MEMCAL SCHOOL 
 LUIBMAMY 
 
 Panaina- Pacific Intern 
 iiXposition Company. 
 
 
Digitized by the Internet Archive 
 
 in 2007 with funding from 
 
 IVIicrosoft Corporation 
 
 http://www.archive.org/details/clinicabacterioOOemerrich 
 
CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 FOR 
 
 PRACTITIONERS 
 
CLINICAL BACTERIOLOGY 
 AND Hy^MATOLOGY 
 
 FOR 
 
 PRACTITIONERS 
 
 6^ v'' • =^ 
 
 WfD'ESTE EMERY, M.D., B.Sc. Lond. ' 
 
 CLINICAL PATHOLOGIST TO KING'S COLLEGE HOSPITAL AND PATHOLOGIST TO THE CHILDREN'S 
 
 HOSPITAL, PADDINGTON GREEN ', FORMERLY ASSISTANT BACTERIOLOGIST TO THE ROYAL 
 
 COLLEGES OF PHYSICIANS AND SURGEONS, AND SOMETIME LECTURER ON PATHOLOGY 
 
 AND BACTERIOLOGY IN THE UNIVERSITY OF BIRMINGHAM 
 
 THIRD EDITION 
 
 PHILADELPHIA 
 BLAKISTON'S SON & CO., 1012, WALNUT ST. 
 
 1908 
 
PRINTED IN ENGLAND 
 
\ ^06 
 
 PREFACE TO THIRD EDITION 
 
 In making such alterations as seemed called for in the prepara- 
 tion of this edition, an attempt has been made to avoid as far as 
 possible any further expansion. It has been thought, however* 
 that a brief account of the methods of preparing bacterial vaccines 
 should be inserted : not that the practitioner should necessarily 
 make them for himself, but so that he may have a general idea 
 of the process. Of the other changes made, the main are an 
 account of making cultures of the blood by the use of an all- 
 glass exploring syringe, fuller details concerning lumbar puncture, 
 the diagnostic value of this operation in uraemia, and the use of a 
 Dewar's flask as an incubator. 
 
 Some of the coloured plates have been redrawn, and Figs. 31, 
 34, and 45 (for which I have to thank Mr. R. Gompertz) added. 
 My thanks are also due to Dr. H. B. Day for Figs. 16, 17, and 
 26, and to Messrs. Down Bros, for Fig. 30. 
 
 W. D'ESTE EMERY. 
 
 June, 1908. 
 
 COMPLIMENTS 
 OF 
 
 kHsto&'s Sob Co.^ 
 
 >i54() r '"" ^^ 
 
PREFACE TO SECOND EDITION 
 
 The success of the first edition of this book, and in particular 
 the numerous proofs I have received that it has been of value to 
 those for whom it was intended — that is, to practitioners who are 
 without training in pathology, and who want a simple guide to 
 refer to — has encouraged me to add to it somewhat, though 
 without altering its general scope. 
 
 The additions to the bacteriological portion are mostly concerned 
 with the examination of materials from special parts of the body — 
 mouth, conjunctiva, etc. — which were insufficiently dealt with in 
 the first edition, and are treated on simple lines ; in most cases 
 the diagnosis by microscopical methods, and in particular by the 
 application of Gram's stain, is given. 
 
 The haematological portion is almost all new, and is written in 
 response to numerous requests for a practical guide to blood 
 examinations, especially their application to the diagnosis of 
 disease. I have attempted to deal with the subject on the same 
 lines as the bacteriological portion — that is, to explain the con- 
 ditions under which a certain examination is indicated, the method 
 employed (in full detail), the deductions to be drawn from the 
 results obtained, and the cautions necessary in interpreting them. 
 It is in respect of these interpretations of blood-counts that I 
 think such a guide as this is most urgently required, as I find 
 there is a lamentable ignorance of the conditions under which 
 blood-counts are required and the methods of drawing deductions 
 from them. The result is that these examinations are often made 
 unnecessarily or omitted when they should be made, that the 
 diagnosis is not helped, and the clinical pathology is not appre- 
 ciated as it ought to be. The great stumbling-block has been 
 
Vlll PREFACE TO SECOND EDITION 
 
 that the blood-count has been thought to give the diagnosis 
 directly, without requiring any mental exertion on the part of the 
 physician or surgeon. It does so in some cases, but in most it is 
 to be regarded as an additional fact to be considered in making 
 a diagnosis — in many cases the chief and most important fact, but 
 one that must always be considered in conjunction with those 
 obtained by ordinary clinical methods. The results of a blood 
 examination are similar to those of any other physical examina- 
 tion ; they are easy to obtain, but often very difficult to interpret, 
 and, just as in the diagnosis of the diseases of the chest or the 
 nervous system, it requires much experience to make use of signs 
 which a student can elucidate. And, as in the case of the other 
 physical signs, the blood-findings are sometimes equivocal. It is 
 about as reasonable to discredit them on that account as to dis- 
 credit percussion of the chest because dulness does not always 
 indicate pneumonia. In spite of these difficulties the examination 
 of the blood is one that is more likely, in many cases, to give a 
 clue to what is actually taking place than is any other. As Cabot 
 puts it, *' The blood is the only tissue which we can easily examine 
 during the life of the patient. Its relations to all other tissues are 
 such that it is typical of them all in a way that no other tissue is, 
 acting on all and being acted on by all." Here I have attempted 
 to give an elementary guide to some of the more important and 
 simple changes, and for further reference the practitioners are 
 recommended to consult one of the largest works on the subject, 
 such as those of Da Costa, Ewing, or Cabot, all of which are 
 admirable and full of valuable information. 
 
 I have to thank Dr. Whitfield for the loan of the very admirable 
 photographs on Plates IV. and VI., and for revising the sections 
 on ringworm, etc., and Professor Schaudinn for the figures of 
 the spirochaete of syphilis shown on Plate V. 
 
 W. D'ESTE EMERY. 
 March, 1906. 
 
PREFACE 
 
 No practitioner who wishes to do his best for his patients and 
 to promote his own interests can afford to neglect any means of 
 cUnical investigation which may help him to arrive at a correct 
 diagnosis, and offer hints as to prognosis and treatment. Pre- 
 eminent among the more recent methods of investigation are those 
 which are applied by the bacteriologist, and it is no exaggeration 
 to say that in many of the infective diseases a diagnosis which is 
 made without a bacterioscopic examination is either mere guess- 
 work or can only be made so late that the patient has suffered 
 unnecessarily in health and the practitioner in prestige. In many 
 cases, however, the investigation requires a considerable amount 
 of technical skill and access to a well-equipped laboratory. The 
 former may, perhaps, be possessed by the rising generation (for 
 bacteriology is now an integral part of the medical curriculum), 
 but it would be unfair to expect every medical man to add the 
 latter to his already expensive equipage. But in many cases the 
 diagnosis can be arrived at by very simple means — a few slides, 
 cover-glasses, and stains, a good microscope (which ought to be 
 considered as essential as a stethoscope), and a very moderate 
 amount of technical skill, will often enable the practitioner to 
 arrive at a correct diagnosis in a very short time. This little book 
 is intended, in the first instance, to show exactly when this may be 
 done, and to provide clear, succinct, and full descriptions of simple 
 methods which may be employed. The descriptions of the opera- 
 tions which the practitioner can carry out for himself are mostly 
 written in the imperative mood, and are intended to be referred to 
 constantly and carried out, step by step, during the process. They 
 represent the instructions which would be given by a teacher when 
 
X PREFACE 
 
 watching a beginner making a simple bacteriological examination 
 for the first time. 
 
 It cannot be too strongly urged that the practitioner should 
 make the examination for himself whenever it is possible for him 
 to do so, and if not, to call in the services of an expert pathologist. 
 The report which is sent from a public laboratory may often be of 
 very considerable value, but it must be remembered that the 
 bacteriologist can only supply facts, and the inferences which may 
 be drawn from those facts will largely depend upon a knowledge 
 of the patient's clinical history and the method in which the 
 material was obtained. The bacteriologist is too often in the 
 position of a detective who has to unravel a mystery from obser- 
 vations made by other people, and has no opportunity of making 
 investigations for himself. A bacteriological examination which 
 is made by one person and interpreted by another, or which is 
 made on material which has passed through more hands than one, 
 loses much of its value, and an investigation made on the spot 
 may be more valuable than one made by a bacteriologist of far 
 greater experience at a distance. 
 
 The methods which are described in this little book are not in 
 all cases the ideal ones, and in some cases they are somewhat 
 different from those which are generally used, but they are simple 
 and efficient. Of course, the simple examinations which are 
 described here would frequently be supplemented by more com- 
 plicated cultural ones by a trained bacteriologist. The methods 
 described here have been taught in the post-graduate classes 
 which were initiated some two years ago in the University of 
 Birmingham. These have already been attended by about a 
 hundred practitioners, who have found these methods of great 
 assistance to them in their everyday practice. They have been 
 selected so as to provide examples of some of the more important 
 operations in constant use in the bacteriological laboratory. The 
 author takes this opportunity of expressing his cordial thanks to 
 Professor Leith for his kind suggestions as to the general scope of 
 the book. 
 
 No apology will be made for the numerous repetitions which 
 will be found in this book. They are essential to its scope, which 
 is to give clear accounts of the processes with as little reference to 
 
PREFACE XI 
 
 Other chapters as possible. In the majority of cases each section 
 is complete in itself. 
 
 These instructions are followed by information as to the inter- 
 pretation of the results which may be obtained, and this informa- 
 tion applies equally whether the medical man has made the 
 examination for himself or has obtained it ready-made from a 
 public laboratory. It, too, commonly happens that practitioners 
 feel themselves aggrieved because they get a negative report (as 
 to the presence or absence of Widal's reaction) on blood taken 
 during the first few days of an illness which turns out to be 
 typhoid fever, or are inclined to discredit bacteriological examina- 
 tions because diphtheria bacilli are found in throats which exhibit 
 no membrane and clear up in a few days without serious symptoms. 
 
 In the second place, there are a few cases in which the investiga- 
 tion had better be made in a public laboratory. In these the 
 questions of what to send and how to send it are fully explained. 
 This is a most important point. A bacteriologist is not a magician 
 who is able to weave a spell if he has a small portion of his 
 victim's anatomy to work upon, and the materials must be taken 
 in the proper way if his results are not to be useless or even mis- 
 leading. This is well seen in the examination of the blood for 
 bacteria. In many cases the blood is drawn in such a manner 
 that it must necessarily be contaminated from the skin during the 
 operation, and is transmitted in vaccine tubes which were almost 
 certainly not sterile before being filled. Under such circumstances 
 the bacteriologist will probably report the presence of streptococci 
 or staphylococci, and the practitioner who does not understand the 
 fallacies of the examination may be led to make a diagnosis which 
 will be disastrous to his own reputation, and may be injurious to 
 the patient. 
 
 Lastly, it need scarcely be said that this is not intended to be 
 a substitute for any one of the numerous excellent works on the 
 science of bacteriology which are current at the present time. 
 The practitioner is strongly recommended to supplement the very 
 meagre details concerning the life-history and pathogenic action 
 of the bacteria which are dealt with here by a study of one 
 of these text-books. Muir and Ritchie's admirable " Manual 
 of Bacteriology," Crookshank's " Bacteriology and Infective 
 
XU PREFACE 
 
 Diseases," Hewlett's " Bacteriology," Klein's " Micro-organisms 
 and Disease," McFarland's '* Text-book upon the Pathogenic 
 Bacteria," or Curtis's " Essentials of Practical Bacteriology," 
 are all suitable for this purpose, and a perusal of any one of them 
 will be both pleasurable and profitable to every medical man. 
 
 Several illustrations have been borrowed from sources mentioned 
 in the text, and for the loan of these the author wishes to express 
 his best thanks to the respective authors and publishers. His 
 best thanks are also due to Messrs. Baird and Tatlock, Swift and 
 Son, Zeiss, Leitz, Hawksley, and Hearson, for kindly providing 
 illustrations of apparatus made by them. 
 
CONTENTS 
 
 PART I 
 BACTERIOLOGY 
 
 Section I 
 APPARATUS AND PROCESSES 
 
 PAGE 
 
 THE BACTERIOLOGICAL MICROSCOPE - - - - I 
 
 STERILIZATION OF APPARATUS, ETC. - - " " 5 
 
 PREPARATION OF CULTURE MEDIA - - "9 
 
 INOCULATION OF CULTURE MEDIA - - - - l6 
 
 INCUBATION OF CULTURES - - - " - l8 
 
 METHOD OF EXAMINING CULTURES - " " - 22 
 
 gram's METHOD OF STAINING- - " " -24 
 
 EXAMINATION OF FILMS — USE OF MICROSCOPE - -27 
 STAINS -------- 28 
 
 CLEANING SLIDES AND COVER-GLASSES - - " 3^ 
 
 PIPETTES - - - - - " -32 
 
 Section II 
 DIAGNOSIS OF CERTAIN DISEASES 
 
 DIPHTHERIA - - - - " " "37 
 
 TETANUS - - - - - - - 45 
 
 THE PNEUMOCOCCUS, PNEUMONIA, ETC. - - "49 
 
 INFLUENZA - - - - " " "52 
 
 ANTHRAX - - - - - - - 54 
 
 TUBERCLE - - - - " " "59 
 
 LEPROSY - - - - - ' - ^5 
 
 ACTINOMYCOSIS, OR STREPTOTHRICOSIS - - ' ^5 
 
XIV CONTENTS 
 
 PAGE 
 
 GLANDERS - - - - - - 68 
 
 TYPHOID FEVER - - - - - 69 
 
 GONORRHCEA - - - - - - - 84 
 
 SYPHILIS - - - - - - 89 
 
 CHOLERA - - - - - - - 91 
 
 PLAGUE - - - - - - 93 
 
 SOFT SORE ...---. 94 
 
 RINGWORM - - - - - - - 95 
 
 OTHER SKIN DISEASES ------ lOO 
 
 Section III 
 
 COLLECTION AND EXAMINATION OF CERTAIN MORBID 
 
 MATERIALS 
 
 THE COLLECTION AND EXAMINATION OF PUS - - - I02 
 
 THE BACTERIOLOGICAL EXAMINATION OF THE MOUTH AND 
 
 FAUCES - - - - - 105 
 
 THE BACTERIOLOGICAL EXAMINATION OF THE NOSE AND 
 
 ACCESSORY CAVITIES - - - - - IIO 
 
 THE CONJUNCTIVA - - - - - - III 
 
 THE SPUTUM - - ' - - - - 113 
 
 THE GASTRIC CONTENTS AND VOMIT - - - "US 
 
 THE URINE - - - - - - - 118 
 
 THE COLLECTION OF FLUIDS FROM SEROUS CAVITIES - - 121 
 
 THE PLEURA - - - - - • - 1 23 
 
 FLUIDS FROM JOINTS - - - - - - 1 26 
 
 LUMBAR PUNCTURE - - - - - - 1 28 
 
 THE BACTERIOLOGICAL EXAMINATION OF THE BLOOD - - 1 38 
 
 EXAMINATION FOR BACTERIA IN FILMS - - - I41 
 
 MALARIA -.-.... 142 
 
 EXAMINATION BY CULTURAL METHODS - - -143 
 
 ESTIMATION OF THE OPSONIC POWER OF THE BLOOD - . - 148 
 
 COLLECTION OF MATERIAL AT POST-MORTEM EXAMINATIONS - 158 
 
 SECTION-CUTTING - - - - - "159 
 
 FIXING MATERIAL FOR CUTTING ... - 161 
 
 SECTION-CUTTING BY THE FREEZING METHOD - - " 163 
 
 STAINING AND MOUNTING FROZEN SECTIONS - - ' - 165 
 
CONTENTS XV 
 
 PAGE 
 
 THE PARAFFIN PROCESS - - - - - 1 66 
 
 DEHYDRATION - - - " " - I 66 
 
 CLARIFICATION - - - - - - 167 
 
 INFILTRATION WITH PARAFFIN - - - - 1 67 
 
 CASTING THE BLOCKS ----- 16^^ 
 
 CUTTING THE SECTIONS - - - - - 1 68 
 
 STAINING AND MOUNTING PARAFFIN SECTIONS - - 170 
 
 PART II 
 HEMATOLOGY 
 
 ESTIMATION OF THE AMOUNT OF H/EMOGLOBIN - "175 
 
 CLINICAL APPLICATIONS - - - - "179 
 
 ESTIMATION OF THE RED CORPUSCLES - - - 181 
 
 ESTIMATION OF THE NUMBER OF LEUCOCYTES - "193 
 
 THE INVESTIGATION OF THE MORPHOLOGY OF THE LEUCOCYTES 
 
 AND RED CORPUSCLES - - - - - 1 98 
 
 FIXATION OF BLOOD-FILMS ----- 202 
 STAINING BLOOD-FILMS FOR THE INVESTIGATION OF THEIR 
 
 CELLS _.-__-- 203 
 
 DIFFERENTIAL LEUCOCYTE COUNT - - - - 2o6 
 
 DIAGNOSTIC APPLICATIONS OF THE BLOOD COUNT AS A WHOLE 2l6 
 
 PART III 
 CYTO-DIAGNOSIS 
 
 CYTO-DIAGNOSIS . - . - - 233 
 
 APPENDIX .-.---- 245 
 
 INDEX - - . - . 247 
 
DESCRIPTION OF PLATE I. 
 
 Fig. I. — Diphtheria bacilH (long form) from a young culture o 
 blood-serum. LofHer's blue, x looo. 
 
 Fig. 2. — Diphtheria bacilli (short form) and Hoffmann's bacillu: 
 From young cultures on blood-serum. Loffler's blu( 
 
 X lOOO. 
 
 Fig. 3. — Pneumococci in sputum from a case of pneumonia 
 Stained with dilute carbol fuchsin and thoroughly washec 
 X 1000. 
 
 Fig. 4. — Anthrax bacilli. The lower portion of the field show 
 bamboo-like chains and spores, and is taken from culture! 
 Methylene blue, x 1000. The portion showing spores i 
 stained with carbol fuchsin, decolorized by brief immersio 
 in methylated spirit, and counterstained in methylene blue 
 
 Fig. 5. — Pus showing streptococci. Stained by Gram's methoc 
 counterstained with eosin. x 1000. 
 
 Fig. 6. — Pus showing gonococci and staphylococci. Stained b 
 Gram's method and counterstained with dilute carbol fuchsir 
 X 1000. 
 
It' 
 
 PLATE r 
 
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 / 
 
 ,4 MS 
 
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 /^ 
 
 Figl 
 
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 Fi<f 5 
 
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 \. 
 
 ft 
 
 
 
 Fi 
 
 •it 
 
 
 
 •5 « O 
 
 Fi<j 6 
 
 Wd-EE. d£ 
 
DESCRIPTION OF PLATE II. 
 
 Fig. I. — Tetanus bacilli from a culture. Stained with dilute 
 carbol fuchsin. x looo. 
 
 Fig. 2. — Tubercle bacilli in sputum. Stained with carbol 
 fuchsin and decolorized in dilute sulphuric acid ; counter- 
 stained with methylene blue, x looo. 
 
 Fig. 3. — Influenza bacilli in sputum. Some are contained 
 within a leucocyte. Loffler's blue, x 1000. 
 
 Fig. 4. — Plague bacilli from a culture. A short chain and some 
 coccoid (involution) forms are shown. Loffler's blue. 
 x 1200. 
 
 Fig. 5. — Vibrio of Asiatic cholera. Dilute carbol fuchsin. 
 X 1000. 
 
 Fig. 6. — Small colony of actinomyces, as it appears in pus. No 
 clubs are visible. Gram's method, x 600. 
 
PLATE II 
 
 I 
 
 FicfJ 
 
 <S 
 
 ' s- 
 
 ap 
 
 J'iy^ 
 
 *::'/ 
 
 ^^ ^ 
 
 ^ t ^ •^ 
 
 J^z^J 
 
 Fiq 4- 
 
 ,^^ 
 
 'HI'. 
 
 Fiq 5 
 
 ^>y 6 
 
 WdEE.delt. 
 
DESCRIPTION OF PLATE III. 
 
 Fig. I. — Diplococciis meningitidis in film, from cerebro-spinal fluid 
 obtained by lumbar puncture in a case of cerebro-spinal 
 fever. 
 
 Fig. 2. — Gonorrhoeal pus, showing numerous polynuclear leuco- 
 cytes, one of which contains gonococci ; a part of a 
 squamous cell also shown. 
 
 Fig. 3. — Pneumobacillus in pus from a case of conjunctivitis. 
 
 Fig. 4. — Pneumococci in sputum from a case of pneumonia. 
 
 Fig. 5. — Micrococcus catarrhalis in sputum from a case of 
 ' bronchitis. 
 
 Fig. 6. — Staphylococci in old and degenerated pus. 
 
 This plate is to illustrate the main morphological differences 
 between the chief pathogenic diplococci and organisms re- 
 sembling them. All the films were stained by Gram and 
 counterstained by carbol fuchsin. In Figs, i, 2, 3, and 5 the 
 organisms were coloured pink ; in 4 and 6 they were violet. 
 The figures are all drawn to the same scale. 
 
PLATE III. 
 
 Si if 
 
 
 
 «0l"f 
 
 Fig. 
 
 Fig. 2. 
 
 ^ «l»?* 
 
 Fig. 3. 
 
 Fig. 4. 
 
 -/ f 
 
 ^1 ^/' 
 
 
 Fig. 5. 
 
 Fig. 6. 
 
DESCRIPTION OF PLATE IV. 
 
 Fig. I. — Streptothrix (actinomycosis) in pus. Many of the 
 filaments are in short lengths, resembling bacilli. From a 
 case of actinomycosis of the pleura. (Gram.) 
 
 Fig. 2. — Film from the tonsillar exudate in Vincent's angina 
 (carbol fuchsin), showing bacilli and spirilla. 
 
 Fig. 3. — Morax-Axenfeld bacillus in pus from conjunctivitis 
 (carbol thionin). 
 
 Fig. 4. — Microsporon furfur in epidermic scale. Stained by 
 method given on p. 96. 
 
 Fig. 5. — Boas-Oppler bacillus, from the vomit in carcinoma 
 ventriculi. (Gram.) 
 
 Fig. 6. — Bottle bacillus (see p. 10 1). (Gram.) 
 
 (Photographs by Dr. A. Whitfield.) 
 
Fig. I. 
 
 Fig. 2. 
 
 Fig. 3. 
 
 Fig. 4. 
 
 Fig. 5. 
 
 Fig. 6. 
 
DESCRIPTION OF PLATE V. 
 
 Fig. I shows Spirochccta pallida (in the centre) and Spirocha;ta 
 refringens (to the left), under very high magnification. 
 
 Fig. 2 shows the same organisms under a yg'^^^^^ oil-immersion. 
 
 [Lent by Professor Schaudinn.) 
 
PLATE V. 
 
 \l " ^i^^r* ' ' 
 
 
 
 H^^B' 
 
 ""; \^ * 
 
 ^^^^^B 
 
 u 
 
 |^^^^^^^^^^^^^^Hp# 
 
 ' 
 
 I^^^^B'' 
 
 
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 [ 
 
 ■f^^ 
 
 t- 
 
 
 Fig. I. 
 
 _^, . 
 
 • t T 
 
 • 'm , 
 
 
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 : 
 
 • 
 
 
 
 J 
 
 
 
 
 
 Fig. 2. 
 
DESCRIPTION OF PLATE VI. 
 
 Fig. I. — Megalosporon edothrix from beard region. Hair-root 
 (below) showing some invasion. Root-sheath (above) with 
 abundant mycehum. 
 
 Fig. 2. — Megalosporon endothrix in hair. Notice the intact 
 cuticle. (Magnified less than Fig. i.) 
 
 Fig. 3. — Megalosporon endothrix in nail. 
 
 Fig. 4. — Microsporon Audouini in hair. Spores outside hair 
 partially removed to show mycelial elements within the 
 hair. 
 
 Fig. 5. — Favus in hair. 
 
 Preparations all stained by the method given on p. 96. 
 
 {Prepared and photographed by Dr. Whitfield.) 
 
PLATE VI, 
 
 
 
 7- '-.>;^ 
 
 
 V 
 
 Fig. I. 
 
 
 Fig. 2. 
 
 i 
 
 Fig. 3. 
 
 Fig. 4. 
 
 Fi... 
 
DESCRIPTION OF PLATE VII. 
 
 Figs, i, 2. — Lymphocytes. 
 
 Fig. 3. — Lymphocyte practically devoid of protoplasm. 
 
 Fig. 4. — Large lymphocyte. 
 
 Figs. 5, 6. — Large hyaline cells. 
 
 Fig. 7. — Polynuclear leucocytes. 
 
 Fig. 8. — Eosinophile leucocyte. 
 
 Fig. 9. — Mast cells (these are from normal blood). 
 
 Figs. 10, 11. — Myelocytes. 
 
 Fig. 12. — Eosinophile myelocyte. 
 
 Fig. 13. — Large mast cell from a case of spleno-meduUary leuco - 
 cythaemia. 
 
 Stained by Jenner and drawn to scale, but relatively slightly 
 larger than the corpuscles on Plate VIII. 
 
© 
 
 Fi<jl 
 
 PLATE VII 
 
 Fi^2 
 
 Fi^ 5 
 
 Fvq 4 
 
 
 Ftg 5 
 
 FijS 
 
 i.;rv>.>^-\ 
 
 J<^i 
 
 F.ff 
 
 >\-. 
 
 ^M 
 
 •^^«i;& 
 
 Ftff8 
 
 
 Fij,9 
 
 :CS^rr:. 
 
 Fi^ JO 
 
 I'-. 
 
 >■.:• 
 
 .\' 
 
 ^•^l:^*^*-^' 
 
 j-iffis 
 
 
 Fiff 11 
 
 ^Vy /J 
 
 Wd'EE.dell. 
 
DESCRIPTION OF PLATE VIII. 
 
 Fig. I. — A normal red corpuscle. 
 
 Fig. 2. — Red corpuscle showing granular basophilia. 
 
 Fig. 3. — A red corpuscle showing polychromatophilia. 
 
 Fig. 4. — Microcyte. 
 
 Fig. 5.— Megalocyte. 
 Fig. 6. — Poikilocytes. 
 
 Fig. 7.— Normoblast. 
 
 Fig. 8. — Normoblast with dividing nucleus from a case of Von 
 Jaksch's anaemia. 
 
 Fig. 9. — Normoblast with vesicular nucleus and polychroma- 
 tophilic stroma, from foetal blood. 
 
 Figs. 10, ti, 12. — Megaloblasts, the last showing polychroma- 
 tophil degeneration. 
 
 Specimens stained by Jenner and drawn to scale, appearing 
 about twice the size as when seen under a ^2"^"^^ lens and 
 IV. eyepiece (Leitz). 
 
PLATE VIII 
 
 Ftgl 
 
 Fi^2 
 
 Fi^5 
 
 Fi<f4 
 
 Ft^ 5 
 
 I'j 
 
 Fi-6f6 
 
 Fv^7 
 
 Fvff8 
 
 e 
 
 F^ff9 
 
 Fi^ lO 
 
 Fi^ n 
 
 Fttf 12 
 
 Wd'EE.ddt 
 
DESCRIPTION OF PLATE IX. 
 
 Fig. I. — Tuberculous pleurisy. Wet preparation stained with 
 methylene blue, showing lymphocytes and red corpuscles. 
 (The nucleoli are not usually so clearly shown, and are not 
 seen in dried films without special staining.) 
 
 Fig. 2. — Pus from knee affected with gonococcal arthritis (early 
 in the disease). The figure will serve to show the appear- 
 ances in an early case of septic pleurisy or empyema. Poly- 
 nuclear cells predominate, but there are a few lymphocytes. 
 The large cells are endothelial, and have ingested some 
 polynuclear leucocytes which show fragmentation of the 
 nucleus. Film dried, fixed with perchloride of mercury, 
 and stained with carbol thionin. 
 
 Fig. 3. — Pus from an old pneumococcic empyema. The cells 
 are polynuclears, but are highly degenerated, and stain very 
 feebly. Stained as in Fig. 2. (All magnified 1000 diameters.) 
 
PLATE IX. 
 
 Fig. I. 
 
 80 
 
DESCRIPTION OF PLATE X. 
 
 Fig. I. — Primary pleurisy, probably due to true rheumatism. 
 Wet preparation stained with methylene blue. The large 
 cells are " active " endothelial cells, many of which are 
 crowded with large refractile granules. Notice the different 
 degrees with which these cells have taken the stain. There 
 are also some polynuclears, lymphocytes, and red blood- 
 corpuscles. 
 
 Fig. 2. — From a case of passive pleural effusion due to cardiac 
 disease. (The appearances in that due to renal disease are 
 identical.) Flat '' passive " endothelial cells often grouped 
 into large plates, some much larger than those shown. A 
 few lymphocytes and red blood-corpuscles (the latter possibly 
 from the puncture). Film stained with carbol thionin. 
 
 Fig. 3. — Cells from a case of ascites due to malignant disease 
 of the ovary. Huge endothelial cells, showing large clear 
 vacuoles. There are numerous red corpuscles, one of which 
 has been ingested by an endothelial cell. Carbol thionin 
 (On same scale as Plate IX.) 
 
PLATE X. 
 
 O 
 
 \^ 
 
 Fig. I. 
 
CLINICAL BACTERIOLOGY 
 AND HEMATOLOGY 
 
 PART I 
 
 BACTERIOLOGY 
 
 SECTION I 
 APPARATUS AND PROCESSES 
 
 THE BACTERIOLOGICAL MICROSCOPE 
 
 The essentials which a microscope must possess in order to 
 render it available for bacteriological work are : 
 
 1. K firm and rigid stand and stage. 
 
 2. A firm, accurate, and delicate fine adjustment. 
 
 A microscope which possesses these may be made available for 
 bacteriological work by the addition of the necessary parts, but 
 one that is deficient in these respects is useless. 
 
 3. A convex and a flat mirror. 
 
 4. An Abbe's condenser and iris diaphragm. 
 
 5. Three lenses, a low power [\ inch or, better, | inch), a high 
 power (J inch or thereabouts), and a y^^-inch oil immersion. 
 
 If the practitioner already possesses a microscope made by a 
 reliable firm, and in good condition, this should be sent to a maker 
 (not necessarily the maker of the microscope in question) or to a 
 bacteriologist for an opinion as to whether or no it is sufficiently 
 firm, and has a fine adjustment good enough, to justify the addition 
 of the other parts. If this is the case it should be fitted with an 
 [Abbe's condenser and an iris diaphragm; the cost should not 
 exceed 30s. or £1. But it is useless to have this alteration made 
 unless the stand is sufficiently steady to carry an oil-immersion 
 
 I 
 
2 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 lens ; and in most cases it is useless to think of having an 
 inefficient fine adjustment altered for bacteriological use. 
 
 There are now many microscopes which are sold at a com- 
 paratively small price, and which will answer every purpose. Of 
 these, Leitz's lib. stand, with the lenses above mentioned, a triple 
 nose-piece (which is so great an advantage that it might almost be 
 called an essential), and two eye-pieces, costs about £i^, and may 
 
 Fig. I. — Bacteriological^Microscope. 
 
 be highly commended. The stand is very firm and strong, the 
 fine adjustment delicate, and the lenses altogether admirable. The 
 only objection to this microscope is that it is rather heavy ; this is 
 an advantage for laboratory work, but it is a disadvantage for a 
 medical man, who may have to examine blood, etc., at the patient's 
 bedside. Swift and Son manufacture a microscope of much the 
 same type at about the same price ; it has all the good points of 
 the Leitz microscope and is somewhat lighter. The same firm 
 
THE BACTERIOLOGICAL MICROSCOPE 3 
 
 also supplies a portable instrument, the cost of which (without 
 lenses) is £^. This is very ingeniously made, and will carry an 
 oil-immersion lens quite well ; it is perhaps the most suitable 
 stand for a practitioner who may have to do work at a distance, 
 for it packs into a very small compass and is not heavy. The 
 same firm also make a microscope designed by Dr. Briscoe, 
 which is even more portable, has a mechanical stage, and packs 
 into a box in which all the requisites for blood-counts are carried. 
 It is especially adapted for blood-work at the bedside. 
 
 Fig, 2. — Bacteriological Microscope. 
 
 The cost of a y\r-inch oil-immersion lens is, roughly speaking, 
 £^. Beck and Watson supply good lenses at £\. 
 
 The cost of the other lenses may be put down at 30s. or less 
 apiece. Leitz's No. 3 (about f inch) costs 15s., and his No. 6 
 (about i inch) costs 30s. ; these are both extremely well adapted 
 for bacteriological work. 
 
 With regard to eye-pieces, it is an advantage to have two, a 
 No. 2 and a No. 4. Higher powers may be used, but it must be 
 remembered that any increased gain in magnification brought 
 
4 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 about by the eye-piece is attended by a loss of definition. The 
 same appUes to the increased magnification obtained by pulling 
 out the draw-tube. 
 
 Fig. 3.— Portable Microscope (Swift). 
 
 Fig. 4. — Portable Microscope (Swift). 
 
 The total cost of a microscope may be put down at £1^, and 
 for this sum a thoroughly efficient instrument can be obtained, 
 while one that will answer every purpose may be bought for 
 
THE BACTERIOLOGICAL MICROSCOPE 5 
 
 decidedly less. The cost of adapting a good stand will be about 
 ^6 IDS. or £'■] (30S. or £1 for the substage arrangement and £^ for 
 the lens), or less if a cheaper oil-immersion lens is obtained. 
 
 A mechanical stage is a very great advantage in haematological 
 work, since it enables a preparation to be moved regularly back- 
 ward and forward and up and down, so that all parts of its 
 surface may be examined systematically. A simple form is all 
 that is required, the verniers being quite unnecessary for most 
 purposes. It can be fitted to most stands. Leitz's can be 
 specially recommended. 
 
 Fig. 5. — Hot-air Sterilizer: Simple Form. 
 
 It need scarcely be said that there are many admirable micro- 
 scopes other than those mentioned above, which have simply 
 been selected as favourable and low-priced instruments of English 
 and Continental manufacture. Amongst others, the microscopes 
 made by Beck, Ross, and Watson are all good, and reasonable 
 in price. 
 
 STERILIZATION OF APPARATUS, ETC. 
 
 Requisites. — i. A thermometer graduated to 200° C. 
 ^ 2. A hot-air sterilizer ; or a cubical biscuit tin, the soldering of 
 which has been replaced by brazing — this must be mounted upon 
 a tripod stand ; or a kitchen oven, preferably a gas oven. 
 
b CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 3. A steam sterilizer ; or a large kitchen steamer — this should 
 be deep enough to contain a litre flask holding a funnel. 
 
 4. A large Bunsen burner or spirit-lamp. 
 
 Bacteria and their spores are ubiquitous, and it is necessary to 
 sterilize all vessels and other apparatus and all culture media 
 before use. The methods which are adopted all depend upon the 
 action of heat ; chemical antiseptics are rarely used in the bacterio- 
 logical laboratory for the sterilization of apparatus, for it would be 
 difficult to remove them completely, and the traces which might 
 remain would prevent the development of those germs which we 
 
 Fig. 6.— Hot-air Sterilizer. 
 
 wished to cultivate. Two chief methods are in use, sterilization 
 by dry heat and by steam; we exclude sterilization by steam under 
 pressure, as this requires special and expensive apparatus, and is 
 never absolutely necessary, though often convenient. 
 
 Dry Heat is used to sterilize all glass vessels (flasks, Petri 
 dishes, test-tubes, pipettes, etc.), cotton-wool, and metal instru- 
 ments. The heat must be continued for at least half an hour, 
 and must not fall below 150° C. as indicated by the thermometer. 
 Another method, which is less reUable than the use of the ther- 
 mometer, but which may be resorted to in an emergency, is to 
 wrap the apparatus loosely in cotton- wool, and to proceed with the 
 
I 
 
 STERILIZATION OF APPARATUS 7 
 
 heating (allowing the temperature to rise gradually) until the outer 
 part of the wool is slightly singed over the whole of the exposed 
 surface. 
 
 The special sterilizer which is used in the bacteriological 
 laboratory consists of a copper or iron oven with double walls 
 and perforated metal shelves. There is a hole in the top, which 
 is fitted with a perforated cork, through which the thermometer 
 passes. The oven is mounted on a stand, and heated by means 
 of a large Bunsen or Fletcher's burner. 
 
 An efficient sterilizer may be made out of a cubical biscuit box, 
 but it will not stand much usage unless the joints are brazed 
 instead of being soldered : this can be done by any tinsmith. It 
 is much better to have the bottom of the box replaced by a sheet 
 of copper, and a sterilizer made in this way will answer every 
 purpose and be fairly durable. A circular hole is cut through the 
 centre of the lid and fitted with a cork bored so as to admit the 
 thermometer. A false bottom or a shelf an inch or so from the 
 bottom will keep the articles which are being sterilized from 
 the heated surface ; the false bottom may be made from a sheet 
 of tin 2 inches longer in two of the sides than the bottom 
 of the box. The extremities of the shorter sides are to be 
 turned down for a length of an inch, and several holes cut in 
 the plate. 
 
 Lastly, the kitchen oven may be pressed into service if no 
 other sterihzer is at hand in an emergency. The apparatus to be 
 sterilized is to be placed on a layer of cotton-wool on one of the 
 shelves, and the temperature is observed by means of the thermo- 
 meter, which should be thrust through the little window which 
 permits of the regulation of the temperature ; or the heat may 
 be continued until the cotton-wool is singed over the whole of 
 the exposed surface. This method is very convenient for prac- 
 titioners sending materials to a laboratory for bacteriological 
 examination. 
 
 A gas oven is even more convenient, as the temperature can 
 be regulated to a nicety. 
 
 All glass apparatus must be thoroughly cleansed and dried 
 before sterilization. The remaining steps differ somewhat in the 
 different cases. 
 
 Flasks are plugged lightly with cotton-wool before being placed 
 in the sterilizer. Bottles may be sterilized in the same way ; 
 they may also be sterilized by boiling. Test-tubes are treated in 
 
8 
 
 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 the same way as flasks. Petri dishes are wrapped round with 
 tissue-paper or filter-paper before being sterihzed. 
 
 Before removing glass apparatus from the sterilizer remember 
 to let the temperature fall gradually, or the vessels may crack. 
 
 If a sterilized test-tube be required in a hurry (as often 
 happens), plug a clean tube with cotton-wool, and hold it with 
 forceps, one blade being inside and one outside the open end. 
 Then heat every part of the tube thoroughly in the flame, taking 
 care the heat is great enough to scorch the plug. 
 
 Cotton-wool is sterilized by being spread out in thin layers on 
 
 the shelves of the apparatus, and 
 the heat is continued until the out- 
 side is singed. 
 
 Metal instruments (knives, scissors, 
 etc.) may be sterilized in the same 
 way and at the same time. They 
 should be wrapped loosely in cotton- 
 wool, and should not be removed 
 from their wrapping until the mo- 
 ment at which they are to be used. 
 
 Steam is chiefly used for the 
 sterilization of culture media before 
 use, and for the destruction of cul- 
 tures when they are done with. The 
 latter purpose, however, is accom- 
 plished more speedily and safely by 
 the addition of a few drops of com- 
 mercial formalin to each tube, or 
 the tubes may be filled with i in 20 
 carbolic, which, however, cannot be 
 relied on to kill spores. 
 The proper steam sterilizer consists of a metal cylinder with a 
 perforated diaphragm 6 or 8 inches from the bottom. It is en- 
 closed in a thick layer of felt or other non-conductor of heat, and 
 is provided with a lid. The space between the bottom and the 
 diaphragm is partly filled with water, which is boiled by means of 
 a Bunsen flame or Fletcher's burner, the apparatus to be sterilized 
 being placed in the chamber above so as to be exposed to the 
 steam. 
 
 An ordinary steamer (such as is used for cooking potatoes or 
 fish) will answer every purpose. In procuring such a steamer 
 
 Fig. 7. — Steam Sterilizer. 
 
PREPARATION OF CULTURE MEDIA 9 
 
 for bacteriological use it is best to choose one that will 
 accommodate a litre flask holding a funnel, as it is often a 
 great convenience in the filtration of fluids which become 
 solid on cooling to carry out the process in an atmosphere of 
 steam. 
 
 Exact details of the way it is used will be given subsequently. 
 
 PREPARATION OF CULTURE MEDIA 
 
 Bacteria are grown in the same way as other plants. A 
 gardener who wishes to grow a plot of a particular plant will first 
 prepare a soil suitable for the growth of that plant, and free it as 
 far as possible of all seeds, roots, etc. He will then sow it with 
 the seeds of the plant in question, and do what he can to expose 
 them to a suitable temperature. An exactly similar process is 
 adopted when we wish to cultivate the smallest of all plants. The 
 soil which we prepare is called the culture medium, and differs in 
 the case of different bacteria ; the process of freeing this soil from 
 bacteria and their spores is called sterilization, and we insure a 
 suitable temperature by means of an incubator, the heat of which 
 is kept constant. 
 
 The culture media which are used for special purposes are 
 almost innumerable, but in the daily routine of the laboratory 
 and for diagnostic purposes, broth, gelatin, agar-agar, and 
 blood-serum, are all that are really necessary in the vast 
 majority of cases. These media may all be bought from any 
 firm of manufacturing chemists, or from any bacteriological 
 laboratory ; and their purchase saves a great deal of work, 
 and is to be recommended for those who only wish to use 
 them occasionally. They are sold in test-tubes, which are kept 
 sterile by being plugged fairly firmly with cotton-wool ; this sub- 
 stance prevents the passage of bacteria as long as it is. kept dry. 
 The tubes are best stored in jars provided with tightly fitting lids, 
 and it is an advantage to place a shallow layer of a solution of 
 perchloride of mercury (or other non-volatile antiseptic) in the 
 bottom of each jar to prevent the medium from drying up. It is 
 scarcely necessary to add that not the smallest trace of the lotion 
 should be allowed to come into contact with the cotton-wool 
 plug of the tubes. Or the medium may be kept from drying 
 up by covering the tubes with indiarubber caps sold for the 
 purpose. 
 
10 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Broth is very easily made, and, as it is the foundation of many 
 other media, the practitioner is strongly advised to prepare it for 
 himself. 
 
 Requisites. — i. Liebig's Extract of Meat. 
 
 2. Peptone. 
 
 3. Common salt. 
 
 4. A dilute solution of sodium carbonate — about i per cent., 
 but the exact strength does not matter. 
 
 5. A large flask, a stirring-rod, and a large glass funnel. 
 
 6. Test-tubes and cotton-wool plugs. The exact size of the 
 tubes is unimportant, but 6 inches by f inch is convenient. The 
 plugs are best prepared from wool which has been previously 
 sterilized by dry heat, and should be fairly firm. The tube, with 
 the plug in situ, must be sterilized by dry heat ready for use. 
 
 7. Litmus-paper. 
 
 Method. — Take i litre of tap-water in the flask, and add 
 5 grammes of Liebig's Extract, 10 grammes of peptone, and 
 5 grammes of common salt, and boil until all are dissolved. Test 
 the reaction by withdrawing a drop of the fluid on the stirring-rod 
 and applying it to a piece of litmus-paper. You will probably 
 find that it is slightly acid. Now add some of the solution of 
 soda, drop by drop, testing after each addition, until the reaction 
 of the fluid is slightly alkaline.* Boil the fluid for half an hour, 
 to coagulate any albumin which may be present. 
 
 Next filter the broth into a sterile flask, passing it through a 
 double thickness of white filter or blotting paper, and plug the 
 flask firmly with sterilized cotton-wool. 
 
 If the broth is to be used for the manufacture of gelatin or 
 agar, it is next sterilized in the flask ; while if it is to be used 
 as it is as a culture medium, it is decanted into tubes and then 
 sterilized. 
 
 In decanting media into tubes be very careful not to .get the 
 plug wet, and not to let any of the medium get on to the upper 
 part of the tube ; otherwise the plug will stick to the tube, and 
 there will be some danger of bacteria from the air " growing 
 through " the fluid contained in the interstices of the plug and 
 contaminating the culture. Ordinary non-absorbent (brown) wool 
 
 * If during the neutralizing process too much alkali is added, then it is 
 necessary to reacidify with dilute hydrochloric acid and reneutralize. The 
 sodium chloride formed makes no practical difference in the medium. 
 
i 
 
 PREPARATION OF CULTURE MEDIA II 
 
 is better than the white absorbent wool, as it is less easily 
 wetted. 
 
 The broth (and other culture medium after being melted) may 
 be poured into the tubes in the following way : A sterilized funnel 
 is united by a short length of indiarubber tubing to a piece of glass 
 tubing drawn out to a point ; the rubber tube is clipped by a 
 spring clip or a pair of pressure forceps. The funnel is now 
 mounted on a retort-stand, filled with the medium, and covered 
 over with a piece of glass. The cotton-wool plug is removed from 
 a test-tube, and the latter placed so that the glass tube attached to 
 the funnel reaches nearly to the bottom. The clip is released, and 
 the requisite quantity of broth (enough to fill the tube to the depth 
 of if or 2 inches) is allowed to run in ; the clip is then reapplied 
 and the tube removed and plugged. This process is repeated 
 until enough tubes have been filled. 
 
 The tubes and the broth which remains over (after having been 
 poured back into the flask and the latter plugged with cotton-wool) 
 are now sterilized. The vessels are placed in the steam sterilizer 
 and exposed to steam for half an hour on three successive days ; this 
 process is called intermittent sterilization, and its rationale is very 
 simple. The first steaming destroys all developed bacteria, and 
 would sterilize the fluid entirely if no spores were present. In 
 the interval between the first and second sterilization most of the 
 spores which may be present will develop into mature bacteria, 
 and these will be killed by the second steaming. The third 
 sterilization is to kill off any bacteria which may not have 
 developed from spores in the first interval. A very similar 
 process is adopted by the gardener in freeing soil from weeds ; 
 the application of chemical weed-destroyers or a thorough hoeing 
 will destroy developed plants, but will not injure seeds which may 
 be contained in the soil, and these processes are repeated, intervals 
 being allowed to permit the development of the plants, until they 
 reach the stage in which they are vulnerable. 
 
 To recapitulate : Mix the ingredients, and heat until they are 
 dissolved, render slightly alkaline, boil half an hour, filter. Then 
 place in sterilized flask or into test-tubes, and sterilize in the steam 
 sterilizer for half an hour on three successive days. 
 
 Nutrient Gelatin is broth which has been solidified by the 
 addition of from lo to 15 per cent, of gelatin ; the former amount 
 is used in the winter, the latter in the summer. For general 
 purposes 12^ per cent, may be used in all cases. 
 
12 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 The Special advantages of gelatin as a culture medium are two- 
 fold. In the first place, a gr^t many organisms grow in or on it 
 in a characteristic way, so that a bacteriologist may be able to 
 identify the organism by inspection of the culture. This arises 
 partly from the fact that some bacteria produce a ferment which 
 digests gelatin just as pepsin does ; these bacteria " liquefy " the 
 gelatin, and the distinction between the bacteria which have and 
 those which have not this property is very important for purposes 
 of diagnosis. Further, some bacteria liquefy rapidly and others 
 slowly, and this is another important point in the identification of 
 a germ. 
 
 In the second place, the gelatin medium may be melted at a 
 temperature (about 25° C.) at which bacteria are not killed. This 
 fact is made use of in the isolation of bacteria from a fluid which 
 contains several species by the process known as " plating." 
 Suppose, for instance, that we find by microscopic examination 
 that a specimen of pus contains two different species of bacteria 
 (perhaps a bacillus and a coccus), and we wish to obtain the two 
 organisms in pure culture so that we can ascertain their nature 
 and properties. We take a tube of gelatin and melt it by placing 
 it in warm water, and then inoculate the medium with a minute 
 quantity of the pus. We then shake it so as to distribute the 
 organisms throughout the melted fluid, and then pour the latter 
 into a flat dish (Petri's plate), so that the gelatin flows out into a 
 thin film and then sets. If our dilution has been properly made, 
 we shall have separated each organism from its neighbours, and 
 each separate germ will grow up into a "colony," which will soon 
 be visible to the naked eye. In all probability we shall be able 
 to see that these colonies are of two kinds : one may liquefy 
 and the other not, one may be coloured and the other colour- 
 less, one may be round and the other angular, etc. Samples 
 of each sort of colony are then transplanted to fresh culture- 
 tubes, and again incubated. An example of this process is given 
 on p. 58. 
 
 A slight modification of this process enables us to make an 
 estimate of the number of living bacteria which is present in a 
 given fluid. To do this we have to follow out the above process, 
 adding a definite measured quantity of the fluid to the culture-tube 
 of liquefied gelatin. The number of colonies which develop is 
 counted, and this gives us the number of bacteria in the sample of 
 fluid. For example, if ^^ c.c. diff"used throughout a tube of 
 
I 
 
 PREPARATION OF CULTURE MEDIA I3 
 
 melted gelatin and poured out into a thin film produced twenty 
 colonies, it follows that i c.c. of the fluid contained 200 bacteria. 
 This is a brief description of the essentials of the method adopted 
 in the quantitative examination of water and other fluids. 
 Requisites for the Mamifacttive of Gelatin. — i. Broth. 
 
 2. Gelatin. (Coignet's gold label gelatin is best, but any good 
 brand will do.) 
 
 3. Dilute solution of sodium carbonate. 
 
 4. Litmus-papers. 
 
 5. Flasks, stirring-rod, funnel, and plugged test-tubes as for 
 broth. 
 
 Method. — Measure the broth and add to it 12 J grammes of 
 gelatin for each 100 c.c. ; allow to soak for an hour or more, and 
 then heat until the gelatin is dissolved. Continue the heat, and 
 render the medium faintly alkaline, just as was done in the 
 preparation of broth. Now filter through a moistened filter-paper. 
 To avoid the setting of the gelatin during the filtration, it is best 
 to use a double-jacketed funnel containing hot water, but if this is 
 not at hand the whole apparatus (flask and funnel) may be placed 
 in the steam sterilizer (the lid being kept off to avoid the drops 
 of condensed water which might otherwise fall into the funnel) 
 or in a wavm (but not hot) oven, and left at a temperature of about 
 40° C, until the process is complete. 
 
 The gelatin which is made by the above process is sufficiently 
 clear for most purposes. A more sightly medium may be made 
 by clarification of the above by white of Qgg. To the medium 
 (after neutralization, but before filtration) add the white of one 
 egg for each 250 or 300 c.c. of fluid, and shake thoroughly. Now 
 boil in the steamer for half an hour, and filter as before. 
 
 Test-tubes are filled with gelatin just in the same way as with 
 broth, and the process must be carried out quickly to avoid 
 solidification of the medium. Some of the test-tubes are allowed 
 to cool in the vertical position, others lying in a sloping position, 
 so that the upper surface of the gelatin forms an ellipse some 
 3 inches long. The former tubes are inoculated by driving a 
 straight platinum needle charged with the material containing 
 the bacteria into the gelatin in the axis of the tube ; cultures 
 made in this way are called "stab cultures." The gelatin 
 " slopes " are inoculated by drawing the charged needle along the 
 surface of the medium, care being taken not to plough it up ; 
 cultures made in this way are called " stroke cultures." 
 
14 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Agar, or Agar-Agar, is the name given to the dried strips of a 
 Japanese seaweed. It forms a jelly which differs from that con- 
 taining gelatin in that it melts at a higher temperature ; nutrient 
 agar, as used in the laboratory, melts just below the boiling-point 
 of water and sets at about 40° C. This is an advantage in the 
 cultivation of most pathogenic bacteria, for these grow (as a rule) 
 best at or near the temperature of the body, the temperature to 
 which they are exposed under natural circumstances ; and at this 
 temperature gelatin would melt. Agar is somewhat difficult to 
 prepare unless the practitioner has an autoclave, and may be 
 bought with advantage. But the following method is not very 
 difficult, and, as agar is perhaps the most generally useful of all 
 media, should be learnt. 
 
 Requisites. — i. Broth. 
 
 2. Agar-agar. This should be cut up into very small pieces with 
 a pair of scissors, or may be bought in powder. 
 
 3. Solution of acetic acid (glacial acetic acid, 2 to 4 c.c. ; water, 
 500 c.c). 
 
 4. A large beaker. 
 
 5. Other apparatus and materials as for gelatin. 
 
 Method. — Weigh out 2 grammes of agar for each 100 c.c. of 
 broth to be used, and soak it in the dilute acetic acid for a quarter 
 of an hour. Now strain off the acid and wash the agar in water 
 until a small piece does not redden blue litmus-paper when pressed 
 upon it. Place the broth in a glass beaker and add the agar. 
 Now place the beaker upon a piece of wire gauze on a tripod 
 stand, and apply a small Bunsen flame or spirit-lamp ; this must 
 be placed so that the flame impinges on a point not far from the 
 side of the beaker. As the fluid is heated it will rise, and a 
 continual circulation will take place, so that the fragments will 
 not stick to the bottom and cause it to crack. When all is 
 dissolved, the hot liquid must be carefully neutralized. It is then 
 allowed to cool to about 50° C, and the white of an egg added for 
 each 500 c.c. of fluid and mixed in thoroughly by being stirred 
 with a glass rod. The whole is then placed in the steamer for an 
 hour, at the end of which time the albumin should be completely 
 coagulated. The beaker and its contents are then allowed to cool 
 gradually, so that the coagulum (retaining all solid particles) may 
 settle to the bottom before coagulation is complete. Perhaps the 
 best method of accomplishing this is to place it in the oven 
 (taking care that the temperature does not exceed 100° C.) after 
 
PREPARATION OF CULTURE MEDIA 15 
 
 the lire has been raked out at night. In the morning the mass 
 will be found to have soUdified, and there will be a coagulum at 
 the bottom. The beaker is then inverted and the mass " turned 
 out " just as a cook turns out a jelly, and the sediment is cut 
 off with a sharp knife. This avoids filtration, which is very 
 troublesome. 
 
 An alternative method is to filter the melted jelly through 
 moistened filter-paper. It is necessary to keep flask and funnel in 
 a steamer (the water of which is kept boiling vigorously) during 
 the whole process, or the jelly will solidify in the outflow tube of 
 the funnel. Or it may be filtered through a double thickness of 
 ordinary surgical lint (non-medicated). It runs through this very 
 quickly, and the funnel need not be kept hot. The resulting 
 medium is not absolutely clear, but sufficiently so for most 
 purposes. 
 
 The agar is again melted and placed in test-tubes ; these are 
 sterilized on three successive days and allowed to set in a sloping 
 position. For certain purposes glucose, glycerin, etc., are added 
 to the agar. The addition should be made to the melted medium 
 just before it is poured into the tubes. 
 
 Solidified Blood-serum is very difficult to prepare, and is 
 best purchased ready for use from a good laboratory. It is used 
 chiefly in the diagnosis of diphtheria by the examination of 
 '' swabs " from the throat. 
 
 Potato tubes are in occasional use, and are easy to prepare. 
 The process is as follows : Take large and sound potatoes and 
 scrub them thoroughly with a nail-brush under the tap. Peel 
 them deeply enough to remove the eyes completely. Then cut 
 them into cylinders a little less than j inch in diameter (if you are 
 using |-inch test-tubes) and as long as possible ; this is best done 
 by means of a cork-borer, but they may be shaped by means of a 
 knife if this is not at hand. Then cut each cylinder in half by a 
 cut running obliquely from end to end ; the shape of each half 
 should be exactly like that of the medium in a sloped gelatin tube. 
 Place the halves in a large vessel of tap-water and allow them 
 to soak all night ; it is a good plan to use running water if 
 possible. 
 
 After this has been done place each half (base downwards) in a 
 test-tube, having previously inserted a small mass of absorbent 
 cotton-wool and enough water to saturate it. Plug the mouth of 
 the tube with cotton-wool and sterilize on three successive days. 
 
l6 CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 INOCULATION OF CULTURE MEDIA 
 
 The method in which this is done varies greatly according to 
 the end in view, and variations of the process now to be described 
 will be mentioned under their appropriate headings. We will 
 suppose that we have to examine a specimen of pus, and wish to 
 make a stroke culture on agar and a stab culture in gelatin. The 
 following must be at hand : 
 
 1. The pus. 
 
 2. A sloped agar tube and a stab gelatin tube. 
 
 3. A Bunsen burner or a spirit-lamp with a tall flame. 
 
 4. A pair of dissecting forceps. 
 
 5. Platinum needles. Each needle consists of a piece of 
 platinum wire about 3 inches long mounted in the axis of a glass 
 
 Fig. 8. — Platinum Needles. 
 
 rod about 6 or 8 inches in length. The wire should be just thick 
 enough not to bend too easily. They are easily prepared. The 
 rod is selected, and the length of platinum wire is held in an 
 ordinary pair of forceps. The end of the glass rod is held in the 
 flame until quite soft ; the end of the wire is then heated to 
 redness, and pushed into the rod to the depth of about J inch, 
 taking care that it is kept in the axis. The whole is allowed 
 to cool, and is ready for use. 
 
 For some purposes we use needles which terminate in a small 
 loop, so that they will retain a drop of fluid. These are prepared 
 in the same way as the straight needles, the free end of the wire 
 being subsequently twisted round a French nail or other suitable 
 object. 
 
 The method is as follows : 
 
 I. Hold the culture-tube you are going to inoculate first between 
 the index and middle fingers of the left hand, pointing the mouth 
 
INOCULATION OF CULTURE MEDIA 17 
 
 of the tube slightly downwards (so that no dust shall drop into 
 it) and to the right. Tubes of solid media should always be held 
 in this position during inoculation ; tubes of liquid media are held 
 in a similar way, but of course their mouths must point upwards. 
 
 2. Put the projecting portion of the cotton-wool plug of the test- 
 tube into the flame so as to singe it ; this is to destroy any germs 
 which may have been deposited upon it from the atmosphere. 
 
 3. SteriHze the points of the forceps by passing them slowly 
 through the flame, and then use them to remove the plug. Place 
 this between the ring and little fingers of the left hand, and put 
 the forceps down. 
 
 4. Take the platinum needle in the right hand, heat the whole 
 of the wire to redness, and pass the lower 3 or 4 inches of 
 
 Fig. 9. 
 
 the glass rod slowly through the flame. Remember that every 
 portion of the needle which goes inside the tube must be sterilized 
 in this way. Allow the needle to cool ; you should have found 
 out how long this will take by a previous experiment. 
 
 5. Dip the tip of the needle into the pus ; pass it into the tube 
 until it reaches nearly to the bottom of the tube (now uppermost), 
 and allow it to rest upon the sloping surface of the medium ; now 
 withdraw it gently, allowing the tip of the wire to trail gently 
 along the whole length of the sloped surface. Do not touch the 
 medium with the glass shoulder of the needle. 
 
 6. Sterilize the needle as before. This step must never be 
 forgotten. 
 
 7. Take the cotton-wool plug in the forceps, put it in the flame, 
 and singe all parts of its surface. Then plug the tube while 
 the wool is still burning. Label it. 
 
 To make a stab culture take the other gelatin tube and proceed 
 as before until you get to step 5. When you have passed the 
 
 2 
 
l8 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 needle into the tube drive it steadily into the medium, taking care 
 not to deviate from the axis of the tube. Finish the process as 
 before. 
 
 All this may seem involved. As a matter of fact it is very 
 simple, and need not take more than a minute to perform. But 
 every step must be carried out, and the whole process must be 
 learnt so thoroughly that it is performed automatically whenever 
 a culture is made. 
 
 INCUBATION OF CULTURES 
 
 The limits between which bacteria can live are very wide ; 
 some grow best at one temperature, others at another, the limits 
 for the great majority of organisms being about i6° C. and 40° C. 
 In practice two temperatures are all that is used for ordinary 
 work. The lower, or so-called " room temperature," is about 
 20° C. (68° F.), and is of most use for those bacteria which grow 
 naturally outside the body — i.e., as saprophytes. The higher, or 
 body temperature, is about 37° C. (98-6 F.), and is the best 
 temperature for the majority of germs which live within the 
 body — i.e., the parasites. It is obvious that gelatin cannot be 
 incubated at this high temperature, as it melts at 25° C. or 
 thereabouts ; but all other media are available. 
 
 The term " room temperature " must not mislead the practi- 
 tioner, for the temperature of many rooms is not constant at or 
 near 20° C. for periods sufficiently long to permit of its use for 
 incubating bacteria. In the laboratory we use an incubator, the 
 temperature of which is regulated by means of an automatic 
 regulator, and remains constant for long periods whatever be the 
 external temperature. It is hardly necessary for the practitioner 
 to purchase one of these. Careful search in the house will 
 usually reveal some cupboard or corner in which the temperature 
 will remain sufficiently near 20° C. for a sufficiently long period ; 
 it is more important that it should not rise above 22° C. than that 
 it should not fall below 18° C, as the former temperature may 
 melt the gelatin, while the latter will only delay the growth of the 
 colonies. It will probably be necessary to find one such place in 
 the hot weather {e.g., the cellar) and another one in the winter 
 {e.g., a cupboard not far from the hot-water pipes). 
 
 It is necessary that cultures which are being incubated should 
 
INCUBATION OF CULTURES 
 
 19 
 
 be kept in the dark, as light is inimical to the development of 
 nearly all bacteria. 
 
 Incubation at the body temperature presents more difficulty. 
 An incubator is almost essential when much work has to be done, 
 and of these Hearson's is by far the best. The smallest size costs 
 about £6 los., and is a thoroughly reliable and durable affair. 
 Cheaper instruments are also procurable: the "Edinburgh" 
 incubator (Alex. Fraser, 22, Teviot Place, Edinburgh) costs about 
 
 Fig. 10. — Incubator. 
 
 £^ I OS. complete. Foreign incubators can be obtained at an even 
 lower price, but are not very durable. 
 
 Much can be done without the use of so expensive an apparatus 
 if the practitioner can find a room in which the temperature keeps 
 approximately constant throughout the twenty-four hours. A tin 
 biscuit box (or any other metal box) is covered with cotton-wool 
 on the top and sides, the bottom being left bare, and mounted on 
 a tripod stand. It is heated by means of an ordinary night-light 
 
 2 — 2 
 
20 
 
 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 (two may be necessary if the weather is cold) shielded from 
 
 draughts by means of a wide lamp-chimney or a tin cylinder 
 
 made out of an ordinary canister. The temperature is observed 
 
 by means of a thermometer projecting through a hole in the lid, 
 
 and the night-light raised or lowered until the temperature reaches 
 
 the desired figure. The whole apparatus should be placed on a 
 
 metal tray containing a small quantity of 
 
 Q water, and put in the middle of the floor, and 
 
 B away from any inflammable materials. This 
 
 yiz^l — Ti will be found to answer admirably, and can 
 
 y^ / ^ easily be fitted up in an emergency. 
 
 It would be better to use a tin box specially 
 made for the purpose, and having a door at 
 the side and a perforated false bottom, so that 
 the culture-tubes do not rest directly on the 
 metal exposed to the flame. This latter had 
 best be made of copper. 
 
 Dr. Bottomley, of Boscombe, informs me 
 that he has used the simple apparatus shown 
 in Fig. 1 1 for five years, and finds it answers 
 admirably in the diagnosis of diphtheria, etc. 
 It is an ordinary square vaseline tin, into the 
 round opening of which is put a cylindrical coffee tin. The 
 outer tin is filled with water, and a thermometer is put into a hole 
 through its lid. The culture-tubes are placed in the inner tin, and 
 the apparatus is heated by a paraffin lamp. 
 
 If the practitioner is fortunate enough to possess a conservatory 
 which is kept at a temperature approximating to that of the body, 
 this will serve admirably. The culture-tubes must be kept in a 
 box which will exclude light. 
 
 In the absence of this a cupboard near the kitchen fire or the 
 hot-water cistern may be found that will answer the purpose ; a 
 thermometer should be placed in it, and examined from time to 
 time throughout the day, and if the temperature does not fall 
 below 30° nor rise above 40°, it will serve at a pinch, though a 
 temperature which is more constant near 37° is desirable. It has 
 to be remembered that we are not now speaking of the incubation 
 of cultures for purposes of research ; we are dealing with methods 
 of cultivation which are necessary for diagnostic purposes, and 
 for these it is usually sufficient if the temperature remains nearly 
 constant at the proper point for some eighteen hours. 
 
 Fig. II. 
 
INCUBATION OF CULTURES 
 
 21 
 
 Another method I have recently adopted is to use a "Thermos " 
 flask, or, what comes to the same thing, a Dewar's liquid air 
 flask. This is nearly filled with water at a temperature of about 
 40° C. and the culture-tube inserted. If a Thermos is used the 
 cap is then applied, if a Dewar's flask a few drops of oil are 
 placed on the surface of the water (to prevent evaporation, which 
 greatly hastens the process of cooling). The water and culture- 
 tube cool very gradually, and most pathogenic organisms will 
 give an excellent growth before this happens. The Dewar's 
 flask should have a capacity of 600 c.c. and an internal diameter 
 at the neck of i inch or so. It can be supported on a jam-pot 
 with a layer of cotton-wool round the neck. I find it cools at the 
 rate of about half or two-thirds of a degree per hour. 
 
 Fig. 12. — Dewar's Flask adapted for Use as Incubator. 
 
 a, Culture tube ; b, vacuum ; c, water ; d, layer of oil ; e, cotton-wool round 
 mouth of jar for support. 
 
 The author once succeeded in making a diagnosis in a case of 
 supposed diphtheria by the following method : The tube of 
 medium was inoculated from the throat, and placed in a jam-pot 
 which was partly filled with water at 37° C. ; a thermometer was 
 also inserted, and the whole placed near the fire. It was watched 
 from time to time, and moved near the fire if the temperature 
 showed signs of falling, and vice vevsa^ until the proper position 
 was found. Next morning there was an excellent growth, and 
 the diagnosis was made with certainty. 
 
 Lastly, the author has heard of a practitioner who was in the 
 
22 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 habit of incubating cultures at the body temperature by carrying 
 them in an inner pocket during the day, and taking them to bed 
 with him at night \* 
 
 METHOD OF EXAMINING CULTURES 
 
 Requisites. — i. Clean slides and cover-glasses. (These must 
 be the thinnest in ordinary use — i.e.^ No. i.) 
 
 2. A platinum needle (straight or loop). 
 
 3. A Bunsen burner or a spirit-lamp with a tall flame. 
 
 4. The stain to be employed (see p. 28). 
 
 5. Canada balsam dissolved in xylol. This should be bought 
 ready for use. 
 
 6. A pair of dissecting forceps. 
 
 7. Strips of white blotting or filter paper. 
 
 Process. — i. Sterilize the needle, and place a small drop of water 
 (preferably distilled) in the centre of a clean slide. 
 
 2. Take the culture-tube in the left hand between the index and 
 middle fingers with its mouth directed to the right and (in the case 
 of a culture on a solid medium) slightly downward. 
 
 3. Burn the surface of the plug in the flame. Remove the plug 
 with the forceps (previously sterilized by being passed slowly 
 through the flame), and place it between the ring and little fingers 
 of the left hand. Lay the forceps down. 
 
 In cases where you are examining the culture for diagnostic 
 purposes only, and do not care if it becomes contaminated during 
 the process, it is unnecessary to take these precautions. The 
 cotton-wool plug may then be removed with the fingers and laid 
 down on the table. As a matter of fact, very few cultures do 
 become contaminated, even if no precautions are taken. 
 
 4. Sterilize the needle in the flame and allow it to cool. 
 
 5. Now introduce the needle into the tube, and take up a small 
 portion of the growth, taking care not to scrape up the surface of 
 the medium as you do so. Most beginners fall into the mistake 
 of taking up far too much of the growth, and preparing a film 
 which is spread so thickly that the individual bacteria cannot be 
 distinguished. 
 
 6. Take the plug up in the forceps, burn its surface in the 
 flame, re-plug the tube and lay it down. 
 
 * Since the above was written an incubator to be used in this way has been 
 invented. 
 
I 
 
 METHOD OF EXAMINING CULTURES 23 
 
 7. Stir the droplet of water which has been deposited on the slide 
 with the tip of the needle, so that the bacteria which it carries 
 are mixed with the water. Now spread out the emulsion thus 
 produced so as to form a patch about J inch in diameter. If it 
 does not spread out uniformly it is a sign that the slide is not clean. 
 
 8. Sterilize your needle. 
 
 g. Allow the film to dry spontaneously. If you have spread it 
 out sufficiently this will take a very short time. 
 
 10. Fix the film by passing the slide slowly through the flame 
 once or twice. This coagulates the albumin present, and the 
 bacteria are now fixed down so firmly that they will not be re- 
 moved by subsequent washing. The exact amount of heat which 
 should be used cannot be stated, as it varies according to the 
 thickness of the slide, etc., and can only be determined by practice. 
 It may be estimated roughly by pressing the finger upon the upper 
 surface of the slide close to the film, but not touching it. The 
 slide should be just uncomfortably warm to the finger, but not hot 
 enough to burn it. 
 
 11. Filter a few drops of the stain on to the surface of the film, 
 and allow it to act for the requisite time. Exact details will be 
 given in each case. 
 
 12. Next wash the slide under the tap, blot it with clean white 
 filter-paper, taking care to avoid rubbing, and warm it gently over 
 the flame until absolutely dry. 
 
 13. Place a drop of balsam on the film, and apply a clean dry 
 cover-glass. 
 
 The preparation is now ready for examination. 
 This also is a process 
 
 which sounds more iniif iiiillrl iiliiiiilli li illllll |||||||pi!l!i^^f 5 ) 
 
 complicated than it 
 
 really is. The steps 
 
 are readily learnt, and 
 
 the whole process (ex- Fig. 13. —Cornet's Forceps. 
 
 eluding the time spent 
 
 in staining, which may be long or short) does not take more than 
 
 two or three minutes. 
 
