it^.C 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. 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 OP 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 1 ,)',)> , ) 3 ^ Copyright, 1920, by CHARLES C. BASS, M.D. AND FOSTER :\r. JOHNS, M.D. < t t I I I 1* . • . ' , t . . . t PRINTKD IN AMF:RICA 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. 424^39 in PREFACE TO FIRST EDITION Several years of teaching Clinical Laboratory Diag- nosis, making laboratory examinations and often interpret- ing the findings, has forcibly impressed ns with the need of such a book as this is intended to be. We take np 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 formulse 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 liere and to state tliat all we know of laboratory diagnosis or any otlier subject lias 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. \Vritten 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 V 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 hst 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 w^iich 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 liglit — Adjustment of the mirror — The Abbe condenser — Focussing — Oil immersion lenses. CHAPTER II Blood 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 HI ]\Ialaria 54 Obtaining bh)od and making {)reparations for examina- tion for malaria })lasmodia — Making the examination and recogiu'tion of j)lasmodia — Descri[)ti()n of malaria plas- mocha — Differentiation of malaria plasmodia — Interpreta- tion of examination of the blood for malaria. • • vn Vlll CONTENTS CIIAPTKH IV PAGE Typhoid Agglutination Tkst 65 Tcchnic — Interpretation of the t^^phoid 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 — INIcthod of ex- amination — Diagnosis of microscopic objects commonly found in urine — Interpretation of urine examinations. CHAPTER VI Gastrk Contents 87 Obtainin"" material to be tested — Test for free HCl and total acidity — Other examinations — Interpretation of the findings in gastric contents. CHAPTER VII Feces 90 Examination for intestinal parasite ova and larvas — Collection of specimens — Making the preparation for examination — Concentration of ova by means of the centri- fuge — Method of examining a slide preparation — Unci- narla americana ova — Ascaris lumbricoides ova — Trichuria trichuris ova — Oxyuris vermicularis ova — Hymenolepis nana ova — Tenia saglnata and tenia solium ova — Larvas of uncinaria and strongyloides — Examination for ameba^ in amebic dysentery — Collection of specimens for examina- tion — Exfimination of unstained material — Technic of staining fimeb;e and examination of stained specimens — Differentiation of pathogenic from non-pathogenic amcbie — Interpretation — Test for occult blood — Technic of test — Interpretation. CHAPTER VIII Prs AND ExruATKs Gkxkkat.ly 108 General remarks — Making the preparation and stain- ing — Application In j)ractlce. CHAPTER IX Sputim 112 Exjuninatloii for tubercle bacilli — Collecting specimens — M;iking the preparation — Staining — Description of tubercle bacilli — Interpretation. CONTENTS IX CHAPTER X PAGE I-EPROSY 120 Technic of obtaining material and making preparations for examination — Staining — Appearance of lepra ba«cilli — Interpretation. CHAPTER XI Spixai. 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 GOXORRHCEA 1'^^ Obtaining material and making preparation — Carbol- fuchsin and meth^^ene blue stain — Gram's staining method • — Appearance of gonococci — Interpretation. CHAPTER XIV Syphilis l-il Examination for Treponema pallida — Obtaining material and making preparation for examination with the darkfield condenser — Examination with the darkfield condenser — India ink preparation — Ditferentiation between Treponema pallida and other spirochetes — Gland puncture — Interpre- tation — Wassermann serum test — ^Materials required — A])- paratus required — iMethod of making the test — Control — Test- — Interpretation. Appendix 163 Complete list of apparatus and material refjuircd. Index 1(39 LIST OF ILLUSTRATIOXS FIG. PAGE 1 — Bauscli & Lomb microscope 2 2 — Spencer *'No. 44H" microscope 3 3 — Proper position at the microscope 4 4 — Learning to look in the microscope witli both eyes open. . 5 5 — I.eitz microscope with ^lazda hunp 5 C — 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 ])lood sj)read 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 3-1 — 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 40 — Cleaning cover-glass 40 41 — Counting chamber resting face downward against the foot of the microscope after it has been cleaned 40 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 44 — 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 — L^neven 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 fortli to facilitate mixing and to hasten agglutination 6G 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 wltli 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 ()1 — T.ookIn<^ for faint cloud In test for albumin 73 62 — Cloud due to .ilhumin in urine as seen in the proper light against a dark background 73 63 — Filling tube with uiine to a depth of 2\(> inches in quanti- tative test for albumin 74* 64 — Preparing fresh IVhllng's solution in testing for sugar. . 75 6'^ — 15oiling Fehling's solution and urine in testing for sugar. . 76 66 — .Vdtling urine from 1 cc. graduated pipette in quantitative test for sugar 77 67 — Klectric centrifuge with Cornell shields 79 68 — Box guartl around electric centrifuge 79 69 — Hand centrifuge with Cornell shields 80 70 — Filling balance tube to height of colunni 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 w^ater 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 wa}" 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 Avith the same magnification for comparison 97 84 — Photomicrograph of ovum of tenia saginata enclosed witliin 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 sha})e 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 IIJ.USTKATIOXS xiil FIG. PAGE 89 — Proper spread of pus or similar material to be stained and examined 110 90 — Tlioroughlv 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 12.3 99 — Bass diphtlieria 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 1 42 108 — Best form of darkfield condenser 143 109 — Diagram showing path of rays through a darkfield con- denser, and a /42 inch oil immersion lens fitted with funnel stop 113 110 — 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 ll'^J 112 — Photomicrograph of Treponema })allida in '\'hancre juice," as seen with the darkfield microscope 146 113 — Proper India ink preparation ll^) 114 — Photomicrographs of India ink ])reparatlons containing spirochetes H ' 115 — Etherizing guinea-pig; preparing to draw })lood 150 116 — Introducing the needle while steadying the parts 150 117 — Drawing blood from guinea-pig 151 XIV IJST OF ILLUSTRATIONS FIG. PAGE 118 — Burrouglis-Wollcoiiic all-glass syringe ^vith platino-irid- iuni 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 12T — 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 w4th saline solution from a ^vater 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 PI.ATE PAGE I — A. Small mononuclear leucocytes B. Large mononuclear leucocytes C. Polymorphonuclear neutrophilic leucocytes 24* II — A. Pohinorphonuclear eosinophilic leucocytes B. Pol^'morphonuclear basophilic leucocytes C. Relative sizes of the normal blood elements 24i III — Neutrophiles — 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 All — Tertian malaria plasmodia 55 VIII — Quartan malaria plasmodia 55 IX — Chemical tests of urine 71 X — Microscopical findings in urine 83 XI — Test of gastric juice for free HCl and total acidity. . 87 XII — Intestinal parasite ova and larvne 97 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 XA'I — Diplococcus intraccllularis — Diplococcus pneumonia? — Bacillus influenzjr — Bacillus tuberculosis 124« XA'II — Diphtheria bacilli from different cases suggesting variation in morphology 133 X^'III — A and B. Pus in acute gonorrlura C. Pus from case of acute non-specific urethritis D. Pus obtained from prostate and seminal vesicles by massage in case of chronic gonorrhcra 139 XIX- — Modified comj)lement fixation test for syphilis 159 PRACTICAL CLINICAL LABORATORY DIAGNOSIS CHAPTER I USE AND CARE OF THE MICROSC OPE 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 microsco2)e. — 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 countrv 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 2)urchased for routine clinical laboratory work only. Either of tlie otlier two serves tlie 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 (IG mm.), No. 6 (4 mm.) and V\-i' (1.8 mm.) oil immersion of N. A. 1.30; Ocular IV (lOX) ; triple nosepiece; Abbe condenser of 1.20 N. A. with condenser iris (haphragni in substage (Fig. 5). E. Leitz, 30 East 18tli Street, New York. Bausch & Lomb "F F S," with objectives 10 nmi., 4 1 'V' 'I^KAcTi:TCAit. CLiy;c\SL LABOKATOKY DIxVGNOSIS Fig. 1. — Baiisch & Lonil) niicroscopc described on page 1. Shown M'ith mechanical stage attached. USE AND CARE Or THE :MICR0SC0PE 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, Xew York. Fig. 2. — SiX'iK-er "No. 11-11." Shown witlionl inocliaiiical stage. Spencer "Xo. 44-IT," with objectives 16 mm., 4 mm. and 1.8 mm. oil immersion; ocuhir lOx; triple nose])iece; Abbe condenser of X. A. 1.20 with condenser iris diaphragm (only) in snbstage (Fig. 2). Spencer T.ens Co., Buffalo, New York. A mechanical stage (Fig. 1) is essential for best work, 4 PRACTICAL CLINICAL LAIJORATOKY DIAGNOSIS and one made by the same manufacturer and suited to the particular microscope stand selected, should be purchased with the microscope. If darkticid work is to be done (and it should be, if diagnosis of -suspected syphilitic lesions is to be made) a darkticid 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 micro8COX>e, — 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 (Mnzdn lamp, QH watt) and light-holder in proper ]io.siti( Leitz microscope described on ]iage 1, also shown witli mechanical sta attached jiosition. 6 PRACTICAL CLINICAL LABORATORY DLVGXOSIS while looking in the microscope with one. Learn to look in the microscope with either eye. Fig. a. — Improper use of roncavo mirror with Al)l)e condenser. Don't make this mistake. 'J'his arrangement may he tlesirahle when tlie liglit is too strong and when using the kjw power lens (16 mm.). Source of llc/hf. — ^V window on the north side of the room is hest. Where electric lights are availahle they give the most uniform light and have the further advantage of USE AND CARE OF THE MICROSCOPE being usable any time of day or night. A mazda lamp with ronnd 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 rnirror. — The mirror has one concave and one plane siu'face. 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- callv 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 brightest light is obtained. The mirror must be readjusted every time the microscope is moved. IVie Abbe condense7\ — The Abbe condenser (Fig. 7) serves the purpose of concen- trating the liglit 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 tliis 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 liaving tlie surface of the condenser approximately as high as tlie surface of tlie stage of tlie microscope. Fxamine your Abbe condenser and learn how it can ])e moved u]) and down, and look out for its getting out of ])lace. There is an iris diaphragm under the condenser witli whicli tlie amount of light must be regu- lated. It is strange how long it takes students to learn to Fig. 7. — Ablie condenser re- moved from the sub-stage. This can easily be slipped out of its carrier. The iris dia- phragm is opened or closed by the lever shown at the left lower side of the picture. 8 PRACTICAL CIJXICAL LABORATORY DIAGNOSIS appreciate tlie 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 Fig. 8. — Illustrntinfr mnnnor in wliicli lipht rnys reflectf'd from the flat mirror arc focussed iijjoii the objeet by the Abbe eondenser in proper })osition. frequently many times in examining a single specimen. Learn tJiis note. Generally speaking, the higher the magni- fication tlic more liglit required. Witli low 2)ower lenses it USE AND CARE OF THE MICROSCOPE 9 Fig. 9. — Compare with Fig. S aiifl note the ioss of light upon the object, wlicre it is desired, resulting from the Ahhe condenser being too low. It often happens that the condenser is slipjX'd down in tlie sleeve, which carries it and is not noticed and the microsco})e is used at this great disadvantage for some time. Watch out for this. is necessary to reduce the light. Also tlie more color an object has the more light is required. Hyaline and un- stained objects require less liglit. 10 PRACTICAL CLINICAL LABOKATOKY DIAGNOSIS A B 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 tij^ of the objective near the object; then look in the microscope and rack upward until the ob- ject apj^ears. Then focus with the fine adjustment. ^la- 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 tlie place selected to examine. Hun 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. ^Vij^jc tlie 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 CAKE 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 low^er 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 w^iping 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 w^hite 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 tliat 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 Ohtaimng hlood for all microscopic examinations, — Some kind of a "blood sticker" is required. A good and con- venient one may be made of a straiglit Hagedorn needle No. 6. The 2^oint of most of them is too long and narrow. 1 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 di'am 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 wlien taking blood from yourself. One should learn to take l)lood and make the S23reads with his own blood. It is (sometimes) more convenient to draw blood from the ear- lobe of patients. The princij^les 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 ai:itiseptics, except, perhaps, in special in- stances. Squeeze the place to be stuck between the tliumb and finger and make a quick stick with the needle while scpieezing. The pressure lessens pain sense. INIost workers can govern tlie depth and accuracy of the sti**k better if they steady the hands by letting them rest upon a table or other surface wlien sticking a finger (Fig. 12), or against the side of the face and neck oT the patient when sticking the ear 12 15L00D 13 Fig. 13.~Squeezing: finger when sticking to lessen pain sense. Fig. 13. — Squeezing the ear-lobe and steadying tlu- hand against the f.ict- wlien making tlie stick. (Fig. 13). The stick should not be deep enongli to cause blood to flow without ])ropcr niani])ulatiou of the siu'round- ing tissue. It sliould be made so (piickly and well that the patient hardly knows when it is done. 14 PKACTICAL CLINICAL LAliOKATOKY 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. 1(). — Squeezing out the blood. Note that the pressure is not made ^ close to the j^uncture. 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. l7.-^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 nuide by the fingers of the left hand. Fig. 18. — Squeezing l)lood from patient's fmgcr. Note that pressure is not made close to the jiuncture. and 18). If you Iiold 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 witli wliicli to make one spread. About one-fourth drop is the proper amount. Stop the pressure 16 PRACTICAL CLINICAL LABOR ATOKY DIAGNOSIS Fig. 19. — Taking up blood from patient's finger. Fig. 20. — Taking up lilood on slide from your own finger. The edge of the slide is first steadied against your left forefinger. and no more blood Avill flow if the stiek 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 tlie skin. Tlie 2)roper 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 ]Mcture erroneously shows the blood about to be taken up on the wrong end of the slide. It should be taken on the lower end. sj^read 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 mani[)ulated, twenty-five to fifty preparations can be made from it if desired. Making hlood spread for differential leucoeyte count, ed'a mi tuition for tnalaria plasmodia, looking for abnormal cells, making typhoid agglntination test, etc. — The advan- 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 wlio 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 ])urpose 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 w^ater and wiped dry with a towel or cloth. Hold by the edges w^hen w^iping. Oily or stained slides may be cleaned by boihng 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 in.ooD 19 Fig. 23. Fig. 24. Fig. 23. — Proper pose of hand and fingers to receive slide with blood on it. Fig. 24. — Slide witli drop of blood on it held in proper position for spreading. is^sk^r?? Pig. 25. — Slides held in i>roj)er position ])reparator\ to spreading blood. 20 PRACTIC^VL CIJNICAL LABORATORY DIAGNOSIS held ill the hand, tlie hlood is spread with another slide held hi the other hand. Place the end of the spreader slide just in front of the drop of hlood and bring it back to it (Fig. 2.5). Hold it at an angle of about thirty de- A B 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. grees (Fig. 26). The blood spreads toward both edges and is all behind the sharjD edge of the end of the spreader slide. Xow, w-ith a rather quick movement, j^ush (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 tliird of tlie slide or a little more. Note (Fig. 22) that the last j)art of the sj^read is considerably thhmer than the first j^art. Such a spread offers fields of any desired thickness. One made with too slow a- motion resulting in a very thin pi-e])aration will always show dragging of the leuccjcvtes, 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. Fig. 28. — The blood has been spread by pushing (n. t pulling) the spreader slide quickly. Staining hJood slides with lVric/]it\< staiti. — There are several good methods of staining blood. We prefer Wright's stain. It is a good one. Wriglit's stain is a solution of a 22 PRACTICAL CLINICAL LABORATORY DIACJNOSIS mixture of eosiiiate of niethvlene blue and eosinate of meth- ft ylene azin*, prepared in a certain way described by J. H. \Vright {Journal A. M. A., Vol. LV, p. 1979), dissolved in 2)ure methyl alcohol. The powder may be purchased in bulk or in the form of tablets (6 tablets in a bottle) made by Burroughs, Wellcome «Sc Co., or the staining solution may be purchased ready for use. Much of the latter sold is unsatisfactory. ft. To make the solution from the powder, dissolve 0.2 gm. in 100 c.c. of Merck's methyl alcohol, highest j^m'ity. 