I . tt l- granules in the diphtheria bacillus, but the granules were quite small. Except the diphtheria bacilli, very few other organisms give this reaction, and it may prove, as Dieudonne remarks, of importance in the differential diagnosis. He, however, adds that Neisser only obtained this reaction in one race of pest bacilli. “In many cases, a diagnosis may be made by microscopic examination alone, as in no other known condition than plague, do bacilli with the morphological characters of the plague bacillus, occur in the lymphatic glands. An examination of the blood will only give positive results in severe cases. And in every instance, on the occurrence of the first suspected case, every care to exclude possibility of doubt should be used before a positive opinion is given.” (Muir and Ritchie.*) * Muir and Ritchie : Bacteriology. 3rd edition, p. 428. 184 CLINICAL DIAGNOSTIC BACTERIOLOGY. Actinomycosis. Actinomycosis, although rare, is just one of those diseases in which a microscopic examination of the pus, sputum, etc., is absolutely essential for its recognition, and as it is, in not a few cases, a curable disease, this is of vital importance. Cultures are quite unnecessary for diagnostic purposes. According to Sheridan Delepine* actinomycotic lesions in man have undoubtedly been repeatedly mistaken for chronic abscesses, tumours, chronic tubercular, or syphilitic lesions. More than half the cases occur in the region of the head and neck ; thus Leith found that out of 430 cases, actinomycosis attacked the head, neck and tongue in 5575%, the abdomen in 2 r6 %, the lungs in 1 3 • 2 5 °/, the skin in 2 '5 % whilst in the remaining 6 ’9 % of cases the primary seat was doubtful. The commonest position in man is the lower jaw. Actinomycosis attacking the lungs simulates bronchitis, broncho-pneumonia, pleurisy, and especially tuberculosis. “ These patients puffer from cough, fever, night- sweats, sometimes haemoptysis, and their expectoration, which is muco-purulent or purulent, contains the ray fungus.” (Wurtz).t Godleell believes that actinomycotic disease of the chest is much more * common than is generally * Sheridan Delepine: “Actinomycosis." Encyclopaedia Medica, 1899. Vol. i., p. 09. t Wurtz : Bact^riologie. Paris, 1897. p. 483. 11 Fowler & Godlee. Diseases of the Lung' : p. 453. ACTINOMYCOSIS. 1 8.5 supposed, and has seen five cases in a little over a year. He says the expectoration in a case of this disease may be simply mucus, or may be purulent, and it is sometimes stated to be rusty like that in pneumonia. Sometimes a patient will say that he has expectorated at one time a large quantity of offensive material. He further remarks that a diagnosis may be made by simply finding, with a lens, or with the naked eye, the yellow particles which, though somewhat like crystals of iodoform, are paler in colour and globular, not flat. They are sometimes present in great abundance, but are not often met with in very large numbers. Godlee makes the following suggestive remark : “ If on opening an abscess which is supposed to be of considerable size, either connected with the rib or pleural cavity, or spine, or kidney, or caecum, the amount of pus is found to be unexpectedly small, and the haemorrhage is very free, suspicion should be aroused ; and, if after a few days of free drainage, it is found that insidious burrowing of the matter is taking place beneath the skin, a careful search should be made for the granules of actinomyces.” It must, however, be remembered that in a small number of cases, tubercle bacilli and actinomycosis may occur together in the lung of the same patient. The character of the discharge, when an abscess has opened, varies somewhat in different positions. It may be viscid, or lumpy, or like ordinary laudable pus, or it may be thin and yet tenacious. The sputum may be fetid, viscid, and yellow, with small greenish masses in it. If left to stand it forms two layers, an upper mucus and a lower gluey layer, in which the fungus may be found. 1 86 CLINICAL DIAGNOSTIC BACTERIOLOGY. Naked Eye characters of the fungus. — Whatever be the nature of the pus or sputum, it will often be seen to contain innumerable minute specks, which consist of the organism embedded in a layer of pus cells. Wurtz* states that it is necessary to examine the pus microscopically as soon as possible, as “the fungus rapidly undergoes alteration, and the character- istic form of the parasite may not be found the next day.” He quotes Guermonprez, who says, that it is often very difficult to make a diagnosis, even with the aid of a microscope. If a little of the pus be spread out on a piece of glass, in a developing dish, or shaken up in a test tube with a little water, the actinomycotic grains will easily be seen, often in large numbers. These vary in colour ; usually, however, they are yellowish or brownish by reflected, and greenish by transmitted, light. According to Bostromt the young actinomycotic granules are transparent-grey, almost like mucus, especially in human beings ; the older granules are opaque-white ; whilst the still older ones are yellowish, yellowish-brown or yellowish-green, in colour, according as the threads or clubs are most prevalent. Those found in the intestinal canal are of a darker colour, owing to the action of the sulphur on them ; whilst the fungi found in cattle are usually of a sulphur yellow tint. They have been likened to grains of iodoform in the pus, but Muir and Ritchie II state that the distinctly * Wurtz : loc. cit., p. 482. t Schlegel : “ Aktinomykose.” Handbuch der path. Mikroorganismen. Vol. ii. , p. 863. || Muir and Ritchie : Manual of Bacteriology. 3rd edition, p. 270. ACTINOMYCOSIS. i37 yellow colour is only occasionally found, “ in fact in the human subject they occur much more frequently as small specks, of semi-transparent appearance, and of greenish-grey tint.” They are generally of a soft tallow-like consistence in man, but in cattle they are not infrequently hard and gritty, owing to the presence of calcareous deposits. The size of these granules varies considerably. Sometimes they are only seen on microscopic examina- tion, being about o'oi to o-2 mm., usually, however, they are quite visible to the naked eye, and attain a diameter of 075 mm. They are described as usually being the size of a grain of sand, pin’s head, or millet seed, seldom as large as a poppy seed or small pea. (Kitt). Microscopic appearance. — A few of these grains are picked out of the discharge, or taken off the dressing with a needle, transferred to a slide, and covered with a cover-glass. Examined with a one- inch objective, they will appear as more or less spherical masses of a pule greyish-yellow or greenish- yellow tint, somewhat like a minute raspberry, with a coarsely granular surface. If the cover-glass be gently pressed down, the grains, which flatten out like specks of tallow, will now be seen to have separated into irregular, or wedge-shaped fragments of a faintly brown colour. If the spherical, oblong, or reniform masses, of which the tufts are composed, be examined with a higher power— e.g., ]/« inch objective— they will appear as a rosette of clubs. These simple means are generally sufficient for diagnostic purposes, and the character of the organism can easily be recognised with a magnification of 100 to 300 diameters. 1 88 CLINICAL DIAGNOSTIC BACTERIOLOGY. Schlegel says that stained film preparations have no advantage for diagnostic purposes, as the character- istic appearance may be lost by the crushing of the tufts, whilst the uninjured tufts may become too opaque when stained. Stained preparations are, however, necessary for the examination of the finer structure of the fungus. When examined under a higher power, either in preparations teased in water, or in three-quarter per cent, salt solution, and mounted in water or glycerine, or in stained film preparations, the following parts may be made out (Plate II. Fig. 9) : — I he centre of the fungus consists of interlacing filaments of o'3 to o’5 /j. in diameter, forming a loose or dense network. \ hese filaments are wavy long threads, often branched, which radiate towards the periphery. They usually stain quite regularly, but in some, especially the older colonies, cocci, spore- like or rod-like bodies may be seen. Among the filaments are seen numerous cocci, measuring o-5 /i in diameter, short bacilli-like rods, or short threads. These small, round cocci may be found in a filament, arranged something like strepto- cocci, or free, in the centre of the young colonies. They have been spoken of as spores or conidia. Probably the cocci and the short rod-forms are developed from a filament, or may themselves grow into longer threads. Outside the central felt-work, composed of filaments and cocci, we have the thickened clubbed endings of the radiating filaments, as well as projecting filaments from the inner zone. # Schlcg'el : loc. cit., p. 864. ACTINOMYCOSIS. I 89 The clubs are pear-shaped bodies, formed by a hyaline swelling of the sheath of the filament, which are arranged in a radial manner ; hence the term ray fungus given to the actinomycotic parasite. They have their thin end directed towards the centre, the thickened end pointing outwards. The clubs vary in size, but measure, on an average, 10 to 60 fj- in length, and up to iom in breadth, and may show branching. The relation of the clubs to the threads is well seen when preparations are stained with orange-rubin, the former being stained crimson, the threads blue. In the younger clubs the protoplasm of the thread may be seen in the interior of the club. The proportion of clubs to threads varies in different species. In man, especially, in rapidly growing colonies, threads with comparatively few, or even no clubs may be found, although, as a rule, both clubs and threads are present, whilst in actino- mycosis in cattle, the clubs generally appear early, and are much more numerous, and the filaments, especially if calcification has begun, often become indistinct. Muir and Ritchie* state that in the human subject the clubs are often fragile structures, which are easily broken down, and may even dissolve in water, and for this reason are well seen in the fresh condition, but may be indistinguishable in hardened preparations. Around the whole actinomycotic follicle a layer of inflammatory cells, sometimes containing giant cells, may be seen, especially in sections. Methods of Starnwg. — Film preparations, or sections, can be stained with various aniline dyes, * Muir and Ritchie : loc. ci/.t 3rd edition, p. 273, IQO CLINICAL DIAGNOSTIC BACTERIOLOGY. and of these Gram’s method, or Weigert's modification, with or without special after - stains, gives most excellent results. For the preparation and staining of sections, I must refer the reader to the larger text-books of pathology and bacteriology. In order to obtain film preparations, some of the small yellowish - greenish nodules, picked out from the pus, sputum, or from the scraping of the growth or granulation tissue, are placed in the centre of a slide, and by means of another slide, the material is spread out. Here it is of more importance to avoid breaking up the structure of the fungus than it is to obtain perfectly thin films. The films, after drying in the air, are fixed by passing them over the flame in the usual way ; or, better, by immersion in alcohol, or alcohol and ether, for a few minutes. Of the single dyes, staining in carbol-thionin for one to two minutes gives very good results. Gram’s method, with after-staining with eosine, is very suitable, as by this means the threads and cocci are coloured violet, the clubs pink. It must be remembered that in actinomycosis in man the threads retain Gram, the clubs do not ; but in cattle, the clubs, as well as the threads and cocci, retain Gram’s stain. Bostrom* stains cover-glass preparations with aniline water gentian violet, as in the first stage of Grams method ; but, instead of then using the iodine solution, he decolourises the preparation in alcohol containing eosine or picric acid. Film preparations, as well as sections, can also be * Encyklopadie der mikroskopischen Technik. 