Corn ^.L+'U'S ell XElnivetsit^ OF THE State doWcQC of agriculture S..i}]i}^... 584 Cornell University Library Essentials of laboratory diagnosis desig 3 1924 003 470 907 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003470907 PLATE 1. AlITHOR's Ol'l-K'E Lahorai'ory For details and description see Appemlix, page 290. ESSENTIALS OP LABORATORY DIAGNOSIS DESIGNED FOR STUDENTS AND PRACTITIONERS BY FRANCIS ASHLEY JAUGHT, M.D. DIRECTOR OF THE LABORATORY OF THE DEPARTMENT OF CLINICAL MEDICXNB AND ASSISTANT TO THE PROFESSOR OF CLINICAL MEDICINE, MBDICO-CHIRUROICAL COLLEOE, BTC, ETC.. PHILADELPHIA, PA, CONTAINING ELEVEN FULL-PAGE PLATES (THREE IN COLORS) AND THIRTY-NINE TEXT ENGRAVINGS. FOURTH REVISED EDITION. PHILADELPHIA: F. A. DAVIS COMPANY, Publishers 1913 COPYRIGHT, 1909 COPYRIGHT, iglO COPYRIGHT, August, 1911 COPYRIGHT, October, 1912 BY F. A. DAVIS COMPAMY Copyright, Great Britain. All Rights Reserved Pliiladelpbia, Pa., U. S, A. Press of F. A. Davis Company 1914-16 Cherry Street PREFACE TO FOyRTH EDITION. The steady advance of clinical medicine and de- velopment of new and practical methods of study demand a constant supervision and alteration in a work devoted to the subject. The fourth edition of this little book has been forced upon the author at this early date, making the fourth edition in as many years. As the work involved in the preparation of a new edition is always far greater than appears in the finished product, it is hoped that this latest edition will.receive the same appreciation as those preceding it. F. A. P. (iii) PREFACE TO FIRST EDITION. A KNOWLEDGE of SO many branches of medicine is required of the medical student of to-day, and as the time at his com- mand is comparatively limited, a manual embodying the essen- tials of the subject such as this aims to do cannot but prove useful. At the same time, it is believed to contain all infor- mation necessary to provide a working outline of clinical lab- oratory methods for the busy practitioner. The book is not intended to take the place of the many excellent and exhaustive text-books on Clinical Medicine, but rather to supplement them, by pointing out to the busy student and practitioner simple and reliable methods by which he may obtain the information desired, without unnecessary expendi- ture of valuable time upon difficult, tedious or untried methods. The author has endeavored in this work to present in as concise a manner as possible a selection of the analytical methods employed in the clinical laboratory, without burden- ing the reader with unnecessary detail, cumbersome methods, etc., many of which are extremely difficult, often requiring considerable knowledge of general chemistry and elaborate apparatus, which may place them beyond the reach of the practitioner. For these reasons it is hoped that this little work will prove equally valuable to the student and the practicing phy- sician. In preparing this work special effort has been made to bring the subjects treated up to date, by the introduction of such new methods as have proven reliable. Some of the (iv) PREFACE. V material is entirely new, and many of the plates and cuts have been prepared from original drawings and photographs by the author. Special mention is to be made- of the plate and draw- ings (Plate VI, Figs. 17 and 18) of the normal relations of the stomach, which were prepared expressly for this work by Dr. George E. Pf abler. The appendix has been arranged to furnish a working basis for the equipment of a clinical laboratory, at the same time affording reference for the preparation of stains, reagents, etc., mentioned in the text. The leading authorities have been freely consulted, and much material has been obtained from such authorities as V. Jaksch, Sahli, Caille, Grawitz, Krehl, Max Braun, Tyson, Abbott, Purdy, Kemsen, and Holland. The author takes this opportunity to express his appre- ciation of the many valuable suggestions received from Drs. Judson Daland and Wm. Egbert Eobertson, and Mr. Geo. B. Johnson, of the F. A. Davis Company; also, to his associate. Dr. Francis J. Dever, for invaluable aid lq the preparation of the manuscript and in correcting the proof. F. A. F. 1831 Chestnut Street, Philadelphia. INTRODUCTORY NOTE. Br JuDsON Daland, M.D. Professor of Clinical Medicine,. Medico-Chirurgical Colleee, Philadelphia. 9 This book should be in the possession of every medical student and most practicing physicians. It contains, as its title implies, the essentials, and only the essentials of those procedures necessary to clinical laboratory diagnosis. The peculiar value of this book resides in the concise and practical manner in which each subject is treated and each test described, and the entire absence of all superfluous data. This book is partieulq,rly well suited, not only to the -med- ical student in the preparation of cases assigned to him for study, but also to practicing physicians, who have grasped the necessity of establishing a small laboratory in connection with every-day work. Those possessing such a laboratory will find it necessary to consult this book at short intervals. The more it is employed the more will its practical value be demonstrated. 1 unhesitatingly recommend this work to medical students, and trust that it will find a place in the laboratory of every prac- ticing physician. (vi) CONTENTS. PAGE Section I. The MicRoscca?E 1 Selection of the Instrument, 1. Care of the Microscope, 1. The Parts of the Microscope, a. The Oil-rmmersion Objective, 5. Apochromatlc Objectives, 5. Illumination, 6. CleaWng the Microscope, 7. The Mechanical Stage, 8. The Warm Stage, 9. The Maltwood Finder, 9. Examination of Urinary Sediments, II. Examination of the Blood, 16. The Camera Lucida, 17. Section II. The Sputum 19 General Considerations, 19. Gross Composition, 20. Color, 21. Reaction, 23. Air Content, 23. Odor, 24. Hemoptysis, 24. Macro- scopic Examination, 25. Microscopic Examination, 26. Preparation of Stained Specimens, 29. Section III. The Blood 31 Chemical Composition, 31. Methods of Obtaining Specimen, 31. Appearance of Fresh Blood, 32. Specific Gravity, 33. The Amount and Reaction, 34. Estimation of Per Cent, of Hemoglobin, 35. Esti- mation of Erythrocyt.es, 37. Hematokrit Determination of Erythro- cytes, 39. Percentage of Erythrocytes, 40. The Color-Index, 41. The Volume-Index, 41. Estimation of Leukocytes, 42. Examination of Fresh Drop, ,43. Staining Methods, 45. hematologic Terms, 47. Varieties of Leukocytes, 48. Differential Couht, 49. Leukodytosis, 50. The Anemjas,, 62. Chlorosis, 54. Leukemia, 54. Spectroscopic Examination, 66. Baoteriologic £:xamination, 58. Spirochsete Pallida, 69. Section IV. Opsonic Method 51 General Consideration, 61. The Theory, 61. Therapeiitic Application, 63. Guides to Treatment, 63. Section V. Blood-Prbssuee 65 Capillary Blood-Pressure, 65. Terms and Definitions, 65. The Prin- ciple of the Sphygmomanometer, 68. Explanation of Circular Com- pression, 69. The Mercury Sphygmomanometer, 70. Application of Sphygmomanometer, 71. Systolic Reading, 72. The Pocket Sphyg- momanometer, 74. Normal Blood-Pressure and Variations, 75. Effect of Alcohol and Tobacco, 79. Pathologic Variations, 80. Blood-Pres- sure in Heart Diseases, 81. Functional Heart Tests, 82. Cardiovas- cular Renal Disease, 84. High-Pressure Diseases, 85. Blood-Pressure During Pregnancy, 86. Blood-Pressure in Acute Infections, 86. Blood-Pressure Tests for Life Insurance, 87. Section VI. Coagulation 80 General Consideration, 89. Wright's Method, 90. Method of Russell & Brodie, 93. Clinical Observations, 94. Section VII. Blood Parasites 96 Plasmodium Malarias, 96. Differential Table, 98. Detection of Organ- ism, 98. Clinical Relations of Plasmodium, 98. Spirochaete of Re- lapsing Fever, 99. Filarla Sanguinis Hominis, 99. (Vii) Vlil CONTENTS. PAGE Section VIII. Animal Pabasitks 101 Definition, 101. Classiflcation, 102. Protozoa, 103. Platyhelminthes, 107. Nematodes, 113. Section IX. Dbtbemination of the Functions of the Stomach. 118 The Gastric Contents— Vomitus, 118. Methods of Obtaining Specimen, 119. Preliminary Preparation of the Patient, 120. Modified Ewald Test-Brealtfast, 121. Teohnic of Removal of Test-Meal, 122. Inflation of the Stomach, 123. Determination of the Capacity of the Stomach, 125. Gastric Lavage, 126. The Acids of Digestion, 126. Estimation of Free Hydrochloric Acid, 129. The Organic Acids, 130. Test for Occult Bleeding, 131. Estimation of Peptic Activity, 132. Estimation of Trypsin, 136. Estimation of the Activity of Rennet, 136. Digestion of Starch and Sugar, 137. Determination of Gastric Activity, 137. Detection of Bile, 137. Test for Motor Functions, 137. Rontgen Ray Examinations, 138. Section X. The Feces 143 Physical Characteristics, 143^ Study . of _ Intestinal Digestion, 144. Determination of Motor Functions of Gastro-Intestinal Tract, 146. Chemical Examination of Feces, 150. Occult Bleeding in Gastro- intestinal Tract, 153. Bacteria and Protozoa in Feces, 154. Clinical Significance of Examinations of Feces, 157. Foreign Bodies, Calculi and Concretions, 158. Section XI. The Urine 161 General Considerations, 161. Description and Importance of the Urine, 163. Physical Characteristics of the Urine, 164. The Amount, 166. The Specific Gravity, 168. Estimating the Total Solids, 171. The Reaction, 172. Chemical Composition of. the Urine, 174. Phosphates, 176. . The Sulphates, 179. The Chlorids, 180. The Organic Constit- uents of the Urine, 181. Urea, 182. Uric Acid, 187. The Urates, 189. Creatinin, 191. Oxalates, 192. Abnormal Constituents of the Urine, 194. Albumin, 194. Glucose, 201. Cammldge Reaction, 209. Polari- metric Method, 210. Significance of Glycosuria, 210. Raw Sugars in Urine, 211. Acetonuria, 212. Diacetic Acid, 212. Oxybutyric Acid, 213. Bile Pigments, 215. Pyuria, 217. Bpithelia in Urine, 218. Tube Casts, 218. Cylindroida, 221. The Inorganic Sediment, 223. Urinary Con- cretions, 225. The Diazo Reaction, 226. Drugs and Poisons in the Urine, 227. Section XII. The Cerebeo-Spinal Fluid 231 The Presence of Cerebrospinal Fluid, 231. "Determination of the Cell- Content, 232. Determination of the Proteid-Content, 234. Interpreta- tion of the Findings, 235. Section XIII. The Body Fluids 2.36 Peritoneal Fluid, 236. Inflammatory Exudate, 236. Transudates, 236. Pleural Fluid, 237. General Considerations, 237. Non-Inflammatory Transudate, 237. Inflammatory Exudate, 237. Method of Making Permanent Stained Specimen, 237. Microscopic Appearance of Stained Specimens, 238. The Pericardial Fluid, 238. The Synovial Fluid, 238. Hydrocele Fluid, 239. Section XIV. Human Milk 240 General Considerations, 240. Physical Characteristics- and Composi- tion, 240. Examination of Milk, 241. Determination of the Fat, 241. Determination of the Sugar, 243. Estimation of the Proteids, 243. Chemical Composition, 243. CONTENTS. ix PAGE Section XV. Bacteriologic Methods 244 The Tubercle bacillus, 244. Differential Diagnosis, 446. Staining Methods, 246. Dlplococcus Paeumonii, 248, Method of Staining Capsules, 249. Bacillus of Pteifter, 249. Bacillus DipHtherise, 250. Gonococcus of Neisser, 261. Gram's Method of Staining, 252. Re- action of Bacteria to Gram's Method, 2631 LoefHer's Methbd of Stain- ing Flagella, 234. Sterilization, 255. Intermittent Sterilization, 259. The Autoclave, 261. Chemical Sterilization and Disinfection, 262. Preparation of Culture Media, 262. Nutrient Bouillon, 263. Nutrient Gelatin, 265. Nutr'ent Agar-Agar, 266. Glycerin Agar, 266. Blood- Serum, 267. Loeffler's Blood-Serum, 269. Technio for Plates and Petri Dishes, 270. The Incubator, 271. Section XVI. Seeodiagnosis 274 Agglutination, 274. Widal Reaction, 274. Discussion of Complement Fixation, 276. Principle and Technic of the Wassermann Reaction, 278. Principle and Technic of the Noguchi Reaction, 280. Preparation of Reagents, 283. Standardization of Reagents, 286. Value of the Tests, 288. Section XVII. Appendix 290 Description of Laboratory Cabinet, 290. Urinalysis Test Set, 291. Report Forms, 293-298. Table of Clinical Terms, 299. Blood-pressure Chart, 300. Apparatus, 301. Chemicals and Reagents, 303. Stains, 308. Identifieation of the Tubercle Bacillus, 317. Antiformin Method, 318. Tubercle Bacilli in the Blood, 319. Tubercle Bacilli in the Feces, 322. Test for Pancreatic Activity, 322. Bile Pigment Reaction, 324. Leukocyte, Differential Count Computing Chart,. 325. Modified Esbach Test, 326. Tables of Weights and Measures, 327-329. Com- parison of 'Thermometers, 330. Index 331 LIST OF ILLUSTRATIONS. PAGE Plate I. Author's Office Laboratory Frontispiece Plate II. Normal and Pathological Blood-cells (Colored) 42 Plate III. Sphygmomanometer, in Position tor Observation 80 Plate IV. Malarial Parasites. (Kolle & Wassermann) 96 Plate V. Intestinal Parasites of Man : 104 Plate VI. Skiagraph. Normal Stomach. Child, Age 4 Years 140 Plate VII. Indican Scale. (Colored) 180 Plate VIII. Uric Acid Crystals with Amorphous Urates. (Peyer.) (Colored). 190 Plate IX. Uric Acid Crystals 224 Plate X. Calcium Oxalate Crystals and Phosphates 224 Plate XI. Uric Acid Crystals, Cholesterin, Cystin, Tyrosin and Leucin 224 riG. 1. American-make Microscope, showing Triple Nosepiece, etc 2 2. Mechanical Stage 8 3. Maltwood Finder 10 3». Camera Lucida 17 4. Bronchial Cast, from case of Fibrinous Bronchitis in service of Dr. Judson Daland (Original) 22 5. Fleischl Hemoglobinometer . . ., 32 6. Thoma-Zeiss Hemocytometer in case. (A. H. Thomas) 36 7. Appearance of Field of Thoma-Zeiss Hemocytometer, when properly Mounted for Counting the Red Corpuscles 38 8. Daland Hematokrit. (A. H. Thomas) 39 9. Puling of Chamber of Thoma-Zeiss Hemocytometer 42 10. Normal Pulse Tracing: Showing relation of Systolic, Diastolic, Pulse Pressure, and Mean. Pulse pressure equals 30 66 11. Diagram of Relations of Armlet to Brachial Artery. Explanation of Systolic Reading 67 11». Diagram of Relation of Armlet to Brachial Artery. Explanation of Diastolic Reading 68 12. Standard Mercury Sphygmomanometer 70 12". Faught Pocket Sphygmomanometer 74 12". Woley's Normal Blood-pressure Table 76 13. Wright's Coagulometer 91 14. Bogg's Coagulometer, diagrammatic representation 93 15 Outfit for Gastric Test-meal Removal, Lavage and Inflation 119 (Xi) xii LIST OF ILLUSTRATIONS. FIG. PAGE 16. Diagrammatic Representation of Arrangement of Bottle for Measuring Cubic Contents of Stomach 125 17. Solid Line shows Form and Relation of typical normal Stomach, with subject in standing posture 139 18. Dotted Line shows Form and Location of normal Stomach during filling. Shaded portion shows Contraction and Change in form of Stomach 140 19. Strasburger Apparatus. (After Steele) 151 20. Various Forms of Urinometers and Urinometer Cylinders 169 21. Westphal Balance. (A. H. Thomas) 170 22. Various Forms of Ureameters 187 23. Urate of Soda and Crystals of Uric Acid, Oxalate of Lime, and Cystin . . 190 24. Esbach Albuminometer 199 25. Binhorn Saccharimeter during Performance of Test 207 26. Polarimeter. (A. H. Thomas) 210 27. "Large White Kidney." X 350. (Lenhartz) 219 28. Chronic Bright's Disease. X 350. (Lenhartz) 219 29. Sedimentation Glasses 220 30. Centrifuge Tubes 223 31. Arnold Steam Sterilizer. (A. H. Thomas) 260 32. Autoclave. (A. H. Thomas) 261 33. Thermostat or Incubator. (A. H. Thomas) 272 34. Portable Clinical Laboratory Set 293 35. New Graphic Chart for Recording Blood-pressure Observations 300 36. A Computing Chart to Facilitate Making o. Differential Leukocyte Estimation 325 I. THE MICROSCOPE. SELECTION OF THE INSTRUMENT. In purchasing a microscope one should consider nothing but the best. This is true economy, for a cheap or inferior in- strument is always an unsatisfactory one which, when once bought, is hard to get rid of, and if later discarded for a better instrument, represents a total loss. The possession of a good microscope is an absolute essential. Its services are required in almost every investigation of modern clinical diagnosis, and there is hardly a chapter in 'this work which does not call for its use. While the finest and most expensive instruments are still of foreign make, there are, nevertheless, many of American make which compare favorably with the -imported instruments. An example of a good American instrument is shown in Fig. 1, and is one of a series manufactured by the Bausch and Lomb Optical Co. CARE OF THE MICROSCOPE. In handling any instrument it is necessary to see that it is always grasped by some one of its solid parts — the base or the standard. Some of the newer makes are provided with an aperture in the standard for this very purpose. If the instrument is to be in daily use it should be kept under a bell jar or in a specially prepared cabinet built above the work table. (See Frontispiece.) Eeference to Fig. 1 will illustrate the mechanism and the different parts with which one should become familiar before attempting to use the instrument. Efforts to use the microscope by any one unfamiliar with its different parts and adjustment should never be permitted, since careless handling may result in serious damage to the objeetivfe or other delicate parts. (1) 2 THE MICROSCOPE. DESCRIPTION OF THE MICROSCOPE. Referring to Fig. 1, the various parts of tlie instrument about to be described may be located. The Ocular, or Eye-Piece, consists of one or more converg- ing lenses, the combined action of which is to magnify the COARSE ADJUSTMENT FINE ADJUSTMENT HANDLE ARM OCULAR DRAW TUBE TUBE TRIPLE NOSEPIECE WITH OB- JECTIVES STAGE IRIS DIAPHRAGM SUBSTAGE CONDENSER MIRROR Fig. i.— American-m.\ke Microsoopb showing Triple Nosepiece and Aperture in Standard to facilitate handling. image formed by the objective. This part is contained in a tube of its own, which telescopes into the top of the barrel, and is the part which the ^eye approaches when viewing the object. DESCKIPTION OF THE MICROSCOPE. 3 Various oculars are usually provided, and are numbered from 1 to 8. As the number advances, the magnifying power of the ocular increases; at the same time the length of the tube decreases. It is advised, when increased magnification is desired, to accomplish this by increasing the power of the ob- jective rather than by using stronger oculars. The clearness and definition of the picture will then be preserved, whereas if the power of the ocular is increased it is usually at the expense of definition. It will be found of great convenience to cement into one of the oculars a fine hair or eyelash. This should rest upon the upper surface of the central diaphragm of the ocular, when it will always be in focus. This serves as a pointer which can be used to single out from a blood or other specimen some special point of interest that it is desired to demonstrate to another person. This is particularly valuable in class demonstration. The Barrel, or Tube, is the large tube of the microscope, which serves as a conductor of the rays as they pass from the objective to the ocular. The Objective consists of a system of converging lenses arranged at the lower end of the barrel, where it forms a mag- nified inverted image of the object. Upon this piece depends the magnifying power of the microscope. Most objectives are designated by numbers running from 1 to 15, these numbers representing the fractions of an inch at which the lens operates. Foreign makes are designated by letters which are not, in any way directly comparable to the numbers of the American objec- tives. For one desiring three objectives, the most useful num- bers will be found to be the 3, the 7 or 6, and the 12-oil-im- mersion, the 3 and 6 or 7 being chiefly employed in bkod-counl^ ing, while the 12th is necessary for blood-examination an,d bac- teriologie work. The Stage is the table fastened below the barrel and in a right-angle plane to it. This serves to retain the object in a vertical plane to the optical axis of the instrument.. The table is provided with spring clips to better hold the slj^e in, ppsiljion during examination. The newest Leitz. scope is fumi^Jiied with a niovable (not mechanical) stage, which, by the operatiq^jjOf two milled screws, is capable of slight movement in all direc- 4 THE MICROSCOPE. tions. This, when a mechanical stage is not at hand, is of great service in searching blood and bacteriologic slides. The Reflector is a small mirror situated below the stage which serves to direct the rays of light upward through the object in the optical axis of the microscope. The reflector has two sides, one carrjang a concave, and the other a plane, mirror. The Sub-Stage Condenser (Abbe's) consists of a system of lenses arranged between the stage and the reflector. The object of this part of the instrument is to collect and condense the rays as they come from the reflector, so that they are focused upon the object, thus furnishing brilliant illumination. The Iris Diaphragm is now universally employed for con- trolling the intensity of the illumination. This is held in the same carrier as the condenser, and is just below it. By means of a lever every gradation of light, from the most intense to absolute darkness, is readily obtained. The proper manipula- tion of this diaphragm constitutes a very important part of the practical knowledge gained from the use of the microscope, and has mtich to do with the success of many investigations. The Adjustments. — The coarse adjustment is the rack-and- pinion mechanism projecting from the upper part of the stand- ard, and is employed to rapidly raise and lower the barrel and its attachments. The fine adjustment is a micrometer screw situated usually below the rack and pinion. This serves the purpose of very gradually raising or lowering the barrel in order to obtain exact focus. The Draw-Tube is a very important adjunct of high-grade' instruments, because by its skilful manipulation slight errors in refraction, due to inequalities in slide or cover-glass, may be icorrected. ' The Nose-Piece, or collar, is fastened to the lower end of the barrel, and permits the attachment of two or three objectives at one time in such a position that, by rotation of the collar, any one of them may be immediately brought into the axis of the instrument. The most important accessories of the microscope are the objectives, and the quality of all microscope work largely de- pends upon their perfection. While the quality of objectives vary much, one cannot go far astray if they are obtained through APOCHROMATIC OBJECTIVES. 5 a reputable supply house or from a well-known manufacturer, such as the Zeiss or Leitz abroad, and the Bausch and Lomb Optical. Co., U. S. A. THE OIL-IMMERSION OBJECTIVE. The oil-immersion objective, or homogeneous system, is so constructed that when in use the pencil of light passing through the object to the objective traverses only media of the same refractive index. This is accomplished by placing between the cover-glass and the end of the objective a medium having the same refractive index as glass. To accomplish this a drop of cedar oil is placed upon the cover-glass, and the objective brought into contact with this, and the observation made through the oil. This class of lens is intended to work only with the oil, and is unsatisfactory when used dry. Frequently it is not convenient to use cedar oil for the preliminary examination often employed to determine the progress of staining, etc. ; here the staining fluid may be washed off and a drop of water used in place of the oil; this will give a sufficiently good picture for the pur- pose, and has the advantage of not interfering with the addition of further stain when desired. The tube or barrel of the microscope is made in two forms, the long or English type being from 8 to 10 inches, and the short or Continental type having a length of 6% inches. The latter is the more desirable form, since the majority of manu- facturers of objectives adjust them for the short tube. APOCHROMATIC OBJECTIVES. This term is applied to a particular variety of lens contain- ing a special kind of glass (containing calcium jflvioride), be- sides the usual crown and flint glass, the object of this being to produce a greater degree of achromatism, thus reducing chromatic aberration. The special value of these objectives when used with a compensating ocular (compensating eye-pieces are specially constructed for use with apochromatic objectives), is as follows: Three rays of the colored spectrum, instead of two, as in case of the achromatic glasses, are focused in the same plane, leaving only a minute tertiary spectrum ; also with these objectives the spherical aberration is corrected for two 6 THE MICROSCOPE. colors in the brightest part of the spectrum, and the objective shows the same correction for marginal rays as for the central part of the aperture. Finally, apochromatic objectives admit the use of highly magnifying oculars. This form of objective is particularly useful in photomierographic work, where it is very important to abolish chromatic aberration. ILLUMINATION. Illumination constitutes a most important factor in the practical use of the microscope, and upon its proper manage- ment depends very much of the efficiency of the work. Direct, unmodified sunlight is unsuited, and should not be employed, for general work. North light is the most uniform and steady, and is to be preferred. The use of artificial light should, as far as possible, be avoided, and when it must be used its character and color may be greatly improved by inserting a piece of blue glass between the reflector and the object. It is alvifays neces- sary to keep the eye close to the ocular and if possible to keep the unemployed eye open. Too much light is always to be avoided, as it is only an added strain to the eyes, accomplishes nothing, and may materially interfere with the efficiency of the work. Oblique Light. — For urinary work the oblique light is best. By this term is meant light in which the parallel rays from the plane mirror meet the optical axis of the microscope at an angle. This form of light may be obtained in the following ways: (a) By placing the reflector to one side of the stage; this results in an oblique ray without materially lessening the strength of the light; the condenser must, of course, be swung out of the way. In microscopes which have a fixed mirror, i.e., only mov- able perpendicularly at right angles to the axis, oblique light may be obtained through the condenser, as follows: (6) First, focus the light upon the object through the condenser; then, lower the condenser until its focus is considerably below the plane of the object; thus the rays emerging from the condenser will decussate and then diverge, so that all but axial rays will fall upon the object in an oblique direction. TO CLEAN THE MICROSCOPE. 7 TO CLEAN THE MICROSCOPE. Should the lenses or oculars become blurred or spotted from dust or dirt, proceed as follows : If the dirt is upon the ocular it will be discovered by rotating the ocular within the barrel while observing the illuminated field of the microscope. If the obstruction is upon the ocular it will be seen to move; if upon the objective it will remain stationary during this manipula- tion. To find an obstruction upon the objective rotate it, when the spot or mark will move with it. Finally, if after testing both the objective and ocular in the manner described above the location of the dirt or dust is not demonstrated, the trouble will then be found upon the glasses or spectacles if the observer happens to wear them. Having located the trouble, remove the affected part and cleanse as follows : ' If careful polishing with bibulous paper or fat-free silk fails to accomplish the desired result, then moisten the silk or paper with a trace of distilled water, using this to aid in the removal, finally polishing dry. For oily or resinous smears a small amount of alcohol may be used. ' Special care should then be exercised to avoid any excess of the solvent which may enter between the individual lenses and, dissolving the cement, ruin the part. After using the oil- immersion it should always be cleaned before leaving the instru- ment. After removing the excess of oil with the silk, wipe off the remainder with a trace of xylol or benzine, immediately wiping dry. Cover-glass preparations may be treated in a similar way. Glass surfaces should never be touched with the fingers, and great care should be exercised to avoid dropping and possi- ble fracture of the oculars and objective. All metal parts of the instrument should be kept free from liquids, particularly acids and alkalies, benzine, xylol, alcohol, turpentine, and chloroform. To clean the mechanical parts, none but the best machine oil should be used, and this only in small amounts and at long intervals. The polished brass requires nothing but occasion- ally polishing with clean chamois. Two keynotes to successful use and preservation of the microscope are: handle with care and keep scrupulously clean. THE MICROSCOPE. THE MECHANICAL STAGE. ()f the )iiany ai x>v^ ^ 1^ y>^ *>* ^»j. "v J^ 1 ^ ^^^Sh 5 »^ |a^ E ' X ^^ \ ^H|^^ w Fig. 4. — Bronchial Cast, from case of Fibrinous Bronchitis IN SERVICE OF DR. JuDSON DaLAND (OrIOINAL). of the lung in Ijroneho-pneumonia, empyema or in bronchitis. If the Ijlood is dark or black it may he due to pulmonary infarction. Most commonly hemoptysis is observed in phthisis, when it recurs intermittently for days or weeks. Finally, it should be remembered tliat malingerers may simulate disease of the respiratory tract by sucking blood from wounds of the gums, lips, tongue or cheeks. (h) Tlcmorrhwiic ftpntniii is oliserved in traumatic or tuberculous hemorrhage, in hemorrhagic infarctions, and in lobar AIR CONTENT. 23 pneumonia. Also in tumors in or near the respiratory tract, and finally in congestion of the pulmonary circulation. Certain derivatives from the blood-pigment produce in the sputum shades very similar to that-of blood — the rusty sputum of pneumonia, for instance. The coloring matter here is partly changed blood and partly a yellowish-red derivative of blood- pigment, about which very little is known. Peculiar' lemon- colored and grass-green shades are also frequently observed in pneumonic sputum. These likewise are due to changed blood- pigment. Such sputa respond to Gmellin's test for bile-pigment (see page 214). A peculiar light-brown sputum is sometimes observed in heart disease, particularly mitral, in which amor- phous blood-pigment is found encapsulated in the alveolar epi- thelium (heart-failure cells) . A type of green sputum has been observed in cases of lung tumor, the nature of the pigment in question is as yet unknown. (i) Extraneous discolor ations. Other noticeable discolora- tions of the sputum are observed from the admixture of inhaled dust-particles. The hlack sputum of miners, and the Hue spu- tum of workers in ultramarine, are examples of this class. Finally, it should be remembered that the sputum voided, following the ingestion of certain food-stuffs, may be discolored from contamination with these occurring in the mouth and pharynx. A greenish discoloration of the sputum is sometimes the result of the activity of certain chromogenic bacteria, espe- cially the Bacillus virescens.^ Yellow and bluish sputa of prob- able bacterial origin have occasionally been observed. REACTION. The reaction of fresh sputum is generally alkaline; it may occasionally be acid, and usually becomes so after standing for some time through decomposition resulting from bacterial growth. AIR CONTENT. Sputum is often more or less foamy or frothy, due to the presence of air. Other things being equal, the content of air in the sputum is greater the finer the bronchi from which the iFrick: Virchow's AroWv.. Vol. cxvi.. 1889. 24 THE SPUTUM. sputiun is derived. The consistence of sputum often has a bear- ing on this. The amount of air contained can easily be deter- mined' by comparing its specific gravity to that of vrater. Air- containing sputum will float; airless sputum virill sink. THE ODOR. Fresh sputum has a rather characteristic, but indescribable, odor. On standing it may acquire a disagreeably nauseating odor from decomposition, resulting from contained bacteria. Freshly voided sputum has a very decided odor in bronchiectasis, purulent bronchitis, tuberculosis, gangrene of the lung, and lung abscess. The disagreeable odor here arises from the activity of putrefactive bacteria. Stagnation of the secretion in cavities favors decomposition. In this vi^ay the foul odor of the expecto- ration in consumptives may be imparted to the breath. Finally, it must be remembered that fecal vomiting and diseased condi- tions of the mouth may be responsible for the odor of the breath and sputum, as will also the ingestion of certain volatile drugs, including alcohol. HEMOPTYSIS. True hemoptysis means the expectoration of an appreciable amount of pure, or nearly pure, blood, not merely sputum tinged with blood. The amount of blood may continue small, and may persist for many days or may, as in the case of rupture of an aortic aneurism, be sufficiently large to cause death in a short time. Before making a diagnosis of hemoptysis it is necessary to exclude blood coming from the nose, pharynx, larynx, buccal cavity, and tonsils. The Common Causes of Hemoptysis. — (a) Pulmonary dis- ease, usually tuberculous. It may also occur in the early stages of lobar pneumonia, abscess, bronchiectasis, gangrene, and can- cer of the lung. (b) Cardiac disease. Here it may be the result of venous obstruction occurring in the course of valvular disease. This is a not uncommon cause of slight but long-continued bleeding. (c) Vascular disease. The most important condition is GROSS EXAMINATION OF SPUTUM. 25 rupture of an aneurism into the respiratory tract. Leakage from the same cause may cause slight but persistent hemoptysis. (d) Diseases of the Mood. Hemoptysis may occur during the course of hemophilia, purpura, leukemia, scurvy, and severe anemia. It has been noted occasionally in the course of some of the exanthemata. (e) Miscellaneous causes. Vicarious menstruation and hysteria. GROSS EXAMINATION OF SPUTUM. Examination should be made both\ipon a white and a black background. Many sputa appear to the naked eye to be homo- geneous — pure mucus, pure pus, pure blood ; but sometimes not only may the sputum of one patient vary, but difEerences may be noted in each expectoration. "Dittri^Ji's plugs" are yellowish-white masses, the size of a mustard-seed, and are easily seefi over a black background. They come from the smaller bronchioles in putrid diseases, especially in putrid bronchitis and pulmonary gangrene. Micro- scopically they are composed of clumps of bacteria and fatty acid crystals. They have a very intense and disagreeable odor. Somewhat similar plugs may be encountered in the sputum in follicular tonsillitis. These plugs should not be confounded with those little masses described by, and known as, Cursch- mann^s spirals. (See page 27.) Formations consisting of fibrin are encountered in certain infections, and should be easily recognized by their white color, tenacious consistence, and sometimes by their shape (casts and molds) . Foreign bodies are sometimes aspirated into the respiratory tract, where they may remain for years without causing any symptoms until a fit of coughing dislodges them and they appear in the sputum. Concretions are sometimes, though very rarely, formed in the lungs during chronic inflammatory conditions. These are accidentally coughed up when they may be found in the sputum. Occasionally these stones may have their origin in the- crypts of the tonsils, or they may be calcified -lymph nodes that have ulcerated into the lung. 26 THE SPUTUM. MICROSCOPIC EXAMINATION OF THE SPUTUM. A microscopic examination of the sputum reveals the pres- ence of cells, elastic fibers, casts, spirals, crystals, and micro- organisms. It is advisable to examine first the fresh unstained sputum, as the presence of fungi or crystals in the sputum, and the nature of many of the cellular elements, can be determined only in this way. Afterward dried and stained cover-glass preparations may be made for more minute and detailed study. Preliminary Examination. — This is necessary in order to locate suspicious particles which may be scattered throughout the large mass of sputum. This is made either with the unaided eye or with a hand-lens. A thin layer of sputum is necessary to successful examiaation. Por this purpose a moderate-size Petri dish and cover is much better than the flat pieces of glass ordinarily employed, which are uncleanly and difficult to handle. A small amount of sputum is placed on the inside of the cover, and the other half of the dish pressed down into this, the rim very successfully preventing the escape of excess of material. The Unstained Specimen. — Por the purpose of isolating any characteristic particles, the sputum should be spread out in a thin layer in the dish, and the material teased out with needles or tooth-picks. Having located a likely particle, it is transferred to a clean slide and flattened out by pressing a cover-glass down " upon it. This should be examined first by the low, and later by the medium, power objective. Appearance, — Most sputum consists, microscopically, of a ground-work of mucous matrix of indefinite structure and appearance, in which are imbedded a variety of microscopic objects, principally cells. 1. Pus cells. The number of these indicates, in a general way, the purulent nature of the specimen. The character of the corpuscles varies greatly. Their size is from 7 to 10 micro- millimeters, they appear more or less granular, are sometimes distinctly pigmented, containing one or more irregular nuclei. The granules are composed some of proteid, some of fat, and some of extraneous debris. 2. Epithelial cells found in the sputum differ from the pus cells, being usually larger in size, and by exhibiting one rather MICROSCOPIC EXAMINATION OF THE SPUTUM. 2T large vesicular nucleus. Various types of epithelia are met in the sputum, and their recognition is of considerable value in locating the origin of the expectoration, although, many times, the conditions to which they have been subjected after being shed have so altered their appearance that little knowledge can be gained from their study, (a) Squamous epithelia are derived from the mouth, the pharynx, and from part of the larynx, (h) The cylindrical epithelium is derived from the nose or from the smaller bronchi, and are seen as pear-shaped or oval cells, some of which possess cilia, (c) Pulmonary, or alveolar epithelium, is oval and measures from 20 to 30 micromillimeters in diameter. 3. "Heart-failure" cells. These are oval or round, pig- mented, alveolar cells. When numerous their presence is said to be indicative of chronic passive congestion of the lungs, usually depending on the failing compensation of cardiac valvular dis- ease. Their presence is, therefore, usually associated with the common signs in the lungs, which are indicative of failing com- pensation, viz. : moist rales, mucous expectoration, and cyanosis. 4. Eosinophiles may occasionally be found in large num- bers associated with Charcot-Leyden crystals in the expectora- tion of bronchial asthma. 5. Red-Mood cells. The appearance of red-blood cells in the sputum will depend largely upon the length of time that they have been shed. As they grow old they become pale, shad- owy, and fragmented. The finding of a few red-blood cells in the sputum is of no diagnostic import. They occur naturally in large numbers in hemoptysis, and are constant and more or less abundant in all inflammatory diseases of the lungs, par- ticularly phthisis. 6. Casts. These may vary in size from those which repre- sent molds of the trachea and larger bronchi to those coming from the smaller bronchioles, and which are from 1/4 to 1% inches long. These smaller casts are the more common, and when present usually require the use of the low-power objective to demonstrate them. These casts usually occur in one of the three following diseases: the largest in diphtheria, medium- size in fibrinous bronchitis, and the smallest in lobar pneumonia. 7. Curschmann's spirals consist of worm-like spirals 1 to 2 centimeters long, and about 1 millimeter wide. They are 28 THE SPUTUM. more or less opaque, and are usually found surrounded by a thick, clear mass of mucus. They frequently show a central, undulating, thread-like core around which are twisted, in a spiral manner, the mucous threads. Entangled in these spirals are usually eosinophiles and Charcot-Leyden crystals. They occur frequently in the sputurii of bronchial asthma, more rarely in phthisis, bronchitis, and in lobar pneumonia. Their presence may be of service in differentiating bronchial from other forms of asthma. Crystals. — (a) These are found usually only when the sputum has been retained within the body for a length of time. Crystals of fat or of fatty acids are most frequently encoun- tered. They appear as long, slender needles, either singly or grouped into fine rosettes or sheaves. They are readily soluble in potassium hydrate or in ether. This solubility is easily de- termined by allowing a little of either fluid to flow under the edge of the cover-glass while observing the crystals in question. (b) Crystals of calcium phosphate may be encountered under conditions of retention and stagnation. (c) Charcot-Leyden crystals are occasionally encountered, particularly in the expectoration of bronchial asthma, and are here accompanied by eosinophiles. They appear as colorless, elongated double pyramids, varying considerably in size. They are often so small that high magnification is required to reveal them. (d) Cholesterin crystals are but rarely seen in the sputum. They occur as transparent, colorless, rhomboidal platelets, with notched or irregular angles and ends. (e) Hematoidin crystals are derived from hemoglobin by a process of decomposition, and occur as needles and rhomboidal platelets of reddish and brownish hue. They are found chiefly in the sputum from old" abscesses or perforating empyemas. (f) Leucin globules and tyrosin crystals are found in putrid sputum from old perforating abscesses or in putrid bron- chitis. (g) Calcium oxalate, in minute octahedral crystals, are occasionally met. Elastic Fibers. — When the lung-tissue is destroyed to any extent by pathologic processes, elastic fibers are apt to be PREPARATION OF THE STAINED SPECIMEN. 29 encountered in the sputum. Their presence in the sputum proves conclusively the occurrence of some destructive process within the lung. Hence their importance ia the diagnosis of tuberculosis of the lung before the appearance of tubercle ba- cilli. Elastic fibers occur also in pulmonary abscess and gan- grene. These fibers are usually detected in a thin layer of sputum examined microscopically. In this examination care must be observed to avoid confusing true elastic fibers with the some- what similar vegetable fibers, which latter are generally larger and less uniformly wavy. To detect particles or shreds of elastic tissue in the sputum, suspicious lumps should be thoroughly mixed with an equal quantity of 20-per-cent. sodium hydrate solution; then a large volume of water is added, and the whole allowed to sediment for a few hours. The sediment is then removed and examined under the microscope for the characteristic fibrillated masses. If elastic tissue is not found by this procedure, the entire quantity of the twenty-four-hour specimen should be boiled with an equal quantity of the sodium hydrate solution. The resulting gelatinous mass is then mixed with several volumes of water, and allowed to sediment. About 15 cubic centimeters of the sediment is now removed with a pipette and centrifuged for fifteen minutes. The final precipitate is now carefully removed and examined as above. Fragments of tumors are occasionally encountered in the examination of sputum. These should be removed and pre- pared for sectioning and staining. PREPARATION OF THE STAINED SPECIMEN. Another suspicious particle having been isolated and re- moved from the mass of sputum, it is transferred to and care- fully spread upon a clean cover-glass. This should then be treated to fixation and staining, the technic of which will de- pend upon the nature of the information sought. (For methods of staining and for differentiation of the organisms by anti- formin, etc., see section on Bacteriologic Methods, page 344.) 30 THE SPUTUM. PULMONARY ACTINOMYCOSIS. This condition, also known as disease of the ray-fungus, occasionally causes disease of the lung, but is exceedingly rare in this country. The characteristic yellowish- or grayish- green granules, if found, are often sufficient for a diagnosis, which should, however, always be confirmed, by microscopic search. In some cases the characteristic microscopic rosettes with clubbed rays are found; in others only branching threads, staining by Gram's method, will be found. Clinically, the course of the disease is similar to that of pulmonary tuberculosis, except that instead of the tubercle bacillus the ray-fungus is found. ECHINOCOCCUS. Earely eehinococcus booklets enter the pulmonary tract and appear in the sputum. They usually originate in abscesses of adjacent organs, particularly the liver. (See section, "Animal Parasites," page 101.) DISTOMUM PULMONALE; SYN. DISTOMUM WESTERMANNL This organism (for classification see page 102) is a not uncommon cause of disease of the lung in eastern countries, particularly in Asia, but may occasionally be encountered in other parts of the world.' The symptoms are not unlike those of pulmonary tuberculosis, for which it- may be mistaken. Its presence is determined by finding the ova in the sputum. These are oval, of a brownish-yellow color, with a fairly thin shell, and measure 0.0875 to 0.1025 mm. in length, and 0.052 to 0.075 mm. in breadth. III. THE BLOOD. THE CHEMICAL COMPOSITION OF THE BLOOD. A GENERAL idea of the composition of the blood may be had from the following table, which is taken from Simon's "Physiologic Chemistry." The calculations are made for 1000 parts by weight. Corpuscles 480 00 parts Water 276.90 " Oxyhemoglobin 193.90 " Stroma, including salts 9.20 " Plasma 520.00 parts Water 477.37 " ' Albnm.ns 35.88 " Extractives 2.39 " Inorganic salts 4.36 " The predominating solid substance in the blood is oxy- hemoglobin; it represents 10 per cent, of the total weight of the blood, 40 per cent, of the weight of the corpuscles, and 65 per cent, of all organic matter present. The mineral constituents comprise sodium, potassium, cal- cium, magnesium, and iron. Fats are present to the extent of from 0.2 to 0.3 per cent. These may be temporarily increased after the ingestion of much fatty food, and also in many pathologic conditions. The plasma normally contains small amounts of oxygen and nitrogen in solution, with varying amounts of carbon dioxide. METHODS OF OBTAINING BLOOD FOR EXAMINATION. The tip of the finger or lobe of the ear are the sites usually selected from which to obtain specimens. In the majority of (31) 32 THE BLOOD. examinations only a small amount — a few drops — is necessary. This is obtained by simple puncture of the skin made with a glover's needle, the half-point of a new steel pen, a Daland lancet (see Fig. 5), or the so-called pistol-knife. The two last-mentioned instruments are to be preferred because they permit of regulation of the depth of the puncture. For larger quantities of blood, a few cubic centimeters or more, it is advisable, provided there is no contraindication, to obtain the specimen by the use of wet cups. It must be borne Fig. 5. — Fleischl Hemoglobinometkr, surrounded by accessories necessary to performance of the test, INCLUDING Daland Lancet. in mind that by this method there is always more or less ad- mixture of lymph. Another method is to draw the blood from a dilated vein into a large sterile antitoxin s)'ringe. Lastly, an ordinary venesection may lie resorted to. The withdrawal of l^lood, if ascptically performed, is prac- tically free from danger and need disturb the patient very little. Before proceeding in any case it is advisable to determine the absence of bciiiopbilia in ilie patient. Appearance of Fresh Blood. — The exuding drop of blood THE SPECIFIC GRAVITY. 33 shows even to the naked eye a number of properties. The redder it iSj the richer it is in oxyhemoglobin; the darker, the greater the amount of reduced hemoglobin. Microscopically, it reveals a great number of cellular elements; these are colored and col- orless discs. The red cells appear as non-nucleated bi-concave discs, measuring on the average 7 micro-millimeters in diameter. Viewed singly through the microscope by transmitted light, they are of pale-greenish hue. The colorless cells or white corpus- cles are, as a rule, somewhat larger than the red cells, and pre- sent either mono- or poly-nucleated protoplasm. The plaques, or Mood-platelets, appear as minute, color- less discs measuring less than half the diameter of the red cells. They usually occur in groups or bunches of half a dozen or more, and are present in normal blood to the number of about 635,000 per cubic millimeter. There are no other morphologic constituents of the blood. Color. — The color of normal blood is due to the presence of an albuminous substance in the corpuscles termed hemo- globin. In the arterial blood it is in combination with oxygen, and is here termed oxy-hemoglobin. In the venous blood a mix- ture of both hemoglobin and oxy-hemoglobin occurs. With a preponderance of oxy-hemoglobin, the blood tends to a scarlet hue; when the hemoglobin predominates, the blood is of a bluish color. Pathologic Oianges in Color. — In coal-gas poisoning the blood is cherry- red. After poisoning from potassium chlorate, aniline, hydrocyanic acid, and nitro-benzol, the blood is brownish-red or chocolate color. In extreme cases of leukemia the blood may have a milky appearance due to the excessive number of white blood cells present. The Odor. — This is characteristic and differs in different species of animals. It is due chiefly to the presence of volatile fatty acids. The Taste. — The taste of blood is salty, but at the same iime insipid. THE SPECIFIC GRAVITY. The specific gravity seems to vary with the amount of hemoglobin. It is influenced by the age and sex of the indi- 34 THE BLOOD. vidual, the process of digestion, exercise, pregnancy, etc. The normal average in adults varies between 1.058 and 1.062. Determination of the Specific Gravity (method of Ham- merschlag). — A cylinder about 10 centimeters in height is partly filled with a mixture of benzol (sp. gr. 0.889) and chlo- roform (sp. gr. 1.526), so that the specific gravity of the mix- ture lies between 1.050 and 1.060. Into this a drop of blood is allowed to fall directly from the finger. It is then brought into suspension by the addition of either a little chloroform or benzol, according to the tendency of the drop to sink or rise in the cylinder. As soon as the drop remains stationary in the fluid the specific gravity of this is taken by an accurate hydrometer (one reading to the fourth decimal should be used). The reading represents the specific gravity of the blood tested. THE AMOUNT. The total amount of blood in the normal adult is said to amount to about one-twelfth or one-fourteenth of the body weight. THE REACTION. The reaction of the blood is slightly alkaline, due to the presence of the mono-sodium carbonate and the di-sodium phosphate in solution in it. The reaction may be roughly determined by drawing a strip of neutral litmus paper, which has been thoroughly moistened with a concentrated solution of common salt, through the blood, and then rapidly washing the corpuscles ofE with the same solution. Owing to the develop- ment 6f certain acids, the alkalinity of the blood rapidly dimin- ishes after it is shed. This fact renders this determination a rather difficult matter. The normal variation of alkalinity is very slight. By accurate titration the normal degree of alka- linity of the blood, under normal conditions, corresponds to from 335 to 360 milligrams of sodium hydrate for every 100 cubic centimeters of blood. ESTIMATION OF HEMOGLOBIN. 35 QUANTITATIVE CLINICAL METHODS. The usual clinical methods applied to the blood in the study of disease are: the estimation of the percentage of hemoglobin, the enumeration of the erythrocytes and the white blood-corpuscles, and a differential count of the various white elements. The apparatus necessary for the performance of these sev- eral examinations are as follows: — ■ I. — A good microscope with 3, 6, and oil-immersion (Viz) objectives. II. — A hemoglobinometer (Gower's, Pleischl's or Sahli's). III. — A Thoma-Zeiss hemocytometer. IV. — Slides, covers, stains, etc. (For complete list see Appendix.) Estimation of the Percentage of Hemoglobin. — Method of Gower: The tip of the finger or lobe of the ear is punctured after having been thoroughly cleansed with alcohol, followed by ether. The first drop obtained is wiped away, and the second, which should flow without assistance either by pressure or rub- bing, is drawn up by suction into the pipette to the 30 centi- meter mark; all adhering blood is wiped from the outside of the tube. The contents are now immediately forced out into the graduated observation tube, which has previously had a few drops of distilled water placed in it (this is to prevent the blood from coagulating on its walls). Be sure that all blood contained in the mixing tube has been washed into the gradu- ated tube. Now, while holding the two tubes side by side directly against the light, add distilled water, drop by drop, until the shade of color is the same in the two tubes. The division on the scale to which the fluid rises will express the per cent, of hemoglobin. Method of Fleischl. — The instrument (Fig. 5) consists of a metal stand having a horizontal table with a circular aper- ture in its center, beneath which is placed a reflector of plaster- of-Paris. Immediately beneath the aperture and above the reflector, a graduated wedge of tinted glass is arranged to move in a horizontal plane by means of a milled thumbscrew. This graduated wedge of glass is shaded to represent varying degrees 36 THE BLOUD, of liemogioljin content, and canies with it a scale indicating the Idood-strengths. For collecting and diluting the blood for examination, a i)ipette and diluting chamber are furnished. The technic is as follows: — By the aid of the jiipetto which is furnislied with the ap- paratus, an exactly determined amount of blood is dissolved in a measured ((uantitv of distilled water (the contents of one-half of tlie diluting chamber, the other half being filled with an ecpial amount of distilled water). The cell thus prepared is then placed over the aperture in the table, and artificial (candle, lamp, or gas) light is directed tlirougli it by means of the re- flector. The part of the wedge is now searched for which accurately com]iares in tint with tlie solution of blood under Fig. 6.— Thoma-Zeiss Hemocvtometer ix C.ise. (A. H, T, Co.) examination, and the numlier of the scale is read olf that corre- sponds h) this point on the glass wedge. (It is a matter of com- mon ex])erience that the grading of these instruments is too 'liigh, and that a specimen of lilood that coi'responds to 90 or 95 on tlie scale is normal.) Imimeration of the Corpuscles. — The Thoma-Zeiss ap- pai'atns consists of a couuting slide and two diluting pipettes, tei'med "melangeni's" (Fig. (i ) . Tliis slide is so constructed that lit contains a chamber in its center having a de]ith (d' \\^i milli- TnetiT. The center of the floor of the cell is di\ided liv line niii-i'oscopic lin(>s into minute squares, the sides of which are |, equals the dilution of the biood, and 400 (all) the small squares counted. Then the nrinber of leukocytes per cul)ic millimeter of undiluted blood will be found iiy dividing tlic number of cells counted by the number of .squares counted and multiplying by the dilution times 4000, viz.: — Let x equal the number of leukocytes per cubic millimeter, and y equal the number of cells counted in 400 small squares. Tlien, ,;;,, x'20 X 4000 = x or, y X 200 ^ X = leukocytes per cnl)ic millimeter of undiluted blood. ^Sahli's DIafrnosis quoting: Capps ; Joe. clt. PLATE II. Zi' /-=%■. ..{■'^^•;.. '00" Normal and Pathological Blood-cells. /, Normal red cell, or erythrocyte ; diameter, 7.5 m. 2, Nucleated red cell, or normoblast: diameter, 7.S /i. j, Megaloblast ; diameter, 8 to 12 M : seen in pernicious anemia. 4, Small lymphocyte ; diameter, 6 to 8 M : average in normal blood, 20 to 26%. 5, Large lymphocyte; diameter, 8 to 13 /" ; average in normal blood, 5 to 9 %. 6, Polymor- phonuclear leukocyte; diameter, 10 to 11 m; average in normal blood, 65 to 70%. 7, Eosinophile ; diameter, 19 to 12 m : average in normal blood, ^ to 2%. 8, Large mononuclear leukocyte ; diameter, 12 to 18 H ; average in normal blood, 1 to 2%. 9, Transitional : diameter, 12 to 17 M : average in normal blood, 2 to 3%. 10, Neutrophilic myelocyte ; diameter, 12 to 20 m; seen in myelogenous leukemia. //. Eosinophilic myelocyte; diameter, 10 to 16 iu; seen in myelogenous leukemia. ■QUANTITATIVE CLINICAL METHODS. 43 Examination of a Drop of Fresh Blood. — If a drop of fresh blood is placed upon a slide and a perfectly clean cover-glass allowed to fall upon this, a fresh preparation for examination will be produced, which may either be examined immediately or if sealed around the edge with a little vaseline, may be car- ried to the laboratory and examined within an hour. With the aid of a 6 objective many interesting points may be observed. First, the shape of the red cells and their hemoglobin staining, also rouleaux formation. Second, the structure of the white corpuscles by which the different varieties may be roughly dif- ferentiated. Third, the hlood-platelets. Fourth, a rough idea of the relative number of red and white cells may be formed. This matter will be discussed more in detail a little later. Studies of the minute structure of the leukocytes cannot be made satisfactorily by this simple method, as some of the forms are present only in small numbers, and are detected with difSculty. Further, prolonged examination of the fresh specimen allows time for changes, which rapidly obscure the identity of the cells ; hence we are obliged to resort to a method of preparing a specimen which shall be permanent and which can be stained as a further aid to differentiation. These meth- ods have the added advantage that they allow the investigator to work quite independently of the presence of the patient, to choose the time and place of examination, and permit at any time of verification and demonstration of the original result obtained. Preparation op the Specimen. — The blood is obtained from the tip of the finger or the lobe of the ear after the man- ner described above. Cover-glasses should previously be pre- pared for reception of the fresh blood as soon as it is drawn. The cover-glasses should be square, of seven-eighths or one inch in diameter, and of special thinness (0.1 to 0.08 mm.). They should be washed carefully with warm water and soap, dried with fat-free gauze, and finally wiped off with ether. This is necessary to insure proper spreading of the blood between the two glass surfaces, which would be prevented by traces of fat or particles of fiber or dust, etc. Just before the glasses are used they should be wiped off with a piece of silk or tissue paper, and thereafter handled with care by the comers, or. 44 THE BLOOD. better still, with forceps. This preparation of the cover-glasses may be done in the laboratory or carried on at the bedside, as desired. The glasses being ready and the finger cleansed and punc- tured, a small drop of blood is allowed to exude without pres- sure. Xow a cover-glass is taken up diagonally between the thumb and finger of one hand, and its center allowed to touch the drop of blood. Immediately taking another cover-glass in the same manner in the other hand, it is allowed to fall upon the drop. If the glasses are perfectly clean and the maneuver properly executed, the blood will immediately spread out in a uniform layer between the two glasses. The two cover-glasses are now separated one from the other by a rapid sliding motion, in the strict plane of their surfaces, without the slightest lifting or tilting. If the drop taken was small enough and the technic prop- erly carried out in every detail, the result will be two uniform smears, covering the larger portion of the surfaces of the cover- glasses which, examined under the microscope, will present a uniform layer of corpuscles, with little if any overlapping of the individual cells. Usually six films are prepared in this manner and allowed to dry in the air without heat. ^Methods of Fixation. — In using the Eomanowski stains no previous fixation of the albuminous film is required. Before applying any of the other stains it is necessary to "fix" the blood-film. If this is not done the corpuscles will be washed from the glass liy the fluids applied. 1. A strip of sheet copper eighteen inches long and two or three inches wide, is placed upon a stand, and the flame from a Bunsen burner placed under one end. After allowing suf- ficient time for the plate to attain a maximum heat the films are placed upon it at a point where a drop of cold water fails to roll off but adheres to the hot metal and steams away. At this point fixation is complete in from one-half to three-quarters of an hour. 2. Fixation may be accomplished by immersion for five to ten minutes in a mixture of 1 part formaldehyde solution and 99 parts absolute alcohol. STAINING METHODS. 45 3. For routine work when fair accurac}', accompanied by speed in obtaining results, is necessary, fixation in the naked flame of a Bunsen burner may be practiced. This is accom- plished by holding the dried smear diagonally between the thumb and forefinger of one hand, film side up, and passing it through the flame a number (five to ten) of times with sufficient rapid- ity to prevent the fingers being burned. Methods of Staining: Eosin and Methylene-Blue. — The fixed film held in the cover-glass forceps is flooded with a %-per-cent. alcoholic solution of eosin, which is gently washed oS with distilled water after the lapse of one or two minutes. The counter-stain of methylene-blue, 1-per-cent. aqueous solu- tion, is then added and allowed to act for from two to five minutes, according to the density of the film. Finally, wash- ing in distilled water, blotting and drying, complete the process prior to mounting and examination. This is an extremely simple, yet efEective and permanent, method of staining blood-smears. Little difficulty will be ex- perienced in obtaining satisfactory specimens. The eosin should not be allowed to act too long, but there is little danger of over- staining with the methylene-blue. Both of these stains are permanent when prepared, and can be kept until exhausted. Properly performed this method gives very vivid and con- trasting pictures, the nuclei of the different white cells fak- ing various shades of blue, pale in the lymphocytes and dark in the polymorphonuclears. The protoplasm appears faintly tinted blue (paler than the nuclei). The eosinophilic granules are a bright pink. The erythrocytes a dusky red. Ehrlich's Triacid Staining Method (for formula and prepa- tion see Appendix). — The smear is taken in the cover-glass forceps and 'a few drops of the prepared stain placed upon it and allowed to remain for about five minutes. There is little danger of over-staining. The specimen is next washed in dis- tilled water and thoroughly dried prior to mounting. If' mounted in Canada balsam it is imperative that this should not contain any chloroform, otherwise the colors will gradually become blurred. The RomanowsTci or "Universal" Staining Method. — Leish- mann's modification of the Eomanowski stain, as made by 46 THE BLOOD. Wright,* is the stain used. This method is altogether quicker and easier than the method of Ehrlich. It requires no fixing fluid or heating apparatus, and gives pictures which are uni- formly superior. Technic. — Allow three or four drops of the prepared stain to fall upon the smear and permit it to remain one-half min- ute, rocking the cover . gently so as to insure an even distribu- tion of the stain. Xo attempt is made to cheek evaporation. At the end of one-half minute add double the quantity of dis- tilled water, i.e., six to eight drops, and allow it to mix with the alcoholic stain. Immediate mixing is hastened by gently rock- ing the cover-glass. The film is now allowed to stain for five minutes. In thick smears ten minutes may be necessary. The stain is now gently washed off with distilled water, and a few drops of water are allowed to rest on the film for one minute, when it should be dried in air and mounted. Appearance of Blood-Films. ^ — ^Erythrocytes, pale pink or greenish semi-transparent. Polymorphonuclears. — Nuclear network, stained a very ruby-red color, or with sharply defined margins. Extra-nuclear protoplasm, colorless. Fine eosinophilic granules, red. Mononuclears. — Nuclei ruby-red with extremely sharp, clear outlines. Extra-nuclear protoplasm, pale eau-de-nid or blue, occasionally showing a few red granules. Lympliocytes. — Same as mononuclears, except that as a rule the nuclei are more deeply stained. Coarse-Grained Eosinophiles. — Nucleus only, red, but not so densely stained. Granules pale pink. Basopliiles. — Granules very densely stained of a very pur- plish-black tint. Nucleus red, but usually more or less meshed by granules overlaying it. Nucleated Red Cells. — Nucleus almost black, with sharp outline, extra nuclear portion gray. Blood Plates. — Deep ruby-red with spiky margins, fre- quently showing a pale blue spherical zone surrounding the red center. Bacilli and Micrococci. — Speaking generally, these stain ' Wright: Jour. Med. Research. Vol. vii, 1902. ' British Medical Journal, Vol. li, pege 757, 1901. TERMS IN COMMON USE IN CLINICAL HEMATOLOGY. 47 evenly blue, but by prolonging the staining period and subse- quently decolorizing with absolute alcohol many interesting variations may be noted with different organisms by which structural details are brought out not generally observed by other staining methods. Malarial Parasites. — The body of the parasite stains blue and its chromatin ruby-red. In the case of the tertian parasite, Schuffner's dots are well marked in the containing red blood- corpuscles. The only weak points in the Romanowski stain are said to be .the deceptive resemblance between megaloblasts, certain lymphocytes and certain myelocytes, and failure to differentiate the basophiles. TERMS IN COMMON USE IN CLINICAL HEMATOLOGY. Anemia. — A condition of the blood in which there is a deficiency in one or more of the normal constituents. Anliydremia. — A deficiency in the normal fiuid of the blood. Basophilic Granulation. — A peculiar granular degeneration of the red blood-cells which is noted in chronic lead-poisoning. Hydremia. — An excess of fluid in the blood. Leukocytosis. — ^An increase above the normal number of white blood-cells. Leukopenia. — A diminution in the number of white blood- cells. Lipemia. — The presence of an abnormal amount of free fat in the blood. Macroblasts.— Nucleated red blood-cells of more than nor- mal diameter. Macrocytes. — Abnormally large red blood-cells. Melanemia. — The presence of free pigment in the blood. Microblasts. — 'Nucleated red blood-cells of abnormally small size. Microcytes. — Abnormally small red blood-cells. Normoblasts. — Nucleated red blood-cells of normal diameter. 48 THE BLOOD. Oligochromemia. — A diminution in the normal amount of hemoglobin. This may occur either independently or coin- cidently with a diminution in the number of red blood-cells. Oligocythemia. — A diminution in the number of red blood- cells. Plethora. — An increase in the total quantity of blood above normal. Poikilocytosis. — This term is applied to the very irregular shape of the erythrocytes observed in certain pathologic condi- tions. Polychromatophilic Degeneration or Anemic Degeneration (Ehrlich). — This is an atypical staining reaction of the eryth- rocytes, the significance of which is not yet definitely deter- mined. Polycythemia. — An increase in the number of red blood- cells as compared with the fluid-content. VARIETIES OF LEUKOCYTES. 1. Normal Leukocytes. — (a) Lymphocytes : These are derived from the lymph-glands, and appear as small, round cells about the size of a red blood-corpuscle, with a large, centrally located nucleus, and a small margin of protoplasm. The nu- cleus stains rather intensely with the nuclear stains (hematox- ylin, methylene-blue, and Bhrlich's triple stain). The proto- plasm is free from granules. (b) Large Mononuclear Leukocytes. — These cells are two or three times as large as a red blood-cell, with a large, usually oval nucleus which is generally eccentrically placed. It stains poorly with nuclear stains. There is a relatively large amount of protoplasm, which is free from granules.. They are derived from the bone-marrow, and may be regarded as the parent type of the polymorphonuclear. (c) POLYNUCLEAR OF POLYMORPHONUCLEAR (NEUTRO- PHILIC Leukocytes. — These are recognized by their multiple, irregular-shaped, or bent nucleus. The nuclei stain very in- tensely, and the protoplasm is densely packed with neutrophilic granules. (d) Eosinophilic Cells. — These resemble the polymor- THE DIFFERENTIAL COUNT. 