION 
WATER 
CALCULATED SODIUM 
CHLORIDE 
1 
0.0 
28 c.c. 
X 
mg. 
per 
100 cc. 
2 
1.0 mg. 
. = 1 c.c. Sj 
27 e.c. 
X + 25 
( C 
( c 
C ( ( C 
3 
3-0 “ 
= 3.0 c.c S. 
25 c.c. 
X + 75 
( c 
( ( 
a a 
4 
5.0 “ 
= 5.0 c.c. S, 
23 e.c. 
X +125 
c c 
a 
( ( ( c 
5 
7.0 “ 
= 7.0 c.c. Sj 
21 e.c. 
X +175 
( c 
c ( 
c ( a 
Where S, 
= a solution containing 1 gram 
of sodium chloride 
per 
liter. 
Preparation of Volumetric Solutions 
A normal solution of an acid contains in a liter 1.008 grams of 
replaceable hydrogen, of an alkali 17.008 grams of hydroxyl. 
The amount of an acid required to make one liter of a normal 
solution is the molecular weight of the acid in grams divided 
by the number of replaceable hydrogen atoms which it contains. 
Similarly the molecular weight of a base divided by the number 
of hydroxyls which it contains is the amount necessary to make 
one liter of a normal solution. 
Equal volumes of a normal solution exactly neutralize each 
other. If one known normal solution is available, others can be 
made indirectly by titration against the known solution. The 
normal solution which can be most easily prepared accurately is 
that containing acid potassium phthalate. This salt is a crystal- 
line substance of high molecular weight and is easily procured 
in pure form. The alkalis can then be made by titration against 
the phthalate solution, and the other acids by titration against 
an alkali. 
The preparation of normal hydrochloric acid by the distillation 
is also a simple and accurate procedure. This method may be 
employed if acid potassium phthalate is not available. 
N/10 and N/100 solutions are made either by dilution of a nor- 
mal solution or by direct titration. 
1. N/10 Sulphuric Acid.—Take of Merck’s anhydrous reagent 
sodium carbonate about 7 or 8 grams and ignite gently in a pre- 
viously weighed platinum crucible, not allowing the heating to 
exceed a dull red in order to avoid the conversion of small 
amounts of carbonate into hydroxide, which may take place at 
high temperatures. The object of the heating is to dehydrate the 254 
CLINICAL LABORATORY METHODS 
, , Fit 66-—B«rette arrangement The reagent bottles are connected in series with the compressed air outlet The 
burettes have three way stopcocks (Fig. 52) and fill automatically. Such an arrangement saves much time in makfmr nrm 
tein-free blood filtrates and in titrations and quantitative chemical procedures. CHEMICAL PROCEDURES AND SOLUTIONS 
255 
salt completely and to decompose any bicarbonate which may be 
present. 
Allow to cool in the desiccator, and on the balance quickly 
remove enough to leave exactly 5.3 grams in the crucible. 
Dissolve this in hot distilled water, rinsing the crucible well. 
Allow the fluid to cool and then make up to exactly 1 liter. 
Take 6.2 c.c. of chemically pure II2S04 (sp.gr. 1.84), dilute 
with four or five volumes of distilled water and allow to cool. 
Transfer to a 2 liter cylinder and add distilled water to the 
mark. Shake well and fill a 50 c.c. burette with the acid and 
another with the sodium carbonate solution, in each case rinsing 
out the burette first with some of the solution. 
Measure 50 c.c. of the carbonate into a beaker, add a few drops 
of methyl orange, and titrate with the acid until a pink tinge 
is noticeable and the addition of a drop of alkali restores the 
neutral color. Repeat until duplicates are obtained differing by 
not more than 0.1 c.c. 
The acid will be found too strong and the amount of water for 
dilution is poured into the cylinder. The amount of water is 
calculated as follows: for example, 
49.5 c.c. of acid neutralizes 50 c.c. of the alkali; then 
c=™ 
n 
1900 x .5 
° = 49.5 
C = 19.2 
C = Number of cubic centimeters of water to be added. 
N = c.c. of solution remaining. 
d == Difference between number of cubic centimeters theoret- 
ically required and number of cubic centimeters actually used in 
titration. 
n = Number of cubic centimeters used in titration. Repeat 
the titration and correction until the two solutions are adjusted 
so as to balance evenly. If the acid is too weak it is simple to 
make it a little too strong again by adding a drop or two of con- 
centrated acid and then diluting to the required degree. 
2. N/l Sulphuric Acid.—A normal solution of sulphuric acid 256 
CLINICAL LABORATORY METHODS 
may be made by the same procedure as that given for the N/10 
acid. Dissolve the 5.3 grams of sodium carbonate in 100 c.c. of 
distilled water. Take 62.0 c.c. of chemically pure H2S04 (sp.gr. 
1.84), dilute with four or five volumes of water, cool and dilute 
to two liters. 
Titrate and correct as indicated above. 
3. Normal and N/10 Hydrochloric Acid.—(Hulett and Bonner, 
Jour. Amer. Chem. Soc., 1909, xxxi, 390.) This extremely accu- 
rate method depends on the fact that when hydrochloric acid 
solution is distilled at 760 mm. pressure the concentration of 
HC1 in the undistilled portion approaches 20.24 per cent. When 
this is reached further distillation yields a distillate also con- 
taining HC1 of this concentration. To prepare stock HC1 solution 
for standards, add to concentrated IIC1 (sp. gr. 1.2) an equal vol- 
ume. of water and bring to a density at 25 degrees of 1.096 by 
addition of more water or concentrated HC1. Distill off three- 
quarters of this mixture. The remaining one-quarter has within 
1 part in 10,000 the following composition: 
Barometric 
Per cent 
Grams of solution 
Grams of solution 
pressure at 
HC1 
to make 1 liter of 
to make 1 liter of 
distillation 
N/10 HC1 
N/l HC1 
770 
20.218 
18.04 
180.4 
760 
20.242 
18.02 
180.2 
750 
20.266 
18.00 
180.0 
740 
20.290 
17.97 
179.7 
730 
20.314 
17.95 
179.5 
4. Normal and N/10 Sodium Hydroxide.—Dissolve 100 grams 
of c.p. sodium hydroxide in 100 e.c. of water and let the solution 
stand. Sodium carbonate is insoluble in such a concentrated 
NaOH solution, and whatever carbonate is present settles to the 
bottom as sediment. For each liter of N sodium hydroxide re- 
move with a graduated pipet 57 c.c. of the clear solution and 
dilute to 1,000 c.c. Standardize against normal acid as described 
on the preceding page; for each liter of N/10 sodium hydroxide 
dilute 5.7 c.c. of the clear solution to 1,000 c.c. and standardize 
against N/10 acid. 
5. N/10 Iodine.—Weigh out 12.685 grams of pure resublimed 
iodine into a small weighing bottle using a porcelain spatula. 
Dissolve 18 grams of pure KI in about 150 c.c. of water. Transfer 257 
CHEMICAL PROCEDURES AND SOLUTIONS 
the iodine to a liter flask washing out the last traces with some 
of the KI solution, which is then poured into the flask. Stopper 
and shake occasionally until dissolved. If necessary a few more 
crystals of KI may be added to aid solution. Dilute to the mark 
and mix well. Keep in glass stoppered bottle in cool dark place. 
Standardize at once against N/10 sodium thiosulphate solution. 
Measure out accurately 25 c.c. of the iodine solution into an Erlen- 
meyer flask, run in sodium thiosulphate until the color is pale 
yellow, then add a few cubic centimeters of a 1 per cent solution 
of starch (preferably soluble starch) and titrate to disappearance 
of blue color. Care should be taken near the end point. 
6. N/10 Potassium Permanganate.—Dissolve 3.162 grams of 
pure potassium permanganate in a liter of distilled water, allow 
to stand a few days, and filter through glass wool. Standardize 
against N/10 oxalic acid solution or against pure dry sodium or 
potassium oxalate. One c.c. of N/10 permanganate is equivalent 
to 7.0 mg. of sodium oxalate. 
7. N/10 Sodium Thiosulphate Solution.—Weigh out 25 grains 
of ordinary c.p. sodium thiosulphate or 24.83 grams of the pure 
dry recrystallized salt. Dissolve in water and dilute to a liter. 
Boiled distilled water must be used. Keep in a bottle with a 
siphon arrangement and carrying a soda lime tube to exclude C02. 
It is best standardized against acid potassium iodate KH (103)2. 
Weigh out accurately 0.3249 gram of acid potassium iodate. 
Dissolve in 50 c.c. of water, heating gently if necessary. Trans- 
fer the solution to a 100 c.c. flask, rinsing the beaker carefully 
and make to mark with water. This solution is exactly decinor- 
mal. Pipet out 25 c.c. into an Erlenmeyer flask, add 1 gram of 
potassium iodide dissolved in a little water, and a few cubic cen- 
timeters of dilute hydrochloric acid. Titrate immediately with 
the thiosulphate solution. When the solution becomes a pale 
yellow add a few cubic centimeters of one per cent solution of 
soluble starch and titrate to loss of blue color. 
8. Normal Acid Potassium Phthalate.—This acid salt may be 
conveniently used as a starting point for the preparation of 
standard acids and alkalis. It can be obtained chemically pure, 
has no water of crystallization, has a high molecular weight, and 
is freely soluble in water. 258 
CLINICAL LABORATORY METHODS 
The salt is dried at 110° to 115° C. to constant weight. A nor- 
mal solution contains 204.14 grams in a liter. 
Calibration of Volumetric Apparatus 
(Medical War Manual No. 6) 
All apparatus used for accurate work must be calibrated. Ex- 
cept for that checked by the Bureau of Standards no commercial 
apparatus is entirely reliable, errors exceeding 1 per cent being 
frequent. 
Flasks are calibrated by weighing into them the amount of 
water necessary to make the desired volume of the temperature 
of calibration. Table LVII shows the weights of water over the 
range of ordinary room temperature which fill a volume of 1 c.c. 
The figures are corrected for the weights of air displaced by the 
water and by the brass weights. The water should be weighed 
to 1 part per 1000, i.e., the water held by a 10 c.c. flask is weighed 
to 0.010 gm., but a liter flask is sufficiently accurate if within 1 gm. 
Table LVII 
TEMPERATURE 
C° 
WEIGHT OE 1 C.C. OF 
WATER IN GM. 
VOLUME OF 1 GM. OF 
WATER IN C.C. 
