“As early as 1874, Gerlach had experimented largely in feeding the milk of tuberculous cows, and had infected calves, pigs, sheep and rabbits in this way. Chauveau in (1874), Klebs (1873), Semmer (1880) and Parrot (1870) fed calves, cats, Guinea pigs, pigs, sheep and a variety of other species of animals with tuberculous flesh or lungs, or saliva, producing tuberculosis in a large proportion of cases. In this manner they escaped the charge of the operation of a wound and of ærial bacteria, and yet succeeded in producing infection. The lesions in this case began in the intestinal mucous membranes and mesenteric glands.
“Another essay in the same direction was made by Tappeiner (1878), who infected rabbits, Guinea pigs and dogs, by enclosing them in boxes and compelling them to breathe tuberculous liquids intimately mixed with the air atomization. In these cases lesions began in the lungs and bronchial and mediastinal glands.[[1]]”
[1]. (From Report to the N. Y. Legislature, 1895. James Law.)
Bacteriology. The bacillus tuberculosis, the essential cause of tubercle, was demonstrated by Koch in 1882. It was at first supposed to be peculiar in its indisposition and tardiness to take a stain, and in its retention of the stain once imparted, even when it is treated with acids. Later observations have shown that these characteristics are common to certain other bacilli, notably to those of leprosy, smegma, and to specimens found in timothy hay and other grasses, cow dung, milk and butter. These furnish sources of fallacy in the milk and butter especially, but when the specimens examined are taken from the interior of a tubercle, lymph gland or serous cavity the danger of error may be practically ignored. What is quite as important is the fact that the tubercle bacillus is subject to evolutionary changes in adapting itself to a habitat in different genera of animals, and to a lesser extent in different parts of the same animal, by which its pathogenesis is modified, but these variations do not materially affect the staining qualities. Still more striking variations have been found in old cultures, ovoid forms (Metchinkoff), club-shaped microbes and filaments (Metchinkoff, Klein, etc.), branching filaments (Fischl, Coppen Jones), divergent club-shaped groups, like actinomyces (Babes and Levaditi). The last named, seen in inoculated rabbits, have been held to establish a relationship between the microbes of tuberculosis and actinomycosis, as the club-shaped cells do not take the usual Ziehl-Neelsen stain for tubercle bacilli, but the Birsch-Hirshfeld’s actinomyces stain. Apart from the rabbit such variations are not likely to prove sources of fallacy in identification of the microbe.
Morphology. As met with in the tubercle of man or ox the bacillus is a minute rod with rounded ends, 1.5 to 3.5μ long, by 0.2μ in thickness. In the ox it is shorter and thicker on an average than in man. They are usually solitary, but two and exceptionally even three or four elements may be united. In stained specimens unstained portions are frequently seen (spores?). When cultivated on blood serum there is a tendency to form elongated rope-like colonies having a waving outline. Other evolution forms have been already noted.
Staining. The resistance to staining is overcome by a heated alkaline watery solution of methylene blue (Koch), or better by a carbolated solution of fuchsin (Ziehl-Neelsen). To a 5 per cent. watery solution of carbolic acid add one tenth of its volume of saturated alcoholic solution of fuchsin. The suspected sputum or scraping from a section of a lesion is spread in a thin layer on a cover glass by drawing over it the straight edge of a second cover glass, dried in the air, and passed three times through an alcohol flame with the smeared side up. It may then be floated on the surface of the staining fluid with the specimen side down, then gently heated for three or four minutes almost to ebulition, drying being prevented by the addition of more of the carbol-fuchsin solution as required. It is then washed in water to remove excess of the staining fluid and treated with a bleaching acid solution (sulphuric acid 5 per cent. or nitric acid 15 per cent.) When decolorized it is well rinsed in two or more portions of a 60 or 70 per cent. alcohol and then in water. Next stain with a 1 or 2 per cent. aqueous solution of methylene blue for one minute and wash off the excess with water. The tubercle bacilli will appear of a deep red and other bacteria blue.
Gabbett’s counter-staining and decolorizing solution is especially useful in economizing time. After staining, as above directed, in the carbol-fuchsin solution for one minute, wash in water, then cover with a solution of 2 grams methylene blue (powder) in 100cc. of 10 per cent. sulphuric acid, until the film has a faintly bluish tint: then wash thoroughly in water and examine. Tubercle bacilli come out red, others blue.
In examination of tissues the microscopic sections are steeped for twenty-four hours in the carbol-fuchsin solution (or 1 hour at a temperature of 45° to 50° C.), then decolorized in a 5 per cent. sulphuric acid for a few seconds, then in a 70 per cent. alcohol: they are then counter-stained in an aqueous solution of methylene blue, washed in water dehydrated with absolute alcohol, cleared up in xyol or oil of cedar, and mounted in Canada balsam.
Biology. The bacillus tuberculosis is notorious for its variant forms, assumed in adapting itself to given environments, and which it retains with considerable tenacity for a time, even when placed in a new and different medium. It is largely held to be an obligatory parasite, and incapable of survival as a saprophyte, yet Straus, Nocard and others, have shown that the form obtained from the tuberculosis of birds can easily be made to live in suitable dead mixtures of organic matter. It is also notorious that the bacillus taken direct from the tubercle of the mammal, and which, as a rule, fails to grow at once in glycerined bouillon, yet, after a first successful culture in the new medium, often adapts itself completely, and thereafter it can in many cases be transferred from bouillon to bouillon, with as great certainty as it could formerly be inoculated from ox to ox. The difference is one of habit and adaptability, rather than any primary and permanent distinction of species. There is every reason to believe that the microbe has lived, and under given conditions can still be made to live as a saprophyte, with a greatly reduced adaptability to parasitic life in the animal, just as we see to-day that it is only with great difficulty transferred from certain genera of animals to certain other genera (from bird to ox and vice versa). Trudeau tells us of a culture of a bacillus tuberculosis from man, inoculated on the rabbit and then cultivated in vitro in successive generations for six years, that was in this way robbed of its pathogenesis for Guinea pigs, which, after inoculation, lived for many months, some two and a half years, and some even recovered. The average duration of life in the Guinea pig, after tuberculosis inoculation from man, rabbit, Guinea pig or recent culture is but seventeen days. The bacillus of human sputum often produces only localized tuberculosis in the ox. Even in the same species and individual the pathogenesis often varies materially. Nothing is more familiar than the slow progress of tuberculosis in the bones and lymph glands of man, on the one hand, and its frequently rapid progress in the lungs, liver and brain, on the other. As inoculated on the lower animals bacilli from the lungs of man are usually more virulent than those from the lymph glands (Creighton, Arloing, F. Craven Moore, Courmont and Denis). Among seven specimens of human sputum, the bacilli in six cases showed a fair average vitality, whereas those of the seventh failed to grow on blood serum of the dog (Theobald Smith). In both man and cow a large proportion of cases of tuberculosis remain localized, the disease lasting for years with little or no appreciable advancement, and not a few subjects appear to make a permanent recovery.
On every hand this evolutionary tendency of the bacillus tuberculosis places itself in evidence, demanding a recognition of the fact, that more or less transient or lasting variations in accordance with previous or existing environment, control to a large extent the transmissibility of the disease among different genera and even among individuals of the same genera and species, and also the fatal progress, or the mild and evanescent issue of the infection.