49 phonuclear cells, except that the small neutrophilic granules are replaced by coarse acidophilic (eosinophilic) granules. These granules are highly refractive, so that they can be readily recog- nized without staining. (e) Mast Cells. — These are cells of the polymorpho- nuclear type with marked basophilic granules, which are quite large, uneven, and irregularly distributed. They are not dis- tinctly stained by the triple stain. 2. Pathologic leukocytes. — (a) Moxonoclear Keutro- PHiLio Cells (Myelocytes) : These are large cells with a large, faintly-staining nucleus, differing from the large mono- nuclear cells of normal blood by the pre'sence of neutrophilic granules in the protoplasm. (b) MONONUCLEAE EOSINOPHILIC CeLLS (EhKLICH's EosI^TOPI-IILIC Myelocytes) : These cells are what their name implies, mononuclear eosinophiles. Very small cells of this type have been termed eosinophilic microcyies. THE DIFFERENTIAL COUNT. The distinguishing characteristics of the various white cells, as brought out by any one of the differential stains above outlined, allow the investigator to separate these into several groups, and thus to estimate their relative numbers which are usually expressed in percentage. The cells for a differential count can be enumerated without the aid of a mechanical stage, but this instrument of precision is a distinct aid in the per- formance of this procedure and should always be used when possible. Select a imiform and not too dense part of a stained smear, and adjust upon the mechanical stage so that the field presents cells in uniform arrangement without any overlapping. ]Srow, by means of the thumb-screws of the mechanical stage, the field is carried back and forth before the eye of the observer, so that the same part of the field is not brought into view more than once (this matter is very simple when a mechanical stage 'is used). As the parade of cells passes before the eye, the white cells are observed, classified, and the number jotted down. This is continued until not less than two hundred (see page 324 for computing chart to facilitate the count), and preferably five hundred or a thousand, cells have been counted. With the total ,50 THE BLOOD. number of cells counted known, and also the number of cells in each class recorded, it is a simple matter to calculate the percentage of each variety of cell in the specimen examined. The Normal Differential Count. — The normal leukocytic count may vary between 5000 and 10,000 white cells per cubic millimeter. The average normal leukocyte count is usually placed at 7500 leukocytes per cubic millimeter. The minimum and maximum number of cells for each type, estimated from the minimum normal (5000) leukocyte count, is given in the following table: — VABIETT. MINIMUM. :j:AXIMUM. Polymorphonuclears 3000 3500 Small lymphocytes 1100 1500 Large lymphocytes 250 350 Eosinophiles 50 100 Mast cells 5 25 The minimum and maximum number, of cells of each cell- type estimated from the maximum normal leukocyte count (10,- 000). VABIBTY. MINIMUM. MAXIMUM. Polymorphonuclears 6000 8000 Small leukocytes 2200 3000 Large leukocytes 500 900 Eosinophiles 100 200 Mast cells 10 50 For the average normal standard for each cell-type we may adopt the following standard : — VARIETY. NUMBER. PEK-CENT. Polymorphonuclears 4875 65 Small lymphocytes 1950 26 Large lymphocytes 525 7 Eosinophiles 75 1 Mast cells 8 0.1 LEUKOCYTOSIS. For convenience in the study of the various forms of leu- kocytosis, they may be divided into two classes : (a) Physiologic leukocytosis, (h) Pathologic leukocytosis. (a) Physiologic Leukocytosis. — The average number of LEUKOCYTOSIS. 51 leukocytes, per millimeter of blood, is normally 7500. For chil- dren the average is slightly more. For weak and poorly nour- ished persons slightly less. The numbers of leukocytes in the peripheral blood of any individual vary from time to time. They (1) may be increased after a hearty meal, especially if it contains much proteid material. Physiologic leukocytosis may also occur (3) during pregnancy, particularly during the latter months of the condition; (3) in the new-bom, and (4) after cold baths. In these so-called physiologic leukocytoses the increase does not usually exceed 30 per cent, of the normal, though in children it may be doubled. A hypo-leukocytosis is said not infrequently to precede a hyper-leukocytosis. (b) Pathologic Leukocytosis. — Many infections cause an increase in the number of white corpuscles in the peripheral circulation. Although the varieties of cells are the same as in health, the relative proportions are usually altered. In the most common form of pathologic leukocytosis, the percentage of lym- phocytes is diminished, while the polymorphonuclears are fre- quently increased from normal (65 per cent.) to 90 or 95 per cent. The polymorphonucha/r leukocytosis occurs especially during inflammatory processes, and above all in those accompanied by purulent exudation. In certain infectioiis diseases, notably typhoid fever and uncomplicated tuberculosis, there is usually no increase in the number of white blood-cells. The origin of the extra leukocytes has not yet been de- finitely determined, as we do not know whether they are derived from the bone-marrow, lymph-glands, or from other tissues. Another form of leukocytosis is characterized by a relative increase in the number of eosinophilic leukocytes. This condi- tion of the blood is notably observed in bronchial asthma, trichi- nosis, and infections with other animal parasites. It is of in- terest to note that in these conditions there usually exist local collections of eosinophiles at the seat of disease. Thus, in the walls of the bronchi and in the exudate in bronchial asthma, and about the embryos in trichinosis. 52 THE BLOOD. The number of white cells in a pathologic leuliocytosis not infrequently reaches 80,000 to 30,000 cells per cubic millimeter, and has been known to reach the enormous number of 168,000 (Grawitz). Leukopenia. — A diminution in the number of leukocytes in the peripheral blood occurs in a variety of conditions. It has been observed in cachexias, in intoxications, many anemias, and in some infectious diseases, notably in typhoid fever and in malaria. In leukopenia, as in leukocytosis, the relative proportions of the various varieties of white cells are usually changed. For example, in typhoid fever there is often a relative increase in the number of lymphocytes. THE ANEMIAS. Anemias are conveniently classified as primary when due to some unknown cause, and in which the blood-changes are, as a rule, of both a quantitative and a qualitative type. And as secondary, when the cause is known and when the blood-changes are usually of a quantitative type only. The Secondary Anemias. — Causes : Secondary anemia is observed after hemorrhage, during pregnancy, during chronic and constitutional diseases, and in poisoning, including that large and vague group of conditions comprising auto-intoxication. In chronic digestive disorders, malignant tumors, tuberculosis, syph- ilis, malaria, and in the difEerent forms of helminthiasis. The Blood-Changes. — The chief blood-changes in sec- ondary anemia consist in a reduction of hemoglobin and a diminution in the number of red blood-cells. Mild forms show no other changes in the blood-picture. Severe forms show poi- kilocytosis, macrocytosis, and microcytosis. The extent to which these conditions are observed corresponds roughly to the severity of the anemia. Further the red corpuscles often manifest a change in reac- tion to the ordinary staining reagents. They stain poorly, un- evenly, and some parts of some cells refuse entirely to take the stain. This condition is termed polychromatophilia. This THE ANEMIAS. 53 change is no indication of the grade of the anemia, as it is observed in the mildest forms of secondary anemia. The white cells in the simple anemias present nothing that is characteristic. Summary. — The essential blood-changes in secondary, or simple, anemias consist in a diminution in the hemoglobin per- centage, and in the number of red blood-cells. The red cells may show polychromatophilic degeneration and poikilocytosis. The Primary Anemias. — General Considerations : Un- like the secondary anemias, the blood-changes in primary an- emias, besides showing any or all of the modifications observed in the former, present very striking and characteristic alterations in the white blood-cells. Progressive Pernicious Anemia. — In contrast to the process in simple anemia, in progressive pernicious anemia, blood-degen- eration in certain portions of the blood-making organs, notably in the bone-marrow, takes place in a manner different from the physiologic. Consequently, in the blood-formative organs and also in the circulation, we note cells often in great numbers that are never seen in the normal blood. These pathologic elements are present in embryonal life ; so in pernicious anemia we speak of the reversion of blood-formation to the embryonal type. The Blood-Changes. — In a typical, well-defined case of pernicious anemia, the first glance at a well-prepared stiined specimen of blood is sufficient to separate it immediately from the class of simple anemias. We find that a large number of erythrocytes have a diameter greater than normal (megalocytes : diameter 15 to 16 microns). These cells by their staining show a great richness in hemoglobin. Careful search always shows some megaloblasts, i.e., nucleated (embryonal) red corpuscles. ISTormoblasts and microcytes are also in evidence. Other changes usually present are poikilocytosis, polychromatophilia, and gran- ular degeneration. The red corpuscles are always notably decreased, and may be less than 30 per cent, of the normal. The percentage of hemoglobin is also diminished, but practically always in a less degree than the red cells. On account of this condition the color-index is very frequently above 1.0. 64 THE BLOOD. CHLOROSIS. Blood-Changes. — The blood when drawn flows .freely from the puncture, and is markedly watery. Hemoglobin estimation shows a decided reduction in the percentage of hemoglobin with- out a corresponding deficiency in the number of red blood- cells. The color-index is therefore low, below 1.0, in contrast to the high color-index observed in pernicious anemia. This particular characteristic is not unique in chlorosis, as other forms of anemia may also show it. Morphologically we find striking changes in the erythro- cytes (many appear as macrocytes), which are pale and are without a distinctly pronounced central umbilication — the cells appear swollen. The particular cells have been designated "chlo- rotic" blood-corpuscles. Severe eases shows poikilocytosis, and nucleated red blood-cells. Polychromatophilia and granular degeneration, which are the genuine phenomena of degeneration, are not observed. The condition of the leukocytes is not uniform. The cells themselves do not show any characteristic changes in this dis- ease. The blood-plaques appear in markedly increased numbers, so that many groups of these cells appear in every field of the fresh preparation.^ These are often very evident in the stained preparation, especially if a basic stain, such as methylene-blue^ has been used. LEUKEMIA. Ganges in the Number of Erythrocytes. — The blood from 'a marked case of leukemia is distinctly watery ; in extreme cases it may be a whitish-red as it emerges from the puncture; this is owing to the great increase in white elements. The microscopic examination of the fresh blood in estab- lished cases, shows even without counting the enormous increase in the white blood-cells. The count of the white corpuscles shows 100,000 to 500,000, or even more, to the cubic millimeter. In some cases of leukemia no noteworthy change in the erythrocytes, either in number or appearance, occurs. As a rule «E. Grawite: Modern Clin. Med., p. 327. LEUKEMIA. 55 they are decreased to about half the normal number. Beside the diminution in these cells the blood contains varytug numbers of normoblasts^ and more rarely megaloblasts. The amount of hemoglobin is diminished^ but the coloring of the individual corpuscles need not be diminished (no altera- tion in the color-index). Changes in the Blood-Plaques. — An increase in the number of the blood-plaques has been noted in a number of cases. Blood Morphology. — The diagnosis of leukemia can fre- quently be made without further consideration in cases which show an extraordinary increase in the number of white elements, but in doubtful cases the presence of the condition can only be determined by an exact examination of the morphology of the leukocytes. Besides noting the great increase in the number of white cells, the polymorphonuclear elements will usually be found the most numerous.^ In other cases the increase is chiefly among the lymphocytes. We may therefore differentiate two forms of leukemia. 1. Lymphatic Leukemia. 2. Leukocytic Leukemia. Since the source of the lymphocytes is from the spleen and from the lymph-glands, and that of the leukocytes from the bone-marrow, the common designation of lymphatic leukemia (lymphemia) and myelogenous leukemia (myelemia) may be ap- plied to these two basic forms of leukemia. As, however, the bone-ma;rrow normally produces typical lymphocytes, and as it has been demonstrated in rare conditions that an overflooding of the blood with lymphocytes may occur through proliferative changes in the bone-marrow without en- largement of the spleen or lymph-glands (myelogenous lymph- emia). It seems preferable to apply the less prejudicial division of the leukocyte forms into "lymphatic leukemia" and "leuko- cytic leukemia" (W. von Leube). Differentiation. — 1. Lymphatic Leukemia: The blood- picture is conspicuous for its great preponderance of large and small lymphocytes, in comparison to the leukocytes. Nucleated red cells (normoblasts) and megaloblasts, although present in 'W. von Leube: Modern Clin. Med., p, 349. 66 THE BLOOD. this form of leukemia, are by no means as abundant as in the leukocytic form. 2. Leukocytic Leukemia. — This is by far the most com- mon form of leukemia. It may easily be difEerentiated from the preceding by the entirely different blood-picture. The in- crease in the white elements is usually very marked. Here, how- ever, the polymorphonuclear leukocytes are greatly increased in the microscopic blood-picture. Neutrophiles and eosinophiles are absolutely always increased (Ehrlich). There is also an increase in the mast cells, which may be twice as numerous as the eosinophiles. Their determination is of the greatest im- portance, since a marked increase in mast cells is observed only in this disease (von Leube). The phenomena which especially characterize leukocytic leukemia, and which result from changes in the bone-marrow (myelogenous leukemia) are the occurrence of neutrophilic and eosinophilic myelocytes. These may be present in enonnous numbers (up to 100,000 per cubic millimeter). The first sight of such a picture simulates the blood-picture of acute lymphemia with its large mononuclear cells. The myelocyte is a large mononuclear cell with an irregular nucleus, surrounded by a considerable amount of protoplasm, in which are either neutrophilic or eosinophilic granulations. Be- sides these immature leukocytes (myelocytes), immature forms of erythrocytes, also originating in the bone-marrow, are found in the circulating blood-stream of patients suffering from leuko- cytic leukemia. There are also normoblasts and occasionally megaloblasts. SPECTROSCOPIC EXAMINATION. Solutions of hemoglobin and its derivative compounds, when examined with the spectroscope, give distinctive absorption bands. The Spectroscope. — Light, when made to pass through a glass prism, is broken up into its component rays, giving the play of rainbow colors known as the spectrum. A spectroscope is an apparatus for producing and observing the spectrum. In brief, the apparatus consists of a base or stand, two horizontal SPECTROSCOPIC EXAMINATION. 57 tubes, and a prism arranged to take the light coming from one and to pass it into the other. Light falls upon the prism through one tube known as the "collinator tube." A slit at the end of this tube admits a nar- row ray of light which, by means of a convex lens in the other end of the tube, is made to fall upon the prism with its rays parallel. In passing through the prism the ray of light is dis- persed by unequal refraction, giving the spectrum. The spec- trum thus produced is examined by the observer through the other tube, which is a telescope. When the telescope is properly adjusted the rays entering from the prism produce a clear pic- ture of the spectrum. If the light used is lamplight, then the spectrum will be continuous, the colors gradually merging one into the other from red to violet. If sunlight is used the spec- trum will be crossed by a number of narrow, dark lines known as "Fraunhofer lines." The position of these lines in the solar spectrum is fixed, and the more distinct ones are designated by the letters of the alphabet, A, B, C, D, B, etc. If, while using artificial light or the solar spectrum, a solu- tion of any substance which gives absorption bands, is placed in front of the slit so that the light is obliged to traverse it, then the spectrum, as observed in the telescope, will show one or more narrow or wide, black bands, which are characteristic of the substance examined and which constitute its absorption spec- trum. The position of these bands may be located by describing their relation to the Fraunhofer lines. While the cost of the spectroscope may prohibit its use by the general practitioner, it is to be found in many laboratories, and its use in certain cases, particularly in poisoning, is abso- lutely essential, since it enables the student to arrive at definite conclusions which cannot be reached in any other way. For ordinary investigation the pocket spectroscope is all that is required. The detection of CO-Hb and of methemo- globin, the first occurring in carbonic-acid poisoning, the second in the various forms of intoxication, particularly with chlorate of potash. The smallest amount of hemoglobin or its deriva- tives may be demonstrated with certainty by this means in cases where ocular examination would leave uncertainty. The determination of CO-Hb (carbon-monoxid hemoglobin) 58 THE BLOOD. may present difficulty on account of the similarity of the spec- trum to that of 0-Hb (oxyhemoglobin). The differentiation is only certain when we observe that the line of CO-Hb does not appear on the addition of reducing agents — for example, ammo- nium sulphate. The CO-Hb is a strong combination, while, on the other hand, the 0-Hb is changed by the reagent to that of reduced hemoglobin. BACTERIOLOGIC EXAMINATION. Both diagnosis and prognosis may, in some cases, be helped by a bacteriologic examination of the blood. As there are quite a number of diseases in which the pathologic agents enter the blood, where diagnoses may thus be corroborated by a simple microscopic investigation of the blood, as in relapsing fever, malaria, and syphilis. In other cases, because of the small total number of organisms, or of the presence of others due to con- tamination, it is advisable to proceed by the more tedious but more certain bacteriologic methods. (Tubercle Bacilli, Method of Eosenberger, etc., see page 317.) THE CLINICAL VALUE OF BLOOD CULTURES.^ "Blood-cultures are most likely to be of value in the fol- lowing diseases : Typhoid fever, pneumonia, other forms of pneu- mococcic infections, and the whole group of septic cases in_ which the sepsis is associated with wounds, pelvic diseases, abortion, puerperal infection, endocarditis, and local diseases of the throat and many other regions. "In infections with typhoid bacillus, the pneumococeus, staphylococcus, and streptococcus, the results are usually posi- tive. "In a case of typhoid the examinations along bacteriologic lines are most likely to be successful between the latter part of ■ the first and the middle of the third week. In sepsis due to the pyogenic cocci positive results are obtained in the majority of cases. "Blood-cultures may clear up doubt in the presence of * Edsall : quoting Longsoope and Evans. THE SPIROCHETE PALLIDA. 59 typhoid fever or pulmonary tuberculosis. They may also be of considerable use in obscure infections in childhood." The Specimen. — In the preparation of blood-cultures it is advisable to puncture a vein in the arm under strict aseptic precautions to withdraw one or two cubic centimeters of blood, and to proceed immediately to prepare cultures or proceed with animal experiments. Eelapsing Fever. — The diagnosis of this disease is only positive when the spirilli are found in the fresh blood. As a rule this examination is easy, for the peculiar curved form of the spirillum precludes confusion with other organisms. Their numbers are usually large and their detection facilitated by their motility. In addition they stain readily with the various aniline colors (as carbol-fuchsin). Malaria. — In this disease the microscopic examination is of extreme practical importance. The organisms may be readily detected in fresh blood at the bedside with the aid of a good microscope. The dried stained specimen, which is usually the more practical to procure in practice, will serve to detect them even when in but small numbers. The specimen for examination is best taken in the afebrile period, shortly before the expected paroxysm. Previous to this the administration of quinine should be withheld for as long a period as possible. THE SPIROCHjiETE PALLIDA. Description of the Spirochsete. — The spirochsete is four- fourteenth microns in length, and at most only one-quarter of a micron in breadth; its form is that of a corkscrew with six to fourteen or even twenty-four turns. It is in active motion when examined in the fresh, and may show this activity for days in salt solution, when the tissues are kept at 20° to 27° C. (Beer). Method of Obtaining a Smear (Roescher). — The surface of the sore is cleaned with alcohol, irrigated with salt solution, and dried. It is then scraped with a needle, care being taken to avoid drawing blood, but bringing out a good deal of serum. Smears are made from the serum, and in it the spirochete will be found. Serum may be drawn from an enlarged gland by 60 THE BLOOD. means of a hypodermic needle and the smears made from this if desired. The Giemsa Method (for preparation of stain see Appen- dix). — The films are fixed in methyl, or ethyl alcohol, and dried ■with blotting paper. The stain is then diluted with distilled water in the proportion of one drop to 1 cubic centimeter of water, and then poured on the specimen. For satisfactory results the stain should remain on the specimen for from one to twenty- four hours. The time for staining may be materially shortened by adding a few drops of a ■M.ooo solution of potassium carbonate to the diluting water. By this expedient the spiroehEete may be demonstrated in fifteen minutes, though for the best results an hour's contact with the stain is advised. Method of Oppenheim and Sachs. — The smears are dried in the air and then, without previous fixation, are flooded with an alcoholic solution of carbol-gentian-violet (for preparation of stain see Appendix). After flooding the preparation it is warmed until it steams for a few minutes only; it is then washed gently in running water, dried, and examined. The spirochsete are stained blue. Method of F. C. Wood (fifteen minutes). — The smears are fixed in strong methyl alcohol and dried with blotting paper. The following stains are then applied in succession : — First, a few drops of a %ooo watery solution of yellow water soluble eosin are spread over the smear by means of a pipette. Second, four or five drops of a %ooo aqueous solution of methylene azur II. The two colors are thoroughly mixed by spreading over the smear and by rocking the cover-glass. This mixture is allowed to act for ten minutes. The stain is then washed off by a strong stream of distilled water, the preparation dried between blotters, mounted, and examined by an oil-immer- sion. For mounting use xylol-dammar, as the ordinary Canada balsam bleaches these preparations. Usually any precipitate which forms can be removed with the stream of water. If espe- cially clean and clear specimens are desired, the precipitate may be removed by momentary immersion in 95 per cent, alcohol, which should be immediately washed off with distilled water. By this method the spirochsete appear a light carmine color. IV THE OPSONIC METHOD. GENERAL CONSIDERATIONS, Up until the last decade of the nineteenth century, we were in the habit of using the phrase "the resisting power of the body." But if we had been asked to say exactly how the body actually did resist disease, we would have been forced to confess our ignorance. Now, however, thanks to the researches of MetchnikofE and Wright, we have some idea of the modus operandi of this mysterious "resistance." And we now know that the resisting power of the body is dependent upon the relation of the co-factors, bacteria and phagocyte, and that this power of the body may be measured for each infective bacteria by the opsonic index. These recent results of Wright's researches have not merely a scientific interest. As so often happens in the history of science these results have a direct practical bearing in this case on the diagnosis and treatment of disease. Wright, Douglas and Eeid have devised a method of esti- mating the opsonic power of the blood, and applying this to the vaccine treatment of bacterial diseases. When bacteria are grown in culture tubes and then sterilized, and this dead culture is used in therapeutic inoculation, it is called a "bacterial vaccine." For example, if a patient has to be treated who has boils which are due to infection by staphylococcus pyogenes, a culture of this staphylococcus is made, then it is killed, and the patient inoculated with a proper dose of this vaccine. If the patient has tuberculous^ glands he is similarly inoculated with a vaccine (tuberculin) consisting of sterilized tubercle bacilli. The Theory. — Two elements come into consideration in the protection of the organism against invading micro-organisms. The leukocytes constitute one of these elements; the antibacterial substances in the body-fluids the other. (61) 62 THE OPSONIC METHOD. (a) Leukocytes. — The leukocytes come into consideration, in connection with the resistance to the bacterial infection, by virtue of the fact that they are capable of ingesting bacteria and disintegrating these by intracellular digestion. We may usually distinguish between "spontaneous" and "induced" phagocytosis. "Spontaneous" phagocytosis is the process of ingestion and destruction which occurs in different media, as physiologic salt solution. It is a slow process and the bacteria ingested are in moderate numbers. It is also irregular, in that the number ingested of different species of bacteria varies. Strikingly different from such spontaneous phagocytosis is the "induced" phagocytosis, which occurs when leukocytes are brought into contact with bacteria which have been, or actually are at the moment, subjected to serum action. The "induced" phagocytosis which occurs under these conditions is distinguished by the following facts : — 1. It is an exceedingly rapid process. 2. With hardly an exception every adult leukocyte is phagocytic. 3. When the supply of organisms is unrestricted the leukocytes will ordinarily fill themselves to absolute repletion. 4. The leukocytes will continue to digest bacteria in a concentration of salt solution which will entirely suppress "spontaneous" phagocytosis. (b) Antibacterial Elements in the Blood. — The anti- bacterial elements which are here in question are of "bacterio- trophic" properties, in that they turn toward and enter into combination with the elements of the bacterial body. When a condition of immunity is conferred upon a patient infected with staphylococci his opsonic activity toward this bacterium is greatly augmented as compared with the normal. Wright and his pupils, as a result of numerous observations, have classified diseases clue to bacterial invasion as follows: — 1. Diseases in which the bacterial process is strictly local- ized or shut off from the IjTuph-channelo, as furunculosis, lupus, and tuberculosis, and in fact almost any chronic infection. The opsonic index in these cases is constantly below normal, owing to the absence of immunizing stimuli emanating from the com- pletely walled-off infective process. ?. Diseases in which the bacterial process is but loosely shut APPLICATION TO TREATMENT. 63 off, especially from the lymph-channels. In these infections, which are generally acute, immtmizing agents from the bacterial process, from time to time, get into the circulation; thus the opsonic index may be normal, above or below normal, at various times. A good example of this class is tuberculosis of the Joints. 3. Diseases in which the bacterial infection is in the blood- stream. Here the opsonic index is usually above normal. To this class belong the infectious diseases: septicemia, endo- carditis, etc. 4. Nomial individuals not subject to any bacterial infection present a constant opsonic power to the various pathogenic bacteria. Application to Treatment. — It is perhaps in a direction to therapeutics that the most important step has lately been taken in opsonic research. The treatment depends upon making certain tests upon the blood whereby resistance of the patient against bacterial infections can be measured, and then if the resistance is found to be subnormal applying the proper stimulus (vaccine) to increase the resistance. The value of the opsonic index in the vaccine treatment of disease is to enable us to apply a treatment at the most favorable point, and to graduate the dose. The opsonic index expresses the power of the blood of one individual to destroy bacteria compared with the same power in a healthy person. The result of the proper administration of bacterial vaccines is an increase in the opsonic index and the gradual production of the state of active immunity. According to Wright the relation of the phases can be accurately determined only by means of the opsonic index. Many observers, however, carry out successful treatment without the aid of the index. To get the best results in all cases a skilled laboratory man and a well-equipped laboratory are necessary. However, excel- lent results may be obtained by the general practitioner with stock or autogenous vaccines prepared by expert laboratory workers. The cases usually treated with vaccine therapy by the estimation of the opsonic inaex are those which have failed with general medical treatment. Guides for the Opsonic Treatment of Disease.^ — 1. The iB. A. Thomas : Jour. A. M. A., Jan. 29, 1910. 64 THE OPSONIC METHOD. diseases contraindieated are diffuse infections characterized by septicemia, pyemia and grave sapremia. 3. Those in which this agent is beneficial or curative are the superficial acute, subacute, and chronic process, especially the latter tvro. 3. The acute cases, in which brilliant results can be uniformly expected, are. acne vulgaris, furunculosis, carbunculosis and subcutaneous abscesses. 4. Subacute and chronic gonorrheal and tuberculosis affections are amenable to vaccine therapy and, • because of the impossibility and impracticability of employing an autogenous bacterin, a reliable stock preparation should be used. 5. Certain acute gonorrheal infections can be benefited. 6. It is doubtful whether tuberculin therapy should ever be employed in very acute tuberculosis. 7. Autogenous bacterins are always to be preferred over the stock preparation, and success or failure fre- quently depends on this fact. 8. Although the duration of the period of greatest potency of bacterins is undetermined it is better to prepare a fresh bacterin every two or four weeks. 9. It is believed that the best effects, particularly in chronic cases, occur when the quantity of bacterin is slowly and cautiously increased during successive inoculations, thereby avoiding hyper- susceptibility or anaphylaxis. 10. Therapy of both bacterins and tuberculins can be satisfactorily executed by keen observance of the clinical symptomatology. Eeliance on the opsonic index as a guide is not only unnecessary, but often actually conducive to erroneous conclusions, owing to its variability. The number of cases successfully treated with bacterial vaccines without control of dosage by the opsonic index will increase with our knowledge of the subject. For the most suc- cessful treatment of certain obstinate, severe or mixed infections, and for diagnosis of certain obscure conditions, the opsonic index will continue to be required. The essentials of vaccine treatment are briefly: 1. Diagnosis. 2. Appropriate dosage. 3. Proper intervals between doses. In these days of scientific medicine and of endowed laboratories for medical researches the busy practitioner should make it a point, even if he finds time for nothing else, to keep himself posted upon the latest results of studies in vaccine therapy. V. THE BLOOD-PRESSURE. Capillary Blood-Pressure. — The pressure of the blood in the capillaries is low, because of the resistance offered to the progress of the blood by the fine bore of the vessels, and because of the relative large cross-sectional area of all the capillaries compared to that of the aorta and great vessels. The capillary, pressure, has been found to be much lower than iu the arteries, and considerably higher than the pressure in the great veins. This pressure has been found to equal that required to sustain a column of from 34 to 54 millimeters of mercury. 1 Clinically the capillary blood-pressure is not of Bufficient importance to warrant a further discussion here. Terms and Definitions. — The Pulse: The pulse is the rhythmically recurring impulse propagated by the systole of the left ventricle and palpable throughout the arterial system. Aeteeial Peessuee. — By arterial pressure is meant" the degree of force exerted by the blood within the vessel. It is primarily dependent on the strength of the heart as measured by its rate and by the volume of blood expelled at each systole, balanced by the elasticity of the vessel walls and capillary resistance. The Systolic Peessuee (Pig. 10). — The systolic pressure as indicated by the sphygmomanometer, represents the pressure within the vessels at the time of systole of the ventricles. The Diastolic Peessuee (Fig. 10). — The diastolic pres- sure represents the ebb to which the arterial pressure falls during cardiac diastole. I, The Pulse Peessuee. Eange oe Amplitude (Pig. 10). — The arterial pulse is caused by variations in pressure within the arterial system caused by the intermittent pumping action of 'Amer. Textbook of Physiology, page 377. (65) 66 THE BLOOD-PRESSURE. the heart. The difference between systolic and diastolic pressures i.e., the variation in pressure occurring within the vessel during a complete cardiac cycle, is termed the pulse pressure. This figure is obtained by subtracting the diastolic from the systolic pressure. The normal pulse pressure ranges between 30 and 40 millimeters of mercury. Variations in the pulse pressure in the same individual constitute a most important part of the study of blood-pressure. It is theoretically possible that the pulse pressure should be influenced in at least three ways : 1. An increase in the amount of blood delivered at each beat of the heart would tend to increase the difference between systolic and diastolic pressures. 2. A rapid emptying of the vessels, the cardiac output remaining the Mean =115 Fig. 10. -Normal PrLSE Tracing: showing relation of Systolic, Diastolic, Pulse Pressure, and Mean. Pulse Pressure Equals 30. same, would tend to increase this difference. This would occur independently of whether the blood was passed onward into the capillaries or was regurgitated into the ventricle. 3. Eigid vessel-walls would increase pulse pressure. If the arteries were rigid tubes, the heart at each systole would be compelled to move the blood in the arterial system as a whole, while during diastole the flow would cease. There would thus be an increase of pressure during systole, while during diastole it must fall rapidly toward zero. The Mean Peessuee. — The mean blood-pressure is valuable chiefly as an indication of the amount of strain to which the heart and larger vessels are subjected. It varies with the pulse pressure, the systolic pressure and the diastolic pressure. To obtain the mean pressure, divide the sum of the systolic and diastolic pressures by two, or add half of the pulse pressure to the diastolic pressure (Fig. 10). SIGNIFICANCE OF PULSE-PRESSURE. 67 Pathologically, the pulse-pressure increases in organic diseases of the kidneys, in arteriosclerosis, and in aortic insnfla- ciency. It diminishes from other organic diseases of the heart, affecting the valves or myocardium. 2 Significance of Pulse-Pressure. — The normal pulse-pressure indicates a normally acting heart, a proper systolic output, and A.— Pressure in "b" 135 mm. He., pressure in "a" 130 mm. Hg.. a is therefore collapsed, pulse cannot pass. B.— Pressure "b" 129 mm. He., pressure in "a" 130 mm. Hg., pulse passes. Fig. 11.— Diagram of Relations of Armlet to Brachial Artery. expl.\nation of systolic reading. a, artery ; b, compressing armlet; c, retaining cuff ; d, tube to manometer ; e, humerus. a normal vascular distribution of blood. Any condition which interferes with the normal flow of blood from the capillaries will tend to increase the systolic pressure, and will produce a greater pulse-pressure. We find therefore an increased pulse- pressure in arteriosclerosis. The increased systolic output of the large heart ia aortic regurgitation produces, during com- pensation, a very large pulse-pressure. According to Gerhardt, ' Elchberg: Jour. A. M. A., Sept. 19, 1908. 68 THE BLOOD-PRESSURE. the sj'stolic blood-pressure in broken compensation may be high, but the pulse-pressure is always diminished and becomes greater, according to the re.ationship of compensation. An increased pulse-pressure in the absence of any other explanation is very suggestive of organic disease of the kidneys. Myocardial de- generation of all kinds is productive of a reduction of the pulse- A.— Systolic pressure in "a "130 mm.Hff., pressure in "b"101 mm. Hg., artery not compressed. B,— Diastolic pressure in "a" 100 mm.Hg., pressure in "b" 101 mm.Hg:,, artery collapsed. Fig. lla.— Diagram of Relation of Armlbt to Brachiai, Artery. Explanation of Diastolic Reading. a, artery ; b, compressing armlet i c , tetaining cufE ; a, tube to manometer ; e, humerus. pressure, and the mean of this is a fair estimation of the state of the myocardium, upon which is based the work test of Graupner. (Test on page 88.) The Principle of the Sphygmomanometer. — Vital tissue is perfectly elastic. Therefore any pressure applied to the surface of the body will be directly transmitted to the underlying structures without loss of force. It is upon this principle that the indirect method of measuring the blood-pressure is based. SYSTOLIC AND DIASTOLIC READING. 69 Pressure is applied to an accessible part of the body over a large blood-vessel such as the brachial. If the amoufit of this pressure is sufficient to overcome the pressure of the blood within the vessel, the vessel will be collapsed and the pulse prevented from passing beyond it. If the amount of the compressing force is measured and expressed in definite terms of weight (as millimeters of a column of mercury) then we can, by applying just sufficient pressure to collapse the vessel, measure the amount of force exerted by the blood in preventing this collapse. In practice the compressing force is obtained by a cautery bulb or a small hand pump, and applied to the arm by means of a hollow, flat, rubber bag. This is applied about the arm and held there by some form of inelastic cuff. A tube communicating with a mercury manometer measures the amount of pressure applied over the vessel. Explanation of the Systolic Beading. — Fig. 11, A and B, shows the relation of the compressing bag to the artery. In Fig. A the pressure within the cuff is greater than the blood-pressure within the artery, which is therefore collapsed and the pulse in the distal end of the vessel cut off. In B the pressure in the cuff has been reduced so that it is a fraction of a millimeter less than the systolic pressure within the vessel. Now at each systole a small amount of blood will pass the constriction and will reach the distal end of the artery, where the wave can be fe't by the palpating finger at the wrist. Explanation of the Diastolic Reading. — Fig. 11a, A and B, represents the conditions existing between the constricting cuff and the vessel at the diastolic time of pressure. A represents a pressure within the cuff less than the systolic pressure in the vessel ; this is insufficient to affect the vessel during the systolic period. B shows the artery and cuff during the diastolic period, when the pressure within the artery is at its lowest point, a fraction of a millimeter less than the pressure within the cuff. Consequently the artery is collapsed at this time. The effect of each succeeding systole is to alternate between a round and flat vessel at the point of compression. This affects the volume of air within the cuff, which is in turn transmitted to the manom- eter and becomes visible in the rhythmic fluctuation of the column of mercury, which is synchronous with the pulse beat. 70 THE BLOOD-PRESSURE. Since the fluctuation will reach a maximum at the time when the pressure" in the cuff is approximately equal to the diastolic pressure in the vessel, we are justified in considering the base of the manometer column at this time a measure of the diastolic pressure within the vessel. The Sphygmomanometer. — Descbiption: Generally speak- ing, there are two types of mercury (fluid column) instrument. One of these employs a vertical tube into which the mercury Pig. 12.— Padght s Mercury Sph-ygmomanometer . A. Inner arm-band; B, outer arm-band; C, tube from arm-band: D, nipple for tube from arm-band; E, ptmip ; P, nipple for pump tube; G, millimeter scale; H, manometer tube; I. link-brace and lock; K, mercury guard cock; L, mercury guard cock ; M, pressure guard cock ; N, release valve. column is forced from a large containing chamber in the base of the instrument. The pressure is measured in millimeters of mercury on an appropriate scale attached to the vertical glass tube. The other employs a glass tube (similar to that first used by Poiseuille) bent in the form of a "U" with the open ends up. This tube is partly filled with mercury and one end connected by means of suitable tubing with the compression part of the apparatus. The pressure is measured upon a suitable scale placed between the two limbs of the tube, and is represented by the THE SPHYGMOMANOMETER. 71 difference in the height of the mercury in the two limbs of the "U" tube. The sphygmomanometer bearing the author's name (see Piate III) is modelled after the type of apparatus employing the "U" tube and is designed to overcome the many shortcomings of the earlier instruments and to furnish an instrument which is easy to use, difficult to get out of order, accurate and as light and portable as is compatible with exactness and strength. The mahogany case, which encloses the complete apparatus, including the arm-band and pump, measures 4 X 4% x 16 inches and weighs 3 pounds and 9 ounces. The lid is hinged at one end and when raised supports the working parts of the apparatus. A spring check allows the lid to be raised to a vertical position, where it is automatically held locked during observation. The "IT" tube is provided with a scale, which has been arranged to give the reading directly in millimeters of mercury, each space representing 2 mm. Hg. The range is from, to 300. A special and distinctive feature of the apparatus is the means of preventing loss of mercury from the manometer tube when the instrument is not in use. This is accomplished by means of two small cocks placed at either extremity of the "U" tube, and which are kept closed when the apparatus is not in use. By eliminating all detachable parts, the time required to make the reading is reduced to a minimum, the only prelimi- naries to the test being to lift the lid, open three cocks and attach two tubes to their respective nipples. To Operate the Mercury Sphygmomanometer (Fig. 13). — The patient should be in a comfortable position, and in a sitting or reclining posture. The instrument should be upon a level surface within easy reach of the examiner. The lid is then raised until it locks in a vertical position. If the tube from the hand bellows is not already connected to the nipple F it should be firmly attached to it. The two mercury guard cocks, K and L, at the ends of the "U" tube should be opened and the escape valve N tightly closed. The hollow rubber bag of the arm-band A should be firmly wrapped around the bared arm of the patient and securely bound there by the leather cuff and straps B. The cuff should be applied snugly, without pressure, as it is not designed to com- 72 THE BLOOD-PRESSURE. press the member, but only to restrain the inner rubber bag while pressure is applied within it. The tube C from the arm-band is attached firmly to the nipple D. The cock in the nipple F is opened. This arrangement forms a continuous closed pneumatic system communicating freely with the manometer tube of the instrument. Now, when pressure is raised in the arm-band, by the pump, the amount of force exerted is indicated by the rise of the right-hand column in the manometer tube H, the height of which will be indicated on the scale G in millimeters of mercury. To Obtain the Systolic Eeading. — ^With one hand find the pulse at the wrist of the arm to which the arm-band has been applied. The fingers should be in a comfortable position and under no circumstances be moved during the observation. Care should also be observed that the pulse is not cut off by undue pressure of the palpating fingers. While the pulse is thus under observation, the pressure in the apparatus is raised by means of the hand bellows or pump until the pressure within the constricting band is sufficient to prevent the impulse from reaching the wrist. When this is ac- complished the cock in the nipple M is closed to eliminate the elastic pressure of the hand bellows. ISTow by a fraction of a turn in the valve N the pressure in the system is slowly released. During this part of the procedure, a close watch should be kept upon the height of the mercury column and for the return of the first pulse beat at the wrist. The level of the mercury column at the instant when the pulse passes the compression-band will "^ represent the systolic pressure of the patient under observation. It is advisable to repeat this procedure a few times to check the correctness of the finding. The diastolic pressure may be obtained in several ways. The method employed will depend upon the character of the instrument used and the method of preference of the operator. The methods will be described in the order in which they have been devised. 1. Visible Method. — This depends on the to-and-fro motion imparted to the mercury in the "U" tube, which occurs after the pressure has fallen below the systolic point. Having deter- THE SPHYGMOMANOMETER. 73 mined the systolic pressure, again raise the pressure to a few millimeters above this point and immediately close the valve M. Now allow the pressure to fall very slowly by releasing the valve N. As the mercury falls below the systolic point it will be noted that it acquires a. rhythmic motion corresponding in time to the pulse. This will be found to gradually increase in ampli- tude up to a certain point, after which it decreases and iinally ceases before zero pressure is reached. During this gradual fall, the base of the mercury column, when the mercury is making the greatest excursion, represents the diastolic pressure. 2. Palpatory Method. — Eaise the pressure within the appa- ratus to the systolic point, then, while keeping the fingers in touch with the pulse, allow the mercury column to gradually fall as in the first method. It will then be noted that at first the pulse is very feeble and thready in character and continues so for a time, and as the pressure falls it will suddenly assume the full, bounding character of the pulse of aortic regurgitation. Immediately this change occurs the height of the mercury columns will represent the diastolic pressure in millimeters of mercury. 3. Auscultatory Method. — This method, first suggested by Sterzing, seems to be growing in popularity, since it appears to be a more accurate means of determining the diastolic pressure than the preceding. The method is as follows: After applying the apparatus in the usual manner .and raising the pressure to obliteration of the pulse, a stethoscope is placed over the brachial artery below the cuff. As the pressure is gradually allowed to fall a pulse tone is heard as the circulation commences. This represents systolic pressure. This tone now undergoes a number of changes until it suddenly becomes very faint and almost im- mediately disappears. The reading of the sphygmomanometer at this moment represents the diastolic pressure. Cautions. — To obtain accurate and reliable clinicali4*data with the sphygmomanometer, it is important that some system- atic technic be adhered to, and that all observations not only on the same patient, but in all cases, be made under as nearly the same conditions as possible. Attention to detail will eliminate largely the errors arising from such factors as position of the Missing Page Missing Page 76 THE BLOOD-PRESSURE. No. 2, p. 181) of examining 1000 healthy subjects between the ages of 15 and 60 years is a very important contribution to our knowledge of normal blood-pressure, the results of which are shown in the accompanying chart (Fig. 126). Except for slight mm 15 TO 30 30 TO 40 40 TO EO 60 TO 60 1 in la r « » o fti IS) n « in (M a » n n n tn . > n * V \ in ^ 01 J U1 iP ■4 irt. J) ifl o , ^ 143 l/ls 147 146 ^ ^ ' ' ^ *■ 144 143 142 141 140 ^ = ^ - ■T v^v J J iMEHP - 139 138 137 136 134 133 132 131 , ^ p" . ^ r" — 129 123 127 126 19"? , ■ .. _ r \\ e \i L Ei ^ ^ -/ L ~ 'fl Ql E Ml, ^ 124 123 122 121 1?0 Ly ^ e f^^ ■X ^ 119 lis 117 116 115 ' ■ ^ ^ _. ,^ -^ 114 113 112 111 no , ^ - " J /• /• 109 loa 107 106 ' , ^ > . ,^ -r . -- i« V- , „ _. _ 104 103 102 101 J.V.P. ~ ■ ^ " f» , ~ ~ "' ^ -' ~" ;^ ^ f ' — _ _ _ ^ ^ ' J M ^ _ •t. _ _ _ _ _ _ _ _ .- _ ^ „ _ _ _ L L Fig. 12b.— Woley's Chart Showino Effect of Age on Blood-Pressdre Giving Mean, High and Low Average. variation, the figures obtained by this observer are in accord with the results given by our best authorities. In children under two years of age, Cook places the normal systolic pressure between 75 and 90 mm. Hg. According to Lauder Brunton {Lancet, Oct. 17, 1898) the maximum pressure POSTURAL VARIATIONS IN BLOOD-PRESSURE. 77 in children between 8 and 14 years is 90 mm. Hg; in youths from 15 to 21 years, from 100 to 120 mm. Hg. As early adult life is passed we have to deal with those normal changes in the cardiovascular-renal system which are the result of the wear and tear of every-day life, and which leave their mark in a lessened arterial elasticity, a lessened functional activity of the kidneys, and by inducing degenerative changes in the myocardium often eventuate in chronic myocarditis. The inevitable result of these changes is a gradual elevation in the normal systolic blood-pressure. We have now to establish new normals by which we may determine the pathological. For this purpose I have devised and employed the following rule: Con- sider the normal average systolic blood-pressure at age 20 to be 120 mm. Hg, then for each year of life above this add one-half of one millimeter to 120. This at the age of 60 means an average systolic pressure of 145 mm. Hg, which coincides closely with the figure given by Woley, Janeway and others. Size and Temperament. — With the standard armlet the factor of size of the individual does not enter. Temperament, on the other hand, does undoubtedly affect the reading, because in the nervous it is often impossible to remove entirely the effect of psychic influence; therefore, allowance must be made for an abnormally high reading which will often fluctuate to a surpris- ing degree in a limited period of time. Diurnal Influence. — A record of blood-pressure taken at frequent intervals during twenty-four hours makes a very strik- ing picture. The influence of this factor is most difiicult to estimate. Janeway suggests a variation of 60 mm. Hg as the extreme. I have not seen it equal this. Authorities agree that the lowest blood-pressure is reached during the early hours of sleep and that a gradual rise occurs toward morning (Brush and Fairweather, Am. Jour. Physiol., vol. v, p. 199). During the day there is a physiologic rise which reaches the maximum in the evening. Posture. — This should not be confounded with the effect of gravity, which may be eliminated by making all observations with the cuff at the level of the heart. Authorities differ ; Jane- way, and Erlanger and Hooker hold that the effect of posture is insignificant, while 0. Z. Stephens {Jour. A. M. A., Oct. 1, 1904) 78 THE BLOOD-PRESSURE. and A. M. Sanford {Jour. A. M. A., Feb. 18, 1898) demonstrate by the examination of a larger number of normal individuals that this has a very constant and definite irifluence. Their observations, with which mine agree, show that there is little alteration in pressure between the standing and sitting postures; occasionally there is a rise of a few millimeters. Be- tween the standing and the recumbent Stephens reports an elevation often amounting to- 30 mm. Hg, while Sanford notes a rise amounting to only half of this. Between the standing and the head down (Trendelenburg) the rise in pressure may reach 35 mm. Hg. These observers note a compensatory lowering of pulse rate, upon which Schapiro has based a test for the func- tional capacity of the heart. Prolonged rest in bed by one accustomed to active exercise, especially if there was a tendency to high pressure, causes a rapid and marked fall with the establishment of a new systolic level. Emotion and Excitement, Including Pain. — In deter- mining the influence of psychic disturbances, temperament plays an important part. The pressure-raising effect of pain, fright, fear, and apprehension must always be recognized. Vasomotor disturbances from sensations of heat or cold and changes in the arm from prolonged pressure of the arm-band must not be ignored. Every effort should be made during the test to elimi- nate psychic and vasomotor disturbances by establishing a proper understanding between the patient and physician, and by completing the test with as little delay as possible. Exercise. — Muscular exertion in the healthy, especially if sharp, may cause an elevation in blood-pressure of from 5 to 15 millimeters. This elevation becomes less marked as the subject becomes accustomed to performing the act or acts. This reduc- tion in susceptibility of the cardiovascular system is one of the beneficial effects of training. When effort is prolonged but moderate in severity (as in walking) the systolic pressure may rise from 5 to 10 millimeters, but soon adjusts itself to a new level upon which additional exertion has little if any effect, until a condition of fatigue is reached. Fatigue from prolonged exertion results in a fall in pressure which becomes greater until a dangerously low pressure DIET; ALCOHOL; TOBACCO. 79 is reached, if the effort is continued (Karrington, Zeitschr. f. hlin. Med., 1903, vol. 1, p. 322). During moderate exercise in a normal healthy person the systolic and diastolic pressures tend to become more widely separated, i.e., the pulse-pressure becomes greater (Krehl, "Clinical Pathology," 1905, third edition). Upon this physiological fact is based the work test of Graupner ("Die Messung. der Herzkraft," 1905). Passive movements and massage, except when causing pres- sure upon the thorax or abdomen, can be prolonged without materially altering the systolic pressure (Bichberg). Diet and Digestion. — ^The difficulties in the way of ac- curately gauging the effect of diet and digestion on blood- pressure are great, because of the difficulty met in eliminating conditions bordering on the pathologic. The effect of overeating, overingestion of fluids including alcohol, insufficient exercise and defective elimination must all be considered and weighed. Observers differ upon this question, some holding that as a result of the normal splanchnic engorgement blood-pressure falls a few millimeters after eating, while others affirm that observa- tion shows a normal rise in pressure amounting to from 10 to 20 millimeters and that the rise may exceed this when large quan- tities of fluid, particularly beer, is taken with the meal. Alcohol. — Clinical evidence so far shows that a moderate amount of alcoholic beverages taken regularly does not ma- terially influence blood-pressure. The irregular use of alcohol occasions first a rise of from 10 to 20 millimeters followed shortly by a fall as the superficial capillaries become dilated. Tobacco.— Little has been added to our knowledge of the effect of smoking on blood-pressure since the contribution of H. A. Hare which appeared about twenty years ago. We find that the moderate use of tobacco in those accustomed to its habitual use causes at first a sedative action with lowering of blood-pres- sure, while if used immoderately the pressure is elevated. A long series of tests by the writer, upon three healthy subjects, showed a fall in blood-pressure from 4 to 10 millimeters after smoking one cigar. If this was followed by a second, the pressure tended to return to the original level, while after from three to five cigars smoked in succession the pressure rose from 2 to 15 millimeters above the original reading. 80 THE BLOOD-PRESSURE. Pathologic Variations in Blood-Pressure. — Por convenience in study we may appropriately divide pathologic alterations m blood-pressure into pathologic high pressure and pathologic low- pressure. Pathologic High Blood-Peessurb.— High pressure per se is not a disease but a phenomenon or symptom, which may accompany a great variety of diseased conditions, including dia- betes, gout, syphilis, chronic lead poisoning, cardiovascular renal disease^ many anomalies of nutrition, etc. Acute Asphyxia and Acute Anemia of the brain (medullary centers) will powerfully stimulate the vasomotors, causing con- traction of the splanchnic area and a rise in blood-pressure. Lead Colic affecting the abdominal vessels is usually asso- ciated with high blood-pressure, as is also the early stage of peritonitis.!'' Continuous high pressure is seen in certain forms of ne- phritis. Thus in primary acute Bright's disease and in nephritis secondary to scarlet fever, there is practically always a marked rise in arterial pressure. A rise amounting to more than 50 millimeters Hg has been observed within 48 hours of the onset of an acute nephritis. !" Elevated pressure is also found in beginning arteriosclerosis of the first part of the aorta and of the splanchnic vessels. Pathologic Low Pressure. — A pathologic depression in blood-pressure may be caused by the depressing influence of cir- culating toxins acting either upon the heart, blood-vessels or con- trolling nervous mechanism or to sudden withdrawal of a large volume of blood from the circulation as in hemorrhage, after venesection, copious diaphoresis, diarrhea, or in shocTc. The lowest blood-pressnre compatible with life has been reported by Neu to be from 40 to 45 millimeters of mercury and this only occurred with subnormal temperature in the moribund state. He has seen recovery after a fall in pressure down to 50 millimeters. It is noted that a moderate and progressively falling pressure occurs in most progressive and prolonged fevers, as in typhoid "Krehl: Loc. cit. " Buttermann : Arch. klin. Med., vol. Ixxiv, page 11. PLATE III. Sphygmomanometer, in Position for Observation. BLOOD-PRESSURE IX DISEASES OF THE HEART. 81 lever. AVlien due to such a cause the depression is rapidly over- come apd disappears as convalescence is established. AMdespread dilatation of the vessels and consequent lower- ing of blood-pressure have been noted in the last stages of arterio- sclerosis. (Krehl.) READY TABLE SHOWING THE USUAL TENDENCY OF BLOOD- PRESSDRE IN MANY DISEASES: HIGH VARIABLE LOW Arteriosclerosis Chronic Bronchitis Anemia Aneina Pectoris Cerebral Tumor (1st stage) Chlorosis Chrome Bright's Disease Neuraigia Cardiac Dilatation Acute Bright's Disease Neurasthenia (true) Diabetes Cirrhosis of the Liver Pleurisy Diarrhea Cerebral Hemorrhage Asthma Exhaustion Dilatation of Aorta Jaundice Pulmonary Tuberculosis Emphysema Obesity (early) Gout Insanity with Mania Shook and Collapse Migraine Rheumatism Starvation Pi egnancy (toxemia) Exophthalmic (Joitre Hemonhage Uremia Menopause Cholera Toxemias Rheumatoid Arthritis in Aortic Regurgitation Children Eclampsia Plumbism Blood-Pressure in Diseases of the Heart, in Valvular Lesions. — In the study of valvular disease of the heart the readings do not seem to have special bearing upon the primary conditions (defective valve) except in cases of aortic regurgitation. This is in part due to the usual complicated nature of the condi- tion, which often includes arterial and myocardial changes and involvement of the kidneys. Aortic Eegurgitation. — The blood-pressure finding in aortic regurgitation is very characteristic. The great pulse-pres- sure occurring in this condition being alniost pathognomonic. Eeferring to the physics of the circulation we find that in aortic regurgitation the left ventricle is called upon to deliver an abnormally large volume of blood into the aorta to supply the demands of the circulation. This is because the heart is required not on'y to furnish sufficient blood for the needs of the body, but must also inject into the aorta at each systole enough surplus to compensate for the regurgitation of a large volume of blood into the left ventricle during diastole. The natural result of a sudden injection of this large amount of blood into the arterial system will be to cause a sudden and great rise in systolic blood- pressure (immediately succeeding systole, the blood disperses in two directions, forward through the capillaries and backward 82 THE BLOOD-PRESSURE. into the ventricle, producing the phenomenon of the water- hammer pnlse). Thus the pressure rapidly falls and the dia- stolic pressure is abnormally low. The combined result of this high systolic and low diastolic pressure is a great pulse-pressure. In the presence of moderate or high-grade generalized arteriosclerosis this phenomenon is further accentuated because the lack of normal elasticity in the arterial system tends to further reduce the diastolic pressure. Chkonic Myocarditis. — The sphygmomanometer is a most valuable means of detecting alterations in the musculature of the heart, often before the development of the usual physical signs. In the physical examination the state of the superficial vessels, together with the pulse-rate and particularly the reaction of the heart to posture and exercise as determined by the sphygmomanometer, is all-important. This latter may be deter- mined by the following tests : — PuNCTiONAL Test. — Moderate exertion raises pressure in normal hearts and this rise is sustained during it if not unduly severe or prolonged. In weakened heart muscle from any cause a primary rise may occur, but is quickly followed by a fall; in the worst a fall occurs from the first. Graupnee's Test. — This is based upon the physiologic fact that a given amount of exercise, such as ten bending movements or running up a flight of stairs, causes an acceleration in the pulse-rate and an elevation in blood-pressure. But the latter does not appear coincidently with the former; or, if, as in some cases, the pressure does rise first, it fails to rise again after the pulse has returned to normal. It is this secondary rise which indicated a good heart muscle. A not too seriously affected heart may show a rise in blood-pressure immediately after the exertion, but with the slowing of the pulse the pressure will be found to have fallen to a level lower than before the experiment. The sphygmomanometer is required for an accurate demon- stration of these changes in pressure, which may be recorded in definite units of measure for future reference and comparison. Shapiro's Test. — ^This is based upon the alteration in pulse-rate occurring in normal individuals on change of posture from the standing to the recumbent. ISTormally, the number of BLOOD-PRESS URp; IN DISEASES OF THE HEART. 83 pulse beats per minute is from seven to ten less in the recum- bent position; but when chronic myocarditis develops this dif- ference tends to disappear, so that in seriously weakened hearts the pulse may be as rapid in the recumbent as in the sitting posture. Cautions. — It is not advisable to apply this test to patients with excessively high Mood-pressure, in those of apoplectic tend- ency, or in those with high-grade arteriosclerosis. The test is unsafe in those with a systolic pressure of 200 millimeters or over. In such cases there is danger of ocular or cerebral hemor- rhage or acute dilatation of heart. The test will be difficult if not impossible of application in women unless all tight clothing is removed. Valvular disease is not necessarily a contraindication to this test, as the condition of the myocardium seems to be the only important factor, except in aortic regurgitation with high pres- sure, so that the presence of valvular lesions need not detract from the value of the information obtained by this test. Tachycardia. — Some eases of tachycardia with demon- strable heart weakness may be the direct result of the hypoten- sion, i'' This is seen in advanced arteriosclerosis, in shock and after large doses of alcohol, chloral and veratrum viride, and as the result of the toxemias of typhoid fever and tuberculosis. Angina Pectoris. — Eeports by Eussell and others seem to indicate that there is not necessarily any elevation in pressure in the interval of attacks. The coincidence of generalized arteriosclerosis, which is one of the causative factors in the pro- duction of this condition, will affect the pressure according to the location and extent of the arterial change. A rise occurs shortly before or coincident to the attack of pain, passing again when the pain ceases. It should not be forgotten that cases of undoubted angina pectoris will be encountered in which there can be demonstrated no elevation in pressure either in the interval or during the attack. It has also been shown that in certain cases accompanied by a constantly high blood-pressure the attacks may be less- "Norris : U. of P. Med. Bull., April, 1908. 84 THE BLOOD- PRESSURE. ened ia number and severitj', or even prevented by prophylactic measures directed toward a reduction of high pressure. Caediovascular Eenai- Disease. — Clinical experience teaches that the conditions of arteriosclerosis and chronic inter- stitial nephxitis are with difficulty treated as separate and dis- tinct conditions. Their correlation is so frequent that we have come to look upon the contraction kidney of chronic interstitial nephritis as but the terminal stage in arteriosclerosis. In the study of these phenomena it is of the utmost impor- tance to recognize that arteriosclerosis pei- se need not, and fre- quently does not, imply a pathologic elevation in blood-pressure. Cases have come under observation in which evidence of arterio- sclerotic changes in the radials was marked; yet the sphyg- momanometer findings showed no evidence of hypertension — in one case hypotension was found. The anatomic changes found in the kidneys is the result of interference in their nutrition incident to alterations in normal blood-supply and normal blood-pressure. As the direct result of the heightened pressure generally found in cases of arteriosclerosis the pulse is usually spoken of as "high tension," "incompressible," or "hard." Associated with this we find the well-known urinary findings of chronic interstitial nephritis and usual'y an accentuated aortic second sound. The sphygmomanometer is of great value in studying the conditions of the circulation in cardiovascular renal disease, particularly in the early stages, when it may furnish a warning of grave danger. Often the individual is unaware that he has departed in any way from the normal. High pressure accom- panied by Email amounts of albumin and the occasional appear- ance of casts, is strong evidence in favor of permanent kidney change. In cases presenting only slight or no alteration in the normal blood-pressure, but with a suspicion of chronic nephritis, it will be necessary before establishing a diagnosis to eliminate transitory changes which may be due to constipation and auto- intoxications. In the absence of any definite physical signs which point to kidney involvement, but with a continuously elevated blood- pressure, which cannot be otherwise accounted for, we are justi- fied in strongly suspecting chronic interstitial nephritis. CLINICAL VALUE OF THE SPHYGMOMANOMETER. 