ft 
 
THE LIBRARY 
 
 OF 
 
 THE UNIVERSITY 
 OF CALIFORNIA 
 
 PRESENTED BY 
 
 PROF. CHARLES A. KOFOID AND 
 MRS. PRUDENCE W. KOFOID 
 
Hamilton's System of 
 
 LEGAL MEDICINE 
 
 [Complete in 2 Vols. 
 
 A Complete work of 
 Reference for Medical 
 and Legal Practitioners 
 
 By ALLAN flcLANE HAMILTON, M.D., 
 
 Consulting Physician to the Insane Asylums of New York City, 
 
 Assisted by LAWRENCE QODKIN, Esq., of the 
 New York Bar, and Others. 
 
 Its list of thirty contributors includes the names of the 
 most distinguished writers arid authorities upon Medical 
 Jurisprudence in America. As a book of reference, with 
 
 its 4000 citations 
 and cases, it will be 
 found an invaluable 
 help to medical men, 
 and to those of the 
 legal profession who 
 desire the aid of the 
 most advanced and 
 sound opinions of 
 practical students of 
 forensic medicine. 
 So much opprobri- 
 um has been attach- 
 mm ed to the word "ex- 
 pert," that it has 
 been the aim of the 
 Editor and his col- 
 leagues to give the 
 work a decided judicial and impartial tone, so that it may 
 be consulted with confidence by all as an authority of the 
 highest order. 
 
 Short articles upon legal subjects by distinguished 
 members of the American Bar are a marked feature of the 
 work. The cases cited are recent, and chiefly American, 
 and up to date in theory and practice. 
 
 THE WORK is complete in two large royal octavo 
 volumes of about seven hundred pages each, illustrated. 
 Fully Indexed by 3202 References. In cloth, per 
 volume, $5.50; full sheep, per volume, $6.50. 
 Sold by subscription. Sent carriage paid on receipt of price. 
 
 E, B. TREAT & CO,, Publishers, 
 241-243 West 23d Street, New York. 
 
AN ATLAS 
 
 OF THE 
 
 Bacteria Pathogenic in Man 
 
 WITH 
 
 DESCRIPTIONS OF THEIR MORPHOLOGY AND 
 MODES OF MICROSCOPIC EXAMINATION. 
 
 BY 
 
 SAMUEL G. SHATTOCK, F.E.C.S., 
 
 Joint Lecturer on Pathology and Bacteriology, St. Thomas' Medical School, 
 
 London ; Pathological Curator of the Museum of the 
 
 Royal College of Surgeons, London. 
 
 WITH AN INTRODUCTORY CHAPTER 
 ON 
 
 BACTERIOLOGY : 
 
 ITS PRACTICAL VALUE TO THE GENERAL PRACTITIONER. 
 
 BY 
 
 W. WAYNE BABCOCK, M.D,, 
 
 Pathologist to the Kensington Hospital for Women; Clinical Pathologist to the 
 
 Medical-Chirurgical Hospital; Demonstrator of Pathology and Bacteriology 
 
 in the Medico-Chirurgical College of Philadelphia. 
 
 SIXTEEN FULL-PAGE COLORED PLATES. 
 
 E. B. TEEAT & CO., 
 
 241-243 West 23d Street, 
 
 NEW YORK, 
 
 1899. Price, 81. OO 
 
COPYRIGHT, 
 E. B. TREAT & CO. 
 
 1899. 
 
J. 
 
 
 Publisher's Note. 
 
 This Atlas was originally published in the "International 
 Medical Annual " in two sections, one in the 1898 issue, the other 
 in the 1899 issue. 
 
 The perfection of the plates illustrating the appearance, under 
 the microscope, of the Bacteria Pathogenic in the human subject 
 was of such a high degree as to evoke the admiration of those 
 familiar with Bacteriology. Then, too, the descriptive text was 
 succinct and practical, and written for the use of physicians desir- 
 ous of keeping up to date, but who have not the time nor inclina- 
 tion to master the elaborate text books on Bacteriology. 
 
 We have had requests that these articles be reprinted in book 
 form, thus giving a sufficiently complete work on the subject to 
 meet ordinary requirements. Having secured a number of sets of 
 the illustrative plates, we have decided to meet these requests. We 
 trust our effort to give the medical profession a small but first-class 
 work both as to authorship, illustrations, press work, etc. at a 
 very reasonable price will meet with a reception such as we believe 
 it to be entitled to. 
 
 The value of the original work has been much added to by the 
 publication of an introductory chapter on "Bacteriology; its Prac- 
 tical Value to the General Practitioner," by W. Wayne Babcock, 
 M.D., of Philadelphia. 
 
 M3517S5 
 
BACTERIOLOGY : 
 Its Practical Value to the General Practitioner. 
 
Bacteriology : Its Practical Value to the 
 General Practitioner. 
 
 BY W. WAYNE BABCOCK, M.D., 
 
 Pathologist to the Kensington Hospital for Women; Clinical Pathologist to the 
 Medico-Cbirurg'cal Hospital; Demonstrator of Pathology and Bacteriology 
 in the Medico- Chirurgical College, of Philadelphia. 
 
 To the general practitioner, bacteriology is offering a con- 
 stantly increasing field of usefulness. From its earliest days, this 
 science has suggested a theoretical basis for treatment, while it has 
 developed, especially during more recent years, numerous prod- 
 ucts of practical remedial value. For the most part it has not 
 been difficult for the physician to avail himself of these advances 
 in treatment. The methods founded upon theory have been par- 
 ticularly popular, and it has been only necessary for the micro- 
 organismal nature of an affection to gain credence in order to 
 have innumerable preparations of real or fancied antiseptic value 
 pressed into service. 
 
 From the earlier vague and theoretic means of combating 
 bacterial invasion, we are now emerging upon a more rational 
 therapeusis, founded upon laboratory investigation and endorsed 
 by clinical trial. Although preventive inoculations and the use of 
 toxins and antitoxins have been adopted with some reluctance, 
 their application is not difficult and their employment is now be- 
 coming general. Coincident with the recent advances in etiology, 
 prophylaxis and therapeutics, and scarcely less important, are the 
 strides made in bacteriologic diagnosis and prognosis. And yet, 
 despite the great value of these latter innovations, it is entirely 
 
 7 
 
g BACTERIOLOGY : 
 
 probable that bacteriology has been of far greater service to the 
 practitioner in the line of treatment than in diagnosis. The 
 scratch of the vaccine lancet or the thrust of the antitoxin needle 
 requires neither erudition nor great technical skill, while in- 
 dulgent manufacturers beg to supply the practitioner with more 
 convenient and refined products. On the other hand, unfortun- 
 ately, the methods of diagnosis have not only required laboratory 
 training but also laboratory apparatus. 
 
 These difficulties are being largely overcome in the medical 
 centers by the establishment of municipal laboratories, but in the 
 more remote districts the general practitioner is left, as usual, 
 self-dependent. There is, moreover, a prevalent impression that 
 the general practitioner requires but little knowledge of bacteri- 
 ology. This needs correction. The specialist is a man who, by 
 exceptional proficiency in a single branch, is exempted from a 
 thorough knowledge of general medicine. Serving in a single 
 field, he exacts service from his confreres in all others, including 
 that of skilled laboratory workers. The true position of the 
 general practitioner is, naturally, quite the reverse, and with the 
 knowledge that "diagnosis is treatment," bacteriology must be far 
 from the least of 'his many accomplishments. He may therefore 
 rejoice in the fact that some of the most important of bacteri- 
 ologic methods now require but simple apparatus, are capable of 
 rapid performance and demand no exceptional skill. Given a 
 good microscope, which may now be considered an essential part 
 of the practitioner's outfit, a very moderate additional expense 
 will provide the essential equipment for many of the very impor- 
 tant diagnostic tests. Indeed, it is now possible without any aid 
 
ITS PKACTICAL VALUE TO THE GENERAL PRACTITIONER. 9 
 
 from the microscope and with no more complicated apparatus 
 than a test tube, to determine the presence and activities of 
 typhoid bacilli in the body; and thus, by a procedure scarcely 
 more difficult than the test for sugar in the urine, diagnose the 
 existence of enteric fever. The single example may serve to il- 
 lustrate the error of a prevalent opinion that bacteriology is 
 hardly accessible to the practitioner as an aid in his daily work. 
 
 In this article I desire to mention a number of the practical 
 advances in bacteriology and to indicate how much of essential 
 value in the various fields they offer to the progressive physician. 
 Certain of these advances may never have a general use; others 
 are as yet very imperfectly developed ; improvements may be ex- 
 pected upon all ; yet we do our patients an injustice if we do not 
 avail ourselves of some of the present benefits. It is convenient 
 to group the bacteriologic advances under the general headings of 
 Etiology, Diagnosis, Prognosis and Treatment. In attempting 
 this, no excuse is offered for mentioning much that is trite to 
 many medical readers. 
 
 Etiology. On referring to the causal relations of bacteria to 
 morbid conditions, we find that there is a large number of dis- 
 eases for which micro-organisms may reasonably be claimed as 
 the inducing factors; a considerable number for which certain 
 bacteria have been described as the etiologic agents, and until 
 within a very recent period but a very moderate number of 
 which definite bacteria have been satisfactorily established as 
 casual. Under this latter class we now have such prominent 
 bacteria as Koch's bacillus of tuberculosis, Eberth's bacillus of 
 typhoid fever, Klebs-Loffler's bacillus of diphtheria, Koch's spiril- 
 lum of cholera, Neisser's diplococcus (gonococcus) of gonorrhea, 
 
10 BACTERIOLOGY : 
 
 Nicolaier's bacillus of tetanus, Obermeier's spirillum of relapsing 
 fever, and Kitasato's bacillus of plague. Scarcely less impor- 
 tant and also well authenticated is the bacillus anthracis of malig- 
 nant pustule, bacillus mallei of glanders, bacillus leprae of 
 leprosy and the streptothrix (?) (ray fungus) of actinomycosis, 
 and of madura foot. To these should be added the well-recog- 
 nized, micro-organismal causes of many septic and suppurative 
 conditions such as the varieties of staphylococci, so frequent in 
 abscesses; the streptococcus found in erysipelas; the bacillus 
 pyocyaneus of green pus ; the bacillus coli communis, frequent in 
 abscesses adjacent to the intestines ; the bacillus aerogenes eapsu- 
 latus, found in a usually fatal form of sepsis associated with 
 gaseous edema, etc. 
 
 Nearly all of these organisms have been proved to be the 
 specific causes of their respective diseases by conforming to the 
 well-known rules of Koch. That is, the organism is found to be 
 constantly present in the bodies of those affected by the disease, 
 may be grown in pure culture outside of the body, when properly 
 inoculated in lower animals it reproduces the disease, and may 
 again be recovered from the animal so infected. 
 
 There are also good reasons for accepting a number of other 
 organisms as specific. Recent investigations corroborate the 
 claims of Sanarelli that his bacillus icteroides is the cause of yel- 
 low fever. 
 
 The bacillus described by Canon and Pfieffer for influenza, 
 and by Canon and Pielicke for measles, are generally accepted. 
 The Lustgarten bacillus of syphilis has not been sustained, but 
 van Niessen claims to -have cultivated from syphilitic blood a 
 bacillus producing characteristic lesions in pigs and monkeys. 
 
ITS PRACTICAL VALUE TO THE GENERAL PRACTITIONER. 11 
 
 The claims made that the bacteria causing mumps, scarlatina 
 and whooping-cough have been isolated, do not, as yet, appear 
 conclusive; while the organisms of such notably infectious dis- 
 eases as typhus and small-pox await discovery. The better 
 knowledge of the bacteria producing the infections has been fruit- 
 ful in improving hygienic measures a subject too extensive to be 
 considered at this time. Not less important have been the re- 
 flections of etiologic advances in improvements of diagnosis, 
 prophylaxis and treatment. 
 
 Bacteriologic Diagnosis. Having ascertained that a micro- 
 organism is invariably found in the body in association with a 
 single disease and with that disease only, the diagnosis of the dis- 
 ease may readily hinge upon the determination of the presence 
 of the bacterium. The chief methods used to determine the char- 
 acter of u micro-organism, 'and thus diagnose the disease, depend 
 upon: (1) Peculiarities in form and arrangement; (2) peculiari- 
 ties of staining; (3) peculiarities of cultural growth; (4) pecul- 
 iarities of effects produced when introduced into the bodies of 
 certain animals; (5) peculiarities in behavior of cultures of the 
 micro-organism when brought in contact with certain fluids from 
 the diseased animals. 
 
 Certain of the higther forms of the vegetable parasites, es- 
 pecially the parasitic moulds and yeasts, are readily recognized by 
 their peculiarities of form -alone. For example, the sacchar- 
 omyces <albicans of thrush and the varieties of aspergilli found in 
 the external ear are readily determined by microscopically ex- 
 amining some of the suspected material in a drop of -an indiffer- 
 ent fluid; while an accurate diagnosis of the varieties of ring- 
 
12 BACTERIOLOGY : 
 
 worm, of favus, of tinea versicolor, may be obtained by removing 
 some of the affected epithelial scales and hairs and examining 
 under the microscope for their respective moulds, after treating 
 with alcohol and ether, and strong caustic potash to remove the 
 
 fat. 
 