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 ^ve to ten minutes. 3. Wash, dry and examine with the oil immersion lens. '' ft 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 wiiich 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 j^oured on the slide should be the minimum that will cover the film w^ell 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 ])egins 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 w^atch for this than to go ])y exact time. Add the water from the w\ater bottle (Fig. 29), the tube from wliich should hang over the staining ])ar and jar, and it should be just long enougli 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. — Diliitiiifr "\Vrij>lit's stain on slide. Slide restinjr n]imi tlie slide-rest over a waste Jar. Water from \vater-l)()tth". 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 mimites, and see wliich is the best. The water used to dilute and to wash with nmst be either distilled or at least of high purity. Rain water is usuallv f^ood. After washing the 2:)reparation it should be promptly dried. First wipe the back of tlie slide and the front around 24 PRACTICAL CIJXICAL LABOKATOKV DIACXOSIS 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. Dcscnpt'ion of flic Jcucocf/fcs found in normal blood. — There are at least ^ve different kinds of leucocytes present in normal blood. Thev are differentiated from each other by tlie size, sliape and staining reaction of tlie nuclei, cytoplasm and granules present in tlie cytoplasm of some of them. The classification which we believe most nearly correct, in the liglit of our present information, is as follows: A. Small mononuclear leucocytes. (Lymphocytes.) These cells (l^late I) have a single round or oval nucleus ^\hich is frequently indented on one side. They have a i'elati\ely small amount of cytoplasm. Some have only a narrow band surrounding the nucleus, while others have ^'er^' 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 cyto^^lasm 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 ^r 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 ei-ytlii-ocytes. 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 dee])ly stained s])ecimens the cyt()])lasm of the older cells a])proaches the lavendci- of the neutrophile, making the differentiation depend largely on size and amount of c^i:oplasm. The nu- cleus varies in s]ia])e fi'om round or oval to slightly lobu- lated. It is often placed more or less concentrically. Some J '- r- A. Polymorphonuclear Eosinophilic Leucocytes. ^ /^ h 1^ %. t#> *..* V ^' ^» ^ / # ¥ 1 ^fett 'f* *1 .* <#• ^^' 0^ •^^' B. Polymorphonuclear Basophilic Leucocytes. j-^ * ilF 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 tlie nuclei of the neutrophiles, but tliere is possibly greater tendency for the nucleus to be divided into distinct lobes (2 or 3). The nuclei are often liorse-slioe shaped. The cytoplasm does not stain, but there are many large granules embedded in it, wliicli take the eosin in a mixture containing eosin, and tlie cells are tlierefore called eosinophilic or eosino- philes, dropping the other cumbersome part of the name. 26 PKACTICAL Cl.lMCAL LABORATORY DIAGNOSIS Thev constitute about 1'^ to -AS 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. Tlie 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 AVright's stain their color is a combination of the dark ])lue methylene blue and the purplish azur. The basophiles constitute less than 1% of all the leucocytes in the blood of normal adults. Method of mailing a differential leucocyte count, — A differential leucocyte count is a determination of the rela- tiye proportion or j^ercentage of the different kinds of leucocytes present. The ordinary blood spread described aboye (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 adyantage. 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 tliicker part of the spread. A good spread has areas, vary- ing from too thick to count well, at the thick end, to un- necessarily tliin, even for tlie beginner, at the other end. It frequently takes a long time for beginners to learn to locate proper ground to study. They do not reahze the extent of the pre])arati()n under such liigli magnification. The student sliould learn early to select tlie 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 diflFerential 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 ^rhicli to mark down the different cells as thev are found. It is not necessarv to write out tlie names of the different cells as these are famihar terms, whicli 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 basoj^hilic leucocytes respectively. Like- wise U mav stand for unclassified leucocvtes. A mark is 28 PRACTICAL CLINICAL LABOKATOllY DIAGNOSIS made opposite tlie proper class for each cell found. For convenience in coiintino- 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: StfllWI Lll El irlll The percentage of each kind of cell can be calculated after anv number has been counted, but it is much better to count exactly one himdred 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 ])iit down eacli cell, he learns to carry several cells "in his head" before stopping to put them down. This saves time. Finallv, one should learn to carry the small mono- niiclcars and neutropliiles "in his head" imtil nearly one liundred have been counted, only stopping to put doAvn the other cells which are few in number. When the number of cells reaches near] if one hundred they are all put down and the tally sheet would look something like this: BLOOD 29 /f 23 zm jr U Ell 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: /S^ 23 Z3 Ull ^l 3 I JBI 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 wiih the left eye in making differential leucocyte counts. The tally can be })Iainly 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 he learned with a little practice and has many advantages. It is worth learning. With good methods and with practice one should he ahle to count one hundred cells in tliree minutes or less time. Somewliat more exact figures can he ohtained by count- ing several separate lumdred cells and taking the average of all tlie counts, but much more depends upon properly made spreads tlian upon counting large mimbers of cells. Xo count of tlie cells on a poor spread in which the larger cells liave 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 tliey vary. When the count is carefully made on a properly made spread and in the part of the spread made before tlicre 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 leucocjites 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: BLOOU 31 S— 20% to 30%. a. L— \% to 8/0 N— 607o 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 ii> 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 numl)er 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 tlieir toxins are ab- sorbed, very large numbers of neutropliiles are present; })ut when there is good drainage, and tlierefore little or no absorption of toxin into the system, there is little or no in- crease of tlie neutrophiles. For instance, staphylococci in 32 PRACTICAL CLIXICAL LABORATORY DIAGNOSIS the appendix or in tlie peritoneal eavity 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 tliere 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- ])hiles before drainage is established, but afterward they fall to near or quite normal in a few hours. An increase in the ])er cent, of neutrophiles indicates therefore absorption of toxin from a pyogenic infection or disease in some part of the l)ody, 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-foui* 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 l)e named malaria plasmodia, typhoid bacilli, tubercle bacilli (unless in very large numbers), and tetanus bacilli. Tlie 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 ])ronchial asthma. There is slight to considerable increase in a variety of parasitic skin diseases. Eosinophiha also occurs in most cases of foreign protein poisoning and ana- phylaxis. Basophiles '^. BM Mature Cells. Myelocytes. • EM NM Myelocytes. NM B upper half of the picture traces backward the development of the polymor- ear leucocytes from myelocytes to mature adult neutrophiles eosinoyhiles and 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 iiathological 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 polymorj)honuclear 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 2:)olymorphonuclear cells, and there must tlierefore 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 tlie abnormal lencocvtes which fall hi one or the other of the above gronps, there are usually a good nianv others in leukemic blood that cannot be cla'ssified. They should all be grouped under the head of unclassified cells. When makino' 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 p?T5<^7?(?^ of j^at^^ological leuco- eytes, — ]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. 33. — Diluting pipette. 0.4 cu.mm 0.04 •• ■• 0.004 •• 0.00025- r^ \y BASS RULING 6544 Fig. 33. — Bas.s 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. a-- 7 ^ rr Fig. 3k — Counting chamber. Cross section, a. Object sbde. b. Counting plate, c. Rectangular plates to support cover-glass exactly 0.1 mm. above the count- ing plate, d. Special thick cover-glass. It consists of a heavy object slide upon which a small oblong counting plate is cemented (Fig. 34) . On either side of the counting plate there is cemented upon the slide a rectangular glass plate so as to leave a gap between the edges of the counting plate and the inside edges of these rectangular plates. The latter are exactly 0.1 mm. higher than the counting plate, so that when a cover-glass is placed over both there will be a space of 0.1 mm. between it and the counting plate. The Bass ruHng (Fig. 35) in the middle of the counting plate consists of a square of 2 mm. side. This is divided into ten rectangles, 0.2 mm. X 2 mm., for counting leucocytes. For counting erythrocytes there are superimposed upon the ruled space at each corner and in the center five squares of 0.2 mm. side, each further divided into 16 small squares of 0.05 mm. side. The cubic contents of the entire ruled space, 0.1 mm. deep, is 0.4 cu. mm. The total cubic contents of the five large squares is 0.02 cu. mm. In addition to this special apparatus a diluting fluid is required. We use Toison's fluid, which permits counting both leucocytes and erythrocytes in the same preparation. The fornmla is: 1? Sodium cliloride 1 gm. Sodium sulpliate 8 gm. Glycerine '. . . . 30 c.c. Methyl violet, jB 0.025 gm. Water 160 c.c. 36 PRACTICAL CLINICAL LABORATORY DIAGNOSIS It should be kept in bulk in a tall, narrow bottle and whenever a count is to be made, enough of the solution should be poured off (into a small bottle) for present use. Do not use dircctUj from stock bottle. Do not return unused Fig. 35. — Bass ruling. solution to stock bottle. If managed as directed the solution usually keeps well, otherwise it may deteriorate from the growth of moulds and yeasts and soon be unsatisfactory. For counting leucocytes only, a 2% solution of acetic acid in water, which destroys tlie erythrocytes and makes the leucocytes easier to see, is useful. It can be made up extemporaneously when required for use, or it may be BLOOD 37 poured from a stock bottle when required as advised above for Toison's solution. An 0.85% sodium chloride solution ("Xormal saline") is a fairly good diluting fluid when the ervthrocvtes onlv are to be counted. Cleaning the pipeite and mahing the dilution. — The pipette must be clean and perfectly dry before use. As- suming that it has been used it is cleaned and dried as follows: (1) Slip off the rubber tube and blow through the tip of the pij^ette to expel any fluid present. (2) Re- place the rubber tube and draw pipette full of water. (3) Fig, 36. — Taking up blood into the diluting pipette from the patient's finger. Note that the operator's right hand is steadied by touching a finger against the finger of the patient. Slip off rubber tube and blow water out. (4) lleplace rubber tube and draw pipette full of alcohol. (5) Slip off rubber tube and blow alcohol out. (6) Replace rubber tube and draw pi2)ette full of etlier. (7) Slip off' rubber tube and remove ether by slinging the pipette somewhat like "shaking down" the mercury in a thermometer. (8) Replace rubber tube and remove last trace of ether by suck- ing air through the pipette when it will be dry and ready for use. Always dry and clean the pipette after use and have it ready for the next time. (\Vhen thoronglily dry the bead in the bulb of the 2)ipette rolls about freely when the pij^ette is shaken.) 38 PRACTICAL CLINICAL LAIJOKATOKY DIAGNOSIS To make the dilution, liave the dihitino' fluid ready. Well up a good sized drop of blood (without squeezing hard) and draw blood (Fig. 36) into the pipette to the Fig. 37. — Filling diluting pipette with diluting fluid. Pipette held high enough that the marks may be easilv seen. Fig. 38. — Revolving pipette to mix contents. Note that pipette is held at about the middle. By holding rubber tube as shown, and revolving it by rolling the fingers, best agitation and mixing is secured. mark 1. Quickly wipe the tip of the pipette to remove any blood on it and draw up (Fig. 37) diluting fluid until the mixture reaches the mark 101, being careful not to get an air bub])le in the pipette. Xow revolve (Fig. 38) the pipette to mix thoroughly and the dilution is ready for use. The counting may be done any time within an hour or two. If to be carried anv considerable distance to the BLOOD 39 laboratory, stretch a rubber band over the ends of the pipette (Fig. 39) to prevent tlie fluid from running out. Fig. 39, — Pipette containing diluted blood. Rubber band stretched over ends to prevent escape of fluid when carrying. Making the iJrepa7'ation. — The counting chamber and cover-glass must be scrupulously clean and free from dust particles. Since the parts of the counting chamber are cemented together it must never be washed with anything except water, but it can be washed with water freely. Care- fully dry it and then wipe it free of dust particles with a handkerchief or similar piece of cloth. If it is held so that the light falls upon it at an angle and so that there is a dark background in the distance, dust particles can be much better seen. These should be j)icked off with the handkerchief wrapped around the end of the finger. Hav- ing it clean, rest it face downward against some con- venient object, as the foot of the microscope (Fig. 40), to prevent dust particles from settling on it before use. Now thoroughly clean the cover-glass. AVash it if neces- sary. Hold by the edges only (Fig. 40) when cleaning. After it has been wiped as clean and free of dust j^articles as possible, with the handkerchief, hold it in proper indirect light with a dark background in tlie distance to see any remaining dust particles whicli sliould tlien be 2)icked ofl^. Now place it, clean side down, upon some convenient dust free surface, like tlie wheel of the mechanical stage (Fig. 41), until required for use. Having the counting chamber and cover-glass ready for use, have convenient some hard glass or metal object (prefera])ly not sharp on tlie edge) with which to press down the cover-glass when needed. The handle of the platinum loop or eye end of the blood sticker needle or 40 PKACTICAL CLINICAL LABOKATOKY DIAGNOSIS Fig. 40. — Cleaning cover-glass. Note that it is held by the edges only. Fig. il. — Counting chamber resting face downward against the foot of the iiiioro- scf)pc after it has been cleaned. Cleaned cover-gli^ss resting upon wheel of nieelianical stage. BLOOD 41 even the homo vial which holds it are convenient for this purpose. Wood or rubber instruments are not good, nor is a lead pencil. Now thoroughly mix the contents of the pipette (rubber tube attached) and blow out two or three drops of fluid Fig. 42. — Squeezing- out diluted blood upon the counting plate (old style counting chamber). Note folding of soft rubber tube. Fig. 43. — First step in placing cover-glass upon the counting chamber. Note that both hands rest (to steady them) upon the table. to wash out the long arm of the pipette. Then quickly squeeze (Fig. 42) onto the center of the counting plate the necessary amount of the diluted blood. A little expe- rience is necessary to get the proper amount. It should never be sufficient to rim over into the moat, but must alwavs more tlian cover the ruled area, l^lace the cover- glass over it at once. The steps in doing this properly are illustrated in Figs. 43, 44, 45, 46. The cover-glass should 42 PRACTICAL CLINICAL LABORATORY DIAGNOSIS now be pressed down (Fig. 47) firmly at each of the four corners. Do not move the cover-glass in doing this. There must never be fluid between the cover-glass and the plates on wliich it rests. The directions sometimes given by manu- facturers of the new style counting chambers to allow the • Fig. 44. — Second step. The cover-glass is tilted up by the pressure of the finger upon the edge that extends a little over the outer plate. Fig. 45. — Third step. Bringing down the cover-glass by pressure with the handle of the jilatiniini loop. fluid to flow under the cover-glass is a mistake and should not be followed. If the surfaces of the plates and cover-glass are perfectly clean and free from dust particles, and if the mani[)ulation has been properly done, Newton's color rings (rainbow colors) will l)e present on each side. These can BT.OOJ) 43 Fig. 46\ — Fourth step. The coA'er-glass has been brought down and the finger has been lifted from it. Fig. 4-7. — Fifth step. Pressing down the eover-gbiss at each earner. Note th;it the hand holding the instrument with whieh pressxire is made is steadied by resting upon the tal)U-, Care must be taken to axoid slip})ing the eover-glass. 