Department of Bacteriology, Pathology and Microscopy. Edited by CHAS. F. CRAIG, M. D. Member of the American Medical Association. Member of the American Microscopical Society, Pathologist to Danbury Medical Society, etc. Editorials. NINTH PAPER. 22. THE BACILLUS TYPHOSUS. uute oval rods, and in cultivations often are arranged in long threads, each thread being formed of many ba cilli. They stain somewhat slowly with the anilin dyes, and often pre sent unstained spaces, which by many, notably Hirschfeld, are held to be spores. In sections the bacilli occur singly. If the bacilli are stained suitably, they are seen to be surrounded by a hyaline sheath from which radiate a number (eight to twelve) of fine flagellæ, which are inserted both laterally and at the ends of the germs. It is by means of these organs that the typhoid bacilli are enabled to move more or less rapidly. The more recent the culture the more rapid the movements. The bacilli so stained, constitute the socalled "spider cells". Variations in the morphology of this organism have not been thought to commonly exist, but in a recent paper Carter, of Johns Hopkins, records some very interesting involution forms, consisting of long rods with unstained bulbous ends, long threads, irregular rods with unstained ends, and very short oval rods. Babes, Chantmesse, Vilchur and Widal have also described involution forms. Historical.-Among the first to describe bacteria as occurring in ty phoid fever may be mentioned Klebs, Klein, and Birsch- Hirschfeld. Their researches date before 1880, and relate almost altogether to the occurrence of germs in sections of typhoid lesions, stained in various ways. The first to describe an organism in this disease was Klebs, who found in all the cases which he examined, twenty-four in number, small rodlike bacilli, growing in long narrow filaments. He succeeded in making cultivations, but in the light of modern research it must have been that his cultures were mixed ones. To Eberth belongs the honor of the discovery of the bacillus of typhoid fever. In 1880, after long and scientific study, he was able to demonstrate the existence of a bacillus in the organs of those dead of typhoid, and embodied his results in two papers.1 He believed them to be identical with those described by Klebs, they being short, stout bacilli, with rounded ends, and staining but indifferently. He found them in cadavers of typhoid patients, in the fæces, intestinal lesions, mesenteric glands, liver, and other organs. About the same time, Koch working independently, confirmed Eberth's discoveries and obtained photographs of the germ. Gaffky was the first to obtain the typhoid bacillus in pure cultures. Other observers, as Meyers and Coats, soon confirmed the results of Eberth and Koch, and conceded the etiological relationship of the bacillus to the disease. To-day there is but one opinion held by the best investigators, i. e., that typhoid fever is due, and due only, to the bacillus typhosus. Description.-The typhoid bacilli are short, thick organisms -2.3μ long, 0.7-0.9μ in breadth-or about one-third the diameter of a red blood corpuscle, and having rounded ends. They occur sometimes as mi 1. Virchow's Archiv. 1880, Bd.. 81; Ibid. 1881. Bd..83. 2. Mitt. aus dem Kais. Gesundheitsamte, 1881, Bd. 1. 3. Ibid, 1884, Bd. 2. A. B. a. Bacillus typhosus. x 1000. b. Spider cells. Typhoid bacilli stained to show flagella. (After Frænkel and Pfeiffer.) Typhoid bacilli are very resistant. Prudden found them to be virulent after being frozen in ice for 103 days, after heating at a temperature of 132.6° C. and after alternate thawing and freezing. Seitz and Latham have found that they are killed by quinine, salicylic acid, calomel, B-naphthol and chlorate of potassium. They are killed by carbolic acid 2-3% solution and bichloride of mercury, 1-5000. Chantmesse and Richard found that slaked lime, four parts in 1000 of water, sterilizes typhoid stools in half an hour. In ordinary water the bacilli retain their vitality for months, and may multiply, as they may, also, in earth. They are capable of growing in milk, and have been found living in butter after a week. Sunlight is a deadly enemy of this germ, pure cultures being killed in from one to seven days when exposed, and if suspended in sterilized water they are killed by direct sunlight in less than an hour. A 4. Bulletin Johns Hopkins Hospital, June, 97. 