'.:■■, . Class Book. CopightN , |120 COPYRIGHT DEPOSIT. PRACTICAL CLINICAL LABORATORY DIAGNOSIS A THOROUGHLY ILLUSTRATED LABORATORY GUIDE EMBODYING THE INTERPRETATION OF LABORATORY FINDINGS, DESIGNED FOR THE USE OF STUDENTS AND PRACTITIONERS OF MEDICINE BY CHARLES C. BASS, M.D. \i PROFESSOR OF EXPERIMENTAL MEDICINE AND DIRECTOR OF THE LABORATORIES OF CLINICAL MEDICINE, THE COLLEGE OF MEDICINE, TULANE UNIVERSITY OF LOUISIANA AND FOSTER M. JOHNS, M.D. ASSISTANT PROFESSOR OF MEDICINE IN THE LABORATORIES OF CLINICAL MEDICINE, THE COLLEGE OF MEDICINE, TULANE UNIVERSITY OF LOUISIANA ILLUSTRATED WITH 133 BLACK AND WHITE TEXTUAL FIGURES AND 19 PLATES IN COLORS SECOND EDITION, REVISED NEW YORK REBMAN COMPANY ^ ^?> Copyright, 1920, by CHARLES C. BASS, M.D. • AND FOSTER M. JOHNS, M.D. m 1 3 192U PRINTED IN AMERICA ©CI.A566545 ! PREFACE TO SECOND EDITION The entire first edition was so quickly exhausted that we feel confirmed in our former opinion that there is great need for just such a simple laboratory guide as this was in- tended to be. We are indeed grateful for the reception it has had. Its adoption and increased use in other schools has been especially gratifying. Judging from the useful purpose the book has served our students, we look for wider use by students — both graduate and undergraduate. In the second edition we have endeavored to correct such errors as have been discovered in the first edition and to bring it up to date in every way. Additions have been made wherever necessary, but we have not departed from our former aim of putting the essential facts in the shortest and clearest form possible. No radical changes have been made. We appreciate the kindness of those who have pointed cut errors in the first edition and made many valuable suggestions. The Authors. m PREFACE TO FIRST EDITION Several years of teaching Clinical Laboratory Diag- nosis, making laboratory examinations and often interpret- ing the findings, has forcibly impressed us with the need of such a book as this is intended to be. We take up only those laboratory examinations that are useful in ordinary, every-day practice and do not include tests that are seldom used or that are practical only for special laboratory workers. Our plan throughout the book is to give only one method of making a test or examination. We select what we con- sider the best, simplest, most practical method of making each test and do not sacrifice space or clearness by giving several tests or methods for obtaining the same information. All apparatus employed are specified, usually illustrated, and when thought to be advantageous, a source of supply considered reliable is given. Reagents are all specified, the formulae given, and an economical and practical source of supply is stated when thought advantageous. Some of the technic is original. Many of the "short cuts" and simplified steps are our own personal adaptation of the work of others. We wish to give full credit here and to state that all we know of laboratory diagnosis or any other subject has been learned either directly or indirectly from the work of others. All the illustrations and colored plates are original. They are accurately drawn and colored, fre- quently with the aid of the camera lucida. It often occurs that a thing can be illustrated better by pictures than by written description. Written descriptions are sometimes too technical to be easily understood or too long for convenience. A single picture often shows as much as can be described VI PREFACE on a page or more of type. The picture can be read at a glance, while the page of print requires minutes to read. In an appendix we give a list of the necessary material and apparatus required for the laboratory diagnosis de- scribed in the text. To do all ordinary laboratory tests one requires only a very limited amount of material and appara- tus, and a knowledge of how to use them. We are not ad- vertising for anybody, but wherever deemed advisable we have not hesitated to recommend apparatus or material of a particular make or from a source considered reliable. In the text we give, as fully as space will permit, the interpretation and relative value of the different findings. It would, of course, be impossible to discuss thoroughly in a work of this kind every possible indication of all the tests. Though the book is intended primarily for students and those doing only the ordinary every-day clinical laboratory work, it is believed that laboratory specialists may also find in it some practical suggestions of value to them. We wish to express our appreciation of suggestions made by many friends and associates, and the opportunities that our positions in the Tulane College of Medicine have furnished which have permitted the experience and ob- servations on which this book is largely based. The Authors. CONTENTS CHAPTER I PAGE Use and Care of the Microscope 1 Selection of a microscope — Correct position of the microscope — Source of light — Adjustment of the mirror — The Abbe condenser — Focussing — Oil immersion lenses. CHAPTER II Beood 12 Obtaining blood for all microscopic examinations — Mak- ing blood spread for differential leucocyte- count, examina- tion for malaria plasmodia, looking for abnormal cells, making typhoid agglutination test, etc. — Staining blood slides with Wright's stain — Description of the leucocytes found in normal blood — Method of making a differential leucocyte count — Interpretation of variation in the pro- portion of the normal leucocytes in the blood — Description of abnormal or pathological leucocytes — Interpretation of the presence of pathological leucocytes — Total leucocyte counts — Special apparatus and material required — Clean- ing the pipette and making the dilution — Making the prep- aration — Counting the cells — Interpretation* of total leu- cocyte counts — Counting the erythrocytes — Estimation of the hemoglobin — Color index — Interpretation of number of erythrocytes, hemoglobin per cent, and color index — Pathological erythrocytes — Interpretation of pathological erythrocytes. CHAPTER III Malaria 54 Obtaining blood and making preparations for examina- tion for malaria plasmodia — Making the examination and recognition of plasmodia — Description of malaria plas- modia — Differentiation of malaria plasmodia — Interpreta- tion of examination of the blood for malaria. Vlll CONTENTS CHAPTER IV PAGE Typhoid Agglutination Test 65 Technic — Interpretation of the typhoid agglutination test. CHAPTER V Urine 70 Collection of specimens for examination — Specific grav- ity — Test of the reaction — Test for albumin — Test for sugar — Test for indican — Test for acetone — Preparation of specimens for microscopic examination — Method of ex- amination — Diagnosis of microscopic objects commonly found in urine — Interpretation of urine examinations. CHAPTER VI Gastric Contents 87 Obtaining material to be tested — Test for free HC1 and total acidity — Other examinations — Interpretation of the findings in gastric contents. CHAPTER VII Feces ; . 90 Examination for intestinal parasite ova and larvae — Collection of specimens — Making the preparation for examination — Concentration of ova by means of the centri- fuge — Method of examining a slide preparation — Unci- naria americana ova — Ascaris lumbricoides ova — Trichuria trichuris ova — Oxyuris vermicularis ova — Hymenolepis nana ova — Tenia saginata and tenia solium ova — Larvae of uncinaria and strongyloides — Examination for amebas in amebic dysentery — Collection of specimens for examina- tion — Examination of unstained material — Technic of staining am.ebae and examination of stained specimens — Differentiation of pathogenic from non-pathogenic amebae — Interpretation — Test for occult blood — Technic of test — Interpretation. CHAPTER VIII Pus and Exudates Generally 108 General remarks — Making the preparation and stain- ing — Application in practice. CHAPTER IX Sputum 112 Examination for tubercle bacilli — Collecting specimens — Making the preparation — Staining — Description of tubercle bacilli — Interpretation. CONTENTS IX CHAPTER X PAGE Leprosy 120 Technic of obtaining material and making preparations for examination — Staining — Appearance of lepra bacilli — Interpretation. CHAPTER XI Spinal Fluid 122 Meningitis — Obtaining material and making preparation — Meningococci — Pneumococci — Tubercle bacilli — Other bacteria — Cells present — Test for globulin increase — Inter- pretation. CHAPTER XII Diphtheria 127 Principles of laboratory diagnosis of diphtheria — Ma- terial required — Making the culture — Incubation — Exam- ination of culture — Making preparations for examination ■ — Staining — Description of diphtheria bacilli — Interpreta- tion. CHAPTER XIII Gonorrhoea 136 Obtaining material and making preparation — Carbol- fuchsin and methylene blue stain — Gram's staining method ■ — Appearance of gonococci — Interpretation. CHAPTER XIV Syphilis 141 Examination for Treponema pallida — Obtaining material and making preparation for examination with the darkfield condenser — Examination with the darkfield condenser — India ink preparation — Differentiation between Treponema pallida and other spirochetes — Gland puncture — Interpre- tation — Wassermann serum test — Materials required — Ap- paratus required — Method of making the test — Control — Test — Interpretation. Appendix 163 Complete list of apparatus and material required. Index 169 LIST OF ILLUSTRATIONS FIG. PAGE 1 — Bausch & Lomb microscope 2 2 — Spencer "No. 44H" microscope 3 3 — Proper position at the microscope 4 4 — Learning to look in the microscope with both eyes open. . 5 5 — Leitz microscope with Mazda lamp 5 6 — Improper use of concave mirror with Abbe condenser. ... 6 7 — Abbe condenser , 7 8 — Abbe condenser in proper position 8 9 — Abbe condenser too low 9 10 — Photomicrographs of the same field, showing effect of proper and improper illumination 10 11 — A good blood sticker 12 12 — Squeezing finger when sticking to lessen pain sense 13 13 — Squeezing ear lobe 13 14 — First step in obtaining blood from yourself after sticking the finger 14 15 — Constricting patient's finger after having made the punc- ture ; 14 16 — Squeezing out the blood. 14 17 — Second step in obtaining blood from the finger 15 18 — Squeezing blood from the patient's finger 15 19 — Taking up blood from the patient's finger 16 20 — Taking up blood on slide from your own finger 16 21 — Taking up blood on slide from ear lobe of patient. . . 17 22 — A good general purpose blood spread labeled with an ordinary lead pencil 18 23 — Proper pose of hand and fingers to receive slide with blood on it 19 24 — Slide with drop of blood on it held in proper position for spreading 19 25 — Slides held in proper position preparatory to spreading blood 19 26 — A. Indicating how blood collects beneath the spreader slide when the latter is held at proper angle 20 B. Indicating how blood collects in front of end of the spreader slide when the latter is held at too great an angle 20 27 — Indicating angle at which slides should be held and direc- tion in which the spreader slide should be moved in mak- ing blood spread 21 x CONTENTS XI FIG. PAGE 28 — The blood has been spread by pushing (not pulling) the spreader slide quickly , . 21 29 — Diluting Wright's stain on slide , 23 30 — Indicating manner of crossing and recrossing blood film in making differential leucocyte counts 27 31 — Looking in the microscope with the left eye in making dif- ferential leucocyte counts 29 32 — Diluting pipette 34 33 — Bass counting chamber 34 31 — Counting chamber. Cross section 35 35 — Bass ruling 36 36 — Taking up blood into the diluting pipette from the pa- tient's finger 37 37 — Filling diluting pipette with diluting fluid 38 38 — Revolving pipette to mix contents 38 39 — Pipette containing diluted blood 39 10 — Cleaning cover-glass 10 11 — Counting chamber resting face downward against the foot of the microscope after it has been cleaned 10 42 — Squeezing out diluted blood upon the counting plate (Tiirck's counting chamber) 41 43 — First step in placing cover-glass upon the counting chamber 41 11 — Second step 42 45— Third step 42 46— Fourth step 43 47— Fifth step 43 48 — Looking at distribution of cells upon ruled plate 44 49 — Even distribution of cells upon the ruled plate 45 50 — Uneven distribution of cells upon the ruled plate 45 51 — Order in which the erythrocytes in the sixteen small squares in one large square should be counted 47 52 — The twelve cells to be counted in this small square are shaded 48 53 — Spreading the drop of water over the blood to dissolve it in making typhoid agglutination test 66 54 — Tilting slide back and forth to facilitate mixing and to hasten agglutination 66 55 — A series of agglutination tests 67 56 — Looking at typhoid agglutination test 68 57 — Looking at typhoid agglutination test at night by the light of a match 69 58 — Testing specific gravity with urinometer 71 59 — Boiling urine in test for albumin 71 60 — Adding acetic acid to urine from a drop bottle in testing for albumin 72 Xll LIST OF ILLUSTRATIONS FIG. PAGE 61 — Looking for faint cloud in test for albumin 73 62 — Cloud due to albumin in urine as seen in the proper light against a dark background 73 63 — Filling tube with urine to a depth of 2% inches in quanti- tative test for albumin 74 64 — Preparing fresh Fehling's solution in testing for sugar. . 75 65 — Boiling Fehling's solution and urine in testing for sugar . . 76 66 — Adding urine from 1 cc. graduated pipette in quantitative test for sugar 77 67 — Electric centrifuge with Cornell shields 79 68 — Box guard around electric centrifuge 79 69 — Hand centrifuge with Cornell shields 80 70 — Filling balance tube to height of column of urine 81 71 — Pouring out sediment and spreading on slide with mouth of the tube 81 72 — Proper spread of urine sediment 82 73 — Proper bottle containing sufficient formed feces for exam- ination 91 74 — Stirring feces in a couple of drops of water on a slide to make properly diluted preparation 91 75 — Proper spread of diluted feces showing also how print may be just read through it 92 76 — Photomicrograph of feces prepared by diluting in the ordinary way and showing two tapeworm ova 93 77 — Photomicrograph of the same specimen of feces prepared by centrifuging 94 78 — Solution of feces made in the bottle in which it was brought to the laboratory 94 79 — Straining diluted feces into centrifuge tube 95 80 — Diluted feces after centrifuging 95 81 — Pouring out and spreading sediment from feces on slide with lip of centrifuge tube 96 82 — Diagram indicating proper method of examining entire preparation 96 83 — Photomicrographs of different ova all taken with the same magnification for comparison 97 84 — Photomicrograph of ovum of tenia saginata enclosed within its vitelline membrane 100 85 — Photomicrograph of hookworm ovum and larva and ovum of trichuria 101 86 — Photomicrograph of anterior end of hookworm larva under high magnification 101 87 — Change of shape and position of an endameba during in- tervals of a few seconds each by ameboid motion 105 88 — Drawing illustrating morphological classification of bacteria 109 LIST OF ILLUSTRATIONS Xlll FIG. PAGE 89 — Proper spread of pus or similar material to be stained and examined 110 90 — Thoroughly draining sediment in bottom of centrifuge tube 110 91- — Picking out favorable material from sputum in a Petri dish 114. 92 — Touching the heated slide to the back of the hand to avoid getting it hot enough to damage the film of pus when fixing with heat 115 93 — Proper way to apply stain to a slide 116 94 — Heating slide in staining with carbol fuchsin 117 95 — Decolorizing with sulphuric acid solution 118 96^Scraping. leprous lesion 120 97 — Introducing the needle in making lumbar puncture with patient in sitting position 122 98 — The spinal fluid is allowed to drop directly into the centrifuge tube 123 99 — Bass diphtheria culture tube 127 100 — Removing cotton plug from culture tube preparatory to inoculating with the swab 128 101 — Inoculating culture media by rubbing swab over surface. . 129 102 — Sterilizing the platinum loop in flame of Bunsen burner. . 132 103 — Removing cotton plug, sterile platinum loop held in hand . 132 104 — Scraping the surface of the culture with the platinum loop to obtain bacteria for examination 133 105 — Proper long thin streak of material from the culture to be stained and examined 133 106 — Decolorizing with alcohol 139 107 — Scraping chancre 142 108 — Best form of darkfield condenser 143 109 — Diagram showing path of rays through a darkfield con- denser, and a /I2 inch oil immersion lens fitted with funnel stop 143 110 — Gas filled Mazda lamp for dark-ground illumination. . . . 144 111 — Illustration of the position of the funnel stop placed in the lens case of M2 inch oil immersion lens for darkfield work 145 112 — Photomicrograph of Treponema pallida in "chancre juice," as seen with the darkfield microscope 146 113 — Proper India ink preparation 146 114 — Photomicrographs of India ink preparations containing spirochetes 147 115 — Etherizing guinea-pig; preparing to draw blood 150 116 — Introducing the needle while steadying the parts 150 117 — Drawing blood from guinea-pig 151 XIV LIST OF ILLUSTKATIOXS FIG. PAGE 118 — Burroughs-Wellcome all- glass syringe with platino-irid- ium needle attached 152 119 — Drawing blood from patient 152 120 — Further distention of veins by clinching the fist 153 121 — Squeezing the arm to further distend the veins 153 122 — Introducing the needle 154* 123— Drawing the blood 154 124 — Making pressure over puncture to prevent bleeding under skin 154* 125— Wood test-tube block 2x4x8 inches 155 126 — Small electric incubator suitable for complement fixation tests on a small scale 156 127 — Showing arrangement of tubes in rack and use of 1 c.c. pipette in measuring serum, antigen, etc., into tubes. . 157 128 — Proper method of washing pipette with saline solution from a water bottle equipped with tube and pinch-cock. 158 129 — Scheme showing distribution of serum, antigen, etc., in the control tubes and the test-tubes 159 130 — Arrangement of work table where electric light is used, showing most of the material and apparatus required in ordinary microscopic work 164 131 — Micro burner, much better than the larger Bunsen burner 165 132- — Proper drop bottle for stains, reagents, etc 167 133 — Two kinds of improper drop bottles 168 LIST OF PLATES PLATE PAGE I — A. Small mononuclear leucocytes B. Large mononuclear leucocytes C. Polymorphonuclear neutrophilic leucocytes 24 / II — A. Polymorphonuclear eosinophilic leucocytes B. Polymorphonuclear basophilic leucocytes C. Relative sizes of the normal blood elements 24 v III — Neutrophils — Eosinophiles — Basophiles — Mature cells— Myelocytes 32 ' IV — Tallquist hemoglobin scale 49 V — Abnormal erythrocytes : Anisocytosis — Poikilocytes — Megaloblasts — Normoblasts- — Granular or stippled erythrocytes — Polychromatophilia 53 " VI — Estivo-autumnal malaria plasmodia 55 / VII — Tertian malaria plasmodia 55 VIII — Quartan malaria plasmodia 55" IX — Chemical tests of urine 71 v X — Microscopical findings in urine 83 XI — Test of gastric juice for free HC1 and total acidity. . 87 ^ XII — Intestinal parasite ova and larvae 97 K XIII — A and B. Bloody mucus in amebic dysentery C. Preparation from pyorrhea lesion 103 XIV— Test for occult blood 105 XV — A and B. Tubercle bacilli in sputum C. Lepra bacilli D. Pneumococci 118 ,. XVI — Diplococcus intracellularis — Diplococcus pneumoniae — Bacillus influenzae — Bacillus tuberculosis 124 ' XVII — Diphtheria bacilli from different cases suggesting variation in morphology 133 l XVIII — A and B. Pus in acute gonorrhoea C. Pus from case of acute non-specific urethritis D. Pus obtained from prostate and seminal vesicles by massage in case of chronic gonorrhoea 139 ^ XIX — Modified complement fixation test for syphilis 159 PRACTICAL CLINICAL LABORATORY DIAGNOSIS CHAPTER I USE AXD CAKE OF THE MICROSCOPE The microscope is used in a large part of the examina- tions made in the clinical laboratory. Therefore it is im- portant to have a good microscope and to know how to use and care for it. The best microscope will be of little service to one who does not know how to use a microscope properly. Selection of a microscope. — It so often occurs that those beginning laboratory work get useless and improper micro- scope equipment that we give here a selection of microscopes of the three standard makes in common use in this country best suited for the work described in this book. The German made Leitz microscope is a somewhat higher grade instru- ment than the American made Bausch & Lomb, or the Spencer, but costs considerably more on account of import duty imposed. It is questionable whether the additional ex- pense is warranted when the instrument is purchased for routine clinical laboratory work only. Either of the other two serves the purpose perfectly, though we have felt a cer- tain preference for the Bausch & Lomb. No serious mis- take will be made, however, in selecting any one of the three specified below. Leitz model "II L," with objectives No. 3 (16 mm.), No. 6 (4 mm.) and 1 /i2 // (1.8 mm.) oil immersion of N. A. 1.30; Ocular IV (10X) ; triple nosepiece; Abbe condenser of 1.20 X. A. with condenser iris diaphragm in substage (Fig. 5). E. Leitz, 30 East 18th Street, New York. Bausch & Lomb "F F S," with objectives 16 mm., 4 1 2 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Fig< l —Bausch & Lomb microscope described on page 1. Shown with mechanical stage attached. USE AND CARE OF THE MICROSCOPE 3 mm. and 1.9 mm. oil immersion; ocular lOx; triple nose- piece; Abbe condenser of 1.20 N. A. with condenser iris diaphragm (only) in substage (Fig. 1). Bausch & Lomb Optical Co., Rochester, New York. Fig. 2. — Spencer "No. 44-H." Shown without hiechanical stage. Spencer "No. 44-H," with objectives 16 mm., 4 mm. and 1.8 mm. oil immersion; ocular lOx; triple nosepiece; Abbe condenser of N. A. 1.20 with condenser iris diaphragm (only) in substage (Fig. 2). Spencer Lens Co., Buffalo, New York. A mechanical stage (Fig. 1) is essential for best work, 4 PRACTICAL CLINICAL LABORATORY DIAGNOSIS and one made by the same manufacturer and suited to the particular microscope stand selected, should be purchased with the microscope. If darkfield work is to be done (and it should be, if diagnosis of 'suspected syphilitic lesions is to be made) a darkfield condenser (Fig. 108) to fit in the substage of the particular microscope and a suitable funnel stop (Fig. Fig. 3. — Proper position at the microscope. Both eyes open. Ill) for the oil immersion lens should be purchased at the same time. If ordered later give the manufacturers the fac- tory number and exact description of your microscope. Correct position of the microscope. — Do not tilt your microscope stand. Sit up straight, close to the table, having the base of the microscope near the edge of the table (Fig. 3). Keep both eyes open. To learn to do this it is best to first turn the head to one side considerably (Fig. 4) and to gradually get it back straight. A few hours' prac- tice is usually sufficient to learn to keep both eyes open USE AND CARE OF THE MICROSCOPE Fig. 4. — Learning to look in the microscope with both eyes open. The head is turned to one side. Fig. 5. — A good light (Mazda lamp, 2.