 Most bacteriologists make their films on cover-glasses and not 
 on slides. The steps are just the same, except that the cover- 
 glasses, being much thinner, naturally require much less heating 
 to fix the film ; they are passed rapidly through the flame three 
 times. It is a great advantage to use Cornet's forceps in working 
 
24 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 with cover-glasses. These forceps are self-retaining, and hold 
 the cover-glass in a horizontal position, so that stains can be 
 poured upon them whilst the forceps are resting on the table. 
 In using them put the cover-glass between the jaws of the 
 forceps, which must clip them a little distance from the corners, 
 otherwise the stain will run into the forceps. Make a point of 
 placing them on the table with the keyhole upwards, so that you 
 will always know on which side of the cover-glass the film has 
 been spread. But the staining can be carried out equally well 
 in a watch-glass, and the cover-glass may be held in dissecting 
 forceps. 
 
 It is far easier and more satisfactory in every way to make the 
 films on the slides. Beginners will find that they will break large 
 numbers of cover-glasses (which must be thin), drop more on the 
 floor, and will be in constant doubt as to which is the film side. 
 With slides these difficulties do not occur, and the use of forceps 
 is quite unnecessary. 
 
 It is not absolutely necessary to use cover-glasses in the 
 preparation of these bacterial films unless they are to be kept for 
 future reference, and except in such cases I have discarded them 
 for years. To examine the unmounted film it is only necessary 
 to dry it thoroughly, and to put the immersion oil (see p. 27) 
 directly on to the stained area. This method is especially useful 
 in searching for the tubercle bacillus, for no one wants to keep 
 films of tuberculous sputum after the diagnosis has been made, 
 and if the necessity did arise a drop of balsam can be placed on 
 the film (without removing the oil) and the cover- glass applied. 
 The one advantage of covering the films at once is that it permits 
 of the use of the low powers, and as this should always be done 
 by beginners, the covering of the preparations has been recom- 
 mended throughout. 
 
 GRAM'S METHOD OF STAINING 
 
 The method of staining described above is available for all 
 organisms, and therein consists its advantage. But other things 
 than bacteria are stained : pus cells, fragments of tissue, debris, 
 etc., will all be coloured, and may obscure, or even be mistaken 
 for, bacteria. Gram's method possesses the enormous advantage 
 that by its use the bacteria are coloured, while other structures 
 (with the exception of particles of keratin and dividing nuclei) 
 
I 
 I 
 
 gram's method of staining 25 
 
 are not. Hence in a film stained in such a way the bacteria are 
 very distinct. 
 
 Gram's method possesses another advantage. It is a selective 
 stain. Some bacteria retain the stain, whilst others do not, and 
 this fact is of great value in diagnosis. The diphtheria bacillus, 
 for instance, stains when treated in the way we shall describe, 
 and if an organism which presents the character of that bacillus 
 does not stain by the process it must be of some other species. 
 We shall append a table of the most important pathogenic 
 bacteria which stain and which do not stain in this way. 
 
 Requisites, — i. AniHne gentian violet, or carbol gentian violet 
 (see p. 30). 
 
 2. Gram's iodine solution (see p. 30). 
 
 3. Absolute alcohol or methylated spirit. 
 
 Process. — i. Spread, dry, and fix the film in the way described 
 above. Stain for two or three minutes in the aniline gentian 
 violet or carbol gentian violet. 
 
 2. Rinse in water to remove excess of stain and flood with the 
 iodine solution, and allow the latter to act for a minute. 
 
 3. Wash off the iodine solution with alcohol, and continue the 
 application of the latter until no more colour comes away. It is 
 best to pour a little alcohol on the slide and rock the latter from 
 side to side for a minute or so, then to pour off the alcohol and 
 add a fresh supply, and continue this until the alcohol comes off 
 colourless." 
 
 The Gram staining proper is then finished, but if the organisms 
 present do not stain by the method the result will be that every- 
 thing is decolorized, and microscopic examination will reveal 
 nothing. This, of course, might prove that the organism did not 
 stain by Gram, but it might also be due to some error in technique, 
 by which no organisms were put on the slide to begin with or got 
 washed off at a subsequent stage. To exclude this possibility 
 proceed to counterstain with some stain very different in colour 
 from the gentian violet : dilute carbol fuchsin is best. Stain in 
 this for a quarter of a minute — not more, for the stain is a very 
 powerful one, and if allowed to act too long may displace the 
 
 * Some books contain the absurd statement that the decolorization is to be 
 carried out until the film is a pale blue or violet colour. No such rule can be 
 given. If there is nothing that retains Gram the film will be colourless when 
 finished ; if it is a thick smear of an organism retaining the stain it may be 3, 
 very deep violet, 
 
26 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 stain from a Gram-staining organism : wash for a minute or so 
 under the tap, dry, and mount. Organisms which retain Gram's 
 stain will be violet, almost black, organisms which do not will be 
 red. Pus, blood, etc., if present, will also be red. This counter- 
 staining by means of a good contrast stain is really almost a 
 necessary sequel to Gram's method, and in practical diagnostic 
 work the two almost always go together. 
 Dry and mount as before. 
 
 The following important bacteria stain by Gram's method : 
 Staphylococci. 
 Streptococci. 
 Pneumococci. 
 Sarcinae. 
 
 The skin coccus (M. epidermidis). 
 The bacillus of anthrax. 
 
 „ diphtheria. 
 
 „ tetanus. 
 
 „ tuberculosis. 
 
 „ leprosy. 
 
 „ Boas-Oppler. 
 
 The various species of actinomycosis fungus or streptothrix. 
 The fungi of ringworm, favus, etc. 
 Yeasts. 
 
 The following important pathogenic bacteria do not stain by 
 Gram's method : 
 The gonococcus. 
 
 Diplococctts intracelhilavis meningitidis (Weichselbaum). 
 Micrococcus catavrhalis. 
 The pneumo-bacillus. 
 The Bacilhts coli communis. 
 The bacillus of glanders. 
 
 „ typhoid fever. 
 
 „ influenza. 
 
 plague. 
 „ soft sore. 
 
 The Bacillus fusiformis. 
 The vibrio of Asiatic cholera. 
 
 The spirilla or spirochsetes of relapsing fever, syphilis, and 
 Vincent's angina, and all other parasitic protozoa. 
 
k 
 
 EXAMINATION OF FILMS — USE OF MICROSCOPE 27 
 
 EXAMINATION OF FILMS— USE OF MICROSCOPE 
 
 Daylight is the best illuminant for microscopic work, and 
 the light reflected from a white cloud opposite the sun is 
 best of all. Direct sunlight is useless, but the light obtained 
 from a ground -glass window on which the sun is shining is 
 very good. 
 
 For work at night the light from an incandescent gas-burner at 
 a distance of 2 or 3 feet is excellent, but an ordinary paraffin 
 lamp will answer quite well. 
 
 Having arranged for a suitable source of light, turn the flat 
 mirror uppermost, and move it about until a beam of light is 
 thrown on to the condenser. Remember : 
 
 In examining stained specimens use a large diaphragm. 
 
 In examining tmstained objects use a small diaphragm. 
 
 You are now about to examine a stained specimen. Place the 
 slide on the stage, putting the stained film in the centre of the 
 aperture, and turn on the low power. Look down the eye-piece, 
 and move the mirror about until the field is brilliantly illuminated. 
 Focus the microscope (using the coarse adjustment) until the 
 image is clearly defined. Now move the slide about until there 
 is a deeply-stained area in the centre of the field. This area will 
 not necessarily be the best for examination with a higher power, 
 but it will serve to catch the eye when focussing the lenses which 
 focus at a short distance from the object. 
 
 Now turn on the high power (the \ inch). Remember that the 
 *' working distance " of all lenses is necessarily less than their 
 focal distance, and that a ^-inch lens focusses at a distance from 
 the object which is decidedly less than \ inch ; so also with the 
 other powers. Lower the lens until it almost touches the object, 
 and screw up the substage condenser as high as it will go. 
 Look down the microscope and focus sHghtly upwards, using the 
 coarse adjustment, until you catch a glimpse of colour ; then 
 focus very slowly until the object is sharply defined. 
 
 After a little practice you will be able to focus downwards on 
 to the film, keeping a sharp look-out for the first appearance of 
 colour, but for beginners the foregoing method is easier and 
 safer. 
 
 Study the object with the high power, and move it about until 
 you find an area where the bacteria are neither too thickly nor 
 
28 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 too thinly spread, and are well stained. Make out as much of 
 their appearance as you are able to do with this power. Very 
 much can be done ; tubercle bacilli, gonococci, and many other 
 bacteria, may be recognised with this power, and the peculiar 
 arrangement of diphtheria bacilli can be seen. 
 
 Apply the clips to keep the slide in place. 
 
 Now raise the tube of the microscope for a short distance, 
 using the coarse adjustment, and place a small drop of cedar oil 
 on the centre of the cover-glass. Lower the tube (using the 
 coarse adjustment) until the nozzle of the lens touches the drop 
 of oil ; then put your head on a level with the stage, and continue 
 to focus downwards, going very carefully, until the lens almost 
 touches the cover-glass. Next look down the microscope, and 
 focus upwards, using the fine adjustment, until you begin to see 
 colour ; then go more slowly until the film is well defined. 
 
 Beginners are strongly urged to adopt this method of focussing 
 an oil-immersion lens until they have acquired a considerable 
 amount of practice. It takes a little time, but this is well repaid 
 by the absence of all danger or injury to lens and cover-glass. 
 After a time you may lower the lens until it touches the oil, and 
 then look down the microscope and continue to lower it with the 
 fine adjustment. 
 
 After use, wipe the front of the immersion lens with a soft silk 
 handkerchief kept specially for the purpose, and put the microscope 
 back into its case. If oil or balsam should get dried on the lens, 
 wipe it with a handkerchief just moistened with xylol or pure 
 turpentine, and then wipe quickly with a dry handkerchief. 
 Never dip the point of a lens into xylol or alcohol. Never 
 remove the front combination of an oil-immersion lens for clean- 
 ing or any other purpose. 
 
 STAINS 
 
 The following stains are all that is really necessary for the vast 
 majority of purposes : methylene blue, basic fuchsin, gentian violet, 
 thionin, and water-soluble eosin. Bismarck brown may also be 
 obtained. Ten grammes of each will last the practitioner for a 
 long time, and this amount costs from yd. to is. They should be 
 of Griibler's make, and can be obtained from Messrs. Baker, 
 Holborn ; Baird and Tatlock, Hatton Garden ; J. J. Griffin, 
 Kingsway, W.C. ; A. Fraser, Teviot Place, Edinburgh; and from 
 
STAINS 29 
 
 Messrs. Southall or Philip Harris, Birmingham. Other firms 
 will also supply stains, but Griibler's should always be specified. 
 
 They are conveniently kept in a saturated solution of absolute 
 alcohol. The following formulae are the most useful : 
 
 1. A Saturated Watery Solution of Methylene Blue. — This does not 
 keep very well, and a fresh amount should be prepared after a 
 month. It is mostly used for staining blood-films, and for staining 
 wet specimens of cells from the pleura, pericardium, etc. ; borax 
 methylene blue will serve every purpose in bacteriological work. 
 Instead of this I now employ acid methylene blue (No. 9). 
 
 2. Loffler^s methylene blue is prepared by adding 30 c.c. of a 
 saturated solution of methylene blue (alcoholic) to 100 c.c. of 
 a I in 10,000 solution of caustic potash. 
 
 The potash solution is prepared thus : Take i c.c. of a 10 per 
 cent, solution of caustic potash and make up to 100 c.c. with 
 water ; shake thoroughly and pour away 90 c.c. ; make up to 
 100 c.c. with water, and again shake. A sufficiently close ap- 
 proximation is made by adding i minim of the 10 per cent, 
 solution to 2 ounces of water. 
 
 This stain keeps fairly well. 
 
 3. Hanson's borax methylene blue, which I now use in place ot 
 Loffler's blue and find much preferable. A stock solution (which 
 keeps well) is prepared by dissolving 2 grammes of methylene 
 blue and 5 grammes of borax in 100 c.c. of water. It is to be 
 diluted with five to ten times its volume of water for use. It 
 may be used whenever Loffler's blue is recommended. In 
 English measures it is methylene blue, grs. xxx. ; borax, grs. Ixxv. ; 
 aq., giii.ss. 
 
 4. Cavbol fuchsin is made by adding a saturated alcoholic solution 
 of fuchsin to carbolic acid lotion (i in 20) until the fluid has lost 
 its transparency, or i part of the saturated solution to 9 of the 
 carbolic acid. This keeps well. 
 
 The following is another formula : 
 
 Fuchsin, i gramme, or grs. xv. 
 I in 20 carbolic, 100 c.c, or giii.ss. 
 Glycerin, 50 c.c, or 3xiv. 
 
 5. The above stain diluted with four or five times its volume of 
 water. Label " Dilute Carbol Fuchsin." It is usually prepared 
 by diluting a little strong carbol fuchsin when required. 
 
30 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 6. A niline gentian violet ^ which is prepared as follows : 
 
 First prepare aniline oil water by shaking water (preferably dis- 
 tilled) with more aniline oil than it will dissolve ; a milky emulsion 
 will result, and this must be allowed to settle for a short time. 
 Then filter it through a double thickness of filter-paper which 
 has been previously moistened with water. 
 
 To 9 parts of the solution thus obtained add i part of saturated 
 solution of gentian violet in alcohol. 
 
 This solution keeps badly, and it is necessary that it should be 
 freshly prepared, as very important inferences are drawn from 
 results obtained with it. The following keeps better, and answers 
 every purpose. 
 
 Carbolic Gentian Violet (a substitute for aniline gentian violet). — 
 Add I part of saturated alcoholic solution of gentian violet to 
 9 parts of a I in 20 carbolic lotion. 
 
 7. Carbol thionin is made by adding i gramme of thionin to 
 100 c.c. of a I in 40 solution of carbolic acid. 
 
 This stain keeps fairly well, but it must always be filtered 
 immediately before use, as crystals which may have a most 
 delusive resemblance to long slender bacilli are frequently de- 
 posited in it. A similar formation of crystals also occurs if the 
 stain be allowed to dry on the slide. 
 
 In cold weather the thionin may crystallize nearly completely out, 
 and the fluid stain very badly. If kept in a warm place for a few 
 hours it will recover its properties, the sediment being redissolved. 
 
 8. Eosin is used in a 4 or 5 per cent, watery solution. This 
 keeps well. Red ink (slightly diluted) will answer most purposes. 
 
 9. Acid methylene blue is prepared by mixing 2 parts of borax 
 methylene blue (vide supra) , i part of glacial acetic acid, and 7 parts 
 of water. It is only used for staining cells, casts, etc., in wet 
 preparations, and has the advantage of dissolving the red blood- 
 corpuscles, which when numerous often obstruct the view of the 
 more important elements. It keeps well. 
 
 Stains should be filtered before use. Where much work is to 
 be done, it is convenient to keep them in bottles which are closed 
 with a perforated cork through which a small glass funnel is 
 placed. A filter-paper is kept permanently in this funnel, and the 
 stain is filtered directly on to the slide or cover-glass. 
 
 Gram's iodine solution may be mentioned here, though it is not a 
 stain. It consists of a solution of iodine, i part ; iodide of potassium, 
 2 parts ; water, 300 parts. It keeps indefinitely. 
 
I 
 
 CLEANING SLIDES AND COVER-GLASSES 3I 
 
 CLEANING SLIDES AND COVER-GLASSES 
 
 Slides and cover-glasses must be absolutely clean when used in 
 the bacteriological laboratory ; it is especially necessary that they 
 should be free from the slightest trace of grease, for this will 
 prevent fluid from spreading out into a thin and uniform film. 
 
 Slides are best cleansed by dropping them one at a time into 
 strong nitric or sulphuric acid, and allowing them to soak for an 
 hour or more. They are then washed in running water for 
 another hour, soaked in strong ammonia for an hour, and kept in 
 alcohol. As methylated spirit is not so good for this purpose, the 
 use of alcohol is rather expensive ; but it is not absolutely 
 necessary, and the slides may be stored in a solution of ammonia 
 (about I in 10) until required. When about to be used, they are 
 to be wiped dry with an old linen handkerchief kept specially for 
 the purpose. This handkerchief should be as old as possible, and 
 should have been washed until it has begun to fall to pieces. 
 
 Another and more rapid method is to place the slides in a thin 
 glass or earthenware vessel and moisten them with methylated 
 spirit, and then to cover them completely with strong commercial 
 nitric acid, placing the vessel in the open air. In a little while 
 the acid will become very hot and emit copious fumes. When 
 the ebullition has ceased, any fat which may be present will have 
 melted, and will form a pellicle on the surface, whilst other organic 
 materials will have been destroyed. The acid is then to be poured 
 off, taking care to remove the pellicle of grease with it, and a 
 stream of water allowed to fall into the vessel until the acid has 
 been washed away. The slides (and the method is a good one for 
 cover-glasses also) may then be placed in absolute alcohol or 
 methylated spirit, and only require drying to be ready for use. 
 
 Another method which I have recently used and found excellent 
 is to boil them in a 5 per cent, solution of lysol ; they may be 
 allowed to remain in this fluid until required, and are then 
 polished with a handkerchief — and it may be pointed out that 
 thorough friction is a sine qua non whatever method of cleaning be 
 adopted. Slides and cover-glasses after use may be soaked in the 
 lysol solution, a pot of which should be kept at hand for the 
 purpose ; they will be sterilized and partially cleaned in a few 
 hours. 
 
 When no properly-cleaned slides are at hand, the following 
 method may be adopted, though it is not so good : Dip the end 
 
32 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 of a clean handkerchief in strong spirit (absolute or rectified) and 
 wipe the slide with it, using a considerable amount of friction. 
 Now dry it with the special handkerchief mentioned above, heat 
 it thoroughly in a smokeless flame, and allow to cool completely. 
 Spread the film on the surface which was exposed directly to the 
 flame. 
 
 Cover-glasses are cleaned in the method advised for slides, and 
 should be stored in strong alcohol smelling strongly of ammonia. 
 They are wiped with the special handkerchief immediately before 
 use. 
 
 When cover-glasses are to be used for covering films spread 
 upon slides (as is generally the case if the method recommended 
 in this book is adopted) it is quite sufficient to wipe them carefully 
 with a clean handkerchief moistened with spirit, and then to dry 
 them. 
 
 After slides or cover-glasses have been cleaned, the utmost care 
 must be taken that they do not come in contact with the skin, or 
 a thin film of grease will be deposited upon them. 
 
 PIPETTES 
 
 Glass pipettes for the collection of pathological fluid for 
 bacteriological examination are very frequently required. They are 
 readily made from a piece of quill glass tubing, and a few should 
 always be kept in stock against emergencies. 
 
 One form consists of a bulb about J inch long, each end of 
 which is drawn out into a narrow tube at least 6 inches long, 
 tapering gradually to the extremities (Fig. 14, a). To make such 
 a pipette, take a piece of glass tubing about 6 inches long and a 
 J inch wide, and heat it in a luminous gas flame at a point J inch 
 or so from the centre. Continue the heat until the glass is 
 thoroughly softened over at least J inch of its length, turning 
 the tube round all the time ; then remove it from the flame 
 and draw the two ends apart with a steady, uniform pull, so 
 that the heated portion draws out into a capillary tube several 
 inches in length. Repeat the process at a point about -J inch 
 from the tapering end of the larger portion of the tube ; heat the 
 bulb, and then seal off both ends of the capillary portion before 
 the bulb cools. 
 
 The pipettes are necessarily sterile, having been drawn out of 
 partially melted glass, and they will remain sterile indefinitely. 
 
PIPETTES 33 
 
 Of course, the exterior of the glass will become contaminated, 
 and it should be passed through the flame before use. 
 
 The ends of the tube being sealed up while the bulb contains 
 heated air, it follows that the bulb will contain a partial vacuum 
 on cooling. This fact is made use of in the collection of 
 specimens. Suppose, for instance, we wish to take some blood 
 from a heart at a post-mortem examination for investigation at a 
 distance. A point on the surface of the heart is first seared with a 
 hot iron to destroy any germs which might be present, and the 
 end of the pipette (still sealed) is thrust through into one of the 
 cavities. It is then broken off by dexterous pressure against the 
 heart wall, and the pipette will fill slowly with the blood. Another 
 
 D 
 
 c 
 Fig. 14. — Pipettes. 
 
 method is to break off the tip of the pipette and to warm the 
 bulb before making the puncture. The fluid will rise as the 
 bulb cools ; or both ends may be broken up and the fluid drawn 
 into the bulb by gentle suction. 
 
 Under any circumstances both ends of the pipette must be sealed 
 up in a flame (the flame of a wax match will answer at a pinch), 
 and the tube labelled. 
 
 Another variety of pipette which was much used for the collec- 
 tion of blood for the purpose of testing the serum reaction for 
 typhoid fever, is drawn out to a point at one end only, the other 
 being left wide and separated from the bulb by a constriction 
 (Fig. 14, b). The open end should be loosely plugged with 
 cotton-wool, and serves as a mouthpiece. The manufacture of 
 these pipettes presents a little difficulty, but a small amount of 
 practice will enable the practitioner to turn out a perfectly 
 serviceable one on occasion. 
 
 Wright's blood capsules (Fig. 15) are the best contrivances for 
 collecting blood when the serum has to be examined, and have 
 quite replaced the pipettes described above. They can be pre- 
 
 3 
 
34 
 
 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 pared easily after a little practice, or may be bought from 
 R. B. Turner, ii, Foster Lane, E.G., at a moderate price. 
 
 A Wright's blood capsule consists of a piece 
 of glass tubing which is drawn out straight to a 
 narrow point at one end, whilst the other, also 
 drawn out to a point, is curved round parallel 
 with the main tube in the shape of a U (Fig. 15). 
 To use it, proceed as follows : Prick the patient's 
 ear, or, if you prefer it, the finger (after the 
 application of a wide indiarubber band), and 
 squeeze out a large drop of blood. Place the 
 tip of the curved end of the pipette in this drop, 
 holding the pipette with the straight end point- 
 ing upward (Fig. 16), and you will find that the 
 blood will run rapidly into the curved tube by 
 capillary attraction ; continue to squeeze out 
 more blood and to suck it up until you have 
 collected as much as you want. It should be 
 quite easy to get the pipette half full. Then gently warm the 
 tapering portion of the straight end of the pipette in a spirit-lamp 
 or match-flame, and afterwards seal the tip (Fig. 17). As the air 
 
 Fig. 15. (About 
 § full size. ) 
 
 which is now imprisoned in the upper part of the pipette contracts 
 it will suck the blood from the curved limb into the body of the 
 pipette, which can be inverted and the blood shaken into the tip, 
 but take care not to do this until the glass is coldj as the serum loses 
 some of its properties when heated. If you are not going to 
 
PIPETTES 
 
 35 
 
 examine the serum for some time seal the other end to prevent 
 evaporation. 
 
 When the blood has coagulated it will begin to contract away 
 from the sides of the tube, the serum being forced out of the clot. 
 When this process is complete there will be a central dark- 
 
 #^c^ 
 
 Fig. 17. 
 
 Fig. 18. 
 A, Before coagulation : B, after, showing clot suspended in clear serum. 
 
 coloured clot suspended in clear serum. The tube can now be 
 notched with a file and broken, and the serum removed with a 
 pipette and used for Widal's reaction, the estimation of the 
 opsonic index, etc. If much serum is required, the pipette can 
 be hung by its crook (straight end downward) in the bucket of a 
 
 3—2 
 
36 CLINICAL BACTERIOLOGY AND H^MATOLOGi^ 
 
 centrifugal machine and centrifugalized. In this way the clot is 
 driven to the bottom and a large crop of serum obtained. 
 
 Since some practitioners seem to have difficulty in collecting 
 the quantity of blood desirable for the opsonic and other tests, a 
 few more notes on the process may be added. I personally prefer 
 to obtain the blood from the ear, and proceed as follows : the 
 lobe is well rubbed^by means of a piece of lint, until it is markedly 
 hyperaemic, and is then punctured on its lower border with a 
 Hagedorn's needle or platino-iridium hypodermic needle (previously 
 sterilized in the flame). This is done by a short, sharp "jab," 
 and should not be felt as pain at all ; I have frequently done it 
 without waking a sleeping child. The needle is then laid down, 
 and if the blood flows out in large drops (as usually happens 
 if the ear has been well rubbed) it is collected without the 
 slightest difficulty, and this is specially likely to be the case if 
 you puncture the ear of the side on which a patient has been 
 lying. If it does not flow readily, take the lobe of the ear 
 between the forefinger and thumb of both hands, and squeeze it 
 gently so that all the blood in the lobe is forced through the 
 puncture, and collect the drop thus formed. When this has 
 been done the lobe of the ear will be full of blood again, and a 
 second drop can be milked out. Repeat this process as often as 
 necessary. There are few patients from whom several pipettes 
 half full cannot be collected from a single slight puncture. 
 
 Many pathologists prefer the finger. In that case an excellent 
 needle can be made by softening a small piece of capillary tubing 
 in the flame and separating the two ends rapidly. This will give 
 two tapering tubes, which can be broken off so as to leave a very 
 sharp point, with which the skin at the side of the nail can be 
 punctured almost painlessly. The ligature is then applied as in 
 the figure, and the patient bends his finger forcibly, squeezing 
 out two or three drops of blood. After these have been collected 
 the bandage must be removed and the hand allowed to hang 
 down, so that the finger refills with blood. It is rebandaged and 
 rebent, and more blood obtained. This process should be learnt, 
 since it is the simplest one by which the practitioner can obtain 
 blood from himself, as is often necessary in opsonic work. 
 
SECTION II 
 DIAGNOSIS OF CERTAIN DISEASES 
 
 DIPHTHERIA 
 
 Diphtheria is a local disease with general symptoms. The local 
 symptoms, are due to the local action of the bacillus which causes 
 the disease, while the general symptoms are due to the toxin or 
 poison which they produce, and which is carried in the blood- 
 stream to the brain, heart, and other organs. Now, the local 
 symptoms are comparatively unimportant, and it is to the general 
 symptoms caused by the toxin that diphtheria owes the greater 
 part of its high mortality. Diphtheria antitoxin neutralizes this 
 toxin (much in the same way as an alkali neutralizes an acid), 
 and prevents it from harming the vital structures ; but it does not 
 repair the harm that the toxin has done. It is obvious, therefore, 
 that we must not make our diagnosis of diphtheria from the 
 general symptoms if the antitoxin treatment is to do any good. 
 The diagnosis is to be made from the local symptoms, and this is 
 what we can rarely do by ordinary clinical methods at a stage 
 sufficiently early to get the full value of the antitoxin treatment. 
 
 The practitioner has a choice of two methods. He may inject 
 all patients who suffer from sore throats which present the slightest 
 resemblance to those seen in diphtheria, or he may employ 
 bacteriological methods of diagnosis. The former method may be 
 applicable in an epidemic of diphtheria, but suspicious throats are 
 common and antitoxin expensive. In most cases it is necessary 
 to have recourse to the second method. 
 
 Most sanitary authorities have now recognised that it is their 
 duty and privilege to provide for the bacterial investigation of 
 supposed diphtheria free of charge to doctor and patient, and 
 supply outfits to be used for taking the material and transmitting 
 it to the laboratory. When the practitioner lives within easy 
 
 57 
 
38 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 reach of the laboratory (so that the swabs may reach it quickly) 
 it is his bounden duty to avail himself of the opportunity thus 
 afforded of getting a free opinion from a specialist. 
 
 But the case of a practitioner living at a distance is somewhat 
 different. Suppose the patient is seen on the first day of the 
 illness, an unusual occurrence except in an epidemic. The swab 
 is taken, despatched by post, and reaches the laboratory on the 
 second day. It is inoculated, and the culture is incubated and 
 examined on the third day, the result reaching the practitioner 
 about noon on that day. Now, the mortality of diphtheria which 
 is treated with antitoxin on the first day is very small — certainly 
 less than 5 per cent. — while the mortality in cases in which its 
 use is not commenced until the third day is much higher — probably 
 from 10 to 15 per cent., or even higher. In other words, from 
 five to ten patients out of every hundred lose their lives if the 
 doctor waits for the result of the bacteriological examination. 
 It is therefore highly advisable that every practitioner should 
 provide himself with a bacteriological microscope, and should at 
 least examine a film prepared directly from the swab and stained 
 in the manner described below. He should also make cultures or 
 send a swab to the laboratory for examination. 
 
 Swabs and outfits are provided by the laboratory where the 
 ^examination is made, or can be bought from most manufacturing 
 chemists and instrument-makers. A swab consists of a steel or 
 copper (aluminium would be better) wire, the extremity of which 
 is covered drumstick fashion with a tightly-fitting roll of cotton- 
 wool. The other end is pushed through a cork, and the whole is 
 contained in a stout glass tube. It is sterilized before use. These 
 swabs may be readily made at home. A test-tube is fitted with 
 a good cork through which is passed a stout steel knitting-needle. 
 This should be long enough to pass nearly to the bottom of the 
 tube when the cork is in place, and the end which is to be 
 outside the tube should be cut off short. The other must be 
 roughened by a few strokes of a file. A small piece of cotton- 
 wool (unmedicated) is then held between the thumb and finger of 
 the left hand, transfixed with the roughened end of the wire, and 
 twisted round it. The swab is now placed loosely in the tube and 
 sterilized by dry heat (see ante, p. 7). It is allowed to cool in the 
 sterilizer, and the cork is pushed home into the tube as soon as it 
 is cold enough to handle. These swabs will keep indefinitely, 
 and a stock of them should always be kept at hand, as they are 
 
DIPHTHERIA 39 
 
 very handy for other purposes. After use the cotton-wool should 
 be burnt off in a Bunsen burner or spirit-lamp, and another 
 piece appHed and the whole resterilized. 
 
 If a practitioner should see a supposed case of diphtheria when 
 he is unprovided with a swab he can readily extemporize one 
 which will answer sufficiently well out of some cotton-wool (non- 
 medicated), a wooden skewer or pen-holder, and a glass phial. 
 The wool is wrapped round the tip of the skewer, and (after the 
 swab has been taken) the latter is placed in the phial in such a 
 position that the cotton-wool does not touch the glass ; the place 
 between the skewer and the neck of the bottle is plugged with 
 cotton-wool. It is not absolutely necessary to sterilize the swabj 
 although it is a very great advantage to do so if time permits. 
 
 The method of taking the swab is of great importance, and must 
 be carried out in full detail. It is necessary that the patient 
 should not have had an antiseptic gargle or application for at 
 least two hours previously. It is also advisable to allow him to 
 drink some beef-tea or boiled water {not milk, for this may contain 
 certain bacilli which closely resemble those of diphtheria) imme- 
 diately before the process. This will serve to cleanse the parts. 
 
 Requisites. — i. A good light. 
 
 2. The swab in its tube. 
 
 3. A tongue depressor. The form which is hinged so as to 
 bend at a right angle is most convenient. 
 
 4. A vessel containing antiseptic lotion or boiling water. 
 Method. — I. Place the patient so as to face the light. If a 
 
 small child, he should be held on his nurse's lap, with a blanket 
 wrapped round his chest so as to restrain his arms. 
 
 2. Loosen the cork in the tube so that the swab may be with- 
 drawn with one hand, and place it at a convenient spot on your 
 right side. 
 
 3. Get the patient to open his mouth, insert the tongue 
 depressor (held in the left hand), and get a clear view of the area 
 chiefly affected. Do not proceed with the process (if it can be 
 avoided) until you have done this. 
 
 4. Take the cork between the finger and thumb of the right 
 hand and pass the swab into the patient's mouth, taking great 
 care not to touch his lips, tongue, or palate. Press it firmly 
 against the area which you wish to examine, and rotate it between 
 the finger and thumb so as to remove some of the secretion, and, 
 if possible, some of the membrane. Withdraw the swab, again 
 
40 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 taking care not to touch any part of the mouth, and replace it in 
 the tube. 
 
 5. Withdraw the tongue depressor, and place it in the anti- 
 septic lotion or boiling water. 
 
 6. Push the cork home into the tube. 
 
 Method of examining the Swab. — This may be carried out by means 
 of stained films, prepared directly from the swabs, or by means 
 of cultures. The former method is less useful than the latter, 
 but we shall consider it first, as it can be performed by anyone 
 who possesses a microscope carrying a yV^nch oil-immersion 
 lens, and often gives valuable information. Moreover, it does 
 not take long, and but little delay is caused. 
 
 Requisites. — i. Clean slides and cover-glasses. 
 
 2. Stains. — Loffler's blue or carbol thionin, and aniline gentian 
 violet. 
 
 3. Gram's iodine solution and alcohol — methylated spirit will do. 
 
 4. Strips of white filter or blotting paper. 
 
 5. Balsam. 
 
 Method. — Prepare a film in the following way : Rub the swab 
 on the middle of a clean slide, so as to spread some of the secretion 
 into a thin layer on the surface. Allow it to dry, and fix it by 
 passing it slowly through the flame, until the upper surface is just 
 too hot to prevent your pressing your finger upon it in comfort. 
 Allow it to cool. 
 
 Now filter a few drops of Loffler's blue or carbol thionin on 
 to the film, and allow it to act for two minutes. Wash under 
 the tap. 
 
 Dry by pressing carefully with strips of blotting-paper, and 
 then in the flame. Place a drop of balsam upon the film, and 
 apply a cover-glass. 
 
 Prepare a second film, and stain by Gram's method (p. 24), 
 counterstaining by dilute carbol fuchsin. 
 
 The films are now examined microscopically (see p. 27). We 
 shall defer the description of the points upon which a diagnosis 
 is to be based until we deal with the examination of cultures. 
 
 Cultural Methods. 
 
 The diphtheria bacillus grows best at or near the body 
 temperature (about 37° C), and flourishes on almost all culture 
 media. But agar is scarcely ever used in growing it for diagnostic 
 
DIPHTHERIA 4I 
 
 purposes ; this medium serves well for the cultivation of a great 
 many organisms, some of which are almost always present in the 
 mouth, so that cultures made upon it are usually very impure. 
 We use a medium which permits the development of the diphtheria 
 bacillus, and inhibits that of most other organisms. The best is 
 solidified blood-serum. ^ 
 
 The method in which the medium is inoculated is as follows : 
 The tube of culture medium, and the tube containing the swab, 
 are held side by side between the index and middle fingers of the 
 left hand, the mouths of the tubes pointing to the right and 
 slightly downwards. The plug of the culture-tube is then singed, 
 removed by means of a pair of forceps, and placed between the 
 ring and little fingers of the left hand. The cork and wire of the 
 swab-tube are now withdrawn, and the cotton-wool plug is inserted 
 into the culture-tube, and passed onwards until it reaches the 
 sloped surface of the medium. It is then rubbed gently on the 
 latter, and twisted round and round so that every part of the swab 
 may come into contact with the medium. If there is a piece of 
 membrane special care should be taken to see that it is rubbed 
 on the surface, for it is here that we are most likely to find 
 the bacilli. The swab is now withdrawn and replaced in the 
 tube, and the cotton-wool plug of the culture-tube singed and 
 replaced. 
 
 The tube thus inoculated must now be incubated for about 
 eighteen hours at a temperature not exceeding 37° C, and is then 
 ready for examination. 
 
 Hewlett has suggested a useful method which may be carried 
 out without any special apparatus, the white of a hard-boiled egg 
 being used as the culture medium. Take a fresh egg and boil it 
 for ten minutes or more, and allow it to cool. Now take a 
 narrow-mouthed wineglass (or a wide-mouthed bottle, which is 
 better), and rinse it out with perchloride of mercury lotion. 
 Sterilize a knife by passing it slowly through the flame, and cut 
 off the top of the egg, care being taken not to cut into the yolk. 
 Invert the egg into the wineglass (which must be narrow enough 
 to prevent the egg from dropping down into it), taking care that 
 none of the lotion touches the cut surface. This is the culture 
 medium, and it is sterilized ready for inoculation. At a pinch 
 it may be incubated in a warm corner near the fire, near the hot- 
 water cistern, or other warm place. 
 
42 CLINICAL BACTERIOLOGY AND H^EMATOLOGY 
 
 Examination of the Cultures. 
 
 1. Naked-eye. — Each living diphtheria bacillus which has been 
 deposited upon the surface of the culture medium and kept at a 
 suitable temperature will develop into a colony of bacilli ; and 
 these colonies are fairly distinctive, being different from those 
 which are formed by most other organisms. The expert 
 bacteriologist can often give an accurate guess as to the presence 
 or absence of diphtheria bacilli by mere inspection of the 
 cultures. The colonies formed by diphtheria bacilli on solidified 
 blood-serum are small round raised spots ; they are variable in 
 size, but rarely exceed that of the head of a medium-sized pin. 
 They are white or grey in colour, and opaque. They do not 
 tend to run together so as to form a uniform film over the 
 surface of the medium, but remain discrete even when closely 
 packed. Some cocci form colonies which closely resemble those 
 of diphtheria, but they rarely become elevated so high above the 
 surface in the same space of time. 
 
 2. Microscopical. — Prepare films by the method described on 
 p. 22 following out all steps in the fullest detail. Stain one of 
 them (step ii) with Lolfler's blue or carbol thionin, allowing the 
 stain to act for two minutes, and the other by Gram's method. 
 
 In removing some of the growth to make the film, remember 
 the facts just stated as to the characters of the colonies of the 
 bacillus, and select a colony presenting those characters (especially 
 that of elevation), if one is present. If there is no apparent 
 growth in the tube take " sweeps " of the whole surface. This is 
 conveniently done by means of a platinum loop shaped like a 
 stirrup, the flat bar being drawn along the surface of the medium 
 from bottom to top, just as a rake is drawn along a flower-bed. 
 
 Now examine your specimens in the way described on p. 27. 
 
 Characters of the Diphtheria Bacillus. 
 
 The following are the chief points which are considered in 
 deciding whether a given stained slide does or does not show the 
 diphtheria bacillus. 
 
 I. The shape of the bacillus is very variable, and this is a 
 feature which often affords us great assistance ; a specimen in 
 which all the bacilli present resemble each other exactly in shape 
 and size is not from a case of diphtheria. Diphtheria bacilli are 
 
DIPHTHERIA 43 
 
 narrow rods ; they are either straight or slightly curved in an arc 
 of a large circle or into an / shape (Plate I., Figs, i and 2). 
 Their ends are usually rounded, but it is not uncommon to find 
 forms with one end or both sharply pointed. Lastly, clubbed 
 forms are to be met with in almost all cultures, though they are 
 most frequent in those which have been incubated for several 
 days ; they may be compared to a note of exclamation (!). 
 
 2. Size. — Two well-marked varieties occur. The long form 
 is about as long as a tubercle bacillus (to compare it with 
 an organism with which the practitioner may readily become 
 acquainted), or somewhat longer ; it is decidedly thicker. The 
 short form is only about half as long and thick in proportion. 
 
 We do not know anything as to the difference in pathogenicity 
 (if any) of the long and the short varieties of the diphtheria 
 bacillus. They appear to "breed true" for long periods, and 
 cases of diphtheria caused by the one appear to have as high a 
 mortality as those caused by the other. 
 
 Hoffmann's bacillus is dealt with subsequently. 
 
 3. Staining Reactions. — The diphtheria bacillus stains readily 
 with all the stains in common use for bacteriological purposes. 
 It usually (but not invariably) stains irregularly, deeply-stained 
 portions alternating with others which remain colourless. This 
 gives rise to a beaded appearance, and forms sometimes occur 
 which can hardly be distinguished from short chains of strep- 
 tococci. When a powerful stain is applied for a long time this 
 appearance may be lost. 
 
 The diphtheria bacillus stains deeply with thionin. This often 
 affords a certain amount of help in the diagnosis, as many other 
 bacilli do not stain nearly so deeply in the same time. 
 
 It stains by Gram's method. A beginner should always test 
 his results in this way : If suspicious bacilli do not retain the 
 violet stain they are not those of diphtheria. 
 
 4. Arrangement. — This is a most characteristic feature, but 
 it is one which is difficult to describe. The old comparison to 
 the strokes which form a Chinese letter is a fairly good one; 
 the bacilli lie in little groups, some lying parallel to one another, 
 and some at various angles with these. The characteristic 
 arrangement is best seen in a specimen made from a pure 
 culture of the short form. 
 
 Before coming to a conclusion as to the presence or absence of 
 diphtheria bacilli from an examination of a stained film, make a 
 
44 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 very thorough search ; if no bacilli are seen, make several more 
 films and examine them. When you see a group of bacilli, 
 examine it carefully, noting each characteristic and comparing 
 it with those described above. 
 
 The beginner is strongly recommended to procure a series of 
 slides of diphtheria bacilli from a bacteriological laboratory, and 
 to study them carefully. 
 
 Hoffmann's bacillus (Plate I., Fig, 2) is probably a modified 
 form of the true diphtheria bacillus, and is frequently met with 
 in throat cultures. It is about as long as the short form of 
 diphtheria bacillus, but decidedly plumper, and is more uniform 
 in shape and size. These bacilli stain uniformly and deeply. 
 They exhibit the same arrangement, and are often grouped in 
 pairs. Clubbed forms do not occur. 
 
 The diphtheria bacilli which occur in films made direct fvom 
 the swab are similar to those seen in cultures, but are often 
 thicker ; they may stain uniformly, and clubbed forms are rare. 
 It is unusual to be able to make an absolute diagnosis as to their 
 nature, but it may be done at times. Yet such an examination is 
 often useful. If suspicious bacilli are present you should inject 
 antitoxin forthwith ; if no suspicious bacilli are seen it is safe to 
 wait for the result of the cultural examination. 
 
 Interpretation of Results. 
 
 The discovery of the diphtheria bacillus in the exudate may mean — 
 
 {a) That the patient is suff'ering from diphtheria. 
 
 {b) That he has sufi"ered from diphtheria and is now con- 
 valescent, but is still infectious. The bacilli may persist for weeks 
 or months, and while they do so the patient must be isolated and 
 treated with antiseptic gargles. 
 
 {c) That he is in danger of acquiring diphtheria if subjected 
 to any influence which lowers his vitality, or which would cause 
 ordinary sore throat in any ordinary person. 
 
 {d) It always means that the person may communicate diphtheria 
 to a susceptible subject. 
 
 The significance of Hofi"mann's bacillus is not yet settled. It is 
 frequently found in the throat when the patient is convalescing 
 from an attack of diphtheria, and sometimes in subjects who after- 
 wards develop diphtheria. It also appears to cause epidemics of 
 sore throat which do not present anything remarkable in their 
 
TETANUS 45 
 
 clinical characters. It is safest to regard any patient who presents 
 these bacilli as being infectious. In other words, in the present 
 state of our knowledge it is best not to draw any distinction 
 between Hoffmann's bacillus and the true diphtheria bacillus, as 
 far as infectivity is concerned." 
 
 A negative result may mean — 
 
 {a) That the patient is not suffering from diphtheria. 
 
 Q)) That the swab did not touch the affected area. 
 
 We exclude errors in technique and observation. 
 
 A sterile culture may mean — 
 
 (a) That an antiseptic was used too soon before taking the swab. 
 
 {h) That the diseased portion of the throat was not touched. 
 Other parts of the mouth contain numerous bacteria, but many of 
 them do not grow well on blood-serum or ascitic agar. 
 
 We again exclude errors arising in the laboratory. 
 
 Whenever the culture-tube remains sterile, the examination 
 should be repeated. 
 
 TETANUS 
 
 The pathology of tetanus is very much like that of diphtheria. 
 In each disease the specific bacilli are localized at or near the 
 region at which they enter the body, and form a toxin which 
 affects distant organs. In the case of diphtheria, as we have seen, 
 the toxin passes by the blood-stream, but in tetanus it creeps from 
 the region where it is produced up the peripheral nerves to the 
 brain and spinal cord. In each case research has shown that an 
 antitoxin is formed which neutralizes this toxin and prevents it 
 from uniting with the cells of the body, but which has not the 
 power of turning it out from such a combination. In other words, 
 tetanus antitoxin, like that of diphtheria, is preventive, but not 
 curative. But here, unfortunately, the resemblance between the 
 two diseases ceases. The local lesion in diphtheria is obvious, 
 and its presence causes a good deal of inconvenience to the patient; 
 
 * Since the publication of the first edition of this book there has been much 
 controversy on this point, but I have seen nothing which leads me to alter the 
 above opinion, though it is certainly true that laboratory evidence of the 
 relation of the organisms appears lacking. It may be noted that those who 
 deny the relation of Hoffmann's bacillus to diphtheria do so, for the most part, 
 on this failure of laboratory evidence, whilst those who look on Hoffmann's 
 bacillus with suspicion do so mainly on clinical grounds. (For an account of 
 other forms of sore throat, see p. 105.) 
 
46 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 he sees a medical man early, and the diagnosis of diphtheria is 
 made before much of the toxin has entered the blood. It is 
 different with tetanus. In this the local symptoms are practically 
 nil ; there may be suppuration at the region of inoculation, but 
 this is so common as not to excite suspicion. The result is that 
 the diagnosis is not made until the appearance of the symptoms 
 referable to the nervous system indicates that the period at which 
 antitoxin might have been used with success has gone by. 
 
 To illustrate this we will imagine the local lesion of diph- 
 theria to be so slight as to be unnoticed by doctor and patient. 
 The result would be that the disease would only be diagnosed when 
 the severe toxsemic symptoms had manifested themselves, and 
 antitoxin would then be almost or quite useless. If it were not 
 for the discomfort and pain caused by the throat lesion of 
 diphtheria, the antitoxin treatment of the disease would have 
 probably been abandoned as useless. 
 
 But tetanus may be diagnosed by means of a bacteriological 
 examination of the local lesion before toxic symptoms have 
 appeared, and in cases where this is done we may safely look for 
 results from tetanus antitoxin which are as good as those obtained 
 from the early use of diphtheria antitoxin ; for the experimental 
 evidence in favour of the one is every whit as great as that in 
 favour of the other. 
 
 Considerations of time would prohibit the bacteriological 
 examination of the multitude of small wounds which are seen by 
 the majority of medical men. But a wound which clinical 
 experience and bacteriological research as to the occurrence of 
 tetanus bacilli outside the body indicates as being one which is 
 likely to become infected with the bacillus in question should be 
 submitted to a careful and prolonged search for the bacillus. 
 These are deep incised and lacerated wounds, especially those of 
 the hand and foot, and particularly if garden earth or horse-dung 
 has been rubbed into the tissues. Wounds made with splinters, 
 rusty nails, or the wads from firearms, should be examined. 
 Severe lacerated and contused wounds in *' run- over" cases, con- 
 taminated by the dirt of the road, must also be regarded with 
 suspicion, for tetanus follows superficial wounds almost as 
 frequently as deep ones. Tetanus may follow a wound which 
 heals up by first intention, but this is unlikely ; suppuration or 
 necrosis of the edges (though not due to the tetanus bacillus itself) 
 is present in the majority of cases. 
 
TETANUS 47 
 
 Examination of Pus from Suspected Cases of Tetanus. 
 
 Requisites. — i. Slides and cover-glasses. 
 
 2. A stiff platinum loop. 
 
 3. Bunsen burner or spirit-lamp. _ 
 
 4. Loffler's blue or carbol thionin. 
 
 5. Materials for Gram's staining. 
 
 6. Balsam. 
 
 If cultures are to be taken, add a pipette (see Fig. 146), a deep 
 tube of agar to which 2 per cent, of grape-sugar has been added 
 previous to sterilization, a flask of water, and a thermometer. 
 
 Method, — Scrape the deeper portions of the wound with the 
 platinum loop, and spread out the secretion thus obtained on the 
 surface of a slide. Prepare several of these slides, and fix the 
 film by heat. Stain some by the simple stain for two minutes 
 and others by Gram's method. 
 
 The bacillus of tetanus is about as long as the tubercle bacillus, 
 and is very slender. It stains by Gram's method. A very 
 characteristic feature is its method of spore- formation. The 
 spores are spherical bodies which are formed at the extremities of 
 the bacilli, giving them the appearance of pins or drumsticks. 
 The spores do not stain by the ordinary stains, and appear as 
 colourless and highly refractile bodies (Plate II., Fig. i). 
 
 The cultures are made in agar to which 2 per cent, of grape- 
 sugar is added, and the needle or pipette used in making the 
 inoculation is plunged deep down into the medium. The bacillus 
 of tetanus is an anaerobe — i.e., it grows only in the absence of 
 oxygen. The stabs are made deep in order to inoculate the 
 material far away from the air, and the glucose is added to absorb 
 any oxygen which may be in the medium. To increase our 
 chances of obtaining this bacillus in pure culture, the material 
 to be examined is to be heated to a temperature which will kill all 
 developed bacteria, but which will not be injurious to spores ; the 
 tetanus bacillus is the only anaerobic organism with a spherical 
 terminal spore which is at all likely to occur in a wound. 
 
 Method. — The inoculations are to be made with a pipette. If 
 the pus which comes from the wound can be drawn up into the 
 capillary tube of a glass pipette such as is described on p. 33, 
 the material should be collected in this way. If this is not the 
 case, the wound must be scraped with a sterilized platinum 
 needle or other suitable instrument, and the material thus obtained 
 
48 CLINICAL BACTERIOLOGY AND H.F.MATOLOGY 
 
 mixed with some boiled water (previously cooled) and then 
 sucked up into the pipette ; the end of the latter is then to be 
 sealed in the flame, care being taken that the material itself is 
 not heated. 
 
 Having filled and sealed the pipette, heat some water in a small 
 flask or large test-tube until it reaches 80° C, as measured by the 
 thermometer ; insert the sealed end of the pipette in the water, 
 and maintain the temperature for ten minutes. The thermometer 
 is to be kept in the water the whole of the time, and the flame is 
 to be taken away when the temperature rises above 80° C, and 
 reapplied when it falls below that point. 
 
 At the end of this time the pipette will contain no living object 
 other than spores. Break off its point and insert it gently into 
 the glucose agar, taking care to keep exactly in the axis of the 
 tube, until the tip of the pipette reaches almost to the bottom of 
 the test-tube. Withdraw the pipette gradually, blowing out its 
 contents as you do so. The spores of the tetanus bacillus (if 
 present) will now be inoculated deep down in the medium, far 
 away from the air. To reduce the supply of oxygen still further 
 it is a good plan to melt some paraffin (a hard candle answers 
 perfectly) and pour a layer an inch thick over the surface of the 
 medium ; or vaseline which has been heated to the boiling-point 
 of water (to sterilize it) may be used. 
 
 The cultures thus made are to be incubated for a few days at 
 the body temperature. After about forty-eight hours the growth 
 begins to appear in the deeper portions of the tube as a series of 
 delicate wavy outgrowths from the central stab. These do not 
 appear in the upper portion of the medium, where the oxygen 
 (which diff"uses down from the air, unless the tube has been 
 sealed with paraffin) hinders their growth. If the culture is pure 
 — and this is not likely to be the case, as these wounds are 
 usually very highly contaminated — no gas will be produced by the 
 growth, whereas the bulk of the other organisms which are 
 likely to occur produce gas. A bubble or two in the course 
 of the growth, therefore, indicates that the culture is not pure 
 tetanus bacillus, but that organism may nevertheless be present. 
 If the tube shows such a growth, it should be submitted to a 
 microscopic examination. It is a good plan to break the tube 
 and to spHt up the cylinder of medium with a knife ; films are 
 made from the growth and stained as above. Spores are formed 
 after about thirty-six hours. 
 
THE PNEUMOCOCCUS, PNEUMONIA, ETC. 49 
 
 The other methods of cultural examination are far more 
 difficult. 
 
 Interpretation of Results. 
 
 If bacilli having the above characters are found in films, the 
 diagnosis of tetanus must not be considered as being absolutely 
 proven, for there are other bacilli which might be mistaken for 
 those under discussion ; but the probability that the patient will 
 develop the disease is so strong that steps should be taken 
 accordingly. The wound should be scraped and thoroughly 
 treated with antiseptics, and antitoxin should be given. If the 
 deeper portion of the glucose-agar stab shows the tree-like 
 growth which has been described and contains slender drum- 
 stick bacilli, the case is strengthened, even although the upper 
 part of the medium is contaminated with other organisms. 
 
 The only way in which the bacilli can be recognized with 
 absolute certainty is by means of animal experiments ; this is a 
 very certain method, and one that can be carried out in the 
 absence of pure cultures. The material is diluted with some 
 broth and divided into two parts, of which one is injected into an 
 animal just as it is, whilst the other is mixed with antitetanic 
 serum and then injected. In a case of tetanus, the first animal 
 will die with tetanic symptoms, whilst the latter will survive or 
 die of sepsis without tetanus. 
 
 THE PNEUMOCOCCUS, PNEUMONIA, ETC. 
 
 The pneumococcus is a very important organism, and one 
 which plays a prominent part in the production of disease. It 
 may occur in the mouth in a healthy person ; hence its recogni- 
 tion in small quantities in the sputum is not of diagnostic value. 
 
 The pneumococcus is a very common cause of disease of the 
 respiratory system. It causes : 
 
 1. Acute lohav pneumonia. Opinions differ as to whether it is the 
 only cause of this disease, though it appears most probable that 
 this is the case. 
 
 2. Lohulav {broncho-) pneumonia. This disease may also be 
 caused by streptococci, staphylococci, diphtheria bacilli, influenza 
 bacilli, plague bacilH, tubercle bacilli, and others. The pneumo- 
 coccus may also occur as a secondary infection in lobular pneumonia 
 due to any of these. 
 
 4 
 
50 CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 3. Pleurisy, either the serous, fibrinous, or purulent varieties, 
 and especially in children. It is important to notice that the 
 prognosis of empyema is better when the disease is due solely 
 to the pneumococcus than when other organisms (streptococci, 
 staphylococci, tubercle bacilli, etc.) are present, and such cases 
 usually recover without resection of ribs. The bacteriological 
 examination of the pus from a pleural cavity may thus lead to 
 results important as to prognosis and treatment. 
 
 4. The pneumococcus may occur as a secondary infection in 
 almost any disease of the lung ; for instance, in the walls of a 
 phthisical vomica. 
 
 The most important primary lesions due to the pneumococcus 
 outside the respiratory system are : 
 
 1. Otitis media (of which it is a very common cause), empyema 
 of the antrum, and of the frontal and other accessory sinuses of 
 the nose. 
 
 2. Erysipelas, which is usually due to another organism, the 
 Streptococcus pyogenes. 
 
 3. Brawny induration of the skin, with or without suppuration. 
 This is uncommon, but I have seen several cases. 
 
 4. Suppuration in any region, especially in infants, in whom it 
 is probably the most common pyogenic organism. 
 
 Pneumococci may escape into the blood from any of these 
 lesions, and may appear in that fluid when there is no obvious 
 primary lesion from which it could have gained access. The 
 most common results are : 
 
 1. Septicemia. 
 
 2. Ulcerative endocarditis. Many other bacteria may cause this 
 disease. 
 
 3. Meningitis. Pneumococcal meningitis may also be due to 
 direct spread from the middle ear. 
 
 4. Arthritis. 
 
 5. Peritonitis. 
 
 The prognosis in these latter affections is on the whole slightly 
 better if they are due to the pneumococcus than if caused by 
 other organisms, except in the case of meningitis, which is usually 
 fatal in a few days. 
 
 In actual practice we have most commonly to search for the 
 pneumococcus in sputum, pus, and blood. In the latter case 
 cultural methods are usually necessary, and we shall defer its 
 consideration for the present. 
 
I 
 
 THE PNEUMOCOCCUS, PNEUMONIA, ETC. 5I 
 
 Sputum. — The examination of sputum may be made in order to 
 make a diagnosis as to the presence or absence of pneumonia, in 
 a case in which the physical .signs are indeterminate, or to 
 estabHsh the nature of a lobular pneumonia. 
 
 The patient must wash out his mouth with water, which should 
 have been boiled and allowed to cool. He must then spit into a 
 clean wide-mouthed bottle, also containing boiled water, and care 
 must be taken that the sputum used for the examination comes 
 directly from the lungs, and is not merely mucus which has 
 collected in the mouth. 
 
 The mass of mucus forming a single " spit " is agitated gently 
 in the water to remove contaminations from the bronchial tubes 
 and mouth ; the water is poured off and more added, and the 
 process repeated several times. Then the mass of mucus is fished 
 out, placed in a watch-glass, carefully opened with a pair of scissors, 
 and a piece about as big as a pea is removed from the centre of 
 the mass with a platinum loop. It is placed on a clean slide, 
 another slide pressed upon it, and the two are slid apart. The 
 films thus obtained are allowed to dry, and fixed by heat in the 
 usual way. 
 
 One is then stained by Gram's method, and counterstained by 
 dilute carbol fuchsin for about a quarter of a minute, washed, 
 dried, and mounted, and the other with undiluted carbol fuchsin. 
 
 The pneumococcus is a diplococcus — i.e., the individual cocci 
 are arranged in pairs. Each coccus has usually an oval or lancet 
 shape, the sharp ends of the two germs pointing away from one 
 another (Plate I., Fig. 3, and Plate III., Fig. 4). Abnormal 
 forms (round cocci, short bacilli, etc.) are frequent. The pneumo- 
 coccus has a capsule when it occurs in the living body or in patho- 
 logical exudates, but not in most cultures. This capsule does not 
 stain readily, and appears in a properly-stained specimen as a 
 clear halo round the two cocci. 
 
 Examine your Gram specimen first. The pneumococci (which 
 will be almost black) should be clearly seen, and you should be able 
 to make out their shape and characteristic arrangement in pairs. 
 
 The general surface of the film is stained pink, while there is a 
 clear and colourless zone round each pair of cocci. This is the 
 capsule, which is rendered distinct by "negative staining," and 
 these appearances will be better seen in a somewhat thick and 
 deeply-stained film, though if the carbol fuchsin has been allowed 
 to act too long the capsule may be stained a faint pink. There 
 
 4—2 
 
52 CLINICAL BACTERIOLOGY AND H^EMATOLOGY 
 
 are special methods of staining which may be used to render the 
 capsule more distinct, but these are usually unnecessary for the 
 diagnosis. 
 
 Interpretation of Results. 
 
 In cases of lobar pneumonia you will probably fmd pneumococci 
 in great quantity, and no other bacteria in a specimen of sputum 
 made in the manner descvihed. If you find many pneumococci in a 
 case of lobular pneumonia the disease may have been caused by 
 another germ, and the cocci in question may have been nothing 
 more than a secondary infection. A specimen should be stained 
 deeply by carbol fuchsin or methylene blue and searched for 
 bacilli resembling those of influenza, etc., and another should be 
 stained for the tubercle bacillus if the clinical aspect of the case 
 suggests the possibility of a tuberculous origin for the disease. 
 
 Pus is examined in the same way, and presents similar appear- 
 ances. Most of the cocci are extracellular, but some are frequently 
 contained in the cells, and may then not retain Gram's stain. 
 
 It is not usually necessary to make cultures, as the pneumococcus 
 is readily recognized in pathological material from its morphological 
 appearance and staining reactions alone. Where cultures are 
 required, agar is about as good a medium as can be used, and it 
 must be incubated at the body temperature. The colonies are 
 visible after twenty-four hours as very small transparent circular 
 masses, which are but slightly raised and show but little tendency 
 to increase in size on further incubation. It is a very delicate 
 organism, and one which readily dies out on ordinary media, so 
 that if a culture is to be " kept going " it must be transplanted 
 every two or three days to a fresh tube. This is one of its most 
 characteristic features. 
 
 INFLUENZA 
 
 In the first edition of this book, in treating of influenza, I followed 
 the usual teaching of bacteriologists and regarded the disease as a 
 specific one, and as being caused in all cases by the influenza (or 
 Pfeiff"er's) bacillus. Recent observations, both in England and on 
 the Continent, have shown that this view can no longer be upheld, 
 unless we greatly restrict the use of the term " influenza," and use 
 it only for those cases in which the bacillus in question is found. 
 This is quite unjustifiable, for the diseases appear to be identical 
 in clinical history ; and in cases in which we should be practically 
 
INFLUENZA ^ 53 
 
 certain of finding Pfeiffer's bacillus if they occurred a few years 
 back, we now find other organisms, especially the Micrococcus 
 catarrhalis. In this uncertain state of bacteriology the results of 
 an examination of the sputum are deprived of much of their value 
 as a means of diagnosis, but the methods will be described, since 
 more information is required,- and this information the general 
 practitioner is usually in the best situation to obtain. 
 
 Methods. — If possible, the sputum should be obtained in a method 
 similar to that recommended in pneumonia, as it greatly facilitates 
 the process and renders the results more trustworthy if the sputum 
 comes directly from the lungs, and is not contaminated with bacteria 
 from the mouth. The nasal secretion may also be examined, and 
 frequently contains the organisms in pure culture and vast numbers; 
 where the nose is affected, better results will be obtained in this 
 way than from the sputum. The mucus may be collected on a 
 diphtheria swab or on a platinum loop, or by means of one of the 
 angled pipettes described on p. no, though it is often too thick to 
 be sucked up into such a narrow tube. 
 
 Films are prepared from the sputum by squeezing a small mass 
 between two slides and sliding them apart. Dry and fix by heat. 
 Two should be prepared. The first should be stained by Gram's 
 method, and counterstained by dilute carbol fuchsin for a quarter 
 of a minute, then washed, dried, and mounted. The other is to be 
 stained more deeply with carbol fuchsin or Loffler's blue — about 
 five minutes with gentle heat in either case. The influenza bacillus 
 stains with difficulty, and may not be seen in the Gram's specimen 
 which is lightly stained with fuchsin. And there is a good reason 
 for not carrying the counterstaining too far in the former case, 
 since if the carbol fuchsin is used for too long a time it may dis- 
 place the violet stain of an organism which retains Gram. 
 
 The influenza bacillus is an extremely minute organism, and one 
 which requires the highest powers of the microscope for its study. 
 It is an extremely minute rod, and it would take from eight to 
 sixteen of these rods to make up the diameter of a red blood- 
 corpuscle. They occur in vast numbers in the sputum or nasal 
 mucus, and are frequently found within the leucocytes, and when 
 in this situation may appear to have a capsule, being contained in 
 vacuoles in the protoplasm (Plate II., Fig. 3). They are often 
 arranged in pairs, in which case they might be mistaken for small 
 but unusually elongated pneumococci, but for the fact that they do 
 not stain by Gram's method. 
 
54 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 These features are sufficient to identify them for dinical 
 purposes. Cultures are difficult to obtain, since the organism 
 only grows in presence of haemoglobin — e.g., on agar tubes streaked 
 with sterile blood (see p. 85). Under these circumstances they 
 form very minute translucent colonies, much like those of the 
 pneumococci, and cultures have a great tendency to die out. 
 
 The M. catarrhalis (Plate III., Fig. 5), the next most frequent 
 cause of clinical influenza, is a diplococcus which does not stain by 
 Gram, and which has a considerable amount of resemblance to 
 the other two non-Gram-staining diplococci, the gonococcus and the 
 meningococcus. It occurs in vast numbers in the sputum and 
 nasal mucus of influenza, in the nasal mucus of a " common cold," 
 and is a common cause of bronchitis of ordinary type. It is also 
 an occasional cause of sore throat, and is not infrequently met 
 with in the examination of supposed cases of diphtheria. The 
 resemblance to the two other organisms named arises from the 
 fact that it is frequently intracellular. There is not usually any 
 difficulty in distinguishing between the three, owing to the difference 
 in their habitat — the gonococcus affecting the mucous membrane 
 of the urethra or cervix, the meningococcus the meninges, and 
 the M. catarrhalis the nose, mouth, and respiratory passages. 
 There are minute morphological differences between the three, 
 and an expert can usually identify them in film preparations 
 from the body, but where there is any question of the nature of the 
 organism present cultures ought to be made. The M. catarrhalis 
 is the only one of the three which will grow on gelatin at the 
 room temperature. 
 
 ANTHRAX 
 
 Anthrax occurs in man in three forms. The most common is 
 cutaneous anthrax, or, as it is sometimes called, malignant pustule. 
 Pulmonary anthrax, or wool- sorter's disease, is much rarer, and 
 intestinal anthrax rarer still. The practitioner will find the greatest 
 assistance from a bacteriological examination in the cutaneous 
 form of the disease ; he may search for the specific bacillus in 
 the sputum in a supposed case of wool-sorter's disease, but he 
 must be careful in his interpretation of his result, as bacilli which 
 might be mistaken by an untrained observer, relying on the 
 morphological appearances alone, sometimes occur in the sputum. 
 The search for bacilli in the faeces in a supposed case of intestinal 
 anthrax must be relegated to an expert. 
 
ANTHRAX 55 
 
 The true nature of a case which is examined post-mortem can 
 easily be determined bacteriologically ; the cut surface of the 
 liver or spleen should be rubbed upon a clean slide, and the films 
 treated secundum art em. They will probably show the bacilli in 
 large numbers. Sections may also be cut and stained by Gram's - 
 method (see p. 172). 
 
 In the later stages of any infection with anthrax the bacilli 
 may be found in the blood. They may be apparent on examina- 
 tion of stained films, or by cultural methods similar to those used 
 in the diagnosis of malignant pustule. 
 
 The anthrax bacillus varies considerably in length, but is 
 always a large organism, and may be considerably longer than 
 the diameter of a red blood-corpuscle. It is much thicker than 
 the bacilli which have been dealt with hitherto, and it is invariably 
 straight. The ends of these bacilli are cut sharply at right angles 
 to the sides of the organism, and may be even somewhat concave ; 
 this is a most characteristic feature. The anthrax bacillus stains 
 by Gram's method (Plate I., Fig. 4). 
 
 In cultures the appearances are somewhat different. Here the 
 bacilli are frequently arranged in long chains which have an 
 appearance which has been compared to that of a bamboo ; chains 
 occur in the blood or in the inflammatory exudate, but are usually 
 much shorter than those seen in cultures. But the most important 
 features in cultures of the anthrax bacillus is the development 
 of spores, which are oval, highly refractile bodies, and lie in or 
 near the centre of the bacilli, one in each. These spores are 
 possessed of tough capsules, which resist the action of the ordinary 
 stains much in the same way as the tubercle bacillus. Thus 
 it happens that in films of a cultivation of the anthrax bacillus 
 which have been stained with such a dye as weak methylene blue 
 the spores are readily seen as colourless and refractile oval areas 
 in the centre of the bacilli, the latter being stained blue. The 
 spores themselves may be stained by a modification of the 
 process used for the tubercle bacillus. The films are first stained 
 by heated carbol fuchsin, which penetrates slowly through the 
 capsule; they are then decolorized by a very rapid immersion 
 in very dilute sulphuric acid (i per cent.) or in methylated spirit, 
 and examined microscopically. If the red colour has been entirely 
 removed from the bacilli, but is still present in the spores, the films 
 are ready to be counterstained by methylene blue ; if not, they 
 must be dipped in the acid or spirit once more and re-examined, 
 
56 CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 When this process is successful, the spores are stained red, and 
 the bacilli blue (Plate I., Fig. 4). 
 
 The presence of spores enables us to isolate the bacilli from most 
 of the organisms with which they are likely to be contaminated, 
 by a very simple process. The spores resist the action of heat 
 just as they resist stains, and for the same reason, and a suitable 
 temperature will kill off all the non-sporing organisms and spare 
 the spores. The latter may then be inoculated at a suitable 
 temperature, and will develop into bacilli. This process, however, 
 is not applicable to the examination of the blood or morbid 
 effusions, as the bacillus of anthrax does not form spores in the 
 living body. In this it differs from the tetanus bacillus, in which 
 the process may be applied direct to the material from the body. 
 
 Investigation of a Supposed Case of Malignant Pustule. 
 
 Requisites. — i. Several glass pipettes; if cultures are not 
 required, one will be enough. 
 
 2. Clean slides and cover-glasses. 
 
 3. Bunsen burner or spirit-lamp. 
 
 4. Loffler's methylene blue ; also the materials for Gram's 
 staining. 
 
 5. Balsam. 
 
 6. Tubes of gelatin if cultivations have to be taken. 
 
 Method, — Break off the extreme tip of one of the glass pipettes 
 and insert into one of the vesicles around the dark papule in the 
 centre of the lesion ; it may be necessary to make a puncture 
 with a sterilized needle before this can be done. If the fluid does 
 not rise spontaneously into the pipette, break off the other end 
 and suck gently, watching the column of fluid so that it does not 
 get into your mouth. It is safer to use a pipette with a wide 
 mouthpiece plunged with cotton-wool (Fig. 14), or, best of all, 
 to suck up the fluid by means of an india-rubber nipple. 
 
 Having obtained a drop or two of the fluid exudate, blow it out 
 on to the surface of a clean slide and spread it out into a film ; 
 prepare as many of these as you can. Allow them to dry, and 
 stain one with Loffler's blue and some by Gram's method. 
 
 Examine with the oil-immersion lens. Make a careful search 
 over the films, looking for large cigarette-shaped bacilli, noting 
 whether they are or are not arranged in chains. Examine the 
 Gram specimens, and see whether the bacilli are to be seen in 
 them also. 
 
ANTHRAX 57 
 
 Interpretation of Results. 
 
 If the case is really one of malignant pustule, the chances are 
 very greatly in favour of your finding the bacilli in large numbers, 
 and the failure to do so tells strongly against a positive diagnosis. 
 