115. temperature of 60° C. kills them in twenty minutes. The bacilli undoubtedly live for a long time in fæces, in which they are most numerous during the first and second week of the disease. Virulent cultures have been made from fæces kept in a sterilized tube for fifteen days, thus showing that in sewage the bacilli may exist virulently for a considerable length of time. Prudden and Ernst have found typhoid bacilli present in water filters, and virulent cultures have been made from such filters. Staining. The typhoid bacilli stain in watery solutions of the aniline colors, but somewhat slowly. This should be borne in mind as good stained specimens are only secured where the stain is allowed to act for at least fifteen minutes. The bacilli give up their color very easily to bleaching agents, and do not stain by Gram's method. Methylene blue is especially good as a staining reagent, proceeding as follows: A little of the material to be examined is smeared upon a cover glass, another drawn over it, and both allowed to dry. They are then placed face downward in a strong watery solution of methylene blue and allowed to stain for ten or fifteen minutes; the cover glasses are then washed in alcohol, and mounted in balsam, or examined directly with the immersion lens. In order to show the presence of flagella, a special stain is employed. Loeffler's method, which is the best, is as follows: Add, until there is produced a violet black hue, an aqueous solution of ferrous sulphate to an aqueous solution of tannin, of the strength of twenty per cent. Then add 4 c.c. of 1-8 aqueous solution of logwood. This is solution No. 1. Solution No. 2 is composed as follows: Sol, caustic soda (1%) part 1. parts 5. The dye is dissolved in the aniline water, and the caustic soda solution added. The prepared cover glass is placed face downward in a watch glass containing Sol. No. 1, which is then held over the alcohol flame and heated until steam arises; the cover glass is then removed, washed in distilled water and alcohol; then a few drops of solution No. 2 is placed upon it, and it is again heated until steam arises, and then washed in alcohol and examined. It is always It is always difficult to secure good specimens of stained flagella, but patient, care ful work is often rewarded by very beautiful results. A word in regard to what may be expected from a microscopic examination of the stools in typhoid. Such an examination, if cultures are not made, is absolutely worthless, as the great number of other organisms in such stools, some of which, notably the bacillus coli communis, are indistinquishable morphologically from the typhoid bacillus, render a microscopic diagnosis impossible without the aid of cultures. The bacilli may be stained in sections by immersing for twenty-four hours in Loeffler's alkaline methylene blue, or carbolic fuchsin, washed in water and dehy drated in aniline oil, after which they are mounted in balsam. Cultivation.-The bacillus typho-sus is ærobic, grows upon all the ordinary nutrient media, and best when freely supplied with oxygen and at a temperature of 30° to 35° C. It can also grow when not supplied with oxygen. Gelatine Plate Cultures.-Upon this media the colonies appear first as round or oval dots, white and regular in outline, but gradually grow yellowish, with a finely notched border and irregular linear lines. The gelatine is not liquefied. Old colonies have a furrowed surface and are iridescent. Stab cultures on gelatine show a slightly yellow, furrowed, iridescent, notched or serrated surface growth, and a darker growth along the line of puncture, with finely dentated borders. Agar-Agar.-Upon this media there occurs a white growth, with lobed or notched border. Blood Serum.--A white layer, showing but dimly, occurs on this media. The serum is not liquefied. Potato Culture.-The cultivation of the bacillus typhosus upon this media is of the greatest importance in distinguishing between it and the bacillus coli communis. Upon potato, at incubation temperature, in about two days a glossy, moist appearance is noted over the innoculated area, and a sense of greater resistance is felt if the innoculating needle is passed over it. Under the microscope the bacilli may be seen moving with wonderful speed. If the potato be made alkaline in reaction, the growth acquires a yellowish color when very old. Upon this media the bacillus coli grows as a luxuriant yellow or brownish yellow layer along the innoculated area, being thick and very apparent, whereas, the growth of the typhoid bacillus is invisible. a Diagnosis Between Bacillus Typhosus and Bacillus Coli Communis.Always present in the intestine and in normal stools, there occurs group of organisms known as the colon group of bacilli, and one of these, the bacillus coli communis, is of great interest, as it is almost indistinguishable morphologically from the typhoid bacillus. Occurring as it does, both in health and disease, it is obvious how important it is to