THE TUBERCLE BACILLUS 491 



dead bacilli led Koch to emplo3 r various methods of extraction of cultures 

 for the manufacture of tuberculin. 



"Old Tuberculin " l (Koch) ("T.A.K.") The first tuberculin made by 

 Koch is produced in the following manner : Tubercle bacilli are grown 

 in slightly alkaline 5 per cent glycerin-pepton bouillon for six to eight 

 weeks. At the end of this time, growth ceases and the corrugated 

 pellicle of tubercle bacilli, which during growth has floated on the 

 surface, begins, here and there, to sink to the bottom. The entire 

 culture is then heated on a water-bath at about 80 C., until reduced to 

 one-tenth of its original volume. It is then filtered either through 

 sterile filter paper or through porcelain filters. The resulting filtrate is 

 a rich brown, syrupy fluid, containing the elements of the original cul- 

 ture medium and a 50 per cent glycerin extract of the tubercle bacilli. 

 While the glycerin is of sufficient concentration to preserve it indef- 

 initely, 0.5 per cent phenol may be added as an additional precaution. 

 Dilutions of this fluid are used for diagnostic and therapeutic purposes. 



"New Tuberculin" 2 (Koch) (TA, TO, TR). Koch believed that 

 the immunity resulting from treatment with the old tuberculin was 

 purelj r an antitoxic immunity, devoid of all antibacterial action. The 

 use of whole dead tubercle bacilli for immunization purposes, however, 

 was impracticable; because, injected subcutaneously, they were not 

 absorbed, and introduced intravenously they were deposited in the lungs 

 and gave rise to lesions. Koch was led, therefore, to resort to more 

 energetic extraction of the bacilli in the hope of procuring a substance 

 which could be easily absorbed and would at the same time give rise, 

 when injected, to antibodies more definitely bactericidal. By extract- 

 ing tubercle bacilli with decinormal NaOH, for three days, filtering 

 through paper and neutralizing, he obtained his TA (alkaline tubercu- 

 lin). This preparation seemed to fulfil some of the hopes of its dis- 

 coverer, but had the disadvantage of often producing abscesses at the 

 points of injection. Koch then resorted to mechanical trituration of 

 the bacilli. The method he subsequently followed for tuberculin pro- 

 duction is now extensively used, and is carried out as follows: 3 



Virulent cultures of tubercle bacilli are dried in vacuo and thoroughly 

 ground in a mortar. Grinding is continued until stained preparations 

 reveal np intact bacilli. (This is done by machinery in all large manu- 

 factories.) One gram of the dry mass is shaken up in 100 c.c. of sterile 

 distilled water. This mixture is then centrifugalized at high speed. 



l Koch, Cent. f. Bakt., 1890; Deut. med. Woch., 1891. 



2 Koch, Deut. med. Woch., 14, 1897. 3 Ruppel, Lancet, March 28, 1908. 



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