1003, p. 8. ANTHRAX. I9I stained with haematoxylin and eosine, or orange -rubin. Crookshank* says that in Gram’s method, with after-staining in orange rubin, we have a test that is as characteristic and useful as the stain for the tubercle bacilli ; the filaments and cocci are stained blue, the clubs crimson, and the tissue elements pink. By this method the prolongation of the blue threads into the clubs can be very clearly seen. The streptothrix madurse, the fungus found in madura foot or mycetoma, is closely allied to the actinomyces. Anthrax. Anthrax is found in man in the form of (1st) a cutaneous disease— the so-called malignant pustule, (2nd) an intestinal disease, and (3rd) as a pulmonary disease, e.g.% woolsorter’s disease or anthracaemia. It is more common in animals, and is conveyed to man from the carcases, skin, hair, and bodies of infected animals. It is possible in many cases to diagnose the disease in man or animals by means of a microscopic examination of stained preparations alone, although it is advisable to confirm this by cultures and inoculations into animals. In support of this statement, Belli says, that in man “a positive diagnosis may be made directly by * Crookshank : Bacteriology and Infective Diseases. London, 1896. p. 433. t Bell: “Anthrax,” Allbutt’s System of Medicine. Vol. ii., p. 535, ig2 CLINICAL DIAGNOSTIC BACTERIOLOGY. the microscopic examination of the fluid for the bacillus anthracis ; or indirectly by inoculating- a white mouse with a drop of serum from a vesicle.” Hewlett* says that in animals it is often possible to' make a diagnosis by means of cover-glass preparations, and “ the stained preparations can be kept and produced in a court of law.” He also remarks that cultivations on agar or gelatine may give positive results when the microscopic examination has been negative. The inoculation of a guinea pig or mouse should be made in cases of doubt, as this, he states, is by far the most certain method of diagnosis, a negative result being nearly as valuable as a positive one. He, however, draws attention to the fact that both cultivations and inoculations may fail to give positive results if the material be old or putrid. Description of the Bacillus Anthracis. If the blood or spleen juice of an animal, or under some conditions, of man, attacked with anthrax, be examined in the fresh and living condition, a large number of rod-like bacilli will be seen amongst the red blood corpuscles. These are in length about the diameter of a red blood corpuscle, and measure, on an average, 4-5 to to 10/i long, and 1 to i^/x broad. They appear as homogeneous structureless cylindrical rods, with, in the fresh condition, rounded ends, and show no sign of movement. They occur singly, or more usually joined together in twos or threes ; longer thread forms, though * Hewlett ; Bacteriology- 2nd edition, p. 192, PLATE II. Fig-. 9. — Actinomyces. The central portion consists of filaments stained blue, and the peripheral part shows the clubs stained pink. Stained by Gram’s method and orange-rubin. Fig. 10.— Spirillum of Relapsing fever; blood film preparation stained with eosine and methylene-blue. Fig. 11. — Plague Bacilli in the pus from a bubo. Stained with eosine and methylene-blue. Fig. 12. — Anthrax Bacilli in pus. Stained with eosine and methylene-blue. Fig. 13. — Tricophyton microsporon, or small spored ringworm in Hair. Fig. 14. — Tricophyton megalosporon, or large spored ring-worm in Hair. Fig. 15. — Achorion Schoenleinii, or Favus fungus, in Hair. Fig. 16. — Microsporon furfur, the fungus of tinea versicolor. [Figs. 13 to 16 after Malcolm Morris.] PLATE H. F .L. Coles, del ANTHRAX. 193 common in cultures, are rare in fresh blood preparations. The anthrax bacillus, as seen in stained film preparations, appears somewhat different from that seen in the fresh condition. It stains easily with any of the ordinary aniline dyes, and retains Gram’s stain. When examined under these conditions, the bacilli are no longer symmetrical rods with rounded ends, as is the rule in fresh preparations, but show some thickening at each end, which is abruptly cut off, and this is actually concave instead of convex, so that, when two bacilli lie together, there is a biconvex space between them. (Plate II. Fig. 12.) Sobernheim* has compared a chain of anthrax bacilli to the appearance of a bamboo cane, with its characteristic thickenings at regular intervals, and says that, although this is not seen in all cases, yet it is often found, and is somewhat characteristic of these bacilli. In blood, except perhaps in swine, chains are never made up of more than five or six segments, whilst in cultures they may be of almost unlimited length. (Hewlett.) Mac6t in his account of the bacillus, says: — “With the aid of a good objective and staining reagents, thin partitions, of which the length is more than double the breadth, are seen separating the bacilli. Often on the other hand, the bacilli making up a chain, appear separated from each other; between the two succeeding elements there occurs a clear space which is irregular in outline, due to the fact that the * Sobernheim : “ Milzbrand.” Handbuch der path. Mikro-organismen.” Vol. ii., p. 9. t Mac£ : Traite practique de Bacteriologie. Paris, 1901. p. 483. 0 194 CLINICAL DIAGNOSTIC BACTERIOLOGY. bacilli are not cut off at right angles, but terminate in a slightly sinuous line. This is a characteristic on which Koch strongly insists, and which he considers as characteristic of the bacillus anthracis. It enables us to distinguish this species from other bacteria, which are likely to be met with in the same conditions. “ It is only seen in fixed and stained preparations, and this suggests that it is due to the action of the reagents used ; but it loses none of its value from that. This characteristic is not ordinarily seen in preparations made from cultures.” Sometimes the bacilli, instead of being arranged in a straight line, lie at right angles one to another. The bacillus anthracis is provided with a capsule. This is not present in all, and Serafini, who first noticed it, said that it was only to be found in fresh material, not in cultures. Other observers, whilst agreed that it is commonly met with in blood and tissues, have found it in some cultures. For the demonstration of the capsule, any of the methods used for staining this structure described under the pneumococcus (page 136) may be used. Under certain conditions, particularly the presence of oxygen, suitable temperature and culture media, the anthrax bacilli form spores. These are never found in the blood or tissues during life, and even some time after death, but are always met with in aerobic cultures, in which nearly every segment of the bacillus contains one. The anthrax spores are extremely resistant, and withstand, according to Koch, boiling for five minutes. The fact that spore formation never occurs within the bodies of animals suffering from anthrax, unless ANTHRAX. X95 the bacilli are exposed to the oxygen of the air, is a matter of vital importance, as carcases of infected animals should not be opened, but buried entire with lime. The examination for diagnostic purposes is usually made from a small piece of the ear which is cut off. Method of Examination. — In man , film preparations should be made from the fluid of the vesicles, from the scrapings of the incised or excised so-called pustule, in cases of the local form of the disease. The blood should be examined, when the disease takes the form of a general infection, either by thin films, or by Ross’s method of making thick films, as described under plague. In animals , films should be made from the blood, or after death in cases in which the animal has been opened, from the juice of the spleen. J. McFadyean* states that : — “ When an anthrax carcass is left unopened, the invasion by putrefactive bacteria is sometimes so complete within 24 hours, that not a single anthrax bacillus can be detected by microscopic examination in any of the organs in the chest or abdomen ; but in the blood of the ears or feet, the anthrax bacilli may be recognisable on the third day after death. “ When an animal is unexpectedly found dead, and anthrax is suspected, if the carcass is already partially putrid, blood from an ear or a foot ought to be examined in preference to spleen pulp, or blood from one of the large veins of the body. At the present time, the material sent to the laboratory for examina- tion in suspected cases of anthrax, is almost always * J. McFadyean : Journal of the Royal Agricultural Society. No. 18. 1894. 196 CLINICAL DIAGNOSTIC BACTERIOLOGY. the spleen or part of it, and in a considerable proportion of cases, a positive opinion cannot be given because of putrefactive changes.” Films should be fixed by immersion for a few minutes in absolute alcohol, or alcohol and ether, but also, though not quite so satisfactorily, with heat. The anthrax bacillus stains readily with any of the aniline dyes, and especially well with Gram’s method. Such films may then be stained with Loffier’s methylene-blue, dilute carbol - fuchsin (1 in 10), or thionin, and also with Gram’s method with eosine in the ordinary way. Films may also be double-stained with eosine and methylene-blue, either separately or combined, as in Jenner’s or Leishman’s method, as described under gonococci and plague. Films made from the malignant pustule generally show the characteristic bacilli in good numbers, but sometimes they are scanty ; whilst in blood films, anthrax bacilli are seldom seen in man, except in very severe cases shortly before death, and even then they may be absent. Possibly, the examination of a considerably larger drop of blood by Ross’s method of making films, may render the diagnosis in this medium more successful. Special Methods of Staining. — The capsule of the bacilli may be shown by using one of the special stains for this purpose — eg ., F'riedlander’s, Johne’s, Klett’s, Muir’s, etc., mentioned under the pneumo- coccus. (See page 136.) Sobernheim states that Zettnow’s modification of Romanowsky’s eosine methylene-blue, stains the bacillus and its capsule exceedingly well. The body of the bacillus is stained blue, its contained chromatin red, and the capsule a pale reddish colour, ANTHRAX. l9l7 Probably, Leishman’s modification of this stain, used as described under the plague bacillus (page 180), would give equally good results. Spores in anthrax bacilli from cultures may be stained with Moller’s* method as follows : — 1. The air - dried film is passed three times through the flame, or placed in absolute alcohol for two minutes. 2. Place in chloroform two minutes, to remove any fat. 3. Wash in water, and treat with 5 / chromic acid for two minutes. 4. Again wash, and drop filtered carbol-fuchsin on the film, and heat this over the flame, just as in tubercle bacillus staining, for one minute. 5. Wash, and decolourise in 5 °/Q sulphuric acid. 6. Wash thoroughly, and counter-stain with a watery solution of methylene-blue. 7. Wash, dry, and mount in Canada balsam. The spores stain red ; the body of the bacillus blue. Sobernheim speaks most highly of A. Klein’s methodt of staining spores as being most reliable for the anthrax bacillus. The following is the pro- cedure : — 1. Equal quantities, about 1 to 2 cc.m., of filtered carbol-fuchsin and of an emulsion of the spore-bearing bacilli in physiological salt solution, are mixed together in a watch glass, which is gently heated over a flame till steam rises, for about six minutes. * Encyklopadie der Mikroskopischen Technik., 1903, p. 901. t Handbuch der path. Mikro-organismen. Vol. i. , p. 424. 198 CLINICAL DIAGNOSTIC BACT ERIOLOGV. 2. At the end of that time, a small drop of the mixture is taken out by means of a platinum loop, and spread in a thin layer over cover-glasses or slides. These are left to dry in the air, and then fixed, by passing them twice through the flame. 3. Decolourise in 1 % sulphuric acid for a few (one to two) seconds. 4. Wash in water. 