distinguish between the two organisms, for so close is their resemblance, that some authorities have gone so far as to consider the coli communis as actually the non-virulent form of the typhoid bacillus. This theory is, I believe, untenable, for there are a great many well-marked differences between the two organisms, if they be studied carefully. Morphologically, the typhoid bacillus is distinguished by its greater length and breadth, its more active movements, and the possession of from four to eight more flagella. These differences could only be detected by the skilled bacteriologist, and are not depended upon in making a diagnosis. The appearance of cultures of both organisms upon special media are of great diagnostic importance. The growth of the two organisms upon potato has already been spoken of, the typhoid growth being invisible, while that of the coli communis is plainly seen as a thick yellow, or brownish yellow layer, and characterized by a disagreeable odor. This media (potato) furnishes a reliable means of differentiating the organisms where pure cultures of a suspected organism can be tested separately. Very many special media have been devised by bacteriologists for diagnosing the typhoid bacillus, notably by Chantmesse, Widal and Holz, but the method of Elsner, is the most practical, and generally useful. In a recent contribution, Richardson,' of Boston, gives his method of preparing Elsner's media, which is so explicit and admirable that I have ventured to copy it verbatim. It is as follows: 1. (a) Prepare carefully and cut into small pieces half a kilogramme of small potatoes. (Small potatoes (Small potatoes are less acid and thus better for our purpose). Or better, grate the potato to a fine pulp. (b) Add a litre of water and let stand twentyfour hours in a cool place. Or, instead, the mixture may be boiled for one and a half hours. In the latter 5 Boston Med. and Surg. Jour., Oct. 28, 1897. case, after boiling, make up to one litre the loss of water by evaporation. 2. Mash thoroughly, and strain through a fine cloth. 3. Boil with 10 per cent. or, 15 per cent. gelatin. 4. Clear with egg. 5. Filter first through cotton and then through paper. 6. Test the acidity with decinormal sodic-hydrate solution. If 10 c.c. are neutralized by from 1.5 to 2.5 c.c. of the sodic hydrate solution, the medium is sufficiently correct as to acidity. If it takes more than 2.5 c.c. to neutralize 10 c.c. of the medium, then the acidity must be reduced by the addition of normal sodic-hydrate solution. 7. Add K. I., one per cent.: (a) to the medium as a whole, or (b) to the individual tubes as used. Use a solution of K. I. so made that one cubic centimetre is equivalent to one gramme of the salt. Upon this media the colon bacillus cultures appear in about twenty-four hours as round brownish colonies, very distinct, while the typhoid cultures only appear after forty-eight hours or more, as pale, translucent, finely granular colonies, or as round colonies resembling, according to Elsner, drops of dew. This method is very serviceable in detecting ty. phoid bacilli in stools, and until recently was generally used for that purpose. In an address at the Fourteenth Congress of the Sanitary Institute, Liverpool, Sept., 1894, Klein, in speaking of the differentiation of the bacillus typhosus and the bacillus coli communis, said: "The three principal cultural characters, however, by which these two organisms can be distinguished best and easiest are: Shake cultures in gelatine, milk cultures and broth cultures. While the bacillus coli rapidly forms gas bubbles in gelatine shake cultures, the typhoid bacillus does not do so; while the bacillus coli curdles milk the typhoid bacillus does not do so; and while the bacillus coli produces indol in both cultures, the typhoid bacillus does not do so." The following table of the peculi. arities of each bacillus is by Klein", and is a valuable one for reference. From the foregoing it will be seen that though the typhoid bacillus and the bacillus coli communis resemble each other very closely morphologically, yet there is a wide difference between their cultural appearances and phenomena. 6. Brit. Med. Jour., 1894, Vol. xi. Typhoid Bacillus (Eberth, Bacterium coli commune Gaffky). 1. Size, length 2-3 breadth 0.7-0.8 (Gaffky). 2. Has active movements. 2. 3. Has 10-12 flagella 3. (Nicolle and Morax), 18-24 (Rémy and Sugg). Produces no indol in 4. peptone water inocu- (To 10 c.c. of the culture 1 c.c. of a 0.02 per Movements more sluggish. Has 8-10 flagella more fragile than those of typhoid (Nicolle and Morax). 1-3(Luksch).4-6 (Rémy and Sugg). Note to 3.