 The similarity between different varieties of bacteria is so 
 great, however, and pathogenic micro-organisms are so frequent- 
 ly aped in shape and grouping by the benign varieties, that this 
 first method of diagnosis, as a rule, is merely corroborative to 
 staining and cultural methods. Thus the most noteworthy pe- 
 culiarity of the tubercle bacilli is their resistance to decolorizing 
 agents ; and yet it is also very important that the organisms ap- 
 pear as short rods, with rounded ends, a slight curve and a ten- 
 dency to be arranged in II, V or X like figures. The simple 
 method of diagnosing this bacillus by staining has proved of chief 
 value in tuberculosis of the respiratory tract, the presence of 
 tubercle bacilli in the sputum being practical proof of the ex- 
 istence of the disease. The same method of diagnosing tuber- 
 culosis in other parts of the body has given less satisfaction. In 
 tuberculosis of the genito-urinary tract, it is often difficult to find 
 the bacilli in stained preparations of urinary sediment, and the 
 bacilli are rarely seen in the pus from tuberculous abscesses. Al- 
 though sputum that has become putrid may continue to show the 
 characteristic bacilli, it is advisable to make preparations of fresh 
 fluids. Urine, especially, should be speedily centrifuged and ex- 
 amined, and it is probably an advantage to use a method which 
 obviates certain of the extraneous urinary substances. While the 
 smegma bacillus and the leprosy bacillus resemble the tubercle 
 
ITS PEACTICAL VALUE TO THE GENERAL PRACTITIONER. 13 
 
 bacillus in staining properties,, it is to be recalled that both of 
 the former bacilli stain more rapidly, decolorize more easily and 
 have different grouping tendencies than the latter. The failure 
 to find tubercle bacilli, of course, does not necessarily negative 
 the presence of the disease, nor may the number of bacilli found 
 bear any relation to the extent of the disease process. 
 
 Gonorrhea, 'at present, is most accurately diagnosed by stain- 
 ing. The organism, which is a rather large diplococcus with 
 flattened surfaces in opposition, and a tendency to be found in 
 groups in or upon the cells, is differentiated from most other 
 diplococci by failing to stain by Gram's method, although readily 
 coloring with the ordinary basic anilin dyes. In subacute or 
 chronic cases the gonococci, having invaded the deeper epithelial 
 layers, may be absent from the discharge except during exacerba- 
 tions. It has, therefore, been suggested that in gleets of obscure 
 nature the "Beer Test" be used ; the visit to Bacchus being fre- 
 quently rewarded by the return of the gonococcus to the dis- 
 charge. 
 
 The staining of cerebro-spinal fluid withdrawn by the lumbar 
 puncture has been a decided aid in diagnosing certain varieties of 
 cerebro-spinal inflammation. The diplococcus intracellularis 
 meningitidis of Weichselbaum, frequent in the epidemic and very 
 fatal form, resembles the gonococcus by its presence in the 
 leucocytes and by being decolorized by Gram's method. The 
 diplococcus of pneumonia has also been found in the fluid of 
 many of the fatal cases. 
 
 In the diagnosis of gastric carcinoma probably the most sug- 
 gestive addition to the stomach contents, apart from the presence 
 
^4 BACTERIOLOGY : 
 
 of detached bits of tumor, is the long immobile, club-shaped 
 Oppler-Boas bacillus. 
 
 The most important recent advance in the diagnosis of puer- 
 peral sepsis is the employment of bacteriologic methods to as- 
 certain the variety of the infecting organism. The fatal infec- 
 tions with the gas bacillus (bacillus aerogenes capsulatus of 
 Welch) and with the septicemia-producing streptococcus should 
 be carefully separated from the milder infections by the colon 
 bacillus and the staphylococcus and the usually localized 
 gonococcus invasions. A sterile glass tube may be inserted into 
 the uterine cavity, some of the lochia withdrawn into the tube, 
 and the tube ends sealed pending the examination of the contents. 
 
 In general surgery the value of staining the fluids from in- 
 fected foci, even during the operation, in order to determine the 
 necessary operative procedure, has not diminished, although the 
 method is not new. 
 
 Certain bacteria exhibit, under varying conditions, altera- 
 tions in their staining properties. Such bacteria may be more 
 distinctively stained after cultivation upon a particular medium. 
 This is so important with the diphtheria bacillus that the diag- 
 nosis is made by staining after cultivation upon Loffler blood- 
 serum mixture. The rapid growth, at incubator temperature. 
 of the irregularly rounded, elevated, porcelain white colonies 
 tending to rapidly coalesce into a diffuse smeary layer, is also 
 quite characteristic of this organism. Eisner, Kashida and Hiss 
 have devised special media 1 for developing characteristic cultures 
 
 1 Piorkowsky (Ber.klin. Woch., 7,99,) has recently recommended a medium 
 composed of normal urine containing 0.5* peptone and 8 3* gelatin. The medium Is 
 Inoculated from the stools, and incubated 20 hours The typhoid colonies appear aa 
 colorless spots of radiating threads, while the colon cultures are sharply defined, 
 round, yellowish colonies. 
 
ITS PEACTICAL VALUE TO THE GENERAL PRACTITIONER. 15 
 
 from the typhoid bacillus, but their practical diagnostic value can 
 hardly compare with the simple method of serum diagnosis. Cul- 
 tures, at present, are of chief value to the practitioner in diagnos- 
 ing diphtheria, and those who have not the advantage of a labora- 
 tory may procure the necessary media,, and with a little training 
 make their own examinations. In lieu of an incubator the inocu- 
 lated tube, in a protecting case, may be carried in an inner pocket, 
 the body supplying the necessary warmth. 
 
 Animal inoculations have proven useful in diagnosing genito- 
 urinary tuberculosis in cases where the bacilli were not shown by 
 staining. Some of the fresh urinary sediment (obtainable from a 
 single kidney by the ureteral catheter) is injected into the ab- 
 dominal wall of a guinea pig that is killed after five weeks and 
 examined for tuberculous lesions. Animal inoculations are also 
 of value in diagnosing the type of many of the infections ; while 
 the subdural inoculation, in a rabbit, of a bit of the spinal cord of 
 the affected animal remains the best method of ascertaining the 
 existence of rabies. 
 
 One of the greatest advances in bacteriologic diagnosis is that 
 dependent upon the tendency of the bacteria of >a given disease to 
 agglutinate, or collect in clumps, and to lose their motility when 
 brought in contact with the serum from a person affected by the 
 disease. Although this method is useful in determining the 
 variety of the bacteria, the presence of infective organisms in 
 drinking water, etc., its greatest value has been in diagnosing 
 disease. 
 
 Being present in over ninety-five per cent, of cases of ty- 
 phoid, it is the most useful diagnostic sign yet discovered. The 
 
16 BACTERIOLOGY : 
 
 method has not been as thoroughly developed in other diseases, 
 but has shown a value in aiding the identification of cases of 
 cholera, glanders, malta fever/ tuberculosis, leprosy, relapsing 
 fever and other diseases. Considering that the method is yet in 
 its infancy, the results obtained have been surprisingly accurate. 
 The test is performed with or without the microscope, the former 
 having the -advantage of quickness and greater accuracy. The 
 main difficulty that the practitioner will find with this method is 
 the difficulty of keeping on hand fresh cultures of the bacilli. The 
 advantages of emulsions of dead bacilli are readily apparent, but 
 thus far they have proven less reliable than the fresh cultures. 
 
 A final, simple and practical method of diagnosis is by the in- 
 jection of bacterial products into the affected animal. Tuber- 
 culin, a glycerin extract of tubercle bacilli, has proved to be a 
 most reliable agent for diagnosing tuberculosis. Unfortunately, 
 its tendency to exacerbate this affection has largely precluded its 
 use in the human family. Mallein has also proven successful in 
 diagnosing glanders in horses. 
 
 Prophylaxis and Treatment. Two main classes of bacterial 
 remedies have been developed, namely, those obtained directly 
 from the micro-organism and those indirectly obtained by the ac- 
 tion of bacterial products upon animals. To the first class be- 
 long the toxins -and vaccines ; to the latter class the antitoxic and 
 bactericidal serums. The former <have been of chief value as 
 prophylactic 'and immunizing agents, but, as yet, have not re- 
 vealed the curative powers of the latter class. Upon the other 
 hand, the antitoxins have more transient immunizing powers 
 than the toxins or vaccines. Protective inoculations or vaccin- 
 
ITS PRACTICAL VALUE TO THE GENERAL PRACTITIONER. 17 
 
 ations are now accessible to the practitioner for small-pox, an- 
 thrax, cholera, plague, hydrophobia, and for a number of diseases 
 of the lower animals as black leg (symptomatic anthrax), hog 
 cholera, ttc. 
 
 The curative effect of the bacterial products of the tubercle 
 bacillus devised by Koch have hardly proven commensurate with 
 their dangers, while the employment of Coley's mixed toxins of 
 prodigiosus and erysipelas for the relief of inoperable malignant 
 tumors has been followed by a cure only in occasional cases. .The 
 antitoxins have shown temporary but valuable immunizing 
 powers in diphtheria and tetanus, and the diphtheria antitoxin 
 has revealed marked curative powers much greater, indeed, than 
 that of tetanus. 
 
 The use of streptococcus antitoxin has been followed by good 
 results in certain cases of sepsis and even small-pox. The results 
 suggest the desirability of greater accuracy in diagnosing the 
 variety of the infecting organism as an aid to the picper treat- 
 ment. 
 
 With a prospect for the future of increased and simplified 
 diagnostic advances, and multiplied and more efficient curative 
 products, the advantage of a more thorough knowledge of bac- 
 teriology to the practitioner are not easily overestimated. 
 
AN ATLAS 
 
 OF THE 
 
 BACTERIA PATHOGENIC IN MAN. 
 
An Atlas of the Bacteria Pathogenic in 
 the Hitman Subject. 
 
 PART I. 
 
 BY SAMUEL G. SHATTOCK, F.R.C.S. 
 
 THE object of the accompanying plates is to present graph- 
 ically from original preparations all the chief bacteria which are 
 pathogenic in the human subject. 
 
 The originals of the drawings (in the Museum of the Royal 
 College of Surgeons, London) have been made under the au- 
 thor's supervision by Mr. G, T. Gwilliam, F.R.A.S., who, from 
 an astronomical experience, is a draughtsman of extreme accur- 
 acy, and they may be relied upon as absolutely faithful repre- 
 sentations of the objects themselves. 
 
 All the preparations have been drawn as viewed under a ~ 
 homogeneous immersion, Leitz, ocular No. 2, tube length 170 
 millimeters, giving a magnification of 680. It is to be noted, 
 however, that in order not to tax the eye, they have in all cases 
 been slightly enlarged in the drawing. The actual magnifica- 
 tion amounts to about 1,000. 
 
 Each illustration gives the chief forms selected from several 
 fields in order to bring out their variations, their differences in 
 size, or their grouping, etc., a result which will dispel the idea 
 of uniformity in these respects that is sometimes gained from 
 representations where such differences are studiously excluded. 
 
 All the drawings have been made from cultures in an active 
 
 21 
 
22 PATHOGENIC BACTERIA 
 
 stage of growth, in order that abnormal, degenerate forms might 
 be eliminated. The precise mode of preparation, the age of the 
 culture, and, when important, its original source, are given un- 
 der the several figures. 
 
 The fungi, which are pathogenic in the human subject, be- 
 long to the botanical subdivision of (1) Schizomycetes (^zVctf, 
 I split, KvHrjS, a fungus) Fission-fungi, or Bacteria; (2) Blas- 
 tomycetes (BXaffroZ, a bud) Budding-fungi, or yeasts; (3) Hy- 
 phomycetes ("TpoZ, a web), Hyphal-fungi, or moulds. 
 
 Strictly speaking, the study of bacteriology does not com- 
 prise that of yeasts and moulds, which are for this reason ex- 
 cluded from some systematic works devoted to bacteriology. 
 Much less could the consideration of animal micro -parasites find 
 a place in a strict system of bacteriology, although, owing to its 
 present small dimensions, this subject is included, and conven- 
 iently so, in at least one standard English work on " Bacter- 
 iology." 
 
 With the exception of thrush, all the illustrations given are 
 limited to bacteria proper or schizomycetes. 
 
 In regard to this classification of pathogenic fungi,* it will 
 be enough to say that bacteria are achlorophyllous vegetable or- 
 ganisms, characterized by a fissiparous method of reproduction, 
 though in a certain number there occurs a second or additional 
 method, viz., spore-formation. 
 