> watt) and light-holder in proper position. Leitz microscope described on page 1, also shown with mechanical stage attached. 6 PRACTICAL CLINICAL LABORATORY DIAGNOSIS while looking in the microscope with one. Learn to look in the microscope with either eye. and when using the low power lens (16 mm.)- Source of light.— A window on the north side of the room is best. Where electric lights are available they give the most uniform light and have the further advantage of USE AND CARE OF THE MICROSCOPE 7 being usable any time of day or night. A mazda lamp with round frosted globe is best. If some such lamp-holder as shown in Fig. 5 is used so the light can be placed near the microscope, a 25 watt mazda lamp will give sufficient light, otherwise use a 50 watt lamp. Adjustment of the mirror. — The mirror has one concave and one plane surface. When used without the Abbe con- denser (which is seldom done in our work), the concave sur- face gives the strongest light by concentrating the rays, while the plane surface gives the least light. These condi- tions are reversed (Fig. 6) when the Abbe condenser is being used. The plane surface should be used, therefore, practi- cally all the time. The mirror must be adjusted so that the best light is directed through the ob- ject. This is determined by tilt- ing the mirror from side to side while looking in the microscope. Xote the point at which the Fig. 7. — Abbe condenser re- . . , . moved from the sub-stage. brightest light IS Obtained. lhe This can easily be slipped out . -1 j • ■ -1 of its carrier. The iris dia- mirror must be readjusted every phragm is opened or closed time the micrOSCOpe is moved. by the lever shown at the left r lower side of the picture. The Abbe condenser. — The Abbe condenser (Fig. 7) serves the purpose of concen- trating the light rays and directing them at a different angle through the object. It is carried in a slip sleeve in the substage. It frequently gets pushed too low in this sleeve and this is a great source of poor illumination. Refer to Figs. 8 and 9 and note that proper illumina- tion is obtained by having the surface of the condenser approximately as high as the surface of the stage of the microscope. Examine your Abbe condenser and learn how it can be moved up and down, and look out for its getting out of place. There is an iris diaphragm under the condenser with which the amount of light must be regu- lated. It is strange how long it takes students to learn to 8 PRACTICAL CLINICAL LABORATORY DIAGNOSIS appreciate the necessity of regulating the light for different objects. In practice the light should be adjusted and regu- lated for practically every slide examined (Fig. 10), and Yia-, 8. — Illustrating manner in which light rays reflected from the flat mirror b are focussed upon the object by the Abbe condenser in proper position. frequently many times in examining a single specimen. Learn this now. Generally speaking, the higher the magni- fication the more light required. With low power lenses it USE AND CARE OF THE MICROSCOPE 9 Fig< 9.-Compare with Fig. 8 and note the ioss of light upon the object, where it is des red, resulting from the Abbe condenser being too low. It often happens that the condenser is slipped down in the sleeve, which carries it and is not noticed and the microscope is used at this great disadvantage for some time. Watch out for this. is necessary to reduce the light. Also the more color an object has the more light is required. Hyaline and un- stained objects require less light. 10 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Fig. 10. — Photomicrographs of the same field. A. Too much light. Note that the hookworm egg is poorly shown and other objects cannot be seen. B. Proper amount of light obtained by reducing size of the opening in the iris diaphragm. Focussing. — Place the part of the object to be examined approximately in the center of the field which can be guessed by its position over the Abbe condenser. With the coarse adjustment bring the tip of the objective near the object; then look in the microscope and rack upward until the ob- ject appears. Then focus with the fine adjustment. Ma- nipulate the fine adjustment with the left hand and the mechanical stage with the right (Fig. 3). Use of oil immersion lenses. — Only the best cedar oil "for immersion" should be used. It gets gummy and col- ored upon long exposure to light and air. Don't use such oil. Don't let dust get into it. Put a small drop of oil on the specimen over the place selected to examine. Run the tip of the oil immersion lens down until it touches the oil. Now focus as with other objectives. After use the oil must be cleaned off. Wipe the lens carefully with a soft linen cloth. A handkerchief is good for this purpose. Never let oil dry on the lens. If you do, remove it with a cloth moistened with xylol. USE AND CARE OF THE MICROSCOPE 11 Cleaning the lenses. — The exposed surfaces of oculars, objectives, condensers and mirrors are likely to have dust particles collect upon them at any time. It is best to wipe them off with a soft linen cloth (handkerchief) just before use each time. Keep the ocular and objectives attached to the microscope all the time, and there is little danger of dust getting on the back lenses of the objectives and lower lens of the ocular. They require cleaning only occasionally. Sometimes the inner surfaces of the lenses in the ocular require cleaning due to a film that forms on them. This you can do. Objectives should be sent to the makers every three to five years or oftener, if necessary, to be cleaned. Don't try to clean them inside. You should clean the back lens of the objectives occasionally by wiping with a suitable piece of cloth wrapped on a small wooden stick or applicator, or with the corner of the cloth rolled into proper shape. Care of the microscope stand. — It is better to keep the microscope assembled and on the table ready for use. A bell jar is a good cover to protect it from dust. Wipe it occasionally with a slightly oily cloth. Wipe off any gummy oil on the bearings and oil them a little with soft tallow or white vaseline. If gummed too badly, remove by wiping the bearings only with cloth moistened with xylol. You can put a little oil in the bearings of the coarse adjustment. If anything gets wrong with the fine adjustment send the microscope to the makers for repairs. Don't try to repair it yourself. Clean and oil the mechanical stage in the same way that you do the stand. Be sure to put the mechanical stage on so that it does not bind and drag on the stage of the microscope. CHAPTER II BLOOD Obtaining blood for all microscope examinations. — Some kind of a "blood sticker" is required. A good and con- venient one may be made of a straight Hagedorn needle No. 6. The point of most of them is too long and narrow. i Fig. 11. — A good "blood sticker." A properly sharpened straight Hagedorn needle No. 6, in 2 dram homo vial. Grind it to a proper short angle. Stick the eye end of the needle into a cork stopper and insert it into a two dram homo vial to carry it in (Fig. 11). The side of the first phalanx of one of the fingers is a convenient place to draw blood from. This is especially true when taking blood from yourself. One should learn to take blood and make the spreads with his own blood. It is (sometimes) more convenient to draw blood from the ear- lobe of patients. The principles of the technic are the same. Wipe the skin dry at the site it is intended to stick. If the skin is dirty, wash it, but it is not necessary nor desirable to sterilize with antiseptics, except, perhaps, in special in- stances. Squeeze the place to be stuck between the thumb and finger and make a quick stick with the needle while squeezing. The pressure lessens pain sense. Most workers can govern the depth and accuracy of the stick better if they steady the hands by letting them rest upon a table or other surface when sticking a finger (Fig. 12), or against the side of the face and neck of the patient when sticking the ear 12 BLOOD 13 Fig. 12. — Squeezing finger when sticking to lessen pain sense. Fig. 13. — Squeezing the ear-lobe and steadying the hand, against the face- when making the stick. (Fig. 13). The stick should not be deep enough to cause blood to flow without proper manipulation of the surround- ing tissue. It should be made so quickly and well that the patient hardly knows when it is done. 14 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Fig. 14. — First step in ob- taining blood from your- self after sticking the finger. The blood is pre- vented from running back by pressure made around the finger by squeezing it in this manner. Fig. 15. — Constricting patient's finger after hav- ing made the puncture, the first step in squeezing out blood. Fig. 10.— Squeezing out the blood. Note that the pressure is not made close to the puncture. BLOOD 15 To squeeze out the blood use both hands. Try to en- tirely surround the end of the fingers (Figs. 14 and 15) or ear-lobe (Fig. 16) with your fingers so that the blood can- not escape back. Your fingers should not be nearer than from one-fourth to one-half inch of the stick (Figs. 17 Fig. 17. — Second step in obtaining blood from one's own finger. Note that pres- sure is not made near the site of the puncture. The pressing fingers of the right hand are held nearly parallel to the bleeding finger, and pressure is made in this manner while the blood is prevented from returning by the con- striction made by the fingers of the left hand. Fig. 18. — Squeezing blood from patient's finger. Note that pressure is not made close to the puncture. and 18). If you hold and press too close to the stick, little blood will be obtained. Hard squeezing dilutes the blood with tissue juices and must be avoided. Squeeze out the proper amount with which to make one spread. About one-fourth drop is the proper amount. Stop the pressure 16 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Fig. 19. — Taking up blood from patient's finger, Fig. 20. — Taking up blood on slide from your own finger. The edge of the slide is first steadied against your left forefinger. and no more blood will flow if the stick was of the proper size and depth. If the skin is dry, the blood will stand up properly as a small round mass, but if it is not dry it will spread out on the skin. The proper amount of blood is BLOOD 17 taken up on a clean slide by touching the slide to it. Do not let the slide touch the bleeding finger. It is best for most operators to steady the hand which holds the slide against the table (Fig. 19), or the other hand (Fig. 20), or the face (Fig. 21) of the patient when taking blood from the ear, before trying to touch the slide to the drop of blood. The blood is now spread out upon the slide and then another proper amount of blood is squeezed out and Fig. 21. — Taking up blood on slide from ear-lobe of patient. This picture erroneously shows the blood about to be taken up on the wrong end of the slide. It should be taken on the lower end. spread upon another slide in the same way. It is best to make at least two good preparations, and preferably three, in every case. If a proper stick is properly manipulated, twenty-five to fifty preparations can be made from it if desired. Making blood spread for differential leucocyte count, examination for malaria plasmodia, looking for abnormal cells, making typhoid agglutination test, etc. — The ad van- 18 PRACTICAL CLINICAL LABORATORY DIAGNOSIS tages of well made blood spreads are sufficient to justify the effort necessary to learn to make them. There are compara- tively few who can make good ones. A general purpose blood spread should have both thick and thin portions, and there should be no dragging of the larger cells. The blood should be spread upon the middle third or a little more of the slide and should not reach to either end (Fig. 22). Fig. 22. — A good general purpose blood spread labeled with an ordinary lead pencil. Have the slides clean and free from chemicals. When not soiled with oily or gummy material and stains, they may be washed in water and wiped dry with a towel or cloth. Hold by the edges when wiping. Oily or stained slides may be cleaned by boiling them in water to which has been added a little washing powder or soap, and subsequently rinsing well. Take up the blood on the slide at about three-fourths of an inch from one end. The slide may be placed upon the table while spreading the blood, or it may be held in the hand. If held in the hand, which is preferable, first hold the hand in the position shown in Fig. 23, ready to receive the slide. Place the end of the slide farthest from the blood drop, between the thumb and finger (Fig. 24), and bring up the little finger to support the other end, or lay it down on the table before you. Whether the slide is lying on the table or BLOOD 19 Fig. 23. Fig. 24. Fig. 23. — Proper pose of hand and fingers to receive slide with blood on it. Fig. 24. — Slide with drop of blood on it held in proper position for spreading. .Fig. 25. — Slides held in proper position preparatory to spreading blood. 20 PRACTICAL CLINICAL LABORATORY DIAGNOSIS held in the hand, the blood is spread with another slide held in the other hand. Place the end of the spreader slide just in front of the drop of blood and bring it back to it (Fig. 25). Hold it at an angle of about thirty de- Fig. 26. — A. Indicating how blood collects beneath the spreader slide when the latter is held at proper angle. B. Indicating how blood collects in front of the end of the spreader slide when the latter is held at too great an angle. gre.es (Fig. 26). The blood spreads toward both edges and is all behind the sharp edge of the end of the spreader slide. Now, with a rather quick movement, push (Fig. 27) the spreader slide toward the end of the lower slide (Fig. 28) spreading out the blood in a thinner or thicker film, according to whether the movement is slow or rapid re- spectively. It is desirable to leave the blood on the middle third of the slide or a little more. Note (Fig. 22) that the last part of the spread is considerably thinner than the first part. Such a spread offers fields of any desired thickness. One made with too slow a motion resulting in a very thin preparation will always show dragging of the leucocytes, which alters the differential count. Allow the blood to dry. The preparation may be labeled by writing BLOOD 21 on the blood with a pencil after it has dried. Such slides will keep for several days unstained, if not convenient to stain them earlier. However, you will generally get the best stains on fresh specimens. Fig. 27.— Indicating the angle at which slides should be held and the direction in which the spreader slide should be moved in making blood spread. J Fig. 28.— The blood has been spread by pushing (net pulling) the spreader slide quickly. Staining blood slides with Wright's stain.— There are several good methods of staining blood. We prefer Wright's stain. It is a good one. Wright's stain is a solution of a 22 PRACTICAL CLINICAL LABORATORY DIAGNOSIS mixture of eosinate of methylene blue and eosinate of meth- ylene azur, prepared in a certain way described by J. H. Wright {Journal A. M. A., Vol. LV, p. 1979), dissolved in pure methyl alcohol. The powder may be purchased in bulk or in the form of tablets (6 tablets in a bottle) made by Burroughs, Wellcome & Co., or the staining solution may be purchased ready for use. Much of the latter sold is unsatisfactory. To make the solution from the powder, dissolve 0.2 gm. in 100 c.c. of Merck's methyl alcohol, highest purity. To make it from the tablets, pulverize and dissolve 6 tablets (contents of one vial) in 75 c.c. of the alcohol. The stain improves with age for a week or two. If kept in well-closed bottles and out of bright light, it keeps almost indefinitely. The technic of staining is as follows : 1. Flood spread with Wright's stain about one minute. 2. Dilute with about two to three times as much water. Allow to stain five to ten minutes. 3. Wash, dry and examine with the oil immersion lens. The object of the first step is to fix the preparation (harden the cells and incidentally to stick the film to the slide) and is accomplished by the methyl alcohol with which the stain is dissolved. The slide should be placed on a slide- rest or staining bar over a waste jar and not held in the hand. The amount of stain poured on the slide should be the minimum that will cover the film well and not evaporate sufficiently to throw down a precipitate on the slide in the one minute it is allowed to act. In about one minute after the stain is put on the slide it begins to change to a purplish color, and to throw down a precipitate. Just at this point is the proper time to add the water. It is better to watch for this than to go by exact time. Add the water from the water bottle (Fig. 29), the tube from which should hang over the staining bar and jar, and it should be just long enough to clear the slide. By noting the amount of staining fluid on the slide one can guess when BLOOD 23 he has added two or three times as much water. It is often necessary to vary the quantity of water added somewhat with different samples of staining solution. It is also some- times necessary to vary the length of time the diluted stain is allowed to remain on the slide, with different samples. Fig. 29. — Diluting Wright's stain on slide. Slide resting upon the slide-rest over a waste jar. Water from water-bottle. A good plan when beginning the use of a stain with which you are not familiar is to stain several slides different lengths of time, say from one to ten minutes, and see which is the best. The water used to dilute and to wash with must be either distilled or at least of high purity. Rain water is usually good. After washing the preparation it should be promptly dried. First wipe the back of the slide and the front around 24 PRACTICAL CLINICAL LABORATORY DIAGNOSIS the spread. Drying may be hastened by fanning the slide in the air or by propping it up on end against some con- venient object. Description of the leucocytes found in normal blood. — There are at least five different kinds of leucocytes present in normal blood. They are differentiated from each other by the size, shape and staining reaction of the nuclei, cytoplasm and granules present in the cytoplasm of some of them. The classification which we believe most nearly correct, in the light of our present information, is as follows: A. Small mononuclear leucocytes. (Lymphocytes.) These cells (Plate I) have a single round or oval nucleus which is frequently indented on one side. They have a relatively small amount of cytoplasm. Some have only a narrow band surrounding the nucleus, while others have very much more. They vary in size from about the diam- eter of an erythrocyte to about twice that size. The nu- cleus stains a beautiful purple in well-stained specimens and the cytoplasm varying shades of deep blue. The nu- cleus and cytoplasm are sharply differentiated. In the cytoplasm of a good many of the lymphocytes, there are from one to several reddish stained granules. They con- stitute about 20% to 30% of all the leucocytes in the blood of normal adults. B. Large mononuclear leucocytes. These cells (Plate I) are larger than the lymphocytes and two to three times the diameter of erythrocytes. Both the nucleus and cytoplasm appear to be less dense than those of the small mononuclear leucocytes, and frequently the cytoplasm is not so clearly differentiated from the nucleus. The proportion of the cytoplasm to nuclear material is much greater than in the small mononuclear leucocytes. The nucleus stains purple and the cytoplasm varying shades of light blue. In deeply stained specimens the cytoplasm of the older cells approaches the lavender of the neutrophile, making the differentiation depend largely on size and amount of