720 Tuberculosis 



extract of cultures of the tubercle bacillus — tuberculin — ^produced 

 the same effect as the dead cultures originally used, and announced 

 the discovery of this substance to the scientific world, in the hope 

 that the prolongation of life observed to follow its use in the guinea- 

 pig might also be true of man. 



The active substance of the "tubercuhn" seems to be an al- 

 buminous derivative (bacterioprotein) insoluble in absolute alcohol. 

 It is a protein substance and gives all the characteristic reactions. 

 It differs from the toxalbumins in being able to resist exposure to 

 i2o°C. for hours without change. Tuberculin is almost harmless 

 for healthy animals, but extremely poisonous for tuberculous ani- 

 mals, its injection into them being followed either by a violent 

 febrile reaction or by death, according to the extent of the disease 

 and size of the dose administered. 



Preparation of Tuberculin. — The preparation of tuberculin is simple. Flasks 

 made broad at the bottom so as to expose a considerable surface of the contained 

 liquid are filled.to a depth of about 2 cm. with bouillon containing 4 to 6 per cent, 

 of glycerin, and preferably made with veal instead of beef infusion. They are 

 inoculated with pure cultures of the tubercle bacillus, care being taken that the 

 bacillary mass floats upon the surface, and are kept in an incubator at 37°C. In 

 the course of some days a slight surface growth becomes apparent about the 

 edges of the floating bacillary mass, which in the course of time develops into a 

 firm, coarsely granular, wrinkled pellicle. At the end of some weeks development 

 ceases and the pellicle sLuks, a new growth sometimes occurring from floating 

 scraps of the original. 



Some bacteriologists prefer to use small Erlenmeyer flasks for the purpose, but 

 large flasks, which contain.from 500 cc. to i liter, are more convenient. The con- 

 tents of a number of flasks of well-grown cultures are poured into a large porcelain 

 evaporating dish, concentrated over a water-bath to one-tenth their volume, and 

 filtered through a Pasteur-Chamberland filter. This is crude tuberculin. 



When doses of a fraction of a cubic centimeter of crude tuberculin are injected 

 into tuberculous animals, an inflammatory and febrile reaction occurs. Superfi- 

 cial tuberculous lesions (lupus) sometimes ulcerate and slough away. The febrile 

 reaction is sufiiciently characteristic to be of diagnostic value, though tuberculin 

 can only be used with perfect safety as a diagnostic agent upon the lower 

 animals. 



From the "crude" or original tuberculin Koch prepared a purified or "refined" 

 tuberculin by adding one and one-half volumes of absolute alcohol, stirring 

 thoroughly, and standing aside for twenty-four hours. At the end of this time a 

 flocculent deposit will be seen at the bottom of the vessel. The supernatant 

 fluid is carefully decanted and an equal volume of 60 per cent, alcohol poured into 

 the vessel for the purpose of washing the precipitate, which is again permitted to 

 settle, the fluid decanted, and the washing thus repeated several times, after 

 which it is finally washed in absolute alcohol and dried in a vacuum exsiccator. 

 The white powder thus prepared is fatal to tuberculous guinea-pigs in doses of 2 to 

 10 mg. It is soluble in water and glycerin and gives the protein reactions. The 

 tuberculin as Koch prepared it is now known as "concentrated" or "Koch's 

 tuberculin," to differentiate it from the "diluted tuberculin" sometimes sold in 

 the shops, which is the same thing so diluted with i per cent, aqueous carbolic 

 acid solution that i cc. equals a dose. The dose of the concentrated tuberculin 

 is 0.4 to 0.5 cc; that of the diluted tuberculin, i cc. 



Tuberculin does not exert the slightest influence upon the tubercle 

 bacillus, but acts upon the tuberculous tissue, augmenting the 

 poisonous influence upon the cells surrounding the bacilli, destroy- 

 ing their vitality, and removing the conditions favorable to bacillary 

 growth, which for a time is checked. This action is accompanied 