85 In the average case of moderate duration the blood-pressure readings will range between 160 and 350 millimeters Hg or even higher. The height of the pressure will be practically con- tinuous except for minor fluctuations caused by constipation, auto-toxemias and the usual physiologic factors. In primary acute Bright's disease occurring in the course of scarlet fever there is practically always a sudden and marked rise in pressure. Buttermann {loc. cit.) has noted a rise of as much as 50 millimeters Hg occurring in 48 hours after the onset of an acute nephritis. In advance nephritis the detection of further sudden elevations will indicate the urgent necessity for immediate reduction in pressure to protect the cerebral vessels from rupture or the occurrence of edema of the brain and coma. Uremic Disturbances. — ^The routine use of the sphygmo- manometer in all cases of nephritis is as important as the routine examination of the urine. Approaching uremic crises are usually indicated by a sudden marked rise in pressure, often before they become evident in any other way.^** In cases of contracted kidney a sudden fall in pressure indicates the giving out of the heart. Cerebral Hemorrhage. — In the majority of cases of cerebral hemorrhage we have to deal with cardiovascular renal disease in which the involvement of the cerebral vessels is but an indicant (usually terminal) in the general arteriosclerotic change. Eecognizing this nntimate relation between these conditions it seems almost unnecessary to note that the control of the high pressure is the most essential feature in the management of cases with an apoplectic tendency. Ocular Hemorrhage. — Fox and Batroff^i report in detail a study of one hundred consecutive cases of ocular hemorrhage in which the blood-pressure test was employed. In 80 per cent, of these cases hypertension was encountered. Forty per cent, of these cases of retinal hemorrhage were accom- panied by chronic interstitial nephritis. Arteriosclerosis was present also in 37 per cent, and parenchymatous nephritis in 13 per cent. These authors are convinced that increased arterial tension ™H. Engel: BerUn. klin. Wochen., Oct. 23. 1908. " Colorado Med., May, 1909. 86 THE BLOOD-PRESSUKE. is an important factor in the causation of other ocular condi- tions, as acute glaucoma. Exophthalmic Goiter. — Norris^^ notes that instability of the blood-pressure in which there occurs frequent and irregular alterations between normal and hyper- and hypo- tension occurs notably in exophthalmic goiter. This condition has also been reported by L. F. Baker.^s These alterations, particularly toward hypotension, may be related to the tachycardia found in this disease either as cause or effect. Pregnancy, Toxemia, and Eclampsia. — The obstetrician of the present day must have constant recourse to the blood-pres- sure test if he would maintain the lead in his profession. The sphygmomanometer now ranks with urinalysis in the examina- tion of pregnant women. In the blood-pressure test we have a most valuable means of detecting early toxemias, which often lead to the eclamptic state. John Cooke Hirst {N. Y. M. J., June 11, 1910) states that the earliest and most constant sign of toxemia in the latter half of pregnancy is a high and constantly rising blood-pressure, and this symptom precedes albuminuria and all the constitu- tional signs of an impending eclamptic attack. Examinations of normal non-pregnant women showing no signs of heart or kidney lesions gave an average systolic pressure with the Faught instrument of 112 mm. He then took the pressure of 100 normal pregnant women, these showing no signs of albumin or any sign of toxemia, and found the pressure to average 118. He found that these figures held good up to ap- proximately seven and a half months, after which date there was a gradual rise, so that in the middle of the last month of the pregnancy a fairer average was 124 mm. ; with subsidence of the uterus the pressure showed a slight fall. Acute Infections. — In the study of infectious diseases, the routine use of the sphygmomanometer offers an almost unlimited field of usefulness, which no physician can afford to neglect; naturally this test offers very little in the way of diagnosis, but in prognosis and as a guide to treatment there is so much posi- tive evidence that it furnishes a constant and most reliable aid. ^ Loc. cit, ■"Jour. A. M. A., Dot. 12, 1907. CLINICAL VALUE OF THE SPHYGMOMANOMETER. 87 The basis for the application of this test is the relation of collapse to the vasomotor sj'stem. Single observations are valueless, because .they lack other figures for comparison; careful daily obpervation should be made and recorded, together with the pulse and temperature, and charted as shown on page 300. Pneumonia may show either a mild hypo- or hyper- tension, depending upon the toxemia. Vasomotor failure is early shown by a falling blood-pressure and a rising pulse rate. This is so uniform that Gibson {Edinburgh Med. Jour., Jan., 1908) for- mulated the following: — "When arterial pressure expressed in mm. of Hg does not fall below the pulse rate expressed in beats per minute, the fact may be taken as an excellent augury, while the converse is equally true." Typhoid Fever, — ^This is a disease showing hypotension, which gradually progresses with the development of toxemia. A pressure of between 90 and 100 may be considered normal in this disease. The pulse-pressure is diminished. The complica- tions of perforation and peritonitis occasion an early and decided rise followed by a secondary fall. Crile {Jour. A. M. A., May, 1905) and Cook and Briggs {Johns Ilophins Hospital Report, 1903) report that a complicating hemorrhage will cause a rapid fall in blood-pressure without an initial rise, by which fact it may be separated from the preceding. Diphtheria. — Low pressure during the invasion and a gradual rise toward normal during the convalescence. Scarlet Fever has little effect upon the blood-pressure curve unless complicated by a nephritis, when hypertension will be noted. Cerebrospinal Meningitis. — Blood-pressure tends to rise during the acute stage, and to return to normal during con- valescence. Sophian {Jour. A. M. A., vol. Iviii, 'So. 12) employs the sphygmomanometer as a guide during fluid removal and the injection of antimeningitic serum, and thereby was enabled to greatly reduce mortality of this disease. In Life-insurance Examinations^^ it is now well recognized ^'"The Insurance Examiner and the Blood-pressure Test," F. A. Faueht, Medical Record, Ausr- 10, 1912. 88 THE BLOOD-PRESSURE. that such pathologic changes maybe present in the cardiovascular and renal systems long before any suggestive symptoms are com- plained of by the individual, or if any complaint is made it is usually attributed to some trivial cause. In the presence of such a beginning arteriosclerosis the blood-pressure need not be greatly increased; an elevation of 30 to 40 mm. above that estimated as normal for the individual is significant and demands explanation. On the other hand, a rise of even this amount should never be hastily assigned to arterio- sclerosis and the risk therefore rejected without further study. It is necessary to recognize in this connection the activity of other and less important factors, such as the alimentary hypertension, so well described by Enssell, occurring in normal vessels due to errors in diet of either quantitative or qualitative origin and responding immediately to the correction of such errors, together with stimulation of the eliminative functions. Of further interest, particularly to the life-insurance examiner, are the so-called physiologic variations, caused by age, sex, mental and physical excitement, fatigue, etc. These must all be taken into consideration in estimating the character and class of the risk. (See page 75.) Such variations need not confuse the examiner, as they all occur within a range sufficiently restricted to prevent them from obscuring the issue. The only one which needs special consideration is the age factor. To determine this, employ the author's formula explained on page 77. High Pressure and Transient Albuminuria. — Probably the most confusing combination of symptoms met is the case which presents a slight hypertension and an occasional trace of albumin in the urine. These cases are best examined at the home or branch office and should be referred there whenever possible, for it is only after very careful and complete examina- tion with repeated urine and blood-pressure tests that a correct conclusion regarding the safety of the applicant can be reached. If after eliminating the possibility of an alimentary hyper- tension a distinct elevation in pressure remains with albumin in the urine, even in occasional minute traces, the risk is doubt- fully good, while if accomplished by accentuation of the aortic second sound or casts the risk is bad and calls for rejection. VI. COAGULATION TIME OF THE BLOOD. GENERAL CONSIDERATIONS. Ti-iE results obtained by improved clinical methods have only recently been reduced to a sufficiently accurate basis to admit of much confidence being placed in them, or to permit of their use in clinical medicine. The time required for coagulation of the blood depends upon many conditions. This fact makes attention to detail and uniformity of technic of the utmost importance. The chief factors influencing the coagulation-time are : 1. The time during which the shed blood remains in contact with the tissues, coagulation being slower from a deep than from a superficial incision; the difference in time may amount to as much as three minutes (Emerson). 2. The amount of pressure applied to the tissues to start the flow. 3. Upon the amount of blood shed. 4. Upon the nature of the vessel receiving the blood and its temperature. Factors of less importance are : time of day, atmospheric humidity, relation of time of shedding to diges- tion, ingestion of drugs, etc. THE SPECIMEN. This should be taken midway between meals, and not after the ingestion of drugs, which may influence coagulation- time. The tip of the finger or the lobe of the ear should be thoroughly cleansed and dried, and a sufficiently large puncture made with a flat-bladed instrument (Daland lancet) to insure free and sufficient flow. METHODS OF DETERMINATION. Milan's Method. — Place a drop of blood upon the center of a large glass slide or watch crystal. After a minute or two gently (89) 90 COAGULATION TIME. tilt the glass from side to side. This is continued at short in- tervals until the drop of blood, which at first assumed a pear shape when the glass was tilted, is seen to assume the form of a blunt and symmetrical cone. This change in the character of the drop indicates the completion of coagulation. The aver- age time of coagulation of normal blood by this method is about five minutes. This method is at best imperfect and uncertain, since none of the modifying factors are controlled. Wright's Method. — This is a far more accurate method, since in a large measure it controls and renders uniform the majority of the variable factors. Wright's coagulometer consists of the following parts (Fig. 13) :- Copper reservoir with numbered grid and rubber mat. Pinchcoek and tube. Thermometer marked 18.5° and 37° C. Twelve pipettes numbered 1 to 12. Wire for cleaning tubes. Lancets in bottle. Aspirating tube. Bottles for ether and alcohol. The Principle of the Method. — A series of tubes of stand- ard caliber filled with a definite amount of blood. The coagula- tion is observed at a standard temperature, and the coagulation- time is determined by blowing the contents of each tube upon a piece of clean white blotter or filter paper, at increasing in- tervals from the time of filling. The tubes are 10 centimeters in length and have an internal caliber of 0.25 millimeters; they are marked at a point 5 cen- timeters from the lower end of the tube {Fig. 1). The coagula- tion-time is usually determined at 18.5° C. This temperature has been, chosen as a standard because it approximates the tem- perature of the hospital ward or bed-room in temperate climates, and because this temperature delays the coagulation sufficiently long to allow the coagulation-time being, even in cases of rapidly coagulating blood, determined with considerable accuracy. In case of slowly coagulating blood it is convenient to carry out the determination at 37° C. (blood-heat). 92 COAGULATION TIME. Method of Filling the Tubes. — The blood which flows from the puncture is drawn into the tube with the least possible delay. A column of blood approximately 5 centimeters in length, is' drawn into each tube, and then this column drawn a few centi- meters from the tip of the tube. No important fallacy will arise if the column of blood be slightly longer or shorter than the 5 centimeters. Before beginning the observation it is well to have the tubes arranged in the order in which they are numbered. In ordinary cases from six to eight tubes are employed. For cases where coagulability is greatly reduced, a larger number of tubes will be required, unless the coagulation-time has been approximately determined by a preliminary test. A watch provided with a second-hand, and paper and pencil for noting the time of filling and blowing out, should be at hand. - If an assistant is available, the task of keeping record of the time may be conveniently committed to him. It is of decided advantage, before commencing, to set the second-hand so that it coincides with the minute markings around the dial. As each drop of blood is drawn, the time (min- utes and seconds) is recorded. It will be found convenient to allow an interval of ten or fifteen seconds between the filling of successive tubes. As each tulae is filled it is returned to the tank. It is not necessary to replace the rubber caps, as the air-space at the top of the tube is sufficient to prevent the water coming in contact with the blood in the tube. The viscosity of the blood will be sufficient to prevent the blood being forced further u.p the tube. During the time that the tubes remain in the reservoir the temperature of the water must be maintained by the addition of hot or cold water from time to time as necessary. After the lapse of exactly two minutes from the filling of tube Xo. 1, it is removed from the water and its contents blown upon a piece of white blotting or filter paper. If the contents cannot be blown out coagulation is noted down as complete. If the contents can be blown out, but shreds of fibrin are seen adhering to the interior of the tube, coagulation is recorded as incomplete. If the contents can be blown out cleanly and im- mediately sink into the paper, coagulation has not yet begun. METHODS OF DETERMINATION. 93 If in the first tube (two minutes) coagulation has been found to be complete, the second tube is removed at the expira- tion of one and three-quarter minutes, and the contents treated in the same manner. If this also is found clotted, the next in the series is tested at a still shorter interval, and so on until a tube is found in which coagulation is incomplete. If the blood in the first tube, after the expiration of two minutes, shows coagulation to be incomplete, a slightly longer interval is allowed before the second tube is blown out, and so on until one is found which shows coagulation to be complete. The same process is carried out if, in the first tube, coagu- lation has not yet begun, except that a still longer interval is allowed to pass before the second tube is blown out. Fig. 14.— Bogg's Coagulometer (A. H. T. Co.) Permissible Margin of Error. — If the directions which have been outlined above are adhered to, the margin of error in the coagulation-time should not be more than five seconds, and should never exceed fifteen seconds. The normal coagulation-time By this method lies between three and five minutes. Method of Russell and Brodie. — This method requires a microscope. The coagulometer consists of a small, moist cham- ber with a glass bottom (Fig. 14), which can be placed upon the stage of the microscope. This is fitted with a truncated cone of glass, which projects downward into the moist chamber. The end surface of this cone is of definite size (about 5 millimeters diameter), and on it is placed the drop of blood, care being taken to see that the drop of blood only just covers the surface ; hence is always of the same size. The cone is then quickly fitted into the moist chamber to prevent alterations due to drying, temperature, etc. Through the side of the moist chamber pro- jects a fine tube, through which, by means of a hand-bulb, a gentle stream of air can be projected against the blood. The 94 COAGULATION TIME. preparation of the slide being completed, the drop of blood is observed with the low power of the microscope. The blowing should be done at as long intervals as possible, and also very gently. The corpuscles will first be seen to move freely as individuals; later, as coagulation begins, the corpuscles will no longer move freely in the drop, but the drop will begin to change shape en masse. Knally, there will occur only elastic motion, and the part of the drop displaced by the current of air will spring back to its original position as soon as the current of air ceases. This is to be taken as the terminal point, as now only can the clot be demonstrated by quickly removing the slide and touching the drop to a piece of dry filter paper. This apparatus is probably the best method yet devised for the determination of coagulation-time. Eecently this instrument has been improved upon by Boggs, who has substituted a metal tube and an improved cone. With the improved instrument Emerson (Clinical Diagno- sis) reports normal variations in the coagulation-time between three and eight minutes, with an average of five minutes, eight seconds. CLINICAL OBSERVATIONS. The following results were obtained by Drs. Eobertson, Ill- man, and Duncan with the Wright apparatus^ : — The normal clotting time was found to be from two minutes, thirty seconds, to two minutes, forty-five seconds, using half blood-heat (18.5° C). Typhoid Pever. — In the acute febrile stage the average clotting time obtained from observations on 61 cases was three and one-third minutes. A lengthened clotting time was found to indicate impending hemorrhage. If, after hemorrhage, the usual shortening of the clotting time does not occur, then an- other hemorrhage is imminent. This shortening of coagulation- time is also noted after hemorrhage accompanying abortion and carcinoma of the uterus. In typhoid fever the clotting time returns gradually to nor- 'Report of Section on Pharmacology and Therapeutics, Araer. Med. Asso., page 51 ei itq.. 1907. CLINICAL OBSERVATIONS IN DISEASE. 95 mal, as the temperature declines with the establishment of con- valescence. In cases showing abnormal clotting time, the administration of the many drugs supposed to favor this condition, both sepa- rately and combined, was found to have no appreciable effect upon the condition. The following averages were obtained from observations on a large nuiriber of patients : — Specific meningitis 3.10 Coal gas poisoning 4.05 Gastric ulcer 4.10 Salpingitis 5.15 Alcoholic gastritis 3.10 Apoplexy 3.05 Carcinoma 4.35 Jaundice 6.30 to 8.30 Rheumatism 3.00 to 4.00 Pneumonia 2.47 Nephritis 2.25 Diabetes 2.55 Exophthalmic goitre 2.45 VII. BLOOD PARASITES. THE PLASMODIUM OF MALARIA. This is the only sporozoa found in the blood which is con- nected with disease in man, although numerous hemosporidia have been reported in many of the lower animals. History of the Malarial Parasite. — This is one of the mo:t interesting chapters in medicine. The parasite was discovered by Laveran in 1880, but it was not until 1885 that Golgi observed that sporulation occurred simultaneously with the malarial paroxysm. Golgi also demonstrated the existence of different species for different types of fever. Life History. — When man is first infected there commences a non-sexual cycle which is completed- in forty-eight or seventy- two hours, depending upon the species of parasite. The sporozoHe bores into a red cell, assumes a spherical form, and continues to enlarge. As it approaches maturity, it shows signs of division into a varying number of spore-like bodies. The para- site at this stage is termed a merocyte. When the merocyte ruptures, these spore-like bodies, or merocytes, each enter a fresh red cell and develop as before. At the time these merocytes rupture, it is believed that a toxin is liberated which causes the malarial paroxysm. The cycle goes on by geometric progression. From the iirst indication of the sporozoite it is usually two weeks before a sufficient number of merocytes rupture simultaneously to produce toxic symptoms (the period of incubation). This cycle is termed schizogony. After a varying time sexual forms develop. These are termed gametes, and show two types, one ■which contains more pigment, has little chromatin, and stains more deeply; this is the female, or macrogamete; in' the other there is little pigment, much more chromatin, and it stains less deeply; this is the male, or microgametocyte. (96) DESCRIPTION OF PLATE IV Malarial Parasites. (Kolle & 'Wa&seYmnnn.^ 1. Two tertian parasites about thirty-six hours old, attacked blood- corpuscles magnified. 2. Tertian parasite about thirty-six hours old; stained by Roman- owsky's method. The black granule in the parasite is not pigment but chromatin. Next to it and to the left is u, large lymphocyte, and under it the black spot is a blood-plate. 3. Tertian parasite, division form nearby is a polynuclear leukocyte. 4. Quartan parasite, ribbon' form. 5. Quartan parasite, undergoing division. 6. Tropical fever parasite (^stivo-autumnal.). In one blood- corpuscle may be seen a smaller, medium, and large tropical fever-ring parasite. 7. Tropical fever parasite. Gametes half-moon spherical form. Smear from bone marrow. 8. Tropical fever parasite, which is preparing for division heaped up in the blood capillaries of the brain. Asexual Forms 9. Smaller tertian ring about twelve hours old. 10. Tertian parasite about thirty-six hours old, so-called ameboid form. 11. Tertian parasite still showing ring fever, forty-two hours old. 12. Tertian parasite, two hours before febrile attack. The pigment is beginning to arrange itself in streaks or lines. 13. Tertian parasite further advanced in division. Pigment collected In large quantities. 14. Further advanced in the division. (Tertian parasite.) PLATE IV. LIFE CYCLE OF THE PLASMODIUM. 97 When the gametes are taken into the stomach of the mosquito (AnophelinEe), the male cell shows tail-like projections which have an active lashing movement, which break oS from the cell carrier and are thereafter termed microgametes. These enter the macrogametes and the combination forms a zygote. The zygote enters the epithelia layer of the stomach of the mosquito, where it continues to enlarge for about one week. When it has reached the size of about 60 microns it is seen to contain hundreds of delicate falciform bodies. The mature zygote now ruptures, and the sporozoites are thrown off into the midgut of the mosquito, whence they make their way to the salivary glands, from which they are introduced into the circula- tion of the person bitten by the mosquito, and then start the non-sexual cycle already described. As the sexual cycle takes place into the mosquito, this insect is the definitive host, while man is the intermediary host. There are three species of malarial parasites : first, P. vivax, or benign tertian, with a cycle of forty-eight hours; second, the P. malarice, or quartan, with a cycle of seventy-two hours, and, third, the P falciform, or estivo-autumnal or malignant tertian, with a cycle of forty-eight hours. Variations in cycle may be produced by infections from mosquitoes on successive nights, so that one will mature and sporulate twenty-four hours before the second. This will give a quotidian type of fever. In the estivo-autumnal infections ac- celeration or retardation in sporulation will cause a very pro- tracted paroxysm lasting from eighteen to thirty-six hours. This tends to give a continued fever instead of the characteristic type. In the diagnosis of malaria one should also examine both the fresh specimen and stain, as each gives valuable information in differentiating species. When time will not permit the examination by both methods, always use the smear stained by Leishmann's stain, as the small, externally situated rings of estivo-autumnal fever may escape notice in a fresh specimen. 98 BLOOD PARASITES. DIFFERENTIAL DIAGNOSIS OF PLASMODIA. TERTIAN. Develops in 48 hours. Pale and indistinct. Actively ameboid. Pigment fine. Pigment actively mo- tile. Pigment light. Full size of corpuscle. Degeneration forms twice the size of corpuscle. Segments 16 to 20. Irregular segments are frequent. Corpuscles large, colorless, and swol- len. QUARTAN. Develops in 72 hours. Sharp outline; re- fracts. Slightly ameboid. Pigment coarse. Pigment slow in move- ment. Pigment dark. Smaller than corpus- cle. Degeneration forms vary; much smaller than tertian. Segments 5 to 12. Beautiful rosettes. Corpuscles shrunken and brassy. ESTI VO- AUTUMNAI . Develops in 24 to 48 hours. Has irregular appear- ance. Actively ameboid. Pigment granules very few. Pigment still. Pigment light. Smaller than corpus- cle. Absent. Segmentation forms not seen in periph- eral blood. Corpuscles shriveled, brassy, not decolor- ized. Forms crescents. It is evident that, while there is considerable similarity among the three varieties of parasites, there are, nevertheless, certain peculiarities and characteristics which enable the expert to quite positively differentiate between them. THE DETECTION OF THE PLASMODIUM. In searching for the organism it is always desirable, when possible, to examine the fresh specimen at the bedside of the patient. If this is impracticable, warm the slide and seal the cover with a little vaseline, and the organisms will retain their motility for at least a couple of hours. If considerable time must elapse before the examination, films should be made, dried, and stained by one of the poly- chrome (Romanowski) stains. Cautions. — Do not have the blood-films too thick ; the individual cells should not overlap; there should be no rouleau formation. Do not give up the search until all the films have been examined thoroughly. A number of fields may show no organisms, then Eome may appear. The specimen is best ob- tained either eight or ten hours before or after a chill, ^s at this time the organisms are most likely to be in the peripheral cir- FILARIA SANGUINIS HOMINIS. 99 culation. Medication should be withheld for as long a time before the specimen is taken as possible. "What to Examine For. — Fresh specimen: Look for red cells containing actively moving black specks (pigment granules and li\ing protoplasm). Unusually pale cells containing clear areas which are irregular and constantly changing shape. Extra large red blood-cells. After a little experience the pigmented organisms are read- ily distinguished. Violent commotion among a group of red cells will direct attention to the flagellate. The hyaline bodies are visually the most difficult to identify without experience. The apparent, but not real (artefact), may be found in large numbers; these are usually distorted and deformed cells that have become altered in the preparation of the specimen. Finally, pigment-bearing leucocytes may be found in excess in malaria. These are, as a rule, polymorphonuclears which have become phagocytic, and have taken up the iron pigment set free by the malarial organisms. SPIROCH.^ETE OF RELAPSING FEVER. The specific organism of relapsing fever may be found in the fresh specimen of blood. Prepare this in the same manner as for the malarial parasite. This organism appears as narrow spiral filaments from 36 to 40 microns in length. They are actively motile, and attract attention by the active movement imparted to the red blood-cells in their vicinity. They are in the peripheral blood only during the fever, when they appear in large numbers. Many may be seen in a single field of the microscope. FILARIA SANGUINIS HOMINIS. There are several forms of this parasite; for special distin- guishing characteristics, larger works must be consulted. (See also page 113.) The most important of the group is the filaria nocturna, whiqh is supposed to be responsible for certain forms of chyluria, elephantiasis, and lymph-scrotum. The adult or parent organism is slender and thread-like, varying fiom three to six inches in length. It inhabits the Iji^mphatics and tissues. 100 BLOOD PARASITES. The embryos average in length about one-seventy-fifth of an inch, and are about as wide as a red blood-cell. The search for the embryos should be made both in the day and the night. Study under lovf power a fresh drop of blood. These embryos are decidedly active, and create a commotion among the neighboring cells. THE TRYPANOSOME. This is the organism of the so-called sleeping sickness. There are a large number of varieties of this organism which infest man and the lower animals. The trypanosoma g^ambiense can be demonstrated in the majority of cases. It is described as varying in length from 20 to 25 microns, by about .2 to 2.5 microns in width. (See e, Plate V.) It is distinguished by a single flagellum which extends through almost the entire length of the organism, and extends beyond its anterior end. When alive it has a slow, spiral, undulating movement. (See page 106.) LEISHMAN-DONOVAN BODIES. These bodies were first found in the spleen of patients who were apparently suffering from an irregular form of malarial fever. The body is small or elliptical, from 2 to 3 microns in diameter, containing chromatin. The bodies occur either free in the plasma or imbedded with others in a matrix or zooglia, often as many as a dozen being in one cluster. They are believed not to occupy the bodies of the "red blood-c'ells. They are not demonstrable in the peripheral blood. Staining. — ^Wright's modification of the Eomanowsky stain : the chromatin dark, the cell-body blue, and the zooglia a fainter mauve. VIII. ANIMAL PARASITES. THE ANIMAL PARASITES OF MAN. By "animal parasites of man" is meant animal organisms which live temporarily or permanently on or in the human body, and which receive their nourishinent therefrom. Temporary parasites include the ectoparasites (epizoa). These may inhabit the skin, conjunctival sac, the mouth, the nose, and the accessory sinuses. A familiar example of this class is the sarcoptes scabiei or itch mite. Most of the permanent or stationary parasites are found in the internal organs, and belong to the class of entoparasites or entozoa. Many of these parasites, such as the tenije, ascarides, and ankylostoma, inhabit man only when mature; others, of which the echinococcus is an example, inhabit man only during a certain period of their existence. Thus man may be either the actual or only the intermediate host. Again, for many para- sites, such as the tenia solium and the tenia saginata, man is the only host. Finally, man may also become the host for para- sites which, as a rule, select some other animal. Thus the cysti- cercus cellulosas, common to the pig and the cat, may occasion- ally be found in man. In this section the classification of Max Braun^ has been adopted, and the majority of the descriptions of the parasites which follow have been abstracted from this work. No attempt has been made to include the rarer forms of parasites, as these are considered to be beyond the scope of this work. ' Enslish Translation of "The Animal Parasites of Man." (Wood & Co., 1906.) (101) 102 ANIMAL PARASITES. CLASSIFICATION OF THE MORE COMMON ANIMAL PARASITES OF MAN. A. Protozoa. Class I. Ehizopodia. — Ameba coli (Loesch). Class II. Flagellata (Mastigophoea). (a) Trichomonas: 1. Trichomonas vaginalis. 3. Trichomonas intestinalis. 3. Trichomonas pulmonalis. (b) Gircomonades: 1. Lamblia intestinalis. 2. Tripanosoma. Class III. Spoeozoa. Coccidia: 1. Coecidium perforans or hominis. 2. Hemosporidia. Class IV. Infusoria. — Balantidium coli or Parame- cium coli. B. Platyhelmintlies (Flat worms). Class I. Teematoda (End). 1. Pasciola hepaticum syn. distomum hepaticum. 2. Distomum pulmonale syn. distomum Westermani. 3. Distomum lanceolatum syn. dicrocelium lanc6o- latum. 4. Distomum hematobium syn. bilharzia, syn. schis- tosomum hematobium. Class II. (Rud.) (a) Boihriocephaloidia: 1. Bothriocephalus latus. syn. tenia lata. (b) Teniidw: 1. Tenia nana. 2. Tenia lanceolata. 3. Tenia solium. 4. Cysticercus acanthotrias. 5. Tenia saginata or mediocanellata. 6. Tenia echinococcus. PROTOZOA. 103 C. Nematoda (Thread worms). 1. Strongyloides (rhabdonema- strongyloides) syn. anguil- lula intestinalis et stercoralis. 2. Filaria sanguinis hominis. 3. Trichocephalus dispar (whip worm). 4. Trichina spiralis. 5 . Ankylostom a duodenale. 6. Uncinaria Americana. 7. Ascaris lumbricoides. 8. Oxyuris vermicularis. A. PROTOZOA. The protozoa is a microscopic living organism. It is mono- cellular and represents the lowest form of animal life. The substance of the body consists of a finely granular, contractile protoplasm, which may be mono- or poly- nuclear. The viscid hyaline entosarc is capable of motion by expansion and con- traction, or by the extension and retraction of pseudopodia, cilia or flagella. Propagation takes place by segmentation or gemmination. CLASS I. RHIZOPODIA. The ameba coli produces the well-known amebic dysentery. The ameba in man is, however, not confined to the intestines, but has been found in the pus of liver abscesses, in pleuritic and peritoneal exudates, in the mucous membranes, and in tumors of the urinary bladder. Characteristics. — This organism is an ameboid body meas- uring from 20 to 30 microns in diameter. It is composed struc- turally of a clear protoplasmic outer portion, ectosare, and a finely or coarsely granular central portion, entosarc, which usu- ally shows a number of clear vacuoles and one or more nuclei {Plate V, a). When living it shows active ameboid movements which are greatly increased if the organism is kept warm. In the living state the cell frequently includes foreign bodies, such as bacteria, pigment granules, and fragments of blood-corpuscles and other cells. 104 ANIMAL PARASITES. Locomotion is accomplished by irregular extension and re- traction of pseudopods, which are thrown out from the periphery. These pseudopods are at first composed of the clear outer por- tion, which, as the projection gradually increases, includes the central granular zone. When surrounded by unfavorable envi- ronment, the organism undergoes a form of change known as the encysted state. In this state the body becomes spherical, and the outer wall thickened, while the division of the cell into two portions is lost, the whole becoming uniformly granular. Method of Examination. — The fecal discharges should be caught in a warm receptacle and immediately transferred to the laboratory for examination. If it is necessary to keep the specimen for a short time, this may be accomplished by imme- diately placing the specimen in a thermostat at body tempera- ture, where it should remain until transferred to the warm stage of the microscope. Preservation of specimens for more than a few hours is unsatisfactory, because even under the favorable circumstances of heat and moisture obtaining" in the thermostat, motility is rapidly lost, and at the expiration of twenty-four or thirty-six hours the organisms are no longer discoverable. The "Warm Stage. — A convenient method of maintaining a warm stage for examination and study of this organism, is as follows: A flat strip of copper or of brass, three inches wide by six or eight inches long, is perforated by a half or three- quarter inch aperture, situated in the center of one end at a distance of about one inch from the free margin. The stage of the microscope should be covered with several thicknesses of asbestos paper or felt, upon which the metal sheet is clamped so that the openings in the stage and in the copper-strip coin- cide. A Bunsen burner or alcohol lamp is adjusted under the outer extremity of the strip, so that when the metal has attained its maximum heat the portion immediately surrounding the aperture is maintained at about body-temperature. A specimen placed upon a slide, in position for examination, will maintain the motility of the ameba for a number of hours. It will be found convenient to examine the spread, first by the low power, then, after locating a suitable portion of the slide, the higher objective may be swung in and the organisms studied in detail. The important aids to successful search are PLATE V. Intestinal Parasites of Man. [RedraiAjn from Max Braun) a, Amebae coli. 4,_ Trichomonas vaginalis, c, Trichomonas intestinalis. d, Lamblia intestinalis. e, Tripanosomes. /, Coccidium hominis. PROTOZOA. 105 a thin and uniform spread of liquid feces, and a low, slightly oblique illumination. CLASS II. FLAGELLATA. a. Trichomonides. 1. Trichomonas Vaginalis (see Plate V, b). — The form of the body is very variable, elongated, fusiform or bean-shaped. It is ameboid. The length varies between 0.015 to 0.025 milli- meters in length, by 0.007 to 0.013 in breadth. The posterior ex- tremity is drawn out into a point and is about one-half the length of the remainder of the body. The cuticle is very thin and the body-substance finely granular. At the anterior ex- tremity there are three or four flagella, which are of equal length, and which are firmly United at their base, but which easily fall oflE. There is an undulating membrane which moves spirally across the body, arising from the base of insertion of the flagella and terminating at the base of the caudal process. The nucleus is vesicular, elongated, and situated in the anterior extremity. Propagation occurs by division. 2. Trichomonas Intestinalis (see Plate V, c). — Some authors believe this organism to be identical with the trichomonas vaginalis. It is described as being 0.01 to 0.015 millimeter in length ; the posterior extremity terminates in a point with a row of cilia. These commence at the anterior extremity and extend over the body. This organism has been found in the urethra of man, the vagina, the large and small intestines of both healthy and sick persons, in the stomach and in the oral cavity. 3. Trichomonas Pulmonale. In all probability this is identical with the preceding. b, Cercomonides. 1. Cercomonas Intestinalis or Lamblia Intestinalis (see Plate V, d). — Length, 0.01 to 0.02 millimeter, and width, 0.005 to 0.012. The flagella are of about equal length (0.009 to 0.014 millimeter). The body is finely granular, and has a very thin cuticle, which does not entirely prevent changes in the form of the body. The very motile tail-like appendages in the frontal 106 ANIMAL PARASITES. plane are flattened; the excavation at the anterior extremity which is directed obliquely forward and with its border project- ing backward. The anterior pair of flagella arise from the ante- rior edge of the peristome; the lateral and median from the pos- terior edge, whereas the tail flagella are inserted at the pos- terior end. The anterior flagella appear to be connected with the nucleus. The nucleus is dmnb-bell shaped and has a nucleolus in each half, and lies anteriorly in the part of the body which is ex- cavated. This organism has encysted stages. These cysts are oval and measure .01 by .007 millimeter. They are surrounded by a fairly thick hyaline layer through which the outline of the creatures can sometimes be seen quite distinctly. Trypanosoma. — The Trypanosome- has a more or less spindle-shaped body, along one border of which runs an undulat- ing membrane. There is a nucleus and a blepharoblast, the latter being located anteriorly as a chromatin staining dot or rod. From this blepharoblast the flagellum proceeds posteriorly bordering the undulating membrane and projecting freely beyond the posterior end. The nucleus is larger, nearer the posterior end, and does not stain so intensely as the bleph- aroblast. T. Gambiense. — This is the trypanosome causing human trypanosomiasis, the later stage of which is known as sleeping sickness. It is from 17 to 28 |U, long, and from 1.5 to 3 ^u wide. It is very difficult to distinguish the human trypanosome from some of the other pathogenic ones by staining methods. It is present in the blood, usually in exceedingly small numbers, and in the lymphatic glands of patients. It is by puncture of the glands that we have the best means of finding the parasites. The parasite stains readily with Wright's stain. The transmitting agent is the Glossina palpalis. It is not known whether this occurs by direct or indirect transmission. At any rate, no tsetse, no trypanosomiasis. CLASS III. SPOROZOA. Coccidia. 1. CocciDiuM HoMiNis OR Peefoeans. — This organism is oval. The fertilized sporont stage is the oocyte (see Plate \, f), and measures .024 to .035 by .003 to .014 millimeter. It is sur- rounded bv an integument with a double outline which has an ^ E. R. Stitt : " Practical Bacterioloey," etc., 1909. PLATYHELMINTHES. 107 opening at the pointed pole. The plasm, which is somewhat coarsely granular, entirely fills the integument or is gathered together in a rounded central mass. The coccidia are evacuated from the bowel in this stage, and sporulate in the open within two or three weeks. The fully developed spores are of a broad fusiform shape and measure .012 to .015 millimeter in length by .007 millimeter broad. They each contain two sporozoites, broad at one end and pointed at the other, forming a bent dumb-bell shaped body. The chromatin mass is called the centrosome, and the extremity of the body which encloses this, is the anterior extremity. A granular residual body lies in the concavity. This or- ganism is found in the intestinal tract,- where it may give rise to violent auto-infection and chronic diarrhea. 2. Hemospoidi^. — These organisms are the cause of ma- laria and have been described in another section (see page 96). CLASS IV. INFUSORIA. Balantidium Coli or Paramecium Coli. — The iody is oval, .06 to 1.0 millimeter in length, by .05 to .07 millimeter in width. The peristome is funnel-shaped or contracted, the anterior end being either blunt or pointed. The ecto- and ento- sarc are distinctly separate, the latter granular, and containing drops of fat, mucus, starch granules, bacteria, and occasionally leukocytes' and erythrocytes. There are usually two contractile vacuoles. The anus opens at the posterior extremity, and the organism contains a macro- and a micro-nucleus. It occurs in the large intestines of man. B. PLATYHELMINTHES (Flat Worms). CLASS I. TREMATODES. 1. DiSTOMDM Hepaticdm (Liver fluh e ) .—Jjength, 20 to 30 milluneters ; breadth 8 to 13 millimeters. The head-cone is 4 to 5 millimeters long, and sharply demarcated from the posterior part of the body. Spines in alternating transverse rows, and extending on the ventral surface to the posterior bor- der of the testes, and on the dorsal surface not quite so far. The spines are smaller on the head-cone than on the posterior part 108 ANIMAL PAEASITES. of the body, where they are discernible by the naked eye. The suckers are hemispherical and near each other. The oral sucker is about 1 millimeter, and the ventral about 1.6 millimeter in diameter. The pharynx, which includes almost the entire esoph- agus, measures .7 millimeter in length and A millimeter in breadth. The intestine bifurcates in the head-cone, and the branches are furnished with blunt sacs directed outwardly. The ovary is ramified and is situated in front of the transverse vitello duct. The shell-glands lie near the ovary and are in the median line. The ova are yellowish-brown, oval, with cap-like lid. They measure .130 to .145 by .07 to .09 millimeter. The average size is .132 by .08. The liver fluke is an inhabitant of the bile- ducts of man. 2. DisTOMUM Pulmonale oe Distomum Westeemanni. — The body is of a faint, reddish-brown color and plump oval shape, with the ventral surface a little flattened. This organism measures 8 to 10 millimeters in length by 4 to 6 millimeters in thickness. It possesses two suckers of equal size (.75 millime- ter). The eggs are oval brownish-yellow, with a fairly thin shell and measure .0875 to .1035 millimeter in length, by .052 to .075 millimeter in breadth, the average being .0935 by .057 milli- meter. Their location is usually in the lung, but they may enter the blood-vessels and be carried to another part of the body. 3. Distomum Lancedlatum or Diceocelium Lanoeo- LATUM. — In the fresh condition this is a yellowish-red organism, the body is flat, almost translucent, with a conical neck at the level of the ventral sucker. This point is marked by a shallow constriction. The length and breadth vary according to the amount of contraction, being usually 8 to 11 millimeters by 1.5 to 2.0 millimeters. The suckers are about one-fifth of the body-length distant from each other, and of about equal size (.23 by .25 millimeter). The eggs are oval with a sharply defined operculum at the pointed pole. They measure .030 by .011 millimeter, and occur in the feces. The parasites reside in the liver. 4. Distomum Hematobium ok Bilhaezia. — The male is PL/^TYHELMINTHES. 109 whitish and measures 12 to 14 millimeters in lengthy but is already mature when 4 millimeters long. The anterior end is 6 millimeters or a little more in length ; the suckers are situated near each other ; the oral sucker is inf undibulif orm ; the dorsal lip being a little longer than the ventral. The ventral sucker is slightly the larger, and is pedunculated. A little behind the ventral sucker the body broadens to a width of 1 millimeter, decreasing, however, in thickness. The lateral edges curl ven- trally, so that the posterior part of the body is almost cylin- drical. The posterior end is somewhat attenuated, and the dorsal part of the posterior extremity of the body is covered with spinous papilli. The females are iiliform, about 30 millimeters in length, pointed at each end and measuring .25 millimeter in diameter. The color of the body varies according to the contents of the intestine (posteriorly they are dark-brown or black). The cuticle is smooth except in the suckers, where there are very delicate spines, and at the tail end where there are larger spines. The anterior part of the body measures from .2 to .3 millimeter in length. The eggs are fusiform and much dilated in the middle. They have no lid and are provided with a terminal spine at the posterior extremity. The eggs measure 0.12 to 1.12 millimeters in length, by .05 to .073 millimeter in breadth. They are yel- lowish in color, slightly transparent, and provided with a thin shell. The spine may sometimes be absent. The eggs apparently vary greatly in size. The organism lives in the portal vein and its branches. CLASS II. a, Bothriocephaloidia. i. BoTHRiocEPHALis Latus OR Tenia Lata. — Length from 2 to 9 or more millimeters. Color, yellowish gray; after lying in water lateral areas become brownish and the rosette of the uterus brown. The head is almond-shaped, 3 to 3 milli- meters in length. Its dorso-ventral axis is longer than its trans- verse diameter; the head is therefore generally flat, concealing the suctorial grooves at the borders. The neck varies in length according to the degree of contraction, and is very thin. There 110 ANIMAL PARASITES. are from 3000 to 4500 proglottides. Their breadth is usually greater than their length, but in the posterior third of the body they are almost square, while among the very oldest some may be longer than they are broad. The eggs are large with brownish shells and small lids. They measure .068 to .071 by .045 millimeter. The proglottides near the posterior extremity of the worm are frequently eggless. b. Teniidae. 1. Tenia Nana. — The worm is 10 to 15 millimeters in length, and .5 to .7 millimeter in breadth. The head is globular and is from .25 to .30 millimeter in diameters. The rostellum has a simple crown consisting of 34 to 30 hooks, which are only .014 to .018 millimeter in length. The neck is moderately long. The proglottides are very narrow, about 150 in number, .4 to .9 millimeter in breadth, by .014 to .030 millimeter in length. The eggs are globular or oval, and measure .030 to .048 millimeter. The oncospheres measure .016 to .019 millimeter in diameter. The worm lives in the intestines; the ova, and proglottides are found in the feces. 3. Tenia Lanceolata or Hymenolepis Lanceolata. — The parasite measures 30 to 130 millimeters in length, and 5 to 18' millimeters in breadth. The head is globular and very small, the rostellum is cylindrical with a crown composed of eight hooks (0.031 to 0.035 millimeter in length). The neck is very short. The segments increase gradually in breadth, but vary little in length. The ova have three envelopes and are oval, measuring 0.050 by 0.035 millimeter. The external envelope is membranous and much wrinkled, the middle one is thick, and the internal one very thin. 3. Tenia Solium or Tenia Vulgaris. — The average length of the entire tapeworm is about 2 to 3 meters, but may be more. The head is globular, 0.6 to 1.0 millimeter in diameter. The rostellum is provided with a double row of hooks, twenty-two to thirty-two in number; large and small hooks alternate regularly. The length of the largest hooks is 0.16 to PLATYHELMINTHES. HI 0.18 millimeter, of the small ones 0.11 to 0.14 millimeter. The average number of proglottides is 800 to 900; they increase very gradually in size. At about 1 millimeter behind the head they are square and have the generative organs fully developed. Segments sufficiently mature for detachment measure 10 to 12 millimeters in length, by 5 to 6 millimeters in breadth. The fully devloped uterus consists of a median trunk with seven to ten lateral branches on each side, some of which are again ramified. The eggs are oval, the egg-shell very thin and delicate. The embryonal shell is very thick with radial stripes ; it is of a pale- yellow color, globular, and measures 0.031 to 0.036 millimeter in diameter. The oncospheres with six hooks are likewise globu- lar, a;nd measure 0.02 millimeter in diameter. 4. Cysticercus Acanthoteias.— This resembles the cysti- cercus cellulose in form and size, but carries on the rostellum a triple crown each consisting of fourteen to sixteen hooks which differ from the hooks of the cysticercus cellulose or of the tenia solium by the greater length of the posterior root process and the more slender form of the hooks. The large hooks measure 0.153 to 0.196 millimeter, the medium-size hooks 0.114 to 0.14, and the small ones 0.063 to 0.07. 5. Tenia Saginata or Tenia Mediocanellata. — The length of the entire worm averages 4 to 8 to 10 meters and more, even up to 36 meters. The head is cuboid in shape, 1.5 to 2 millimeters in diameter. The suckers are hemispherical (0.8 millimeter), and are frequently pigmented; There is a sucker-like organ in place of the rostellum, and this is also frequently pigmented. The neck is moderately long and about half the breadth of the head. The proglottides average more than 1000, and gradually increase in size. from the head back- ward. The detached mature segments are exactly like pumpkin- seeds — they are about .16 to 20 millimeters long, by 4 to 7 milli- meters broad. There are twenty to thirty-five lateral branches at each side of the uterus, and these often again ramify. The eggs are more or less globular, the egg-shell frequently remains intact, and carries one or two filaments. The embryonal shell is thick, radially striated, transparent and oval. It is 0.3 to 0.4 millimeter in length, by 0.02 to 0.03 millimeter in breadth. 112 ANIMAL PARASITES. This worm in the adult condition dwells exclusively in the intestinal canal of man. The corresponding cysticercus occurs in the ox and steer. 6. Tenia Echinococcus. — This worm measures 3.5 to 5 or 6 millimeters in length; the head is 0.3 millimeter in breadth, and has a double row of twenty-eight to fifty booklets on the rostellum. The size and form of these booklets vary. The larger ones are 0.040 to 0.045 millimeter in length, the smaller ones are 0.030 to 0.038 millimeter. The suckers measure 0.13 millimeter in diameter. The neck is short, behind which there are only three or four segments, the posterior of which is about 2 millimeters in length and 0.6 millimeter in breadth. The ovary is horse-shoe shaped, with the concavity directed back- ward. The median trunk of the uterus is dilated when filled with eggs, and instead of lateral branches has lateral protuberances. It is not uncommon for the eggs to form local heaps. The em- hryonal shell is moderately thin with radiating fibers, is almost globular, and measures 0.030 to 0.036 millimeter in diameter. The mature parasite lives in the small intestine of the domestic dog and the wolf, and from them, the dog chiefly, they are transmitted to man. Diagnosis of Cestodes. — ^The microscopic examination of the feces should never be neglected when the presence of tape- worm is suspected. Often by careful, frequently repeated exami- nations, insistent symptoms, referable to the digestive tract, the nervous system or the general nutrition, may be cleared up by the finding of segments or ova in the feces. As the uterus of the tenia has no exit, the eggs can only find egress when the mature proglottide is injured. In the case of the tenia saginata the discharge of eggs is almost the rule. The proglottides whejj discharged are usually without eggs. The eggs of the solium and the saginata are only distinguished by their size. In examining the stools for evidences of tapeworms, one must be careful not to confound remnants of undigested food, mucous casts, and shreds of tendon with the proglottides. The proglottides, after being soaked in water, assume their character- istic form. As a rule the microscopic determination of ova is a more certain means of diagnosis than the macroscopic seg- ments. NEMATODES. 113 To determine from the shape of the proglottide which variety of worm is present, it is advisable to fix the segment between two glass slides. The proglottide of the tenia solium is more delicate and more transparent than the tougher segments of the tenia saginata. In the former the branching uterus is more plump, and the number of lateral twigs- are from seven to ten, while the uterus of the tenia saginata shows from twenty to thirty or more. C. NEMATODES (Thread Worms). Strongyloides Intestinalis or Anguillula Intbsti- NALis ET Steecoralis. — 1. (a) The parasitical generation (anguillula intestinalis) measures 2.3 millimeters in length by 0.034 millimeter in breadth. The cuticle is finely transversely striated. The mouth is surrounded by four lips, the esophagus is almost cylindrical and is a quarter the length of the body. The anus opens just in front of the pointed posterior extremity. The eggs measure 0.050 to 0.58 millimeter in length, and 0.030 to 0.034 in breadth. (b) The free-living generation (anguillula stercoralis) is sex- ually differentiated. The body of the male is cylindrical, smooth, somewhat more slender at the anterior extremity, and pointed at the tail end. The mouth has four lips and the esophagus a double dilatation. The males measure 0.7 by 0.035 millimeter, and carry the posterior extremity curled up. The two spicules are small and much curved. The females measure 1.0 millimeter in length or a little more, 0.05 millimeter in breadth. The tail end is straight and pointed. The yellowish thin-shelled ova measure 0.07 millimeter in length by 0.045 millimeter in breadth. 2. PiLARiA Sanguinis Hominis (filaria lancrofti or filaria nocturna). — The male is colorless and measures 40 millimeters in length and 0.1 millimeter in diameter. The cephalic ex- tremity is a little thickened, the posterior extremity is bent and rounded, but is not twisted cork-screw like. The anal orifice opens 0.138 millimeter in front of the posterior border. The female is brownish, 7.8 to 8.0 millimeters in length and 0.21 to 0.28 millimeter in breadth. The cephalic and caudal extremi- 114 ANIMAL PARASITES. ties are rounded. Almost the entire body is occupied by the two uteri, from which the larvae emerge early. The length of the larvcB average 0.13 to 0.3 millimeter, their breadth 0.007 to 0.07 millimeter. They are surrounded by a delicate protective investing membrane which is not quite close to them. The lymphatic vessels in various parts of the body are doubtless the normal habitat of the adult worms, but these have also been found in the left ventrice of the heart. The young eva, by means of the lymph-stream, reach the blood and are distributed with it through the body. They also pass through the vessel walls and may be found in the fluid of the glands of the body. The larvae are first found in infected patients only in specimens of blood that have been taken after sunset. Their number increases considerably until after midnight, and after that time begin to diminish. From mid-day until evening no filariae are found in the peripheral blood. 3. Teichocepi-ialus Dispak ok Ascaris Thichiuea. — The male measures 40 to 45 millimeters in length, the spiculum is 2.5 millimeters long and lies within a retractUe pouch beset with spines. The female measures 45 to 50 millimeters in length, of which two-fifths appertain to the posterior part of the body. The ova are barrel-shaped and have a thick, brown shell which is perforated at the poles. Each opening is closed with a light- colored plug. The eggs measure 0.05 to 0.054 millimeter in length and 0.033 millimeter in breadth. They are deposited before segmentation. This worm usually lives in the cecum of human beings, and is occasionally found in the vermiform ap- pendix, in the colon, and exceptionally in the small intestine. Usually only a few are present, and they do not cause any par- ticular disturbance. The development of the eggs is completed in water or in moist soil, and occupies a longer or shorter period according to the season. The eggs and larvffi possess great powers of resist- ance, and have been known to remain as long as five years in the egg-shell without losing their vitality. 4. Trichina Spiralis. — The male measures 1.4 to 1.6 mil- limeters in length and 0.04 millimeter in diameter. The ante- rior part of the body is narrowed, the orifice of the cloaca is terminal and lies between the two caudal appendages; behind NEMATODES. 115 there are four papillae. The female measures 3 to 4 milliiiieterB in length and 0.06 millimeter in diameter; the anus is terminal. Trichina spiralis occupies in its adult stage the small intestines of men and of various mammals, including the domestic rat, domestic pig and domestic dog. HisTOEY OF Development of Trichina Spiralis. — Shortly after entering the intestines the encysted trichinae, escape from their capsules, and then enter the duodenum and jejenum where they become adult. During this period they do not greatly increase in size. The males grow from 0.8 to 1.0 millimeter, the females from 1.5 to 1.8 millimeters. Soon after copulation, which takes place in the course of two days, the males die off, and the females, which soon attain the length of 3.0 to 3.5 millimeters, either bo"re more or less deeply into the villi or penetrate the mucous membrane and enter the lym- phatic spaces. Here they deposit their young which, according to Leuckart, average at least 1500. The migrations are mostly passive, the larvae being carried along by the lymph-stream or by the circulating blood. The young brood is distributed throughout the entire body, but the conditions necessary to its further development, are found only in the transversely striated muscle. On the ninth or tenth day after infection the first trichinffi have reached their destination, but further invasions are constantly taking place. Two or three weeks after infec- tion the^ spirally rolled up trichinae have grown to 0.8 to 1.0 millimeter, and in their vicinity the muscle fibers are swollen. The capsule is formed by the inflamed connective tissue pro- ducing the cystic membrane. The cysts are lemon-shaped and usually lie with their longitudinal axis in the direction of the muscle fibers. On an average they measure 0.4 millimeter in length by 0.25 millimeter in breadth. 5. Anktlostoma Duodenale or TJncinaria Duodenale. — The body is cylindrical, attenuated anteriorly, and of a slightly reddish color. In the oral cavity on the ventral surface, close behind the orifice, are four hook-like teeth directed back- ward; on the dorsal surface there are two teeth directed for- ward. The males measure 8 to 10 millimeters in length, and 0.4 to 0.5 millimeter in breadth. The bursa has two large lateral and one small dorsal alar processes. The females measure 18 to 116 ANIMAL PAEASITES. 18 millimeters in length, and the caudal extremity has a small spine. The eggs are elliptical and have thin shells; they meas- ure 0.033 to 0.045 millimeter in breadth, and 0.055 to 0.065 millimeter in' length. The ankylostpma duodenale lives in the duodenum, and may rarely be found in the first part of the jejunum. 6. Uncinaeia Americana can readily be distinguished from the preceding worm. It is shorter and more slender. The male worm measures from 7 to 9 millimeters in length by 0.3 to 0.35 in diameter; the female 9 to 11 millimeters in length by 0.4 to 0.45 millimeter in diameter. The buccal capsule is much smaller, and presents an irregular border; instead of four ven- tral hook-like teeth, it is provided vi^ith a vertical pair of promi- nent semilunar plates similar to those of a dog hook-worm. The pair of dorsal teeth is likewise represented by a pair of slightly developed chitinous plates of the same nature. The eggs are larger than in IT. Duodenale ; they measure 64 to 75 micromillimeters by 36 by 40 micromillimeters in breadth. So far this worm has been found only in man; its anatomical habitat is the small intestine. 7. AsCABis LuMBRicoiDBS. — The coloring in the fresh stage is reddish- or grayish-yellow. The body is of an elongated spindle shape and the dorsal oral papillae carries two papillae of sense and the two ventral oral papillae are papillae of sense. The male measures from 15 to 25 centimeters in length and about 3 millimeters in breadth. The posterior extremity is conical and bent ventrically into a hook. The spicules measure 3 millimeters in length and are curved and broadened at their free ends. On each side of the orifice of the cloaca are seventy to seventy-five papillae, of which seven pairs are post-anal. The female measures 30 to 40 centimeters in length and about 5 millimeters in diameter, the posterior extremity is conical and straight. The vulva is at the border between the middle and posterior thirds of the body, from which the two uterine tubes pass straight to the posterior end of the body. The convoluted ovaries measure ten times the length of the body. The ova are elliptical with a thick transparent shell and an external coating of albumin which forms protuberances. The ova measure 0.05 to 0.07 millimeters in length and 0.04 to 0.05 NEMATODES. 117 millimeters in breadth; they are deposited before segmentation. The albuminous coating is stained yellow by the coloring matter of the feces. This worm is one of the most frequent parasites of man, and is distributed over all parts of the world. 8. OxYDRis Veemiculaeis or Ascaeis Veemicularis. — Color, white; the attenuated cuticle forms swellings at the anterior end which extend some distance back along the middle of the ventral and dorsal surfaces. There are three small re- tractile labial papillae around the mouth. The male measures 3 to 5 millimeters in length, and shortens on death. The poste- rior extremity of the body is rolled ventrally and presents pa- pillse. The female is 10 millimeters in length and 0.6 milli- meter in diameter. The anus is about 3 millimeters in front of the tip of the tail ; the vulva is in the posterior third of the body. The eggs are oval, thin-shelled, and measure 0.05 by 0.02 millimeter. They are deposited with embryos already de- veloped, and are seldom found in the feces. IX. DETERMINATION OF THE FUNCTIONS OF THE STOMACH. THE GASTRIC CONTENTS, The significance of the term gastric contents, in the fol- lowing pages, is taken to mean the material found in the stomach and extracted by the gastric tube at the expiration of a fixed and definite period after the ingestion of a test-breakfast. ^ THE VOMITUS. It is not good practice to utilize vomitus for the purpose of chemical analysis. Such material is usually of very uncertain composition, being contaminated with mucus from the upper part of the tract; further, the amount or composition of the food previously ingested is an unknown factor, affecting mate- rially the quantitative and qualitative findings. However, it may be found of decided advantage to test all vomited matter for the presence or absence of acidity and free hydrochloric acid. In the presence of suspected cancer the vomited material may be searched for necrotic tissue shreds or sarcinse. METHODS OF OBTAINING SPECIMEN FOR EXAMINATION. The usual apparatus employed to remove the test-meal comprises the well-known gastric tube of soft red rubber, fitted at one end with a soft rubber funnel, and containing near this a bulbous expansion without valves. A recent modification of and improvement over this is the large bulb devised by Ewald. This bulb is sufficiently large to contain the total quantity of ' The Ewald test-breakfast consists of an ordinary roll weigrhing about 35 ^rams, and 300 cubic centimeters of water or weai^ tea without mUk or sugar, (118) METHODS OF OBTAINING SPECIMEN. 119 material removed, thus overcoming in a measure the ditficulties of the smaller bulb. These two methods are usually successful in obtaining the xlesired material for examination, but are dif- ficult to manage and possess a decided disadvantage in that they Fig. 15.— Complete Outfit for Gastric Test-meal Removal, Lavage and Inflation. do not provide for the important procedure of lavage and. infla- tion. Also, tubes of irregular caliber are difficult to cleanse, and unless great care is exercised in this direction, they may become a source of contamination, if not carriers of infection. 120 THE STOMACH. Some years ago Dr. Judspn Daland adopted for this pur- pose two large open-mouth bottles and a double-action Davidson bulb ; these are used in conjunction with the plain gastric tube. With this arrangement, aided by an assistant, it is possible to rapidly and cleanly obtain a sample of gastric contents (in con- siderably less than a minute), and to follow immediately with lavage and inflation if desired. To those who have used this method with uniform success in both private and hospital practice, its advantage is evident, the only drawback being the almost absolute necessity for trained assistance, this beiag occasioned by the complicated nature of the apparatus, which requires a second pair of hands to manipu- late the bottle and its connections .during the passage of the tube. To overcome this drawback the author has devised a reversing valve (see Fig. 15), With this valve it is unnecessary to make any change in the tube connections when once the apparatus is set up. The perfected device has been worked out with great care, having in mind the necessity of a simple mechanism capa- ble of beiag cleansed and kept in repa,ir without difficulty. PRELIMINARY PREPARATION OF THE PATIENT. The preliminaries leading up to the extraction of the test- meal should be as nearly uniform in every case as possible. By adopting a definite routine and adhering to it we eliminate, in a great measure, the errors which would otherwise creep in and lesson the value of the findings. The adoption of the following rules will enable the examiner to obtain a series of reports in one case or in groups of cases of far greater value than could be obtained by an irregular technic: — ■ First. — It is advisable that all medication should be with- held until after the gastric analysis has been made. If this is impossible, then a period of days should elapse before making the test, during which all treatment is stopped. Second. — The test-meal should be given on an empty stom- ach; either one which has been emptied by fasting or by pre- vious lavage. MODIFIED EWALD BEEAKFAST. 