cytoplasm. The nu- cleus varies in shape from round or oval to slightly lobu- lated. It is often placed more or less concentrically. Some late I. \. Small Mononuclear Leucocytes B. Large Mononuclear Leucocytes. U«a l \4ift C. Polymorphonuclear Neutrophilic Leucocytes. Plate II. t>» A. Polymorphonuclear Eosinophilic Leucocytes. «c ;*•" *>-Vi ft * W ■ ■5*5* * ' ■ - v j ' ■** . i V ^* k ? #0 B. Polymorphonuclear Basophilic Leucocytes. C. Relative Sizes of the Normal Blood Elements. BLOOD 25 of the large mononuclear cells contain no granules, but most of them do show from a few to many purple-stained gran- ules in their cytoplasm. These vary in size between that of the granules in neutrophiles and those in eosinophiles. The large mononuclear leucocytes constitute about 1% to 8% of all the leucocytes in the blood of normal adults. C. Polymorphonuclear neutrophilic leucocytes. These cells (Plate I) are from two to nearly three times the diam- eter of erythrocytes. The nucleus stains purple and is polymorphous, every one being somewhat different in shape from every other one. The outline of the nucleus is very irregular, and most of them are more or less lobulated. When studied carefully under high magnification the nucleus appears to consist of a string or ribbon of nuclear material wadded up in the cytoplasm of the cell. Some- times the bands connecting the lobes or masses are so very thin that they can be made out only with difficulty. In other (younger) cells the nucleus is horse-shoe shaped. The cytoplasm does not stain, but it contains many fine granules which do stain purple, some taking more of the red than others. The fact that the cytoplasm of these cells had no special affinity for either the basic or the acid stains then employed led to their being called neutrophilic or neu- trophiles. In speaking of them it is common to call them neutrophiles, dropping the other cumbersome part of the name. The polymorphonuclear neutrophilic leucocytes con- stitute about 60% to 70% of all the leucocytes in the blood of normal adults. D. Polymorphonuclear eosinophilic leucocytes. These (Plate II) average very slightly larger than the neutro- philes. Their nuclei resemble very much the nuclei of the neutrophiles, but there is possibly greater tendency for the nucleus to be divided into distinct lobes (2 or 3). The nuclei are often horse-shoe shaped. The cytoplasm does not stain, but there are many large granules embedded in it, which take the eosin in a mixture containing eosin, and the cells are therefore called eosinophilic or eosino- philes, dropping the other cumbersome part of the name. 26 PEACTICAL CLINICAL LABORATORY DIAGNOSIS They constitute about 1% to 4% of all the leucocytes in the blood of normal adults. E. Polymorphonuclear basophilic leucocytes. These cells (Plate II) are slightly smaller than the neutrophiles. The nucleus stains faintly and the lobes are less distinct. It more than half fills the cell. The cytoplasm does not stain. There are a variable number of large coarse granules which seem to project from the surface. They take the basic stains like methylene blue and on account of this affinity for basic dyes the cells are called basophilic, or baso- philes. The latter term is generally used. These granules, as well as a few finer granules sometimes in the cytoplasm, are also slightly azurphilic and in specimens well stained with Wright's stain their color is a combination of the dark blue methylene blue and the purplish azur. The basophiles constitute less than 1% of all the leucocytes in the blood of normal adults. Method of making a differential leucocyte count. — A differential leucocyte count is a determination of the rela- tive proportion or percentage of the different kinds of leucocytes present. The ordinary blood spread described above (Fig. 22), stained with Wright's stain, is suitable for this purpose. Differential counts are made with the oil im- mersion lens. A mechanical stage is an advantage. Begin- ners and those not thoroughly familiar with the different cells should make their counts on a comparatively thin part of the spread where it is not more than one cell thick. After more experience, more rapid counting can be done on the thicker part of the spread. A good spread has areas, vary- ing from too thick to count well, at the thick end, to un- necessarily thin, even for the beginner, at the other end. It frequently takes a long time for beginners to learn to locate proper ground to study. They do not realize the extent of the preparation under such high magnification. The student should learn early to select the proper part of the preparation. The great difference can be best ap- BLOOD 27 predated by looking over the spread carefully from one end to the other. Whenever favorable ground is located move toward one edge of the spread until the edge is reached (Fig. 30), keeping tab of all leucocytes observed. Then move toward one end or the other at least the width of the microscope field, and then across the spread to the opposite edge. In this way cross the spread back and forth, always on new ground, until the desired number of cells have been counted. Fig. 30. — Indicating manner of crossing and recrossing blood film in making differential leucocyte counts. No cell is counted twice. All cells passed over must be counted. If they cannot be classified, put them down as unclassified cells. At first a good many that cannot be classified are found, but with experience the student is soon able to classify practically all the cells found in normal blood. In counting, a tally sheet should be provided on which to mark down the different cells as they are found. It is not necessary to write out the names of the different cells as these are familiar terms, which can as well be indicated by proper abbreviations. S, may stand for small mono- nuclear leucocytes; L, for large mononuclear leucocytes, and N, E and B, for polymorphonuclear neutrophilic, eosinophilic, and basophilic leucocytes respectively. Like- wise U may stand for unclassified leucocytes. A mark is 28 PRACTICAL CLINICAL LABORATORY DIAGNOSIS made opposite the proper class for each cell found. For convenience in counting up, it is best to tally with the mark for every fifth cell of any kind. After a few cells have been counted the tally sheet will look something like this: situ mi i Lll JVM IU1IU IN BUI si B irlll The percentage of each kind of cell can be calculated after any number has been counted, but it is much better to count exactly one hundred cells, in which case the number of each kind of cell counted is also the percentage, and no other calculation is necessary. After a little experi- ence one learns to stop and add up when nearly one hundred cells have been counted. The number required to make the one hundred is now found in the specimen and then added. At first this is slow work, but one should practice to acquire speed. After a little time, instead of stopping to put down each cell, he learns to carry several cells "in his head" before stopping to put them down. This saves time. Finally, one should learn to carry the small mono- nuclears and neutrophiles "in his head" until nearly one hundred have been counted, only stopping to put down the other cells which are few in number. When the number of cells reaches nearly one hundred they are all put down and the tally sheet would look something like this: BLOOD 29 J* 23 zm jru £11 m On adding up it is seen that three cells are required to make one hundred. These are. found and added and the tally sheet completed. It would look as follows: /? 23 23 £JW, S JT U + 2 &S £11 + 1 3 31 I Another important short step in making differential leucocyte counts is to look in the microscope (Fig. 31) with "* Fig. 31. — Looking in the microscope with the left eye in making differential leucocyte counts. The tally can be plainly seen with the right eye with- out moving the head. 30 PRACTICAL CLINICAL LABORATORY DIAGNOSIS one (the left) eye and look at the tallying with the other (the right) eye, without moving away from the microscope. This can be learned with a little practice and has many advantages. It is worth learning. With good methods and with practice one should be able to count one hundred cells in three minutes or less time. Somewhat more exact figures can be obtained by count- ing several separate hundred cells and taking the average of all the counts, but much more depends upon properly made spreads than upon counting large numbers of cells. No count of the cells on a poor spread in which the larger cells have been dragged off, can tell the proportion of the cells in the original blood. The student should count several separate hundreds on the same spread and on different spreads of the same blood and see for himself how much they vary. When the count is carefully made on a properly made spread and in the part of the spread made before there was any dragging of cells whatever, there is very little variation indeed. In practice, therefore, a properly made count of one hundred cells is sufficient for practical pur- poses. Interpretation of variation in the proportion of the nor- mal leucocytes in the blood. — It should be understood that whenever the total number of a given kind of leucocyte is increased or decreased the percentage is correspondingly increased or decreased accordingly, but the percentage of the other cells is decreased or increased though their total number may remain exactly the same. Frequently what may be a low percentage for a given cell may in fact not indicate any actual loss of such cells, but on the contrary an increase in other cells, or vice versa. The percentage of the different cells in the blood of normal adults should be memorized and "at your tongues end" at all times. It is: BLOOD 31 S— 20% to 30%. L— 1% to 8%. X— 60% to 70%. E— 1% to 4%. B— to 1%. In children under five years of age there are usually from 40% to 50% small mononuclear leucocytes. Often there are even more in very young children. The per cent, of neutrophiles is correspondingly less. With increased age the proportions gradually change toward those of the cells in adult blood which are reached soon after puberty. Most women have a little larger per cent, of small mononuclear cells and a correspondingly smaller per cent, of neutrophiles. It is not at all uncommon to find these cells to be more than 30% of all of the leucocytes in the blood of perfectly well women. The small mononuclear leucocytes are in- creased in those diseases in which there is irritation of the lymph tissues of the body, and especially those in which the disease is fought or resisted largely by this kind of cell. Syphilis and tuberculosis and typhoid fever are* examples of such diseases, in all of which there is more or less in- crease of the per cent, of the small mononuclear leucocytes present. The large mononuclear leucocytes vary so much in health and also under influences not now recognized that slight variations have no special significance. They are greatly increased in acute lymphatic leukemia. The neutrophiles are the phagocytes of pyogenic bacteria and their number in the blood is increased in the presence of any disease caused by pyogenic bacteria, provided, of course, nature is able to respond to the call and furnish them. When pyogenic bacteria are present in a tissue from which drainage is poor or impossible and their toxins are ab- sorbed, very large numbers of neutrophiles are present; but when there is good drainage, and therefore little or no absorption of toxin into the system, there is little or no in- crease of the neutrophiles. For instance, staphylococci in 32 PRACTICAL CLINICAL LABORATORY DIAGNOSIS the appendix or in the peritoneal cavity would give rise to a very high neturophile count, while the same bacteria on a skin lesion or other surface lesion would give rise to little or no increase; gonococci in a joint would give rise to a high count, while there occurs little or no increase in simple infec- tion of the urethra with the same bacteria; streptococci in a middle ear abscess give rise to great increase of neutro- phils before drainage is established, but afterward they fall to near or quite normal in a few hours. An increase in the per cent, of neutrophiles indicates therefore absorption of toxin from a pyogenic infection or disease in some part of the body, but cannot point out the tissue involved or the par- ticular bacteria concerned. All other things being equal, the less drainage, the higher the count. A very small focus of disease in a tissue from which there is no drainage, like, for instance, in bone, under periosteum, in the middle ear, etc., may give rise to a high neutrophile count. A high neutrophile count indicates, therefore, not only disease proc- ess due to pyogenic bacteria, but also that drainage is poor or lacking. About ninety-four is the highest neutrophile percentage that is often seen. The neutrophiles are reduced or at least not increased in diseases due to non-pyogenic bacteria, or those not due to bacteria of any kind. Of the common pyogenic bacteria we may mention pneumococci, streptococci, meningococci, staphylococci of almost all kinds, and gonococci. Of the com- mon disease-producing organisms that are non-pyogenic may be named malaria plasmodia, typhoid bacilli, tubercle bacilli (unless in very large numbers), and tetanus bacilli. The eosinophiles are usually increased in intestinal para- site infections, especially hookworm infection, in which they sometimes reach 15% to 20% or more. They are increased in trichinosis, sometimes to 75% or more. There is usually great increase during and soon following an attack of true bronchial asthma. There is slight to considerable increase in a variety of parasitic skin diseases. Eosinophilia also occurs in most cases of foreign protein poisoning and ana- phylaxis. Plate III. EM Basophiles. BM '' : j£gW Mature Cells Myelocytes. Myelocytes. The upper half of the picture traces backward the development of the polymor- phonuclear leucocytes from myelocytes to mature adult neutrophiles eosinoyhiles and bosoyhiles. N-neutrophile; NM-neutrophilic myelocyte; E-eusenoyhile ; EM-euseno- philic myelocyte; B-bosophile: MB-bosophilic myelocyte. BLOOD 33 The eosinophiles are decreased by pyogenic infection and frequently none are found. An increase of the neutro- philes with marked decrease of the eosinophiles is known as "Simon's septic factor" and is a very strong indication of pyogenic disease or sepsis. The basophiles are increased to large proportions in some cases of myelogenous leukemia. Description of abnormal or pathological leucocytes. — The neutrophiles, eosinophiles and basophiles found in the normal blood are the descendants of myelocytes which are normally present in bone marrow and other blood-making tissue of the body. In certain diseases, notably myeloge- nous leukemia, myelocytes in large numbers appear in the circulating blood. Within certain limitations most of them can be grouped under one or the other of the following three heads: A. Neutrophilic myelocytes. These (Plate III) are large cells, having a single round, or oval-shaped nucleus, and neutrophilic granules, which are like the granules in polymorphonuclear neutrophiles, in the cytoplasm. The cytoplasm sometimes takes more or less of the methylene blue in Wright's stain. B. Eosinophilic myelocytes. These (Plate III) are large cells having a single round or oval nucleus, and eosinophilic granules which are like the granules in poly- morphonuclear eosinophiles, in the cytoplasm. The cyto- plasm usually takes more or less of the methylene blue in Wright's stain. C. Basophilic myelocytes. These (Plate III) are large cells having a single round or oval nucleus and basophilic granules which are like the granules in polymorphonuclear basophiles in the cytoplasm. It should be appreciated that the myelocytes are simply young cells that have appeared in the circulation before they had aged into the polymorphonuclear cells, and there must therefore be a good many cells in such a blood that are on the border line (Plate III) between them. 34 PRACTICAL CLINICAL LABORATORY DIAGNOSIS In addition to the abnormal leucocytes which fall in one or the other of the above groups, there are usually a good many others in leukemic blood that cannot be classified. They should all be grouped under the head of unclassified cells. When making a differential leucocyte count and myelocytes are found present, one should add to the usual tally sheet a head for each variety, as, for instance, MN, ME and MB. Interpretation of the presence of pathological leuco- cytes. — Myelocytes (approaching the adult polymorphonu- clear neutrophile cell type) may occasionally be found in small numbers in blood in which there is a considerable septic leucocytosis, and sometimes when there is no leuco- cytosis. They are generally counted in and considered with the neutrophiles. Large numbers are not found except in some form of myelogenous leukemia. Total leucocyte counts. Special apparatus and material required. — The special apparatus required are: A. Dilut- ing pipette ( Fig. 32 ) . This should provide for diluting the blood one hundred times. B. Counting chamber (Fig. 33). The counting chamber devised by one of us (Bass) has Fig. 32. — Diluting pipette. 0.4 cumm 0.04 •• •• 0.004 •• 0.00025- r\ ^/ n BASS RULING Fig. 33. — Bass counting chamber. BLOOD 35 advantages over all others. The chief advantage lies in the simplicity of the ruling. This same Bass ruling is now supplied on the several different styles of counting chambers. , .^^■Hf^^^" ' TT^l Fig. 46. — Fourth step. The cover-glass has been brought down and the finger has been lifted from it. Fig. 47. — Fifth step. Pressing down the cover-glass at each corner. Note that the hand holding the instrument with which pressure is made is steadied by- resting upon the table. Care must be taken to avoid slipping the cover-glass. 44 PKACTICAL CLINICAL LABORATORY DIAGNOSIS be seen best by looking at the top of the preparation held toward the light of a window or door and just a little below the level of the eyes. Unless they are present the preparation is not a good one. Do not tilt the 'prepara- tion. Hold it level. Now lay it down for two or three minutes during which time all the cells settle onto the. surface of the counting plate. In spite of good technic and usually due to slow manipu- lation, the cells sometimes settle unevenly and the next Fig. 48. — Looking at distribution of cells upon ruled plate. Dark background directly in front and in the distance. Light from window at left. step is to examine it to determine this question. Hold the preparation (level) above the level of the eyes (Fig. 48), having the light fall upon it from above, but look at it from below with a dark background in the distance. The evenness (Fig. 49) or unevenness (Fig. 50) can be seen, and if the cells are not evenly distributed the preparation is not good. Counting the cells. — Reduce and adjust the light of the microscope so that the ruling can be seen best. Count with the 4 mm. objective. Find the upper left hand corner of the ruled area (in fact the lower right hand corner; images are BLOOD 45 ■-■ ,.:-.'