--Some of my specimens of bacteria coli commune have six flagella, most have only two or three. They appear to be very easily broken off. The bacilli adhere very tenaciously together in agar cultures. In drop cultivations a better result is obtained, but even then the number of flagella varies much more than in the specimen of typhoid. The bacillus was from a diarrhoeal stool. The diagnosis by flagella may be made from my specimen.-J. R. produced in Indol forty-eight hours at 37° in peptone water. 5. Causes no formation of 5 Causes abundant evolu ex gas in media contain- 6. No evolution of gas in 6. tures. 7. Does not curdle steril- 7 ized milk (Chantemesse and Widal). 8. In cultivations in neu- 8. tral whey an acidity equal to 2-3 per cent. in volume of 1-10th normal sodium hydrate solution is produced (Petruschky), 9. Growth typical on pota- 9. to acidified with 0.3-1 per cent, solution of tartaric acid or monosodium phosphate (Ferrati). 10. Grows more slowly on 10. Gelatine. 11. Does not split up 11. amygdaline in bouillon (Péré). 12. In fresh bouillon an 12. acid reaction remains for some days (Péré). 13. Does not grow in bouil- 13. lon containing 1-7000th part of formalin (by volume) (Schild). 14. The surface of gela- 14. tine upon which typhoid bacilli have already grown no longer grows them when again inoculated (Wurtz). tion of gas in media containing sugar. Bubbles surrounding the colonies in gelatune "shake cultures." Curdles sterilized milk. In similar cultivations an acidity equal to 7-8 per cent. of alkali is produced. Growth on potato thus accidified-a thick yellow layer (Ferrati.) Grows more quickly on gelatine. In bouillon cultures amygdaline is split up into glucose and hydrocyanic acid Péré). In fresh bouillon the reaction, at first acid, becomes alkaline in five days (Péré). Grows in bouillon containing 1-7000th part (by volume) of formalin (Schild). Will grow on a stratum of gelatine on which typhoid bacilli have previously grown (wurtz). Widal's Serum Reaction.-In the recent discovery, by Widal, of the serum reaction we have become possessed of a most valuable, and easily applied method of diagnosing typhoid fever. In recent paper, I have given the principles governing this a method and my own experience with it, but here only a very brief account can be given of it. A mass of literature has accumulated relating to this subject, and very many observers have tested the method, notably Widal, Pfeiffer, Durham, Wyatt Johnston, Welch, Park, Courmont, Stern, Da Costa, C. Frænkel, and Green, all agreeing that it is of greatest diagnostic value. Widal first called attention to the fact that the blood serum of a person suffering from typhoid or convalescent from it, causes loss of motion, and agglutination of typhoid bacilli when it is added to cultures. He was the first to use this method in diagnosing the disease, by mixing a drop of blood from a suspected case with ten or fifteen drops of a bouillon culture, and examining under the microscope. To Johnston, of Montreal, belongs the credit of making the method available in clinical work, by the discovery that dried blood, even though kept for a long period of time, when moistened with water and added to the prepared culture, produced the same result as fresh blood serum. That this is true has been proven by a large number of observations and the health departments of various cities have made a routine practice of making the serum diagnosis of typhoid from blood dried upon slips of paper or mica. All that is needed in making the test by Johnston's method is a good microscope with one-fifth to oneeighth inch objectives, a pure bouillon or agar culture of the typhoid bacillus, some cover glasses and slips, a platinum-wire loop, and a drop of dried blood from the case to be examined. With this simple outfit, combined with the necessary knowledge of bacteriological technics, it is safe to say that in the vast majority of cases typhoid fever may be accurately diagnosticated as early as the end of the first week. That this is of immense value to the clinician goes without saying, and if the method is as successful as at present it seems to be, we may look for valuable results, especially in the differentiation of the numerous continued fevers. Fresh cultures taken from month old stock cultures give the best result with the dry blood method, and only those from twelve to twentyfour hours old should be used. As to the degree of dilution, one to ten is best if the examination is positive, but where doubt exists, one to fifty is more certain. There is a chance of a reaction appearing in cases not 8. La Semaine médicale, June 26, 1896. typhoid, where the degree of dilution is from one to ten to one to fifteen. In describing the method of application