44 TKACTICAL 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 tlie level of the eyes. Unless they are present the preparation is not a good one. Do not tilt the prepara- Hon. Hold it level. Now lay it down for two or three minntes during Avhieh time all the cells settle onto the surface of the countiug jDlate. In spite of good teclmic and usually due to slow manipu- hition. tlie 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 ])reparation (level) above the level' of the eyes (Fig. 48), having the light fall upon it from above, but look at it from below witli a dark background in the distance. The evenness (Fig. 49) or unevenness (Fig. 50) can be seen, and if J:he cells are not evenly distributed the preparation is not good. Couuiiiuj the cells. — Reduce and adjust the liglit of the microscope so that the ruling can be seen best. Count w^ith the 4 mm. objective. Find the upper left hand corner of the ruled area (in fact the lower riglit hand corner; images are BLOOD 45 Fig. ."iO. — Uneven ditrtrihution of cells upon the ruled ]ilate. Note thin nrea at top and thick area on the right. 46 PRACTICAL CLINICAL LAIJOKATOKY DIAGNOSIS reversed in the niicroseope) . Place the end of the first rectangle in the center of the field and move toward the riii'ht, coiintino- all the leucocytes found in the rectangle. When the end is reached drop down until the next rectangle is in the center of the field and move in the opposite direc- tion until all the cells in it are counted. Drop down to the next and count all tlie leucocytes in it in the same way. Thus continue until all the leucocytes in the ten rectangles have heen counted. Make the rule of counting in any cell found uj)on the left or upper boundary line of any square or rectangle when counting the cells in that area, but count out any cell found upon the right and lower boundary lines of a given rectangle or square, when counting the cells in it. The leucocytes are stained violet when Toison's fluid is « used. They are clear, many-shaped, prominently appear- ing bodies when acetic acid is used. The small ruling is disregai'ded when counting the leucocytes. ^Multiply the number of leucocytes counted in the entire ruled space by 250, which gives the number of cells per cu. nnn. Interpretation of total leucocyte counts. — Blood of nor- mal individuals contains 5,000 to 8,000 leucocytes. There is a slight increase during digestion, and children's blood gives slightly higher values than adults'. The total number of leucocytes is increased in leukemia sometimes to very high figures. Seven hundred thousand has been recorded in myelogenous leukemia, but 400,000 to 500,000 is an average high count. In lymphatic leukemia the count seldom reaches 200,000; 90,000 or 100,000 being an average high count. The total number of leucocytes is increased in those diseases in which there is increase in the percentage of y)articular cells as a result of irritation of the tissue which prcxhiccs them, or as a result of the demands of nature for the pai'ticulai" cells to antagonize the disease process pres- ent. For instance, there is usually slight increase in syphilis during the stage of lymph tissue involvement. In the BLOOD 47 worst cases of disease due to pyogenic bacteria and in which much toxin is absorbed, the total leucocyte count may be increased to 40,000, or even 50,000, but these figures are rare. Naturally the count depends not only o o C) a ■\ o o O^Oo 'O Q o -^ %0 8 ocg o O O ^ Q U ^ ^-"^^ a o e- ^?T^5?^ 09^0 Q O "\ 0^=^ a o^ o ■Q¥^ Qt^ o ^ Ooo Oc^- Oo o Q o /.op 0^0 .^ ' ^ Q '^ O c:^c-,n o ^ o oc oo c?, -^ Q O (^ ^ 3V O O ^d OO CJ a (7 ^ ^ o a <^ Fig. 51. — Arrow iiidicates order in wliicli 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 incHvidual to meet the demand. Long-continued disease often tends to exhaust the caj^acity 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 onlv tlie general tendency and cannot be exactly correct in • C7 ft ft all cases, and in all combinations of disease. The total leucocyte count sliould be interpreted in the light of the (^tlier 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 o o. (^ o cf o Fig. 0-2. — 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 ^\e large squares, — one at each corner of the ruled space and one in the center, each ruled into sixteen small squares. Count the erytlirocytes 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 BLOITING PAPER REQUIRED AND THE AKRANGE- MENT OF THE COLORED SCALE Tlip colors liorp shown arc not exactly tlio same sliade aa in tlie oriojinal, they do not correspond to the difrprent pprccntafifps of licmoijlobin and can- not be used to ascertain or mpasnre tlie nuantitv of liemotjlobin. BLOOD 49 square count i?i (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 ])l()od is an expression of the average amount of hemoglo])in per erytlirocyte, tlie 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 fiivst two figures on the left hand side of the number of erythrocytes per cu. mm. by 2, 50 PKACTICAI. CIJNICAI. I.AliOKATOKY DIAGNOSIS unless it is less tliaii one million, when only the first figure would be multiplied by 2. Interpretation of number of erythrocytes, hemoglobm per cent, and color indcLV. — In health, females have about 4, .500, ()()() to ,5,000.000 erythroeytes per cu. mm., and males have about .5,000,000 to 5,500,000. Five million is generally considered the average. The liemoglobin in blood of normal individuals is about lOO^c, 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^ o 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, usuallv 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 hiffh as from 7,000,000 to even 10,000,000 per cu. mm. The hemoglobin percentage is increased by any of the a])ove influences that increase the erythrocyte count. The color index would tlierefore 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 ft..' ft..' -' 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 somew^hat 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 greath" 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 erytlirocytes 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. Tlie color index is nearly always below the normal hmits in secondary anemia. In long standing secondary anemia witli low hemo- globin and erythrocytes the color index may approach or reach normal figures. The lowest color index occurs in chlorosis. Pathological erf/tJirocf/fcfi. — Tlie shape of normal erytli- rocytes is that of a ])iconcave (hsc. Sometimes tliere is great variation in tlie shape of many of the cells. This con- dition is known as poikilocytosis and such abnormally shaped erythrocytes are ])()ikilocytes. (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 iiiicrocytes. 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 j^rimary 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. Xuclei of ervthrocvtes 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 manv 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 ervthrocvtes 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 basopliilic 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 sliould be clearly understood that there may be many different c()ml)iiiations or variations from the normal in a single cell or in the cells in a given blood. For instance, a megalocyte may ])e nucleated and therefore be a megalo- blast, it may be a poikilocyte, it may be stippled and it may % ^■'>. "V. Anisocytosis. r- P^oikilocytes. It Jft Megaloblasts. ^^ -^j^m^ ^b. ■• • • % Aormobiasts. ^^ HP .J £■ t .*ji* ■'.pfti ^^ iranular or Stippled Erythrocytes. % Polychromatophilia. 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 MAI.AKIA Ohtd'ining blood and ?naking preparations for eocamina- tioji for nudaria 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. Mailing the eocamination and recognition of plasmodia, — Searcli for plasmodia only in f)arts of the preparation where the staining is good (nuclei of the mononuclear leucocytes are })urple 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 w^here the cells are more tlian one cell thick. After sufficient experience you can recognize plasmodia satisfactorily w^here the spread is two or tliree cells thick and of course at a considerable saving of time, since the thicker the blood film the more plasmodia arc ])rc.^'iit in a given area. IMalai'ia plasmodia are in (or on) tlie erythrocytes, and no object sliould be considered as ])r()bably aplasmodium uidcss it Is so situated. It is true that many plasmodia, es2)ecially old gametes, are in (or on) little more than 54 # V. •s 0. J5^.v •J ^*V ^' \ ,-^ ^ i \ ( W^' c^ Varieties of Schizonts found in the Peripheral Circulation. ^^A^fi.m,. ^ r VC^^^ ^:. -/ Gametes. V O 02 C> »— 1 QQ c3 O o H O o 12^ f3 ?H DO (1) O t) O r ^ £ r ■■•■ ->, erf Eh •r ^ o O o o p ^ S § 1-:; 1— 1 O 1— 1 o ^ 'P S o 1^ P C3 O 4-' H ^ ^ ? «r p:i o o P^-n W o [I^ t^ r=^ - P^ Q r-; C^ . C/2 - ^ 2 ^ <^ c/2 < ^3 ;i^ ^ r-H kI; ^ rr tH o <1 C/2 1— 1 Q 32 . O Ph Eh O o o o ^ be O H fcrH o o ^ rt ::? rt o H 1 ^ ^^ r-' <^ ;z; ^r- --^ O P=: o t: -P o d ^ r: o oj ^ ^ o T^ "^ ^H /- -J U2 2 ^ H 1 ? ^- ^' M -'• ^ CJ C5 ;^ '^ "zz -M iz; ^ r- y; .O l-H ;r .-=; r; « H p ^^ o < o 2^ o l-H :3 ;^ - t— 1 2 -• 2 ^ r^ r ;^ rt P^ -h^ 2 'S ^ ^ PQ r- ^ ^ ^ :5 2 t: EH c3 ?:: rt l' 03 1 ^ ^ 1 0. 0) 1 o 03 0/ Q 03 ■S a o S-i ^ Sh 2 o o O o CJ o 3J c c C d U h-i h-i l-H HH 1— t (« u 1 b 03 b U 1— t cS Q -2i 3 \ % s 01 ^ rt < O o o u .J < H Z u K a o 03 m !c c o .£ 'in o W ca P. o 3 -t-5 tj: f-^ m « o 4; c a; 03 o Q (C cc CO cc a cS cC C5 O) m a> O ^ u ^ U o o o o c C C C 1—1 Hi 1— 1 Hi m 3 C o c o E bo 0) w 03 (i O C m cS 3 C O C o E w o O) o Z ■< H O e3 >j o ^ -^ o tL ^ u >'5 1—1 c o o 2 tc s •- c u »■ "^ h^ o r 6 o o o ci 03 fcc c t4 ^ ti =3 ti) ;3 o « >, ^3 o ^ -t-s -t-> ^?, WW S ?r 'bb CS cc r t- tt C o s CC o ^ o" <1^ n-, « 03 03 03 O) t» 03 3^ 03 03 03 cS rt -tf CS a a; es tx 4) o es o V CS o o o r- c C C »— 1 1— ( >— ( 1— 1 h-i 1— I • ^ 03 13 »-> 03 > 03 03 03 c rt rt ;- -r^ B- ■i w 03 S ^ QJ QJ O >H ^ J2 z o H Q Z o (J Of o a m < a m > 03 w m *-< PQ -.— -^ 1 ^^ es 01 £ ^-H o ^^•^^ O o i 03 O) OS Oi 03 .2 "S o m w e3 o 03 '-4-> 'So c 03 itliias ck) <1 03 0) 03 0; o o O f^^ '^ •*-« o -M tc '-M O eS 0^ .—1 4_j 0; -*-> 03 03 O) »1 O o £ rt O ;^ 03 c cS be ci ^ ^ *"] pj^ r^i 03 • ^^ O O O O ^ ^ O O w w w eS m S cS o 56 o m u o >,. e: F^ p-- tc P o Oi cc tK -(J eS ci cS o O (H ;-> ^ u g bfc « 1— 1 cc t/3 a; O C r-5 (U m w cS rt c o s- ^H o o ^N C h-^ t— 1 03 o 0) Q) CO CO eS CS ;- « o C C 1— 1 1— 1 1=: (D O CO m c3 d O a; Vm Wi u o C C H-l I— I O o o -t-5 c ^H 1 eS a o OJ r^ u '^■' bC ? ■+J c ^^ fi -u o • i-H « Tt-g :3 3 P— 1 ^ , CS 0; 03 0; >. 03 r- ^ 5r o: <3j rt c; 't; o o o ^ fc- Si S- G o O rS o C r^ c »-> 1— 1 (—1 H- 1 o m > c o o c e« ^ be o c a» •F^ ui -<-> s -tj ^^ ^ ^ , Oi tr 0) 03 cS tC eS C CS O) ^- -t-> h O 02 o c Hi r o o 03 ^ u a> o CO ri CS H a go 1 CS l-> _^ . 0) o O; -«-) '5 c ■ — ' 3 >-. * • FN < ^-^ -»-> 03 - — - " — ■ ee --; CS • F-» 03 03 8 t- 03 • fH ,c CS '^ o > .2 *-*-> -i-> c '2 '+2 •-* F', cS F— 1 03 CS CS c c w K s O O O 1 .F. 0; cS ._ o: j; O 03 W 03 "t; >— '^- ^^ C *" CS I- F= -r C Cj - CO .- c s c o X 03 -r Ca:E-^C c; • ^N 03 hJ >^^ •-^ f:^ rr. _^03 ci titi cos 12 C F- CS 2;, tL £-« C 1— i 1— ( H-1 w o 03 fO o I— I CS .S 03 57 o 91 Q. O en CO CO .a 3 be u 2: o o Ea o o o H Z H « 03 IS o. o ,c *5 o 09 O u 3 m u 13 3 o c o g be CO u o CO ci s c a; CO 0) CO Ol ~>^ o o 'a! to a '3 d o Ph 05 o o CO a; en a; P 0) OJ CO cs >> >» -^ o 93 > 3 cS S 03 cj s ?^ o; o; >- b ^- t o be o be c C — 1 1— I m 0) CO a; in 3 C o a o a m CO eS u C 01 ^ > ■^ . o -+J o c rt - rr -.> 'd c! a> o o; ^H cs ^ ^ Ol o be ti CO fn o 't; CO ce o 2 O u o o D < O 1 o -p OJ O o o c c o -p (M rt ^H a> o be o 0) o CO o o w ^H c i -p Z o ^ o S" o be^ 0) o Ui O 0/ ^ 1—1 o CO 73 O t- o o o c ^o CO 0) ci ^ >-> O) C o ^ 0> r- r— S-, '^ Ol Ol be 03 CO » O C/3 -»J 03 J- 03 OJ CO Te 2^ r- rt a p— 4 ^ t^ i? o CO CO 3 o ^'^ ^ 9 n 03 0) o r^ CO CO c ci C £- H ^N eS 03 03 P m c Si « fc « .iS t— 1 O C 1— 1 2 O H z o u to < u CO es 0) ce CS £&< o s ^ r 'S o O cS CS 0; •4-9 o o o .5 c c5 ce C a- ^ ^ ^ '^ cs _d £'i PH eS •^ - cs '- c5 o ^ t— 1 k— 1 r<; . (^ L o Si CO be 2 O; CO CO pi p-* o < cS E 03 0^ '-M TS 0^ X "^ f'. 03 ^ 1^ 58 en m 0) s 0) 4) CO en Ki Cj OJ O) ;-i u o o OJ OJ Q o 0) CO a; a; o o m (V OS CO 0) OJ CO CO OS eS 0) >^ cS '^ C CO 0^* ■" ;3 a) «^ ^v OJ a; 05 CO 0) CO to es cS SC CS c3 OJ a- rt OJ a> ^ ^H -M ^ !m o a CO o O C r^ c r- t— 1 hH 1— ( h— 1 CO o o 3 S £ - ^ c — o c t/: CO a c tc c • ^^ Sm 3 rs be -u cS ^d^a m be ^_, >» x ^ cs a; «_■' K C 03 c a> o (-. a; o CO S h- 1 (V 03 C3 m u u C C o: 03 fS 0/ 'A 1 03 ci s '^ c 03 c5 n3 d = .<» d ^ '5 » ^_> o S CO '^ bt '■Z "o >y ^* • — -t-> ^~ o n r-t o C oi bc 0; 0; C CO 03 O -M CS £ o V u Cj ei S >. > ^ ^ cs t) '^ cz. 01 I eS O O -P -P S 03 c a c >- *> 03 a) ce S ■p 8 c/: O O I — 1 cZ 3 03 T. c 59 z p o n H >> O o o 1-3 < H H 6S H fa o 03 aj 15 O c o H 0) ec u 9 a o u 3 m u s 3 C o c o bo ;^ CIS 0) CO in h S3 0) u 3 C O ^ 03 ;^ ^1^ o c« E ^ u C w 1— 1 CG w Z D O o o o u < H O H a- u o .2 IT' ^ a) . C K J' "^ CS fct . OKI". i. -M ^- O tK CS 1 t o ^-^ s 03 » -M fct: cs ce eS ^ -p o CO S '^ -^ Cfi O 1^ S S5 o (D a; 3 CO 03 (D to cc eS rt -»^ eS ^ 0; cc a! tr. •^ t- ^ C o a< o .!^ Q Ht 1—1 z o z o u o: o a < CO i^ 3 u <1 ~-' m m 03 . — . C ^- -- ^ o .;: ^O Is ri r-i 9 00 «3 o »-• ^ " o -2 S c^ ^ ^ ^ be ;3 J , ^ o ^ o o ^-< 1^ C3 r— o «4-l be c .X Zl !R c^ c — f^ ^ r- Q o ;- fM H H i-> ?^ 60 -'i:-'^ 4 •• .^ '^ H^ ^^'i \ ; >* > I \^- 'L >v«^- V r«K :»r ^ ^-/--V^-i ^^•' \ .^ v>-'- '^ ^.^ • i^ V 4 . ^'^^ *» • r< Schizonts. •^rv-V-^ JV? *»:, »-'«^^« ...-^ ^ f if*. *^-^*'"">« ■x>... .*&.. ♦'*&•''■ :^ 4^%. .^■-.'^" :J<^, ;i. vl^'? ■'^: 4.^*^ ^- Schizonts. { 1%* •n;»* Gametes -^^.i ' ^ ':^j» r^**-^ m *#%-; '^^^ .* ^'i »■*.' •i'^f'^^' Quartan Malaria Plasmodia. MALARIA 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 j)i'esent 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, wiiether 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. Descriiotion 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 A^'I, Plasmodium falciparum) ; (2) tertian (Plate VII, Plas- modium vivaoc) and (3) quartan (Plate VIII, Plasmo- dium malarice) . They all grow and reproduce in the blood of man in the same general way. The smallest Plasmodium consists of a tliin round wall of basophilic protoplasm enclosing a round or oval mass that does not stain. This gives the appearance of a ring. Tliere 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 beeonie more numerous until the develop- ment of the parasite is complete. An hidividual Plasmo- dium reaches maturity in about forty-eight hours in the case of estivo-autumnal and tertian, and in seventy-two liours in the case of the quartan j^arasite. The chromatin granule grows in mass for about half the developmental l^eriod, 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 tlie end of the period division of the cytoplasm of the organism occiu's, 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 i^lasmodium is called a "rosette." Finally the capsule of the rosette ruptures and each segment or young Plasmodium, known as a vierozoite, 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 j)lace without sexual influence and is known as schizogoiu /. A-sexual plasmodia (of any age) are schizonts. After a person has been infected with malaria plasmodia more tlian two or three wxeks, and as a result of in- fluences not now well understood, certain plasmodia, in- stead of develojoing through the a-sexual cycle of schizo- gony, become sexually differentiated and take somewhat different form. These are supposed not to be caj^able of reproduction in man. The males are capable of giving rise (in the mosquito) to microg'ametes, and tlie 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 extremelj^ rarely) and therefore the onlv 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 rea23pear 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 plasviodia. — 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 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Schizonts in the ripheral blood. Shape of gametes. EsTivo- Autumnal pe-' Ring-s only. Shape of outline of schizonts in periph- eral blood. Influence upon color of erythrocyte. Influence upon size of erythrocyte. Pigment in schizonts and gametocytes. Schuffner's granules.* Number of segments in rosette. Period of a-sexual de- velopment. Tertian Crescentic and oval. Rings. May have two chromatin granules. Not changed. Not changed. Mediumtocoarse. (Not present.) (24 to 32.) (48 hours. ) All sizes. Round or oval. Irregular after ring stage passed. Faded, pale. Enlarged. Very fine. Often present. 32. 48 hours. Quartan All sizes. Round or oval. Regular after ring shape is passed. Darker. Normal red color inten- sified. Reduced. Coarse. Not present. 8. 72 hours. * Red staining granules in the erythrocyte containing the parasite. Intcri)retatio7i of eooamination of the blood for malaria. — There are always sufficient plasmodia in the j)eripheral 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 l)lood and to determine that the patient has not some plasmodia in his body. If he had enough to make him sick, however, thev could be found readily. It should ])e aj)preciated tliat the number of plasmodia present in the ])l()o(l 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, witliin a week or two. Place on the blood one drop of water. Carefully spread this over the film of blood 0.5 66 PRACTICAL CLINICAL LABORATORY DIAGNOSIS (Fig. 53) witli tlie 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. o3. — Spreading the drop of Avater 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 1 Fig. ."Jt. — Tilting slide baek and forth to faeilitate mixing and to hasten agglutination. forth. This also liastens tlie reaction. If the blood con- tains tlie specific agglutinin for typhoid bacilli, the milhons of bacilh present soon begin to collect together, first in small TYPHOID AGGLUTIXATIOX 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 Fi^. 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 tlie course of the disease. A few do not give positive reactions at any time. Usually 68 PRACTICAL CLIXICAL LAliORATOKY DIAGXOSIS 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. — Ix)oking at ty]ihoi(l agglutination test. The granules can be seen best when tliere 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 fe^v 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 2:)aticnt lias not tyi)hoid. Its nega- tive value is in proj^ortion 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 spccitncfis for examination. — The only proper sj^eciiiien 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 S2)ecimens 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 houi's 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 microsco^Dic 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 ])efore 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 • y< 1 ^H h I ^M ' 1 ^ ir 1 ( fl J 'LATE IX ( HEMICAL TP:STS OF URINE Tube 1. 