5. Counter-stain in dilute methylene-blue solution, without heat, for three or four minutes. Sobernheim suggests that if the above mixture is made in a test-tube, and heated over a gas flame, the staining is more rapid and intense, without in any way injuring the form of the bacilli. The Differential Diagnosis. — The most probable organism met with in disease in man which might be mistaken for the anthrax bacillus, is the bacillus of malignant oedema. The Bacillus of Malignant (Edema, or vibrion septique of Pasteur, occurs in man in a condition of inflammatory oedema associated with emphysema, and ultimately followed by gangrene, which is known as malignant oedema. The bacillus, which is about 3 u long and 1 m broad, occurs in the form of rods or long filaments, some of which are motile. It forms spores, which are generally situated in the centre, sometimes towards the end, of the bacillus, and these are oval in shape, and somewhat thicker than the body of the bacillus. They do not stain by Gram’s method. Spores never occur in the thread forms, only in the rod-shaped bacilli. Comparing this bacillus with the anthrax bacillus, it RELAPSING FEVER. 199 will be seen that the bacillus of malignant oedema is somewhat smaller in size ; it has not the rigid cylindrical appearance ; it possesses large oval spores, even in tissues, which cause the body of the bacillus to swell out in the centre, or towards the end ; it possesses flagella, and is motile ; it has no capsule, and does not stain with Gram’s method. The absence of a capsule is, according to Sobernheim, an important point in its differential diagnosis from the bacillus of anthrax. The Spirillum of Relapsing Fever, or the Spirochsete Obermeieri. This is a good example of a disease which can only be diagnosed with absolute certainty by means of the microscope. Relapsing fever has an incubative period of about seven days, at the end of which an attack of fever occurs, lasting usually from five to seven days. This is abruptly terminated by a crisis, and is followed by an apyrexial period, or “first period of apyrexia,” which usually persists for a week. At the end of this time a relapse, or second paroxysm, comes on, and is similar to, but is generally a day or two shorter, than the first attack. This in turn is followed by a “second period of apyrexia,” which usually coincides with that of convalescence. It is common to see two attacks of fever ; much rarer to find one, or three, or four paroxysms. 200 CLINICAL DIAGNOSTIC BACTERIOLOGY. The specific organisms appear in the blood shortly before an attack, rapidly increase in number during the pyrexia, and begin to disappear just before the crisis. 1 hey may be found in very large numbers during the fever, but are entirely absent, or practically so, from the blood during the apyrexial period. They reappear at the next paroxysm, but disappear when that is over. There is no relation between the number of spirochsetes found in the blood and the intensity of the attack. (Wladimiroff.)* The spirochsetes are long, slender, spiral or sinuous filaments, measuring io, 20, or 40 fi, or more in length, and at most 1 m in breadth. The number of spiral turns varies from six to twenty. (Plate II. Fig. 10.) “ In fresh blood, the spirillum is seen to be flexible and very active. Its movements are progressive, with undulations passing wavelike along from one extremity to the other. It is so fine, that under a low power its presence is only revealed by the commotion amongst the blood corpuscles, which by the rapid movement of the spirilla are thrust violently aside.” (Wesbrook).t The ends of the spirillum are sharp and tapering. Heydenrich distinguishes three movements in the spirilla, a twisting on the long axis, bending to one or other side, or a forward or backward movement of the whole organism. Wladimiroff says that although many text books mention that the spirillum possesses flagella, this has not been proved. * Wladimiroff : “ Riickfallfieber.” Handbuch der path. Mikro- organismen. Vol. iii., p. 78. t Wesbrook : “ Bacteriology of Relapsing Fever.” Allbutt's System of Medicine. Vol. i., p. 951. relapsing Lever. 201 The spirilla have a considerable degree of vitality outside the human body. Heydenrich found that their motility could be preserved for 2\ to 14 days, according to the temperature at which they were kept, and in the bodies of those who had died during the crisis, they were found in a postmortem made 40 hours after death, although all their movement usually ceases 24 hours after the death of the patient. In the blood of the living, spirilla may be found some hours before the crisis, and may persist through a pseudo-crisis, but after a true crisis they generally disappear rapidly, and are not to be found in the apyrexial period between two true attacks. (Wesbrook). According to Carter, they may at their height be in the proportion of Y20 to Yio the number of the red blood corpuscles. Method of Examination. — The blood may be examined in the fresh state, when the movements of these characteristic organisms can be seen, or in dry stained film preparation. To demonstrate them in the fresh condition, the lobe of the ear, or tip of the finger of a patient during the febrile period, is pricked, and the exuding drop of blood, caught on a clean cover-glass, held by forceps, is placed with the drop downwards on a slide, and the blood allowed to spread out by the weight of the cover-glass. Any movement of the red cells should attract attention, and with a suitable magnification, and with the iris diaphragm somewhat closed, these very transparent motile organisms may be seen moving between the corpuscles. Sometimes, even with a high magnification, it is not easy to detect the spirilla, especially when the corpuscles are thick, and even in the clear spaces, 202 CLINICAL DIAGNOSTIC BACTERIOLOGY. th ese fine threads have much the same refractive index as the medium in which they move, and can be overlooked. If it is desired to preserve the spirochsete, the blood should be collected in pipettes, and the end of these sealed in the flame. Mamurowski places a drop of Muller’s fluid on the finger, and pricks through this, so that the exuding blood mixes with the fluid, and is fixed at once. This is said to give very good results. Weigert uses for the same purpose salt solution or osmic acid, and points out that the skin should not have been previously cleansed with alcohol, as this may cause coagulation of the blood, and so obscure the organisms. Leptochinsky spreads thin films of blood, and examines these dry, without any further treatment, and in dry preparations the spirilla are said to be easily seen, but even here if the film be thick they may be obscured. Albrechts treats the air-dried films several times with acetic acid, and then washes and dries them ; by this means the haemoglobin is removed, and only the nuclei of the leucocytes with their granules and the spirochaete are seen. The more active the movement of the spirilla before the film is made, the more cracked and irregular will they appear. Staining. — Air-dried films, after fixation in alcohol and ether, may be easily stained with any of the ordinary aniline dyes, e.g., thionine, methylene-blue, fuchsin, etc. They may also be double-stained with eosine and methylene-blue, separately or combined, by Jenner’s or Leishman’s stain, as described under gonococci. The films of blood may be either the ordinary thin PARASITIC FUNGI. 203 preparations, in which all the elements of the blood, as well as the parasite, are seen, or Ross’s method of preparing thick films may be used, and either of these preparations can be very satisfactorily stained with Leishman’s modification of Romanowsky’s stain, as described in detail under plague bacilli (page 180). Parasitic Fungi, affecting the SKin and Hair. The following fungi are those with which we are chiefly concerned from a diagnostic point of view. In ringworm we find the small spored fungus, Microsporon Audouini, or the large spored fungus, Trichophyton megalosporon : in favus the Achorion Schoenleinii : in tinea versicolor the Microsporon furfur. Other rarer forms, viz., the Microsporon minutissimum causing erythrasma, and Tinea imbricata, a tropical form of ringworm, also occur. Microsporon Audouini, or Tricophyton Microsporon. This, the small spored form, is by far the most frequent, causing 80 to 90 % of all forms of ringworm found in London. It attacks the scalp, face, and neck of children, not adults. It is important to distinguish it from the other forms, as it is very refractory to treatment, and practically only attacks children. With the naked eye it will be seen that almost every hair over an infected patch is diseased, fragile, or broken off, leaving comparatively long stumps. 204 CLINICAL DIAGNOSTIC bacteriology. The stumps have a white parasitic sheath extending over them throughout the intra-follicular portion, and for 3 mm. beyond the exit of the hair (Colcott Fox).* Microscopically. This is a small fungus affecting both the inside and outside of the hair. The fungus on the outside consists of spores over the bulb, and extending round the shaft of the hair up to the broken end, like a bark. Inside, the fungus forms segmented mycelia, which may be beaded. These mycelia attack chiefly the superficial parts of the hair, but terminate just above the bulb in a very characteristic fringe, and it is at this point that the hair is very apt to break. In the scrapings the fungus is seen as coarse and irregularly branching mycelia, which, although varying in length, are generally short. The branching usually takes place at right angles to the main thread. According to Sabourand the spores of this variety measure about 2 to 3 u. Jamieson says that the mycelium is so fragile that it is seldom visible, but its special position is in the substance of the hair, and it forms an enveloping sheath round it. (Plate II. Pig. 13.) Spores, he remarks, are found both inside and outside the hair, in large masses. Trichophyton Megalosporon, or large=spored Ringworm. The spores in this form are larger, measuring 7 to 8 jj. in diameter, and mycelium is always distinctly seen. It attacks the body, beard, nails, and sometimes the scalp, causing tinea barbae and corporis, and also some forms of tinea capitis, in which position it is * Colcott Fox: Brit. Med. Journal, 1897. p. 876. PARASITIC FUNGI. 205 always much easier to cure than the small spored variety. (Plate II. Fig. 14). It is easily distinguished from the T. microsporon by the large size of the spores and the presence of mycelium, but it may, on this account, be confused with the parasite of favus. (Jamieson).* By some writers it is divided into the endothrix and ectothrix. Morris, however, thinks this is based merely on its accidental position, and may only depend on the degree of invasion. Both forms attack the root first and grow upwards, the mycelia are regularly jointed and branch dichotomously, and the spores are arranged in chains. Colcott P'ox says that the endothrix form is distinguished with the naked eye, by the fact that there is no white sheath like the T. microsporon, and the stumps break off lower down, even in the mouth of the follicle, and become dark coloured and swollen. The spores are placed round the hair, sometimes inside— sometimes outside, or both. Malcolm Morrist states that whilst tinea circinata, ringworm on the body, has a characteristic appearance clinically, the discovery of the fungus is not always easy, even to an expert. On the other hand, cases of ringworm of the scalp are sometimes difficult to diagnose by their clinical appearance, and can only be determined with certainty by a microscopic examination. Method of Examination. — The affected hairs, which are broken, bent, or sometimes display a powdery sheath, are pulled out with a pair of forceps. It is of no use to pull out hairs indiscriminately. * Jamieson : Diseases of the Skin. Edin., 1894. 