 Yeasts, whilst again they may multiply by spore-formation, 
 do so as an ordinary method by budding, i.e.) in place of the 
 cell becoming symmetrically partitioned into two or more ele- 
 
 * The absence of chlorophyll, which has commonly served as a basis for defining 
 the group of fungi, is not held by Sachs as sufficiently fundamental for such a sub- 
 division; he classes with achlorophyllous schizomycetes certain chlorophyllous 
 forms which multiply by a similar method, under the name of schizophyta, etc. 
 
OF THE HUMAN SUBJECT. 23 
 
 ments, there grow out one or more processes or buds, which, 
 after increasing to a certain size, may become abstricted off and 
 disconnected from the parent cell, or whilst acquiring an inde- 
 pendent cavity, may remain connected with it and generate 
 further elements by the same plan. The hyphomycetes occupy 
 a higher position than either of the foregoing groups, in that 
 they present distinct organs of fructification, and are capable, 
 moreover, of a proper sexual process of propagation. 
 
 Not that these subdivisions are more clearly defined than 
 are others in biology. 
 
 The branching filamentous forms, at times met with in the 
 tubercle and tetanus bacillus, make an approach toward the 
 mycelial productions of moulds; and in actinomyces and strep- 
 tothrix Madurae, a dense and branched mycelium is regularly 
 developed in artificial cultures. 
 
 The microscopic characters of bacteria are in scarcely any 
 case sufficient to allow of their identification. It is by the en- 
 semble of their cultural characters on different media, their chem- 
 ical products, the results of experimental inoculation upon ani- 
 mals, that identification is possible, and to these must be added 
 the effects produced upon bacteria by the action of specifically 
 immunized sera. 
 
 Accumulated observations have disclosed a bacterial king- 
 dom of such dimensions that to determine or identify any given 
 individual is a matter of increasing difficulty. The problem of 
 identification is reduced, however, within comparatively easy 
 limits when those organisms which are pathogenic are alone con- 
 sidered, and still more so when attention is restricted to those 
 that cause disease in the human subject. 
 
 The fundamental argument adopted by Darwin for the 
 
24 PATHOGENIC BACTERIA 
 
 theory of evolution, viz., the impossibility of defining species, 
 holds equally through microscopic and macroscopic forms of life. 
 Nevertheless, of the different methods of classifying bacteria, 
 those founded upon form are the only ones at present practica- 
 ble. Of such classifications there are almost as many as there 
 are authors. For all practical purposes, however, the patho- 
 genic bacteria are reducible to spheres and rods; the latter being 
 of the most various lengths, at times produced into long fila- 
 ments, which may be straight or spirally twisted, or, in a few 
 instances, branched. 
 
 As to the terms bacterium and bacillus, the former is now 
 obsolete; every straight, rod-shaped organism is a bacillus. If 
 the first is still employed, it is only in deference to pre-estab- 
 lished usage; the early distinction between the two, founded as 
 it was upon difference in length, is rendered valueless by the var- 
 iations in this respect presented by one and the same organism; 
 in the more highly pleomorphic examples, the shortest forms of 
 bacilli may be indistinguishable from spheres or cocci. The pro- 
 posal to name as "bacilli" straight, rod-like organisms which 
 form spores, and "bacteria" those that do not, has not been 
 followed, since there is nothing whatever in the terms indicative 
 of such a difference. Besides the straight rod or bacillus, the 
 other chief forms of pathogenic bacteria are the spherical, or 
 cocci; and the twisted rod or spirillum. 
 
 It may be observed, in passing, that micrococci are not in 
 all cases geometrically spherical. The faces of subdivision are 
 very frequently flattened so long as the resulting elements re- 
 main united, whatever disposition the grouping takes whether 
 in twos (diplococci), fours (tetracocci), eights (sarcinacocci), or 
 linear series (streptococci); it is not rare again to find the ele- 
 
OF THE HUMAN SUBJECT. 25 
 
 ments of a chain or streptococcus elongated in the direction of 
 its length, i.e., oval in place of spherical. 
 
 The microscopical examination of bacterial cultures may be 
 reduced to a comparatively simple technique. Bacteria may be 
 examined after being dried, or in their natural condition. The 
 advantage of the first method is that the preparations are perma- 
 nent, and to this must be added the greater facility with which 
 they admit of being drawn or photographed. Under the other, 
 the organisms suffer no reduction in size or alteration in form, 
 nor can appearances artificially produced be mistaken for such as 
 are natural. There is the same difference, in short, as between 
 the study of plants growing in a garden and those dried in a 
 herbarium. The second method is, therefore, in all cases pref- 
 erable; obviously for the study of growth and reproduction it is 
 absolutely necessary. 
 
 The Hanging Drop. Examinations in the natural state are 
 carried out by means of the hanging drop, which may be de- 
 scribed in detail as being the most beautiful of all methods, and 
 as expeditious as any. 
 
 If a culture on agar or gelatine (when this is not liquefied 
 by the growth) is to be examined, a thin, narrow ring of vaseline 
 is painted around, but a short way from the depression of a hol- 
 low ground slide; a single 6'se or loop of distilled water is then 
 transferred to the centre of a square or circular cover-glass (No. 
 1 thickness). For the sake of those unacquainted with such 
 matters, it may be said that the 6'se or loop is a small eyelet 
 made at one end of a piece of platinum wire about two and a 
 half inches in length, the other end of which is pressed into the 
 end of a piece of solid glass rod, seven inches long, softened in the 
 flame of a Bunsen burner; the diameter of the eyelet, which is 
 
26 PATHOGENIC BACTERIA 
 
 made by bending the wire with the end of a small-pointed pair 
 of forceps into a complete circle, should be two millimeters 
 (about ^ inch), and in all cases it is to be sterilized by raising it 
 to a red heat in the flame before and after use (Fig. I). After 
 
 Fig. 1. The 6se or platinum loop, the length of the wire and glass rod reduced. 
 
 sterilization in the flame, the loop is inserted into the culture 
 tube, applied to the growing edge of the culture, withdrawn, 
 and immediately transferred to the drop of water on the cover- 
 glass, in which it is moved until slight turbidity appears; the 
 excess of the culture on the loop is now burnt off in the flame, 
 and the bacteria thoroughly distributed on the cover-glass by its 
 means, the drop being extended so as to cover an area of about 
 four millimeters in diameter. The cover glass is then seized at 
 the edge in a small pair of forceps, turned over, and applied to 
 the hollow ground slide so that the minute drop of fluid lies 
 over the center of the hollow space in the slide without coming 
 into contact with the latter; the gentlest pressure made around 
 the border of the cover-glass will then render the moist chamber 
 completely air-tight (Fig. 2). A moist chamber may be impro- 
 
 Fig. 2. Section of hollow ground slide with cover-glass in situ, slightly re- 
 duced. 
 
 vised, however, in a very effective manner on an ordinary flat 
 slide, as follows: Cut a piece of thick blotting-paper an inch 
 square, fold it, and cut away a small semi-circle from the center 
 of the folded border, open it out, wet it, and lay it on the slide; 
 
OF THE HUMAST SUBJECT. 27 
 
 the cover glass having been prepared exactly as before is inverted 
 so that the hanging drop lies over the centre of the circular hole 
 in the blotting paper (Fig. 3). 
 
 Fig. 3. Section of slide and moist chamber prepared with blotting-paper 
 slightly reduced. 
 
 If the examination be prolonged, it is necessary to moisten 
 the paper from time to time. For the study of such prepara- 
 tions, much of the light must be cut off with the diaphragm, 
 assuming that the Abbe condenser and plane side of the mirror 
 are used; unstained organisms are otherwise scarcely discernible. 
 
 The most suitable area for observation is the edge of the 
 drop where the film of fluid is thin, and where there is what may 
 be called a "still" layer in which an accumulation of the bac- 
 teria takes place, and where their movements (whether active or 
 Brownian) are less lively, and eventually cease. 
 
 The best plan of finding the edge is to place the slide with 
 the hanging drop centrally on the stage of the microscope, 
 transfer a droplet of cedar oil exactly over the hanging drop, 
 and screw down the coarse adjustment until the oil rises up to 
 meet the object glass ; the rest of the focussing is then carried 
 out by means of the fine adjustment which is screwed until 
 some of the bacteria come into focus ; the slide is then moved in 
 any one definite direction until the edge of the drop is reached. 
 Besides the forms, the motility of the bacteria is to be deter- 
 mined by the hanging drop. For the latter purpose, the use of 
 broth cultures is preferable to those made on agar ; nevertheless, 
 
V?8 PATHOGENIC BACTERIA 
 
 if a preparation of the typhoid bacillus, e.g., is made from an 
 agar culture of twenty-four hours' growth, in the way described, 
 there is no difficulty in observing the inotility of the microbe, 
 though the movements are not so active as in a broth culture. 
 Examined in the hanging drop, it is to be noted that even non- 
 motile bacteria present marked passive or Brownian move- 
 ments ; these are distinguishable by the fact that no real loco- 
 motion takes place, the movements being of a to-and-fro or 
 oscillatory character. 
 
 Coloration of the Hanging Drop. The hanging drop having 
 been so viewed, may next be advantageously stained. It is not 
 necessary for this purpose to make a new preparation. The 
 cover-glass is removed and turned over on a piece of filter paper, 
 so that the drop is again brought uppermost. 
 
 The best staining fluid is a dilute solution of gentian violet, 
 made by adding .5 c.c. of an aqueous solution of gentian violet 
 (gentian violet 2.25 grammes, distilled water 100 c.c.) to 10 
 cubic centimeters of distilled water. The dilution may be ar- 
 rived at sufficiently well by placing a few drops of the gentian 
 violet solution in a test tube and adding distilled water until the 
 fluid is sufficiently dilute to allow of being seen through when 
 held between the eye and the light. The sterilized 6se is dipped 
 into this, and the drop of dye mixed on the cover- glass with the 
 hanging drop already there ; the cover-glass is then again in- 
 verted and replaced over the hollow slide or upon the blotting- 
 paper of the moist chamber. The ordinary histological method 
 of running in the dye from one side of a wet preparation made 
 without hollow slide or moist chamber, is followed by too much 
 displacement. 
 
 The action of the dye is easily studied in all its grades in 
 
PLATE XXIII 
 
 s 
 
 >4/6/ca/s. 
 
 MEDICAL ANNUAL, 1898 
 
PLATE XXIM. 
 
 SACCHAROMYGES ALBIOANS. THE BLASTOMYCES OR YEAST-CAUSING THRUSH. 
 
 The drawing is made from a hanging drop of a broth culture which had been 
 incubated at 37* C. for 24 hours. 
 
 There is shown on the left and upper part of the figure a series of simple oval cells ; 
 and below these, similar cells from which the formation of a second element is pro- 
 ceeding ; this appears as a minute bud growing out from one of the ends or side of the 
 parent cell. Where the cells lie in groups their opposed faces are flattened or facetted, 
 an extensive group resembling a mosaic. The process whereby a filamentous stage is 
 reached consists in the elongation of the cells, which continue to give rise to others 
 from apex and sides until a complex branching system results. The chief secondary 
 branches arise from the nodes of the main filaments, and from the former a tertiary 
 set of smaller cells, and so forth. 
 
 On the right hand side are shown four cells, the protoplasm of which has been tinted 
 with a dilute aqueous solution of gentian violet ; they exhibit a certain number of 
 spaces filled with fluid, or vacuolea, analogous to those holding sap in higher plants ; 
 and spherical granules, the function of which is not yet known, possibly zymogenic 
 in nature. 
 
 In the elongated or filamentous cells the vacuoles are sometimes of considerable 
 length, and can be readily seen in tbe unstained condition. 
 
PLATE XXIV 
 
 Fig. 1. Typhoid Bacillus. 
 
 Fig. 2. Typhoid Bacilli. 
 
 MEDICAL ANNUAL, 1898 
 
PLATE XXIV. 
 
 FIG 1. TYPHOID BACILLUS. 
 
 Prom a culture 24 hours' old, incubated at 87 C., and made on the surface of agar. 
 Stained with carbol fuchsin, washed in water acidulated with acetic acid. Straight or 
 very slightly curved rods, the shortest of which appear as oval : the bacilli present 
 great variations in length and breadth: parallel grouping is fairly common. The 
 filamentous forms exhibit no trace of a segmented or composite structure. 
 
 FIG. 2. TYPHOID BACILLI. 
 
 Five typhoid bacilli, from a dried preparation stained by Van Ermengen's silver 
 method, and showing the long wavy flagella arising around them. The largest number 
 shown is seventeen, but this may be exceeded. 
 
PLATE XXV 
 
 Fig. 1. Colon Bacillus. 
 
 Fig. 2. Colon Bacillus. 
 
 MEDICAL ANNUAL, i8(>8 
 
PLATE XXV. 
 FIG. 1. COLON BACILLUS. 
 
 From a culture in a simple aqueous solutiou of Peptone, 24 hours' growth, incubated 
 tit 37 G. 
 