'i'est for albuiiiiii. No cloud after hdiliii;^^ and addition of acetit aci'l. 'lube 2. 'Jest for all)iiiiiin. Cloud })roduced by boiling and remaining after addition of acetic acid, thus showing jircsence of albumin. Tube 3. Test for indican. ^Negative reaction. C'lilorofoim not colored. Tube 4. Test for indican. Positive reaction. ( hloroform colored blue. Tube o. Jest for sugar with l<\'hling's solution. Negative reaction. No change in color. Tube (i. Test for sugar with J'ehling's solution. I'ositive 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 S. "^1 est, for JlfffmiP Posi + ivp rpupfinn ATn «liaanr»pn7'!iTir»p» nf rt^il URINE 71 Physical Examination Sjjecific gfrwiti/. — A urinonieter (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. r 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 jDaper changes to blue. Test for alhumm. — (A) Qualita- tive. Fill a test-tube three-fourths full of urine. Hold bv 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), Fig. 58. — L^rinometer. Testing specific grav- ity. Note curve of meniscus. Fig. 50. — Roiling urine in test for albumin. 72 PRACTICAL CLINICAL LAIJOKATOKY DIAGNOSIS varying in intensity with the anionnt present, will appear. Phosphates also prodnce 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. fiO. — Adding acetic acid to urine from a drop bottle in testing for albinnin. 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 tliat is absolutelv accurate. The one here « given is accurate enougli for practical purposes. Fill a test-tube with urine to a depth of 2^/) inches (Fig. 65). Add a])out one-fourth as much of a 10% solution of potas- sium ferrocvanide and about 1 c.c. of eclacial acetic acid. URINE 73 Fig. (il. — Looking for faint cloud in test for albumin. Dark background with light falling on the tube from the side. Fig. V-2. — Cloud due to albumin in urine as seen in the proper light against a dark background. 74, PRACTICAL CIJXICAL I.AHOKATOKY 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. Eiach one-quarter inch of sediment represents 10% of moist albumhi. Smaller quantities may be a2)proximated. The formula for the po- tassium ferrocvanide solution is: I^ Potassium ferrocvanide 10 gms. Water, to make 100 c.c. ]Mix. Fig. 63, — Filling tube with urine to a depth of QV2 inches in quantitative test for albumin. The tube is held l)v the side of an ordinary inch rule. Test for sncjar. — (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 u])on 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: URIXE 75 Sodium-potassium tartrate 173 grams Sodium hydroxide 125 fframs » c5 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 se23arate 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- Fig. 61. — 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-l)ottlcs 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, liold the finger as a marker (Fig. 04) at the ])lace wliere the solution will rise to when approximately an equal volume of the other solu- 76 PRACTICAL CLINICAL LABORATORY ])IAGXOSIS Fig. 65. — Boiling Fehling's solution and urine in testing for sugar. tion is added. Add the other sohition, shake, and we now liave about 1 c.c. of a mixture of approximately equal parts of the two dif- ferent solutions, wdiich con- stitutes Fehling's test solu- tion. Heat the solution to boiling over a B u n s e n 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 023aque yellow color (Plate IX) and shortly after a dense vellowish-red sediment falls to the bottom. Shake the tube while heating to prevent boihng or "popping" out. Xo 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 tlu'ough 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 ])ipette 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 wiie^'e all tlie 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 disa])})ears. Now read off on the pipette the amount of ui-ine used and calcuhite tlie per cent, of sugar indicated by the test. One c.c. Fehling's solution requires 0.005 gi'am 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 -^ 0.08 = 6l4> the per cent, of sugar indicated. If a large amount of glucose is present it is better to dilute the urine, say ten Fig. 6hth of laboratory awkwardness to allow m'ine 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. Xo cover-glass is necessarv. Method of Lwaminat'ion, — Examine wdth the low powxr (IG mm.) lens. It will occasionally be necessary to swing m place the high dry lens (4 mm.) to examine under higher magnification some particular object about wdiich doubt exists. Here as ^vell as in other places it is important to have a high j^ower lens with a long w^orking distance, such as the B. and L., 4 mm. of N. A., 0.65 (and not one of 0.8.5) 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 w^ay. Do not try to do routine laboratory work under such a handicap. Place one corner of the preparation in j)osition for ex- amination and by means of the mechanical stage move the slide from end to end, dropping dow^n the width of a field each time until the entire specimen is examined. This re- (juires 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). Diacjiioms of microscopic objects commonhf found in vrinC. — Ked ])lood cells (Plate X, b) are usually more or less alter(fd bv the urine. Thev may be swollen or more or less faded, or they may be crenated. They appear green- ish instead of red. Thev mav be differentiated from other objects when in doubt by mixing a little acetic acid with PLATE X MI(TU)S( OinCAL FTXDTXr.S TX TTRTXE a. Low powor fi"'l(l of iirino scdiniont. containing; a few epithelial cells, us cells, red blood cells and tlirce hyaline casts. b. Hifrhor niagnifif-ntion. Fifhl contains ])us cells, red blood cells and few bacilli. c. Hyaline casts. Xote irregularity in size and shape of different casts, wo epithelial cells are shown. URINE 83 the material on the slide. Red blood eells are dissolved, wliile other cells can be seen better. Pus cells (Plate X, b) are to be recognized by their granular protoplasm and their nuclei which can be made out upon proper adjustment of the light. When in doubt add a drop of acetic acid to bring out the nuclei better or the urine may be drained off after the cells settle on the slide and the specimen stained. (Technic, page 140.) Epithelial cells (Plate X, a) vary much in their size, shape and appearance according to what part or parts they come from. They are to be recognized by their distinct relatively small single nuclei. Casts (Plate X, a, c, d, e) are of several varieties. They are casts of uriniferous tubules and therefore vary in size and shape according to the size and shaj)e of the differ- ent tubules in which they are formed. As the casts are forced out of the tubules thev are broken more or less so that some are short pieces w^iile others are very long and many others are of intermediate lengths. Some are two or three times as thick as others. The basis of all casts is a hvaline material. Many of them consist of this material onlv and are called hvaline casts. Sometimes the disease involving the epithelial cells lin- ing the tubule where a cast forms results in the loss of the cement substance holding the cells in place and some of them adhere to the cast when it is forced out. These cells are usually swollen and colored more or less yellowish or brownish. Such casts are called epitlielial casts. An epi- tlieh'al cast is a hyaline cast onto whicli epithelial cells are attached. If such damaged cells are not carried out with casts they may continue to degenerate and break up into coarse granules. These adhere to casts that form in the tubules and such casts are now called coarse granular casts. The granules vary from a light yellow to dark brown in color. A coarse granular cast is a hyaline cast with coarse granules adhering to or imbedded in it. 84 PRACTICAL CLINIC^iL LABORATOllY DIAGNOSIS Unless the granules are carried out while they are coarse they continue to hreak up into finer granules and casts forming in a tuhule where such a process is going on would have some fine granules on them. In fact, granules are actually imbedded in the casts. Such casts are called fine granular casts. A fine granular cast, therefore, is a livaline cast with fine c^ranules on or in it. Sometimes the degeneration of the lining cells of tubules which generally gives rise to epithelial casts, coarse granular and fine granular casts is a fatty degeneration instead of granidar degeneration and casts formed in such tubules would then liave fat droplets on or in them. Such casts are called fattv casts. A fatty cast is a hyaline cast with fat droplets on or in it. It sometimes occurs that red blood cells pass into tubules where casts are forming and adhere to them. Such casts are called red blood cell casts. Likewise occasionally pus cells may be attached to casts where they form. Such casts are called pus cell casts. There are often various combinations of the above differ- ent varieties of casts. For instance, a single cast may have epithelial cells, coarse granules, fine granules, and fat drop- lets on or in it. It often occurs that one end of a cast may be hyaline while the other may be loaded with granules. Rarely casts consist of a highly refractile amyloid ma- terial instead of the usual hyaline material. Such casts are called amyloid casts. In addition to the above true casts there are present in most fresh specimens of urine a variable number of shreds or cylindroids (Plate X, f ) of a hyaline material very much resembling hyaline casts. They are irregular in shape and size and one end is usually very thin. They are much narrower in places than in others and do not give one the impression that they are true casts of the uriniferous tubules. The dividing line between hyaline casts and some of these cylindroids is diihcult to draw. In fact one must often URINE 85 feel in doubt about some of them. When in doubt give the patient the benefit of the doubt. Interpretation of urine examinations. — Specific gravity is high (above 1025) in most cases of glycosuria and very low in various nervous conditions. A low specific gravity (1000 to 1010) with inability to concentrate occurs in in- terstitial nephritis. Normal values or higher occur in paren- chymatous nephritis. The S23ecific gravity is susceptible of great variation in health according to the amount of water taken in and the amount of solids excreted. The reaction of normal urine is acid. Alkaline urine results from an almost exclusive carbohydrate diet. De- composition of urine, either in the bladder or after it is voided, usually changes the reaction to alkaline. Albumin in urine is derived from the blood serum and when present indicates that on account of some influence serum is passing through the kidneys. High blood pres- sure often causes a small amount of albumin in the urine. The chief cause of albuminuria is disease involving the kidneys. Nephritis is accompanied by albuminuria, the amount of albumin varying greatly with the different forms of nephritis and in different individuals. Albumin in the urine is also produced by inflammatory disease accompanied by suppuration involving the mucous membrane of any part of the urinary tract. The albumin is derived from the in- flammatory exudate and naturally varies in amount greatly. Sugar (glucose) in the urine results from diseases and conditions involving the metabolism of carbohydrates. Ex- cessive eating of sugar may produce it temporarily in in- dividuals who are apparently healthy. Diseases and injuries involving the floor of the fourtli ventricle are usually ac- companied by glycosuria. The cliief cause of glycosuria is diabetes mellitus, a disease of the pancreas involving the islands of Langhans. An excess of indican in the urine indicates absorption of products of ])utrefaction of animal proteids in the ali- mentary tract. Intestinal obstruction or stasis is an im- « portant cause. 86 PRACTICAL CLINICAL LAliOKATOKY DIAGNOSIS Acetonuria indicates "acid-intoxication," wliatever that is. Red blood cells in the urine sliow that hemorrhage is going on somewhere along the in-inary tract. The presence or absence of ervthrocvtes in the m'ine is very valuable in differentiating ''renal colic" from other pain due to disease in other organs in the same region. Pus cells in urine indicate suppuration somewhere along the genito-in-inary tract. There are a few or many pus cells from the vagina and vulva in the urine of most w^omen. Epitlielial cells are present in all specimens of urine. ]Many more are present in urine from females. A few hyaline casts are present in the urine of most people past the age of fifty, especially if the blood j^i'essure is high. A few may also be found in the urine of persons who have not any recognizable disease. Hyaline casts there- fore do not indicate necessarily any disease of the kidney. On the other hand, they are j^resent and usually in large numbers in nej^hritis of all types. Epithehal and coarse granular casts indicate acute nephritis. Fine granular casts are present in all forms of parenchymatous nephritis, but generally sjjeaking they in- dicate somewhat less acute disease than the coarse m-anular and ej^ithelial cell casts do. Pus cell casts indicate production of pus in the tubules. Red blood cell casts indicate that blood cells are leakino* through the walls of the tubules at the time and place casts are being formed. Cylindroids have no pathological significance that we know of. PLATE XI TEST OF GASTRIC JUICE FOR FREE PICl AND TOTAL ACIDITY Tube 9. Clear gastric juice. Tube 10. Change of color, upon addition of dimethylaminoazobenzol, indicating free HCl. lube 11. HCl has been neutralized by the addition of decinormal sodium hydroxide solution. There is usually a yellowish tint, due to organic acids. ''"iibe 12. NfMitralization of all acids (total acidity). Color produced by phcnolplithalt'in iridifntor. CHAPTER VI GASTRIC CONTEXTS Ohi (lining material to he tested. — After the patient has fasted twelve hours (over night) or longer, give a test meal, consisting of two or three crackers or a slice of bread and one or two glasses of water. The food should be thoroughly masticated. After three-fourths to one hour remove the stomach contents with the stomach-tube. A tube with a bulb to make suction with is quite an advantage. Test for free HCl and total aciditij. — Obtain some clear gastric juice by centrifuging a tube of the gastric contents just removed. The heavy particles go to the bottom. The froth, etc., rises to the top. Remove one c.c. of the clear fluid with a graduated pipette (1 c.c.) and place in an- other test-tube. Add one drop of 0.2% solution of dimeth- vlaminoazobenzol in alcohol. Formula: « Dimethylaminoazobenzol 0.2 gm. Alcohol (95% ) 100 c.c. Mix. This solution keeps well. If free HCl is present a ])riglit cherry red color (Plate XI, 10) will appear at once. Now add one drop of 0.2% solution of phenolphthalein in alcohol. Formula: Phenolphthalein 0.2 gm. Alcohol (95% ) 100 c.c. Mix. Tliis solution keeps well. Xo considerable cliange occurs. It is now ready for titration witli decinormal alkali solution, the dimethvlami- noazobenzol serving as an indicator for tlie free HCl and the phenolphtludcin for the total acidity. 87 88 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Xow take up in a 1 c.c. pipette graduated in hundredths 1 c.c. of decinornial sodium liv(h*oxide sohition. Formula: Sodium hydroxide 0.4 gm. Water 100 c.c. Mix. Add this slowly to the fluid, shaking as each 0.1 c.c. or less is added. Xote the amount of alkali required to just cause the disappearance of the cherry red color (Plate XI, 11). This indicates the total free HCl present. Con- tinue to add the alkali solution until a permanent rose color (Plate XI, 12) appears. This indicates the total acidity and the total amount of alkali used should be noted. The degree of acidity is expressed in terms of the number of c.c. of the decinornial solution, that would be required to neutralize 100 c.c. of gastric fluid. Each one hundredth c.c. in our test represents one degree. For instance, if 0.35 c.c. of the decinornial solution is required in the first titration the total free HCl would be 35, and if 0.65 c.c. (in all) is required before the second reaction is obtained, the total acidity would be 65. Other eocaminaiions. — Test gastric contents for occult blood in the same w^ay as feces are tested for it, see page 107. Blood and pus cells can be recognized by microscopic examination. Look first at a specimen of the material witli the high dry objective. If in doubt as to the diagnosis of pus cells, make a spread on a slide, and stain (technic, page 112) and examine with the oil immersion lens. Interpretation of the jindings in gastric contents. — The free HCl in gastric contents of normal individuals is usu- ally between 25 and 50, by this test. Above 50 indicates hyperclilorliydria and occurs in neuroses, most cases of gastric ulcer and beginning clironic gastritis. Values below 25, hypoclilorhydria, are usually found in early carcinoma and in most conditions associated with general systemic depression, including some neuroses. Absence of free HCl, GASTRIC CONTENTS 89 achlorhydria, occurs in most cases of advanced gastric cancer and far advanced chronic gastritis. Total acidity is due to HCl, free or combined, to acid salts and in pathological conditions to organic acids. Nor- mally it ranges from 40 to 70. Variations above or below these figures have the same indications as variations in the free HCl, provided there is a corresponding increase or decrease of the free HCl present. Whenever the free HCl is very low or absent and the total acidity is high, it indi- cates fermentation with the production of organic acid, such as occurs in advanced carcinoma and far advanced chronic gastritis. CHAPTER VII FECES EiVaJtrinatioji for intestinal jxirasite ova and larvce. — All the intestinal worms either lay eggs or deposit larvae in the intestinal eanal. These mav be fonnd hi the feces, ^lost of the worms prodnce very large nnmbers of ova or larviu. They are characteristic of the particular kind of worm that produces them. Wliether a given individual is host for intestinal worms can therefore be determined bv microscopic examination of the feces for ova and larvae. The kind of worms present can also be determined by the same means. Collection of specimens. — All of the intestinal worms commonlv found in this country inhabit the small intestine and cecum, except oxyuris, which inhabits the large intes- tine. The ova and larvae being deposited high up in the intestine are thoroughly distributed in the feces. Oxyuris ova are sometimes more numerous on the surface of formed stools. Feces of normal consistency are most suitable for examination. Liquid feces generally contain fewer ova, but they usually contain a sufficient number for diagnostic purposes. One or two drams of feces is a sufficient quantity. Larger quantities are objectionable and sometunes un- j)leasant and embarrassing. Unless patients are instructed as to the quantity desired and how to collect it they some- times bring to the laboratory very large quantities and in the most inappropriate containers. A fruit jar full of li(iuid feces is not at all uncommon; and an unwieldy pack- age it is. Or a })ottle is completely filled with fermenting feces and tightly sto])])ere(l. The shaking dui'ing transpor- tation hastens gas formation, and whenever the stopper is loosened it often shoots out, scattering feces about the labo- ratory and sometimes on the operator. A wide mouth two 90 lECES 91 ounce bottle (Fig. 73) with a new cork stopper is a very appropriate container. The patient may l)e instructed to obtain such a bottle from the drug store, or it should be furnished him at the time the specimen is requested. At the time the con- tainer is supplied and the speci- men requested it is well to tact- fully tell the patient that only a small quantity — "about so much" — is desired, and not to fill the bottle full. Specimens should be submitted for examination within twenty-four hours of the time they were passed whenever practical, to Fig. 73.