4th Edition, p. 554. t Malcolm Morris: “Diseases of the Skin." London, 1903, 206 clinical diagnostic bacteriology. Duckworth and Behrend’s chloroform test may help materially in the selection. If a few hairs from a case of ringworm of the head are placed on a slide, and a few drops of chloroform added, it will be seen, when the latter has evaporated, that all the hairs containing spores, or those in which the structure has been broken up by the parasite, assume a chalky whiteness. If chloroform be dropped on the head, after its evaporation, the skin of the scalp becomes white, and with a lens the diseased hairs are also seen to be white, whilst the normal hairs retain their usual colour. This reaction is more distinct in dark than in fair hair, and by its means the affected stumps may be seen and extracted. Jamieson* states that neither in chronic eczema, favus, or in any other condition is such an alteration produced by chloroform. In ringworm of the beard, it is necessary to examine several stumps, and if crusts are present, suitable stumps may sometimes be found on their under surface. In ringworm of the body, the margins of the rings should be somewhat deeply scraped with a scalpel, and the scales so obtained examined. In ringworm of the nails, the affected part should be deeply scraped with a sharp knife, and the scrapings submitted to strong liquor potassse for some time. The hairs, stumps, or scales are placed on a slide, and if any ointment has been used on the affected part, treated with a little ether for a minute or so, and then with a few drops of filtered liquor potassse. The slide should now be heated over a spirit lamp for half a minute, when the hairs, etc., may be * Jamieson : loc. cit.y page 560. PARASITIC FUNGI. 207 transferred to a watch glass of distilled water, and thence on to a slide, covered with a drop of glycerine, and a cover-glass put on the preparation. Sabourand uses a stronger solution of potash — viz., 40 %, both for the hair and nails. In many cases I find it more convenient, especially when many scales are present, to remove the excess of potash after the preparation has been warmed, by means of filter paper, and then examine in glycerine, or even in the liquor potasste itself. This is all that need be done for diagnostic purposes, and the only possible fallacy is to mistake some of the small fat globules for spores. For a detailed examination of the structure of the parasites, and for permanent preparations, the fungus should be stained. Malcolm Morris’ Method, slightly modified. Hairs from the margin of a suspicious patch are extracted, and the root and shaft end about a quarter of an inch long, are cut off. These are washed in ether to remove the fatty matter, and are then stained with carbol - fuchsin, or carbol- gentian-violet, either on the slide, or in a watch- glass for about half - an - hour, during which the preparation should be heated for five minutes or so. Morris points out that the small spored fungus stains very quickly in about five minutes, whilst the large spored form takes much longer. Pour off the stain, and add Gram’s solution of iodine to the preparation, and let it remain five or ten minutes. Dry the preparation with filter paper, and decolourise for a few seconds with aniline oil, or “ with a mixture 20 8 CLINICAL DIAGNOSTIC BACTERIOLOGY. of two to four drops of nitric acid in aniline for ten to fifteen minutes.” It may, instead, be placed in aniline oil to which iodine has been added, until the solution has a deep mahogany colour. It is then washed in xylol, and mounted in Canada balsam. A simpler method of making a permanent stained film is, after making a diagnosis in liquor potassm, to remove the cover-glass, and gently wash the preparation with water, or with 1 5 % alcohol in water, remove the fluid with filter paper, and then let the preparation dry in the air. When thoroughly dry, it may be treated as an ordinary film preparation, fixed by heat or alcohol, and stained by Gram’s method, or its modification. Achorion Schonleinii. Favus is very rare in England, but until recently not uncommon in Scotland. In order to prepare a specimen for microscopic examination, the hairs and crusts from a favus cup or scutulum, as it is called, should be treated in exactly the same way as was described for ringworm. When teased out with liquor potassae the favus cup is seen to consist of branching mycelia and spores, mixed with granular debris and pus corpuscles. The spores are round or oval, measuring 3 to 8 m long, and 3 to 4 /x broad, and may occur singly or in chains. The centre of a favus cup is mainly composed of spores, the margin of mycelial threads. The mycelium, which is made up of shorter pieces than the tricophyton, forms a dense feltwork, in which spores are found. Its protoplasm is somewhat granular, and the breadth of the filament variable, PARASITIC FUNGI. 209 It will be noticed that the structure of the affected hair is not as much broken up by this parasite as in ringworm, and the mycelium runs for the most part in the long axis of the shaft. (Plate II., Fig. 15). “ Hairs, affected with favus, have none of the ragged outline, nor the dissected aspect that those in tinea tonsurans have, and consequently they, when they do break, do so at a much greater distance from the skin.” (Jamieson). The parasite of favus in the hair is distinguished from that of ringworm of the head, by the absence of the chalky whiteness when chloroform is applied, there is more mycelium and fewer spores, and the spores are more generally oval than round. Microsporon furfur. If the somewhat characteristic pale fawn or brown patches of pityriasis or tinea versicolor are scraped with a knife, and the scales so obtained placed on a slide, covered with a drop of liquor potassce, and the cover-glass applied, this fungus will be readily recognised. Should it be desirable to make permanent prepara- tions, the following method, recommended by Joseph and Loewenbach* may be used. It is equally applicable for ringworm, and for the microsporon minutissimum of erythrasma. The epithelial scrapings are subjected to the action of a mixture of alcohol and ether for twenty-four hours, then treated with glacial acetic acid for about five minutes, and placed on a slide, where the pieces * Joseph and Loewenbach : Technique histologique appliqu^e la Dermatologic : Translated by Minne. Paris, 1902, p. 119. P 2 IO CLINICAL DIAGNOSTIC BACTERIOLOGY. are teased out with needles. The slide is then held over a flame, in order to slowly evaporate the acetic acid, and the preparation allowed to dry in the air. They may then be treated as dry film preparations, and stained with methylene-blue or by Gram’s method. Microscopically, favus is at once distinguished from a secondary syphilide, which it somewhat resembles, by the characteristic appearance of interlacing mycelium, inclosing groups of round spores. (Plate II. Fig. 16.) The mycelium consists of short thick curved hyphae, measuring about 7 to 13/* long, and 3 to 4 m broad. The spores, which are round, occur in clusters or groups, generally singly, seldom in chains, and measure 4 to 7 ju in diameter. Plaut* says that in stained preparations, darkly coloured globules may be seen inside the spores, lying close to, but not actually in contact with the capsule, whilst the rest of the protoplasm is either colourless or faintly stained. Not infrequently, the spores are seen to have ruptured, setting free the deeply stained granules. Microsporon minutissimum. Erythrasma, a rare disease characterised by the presence of brown patches, especially in the genito- crural region, in the axilla, and between the nates, is associated with a parasite very like the M. furfur, except in the minute size of the spores and filaments. According to Jamieson, to demonstrate it properly, carefully stained preparations and a magnification of 700 diameters are necessary. The mycelia, very fine twisted filaments which form * Plaut : “ Die Hyphenpilze." Handbuch der path. Mikro-org'anismen, Vol. i., p. 652, SERUM DIAGNOSIS. 2 I I figures of S or V, are branched and distinctly septate, a single member of which measures, according to Sabourand, 5 to 15 m long, and O’S to 13 broad. Among the branched filaments are found groups of exceedingly small, round, or somewhat angular spores. It is distinguished from M. furfur by the small size of the elements, and both microsporon furfur and minutissimum are distinguished from all the other skin parasites by the fact that the spores occur in groups, and are never distributed uniformly throughout the mycelium. Serum Diagnosis. If the blood serum, in certain dilution, from a patient suffering from typhoid or Malta fever be added to an emulsion of living typhoid or Malta bacilli, these organisms will be seen under the microscope to be aggregated or collected into clumps, the agglutination reaction If the experiment has been performed in suitable glass tubes, a sedimentation reaction occurs, and this is visible to the naked eye. Widal’s agglutination or sedimentation reaction may, therefore, be either a microscopic rapid or a macroscopic slow test. 2 I 2 CLINICAL DIAGNOSTIC BACTERIOLOGY. 1 he method is one of the greatest use for the practical diagnosis, particularly of typhoid and Malta fever. Those who have at hand cultures, media, and an incubator, use fresh cultures of bacilli, but, as Widal pointed out, the diagnosis can be made with an emulsion of dead bacilli. Collection of the Blood. — The simplest method of obtaining blood serum, is that suggested by Cabot.* The most dependent part of the lobe of the ear is punctured with a triangular pointed needle, and the ear is then “ milked strongly downwards towards the punctured spot,” into a very small test-tube, which need not be sterilized. In this way, fifteen drops of blood are easily obtained, and will yield two or three large drops of serum. As soon as the blood has coagulated — a matter of two or three minutes — the clot is separated from the sides of the test-tube by means of a piece of wire, or blade of a penknife, and the serum then escapes and can be collected. Blood may also be collected in capsules, as Wrightt has suggested. These are made by heating a piece of glass tubing in two places, and drawing it out into capillary tubes, one end of which is bent up so as to form an acute angle with the capsule. The capsule is half filled with blood by capillarity and gravity. Both ends of the capsule are sealed by heating in the flame of a match or spirit lamp. When the serum has separ- ated from the clot, it is aspirated into a capillary tube. The blood may also be collected on the surface of a slide, piece of note paper or blotting paper, and afterwards dissolved in a drop of water. * Cabot : Serum Diagnosis : 1899. t Wright : Brit. Medical Journal, Feb. 5th, 1898. p. 355, SERUM DIAGNOSIS. 213 The next step depends on whether we have at hand an incubator, media, and a reliable culture of the typhoid bacilli. If so, a tube of neutral bouillon is inoculated with some of the latter, and incubated for not more than 24 hours, at a temperature of 37 °C. The bacilli should be actively motile and free from clumps, and in order to determine this, a drop of the bouillon culture should therefore be examined under the microscope. If clumps are present, some of the culture should be filtered through a filter paper, moistened with distilled water, as suggested by Hewlett. Sheridan Delepine* says : — “ When the typhoid bacillus is reinoculated in neutral bouillon daily for weeks or months, there comes a time when, all of a sudden, all the cultures will clump spontaneously on the addition of any serum.” Having thus obtained serum, and an evenly turbid bouillon culture, the next thing is the dilution of the serum. Dilution of the Serum. — The question of the degree of dilution advisable, should always be considered with the necessary time limit, and Hewlett! states that “the dilution should not be less than 1 in 30, nor for convenience, more than 1 in 50, with a time limit not exceeding one hour.” Muir and Ritchie[| say that if serum diluted 1 in 30 causes clumping in half-an-hour, it is certainly from a case of typhoid, and doubt should exist if a lower dilution or a longer time is necessary. * Sheridan Delepine : Brit. Medical Journal , 1897. Vol. i. , p. 969. t Hewlett : Bacteriology, 190‘2. 2nd edition, p. 278. || Muir and Ritchie: Bacteriology. 