 The figure is introduced to show the "end-staining" often observed in bacilli, the 
 causation of which is not fully understood. The interest attaching to the present 
 specimen, is that it demonstrates the effect of one particular factor in leading to this 
 result. It has been noticed by Mr. H. C. Haslam that the "end-staining" occurs 
 regularly in the colon bacillus if it is grown in alkaline media. 
 
 In an unstained hanging drop corresponding appearances present themselves, the 
 ends of the bacilli being darker and more granular than the centres. 
 
 FIG. 2. COLON BACILLUS. 
 
 From an agar culture of 48 hours' growth, incubated at 37 C. 
 
 Showing the usual form of the Colon Bacillus : plump straight rods with rounded 
 ends, variable in length and thickness, the shortest appealing as oval. Parallel 
 grouping occurs. As there are many varieties of Colon Bacillus, it is necessary to take 
 one as a standard ; that selected is from a culture of Professor Bscherich's (the 
 original describer of the bacillus). A subculture of this particular organism was 
 obtained from Professor Bscherich by Mr. Wallis Stoddart, to whom the author is 
 indebted for a sample of the strain. 
 
PLATE XXVI 
 
 Fig. 1. Staphylococcus Pyogenes Aureus. 
 
 Fig. 2. Plague Bacillus. 
 
 MEDICAL ANNUAL, i8 9 8 
 
PLATE XXVI. 
 FIG. 1. STAPHYLOCOCCUS PYOGENES AUREUS. 
 
 From the edge of a growing culture on agar. 
 
 Carbol fuchsin, washed in acidulated water. The cocci occur in clusters or 
 botryoidal groups (2ra<J>vA?- bunch of grapes), here and there in short chains, 
 twos, or singly. The individuals in the groups vary in size, indicating probably 
 differences in age ; in some of the groups the process of division appears in a 
 diplococcus with flattened sides, the elements of which have not yet parted and 
 become spherical. 
 
 FIG. 3 PLAGUE BACILLUS. 
 
 Agar culture, incubated 37 C.; the advancing edge of a three days' growth. 
 
 Carbol fuchsin, washed in acidulated water. Short rods, mostly of oval form, 
 often in pairs, side by side. Here and there slightly curved, longer forms occur, 
 some of remarkable thickness. 
 
 The original source of the bacillus was a Lascar admitted into the Seamen's 
 Hospital, Albert Dock, from a P. and O. steamer arriving from Bombay; the 
 bacteriological diagnosis was made by Dr. R. T. Hewlett; inoculations carried 
 out upon guinea pigs caused d^ath with typical symptoms. 
 
 The forms examined in the blood of animals experimentally inoculated with 
 this particular bacillus showed marked "end-staining"; this is entirely absent 
 in the specimen prepared from the agar culture. 
 
PLATE XXVII. 
 
 GONOCOCCUS. 
 
 A cover glass preparation of urethral pus, made by means of the loop, from 
 the ureihral meatus in a case two days after infection. 
 
 Carbol fuchsin, washed in acidulated water. Two flattened epithelial cells 
 from the urethra, and one " polynuelear " leucocyte are represented. The stain 
 has tinted the bodies of the epithelial cells, but not that of the pus cell. The 
 cocci, without exception, occur in pairs with flattened or slightly concave faces; 
 here and there is a group of four. The diplococci are set in groups or colonies 
 which lie outside and unconnected with the cells; elsewhere, upon and within 
 them. The groups connected with the epithelial cells are surrounded with an 
 uncolored zone of ground substance indicating their location within the cell 
 protoplasm. Those about the nucleus of the leucocyte are in cell protoplasm. 
 
PLATE XXVtt 
 
 Gonococcus. 
 
 MEDICAL ANNUAL, 16 
 
PLATE XXVIII. 
 Fio. 1. ANTHRAX BACILLUS. 
 
 Agar culture, incubated at 87 C.; of 24 hours' age : the films treated with 20 per 
 cent, acetic acid before staining with aqueous solution of gentian violet. 
 
 The lowest filament is as yet sporeless ; some of its component segments are but half 
 the length of others, and separated by a narrower interval, indicating that fission and 
 interstitial elongation are proceeding in the filament. 
 
 In the others, various stages of spore-formation are shown. The spores appear at 
 first as minute unstained points at or near the centre of the segments, the capsule of 
 tha spores preventing the penetration of the dye. When fully grown, the spores dis- 
 tend the wall or "sheath" of the bacillary cell, from which they are subsequently set 
 free, as shown at the highest part of the figure. In many of the sporulating segments 
 there is a minute second point, probably an abortive second spore, since the fully 
 developed spore in in most instances not strictly central, but nearer one end of the 
 spore-bearing segment, and this whether the latter presents a second point or not. 
 
 The staining or the spores is best carried out by means of hot carbol fuchsin, and 
 the use of acid and methylene blue as described under the Tubercle bacillus. 
 
 FIG. 2. ANTHBAX BACILLUS. 
 
 Broth culture of 24 hours' growth, incubated at 87 C. Carbol fuchsin, washed in 
 acidulated water. 
 
 Very few spores were found in the preparations, and none are present in the 
 filaments figured. The spore formation takes place only under the free access of gaseous 
 or atmospheric oxygen. The filaments adhere in tresses or strands. 
 
 Active subdivision is taking place in the segments, the ends of which remain straight 
 or squarely cut. 
 
PLATE XXVUl. 
 
 \ \ 
 
 \ 
 
 Fig. 2. Anthrax Bacillus. 
 
 MEDICAL ANNUAL, 
 
PLATE XXIX. 
 ANTHRAX BACILLUS. 
 
 ,*^ eSS *r i PreP"?. 4 ' 011 " of a small colony on agar in a Petri capsule; lowly 
 magnified ; the actual diameter of the colony is one millimetre. 
 
 Aqueous solution of gentian violet, washed in acidulated water 
 or ha1ry p rocelse? Stl f * feltw rk f wavy fllamen <*> extending at the margins in tufts 
 
 allowing the latter to fal1 on the colo >- 
 
PLATE XXIX 
 
 
 Anthrax Bacillus. 
 
 MEDICAL ANNUAL, 1898 
 
PLATE XXX. 
 
 TUBERCLE BACILLUS. 
 
 A preparation of Tubercular sputum, distributed over the cover-glass in the fresh 
 state. Carbol fuchsin, heated on the cover, the latter then transferred to a watch 
 glass of 25 per cent, hydric sulphate till decolourized, washed in water, and then 
 counter-stained with aqueous solution of inethylene blue in the cold, washed again in 
 water, and allowed to dry. Tubercular sputum is best examined when no antiseptics 
 have been used for its- disinfection. It should be spread out in a thin layer over a 
 sheet of glass on a black surface, or one of the black-bottomed plates made for the 
 purpose; the most purulent foci are then isolated with needle and forceps, or cut out 
 with scissors and forcibly broken up and distributed on a series of cover-glasses with 
 the back of the forceps, whilst the covers are steadied by the pressure of a needle. 
 Passing the films through the name is not necessary. If the cover-glasses are not 
 heated after the carbol fuchsin has been filtered on to them, the dye must be allowed to 
 act for a considerably longer time fifteen minutes: if heated, they must be held over 
 a low flame in a small pair of forceps of which the ends have been somewhat sharply 
 bent, in order that the dye may not be conducted to the under side of the glass. 
 
 The rationale of this particular method is as follows: the carbol fuchsin stains all 
 the different microbes present ia such a film ; with the exception of the tubercle 
 bacilli, all are afterwards decolourized by the action of the hydric sulphate, to be 
 re-dyed, together with the cell nuclei, by the methylene blue. This peculiar resistance 
 to the action of the acid is common to the tubercle, leprosy, and smegraa bacilli. 
 
 In the examination of pure cultures of the bacillus, this differential method is 
 unnecessary, and the organism may be stained simply with carbol fuchsin, or by 
 Gram's method, etc. 
 
 In addition to the " polynuclear" leucocytes (pus cells), there are shown in the 
 figure two squamous epithelial cells derived from the upper part of the respiratory 
 passages ; the body of these is faintly tinted with the blue. 
 
 Many of the tubercle bacilli occur in small grotesque groups, that have been 
 likened to Chinese characters. 
 
 The bacilli are mostly slightly curved, their extremities rounded, and their proto- 
 plasm segmented, so that the microbes appear " beaded." The beading is not invariable 
 in such preparations of sputum. 
 
PLATE XXX 
 
 Tubercle Bacillus. 
 
 MEDICAL ANNUAL, 2898 
 
OF THE HUMAN SUBJECT. 47 
 
 such a hanging drop, motile bacteria retaining their power of 
 movement whilst the coloration is proceeding in them. 
 
 To Make a Permanent Preparation of the Hanging Drop. 
 Lastly, a permanent specimen may be made from the same prep- 
 aration by removing the cover-glass, placing it again with the 
 drop upwards on a piece of filter paper beneath a watch-glass or 
 bell-jar, and allowing it to dry ; after which a small drop of 
 Canada balsam (dissolved in xylol) is placed on a slide, a second 
 small drop on the dried film, and the latter then allowed to fall 
 on the slide in the usual manner. Or, after the examination of 
 an unstained hanging drop, this may be allowed to dry in the 
 same way and treated in the manner to be presently described 
 under the dry method. 
 
 The dyeing of hanging drops made from broth cultures (in 
 which case, of course, no water need be placed on the cover - 
 glass) is not quite so satisfactory as those from agar, in conse- 
 quence of an extremely fine colored precipitate appearing in the 
 fluid, probably a proteid present in the broth, which unites with 
 the dye. This precipitate is unconnected with the growth of 
 the bacteria, as it appears if sterile broth is mixed with the 
 diluted dye and examined as a hanging drop. Nevertheless, in 
 such broth preparations when colored, many fields may be found 
 devoid of adventitious granules, which, it may be noted in pass- 
 ing, exhibit lively Brownian movements. 
 
 The examination of jelly that is being liquefied by the 
 growth of an organism is conducted precisely as that of a broth 
 culture ; and admirable preparations may be obtained by color- 
 ing the hanging drop with dilute solution of gentian violet in 
 the manner already described. 
 
 Of all the methods of bacterial microscopy, the hanging 
 
48 PATHOGENIC BACTERIA 
 
 drop, with few exceptions, is the best ; the important method of 
 Gram, for instance, cannot be carried out except on a dry prep- 
 aration. 
 
 The Dry Method. The more minute attention paid to de- 
 tails, the more satisfactory will be the results. 
 
 To commence with, then, the cover-glasses (No. 1 thick- 
 ness) must be properly clean. They are to be moved about in a 
 capsule or other flat vessel of hydrochloric acid, washed in com- 
 mon water and then kept in a wide-mouthed stoppered bottle of 
 absolute alcohol, to which glacial acetic acid has been added in 
 the proportion of 10 per cent. The addition of acid will be 
 found to prevent the cover-glasses receiving too high a polish 
 when wiped, a result which greatly interferes with the equable 
 distribution of the bacteria over the surface. Three cover-glasses 
 having been wiped dry, a loop of distilled water is placed on 
 each ; the loop should be drawn a short way over the surface, 
 and if it is found that the water does not adhere to the glass, 
 but returns again to a bead, the cover must be replaced in the 
 acidulated alcohol and wiped afresh. The sterilized loop having 
 been inserted into the culture tube and a small quantity of the 
 growing edge withdrawn, it is applied to the drops of water in 
 succession, and the residue having been burned off in the flame, 
 the loop is used to distribute the turbid drop over the entire 
 face of each of the cover-glasses, which are then allowed to dry; 
 the distribution is facilitated by breathing on the surface, and 
 the glasses are best steadied by the pressure of a needle. The 
 prepared side is to be kept upwards throughout all the subse- 
 quent manipulations. The time-honored custom is now to pass 
 the covers thrice across the flame, with the object of coagulating 
 the albumen and fixing the films. Although so generally 
 
OF THE HUMAN" SUBJECT. 49 
 
 adopted, it is in reality wholly unnecessary ; if the transit 
 through the flame is made sufficiently slowly to really heat the 
 glass, there is a risk of inducing still further shrinkage of the 
 bacteria. Not only is the practice unnecessary for preparations 
 made from agar cultures, but from careful comparative experi- 
 ments, the author has found it equally without advantage in the 
 case of such as are made from liquefied gelatine or from broth ; 
 the film will remain adherent even if the dye has to be heated 
 on the glass, provided always that it is not afterwards violently 
 washed beneath a stream, but in a vessel, of water ; to such cul- 
 tures the addition of water is not required ; the loop is simply 
 dipped into the fluid and distributed over the cover-glass. 
 
 The next procedure is that of staining For this purpose 
 aniline dyes are alone used ; none others are sufficiently intense. 
 The three chief are carbol fuchsin, aqueous solution of gentian 
 violet, and aqueous solution of methylene blue ; for special pur- 
 poses modifications are necessary, the most important of which 
 are Gram's method and those for staining spores, capsules, and 
 flagella. Of these dyes carbol fuchsin* holds the first place, and 
 takes the same position for bacterial staining that hematoxylin 
 does for histological : it not only dyes all known bacteria, but 
 gives, as a rule, cleaner preparations, in consequence of its being 
 more readily washed out from extraneous material. 
 
