738 Tuberculosis 



also occur when the secondary inoculation is made with 

 killed cultures of the bacilli. 



It was also observed that if the material used for the 

 secondary injections was not too concentrated and the 

 injections not too often repeated (only every six to forty- 

 eight hours), the animals treated improved in condition, 

 and continued to live, sometimes (Pfuhl) as long as nineteen 

 weeks. 



Tuberculin. Koch also discovered that a 50 per cent, 

 glycerin extract of cultures of the tubercle bacillus tuber- 

 culin produced the same effect as the dead cultures orig- 

 inally 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 " tuberculin " seems to be 

 an albuminous 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 120 C. for hours without 

 change. Tuberculin is almost harmless for healthy animals, 

 but extremely poisonous for tuberculous animals, its injec- 

 tion into them being followed either by a violent febrile 

 reaction or by death, according to the extent of the dis- 

 ease 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 sur- 

 face 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 cul- 

 tures 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 sinks, a new growth some- 

 times 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 c.c. to i liter, are more 

 convenient. The contents 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. Superficial tuberculous lesions (lupus) sometimes 

 ulcerate and slough away. The febrile reaction is sufficiently character- 

 istic to be of diagnostic value, though tuberculin can only be used with 

 perfect safety as a diagnostic agent upon the lower animals. 