3rd edition, p. 326. 2 14 CLINICAL DIAGNOSTIC BACTERIOLOGY. The dilution may be made with neutral bouillon, normal serum, or most conveniently with physiological salt solution. The easiest method of measuring the half dilution, eg ., i in 15, is by means of a graduated capillary pipette, such as the white blood corpuscle pipette. It can also be done in ordinary capillary pipettes, as Wright has suggested. The serum is drawn up to a point marked on the stem of the pipette, and this is blown out into a watch glass. Equal quantities of normal salt solution are drawn up to the same mark, until the necessary dilution is obtained. The dilution can also be made with a platinum loop, as Delepine suggests. One drop of the diluted serum is then mixed on a slide with one drop of the bouillon culture, covered with a cover-glass, or a hanging drop preparation may be made, and examined under a magnification of about Ye inch objective. When examined thus, it will be seen that the bacilli are very soon motionless, in a short time they begin to collect into little heaps, and at the end of half-an-hour or so, very few free bacilli will be seen outside the clumps. Sometimes a bacillus at the margin of a clump will appear spinning round one of its ends. When the reaction is feeble, or just about to begin, the bacilli are drawn into very loose groups and do not actually touch one another, and the movement of the other bacilli may not actually cease, but become sluggish. At the end of the time limit — half to one hour — the clumping will be quite distinct if the case be one of typhoid fever. Instead of living typhoid bacilli, an emulsion made SERUM DIAGNOSIS. 215 from dead cultures can be used in exactly the same way as just described, but of course no movement is present, and one must look for clumping of the dead bacilli. Dead cultures are usually employed in the sedimenta- tion methods. In addition to this method, which is usually spoken of as the microscopic or rapid one, a macroscopic or sedimentation method may be used with living or killed cultures of these bacilli. Wright and Semple* have described in detail the method of serum diagnosis, by dead cultures, of typhoid and Malta fever. As they say, the diagnosis can by this means be made by any practitioner at home or abroad, without the need of an incubator, cultures, or even a microscope. “He will be able to carry about these cultures without risk, and he will not need to take precautions against infection when he is employing them in the serum diagnosis of doubtful cases of fever. His whole equipment in connection with serum diagnosis may, in fact, be narrowed down to a supply of these dead bacteria, a small supply of glass tubing of, say, 3/ig to 1/i inch diameter, and the blowpipe apparatus which is described.” At my request the Director of the Lister Institute of Preventive Medicine, Chelsea Gardens, S.W., has kindly promised to supply, at a very small cost, dead cultures of these bacteria ; and sero-sedimentation tubes and capsules for collecting the blood (full directions for making these will be found in The Lancet , Vol. I., 1903, and the Brit. Med. Journal , Vol. I., Feb. 5, 1898) may, if it is not convenient to make them at * Wright and Semple : Brit. Med. Journal , 1897. Vol. I., p. 1214. 216 CLINICAL DIAGNOSTIC bacteriology. home, be obtained from A. E. Dean, Jun., 73, Hatton Gardens, E.C. The following is, briefly, the method adopted in using sedimentation tubes. One of the tubes is marked on the stem with a red wax pencil, or ink, and the undiluted serum drawn up to this mark. This is then blown out into a watch glass, and normal saline solution is drawn up to the same point four times, and this blown out and mixed with the serum, so that we have now a dilution of 1 in 5. With this 1 in 5 dilution, we can, by similar means, make a 1 in 15 diluted serum, and equal quantities of this and the dead culture fluid are drawn into the capillary sedimentation tube, mixed in the bulb of the same, and the mixture again driven down into the capillary stem, The orifice of the latter is then sealed in the flame, and the tube placed in a vertical position for twelve hours. A control tube of equal volumes of the bacterial culture and normal saline solution should be made at the same time. When examined at the end of the specified time, the fluid in the control tube has remained turbid throughout. In the tube containing the typhoid serum, the upper layer of the fluid has become clear, and a flocculent precipitate has fallen to the bottom of the tube. “A less complete positive reaction would have manifested itself in the formation of a precisely similar flocculent precipitate in an incompletely clarified fluid.” It is advisable in using the sedimentation tubes to use serum without any corpuscles, and normal saline solution, and not distilled water, as these may cause a little confusion. Prof. Wright* has recently said that “if we accept * A. E. Wright: Lancet , Vol. II., 1903. July 25. SERUM DIAGNOSIS. 2 I 7 as adequate an agglutination altogether inconspicuous to the naked eye, and if we allow a considerable interval for its appearance, we must exact a high degree of serum dilution. But reflection will make it clear that we can maintain exactly the same strictness in our standard, if we relax in the matter of our dilutions, and become correspondingly more exacting in other respects. Our exactions may, for instance, take the form of demanding that the agglutination effect shall manifest itself instantaneously, that the agglutination effect shall make its appearance in dense bacterial suspension, and that it shall be so pronounced as to discover itself to the naked eye.” By this means, he says, “ we dispense with all necessity for a microscope, and we obtain our results with an absolute minimum of delay— in the case of an ordinary typhoid serum, practically instantaneously. I may note that such an instantaneous and macroscopically visible agglutination, obtained in a mixture of equal parts of undiluted serum and typhoid culture, is, in my experience, quite as conclusive of the diagnosis as an agglutination visible ' under the microscope after a quarter-of-an-hour in a i in 50 dilution of the serum.” In a private communication, Prof. Wright, speaking of the dilution advisable for use with the dead typhoid cultures, says : — “ I think dilution of the serum for purposes of diagnosis of typhoid is unnecessary.” The serum diagnosis of Malta fever may be carried out with dead cultures in the same way as the above. At present, the serum diagnosis of tubercle by an emulsion of tubercle bacilli cannot be considered as a practical diagnostic method. On this subject, Wright* * Wright : “A note on the Serum Reaction of Tubercle.” Lancet , 1903. Vol. i., May 9. 2 I 8 CLINICAL DIAGNOSTIC BACTERIOLOGY. says “ Bearing in mind, in particular, the fact that tuberculous infections are for the most part purely localised infections, we have little reason to expect to derive much assistance in the diagnosis of tubercle from the indications of the serum sedimentation reaction. In point of fact, in contrast with what is the case in septicaemic diseases, such as typhoid and Malta fever, very little diagnostic importance attaches to the negative result of a serum sedimentation test in the case of tubercle. Important, on the other hand, in connection with the serum sedimentation reaction, is the fact that it gives, in the case where the patient is being subjected to therapeutic inocula- tions of tubercle vaccine, some indication of the extent to which anti-bacterial substances are being elaborated in the organism.” In the diagnosis of typhoid it must be remembered that occasionally, though very rarely, the agglutination reaction may be absent throughout the whole course of a definite attack of enteric fever. Generally speaking, the reaction is not obtained earlier than the sixth or seventh day of the fever, and sometimes considerably later than this. The serum of a patient who has had an attack of typhoid, or who has been inoculated with Wright’s serum, may show the reaction for a very considerable time after, and it is of importance to remember this fact in making a diagnosis of a doubtful case. If the blood fails to give a reaction when tested on three occasions, at intervals of a week, it is improbable that the case is one of typhoid. (Hewlett.) Certain strains of typhoid bacilli, and certain condi- tions of the culture media, may give somewhat variable results. The serum in some cases of illness due to SERUM DIAGNOSIS. 2 19 the para - typhoid or para - colon bacilli, which really belong to the bacillus enteritidis group of organisms, may occasionally give an agglutination reaction, and even normal serum will generally agglutinate in a dilution of 1 in 3 or 4, and sometimes in greater dilutions. (Hewlett.) Notwithstanding these facts, the serum diagnosis of typhoid fever is of extreme value, and now that dead cultures of typhoid bacilli can be so easily obtained, and the method so simplified, it is within the reach of all practitioners. Ficker’s Method* of Serum Diagnosis of Typhoid. Ficker, of the Hygienic Institute of the University, Berlin, realising the importance of the Gruber-Widal serum test of typhoid fever, has simplified the process, and adapted it to the wants of the practitioner. In order to render the method as practicable as possible, he has kept the following points in mind : — “ 1. The use of living typhoid cultures for the reaction must be dispensed with, as the physician does not possess the necessary outfit for work with infectious cultures. “ 2. The preparation containing the specific agglu- tinating body must be stable, and free from living typhoid bacilli. “3. It must not tend to clear spontaneously during the time necessary for the reaction. * M. Ficker: Ueber ein Typhusdiagriosticum. Berliner Klin IVochenshr. 1903. No. 45, Nov. 9, p. 1021. 2 20 CLINICAL DIAGNOSTIC BACTERIOLOGY. “4. The reaction must be quite evident to the naked eye, and absolutely definit e in its result. “ 5. The reaction must not require continued observation for two hours, as the usual laboratory method does, as this loss of time is not possible in practice. “6. It must be carried out at ordinary room temperature, without the necessity of an incubator. “ 7. The preparation must be influenced by the measured dilutions of serum, in precisely the same way, and in the same degree as is the living suspension of the typhoid bacillus.” Picker states that he has succeeded in preparing a fluid which is capable of replacing the culture of living typhoid bacilli. The third and seventh conditions were very difficult to fulfil, but he has managed to overcome these. His diagnostic preparation is a slightly turbid sterile fluid, which can be kept for more than nine months, if preserved in a dark and cool place. It ought to be shaken up from time to time and especially before use. He advises that the serum which is to be examined should be diluted with ten times its volume of sterile physiological salt solution, and o‘2 cc.m. and o’i cc.m. of the diluted serum, measured with a graduated pipette, is placed in pointed glass tubes, labelled No. 1 and No. 2. The tube No. 1 contains o'8 cc.m. of the diagnostic fluid, and No. 2 tube, 0 9 cc.m. A third tube contains 1 cc.m. of the diagnostic fluid, without the addition of any serum. The tubes are then corked up, and the contents well shaken. They are SERUM DIAGNOSIS. 