121 Third. — The volume and composition of the test-meal should be uniform. The particular meal employed to be deter- mined by the operator according to his preference. Fourth. — ^The removal of the meal should (depending on its composition) be accurately timed.; this time to be measured from the beginning and not from the end of the meal. Fifth. — The test-meal should be removed without dilution if possible. If dilution should be necessary to accomplish re- moval, the amount of water used should be definitely knoVn, and the total withdrawn must exceed the amount used. If this rule is not observed, all investigations of a quantitative nature are valueless. Sixth. — The tube should remain within the esophagus only long enough to allow time to compress the bulb. If much time elapses after introduction before removal of the contents, the hypersecretion of mucus occasioned by the presence of the tube will alter the composition of the sample and possibly result in erroneous conclusions. A tube of relatively large diameter is to be preferred, being easier of insertion, less uncomfortable to the patient, and in- suring greater success in obtaining a sample. A suitable tube should measure between three-eighths and one-half inch outside diameter, and should be provided with both a terminal and lat- eral opening in the gastric end. A lubricant for the tip of the tube is unnecessary. The contact of the tube with the patient's pharynx immediately ex- citing sufficient flow of mucus for this purpose. This is prefer- able to either nauseating oil or hygroscopic glycerine. In case of great reflex excitability of the pharyngeal constructors, this region may be first sprayed with a dilute solution of cocaine. Modified Ewald Breakfast. — Of the test-meals employed, one of generally useful composition includes the white of two eggs poached or soft boiled, without yelks and without season- ing; two pieces of toast without butter (the slightest trace of butter or fat will cause lactic and butyric acid fermentation), and a cup of tea without milk or sugar. This meal should be removed at the expiration of one hour when, under ordinary circumstances, there will be recovered be- tween 30 and 90 cubic centimeters. 122 THE STOMACH. The component parts of the apparatus are as follows: — A plain gastric tube without bulb or funnel. A double-action Davidson hand-bulb. A large rubber stopper having two perforations. Two wide-mouth bottles containing more than a liter each, and graduated in cubic centimeters.^ A short length of large glass-tubing and some one-quarter inch rubber-tubing. ' The reversing valve. Technic of Removal. — Fill one graduated bottle to the 500 cubic centimeter mark with warm, sterile water. Tit the double perforated stopper with the glass tube and the reversing valve. Place the stopper firmly in the empty bottle and attach the gas- trie tube to the glass tube, and the two ends of the double action bulb to the two horizontal tubes of the valve. Finally, ascertain the direction of the air-current through the valve by making a few pressures on the bulb. Set the valve to make negative pres- sure withiQ the bottle. The tube should now be passed, with the patient preferably in the sitting posture. As soon as the tube enters the cardiac orifice, a few quick pressures are made on the bulb. This de- velops a slight degree of negative pressure within the bottle, when the gastric contents will immediately flow into the bottle. Sudden stopping of the flow from occlusion of the tube by par- ticles of food or mucus, may immediately be removed by mo- mentarily reversing the lever. This will cause a small portion of the gastric contents to return through the tube, effectually washing out the obstruction. This simple maneuver may be repeated as frequently as necessary to obtain a sufiicient speci- men. In the event of failure to obtain sufficient material by this means, the difficulty will usually be overcome by the introduc- tion of a measured amount of water. To accomplish this the stopper with all its connections is removed from the bottle and fitted into the bottle containing the 500 cubic centimeters of warm water. The valve is set to make positive pressure within 2 These special bottles are not necessary for practical purposes ; any largfe, open mouth bottle of sufficient quantity, such as a quart milk bottle, may be substituted. A mark must be made at the measured 500 cubic centimeter mark on one bottle. INFLATION OF THE STOMACH. 123 the bottle, and by means of the bulb about 400 cubic centimeters are run into the stomach; then by reversing the valve the whole is withdrawn. It is necessary to recover more than a total of 500 cubic centimeters if any determinations of a quantitative nature are to be made. If it has been necessary to resort to the introduction of water to effect the removal of the test-meal, allowance must then be made in the final calculations, so that the results will repre- sent the quantities in pure gastric contents. For Example. — Suppose after employing 500 cubic centi- meters a total of 550 cubic centimeters is recovered, of this amount only 50 cubic centimeters represent actual gastric con- tents, or one part in every eleven. It will be necessary then to multiply any figures obtained in the calculations of acidity by the factor eleven in order to express the results in terms of undiluted gastric contents. Inflation of the Stomach. — Next to the x-ray, probably the best method of determining the size, shape, and location of the stomach is by the introduction of air. To accomplish this two methods are available. Of these, the second is greatly to be preferred for reasons to be stated later : — 1. The first consists in administering one dram of sodium bicarbonate dissolved in a little water, to be immediately fol- lowed by an equal quantity of tartaric acid, also in solution. The combination of these causes the evolution of carbon dioxide gas within the stomach, which immediately distends that organ. This method is open to serious objection, because the quantity of gas produced cannot be controlled and over-production, be- sides causing great discomfort if not doing actual damage, may result in hemorrhage and great cardiac embarrassment. On the other hand, sufficient gas may not be evolved to completely distend the stomach, and thus its full size and shape fail to be accurately determined. In the light of these facts it would seem best that this method be permanently abandoned for the following, which is more in accord with the principles of scien- tific medicine. 3. The second method of inflation is accomplished through the stomach tube by means of a Davidson bulb. This simple combination may be employed with safety and accuracy, and 124 THE STOMACH. even in the absence of the graduated bottles and reversing valve can be made to serve. A greater refinement in the technic is attainable with the aid of bottles and valve above described. Their use may con- veniently follow the removal of the test-meal. With this outfit in addition to inflation we may also roughly measure the cubic contents of the distended stomach, by introducing a measured quantity of air. Technic of Inflation. — By inflation the position of the stom- ach may be accurately outlined. After the test-meal has been removed the patient is placed in the semi-recumbent posture, and the empty stomach is outlined as accurately as possible by auscultatory percussion. Air is then introduced into the stomach through the tube in sufficient quantity to produce a distinct change in the auscultatory percussion-note. The quantity of air required to accomplish this is quite small, not sufficient to alter the relation of the organ to surrounding viscera, as is the case when the stomach is ballooned with air. By the proper working of the valve and the bulb on the apparatus, we can change the gastric note at will, thereby being able to differentiate abso- lutely between gastric and colonic tympany. By this method gastroptosis can be absolutely determined, the author having in many instances had the results confirmed by the x-ray. Contraindications to Inflation. — ^These are the same as for the use of the stomach-tube itself, viz. : Myocardial degen- eration, with or without endocarditis ; angina pectoris, aneurism, advanced vascular degeneration, hemorrhage from any part of the body, and all diseases in which hemorrhage is likely to occur. Gastric ulcer is, of course, a contraindication, especially when hemorrhage has been noted. Another contraindication, and one which is not sufficiently emphasized, is neurasthenia and allied mental states with gastric symptoms. In these diseases the symptoms referable to the stomach are not due to organic change in that organ, but are psychic in origin. The use of the tube in these cases serves but to reinforce the idea of disease of the stomach, and renders a cure more difficult and occasionally im- possible. This, of course, refers to those cases which are ob- viously psychic in origin. In certain cases it is not possible to INFLA.TION OP THE STOMACH. 125 determine whether the disease is organic or not; here the tube would have to be used for diagnostic purposes. It has been sug- gested that the unpleasantness of the passage of the tube itself will act as a curative agent. Experience teaches that usually more harm than good will come from the use of the tube with this intent. To Determine the Capacity of the Stomach by Inflation. — To accomplish this the apparatus employed in the preceding de- _zi>» ,c,c 6 so — £■ 10 "if >o ~3 ID c.c. Fig. 16.— Diagrammatic Representation of Arrangement of Bottle FOR Measuring Cubic Contents of Stomach. scription must be augmented by the addition of a second double- perforated rubber stopper and a second section of glass-tubing, arranged in a similar manner to the first. The apparatus is to be set up as follows (see Fig. 16) : The extremity of the gastric tube is attached to the short glass-tube of the second stopper, and a section of tubing made to join the long glass tube of each stopper. The bottle belonging to the valve and bulb is filled with water to the 1000 cubic centimeter mark, and both stoppers placed in their respective bottles. Now, with the tube in the stomach and the valve set for compression, 126 THE STOMACH. the water is gradually forced over into the second bottle. This in turn displaces the air, and forces it into the stomach. When the patient indicates that the stomach is full, the amount of water displaced from the first bottle will equal the amount of air forced into the stomach, and can be read in cubic centimeters from the scale on the bottle. (See diagram.) The valve should now be reversed and the air withdrawn. Technic of Gastric Lavage. — Prepare a large pitcher of sterile water at body temperature, and a receptacle for waste. (If desired normal saline or dilute alkaline water may be sub- stituted.) Place 500 cubic centimeters of the wash-solution in one bottle, and by means of the valve and bulb force a few hun- dred cubic centimeters into the stomach; then, after allowing it to remain for a few moments, reverse the valve and withdraw as much as possible. Discard the returned water and repeat this process until the wash-water returns clear. A quantity of this saline or alkaline solution may be allowed to remain when the tube is finally withdrawn. Indication for the Use of the Gastric Tube. — The tube is positively indicated in cases of carcinoma associated with py- loric stenosis. The result here is decided relief from distressing symptoms, and often actual prolongation of life. In certain cases of pernicious anemia, especially in the later stages, stagnation of food gives rise to distressing symptoms, and by allowing absorption of toxins hastens the progress of the disease. Also, in cases where poison has been taken into the stom- ach in toxic doses, the stomach tube may be of great service in affording prompt removal. THE ACIDS OF DIGESTION. Under conditions of health, after from ten to fifteen min- utes following the ingestion of food the gastric contents are acid, due to the presence of free acids or acid salts. At this time the free acid recognized is lactic acid. Up to thirty-five or forty minutes lactic acid predominates, and only traces of HCl can be detected. Shortly after this the lactic acid disap- ACIDS OF DIGESTION. 127 pears and only HCl remains, so that at the end of one hour no lactic acid can be demonstrated. Hydrochloric acid is. actually present from the beginning, but its presence is masked by the excess of lactic acid and the HCl combined with bases. Free HCl increases with the progress of digestion until it reaches 0.15 to 0.80 per cent, after a light meal, or from 0.30 to 0.33 per cent, after an abundant meal. CHEMICAL COMPOSITION OF THE GASTRIC JUICE (containing water with saliva). Water 994.40 parts. Solids 5.60 " Organic material 3.10 Mineral salts 2.30 Sodium chlorid 1.46 Calcium chlorid 0.16 Potassium chlorid 0.55 Ammonium chlorid Trace. Calcium phosphate Trace. Magnesium phosphate Trace. Iron 0.12 Free HCl 0.20 FREE ACIDS. For the simple qualitative demonstration of free acids, organic and inorganic, the congo-red and tropeolin papers are convenient. The former turns dark-blue, the latter dark-brown, when moistened with a solution containing free acids, but neither of these react to acids which are combined with bases. Detection of Free HydrocMoric Acid. — For the qualitative determination of the presence of free HCl, a number of tests are available. Tests. — 1. Toffee's Dimethtl-amido-azobenzol : For this test either the 0.5-per-cent. alcoholic solution of this chem- ical can be used, or for convenience filter-paper may be soaked in the 0.5-per-cent. solution, allowed to dry, and then kept bot- tled for use. This solution is' delicate for 0.003 per cent. HCl. Combined HCl as well as acid salts and inorganic acids, in the concentration in which they occur in the stomach, will not 128 THE STOMACH. cause this solution or the prepared paper to become pink. A pink reaction denotes the presence of free hydrochloric acid. 2. GtUnzberg's Phloroqlucin Vanillin (for reagent see Appendix). — This reagent, if active, is of a pale-yellow color. It darkens and deteriorates on exposure to light, so should be kept in a dark-colored bottle or prepared fresh each time that it is required. A drop or two of this reagent is placed in a porcelain dish, together with an equal amount of filtered gastric contents, and heat gently applied until the liquid has evapo- rated. If HCl be present a rose-red color will appear at the margin of the evaporating fluid. This test is unmistakable and is most delicate, demonstrating the presence of free HCl in the proportion of 1 to 10,000 or 0.005 per cent. The reaction is not interfered with by albuminates, by salts present in the usual amount, or by organic acids. QUANTITATIVE ESTIMATION OF TOTAL ACIDITY. The reaction of the filtered gastric Juice being determined by the congo-red or the tropeolin papers, the total acidity is next determined by titrating against a decinormal sodium hydrate solution. Technic. — A Mohr's burette is filled to the "0" mark with standardized decinormal sodium hydrate solution. To 10 cubic centimeters of filtered gastric Juice in a por- celain dish, 10 cubic centimeters of distilled water is added, and one or two drops of a 1-per-cent. alcoholic solution of phenol- phthalein added as an indicator. (This indicator is inactive in the presence of carbon dioxid.) In the presence of free acids this mixture is colorless. The sodium hydrate is now run in slowly with constant stirring, until the rose-color, which appears on the addition of each drop of alkaline solution, no longer dis- appears nor is intensified by further addition of the sodium hydroxid solution. As a rule the acidity of the gastric contents, one hour after the ingestion of the test-breakfast, requires from 4 to 6 cubic centimeters of decinormal NaOH to neutralize 10 cubic centi- meters of gastric contents. For example, suppose 5.2 cubic centimeters were required to neutralize 10 cubic centimeters of FREE HYDROCHLORIC ACID. 129 gastric contents (this is within normal limits). The result of the estimation is usually expressed in parts of decinonnal NaOH per 100 parts of gastric filtrate. This expression is easily ob- tained by moving the decimal point one place to the right. The result in the above example would therefore be recorded as 52 parts of decinonnal NaOH per 100 parts of gastric contents. QUANTITATIVE ESTIMATION OF FREE HYDRO- CHLORIC ACID. To 10 cubic centimeters of filtered gastric juice add an equal quantity of distilled water, and one or two drops of Topfer's reagent. This mixture placed in a porcelain dish is titrated with a decinormal solution of NaOH until the pink color of the solu- tion has been entirely removed and is replaced by pale-yellow. Suppose, for example, 6 cubic centimeters were the amount required to neutralize the free HCl contained in 10 cubic centi- meters of gastric contents. To obtain the percentage of HCl in the gastric contents the following calculations are required: — One cubic centimeter of decinormal NaOH solution is equivalent to 0.00365 gram of HCl (decinormal NaOH is equiva- lent to 4 grams of NaOH dissolved in exactly 1000 cubic centimeters of distilled water ; each 1 cubic centimeter of this solution should exactly neutralize 0.00365 gram HCl). There- fore 6 X 10 would equal the number of cubic centimeters of NaOH solution required for every 100 cubic centimeters of gastric contents, and the result, 60 x 0.00365, would equal the percentage of HCl in the specimen under examination, which would be 0.219 per cent. HCl. Causes af Lowered Gastric Secretion. — I. In acute and chronic gastric inflammation. II. Atrophy of the gastric mucosa from any cause. III. General functional depression. IV. Expression of a gastric neurosis. V. Congenital idiosyncrasy. DETERMINATION OF ORGANIC ACIDS. These include lactic and acetic, and the true fatty- acids, particularly butyric. Acetic and fatty acids are not found dur- 130 THE STOMACH. ing normal digestion, and if present, as they sometimes are, have either been introduced with the food or have been produced by fermentation of carbohydrates set up in the stomach by bacteria introduced with the saliva. The physiologic presence of lactic acid during the first stage of gastric digestion, may be due either to its formation within the stomach, or from its having been introduced with the food, as in balced bread. Ufi'leman's Test for Lactic Acid. — The addition of a few drops of filtered gastric contents to Uffleman's reagent (see Appendix) in a test-tube will, in the presence of lactic acid, change the amethyst-blue to a canary-yellow. This test is posi- tive in the presence of 1 part of lactic acid in 30,000. Sources of Error. — Lactates cause the same reaction. This is, however, immaterial, since we desire to recognize lactic acid whether free or combined. The reaction also takes place with alcohol, sugar, and certain salts, particularly the phosphates, but rarely in their usual concentration after- the test-breakfast. The fatty acids, particularly butyric, strike a tawny-yellow color with a reddish tinge with Uffleman's reagent. The reac- tion is positive with 1 part in 3000. Fatty acids may also be detected by heating to boiling a few cubic centimeters of gastric filtrate in a test-tube, over the mouth of which a strip of moist neutral litmus paper is placed. On this the vaporized volatile acid will produce the usual change. Acetic acid is easily recognized by its odor, but it may also be detected by neutralizing a watery residue after the re- moval of an ethereal extract, with sodium carbonate, and then adding neutral ferric chlorid solution. In the presence of acetic acid a striking blood-red color will be produced. A simi- lar reaction occurs in the presence of formic acid, but this acid is never present in gastric juice. Alcohol. — This is sometimes formed in the stomach during intense yeast fermentation. This may be detected by the Lis- hen's iodoform test, applied as follows: To a portion of the gastric filtrate add a small portion of liquor potassii, and then a few drops of a solution of iodine and potassium iodide in water. (See Appendix.) If alcohol be present a yellowish scum gradu- ally occurs on the surface, which is readily recognized as iodo- TESTS FOR OCCULT BLOOD. 131 form by its odor. The same reaction occurs in the presence of acetone, but occurs more rapidly. Propeptone and Peptone. — These are products of albumin digestion, and when present indicate the activity of this part of the gastric function. A few drops of filtrate added to hot Pehling's solution produces a purplish color, if they are present. Starch. — The addition of a drop of Lugol's solution to a piece of filter paper upon which some unfiltered gastric contents has been allowed to fall will, in the presence of starch, yield a blue reaction. This reaction is intensified by the addition of a drop of crude nitric acid. MICROSCOPIC EXAMINATION. Examination of the unfiltered gastric contents under low power will reveal the eccentrically marked oval starch grains, the budding yeast, cells, food debris, and epithelial cells, etc. The presence of leukocytes in the washed stomach-contents is strong evidence in favor of gastric carcinoma. TESTS FOR OCCULT BLOOD. Blood may be intermittently present in the gastric contents or gastric vomitus as a result of one of the following condi- tions : — I. Ulcer of the stomach or intestines. II. Benign pyloric stenosis. III. Spasm of the pylorus. Occult blood is usually constantly present in cases of malignant disease of the stomach or esophagus. Boas Modification of the Webek Test. — To 15 cubic centimeters of gastric contents, add an equal amount of ether and thoroughly agitate. To this mixture add 3 to 5 cubic centi- meters of strong glacial acetic acid, and again agitate. Now allow the mixture to settle and decant 10 or 15 cubic centimeters of the clear supernatant liquid, and to this add 4 or 5 cubic 132 THE STOMACH. centimeters of ozonized oil of turpentine. In the presence of blood a violet or blue color will appear, which is intensified by the addition of chloroform. (See also test on page 154.) Acetic Acid Ethek-Guaiac Test. — To 10 cubic centi- meters of gastric contents add 10 cubic centimeters of ether and 5 cubic centimeters of strong acetic acid. Thoroughly shake, and add two or three grains of powdered gum guaiac, and Again agitate ; allow to settle, and then add a few drops of fresh solu- tion of hydrogen dioxid. In the presence of blood a purple or blue ring will appear at the line of contact, or the solution grad- ually assume a grayish-blue color. ESTIMATION OF PEPTIC ACTIVITY. The digestive power of the filtered gastric contents from the recovered test-meal depends upon, first, the amount of pepsin contained, and, second, upon the amount of free acid present, particularly the free hydrochloric acid. Artificial digestion is the only means at our command by which we may determine the peptic activity of the gastric juice. Method op Bvfald. — Prepare from the albumin of eggs, which have been boiled just sufficiently to cause firm coagula- tion, small discs or squares by first cutting thin slices of the coagulated albumin, and from these slices the cubes or discs. These bits of albumin may be prepared in bulk and preserved in glycerin, which is carefully washed off before using. The Test. — Place an equal quantity (5 to 8 cubic centi- meters) of filtered gastric juice in each of four test-tubes, add to each tube one or two pieces of the prepared albumin ; then. To tube 1 add nothing. To tube 2 add one drop of HCl. To tube 3 add four grains of pepsin. To tube 4 add the above quantities of HCl and pepsin. These tubes are now placed in an incubator at 37° C, and from time to time examined to note the progress of liquefaction of the albumin. By comparison we can roughly determine whether digestion is progressing normally in the unaltered tube, and whether pepsin, hydrochloric acid, or both, are necessary to accomplish it. ESTIMATION OF PEPTIC ACTIVITY. 133 The results obtained from this investigation can only be considered in the light of an approximate indication of the peptic activity of the material tested. According to Nierenstein and SehifE^ it is necessary to dif- ferentiate three factors in order to arrive at an adequate idea of the significance of these pepsin determinations. These are: the diluting secretion, the pepsin secretion, and the hydrochloric acid secretion, all three of which must be regarded as distinct expressions of the secretory activity of the glandular parenchyma. Following a better knowledge of the activity of these dif- ferent factors, two improved modifications of the method of pepsin determination have been advanced. These are the meth- ods of Hammerschlag and of Mett.* Method op Hammehschlag. — Briefly outlined the method depends upon the activity of a few centimeters of gastric juice upon a 1-per-cent. filtered solution of egg albumin. Two test- tubes, one filled with the albumin solution alone and the other with the albumin solution, plus the gastric filtrate, are incu- bated for an hour at 37° C. At the expiration of this time the albumin content of each tube is estimated volumetrically, ac- cording to the method of Esbach (see page 198). The differ- ence between the albumin precipitate in the two tubes is equal to the amount of albumin digested, and therefore is a measure of the peptic activity of the gastric juice. The objections to this method are, that the Esbach method is not very accurate, and also that albumoses are partly precipitated by the reagent. How- ever, this method is sufficiently accurate for the ordinary clinical purposes, and when the Mett method cannot be followed, may be adopted. It would be better, however, to employ a method which employs diluted gastric juice, as in the Mett method. Mett's Method of Peptic Dbtehminatiojt. — Glass ca- pillary tubes from 1 to 3 millimeter^ diameter and of convenient length, are filled by suction with the fluid portion of egg albu- min. In order to avoid accidental variation in the egg albumin, it is better to use the albumin of several eggs mixed. These tubes as they are filled should have their ends plugged with 'Arch. t. Verdauungskrankh, Vol. iii. 1902. *Sahli's Diagnosis. ' J. A. D., Petersburff, 1889, from Palow's Laboratory. 134 THE STOMACH. bread crumbs to prevent loss of contents before coagulation. After filling they are placed horizontally in a simmering water bath, where they are allowed to remain for from four to five minutes. While boiling, the tubes should be kept in motion to insure uniform coagulation of the albumin. When freshly made, the albumin tubes contain innumerable bubbles which, however, gradually disappear, after four or five days the tubes are ready for use. After boiling, the tubes are wiped dry and the ends closed with sealing wax or stick lac to prevent drying. Thus prepared a stock of tubes may be kept indefinitely, but it should be ascertained before using that the albumin is still evenly in contact with the walls of the tube ; if they have dried out they are not fit for use. These tubes are cut as needed into lengths of from 3 to 3 centimeters. This cutting is best accomplished by nicking the glass with a small triangular file, when a quick bend at this point will usually cause accurate fracture of both tube and albu- min. Portions containing air bubbles or showing irregular fracture, should be discarded. Technic.^ — For quantitative determinations of the peptic activity of the filtered gastric contents, employ a dilution of 1 to 16, using as a diluent ^ HCl, using for each test 1 cubic centimeter of contents and 15 cubic centimeters 30th normal HCl. This mixture is placed in a covered Stender'^ dish after two Mett's tubes have been placed in it, and the specimen allowed to digest in an incubator (37° C.) for twenty-four hours. At the end of this period the digested cylinders of egg albumin at each of the four ends are measured ofi' and the average reading calculated. The relative amount of pepsin is then obtained by squaring the result (Shurtz's law), and if desired multiplying by the dilution, e.g., 16. For making the measurements a pair of callipers with a vernier reading 0.1 of a millimeter, is con- venient. A lens is useful principally for reading the vernier. One end of the tube is placed against one jaw of the calliper, and the other is separated until its end is Just visible through = After Sailer and Farr, U. of Pa. Med. Bui., Oct. 1906. ' A Stender dish is similar to a Petri dish, but is deeper, with a flat ground glass lid. ESTIMATION OF PEPTIC ACTIVITY. 135 the opalescent edge of albumin. If the tube is at all oblique the shortest side is taken, while if the albumin is at all uneven, the highest point to which digestion has extended is taken. In place of the callipers the ordinary mechanical stage, which is usually fitted with a vernier scale, may be used. Nierenstein and SchifE have proven that a dilution of at least 1 to 16 is absolutely necessary if we wish to obtain a rela- tive idea of the quantity of pepsin as estimated from the diges- tion length. This is because with a dilution of less than 16, the length of digestion decreases as the length of time increases, owing to the concentration and activity of the inhibiting sub- stances. This great dilution also decreases the amount of pepsin in the mixture, so that the digestion length is kept within the limits of Shurtz's law (3.6 millimeters in twenty-four hours). As there are instances when this dilution is not sufficient (with very active pepsin solutions), it may become necessary when the digestion length exceeds 3.6 millimeters with the 16-fold dilution, to repeat the pepsin test with a dilution of 1 : 33 ; then by squaring the length as before, and multiplying this quantity by 32, the relative amount of pepsin in the undiluted gastric contents is obtained. It is clear that in this estimation the unit of the relative amount of pepsin will be that quantity of pepsin by which 1 millimeter of albumin in the Mett tube will be digested in twenty-four hours with an acidity of 0.18 HCl. In this determination we do not consider the absolute quantity of pepsin, but simply the degree of concentration, for the result of Mett's method is the same whether large or small quantities of digestive substances are employed. ^ Summary. — Conclusions of Sailer and Farr regarding varia- tions due to alterations in technic^: — 1. Difference in the caliber of the tubes. The variations in the reading of the two ends of one tube were as great as between tubes of different caliber for tubes between 1 and 2 millimeters. In tubes greater than 2 millimeters the rate of digestion seemed to be uniformly greater in tubes of larger diameter. 2. The age of the tubes. Provided there is no separation ' Sahli's Diagnosis, fourth edition, 1905. " Sailer and Farr, he, cii. 136 THE STOMACH. of the albumin nor putrefactive softening, age does not seem to affect the tubes. The method of preparation tends to make them sterile. Tubes that are too fresh (less than four or five days old) are said to digest more rapidly than tubes that are "ripe.'' 3. Variability in the digestion of albumin from different eggs is an unimportant factor. 4. The degree of digestion within certain limits is said to vary directly with the duration of digestion. 5. Variations in the temperature of the incubator. This is undoubtedly an important factor, but there are no definite data to offer. 6. The effect of variations in the acidity of the original specimen. This has been provided against in the technic of the method, since the use of so large an amount of diluting acid solution tends to render the acidity of the diluted specimen uniform in every instance. ESTIMATION OF TRYPSIN. See Appendix, page 323, for Method of Mett, etc. ESTIMATION OF THE ACTIVITY OF RENNIN OR MILK-CURDLING FERMENT. Xoi-mal gastric juice contains, besides hydrochloric acid and pepsin, the rennin ferment as a natural secretory product of the gastric mucosa. Eennin possesses the property of coagu- lating milk without the presence or assistance of acids. Method of Leo. — To 10 cubic centimeters of fresh, uncooked neutral or amphoteric milk, add from two to five drops of filtered gastric juice, and place the mixture in an incubator at 37° C. If rennin is present in normal amount, curdling should take place in from ten to fifteen minutes. In this process the slight amount of acid contained in the gastric filtrate is insufficient to cause coagulation. If curdling takes place very slowly it is questionable whether this change is due to the action of the rennin or to the formation of lactic acid, so to be exact, the reaction of the mixture should be taken before and after curdling has occurred. The rennin reaction is certain to have occurred only in the presence of an unchanged reaction. If coagulation does not occur within an hour rennin can be considered absent. As a further guide it may be remem- MOTOR FUNCTrON OF THE STOMACH. 137 bered that the characteristic curd from rennin is a cake of casein floating on clear serum, while the curd from lactic acid is lumpy and broken. DIGESTION OF STARCH AND SUGAR. During digestion starch is converted into grape-sugar, while cane-sugar is converted into invert-sugar (a mixture of cane- and grape-sugar). Starch is recognized by the deep blue color produced by the addition of a dilute solution of iodine or Lugol's solution. This reaction grows less vivid as the starch is converted. If starch digestion is unduly delayed or does not occur, we may infer hyperacidity of the gastric juice. DETERMINATION OF PANCREATIC ACTIVITY. See Appendix, page 323, for Method of Volhard. DETERMINATION OF THE RATE OF ABSORPTION FROM THE STOMACH. Penzoldt's Method. — ^A capsule containing potassium iodide (one and one-half grain) is swallowed. The appearance of the iodine reaction in the saliva indicates that absorption has occurred from the stomach. To test for the iodide in the saliva, paper moistened with starch paste and dried is used. After the capsule has been swallowed, the paper is moistened with saliva at short regular intervals, and then touched with a glass rod dipped in commercial (better, fuming) nitric acid. Upon the appear- ance of iodine in the saliva the characteristic blue reaction occurs. When absorption is normal this reaction is usually positive in from ten to fifteen minutes; but if absorption is delayed, the reaction may be slow in appearing or occur not at all. DETECTION OF BILE. See Appendix, page 324. TEST OF THE MOTOR FUNCTION OF THE STOMACH. If attempted extraction of a full meal six hours after in- gestion fails when properly performed, or if nothing can be 138 THE STOMACH. recovered from an Ewald test-breakfast after two and one-half hours, the motor function of the stomach may be considered normal. A SECOND METHOD is to administer a large dose (ten or fifteen grains) of salol and test the urine at definite periods for the appearance of the products of its decomposition. The com- ponents of salol, carbolic and salicylic acid, are separated in the alkaline juice of the small intestine. They remain unchanged and undissolved during gastric digestion. Salicylic acid is readily detected in the urine by the violet color produced by the addition of neutral ferric chlorid solution. This test is con- veniently performed by moistening filter-paper and bringing a drop of the ferric chlorid solution in contact with it. If gas- tric peristalsis is normal salicylic acid should begin to appear in the urine from forty to sixty or seventy-five minutes after ingestion of fifteen grains of salol. loDOFOBM Method. — Give with the test-breakfast one gram of iodoform in a well sealed capsule. The iodoform being in- soluble in the gastric juice, will not be absorbed by the stomach, but will be passed on by peristaltic action to the intestine. By the action of the fluids of the duodenum the iodoform is decom- posed with the formation of soluble sodium iodide. The demon- stration of the iodide in the saliva by means of starch paper and nitrous acid, will indicate the time when the gastric contents is being discharged into the intestine. Iodine should normally be detected in the saliva in from one hour to one hour and a half after the ingestion of the capsule.- ROENTGEN RAY EXAMINATION. With the advent of the Eoentgen ray and its practical appli- cation, it has become an invaluable aid to diagnosis. A number of competent men have applied this agent in the study of the digestive tract, and have reached so many valuable conclusions pertaining to the size, location, motility, etc., of the stomach, that the examination of a patient suffering with any disorder of the digestive tract must be considered incomplete unless a Eoentgenologic examination has been made. The following quotation from an article by an expert in ROENTGEN RAY EXAMINATION. 139 this method of examination and treatment shows the present attitude of the profession upon this point^O; — "The superiority of this method of examination over others is recognized, I think, by all who have investigated suifieiently, and the subject has reached a stage of development when no ease Pig. 17.— Solid Line shows form and relation of typical normal Stomach, with subject in standing posture. dotted line .shows altera- TION IN POSITION OF Stomach, the result of contrac- tion OF the Abdominal Muscles. A, Location of Ensiform. B, Umbilicus. C, Cardia. D, Pylorus. involving chronic ailment of the alimentary canal is completely investigated until there has been a Eoentgenologic examination by a competent man." From a careful examination of a number of normal individuals by the same observer, the following outline » G. E. Pfahler, Jour. Amer. Med. Asso., Deo. 21, 1907. 140 THE STOMACH. may be considered to be that of a normal stomach in a healthy individual : — When empty or moderately filled the stomach (Figs. 17, 18 and Plate VI) occupies the left side of the abdomen and extends from the inner two-thirds- of the left dome of the diaphragm to the median line just above the umbilicus. The upper two- D Fig. 18.— Dotted Line shows form and location of normal Stomach. Shaded portion shows contraction and change in form OF Stomach during filling. A, Ensiform. B, Umbilicus. C, Cardia. D, Pylorus. E, Upper Pole. Pole. G, Showine place where Peristaltic Waves begin. F, Lower thirds is almost vertical, and the lower third is almost hori- zontal, making the general direction of the stomach slightly oblique. In the normal stomach the pylorus is on a level with the lower pole. No difference has been recognized between the normal type in children and in adults. PLATE VI. Normal Stomach. Child, Age 4 Years. Instantaneous Postero-anterior Exposure in Standing Posture. Shows Normal Stomach in Normal Position and its Relation to the Heart, Diaphragm, Spinal Column and Pelvis. A. Ensiform Cartilage. B. Umbilicus. Arrow Indicates Location of Pylorus. [Radiograph hy Dr. G. E. Pfahler.) ROENTGEN RAY EXAMINATION. 141 The stomach of the average individual, in the standing position, extends to or below the umbilicus. It is vertical for more than its upper two-thirds. It occupies the left side of the abdomen, except when distended. The pyloric portion extends for one to two inches beyond the median line to the right. The stomach is normally a very movable organ, as shown by the fact that about two-thirds of it crosses the median line when the patient lies upon the right side. When distended with food the pylorus is carried downward and to the right. In summing up the results of his studies of the alimentary canal by the Eoentgenologic method. Dr. Pfahler arrives at the following conclusions pertaining to its utility in diseases of the stomach : — "1. The Eoentgen examination will demonstrate obstructive disease anywhere along the alimentary canal, and much informa- tion concerning its character may be obtained. "2. The Eoentgen method is probably the most useful in the study of the stomach. By this means the size, form, position, motility, effects of massage, respiratory movements, abdominal contractions, peristaltic action, and the effect of food can be studied. "3. The Eoentgen rays are valuable in collecting additional data in the diagnosis of carcinoma of the stomach, but must not be depended upon to make an absolute diagnosis. "4. The Eoentgenoscopic and the Eoentgenographic method of examination each has its advantage. Eoentgenoscopically we study the motion of the viscera, while Eoentgenographically we make accurate records and often obtain finer detail." Tbchnic of the Mkcjiod.— Preparation of the Patient: The stomach and bowels should be empty, if possible. A purga- tive should be given the day before, and if the bowels are not moved an enema should precede the examination. Depending on the information desired, an ounce of bismuth may be given with the full meal, and this mixed with the food by massage or muscular contraction, or the bismuth-kefir mixture (bis- muth subcarbonate, one ounce; kefir, one pint) may be given. When carcinoma is suspected, no food should be given be- fore the examination because the lodgment of food in the stom- ach may give a picture suggestive of neoplasm. Men need no 142 THE STOMACH. special instructions regarding dress. Women should wear a kimona or special muslin gown. This special muslin gown hangs from the shoulders and should be slit in front at the level of the pubes and posteriorly in the lumbar region for the dis- tance of ten inches. These slits are closed by strings. Through them the necessary land marks are determined, and the cents are attached to the ensiform cartilage and the umbilicus by adhesive plaster. Apparatus. — Besides the usual apparatus for Eoentgen- ographic examination, appliances are necessary which will enable the operator to make the observations and negatives in any posi- tion. Eoentgenoscopic apparatus is also necessar}', and a dark room which is absolutely dark. Dr. Pfahler has recently adapted a dark room for this purpose. All the apparatus is in an adjoin- ing room. The rays are passed through a door into the dark room before reaching the patient. Therefore all light is excluded and the patient is not frightened by the noise of the machine, nor is there danger of the patient touching the wires. In addi- tion this door cuts off the soft rays and make it practically im- possible for the skin of the patient to be burnt during the observations. It is necessary in order to study the shadows on the screen that the operator should remain in the dark room for fifteen minutes before beginning the examination. In order to study both the stomach and the colon at the same time, an ounce of bismuth mixture may be given and allowed to pass to the colon. After twenty-four hours another ounce of bismuth is given, when the outlines of the stomach and colon will appear in their relative positions. X. THE FECES. PHYSICAL CHARACTERISTICS. The Number. — One stool per day is the normal average for a healthy adult. Three daily, or one in forty-eight hours, may be normal for some individuals, and not incompatible with health. The Reaction. — Whether the stools be acid or alkaline is of no special clinical importance. In adults the reaction is usu- ally alkaline, sometimes neutral, but rarely acid. Acid stools are the rule in infants. The Amount. — The amount varies in proportion to the amount of solids ingested. A preponderance of vegetable food usually produces a large quantity, while animal food leaves com- paratively little residue. The average daily amount of feces varies between 60 and 250 grams, of which 75 per cent, is water. To weigh solid feces ascertain the weight of both the feces and their container, then weigh the container empty, and sub- tract the latter from the former weight, which will represent the weight of the contained feces. If the feces are liquid they may be measured, and the amount expressed in cubic centi- meters. The Odor.- — The disagreeable odor is largely due to the presence of indol and skatol, but may be further increased by hydrogen sulphide, methane, and methyl-mercaptan. The Consistence; — This varies greatly and depends largely upon the amount of fluids ingested, the temperature, the climate, and the condition of the digestive tract. In man the usual form is the characteristic plastic cylinder. Clinically expressed, the consistence of the feces may be liquid, mushy, or solid or formed. Serous Stools. — ^These are composed of fluid without fecal matter, and are of considerable diagnostic importance. Such (143) 144 THE FECES. stools are characteristic of Asiatic cholera, cholera morbus, chol- era infantum, and poisoning by antimony. In cancer of the rectum evacuations are small, frequent, and serous. Arsenic poisoning and acute catarrhal enteritis may produce this form of stool. Poisoning by toad-stools is also a frequent cause of serous stools. The Color. — The color varies with the character of the food ingested, and is usually little influenced by the decomposition products of the biliary pigments. In adults, the color usually varies from a light to a dark-brown. Lack of color in the stools may occur in health, owing to the over-oxidation of the coloring matter into colorless products of bilirubin. Such stools do not necessarily indicate the presence of large amounts of fat nor obstruction of the biliary passages. In infants, fat and undigested milk produce a whitish-color tinged with bile-pigments. Green stools are seen after taking calomel. After expo- sure to the air such stools are usually acid. Yellow stools may be caused by the ingestion of santonin, rhubarb, or senna. Typhoid stools are yellowish-brown and have received the name of "pea-soup stools." White or clay-coloeed stools frequently denote the ab- sence of bile, as in obstructive jaundice. Very dark or black stools may result from the administra- tion of iron, manganese or bismuth, or from the ingestion of much meat, blackberries or red-wine. "Tahey" stools usually denote hemorrhage. EXAMINATION OF INTESTINAL DIGESTION BY MEANS OF GLUTOID CAPSULES. Sahli recommends the use of glutoid capsules which are made from gelatin hardened in formaldehyde. These either do not dissolve in the gastric juice at all, or only after a consid- erable time, but are rather quickly soluble in the normal intes- tinal juices. 1 These are preferable to the keratin coated pills which were originally recommended, and are useful to diagnose the condition of intestinal digestion, e.g., the pancreatic func- 1 Deutoh. Med. Woch., No. 1, 1897. DETECTION OF IODINE. 145 tion. They are filled with some substance which does not diffuse through the capsule wall, and whose absorption may be studied by an examination of the saliva or the urine. For diagnostic purposes glutoid capsules, containing two grains (0.13 gram) of iodoform and four grains (0.26 gram) salol, are con- venient. In order to make the conditions of the test as uniform as possible in regard to the length of time the capsules remain in the stomach and in regard to the degree of digestive stimulation, it is advisable to administer the capsule with the test-breakfast. Experience has shown that normally, under the best conditions, i.e., normal gastric motility, normal intestinal digestion, and normal intestinal absorption, iodine reaction may be expected to appear in the saliva, and salicyluric reaction in the urine in from four to six hours. If one wishes to test the accuracy of any particular lot of capsules, it will be necessary to prove that this reaction time is obtained in healthy normal individuals. Only rough approxi- mate differences in the time of the reaction are of clinical im- portance, so that it is sufficient to examine the saliva and the urine after six, ten, and twenty-four hours. The best results from this.:method are obtained by administering the capsule in the morning upon an empty stomach, and then, four hours later, allowing the patient to resume his meals as usual. The speci- mens of urine and saliva may, of course, be saved, and exam- ined afterward. DETECTION OF IODINE IN THE URINE OR SALIVA. A few cubic centimeters of the fluid to be examined are boiled with a small piece of starch about the size of a pea, until the latter is v!issolved. After cooling, the mixture is carefully overlaid upon nitric acid. If iodine is present a blue-violet ring, that gradually disappears, is formed at the line of junction of the two fluids. A SECOND METHOD is to add to the urine a few drops of crude nitric acid and a less number of drops of chloroform, and agitate the mixture gently. If iodine be present the chloroform, which settles to the bottom, will be tinged rose-red or violet. 146 THE FECES. Both of the above tests are very delicate. But if the urine contains a very slight trace of iodine the chloroform-test is not very conclusive, since indol, skatol, and urorosein pigments may tinge the chloroform red. DETECTION OF SALICYLURIC ACID IN THE URINE. Dilute ferric chloride is added to the urine drop by drop. If the latter becomes a more or less intense violet the reaction is positive. DETERMINATION OF THE MOTOR FUNCTIONS OF THE GASTRO-INTESTINAL TRACT. Method of Adolph Schmidt.^ — Schmidt demands two con- ditions for a satisfactory clinical method of examining the feces : 1. A knovif ledge of vi^hat a normal stool should be under a certain diet, involving the tise of a "test-diet." 2. The methods of examination, must be so jsimple that they are vi^ithin the reach of every physician. The Test-Diet. — The requirements are: (a) That it be nutritious enough to furnish calories sufficient fo^he body's needs, (h) That it be so constituted that it can be readily ob- tained in any household or hospital dietary, (c) That it con- tains a known amount of its constituents, so that variations in digestion and absorption can be detected in the stool. Schmidt suggests the diet which is given below, followed by the approximate equivalents as sugested by Steele: — Milk, 1.5 liters (3% pints). Zwieback, 100 grams (3 ounces well-dried toast). Two eggs. Butter, 50 grams (1% ounces). Beef, very rare or raw, 125 grams i}/^ pound). Potatoes, 190 grams (6 ounces). Gruel made from 60 grams oat-meal (2% ounces). Sugar, 20 grams (i/i; ounce). This may be given somewhat as follows : — ' Quoted by Steele, Medical News, Dec, 1905. DETERMINATION OF MOTOR FUNCTIONS. 147 Breakfast. — Two eggs, half of the amount of toast and butter, 3 glasses of milk, oatmeal, and sugar. Dinner. — The steak and potatoes, % of the amount of toast and butter, 1% glasses of milk. Supper. — ^Two glasses of milk, remainder of toast and butter. This diet is sufficient for the needs of the average patient, and while not offering much variety, is only taken for two or three days. A capsule containing five grains of charcoal is given with the first meal, and no examination made until the charcoal (black) appears in the stool. The amount of each article composing the day's dietary must first be accurately determined and compared to the con- tents of convenient measures, so that the articles need not be weighed every meal. This does not apply to the meat and potatoes which should be weighed each day. The Pekiod of Passage. — The period of time required for food to pass through the intestinal tract can easily be deter- mined by watching for the black in the stool. It is quite as necessary to know the time required for the passage of chyme- through the gastro-intestinal tract, as . it is to ascertain how often the stools occur, since the two are in no way identical. It has been shown in chronic colitis with several watery stools a day, that the period of passage may be normal, and that peristalsis is decidedly increased only in the colon. According to Strass, using a diet similar to Schmidt, the time required for food to pass through the alimentary canal varies normally be- tween ten and thirty hours. Under abnormal conditions this may vary between four and forty-eight hours. The Specimen. — After the appearance of the charcoal, which indicates the beginning of the passage of the test-diet, a portion of one passage is collected in a clean receptacle and transferred to a clean, wide-mouthed bottle, and conveyed to the laboratory for examination. Much can be determined from a naked-eye examination of such a mass. The technie for this examination, as outlined by Steele, is as follows: — Take a piece of formed stool about the size of an English walnut or an approximate equivalent of liquid feces, and rub it 148 THE FECES. up in a mortar with distilled water until quite smooth and liquid. Part of this is then poured into a Petri dish and examined in a good light on a dark background. A little experience will enable the observer to acquire a large amount of information about the composition of the stool by this macroscopic examination. The Gross Appearance of Normal Stools. — In normal diges- tion nothing should be seen by the naked eye, save a varying number of brown points (oatmeal husks), cellulose and indigest- ible parts of food, and occasionally sago-like grains that look like mucus, but which are found upon microscopic examination to be grains of potato. If, on the addition of the water, fat globules are seen, they must be considered pathologic unless the patient is known to have ingested excessive amounts of fats, as olive or castor oil. Gross Appearance of Pathologic Stools. — (a) Mucus in large or small flakes is not affected by rubbing in the mortar. The mucus appears as glassy, translucent flakes often stained yellow by bile-pigment. Doubtful cases may be decided by the microscope. (b) Pus in sufficient quantity to be recognized macro- scopically, will have the characteristics of pus in any other region. It usually appears as small, yellowish streaks or collec- tions throughout the stool. (c) Blood, if in appreciable amount, will appear as dark reddish-brown or black; if from high in the digestive tube, becoming more nearly the color of fresh blood as the source of origin approaches the lower end of the bowel. (d) Parasites. — Segments of tape-worm, also thread- and seat-worms, may be detected in varying numbers in patients so afflicted. (e) Foreign bodies having entered the digestive tract, if indigestible and not impeded in their progress, may be recovered from the stool at varying periods after ingestion. (f) Calculi from the various glands situated along the di- gestive tract, or from the intestii al canal itself (enteroliths), may be found in the feces, and appear as calcareous masses of various sizes and composition, depending upon their origin and MICROSCOPIC APPEARANCE OF NORMAL STOOLS. 149 (g) Remnants of connective tissue and sinew from beef- steak. These can be detected by their whitish-yellow color and toughness, by which they can be distinguished from mucus. In case of doubt the piece should be examined microscopically with a drop of acetic acid. Connective tissue then loses its fibrous structure, while mucus becomes more thread-like. Small, single pieces of connective tissue can be found in normal stools. (h) Remnants of Muscle-Fiber. — These appear as small reddish-brown threads or small irregular lumps. (i) Remnants of Potato. — These appear like boiled grains of tapioca and may easily be confused with mucus. The micro- scope will show the true nature of these bodies. (j) Large crystals of triple phosphate occur in foul stools, and can be recognized by their shape and by their solubility in all acids. Microscopic Appearance of Normal Stools. ^ — Three slides are prepared from the liquid feces. The first is merely a drop of the material to be examined by both low and high power. The second slide is prepared by mixing a drop of acetic acid and a drop of the liquid feces upon a slide, heating it to boiling and then putting on a cover-glass. The third slide is prepared by mixing a drop of liquid feces with a drop of Lugol's solution. MiCKOScopic Examination of Slides. — Slide 1 will show (a) single small muscle fibers colored yellow with cross striation. Visible with a Leitz 3, but showing better with higher power. (I) Small and large yellow crystals of salts of the fatty acids, (c) Colorless (gray) particles of soap, (d) Single potato cells without distinguishable contents, (e) Particles of oat- meal and grain husks. Slide 2. — A general idea of the fat-content of the stool may be obtained. Upon cooling, small drops of fatty acids may be found covering the whole preparation. The large crystals of salts of the fatty acids are broken up by the acetic acid, and fatty acids liberated. If the slide is heated again and exam- ined hot, the fatty acids will be found to run together in drops which, as the slide cools, break suddenly apart. ^Teohnio of E. Button Steele, Medical News, Dec. 16, WOi 150 THE FECES. Slide 3. — Here should be found violet-blue grains in some of the potato cells, and small single blue points, probably fungi or spores. Pathologic Microscopic Findings. — Slide 1. (a) Muscle fibers in excess, perhaps with yellow nuclei, (b) Neutral fat drops, or fatty acid, in soap crystals, (c) An excess of potato cells with more or less well-preserved contents, (dj Parasite eggs, mucus, connective tissue, pus, etc. Slide 2. — Patty-acid droplets in excess. Slide 3. — Blue starch grains in the potato cells or free, oat- meal cells, fungi, spores or mycelia. FoKMED Elements in the Feces. — (a) Blood. Eed cells, if recently shed, may be distinguished by their characteristic form, or if disintegrated, only masses of brownish-red amorphous hematoidin will be found. In a certain percentage of cases char- acteristic crystals of hematoidin will be found. (b) Epithelial Cells. — These are normally present in mod- erate numbers and represent the natural desquamation from the intestinal canal. They are more or less disintegrated, depending to some extent upon their height of origin in the intestinal canal, and upon the length of time they have remained free in the digestive tube. In catarrhal conditions they may be present in very large numbers, when they may assume diagnostic impor- tance. (c) Pus Cells. — These rapidly undergo decomposition, so that even when numerous and coming from a comparatively short distance above the rectum, they may be beyond recogni- tion. The characteristic pus cell appears as a small round or slightly oval granular body. The presence of very much pus in the stools is indicative of rupture of an abscess into the intes- tinal tract. CHEMICAL EXAMINATION. This comprises only five routine tests: 1. The reaction. 2. The sublimate test for the condition of the bile salts. 3. The fermentation test. 4. The test for "lost albumin." 5. The test for occult blood. 1. The Eeaction. — This is quite difficult to get with the ordinary litmus paper. It can be easily determined by dropping CHEMICAL EXAMINATION. 151 Fig. 19- -.Strasburger Appar.^tus. showing arrangement of Bottles FOR Fermentation Test of Feces. (After Steele.) 162 THE FECES. a little softened fecal matter into 5 or 10 cubic centimeters of weak watery solution of neutral litmus, shaking it and noticing the color reaction. 3. The Sublimate Test. — This consists of taking a few cubic centimeters of liquid feces and mixing with an equal amount of a saturated watery solution of HgCl2. A normal stool will quickly turn a pinkish-red, indicating the presence of hydrobilirubin, which will be more intense the fresher the material. A green color is pathologic and indicates the presence of unchanged bile-pigment. 3. Fermentation Test. — Described by Steele, using his modification of Strasburger's apparatus. The apparatus consists of a two-ounce, wide-mouth bottle A (see Fig. 19). This is fitted with a perforated cork through which runs a tube to the test-tube B, which is also fitted with a rubber cork with two perforations. A bent tube runs from the tube B to the test-tube C, to allow for the escape of air. Each tube has a capacity of a little more than 30 cubic centi- meters when fitted on the corks. The apparatus is simple and easily constructed, and broken parts can be replaced readily. The Test. — About five grams of solid feces, or an equivalent of liquid feces, are rubbed up with a little distilled water and placed in the main bottle A. This is filled with sterile water, the tube is filled with water and fitted into place (not neces- sarily full), and tube C is then fitted on empty. The reaction is carefully noted before the test is started. The apparatus is then stood in a warm place — best in an incubator at 37° C. for twenty-four hours. If gas forms by fermentation in A it will rise into B, and the amount will be indicated by the amount of water displaced into C. Normally the fermentation test should show practically no gas, and the original reaction of the material should be unaltered in twenty-four hours. If more than one- third of the tube C is filled, it is pathologic. If the reaction after twenty-four hours is decidedly more acid, it is a carbo- hydrate fermentation. If alkaline, and with a foul smell, it is a fermentation of the albumins. 4. Test for Lost Albumin. — A portion of softened stool is filtered (a slow and difficult process). The filtrate is shaken with silicon and refiltered, then is saturated with acetic acid to BLOOD IN THE STOOL. 163 bring down the nucleo-proteids, and finally a drop of potassium ferrocyanide solution is added. A decided precipitate indicates albumin. A positive test shows only that there is a decided diminution in albumin digestion. BLOOD IN THE STOOL. If bleeding has its origin in the upper part of the digestive tract (stomach or small intestine), it is so altered by the action of the digestive fluid that, by the time it finally appears in the stool, it has a black or brownish-black appearance which has been likened to tar or coffee grounds. However, if the hemor- rhage be large and the peristalsis very active, blood may appear almost unchanged in the stool, even when of high origin in the digestive tube. Eelatively small amounts of blood may be so changed and mixed with the feces that they, cannot be detected by the naked eye or by the microscope. This is termed "occult" blood, and is only recognizable by chemical means. Preliminary Technic. — Owing to the possibility of the pres- ence of a positive reaction, resulting from the ingestion of hemo- globin-containing food in a normal individual, it becomes neces- sary to restrict the diet in order to eliminate this possible source of error, and to limit the test-diet by the administration of a capsule containing five or ten grains of charcoal, and to watch the stools for the appearance of the black discoloration due to the passage of the charcoal. Only after the appearance of the charcoal in the feces should a specimen be taken for the test for occult blood. Steele* recommends a "liquid diet," including milk and broths, or a "semi-liquid diet" composed of milk, eggs, and toast, to which may be added moderate amounts of the ordinary winter vegetables. Eed meats and beef juice should positively be withheld. Steele has shown that iron, either in the organic or inorganic form, will even in large doses not affect the reaction. As a preliminary to the test, it is necessary to eliminate as fully as possible extraneous sources of blood. Thus tuber- ■• Amer. Jour. Med. Sci., July, 1905 154 THE FECES. culous ulcer, typhoid fever, hemorrhoids, fissure, and pur- pura should be excluded. Also ingestion of carmine, swallowed blood from any cause, hemoptysis, epistaxis, and menstruation. Technio of Test.5 — If the feces are solid they must be softened with distilled water. To 5 or 6 cubic centimeters of liquid feces, in a wide-mouthed cork-stoppered bottle, add about thrice as much ether and agitate or thoroughly mix by shaking. Then add a few grains of powdered guaiac and again agitate. Pollow this by 5 cubic centimeters of glacial acetic acid (99.4 per cent.), and still again agitate. Allow this mixture to stand until the solid particles settle to the bottom, and then decant into each of two test-tubes 5 cubic centimeters of the supernatant liquid. One test-tube should be kept as a control. To the other add 1 or 2 cubic centimeters of a fresh solution of hydrogen dioxid. If a bluish discoloration occurs either at the line of contact of the two solutions or throughout the mixture, the reaction is positive. Significance of the Occult Blood. — According to Boas occult blood is constantly present in carcinoma of the gastro- intestinal tract; intermittently present in gastric and duodenal ulcers; occasionally in stenosis of the pylorus, and is absent in gastritis, hyperchlorhydria, and in the gastric neuroses. BACTERIA AND PROTOZOA IN THE FECES. There are many varieties of bacteria in the feces. They have been estimated to amount to about one-third of dried feces. Some are harmless at all times; others which are, under ordi- nary conditions, harmless may develop pathogenicity under cer- tain circumstances. Bacillus Coll Communis. — This organism is constantly pres- ent in the feces, and is normally non-pathogenic. It has, how- ever, been found in pure culture in cases of appendicitis, empyema of the gall-hladder, pyelitis, and cystitis. Morphology.^ — It is a rod with rounded ends, sometimes so short as to appear almost spherical, while again it is seen with ^ From Professor Daland's Laboratory. "Abbott's Bacteriology. BACTERIA AND PROTOZOA IN THE FECES. 155 very much longer threads. It may occur as single cells or joined together in pairs, end to end. It is motile, and does not form spores. It stains with the ordiaary alkaline dyes, and is decol- orized by Gram's method. Compared to the typhoid bacillus, it will be found to be less motile; grows more rapidly on gelatine colonies, and lux- uriantly on potato (typhoid growth usually invisible). Colon bacillus coagulates milk in incubator from thirty-six to seventy- two hours ; typhoid does not. Colon bacillus decomposes sugar solutions; typhoid does not.'^ Bacillus Typhosis. — ^This is a distiuctly pathologic bacte- rium, and is the specific cause of typhoid fever. Its appearance in the stools is scanty, but usually can be isolated by appropriate methods during the first few days of the disease. Pratt, Peabody, and Long^ obtained it from the stools of only 31 per cent, of febrile cases, and none in twenty-one con- valescents examined by them. They state that it occurs most in the blood, and that it does not develop in the intestinal eon- tents except under unusual conditions. The bacillus in the in- testines comes chiefly from the gall-bladder; frequently it is found in the urine in larger numbers than in the feces, and is found in greatest numbers in feces containing blood. MoEPHOLOGY. — The bacillus is about three times as long as it is broad, with rounded ends. Its length may vary greatly, but its width remains fairly constant. It is actively motile, and Loeffler's method of staining will show it is possessed of many delicate flagelli (see methods of staining). Its growth on potato usually is invisible. It does not cause coagulation of milk (colon bacillus does). Owing to the tend- ency to retraction of the protoplasm from the cell-envelope, and the consequent production of vacuoles in the bacilli, the staining is often irregular. It stains with the ordinary aniline dyes. Bacillus lactis aerogenes and the bacillus proteus vulgaris, are probably always present in the stools. They are probably pathologic in many eases of cholera infantum. >For methods of platingr, isolating, and culture, see works on bacteriology. 'Jour. Amer. Med. Asso., Sept. 7, 1907. 156 THE FECES. Streptococcus aerogenes. — ^I'his is an etiologic factor in certain cases of entero-colitis. Bacillus Dysentericus (Shiga bacillus) is the cause of one form of infantile dysentery, and is generally present in the dis- charges of such patients showing blood and mucus. The Comma Bacillus. — This is the infective agent in Asiatic or true cholera. MoKPHOLOGY. — It is a slightly curved rod of a length of from one-half to two-thirds that of the tubercle bacillus, but thicker. Its curve is never very marked, it may be nearly straight. It is a flagellated organism, but has only one flagellum attached to one end. It is actively motile. It does not form spores. Cultural Characteristics.^ — On gelatine plates at room- temperature, its development can be observed after as short a period as twelve hours. It rapidly liquefies gelatine. It is strictly aerobic. It takes the ordinary stains. Diagnostic Method. — A smear is made from one of the small, slimy particles found in the semi-fluid evacuations, dried, fixed, and stained in the ordinary way. If upon microscopic examination only curved rods, or curved rods greatly in excess of all other organisms, are found, the diagnosis of Asiatic cholera most probably is correct. Tubercle Bacillus. — This organism probably is always pres- ent in all cases of intestinal tuberculosis. Diagnostic Teohnic (E. C. Rosenberger's). — A selection of "any part of feces is made, there being no effort at a selection of any particular mass or portion. If the stool is solid a small mass is mixed with sterile water. After drying and fixing, the spread is stained with carbol fuchsin for fifteen or twenty minutes, cold. The excess of stain is drained ofE and Pappen- heim's solution (see Appendix) is applied, and when the preparation is the color of the counter stain (methylene-blue) thorough washing in distilled water is resorted to, the spread being then dried and mounted in balsam. The most important point in the technic is to obtain a spread the color of the counter stain with not a particle of the carbol-fuchsin showing to the naked eye."** " Solis-Cohen : New York Med. Joui-., Aue. 21, 1907. CLINICAL SIGNIFICANCE OF EXAMINATIONS. 157 It is alleged, but not conclusively determined, that no other acid- or alcohol-fast bacilli will withstand this method except- ing the tubercle bacillus. Ameba Dysenterise.