•'" •'. '-'■' }■•' ..:£-" ^■ a : ' ^ OcTOo d»" o^^lo^ ~\ .o Q o c° Q (^CWl O o >Tq%o & $tf £ o MO Q O v ^rO TT P6° o 9pP o voir yo ?9aO ] o o ° X O J " ,^-ys o^°:u oo (7 o o Di o. £L Fig. 51. — Arrow indicates order in which the erythrocytes in the sixteen small squares in one large square should be counted. We count 200 cells here. upon the disease or toxin which calls out the cells, but also upon the capacity of the individual to meet the demand. Long-continued disease often tends to exhaust the capacity to respond. At the end of this chapter we give a list of the common 48 PRACTICAL CLINICAL LABORATORY DIAGNOSIS diseases and their influences upon the differential and total leucocyte count. It should be understood that this gives only the general tendency and cannot be exactly correct in all cases, and in all combinations of disease. The total leucocyte count should be interpreted in the light of the other clinical facts in the case as well as the differential leucocyte count. Counting the erythrocytes, — The same apparatus is used as in making total leucocyte counts. Use either Toison's oGqo o Fig. 52. — The twelve cells to be counted in this small square are shaded. Those not shaded are not counted. fluid or 0.85% salt solution as diluting fluid. Dilute the blood one hundred times. When Toison's fluid is used the leucocytes and erythrocytes may be counted in the same preparation. In either case make the preparation in the counting chamber as described for counting leucocytes. In the Bass ruling (Fig. 35) there are five large squares, — one at each corner of the ruled space and one in the center, each ruled into sixteen small squares. Count the erythrocytes in each of these large squares. Begin at the upper left hand corner and count all the erythrocytes in one small square after another (Fig. 51) until all are counted. Much time can be saved by counting by twos or fives instead of by ones. When considering any given PLATE IV. TALLQUIST HEMOGLOBIN SCALE SHOWING THE SPECIAL BLOTTING PAPER REQUIRED AND THE ARRANGE- MENT OF THE COLORED SCALE The colors here shown are not exactly the same shade as in the original, they do not correspond to the different percentages of hemoglobin and can- not be used to ascertain or measure the quantity of hemoglobin. BLOOD 49 square count in (Fig. 51) all cells that touch the left or upper lines, and count out all that touch the right and lower lines. Do not stop to write down the cells until all in a large square have been counted. Add up the number of cells counted in the five large squares. The total number of cells counted in the five large squares multiplied by 5,000 equals the number of cells per cubic millimeter. Estimation of the hemoglobin, — The Tallquist scale con- sists of a series of ten red colored cards (Plate IV), each representing different percentages of hemoglobin in steps of ten and ranging from 10% to 100%. The colored cards are mounted on one sheet and bound in book form, with several pages of soft white filter paper. Each colored card is perforated in the middle with a hole about one- fourth inch in diameter. Well up a good drop of blood without much squeezing and touch a piece of the white paper to it. A spot on the paper is thus saturated with blood. Wait just a moment to allow the blood to spread through the paper by capillarity as far as it will and then promptly compare it with the different shades of the color sheet, holding the paper behind the color and viewing the blood through the holes in the color card. Pass quickly from one to the other until the blood is matched with a shade on the color sheet and note what per cent, it repre- sents. This is the estimate of percentage of hemoglobin in the blood. Accuracy improves with practice. Sometimes the color seems to fall between two cards or shades, and in such case the estimate should be made accordingly. For instance, if it seems to fall between 80% and 90% we would call it 85%. Color index. — The color index of blood is an expression of the average amount of hemoglobin per erythrocyte, the normal being represented by 1. Divide the percentage of hemoglobin by the percentage of erythrocytes. The quotient is the color index. A quick way to find the per cent, of erythrocytes is to multiply the first two figures on the left hand side of the number of erythrocytes per cu. mm. by 2, 50 PKACTICAL CLINICAL LABORATORY DIAGNOSIS unless it is less than one million, when only the first figure would be multiplied by 2. Interpretation of number of erythrocytes, hemoglobin per cent, and color index. — In health, females have about 4,500,000 to 5,000,000 erythrocytes per cu. mm., and males have about 5,000,000 to 5,500,000. Five million is generally considered the average. The hemoglobin in blood of normal individuals is about 100%, but there is a slight variation above, or especially be- low, in most people. Reading with the Tallquist scale we may consider values of 80% to 110% within the bounds of technical variation and the variation of normal blood. The color index of the blood of normal individuals is theoretically 1, but there is a slight variation above or below this in apparently healthy individuals. Indices of from 0.8 to 1.15 are within the limits of the variation in the blood of normal individuals and of technical variation. The number of erythrocytes may be increased by any disease or influence that removes considerable water from the blood, provided, of course, a corresponding amount is not taken in at the same time. Acute diarrhea, profuse sweating, etc., may lead to increase in the total number of cells per cu. mm. Abstinence from fluids leads to the same condition. Profuse diuresis, such as occurs in advanced diabetes, usually leads to concentration of the blood cells. Decompensation in various heart conditions tends to con- centrate the blood and thereby increase the erythrocyte count. In addition to these various dehydrating influences or diseases which may lead to an increased number of erythro- cytes, there is a disease, idiopathic polycythemia, in which the total erythrocyte count sometimes reaches as high as from 7,000,000 to even 10,000,000 per cu. mm. The hemoglobin percentage is increased by any of the above influences that increase the erythrocyte count. The color index would therefore be unchanged. The number of erythrocytes is decreased in all anemias. The decrease may be much or little according to the grade BLOOD 51 and kind of anemia. Counts as low as 500,000 sometimes, but very rarely, are met. The kind of anemia is indicated and the diagnosis much aided by a consideration of both the erythrocyte count and the hemoglobin percentage — the color index. The number of erythrocytes is reduced in the primary anemias as, for instance, pernicious anemia, to sometimes 1,000,000 cells or less; and though the hemoglobin is also always below normal, the reduction of the hemoglobin is not so great as that of the erythrocytes. This gives rise to a high color index (1.15 to 1.65 or more). A color index well above the limits of technical and normal variation strongly indicates pernicious anemia. The number of erythrocytes is somewhat reduced in chlorosis, but usually the reduction is only moderate. In fact, a great many cases of chlorosis have more than 4,000,- 000 erythrocytes. The hemoglobin is greatly reduced in this disease and this gives rise to a very low color index. Such indices as 0.35 to 0.5 are common, and strongly indi- cate chlorosis. In secondary anemia, due to loss of blood or to disease, there is more or less reduction in the number of erythrocytes according to what may be the cause in the particular case. The hemoglobin is reduced more than the number of erythrocytes, giving rise to a low color index. The color index is nearly always below the normal limits in secondary anemia. In long standing secondary anemia with low hemo- globin and erythrocytes the color index may approach or reach normal figures. The lowest color index occurs in chlorosis. Pathological erythrocytes. — The shape of normal eryth- rocytes is that of a biconcave disc. Sometimes there is great variation in the shape of many of the cells. This con- dition is known as poikilocytosis and such abnormally shaped erythrocytes are poikilocytes. (Plate V.) Sometimes there is great variation in the size of the eryth- rocytes. Some are much smaller than the normal and are 52 PRACTICAL CLINICAL LABORATORY DIAGNOSIS called microcytes. Others are much larger than the normal size and are called megalocytes. This condition of great variation in the size of the erythrocytes is called anisocytosis. (Plate V.) Normal erythrocytes in the circulating blood never have nuclei. In certain blood diseases, especially the primary anemias, a few to very many nucleated erythrocytes (Plate V) are present. The nuclei vary in size. They are round or oval, sometimes presenting two or more lobes and in a good many of the larger ones a reticulated structure can be made out. This gives some suggestion of the spokes of a wheel. Nuclei of erythrocytes stain dark blue to almost black with Wright's stain and usually appear to be separate from or lying upon the cell to which they belong. When very large erythrocytes (megalocytes) contain nuclei they are called megaloblasts. A normal size erythrocyte containing a nucleus is a normoblast. We sometimes find free nuclei. In anemia, due to certain poisons, notably chronic lead poisoning, pernicious anemia, the leukemias, and to a less extent malaria, a few to many of the erythrocytes contain basophilic (blue staining) granules. These vary much in size and number from a few coarse granules to many very fine granules. Such cells are known as stippled, or granu- lar erythrocytes. (Plate V.) In normal blood all the erythrocytes take about the same shade of color when stained with Wright's (or other poly- chrome) stain, but in pathological blood, certain cells are often basophilic and take more or less of the blue. These cells are known as basophilic erythrocytes and the condi- tion of the blood as polychromatophilia. (Plate V.) It should be clearly understood that there may be many different combinations or variations from the normal in a single cell or in the cells in a given blood. For instance, a megalocyte may be nucleated and therefore be a megalo- blast, it may be a poikilocyte, it may be stippled and it may Plate V. Anisocytosis. m Poikilocytes P# • * i Megaloblasts. * % + • % ; # Granular or Stippled Erythrocytes || 'i Polv 'vbilia. irocytes. BLOOD 53 be basophilic. In fact, many of the nucleated erythrocytes are basophilic or stippled or both. Interpretation of pathological erythrocytes. — Nucleated erythrocytes, poikilocytosis and anisocytosis occur chiefly in the primary anemias (pernicious anemia, leukemia, etc.). In considering poikilocytosis and anisocytosis due allowance must be made for the variation in size and shape that oc- curs, especially at the edge of slide preparations of normal blood. In the pathological condition the abnormality exists in all parts of the preparation. Stippled erythrocytes suggest lead poisoning, or pri- mary anemia (and rarely malaria). Polychromatophilia suggests primary anemia (and chronic malaria). In many cases pathological erythrocytes appear in showers lasting for a few days, and then almost disappear for an equal or a longer period. The absence of pathological erythrocytes in a case of anemia is evidence (not conclusive) of secondary anemia. Secondary anemias of long duration occasionally tend to approach the blood picture of pernicious anemia. CHAPTER III MALARIA Obtaining blood and making preparations for examina- tion for malaria plasmodia. — There are more malaria plas- modia in the peripheral blood during the first six to eight hours following the onset of each paroxysm than at any other time, but there are sufficient plasmodia at all times for a diagnosis to be made in practically all cases of malaria having fever at the time. The time to obtain specimens of blood for examination is therefore whenever malaria is sus- pected. Do not wait until any special time with reference to the occurrence of paroxysms. Make an ordinary blood spread (see page 22). The specimen may be stained and examined at once or at your convenience, according to the demands of the case. Stain with Wright's stain. Making the examination and recognition of plasmodia. — Search for plasmodia only in parts of the preparation where the staining is good (nuclei of the mononuclear leucocytes are purple and the cytoplasm definite blue) and where the cells are not too thick. Prior to the acquisition of much ex- perience with malaria plasmodia never examine where the cells are more than one cell thick. After sufficient experience you can recognize plasmodia satisfactorily where the spread is two or three cells thick and of course at a considerable saving of time, since the thicker the blood film the more plasmodia are present in a given area. Malaria plasmodia are in (or on) the erythrocytes, and no object should be considered as probably a plasmodium unless it is so situated. It is true that many plasmodia, especially old gametes, are in (or on) little more than 54 Plate VI. fl&tfl m ■3 ■'■ ■■y£ V-S i» a : .%' f* \ r Varieties of Schizonts found in the Peripheral Circulation. If. "«. Gametes. o Autumnal Mala modia." © 03 © QQ cd o o H o © 2; 05 O O p ©r-=5 ,Q o g^ S o Ph ho? ^ "S ^ £*5 o o o S 2 02 ^ 03 p w HH <^ H #2 O H- 1 Eh HH Q o ^ TO © +jj w ^ S « « o O ft^ © © W o N -H> ,-Cj ^ Q S^ S . ^H Q «3 H°Ofl Q QQ £j . % ^ H hM u H £ HH O ^ ° £ d © ° 3 5 g s S ^ Is O H- 2 ^ w o s/2 Eh O DC ^ 2 hH __, +j -TJ +J 2, 2 £ ~ I— i ° J °3 K^ H h-1 pq -1 T? © +j Eh C3 C3 0) co c3 ^ CD CD rd J3 CD +3 CD CD Fh CD Fh CO rH 03 CO CD CO CD ^.^ 03 o3 ^C c3 T3 CD CD O CD O ?3 g ft s a co fl pj H- 1 CD pj o < O CD •+3 CO co < +3 CD CD CO T3 T3 -P d a CD CD P^ r=l Pn Ph < CO c^° >? CO p '£ -P S feJD -P H 03 03 CO o e p 2 tx) 5 s ^ b£rf CO ^ 03 03^ fa CO *-• 03 CD =5 CO » » CD CO 03 co 03 CO oT CO e« ™ oa << H O c3 3 C« CD P a oS 03 03 03 Q cd 03 03 03 03 O 03 o CD O 03 03 03 03 ^ ^ 2 3 d d d Pi rt c P t— 1 HH 1-1 h- 1 1—1 M M 1— 1 ( , 2 a ^ o •2 03 s a 03 s > 'o O o Fh 43 'oT -p p 03 CO > 03 a *CO Ph o M O fa- ce 'co a X c3 s o CO '5 co 'So a S3 CO CO CO CO . (s3 'rS "CT Sh*3 'co 88 o CO 'co 03 co 03 a w o 03 fa w Q co d 'o O o 03 -3 £ s O '-M 3 03 CO 03 A 8 H u "o o | rt "o ^H CO fl c8 bxj P* 3 eg K*a 03 '3 M O O O U O U 3 O Q ft ft ft H 56 1 03 co d o> 03 03 A o> co d 03 J-C o 03 o it* CO PS oT CO -p 03 03 § ^ I— i Increase in most cases. Highest in the order named 03 CO d 03 Jh 03 03 A 03 eS 03 *H CJ Pj 1— 1 03 co d 03 O 03 CO ei 03 rH 03 c 03 CO cd 03 f-i 03 d 03 CO e$ 03 (h 03 Pi M 03 03 Jh 03 PI 1— 1 03 CO c3 03 t-i 03 PI 1— 1 03 03 Jh 03 PI 1—1 03 CO o3 03 03 © © -P cS S-. 03 O 3 u a) o> o So M -p cS 03 fH 03 oT co 03 P! h- 1 P) 03 -P o -bio'-g 2h 03 CO *£ 03 rC PI M o -p ,4 be tO 43 03 r* CO 03 ci T3 03 O & s 1 03 CO c 03 tt V c 03 > 1 be .S 'u 03 CO 03 d be 03 d t-< -p 03 CO PI 03 PI o s Ph o 03 oT CO oS 03 o § j>» "d pi CO 03 O CO rt ej H 03 U U | e3 Fh 03 o S co eg --v 03 03 O > CO co "to 03 03 <{ CO -p 'ft -p CO cS o 03 *8 o o to ^p °G -p CO d o c3 o PI o O Gonorrhoea! Arthritis Epididymitis Orchitis, etc. -p o O Helminthiasis Hookworm Strongyloides Tricocephalus Oxyuris Ascaris Tenia 03 > CO CO •rt co ."£ 83 03 CO c3 03 CO t3 co be O w c 03 PI > r-l 03 !> -+^> ^-p cti c3 ^ 03 « o> fn fH ^ be 5 &J0 fi M fee P 'd P o p ^>> . o -M 05 O) cd ce _, CD o3 Gi » O 03 CJ ^ d ft ^ v^ ^ o o &C f-i p Pi cS <3 u P ■+J p 03 © © o © © -p OJ f-t o b£ © flj o CO © rt o © o 1 — 1 £ i— i II ^^ £ © o 03+-> 0Q 03 Pi r5 ^ ^ Pi <*< r-i -p ai ^ 2? bD O 5Q o r^ T rP S C- w -^ o ■+3 e6 ri d S3 efl s p s $ £ pi? 03 Pi r^ pi 03 J 1-3 ^ 58 'p Pi c3 ce o 03 rO Ph_ -" a cd .s 0) 02 © cd bJD © cd s- -i-3 © 02 P I— 1 -i-3 cd © 5-. bJO vb lid P 02 oT 02 cd © © p 0^' - CD 02 cd 03 J-l © oT 03- 02 cd a ■— 1 -P CS cj Sh S3 03 ■— c ps P i—i Increase only moderate or not at all until pustulation, when moderate increase occurs Increase, slight during secondary stage C3 CO cd ^H 03 P 1—1 'S BO ? - fa c -^ +; o G cd s-. o3 c3 '5 o £ pa © P S p be © © | 3 p cd 02 02 £ ^ -a p-i 02 "S3 c cd P c © cd QQ cd cd © Fl P CJ Septicemia due to sta- phylococci, strepto- cocci and pneumo- coccal o = 02 IS 03 -t-J P O «j CO 'co p 59 w H £ P O O H H S* O u a, P 3 H .5 *g fc @ S ° flu Q B <® 2 M H P ^J rrl 0> til rrl ri -P au OS o S eg u > 81 » O 3 ^ .5 •§£ H s II a3 fcfi 60 Plate VII. * * . y T# < 1 • ^si > #^'3 #c s# =€:-i Schizonts. 3tfS ': I Gametes. '-:, Plate VIII. £0 1& ft ft* 4^ Schizonts. *> Gametes. Quartan Malaria Plasmodia. MALAKIA 61 "shadows" of erythrocytes, the cells having faded to a great extent. The cytoplasm stains blue, the chromatin stains the usual red, like chromatin in the nuclei of cells, platelets, etc. Pigment granules when present do not stain but have a light to dark brown color. Do not expect to find malaria plasmodia well stained in preparations where the leucocytes and platelets are not well stained. Unless the nuclei of leucocytes have the proper reddish tint due to proper staining of the chromatin in them, the chromatin of the plasmodia will not be properly and characteristically stained. In examining a blood specimen for malaria plasmodia one should acquire the habit of noting, upon first look at the preparation, whether the staining is good and not look for plasmodia in any but well stained preparations and good areas of such preparations. The number of plasmodia present varies very much. Often there may be several plasmodia in every field, while in other cases there may be so few that a search of several minutes may be required before a single plasmodium is found. Description of malaria plasmodia. — There are three distinct species of malaria plasmodia now known to infect man, and it is highly probable that it may be found that still others exist. They are (1) Estivo-autumnal (Plate VI, Plasmodium falciparum) ; (2) tertian (Plate VII, Plas- modium vivaoc) and (3) quartan (Plate VIII, Plasmo- dium malaria?). They all grow and reproduce in the blood of man in the same general way. The smallest Plasmodium consists of a thin round wall of basophilic protoplasm enclosing a round or oval mass that does not stain. This gives the appearance of a ring. There is one, and sometimes there are two granules of red-stained chromatin at one side of the ring, giving rise to the designa- tion "signet ring." As the plasmodium grows the erythro- cyte is slowly consumed and after about twelve to twenty- 62 PRACTICAL CLINICAL LABORATORY DIAGNOSIS four hours there appear light to dark brown granules of pigment. These become more numerous until the develop- ment of the parasite is complete. An individual Plasmo- dium reaches maturity in about forty-eight hours in the case of estivo-autumnal and tertian, and in seventy-two hours in the case of the quartan parasite. The chromatin granule grows in mass for about half the developmental period, and after this begins to divide. Division goes on slowly until at the end of the period it has divided into the number of divisions peculiar to the particular species. To- ward the end of the period division of the cytoplasm of the organism occurs, and now the whole plasmodium is made up of a number of separate chromatin granules each sur- rounded by its own cytoplasm. Such a mature, or seg- mented plasmodium is called a "rosette." Finally the capsule of the rosette ruptures and each segment or young plasmodium, known as a merozoite, is capable under favor- able conditions of attaching itself to an erythrocyte and passing through a similar cycle of development, giving rise in turn to another crop of merozoites. This kind of re- production takes place without sexual influence and is known as schizogony. A-sexual plasmodia (of any age) are schizonts. After a person has been infected with malaria plasmodia more than two or three weeks, and as a result of in- fluences not now well understood, certain plasmodia, in- stead of developing through the a-sexual cycle of schizo- gony, become sexually differentiated and take somewhat different form. These are supposed not to be capable of reproduction in man. The males are capable of giving rise (in the mosquito) to microg'ametes, and the females to macrogametes. They are capable of reproducing by sporogony in the mosquito. Sexual plasmodia in the blood are correctly called gametocytes, but in common usage the term gamete is used. Estivo-autumnal gametocytes are crescent shaped, but tertian and quartan gametocytes are round or oval and resemble very much the developing, al- MALARIA 63 most mature schizonts. There is no division of the chro- matin in gametes. Estivo-autumnal schizonts disappear from the peripheral circulation and lodge in the capillaries when they are about twelve hours old. They do not reappear in the peripheral blood until after segmentation (except extremely rarely) and therefore the only estivo-autumnal schizonts found in peripheral blood are the ring forms. The tertian and quartan schizonts begin to disappear from the peripheral circulation after about two-thirds of their period of development has passed, but on account of the fact that they are capable of ameboid movement they often pass out of the capillaries in which they lodge and reappear in the peripheral circulation. It frequently occurs that full grown schizonts and rosettes of these species are found in the peripheral circulation. This fact that all ages of tertian and quartan schizonts are present in peripheral blood and only the ring stage of estivo-autumnal schizonts constitutes a most valuable means of differentiation between the different species. The gametes of all species appear in the peripheral blood. Differentiation of malaria plasmodia. — The rings of dif- ferent species look very much alike and it is not practical to diagnose species by the rings. The very small rings found in pernicious estivo-autumnal malaria may be dif- ferentiated from those of the so-called benign estivo- autumnal or tertian and quartan parasites. They are extremely small and are often overlooked. The presence of other size parasites than rings shows either tertian or quartan infection. The absence of them indicates that the rings are estivo-autumnal. Infection with more than one species occurs, but is rare. The most useful differential points are shown in the fol- lowing table: 64 PKACTICAL CLINICAL LABORATORY DIAGNOSIS Estivo- Autumnal Tertian Quartan Schizonts in the pe- Rings only. All sizes. All sizes. ripheral blood. Shape of gametes. Crescentic and oval. Round or oval. Round or oval. Shape of outline of Rings. May have Irregular after Regular after ring schizonts in periph- two chromatin ring stage shape is passed. eral blood. granules. passed. Influence upon color of Not changed. Faded, pale. Darker. Normal erythrocyte. red color inten- sified. Influence upon size of Not changed. Enlarged. Reduced. erythrocyte. Pigment in schizonts Medium to coarse. Very fine. Coarse. and gametocytes. Schiiffner's granules.* (Not present.) Often present. Not present. Number of segments in (24 to 32.) 32. 8. rosette. Period of a-sexual de- (48 hours. ) 48 hours. 