 The cover-glass should be quite covered with a high bead of 
 the stain. This may be done from a drop bottle, but the author 
 invariably filters the stain directly on to the cover-glass through 
 a small cone of filter-paper held across its overlapping parts in a 
 pair of forceps. The stain having remained on from five to fif- 
 
 * Fuchsin, 1 gram: absolute alcohol, 10 c.c. Dissolve and add lOOc.c. of a 5 
 per cent, aqueous solution of carbolic acid. 
 
50 PATHOGENIC BACTERIA 
 
 teen minutes, the cover-glass is taken up with a fine pair of 
 pointed forceps, tilted to throw off the stain, and at once gently 
 submerged in a saucer of water faintly acidulated with acetic 
 acid ; the washing may be completed in a second or a third 
 saucer, and finally in unacidulated water. The degree of acidity 
 can be arrived at by pouring a few drops of glacial acetic acid 
 into a saucer, inverting it to get rid of the excess, and then fill- 
 ing with water, common or distilled a solution of about .5 c.c. 
 glacial acetic acid to 70 c.c. of water. 
 
 The washing is preferably carried out in this way rather 
 than beneath a stream, as the latter tends to displace the film ; 
 nor is the excess of the dye so well removed by water without 
 the acid. 
 
 Fig. 4. Sheet of lead bent at right angles, with filter-paper folded upon it, and 
 cover-glass stood edgewise to drain without the film coming in contact with the 
 paper. 
 
 After the final washing the cover-glass is allowed to drain 
 and to dry by placing it obliquely with the prepared surface 
 downwards, against a sheet of filter-paper folded at right angles 
 over a piece of lead (Fig. 4). It may be more rapidly dried by 
 pressing between filter-paper, but at the risk of damaging the 
 film and transferring fibers to it. When dry, the preparation is 
 completed by mounting it on a slide with a xylol solution of 
 Canada balsam. 
 
 In a certain few cases (as in agar cultures of the anthrax 
 
OF THE HUAIAH SUBJECT. 51 
 
 bacillus) this method does not satisfactorily clear the prepara- 
 tion, i.e., decolorize the ground substance in which the bacteria 
 lie. 
 
 Under such circumstances, the cover-glasses, after being 
 prepared with the bacteria and allowed to dry, are to be left for 
 ten minutes in a capsule or saucer of 20 per cent, solution of 
 acetic acid ; after which they are washed in distilled water, al- 
 lowed to drain and to dry, edgewise, on filter-paper, and after- 
 wards stained in the usual manner, the subsequent washings 
 being carried out without acetic acid. It is not requisite to pass 
 the films across the flame at any stage of the procedure, even if 
 the cover-glasses have been prepared from broth cultures. Even 
 this at times fails to give clean preparations (as, e.g., in the case of 
 the glanders bacillus) ; the use of saturated alcoholic solution of 
 gentian violet, left on the cover- glass for an hour (beneath a 
 watch glass), will often then give satisfactory results ; the wash- 
 ing in such circumstances should be carried out rapidly in water 
 to which no acetic acid has been added. 
 
An Atlas of the Bacteria Pathogenic in 
 the Human Subject. 
 
 PART II. 
 
 As stated in Part L, the object of this Atlas is to present 
 graphically from original preparations the chief bacteria which 
 are pathogenic in the human subject The figures now intro- 
 duced comprise nearly all the remaining bacteria of interest. 
 Certain are omitted Amongst such omissions may be named 
 the bacillus of influenza, diplococcus intra-cellularis, bacillus 
 enteritidis, bacillus melitensis, streptothrix pseudo-tuberculosa, 
 etc. ; and time will surely add yet others as causative agents, es- 
 pecially in the group of infective granulomata. 
 
 Amongst the micro-organisms depicted in the present series, 
 two of the most important in practical medicine are the typhoid* 
 and diphtheria bacilli; and a succinct statement may be made 
 in regard to the bacteriological diagnosis, firstly of typhoid fever, 
 and secondly of diphtheria. 
 
 Typhoid Fever. Although the bacteriological diagnosis 
 of typhoid during life can be made from the discovery of the 
 typhoid bacillus in the stools or the urine, such methods are of 
 little practical avail, necessitating, as they do, a well- equipped 
 laboratory and the expenditure of much time. 
 
 Widal's Reaction. However interesting it might be to give 
 an account of the observations of previous investigators which 
 led up to Widal's application, neither space nor the main object 
 of the present article will allow of it. Widal was, however, the 
 first to demonstrate that a reaction, which had been previously 
 
 * Other illustrations of this are given in Part I. 
 
 53 
 
54 PATHOGENIC BACTERIA 
 
 shown to take place between the serum of animals experimen- 
 tally immunized against certain diseases and cultures of the spe- 
 cific bacilli producing such, could be obtained during the period 
 of infection, and in this way serve as a means of clinical diagno- 
 sis. If thte blood serum of an animal which has been rendered 
 immune against, e. g., the typhoid bacillus be added in a tost 
 tube to a living broth culture of the same microbe, the bacilli in 
 the culture rapidly cohere and subside to the bottom of the tube. 
 This is known as " clumping" or "agglutination," and is taken 
 to indicate the presence of an agglutinating substance or " agglu- 
 tinin" which has formed in the blood in consequence of the 
 presence of the bacilli experimentally introduced into the ani- 
 mal. The blood serum acquires this property, however, before 
 immunization is established, i. e., during the progress of infec- 
 tion itself, and the property may be utilized as a means of diag- 
 nosis, for the important reason that the serum of a typhoid 
 patient, whilst it will agglutinate typhoid bacilli, will not agglu- 
 tinate those of other kinds. What is true in the case of typhoid 
 is true also of other bacterial diseases. The phenomenon admits 
 of two applications; a disease may be diagnosed by the action of 
 the blood serum upon a known bacillus; or a bacillus may be 
 diagnosed by the action of a known "immunized serum." It 
 is to the former that importance attaches in practical medicine 
 and surgery. 
 
 After this brief explanation of the rationale of Widal's re- 
 action, the method of carrying it out may be described. 
 
 The broth culture of typhoid bacillus used must not be 
 more than of twenty-four hours' growth, and must be grown in 
 the incubator at 37 C, preferably in a broth that is not alka- 
 line, but amphoteric (giving simultaneously an acid and an alka- 
 
OF THE HUMAN SUBJECT. 55 
 
 line reaction to blue and red litmus paper). There arises even 
 at this point the first possible source of fallacy. If such a broth 
 culture be examined by the hanging-drop method (fully de- 
 scribed in the previous volume), clumps of bacilli are not rarely 
 encountered, and these, at times, of considerable size. In all 
 cases, then, the broth culture is to be first passed through a 
 small double cone of filter-paper into a watch-glass, in order to 
 remove any such clumps present; and after this, a hanging drop 
 is to be prepared for comparison with the result obtained when 
 the reaction is tried for. One important difference to be ob- 
 served in the microscopic preparations is that although in the 
 broth culture clumps may be met with, the field between con- 
 tains varying numbers of bacilli in active movement; whereas 
 when clumping occurs from the action of the serum of a typhoid 
 patient, the bacilli become motionless between the bacterial isl- 
 ands, if any remain incoherent. 
 
 How to test the Blood Serum. The reaction is obtainable 
 quite early in the course of the disease, as early as the fifth day, 
 and it persists during convalescence, but for an extremely vari- 
 able period afterward. Not rarely the reaction will continue for 
 a year, but it may last many years, and might without enquiry 
 into a patient's history be erroneously taken to prove the exist- 
 ence of typhoid on the occasion of some subsequent illness sug- 
 gestive of it. 
 
 The blood to be used in the test is withdrawn either from, 
 the lobe of the ear or from the back of the finger near the root 
 of the nail; and the puncture is best made by means of a surgi- 
 cal or other neede with a cutting edge as well as a sharp 
 point. 
 
 If the test cannot be carried out at the time, the blood must 
 
56 PATHOGENIC BACTERIA 
 
 be collected in a pipette of the kind represented in the adjoin- 
 ing wood cut (Fig. 5), by breaking off the sealed ends of the 
 
 Fig. 5. Pipette containining blood which has separated into clot and serum, the 
 former occupying the lower half of the bulb (nat. size). 
 
 capillary to be on either side of the bulb, and applying one end 
 to the issuing blood. When the expanded part is filled, the ends 
 are hermetically sealed in the edge of the flame of a spirit-lamp. 
 Blood so stored can be tested at leisure, and (if kept in the dark) 
 retains its qualities for long periods. Even if dried the blood 
 will provide the reaction; for this purpose it is collected on a 
 series of cover-glasses, which, after being allowed to dry, may 
 be, if necessary, posted to an expert for examination. If dried 
 blood is used, a solution of the specific substances in it is ob- 
 tained by means of distilled water; but the method is an inferior 
 one, owing to the difficulty of estimating the dilution reached 
 a matter of cardinal importance. 
 
 Nine loop-fulls* of the filtered broth culture of typhoid are 
 placed separately and fairly close together on an ordinary micro- 
 scopic slide, the loop or ose being introduced as many times into 
 the broth. The platinum wire is now sterilized in the flame, 
 and with it a single loop of the blood serum is transferred to the 
 slide and well mixed up with the whole of the nine droplets of 
 broth culture. If the test is carried out on the spot, a few drops 
 of blood may be allowed to flow from the lobe of the ear or fin- 
 ger, and to clot in a small test tube or a watch-glass; the serum 
 so furnished will be ample. 
 
 * For a figure of the " loop," see p. 26. 
 
OF THE HUMAK SUBJECT. 57 
 
 If the blood to be used has been stored in a pipette, the two 
 ends of this are broken off, and the contents blown gently on to 
 a slide. A hollow-ground slide having been prepared with a 
 ring of vaseline, and a clean cover-glass (before commencing the 
 proceedings just described), a single ose of the admixed broth 
 and sernm is placed on the center of the cover glass and gently 
 spread out so as to cover an area about 4 millimeters in diameter; 
 the cover is then inverted, placed over the hollow of the slide, and 
 gently pressed at the margin so as to render the enclosed space 
 quite air tight. The preparation is now placed beneath the 
 microscope and and examined, with a ^ homogeneous immer- 
 sion, or a ^ objective, which answers perfectly well for a study of 
 the result. As the preparation is unstained, much of the light 
 (if -y^- is used) must be cut off by means of the diaphragm the 
 bacilli are otherwise scarcely visible. 
 
 If WidaFs reaction ensue, it is seen in the movements of the 
 microbes becoming sluggish and ultimately ceasing, whilst they 
 become at the same time aggregated into clumps of the kind 
 represented in Fig. 1, Plate XVIII. 
 
 The time allowed for the observation should be half an 
 hour. If no reaction has ensued within this time, the result is 
 to be reckoned negative, and the existence of typhoid may be 
 excluded, not with absolute certainty, but with very high proba- 
 bility. In the case of such a negative result, similar examina- 
 tions must be repeated during the course of 'the disease, as the 
 reaction, for causes not known, is in some cases delayed. If, 
 however, the bacilli become motionless, even without any 
 marked clumping, or if they become motionless, and clump, the 
 result is to be reckoned as positive. 
 
 Before deducing the existence of typhoid in these circum- 
 
58 PATHOGENIC BACTERIA 
 
 stances, nevertheless, the result of farther dilutions must be 
 tested, for the reason that the reaction ensues in certain cases in 
 which typhoid does not enter into the question, and, moreover, 
 that the blood of healthy persons possesses at times an unusual 
 degree of the agglutinizing power which is normally present. 
 
 The diagnosis, it may be said, becomes strengthened in pro- 
 portion as the reaction persists on dilution. In certain instances 
 it has been ascertained that a dilution of 1 to 5,000 will yet suf- 
 fice for its production, and even a considerably further dilution 
 than this. The test is to be repeated, therefore, with a dilution 
 of 1 in 20 (i.e., 1 part of serum to 19 of the broth culture), and 
 if still observed, with a dilution of 1 in 50. The last limit may 
 be held to suffice for the exclusion of other possible sources of 
 phenomenon, and to establish a diagnosis of typhoid. The dilu- 
 tion of 1 in 20 is best made by placing nineteen loops of the broth 
 separately on a slide, and mixing with a single loop of the serum; 
 that of 1 in 50, by diluting 1 loop of serum with 10 of distilled 
 water, and mixing one loop of this with four loops of the broth 
 culture. 
 
 After use, all the slides, cover-glasses, and other materials 
 are to be disinfected in a 1 in 20 carbolic acid solution. 
 