— This is now generally conceded to be the specific cause of the so-called amebic dysentery. In typical cases the stools contain much blood-stained mucus, containing large numbers of these amebae. These parasites so resemble epithelial cells that a positive diagnosis can only be reached when they are seen under the microscope to move and to extend their pseudopods. To keep them alive sufficiently long for examina- tion, the feces must be caught in a warm pan and a portion im- mediately transferred to a warm microscope slide for examina- tion. The organism is from 8 to 50 microns ta diameter (see Plate I). To develop the nucleus the organisms should be obtained fresh; they are killed by the addition of a few drops of acetic acid or corrosive sublimate (sat. watery solution) while on the microscope stage. The nucleus is spherical and about 5 microns in diameter (see page 103). For intestinal worms, ova, etc., see section on "Animal Parasites." THE CLINICAL SIGNIFICANCE OF THE EXAMINATIONS. Mucus. — As a rule, the appearance of mucus in the stool indicates the presence of inflammation of the mucous membrane, and is the one trustworthy sign of that condition (Steele). There are two conditions in which mucus has no significance: 1. When thin mucus spreads over the surface of a hard stool. 2. The so-called mucus-colitis with the discharge of mucus casts. Bile-Pigment. — ^A green color of part or all the stool (by the sublimate test) is pathologic, except in children. It means too short a period of passage through the intestines. A fresh normal stool should give a pink reaction with HgCl2. If a color reaction of any kind is absent it indicates either absence of bile from the intestine, a very fatty stool, or the reduction of hydrobilirubin (urobilin) into leucohydrobilirubin, a colorless product (Steele). Fat. — It will require some practice in the use of the diet to tell whether there is an excess of fat in the stool or not. As the 158 THE FECES. normal amount of fat varies within wide limits in normal feces, only a great excess can be considered abnormal. Kemnants of Meat. — Normally there should be only micro- scopic particles of connective tissue and muscle fiber. An excess of either is often visible to the naked eye. Excess of connective tissue means insufficient gastric diges- tion, because fibrous tissue is digested in the juices of the stom- ach only. Excess of undigested muscle-fiber means disturbance in intestinal digestion, and probably means trouble in the upper part of the small intestine, as follows: (a) Indicating a total lesion of the pancreas and absence of proteolytic and tryptic digestion, (h) Trypsin may be present, but its activating en- zyme, enterokinase, may be absent, (c) The period of passage of the food through the intestine may be so rapid that no time is given for its digestion, (d) Large pieces of undigested mus- cle may be present, because gastric digestion is imperfect and the connective-tissue framework of the meat has not been prop- erly removed. Pathologic carbohydrate fermentation means poor starch digestion and indicates, as a rule, disturbance in the small intes- tine, which is usually due to insufficiency in the suecus entericus. Pathologic albumin fermentation means a large remainder of albumin in the feces, and indicates, in Schmidt's experience, serious trouble, usually some anatomic change in the mucous membrane of the small intestine (Steele). FOREIGN BODIES, CALCULI, AND CONCRETIONS. An attack of biliary colic or gall-stones may be followed by the appearance, from time to time, of gall-stones in the feces, or they may appear without the previous occurrence of any symptoms referable to the liver or its appendages. In search- ing for them, the feces should be rendered thoroughly fluid by rubbing in water and then by passage through a sieve. Various forms of simple and complicated apparatus are upon the market, and are known as stool-sieves. A very satisfactory home-made substitute is made by filling in a wire-frame with two or three layers of gauze or cheese-cloth, the wire-frame being arranged to fit around the rim of the bowl of the closet. To be certain of FOREIGN BODIES, CALCULI, AND CONCRETIONS. 159 not missing the stones it is necessary to examine all movements for at least fourteen days following the cessation of the attack of colic. Even a careful and prolonged search may fail to find the concretion in certain undoubted eases of gall-stone disease. Sometimes because the stone causing the symptoms w^as not passed, but after being jammed in the neck of the bladder finally returned into the cavity of the gall-bladder itself. Again, the stone may be retained for a long time in a fold or diverticulum of the intestinal tract, and finally the concretion may have fallen to pieces in the intestine. Another explanation of why the stones fail to appear is that typical attacks of colic may be due to in- flammation without the presence of gall-stones. Gall-stones are concretions which form in the biliary pas- sages. They vary in size from a pin-head to that of a pigeon's egg or even larger. They are composed chiefly of eholesterin and calcium-bilirubin in varying proportions; besides these there are present minute amounts of other bile-oxidation prod- ucts and calcium-carbonate. A predominance of eholesterin produces a light-, and of calcium-bilirubin a dark-colored stone, the absolute color varying usually between a light- or dark-brown to a dark-olive green. Stones vary greatly in hardness, and on cross-section usually show distinct concentric layers of crys- talline substance, sometimes of different colors. The sur- faces may present a beautiful faceted formation from attrition between a number of stones lying in the bladder, or they may be irregularly granular. It is important not to confound other intestinal concretions with gall-tones. Woody bits of food, particularly the cores of pears, have been termed pseudogall-stones. The microscopic examination of bits of these scraped off with a knife, present the picture of characteristic wood cells. Chemical examination will also prevent mistake (see below) . So-called biliary sand, in most instances, consists of these small pseudogall-stones. The existence of true biliary sand has not yet been conclusively proven. Another kind of pseudogall- stone consists of balls of fat and fatty soaps which are not easily melted. They are found in the stools after the administration of large amounts of olive oil, as in a favorite method of treating cholelithiasis. 160 THE FECES. For chemical examination gall-stones are at first dried, then powdered and treated with alcoholic ether to extract the choles- terin. This may then easily be recognized by allowing the extract to evaporate upon a watch-glass, when the characteristic glistening rhomboids of cholesterin crystallize out, and may be easily recognized by the microscope. After extraction with alcohol and ether the residue is treated, while cold, with very dilute potassium hydroxid. If the powder contains calciiim- bilirubin, a yellow solution will be obtained which gives Gmel- lin's reaction. The much rarer panceeatic concretions diSer from gall- stones in that they contaia no bile-coloring matter, and are com- posed chiefly of calcium-carbonate, which dissolves readily with effervescence in hydrochloric acid. Intestinal Stones or Fecal Concretions. — These are sup- posed to play an important part in exciting attacks of appendi- citis, but seldom appear in the stools. They consist almost exclusively of ammonio-magnesium phosphate (triple phos- phate), and should be examined after the manner of urinary calculi (see page 226). 1° ^°Sahll's Dlasrnosis. XL THE URINE. PART I. GENERAL CONSIDERATIONS. The normal constituents of the urine are usually tested for by quan titati ve methods, since we are concerned with the actual amount of these substances and not with their presence or ab- sence from a given sample. In testing for abnormal constitu- ents, on the other hand, we are concerned, as a rule, with their presence_or_absence, though in certain instances we may desire to know the absolute quantity of these abnormal substances. The Sample. — If we are to make a qualitative examination for abno rinal co nstituents in a single sample of urine, it is best to collect a specimen about thre e hour s after the ingestion of a hearty meal (dinner), as such a sample is most likely to con- tain the substances sought (usually alb umin or su gar). Cases which at times show a trace of |lbumin will usually shoXsr an incre ase late in j he day or after ac tive exe rcise, and the collection of specimens should be timed to meet these conditions. Method op Collection of Specimens. — The following is a copy of the directions furnished by Prof. Judson Daland for the instruction of his patients: — DiBECTioNS FOE COLLECTING Ueine. — The Urine should be collected in a perfectly clean vessel and four ounces sent to the laboratory. Wide- mouthed four-ounce bottles especially adapted for this purpose should be obtained at the drug stores, and when possible the urine should be directly passed into the bottle. 1. The evening specimen is to be obtained in the following manner. Empty the bladder immediately before the evening meal and discard this ^ urine. From the urine first passed after the evening meal, take four ounces and note the hour when voided. 2. The second specimen is obtained from the urine first passed upon arising in the morning. Note the hour when the urine was passed. To obtain the total quantity -of urine passed in 2Jf hours. On the day when the observation is begun, at a definite hour, empty the bladder, " (161) 162 THE URINE. and discard this urine. All the urine passed afterwards is to be collected in a suitable, clean, dust-proof receptacle and kept in a cool place. The following day at the same hour, when the bladder was first emptied and the urine discarded, again empty the bladder. This urine should be added to complete the total amount for 24 hours, which should be ex- pressed in ounces. After the total amount of urine has been collected and thoroughly mixed, se nd four ounces of the mixture. Example. Observation began on January 1st., at 8 A.M. The blad- der is emptied at 8 A.M., this urine is discarded; the urine passed dur- ing the day and night are saved. The next morning, January 2nd., at 8 A.M., the bladder is again emptied, and this urine is added to complete the totaf quantity for 24 hours. A label on which is written the name, date, and time when the urine is passed should be pasted on the bottle. The Twenty-four Hour Specimen.- — For accurate results, particularly by quantitative methods, it is necessary to examiae a portion of the mixed urine voided during twenty-four hours. Such a specimen should be examined within six or twelve hours after the collection is complete; this will usually prevent errors 'due to the processes of decomposition and putrefaction which might destroy the formed elements. Catheterized Specimen. — Catheterization is often resorted to in order to obtain urine free from contamination, which might /enter it from the lower part of the urinary tract. For specimens of uriue from one kidney the Harris segregator may be used, or in the cases of women, the ureters may be catheterized. Urine kept at room-temperature, particularly in summer ■'time, readily undergoes decomposition and putrefaction. These changes render it unfit for examination. DECOMPOSITION CHANGES IN NORMAL URINE. If no method of preservation of the sample is employed, the fre sh uri ne, which is e lgar of a cid reaction and showing no deposit, will gradually undergo the following changes: — 1. The lower part grows cloudy from sedimentation of mucus, cells and other detritis, the urine is still acid. 2. This sediment gradually settles to the bottom and may show minute cr ystals of uric acid, the urine is less acid. 3. Uniformly cloudy from beginning, precipitation of phos- phates. The urine is very faintly^acidjorjieutKir 4. Very tuAid-feem-yc ficipitation of phosphates and devel- opment of bacteria. Copious sediment of triple and amorphous phosphates, bacteria, ammonium urate, and epithelial debris. Alkaline reaction and ammoniacal odor. DESCRIPTION AND IMPORTANCE. 163 PRESERVATION OF SAMPLE. When, for any reason, it becomes necessary to delay the examination of urine past the time when decomposition changes usually occur, these changes may be retarded in a number of ways: 1. By refrigeration. 2. By the addition of two or three grains of chloral for each ounce of urine. 3. By the addition of ten drops of 4-per-cent. formaldehyde solution for each ounce of urine. 4. By shaking with chloroform in the amount of five drops to the ounce of urine. DESCRIPTION AND IMPORTANCE OF THE URINE. The urine is an aqui«)us solution of the by-products of metabolism, so far as t)rese are not excreted or eliminated by the lungs, bowels, op^^fein. It is the most important excretory product of the body, and is the medium through which the end- products of nitrog enous metabolism and the soluble m ineral sal ts- are almost exclusively excreted under normal conditions. Ab- normal products of metabolism and many substances which have found their way into the circulation from without and which are foreign to the body, are likewise carried out in solution. '^ Not less than 50 per cent, of the total fluid ingested daily is excreted as urine. / General Characteristics. — Normal urine is perfectly trans- parent when voided, but soon becomes turbid, and on standing deposits a light flocculent sediment composed of a mucinous body and a few epithelial cells and leucocytes. If the urine is kept cold and care is exercised to exclude the entrance of micro-organ- isms, the upper portion of the urine will remain clear indefln-^ itely. Ammoniacal fermentation, due to the activity of the iac- terium urea and the micrococcus urea, causes cloudy urine from the precipitation of the phosphates. Bacteria-free urine may, in winter, become cloudy because the contained urates are less soluble in cold urine than in warm- urine. On standing the urates settle to the bottom, and the supernatant liquid remains clear as long as bacterial contamina- tion does not occur. Passage of Turbid Urine. — In man the passage of turbid urine is always abnormal, except during the first days of life. 164 THE URINE. when the turbidity is due to the profuse desquamation of epithe- lial cells and the relatively large amount of urates. PHYSICAL CHARACTERISTICS OF THE URINE. The Color. — The c olor of urine normally varies from a light yellow to a dark amber. This is largely influ enced by the con- cent ration of the sec retion and by the reaction. The pigmenta- tipn is due chiefly to the presence of a substance called uro;:^ /dhrome (derived from the biliary pigments) and indoxyl potas- smm sulphate (indican). Aci d urine is always darker than alka- l ine; the color is naturally lighter when the excretion is abundant than when it is scant. Deviations from the noemal color are notably observed in certain diseases and during the administration of certain drugs. It may also occur in apparently healthy individuals in consequence of certain undetermined anomalies of metabolism. Pale urine may occur as a neurosis or during the course of certain nervous diseases, particularly in epilep sy and hysteria. It may be a symptom of chro nic nephritis and" d iabetes. Darh urine, which is clear, occurs in the course of most acute fevers, and is due to the presence of uro-erythrin. A smoky color denotes the presence of decomposed blood, as in acute nephritis. Blood-red or pinh urine usually denotes the presence* of fresh blood. Eeeently Bar and Duaney^ have called attention to a fal se bloody uri ne due to the activities of a pseudo- membranous and chromogenic bacterium. Yellow-brown or greenish urine suggests the presence of bile. Browni sh urin e occurs in melanosis and after the inges- tion of rhubarb, senna, or tannic acid. Smoky-hrownlirine indicates the presence of the end-prod- ucts of ingested carbolic acid or its analogues. Pale o reenish w ine, with a high specific gravity, usually indicates__glucose. White urine denotes the presence of pus or chyle. Whitish turiiSity Aenotes: 1. Pus. 3. Phosphates. 3. Am- monium urate. ' Le ProgrSs Medioale, Mar. 24, 1906. PHYSICAL CHARACTERISTICS. 165 Urine which Darkens on Standing. — Some urine, after standing for a variable period, becomes dark-brown or black. This may be due to the presence in the specimen of melanin or of phenol. Non-pathologic phenol urine or "carbol urine" may arise from medicinal or surgical treatment with phenol or closely allied compounds. Under these conditions the end-products in the urine usually appear as resorcin. Such urine, if alkaline when voided, rapidly darkens on exposure to the air, and if acid becomes dark more slowly, as the alkaline reaction gradually develops. Under certain pathologic conditions the uriae may darken on exposure to the air, owing to the presence of alkapton. This condition is known as alkaptonuria. A similar darkening in pathologic urine may result from the presence of a coloring matter termed melanogen. Test. — To differentiate between the phenols and melan- ogen: Add bromine water to the suspected urine. In the presence of phenols there will be produced a permanent, yel- low precipitate. A primary yellow precipitate which gradually darkens, becoming finally brown or black, indicates melanogen. Corroborative Test. — Add to the fresh undarkened urine a few drops of dilute solution of neutral ferric chloride. A violet discoloration denotes phenols, brown or black denotes melan- ogen. It must be remembered that urine containing alkapton will give many of the chemical tests for sugar which depend on a reduction process. The Odor. — Freshly passed normal urine has a peculiar characteristic aromatic odor, resulting from the contained \ volatile acids. Decomposing urine has the characteristic odor due in part to the free ammonia resulting from the decomposi- tion of urea. Urine which is ammoniacal when freshly passed, points to a pathologic fermentation occurring in the bladder, usually ac- companying cystitis. A putrid odor denotes putrefactive change occurring iu pus ^ or other albuminous substances. An odor resembling acetonj : is occasionally observed and denotes diabetes mellitus. 166 THE UEINE. Urine containing cystin may, upon standing, develop the odor of HTDEOGEN SULPHIDE. Some articles of food, as asparagus and onions, and certain aromatic medicines, as turpentine and copaiba, may give char- acteristic odors. THE AMOUNT. The average daily excretion of nrine in the United States is somewhat less than in foreign beer-drinking countries. A fairly normal average for the United States may be stated to be between 1200 and 1600 cubic centimeters, or about fifty ounces for men, while for women it is slightly less. Generally speaking, the amount will vary in inverse ratio to the insensible perspira- tion: hot weather diminishes and cold weather increases the amount. Profuse perspiration, sweating, vomiting, and diar- rhea all decidedly diminish the amount. Children, and espe- cially nursing infants, on account of the preponderance of liquid in their food, excrete a proportionately larger amount of urine, compared with body-weight, than do adults. The normal amount of urine passed in twenty-four hours is consequently subject to wide variations, depending on the amount of fluid ingested, the character and the quantity of the food, the process of digestion, the blood-pressure, the surrounding temperature, the emotions, sleep, exercise, age, sex, and body- weight. During repose much less nrine is excreted than during activity, hence the excretion during the night is less than during the day. The maximum secretion is usually observed during the first few hours succeeding a hearty meal. Artificially the excretion may be i ncr eased ^b;^ substances _which have a tendency t o Ta.ise_ liioQA^iiiiitiU.i'ii,'''as'tea, coffee, and alcohol. ManY^rngs bring about the same result. The most important of the medicinal diuretics are digitalis, squill, broom, juniper, nitrous ether, urea, etc. Disti lled w ater also possesses distinct diuretic properties. The quantity of urine in pathologic conditions depends, first on the condition of the secreting re nal parench yma and, sec- ondly, upon the condition of the bloo d-curr ent in the kidneys. It will therefore be affected in general by circulatory disturb- ances, as well as by disease of the kidney itself. To appre- VARIATIONS IN THE AMOUNT. 167 ciably alter the amount of the excretion, b oth kidneys must be diseased, for if one kidney be healthy it will assume vicariously the function of the other organ. This compensatory action regularly occurs after extirpation of one kidney. As a rule the more acute the nephritis the more the excre- tion of urine will fall below normal, while the more chronic the process the more will the amount exceed normal. This increase reaches its maximum in the true contracted kidney where the volume excreted in twenty-four hours may be very great. Dis- eases of the heart and lungs, leading to chronic passive conges- tion, will diminish the total amount of the lirine, a condition evidently dependent upon interference in the renal circulation. It is evident, therefore, that the determination of the total output of the kidneys and an observation of the variation in the amount during the course of disease, may be of great diagnostic and prognostic value. The increased excretion of urine following convulsions, par- ticularly the hysterical-variety and after attacks of angina p ec- toris, is probably dependent upon some v asomoto r disturbance. Many alterations in the volume of the urine depend upon quan- titative and qualitative variations in the substances eliminated, and are primarily induced by disturbances in metabolism. Anuria, or entire failure to void urine, may be due either to complete suppression of the secretion in the kidneys or to obstructions in the urinary tract. Hydruria. — This is a state of the urine in which the fluid is increased out of proportion to the solids, and is usually asso- ciated with an increase in the total twenty-four hours' output. Polyuria. — This term is applied to an increase in the elimi- nation of the urine as a whole, both fluids and solids. Oliguria is applied to a diminution in the total excretion of the urine. Polyuria may be noted in the following conditions : 1. Dia- betes mellitus. 2. Diabetes insipidis. 3. Chronic interstitial nephritis. 4. Amyloid disease of the kidney. Oliguria is met under the following circumstances: 1. Valvular heart-disease. 3. Diminished blood-pressure (see sec- tion on blood-pressure). 3. Acute articular rheumatism. 4. Chronic parenchymatous nephritis. 5. Acute congestion and 168 THE URINE. inflammation of the kidneys. 6. Decreased cell activity in shock. 7. Failure of nutrition preceding death. SPECIFIC GRAVITY. The average specific gravity of normal urine of 1500 centi- meters (fifty ounces) volume for twenty-four hours, is about 1030. Slight variations (1015 to 1038) from this standard are consistent with perfect health and depend chiefly upon the char- acter of the food ingested, the quantity of water taken, and upon the state of metabolism. Under certain conditions of apparent health, as after a hearty meal, the specific gravity of the indi- vidual specimen may be as high as 1035, and after excessive in- gestion of fluids it may temporarily fall to 1005. Under pathologic conditions the specific gravity may vary between 1001 and 1055 or even higher. If the diet consists largely of nitrogenous food it will furnish a relatively larger amount of solids; in consequence the specific gravity of the urine will be increased. Active muscular exertion also tends to raise the specific gravity by increasing tissue catabolism. Co- pious diaphoresis may bring about a concentration of the urine by diminishing the amount. Fasting has a similar effect. The specific gravity usually varies inversely with the volume both under normal and pathologic condition. A scanty urine is more concentrated than a profuse one. Diabetes mellitus forms an exception to this rule, since the presence of sugar produces a high specific gravity in spite of the excessive amount of the excretion. This fact is so characteristic that a tentative diag- nosis may be made upon this alone. From the foregoing it is evident that, from a clinical stand- point, it is necessary to consider the specific gravity, the total solids, and the total volume together, as they are intimately and logically related to each other, and that the determination of the specific gravity is of greatest value and significance when the total volume of the urine for twenty-four hours is known. Also that the chief clinical value of the study of the amount and of the specific gravity is to aid in the estimation of the total solids of the urine (see below). The specific gravity of the urine is usually determined with SPECIFIC GRAVITY. 169 the aid of a hydrometer or urinometer, but is more accurately determined by the Westphal balance. Only approximately cor- rect results may be obtained with the urinometer. The Use of the Urinometer. — The scale of the urinometer is usually marked in regular intervals from 1000 to lOGO (Fig. 20). To insure ease and accuracy in reading, these markings should not be too close together. i\Iany urinomcters are inaccurately made, so that before purchasing an instrument it is always well to compare it with a standard instrument, or at least to ascertain that it floats at the 1000 mark in distilled water at the standard temperature (15° C. or 60° F.). Although a Fig 20— Various Forms of Urinometers and Urinometer Cylinders. large instrument is more accurate, a small one, reqriiring less nrine, is more convenient and must frequently be employed for lack of sufficient urine in which to float the larger instru- ment. To determine the specific gravity of the urine : After allow- ing the urine to cool to room temperature (about 60° F.), it is poured into the urinometer tube { the urinometer and an appro- priately sized glass cylinder are usually sold together), and the urinometer immersed in the urine ; then with the eye on a level with the surface of the urine, the division of the scale is read off' which corresponds to the lowest part, of the curve of the meniscus. To insure accuracy the containing cylinder should be sufficiently large to allow the urinometer to float freely and not come in contact with the sides. All bubbles and froth should 170 THE URINE. be removed from the surface of the fluid by means of filter-paper. If the urine is cooler than 15° C, one-third of a urinometer unit should be subtracted for every degree centigrade below the standard temperature. If warmer an appropriate addition should should ])e made. Since the specific gravity of individual urine specimens vary greatly during twenty-four hours, it is nec- essary tliat the specific gravity should be taken from a mixed specimen of the twenty-four hours' collection. A reading taken from any single specimen is of little clinical significance. If tlie specimen is too small to work with in the ordinary way, it is a very simple matter to dilute with a known propor- tion of distilled water. Estimate the specific gravity of the Fig. 21.— Westphal Balance. (A. H. Thomas.) diluted urine and tlien calculate the specific gravity of the spe- cimen at least ajiproximately. The Westphal balance (see Fig. 21) is an extremely ac- curate metliod of determining the specific gravity, carrying the reading to the fifth figure (fourth decimal). The method of employing the Westphal balance is as follows: — When the instrument is mounted the glass plummet which is suspended from one end of the l)eam will Iwlance in distilled water at lo° C. and represents 1. Now pour the urine into the jar until tlie twist in the platinum wire is Ijclow the surface. Weighing is accomplished as follows: Place the first rider upon the end of tlic lieam aliove the float, then place the second rider in the first notch to the left on tlie scale on the beam. If the plummet rises place the rider on the second notch. If now tlie METHOD OF ESTIMATING THE TOTAL SOLIDS. 171 beam balances and the temperature of the urine is 15° C, the specific gravity of the urine is exactly 1030. If the float still rises take the third rider and find the notch in which the beam balances or nearly so. If the beam balances with the third rider in the forth notch, the specific gravity is exactly 1034. Should, however, the float still slightly rise, take the fourth rider and find the exact balance, and if this is in the sixth notch the specific gravity is exactly 10346. In other words, the second rider (in size) gives the third figure, the third rider the fourth (third decimal), and the fourth rider the fifth figure or the fourth decimal. With a little practice determinations with the West- phal balance are rapidly and accurately made. Care should be exercised to see that no air bubbles become attached to the cord or float, and that temperature corrections are made as directed below. Corrections for Temperature. — The temperature of the urine immediately after being voided, ranges from 85° to 95° F. (39.5° to 35° C); therefore in taking the specific gravity of fresh urine its temperature must be observed, and for every seven degrees Fahrenheit that the thermometer indicates above the temperature at which the instrument is standardized, one degree should be added to the specific gravity, as indicated by the instrument. METHOD OF ESTIMATING THE TOTAL SOLIDS. The accurate chemical method of weighing is too difiicult and tedious for general clinical work, descriptions of which may be found in works on physiologic chemistry. The normal average weight of solids is sixty-five grams. The specific gravity is a pretty accurate index of the amount of solids excreted by the urine when this has been determined from the twenty-four hours' urine. The solids excreted in one litre of urine may be approxi- mated in grains by multiplying the last two figures of the spe- cific gravity by 3.3337 ( Vierodt's factor) . Trapp's Formula. — Multiply the last two figures of the specific gravity by two, and the result will represent the parts of solids in one litre of urine. Example : If the specific gravity 172 THE URINE. is 1023, then 23 times 2 will equal 46 parts of solids per 1000 cubic centimeters. Bird's Formula. — The last two figures of the specific gravity about represent the grains of solids in a fluidounce of urine tested. Thus, a specific gravity of 1022 would contain approxi- mately twenty-two grains of solids per ounce of urine. Metz's Formula. — Multiply the last two figures of the spe- cific gravity by .00233, and multiply this product by the total twenty-four hours' volume in cubic centimeters. The final product will be the total weight of solids expressed in grams. Example: Specific gravity = 1024: then 24 x .00233 x 1500 cubic centimeters equals 8'^.27 grams of solids in twenty-four hours' excretion. THE REACTION. The reaction of the twenty-four hours' urine is usually acid, sometimes amphoteric, and rarely alkaline. The normal acidity is not due to the presence of free acids, but to acid salts, chiefly acid phosphates. Primarily the reaction of the urine depends upon the character of the diet. Increase in ingestion of food- stuffs containing alkaline salts (vegetables), or when such salts are formed within the body from organic acids contained in the food (fruits), will tend toward an alkaline reaction when the urine is voided. The elimination of alkaline urine in this in- stance is due to the presence of a fixed alkaline, and therefore cannot be considered pathologic. It has already been stated that the acid reaction of urine is normally due chiefly to the acid phosphates. Besides these a certain amount of the neutral phosphates of calcium, ammonium, and of sodium usually occur, and it may happen that the acid and neutral phosphates are present in such proportions that the sample will turn blue litmus red and red litmus blue. Such a urine is said to be amphoteric. Urine, when allowed to remain exposed to the air at room- temperature, gradually undergoes ammoniacal fermentation. This is due chiefly to the action of certain micro-organisms upon urea, which is decomposed by a hydrolytic process into ammonia, carbon dioxid, and water (see page 162). THE REACTION. I73 The result of this development of an alkaline reaction is that the soluble phosphates of the alkaline earths are precipi- tated as tri-calcium phosphate and ammonio-magnesifiun phos- phate, at the same time the soluble urates are transformed into the insoluble ammonium salt (see Fig. 23). Test. — In order to determine whether the alkalinity of a given specimen is due to a fixed or to a volatile alkali (am- monia), a strip of red litmus is clamped into the cork of the bottle so that it does not touch the liquid but remains dry. Volatile alkali will gradually turn the paper blue, while a fixed alkali will not affect the paper unless the alkaline fluid touches it. In specimens where the degree of alkalinity or acidity is so slight that the litmus-paper test leaves the examiner in doubt, the uncertainty may be eliminated by the following expedient: Take two pieces of litmus-paper, one red and the other blue; allow the urine to come in contact with but half of each strip of paper; then with distilled water allow the whole extent of both strips to become wet. This eliminates any change in the paper due to the presence of moisture alone, and brings out sharply slight changes in the color of the litmus-paper by allowing comparison between the adjacent portions of each strip of paper. Increased Acidity. — The urine is frequently found hyper- acid in: — 1. Fevers. 2. Inflammations of the liver. 3. Hyperchlorhydria. 4. Acute rheumatism. 5. Lithemia. 6. Neurasthenia. Alkaline urine may be passed under the following condi- tions : — 1. Undue retention of urine in the bladder. 2. When there is residual urine. 3. Presence of urea-decomposing bacteria in the bladder. 4. Chlorosis. 5. Organic nervous diseases. 6. In marked degrees of general debility. 174 THE URINE, ESTIMATION OF ACIDITY OR ALKALINITY OF THE URINE. Reagents. — (a) A one-tenth normal solution of NaOH (1 cubic centimeter equals 0.004 gram NaOH). (b) A one-tenth normal solution of HCl (1 cubic centi- meter equals 0.00365 gram HCl). Since these solutions are difficult and tedious to prepare, it may be well to obtain them ready-made from reliable chemical- supply houses. The Test. — If the urine has an acid reaction, measure 100 cubic centimeters into a beaker, and add a few drops of a litmus solution. Now add from a burette, drop by drop, sufficient of the NaOH solution to produce an alkaline reaction. State the acidity in this wise : 100 cubic centimeters of urine requires "X" cubic centimeters of one-tenth normal NaOH to render alkaline to litmus. Put in place of "X" the number of cubic centimeters of NaOH used in the examination. If the urine to be tested is alkaline, then first add to the 100 cubic centimeters exactly 20 cubic centimeters of the one- tenth normal HCl solution. Stir well, and then proceed as in case of the acid urine. State the result as follows : 100 cubic centimeters of urine requires "Y" cubic centimeters of one- tenth normal HCl to render alkaline to litmus. Put in place of "Y" the difference between 20 cubic centimeters and the number of cubic centimeters of NaOH used. CHEMICAL COMPOSITION OF THE URINE. A general idea of the chemical composition of the urine and of the quantitative variation of the individual components may be had from the accompanying table (after Simon). The indi- viduals from whom the urine was obtained were adults. Their habits, diet, etc., may be taken to be that of the average Ameri- can city dweller. It must be borne in mind that tables of chemical composi- tion are based upon averages taken from a large number of complete analyses, the composition of which, even in perfect health, covers a greater range of variation than indicated in the following table : — INORGANIC CONSTITUENTS. 175 CHEMICAL ANALYSIS OF THE URINE (Simon). Grams. Water 1200 to 1700 Solids 60 Inorganic solids 25.0 to 26.0 Sulphuric acid (H2SO4) 2.0 to 2.5 Phosphoric acid (P2O5) 2.5 to 3.5 Chlorine (NaCl) 10.0 to 15.0 Potassium (K2O) 3.3 Calcium (CaO) 0.2 to 0.4 Magnesium (MgO) 0.5 Ammonia (NH3) 0.7 Fluorids, nitrates, -etc 0.2 Organic solids 20.0 to 35.0 Urea 20.0 to 30.0 Uric acid 0.2 to 1.0 Xanthin bases 1.0 Creatinin 0.05 to 0.06 Oxalic acid 0.05 Conjugate sulphates 0.12 to 0.25 Hippuric acid 0.65 to 0.7 Volatile latty acids 0.05 Other organic solids 2.5 THE INORGANIC CONSTITUENTS OF THE URINE. The inorganic constituents of the urine represent the excess of mineral salts that find their way into the blood from the digestive tract or which develop within the body during the processes of metabolism, especially during albumin decomposi- tion. We therefore find that exercise and the ingestion of large amounts of food^ as well as the increased cell activity occurring in acute fevers, lead to an increased elimination of salts, and conversely smaller amounts of salts are eliminated when the intake of food in general is restricted. These statements apply particularly to the phosphates. The bases which are found in the urine in combination with hydrochloric acid, phosphoric acid, and sulphuric acid, are chiefly sodium, potassium, calcium, magnesium, and ammonium. It is believed that the mono-acid phosphates of the alkaline earths are held in solution by sodium chlorid, and also by the di-acid sodium phosphates, to which- latter the acidity of the urine is largely due. While the greater portion of the sulphuric which results 176 THE URINE. from albumin decomposition is found in the urine combined with inorganic bases, a variable fraction also occurs united with cer- tain aromatic substances which are developed in the intestines during putrefaction and decomposition. The resulting bodies are spoken of as the ethereal or conjugate sulphates. They com- prise the alkaline salts of indol, skatol, and phenol. The Phosphates. — The amount of phosphoric acid excreted by the healthy individual in twenty-four hours ranges from 2.3 to 3.5 grams, the average being 2.8. In the urine the phosphoric acid is found in part combined with, the alkaline earths — earthy phosphates — and in part with the alkalies as the alkaline phos- phates. The alkaline bases represent about two-thirds of the total phosphoric acid in combination. The earthy phosphates are insoluble in water, but are soluble in dilute acids. They comprise the phosphates of calcium and magnesium. The calcium phos- phates predominate. In acid urine the earthy phosphates are in solution, while in alkaline urine they are precipitated. They are thrown down by heat, and also when the reaction of the urine heeomes alkaline, whether from a course of internal alkaline medication or from the putrefaction of the urea of the urine. If, during decomposition of the urine, the contained acid phos- phates are acted upon by the ammonia resulting from urea decomposition, ammonium-magnesium phosphate (triple phos- phate) is formed and appears in the urine as the characteristic prismatic or coffin-lid crystals. The alkaline phosphates comprise the phosphates of sodium and potassium; of these the sodium is the more abundant. These, unlike the earthy phosphates, are easily soluble in water and in alkaline fluids. The alkaline phosphates form the chief bulk of urinary phosphates. The normal urinary acidity depends upon the presence of these acid phosphates, and not upon the presence of free acid. While the bulk of the phosphates in the urine is derived from the decomposition of food-stuffs, a part also is derived from the breaking down of the highly complex organic bodies: lecithin and nuclein. Clinically, the execretion of phosphoric acid and its determi- nation is of very little significance, since it is so largely dependent upon the influence of diet, exercise, etc. INORGANIC CONSTITUENTS. 177 Detection. — If neutral or alkaline urine is heated in a test-tube, a precipitate will be formed which will be found to consist of earthy phosphates. Such a precipitate might be caused by the presence of albumin. To remove the doubt add a few drops of dilute (10 per cent.) acetic acid, when a precipitate due the phosphates will immediately disappear, while if albuminous the cloud will remain or may be increased. Test for Earthy Phosphates. — To a few cubic centi- meters of urine in a test-tube add a few drops of liquor potassii and boil. The earthy phosphates will be thrown out of solu- tion, and may, after settling, be collected upon a filter. Now, to the filtrate add one-third of its volume of "magnesia mix- ture" (for formula see Appendix). The precipitate thus formed represents the phosphoric acid that was in combination with the alkaline bases, combined now in the form of ammonium- magnesium phosphate. Estimation of the Phosphates by the Centrifuge. — In a graduated percentage centrifuge tube (see Fig. 30) mix 10 cubic centimeters of urine with 5 cubic centimeters of "magnesia mixture"; invert several times to thoroughly mingle. Eevolve in the centrifuge (Figs. 36, 37 and 38) for three minutes. Read off every one-tenth cubic centimeter of precipitate as 1 per cent, by bulk of total phosphates. The average normal percentage by this method is eight. Roughly, each one-tenth cubic centimeter of sediment is equal to about 0.0335 per cent, by weight of P2O5. Determination of the Total Phosphoric Acid. — For this determination the following solutions are required: — 1. A standard solution of uranium nitrate: 30.3 grams of uranium nitrate are dissolved in 1000 cubic centimeters of dis- tilled water, then each cubic centimeter of this mixture is equivalent to 5 milligrams of phosphoric acid. 3. Sodium acetate solution: 100 grams of sodium acetate are dissolved in 900 cubic centimeters of distilled water, and to this 100 cubic centimeters of acetic acid are added. 3. Saturated solution of potassium ferrocyanide. Mdhod. — Fifty cubic centimeters of urine are placed in a beaker and 5 cubic centimeters of the sodium acetate solution added. The mixture is warmed over a water-bath and uranium 12 178 THE URINE. nitrate solution added from a burette as long as a precipitate is formed. If the formation of precipitate is not easily recog- nized, a drop of the potassium ferrocyanide solution may be added; then, as long as a brown color does not appear where the drop falls, the uranium nitrate solution should continue to be added. The end point in the precipitation reaction is reached when a reddish-brown discoloration appears upon the addition of a drop of the uranium nitrate. The quantity of uranium nitrate solution employed to accomplish complete pre- cipitation is now read off from the scale on the burette, each cubic centimeter of which will equal 5 milligrams of phosphoric acid. This number and the 50 cubic centimeters of urine em- ployed furnished the working bases for calculating the per- centage. The presence of sugar or albumin does not interfere with this reaction. Separate Estimation of the Earthy and Alkaline Phosphates. — Two hundred cubic centimeters of urine in a beaker are rendered alkaline by the addition of ammonium hydroxid, and set aside for a few hours. The earthy phos- phates are thus precipitated and may be collected upon a filter- paper, and after washing with dilute ammonia (1 : 3) are trans- ferred to a beaker, where they are dissolved with as little acetic acid as possible. Distilled water is then added so as to malce the total volume approximately 50 cubic centimeters, when the solution is boiled and then titrated as above. In a second por- tion of urine the total phosphates are determined as outlined above. Then the difference between the two results will repre- sent the quantity of phosphoric acid present in combination with the alkalies. Significance. — As has been shown above, the phosphates are dependent upon many uncertain factors which are determined with difficulty and are of little clinical significance. When in a given case the phosphates are constantly thrown out of solution, it may be taken as an indication that the formation of gravel or calculus is impending. If the usual signs of ammo- niacal fermentation are present, the significance is plain. THE SULPHATES. 179 THE SULPHATES. General Considerations. — The major portion of the sul- phates appearing in the urine are derived from the food, and comprise the simple mineral sulphates of sodium and potassium. Only a small portion exists in organic combination as the ethe- real or conjugate sulphates. The three predominating conjugate sulphates are: Phenol potassium sulphate, indoxyl potassium sulphate (indican), and skatoxyl potassium sulphate. The mineral sulphates comprise about nine-tenths of the total sulphates in the urine. Test foe Mineral or Preformed Sulphates. — Add a few- drops of acetic acid (to prevent the formation of barium phos- phate) to a test-tube half full of urine. Now, upon the addition of a solution of barium chlorid a white precipitate of insoluble barium sulphate will be formed. This precipitate varies in density from a faint white cloud to a bulk that gives a thick creamy consistence to the whole mixture. One can roughly deter- mine by the amount of precipitate as compared with the known normal standard, whether the sulphates are increased or not. Test for Conjugate or Ethereal Sulphates. — Mix equal quantities of alkaline barium chlorid (see Appendix) and urine in a test-tube. After allowing a few minutes for the precipitate to form the mixture is filtered. This process precipi- tates both phosphates and preformed sulphates. The filtrate is now acidified with 5 cubic centimeters of a (one-fifth vol.) HCl solution and then boiled for some time. The presence of ethe- real sulphates is indicated by a reddish discoloration of the fluid which in addition becomes turbid. Potassium Indoxyl Sulphate or Indican. — As this substance represents the characteristic ethereal sulphate, it may be taken as an indicator for the whole group, and the tests for this sub- stance used for estimating the relative amount of the whole group of ethereal sulphates in the urine. The several tests for indican are based upon the fact that an excess of HCl will liberate the indoxyl, which can then, by the addition of an' oxidizing agent, be converted into indigo-blue, and fbially this can be recognized in small amounts by extraction from the bulk of urine with chloroform. 180 THE URINE. Test foe Indican (modified Jaffee). — Take 20 cubic centi- meters of filtered urine in a test-tube and add 3 or 4 -cubic centimeters of chloroform, and one drop of a 1 per cent, solu-- tion of potassium chlorate, and finally 30 cubic centimeters of HCl. This mixture is to be thoroughly agitated and mixed by pouring repeatedly from one test-tube to another. This should be repeated at intervals of two or three minutes covering a period of ten minutes. The presence of indican is indicated by a blue discoloration of the chloroform. In the presence of a large amount of indican the chloroform vrill appear almost black, while the whole mixture will assume a dusky bluish-red color. (For comparative color-scale see Plate VII.) While a trace of indican cannot be considered pathologic, there are, nevertheless, many specimens, possibly one-third, which fail to show any discoloration of the chloroform. Caution. — An excess of oxidizing agent, either in volume or strength, will prevent a positive reaction through over- oxidation of the indoxyl compound. 3. An excess of chlo- roform will result in too great dilution of the indigo, causing failure to detect traces of indican. 3. A reddish discoloration of the chloroform is not due to indican. Such a reaction occurs in the urine of patients who are taking iodide, and possible bro- mide. The simultaneous occurrence of a red and a blue re- action, will produce a color bordering on purple. This possible source of error may be removed by the addition of a few drops of a 10-per-cent. solution of sodium thiosulphite, which will bleach to pink color due to iodine. Clinical Significance of Indican. — The presence of more than a trace of indican probably denotes the existence of undesirable decomposition in the intestinal tract. The occur- rence of a positive indican reaction does not necessarily indicate constipation. THE CHLORIDS. The quantity of chlorids in the urine is usually decreased' in: 1. Most febrile diseases. 3. Nephritis. 3. Wasting dis- eases. 4. Pneumonia. Approximate Estimation of the Chlorids. — To a few cubic centimeters of urine in a test-tube, add a few drops of nitric >^4!: -J u a « < fH H M W J **! ^5 ORGANIC CONSTITUENTS. 181 acid, boil and filter to remove the albumin. Add to the filtrate a few drops of a 10-per-cent. solTition of silver nitrate. The abundance of the white cloud will roughly indicate the amount of chlorids present. Accurate Methods of Quantitative Determination. — Method of Salkowski-Volhard. Take 10 cubic centimeters of urine in a beaker and dilute with 50 cubic centimeters of distilled water, ■and then treat with 4- cubic centimeters of concentrated nitric acid and 15 cubic centimeters of a standard silver nitrate solu- tion. The mixture is then further diluted with distilled water up to 100 cubic centimeters, and after thorough agitation is passed through a dry filter. In a carefully measured portion of the filtrate the excess of silver is carefully titrated with a solu- tion of potassium sulphocyanide of such a strength that 35 cubic centimeters corresponds to 10 cubic centimeters of the standard silver solution. A few drops of a saturated solution of amraonio-ferric aJum serve as an indicator. The amount of silver solution employed to precipitate the chlorids in the 10 cubic centimeters of urine is then calculated. The number of cubic centimeters necessary for this precipitation is multiplied by 0.01, which will indicate the amount of chlorids in 10 cubic centimeters of the urine calculated as sodium chlorid. The presence of albumins or of sugar does not interfere with this reaction. THE ORGANIC CONSTITUENTS OF THE URINE, GENERAL CONSIDERATIONS. The organic constituents of the urine comprise the normal end-products of nitrogenous metabolism within the body, also various products of albuminous putrefaction which have found their way into the general circulation from the intestinal tract. Finally certain pigments which bear a relation to the normal blood-pigment, and various substances of obscure origin are en- countered. Under abnormal conditions we may meet with normal constituents of the blood which do not ordinarily appear in the urine. Lastly, in pathologic conditions, we meet various products of abnormal metabolism. ,. ^ 182 THE URINE. UREA. Urea was first synthetically prepared from ammonium eya- nate in 1838 by Wohler. Formerly it was supposed that urea resulted from uric acid through a process of oxidation, and that this was its only source. Now this error is well known, and while it is recognized that the formation of urea from uric acid is possible and that a small portion of the total amount may be derived in this manner, modern research has shown that in man urea is largely derived from the destruction of the nucleins within the body, and that the sources of the nucleins are both the tissue cells and the cells of animal foods ingested. It has been repeatedly shown that during the decomposi- tion of albumins by means of acids and alkalies, as during the process of tryptic digestion and albuminous decomposition, a large amount of mono-amido-acids results. And it is supposed that these bodies probably represent the intermediary products in the transformation of albuminous nitrogen into urea. Under certain pathologic conditions these acids may appear in the urine, and when they are there noted the elimination of urea is much dirriinished. In health, however, this does not occur, and on examination of the different tissues of the body such acids are found only in traces. We must conclude, therefore, that these acids, supposing them to occur as the primary products of albuminous decomposition within the body, are transformed at once into other substances, which in turn give rise to urea. It has also been shown that the amido acids yield carbamic acid on oxidation, and that on alternate oxidation and reduction urea can be produced from the ammonium salt (ammonium carbamide). While it is generally assumed that urea is largely a-eferable to a transformation of the mono-amido-acids into ammonium carbamate, and while it has also been shown that such a transformation does actually occur, it must be remem- bered that at best only traces of these amido acids are found in the tissues. From these conclusions it is reasonable to believe that the greater portion of albuminous nitrogen is set free from the vari- ous organs of the body in the form of an ammonium salt of para- laetic acid, and that this salt is now generally conceded to be the antecedent of urea. UREA. 183 It is probable that a certain amount of urea is produced in the body in a number of ways, and there is ground for the belief that its formation is not confined to a single organ. The greater part is, without doubt, produced in the liver. In corroboration of this fact it has been repeatedly shown that in disease of this organ, when accompanied by extensive destruc- tion of the glandular elements, a diminished amount of urea is found in the urine, while ammonia and lactic acid are found in increased amounts. In certain cases of this class as much as 37 per cent, of the total urinary nitrogen has been found in the urine in the form of ammonia. If we accept the modern doctrine that urea originates not only in different ways, but that it may also be formed in other organs beside the liver, then we can understand why it is that in certain diseases of the liver the diminution in the amount of urea excretion is not always proportionate to the extent of parenchymatous degeneration, and that no case has yet been reported in which the excretion of urea has ceased altogether. Nitrogenous Equilibrium. — The albumins are the ultimate source of the urea, and according to Pettenkof er they exist within the body in two forms, viz. : as organized albumin, which is built up into tissues, and the so-called circulating albumin, which is present in excess of the actual requirements of the body and which may be broken down directly and eliminated in the urine without ever having entered into the construction of the body- proper. This latter portion of the body albumin is said to fur- nish the bulk of the urea, while the fixed tissue albumin repre- sents the minor but more uniform source. The actual amount eliminated will, therefdre, be primarily dependent upon the quan- tity ingested. Experiment has shown that under normal conditions of average diet the total urinary nitrogen is practically equivalent to the quantity ingested, barring a small fraction, which escapes digestion and appears in the feces. Such a condition is spoken of as the nitrogenous equilibrium of the body. Of this relation infinite variations exist, which may even vary from time to time in the same individual. If the amount of nitrogenous food is suddenly diminished the amount of urinary nitrogen will also decrease; then if the reduced ingestion remains constant the 184 THE URINE. nitrogen output, while lowered, will at the same time tend to remain level. If, on the other hand, the nitrogen intake is in- creased, an increased nitrogen elimination speedily follows, but here a certain fraction will be retained in the body and gradually a higher level of equilibrium will be established. There are natural limits to this power of accommodation of the system to nitrogenous ingestion and elimination, so that we may find a point which varies in different individuals where a further nitrogen ingestion does not lead to a higher level of nitrogenous equilibrium, and when, consequently, a further re- tention of nitrogen does not occur. Overfeeding then results in various digestive disturbances. Diarrhea and vomiting may occur through nature's effort to protect the body from a further increase in circulating nitrogen. Underfeeding, on the other hand, generally leads to in- creased destruction of the organized albumins. Although for a while the body's store of fats and carbohydrates is capable of protecting the body against an undue loss of nitrogen in this direction, still if the reduced intake of nitrogen continues suf- ficiently long, death finally ensues. From the fact that the level of nitrogenous equilibrium varies in different individuals and in the same individual from time to time, it follows that the amount of urea excreted must also vary according to the same irregular manner. Any figures, ■therefore, which are supposed to indicate the amount of urea •eliminated, can be of little and uncertain value unless the actual ■state of the individual's health is known, also his body-weight, habits as to exercise, the amount of nitrogenous food ingested, -etc. Only when we are in possession of an accurate knowledge of these several factors can we say whether the urea excretion is or is not normal. Certain figures have been compiled by physiologists to indi- cate the amount of nitrogen ■ivhich should enter into the com- position of the diet and from which we may approximately calculate the amount of urea that should be excreted. By esti- mating this, or still better, by determining the total nitrogen elimination, we can then determine whether or not the indi- vidual is consuming the proper amount of nitrogenous food in his dietary. While such figures may serve as a general guide, UREA. 185 they have mostly been constructed without due regard for the factors above indicated, and should not, therefore, be too im- plicitly relied upon. According to Simon, among the well-to-do classes the elimi- nation of from twenty to twenty-five grams of nitrogen in twenty-four hours is about normal, taking the average body- weight of the individual into consideration. A smaller amount is not infrequently met in persons of sedentary habits who may appear to be in perfect health. Urea in Disea^. — While extensive variations occur in the urea excretion of health, still greater variations from the average standard are noted in disease. But here also should be taken into account the amount of nitrogen ingested in relation to the body-weight. An increased elimination of urea referable to the destruc- tion of organized albumins is frequently observed, but this may in cases be obscured by a deficient nitrogen ingestion un- less the total intake of the latter is not definitely known. It is of interest here to note that in certain diseases of the liver in which there is great destruction in the parenchyma, the amount of urea may be markedly diminished, even when a fairly abundant supply of nitrogen is taken in. This will be found due largely to the interference in urea synthesis, and as a sec- ondary result we find that in these cases a considerable portion of the nitrogen appears in the urine in the form of ammonium salts of paralactic acid (see page 182) and of carbamic acid; in extreme cases as mono-amido-aeids and as leucin and tyrosin. Properties of Urea. — Urea crystallizes in colorless, quadri- lateral, or six-sided prisms with oblique ends, or when rapidly crystallized in delicate white needles which melt at 132° C, but which are probably decomposed at a temperature of 100° C. They contain no water of crystallization, and are permanent in the air, and easily soluble in cold water, in which they form a neutral solution. With nitric acid, urea unites to form urea nitrate, which crystallizes out in octahedral lozenge-shaped or hexagonal platelets. Urea nitrate is readily soluble in distilled water, but is soluble less readily in water acidified with nitric acid. Its formation is frequently observed when urine is exam- ined cold, with nitric acid, for the presence of albumin. On 186 THE URINE. heating, the crystals are decomposed without leaving any residue. Detection of Urea. — 1. To detect urea place a drop or two of the euspected fluid upon a glass slide, and after adding a drop or two of nitric acid, warm gently. If urea is present, after partial evaporation and cooling, the microscope will show the characteristic crystals of urea nitrate. These are either rhombic or hexagonal plates, frequently overlapping like shingles on a roof. Their acute angles measure 82°. 2. Add to the suspected fluid, in a test-tube, a few drops of fresh sodium hypobromite. A rapid evolution of gas will indicate the presence of urea. Since clinical observations are concerned in the total out- put of urea, it becomes necessary to estimate the quantity or percentage from a sample of twenty-four hours' urine. Under normal conditions of average health the percentage of urea is two. The average daily excretion of urea is forty grams or five hundred grains, or about half the weight of the total solids. Quantitative Estimation of Urea. — This quantitative esti- mation is determined by calculation from the observed volume of nitrogen gas evolved from a measured quantity of urine by a process of decomposition. For practical purposes one gram of urea is estimated to furnish 37 cuhic centimeters of nitrogen gas. The decomposition of the urea contained in the meas- ured volume of urine is accomplished by means of an alkaline solution of sodium hypobromite (see Appendix for formula of Knop's solution). The test is best conducted in a Doremus- Hinds ureameter (see Fig. 22). The Test. — First pass some urine through the small tube to wet the stop-cock, then fill the large tube with Knop's solu- tion, and the smaller side tube with urine exactly up to the cubic centimeter mark. Now, drop by drop, allow exactly 1 cubic centimeter of urine to pass into the reagent in the large tube. When the bubbles of gas (nitrogen) cease to rise, the fraction of a gram of urea may be read directly from the grad- uated scale on the larger tube. Example. — Suppose that after admitting exactly 1 cubic centimeter of urine the level of the fluid stands at the 0.018 URIC ACID. 187 mark. Then the 1 cubic centimeter of the sample contained 0.018 gram of urea. To obtain the total amount of urea ex- creted in twenty-four hours, it is only necessary to multiply this figure by the number of cubic centimeters in twenty-four hours' urine. URIC ACID. General Considerations. — f ric acid, like urea, is nitrogen- ous. The normal proportion of uric acid to urea is as 1 : 45. In health it exists in solution as sodium and as potassiimi urate. A healthy adult excretes 0.8 to 1.0 gram of uric acid in the course of twenty-four hours. The amount increases physiologically Fig. 22.— Various Forms of Ureameters. with increased ingestion of food, and pathologically with in- creased nitrogen metabolism in about the same proportions as urea. The amount of uric acid in urine varies directly with the specific gravity, so that the last two figures of the specific gravity (calculated to four places), multiplied by two, give approxi- mately the number of centigrams of uric acid in the litre. The daily excretion of uric acid is increased in fevers and in leu- kemia. The relation of the elimination of uric acid to attacks of gout and the so-called uric acid diathesis, is still an unsettled question. Of necessity the elimination of uric acid is increased by the ingestion of uric acid and other purin bodies, as well as by foods rich in nuclein (rich in cells). Properties of Ilric Acid. — ITric acid is practically insoluble in cold water, requiring 18,000 parts of water to dissolve 1 part 188 THE URINE. of uric acid. It is freely soluble in alkalies and in solutions of the carbonates. Pure uric acid crystals are colorless, transparent platelets, the angles of which are frequently irregular and rounded off. Such crystals are occasionally seen in freshly voided urines, but more commonly they are found in the form of brownish-yellow, whetstone-shaped crystals, occurring singly or in groups (see Plate IX, a and &). Uric acid once deposited remains undissolved in acid urine. Owing to the inclusion of the urinary pigments in the crystals, these, when present in appreciable quantity, are spoken of as "brick-dust." Microscopic Appearaoice. — The reddish specks observed by. the naked eye are found, upon microscopic examination, to be various modifications of rhombic prisms (see Plate IX, c, d, e and f) . The simpler forms have some resemblance to the con- ventional lozenge or whetstone. Significance. — If these crystals deposit in appreciable quan- tities soon after micturition, it may be considered as a sign of impending gout or gravel formation. Such a deposit, however, is not necessarily evidence that the elimination of uric acid is excessive. Isolation of Uric Acid. — To 200 cubic centimeters of urine add 10 cubic centimeters of HCl, and let stand for twenty-four hours; the uric acid will then have settled to the bottom of the container, from which it may be collected by decanting, filtering, and finally washing in cold water. Oualitative Tests for Uric Acid. — Mukexid Test : Put the solution supposed to contain uric acid or urates in a porcelain dish, add a drop of nitric acid and evaporate to dryness. After cooling, allow a drop or two of ammonia water to come in contact with the residue. The. presence of uric acid or urates will be shown by bright blue or violet (murexid) color. Schiff's Test.- — Having a residue prepared as above, or crystals supposed to be uric acid, dissolve in a test-tube with the aid of a solution of sodium carbonate. Moisten some filter-paper with a 10-per-cent. solution of silver nitrate. Into the center of this paper allow a few drops of the uric acid sodium carbonate solution to fall. In the presence of uric acid the silver nitrate will be reduced to black metallic silver. Approximate Quantitative Determination. — According to THE URATES. 189 Gubler^ the amount of uric acid in urine may be approximately determined by stratifying the specimen of urine to be tested upon a layer of nitric acid contained in a test-tube, so that the volume of urine to the volume of nitric acid is as 3:2. After a short interval uric acid crystals will separate out as a cloudy white ring at the line of junction of the two fluids. If the amount of uric acid in the specimen is increased, the precipita- tion will be plain in five minutes or less. If diminished it will not appear until later. This determination is of value only when the daily excretion of urine is approximately normal, and if it is diminished in amount it should be diluted with water up to the average amount before applying the test. Obviously the conclusions derived from such a method should be given very slight weight clinically, and then only when the estima- tion is made with a part of the twenty-four hours' collection, properly diluted as mentioned above. Albumin must first be removed by slight acidulation with dilute acetic acid, boiling and filtering, after which the test should be applied when the urine has cooled to room-temperature. THE PURIN BASES IN THE URINE. These comprise xanthin, hypoxanthin, heteroxanthin, para- xanthin, guanin, and adenin. These bases are normally present in the urine and comprise approximately one-tenth as much as the normal uric acid. The amount of purin bases normally found in the urine in twenty-four hours varies between 0.028 and 0.058 gram. There is at present no practical clinical method of estimating them. THE URATES. General Considerations. — When the urine cools the urates may settle to the bottom of the container as a cloudy reddish- yellow precipitate. This is most likely to occur when the urine is scanty, concentrated, and highly acid. This condition often obtains in fevers, and in congestion or inflammation of the kid- ^Laquer: Schmidt's Jarhbucher. Vol. ccxkxvI, No. 10. 190 THE ITRINE. neyg. Tin.' sediment tlien presents a faii-ly characteristic appear- ance, being ela^y-eolored, reddisli-yellow, or rose-red. It may adliore to tlie walls of the \'essel as a line, reddish coating. The ordinary uratic sediment consists of a mixture of the urates of sodium, potassium, calcium, nuignesium, and ammo- nium. Sodium urate predominates. With the exception of ammonium urate, these urates only appear in acid urine. Uratic pediments often contain a few crystals of uric acid which have been formed during the double rearrangement of molecules which resulted in the precipitation of the urates. If Fig. 23.— Urate of Soda and Crystals of Ueic Acid (a), Oxalate OF Lime (o), and Cvstin (<). ■ 35U. urine containing a uratic sediment is decomposed by amnio- niacal fermentation, tJie sediment will be changed to acid-am- juoninm urate, and this latter is the only urate sediment which occurs in alkaline urine. If freshly \oided urine is kept in a cool place, the precipi- tation of urates will occur rapidly. This same precipitation occurs at a higher temperature if the amount of urates is in excess of normal or if the urinary acidity is decreased. This ]ireciiiitate is readily distinguished from ])hosphates by its prompt disappeaj-ance upon the application of heat. Qualitative Tests.—!. TTalf fill a test-tube with turbid urine and ajiply lieat to the upper part. If the turbidity is due to the PLATE VIII. Urjc Acid Crystals with Amorphous Urates. / 450. (After Peyer. ) T CREATININ. 191 presence of urates, the heated portion of the urine immdiately becomes clear. 