72 hours. velopment. * Red staining granules in the erythrocyte containing the parasite. Interpretation of examination of the blood for malaria, — There are always sufficient plasmodia in the peripheral blood to enable one who is competent to find them in a search of ten minutes or less in the case of all persons who have active malarial fever. It is possible, therefore, to de- termine positively that a given case of fever is (or is not) due to malaria. If quinine has been taken by the patient during the 48 hours previous to the time the blood is taken tor examination it may cause the disappearance of plas- modia, and the question as to whether quinine has been taken recently should always be raised in interpreting negative findings. It is often possible to find plasmodia in the blood of people who have chronic or latent malaria by thorough and repeated examinations of the blood, but it is not possible to examine the blood and to determine that the patient has not some plasmodia in his body. If he had enough to make him sick, however, they could be found readily. It should be appreciated that the number of plasmodia present in the blood is not always an indication of the clinical symptoms or vice versa. CHAPTER IV TYPHOID AGGLUTINATION TEST This is a test to determine the presence or absence in the blood of a specific substance, agglutinin, which has the property of causing typhoid bacilli to agglutinate or collect together in clumps. Typhoid agglutinin will agglutinate either living or dead typhoid bacilli. The method of making the test given below is as reliable as any other when done with corresponding accuracy and reliable material, and it has many advantages. The special material required is a suspension of dead typhoid bacilli, ten billion per c.c. in distilled water, and killed and preserved with 1% commercial formalin. A 24 hour growth of typhoid bacilli on neutral agar is used in making it. This material can be made by any competent bacteriologist. This material keeps well for at least several years if kept tightly corked. Shake before using. When a vial of it is frequently opened and used from, it should be discarded and a new supply obtained every six to twelve months. An ordinary medicine dropper and a wooden tooth- pick are also needed. Make the regular blood-spread on a slide, using approxi- mately one-fourth drop of blood. This may be tested at once at the bedside, or it may be tested at any time at your convenience, within a week or two. Place on the blood one drop of water. Carefully spread this over the film of blood 65 66 PRACTICAL CLINICAL LABORATORY DIAGNOSIS (Fig. 53) with the end of a pick or other suitable instru- ment. Tilt the slide from side to side to hasten the dis- solving of the blood. A clear solution of approximately Fig. 53. — Spreading the drop of water over the blood to dissolve it in making typhoid agglutination test. one-fourth drop of blood in one drop of water is thus made. Now add one drop of the suspension of typhoid bacilli and mix by tilting the slide from side to side (Fig. 54) and from end to end, causing the mixture to flow back and Fig. 54. — Tilting slide back and forth to facilitate mixing and to hasten agglutination. forth. This also hastens the reaction. If the blood con- tains the specific agglutinin for typhoid bacilli, the millions of bacilli present soon begin to collect together, first in small TYPHOID AGGLUTINATION TEST 67 grayish clumps (Fig. 55) appearing as a fine granular sedi- ment. Later the clumps of bacilli get coarser and are readily recognized (Figs. 56 and 57). The reaction takes place and is complete within two minutes. When the test is negative no such granular sediment forms. Practice with known typhoid and known negative blood until familiar with the test. Make many such tests until familiar with the appearance of positive and negative re- actions. Do not mistake dust particles for agglutinated Fig. 55. — A series of typhoid agglutination tests, (a) Negative test, (b) Weak positive reaction, (c) Moderate positive reaction. (d) Strong positive reaction. bacilli. There occur doubtful reactions in this test just as in all other tests for specific antibodies which vary in amount from none to sufficient to produce definite positive reactions. Interpretation of the typhoid agglutination test. — About 10 to 20% of all cases of typhoid give positive reactions by the end of the first week. About 70% give positive reactions by the end of the second week. More than 90% give posi- tive reactions at some time during the course of the disease. A few do not give positive reactions at any time. Usually 68 PRACTICAL CLINICAL LABORATORY DIAGNOSIS the reaction may be obtained until after the fever sub- sides. The reaction gradually gets weaker as time passes following convalescence, and after three to six months the Fig. 56. — Looking at typhoid agglutination test. The granules can be seen best when there is a dark background in the distance and the light falls upon the specimen from the side. reaction is weak or negative in the majority of cases. A few give positive reactions for a year or two. A positive reaction, if accompanied by clinical evidence, is practically diagnostic of typhoid. A negative reaction does not prove that the patient has not typhoid. Its nega- tive value is in proportion to the duration of the disease. Given a case of fever (possibly typhoid) of two weeks' duration, for instance, the indication of a negative reaction TYPHOID AGGLUTIXATIOX TEST 69 Fig. 57. — Looking at typhoid agglutination test at night by the light of a match held in the right hand beneath and to the side of the specimen. would be 70% that it is not typhoid. Here, as in most other laboratory findings, the test is of most value when interpreted in connection with the clinical evidence. The same test and technic may be used in the diagnosis of paratyphoid fever provided one uses similar suspensions of paratyphoid bacillus A, and paratyphoid bacillus B, in place of the suspension of typhoid bacilli. CHAPTER V URINE Collection of specimens for examination. — The only proper specimen of urine for examination is a recently voided specimen, except perhaps when it may be desirable to make a quantitative examination of the total urine voided during twenty-four hours. No method of preservation will keep specimens in suitable condition for examination. Dif- ferent specimens keep variable lengths of time. Some are not good after an hour or two, while others are satisfactory for at least ten or twelve hours, and sometimes longer. As specimens get a few hours old, contaminating bacteria, which often multiply very rapidly, alter the formed elements, such as blood cells, casts, etc., that may be present, cause the crys- tallization and precipitation of the salts and effect, by fermen- tation or otherwise, such substances as sugar and albumin that may be present. Sometimes the amorphous or crystal- line sediment that forms in a short time is so great that it renders microscopic examination impractical or at least unre- liable. Do not examine old specimens unless as a last resort. Patients who come to the laboratory or office should void urine there for examination, but if inconvenient they may be instructed to void just before leaving to come to the office and to bring a sample of the fresh urine only. Speci- mens to be sent to the laboratory should be voided just be- fore they are sent. Examination of the early morning urine is sometimes the most valuable. Instruct the patient to void in a clean vessel or urinal and to send about four ounces in a clean bottle. Larger quantities are unnecessary. If significance is to be attached to the presence of pus, instruct the patient to void if possible into two clean glasses and to send two to four ounces from the second glass in a clean bottle for examination. Females should be instructed further to thoroughly wash the vulva with soap and water before voiding. Some physicians take precautions in all cases as routine. However, catheterized specimens are the only kind from females that can be depended on as certain not to contain pus and acid-fast bacilli from the vulva. 70 r 9 - 1 1 ' H I $. I ■': *;'";'' ■".■'.;■ J f ^ ! i 1 I s PLATE IX CHEMICAL TESTS OF URINE Tube 1. Test for albumin. No cloud after boiling and addition of acetit acid. 'lube 2. Test for albumin. Cloud produced by boiling and remaining after addition of acetic acid, thus showing presence of albumin. Tube 3. Test for indican. Negative reaction. Chloroform not colored. Tube 4. Test for indican. Positive reaction. Chloroform colored blue. Tube 5. Test for sugar with Folding's solution. Negative reaction. No change in color. Tube G. Test for sugar with Fehling's solution. Positive reaction. Yellowish red color due to reduced copper. Tube 7. Test for acetone. Negative reaction. Disappearance of red color upon addition of acetic acid. Tube 8. Test for acetone. Positive reaction. No disappearance of red color upon the addition of acetic acid. URINE Physical Examination 71 Specific gravity. — A urinometer (Fig. 58) is required. One of the cheaper grades is sufficiently accurate for ordi- nary purposes. Fill the tube nearly full of urine so that it runs over (in the sink) when the float is put in. This makes the read- ing easier. Read at the bottom of the meniscus. Chemical Examination Test of the reaction. — Dip a strip of blue and a strip of red litmus paper in the urine. If acid, the blue paper changes to red. If alkaline, the red paper changes to blue. Test for albumin. — (A) Qualita- tive. Fill a test-tube three-fourths full of urine. Hold by the lower end in the hand. Boil (Fig. 59), for a minute or two, approximately the upper one inch of the column by holding in the flame of a small Bunsen burner or al- cohol lamp. Revolve and agitate while boiling to prevent boiling over. If albumin is present, a cloud (Plate IX), A Fig. 58 Urinometer. Testing specific grav- ity. Note curve of meniscus. Fig. 59. — Boiling urine in test for albumin. 72 PKACTICAL CLINICAL LABORATORY DIAGNOSIS varying in intensity with the amount present, will appear. Phosphates also produce a cloud upon boiling. Now add a few (4 or 5) drops of glacial acetic acid (Fig. 60). If the cloud is due to albumin it will be intensified. If it is due to phosphates it will be cleared up. A faint cloud due to only a trace of albumin can best be seen by holding, Fig. 60. — Adding acetic acid to urine from a drop bottle in testing for albumin. the tube (Fig. 61) so that it has a dark background a few feet away and has light (from window or otherwise) fall- ing upon it from the side (Fig. 62). (B) Quantitative. There is no practical quantitative test for albumin that is absolutely accurate. The one here given is accurate enough for practical purposes. Fill a test-tube with urine to a depth of 2% inches (Fig. 65). Add about one-fourth as much of a 10% solution of potas- sium ferrocvanide and about 1 c.c. of glacial acetic acid. URINE 73 Fig. 61. — Looking for faint cloud in test for albumin. Dark background with light falling on the tube from the side. Fig. C2. — Cloud due to albumin in urine as seen in the proper light against a dark background. 74 PKACTICAL CLINICAL LABOKATORY DIAGNOSIS Shake and allow to stand a few (2 to 5) minutes. Cen- trifuge until the coagulated albumin has collected well in the bottom of the tube. Measure this. Each one-quarter inch of sediment represents 10 % of moist albumin. Smaller quantities may be approximated. The formula for the po- tassium ferrocyanide solution is: 5 Potassium ferrocyanide 10 gms. Water, to make 100 c.c. Mix. Fig. 63. — Filling tube with urine to a depth of 2% inches in quantitative test for albumin. The tube is held by the side of an ordinary inch rule. Test for sugar. — (A) Qualitative. There are several tests to select from. We give Fehling's test here because it is the one generally required by life insurance companies, for whom no doubt many of our readers will be called upon to make examinations. We require two separate stock so- lutions, Fehling's alkaline solution and Fehling's copper sulphate solution. The formula for Fehling's alkaline solu- tion is : URINE 75 Sodium-potassium tartrate 173 grams Sodium hydroxide 125 grams Water 500 c.c. Mix and filter. The formula for Fehling's copper sulphate solution is: Pure crystals of cupric sulphate. . . . 36.4 grams Water 500 c.c. Mix and filter. These two separate solutions keep well but when mixed together they do not keep more than a few days. It is therefore desirable to prepare the test solution at the time a test is made by mixing equal parts of the alkaline solu- * v Fig. 64. — Tube contains Fehling's copper solution. Adding equal amount of alkaline solution, using finger as a marker. tion and the copper solution. Keep the separate solutions in glass stoppered drop-bottles. The drop-bottles are more convenient to pour the solutions from. To make a test, drop into a test-tube a few drops, not more than one-half c.c, of one solution, hold the finger as a marker (Fig. 64) at the place where the solution will rise to when approximately an equal volume of the other solu- 76 PRACTICAL CLINICAL LABORATORY DIAGNOSIS tion is added. Add the other solution, shake, and we now have about 1 c.c. of a mixture of approximately equal parts of the two dif- ferent solutions, which con- stitutes Fehling's test solu- tion. Heat the solution to boiling over a Bunsen burner (Fig. 65) or alcohol lamp, then add about one- half as much urine and boil again. If sugar is present the solution assumes an Fig. 65.-Boiling Fehling's solution opaqile yellow Color (Plate and urine in testing for sugar. r ^ J v IX) and shortly after a dense yellowish-red sediment falls to the bottom. Shake the tube while heating to prevent boiling or "popping" out. No tube-holder is necessary. (B) Quantitative test for sugar. Measure one-half c.c. of each of the Fehling stock solutions into a test tube by means of a one c.c. graduated pipette. Wash the pipette which should be graduated in hundredths by running water through it. Take up some of the urine to be tested in the pipette and after noting the starting point add a few hundredths of a c.c. to the solution at a time (Fig. 66), boiling a little between each addition. Touch the tip of the pipette to the side of the tube so that all urine let out will flow into the tube. The yellowish red precipitate forms and finally the solution loses its blue color at the point where all the copper is oxidized. Complete reduction of the copper is best determined by centrifuging the tube to throw down the precipitate and noting when the blue color disappears. Now read off on the pipette the amount of urine used and calculate the per cent, of sugar indicated by the test. One c.c. Fehling's solution requires 0.005 gram of glucose to remove the blue color, and therefore URINE 77 the amount of urine used contains 0.005 gram of glucose. To calculate the per cent, of glucose present by a short rule, divide 0.5 by the amount expressed in c.c. of urine used. The quotient is the per cent, of glucose present. For ex- ample, if 0.08 c.c. of urine is required, 0.5 -r- 0.08 = 6^, the per cent, of sugar indicated. If a large amount of glucose is present it is better to dilute the urine, say ten r Fig. 66. — Adding urine from 1 c.c. graduated pipette in quantitative test for sugar. times with water and to test this diluted urine. Of course the amount of sugar present in the diluted urine must be multiplied by the number of times the urine was diluted, to determine the amount in the undiluted urine. Test for indican. — Place about 3 c.c. of urine in a test- tube. Add an equal quantity of hydrochloric acid and about 1 c.c. Fehling's copper solution. Shake vigorously for one- half minute and add a few drops of chloroform. Shake well 78 PRACTICAL CLINICAL LABORATORY DIAGNOSIS again and allow the chloroform to settle to the bottom. If indican is present the chloroform assumes a blue color ( Plate IX), varying in intensity with the amount present, but it remains uncolored if indican is not present. A red color indicates the presence of iodides. Test for acetone. — To about 5 c.c. of urine in a test-tube add an amount of sodium nitro-prusside about as large as the head of a match, shake to hasten solution and add one- half to one c.c. of Fehling's solution (Alkaline). The mix- ture at once turns a cherry red (Plate IX), which rapidly changes to yellow in the absence of acetone while the color changes much more slowly (3 to 5 minutes or longer) if acetone is present. Immediately after the alkaline solution has been added and the tube shaken and before the red color has had time to fade, add a few drops (2 or 3), of glacial acetic acid. The color promptly disappears in the absence of acetone, but is changed slightly to a darker or purplish red when acetone is present. Microscopic Examination Preparation of specimens.- — The objects which we ex- pect to find by microscopical examination are suspended in the urine and must be concentrated by means of a centri- fuge. Satisfactory examination is impossible otherwise. The electric centrifuge is the best ( Fig. 67 ) . It should have a guard (Fig. 68) around it to protect against acci- dents. A good hand centrifuge (Fig. 69) does well in the hands of those who take proper care of them and run them right. They do not last long if run with tubes of unequal weight in them or if turned by jerks at the very highest speed possible. Neither is necessary. The centrifuge should be supplied with square bottom Cornell shields with rubber washers in the bottom. Do not accept any other. No spe- cial centrifuge tubes are required. Use the regular 12 mm. X 115 mm. lipless tubes. However, with low speed and poor centrifuges the special narrow bottom tubes and cor- responding shields are better. Agitate the urine to stir up URINE 79 Fig. 67. — Electric centrifuge with Cornell shield.' Fig. 68. — Box guard around electric centrifuge. 80 PEACTICAL CLINICAL LABORATORY DIAGNOSIS any sediment that may have settled to the bottom of the container and fill the tube about two-thirds full. Select an- other tube as nearly the same size as possible and fill it with water to exactly the same level (Fig. 70), holding them side by side for comparison. If two specimens are to be centrifuged each tube may be filled with urine and (one) labelled. Centrifuge at full speed one-half to two minutes. . i Fig. 69. — Hand centrifuge with Cornell shields. Longer is unnecessary, except when endeavoring to throw down bacteria. This cannot be done satisfactorily with the hand centrifuge and requires several minutes with the elec- tric centrifuge. If not sufficient sediment is obtained from the first tube the supernatant fluid may be poured off, leav- ing the sediment in the bottom, the tube refilled with urine, and centrifuged again. In this way all the suspended ob- jects in several tubes of urine may be obtained if desired, but generally one tube is sufficient for all practical pur- poses. URINE 81 Fig. 70. — Filling balance tube to height of column of urine. After centrifuging, pour off the supernatant fluid. The sediment and a drop or two of urine remain. Shake the tube so as to break and stir up the sediment and pour it out on a slide. Spread out properly on the slide with the edge of the mouth of the tube. (Fig. 71.) A proper Fig. 71. — Pouring out sediment and spreading on slide with mouth of the tube. No pipette is necessary as is also the case with the sediment from feces. 82 PKACTICAL CLINICAL LABORATORY DIAGNOSIS spread (Fig. 72) will not extend to the edges or end of the slide. It is the heighth of laboratory awkwardness to allow urine to run over onto the microscope stage, which will Fig. 72. — Proper spread of urine sediment. occur if it is spread to the edge or end. No cover-glass is necessary. Method of examination. — Examine with the low power (16 mm.) lens. It will occasionally be necessary to swing in place the high dry lens (4 mm.) to examine under higher magnification some particular object about which doubt exists. Here as well as in other places it is important to have a high power lens with a long working distance, such as the B. and L., 4 mm. of N. A., 0.65 (and not one of 0.85) or the Leitz No. 6 (and not No. 7). Those who, through mistake or otherwise, have purchased lenses with short focal length should exchange them or procure proper ones in some way. Do not try to do routine laboratory work under such a handicap. Place one corner of the preparation in position for ex- amination and by means of the mechanical stage move the slide from end to end, dropping down the width of a field each time until the entire specimen is examined. This re- quires only a minute or two where the examiner is competent. The light of the microscope must be adjusted (reduced) so that hyaline objects and those with little color may be seen well (Fig. 10). Diagnosis of microscopic objects commonly found in urine*. — Red blood cells (Plate X, b) are usually more or less altered by the urine. They may be swollen or more or less faded, or they may be crenated. They appear green- ish instead of red. They may be differentiated from other objects when in doubt by mixing a little acetic acid with PLATE X MICROSCOPICAL FINDINGS TV URINE a. Low power fi"l* dim » ■** Diplococcus Pneumoniae. Pneumococci !