152 TUBERCULOSIS 



(i) The preparation of the culture medium (glycerin- 

 ated bouillon), distributing it in suitable flasks and inoculating 

 it with the growth from a pure culture of tubercle bacteria. 



(2) The flasks are placed in an incubator at a tempera- 

 ture of about 37° C. where they remain until the growth 

 ceases. The length of time necessary to accomplish this 

 depends upon the age and condition of the culture from which 

 the inoculations were made. From four to ten weeks are 

 usually required. 



(3) After the maximum growth is attained, the cultures 

 are sterilized by heat, either by boiling in a closed water bath 

 or heating to a higher temperature in an autoclav. 



( 4) After sterilization, the cultures are filtered to remove 

 all of the dead bacteria, and then the filtrate is evaporated over 

 a water bath to the desired degree. 



(5) The concentrated liquid is passed through a Pasteur 

 or Berkefelter filter, standardized, bottled and labeled for dis- 

 tribution. It should be perfectly clear although its color may 

 vary. If it is cloudy it should be rejected. 



It will be seen from the method of preparation that tuber- 

 culin cannot possibly contain living tubercle bacteria. It is 

 heated on two occasions to a temperature and for a length of 

 time far in excess of that required to destroy them besides 

 being passed through a filter capable of removing all bacteria. 



The original tuberculin or lymph of Koch was concen- 

 trated to one-tenth of the volume of the saturated culture. 

 This gave a thick, syrupy liquid owing to the presence of the 

 glycerine. The diagnostic dose which came to be recom- 

 mended for cattle of medium weight was 0.25 c.c. On account 

 of its consistency as well as the minuteness of the dose, it was 

 found to be practicable to dilute this quantity with seven parts of 

 a diluent. A weak solution of carbolic acid was ordinarily used. 

 The difficulties and the danger of contamination involved in 

 making the dilutions in the field led to the method of diluting 

 the tuberculin in the laboratory before sending it out. This 

 has been the practice of the Bureau of Animal Industry for a 

 number of years. Equally as good results are obtained b}' 

 concentrating the saturated culture to the point where 2C.c. 