22 1 then set aside at ordinary room temperature, but excluded from light. After ten, twelve, or fourteen hours, the results will be clear ; after twenty hours, a certain result ought not to be expected. The positive reaction, seen by holding the tubes against a dark background, or in front of the light, consists in a distinct clearing up of the turbidity of the fluid, while if pointed glass tubes have been used, the clumping together of the agglutinating substance into balls will also be seen. Ficker claims that his preparation enables the prac- titioner to carry out the agglutination test for enteric fever without living cultures, without an incubator, and without a microscope, and says that the results which have been obtained in some large clinics testify that the method is reliable. The diagnostic fluid and complete outfit can be obtained from E. Merck, 1 6, Jewry Street, E.C. J. Meyer* has compared the results obtained by Ficker's preparation with that of Widal’s method, in which living cultures are used. He finds that both methods gave exactly the same results, and control tests on non-typhoid blood were equally satisfactory. He thinks that Ficker’s method is simple to carry out, and is not apt to give discordant results owing to differences in the activity of the cultures used, as is the case in the Widal reaction, done with living typhoid bacilli. Meyer : Berliner klin Wochenshr. 1904, Feb. 15, 222 CLINICAL DIAGNOSTIC BACTERIOLOGY. Cytodiagnosis. Cytocliagnosis, a term applied to the determination of the nature and variety of cellular elements found in an exudation, has been elaborated largely by Widal and Ravaut, in France, and Wolff, in Germany; and numerous communications have appeared on the subject since 1900. It has already proved to be a valuable additional aid to diagnosis in England, and although it does not properly belong to the domain of bacteriology, yet it is intimately associated with the bacteriological examinations of exudations in general. In the fluid, drawn off from a case of pleurisy with effusion, it is well known that the search, for example, for the tubercle bacillus, is very difficult and usually unsuccessful, and yet very valuable assistance may sometimes be obtained by an examination of the cellular elements which may be present in it, and the results so obtained may materially assist in the diagnosis, prognosis, and treatment of the case. In addition to pleural effusions, the cerebro-spinal, pericardial, peritoneal, and arthritic fluids can be examined. The fluid to be examined may be that drawn off by an aspirator, or an aseptic syringe. Any of the ordinary hypodermic or diagnostic syringes in common use answer very well, but they should be provided with a sharp needle of about two to three inches long, preferably made of platinum iridium, as this can be sterilized in the flame without injury. CYTODIAGNOSIS. 223 If a bacteriological examination is also to be made of the fluid, the whole syringe should be sterilized, but otherwise it will be sufficient to sterilize the needle only. Of course the skin of the part should be washed with soap and water, then with some antiseptic, as is ordinarily done. The position at which the puncture is made, depends on the nature of the case, and need not be further specified here. If the fluid so obtained is purulent, a few films of the pus should be spread in the usual way, and may then be fixed and stained. In pus, especial attention should also be paid to any bacteria present. If the fluid is serous , and is seen to contain a fair number of cellular elements, it will not be necessary to centrifugalise it. It should be poured into a conical glass (that recommended for obtaining urinary deposits, described under tubercle bacilli in urine, being particularly convenient), and films made from the sediment. If the fluid be apparently very free from cells, it may either be placed in the conical glass (Fig. 3. page 103), especially if there is a large quantity of it, e.g., from a pleurisy with effusion, or it may be necessary to centrifugalise it. In order to prevent coagulation, Widal and others defibrinate it before placing it in the centrifuge. The fluid, immediately after it is withdrawn from the body, is placed in a bottle or strong flask, containing a number of glass beads, and thoroughly shaken until a clot is formed, which takes place after about fifteen minutes to an hour. If the exudation fluid has already undergone coagulation, it is poured with the clot into the flask containing the glass beads, and shaken up for about twelve minutes as before. 2 24 CLINICAL DIAGNOSTIC BACTERIOLOGY. Wolff* says that the fluid should be examined as soon as possible after it has been withdrawn. If this is impossible, coagulation may be prevented by the addition of citrate of soda. He does not recommend Widal’s method. The sediment obtained in either of these ways is spread out on slides in the usual method of preparing films, allowed to thoroughly dry in the air, and they can then be fixed. The method of fixing and staining is practically the same as that for blood films generally. The air-dried films can be conveniently fixed by placing them in alcohol and ether for about one to ten minutes, and then stain them with eosine and methylene separately, or together with Jenner’s stain. In the latter case, it is essential that the films should not have been previously fixed, but merely dried in the air. Unfixed air-dried films may also be stained with Leishman’s modification of Romanowsky’s method. These three methods have been described under the gonococci and plague bacilli. By far the simplest plan for those who are not accustomed to these aniline dyes is to stain the films with eosine and hsematoxylin in the following way : — Films, fixed by immersion in equal parts of alcohol and ether for a few minutes, are placed in a dilute water-alcoholic solution of eosine — viz., eosine, i part ; water, ioo parts ; alcohol, ioo parts — for about a minute. They are then washed in water and dried. Hsematoxylin, Bohmer’s or Delafield’s solution, which can be bought ready made, is then filtered on to the slide, and allowed to remain ten to sixty seconds, * Wolff : “ Exudate and Transsudate.” Encyklopiidie der Mikroskopischen Technik, Berlin, 1903. p. 302, CYTODIAGNOSIS. t 225 according to the staining power of the solution. The film is then washed in water, and examined whilst wet, to see if the blue of the haematoxylin in the nuclei of the leucocytes is sufficiently deep ; if not, they are again exposed to the haematoxylin. The films are then well washed, and even allowed to soak, in tap water, which is sufficiently alkaline to develope the blue of the haematoxylin, dried, and mounted in oil or balsam. I usually stain three such films — one with eosine and haematoxylin, one with Jenner’s, and one with Leishman’s stain. The elements found in films of the sediment will be the leucocytes of the blood, especiadly the polymorphonuclear and lymphocytes of varying size, probably also some red blood corpuscles and endothelial cells. The leucocytes found will, in fresh effusions, exactly resemble those of normal blood, but in older exudations they will probably show considerable altera- tion, the protoplasm having undergone disintegration or degeneration, and the nuclei have often lost their intranuclear network, and stain indifferently, or some- times deeply, with the basic dyes ; but, as a rule, it will not be difficult to classify them, provided that the pus or fluid has not been kept too long before it is examined. In the following account of the changes found in the exudations, I have made free use of a small work by Labbd : “ Le Cytodiagnostic.” Normal serous fluid, taken from a serous cavity, contains white and red blood corpuscles, the former being considerably in excess of those found in the blood. The white cells consist of multinucleated or Q 226 CLINICAL DIAGNOSTIC BACTERIOLOGY. polymorphonuclear leucocytes — i.e., cells with neutro- phile granules and many lobed nuclei ; uninucleated leucocytes or lymphocytes of varying size, containing one relatively large nucleus and no granules ; and eosinophile leucocytes, containing large granules in their protoplasm, which stain deeply with the acid aniline dyes such as eosine. A detailed description of these elements has been given elsewhere.* In addition to the leucocytes, a few endothelial cells will generally be seen. Serous fluids from the pleural or pericardial cavity have much the same characters, whilst those from a joint generally contain fewer cells. (4) Pleural Effusions. 1. Primary Tuberculosis of the Pleura is at its height generally associated with a great increase in the number of the lymphocytes, mixed with a considerable number of red blood corpuscles. (Widal, Litten, and Wolff). During the first few days of the effusion, large uninucleated cells which have been regarded as desquamated epithelial cells, or as large uninucleated leucocytes, may be found in small numbers, but these disappear in a few days. Multi nucleated and eosinophile cells may also be present during this early stage, but they never, according to Widal and Ravaut, exceed 10% of the elements present. 2. Secondary Tuberculosis of the Pleura , associated with pulmonary tuberculosis, is characterised by the small number of cells present, and by the alteration of these. They have undergone fatty degeneration * Coles : The Blood.'1 Churchill : 2nd edition, 1902. CYTODIAGNOSIS. 227 and appear irregular, filled with vacuoles, and contain granules which stain black with osmic acid. Red blood corpuscles are present in very small numbers, the lymphocytes are deformed, and the multinucleated cells are so altered that, according to Labbe, they are only recognisable by their neutrophile granules. “ The nucleus of the multinucleated cells have undergone a degenerative change, which is spoken of as ‘pycnose.’ It is broken up into several ball-like masses, which stain deeply and uniformly, and appear completely separated from one another. Endothelial cells united in masses are never met with. Generally in these cases, the background of the preparation is deeply stained, and it is very difficult to distinguish the outline of the leucocytes, the . protoplasm of which stains the same colour as the background.” (Labbe).* According to some authorities, secondary pleurisies tending towards recovery, become lymphocytic, but when they run into a chronic condition, as is usually the case, the exudation only contains degenerated and altered elements. Generally speaking, tubercular exudations are very constant in their cellular nature, but sometimes in the secondary tubercular pleurisies there may be a marked increase in the percentage of the eosinophile cells. 3. Aseptic Pleurisy in Cardiac and Renal Disease. — These are essentially characterised by the presence of endothelial cells. These are usually found in groups of eight to ten elements, so fused together that the margins of the cells are usually obliterated, and the individual cells only recognised by their nuclei, which * Labbe : Le Cytodiagnostic. Paris,/ 1903, p. 46. 228 CLINICAL DIAGNOSTIC BACTERIOLOGY. are generally not very distinctly stained. Sometimes the epithelial cells occur singly or in pairs, and in cases of recent hydrothorax the endothelial cells are occasionally so abundant as to cover the whole field of the microscope. Later on lymphocytes may appear, and the fused masses of endothelial cells diminish somewhat in number, become dropsical, hyaline, and disintegrate. In congestion or infarcts of the lung occurring in the course of heart disease, red blood corpuscles and multinucleated leucocytes are added to the endothelial and lymphocytic cells. 4. Cancerous P/eurisy. — The cellular elements here are inconstant. Sometimes malignant cells may be found in the fluid. These are larger than the endothelial cells with which they are often associated, and frequently contain granules and vacuoles. Their nuclei, which are very large and oval, often show two or three very large and distinct nucleoli. In other cases, merely groups of endothelial cells, lymphocytes, multinucleated cells, and generally a large number of red blood corpuscles are seen. In a case of pleurisy occurring during secondary syphilis, Widal & Ravaut found 35 % of endothelial cells, 22 % lymphocytes, 37 % large uninucleated eosinophile cells, and 6 °/ of large uninucleated cells with neutrophile granules. It may be noticed that the last two elements do not occur in the blood in health, only in the marrow. They are characteristic of leucocythaemia when found in the blood. 5. Septic Exudations. Pleurisy due to Pneumococci. — These are characterised by the presence of multi- nucleated leucocytes, which are present in numbers directly proportional to the number of pneumococci. CYTODIAGNOSIS. 