«i ; Bacillus Tuberculosis. Preparations of cerebrospinal fluid from meningitis due to different bacteria. All specimens stained with carbol-fuchsin and methylene blue except the one containing tubercle bacilli which is stained with carbol-fuchsin decolorized with acid and counter- stained with methylene blue. SPINAL FLUID 125 found in the cerebrospinal fluid. This may occur especially in traumatic meningitis. Cell counts. — To make a total cell count in spinal fluid make a preparation in the Bass counting chamber, count the cells as in counting leucocytes in blood and multiply the number counted on the ruling by 2.5. This gives the num- ber per cubic millimeter. Usually no dilution is necessary in counting the cells in those fluids in which cell counts are required — tuberculosis; poliomyelitis and syphilis, especially. If there are too many cells to count in an undiluted speci- men, the fluid may be suitably diluted with Toison's fluid or salt solution, in which case the count must be multiplied by the dilution and also 2.5 to get the total number per cubic millimeter in the undiluted fluid. To make a differential cell count, drain off the super- natant fluid and make a spread on a slide of the cell sedi- ment collected by centrifugalization. Stain either with Wright's stain or with the carhol-fuchsin and methylene- blue stain, and count as* in counting blood. Test for globulin increase. — Put saturated aqueous so- lution of ammonium sulphate in a test-tube to a depth of about one inch. Overlay this carefully with spinal fluid which has been cleared of cells by centrifuging. A good way is to allow the fluid to flow down the side of the tube from a pipette. An increase of globulin is shown by a ring varying from a very faint whitish ring appearing not later than one hour to a very heavy ring appearing immediately. Interpretation. — The presence of meningitis and the causative organism can usually be diagnosed with satisfac- tion by proper microscopic examination. It sometimes happens that there are so few meningococci or tubercle bacilli present that these organisms may not be found. When no pus cells are present there is no meningitis pro- duced by pyogenic organisms. When many pus cells are present and no bacteria found, the case is usually meningo- coccal meningitis. Normal spinal fluid contains up to 10 cells per cubic 126 PKACTICAL CLINICAL LABORATORY DIAGNOSIS millimeter, and these are all mononuclear cells. The count in poliomyelitis usually runs 100 to 300 cells with 5% to 10% neutrophiles. In tubercular meningitis the count usu- ally runs 300 to 1000 cells and approximately 100% mo- nonuclears. In tabes there are usually 90 to 300 cells per cubic millimeter, and in paresis about 30 to 90, practically all of which are mononuclears. In meningitis due to any pyogenic organism the total count is enormously increased, nearly all the cells being neutrophiles. Increase of globulin indicates some meningeal infection or disease, including poliomyelitis and syphilis. CHAPTER XII DIPHTHERIA Principles of laboratory diagnosis of diphtheria. — It is possible to make a practical diagnosis of diphtheria bacilli in the laboratory by reason of the fact that when grown upon certain culture media (Loffler's blood serum) they grow much more rapidly than the other bacteria with which they are associated in the nose, mouth and throat, Fig. 99.— Bass diphtheria culture tuhe. The tube on the left has been broken open and the swab has been straightened out ready for use. and that on this media they show characteristic mor- phology not shown by any other bacilli likely to be found in these places. Material required. — A tube of Loffler's blood serum and a sterile swab are required. Tubes and swabs are furnished by city and state board of health laboratories, but they soon deteriorate. A special hermetically sealed tube containing the swab (Fig. 99) also was devised by 127 128 PRACTICAL CLINICAL LABORATORY DIAGNOSIS one of us (Bass) and is to be preferred especially by those who have to buy their culture tubes. This tube is small, convenient, and keeps indefinitely. One doing general or special practice of medicine who is likely to see cases of probable diphtheria should include one or more of these tubes in his armamentarium. Any doctor equipped with Fig. 100. — Removing cotton plug from culture tube preparatory to inoculating with the swab. a microscope and not having access to a general bacterio- logical laboratory should keep such tubes on hand. Making the culture. — In cases where diphtheria is sus- pected there often is membrane or other local evidence of the disease, either on the tonsils, uvula, fauces or posterior nares. Often, however, no membrane is recognized, but still diphtheria is suspected and a laboratory examination for diphtheria bacilli is desired. It is desirable to obtain material to be planted on the culture media from the sur- face of the diseased mucous membrane, and as free as DIPHTHEKIA 129 possible from bacteria and secretions from any other part. Most people who are old enough to try can show the pharynx without the tongue depressor being used, by open- ing the mouth wide and making a gagging effort. At this moment the swab should be rubbed over the surface of the diseased part. In the case of a young child use of the tongue depressor is necessary. In nasal diphtheria the Fig. 101. — Inoculating culture media by rubbing swab over surface. Note how cotton plug is held. swab should be passed into the nose and some of the secre- tion secured. Now, holding the culture tube in the left hand and the swab in the right, remove the cotton plug from the tube (Fig. 100), rub the swab over the surface (Fig. 101) of the culture media gently and then replace the plug. The best way to dispose of the infected swab is to throw it in the fire if one is convenient, or it may be returned to the empty end of the tube and all wrapped in paper to be destroyed at a more convenient time. 130 PRACTICAL CLINICAL LABORATORY DIAGNOSIS In spite of the duty the physician owes to his patient to be prepared for such work as this whenever it is indicated, it often happens that a culture tube is not at hand when the suspicious case is first seen. Instead of waiting until the next visit to get a culture tube, an extemporaneous swab may be made by winding a little absorbent cotton on a suit- able stick and taking the culture with it. The inoculated swab is placed in a dry, clean, small, empty bottle or test- tube or in an envelope, and is carried to the office or labora- tory, where a culture tube is inoculated in the way described above. Incubation. — The inoculated tube or culture, which is usually made at the bedside of the patient, should be started to incubating at once. No special laboratory facili- ties or incubator are required. The tube should be wrapped with paper to insure against the plug coming out and placed in an inside (vest) pocket where the heat of the body keeps it warm enough to favor the growth of diph- theria bacilli. This is as good as an incubator. The tube may be kept warm in the pajama pocket during -the night. Incubation is necessary, because the bacilli grow very slowly and have not their characteristic morphology unless the culture is kept warm. This method of incubation is espe- cially advantageous because it starts from the time the cul- ture is first taken and it may permit the diagnosis to be made several hours earlier. Examination of culture. — Whenever many bacilli are present they usually have grown to sufficient numbers in six or eight hours and have their characteristic morphology so that they may be recognized upon examination. No visi- ble growth will be present at this time. If the exigencies of the case demand very early diagnosis the culture may be examined after six hours and very often the diagnosis can be made. We have occasionally been able to make the diagnosis in four hours. If no diphtheria bacilli can be found upon early examination, the incubation should DIPHTHERIA 131 be continued and subsequent examination made. An ex- amination should be made after eighteen to twenty-four hours incubation before a final negative diagnosis is made. Whenever very few bacilli are present it may require this long for sufficient bacilli to grow for them to be found. After twenty-four hours other bacteria which may be present often overgrow the diphtheria bacilli and prevent satisfactory diagnosis. It is therefore desirable to make the final examination by the expiration of this period. Making preparations for examination. — A good plati- num loop is very valuable in examining the culture. Often the loop of the wire is too large, not smooth, or other- wise improper. The loop can be properly formed and shaped by bending the end of the platinum wire snugly one time around the end of a smoothly sharpened lead pencil just where the lead disappears under the wood. After the loop has been formed, bend it back sharply in order to bring the center of the loop in line with the straight wire. Once a good loop is provided, take care not to bend it out of shape, as many do. The growth on the culture media is not fluid enough to spread well on a slide and it is an advantage to dilute it (on the slide) with some water. Place approximately the smallest quantity of water that the platinum loop will hold near the middle of the side. Now hold the culture in the left hand and the platinum loop handle in the right. Sterilize the loop (Fig. 102) by heating red (white) hot in the Bunsen-burner or alcohol flame. Remove the cotton plug (Fig. 103) from the tube and draw the loop over the surface (Fig. 104) of the culture, endeavoring to scrape off any growth present. Often little or no growth can be seen. Stir the material removed from the culture in the water on the slide and spread out in a thin long streak (Fig. 105). Too thick smears are not good. Now flame the loop again and replace the cotton plug. As soon as the preparation dries it is ready to be stained. 132 PRACTICAL CLINICAL LABORATORY DIAGNOSIS ± Fig. 102. — Sterilizing the platinum loop in flame of Bunsen burner. Note proper size and shape of loop. Fig. 103. — Removing cotton plug, sterile platinum loop held in hand. DIPHTHERIA 133 Staining. — The steps in staining are: 1. Fix with heat. 2. Cover the entire spread with Loffler's methylene blue Fig. 104. — Scraping the surface of the culture with the platinum loop to obtain bacteria for examination. and allow to stain approximately one-half to one minute. Wash, dry, and examine with oil immersion lens. Fig. 105. — Proper long thin streak of material from the culture to be stained and examined. To fix, pass through the flame slowly, film side up, two or three times. Don't get it hotter than can be borne on the back of the hand ( Fig. 92 ) . 134 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Loffler's methylene blue is a valuable routine laboratory stain. The formula calls for: Saturated alcoholic solution of methylene blue (Griibler's) 30 c.c. Solution of potassium hydroxide, 1 to 10,000 100 c.c. Mix. If the American made methylene blue is used, the fol- lowing formula is better : Methylene blue 0.5 gm. Grain alcohol 30 c.c. Solution of potassium hydroxide, 1 to 10,000. . . .100 c.c. Mix. A good way to make up the alcoholic solution of methylene blue is to put 10 grams of methylene blue in a 100 c.c. (4 oz.) bottle and nearly fill with alcohol (95%). Shake thoroughly and allow to settle. This keeps, as does also the staining solution. After removing a part of the saturated alcoholic solution of methylene blue for use the bottle may be refilled with alcohol and more solution will be ready for use again. Sometimes it may be desirable to stain suspected diph- theria preparations with Gram's stain also in doubtful cases. See page 138 for the technic. Diphtheria bacilli are Gram positive, and the darker staining of the polar bodies shows these up well. Description of Diphtheria Bacilli When taken directly from the seat of disease or when grown on ordinary media diphtheria bacilli cannot be differ- entiated by their morphology from many other species of bacilli likely to be found present in the mouth, nose and throat ; but when grown on the Loffler's blood serum media they present a morphology quite characteristic of them and a small group of so-called diphtheroid bacilli (Plate Plate XVII. * V * - V t rf> / V \ ^-^ * - >/ • I *• 2* a / ^ % v - « ^k h>- ■ 1 > 1 \x ( '•■ r i Diphtheria bacilli from different cases suggesting variation in morphology. All of the specimens were stained with Loffler's methylene blue. One was counter- stained with safranin. Note that the polar bodies are shown better. DIPHTHERIA 135 XVII). In the first place they are bacilli and no other organism found need be considered as possibly diphtheria. They vary in their size from small up to very large. Some will be iive or ten times longer or larger than others. Many are club-shaped, others are more or less lanceolate-shaped on one or both ends. Most of the diphtheria bacilli are more or less beaded and in many this is so marked as to make the bacilli look somewhat like chains of streptococci. In addition to the beaded condition a variable proportion of the bacilli contain one, two, three or four much darker staining granules, called polar bodies. When only one or two are present in a bacillus they are usually situated near its ends. These polar bodies are much more numerous in some specimens than in others. They are fairly well shown with the methylene blue stain, but they are shown much better by counterstaining with a red stain such as safranin or carbol-fuchsin (Plate XVII, D). After staining the whole preparation with Loffler's methylene blue, stain one end with 1% safranin solution one-half minute, or with carbol-fuchsin on and off; wash, dry and examine with oil immersion lens. It is seldom that any other diphtheroid organisms show these polar bodies and this is a fairly re- liable differential point, but not infallible. In most cases cultures that contain diphtheria bacilli contain other bacteria, cocci and possibly bacilli. Pure cul- tures are seldom obtained from diphtheria lesions. Interpretation. — Diphtheroid bacilli, Gram positive and in the presence of clinical evidence of diphtheria are for all practical purposes diphtheria bacilli. A properly taken cul- ture properly incubated and examined always shows diph- theria bacilli in the presence of the disease. In the absence of any clinical evidence whatever one should look with doubt upon diphtheroid bacilli. CHAPTER XIII GONORRHOEA Obtaining material and making preparation. — During acute gonorrhoea there is a great deal of pus formed, con- taining many gonococci. If the patient is a male the pus should be wiped from the meatus in order to get rid of the other bacteria that may be growing in the pus after it is exposed to external contamination. A fresh drop is brought to the meatus by "stripping" along the urethra. When there is very little pus being formed this is espe- cially necessary. The fresh pus squeezed from the follicles in the urethra is much more desirable than old pus that has been standing under the prepuce or even in the urethra. When the urethritis is far back, of course it is necessary to "strip" from far back. In chronic gonorrhoea where there is very little pus formed and most of that in the pos- terior urethra, seminal vesicles and prostate, this pus can usually be obtained by massaging and forcing it forward with the finger through the rectum. In acute gonorrhoea in females the external pus should be removed from the orifice of the vagina and meatus, and fresh pus obtained from higher up. There are many other bacteria in the outer pus and gonococci often cannot be demonstrated in it with satisfaction. The best material for examination for gonococci in acute cases is obtained by in- troducing a finger into the vagina and "stripping" the urethra forward. In chronic cases pus may be obtained from the cervical canal or glands through a vaginal speculum. Sometimes it may be desirable to examine the urine for gonococci which are present in gonorrhoeal cystitis. Re- cently voided urine is absolutely necessary. Centrifuge the 136 GOXORRHCEA 137 urine until the pus cells are thrown to the bottom of the tube. If not enough sediment is collected the supernatant urine can be poured off and the tube refilled and centrifuged again. This may be repeated several times if necessary, until suffi- cient pus sediment is secured. Pour off all the supernatant urine and drain well to get rid of all urine. By exercising care the last drop can be drained off, leaving thick pus in the bottom of the tube. With a platinum loop or other suitable instrument make a proper spread of this on a slide. A spread approximately one-fourth inch wide and one and one-half inches long is ideal. The proper thickness can be learned with a little experience. The ideal spread is only one cell thick and the cells should be well separated and not all packed together. All pus should be spread with a single stroke to avoid damaging the cells. Carbol-fuchsin and methylene blue stain. — The best routine stain for gonorrhoeal or any other kind of pus or exudate is a combined stain, carbol-fuchsin and mythylene blue. It shows up the cells and bacteria to the greatest advantage. The steps are: 1. Fix the dry film with heat. 2. Cover film with carbol-fuchsin for a few seconds. Wash. 3. Cover film with Loffler's methylene blue, ^ to 1/2 minute. Wash, dry, and examine with oil immersion lens. To fix, pass the slide, film side up, slowly through the flame of a Bunsen burner or alcohol lamp two or three times. Do not heat any more than can be borne to touch the back of the hand. The stain should cover the film only and not the entire slide. The best way is to touch the dropper of the drop bottle, with a drop of stain on it, to the film and spread (Fig. 96) the stain over the film as it is allowed to flow out of the bottle. Gram's staining method. — Many of the bacteria that may be mistaken for gonococci are stained by Gram's method while the gonococcus is not stained by this method and there- 138 PRACTICAL CLINICAL LABORATORY DIAGNOSIS fore is called Gram negative. Gram's stain does not show up the morphology of bacteria as well as the carbol-fuchsin and methylene blue stain does, and therefore is to be used largely for further test or source of evidence. Gram's stain should be made only after the other has been made and intracellular diplococci have been found. In most cases the picture is so definite that in practice the Gram stain is not required. The technic of Gram's stain as we do it is: 1. Fix with heat. 2. Stain film with carbol-gentian violet about two minutes. 3. Pour off carbol-gentian violet and cover film with Gram's solution one minute. 4. Decolorize with alcohol. Wash. 5. Counterstain with one per cent, safranin solution one-half minute. Wash, dry and examine with the oil immersion lens. The carbol-gentian violet should be made according to Czaplewsky's formula, which is: Gentian violet 1 gram. Liquefied carbolic acid 5 c.c. Neutral glycerin 50 c.c. Water to make 100 c.c. Mix. This stain keeps indefinitely. Instead of washing off the carbol-gentian violet with water the excess should be washed off with a few drops of Gram's solution. The formula for Gram's iodine solu- tion is: Iodine 1 gram. Potassium iodide 2 grams. Water to make 300 c.c. Mix. This solution keeps well. Alcohol from 95 to 100% is all right to decolorize with. Hold the slide by one end. Let the other end be lower and GOXOKRHCEA 139 drop (Fig. 106) alcohol on the upper end of the film so that it runs over the film and off at the lower end of the slide. Only a dozen or two drops are necessary to decolorize. Whenever decolorization is complete it will be noted that the drops of alcohol as they run off are not colored as was the case before. The alcohol is washed off to facilitate the Fig. 106. — Decolorizing with alcohol. The slide is held so that the alcohol which is dropped on it flows over the film and off at the end. Tilting the slide from side to side facilitates the decolorization. counterstaining with an aqueous stain which does not mix well with the alcohol. If we should examine the preparation under the micro- scope after decolorization we would find all Gram positive bacteria stained a very deep violet and the Gram negative bacteria unstained. Unstained bacteria are difficult to see and we therefore stain with a stain that contrasts well with the violet. The unstained objects take the counterstain and are now fairly readily seen. Of the several available 140 PKACTICAL CLINICAL LABORATORY DIAGNOSIS counterstains we choose a one per cent, aqueous solution of safranin.. The formula is : Safranin 1 gram. Water to make 100 c.c. Mix. This stain keeps well. Appearance of gonococci. — Gonococci are readily phago- cyted by the polymorphonuclear leucocytes and therefore a large portion of those present in pus are within the pus cells (Plate XVIII) . There are no other bacteria for which gonococci are likely to be mistaken that are found intra- cellular to the same extent. Though some gonococci are to be found outside of cells it is best not to call anything a gonococcus that is not intracellular. The number in one cell varies from one to twenty or thirty or more pairs of cocci. The cells packed with diplococci found in acute gonorrhoea are so typical that one who is familiar with them could hardly make a mistake. Many of the pus cells have no gonococci in them. In chronic gonorrhoea one may look over many fields of the microscope before he finds a pus cell with gonococci in it, while in acute gonorrhoea practically every field has them in it. With the carbol-fuchsin and methylene blue stain gono- cocci stain blue, while with the Gram stain they take the red counterstain. Gonococci are always in pairs — true diplococci. The division between the two coffee-bean shaped organisms can be made out in most of them, but of course in some they are not seen in the proper position for the division to be made out. Interpretation. — Intracellular, Gram negative, diplo- cocci from the urethra are for practical purposes gonococci, especially when typically arranged in the cells. In the ab- sence of such diplococci an acute urethritis is not gonor- rhoea. It is not possible to determine beyond doubt that gonorrhoea (chronic) is not present in a given case, but thor- ough, repeated, negative examinations are strong evidence that it is not. Plate XVIII. .'8 A. c. i It ^ PP B. ii& » K* D. A. Pus in acute gonorrhoea. Carbol-fuchsin and methylene blue stain -K. Fus in acute gonorrhoea. Gram's stain. C. Pus from case of acute non-specific urethritis. Many staphylococci present. Carbol-fuchsin and