ACID-FAST GROUP 319 



the course of four weeks at blood-heat the surface of the broth is 

 covered with a heavy, wrinkled pellicle. By the end of eight weeks 

 it is ready for the preparation of the tuberculin. The contents of 

 several flasks are then united and placed in a porcelain evaporating 

 dish on a water-bath. The material is concentrated to about 

 one-tenth of its original bulk, when the glycerin content becomes 

 about 40 per cent. This constitutes the tuberculin commonlj' used. 



Tuberculin of Denys. This investigator believed that the 

 efficiency of the tuberculin was impaired by the heat used in con- 

 centration. He filtered unheated broth cultures through porcelain 

 and utilized the filtrate. 



The tuberculol of Landmann, the tuberculocidin or antiphthisin 

 (A. P.) of Klebs, the oxytuberculin of Hirschfelder are all tuber- 

 culins prepared by various modifications of the original Koch 

 method, such as repeated extraction of bacilli at different tempera- 

 tures, treatment with H 2 O 2 , etc. 



Koch's purified tuberculin is prepared by adding lj volumes of 

 absolute alcohol to the crude tuberculin and allowing the mixture 

 to stand twenty-four hours, collecting the precipitate on a filter 

 and washing in 60 per cent, alcohol, and finally drying in a desic- 

 cator at 100. This material is diluted in water or glycerin before 

 use, being soluble in these. 



Tuberculin R. or T. R. of Koch. Young virulent cultures of 

 tubercle bacilli are dried in a vacuum, then ground in an agate 

 mortar or ball mill for a considerable period. The resultant pow- 

 der is next suspended in water, shaken, and centrifuged for half 

 to three-quarters of an hour at a speed of 4000 revolutions per 

 minute. The bacterial fragments are thrown to the bottom. The 

 portion of the tubercle bacilli that remain in solution Koch termed 

 Tuberculinum O. (T. O.); the portion which is precipitated was 

 called Tuberculinum R. (T. R.). Koch dried this T. R., reground 

 it, suspended and centrifuged to get rid of all the T. O. The T. 

 R. was originally planned for use in immunization. This residual 

 material was found to be largely free from the toxic action of other 

 tuberculin and of the T. O. This preparation has not come into 

 use, inasmuch as there is always a possibility that living virulent 

 organisms may survive the treatment and produce disease when 

 injected. 