2. To some urine in a test-tube add some liquor potassii, when the turbidity due to urates promptly disappears. Microscopic Appearance of Urates. — The uratie deposit is composed of fine, somewhat regular granules, usually occurring in groups; sometimes the granules show spiny projections. Ammonium urate occurs only in alkaline urine, and is generally accompanied by a copious precipitation of triple phosphates. Ammonium urate appears as opaque brownish-red spherules with or without projecting spines. (See Fig. 23.) Significance. — The excess of urates is of no special im- portance ; they are increased in most conditions, accompanied by fever, and in many disturbances of metabolism. HIPPURIC ACID. Hippuric acid, in combination with alkaline bases, is a nor- mal constituent of the urine. The average quantity eliminated in twenty-four hours is one gram. This amount may be in- creased by exercise, by a vegetable diet, and by ingestion of ben- zoic acid. In cases where the total excretion of urine is gi-eatly diminished, hippuric will be spontaneously thrown out of solu- tion. This is, however, a rare occurrence, because hippuric acid is readily soluble in water. Microscopic Appearance. — The crystals are characteristic rhombic prisms, resembling in a measure the coffin-lid crystals of triple phosphates. Hippuric acid crystals may, however, be distinguished by the fact that they are precipitated in acid urine only, and also because they do not dissolve on the addi- tion of acetic acid. Phosphates, on the other hand, are precipi- tated only in neutral or alkaline urine, and are readily soluble in dilute acetic acid. CREATININ. This normal urinary constituent is present in the twenty- four hours' specimen to the amount of one gram. Creatinin is derived from the creatin of muscle. It is distinguished in the ctrine by its union with mercuric chlorid, with which salt it 192 THE URINE. forms insoluble compounds. Similar characteristic compounds are formed with zinc chlorid and silver nitrate. The zinc chlo- rid combination has a characteristic appearance, by which this substance may be recognized. Under the microscope the zinc chlorid combination of creatinin appears as minute needles arranged in balls and rosettes. Creatinin reduces alkaline copper solutions, and therefore affects, in a slight degree, the accuracy of the quantitative sugar estimations which depend upon the reducing power of sugar-containing urine. OXALIC ACID. Oxalic acid is normally present in the urine only in very small amounts and it appears in combination with calcium as cal- cium oxalate. The normal amount of this substance in a urine of normal volume is held in solution by the acid sodium phosphate. Since oxalic acid requires 500,000 parts of water to effect solu- tion of 1 part calcium oxalate, even the slightest variation in the normal relation results in its appearance as a deposit. Arti- ficially the crystals may be thrown down by carefully neutral- izing the specimen with dilute ammonia water, or by simply allowing the urine to stand for a time exposed to the air. The sediment of calcium oxalate is usually so scant that it is only recognized by the aid of the microscope. The more rapid the formation of these crystals in the urine after voiding, the smaller will the individual crystals appear ; this will, in a way, indicate whether or not the presence of the crystals is due to a pathologic increase in the oxalic a«id content, or merely to a change of reaction (loss of acidity) from standing. The typical cal- cium oxalate crystal is a perfect octahedron without color, in- soluble in acetic acid, but freely so in hydrochloric acid. (Plate X, a and b.) The presence of crystals of the oxalates in the urine does not necessarily imply a pathologic increase in the total quantity of oxalic acid, since an increase in oxalic acid elimination can only be determined by a careful quantitative estimation from the twenty-four hours' specimen. It is probably that "oxaluria" is often diagnosed on insufficient grounds; a too hasty assump- tion, based on the finding of a few crystals, should be guarded against. LEUCIN AND TYROSIN. 193 Microscopic Appearance. — Oxalates are recognized in one of two forms: perfect octahedra, or in hour-glass and dumb- bell forms. Si^ificance. — These deposits sometimes follow the eating of stewed rhubarb or pther acid fruits containing oxalates. Usually their existence in freshly voided urine indicates imper- fect oxidation of retarded metabolism within the economy. CYSTIN. This substance, in exceedingly small amounts, is a nor- mai constituent of urine. It is almost insoluble in water, and in the very rare cases where it is present in increased amount, it is deposited. To the naked eye the deposit is abun- dant and somewhat resembles that of urates. It is, however, not dissolved by heat or by the vegetable acids, but is readily dis- solved in ammonia water. The ammoniacal solution exposed on a slide and examined under the microscope, slowly develops crystals in the form of hexagonal plates. (Plate XI, d.) Iodo- form, which has found its way into the urine from surgical dressings, may be mistaken for cystin. Tests. — When urine containing cystin undergoes decompo- sition, it develops the odor of hydrogen sulphide. When boiled with a solution of lead oxid in sodium hydrate, black lead sul- phid is formed. LEUCIN AND TYROSIN. These are present in the urine in cases of. acute yellow atrophy of the liver, typhoid fever, and phosphorus poisoning. (Plate XI, e and/.) To examine for these substances, the urine must be concen- trated on a water-bath, and the cooled liquid examined under the microscope. Microscopic Appearance. — Leucin appears as greenish-yel- low spheres with concentric markings or radiating spines. Tyro- sin as feathery sheaves, which resemble the frayed end of a rope. 194 THE URINE. PART II. ABNORMAL CONSTITUENTS OF THE URINE. ALBUMIN. General Considerations. — Albumin is probably present in minute quantities in all urine, since urine always contains a variable number of cellular elements derived from the urinary tract. Thus, even under normal conditions, by means of suffi- ciently delicate tests, we can always demonstrate the presence of albumin. This amount is, however, so small that it returns a negative result with the tests commonly employed in the clin- ical laboratory for the detection of albumin in urine. There- fore the presence of demonstrable amounts of albumin, b}- the tests now to be described, must be considered as variations from the normal. The "normal" albumin is nucleo-aTbumin, and its clinical significance is slight, even when present in an appre- ciable quantity. Several other albumins are found in the urine, of which serum-albumin and serum-globulin are of the greatest clinical importance, and the term albuminuria is understood to indi- cate the presence of one or the other, more often of both, in the urine. Other albumins which may be found in the urine, be- sides those referred to above, are albumose and fibrin. The diagnostic and clinical importance of albumosuria have not yet been determined. Traces have been found in many of the infectious fevers. A large albumosuria may be significant of multiple myelomata, since in this affection large amounts are frequently observed. It has also been found in osteomalacia and in nephritis. Fibrin, when present, indicates the direct entrance of blood-plasma into the urinary tract. It must be distinguished from blood-clots, which come under the head of hematuria. Causes of Transient Albuminurias. — Physiologic oh Func- tional Albuminuria: The presence of easily demonstrable amounts of albumin can hardly be considered normal under any circumstances. However, the presence of a functional albu- ABNORMAL CONSTITUENTS. 195 minuria is recognized by . some authorities. An albuminuria does, however, occur, in which the organic change in the kidney, if there be any, is so slight and evanescent as to be unimportant. This form of albuminuria, compared with that caused by severe and permanent kidney change, may, with propriety, be termed functional, but never, physiologic. Functional. — Albuminuria may occur after violent exer- cise, after a cold bath, after severe mental strain, and after over- eating of proteid food-stuffs, particularly eggs. Whether or not the disturbance giving rise to the albumin- uria is insignificant and transient (renal hyperemia or anemia), or partakes of the gradual and progressive variety due to perma- nent alteration in kidney structure, to which the term chronic nephritis is applied, must be determined by the history, symp- toms, and progress of the case. Fevers. — Here the occurrence of albumin in the urine is, in all probability, dependent largely upon the intensity of the infective process, causing irritation of the kidney epithelium, incident to the passage of toxic substances circulating in the; blood, through the glomeruli and tubules of the kidneys; the inflammatory process so induced being indicated by the amount and duration of the albuminuria. Specific Blood-Changes. — The nature of the changes in the blood occurring in scurvy, peliosis, purpura, hemophilia, and pernicious anemia, is obscure in relation to the production of albuminuria. Foreign Substances in the Blood. — This form of albu- minuria is seen after the excessive ingestion of lead, mercury, ether, chloroform, etc. These probably produce the albuminuria by .irritation of the kidney through efforts of that organ at elimination of the substances themselves, or their oxidation products. CiECULATOET DISTURBANCES. — Here the passive congestion occurring in cardio-vascular and pulmonary diseases directly affects the permeability of the renal epithelium. N"eurotic. — ^This term applies to albuminuria occurring during or immediately succeeding attacks of apoplexy, migraine, tetanus, delirium tremens, and certain head injuries. It is prob- ably toxic in origin. 196 THE URINE. Extea-Eenal. — The presence of blood, pus, chyle or lymph, which has entered the urinary tract in appreciable quan- tities, will cause albumin to appear in the urine. Eecently attention has been called to a form of albuminuria resulting from abnormalities in the function of the genital tract. This form comes and goes irregularly, and does not show the pres- ence of spermatozoa.^ In the majority of the above conditions the quantity of albumin is small and varies from day to day. Under these cir- cumstances the relative clinical importance of the finding will be determined by microscopic examination of the urine, aided by the history and course of the disease in which it occurs. These small and transient albuminurias are of considerable importance from the standpoint of life insurance, since too much importance is frequently placed on the finding of a trace of albumin, particularly in a single specimen, which is usually voided succeeding a full meal. ftualitative Tests. for Albumin. — In detailing the following methods for determining the presence of albumin in the urine, no attempt will be made to offer a great variety of tests. The principle involved in the majority is the same, and depends on the coagulation of albumin in the specimen by heat or by chem- ical reagents. Before proceeding to make the tests, the urine must be filtered or centrifugated to remove the turbidity. If this is due to large numbers of bacteria it cannot be entirely removed by this process. The persistence of a bacterial turbidity will inter- fere with the delicacy of the tests. 1. Boiling and Acetic Acid. — This is the best and sim- plest test for routine laboratory work. If albumin is found it is well to corroborate the finding by another method. The urine should not be alkaline in reaction. J'he Test. — Fill a narrow test-tube almost to the top with clear urine, boil the upper third, being careful to avoid too much agitation. In the presence of albumin, or the neutral earthy phosphates, or of both, a white cloud will appear in the heated area. Now, upon the addition from a pipette of a few drops of 2 William Glenn Young : New York Med. Jour.. Jan, 5. 1907. ALBUMIN. 197 10 per cent, acetic acid, the cloud, if due to phosphates, will vanish, while if caused by albumin it will remain and will per- haps be increased in density. The ^chief advantages of this method are its simplicity and delicacy, and the fact that it allows a comparison between the treated and untreated urine in the same tube. The test is most satisfactory when performed in strong daylight with the tube held close to a dull-black back- ground. Caution. — At times the addition of more acid to a urine already acid will convert the albumin into an acid albuminate, which is not coagulable by heat. To overcome this the acetic acid should always be dilute (10 per cent.), and should never be added to acid urine before boiling, and then only in suffi- cient quantity to produce the desired reaction. 2. NiTKid Acid by Contact. — ^This test is best performed with the Meeker albumin tube. . The Test (with the Meeker tube). — Allow about one-half inch of warm, clear urine to enter the tube through the small curved tip. Place the forefinger over the upper end of the tube to retain the urine, and dry with care the outside of the tube; plunge it immediately into nitric acid of sufficient depth to allow the acid to flow in and elevate the urine above it. If this is done with care the line of demarkation between the urine and the acid will be very sharp, and the presence of albumin will be shown by a white line at the plane of contact. Five minutes should be allowed for the appearance of the reaction, the delicacy of which is increased if both the urine and the acid are pre- viously warmed to about 60° C. (a temperature too high to be endured by the hand for more than a few seconds). Sources of Error. — If a cloud appears at the line of con- tact in a urine known to contain an excess of urates, this source of error can be eliminated by diluting the urine with one or two volumes of water, and repeating the test. Nitric acid precipitates certain resins which appear in the urine after it has been administered for medicinal purposes. If these cannot be excluded another test should be employed. If an excess of phosphates exist they may be held in solution by the addition of a few drops of a lO-per-cent. solution of acetic acid. 198 THE URINE. Nucleo-albumin may be held in solution by the addition of one-sixth volume of a saturated solution of NaCl, and then pro- ceeding as before. Puedy's Test. — Mix a half inch of Purdy's reagent (see Appendix) with four inches of urine in a six-inch test-tube; if a whitish cloud appears either at once or after standing, the urine contains albumin. Tanhet's Test. — Acidify a quantity of urine with dilute acetic acid; if mucin produces a cloud, filter and to the filtrate add about 5 cubic centimeters of alcohol and heat slightly. Now substitute this prepared urine for the urine used in the description of the nitric acid contact-test, using Tanret's reagent in place of the nitric acid (see Appendix) ; the pres- ence of a white line at the levfel of contact denotes albumin. Quantitative Estimation of Albumin. — Method of Esbaoh : For clinical purposes as a' ready means of comparing the albu- min content in a given case or a number of cases, this method will be found applicable and convenient. The amount of gravitated precipitate should never be con- founded with the actual percentage of albumin as determined by accurate weighing methods, which rarely amounts to more than 4 or 5 per cent., while by the method of Esbach the moist precip- itate may exceed half of the bulk of the fluid in the tube. The Test. — Fill the Esbach albuminometer tube (see S'ig. 24) with urine to the line marked "U," and fill with Esbach reagent (for preparation see Appendix) to the line marked "R." The tube is then closed with a rubber stopper, and the liquid thoroughly mixed by repeated inversion without shaking, and is then set aside in a vertical position for twenty-four hours, when the layer of precipitated proteid will have settled to the bottom. The granulations indicate the grams of proteids in the liter. If the amount of proteid is large, it is possible to dilute the urine with two or more parts of distilled water before beginning the test, and then to correct the final reading by the dilution. To insure accuracy the tube ehould stand in a temperature of about 15° C. (60° F.). Very small amounts of albumin less than 0.05 per cent, will not sediment, so cannot be estimated by this method. ALBUMIN. 199 Albumosuria. — Albumose appears in the urine as a part product of proteid metabolism. It is a pre-peptone which ap- pears in the blood and whicii can be produced by artificial diges- tion. Pathologically, its continued and marked presence in the urine usually denotes multiple myelomata. It has also occasionally been noted in syphilis, pneumonia, peritonitis, and cholera. Fig. 24.— Esbach Albujiinometi:r. Appearance of tube at end of 24 hours, when reading should be made. The Test. — Add concentrated nitric acid to the urine, agi- tate, and allow to stand in a cool place. A heavy precipitate will occur, which, upon heating, disappears, only to reappear on cool- ing. At the same time tlie mixture gradually assumes an intense yellow color. Albumin of Bence-Jones. — Tliis sulistance lias been repeat- edly found in cases of multiple myelitis. Its exact nature is 200 THE URINE. not definitely known, but it is generally regarded as an albumose. Its reaction to heat and nitric acid is similar to that of albu- mose. The test for its positive identification is complicated and difficult, and will hardly be required of the clinical laboratory worker. Nucleo-Albumin. — Nucleo-albumiri, beyond a faint trace, will give the same reactions as true blood-albumins. Its pres- ence may be inferred when there is an albumin reaction in a specimen which shows an absence of renal elements with an excess of epithelia of bladder and urethral type. Test. — To dilute acid urine add an excess of acetic acid, stand aside, and gradually a faint cloud will appear. DiFFEEENTiAL Test. — If the preceding test gives a positive reaction, take a fresh portion of the same specimen, and first add one-sixth volume of a saturated sodium chlorid solution, and then some dilute acetic acid. The presence of sodium chlorid will prevent the precipitation of nucleo-albumin in the last test. Fibrin, when present in the urine, usually occurs in suf- ficient amounts to form clots, which are easily distinguishable. When the amount is small it may become necessary to determine the presence of this substance by chemical means. Test for Serum-Globulin. — Add to some urine contained in a small beaker sufficient ammonia water to produce a slightly alkaline reaction; a cloud of phosphates will appear and will settle to the bottom. As soon as a layer of clear urine appears, decant this into a test-tube. Mix this clear urine with an equal quantity of a clear saturated solution of ammonium sulphate. If a flocculent precipitate appears in a few minutes the albumin present is serum-globulin. Test for Serum-Albumin. — Take the final solution ob- tained in the foregoing test and filter it free from, serum-globu- lin. Heat the filtrate to boiling, and add one-tenth volume of strong nitric acid. If a precipitate now appears the urine con- tains serum-albumin. Proteose. — Mix about 50 cubic centimeters of urine con- tained in a beaker with 5 cubic centimeters of strong nitric acid. Heat the solution rapidly to about 80° C. If a pre- cipitate appears, prepare a hot filter-paper by pouring boiling water through it in a funnel. Now filter the hot mixture of GLUCOSE. 201 urine and nitric acid. Add to the clear, hot filtrate its own volume of a clear, saturated solution of sodium chlorid. Allow the mixture to cool. If any precipitate appears in this cooled mixture then proteose was present in the specimen of urine under examination. GLUCOSE. General Considerations. — A trace of sugar can at times be detected in apparently normal urine from healthy subjects by special tests of great delicacy. The ordinary tests employed in clinical medicine, however, do not react to these minute amounts of sugar, hence the findings of glucose by the tests about to be outlined must, in every ease, be considered pathologic. The reducing power of normal urine is, in part, due to the activity of uric acid and ereatinin, and is equal to about a 0.1- per-cent. solution of glucose. Under pathologic conditions the reducing power of urine may be enormously increased, due to the abnormal presence of glucose. This may be present up to as much as fifty grains to the ounce (10 per cent.). The detection of sugar in the urine is based upon a knowl- edge of the following properties of glucose: 1. In hot alkaline solutions, reduces the oxides of copper and bismuth. 2. With brewer's yeast, ferments, forming alcohol and car- bon dioxid. 3. Enters into chemical combination with phenylhydrazin to form characteristic insoluble crystals of glucosazone. 4. Deflects polarized light to the right. In the application of all tests but the fermentation-test, it is necessary to use urine free from albumin. If this be present it must first be removed by the addition of dilute acetic acid and boiling to cause precipitation. This is then filtered out, and the filtrate used in the tests for sugar. Reduction Tests. — Method of Fehling^ (for preparation of reagent see Appendix) : The sample of urine to be tested is first examined carefully for the presence of albumin. If this be 3 The suggrestions for the performance of this test are taken from a communication by C. W. Louis Haolier, M.D., appearing in the Jour. Amer. Med. Asso., page 252, Jan. 25. 1908. 202 THE URINE. found it must be removed by heat and acetic acid, and the filtrate used for the test. About 4 mhic centimeters of freshly pre- pared Fehling's solution are diluted with three parts of water, and brought just to the boiling point in a clean test-tube. Immediately two drops of proteid free urine are added, and the tube shaken vigorously. If the urine contains more than 2 per cent, of sugar, a yellowish-red precipitate immediately appears, changing rapidly to a brownish-red or bright red, which settles out slowly, leaving a greenish-blue supernatant fluid. If the reaction be positive, it is better to roughly dilute the urine with two or four parts of water, and to perform the test again. With the strengths of dextrose from % to 2 per cent., two drops of urine, as stated above, will cause a characteristic pre- cipitation of red suboxid of copper, about which there can be no doubt. With smaller percentages the change may come slowly, but even then heat need not be applied after the addition of the urine. Here little change may be seen in the Fehling's solution by transmitted light, but by reflected light a brownish-red tint is evident, which finally develops into a decided light-red pre- cipitate, gradually settling out and leaving a clear, blue, super- natant fluid. The pentoses, lactose, and even maltose, by this method, do not give the characteristic reaction. Urines showing this reaction always give a reaction with Nylander's solution (see next page). If no change occurs in the Fehling's solution after the addi- tion of the two drops as above, the solution may then be warmed just to the boiling point, and from two to four drops of urine added and the mixture observed. This should be repeated until in all twenty drops of urine have been added. If no change occurs at this point, the urine does not contain dextrose to the extent of 0.5 per cent. Cautions. — The disadvantages of using large amounts of urine (an equal or even one-half volume of the Fehling's solu- tion) are two-fold. In the first place, the strongly alkaline cop- per solution with urine of high specific gravity throws down a more or less voluminous precipitate of cupric phosphate, which takes out the blue color of the solution, and changes it to a dirty, dark green. In the second place, the alkaline solution, acting on the ammonium salts in the urine, liberates sufficient ammonia to GLUCOSE. 203 hold in solution small amounts of the red suboxide of copper, obviously interfering with the detection of' traces of dextrose. Very frequently under these conditions, and especially if the solution has been boiled, even for a few seconds after the addi- tion of the urine, the greenish solution slowly takes on a dirty, yellow-brown color, then become turbid, and, eventually, on standing, there separates out a finely divided opalescent, green- ish-yellow precipitate, which only settles out completely after the tube has been allowed to stand over night. In certain cases this reaction does not begin until a few seconds have elapsed after the addition of the urine. Such a reaction may be termed a pseudo-reaction. Fallacies. — Neglect of the above precautions will result in the occurrence of the pseudo-reaction in about 5 per cent, of nor- mal urines examined. The following substances, if present in the urine in more than normal amounts, will result in partial reduction of Feh- ling's solution. Uric acid, creatinin, hippuric acid, mucin, hypoxanthin, and alkapton. Also the pi-eseuce of the oxidation products of the following drugs : The alkaloids, arsenic, carbolic acid, and hexamethylamin (urotropin). The latter substance does not ordinarily reduce bismuth. Bottger's Bismuth Test. — TJrine containing coagulable proteids must first have them removed by heat and acetic acid. Mix in a clean test-tube equal volumes of urine and liquor po- tassii, and add a few grains of bismuth subnitrate and boil. In the presence of glucose the white powder in the bottom of the tube will gradually become gray, brown, or black. Nylandbe's Bismuth Test. — ISTylander modifies the pre- ceding test by substituting for the subnitrate a special reagent containing bismuth (for reagent see Appendix). Albumin, if present in the urine, must be removed in the usual way. To 10 cubic centimeters of ISTylander's reagent, add 1 cubic centi- meter of urine and bring to the boiling point. A brown or black discoloration of the liquid denotes sugar. Fallacies of bismuth tests. A slight reduction of bismuth may be caused by turpentine, eucalyptus, or the ingestion of large amounts of quinine. Albumin and sulphur compounds in urine produce a black precipitate. 204 THE URINE. Delicacy. — These tests show the presence of glucose in 0.025-per-cent. solution. Tbommee's Test. — To half inch of urine in a test-tube, add one inch of liquor potassii and a few drops of a 10-per-eent. solu- tion of copper sulphate sufficient to cause blue discoloration with slight cloudiness. Heat this mixture to the first signs of boil- ing. If sugar be present the copper will be reduced to yellow- or red-oxid. Slight precipitations, which may occur after pro- longed boiling, are no proof of sugar. Delicacy. — This test, if carefully performed, will demon- strate the presence of sugar in 0.025 per cent, solution. Modifications op Thommer's Test (Simrock). — Mix equal parts of the Simrock reagent (see Appendix) and urine in a test-tube and boil ; the presence of glucose will be indicated by a yellowish or red discoloration and precipitate. Phentlhtdeazin Test. — This test depends upon the pro- duction of characteristic crystals of phenylglucosazone by combi- nation of glucose and phenylhydrazin in hot solution, and their recognition with the aid of a microscope. The Test. — Pill a beaker half full of water and warm upon the wire-gauze over a low Bunsen flame. Place a test-tube, con- taining the urine, which has been acidulated with a few drops of dilute acetic acid, in the beaker. While waiting for this to warm prepare the following solution (this must be made fresh, as it does not keep well) : Weigh out one gram of, phenylhy- drazin hydrochlorid, and two grams of sodium acetate. Mix the salts and dissolve in 10 cubic centimeters of distilled water acidulated with two drops of dilute acetic acid. Filter to clarify. After the water in the beaker has boiled for five minutes, observe the urine in the tube; if it has become turbid, filter. To 10 cubic centimeters of hot urine in a clean test-tube, add 5 cubic centimeters of the filtered reagent, replace in a beaker of boiling water, and continue boiling for one hour. Then cool quickly by immersing the tube in cold water. If sugar was present in the urine a crystalline precipitate will appear. This should be taken up in a pipette and transferred to a microscope slide for exami- nation. The characteristic crystals of phenylglucosazone are fine, faintly yellow needles arranged in the forms of fans, rosettes, and sheaves. QUANTITATIVE ESTIMATION OF GLUCOSE. 205 Fallacy. — Levulose, if present in the urine will produce similar crystals; these can be differentiated by a comparison of the fusing points of the two compounds. Phenylglucosazone fuses at 204° C. Phenyllevulosazone fuses at 150° C. Delicacy. — Glucose forms characteristic crystals when pres-» ent in 0.025 per cent, solution. QUANTITATIVE ESTIMATION OF GLUCOSE. Volumetric Determination by Fehling's. — Measure into a beaker or porcelain dish 10 cubic centimeters of accurately pre- pared Fehling's solution, and 40 cubic centimeters of distilled water. Heat the mixture over wire gauze to boiling. Prepare a dilute solution of suspected urine by adding nine parts of distilled water to one part of urine. This mixture is placed in a burette. N"ow to the boiling solution add the dilute urine, a few drops at a time, from the burette. Continue adding, boil- ing, and stirring until the blue color of the Fehling's sohition completely disappears when viewed by transmitted light. Note accurately the number of cubic centimeters of urine used. Fehling's solution is a standard solution of copper sulphate, 10 cubic centimeters of which is exactly decolorized by 0.05 gram of glucose. Example. — Suppose 8 cubic centimeters of diluted urine were used in decomposing 10 cubic centimeters of Fehling's solution. Then, of undiluted urine, 0.8 cubic centimeter was required. This amount then contained 0.05 gram (10 x 0.005) glucose. To calculate the percentage of sugar in the sample of urine : 0.8 : 0.05 : : 100 : X, which, in this case, "X" equals 6.25 per cent. To calculate the grams of glucose voided in twenty-four hours: If 1500 cubic centimeters of urine were voided in twenty-four hours, then 0.8 : 0.05 : : 1500 : X, which equals 93.75 grams glucose. Caution. — The dilute urine should be added very slowly with frequent boilings and examinations by transmitted light to avoid passing the end reaction. Robert's Differential Density Method. — This method de- pends for its result upon the alteration in density oecuning from the fermentation of saccharine urine. 206 THE URINE. The Test. — Mix thoroughly about 120 cubic centimeters of urine with half a cake of compressed yeast. Take and record accurately the specific gravity of this mixture. Set aside in a warm place (thermostat preferred) for twenty-four hours, and at the expiration of this time again take the specific gravity •and subtract the second reading from the first. Bach degree of the remainder (showing density lost) represents approxi- mately one grain of sugar to the ounce. To obtain the per- centage of sugar in the specimen, multiply the degrees of density lost through fermentation by the factor 0.2J 9. Example. — Specific gravity before fermentation 1041 Specific graviiy after fermentation. 1023 Degrees of density lost 18 This is approximately the grains of glucose in each ounce of urine 18x0.219 equals 3.94^ glucose. This test is easily performed, and is conclusive evidence of the presence of glucose. It is, however, not strictly accurate, and is not to be relied upon when the amount of glucose present is less than 0.5 per cent. FERMENTATION SACCHARIMETER. None of the substances found in the urine, which give the reduction reactions resembling glucose, are susceptible of alco- holic fermentation. This fact establishes the accuracy of the test. The test itself depends upon the fact that when sugar- containing urine is mixed with a quantity of brewer's yeast and kept in a warm place for twenty-four hours, it will ferment, giving off bubbles of CO2, and at the . same time forming alcohol. By measuring the amount of carbon dioxid gas, formed during this fermentation-process, we are enabled to estimate the percentage and amount of sugar contained in the specimen un- der examination. A specially shaped and graduated tube (see Fig. 25) is usually employed in this test. It is known as the Einhorn saecharimeter. The upright tube is graduated from its closed upper end downward from 0.1 to 1.0 per cent. It will not estimate sugar in amounts less than 0.1 per cent., and owing to the varying solubility of the various gases of the urine, depend- FERMENTATION SACCHARIMETEE. 207 ing on differing reactions and the density, it cannot be regarded as absolutely accurate. The Test. — Vrines containing less than 5 per cent, of glucose must be diluted iive times with water. Urines contain- ing more than 5 per cent, must be diluted ten times before pro- ceeding with the test. Fig. 25. — Einhorn Saccharimeter in Use. A shows Gas forrned after Twenty-four Hours from a Diabetic Urine containlDgr 0.27 Per Cent, of Sugar (Dilution 1 to 10). B, Control with Normal Urine. Mix in a large test-tube about 20 cubic centimeters of properly dihited urine with one-twelfth of a cake of fresh, compressed yeast. Completely fill the upright tube of the saccharimeter with this mi.xture, so as to exclude all air from the graduated tube. Fill a second saccharimeter with yeast dissolved in distilled water or with urine known to be free from 208 THE URINE. sugar. These tubes are to be kept in a warm place and allowed to remain undisturbed for from eighteen to twenty- four hours. By this time the sugar-containing urine will be found to have been displaced by gas in the vertical tube. The percentage corresponding to the level of the fluid will represent the percentage of sugar in the diluted urine. This figure mul- tiplied by the dilution, will represent the percentage of sugar in the specimen under examination. Cautions. — The urine must be faintly acid, and must be so diluted that the specific gravity will be less than 1008, and when diluted must contain less than 1 per cent, of glucose. If any gas is liberated by the yeast or fluid in the control-test, this amount must be subtracted from that indicated in the test-mix- ture before computing the percentage. The internal administration of mercuric chlorid, iodoform, salicylic acid, hexamethylamin, quinine, and other antiseptic drugs will iiihibit fermentation, and therefore must be excluded before testing. PURDY'S QUANTITATIVE METHOD. Into a beaker or boiling flask of 350 cubic centimeters capacity put 35 cubic centimeters of Purdy's reagent (see Appendix) and 70 cubic centimeters of distilled water. Boil steadily over a wire gauze, and add urine slowly from a burette until the blue color begins to fade ; now proceed cautiously, and after the addition of each drop wait for a few seconds to see if the end reaction is complete. Continue adding until the solu- tion of the reagent is colorless and transparent. To obtain this result the total amount of urine employed must have contained 0.02 gram of glucose. Example. — Suppose the amount of urine employed to com- pletely decolorize the reagent was 4.5 cubic centimeters, then 4.5 cubic centimeters of urine contained 0.02 gram of glucose; or 1 cubic centimeter of urine contains 0.0044 gram of glucose. This multiplied by 100 will give the percentage of glucose which is equal to 0.44. If the color of the reagent is changed by less than 4 cubic centimeters of urine, it is best to dilute the urine with one or two parts of water, and then multiply by this factor to obtain the final result. CAMMIDGE REACTION. 209 This method is rapid, the technic is simple, and the end- reaction definite and sharp. The reagent is stable. CAMMIDGE REACTION.* This reaction is based upon the presence of certain, at present unknown, substances, which occur in the urine in pan- creatic disease associated with fat necrosis, the detection of which is based upon some special reactions in which phenylhy- drazin hydrochlorid plays an active part. Many observers have followed the methods of Cammidge in the determination of this reaction and its relation to disease of the pancreas. Caminidge himself realizing the non-specifinity of the original A- and B-reactions, lat6r added a third or C- reaction, in an effort to render the findings more conclusive. In spite of this we are as yet unable to place any great confidence in the findings of this test. The results obtained by a number of careful and competent investigators being so at variance that it seems doubtful if this test will ever be established upon a sure and practical foundation. The difficulty may be due in part to the inherent difficulties in the technic itself and to the extreme delicacy of the reaction. The slightest variation in the performance of its various steps often rendering valueless a large amount of tedious and time- consuming work, we must conclude, after a careful analysis of the available, clinical and experimental data pertaining to this reaction and its relation to the diagnosis of pancreatic disease, that it is still in an experimental stage, and that many uncertain and negative results in undoubted lesions of the pancreas make it impossible to place a definite value upon the findings, this together with the difficulty with the test itself removes the Cam- midge reaction as an available clinical procedure from daily use by the clinician. * See " Diseases of Pancreas," Robson and Cammidefe, 1907, for farther information . Also Schumm and Heyler : Munch. Med. Wochen., Feb. 14. 1909 ; Jour. A, M. A,, Aug. 22, 1908 ; J. E. Schmidt : Mitteilungen aus den Grebzgenbieten der Med. und Chir., Jena, July 24, 1909; Speese and Goodman: N. Y. Med. Jour., Aug:. 14, 1909; Cammidge: Lancet, March 19, 1904; Mayo Robson : Lancet, page 773, 1904. 210 THE URINE. POLARIMETRIC METHOD. Because of its ability to turn the ray of polarized light to the right, glucose is called dextrose. The accurate determination of glucose in urine may be made by those possessing polari- scopes. The degree of dextro-rotation can be read on a gradu- ated scale and calculated as percentage or grams of glucose. Since albumin in solution deflects the polarized ray to the right, this must first be removed by acidulating, boiling, and Fig. 26.-roLARiMETER. (A. H. T. Co.) filtering. To make an accurate estimation the urine must be first decolorized by shaking with a piece of lead acetate and filtering. Delicacy. — Instruments vary in delicacy. Usually dis- tinct dextro-rotation may be detected when glucose is present in 0.025 per cent. Fallacy. — JIaltose, which rarely occurs in the urine, if present, will deflect the ray even more than will glucose. (For description of technic see larger works on clinical chemistry or polarimetry.) CLINICAL SIGNIFICANCE OF GLYCOSURIA. The jircsence of glucose in the urine is pathologic. If the amount l)e abundant and persists, associated with copious water- drinking and eating, while the patient is at the same time emaciating, it is indicative of diabetes mellitus. The urine in this condition is usually pale with a fruity odor, and while over- PENTOSURIA. 211 abundant, has a high specific gravity. As much as 10,000 cubic centimeters of urine may be excreted in twenty-four hours, at the same time the specific gravity may be maintained at 1050. Temporary Glycosuria. — The appearance of small amounts of sugar may occur transiently in the urine after excessive inges- tion of sugar; and reducing substances, such as glyeuronic acid, may give a spurious positive reaction. Copper reduction by such substances is not corroborated by either fermentation or the phenylhydrazin tests. Glycosuria also may accompany certain diseases of the brain, spinal cord, and pancreas, or be a transitory accompani- ment of phthisis, pneumonia, cirrhosis, or cholera. LACTOSURIA. Milk sugar occurs occasionally in the urine of nursing women, particularly toward the end of lactation. It will reduce Fehling's and Nylander's solutions, -but returns a negative result with the phenylhydrazin- test. It ferments very slowly. MALTOSURIA. Maltose has been found in the urine of diabetic patient-, and when present reduces copper and ferments with yeast. Its occurrence- is sufficiently rare to be disregarded in the clinical laboratory. PENTOSURIA. Diabetics and morphine habitues occasionally show in the urine traces of pentoses. These will reduce copper, but will not ferment. Newmann's Orcin Test. — This test may be employed to dis- tinguish between the pentoses, the glycuronates, and dextrose. Technic. — Three cubic centimeters of urine are treated with ten drops of a 5-per-cent. alcoholic orcin solution and 10 cubic centimeters of glacial acetic acid. The mixture is now brought to the boiling point and allowed to cool. After cooling concentrated sulphuric acid is added, drop by drop, and shaken until about twenty drops have been added. In the presence of pentoses the color becomes olive-green, glycuronates turn the solution violet, and dextrose gives a carmine color. 212 THj: URINE. ACETONE. Usually in advanced diabetics^ sometimes in fever or in perfect healthy particularly in patients whose diet is rich in pro- teids, the urine may have an ethereal odor, and will give posi- tive reactions to tests for acetone. In the course of a case of diabetes a decline in the percent- age of sugar, accompanied by lowered specific gravity, but with- out corresponding improvement in the general symptoms, may indicate . impending coma. At this time acetone will be found in the urine in increasing amounts, with or without diacetic acid and oxybutyric acid. Ethylene-Diamin-Hydrate Test. — This is the simplest and is probably the most satisfactory for clinical purposes. Test. — Place in a clean test-tube about 5 cubic centimeters of distilled water, and to this add about one grain of sodium, nitro-prusside ; shake to effect soluticii. Add to this an equal amount of suspected urine and thoroughly mix. Gently overlie the mixture with a few drops of a 10-per-cent. solution of ethylene-diamin-hydrate. A pink or ruby-red color at the zone of contact denotes the presence of acetone. A faint white cloud, which appears on the addition of the reagent, does not denote acetone. The solution of sodium nitro-prusside must be freshly prepared. Legal''s Test. — To 5 cubic centimeters of urine in a test- tube, add a few drops of a freshly prepared solution of sodium nitro-prusside and a few drops of sodium hydrate solution. A red color will appear in all urine, which soon changes to yellow. If this is now overlaid with strong acetic acid a change in color at the line of contact from yellow to carmine or purplish-red indicates acetone. DIACETIC ACID. Diacetic acid does not occur in the urine of healthy individ- uals upon an ordinary diet, except possibly in very small quan- tities. It has been observed pathologically, usually in combina- tion with ammonia, in severe forms of diabetes, in fevers, in metabolic disturbances and their coincident autointoxications, in gastric carcinoma and in alcoholism. OXYBUTYRIC ACID. 213 Since acetone is derived from diacetie acid and the condi- tions requisite for the formation of diacetie acid are identical with those for the formation of acetone, the two substances are almost always found in conjunction. If little diacetie acid is formed it is all transformed into acetone, if much is formed then both substances will be found in the urine. Gerhardt's Diacetic Fereic Chloeid Eeaction. — Accord- ing to the customary directions in the text-boolfs for the per- formance of the Gerhardt test for diacetic acid (aceto-acetic acid), on the addition of the ferric chlorid solution to the urine a marlced and obscuring precipitate of phosphates is usually obtained. This may be avoided by the chemist's simple exped- ient of adding the urine drop by drop to 10 to 15 cubic centi- meters of the ferric chlorid solution.^ In this vi^ay it is also possible to get a rough idea of the amount of the aceto-acetic acid present. If this is present in quantity it will give a strong Bordeaux-red coloration with the addition of a very few drops of the urine; otherwise this coloration will not be present until 1 or 3 cubic centimeters of the urine has been added. Sulpho- cyanids, sodium acetate, salicylic acid, antipyrin, thallin and aromatic substances may produce a similar red color. For this reason the presence of diacetic acid should be assumed only after positive results have been obtained by the two following control tests :^ 1. Boil the urine employed for the test and the red color should be very much fainter, because boiling gradually trans- forms diacetic acid into acetone. 2. The urine is acidified with sulphuric acid and then some ether is added. If this is shaken with a diluted solution of ferric chlorid the aqueous layer will turn red. OXYBUTYRIC ACID. This substance cannot be readily detected by chemical means unless a long and difficult preliminary process is resorted to in order to separate it from the other urinary constituents. These methods will be found in larger works on clinical and physio- logic chemistry. 5 P. H. Church : Jour. A. M. A., Oct. 2, 1909. «Sahli: " Diagnostic Methods," 1907. 214 THE URINE. BILE PIGMENTS. General Considerations. — Bile pigments are never found in the urine under normal circumstances. As a rule the freshly voided urine only contains the oxidized derivative, bilirubin. If a cystitis should exist, then subsequent oxidation products, biliverdin, bilifuscin, biliprasin, and bilihumin may also be encountered. Bile-containing urines present a more or less characteristic appearance ; their color may vary from a bright golden-yellow to a dark greenish-brown. On microscopic examination it is usual to find the morphologic elements of the urine stained yellow or reddish-brown. The same color may be imparted to the foam on shaking. Gmellin's Test for Bile-Pigments. — Put some yellow nitric acid in a test-tube and gently overlay it with the suspected urine". In the presence of bile-pigment a play of colors will be observed at the zone of contact. Green will be seen nearest the urine and orange in the upper part of the nitric acid. This test is exceed- ingly sensitive, indicating the presence of bile-pigment in a dilu- tion of 1 to 80,000. Nitric Acid Paper Test. — Moisten a piece of white filter or blotting paper with the suspected urine, and place it on a glazed tile or slab of porcelain. Allow one or two drops of commercial nitric acid to fall into the center of the wet paper. In the presence of bile-pigment concentric rings of blue, violet, green, and yellow will appear. A slight red reaction cannot be considered positive, as it may be produced by other substances than bile-pigment. Import. — When bile is not freely and normally discharged from the bile-passages, the coloring matter from the retained bile is absorbed by the lymphatics, the various body tissues be- come stained with it, and it is partly eliminated in the urine. BILE ACIDS. The presence of bile-acids in the urine has the same clinical significance as the presence of biliary pigment. The tests for the acids are fraught with considerable difficulty, and do not com- pensate in their significance for the time and energy expended. HEMATURIA. 215 It will, therefore, usually be found sufficient to determine the presence of bile-pigments. UROBILIN. General Considerations. — Urobilin is closely allied to the normal urochrome, but is an abnormal product, probably result- ing from the action of reducing agents upon the normal urinary pigment. It is said to be identical with the stercobilin of the feces, and its behavior to chemical reagents can be tested by testing an alcoholic extract of feces. Test for Urobilin. — (a) Mix some urine in a test-tube with an equal amount of a 10-per-cent. solution of zinc acetate. Filter off the precipitate, and the filtrate, in the presence of a demon- strable quantity of urobilin, will show a beautiful greenish fluorescence. With the polariscope the urobilin in this solution gives a distinct absorption spectrum. (b) Fill a clean test-tube three quarters full of urine, and add one drop of strong hydrochloric acid ; to this add about one- sixth volume of amy lie alcohol, and after shaking slowly eight or ten times allow to separate by standing. Pour off the super- natant fluid and add to it thrice its volume of alcohol. Prepare separately a 5-per-eent. solution of zinc chlorid, and add one drop of this to the alcoholic extract of the urine; finally add one drop of ammonium hydroxid. Zinc hydrate will be pre- cipitated, which should be filtered off. In the presence of urobilin the fiKrate will priesent a beautiful green fluorescence. Significance. — Urobilin has been detected in cases of hepatic cirrhosis, malarial anemia, carcinoma, Addison's disease, and pancreatic disease with acholic stools. HEMATURIA. General Considerations. — Blood may gain access to the urinary tract in many ways, and appear in the urine in varying amounts, from the smallest trace, demonstrable only by chem- ical means, to sufficient to produce bloody urine with clots. Blood from the kidney is usually well mixed with the urine, to which it imparts a brownish smoky hue. Under the micro- scope tube-casts of blood-cells may be found. 216 THE URINE. Blood from the ureter' may be well-mixed with the urine, or may appear in characteristic worm-like clots. Blood from the bladder may, or may not, show irregular clots. When recently shed it imparts a bright red color to the urine; it is frequently accompanied by much mucus and large, flat epithelial cells in great numbers. Blood from the prostate, examined by the three-glass test, appears in the first and third glasses only. Blood from the urethra appears in the first glass, and is frequently clotted. Urine may show the presence of blood in the absence of any demonstrable lesion of the genito-urinary tract. It has been noted after the ingestion of strawberries, gooseberries, or a large amount of rhubarb. The blood may be contaminated from menstrual discharge. This possibility should always be borne in mind, and false con- clusions guarded against. Microscopic Appearance. — The most convenient method of demonstrating blood in the urine is by microscopic examination of the centrifugated or sedimentated sample. The corpuscles in ordinary acid urine maintain their characteristic bi-concave shape for a number of days, if decomposition and putrefaction are prevented. If the urine is of high specific gravity, the cells rapidly become crenated. On the other hand, if the urine be alkaline or becomes so after voiding, the corpuscles will appear swollen, shriveled, or shadowy. Urine containing more than a trace of blood is albuminous. Test for Occult Blood in the Urine. — To 10 cubic centi- meters of urine in a large test-tube, add about twice as much ether and agitate thoroughly by pouring from one test-tube to another several times. To this add a few grains of powdered gum guaiac, and again agitate. Next add 5 cubic centimeters of glacial acetic acid (99.4 per cent.) and again agitate; allow this to settle and then pour off the supernatant liquid and divide equally between two test-tubes; set one aside for a con- trol, and to the other add about 2 cubic centimeters of fresh hydrogen dioxid from a pipette, making an effort to have it settle to the bottom as a distinct layer. If a bluish discolora- PYURIA. 217 tion appears either at the zone of contact or throughout the mixture, the presence of blood is demonstrated. HEMOGLOBINURIA. This term signifies the presence of hemoglobin in the urine free from blood-corpuscles. Besides the foregoing test it may be demonstrated by means of the spectroscope. Faintly-acid urine containing traces of hemoglobin will give two character- istic absorption bands of oxyhemoglobin. By the addition of a minute quantity of ammonium sulphid, the spectrum is changed to that of reduced hemoglobin. Hemoglobinuria occurs in a variety of conditions : scurvy, .pyemia, purpura, typhus fever, poisoning from arsenic, phos- phorus, carbolic acid, chloral, and potassium chlorate. There has also been noted a periodic form of obscure origin. PYURIA. Pus being an albuminous fluid, will cause urine containing more than a minute quantity to respond to the tests for albu- min. Urine containing much pus is turbid, and deposits on standing a white or greenish-white sediment which is insoluble in heat and in dilute mineral acids. The addition of hydrogen dioxid produces rapid effervescence. Microscopic examination of pus-containing urine will re- veal the characteristic pus corpuscles. These are spheric, granu- lar, and highly refractile. The nucleus is usually obscured or disintegrated. Donne's Test for Purulent Sediment. — ^To the suspected sediment add a small piece of caustic potash, and stir with a glass rod. Pus will become thick, tough, and gelatinous, while mucus will become flaky and thin. Significance of Pyuria. — The presence of pus in the urine indicates the presence of an inflammatory process in the genito-urinary tract, the location of which can, in some measure, be determined by the character of the associated epithelial cells (see page 218). 218 THE URINE. EPITHELIA The epithelial cells found in urine may come from any part of the genito-urinary tract. Their forms vary greatly, and with a knowledge of the characteristic cells belonging to the different regions, it is possible, with more or less certainty, to determine their origin by their appearance. The puss cell may be taken as the standard of size. Cells from the tubules of the kidney are round and about one-third larger than a pus cell. From the pelvis of the kidney, twice the size of a pus cell and cuboidal or pear-shaped. Prom the ureter, round and slightly smaller than from the pelvis. From the bladder, flat and square. These are the largest cells encountered, except those from the vagina. From the urethra, smaller than from the bladder ; they may be cuboidal or columnar. All epithelial cells are granular and .contain a relatively small nucleus. TUBE CASTS. General Considerations. — In the presence of albuminuria or hematuria, microscopic examination of the urinary sediment will, as a rule, reveal the presence of tube casts. Occasionally some varieties of casts will be found in urine which show neither albumin or blood. The Sediment. — In order to obtain a sediment for micro- scopic examination, some method of precipitating and of con- centrating this precipitate is necessary for a conclusive exami- nation. A centrifuge is the most rapid as well as the safest method to employ. This process only requires from 15 to 30 cubic centimeters of urine, and can be accomplished in a few minutes. After centrifugation a part of the supernatant urine is poured oif and then, with the aid of a pipette with a small point, a drop or two is drawn up and transferred to a clean microscope slide for examination. The method of employing a conical glass may be used if a centrifuge is not at hand. Sedimentation by this means re- quires a number of hours, and, if care is not taken to prevent TUBE CASTS. 219 bacterial contamination and if the specimen is not kept in a cool place, the specimen may decompose and the morphologic ele- ments be destroyed. The sedimentation glass is valuable for roughly estimating the amount of gross sedimentation in phosphaturia, pyuria, etc. (Fig. 29.) Fig. 27.— "Laege White Kid- ney." X 350. h. Hyaline oast. g. Spiral cast. w. Waxy cast. /. Fat-granule cast with: n, Jat needles. Still finer needles oi this type upon the neighboring fat-granule spherule. k. Fat-granule cell. I, Leukocyte, s, Vaginal epithelium. (, Fat- droplets. Fig. 28.— Chbonio Bbight's Dis- ease (Chronic Parenchym- atous AND Interstitial Nephritis). X 360. h, g, e, w. Hyaline, granular, ephithelial, and waxy casts, ep, Renal epithelium, vep, Quite uni- formly fatty renal epithelium. Varieties of Casts. — Hyaline casts are almost transparent and appear as ground glass. They have a delicate but definite outline, and are quite friable. Strong illumination of the field may obscure them entirely, as they are very delicate in outline and structure. They are best seen with the plane side of the reflector, and side illumination which should not be too bright. The particular significance of hyaline casts is not yet posi- 220 THE URINE. tively settlelormaI variations. 3.00 to 5.00 per cent. 6.00 to 7.00 1.00 to 2.25 0.18 to 0.25 89.82 to 85.50 100.00 to 100.00 DETERMINATION OF THE FAT. 241 The composition and food value of human milk are im- paired when the mother is unhealthy. It is aifected injuriously when there exists undue emotional excitement. It will contain an appreciable amount of certain medicines ingested by the mother, which may affect the infant. Examination of the Milk. — The exact determination of the composition of human milk is only to be determined by a com- plete chemical analysis. There are, however, many variations from the normal which the physician may readily ascertain for himself by simple methods of examination. The Quantity. — This may be determined roughly by using the breast-pump, although this is not reliable for many reasons. With sufficiently sensitive scales the physician may, by weigh- ing the infant before and immediately after nursing, determine whether it is getting only one or two, or four or five ounces. The average daily quantity secreted by woman is one liter or two pints. The Eeaction. — Test by litmus paper. The reaction should he, alkaline or amphoteric, but never acid when freshly drawn. A spontaneous change occurs after milk has stood for some time in a warm place ; it coagulates or sours, and becomes acid. This is due to the change of milk-sugar (lactose) into lactic acid. This occurs through the agency of the bacterium lactis. The casein normally is held in solution by the alkaline phos- phates, the acid changes the reaction, and hence causes the casein to be precipitated. Specific Gravity. — This may be taken by a small hydro- meter which is graduated from 1010 to 1040. The specific gravity is lowered by fat, but increased by the other solids. MiCEOSCOPiCALLY, milk is found to be composed of minute brilliant, oil globules encased in a thin envelope of casein. Immediately after delivery the milk is relatively poor in casein, but rich in fatty matter, which exists in considerable amount in the form of colostrum riiasses. The microscope also reveals the presence of colostrum corpuscles, blood, pus, epithelium, and granular masses. Colostrum corpuscles are abnormal after the twelfth day ; blood and pus are always abnormal. Determination of the Fat, — The simplest method is by 242 HUMAN MILK. means of the cream gauge. This consists of a graduated test- tube with a foot, and a ground-glass neck and stopper. The tube is filled to the zero mark with freshly-drawn milk and allowed to stand at room-temperature for twenty-four hours, when the percentage of cream is read off directly from the grad- uated scale. The relation of cream to fat is approximately five to three. Thus 5 per cent, cream equals 3 per cent, fat, etc. Cestteifuge Method. — The use of a specially graduated centrifuge tube is more accurate. These tubes may be used in the ordinary centrifuge for urine. Only 6 cubic centimeters of milk are required for this test. If carefully conducted the test' is nearly as accurate as the chemical analysis. It gives results accurate to within one-fifth of 1 per cent. In the usual apparatus two pipettes are supplied with the centrifuge tubes, one of 5 cubic centimeters capacity, marked milk, the other holding 1 cubic centimeter up to a mark, for introducing the alcoholic solution. To determine the fat by this method, 5 cubic centimeters of the sample is introduced into the tube by means of the pipette marked "milk," 1 cubic centimeter of the alcoholic solu- tion (solution-A) is added, and the tube well shaken. Then, by means of any large pipette, solution-B is added little by little until the tube is filled to the zero mark. It is then placed in the centrifuge and rotated very rapidly for four or five minutes. This will bring the fat to the top in a clear yellowish layer which can be read off in direct percentage by the scale on the neck of the tube. A few drops of water may be added, if necessary, to correct the level of the top of the fluid. If the milk should be richer than 5 per cent, for it will be necessary to dilute the sample by an equal quantity of water, proceed with the test as above, and finally multiply the result by two. Solution-A consists of: — Amylic alcohol .^. 37 parts by volume. Methyl alcohol 13 parts by volume. Hydrochloric acid 50 parts by volume. This solution may be kept for a short while ; if it turns dark it is worthless. Solution-B consists of sulphuric acid, specific gravity 1832. ESTIMATION OF THE PROTEIDS. 243 Determination of the Sugar.^ — The percentage of sugar is nearly constant, so it may be ignored in the usual clinical in- vestigation. Estimation of the Proteids. — There is no simple method of determining the percentage of proteids. If we regard the sugar and salts as constant, or so nearly so as not to effect the specific gravity, we may form an approximate idea of the pro- teids from a knowledge of the specific gravity and the percentage of fat. We may thus determine whether they are greatly in ex- cess or very scanty. The specific gravity then will vary with the proportion of proteids, directly, and inversely with the pro- portion of fat, i.e., high proteids, high specific gravity, low fat, low specific gravity. The application of this principle will be seen by reference to the accompanying table. ^ Composition of Woman's Milk. Specific gravity at 70° F. Cream 24 hrs. Average 1031 7 % Normal variation 1028-1029 8% to 12% Normal variation 1032 5 to S% Abnormal variation. Low (below 1028) High (above 10%) Abnormal variation. Low (below 1028) Low (below 5%) Abnormal variation. High (above 1032) High Abnormal variation. High (above 1032) Low Proteids Calculated. 1.5%. Normal (rich milk). Normal (fair milk). Normal or slightly below. Very low, very poor milk. Very high, very rich milk. Normal or nearly so. • From Holt's "Pediatrics. XV. BACTERIOLOGIC METHODS. By a gradual process of evolution and development the field of clinical medicine has so enlarged its borders that to-day the laboratory worker no longer confines his investigations to examination of the blood, urine, and sputum, etc., but must pos- sess a working knowledge of bacteriology and be familiar with bacteriologic technic to the extent that he may be able to obtain, differentiate, and recognize the commoner pathogenic organ- isms. This section will confine itself to a brief outline and dis- cussion of the essentials of laboratory technic, referring the worker, who would delve further into this fascinating field, to works devoted to bacteriology. The Tubercle Bacillus. — ^This is the most important organ- ism, from a clinical standpoint, at least, which is encountered in the field of clinical medicine. The tubercle bacillus presents great difficulties in the way of artificial cultivation, and the results of such procedures are so unreliable that they are rarely depended upon for purposes of identification, the diagnosis usually being made iipon the greater acid-fast properties of this organism as compared with all others; the final corroboration of the diagnosis being made by animal inoculation, for which purpose the guinea-pig, owing to its great and uniform susceptibility, is commonly employed. The tubercle bacillus is, in the strict sense of the word, a parasite which finds conditions entirely favorable to its growth and reproduction only in the body. On artificial media the bacilli, even when transferred directly from the human body, grows only imperfectly or not at all. For ARTIFICIAL cultivation of the tubercle bacillus, the best results are obtained by the use of some blood-serum medium (see page 267). The finding of tubercle bacilli in the sputum is positive (244) STAINING METHODS. 245 evidence of pulmonary, bronchial or laryngeal tuberculosis. On the contrary, their absence, after carciful search, even of a num- ber of preparations, cannot be considered absolute negative evi- dence. If, after careful search of suspicious particles taken from the sputum of a suspect, whose clinical history and symptoms are strongly suggestive of the disease, the organism is not found, the following devices may be resorted to : — A. The whole amount of a twenty-four hours' specimen of sputum, to which no antiseptic solution has been added, should be placed in a porcelain dish or glass beaker, and stirred with a glass rod until quite thin and diffluent. This should then be stood aside for a few hours to settle, when the lowest portion of the fluid is taken up in a pipette and transferred to a centri- fuge-tube, where it is rotated for from fifteen minutes to half an hour. From the bottom of the centrifuge-tube a small amount of the sediment is transferred to a cover-glass or slide for fixing and staining, B. A considerable quantity of sputum is mixed with an equal quantity of water, and a few drops of a 10-per-cent. solu- tion of sodium hydrate is added. This mixture is then heated until homogeneous, when it is cooled, sedimented, and centri- fuged, and examined as outlined above. Microscopic Appearance. — The tubercle bacillus is a delicate rod usually appearing in stained specimens with a beaded in- ternal structure. It may be straight, but is usually slightly curved in its long axis. Its length is very variable, some being short, others quite long, though never as long threads. Its aver- age length varies between .2 and 5 micro-millimeters, and it is usually very slender. In sputum the organisms may occur singly or in groups of from three to half a dozen or more. Staining Peculiarities. — The recognition of the tubercle ba- cillus depends upon a special method by which they alone are stained. Unstained they cannot be differentiated from the other organisms which may be present. The ordinary methods employed for staining bacteria are not suitable, so that special technic has been devised and is now regularly employed to render their recognition less difficult. 246 BACTERIOLOGIC METHODS. In these methods advantage is taken of the fact that cer- tain substances increase the activity of staining by aniline dyes. With the tubercle bacillus this is accomplished with carbolic acid. Another important point is that these organisms, when once stained, give up their color only with great difficulty, so that agents, which will decolorize all other bacteria in the course of a few minutes, will have no appreciable effect upon the tubercle bacillus. It is upon these two peculiarities that we rely in dif- ferentiating this organism. Differential Diagnosis. — While the peculiar micro-chemical reaction toward staining reagents is unually considered to be unique with the tubercle bacillus, it should be remembered that at least three other species of bacteria, when similarly treated, react in the same way. This fact is particularly important in connection with the microscopic examination of urine and patho- logic secretions from the genito-urinary tract, and from the rec- tum. Here is commonly encountered the smegma iacillus which is the next most important member of the group of acid-fast organisms. Acid-fast bacilli have been found in the sputum and about the teeth and tonsils in a case of non-tubercular dis- ease of the lung.i While these other organisms have the same acid-fast prop- erty as has the tubercle bacillus, they appear so rarely that seri- ous mistakes are not likely to occur. The method of Pappen- heim (see page 247) may be relied upon to overcome this diffi- culty. This method colors the tubercle bacillus red and the smegma bacillus blue. Staining Methods. — Ziehl-Nielsen : Place a few drops of the carbol-fuchsin solution (for preparation of stain see Appendix) upon the fixed cover-glass preparation, and hold over a low Bunsen flame until steam begins to rise. Do not boil. ■Continue the steaming process for from three to five minutes, pour off excess of stain, and wash in water. It is important to prevent the staining reagent from reaching the under surface 'of the cover-glass. If this is permitted the pigment will become dry and burned into the glass, when it will successfully resist all efforts at complete decolorization. Decolorize with acid-alcohol ' Pappenheim : Berlin, klin. Wooh., No. 37, p. 809, 1898. STAINING METHODS. 247 solution (see Appendix) or with 25 per cent, sulphuric acid. De- colorization should be continued until the red color has entirely disappeared from the specimen ; if this process is not thoroughly done the finished slide, so far as its diagnostic value is con- cerned, will be worthless. The specimen is then washed in water, and counterstained for from one to three minutes with a l-per-cent. aqueous solution of methylene-blue, after which it is washed, dried, and mounted for microscopic examination. By this method the tubercle will appear as the characteristic red rods upon a blue background. A field which reveals other red objects has been insufiiciently decolorized and should be dis- carded for a more carefully prepared specimen. Gabbett's. — Flood the dried and fixed specimen with caj"- bol-fuchsin and steam for three minutes; then pass directly to the acid methylene-blue stain (see Appendix) for one minute. Finally wash, blot, dry in the air, and mount. This field, if properly prepared, should have the same appearance as that prepared by the Ziehl-Nielsen method. Pappenheim recommends the following technic : 1. Stain in carbol-fuchsin by steaming near the boiling point for three or four minutes. 