-Proper bottle contain facilitate the recognition and dif- ferentiation of strongyloides larvae, but much older specimens are satisfactory to examine for ova. Making the preparation for eocamination. — Place two drops (not more) of water in the middle of a slide. Take ing sufficient formed feces for examination. Fig. 7t. — Stirring feces in a couple of drojxs of water on a slide to make l)roi»erly (lihiicd preparation. u]) a (juantity of feces about tlie size of a match liead on the end of an ordinary wooden tootlipick. Stir the feces (Fig. 74) in the water on the shde until sufficient has been 92 PRACTICAL CLIXIC^VL LABOKATOKY DIAGNOSIS dissolved oft' to produce the proper concentration of feces for examination. The material slionld be properly spread out on the slide at the same time. Do not let it come to tlie edge or end of the slide (Fig. 72). The solution of feces should be about as tliick as will just barely permit ordinary print to be read tlirough it (Fig. 75). It is not neces- .. of^\\a<.hinKloii 3Mcr«onnI -,n.I 'I...,, m |..-.r:i,i,..l..gy. I,kI» i.|,...l pait-r.. uill „.,t ..„..,IK t '•III I '.T,- ,.•■ ,n. ,.,...„. .1. ..- . . ' . •'- • f'^: ;> l.ic.,i;v'.i. . ""• ■' l' "' i >." f r.M > } n.i., i . niif n , . i iiv,!. ' , .' .,..^ j„ , xi.crK-n^, ■J, D 4iir ~-> I'LA'IK XI i IXl'ESTTXAL PARASITE OVA AXD LARV.^ A. Unoinarifi ova' in difToront stapos of developnipnt up to larvae. ]i. Uncinaria larva. Xote lonjif l)uecal capsule. ('. Stroiij^vloidcs larva. Xotv sliort fniccal C'aj)snlc. I). Oxyuris ova in (lillVrcnt stages "f (Icvclopinnit u]) in larval stage. r)ne is seen on end. E. Oxyuris larva. Xote long pin-like tail. F. Tenia saginata "ova." 'I liose on tlie riglit have a tliin, delicate cover- inor vvliieh is frcniicTif Iv ween in fresh oreoarations. FECES 97 moved. Whenever the end of the preparation is readied drop doAvn the width of the field and move in the opposite direction. Upon reaching the end, drop down the width of the field as before, and so continue until the entire prepa- ration has been examined. In this way the entire S2)read of material may be looked over in a few minutes. After one has acquired exjDerience the slide may be moved quite rapidly, only hesitating or stopping now and then to focus on and examine more closely some suggestive object. Once in a while it may be necessary to turn in j)lace the high /-\ / / « ^'I'C-A / V X A 1 • , - v ^ ■ 1 • • ^.l -V ■■- ";-. • -'".■' \ . ^ ( «iii Fig. 83. — P'noiomicrographs of diiferent ova all taken with the same magnifica- tion for comparison, a. Uncinaria americana. b. Oxyuris vermiciilaris. c. Trichuria trichuris. d. Tenia saginata. e. Ascaris lumbricoides. power objective (4 mm.) to study more closely, under higher magnification, some doubtful object, but this lens should not be used for routine examination. The field covered by this lens is so much smaller that it would take entirely too much time to look over a given preparation. Uncinaria avieiicana ova, — Uncinaria ova (Plate XII, a) are oblong, round-ended, and measure about 38 microns in diameter and 60 microns m length (Fig. 83). They consist of three distinct parts — the shell, the nucleus or yolk, and the clear space filled with albumin between the yolk and the shell. An object should not be diagnosed to be an ovum unless these three separate parts can be made out. The shell appears as a distinct narrow line. It is regu- lar in outline. The nucleus is made up of granular material. The granules are proba])ly colorless, but the mass appears 98 PRACTICAL CLINICAL LABORATORY DIAGNOSIS gray or dark gray, according to the light used. The nucleus usually shows a certain amount of development varying from division into two segments up to embryo formation. JNIost ova, however, in fresh specimens are in the two to eight segment stage. The nucleolus, a clearer spot, can usually be recognized in the segments. The space between the shell and tlie nucleus is of diagnostic importance. The nucleus is irregular in outline, according to the stage of division, but the shell remains regular. This is in con- trast to certain vegetable cells and other objects which have an outer membrane and a nucleus, suggesting ova. In these objects the outer membrane dips in wherever there are indentations in the nucleus, and this gives the object an irregular outline. They also vary in size considerably, wliile the size of ova of any given kind of worm is fairly uniform. Ascaris lumbricoides ova, — The ova of the ascarides (Plate XII, J) that infect man vary considerably in size and shape. They measure about 64 by 52 microns. The outer membrane proper is covered with a thick layer of gelatinous material, which is very irregular in outline and gives the ovum a very rough nodular appearance. The color of this varies from a very light to a very dark brown. Ova are sometimes found from which this outer coat has been partially or completely torn . The nucleus cannot be seen as well as the nucleus of the clear ova, largely because of the thick colored outer coating. There can, however, always be made out a clear space be- tween the shell projier and the nucleus upon proper adjust- ment of the light. Trichuiia trichuris ova. — These ova (Plate XII, I) are very characteristic in appearance and should never be mis- taken. They are oblong, measure about 52 by 25 microns and vary in color from liglit brown to veiy dark brown. There is a knob of lighter-colored material on either end. The nucleus and clear space can be made out in most specimens, but the thick, dark-colored shell prevents their 'f ^f FECES 99 being seen as satisfactorily as in the case of some of the more transparent ova — uncinaria, for instance. Ocvyims vermiciilafis ova. — These ova (Plate XII, D) are flattened on one side and oval on the other. They meas- ure about 50 by 30 microns. The shell is thicker than that of uncinaria ova, and is shiny in appearance. The nucleus and clear albumin space are plainly seen. Empty egg-shells are often found. They contain a smaller mass of bacteria or debris in place of the nucleus. Larvse (Plate XII, E) are sometimes found, and are to be recognized by the long, sharp, pinlike tail. The eggs of oxyuris are deposited in the rectum and in the folds around the anus and vagina. Therefore, much larger numbers of eggs can be found in material properly collected from these parts than in the feces. A good way to collect material for this purpose is to scrape the surface of the parts with some suitable instrument, like, for in- stance, the end of a microscope slide. Dilute the material collected with a drop of water on a slide and examine. Both ova and larvse are usually found. Hymenolejns nana ova. — These ova (Plate XII, H) measure about 48 by 36 microns. They have an outer and inner membrane, each of which is quite thin and appears as a distinct line. The space between the two membranes is clear, except for a few stride, which run irregularly longi- tudinally. The nucleus is light gray and contains 4 to 6 very plain booklets. The space between the inner mem- brane and the nucleus is not very large and looks clear. Tenia saginata and tenia solium, ova. — These ova (Plate XII, F-G) are oval, brownish or grayish colored, and measiu'e about 36 by 30 microns. There is an inner and an outer membrane, and the space between contains a great many radial striations. On account of the color of the covering the nucleus is not so well seen. Hooklet-like objects are present in both. The ova of both s})ecies look almost alike. These ova are often seen covered by a vitel- line membrane (Fig. 84). 100 PRACTICAL CLIXICAL LABORATORY DIAGNOSIS JLarvce of nncinaria and strongyloides. — After feces containine^ nncinaria ova have been ont of the body for twenty-four hours or more some of the ova are hkely to Fig. 84. — Photomicrograph of ovum of tenia saginata enclosed within its vitelline membrane. This membrane is usually soon lost after the ova are expelled into tlie feces, and most specimens do not show it. hatch (Fig. 85) if the conditions are favorable. The larvae (Plate XII, B) are about 0.21 mm. long by 0.02 mm. thick. They wiggle about for a w^iile, but usually die in undiluted feces in a few hours. In this stag-e thev are known as rhabditiform. Thev look very much like the rhabditiform larv« of strongyloides. The ova of strongyloides are deposited by the adult worm in the glands of Lieberkuhn, and there hatch, giving rise to rhabditiform larva (Plate XII, C), which are passed in the feces. They wiggle about actively, but often many of them die after a few hours. They are about the same size as nncinaria larvae, and look almost exactly like them. The most practical differential points between nncinaria larviu and strongyloides larvje for one not especially familiar w^ith these parasites are: 1. Whenever nncinaria larvae are present there are also many uncinaria ova present. Strongyloides larvae are not accompanied by tlie presence of ova except in cases of double infection. FECES JO » 1 > " 4 v^V j' ■> Fig. 85. — Photomicrograph of hookworm ovum and larva and ovum of trichuria. 2. Uncinaria larvse are not present in feces under twen- ty-four hours after they are passed. Strongyloides larvae are present from the first. 3. The buccal capsule of strongjdoides larvae is very Fig. 86. — Photomicrogrnph of anterior end of hookworm larva under high niagJiification. Note long buccal capsule. 102 r-tACTICAL CLINICAL LAIJOKATOKY DIAGNOSIS short, wliile tliat of uncinai-iii larvii? (Fi<]f. 8()) is rela- tively long. EiVaminaiion for amchcc in amebic dyscntcrij. — A large part of tlie cases of dysentery occnrring in this country, and especially tliose occurring in tlie tropics, are due to pathogenic anieba\ There are many species of amebfe, only a few of wliicli live as parasites upon other animals. They are thci-efore known as endamebge. There is at present considerable difference of opinion among authorities on the subject, as to whether there are one or more species of endameba? found in the intestine of man. It is certain that a supposedly harmless ameb^ — ameba coli — is very fre- quently found in the discharges from man, and that this species is quite different from the pathogenic species which cause dysentery. In view of the present unsettled state of the classification and nomenclature relating to the path- ogenic amebge, we give methods of diagnosing and differ- entiating the pathogenic species from nonpathogenic spe- cies onlv, and do not enter further into the classification. Collection of specimens for eccamination, — It should be imderstood that the disease process of amebic dysentery consists chiefly of idceration of varying degree and varying extent in the colon, and that the endamebcT are present and reproducing in and on the ulcerating tissue. There is more or less pus and mucus being thrown off from the diseased tissue all tlie time, and this contains the endamebse which we desire to find. During a period of acute active disease there may be many discharges daily, consisting chiefly of tliis mucus and pus usually containing more or less blood. This is ideal material for examination. Cases of acute dysentery can have such actions by making the effort at most any time, and furnish the proper fresh specimen for examination. In the subacute or quiescent stage there may ])e only an occasional particle of the bloody or puru- lent mucus mixed with the fecal stool. The examination must be made within one or two hours of the time the material was discharged, as endameba3 rapidly die and FECES 103 their appearance changes upon exposure to an\ During cold weather the specimen must be examined even more promj^tly, unless it is kept warm artificially in some way. Keeping the bottle containing the specimen in warm water is a good way to keep it warm until the laboratory is reached. It is always better, whenever practical, to have the patient come to the laboratory and there pass the stool into a pan or other vessel for immediate examination, or bring the microscope to the j^atient. The next best thing is, whenever patients are not near the laboratory, to make several proper slides from selected particles of bloody or purulent mucus and allow them to dry. These may be satisfactorily examined for endamebas at any subsequent convenient time by staining them, provided one is at all familiar with stained endamebee. It should be remarked here that endameb^e may be demonstrated in purulent material from abscesses of the liver and other parts of the body. They are present in largest numbers, however, in the walls of such abscesses. Eocmnination of unstained material. — With an ordinary wood toothpick held in each hand pick out particles of the bloody purulent mucus, which j)i'obably came from ulcers, and place on a slide. Spread just sufficiently to make a preparation of proper thickness to examine, but not so thin that it will dry out rapidly. By prompt work a proper examination can be made before it dries out. It is better to spend a short time examining each of several specimens j^repared from different parts of the stool tlian a long time on one preparation. Those w^ho cannot work rapidly enough and those who prefer it, sliould use cover-ghisses. Put a cover-glass on tlie material on the slide and make sliglit pressure, if necessary, to tliln out the material, but do not make too thin. The liigh, dry objective is generally used to exanu'ne for endameba% but witli a little practice they can be seen well witli the low power lens (10 mm.), and the time required to look over a given area is mucli less. The light must be reduced until the hyaline objects 104 PRACTICAL CLINICAL LAIJORATORY DIAGNOSIS on the slide are clearly seen. Pus and blood cells are readily recof^nized. EndaniebcT (Plate XIII, a) appear to be larger cells containing coarse granules and blood cells in the endosarc, surrounded by a clear ectosarc. In actiye endaniebtT the ectosarc is constantly changing its shape. It may throw out a pseudopod into which the endosarc flows. Another pseudopod may be projected in another direction, and either be withdrawn or the ectosarc moyes into it. These moyements (Fig. 87) are slow and are known as ameboid motion. The endameba may change its position slowly, and finally move out of the field. Technic of staining amehce and examination of stained specimens. — IMany different methods of staining have their indiyidual adyantages for special purposes. The best, most practical method for ordinary diagnostic purposes is the carbol-fuchsin and methylene blue stain for pus and exu- dates, giyen on page 140. The ectosarc (outer portion) and endosarc (inner portion) are well differentiated (Plate XIII, b). The stain varies somewhat, but the ectosarc is usually more or less purple, while the endosarc stains varying shades of blue. The nucleus is not specially well stained by this method, but is well enough stained for present purposes. It stains red. The endosarc contains from one to several blood cells or fragments of blood cells, and usually a few bacteria. Occasionally a very small endameba may be found which contains no blood cells. Large endameba are two, three or four times the diameter of polymorphonuclear neutrophile pus cells, which can usually be found present in such preparations for comparison. The endameba found in dysentery average considerably larger than those found in alveolodental pyor- rhea (endameba^ buccalis) (Plate XIII, c). Differentiation of jjathogenic fi^oin iion-pathogenic ame- hce. — The non-patliogenic ameba? found in feces are on the average smaller than the pathogenic species, but otherwise resemble them very much in their movements and ofeneral appearance. The striking difference is that the pathogenic -,-'."'?*",' ^- } ^ sV"^ ^ :3i* B. %y- •»r- 1 IsW X^' v*^' rt 9 «> # !-• ' ; % ♦f • •- r." :9 c. A. Typical appearance of bloody mucus in amebic dysentery showing endamebae. High mamification. 8 10 11 12 Fig. -C'liange of slinjie and position of an rndanu'lia during intervals of a few seconds each bv ameboid motion. 105 lOf) PRACTICAL CLINICAL LABOKATOKY DIAGNOSIS species phaoocyte blood cells, while the others do not, except possibly occasionally. For practical purposes this is the best differential point, and the only one necessary. Ameba? containing blood cells may be considered path- ogenic endanieb^. Interpretation. — In active or acute amebiasis pathogenic endamebcT? can always be found upon proper examination. During a quiescent stage of the disease, when few ulcers remain and when they are almost healed, it may be impos- sible to find endameba? because of the very small number present, and the absence of the kind of material in which they may be found. It therefore may not be possible to determine by a single microscopic examination that a pa- tient has not quiescent amebic infection, but it is possible to determine that an acute case of dysentery is or is not due to endameb^. Test for oecult blood. — Hemorrhage mav occur so hio-h m the alimentary tract that the blood is so altered that it cannot be recognized in the feces either by macroscopic or microscopic examination. Such blood is called occult blood. Whenever practical, special precautions should be taken to avoid outside sources of blood that would give a positive reaction, such for instance as eating food con- taining blood, hemorrhages from the mouth and nose, or hemorrhoids, etc. A purgative followed by a meat-free diet a day or two before the specimen is collected insures against blood from food. Technic of test.