35 
0.0081 
1.0019 
1G 
0.0070 
1.0021 
17 
0.0977 
1.0023 
18 
0.0076 
1.0024 
10 
0.0074 
1.0026 
20 
0.0072 
1.0028 
21 
0.0070 
1.0030 
22 
0.0067 
1.0033 
28 
0.0065 
1.0035 
24 
0.0963 
1.0037 
25 
0.9960 
1.0040 
2G 
0.9958 
1.0042 
27 
0.9955 
1.0045 
28 
0.9952 
1.0048 
20 
0.9949 
1.0051 
Burettes are calibrated by allowing them to deliver distilled 
wrater, 2 c.c. at a time, into a bottle and weighing the water. The 
bottle should contain a layer of paraffin oil a few millimeters 
thick. This floats on top of the water and prevents loss by evap- 
oration. It is not necessary, therefore, to stopper the bottle. The CHEMICAL PROCEDURES AND SOLUTIONS 
259 
grams of water noted are multiplied by the volume of 1 gram at 
the temperature observed. If the results do not agree to within 
0.05 c.c. (for a 25 to 50 c.c. burette) with the readings, the cor- 
rections should be plotted on a sheet of coordinate paper, which 
is hung by the burette for reference. 
The accompanying figures (Table LYTII) for the first 10 c.c. 
of a burette serve as an example. 
Table LV1TI 
BURETTE 
READING 
C.C. 
WEIGHT Ob' WATER 
DELIVERED AT 22 °C. 
GM. 
VOLUME OP WATER 
DELIVERED 
(—\vt. x 1.0033) 
C.C. 
CORRECTION 
TO BURETTE 
C.C. 
2 
2.000 
2.006 
plus 0.01 
4 
4.002 
4.008 
l C 
0.01 
6 
6.009 
6.017 
( c 
0.02 
8 
8-020 
8.050 
(( 
0.05 
10 
10.020 
10.050 
(( 
0.05 
The plus sign indicates that each correction is to he added to 
the observed reading in order to give the actual volume of liquid 
delivered. Were the volumes of water delivered at any points 
less than indicated by the burette readings the corresponding 
corrections would be indicated by minus signs. 
Pipettes are calibrated by filling to the mark with distilled water 
and discharging into a weighing bottle. The water delivered 
should be weighed to within 1 part per 1000. If the mark is not 
accurate a correct one should be made with a wax pencil, subse- 
quently etched in and indicated by an arrow. 
Pipettes may be calibrated for either drainage or blow-out deliv- 
ery. For drainage the tip of the pipette is allowed to touch the side 
of the receiving vessel as delivery is finished and a drop of liquid 
remains in the tip. For blow-out delivery this final drop is ex- 
pelled. The expulsion is conveniently effected by closing the 
upper end of the pipette with the right forefinger and warming 
the bulb by gripping it with the left palm. The expansion of 
air in the bulb forces the last drop of water out of the tip. For 
all pipettes below 5 c.c. blow-out delivery should be used. Unless 
all of the pipettes in the laboratory are calibrated for either blow- 
out or drainage delivery, each pipette must be etched “Blow-out” 
or “Drainage.” 260 
CLINICAL LABORATORY METHODS 
Use of Indicators 
Indicators are chemical substances possessing an intense color 
which changes in the presence of acid and alkali. A small amount 
of the indicator will tinge a large quantity of fluid. The change 
in color of the indicator is due to the fact that its color is de- 
pendent upon the degree of its dissociation, which in turn is 
determined by the presence of free acid or free alkali. 
Different indicators change color at greater or less hydrogen- 
ion concentration. The indicator selected for any titration should 
C.C io NOWWU ftDT5FP TO <0«- lNK>RrAAl» ftClP 
Fig. 67.—Titration curves of hydrochloric acid and acetic acids, showing the gradual 
color change of methyl red and phenolphthalein within their Ph zones of transformation. 
Curves are approximate only. (After Clark.) 
be one which gives a sharp color reaction which is sensitive to the 
form of acidity to be determined. 
If we use Sorenson’s expression PH to indicate the logarithm of 
the reciprocal of the H-ion concentration, at the neutral point 
PH is 7; values of PH greater than 7 are on the alkaline side; 
values less than 7 are on the acid side of neutrality. 
A general idea of the principles applied in the use of indicators 
for titration may be gained by the comparison of the titration of 
hydrochloric acid with sodium hydroxide and of acetic acid with 
sodium hydroxide (Clark). In Fig. G7 the curves show the PH CHEMICAL PROCEDURES ANI) SOLUTIONS 
261 
(logarithm of the reciprocal of the hyclrogen-ion concentration) 
plotted as ordinates as equal concentrations of hydrochloric and 
acetic acids respectively are neutralized with successive quanti- 
ties of NaOlI (abscisses). 
At the right of these curves are indicated the conduct of methyl 
red and of phenolphthalein within the PH zones of their color 
changes. It will be seen that in the titration of hydrochloric 
acid either indicator would show a sudden color change with the 
slightest addition of alkali when practically complete neutral- 
ization is approached. On the other hand in the acetic acid solu- 
tion methyl red undergoes a slow color change in a PH zone within 
which acetic acid is only partly neutralized. Only by the use of 
an indicator changing color at a much lower hydrogen-ion con- 
centration (higher PH) will the end point of this titration be 
reached. A similar set of curves might be drawn to show similar 
relations in the titration of alkalis with acids. It will be noted 
in the cases cited that phenolphthalein is serviceable in both 
cases. 
It is simply the extension of such considerations which leads 
to the choice of phenolphthalein for the general titration of acids 
by bases. Similar considerations lead to the choice of methyl 
red in general titrations of alkalis with acids. In many cases, 
however, the choice must be determined by a close study of the 
acid or base and the special conditions employed. 
Only indicators which change color well on the acid side at 
PH = 5 or less, such as methyl red, can be used for titrating 
strong acids with weak bases, and for the titration of alkalis in 
the presence of carbonic acid. Likewise for the titration of 
strong bases with weak acids the indicator must change at PH = 8 
or more such as phenolphthalein. Almost any indicator can be 
used in the titration of strong bases such as NaOII against strong 
acids, as one drop of the solution will carry the hydrogen-ion 
concentration so far beyond neutrality that the turning point 
of any common indicator will be passed. 
Phenolphthalein is unsatisfactory in the presence of ammonium 
salts so cannot be used as an indicator in ammonia titration. In- 
dicators which change at PH = 3 to 4 can be used for the titra- 
tion of mineral acids in the presence of weak organic acids, as at 262 
CLINICAL LABORATORY METHODS 
INDICATOR 
COLOR 
PH AT WHICH 
FORM IN AVHICII 
INDICATOR IS 
EMPLOYED 
SPECIAL USE 
CHEMICAL NAME 
COMMON NAME 
ACID 
ALKALINE 
COLOR CHANCES 
Dimethyl-amino-azo- 
benzene 
Topfer’s reagent 
Eed 
Yellow 
3-4 
0.4 per cent in 
95 per cent al- 
cohol 
Titration of mineral 
acids in presence of 
organic acids (HC1 in 
stomach contents). 
Dimethyl-amino-azo- 
benzene-sulphonate 
Methyl orange 
Eed 
Yellow 
3-5 
0.1 per cent sol. 
in 50 per cent 
alcohol 
Titration of mineral 
acids in presence of 
carbonic acid. 
Diazo compound of ben 
zidine and naphthionic 
acid 
Congo red 
Blue 
Eed 
4-5 
0.5 per cent sol. 
in 95 per cent 
alcohol 
Titration of weak bases, 
(ammonia) with min- 
eral acid. 
Sodium-alizarin-sul- 
phonate 
Alizarin red 
Yellow 
Eed 
5-6 
1.0 per cent so- 
lution of Na 
salt in 50 per 
cent alcohol 
Titration of weak bases, 
(ammonia) with min- 
eral acid. 
Ortho carboxy-benzene 
azo-dimethyl-aniline 
Methyl red 
Eed 
Yellow 
4-6 
0.02 per cent sol. 
in 60 per cent 
alcohol 
Titration of bases with 
strong acid 
Litmus 
Eed 
Blue 
About 7 
Paper 
Phenolphthalein 
Color- 
less 
Eed 
8-9 
1.0 per cent so- 
lution in 95 
per cent alcohol 
Titration of organic 
acids with mineral al- 
kali. 
Table of Indicators1 
Table LIX 
‘Abbreviated from Medical War Manual, No. 6. CHEMICAL PROCEDURES AND SOLUTIONS 
263 
such an end point the weak organic acid exerts but little influence 
on the titration results. 
Table LIX summarizes the important points about the indi- 
cators commonly employed in chemical titrations and shows the 
use for which each is best adapted. A special set of indicators 
is used in the determination of the hydrogen-ion concentration 
of biological fluids (see page 239). 
Preparation of Dakin’s Solution 
1. From Liquid Chlorine and Sodium Carbonate—Dakin’s solu- 
tion is to be made routinely from liquid chlorine and sodium 
carbonate. 
Fig. 68.—Apparatus for making Dakin’s solution from liquid chlorine. (Courtesy of 
Wallace and Tiernan Co.) 
Procedure.—Prepare a solution containing 18.0 grams anhy- 
drous sodium carbonate (or 21.0 grams monohydrate sodium car- 
bonate) per liter in distilled water. Run in chlorine through 
diffusor (Pig. 68) until a sufficient quantity has been introduced 
as indicated by the gauge. The gauge indicates the number of 
cubic centimeters of Dakin’s solution made per minute. 
Titration.—Measure 10 c.c. of Dakin’s solution into beaker 264 
CLINICAL LABORATORY METHODS 
containing 50 c.c. of water. Add 5 c.c. of 10 per cent potassium 
iodide solution and 2 c.c. of glacial acetic acid. Then run tenth 
normal sodium thiosulphate solution in the flask until the decol- 
orization of the solution is complete. 
The number of cubic centimeters of tenth normal thiosulphate 
required to decolorize the solution multiplied by the factor 0.0372 
gives the percentage of sodium hypochlorite. The solution must 
titrate between 0.45 and 0.50 per cent sodium hypochloride. 
Table LX shows the percentage of hypochlorite corresponding 
to the amount of thiosulphate used. 
Table LX 
C.c. N/10 sodium 
thiosulphate used 
Per cent of sodium 
hypochlorite in solution 
12.1 
0.4507 
12.2 
0.4545 
12.3 
0.4582 
12.4 
0.4619 
12.5 
0.4656 
12.0 
0.4694 
12.7 
0.4731 
12.8 
0.4768 
12.9- 
0.4805 
13.0 
0.4843 
13.1 
0.4880 
13.2 . 