methylene blue stain. D. Pus obtained from prostate and seminal vesicles by massage in case of chronic gonorrhoea showing a few gonococci. Carbol-fuchsin and methyl blue stain. ene CHAPTER XIV SYPHILIS I. Examination for Treponema pallida, — There are two simple practical methods of demonstrating Treponema pal- lida: with the darkfleld microscope and by preparing with India ink. The former is far superior to the latter, but re- quires from about fifteen to thirty dollars worth of extra apparatus. There is hardly any question, however, but that those who assume the responsibility of the diagnosis of syphilis are under solemn obligation to their patients to the extent of either being prepared to make such examinations or to have them done by others who are prepared to do so. The diagnosis by inspection of early lesions of syphilis often remains in doubt, but it can almost always be made with certainty by proper microscopic examination. Obtaining material and making preparation for examina- tion with the darkfield condenser. — The Treponema pallida is present usually in very large numbers in all chancres, mucous patches, condylomata, adjacent swollen lymph nodes and in smaller numbers in other syphilitic skin lesions and most other syphilitic lesions of the body. It must be under- stood that the organisms are in the tissue and not on it. In order to obtain material containing them, we must obtain it from the diseased tissue. In the case, for instance, of a chancre, it is necessary to obtain material from the hard syphilitic tissue and not simply from the indurated or ul- cerated tissue over or around it. What is needed is "juice" from the diseased tissue and the scraped up tissue itself. It is very necessary to avoid getting a large amount of blood with the material. To obtain proper material (Fig. 107) grasp the chancre between the thumb and forefinger and 141 142 PRACTICAL CLINICAL LABORATORY DIAGNOSIS make sufficient pressure to drive out the blood, as is shown by the anemic appearance of the tissue. Maintain the pres- sure and with a scalpel scrape down into the hard chancre tissue. Scrape up some of the tissue and squeeze out some "juice" of the tissue. Either transfer these to a slide with the scalpel or touch the slide to the drop of fluid that has been squeezed out. Sometimes patients will not endure the pain produced. In case of an ulcerated lesion a drop of a one per cent, cocaine solution may be applied to the ulcer to anesthetize it, or infiltrate the tissue beneath the chancre with a one-half per cent, solution of cocaine. In the case of Fig. 107. — Scraping chancre. Sufficient pressure is maintained to render the tissue anemic. skin lesions on any part of the body the same technic is followed. Whenever lesions in the mouth are so located on the lip or tongue that they can be manipulated in the same way, it should be done. Otherwise, it is better to pinch off a little of the diseased tissue with a suitable instrument, rinse it in salt solution and then to crush it on a slide with the end of another slide and obtain proper fluid from the diseased tissue. The diagnosis is less dependable whenever the lesion is in the mouth on account of the possibility of finding the nonpathogenic spirochetes commonly present in the mouth and mistaking them for Treponema pallida. If the scraped-up material is too thick it may be diluted with a little salt solution. Cover the small quantity (about one- fourth drop) of " juice" from the diseased tissue with a SYPHILIS 143 cover-glass and it is now ready for examination with the darkfield condenser microscope. Examination with the darkfield condenser. — There are several makes of darkfield condensers on the market. Some Fig. 108. — Best form of darkfield condenser. It fits in place of the Abbe condenser of the make of microscope for which it is intended. are placed on top of the stage of the microscope and are interchangeable or usable on any microscope. Others, which are the best (Fig. 108), are made for each particular make of microscope and fit in the sleeve in the place of the '%^J:k Fig. 109. — Diagram showing path of rays through a darkfield condenser, and a 3/52 inch oil immersion lens fitted with funnel stop. Abbe condenser. In the use of any make of darkfield condenser the Abbe condenser must be slipped out or swung out, according to the make of microscope being used. The principle (Fig. 109) of darkground illumination is that by means of a central stop no direct light is permitted 144 PKACTICAL CLINICAL LABORATORY DIAGNOSIS to pass through the object, while by means of a system of lenses and reflectors the object is illuminated by strong light reflected from the sides and at an angle. In this way the particles suspended in the fluid are shown as very bright objects against a dark or black background. A very strong light is necessary. Direct sunlight is per- fect when available. Those who do not have many of such examinations to make, and those who have not electric cur- rent, may find it a fairly satisfactory source of light. In Fig. 110. — Gas-filled Mazda lamp for dark-ground illumination. May be screwed in ordinary light socket. most instances a time for the examination can be set when the sun is shining. Where electric current is available and considerable use of this method of examination is made a gas-filled Mazda lamp with concentrated filament (Fig. 110) or a small arc lamp will be found more satisfactory. Slides and cover-glasses of proper thickness should be used. If the manufacturers have not stated the require- ments for the instrument you have with the general direc- tions write them for the information, or better, for a supply of a box of slides and cover-glasses suitable for your ap- paratus. SYPHILIS 145 To examine the specimen, place a drop of immersion oil (or water) on the darkfleld condenser which must have previously been centered. This is done by moving it with the set-screws while observing the small ring in its center, under the low power of the microscope. The examination may be made with the high dry lens, but the treponemas appear quite small under this magnification. The oil im- mersion lens gives the most beautiful field, but is a little Fig. 111. — Illustration of the position of the funnel stop placed in the lens case of y 12 inch oil immersion lens for darkfleld work. more difficult to use. It must have a funnel stop (Fig. 111). The makers will furnish the stop for a few cents. Stops are already supplied with some microscopes. There is a great deal of Brownian movement of all the small particles suspended in the fluid under examination and we now see many things that cannot be seen by direct illu- mination. The suspended particles have more or less dancing motion. Spirochetes (Fig. 112) are seen in living, active state. They apparently revolve in corkscrew fashion and also move laterally. They have little locomotion but are carried about more or less by currents. One should practice on preparations made from material taken from between the teeth which usually contains more or less spirochetes of other species. 146 PKACTICAL CLINICAL LABORATORY DIAGNOSIS India ink preparation. — Only a small portion of the spirochetes present can be seen in an India ink preparation. It is important to have a good quality of India ink. The Gunther Wagner brand is a good one. The "juice" from the suspected tissue is mixed on a side with about an equal Fig. 113. — Photomicrograph of Treponema pallida in "chancre juice," as seen with the darkfield microscope. amount of India ink, and spread upon the slide. A wood toothpick is convenient to mix and spread with. A little ex- perience is necessary to learn just what thickness to make the preparation, but it is a good idea to make some thin and some thick areas (Fig. 113). Practice with material scraped from the edge of your own gums between the teeth, Fig. 113. — Proper India ink preparation. Note variation in thickness of spread. which generally contains the Spirocheta Dentimn. Allow the preparation to dry and examine with the oil immersion lens, using strong light. Spirochetes (Fig. 114) appear as perfectly clear spirals, against the black granular back- ground. Other objects, such as bacteria, blood cells, etc., are also shown. Fig. 114. — Photomicrographs of India ink preparations containing spirochetes. a. Treponema pallida. Compare the red blood cells. b. Treponema pallida. c. Spirocheta refringens. d. Treponema microdentium and Treponema macrodentium. 147 148 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Differentiation between Treponema pallida and other spirochetes. — We hardly think it wise for most observers to undertake to differentiate Treponema pallida from some of the other spirochetes upon morphological differences. There are spirochetes in the mouth, nose, throat, rectum and vagina of a large per cent, of all persons, but there are no non-pathogenic species on the skin. Spirochetes found in scrapings from lesions not of mucous membranes are therefore most certainly either the pallida or, in the very rare tropical disease, yaws, the Treponema pertenuis. Spirochetes found in material from lesions of mucous mem- branes are quite likely to be the common species found in the orifices of the body. If, however, the precaution is taken to thoroughly cleanse the surface before scraping, these are less likely to be encountered. The Treponema pallida have from about six to ten or more turns, while most of the other spirochetes have fewer. The pallida is small and the turns are short. There are five or more turns to the diameter of an erythrocyte. This is a convenient (though not absolute) test, because in most speci- mens we have a few erythrocytes to which any spirochetes found may be compared. Gland puncture. — The enlarged glands, inguinal and others, in early syphilis usually contain very many trepo- nemas, and gland puncture usually furnishes a little fluid very rich in them. The puncture is a very simple operation. A good hypodermic syringe and No. 24 needle are needed. The syringe and needle should be dry. Sterilize the skin over the intended site of puncture with tincture of iodine (an area one-fourth to one-half inch in diameter is large enough) and try to carry the point of the needle into the center of the gland. Make suction and rotate the needle a little. Stop the suction and withdraw the needle. Make preparations of the gland "juice" and examine in the same way as material obtained from other sources. Interpretation. — Small spirochetes from a gland punc- ture or from a lesion not on a mucous membrane are for SYPHILIS 149 practical purposes Treponema pallida and practically make a diagnosis of syphilis. A competent examination of a sus- pected lesion with the darkfield condenser for treponemas, if negative, is almost proof that it is not syphilis, but it is not infallible proof to this effect. II. Wassermann serum test. — It is not within the scope of this book to enter into the fundamental principles in- volved in the serum test for syphilis. We do not describe the original Wassermann technic. Those who desire to use this and to understand the principles of the test are referred to books on serology. We will, however, describe a simple, modified method of making the complement fixation test for syphilis which can be carried out by persons with very little laboratory equipment, and without much experience in this kind of work. We will give the exact technic, every step, but will not undertake to explain the reason for every- thing that is done. Materials required. — (1) We require an alcoholic solu- tion of antigen. This is kept as stock and a small amount is diluted with salt solution whenever a test is to be made. Antigen may be obtained from commercial sources. The dose must be known. It must be known how much it must be diluted, so that 0.2 c.c. contains the proper dose when the hemolytic unit is 0.1 c.c. of 12%% washed guinea pig's blood and the amount of active human serum necessary to hemolize this quantity of cells. The source of supply may furnish this information or we may work it out ourselves. All that is necessary is to make tests with known positive and known negative bloods, employing different quantities of antigen solution. In this way we may determine the smallest quantity that will give positive reactions with positive blood, and the largest quan- tity that will give negative reactions with negative blood. Usually there is a good margin between them, and we "split" the difference. A good antigen will require to be diluted about fifty to two hundred times. At present, antigen with the dose 150 PRACTICAL CLINICAL LABORATORY DIAGNOSIS stated can also be obtained from Dr. F. M. Johns, P. O. Box 770, New Orleans, La., for $2.00 in advance for suffi- cient to make about one hundred tests. (2) Washed guinea-pig blood cells, 12%%. Etherize Fig. 115. — Etherizing guinea-pig preparing to draw blood. Cup with cotton to hold ether makes the best "cone" for this purpose. a guinea-pig (Fig. 115). Thrust the needle attached to an all-glass syringe directly into the pig's heart (Fig. 116). Dissecting one or two previously will serve best to tell where the heart is located. The needle should be not larger than Fig. 116. — Introducing the needle while steadying the parts. SYPHILIS 151 No. 24. It is not necessary to have the needle and syringe sterile. Draw 1 c.c. of blood (Fig. 117). Put this in a test-tube with 6 or 8 c.c. of salt solution. Centrifuge until the cells are collected at the bottom. Pour off the super- natant fluid and refill to make 8 c.c. This is called washed Fig. 117. — Drawing blood from guinea-pig. guinea-pig cells, 12%%. It should be made fresh for each day's work. (3) Salt solution, 0.9%. Dissolve 9 gm. sodium chloride in 1,000 c.c. water. It is convenient to keep this in a bottle with tubing, pinch-cock, etc., arranged on a shelf like the water bottle outfit used in staining slides (Fig. 130). (4) Patient's blood to be tested. We collect from 5 to 10 c.c. for this purpose from a vein usually at the bend of the elbow. The best apparatus to draw it with is an abso- lutely dry, all-glass syringe (Fig. 118) and Xo. 24 platino- iridium needle. It is not necessary that they should be steril- ized. Sterilize the needle just before use by mopping it with tincture of iodine. Blood can be drawn with the needle alone by inserting it into the distended vein and allowing 152 PKACTICAL CLINICAL LABORATORY DIAGNOSIS the blood to flow into a test-tube, but this is not so satis- factory. With the patient sitting, or if in bed lying, and the operator sitting in proper position, have patient grasp Fig. 118. — Bur- roughs, Well- come all -glass syringe with platino - iridium needle attached. There are other good and cheaper all-glass syringes of the Luer type on the market now. Fig. 119. — Drawing blood from patient. Arm hanging down. Patient grasping above the Hanging, uuwu. j. auciu gia elbow to distend the veins. the arm above the elbow (Fig. 119). This is better than tying something about the arm. If held properly, this prevents the return flow of blood which distends the veins. Have him clinch the fist (Fig. 120) of the arm to be bled. This further distends the veins. If still not distended suffi- ciently, you can force more bipod into them by gripping (Fig. 121) the arm just below the elbow. Now apply tincture of iodine to an area about the size of a silver dime SYPHILIS 153 over the point selected to stick the needle in. With a finger of the left hand pull (Fig. 122) the skin tight over the vein and, holding the syringe in the right hand, which is steadied Fig. 120. — Further distention of veins by clinching the fist. Iodine has been applied to the skin over the vein selected for puncture. Note the small area. against the arm of the patient, stick the needle at the proper angle into the vein. By deliberation and accuracy it is easy to strike the vein almost every time. Do not stick through. Fig. 121. — Squeezing the arm to further distend the veins. Now draw (Fig. 123) the desired quantity of blood. Take care not to pull the needle out or stick it through the vein while drawing the blood. Instruct patient to "let go" his grip. Then quickly withdraw the needle and make pressure (Fig. 124) over 154 PKACTICAL CLINICAL LABORATORY DIAGNOSIS r % Fig. 122. — Introducing the needle. Note proper angle of needle and manner of steadying the hand. Note also how the skin is pulled tight over the vein and in the opposite direction to the stick of the needle. . jf Fig. 123. — Drawing the blood. Fig. 124. — Making pressure over puncture to prevent bleeding under skin. SYPHILIS 155 the puncture for a minute or two with a corner of a towel or piece of gauze. This prevents subcutaneous hemorrhage of a drop or two that otherwise often occurs. Now remove the needle from the syringe and force the blood into a clean, dry test-tube before it has time to clot in the syringe. Do not use a syringe that is also used for giving medicines by injection if avoidable. Remove the iodine stain from the arm of the patient with a few drops of alcohol. Clean up the syringe before the blood clots in the needle. The blood should be tested in from two to twenty-four hours after it is drawn. It is not necessary to keep it on ice if it will be tested within twenty-four hours. Such specimens may be sent to other laboratories by mail for examination when you are not prepared to make the test yourself. Stopper the tube with a new cork, pack carefully in a mailing case, and send by special delivery, first class, sealed mail. Do not register or send by parcel post. Do not send to arrive on a Sunday or a holiday for fear of delay in delivery. Apparatus required. — (1) All-glass syringe, 5 (or 10) c.c, with a one inch platino-iridium needle No. 22 for taking blood from patient and a 2 c.c. all-glass syringe with No. 24 platino-iridium needle for bleeding guinea-pigs. These needles can be sterilized just before using by mopping with Fig. 125.— Wood test-tube block 2x4x8 inches. Holes may be bored to accommodate different size tubes. tincture of iodine. They are quite soft, and therefore are likely to get dull, but can be resharpened on a small fine- grained stone. 156 PRACTICAL CLINICAL LABORATORY DIAGNOSIS (2) Two or three dozen plain lip less test-tubes, 12 x 115 mm. outside measure, should be provided. These are the same kind of tubes used in the other work described in this book. They are adopted for purposes of uniformity. (3) Two (one would do) graduated pipettes, 1 c.c. graduated in hundredths. The graduation should not come to the end or tip. (4) Test-tube rack, double row, bored to accommodate two rows of six of these tubes each. See Fig. 127. A block of wood 2x4x8, with a double row of holes bored one inch deep in it, makes a splendid rack (Fig. 125). Fig. 126. — Small electric incubator suitable for making complement fixation tests and other laboratory work by one who does not have more elaborate equipment. An incubator is convenient because the reaction is more active at 98° F. A small electric incubator (Fig. 126) is suitable, and they are now comparatively inexpensive. SYPHILIS 157 METHOD OF MAKING THE TEST 1. Set up in the rack two rows of five tubes each (Fig. 127). 2. Having previously centrifuged the patient's blood in order to separate the serum from the clot, now place in the Fig. 127. — Showing arrangement of tubes in rack and use of 1 c.c. pipette in measuring serum, antigen, etc., into tubes. Note end of pipette touching side of tube. first tube of the first row 0.03 c.c. of serum. In the second put 0.04 c.c. In the third put 0.05 c.c. In the fourth put 0.06 c.c. In the fifth put 0.07 c.c. In measuring small quantities into tubes from a pipette allow the end of the pipette to touch the side of the tube (Fig. 127). Place the same amounts in the corresponding tubes of the second row. 3. Put 0.2 c.c. salt solution in each tube in the first row. 4. Put 0.2 c.c. diluted antigen in each tube in the second row. The stock alcoholic solution of antigen has already 158 PRACTICAL CLINICAL LABORATORY DIAGNOSIS been diluted with salt solution sufficiently that 0.2 c.c. con- tains the proper dose of antigen. The dilution would he made by placing a small quantity of antigen (say 0.05 c.c.) in the necessary amount of salt solution. Fig. 128. — Proper method of washing pipette with saline solution from a water bottle equipped with tube and pinch-cock. We now have duplicate rows of tubes containing gradu- ally increasing quantities of the patient's serum. Those of the first or control row contain no antigen, while those of the second or test row contain antigen. Shake them so as to mix their contents. Allow them to stand five to ten minutes if kept in an incubator at 98.6° F., or double that nnnnn r test A &■ I 'pi n n n n I' f D | ■ - *fl PLATE XIX MODIFIED COMPLEMENT FIXATION TEST FOR SYPHILIS A. Negative reaction. B. Negative reaction. C. Doubtful positive reaction. D. Positive reaction. E. Strong positive reaction. SYPHILIS 159 length of time if kept at a warm living room temperature. 5. Place in each tube 0.1 c.c. of the washed guinea-pig cells. It is important to shake the tube containing the sus- pension of cells, because they tend to settle to the bottom. CONTROL Tube 1 0.03 c,c. 0.2 o.'i 2 3 •4 5 Pt. Serum Saline solution Guinea-pig cells. . . . 0.04 c.c. 0.2 6'i'" 0.05 c.c. 0.2 6:i" 0.06 c.c. 0.2 6!i 0.07 c.c. 0.2 6!i TEST Tube 1 2 3 4 5 Pt. Serum Antigen solution . . . 0.03 c.c. 0.2 6!i 0.04 c.c. 0.2 o'.i 0.05 c.c. 0.2 6!i" 0.06 c.c. 0.2 o'l 0.07 c 0.2 c. Guinea-pig cells. . . . 