2. Pour off the excess of carbol-fuchsin and treat without washing with Pappenheim's solution (see Appen- dix), pouring it slowly three or four times over the preparation, and allow it to drain off. 3. Wash in water, dry, and mount. Duration of entire procedure from three to five minutes. Czaplew^sky's Method.2 — This method employs the fol- lowing solutions: (a) One gram of fuchsin is dissolved in 5 cubic centimeters of liquid carbolic acid in a dish; 50 cubic centimeters of glycerin are then added with constant stirring, and -finally dilute with water to 100 cubic centimeters. The solution is said to keep extremely well, and does not need to be filtered, (l) Ebner's decolorizing solution (see Appendix). Method. — 1. Stain, with the aid of heat, for three or four minutes. 2. Decolorize by treating the specimen alternately with Ebner's fluid and distilled water. 3. Counter-stain with methylene-blue for one minute. 4. Wash, dry and mount in Canada balsam. The chief advantage of this method is said to 2 Hyg. Rundschau, No. 21, 1896. 248 BACTEKIOLOGIC METHODS. be that all acid-resisting bacilli but the tubercle bacillus are decolorized, and therefore cannot be confused with the bacillus in question. MICROCOCCUS LANCEOLATUS. The presence of Frankel's diplococcus in the sputum of patients suffering with croupous pneumonia, is fairly constant, so that its demonstration is of considerable diagnostic impor- tance. They appear as elongated lanceolate cocci, usually ar- ranged in pairs with their bases approximated. They are sur- rounded with a faintly staining capsule which, in dry prepara- tions, does not usually take the stain at all, although the ordi- nary method employing methylene-blue has occasionally, in the author's experience, demonstrated a faint but distinct capsule._ The organism is supposed to be the cause of lobar pneu- monia, but must not be confounded with the other diplococci occurring in the sputum, more especially with Friedlander's bacillus. The latter also possesses a capsule, but has nothing whatever to do with the production of lobar pneumonia, though occasionally they may accidentally be present. Friedlander's organism, when highly magnified, will be found to be a short rod. Cultural characteristics will also serve to differentiate, as will also Gram's staining method, which decolorizes Fried- lander's and stains Frankel's organism. The following modification of Gram's method will be found satisfactory (for preparation of staining reagents see Appendix) : Hold the fixed and dried cover-glass preparation in the forceps and flood with carbol-gentian violet; allow this stain to act for from, three to four minutes. Wash and trans- fer to the iodine-potassium-iodide solution for from one to two minutes. Next wash in alcohol until the apparently dirtily- stained film is decolorized. Transfer to absolute alcohol, then to oil of cloves, and finally mount in balsam. By this method the organism appears as a dark-blue or violet diplococcus. Fried- lander's organism will remain unstained. A very useful method for differentiating Frankel's coccus is that devised by W. Wolf. By this method the dry preparation is first stained in aniline water saturated with fuchsin, and then placed for one or two minutes in a dilute watery solution BACILLUS OF INFLUENZA. 249 of methylene-Uue. The cocci will now be found stained blue, the capsule rose-red, and the body of the specimen purplish-red. Method for Staining Capsules. — Prepare the cover- glass smear in the usual way, then without drying flood with glacial acetic acid. At the expiration of one minute pour off the excess of acid, and without washing flood the specimen with aniline water gentian-violet (Koch-Ehrlieh), which should be allowed to act for four minutes, when the excess of stain is poured off and a fresh portion added, which is allowed to act for another two minutes. The cover-glass is now washed in two or three changes of normal saline solution (it may be found necessary to employ a saline solution of 1.5 to 2.0 per cent.), after which it is blotted, dried, and mounted in the usual way. By this method, the capsule will appear as a faintly tinted halo about the diplocoecus. The absence of this cocci practically excludes the diagnosis of lobar pneumonia, although its demon- strated presence is by no means positive evidence in the other direction, because this organism has repeatedly been demon- strated in the sputum and mouth secretions of healthy indi- viduals. BACILLUS OF INFLUENZA OR PFEIFFER'S BACILLUS. A small slender bacillus occurring usually in very great numbers in the nasal secretion of fresh attacks of true influenza, but not found in the ordinary short attacks of prostration accom- panied by coryza, which is at present designated influenza or "grippe," largely for lack of a more definite diagnosis. Mor- phologically, it is a very small rod appearing frequently in pairs ; in parts of the secretion this organism may be found in what is practically a pure culture, occurring both within and without the leukocytes. Their length is usually from two to three times their width, they rarely form chains. The ends of the rods are rounded off, and if imperfectly stained (they stain with diffi- culty) the ends will be more deeply stained than the center, giving an appearance not unlike a diplocoecus with just a sug- gestion of a capsule. According to recent authority this bacillus does not possess a capsule, the deceptive appearance being prob- ably a staining- peculiarity of certain cells. The bacillus is non- motile, and can only be cultivated upon special media contain- 250 BACTERIOLOGIC METHODS. ing hemoglobin. This characteristic will readily serve to dif- ferentiate it from the colon bacillus and other organisms of sim- ilar appearance. An easily made and satisfactory medium is prepared by spreading a little fresh blood upon the surface of ,an ordinary agar slant and inoculating this with the infected material. This organism only develops on artificial media be- tween the temperatures of 26° and 43° C, growing best at body- temperature. Upon the blood-agar slant incubated at 37° C, there will develop minute, transparent, watery colonies that are without structure, somewhat resembling droplets of due. They ^re usually discrete, and show little or no tendency to coalesce. Staining. — One of the best methods of staining is with a -dilute watery solution of Ziehl's carbol-fuchsin (the color of the solution should be pale red). This solution should be al- lowed to act for five minutes. This organism is decolorized by 'Gram's method. A second method of staining is with Loeffler's methylene- .blue (for preparation see Appendix). This stain should be .allowed to act for five minutes. Then wash in water, mount, .and examine for blue organisms. BACILLUS OF DIPHTHERIA. From the grayish white deposit on the fauces of a diph- theritic patient, prepare a series of cultures in the following way : — ■ Have prepared a few tubes of Loeffler's blood-sermn. Pass .a stout platinum needle which has previously been sterilized into ihe membrane and rub it around there ; then being careful that it touches nothing else, rub it carefully over the surface of two tubes. The tubes are then immediately replugged and placed in the incubator. If the case be one of true diphtheria, the tubes Tvill be ready for examination the following day. The blood-serum mixture is to be preferred to the ordinary plate method because the organism of diphtheria grows better on this medium than upon any other; it is also a difEerential method in a general sense, because other organisms do not grow well on Loeffler's serum, hence a luxuriant growth at the expira- tion of twenty-four hours should always be considered diph- theritic until proven otherwise. GONOCOCCUS OF NEISSER. 251 Appearance. — After twenty-four hours the tubes present a •characteristic appearance. Their surfaces are marked by more or less irregular patches of white or cream-colored growths, which is usually more dense at the center than at the periphery. Staining. — From this culture smears are made upon clean •cover-slips or slides, dried and fixed in the usual way, and then stained with Loeffler's alkaline methylene-blue. There will now be seen, in a typical case, upon microscopic examination, slightly ■curved bacilli of irregular size and outline. In some cases they will be more or less clubbed at one or both ends; sometimes they are spindle shaped or may present curved edges. They are rarely or never regular in outline. Many of these irregular rods are seen to be marked at circumscribed points in which their protoplasm is deeply stained. This irregularity in outline and appearance is the morphologic characteristic of the bacillus of diphtheria. THE GONOCOCCUS OF NEISSER. On microscopic examination the pus from an acute case of gonorrhea will show numerous small bodies, usually arranged in pairs. These cells will be found both within the protoplasm of some of the pus cells. The cells containing the gonococcus are usually crowded with the organisms, though the majority of pus cells do not contain them. This organism, on account of its frequent arrangement in pairs, is often called the diplococcus of gonorrhea. It is always found in gonorrheal pus and often persists into the convalescent stage after the external discharge has disappeared. It is easily detected in the pus of acute in- Tasion, while in the subacute and chronic conditions its detec- tion is often a matter of considerable difficulty. Cultivation. — It does not grow upon the ordinary culture media, and can only be isolated in culture through the employ- ment of special methods. Blood or blood-serum is a necessary ■constituent of all media for the artificial cultivation of this ■organism. Some investigators have been successful in growing it upon other body fluids such as ascites fluid, pleural effusion, and the fluid from ovarian cysts. A useful medium may be p)repared by mixing equal parts of human blood-serum with ordi- nary sterilized nutrient agar-agar. This is accomplished by 252 BACTERIOLOGIC METHODS. liquefying the agar maintaining it at a temperature of 50° C. until after the mixture is made, after which it is allowed to solidify. Distinguishing Characteristics. — 1. It is seen practically always in the form of a diplococcus, having the characteristic biscuit form with the long diameters of the individual cells apposed. 2. In gonorrheal pus some organisms are practically always found within the protoplasm of some of the cells. 3. It stains readily with the ordinary staining reagents, but it is promptly decolorized by Gram's. 4. It fails to develop on the ordinary artificial media (sepa- rating it from the diplococcus intracellularis meningitidis which grows freely). 5. It has no pathogenic properties for the lower animals. Staining. — The ordinary stains are satisfactory, one of the simplest being a 1- or 3-per-cent. aqueous solution of methy- lene-blue which gives a very clear picture. Most important as a differential method is its failure to retain its color when treated by Gram's method. SPECIAL STAINING METHODS. Greun's Method. Method. — Objects are first treated with an aniline water solution of gentian-violet which is made after the formula of Koch-Ehrlich (see Appendix for preparation of stain). After staining in this solution for fifteen to thirty minutes the excess of stain is drained off and the film treated for five minutes with Gram's iodine solution (see Appendix). They are next trans- ferred to alcohol and thoroughly rinsed. If careful examina- tion at this point reveals any violet color in the film, it must again be treated with the iodine solution until all violet color is removed. After a final washing in alcohol the specimen may be mounted and examined or a counter-stain of carmine may first be employed. "Wright's Modification. — Stain for one minute in carbol- gentian-violet (see Appendix). Wash in water from thirty GRAM'S METHOD. 253 to sixty seconds. Lugol's solution is then allowed to act upon the specimen for one to three minutes. Wash and dry. Dif- ferentiate with aniline-xylol (2:1) to which 1.5 per cent, of acetone has been added for one or two minutes. Wash with xylol, dry, and counter-stain with dilute carbol-fuchsin (1:10) for about one minute, during which the specimen should be warmed slightly. The specimen is finally washed, dried, and mounted for examination. The process of decolorizing is only a relative one, some bacteria decolorizing more readily than others, so that much depends upon the intensity of the decolorizing reagent, and also upon the time during which it is allowed to act. The counter- stain method with dilute carbol-fuchsin is a differentiated proc- ess indicating those organisms that have been decolorized. All decolorized organisms by this method take on a red color. This counter-stain is of particular value where pictures of a number of bacteria are made, as in sputum examination. The following organisms retain the violet stain by Gram's methods : — Streptococci. Staphylococci. Bacillus tuberculosis. Bacillus anthracis. Bacillus aerogenes capsulatus. Bacillus diphtherisB. Diplococcus pneumonia3 (Prankel's). Bacillus tetanus. The following organisms are decolorized by Gram's method : — Diplococcus meningitidis intraeellularis. Bacillus pyoeyaneus. Micrococcus gonorrhceffi (Keisser's). Bacillus malignant cedema. Bacillus influenzae. Bacillus typhosus. Bacillus cholerse. Bacillus pneumoniae (Friedlander's). 254 BACTERIOLOGIC METHODS. Loeffler's Method of Staining Flagella. It is essential that the bacteria be evenly and not too nu- merously distributed over the cover-slip. Preparation of Cover-Slip. — The glasses must be perfectly clean. Lay six cover-slips on an even surface, and place in tlie center of each a small drop of distilled water. Prom the mate- rial to be examined, transfer a minute quantity to the drop of water on the first cover-slip; from this one transfer a minute quantity to the second, and so on to the sixth. This insures a varying dilution of the organisms in the different preparation. They are then all spread, dried, and fixed in the usual way. The cover-slip preparations are next warmed in Loeffler's mor- dant (see Appendix). A few drops of this solution is placed upon the preparation which is held over a low Bunsen fl.ame until it begins to steam. It should not be boiled. After steaming for a few moments the mordant is washed off with water and then with alcohol. The bacteria are now stained in the Koch-Bhrlich aniline water- fuehsin solution (see Appendix). When treated in this way various bacteria behave differ- ently, the flagella of some staining readily, others require the addition of an alkali in varying proportion to obtain the best results, others again stain best after the addition of an acid. To meet these conditions an exact 1-per-cent. solution of caustic soda in water must be prepared, and also a solution of sulphuric acid of such strength that 1 cubic centimeter will exactly neutralize 1 cubic centimeter of the alkaline solution. For the different bacteria which have been studied by this method Loeffler recommends that one or the other of these solu- tions be added to the mordant before using, in the following proportions : — Of the Acid Solution. For Spirillum eoneentricum, no addition of either acid or alkali. . For Spirillum cholera Asiaticse, % to 1 drop to 16 cubic centimeters of the mordant. For Spirillum Metchnieovi, 4 drops of acid to 16 cubic centimeters of the mordant. For Spirillum rubrum, G drops of acid to 16 cubic centimeter of the mordant. STERILIZATION. 255. For Bacillus pyoeyaneus, 5 drops of acid to 16 cubic centimeters- of the mordant. Of the Alkaline Solution. For Bacilhis mesentericus vulgaris, 4 drops of alkali to 16 cubic centimeters of mordant. For Bacillus Micrococcus agilis, 20 drops of alkali to 16 cubic centi- meters of mordant. For Bacillus typhosus, 22 drops of alkali to 16 cubic centimeters of mordant. For Bacillus subtilis, 28 to 30 drops of alkali to 16 cubic centi- meters of mordant. For Bacillus malignant edemae, 36 to 37 drops of alkali to 16 cubic- centimeters of mordant. For Bacillus symptomatic anthrax, 35 drops of alkali to 16 cubic centimeters of mordant. The drops used run 22 to the cubic millimeter. STERILIZATION. General Considerations. — Acquaintance with the funda- mental principles of sterilization and of disinfection are abso- lutely necessary to the successful performance of all bacterio- logic investigations. The term sterilizaiUon, as commonly em- ployed, implies the absolute destruction of bacterial life by heat, while the term disinfection is commonly applied to accom- plishing the same end through the agency of chemical sub- stances capable of destroying bacterial life. Strictly speaking, the term sterilization implies the com- plete destruction of the vitality of all micro-organisms that may be present in or on the substance or substances to be sterilized. Such a result can obviously be accomplished by either thermal or chemical means, while disinfection need not, of necessity, de- stroy all living organisms that are present, but only those having the power of infecting or of producing disease, and may or may not, as the case may be, cause complete destruction of bacterial life, as in sterilization. It is therefore possible to accomplish both disinfection and sterilization by either chemical or thermal means. In the laboratory the employment of these different terms depends upon and is governed lay circumstances. It is, of course, essential that all culture media should not only be absolutely 256 BACTERIOLOGIC METHODS. free Irom all bacteria, whether they are pathogenic or not, but also from their spores. In a word, they must be sterile. At the same time it is equally essential that the original chemical com- position and physical properties of the media should remain un- altered by the process. It is self-evident, therefore, that steriliza- tion of such substances by means of chemical agents, is out of the question, for while this method would destroy all bacterial life, it would not only alter the chemical composition of the media, but by becoming inseparably mingled with the media, would, by its continued presence, effectually prevent the growth of bacteria in the material for all time; that is to say, after having performed its function as sterilizer, it would by its con- tinued presence exercise its function as an antiseptic, and render the material useless as a culture medium. Exceptions to this general rule are found in certain volatile substances such as alco- hol and ether which, after having performed their bactericidal powers, may be completely driven off by the application of heat. Sterilization by Heat. — Sterilization by means of high tem- peratures may be accomplished in a variety of ways : 1. By dry sterilization, which is accompanied by subjecting the articles to adequate degrees of heat in a properly constructed oven. 2. By subjecting them to the influence of live steam at 100° C. 3. By subjecting the substances to steam under pressure. When employing steam under pressure, the temperature to which the articles are subjected will depend upon the pressure developed — the greater the pressure the higher the temperature. Sterilization by Dry Heat. — This method has the following disadvantages which limit its applicability : 1. The temperature must be relatively high and the period of exposure long as com- pared to moist heat (steam). 2. The penetration of dry heat into substances to be sterilized is much less thorough than that of steam. 3. Many substances of vegetable and animal origin are rendered valueless by the temperature required for dry ster- ilization. Successful sterilization by dry heat cannot usually be accom- plished by a temperature lower than 150° C, and this tempera- ture must be continued for not less than an hour. In general, it may be said that dry sterilization is only suited for sterilization of such substances as glassware, dishes, flasks, test-tubes, pi- STERILIZATION. 257 pettes, etc-., and for such metal instruments as are not injured by heat. Steam or moist heat sterilization possesses great penetrating power, and is much more rapid and thorough than the above method, and, further, it is far less likely to destroy the material so treated. This method should be employed for sterilizing all culture media, fabrics, cotton, wood, and organic material in general. Aside from the relative applicability of the two methods, their mode of action toward the organisms to be destroyed is very different. The penetrating power of steam renders it far more efl&cacious than dry heat. The spores of several organisms which are destroyed by exposure to steam for a few minutes, resist the destructive action of dry heat at a higher temperature for a longer period of time. The method of applying heat for sterilization depends chiefly upon the character of the substances to be sterilized. The application of dry heat is always continuous, i.e.^ the objects to be sterilized are simply exposed to the proper temperature for the requisite time necessary to destroy all living organisms and their spores which are either in or upon them. With steam, on the other hand, the articles to be sterilized are frequently of such a nature that prolonged sterilization would injure them. For this reason it has been found desirable to subject such ob- jects to the influence of steam intermittently for a number of short periods. The PHiNCiPLE involved in the intermittent method de- pends on the differing powers of resistance to heat displayed by different organisms in different stages of their development. During the life of many bacilli they enter a stage during which their resistance to both chemical and thermal agents is mate- rially increased. This increased power of resistance is possessed by the organisms when they are ia the spore or resting stage. Some spores of certain organisms have been encountered which retain the power of germination after an exposure of more than an hour to the temperature of boiling water. This difference in the thermal heat-point of bacteria and their spores is taken advantage in the process of sterilization known as the fractional or intermittent method. 17 258 BACTERIOLOGIC METHODS. As all culture media depend for their usefulness upon more or less unstable organic compounds, the effort of sterilization is to destroy the organisms in the shortest possible time by expo- sure to least possible amount of heat. This is accomplished by subjecting them to a temperature at a time when the bacteria are in the vegetative or growing stage. In order to develop any existiag spores the media, during the intervals between steriliza- tion, should be kept under such conditions of temperature and moisture as will favor the process of vegetation (room-tempera- ture) . During the first application of heat the mature vegetative forms are destroyed, while certain spores which may be present resist this treatment and survive the temperature. Now the sterilization is discontinued and the media is allowed to remain for a time, usually twenty-four hours at room-temperature. During this time those spores which resisted the first heating have conditions favorable to germination. A second short ex- posure kills this crop of bacilli, when a second rest, followed by a third short exposure, kills the remaining organisms and the media will usually be found sterile. It should be remembered that while all spores which are present are not killed by the first exposure, still their power of germination may be so inhibited by the exposure to 100° C. that their germination is delayed, that they cannot possibly germinate during the twenty-four hours' intervals. Experiment has shown that the fractional process gives the best results when the objects are subjected to the action of live steam (steam at ordinary atmospheric pressure) for fifteen minutes on each of three consecutive days, and that during the intervals the cultures should be maintained at a temperature between 25° and 30° C. The substances thus treated will re- main sterile for an indefinite time, .provided they are not exposed to the re-entrance of micro-organisms. An occasional exception will be noted when, after careful treatment as above outlined, certain species of spore-forming bacteria will not have been entirely destroyed by this method. These are usually of the non-pathogenic group of the so-called soil organisms. Finally it must be bom in mind that this method is only STERILIZATION. 259 applicable to substances capable of presenting conditions favor- able to spore germination, and that dry substances, such as in- struments, apparatus, or organic materials, in which decomposi- tion has set in, where the natural conditions favorable to spore germination are absent should not be treated by this method, but must be subjected to higher temperatures for longer periods of time. Intermittent Sterilization (at low temperature). — The process of intermittent sterilization at comparatively low tem- peratures is based upon the principle outlined above, but differs in the details of its application as follows : 1. It requires a greater number of exposures to accomplish complete sterilization. 2. The temperature at which it is accomplished is not above 68°-70° C. It is employed for sterilization of easily decomposible ma- terials and those which would be rendered useless by the appli- cation of steam at 100° C, but which are unaltered by the tem- perature employed. This method is applicable for sterilization of certain albuminous media where, it is desirable to retain their fluid condition during sterilization and which would be coagulated by exposure to higher temperature. This process requires that the temperature employed should be between 68°-70° C, and that an exposure of one hour should be made each of six consecutive days. During the intervals the material is kept at a temperature between 35°-30° C. to favor germination of any spores that may be present. Direct Steam Method. — Sterilization by means of steam is also accomplished by what is known as the direct or continuous method. By this process both mature organisms and their spores are destroyed by a single exposure to steam at zero pressure — live or steaming steam. The sterilization is accomplished by a single exposure of one hour. Steam-Pressure Method (by Autoclave). — By employing steam under pressure we are able, by increasing the temperature, to materially shorten the time necessary to accomplish complete sterilization. By employing a pressure of approximately one atmosphere (15 pounds) a temperature of about 123° 0. is obtained. This is sufficient to accomplish complete sterilization by one exposure of fifteen minutes. 260 BACTERIOLOG IG METHODS. When this iiietliod is eiiiplnyed it will occasionally be found that the coagidatii]g power of gelatin is reduced, and that it becomes slightly cloudy, wliile in agar-agar a fine, flaky precipi- tate is noticed. For accurate time and temperature exposures this is a \ cry \uicertain method. Oljviously the material is sub- ject to active temperatures during the heating up as well as during the cooling process, besides the actual time during which tlie maximum pressure is maintained. i\lso, if great care is not Fig. 31.— Arnold yiEAM .Sterilizkr. (A. H. T. Co.) observed to prevent premature opening of the autoclave, a sudden, rajiid ebullilion of the fluid occurs which, in the case of test-tulies, tlie media may comjileielv lioil away. AVhiie iliis metliod of sterilization is nof well suited for delicate exiieriuu^uts where a de(iiii(e tiun- exposure to definite tem])erature is of importance, si ill, for general lal)oratory pur- prises, it has mnch io rci-omniend it in the way of a tinu^ saver, and in the cei'taintv willi which sterilization is accom]ilished. TJie a|-iparatus designrd io sterilize under pressui'c is termed an autoclave. THE AUTOCLAVE. 261 Practical Application of the Method. — 1. The Arnold Steam Sterilizer (Fig. 31) : This is probably the best steril- izer for general laboratory purposes, since it is simple and eco- nomic in its operation. The difference between this apparatus and ■ the original sterilizer devised by Koch is that it provides for the condensation of the steam after its escape from the Fig. 32.— Autoclave. (A. H. T. Co.) sterilizing chamber, and returns the water of condensation auto- matically to the reservoir. 2. The Autoclave. — The advantage of this method is so well recognized that its use has practically superseded the inter- mittent method with live steam, except when the temperature developed by the autoclave is sufficient to destroy the materials subjected to it. By this plan sterilization is accomplished in fifteen minutes by exposure to steam under the pressure of one atmosphere. 262 BACTERIOLOGIC METHODS. The autoclave (see Fig. 32) embodies the same principle as the steam sterilizer. It provides for the generation of steam within a chamber capable of being hermetically sealed after the introduction of the substances to be sterilized. The chamber is fitted with a safety-valve arranged for regulating the degree of pressure. A thermometer passes through the wall and enters the chamber, thus allowing the temperature to be followed and the pressure properly regulated. HoT-AiR Sterilizer. — The hot-air sterilizers employed for this work are simply double-walled boxes of Swiedish iron, having a double door and a copper bottom. They are fitted with proper openings in the walls to permit circulation of the heated air. The heat is obtained from the flame of a Bunsen burner applied directly to its bottom. These bottoms are usually constructed of copper, and because they readily burn out, are now made so that they may easily be replaced. Properly constructed sterilizers with removable bottoms may now be obtained and should be sought when purchasing, as they will save much annoyance and not a little expense. Sterilization by this method is accomplished in from a half to one hour at a temperature of from 150°-180° C. CHEMICAL STERILIZATION AND DISINFECTION. It is possible by means of certain chemical substances to destroy all bacteria and their spores, or by the same means to remove all their pathogenic properties; in other words, to dis- infect them. When it is desirable to use chemical disinfection in the laboratory, successful results may be obtained by employing a 33- to 40-per-cent. solution of carbolic acid. Under ordinary circumstances this will accomplish the result in from twenty minutes to half an hour. It is, however, not reliable for the destruction of spores of resistant organisms, such as the spores of the anthrax bacillus. All materials and issues containing infectious organisms should be burned and all other cloths, test-tubes, flasks, dishes, etc., should be boiled in a 2-per-cent. soda (ordinary washing soda) for a half-hour, or should be exposed in the steam ster- ilizer for the same length of time. PREPARATION OF CULTURE MEDIA. 263 Intestinal evacuations are best disinfected with chlorinated lime, which .shonld contain at least 0.35 per cent, of free chlo- rin. This solution should be mixed in equal parts with the material to be disinfected, and then shonld be allowed to stand for one or two hours before being disposed of. Sputum in which tubercle bacilli may be present, as well as vessels containing it, and the eating utensils of , tuberculous patients, should be boiled with a 2-per-cent. soda solution for from a half to one hour, or should be exposed in the steam ster- ilizer for the same period. PREPARATION OF CULTURE MEDIA. Bouillon. — Five hundred grams of freshly chopped, lean beef, free from fat and tendons, are soaked in a liter of water for twenty-four hours, during which time the temperature of the toixture is kept low by surrounding it with ice. At the expiration of this time the mixture should be strained through coarse muslin until a liter of fluid has been recovered. To this now add ten grams of dried peptone and five grams of common table salt. It is then to be rendered neutral or slightly alkaline by the addition of a few drops of a saturated sodium carbonate solution. The flask containing the mixture is then placed either in the steam sterilizer or upon a water-bath and kept at the boiling point until all the albumin has been coagu- lated, and the fluid portion is clear and of a pale-straw color. It is then filtered through a folded filter-paper and finally ster- ilized in the steam sterilizer by the fractional method (see page 259). This is the original method of Koch which has been modified and improved in the following ways: — Neutralization. — Ordinarily this is accomplished by the addition of a saturated solution of sodium carbonate, and the reaction determined by red and blue litmus paper. This sodium carbonate solution is not so good, however, as a strong solution of sodium or potassium hydroxid, because the carbonic acid arising during the process of neutralization with the sodium carbonate frequently produces a temporary acid reaction which later disappears on boiling. Exact titration with an 0.4-per-cent. solution of sodium hydrate, obviates this diSiculty. The process 264 BACTEEIOLOGIC METHODS. is applied only after the bouillon has been deprived of all its coagulable albumin by boiling and has again been reduced to room-temperature. Technic. — First ascertain the exact volume of the fluid. From this sample take exactly 5 or 10 cubic centimeters and add a few drops of a 1-per-cent. alcoholic solution of phenol- phthalein as an indicator. The 0.4-per-cent. alkaline solution is placed in a graduated burette, and the solution to be tested in a porcelain dish or casserole. 'Now add the alkaline solution, drop by drop, until the bouillon turns a faint rose color. A second measured quantity of bouillon is treated in the same manner as a check, and if the amount of sodium hydrate solu- tion required to cause neutralization is the same or only slightly different, a simple calculation will indicate the amount of soda solution required to neutralize the bulk of the medium. Thus, if for 10 cubic centimeters of the bouillon would be required 1.5 cubic centimeters of the 0.4-per-cent. sokition of sodium hydrate, then for the remaining 980 cubic centimeters (original volume 500 cubic centimeters), it would reqiiire 98 times 1.5 cubic centimeters of the soda solution to neutralize the total amount of bouillon, or 147 of the 0.4-per-cent. sodium hydrate solution would have to be added to the 980 cubic centimeters of bouillon to accomplish neutralization. To avoid over-diluting the bouillon by the weak alkaline solution, it is better to employ for this purpose a 4.0-per-cent. solution of NaOH, of which only 14.7 cubic centimeters would be required. Not infrequently the filtered neutralized and sterilized bouillon will be found to contain a fine flocculent precipitate. This may be due either to an excess of alkalinity or to incom- plete precipitation of the albumin. The former may be corrected by the addition of a little dilute acetic or hydrochloric acid, fol- lower by a second boiling, filtering, and sterilization. If due to imperfect precipitation of the albumin this may be corrected by reboiling and filtering. 2. The substitution of prepared meat extract for the fresh extract is now almost universal. Any good stock meat extract will answer the purpose, and should be used in the strength of from two to four grams to the liter of water. Peptone and PREPARATION OF CULTURE MEDIA. 265 sodmm ehlorid are added, as in the original method of prepara- tion. The advantages of the meat extract are a decided shorten- ing of the time required for preparation of the media, and the production of a more uniform material. Nutrient Gelatin. — ^In making nutrient gelatin the bouil- lon is prepared first as outlined above, except that the reaction is corrected only after the gelatin has been completely dissolved. The reaction of the gelatin of the shops is frequently quite acid, entailing the addition of considerable more alkaline solution than required for the bouillon alone. The gelatin is added in sufficient quantity to make a 10- or 12-per-cent. solution. Its complete solution is accomplished either on a water-bath or over the bare flame. In the latter instance it must be constantly stirred to prevent burning the gelatin on the bottom of the pan. When the gelatin is com- pletely dissolved it can readily be filtered through an ordinary folded filter paper in a glass funnel. It not infrequently happens that in spite of the most careful teehnic the filtered gelatin is not perfectly clear; then clarification is required. For this pur- pose the mass must be redissolved, and when the temperature *s between G0° and 70° C. the white of an egg, which has been beaten up with about 50 cubic centimeters of water, is added and thoroughly mixed in. This mixture is then again brought to the boiling point until coagulation of the egg albumin occurs. This process results in large, fiaky coagula of albumin, which it is best not to break up, as fine fiakes of albumin will clog the filter-paper and materially interfere with the process of filtra- tion. The filter-paper should always be moistened before filtering. If this is not done, the pores of the filter-paper will become clogged with gelatin and coagulated albumin, which will greatly interfere with rapid filtering. Gelatin should not, as a rule, be boiled for more than fifteen minutes, nor left in the steam sterilizer for more than thirty minutes, otherwise its property of solidifying will be impaired. As soon as the preparation of the gelatin is completed it should be sterilized in the steam sterilizer for fifteen minutes on 266 BACTERIOLOGIC METHODS. three consecutive days. The mouths of the containing flask or test-tubes should be completely plugged with raw cotton. Nutrient Agar-Agar.— The preparation of this is difficult and tedious, and frequently fails from lack of patience or of experience, or both. Every worker has some slight modification of his own, but if, according to Abbott, the following directions are carefully carried out, the product will usually be satis- factory. Prepare bouillon in the usual way. As agar-agar re- acts neutral or faintly alkaline, the neutralization of the bouil- lon may be accomplished before the agar-agar is added. Finely chopped or powdered agar-agar is added to the bouillon in the proportion of 1 to 1.5 per cent. The mixture is then placed in an agate or earthen-ware boiler, and the height of the fluid, before boiling, marked upon its inside. If a liter of the medium is being made add about 375 cubic centimeters or more of water, and boil slowly for about two hours or until the excess of water that was added has been evaporated. Do not allow the fluid in the vessel to fall below the original level. If this occurs water must be added to bring the final amount up to one liter. At the expiration of two hours remove from the fire and cool rapidly by immersion in a pan of cold water. Stir constantly until the temperature of the mass has fallen to about 70° C, then add the white of one egg that has been previously beaten up in about 50 cubic centimeters of water. Mix this in well and allow to boil for half an hour longer, keeping the fluid up to the original one liter mark. The fluid is now easily filtered at room-temperature through a heavy folded filter. If properly prepared and filtered through a properly folded filter-paper, it should pass through at the rate of one liter in from fifteen to twenty minutes. Glycerin Agar-Agar. — The nutrient properties of agar- agar for certain organisms is greatly increased by the addition of 5-per-cent. glycerin. If glycerin is added to the agar-agar, it should be done after filtration and before sterilization. If after filtration the medium is found to contain flocculi, investigate the reaction. If it is quite alkaline it must be neu- tralized, boiled, and filtered again. If the reaction is neutral PREPARATION OF CULTURE MEDIA. 267 or only faintly acid, dissolve and again clarify with egg albumin as directed above. The most important feature of all media apart from their proper preparation is the reaction. They must be neutral or only very faintly alkaline to litmus, as but few organisms de- velop well on acid media. Blood-Serum. — For the preparation of a small amount of blood-serum for culture media purposes, it may be obtained from small animals under such aseptic precautions as will guard against gross contamination. For laboratory purposes, where large amounts are required, it is best obtained from the slaughter houses. Under these conditions a certain amount of contamina- tion is unavoidable, though its extent may be limited by observ- ing certain precautions. The original method of Koch with a few slight variations is as follows: — The animal from which the blood is to be obtained should be suspended by the hind legs so that its head is a few feet from the floor. The head should be held back when, with one sweep of a sharp knife, the throat is completely cut through. The blood as it spurts from the vessels should be collected in large glass jars which have been previously sterilized and dried with alcohol and ether. The jars should be provided with close- fitting cover and clamps capable of hermetically sealing them (large museum specimen Jars will be found very satisfactory for this purpose). Prom two-gallon jars of blood there is usually recovered from 500 to 700 cubic centimeters of clear serum. The jars having been filled with blood, their covers are re- placed loosely and then they are allowed to stand quietly for about twenty minutes until clotting has begun. At the expira- tion of this time a clean glass rod is passed about the edges of the surface of the forming clot to break up any adhesions that have formed to the sides of the jar. The covers are now replaced and clamped down tightly, then with as little agitation as possible the jars are transferred to an ice chest where they should remain for from twenty-four to forty-eight hours. The temperature of the ice chest should be only sufl&eiently low to prevent bacterial growth, but not so cold as to prevent coagula- tion. When the jars are removed from the ice chest a firm clot 268 BACTERIOLOGIC METHODS. will be found in the bottom of the jars. The serum is drawn off with a sterile pipette or syphon, and transferred to sterile glass cylinders. These cylinders are now placed on ice for an- other twenty-four hours, when the corpuscles will have settled to the bottom, leaving the serum above quite clear. This is then ready to be pipetted off into sterile test-tubes, about 8 cubic cen- timeters in each tube, or into flasks of 100 cubic centimeters' capacity. It is now ready for sterilization. This is accomplished by the intermittent method at a temperature of 70° C. for a period of one hour on five consecutive days. During the intervals it should be kept at room-temperature. After sterilization the tubes may be allowed to remain fluid or they may be coagulated by a short exposure to 80° C. For solidifying, the tubes should be placed in an inclined position in order to secure the greatest possible surface from the quantity of serum employed. The process of solidification requires constant attention if good results are to be obtained. No rule can be laid down for the time required to accomplish it, as this is not constant. Too high temperature and too rapid solidification results in an opaque and inelastic medium. AVhen solidification is complete the tubes may be retained in a vertical position, and unless for immediate use must be protected from drying. This may be done by burning off the superfluous ends of cotton and covering them with sterile rubber caps; or what is just as satisfactory and far cheaper, sterilized corks may be pushed down upon the cotton plugs. Owing to the employment of large quantities of serum, principally for the detection of diphtheria, the tedious method of Koch has been largely superseded by a number of more rapid modifications. Method of Councilman and Mallory. — By this method the serum is more quickly prepared. Eigid precautions against con- tamination of the blood during collection are not necessary, and the resulting medium, while neither transparent or translucent, fully meets the ordinary requirements of bacteriology. By this method the serum is decanted directly into sterile test-tubes as soon as obtained; it is then firmly coagulated in the slant position by exposure in a dry-air sterilizer at from 80° to 90° C. It is then immediately sterilized in the steam sterilizer PREPARATION OF CULTURE MEDIA. 269 at 100° C. for fifteen minutes on three consecutive days, as is the case with other media. Loeifler's Blood-Serum Mixture. — This mixture consists of one part neutral meat-infusion bouillon, containing 1 per cent, of grape-sugar and three parts blo'od-serum. The mixture is placed in test-tubes, sterilized, and solidified in exactly the same manner as described under blood-serum preparation, except that it requires a longer time and a higher temperature for coagula- tion. PREPARATION OF TUBES, FLASKS, ETC., FOR CULTURE MEDIA. While the media are in the course of preparation it is well to get the tubes, flasks, pipettes, etc., ready for their reception. These must be absolutely sterile. To this end both old and new tubes should first be boiled for a half-hour in a 2-per-cent. soda solution, then carefully swabbed out with appropriate bristle brushes. After rinsing in clear water they are immersed in a 1-per-cent. solution of hydrochloric acid for a few minutes, then rinsed again and stood, round end up, to drain. When dry they are plugged with raw cotton carefully inserted so that there are no cracks^or openings in the occluding cap. The plug should fit neither too loosely nor too tightly, but should fit firm enough to hold the weight of the tube when lifted by the protruding cotton. The tubes thus plugged are then placed upright in wire baskets and heated for one hour in the hot-air sterilizer to a temperature of 160° C. Tubes so prepared, if undisturbed, will remain sterile for an indefinite period. Filling Tubes. — The tubes are best filled with the aid of a separating funnel, though ii not convenient the tubes can, with a little care, be successfully filled directly from the flasks. It is not necessary to sterilize the funnel as the media in the tubes is to be steriliz;ed as soon as they are filled. In any case, care should be observed to prevent any of the medium from coming in contact with the mouth of the tubes, which would cause the cotton plugs to adhere to it, making them hard to remove, presenting a very untidy appearance and materially in- terfering with subsequent manipulations. After.filling, the tubes are ready for final sterilization. This 270 BACTERIOLOGIC METHODS. is accomplished in the steam sterilizer by the three-day frac- tional method. TECHNIC FOR PLATES AND PETRI DJSHES. Plates. — The plate method can be employed with both agar and gelatin, but cannot be practiced with blood-serum, because the latter when once it is solidified cannot again be rendered liquid. Plates are usually referred to as a set. This term includes three separate plates each representing a mixture of the organ- ism in a state of greater dilution. The plates are numbered 1, 2, and 3. A set of plates may be prepared as follows : Three tubes, each containing the requisite amount of gelatin or agar- agar, are placed in a water-bath and warmed until the medium is fluid. Agar-agar becomes fluid at about the temperature of boiling water; gelatin is fluid between 35° and 40° C. In the case of the agar-agar tubes, after liquefying they must be cooled to 40° C, at which temperature they remain fluid while the organisms are introduced. If this cooling is omitted the tem- perature of the medium when the organisms are inoculated will be sufiiciently high to destroy their vitality. The medium now being liquid and of a proper temperature, the material containing the organisms is taken up on a sterile platinum wire-loop and transferred to tube 1, where it is thor- oughly disintegrated and mixed by rubbing against the side of the tube. The more carefully this is done the more uniform' will be the distribution of the organisms and the better the final re- sults. The loop is now again sterilized by passing through the flame, and when cool three loops full from tube 1 are trans- ferred to tube 3, where they are carefully stirred in. Again, the wire is sterilized and the same manipulation carried out be- tween tubes 2 and 3. This completes the dilution. During these manipulations, which must be done rapidly if agar is employed, the temperature of the water bath must be kept between 39° and 43° C. If the temperature falls below 38° C. the agar-agar will become solidified and can then only be reliquefied by the application of heat sufBcient to destroy the organisms introduced. After inoculation the contents of these tubes are poured out THE INCUBATOR. 271 upon the sterilized plates, cooled, and incubated for twenty-four or forty-eight hours. Petri Dish Method. — This process materially simplifies the original technic of the plate method. It consists in substituting for the flat plates of glass small, round double-glass dishes having about the same surface area as the original plates. The inocu- lated and liquid media is poured directly into these, their covers are immediately replaced, and they are set aside to cool. In all other respects the process is the same as Koch's original plate method. These dishes have vertical sides which prevents over- flowing of the medium. A convenient size for this method nieas- ures about 12 centimeters in diameter, but dishes of other sizes are easily obtainable. The dishes are readily sterilizable by hot air or steam, and have the great advantages that the danger of contamination is reduced to a minimum, since after sterilizing the plates do not have to be separated until the pour- ing on of the medium, and then only for a moment. THE INCUBATOR. After the plates have been made and solidified they should be transferred to an incubator where a uniform and favorable temperature may be maintained. Various types of incubators have been devised, but since the principle and purpose of all is the same, a general description of their construction and method of employment is all that is required. The incubator or thermostat (Fig. 33) consists essentially of a copper chamber of convenient size provided with double walls, between which heated water circulates. The incubator chamber has a close-fitting door of heat-proof construction, and usually within this is a second door provided with a glass front, which permits inspection of the interior of the incubator with- out actually opening the chamber, and so reducing the tem- perature. The whole apparatus is set upon an enclosed base and is covered with asbestos board to prevent loss of heat from radiation. In the top of the chamber is a small opening fitted with a perforated cork through which a thermometer projects into the interior. Two other openings are provided, one for the thermometer which records the temperature of the circulating water, the other for the thermo-regulator. At one side of the 272 BACTERIOLOGIC METHODS. apiKiratus is a vei'lieal water gauge provided with an ripper opening for the introduction of water, and a stop-cocli below for drawing off tlie water when occasion required. "W'lien in opera! inn tlie apparatus sliould i.ie liept full of water; ntlierwise. tlie ol)ject of the water jacket will be defeated and tlie temperature of the interior of tlie chamber will not be maintained. TTeat is supplied to the incubator by a gas burner Fig. 33.— Thermostat or Incubator. (A. H. T. Co.) placed witliiu the inclosed space helow the chamlicr. The par- ticular ffirm of burner usually employed is known as "Koch's safety ])ui'ner,'' \\'lii(h is so constructed that should liy accident the light 111' extinguished, tlie flow of gas would be almost im- ]iiediati'l\' sliiit (jII'. An ordinary blunsen ])urner, well protected from sudden gusts ni air, will ser\e the jiurpose eqaallv as well. The Thermo-Kegulator. — The ePficiency of tlie thermostat de|i('uils ii|i(ii! tile ]ir(i|)er and unil'drni teiii]HU'alure which is niaiiit;nned liy (lie iliermo-regubitor. A satisfactory regrdator sliould iirrniit of a fluctuation of not more tlian 0.2° C. in the lenij)ej-al lire ^^■iillin tlie cliamljcr of tlu' a|i|iaratus. THE INCUBATOR. 273 The commonest form of regulator is constructed upon the principle involving the expansion and contraction of fluids under the influence of heat and cold. By means of such expansion and contraction the amount of gas passing from the source of sup- ply to the burner is modified as the temperature of the, water in the incubator rises or falls. For the successful employment of clinical laboratory meth- ods, the thermo-regulator is useful, but not essential. In a room of fairly uniform temperature the flow of gas can be regulated by hand until the internal temperature of the incubator is 37° C. After this, if the water level is maintained and the apparatus protected from protracted change in the surrounding tempera- ture, the variation in the internal temperature will be so slight that it need not be considered. XVL SERODIAGNOSIS. WIDAL REACTION. Agglutination. — We know that in the natural infections of man j)eculiar changes occur in the blood-serum due to the influ- ence of specific bacteria or their soluble toxins. Among these, the most familiar occurs in typhoid fever and is shown by the agglutination of these motile organisms in the Widal reaction. Widal Reaction. — Definition : Agglutination in a bac- terial sense refers to the clumping or precipitation of micro- organisms by the action of the serum. This is the so called Gruber-Widal reaction. Xormal serum will agglutinate many bacteria, as typhoid, colon, pyocyaneus and dysentery, but not the streptococcus and many others. This agglutination only occurs in low dilutions, though the typhoid has been found to agglutinate in dilutions of 1 to 30. This point is to be remembered in practical diagnosis. Technic. — Two methods: 1. The macroscopic or naked eye observation of the clumping and sedimentation of a homo- geneous suspension- of bacteria in a test tube. 2. The micro- scopic observation of the clumping of the organisms when mixed in diluted serum and mounted in a hanging-drop preparation. For the hanging drop it is necessary to have a slide with a concave depression in the middle. A drop of the serum under examination is placed in the center of a cover-glass, which is then placed drop-side down over the depression, its edges being sealed by vaseline or paraffin. Tn this preparation with the aid of a microscope the loss of motility incident to agglutination is readily observed. Different typhoid cultures vary in their susceptibility to clumping so that eacli observer should make himself familiar with the peculiarities of liis own cultures. (274) WIDAL REACTION. ' 275 In order to have fresh typhoid cultures always at hand it is best to transfer glycerin-agar cultures every eight to fourteen days, or otherwise cultures degenerate and die off. A bouillon culture is prepared from the water of condensation of the agar- culture 24 hours before the test is to be made and is incubated at room temperature. The Test. — A simple method that is practiced by many boards of health and in private laboratories is to dry a few drops of patient's blood on a piece of paper and transmit that to the laboratory for examination (see appendix for illustration of Philadelphia Widal Test Blank). The Serum Dilution. — It is usually held that a dilution of one to forty or one to fifty with normal salt solution is sufficient to eliminate the possibility of false agglutination by normal sera, and at the same time sufficiently low to permit of the good reaction to nearly all typhoid cases, excepting possibly advanced convalescents. Loop Dilution. — A convenient method of measuring the culture and serum is to employ a fine wire loop such as used in bacteriologic work. One loop full of serum placed on a slide and diluted with ten loops of water will give a dilution of 1 to 10. By this means minute quantities of serum or blood can quite accurately be reduced to the desired dilution. If agglutination occurs in a microscopic preparation, we note, with the aid of the high power, that, in the course of from fifteen minutes to an hour as the micro-organisms swim about, a few as they come in contact have a tendency to remain in this relation. In the course of a few more minutes other organisms join this group and other groups form throughout the field; motility becomes gradually less until it ceases entirely in the typical reaction. The complete change occurs in from six to eight hours. No less than five iacteria mv^t hecome permanently agglutinated to constitute a positive reaction. The test is most decisive when large masses of permanently agglutinated organ- isms are formed which can be seen with the lower power. 276 SEEODIAGNOSIS. THE PRINCIPLES OF THE WASSERMANN AND NOGUCHI REACTIONS, AND THEIR COMPARATIVE VALUE TO THE CLINICIAN.' Since the appearance of the original communications of Wassermann, Neisser and Bruck announcing a new method of syphilitic diagnosis, medical literature both in Europe and in America has been literally flooded with papers dealing with this subject, and it is highly probable that no other single topic has received so much consideration from so many workers in the various fields of medicine. A very great deal of this work has been of the most careful and painstaking character. While the subject is a relatively narrow one the significance of a luetic infection is such that every aspect of the question of serum diagnosis has been subjected to the most careful scrutinj^ and has been investigated from both the clinical and experimental points of view, and now, after over three years, certain facts have been definitely determined. It is perhaps as well at the outset to say, however, that neither of the reactions to be described can be regarded as absolutely specific. The Wassermann reaction, based upon an ingenious prin- ciple, worked out by Bordet and Gengou, gives to the experienced, laboratory worker a very satisfactory means of diagnosticating syphilitic affections, even in individuals who were infected years ago, of course excluding a dormant condition of patients at the end of a successful treatmentT In order to be able to grasp the steps in the Wassermann reaction it is indispensable to be acquainted with the steps involved in demonstrating antibodies of any kind formed acci- dentally (by disease) or purposely (immunizing) in the body fluids of an animal. Ehrlich showed long ago that in the demonstration of antibodies, or, as he called them, amboceptors, three distinctly difl'erent substances are required in order to form a complete reaction : First, the cell or poison against which we wish to immunize, or, more plainly, against which we desire to obtain an antibody; second, the antibody (or amboceptor) obtained by repeated injections of the special cell or poison into 1 Kaplan ; Am. Jour. o£ Med. Sci., Jan., 1910. HEMOLYSIS. 277 the rabbit (or any other suitable animal) ; third, a completing substance — the complement. This latter substance is present in variable quantities in the sera of all animals, its quantity being rather constant in guinea-pigs. It is destroyed by heating various sera to 56 or 57 degrees centigrade for one-half hour, and is similarly affected by various other physical agents. This is not the case with antibodies which are comparatively thermo- stable. We have then three factors: (1) A cell to be destroyed or a poison to be neutralized; (3) a substance capable of doing this — the amboceptor or antibody; and (3) a completing sub- stance, without which the reaction cannot take place — the complement. Ehrlich and others in order to impress the reaction upon the minds of men interested in immune processes, made use of diagrams. To make it still more familiar, let a lock represent the cell, a key which fits it the antibody, and the hand that will turn the key the complement. By giving a lock to a smith we can get a key made to fit the specific lock exactly. When we inject cells we can get an antibody which exactly fits the cell injected, and the same is true when we inject a bacillus or a poison. All these substances capable of producing antibodies (antibody generators) are known as antigens. To determine whether a bacterium was killed or a poison neutralized by being exposed to the action of a specific ambo- ceptor, is not as simple a process as the demonstration of the destruction of red blood-corpuscles by an amboceptor directed against them. A suspension of red blood-corpuscles minus amboceptor and complement gives an opaque red mixture; when we add the amboceptor plus complement and incubate at 37° C. the opacity disappears and a clear red fluid results. It is apparent that hemolysis or destruction of red cells is a phenomenon that can readily be seen in vitro, and its presence signifies that the three substances spoken of above are present in the test-tube. If any one of the three is not there, or is present in an inactive state, the red cells will remain unaffected and the mixture will retain an opaque, red color. Hemolysis. — The phenomenon known as hemolysis depeiids upon the destruction of red blood-corpuscles. There are many reagents capable of doing this, such as distilled water, acids, and 278 SEEODIAGNOSIS. alkalies. It is also possible to form in ■warm-blooded animals substances which will bring about hemolysis against certain red blood-cells. This is accomplished by injecting an animal (a rabbit or goat) with the cells of a sheep or any other animal. The serum from such a rabbit, when brought in contact with the cells of a sheep will cause the mixture to become clear (hemo- lysis). The same amount of serum from an untreated rabbit, will have no effect on a similar suspension of sheep cells. The substance produced in the rabbit's serum is known as the anti- sheep amboceptor, and together with sheep cells and complement (from a guinea-pig) is known as a hemolytic system. We learn from the above exposition that in order to prove the presence or absence of certain antibodies, we make use of the phenomenon of hound or unbound complement, utilizing a hemolytic system simply as an indicator. Exactly the same prin- ciple is applied to the serum diagnosis of syphilis. Luetic Antigen. — Unable to produce a growth of spirochjete pallida upon any culture medium, we have to be contented with organic extracts containing them in greatest numbers, for this purpose the liver of the luetic fetus fills the requirement. The extract obtained is known as leutic antigen, and need not con- fuse anybody, for we know that an antigen is a body capable of forming antibodies. If an individual has had syphilis some years ago, he would also have syphilitic antibodies in his serum which, when brought in contact with the extract from the syphi- litic liver, would invariably bind complement, and a hemolytic system (sheep cells or any other red cells with the corresponding amboceptors) will not be affected, because the complement had been bound previously. Principle and Technic of the Wassermann Reaction. — As mentioned before, antibodies will attract complement if the antigen responsible for their formation is present in the same test-tube. In the Wassermann reaction a serum containing anti- bodies capable of uniting with the antigen is used (substance containing lipoids) and thus deviating the introduced comple- ment, will not permit hemolysis to occur, if sheep cells and their antishecp amboceptor are snbfequently placed in the same tube, as for obvious reasons, the complement was bound or deviated previously by conditions suitable for such an interaction. If WASSERMANN REACTION. 279 the patient's serum does not contain the required antibody, the introduced complement will remain unbound and in a fit condi- tion to destroy the sheep cells when subsequently introduced with their antisheep amboceptor. Modus Opeeandi. — (1) Obtaining blood from patient: A fairly stout piece of rubber tubing is placed a little above the elbow and held snugly in place by an artery clamp. Do not obliterate the pulse. This brings into prominence the veins at the bend of the elbow. To a stout hypodermic needle (use a 19 bore — one and one-half inch needle) attach a two-iach piece of rubber tubing. Holding the free end of the rubber tubing in an ordinary sterile test-tube, quickly plunge the needle into the most prominent vein; if expertly done, the patient will hardly feel it and the blood will immediately begin to flow. About 6 to 10 cubic centimeters of blood is withdrawn and placed in the ice box over night' to coagulate. The serum separates and may be pipetted off absolutely clear without cells. It is advisable to take the blood as far from a meal as possible, as proximity to a meal makes the blood lipemic, interfering with perfect working conditions. (2) Having obtained 1 or 3 cubic centimeters of clear serum, it is placed in a test-tube in the thermostat at 56 degrees for one hour. Care must be taken not to permit the heat to rise too high (over 58 degrees). (3) After this, 0.2 cubic centimeter is placed in each of two test- tubes, one the test, the other the control. (4) To each is now added 0.1 cubic centimeter fresh complement. (5) To the test portion is added one unit of antigen. The control does not receive any antigen. (6) Each tube receives now 3 cubic centi- meters of a 0.95 per cent. NaCl solution. In order to be able to judge properly the correctness of the procedure, the more controls one has the better; it is therefore necessary to compare the serum to be tested with two sera from known positive and negative bloods. (7) Shake every tube well and place in incu- bator at 37 or 38 degrees for one hour. During this time, if the serum is luetic the antibodies present will, together with the antigen, bind the complement and render it inactive for hemoly- sis. (8) After one hour- incubation each tube receives two units of amboceptor and 1 cubic centimeter of a 5 per cent, suspension of sheep cells in 0.95 NaCl. The tubes are again vigorously 280 SERODIAGNOSIS. shaken and placed in the incubator at 37 degrees and inspected after ten minutes. If the reagents are properly adjusted hemoly- sis begins in the control tubes in fifteen to twenty minutes, and careful watching becomes a very essential point at this stage of the test. As soon as the control is completely hemolyzed the tubes are to be compared; only those should be pronounced negative that show a transparent fluid the same as the control. Permitting the tubes to stand undisturbed in a cool place (15 to 17 degrees C.) for twenty-four hours shows in the positive test a deposit of red cells, the size of the deposit depending upon the severity of the infection or proximity to the initial lesion as well as upon the degree of balance of the reagents used. Usually a markedly positive serum gives at the end of twenty-four hours a clear supernatant fluid of a light pink hue with a Bordeaux red accumulation on the bottom of the tube. The weaker the reaction, the redder the supernatant fluid and the scantier the deposit of cells. In testing more than one serum, the reaction in each individual test must be considered as finished as soon as the controls are completely hemolyzed, in which case the two tubes are immediately removed to a cool place. Principle and Technic of the IJoguchi Reaction. — This is same as in the Wassermann, excepting that the amboceptor is directed against human cells. It also facilitates the handling of reagents, as they are mostly paper soaked in the antigen and amboceptor. These do not readily deteriorate, as is the case with fluid biological reagents. The serum does not need inacti- vation at 56° C. Modus Operandi. — (1) With a capillary pipette allow one drop of fresh serum to fall into a narrow (1 centimeter lumen) test-tube. The pipette is not to be' used for any other serum. (2) Add 0.05 cubic centimeter fresh complement. (3) To the front row (rear row for control) add one piece (more or less, depending on the titre) of antigen paper. (4) Prepare a suspension of human cells 1 drop of blood to 4 cubic centimeters NaCl 0.95 per cent. It is best to prepare about 60 cubic centi- meters of NaCl solution and allow 15 drops of blood to fall from the experimenter's finger into the solution. The human cell suspension is placed over night in the ice box. Next morning the supernatant clear salt solution is pipetted off and a fresh NOGUCHI REACTION". 