0.4917 
13.3 
13.4 
0.4992 
Tests for Alkalinity.—A. With powdered phenolphthalein. A 
few crystals of powdered phenolphthalein are dropped on the 
surface of about 5 c.c. of the solution, and the solution vigorously 
shaken. The solution must remain colorless. B. With alcoholic 
solution of phenolphthalein. About a half cubic centimeter of 
alcoholic solution of phenolphthalein is added to 5 c.c. of the 
Dakin’s solution. A red color should develop, but quickly dis- 
appear. 
If the solution does not titrate between 0.45 and 0.50 per cent 
sodium hypochlorite, or gives a reaction with powdered phenol- 
phthalein, or fails to give a flash of color with alcoholic solution 
of phenolphthalein, it should be discarded. Store in dark bottles 
in a cool place. 265 
CHEMICAL PROCEDURES AND SOLUTIONS 
Each time a portion is dispensed, it should be titrated, and 
the bottle labeled as follows: 
Dakin’s Solution 
per cent sodium hypochlorite on 
Titration 
2. From Chlorinated Lime, Sodium Bicarbonate and Sodium 
Carbonate—If liquid chlorine is not available, the Dakin’s solu- 
tion may be made by the following method. 
Titration of Chlorinated Lime.—Weigh out 20 grams of an 
average sample of chlorinated lime; mix as completely as possible 
with one liter of tap water, and leave in contact for 6 hours, 
shaking from time to time. 
Measure exactly 10 c.c. of the clear fluid; add 20 c.e. of a 10 
per cent solution of potassium iodide, and 2 c.c. of acetic acid. 
Add a decinormal solution of sodium thiosulphate until decolora- 
tion is complete. 
The number of cubic centimeters of thiosulphate solution re- 
quired for complete decoloration, multiplied by 1.775 gives the 
weight of the active chlorine contained in 100 grams of the chlor- 
inated lime. 
The figure being known, it is applied to the accompanying 
Table LXI 
TITER OF 
CHLORINATED 
LIME 
CHLORINATED 
LIME 
GM. 
ANHYDROUS 
SODIUM CARBONATE 
GM. 
SODIUM 
BICARBONATE 
GM. 
20 
230 
115 
96 
21 
220 
110 
92 
22 
210 
105 
88 
23 
200 
100 
84 
24 
192 
96 
80 
25 
184 
92 
76 
26 
177 
89 
72 
27 
170 
85 
70 
28 
164 
82 
68 
29 
159 
80 
66 
30 
154 
77 
64 
31 
148 
74 
62 
32 
144 
72 
60 
33 
140 
70 
59 
34 
135 
68 
57 
35 
132 
66 
55 
36 
128 
64 
53 
37 
124 
62 
52 266 
CLINICAL LABORATORY METHODS 
table, which will give the quantities of chlorinated lime, of 
sodium carbonate, and of sodium bicarbonate, which are to be 
employed to prepare 10 liters of the solution. 
To prepare 10 liters of the solution: 
1. Weigh exactly the quantities of chlorinated lime, sodium 
carbonate and sodium bicarbonate, which have been determined 
in the course of the preceding trial. 
2. Place in a 12 liter jar the chlorinated lime and 5 liters of 
ordinary tap water, agitate vigorously for a few minutes, and leave 
in contact for from 6 to 12 hours, overnight, for instance. 
3. At the same time dissolve, cold, in the 5 other liters of water 
the sodium carbonate and the bicarbonate. 
4. Pour all at once the solution of the sodium salts into the jar 
containing the maceration of chlorinated lime, agitate vigorously 
for a few moments, and leave it quiet to permit the calcium car- 
bonate to settle as it forms. At the end of the half hour, siphon 
the liquid and filter it through double paper to obtain an entirely 
limpid product which must be protected from light. 
Titrate and test for alkalinity as indicated above. 
Preparation of Nessler’s Solution (Folin Method) 
This reagent is essentially a solution of the double iodide of 
mercury and potassium (Hgl2, 2KI) containing sodium or potas- 
sium hydrate. A stock solution of the double iodide is best pre- 
pared as follows: 
Transfer 150 grams of potassium iodide and 110 grams of 
iodine to a 500 c.c. Florence flask; add 100 c.c. of water and an 
excess of metallic mercury, 140 to 150 grams. Shake the flask 
continuously and vigorously for seven to fifteen minutes, or until 
the dissolved iodine has nearly disappeared. The solution becomes 
quite hot. When the red iodine solution has begun to visibly 
pale, though still red, cool in running water and continue shak- 
ing the solution until the reddish color of the iodine has been 
replaced by the greenish color. Decant and wash the sediment. 
Dilute the solution and washings to a final volume of 2 liters. 
If the cooling was begun in time the resulting reagent is clear 
enough on immediate dilution with 10 per cent alkali and water. CHEMICAL PROCEDURES AND SOLUTIONS 
267 
Prepare the final Nessler solution as follows: 
From completely saturated caustic soda solution containing 
55 grams of NaOH per 100 c.c., decant the clear supernatant liq- 
uid, and dilute to a concentration of 10 per cent. Determine by 
titration that a 10 per cent solution has been obtained. 
Introduce into a large bottle 3,500 c.c. of 10 per cent sodium 
hydroxide solution, add 750 c.c. of the double iodide solution and 
750 c.c. of distilled water, giving 5 liters of Nessler’s solution. 
In the absence of modifying circumstances such as much acid 
or alkali, this reagent should be added in the proportion of 10 c.c. 
per 100 c.c. of volume to which the Nesslerized solution is to be 
diluted. As a general rule the volumetric flask should be at least 
two-thirds full before adding the Nessler reagent, otherwise, tur- 
bid mixtures may be obtained. 
(Benedict, S. R.: Jour. Biol. Chem., 1914, xviii, 182) 
Preparation of Creatinin 
To 10 liters of undecomposed urine in a large precipitating 
jar add with stirring a hot solution of 180 grams of picric acid 
in 450 c.c. of boiling alcohol. Allow to stand overnight and 
syphon off the supernatant fluid. Pour the residue upon a large 
Buchner funnel, drain with suction, wash once or twice with cold 
saturated picric acid and suck dry. Treat the dry or nearly dry 
picrate in a large mortar or evaporating dish with enough con- 
centrated HC1 to form a moderately thin paste (about 60 c.c. of 
acid for each 100 grams of picrate) and stir the mixture thor- 
oughly with the pestle for 3-5 minutes. Filter with suction on 
a hardened paper, and wash the residue twice with enough water 
to cover it, sucking as nearly dry as possible each time. Trans- 
fer the filtrate to a large flask and neutralize with an excess of 
solid magnesium oxide (the “heavy” variety is best). Add this 
oxide in small portions with cooling of the flask under running 
water between the additions. Neutralization of the acid will be 
indicated by a bright yellow color of the mixture, or litmus 
paper may be used to test it. Filter with suction. Wash the 
residue twice with water. Immediately add a few cubic centi- 
meters of glacial acetic acid to the filtrate to make it strongly 268 
CLINICAL LABORATORY METHODS 
acid. Pay no attention to any precipitate that may form, but 
dilute the solution with about 4 volumes of 95 per cent alcohol. 
After 15 minutes filter off the slight precipitate which forms. 
Treat the final filtrate with 30-40 c.c. of 30 per cent zinc chloride. 
Stir and let stand overnight in a cool place. Pour off the super- 
natant liquid and collect the creatinine zinc chloride on a Buchner 
funnel, wash once with water, then thoroughly with 50 per cent 
alcohol, finally with 95 per cent alcohol and dry. A nearly white, 
light crystalline powder should be obtained. The yield should be 
90-95 per cent of the original creatinine (usually about 1.5-1.8 
grams of creatinine zinc chloride per liter of urine). 
Recrystallize the creatinine-zinc chloride by treating 10 grams 
with 100 c.c. of water and about 60 c.c. of normal sulphuric acid, 
heating the mixture until a clear solution is obtained. Add about 
4 grams of purified animal charcoal, continue boiling for about 
a minute, filter with suction through a small Buchner funnel, 
pouring the filtrate back on the filter three or four times until it 
runs through perfectly colorless. Wash residue with hot water 
and transfer the total filtrate to a beaker and while hot treat 
with a little strong zinc chloride solution (3 c.c.) and with about 
7 grams of potassium acetate dissolved in a little water. After 
ten minutes dilute with an equal volume of alcohol, and allow 
to stand in a cold place for some hours. Filter off the crystalline 
product. To remove the small amount of potassium sulphate 
which it contains stir up with twice its weight of water, filter, 
wash with a little water and then with alcohol. The preparation 
should be snow white. Yield, 85-90 per cent. 
Place the finely powdered recrystallized creatinine zinc chlo- 
ride in a dry flask and treat with seven times its weight (by vol- 
ume) of concentrated aqueous ammonia. Warm slightly and agi- 
tate gently until a clear solution is obtained, care being taken to 
drive off no more ammonia during the warming than is necessary 
to obtain a clear solution. Stopper the flask, allow to cool, place 
in the ice box for an hour or more. Pure creatinine crystallizes 
out. It may be recrystallized from boiling alcohol or concen- 
trated ammonia, but this is usually unnecessary. The product is 
perfectly pure and can be used as a standard in the quantitative 
determination of creatine and creatinine. CHEMICAL PROCEDURES AND SOLUTIONS 
269 
Method for Purification of Picric Acid 
(Folin and Doisy: Jour. Biol. Chem., 1916-17, xxviii, 349) 
Transfer about 600 gm. of wet picric acid, or about a pound of 
dry picric acid, to a large beaker (capacity not less than 4 liters). 
Pour on boiling water until the beaker is nearly full and add 
200 c.c. of saturated (50 per cent) sodium hydroxide solution. 
Stir, and if necessary heat again until all the picric acid has dis- 
solved, yielding a deep red picrate solution. To the hot solution 
add rather slowly, with stirring, 200 gm. of sodium chloride. 
Cool in running water to about 30° C., with occasional stirring. 
Filter on a large Buchner funnel and wash a few times with 5 
per cent sodium chloride solution. Transfer the picrate to the 
large beaker, fill with boiling water, and when the picrate is 
dissolved add, with stirring, first 50 c.c. of 10 per cent sodium 
hydroxide solution, and then 100 gm. of sodium chloride. Cool 
to 30° C., with stirring, filter, and wash with sodium chloride 
solution, as before. Repeat the solution and precipitation of the 
sodium picrate twice more, but for the last washing of the last 
precipitated picrate use distilled water instead of sodium chlo- 
ride solution. 
