354 MICRO-ORGANISMS AND DISEASE [chap. 



Koch has shown that by subcutaneous inoculation, by 

 inhalation, inoculation into the peritoneum, the anterior 

 chamber of the eye, &c., of artificial subcultures far 

 removed (by many generations) from the original source, 

 typical tuberculosis is produced in all animals susceptible to 

 tubercle (guinea-pigs, rabbits, dogs, rats, mice), and that of 

 course the tubercular deposits in these experimental animals 

 again contain abundantly the tubercle bacilli ; thus the final 

 and exact proof that the tubercle bacilli are the vera causa 

 of the tubercular process was definitely established. The 

 intravascular and intra-peritoneal injection produced the 

 most striking and rapid results. 



Although the growth on glycerine Agar mixture is copious, 

 it yet has this drawback, that by continued subculture the 

 virulence of the bacilli is worn off. The first subcultures 

 act virulently, inasmuch as they produce on inoculation into 

 guinea-pigs general tuberculosis ; thus even with a fourth 

 and fifth subculture I have succeeded in producing the 

 same results as by directly using sputum or bovine tubercle, 

 but after the eighth or tenth generation I have not succeeded 

 in producing general tuberculosis and death of the guinea- 

 pigs by inoculation. I have found that if from an Agar- 

 glycerine culture, which, owing to age or subcultures, has 

 lost completely its virulence, new cultures are established in 

 alkaline beef broth, to which a piece of boiled white of egg 

 is added, these acquire rapidly again a somewhat virulent 

 character. 



Also on Agar ascites fluid with glycerine (see a former 

 chapter) the original virulence can be re-established. It 

 ought to be also stated that by continued subculture in 

 glycerine broth or in glycerine Agar the growth becomes 

 more abundant and makes its appearance in much shorter 

 time. 