 Cultural Methods. — The fluid for examination is taken in exactly 
 the same way as that described above, but the isolation of the 
 organisms will be greatly facilitated if antiseptic methods are 
 employed to prevent contamination with skin bacteria. To this 
 end the surface of the lesion should be washed gently with 
 carbolic or perchloride lotion, and then (very thoroughly) with 
 alcohol or methylated spirit to remove the antiseptic. The 
 surface is then allowed to dry. 
 
 If the material is to be transmitted to a pathologist for examina- 
 
 FiG. 19. — Petri's Dish. 
 
 tion (and this is the wisest course to adopt, as animal inoculations 
 are almost necessary for the absolute identification of the bacillus), 
 the fluid must be carefully sucked up into the bulb, and both ends 
 of the pipette carefully sealed. 
 
 If the examination is to be made at home, the best way is to 
 make two inoculations in gelatin. The first should be a stab 
 culture, and may be made with the pipette direct ; or the fluid 
 may be blown out into a watch-glass or on to the surface of a 
 slide (in either case sterilized by being heated in the flame and 
 then allowed to cool), and the stab made by dipping the end of a 
 straight platinum needle into the fluid, and then driving it into the 
 gelatin. 
 
 The other culture is made with the pipette ; this is driven into 
 the gelatin in a tube and the contents blown out. The gelatin is 
 then immersed in warm water until it is melted, and poured out 
 into a Petri's dish (Fig. 19) previously sterilized by dry heat and 
 allowed to cool. 
 
 Both cultures are incubated at a temperature of about 20° C. 
 
 In about two days the gdatin stab-tube will show a very 
 
58 
 
 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 characteristic appearance if the anthrax bacilli are present in 
 pure culture. The growth takes place in Hnes which project 
 nearly at right angles to the line of inoculation, and grow more 
 vigorously the nearer they are to the surface. The result is the 
 development of a culture which has a strong resemblance to an 
 inverted fir-tree (Fig. 20). In another day or two the gelatin will 
 begin to show a certain amount of liquefaction, which begins at 
 the surface. 
 
 The appearances in the plate culture are 
 perhaps not quite so characteristic, but they 
 are manifested in impure cuhures. The 
 young colonies of anthrax bacilli have a 
 whorled appearance, which has been com- 
 
 FlG. 21. 
 
 -Young Colony of Anthrax Bacillus 
 (x 15). (Crookshank.) 
 
 Fig. 20. -Stab Cul- p^red to a barrister's wig or to the head of 
 
 TURE OF Anthrax ^ '=' 
 
 Bacillus. (Crook- Medusa (Fig. 21). The plate should be placed 
 
 shank.) upon the stage of the microscope, and 
 
 examined for these colonies with the low 
 power. If one is found, a clean cover-glass should be pressed 
 upon it, lifted up with a needle, so as to bring up the colony with 
 it, fixed by heat, and stained with carbol thionin or methylene 
 blue. The colonies are most characteristic after two days' 
 incubation ; at a later period the gelatin is liquefied and spores 
 are formed. 
 
 A culture which presents these cultural and morphological 
 appearances may be considered to be one of anthrax with almost 
 absolute certainty, though other tests (notably animal tests) would 
 be applied in a laboratory. 
 
TUBERCLE 59 
 
 TUBERCLE 
 
 The diagnosis of tuberculosis by bacteriological methods (in 
 the case of most morbid exudates) is within the reach of every 
 practitioner ; cultural methods are not used, and the recognitioiT 
 of the bacillus is rapid, easy, and certain. 
 
 The bacilli may be sought for in sputum, urine, pus, faeces, or 
 any other morbid material. We will first describe the method of 
 staining which should be adopted, then the characters on which 
 the recognition of the bacillus depends, and, lastly, the methods 
 by which the films are prepared from the various materials. 
 
 Staining the Tubercle Bacillus. 
 
 Requisites. — i. Slides, cover-glasses, forceps, and balsam. 
 
 2. A Bunsen burner or spirit-lamp. 
 
 3. Dilute sulphuric acid — about 20 per cent. — contained in a 
 wide-mouthed bottle or in a jar. This must be large, enough to 
 admit a slide, but not large enough to permit it to fall down to 
 the bottom. 
 
 4. Carbol fuchsin. 
 
 5. Methylene blue. (Saturated watery solution or Loffler's blue.) 
 
 6. A metal (iron or copper) plate. The exact dimensions do 
 not matter, but 8 by 4 by ^ inches is a convenient size. It should 
 be mounted upon a tripod. This slab is not absolutely necessary, 
 but it is a very great advantage. 
 
 Method. — We will suppose that the film has been prepared by 
 one of the methods described subsequently, allowed to dry, and 
 fixed by heat. 
 
 1. Place the slide upon the metal plate, and heat the latter by 
 the flame. Flood the slide with carbol fuchsin, and let the heat 
 continue until the stain steams, but do not allow it to dry up ; let 
 this go on for from three to five minutes. If the stain shows 
 signs of drying up, add a little more ; if it begins to boil, slide it 
 along the plate away from the flame, or remove the latter for a 
 short time. 
 
 If you have no metal plate, it is possible to hold the slide with 
 a pair of forceps, but in this case the film is most conveniently 
 made on a cover-glass. 
 
 Remember not to let the stain dry up. 
 
 2. Remove the slide from the plate with the forceps, and wash 
 it under the tap or in a bowl of water. 
 
6o CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 3. Put it into the bottle containing the dilute acid. After three 
 or four minutes withdraw it and again wash. If much pink 
 colour comes back, re-insert it in the acid for a short time, and 
 again wash. The process must be repeated until the film only 
 shows a slight pink tinge. 
 
 4. Now apply the methylene blue for half a minute or so. 
 
 5. Wash, dry with blotting-paper, and then by gentle heat. 
 Apply a drop of balsam, and cover. 
 
 Another Method. — Filter into a test-tube sufficient carbol fuchsin 
 to flood the film, boil it over the Bunsen or spirit-lamp, and pour 
 it on to the film whilst still boiling. Let it act until it is cool, 
 when the bacilli will be found to be stained, and the process of 
 decolorization may be proceeded with. 
 
 The times which are given above may be considerably shortened 
 in practice, but I do not advise this until considerable skill is 
 acquired. Bacilli are much more easy to recognize if they are 
 deeply stained ; this is the reason for the prolonged staining, 
 which may appear unnecessary to some. The prolonged de- 
 colorization is an advantage, since it insures that the tubercle 
 bacilli shall be the only things left stained red ; if you leave the 
 preparation in the acid for a short time, you are more likely to get 
 crystals of carbol fuchsin, stain retained in deep scratches of the 
 glass, etc., all of which a beginner may easily mistake for bacilli. 
 The counterstaining with methylene blue may be shortened or 
 omitted altogether, though this is not advisable, as it is then more 
 difficult to focus the film. 
 
 Recognition of the Tubercle Bacillus. 
 
 The tubercle bacillus is about half as long as a red blood- 
 corpuscle is wide, or rather longer, and is very slender. It is 
 straight or slightly curved, and is variable both in shape and in 
 size (Plate II., Fig. 2). 
 
 We recognize it by means of a staining reaction. Tubercle 
 bacilli contain a considerable amount of fat, and this prevents 
 them from staining readily with ordinary stains. In the process 
 described above we used fuchsin, which is a very powerful stain, 
 and added a mordant (carbolic acid), which increases its penetrative 
 properties. Even with this staining is very slow, so that we 
 heated the specimen. 
 
 The fat which prevents the bacilli from staining also prevents 
 
TUBERCLE 6l 
 
 the stain from being removed by such substances as acids and 
 alcohol. In stage 3 of the above process we aim at allowing the 
 acid to act until it has removed the fuchsin from everything 
 except the tubercle bacilli. The methylene blue is a counter- 
 stain, and colours all organisms, pus cells (especially their nuclei), 
 epithelial cells, and shreds of lung-tissue — in fact, everything 
 except the tubercle bacilli. The latter appear as slender red rods, 
 which often show the irregular staining which has been described 
 as occurring in the diphtheria bacillus. 
 
 Now, " acid-fast " bacilli are very rare, though they have been 
 found in unexpected situations of late years. Only three such 
 bacilli need be taken into consideration in dealing with human 
 pathology. These are the tubercle bacillus, the leprosy bacillus, 
 and the smegma bacillus. The bacillus of leprosy would rarely 
 lead to mistakes in this country ; it is recognized by the fact that 
 it is straighter and more uniform than the tubercle bacillus, and 
 by the fact that it resists decolorization more powerfully than the 
 tubercle bacillus. The smegma bacillus may occur in the urine, 
 and lead to mistakes unless the sample examined was drawn off 
 per catheter. It is distinguished by the fact that it is readily 
 decolorized by alcohol (absolute alcohol or methylated spirit), 
 while the tubercle bacillus is not. In staining a film from the 
 urine, we decolorize in spirit for a quarter of an hour after the 
 acid and before the methylene blue — i.e., between stages 3 and 4 
 in the above description. 
 
 In searching for the tubercle bacillus, the J-inch lens will serve, 
 though an oil-immersion lens is an advantage. 
 
 Method of Collecting the Sputum. 
 
 This is of some importance, and the method recommended 
 should be carried out in all cases. 
 
 Get the patient to wash out his mouth thoroughly with warm 
 water before going to bed. Let him spit into a clean bottle, jar 
 or tin, and employ only the sputum coughed up before food is 
 taken in the morning. 
 
 It is often impossible to get sputum from children, but in them 
 it is often possible to pick masses of sputum out of the vomit and 
 to demonstrate tubercle bacilli therein ; and when no such masses 
 are seen, bacilli may occasionally be found if the whole vomit be 
 carbolized in the manner described subsequently and the sedi- 
 
62 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 ment examined. In some cases in which there is no vomit it is 
 occasionally justifiable to give an emetic for the purpose of 
 securing sputum and establishing the diagnosis. 
 
 Method of Preparation of the Film. 
 
 Sputum. — Pour the sputum into a Petri dish or watch-glass, 
 and place the vessel upon a dark surface. Examine it closely, 
 looking out for small yellow particles ; these consist of caseous 
 material, and will probably contain tubercle bacilli in large 
 numbers. The advantage of getting the patient to wash out his 
 mouth and using only fasting sputum is obvious, for particles of 
 food may present exactly the same appearance. 
 
 Having found such a mass, pick it out by means of a platinum 
 loop or pair of forceps, and transfer it to the middle of a clean 
 slide. Now place another slide on the top of the first, squeeze 
 them together, and then slide them apart. You should get two 
 good uniform films. Allow to dry spontaneously, and fix by heat. 
 
 If there are no caseous masses, pick out a mass of the sputum 
 at random and proceed as before. 
 
 If no bacilli are found at the first examination, and you still 
 suspect tubercle, proceed as follows : Half fill an ordinary 
 medicine bottle with carbolic lotion (i in 20), and add a drachm or 
 two of the sputum. Shake thoroughly for a few minutes, and 
 place the bottle where you can give it an occasional shake during 
 the next few hours. Then pour the milky emulsion which results 
 into a conical urine-glass, and allow it to stand for twelve hours or 
 more. Remove some of the deposit which will form with a 
 pipette, and spread it into a thin film on a slide. Dry and fix. 
 
 Another method which may be used in difficult cases is to 
 digest the sputum with pepsin and PI CI— a pinch of the former 
 and sufficient of the latter to make the fluid faintly acid — in an 
 incubator for a couple of hours, shaking occasionally, and then to 
 boil the digested material to prevent further action. The result 
 may be centrifugalized or allowed to deposit, and the sediment 
 examined. 
 
 Urine may be centrifugalized or allowed to stand without 
 previous addition, but better results are obtained if carbolic acid 
 (Hquefied or in crystals) is added to the urine in amount sufficient 
 to convert it into a i in 20 solution. This is allowed to deposit or 
 (better) is centrifugalized. Films are prepared from the deposit. 
 
TUBERCLE 63 
 
 Remember that they should be left in alcohol for a quarter of 
 an hour or so after staining. 
 
 Pus is best carbolized in the same way as sputum ; if very 
 thin it may be treated like urine. The tubercle bacilli will 
 rarely be found in pus unless it is examined soon after the 
 abscess is opened, but may be detected by inoculation experi- 
 ments for long periods. 
 
 Clear exudates are more difficult to examine, and, as they usually 
 contain bacilli in very small numbers only, a negative result 
 should not be given too much weight. (For the best method to 
 employ, see p. 121.) The examination is best made in a bacterio- 
 logical laboratory, as decisive results can only be obtained by 
 animal experiments. Collect the f^uid in a bottle which has 
 previously been boiled in water for half an hour and allowed 
 to cool. Cork it with a cork which has also been boiled. 
 Add no antiseptic, and forward it to the laboratory as soon as 
 possible. 
 
 Milk may be examined in the same way as urine, films being 
 made from the cream as well as from the deposit. These films 
 are fixed, soaked in ether to remove fat, and again fixed. They 
 are then stained as before, and it is advisable to pass them 
 through alcohol. 
 
 The results obtained by this examination are somewhat un- 
 certain, as some of the other acid-fast bacilli found in milk are 
 almost exactly like the tubercle bacillus, and animal experiments 
 are necessary for definite proof. 
 
 When fceces are to be examined, the best plan is to administer 
 opium in amount sufficient to cause constipation. The surface of 
 the scybalous motions which result are to be scraped off and 
 stained in the usual way. 
 
 Interpretation of Results. 
 
 The finding of tubercle bacilli in the sputum is conclusive 
 evidence of tuberculosis of the lungs, but no information as to 
 prognosis can be drawn from the numbers which are present ; 
 they may occur in great quantities in the sputum from patients 
 who are doing well, and the author has found enormous numbers 
 in the sputum of a person who had presented no symptoms of the 
 disease for eight years, and was apparently cured. But a person 
 in whom the bacilli are present is always in danger of a re- 
 
64 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 crudescence of the disease, a«^ may he a source of infection. Absence 
 of the bacilli does not disprove the diagnosis of tuberculosis ; 
 bacilli do not appear in the sputum until the lung-tissue in which 
 they occur breaks down, and are therefore absent in the early 
 stages of acute tuberculosis. 
 
 In some cases of ordinary chronic phthisis bacilli may occur in 
 the sputum in very scanty numbers, and may be missed unless a 
 very careful search is made. Bacilli should not be considered as 
 being absent until well-stained films have been examined for 
 at least half an hour, and the examination repeated on several 
 occasions. It is in these cases that the carbolic method of 
 examination is so useful. 
 
 The finding of tubercle bacilli in the urine is practically abso- 
 lute proof of tuberculosis of some part of the urinary tract, 
 probably the kidneys or bladder. Absence of bacilli implies 
 nothing unless the examination has been made very thoroughly 
 and repeated several times at intervals. Then it affords pve- 
 sumptive evidence that the urinary passages are free from the 
 disease. 
 
 The same is true of the examination of pus. Tubercle bacilli 
 rarely occur in inflammatory exudates except in very small 
 numbers, and can often only be demonstrated by animal experi- 
 ments. If you examine pus from a chronic abscess and find no 
 organisms of any kind, it is almost certain that the process is a 
 tuberculous one, and the negative evidence obtained by the failure 
 to find tubercle bacilli should not be allowed to carry much 
 weight. The same is true for the clear exudates. 
 
 In suspected cases of tuberculosis, in which no infective 
 material is forthcoming, there are two methods which may be 
 used : (i) The estimation of the opsonic index (see p. 148), and 
 (2) the use of Koch's old tuberculin. This is usually considered to 
 be too dangerous for practical use, but if a small dose only be given 
 (tu^ c.c), and if it is not employed on cases in which there is a 
 mixed infection, I believe the danger to be practically nil. The 
 tuberculin is sold in i-c.c. bottles. For use, add the contents of 
 one of these to 200 c.c. (about gvii.) of recently boiled and cooled 
 normal saline solution. Mix well and inject i c.c. (ni^xvii.) of 
 the fluid hypodermically. In a positive case there will be a sharp 
 reaction, the temperature rising to 103° or more, and remaining 
 elevated for a day or so. 
 
 Information may also be obtained in some cases by a blood- 
 
LEPROSY 65 
 
 count or by cyto-diagnosis (see pp. 227 and 237). Calmette's 
 ophthalmo -reaction may also be used, but is by no means devoid of 
 danger. 
 
 LEPROSY ^ 
 
 The leprosy bacillus resembles that of tubercle, but it is 
 somewhat straighter and more uniform. It occurs in leprous 
 lesions ip great profusion, and its discovery does not present any 
 difficulty. It has never been cultivated. 
 
 In a suspected case of leprosy films should be made from the 
 nasal discharge, for the nasal cavities are very frequently affected. 
 Indeed, it seems highly probable that the primary lesion through 
 which the bacilli gain access to the body is in the nose in most 
 cases. A small portion of one of the leprous nodules may also be 
 removed, and films made by rubbing the cut surface against a 
 clean slide. If there is an ulcer, films may be made from the 
 secretion from it. 
 
 Films should be stained by the method which we have recom- 
 mended for the tubercle bacillus. If bacilli are present in large 
 quantities, the case is almost certainly one of leprosy, for tubercle 
 bacilli are never found in similar situations except in scanty 
 numbers. If a doubt should arise as to the identity of the 
 bacilli, advantage should be taken of the fact that the leprosy 
 bacillus retains the fuchsin even more firmly than the tubercle 
 bacillus when exposed to the action of an acid. A film from the 
 suspected material should be spread at one end of the slide, and 
 some sputum known to be rich in tubercle bacilli at the other ; 
 the whole should be stained by hot carbol fuchsin, and decolorized 
 by being immersed bodily in 25 per cent, sulphuric acid for half 
 an hour. If the tubercle bacilli are decolorized, any bacilli which 
 have retained the red colour are almost certainly those of leprosy. 
 If the tubercle bacilli are not decolorized, a fresh specimen should 
 be prepared and immersed in the acid for a longer period. 
 
 ACTINOMYCOSIS, OR STREPTOTHRICOSIS 
 
 Actinomycosis is very closely aUied to tuberculosis ; the lesions 
 appropriate to the two diseases are almost identical in histological 
 appearance, and the granuloma which occurs in actinomycosis 
 goes on to fibrosis or to the formation of " cold abscesses " just as 
 a tubercle may do. The formation of fibrous tissue is most 
 
 5 
 
66 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 marked in cattle, and in them the disease is more chronic ; 
 suppuration is more common in man, and the disease runs a more 
 rapid course. 
 
 The pus from an actinomycotic abscess is often viscid, and 
 contains a greater or smaller number of small greenish, yellow, or 
 brownish nodules. They are about as large as the head of a very 
 small pin, and are quite opaque ; under the low power of the 
 microscope such a granule has a coarsely granular appearance, 
 and looks something like a raspberry. If nodules presenting 
 these appearances are found in any specimen of pus, whatever 
 be its origin, a careful microscopic examination should be made 
 to determine its nature. This is not difficult. 
 
 Method. — Place some of the pus which contains these granules 
 on a clean slide, and press another slide upon it so as to crush 
 the granules ; dry, fix, and stain by Gram's method. Do not 
 counter-stain. 
 
 Tumours removed or incised at an operation, or organs removed 
 at a post-mortem examination, should have their cut surfaces 
 rubbed upon the surface of a slide, and the film thus obtained 
 treated in a similar way ; or they may be scraped, and the 
 scrapings spread on a slide. Sections may also be cut, but are 
 not usually necessary for the diagnosis. 
 
 Examination of the Specimens. 
 
 Actinomycosis of cattle is caused by the ray fungus, an 
 organism which derives its name from the star-shaped colonies 
 which it forms whilst growing in the tissues. It consists of two 
 chief parts ; the central portion of the colony is formed of a 
 network of narrow filaments, which have a radial arrangement 
 at the periphery (Plate II., Fig. 6). In this part small bodies 
 which have the appearance of cocci may often be seen. The 
 outer zone consists of the clubs which (when present) are so 
 characteristic. These clubs are flask-shaped expansions of the 
 sheath of the radial filaments already mentioned, and are arranged 
 with their narrow extremities pointing inwards. They are not 
 generally present in man, and when present are often badly 
 developed ; they are much more common and more perfect in the 
 ox, where the disease is more chronic and pus-formation rare. 
 
 The reason for this is, perhaps, partly that man is less resistant 
 against the organism, but probably the chief factor is the difference 
 
ACTINOMYCOSIS, OR STREPTOTHRICOSIS 67 
 
 in the fungus present. The organism in bovine actinomycosis is 
 the ray fungus, which is described above ; but in man there are 
 numerous species of fungus which can bring about infection and 
 cause the disease known cUnically as actinomycosis : this disease 
 is therefore not a specific entity, due to a single cause, but a 
 group of alHed diseases, just as suppuration is. The organisms 
 in question are all members of the genus Streptothrix, and consist 
 of long filaments of mycehum, which differ from the bacteria in 
 showing true branching and in breaking up into " chain spores," 
 resembling chains of cocci. The different species vary very 
 greatly in cultural characters, but there are also marked 
 differences in the appearances met with in films of pus, etc. In 
 what may be regarded as typical cases, the nodules described 
 above, when flattened out and stained by Gram's method, show a 
 central portion consisting of a tangled mass of narrow mycelial 
 threads, some of which may show the degeneration into chain- 
 spores, and look like streptococci, whilst there ..may or may not 
 be a peripheral portion showing a radial arrangement (Plate II., 
 Fig. 6, which was drawn from a remarkably perfect specimen). 
 These colonies vary greatly in size, but do not usually fill up 
 more than half the field of an oil-immersion lens, so that it is best 
 to search for them in Gram specimens (not counterstained) with a 
 low power, and to turn successively the ^ and ^^ on to any small 
 violet masses which may be seen. In other cases the fungus will 
 assume the form of threads in masses without any definite 
 arrangement, and in others the threads will be isolated ; in either 
 case it will usually be possible to find threads showing true 
 branching or chain spores, and this is sufficient for the diagnosis. 
 In yet others the bulk of the mycelium splits up into short lengths 
 greatly resembling bacilli, and when this happens the diagnosis 
 may be missed unless a careful search happens to reveal an 
 unbroken piece of mycelium (Plate IV., Fig. i). 
 
 Cultures are usually difficult to prepare, and are not much help 
 in the diagnosis. 
 
 The importance of making this examination as a routine method 
 in all cases in which the diagnosis is not absolutely clear must be 
 strongly urged on all practitioners, since an accurate diagnosis of 
 actinomycosis may be followed in many cases by a cure by means 
 of large doses of iodide of potassium. Actinomycosis has a habit 
 of turning up when least expected ; thus, I have found it 
 accidentally in three cases in the sputum where the true diagnosis 
 
 5—2 
 
68 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 was not suspected ; once in an enlarged tonsil ; once in an appar- 
 ently typical case of cancer of the breast, etc. 
 
 The films should be carefully examined for the presence of 
 these structures. Clubs are not likely to be found in the pus, 
 and their absence does not tell against the diagnosis ; the dense 
 felted network of filaments retaining Gram's stain is what is to be 
 looked for, and its presence is quite sufficient for a diagnosis. 
 Fortunate specimens may show a complete colony, with its 
 irregular network in the centre and the radial arrangement of the 
 fibres on the periphery, or there may be mere fragments of 
 mycelium. 
 
 GLANDERS 
 
 Glanders is one of the infective granulomata, and is closely 
 allied to tuberculosis ; it differs, however, in running a more rapid 
 course and in the greater tendency which the specific lesions 
 exhibit to undergo suppuration. It is caused by the B. mallei, 
 an organism which is nearly as long as the tubercle bacillus, 
 and decidedly thicker. It stains readily with all stains, and is 
 easily decolorized ; it loses its stain when treated by Gram's 
 method, and does not form spores. 
 
 The bacteriological diagnosis of the disease is not easy, and 
 should be referred to a bacteriologist. A quantity of the discharge 
 from a suspected case should be taken with aseptic precautions, 
 and transmitted as soon as possible in a test-tube or bottle which 
 has been sterilized by dry heat or by boiling. Pus had better be 
 sent in a pipette. 
 
 Where abscesses are opened, cultures taken direct from the pus 
 may possibly contain the bacillus in pure culture. In this case it 
 may be identified by the characters of its growth upon potato. 
 The colonies have the colour and appearance of honey at first ; 
 they grow very rapidly, coalesce, and the potato is soon covered 
 with a moist-looking film, which afterwards becomes brown, the 
 surface of the medium in the neighbourhood becoming greenish- 
 brown. If cultures from pus grown on potato exhibit these 
 appearances, and contain a short and thick bacillus which does 
 not stain by Gram's method, the case may be diagnosed as being 
 probably one of glanders, even although the culture be not a 
 pure one. 
 
TYPHOID FEVER 69 
 
 TYPHOID FEVER 
 
 Typhoid fever is caused by a bacillus which is variable in 
 length, though usually short (about half as long as a tubercle 
 bacillus) and thick, its length being only about three times its 
 breadth ; very long forms also occur, but in small numbers. It 
 does not form spores, and it does not stain by Gram's method. 
 It is actively motile ; when a culture of the organism in a fluid 
 medium is examined under the microscope, the bacilli can be 
 seen darting rapidly about in all directions. The bacillus owes 
 its motility to the possession of a large number of long, wavy 
 flagella, which can only be seen after special and difficult staining 
 processes. 
 
 The B. coli communis, the most plentiful organism of the in- 
 testine in man and animals, bears a very close resemblance to 
 the typhoid bacillus, and can only be distinguished therefrom by 
 the application of several cultural and chemical tests, the perform- 
 ance of which takes a considerable amount of time. This renders 
 it very difficult to diagnose typhoid fever by methods similar to 
 those which are in use for the other diseases mentioned — i.e., by 
 the demonstration of the specific organism. Suppose, for instance, 
 that we were to attempt to determine the nature of a case of 
 diarrhcea by a search for the typhoid bacillus in the stools. For 
 every typhoid bacillus which we should encounter we should find 
 a great many colon bacilli, and we should only be able to dis- 
 tinguish the one from the other by a prolonged and careful 
 examination of pure cultures. It is quite certain that the disease 
 might be diagnosed in this way ; indeed, it has been done, but the 
 task is an extremely difficult one, and the diagnosis would be 
 delayed for a considerable period.* 
 
 In other regions in which the typhoid bacillus occurs during an 
 attack of typhoid fever the search is usually facilitated by the 
 absence of other organisms, especially by the absence of the 
 B. coli. The specific bacillus occurs in the blood, spleen, spots, 
 mesenteric glands, liver, and frequently in the urine. 
 
 It may often be demonstrated in the Mood, and the method is 
 now acquiring some importance from the fact that positive results 
 are found to be very frequent if the examination is made early in 
 
 * Modern methods have greatly faciUtated the task, but even now it is only- 
 used when other methods are unavailable, or for certain special purposes. 
 
70 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 the disease — i.e., within the first week. Thus it is available before 
 the Widal reaction appears, and should be used wherever a 
 positive diagnosis is required at a very early date. The blood is 
 drawn direct from a vein by one of the methods described on 
 p. 144, and at least 5 c.c. should be taken, whilst 10 c.c. gives a 
 greater probability of a successful result. The best culture 
 medium to employ is broth to which -5 to i per cent, of sodium 
 citrate has been added ; this prevents the coagulation of the 
 blood and the consequent entanglement of the bacilli in the clot, 
 which, when it occurs, greatly delays the appearance of the 
 growth. The amount of blood indicated above should be 
 inoculated into 100 c.c. of citrated broth and incubated at 37° C. 
 Growth will probably be seen in twenty-four hours or less. Un- 
 fortunately, typhoid bacilli fresh from the body often fail to 
 agglutinate with typhoid serum, and only acquire that property 
 after cultivation for several generations on artificial media : this, 
 the best test, is not always conclusive. But all cases clinically 
 resembling early typhoid which give a culture of motile, non- 
 sporulating. Gram-negative bacilli of the morphological characters 
 described above, should be regarded as early typhoid, and in all 
 probability the appearance of the Widal reaction will soon settle 
 the matter. The other possible diagnoses are extremely unlikely. 
 The further identification of the bacillus is a matter of some 
 difficulty for which expert help should be obtained, or one of the 
 larger manuals of bacteriology consulted. 
 
 The mesenteric glands and liver are, of course, not available 
 or the purpose of diagnosis. 
 
 The demonstration of the bacilli in the urine is sometimes quite 
 easy, and might be of some diagnostic value. But they do not 
 always occur in this excretion, and the B. coli often does so, and it 
 is not easy to distinguish between the two. 
 
 The only way in which typhoid fever can be diagnosed with 
 ease and certainty by a demonstration of the specific bacillus is 
 by an examination of material drawn directly from the spleen by 
 means of a hypodermic needle. The organism occurs constantly in 
 this situation, and its demonstration is not difficult. The necessary 
 operation, however, is by no means devoid of risk, and is now 
 generally abandoned. 
 
 This brings us to the method in which typhoid fever is now 
 usually diagnosed by the bacteriologist — Widal's reaction. This 
 
TYPHOID FEVER 7I 
 
 reaction is a special example of a general law which was discovered 
 by Durham and others, and which is to the effect that the blood- 
 serum of a person who has been through an attack of a bacterial 
 disease will cause the specific organism of that disease to collect 
 into clumps. For instance, if we take a broth culture of the vibrio 
 of Asiatic cholera (which is turbid and opalescent) and add to it a 
 small quantity of blcod-serum from a patient who has recovered 
 from an attack of cholera, we shall find that the culture becomes 
 clear, a sediment collecting at the bottom of the tube ; and if we 
 examine this sediment we shall find that it consists of felted 
 masses of the vibrios. This reaction is a general one, and is 
 given in most, if not all, bacterial diseases. But Widal, Griin- 
 baum, and others, working independently about the same time, 
 showed that, whereas in many diseases it is a reaction of immunity 
 {i.e., does not occur until late in or after the disease), in typhoid 
 fever it is a reaction of infection, and occurs so early in the course 
 of the disease that it is of great value in diagnosis. 
 
 The test is applied by adding a small quantity of the serum 
 from the patient suspected to be suffering from the disease to a 
 larger amount of a young culture of typhoid bacilli, and watching 
 whether the appearance of the culture undergoes any change : it 
 may be watched under the microscope or by the naked eye, the 
 technique differing in the two cases. The microscopic method is 
 rapid and requires a very small amount of blood, and is now 
 generally used. The macroscopic method is perhaps somewhat easier 
 for a beginner, but it takes a longer time and requires a larger 
 amount of blood-serum. 
 
 The technique in either case is readily learnt, and can be 
 carried out with very little difficulty if the materials are at hand. 
 But the test is one which it is seldom advisable for the home 
 worker to attempt, as it requires a young culture of typhoid 
 bacilli. We shall, however, describe the process, as some may 
 carry it out during an epidemic, or if they are living where they 
 have not access to a public laboratory. The process has now 
 been greatly facilitated by the demonstration of the fact (by 
 Widal) that the reaction is given with dead cultures of the bacilli. 
 These cultures can be obtained from any bacteriological laboratory, 
 and will keep. They are prepared by adding 4 or 5 c.c. of 
 the following fluid to an eighteen-hours-old culture of typhoid 
 bacilli, scraping off the growth, pipetting the emulsion into a 
 
72 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Sterile test-tube, and heating to 60° C. for half an-hour to ensure 
 sterility : 
 
 Normal saline solution (o*8 per cent.) - 450 c.c. 
 
 Carbolic acid (liquefied) - - - 1-25 c.c. 
 
 Glycerin - - - - - ^o c.c. 
 
 Boil and allow to cool immediately before use.* 
 
 The bacilli gradually separate from an emulsion prepared in 
 this way, but are distributed throughout the fluid by shaking. 
 With a supply of this culture at hand the practitioner can apply 
 the test at home without difficulty. 
 
 Collect the blood from the ear, as described on p. 34, taking 
 care to get a Wright's pipette about half full. The test may be 
 applied to a dry drop of blood, but in this case there is no 
 possibility of making an accurate dilution, and unless a fair 
 quantity of serum is at hand it is impossible to perform the test 
 by the macroscopic method. 
 
 If a pipette is not at hand, it is easy to manufacture one out 
 of any piece of glass tubing which may be available. 
 
 Method of performing Widal's Reaction by the 
 Microscopic Method. 
 
 Requisites. — i. A young culture (not more than eighteen hours 
 old) of typhoid bacilli on agar. 
 
 Where dead bacilli are to be used, this is to be replaced by a 
 culture obtained from the laboratory and prepared in the manner 
 described. 
 
 2. A small funnel provided with a double thickness of white 
 filter-paper. This is unnecessary if dead cultures are to be used. 
 
 3. Three clean watch-glasses. 
 
 4. A platinum loop. This should be made of fine wire and 
 have a loop (which must be completely closed) about j^^ inch in 
 diameter. 
 
 5. A hollow-ground slide. This is an ordinary slide having a 
 well about \ inch in diameter sunk in its centre. If it is 
 not at hand a cell may be built up on an ordinary slide. Take a 
 piece of thin card i inch square and cut out a square \ inch in 
 diameter from its centre. Fix this perforated square down on to 
 the slide with vaseline or immersion oil. 
 
 * J. H. Borden, Proc. N. Y. Path. Soc, 1904. 
 
TYPHOID FEVER 
 
 73 
 
 6. Thin cover -glasses. 
 
 7. The microscope. The test can be carried out quite well 
 with a J-inch lens. 
 
 Process — i. Making the Emulsion.— Vom a small quantity of 
 tap-water into the culture-tube, or, better, scrape off some of the 
 growth and mix it with some water in a watch-glass. In either 
 case stir it round with the platinum needle for a few minutes, so 
 that the bacilli are evenly distributed throughout the water and 
 form an emulsion. 
 
 Next take the hollow-ground slide and paint a ring of immersion 
 oil round the well (Fig. 22, h). If you are using a built-up cell, 
 
 Fig, 22. —Hanging-drop Preparation. (Crookshank.) 
 
 paint the top of the card with the oil. In either case vaseline 
 may be used. 
 
 Place a drop of the emulsion on a clean dry cover-glass, and 
 invert the hollow-ground slide over it ; press it down so that the 
 oil round the well adheres to the cover-glass ; now invert the 
 slide, and you will have a hanging-drop specimen. The bacilli will 
 be contained in the droplet of water (Fig. 22, a) which hangs 
 from the lower surface of the cover-glass : this will not dry it up 
 if the seal made by the oil is perfect. 
 
 Place the specimen under the microscope and examine it with 
 the low power, using the fine adjustment and stopping down 
 the diaphragm. Focus until the surface of the cover-glass is 
 distinctly seen, and then move the slide about until the edge of 
 the hanging-drop runs across the centre of the field. Then turn 
 on the i-inch lens and open and shut the diaphragm until the 
 field is faintly illuminated ; the exact amount of light required 
 can only be learnt by experience. 
 
 Now focus up and down very carefully, using the fine adjustment, 
 
74 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 until you see a line running across the field and dividing it into 
 a lighter and a slightly darker portion. This is the edge of the 
 drop. Focus a little deeper ; you should see numerous small 
 unstained bacilli, and if these are not visible it probably indicates 
 that the illumination is not right. Open and shut the diaphragm, 
 keeping a sharp look-out down the microscope all the time. It 
 may help matters to lower the condenser for a short distance. 
 
 Having obtained a clear view of the bacilli, examine them for 
 motility and absence of clumps, and see whether they are present 
 in proper proportion to the amount of fluid. 
 
 If the culture is in good condition, the bacilli should be seen 
 darting about in all directions ; but if the movement is but 
 sluggish, the reaction may still be obtained. If the specimen is 
 kept for a short time in a warm place or in the incubator, the 
 movements will usually become more rapid. It is hardly 
 necessary to say that when dead cultures are used there will be 
 no movements of translation, though the bacilli may show 
 oscillatory (Brownian) movements. 
 
 The specimen must be searched thoroughly for clumps of 
 bacilli, and if these are present the emulsion must be filtered 
 through a double thickness of white filter-paper. This examina- 
 tion for clumps is a most important part of the process, and must 
 be attended to whether dead or living cultures are in use. 
 
 Next see that the emulsion is neither too thin nor too thick. 
 No definite rules can be given, but if there are very few bacilli 
 in the field a further supply of growth must be added to the stock 
 of emulsion, and a further specimen examined. If the bacilli are 
 thickly crowded together, the emulsion must be diluted with a 
 little water and re-examined. 
 
 When you are satisfied that the emulsion is right, slide off the 
 cover-glass and drop it into some antiseptic lotion ; of course, this 
 is unnecessary if dead cultures are used. 
 
 2. Making the Dilution. — You are now about to dilute a drop of 
 the serum from the patient with a known multiple (in this case 
 thirty times) of its bulk of the emulsion which you have just 
 prepared. To do so you will take advantage of the fact that the 
 platinum loop, if dipped into a fluid and pressed against a surface, 
 so that every part of the loop touches that surface, will deposit a 
 drop of fluid of definite size. You are about to mix one loopful 
 of the serum with twenty-nine loopfuls of the emulsion just 
 prepared and examined. 
 
TYPHOID FEVER 
 
 75 
 
 Blow the blood from the pipette out on to a watch-glass (to do 
 this it will be necessary to break the tip of the pipette), and tilt 
 the latter so that the serum flows away from the coagulum. Now 
 take a loopful of the serum and place it on another watch-glass, 
 taking care to put the loop flat on the surface of the glass ; this 
 is done more easily if the wire is slightly bent, or if a flat side is 
 used instead of the watch-glass. 
 
 Next heat the platinum loop in the flame; this is to burn off any 
 blood which might remain on it and contaminate the emulsion. 
 Take up a loopful of the emulsion, and place it on the watch-glass 
 by the side of the drop of serum, but not touching it. Repeat this 
 until you have placed twenty-nine drops of emulsion round the 
 
 Fig. 23. 
 a, Negative Widal's reaction ; b, positive Widal's reaction. 
 
 serum. Mix the whole together by stirring them thoroughly with 
 the platinum loop, place a droplet of the mixture on a clean cover- 
 glass, making a hanging-drop specimen, and examine as before. 
 
 If the blood comes from a case of typhoid fever (with certain 
 restrictions which will be discussed below), the microscopic 
 appearances will be quite diflerent from those seen in the drop of 
 emulsion which was previously examined. The bacilli will no 
 longer swim about rapidly in all directions ; 'they will become 
 paralyzed, and remain quite motionless. Further, they will collect 
 into clumps, each clump consisting of a larger or smaller number 
 of bacilli, arranged in a felted network, resembling that seen in 
 a heap of " spellicans " (Fig. 23, b). This is the complete positive 
 reaction ; it consists of two parts, clumping and paralysis, and is 
 given only (in the dilution used) by the blood of a patient who is 
 suffering or who has sufl'ered from typhoid fever. If this is not 
 the case, the bacilli will continue to move about just as before, 
 and will not collect into clumps. 
 
76 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 In the process which has been described above, the blood has 
 been diluted to thirty times its volume, and this is the best dilution 
 to use for diagnostic purposes. But the reaction is given earlier 
 if a lesser dilution (one in ten) is used, though there is then a 
 greater chance of fallacy. 
 
 Sometimes the reaction takes place almost as soon as the serum 
 is added. At other times it is delayed, and for these it is necessary 
 to fix a time limit. With a dilution of i in 30, one hour is a 
 safe time-limit to adopt, and if the reaction takes place after 
 this the result should be looked upon with great suspicion, and 
 the test reapplied after a day or two. 
 
 Exactly similar processes are carried out if dead cultures of 
 typhoid bacilli are used, but here the emulsion should be sent out 
 from the laboratory free from clumps, and containing exactly the 
 right number of bacilli, so as to be ready for immediate use. But 
 the practitioner is urged not to trust to such an emulsion without 
 making a hanging-drop, and examining it just before making the 
 test. 
 
 If dead cultures are used, it is advisable to use a rather less 
 degree of dilution than in the above process. A dilution of i in 
 20 will answer perfectly. The time-limit is the same. 
 
 Interpretation of Results. 
 
 A positive result may mean : 
 
 1. That the patient is suffering from typhoid fever. 
 
 2. That he has suffered from typhoid fever within a certain 
 period before the blood was taken. The hypothetical substance 
 which we believe to be the cause of the reaction (agglutinin) con- 
 tinues to be formed, or remains in the blood, for some time after 
 complete convalescence from typhoid fever; the reaction has 
 been known to persist for seven or eight years, and probably 
 usually does so for about two. This fact must be remembered 
 in interpreting the results obtained from Widal's test. If the 
 patient has suffered from typhoid fever, or from an obscure 
 illness, which might possibly have been typhoid fever, a year or 
 two previously, the positive reaction should be regarded with 
 suspicion. 
 
 In such cases the test should be carried out so that the smallest 
 dilution which will cause clumping can be ascertained, and the 
 test repeated in two or three days. If, for instance, we found 
 
m 
 
 TYPHOID FEVER 77 
 
 at the blood clumps only in a dilution of i in 20 on one 
 day, and in a dilution of i in 100 three days later, this affords 
 a certain proof that the reaction is due to a present attack 
 of typhoid fever, and is not due to one which took place at a 
 previous date. This investigation should be entrusted to an 
 expert bacteriologist, and plenty of blood sent on each occasion. 
 
 A negative result may mean : 
 
 1. That the patient is not suffering from typhoid fever. 
 
 2. That he is suffering from typhoid fever, but the date is too 
 early for the appearance of the reaction. The reaction sometimes 
 occurs on the fifth or sixth day, usually after the tenth day, and 
 in all but a very small number of cases before the end of the 
 second week. If the onset of the disease (as far as it can be 
 fixed) is less than this, the examination should be repeated after 
 two or three days. 
 
 3. In a very small number of cases the reaction is delayed still 
 further, and if the patient dies may not occur at all. These cases 
 are usually severe ones, and do not present any difficulty in 
 diagnosis. Sometimes the reaction is delayed well into the con- 
 valescence in mild attacks, but this is exceedingly rare. 
 
 Macroscopic Method. 
 
 The macroscopic method can be carried out with a young living 
 culture on agar, or with an emulsion of dead bacilli. The technique 
 is not so difficult as in the previous process, and no apparatus is 
 required other than a piece of narrow glass tubing, from which to 
 make pipettes. 
 
 Requisites. — i. A young culture on agar, and some normal 
 saline solution ; or a dead emulsion of typhoid bacilH. 
 
 2. Special glass pipettes. These are to be made from a piece 
 of glass tubing which should not be more than ^ inch in internal 
 diameter and about 10 inches long. The central 2 inches of this 
 tube are to be heated in a flat gas flame until thoroughly softened, 
 then withdrawn, and then the two ends are to be drawn steadily 
 apart. The softened portion is to be drawn out into a long, narrow 
 portion (rather thicker than a vaccine-tube) about 6 inches long 
 and of nearly uniform diameter. The full diameter of the tube 
 should pass rapidly into this narrow portion ; it should not taper 
 gradually (Fig. 14, c). The two pipettes thus obtained are to be 
 broken apart, leaving about 3 inches of the narrow tubing attached 
 to each. The wide end of the tube is to be plugged fairly firmly 
 
78 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 with cotton-wool. This serves a double purpose : it prevents any 
 of the fluid from getting into the mouth, and it offers a certain 
 amount of resistance to the escape of the fluid, and thus renders 
 the pipettes more easy to fill. 
 
 Process. — Prepare an emulsion just as before, making a drachm 
 or so, and filtering it. An emulsion of dead bacilli obtained from 
 a laboratory should be ready for immediate use. Insert the tip of 
 the pipette into the blood-serum (of which there should be several 
 drops), and aspirate it gently into the tube, avoiding air-bubbles ; 
 if these gain access, blow out the serum and begin again. It is 
 easier to see what you are doing if you use a piece of indiarubber 
 tubing (such as is used with a haemocytometer) through which to 
 apply suction. 
 
 Having drawn up enough serum to form a column about 2 inches 
 in length in the narrow portion of the tube, lay the pipette on its 
 side, and make a mark with ink or with a grease pencil to show 
 how high the serum reaches. Now suck the column of fluid a 
 little way into the tube, and insert the tip of the pipette into the 
 emulsion ; suck the latter up the tube until it reaches to the 
 mark. This will give you the same amount of emulsion as of 
 blood-serum ; the two fluids will be separated by a short column 
 of air. Now withdraw the tip for a moment and suck up another 
 small quantity of air ; dip it into the emulsion and suck it up to 
 the mark again. This will give you twice the amount of emulsion 
 as of blood-serum, the three portions of fluid being separated by 
 air (Fig. 24). Repeat this process until you have nine times the 
 amount of emulsion as of serum ; then suck the fluid still further 
 from the tip of the pipette, and seal the latter in a flame. 
 Probably by this time the fluids will have mixed together ; if not, 
 tap the tube gently (holding it upright) until the air-bubbles 
 which you have sucked up make their escape and the fluid forms 
 a continuous column. 
 
 Fill another tube to a similar height with emulsion (for a 
 control), and place the two side by side in an upright position for 
 twelve hours. 
 
 Now examine the fluid in the pipettes. If the emulsion has 
 been made from a living culture, the control pipette {i.e., that to 
 which no blood has been added) will probably remain turbid ; if 
 an emulsion of dead bacilli has been used, it will have become 
 clear, and the bacilli will form a uniform, even layer at the 
 bottom of the pipette. 
 
TYPHOID FEVER 
 
 79 
 
 ^Trdh 
 
 Compare the control-tube with the pipette to which blood has 
 been added. If the reaction is negative, the appearances will be 
 exactly the same in each ; but if the reaction is positive, the bacilli 
 will fall to the bottom in flocculent _^ 
 
 granular masses, which are similar 
 whether living or dead bacilli have 
 been used. In the former case there 
 is no difficulty in determining whether 
 the reaction is present or absent, for 
 the fluid will be clear if it is positive, 
 turbid if it is negative (Fig. 25). The 
 dead emulsion presents more diffi- 
 culties ; you have to compare the 
 granular deposit which occurs in a 
 positive reaction with the more uniform 
 deposit which occurs in the control- 
 tube. If there is any doubt, the best 
 plan is to break off the tip of the pipette 
 and blow out some of the deposit ; 
 examine it under the microscope after 
 applying a cover-glass. In a positive 
 case the bacilli will be seen to be col- 
 lected into clumps ; in a negative case 
 they will remain discrete. 
 
 This process is a modification of that 
 described by Wright (Brit. Med. Jouvn.^ 
 P- 355' 1898), to which the reader is 
 referred for fuller details. 
 
 It is advisable to use a dilution of 
 I in 20 if the macroscopic method is 
 used. 
 
 I now use dead cultures almost ex- 
 clusively in routine work. They are 
 always ready, and they do not vary in 
 sensitiveness as Hving cultures do. The 
 method I now use is simplicity itself, 
 and is specially suited for general 
 practitioners, since it requires abso- 
 lutely no manipulative skill and gives absolutely accurate 
 results. 
 
 The method is, briefly, as follows : I prepare a number of small 
 
 Fig. 24. — Showing Method 
 OF FILLING Pipette. 
 
 a Is the mark on the tube ; 
 b, the serum ; c, c, c, the 
 columns of emulsion, sepa- 
 rated by the air-bubbles, 
 d, d, d; ^ is the cotton- 
 wool plug. 
 
8o 
 
 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 sedimentation-tubes containing a definite bulk of killed typhoid 
 emulsion and sealed at each end ; each tube also contains a small 
 amount of mercury. Accompanying each of these tubes there is 
 a short length of capillary tubing (about i inch long) ; this will 
 hold a definite fraction (|, y^, gV* ^tc, according to requirements) 
 of the amount of emulsion in the sealed tubes. To use them, 
 collect the blood in the ordinary way in a Wright's blood capsule, 
 pipette, etc., and let it coagulate. It is necessary to get clear 
 serum for the test, but it is usually quite easy to do this, even 
 
 Fig. 25. — Positive and Negative Widal's Reaction : Macroscopic 
 
 Method. 
 
 The fluid in the left-hand pipette (negative) is still turbid. The right-hand 
 pipette (positive) contains clear fluid, and has a sediment at the bottom. 
 The lower portions of the pipette have been removed. 
 
 without a centrifuge. File and break the pipette so that the 
 serum is exposed ; then take the capillary tube and touch the 
 surface of the serum, which will ascend the tube and just fill it. 
 File and break off the tip of the sedimentation-tube (at fe, Fig. 26), 
 drop in the capillary tube full of serum, reseal the tip of the tube, 
 and allow it to cool. When cold, shake it violently for half a 
 minute (when the mercury will act as a stirrer), and put it to 
 stand in a vertical position. The whole process takes about two 
 minutes, and is about as easy as testing for albumin. In positive 
 cases the first indication of a reaction may be seen in about an 
 hour, and it is usually complete in four or five. The fluid first 
 
TYPHOID FEVER 
 
 8l 
 
 shows a slight granularity, which becomes more and more marked, 
 minute greyish flocculi separating themselves from the uniformly 
 turbid emulsion. These flocculi gradually get bigger and settle 
 slowly at the bottom of the tube, leaving the supernatant fluid 
 almost clear. The emulsion will settle by itself, but very slowly, 
 and falls as a powdery deposit quite different from the flocculi. 
 An unused sedimentation-tube (shaken immediately before use) is 
 put side by side with the one containing the serum, and used as a 
 control ; the two are inspected from time to time, and if there is 
 
 Fig. 26. 
 
 a, Tube containing emulsion of dead typhoid bacilli and globule of mercury ; 
 b, showing method of inserting the serum in a short length of capillary 
 tubing ; c, a positive reaction ; d, the control. 
 
 no obvious difference in five or six hours the reaction is absent. 
 The presence of the reaction in a dilution of i in 20 is fairly 
 definite proof of infection. 
 
 The tubes are a little trouble to prepare, but a stock sufficient 
 to last for months may be made at the same time. Prepare 
 the typhoid emulsion in the method already described (p. 72). 
 Next prepare some tubes of the shape shown on Fig. 26, 
 sterilize them by dry heat, and place a globule of mercury in 
 each. Then prepare a couple of Wright's pipettes out of a 
 
 6 
 
82 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 piece of glass tubing about as thick as a lead-pencil and 
 about 4 inches long. This is done by holding the tube between 
 the finger and thumb at each end and softening it thoroughly in a 
 blowpipe or Bunsen flame, rolling it round and round the while. 
 When the central inch or more is quite plastic, the tube is removed 
 from the flame and the two ends drawn slowly apart with a steady, 
 uniform pull, and held apart until the glass is quite set. If this is 
 quite successful, the softened portion should be pulled out into a 
 tube about as wide as a steel knitting-needle, fairly uniform in 
 diameter, and if possible 2 feet long : this will make two pipettes 
 each a foot long. To separate them, melt the central portion in a 
 very small flame (such as that of a wax vesta), and pull them 
 apart rather quickly. If the capillary portion of your tube is 
 much less than 2 feet long it will only serve for one pipette, and 
 the place where it is melted must be close to one end. In either 
 case the result should be a pipette Hke that in Fig. 36. 
 
 The most important part of the pipette is the tip, which is 
 represented, magnified three or four times, below the pipette. It 
 must narrow somewhat suddenly into a very fine capillary tube about 
 J inch long, so as to oppose a considerable amount of friction to 
 the movement of a column of fluid in the tube. If the point is a 
 good one, you will have absolute control over the column, and 
 will be able to suck it up to a given point by means of the india- 
 rubber nipple with absolute precision. If the capillary tip is too 
 narrow or too wide, draw it out again until you have one that is 
 entirely satisfactory. 
 
 This is now graduated into equal parts in the simple and 
 accurate method invented by Wright. Take a few drops of 
 mercury in a short test-tube, and cover it with a little water. 
 Put a mark with a grease pencil or with pen and ink on the 
 pipette about i inch from the tip : this is for the unit. Then 
 take the nipple between the thumb and finger of the right hand, 
 squeeze it, insert the tip in the mercury, and suck up the latter 
 exactly to the mark. Then withdraw the tip from the mercury 
 into the water and suck that up to the mark ; the mercury will 
 rise in front of it. When this is done, replunge the tip into the 
 mercury and suck up a second column. Repeat this process until 
 you have filled the tube with alternate layers of mercury and water, 
 each of exactly the same bulk (Fig. 27). When this is done, 
 remove the tube from the fluid and place it flat down on the table, 
 still keeping suflicient pressure on the nipple to retain the fluids 
 
 1 
 
TYPHOID FEVER 
 
 83 
 
 in the same place, so that the junction of the lowest two columns 
 corresponds exactly to the unit mark. Then get a second person 
 to make an ink or grease-pencil mark at the junction of each of 
 the columns. Expel the fluids, and you will have a Wright^s 
 pipette graduated into equal parts. 
 
 If you cannot suck up enough alternating layers to fill the tube, 
 suck up as many as you can, remove the pipette, and mark it as 
 before. Then relax the pressure so that the whole column is 
 drawn upward, and adjust it until the lowest column just coincides 
 with the uppermost mark you have just made, and the rest of the 
 
 Fig. 27.— Showing Method of Graduating a Wright's Pipette in 
 Equal Parts. 
 
 column extends into the ungraduated part of the tube ; make 
 marks against the intersections as before. 
 
 Now proceed to put the emulsion into the sedimenting-tubes 
 with the mercury. If you wish to prepare tubes to give you a 
 dilution of i in 20 (and this is the most convenient for general 
 work), put as much as will fill nineteen divisions of the pipette, 
 sucking it up into the pipette by using an indiarubber nipple. If (as 
 is likely) your pipette does not hold 19 units, it will be necessary 
 to take two or more pipettes full — say 10 and 9 units respectively. 
 Then seal the open end of the sedimenting-tube in the flame, and 
 heat the whole to 60° C. for half an hour to insure sterility. 
 
 Lastly, cut the pipette up into lengths by notching it with a file 
 
 6—2 
 
84 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 opposite the marks. This will give you a series of short capillary 
 tubes each holding Jy of the volume of the typhoid emulsion in 
 the pipettes. These are to be used for the serum, as already 
 described. 
 
 GONORRHCEA 
 
 Nothing is more certain than the fact that gonorrhoea can only 
 be diagnosed by bacteriological methods ; and every practitioner 
 is very strongly urged to practise himself in these methods and to 
 employ them in all cases. Leaving out of account the confidence 
 which the certainty of a direct diagnosis inspires, there is always 
 the possibility that legal questions may arise, and a practitioner 
 who made a diagnosis of gonorrhoea without employing the only 
 means by which that disease can be diagnosed would make a 
 poor show in cross - examination. Lastly, a bacteriological 
 examination will often tell us that the disease is merely lying 
 latent and is still infective when apparently cured ; but not the 
 reverse, for it is not safe to assume that the disease is cured 
 because no gonococci are found. 
 
 The gonococcus chiefly aff'ects mucous surfaces : the urethra 
 in the male, the urethra and cervix uteri in the female, and the 
 conjunctiva in both sexes. These are the regions in which the 
 primary lesion usually occurs, and it may extend by continuity to 
 more distant parts. 
 
 In the male it may involve the prostate, the vesiculae seminales, 
 and the bladder. It is doubtful whether gonorrhoeal epididymitis 
 is due to this organism or to another. 
 
 In the female the inflammation of the urethra may extend to 
 the bladder. The inflammation of the cervix may extend to the 
 mucosa of the uterus, and thence to the Fallopian tubes (causing 
 pyosalpinx), to the mouths of the tubes (causing local adhesive 
 peritonitis, which probably results in sterility), or to the peri- 
 toneum, where it may cause general peritonitis. 
 
 The gonococcus may escape into the blood from any of these 
 lesions, and the results of this occurrence are arthritis, ulcerative 
 endocarditis, or meningitis ; the two latter are rare. 
 
 The search for the gonococcus may have to be made : (i) in 
 urethral pus from either sex ; (2) in pus from the cervix uteri ; 
 (3) in pus from the conjunctiva ; (4) in pus from the meninges, 
 tubes, peritoneum, or other region, whether removed by operative 
 measures or at a post-mortem examination ; (5) in the blood ; or 
 
GONORRHCEA 85 
 
 (6) in the urine. It is to be noticed that the gonococcus rarely, 
 if ever, attacks the vagina, except in young children, and that in 
 cases of vaginitis the cervical secretion should be examined. 
 
 In the vast majority of cases cultural examinations are quite 
 unnecessary. This is fortunate, for the gonococcus does not grow 
 readily on artificial media. It requires for its cultivation the 
 presence of haemoglobin, and in practice the simplest method 
 (should cultures be required for any purpose) is to smear sterile 
 blood over the surface of an ordinary agar tube and inoculate that 
 with the material to be examined. To prepare these tubes, 
 sterilize the tip of the finger with carbolic lotion, washing off the 
 latter with alcohol or ether ; then prick the finger and squeeze 
 two or three drops of blood into the tube. It will run down the 
 medium and mix with the water of condensation at the bottom. 
 Put the tube in the incubator for twenty-four hours to see if it is 
 sterile. This will probably be the case, as the living leucocytes 
 and fresh serum are probably sufiicient to kill the few stray 
 bacteria that may have entered. It is then ready for use, and at 
 the time of inoculation smear the blood over the surface of the 
 agar with the loop. The colonies are very small and translucent 
 (like those of the pneumococcus), and readily die out. The 
 organism has well-marked morphological characters, and the 
 deductions drawn from these characters need only be corroborated 
 in cases of generalized infection or of meningitis, in which the 
 results are to be published (as they should be), and must, therefore, 
 be proved beyond doubt. In such cases the services of a bacteri- 
 ologist should be called in if possible; or the material may be 
 collected in pipettes with the most careful precautions as to 
 asepsis, and forwarded at once. 
 
 Method of Making the Films. 
 
 The pus is to be spread out into thin films at the time at which 
 it is taken, and this is true whether the practitioner intends to 
 make the examination for himself or is about to send the material 
 to a bacteriologist. Gonorrhoeal pus should never be collected 
 on a piece of cotton-wool or enclosed in vaccine tubes. 
 
 The films are to be made thus : Take two clean slides and 
 place two or three platinum loopfuls of the pus on the centre of 
 one of them ; sterilize the needle and lay it down. Now take the 
 other slide and apply its centre to the pus, and allow it to fall on to 
 
86 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 the first slide by its own weight ; do not squeeze the slides 
 together. Then slide them apart, keeping each in its own plane 
 until they are entirely separated. This will give you two excellent 
 films. Allow them to dry and fix them in the fiame. 
 
 The films may also be made on cover-glasses, exactly the same 
 process being adopted, except that it will be necessary to squeeze 
 the two lightly together. The fixation is accomplished by passing 
 the cover-glasses rapidly through the flame. 
 
 These are the methods by which films are spread in all cases ; 
 the way in which the pus should be obtained varies somewhat 
 with the nature of the case. 
 
 In the male it is advisable to cleanse the meatus and to reject 
 the first drop of pus, taking the second with a platinum loop and 
 proceeding as before. Antiseptic precautions are entirely un- 
 necessary, as no attempt is to be made to get cultures. If the 
 patient is suffering from phimosis, and there is a purulent 
 discharge, which may be due to gonorrhoea, chancre, soft sore, or 
 to a non-specific balanitis, a similar method is adopted ; but here 
 many films should be taken, as a prolonged search may be 
 required. If the patient suffers from a slight discharge in the 
 early morning, the best plan is to give him two clean slides. 
 These are to be smeared across the meatus whilst wet with the 
 discharge, and allowed to dry. 
 
 In the female it is necessary to obtain the pus directly from the 
 urethra ; it may be expressed by the finger in the vagina. The 
 first drop should be rejected. 
 
 If the patient is suffering from cervicitis or endometritis, the 
 pus should be taken direct from the cervix, a speculum being 
 used, and the pus being removed by a platinum loop or probe. It 
 is necessary to emphasize the fact that the material must be from 
 these regions if a negative result is to be of any value. The flora 
 of the vagina in all cases of discharge is so extraordinarily 
 abundant that it is almost impossible to recognize the gonococcus 
 with certainty in such material. 
 
 It is absolutely necessary that you should spread the films 
 at once, even if you are having the examination made at a distance. 
 It is next to useless to send pus dried on linen, cotton-wool, a 
 Volkmann's spoon, or even in a thick layer on a slide. The 
 diagnosis may be made from material sent in this way, but the 
 difficulties are much greater, and in some cases the results are 
 less certain. 
 
GONORRHOEA 87 
 
 Preparation of films from conjunctival pus presents no diffi- 
 culties. The same is true of pus from the tubes or other internal 
 regions, whether it is exposed by operative interference or at a 
 post-mortem examination. 
 
 Instructions for the examination of the blood are given subse-_ 
 quently. A considerable number of films should be taken, as the 
 cocci are present in but very small numbers. 
 
 The urine may be examined in the female if a local examination 
 is not considered advisable, or in the male to obtain evidences as 
 to whether the disease is cured or not. The morning urine should 
 be examined. It should be mixed with a small quantity of carbolic 
 lotion or other antiseptic and allowed to settle for twenty-four 
 hours ; it is much better to use a centrifuge if one is available. 
 In cases where we require evidences as to cure after an attack of 
 gonorrhoea, the urine is examined after gentle massage of the 
 prostate. 
 
 Staining of Films. 
 
 One film is to be stained by a simple stain such as methylene 
 blue or carbol thionin. The other is to be stained by Gram's 
 method, and then in dilute carbol fuchsin for half a minute. 
 
 Examination of Films. — First take the specimen in which the 
 simple stain has been used, and examine it with the oil-immersion 
 lens. You will see that it shows numberless cells with very 
 irregularly lobed nuclei ; these are the pus cells or polymorpho- 
 nuclear leucocytes. There will also be some flat squamous 
 epithelial cells. 
 
 The gonococci will be stained even deeper than the cell nuclei, 
 and will be mostly contained within the pus cells. If you see a 
 cell which contains numerous small blue or violet granules, bring 
 it into the centre of the field and examine it more thoroughly, to 
 see whether the granules have the characters of the organism 
 which we are about to describe. 
 
 The gonococcus is a large diplococcus, each component of the 
 pair being shaped like a kidney, the hilum being turned toward 
 that of its fellow. Single forms (which may be rounded) and 
 tetrads are sometimes seen. It does not stain by Gram's method, 
 and this is one of its most important features. Another important 
 point is its arrangement ; during the height of an attack of 
 gonorrhoea it is almost entirely intracellular, being contained 
 within the polymorphonuclear leucocytes. Further, several pairs 
 
88 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 occur in each cell, and the great majority of cells are entirely 
 devoid of cocci (Plate III., Fig. 2). 
 
 If the organism which you find possesses these characteristics, 
 turn to the specimen which has been stained by Gram's method 
 and counterstained by carbol fuchsin (Plate I., Fig. 6). In this 
 all bacteria which retain Gram's stain will be coloured violet, 
 while organisms which do not retain it will be red. You must, 
 therefore, search for groups of diplococci contained within the 
 cells and possessing the above characteristics. They will not be 
 so prominent as in the other specimen, for the cells, nuclei, etc., 
 will be coloured red also, and the contrast is not so great. But if 
 the case is one of gonorrhoea, you will find them after a careful 
 search. 
 
 If the films came from a female, you will probably find the 
 appearances masked by numerous other organisms, especially by 
 the B. vagina, a rather large bacillus (somewhat resembling 
 that of anthrax), which stains by Gram and is often present in 
 large numbers, even if the film was taken in the manner mentioned. 
 Yet in a positive case you will probably find the cells packed with 
 non-Gram-staining diplococci having the above characters without 
 much difficulty. 
 
 Interpretation of Results. 
 
 You are justified in considering a case to be one of gonorrhoea 
 if in films made from the pus — 
 
 1. Large kidney -shaped diplococci are present. 
 
 2. These cocci occur within the pus cells, and at least four pairs 
 (Foulerton) in each affected cell. 
 
 3. The vast majority of cells are entirely free from cocci. . 
 
 4. The organisms in question do not stain by Gram's method. 
 This is an absolutely essential point. 
 
 If cocci are present which answer to the above description, 
 except that they are not enclosed within cells, the case may still 
 be one of gonorrhoea. The gonococcus is frequently extracellular 
 during the early stages of an attack of urethritis, and, though to a 
 less extent, during its involution, whilst cases sometimes occur in 
 which a considerable number of the cocci lie free during the 
 whole course of the disease. 
 
 In the interpretation of films from a female, the above criteria 
 must be insisted on very stringently. In the extraordinary assort- 
 ment of bacteria met with, if there is an admixture of the vaginal 
 
SYPHILIS 8g 
 
 secretion, you may often find organisms resembling the gono- 
 coccus in some points, but not in all. These yield one of the 
 most troublesome problems that the clinical bacteriologist has 
 to face. 
 
 SYPHILIS 
 
 It is now abundantly proved that the Spirochata (or Treponema) 
 pallida of Schaudinn is the actual cause of syphilis, and the 
 diagnosis of this disease by the recognition of the causative agent 
 is now practicable in some cases. The organism is a spirochaete, 
 in shape resembling a spirillum, but probably of animal nature 
 {i.e. J a protozoon). It is very small, or rather very narrow, and 
 stains with great difficulty : it is this fact which has led to its having 
 been overlooked previously. In length it is about equal to the 
 diameter of a red corpuscle, either more or less ; it is made up of 
 about eight or ten close-set curves, and it has sharp ends. These 
 facts are very important, for there are numerous spirochaetes some- 
 what similar in appearance, which are frequently found in ulcers 
 of all sorts, in the mouth, etc., and which have no doubt been 
 frequently mistaken for Schaudinn's organism (Spiroch^ta pallida). 
 The main difference is that in the other common spirochaetes (one 
 of which, the commonest, is called 5. refringens, and is closely 
 allied to, or identical with, that of Vincent) the curves are wider. 
 For example, if we found two spirochaetes exactly as long as a 
 red corpuscle is wide, and in one there were eight complete curves 
 and in the latter only three or four, the former would probably be 
 pallida, the latter refringens (Plate V.). The former is also said 
 to look stiffer and to be less easily bent. The staining reactions are 
 different in the two cases, refringens being stained, though not 
 deeply, with borax-methylene blue or dilute carbol fuchsin, whilst 
 the latter is not. ' 
 
 Method. — The examination may be carried out on scrapings 
 from a supposed chancre, secondary ulcer, condyloma, etc. ; on 
 juice obtained from an enlarged lymphatic gland by puncture with 
 a hypodermic needle ; on the blood expressed from a secondary 
 rash after puncture of the skin, or, according to some authors, in 
 fluid from a blister raised on or near a lesion of such a rash. It 
 appears to be especially abundant in the pemphigoid rashes of 
 hereditary syphilis. It is very rarely found in gummata or in 
 any tertiary lesion. 
 
 Spread the material thus obtained in a very thin layer on a 
 
go CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 scrupulously clean slide, allow to dry, and fix by gentle heat — 
 i.e.f such that it does not get uncomfortably hot to the finger. 
 
 Prepare a mixture of lo c.c. tap- water (or of distilled water 
 + I drop of a I in i,ooo solution of potassium carbonate), and 
 lo drops of Giemsa's stain, w^hich must be bought ready pre- 
 pared. In mixing the two avoid violent agitation ; add the stain 
 to the water in a test-tube, stopper the latter with your thumb and 
 invert it slowly once or twice. Do not mix it until you are ready 
 to proceed with the staining. 
 
 Take the slide in a perfectly clean pair of forceps, flood it with 
 the stain, and heat until it just begins to steam ; remove it from 
 the flame and in fifteen seconds pour off" the solution, replace it 
 quickly with fresh and heat again, again removing it when steam 
 rises and allowing the action to go on for fifteen seconds. Do 
 this four times in all, allowing the action to go on for one minute 
 on the last application. Then wash in tap- water or in distilled 
 water with a drop or two of potassium carbonate solution, blot, 
 dry, and mount. 
 
 Another method is to place the slide face downwards in a Petri 
 dish, supported on two slips of glass. The dish is then filled with 
 a mixture prepared as above, and the staining allowed to go on 
 for twelve to twenty hours in the cold, or three to four hours in 
 the incubator, the dish being covered to prevent evaporation. If 
 the slide is inserted face upwards, it will probably be covered by 
 a fine red precipitate. 
 
 The most minute amount of acid is fatal to the process ; hence 
 all instruments must be dry and clean, and distilled water (which 
 often contains traces of acids) should be avoided. 
 
 Examine the film with the ^^ and your highest eyepiece, taking 
 great care to get a good light, white if possible. 
 
 Another method, with which I have been fairly successful, and 
 which is strongly recommended by Herxheimer, consists in 
 fixing as before, and allowing the film to stain for twenty-four 
 hours in a I in i,ooo solution of Nile blue in distilled water. The 
 spirochaetes are stained a fine blue, and are readily recognizable. 
 
 In making the search a good lens and good light are always, 
 and much patience frequently, necessary ; the spirochaetes may 
 be but one or two on a film, or there may be several on one field 
 of the microscope. Very occasionally they are matted together 
 in a dense mass. Having found a spirochaete, proceed to see if 
 it resembles the pallida or the refringens ; note especially its 
 
CHOLERA 91 
 
 ength (comparing it with a red blood-corpuscle) and the[number of 
 turns of which this distance is made up. If there are about six 
 or eight turns to this distance, it is almost certainly pallida ; if 
 there are fewer, it is not. Examine its ends, and see whether they 
 taper off to a point or terminate abruptly. 
 
 It is also possible to see the spirochaete unstained, when it is 
 actively motile, but it is not possible to distinguish it from its 
 congeners in this way without much practice. In point of fact 
 practice is an essential in the diagnosis of syphilis by the recognition 
 of the spirochaete, and the practitioner is recommended to identify 
 the organism as often as he can in undoubted cases before 
 attempting its recognition in doubtful ones for diagnostic purposes. 
 