Dissolve the purified picrate in the same large beaker, with 
boiling distilled water, and filter hot on a large folded filter, 
collecting the filtrate in a large flask. To the hot filtrate add 
100 c.c. of concentrated sulphuric acid, previously diluted with 
about two volumes of water. The liberated picric acid begins 
to come out at once. Put a beaker over the mouth of the flask 
and cool under running tap water to about 30° C. Filter with 
suction as before and wash free from sulphates with distilled 
water. 
Preparation of Solutions for Intravenous Use 
1. Physiological Salt Solution.—Dissolve 25.5 grams of chem- 
ically pure sodium chloride in three liters of distilled water. 
Filter through paper. 
Scrub thoroughly a dozen 12 ounce bottles. Rinse with tap 
water and then with distilled water. Measure 275 c.c. of the 
salt solution into each bottle. Plug the mouth with cotton and 
gauze. Autoclave for 15 minutes at 20 pounds pressure. 270 
CLINICAL LABORATORY METHODS 
Label as follows: 
Sterile Physiological (0.85%) Salt Solution for Intravenous Use. 
Prepared By 
2. Glucose Solution.— 
Weigh 750 grams of pure glucose and dissolve with the aid of 
heat in about 1500 c.c. of distilled water. Make up to three 
liters with distilled water. Filter through cotton to remove major 
part of flocculent material, then filter through paper. Thoroughly 
scrub bottles of 500 c.c. capacity. Rinse well with tap water and 
then with distilled water. Measure 275 c.c. of the 25 per cent 
glucose solution into each bottle. Plug the mouth with cotton 
and gauze. Autoclave for fifteen minutes at twenty pounds 
pressure. 
Label as follows: 
Sterile 25% Glucose Solution for Intravenous Use. 
Prepared By 
3. Sodium Bicarbonate.—Dissolve 75 grams of chemically pure 
anhydrous sodium carbonate in three liters of freshly distilled 
water. Filter through paper. Add several drops of phenol- 
phthalein solution. 
Place in the flask a Folin absorption tube connected by rubber 
tubing to a calcium chloride drying tube filled with absorbent 
cotton. Stopper the flask with cotton. Place the flask with the 
entire apparatus in the autoclave and heat under 20 pounds 
pressure for 15 minutes. After cooling connect the drying tube 
with the carbon dioxide tank and bubble gas through until the 
solution becomes colorless. This converts the 2.5 per cent sodium 
carbonate solution into a 4 per cent sodium bicarbonate solution. 
Distribute this sterile bicarbonate solution into sterile 500 
c.c. bottles, putting 275 c.c. in each bottle. Replace the sterile 
stoppers and store in cool place. 
Label as follows: 
Sterile 4% Sodium Bicarbonate Solution for Intravenous Use. 
Prepared By CHEMICAL PROCEDURES AND SOLUTIONS 
271 
Preparation of Sodium Citrate Solution for Blood Transfusion 
Dissolve 25 grams of chemically pure crystalline sodium citrate 
in one liter of freshly distilled water. This makes a 2.5 per cent 
solution. Filter through paper if not perfectly clear. 
Scrub thoroughly ten 100 c.c. bottles. Rinse with tap water 
followed by distilled water. Fill with the citrate solution. Stop- 
per with cotton, and autoclave for 15 minutes at 20 pounds 
pressure. 
Label as follows: 
Sterile 2.5% Sodium Citrate for Blood Transfusion. 
Prepared By 
Preparation of Antiformin 
1. From Chlorinated Lime.— 
Sodium carbonate 600.0 grams 
Chlorinated lime 400.0 grams 
Distilled water 4,000.0 c.c. 
Dissolve the sodium carbonate in 1,000 c.c. of distilled water. 
Triturate thoroughly the chlorinated lime in the remainder of 
the water. Filter. Mix the two and filter again. 
To the solution so prepared add an equal volume of 15 per cent 
sodium hydroxide. 
2. From Liquid Chlorine.—If liquid chlorine is available 
the antiformin solution may be made by running the chlorine 
gas into a solution containing 75 grams of sodium carbonate and 
75 grams of sodium hydroxide per liter until the solution titrates 
about 2 per cent sodium hypochlorite. 
Methyl Violet Shellac (for Marking Glassware) 
Best white shellac 10 gr. 
Alcohol, 95% 20-25 c.c. 
Methyl-violet 0.1 gr. 
Apply with a small brush or a tooth pick. 272 
CLINICAL LABORATORY METHODS 
Bichromate Cleaner for Glassware 
Potassium bichromate (powdered) 200 gr. 
Water distilled up to 1500 c.c. 
Sulphuric acid concentrated 500 c.c. 
Stopcock Grease (Boothby) 
Wash ordinary black rubber tubing in water to remove the 
talc. Cut in small pieces and add slowly to an equal quantity 
of lanolin which has been melted. 
The rubber is allowed to melt until the resulting mixture is 
free from lumps. 
Foam Killer 
Twenty per cent solution of rosin in turpentine, CHAPTER XIII 
HISTOLOGICAL TECHNIC 
Preparation of Permanent Sections for Histological Examination 
Reagents.— 
1. Delafield’s Hematoxylin.— 
Hematoxylin crystals 4 grams 
Alcohol—95% 25 c.c. 
Saturated (11%) aqueous solution of 
ammonia alum 400 c.c. 
Add the hematoxylin dissolved in the alcohol to the alum solu- 
tion, and expose the mixture in an unstoppered bottle to the 
light and air for two to three weeks, filter and add: 
Glycerine 100 c.c. 
Alcohol—95% 100 c.c. 
Allow the solution to stand in the light until the color is suffi- 
ciently dark, then filter and keep in a tightly stoppered bottle. 
So long as it is good it has a purplish tinge. 
2. Formaldehyde Fixing Solution.— 
Formalin (commercial) 100 c.c. 
Water to 1000 c.c. 
3. Zenker’s Fixing Solution.— 
Potassium bichromate 2.5 grams 
Mercury bichloride 5.0 grams 
Water 100 c.c. 
Five c.c. of glacial acetic acid is added to 100 c.c. of this stock 
solution just before use. 
4. Ammonia Water.—Add 5 c.c. concentrated ammonia to 95 
c.c. distilled water. 
5. Acid Alcohol.—Add 1 c.c. concentrated hydrochloric acid 
to 100 c.c. 95 per cent alcohol. 
6. Eosin.—Use saturated solution in 95 per cent alcohol. 
273 274 
CLINICAL LABORATORY METHODS 
7. Carbol—Xylol.— 
Carbolic acid crystals (melted) 1 part 
Xylol 3 parts 
8. Mayer’s Glycerine Albumen.—Mix equal parts white of egg 
and glycerine. Beat the mixture thoroughly, filter and add one 
per cent sodium salicylate as preservative. 
Procedure.—(1) Fixation.—Specimens will be sent directly to 
the laboratory from the operating room. Enter at once in the 
Surgical Pathological Record Book, the patient’s name, case 
number, name of the operator, the operation, and the nature of 
the specimen received. 
If small the specimen is placed in 10 per cent formalin. Sec- 
tions of characteristic portions of larger specimens are cut and 
placed in 10 per cent formalin. If the entire specimen is to 
be saved, it is to be placed in Kaiserling’s solution. 
When the specimen consists of uterus and adnexa, cut at 
least one block from uterus showing endometrium, one from 
each tube and one from each ovary. Two sections are to be cut 
from an appendix, one near the middle, and one from the distal 
end. One section is taken through the middle of each tonsil, 
cutting at right angles to the mucous membrane, and from upper 
to lower pole. 
The specimen is allowed to remain in the formalin solution 
overnight. 
(2) Embedding and Cutting.—After fixation, tissue blocks, not 
over 3 millimeters in thickness, are cut and embedded as follows: 
1. Acetone I 2 hours 
2. Acetone II 2 hours 
3. Acetone III Overnight 
4. Chloroform I 1 hour 
5. Chloroform II 1 hour 
6. Saturated solution of paraffin 1 hour 
in chloroform at room tem- 
perature 
7. Paraffin I in oven at 60° C. 1 hour 
8. Paraffin II in oven at 60° C. 1 hour 
Fill a small paper box with freshly melted paraffin and arrange 
the piece of tissue in the box with a hot needle. Place the sur- 
face of the block from which the bottom is to be cut, on the 
bottom of the box. HISTOLOGICAL TECHNIC 
275 
Cool as quickly as possible. 
Remove paper and turn block of tissue and fasten to a small 
wood block. Number block with pencil. 
Cut sections in microtome about ten microns thick and drop 
on surface of a pan of water at about 48° C. 
Rub a small amount of Meyer’s glycerine albumen on slides 
with the finger and push flat sections to two clean slides. Stand 
on end to drain, and then place in incubator overnight. 
(3) Staining.—Sections are stained as follows: 
1. Xylol I 2 minutes 
2. Xylol II 2 minutes 
3. Absolute alcohol I 2 minutes 
'4. Absolute alcohol II 2 minutes 
5. Alcohol 95% 2 minutes 
6. Alcohol 70% 2 minutes 
7. Hematoxylin (Delafield’s) 5 minutes 
8. Distilled water 2 minutes 
9. Acid alcohol until brick red 
10. Ammonia water until blue 
11. Alcohol 70% 2 minutes 
12. Sat. alcoholic solution of eosin 2 minutes 
13. Alcohol 95% I 2 minutes 
14. Alcohol 95% II 2 minutes 
15. Carbol-Xylol 2 minutes 
16. Carbol-Xylol 2 minutes 
17. Xylol 2 minutes 
18. Mount in Canada balsam 
Frozen Sections 
Temporary sections can be quickly prepared for examination 
by the following technic (Wilson: Jour. Lab. and Clin. Med., 
1915, i, 41.) 
Reagents.—1. Unna’s Methylene Blue prepared by Terry’s 
method as follows: 
Place in a petri dish 0.5 gram of methylene blue, (Bausch and 
Lomb), 0.5 gram of potassium carbonate (Merck), and dissolve in 
50 c.c. of distilled water. Place in incubator at 37° C. 
Each day the water lost by evaporation is made up by the addi- 
tion of distilled water. 
Leave in incubator six days. The stain should ripen in this time. 
Test on section of uterus. All the nuclei should be sharply 
stained and the smooth muscle should stain a sharp and beautiful 
purple. 276 
CLINICAL LABORATORY METHODS 
2. Brun’s Glucose Medium.— 
Glucose 40 gr. 
Glycerine 10 c.c. 
Camphor 10 gr. 
Water 140 c.c. 
Mix and filter. 
3. Dextrin Solution.—Dissolve dextrin in distilled water until 
the solution is the consistency of thin molasses. Add 0.5 per 
cent phenol. 
Procedure.—1. Freeze bits of fresh tissue, not more than 2x10 
mm. in the dextrin solution and cut sections 5 to 15 microns thick. 