—M3.^e a thick solution of the material in water, unless it is already sufficiently liquid. Ordinary foi-med feces would be diluted with about ^lxq times as mucli water. AVhenever specimens are contained in a proper bottle container, the best way to make the solution IS to add some water in the bottle, replace the stopper, and shake until enough of the feces has been dissolved to make the proper consistency. It is not necessary that all should ))e dissolved. Place in a test-tube a little benzidine (crys- tals). A dip on the point of a knife about half the size PLATE XIV TEST FOR OCCULT BLOOD Tube I. Xetrative reaction. Tube TL Positive reaction. FECES 107 of a i^ea is ample. Add a few (5 or 6) drops of glacial acetic acid. Shake until benzidine is dissolved. Pour in about one-half to one c.c. of the diluted feces. Shake to mix and then add a few drops (10 or 15) of fresh peroxide of hydrogen. It is better to allow this to trickle down the side of the tube and overlay the material being tested. If occult blood is 2^i*esent, a dark blue ring (Plate XIV) at the zone of contact forms within a minute or two. If none was present, no blue color appears. If the tube is shaken, the whole mixture turns blue, but the ring test is generally more striking. Interpretation. — A positive test show^s the presence of occult blood. Occult blood is present in most cases of cancer of the stomach and intestines all the time, and occasionally in cases of ulcer. Other possible sources of blood must be kept in mind in interpreting a positive test. A negative test showing the absence of occult blood is valuable evidence against the existence of cancer, which is the chief value of the test. It is little evidence against ulcer, but whenever tests made on several different davs are all negative, ulcer is usually not present. CHAPTER VIII PUS AND ]:Xin)ATES GENERALLY General remarks. — It is often desirable to know what kind of bacteria, cells, etc., are present in various kinds of material, such as pus, exudates, etc. It is not considered witliin tlie scope of this book to go into bacteriology except to describe tliose simple methods of investigation that can be carried out by those not equipped with a bacteriological laboratory, but having the simple comparatively inexpensive laboratory equipment suggested in this book. There are certain simple tests that may be made which enable one to determine in many cases what group of organisms bacteria found belong to. One of the distin- guishing features of germs is their shape (Fig. 88). We have ])acilli which are rods of various shapes two or more times as long as they are thick, some of which are motile, and cocci which are round or oval, the length being less til an twice the breadth. The cocci are further divided according to their arrangeipent with relation to each other into diplococci, arranged in pairs; staphylococci, arranged in bunches (graj^e bunch), and streptococci, arranged in chains. Still other classification according to arrangement, such as strepto-bacilli, strepto-diplococci, etc., is sometimes made. Classification according to morphology is based upon stained specimens. MaJxinr/ the preparation and staining. — When obtain- ing pus or exudates for examination with a view to discov- ering the causative germ, it is very important to obtain material from the seat of the disease process. For instance, it mav be that there are manv bacteria at the surface of a lesion which have nothing to do with the cause of it, but are 108 Fifr. 8S. — Drawing lllustratiiifi: inori^liolofiical classification of bacteria* a. Staphylococci. c. I)i])l()C()cci. e. Strcptohacilli. g. Spirillae or curved bacilli. b. Streptococci. d. Hacilli. f. Diphtheroid bacilli. h. Si)irochetae. 109 110 PRACTICAL CLINICAL LABORATORY DIAGNOSIS simply growing secondarily in the pus and exudate. These bacteria may be much more numerous in the material at the surface, while in the diseased tissue none are to be found. The material should be properly spread upon a slide (see Fig. 89) and allowed to dry. AVhenever it is Fig. 89. — Proper spread of pus or similar material to be stained and examined. There are thin and thick areas affording any thickness desired. desirable to examine a very fluid material for bacteria, it should be centrifugahzed to concentrate the bacteria and cells. The supernatant fluid is poured and drained off J Fig. 90. — Thoroughly draining sediment in bottom of centrifuge tube. Every droj) of fluid is poured off. thoroughly (Fig. 90) and some of the sediment spread upon a slide with a platinum loop. The best routine stain for such preparations is the carbol-fuchsin and methylene-blue stain (for technic, see page 137). To determine the presence or absence of acid- fast bacteria, use the carbol-fuchsin, acid and methylene- blue stain (for technic, see page 115). To determine the reaction of the ])acteria to Gram's stain, use Gram's method (for technic, see page 138). PUS AND EXUDATES GENERALLY 111 To determine whetlier bacteria are motile or non-motile, put a drop or less of the fluid material on a slide; or, if the material is too tliick, dilute some of it on a slide with normal salt solution. Xo cover-glass is required except in rare instances. Examine with the "hiffh dry" lens with reduced light. The beginner often mistakes Brownian motion for actual motility. In Brownian motion the objects aj^pear to be dancing and moving actively; but, barring the influ- ence of cin'rents in the fluid, they do not move from place to place. On the other hand, motile bacteria do move (swim about) from place to place. It is good practice to examine a few old specimens of urine for bacteria. Application in practice. — By making the simple tests of morphology, motility and staining reaction, one often gains valuable information as to what class of bacteria are present and, what may actually be of more value, what bacteria are not present. Below we give a short list of common bacteria, with their staining reaction, morphology and motility. CHAPTER IX SPUTUM ELXaiiunation for tubercle bacilli. — The practical labora- tory diagnosis of pulmonary tuberculosis rests upon the demonstration of acid-fast bacilli in the sputum. There are several other acid-fast bacilli that resemble tubercle bacilli morphologically and otherwise, and therefore the laboratory diagnosis is not absolute. It is true that the identity of the bacilli could be determined by sufficient laboratory investi- gation, but the time and facilities required prevent it from being j)ractical for ordinary purposes. Collecting specimens, — Many a failure to find tubercle bacilli in cases of tuberculosis has resulted from the exam- ination of improper specimens, such as saliva or secretion from the posterior nares containing no sputum from the lungs whatever. At the time specimens are requested for examination the patient should be informed that material that comes up from the lungs is wanted, and not saliva and nasal secretion. A mixture of several expectorations is desirable. Most patients raise more sputum in the early morning, after a period of sleep and rest, than at any other time of day. Such sputum usually contains more tubercle bacilli than that raised at other times of day. A clean, wide-mouth bottle, with new cork stopper, makes an appropriate container. Unless the container is furnished or specified, specimens are likely to be brought in the most inappropriate things, such as pasteboard boxes, small tin boxes, etc. Too small a quantity for the most thorough examination is likely to be brought, unless the patient is instructed. Owing to chemical and bacteriological changes that 112 Name 3U3 albus. :us aureus. ?us citreus- is pyogenes. IS luueosus. ;s pneumoniae ( pneumococcus ) . meningitidis ( meningococcus) gonorrhoeae ( gonocoecus ) . cntarrhalis. tetracrena. hracis (anthrax bacillus). tilis. itheriae ( Kleb's-Loffler-bacillus ) udo-diphtheria\ (colon bacillus). eritidis. hosus (typhoid bacillus). atyphosus (paratyphoid A or enteriae. i-osus eapsulatus. Lis (plague bacillus) ']iink cyt'" (Koch-Weeks mi (tetanus bacillus) tussis. bacillus. cvaneus. nceroideus (bacillus of erculosis (tul)ercle bacillus). !gma? (smegiua bacillus) . tter or gra^^s]. •ae (Hansen's bacillus). olerae (cholera vibrio) . !^^ORPIIOLOGY Small, oval or round organisms; usually in clusters. Small, oval or round organisms; usually in clusters. Small, oval or round organisms; usually in clusters. Small, oval or round organisms; usuallv found in chains. Large, oval organism, encapsulated; in chains. Slightly elongated cocci in pairs, surrounded by cap- sule. Often in chains. Bean-shaped cocci, in pairs. Bean-shaped cocci, in pairs. Bean-shaped cocci, in pairs. Bean-shaped cocci, usually occurring in fours. Large size. Large rods, single or in chains. Large rods, sinirle or in chains. Beaded or granular rods, or clubs. Beaded or granular rods, or clubs. Short rods. Short rods. Short rods. Short rods. Short rods- Short rods., capsule. Short, plump rods, with a clear center occasionally < Small, thin rods. Long, slender rods, with one end knobbed. Small, short oval rods. Large, oval rods. Small, slender rods — varying in size — often chains. Long, til ill rods. Slender, often slightly curved, rods. Slender, often slightly curved, rods. Slender, often slightly curved, rods. Slender, often slightly curved, rods. Short, slightly twisted rods, or comma-shaped. Staixinci Reaction- Where Orgaxlsms May Usually be DE^roxsTRATED Gram -f Pus from local infections. Noimal skin. Gram + Pus from local infections. Normal skin. Gram + Pus from local infections. Normal skin. Gram -j- Virulent local infections, showing spread via tlie lympliaties. Gram + Respiratory mucous membrane. Gram + Respiratory mucous membrane, and especially in sputum from lobj Gram — Spinal fluid in cerebro-spinal meningitis. Gram — Pus from gonorrhceal urethritis, vaginitis or ophthalmia. Gram — Respiratory or vaginal mucous membrane. Gram -f Respiratory or vaginal mucous membrane. Gram + Blood of anthrax (charbon) patients. Gram -f Frequent contamination of blood, media, urine, etc. Gram + Suitable cultures from diplitheritic lesions in respiratory tract. Gram -f Respiratory mucous membrane. Gram — Urine, feces, sputum, etc. Gram — Urine, feces, sputum, etc. Gram — Suitable cultures from blood, urine and feces of typhoid patients. i Gram — Suitable cultures from blood, urine and feces of patients with fever. Gram — Sviitable cultures from feces of patients with bacillary dysentery Gram + Respiratory mucous membrane. Gram — Enlarged lymph glands, or suitable cultures from blood in plagii Gram — Conjunctival pus in "pink-eye." Gram + The primary lesion of tetanus (lockjaw). 1 Gram — In sputum from ])atients with pertussis ( wlioojung-cougli ) , 1 Gram — Stomach contents in reduction or absence of HCl. 1 1 Gram — "Bkie-pus" infection of wounds. 1 1 Gram — Pus from non-syphilitic ulcerative conditions of genitalia (chancer Acid fast. Sputum, or pus, from lesions of tuberculosis. Acid fast. Material collected from under tlie male foreskin, or female genita Acid fast. Butter, greasy foods and fresh vegetables. Acid fast. Scraping from the nose, or nodules, or lesions of leprous patients. SPUTUM 113 take place in sputum uj^oii standing, the characteristic staining reactions by which tubercle bacilli are recognized are frequently altered. In some specimens the bacilli begin to lose their staining characteristics in twenty-four to forty- eight hours. Specimens of sputum, therefore, should not be more than twenty-foin- hours old — the fresher the a.' better. Maldng the i^rcparation. — Tubercle bacilli are not pres- ent in all parts of a specimen of sputum. It is therefore necessary to make the preparation for examination from the kind of material most likely to contain bacilli. INIanv a failure to find tubercle bacilli in specimens containing them has resulted from examination of preparations made of material fished out of a bottle, jar or other container with a platinimi loop or other improper instrument. Pour out a proper quantity of the sputum into a Petri dish (we do not know of anything else as good). It is an advantage to set this on the table and place something (the cover of the dish) under one edge so as to tilt it and allow the sputum to flow to one side, leaving the remaining portion of the bottom uncovered by r.putum. This furnishes a convenient surface onto which j^articles may be drawn and teased out in order to separate the desirable from the un- desirable material. Ordinary wood toothpicks are best for this purpose, as well as to spread the material upon the slide with. Use two picks (Fig. 91), one in each hand, and do not try to pick out material for examination with only one. Select the gray, purulent particles to be seen in the sputum. With the picks drag them onto the uncovered part of the bottom of tlie dish. Tease and separate from adher- ing mucus, saliva, froth, etc. Transfer to a slide. Get out several other particles from other parts of the sputum mass, mix and spread all out on tlie slide. Thus is made a prepa- ration representing favorable-looking material from several different parts of tlie sputum and much more likely to con- 114 PRACTICAL CIJXICAL LABORATORY DIAGNOSIS tain tubercle bacilli than one of similar size made from any one part of the sputum. The spread of material on tlie slide should be compara- tively large, but it shoidd never reach the ends or edges of the slide. Burn the contaminated ends of picks and discard. The preparation must now be allowed to dry. There is no harm in warming the bottom side of the slide to hasten drying. It is not necessary that the very thick masses Fig. 91. — Picking out favorable material from sputum in a Petri dish. Note use of two toothpicks. sometimes present on such preparations should be 2)erfectly dry before staining, as these are not examined. Staining. — Tubercle bacilli are surrounded by a fatty caj^sule or covering. This is what gives them their most characteristic staining property known as acid-fast. This fatty covering is not readily penetrated by ordinary stains, and it is also resistant to alcohol and mineral acids. Bv employing a mordant with the stain and by the aid of heat tlie ])acilli can be stained within their fatty covering. The mordant usuallv em])loved is carbolic acid. When once • J. • stained, tubercle bacilli are not decolorized by mineral acids like other bacilli that have no fatty covering, because the acid cannot penetrate the covering. Tliis distinguishes tubercle bacilli front most other bacilli and the different steps in the staining technic have this object in view. SPUTUM 115 The different steps are as follows: 1. Fix with heat. 2. Stain with carhol-fiichsin phis heat one minute. 3. Decolorize wuth mineral acid. Wash. 4. Coiinterstain with Loffler's methylene blue. Wash, dry, and examine with oil immersion lens. The fixation is accomplished by passing the slide slowly, film side up, through the flame of a Bunsen burner or Fig. 9:2. — Touching; the heated slide to the back of the hand to jndge the tempera- ture and avoid getting it hot enough to damage the fihn of material to be examined when fixing with heat. alcohol lamp two or three times. Do not get the slide hotter than it can be borne on the back of the hand (Fig. 92). Use a very small flame. Hold tlie slide in the hand when fixing it. There are several formula? for making the carbol-fuchsin stain. None are better tlian Czaplewsky's. It keeps in- definitely and some otliers do not. Wlien ordering, specify carbol-fuchsin, Czaplewsky's fonnula. The fornmla is: Basic fuchsin 1 gm. Carbolic acid, liquefied 5 c.c. Neutral glycerine 50 c.c. Water to make 100 c.c. 116 PRACTICAL CLINICAL LABORATORY DIAGNOSIS The inoTedients are added and mixed in the order given. The fuehsin can be purchased in original 10 graninie bottles and tlie stain can be made up nuicli more cheaply than the })rice at which tlie staining solution usually sells. How- ever, one ounce of the staining solution, if properly used, is sufficient to stain more than one hundred specimens, and it may be found more convenient to buy the prepared stain in manv instances. This, like the other stains, should be kept in a proper drop bottle for use. Hold the slide by the edges near the end, between the thumb and first finger of one hand, and put on the stain with the other hand. Allow one drop of Fig. 93. — Proper way to ap]>ly stain to a slide. Spread the stain over the material to be stained as it flows from the drop bottle. the stain to flow on the film and by means of the lip of the drop bottle spread (Fig. 93) it over the film, allowing more stain to flow out of the bottle as needed. A quan- tity equal to tw^o, or at the most three, drops is sufficient for any slide. The stain should not be spread to the edge of the slide. It is extremely awdvward to flood the slide with stain so that it runs off on the hands, table, etc. After the stain has been placed on the slide it should he heated by passing it slowly through the flame two or three times. It must not be boiled. Just let it get hot is sufficient. Forceps or special slide-holders are not necessary to hold the slide with while heating (Fig. 94). Anybody who can't heat such a slide properly while hold- ing it with his fingers without burning his fingers or spill- ing tlie stain about, needs laboratory instruction or practice SPUTUM 117 and not a pair of forceps. After heating, the slide may be placed on the slide-rest over the sink or waste jar for a minute, or it may be held in the hand. We use a two and one-half per cent, solution of sulphuric acid to decolorize with. Weaker solutions decolorize more slowly. Much stronger solutions are more likely to alter other bacteria and cells present in the preparation. The acid solution may be made by placing 2.5 c.c. of sulphuric acid in a measuring cylinder and adding sufficient water to make 100 c.c. Pour the stain off of the slide and drop on several drops Fig. 94. — Heating slide in staining witli carbol fnchsin. Slide holders are unnecessary. Use a small flame and small amount of stain. of the acid solution. Allow the first few drops to run off (Fig. 9.5), and wasli off the excess of stain that remained on the slide. Note that the red color of the film fades and in less than a minute l)ecomes a very light pink or purple and ceases to fade more. It is now decolorized. There is no necessity to continue tlie decolorization process, but no harm would result from the application of this strengtli acid solution for several minutes. By this decolorization the stain is taken out of all other bacteria, cells, etc., but if tubercle bacilH or other acid-fast bacteria are present they retain the stain. The slide is washed by running water over it. 118 PKACTICAL CLINICAJ. LAHOUATOKY DIACJXOSIS This completes tlie staining process so far as the tuber- cle bacilli are concerned, l)ut the red stained bacilli show up better against a contrast-stained ])ackground, and it often is desirable to have other bacteria and cells on the slide stained so thcv can be seen at the same time. Loffler's metliylene blue is an excellent counterstain for this purpose. It should be put on and spread over the fihn m the same Fig. 95. — Decolorizing with siilplniric acid solution. The slide is held so that the fluid flows over the i)rei)a ration and off at the end or corner. vvav as the car])ol-fuclisin. It usuallv stains suificientlv in • « » less than one-half minute. Now wash well. Wipe the l)ack, ends and sides of the slide dry. Take up any excess of water })y "blotting" with the towel (or paper) and dry by warming it over the flame. Don't get it too hot. No cover-glass is required. Place some immersion oil on tlie slide and examine with tlie oil immersion lens for the red-stained tubercle bacilli. ■r Xy i» — - . <• \ B. >\ h. jf^ !f«fe "* / J ^:^ y •■^ p^^' "A.