 Diphtheria. For the bacteriological investigation of a 
 supposed case of diphtheria it is necessary, firstly, to make a 
 microscopic examination of a culture from the throat or nasal 
 passages ; and secondly, if the investigation is to be complete, 
 to inoculate animals with a pure culture in order to test the de- 
 gree of virulence which the bacillus possesses, i.e., both the 
 morphological and the physiological characters of the microbe 
 should be determined. 
 
 The inoculation of test tubes for the purpose of dia^ nosis 
 
PLATE XV [II 
 
 
 Fig. 1.W!dal'8 Reaction. 
 
 MEDICAL ANNUAL, 1899. 
 
PLATE XVIII. 
 
 FIG. i. WIDAL'S REACTION. 
 
 This fi-ure is given to show the bacillary clumps which form when the blood serum of a 
 p.uient sufn_nng from typhoid (ever is added to a living broth cultiue of the typhoid bacillus, 
 the phenomenon being known as Widal's reaction. 
 
 In the case of the preparation figured, i part of blood serum was added to 19 ot a twenty- 
 four hours' (incubated) broth culture. The blood had been collected in a pipette and 
 allowed to clot ; a certain number of red corpuscles are admixed with the serum. 
 
 The clumping or agglutination of the bacillus is readily observable under i-Ctri objective, 
 though the clump represented was drawn under i-i2th oil immersion, and is magnified about 
 TOGO times. 
 
 The method of carrying out the test for the diagnosis of typhoid fever is ully given in 
 the text. 
 
 FIG. 2. BACILLUS OF LEPROSY. 
 
 The figure represents three highly vacuolated enclothelial cells from a lymphatic gland 
 secondarily infected in a case of leprosy of the tongue. The cells occurred along with others 
 of similar character in irregular groups scattered throughout the gland. The notable 
 vacuolation (regularly seen in such "leprous cells") is possibly due to an abundant 
 formation of digestive fluid secreted by the cell that it may destroy and utilise the bacilli. 
 Highly vacuolated cells at times hold extremely few bacilli, possibly as a result of such a 
 process of destruction. The bacilli mostly lie in the septa between or around the vacuoles, 
 though when the vacuole is not viewed in strict optical section they appear to lie within. 
 
 The leprosy bacilli are slender, straight, or slightly curved rods, very uniform in breadth, 
 and fairly so in length, and they closely correspond in size and general character with those 
 of tuberculosis, as the latter are met with, e.g:, in phthisical sputum. They present a markedly 
 beaded appearance arising from protoplasmic segmentation. They give, again, the same 
 common staining reaction, in this resembling, moreover, the bacillus of Mnegma ; i.e., after 
 being dyed with carbol fuclisine the bacilli resist decolorisation in a 25 per cent, mixture 
 of sulphuric acid in distilled water. The smegrna bacillus is not infrequently present in the 
 urine of both sexes, and may be mistaken for that of tubercle. This error can be avoided by 
 examining the urine drawn off by catheter, the smegma bacillus being in this way excluded. 
 And to select one of many differential staining methods, though dyed with carbol fuchsine, 
 the colour of the smegma bacillus is discharged in a mixture of 20 per cent, nitric acid in 
 alcohol, whilst that of the tubercle bacillus is retained. For the reliable examination of 
 urine a centrifuge is indispensable. 
 
 The sections of the leprous gland were stained for fifteen minutes (without heat) in carbol 
 fuchsine, passed through 25 per cent, sulphuric acid, washed in water and counterstained for 
 five seconds in a i per cent, aqueous solution of methyl blue, after which they were passed 
 through water, absolute alcohol, oil of cloves, and mounted, finally, in a solution of Canada 
 balsam in xylol. For the counterstaining of tissue methyl not methyler.e blue, is to be used ; 
 the latter is almost entirely removed by the subsequent immersion of the section in alcohol. 
 The counterstaining of cover-glass films of phthisical sputum is satisfactorily carried out by 
 means of an aqueous solution of methylene blue. (See the previous volume.) 
 
 The Leprosy bacilli are not in all cases located within the cells of a tissue : they may lie in 
 a ground substance or gloea occupying the lymph spaces. 
 
PLATE XIX 
 
 f/g. r. Diphtheria Bacillus (typical form}. 
 
 * 
 
 
 \ 
 
 '"'... ... <. 
 
 " -; '- 
 
 2. Diphtheria Bacillus (atypical). 
 
 Fig. 8. Diphtheria Bacillus (atypical). Fig. 4. Hoffmann's Pseudo-Diphtheria Bacillus. 
 
 MEDICAL ANNUAL, 1899. 
 
PLATE XIX. 
 
 BACILLUS OF DIPHTHERIA. 
 
 FIG. i. THE "TYPICAL, 1 " KLEBS-LoFFLER," OR "LONG" DIPHTHERIA 
 
 BACILLUS. 
 
 Fro n a culture on Loffler's blood-serum ot twenty-two hours' age, incubated at 37 C. The 
 growth was the third sub-culture of the original from the throat. Stained with Lijffler's 
 methylene blue, washed in tap water. The case from which the culture was raised concerned 
 a boy (P. R.J, admitted to St. Thomas's Hospital, October iSih, 1897, with inflamed throat ; 
 there was much membrane on the tonsils, and in the larynx as evidenced by the stridor and 
 retraction of the chest. Tracheotomy was performed, the tube being removed on the founh 
 day. Four thousand units of diphtheria antitoxin were administered by subcutaneous 
 injection. Until the early part of November progress had been favourable ; on November 24 th 
 palatal paralysis was noted, the voice acquiring a nasal twang; this slowly improved. The 
 knee-jerks were absent on November agth, and still absent on December 24th, but the patient 
 could at that date swallow without regurgitation. 
 
 The Bacilli are straight or slightly curved rods of varying length and thickness, often set in 
 parallel groups of two or more. The designation of "long" implies that many, not all, are of 
 conspicuous length. One end (or both) may be enlarged or bulbous ; in the absence of this, 
 the ends are abruptly rounded without tapering. The bacilli present a notable segmentation 
 of protoplasm, which is divided into deeply stained parts ; these, which may be far from 
 equidistant, are in some instances flattened across the long axis of the rod, in others, spherical. 
 The extremities of the bacillus correspond with terminal segments. In -some of the shorter 
 rods only terminal parts are dyed end or polar staining. 
 
 The primary forms appear as short uniformly stained bacilli, with ends slightly smaller than 
 the rest of the rod ; these undergo transverse division, before the completion of which they 
 appear as diplo-bacilli with the opposed ends flattened. Segmentation rapidly takes place, 
 the shortest forms exhibiting onh end-staining ; the hitter are distinguishable from true 
 diplo-bacilli by the absence of tapering free ends and the length of unstained centre. These 
 forms of the diphtheria bacillus are classed as " typical," since they are commonly associated 
 with the typical, more virulent examples of diphtheria; yet not invariably so, for not only 
 may ''atypical" forms be highly virulent, but similar "typical" or "long" forms may possess 
 little palhogenicity (as tested upon the guinea-pig), and occur in cases which clinically present 
 no other features than those of sore throat, unaccompanied even with any marked malaise. 
 
 Fu;. 2. "ATYPICAL" OR "SHORT" DIPHTHERIA BACILLI. 
 A culture of thirteen hours' growth on Loffler's blood serum, carried on !rom one (Viennese) 
 of the virulent strains in use at the Conjoint Laboratories of the Royal College of Physicians 
 and Surgeons, London, for the preparation of toxin employed in the production of diphtheria 
 antitoxin from the horse. Besides a few segmented forms are others of an "atypical " kind, 
 commonly ranged in parallel collections of two or more ; these mostly taper off at the 
 extremities, presenting a deeply stained centre, which is usually divided by an uncoloured 
 narrow line indicative of an incompleted transverse fission, the elements being double, or 
 diplo-bacilli. Beyond the deeply stained centre the bacillus is of a lighter blue. Here and 
 there a pyrifonn element occurs, more deeply stained towards the larger end ; or short deep'y 
 stained spindles. 
 
 FIG. S.-A SECOND EXAMPLE OF THE "ATYPICAL" OR "SHORT" 
 DIPHTHERIA BACILLUS. 
 
 From a virulent case of the disease. Diplo-bacilli in parallel groups ; beyond their opposed 
 deeply stained central ends the component elements taper off and are but lightly coloured. 
 Pear-shaped forms are present, and a few which exhibit polar and segmented staining. From 
 its staining reaction the form has been named the "sheath" variety by Dr. J. Eyre, to whom 
 the author is indebted for the preparation from which the figure has been drawn. The " sheath/' 
 variety occurs, as a rule, in the milder types of diphtheria. It is but rarely met with, and is 
 not strictly stable ; if grown for some days upon alkaline potato and again sown on blood- 
 scrum, it acquires the segmented characters of the "typical" form. 
 
 Another atypical pathogenic variant has been described of the same form as the above, but 
 staining uniformly and deeply throughout. 
 
 FIG. 4 . HOFMANN'S BACILLUS. 
 
 Culture of twenty-four hours' on agar, incubated at 37 C. Stained with Loffler's blue, 
 washed in tap water. The culture was carried on from one isolated by Dr. E. A. Peters, 
 to whom the author is indebted for a sample of the strain. Diplo-bacilli, occurring in 
 parallel groups of two or more. The elements composing a single diplo-bacillus are short, 
 squat, wedge-shaped, with opposed bases, and stain uniformly throughout. They are 
 markedly shorter than the atypical or short varieties of the diphtheria bacillus, and relatively 
 broader at their bases. In older cultures segmented and irregular involution forms may be 
 encountered. Hofmann's bacillus (sometimes named a pseudo-diphtheria bacillus) is not 
 found as a cause of true diphtheria in the human subject. Hence, though isolated in many 
 forms of sore throat, such lesions are not to be regarded as diphtheritic. The bacillus, may, 
 however, be associated with the " typical," " Klebs-Loffler," or " long" diphtheria bacilli in 
 diphtheritic affections, but under such circumstances its presence may be regarded as of 
 secondary significance. 
 
PLATE XX 
 
 - 
 
 1. Proteus Vulgarts., 
 
 Fig. 2. Proteus Vulgar!* (the early 
 filamentous phase). 
 
 MEDICAL ANNUAL, i8gg. 
 
PLATE XX. 
 
 BACILLUS PROTEUS VULGARIS. 
 
 This organism is introduced as one of the most common of those causing putrefaction, 
 though different varieties of the bacillus coli are almost as ubiquitous. 
 
 Although by some, putrefactive organisms are not classed as pathogenic, because they are 
 not the causes of any process differentiated clinically as a disease, they are pathogenic in 
 the wider and truer sense, since putrefaction plays so important a part in the sepsis of 
 wounds and in the toxa;mia accompanying cancrum oris, the ulceration of extensive carcino- 
 mata of the alimentary and respiratory passages and conditions of a like kind. 
 
 FIG 2. 
 
 An '' Impression preparation 1 ' from a gelatin culture of eighteen hours. 
 
 Carbol fuchsine, washed in water weakly acidulated with acetic acid. The culture was 
 made by streaking a Petri capsule (after the jelly poured into it had set) with a straight 
 platinum wire infected from a pure culture of the organism. The impression preparation is 
 obtained by allowing a cover-glass to fall upon some part of the streak of growth and then 
 gently raising it by one edge, when the line of culture is brought away adhering to the under 
 side of the glass ; the specimen is then stained in the usual way. 
 
 The preparation shows the early or filamentous stage in the growth of the bacillus, which 
 is highly pleomorphic, whence its name of Proteus. 
 
 The streak (of which one edge is depicted) consists of long, closely-matted, unbranched 
 filaments arranged in strands. As seen at the free margin, some are sharply re-curved upon 
 themselves. 
 
 The organism figured was isolated from macerating muscle, the actual material being beef 
 steak, which was minced and incubated in distilled water at 37 C. 
 
 FIG. i. 
 
 Ail impression preparation of a similar streak culture at a later stage when the gelatin 
 was in process of liquefaction. Carbol fuchsine, washed in acidulated water. 
 
 The filaments have mostly divided into short rods, often constricted across the middle or 
 in pairs end-to-end as diplo-bacilli. Here and there longer rods occur and filamentous 
 forms, but the longer of the latter have not been introduced. 
 
PLATE XXI 
 
 -\' 
 
 I 
 
 ** N. 
 
 U 
 
 o< ' 
 
 : i 
 
 Fig. 1. Bacillus of Glanders. 
 
 . 2. Bacillus Tuberculosis, in a pure 
 culture. 
 
 MEDICAL ANNUAL, 1899. 
 
PLATE XXI. 
 
 FIG. i. -BACILLUS OF GLANDERS (BACILLUS MALLEIJ. 
 
 From the growing edge of a culture on glycerin-agar of three days' growth, incubated 
 at 37C. 
 
 Carbol fuchsine, washed in diluted acetic acid. The culture was made from a potato growth 
 of characteristic honey-yellow colour, raised from a glandered horse, the potato culture being 
 the second remove only from the original. 
 