2. Remove sections from knife with tip of finger and allow 
them to thaw thereon. 
3. Unroll sections with camel’s hair brush or glass lifter in 
1 per cent sodium chloride solution. 
4. Stain 10 to 20 seconds in Unna’s polychrome methylene blue. 
5. Wash momentarily in 1 per cent sodium chloride solution. 
6. Mount in Brun’s glucose solution. 
Permanent frozen sections may be made by the following 
technic: 
Slices of the tissue (not over 2 or 3 mm. thick and 1 cm. 
square) are cut with a sharp scalpel and dropped into 10 per cent 
formalin heated to about 40° C. After about ten minutes the 
preparation is removed from the formalin and frozen, and the 
sections are placed in water and then on the slide. The water is 
drained off with blotting paper and the sections are covered 
with absolute alcohol and brought into close contact with the 
slide by careful pressure with piece of “Royal” blotting paper. 
A few drops of a very thin solution of celloidin dissolved in equal 
volumes of alcohol and ether are then flowed over the section. 
The excess of celloidin is drained off and the celloidin allowed to 
set. This takes only a few seconds, and under no circumstances 
should the preparation be allowed to dry. As soon as the 
celloidin is set the slide is gently dipped in water to wash away 
the alcohol and ether. It is then placed in a solution of Dela- 
field’s hematoxylin diluted 1 to 10 with distilled water. In a 
few moments it is sufficiently stained, and is then rinsed in a 
jar of tap water until all excess of dye is washed away. The HISTOLOGICAL TECHNIC 
277 
slide carrying the section is then placed in a closed jar contain- 
ing saturated alcoholic solution of eosin. A strength of 1 to 
1000 is sufficient. After a few seconds it is rinsed in alcohol and 
transferred to 95 per cent alcohol. After remaining about five 
minutes in this the slide is transferred to a fresh bath of 95 
per cent alcohol until all the water is removed. It is then cleared 
with carbolxylol. Care must be taken to wash out all the car- 
bolxylol by treatment with several baths of xylol, as phenol de- 
colorizes the specimen. When completely dehydrated and cleared 
the section is covered with balsam, and cover pressed down 
upon it. 
Mallory’s Stain for Connective Tissue Fibers 
1. Fix in Zenker’s fluid. 
2. Embed in celloidin or paraffin. 
3. Stain sections in a 0.5 per cent solution of acid fuchsin five 
minutes or longer, depending on the freshness of the tissue. 
4. Transfer directly to the following solution and stain from 
fifteen to twenty minutes or longer: 
5. 
Anilin blue soluble in water 0.5 
Orange G 2.0 
One per cent aqueous solution of phos- 
phomolybdic acid 100.0 
6. Wash and dehydrate in several changes of 95 per cent 
alcohol. 
7. Clear in xylol. 
8. Mount in balsam. 
The fibrils and reticulum of connective tissue, amyloid, mucus, 
and certain other hyaline substances stain blue; nuclei, cyto- 
plasm, fibroglia fibrils, axis cylinders, neuroglia fibers, and fibrin 
red; red corpuscles and myelin sheaths yellow; elastic fibers pale 
pink and yellow. The various structures do not stain with equal 
intensity, so that certain ones are brought out with great sharp- 
ness. This is particularly true of the connective tissue, and of 
fibrin and smooth and striated muscle fibers. 278 
CLINICAL LABORATORY METHODS 
Technic for Staining Mitochondria (Bensley) 
1. Fixation.—The fixing solution is freshly made just before 
use as follows: 2.5 per cent potassium bichromate, 8 c.c.; 4 
per cent osmic acid, 2 c.c.; glacial acetic acid, one drop. 
The tissue must be perfectly fresh and should be cut into pieces 
not larger than 1 cm. cube and placed in the fixing fluid 24 hours. 
Wash in running water for 24 hours. 
2. Dehydration and Embedding.—Fifty per cent alcohol 12 
hours; 70 per cent and 95 per cent alcohol 24 hours each; absolute 
alcohol 6 to 12 hours; half absolute and xylol 6 hours; xylol I, 
3 hours; xylol II, 3 hours; paraffin 60° C. 3 hours; cut sections 
not more than 4 microns thick. 
3. Staining.—(1) Pass the sections, mounted on slides, down 
through toluol, absolute, 95, 70 and 50 per cent alcohol to dis- 
tilled water. 
(2) One per cent aqueous solution of potassium permanganate 
1 minute. 
(3) Five per cent aqueous solution of oxalic acid also 1 minute. 
(4) Kinse in several changes of distilled water about a minute. 
Incomplete washing prevents the staining with fuchsin. 
(5) Stain in Altman’s anilin fuchsin, which is made up as 
follows: Make a saturated solution of anilin oil in distilled water by 
shaking the two together. Filter and add 30 grams of acid 
fuchsin to 100 c.c. of the filtrate. The stain should be ready to 
use in about 24 hours. It deteriorates in about a month. Stain for 
5 minutes in the acid fuchsin solution which has previously been 
warmed to 60° C. 
(6) Dry off most of the stain with a towel and rinse in dis- 
tilled water so that the only stain remaining is in the sections. 
If a large amount of the free stain remains it will form a 
troublesome precipitate with the methyl green; on the other 
hand if too much of the stain is removed the coloration of the 
mitochondria will be impaired. 
(7) Allow a little 1 per cent methyl green, added with a 
pipette, to flow over the sections, holding the slide over a piece 
of white paper so that the colors may be seen. Apply the methyl 
green for about 5 seconds at first and then modify the time to 
suit the needs of the tissue. HISTOLOGICAL TECHNIC 
279 
(8) Drain off the excess of stain and plunge the slide into 95 
per cent alcohol for a second or two, then rinse in absolute for 
the same length of time, clear in toluol, and mount in balsam. 
The mitochondria stain a bright crimson. 
Stain for Tubercle Bacilli in Tissues 
1. Delafield’s hematoxylin, 20 to 30 minutes; 
2. Water, 30 minutes; 
3. Carbol fuchsin, 1 hour at 37° C. (incubator) ; 
(Place slide in petri dish and flood it; then cover to 
prevent evaporation.) 
4. Decolorize in acid alcohol, 1 minute; 
Hydrochloric acid 1 c.c. 
95% alcohol 70 c.c. 
Water 30 c.c. 
5. Wash in 70 per cent alcohol, 2 to 3 minutes; 
6. Wash in water; 
7. Lithium carbonate solution until section takes blue color; 
Sat. sol. lithium carbonate 1 part 
Water 10 parts 
8. Wash in water, 5 minutes; 
9. 95% alcohol, 5 minutes; 
10. Absolute alcohol, 5 minutes; 
11. Xylol, 5 minutes; 
12. Mount in gum dammar. 
To obtain the best results the material should be fixed in 95 
per cent alcohol, though formalin preparations occasionally stain 
well. After fixation in Zenker’s solution, the bacteria do not 
stain satisfactorily. 
Gram-Weigert Method for Demonstration of Gram-Positive 
Bacteria in Tissue 
Carry section into water after removing paraffin as for eosin- 
hematoxylin stain. 
1. Stain twenty minutes to one hour in lithium carmine. 
2. Acid alcohol (do not wash in water) until as seen under the microscope 
the nuclei alone are sharply stained. 
3. Water. 
4. Sterling’s gentian violet five to ten minutes. 
5. Wash quickly in water. 
6. Gram iodin solution two to five minutes. Blot dry on slide. 
7. (Do not wash). 
8. Anilin oil and xylol (equal parts) until clouds of blue no longer wash 
out of the section. 
9. Xylol two changes. 
10. Balsam and cover-slip. 280 
CLINICAL LABORATORY METHODS 
Lithium carmine is made as follows: 
Carmine 2.5 to 5 grams 
Saturated aqueous solution of carbonate 
of lithium 100 c.c. 
Thymol a crystal 
Dissolve the carmine in a small quantity of 95 per cent alcohol, 
bring the lithium carbonate solution to a boil, mix and filter. 
Kaiserling’s Method of Preserving Gross Specimens 
1. Fixation for one to five days in: 
Formaldehyde 200 c.c. 
Water 1000 c.e. 
Nitrate of potassium 15 grams 
Acetate of potassium 30 grams 
Change the position of the specimen frequently, using rubber 
gloves to protect the hands from the injurious effect of the for- 
maldehyde. The time of fixation varies with the tissue or organ 
and size of the specimen. 
2. Drain and place in 80 per cent alcohol one to six hours, and 
then in 95 per cent alcohol for one to two hours, to restore the 
color, which is somewhat affected in the fixing solution. 
3. Preserve in: 
Acetate of potassium 200 grams 
Glycerine 400 c.c. 
Water 2000 c.c. 
Exposure to light gradually affects the colors. The process of 
fixation should be performed in the dark, and the specimens when 
preserved should be kept in the dark except when on exhibition. 
If it seems desirable to cut a thin slice from the face of a 
specimen, this should not be done until the preparation has been 
in the preservative fluid two weeks. The specimen may then 
be placed in alcohol for one to two hours to brighten up the 
colors. 
It is advisable to add camphor, thymol, carbolic acid (one 281 
HISTOLOGICAL TECHNIC 
per cent), or some other preservative to the third solution to 
prevent the growth of molds. 
Mixture for Sealing Museum Jars 
Warm 200 grams of asphalt (Merck) and dissolve the melted 
asphalt in 200 e.c. of linseed oil and 400 c.e. turpentine. 
Warm, and with a small brush apply the cement to the ground 
surface of the jar. CHAPTER XIV 
EXAMINATION OF MILK AND WATER 
Bacteriological Examination of Water 
Collect the sample of water in sterile container, with a sterile 
pipette. If the specimen is collected from a tap, allow the water 
to run several minutes before obtaining the specimen. 
With a sterile pipette place 1 c.c. of the water in a petri dish. 
Ten cubic centimeters of melted agar at a temperature of 40° C. 
are then added to 1 c.c. of water in the petri dish. The medium 
and sample are thoroughly mixed and uniformly spread over the 
bottom of the dish by tilting or by rotation of the dish. 
Incubate for twenty-four hours at 37° and count colonies. 
In each of five Dunham fermentation tubes (Fig. 54) contain- 
ing lactose broth, place 10 c.c. of water with sterile pipette and 
incubate at 37° C. 
If 10 per cent of gas is formed within twenty-four hours, re- 
port as positive for B. coli group. If no gas is formed in twenty- 
four hours—or if the gas formed is less than 10 per cent, incubate 
for forty-eight hours. Subculture on Endo medium and identify 
organisms as belonging to B. coli group before reporting as 
positive. (Page 204.) 