229 These leucocytes, often associated with the presence of endothelial cells, may swell up and appear very like a large uninucleated leucocyte. When such a pleurisy tends towards recovery, the multinucleated elements disappear, and some lymphocytes present themselves. When, on the other hand, it goes on to suppuration, the multinucleated leucocytes become very abundant, their nuclei divide up into balls (pycnosis), and they might be mistaken for lymphocytes, except for the presence of neutrophile granules in the protoplasm. Pletirisy due to Streptococci is associated with a large number of multinucleated and isolated endothelial cells. It will thus be seen that the general rule is that tubercular disease is associated with lymphocytes, pyogenic diseases with multinucleated leucocytes, and the reaction to mechanical irritation with endothelial cells. (B) Peritoneal Fluid. The cellular elements found here are not nearly as constant as in the pleural exudation. In ascites due to mechanical causes, endothelial cells and a few leucocytes are found. In malignant disease of abdominal organs, occasion- ally cancer cells may be met with in the peritoneal fluid, but more frequently endothelial cells are found. In tubercular peritonitis, sometimes only lymphocytes are seen, but Widal and Ravaut have generally found multinucleated cells. ( c) Cerebro=spinal Fluid. Lumbar puncture, as devised by Quincke, was used for therapeutic as well as diagnostic purposes. It is, 2 3° CLINICAL diagnostic IJACTERIOLOGV. with ordinary care, quite a safe little operation, but it must be remembered that it is, from all accounts, somewhat dangerous to withdraw a large amount of fluid — i.e, over an ounce, in cases of cerebellar tumour. I would refer the reader, for details of the method of its performance, either to Labbd’s book already mentioned, or to two articles on cytodiagnosis in nerve disease by Dana and Hastings, and also Fraenkel, in the same number of the Medical Record (New York, Jan. 23, 1904, page 121, et seq.) The fluid which comes through the needle need not be defibrinated, but, as it contains very few elements, it should be immediately centrifugalised. Films are prepared from the sediment, and stained exactly as previously described. The cerebro-spinal fluid in health, or even in acute diseases which are unattended with meningitis, contains no cells, or, at most, a few red blood corpuscles and leucocytes. In tubercular meningitis the cerebro-spinal fluid contains a varying number of cells, which are usually nearly all lymphocytes, with a very few multinucleated leucocytes and red blood corpuscles. In acute menin- gitis, due to the pneumococcus, meningococcus, or streptococcus, the fluid contains only multinucleated leucocytes mixed with lymphocytes and red blood corpuscles. According to Labbe, in fatal cases the multi- nucleated leucocytes persist till death, whilst in a curable case these cells persist during the acute stage, and are gradually replaced by uninucleated cells, and later on, when recovery has taken place, the fluid has returned to its normal appearance. Each relapse is attended with an increase in the CYTODIAGNOSIS. 23I multinucleated leucocytes. From this it will be seen that if cerebro-spinal fluid was taken from a case somewhat late in its course, and lymphocytes found, this might be thought to indicate a tubercular condi- tion. To determine whether the case was one of tubercular or recovery from an acute meningitis, one must be guided by the clinical features of the case, and also by an examination of stained films made from the sediment for the specific organism, remember- ing, however, that the tubercle bacillus is not readily found in this fluid. In tabes, and in general paralysis, the cerebro- spinal fluid contains a large number of cells, especially lymphocytes, some large uninucleated leucocytes and endothelial cells. Generally speaking, the occurrence of lymphocytosis is fairly constant. As a general rule Labbe states that in organic diseases of the nervous system in which the meninges are in a state of inflammation, when the process is acute, a multinucleated leucocytosis is the rule, and this is followed, on its recovery, by, at first, an uninucleated leucocytosis, and finally, a normal condition. When the process is chronic, lymphocytosis is the rule, e.g., in syphilis. Peripheral neuritis, or cerebral tumours, which do not touch the cortical portion of the brain, cause no meningeal irritation, and therefore lymphocytosis is absent. INDEX. Abscess, bacteria in ... PAGE 171 Bacillus of leprosy ... PAGE 26 Absolute Alcohol, fixing by 3 >> malignant oedema 198 Achorion Schonleinii CO O n J1 plague 175 Acid-alcohol 69 pneumonia 134 Acid-fastness, cause of 22 >> pseudo-diphtheria 158 Acid-fast bacteria 12 11 pseudo - tubercu- „ „ in human excretions 48 losis ... soft sore ... co r\ Acid-fast streptothrix 54 syphilis ... 129 „ structures ... 22 Timothy grass ... 36 Actinomyces, method of 189 J) tubercle 16 staining 1) typhoid ... 21 1 Actinomyces, microscopic characters 187 „ xerosis ... Balsam, Canada 158 7 1 Actinomyces, naked - eye characters 186 Bichromate solution ... Actinomycosis ... ... 184 Agglutination... ... ... 21 1 Alcohol as a decolouriser ... 66 Alcohol and ether, for fixing 3 Aniline water ... ... ... 8 Aniline water gentian violet 8 Anthrax bacillus 191 diagnosis of 198 examination of ... 195 staining ... 196 Arthritis, gonorrhoeal Avian tubercle 1 18 23 Biedert’s sedimentation method ... 98 Bipolar staining 144, 176, 182 Bismark brown .123 Blood film spreading ... 180 „ „ Ross’s method 181 Blood serum ... 212 Bottles for staining . 5 Bronchitis •• 133, H5 Bubonic plague 1 75 Bunge and Tranteroth’s Method ... 7 1 Bacteria in pus 171 „ stained by Gram ... 10 „ not stained by Gram 10 Bacillus of anthrax ... ... 192 ,, diphtheria ... 154 „ influenza... ... 143 Burroughs Wellcome’s stains 6, 12 1 Butter, acid-fast bacilli in ... 45 Canada balsam 7 Cancerous pleurisy 228 Capsule staining ... ... 135 „ „ Friedlander’s method ... 138 » „ Johne’s method 137 234 CLINICAL DIAGNOSTIC BACTERIOLOGY. PAGK Capsule Staining Klett’s method... '38 „ „ Ribbcrts’ method 138 Carbol-fuchsin 4, 61 „ -gentian violet 9 „ -methylene-blue 4 „ -thionin 4,9 Cerebro-spinal fluid ... 229 „ ,, meningitis 15 1 ,230 Chancre, bacillus of soft 127 Chloroform test for ringworm 206 Cladothrix asteroides 56 Claudius’ modification of Gram 9 Cleaning slides and cover- glasses ... 1 Clearing films... 7 Condenser substage ... 1 1 Coverglass films 3 „ cleaning ... 1 Crouch’s method of staining 168 Cytodiagnosis 222 ,, of pleural effusions 226 Dahlia .. 168 Degree of resistance of acid- fast bacilli 60 Differentiation of acid-fast bacilli 85 Diphtheria bacillus ... '54 „ „ cultures... 162 „ „ staining... 163 „ membrane '59 „ pseudo-bacillus ... 158 „ streptococci in ... 156 Diplococcus gonorrhoeas 1 1 1 ,, intracellularis meningitidis ... '5' „ pneumonias '3' Drying films ... 6 Ducrey’s bacillus of soft sore 127 Empyema 133, Eosine 120, Ermengen’s solution Erythrasma ... Examination of fa;ces for tubercle bacilli Examination of sputum for tubercle bacilli ... • ... Examination of urine for tubercle bacilli Faeces, tubercle bacilli in ... Favus Films, blood ... „ clearing » drying ,, fixing „ for cytodiagnosis „ mounting „ spreading Fixing films ... Frankel’s pneumococcus Frankel and Gabbet’s method for tubercle bacilli Frankel and Pappenheim’s methodfortubercle bacilli Friedlander’s capsule stain ... „ pneumo-bacillus Fuchsin carbol- Genital organs, bacteria in ... Gentian violet, aniline water „ „ carbolic Gleet, gonococci in Gonorrhoea, complications in „ in female ,, in male Gonococcus ... ,, diagnosis of „ examination of ... „ staining PAGE 1 '39 , 224 1 210 107 89 99 107 208 180 7 6 3 224 7 2 3 '3' 93 72 133 '34 4, 61 129 8 9 1 12 1 18 "3 1 1 1 1 1 1 126 1 19 119 INDEX. 235 PAG E Gram’s method ... & „ „ for gonococci ... 122 „ „ modifications of 9 Grass i., or Timothy bacillus 36 Grass ii. bacillus ... ... 39 „ „ cultivation of 39 ,, „ inoculation of 41 Haematoxylin 224 Hair, examination of 205 Honsell’s method 69 Influenza bacillus ... ... >43 „ „ culture ... 148 „ „ diagnosis of 149 „ ,, examinationof 146 Iodine solution for Gram’s method ... ... ...8, 123 Iodine solution for Neisser’s method ... ... ... 166 Jenner’s stain ... ... ... 121 Johne’s capsule stain ... 137 Ketel’s sedimentation method 98 Klebs-Loffier bacillus ... 154 Klein’s spore stain ... ... 197 Klett's capsule stain 138 Kiihne’s methylene-blue ... 4 Leishman’s stain ... 121,180 Lens, oil immersion ... ... 10 Leprosy bacillus ... ... 26 „ cultivation ... 29 „ diagnosis of ... 28 „ examination of 28 „ „ staining ... 26 Leptothrix buccalis ... . 1 66 Loffler’s bacillus ... ... 154 „ methylene-blue ... 4 Lugol’s iodine solution ... 8 Lupus, tubercle bacilli in ... no Madura foot ... Malignant oedema ... „ pustule ... Mai tafever, serum diagnosis of Manure or mist bacilli Meningitis, cerebro-spinal 151. „ tubercular Meningococcus Method of differentiating acid- fast bacilli Method of staining tubercle bacilli ... ... ...8 Methylene-blue, borax 13 1 „ „ carbol- „ „ Kiihne’s ,, „ Loffler’s Micrococcus catarrhalis ,, gonorrhceae ... ,, lanceolatus „ meningitidis ... „ pyogenes „ tetragenus Microsporon Audouinii ,, furfur ... „ minutissimum... Mist or manure bacillus Moeller’s acid-fast bacilli ... „ spore stain Morris’ method for ringworm Mounting films Muir’s method of capsule staining ... Neisser’s granules in diph- theria bacilli Neisser’s granules in grass ii. bacilli Neisser’s granules in plague bacilli Neisser’s reaction „ „ modification of PAGE 1 9 1 198 191 2 1 1 43 , 230 230 '5i 85 5,9i , '79 4 4 4 ■43 1 1 1 131 151 '7' '73 203 209 210 43 '5 197 207 7 '36 164 74 '83 164 165 CLINICAL DIAGNOSTIC BACTERIOLOGY. 236 PAD E Neisser’s reaction, value of... 167 Nitric acid as a decolouriser 65 Orange rubin ... 191 Pappenheim’s rosolic solution 72 121, 180 „ stain for gonococci 125 Rosolic acid solution . . , 72 „ „ tubercle Ross’ method for blood films 181 bacilli... 72 Roux’ stain 169 Peritoneal fluid 229 Pfeiffer’s bacillus M3 Piorkowski’s stain 169 Saffranine 166 Plague bacillus 175 Sahli’s methylene-blue . .. 131 „ „ diagnosis of ... 182 Sarcina ... 174 „ „ examination of 1 77 Schaiiffler’s stain ... 170 „ „ staining 178 Serous effusions, cytodiagno- Pleural effusions 226 sis ot ... 222 Pleurisy, aseptic ... ... 227 „ cancerous ... ... 228 „ cytodiagnosis of ... 226 „ pneumococcic 133,228 „ tubei'cular ... 110,226 Pneumobacillus, F riedlander’s 1 34 „ Diagnosis ... 140 Pneumococcus, Frankel’s ... 131 „ „ diagnosis 140 „ „ staining 135 Pneumonia 133, 14 1 Polar staining ...144, 176, 182 Pseudo- diphtheria bacillus ... 158 Pseudo-tubercle bacilli ...12,48 „ „ „ compared 58 Pus, bacteria in ... ... 171 „ examination of ... ... 174 „ tubercle bacilli in ... no Pyogenic organisms 171 Reaction, Neisser’s ... ... 164 „ Widal’s 2 1 1 Relapsing fever, spirillum of 199 Renal tuberculosis ... PAGK ... 99 Ribbert’s capsule stain ... 138 Ringworm ... 203 Romanowsky’s stain... ... 179 Leishman’s modification of Serous effusions, tubercle bacilli in ... ... ... no Serum diagnosis ... ... 211 ,, Ficker’s method 219 „ Wright’s method 215 Slides, cleaning 2 Smear preparations ... ... 2 Smegma bacillus ... 30, 79, 102 „ „ cultivation 33 „ ,, differential / 35 diagnosis \ 103 in smegma staining / 30 l 79 reaction... 79 Smith’s method for pneumo- cocci ... ... ... 139 Soloicl stains 6, 121 Spirillum of relapsing fever 199 Spiroclnete Obermeieri ... 199 „ „ examina- tion of 201 Spore staining ... ... 197 Sputum — Examination for influenza li 146 237 INDEX. PAGE Sputum — Examination for pneumococci 135 „ tubercle B ... 89 „ by sedimentation 97 Spreading films 9i Staining bottles 5 „ capsules 136 „ methods 3 „ spores 197 „ tubercle bacilli 61,85,91 Stains ... 4 Staphylococcus pyogenes ... 171 Streptobacillus of soft sore ... 127 Streptococcus pyogenes 172 „ in tubercular sputum 95 Streptothrix, acid-fast 54 „ bovis ... 57 „ Eppinger’s ... 