 CHOLERA 
 
 The diagnosis of cholera can only be made on clinical grounds 
 alone during an epidemic, as other diseases present almost 
 identical symptoms and course. The importance of making a 
 correct diagnosis arises less from the interests of the patient than 
 from those of the general public ; if the case is one of true Asiatic 
 cholera, the sanitary authorities must be notified and the fullest 
 precautions taken to prevent the spread of the disease. In all 
 suspicious cases a quantity of the rice-water stools (in a bottle 
 sterilized by boiling or by dry heat and securely packed) should be 
 forwarded at once to a public laboratory. Meanwhile the 
 diagnosis may be established with a fair amount of certainty by 
 the following simple tests : 
 
 1. Take a platinum loopful of the dejecta and spread it in a 
 thin film on a clean slide ; dry, fix, and stain with carbol fuchsin 
 for three minutes ; wash, dry, and mount. 
 
 2. Prepare another film and stain by Gram's method. 
 
 Examination of the Films. 
 
 The spirillum of Asiatic cholera is about half as long as a 
 tubercle bacillus, or rather longer, and much thicker. It is 
 slightly curved : hence the name of the " comma bacillus." It 
 looks very like a caraway seed (Plate II., Fig. 5). 
 
 In the carbol fuchsin specimen vast numbers of these curved 
 rods will be seen ; probably few other organisms, if any, will be 
 present if the case is one of true cholera. Two or more rods 
 may often be found joined together with their concavities turned 
 
92 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 in Opposite directions, giving the whole the appearance of a 
 very elongated spiral. In the stools (but not usually in cultures) 
 the individual rods have frequently a parallel arrangement, pre- 
 senting the appearance of " shoals of fish swimming up stream." 
 
 If you see these appearances examine the Gram specimen. 
 Very few organisms will be visible, as the cholera vibrio does 
 not retain the stain when treated in this way. 
 
 If vibrios having the above characters are present, proceed as 
 follows : 
 
 Take two or three small flasks (preferably sterilized by heat), 
 and add to each loo c.c. of water, i gramme of peptone, and 
 J gramme of common salt ; boil thoroughly, and allow to cool. 
 This forms a culture medium in which the cholera vibrio will 
 grow very rapidly, and other organisms far more slowly. 
 
 Inoculate each flask with a loopful or two of dejecta, plug 
 each with cotton-wool, and incubate for eight to twelve hours at 
 37° C. If cholera vibrios are present the cultures will conform to 
 the following tests : 
 
 (a) There will be a film on the surface. This will be more 
 marked after a few hours longer. 
 
 {b) This scum will present the microscopic appearances 
 described above, except that the vibrios are usually somewhat 
 straighter than those which occur in the stools, and the " fish-in- 
 stream" arrangement is not marked. They will not stain by 
 Gram's method. 
 
 (c) The addition of a small quantity of pure strong sulphuric 
 acid will give a pink or crimson tint. This is the " cholera-red " 
 reaction, and is caused by the action of sulphuric acid on indol 
 in the presence of a minute quantity of a nitrite; many other 
 organisms {e.g., the B. coli) produce this colour after the addition 
 of a nitrite, very few without it. The cholera vibrio produces 
 nitrites as well as indol. 
 
 Interpretation of Results. 
 
 In a case in which the above phenomena are observed, the 
 inference that the patient is suffering from true Asiatic cholera is 
 so strong that the authorities should be notified and the fullest 
 precautions taken. 
 
 A case in which they are absent is almost certainly not one of 
 true cholera. 
 
PLAGUE 93 
 
 PLAGUE 
 
 The bacteriological diagnosis of plague should be made by an 
 expert ; not because it is difficult, but because so much hinges 
 upon it — at least, in this country. A brief account of the method 
 by which a practitioner who was unable to obtain expert help 
 might proceed may not be out of place. 
 
 The plague bacillus is a short and rather thick rod which occurs 
 in vast numbers in the bubo, in the blood, and in the internal 
 organs. It does not stain by Gram's method, and when stained 
 by other processes it often exhibits a characteristic polar staining, 
 the ends of the bacillus being coloured deeply, whilst the inter- 
 vening portion remains colourless (Plate II., Fig. 4). It might 
 be mistaken for a diplococcus ; it could not be mistaken for 
 the pneumococcus (to which it has some resemblance), as that 
 organism stains by Gram. Degenerate forms which resemble 
 cocci, etc., often occur in cultures, but are seldom met with in 
 the body during life. 
 
 The diagnosis may be made by an examination of fluid 
 aspirated from the bubo or of the blood. In bubonic cases the 
 former method should always be adopted, as the bacilli are 
 present therein in vast numbers, and generally in pure culture ; 
 the amount of fluid which has to be removed is very small, even 
 if cultures have to be taken. 
 
 When this is not the case two films should be made in the way 
 already described, fixed and stained, the one by dilute carbol 
 fuchsin or Loffler's blue, and the other by Gram's method. If 
 the bacilli are present, they will appear as short oval rods which 
 may or may not exhibit the polar staining ; if the specimen has 
 been stained for the proper length of time (about two minutes), 
 most of them will do so, but in any case it will most likely be 
 present in a few. The Gram specimen will not show these rods ; 
 there may be a few pus cocci present as a secondary infection. 
 
 The blood is examined by any of the methods to be described 
 subsequently, and a very careful search made, as the numbers of 
 the bacilli may be comparatively scanty. 
 
 If a careful examination of stained films made from a bubo 
 does not show the organisms having the above characters, it is 
 probably safe to say that the case is not one of plague. 
 
94 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 SOFT SORE 
 
 A very strong case has been made out in favour of the view 
 that the bacillus described by Unna and by Ducrey is actually 
 the cause of soft sore, though the proof does not rest upon the 
 solid ground demanded by Koch. The bacillus has never been 
 cultivated outside the body, and the chief proof of its pathogenicity 
 rests upon the fact of its invariable presence in true soft sores. 
 It is hardly correct to say that it is invariably absent from other 
 sores, unless, indeed, we extend somewhat our conception of what 
 a soft sore really is ; for the author has found bacilli which were 
 indistinguishable from these in sores which were exactly like 
 those which follow herpes preputialis, though more severe than 
 is generally the case. But it is quite possible that future research 
 will show that the bacillus varies greatly in virulence, and that it 
 is only under certain circumstances that it produces typical soft 
 sores. At present all we can say about the diagnostic value of 
 the organism is that its presence in the secretion from an ulcer 
 affords strong evidence that the case is really one of chancroid, 
 and that its ab.ence almost certainly nullifies such a diagnosis. 
 It is scarcely necessary to say that syphilis and soft sore (or 
 gonorrhoea and soft sore) may be inoculated at the same time, 
 and the lesions appropriate to both diseases may be present 
 simultaneously. 
 
 The bacillus in question is a short straight rod, less than a 
 quarter the length of the tubercle bacillus, and not much longer 
 than the bacillus of influenza. It is rather thick in proportion to 
 its length, its length being only about three times its breadth, and 
 it has rounded ends, which often stain more deeply than the 
 centre of the bacillus. It does not stain by Gram's method ; it 
 stains, indeed, with some difficulty, and powerful stains (such as 
 dilute carbol fuchsin or Loffier's blue) should be used. It is best 
 demonstrated in films made from the deeper parts of a typical 
 soft sore, for the superficial parts contain bacteria of all sorts, 
 and the recognition of Unna's bacillus is not easy unless it is 
 obtained in large numbers. 
 
 The bacteriological examination for this bacillus has most often 
 to be made in cases of urethral sore, or of a sore concealed 
 beneath a phimosis. The method of obtaining the specimen is 
 the same in both cases. A fairly stiff platinum loop is inserted 
 
SOFT SORE — RINGWORM 95 
 
 beneath the prepuce, or into the urethra, and moved gently about 
 until the most tender spot is found. This should be scraped as 
 forcibly as the patient will allow, and the loop withdrawn, care 
 being taken that the mass of secretion is not wiped off in so doing. 
 Several films should then be made, the secretion being rubbed up 
 on the slide with a drop of water. They should be stained with 
 either of the stains mentioned above for five minutes or more : 
 it is an advantage to warm them gently. They are then rinsed 
 in water, dried and mounted, and examined thoroughly with an 
 oil-immersion lens. 
 
 When buboes occur in the course of soft sore, the pus they 
 contain should be examined for this organism as soon as they are 
 opened ; the interest in this is chiefly scientific, for opinions are 
 divided as to whether they are caused by this germ or by pyogenic 
 bacteria. In three cases examined by the author it was found (in 
 very scanty numbers) on one occasion, staphylococci on another, 
 and no bacteria of any sort in the third. 
 
 RINGWORM 
 
 An examination of the hair and scales from the skin is of very 
 great value to the dermatologist. It settles conclusively the 
 question whether a patient is or is not suffering from ringworm, 
 and often gives important information as to prognosis, and may 
 enable us to say whether the disease has probably been derived 
 from a human source, or has been contracted from one of the 
 lower animals. 
 
 Where the mere diagnosis of ringworm is in question, an 
 examination of the hair or scales after soaking in liquor potassae 
 is often sufficient. The materials are placed in a drop of the 
 solution on a slide, and covered with a cover-glass ; after a quarter 
 of an hour or so the specimen is carefully examined under the 
 microscope, using a J-inch lens and a small diaphragm. The 
 spores appear as spherical or oval, highly refractile bodies, which 
 can hardly be mistaken for anything but fat globules. This 
 possible source of fallacy may be removed by soaking the hair 
 in ether before applying the liquor potassae. 
 
 This is a rapid and simple method, but it does not enable us to 
 diagnose the nature of the fungus with certainty, except in very 
 typical cases, and when spores are scanty they are readily over- 
 looked. It is a very great advantage to employ some method of 
 
96 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Staining. These are by no means difficult, though they are some- 
 what tedious. Two processes, both modifications of Gram's method, 
 will be given. They are not difficult to apply, but are very slow. 
 
 Method of Staining Hairs, Scales, etc 
 
 Requisites. — i. Aniline gentian violet^ carbol gentian violet, or 
 carbol fuchsin. 
 
 2. Gram's iodine solution. 
 
 3. Aniline oil, to which a little strong hydrochloric acid has been 
 added, two or three drops to a quarter of a test-tube full of the oil. 
 Prepare when required, and shake. 
 
 4. Aniline oil. 
 
 5. Xylol. This is not necessary unless the specimens are to 
 be kept permanently. 
 
 6. Blotting-paper. 
 
 7. Slides, cover-glasses, and balsam. 
 
 Process. — Take a broken hair or stump from the edge of the 
 suspected area, and cut off all the free portion, except a piece 
 about \ inch long. The root and the part of the shaft next 
 to it are all that are to be examined. Several of these hairs can 
 be stained at the same time. 
 
 Stain in a watch-glass full of stain, or on a slide, for a quarter 
 of an hour, or longer — the longer the better ; it is an advantage to 
 warm the stain gently, and it will penetrate better if the hairs 
 have been previously washed in ether, though this is not absolutely 
 necessary. 
 
 Remove the hairs from the stain, and place them on a slide ; if 
 already on a slide, pour off the stain. Dry them with blotting- 
 paper, and pour on the iodine solution ; allow it to act for five 
 or ten minutes. It is an advantage to blot the specimen after a 
 minute or two, and apply a fresh lot of the solution. Blot thoroughly. 
 
 Now decolorize in the solution of hydrochlorate of aniline in 
 aniline oil. This should be poured off and replaced occasionally, 
 and the specimen examined under a low power of the microscope. 
 When the decolorization appears to be complete (it may take 
 many hours), pour off the solution and replace it by aniline oil ; 
 allow this to act for an hour or more, renewing it occasionally. 
 
 If the specimen is not to be kept permanently it may now be 
 mounted in balsam and examined at once. If it is to be kept the 
 
RINGWORM 97 
 
 aniline oil must be washed out by several applications of xylol. 
 Mount in balsam. 
 
 Adamson's method gives good results, but is somewhat more 
 tedious. _ 
 
 Requisites. — i. Liquor potassae. 
 
 2. Dilute alcohol — about 15 per cent. 
 
 3. Aniline gentian violet or its substitutes. 
 
 4. Gram's iodine solution. 
 
 5. Aniline oil. 
 
 6. Xylol. 
 
 7. Blotting-paper. 
 
 8. Slides, cover-glasses, and balsam. 
 
 Process. — Prepare the fragments of hair as before, rejecting the 
 free portions. Place them on a slide, add a drop or two of liquor 
 potassae, and apply a cover-glass. Allow the liquor to act for a 
 quarter of an hour or twenty minutes. 
 
 Now place a large drop of dilute spirit at one edge of the cover- 
 glass, and a piece of blotting-paper at the opposite edge ; this 
 will suck up the potash, and the spirit will run in and replace it. 
 After a few minutes lift over the cover-glass, and wash the hairs 
 gently in more spirit. This will harden them. Dry. If epithelial 
 scales are being examined, they may be fixed to the slide or cover- 
 glass by heat in the usual way. 
 
 Stain in aniline gentian violet for half an hour or less. 
 ■ Pour off the stain, blot gently, and pour on Gram's iodine 
 solution. Allow this to act for five minutes. Blot again. 
 
 Decolorize with aniline oil, pouring it off and applying a fresh 
 lot from time to time. The process may take an hour or more, 
 and the specimen should be left under the microscope and ex- 
 amined occasionally. 
 
 When the decolorization is complete {i.e.^ when the colour is 
 seen to be present in the fungus only), blot gently, and wash 
 thoroughly with xylol. Mount in balsam. 
 
 The specimens are to be examined under a ^-inch objective. 
 A higher power is unnecessary. 
 
 There are certainly three, and possibly more, species of ring- 
 worm which occur in England, and the fungus of favus is closely 
 allied, and is demonstrated by the same process. 
 
 The MicrospoYon Audouini is the most common species of ring- 
 worm fungus in this country, being responsible for about 80 or 
 90 per cent, of all cases. It is the small-spored fungus, and 
 
 7 
 
98 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 it may be distinguished by the fact that its spores are arranged 
 in an irregular mosaic, and not in chains. Its mycelium, which 
 consists of long threads, lies in the interior of the hair, whilst 
 the spores form a thick mass outside (Fig. 28, and Plate VI., 
 Fig. 4). This sheath of spores projects a short distance above 
 the surface, and may often be seen with the naked eye. The 
 outside of the hair is destroyed, and the surface of the hair 
 eroded ; the former feature serves to differentiate it from all 
 other varieties of ringworm, and from favus. 
 
 This fungus usually attacks the scalp in children, and in them 
 it dies out spontaneously about the age of sixteen, whilst other 
 
 Fig. 28. — MiCROSPORON Audouini.* 
 
 varieties of ringworm often do not. It is very rare in adults, and 
 it rarely attacks other regions than the head. It is thought by 
 some to be always caught from a human case of the disease, but 
 there are reasons for thinking that it may be derived by infection 
 from the horse or cat. 
 
 The most important clinical fact about the M. Audouini is that 
 ringworm caused by it is extremely intractable, and may run a 
 prolonged course in spite of the most skilful treatment. 
 
 The Trichophyton (or Megalosporon) endothrix attacks the interior 
 of the hairs, and forms long chains ; it always involves the hair just 
 inside the cuticle, being in reality an endo-ectothrix. Its spores 
 are somewhat larger than those of the preceding species, but the 
 difference is not great ; the organism is most easily recognised by 
 the chain-like arrangement of its spores, and by the fact that 
 
 * Fig. 28 is from Curtis's " Essentials of Bacteriology " (Longmans), 
 
RINGWORM 99 
 
 they lie within the hair, the cuticle of which usually remains 
 intact (Plate VI., Fig. 2). 
 
 This fungus is a rare cause (in this country) of ringworm of the 
 scalp, and the disease caused by it appears to be somewhat easier 
 to cure than that due to the microsporon, though opinions on this 
 point are divided. It also attacks other regions of the body, 
 causing tinea circinata, or eczema marginatum. 
 
 It always appears to be derived from a human case, and never 
 by infection from animals. 
 
 The T. ectothfix (like the microsporon) forms a sheath round 
 the outside of the hair, to which it is closely applied, like the bark 
 to a tree, but, unlike the small-spored variety, does not destroy 
 the cuticle. The spores are about as large as in the preceding 
 species, and are arranged in chains ; this fact, together with the 
 position of the fungus with regard to the hair, will enable a 
 diagnosis to be made (Plate VI., Fig. i). 
 
 This fungus is a rare cause of ringworm of the scalp, and the 
 disease it causes is readily cured. It also causes ringworm of 
 the body and of the beard region, which the endothrix does not 
 attack. According to Sabouraud (to whose work on these 
 organisms we owe most of our knowledge on the subject), kerion 
 is always caused by this organism, but this is not generally 
 accepted. It appears, however, to be a fact that suppurative 
 lesions (folliculitis, kerion, etc.) are usually caused by this fungus. 
 
 This species is often derived from one of the lower animals, 
 especially from the horse, cat, and dog. 
 
 Favus is caused by a closely allied organism, the Achovion 
 Schonleinii. This may be demonstrated by either of the processes 
 already described. It affects the skin in two ways : by the forma- 
 tion of the characteristic scutula and by the ringworm-like invasion 
 of the hair. The scutulum is composed of vertical mycelial fila- 
 ments, which branch, and which appear to be composed of short 
 rods. There are often oval spores at the free ends of these 
 branches, and, according to Sabouraud, branching into three occurs 
 (trichotomous branching), and is very characteristic, though diffi- 
 cult to see. The radiation of several filaments from one point, 
 and the fact that these appear to be made up of short lengths, 
 gives rise to an appearance which has been compared to that of 
 the metatarsal bones, and the term " favic tarsus" has been 
 appUed. 
 
 When favus affects the hair (Plate VI., Fig. 5), the cuticle 
 
 7—2 
 
TOO CLINICAL BACTERIOLOGY ANG HEMATOLOGY 
 
 remains intact, and the inner portion of the hair is packed with 
 long waving filaments, whilst the outside (under the cuticle) is 
 covered with short "elements" — the exact nature of which is 
 doubtful — of every shape and size. 
 
 The identification of the variety of organism which is present 
 is not usually difficult. The first point to be looked to is the 
 arrangement of the spores. If these form an irregular mosaic, 
 the microsporon is present ; if they form filaments somewhat 
 resembling those of a streptococcus, the organism is a tricho- 
 phyton. Then look to see whether the cuticle is intact, and 
 whether the fungus invades the interior of the hair. This 
 can usually be determined by focussing up and down until 
 you see an *' optical section " of the hair in question. It is 
 important not to be deluded by the fact that the fungus which 
 lies on the outside of the hair will appear to be inside it if a 
 surface view only is taken. 
 
 Ringworm of the Nails. 
 
 The nails may be affected by either of the trichophytons or by 
 favus. The diagnosis of the presence of ringworm may usually 
 be made by the examination of pieces of the nails after soaking in 
 liuqor potassae, but a prolonged examination of many pieces must 
 be made before their absence is assumed. The diagnosis of the 
 variety present can only be made by cultures, and is not of cHnical 
 importance (Plate VI., Fig. 3). 
 
 OTHER SKIN DISEASES 
 
 In seborrhoeic affections the bottle bacillus of Unna is constantly 
 present, and as it does not occur at ail frequently in other diseases, 
 if at all, it is a useful test between seborrhoeic dermatitis and 
 psoriasis, especially on the scalp. It is a moderately large 
 organism which consists of an oval, spore-like body attached to a 
 short neck or handle. The former stains faintly, especially in the 
 middle, where it seems to have a clear vacuole, whilst the handle 
 stains deeply. It is very easily recognised after it has once been 
 seen ; it is perhaps the only bacterium which can be positively 
 identified by microscopic means alone. It stains by Gram. 
 
 To search for it, take a scale or two from the affected region and 
 grind it in a drop of water between two slides until reduced to a 
 
OTHER SKIN DISEASES TOI 
 
 pulp. Allow some of this pulp to dry on one of the slides, fix, 
 stain by Gram's method, and do not counterstain. If the film is 
 very greasy, so that the stain does not wet it, warm the slide 
 gently and allow a few drops of ether to flow over the film, fix^ 
 again, and proceed as before. Examine the preparation under 
 a j-^^-inch. The presence of the bottle bacillus is almost conclusive 
 evidence in favour of seborrhoea, as against psoriasis, syphilis, etc. 
 
 The figure (Plate IV., Fig. 6) I owe to the kindness of 
 Dr. Whitfield. It is from an impure culture, the only one that 
 has ever been obtained. 
 
 Tinea Versicolor. — There is usually no difficulty in the diag- 
 nosis of this disease by ordinary clinical methods. Where there is 
 any doubt, one of the scales may be removed and examined in liquor 
 potassae, or by any of the methods described for ringworm. 
 The fungus — M. furfur — is readily detected under a |^-inch. It 
 consists of rather wide mycelial threads, branching and inter- 
 lacing, with masses of refractile spore, looking like bunches of 
 grapes (Plate IV., Fig. 4). 
 
SECTION III 
 
 COLLECTION AND EXAMINATION OF CERTAIN 
 MORBID MATERIALS 
 
 THE COLLECTION AND EXAMINATION OF PUS 
 
 When a simple microscopical examination has to be made, the 
 collection of pus presents no difficulties, as the few bacteria 
 which may gain access from the skin or the air will not lead to 
 error. The case is otherwise where cultural examinations have 
 to be made, or where the material has to be taken to a laboratory. 
 Here the material should be collected in a pipette. This is to 
 be at hand when the abscess is opened ; both ends are to be 
 broken off and passed through the flame two or three times. The 
 pipette should then be held by an assistant, or put with the end 
 which is to be inserted into the pus projecting over the side of the 
 table, so as not to come into contact with any object. 
 
 When the abscess has been opened, a considerable quantity of 
 pus should be allowed to flow out, and the sterilized pipette is 
 then to be passed through the incision (care being taken to avoid 
 contact with its sides) and the pus carefully sucked up into the 
 bulb. The fluid thus obtained may be used to inoculate cultures 
 there and then, or both ends of the pipette may be sealed in the 
 flame. 
 
 The Examination of Pus. 
 
 The organisms which may cause pus are extremely numerous, 
 the most important being streptococci, staphylococci, the pneumo- 
 coccus and the gonococcus, the bacilli of typhoid fever, tubercu- 
 losis, and glanders, the B. coli communis, the B. pyocyaneus (the 
 organism which produces blue pus), and the fungus of actino- 
 mycosis. In the majority of cases the organism which is present 
 in a given sample of pus can be determined by a microscopic 
 examination of films prepared in the usual way and stained by a 
 
 1 02 
 
THE COLLECTION AND EXAMINATION OF PUS I63 
 
 simple stain, such as carbol thionin. A specimen should also be 
 stained by Gram's method and the results compared. 
 
 When cultural examinations are required, the best plan is to 
 make stroke cultivations on agar in the manner described on 
 p. 16, and incubate them for twenty-four hours at the temperature" 
 of the body. The appearances of the colonies will be similar to 
 those described as occurring in cultures made from the blood, to 
 which the reader is referred. It is to be noted, however, that the 
 gonococcus will not grow under such circumstances unless the 
 surface of the medium has previously been coated with blood. 
 
 Another method is to make gelatin plates. This is a very 
 simple matter if the materials are at hand. 
 
 Requisites. — i. Two or three tubes of gelatin. 
 
 2. Two or three sterilized Petri dishes. 
 
 3. A platinum needle — a loop will be best. 
 
 Process.— Inoculate a gelatin tube in the manner described on 
 p. 16, and then melt it by immersion in warm (not hot) water. 
 
 Distribute the pus throughout the melted gelatin by rolling the 
 tube between the hands, and by tilting it from side to side. Do not 
 shake, and do not let the melted gelatin touch the cotton-wool plug. 
 
 Take a loopful of the gelatin and transfer it to a second culture- 
 tube. Melt the gelatin in this and mix as before. Proceed to 
 inoculate a third tube from the second one if you think it probable 
 that the pus is very rich in organisms. 
 
 Now take the first tube and singe the projecting part of the 
 wool plug, and heat the mouth of the tube in order to destroy any 
 germs which may be upon it ; allow it to cool. 
 
 Place the Petri dish on the table in front of you, and raise the 
 lid sufficiently to allow you to insert the end of the test-tube ; do 
 this, and tilt the latter so that the melted gelatin flows into the 
 dish. Immediately replace the lid, and tilt and roll the dish until 
 the gelatin forms an even film over its whole lower surface. 
 Place it on a flat table to set. Repeat the process with the other 
 tubes. Incubate at about 20° C. for two or three days. Examine 
 the dishes, placing them on the stage of the microscope and using 
 the low power. Each organism will have grown into a small 
 colony, which will resemble those which are described in the 
 section on the blood. There will be slight differences, but not 
 enough to lead to error if the examination of the colonies is 
 supplemented by an inspection of stained films. 
 
 The pneumococcus, gonococcus, the fungus of actinomyces, and 
 
104 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 the tubercle bacillus, will not grow on these plates ; the strepto- 
 coccus and the bacillus of glanders will grow feebly, if at all. 
 
 In a day or two longer the plates will, in some cases, be found 
 to have undergone a decided change. If liquefying organisms are 
 present the colonies will soon become depressed below the general 
 surface of the medium, and will be surrounded by haloes which 
 consist of liquefied gelatin. This will happen with the staphylo- 
 cocci and the B. pyocyaneus ; not with the streptococci, the 
 typhoid bacillus, nor with the B. coli. 
 
 The bacillus of blue pus can readily be distinguished from the 
 staphylococci by its morphological appearance (it is a slender rod), 
 and by the fact that the gelatin round the colony is coloured blue 
 or bluish-green, the growth itself being nearly white. 
 
 Interpretation of Results. 
 
 The chief practical value of the bacteriological examination of 
 pus is derived from the fact that if specific vaccine treatment (on 
 Wright's system) is to be used, the vaccine must be prepared from 
 the organism which is causing the disease. If a patient is suffer- 
 ing from a staphylococcic lesion, it is not much use inoculating him 
 for pneumococci, or vice versa. I have seen and obtained such 
 excellent results in some cases by treatment of this nature that 
 very little doubt remains in my mind that the method is one which 
 will be widely used in the future. Except for this the results 
 obtained by a study of the bacteria in pus are more of scientific 
 interest than of practical importance. It is the situation of the 
 collection of pus rather than the bacteria causing it which 
 influences treatment and prognosis. A list of the more important 
 results which are produced by the chief pyogenic bacteria may be 
 of interest. 
 
 Staphylococci are the chief producers of localized suppuration in 
 the skin — such, for instance, as that which occurs in boils, 
 carbuncles, impetigo, folliculitis, etc. They may cause abscesses 
 in any part of the body, and may also give rise to general infec- 
 tions, ulcerative endocarditis, etc., though this is rare. 
 
 It is in the localized skin affections of staphylococcic origin 
 especially that good results are obtainable by specific vaccination, 
 and a cure may often be obtained in cases which are very intract- 
 able by other methods. 
 
 Streptococci usually cause spreading inflammation of the type of 
 erysipelas or cellulitis. They are common causes of osteomyelitis 
 
THE COLLECTION AND EXAMINATION OF PUS I05 
 
 and suppurative and septicaemic or pyaemic processes connected 
 with the puerperium. 
 
 In general infections which are due to streptococci the use of 
 antistreptococcic serum is indicated, and may offer the only hope 
 for the patient. 
 
 The pneumococcus often produces suppuration in connection with 
 the respiratory system, especially empyema. It also causes many 
 cases of suppurative otitis media and meningitis. 
 
 The bacillus of typhoid fever sometimes causes abscesses in con- 
 nection with the bones after (sometimes long after) typhoid fever. 
 It has been found in other suppurative conditions, e.g., empyema. 
 
 The tubercle bacillus gives rise to "cold abscesses," usually in 
 connection with bone. The suppuration which occurs in the walls 
 of phthisical vomicae is due to other bacteria, chiefly streptococci 
 and staphylococci. The pus in true tuberculous abscesses is thin 
 and watery, like milk and water, and often contains small caseous 
 masses. The cells are usually mostly lymphocytes. In most 
 cases it is perfectly easy to find tubercle bacilli in this pus if it is 
 examined when the abscess is first opened, whereas afterwards 
 none may be found after a very long and painstaking search. 
 
 The bacillus of glanders only causes suppuration in the specific 
 lesions of the disease when these run an acute course. 
 
 The B. coli communis is the chief cause of suppuration occurring 
 in connection with the abdominal viscera, especially of peritonitis 
 due to perforation of the intestine and appendicitis. It frequently 
 attacks the urinary passages, causing cystitis, etc. 
 
 The B. pyocyaneus causes blue pus, usually in connection with 
 the skin or subcutaneous tissues. 
 
 The fungus of actinomycosis has been dealt with already. 
 
 THE BACTERIOLOGICAL EXAMINATION OF THE 
 MOUTH AND FAUCES 
 
 The method of examination of the morbid products of the mouth 
 and fauces in diphtheria has been explained in full, and the 
 methods which are used in other conditions are similar in nature. 
 The more important of these allied conditions are : 
 Simple angina and follicular tonsillitis. 
 Vincent's angina. 
 Scarlatinal angina. 
 Thrush. 
 Syphilitic angina. 
 
I06 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Methods. — An examination of a film stained by a simple stain, 
 and of a second prepared by Gram's method, is usually all that 
 is necessary, but it is advisable to be prepared to make cultures 
 subsequently if thought requisite. If the patient is seen at some 
 distance from the laboratory, the material is best collected on a 
 sterilized swab such as is used for diphtheria in the method de- 
 scribed on p. 38, taking great care to rub it on the affected area.. 
 When this is brought to the laboratory, two smears are to be 
 made on slides which have just been sterilized by being heated in 
 the flame, and which have been allowed to cool. As soon as the 
 films have been prepared the swab is to be returned to its sterile 
 tube and kept in readiness for the preparation of cultures, should 
 they be required. It is better to take a second swab, and to keep 
 it until the films have been examined. 
 
 When the patient can be brought to the laboratory it is more 
 convenient to collect the material with a platinum loop. A good 
 loopful of the material is removed, laid on a clean slide, and two 
 films prepared by pressing a second slide firmly on the first and 
 sliding them apart. If there is any difference between the two, 
 the thicker is used for staining by Gram's method. This is fixed 
 in the flame in the usual way, stained by aniline gentian violet 
 (3 minutes), rinsed, fixed in Gram's iodine solution, decolorized in 
 methylated spirit or absolute alcohol until no more colour comes 
 out, stained in dilute (i in 5 or i in 10) carbol fuchsin for a quarter 
 of a minute, washed, and dried. The other film is best stained by 
 carbol thionin, but Loffler's blue answers very well. 
 
 The examination of the films is made at once, and will show 
 whether a cultural examination is necessary, and if so, what 
 medium should be used. Thus, if Gram-staining baciUi are 
 present, diphtheria is suspected, and cultures should be made on 
 blood-serum. 
 
 Simple Angina and Follicular Tonsillitis may be due to 
 streptococci, staphylococci, pneumococci, or the Micrococcus catar- 
 rhalis. These are readily detected in the smears, the first as longer 
 or shorter chains of cocci, the second as cocci which are isolated 
 or in small groups and often contained in the leucocytes, the third 
 as pairs of cocci with a more or less marked lanceolate shape 
 and a capsule: all these stain by Gram. The M. catarvhalis 
 (Plate III., Fig. 5) is recognisable by its shape (kidney-shaped, or 
 a sphere with a segment cut off), by its being larger in size than 
 the staphylococcus, by its being frequently intracellular, and by 
 
BACTERIOLOGICAL EXAMINATION OF MOUTH AND FAUCES IO7 
 
 its not staining by Gram's method. Here a warning already given 
 must be repeated : it is not safe to conclude that an organism 
 does not stain by Gram because the intracellular bacteria are 
 decolorized, and extracellular ones should be sought for, and Ynll__ 
 usually be found. 
 
 If no organisms but the above cocci are found after a very 
 careful search, the conclusion is that the case is probably either 
 simple angina, follicular tonsillitis, ulcerative tonsillitis, or scarlet 
 fever. But it must be remembered that, although a good large 
 area of film has been searched, in reality but a very small volume 
 of secretion has come under observation — probably less than a 
 cubic millimetre — and that diphtheria bacilli may be present ; it 
 is therefore advisable to make a culture on blood-serum before 
 giving a definite diagnosis. 
 
 No conclusions as to the cause of the angina or as to its prog- 
 nosis can be given from the discovery of the causal organism. 
 The presence of a streptococcus renders it more likely that the 
 disease is scarlet fever, but certainly does not prove it. Nor does 
 the examination give much help as to treatment. If the disease is 
 due to a streptococcus and the symptoms are severe, antistrepto- 
 coccic serum may be given, the local treatment being continued 
 as usual. 
 
 Vincent's Angina is a very interesting form of sore throat 
 recently described by Professor Vincent, of Paris, and is especially 
 important since (i) it closely resembles diphtheria, and the two may 
 be readily confounded, and it also may easily be confused with a 
 syphilitic lesion; and (2) it is readily cured by appropriate treat- 
 ment — friction twice daily with a tampon soaked in tincture of 
 iodine. It has attracted very little attention in this country, yet 
 it does not seem to be rare ; I have now seen many cases. 
 One was of great severity, and was associated with the forma- 
 tion of an abscess in the tonsil, and subsequently of another 
 in the soft palate. 
 
 Vincent describes two forms : 
 
 I . An ulcero-membranous variety, which commences with fever 
 and general malaise, and with redness of a tonsil or of a pillar of 
 the fauces. In a day or two a grey or yellowish false membrane 
 appears on the injected area ; it is soft and but slightly adherent, 
 and when removed the mucous membrane is found to be ulcerated. 
 As the disease proceeds the membrane increases in thickness, and 
 a deep ulcer is formed. The breath is foetid and the tongue 
 
I08 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 furred, salivation occurs, and deglutition is painful. The sub- 
 maxillary glands may be enlarged. In most cases the patient 
 recovers in a week or fortnight, but the affection may become 
 chronic and last a month or more. 
 
 More severe forms occur in which the soft palate, uvula, 
 tongue, etc., are invaded, and ulceration also occurs. In some 
 cases there is a scarlatiniform rash, which might lead to the 
 diagnosis of scarlet fever. 
 
 2. The diphtheroid form is rarer, occurring only in 2 per cent, 
 of Vincent's cases. The onset is accompanied by a little fever, 
 some difficulty in swallowing, and foetor of the breath. Locally, 
 the mucous membrane is inflamed and injected, and a whitish 
 membrane is formed ; it is thin at first, but becomes thicker, and 
 when removed leaves an ulcerating or bleeding surface, but the 
 ulceration is less than in the other form. The disease runs a 
 shorter course, recovery occurring in four to eight days. This is 
 the form which so closely resembles diphtheria, and which, I have 
 no doubt, has often been mistaken for it, even after a superficial 
 bacteriological examination of the membrane. 
 
 The diagnosis is made from films prepared from the swabs, 
 stained by Gram's method and counterstained by carbol fuchsin. 
 In the more common form (the ulcero-membranous) two very 
 interesting organisms will be found — a bacillus and a spirillum 
 (Plate IV., Pig. 2).'' In the diphtheroid form the bacillus is 
 present, but not the spirillum ; in either form of the disease the 
 characteristic organism or organisms may be associated with 
 streptococci, staphylococci, etc., and these secondary infections 
 may give rise to grave complications. 
 
 The B. fasiformis, which occurs in both forms of the disease, 
 may be found, in small numbers, as a normal inhabitant of 
 the mouth, and occurs in myriads in the disease. It varies in 
 size, but is on the whole a large bacillus, about as long as the 
 diameter of a red blood-corpuscle, or even longer. Typically it 
 has both ends pointed, giving it the shape of a greatly elongated 
 spindle, but other forms always occur, and may even constitute 
 the majority of the bacilli present. It often contains two or three 
 clear vacuoles, which may not be noticed if the staining is too 
 deep. Both the bacillus and the spirillum are usually actively 
 motile, and it is a good plan to check the results of the examina- 
 
 * It is possible that these are different stages of the same organism, a 
 protozoon. 
 
BACTERIOLOGICAL EXAMINATION OF MOUTH AND FAUCES lOQ 
 
 tion of the stained films by mounting a fresh wet specimen 
 between slide and cover-glass, and examining it under the oil- 
 immersion lens. 
 
 The B. fusiformis plays a very important part in many in- 
 flammatory and ulcerative conditions in and about the mouth 
 and adjacent cavities, the teeth, etc. For instance, in association 
 with the same spirillum it is present in the pus of pyorrhoea 
 alveolaris. The lesions it causes are all associated with a foetid 
 odour, and the cultures (which are very difficult to obtain) have a 
 similar smell. 
 
 The spirillum occurs only in the ulcero- membranous form, 
 and is present in vast numbers, usually even more plentifully 
 than the bacillus ; a well-prepared specimen is one of the most 
 striking and characteristic objects to be seen in the whole range 
 of bacteriology. It is much longer than the bacillus, very thin, 
 and either wavy and irregular in outline or thrown into definite 
 corkscrew curves. These are better seen in a wet specimen, 
 though here the active motility of the organism often makes it 
 impossible to make out its exact shape. It usually stains badly, 
 and I have missed it in specimens rapidly stained with weak 
 stains : dilute carbol fuchsin stains it very well in a quarter of a 
 minute. In one or two specimens I found the spirilla broken up 
 into chains of very minute granules, so that they resembled long 
 chains of very minute streptococci. In each case it was late in 
 the disease, so that they may have been degeneration forms. 
 
 The spirillum differs from that of syphilis in that it is much 
 larger and is easily stained by ordinary dyes, and the two could 
 hardly be mistaken. It is to be noted, however, that the 
 B. fusiformis is not uncommon in syphilitic lesions. 
 
 Scarlatinal Angina cannot be diagnosed with certainty from 
 other forms of sore throat by bacteriological methods. It is usually 
 due to a streptococcus which occurs in very long chains and is 
 somewhat characteristic, but which cannot be diflferentiated from 
 other forms of streptococci by simple means. 
 
 Thrush is usually easily recognisable, but when this is not the 
 case a Gram-stained specimen of the membrane will immediately 
 settle the diagnosis. The specific organism, the Oidium albicans 
 or Saccharomyces albicans, is a mould which appears in the form 
 of large and thick branching mycelial filaments which stain deeply 
 by Gram's method, and which are interspersed with large round 
 or oval spores, which also stain readily and deeply. The organism 
 
no CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 can be readily diflferentiated from the bacterial filaments which may 
 occur in the mouth (leptothrix, etc.) by its relatively enormous size. 
 When cultivated for a day or two on ordinary media mycelium 
 formation does not take place, or only to a very limited extent, 
 and the oval or spherical spores are often mistaken for yeasts. 
 
 Syphilitic Angina may be recognised by the identification of 
 the Spirillum pallida^ but some caution is necessary, since non- 
 pathogenic spirilla are frequently present in the healthy mouth. 
 The films stained by Giemsa's stain must be very carefully 
 compared with others stained by simple dyes (thionin or methylene 
 blue), to make sure that the organism found is not coloured by 
 ordinary means. 
 
 THE BACTERIOLOGICAL EXAMINATION OF THE 
 NOSE AND ACCESSORY CAVITIES 
 
 In health the nasal mucous membrane is sterile except for that 
 portion in close proximity to the orifices ; the vibrissae are 
 especially contaminated with air-borne organisms, and contact 
 with them must be avoided if cultures are being taken. 
 
 Methods. — In most cases a simple microscopical examination of 
 the mucus, muco-pus, or pus from the nose is sufficient, and the 
 material may be taken from the patient's pocket-handkerchief 
 immediately after he has blown his nose. Where cultures are 
 required the methods are more difficult, and the material must be 
 collected as near as possible to the region where it is secreted ; 
 this is especially the case in the examination of the pus from 
 cases of empyema of the antrum, frontal sinus, etc. I have 
 found the most convenient instrument is a long capillary pipette 
 of rather wide calibre, and bent to an angle of about 135 degrees, 
 at a point some 4 inches from its tip, which must be carefully 
 rounded in the flame, so as not to injure the mucous membrane. 
 It is provided with an indiarubber nipple, and is readily prepared 
 from one of the straight pipettes described on p. 151, by heating 
 it gently in a spirit-lamp, at a point about 4 inches from the tip, 
 until the glass is just softened, and then allowing the distal end 
 to fall until the proper angle is reached. The pipette may be 
 sterilized by passing it rapidly through the flame, taking care not 
 to melt it. 
 
 To use it, seat yourself opposite to the patient, insert a nasal 
 speculum sterilized in carbolic acid or in the flame, and examine 
 the nose in the ordinary way by means of a beam of light 
 
BACTERIOLOGICAL EXAMINATION OF THE NOSE, ETC. Ill 
 
 directed up the nose from a laryngoscope mirror. Insert the tip 
 of the pipette, taking great care not to touch the vibrissae or the 
 mucous membrane near the orifice, and pass it upward until it 
 comes in contact with the pus. Then squeeze the nipple and 
 allow it to expand again, slightly moving the tip of the pipette 
 about in the pus, and taking care not to bring it in contact with the 
 mucous membrane. In most cases you will be able to suck up 
 a few drops of pus ; sometimes it is very thick and turbid, and will 
 not enter the pipette, and in this case you must use a platinum 
 loop, which is not nearly so efficacious or so easy to use. But in 
 
 Fig. 29. — Angled Pipette for Collecting Pus from the Nose. 
 
 most cases the angled pipette will be found available after a very 
 little practice. 
 
 The organisms most frequently found in inflammation of the 
 general surface of the mucous membrane of the nose are the 
 diphtheria bacillus, the influenza bacillus, the M. catarvhalis, 
 and, less commonly, the pneumococcus. These do not call for 
 further mention. In early cases of leprosy the bacilli may be 
 found in the nose, apparently before they are present elsewhere 
 in the body, and the diagnosis may be made from their recognition. 
 
 In suppuration of the antrum and other sinuses the bacteriology 
 is very variable. In disease of the antrum due to carious teeth 
 the B. fusiformis^ mixed with numerous other organisms, may be 
 found. In other cases the commonest bacteria are pneumococci, 
 streptococci, staphylococci, and the M. catarrhalis, usually unmixed 
 in each case ; the subject, however, has not yet been sufficiently 
 investigated. 
 
 THE CONJUNCTIVA 
 
 The method of examination is very simple. A little pus is taken 
 with a platinum loop and films prepared. One of these is stained 
 with carbol thionin or Loffler's blue, and the other by Gram 
 followed by dilute carbol fuchsin. 
 
112 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 The most common causes of conjunctivitis are the gonococcus, 
 the pneumococcus, the Koch-Weeks bacillus, and the bacillus of 
 Morax and Axenfeld. In addition to these, certain other bacteria 
 must be briefly mentioned. 
 
 The method for the recognition of the gonococcus need not be 
 further described (see p. 84), nor is it necessary to point out the 
 danger of this form of conjunctivitis, nor the fact that prompt 
 measures must be taken if the eye is to be saved. 
 
 Other organisms somewhat resembling the gonococcus some- 
 times occur in the conjunctiva, but I have never seen or heard of 
 a case in which they were absolutely identical in appearance and 
 distribution, and present in numbers sufficient to lead to errors in 
 diagnosis. 
 
 The pneumococcus is not a common cause of conjunctivitis, and 
 when it occurs the prognosis with regard to the involvement of 
 the cornea is not serious. 
 
 The bacillus of Koch and Weeks is extremely minute, and has 
 a close resemblance to the influenza bacillus. It is very thin in 
 proportion to its length, does not stain by Gram, and is fre- 
 quently intracellular, the cells then containing large numbers of 
 bacilli, though it may be necessary to search over a considerable 
 area of film before an aff"ected cell is seen. It is advisable to 
 search for the organisms in a thionin or Lofller's blue specimen, 
 as it will probably be more distinct than in the Gram specimen 
 counterstained with carbol thionin. 
 
 If the two occurred in the same region, it would probably be 
 indistinguishable from the influenza bacillus ; they differ, how- 
 ever, in cultural characters. 
 
 It causes the common self-limited variety of acute or chronic 
 conjunctivitis. There is no danger that the cornea may become 
 infected ; the disease is very contagious. 
 
 The bacillus of Morax and Axenfeld (Plate IV., Fig. 3) causes the 
 dry conjunctivitis which occurs especially along the edges of the 
 eyelids and at the angles of the eye, and which does not tend to 
 cure in the absence of appropriate treatment. The secretion is 
 usually very scanty, and not purulent ; it is best collected from 
 the caruncle, and it may be necessary to use a capillary pipette 
 for the purpose. 
 
 It is readily recognised as a rather large, broad bacillus, with 
 the sides parallel and the angles slightly rounded, and two bacilli 
 are often seen with their ends approximated together. It is most 
 
THE SPUTUM 113 
 
 frequently extracellular, but some bacilli are frequently seen within 
 the cells. Gram's stain is not retained. It grows readily on blood- 
 serum, which is rapidly liquefied. 
 
 Of the rarer causes of conjunctivitis, the staphylococcus, strepto- 
 coccus, and pneumobacillus may be mentioned. The latter may 
 be recognised by the presence of a capsule surrounding a bacillus 
 which greatly resembles an elongated pneumococcus, which does 
 not retain Gram (Plate III., Fig. 3). 
 
 Diphtheria affects the conjunctiva, and its clinical recognition is 
 not usually difficult. But the bacteriological diagnosis (without 
 the use of animal inoculations) is complicated by the fact that 
 an organism — the xerosis bacillus — which closely resembles the 
 diphtheria bacillus may occur in the conjunctiva either in health 
 or in disease. It does not appear that the two can be differen- 
 tiated with certainty by morphological appearances, staining 
 reactions (the xerosis bacillus ol'ten shows polar granules), or 
 even by cultural tests. 
 
 In tuberculosis of the conjunctiva it is usually necessary to excise 
 a piece of the lesion, and cut sections, but bacilli have been 
 detected occasionally in scrapings. 
 
 THE SPUTUM 
 
 The chief applications of bacteriology to the examination of the 
 sputum have been mentioned already, but it seems advisable to 
 add a few general words on the subject. 
 
 The selection of the material is of prime importance, since it 
 is obviously useless to expect an examination of the secretion 
 of the mouth and fauces to yield information as to the state of 
 the lungs. Where the sputum is copious the danger of this error 
 is not great ; in advanced phthisis the contents of a sputum-cup 
 taken at random will usually show signs of tubercle bacilli. Where 
 the sputum is but scanty the possibility is great, and in this case 
 the patient should be supplied with a clean (and, if possible, sterile) 
 sputum-cup, to be used only for collecting the material for examina- 
 tion, and he must be instructed to use it only after a paroxysm 
 of coughing, and when he distinctly feels the sputum come up from 
 the chest. If the patient is not sufficiently intelligent, the task of 
 collecting the sputum may be entrusted to a nurse, to whom the 
 importance of securing material directly from the lungs has been 
 explained. 
 
 8 
 
1 1 4 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Where it is necessary to work with material which has been 
 collected without any precaution, the microscopical nature of 
 the cells may afford a clue as to its origin. Mucus or muco-pus 
 derived from the mouth, pharynx, etc., is characterized by containing 
 squamous cells ; these are of large size, flattened, have a compara- 
 tively small nucleus, and are often collected into groups of three 
 or four, with a distinct tessellated arrangement. They contain 
 granules (of keratin or an allied substance) which stain deeply by 
 Gram's method ; they are also the last substances (other than 
 acid-fast bacilli) to be decolorized by the acid in the Ziehl-Neelsen 
 process, and when the decolorization has not been carried out 
 quite completely, may remain stained in the form of pinkish 
 plaques. The sputum which comes from the bronchi may be 
 characterized by the presence of columnar cells, which are occa- 
 sionally found to be ciliated, but in most cases this sign fails, and 
 the sputum consists of mucus enclosing polynuclear leucocytes 
 and no characteristic cells of any kind. The same is true of the 
 sputum from "the lungs : there is frequently nothing to dis- 
 tinguish it from that of other regions, and this is especially the 
 case when it is derived from a cavity which is lined with pyogenic 
 membrane. But sometimes the sputum contains very charac- 
 teristic alveolar epithelial cells. These are derived from the lining 
 of the alveoli, the cells of which lose their flattened shape and 
 become spherical in many pathological conditions. They are large 
 round or oval cells, much larger than the polynuclear leucocytes, 
 and have clear protoplasm and a round or oval nucleus, which is 
 often placed eccentrically. They are actively phagocytic, and 
 their contents give an important clue to the nature of the patho- 
 logical process at work in the lungs, since they are derived directly 
 from the alveoli. In pneumonia they may be seen to contain 
 pneumococci, and this is especially the case after the crisis ; in 
 congestion, and especially passive congestion, of the lung they are 
 packed with red corpuscles, often in various stages of destruction, 
 or with granules or crystals of altered blood-pigment. In diseases 
 due to dust the cells contain fragments of the dust in question, 
 but it must be remembered that particles of coal-dust are often 
 found in them in normal conditions in city dwellers. 
 
 In asthma the characteristic Curschmann's spirals may be seen, 
 and the cells will be found to consist almost entirely of eosinophile 
 leucocytes. 
 
THE GASTRIC CONTENTS AND VOMIT II5 
 
 THE GASTRIC CONTENTS AND VOMIT 
 
 The bacteriological examination of the stomach contents is not 
 of much importance except in one case — i.e., in the differential 
 diagnosis of carcinoma ventriculi and simple dilatation of the 
 stomach. For this purpose it is usually sufficient to examine the 
 vomit, or, if vomiting does not occur, the gastric contents removed 
 by a stomach- tube. It is quite unnecessary to give a test meal, 
 though this is advisable if a chemical examination is to be made ; 
 this should be done in all doubtful cases, but since the methods 
 requisite are outside the scope of this work, only a couple of 
 simple tests will be given. 
 
 The examination is carried out quite simply by the wet method. 
 Two or three drops of the vomit (avoiding undigested food or 
 mucus, since the latter is likely to be derived from the mouth or 
 pharynx) are placed on a slide, and either examined just as they 
 are, or a drop of watery methylene blue, gentian violet, or other 
 stain, is added and stirred well in, and the mixture covered with a 
 cover-glass. If there is an excess of fluid (so that the cover-glass 
 is floated up), it may be sucked up by means of a piece of blotting- 
 paper; and if an oil-immersion lens is to be used, it will be 
 advantageous to seal the cover-glass down by means of melted 
 paraffin applied by a hot iron, so as to prevent it from being 
 lifted up by the suction of the oil between the lens and cover- 
 glass. If no positive results are found in the first specimen 
 examined, two or three more should be made, as the characteristic 
 organisms are often not distributed uniformly throughout the 
 vomit. Subsequently it may be necessary to prepare dried films 
 to determine whether the organisms stain by Gram. 
 
 In carcinoma of the stomach the characteristic features are 
 (i) the presence of the Boas-Oppler bacillus, (2) the usual absence 
 of sarcinae, and (3) the absence of hydrochloric and presence of 
 lactic acid. In addition there may be blood, pus, and fragments 
 of tumour ; the latter are very rare and difficult to diagnose with 
 certainty. 
 
 The Boas-OppUv bacillus, or B. geniculatuSf is very characteristic 
 of carcinoma of the stomach, though it does not occur in every 
 case. It is very rare in other conditions ; I have seen it once in 
 a case of simple chronic gastritis, but here it was present in 
 numbers which were very scanty as compared with the profusion 
 
 8—2 
 
Il6 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 in which it occurs in malignant disease, and it would have been 
 overlooked on a casual examination. It is a bacillus of large size, 
 and has a tendency to grow into long threads, which are readily 
 visible under a J-inch lens (Plate IV., Fig. 5). In a wet specimen 
 these threads usually seem continuous, but on examining a 
 dried and stained specimen, they may be seen to be composed of 
 bacillary segments, much like the chains of the anthrax bacillus. 
 In a wet unstained specimen these threads are very easily 
 recognised, as they are highly refractile and may have a slow, 
 crawling motion, or may be non-motile : most usually the latter. 
 Another characteristic feature is the presence of an obtuse angle 
 in some portion of the length of many of the threads, whence the 
 name B. geniculatus is derived. The organism does not form 
 spores. It stains by Gram, and I have noted in several cases the 
 presence of a phenomenon which is very rare amongst bacilli, 
 that of longitudinal "fission in a small number of the chains, 
 which thus come to form double rows of bacilli in close lateral 
 approximation. That this is not a mere effect of staining is 
 shown by the fact that occasionally the two bacilli at one end of 
 this double thread will turn away from one another, so that it 
 looks as if there were true branching at the extremity of the 
 filament. If this turns out to be the case in all specimens, it will 
 be a most important means of identifying the organism.* 
 
 Cultures may readily be obtained on media rather highly 
 acidified with lactic acid, but are not requisite for the identifica- 
 tion of the bacillus. 
 
 In addition to the Boas-Oppler bacillus, the vomit may contain 
 a few yeasts, distinguished by their large size, oval or spherical 
 shape, and by the way in which they reproduce themselves by 
 budding ; there may also be a few bacteria of other sorts. But 
 in many cases the bacilli are present in a state of almost absolute 
 purity, and in large numbers, so that they form tangled masses. 
 
 In nearly all cases in which the bacillus is found the vomit 
 contains no hydrochloric acid, or only a trace. To test for it 
 filter some of the vomit and place a drop or two of the filtrate on 
 a white porcelain tile ; place a drop of a 0*5 per cent, alcoholic 
 solution of dimethyl-amido-azo-benzol close to it and let the two 
 gradually mix. If physiologically active HCl is present, even in 
 very small amount, a transient pink colour will be produced. The 
 
 * Note to Third Edition. It is not, though I have since seen it two or 
 three times. 
 
THE GASTRIC CONTENTS AND VOMIT II7 
 
 commonly recommended phloroglucin and vanillin test does not 
 demonstrate the presence of HCl in combination with proteids, 
 and since this is usually the form in which it occurs in the 
 stomach contents the failure to obtain the phloroglucin and 
 vanillin reaction is not of the slightest importance in diagnosis. 
 The use of the latter test is, I think, one of the chief reasons for 
 the common idea that the absence of HCl is of very little value 
 in the diagnosis of malignant disease. Using the dimethyl-amido- 
 azo-benzol test, I have rarely found it fail, and in the only case I 
 remember in which the acid was found present in considerable 
 amount the growth was found at operation to be spreading out- 
 wards, and hardly to involve the mucous membrane at all. But 
 these tests were mostly made on the fluid obtained after a test 
 meal, and not on vomits. 
 
 Lactic acid usually occurs in the absence of free hydrochloric 
 in cancer of the stomach, but its presence is in itself a fact of 
 little value, since it occurs in other conditions. To test for it in a 
 vomit it is necessary to extract the acid by thoroughly shaking up 
 some of the fluid with ether, pipetting off the latter, and then 
 allowing it to evaporate. The acid is contained in a state of com- 
 parative purity in the residue, which is then dissolved in water and 
 tested in the following way ; To half a test-tubeful of i in 40 
 carbolic add one or two drops of liq. ferri perchlor. A fine 
 amethyst colour will result, and will be changed to a bright 
 canary-yellow on the addition of the solution of the ethereal 
 extract, if the latter contains lactic acid. It is not sufficient to 
 apply this test to the filtrate of a vomit direct, although this is 
 permissible in the case of the fluid removed from the stomach 
 after a test meal. 
 
 The vomit in cases of simple dilatation of the stomach usually 
 contains a variety of bacteria, yeasts, etc., but the most character- 
 istic organisms are the sarcinae, a group of cocci which have 
 very definite microscopical characters. They have the property 
 of dividing by three successive divisions in the three planes of 
 space (at right angles), and the eight resulting cocci do not com- 
 pletely separate from one another. The result is the formation of 
 a group of eight cocci which form one mass, having the shape of 
 a bale of soft material tied round by three tightly drawn cords 
 at right angles to one another. Successive divisions take place 
 parallel to these, and a very complex colony results. The sarcinae, 
 as a rule, are decidedly larger than ordinary cocci, though not as 
 
ri8 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 large as yeasts ; the different cocci of each group usually vary in 
 size amongst themselves, the younger forms being the larger. 
 Most of them stain by Gram, but this test is hardly necessary, 
 as they may be readily recognised in unstained wet prepara- 
 tions. 
 
 They occur in profusion (often mixed with yeasts) in many 
 cases of simple dilatation of the stomach, though not in all. 
 They occur in other conditions, but are very rarely found in cases 
 of carcinoma, and this is the only importance attaching to them. 
 
 THE URINE 
 
 The more important investigations in which the urine has to 
 be examined have been mentioned already. See p. 62 (tubercle 
 bacilli) and p. 87 (gonococci). 
 
 Methods. — Where the examination is to be microscopical and 
 not cultural it is not usually necessary to use a catheter specimen. 
 
 The urine is to be passed directly into a sterilized vessel (or at 
 least a clean and dry one), the first portion being passed into 
 another vessel and rejected. 
 
 The examination should commence by the inspection of a 
 hanging-drop preparation, first under the \, then under the y\r. 
 This will enable you to recognise the presence of motile bacilli, 
 streptococci, other cocci, etc. ; pus, blood, epithelial cells, etc., 
 and crystals. 
 
 In most cases it is not absolutely necessary to centrifugalize 
 the urine, since the bacteria are commonly present in large 
 numbers, but it is an advantage when this can be done. It is 
 not advisable to allow the urine to deposit spontaneously, as there 
 are many chances of accidental contamination, and many common 
 bacteria grow with great rapidity in urine. 
 
 Then pass on to film preparations, preferably from the centri- 
 fugalized deposit. Prepare films in the ordinary way and stain by 
 Gram, counterstaining by dilute carbol fuchsin. This will enable 
 you to study the organisms more closely, and to see whether 
 they retain Gram or not : most of them do. The staphylococcus 
 (which appears in the urine mostly as diplococci) and the M. uvece 
 do so, and the gonococcus and the gonococcus-like diplococcus 
 which causes cystitis are decolorized. 
 
 Where cultures are required, a catheter specimen must be used. 
 
THE URINE iig 
 
 The catheter must be boiled, and the urinary meatus sterilized. 
 The first portion of the urine is to be rejected, and a small 
 quantity of the last part collected in a sterile test-tube, and 
 the plug immediately replaced. A small quantity only is re- 
 quired. ^ . 
 
 Cystitis, Pyelitis, Etc. — These may be due to many organisms, 
 either pure or mixed, and there is but little practical interest in 
 their recognition, except in cases in which the vaccination treat- 
 ment is to be tried. There is, unfortunately, no method of 
 distinguishing between cystitis and pyelitis by the examination 
 of the urine. The chief bacteria causing suppuration in the 
 urinary passages are : 
 
 B. Coli. — This is perhaps the commonest form, and in cystitis 
 or pyelitis due to it the urine remains acid unless other organisms 
 gain access. 
 
 The organism can usually be identified with a fair amount of 
 certainty by an examination of an unstained hanging-drop prepara- 
 tion, when numerous short bacilli will be seen in active movement, 
 and the fluid will be found to contain pus cells. Follow this 
 examination by making a film of the urine, staining by Gram and 
 counterstaining by dilute carbol fuchsin, when the bacilli will be 
 seen stained red. These appearances in an acid urine raise 
 strong presumptive evidence of B. coli, but are not conclusive, as 
 the typhoid bacillus is almost identical. Where cocci or other 
 organisms are present, the urine may be alkaline in spite of the 
 presence of B. coli. 
 
 Proteus Vulgaris. — This is one of the common organisms of 
 suppuration, and produces cystitis with an alkaline urine. It 
 often occurs in conjunction with B. coli, and the urine is alkaline 
 in this case also, the proteus being more powerful as a producer 
 of alkali (by the ammoniacal decomposition of urea) than the 
 B. coli is of acid. 
 
 It closely resembles B. coli in appearance, and is motile ; the 
 chief points of difference are that it is more irregular in size and 
 forms longer threads, and that some of the bacilli often fail to 
 decolorize by Gram. In cases where the two organisms are 
 present it is impossible to distinguish the one from the other, 
 either in a hanging-drop or in stained films. 
 
 If it is necessary to know whether B. coli or B. typhosus is 
 present along with Proteus vulgaris in an ammoniacal urine, the 
 only method is to plate out some of the urine on gelatin 
 
120 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 (see p. 103), making several plates with different dilutions; as a 
 rule, a minute trace of the urine is all that is necessary. Typhoid 
 and coli form small, semi-translucent, greyish colonies which do 
 not tend to spread very much, whilst proteus forms small grey 
 colonies which form radiating branches and which liquefy the 
 gelatin, forming a saucer-like excavation with a little white mass 
 of growth in the centre. 
 
 Streptococci occur occasionally as independent causes of pyelitis 
 or cystitis, and in these cases I believe the prognosis to be some- 
 what worse than in the other forms. 
 
 Staphylococci usually occur as a secondary infection of other 
 forms of cystitis. The M. tirece is a frequent cause of ammoniacal 
 decomposition of the urine, and is often mistaken for a staphylo- 
 coccus ; the two are indistinguishable under the microscope, 
 but staphylococci liquefy gelatin rapidly and M. uvece does so 
 slowly. Their separation is of no clinical importance in these 
 cases. 
 
 The M. uvece appears to be identical with the common skin- 
 coccus, w^hich is the most common and characteristic organism of 
 the epidermis. It is a coccus which very frequently occurs as a 
 contamination in cultures, and, as a consequence, has been 
 accused of causing an abundance of diseases, including cancer. 
 
 Gonococci. — When these are found in the urine it is necessary 
 to consider whether they come from the bladder or urethra. To 
 do so it usually suffices to see whether they occur in all parts of 
 the urine, and not simply in the first portions, which wash the 
 pus from the urethra. 
 
 A caution is necessary in the diagnosis of gonorrhoeal cystitis, as 
 some cases of inflammation of the bladder (with acid urine) are due 
 to an organism closely resembling the gonococcus, but a little larger 
 and more variable in size : I believe it to be M. catarrhalis. In 
 cases where gonorrhoeal cystitis is suspected make a culture on 
 ordinary agar or on blood-serum. The diplococcus in question 
 grows readily, the gonococcus does not. 
 
 Typhoid Bacilli. — These are rare causes of cystitis, but are 
 commonly found in the urine during and after an attack of 
 typhoid fever. When they produce cystitis the urine remains 
 acid. 
 
 They cannot be distinguished from B. coli except by rather 
 complicated cultural tests or, less certainly, by means of the 
 agglutination reaction. In cases where they are suspected the 
 
THE URINE 121 
 
 urine must be plated out on gelatin, and several small greyish, 
 semi-transparent, non-liquefying colonies picked out with a straight 
 platinum wire and each transplanted to a fresh agar tube. After 
 twelve to eighteen hours' incubation there should be a delicate 
 greyish growth, with a slight tendency to spread from the line of 
 inoculation. This is to be tested by performing Widal's reaction 
 with it, using the blood from a patient who is suffering or has 
 suffered from typhoid fever, and whose blood is known to give a 
 Widal's reaction in a high dilution— say, i in 200. If the blood 
 clumps the cultures you have obtained from the urine when used 
 in the same dilution, the organism is almost certainly the typhoid 
 bacillus. 
 
 The importance of this examination is that the typhoid bacillus 
 may be excreted in the urine for years after an attack of typhoid 
 fever, causing no symptoms, but remaining a very potent cause of 
 infection. Since this is a source of such danger to others it is 
 advisable to make use of the services of an expert (who will use 
 better methods than that described) in the examination of a 
 suspected case. The escape of the bacilli may usually be 
 stopped by the administration of urotropin. 
 
 Bacteriuria, or Bacilluria, is a term which should be 
 restricted to the escape of bacteria in the urine without the 
 presence of pus and without clinical evidence of cystitis, pyelitis, 
 etc. The organisms may be very variable ; B. coli is perhaps the 
 most common. 
 
 The disease may be diagnosed when a urine which contains 
 numerous bacteria is found on several occasions to be free 
 from pus. 
 
 THE COLLECTION OF FLUIDS FROM SEROUS 
 CAVITIES 
 
 A bacteriological examination of the inflammatory exudates 
 which collect in the various cavities of the body often yields 
 important information as to the nature of the morbid process, 
 suggests treatment, and influences our views as to the prognosis 
 of the condition. This is especially the case with the fluids which 
 collect in the pleura, the membranes of the brain and cord, and 
 the joints. In some cases all the needful information may be 
 obtained by the examination of stained films, cultures being un- 
 necessary ; and in these cases no antiseptic or aseptic precautions 
 
122 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 (other than those which are dictated by the interests of the 
 patient) are necessary. But in the greater proportion of cases 
 this is not enough, and cultures must be obtained. To this end 
 it is absolutely essential that the most scrupulous precautions should 
 be taken against contamination of the fluid by the organisms 
 which are constantly present in the air and in the skin, or the 
 results will be worthless. The precautions taken must be as 
 complete as those which are used before an operation upon a 
 joint. Indeed, a fresh precaution has to be taken, for whereas 
 the presence in the skin of a small quantity of an antiseptic 
 would not be detrimental to a surgical operation, it might, by 
 getting into the fluid, nullify a bacteriological examination. 
 Hence the skin must be aseptic, and free from any antiseptic 
 chemical. 
 
 The technique, as far as aseptic precautions are concerned, is 
 as follows: The skin at the region to be punctured is first 
 thoroughly cleaned with soap, hot water, and (if the patient can 
 stand it) a nail-brush. Then layer after layer of some reliable 
 antiseptic lotion is painted on, each layer being allowed to soak 
 in before the next is applied. The most suitable lotions for the 
 purpose are perchloride of mercury (i in i,ooo), biniodide 
 of mercury (i in 500 of methylated spirit), or carbolic acid 
 (i in 20). 
 
 After being allowed to act for at least ten minutes, the skin is 
 to be thoroughly cleansed with methylated spirit ; this should be 
 rubbed in with a piece of cotton -wool, and should be poured 
 copiously over the area. The operation may now proceed. 
 
 Where possible it is preferable to apply a dressing of lint 
 soaked in one of the above lotions (which need be of only half 
 the strength) for a few hours. 
 
 The puncture may be made by using some sort of exploring 
 syringe, or a hollow needle without any means for aspiration. 
 The former is used for the removal of fluid from the pleura 
 or synovial cavity, the latter in performing lumbar puncture. 
 But it is necessary that the whole of the instrument used should 
 be rendered sterile by heat; chemical antiseptics are as a rule 
 inadmissible. In the case of a hollow needle no difficulty occurs ; 
 some hypodermic syringes, however, will not stand boiling, and 
 these are useless for the purpose, as the apparatus which is to 
 be used must be boiled for at least five minutes. 
 
 We shall now deal with the most important cavities of the 
 
THE PLEURA 123 
 
 body, describing the methods to be employed in the investigation 
 of the inflammatory exudates which they may contain, and the infer- 
 ences which may be drawn from the results of the examination. 
 
 THE PLEURA 
 
 There is but little to be said about the method to be employed 
 in the collection of fluid from the pleural cavities. The most 
 careful antiseptic precautions are to be taken, and the region to be 
 punctured should be decided by consideration of the physical signs. 
 
 The examination of the fluid thus obtained may be either 
 microscopical, cultural, or by injections into animals. If the 
 latter are required (and inoculation should be performed in all 
 cases where a tuberculous origin is suspected) a considerable 
 quantity of the fluid — an ounce or more — should be enclosed in 
 a bottle which has been sterilized by boihng, and forwarded at 
 once. A drachm or more of lo per cent, sodium citrate solution 
 (boiled) should be added to prevent coagulation. 
 
 Where the diagnosis is to be made by cultural methods, and 
 the cultures are not to be made on the spot, the fluid is best 
 stored or sent to a laboratory in pipettes. These are to be filled 
 from the syringe direct ; the needle is to be removed, and the end 
 of the pipette (sterilized by being passed through the flame) is 
 passed into the fluid, and filled by gentle suction at the other end. 
 Each end is then sealed in a flame, care being taken not to heat 
 the fluid. Two or three such tubes should be sent. 
 
 Clear fluid from the chest rarely shows any micro-organisms on 
 microscopical examination. Cultures are usually sterile ; where 
 streptococci or pneumococci are found the inflammation is likely 
 to pass on into suppuration. The great majority of these cases 
 of "simple " acute pleurisy are really due to the tubercle bacillus, 
 but their true nature is difiicult to determine except by inoculation 
 experiments. A cytological examination of the fluid should be 
 made, and in tuberculous cases the cells present will usually be 
 found to be lymphocytes, with an occasional admixture of blood- 
 corpuscles (see p. 237). If the fluid does not clot spontaneously, it 
 may be very thoroughly centrifugalized, and films prepared from 
 the deposit and stained for tubercle bacilli in the ordinary way ; 
 but they are not always found even in true cases of tuberculous 
 pleurisy. Where the fluid coagulates spontaneously, the best 
 plan is to allow the clot to retract until it has shrunk to a small 
 
124 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 volume, to remove it from the rest of the fluid, allowing all that will 
 to drain away, and then to digest the fibrinous mass in an artificial 
 digestion mixture (pepsin and o*2 per cent. HCl) until completely 
 dissolved. The resulting fluid is now centrifugalized or allowed 
 to stand for a day or so (in which case some thymol should be 
 added to prevent excessive growth of bacteria), and films prepared 
 from the deposit, which will contain the tubercle bacilli. The 
 advantage of this method is that all the bacilli in four or five 
 ounces of pleuritic fluid may be entangled in the clot, and con- 
 centrated into a comparatively small bulk. The tubercle bacilli 
 resist peptic digestion for a long time, but other organisms do 
 not, and the method is not available for them. 
 
 Purulent pleurisies (empyemata) may be caused by many 
 organisms, the most common being the pneumococcus, strepto- 
 cocci, staphylococci, and the tubercle bacillus. 
 
