350 BA CTERIOL OGY. 



the upper layers of the cotton with either sealing- 

 wax or paraffin of a high melting-point ; or by insert- 

 ing over the burned end of the cotton plug a soft, 

 closely fitting cork that has been sterilized in the 

 steam sterilizer just before using (Ghriskey). This 

 precaution is necessary because of the slow growth of 

 the organism. Under the most favorable conditions 

 tubercle bacilli directly from the animal body show no 

 evidence of growth for about twelve days after inocu- 

 lation upon blood-serum, and, as they must be retained 

 during this time at the body-temperature 37.5 C. 

 evaporation would take place very rapidly and the 

 medium would become too dry for their development. 



If these primary efforts result in the appearance of a 

 culture of the bacilli, further cultivations may be made 

 by taking up a bit of the colony, preferably a moder- 

 ately large quantity, and transferring it to fresh serum, 

 and this in turn is sealed up and retained at the same 

 temperature. Once having obtained the organism in 

 pure culture, its subsequent cultivation may be con- 

 ducted upon the glycerin-agar-agar mixture ordinary 

 neutral nutrient agar-agar to which from 4 to 6 per cent, 

 of glycerin has been added. This is a very favorable 

 medium for the growth of this organism after it has 

 accommodated itself to its saprophytic mode of exist- 

 ence, though blood-serum is perhaps the best medium 

 to be employed in obtaining the first generation of the 

 organism from tuberculous tissues. 



The organism may be cultivated also on neutral milk 

 to which 1 per cent, of agar-agar has been added, also 

 upon the surface of potato, and likewise in meat-infu- 

 sion bouillon containing from 4 to 6 per cent, of 

 glycerin. 