 The bacillus (which is very difficult to stain with certainty in the tissues) may be stained 
 in cover-glass films with aqueous solution of gentian violet (ten minutes), followed by washing 
 in i in 10,000 caustic potash solution and afterwards tap water ; or, by means ot alcoholic 
 solution of gentian violet for one hour, rapidly washed in water. The action of Loffler's 
 blue, for cover-glass films is uncertain. 
 
 Somewhat slender rods of varying length and thickness, straight or slightly curved, the 
 shortest appearing as ovals, the longer as unsegmented filaments which are commonly less 
 deeply stained ; some of the filaments present were longer than those figured. Here and 
 there end-to-end pairs are met with. In some of the rods one or more minute, sharply- 
 defined, circular vacuoles are present. A few of the bacilli present a inoniliforin outline, but 
 none any distinct segmentation of protoplasm or " beading," as they may in the tissues. 
 
 The organisms tend to cohere in clusters, some of the smaller of which are selected in the 
 illustration. 
 
 FIG. c.-TUBERCLE BACILLUS. 
 
 Pure culture on Luffler's blood-serum, incubated at 37 C., carried on from a growth on 
 glycerin-agar which was raised from the lymphatic gland of a guinea pig, inoculated from a 
 case of tubercular pleurisy in the human subject. 
 
 The growth on the serum progressed slowly and took the form of a thin, white film in 
 which were sparsely scattered, thicker, more opaque areas. 
 
 Stained with ca.'bol fuchsine warmed on the cover-glass, and treated with 25 per cent, 
 sulphuric acid in a watch-glass. If used in the cold, the dye should be allowed fifteen 
 minutes. 
 
 The culture consists of straight and slightly-curved rods with rounded ends, and of varying 
 length, the shortest hardly more than oval. The bacilli tend to occur in small parallel 
 groups arranged in irregular lines, or set at .various angles to one another in away suggestive 
 of Chinese characters. They exhibit none of the segmentation, or " beading," so commonly 
 presented in phthisical sputum (see Plate XXX in the previous volume). 
 
 Branching Forms. On the right are shown, from the same culture, two examples of the 
 filamentous forms, which recall the branched mycelium of the hyphal-fungi or moulds. 
 
 The colouration of the bacilli (stained by Gram's method) is almost limited to the minute 
 spherical granules lying within the bacillary cell._ _ 
 
 Branching forms are at times met with in phthisical sputum. 
 
PLATE XXII. 
 
 FIG. i. -BACILLUS OF RHINOSCLEROMA. 
 
 From a streak culture on agar, of forty-eight hours' growth, incubated at 37 C. 
 
 Carbol fuchsine, washed in dilute acetic acid. The figure shows a group of bacilli united 
 into a zooglcea by an abundant ground substance which is faintly stained with the dye. 
 The micro-organism consists of spherical elements occurring 'singly, but most frequently in 
 pairs or short chains. Rod forms may also be met with. 
 
 As showing the essential similarity of ground-substance, or gloea, and, that which con- 
 stitutes a bacterial capsule, it will be noticed that whilst at the periphery, where active 
 multiplication is proceeding, the bacilli lie closely embedded in this substance, more 
 centrally, where the latter has accumulated in larger amount, it has parted into areas 
 appertaining to definite groups, showing the divided share which the organisms have taken 
 in its production. At the lower end of the figure, near its middle, there is a diplo-bacillus 
 quite isolated from the neighbouring mass, and furnished with a capsule proper to itself. 
 
 FIG. 2. STREPTOTHRIX ACTINOMYCES. 
 
 Broth culture, of seven days' age. incubated at 37 C. 
 
 In broth the growth occurs as small spherical colonies, which are best examined by being 
 teased out on a cover-glass after haying been washed in distilled water. The preparation is 
 allowed to dry and may be then stained with carbol fuchsine or aqueous solution of gentian 
 violet ; in the former case the subsequent washing is carried out with acidulated water, in the 
 latter with tap water, or. what is better, with a i in 10,000 solution of caustic potash, 
 followed by tap water. 
 
 The culture consists solely of long, interlacing, slightly wavy filaments which give off 
 lateral shoots. The branches vary in length according to their age, taking, on their first 
 appearance, the form of minute excrescences. 
 
 Cultures of the streptothrix Madura? (the cause of mycetoma or Madura disease) are indis- 
 tinguishable in microscopic features from streptothrix actinomyces. 
 
 As found in the tissues of oxen affected with actinomycosis, the filaments, as a rule, 
 terminate in clubbed enlargements, and radiate from a central mass. In the lesions of the 
 human subject the clubs are by no means regularly present, nor is a radial disposition of the 
 filaments always obvious. 
 
 Such differences possibly indicate that the streptothrix causing the disease known clinically 
 as actinomycosis in man and the lower animals is not always identical, the existence of 
 pathogenic varieties (which have received special names) having been demonstrated by 
 means of artificial cultures. 
 
 Owing to its anomalies, the classification of this group of organisms has been long a 
 subject of controversy, which has been terminated, at least for a while, by raising it into a 
 distinct class of fungi under the name of streptothricise. 
 
 The group includes non-pathogenic as well as pathogenic forms, and comprises micro- 
 organisms which, like hyphal-fungi or moulds, form a mycelium of branching filaments 
 originating from spherical spores ; certain of the filaments (like the aerial hyphse of moulds) 
 ru>e into the air from the mycelium and produce at their extremities chains of spherical 
 elements comparable to spores, though of a different morphological nature from, e.t;,, the 
 endo-spores of bacilli. 
 
PLATE XXIL 
 
 >?->'..,. 
 
 ^w 
 
 ../: ;; /" ; ' 
 
 ;: .. , - > J -^ 
 
 Fig. 2.Streptothrix Actinomyces. 
 
 MEDICAL ANNUAL, 
 
PLATE XXIII. 
 
 FIG. i. -STREPTOCOCCUS PYOGENES. 
 
 Broth culture of forty-eight hours' growth, incubated at37C., from the pus of one of the 
 subcutaneous abscesses which arose during the course of puerperal septicaemia. 
 
 Carbol fuchsine, washed in dilute acetic acid. The chain on the right hand side of the 
 figure is added from a cover-^lass preparation of the pus of an axillary abscess which showed 
 large numbers of such without any staphylococci. The micro-organism, which is best 
 studied in broth cultures, presents itself as micrococci arranged in chains of varying length. 
 The component elements are almost everywhere Hattened, and occur in pairs, showing that 
 active division is in progress throughout the chain. Here and there elements occur which 
 are oval or elongated in the direction of the chain, and all transitions may be traced between 
 such and the pairs of flattened cocci resulting from their sub-division. Wedge-shaped 
 forms may be met with in adaptation to the pressure arising at sudden bends. At times 
 division of a component co cus takes place in the long axis of the chain, as may be seen in 
 that on the right hand side. This mode of fission may become a source of lateral branching 
 should the process of sub-division continue in parallel planes. 
 
 At times certain of the cocci will divide cross-wise into four. Some of the chains are 
 very slender, and different parts of the same chain may present marked differences in respect 
 of breadth or thickness. 
 
 FIG. 2. BACILLUS OF QUARTER-MVIL, OR SYMPTOMATIC ANTHRAX. 
 
 Cultivation in 2 per cent, glucose broth, incubated at 37 C., of twenty-four hours' growth. 
 
 The organism, like those of malignant oedema and tetanus, is a strict anaerobe, i.e., it 
 grows only in the absence of gaseous oxygen. The culture was grown in an atmosphere of 
 nitrogen by Buchner's method of removing the atmospheric oxygen with pyrogallate of 
 potassium. Carbol fuchsine, washed in water weakly acidulated with acetic acid. 
 
 The growth at this stage consists of rods of varying length produced either into simple 
 or segmented filaments. The elements composing the filaments are by no means of regular 
 length, in consequence of a continuance of their sub-division. 
 
 The breadth is less than that of the anthrax bacillus, and the apposed ends of the segments, 
 in place of being squarely cut (as in the latter ; see preceding volume), are rounded. 
 
 It must be here observed, however, that in the bacillus of anthrax squareness of the 
 apposed ends may be as wanting as in either malignant oedema or quarter-evil. 
 
 At the end of twenty-four hours few sporulating rods were present in the culture. 
 
 By the third day abund ;nt spore-formation had taken place, as is shown in Fig. 3. The 
 preparation consists of straight rods, large numbers of which are sporing. Pairs of rods 
 joined end-to-end are not infrequent. The spore forms, as a rule, at one of the extremities 
 of the rod, which it considerably exceeds in diameter so as to give rise to a drum-stick 
 appearance, much as in the tetanus bacillus, except that the spores are oval in place of 
 being spherical. 
 
 The formation of the spore is first evidenced by a swelling of the end of the rod : in this 
 enlargement the unstained spore subsequently appears. 
 
 This organism is closely like that of malignant cedema both in its cultural characters and 
 its. morphology. In the bacillus of malignant cedema, however, the spores, in place of 
 bsinsr terminal, form towards the centre of the rods. The bacillus of quarter-evil has not 
 yet been identified as a cause of disease in man ; that of malignant redema not infrequently 
 has. 
 
 Stain : Aqueous solution of gentian violet, washed in i in 10,000 solution of caustic potash, 
 followed by tap- water. 
 
PLATE XXIII 
 
 ?*... 
 
 '*""" 
 
 : 
 * v 
 
 ~ * 
 
 * 
 '-* 
 
 .!'." 
 .t" > " 
 
 / r 
 
 F/g. 1. Streptococcus Pyogenea. 
 
 Fig. 2.~Bocitlu8 of Quarter Evil. 
 
 
 /' 
 
 . 3. Bacillus of Quarter Euil 
 (sparing stage). 
 
 MEDICAL ANNUAL, 
 
PLATE XXIV 
 
 FIG. i. DIPLOCOCCUS PXEUMONL-E. 
 
 Culture on Loffler's blood-scrum, of twenty-four hours' age, incubated at 37 C. 
 
 Stained by Gram's method. The cover-glares having been prepared in the usual way, 
 are immersed ina watch-glass of aniline gentian violet for ten minutes, passed through water, 
 and then placed in Gram's iodine solution for five minutes, after which they are washed in 
 alcohol until no further colour comes away ; they are then placed on edge to dry, and finally 
 mounted in xylol balsam. 
 
 The cocci occur singly and in pairs, but mostly grouped in rows of varying length. In the 
 particular strain shown (which was isolated by Dr. J. W. Wash bourn and Dr. J. Eyre, 
 and found highly virulent when tested upon animals' the chain formation is unusually pro- 
 nounced, the organism being a streptococcal variant. 
 
 Considerably longer chains than the longest depicted were present. The cocci composing 
 the chains are actively subdividing, as evidenced by the flattened pairs of which they so 
 largely consist. Here and there unusually large elements occur in which sub-division has 
 not yet taken place. 
 
 In pure cultures the organism is unprovided with the capsule which it presents when 
 studied in the sputum and pulmonary tissue in cases of acute pneumonia. 
 
 FIG. 2. 
 
 A preparation of the heart-blood of a rabbit expejimentally infected with the foregoing 
 strain of diplococcus pneumoniae by intra-peritoneal injection. All the organisms, whether 
 single cocci or pairs, are surrounded with a thick capsule. The specimen was dyed with 
 the following modification of dahlia stain devised by Dr. A. MacConkey : Dahlia, '5 grms. ; 
 methyl green (oo crystal), 1*5 grms.; saturated alcoholic solution of fuchsine, 10 c.cm. ; 
 distilled water to 200 c.cm. 
 
 The dahlia and methyl green are rubbed up in a mortar with part of the water until 
 dissolved, the fuchsine is then added, and, finally, the rest of the water. The cover-glass, 
 after the dye is placed upon it, is held over a low flame until the steam rises, placed asicl- 
 for five minutes, washed, allowed to dry, and, finally, mounted in xylol balsam. 
 
 The appearance of the capsule under the conditions of the experiment and in the human 
 tissues possibly marks a defensive formation on the part of the bacterium to protect it against 
 the action of the cells and body fluids (Louis Jenner). 
 
 FIG. 3.-TETANUS BACILLUS. 
 
 Culture in glucose broth, grown anaerobically at 37 C. ; stained with gentian violet. 
 
 Slender rods and simple filaments. Most of the rods have sporulated, the spore being of 
 spherical form and situated at one end of the rod which, in consequence, acquires the 
 appearance of a drum-stick. Branching filaments may be met with, as in the case of the 
 tubercle bacillus. 
 
PLATE XXIV 
 
 s 
 
 i y 
 .. '* 
 
 -v % 
 
 t *. .. 
 
 \ ,.--% " ; v 
 "', '," X"- /-""" 
 
 * * . : 
 * . / 
 
 % -' 
 
 Fig, 1.Diplococcus Pneumonice (pure culture, 
 encapsulated). 
 
 % 4^i A 
 1 I * 
 
 . 2.Diplococous Pneumonia (capsulated 
 condition). 
 
 . 3. Bacillus of Tetanus. 
 
 MEDICAL ANNUAL, 2899. 
 