281 quantity of NaCl is added (about 55 cubic centimeters) to the cells in the beaker. Of this cell suspension add 1 cubic centi- meter to every tube in the rack. (5) Incubate for three-quarters or one hour at 38 or 39 degrees, preferably in a large dish of warm water. Occasionally shake the tubes, to insure proper solution of the biological substance on the antigen paper. (6) Add to each tube (after incubation), front and back rows, one piece of amboceptor paper more or less, the quantity depending on the titfe) and replace in the incubator, observing the result after ten minutes, and watching carefully the controls. It will be noted in about fifteen minutes, more or less, that the rear row begins to get clear, and when complete transparency is obtained the test and control tube are to be removed to a cool place and observed. If the reaction is positive, then the front tube (test) will be opaque, in marked contrast to the control, which is transparent. For convenience of observation, make use of a fine sealed tube (about 1 millimeter in diameter) filled with black ink, which, when placed behind the control, will appear as a clear black line, whereas the positive tube will not show the black line, or it appears as a dim shadow — depending on the strength of the reaction. It has been stated that a positive Noguchi test and a negative Wassermann is often due to the presence in the patient's serum of antisheep amboceptors. It is not necessary to perform this test with every serum as a control. Only sera giving the above results need be subjected to a verification. To demonstrate the antisheep amboceptor, place 1 cubic centimeter of a 5 per cent, suspension of sheep cells in a test-tube, add 0.3 cubic centi- meter of patient's serum and 0.1 cubic centimeter complement, add 3 cubic centimeters of ISTaCl solution, place in incubator, and observe. If the amboceptor is present, the cells will dissolve and the mixture become transparent. The time consumed depends upon the number of amboceptor units present. CoNTEOLS. — In the Wassermann and Foguchi reactions it is of vital importance to have every possible error excluded. The substances to be controlled are the antigen, the amboceptor, and each individual serum. The Antigen Control. — This biological reagent, as is known, can per se inhibit hemolysis. To measure the degree of such 282 SERODIAGNOSIS. interference, a tube containing a well-known normal serum, plus antigen, plus complement, and antisheep amboceptor plus sheep cells ought to hemolyze in about twenty to thirty minutes. No reaction is to be considered as finished before the antigen control tube is completely hemolyzed. The Amboceptor Control. — Upon the ef&ciency of the anti- sheep amboceptor depends the rapidity of hemolysis of the sheep cells. It is therefore necessary to establish the amboceptor effi- ciency in a separate tube containing sheep cells, plus complement, plus antisheep amboceptor. It is not essential to add normal serum. The tube containing the above ingredients is always the first to hemolyze, requiring about fifteen to twenty minutes for a complete hemolysis. Control for Each Serum. — Every serum more or less has the power to interfere with hemolysis to a slight degree. In order to control the factor of individual inhibition, every serum tested is placed in each of two tubes, the front tube contains the antigen and all other biological reagents, the rear tube receives everything but the antigen. This shows the degree of individual inhibition as compared with the tube containing the amboceptor control. Efficiency of the Entire System. — For this a well-known luetic serum is utilized. The reaction is to be positive, and hemolysis should not occur in the front tube, even if exposed to incubation temperature for hours after the controls hemolyzed. Equipment. — At least one dozen or even more of Mohr's pipettes, 1 cubic centimeter, graduated into %oo- One dozen 10 cubic centimeter pipettes, graduated into %o- One gross of ordinary test-tubes. One gross of test-tubes 1 centimeter in diameter, 13 centimeters high. One-quarter dozen of graduated cylinders, 50 cubic centimeters; one-quarter dozen 100 cubic centimeters. Two 50 cubic centimeters measuring flasks with glass stoppers. A few pounds of glass tubing, 5 millimeters bore, to make capillary pipettes. One-half dozen test-tube racks for Wassermann tubes; one-half dozen test-tube racks for Noguchi tubes. A piece of rubber tubing for tourniquet. One artery clamp for above. One dozen h3'podermic needles, 19 bore. One thermostat regulated at 57 degrees and one regulated at 37 degrees. One electric centrifuge. Labels and pencil for writing PREPARATION OF ANIMALS. 283 on glass. One tall glass jar for flushing through used pipettes, height to be greater than any pipette used. One dozen Petri dishes. One dozen beakers, 100 cubic centimeters capacity. Two fine forceps, and two Hagedorn needles. One package of quanti- tative filter paper. One razor (for killing guinea-pigs). One 15 cubic centimeter Luer syringe. Peeparation of Animals. — ■ Antisheep Amboceptors : Several healthy rabbits (not less than four) receive every fifth day 1, 2, 3, 4 and 6 cubic centimeters of well-washed sheep cells. This number of rabbits is used, as one or two may die during the injection weeks. The cells are obtained from the slaughtei* house and immediately defibrinated with a wire defibrinator or glass beads. In order thoroughly to wash the cells a high speed centrifuge is necessary, capable of making at least 3000 revolu- tions to the minute. ' Two of the centrifuge tubes are filled with the fluid sheep blood, it being advisable, in order not to spoil the centrifuge, to have them of equal weight. The first eentrifugali- zation brings the cells to the bottom, and the clear supernatant serum is pipetted off. The cells are now mixed with 0.95 per cent. NaCl solution and centrifugalized again, and the super- natant clear fluid is again pipetted off; this is repeated three times. The cells are now approximately serum-free. The entire quantity of cells in the tube is now brought up to its original volume with 0.95 per cent. NaCl solution, and of this, 2 cubic centimeters is used for the first injection. With a sterile glass syringe this quantity is injected into the peritoneal cavity, having previously shaved and cleaned the puncture area. Cotton and collodion prevent wound infection. This procedure is repeated five days later with 4 cubic centimeters of cells brought to its original volume, etc., until each animal has been injected five times. Nine days having elapsed since the fifth injection, the serum of the rabbit contains now a high lytic power against the red blood-corpuscles of the sheep. The rabbit is killed and its serum used. In the Wassermann reaction 1 cubic centimeter of a 5 per cent, suspension of well-washed sheep red cells in 0.95 per cent. NaCl solution is the standard dose for each test. It is evident, therefore, that in order to test the power of our rabbit serum (antisheep amboceptor, as it is now called) we must use this 284 SERODIAGNOSIS. quantity of sheep cells. .Into each of six test-tubes is placed 1 cubic centimeter of a 5 per cent, suspension of sheep cells in NaCl 0.95 per cent. These are marked from 1 to 6, and to each is added 0.1 cubic centimeter of fresh guinea-pig serum (this is Imown as the complement serum and is the quantity used in the Wassermann test). We now add to test-tube (1) 1 cubic centi- meter of a 1 to 200 solution of our amboceptor. To test-tube (2) we add 1 cubic centimeter of 1 to 400; to test-tube (3), 1 cubic centimeter of 1 to 800; to test-tube (4), 1 cubic centimeter of 1 to 1600; to test-tube (5), 1 cubic centimeter of 1 to 3200. This is placed into the thermostat at 37 degrees and the result noticed after fifteen minutes, thirty minutes, up to two hours. It will be seen that in fifteen minutes test-tube (1) is clearing up, or is clear (hemolysis) ; this would indicate that 1 cubic centimeter of a 1 to 200 solution of our amboceptor is capable of destroying in fifteen minutes 1 cubic centimeter of a 5 per cent, suspension of sheep cells. This proportion — 1 to 200 — is too strong, and may give negative results with some positive sera. The unit strength of the antisheep amboceptor is usually twice the quantity capable of hemolyzing the 1 cubic centimeter of cells in two hours. If 1 to 1600 shows hemolysis after two hours and 1 to 3200 does not, then 1600 divided by 2 is the strength of the amboceptor, one unit equals 1 to 800. It is best to run two series of titration — one like the above, the second beginning with 1 to 250, 1 to 500, 1 to 1000, etc., so that a proper mean can be established and a more exact unit made. In table form the above is expressed as follows : — Sheep cells. Cells hemolyzed in Tube 1 amboceptor = 1 to 200 " 2 " =1 to 400 " 3 " =1 to 800 " 4 " =1 to 1600 " 5 " =1 to 3200 Complement 0.1 0.1 0.1 0.1 0.1 5i 1 c.c. 15 minutes 30 minutes 50 minutes IH hours 2 hours Strength of 1 unit, 1 to 16 00 ; dose for 1 test, ] to 800. Date A full-grown rabbit usually furnishes from 50 to 60 cubic centimeters of serum. This is to be kept in a glass-stoppered flask in the ice box (lower compartment) . The hemolytic power does not indefinitely remain the same as in the beginning; it is. PREPARATION OF REAGENTS. 285 therefore, necessary to establish the titre at least once every week, and to make up the dilutions aecordiiigly. These dilu- tions are to be prepared on the day of testing. The rabbit serum does not have to be inactivated to get rid of the complement in it, as the quantity of serum used is too small to influence in any way the resulting outcome of the test. Preparation of Complement. — A full-grovm guinea-pig is held over a Petri dish, and after having it narcotized, the blood- vessels of the neck are severed with a razor. Suspended by the hind legs the animal is exsanguinated, and the collected blood is permitted to remain at room temperature for at least three hours. The serum collects in large drops and may be pipetted off, or the coagulum plus the serum is placed in a centrifuge tube and after five minutes centrifugalization the supernatant serum is pipetted off into a sterile test-tube; but such a serum is not as reliable as when left for three hours with its cells. About 6 cubic centimeters of complement is obtained from one guinea-pig. Preparation of Sheep CeZZs.— Obtained from the slaughter house, the cells are washed three times with 0.95 per cent. NaCl solution, and 1 or 3 cubic centimeters is mixed with 30 or 40 cubic centimeters of salt solution, making a 5 per cent, suspen- sion of cells. Preparation of Antigen. — The fresh liver of a luetic fetus or the liver of any baby cadaver is chopped up very finely, and the mass is spread on a few Petri dishes and dried. The drying process is hastened by a current of air produced by an ordinary electric fan. Lately, not only baby livers, but also the livers of dogs, the hearts of guinea-pigs, and other organs were used to make antigen. The usefulness of the antigen is only established when in actual standardization it is found serviceable and works faultlessly with decidedly syphilitic and unquestionably normal sera. Consequently it makes little difference whether one uses the extract obtained from the liver of a syphilitic fetus or from the heart of a guinea-pig, provided they are well titrated. It is better — according to German workers — to use more than one extract, and have a series with well standardized luetic liver antigen, one with guinea-pig heart, and another with dog liver, or normal human liver. To proceed with the making of 286 SERODIAGNOSIS. antigen the obtained dried liver is rubbed into a powder and kept in an exsiccator over CaClg in a cool, dark place. According to Tscliernogubow, such a powder is serviceable for a very long time. Of this powder, 0.5 gram is extracted at room tempera- ture or in an ice box with 25 cubic centimeters of 95 per cent, alcohol for twenty hours, then filtered, and the filtrate used for experiments. For the actual Wassermann test, one part of this opalescent filtrate is diluted with five parts of 0.95 per cent. NaCl, and 0.5 to 1 cubic centimeter used for each test-tube, the dose depending upon the established titre. The above process extracts from the liver substances soluble in alcohol, chiefly bodies of a fatty nature (lipoids) . There are other means of obtaining lipoids, the above being one of the simplest, having also in view the preservation of the antigen in an active form. The liver, instead of being dried and powdered, may be directly extracted with five volumes of absolute alcohol, and the extract obtained by driving the alcohol off at a temperature not higher than 40° C. or with the electric fan. The obtained extract is much more powerful than the above, is soluble in ether, from which NaCl solutions are made for use. The titre is established carefully as follows : Titration of Antigen. — The unit dose of antigen must be of such a strength that one unit will completely inhibit hemolysis of 1 cubic centimeter of a 5 per cent, suspension of sheep cells, with 0.3 cubic centimeters of a known luetic serum plus 0.1 cubic centimeter of complement ; provided double this dose does not interfere with the complete hemolysis of cells using a known normal serum and complement. Table op ANTiSEif Standardization. Luetic Series Normal Ser ies E.-ich tube contains syphilitic serum, Each tube contains normal serum , 0.2; complement, 0.1: cells, 1 c,c. 5;( ; am- 0.2, complement, 0.1 : cells, 1 c.c, 5^ ; am- bocepter, 2 units. boceptor, 2 units. Hemolysis Tube 1, AnliKen, 0.025 1 hour 15 min. Antisren, 0.025 15 minutes " 2, 0.05 1 " 25 ■• " 0.05 15 " 3, 0.075 2 " 50 " ■• 0.75 15 " 4, 0.101 No hemolysis af- " 0.10 20 ■• " 5, 0.15 ter 24 hours in " 0.15 30 " 6, 0.20 J incubator. " 0.20 35 Dose of 1 unit 0.1 cc. PREPARATION OF REAGENTS. . 287 From the above facts it is evident that the dose next to the largest hemolyzing dose is the strength of one unit, or 0.1 cubic centimeter. It is also apparent that 0.2 cubic centimeter, or a double dose, will not inhibit hemolysis when used with a normal serum. In establishing the unit dose of antigen as well as antisheep amboceptor it is of utmost importance to titrate two or three times in order to get as uniform results as possible, and only uniform work will enable one to come to a proper conclusion as to which is the necessary dose. For establishing the strength of the antigen and amboceptor, well-known fresh luetic and normal sera are to be used, as well as fresh suspension of cells and fresh complement. Before using the standardized reagents it is advisable to -perform two or three actual tests with well-known positive and negative sera. After this the substances may be considered safe for use. The above lines will give one a fair idea concerning the preparation of the biological reagents for the Wassermann reaction. For the Foguchi reaction it will be necessary, first, •to acquaint the reader with the principles involved, and then the preparation of reagents will follow. Preparation of Eeagents. — Antihuman Amboceptor: Babbits are injected with human cells the same as the sheep rabbits. After nine days the rabbits are killed, their serum collected and disposed of as follows : The fluid amboceptor loses strength on standing, so much so that it may not contain one- fourth of its original power a month after the first titration. In a dry state it can be used for a very long time without losing its strength. Prepare antihuman amboceptor by cutting quantitative filter paper in 5 millimeter squares. These squares are stuck onto pins fastened to a cardboard. With a very fine capillary pipette (as fine a one as can be made) one drop is blown on each piece of filter paper and placed in the thermostat for drying. In half an hour the papers are dry and fit for use. By this method each square receives exactly the same quantity of serum, and is not subject to differences in dissemination which must be considered when the serum is blown on a larger piece of filter paper and cut subsequently in 5 millimeter squares. The method takes longer, but the difference is worth while, for each 288 SERODIAGXOSIS. square holds exactly the same quantity of amboceptor. In the test one piece of this amboceptor is serviceable. Titration op Antihumau Amboceptors. Tube 1, Amboceptor, K piece " 2, " 1 ■■ 3, " 1% •• " 4, ■■ 2 Human cells 1 drop to 4 CO. NaCl. 1 c.c. 1 c.c. 1 c.c, 1 c.c. Complement. 0.05 0.05 0.05 0.05 Hemolysis in 2 hours. 20 ■• 11 " Value of the Noguchi Test. — Workers with the Wasser- mann reaction often could not explain why a positive result could not be obtained with some true luetic sera. Later it was demonstrated that this was due to the presence in the human serum of substances capable of dissolving the red blood-corpuscles of the sheep ; in other words, some human sera contained anti- sheep amboceptors. •Sometimes the quantity of antibody is so small that a goodly portion of the complement escapes unbound and does its work by bringing about partial hemolysis; that is the condition of affairs taking place in some weak reactions. If such a serum contained antisheep amboceptors, they would have enough com- plement to cause hemolysis and render the result negative. In the Noguchi test this cannot take place, for the hemolytic system used consists of human cells plus antihuman amboceptor, and, naturally, the human organism does not contain antihuman amboceptors. Luetic sera containing antisheep amboceptors will give a negative Wassermann but a positive Noguchi test. As a result of much work with the Wassermann and Noguchi tests, Kaplan is able to give a fair opinion as to its uses. Shortly stated, the two reactions are of the foremost importance to the clinician, and so far as accuracy is concerned, they almost occupy the first place among our means of detecting diseases. The Wassermann reaction gives a negative result in 8 or 9 per cent, of syphilitic sera. This rather undesirably high per- centage of error is reduced to 1 to 1.5 per cent, when using the Noguchi and the Wassermann combined. VALUE OF TESTS. 289 Never render a decision after one test. Always perform two Wassermann and two Koguchi tests on different days, using the same serum. It is also to be borne in mind that a fairly marked Wassermann reaction, 99 times out of 100, means syphilis, and that a negative Noguchi the same number of times means no syphilis. The two methods are very decisive, but in opposite ways; and used together, carry with them an assurance which no amount of thoroughness and precision will replace if only one method is used. The laboratory worker, being responsible to the clinician for his statements, ought to be in a position to help him con- siderably, but only when his work has been carried out very carefully, unbiased by personal opinion, and submitting the result of a delicate test as read from the test-tube. It is advisable to work with both methods, the Wassermann and Noguehi, as one is a check on the other, which, if properly performed, should give correct reports in 98 per cent, of eases. Questionable reactions are not to be used for diagnosis, and if a serum does not react strongly after a number of repetitions of the test, the diagnosis is to be left to the clinician. Exceptionally strong reactions are obtained in untreated cases of general paresis with both tests, as well as in primary sores four weeks after infection. XVII. APPENDIX. DESCRIPTION OF OFFICE LABORATORY CABINET. For those who are daily employing the methods of clinical cine a detailed description of the author's laboratory cabinet be of interest. As illustrated in frontispiece, this cabinet is ructed of white pine or poplar, of half-inch boards, except owest shelf or table which is one-inch in thiclcness. This sasily be constructed by a carpenter and finished in white el. Its adoption will be found particularly useful to those not nate enough to have a room to devote to laboratory 3ses. The cabinet itself is not at all unsightly, and may stened to the wall in. the consulting room, preferably near shstand, as running water should be easily accessible when ble. For those who possess the advantage of a well-equipped atory, it will be found extremely convenient for preliminary 1 examinations of specimens as soon as they are received. The cabinet occupies a wall space of 22 ins. broad by 28 high, and projects outward from the wall 18 ins. The es are 6% ins. deep, the top shelf providing space for a n bottle of distilled water with syphon-tube attachment, place for storage of large stock reagent bottles, beakers, I, dishes, etc. The compartment furnished with a glass door r the microscope and accessories, where they may be safely under lock and key. The shelf below the top provides space for 24 2-ounce re- ; bottles or a less number of larger ones. The drawer im- ately below this holds filter paper, Petri dishes, forceps, pipettes, etc., while the small compartment to its right is (290) OFFICE LABORATORY CABINET. 291 very convenient for a few test-tubes, cover-glasses, slides, and litmus paper. The table, which measures 18 by 21 ins., is sufficiently large to contain the necessary racks, stands, etc. Passing through the table to its extreme left is a gas-cock with nipple for the attach- ment of a Bunsen burner. Such a cabinet will provide all the necessities for the usual examinations of urine, gastric contents, and sputum. By modi- fication and amplification of this idea the cabinet can be adapted to all the special examinations employed in clinical medicine. EXACT DIMENSIONS OF CABINET. Total height, 28 ins.; total width, 22 ins.; depth of table, 18 ins. ; depth of shelves, 6% ins. ; height of drawer base above table, 8 ins.; size of drawer, 6 by 10 by 4 ins. ; microscope com- partment, 8 ins. wide by 14 ins. high; height from table to top- shelf, 24 ins. The wood should be carefully selected, free from knots, and thoroughly seasoned. It should be joined by screws and glue to prevent warping and separation of the joints and seams. Two coats of good white paint, followed by the same number of white bath-tub enamel, will complete the construction. The door and drawer-knobs are of glass, and the microscope compartment is fitted with a lock. Since the construction of this cabinet several years ago, numerous suggestions for improvement have been received; and among these, the construction of a large, shallow drawer to fit under the loose shelf, which will be very convenient for storing burettes and pipettes without materially increasing the size of the piece. A wooden or metal burette stand and filter holder could be easily attached to the under surface of the second shelf and so arranged that when not in use it could be swung under the shelf and out of the way. Pig. 34 shows a semi-portable laboratory cabinet containing reagents and apparatus sufficient to perfonn a majority of the simpler tests employed in the examination of uncertain sputum, gastric contents, and feces; also for the preparation of micro- scopical specimens of the blood and of bacteria. 292 AITENDIX. Tins may he ol.tained m tbe open market and will be found ol oveat service to those desiring to follow clinical n,etliods m connection with the study of their eases. It is finished m quar- tered oak and presents a not unsightly appearance. 1 he dunen- siims are appro.xiuiately 13 fiy 15 by '2(\ ins. Fig. 34 -Portable Clinical Laboratory Set. aftfr SUGGKSTKD BY THE AUTHOR. (G. P. P. & SON '-"■ ' Plan FORMS OF CLINICAL REPORTS. 293 URINALYSIS. Report of Clinical Laboratory. tCTERISTICS. Physical CHARi Quantitative Determinations. Quantity in l 24 hours. J c.em. r Heat, 10% Acetic Acid. Albumin-| Heller's Nitric Acid. LHeat and Nitric Acid. oz. Color, Appearance, Odor, r Fehling's. Sediment : — Glucose J Phenylhydrazin. Mucus, .Bottger's Bismuth. Urates, Phosphates, Indican, Uric Acid, Skatol, Pus, Acetone, Specific Gravity, Bile Pigments, Reaction, Blood, Diazo, Aceto-acetic Acid, B-Oxybutyric Acid, Drug Reactions, Cammidge Reaction, Quantitative Determination. Albumin (Esbach), (Purdy), Glucose (Fehling's) , (Fermentation), Urea, Ethereal Sulphates, Chlorids, Microscopic Examination. (Centrifugated), (SedimentedV ' Hyaline, Erythrocytes, Granular, Leukocytes, Patty, Pus cells, Casts.- Epithelial, Epithelia, Blood, Spermatazoa, Waxy, Bacteria, Bacterial, Yeast Spores, Cylindroids, Trichomonides. Mucus, Voided, A.M .P.M Examined, A.M P.M Exami ned by , 394 APPENDIX. SPUTUM EXAMINATION. Report of Clinical Laboratory. Physical Chaeacteeistics. Quantity in 24 hours. ■ c.cm. oz. Color, Odor, Consistence, Blood, Character, Serous, Mucous, Muco-purulent, Purulent, Casts, Spirals, Microscopic ExAMiifATioN. Tubercle Bacilli, Blood-Cells, Micrococcus Lanceolatus, Pus Cells, Staphylococci, "Heart Failure" Cells, Streptococci, Hematin Crystals, Bacilli, Elastic Fiber, Actinomyces, Cursclimann's Spirals, Yeast, Casts, Obtained Examined < Examined by FORMS OF CLINICAL REPORTS. BLOOD EXAMINATION. Report of Clinical Laboratory. Geneeai, Examination. Color, {Estimated, Wright's, Bogg's, Viscosity, Flow from Puncture, Specific Gravity, {Thoma-Zeiss per c.mm., Estimated by Daland Hematokrit, Percent., {Fleisohl's Hemoglobinometer, Sahli's Hemoglobinometer, Tallqvist's Scale, Color Index, Plaques, Leukocytes, Microscopic Examination. Erythrocytes. Color, Maerocytes, Polychromatophilia, Microeytes, Granular Degeneration, Normoblasts, Poikilocytosis, Leukooytes, Differential Count, Number of cells counted. Polymorphonuclears, Large lymphocytes. Small lymphocytes, Eosinophiles, Myelocytes, Parasites, Bacteria, Hematin Crystals, Date Examined by .... 296 APPENDIX. GASTRO-ANALYSIS. Report of Clinical Laboratory. Vomitus, Test meal Composition: — Ingested, Extracted, Amount Recovered, Dilution, Physical Charactekistics. Sediment containing: — Color, Blood, Odor, Bile, Consistency, Mucus, Reaction, Chemical Determinations. Total Acidity, Butyric Acid, Free H3'droehloric Acid, Acetic Acid, Combined " " Blood, Lactic Acid, Bile, Protealysis, Microscopic Examination. Starch Grains, Meat Fiber, Epithelia, Erythrocytes, Leukocytes, Oppler-Boas Bacillus, SarcinsB, Bacteria, Necrotic Tissue, Parasites, Date Examined Examined by FORMS OF CLINICAL REPORTS. 397 BLOOD-PRESSURE DETERMINATIONS. Clinical Report. f Faught, Apparatus^ L Stanton, Width of Cuff cm. Part examined, Right, Left, Posture,, Pulse Rate, Systolic mm.Hg. Diastolic mm.Hg. Pulse Pressure mm.Hg. Mean Pressure mm.Hg. Remarks. Time of Day. A. M P.M., Date Examined by 2t>8 APPENDIX. EXAMINATION OF THE FECES. Eeport of Clinical Laboratory. Physical Chabacteeistics. JS'umber of Stools Mucus, in 24 hours. Parasites, Amount in 24 Hours. Crystals, Color, Odor, Consistence, Blood, Pus, Calculi, Connective tissue, Muscle fiber. Foreign bodies. Chemical Examination. Blood, Hydrobilirubin (sublimate test). Fermentation, "Lost" Albumin. Erythrocytes, Pus cells, Epithelia, Crystals, Meat Fiber, Connective Tissue, Starch Cells, Free Fat, Fattv acids. MicBOSCOPic Examination. Tubercle Bacillus, Shiga Bacillus, Comma Bacillus, B. Aerogenes Capsulatus, Amebse, Parasites, Obtained Examined Examined by CLINICAL TERMS. 299 CUNICAL TERMS. Owing to the various and uncertain terms employed to designate the amounts of substances found, in clinical investiga- tions the following scheme has been successfully emjjloyed in our laboratory. Terms to be used in expressing the results of examinations of specimens in laboratory ^ : — ^Questionable trace. Albumin, Sugar, Very faint trace. Faint trace Indican, Trace. Acetone, i Strong trace. Bile, Moderate amount. Blood, etc. Large amount. Very large amount. Excessively large amount For Sediments use: — Occasional. Few. Moderate number. Many. Very many. Excessive!; Y large number. ' After Judson Daland. 300 APPENDIX. BLOOD-PRESSURE CHART. A New Graphic Chart for Recording Blood-Pressure Observations. — ^This chart will be found very valuable in con- nection with serial studies of blood-pressure where it is desired PULSE, TEMPERATURE AND BLOOD PRESSURE CHART D«liMd br Prucb A. Fn|H M. D. OCCUPATION S klM'THAM^ , CMdllTMO.- .V"'^ ADORM^J/i-cSs* a. Pl*GWOBIB I ■w ■/»• 'd.r.Oii^t -i^^^ ISr Zhl M 3C }t W )J 31 ti V3 Pig. 35.— Specimen of a New Graphic Chaxt for Recording Blood Pressure Observations, Arranged for Daily, WteEKDY or Irregular Observation Periods. Spaces Equal 6 Millimeters. to follow from time to time its fluctuations. It is constructed along the lines of a temperature chart. The changes in blood- pressure can be readily followed, and the effect of treatment more certainly demonstrated. APPARATUS. 301 APPARATUS. The following apparatus!^ will be found necessary to con- duct the examinations described in this book : — FOR Burette and pinch-cock. Pipette, 1 c.c. Pipette, 5 c.c. Pipette, 10 c.c. Plask, 1000 c.c. Plask, 100 c.c. Graduated cylinder, 50 c.c. TJrinometer and jar. TJreometer, Doremus. Albuminometer, Bsbach. Plain glass pipettes. Glass stirring-rod. Porcelaifa dishes. Porcelain tile. Beakers. URINALYSIS. Sedimentation glasses. Covers for glasses. Funnels. Test-tubes, assorted sizes. Centrifuge. Plain centrifuge tubes. Graduated Purdy tubes. Bunsen burner and hose. Tripod. Wire gauze. Burette stand. Filter paper. Microscope. Westphal Balance. FOR BLOOD AND Gower's, Fleichl's or Sahli's hemoglobinometer. Thoma-Zeiss hemocytometer. Drop-bottles and droppers. Hypodermic syringe. Sphymomanometer. Daland lancet. Microscope and accessories. BLOOD-PRESSURE. Microscope slides. Cover-glass, % in. diameter. Mechanical stage. Spectroscope. Wright's blood-coagulation ap- paratus or Bogg's modifica- tion of Eussell and Brodies' apparatus. FOR STOMACH EXAMINATION. Two Mohr's burettes (35 and 50 cubic centimeters) will be required. Dimethyl-amido-azo.benzol paper. Dimethyl-amido-azobenzol (Topfer's reagent). '*A complete price list of reagrents and apparatus noted above may be obtained from Geo. P. Pilling & Son Co., Twenty-third and Arch Streets, Philadelphia. 302 APPENDIX. Deci-normal sodium hydrate. Phenol-phthalein (1 per cent, alcoholic). Sol. neutral ferric chlorid. Congo-red paper. Starch paper. Powdered pepsin. Sodium carbonate. Potassium hydrate, 10 per cent. sol. Egg albumin in cubes or buttons preserved in glycerin. Mett's Capillary Albumin Tubes. FOR FECES, CEREBRO-SPINAL FLUID AND MILK. Stool sieve. Breast-pump. Hydrometer, 1010 to 1040. Graduated cream-gauge. Cream centrifuge tube and pipettes. Capillary tube, heavy glass. Diameter, 0.05 mm. Length, 800 to 1000 mm. FOR BACTERIOLOGIC AND OPSONIC WORK. Incubator. McFarland & L'Engle's neph- 1 pr. balances accurate to 0.2 elometer. gram. Arnold steam sterilizer. Bunsen burner. Thermo-regulator. Thermometer registering 200° Eubber tubing. C. Platinum wire loops, glass Thermometer graduating in handle. tenths. Eegistering from Sterile bouillon in tubes. to 50° C. Agar slants. Large watch-crystals. Capillary pipettes. Pure culture of typhoid bacil- Hanging drop slides. lus. CHEMICALS AND REAGENTS. 303 CHEMICALS AND REAGENTS. The following chemicals and reagents will he required to carry out the examinations outlined in this work: — '■ CHEMICALS— FOR URINALYSIS. Acid, nitric. sulphuric. hydrochloric C. P. hydrochloric deci-normal. acetic, glacial. acetic, 10 per cent. salicylic. Ammonium hydrate. Potassium hydrate (sticks). " 20 per cent, solution. Sodium hydrate (sticks). " 40 per cent, solution. " deci-normal solution. Silver nitrate, standard sol. Copper sulphate, 1 to 10 sol. Ammonium sulphate, sat. sol. Sodium chlorid, sat. sol. Litmus papers. Ethyl alcohol. Methyl alcohol. Amylic alcohol. Potassium chlorate, 1 per cent. sol. Lead acetate (tribasic) . Sodium acetate. Potassium ferrocyanid. Zinc chlorid, 5 per cent. aqu. sol. ' Potassium acetate. Chloral. Sodium carbonate. Neutral ferric chlorid. Bajium chlorid, standard sol. Sodium nitroprussid. Solution hydrogen dioxid. Bromine water. Phenylhydrazin hydrochlorid. Ethylene diamine hydate, 10 per cent, aqueous. Zinc acetate, 10 per cent, al- coholic sol. Phenolphthaiein, 1 per cent, alcohol, sol. Powdered guaiac. Orcin. Chloroform. Lead carbonate. FOR BLOOD. Normal saline solution. Benzol. Xylol. Chloroform. Litmus paper, red and blue. Canada balsam. Xylol balsam. Xylol dammar. Ether. % per cent, acetic acid. 2y2 per cent, potassium bi- chromate. 304 APPENDIX. ' REAGENTS— FOR URINE. Magnesia Mixture: — Ammonium ehlorid 1 part. Magnesium sulphate 1 part. Ammonia w"ter 1 part. Water 8 parts. The salts are dissolved in the water and the ammonia water then added. Knop's Solution: — Bottle "A"— Sodium hydrate sol 1 : 35 Bottle "B"— Bromine 1 part. KBr 1 part. Water 8 parts. For the test add 1 part of the bromine solution to 15 or 20 parts of the sodium hydrate solution. Pukdy's Reagent: — Potassium ferrocyanid 10 parts. Strong acetic acid 10 parts. Water 10 parts. Tanret's Reagent: — Dissolve 33.1 grams potassium iodid in 300 cubic cen- timeters of water. Add 13.5 grams powdered mercuric ehlorid and water, stirring until the red precipitate first formed has been dissolved. Dilute to 900 cubic centimeters with water and add 100 cubic centimeters strong acetic acid. Allow to stand twelve hours, and then decant from precipitate and use clear solution. This forms a solution of mercuric-potassium iodid in dilute acetic acid. Esbach's Reagent: — Picric acid 10 grams. Citric acid ■. 30 grams. Water 1000 c.c. CHEMICALS AND REAGENTS. 305 Fehling's Eeagent: — The reagent consists of two solutions which are kept in separate bottles until mixed immediately before using. Solution "A" — Copper sulphate 34.64 grams. Water 500.00 c.c. Solution "B" — Sodium-potassium tartrate 173.00 grams. Sodium hydroxid 135.00 grams. Water 500.00 c.c. These solutions are used in equal parts for the test. Nylandek's Reagent: — Bismuth subnitrate 2 grams. Rochelle salt 4 grams. Sodium hydroxid (8 per cent, sol.) 100 c.c. Trommee-Simrock Reagent : — Copper sulphate 2 grams. Potassium hydroxid (5 per cent, sol.) .... 150 c.c. Glycerin 15 c.c. » Distilled water 15 c.c. Puedy's Reagent for Sugar: — Copper sulphate 4.73 grams. Glycerin 38.00 c.c Water 300.00 c.c. These should be dissolved in the water by gentle heat. Potassium hydroxid 33.50 grams. Water 300.00 c.c. Dissolve separately and then add to the copper solution. When cold add: — Ammonium hydroxid (strong) 450.00 c.c. Water, q. s 1000.00 c.c. DiAzo Reagent: — This reagent consists of two solutions which are kept separate until mixed for the test. Solution "A" — Sulphanilic acid 1.0 gram. Hydrochloric acid (con.) . . . 50.0 c.c. Water 1000.0 c.c. Solution "B"— Sodium nitrite 1.0 c.c. Water , 300.0 c.c. Proportion for test: "A," 5 c.c. "B," 3 drops. 20 306 APPENDIX. FOR BLOOD. Hayem's Diluting Solution for Counting Eed Blood- Cells : — Mercuric bichlorid 0.5 gram. Sodium sulphate 5.0 grams. Sodium chlorid 1.0 gram. Aq. dest ; 200.0 e.c. Toisson's Solution for Simultaneously Counting Bed and White Cells: — Methyl violet 0.05 gram. Neutral glycerin 30.00 e.c. Aq. dest 80.00 e.c. Mix and add : — Sodium chlorid 1.00 gram. ■Sodium sulphate 8.00 gram. Aq. dest. . 80.00 gram. Filter. Twelve minutes required to stain white blood- cells. Fixing Methods: — 1. Equal parts of absolute alcohol and ether. The specimen should be immersed in this for half to two hours. This method is particularly good for malarial parasites and degeneration in blood-cells. 2. Absolute alcohol for five minutes. 3. Flemming's solution: — Chromic acid^ 1 per cent 15 parts. Osmic acid, 2 per cent 4 parts. Glacial acetic acid 1 part. The blood-specimens, as soon as they are made, before they have time to air-dry, are plunged into this solution and allowed to remain for ten minutes. They are then washed in running water for ten minutes and dried. This solution is very useful for demonstrating the chromatin of nuclei. 4. Vapors of formaldehyde. 5. Heat. CHEMICALS AND REAGENTS. 307 6. Wood alcohol. This is coming rapidly in favor, as it can be mixed with the stain, thus reducing the time of preparing specimens. FOR GASTRIC ANALYSIS. Uffleman's Ebagent: — Carbolic acid (4 per cent.) 10 c.c. Water 20 c.c. Liquor f erri chloridi 1 drop. This solution should be a clear amethyst color, and should be prepared fresh for use. Lugol's Iodin Solution: — lodin ; 1 part. Potassium iodid 2 parts. Water 50 parts. Gunzbebg's Phloeoglucin Vanillin : — Phloroglucin 2 gms. Vanillin 1 gm. Alcohol 30 c.c. This solution, if active, is pale yellow. It darkens and deteriorates with age, especially on exposure to light, so should be kept in colored bottles and made fresh from time to time. FOR CEREBRO-SPINAL FLUID AND MILK. Saturated aqueous solution of methyl violet. (R 5.) Ceeam Testing Solutions : — "A" — Amylic alcohol 37 parts by volume. Methyl alcohol 13 parts by volume. Hydrochloric acid 50 parts by volume. B"— Sulphuric acid. sp. gr. 1832. 308 APPENDIX. STAINS. Ehrlicii's "Teiacid" Stain: — Orange G 13.0-14.0 c.e. Acid fuchsin , 6.0- 7.0 e.c. Distilled water 15.0 c.e. Methyl green 25.5 e.c. Alcohol 10.0 c.e. Glycerin 10.0 c.e. The three stains, orange G, acid fuchsin, and methyl green, are prepared in saturated aqueous solutions, and then mixed in the above amounts while being shaken thoroughly. EosiN AND Methylene-Blue : — Eosin 0.5 per cent, in 70 per cent, alcohol. Stain for a few minutes, wash, blot, and apply. Methylene-blue 1 per cent, aqueous. Stain for a few minutes. ■ Chezinsky Stain : — ■ Methylene-blue, sat. aq. sol 40 c.e. Eosin, 0.5 per cent, in 70 per. cent, alcohol. ... 20 c.e. Distilled water 40 c.e. The specimens are fixed in absolute alcohol for from five to thirty minutes, and stained in a thermostat at 37° C. for from three to six hours. Hematoxylin-Bosin (Bhrlich's mixture) : — Eosin (crystals) 0.5 gram. Hematoxylin 2.0 gram. Absolute alcohol, 1 Distilled water, I of each 100.0 gram. Glycerin, J Glacial acetic acid 10.0 gram. Alum in excess. This mixture should be allowed to stand for several weeks before it is ready for use. The specimens are stained in from half to two hours. Caebol Gentian-Violet : — Gentian-violet, cone, alcoholic sol 10.0 c.e. Carbolic acid, 5 per cent, watery sol 100.0 e.c. STAINS. 309 GiEMSA Impeoved Stain : — Aziir II eosin 3-.0 gram: Azur II 0.8 gram. Exsiccate, pulverize, and sift. Dissolve in chemically pure glycerin at 60° C \...^. 250.0 c.c. Wheii solution is complete add: — ■ Methyl alcohol at temperature 60° C..' 350.0 c.c. Shake well, allow to stand for twenty-four hours, then filter. Decolorizing Solutions : — I. Acetic acid, 0.5 to 5.0 per cent, watery solution. II. Mtric acid, 30 to 30 per cent. III. Acid alcohol: — Sulphuric acid (cone.) 30 drops. Alcohol (95 per cent.) 50 c.c. Water 150 c.c. Loefflee's Alkaline Methtlene-E|ltje : — Concent, alcohol solution methylene-blue . . . 30;0 c.c. Potassium hydrate (%oooo) 100.0 c.c. Ziehl's Caebol-Fuchsin : — Fuchsin in substance 1 gm. Carbolic acid (cryst.) 5 gm. Alcohol (96 per cent.) 10. c.c. toistilled water 100. c.c. Or it may be prepared by adding to a 5 per cent, watery solution of: carbolic, acid a saturated alcoholic solution of fuchsin until a metallic. luster appears, on the surface of the liquiA, : , ^' • ' . . ' ' Gabbott's Method: — A — Fuchsin 1 gm. Absolute alcohol ., 10 c.c. , Carbolic acid. (5 per cent.) 100 c.c. B — Mfethylene-blue 3 gm. . Sulphuric, acid (35 per cent, sol.) 100 c.c. 310 APPENDIX. Gram's Iodine : — lodin 1 gm. Potassium iodid 2 gm. Distilled water 300 c.c. Koci-i-Ehelich Gentian- Violet : — Take distilled water, 100. cubic centimeters, and add anilin oil, drop by drop, until the solution has an opalescent appearance. The vessel containing the mixture should be thoroughly shaken after the addition of each drop. It is then filtered through moistened filter paper until the filtrate is clear. To 100 cubic centimeters of the filtrate add 10 cubic centi- meters of absolute alcohol and 2 cubic centimeters of concen- trated solution of gentian-violet. Unna's Orcein Stain: — Orcein in substance 1 gm. Hydrochloric acid 1 c.c. Absolute alcohol ' 100 c.c. Reagents for Staining Plagella : — Mordant. Tannic acid (20 ac. to 80 water) 10 c.c. Ferric sulphate cold sat. sol 5 c.c. Fuchsin sat. watery sol 1 c.c. Adjuvants. Sodium hydrate 1 per cent, aqueous solution. Sulphuric acid (1 c.c. equal 1 c.c. of 1 per cent. NaOH). Pappenheim's Solution : — Corallin 1 part. Absolute alcohol 100 parts. Add to the above solution methylene-blue in bulk to sat- uration. Finally add 20 parts of glycerin. Loefflbk's Mordant : — Tannic acid solution (3 parts acid, 80 parts water) 10. c.c. Ferrous sulphate saturated solution 5. c.c. Fuchsin saturated aqueous solution 1. c.c. STAINS. 311 Ebnke's Fluid: — Hydrochloric acid 2.5 com. Sodium chlorid, C. P 3.5 com. Distilled water 100.0 c.cm. Alcohol, 95 per cent 500.0 c.cm. Labeling Smeaes : — As it often occurs that a number of slides are made at one time, or a number of slides from one patient are. taken at dif- ferent hours, it is necessary that such slides should be labeled at once. The most convenient method is that of writing' on the end or back of a slide with ordinary ink. This should be quite dry before the staining process is begun, then there will be no fear of it washing off. Another method suggested by Powell is : After making a dry film, the name, date, and other necessary information are scratched on the film, with the head or point of a needle, the film used being so extensive that the writing in no way interferes with subsequent study. In place of the needle, E. H. von Ezdorf2 suggests the use of an ordinary black lead pencil, preferably soft. The label thus made on the blood film being a carbon deposit remains permanent and is not affected by the staining and washing of the slide. A New and Stable Solution op Gentian-Violet: — ■ The decided tendency of the average gentian-violet solution to decompose, especially in warm weather, is a difficulty fre- quently encountered by the laboratory worker. The result is loss of the entire solution and riiuch time required to make a new one. The following suggestions are practical and obviate this difficulty. Dr. E. Burvill-Holmes has had success with the addition of 3 to 5 per cent, of glycerin to the stain which improves its stability if kept in a dark, cool place. Muir and Ritchie recommend the use of phenol water 1 part in 10. These methods while being improvements do not prevent decomposition of the stain. Eobert Kilduffe recommends the following preparation which he has found to work admirably. Two stock solutions are employed : A. 5 cubic centimeters 40 per cent, formalde- hyde are added to 95 cubic centimeters of distilled water. B. » Jour. A. M. a., Jan. 8, 1910. 312 APPENDIX. Saturated alcoholic solution of gentian-violet. Mixing these in the proportion of 25 parts of B and 75 parts of A such a solu- tion has been kept for a long time at ordinary temperatures with- out deterioration. The advantages of this solution are said to be: (1) It does not decompose. (2) Moulds cannot grow in it. (3) No modification of technic necessary. (4) Preparations made with it are sterile. Univeesal Staining Method : — The discovery of the process whereby two or more colors could be chemically combined in one staining reagent, marked a great advance in the field of hematology. It affords greater opportunity for more detailed study of the minute structure of the cells of the blood, which in turn has resulted in a better classification of the different elements through the differentia- tion of new and distinct varieties which, until recently, have been unrecognized. Of these combination stains the Polychrome or "universal" staining method is by far the best and most prac- tical, and is now rapidly superseding the older and more cum- bersome methods. For this reason it becomes a matter of con- siderable importance, almost a necessity, that the clinical worker should have at hand one or more of these stains ready for imme- diate use when occasion requires. Unfortunately the preparation of this class of stain in- volves considerable expenditure of time, and demands no small amount of chemical knowledge and manipulative skill. These factors combine to limit the preparation of these stains to a comparatively small number of experienced workers, while the majority of the profession is dependent upon unstable liquid stains, obtainable through the supply houses, the composition of which is frequently so variable as to render the results value- less. Further, all aniline stains of this character are prone to decomposition when kept even for a moderately long time in solution. Fortunately the demand for a uniform and reliable stain has recently been met by a London Pharmaceutical HousBj^" who now carry in stock a number of ver}' uniform and perfectly re- liable stains under the name of "Soloid" brand. These stains are very carefully prepared and their composition practically ^•^ Burroughs Wellcome & Co., with a branch at 45 Lafayette Street, New York City. STAINS. 313 uniform. A definite quantity of the dried stain is compressed into a tiny tablet and dispensed in vials containing six. Each package is accompanied with information indicating the proper dilution and best working conditions for that particular stain. The "Soloid" stains permit of the preparation of small quantities of liquid stain whereby waste through evaporation or decomposition is reduced to a minimum, at the same time the results obtained, as far as the author has employed them, are in every way satisfactory. The following is abstracted from the last descriptive cata- logue of the above company: — The majority of the stains are employed in alcohol in solu- tion, and the different alcohols commonly used are here described. Absolute Alcohol contains not less than 99 per cent, by weight of pure ethyl alcohol, C2H5OH. Alcohol of a Stated Peecbntage, e.g., 50 per cent, alco- hol, means a mixture with water which contains the stated per- centage, i.e., 50 per cent, by volume of pure ethyl alcohol. Methyl Alcohol is a pure substance, CH3OH, prepared by the purification of commercial wood spirit. Commercial methyl alcohol, which is impure, must not be employed in the preparation of Jenner's, Leishman's, or Eomanowski stains. The amounts of distilled water and absolute alcohol re- spectively, required to produce saturated solution of certain dyes in common use, are indicated in the following table : — "Soloid" product of 0.1 em. of dye Water (CO.) Alcohol (c.c.) 7 7 10 2.5 7 7 2 1 5 1 7 7 5 . 10 Bismarck -Brown, pure. Fuchsin, Gentian-Violet, Hematoxylin, Methyl Violet, Methylene-Blue, Thionin Blue, Aqueous dilutions of the above, containing 5 to 10 per cent, of these saturated solutions, are wett adapted for ordinary staining purposes. Various other solutions, ready for imme- diate use, may be prepared from "Soloid" Microscopic Stains according to the following directions: — 314 APPENDIX. EosiN. — To obtain a solution of eosin suitable for general staining, one "Soloid" product may be dissolved in 30 cubic centimeters of 50 per cent, alcohol. This gives a 0.5 per cent, solution. Loeffler's Alkaline Methylene-Blue. — Dissolve one "Soloid" methylene-blue in 7 cubic centimeters of absolute alcohol, and add 25 cubic centimeters of distilled water, to which one drop of Liqiwr Potassae U.S. P. has been added. Aniline Gentian- Violet. — Dissolve one "Soloid" gen- tian-violet in 7 cubic centimeters of absolute alcohol, and add 63 cubic centimeters of a freshly filtered saturated solution of aniline oil in distilled water. Caebol Gentian- Violet. — Dissolve one "Soloid" gentian- violet in 7 cubic centimeters of absolute alcohol, and add 63 cubic centimeters of a 1 per cent, aqueous solution of carbolic acid. ZiEHL^s Carbol-Fuchsin. — Thoroughly powder and dis- solve one "Soloid" fuchsin in 3 cubic centimeters of absolute alcohol, add 22 cubic centimeters of 5 per cent, aqueous solu- tion of carbolic acid ; shake well, and filter before using. Gram's Iodine Solution. — Dissolve one "Soloid" product of reagent A in 10 cubic centimeters of distilled water, add one of reagent B, and when solution is complete, dilute to 15 cubic centimeters with distilled water. Caebol Thionin Blue. — Dissolve one "Soloid" thionin blue in 100 cubic centimeters of a 5 per cent, aqueous solution of carbolic acid. Borax Methylene-Blue. — Dissolve one "Soloid" borax methylene-blue in 10 cubic centimeters of distilled water. Delafield's Hematoxylin. — Dissolve one "Soloid" hema- toxylin (Delafield) in 10 cubic centimeters of a 25 per cent, solution of glycerin in water. Eosin- AzuR (for Giemsa staining with one solution). — Dissolve one "Soloid" product in 5 cubic centimeters of pure meth)i alcohol. Eosin-Methylexe-Blue (Louis Jenner's Stain). — Dis- solve one "Soloid" product in 10 cubic centimeters of pure methvl alcohol. STAINS. 315 RoMANOWSKY Stain (Leishman's Modification). — Dissolve one "Soloid" product in 10 cubic centimeters of pure methyl alcohol. Sodium Caebonate. — When employed in the preparation of Romanowsky stain, dissolve one "Soloid" product (0.05 gram) in 10 cubic centimeters of distilled water, and add one "Soloid" methylene-blue, 0.1 gram (for the method of prepa- ration and use, see British Medical Journal, September 31, 1901, page 757). Biondi-Bhelich-Heidenhain Teiple Stain. — Dissolve one "Soloid" Ehrlich triple stain in 25 cubic centimeters of distilled water; one "Soloid" acid fuschin in 2 cubic centi- meters of distilled water, and mix. The mixture is ready for use and keeps well. ToisoN Blood Fluid. — For the preservation of blood cor- puscles and the counting of the same. Dissolve one "Soloid" product in 3 cubic centimeters of glycerin and 16 cubic centi- .meters of distilled water. The solution should always be filtered immediately before use. The Polycheome Methylene-Blue-Eosin Stains (Eo- MANOWSKi). — There are about fifteen different modifications of this stain. The majority of them are difficult of manufacture, even by an expert, sO' it is recommended that they be bought ready-made from the laboratory apparatus and supply houses which make them. For those who desire to make this stain for themselves, the following modification by Hastings is appended^ as one which is comparatively simple of manufacture.^ All the Eomanowski stains are made with wood alcohol, which, during the first portion of its application, acts as a fixative. Hastings' Stain. — The dry stains necessary are eosin (water solution) yellow (Grubler), and methylene-blue (Ehr- lich's rectif.) (Grubler). ' Hastings : Johns Hopkins Hospital Bulletin, 1905. 316 APPENDIX. Solution "A" — Eosin 1 per cent, aqueous. Solution "B" — Alkaline methylene-blue 1 per cent, aqueous. Solution "C" — Methylene-blue 1 per cent, aqueous. Solution "A" may be kept ready-made, solutions "B" and "C" must be made fresh. To prepare solution "B," use a warm 1 per cent, solution of dry powdered sodium carbonate. Add to it one per cent, of methylene-blue powder, and heat over a water bath for 15 minutes. Add 30 cubic centimeters of water for each 100 cubic centimeters of the original fluid, and heat again for 15 minutes. Then pour off the solution from the residue and divide into two equal parts. To one part add enough 12.5 per cent, acetic acid solution to make a faintly acid reaction. This is best determined by taking a piece of blue litmus paper and allowing a drop to fall upon it, taking as the end reaction the point at which the margin of the drop after absorption in the paper shows a faint pink. Then add the remaining unneutralized por- tion to this. , To mix the stain use distilled water 1000 cubic centimeters. Solution "A," 100 cubic centimeters; solution "B," 200 cubic centimeters; solution "C," 70 to 80 cubic centimeters. In add- ing solution "C" put in 70 cubic centimeters at once, and stir well ; if no precipitate appears, add 1 cubic centimeter at a time until one does appear. After the precipitate appears the stain is allowed to stand for half an hour and then filtered through one filter. Forced filtration is generally necessary. The dry residue is removed from the paper and pulverized. It may be kept in this form or dissolved in Merck's pure methyl alcohol. Seven- to nine-tenths of a gram of dried stain is usually obtained. Three-tenths of a gram is dissolved in 100 cubic centimeters of alcohol for the staining solution. In dis- solving the stain it must be rubbed up with the alcohol in a mortar, as the powder is with difficulty soluble. If there are more than, nine-tenths of a gram of the dried stain obtained, the preparation is useless, and should be begun again. TUBERCLE BACILLI IN THE SPUTUM. 317 For each new lot of stain made up, one must determine the relative proportion of stain and water used in staining, and the relative lengths of time during which the pure and diluted stain is allowed to act. Usually two drops of stain on the smear for one minute and then four drops of water added and allowed to act for four minutes gives the best result. For uniformity in dropping a dropper should be used. All polychrome methylene-blue stains require experiment, since different mixtures by the same method require slight variations in their use. These must be ascertained by trial. Use distilled water to wash the specimen, since tap-water may ruin it. TUBERCLE BACILU. THE IDENTIFICATION OF TUBERCLE BACILLI IN THE SPUTUM BY THE DOUBLE METHOD OF ELLERMANN-ERLANDSEN. H. Kogel^ describes this method, as follows: (1) One volume of sputum (10-15 cubic centimeters) is mixed in a stoppered glass bottle with one-half its volume of 0.6 per cent, sodium carbonate solution. The mixture now stands twenty- four hours in the thermostat at 37 degrees C. (3) The greater part of the supernatant fluid is decanted and remainder is cen- trifugated in a graduated centrifuge tube. The fluid is poured off. (3) Four volumes of 0.85 per cent, sodium hydroxide are added to one volume of the precipitate. After very carefully agitating, this is raised to a boil. (4) Centrifugate and make smear of sediments. The result of this treatment is that practically the entire bacillary content of the whole amount of sputum is spread on one or two slides and 20 to 30 times the number of tubercle bacilli occur per field. The method takes time. In an old sputum autodigestion may have gone far enough to make the first stage of the procedure unnecessary. Very thick sputa may require 48 hours in the thermostat. Very purulent sputa give poorer results than slimy ones. These must be left longer in ^Deutsche Med. Wochen., Dec. 2, 1909. 318 APPENDIX. the incubator and must be boiled longer, with larger amounts of the caustic. A powerful centrifuge is required, and the centrif- ugation must be continued long enough to precipitate com- pletely all the solid matter. The final precipitate consists almost entirely of bacteria. A glance is often all that is required for a diagnosis. Specimens which in the usual smear showed 10 tubercle bacilli to the field, by the double method showed 300 to 400. As a rule, 15 to 30 times, as many were seen. Of 105 specimens of sputum examined, by the usual method 21 were positive. Of those negative, 8 by the double method gave posi- tive results, or an increase of 8 per cent. ANTIFORMIN ISOLATION OF TUBERCLE BACILLL This method is employed to facilitate the detection of tubercle bacilli in pus, urine, sputum, exudates, etc. Antiformin may be bought or may be made by mixing equal parts of Javelle water and sodium hydrate. According to 0. Seeman''5 the sputum should be diluted with 10 to 15 times its volume of this mixture. The pus is rendered homogeneous, which change may be hastened by stirring. In a few minutes to a half hour after standing a clear fluid with a sediment is obtained. This sediment is best removed by decanting the clear fluid and centrifugating the remaining few cubic centimeters. The sediment thus obtained should be washed two or three times with distilled water in the centrifuge to remove the excess of alkali which might interfere with drying and staining the smear. If any difficulty is experienced in making the smear, adhesion may be facilitated by moistening the slide with a little fresh sputum or a few drops of albumin water (one part egg white, ten parts distilled water, two parts 1 per cent, formaldehyde) . This author further states that the tubercle bacilli are not killed by 15 per cent, antiformin in one hour, so that in this method it may be employed for animal inoculation. The method is also said to be valuable in differentiating typhoid fever, malaria and sepsis if applied to the blood, as the tubercle bacilli in the antiformin fluids are found in pure culture all other organisms having been dissolved. * Berlin. Klin. Wochen., April 15, 1909. TUBERCLE BACILLI IN THE BLOOD. 319 E. Burvill-Holmes'' employs the following technie : — "If the specimen of sputum is not large I mix it with an equal quantity of a 30 per cent, solution of antiformin in any sterile receptacle, usually a large test-tube or better, centrifuge tube. This is thoroughly shaken until all the nummular masses are dissolved. The mixture will take on a brownish color when this is complete. A little alcohol (95 per cent.) is now added as I have found that without it owing to, I presume the different densities of the two substances, the bacteria are not well thrown down. The tube is then placed in an electric centrifuge and centrifuged for 10 minutes at high speed. The mixture, except- ing that at the very bottom is pipetted off, and some normal NaCl is added to the small portion at the bottom of the tube and this is thoroughly mixed and again centrifugated for 5 minutes. Again all except the very bottom, which is placed on a clean glass slide upon which preferably a little Meyer's albumin has been smeared, is pipetted off. The slide is stained in the usual way." TUBERCLE BACILLI IN THE BLOOD. The following remarks dealing with the subject of Tubercle Bacilli in the blood are inserted mainly to give students a brief insight into the most interesting and, if corroborated, most important discovery. A year and more has elapsed since Eosenberger presented to the Pathological Society of Philadelphia a communication con- taining evidence of the presence of tubercle bacilli in the blood of practically all cases of tuberculous infection, including not only advance cases but also the most incipient. Pew communi- cations in recent years have aroused such widespread interest. Dr. Eosenberger's report was the logical outcome of a progressive series of studies leading to this final conclusion. Prom a careful examination of his preliminary reports it is evident that his studies were conducted with great pains and perseverance and that he arrived at his final conclusion, that tuberculosis is primarily a baeteriemia which is followed by secondary localiza- tion of the organisms, only after the most careful researches. This conception was so revolutionary that it could not be ^ Personal communication. 320 APPENDIX. accepted without much hesitation and without adequate con- firmation. It has long been known that tubercle bacilli are at times present in the blood. This is shown by the occasional occurrence of primary lesion of the bones, joints and deep-seated organs^ and of occasional cases of primary miliary tuberculosis. Following this announcement many investigators repeated these studies according to the methods of Eosenberger, from both the clinical and experimental aspects. So that during the past year many reports upon this subject have appeared in literature. Up to this writing the great bulk of evidence fails to substantiate this theory. The following references are inserted as the fair examples of results by competent investigators. From the clinical aspect Doctors Hewat and Southerland" obtained one positive in twenty-two examinations of twenty cases of tuberculosis. Schnitter^ obtained fifty per cent, positive in a series of cases of extra pulmonary tuberculosis. Bond Stow'' in ten cases of incipient tuberculosis found no tubercle bacilli in the blood, but had obtained six positive findings in eighteen advance cases. A. E. Taussig^'' examined six cases of tuberculosis all of which were negative and in which guinea-pig inoculations with the blood were all negative. EosenbergH examined twenty cases without a positive finding. M. A. Dailey^^ examined seventeen tuberculous cattle by guinea-pig inoculations without producing the disease. Stain smears of the blood showed an occasional acid-fast bacterium. Doctors Schroeder and Cottoni^ examined forty-two tuber- culous cattle. They inoculated one hundred and four pigs only one of which finally showed lesions resembling slightly tuber- culosis, llicroscopie examinations were all negative. With this strong evidence against the bacteriemic theory of tuberculosis several investigators attacked the problem from another aspect. Their efforts being directed towards finding pos- ' British Med. Jour., Oct. 6. 1909. 'Deutsche Med. Wochen.. Sept. 9, 1909. »Mea Rec, Dec. 11, 1909. "Interstate Med. Jour., Oct., 1909. "Med. Rec, Nov. 13, 1909. '-Boston Med. and Surgr. Jour., Sept. 2, 1909. "Arch. o£ Internal Med., Aug., 1909. TUBERCLE BACILLI IN THE BLOOD. 321 sible sources of contamination or flaws in the teehnic. Walter V. Bremmi* showed that the occurrence of acid- and alcohol- resist- ing rods in water, even when distilled, are not uncommon and unless great care is taken to exclude this contamination the results may be of no value. On November 11, 1909, E. Burvill-Holmes reported find- ing acid-fast bacilli in the distilled water in his laboratory and in the Bryn Mawr Hospital. The failure of competent observers to substantiate Kosen- berger's findings on the one hand (even by animal inoculation) and on the other, the probability that bacilli regarded' as tubercle may have been introduced accidentally into the stains, etc., warrant the conclusion that as yet we have no positive evidence of an almost constant occurrence of tubercle bacilli in the blood of ordinary cases. It would seem that the whole subject of the differentiation of acid-fast bacilli is one that calls for more thorough investigation by bacteriologists, as there are many other organisms like the grass or hay bacillus almost universally present, which are with difficulty decolorized by acids. In addition we may encounter the smegma bacillus, Lusehgarten's bacillus, the bacillus of leprosy and other micro- organisms which have this peculiarity in common. It would, therefore, be rash indeed to pronounce any acid-fast bacillus as tubercle in the absence of corroborative clinical evidence or culture tests. On the contrary there seeifis every reason to believe that the tubercle bacillus does not often circulate in the blood in quanti- ties capable of ocular demonstration or by animal experimenta- tion, and that at the present time the theory that tuberculosis is a primary bacteriemia is unsupported by reliable evidence. Through the courtesy of Dr. Handle C. Eosenbergeris the originator of the test, I am enabled to give in full the methods employed by him in isolating these acid-fast organisms from the blood and feces. The Method. — The Blood: Take five or ten cubic centi- meters of blood from a vein in the arm of the patient (after having cleansed the part thoroughly) . The blood is best drawn "Jour. A. M. A., Sept. 18, 1909. 15 Personal communication. 322 APPENDIX. through a large hypodermic needle fitted with a short piece of rubber tubing, which is held so as to extend into a sterile test- tube or centrifuge-tube. The tube should contain five cubic centimeters of a sterile two-per-cent. solution of neutral sodium citrate to prevent the least coagulation. Antiformin is now .added (pure) until the blood is entirely destroyed. Only a very small quantity of antiformin is needed and should be added, a few drops at a time. The specimen is now centrifugated for twenty minutes, the supernatant fluid carefully poured off and the small precipitate carefully washed with sterile distilled water to remove the antiformin, again centrifugate, collect the precipitate on a perfectly clean slide, dry, fix, and stain for five or ten minutes with cold carbol-fuchsin ; wash in water and then apply Pappenheim's solution. The slide is left in Pappen- heim's solution for two or three minutes, washed with water and the stain again applied for five minutes; this procedure is repeated until the slide actually receives twenty minutes inter- mittent immersion in the Pappenheim's solution. Wash in water, dry and mount, or examine ijnmediately. without mount- ing for red bacilli. The Feces. — Make a spread from any portion of the stool upon a clean slide; dry, fix and stain as above (for blood). 11 acid-fast organisms are not fotmd by this method, take about five grams of the specimen and add pure antiformin, one part antiformin to four or five parts feces, and allow this to act for from fifteen to twenty minutes; then add sterile distilled water; centrifugate; wash the sediment with distilled water; again centrifugate; collect the sediment upon a clean slide, and spread and stain as before. As a rule by this procedure all other bacteria are destroyed so that the only organisms found will be those of tuberculosis. GASTRIC EXAMINATION. Determination of Pancreatic Activity. — The determination of pancreatic activity by the Oil Test Meal is based upon the experimental work of Boldiroff, who found that the introduction of oil into the stomach of dogs caused regurgitation of the duodenal contents in which pancreatic ferments could be demon- GASTRIC EXAMINATION. 323 strated. Volhard devised a method of demonstrating trypsin and applied it to clinical uses. This method has recently attracted sufficient attention to warrant a brief consideration of the method at this time, although the findings have not yet been proven conclusive. The clinical applicaiioii as advocated by C. B. Farr^^ is as follows : — From 100 to 200 cubic centimeters of olive oil or cotton- seed oil are administered through the stomach tube, into the fasting stomach. Food-remains or fluid if found in the stomach should first be removed through the tube. At the expiration of half an hour the contents of the stomach is aspirated; at this time there is usually recovered only a few cubic centimeters of a whitish mucoid fluid, in others as large amount of pale green or dark green fluid is obtained. The tests are carried out upon the fluid without filtration. Eeliable material for the trypsin test is not always forth- coming as, while practically always some fluid is recovered, fre- quently this is nothing more than a small amount of retained or freshly secreted gastric Juice or mucus. Farr suggests that the best criterion as to the character of the fluid should be its color, indicating the presence or absence of bile, and its response to the action toward ferments. I would suggest that the reaction would be also of great value in deter- mining the origin of the fluid. Methods. — Tlie Mett Method (Fully described on page 133). This may be successfully employed, the only modifica- tion being the substitution of a decinormal solution of sodium carbonate for the HCl as described; particles of fibrin are placed in the dishes to detect tryptic digestion. Method of Gross. — A 0.1-per-cent. solution of casein in a O.l-per-eent. sodium carbonate solution is prepared and 10 cubic centimeters placed in each of several test tubes. The trypsin solution, serially diluted. with water is added in 1 cubic centi- meter amounts to the several tubes and all are incubated at 37° C. for 24 hours. At the end of this time 1 per cent, acetic acid is added to each of the tubes and the dilution noted in the "Jour. A. M. A., Dec. 11, 1909. 324 APPENDIX. tube in which cloudiness last appears. If the casein is com- pletely digested no cloud appears. Quantitative Method. — Up to this time no practical method of determining the amount of trypsin present in a given specimen has been found, so that we are only to say whether trypsin is present or absent. The Clinical Application. — ^There is yet not sufficient clini- cal data at hand from which to draw any conclusive findings. Lewinski^''' and others believe that the absence of trypsin after this procedure shows either pancreatic insufficiency or pyloric spasm. This may be true, but the evidence is as yet insufficient. It would be safer to say that the presence of trypsin in the oil test-meal would exclude any disease involving to a marked degree the activity of the pancreatic gland. A New Reaction for Bile Pigments in Gastric Contents. — A. V. Torday and A. Klier^^ in working with the stomach- contents of a Jaundiced patient found that the methyl-violet, with which they were testing for free hydrochloric acid, gave a red color instead of the usual blue. The urine of a jaundiced patient gave the same reaction. Investigating further, they found that various staining fluids gave similar reactions with the bile-stained urine. Pure bile pigments have not been studied. They suggest the following : — Test. — The method consists of adding one drop of a 1-per-cent. solution of the dye to 15 cubic centimeters of water, and to this 1 cubic centimeter of gastric filtrate. According to these observers the delicacy of these tests were found to be about twice as great as the iodine or the Gmelin test. BLOOD. A Computing Chart for the Differential Leukocyte Count. — The accompanying chart (Fig. 35) has been suggested by Dr. Osmond^^ to simplify the work and remove the sources of error in the differential count. The chart is inexpensive and can be made out of a piece of ground glass or of slate suitably ruled and ■"Lewlnski; Deutsche. Med. Wochen., Sept. 10, 1908. " Med. Rec. , Oct. 2, 1909. "E. A. Osmond : Jour. A. M. A., Jan. 8, 1910. DIFFERENTIAL LEUKOCYTE COUNT. 325 marked in ink. The temporary markings are made in pencil and can be readily erased. It is figured out on a basis of two hundred cells and the marks on the left indicate the various types of cells encountered. At the bottom are the calculated ooe ^~ ~~ ~" 001 ni 061 B8 S8I 091 06 »[ oil «8 C9I 091 08 SSI OBI St Sfl Oil ot !8I OSl