and the phenomena observed, in using this test, I shall quote largely from my paper before referred to. The following method I have used with entire satisfaction: Upon a well-warmed glass slide, as used in microscopic work, place a small drop of distilled water. With the platinum needle take a very minute portion of a typhoid culture on agar and mix thoroughly with the distilled water upon the slide. Now moisten the dried blood drop and add a small portion to the drop of culture upon the slide and cover gently with a cover glass, being careful not to press it forcibly down upon the preparation. Examine with a one-fifth to oneeighth-inch dry objective. I have found the one-eighth-inch dry objective most satisfactory in this work, and believe that the use of an immersion lens would tend to falsify the microscopic picture unless a very shallow cell were used. It will also be noticed that I speak of an agar culture of the bacillus instead of bouillon. I have used the agar culture with entire satisfaction, and prefer it simply because it is in a solid medium and easier to handle. The only thing to be observed in using it is to be careful and take a very minute portion of the growth upon the needle in transferring it to the water on the slide. Upon watching such a prepared specimen, supposing that typhoid is not present, the bacilli are seen to be in active motion, darting here and there over the field, and nowhere joined together in any number. Often two will be seen to be attached, but groups of them are not to be seen. On the other hand, if the blood has come from a typhoid patient, in the course of from five to fifteen minutes, if the disease be well advanced -longer, if earlier in the diseasethe bacilli will be seen to become sluggish in their movements, gradually collect in small groups, and finally become agglutinated in clumps containing numerous bacilli, all movement having ceased. The serum of the first week of convalescence is said to produce the most marked reaction. In some cases the reaction takes place very slowly, while in others the bacilli will be seen to group and lose their motility almost as soon as the blood is added to the prepared culture. With this test properly performed I have never seen a reaction take 9. New York Med. Jour.,Feb. 6, 1897, p. 186. place in any but typhoid cases, save in one instance, a case of pneumonia, but which had had typhoid some months before, and I believe that we have in Widal's test an easily applied and very valuable diagnostic means for typhoid fever; in fact, the most valuable means that has, as yet, been discovered. As to whether or no the colon bacillus will react to typhoid serum, it may be said that the most careful observers agree that agglutination of colon bacilli by typhoid serum only occurs very rarely, and is of no diagnostic importance. It should be remembered that there are on record rare cases of typhoid which do not respond to Widal's test. Experimental.- Typhoid bacilli are always present in the stools of typhoid patients, and they have also been demonstrated in the blood and urine. After death they are found in Peyer's patches, mesenteric glands, liver and spleen. Inoculation of cultures of the typhoid bacillus into animals does not produce a typical typhoid as seen in man, as it is probably a fact that animals do not suffer from the disease as it occurs in man. It is a fact, nevertheless, that animals inoculated do often sicken and show symptoms of typhoid, and the bacilli may be found in the internal organs. These facts have been confirmed by numerous experimenters. Frænkel inoculated rabbits, which became sick. and some died; the spleen, Peyer's patches and certain glands showed tumefaction, and typhoid bacilli were found in the affected organs. Similar results have been obtained by Simmonde, Peiper, Chantmesse and Widal, and Cygnoeus. Intravenous injection of pure cultures in rabbits kills them in twenty-four hours, and the bacilli are found in the blood, urine, and excreta. Eberth claims that the bacilli are able to penetrate the placenta and reach the foetus. Poisonous Products. The bacilli elaborate elaborate poisonous products, of which Brieger has isolated a diamine, and Frankel and Brieger a toxalbumin. The poison isolated by Brieger produces great weakness, dilated pupils, feeble respiration, diarrhoea, and death in twenty-four hours. The toxalbumin produces paralysis, a reduction in temperature and at last, death. Immunity.-Brieger, Kitasato,Stern, and Widal are among those who have proven that an animal can be rendered slightly immune to the disease by graduated injections of the cultures of the bacillus, and that the serum of such an animal when inject |