 The pneumococcus is readily demonstrated by a microscopical 
 examination, the method to be employed being the same as that 
 previously described. 
 
 The pus in these cases is thick and creamy, and of a greenish 
 colour ; after it has stood for some time a thin layer of a greenish 
 fluid appears upon the surface. 
 
 When an empyema is due to the pneumococcus alone, no other 
 organisms being present, the prognosis is distinctly better than in 
 cases in which other organisms are present, and the patient often 
 recovers "after simple aspiration. This is especially the case in 
 children, in whom empyema is due to this organism in a very 
 large number of cases, certainly over 90 per cent. 
 
 If cultures are made in pneumococcic cases, it may be noted 
 that occasionally very few of the organisms appear to grow into 
 colonies, as far as can be judged by a comparison between the 
 numbers of cocci present in the films and of colonies on the tubes. 
 This indicates that the majority of cocci are dead, and this makes 
 the prognosis better. The prognosis is also good in cases in which 
 very few cocci are present, and in those in which the cocci that 
 are present are largely contained in the leucocytes ; in this case 
 they may lose their power of retaining Gram's stain. 
 
 The streptococcus is also readily demonstrated by a simple micro- 
 scopical examination ; it grows readily on agar, forming small 
 round colonies, which do not tend to coalesce and are more opaque 
 in the centre than at the periphery. 
 
 The pus is not generally very thick, and has a yellow colour 
 
THE PLEURA I25 
 
 It separates into two layers, the upper transparent layer being 
 much more abundant than is the case with pneumococcic pus. 
 
 This form of empyema is rare in children, but is perhaps, on 
 the whole, the commonest one in adults. The prognosis is much_ 
 worse than in the pneumococcic cases, and thorough drainage and 
 resection of the ribs is essential. 
 
 Staphylococcic empyemata, according to Netter, are very rare ; 
 the single case in which he found the staphylococcus alone was 
 secondary to ulcerative endocarditis. He also states that when 
 this organism is found in the pus tubercle bacilli are often present as 
 well. The prognosis of these cases, therefore, appears to be bad. 
 
 The Uihevcle bacillus is responsible for a comparatively small 
 number of cases, and the results of operative interference are 
 not gratifying. The prognosis is worse than in any other form of 
 the disease. 
 
 The pus is usually white in colour^ and thin and watery. It 
 may contain small masses of white caseous material. The leuco- 
 cytes which it contains are nearly all lymphocytes, unless a 
 secondary infection with pus organisms has taken place. 
 
 The diagnosis may be made from a careful microscopical 
 examination, but to this end it must he careful^ as the bacilli are 
 present in but scanty numbers. 
 
 If no organisms are found after a thorough microscopical ex- 
 amination, the inference is that the case is tuberculous. If a 
 cultural examination is also negative the inference becomes 
 almost a certainty. 
 
 The empyemata arising from rupture of the oesophagus, stomach, 
 intestine, etc., into the pleura, in those due to an external wound 
 with free contamination of the membrane and its contents, and in 
 those due to the rupture of very foul tuberculous vomicae, contain 
 vast numbers of organisms of all kinds — bacilli, cocci, etc. — mixed 
 together. These fluids usually smell badly, and are of very evil 
 omen. 
 
 Lastly, in a few cases other organisms, such as the typhoid 
 bacillus, may be found. 
 
 Having these facts in view, the practitioner is recommended to 
 proceed to examine cases of purulent pleurisy in the following 
 manner : The pus is to be withdrawn with a hypodermic needle 
 or exploring syringe, and a few drops deposited at once on the 
 surface of a culture-tube of agar, spread well over the surface with 
 a platinum loop, and incubated at the temperature of the body. 
 
126 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 The microscopical examination is made in the manner described 
 for pus, a simple stain and also Gram's stain, with dilute carbol 
 fuchsin as a counterstain, being used. The presence of strepto- 
 cocci, staphylococci, and pneumococci will be revealed ; bacilli 
 may be present, and in this case it should not be forgotten that 
 the tubercle bacillus stains by Gram's method. If no organisms are 
 found in these films, or if there are organisms which resemble the 
 tubercle bacillus in general appearance, another specimen should 
 be submitted to prolonged staining in hot carbol fuchsin and de- 
 colorization in dilute sulphuric acid, and thoroughly searched for 
 the tubercle bacillus. If the result is negative, several other films 
 should be searched. 
 
 The cultures are to be examined after twenty-four hours' incu- 
 bation. The pneumococcus will produce tiny colourless colonies 
 on the surface of the agar ; the streptococcus forms similar small 
 colourless colonies, but these are distinctly more opaque in the 
 centre ; staphylococci form opaque white or yellowish colonies 
 which, after longer incubation, spread out, coalesce, and cover the 
 surface of the agar with an even film like a streak of paint ; and 
 the tubercle bacillus does not develop. Films should be made 
 from the cultures, stained and examined. The cultural examina- 
 tion is of great value, but much can be made out by the examination 
 of stained films made directly from the pus. 
 
 FLUIDS FROM JOINTS 
 
 The technique of the process of withdrawing these fluids is 
 exactly the same as in the case of pleurisy ; the needle will 
 naturally be inserted at a point where there is definite evidence of 
 the presence of fluid, and where it lies near the surface. 
 
 The bacteriological examination is conducted on exactly similar 
 lines. A few drops of the fluid should be allowed to flow on to 
 the surface of a sloped tube of agar, and the culture obtained 
 after twenty-four hours' incubation examined in the manner 
 already described. Films should also be made directly from the 
 fluid, and some stained by Gram's method and others by a simple 
 stain such as carbol thionin. 
 
 A great number of organisms may be present : the streptococci, 
 staphylococci, the pneumococcus, gonococcus, and tubercle bacillus, 
 are the most important. The coccus which has been described 
 by several observers as the cause of acute rheumatism cannot be 
 considered as of diagnostic importance at present ; the same remarl^ 
 
FLUIDS FROM JOINTS I27 
 
 applies to the bacillus which is possibly the cause of rheumatoid 
 arthritis. 
 
 Streptococci are readily distinguished on microscopical examina- 
 tion, and may be present even if the fluid is perfectly clear. When 
 they are present in a joint which is not the seat of a perforating 
 wound, they indicate a general infection with the streptococcus, 
 ulcerative endocarditis, etc., and the prognosis is most grave. The 
 author was enabled to diagnose a case of streptococcic septicaemia 
 a few hours after the onset of symptoms by finding numerous 
 chains in a single drop of clear fluid aspirated from the knee-joint. 
 The clinical aspect was at that time very similar to that of severe 
 rheumatism, and the case had been so diagnosed. 
 
 In such cases the use of antistreptococcic serum offers some 
 hope to the patient, and should be tried. 
 
 Staphylococci are generally found in cases of arthritis due to 
 perforating wounds, or in the course of a general infection. They 
 may also occur along with the gonococcus in cases of gonorrhoeal 
 arthritis. 
 
 The pneumococcus occurs in general infection from a primary 
 focus in the lung, middle ear, etc., or in the course of ulcerative 
 endocarditis, and may also occur as a primary infection — at least, 
 in cases in which no other lesion is found. The prognosis appears 
 on the whole to be fairly good in these cases of suppurative 
 arthritis of pneumococcic origin, and complete recovery with a 
 fully movable joint may occur. 
 
 The gonococcus occurs in some cases of gonorrhoeal arthritis ; it 
 may be present in pure culture, or it may be mixed with other 
 organisms, especially the pus cocci. In other cases of gonorrhoeal 
 arthritis no bacteria are found, either microscopically or on cultural 
 examination, and in these the bacteria have probably died out 
 before the fluid was withdrawn or are localized deep down in the 
 tissues. 
 
 The tubercle bacilhis may be found in cases of tuberculous syno- 
 vitis, but it is more probable that the most careful search will be 
 unsuccessful. If bacilli having the general appearance of this 
 organism are found in the Gram specimen, the carbol fuchsin 
 method of staining should be applied to a fresh film. 
 
 Fluid from a joint may be sterile in cases of tubercular synovitis, 
 gonorrhoeal arthritis, synovitis due to an aseptic injury, rheumatism, 
 gout, or in rheumatoid arthritis, etc. 
 
128 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 LUMBAR PUNCTURE 
 
 Fluid may be removed from the spinal meninges for a bacterio- 
 logical or other examination by means of Quincke's lumbar 
 puncture. The information furnished by this means is often of 
 very great value ; in fact, Osier says that *' during the past ten 
 years no single measure of greater value in diagnosis has been 
 introduced." The process is simple, easy, and entirely devoid of 
 danger, and can be carried out without an anaesthetic. I have 
 now performed this operation over three hundred times, and have 
 only seen bad after-effects (in all cases temporary) in three or four 
 cases, in all of which eucaine had been used as a local anaesthetic. 
 I have therefore abandoned the use of this drug, and now employ 
 only ethyl chloride locally. On the other hand, a very large 
 amount of benefit is often derived from it, and it must be regarded 
 as the most useful therapeutic agent at our disposal in the treat- 
 ment of meningitis, especially chronic forms ; one case at least of 
 tuberculous meningitis has been cured by repeated punctures. 
 It acts, of course, by relieving the high intracerebral tension and 
 by removing some of the toxin-containing cerebro-spinal fluid. 
 In basic meningitis I have seen several cases in which the 
 patient's life was saved by it : the urgent pressure symptoms 
 were relieved on several occasions, the relief lasting a few days, 
 and he was thus enabled to tide over the attack, uninterrupted 
 and complete recovery resulting. In a case in which I performed 
 the operation recently a single puncture was followed by imme- 
 diate relief, though the symptoms were most grave, and there has 
 been at present (after a month) no recrudescence. Great temporary 
 relief may also be given in the severe headache of uraemia and 
 chlorosis. 
 
 It should never be done on a " walking " patient, since if too 
 much fluid is drawn off headache or vomiting are said to result ; 
 these soon pass off if the patient rests in bed. 
 
 Requisites. — i. A suitable needle. In children the spinal 
 meninges will be reached at a depth of 3 to 4 centimetres (roughly 
 I to i^ inches), whilst in adults the depth may be twice as great. 
 The needle should not be less than 2I inches long for an infant 
 and 4 inches for an adult, and should be sharp and strong. An 
 antitoxin needle will usually serve very well. A syringe is 
 .unnecessary, and the fluid should never be sucked out of the 
 
LUMBAR PUNCTURE • I29 
 
 spine, or injury to the nerve-roots may result. The needle should 
 always have a sharp and stiff wire inside it when the puncture is 
 made, otherwise it may become blocked by a shred of fibrous 
 tissues picked up during its passage through the parietes, and 
 time may be lost in sterilizing a wire with which to clear it. 
 Another use for the wire arises from the fact that when the 
 intracerebral pressure is low the needle may push the membrane 
 in front of it, instead of perforating it : when this is the case a 
 short sharp push with the wire will probably puncture the 
 membrane, and the point of the needle will slip in. A piece of 
 steel wire is best. 
 
 Since cultures may have to be taken, it is advisable to have 
 some sort of a handle which may be sterilized with the needle and 
 avoid any necessity for a very elaborate sterilization of the hands. 
 I use a pair of artery forceps, one blade of which is passed inside 
 the barrel and the other outside, and the instrument clipped on. 
 
 Fig. 30. — Author's Needle for Lumbar Puncture. 
 
 The two are sterilized together and the forceps used as a handle, 
 and not removed until the puncture has been made. Where 
 a sterilizer large enough to take the two instruments fixed 
 together is not at hand the needle may be placed point down- 
 ward in a test-tube half full of water, and the forceps inserted 
 as far as it will go by its side. The tube is boiled for ten 
 minutes and the needle removed by clipping it with the forceps, 
 as above. The handle will not be sterilized, but that does not 
 matter. 
 
 I have devised a curved needle with a handle fitting into a 
 socket (Fig. 30), which also answers well. It has the advantage 
 that it can be put in a wide tube plugged with cotton-wool, 
 sterilized by dry heat, and taken to the bedside ready for 
 immediate use. 
 
 2. Materials for disinfection of the patient's skin and (if cultures 
 are to be taken) the hands of the operator. Hot water, soap, 
 alcohol, ether, perchloride lotion (i in 1,000). 
 
 9 
 
130 CLINICAL BACTERIOLOGY AND H.^MATOLOGY 
 
 3. Apparatus for boiling the needle in a dilute solution of 
 washing soda.* 
 
 4. Spray for local anaesthesia. If this is used the hard plaque 
 of skin adds considerably to the difficulty of the operation. If, 
 however, the region be frozen twice, and allowed to thaw after 
 each freezing, the skin will be found to have resumed its normal 
 texture and to be very fairly anaesthetic. 
 
 5. Two or three test-tubes sterilized by dry heat and plugged 
 with dry cotton-wool. 
 
 6. If cultures are to be taken, the tubes of medium should be 
 inoculated at the time of the operation if possible. The medium 
 required will depend to a great extent upon the nature of the 
 organism which is expected. If there are no indications upon 
 this point, the most suitable is solidified blood-serum, but in 
 default of this ordinary agar will answer well. If the case is 
 thought to be one of cerebro-spinal fever, the most suitable 
 medium for the cultivation of the specific organism (Weichsel- 
 l)aum's Diplococcus intvacellularis) is alkaline 5 per cent, glycerin- 
 agar, and a couple of tubes of this should be at hand, as well as 
 blood-serum or ordinary agar. 
 
 Process — i. Preliminary. — As in removal of fluids for bacterio- 
 logical examination from other parts of the body, it is better if the 
 skin can be sterilized some hours before the operation, and a pad 
 soaked in an antiseptic fluid kept on the area until the last 
 moment. This is usually impracticable, and the process will be 
 described as if it were performed at a single visit. 
 
 Put the needle to boil in a weak solution of washing-soda, and 
 proceed to the disinfection of the patient's back. When the 
 needle has boiled for five minutes, remove the vessel from the 
 flame and allow it to cool without removing the needle. 
 
 Place the patient on his left side, and find the processes of the 
 second, third, and fourth lumbar vertebrae. A line drawn between 
 the upper points of the iliac crests usually cuts the spine at the 
 upper edge of the spinous process of the fourth lumbar vertebra. 
 Scrub the skin in this vicinity with soap and hot water ; wash the 
 region with alcohol and then with ether, and allow it to dry ; paint 
 on several layers of perchloride lotion, allowing each to soak in 
 before the next is applied ; cover the region with a piece of lint 
 
 * If possible the needle should be sterilized by dry heat previous to the 
 operation, and kept in a tube plugged at both ends with cotton-wool, as in the 
 method recommended for the collection of blood for bacteriological examination. 
 
I 
 
 LUMBAR PUNCTURE , I3I 
 
 soaked with lotion, and proceed to disinfect your hands. Lastly, 
 pour some alcohol on to the skin of the patient's back to wash off 
 the excess of the antiseptic. 
 
 2. Operation — Position. — Get the patient (still lying on his left" 
 side) to draw up his knees so as to flex his back somewhat, and to 
 turn partly over on to his face. 
 
 Identify the processes of the third and fourth lumbar vertebrae, 
 and mark the centre of the space between them by means of the 
 index-finger or thumb of the left hand. If local anaesthesia is to 
 be employed freeze the skin round a point about J inch to the 
 right of the middle line, opposite the spot marked by your 
 
 Fig. 31. — Operation of Lumbar Puncture with Patient in 
 Sitting Position. 
 
 left finger or thumb. Take the needle in the right hand, holding 
 it like a pen, and enter it at a point level with the centre of the 
 interspace, and i centimetre (a little less than J inch) to the 
 right of the middle line. Direct it forwards, slightly upwards, and 
 slightly inwards, and press it in with a steady and uniform pressure ; 
 this must be applied accurately in the axis of the needle, or the 
 latter may bend and take a wrong direction. 
 
 If the needle strikes against bone withdraw it almost completely, 
 and push it on again after changing its direction slightly. If bone 
 is again encountered it may be advisable to try again in the inter- 
 space between the second and third processes. 
 
 9—2 
 
132 . CLINICAL BACTERIOLOGY AND H.53MATOLOGY 
 
 If the patient is able to do so without harm, it is much easier 
 to perform the operation with him sitting in a chair leaning over 
 the back or on the edge of the bed with his head nearly between 
 his knees, so that the spine is bent into a convex curve ; by this 
 means the spinous processes are rendered more prominent and 
 the spaces between the laminae are made wider (see Fig. 31). The 
 operation is thus made much more easy, and the method is usually 
 preferable in cases of tabes or general paralysis, where the fluid 
 is collected for a cytological examination only, and the patient is 
 in good general health. But the method with the patient on his 
 side should also be learnt. 
 
 3. Collection of Fluid and Inoculation of Media. — The first few 
 drops of fluid which escape may be stained with blood ; in this 
 case they should be rejected. Allow a few drops of the fluid to 
 flow directly on to the surface of the medium without touching the 
 glass. Collect also some of the fluid (i to 2 drachms) in the 
 sterilized empty tube. If no fluid flows through the needle, it is 
 presumptive evidence against the presence of acute meningitis. A 
 *' dry tap" may, however, occur from plugging of the needle with 
 fibrin, or from its point coming in contact with a nerve root 
 (Osier), and in some cases of meningitis the purulent exudation is 
 too thick to flow through the needle. , In one case in which no 
 fluid could be obtained by repeated punctures the venous sinuses 
 of the brain were found to be thrombosed : a frequent cause in 
 meningitis is the closure of the foramina in the roof of the fourth 
 ventricle. 
 
 The force of the flow should be noted. In health it flows 
 out slowly, whilst in meningitis it runs faster, and may even spurt 
 out a foot or more; the same thing may happen if there is a 
 cerebral tumour, uraemia, or other cause of increased pressure. 
 
 4. Examination of the Fluid. — {a) Naked-Eye. — When meningitis 
 is present the fluid is always more or less turbid, and some 
 observers hold that the turbidity is greater in proportion to the 
 severity of the case. Osier has pointed out that the fluid may be 
 alternately turbid and clear, being clear during the remissions and 
 turbid during the exacerbations of the disease. Blood-stained 
 fluid may occur in meningitis or from haemorrhage into the 
 cerebral or spinal meninges apart from inflammation."* The 
 presence of clear fluid aflbrds strong evidence of the absence of 
 meningitis, but in tuberculous meningitis the amount of turbidity 
 
 * See also p. 242. 
 
LUMBAR PUNCTURE I33 
 
 may be very slight. It should be estimated by comparing the 
 fluid with some distilled water in a clean test-tube of the same 
 size in the two cases. 
 
 (b) Microscopical —Prepare films of the exudate in the manner^ 
 recommended on p. 135 if the fluid is thin and watery; if it is 
 thick and purulent, treat it Hke ordinary pus. Stain by any of the 
 methods recommended for the examination of the blood (Jenner's 
 stain being most convenient), and examine.* 
 
 The presence of leucocytes (except in very small numbers) 
 indicates meningitis. If the bulk of the leucocytes are lympho- 
 cytes (indicated by their small size, large, circular, deeply staining 
 nuclei, and absence of granules) the presumption is that the case 
 is one of tuberculous meningitis. In acute meningitis due to other 
 bacteria the chief cell is the polynuclear leucocyte ; this may be 
 recognised by its larger size, its twisted (apparently multiple) 
 nucleus, and, if the staining method has been appropriate, by the 
 presence in its protoplasm of minute granules which stain with eosin. 
 The fluid may also contain red blood-corpuscles and shreds of fibrin. 
 
 {c) Chemical. — Cerebro- spinal fluid removed from a person who 
 is not suffering from meningitis contains a very minute amount 
 of albumin, while when the meninges are inflamed the quantity 
 is greatly increased. The method of testing these small amounts of 
 albumin quantitatively is hardly within the reach of practitioners; 
 if a considerable amount of fluid has been obtained, a small 
 quantity should be tested by heat and acetic acid, and the amount 
 of opacity noted. This should be very slight, not more than what 
 would be called a " faint haze " in urinary work. 
 
 Another test which has almost escaped notice, but which I 
 regard as one of the most important, is that for the presence of 
 sugar. For this (as for the testing for albumin) the clear super- 
 natant fluid left after centrifugalization should be used. In health, 
 sugar is present in considerable amount (about 0*06 per cent.), and 
 Fehling's solution is vigorously reduced ; in meningitis, sugar is 
 either reduced to a trace or, much more frequently, completely 
 absent. I have only found this test fail once in about a hundred 
 cases (even in this one the diagnosis was never made absolutely 
 certain), and regard it as the most certain single sign of meningitis.! 
 
 * The cytology of the cerebro-spinal fluid is dealt with more fully under the 
 heading of Cyto-diagnosis. 
 
 t Since writing the above I have met with several cases of tuberculous 
 meningitis in which sugar was present. The reduction of the Fehling is, 
 however, but slight, and the oxide may not appear for several minutes. 
 
134 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 [There are also characteristic chemical changes in uraemia, to 
 which I have paid a good deal of attention of late. In health the 
 cerebro-spinal fluid contains about 0*035 to 0-04 per cent, of urea, 
 07 per cent, of chlorides, and has a freezing-point of —0-56° C, or 
 thereabouts. In renal disease in which the kidneys are acting 
 well these figures are but slightly disturbed, but in uraemia there 
 is abundant evidence of retention of soluble substances. The urea 
 increases greatly — the maximum I have seen being 0-4 per cent. — 
 the chlorides may rise to i per cent., and the freezing-point is 
 depressed. I believe this to be the simplest test of the functional 
 capacity of the kidney, the chemical examinations being easy in a 
 substance of such simple constitution as the cerebro-spinal fluid. 
 It is occasionally of value in patients found unconscious. In 
 one such case I was able to exclude uraemia definitely, though 
 the urine contained albumin and casts : it turned out to be a case 
 of poisoning. In uraemia there is often the additional advantage 
 of its affording a relief to some of the symptoms. Further details 
 are outside the scope of this work.] 
 
 (d) Bacteviological. — The chief organisms which cause acute 
 meningitis are given in the following table, which is modified 
 from one given by Osier : 
 
 Primary {i.e., not dependent on an obvious lesion elsewhere in the body). 
 
 1. Cerebro-spinal fever— 
 
 ,,,^ . , . IWeichselbaum's diplococcus, or meningococcus. 
 (6) Epidemicj 
 
 2. Pneumococcic — 
 
 {a) Pneumococcic infection of meninges alone, not de-' 
 pendent on disease of distant parts of the body. 
 
 {b) Pneumococcic infection of meninges occurring as |.Pneumococcus. 
 part of a general septicaemia without obvious 
 primary lesion. 
 
 Secondary. 
 
 A. To direct extension from local disease of the cranium, middle ear, fossae, 
 
 spinal column, etc. 
 Pneumococcus. 
 Staphylococci. 
 Streptococci, etc, 
 
 B. To septicaemic infection due to disease in a distant part of the body. 
 
 {a) Pneumococcic — 
 
 Secondary to pneumonia, endocarditis, etc. 
 Pneumococcus. 
 
LUMBAR PUNCTURE I35 
 
 (b) Pyogenic — 
 
 Secondary to abscesses, etc., and occurring as a part of a general 
 infection. 
 Staphylococci. 
 Streptococci, etc. _ 
 
 (c) Gonorrhoeal — 
 
 Secondary to gonorrhoea. 
 Gonococcus (rare). 
 
 (d) Tuberculous- 
 
 Secondary to tuberculosis of other regions. 
 Tubercle bacillus. 
 
 (e) Miscellaneous — 
 
 Secondary to typhoid fever. 
 
 Typhoid bacillus (rare). 
 Secondary to influenza. 
 
 Influenza bacillus (rare). 
 Secondary to anthrax, etc. 
 
 Anthrax bacillus, etc. (rare). 
 
 Of these, the organisms which are most likely to occur are : 
 
 The tubercle bacillus ) ,■ r ^ . . e 
 
 I accounting tor about go per cent, of cases. 
 Meningococcus > 
 
 The pneumococcus. 
 
 Streptococci and staphylococci in secondary cases due to spread from 
 
 ear, etc. 
 
 And this shows roughly the order of frequency. 
 
 Preparation of Films. — If the fluid is thick and purulent, films 
 should be prepared, dried, and fixed in the ordinary way. If the 
 fluid is thin and watery, it should be centrifugalized or allowed 
 to stand for some hours. A certain amount of coagulation will 
 take place, and the sediment which collects will contain the bulk 
 of micro-organisms. The clear fluid should be poured off and 
 used for testing chemically, and the sediment used for the pre- 
 paration of films, of which several will be required. The subse- 
 quent examination will depend to some extent upon the nature of 
 the organism which is probably present ; for general purposes 
 stain one or more films with Loffler's methylene blue (two 
 minutes), wash, dry, mount, and examine. 
 
 Streptococci and staphylococci will be readily recognised by 
 their morphological characters. If diplococci are present they 
 may be pneumococci, Weichselbaum's diplococci, or gonococci. 
 Stain a film by Gram's method and counterstain in dilute carbol 
 fuchsin in the method described for the gonococcus. Pneumococci 
 will retain the violet stain, while Weichselbaum's organism and 
 gonococci will be coloured red. 
 
136 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Weichselbaum's Diplococcus meningitidis intvacellulavis is now 
 generally considered to be the specific cause of cerebro-spinal 
 fever. It is a diplococcus which varies very considerably in size, 
 but is usually rather smaller than the gonococcus. The opposed 
 surfaces of two cocci making up the pair are usually somewhat 
 flattened, but this is not so marked as in the latter organism. It 
 is often contained within the polynuclear leucocytes, but is not 
 grouped in large numbers in a single cell — others being free — as 
 is usually the case with the gonococcus (see Plate III., where the 
 two organisms are contrasted). The two also differ in their cultural 
 characters and in their pathogenicity to animals. If any question 
 should arise as to which of the two is present in the meningeal 
 exudation in a case in which no cultures have been taken, other 
 evidence of gonorrhoeal infection should be sought for. 
 
 Still's diplococcus of posterior basic meningitis cannot be 
 distinguished from the D. meningitidis by its morphological 
 characters alone, and many bacteriologists think the two 
 organisms are in reality identical. The truth seems to be that 
 the organism which we call the meningococcus is a very variable 
 one, some forms being much more easily cultivated than others, 
 and also differing in a few other minute points. Yet these forms 
 appear to occur indiscriminately in true epidemic cerebro-spinal 
 meningitis and in posterior basic meningitis, and these diseases 
 are quite indistinguishable on pathological grounds. 
 
 The Rarer Causes of Meningitis. — The bacilli of typhoid fever, 
 anthrax, influenza, etc., may also be recognised in the methylene 
 blue specimen, and should be identified (if possible) by a careful 
 study of their morphological appearances and reaction to Gram's 
 stain. 
 
 If no organisms are found in the methylene blue specimens 
 after a careful search, and if the characters of the fluid are such 
 as indicate that meningitis is present, the presumption is that the 
 case is one of tuberculous meningitis. Films should be stained 
 in the method already described and carefully searched ; the 
 bacilli are present in very scanty numbers, and many films may 
 have to be examined before one is found. "^ 
 
 * Lenharz adds a shred of clean cotton-wool to the fluid. This sinks slowly 
 to the bottom, and is withdrawn after some hours, spread on a slide, dried, 
 and stained for tubercle bacilli. The author has had no experience of this 
 method, but Mr. Leedham-Green informs him that it is of considerable value. 
 (See also p. 138). 
 
LUMBAR PUNCTURE I37 
 
 Cultural Examination. — The tubes which have been inoculated 
 by allowing the fluid to drop directly on to the surface of the 
 medium are to be incubated for twenty-four hours at the body 
 temperature. Streptococci, staphylococci, pneumococci, and the 
 rarer organisms, will probably have developed by this time, and 
 will have formed colonies such as have been previously described. 
 Weichselbaum's diplococcus forms (on blood-serum) " round, 
 whitish, shining, viscid-looking colonies, with smooth, sharply 
 defined outlines which attain a diameter of i to if millimetres 
 in twenty-four hours." The colonies on agar are similar, but 
 slightly larger, and the growth may become confluent. 
 
 If no colonies appear on blood-serum or agar at the end of 
 forty-eight hours, the case is probably due to the tubercle bacillus 
 or the gonococcus. In some cases of cerebro-spinal fever the 
 diplococci in the exudate are all dead, and cultures remain sterile. 
 
 Interpretation of Results. 
 
 The discovery of Weichselbaum's diplococcus indicates that 
 the case is one of cerebro-spinal fever. The chief importance in 
 making the diagnosis (apart from the fact that it may throw light 
 upon the occurrence of several cases of meningitis within a short 
 space of time by proving the existence of an epidemic) arises from 
 the fact that the prognosis is decidedly better than in other forms 
 of meningitis. Roughly speaking, about 40 per cent, of cases 
 recover, though frequently with mental or other defects. It is in 
 these cases that repeated lumbar puncture is of therapeutic value ; 
 it should be done whenever pressure symptoms are urgent. In 
 these cases also great benefit can be obtained from vaccine treat- 
 ment. The vaccine should be prepared from the patient's own 
 culture, and its use carefully controlled by frequent examinations 
 of the opsonic index. The method is, of course, useless in very 
 rapid cases, but very good results are obtainable in the more 
 chronic ones. In some the immediate benefit resulting from each 
 injection is most marked. 
 
 Meningitis due to the pneumococcus may arise from dissemina- 
 tion from pneumonia or other pneumonic lesion, by spreading 
 from the middle ear, etc., or may be primary. The examination 
 of the exudate throws no light upon this point, and the cause of 
 the infection must be sought for on ordinary clinical lines. 
 
 Tuberculous meningitis is proved by the presence of tubercle 
 bacilli in the fluid, and is indicated by sterile cultures, absence of 
 
138 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 bacteria from the stained films, and predominance of lymphocytes. 
 In these cases the fluid often undergoes a very slight coagulation, 
 delicate cobweb-like threads being observable after some hours. 
 This is in itself strong evidence of tubercle, and if the deHcate 
 coagulum can be withdrawn, dried on a slide, and stained by 
 Ziehl-Neelsen's method, there is a fair chance of finding bacilli 
 entangled with it. This is not very easy to do, the best method 
 being to fish it out with a very fine piece of capillary tubing no 
 thicker than a pin. It is easy enough to pick it out with a 
 platinum needle, but almost impossible to get it off the latter on 
 to the slide. 
 
 The other varieties of meningitis do not call for special mention. 
 
 The chief value of lumbar puncture to the surgeon is that it 
 enables him to diagnose a concomitant meningitis (indicating the 
 uselessness of an operation) in cases of lateral sinus thrombosis 
 and cerebral abscess. The fluid usually becomes bloody within 
 twenty-four hours of a fracture of the base of the skull or lacera- 
 tion of the brain. This may assist in the diagnosis of obscure 
 injuries, or of the cause of a case of unconsciousness in which no 
 history can be obtained. 
 
 Haemorrhage into the meninges is indicated by the withdrawal 
 of blood-stained fluid, but it must be remembered that the first few 
 drops may contain a small quantity of blood which has entered 
 the needle during its passage through the tissues, while the rest 
 is clear. Blood-stained fluid may occur in meningitis, and should 
 be submitted to a full examination for leucocytes and bacteria. 
 
 THE BACTERIOLOGICAL EXAMINATION OF THE 
 
 BLOOD 
 
 The bacteriological examination of the blood is not so important 
 as might be thought, as it is only in a very few diseases that 
 pathogenic bacteria are present in the circulation in such quantities 
 as to render the search for them in the minute amounts which are 
 withdrawn for examination at all promising. The method is 
 becoming of more importance daily, since promising results have 
 been obtained in the treatment of septicaemic diseases by means 
 of specific vaccines, which, to get the full advantage of the 
 process, should be obtained from cultures of the patient's own 
 bacteria. 
 
 The chief organisms which have been found in the blood are : 
 
THE BACTERIOLOGICAL EXAMINATION OF THE BLOOD I39 
 
 I and 2. Streptococci and Staphylococci. — These are found in cases 
 of septicaemia, pyaemia, ulcerative endocarditis, etc. ; they always 
 indicate an extremely bad prognosis. The chief importance which 
 attaches to the discovery of these organisms is that it absolutely 
 settles the diagnosis (always provided that there are no errors in 
 technique), and that it indicates whether the use of antistrepto- 
 coccic serum is advisable or not ; it is useless in cases of septi- 
 caemia, etc., which are not due to streptococci. 
 
 A word of warning is necessary in the interpretation of results 
 which indicate that staphylococci are present in the blood. These 
 
 Fig. 32. — Spirillum of Relapsing Fever. 
 
 organisms are constantly present in the skin, and may be found in 
 film preparations or in cultures, unless rigid antiseptic precautions 
 are taken. Streptococci may also occur as contaminations of 
 cultures, but rarely occur in film specimens. 
 
 3. Anthrax Bacilli. — These may be detected with ease and 
 certainty, but they are never found in the blood until it is too 
 late to save the patient. 
 
 4. Tubercle Bacilli. — These are only present in very scanty 
 numbers, and are very difficult to detect. The diagnosis of 
 miliary tuberculosis is to be made by other methods, chiefly by 
 that of exclusion. 
 
 5. The pneumococcHs is found in severe cases of pneumonia 
 (probably it might be found in most cases if a sufficiently large 
 quantity of blood were examined) and in septicaemia and ulcerative 
 
140 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 endocarditis when due to this organism. When found in the blood 
 by ordinary methods it always indicates a bad prognosis, and sug- 
 gests the use of antipneumococcic serum. The identification of 
 the organism is easy and certain if present in quantities sufficient 
 for it to be found in films. 
 
 6. Typhoid bacilli. (See p. 69.) 
 
 7. The bacillus of glanders may be found in acute cases of that 
 disease, but its isolation and identification are matters for an 
 expert. 
 
 8. The infltienza bacillus is present in some or, according to some 
 authorities, all cases of influenza. It may be searched for in films, 
 but no importance should be attached to a negative result. 
 
 9. The bacillus of plague. — This organism is often present in the 
 blood in relatively large numbers, and the disease can usually 
 be diagnosed after a careful search through a number of suitably 
 stained films. But the investigation of a drop of fluid drawn 
 from the bubo (if one is present) permits of an easier and earlier 
 diagnosis. The blood examination is of most value in the pul- 
 monary and septicaemic forms of plague. 
 
 10. The spirillum of relapsing fever is easily found, for it possesses 
 well-marked characters and is present in great numbers. The 
 diagnosis of relapsing fever cannot be made until it has been 
 demonstrated (see Fig. 32). It is best seen by mounting a small 
 drop of blood between slide and cover-glass, and examining it in a 
 perfectly fresh state, when the spirilla are easily found from the 
 commotion they cause by pushing aside the red corpuscles. 
 
 11. The gonococcus has been found in the blood in a few cases 
 of ulcerative endocarditis. Its detection by cultural methods is 
 very difficult, and the services of a bacteriological expert should 
 be called in if the characteristic cocci are not found in blood-films 
 in a case in which the diagnosis of gonorrhoeal ulcerative endo- 
 carditis is probable, as further information upon this point is 
 greatly needed. We may point out that ulcerative endocarditis, 
 septicaemia, etc., supervening in the course of an attack of gonor- 
 rhoea, are not necessarily due to the gonococcus. Any pathogenic 
 bacteria may enter through the lesion of the mucous membrane 
 which the gonococcus has caused. 
 
 12. The B. coli is present in some cases of septicaemia. 
 
 1 
 
EXAMINATION FOR BACTERIA IN FILMS I4I 
 
 EXAMINATION FOR BACTERIA IN FILMS 
 
 This is the easiest method in which bacteria may be found in 
 the blood, and it does not require such a rigid antiseptic technique 
 as is necessary if cultures are to be taken. The films are prepared 
 and fixed in one or other of the methods which will be described 
 subsequently (see p. 198), the only point worthy of notice being 
 that the skin must be very thoroughly cleaned; it may be scrubbed 
 with soap and a nail-brush, using plenty of hot water. The films 
 need not be very thin and even. 
 
 The method of staining will depend upon the organism which 
 is likely to be found, and more especially whether it stains by 
 Gram's method. This is so important in this connection that a 
 repetition of a previous table will not be out of place. 
 
 Gram's Method. 
 Stained. Unstained. 
 
 Streptococci. Typhoid bacilli. 
 
 Staphylococci. Bacillus of glanders. 
 
 Bacillus of anthrax. Bacillus of influenza. 
 
 Bacillus of tubercle. Bacillus of plague. 
 
 Pneumococcus. Bacillus coli. 
 
 Spirillum of relapsing fever. 
 
 Gonococcus. 
 
 If the organism which is present appears in the first list, the 
 staining process is simply that which we have described previously, 
 and the organism will be stained dark blue or violet, and the other 
 structures will be unstained. 
 
 If the bacteria which are present in the films do not stain by 
 Gram's method the matter is more difficult, for any stain which 
 colours them will colour the nuclei of the leucocytes also. Jenner's 
 stain may be used, or the film may be stained by eosin and methy- 
 lene blue separately. The organisms will then be stained blue. 
 Carbol thionin is even more suitable, as the colour which it 
 imparts to the nuclei of the leucocytes is not deep and the red 
 corpuscles are merely tinged. This is the strain which we recom- 
 mend for general use, and in cases in which the nature of the 
 organism (if one be presented) is entirely unknown. 
 
 If bacteria are detected by any of these methods their nature 
 must be recognised by a consideration of their morphological 
 features and staining reactions. 
 
142 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 MALARIA 
 
 The blood in a suspected case of malaria may be examined 
 fresh or in stained films. Of these methods the former is the 
 better, and should be used if possible. An examination of stained 
 specimens should also be made, and is convenient, as it can be 
 performed away from the patient and at leisure. 
 
 Fresh films are made by touching a drop of blood on the 
 patient's finger with the centre of a perfectly clean cover-glass, so 
 as to remove an extremely small quantity of blood. This cover- 
 glass is then allowed to fall on to a clean slide, so that the droplet 
 of blood may be spread out by capillary attraction and by the 
 weight of the cover-glass, just as is the case in the method of 
 making blood-films to be described subsequently. But the slide is 
 not separated from the cover-glass; they are examined just as 
 
 Fig. 33 — Malarial Parasites in the Blood. 
 The dark area shows the parasite as it appears when stained with thionin. 
 
 they are, a ring of vaseline being painted round the edge of the 
 cover-glass to prevent evaporation. 
 
 The specimen is examined with a i-inch objective, and a place 
 found in which the corpuscles are spread in a single layer ; this 
 part is then searched thoroughly with a jV-inch oil-immersion 
 lens. The parasites are seen as pale, irregularly shaped bodies 
 with indistinct margins, which occupy the interior of the red 
 corpuscles, and show amoeboid movements of greater or less rapidity. 
 When the parasites are older they occupy a larger space in the 
 corpuscles, and there are granules of dark pigment around their 
 periphery. These granules are often the first indications of the 
 presence of parasites in the examination of an unstained specimen. 
 At a still later stage the granules will be found in the centre of 
 the corpuscle (the haemoglobin of which is now almost entirely 
 removed), and the parasite will show segmentation into a larger 
 or smaller number of spores by lines which have a radial arrange- 
 ment and give the whole an appearance resembling that of a 
 
MALARIA 143 
 
 marguerite daisy. These are only found when a rigor is 
 "mminent. 
 
 Crescents are found in the aestivo-autumnal formfof malaria ; 
 they are crescentic bodies with rounded " horns," and contain a" 
 ring of pigment granules in the centre. They cannot be mistaken 
 for anything else, and if a single one is found it affords conclusive 
 proof that the patient has been infected with malaria. 
 
 Films for staining are made in the ways already described, and 
 must be thin and perfect. They may be fixed by any of the 
 methods we have recommended, the alcohol-ether and the alcohol- 
 formalin methods being perhaps the best. They may be stained 
 by Jenner's stain, or by eosin and methylene blue used separately; 
 the parasites are stained pale blue and the corpuscles bright red. 
 
 A simpler stain is that recommended by Rees {Practitioner^ 
 March, 1901), involving the use of carbol thionin, prepared by 
 dissolving 1-5 grammes of thoinin in 10 c.c. of absolute alcohol 
 and 100 c.c. of a 5 per cent, solution of carbolic acid. This is to 
 be kept for at least a fortnight, and diluted with four times its 
 bulk of distilled water immediately before use. Staining is com- 
 plete in about ten minutes. Ordinary carbol thionin answers 
 very well indeed. Thionin stains the red corpuscles a faint green, 
 nuclei blue, and the parasites an intense purple. 
 
 In a suspected case of malaria the search should not be 
 abandoned in less than half an hour, or, in the case of an 
 inexperienced observer, much longer. 
 
 A fuller description of the parasite and the differences between 
 the forms which are present in the various forms of the disease 
 is beyond the scope of this work, and the reader is referred to the 
 admirable special number of the Practitioner mentioned above. 
 
 EXAMINATION BY CULTURAL METHODS 
 
 This is a much more difficult matter. The difficulty arises 
 from the abundant bacterial flora of the skin ; unless the most 
 thorough antiseptic precautions have been taken the results are 
 ahsoMely useless. They are worse than useless — they are mis- 
 leading. A case of ulcerative endocarditis, for example, might 
 be due to streptococci, but might be attributed to staphylococci on 
 the strength of an inadequate bacteriological examination. For 
 this reason we are chary of recommending this method of 
 diagnosis in any hands other than those of an expert, and must 
 
144 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 urge the practitioner not to attempt it unless he is prepared to 
 carry it out properly in the most minute detail. It is in particular 
 absolutely useless to attempt to obtain cultures with the blood 
 taken from a skin puncture ; it must be drawn direct from a 
 vein, and a large quantity (not less than i c.c, and preferably 
 4 or 5, or even lo) must be employed. 
 
 One plan is to use a hypodermic needle, and to plunge it 
 directly into a vein.* The process may be carried out as 
 follows : 
 
 Requisites. — i. An all-glass hypodermic syringe of at least 5 c.c, 
 and better 10 c.c, capacity, preferably furnished with platino- 
 iridium needle. 
 
 2. Means of sterilizing the above. I personally keep the 
 syringe always sterilized with carbolic lotion (i in 20). After use 
 at any time it is washed out with the lotion, a little of which is 
 allowed to remain in the barrel, which is thus always sterile. 
 Before use the antiseptic has to be removed, and this is effected 
 by filling the syringe two or three times with sterile nutrient 
 broth, a spare culture-tube being taken for the purpose. This 
 gets rid of the waste of time involved in boiling, and the necessity 
 for carrying cumbrous sterilizers. 
 
 Another method is to boil the instrument for ten minutes. 
 
 Or Wright's method may be adopted. Oil is heated to 150° in 
 a metal capsule, and the syringe washed out two or three times 
 with the hot oil. If no thermometer is at hand the temperature 
 of the oil can be estimated roughly by dropping a crumb of bread 
 into it. If the crumb gives off bubbles, the temperature is 100° or 
 above ; it is turned brown at 1 50°. 
 
 3. Materials for sterilizing the skin are described under the 
 heading of Lumbar Puncture. 
 
 4. A narrow bandage or piece of tape. 
 
 5. A spirit-lamp (not indispensable). 
 
 6. Culture Tubes. — On the whole broth is the most suitable 
 media, and rather large tubes, containing about 25 c.c. of broth, 
 the best to use. One or two cultures on agar should also be 
 taken. 
 
 7. Collodion, to be applied to the puncture after the operation. 
 Method. — Apply the narrow bandage to the upper arm suffi- 
 
 * Dr. Horder has applied this method with great success, and has fully 
 described his technique and results in the Practitioner, November, 1905, to 
 which the reader is referred for further information. 
 
EXAMINATION BY CULTURAL METHODS t45 
 
 ciently tightly to obstruct the venous circulation, but not tightly 
 enough to check that in the artery ; if the former do not become 
 prominent, the patient should be made to hang his arm down, 
 and to clench and relax his fist. 
 
 Select a large vein in the antecubital fossa, and choose, if 
 possible, one that is superficial (as shown by its blue colour), not 
 merely prominent, since a deep vein may slip in front of the 
 needle. Avoid, if you can, a vein lying near an artery. Proceed 
 to sterilize the skin in the ordinary way. 
 
 Next take the sterilized syringe and steriUze the point of the 
 needle in the flame of the spirit-lamp. Proceed to make the 
 
 Fig. 34. — Collection of Blood Direct from Vein. 
 
 puncture as shown in the illustration (Fig. 34). Direct the point 
 of the needle away from the patient's body, so that it faces the 
 blood-flow, and enter it at a point about \ inch from the vein, at 
 one or other side. (This diminishes the chance of subsequent 
 leakage, and possibly of sepsis, organisms picked up by the 
 needle being wiped off" during the passage of the latter through 
 the tissues.) Press it gradually onwards until the needle pierces 
 the wall of the vein, when, in most cases, the blood will rise in 
 the syringe, slowly pressing out the piston before it. If it does 
 not you may make very gentle suction with the piston : it must 
 not be forcible, or the wall of the vein will be sucked in and act 
 as a valve. 
 
 In most cases the only difficulty to arise will be in entering the 
 vein, which may slip in front of the needle if the intravenous 
 
 10 
 
146 
 
 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 pressure is low or the instrument blunt. In this case the vein 
 
 may be made tenser by gently massaging blood in it towards the 
 
 bandage and retaining it there by a finger pressed on to the vessel. 
 
 As soon as the requisite amount of blood has been obtained, 
 
 remove the bandage from the upper arm, and 
 
 then withdraw the needle ; if you leave it on 
 
 longer there may be a considerable amount 
 
 of haemorrhage into the tissues, which does 
 
 no harm, but leaves an unsightly bruise. 
 
 Next make the cultures as follows : Expel 
 nearly all the blood into one of the broth 
 tubes and shake gently, and then put about 
 I c.c. into the agar tube, and place the latter 
 in an incHned position, so that the blood will 
 clot in an even film over the surface. 
 
 Seal the puncture in the skin with col- 
 lodion. If there is haemorrhage into the 
 tissues, bandage the forearm evenly from 
 below upwards. 
 
 Undoubtedly the simplest and best of all 
 methods is that described by James and 
 j|, :J Tuttle {Report of the Presbyterian Hospital, 
 
 New York, 1898). " A piece of glass tubing 
 4I inches in length, and ^ inch in diameter, is 
 drawn out to a tapered end, and ground to fit 
 the cap of a rather fine hypodermic needle. 
 The larger end of the tube having been 
 stopped with a cotton plug, the whole is then 
 placed in a larger tube, and both ends of this 
 are similarly plugged with cotton "**" (Fig. 35). 
 ** The apparatus is then sterilized by dry 
 heat. In using it the inner tube with needle 
 attached is removed ; the skin over one of the 
 
 Bacteriological 
 Examination. 
 
 Fig. 35. — Pipette 
 for Collection 
 of Blood for most prominent veins of the anterior surface 
 
 of the forearm, near the bend of the elbow, is 
 selected, a piece of rubber tubing or a few 
 turns of a bandage being passed round the arm above with 
 moderate pressure, in order to produce distension of vessels. The 
 needle is then plunged into the vessel, and generally blood begins 
 to flow by the blood-pressure itself, but any quantity desired may 
 * These may be obtained from F. Ash, Edmund Street, Birmingham. 
 
EXAMINATION BY CULTURAL METHODS I47 
 
 be obtained by making gentle suction, either by applying the 
 mouth directly to the end of the tube where it is stopped with 
 cotton, or through the medium of a small piece of rubber tubing 
 slipped over it. 
 
 " By the above instrument vein punctures have been made in 
 about 150 cases of a variety of diseases. At no time was any 
 difficulty experienced in obtaining the amount of blood desired, 
 which was generally about i c.c. In a few instances it was 
 necessary to try two punctures before securing a free flow 
 through the needle ; in no case was there any local reaction 
 whatever at the seat of puncture, nor did the patient complain of 
 pain and annoyance." 
 
 I used this method for four years, and found it by far the best 
 for clinical work. The needle is carried ready sterilized, and no 
 time is wasted in boiling it, and the whole process may be per- 
 formed in five or ten minutes. The main disadvantage is that 
 it requires a special instrument, whereas an all-glass exploring 
 syringe should be always available. 
 
 The advisability of employing some such method in which 
 the blood is drawn directly from a vein in place of the simple 
 skin puncture is very apparent from the researches of Kiihnau 
 [Zeitschvift f. Hyg. und Infct., 1890), who made parallel series of 
 experiments by the two methods. He found that in cases in 
 which the blood drawn directly from the vein remained sterile 
 growth (mostly streptococci or staphylococci) occurred in as many 
 as 90 per cent, of cultures inoculated from skin punctures, though 
 the most careful antiseptic precautions were used. 
 
 The cultures thus obtained are incubated at the body tempera- 
 ture, and examined from day to day. The blood in the broth 
 tube will coagulate, and the appearance of growth may be delayed 
 by the entanglement of the colonies in the clot ; sooner or later, 
 however, the clear fluid will become turbid if bacteria are present, 
 and subcultures can be made on agar or blood-serum, and films 
 examined. 
 
 If colonies appear on the agar tube, they are to be carefully 
 examined with a lens, and their characters noticed. The 
 organisms which will be most likely to develop are streptococci, 
 staphylococci, anthrax bacilli, pneumococci, typhoid bacilli, the 
 bacillus of plague, or the B. coli ; the gonococcus may also 
 develop, for it will obtain the haemoglobin necessary for its 
 development from the blood itself. 
 
 10 — 2 
 
148 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Streptococci form small white colonies which show no tendency 
 to run together to form a film. The centre of each colony is 
 more opaque than its periphery. 
 
 Staphylococci form a more or less uniform film, the colonies 
 extending laterally and fusing together. The growth is opaque, 
 and is of a dead white, lemon, or orange colour, according to the 
 nature of the staphylococcus present (albus, citreus, or aureus). 
 
 Anthrax bacilli form small white colonies, having the " barrister's 
 wig " appearance already described. 
 
 The colonies of the pneiimococcus are small flat white points, 
 which do not tend to fuse together. They are difficult to see 
 when they are young, and, in case of doubt, the tube should be 
 returned to the incubator. 
 
 The colonies of the typhoid bacilli and the B. coli are whitish 
 and opalescent. They usually have an angular or poly- 
 gonal appearance when small, and tend to run together when 
 older if they are thickly set. Their discrimination must be left 
 to an expert. 
 
 The bacillus of plague forms white colonies which are circular or 
 have a crenated outline ; they tend to run together, and form a 
 uniform film over the surface of the medium. 
 
 The gonococcuSf if it develops, forms very minute transparent 
 colonies which have been compared to droplets of dew. They do 
 not become confluent. This organism will not grow if trans- 
 planted on to the surface of ordinary media, unless a film of blood 
 be previously spread over it. 
 
 After cultures have been obtained they are to be examined 
 microscopically by the method described on p. 22, and the morpho- 
 logical appearances compared with those of the pathogenic 
 organisms which we have enumerated. It is especially important 
 to test whether the organism which has been isolated stains by 
 Gram's method or not. 
 
 ESTIMATION OF THE OPSONIC POWER OF THE 
 
 BLOOD 
 
 A very important branch of blood-work, and one that seems 
 destined to be of great value in the future, has been introduced by 
 Wright, who has demonstrated the presence in the blood of sub- 
 stances which he calls opsonins, and which have the power of 
 acting on pathogenic bacteria and altering them so that they can 
 
ESTIMATION OF THE OPSONIC POWER OF THE BLOOD 149 
 
 be taken up and digested by leucocytes. These substances are of 
 great importance in that they appear to be the chief agents in the 
 production of some forms of immunity. Take, for instance, the 
 defence of the body against staphylococci. Leucocytes have no 
 power of taking up these organisms, and if the protection of the 
 body were entrusted to them alone a slight staphylococcic lesion 
 would be a very serious matter. But the blood contains a certain 
 amount of antistaphylococcic opsonin — a greater amount in some 
 persons and less in others — and this, by combining with the 
 staphylococci, renders them easily attacked by the leucocytes. It 
 follows that where we can measure the amount of opsonin present 
 we can form some estimate of the patient's resisting power against 
 the organism in question. It is found, for instance, that the serum 
 of patients in the early stages of staphylococcic diseases, such as 
 pustular acne or boils, is usually very deficient in antistaphylococcic 
 opsonins, whilst when cure takes place the amount rises above 
 normal. These opsonins are probably specific — i.e., each organism 
 has its own appropriate opsonin : that for tubercle, for example, 
 is devoid of action on staphylococci, and vice versa. 
 
 The method given is practically that used by Wright. It is a 
 general method, and is available for almost any organism, the only 
 points of difference arising in the preparation of the emulsion of 
 bacteria, which differs somewhat with the various organisms. 
 The method is a relative one. Two tests are made, one with the 
 serum of the patient, and one with that of a healthy person, and 
 the results of the two are compared in the manner to be described 
 subsequently. 
 
 The process is not altogether an easy one, and requires a con- 
 siderable amount of patience and some practice. Yet I know that 
 several practitioners have been able to accomplish it, and as the 
 test is of great importance and interest, and as it requires little 
 in the way of apparatus, it seems right to give a description of 
 it here. 
 
 The requisites are — 
 
 1. The serum of the patient to be tested. 
 
 2. That of the healthy person taken as a control. 
 
 These are best collected in Wright's curved pipettes (see p. 34). 
 They must be taken at approximately the same time (within a 
 few hours), since the opsonin gradually becomes inert. The test 
 should be made not more than three or four days after the blood 
 has been taken. 
 
150 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 3. The emulsion of bacteria. In the case of tubercle an emul- 
 sion of dead bacilli in normal saline solution is employed. This 
 can be obtained ready prepared from Messrs. Allen and Hanbury. 
 If you wish to prepare it for yourself, it is necessary to take a 
 culture of tubercle bacilli in glycerinated broth ; incubate for two 
 months ; boil to kill the bacilli ; filter through filter-paper ; wash 
 with normal saline solution ; let the bacillary mass drain as dry 
 as possible, and then place it in a sterile tube and immerse in 
 boiling water for half an hour to make certain of its sterility. 
 The yellowish mass thus obtained will keep indefinitely, and will 
 serve for many tests. To prepare the emulsion from this, take a 
 small portion (about as big as a grain of rice) and place it in a 
 small agate mortar, and grind it up with the pestle ; then add 
 normal saline drop by drop until about 2 c.c. have been added, 
 continuing to grind meanwhile. This gives an emulsion which 
 contains isolated bacilli as well as clumps. These latter must be 
 got rid of, and to do this it is necessary to centrifugalize for three 
 or four minutes. 
 
 The staphylococcic emulsion is prepared by taking an agar 
 culture not more than twenty-four hours old, adding some normal 
 saline solution, and shaking gently so as to wash off the growth. 
 When the emulsion is made it must be pipetted off into a small 
 tube and centrifugalized for a few minutes. The emulsion must 
 not be too thick, otherwise the leucocytes will take up an uncount- 
 able number of cocci ; the proper density can only be judged by 
 experience, but the emulsion should only be faintly opalescent. 
 Emulsions of pneumococci and other organisms are made in the 
 same way. 
 
 4. An emulsion of living leucocytes. To prepare this take 
 about 10 c.c. of normal saline solution containing J per cent, of 
 sodium citrate, to prevent the coagulation of the blood. This must 
 be freshly prepared (or kept sterile, which is inconvenient), and 
 the simplest method is to use " soloids " prepared for the purpose 
 by Messrs. Burroughs and Wellcome (No. 2,456) ; one of these 
 dissolved in 10 c.c. of water will yield the solution required.* 
 This is put into a centrifugalizing-tube and warmed to blood-heat. 
 A healthy person is then pricked in the ear or finger, and his 
 blood is allowed to drop into the fluid until i c.c. or more has 
 been collected. This is then put into the centrifuge, very exactly 
 counterbalanced, and centrifugalized until all the corpuscles have 
 
 * This very convenient method was suggested by Dr. Whitfield. 
 
ESTIMATION OF THE OPSONIC POWER OF THE BLOOD 15I 
 
 come to the bottom and the supernatant fluid is left clear. If the 
 deposit is closely examined the red corpuscles will be seen to be 
 at the bottom, whilst above them there is a thin whitish layer of 
 leucocytes. Then, with a capillary pipette armed with an india- 
 rubber nipple, the whole of the clear fluid is to be pipetted off" as~ 
 close as possible to the leucocyte layer, but without disturbing the 
 latter. Next, this layer and the upper quarter or so of the column 
 of red corpuscles (which also contain leucocytes) are to be 
 pipetted off and put into a small tube, and thoroughly mixed 
 together by repeatedly sucking them into the pipette and then 
 expelling them. The result is an emulsion of living leucocytes 
 mixed with red corpuscles. The presence of the latter is a decided 
 advantage. (It is advantageous, though not absolutely necessary, 
 to rewash the leucocytes in saline solution in order to get rid of 
 the citrate of sodium.) 
 
 5. Two Wright's pipettes. These are drawn out from a piece 
 of ordinary glass tubing about 4 inches long and about as thick 
 as a lead-pencil. This is held at each end, and the central portion 
 is thoroughly softened in a Bunsen or blowpipe flame, the tube 
 being turned round the while. When quite soft the tube is 
 removed from the flame, and the ends then pulled firmly and 
 steadily apart until the softened portion is pulled out into a thin 
 tube (about the thickness of a steel knitting-needle or a little less, 
 and a foot or more long). This will give two pipettes, and to 
 separate them melt the central portion in a small flame, such as 
 that of a wax vesta, and when the glass is softened pull them 
 quickly apart. The whole pipette should be like this — 
 
 Fig. 36. 
 
 The lower figure represents the point, which must have as 
 nearly as possible the shape represented in the figure. The ease 
 and accuracy of the process depends in great measure on this 
 being the case. 
 
 The Process. — i. Prepare a pipette by placing an indiarubber 
 nipple on the thick end. Then with a grease pencil or with pen 
 and ink make a transverse line about i inch from the pointed end. 
 
152 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 The volume of fluid contained in the tube between the point and 
 this mark is spoken of as the unit. 
 
 2. Having the patient's serum, the emulsion of leucocytes, and 
 the emulsion of bacteria, ready in front of you, take the pipette 
 between the index-finger and thumb of the right hand, and com- 
 press the nipple. Immerse the point beneath the surface of the 
 emulsion of bacilli and relax the pressure on the nipple until the 
 emulsion has risen exactly to the mark, so that you have drawn 
 up one unit ; then remove the point from the fluid and relax the 
 pressure again, so that a small column of air will be sucked up. 
 This will be quite easy if the point is a good one ; otherwise it will 
 be difficult or impossible, as the column of fluid will either refuse 
 to stir or will oscillate violently. 
 
 Next immerse the point in the emulsion of leucocytes and draw 
 
 Fig. 37. 
 
 Fig. 38. 
 
 up one unit. This will be separated from the emulsion of bacteria 
 by the short column of air. Remove the point from the emulsion 
 and draw up a second column of air ; reinsert it and draw up 
 a second unit of leucocytes, and then a third column of air. 
 
 Lastly, draw up one unit of the serum. You will then have in 
 your pipette (counting from the nipple towards the point) one unit 
 of bacterial emulsion, a column of air, a unit of leucocytes, a 
 column of air, a second unit of leucocytes, a column of air, and, 
 lastly, a unit of serum (Fig. 37)." 
 
 3. Put the point of the pipette on to a clean slide and express 
 the whole of its contents, and mix them well together, sucking 
 them repeatedly into the pipette and expelling them. When 
 thoroughly mixed suck them into the pipette, suck up a short 
 column of air, and seal the tip in the flame (Fig. 38). 
 
 Then place the pipette point downwards in the incubator at 
 35° to 37° ^-j noting the time exactly, and proceed to prepare a 
 second pipette in exactly the same way, using the same emulsions 
 
 * It is not necessary to take more than one unit, and where many estimations 
 are being made one only should be employed, as less ' ' cream ' ' is required. 
 
ESTIMATION OF THE OPSONIC POWER OF THE BLOOD I53 
 
 of bacteria and leucocytes, but the control serum instead of the 
 patient's. Place this in the incubator by the side of the other, 
 noting the time at which you do so. When no incubator is at 
 hand the tubes may be placed in a vessel of water, which can be 
 kept at blood-heat for the necessary time (fifteen minutes) witlT 
 very little trouble, or the Dewar flask mentioned on p. 21 may be 
 used. 
 
 When each pipette has been incubated for a quarter of an 
 hour, remove it from the incubator, break off the end, fit the 
 nipple to the thick end, and expel the contents on to a clean 
 slide. Next mix them thoroughly together. Then prepare suit- 
 able films in the usual manner. The best method for this is 
 the one given on p. 198, in which the film is spread on two cover- 
 glasses. A small drop of the mixture is placed on one of the 
 cover-glasses held in the left hand in the manner described, the 
 second glass applied, and the two slid apart. The process is very 
 easy, and if the cover-glasses have been properly cleaned two 
 excellent films will result. 
 
 Most bacteriologists spread their films on slides, using a second 
 slide as a spreader. I am convinced that this method is not so 
 good as that given above. It is true that it renders the counting 
 somewhat easier, since the leucocytes are all collected together at 
 the end of the film, whereas with the cover-glass method they are 
 scattered all over the surface. But the counts by the latter 
 method are much more uniform, the leucocytes more defined, and 
 there is less danger of bacilli being ground mechanically into the 
 cells ; there is always a considerable margin of error in the pro- 
 cess, and I believe that the extra amount of accuracy obtainable 
 in this way is quite worth the additional trouble involved. 
 
 The films have next to be stained. When the organism in 
 use is the staphylococcus, pneumococcus, etc., Jenner's stain is as 
 good as any ; or the film may be fixed with formalin (p. 203) or 
 perchloride of mercury (p. 203), and stained with carbol thionin. 
 In the case of tubercle bacilli it is best to fix with saturated solu- 
 tion of perchloride of mercury (one or two minutes), wash, stain 
 in the ordinary way with hot carbol fiichsin, decolorize for half to 
 one minute in 2^ per cent, sulphuric acid in methylated spirit, and 
 to counterstain for about five minutes in borax methylene blue, 
 or in Delafield's haematoxylin. It is necessary to get the proto- 
 plasm of the leucocytes clearly defined, so that a powerful stain 
 is necessary. Washj dry, mount. 
 
154 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Lastly, the films are examined with the oil-immersion lens. The 
 polynuclear leucocytes will be found to contain the bacteria, and 
 it will be necessary to count the number in each of fifty leucocytes 
 in both your preparations — i.e., in that made with the patient's 
 serum and in the control made with that from a healthy person. 
 The ratio between the two gives the opsonic index. For example, 
 in one case the number of tubercle bacilli contained in fifty poly- 
 nuclear leucocytes taken at random amounted to 78. In the 
 control specimen from a healthy person the same number of poly- 
 nuclears contained 172. The ratio xV% = o*45 gives the opsonic 
 index ; it shows that the patient has less than half the normal 
 amount of opsonin, and probably, therefore, less than half the 
 normal resisting power to the tubercle bacillus. 
 
 The diagnostic value of the examination is considerable. In 
 acute infections the index is almost always low to the organism 
 causing the disease, but normal to others. For example, in 
 a case of severe furunculosis the index to staphylococci was 
 0-65, and in a case of pustular acne 07. In a case of septicaemia 
 due to streptococci the index (tested with a culture of strepto- 
 cocci obtained from the patient's blood) was o-6 on several 
 occasions. 
 
 When spontaneous cure takes place it is accompanied by, and 
 is apparently due to, an increase of the opsonic power of the blood. 
 Thus, in pneumonia the opsonic index tested with pneumococci is 
 below normal until the crisis is reached, when there is a sudden 
 rise above normal, so that the patient is for a short period more 
 resistant than the healthy person. In staphylococcic lesions the 
 rise is as a rule more gradual and irregular, and the lesions may 
 persist when the index is high, though in most cases this heralds a 
 rapid improvement. 
 
 The behaviour of the opsonic index in tuberculosis is very in- 
 teresting. The main feature is that it is very variable, especially 
 in patients in whom the disease is progressing rapidly and in those 
 who are taking exercise. This variability is supposed to be due 
 to auto-inoculation, bacilli being detached from the lesions and 
 lodged in the tissues, where they act as small doses of vaccine. 
 As a general rule it is thought that (in lupus especially"^) a high 
 index is of favourable import, and vice versa, but there is not the 
 same more or less direct relation between a high index and the 
 process of cure that there is in pneumococcic and some other 
 * Bulloch, Trans. Path. Soc, 1905. 
 
ESTIMATION OF THE OPSONIC POWER OF THE BLOOD 155 
 
 infective processes. A patient may die of tuberculosis whilst his 
 opsonic index is high. This seems paradoxical, but the conditions 
 are exceedingly complex, and the symptoms of pulmonary tuber- 
 culosis are due almost as much to the other organisms (strepto- 
 cocci, etc.) as to the tubercle bacilli itself. In miliary tuberculosis 
 the index is often high, and shows great variations in a short time. 
 I believe you are almost safe in diagnosing tubercle if the index 
 is below 0-8 or above 1*2, and if it shows marked variations from 
 day to day the probability is still greater. 
 
 A low opsonic index towards a given organism, therefore, 
 denotes either (i) an infection with that organism, or (2) a low 
 power of resistance, so that if the patient is exposed to infection 
 invasion will readily take place. In such cases he should be care- 
 fully shielded from exposure, and the general health improved by 
 fresh air, careful feeding, tonics, etc. 
 
 A high opsonic index {i.e., one decidedly above normal) usually 
 indicates that the patient has had an attack of the disease caused 
 by the organism in question, and has overcome it. Normal 
 persons differ very little amongst themselves ; for instance, in a 
 series of healthy persons, if the average index be taken as normal, 
 it is unusual to find one below 0-95 or above i'05. 
 
 A study of the opsonic index is the basis of Wright's method 
 of treatment by means of vaccines. In carrying this out the 
 patient's opsonic index is raised by injections of the organisms 
 which cause the disease (carefully sterilized) in appropriate doses. 
 These are given in a healthy part of the body, where the tissues 
 are stimulated to produce opsonins against the organisms intro- 
 duced, and these opsonins are carried in the blood to the lesion. 
 The reason why it is necessary to estimate the opsonic index of 
 the blood from time to time in carrying out this process is that 
 the injection causes it to fall for a variable time (usually a few 
 days — the negative phase), and if a second injection be given 
 before this fall has gone off and been succeeded by a rise above 
 the initial level, harm rather than good will follow. 
 
 The method of preparing these vaccines is roughly as follows : 
 The cultures should in all cases where practicable be prepared 
 from the organisms isolated from the patient himself. "Stock" 
 vaccines may do good in some cases, especially, perhaps, in 
 staphylococcic diseases ; but in diseases due to streptococci, B, coli, 
 etc., they are not nearly so likely to succeed as vaccines specially 
 prepared : these organisms differ slightly in different cultures, and 
 
156 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 a vaccine prepared from one variety may have little action on 
 another. The culture should be made on agar (or blood-agar in 
 the case of gonococci, etc.), and should be one or two days old. 
 To each culture- tube about 2 to 5 c.c. of sterile normal saline 
 solution are added, and the bacteria scraped off by means of a 
 platinum needle and the tube gently shaken, so that the organisms 
 are emulsified. The number of bacteria in this emulsion must 
 next be counted. Wright's method is usually employed, and is 
 carried out by mixing the bacterial suspension with blood in known 
 proportions, making films, and counting the red corpuscles and 
 bacteria in the same field of the microscope, so as to obtain the 
 relative proportions of the two. The number of red corpuscles 
 per cubic millimetre is known (or can be counted), and from that 
 the number of organisms can be calculated. Proceed as follows : 
 Take an opsonin pipette, and draw up one volume of blood from 
 a healthy person, then a bubble of air, then one volume of citrate 
 solution (p. 150), then one volume of the emulsion. Mix them 
 together on a slide, make films on two cover-glasses, dry, and 
 stain by Jenner's method. Proceed to count the red corpuscles 
 and bacteria on several fields of the microscope : this will be easier 
 if you rule four ink lines enclosing a square on the lower lens of 
 your eyepiece, and count the objects lying therein. When you 
 have done this, move the slide so as to get a fresh film, and count 
 again. Do this a large number of times, and add the totals of the 
 corpuscles and of the bacteria, and calculate the ratio between the 
 two. (Thus in one case the total red corpuscles on the various 
 fields were 18, 15, 20, 21, 14, 17, 10, and 15 ; and the bacteria 37, 
 26, 31, 40, 25, 30, 32, 36 : the totals were 130 and 257, or, roughly, 
 I to 2.) The calculation of the number of bacteria per cubic 
 millimetre is then easy, and that multiplied by 1,000 gives the 
 number per c.c. 
 
 Next the emulsion has to be sterilized by heat. It is placed in 
 a test-tube, which is drawn out and sealed in a blow-pipe flame 
 and completely immersed in a water-bath at 60°. After one hour it 
 is removed, and a small amount placed on a suitable culture 
 medium and incubated (in order to ascertain its sterility), and the 
 tube re-sealed. If sterile, it is now to be diluted with a 0-25 per 
 cent, solution of lysol or carbolic acid in sterile normal saline 
 solution to such an extent that the dose required is made 
 I c.c. Thus an emulsion of staphylococci was found to contain 
 2,500,000,000 cocci per c.c. The dose required was 500,000,000, 
 
ESTIMATION OF THE OPSONIC POWER OF THE BLOOD I57 
 
 SO that I part of the emulsion was diluted with four of 0*25 per cent. 
 lysol. Lastly, it is pipetted off with a i c.c. pipette or hypodermic 
 needle (of course, sterile) into i c.c. ampoules, previously sterilized 
 by heat. 
 