0.1 Fig. 129. — Scheme showing distribution of serum, antigen, etc., in the control tubes and the test-tubes. Dotted lines represent incubation. Shake the tubes gently to mix and allow to incubate or stand ten to twenty minutes (Fig. 129). 6. Read the reaction (Plate XIX) . In the control tubes it will be noted that hemolysis has taken place except per- haps in the first, or first and second, or possibly in the first, second and third tubes. Hemolysis is indicated by the cloudy suspension of cells changing to a clear solution. One or more of the tubes will usually show partial hemolysis. In case of a negative (non-syphilitic) serum the tubes of the test row will show practically exactly the same amount of 160 PKACTICAL CLINICAL LABORATORY DIAGNOSIS hemolysis as in the control. A slight amount of anti- hemolytic influence is exerted by the antigen, however, and we therefore read the reaction in the test-tube next above the lowest control tube in which hemolysis has taken place. If, for instance, tube number three is the lowest in which complete hemolysis has occurred in the control series, we read the reaction in the fourth tube of the test series. If hemolysis is complete the reaction is negative, but if hemol- ysis is slight or not present, then the reaction is positive. The stronger the reaction the larger will be the number of tubes in the test row in which hemolysis is partial or absent. If no hemolysis is present in the first tube above, and those above it show some hemolysis, we would call it positive. If there is no hemolysis in the first two tubes above, we would call it strong positive. A certain amount of experience and judgment are, of course, necessary in this as in other tests of this nature. There will be some doubtful reactions, as occurs with other serum tests. When in doubt, give the patient the benefit of the doubt, according to the clinical evidence. The modified Wassermann test described above is not applicable to spinal fluid because of absence of native com- plement. For technic and interpretation of the globulin test and cell counts in syphilis, see Chapter X. Interpretation. — The above method of making the test is as reliable as the original or Wassermann, when done by competent and careful laboratory workers and with reliable antigen. It is so simple that it can be done with a consid- erable degree of satisfaction by many who are not familiar with the technic and principles of the original Wassermann. Practically all cases of active syphilis after the appear- ance of the secondaries give positive Wassermann reactions. Practically all non-syphilitic persons give negative reac- tions. As the disease improves under treatment or other- wise, or as it becomes chronic, there is less and less chance of the blood giving a positive reaction. If the reaction is positive it is weaker. Many cases of tertiary syphilis and SYPHILIS 161 uncured syphilis give negative reactions. On account of the fact that there are some syphilitics who give negative reactions and an occasional non-syphilitic who gives a posi- tive reaction, the test cannot be relied upon for infallible diagnosis of the disease or for the contrary diagnosis. It must be considered in connection with the clinical evidence in the case for it to be most valuable. When thus considered it is often of much value. The test is usually negative before the appearance of secondaries and should not be allowed to take the place of the much more reliable examination for Treponema pallida. APPENDIX LIST OF APPARATUS AND MATERIAL REQUIRED 1. Microscope complete with mechanical stage attached, Figs. 1, 2 and 5. 2. Dark field condenser if diagnosis of suspected syphilitic lesions is to be made, Figs. 108, 109 and 111. 3. Gas filled Mazda lamp if darkfleld work is to be done. The No. 1782 lamp with rheostat for 110 volt D. C. or A. C. circuits sold by Bausch & Lomb Optical Co., is recommended. Fig. 110. 4. Mazda lamp, 25 watt, round frosted globe, if electricity is available and you care to use this somewhat more satisfactory source of light. See p. 6 and Fig. 5. 5. 1 "blood sticker," p. 12, Fig. 11. 6. 1 (or 2) box of 50 microscope slides, medium thick- ness, white glass, ground edges. Sometimes slides are sold that have more or less discoloration in the center. If not perfectly clear throughout, don't accept them. 7. 1 box of 100 cover-glass, 7 /g in. square, No. 2. Some- times cover-glasses are sold that are cloudy in the center. Don't accept them. 8. 100 c.c. grain alcohol (95%). 9. 1 tube (6 tablets) Burroughs, Wellcome & Co. "soloid" tablets for making Wright's stain. This is sufficient to make 90 c.c. of Wright's stain. Make up only 30 c.c. at a time. See p. 22. 10. 100 c.c. Merck's methyl alcohol, highest purity, to make Wright's stain. See p. 10. You can economize in the end by buying an original package of 500 c.c. bottle. If kept tightly corked it keeps indefinitely. 163 164 APPENDIX 11. 1 half -gallon water bottle, tubing and Mohr's pinch cock, arranged as shown in Fig. 130. A piece of glass tubing is so shaped that one end may be car- ried to the bottom of the bottle, while the other turns down on the outside of the bottle. Three or Fig. 130. — Arrangement of work table where electric light is used, showing most of the material and apparatus required in ordinary microscopic work. It is preferable to have the centrifuge located on another table or shelf and not as here shown. Note arrangement of water bottle, waste jar and slide rest across it. Note that the tip of the tube from the water bottle is about one inch above the slide rest. Have yours so arranged. The tube may be all rubber except the bent glass tube that goes to the bottom of the bottle and the tip, instead of the intermediate glass tubing as here shown. four feet is high enough for the water bottle to be placed. The balance of the tube may be rubber, except the tip. In ordering, specify "water bottle, tubing and pinch cock, described in Clinical Labora- tory Diagnosis, Bass and Johns, Fig. 130." APPENDIX 165 12. 1 diluting pipette for counting blood, 1 to 100, some- times called red cell pipette. Fig. 32. 13. 1 Bass counting chamber, Fig 33. Bausch & Lomb Optical Co. and other makers. 200 c.c. Toison's fluid, p. 35. 14 15. 16. 18 19 Tallquist hemoglobin scale. Fig. 54. bottle (2 drams) suspension of typhoid bacilli for making Bass-Watkins agglutination test for typhoid. See p. 65. Dr. F. M. Johns, P. O. Box 770, New Orleans, will furnish this for $1.00 — cash in advance. 17. 1 medicine dropper, plain (for use in making typhoid agglutination test). box ordinary hardwood toothpicks, best quality. dozen test-tubes, lip less, 12 mm. x 115 mm., outside measure. Must be of good quality. If you expect to make the modified Wassermann test described in this book, you should get 3 dozen of these tubes. No other test-tubes are required for the work de- scribed in this book. 20. 1 micro Bunsen burner and 3 feet of pure gum tubing suitable for it. See Fig. 131. Don't get the large regular size Bunsen burner. If you have not gas connections in your laboratory, get an alcohol lamp with glass cap to fit over the burner, in place of the Bunsen burner. Fig. 131. — Micro burner, much better than the larger Bunsen burners. 21. 1 Urinometer, ordinary form. Fig. 58. 22. 1 tube litmus paper strips, red. 23. 1 tube litmus paper strips, blue. 24. 100 c.c. glacial acetic acid. 166 APPENDIX 25. 100 c.c. potassium ferrocyanide solution, 10%. See p. 72. 26. 100 c.c. Fehling's alkaline solution. See p. 74. 27. 100 c.c. Fehling's copper sulphate solution. See p. 74. 28. 1 (better 2) graduated pipette, 1 c.c, graduated in hundredths. 29. 50 c.c. hydrochloric acid, C.P. 30. 50 c.c. chloroform. 31. 10 grms. sodium nitro-prusside. (This is used only in testing urine for acetone.) 32. 1 hand centrifuge (Fig. 69), with Cornell shields (do not accept any other) and rubber washers in these. See p. 79. If electricity is available, it is better to get an electric centrifuge. Get the Purdy centri- fuge, also with Cornell shields. (Fig. 67.) In order- ing state whether for direct or indirect current and whether for 110 or 220 volt current. 33. 30 c.c. dimethylaminoazobenzol solution, 0.2% in al- cohol. See p. 87. Indicator in testing gastric con- tents. 34. 30 c.c. phenolphthalein solution, 0.2% in alcohol. See p. 87. Indicator in testing gastric contents. 35. 100 c.c. decinormal sodium hydroxide solution. See p. 88. Should be kept in glass stoppered bottle. Used in testing gastric contents. 36. 1 glass funnel, 2% in. 37. 10 gm. benzidin (crystals). 38. 1 Petri dish with cover, 4 in. 39. 100 c.c. carbol-fuchsin Czaplewsky's formula. Accept no other. See p. 115. 40. 100 c.c. sulphuric acid solution, 2%%. See p. 117. 41. 6 Bass diphtheria culture tubes, hermetically sealed. Accept no other. See p. 127. (H. K. Mulford k Co. or Parke, Davis & Co.) 42. 1 platinum loop in glass rod handle, No. 26 wire. See p. 131. 43. 100 c.c. Loffler's methylene blue solution. See p. 134. APPENDIX 167 44. 50 c.c. earbol-gentian violet Czaplewsky's formula. Ac- cept no other. See p. 136. 45. 50 c.c. Gram's iodine solution. See p. 136. 46. 50 c.c. safranin solution, 1%. See p. 138. 47. 1 bottle Gunther- Wagner Liquid Pearl India Ink. You will need this only provided you do not get a darkfield condenser, as you should do. 48. Funnel stop for your oil immersion objective if you get a darkfield condenser. Figs. 109 and 111. This should be ordered for your particular microscope from the manufacturers. Give them the factory num- ber of your microscope and the objective. 49. Antigen for making complement fixation test for syph- ilis, described in this book, if you expect to make this test. See p. 150. 50. 1 All glass syringe, 5 c.c. (or 10 c.c), with platino- iridium needle, No. 24. 51. 2 (1 if you don't attempt serum test for syphilis) test- tube racks to accommodate 10 or 12 half -inch test- tubes (Fig. 127), or bore them in a block of wood. Fig. 125 shows a block bored with different size holes, and suggests the possibilities. Fig. 132. — Proper drop bottle for stains, reagents, etc. 52. 12 "TK" drop bottles, with flat top. (Fig. 132.) Ac- cept no other. (Fig. 133.) 30 c.c. These are for your stains and reagents. 168 APPENDIX 53. 30 c.c. peroxide of hydrogen, to use in test for occult blood. (You may already have this in your office for other purposes.) Fig. 133. — Two kinds of improper drop bottles. 54. 100 c.c. saturated aqueous solution of ammonium sul- phate. INDEX PAGE Abscess of liver, leucocyte count in 57 Acetone in urine, test for 78 Actinomycosis, leucocyte count in 55 Addison's disease, leucocyte count in 55 Agglutination test for para-typhoid 65 for typhoid 69 Albumin in urine, test for 71 Amebae, collection of specimens for examination for/ 102 differentiation of pathogenic from non-pathogenic 104 examination of unstained material for 103 examination for, in dysentery 102 interpretation of examination for 106 technic of staining 104 Anemia, pernicious, leucocyte count in 55 post-hemorrhagic, leucocyte count in 55 Anisocytosis 53 Antigen in complement fixation test for syphilis 149 dose 149 source of supply 149 Apparatus required in testing blood for syphilis 155 Appendicitis, leucocyte count in 55 Arthritis, acute, leucocyte count in 55 Ascaris infection, leucocyte count in 57 Ascaris lumbricoides, ova of in feces 98 Asthma, leucocyte count in 55 Bacteria, staining reaction, motility and morphology of Ill in cerebrospinal fluid 124 morphological classification 109 Basophiles 26 Bass counting chamber 34 Bass diphtheria culture tube 127 Blood, collection of for test for syphilis 151 obtaining for all microscopic examinations 12 occult in feces, test for 106 occult in gastric contents, test for . 88 sending specimens by mail to be tested for syphilis 155 Blood spreads, keeping unstained 22 labeling 18 making 17 Blood staining 22 "Blood sticker," a good 12 Bronchiectasis, leucocyte count in 55 Bronchitis, leucocyte count in 55 Burns, leucocyte count in 56 169 170 INDEX PAGE Carbol-fuchsin and methylene blue stain for pus and exudates, technic 137 Carbol-fuchsin stain, Czaplewsky's 115 Carbol- gentian violet stain, Czaplewsky's 138 Carcinoma, leucocyte count in 56 Casts, in urine 83 Centrifuge, use of in examining feces 92 use of in examining urine . 79 Cerebro-spinal fluid, cells in 124 interpretation of examination of „ . 126 Chancre, scraping to get material for examination 142 Children, percentage of leucocytes in 31 Chlorosis, leucocyte count in 56 Cholangitis, leucocyte count in 56 Cholecystitis, leucocyte count in 56 Cholelithiasis, leucocyte count in 56 Cholera, Asiatic, leucocyte count in 56 Cirrhosis of liver, leucocyte count in 56 Color index of blood 51 interpretation of 51 Complement fixation test for syphilis, interpretation of 160 modified method of making 1 19 Condenser, the Abbe 7 Counting chamber for blood cells, Bass' 34 Counting leucocytes 44 Culture tubes for diphtheria, Bass' 127 Cylindroids, in urine 84 Cystitis, acute, leucocyte count in 56 Czaplewsky's formula for carbol-fuchsin 115 carbol-gentian violet 141 Darkfield condenser, use of 143 Darkground illumination 143 Dengue, leucocyte count in 56 Diabetes, leucocyte count in 56 Digestion, heavy meal, leucocyte count during 56 Diphtheria, examination of culture for 130 incubation of culture , . 130 leucocyte count in 56 method of making culture 128 principles of laboratory diagnosis of 127 Diphtheria bacilli, description of 134 staining 133 Diphtheria culture, interpretation of 135 making preparation from 131 staining preparation from 133 Eclampsia, leucocyte count in 56 Endamebae. (See Amebee.) IXDEX 171 PAGE Endocarditis, leucocyte count in 57 Eosinophils 25 Epididymitis, gonorrhoea^ leucocyte count in 57 Erysipelas, leucocyte count in 57 Erythrocyte counts, calculating the number per cmm. in 49 interpretation of 51 total 48 Erythrocytes, abnormal or pathological 53 basophilic 54 method of counting 48 pathological interpretation of 54 stippled or granular 54 Feces, collection of specimens of 90 concentration of ova in by means of centrifuge . . . 92 examination of for intestinal parasite ova and larvae 90 larvae of uncinaria and strongyloides in 100 making preparation for microscopic examination 91 method of examining slide preparation of 95 occult blood in, interpretation of 107 occult blood in, test for 106 ova of ascaris in 98 ova of hymenolepis nana in 99 ova of oxyuris vermicularis in 99 ova of tenia in 99 ova of trichuria in 98 ova of uncinaria in 97 Fehling's solution 75 Filariasis, leucocyte count in 57 Funnel stop 145 Gametes 63 Gametocytes 63 Gastric contents, examination of 87 interpretation of examinations of 88 test for free HC1 in 87 test for occult blood in 88 test for total acidity in 87 Gastritis, leucocyte count in 57 Gland puncture in diagnosis of syphilis 148 Gonococci, carbol-fuchsin and methylene blue stain for 137 description of 140 Gram's stain for 137 interpretation of examination for 140 in urine 135 Gonorrhoea, in females 136 laboratory diagnosis of 136 leucocyte count in 57 obtaining material and making preparation for examination in. . 136 172 INDEX PAGE Gout, leucocyte count in 57 Gram's solution, formula 138 Gram's staining method 137 Guinea-pig, bleeding 150 Helminthiasis, leucocyte count in 57 Hemoglobin, estimation of . . . . 50 per cent., interpretation of 51 scale — Tallquist's 50 Hepatitis, leucocyte count in 57 Hodgkin's disease, leucocyte count in 57 Hookworm, infection, leucocyte count in 57 ova of in feces . . • 97 Hymenolepis nana, ova of in feces 99 Illumination, darkground 143 Incubator or waterbath, for complement fixation tests 156 India ink, use of in examining for spirochetes 146 Indican in urine, test for 77 Influenza, leucocyte count in 57 Intestinal obstruction, leucocyte count in 57 parasite infections, eosinophiles in 32 Kala-azar, leucocyte count in 58 Lamp, Mazda, as source of light in use of microscope 5 Larvae, differentiation between uncinaria and strongyloides 100 of strongyloides 100 of uncinaria 100 Lead poisoning, stippled erythrocytes in 53 Lenses, cleaning 11 oil immersion, use of 10 Lepra bacilla, description of 121 interpretation of examination for 121 staining 121 Leprosy, microscopic diagnosis of 120 obtaining material and making preparation from for examina- tion 120 Leucocyte count, differential 26 influence of infection with pyogenic bacteria upon 31 interpretation of , 30 number cells necessary to count in 30 Leucocyte count, total, acid solution as diluting fluid in making. . 36 apparatus and material required 34 calculating number per cmm. in 46 cleaning pipette 37 interpretation of 46 Leucocyte count, making the dilution in . 37 making preparation for 39 Toison's solution as diluting fluid in making 35 INDEX 173 PAGE Leucocytes, abbreviation to be used in making differential count of. 27 abnormal or pathological 33 description of found in normal blood 24 interpretation of presence of pathological 34? large mononuclear 24 percentage of different, in normal adults 30 percentages in children 31 polymorphonuclear basophilic 26 polymorphonuclear eosinophilic 25 polymorphonuclear neutrophilic 25 small mononuclear 24 variations in the proportion of 31 Leukemia, leucocyte count in 58 stippled erythrocytes in 54 Light, regulation of in use of the microscope 8 source of in use of the microscope 6 Loffler's methylene blue stain, formula 137 Lumbar puncture 122 Malaria, leucocyte count in 58 Malaria, plasmodia, description of 61 differentiation of 63 examination for 61 interpretation of examination of blood for 64 making preparations to be examined for 61 staining 62 time to examine for 61 Mastoiditis, leucocyte count in 59 Mazda lamp, gas-fitted 144 Measles, leucocyte count in 58 Megaloblasts 52 Megalocytes 52 Meningitis, cerebro-spinal 125 leucocyte count in 58 obtaining material for examination for 125 Meningococci, in cerebro-spinal fluid 124 Merozoites 62 Methylene blue stain, Loffler's, making 137 Microscope, adjustment of mirror in use of 7 care of stand of 11 focussing 10 light in the use of 6 model of Bausch & Lomb make recommended 1 model of Leitz make recommended 1 model of Spencer make recommended 1 selection of a 1 use and care of the 1 Mumps, leucocyte count in 58 Myelocytes, basophilic 33 eosinophilic 33 neutrophilic 33 174 INDEX PAGE Myxedema, leucocyte count in 58 Nephritis, leucocyte count in 58 Neutrophiles 25 Newton's color rings, looking for in blood preparation 42 Normoblasts 53 Occult blood, test for in feces 107 test for in gastric contends 87 Orchitis, gonorrhoea!, leucocyte count in 57 Otitis media, leucocyte count in 59 Oxyuris infection, leucocyte count in . 57 vermicularis, ova of in feces 99 Pellagra 59 Pneumonia 59 Poikilo^ytosis 53 Polychromatophilia 53 Pregnancy 59 Pyelitis 59 Pyelonephritis 59 Red blood cells. (See Erythrocytes.) Round worm, ova of in feces 99 Safranin as a counterstain " 140 Sarcomatosis, leucocyte count in 59 Scarlatina, leucocyte count in . . 59 Schizogony 62 Schizonts 62 Scurvy, leucocyte count in 59 Septicemia, leucocyte count in 59 Simon's septic factor 33 Slides, cleaning 18 Smallpox, leucocyte count in 59 Spinal fluid . 122 cell counts in 125 interpretation of findings in . 125 meningococci in 124 obtaining material for examination of 122 other bacteria in 124 pneumococci in 124 test of for globulin increase 125 tubercle bacilli in . 124 Spirocheta. (See Treponema.) dentium 146 refringens 147 Spirochetes, movements of . 145 Sputum, collecting specimens of • • 112 examination of for tubercle bacilli 112 interpretation of examination of 121 Stain, Wright's 22 INDEX 175 PAGE Strongyloides infection, leucocyte count in 57 Sugar in urine, test for 74 Syphilis 141 apparatus required in testing blood for 155 collection of blood to be tested for 141 examination for Treponema pallida in 148 gland puncture in diagnosis of 148 interpretation of complement fixation test for 160 leucocyte count in 59 sending specimens of blood by mail to be tested for 155 Wassermann serum test in 149 Tallquist's hemoglobin scale 49 Tenia infection, leucocyte count in 57 ova of in feces 99 Test meal, in gastric analysis 87 Test-tube block, wood 156 Toison's fluid 35 Tonsillitis, leucocyte count in 59 Total acidity, in gastric contents 87 Treponema macrodentium 147 Treponema microdentium 147 Treponema pallida, differentiation of from other spirochetes 148 examination for 141 interpretation of examination for 160 Treponema pertenuis 148 Trichinosis, leucocyte count in 60 Trichuria trichuris, ova of in feces 98 Tricocephalus. (See Trichuria trichuris.) infection, leucocyte count in 57 Tubercle bacilli, description of 124 in cerebrospinal fluid 125 in sputum 114 making preparation for examination for 115 staining 116 Tuberculosis, leucocyte count in 60 Typhoid, agglutination test for 65 agglutination test, interpretation of 67 leucocyte count in _. 60 Typhus fever, leucocyte count in 60 Uncinaria, ova of in feces 97 Uremia, leucocyte count in 60 Urine, albumin in, test for, qualitative 71 albumin in, test for, quantitative 72 acetone in, test for 78 casts in 83 collection of specimens of for examination 70 cylindroids in 84 epithelial cells in 83 indican in, test for 77 interpretation of examinations of 85 176 INDEX PAGE Urine, microscopic examination of , 73 preservation of specimens of 70 pus cells in 83 quantitative 76 reaction of 71 red blood cells in 82 specific gravity of 71 sugar in, test for, qualitative 74 Wassermann reaction, interpretation of 160 Whipworm, ova of in feces 99 Whooping cough, leucocyte count in 60 Women, small mononuclear leucocytes in blood of 31 Wright's stain * 22 Yellow fever, leucocyte count in 60