Examination of Milk and Cream 
Collection of Sample.—Mix the milk or cream thoroughly in 
container by shaking or by pouring from one container to the 
other several times. Remove sample in a sterile bottle. 
Bacterial Count.—With a sterile pipette withdraw 1 c.c. of 
milk from the sample bottle and introduce into a bottle con- 
taining 99 c.c. of sterile water, thus making a 1-100 dilution. 
Mark bottle 1/2. 
One cubic centimeter of this dilution is transferred to a second 
bottle containing 99 c.c. of sterile water, giving a dilution of 
1-10,000. Mark bottle 1/4. 
282 EXAMINATION OF MILK AND W A TICK 
283 
Introduce 0.1 c.c. of the 1/2 dilution into a sterile petri dish, 
marking the plate 1/3. Put 1 c.c. of the 1/4 dilution into a second 
plate and mark 1/4. 
Pour enough melted meat extract agar cooled to 45° C. into 
each plate to well cover the bottom. Mix the agar and sample 
well by rotating and tilting the dish. 
Incubate 48 hours at 37° C. and count the colonies of bacteria. 
Each colony on the 1/3 plate represents 1000 bacteria per cubic 
centimeter and each on the 1/4 plate represents 10,000 bacteria 
per cubic centimeter. 
Put 0.1 c.c. of the original milk or cream into a lactose fer- 
mentation tube (Fig. 54) labeling it 1/1; 1 c.c. of the 1/2 dilution 
Fig. 69.—Babcock test bottle for determining the fat in milk. 
into a second tube and marking it 1/2; 0.1 c.c. of the same dilu- 
tion into a third, marking it 1/3 ; and 1 c.c. of the 1/4 dilution 
into a fourth tube, labeling it 1/4. 
Incubate the tubes for 24 hours and examine for gas formation. 
Ten per cent or more gas in the fermentation tubes represents 
respectively 10, 100, 1000 and 10,000 of B. coli per cubic centi- 
meter of milk. If desired, the organism causing the fermentation 
may be isolated in pure culture and further tests made to verify 
the assumption that it is the bacillus coli. 
Determination of Butter Fat.—With a pipette deliver 17.6 c.c. 
of well mixed milk into a Babcock bottle (Fig. 69). Add 17.5 
c.c. commercial sulphuric acid using the acid measure. Mix well. 284 
CLINICAL LABORATORY METHODS 
Centrifuge at 1200 revolutions per minute for five minutes. Add 
hot water to neck of bottle. Centrifuge two minutes and add hot 
water to about the 7 per cent mark on graduated tube of the 
bottle and two drops of alkanet root in glymol to facilitate 
the reading. Centrifuge one minute. The per cent of butter 
fat is read off directly from the scale. In determining the 
butter fat in cream, mix the sample Avith equal parts of water 
and proceed as with milk except that 18 c.c. of the diluted 
cream is used instead of 17.6 c.c. INDEX 
A 
Acetone bodies in blood, quantita- 
tive determination of, 
162 
in urine, qualitative tests for, 
20 
quantitative determination 
of, 62 
Acid alcohol for Ziehl-Neelsen 
stain, 196 
Acid digestion mixture for total 
nitrogen, 44 
Acidosis, blood chemical findings 
in, 126 
toleranee to sodium bicarbonate 
as test for, 72 
Agar blood, 203, 204 
brilliant green, Krumwiede’s, 205 
dextrose brain, 206 
double sugar, Bussell’s, 205 
Endo’s, 204 
glucose, 203 
meat extract, 203 
meat infusion, 202 
Albumin in exudates, determination 
of, 226 
in urine, method of recording, 20 
qualitative tests for, 19 
quantitative determination of, 
41 
Alkali tolerance test, 72 
Alizarin, preparation of, 262 
Amboceptor for Wassermann, prep- 
aration of, 177 
titration of, 179, 182 
Ammonia in urine, quantitative de- 
termination of, 48 
Ameba coli, examination of stool 
for, 84 
histolytica, examination of stool 
for, 84 
table for differentiating, 86 
Andrade indicator, preparation of, 
195 
Animal inoculation for tuberculosis, 
213 
Anisocytosis of erythrocytes, 112 
Antiformin, preparation of, 271 
use of, in examining sputum, 80 
Antiformin, use of—Cont’d 
in examining stools, 216 
in examining urine, 216 
Apparatus, volumetric, calibration 
of, 258 
Ascar’s lumbricoides, ova of, 83 
Ascitic fluid, examination of, 226 
Autogenous vaccines, preparation 
of, 230 
preservation of, 231 
standardization of, 231 
sterilization of, 231 
Avery method for typing pneumo- 
coccus, 191 
B 
Babcock method for determining 
fat in milk, 283 
Bacilli, gram negative, table for 
differentiating, 214 
Oppler-Boas, in gastric contents, 
77 
Bacillus, diphtheriae, Neisser’s stain 
for, 196 
Ponder’s stain for, 197 
tuberculosis, animal inoculation 
for, 213 
in feces, staining for, 216 
in spinal fluid, 222 
in sputum, 80 
in urine, 216 
typhosus, reaction of Bussell me- 
dium to, 205 
stool culture for, 216 
Bacterial count, wound, 232 
of milk, 282 
Bacteriological examination of 
feces, 216 
of spinal fluid, 221 
of transudates and exudates, 
227 
of urine, 212 
Barfoed’s solution, preparation of, 
19 
test for monosaccharides, 31 
Barometer coefficients, table of, 169 
Beef tape worm (see Taenia sagi- 
nata) 
285 286 
INDEX 
Benedict’s method for preparing 
creatinine zinc chloride, 
267 
for determination of uric acid 
in blood, 135 
for determination of uric acid 
in urine, 49 
for quantitative determination 
of glucose in urine, 37 
qualitative test for glucose in 
urine, 20 
solution for sugar determination, 
preparation of, 141 
qualitative, 81 
quantitative, 37 
Benzidine reaction for blood in 
feces, 81 
Bichromate cleaner for glassware, 
272 
Bile salts, tests for, in urine, 22 
in blood plasma, 123 
medium for blood culture, 209 
pigments, in blood plasma, 122 
in feces, 81 
in urine, 21 
Bismarck brown, preparation of, 195 
Bleeding time, determination of, 117 
Blood, agar, 203 
bile pigments in 122 
bleeding time of, 117 
carbon dioxide combining power 
of, 163 
casts in urine, 28 
chemical changes in various path- 
ological conditions, 126 
examination of, 125 
determination of calcium, 158 
of carbon dioxide combin- 
ing power, 163 
of chlorides, 145 
of cholesterol, 148 
of creatine, 140 
of creatinine, 138 
of nonprotein nitrogen, 131 
of oxygen combining ca- 
pacity, 171 
of phosphates, acid solu- 
ble, 154 
lipoid, 154 
total, 152 
of sugar, 141 
of sugar tolerance, 144 
of urea nitrogen, 133 
of uric acid, 135 
obtaining blood for, 125 
standardization of, 249 
chlorides, determination of, 145 
Blood—Cont’d 
coagulation time of, determina- 
tion of by Howell ’s 
method, 116 
by Lee and White’s method, 
117 
by capillary tube method, 117 
corpuscles, enumeration of, 90 
counting, 90 
apparatus for, 90 
culture, sodium citrate solution 
-for, 194 
culture, 210 
erythrocytes, determination of 
resistance to salt solu- 
tion, 113 
reticulated, 115 
vital staining of, 115 
fresh, examination of, 105 
in feces, 81 
films, preparation of on cover 
slips, 105 
staining of, 106 
filtrate, protein free, preparation 
of, 129 
in gastric contents, 77 
grouping of, 191 
hemoglobin content of, 101 
matching of, 192 
platelets of, counting, 95 
plasma, qualitative tests, for bile 
pigments, 122 
for bile salts, 123 
for urobilin, 124 
serum reactions, Widal tests, for 
paratyphoid infection, 
188 
for typhoid infection, 188 
Wassermann test for syphilis, 
175 
staining of, Ehrlich’s method, 107 
Wright’s method, 106 
vital, 115 
sugar in diabetes mellitus, 126 
sugar tolerance test, types of 
curves obtained, 145 
transfusion, blood tests prelimi- 
nary to, 191 
in urine, tests for, 21 
serum, Loeffler’s, 207 
Bock-Benedict colorimeter, 243 
Brilliant green agar, Krumwiede’s, 
205 
Broth, carbohydrate, 208 
dextrose brain, 206 INDEX 
287 
Broth—Cont’d 
glucose, 208 
ascitic fluid, 209 
meat extract, 204 
meat infusion, 204 
sugar free, 208 
Bruns’ glucose medium, 276 
Buffer solution for blood stains, 107 
C 
Calcium in blood, determination of, 
158 
table for calculating, 161 
in urine, determination of, 60 
Calcium carbonate, crystals of, in 
urine, 27 
Calcium oxalate, crystals of, in 
urine, 27 
Capsules, stains for, 197 
Carbol-fuchsin, Ziehl-Neelsen, 196 
Carbol-thionin, preparation of, 194 
Carbol-xylol, 274 
Carbon dioxide combining power of 
blood, determination of, 
163 
table for calculating, 170 
Casts in urine, 28 
Cestodes, classification of, 83 
Charcot-Leyden crystals in sputum, 
80 
Chlorides in blood, determination 
of, 145 
table for calculating, 148 
method for checking, 252 
in urine, determination of, 41 
table for calculating, 43 
Cholesterol in blood, determination 
of, 148 
method for checking, 252 
table for calculating, 150 
Citron’s scale for reading the Was- 
sermann test, 186 
Coagulation time of blood, 116, 117 
Cocci, gram negative, table for dif- 
ferentiating, 215 
Colloidal gold reaction in spinal 
fluid, 222 
types of abnormal reactions, 227 
Color index, definition of, 103 
method of determining, 103 
Colorimeter, Hellige, 98 
Bock-Benedict, 243 
Duboscq, 243 
use of, 243 
Congo red, preparation of, 262 
Creatine in blood, determination of, 
140 
in urine, determination of, 53 
Creatinine, in blood, determination 
of, 138 
method for checking, 251 
table for reading, 139 
in urine, determination of, 51 
table for calculating, 53 
preparation of, 267 
Creatinine zinc chloride, prepara- 