 The dosage varies with different bacteria and with differeril 
 cultures, and the following are approximate only. With staphy- 
 lococci it varies between 50,000,000 and 1,000,000,000, 500,000,000 
 being a good average. Meningococci may be given in the same 
 doses, or rather smaller : with a young child I commence with 
 25,000,000. B, coli is usually rather toxic, and may cause a good 
 deal of local and general reaction, and the first dose should not 
 exceed 20,000,000 ; it may subsequently be increased somewhat 
 if thought necessary. With pneumococci the dose may be 
 50,000,000 to 100,000,000 or more, and with gonococci 50,000,000 
 to 250,000,000. 
 
 The dose of tuberculin (T. E. or T. R.) is y^L^ milligramme to 
 jjqL^ milligramme. The material is bought in bottles containing 
 I to 5 C.C., with the strength stated on the label, and dilutions 
 prepared with 0*25 per cent, normal saline solution, previously 
 sterilized. 
 
 As regards the question as to the necessity of controlHng the 
 injections by observations of the opsonic index, it is difficult to 
 speak with confidence. With staphylococci it is probably un- 
 necessary, and a small dose every week, or a larger one every 
 fortnight, will usually yield good results. In the case of tubercle 
 I am doubtful as to its advantage, but usually carry it out for one 
 or two injections in order to make sure that I am not dealing with 
 a patient with a very long negative phase, in which case I lower 
 the dose. In the other organisms I prefer, if possible, to take the 
 index regularly, and I believe this is especially desirable in the 
 case of meningococcic infections. 
 
 The results of this method of treatment are extremely good 
 in the case of staphylococcic lesions (boils, acne, etc.), though 
 in some chronic cases a long course is necessary. In the 
 complications and sequelae of gonorrhoea (iritis, arthritis, etc.) 
 great benefit is also obtained, and some chronic pneumococcic 
 lesions (such as sinuses left after empyemata) heal very rapidly 
 after one or two injections. Chronic cystitis and pyelitis due to 
 B. coli are sometimes cured, but more often the symptoms are 
 greatly reUeved without' actual cure being obtained. Some cases 
 of ulcerative endocarditis and septicaemia have been completely 
 
158 CLINICAL BACTERIOLOGY AND H.^MATOLOGY 
 
 cured, and the other applications of the process are too numerous 
 to mention here. In general we may say that it should be tried 
 in all chronic infective diseases that do not yield readily to easier 
 methods. 
 
 In tuberculosis the results have been on the whole disappointing, 
 and the method should be reserved as an adjuvant to other 
 treatment, or for lesions (tuberculous iritis, ulceration of the 
 bladder, etc.) in which direct surgical treatment is unavailable. 
 Tuberculous sinuses often heal well after a few injections, and 
 tuberculous ulceration of the skin does better than ordinary lupus. 
 
 COLLECTION OF MATERIAL AT POST-MORTEM 
 EXAMINATIONS 
 
 The saprophytic bacteria which occur in such vast numbers in 
 the skin and alimentary canal during life undergo very rapid 
 multiplication after death ; hence, in cases where bacteriological 
 examinations have to be made, the sectio should be performed as 
 soon as possible after death. 
 
 The materials which should be examined in all cases are the 
 heart-blood, the spleen, and the liver, and the following methods 
 are to be employed : 
 
 The heart-blood should be collected in the method which has 
 been described previously (see p. 33), and cultures may be made 
 upon the spot, or the pipettes sealed at both ends and taken to the 
 laboratory. 
 
 The spleen may usually be examined in the same way. If it is 
 so firm and hard that no fluid rises into the pipette, it should be 
 treated in the same way as the liver. 
 
 Cultivations should be made from the liver at the time when the 
 autopsy is performed. The organ should be cut in half, and a 
 small portion of the cut surface deeply seared with a hot iron. 
 This area is then to be perforated with a stout platinum needle, 
 and the culture media inoculated at once. 
 
 If the material has to be taken to a distance, and no culture- 
 tubes are at hand, a different course must be adopted . The simplest 
 way is to cut out a cube of liver substance from the centre of the 
 organ, and to sear every part of its surface^ with the fiat of a red- 
 hot knife. The block (which may be about as large as a lump of 
 sugar) must be dropped at once into a sterilized bottle. Another 
 
SECTION-CUTTING I59 
 
 plan is to sear the surface of the block, and then to tie a piece of 
 string round it and dip it quickly into melted paraffin (a candle 
 will do), and allow the coating to set ; the dipping is to be 
 repeated several times, and the specimen (string and all) may^ 
 then be packed without further precautions. In any case it must 
 reach the laboratory as soon as possible. 
 
 Where cultural examinations are not required, small portions 
 of the organs should be placed in a suitable hardening fluid as 
 soon as possible. Equal parts of methylated spirit and water is 
 perhaps as good as anything, and, in the absence of this, undiluted 
 whisky or other spirit answers equally well (see p. 161). 
 
 Other solid organs are treated in the same way. Fluids (pus, 
 the contents of cysts, pericardial or other fluid, etc.) should be 
 collected in pipettes in the manner adopted for the heart-blood. 
 
 SECTION-CUTTING 
 
 The methods employed in section-cutting are somewhat outside 
 the scope of this work, inasmuch as sections are rarely necessary 
 for the purposes of bacteriological diagnosis, and I have attempted 
 to give the simplest possible methods in all cases. The presence 
 of bacteria in the tissues can usually be demonstrated by the simple 
 processes of smearing the cut surfaces of tissues on clean slides 
 or cover-glasses, and treating the films thus obtained by the fixing 
 and staining methods previously described. If, for instance, we 
 have to search for tubercle bacilli in tuberculous glands, it is 
 usually sufficient to smear the cut surfaces of the glands on a slide, 
 dry, fix by heat, and stain in the same way as sputum is stained 
 for the tubercle bacillus. If anthrax bacilli were being looked for 
 in the liver or other organ removed post-mortem, the same 
 method of procedure would be adopted, except that Gram's 
 method of staining would be used. So also for typhoid bacilli in 
 the spleen, where the film would be stained with a simple stain 
 such as thionin or Loffler's methylene blue. 
 
 It seems advisable, however, to give a short general account of 
 the processes involved in section-cutting, for they are by no means 
 difficult, and do not require very elaborate apparatus. Further, 
 the same methods of section-cutting are used for investigating 
 the nature of tumours, etc., and this is done already by many 
 practitioners and should be done by still more. 
 
l60 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Slices of organs or tissues which are to be cut have first to be 
 fixed. The process of fixation consists essentially in the applica- 
 tion of some agent which brings about coagulation of the com- 
 ponent proteids with as little distortion of the morphological 
 elements as possible ; if this step were not carried out the 
 subsequent processes would be liable to cause alterations in the 
 shape, size, and appearance of the cells and fibres. There are 
 two chief methods of fixation, that involving the use of chemical 
 substances, and that involving the use of heat. The processes 
 which are used in fixing the tissues harden them at the same time ; 
 this is necessary, for fresh tissue would yield before the sharpest 
 knife, and could not be cut into thin sections. These processes are 
 always carried out, no matter what method of section cutting is to 
 be adopted. 
 
 In cutting sections it is necessary that the material should be 
 sufficiently firm and homogeneous in consistency. The former is 
 secured to some extent by the process of hardening, but a 
 properly hardened block is rarely firm enough to permit of its 
 being cut into sections without further preparation. Further, it 
 almost invariably happens that some parts of the material are 
 firmer or harder than others ; and if such a substance were cut 
 the harder parts might be sufficiently firm, whilst the softer parts 
 would simply crumble before the knife. There are two methods 
 of overcoming this difficulty — freezing and embedding. 
 
 The freezing process is very simple, and it is one which can easily 
 be carried out at home. The sections which it yields are usually 
 quite sufficient for purposes of histological research (the diagnosis 
 of tumours, etc.), but they are rarely sufficiently thin for a proper 
 demonstration of the bacteria which they may contain. The 
 sections are cut more easily by the freezing than by the paraffin 
 process, but they are decidedly more difficult to manipulate. 
 
 In the freezing process the block or tissue after fixing and 
 hardening is dipped, or, better, soaked for some hours, in a thick 
 solution of gum arable. It is then placed on the plate of a micro- 
 tome and frozen until the tissue assumes the consistency of fairly 
 hard cheese, and can be cut into thin sections. 
 
 The embedding process should be called the infiltration process ; 
 the tissue to be cut is infiltrated throughout with some firm 
 substance, and not merely embedded therein. Two embedding 
 materials are in general use — paraffin and celloidin. The latter 
 will not be described, as it is only necessary for special work, and 
 
FIXING MATERIAL FOR CUTTING l6l 
 
 for ordinary purposes cannot compare with paraffin for beauty of 
 results and facility of application. 
 
 In the paraffin process the tissue is infiltrated throughout with 
 hard paraffin (such as is used for the better varieties of paraffin 
 candles), so that every cell and every fibre is permeated and 
 supported on every side. To do this requires a number of 
 processes. It would be of no use to immerse the block of tissue 
 in the paraffin just as it is, for the paraffin would not wet it, much 
 less soak into it. The water is first removed; and this is done by 
 soaking the material in absolute alcohol. But alcohol does not 
 -dissolve paraffin or mix with it ; it is therefore necessary to remove 
 it by means of some fluid which will mix with it on the one hand 
 and paraffin on the other. Of these there are many : xylol, 
 chloroform, benzine, cedar oil, and many more are in use for 
 special purposes. Chloroform answers most purposes, and is to 
 be generally recommended. The block of tissue is now ready to 
 be soaked in melted paraffin ; it is kept in a bath of this substance 
 until the chloroform has been entirely driven off and replaced by 
 the paraffin. The whole is then allowed to cool, is shaped into 
 suitable blocks, and is then ready for cutting. 
 
 We shall now describe the processes in fuller detail. 
 
 FIXING MATERIAL FOR CUTTING 
 
 These processes must be understood by all practitioners, even 
 although they do not intend to cut sections for themselves. It 
 happens to every medical man to find it necessary to send tumours, 
 etc., to a laboratory to obtain a pathological diagnosis ; and in 
 very many cases the materials are treated in a way which 
 absolutely prevents good sections being obtained. Many fixing 
 fluids are in use, and any of them may be selected, but it is 
 absolutely necessary that the material to be investigated should be 
 cut into small pieces and put into a large bulk of the fluid at once. 
 This is especially necessary in the case of material removed at a 
 post-mortem examination, where the tissues and organs have 
 already undergone alteration. 
 
 As regards the size of the slices which are to be placed in the 
 hardening fluid, it is sufficent to say that they should never 
 exceed -^ inch in thickness, and if perchloride of mercury is used 
 should be even thinner. The other dimensions of the block are 
 of less importance. 
 
 II 
 
ibz CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 The bulk of the fluid in which the block is placed should be at 
 least twenty times that of the block, and it is not advisable to 
 place two blocks in the same vessel. 
 
 The fluids which we shall recommend for this purpose are : 
 
 1. Perchloride of mercury in normal saline solution. This is 
 prepared by dissolving common salt in water in the proportion of 
 7 grammes to a litre (about 3 J grains to the ounce), and saturating 
 this solution whilst hot with perchloride of mercury. The 
 solution must be allowed to cool completely ; as it does so, 
 crystals of the mercury salt will separate out. 
 
 This fluid fixes completely in twenty-four hours, or less, and 
 gives most excellent results. Its powers of penetration are not 
 very great, so that slices of tissue which are to be fixed in it 
 should be thin. 
 
 The after-treatment of the blocks fixed in this fluid must be 
 described briefly. They are allowed to remain in the solution for 
 twenty-four hours and no longer, and are then washed for twenty- 
 four hours in running water to remove the perchloride of mercury. 
 They are then passed through the various strengths of spirit (as 
 will be described subsequently), a little tincture of iodine being 
 added to each to remove any mercury which may still remain. 
 The other steps are the same as those which are used if other 
 methods of fixation have been adopted. 
 
 2. Formalin. This should be used in a 5 per cent, solution in 
 normal saline solution (o'8 per cent.). It yields very good results, 
 and is perhaps the fluid which can be most warmly recommended 
 to a practitioner who is going to send his material to a public 
 laboratory. ■'^ The fluid has very great powers of penetration, and 
 the slices may be much thicker than we have recommended. 
 The one objection to the fluid is that it interferes somewhat with 
 the way in which the sections stain. 
 
 3. Alcohol is a very good fixing fluid. When it is used the 
 blocks should be cut small and placed at once in methylated 
 spirit diluted with an equal quantity of water. 
 
 * Formalin should not be used for tissues which are to be searched for the 
 tubercle bacillus, as it prevents the decolorizing action of the acid. 
 
SECTION-CUTTING BY THE FREEZING METHOD 
 
 163 
 
 SECTION-CUTTING BY THE FREEZING METHOD 
 
 Sections which are prepared by the freezing method are rarely 
 as thin as those prepared by one or other of the infiltration- 
 processes, but are prepared very rapidly, and are often sufficient 
 for diagnostic purposes, where rapidity is the first consideration. 
 
 The blocks of tissue must be hardened before being cut, any 
 of the above fluids being applicable ; where alcohol is used it 
 must be washed out in water, as it will not freeze. Where 
 more rapid work is required the best method is a modification of 
 the old boiling process, as revived by Mr. Strangeways. The 
 slices of tissue from which sections are to be cut are thrown at 
 once into boiling water, and allowed to boil vigorously for two or 
 
 Fig. 39. — Cathcart's Microtome arranged for cutting Frozen Sections. 
 
 three minutes ; the water must be actually boiling when the 
 tissues are added, and the bulk used should be large as compared 
 with the block. The tissues are then rapidly cooled by being 
 thrown into cold water, and are then ready for cutting. The 
 outer surface of the block should be rejected. 
 
 This method of fixation leads to a little distortion of the tissues 
 and alters any blood which they may contain, but it is very good 
 for diagnosing tumours. It is invaluable in the post-mortem 
 room, and for diagnosis of the nature of a tumour during opera- 
 tion. In skilful hands a section may be cut, stained, mounted, 
 and a diagnosis made in ten minutes ; or if no process of fixation 
 
 II — 2 
 
l64 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 by boiling be used, in much less. I have cut, stained, mounted, 
 and diagnosed a tumour within three minutes. 
 
 A microtome is necessary for the successful cutting of sections, 
 and the Williams and Swift patterns are those in general use for 
 the freezing process. AVe shall recommend the practitioner who 
 intends to take up this branch of work to procure a Cathcart 
 microtome, which is exceedingly cheap (it costs about a guinea) 
 and answers admirably. The great advantage of this machine is 
 that it will serve for cutting sections in paraffin as well as for 
 frozen sections. 
 
 The blocks of tissue which are to be cut are dipped in a thick 
 and syrupy solution of gum arabic ; if time is no object it is a 
 great advantage to soak them in this for several hours. A block 
 is then placed on the corrugated plate of the microtome and 
 frozen by means of the ether spray which impinges upon it. 
 When the mass is nearly frozen a section is taken off by means 
 of a razor which is ground flat on one side, or the special knife 
 which may be obtained with the apparatus ; it is better to 
 moisten the upper surface of the knife with a little of the gum. 
 The section is carefully removed with a camel's-hair brush and 
 placed in a large vessel of clean water, so that the gum may be 
 dissolved out of it, and is then ready for staining. The block is 
 then raised by means of a very slight turn of the large milled 
 head under the apparatus, and another section cut. 
 
 The mass must not be frozen too hard ; if this has been the 
 case, the necessary thawing will be hastened by gently breathing 
 on the block. If it begins to thaw, a few squeezes of the bellows 
 will bring it to the proper consistency. 
 
 For extremely rapid work, the best method of freezing is by the 
 use of liquid carbon dioxide : this, however, is difficult in private 
 practice, owing to the unportability of the cylinders. The best 
 method is to use ethyl chloride or ansestile in metal cylinders, 
 such as are used for local anaesthesia. The best method is as 
 follows : Place a few drops of water on the plate of the microtome, 
 and freeze it solid by the ether spray whilst the surgeon is 
 removing the tumour ; cut a suitable slice off the latter and place 
 it on the layer of ice thus formed ; direct the ethyl chloride spray 
 downwards on to the slice, which will be frozen in a few seconds. 
 Unless the water be previously frozen on the plate of the microtome, 
 the block of tissue is very liable to slip, the lower portion being 
 frozen last. 
 
STAINING AND MOUNTING FROZEN SECTIONS 165 
 
 Where very rapid work is required it is not advisable to stain 
 the sections in the method described in the next paragraph, since 
 it takes too long. A simple stain (such as watery methylene blue) 
 is used, the staining being done on the slide, a cover-glass applied, 
 and the excess of stain removed by means of blotting-paper, ft 
 is necessary to acquire a considerable amount of experience of 
 this method before using it for diagnosis, as the appearance 
 of sections prepared in this way and examined in a watery fluid is 
 very different from that which they have when double-stained and 
 mounted in balsam. 
 
 STAINING AND MOUNTING FROZEN SECTIONS 
 
 These processes are best carried out in watch-glasses. No 
 attempt will be made to describe the methods by which frozen 
 sections may be stained for the purposes of bacteriological research, 
 for they are not so suitable as paraffin sections for this purpose. 
 We shall describe the process of staining in haematoxylin (with 
 or without eosin as a counterstain) and mounting in balsam. 
 
 The requisites are : Five watch-glasses containing respectively 
 haematoxylin, watery solution of eosin (about i per cent.), alcohol 
 (50 per cent.), absolute alcohol, and clove oil ; a saucer or other 
 vessel containing water to which a few drops of ammonia have 
 been added ; several strips of thin writing-paper, each about 
 I inch wide and 2 inches long ; some needles, which may be 
 mounted in handles ; slides, cover-glasses, and balsam. 
 
 A section is to be removed from the bowl of water in which it 
 is floating by means of one of the strips of paper ; this must be 
 inserted under it, and the section " pinned " in place upon it by one 
 of the needles. A special section -lifter may be used, but is not so 
 good. It is then transferred to the watch-glass containing the 
 haematoxylin solution, and the staining process is allowed to go 
 on for a minute or two, a fresh section being manipulated whilst 
 it is taking place. The first section is then removed in the same 
 way as before, and placed in the water containing the ammonia ; 
 it soon turns blue, and when this is the case it is ready to be 
 transferred to the eosin, then into the dilute alcohol, the absolute 
 alcohol (where it should remain for a minute or more), and finally 
 into the oil of cloves. It is then ready to be mounted in balsam* 
 A convenient way in which a section can be transferred to a slide 
 is as follows : The section is carefully spread out whilst in the 
 
l66 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 oil of cloves, two needles being used for the purpose, and a slip of 
 paper insinuated beneath it. This strip of paper is then drawn 
 slowly out of the liquid, and any folds or creases which may be in 
 the section straightened out with the needles, the excess of the 
 oil of cloves being allowed to drop off whilst this is taking place. 
 The strip of paper is then inverted (the section remaining adherent 
 to the under surface), placed upon a clean slide, and pressed firmly 
 upon it ; the pressure squeezes out the greater part of the oil, so 
 that the section adheres to the slide, and the paper can be stripped 
 cautiously from it. A drop of balsam is then applied, the section 
 covered with a cover-glass, and examined under the microscope. 
 
 It is a great advantage to rinse the section in distilled or clear 
 rain-water after removing it from the haematoxylin. 
 
 The solution of haematoxylin is best bought ready made, as its 
 preparation is somewhat difficult. Delafield's solution is the best 
 for general work. 
 
 A counterstain is not really necessary for diagnostic purposes, 
 and its omission hastens the process somewhat. 
 
 THE PARAFFIN PROCESS 
 
 Tissues which are to be cut in paraffin may be hardened in any 
 of the fluids mentioned above. They are then dehydrated, cleared 
 in chloroform or other fluid which mixes with alcohol and dissolves 
 paraffin, and finally soaked in a mixture of hard and soft paraffin 
 kept just at the melting-point. This paraffin should be obtained 
 specially for the purpose ; the Cambridge paraffin is the best. It 
 is made in two varieties, the soft, which melts at 48° C, and the 
 hard, which melts at 55° C. The amounts of each which should 
 be used for embedding depend upon the external temperature : in 
 very hot weather hard paraffin may be used alone, while under 
 average circumstances a mixture of equal parts of each is best. 
 
 We shall now proceed to describe the various processes seriatim. 
 
 Dehydration. — This is very simple. The blocks of tissue are 
 placed in weak spirit for a few hours or for a day, then changed 
 into stronger spirit, and so on until absolute alcohol is reached. 
 The slower this process is carried out the better will be the results ; 
 in practice the strengths of the successive lots of spirit used may 
 be 40 per cent., 75 per cent., and the strong methylated spirit, and 
 the block may remain in each for twelve hours. Lastly, it goes 
 into two successive lots of absolute alcohol. 
 
THE PARAFFIN PROCESS 167 
 
 In all cases the amount of fluid must be greatly in excess. It 
 is useless merely to cover the block with the spirit. 
 
 Clarification. — In the next step the alcohol is removed from the 
 tissue and replaced by some fluid which will dissolve paraffin. 
 Fats are dissolved out from the tissues at this stage. — 
 
 This step is also very simple. The blocks are passed directly 
 from absolute alcohol into chloroform, and allowed to remain 
 there for twelve to twenty-four hours, according to their size. It 
 is not necessary to use a preliminary bath of a mixture of alcohol 
 and chloroform. 
 
 It is a good plan to place the bottle containing the block in a 
 warm place with the cork out for an hour or so before proceeding 
 further, as by so doing the last traces of the alcohol will be removed. 
 
 Infiltration with Paraffin. — This is the stage which presents most 
 difficulties to the home-worker, for it is necessary to keep the 
 
 Fig. 40. — L-sHAPED Moulds for embedding in Paraffin. 
 
 block of tissue soaked in paraffin which is just melted for at least 
 twelve, and more often twenty-four, hours. To do this properly 
 involves the use of some sort of incubator. This might possibly 
 be rigged up out of a tin biscuit-box in the manner already 
 described, though considerably more heat would be necessary, as 
 the paraffin melts at about 50° C. But the writer has often 
 embedded the blocks by placing them in bottles containing the 
 paraffin at such a distance from the fire that the paraffin is never 
 completely melted, but always shows a thin solid layer on the 
 surface. To do this it is only necessary to look at the bottle 
 occasionally, and move it a little further from the fire if the 
 paraffin is completely melted, and vice versa. The process may 
 be stopped at night without any harm resulting, and if the soaking 
 only continues for a few hours at a time it is of no consequence so 
 long as the total period is made up. 
 
 Casting the Blocks. — Special metal moulds are used in the labora- 
 tory (Fig. 40). A pill-box will do quite well. A small amount of 
 melted paraffin is poured into the box, and the piece of tissue is 
 
l68 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 taken from the bottle containing the melted paraffin with a pair of 
 forceps (previously warmed, so as to prevent the paraffin from 
 setting upon the points), and placed in the paraffin in the pill-box. 
 It is necessary to see that the surface from which sections are to 
 be cut should be placed downwards. The box is then filled up 
 with melted paraffin, and placed in a cool place or surrounded with 
 water. The moment a firm film has formed over the surface the 
 whole is plunged in cold water to hasten the setting of the paraffin ; 
 the more rapidly this takes place the better will the block cut. 
 When the paraffin mass has hardened completely throughout, it 
 is trimmed into shape, taking care that the edges of the surface 
 which is to be cut are accurately parallel. 
 
 Cutting the Sections. — For cutting sections in paraffin no 
 microtome can be compared with the Cambridge Rocker, but 
 very excellent results can be obtained by the use of the 
 
 ■ 
 
 Fig. 41. — Clamp for holding Wooden Block with the Paraffin Block. 
 
 Cathcart microtome already mentioned. The paraffin block 
 containing the piece of tissue is mounted on the freezing-plate 
 of the microtome (which must be heated, and the lower surface 
 of the block pressed upon it), and the sections cut in the manner 
 described ; a very sharp knife is essential, and the stroke must be 
 quicker and sharper than is the case when frozen sections are 
 being cut. In another form of the microtome a special inner 
 tube is provided for cutting sections by the paraffin process. 
 The blocks are retained in place by a clamp, and appear in the 
 same position as that occupied by the mass of frozen gum. As 
 the paraffin is not sufficiently hard to be gripped by this clamp, 
 they must first be mounted on a piece of wood of a suitable size 
 and shape. This can be cut out of a piece of firewood, and 
 should have one surface left rough ; this surface must be dipped 
 in melted paraffin, and the under surface of the block partially 
 melted in the flame and pressed firmly upon it. The piece of 
 wood is then to be placed in the jaws of the clamp, and the screw 
 tightened up. 
 
THE PARAFFIN PROCESS l6g 
 
 In the Cambridge Rocker and in some other forms of micro- 
 tomes the sections adhere to one another at the edges, and form 
 long ribbons as they are cut. In the Cathcart microtome this is 
 not the case, and each section must be dealt with separately ; it 
 is to be removed carefully from the knife-blade with a cameFs-^ 
 hair brush or a finely pointed pair of forceps, and placed upon 
 the surface of a bowl of water, just hot enough to warm the 
 paraffin without melting it. When this is done the sections will 
 spread out and lose all the creases, and are then ready to be 
 mounted on slides or cover-glasses. 
 
 It often happens that the sections roll up on the knife. In this 
 case they must be placed on the surface of cold water, and an 
 attempt made to straighten them out by careful brushing with a 
 camel's-hair brush ; when fairly flat they are to be lifted up on 
 a slide or piece of paper (dipped into the water and insinuated 
 below them), and transferred to the hot water as before. But 
 the rolling of the sections may often be prevented by sharpening 
 the knife, by re-embedding the tissues in harder or in softer 
 paraffin according to the weather, or by varying the angle which 
 the knife edge makes with the glass runners of the microtome. 
 These devices can only be learnt by experience. 
 
 When the sections are flattened out on the surface of the hot 
 water, they are ready to be mounted upon slides or cover-glasses : 
 slides are by far the best for beginners. The slides (or cover- 
 glasses) must be perfectly clean, and are best kept in methylated 
 spirit until they are to be used, and the spirit not wiped ofl". 
 Each slide is then inserted separately into the water in an oblique 
 position, and the section moved until it lies over the centre ; the 
 slide is then raised out of the water, and carries the section out 
 with it. 
 
 The excess of water is now to be removed by a piece of blotting 
 or filter paper, and the slide placed in the warm incubator for a 
 few hours. At the end of this time the sections will adhere by 
 atmospheric pressure (like a boy's leather sucker to a stone), and 
 will not come off in the subsequent processes. If an incubator 
 is not at hand the slides may be placed near the fire (protected 
 from dust), and kept at the body temperature or a little higher for 
 a few hours ; the exact temperature does not matter^ and no harm 
 will result if the paraffin melts, provided that the section has 
 previously become dry. 
 
 In the older methods of fixing sections to the slides various 
 
170 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 forms of cements had to be used, and were a great disadvantage. 
 They are quite unnecessary except for sections of the central 
 nervous system ; if these are being dealt with the slide must be 
 coated with a very thin layer of a solution of egg-albumin in water 
 before the section is laid upon it. The process is then exactly 
 the same as before. 
 
 STAINING AND MOUNTING PARAFFIN SECTIONS 
 
 We will suppose that the sections have been cut, flattened out 
 on hot water, and caused to adhere to slides, and shall describe 
 in general terms the steps through which they must be taken 
 before they are ready for examination. In the first place, it is 
 obvious that the paraffin, which permeates all parts of the 
 section and surrounds it on all sides, must be removed, and 
 this is done by pouring xylol, benzine, or turpentine upon it. 
 At least two supplies of the fluid should be used, and it should 
 be allowed to act for at least two minutes, the slide being 
 rocked all the time. We have now removed the paraffin, and 
 the next step is to remove the xylol or other solvent ; this is 
 done by means of absolute alcohol. At least two lots should be 
 used, and it should be allowed to act for two minutes. The sHde 
 is then washed in water, and is ready for staining. When the 
 section is wet with xylol it will be quite transparent; this is 
 because the refractive index of the xylol is almost the same as 
 that of glass, and the rays of light which come through the section 
 are not bent. But when the alcohol is added the section will 
 suddenly become opaque, and for the opposite reason. 
 
 If there is a milkiness on the section or slide when the water 
 is poured on, it is a sign that the xylol has not been completely 
 removed ; xylol will not mix with water, and forms an emulsion 
 with it. If this should happen, you must give the section another 
 dose of absolute alcohol, and rewash in water. 
 
 It is an advantage to wipe the surface of the slide (of course 
 avoiding the section) before going from one fluid to another. 
 
 A cardinal rule in dealing with paraffin sections is never to let the 
 section get dry from the moment the first dose of xylol is added 
 until the final mounting in balsam. 
 
 The methods of staining which are in use are legion, and it 
 would be far beyond the scope of this book to describe even a 
 few of those which are used in histological work, and to give 
 indications for their use. It will be sufficient to describe (i) a 
 
STAINING AND MOUNTING PARAFFIN SECTIONS 171 
 
 method suitable for the diagnosis of tumours, etc., and for 
 ordinary histological purposes ; (2) a method of staining to demon- 
 strate bacteria which stain by Gram's method ; (3) a method for 
 bacteria which do not stain by Gram's method ; and (4) the 
 process for demonstrating tubercle bacilli in the tissues. ~ 
 
 I. Staining sections for histological purposes: 
 
 1. Xylol, two lots (to remove paraffin). 
 
 2. Absolute alcohol, two lots (to remove xylol). 
 
 3. Water (to remove the alcohol). 
 
 4. Stain with haematein (or haematoxylin*) for ten minutes or 
 more, according to the nature of the specimen and the con- 
 dition of the stain. The exact length of time can only be learnt 
 by trial, but ten minutes will be about right. Rinse in distilled 
 water. 
 
 5. Wash thoroughly in tap- water, continuing the washing until 
 the sections have a decidedly blue tinge. The haematoxylin 
 compounds are very much like litmus, being red in presence of 
 acids and blue in presence of alkalies ; the sections are to be 
 coloured blue, and the necessary alkali is contained in the tap- 
 water. It will hasten the process to rinse them in a very dilute 
 solution of ammonia, or, best of all, a saturated solution of lithium 
 carbonate. 
 
 6. Stain in watery eosin for a minute or so. This is the 
 coitntevstain. The haematin will stain all nuclei blue, but will 
 scarcely tinge anything else ; the eosin is added to stain other 
 structures a pale pink, and thus make them more visible. It 
 stains almost instantaneously. 
 
 7. Wash off the eosin under the tap. 
 
 The sections are now stained. But they are opaque, and not 
 in a suitable condition to be examined under the microscope, and 
 are to be rendered transparent by being mounted in balsam. 
 Now, this cannot be done in the same way as was used in the 
 mounting of films, for the drying would cause the sections to 
 shrivel and obscure their structure. The water is to be removed, 
 it is true, but by the use of absolute alcohol ; at least two lots 
 should be used, and the slide rocked from time to time. Then 
 the alcohol (which will not mix with balsam) is to be removed by 
 the use of xylol, balsam added, and the section covered with a 
 cover-glass. The remaining steps are therefore : 
 
 8. Absolute alcohol, two lots (to dehydrate). 
 
 * Delafield's haematoxylin is the best for ordinary work, and is best bought 
 ready made. 
 
172 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 9. Xylol, two lots (to render the section permeable to 
 balsam). 
 
 10. Balsam and a cover-glass. 
 
 The last three steps are practically the same as the first three, 
 but in the reversed order, and similar phenomena are seen. The 
 section is opaque whilst wetted with the alcohol, and becomes 
 transparent when the xylol is added, and this transparency is the 
 proof that the steps have been carried out properly. If the 
 section looks opaque when held against a perfectly dark back- 
 ground, an additional dose of alcohol must be used, and the 
 xylol applied again. 
 
 11. Gram's method as applied to sections, suitable for sections 
 of diphtheritic membrane, organs containing anthrax bacilli, 
 streptococci, staphylococci, etc. : 
 
 1. Xylol, two lots. 
 
 2. Absolute alcohol, two lots. 
 
 3. Water. 
 
 These steps are always the same with paraffin sections, no 
 matter what stains are to be used subsequently. 
 
 4. Aniline gentian violet — five minutes. 
 
 5. Gram's iodine solution — three minutes or more. 
 
 6. Absolute alcohol or methylated spirit — until no more colour 
 comes out. This step is best carried out as follows : Hold the 
 slide by one end, keeping the fingers clean by using a duster or 
 pair of dissecting forceps, and pour a little spirit on the section ; 
 rock it gently from side to side and notice the clouds of colour 
 which it takes up. After a little time pour off the spirit and add 
 a fresh lot ; repeat the rocking, and pour off again. Do this 
 until the spirit comes away quite clean, and does not take up any 
 colour from the section. This may take a long or short time, and 
 no definite rules can be laid down. 
 
 In some cases decolorization can be carried out best by the 
 use of clove oil. This is applied when the section is wet with 
 absolute alcohol (for it will not mix with water), and must be 
 entirely removed by the same fluid before the section is 
 mounted, or it will cause it to fade. Clove oil is a very powerful 
 decolorizing agent, and requires careful use, or the colour may 
 be removed from the bacteria. 
 
 7. Eosin — half a minute or more. This is a counterstain, and 
 is used to demonstrate the structural elements, which are not 
 coloured by the gentian violet. It may be omitted in some cases. 
 
STAINING AND MOUNTING PARAFFIN SECTIONS I73 
 
 8. Absolute alcohol — two lots (to remove the water). 
 
 9. Xylol — two lots, or until the section becomes transparent. 
 
 10. Balsam and a cover-glass. 
 
 This method of staining is very easy of application, and the 
 results are exceedingly beautiful. Bacteria which take the stain 
 are coloured blue or violet, and actively dividing nuclei and 
 keratin are stained in the same way, while all other structures 
 are stained pink. 
 
 III. Method for bacteria which do not stain by Gram's method, 
 suitable for sections of typhoid ulcers, lymphatic glands con- 
 taining plague bacilli, etc. 
 
 The problem before us in this case is not at all easy of solution. 
 In the first place, the stains which colour the bacteria also colour 
 the tissues, especially the cell nuclei ; the bacteria are easy to 
 stain, but it is difficult to stain a section in which there is good 
 dififerentiation. In the second place, the stains which are used for 
 bacteria are all soluble in alcohol : but alcohol is used to dehydrate 
 the sections. The following method will be found to serve fairly 
 well in most cases, though it requires a certain amount of practice 
 for its successful accomplishment. 
 
 I, 2, and 3. Xylol, alcohol, and water, as before. 
 
 4. Stain in carbol thionin for ten minutes or a quarter of an 
 hour. 
 
 5. Wash in running water for ten minutes or longer. This 
 removes the stain from the tissues before decolorizing the 
 bacteria, and a fairly differentiated specimen may be obtained if 
 the processes of staining and washing are carried out for suitable 
 lengths of time. 
 
 Unna's polychrome methylene blue may be used in a similar 
 manner, and gives even better results. The staining should be 
 continued for about ten minutes, and decolorization effected by 
 very short immersion in dilute acetic acid (about J per cent.), 
 followed by a good washing in pure water. 
 
 6. Remove as much water from the section as you can without 
 actually drying it by the cautious use of clean blotting-paper. 
 Then apply aniUne oil until the section becomes perfectly trans- 
 lucent. Aniline oil mixes with water on the one hand and xylol 
 on the other, and can be used for dehydration just as alcohol 
 was ; the process is slower, and several lots of the oil must be 
 used. 
 
 7. Wash off all the aniline oil by successive applications of 
 
174 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 xylol. The permanence of the preparation will depend on the 
 thoroughness with which this step is carried out. 
 
 8. Balsam and a cover-glass. 
 
 IV. Staining sections to demonstrate the tubercle bacillus ; 
 applicable to the leprosy bacillus also. 
 
 I, 2, and 3. Xylol, alcohol, and water, as before. 
 
 4. Carbol fuchsin heated until the steam rises for five minutes 
 or longer, care being taken that the section does not dry up. Or 
 the slide may be immersed in the stain and kept in a warm place 
 for twenty-four hours. 
 
 5. Dilute sulphuric acid until only a faint pink tinge appears 
 after washing. This will generally require an immersion of ten 
 minutes or more. 
 
 6. Methylene blue for three or four minutes. Some of the 
 stain comes out in the alcohol, so that the section must be stained 
 more deeply than will be required ultimately. 
 
 7. Rinse off the blue stain in water, and then remove the greater 
 part of the latter with blotting-paper ; this is to render the dehy- 
 dration more rapid. 
 
 8. Absolute alcohol, two lots in rapid succession. 
 
 9. Xylol. 
 
 10. Balsam and a cover-glass. 
 
PART II 
 
 h^:matology 
 
 ESTIMATION OF THE AMOUNT OF HEMOGLOBIN 
 
 The best form of haemoglobinometer now obtainable is un- 
 doubtedly Haldane's ; it was introduced since the first edition 
 of this book (in which Cowers' and Oliver's were described) was 
 published, and is now generally used. It is as simple and cheap 
 
 Fig. 42.— Gowers' H^moglobinometer. 
 
 as Gowers', and almost as easy to use, and gives much more 
 accurate results. In Gowers' method the margin of error is very 
 considerable, though it will answer perfectly well when we simply 
 wish to see whether a patient is improving under treatment, but 
 must not be relied on for absolute results. Oliver's haemoglo- 
 binometer is a little more difficult to use, but is somewhat easier 
 to read ; its price prevents it from coming into universal use. 
 Gowers' H^moglcbinometer consists of two tubes mounted 
 
 17s 
 
176 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 in a small stand. One of these tubes is filled with a jelly tinted 
 to represent the colour of normal blood of a certain degree of 
 dilution. The other is graduated into a hundred parts, the 
 graduation being such that when 20 cubic millimetres of normal 
 blood are diluted with water up to the 100 mark, the colour of the 
 two tubes should be exactly the same. A pipette measuring 20 
 cubic millimetres and a dropping-bottle (which is to be filled with 
 water) are also provided. 
 
 Method of Use. — Place a few drops of water (preferably, but not 
 necessarily, distilled) in the graduated tube. Draw the blood in 
 the usual way. Apply the tip of the measuring pipette to the 
 drop, and suck gently until the blood reaches up to the mark. 
 Now put the tip of the pipette into the small quantity of water in 
 the bottom of the graduated tube and blow out the blood ; this 
 will sink to the bottom of the tube ; now raise the tip of the 
 pipette into the supernatant layer of clear water; suck water 
 up the pipette until it reaches above the mark, and blow it out ; 
 repeat this process until the blood is thoroughly washed out 
 from the tube. Take great care not to withdraw any of the 
 diluted blood when removing the pipette. Finally shake the tube 
 so as to mix the blood and water thoroughly. 
 
 Place the two tubes side by side on a sheet of white paper in 
 front of a well-lighted window which is not exposed to direct 
 sunlight ; look at them by the light which is reflected from 
 this paper, and add water from the pipette belonging to the 
 dropping-bottle, drop by drop, until the colour in the two tubes 
 is exactly the same. Read off the height of the column of diluted 
 blood ; this gives the percentage amount of haemoglobin. 
 
 Haldane's H^moglobinometer is similar in principle, but 
 here the standard tint consists of a sealed tube containing a solution 
 of carboxy-hsemoglobin. The rest of the apparatus is exactly like 
 Gowers', and the method of use is similar, except that, after the 
 20 cubic millimetres of blood have been diluted with the few drops 
 of water in the comparison-tube, it is to be converted into CO haemo- 
 globin by saturation with ordinary coal-gas. To do this, take the 
 curved tube supplied with the apparatus, fit it to an ordinary gas- 
 burner, and insert the other end of the tube in the comparison-tube, 
 taking care not to touch the solution of blood : turn on the gas, and 
 allow it to run into the tube for some time. When the comparison- 
 tube is filled with gas remove it, close it quickly with the finger, 
 and shake gently for a minute or two ; if you wet your finger 
 
ESTIMATION OF THE AMOUNT OF HAEMOGLOBIN I77 
 
 with the diluted blood, wipe it off carefully on to the top, so as to 
 avoid loss. 
 
 The remaining steps are again like those in Gowers' haemo- 
 globinometer, but with this difference : that you are comparing 
 two solutions of the same substance. These are very easy to match, 
 and the exact quality of the light does not matter, so that the 
 method may be used by any artificial light. 
 
 I find it convenient to saturate the water in a bottle with 
 CO by bubbling coal-gas through it for some minutes. The 
 haemoglobin is then converted into CO haemoglobin in the process 
 of dilution, no further gassing is necessary, and the procedure is 
 exactly like Gowers' in all respects. The solution will keep for a 
 day or two if well stoppered. 
 
 Oliver's H^moglobinometer differs from that of Gowers in 
 that the degree of dilution is constant and the colour of the diluted 
 blood is read off by comparison with a seriesof care fully graduated 
 standards. It consists of (i) a capillary glass tube with thick 
 walls and ground ends, one of which is flat and the other pointed : 
 this tube is mounted in a metal handle, the other end of which 
 serves as a stirrer (Fig. 43, c) ; (2) a small cell with an opaque 
 white bottom, and provided with a cover-glass which has a slight 
 bluish tint {e) ; (3) a series of twelve coloured glass discs mounted 
 over an opaque white background (a) ; (4) certain small pink glass 
 discs used as riders ; (5) a short glass pipette with an indiarubber 
 nipple at one end and a short length of indiarubber tubing at the 
 other (d) : the latter fits over the pointed end of the capillary tube 
 mentioned first ; and (6) a small wax candle such as is used for 
 Christmas-trees. A camera-tube lined with a green material 
 is used to screen the eyes whilst the comparison is being 
 made. 
 
 Method of Use. — Prick the patient in the usual way. Apply the 
 polished end of the capillary tube to the drop of blood ; this will 
 completely fill the tube, being drawn up by capillary attraction. 
 When quite full, wipe both ends of the tube with the fingers, and 
 apply the end of the glass pipette (previously filled with water) to 
 the pointed end of the capillary tube. Now squeeze the nipple 
 gently, so as to force the blood and (subsequently) the water drop 
 by drop into the cell. Interrupt the process occasionally, and stir 
 the contents of the cell with the metal handle of the measuring- 
 tube. Continue to add water until the cell is exactly full : this is 
 the first step which presents the slightest difficulty. Apply the 
 
 12 
 
178 
 
 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 cover-glass ; this must not enclose any air under it, nor cause any 
 of the diluted blood to flow into the moat round the cell. 
 
 Fig. 43.— Oliver's H^moglobinometer. 
 
 The specimen is now ready for comparison with the standards. 
 It is to be taken into a dark room and examined by the light of 
 
CLINICAL APPLICATIONS I79 
 
 one of the candles. This is to be placed in front of the observer 
 at a short distance from the specimen and standards, which must 
 lie side by side. 
 
 The viewing is best done by means of a camera-tube which 
 folds into the box containing the whole apparatus. It terminate 
 in a diaphragm which is perforated by two small holes, one of 
 which is to be placed over the centre of the specimen and the 
 other over the centre of the standard. The latter is to be moved 
 about until a disc is found which nearly or quite corresponds in 
 colour with the diluted blood in the cell. If the correspondence 
 is exact, the process is at an end ; the number against the disc in 
 question represents the percentage amount of haemoglobin. If 
 there is no disc which exactly matches the specimen, the latter is 
 placed against the disc which is nearest to it, but not so deep in 
 colour. For example, if we found that the specimen was darker 
 than the disc numbered 50, but paler than that numbered 60, then 
 it would be placed opposite to 50. A slip of colourless glass is 
 then applied over the specimen, and riders over the standard disc, 
 until an exact match is obtained. If, in the case mentioned above, 
 we had to add a rider marked 5 to the standard to bring about an 
 exact match, the percentage amount of haemoglobin in the blood 
 would be 55. 
 
 It is an advantage to place cell and standards side by side 
 rather than one above the other, for the upper and lower portions of 
 the retina differ in sensitiveness to colour, whilst the sides do not. 
 
 CLINICAL APPLICATIONS 
 
 I. It is impossible to estimate even the presence of anaemia, to 
 say nothing of its degree, without an examination of the amount of 
 haemoglobin. I have been repeatedly asked by highly skilled 
 clinical observers to examine cases presenting all the appearances 
 of anaemia in whom the blood has been in every respect normal. 
 The examination, therefore, should be made in all cases of supposed 
 anaemia, and the diagnosis should not be considered as established 
 until this has been done. 
 
 The recognition of the degree of anaemia is advisable, in that 
 it permits the effect of treatment to be watched and ineffective 
 remedies to be discontinued. It also affords a guide as to prog- 
 nosis, for if a patient's amount of haemoglobin increases during, 
 
 12 — 2 
 
l80 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 say, the first week of treatment, a good prognosis may be given, 
 although there are no other signs of improvement, and some idea 
 as to the time necessary to effect a complete cure may be obtained. 
 
 2. Apart from an ordinary anaemia, the estimation of the haemo- 
 globin may give an important clue as to the presence or absence 
 of other diseases. For instance, in severe sepsis there is usually 
 a very marked and rapid fall in the amount of haemoglobin, due to 
 the destruction of the red corpuscles by the toxin of the infective 
 organisms. This is especially valuable in that it occurs in severe 
 infections in which the leucocytes often do not undergo character- 
 istic alterations (p. 222). Thus, if an increasing anaemia is found 
 in a patient during the puerperium, it points strongly to puerperal 
 fever, and the prognosis is bad, assuming, of course, that there is 
 no haemorrhage or other cause of anaemia. 
 
 In interpreting the results of this examination you must re- 
 member that when there is severe diarrhoea, sweating, polyuria, 
 or any other symptom in which there is a great loss of water, the 
 blood may be temporarily concentrated, and the amount of haemo- 
 globin (and of red corpuscles, but not of leucocytes, or not to a 
 proportionate extent) may appear to rise. Discount this in giving 
 a good prognosis in septic conditions from the increase in the 
 haemoglobin. A similar concentration may occur from mitral 
 disease or venous stasis from any cause. 
 
 3. A fall in the amount of haemoglobin in a case watched from 
 day to day indicates haemorrhage, and is occasionally valuable in 
 the differential diagnosis of internal haemorrhage — e.g., in cases of 
 ruptured tubal gestation. 
 
 4. In malaria there is a fall in the amount of haemoglobin, very 
 rapid and sudden in the early stages, and often marked, though 
 less rapid, in the later ones. The fall is often to an extent only 
 equalled in acute sepsis. This may be of much diagnostic value. 
 In typhoid fever and in the other diseases for which malaria may 
 be mistaken the anaemia is usually developed much more slowly, 
 if at all. 
 
 5. Considerable help is afforded in the diagnosis of syphilis by 
 Justus's test, which is based on the fact that in the primary and 
 secondary stages of that disease the red corpuscles are abnormally 
 easily destroyed by the action of mercury, provided that that drug 
 has not been previously administered. The test is also of value 
 in the diagnosis of hereditary syphilis. 
 
 To use it, estimate the haemoglobin as accurately as possible, 
 
ESTIMATION OF THE RED CORPUSCLES l8l 
 
 and then give the patient a single large inunction of mercury, 
 thoroughly rubbed in : Justus recommends 3 grammes (45 grains) 
 for an adult, i gramme for an infant. About twenty-four hours 
 afterwards estimate the haemoglobin again. In a positive case there 
 will be a fall of to to 20 per cent., and this fall may continue foF 
 a day or two, and then give place to a gradual rise. 
 
 The test should not be employed when there is marked anaemia 
 to begin with, as in these patients the blood-corpuscles seem very 
 sensitive to the mercury, even when the disease is not syphilitic. 
 With these exceptions, I believe the test to be of very great value, 
 though more importance is to be attached to a positive than to a 
 negative result. 
 
 ESTIMATION OF THE RED CORPUSCLES 
 
 The best apparatus for the estimation of the number of 
 corpuscles (whether red or white) is the Thoma-Zeiss haemocyto- 
 meter. It should be provided with two pipettes, one for counting 
 the red corpuscles and one for the leucocytes. The latter is rarely 
 used and need not be procured. 
 
 Examine the pipettes. Each has a small bulb containing a 
 little glass ball, and a stem which is graduated into several parts 
 below the bulb, and has a single transverse graduation above it. 
 
 The pipette intended for use in counting the leucocytes may be 
 distinguished by the fact that it has the figure 1 1 over the single 
 transverse graduation above the bulb. 
 
 There are two sorts of pipettes used for counting the red 
 corpuscles. In the one form the stem below the bulb is divided 
 into ten parts, the upper one (nearest the bulb) being marked i, 
 and the middle one 0*5 (Fig. 44, 5). In the other one the same 
 portion of the stem is graduated into three portions numbered -j-l^t 
 jIq, and 2 Jo ; the figure mentioned first is placed nearest the bulb. 
 These pipettes are used in the same way, and it is quite immaterial 
 which is obtained ; we shall describe the use of the first form. 
 
 The rationale of the method is this : Blood is sucked up to one 
 of the divisions on the lower part of the stem, and then an inert 
 diluting fluid is drawn up to the single mark above the bulb, and 
 the two mixed by rotating the whole apparatus for a minute or 
 two. This gives us a dilution of blood of definite strength, the 
 exact amount of dilution depending upon the amount of blood 
 which was taken. Thus, if blood had been drawn up to the 
 
l82 
 
 CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 figure I, we should have a dilution of i in loo, while if blood had 
 been drawn up to the figure 5 the dilution would be 0*5 in 100, 
 or I in 200, and so on. In the case of the other form of pipette 
 the dilution is read off directly from the figures on the lower stem. 
 
 The diluted blood thus obtained is spread out in a film of a 
 definite known thickness on the slide supplied on the instrument 
 (Fig. 44, a). This is ruled in squares, and the squares are of 
 known size. The amount of blood lying upon each square is thus 
 known, and the number of corpuscles which lie upon it being 
 counted under the microscope, all the data for the calculation are 
 obtained. 
 
 In blood examinations it is absolutely necessary that all points 
 
 C Zeiss 
 Jena. 
 
 Fig. 44. — Thoma's H^mocytometer, 
 
 in the technique should receive the most careful attention, or the 
 result will be worse than useless. For this reason we shall 
 describe each step in the process at some length, and advise the 
 practitioner to make several estimations before placing any 
 reliance whatever on his results. 
 Requisites. — i. The haemocytometer. 
 
 2. A needle suitable for obtaining a small quantity of blood. 
 A straight Hagedorn's needle (about 2 inches long) is the very 
 best that can be used, and an ordinary hare-lip pin will answer 
 very well. It is best to use a needle with aflat cutting-point, and 
 not a round or triangular one, as the prick is less painful. 
 
 3. Diluting fluid. There are a good many formulae for this, 
 and some are rather complicated. Isotonic saline solution (com- 
 mon salt o'8 or thereabouts) will answer perfectly ; it is advisable 
 to add to it a small quantity of some stain, methyl violet being 
 
Estimation oi^ the rEd corpuscles 183 
 
 the best, although gentian violet will do very well. This colours 
 the leucocytes, so that they are readily distinguished from the red 
 corpuscles. ■**■ 
 
 4. A microscope having a J-inch lens which will focus through 
 the thick cover-glass supplied with the haemocytometer. If the 
 examination is not to be made by the bedside, a strong indiarubber 
 band a little shorter than the pipette should be carried. 
 
 Process. 
 
 I. Pricking the Patient. — The blood may be procured from the 
 convex border of the lobe of the ear or from the lateral surface 
 of the last phalanx of the finger. The advantage of the former 
 situation is that the pain is very slight, the skin being thin, and 
 that the patient cannot see what you are doing, and is not likely to 
 start at the critical moment. It is to be recommended for children 
 and nervous women. The advantage of the finger is that the 
 skin is free from hairs, and these are objectionable in the prepara- 
 tion of films by the cover-glass method ; an additional advantage 
 is that the patient can put his hand into the position most con- 
 venient to you, and you have not to lean over him. 
 
 The area of the skin to be punctured may be washed with soap 
 and water and then with pure water, and wiped dry, but this is 
 not really necessary. It is necessary, however, to rub the patient's 
 ear or finger well with a towel or piece of lint, so as to make it 
 hypersemic ; unless you do this you may have difficulty in 
 collecting sufficient blood, especially if the skin is cold. The 
 needle is sterilized by being passed slowly through the flame of a 
 spirit-lamp or Bunsen burner ; the area of skin to be pricked is 
 taken between the finger and thumb of the left hand, and a rapid 
 and fairly deep stab made with the needle. The skin is then 
 released, and a drop of blood allowed to exude ; this is wiped away, 
 and the next drop which oozes out is used for examination. 
 
 The skin must never be pinched when blood is being with- 
 drawn for this examination ; the blood must always be allowed 
 to flow out naturally, but if a flat needle be used, the edges of the 
 cut made by it may be held apart by gentle pressure with the 
 finger and thumb. 
 
 * The following formula is better : Distilled water, i6o c.c. ; glycerin, 
 30 c.c. ; sodium sulphate, 8 grammes ; sodium chloride, i gramme ; methyl 
 violet, a trace (Toison's fluid). 
 
184 CLINICAL BACTERIOLOGY AND H.?iMATOLOGY 
 
 2. Filling the Pipette. — The degree of dilution is determined by 
 the number of corpuscles per cubic millimetre which you expect 
 to find. If the patient is anaemic, use i in 100 ; if he has approxi- 
 mately the normal number of corpuscles, or if you have reason 
 to think that they are present in increased quantities, use a 
 dilution of i in 150 or i in 200. 
 
 In most cases you will find it advisable to count the red and 
 the white corpuscles in the same specimen, and if this is the case, 
 use a dilution of i in 100, whether you expect the patient to be 
 anaemic or not. It is less easy to count the reds (if numerous) 
 with this low degree of dilution than with a higher one, but it is 
 not really difficult, and if you use a higher degree of dilution 
 considerable error will be introduced into the leucocyte count. 
 
 Having decided upon the degree of dilution, insert the tip of 
 the pipette into the drop of blood lying on the skin, take the bone 
 mouth-piece attached to the indiarubber tube in your mouth, and 
 suck the blood up to the appropriate mark. If air-bubbles gain 
 access, blow the blood out and begin again quickly. If you over- 
 shoot the mark, remove some of the blood by touching the tip of 
 the pipette against some lint or absorbent cotton-wool. Be 
 careful, also, to wipe off any blood there may be on the outside 
 of the tip. Place the tip of the pipette in the diluting fluid ; a 
 small quantity should be poured out into a watch-glass or other 
 suitable vessel, so as to avoid any possibility of allowing some 
 blood to escape into the stock bottle, and invalidating a sub- 
 sequent observation. Suck the diluting fluid slowly into the 
 pipette until it reaches the single mark above the bulb ; rotate the 
 pipette between the finger and thumb as you do so. 
 
 Now remove the pipette from the diluting fluid, place the tip 
 of the finger over the aperture of the pipette (Fig. 44, S), and 
 proceed to mix the contents by rotating the pipette and by turning 
 it over and over. 
 
 If the examination is to be made at a distance, remove the 
 indiarubber tube and stretch an indiarubber band over it, so as 
 to close both apertures of the pipette. It is advisable to make the 
 examination in a few hours, otherwise considerable errors may 
 creep in. 
 
 3. Preparation of the Specimen. — The slide which is supplied with 
 the instrument consists of a thick and perfectly flat slip of glass 
 (Fig. 44, 0), on which is cemented a glass square having a round 
 hole in its centre {W). In the centre of the hole thus left there 
 
 \ 
 
ESTIMATION OF THE RED CORPUSCLES 185 
 
 is a circular disc of glass (B) ; this inner disc is made of glass 
 which is exactly y\j millimetre thinner than that of which the 
 outer glass is constructed. When the whole cell is covered 
 with a perfectly flat cover-glass (D) there will, therefore, be a 
 space exactly y^ millimetre between the lower surface of this" 
 cover-glass and the upper surface of the central disc ; this space 
 is to be filled with the diluted blood. 
 
 Slide and cover-glass are to be wiped clean with a soft hand- 
 kerchief moistened with water {not alcohol or xylol, which may 
 spoil the former), and then thoroughly dried ; there must not be 
 the minutest particle of dust on any part of the surface. 
 
 The slide and cover-glass being ready, mix the contents of the 
 pipette as you did before (this must always be done immediately 
 before making the specimen, no matter how carefully it had been 
 done a short time previously), and blow out about half of the 
 fluid in the bulb ; this is to wash the diluting fluid out of the 
 lower part of the stem. Now clip the indiarubber tube firmly 
 between your finger and thumb, so as to prevent the access 
 of air, and therefore the escape of fluid, and wipe the tip of the 
 pipette from all fluid ; this may be done with the forefinger. 
 Place the tip of the pipette on the centre of the central disc of 
 the slide, and relax your pressure on the indiarubber tube so 
 as to allow a small drop of fluid to escape ; this is perhaps the 
 most difficult part of the process, and the exact amount which 
 must be allowed to fall on to the slide can only be learnt by 
 experience. 
 
 Cover the slide in this way : Place your finger at the side on 
 the glass square on the slide, and apply the cover-glass, letting it 
 rest against your finger ; lower it gently in place with a needle or 
 other suitable object. When it is in place press it gently with 
 the needle at each corner in succession, and look at it obliquely, 
 so as to see the light reflected from the surface. If the slide and 
 cover-glass are in sufficiently close contact, you will see Newton's 
 rings (looking like the eye of a peacock's feather) round the point 
 at which you are applying pressure. If you do not see this, the 
 inference is that there is some dust between the slide and cover- 
 glass ; you must clean both and begin again. 
 
 It is a great advantage to clip the cover-glass to the counting- 
 chamber until the corpuscles have had time to settle. When this 
 has taken place the depth of the chamber is immaterial, and it 
 does not matter if it rises somewhat. The simplest method is to 
 
l86 CLINICAL BACTERIOLOGY AND H^MATOLOGV 
 
 use four Cornet's forceps, or even only two (applied at opposite 
 sides), as in the figure. Where this is done an ordinary No. i 
 cover-glass may be used instead of the special thick one. 
 Newton's rings should appear round the tips of the forceps, as 
 shown in the illustration. The preparation should stand for five 
 minutes to allow the corpuscles to settle, when the forceps are 
 removed and the count made (see Fig. 45). 
 
 If you have taken the right amount of fluid, the drop should 
 extend exactly to the edge of the central glass disc, but should 
 not run over into the " moat " (Fig. 44, ;'). If this happens, or if 
 there are any bubbles under the cover-glass, you must begin again. 
 
 Fig. 45. — Counting Chamber with Cover-glass clipped into Position. 
 
 If the drop does not quite extend to the edge of the central disc, 
 no great harm is done. 
 
 4. Focussing the Specimen. — This is somewhat difficult for be- 
 ginners, and merits a short description. Place the slide under the 
 microscope, taking care to get it accurately centred, and examine 
 it with the low power. You will find that the central disc is ruled 
 into squares like a chess-board (Fig. 44, c). Get these squares 
 into the centre of the field (see Fig. 46). 
 
 Do not forget you are dealing with an unstained object ; use a 
 flat mirror and a small diaphragm. The examination is often 
 easier if artificial light is used. 
 
 Now turn on the high power (i-inch or i-inch), and screw it 
 
ESTIMATION OF THE RED CORPUSCLES 
 
 187 
 
 downwards until it almost touches the cover-glass ; look down the 
 microscope and focus gently upwards, using the fine adjustment, 
 and keeping a careful look-out for the rulings. 
 
 Some i-inch lenses focus too near the object to be of any use. 
 If this is the case, you must either get an objective specially for 
 the purpose, or a cover-glass which is hollowed out in the centre. 
 These can be bought from the same place as the haemocytometer. 
 
 A 
 
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 iii^y 
 
 v?/- 
 
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 Fig. 46. — The "Bars" which are to be Counted are Shaded. 
 
 If the ruUngs of the slide are indistinct, they may be darkened 
 by rubbing them with a very soft lead-pencil. 
 
 5. Counting the Corpuscles. — Move the slide about until you have 
 come to one corner (preferably the left upper corner) of the ruled 
 area. You will see that each fifth space is marked off by a line 
 running down its centre ; this is to guide the eye and facilitate 
 counting. The whole square consists of four hundred small 
 
i88 
 
 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 squares, twenty along each side. You have to count at least a 
 hundred of these small squares. The simplest way to do this is 
 to count five " bars " of twenty each, each bar extending right 
 across the ruled " chess-board." The bars selected should be as 
 far as possible apart from one another, so as to get a good 
 average. In practice it is simplest to take the top one, and all 
 the others that have a double ruling ; when you have counted 
 these you will have counted five rows of twenty each at equal 
 distances from one another, which will give you a very fair 
 average. (See Fig. 46, where the bars which are to be counted 
 are shaded.) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 S'n 
 
 
 
 
 
 
 
 
 
 
 a 
 
 
 .JB 
 
 
 
 
 
 
 
 
 
 
 t 
 c 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Fig. 47. — Showing Method of Counting Red Corpuscles. 
 
 a, a, a, are counted in square A ; b, b, inB ; c in C. In this method each 
 corpuscle is counted once, and once only. 
 
 In counting these bars count all the corpuscles which are lying 
 on or touching the top line, as in the bar you are counting, also 
 those which are lying on or touching the extreme left-hand 
 limit of the bar. Exclude those on or touching the lower and 
 right-hand lines. The reason for this will appear subsequently. 
 
 This is the most convenient way when you have no assistant 
 to take down numbers, since you only have to remove your eye 
 from the microscope for the purpose of recording results five 
 times in the whole process, which takes about five minutes or 
 less. It is, however, rather more accurate to take down the 
 count square by square, dictating the number to an assistant, who 
 tells you when you have counted a hundred ; you are less likely 
 
ESTIMATION OF THE RED CORPUSCLES l8g 
 
 to make errors in the count by this method. If you follow this 
 method in place of that given above, the following notes should 
 be taken into consideration. 
 
 If you exclude the spaces which are thus marked with a double 
 line, the whole area will be marked out into a series of large" 
 squares, each consisting of 4x4=16 smaller squares (Fig. 47). 
 It is convenient to count the smaller squares in these groups of 
 sixteen. At least a hundred of the smaller squares — i.e.y six of 
 the large groups and four small squares — should be counted. 
 
 In counting one of the smaller squares it is convenient to begin 
 with the corpuscles which are lying in the middle of the square, 
 and then to count those which are lying on the lines. In dealing 
 with these you count those which are lying on the tipper and left- 
 hand lines as being within the square, and those that are on the 
 lower and right-hand lines as being without it ; if you like you may 
 reverse this, but you must keep to the same method throughout 
 (see Fig. 47). 
 
 A few white corpuscles will be met with in every case, while 
 if the blood was taken from a patient with leucocytosis or leuco- 
 cythaemia there will be many. They may be distinguished from 
 the red corpuscles by their greater refractivity, or, if a stain has 
 been used in the diluting fluid, by their being faintly tinged. It 
 is scarcely necessary to say that they should not be counted. 
 
 6. The Calculation. — The best way of calculating the number of 
 corpuscles present from the data thus obtained is the following : 
 
 First add up the number of corpuscles in all the squares which 
 you have counted, and divide the sum by the number of squares 
 counted. This gives the average in each square. 
 
 Now the space enclosed between each square and the 
 cover-glass above it is ^^ millimetre deep, -gV millimetre 
 wide, and ^^ millimetre long ; its cubic capacity is therefore 
 TU ^ ^V ^ 2V = ToW cubic milUmetre. Therefore the ^ ^Vcy P^^^ of a 
 cubic millimetre contains the number of corpuscles which we 
 have already found as the average. 
 
 But the square contained diluted blood ; if the amount of 
 dilution was i in 100, the amount of blood contained in the space 
 over each square was yj^^ part of ^xrVir cubic millimetre. 
 
 Therefore the number of corpuscles which has been determined 
 as being the average per square is contained in 4^V(r ^^ twu cubic 
 millimetre of undiluted blood, the dilution being taken as i in 100. 
 
 Hence the number of corpuscles in i cubic millimetre of 
 
igO CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 undiluted blood is obtained by multiplying the average per square 
 by the number which expresses the dilution (in this case loo), 
 and then by 4,000. 
 
 It may be expressed as a formula, thus : 
 
 If n is the total number of corpuscles counted, 
 
 s is the number of squares counted, 
 
 and if the dilution is i in d, 
 
 then the number of corpuscles per cubic milli- 
 
 metre is — x a x 4,000. 
 s 
 
 Example. — Suppose that we have counted 100 squares, and 
 have found that they contain 1,200 corpuscles, then the average 
 per square is 12. 
 
 Then -^-^jj cubic millimetre of diluted blood contains 12 cor- 
 puscles. 
 
 Or, Y^(y of tttVo '^^^diliited blood contains 12 corpuscles, 
 supposing the dilution was i in 100. 
 
 Therefore i cubic millimetre of undiluted blood contains 
 12 X 100 X 4,000 = 4,800,000 corpuscles. 
 
 Or by the formula — 
 
 Number of corpuscles per cubic millimetre : 
 
 1200 o 
 
 X 4,000 X 100 = 4,000,000. 
 
 100 
 
 Where the dilution is i in 100 (as is recommended, since it 
 enables the red corpuscles and the leucocytes to be counted in one 
 specimen) the calculation can be simplified still further. Add 
 up the number of corpuscles in the hundred squares counted and 
 multiply by 4,000. If you count 200 squares, multiply by 2,000, 
 and so on. 
 
 In a normal count there are 1,250 in the hundred squares counted ^ 
 250 in each ** bar " of twenty squares, and 12 J in each small square, with 
 the dilution of 1 in 100. A knowledge of these facts will enable the 
 approximate condition of the blood to be obtained at a glance. 
 
 The beginner is strongly advised to work out the problem at 
 full length until he has become absolutely familiar with the 
 reason for all the steps. 
 
 Clinical Applications. 
 As this is more tedious than the estimation of the haemoglobin, 
 and is really less important in the recognition of anaemia, it may 
 often be omitted in clinical work. When possible it should be 
 
ESTIMATION OF THE RED CORPUSCLES I9I 
 
 done, as it serves as a useful check on the results obtained by the 
 estimation of haemoglobin. The normal numbers in health are 
 taken to be 5,000,000 red corpuscles per cubic milHmetre in adult 
 males, and 4,500,000 in adult females. As a matter of fact, these 
 figures are very frequently exceeded. In newly born children the- 
 number is about 5,250,000, and in older children about 5,000,000 in 
 both sexes. Any decided fall from these figures indicates anaemia. 
 
 The number of red corpuscles per cubic millimetre is increased in 
 any condition in which the total volume of the blood is diminished 
 by loss of the fluid portion of the blood — e.g., in severe diarrhoea. 
 
 This fact is occasionally of diagnostic value. For example, the 
 red corpuscles appear more numerous in typhoid fever, especially 
 in the earlier stages of the disease : the figure may exceed 
 6,000,000. Later in the disease a fall takes place, though it is 
 never very great, and if in a continued fever the red corpuscles 
 are less than 3,000,000, the diagnosis of typhoid is unlikely. 
 This is very different to what happens in malaria, where there 
 is great and progressive destruction of the red cells, and a figure 
 below 2,000,000 very common. This may be of value in diagnosis 
 in cases where the parasites cannot be found, and Widal's reaction 
 fails or cannot be tried. 
 
 On the other hand, care must be taken not to mistake this 
 concentration of the blood for an actual improvement — e.g., if in a 
 case of septic infection, puerperal fever, etc., the number of reds 
 shows a sudden rise, the question of whether the blood has been 
 concentrated by profuse diarrhoea, sweating, etc., must be inquired 
 into before the findings raise hope of a speedy recovery. Mistrust 
 all results showing an increase of more than 100,000 red corpuscles 
 a day. Such rapid improvement does occur, but is unusual. 
 
 The red corpuscles are also very numerous (up to 10,000,000) 
 in congenital cardiac disease with cyanosis, and as an obscure 
 primary condition, and in venous stasis from mitral disease or 
 any other cause. 
 
 A decrease of the number of red cells indicates anaemia, and the 
 numbers may be taken as a criterion of the degree of anaemia 
 present. But this is not so accurate a test as the percentage of 
 haemoglobin, since it is the quantity of this substance that is of 
 importance, the number of parcels into which it is divided being 
 of comparatively little moment. The grade of anaemia, therefore, 
 should be expressed by the percentage of haemoglobin, not by the 
 number of corpuscles. This is of some importance, since in the 
 
192 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 cases of typhoid fever referred to above the haemoglobin is usually 
 slightly lowered, showing that there is anaemia, even where the 
 number of corpuscles is abnormally high. 
 
 In the diagnosis of the nature of an anaemia the enumeration of 
 the red corpuscles is necessary, and the result is to be considered 
 in connection with the percentage of haemoglobin. The colour- 
 index , or index of corpuscular richness, is the figure which indicates the 
 richness of each corpuscle in haemoglobin, the normal figure being 
 unity. It is obtained by dividing the amount of haemoglobin by 
 the number of corpuscles, each being expressed as a percentage 
 of the normal amount. In health there are 5,000,000 red 
 corpuscles = 100 per cent, of normal and 100 per cent, of haemo- 
 globin. The index is therefore : 
 
 100 
 
 100 ~^' 
 If we found a case in which the red corpuscles were 4,000,000 
 ( = 80 per cent, of normal) and the haemoglobin 40 per cent., the 
 colour-index would be : 
 
 40 
 
 8o = °-5- 
 
 In another case we might find : — Red corpuscles 1,000,000 
 ( = 20 per cent, of normal), and haemoglobin 25 per cent. The 
 colour-index is : 
 
 -^ = I-2S. 
 20 -' 
 
 These figures would be suggestive of chlorosis and pernicious 
 anaemia respectively. 
 