PLATE XXV. 
 
 FIG. i. BACILLUS PNEUMONI/E (FRIEDLANDER). 
 
 From a streak culture i>n agar, of twenty- four ho\\rs' growth, incubated at 37C. 
 
 Stained wii h aqueous solution of gentian violet, washed in caustic potash solution i part 
 to '0,000), followed by tap-water. 
 
 Thick rods of varying length, the shortest appearing as ovals or even as coccus fornih or 
 spheres. Fairs of oval (or spherical) elements, end-to-end nre not uncommon. In some <( 
 the shelter form-; one or more circular vacuoles are present. 
 
 In pure cultures the micro-organism has no capsule such as it presents when found in the 
 sputum or lung in acute pneumonia 
 
 It is not stained by Grain's method, and in this contrasts with the diplococcus pneumoni.c 
 which is found in othi-r cises of acute pneumonia, in the sputum and pulmonary tissue, etc. 
 
 FIG. 2. 
 SPIRILLUM OF ASIATIC CHOLERA (KOCH'S "COMMA BACILLUS "V 
 
 From a streak culture on agar, of twenty-four hours' growth, incubated at 37 C. 
 
 Aqueous solution of gentian violet, washed in i in 10,000 caustic potash solution, followed 
 t>y tap-water. 
 
 A group of typical, slighty-cur\ed rods is shown at the lowest part of the figure ', others 
 are selected to show varieties of form. The rods vary in length as well as in thickness. 
 Some are sharply curved like the letter C ; others are curved in opposite directions and 
 present two bends like a shallow S. 
 
 Some of the bacilli exhibit one or two circular vacuoles ; the vacuole may occupy one end 
 of the rod, which may be slightly enlarged. Although commonly rounded, the extremities of 
 the bacilli may be bluntly pointed. In addition to the simple, curved rods, there are longer 
 filamentous, undulatory forms of different lengths; these present no traces of a segmented 
 or composite structure. The proper spirillar or twisted feature of the filamentous forms is 
 appreciable only in a hanging drop made, e.g., from a broth culture. In such a drop even 
 the longest forms are twisted, as appears by the alternation of parts within and out of focu. 
 In 'iried preparations the curves are reduced to a single plane, the filament becoming 
 undulatory or serpentine without spiral twist. 
 
PLATE XXV 
 
 /-. * 
 
 5 
 
 ' & 
 
 \ * 
 '\ 
 
 ->> 
 
 Fig. l.Baeillus Pneumonia. FRIEDLANDER. 
 
 Fig. 2. Spirillum of Asiatic Cholera ("Comma 
 Bacillus "). 
 
 MEDICAL ANNUAL, 1899. 
 
OF THE HUMAN SUBJECT. 77 
 
 is carried out as follows: The best culture medium is Lbffler's 
 blood serum, though ascitic or pleuritic, or even hydrocele fluid 
 answers sufficiently well ; the medium is solidified by heat in 
 test tubes placed on the slant to furnish a surface on which the 
 microbes may be sown. The serum slant may be inoculated by 
 means of the platinum ose ; this having been first sterilized in 
 the flame is drawn over the tonsil or pharynx (over the mem- 
 branous exudation if such is present) and is then rubbed over 
 the whole surface of the medium so as to distribute the organ- 
 isms present as widely as possible. Or the serum may be more 
 effectively inoculated by means of a small swab of cotton. 
 The swabs for this purpose are made by passing a strip of cotton 
 through an eye at the end of a piece of stout copper wire 
 around which the material is then twisted for a short distance 
 
 c 
 
 Fig. 6. A reduced figure of the swab, etc., described in the text. 
 
 (Fig. 6). The opposite end of the wire is bent to afford a hold, 
 and a second piece of cotton is wrapped around near this as a plug 
 which closely fits the mouth of the test tube, into which the 
 swab is inserted. 
 
 A set of such tubes may be sterilized by being heated for an 
 hour in the hot-air sterilizer at 150 0, and can be kept ready 
 for use. When about to be employed the swab is removed from 
 the tube and applied to the most promising area of the throat ; 
 it is then introduced into the culture tube and gently moved 
 over the surface of the serum The swab may then be destroyed 
 
78 PATHOGENIC BACTERIA 
 
 in the flame. After inoculation the culture tube is incubated 
 at 37 C, having been first capped to prevent any drying of the 
 medium. Incubation of the tube is essential, and without an in- 
 cubator the further investigation is preferably handed over to an 
 expert or to one of the Associations or Institutes where such 
 work is carried out. 
 
 Culture tubes and swabs may be obtained, moreover, from 
 many such sources. After twenty-four hours' incubation the 
 growth of the diphtheria bacillus appears as small hemispherical, 
 greyish, shining colonies; where such have arisen so closely as to 
 coalesce the circular outline is wanting. As a rule, colonies of 
 other bacteria develop concurrently, such, e. g., as staphylococcns 
 pyogenes aureus, staphylococcus pyogenes albus, streptococcus 
 pyogenes, sarcina lutea, or forms of yeasts. The colonies of the 
 diphtheria bacillus cannot be distinguished with any certainty 
 by their macroscopic characters, though yellow colonies may be 
 ignored. If no suspicious colonies appear for individual exam- 
 ination, the 6se is swept over the culture and the material trans- 
 ferred to cover-glasses, on each of which a drop of distilled water 
 has been previously placed, the examination being made in the 
 dried state, by the technique fully described in Part I. 
 
 Labor may be saved by using a single, long cover-glass on 
 which a series of such preparations may be made and stained, in 
 place of separate circles or squares. 
 
 The films having been allowed to dry, they may be stained 
 with carbol fuchsine or by Gram's method, but as satisfactorily 
 and simply with Loffler's methylene blue as with any other 
 dye: 
 
 Concentrated Alcoholic solution I Solution of Potash (1-10,000) 
 
 of Methylene Blue 30 parts | 100 parts 
 
 The stain is allowed to act for five minutes and washed off 
 
OP THE HUMAN" SUBJECT. 79 
 
 in tap water, the cover-glasses being then placed on edge to dry 
 or gently pressed between filter paper; when quite dry they are 
 mounted in xylol balsam. 
 
 The diphtheria bacillns is depicted and described on Plate 
 XIX. 
 
 If no diphtheria bacilli are found, a second series of prepa- 
 rations should be made from another 6se of the same culture, 
 and if a negative result is again obtained, a third set. Where 
 any doubt enters into the morphological diagnosis it is impera- 
 tive to test the virulence of the bacillus upon animals. With 
 this object a tube of broth is inoculated from a colony of the ba- 
 cillus, and after forty hours' incubation 2 cubic centimeters of 
 the shaken culture are injected into the subcutaneous tissue of 
 the anterior or lateral abdominal wall of a guinea pig weighing 
 500 grams. The broth used for the culture is prepared from 
 minced veal which is allowed to ferment in order to remove the 
 glucose present in it, and by so doing to reduce the amount of 
 acid formed by the bacillus, the production of which acts dele- 
 teriously upon the micro-organism and diminishes the amount of 
 toxin elaborated by it. 
 
 It must be borne in mind that diphtheria bacilli present all 
 grades of virulence, the virulence at one end of the scale dimin- 
 ishing until it entirely vanishes. 
 
 There are, that is to say, diphtheria bacilli of typical mor- 
 phological form which have, presumably, lost their virulence, 
 and the injection of a broth culture of which produces neither 
 local nor general results in the animal upon which they are 
 tested. In the lower grades of virulence a local swelling only 
 results; in the higher, death ensues in from twenty- four to forty- 
 eight hours; in the case of very high degrees of virulence, much 
 
80 PATHOGENIC BACTERIA 
 
 smaller doses of such a broth culture are lethal within the same 
 time; death may be deferred for seven or even twenty-one days 
 when the virulence is low. 
 
 The crucial test, however, as to whether a bacillus is truly a 
 diphtheritic one is not, strictly speaking, its possession in gen- 
 eral of a pathogenic property, but of one so specific that the lo- 
 cal or general action of a broth culture is inhibited by the pre- 
 vious injection of the animal with anti-diphtheritic serum. 
 
 The true diphtheria bacilli, again, are characterized physi- 
 ologically by producing an acid reaction in a 1 per cent, glucose 
 broth. This reaction is not given, e.g., by Hof mann's bacillus 
 a form sometimes present along with that of true diphtheria, or 
 met with in cases of sore throat where none of the true forms 
 occur (see Plate). 
 
 Lastly, it is a fact, with much practical bearing, that bacilli 
 having the morphological characters of those of diphtheria, and 
 highly virulent, as tested upon the guinea pig, may be isolated 
 from the throats of persons, who themselves exhibit no disease 
 and have not suffered from diphtheria; since such individuals, 
 whilst themselves immune, may serve as carriers of infection. 
 This has been particularly shown in the case of outbreaks of 
 diphtheria in schools. 
 
 And, what is equally important, after a diphtheritic patient 
 has quite recovered from the disease, that is to say, after having 
 acquired an immunity from the disease by reason of having had 
 it, he may bear about diphtheria bacilli in the throat for months, 
 harmless enough to himself, but capable of conveying the dis- 
 ease to others. It becomes, hence, strictly necessary to make 
 repeated bacteriological examinations of the throat after conva- 
 lescence is established; and if the patient is to be no longer a 
 
OF THE HUMAN SUBJECT. 81 
 
 source of danger to others, his isolation may be maintained so 
 long as the bacilli persist. 
 
 Formulae of the stains referred to in this volume : 
 
 Carlol fuchsin (Neelsen's solution'): 
 
 Fuchsin 1 gram 
 
 Absolute Alcohol 
 
 Aqueous solution of Carbolic 
 
 10 cubic centimeters (c.c.) 
 
 Aqueous solution of methylene Hue: 
 
 ylene Blue 2 
 
 lute Alcohol 15 c.c. 
 
 Acid (5 per cent.) 100 c.c 
 
 Methylene Blue 2 grams | Distilled Water 85 c.c. 
 
 Absolu 
 
 Loffler's methylene blue : 
 Concentrated alcoholic solu- 
 
 tion of Methylene Blue 
 
 30 c.c. 
 
 Solution of Caustic Potash in 
 distilled water (1 in 10,000) 
 100 c.c. 
 
 Aqueous solution of gentian violet : 
 Gentian Violet 2.25 gram | Distilled Water -100 c.c. 
 
 Or the following may be used, except for the coloration of 
 the hanging drop (see p. 28). 
 
 Gentian Violet 1 gram I Distilled Water 80 c.c. 
 
 Absolute Alcohol 20 c.c. | 
 
 As gentian violet is a basic dye, the washing of cover-glasses 
 after staining with an aqueous solution of this re-agent is best 
 carried out in tap water, which is naturally slightly alkaline; 
 distilled water extracts more of the dye, and acidulated water 
 yet more. Better than tap water, however, is a solution of caus- 
 tic potash in distilled water, one in 10,000, the use of which for 
 this purpose was devised by Mr. Arthur Mead. The cover- 
 glasses, after staining, are passed through two saucers of the 
 potash solution, and finally through tap water. 
 
82 PATHOGENIC BACTERIA. 
 
 A series of comparative trials has shown that the violet is in 
 this way rendered more intense, and the result of the stain more 
 certain. Or the potash, as suggested by the author, may be 
 added to the dye. The following formula of Mr. Mead's gives 
 excellent results, the specimens being washed in tap water after 
 the use of the stain. 
 
 Potassic gentian violet : 
 Gentian Violet . 1 gram 
 Absolute Alcohol 20 c.c. 
 
 Caustic Potash Solution in dis- 
 tilled water (1 in 10,000) 
 
 100 c.c. 
 Concentrated alcoholic solution of gentian violet : 
 
 Gentian Violet 25 gram | Absolute Alcohol 100 c.c. 
 
 Gram's method : * 
 
 (1) Aniline gentian violet : 
 
 Concentrated alcoholic solu- I Aniline Water 1,000 c.c. 
 
 tion of Gentian Violet 11 c.c. | 
 
 The solution is to be freshly made, and filtered before use. 
 Aniline water is prepared by well shaking 4 c.c. of pure aniline 
 with 100 c.c. of distilled water, and twice filtering through 
 paper, first moistened with distilled water. 
 
 (2) Iodine solution : 
 
 Iodine 1 gram I Distilled Water 300 c.c. 
 
 Iodide of Potassium 2 
 
 The preparation of distilled water for the purpose of 
 making cover-glass films (described on p. 26), may be ex- 
 peditiously carried out by allowing the steam of a beaker or 
 kettle to condense on the clean under side of a flat capsule, par- 
 tially filled with cold water, and afterward quickly inverting the 
 latter, when ample will be found on the side brought uppermost. 
 
 * [The thin smear is dried on the cover-glass, fixed by heat, stained with aniline 
 gentian violet for five minutes, immersed in the iodine solution one or two minutes, 
 washed in strong alcohol until nearly colorless, dried and mounted with balsam. 
 
I.U