tion of, 267 
Crystals in urine, 23 
Cultures, blood, 210 
eye and ear, 212 
miscellaneous, 212 
nose and throat, 212 
sputum, 211 
stool, 211 
urine, 212 
Curschmann’s spirals in sputum, 80 
Cylindroids in urine, 29 
D 
Dakin’s solution, preparation of, 
from liquid chlorine, 263 
from chlorinated lime, 265 
Dark-field examination for spiro- 
cheta pallida, 228 
Diabetes mellitus, blood findings in, 
126 
chart of glucose tolerance in, 
145 
Diacetic acid in urine, tests for, 21 
Dialyzing sacs, preparation of, 128 
Dibothriocephalus latus, ova of, 83 
Diphtheria bacillus, Neisser’s stain 
for, 196 
Ponder’s stain, 197 
virulence test for, 213 
Duboscq colorimeter, 243 
Duke’s method of determining 
bleeding time, 117 
Duodenal contents determination of 
urobilin and urobilino- 
gen in, 77 
Dunham’s fermentation tubes, 209 
peptone solution, 207 
E 
Ear culture, 212 
Ehrlich’s reagent, preparation of, 19 
triple stain, preparation of, 107 
use of, 108 
Elastic fibers, examination of spu- 
tum for, 80 288 
INDEX 
Endo’s agar, 204 
Entamebae coli, examination of 
stool for, 84 
histolytica, examination of stool 
for, 84 
table for differentiating, 86 
Eosinophiles, 108 
Epithelial casts in urine, 28 
cells in urine, 23 
Erythrocytes, count of, normal, 94 
color index of, 103 
enumeration of, 92 
apparatus for, 90 
nucleated, 112 
resistance of, to hypotonic salt so- 
lution, 113 
saturation index of, 104 
in urine, 21 
volume index of, 104 
Esbach’s tube for determining al- 
bumin in urine, 41 
Exudates, determination of albumin 
in, 226 
examination of, 226 
Eye culture, 212 
F 
Fatty casts in urine, 28 
Feces, bacteriological examination 
of, 211 
for tubercle bacillus, 216 
chemical examination of, for bile 
pigments, 81 
for occult blood, 81 
for reaction, 84 
culture, 211 
examination of, routine, 81 
macroscopic examination of, for 
blood, 81 
for color, 81 
for gallstones, 82 
for mucus, 81 
microscopic examination for ame- 
bae, 84 
for blood, 84 
for ova, 84 
for parasites, 84 
for pus, 84 
urobilin and urobilinogen, quan- 
titative determination of, 
87 
Fehling’s solution, preparation of, 
19 
test for sugar in urine, 19 
Fermentation test for sugar in 
urine, 31 
Fluids, puncture, examination of, 
226 
Foam test for bile, 21 
killer for aeration procedures, 
272 
Folin and Wu’s formula for Ness- 
ler’s solution, 266 
method for determining blood 
creatine, 140 
creatinine, 138 
sugar, 141 
Fragility of erythrocytes, determi- 
nation of, 113 
Frozen sections, preparation of, 275 
G 
Gastric juice, blood in, 77 
mercury in detection of, 232 
microscopic examination of, 77 
Oppler-Boas bacillus in, 77 
pus in, 77 
qualitative chemical tests: 
for blood, 77 
for free hydrochloric acid, 75 
for lactic acid, 76 
quantitative chemical tests: 
for free hydrochloric acid, 76 
for hydrochloric acid deficit, 
76 
for total acidity, 76 
routine examination of, 75 
sarcinae in, 77 
test meal for, 75 
yeast in 77 
Gelatin, meat extract, 207 
Gentian violet, Sterling’s solution, 
195 
Geraghty and Rowntree’s test for 
renal function, 70 
Gerhardt’s test for diacetic acid in 
urine, 21 
Giffin and Sandford’s method for 
determining resistance of 
erythrocytes to hypotonic 
salt solution, 113 
Glucose, agar, 203 
tolerance test, 144 
in urine, qualitative test for, 20, 
29 
quantitative determination of, 
39 
table for calculating, 40 
in blood, Folin and Wu’s method 
for determining, 141 
table for calculating, 143 
Glycuronates, Tollens’ test for, 32 INDEX 
289 
Gonococcus, examination of smears 
for, 216 
Gram’s iodine solution, 195 
stain, 195 
Gram negative bacilli, fermentation 
reactions of, 214 
cocci, fermentation reactions 
of, 215 
Gram-Weigert stain for bacteria in 
tissues, 279 
Granular casts in urine, 28 
Guaiac test for blood, in feces, 81 
in gastric contents, 77 
in urine, 21 
Guinea pig, technic for bleeding 
from heart, 179 
Gunzburg’s reagent, preparation of, 
75 
H 
Hayem’s diluting fluid, 90 
Hays’ test for bile salts in urine, 
22 
Heller’s test for albumin in urine, 
19 
H|ellige colorimeter, determination 
of hemoglobin with, 98 
method for calibrating, 98 
Hematoxylin, Delafield’s, 273 
Hemoeytometers, ruling of, 91 
pipettes, 90 
Hemoglobin, chart showing amount 
of, at different ages, 101 
determination of, methods for, 98 
comparison of, 97 
acid hematin with Hellige col- 
orimeter, 98 
Sahli’s, 98 
Van Slyke’s gasometrie, 171 
Hiss’ capsule stain, 197 
Hookwmrm, new world species (see 
Necator americanus) 
old world species (see Anchylos- 
toma duodenale) 
Hopkins’ method for standardiza- 
tion of vaccines, 231 
Howell’s method for determining 
coagulation time, 116 
Huntoon’s capsule stain, 198 
Hyaline casts in urine, 29 
Hydrobilirubin in feces, test for, 
81 
Hydrochloric acid deficit in gastric 
juice, 76 
free in gastric juice, 76 
Hydrogen-ion concentration, method 
for determining, 
in blood, 127 
in media, 200 
in biological fluids, 234 
in urine, 36 
Hymenolepis nana, ova of, 83 
I 
Identification of reducing sub- 
stances in urine, 29 
Index, McLean’s of kidney func- 
tion, 69 
Indican in urine, tests for, 21 
Indicators, use of, 260 
table of, 262 
Intestinal parasites, classification 
of ova of, 83 
preservation of, 88 
Intravenous solutions, preparation 
of, 269 
K 
Kaiserling’s method for preserving 
specimens, 280 
Kidney function, McLean’s index 
of, 69 
Mosenthal test meal of, 67 
phenolsulphonephthalein test 
of, 70 
Krumwiede’s brilliant green agar, 
205 
L 
Lactic acid in gastric juice, test 
for, 76 
Lactose bile medium, 209 
Lactose in urine, Rubner’s test for, 
34 
Lange colloidal gold test, chart 
showing typical reac- 
tions, 227 
preparation of solution, 222 
reading reaction, 226 
technic of test, 225 
Lee and White’s method for deter- 
mining the coagulation 
time of blood, 117 
Leucocytes, differential count of, 108 
enumeration of, 95 
eosinophilic, 108 
in sputum, 79 
large mononuclear, 112 
normal number of, 95 290 
INDEX 
Leucocytes—Cont’d 
polymorphonuclear basophile, 112 
eosinophile, 108 
neutrophile, 108 
reaction of, with oxydase stain, 
117 
small mononuclear, 112 
table for differentiating, 109 
transitional, 112 
Leucocytosis, 95 
Leucopenia, 95 
Levulose, Seliwanoff’s test for, 32 
Litmus milk, 207 
Litmus, preparation of, 262 
Loeffler’s blood serum, 207 
methylene blue, 196 
Lugol’s solution, preparation of, 18 
M 
McLean’s index of kidney function, 
69 
Macrocytes, 112 
Magnesium, in urine, determination 
of, 61 
normal quantity, 35 
Malaria, examination of blood for, 
119 
chart showing appearance of or- 
ganisms in fresh blood, 
120 
in stained blood, 121 
Mallory’s stain for connective tis- 
sue, 277 
Marshall’s method for determining 
urea in urine, 46 
Mayer’s glycerine albumin, 274 
Meat extract agar, 203 
broth, 204 
gelatin, 207 
infusion broth, 204 
Media, preparation of, 202 
titration of to definite hydrogen- 
ion concentration, 200 
Medium, agar, ascitic fluid, 209 
blood, red, 203 
brown, 204 
Endo’s, 204 
glucose, 203 
meat extract, 203 
meat infusion, 202 
blood serum, Loeffler’s, 207 
carbohydrate, 208 
dextrose brain, 206 
Dunham’s peptone, 207 
Endo’s, 204 
Medium—Cont’d 
Krumwiede’s brilliant green 
agar, 205 
lactose bile, 209 
Loeffler’s, 207 
litmus milk, 207 
meat extract broth, 204 
gelatin, 207 
Petroff’s, 208 
potato, 206 
Russell’s double sugar, 205 
sugar free broth, 208 
Megaloblasts, 112 
Mercury, detection of, in gastric 
juice, 232 
in feces, 232 
in urine, 232 
Methyl orange, preparation of, 262 
Methyl red, preparation of, 262 
Methyl violet shellac, 271 
Methylene blue, Loeffler’s, 196 
Unna’s, 275 
Microblasts, 112 
Milk, dairy, bacterial count of, 282 
determination of butter fat, 283 
litmus for medium, 207 
Mitochondria, technic for staining, 
278 
Mosenthal test for renal function, 
67 
table for, 68 
Mould in sputum, 79 
Mouse method for typing pneumo- 
coccus, 189 
Museum jars, mixture for sealing, 
281 
Myeloblasts, 113 
Myelocytes, 113 
N 
Necator americanus, ova of, 83 
Neisser stain for diphtheria bacilli, 
196 
Nephritis, chemical blood findings 
in, 126 
Nephelometer, use of, 246 
Nessler’s solution, preparation of, 
266 
Neubauer ruling of liemocytometers, 
91 
Neutral red, use of in identifying 
amebae, 85 
Nitrogen, total, in urine, determi- 
nation of, 44 
normal quantity, 35 INDEX 
291 
Nitrogen—Cont’d 
nonprotein in blood, determina- 
tion of, 131 
method for checking, 250 
table for calculating, 123 
Nitric acid ring test for albumin 
in urine, 19 
Normoblasts, 112 
Nose culture, 212 
Nucleated red blood cells, 112 
Nylander’s reagent, preparation of, 
18 
test for sugar in urine, 29 
O 
Obermeyer’s reagent, preparation 
of, 18 
test for indican, 21 
Oppler-Boas bacillus in gastric 
juice, 77 
Oxydase reaction of leucocytes, 117 
Oxygen capacity of blood, deter- 
mination of, 171 
table for calculating, 173 
Oxyuris vermicularis, ova of, 83 
P 
Pandy’s test for globulin in spinal 
fluid, 220 
Parasites in feces, 84 
ova of, classification, 84 
preservation of, 88 