 The percentage of red corpuscles is obtained from the absolute 
 count by multiplying the millions figure and the hundred thousand 
 figure by two (since the normal is 5,000,000, the said figures of 
 which become 100 when multiplied by two). Thus, 2,500,000 
 = 50 per cent. ; 900,000 =18 per cent., etc. The same rule may be 
 used for women, for the slightly lower normal total of reds is 
 accompanied by a smaller amount of haemoglobin. 
 
 The following are the general rules (to which exceptions are 
 rare) for the interpretation of the colour-index : 
 
 1. An index decidedly above unity indicates pernicious anaemia, 
 and usually in this disease the greater the anaemia the higher the 
 index. 
 
 2. In chlorosis the index is greatly diminished : 0*2 has been 
 recorded, and the average is about 0*5. The exact figure throws 
 
ESTIMATION OF THE NUMBER OF LEUCOCYTES 
 
 193 
 
 no light on the severity of the case, which must be estimated by 
 the amount of haemoglobin. 
 
 3. In anaemia due to a single large haemorrhage the index is i 
 at first, both haemoglobin and corpuscles being, of course, lost in- 
 equal proportions. As the blood begins to be regenerated it falls 
 somewhat, not usually lower than 0-9. In anaemia due to multiple 
 haemorrhages, infectious diseases, poisoning, malnutrition, etc. 
 (secondary ancBmia), blood loss and regeneration are taking place 
 simultaneously, and the index falls to 0-9, o-8, or even lower. 
 
 ESTIMATION OF THE NUMBER OF LEUCOCYTES 
 
 In clinical work it is quite sufficiently accurate to count the leuco- 
 cytes in the same preparation as was used for the red corpuscles, 
 and this is a great saving of time and trouble. Proceed as follows : 
 
 Having focussed the rulings on the slide, move the draw-tube 
 
 
 ^ 
 
 
 
 
 
 ^-v 
 
 N 
 
 
 / 
 
 
 
 
 
 
 N 
 
 / 
 
 
 
 
 
 
 
 
 \ 
 
 / 
 
 
 
 
 
 ( 
 
 ) 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 I 
 
 \^ 
 
 
 
 
 
 
 
 
 
 
 / 
 
 \ 
 
 
 
 
 
 
 
 
 / 
 
 
 \ 
 
 
 
 
 
 
 J 
 
 
 \\ 
 
 
 
 
 
 J 
 
 \y 
 
 
 Fig. 48. — Showing Field of Microscope adjusted so that its Diameter 
 IS Equal to that of Eight Squares. 
 
 of the microscope up and down until the upper and lower limits of 
 the field of the microscope coincide exactly with two of the hori- 
 zontal lines, and count the number of spaces (each enclosed between 
 two horizontal lines) in the diameter of the field. Using a ^-inch 
 objective it will be found possible to arrange matters so that these 
 are eight in number, and this will be found convenient, though any 
 other number will do. The essential thing is that the upper and lower 
 borders of the field shall coincide exactly with the rulings. We will 
 suppose that the number is eight. Then the diameter of the field 
 of the microscope is equal to eight times the length of a side of 
 a square, and its radius is equal to four times the length of a side 
 of a square. The total area of the field of the microscope is there- 
 
 22 22 
 
 fore 4 X 4 X ~- (ys x jr, where ir is taken as — -), or 50 and a frac- 
 
 13 
 
194 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 tion. Practically, therefore, when we look down the microscope 
 after it has been adjusted in this way we are looking at fifty 
 squares ; and this fact enables us to dispense entirely with the 
 rulings, and count over the whole area of the disc with great 
 rapidity. The slide is placed in position, and all the cells which 
 are seen in the field counted and the result noted down, or, 
 preferably, dictated to someone else. The slide is then moved on 
 until a perfectly fresh portion of the field comes into view ; it is 
 advisable to go too far rather than not far enough. For this 
 purpose (as for a great deal of blood-work) a mechanical stage is 
 a great advantage. In this way 4,000 squares — i.e.^ eighty fields — 
 may be counted in a very short time. 
 
 It is a very great advantage to be able to dictate these numbers to 
 an assistant, who will tell you when forty fields have been counted. 
 In most cases this will be enough, but if the numbers come out 
 irregularly — ix.^ several in one field and none in others — it is best 
 to count eighty fields or to make a fresh preparation. 
 
 With the arrangement recommended — that is, with a field eight 
 small squares in diameter — you can tell at a glance whether there 
 is or is not leucocytosis. // the leucocytes average one pev fields 
 they are 8,000 per cubic millimetre^ a normal count ; if two per 
 fields they are 16,000 per cubic millimetre^ a moderate leucocytosis ; 
 if three per fields they are 24,000 per cubic millimetre, a high 
 leucocytosis. 
 
 The calculation in this case is very simple. If you have 
 counted eighty fields, the total number is the number of leucocytes 
 in 80 X 50 = 4,000 small squares. Now this is the number of 
 small squares in a cubic millimetre, so that the number only 
 requires to be multiplied by the dilution, in this case 100, to give 
 the number of leucocytes per cubic millimetre. If you have 
 counted eighty squares, therefore, add up the result and put on 
 two noughts; if you have counted forty squares, multiply the 
 result by two and then put on two noughts. Thus, if there are 112 
 leucocytes in 40 fields, the number per cubic millimetre is 22,400. 
 
 Where very great accuracy is required the special diluting 
 pipette should be used.-'' 
 
 All the steps are similar to those just described at full length, 
 except that a different diluting fluid is used. 
 
 * I allow this statement to stand, but have now great doubts as to its 
 corre2tness, and believe that the results obtained by the field method are 
 even more accurate, especially in unpractised hainds, 
 
ESTIMATION OF THE NUMBER OF LEUCOCYTES ig5 
 
 The diluting fluid is one which destroys ("lakes") the red 
 corpuscles, but does not injure the leucocytes. It consists of a 
 0-3 or 0*5 solution of acetic acid (glacial) in water ; it is better to 
 add a small quantity of methyl violet or gentian violet, so that the_ 
 leucocytes are stained and thereby rendered more prominent. 
 This solution is best prepared fresh, or at any rate kept in a well- 
 stoppered bottle. 
 
 The pipette is distinguished from that used for the red corpuscles 
 by its having the number 1 1 above the bulb. This indicates that 
 if blood be sucked up to the mark i below the bulb, and diluting 
 fluid up to the transverse mark above the bulb, the dilution will 
 be I in 10, and so on. 
 
 The blood should be sucked up to the mark i if a great excess 
 of leucocytes is not expected. If the case is one of leucocytosis, 
 a greater dilution is better ; whilst if there is a great excess of 
 leucocytes (such as occurs in severe leucocythaemia), it is best to 
 use the red corpuscles pipette with a dilution of i in 100, but 
 employing the acetic acid diluting fluid. Then proceed to make 
 the preparation, and count by the field method as above. 
 
 Immediately after use the pipettes must be thoroughly cleaned. 
 The fluid which remains in the bulb must be blown out, and for 
 this purpose, as well as for the subsequent washings, it is an 
 advantage to reverse the position of the indiarubber tube, so that 
 the fluid may be blown out through the upper part of the pipette, 
 this being the wider. The whole pipette must now be filled with 
 water (preferably distilled), and the water blown out. This process 
 is repeated, using absolute alcohol, and allowing it to run out of 
 the pipette without blowing it. Lastly, fill the whole pipette with 
 ether, remove the indiarubber tube, replace it with the tube of an 
 ordinary spray (such as is used for scent fountains, throat sprays, 
 etc.), and pump air through until the apparatus is absolutely dry. 
 You can tell when this has happened by the fact that the ball 
 inside the bulb will emit a clear ringing sound when the pipette 
 is shaken. It is useless to attempt to dry the tube by blowing 
 through it from the mouth. 
 
 If blood has coagulated within the apparatus, it must be digested 
 out. Fill the whole with an artificial digestion fluid (pepsin and 
 very dilute hydrochloric acid), and place it in a test-tube of the 
 same fluid in a warm place for twenty-four hours. Then try to 
 clean it as before, and repeat the digestion if this is impossible. 
 
 13 — 2 
 
196 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Clinical Applications. 
 
 The clinical applications of the leucocyte count are so wide 
 that it is hardly possible to summarize them here ; it is more 
 convenient to refer to each special case separately under the 
 heading of the blood-count as a whole. For example, in dealing 
 with typhoid fever the leucocyte count is explained and the points 
 on which a diagnosis is framed are given, which, in a case in 
 which there is a doubt as to the diagnosis between this con- 
 dition and pneumonia, may be referred to in conjunction with the 
 account of the latter. 
 
 It will be convenient, however, to give a list of the usual 
 counts met with in certain diseases, classified under five headings 
 in respect of the number of leucocytes to be expected in them. 
 But these figures must always be considered with reference to 
 the fuller accounts of the diseases in question. 
 
 I. Enormous Leucocytosis (100,000 to 1,000,000). — Such 
 figures are practically only met with in myelogenous or lymphatic 
 leucocythaemia, though suppuration, pneumonia, and hooping- 
 cough may very rarely approximate thereto. 
 
 II. High Leucocytosis (20,000 to 100,000). — Suppuration in 
 all situations and of all kinds, the degree of the leucocytosis being 
 a measure of the virulence of the organism and the resisting 
 power of the patient. 
 
 Pneumonia, in which the same facts hold. 
 
 Hooping-cough. 
 
 Meningitis, especially suppurative meningitis, whether cerebro- 
 spinal, pneumococcic, etc. In tuberculous meningitis there may 
 be as many as 25,000 leucocytes. 
 
 Pleurisy, especially empyema. A leucocyte count of 20,000 
 does not exclude tuberculous pleurisy, but tells strongly against it. 
 
 Scarlet fever. 
 
 Diphtheria. 
 
 Hemorrhage. — After a very severe haemorrhage, whether internal 
 {e.g., rupture, extra-uterine gestation) or external {e.g., from 
 gastric ulcer), figures over 20,000 are occasionally seen, but 
 usually rule lower (12,000 to 15,000). The number usually falls 
 in a few days. 
 
 Similar figures are seen immediately before death, especially if 
 the death is lingering, and very high figures are occasionally seen. 
 It is sometimes of value in prognosis. 
 
ESTIMATION OF THE NUMBER OF LEUCOCYTES I97 
 
 In ovarian cysts with twisted pedicle, in intussusception and 
 volvulus, the figures may exceed 20,000. 
 
 III. Moderate Leucocytosis (10,000 to 20,000). — Here come 
 mild cases of almost all the diseases mentioned above. Thus, a 
 very mild case of pneumonia may show a leucocytosis within 
 these limits, or be normal. 
 
 Inflammatory. — Leucocytosis in inflammatory conditions other 
 than suppuration rarely exceeds 20,000, and does not often reach 
 16,000 : this is a most important diagnostic feature. 
 
 Rheumatism, in the absence of complications. 
 
 Tonsillitis, except when very severe, when higher figures may 
 be seen. • 
 
 Secondary anamia (see p. 217). 
 
 Gout. — Here the condition is normal during the interval, with a 
 slight leucocytosis during the attack. 
 
 Small-pox, during the pustular stage. In severe cases the count 
 may exceed 20,000, and figures much above this indicate a bad 
 prognosis. 
 
 Perforation in Typhoid Fever. — Here the leucocytosis usually 
 attains 15,000 in less than an hour, and may go much higher. 
 
 Secondary syphilis in most cases. 
 
 Malignant Tumours. — There is often, but not invariably, a slight 
 rise in the leucocytes with malignant tumours, but it is hardly 
 marked in the early stages. 
 
 In addition to these pathological leucocytoses, there are two 
 physiological conditions associated with a moderate leuco- 
 cytosis. 
 
 Digestion. — Under normal conditions there is a rise of i ,000 to 
 3,000 soon after a meal. This must always be remembered in 
 interpreting a leucocyte count. A figure of 12,000 obtained in 
 a patient an hour or two after a full meal does not neces- 
 sarily indicate disease. If possible, examine the patient when 
 fasting. 
 
 Pregnancy. — The figures rise gradually toward the end of 
 pregnancy, and at term may reach 15,000 or even higher. 
 Recollect this in interpreting counts in pregnancy or soon after 
 parturition. 
 
 IV, Normal Counts (5,000 to 10,000, average 7,500) are 
 met with in a variety of conditions, but in comparatively few 
 that are attended with pyrexia. Of these the most important 
 are : 
 
198 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Tuberculosis, except tuberculous meningitis (15,000 to 25,000), 
 and occasionally in tuberculous pleurisy (10,000 to 20,000). 
 
 Typhoid fever. 
 
 Malta fever. 
 
 Measles. 
 
 Malaria. 
 
 Mumps. 
 
 Varicella. 
 
 Pernicious ancsmia. 
 
 Chlorosis. 
 
 Primary syphilis. 
 
 Influenza. • 
 
 In any of these there will be leucocytosis if an inflammatory 
 complication is present. Thus, in typhoid fever with perforation 
 or pneumonia the count is raised. 
 
 Very severe sepsis. 
 
 V. A Lowered Count, or Leucopenia (under 5,000). — This 
 is not met with very frequently, and all the cases are included 
 under IV. The chief are typhoid fever, pernicious anamia, chlorosis, 
 influenza, malaria, and uncomplicated tuberculosis. It is also found in 
 starvation and malnutrition. 
 
 THE INVESTIGATION OF THE MORPHOLOGY OF 
 THE LEUCOCYTES AND RED CORPUSCLES 
 
 To study the morphology of the leucocytes and red corpuscles 
 it is necessary to prepare thin and even films of the blood, and to 
 submit them to appropriate methods of staining. 
 
 Method of Preparing Films — I. With Cover-glasses. 
 
 This is the best method for ordinary purposes, and, if the in- 
 structions are carried out exactly, is a very easy one. 
 
 Requisites. — i. Perfectly clean cover-glasses. They should be 
 cleaned by one of the methods described on p. 31, and kept in 
 spirit. Immediately before they are required for use they must 
 be removed with a clean pair of forceps and dried with an old 
 and soft handkerchief. They may then be passed rapidly through 
 the flame, and allowed to cool. 
 
 I prefer No. 2 cover-glasses for this examination, as much 
 better films are made on them than on thin ones, which are 
 
MORPHOLOGY OF LEUCOCYTES AND RED CORPUSCLES IQQ 
 
 liable to bend under the powerful suction exerted by the capillary 
 attraction of the drop of blood when spread out thin. 
 
 2. A needle for drawing blood. 
 
 (3. A platinum loop having a diameter of about y^^j inch is 
 sometimes useful, especially to a beginner.) 
 
 Rub the patient's ear or finger thoroughly with a piece of lint or 
 a towel, so as to make it hyperaemic. Prick it, and wipe away 
 the first drop of blood. Then allow another drop to exude ; if 
 necessary, you may squeeze it a little. 
 
 Take a cover-glass between the first finger and thumb of the 
 left hand, holding them by the opposite angles, and take another 
 between the first finger and thumb of the right hand, holding it 
 by adjacent angles (Fig. 49). 
 
 Fig. 49. 
 
 Touch with the upper surface of the cover-glass in your left 
 hand the drop of blood on the patient's skin, so as to remove a 
 very small droplet. This is the most difficult step : you must 
 not get too much or too little blood, otherwise the films will be 
 useless. It is advisable to avoid letting the cover-glass touch the 
 patient's skin. 
 
 Now put the right-hand cover-glass over the left-hand one, the 
 centres coinciding ; lower the upper (right-hand) one until the 
 droplet of blood just touches it, and then let go (Fig. 50). 
 
 You will see the droplet of blood spread itself out by capillary 
 attraction between the two cover-glasses. 
 
 At this stage you will see whether you have taken the right 
 amount of blood or no. If you have, the drop will spread out, 
 still retaining its circular shape, until it approaches the octagon 
 formed by the intersecting edges of the two cover-glasses 
 (Fig. 50) ; if you have taken too little it will not reach so far, 
 and if you have taken too much it will extend further, and the 
 upper cover-glass will float loosely on the lower. 
 
200 
 
 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 It is necessary to lay great emphasis on the fact that the cover- 
 glasses must not be squeezed together, but must simply come 
 together by capillary attraction. 
 
 When the drop has ceased to spread take hold of the upper 
 cover-glass with the finger and thumb of your right hand and 
 slide the two apart, keeping them in the same plane; this is 
 
 Fig. 50. 
 
 readily done, since the cover- glasses are free to turn, being only 
 held loosely at the points. If you do not do this, either the cover- 
 glasses will break, or else the upper cover-glass be lifted from the 
 lower one, and the film will resemble the marks left on a knife which 
 has been pressed on butter and lifted off; such films are useless. 
 Here, again, you find whether you have taken the right amount 
 
 Fig. 51. 
 
 of blood. If you have taken too little, the cover-glasses will be 
 very difficult to separate ; it may, indeed, be impossible to do so 
 without breaking them. If you have taken too much, they will 
 separate with great readiness, and the blood will spread in uneven 
 smears instead of forming a uniform film. 
 
 I used formerly to recommend the use of forceps for holding 
 the cover-glasses in making these films. The only advantage is 
 that it avoids " steaming " the cover-glasses by the condensation 
 
MORPHOLOGY OF LEUCOCYTES AND RED CORPUSCLES 20I 
 
 of moist are from the fingers, which may distort the corpuscles. 
 Except when the operator's fingers are very moist it is quite 
 unnecessary, and perfect films may be obtained with the cover- 
 glasses held in the fingers. 
 
 The blood may be taken by means of the platinum loop, and' 
 this is a good plan, as all danger of smearing the cover-glass 
 upon the skin is avoided. If several films are to be taken, a 
 number of platinum loops should be provided, as the blood upon 
 them soon coagulates. The exact size of the loop can only be 
 learnt by experiment, and when one has been found to deliver 
 a drop of the right size it should be kept entirely for this work 
 and carefully protected from injury. 
 
 Fig. 52. — Method of spreading Films with Cigarette-paper. 
 
 II. Method with Cigarette-papers. 
 
 Requisites. — i. Perfectly clean slides. 
 
 2. Some fairly stiff cigarette-papers cut in half longitudinally. 
 Paper which is decidedly ridged or ribbed will not answer. 
 
 3. Needle. 
 
 Method. — The patient is pricked, and the first drop of blood 
 wiped away as before. One of the half strips of cigarette-paper 
 is now held in the right hand, the index-finger being placed 
 above the strip, and the edges held between the thumb and 
 index-finger and the index and middle fingers respectively ; this 
 converts it into a gutter, the convex edge of which is downward. 
 The edge of this gutter which points away from you (and which 
 is formed by a machine-cut edge of the paper) is now dipped into 
 the drop of blood, and a small quantity picked up on its lower 
 surface. This lower surface is then placed on a clean slide 
 parallel to one of its shorter edges and about J inch from it, and 
 pressed gently upon it so as to flatten out the paper gutter ; as 
 this flattens out the edge of the drop of blood on its under 
 surface will follow it. The strip of paper is now drawn towards 
 
202 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 the other end of the sUde with a steady uniform movement, and 
 in doing so the drop of blood is spread out into a long uniform 
 film. In this way a film J inch wide and 2 inches long can be 
 made on a single slide. A fresh piece of paper is to be used for 
 each specimen (see Fig. 52). 
 
 The author is of opinion that it is best to adopt the cover-glass 
 method, as he has found that it presents fewer difficulties for 
 beginners ; this is not the universal experience, and it is a good 
 plan to try both, and adopt that with which you get the best 
 results. 
 
 Films are sometimes spread on one slide by means of another, 
 which is used as a spreader in much the same way as the 
 cigarette-paper described above. This is very good for malaria 
 parasites, and for alterations in the red corpuscles, but is useless 
 for making differential counts, as some of the leucocytes are 
 carried along with the spreader and left at the end of the film. 
 
 Fixation of Blood-films. 
 
 If films are required simply for bacteriological purposes {i.e., to 
 search them for bacteria), they may be fixed by passing them 
 three times through the flame, just as if they were ordinary films. 
 This, however, is not to be recommended in the study of the 
 cells of the blood or of the parasite of malaria, though it answers 
 very well in skilful hands. Three methods should be learnt : the 
 method by heat, the alcohol and ether method, and the formalin 
 method. Of these, the first is necessary if Ehrlich's triacid stain 
 is to be used, but the others are perhaps better for other stains. 
 In addition to these we must mention that if Jenney's stain is used 
 a preliminary fixation is unnecessary, as the fluid fixes the film and 
 stains it at the same time. This is the method of blood examination 
 which is most suitable for practitioners, and it is doubtful whether 
 it is not also the best for the most accurate and delicate scientific 
 research. 
 
 I. Method of Fixation by Heat. — Slides or cover-glasses to be 
 fixed by this method must be exposed to a temperature of 120° C. 
 for about five minutes — slides requiring a slightly longer time 
 than cover-glasses. 
 
 The ideal way is to use a dry-air sterilizer (see p. 5), to place 
 the films in it whilst cold, to heat up to 120° C, and then to turn 
 out the gas. In the absence of this apparatus a metal slab or 
 plate such as has been recommended for use in staining the 
 
FIXATION OF BLOOD-FILMS 203 
 
 tubercle bacillus answers well. It is mounted upon a tripod 
 stand, and the heat applied at one end. After a time the tem- 
 perature of various portions of the plate is tested by the 
 application of a few drops of water ; the point at which the drop 
 assumes the "spheroidal state" {i.e., takes the form of a sphere, ~ 
 and does not wet the plate) is about the right point to use. The 
 slides or films are placed at this point for the appropriate time. 
 
 II. Fixation by Perchloride of Mercury. — Flood the film with a 
 saturated watery solution of perchloride of mercury, allow to act 
 for a minute, and wash for a minute under the tap, or by washing 
 it in a vessel of water. 
 
 This method of fixation is about the best that can be used for 
 general purposes. 
 
 III. Fixation by Alcohol and Ether. — This is very simple ; the 
 films are placed in a mixture of equal parts of alcohol and ether 
 for at least half an hour. 
 
 This method of fixation is good, but slow. 
 
 IV. Fixation by Formalin. — There are several methods by which 
 the fixative action of formalin can be used for blood-work. Of 
 these, the use of a mixture of i part of formalin with 9 parts 
 of absolute alcohol answers perfectly. The films are immersed in 
 this for half a minute, and then washed thoroughly under the tap. 
 
 This method of fixation is both good and rapid. 
 
 Staining Blood-films for the Investigation of their Cells. 
 
 There are a great many methods of staining blood-films, and 
 all depend upon the division of stains into two varieties, the acid 
 and the basic. All the stains which are used in this branch of 
 histology are salts, and in some of these salts the acid radicle 
 does the staining, in others the basic. 
 
 Acid stains are those in which the colouring property resides in 
 the acid of the salt. A familiar example is picrate of potash, a 
 yellow stain in which the picric acid is the active ingredient. 
 The acid stains in chief use are eosin, acid fuchsin, and orange 
 G. Substances which stain with an acid stain after suitable 
 exposure to a mixture of an acid and a basic stain are called 
 oxyphile, or, from the frequent use made of eosin as an acid stain, 
 eosinophile. 
 
 Basic stains are those in which the colouring property resides 
 in the basic radicle of the salt ; they include all the stains which 
 are in use for staining bacteria, and they all colour the nuclei of 
 
204 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 cells. The most important are methylene blue, methyl green, 
 and toluidin. Ordinary basic fuchsin used in staining the tubercle 
 bacillus belongs to this group, as do haematoxylin, carmine, etc. 
 
 We shall describe three methods of staining, and these are 
 sufficient for all purposes of diagnosis. They are : (i) Ehrlich's 
 method with his triacid stain ; (2) Jenner's method ; and 
 (3) eosin and methylene used separately. Of these, the second 
 method is the simplest, and all that is necessary in the vast 
 majority of cases. The third method is an emergency one, for 
 use when Jenner's stain is not at hand : the first is now almost 
 obsolete, but occasionally useful when a study of the granulations 
 of the leucocytes is of importance. 
 
 1. Ehrlich's stain consists of a mixture of acid fuchsin, orange 
 G, and methyl green dissolved in water, glycerin, and alcohol. 
 It is difficult to prepare, and should be purchased from a trust- 
 worthy maker. Its use is very simple. The film is fixed by 
 heat in the manner already described, and the stain is poured on 
 to it, and allowed to act for five minutes. The film is then 
 washed, dried with blotting-paper, and then by gentle heat, and 
 mounted in balsam. 
 
 Nuclei are stained green, red blood-corpuscles orange, and 
 eosinophile granulations bright red. The small eosinophile 
 granulations which are present in the polymorphonuclear cells 
 (the neutrophile granulations of Ehrlich) are stained a purplish or 
 coppery colour. The basophile granulations are unstained. 
 
 This stain is not suitable for the parasite of malaria, nor for 
 bacteria. 
 
 2. Jenner's stain consists of a solution of a compound of eosin 
 and methylene blue in methyl alcohol. It must be bought 
 ready prepared. Nothing could be more simple than the way in 
 which it is used ; no preliminary fixation is necessary, the film 
 being allowed to dry and flooded with the stain. After a period 
 of from a minute and a half to three minutes, the stain is washed 
 off by waving the film to and fro in distilled or rain water for a 
 few seconds, and the specimen dried by blotting it between two 
 pieces of clean blotting or filter paper, allowed to get quite dry, 
 and mounted in balsam or cedar-oil, and the specimen dried and 
 mounted.* 
 
 * It has been objected that Jenner's method does not always give good 
 results, and that the above is an insufficient account of the process. Provided 
 that the stain is good (Grubler's can always be relied on, and will keep for 
 
STAINING BLOOD-FILMS 205 
 
 After the use of this stain nuclei are stained blue, red corpuscles 
 red, eosinophile granules red, and basophile granules violet. The 
 descriptions of the leucocytes and abnormal red forms, which are 
 appended, are all based on the appearances seen in films stained 
 by this method, from which also Plates VII. and VIII. were drawn. 
 
 Jenner's stain is suitable for a study of the parasite of malaria, 
 which it stains blue. It may be used for the detection of bacteria. 
 
 Leishmans stain is similar in some respects to Jenner's, and is 
 used as follows : The film is flooded with the stain, which is allowed 
 to act for two minutes ; two or three drops of distilled water are 
 then added, and the process allowed to continue for two or three 
 minutes longer. It is then washed, dried, and mounted as above. 
 The colours of films stained by this method differ from those in 
 which Jenner has been used, the main point being that the nucleus 
 of the leucocytes is a fine purplish red ; the leucocytes, however, 
 can be easily recognised from the coloured plates. 
 
 Leishman's method is the best for working with malaria and all 
 parasitic protozoa, the nuclei of which are stained a bright red. 
 For ordinary blood-work I personally prefer Jenner (perhaps 
 because I am more used to it), and the practitioner is recommended 
 to choose one process and stick to it. If he is likely to need it for 
 malaria or other parasites, Leishman's stain should be used. 
 
 3. Eosin and methylene blue used separately. 
 
 In this method the films are to be stained with the eosin first, 
 and then with the methylene blue. Its successful application 
 requires a certain amount of practice. 
 
 The eosin used must be in watery solution, and the exact 
 strength does not matter : 4 per cent, is a convenient strength to 
 use. Most specimens of red ink (slightly diluted) will do quite 
 well. The films are to be stained in this solution for three or 
 four minutes ; no harm will result if they are left in much longer. 
 They are then washed and immersed in a saturated watery 
 solution of methylene blue. This is the difficult part of the pro- 
 cess, for no general rule can be given as to the length of time for 
 which this stain must be applied ; it may be ten seconds, or it 
 may be two or three minutes. The only safe way is to stain the 
 
 several months after being opened, if kept well corked), the method described 
 will always succeed if well-spread films are used. This is essential. But even 
 with bad films (and very bad ones are sent me at times) the results are always 
 sufificiently good to allow a differential leucocyte count to be made. 
 
206 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 film for a quarter of a minute, wash it, and then examine it under 
 the low power of the microscope. If the film is properly stained, 
 the nuclei of the leucocytes will be seen as blue points, which can 
 be distinguished with great ease with the |-inch objective. If 
 they are not visible, the methylene blue must be applied for about 
 a quarter of a minute more and the examination repeated. 
 When the nuclei are seen to be well stained the film is dried 
 and mounted. 
 
 This process gives results which resemble those afforded by 
 Jenner's stain, except that the fine eosinophile granulations in the 
 polymorphonuclear cells are always less obvious and often quite 
 invisible. It is also suitable for malarial parasites and bacteria. 
 
 The practitioner is recommended to practise this method of 
 staining, as it does not require any reagents which are not to be 
 found in every well-stocked surgery. The watery solution of 
 methylene blue which is used as a counterstain for the tubercle 
 bacillus and some red ink are all that are necessary. 
 
 DIFFERENTIAL LEUCOCYTE COUNT 
 
 The following varieties of leucocytes are to be recognised, the 
 description in each case being taken from a preparation stained by 
 Jenner's method. 
 
 A. Cells devoid of Granules. 
 
 I. Lymphocytes (Plate VII., Figs, i, 2, 3, 4) are variable in size, 
 some being about as big as a red corpuscle, others nearly twice this 
 size. Each has a single nucleus, which is circular or nearly so, 
 and which stains a deep blue. The protoplasm forms a narrow 
 band round the nucleus, and also stains blue, often more deeply 
 than the nucleus. 
 
 Variations of these cells occur. In some cases the whole seems 
 to stain uniformly, in which case it is probably a free nucleus 
 (Plate VII., Fig. 3). In others the protoplasm appears to be 
 studded with blue granules, which often lead beginners astray. 
 They are not true granules, but knots in the protoplasmic 
 network. 
 
 In healthy adults they average about 25 per cent, of all the 
 leucocytes present, varying between 22 and 28 per cent. In 
 childhood they are more numerous — up to 60 per cent. 
 
DIFFERENTIAL LEUCOCYTE COUNT 207 
 
 The small forms are the more numerous, but as no trustworthy 
 diagnostic information can be drawn from the proportions of the 
 large and small forms, they are usually counted together. Very 
 large forms are often abundant in acute lymphatic leucocy- 
 thaemia. 
 
 Large Hyaline or Large Mononuclear Leucocytes (Plate VII., 
 Figs. 5, 6). — These are the largest cells met with in normal 
 blood, and may have a diameter two and a half that of a red 
 corpuscle. They have a round, oval, kidney-shaped, or twisted 
 nucleus, which stains less deeply than those of the lymphocytes, 
 and has often a purplish colour. The protoplasm is relatively 
 more abundant than in the lymphocytes, and stains very faintly 
 of a bluish or purplish colour. It often has false granules similar 
 to those of lymphocytes, but no true ones. 
 
 It occurs in small numbers (i to 4 per cent.), and variations are 
 of little diagnostic importance. 
 
 B. Cells containing True Granules in their Protoplasm. 
 
 I. Polynuclear Leucocytes (Plate VII., Fig. 7). — These are rather 
 larger than a red corpuscle, and have a nucleus which is twisted 
 into various irregular shapes ; it is often deeply lobed, so that it 
 appears to be multiple, but is always really single except in de- 
 generated forms. 
 
 It contains in its protoplasm numerous very fine granules of a 
 substance which stains pink with the eosin in Jenner's stain, and 
 which are therefore considered by many English pathologists to 
 be fine eosinophile granules. With triacid stain they are a sort 
 of copper colour, quite unlike the large eosinophile granules, and 
 are frequently spoken of as neutrophile. The term is a convenient 
 one, whatever the scientific justification for it. 
 
 They vary in numbers between rather wide limits. In the 
 adult 70 per cent, is a fair average, but they may be much higher, 
 and approach 80 per cent. In childhood they are much less 
 numerous (30 to 40 per cent.). 
 
 la. Myelocytes or Neutrophile Myelocytes. — These are the mother- 
 cells of the above, and do not occur in the blood in health, but 
 may be found in the bone-marrow. There are two varieties. 
 
 CorniVs myelocyte is usually a very large cell with a large, faint- 
 staining round or kidney-shaped nucleus, often placed decidedly 
 to one side of the cell or even touching the periphery. The pro- 
 
208 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 toplasm is relatively scanty and contains neutrophile granules, 
 often in very small numbers and of feeble staining power. It is 
 often necessary to use triacid stain to demonstrate them ; neglect 
 to do this may lead to their being confounded with large lympho- 
 cytes or hyaline cells (Plate VII., Fig. ii). 
 
 Ehrlich's myelocyte is similar to the above, but smaller ; it is 
 usually rather larger than a polynuclear. It has a round or oval 
 nucleus, which is often central or but slightly excentric, and which 
 stains deeper than that of Cornil's myelocyte, but not so deeply 
 as that of a polynuclear. Its neutrophile granules are usually 
 distinct (Plate VII., Fig. lo). 
 
 Cornil's myelocyte is probably derived from Ehrlich's by a 
 process of degeneration, especially dropsy of the nucleus. 
 
 2. Eosinophile Leucocytes (Plate VII., Fig. 8). — These are about 
 as large as a polynuclear, and have a nucleus which is usually 
 bilobed, but which may be more distorted. The chief feature of 
 the cell is the presence of numerous relatively large granules 
 which stain brilliantly with the eosin : they are spherical in shape 
 and very uniform in size. They average about i to 4 per cent, of 
 the leucocytes in health, but slightly higher or lower figures may 
 occur. 
 
 2a. Eosinophile myelocytes (Plate VII., Fig. 12) do not occur in 
 healthy blood, and bear the same relation to the eosinophile cells 
 as ordinary myelocytes to the polynuclear cells. They are dis- 
 tinguished from eosinophiles by their larger size and relatively 
 large circular nucleus. 
 
 3. Basophile Cells or Mast Cells (Plate VII., Fig. 9). — These are 
 about as large as polynuclears, and have twisted (usually trilohed) 
 nuclei, which occupy more of the cell than do those of the poly- 
 nuclears. They have a comparatively small number of granules, 
 which (unlike those of the other leucocytes) vary in size and stain 
 blue or purpHsh-blue with Jenner. The granules do not stain at 
 all with triacid. 
 
 They are often not found in persons in robust health, but in the 
 average hospital patient occur in small numbers - usually less 
 than f per cent. 
 
 -^a. Large Mast Cells (Plate VII., Fig. 13). — These occur only in 
 the blood in myeloid leucocythaemia, and are very characteristic 
 of that condition. They may possibly be eosinophile myelocytes 
 with degenerated granules. 
 
DIFFERENTIAL LEUCOCYTE COUNT 209 
 
 Method of making a Differential Count. 
 
 Having prepared a film by any of the methods previously 
 described, the next step is to make a differential count in the 
 following way : Focus the film under the microscope, using an 
 oil-immersion lens ; when you have had sufficient experience it 
 is quite easy to make the count with a J-inch lens, which is 
 quicker, but not advisable for beginners. Then note down the 
 nature of each leucocyte as you come to it, moving the film 
 across the stage of the microscope from end to end, and then 
 moving it a little way upwards or downwards and returning in 
 the opposite direction, so as never to pass over the same part of 
 the film twice. The simplest way of noting down the leucocytes 
 is to assign single letters to each variety, P for polynuclear, E for 
 eosinophile, etc., and to put these down in blocks of five each 
 way, thus : 
 
 P P P LE 
 
 PP LL H 
 
 LP P P P 
 
 PL LPL 
 
 P P P L P 
 
 In this way you can tell at any time how many leucocytes you 
 have counted. It is much quicker to dictate the numbers to a 
 second person, who takes them down as described ; you do not 
 then have to look constantly from the microscope to the paper, 
 and vice versa^ and the whole process takes a very few minutes, 
 unless the leucocytes are very scanty. 
 
 For most clinical purposes 400 leucocytes will be enough to 
 count, though where very great accuracy is required 1,000 is 
 not too many. Having counted the required number, proceed to 
 count the numbers of P's, L's, etc., and reduce them to a 
 percentage. 
 
 Whilst making the differential count keep a sharp look-out for 
 abnormal leucocytes, abnormal red corpuscles (see p. 213), 
 parasites, etc. The collections of blood platelets which form 
 such a prominent feature in some blood films should not be 
 confounded with anything else, as they are quite characteristic, 
 though a single blood platelet lying on the top of a red corpuscle 
 may look very like a young malaria parasite. Each platelet is a 
 very small mass which stains blue or purple with Jenner's stain, 
 and often appears hollow or irregular in shape, and they are often 
 grouped in masses of quite large size. 
 
210 CLINICAL BACTERIOLOGY AND HiEMATOLOC^ 
 
 Alterations in Disease. 
 
 I. Lymphocytes, — They may be relatively increased {e.g., to 
 figures above 30 per cent.) when the total figures are normal or 
 raised. These have different meanings. 
 
 Lymphocytosis with normal or lowered total counts occurs in 
 pernicious anaemia, typhoid fever, uncomplicated tuberculosis 
 (but not always), in some cases of purpura (so-called idiopathic 
 purpura), and occasionally in syphilis, Hodgkin's disease, and 
 some other diseases. 
 
 Lymphocytosis with a high total count occurs as a normal 
 condition in infancy, and is accentuated both as regards the 
 percentage of lymphocytes and the total number of leucocytes in 
 almost all infantile diseases, especially rickets and hooping-cough. 
 
 In adults a very high total count (100,000 or more) with a very 
 high percentage of lymphocytes (up to 99*5 per cent.) indicates 
 lymphatic leucocythaemia. Smaller increases sometimes occur 
 in other diseases. 
 
 A relative dimimition of the lymphocytes frequently occurs as a 
 result of the increase of other leucocytes, rarely as a true absolute 
 diminution. 
 
 Large Hyaline Cells. — An increase or decrease of these is 
 occasionally observed, but so erratically that it is of no use in 
 diagnosis. 
 
 Polymiclear Leucocytes. — This is the most common cell to undergo 
 increase, so much so that in the list of causes of leucocytosis 
 given on p. 196 it may be assumed 'that the increase is due partly 
 or entirely to an increase of polynuclears, unless the opposite is 
 stated here. 
 
 One special case needs a reference. In severe sepsis with a 
 normal or diminished polynuclear count it is usual for the 
 polynuclears to be relatively increased, just as they are when 
 the total count is raised. This is very important in diagnosis, 
 for in cases where sepsis is suspected a normal leucocyte count 
 must always be followed by a differential count, and if there is a 
 relative increase {e.g.^ 85 per cent, or more) of polynuclears it 
 must be regarded as a bad sign. A relative increase of poly- 
 nuclears with a normal or moderately raised total count may also 
 occur in malignant disease. 
 
 A form of degeneration of the polynuclears requires notice, as 
 it is of some practical value. This is the iodine or glycogenic 
 
DIFFERENTIAL LEUCOCYTE COUNT 211 
 
 reaction. To test for it make a film in the ordinary way, dry 
 and mount it without fixation in the following mixture : 
 
 Iodine i part. 
 
 Iodide of potassium - - - - 3 parts. ^ — 
 Saturated watery solution of gum acacia loo parts. 
 
 This should not be used more than a fortnight after it has been 
 made. A simpler but equally good method is to expose the film 
 to the fumes of solid iodine for a few hours. This can be done by 
 fixing the film (without previous treatment of any sort) at the top 
 of a wide-mouthed bottle containing the substance ; it is then 
 mounted in oil or balsam. Allow it to act for a quarter of an 
 hour or more, then examine with a yV-inch lens in a white light 
 — daylight if possible. In cases where the reaction is present 
 a variable number of the polynuclears will be found to contain 
 granules or masses of a reddish-brown or mahogany colour ; 
 sometimes there are large masses, and sometimes almost the 
 whole of the protoplasm appears brown. 
 
 The importance of this reaction is that it usually occurs, and 
 is indeed very marked, in the cases of severe sepsis in which 
 there is no increase of the total leucocyte count (see p. 198), as 
 well as in suppuration. It occurs in other conditions, such as 
 pneumonia, hooping-cough, uraemia, etc. In many cases a small 
 percentage only of the cells is aff'ected, and a good search must 
 be made. 
 
 The fact that it occurs in so many diseases detracts somewhat 
 from its value in diagnosis, but when the question is simply the 
 presence or absence of pus — e.g.j in appendicitis — a positive result 
 will usually indicate that suppuration has occurred, and vice versa. 
 Hence it is sometimes of value when the leucocyte count yields 
 uncertain results — i.e., figures between 16,000 to 20,000 per cubic 
 millimetre. 
 
 Myelocytes. — Ehrlich's myelocytes occur in small numbers in 
 many infectious diseases, especially diphtheria, and occasionally 
 in forms of anaemia, but they are only present in large numbers 
 in myeloid leucocythaemia. Cornil's myelocytes are practically 
 limited to the latter condition, where the two forms frequently 
 together make up 60 per cent, of all leucocytes. 
 
 Eosimphiles. — An increase of eosinophiles (eosinophilia) occurs 
 to a small extent in numerous diseases, and is of diagnostic 
 importance in the following : 
 
 I. In diseases due to animal parasites. Here the increase may 
 
 14 — 2 
 
212 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 be very great, as in trichinosis, where the eosinophiles usually 
 form 40 to 80 per cent, of all leucocytes, a fact of great import- 
 ance, and absolutely distinguishing the disease from typhoid 
 fever, rheumatism, and other diseases which may be confounded 
 with it ; rarely, however, there may be no eosinophilia, so that 
 its absence does not definitely exclude the disease. In bilharzia 
 disease there is often, though not always, a mild eosinophilia. 
 There is practically always an increase in ankylostomiasis. It 
 may be moderate, or may reach 70 per cent. This fact enables 
 infected individuals to be picked out of a gang of workmen with 
 much less trouble than by an examination of the faeces. In 
 diseases due to other intestinal worms (ascarides, oxyuris, taeniae), 
 there may or may not be eosinophilia. 
 
 In hydatid disease there is frequently a moderate eosinophilia, 
 and sometimes a great one. This is important in the diagnosis 
 between hydatid and abscess of the liver, since in the latter 
 condition the eosinophiles are usually scanty. In such a case 
 the higher the count is above 4 per cent., the more likely is the 
 disease to be hydatid, and vice versa. 
 
 In the only case of cysticercus I have seen the eosinophiles 
 ranged between 5 and 7 per cent. 
 
 2. In extensive skin diseases, especially pemphigus and 
 urticaria, there is often a great increase, but it occurs in so many 
 conditions that its diagnostic value is but slight (see p. 232). 
 
 3. Diseases of the lungs. — In true asthma there is during the 
 paroxysms, and for a short time after them, a very decided 
 increase — to 10 per cent, or so. The cells in the sputum, too, 
 are practically all eosinophiles. This does not occur in the other 
 spasmodic diseases which so mimic true asthma, and is often of 
 critical importance in the diagnosis. Eosinophiles are never 
 found in the sputum in tuberculosis. 
 
 4. Myelogenous leucocythcemia. — Here there is an enormous abso- 
 lute increase in the total numbers of the eosinophiles, counting the 
 eosinophile myelocytes with them ; the percentage may not be 
 greater than in health. 
 
 This is one of the most constant signs of the condition. 
 
 A decrease of eosinophiles occurs in nearly all inflammatory 
 leucocytoses, especially in pneumonia, where a careful search over 
 many films may fail to reveal the presence of a single cell. Their 
 reappearance in any of these diseases is of distinctly good omen, 
 and one on which I place a good deal of reliance. It is not of 
 
DIFFERENTIAL LEUCOCYTE COUNT 213 
 
 much value in pneumonia, as it does not occur till after the crisis, 
 but in chronic suppurative diseases, etc., a gradual increase in 
 the eosinophiles often heralds improvement. Neusser holds that 
 the same is true in tuberculosis also. - 
 
 Eosinophile Myelocytes.— The presence of these is practically 
 diagnostic of myelogenous leucocythaemia. 
 
 Mast Cells. — These only occur in fractional percentages in any 
 disease, except myeloid leucocythaemia, where 5 or lo per cent, is 
 common ; this is perhaps the most definite, constant, and readily 
 recognised sign of the disease. The large forms, with circular or 
 kidney-shaped nuclei, are practically only seen in this condition. 
 
 Morphological Changes in the Red Corpuscles. 
 
 Normal red corpuscles (Plate VIII., Fig. i) hardly need 
 description, and the practitioner can readily make himself 
 familiar with their appearance. They are strongly oxyphile, 
 staining pink with the eosin in Jenner's stain, and with acid 
 fuchsin in the triacid. It is especially important to become 
 familiar with their size, since modifications in this respect are 
 very important in the diagnosis of certain forms of anaemia. 
 
 Abnormal Forms of Red Corpuscles. 
 
 Two great classes are to be recognised — those which retain 
 the characters of a normal red corpuscle, in that they have no 
 nuclei, and those which resemble the embryonic corpuscles in 
 being nucleated. The form^er, where they have special designa- 
 tions, have names terminating in -cyte (microcyte, megalocyte, 
 etc., based on the type of the normal corpuscle, which is called 
 erythrocyte, xanthocyte, or normocyte, convenient terms that 
 have never gained wide currency), whilst the nucleated forms 
 have names ending in -blast. 
 
 Abnormal Non-nucleated Forms (-cytes). 
 
 1. Microcytes (Plate VIII., Fig. 4) are red corpuscles which are 
 decidedly below the normal in size, but are otherwise normal. 
 They are indications of anaemia, but, as they may occur in any 
 severe anaemia, and do not serve to indicate its type, their recog- 
 nition is not a matter of much importance. 
 
 2. Megalocytes ov Macvocytes (Plate VIII., Fig. 5). — These are 
 large corpuscles, and the name should be restricted to forms 
 
214 CLINICAL BACTERIOLOGY AND H.EMATOLOGY 
 
 that are at least one and a half times as broad as a normal 
 corpuscle ; they may be much larger. 
 
 It is important to learn to recognise them, since their presence 
 is an almost constant sign of pernicious anaemia, and it is rare 
 to find them in any considerable numbers in other conditions. 
 When you are frequently examining blood-films with the same 
 lens and eyepiece, you will soon be able to pick out forms of 
 abnormal size. When you have not had this experience it is 
 advisable to make a film of normal blood for comparison, and to 
 look first at one and then at the other. A still better method 
 (due to Shattock) is to mount the two films (the normal and 
 abnormal, both stained) face to face, and then to mount the pair 
 on a slide, and examine them with an oil-immersion lens ; a slight 
 turn of the fine adjustment will enable you to pass at once from the 
 one to the other, and to compare the diameters of the corpuscles 
 with some degree of accuracy. Another useful method is to 
 employ the counting-chamber of the haemocytometer, and an 
 idea of the diameter of the reds should be gained in all cases of 
 pernicious anaemia whilst the count is being made. The diameter 
 of one of the small squares is almost exactly six times that of a 
 normal red corpuscle. If you look at the squares with a central 
 ruling, you will be able to compare any corpuscles which may be 
 in them with the semi-diameter ; the one should be one -third of 
 the other, and you may consider as a megalocyte any corpuscle 
 which is one-half or more of this semi-diameter. 
 
 3. Poikilocytes (Plate VIII., Fig. 6) are deformed corpuscles, 
 and are typically shaped like a pear, but may be kidney-shaped 
 or quite irregular. They may be about as large as a normal 
 corpuscle, or smaller, or larger, and they may stain abnormally. 
 Poikilocytes are more common in pernicious anaemia than in 
 other diseases, but are not of much diagnostic importance, since 
 they only occur in advanced stages of the disease, long after the 
 diagnosis should have been made. To recognise them, put a 
 small drop of blood on a perfectly clean slide, apply a cover-glass, 
 and examine at once ; do not identify poikilocytes in dried films 
 or in the counting-chamber of the haemocytometer until you have 
 had a good deal of experience, as accidentally injured and con- 
 torted forms may occur in either. 
 
 4. Polychvomatophil Degeneration (Plate VIII., Fig. 3). — In this 
 condition the corpuscle (which may otherwise be normal or 
 abnormal in other ways), instead of being strictly acidophile in 
 
DIFFERENTIAL LEUCOCYTE COUNT 215 
 
 its staining reactions, stains with the basic stain to a greater or 
 less extent ; thus, with Jenner's stain it stains a variable mixture 
 of pink (from the eosin) and blue (from the methylene blue). It 
 may be lilac, purplish, or almost pure blue. The change is 
 readily recognisable in ordinary films stained by Jenner's method. 
 
 It is especially common in pernicious anaemia and in von 
 Jaksch's anaemia of children, but may occur in almost any form 
 of anaemia, if very severe. It is not a very important diagnostic 
 feature, but its presence always constitutes a bad sign. 
 
 5. Granular degeneration (Plate VIII., Fig. 2) takes the form of 
 numerous granules of varying size, which occur in the red cor- 
 puscles, and which stain almost black with the basic portion of 
 the stain ; the rest of the corpuscle often shows polychromatophil 
 degeneration. 
 
 It occurs also in any severe anaemia, especially in von Jaksch's 
 anaemia, where corpuscles in which it occurs may be extremely 
 plentiful. Except where this occurs it is not of much diagnostic 
 importance, unless it is true that it occurs as a very early and 
 constant sign in lead-poisoning. 
 
 Nucleated Forms (-blasts). 
 
 I. Normoblasts (Plate VIII., Figs. 7, 8, 9). — These are corpuscles 
 which resemble the normal in shape and size, but which have a 
 nucleus. This is central, large in proportion to the corpuscles, 
 surrounded by a comparatively narrow band of stroma, and 
 circular; in some cases it is double or multiple. Normoblasts can 
 usually be recognised with ease from any other cells which occur in 
 the blood, from the fact that the nucleus stains very deeply — more 
 deeply than any other found in the blood. It frequently happens 
 that the narrow ring of stroma may show polychromatophil de- 
 generation and stain blue ; in this case it is difficult to distinguish 
 the cell from a lymphocyte, but the deeply stained, almost black, 
 nucleus should prevent mistakes. 
 
 Normoblasts are the cells from which the normal blood- 
 corpuscles are formed, but in health they are confined to the 
 bone-marrow, except in very young infants, in whom a very few 
 may be found in the circulation. Their presence in the blood of 
 older persons indicates an anaemia of some severity, and that thia 
 anaemia is being combated in a normal way ; the bone-marrow is 
 so active that some of its normoblasts overflow into the circulating 
 blood. They are therefore rather a good sign than otherwise. 
 
2l6 CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 Occasionally you may find them in very large numbers in the 
 blood of a case of anaemia, especially in chlorosis : this is called a 
 "blood-crisis," and when it occurs the patient will improve very 
 rapidly. If you want to count their numbers, the simplest way is 
 as follows : Count the leucocytes in the way already described, 
 and calculate the number per cubic millimetre. Then take 
 a stained film and count 400 or 500 leucocytes, noting how many 
 normoblasts you see whilst doing so. A simple calculation will 
 give the number of normoblasts per cubic millimetre. Thus, 
 if there are 8,000 leucocytes per cubic millimetre, and 72 normo- 
 blasts are seen whilst counting 500 leucocytes, the total number 
 
 of normoblasts per cubic millimetre is 7^ ^ >QQQ ^ 1,152. 
 
 500 
 
 2. Megalohlasts (Plate VIII., Figs. 10, 11, 12). — The recog- 
 nition of these is of the utmost importance, as if they are 
 present in any appreciable numbers in the blood of an adult 
 the diagnosis of pernicious anaemia is almost a certainty, and 
 if a single example is found the diagnosis is highly probable. 
 They occur very rarely in adults in other conditions, but are 
 more common in severe anaemia in childhood, especially in 
 von Jaksch's anaemia, where they may be plentiful. 
 
 In size megaloblasts resemble megalocytes, but may be even 
 'arger ; very large forms are called gigantoblasts, and may have 
 a diameter nearly three times as great as a normal corpuscle. 
 A megaloblast has a nucleus which is usually larger than that of 
 a normoblast, though it may be smaller in proportion to the size of 
 the cell ; it is sometimes double or multiple. It stains much less 
 deeply than the nucleus of a normoblast — in fact, it may stain so 
 faintly that it is overlooked. Except for this the only mistake 
 commonly made by beginners is to confuse a megaloblast with 
 polychromatophilic stroma (which frequently occurs) with a large 
 hyaline leucocyte. 
 
 DIAGNOSTIC APPLICATIONS OF THE BLOOD- 
 COUNT AS A WHOLE 
 
 In this chapter a brief outline of the chief practical applications 
 of the blood-count will be given, with especial reference to cases 
 where it is of use in the discrimination between two diseases 
 which are difficult to distinguish clinically. 
 
 HAEMORRHAGE. — After a severe haemorrhage there is at first no 
 alteration of the blood ; the total volume is diminished, but the 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 217 
 
 part that remains is normal. In a very short time, however, fluid 
 is absorbed from the tissues so as to make up the normal volume, 
 and in consequence the red corpuscles and haemoglobin fall in the 
 same proportion. With this there is usually a leucocytosis, high 
 figures (20,000 or more) being sometimes reached ; do not forget 
 this in dealing with a blood-count in a patient who has recently 
 had a haemorrhage. As the process of regeneration continues, the 
 improvement shows itself first in an increase of the red corpuscles, 
 so that the colour-index falls slightly (to about 0-9). 
 
 The anaemia from repeated haemorrhages is one of the varieties 
 of secondary anaemia. 
 
 Secondary Anemia. — This term is used for anaemia which is 
 due to any definitely recognised cause — haemorrhage, malnutrition, 
 sepsis, intoxications {e.g., lead-poisoning), etc. — that is to say, it 
 includes all cases of anaemia except those of unknown pathology, 
 such as chlorosis and pernicious anaemia. 
 
 In secondary anaemia there is a reduction of red corpuscles to 
 an extent dependent on the potency and continuance of the cause, 
 and a slightly greater reduction of the haemoglobin, so that the 
 colour-index is lowered. It does not usually fall below 0*7, 
 and 0-8 may be taken as a fair average. Anything below this is 
 rarely met with apart from chlorosis. The red corpuscles are 
 usually practically normal in appearance ; normoblasts are rare, 
 and their presence is a good sign. 
 
 The leucocytes, especially the polynuclears, are usually slightly 
 increased, and this is the chief or only means of distinguishing a 
 secondary anaemia with low colour-index from chlorosis, in which 
 the leucocytes are normal or reduced. 
 
 Pernicious Anemia. — The feature which usually first raises 
 suspicion of pernicious anaemia is the high colour -index. It is 
 usually over i, and, taking the average of a number of cases, 
 it increases in proportion to the amount of reduction of the 
 corpuscles, thus : 
 
 Red Corpuscles. 
 
 Average Colour-index. 
 
 Pernicious. | Secondary. 
 
 1 
 
 Under 500.000 
 500,000 to 1,000,000 
 1,000,000 to 2,000,000 
 2,000,000 to 3,000,00D 
 3,000,000 to 4,000,000 
 
 x-6 
 
 1-4 
 
 1-23 
 I '2 
 0-99 
 
 077 
 0-82 
 0-82 
 
2l8 CLINICAL BACTERIOLOGY AND HiEMATOLOGY 
 
 When you find a case with a high colour-index, it should 
 immediately raise a suspicion of pernicious anaemia. Look 
 again at the preparation in which you have counted the reds in 
 the Thoma-Zeiss haemocytometer, and look for unusually large 
 corpuscles (megalocytes), which, according to Ewing, should form 
 35 per cent, of all corpuscles, or the diagnosis is to be made with 
 caution. Then count the leucocytes in the same preparation. 
 Leucopenia is very characteristic ; if the number exceeds 6,000, 
 pernicious anaemia is unlikely, unless inflammatory complications 
 are present. Then make a differential count on a stained film, 
 looking out for megaloblasts and nonnohlasts as you do so ; in 
 pernicious anaemia there is almost always a relative lymphocytosis, 
 and the diagnosis is unlikely with the lymphocytes much below 
 40 per cent. If you have not yet seen a megaloblast, continue to 
 search for them, as they are usually present in pernicious anaemia 
 of moderate severity, and comparatively rare in other conditions, 
 except in children. The significance of the discovery of a single 
 megaloblast will depend on the other findings ; if these point to 
 pernicious anaemia, the megaloblast may be taken as clinching 
 the diagnosis, but if they are not of this nature its importance is 
 much less. Do not exclude pernicious anaemia because no 
 megaloblasts are found. According to Ehrlich and others they 
 always exceed the normoblasts in numbers, but this is not a safe 
 guide, as in some cases you may find normoblasts alone on some 
 occasions, and megaloblasts and normoblasts in differing pro- 
 portions on others. Polychromatophil and granular degeneration 
 and poikilocytosis are common in advanced stages of the disease, 
 but the diagnosis ought to have been made before their appearance. 
 
 The diagnosis from secondary ancemia rests on the high colour- 
 index, the leucopenia, lymphocytosis, nucleated corpuscles and 
 megalocytes, and is usually easy. Anaemia associated with 
 intestinal parasites may resemble idiopathic pernicious anaemia in 
 every respect except in that the former is accompanied by 
 eosinophilia. In pernicious anaemia the eosinophiles are usually 
 low, and if they exceed 4 per cent, the faeces should be searched 
 for the ova of parasites (especially ankylostoma, bothriocephalus 
 and oxyuris). The anaemia of carcinoma of the stomach and other 
 gastric diseases may closely resemble pernicious anaemia, but in 
 most cases there is a high leucocyte count, with increase of 
 polynuclears and diminution of lymphocytes. 
 
 Chlorosis. — Here there is a moderate reduction of the red 
 
 I 
 
DMGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 2ig 
 
 corpuscles and a great reduction of the haemoglobin ; the colour- 
 index falls, therefore, and 0-5 may be taken as an average, 
 though much lower figures occur. The corpuscles are pale, but 
 abnormal forms are rare. Normoblasts are very rare, but when_ 
 they occur usually herald a rapid improvement. The leucocytes 
 are normal in numbers, or there may be leucopenia. 
 
 It may be confounded with various forms of secondary anaemia, 
 but in them the colour-index is usually higher, and the leucocytes, 
 especially the polynuclears, tend to be increased. 
 
 Myelogenous Leucocyth^mia (" spleno-medullary"). — There 
 is an enormous increase in leucocytes, which in an average case 
 may amount to 400,000. All varieties of leucocytes are increased 
 in absolute numbers, but the increase mainly affects the poly- 
 nuclears and the eosinophiles ; the lymphocytes are relatively so 
 scanty that they may be difficult to find. In addition, there are 
 abnormal cells : eosinophile myelocytes and large cells with 
 basophile granulations, cells occurring in practically no other 
 disease, and both Ehrlich's and Cornil's myelocytes. 
 
 There is usually anaemia of the secondary type, and normoblasts 
 are numerous. 
 
 The whole picture is most characteristic, and can hardly be 
 mistaken for anything else. 
 
 Lymphatic Leucocyth^mia. — There is no difficulty in the 
 recognition of a typical case of chronic lymphatic leucocy- 
 thaemia ; the leucocytes are enormously increased (100,000 to 
 1,000,000), and consist almost entirely of lymphocytes (often up 
 to 99 per cent.) : in many cases the large forms predominate. 
 There is also a varying degree of secondary anaemia. 
 
 In an adult these appearances are quite characteristic, but in 
 childhood similar counts may be seen in a variety of conditions, 
 such as hooping-cough, broncho-pneumonia, etc., though it is 
 rare to find a figure as high as 100,000. 
 
 In some acute cases of lymphatic leucocythaemia similar 
 appearances occur. In others the increased percentage of 
 lymphocytes is present, but not the total increase ; thus, in one 
 case fatal in a few weeks I never found more than 10,000 
 leucocytes, in which the lymphocytes varied between 76 per cent, 
 and 100 per cent. These cases are very difficult to diagnose 
 from acute tuberculosis of the lymphatic glands. In the latter 
 case the total count may be expected to be low, in the former 
 slightly raised ; in tubercle the percentage of lymphocytes rarely 
 
220 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 exceeds 50, whilst 75 per cent, at least would be required before 
 the condition under discussion could be diagnosed. 
 
 Other diseases (especially acute purpura haemorrhagica) give 
 very high lymphocytoses in adults, but as they are quite different 
 clinically the blood-count would not be misleading. 
 
 Hodgkin's Disease. — There is little doubt that several diseases 
 are included under this heading. In the true Hodgkin's disease 
 there is at first no change in the blood, not even anaemia ; in one 
 case I examined at intervals for over a year the red corpuscles 
 were always above 5,000,000, and the haemoglobin above 100 per 
 cent. Later, there is anaemia of secondary type, with a slight 
 leucocytosis, with or without a moderate increase of polynuclears. 
 In other cases there may be anaemia from the first, a normal or 
 diminished number of leucocytes, and a relative increase of 
 lymphocytes of moderate amount ; these cases are probably more 
 closely allied to lymphatic leucocythaemia, and for them the term 
 " pseudo-leukaemia " might be used. I believe them to be of more 
 rapid course than true Hodgkin's, but the two cannot be definitely 
 separated on clinical grounds alone. 
 
 The diagnosis between these forms of enlarged glands and 
 those due to tuberculosis cannot be made by a blood-count. The 
 opsonic index to tubercle may be of assistance. 
 
 Lymphosarcoma is also associated with a practically normal 
 blood condition, and cannot be diagnosed from aleukaemic leuco- 
 cythaemia and Hodgkin's disease. 
 
 Splenic Anemia. — It is very doubtful whether this disease is 
 really a distinct entity, and it is certain that many of the reported 
 cases have nothing in common but the accidental and not unusual 
 concomitance of anaemia and an enlarged spleen. In the cases in 
 which the name may fairly be applied there is idiopathic enlarge- 
 ment of the spleen, with anaemia ; the latter is usually of medium 
 grade, figures under 2,500,000 being uncommon, and the colour- 
 index is moderately low (07 to o'g). In severe cases there may 
 be numerous normoblasts, poikilocytes, and polychromatophil 
 degeneration of the red corpuscles. The only feature that can be 
 considered as characteristic is the frequent presence of leiicopenia 
 with relative lymphocytosis ; there may be a few myelocytes. 
 
 It may be confounded clinically with myelogenous leucocy- 
 thaemia or pernicious anaemia, but is readily distinguished by the 
 blood-count. In secondary anaemia with an enlarged spleen (such 
 as occurs in malignant disease, infective processes, etc.) there will 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 221 
 
 probably be a polynuclear leucocytosis. Hodgkin's disease with 
 an enlarged spleen and without palpable glands may be indis- 
 tinguishable from splenic anaemia, and probably some of the 
 recorded cases have been of this nature. — 
 
 Anemia in Infancy. — The rules for the interpretation of blood- 
 counts in adults are not applicable in infancy, where the conditions 
 of blood formation are so different. In particular the presence of 
 nucleated red corpuscles, and especially of megaloblasts, is of little 
 importance, and frequently occurs in conditions insufficient to 
 call for their appearance in older patients. The colour- index is 
 extremely variable ; it usually tends to be very low, although this 
 does not indicate a disease having any connection with chlorosis, 
 a disease which does not occur in infancy. On the other hand, a 
 high colour-index is not infrequent, and does not necessarily 
 point to pernicious anaemia, which is excessively rare. Lastly, 
 degenerative changes, such as poikilocytosis, granular degeneration, 
 and polychromatophilia, are very common, occur in comparatively 
 mild grades of anaemia, and have not the serious import they have 
 in the adult. 
 
 Leucocytosis in Anamia in Infants. — This is very common, 20,000 
 to 60,000 being frequently met with ; in most cases there is a 
 predominance of lymphocytes, and the presence of myelocytes is 
 quite common, rendering the diagnosis from leucocythaemia a 
 matter of some difficulty. As a general rule, the presence of a 
 high leucocytosis in infantile anaemia is a bad sign, and indicates 
 a worse prognosis than if it is absent. 
 
 Secondary Ancsmia in Infancy {Syphilis, Rickets , Scurvy, Tubercle, 
 etc.), — There are no characteristic changes by which the different 
 causes of secondary anaemia can be recognised. The colour-index 
 is usually low (especially, perhaps, in syphilis), normoblasts are 
 not uncommon, an occasional megaloblast may be seen, and there 
 is often leucocytosis, with increase of lymphocytes. 
 
 Von Jaksch's Anmnia {Anmnia Infantum, Pseudo-leukcsmia). — It is 
 uncertain whether this is to be considered as a definite disease, as 
 an intermediate form between pernicious anaemia and leucocy- 
 thaemia, or as a form of secondary anaemia with somewhat 
 characteristic blood changes. I am rather inclined to the belief 
 that it does represent a definite blood disease, but that it is 
 frequently associated with, and perhaps due to, other diseases 
 such as syphilis, rickets, tubercle, or gastro-intestinal diseases. 
 The blood changes are — (i) An excessive grade of anaemia, usually 
 
222 CI.INICAL BACTERIOLOGY AND HEMATOLOGY 
 
 associated with a low colour-index ; in some cases, however, it 
 may be high, and I have seen it as high as i"8. (2) High 
 leucocytosis, often 50,000 or more, with extraordinary changes in 
 the leucocytes, so that they can hardly be classified on the usual 
 lines ; myelocytes are not uncommon. (3) Striking and profound 
 changes in the red corpuscles, the most important being the 
 presence of numerous normoblasts, many of which show dividing 
 miclei (Plate VI 1 1., Fig. 8) ; megaloblasts and atypical forms 
 occur, but are less numerous. The non-nucleated red corpuscles 
 show all forms of degeneration, poikilocytes, megalocytes, and 
 microcytes being present, whilst many are affected with granular 
 basophilia (Plate VIII., Fig. 2) or polychromatophilia (Plate VIII., 
 Fig. 3). In severe cases the majority of the red corpuscles may 
 be abnormal, and the appearance of the stained films is very 
 extraordinary. 
 
 The prognosis of these cases is fairly good if proper treatment 
 be adopted — much better than in the primary blood diseases with 
 which they might be confounded. 
 
 Sepsis, Suppuration, and SEPTic^MtA. — In most cases of infec- 
 tion with septic bacteria, whether local or general, there is marked 
 leucocytosis, due especially to an increase in the polynuclear 
 leucocytes. For instance, in an ordinary case of appendicitis 
 of average severity we may expect the number of leucocytes to 
 rise gradually to 25,000 or 30,000, about 90 per cent, being poly- 
 nuclears. At the same time there is usually a moderate fall in 
 the amount of haemoglobin and in the number of red corpuscles. 
 In practice we have to consider three types, in each of which the 
 blood-counts vary. 
 
 I, Very Severe Cases.- — Puerperal and other forms of septicaemia, 
 general septic peritonitis due to very virulent bacteria, especially 
 in an enfeebled patient, etc. 
 
 Here there may be but slight leucocytosis, and this, taken in 
 conjunction with the patient's general condition, is not a good 
 sign, but a bad one. In many cases the total number is within 
 the normal limits, but whether this is the case, or whether the 
 leucocytes are slightly increased, a clue to the condition will be 
 given by the fact that the polynuclears show their usual increase, 
 and the glycogen reaction is present and often very marked. 
 
 The haemoglobin, however, gives much more valuable indica- 
 tions ; the severer the case the more rapidly it falls, and vice versa. 
 The same information can be obtained, though not so well, from 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 223 
 
 the variation in the red corpuscles. Thus, in a case of severe 
 sepsis of any sort the red corpuscles and haemoglobin may fall 
 enormously in a few days. This is a very valuable test for 
 puerperal fever. Under ordinary circumstances the amount oT 
 blood lost at parturition should not lower the corpuscles below 
 4,000.000, and if a count greatly below this is found in a few days' 
 time, there having been no severe haemorrhage in the meantime, 
 the diagnosis of septic infection is probable, whatever be the 
 numbers of the leucocytes ; if at a later date the numbers are 
 lower still, the diagnosis is almost certain. The haemoglobin 
 may fall 10 per cent, per day, or even more in a severe 
 case. 
 
 Conversely, a rise in the number of red corpuscles and haemo- 
 globin is a good sign when the existence of septic infection is 
 certain, whatever be the clinical condition ; a cessation of the fall 
 is good also, but to a less extent. (Beware, however, of mistaking 
 a concentration of the blood from diarrhoea or profuse vomiting 
 for a true rise.) As an example I may quote the case of a patient 
 under Dr. Hayes in King's College Hospital, in whom about a 
 fortnight after parturition there were 1,306,000 reds, 26 per cent, 
 haemoglobin, 13,400 leucocytes, of which 87 per cent, were 
 polynuclears. Here 2,700,000 reds had been lost in two weeks, 
 indicating a very severe sepsis. Her condition appeared desperate, 
 yet in a week the reds had increased to 2,750,000, in a fortnight 
 more to 3,760,000, when for the first time she showed clinical 
 improvement. In nine days more they had reached 4,000,000, 
 and the patient was out of danger. Here a good prognosis was 
 given solely on the blood-count. 
 
 In these cases a great diminution or total disappearance of the 
 eosinophiles is a bad sign, their reappearance a good one. 
 
 2. Suppuration. — Where the sepsis is localized and not so severe, 
 so that a limited focus of suppuration occurs, there is a leucocy- 
 tosis which, in round figures, exceeds 20,000, and the increase is 
 mainly due to a rise in the polynuclears. Various writers give 
 different figures as that above which pus is indicated : some take 
 15,000, which I find to be reached fairly often when there is no 
 pus ; others 25,000, which is frequently not reached when sup- 
 puration has occurred. The number 20,000 has been taken as 
 the result of a considerable amount of experience of all forms of 
 suppuration, and will prove a correct indication in at least 90 per 
 cent, of all cases. It is especially useful in appendicitis, in which 
 
224 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 it is an almost certain guide. The glycogen reaction is usually 
 present, and is a valuable confirmatory test. 
 
 The following considerations must be remembered : 
 
 (a) The increase of leucocytes to the figures mentioned above 
 only occurs when the pus is pent up, not when it occurs on a 
 free surface and can escape. I have twice seen a rapid fall of 
 the leucocytes due to rupture of an appendicitic abscess into the 
 intestine. 
 