- .'^ 48^W^- w^ r^v§v C. ^^m^ and B. Tubercle bacilli in sputum stained with carbol-fuchsin-acid- tie blue stain. Xote greater amount of beading in B than in A. Lepra bacilli in material scraped from leprous lesion. Carbol-fuchsin- ihylene blue stain. Note the large number of bacilli present and the m-iofi/-. loi-vT-i r-ollcr rill^rl ii7itVi •frx* r»o/-«ilii SPUTUM 119 Description of tubercle bacilli. — The bacilli (Plate XV, A-B) may be present in large numbers and in almost every field, or they may be so few that one or more are found only after several minutes' search. There often are clumps of several bacilli, but usually the number is small and many are found separate from others. It is common to see two or more bacilli lying parallel to or somewhat across each other. Two may be seen lying end to end. Most tubercle bacilli are slightly curved, and in many specimens they are slightly beaded in appearance. Interpretation. — It seldom occurs that any acid-fast bacilli are found in the sputum except tubercle bacilli. For all practical purposes acid-fast bacilli found in sputum, in the presence of clinical evidence of tuberculosis, are tubercle bacilli. A thorough examination of a proper specimen of sputum without finding any acid- fast bacilli is evidence that the patient has not tuberculosis advanced to the stage in which there is breaking down of lung tissue. It is not, however, proof of this, since it often occiu's that bacilli are found only after repeated examinations. It is not possible, therefore, to determine with certainty that a patient lias not tuberculosis by not finding tubercle bacilli, no matter how thorough the examination. CHAPTER X LEPKOSY Tcchnic of obtaining material and making 7?/T;;r/rrt//o??5 for CiVamination. — Lepra bacilli are present in the tissues of leprous lesions, and in the case of tubercular leprosy tliev are almost incredibly numerous. Since the bacilli are Fig. 9(). — Scraping leprous lesion. Bleeding is prevented by pressure of the fingers. cliiefly in the fixed tissue cells, it is necessary to scrapie up the tissue and examine the scrapings for the bacilli. Most lepra lesions are anesthetic, and no pain is produced. Catch up and squeeze (Fig. 9()) the suspected tissue between the thumb and finger. AVith a scalpel scrape down into the tissue and secure scraped-up tissue fairly free from blood. This is accomplished by keeping tlie area anemic l)y the ])ressure of the fingers. The edge of the lesion often con- tains the most bacilli. When tubercles are present, scrape them. Spread the scraped material on a slide and allow to dry 120 LEPROSY 121 Staining. — Lepra bacilli are acid fast and are to be stained exactly like tubercle bacilli (see page 114). Appearance of lepra bacilli. — Lepra bacilli (Plate XV, c) look like tubercle bacilli, to ordinary observation at least, but in most specimens the very much larger numbers are striking. Many of the bacilli are seen arranged in masses or in the so-called lepra cells. There is no staining or morphological difference between lepra bacilli and the tuber- cle bacilli that permits differentiating them. Interj)retation. — Large numbers of acid-fast bacilli, es- pecially if grouped like lepra bacilli, found in material scraped from tissues showing clinical evidence of leprosy, are for practical purposes lepra bacilli. The absence of acid-fast bacilli in scrapings of a tissue indicates very strongly, but does not prove infallibly, that the tissue is not leprous. Lepra bacilli are almost always found in the nodular type and ulcerative lesions. The anesthetic leprous ery- themas on the cutaneous surfaces are sometimes due to lo- calization of bacilli along the nerve trunks and the lesion itself may not show any bacilli. CHAPTER XI SPINAL FLUID Meningitis. — The different forms of cerebro-spinal men- ingitis can usually be easily diagnosed by proper microscopic examination of cerebro-spinal fluid obtained by lumbar puncture. Since we now have a valuable specific remedy I'^ig. 97. — Introduciiip: tlie needle in makiiiji- IvMuhar ]ninotiire with patient in sitting- ])osition. The skin has been sterilized at the site of puncture with tincture of iodine. for at least one form (meningococcic), exact diagnosis be- comes of great importance. Obtaining material and making j)rei)aration. — It is not considered within the scope of this book to describe the teclinic of lumbal' puncture (Fig. 97). We would state, however, that it is a very simple operation, and easy to perform. The patient shoidd be tiu'ued from side to side a few minutes before the puncture is made, in order to 122 SPINAL FLUID 123 stir up the cells in the canal, which tend to settle to the lower side when the patient lies in one position for some time. The quantity of fluid drawn for diagnostic purposes should be 10 c.c. It should be allowed to drop (Fig. 98; Fig. 98. — The spinal fluid is allowed to drop directly into the centrifuge tube. directly from the lumbar puncture needle into two clean test-tubes or small bottles. The first one is likely to con- tain some blood cells. Therefore, the second one should be used for examination, especially in making cell counts and globulin tests. The sooner tlie examination is made after the fluid is drawn tlie better, except in the case of tubercu- lar meningitis, in which it is better to wait two or three hours for tlie pellicle of fibrin to form. Sometimes the fluid is very cloudy and purulent, in which case smears slioidd l)e made at once whefl^it is drawn. When not quite cloudy it is necessp.ry to centrifugalize the 124 PRACTICAL CLINICAL LABORATORY DIAGNOSIS specimen, in order to concentrate the cells and bacteria. Centrifugalize long enough to thoroughly settle the sus- pended cells, etc., to the bottom. Pour off the supernatant clear liuid and drain well (Fig. 90). With platinum loop make proper spreads of the sediment on slides. A spread about one-quarter of an inch wide and one inch or more long is satisfactory. Allow these to dry in the air and then stain. Usually a 2)ellicle of fibrin forms in fluid from tu- ])crcular meningitis upon standing for an hour or two. As this contracts it gathers tubercle bacilli in its meshes, if any arc present. This should be "fished" out and spread upon a slide or slides for examination. If no pellicle forms a cen- trifugalized specimen should be examined. A high speed centrifuge will throw down tubercle bacilli fairly well. The best stain for all kinds of pus is the carbol-fuchsin and methylene-blue stain (see page 137 for the technic). It is usually desirable to make a Gram's stain also (see page 138 for the technic). Whenever tubercle bacilli are to be looked for, the carbol-fuchsin, acid and methylene- l)lue stain is to be employed (see page 114 for the technic). Meningococci. — Meningococci (Plate XVI) are intra- cellular (and extracellular) coffee-bean-shaped diplococci. In some specimens they are quite numerous, but in many others they are not. Frequently a search of many minutes is required to find a few pairs of cocci. It is a good idea not to diagnose as meningococci any organis^m that is not intracellular. The meningococcus is Gram negative. Pncumococci. — In pneumococcal meningitis the pneu- mococci (Plate XVI) are usually very numerous. They are chieflv extracellular, but many are intracellular. The lanceolate-shaped diplococcus, which is Gram positive, is (juite characteristic. Tubercle haciJli. — Tubercle bacilli from tlie cerebro- s})inal Huid (Plate XVI) stain and look like the same or- ganism from other sources. They are usually not numerous. Other hTicteria. — Other bacteria of various species may be, in rare instances, the cause of meningitis, and may be I. . Diplococcus Intracellularis. Menincococci trk^ Diplococcus Pneumoni*. Pneumococci ■■^^^ Bacillus Influenzae. M V. i % Bacillus Tuberculosis. Preparations of cerebrospinal fluid from meningitis due to different bac All specimens stained with carbol-fuchsin and methvlene blue exceot the one contf % 1 SPINAL FLUID 125 found in the cerebro-spinal 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 carbol-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 bacilH present that these organisms may not be found. Wlien no 2)us cells are present tliere 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 PRACTICAL CLINICAL LABORATORY DIAGNOSIS millimeter, and these are all mononuelear cells. The count ill poliomyelitis usually runs 100 to 300 cells with 5% to 10^ c neutrophiles. In tuhercular meningitis the count usu- ally runs ;300 to 1000 ceils and approximately 100% mo- nonuclears. In tahes there are usually 90 to 300 cells per cubic millimeter, and in paresis about 30 to 90, practically all of wliich 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, I Fig. 99. — Bass diphtlieria culture tube. Tlie tube on the left lias been broken open and the swab has been straightened out ready for use. and tliat on this media thev show cliaracteristic mor- phology not sliown by any other bacilli likely to be found in these places. Material required. — A tube of Loffler's blood serum and a sterile swal) are required. Tubes and swabs are furnislied l)v city and state board of liealth laboratories, but they soon deteriorate. A special hermetically sealed tube containing tlie swal) (Fig. 09) also was devised by 127 128 PRACTICAT. CLINICAL LA150UAT0RY 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 diphtlieria sliould 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. jSIdldug 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 (hplitheria 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 DIPHTHERIA 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 r Fig. 101. — Inoculating: culture media by rubhinjr swab over surface. Note how cotton plug is iiehl. swab should be passed into the nose and some of tlie secre- tion secured. Now, holding the culture tube in tlie left hand and the swab in tlie riglit, remove tlic cotton ])hig from tlie tube (Fig. 100), rub tlie swab ovci* tlic surface (Fig. 101) of the culture media gently and tlicn replace the ])liig. Tlie best way to (hs])()sc of tlie infected swab is to tlirow it in tlie tire if one is convenient, or it mav • be returned to the empty end of tlie tube and all wrap^^ed in paper to be destroyed at a more convenient time. 130 PRACTICAL CLINICAL LABORATORY DIAGNOSIS In spite of tlic duty the physieian owes to his patient to be prepared for sueli work as this whenever it is indieated, it often liappens that a culture tube is not at hand wlien the suspicious case is iirst seen. Instead of waiting until the next visit to get a culture tube, an extemporaneous swab may be made bv 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. Incuhation. — The inoculated tube or culture, wdiich 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 WTapped with paper to insure against the plug coming out and placed in an inside (vest) pocket w^here the heat of the ])ody keeps it warm enough to favor the growth of diph- theria ])acini. This is as good as an incubator. The tube may ])e kept w^arm in the pajama pocket during the night. Incubation is necessary, because the bacilli grow very slowly and have not their characteristic morphology unless the cidture is kej^t warm. This method of incubation is espe- cially advantai^-eous because it starts from the time the cul- ture is first taken and it may permit the diagnosis to be made several hours earlier. Eocamliudlon of ciiUure. — Whenever many bacilli are })resent they usually liave grown to sufficient numbers in six oi- eight hours and have their characteristic morphology so that tlicy may be recognized upon examination. No A^isi- ble gi-owth will be ])resent at this time. If the exigencies of the case demand very early diagnosis tlie culture may • • CI' * be examined after six hours and very often the diagnosis can ])e made. We have occasionally been able to make the diagnosis in four hours. If no (li])]itheria 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 twentv-four hours other bacteria which mav 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. Malxing preixirations 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 shde and it is an advantage to dilute it (on the shde) with some water. Place approximately the smallest quantity of water that the platinum loo]) will hold near the middle of the side. Now hold tlie culture in the left hand and the platinum loop handle in tlie right. Sterilize the loop (Fig. 102) by lieating red (white) liot in the Bunsen-burner or alcohol flame. Kcmove the cotton plug (Fig. 10:5) from the tube and draw the loop over the surface (Fig. 1()4<) of the culture, endeavoring to scra])e oft' any growtli present. Oi'lcn little or no growth can be seen. Stir tlie material removed from the culture in the water on the slide and s])read out in a thin long streak (Fig. 105). Too thick smears are not good. Now flame the loop again and replace the cotton ])lug. As soon as the preparation dries it is ready to be stained. 132 PRACTICAL CIJXICAI. LABORATORY DIAGNOSIS Sterilizing the platinum loop in flame of Bunsen burner. Note proper size and shape of loop. Fig. 103. — Removing cotton plug, sterile i)latinum 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 tlie culture to be stained and examined. To fix, pass tlirouo'li tlic flame slowly, film side up, two or three times. Don't get it hotter than can be borne on the back of the liand (Fig. 92). 134 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Loffler's methylene blue is a viiluable 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: IVIetliylene 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 a. 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 ])y their mor])hol()gy from many other species of bacilh hkely to be found present in tlie mouth, nose and throat; but wlien grown on the Loffler's ])l()od scrum media they present a morj)hology quite cliaracteristic of them and a small group of so-called diphtheroid bacilli (Plate »\. A^H r-N I'' -:/ w% ^ ,1 -^' » " . 1>5» ''7 ; k '"'^^ ./^.^ ^^^)^, r^'ifr- R* '/a V '^' 4 " ^k .' ; A-'mUlP^ 9^% . ^ i ^ ^ ^ :f^ r « / ' -^ < . '4 :» ' W| •t \ ^^- 'i i • .1 ^ tw-"*^' ♦,. - 1 . . ,^ «.- n ;?S^ f * <>-< c A. Pus in acute g^onorrhoea. Carbol-fnchsin and met'hylene blue stai B. Pus in acute gonorrhoea. Gram's stain. C. Pus from case of acute non-specific urethritis. Many staphyl< CHAPTER XIV SYPHILIS I. Eocamination for Treponema pallida. — There are two simple practical methods of demonstrating Treponema pal- lida: with the darkfield 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 darh field condenser. — The Treponema pallida is present usually in very large numbers in all chancres, mucous patclies, 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 tliem, we must obtain it from the diseased tissue. In the case, for instance, of a chancre, it is necessary to o])tain material from tlie hard sypliilitic tissue and not sim])ly from tlie indurated or id- cerated tissue over or around it. AVliat 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 witli the material. To obtain pr()])er material (Fig. 107) grasp the chancre between the thumb and forefinger and 141 142 PRACTICAL CLINICAL LABOKATOUY DIAGNOSIS make sufficient pressure to drive out tlie blood, as is shown by the anemic appearance of the tissue. JNIaintain the pres- sure and with a scal2)el scrape down into the hard chancre tissue. Scrape up some of tlie tissue and squeeze out some "juice" of the tissue. Either transfer these to a slide with the scalpel or toucli the slide to the drop of fluid that has l)een 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 anestlietize 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 jiart 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 tlie diseased tissue wdth a suitable instrument, rinse it in salt solution and then to crush it on a slide w^ith tlie end of another slide and obtain proper fluid from the diseased tissue. The dia<^n()sis is less dependable whenever the lesion is in the mouth on account of the possibility of findino^ the nonpatliogenic spiroclietes commonly present in the mouth and niistakino- tliem for Treponema ])anida. If tlie scraped-up material is too thick it may be diluted with a little salt solution. Cover the small (juantity (about one- fourth drop) of "jnlce" from the diseased tissue with a SYPHILIS 143 cover-glass and it is now ready for examination with the darkfield condenser microscope. Eccamination with the darh field 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 Fig. 109. — Diagram sliow iiig path of rays through a darkfield condenser, and a W2 inch oil immersion lens fitted with funnel stoj). Abbe condenser. In the use of anv make of darkfield ft condenser the iVbbe condenser must be slipped out or swung out, according to the make of mici'oscope being used. The ])rinci])le (Fig. 100) of darkgi-ound illumination is that by means of a central stoj) no direct liglit is permitted 144 PRACTICAL 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 sliown 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. — Cias-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 vou liave with the o^eneral 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 14<5 To examine the specimen, place a drop of immersion oil (or water) on the darkfield 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 sto}) placed in the lens case of 1,12 i^^'h oil immersion lens for darkfield 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 suspe;nded in tlie^fluid under examination and we now see many thinos that cannot be seen bv direct illu- mination. The suspended particles have more or less dancing motion. Spirochetes (Fig. 112) are seen in hving, active state. They apparently revolve in corkscrew fasliion and also move laterally. They have little locomotion but are carried about more or less by currents. One sliould practice on preparations made from material taken from between tlie teetli wliich usually contains more or less spirochetes of other species. 