Pentose in urine, tests for, 32 
Peptone solution, Dunham’s, 207 
Petroff’s medium, 208 
Phenolphthalein, preparation of, 
262 
Phenolsulphonephthalein test for 
renal function, 70 
Phenylhydrazine test for sugar in 
urine, 29 
Phloroglucin test for pentose, 32 
Phosphate, ammonium magnesium 
crystals of, in urine, 27 
calcium in urine, 27 
determination of, in blood, 151 
table for calculating, 157 
in urine, 58 
“triple” (see ammonium phos- 
phate) 
Physiological salt solution, prepa- 
ration of, 194 
Picric acid, purification of, 269 
testing of, 51 
Plasmodium of malaria, falciparum, 
120, 121 
malaria, 120, 121 
table showing differentiation of 
types, 120, 121 
vivax, 120, 121 
Platelets in blood, enumeration of, 
95 
Pleural fluid, examination of, 226 
Pneumococcus, determination of 
types, 189 
Poikilocytosis, 112 
Polariscopic method for determin- 
ing sugar in urine, 39 
Polychromatophilia, 113 
Ponder’s stain for diphtheria ba- 
cillus, 197 
Potato medium, 206 
Protein free blood filtrate, prepara- 
tion of, 129 
Punctate basophilia, 113 
Pus casts in urine, 28 
in feces, 84 
in urine, 22 
R 
Reaction of media, adjustment of, 
200 
Red blood cells, color index of, 103 
saturation index of, 104 
volume index of, 104 
Rieder’s cells, 113 
Ross-Jones’ test for globulin in 
spinal fluid, 220 
Russell’s double sugar agar, 205 
S 
Safranin for counterstain, 195 
Sahli’s method for the determina- 
tion of hemoglobin, 98 
Sarcinae in gastric contents, 18 
Saturation index, 104 
Scott-Wilson reagent, preparation 
of, 18 
test for acetone in urine, 20 
Sections, frozen, 275 
Sections, preparation of, for histo- 
logical examination, 273 
Sediment in urine, examination of, 
22 
Seliwanoff’s test for levulose, 32 
Shellac, methyl violet, 271 
Sodium bicarbonate solution, intra- 
venous, preparation of, 
270 292 
INDEX 
Sodium citrate solution for blood 
culture, 194 
Sodium hydrate solution normal, 256 
Solutions, intravenous, preparation 
of glucose, 270 
physiological salt, 269 
sodium bicarbonate, 270 
sodium citrate, 271 
normal, preparation of, acid po- 
tassium phthalate, 257 
hydrochloric acid, 256 
iodine, 256 
potassium permanganate, 257 
sodium hydroxide, 256 
thiosulphate, 257 
sulphuric acid, 255 
Spinal fluid, bacteriological exami- 
nation of, 221 
cell count of, 221 
colloidal gold reaction in, 222 
examination of, routine, 220 
for tubercle bacillus, 222 
globulin, tests for excess, 220 
Wassermann reaction of, 186 
Spirals, Curschmann’s in sputum, 
79 
Spirocheta pallida, dark-field exam- 
ination for, 228 
method for staining, 198 
Sputum, antiformin method of ex- 
amining, 80 
collection of specimen, 79 
culture, 210 
Curschmann’s spirals in, 79 
elastic fibers in, 79, 80 
examination of, for tubercle ba- 
cilli, 80 
fresh, 79 
routine, 79 
gross appearance of, 79 
microscopic examination of, 79 
pneumococcus in, typing of, 189 
Staining solutions, stock, 194 
Sterilizing, hot air, 199 
steam, 199 
Sterling’s gentian violet solution, 195 
Stomach contents (see gastric juice) 
Stools (see feces) 
Stopcock grease, 272 
Streptococci, classification of, 217 
Sugar in blood, determination of, 
141 
method for checking, 250 
table for calculating, 143 
in urine, qualitative tests for, 20, 
29, 31 
Sugar, in urine—Cont’d 
quantitative tests for, Bene- 
dict’s, 37 
polariscopic, 39 
tolerance test, blood, 144 
Sugar-free broth, 208 
Sulphates in urine, determination 
of, 55 
normal quantity, 35 
T 
Taenia saginata, ova of, 83 
solium, ova of, 83 
Test meal, gastric, 75 
Mosenthal, nephritic, 67 
Throat culture, 212 
Tollens’ test for glycuronates, 32 
Topfer’s reagent, preparation of, 
262 
Transfusion of blood, tests prelimi- 
nary to, 191 
Transitional leucocyte, 112 
Transudates and exudates, exami- 
nation of, 226 
Trichocephalus dispar, ova of, 83 
Tsuchiya’s method for quantitative 
determination of albu- 
min in urine, 41 
Tubercle bacillus, animal inocula- 
tion for, 213 
in sputum, 80 
in stool, 216 
in tissues, stains for, 279 
in urine, 216 
Turck’s solution, preparation of, 90 
Typhoid fever, Widal test in, 188 
U 
Unna’s methylene blue, 275 
Urate, ammonium, in urine, 28 
Urea nitrogen, in urine, quantita- 
tive determination, 46 
normal quantity, 35 
in blood, quantitative determi- 
nation, 133 
method for checking, 250 
table for calculating, 135 
Uric acid, crystals of, in urine, 23 
in blood, quantitative deter- 
mination of, 135 
method for checking, 251 
table for calculating, 137 
in urine, normal quantity, 35 INDEX 
293 
Uric acid, in urine—Cont’d 
quantitative determination of, 
49 
table for calculating, 51 
Urine, acetone in, quantitative de- 
termination of, 62 
tests for with sodium nitro- 
prusside, 21 
with Seott-Wilson reagent, 
20 
acidity of, by titration, 36 
albumin in, qualitative test for, 
heat and acetic acid, 19 
Heller’s nitric acid, 19 
quantitative determination of, 
41 
ammonia in, determination of, 48 
normal quantity, 35 
ammonium urate in, 28 
bacteriological examination of, 
212 
for tubercle bacilli, 216 
bile salts in, test for, 22 
foam test, 22 
Hays’ sulphur test, 22 
pigments in, tests for, 21 
foam test, 21 
iodine test, 21 
Rosenbach test, 21 
blood in, test for, 21 
calcium in, determination of, 60 
normal amount, 35 
carbonate in, 27 
casts in, blood, 28 
epithelial, 28 
fatty, 28 
granular, 28 
hyaline, 29 
pus, 28 
waxy, 29 
chemical composition of normal, 
35 
chlorides in, normal quantity, 35 
quantitative determination, 41 
table for calculating, 43 
creatine in, normal quantity, 35 
quantitative determination, 54 
creatinine in, normal quantity, 35 
determination of, 51 
table for calculating, 53 
crystals in, of ammonium mag- 
nesium phosphate, 27 
of ammonium urate, 28 
of calcium oxalate, 27 
of calcium carbonate, 27 
of calcium phosphate, 27 
of calcium sulphate, 27 
Jrine, crystals in—Cont’d 
of sodium urate, 28 
of uric acid, 23 
culture, 212 
cylindroids in, 29 
diacetic acid in, qualitative test 
for, 21 
quantitative determination, 64 
epithelial cells in, 23 
examination of, microscopic, 22 
qualitative, 17 
quantitative, 35 
routine, 17 
hydroxybutyrie acid, quantitative 
examination, 64 
indican, tests for, 21 
magnesium in, determination of, 
61 
normal quantity, 35 
mercury in, detection of, 232 
nitrogen in, determination of 
total, 44 
normal quantity, 35 
table for calculating, 47 
phosphates in, determination of, 
58 
normal quantity, 35 
preservation of, 35 
pus in, 22 
reagents, 18 
red blood cells in, 22 
reducing substances in, identifica- 
tion of, 29 
specific gravity, 17 
sugar in, qualitative tests, Bene- 
dict ’s, 20 
Fehling’s, 19 
fermentation, 31 
phenylhydrazine, 29 
Nylander’s, 29 
quantitative tests, Benedict’s, 
37 
polariscopic, 39 
sulphates in, determination, of, 55 
normal quantity, 35 
urates in, 23 
urea nitrogen in, determination 
of, 46 
normal quantity, 35 
uric acid, determination of, 49 
normal quantity, 35 
urobilin in, qualitative tests for, 
21 
quantitative determination, 73 
urobilinogen in, qualitative tests 
for, 22 
quantitative determination, 73 294 
INDEX 
Urobilin in blood plasma, 124 
in urine, qualitative test for, 21 
quantitative determination of, 
73 
in feces, quantitative determina- 
tion, 87 
in duodenal contents, quantitative 
determination, 77 
Urobilinogen in duodenal contents, 
quantitative determina- 
tion, 77 
in feces, quantitative determina- 
tion of, 87 
in urine, qualitative test for, 21 
quantitative determination of, 
73 
V 
Vaccines, autogenous, preparation 
of, 230 
preservation of, 231 
standardization of, 231 
sterilization of, 231 
Van Slyke and Cullen’s method for 
determining the carbon 
dioxide combining pow- 
er, 163 
urea nitrogen in urine, 46 
in blood, 133 
Van Slyke’s gasometric method for 
determining hemoglobin, 
171 
Vital staining of blood, 115 
Vogel and Lee’s method for deter- 
mining mercury in ex- 
cretions, 232 
Volume index of red blood cells, 104 
Volumetric apparatus, calibration 
of, 258 
solutions, preparation of, 253 
W 
Wassermann reaction, amboceptor, 
hemolytic, preparation 
of, 177 
Wassermann reaction, amboceptor— 
Cont’d 
titration of, 179, 182 
antigens, preparation of, 179 
titration of, 180 
complement, dilution of, 179 
preparation of, 179 
titration of, 183 
diagnostic test, 184 
general considerations, 175 
glassware, preparation of, 175 
reading diagnostic test, 186 
reagents, titration of, prelimi- 
nary to diagnostic test, 
181 
saline solution for, 176 
serum for, preparation of, 179 
sheep cells for, 176 
with spinal fluid, 186 
Water, bacteriological examination, 
2'82 
Waxy casts in urine, 29 
White blood cells (see leucocytes) 
Widal reaction, interpretation of 
results of, 188 
preparation of reagents, 187 
technic of, 188 
Wound bacterial count, 232 
Wright and Kinnicutt’s method for 
counting blood platelets, 
95 
Wright’s stain, preparation of, 106 
use of, 107 
Y 
Yeast cells, in gastric juice, 77 
Z 
Zenker’s fixing solution, 273 
Ziehl-Neelsen’s carbol fuchsin, 196 
method for staining the tubercle 
bacillus, 80, 196