 (b) Where the spread of the suppuration ceases (due to the death 
 or latency of the organisms it contains) the leucocytosis gradually 
 subsides, and an old, thick-walled collection of pus in the tissues 
 may give a normal count. This is especially common in gonor- 
 rhoea! pyosalpinx ; the gonococcus dies out rapidly, but the pus 
 remains, and unless you examine the case early there will be no 
 leucocytosis, or but little. 
 
 (c) The leucocytosis gives no indication of the seat of the pus ; 
 there may be a small abscess in the body far from the region 
 under suspicion. 
 
 (d) The height of the leucocytosis gives no indication of the 
 size of the abscess or of its severity. 
 
 (e) In interpreting a high leucocytosis to mean pus, you must 
 exclude the other causes of a similar blood condition — pneumonia, 
 etc. Thus, in a severe abdominal disease leucocytosis may be 
 due to an ovarian cyst with a twisted pedicle, a ruptured tubal 
 gestation, etc. 
 
 (/) Cold (tuberculous) abscesses do not usually give a poly- 
 nuclear leucocytosis; if there is one, it indicates a secondary 
 septic infection, and is a decidedly bad sign. 
 
 3. In moderate cases of septic or other form of inflammation, sup- 
 puration of free surfaces, etc., there is usually a moderate 
 leucocytosis (up to 18,000), with increase of the polynuclears. 
 
 Typhoid Fever. — In the early stages there is usually some 
 concentration of the blood, the red corpuscles often exceeding 
 6,000,000 ; at a later period they fall somewhat, but rarely below 
 4,000,000. In most other diseases (malaria, septicaemia, tubercu- 
 losis, etc.) for which typhoid is likely to be mistaken the rule is to 
 find marked diminution of the red corpuscles; when these are high 
 with a fever of some duration typhoid fever should be suspected. 
 
 The leucocytes are usually normal or diminished in numbers 
 (3,000 to 6,000), and there is often slight excess of lymphocytes 
 (average about 50 per cent.). This is not unlike what occurs in 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 225 
 
 tubercle and malaria, but is of diagnostic value in distinguishing 
 typhoid from septicaemia, in which case there may be no excess 
 of leucocytes, but there is usually a relative excess of polynuclears. 
 The number of leucocytes in typhoid is of some value in prog- 
 nosis ; the lower the count, the more severe the case, though to 
 this rule, as to all others in blood-work, there are exceptions. 
 
 In the later stages of the disease the diagnosis is best made by 
 Widal's reaction. 
 
 Complications. — Here the condition of the patient must be taken 
 into account ; a complication {e.g., perforation) which causes a 
 rapid and marked leucocytosis in a patient who has not been 
 greatly enfeebled by a long and severe illness may cause no 
 increase, or even a diminution, in an exhausted subject. If this 
 rule is forgotten, the indications from the blood-count may be 
 unjustly stigmatized as misleading. 
 
 Perforation, — There is a rapid increase of leucocytes, which is 
 said to occur in half an hour ; the total number may be 15,000 or 
 more. In a patient who is not greatly exhausted this is a very 
 sure sign ; exceptions do occur, but are not frequent. 
 
 HcEmorrhage. — If a count has been made a short time previously, 
 a fall in the red corpuscles may be noticed in some cases, but does 
 not always seem to occur. There may also be slight leucocytosis; 
 this is only of importance in that it leaves the diagnosis of 
 perforation or haemorrhage doubtful, though raising the pre- 
 sumption that one or other has occurred. As a rule it is not 
 high, and the more it rises above 15,000, the more likely is 
 perforation to be the cause. 
 
 Pneumonia. — When it occurs early in the disease there is usually 
 a slight leucocytosis ; when it occurs later there may be none. 
 Do not exclude pneumonia, therefore, because of its absence. 
 
 It follows from the above that a raised leucocyte count in 
 typhoid fever always points to a complication of some sort, but 
 does not necessarily indicate its nature. 
 
 Pneumonia. — Here the results of blood examinations are 
 fairly constant, and of much value in diagnosis and prognosis. 
 In ordinary cases there is marked leucocytosis, due entirely to 
 an increase of the polynuclears, which may reach 95 per cent. 
 There is also moderate secondary anaemia. 
 
 According to Ewing, who has had much experience on the 
 subject, the grade of leucocytosis is roughly proportionate to the 
 extent of the lesion. The following are his averages : 
 
 15 
 
226 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Average Leucocytes. 
 
 1 lobe affected . . _ . 20,000 
 
 2 lobes ,, .... 22,700 
 
 3 » n - . - . 25,000 
 
 4 „ „ .... 27,000 
 
 But higher counts, often much higher, are frequently seen. 
 
 These figures serve to exclude typhoid fever, tuberculosis, 
 acute tuberculous pneumonia, and influenza, which do not cause 
 leucocytosis ; a true lobar pneumonia implanted on the latter 
 raises the leucocyte count, a lobular one does not. 
 
 In a few cases of pneumonia there is no leucocytosis, but these 
 hardly detract from the value of the sign. They are (i) very 
 mild cases, and (2) very severe ones, usually rapidly fatal, in 
 which the system fails to react to the infection ; the iodine 
 reaction of the leucocytes is well marked in these. A low count 
 in pneumonia, therefore, may be a good or bad sign : which it is 
 can be told by a glance at the patient. A moderate leucocytosis 
 which gradually declines is a bad sign in a case of any severity. 
 
 As a rule the leucocytes fall to nearly normal at the crisis, 
 sometimes a little before ; in such cases the crisis may be pre- 
 dicted, and a sudden fall to normal after a week or so is of 
 very good omen. If the leucocytes remain up after a crisis it is 
 most likely to be due to empyema. 
 
 Malaria. — Here, of course, the diagnosis should be made by 
 finding the specific micro-organism in the blood (p. 142). 
 Where this cannot be done the case may still be one of malaria, 
 and the blood-count may aid in the diagnosis. There is anaemia, 
 often coming on rapidly and attaining very low figures. There 
 is no leucocytosis, and according to many observers there is a 
 great increase in the large lymphocytes, which almost always 
 become more numerous than the small ones. This test is not 
 interfered with by the administration of quinine, which renders 
 the parasites difficult or impossible to find. 
 
 Scarlet Fever and Measles. — In the former there is, except 
 in the very mildest cases, a marked leucocytosis ; in the latter the 
 blood is normal in the absence of pneumonia or other complica- 
 tions. In scarlet fever the leucocytes range from 10,000 to 40,000, 
 and according to some authors the prognosis is very bad in cases 
 showing more than 30,000 ; there is an excess of polynuclears 
 (80 to 90 per cent.), which is very noticeable in children, where 
 there is usually a high proportion of lymphocytes. 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 227 
 
 German Measles is not accompanied by leucocytosis. 
 
 Hooping-cough. — There is a high grade of leucocytosis (20,000 
 to 60,000), due mainly to an increase of lymphocytes. This is said 
 to occur before the hooping occurs and to be of diagnostic value, 
 but leucocytosis with lymphocytosis is so common in children 
 that little value should be attached to it unless really high figures 
 are found. 
 
 Influenza. — Here the blood-count may be of value, since, in 
 contradistinction to the majority of acute febrile diseases, there 
 is no leucocytosis if complications are absent. In other febrile 
 diseases of rapid onset — pneumonia, tonsillitis, rheumatic fever, 
 septic affections, plague, etc. — leucocytosis is almost constant. 
 
 Rheumatism. — Except in the very mildest cases there is leuco- 
 cytosis, and, according to Turk, Ewing, and others, when there 
 are more than 20,000 there is almost certainly some complication, 
 such as endocarditis, pericarditis, pneumonia, or hyperpyrexia. I 
 believe this may be taken as a safe general rule, though excep- 
 tions do occur. 
 
 Turk believes that a clue to prognosis may be got from the 
 percentages of eosinophiles present ; with a proportion above the 
 normal the case is likely to be a mild one. 
 
 Tuberculosis. — There is usually marked anaemia of the 
 secondary type, but in cases with sweating and diarrhoea this 
 may be masked by the concentration of the blood ; an apparent 
 improvement in this respect may in reality be a bad sign. The 
 leucocytes are usually normal, though the lymphocytes may be 
 rather high. 
 
 Where secondary septic infection takes place — e.g.^ in a vomica 
 — the blood is that of sepsis ; there is a variable leucocytosis, 
 excess of polynuclears, and advancing anaemia. 
 
 The blood-count is not of much value in the diagnosis of 
 tubercle ; it is of some value in prognosis. Increase in the poly- 
 nuclears and in the grade of anaemia are bad signs in phthisis. 
 
 There are one or two precautions to be noted in special cases. 
 In tuberculous empyema there is frequently a secondary infection, 
 and the presence of a leucocytosis does not show that the disease 
 is not tuberculous ; the same thing applies to tuberculous abscesses 
 in other parts, including the joints. 
 
 Tuberculous meningitis appears to offer the most marked 
 exception to the rule, that uncomplicated tubercle does not cause 
 leucocytosis. Here there is often moderate leucocytosis, and 
 
 15—2 
 
228 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 according to Horder it may reach 25,000. I have several times 
 met with 20,000 or thereabouts. Tuberculous pleurisy is usually 
 without marked leucocytosis, but occasionally the figures may reach 
 18,000 to 20,000. 
 
 Syphilis. — There is usually progressive secondary anaemia with 
 moderate leucocytosis (12,000 to 16,000), due mainly to increase of 
 lymphocytes, often of the large type, but the figures are too incon- 
 stant to be of much value in diagnosis (see p. 180, Justus's test). 
 
 Purpura Hemorrhagica. — There is naturally advancing 
 anaemia, which may be associated with a low or normal colour- 
 index. In the most common type of case, in which the prognosis 
 is relatively good, there is also the usual slight polynuclear leuco- 
 cytosis met with in secondary anaemia, or it may reach a very 
 high grade. In some cases, however, in which the prognosis is 
 extremely bad, there is leucopenia with lymphocytosis. In one 
 case (under Dr. Dalton at King's College Hospital) which was 
 fatal in a few days the leucocytes were 2,000 and the lymphocytes 
 over 95 per cent. A blood - count should always be made in 
 purpura haemorrhagica, when these cases (probably quite different 
 in nature) may be diagnosed early and their gravity recognised.* 
 
 Malignant Tumours. — These are frequently associated with a 
 moderate leucocytosis, with increase of the polynuclears, and slight 
 anaemia. The leucocytosis is said to be more marked in the sar- 
 comata than in the carcinomata. These facts are very rarely of 
 value in diagnosis, since whilst the growth is small and removable 
 the blood is usually normal, except in an ulcerated carcinoma of 
 the gastro-intestinal tract, and in any case there are so many 
 examples of malignant growth with normal blood, and so many 
 causes of slight leucocytosis, that its presence is not much help. 
 Malignant tumours of the oesophagus are occasionally unaccom- 
 panied by leucocytosis, but most follow the general rule. > 
 
 Cancer of the Stomach. — The frequency with which difficulty 
 arises in the early diagnosis of this condition renders any assist- 
 ance important, and although the indications given by the blood- 
 count are not conclusive, they are helpful in conjunction with the 
 clinical examination and the investigation of the vomit (p. 115). 
 In some cases the blood is normal, but usually there is one of 
 two conditions : either a marked secondary anaemia with a rather 
 low colour-index (averaging 0-63 according to Osier and McCrae), 
 
 * Some at least of these cases are really acute lymphatic leucocythaemia 
 without glandular lesions. 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 22g 
 
 with slight polynuclear leucocytosis — about 12,000 to 18,000, of 
 which 80 to 90 per cent, are polynuclears ; or a condition closely 
 resembling pernicious anaemia, with a high colour-index, megalo- 
 cytes, and occasionally megaloblasts. In the latter case the_ 
 diagnosis from true pernicious anaemia may usually be made by 
 the fact that in carcinoma ventriculi there is polynuclear leucocy- 
 tosis instead of the leucopenia with lymphocytosis of pernicious 
 anaemia. The blood finding in this case is very suggestive. 
 
 According to some authors there is no digestion leucocytosis in 
 cancer of the stomach, whilst there is in other diseases. There 
 appear to be numerous exceptions to this rule, though it is true 
 in the majority of cases, and might be allowed some weight in 
 forming a diagnosis. To test for it enumerate the leucocytes in 
 a patient who has eaten nothing since the previous day ; let him 
 take a meal (of which meat should form part), and repeat the 
 examination in three or four hours' time ; a rise of 2,500 to 3,500 
 may be considered normal. 
 
 Ulcer of the Stomach with haemorrhage leads to secondary 
 anaemia, but, unlike carcinoma of the stomach, is usually un- 
 associated with leucocytosis. There are exceptions to this rule, 
 and when the ulcer reaches the peritoneum and causes local 
 peritonitis, or ruptures and causes general peritonitis or localized 
 abscess, leucocytosis occurs. 
 
 Cirrhosis OF the Liver. — In ordinary uncomplicated alcoholic 
 cirrhosis there may be anaemia, but there is no leucocytosis, or at 
 most very little. In most of the diseases for which it may be 
 mistaken an ordinary polynuclear leucocytosis is present. 
 
 Hanot's cirrhosis is said to be accompanied by leucocytosis, 
 but, as this may be intermittent, the diagnostic value of the test 
 cannot be great in cases where no leucocytosis is found. 
 
 Abscess of the Liver and Hydatid Cyst. — The former disease 
 is usually associated with leucocytosis, the latter is not, though here, 
 again, there are exceptions. Some importance should be attached 
 to the simple count, but more to the differential count, since in 
 hydatid the eosinophiles are usually raised (57 per cent, has been 
 recorded, but 6 to 8 per cent, is more usual), and in ordinary 
 septic disease are absent, low, or normal, and these rules apply 
 w^hether the leucocytes are normal or increased. 
 
 Peritonitis and Appendicitis. — See p. 222, Sepsis and Sup- 
 puration. 
 
 Pleurisy and Empyema. — With a mechanical pleural effusion 
 
230 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 (cardiac or renal) there is no leucocytosis. With simple non-tuber- 
 culous pleurisy the blood is usually normal, and the same holds 
 in the tuberculous cases, but here the leucocytes are occasionally 
 raised to 18,000 or more. Such cases may be distinguished from 
 pneumonia or empyema by the presence of the iodine reaction in 
 the latter and its absence in pleurisy. Empyemata are always 
 associated with a high leucocytosis, except sometimes in the 
 tuberculous forms. 
 
 Asthma. — True asthma has a very characteristic blood condition. 
 During an attack there is a leucocytosis of moderate grade, with 
 a great increase of eosinophiles ; 10 per cent, may be taken as an 
 average, though much higher counts have been recorded. In the 
 intervals the total numbers are normal, but there is usually a 
 moderate eosinophilia, 5 to 7 per cent, or more, and I have found 
 a slight increase of mast cells (about i per cent.), which is so 
 rare a phenomenon that it may be of some diagnostic value. 
 Eosinophilia rarely occurs associated with other forms of spas- 
 modic dyspnoea, and its presence serves to diagnose asthma from 
 cardiac or renal dyspnoea — often a matter of great importance — 
 or from dyspnoea due to pressure on the bronchi, trachea, etc. 
 
 The sputum in asthma is usually characteristic, and contains 
 the peculiar spirals and vast numbers of eosinophile cells ; these 
 are rarely seen in cases of bronchitis, but never in any numbers 
 in other diseases. 
 
 Bronchitis and Broncho-pneumonia. — With simple bronchitis 
 there is the usual inflammatory leucocytosis, usually about 12,000 
 to 14,000. In broncho-pneumonia the count is much higher — 
 20,000 or more. This applies to children as well as to adults. 
 
 Endocarditis. — Not much help can be obtained from the 
 leucocytes in the diagnosis between the simple and the malignant 
 form, since in either case there may be a normal or slightly raised 
 count. But in malignant endocarditis there is usually a rapidly 
 increasing anaemia of secondary type. The true test, however, is 
 the bacteriological one ; the blood is sterile in simple endocarditis, 
 whereas organisms are usually found in ulcerative cases, though 
 more than one examination may be necessary. 
 
 Valvular Lesions require brief mention, since their presence 
 causes alterations which might cause embarrassment in the diag- 
 nosis of other conditions if unrecognised. With mitral lesions, if 
 not fully complicated, there is a tendency for an increase in the 
 red corpuscles (due to venous stasis) which may reach 8,000,000. 
 
DIAGNOSTIC APPLICATIONS OF THE BLOOD-COUNT 23X 
 
 In morbus caeruleus it may be still higher — 10,000,000 or more. 
 In aortic disease, on the other hand, there is a tendency to slight 
 anaemia. The leucocytes remain normal in both cases. 
 
 Puerperal Fever. — The great difficulty in interpreting blood 
 findings in the puerperium is the fact that the blood is not normaF 
 immediately before child-birth. The change affects the leucocytes, 
 which are increased, it may be as high as 36,000 (Cabot) ; this is a 
 very unusual figure, and on the average the numbers do not exceed 
 16,000 in primiparae and 12,000 in multiparae. Leucocytoses within 
 these limits are to be looked on as being probably normal, and not 
 as indicating sepsis. More information may be gained by counts 
 at intervals. The figures should decrease rapidly after delivery, 
 becoming normal in less than a fortnight, and if a count remains 
 the same on two successive days an inflammatory process is 
 suggested, and a definite rise constitutes almost absolute proof. 
 It is in these cases especially that the glycogen reaction is of 
 value, and its presence should outweigh that of the total numbers. 
 The differential count is not of much value, since the polynuclears 
 are increased in normal pregnancy, unless these cells reach 90 per 
 cent, or more. 
 
 The chief reliance is to be placed on the haemoglobin. It 
 should be normal or slightly reduced at the end of pregnancy, 
 fall in proportion to the haemorrhage at parturition and for a day 
 or two after, and then be rapidly regenerated. Under normal 
 circumstances it should not be much below 70 per cent, nor the 
 red corpuscles much below 4,000,000. Figures much below these 
 (unless there has been great haemorrhage, or unless it has been 
 repeated) raise suspicions of sepsis, whilst an observed fall is 
 almost definite proof. This has also much value in prognosis 
 (see p. 223). 
 
 Perimetritis, Parametritis, etc. — Here the usual relations 
 hold good. There is a moderate leucocytosis in a non-suppurative 
 lesion, a high one when suppuration occurs. The figures are 
 usually somewhat lower than in other parts of the body, and 
 T 8,000 may be taken as fairly definite evidence of pus, provided 
 other sources of leucocytosis can be excluded. 
 
 Other Pelvic Swellings. — The blood-counts in these cases 
 have to be interpreted with much caution, and are often very 
 equivocal. Thus, pyosalpinx is usually associated with the ordinary 
 signs of pus, but the cases are frequently tuberculous, when there 
 is no leucocytosis, or gonorrhceal, when there is only leucocytosis 
 
232 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 if the count is made whilst suppuration is in progress. The sterile 
 collections of pus left after an attack of gonorrhceal salpingitis 
 do not cause leucocytosis. A normal count, therefore, does not 
 exclude pus in the tubes. Similarly, there are exceptions to the 
 rule that simple ovarian tumours and cysts are associated with 
 normal blood. Where there is inflammation and formation of 
 adhesions there is slight leucocytosis, where there is much peri- 
 toneal irritation a higher one, and with twisting of the pedicle 
 very high figures may be reached. These latter are liable to be 
 mistaken for acute peritonitis, but the leucocytes do not give the 
 iodine reaction. Further, malignant ovarian tumours usually cause 
 a slight leucocytosis and decrease of reds ; this has been suggested 
 as of diagnostic importance, but it might be due to so many other 
 conditions that not much reliance can be placed on it. A normal 
 condition of the blood, however, would be some evidence against 
 malignancy. In tubal gestation the blood is normal. After rupture 
 there is anaemia and increase of the leucocytes, which may reach 
 24,000 or more. 
 
 Pemphigus, Dermatitis Herpetiformis, and Erythema 
 Multiforme, are associated with a high percentage of eosino- 
 philes, and this is of importance in the diagnosis of these diseases 
 from local infective processes, which they often closely resemble. 
 The eosinophiles in the former group of diseases may be expected 
 to exceed 10 per cent., and may be much higher, and there may 
 be a high leucocytosis. 
 
PART III 
 
 CYTO-DIAGNOSIS 
 
 Cyto-diagnosis is the diagnosis of the cause of exudates by the 
 recognition of the cells which they contain. It may be regarded 
 as a branch of haematology, though the cells which are 
 encountered are not wholly those of the blood. Its results are 
 less certain than those obtained by the recognition of the organism 
 (if any) present in the exudate, but are often easier to obtain ; 
 they are more certain than those obtained by an examination of 
 the blood, as easy to obtain, and in most cases very much easier 
 to interpret. As the methods are very simple and require no 
 special apparatus (though a centrifuge is a very great help), they 
 are within the reach of all practitioners, and a cytological examina- 
 tion should always be made when fluid is withdrawn from the 
 chest, abdomen, etc., whether for diagnosis or treatment. 
 
 Method of collecting the Cells. — No description will be 
 given of the methods of obtaining the exudate, as those which 
 are not in general use have been dealt with already. 
 
 If the fluid does not clot spontaneously, it is only necessary to 
 centrifugalize a portion (as much as the tube will hold — about 
 lo c.c.) for five minutes or so, and then to invert the tube and pour off" 
 as much of the supernatant fluid as will come away. The sediment 
 will be left, and a drop or two of fluid will run back down the 
 sides of the tube. These must be thoroughly mixed in with the 
 deposit so as to form a uniform emulsion. 
 
 In the absence of a centrifugal machine, allow the fluid to 
 stand for twelve hours or so to settle, adding a crystal of thymol 
 to prevent decomposition. Then remove some of the deposit 
 with a pipette ; you cannot invert the tube in this case, as the 
 sediment is not so compact, and will pour out. 
 
 233 
 
234 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 When the fluid has coagulated, put it in a strong bottle with 
 some glass beads or balls, or fragments of glass of any sort, and 
 shake for ten minutes. This will break up the coagulum and set 
 most of the cells free. Allow the fluid to stand for a minute or 
 two so that the pieces of fibrin may settle ; then decant the 
 supernatant fluid into the centrifugalizing or sedimenting tube, 
 and proceed as before. 
 
 Where the fluid is pus no preparation is usually necessary. If 
 it clots it does so very feebly, and in this case a little stirring 
 with a platinum loop will set free plenty of cells for examination, 
 or you may take up the clot with a loop and rub it on the slide or 
 cover-glass. 
 
 Method of preparing the Specimen for Examination. — 
 The specimens may be examined wet or dry. In most cases the 
 former method is best, as it is quicker, and often yields information 
 which cannot be obtained by a dry specimen. The preparations, 
 however, do not keep, and where permanent ones are required 
 the method is inapplicable. 
 
 Wet Method. — Place one drop of watery methylene blue or 
 borax methylene blue on a slide and add two or three drops of 
 the emulsion of cells. Stir with the platinum loop or needle, 
 allow the mixture to stand for two or three minutes, and then 
 apply a cover-glass. The cover glass may be cemented to the 
 slide by means of melted paraflin applied with a hot iron rod ; it 
 is best to do this if the oil-immersion lens is to be used, otherwise 
 the suction of the lens may lift up the cover-glass. 
 
 Or, put two or three drops of the emulsion on a slide, cover, 
 and examine without staining. Then put a drop or two of acid 
 methylene blue (see p. 30) on the slide just touching the cover- 
 glass ; it will pass in by capillarity, and at different distances 
 from the edge you will get an unstained area, an area where the 
 stain is faint, but very selective, and a deeply stained area. The 
 middle zone is best to examine. The red corpuscles, if present, 
 will be dissolved by the acid, and after a few minutes the cells 
 will be stained with great distinctness. 
 
 Dry Method. — Prepare films on the slide or cover-glass 
 (p. 199), using the emulsion exactly as if it were blood. This 
 may be stained by Jenner's method (p. 204) or fixed and stained 
 subsequently. Any of the fixing and staining methods described 
 for blood may be used, but I prefer to allow the film to dry, fix 
 with saturated solution of perchloride of mercury for a minute 
 
CYTO-DIAGNOSIS 235 
 
 or two, wash, stain in carbol thionin for two minutes, wash, dry 
 and mount. This renders everything very distinct except (some- 
 times) the nucleoli of the endothelial cells and the granules of the 
 polynuclears. Where the latter have to be inquired into (which 
 rarely happens) the film must be fixed with heat and stained with 
 triacid (p. 204). 
 
 Cells met with in Exudates. 
 
 Leucocytes derived from the blood are present in the majority 
 of exudates, and in most cases they are of ordinary appearance, 
 and readily recognised from the descriptions already given. The 
 polynuclears, however, may undergo various forms of degeneration, 
 and become so altered that their nature may be difficult to make 
 out. This occurs mainly in old exudates, especially in pus. 
 There are three chief forms : 
 
 (a) In some cases the nucleus undergoes fragmentation — i.e.^ 
 breaks up into several isolated masses, so that the cell becomes 
 truly polynuclear (Plate IX., Fig. 2, where the ingested leuco- 
 cytes are fragmented). These masses stain deeply, and the cell 
 is easy to recognise, but it often happens that the fragments 
 of the nuclei are set free, and may then be mistaken for small 
 lymphocytes. If a specimen be stained by the triacid stain, they 
 may often be distinguished by a few granules which remain 
 adherent to the nucleus. 
 
 (b) Dropsy of the nucleus, which converts it into a large circular 
 or reniform mass which stains faintly. In this case the cell 
 resembles a large lymphocyte, large hyaline leucocyte, or myelo- 
 cyte. It may usually be distinguished from the former by the 
 presence of granulations, and the latter is not known to occur 
 in exudates except in cases of leucocythaemia. 
 
 {c) Fatty degeneration of the cell, shown by the occurrence of 
 clear refractile granules with sharp contour. These are only 
 seen in wet preparations. In this case the cell usually undergoes 
 severe degenerative changes or even complete solution, and in 
 old pus it may be difficult to make out any definite cells at all 
 (Plate IX., Fig. 3). 
 
 As a matter of fact, these degenerative changes rarely cause the 
 slightest difficulty in diagnosis. It frequently happens that no 
 pathologist is able to say definitely what is the nature of any 
 particular cell, but the nature of the cells as a whole is usually 
 obvious at a glance. 
 
236 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 Red corpuscles occur frequently, especially in tuberculous and 
 malignant exudates, which may be definitely haemorrhagic. It is 
 necessary to distinguish corpuscles belonging to the exudate, 
 which are intimately mixed with it, from those derived from the 
 puncture, in which case the blood is most marked at the beginning 
 or end of the flow. 
 
 Endothelial cells are very important, and it is necessary to be 
 able to recognise them at once. In certain passive exudates 
 (due to cardiac and renal disease) these cells occur as large flat 
 plates, exactly as if the endothelial coat had been scraped ofi" the 
 pleura (Plate X., Fig. 2). They are then seen to be much larger 
 than the largest of leucocytes, and to have a diameter three or four 
 times that of a red corpuscle. Each has a nucleus (sometimes 
 more) which does not usually stain very deeply, protoplasm which 
 stains more faintly still, and one or more nucleoli which stain 
 very deeply in wet preparations, less so in dry ones. 
 
 These cells are often grouped into" placards" (Plate X., Fig. 2), 
 the edges of adjoining cells fitting into one another like those of the 
 counties on a map. The groups of cells thus formed are always 
 flat, and careful focussing up and down shows that they consist 
 of a single layer of cells — an important fact, as it distinguishes 
 them from masses of cells of a malignant growth. 
 
 Endothelial cells are very phagocytic, and ingest bacteria, red 
 corpuscles (Plate X., Fig. 3), leucocytes (Plate IX., Fig. 2), etc. 
 They often undergo fatty degeneration (Plate X., Fig. i) or 
 general degeneration, shown by their very faint staining ; complete 
 solution of the protoplasm may occur, and the nucleus be set free. 
 It may then be mistaken for a lymphocyte. 
 
 Where inflammation takes place in a serous membrane the 
 first thing that happens is that the endothelial cells are set free, 
 so that they are always found with the leucocytes in the early 
 stages of pleurisy or peritonitis. If the inflammation is severe 
 they are destroyed, and the fluid at a later date does not contain 
 them. If the inflammation is less intense they are stimulated to 
 growth, and the young proliferating forms are often very similar 
 to the large lymphocytes and hyaline leucocytes. They vary 
 greatly in size, forming a continuous series between a cell as large 
 as the large lymphocyte to one as large as the plates described 
 above. They are round or oval, not mutually adapted, as in the 
 endothelial cells which have desquamated in passive exudates ; 
 sometimes two hemispherical cells may be found in apposition 
 
CYTO-DIAGNOSIS 237 
 
 (Plate X., Fig. i). The smaller {i.e. ^ younger) the cell, the smaller 
 is the ring of protoplasm in proportion to the size of the nucleus, 
 and the more deeply does it stain. Cells of the type described 
 above will be referred to as '' active " endothelial cells, in contra- 
 distinction to the " passive " plaques of desquamated endothelium. 
 Malignant Cells. — These cannot be distinguished with certainty 
 from some types of endothelial cells— at least, I must confess 
 myself unable to do so. Cells in mitosis are, of course, very sug- 
 gestive, but very rare, and there is no reason why they should not 
 occur in ordinary active endothelium. But malignant cells may 
 occur grouped in a characteristic way (p. 239). 
 
 Pleuritic Effusions. 
 
 It is in these that cyto-diagnosis is of chief value, and its results 
 most trustworthy. A diagnosis based on the subsequent rules 
 will rarely be found erroneous. 
 
 Tuberculous Pleurisy. — Two forms are to be recognised : 
 the primary, the so-called idiopathic form, in which the prognosis 
 is good as regards immediate recovery, but which indicates a 
 great probability that the patient will subsequently become 
 phthisical ; and the secondary, which is due to the extension of a 
 tuberculous lesion to the surface of the lung, and is probably due 
 to tubercle plus mild sepsis. 
 
 Primary Tuber cidotis Pleurisy. — The fluid is fairly clear, yet 
 yields numerous cells on centrifugalization. It usually clots 
 spontaneously. 
 
 The cells are almost all lymphocytes, and there are some red 
 blood-corpuscles (Plate IX., Fig. i). There may also be a 
 few large endothelial cells, flat plates with a well-marked nucleus, 
 and often one or more nucleoli ; their characters will be described 
 more fully subsequently. They are cells which have been desqua- 
 mated from the pleura, and play no part in the pathological process. 
 
 In cases examined at an early stage there may be a few poly- 
 nuclear cells — up to 15 per cent. As the case progresses, these 
 become fewer and fewer, and after the first week only isolated 
 specimens can be seen. The diagnosis may be clinched in some 
 of these cases by the demonstration of the tubercle bacillus. 
 
 Secondary Tuhercidous Pleurisy. — In this case the lymphocytes are 
 mixed with polynuclear leucocytes in approximately equal proportions ; 
 this is a reason for thinking that there is a septic element super- 
 added to the tuberculous one. As the case progresses, the septic 
 
238 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 organisms may gain entrance in larger numbers, and the case 
 would become indistinguishable from an ordinary empyema unless 
 a cytological examination had been made early; or, and this is 
 more usual, the polynuclears may gradually disappear, and the 
 case become a simple tuberculous one. 
 
 In tuberculous pyopneumothorax the polynuclears greatly pre- 
 dominate, but lymphocytes are usually present in fair numbers. 
 The lesion is due to the bursting of a vomica or abscess that is 
 almost necessarily septic. 
 
 "Septic" Exudates {i.e.^ those due to the pneumococcus, 
 streptococcus, gonococcus, and similar pyogenic bacteria). — The 
 characteristic cell is the polynuclear leucocyte. In the early 
 stages the films show these cells in large numbers, and they are 
 mixed with red corpuscles and with endothelial cells of active 
 type (Plate IX., Fig. 2). The pathogenic organism may be 
 distinguished either in films or in cultures. The process may 
 evolve on one of two lines, and in either case the cytology is 
 fairly characteristic. 
 
 {a) The process is mild, and recovery takes place ; this is most 
 likely to occur when the inflammation is due to the gonococcus 
 (in joints especially) or to the pneumococcus in a strong subject. 
 The polynuclears and endothelial cells become more and more 
 scanty, and lymphocytes make their appearance in increasing 
 numbers. The discovery of these cells in a septic exudate is a 
 good sign ; the discovery of the pathogenic organism mainly or 
 entirely within the cells is another. 
 
 (h) The process may pass on to suppuration. In this case the 
 endothelial cells become less and less abundant, and the poly- 
 nuclears become more numerous and undergo the various forms 
 of degeneration described above (Plate IX., Fig. 3). 
 
 Cryptogenic Pleurisy, possibly due to True Rheumatism. 
 — In this case the predominating cell is the active endothelial cell 
 in various stages of fatty degeneration, and in addition there is a 
 comparatively small number of all the leucocytes in approxi- 
 mately the same proportions as in the blood, and a few red 
 corpuscles. The exudate is sterile. These appearances are 
 found in the very rare cases of true rheumatic pleurisy, and may 
 be regarded as a good sign, in that they do not indicate a tuber- 
 culous or septic origin (Plate X., Fig. i). 
 
 Pleuritic Exudates due to Malignant Disease. — The 
 appearances vary, and a definite diagnosis cannot always be 
 
CYTO-DIAGNOSIS 239 
 
 made. There is no criterion (other than certain mitoses, which, 
 as far as I know, have never been made out in an exudate) 
 by which an isolated mahgnant cell can be distinguished from an 
 active endothelial cell. In some cases, however, the masses qL 
 cells, which can be seen to be solid and several cells thick/'' occur 
 in the exudate mixed with red corpuscles and a few leucocytes. 
 These cells are variable in size, usually stain deeply, and often 
 have a well-marked nucleolus. In the figure shown (which comes 
 from the ascitic fluid in a case of carcinoma of the ovary) the 
 resemblance to an alveolus of carcinoma as seen in a section is 
 very distinct (Fig. 53). 
 
 Fig. 53. — Malignant Masses in Ascitic Fluid, 
 
 In other cases, and much more frequently, these masses are 
 absent, and their place is taken by large endothelial cells (often 
 many times larger than a red corpuscle), which can be seen in a 
 wet preparation to be in various stages of fatty and other forms 
 of degeneration, and which in dry preparations are found to be 
 extensively vacuolated (Plate X., Fig. 3), and often contain 
 ingested red corpuscles. These may be arranged in masses, are 
 mixed with red corpuscles, and perhaps a leucocyte or two. 
 
 Some writers consider these cells to be growth-cells, but on 
 what grounds I do not know. When they form masses they are 
 always one cell thick, never solid alveoli ; and, more conclusive, 
 
 ♦ As shown by focussing up and down whilst examining a wet specimen, so 
 as to obtain a series of " optical sections." 
 
240 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 when an opportunity is obtained of examining the cells of the 
 growth post-mortem, they are often absolutely different from 
 those found in the exudate during life. 
 
 In yet other cases the endothelial cells are of the ordinary 
 passive type, but a suspicion of the nature of the growth may be 
 obtained from the number of red corpuscles present. 
 
 " Mechanical " Exudates (i.e., those due to cardiac disease, 
 pulmonary congestion, or renal disease). — The deposit from the 
 exudate is usually very scanty, and consists of large flat masses 
 of passive endothelial cells, there being often many cells in 
 one large plate ; their outlines may be indistinct. There is 
 usually nothing else, but there may be a few red corpuscles or 
 leucocytes. 
 
 Pleurisy Secondary to Infarcts. — Endothelial cells mixed 
 with much blood and with many polynuclear leucocytes have 
 been described, but I have no personal experience of the condition. 
 
 Peritoneal Exudates. 
 
 These are very equivocal, and often difficult or impossible to 
 interpret. The ultimate conditions leading to the production of 
 the cells are doubtless the same in the peritoneum as in the pleura, 
 but here the fluid is in close proximity to the intestine, and liable 
 to constant mild infective processes. These call forth a poly- 
 nuclear leucocytosis, which is very common in ascitic fluid, and 
 devoid of the significance which it has in the pleura. 
 
 Tuberculous Peritonitis may be accompanied by a pure 
 lymphocytosis, the cells becoming extraordinarily abundant, so 
 that the fluid may be turbid, or there may be polynuclears in a 
 practically pure state. I do not think the condition can be diag- 
 nosed unless tubercle bacilli are found. 
 
 Septic Peritonitis. — The cells are all polynuclears, except in 
 the early stages, in which a few endothelial cells and red corpuscles 
 may be found. The diagnosis is to be made by the discovery of 
 the organism, which is usually easy. 
 
 Mechanical Ascites {i.e., that due to cirrhosis of the liver, 
 renal disease, cardiac disease, etc.). — Here endothelial cells, often 
 in masses, are almost always present, and sometimes practically 
 unmixed with other cells. But chronic inflammation of the 
 peritoneum, with or without mild sepsis, is frequently present, 
 and polynuclears and lymphocytes frequently occur. 
 
C\TO-DIAGNOSIS 24I 
 
 Malignant Disease. — Here the large vacuolated endothelial 
 cells shown on Plate X., Fig. 3, may occur, and are extremely 
 suggestive, though they cannot be taken as definite proof. Rarely 
 you may find definite malignant masses, which, of course, settles 
 the diagnosis. In other cases there may be numerous polynuclear 
 cells, and in yet others mostly lymphocytes. The presence of an 
 abundance of red cells is suggestive, provided you can be sure it 
 does not come from the puncture, in which case it will be most 
 abundant at the beginning of the flow. It will be apparent that 
 the diagnosis of malignancy cannot be made in the majority of 
 cases by the cytology of the ascitic fluid. 
 
 The Meninges. 
 
 Here the technique is somewhat different. Clotting is not so 
 likely to occur, and when it does so is much slower, so that if the 
 fluid can be examined in any reasonable time there is no necessity 
 to break up the clot. This is fortunate, since the number of 
 cells present is of importance, and they cannot be counted in a 
 specimen which has coagulated. My own method is to count the 
 cells directly, without concentration and without dilution, in a 
 Thoma-Zeiss counting-chamber, by the method used for the 
 leucocytes and described on p. 193. If the specimen has had time 
 to sediment, shake it thoroughly ; then place a loopful or two on 
 the counting-chamber, cover it, getting Newton's rings, and allow 
 to settle. Then arrange the microscope so that the diameter of 
 the field is equal to that of eight small squares, and proceed to 
 count the leucocytes on forty or eighty fields ; in the former case 
 the result multiplied by two gives the number of cells per cubic 
 millimetre, whilst if eighty are counted the number is given 
 direct, no calculation being necessary. (There is no dilution of 
 the fluid, and you have counted the actual number in ^ or i cubic 
 millimetre.) The only difiiculty arises if red corpuscles are 
 present ; they may be distinguished by being less granular and 
 less refractile than the leucocytes, and are not to be counted. 
 
 The following rules may be taken as approximately correct for 
 the numbers of cells met with in various conditions. In health 
 there may be none, and never more than single figures per cubic 
 millimetre : the average is perhaps one or two. In " aseptic " 
 meningitis — i.e., that due to syphilis, or that which occurs in tabes, 
 general paralysis, some forms of herpes, and in almost any organic 
 lesion involving the meninges — the number per cubic millimetre 
 
 16 
 
242 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 is expressed in two or three figures, and usually ranges between 
 50 and 200. In tuberculous meningitis the numbers are higher, and 
 1,000 is perhaps the average, but it may go much higher, and in 
 ** septic " meningitis^ including cevehvo -spinal meningitis, the numbers 
 are very large, often running into tens or even hundreds of 
 thousands. 
 
 Having counted the leucocytes, proceed to centrifugalize the 
 fluid, and examine films from the deposit by the wet or dry method. 
 The former shows the cells more clearly, and is to be preferred 
 when a cytological examination only is required, as in the diagnosis 
 of tabes or general paralysis. Where bacteria are to be looked 
 for, dried films should be made and stained by Jenner's method, 
 or fixed with perchloride and stained by thionin. Then proceed 
 with the chemical examination of the fluid already described. 
 
 Normal fluid occurs in any nervous disease not attended by an 
 organic lesion of the meninges : deep cerebral tumours, hysteria, 
 deep cerebral haemorrhages, peripheral neuritis, epilepsy, syringo- 
 myelia, etc. 
 
 In cerebral tumour the fluid may be under excessive pressure, so 
 that it squirts out of the needle ; in such cases there may be great 
 relief to the headache after the withdrawal of a considerable 
 amount of fluid. With a cortical tumour there is usually slight 
 lymphocytosis. According to some writers, the pressure is 
 moderately raised in epilepsy. 
 
 Aseptic meningitis, using the term to indicate that there are no 
 cultivable organisms present, occurs in syphilis, tabes, general 
 paralysis, superficial gummata and other tumours, insular sclerosis, 
 chronic alcoholic meningitis, hypertrophic pachymeningitis, acute softening, 
 some cases of herpes, etc. In these cases we may expect to 
 find a hundred or two leucocytes — practically all of which are 
 lymphocytes — per cubic millimetre. In addition there is usually 
 a slight excess of albumin, and sugar is present, though some- 
 times reduced in amount. 
 
 A slight lymphocytosis, therefore, does not in itself give a clue 
 to the diagnosis unless it rests between two conditions, one of 
 which causes lymphocytosis, whilst the other does not. Thus, if 
 the diagnosis is either insular sclerosis or hysteria, the presence 
 of a moderate leucocytosis tells strongly in favour of the former. 
 Similarly in the differential diagnosis between tabes and peripheral 
 neuritis, and between general paralysis and most of the diseases 
 which it simulates. It is to be noted that excess of lymphocytes 
 
CYTO-DIAGNOSIS 243 
 
 in the cerebro-spinal fluid is a very early and a very constant finding 
 in these affections, and often occurs long before the diagnosis can 
 be made by ordinary clinical methods. Its absence is most im- 
 portant as a negative test ; its presence is only equally important . 
 if the diagnosis certainly lies between a disease in which this sign 
 is present and one in which it is absent. 
 
 Syphilis. — In syphilis without involvement of the meninges 
 the cerebro-spinal fluid remains normal, but with the slightest 
 involvement of these structures a lymphocytosis occurs. 
 
 Tuberculous Meningitis has been described already. The 
 lymphocytes may be so numerous as to make the fluid very 
 slightly turbid as compared with pure water, and may number 
 10,000 per cubic millimetre. There is a slight excess of 
 albumin, no sugar, and sometimes albumose is present. Occa- 
 sionally the films show a few polynuclear cells, but they are never 
 numerous. 
 
 HEMORRHAGE. — With a deep cerebral haemorrhage the fluid is 
 usually clear for two or three days, and then tinged with blood and 
 blood pigment ; the time necessary for this to occur depends on 
 the depth of the haemorrhage from the surface or from the 
 cerebral ventricles. 
 
 When you find blood in the cerebro-spinal fluid, make sure that 
 it does not come from the parietes. If this is the case, it will be 
 most abundant or limited to the commencement of the flow, and 
 if much is present the fluid will coagulate. If it is due to a lesion 
 it will be intimately mixed with the fluid, and will not coagulate. 
 After blood has been present in the cerebro-spinal fluid for about 
 two days, part of the haemoglobin is converted into a yellow 
 pigment ; if, therefore, after centrifugalization the supernatant 
 fluid is yellow, it indicates that the blood was actually present in 
 the cerebro-spinal fluid whilst in the body, and that the haemor- 
 rhage had occurred two days or more previously. If the super- 
 natant fluid is colourless, the blood came from the puncture, or 
 had only been recently effused. 
 
 If, as sometimes happens, you can only get a few drops of fluid 
 from a lumbar puncture, it is not wise to attach any importance 
 to the presence of a moderate amount of blood. The needle 
 always picks up some corpuscles in its passage through the 
 parietes, and these may be quite obvious if not diluted with a 
 considerable amount of fluid. 
 
 Large endothelial cells containing numerous red corpuscles 
 
 16 — 2 
 
244 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 (similar cells to those shown on Plate X., Fig. 3) may appear in 
 the fluid after a cerebral haemorrhage. They do not make their 
 appearance until three or four days after the blood has been 
 poured out. 
 
 H(smovrhage into the meningeal cavities or into the ventricles is, of 
 course, accompanied by blood in the cerebro-spinal fluid. This 
 may be found in cases of fracture of the skull, especially of the 
 base, or of the spinal column, or in contusion of the brain, and it 
 is worth noting that in some cases there has been great reUef of 
 the symptoms after the fluid has been drawn off". Lumbar 
 puncture should always be remembered as a means of diagnosis 
 in patients found unconscious. 
 
 "Septic" Meningitis — i.e.^ that due to the meningococcus, 
 pneumococcus, streptococcus, typhoid bacillus, etc. — is character- 
 ized by the presence of large numbers of polynuclear cells in the 
 fluid (see p. 133). In all cases the numbers tend to be more 
 numerous than in tuberculous meningitis, and may run into 
 hundreds of thousands per cubic millimetre, in which case the 
 fluid resembles watery pus. 
 
 The importance of this fact is very great, and the discovery of 
 polynuclear leucocytoses has led to the discovery that mild cases 
 of this affection occur which are hardly diagnosable clinically, and 
 which may be completely cured. According to several French 
 authorities, the first indication of commencing cure is the appear- 
 ance of lymphocytes, which must therefore be looked on as a 
 good sign ; after a time the polynuclears become less and less 
 numerous, and finally the lymphocytes alone remain, and may 
 persist for some time after cure has taken place. I have watched 
 several septic cases recover, and have seen this occur two or 
 three times. The appearance of lymphocytes in an acute septic 
 meningitis may therefore justify a good prognosis. 
 
APPENDIX 
 
 The following tables may be useful if stains, etc., have to be 
 prepared when the metric weights and measures are not at hand. 
 The equivalents given are not absolutely accurate, but are 
 sufficiently near for most purposes : 
 
 I Litre - - - - 35I fluid ounces. 
 
 100 c.c. - - - - 3! fluid ounces. 
 
 I Gramme - - - i5'43 grains. 
 
 I Fluid ounce - - - 28*4 c.c. 
 
 I Fluid drachm - - y^ c.c. 
 
 I Grain - - - - 0*064 gramme. 
 
 To convert grammes per litre into grains per ounce, multiply by 
 the factor ^^. Thus, 10 grammes per litre = 10 x ^^^ = 4-4 grains 
 per ounce. 
 
 To convert cubic centimetres per litre into minims per ounce, 
 multiply by the factor |f. Thus, 25 c.c. per litre = 25 x ^f = 12 
 minims per ounce. (A close approximation is given by multiplying 
 
 To convert degrees Centigrade into degrees Fahrenheit, multiply 
 by the factor | and add 32. 
 
 II 
 
 Neisser's Method of Staining the Diphtheria Bacillus. 
 
 The following method has been placed in the Appendix, as 
 there is some doubt as to its value. It is a method by which the 
 so-called polar bodies are stained with methylene blue, while the 
 rest of the organism is coloured a faint yellowish-brown with 
 Bismarck brown. 
 
 Cultures should be made on blood-serum, and should not be less 
 
 24s 
 
246 CLINICAL BACTERIOLOGY AND HEMATOLOGY 
 
 than nine nor more than twenty -four hours old. Films are spread 
 in the ordinary way and stained for half a minute in — 
 
 Methylene blue . . . i gramme. 
 
 Alcohol (96 per cent.) - - 20 c.c. 
 
 Glacial acetic acid - - - 50 c.c. 
 
 Water ----- g^o c.c. 
 
 They are then washed and treated for half a minute with — 
 
 Bismarck brown . - . 5 grammes. 
 
 Water ----- 1000 c.c. 
 
 The polar bodies are small spheres which are contained in the 
 bacilli, there being usually two in each bacillus, one at each end. 
 In a film specimen of the true diphtheria bacillus stained in this 
 way they appear as very minute dark blue or black dots, which 
 may easily be mistaken for cocci ; the bodies of the bacilli are 
 often almost invisible. According to some authorities, the 
 presence of these granules in young cultures of bacilli which 
 present the morphological characters of the diphtheria bacillus is 
 proof of their virulence, whilst their absence proves the cultures 
 to be of the non-virulent "pseudo-diphtheria" bacillus. The 
 method can also be applied to films made directly from the swabs, 
 and recent researches seem to prove that the results thus obtained 
 are of considerable diagnostic value. 
 
 If this method be adopted the films must be very carefully 
 searched, as it often happens that characteristically stained bacilli 
 may be seen in one part of the field, while they are entirely absent 
 elsewhere. This is especially true of films made direct from the 
 swabs. 
 
 The author is inclined to attach very considerable importance 
 to a positive result obtained with this method of staining, but 
 would not consider a negative result as indicating the absence of 
 the diphtheria bacillus. 
 
 Ill 
 
 Leishman's Method of Staining the Spirochete of 
 Syphilis. 
 
 I have seen better specimens of the spirochaete obtained by 
 this method than by any other, but rny own results have been less 
 satisfactory.. Prepare films in thp, usual manner, allow them to 
 
APPENDIX 247 
 
 dry, and fix by means of methyl alcohol for five minutes ; then 
 pour off the spirit and allow the films to dry again. Next spread 
 a little fresh blood-serum (obtained from a Wright's pipette in the 
 usual way) in a thin film over the specimen, using the end ^f 
 a slide as a spreader. When dry, flood the slide (or cover-glass) 
 with Leishman's stain diluted with an equal volume of distilled 
 water. Allow it to act for one hour, wash, blot, and dry. 
 
 IV 
 
 Demonstration of Tubercle Bacilli in Clots from 
 Pleuritic Fluid, etc — Improved Method. 
 
 This method yields better results than the simple digestion of 
 the clot previously referred to. Remove the clot from the fluid 
 and place it on a large filter-paper on a funnel, so that the fluid 
 can drain away. When it has shrunk to a small size remove it 
 and place it in a large volume of water (not necessarily distilled), 
 and keep in gentle movement for a few minutes. Repeat the 
 process two or three times, so as to wash away all haemoglobin, 
 soluble proteid, etc., from the clot. Place the latter in a clean 
 (preferably new) test-tube, add 4 to 5 c.c. of water, a drop of 
 dilute (i to 10) HCl, and a pinch of pepsin, shaking until the 
 latter is dissolved. Keep it in the incubator until the clot is com- 
 pletely dissolved, adding a crystal of thymol to prevent excessive 
 bacterial growth. Then shake the fluid, put it in a clean centri- 
 fugalizing tube, and centrifugalize thoroughly. Prepare films 
 from the exudate and stain in the usual way, taking great care to 
 avoid over-decolorization j one or two dips in 2J per cent, sul- 
 phuric acid will usually suffice. 
 
 The clot from a pint or more of fluid can be treated in this 
 way ; the more the material taken, the greater the chance of 
 success. 
 
 OF 
 
 P. MUs Sun 6. 
 Mmm. w "' 
 
INDEX 
 
 Abscess, bacteriology of, 102 
 
 cold, 105 
 
 collection of pus from, 102 
 
 leucocytosis in, 223 
 
 of liver, 219 
 Achorion Schonleinii, 99 
 Acid-fast bacilli, 61 
 Acid, hydrochloric, test for, 116 
 
 lactic, test for, 117 
 
 stains, 203 
 Actinomycosis, 65 
 Adamson's staining method, 97 
 Agar, 14 
 Anaemia, 179, 191 
 
 in infancy, 221 
 
 pernicious, 217 
 
 secondary, 217 
 
 splenic, 220 
 
 Von Jaksch's, 221 
 Anaerobic bacteria, 47 
 Angina, 106 
 
 scarlatinal, 109 
 
 simple, 106 
 
 syphilitic, no 
 
 Vincent's, 107 
 Aniline gentian violet, 30 
 Ankylostomiasis, 212 
 Anthrax, 54 
 Antitoxin, diphtheria, 37 
 
 tetanus, 45 
 Antrum, suppuration of, in 
 Aortic lesions, blood in, 231 
 Appendicitis, 212, 229 
 Arthritis, bacteriology of, 126 
 
 gonorrhoeal, 84, 127 
 
 tuberculous, 127 
 Ascites, cytology of, 240 
 Asthma, blood in, 230, 212 
 
 sputum in, 114 
 
 Bacilluria, 121 
 Bacillus, acid-fast, 61 
 
 of anthrax, 54 
 in blood, 139 
 
 of Boas and Oppler, 115 
 
 Bacillus, "bottle," 100 
 
 coli, 69, 105 
 
 in urine, 119 
 
 comma, 91 
 
 of diphtheria, 40, 42 
 
 Ducrey's, 94 
 
 fusiformis, 108 
 
 geniculatus, 115 
 
 of glanders, 68, 105 
 
 Hoffmann's, 44 
 
 of influenza, 52 
 
 of Koch and Weeks, 112 
 
 of leprosy, 65 
 
 mallei, 68, 105 
 
 of Morax and Axenfeld, 112 
 
 Pfeiffer's, 52 
 
 of plague, 93 
 
 pyocyaneus, 102, 104, 105 
 
 of smegma, 61 
 
 of soft sore, 94 
 
 of tetanus, 47 
 
 of tubercle, 60. See also Tubercle 
 
 of typhoid fever, 69, 105 
 in blood, 69 
 in urine, 119 
 
 vaginae, 88 
 
 xerosis, 113 
 Bacteriuria, 121 
 Basic stains, 203 
 Basophile cells, 208 
 Bilharzia disease, blood in, 212 
 Bismarck brown, 246 
 Blood, bacteriology of, 138 
 
 collection of, for serum, 55 
 
 crisis, 215 
 
 cultures, 69, 138 
 
 -films, fixation of, 202 
 preparation of, 198 
 staining of, 203 
 
 opsonic index of, 148 
 
 platelets, 209 
 
 -serum culture medium, 15 
 Boils, 104 
 
 Borax methylene blue, 29 
 Bottle bacillus, 100 
 
 248 
 
INDEX 249 
 
 Bottles, sterilization of, 7 
 Bouillon. See Broth 
 Bronchitis, 230 
 Broncho-pneumonia, 230 
 Broth, preparation of, 10 
 Bubo in plague, 93 
 in soft sore, 95 
 Bulloch, 154 
 
 Capsules of pneumococci, 51 
 
 Wright's blood, 33 
 Carbol fuchsin, 29 
 
 gentian violet, 30 
 
 thionin, 30 
 Carcinoma ventriculi, blood in, 228 
 Catarrhalis, micrococcus, 53, 54 
 Cathcart's microtome, 163 
 Cells, alveolar, 114 
 
 bronchial, 114 
 
 endothelial, 236 
 
 malignant, 237 
 
 squamous, 114 
 Cerebral tumour, lumbar puncture 
 in, 242 
 cerebro-spinal fluid in, 242 
 Cerebro-spinal fluid, 132 
 
 cytology of, 133, 241 
 
 fever, 134. 135 
 Cleaning blood pipettes, 195 
 
 cover-glasses and slides, 31 
 Chlorosis, 218 
 Cholera, 91 
 
 Clumping of bacteria, 71 
 Cirrhosis, blood in, 229 
 Colour-index, 192 
 Comma bacillus, 91 
 Conjunctiva, bacteriology of, 11 1 
 Cornet's forceps, 23 
 Cornil's myelocytes, 207 
 Corpuscles, red, enumeration of, 181 
 
 morphology of , 213 
 Cotton-wool, sterilization of, 8 
 Cover-glasses, cleaning, 31 
 Crescents in malaria, 143 
 Cultures from blood, 143 
 
 from cerebro-spinal fluid, 137 
 
 from pus, 103 
 
 from throat, 106 
 
 examination of, 22 
 
 incubation of, 18 
 
 inoculation of, 16 
 
 media, 9 
 
 stab and stroke, 13 
 
 sterilization of, 8 
 Cysticercus, eosinophilia in, 212 
 Cystitis, bacteriology of, 119 
 Cyto-diagnosis, 233 
 
 Degeneration, glycogenic, 210 
 of leucocytes, 235 
 of red corpuscles, 214 
 
 Dermatitis herpetiformis, blood in 
 
 232 
 Dewar's flask, 21 
 Differential leucocyte count, 209 
 Diphtheria, 37 
 Diplococcus meningitidis intracell«-- 
 
 laris, 136 
 Ducrey's bacillus, 94 
 Durham, 71 
 
 Ehrlich's myelocytes, 208 
 
 triacid stain, 204 
 Embedding in paraffin, 160, 166 
 Empyema, 124, 230 
 Endocarditis, blood in, 230 
 
 ulcerative, 50 
 Endothelial cells, 236 
 Enteric fever. See Typhoid fever 
 Enumeration of bacteria in water, 12 
 in vaccines, 156 
 
 of leucocytes, 193 
 
 of red corpuscles, 181 
 Eosin, 30, 203 
 Eosinophile leucocytes, 208, 211 
 
 myelocytes, 208, 213 
 Eosinophilia, 211 
 
 Epilepsy, cerebro-spinal fluid in, 242 
 Erysipelas, 50 
 Erythema multiforme, 232 
 Ewing, 225 
 
 Faeces, tubercle bacilli in, 63 
 Fauces, bacteriology of, 105 
 
 cells from, 114 
 Favus, 99 
 Fever, puerperal, 223, 231 
 
 relapsing, 140 
 
 scarlet, 226 
 Films, blood, fixation of, 202 
 preparation of, 198 
 staining of, 203 
 
 of exudates, 234 
 
 of sputum, 62 
 Fixation of blood-films, 202 
 
 of tissues, 160, 162 
 Flasks, sterilization of, 7 
 Follicular tonsillitis, 106 
 Folliculitis, 104 
 Foulerton, 88 
 
 Fragmentation of nucleus, 235 
 Freezing process, 160, 163 
 Fuchsin, carbol, 29 
 
 Gastric contents, examination of, 115 
 Gelatin, 13 
 
 liquefaction of, 12, 58 
 General paralysis, cerebro-spinal fluid 
 
 in, 241 
 Gentian violet, aniline, 30 
 German measles, 227 
 Giemsa's stain, 90 
 
250 
 
 INDEX 
 
 Glanders, 68 
 Glycogen reaction, 210 
 Gonococcus, 84 
 
 in blood, 140, 145 
 
 in conjunctiva, 112 
 
 in joints, 127 
 
 in meninges, 134 
 
 in urine, 120 
 Gowers' haemoglobinometer, 175 
 Gram's iodine, 30 
 
 method, 24, 172 
 Granular degeneration, 215 
 Griinbaum, 71 
 
 Haematoxylin, staining by, 165. 171 
 Haemocytometer, Thoma's, 182 
 Haemoglobin, estimation of, 175 
 Haemoglobinometer, Gowers', 175 
 
 Haldane's, 176 
 
 Oliver's, 177 
 Haemorrhage into meninges, 243 
 Hairs, ringv^^orm in, 95 
 Haldane's haemoglobinometer, 176 
 Hanging-drop preparation, 73 
 Hanot, 229 
 
 Herpes, cerebro-spinal fluid in, 241 
 Herxheimer, 90 
 Hevi^lett, 41 
 Hodgkin's disease, 220 
 Hoffmann's bacillus, 44 
 Hooping-cough, 227 
 Horder, 228 
 
 Hyaline leucocytes, large, 207 
 Hydatid disease, blood in, 212 
 Hydrochloric acid, test for, 116 
 Hysteria, cerebro-spinal fluid in, 242 
 
 Impetigo, 104 
 Incubation of cultures, 18 
 Incubators, 19 
 Infancy, anaemia in, 221 
 Influenza, 52 
 
 blood in, 227 
 Inoculation of cultures, 16 
 Iodine reaction, 210 
 
 Gram's, 30 
 
 Jenner's stain, 204 
 Joints, fluids from, 126 
 Justus's test, 180 
 
 Kerion, 99 
 
 Koch-Weeks bacillus, 112 
 
 Kiihnau, 147 
 
 Lactic acid, test for, 117 
 Large hyaline leucocytes, 207 
 Lead-poisoning, blood in, 215 
 Leishman's stain, 205 
 Leprosy, 65 
 Leptothrix, no 
 
 Leucocytes, enumeration of, in blood, 
 
 193 
 in cerebro-spinal fluid, 241 
 
 morphology of, 206 
 Leucocythaemia, 219 
 Leucocytosis, 196 
 Leucopenia, 198 
 
 Liquefaction of gelatin, 12, 58, 104 
 Liver, abscess of, 229 
 
 cirrhosis of, 229 
 
 examination of, 158 
 
 hydatid, blood in, 229 
 Locomotor ataxy. See Tabes 
 Loffler's blue, 29 
 Lumbar puncture, 128 
 Lupus, opsonic index in, 154 
 Lymphocytes, 206, 210 
 Lymphocytosis, 210 
 
 of cerebro- spinal fluid, 210, 242 
 Lymphosarcoma, 220 
 
 Macrocytes, 213 
 Malaria, blood in, 226 
 
 parasites of, 142 
 Malignant disease, blood in, 228 
 
 exudates, cells of, 237, 239 
 
 pustule, 54 
 Manson's methylene blue, 29 
 Mast cells, 208, 213 
 McCrae, 228 
 Measles, 226 
 
 German, 227 
 Media, culture, preparation of, g 
 Megaloblasts, 215 
 Megalocytes, 213 
 Megalosporon ectothrix, 99 
 
 endothrix, 98 
 Meningitis, 132, 134 
 
 tuberculous, 133, 137, 227 
 Meningococcus, 136 
 Methylene blue, 29 
 acid, 30 
 Loffler's, 29 
 Manson's, 29 
 Micrococcus catarrhalis, 53, 106 
 
 epidermidis, 26 
 
 ureae, 120 
 Microcytes, 213 
 Microscope, choice of, i 
 
 use of, 27 
 Microsporon Audouini, 97 
 
 furfur, loi 
 Microtomes, 163, 168 
 Milk, tubercle bacilli in, 63 
 Mitral lesions, blood in, 230 
 Mononuclear leucocytes, large, 207 
 Morax-Axenfeld bacillus, 112 
 Morbus caeruleus, blood in, 231 
 Mouth, bacteriology of, 105 
 Mycelium, 67 
 Myelocytes, 207, 211 
 
INDEX 
 
 251 
 
 Myelocytes, eosinophile, 208, 213^ 
 Myelogenous leucocythaemia, 219 
 
 Nails, ringworm of, 100 
 Needles, platinum, 16 
 Negative staining, 51 
 Neisser's method, 245 
 
 stain, 245 
 Nile blue, 90 
 Normoblasts, 215 
 Nose, bacteriology of, no 
 Nutrient gelatin, 11 
 
 Oidium albicans, 109 
 
 Oil-immersion lens, 28 
 
 Oliver's haemoglobinometer, 177 
 
 Opsonic index, 148 
 
 Osier, 228 
 
 Osteomyelitis, 105 
 
 Otitis media, 50 
 
 Ovarian tumours, blood in, 232 
 
 Paraffin process, 166 
 
 Parametritis, 231 
 
 Parasites, animal, 211 
 
 Pemphigus, 212, 232 
 
 Perimetritis, 231 
 
 Peritoneal fluid, cytology of, 240 
 
 Peritonitis, 50 
 
 blood in, 213, 229 
 
 tuberculous, 240 
 Petri dishes, sterilization of, 8 
 Pfeiffer's bacillus. See Influenza 
 Pipettes, 32 
 
 Wright's, 151 
 Plague, 93 
 Platelets, blood, 209 
 Plating, isolation of bacteria by, 12, 
 
 51, 103 
 Platinum needles, 16 
 Pleurisy, 50, 123 
 
 blood in, 229 
 
 cytology of, 237 
 
 due to infarcts, 240 
 
 malignant, 238 
 
 rheumatic, 238 
 
 septic, 238 
 
 tuberculous, 123, 237 
 Pneumococcus, arthritis due to, 126 
 
 in blood, 139 
 
 capsules of, 51 
 
 characters of, 51 
 
 cultivation of, 126 
 
 empyema due to, 124 
 
 lesions due to, 49 
 
 meningitis, 134 
 Pneumonia, blood in, 225 
 
 lobar, bacteriology of, 49 
 
 lobular, bacteriology of, 49 
 
 opsonic index in, 154 
 Poikilocytes, 214 
 
 Polar bodies, 246 
 
 staining, 93 
 Polychromatophil degeneration, 214 
 Polynuclear leucocytes, 207, 210 
 Post-mortem examinations, 158 
 
 Potato culture medium, 15 ^ 
 
 Proteus vulgaris, 119 
 Pseudo-leukaemia, 230 
 Psoriasis, loi 
 
 Puerperal fever, blood in, 231 
 Pulmonary anthrax, 54 
 Purpura haemorrhagica, 228 
 Pus, actinomycotic, 66 
 
 bacteriology of, 102 
 
 collection of, 102 
 
 pleuritic, 124 
 
 pneumococcic, 50 
 
 tuberculous, 63 
 Pustule, malignant, 54 
 Pyelitis, 119 
 Pyosalpinx, 231 
 
 Ray fungus, 66 
 
 Red corpuscles, enumeration of, 181 
 
 morphology of, 213 
 Relapsing fever, 140 
 Rheumatic pleurisy, 238 
 Rheumatism, 227 
 Ringworm, 95 
 
 staining of, 96, 97 
 Rocking microtome, 168 
 
 Sabouraud, 99 
 
 Saccharomyces albicans, 109 
 Sarcinae, 117 
 Scarlatinal angina, 109 
 Scarlet fever, blood in, 226 
 Schaudinn, 89 
 Seborrhoea, loi 
 Section cutting, 159 
 
 staining, 165, 170 
 Sepsis, 210, 222 
 Septicaemia, 50 
 
 Serous cavities, fluids from, 121 
 Skin diseases, eosinophilia in, 212, 
 
 232 
 Slides, cleaning, 31 
 Smegma bacillus, 61 
 Soft sore, 94 
 Spirillum of relapsing fever, 140 
 
 Vincent's, 109 
 Spirochaeta pallida, 89 
 
 refringens, 89 
 Spleen, examination of, 158 
 Splenic anaemia, 220 
 Spleno-medullary leucocythaemia, 219 
 Spores, II, 48, 55 
 Sputum, collection of, 61, 113 
 
 cytology of, 114 
 
 in influenza, 53 
 
 pneumococci, 51 
 
252 
 
 INDEX 
 
 Sputum, tuberculous, 60 
 Stab and stroke cultures, 13 
 Staining, negative, 51 
 Stains, 28 
 
 acid, 203 
 
 basic, 203 
 
 Ehrlich's, 204 
 
 Jenner's, 205 
 
 Leishman's, 205 
 
 triacid, 204 
 Staphylococci, 104 
 
 in blood, 139, 148 
 
 in joints, 127 
 
 in meningitis. 134 
 
 in pleura, 125 
 
 in urine, 120 
 Sterilization, 5 
 
 of cultures, 8 
 
 by dry heat, 6 
 
 intermittent, 11 
 
 of skin, 122, 130 
 
 by steam, 8 
 Stomach, carcinoma of, 115, 228 
 
 dilatation of, 117 
 Streptococci, 104 
 
 in blood, 139, 148 
 
 in joints, 127 
 
 in meningitis, 134 
 
 in pleura, 124 
 
 in urine, 120 
 Streptothricosis, 65 
 Suppuration. See Pus 
 Swabs, 38 
 Syphilis, 89 
 
 blood in, 228 
 
 cerebro-spinal fluid in, 241 
 
 Justus's test, 180 
 Syringomyelia, cerebro-spinal fluid 
 in, 242 
 
 Tabes, cerebro-spinal fluid in, 241 
 Test-tubes, sterilization of, 7, 8 
 Tetanus, 47 
 Thionin, 30 
 Throat swabs, 38 
 Thrush, log 
 Tinea versicolor, loi 
 Toison's fluid, 183 
 Tonsillitis, 106 
 Treponema pallidum, 89 
 Triacid stain, 204 
 Trichinosis, 212 
 Trichophyton ectothrix, 99 
 endothrix, 98 
 
 Tubal gestation, blood in, 232 
 Tubercle, bacillus of, 60 
 ■ in blood, 139 
 
 in conjunctiva, 113 
 
 in joints, 127 
 
 in meninges, 134, 137, 241 
 
 in pleura, 125 
 
 in pus, 105 
 
 in sputum, 60 
 Tuberculin, 64, 157 
 Tuberculosis, blood in, 227 
 
 opsonic index in, 154 
 Tuberculous meningitis, 134, 137,242 
 
 pleurisy, 125, 237 
 Tumours, malignant, blood in, 228 
 Turk, 227 
 Typhoid fever, 69 
 
 blood in, 213 
 
 Widal's reaction, 70, 72, 77 
 
 Ulcer of stomach, 229 
 Ulcerative endocarditis, 50, 104 
 Unna's bacillus, 94 
 
 bottle bacillus, 100 
 Uraemia, 134 
 Urine, bacteriology of, 118 
 
 gonococci in, 87 
 
 tubercle bacilli in, 62 
 Urticaria, 212 
 
 Vaccines, 104, 155 
 Vagmae, bacillus, 88 
 Vaginitis, 88 
 Valvular disease, 230 
 Vibrio of cholera, 91 
 Vincent's angina, 107 
 Vomit, examination of, 115 
 Von Jaksch's anaemia, 221 
 
 Water, examination of, 12 
 
 Weichselbaum, 136 
 
 Weights and measures, 245 
 
 Whitfield. loi 
 
 Whooping cough. See Hooping 
 
 cough 
 Widal's reaction, 70, 72, 77 
 Wool-sorter's disease, 54 
 Wright's blood capsules, 33 
 opsonic method, 149 
 pipettes, 151 
 
 Xerosis bacillus, 113 
 
 Yeasts, 116 
 
 H. K. Lewis, 136, Gower Street, London. IV.C. 
 
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