146 PRACTICAL CLINICAL LAliOKATOKY DIAGNOSIS India ink preparation. — Only a small portion of the spirochetes present can be seen in an India ink preparation. It is important to liave a good qnality of India ink. The Gnntlier Wagner brand is a good one. The '' juice" from the suspected tissue is mixed on a side with about an equal Fig. 11;?. — Photomicrograph of Treponema pallida in "chancre juice," as seen with the darkfield microscope. amount of India ink, and sjDread 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 tlie 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, l"i;r. 113. — Proper India ink preparation. Note variation in thickness of spread. wliicli generally contains tlie Spirochcta Dentin m. Allow the preparation to dry and examine with the oil immersion lens, using strong light. S])ir()chetes (Fig. 114) appear as j)erfectly clear spirals, against the black granular back- ground. Otlier objects, such as bacteria, blood cells, etc., are also shown. ^-1 Fig:. 114-. — Pliotoniirropr.'iplis of Tndin ink proparntions containing: spirochetes. a. Tr<'])()n(in;i pallida. Compare the red blood cells. b. Trejjoneina j)allida. e. Si)irocheta refringens. d. Treponema niicrodentium and Tre])onema macrodentium. 148 PKACTICxVL CIJXICAI. LABOKATOKY DIAGNOSIS Diffcrcniidfion hctii^ccn Treponema pdJlida and other S'pirocJictcs. — AVe luii'dly think it wise for most observers to undertake to differentiate Treponema pallida from some of the other spiroehetes upon morphok)gical differences. There are spirochetes in the mouth, nose, throat, rectum and vagina of a ku'ge i)er cent, of all persons, but there are no iion-2)atho(>enic species on the skin. Spirochetes found in scrapings from lesions not of mucous membranes are therefore most certainly either the j^allida or, in the very rare tropical disease, yaws, the Treponema p)ertenuis. Spiroclietes 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 sj^eci- mens we have a few erythrocytes to which any spirochetes found may be conq^ared. Gland puneture.—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 Xo. 24 needle are needed. The syringe and needle should ])e 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 tlie point of the needle into the center of the gland. ]Make suction and rotate tlie needle a little. Stop the suction and witlidraw tlie needle. Make preparations of the gland "juice" and examine in the same way as matei'ial obtained from otiier sources. Interpretation. — Small s])iroclietes from a gland })imc- ture or from a lesion not on a mucous membrane are for SYPHILIS 149 practical j^ui'poses 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. Wassermanii seruin 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 tliis way we may determine the smallest (piantity that will give positive reactions w^ith positive blood, and the largest quan- tity tliat will give negative reactions witli 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, Xew Orleans, La., for $2.00 in advance for suffi- cient to make about one hundred tests. (2) Washed guinea-pig blood cells, 121/^7^- Etherize J-'ig. 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 P'ig. IIG. — Tntroduring the needle while steadying the i)arts. 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, 12l/4%. It should be made fresh for each (lav'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 witli tubing, pinch-cock, etc., arranged on a slielf 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 pur])ose from a vein usually at tlie bend of tlie el])()w. The best apparatus to draw it witli is an abso- lutely dry, all-glass syringe (Fig. 118) and Xo. 24 platino- iridium needle. It is not necessarv that thev sliould be steril- ized. Sterilize tlie needle just before use by mopping it with tincture of iodine. Blood can be drawn with the needle alone by inserting it into tlie distended vein and allowing 152 PRACTICAL 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 i ■If-: - / J, 5 -L' 2£ - o ■ r- Fig. 118. — Bur- ro u g h s , A\' <- 1 1 - come all - glass s y r i II g e w i t h ))latino - iridium needle attached. There are other good and cheaper all-glass syringes of the Luer type on the market now. Fig. 110. — Drawing blood from patient. Arm hanging down. Patient grasping above the 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 bl^)0(l 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 1 I ■ 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. 131, — Squeezing the arm \n fnrthi-r distend tlic veins. Now draw (Fig. 123) the desired (juantity 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" liis grip. Then quickly withdraw the needle and make pressure (Fig. 124) over 154 PRACTICAL CLINICAL LABORATORY DIAGNOSIS Fig. 12-2. — 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. Fig. 123. — Drawing the blood. Fig. 121. — Making jiressure 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 di'awn. It is not necessary to keep it on ice if it will be tested w^ithin tw^enty-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 Xo. 22 for taking blood from patient and a 2 c.c. all-glass syringe with No. 2-t platino-iridium needle for bleeding guinea-pigs. These needles can be sterilized just before using by mopping with Fig. 12'). — Wood test-tube Mock ;? x 1 \ S inches. Holes may be bored to acconunodate ilittVrent .size tubes. tincture of iodine. Tliey are quite soft, and therefore are likely to get dull, but can be resliarpened on a small fine- grained stone. 156 PRACTICAL CLINICAL LABOKATOKY DIAGNOSIS (2) Two or three dozen plain lipless test-tnbes, 12 x 115 inni. ontside 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 ^Hpettes, 1 c.c. graduated in Inindredths. The graduation sliould 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). J'ig. 1J(). — Small electric incubator suitable for making complement fixation tests and other laboratory work by one who does not have more elaborate equipment. xVn incubator is convenient because the reaction is more active at 98' F. A small electric incubator (Fig. 12(3) is suitable, and they are now comparatively inexpensive. SYPHILIS 157 METHOD OF MxYKIXCJ 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 F Eig. 1;2T. — Showing- arrangement of tubes in rack and use of 1 c.c. pijiette 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 tliird ])ut 0.0.5 c.c. In tlie fourtli put 0.0() c.c. In the fifth put 0.07 c.c. In measuring small ([uantities into tubes from a pipette allow tlie end of the pipette to touch tlie side of tlic 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 alcoliohc sohition of antigen has ah'eady 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 be made by jjlacing a small quantity of antigen (say 0.05 c.c.) in the necessary amount of salt solution. «V>V'^'>'--^f^i^!'H*?F^'Ti-=''"^;"'''''-^'^3??H^"-^-'",':".-*'W Fi^. 1;2H. — Proper nietliod of washing pipette with saline solution from a M'ater bottle equi})ped with tube and pinch-cock. We now have duplicate rows of tubes containing gradu- ally increasing quantities of the patient's serum. Those of tlie 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 hve to ten minutes if kept in an incubator at 98.6° F., or double that COnrfcr»oi test *^ 1 1 1 1 1 1 1 I > 1 1 J 1 1 1 1 W } *^- V ■■ IBB KSM km lem iiuLJ 1 1 PLATE XIX ODIFIED COMPLEMENT FIXATION TEST FOR SYPHILIS Negative reaction. Negative reaction. Doubtful positive reaction. 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 ' 2 3 4 5 Pt. Serum Saline solution Guinea-pig cells. . . . 0.03 c,c. 0.2 O.'i 0.04 c.c. 0.2 6!i 0.05 C.C. 0.2 0.1 0.06 C.c. 0.2 OA 0.07 c.c. 0.2 b'.i TEST Fig. 1:39. — 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 liemolysis lias taken place except jjer- haps in the first, or first and second, or possibly in the first, second and tliird tubes. Hemolysis is indicated by the cloudy suspension of cells changing to a clear solution. One or more of the tubes will usually sliow ])artial liemolvsis. In case of a negative (non-sy])liilitic) serum the tubes of the test row will show practically exactly the same amount of i()0 PKACTICAL CLINICAL LAliOKATOKY J)LVCJNOSlS hemolysis as in the control. A slight amount of anti- liemolytic influence is exerted by the antigen, however, and wc 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 Iicmolysis has occurred in the control series, we read the reaction in the fourtli tube of the test series. If Iicmolysis is complete the reaction is negative, but if hemol- ysis is slight or not present, then the reaction is positive. The stron<>er tlie 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 ai)ove it show some hemolysis, we would call it positive. If tliere is no hemolvsis 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 l^ecause of absence of native com- plement. For teclinic and interpretation of the globulin test and cell counts in syphilis, see Chajiter X. Interpretation. — The above method of making the test is as reliable as the original or Wassermann, when done by com})etent 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 w^ith 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-sypliilitic 2)ersons give negative reac- tions. As the disease improves under treatment or other- wise, or as it becomes chronic, tliere is less and less chance of tlie ])lo()d giving a positive reaction. If tlie reaction is positive it is weaker. INIany cases of tertiary syphilis and SYPHILIS 161 unciired syphilis give negative reactions. On account of the fact that there are some syphihtics 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. ^licroscope 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 darkfield 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 vou care to use this somewhat more satisfactory source of hght. 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, % in. square. No. 2. Some- times cover-o-lasses are sold that are cloudv in the center. Don't accept them. 8. 100 c.c. grain ak'oliol (95%). 9. 1 tube (6 tablets) Burroughs, Wellcome & Co. "sok)i(r' tablets for making AVriglit's stain. This is sufficient to make 90 c.c. of Wriglit's stain. ]Make up only 30 c.c. at a time. See p. 22. 10. 100 c.c. IVIerck's methyl alcohol, higliest purity, to make AVright'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. 1 0.3 164 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 ])refera])le 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 tij) 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. 14. 200 c.c. Toison's fluid, p. 35. 15. 1 Tallquist hemoglobin scale. Fig. 54. 16. 1 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). 18. 1 box ordinary hardwood toothpicks, best quality. 19. 1 dozen test-tubes, lipless, 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 f)aper 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. Fcliling'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, 21/0 in. 37. 10 gm. benzidin (crystals). 38. 1 Petri dish with cover, 4 in. 39. 100 c.c. carbol-fuchsin Czaplewsky's fornmla. Accept no other. See p. 115. 40. H)0 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. JNIulford & Co. or Parke, Davis & Co.) 42. 1 platinum loop in glass rod handle, Xo. 26 wire. See p. 131. 43. 100 c.c. I^offler's methylene blue solution. See p. 134. APPENDIX 167 44. 50 c.c. carbol-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 vou 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 syphihs) 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. 52. Fig. 13J. — Pr()|)(r drop l)()ttlc for stains, reagents, etc. 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. ( Vou niiiy 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 6o for typhoid 69 Albumin in urine, test for 71 Amebae, collection of specimens for examination for 102 differentiation of pathogenic from non-pathogenic 10 i 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 cerebros])inal fluid 124 morphological classification 109 Basophiles -6 Bass counting chamber '^4 Bass diphtlieria 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 s])ecimens by mail to be tested for sypliilis 155 Blood spreads, keeping unstained 2- labeling 1 ^ making 1'^ Blood staining -2 "Blood sticker," a good • • 12 Broncln'ectasis, leucocyte count in '5-5 Bronchitis, leucocyte count in ^^ Burns, leucocyte count in '^o 169 170 INDEX PAGE Carbol-fiiclisin and mctliylene blue stain for ])iis and exudates, teelniic 137 Carbol-fuclisin stain, Czaplewsky's 115 Carbol-gentian violet stain, Czaplewsky's 138 Careinouia, leueoeyte eount in 56 Casts, in urine 83 Centrifuge, use of in examining feees 92 use of in examining urine 79 Cerebro-s])inal fluid, eells in 121. inter})retation of examination of 126 Chancre, scra})ing to get material for examination 14-2 Children, percentage of leucocytes in 31 Chlorosis, leucocyte count in 56 Cholangitis, leucocyte count in 56 Cholecystitis, leucoc^'te 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 14<9 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 Diplitheria, examination of culture for 130 incubation of culture 130 leucocyte count in 56 method of making culture 128 prinei]iles of laboratory diagnosis of 127 Dipl)th(ria bacilli, d("scri))ti()n of 13 1< staining 133 Diphtheria culture, inter))retation of 135 making preparation from 131 staining preparation from 133 Eclampsia, leucocyte count in 56 Endamebae. (See Ameba'.) IXDKK 171 PAGE Endocarditis, leiicoc^'te count in 57 Eosino})hile.s 25 Epididymitis, gonorrhoeal, 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 5;j basophilic 54 method of counting 48 pathological interpretation of 5 t stij^pled or granular 54 Feces, collection of specimens of 90 concentration of ova in bv 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, leucocvte count in 57 Funnel stoj) I lo Gametes 63 Gametocytes 63 Gastric contents, examination of 87 inter])retation of examinations of 88 test for free HCl in 87 test for occult blood in 88 test for total acidity in 87 Gastritis, leucocyte count in 57 Gland puncture in diagnosis of sy])liilis 11-8 Gonococci, carbol-fuchsin and methylene blue stain for 137 description of ^ ^^ Gram's stain for ^^7 interpretation of examination for ItO in urine l*^^ Gonorrlid'a, in females I'i6 laboratory diagnosis of 136 leucocyte count in 57 obtaining material and making jjreparation 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 Hodffkin's disease, leucocyte count in 57 Hookworm, infection, leucocyte count in 57 oya of in feces 97 Hymenolepis nana, oya of in feces 99 Illumination, darkground 143 Incubator or waterbath, for complement fixation tests 156 India ink, use of in examining for sjiirochetes 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 Laryae, 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, microscoj^ic diagnosis of 120 obtaining material and making preparation from for examina- tion 120 Leucocyte count, differential 26 influence of infection with pyogenic bacteria upon 81 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 21- variations in the proportion of 31 Leukemia, leucoc^'te count in 58 stippled erythrocytes in 5 i 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, plasmoJia, 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 Megalocvtes 52 Meningitis, cerebro-spinal 125 leucocyte count in 58 obtaining material for examination for 125 Meningococci, in cerebro-spinal fluid 121 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 Bauscli Sz 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 Mum})s, leucocyte count in 58 Myelocytes, basophilic 33 eosinophilic 33 neutro})liilic 33 174 INDEX PAGE Myxedema, leucocyte count in 58 Nepliritis, leucocyte count in 58 Neutrophiles 25 Newton's color rings, looking for in hlood ])reparation 42 Normoblasts 53 Occult blood, test for in feces 107 test for in gastric contents 87 Orchitis, gonorrlia'al, leucocyte count in 57 Otitis media, leucocyte count in 59 Oxvuris 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 14-0 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 Small])ox, leucocj'te count in 59 Spinal fluid 122 cell counts in 125 inter])retation 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 1 24 Spirocheta. (See Treponema.) dentium 146 refringcns 147 Spirochetes, movements of 145 Sputum, collecting specimens of 112 examination of for tubercle bacilli 112 inter])retation 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 ai)paratus 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 1 i8 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 Ill making preparation for examination for 115 staining 116 Tuberculosis, leucocyte count in 60 Typlioid, 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 (50 Urine, albumin in, test for, qualitative 71 albumin in, test for, quantitative 72 acetone in, test for 78 casts in 83 collection of sjiecimens of for examination 70 cylindroids in 81 epitliclial cells in 83 indican in, test for 77 interpretation of examinations of 85 176 INDEX PAGE Urine, microscopic examination of , 78 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 Wrijrlit's stain ' 22 Yellow fever, leucocyte count in ..,..., 60 14 DAY USE RETURN TO DESK FROM WHICH BORROWED » This Dook is due on die last date stamped below, or on the date to which renewed. Renewed books are subjert to immediate recall. LD 21-50m-4,'63 (D6471sl0)476 General Library University of California Berkeley fimsm wiim "V m •»i ./«!;■»: ,• . ^ ■,'■' ^'■^ ' ']^ y-''' ■; ^""V ; f- I