486 MICROBIOLOGY OF SPECIAL INDUSTRIES. 



made from attenuated as well as from virulent cultures. The organism 

 is inoculated into beef bouillon to which 5 per cent glycerin has been 

 added. The culture medium is usually distributed in flasks and the 

 tubercle organisms, when inoculated, are carefully placed on the surface 

 of the medium. The cultures are incubated at a temperature of 37 

 to 38 for six to ten weeks or longer, during which time a heavy growth 

 slowly spreads over the 'surf ace of the medium and finally falls to the 

 bottom of the flasks. In the successful preparation of tuberculin it is 

 important that the cultures should remain undisturbed, having access 

 to plenty of air, that the incubator temperature should be constantly 

 maintained without fluctuations, and that the organisms should be allowed 

 to grow until they have completely elaborated the active "tuberculinic" 

 substance. After the growth is complete, the cultures are removed 

 from the incubator and sterilized in streaming steam. The killed 

 cultures are then evaporated over a water bath to one-tenth the original 

 volume, the bacteria are removed by passing the cultures through paper 

 and a Berkefeld filter and a preservative is added. For cattle the dose 

 of tuberculin concentrated by evaporation to one-tenth the original 

 volume, is 0.25 c.c. to 0.7 c.c. Because of the fact that the material is 

 thick and syrupy in consistency and the dose is inconveniently small, 

 it is usually diluted with seven parts of weak carbolic acid solution, or it 

 may be evaporated to four-fifths the original volume and preserved by 

 the addition of i per cent carbolic acid. 2 c.c. of the diluted tuberculin 

 is used as the dose for cattle. The product should be tested for activity 

 by treating known tuberculous animals with the tuberculin under test. 

 The presence of typical reactions in tuberculous animals indicates the 

 reliability of the product. 



In human, as well as in veterinary practice, tuberculin may be applied 

 as a diagnostic agent in various ways. In addition to the hypodermic in- 

 jection of tuberculin (old), as described above, the methods of Calmette,* 

 von Pirquet and Moro may be used. Calmette's ophthalmo test consists 

 in the instillation in the eye of Koch's purified or refined tuberculin. 

 Purified tuberculin is prepared by treating the original tuberculin with 

 absolute alcohol, washing and drying the precipitate. One drop of a 

 i per cent solution of purified tuberculin is placed in the eye. A positive 

 reaction is manifested by a congestion of the palpebral and ocular con- 

 junctiva a few hours after the application of the tuberculin. The method 



* Calmette: Presse Medicale, 1907, 15. 