56 „ madurae 191 „ resistance to acids 82 Sulphuric acid as decolouriser 62 Suppuration 17' Syphilis 129 Thionin, carbol 4, 9 „ Nicolle's 4 Timothy grass bacillus 36 „ „ culture 37 „ „ inoculation... 38 Tinea ... PAGE 203 Tranteroth & Bunge’s method 7i Tricophyton megalosporon ... 204 „ microsporon 203 Tubercle bacilli 16 „ „ avian 23 „ „ cultures ... 19 „ „ staining reaction 20 Tubercular faeces 107 „ sputum ... 89 „ urine 99 Tuberculosis, pseudo- 52 „ serum, diagnosis of 217 Typhoid, agglutination re- action in 2 r 1 „ serum diagnosis... 21 r Urinary sediment, fixing 3 104 Urine, examination for tubercle B ... 99 „ in renal tubercle ... 99 „ smegma, bacilli in .. 102 Vesuvin 123 Wahl’s stain for gonococci ... 124 Washing films 6 Weigert’s method 10 Widal's reaction 21 1 Woolsorter’s disease... 191 Xerosis bacillus 158 Xylol for clearing ... 7 Ziehl-Neelsen’s fuchsin 4, 61 STEAM PRESS, CIRENCESTER. No. 3. London , 7, Great Marlborough Street, A pril , 1 904. A SELECTION FROM J. & A. CHURCHILL’S CATALOGUE, COMPRISING MOST OF THE RECENT WORKS PUBLISHED BY THEM. N.B. — /. iSr5 A. 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The Veterinarian’s Pocket Re- membrancer : being Concise Direc- tions for the Treatment of Urgent or Rare Cases. By George Armatage, M.R.C.V.S. Second Edition. Post 8%to, 3s- Chauveau’s Comparative Anat- omy of the Domesticated Ani- mals, revised and enlarged, with the Co- operation of S. Arloing, Director of the Lyons Veterinary School. Edited by Geo. Fleming, C.B., LL.D., F.R.C.V.S., late Principal Veterinary Surgeon of the British Army. Second English Edition. 8vo, with 585 Engravings, 31s. 6d. general editorship of Chalmers 14 Volumes, ^13 12s. net. LOADON: 7, GREAT MARLBOROLGH STREET. INDEX TO J. & A. CHURCHILL’S LIST. Allen's Chemistry of U rine, 1 1 Commercial Organic Analysis, 13 Andrewes’ Disinfection and Sterilisation, 4 Armatage’s Veterinary Pocket Remembrancer, 14 Ballantyne’s Antenatal Pathology and Hygiene, 2 Barnes (R.) on Obstetric Operations, 3 on Diseases of Women, 3 Beale (L. S.) on Liver, 6 Slight Ailments, 6 Urinary and Renal Derangements, 12 Beale (P. T. B.) on Elementary Biology, 2 Beasley’s Book of Prescriptions, 5 — Druggists' General Receipt Book, 5 Pharmaceutical Formulary, 5 Bell on Sterility, 4 Bentley and Trimen’s Medicinal Plants, 5 Bentley’s Systematic Botany, 5 Berkart’s Bronchial Asthma, 6 Bernard on Stammering, 7 Berry’s (Jas.) Thyroid Gland, 9 (R. J.) Regional Anatomy, 1 Bigg’s Short Manual of Orthopaedy, 9 Birch’s Practical Physiology, 2 Bloxam’s Chemistry, 12 Laboratory Teaching, 12 Bousfield’s Photo-Micrography, 14 Bowlby’s Injuries and Diseases of Nerves, 9 Surgical Pathology and Morbid Anatomy, Brockbank on Gallstones, 7 Brown’s (Haydn) Ringworm, 11 (Campbell) Practical Chemistry, 13 Bryant's Practice of Surgery, 8 Bulkley on Skin Diseases, 10 Butlin’s Operative Surgery of Malignant Disease, 11 Malignant Disease of the Larynx, 11 Buzzard’s Diseases of the Nervous System, 7 Peripheral Neuritis, 7 Simulation of Hysteria, 7 Cameron’s Soaps and Candles, 14 Carpenter and Dallinger on the Microscope, 14 Cautley’s Infant Feeding, 4 Charteris’ Practice of Medicine, 0 Chauveau’s Comparative Anatomy, 14 Churchill’s Face and Foot Deformities, 9 Clouston’s Lectures on Mental Diseases, 3 Clowes and Coleman’s Quantitative Analysis, 13 — — Elmntry Practical Chemistry, 13 Clowes’ Practical Chemistry, 13 Coles on Blood, 6 Cooke’s Electric Lighting, 14 Cooley’s Cyclopaedia of Practical Receipts, 14 Cooper on Syphilis, 12 Cooper and Edwards’ Diseases ot the Rectum, 12 Corbin and Stewart's Physics and Chemistry, 12 Cripps’ (H.) Ovariotomy and Abdominal Surgery, 9 Cancer of the Rectum, 12 Air and Faeces in Urethra, 12 Cripps’ (R. A.) Galenic Pharmacy, 4 Cuff” s Lectures to Nurses, 4 Cullingworth’s Short Manual for Monthly Nurses, 4 Dalby’s Diseases and Injuries of the Ear, 10 Short Contributions, 10 Dana on Nervous Diseases, 7 Darier’s Ocular Therapeutics, 10 Day on Headaches, 8 De Langenhagen on Entero-Colitis, 8 Dibdin’s Photometry, 14 Duncan (A.), on Prevention of Disease in Tropics, 5 Dunglison’s Dictionary of Medical Science, 12 Edwards’ Chemistry, 14 Ellis's (T. S.) Human Foot, 9 Encyclopaedia Medica, 14 Fagge’s Principles and Practice of Medicine, 6 Fayrer's Climate and Fevers of India, 5 Fenwick (E. H.), Electric Illumination of Bladder, 11 Tumours of Urinary Bladder, 11 Ulceration of Bladder. 11 Symptoms of Urinary Diseases, 1 1 Atlas of Electric Cystoscopy, 11 ; Obscure Diseases of Urethra, ix Fenwick’s (S.) Medical Diagnosis, 6 Outlines of Medical Treatment, 6 Ulcer of the Stomachand Duodenum, 6 Fenwick’s (S.) Cancer of Stomach, 6 Fink's Operating for Cataract, q Fowler’s Dictionary of Practical Medicine, 5 Fox (G. H.) on Skin Diseases of Children, 10 Fox (Wilson), AtlasofPathological AnatomyofLungs, 6 Treatise on Diseases of the Lungs, 6 Frankland and Japp’s Inorganic Chemistry, 12 Fraser's Operations on the B rain, 8 Fresenius Qualitative Analysis, 13 Quantitative Analysis, 13 Galabin’s Diseases of Women, 3 Manual of Midwifery, 3 Gardner’s Brewing, Distilling, and Wine Manuf., 14 Glassington's Dental Materia Medica, 10 Godlee’s Atlas of Human Anatomy, 1 Goodhart’s Diseases of Children, 4 Gorgas’ Dental Medicine, 10 Gowers' Diagnosis of Diseases of the Brain, 7 Manual of Diseases of Nervous System, 7 Clinical Lectures, 7 Subjective Sensations and other Lectures, 7 Epilepsy, 7 Medical Ophthalmoscopy, 7 Syphilis and the Nervous System, 7 Granville on Gout, 7 Gray’s Treatise on Physics, 14 Green’s Manual of Botany, 5 — - — — Vegetable Physiology, 5 Greenish's Materia Medica, 4 Foods and Drugs, 4 and Collin’s Vegetable Powders, 4 Groves, Thorp, and Dibdin’s Chemical Technology, 14 Guy's Hospital Reports, 7 Habershon’s Diseases of the Abdomen, 7 Hadley on Nursing, 4 Haig’s Uric Acid, 6 Diet and Food, 2 Hamer’s Manual of Hygiene, 2 Harrison’s Urinary Organs, 11 Hartridge’s Refraction of the Eye, 9 Ophthalmoscope, 9 Hawthorne’s Galenical Preparations ot B.P., 5 Heath’s Injuries and Diseases of the Jaws 8 Minor Surgery and Bandaging, 8 Operative Surgery, 8 Practical Anatomy, 1 Clinical Lectures, 8 Hedley’s Therapeutic Electricity, 5 Heusler on the Terpenes, 13 Hewlett’s Bacteriology, 3 ■ — Serum Therapy, 3 Hill on Cerebral Circulation, 2 Holden’s Human Osteology, 1 Landmarks, 1 Horrocks’ Bacteriological Examination or Water, 3 Hovell’s Diseases of the Ear, 10 Hughes and Keith’s Practical Anatomy, 1 Impey on Leprosy, 10 Ireland on Mental Affections of Children, 3 Jacobson’s Operations of Surgery, 8 Jellett’s Practice of Midwifery, 3 for Nurses, 3 Gynaecology, 3 Jessop’s Ophthalmic Surgery and Medicine, 9 Johnson’s (A. E.) Analyst’s Companion, 13 Journal of Mental Science, 3 Kelynack’s Pathologist’s Handbook, 1 Keyes' Genito-Urinary Organs and Syphilis, 12 Knuthsen on Hiccough, 8 Kohlrausch’s Physical Measurements, 14 Lane’s Rheumatic Diseases, 7 Lawrie on Chloroform, 4 Lazarus-Barlow's General Pathology, 1 r — 7~ Pathological Anatomy, 1 Lee s Microtomist’s Vade Mecum, 14 Leftwich’s Syphonage, 6 Lewis and Balfour’s Public Health, 2 Lewis (Bevan) on the Human Brain, 2 Liebreich (O.) on Borax and Boric Acid, 2 Ltebreich’s (R). Atlas of Ophthalmoscopy, 10 Lucas s Practical Pharmacy, 4 Luke’s Anaesthetics, 4 MacMunn’s Clinical Chemistry of Urine, 11 f Continued on the next page. LONDON: 7, GREAT MARLBOROUGH STREET. Index to J. & A. Churchill’S List — continued. Macnamara’s Diseases and Refraction of the Eye, g Diseases of Bones and Joints, 8 McNeill’s Epidemics ajid Isolation Hospitals, 2 Malcolm's Physiology of Death, 9 Marsh’s Clinical Essays and Lectures, 8 Martin’s Ambulance Lectures, 8 Maxwell’s Terminologia Medica Polyglotta, 12 Maylard’s Surgery of Alimentary Canal, g Mayne’s Medical Vocabulary, 12 Microscopical Journal, 14 Mills and Rowan’s Fuel and its Applications, 14 Moore's (N.) Pathological Anatomy of Diseases, 1 Moore’s (Sir W. J.) Family Medicine for India, 5 — Manual of the Diseases of India, 5 Morris’s Human Anatomy, 1 Anatomy of Joints, 1 Murray’s Hare-Lip and Cleft Palate, 8 Nettleship’s Diseases of the Eye, 9 Notter's Hygiene. 2 Oliver’s Abdominal Tumours, 3 Diseases of Women, 3 Ophthalmic (Royal London) Hospital Reports, 9 Ophthalmological Society’s Transactions, 9 Ormerod's Diseases of the Nervous System, 7 Parkes’ (E.A.) Practical Hygiene, 2 Parkes’ (L.C.) Elements of Health, 2 Parsons’ Ophthalmic Optics, 9 Paton’s (Noel) Physiology, 2 Pavy’s Carbohydrates, 6 Pereira's Selecta e Prescriptis, 5 Pitt-Lewis’s Insane and the Law, 3 Proctor’s Practical Pharmacy, 4 Pye-Smith’s Textbook of Medicine, 6 Diseases of the Skin, 10 Ramsay’s Elementary Systematic Chemistry 13 Inorganic Chemistry, 13 Richardson’s Mechanical Dentistry, 10 Richmond’s Antiseptic Principles for Nurses, 4 Roberts’ (C. H.) Gynaecological Pathology, 3 Roberts’ (D. Lloyd) Practice of Midwifery, 3 Robinson’s (Tom) Eczema, 11 Illustrations of Skin Diseases, 1 Syphilis, n Ross’s Diseases of the Nervous System, 7 St. Thomas’s Hospital Reports, 7 Sansom’s Valvular Disease of the Heart, 7 Savill’s Clinical Medicine, 6 Schofield’s Force of Mind, 3 Scott’s Atlas of Urinary Deposits, 11 Shaw’s Diseases of the Eye, 9 Shaw-Mackenzie on Maternal Syphilis, 12 Short Dictionary of Medical Terms, 12 Silk’s Manual of Nitrous Oxide, 10 Smith’s (Ernest A.) Dental Metallurgy, 10 Smith’s (Eustace) Clinical Studies, 4 Disease in Children, 4 Wasting Diseases of Infants and Children, 4 Smith’s (F.) Bullet Wounds, 8 Smith’s (F. J.) Medical Jurisprudence, 2 Smith’s (J. Greig) Abdominal Surgery, 8 Smith’s (Priestley) Glaucoma, 10 Snow’s Cancer and the Cancer Process, 11 Palliative Treatment of Cancer, 11 Reappearance of Cancer, 11 Solly’s Medical Climatology, 8 Southall’s Materia Medica, 5 Squire’s (P.) Companion to the Pharmacopoeia, 4 London Hospitals Pharmacopoeias, 4 Methods ana Formulae, 14 Starling’s Elements of Human Physiology, 2 Sternberg’s Bacteriology, 6 Stevenson and Murphy’s Hygiene, 2 Sutton’s (F.) Volumetric Analysis, 13 Swain’s Surgical Emergencies, 8 Swayne’s Obstetric Aphorisms, 3 Taylor’s (A. S.) Medical Jurisprudence, 2 Taylor’s ( F.) Practice of Medicine, 6 Thin’s Cancerous Affections of the Skin, 10 Pathology and Treatment of Ringworm, 10 on Psilosis or “ Sprue,” 5 Thompson’s (Sir H.) Calculous Disease, 11 Diseases of theUrinaryOrgans, 11 Lithotomy and Lithotrity, 11 Stricture of the Urethra, 11 Tumours of the Bladder, 11 Thorne’s Diseases of the Heart, 7 Thresh’s Water Analysis, 2 Tilden’s Manual of Chemistry, 12 Tirard’s Medical Treatment, 6 Tobm’s Surgery, 8 Tomes’ (C. S.) Dental Anatomy, 10 Tomes’ (j. and C. S.) Dental Surgery, 10 Treves and Lang’s German-English Dictionary, 12 Tuke’s Dictionary of Psychological Medicine, 3 Turner on Sinuses of the Nose, 10 Tuson’s Veterinary Pharmacopoeia, 14 Valentin and Hodgkinson’s Practical Chemistry, 13 Vintras on the Mineral Waters, &c., of France, 8 Wagner’s Chemical Technology, 14 Wallace on Dental Caries, 10 Walsham and Spencer’s Surgery, 8 Waring’s Indian Bazaar Medicines, 5 Practical Therapeutics, 5 Watson’s Diseases of Eye, 0 Watts’ Organic Chemistry', 12 West’s (S.) How to Examine the Chest, 6 White’s (Hale) Materia Medica, Pharmacy, &c., 4 Wilks’ Diseases of the Nervous System, 7 Wilson’s (Sir E.) Anatomists’ Vade-Mecum, 1 Wilson's (G.) Handbook of Hygiene, 2 Wright’s (H.), Malarial Fevers of Malaya, 5 Beri-beri, 5 ynter and Wethered's Practical Pathology', 1 Year-Book of Pharmacy', 5 N.B.—J. $ A. Chut chills larger Catalogue of about 600 works on Anatomy , Physiology , Hygiene, Midwifery, Materia Medica, Medicine, Surgery, Chemistry, Botany , fyc. fyc., with a complete Index to their Subjects, for easy reference will be forwarded post free on application. AMERICA. — J. 4’ A. Churchill being in constant communication with various publishing houses in America are able to conduct negotiations favourable to English Authors. LONDON: 7, GREAT MARLBOROUGH STREET. / > I ■» -t-t-F’rr r-f:rr rrr r m' ri V i'trrwtj-y .■ .■ /•/ , jy / / / | *ipwanpt»nra^ff/- > 1 .■v^ /.*•/. • . rr> £' ?s s .*.*.*.• s .*ss /.*.•.• ’AWifiWS. '■ /•/ i i- 4 v • x /.' / •> i£*K -&-.V >: .V .*>**« ' V * : «*V> ,y f. •'.' / 0\'\ \.S \ «Hv>^vv>»%^>^vvvv>).vwvi>vv,.-%vv vv »w> m- '• '• :.v v >'-:• . 1, » wh.\ 1