484 PATHOGENIC MICROORGANISMS 



the acute infection which may be produced by the contaminating 

 organisms and succumb at a later date (four to six weeks) to the 

 tuberculous infection. The bacilli may then be obtained, after sterile 

 dissection, by making cultivations from lymph nodes or other tubercu- 

 lous foci which contain only tubercle bacilli. When isolation from 

 sputum is attempted, whether directly or by means of animal inocula- 

 tion, the sputum may be rendered comparatively free from contaminat- 

 ing bacteria by a process of washing devised by Koch. The sputum is 

 thoroughly rinsed in running water to free it from its outer covering of 

 pharyngeal mucus. It is then washed in eight or ten changes of sterile 

 water. The material selected for cultivation is taken from the center 

 of the washed mass, if possible from the small flakes of caseous material 

 often visible in such sputum. 



On blood serum at 37.5° C, colonies usually become visible at the 

 end of eight to fourteen days. They appear at first as small, dry, gray- 

 ish-white, scaly spots with corrugated surfaces. After three or four 

 weeks' cultivation, these join together, covering the surface of the 

 medium as a dry, whitish, wrinkled membrane. Coagulated dog serum 

 is regarded by Theobald Smith ^ as one of the most favorable media 

 for the growth of tubercle bacilli. 



Slants of agar, to which whole rabbit's blood has been added in 

 quantities of from 1 to 2 c.c. to each tube, make an excellent medium for 

 this bacillus, both for isolation and continuous cultivation. 



Cultivation methods were simplified by the discovery by Roux 

 and Nocard that growth can be obtained upon media to which glycerin 

 has been added. Upon glycerin-agar (glycerin 3 to 6 per cent), at 

 37.5° C, colonies become visible at the end of from ten days to two 

 weeks, at first as dry, white spots; later, as delicately corrugated 

 membranes. 



Glycerin bouillon (made of beef or veal with pepton one per cent, 

 glycerin six per cent, and rendered slightly alkaline) is an extremely 

 favorable medium. It should be filled, in shallow layers, into wide- 

 mouthed fiasks, since free access of oxygen is essential for growth. 

 Transplants to this medium should be made by carefully floating flakes 

 of the culture upon the surface. In this medium the bacilli will spread 

 out upon the surface, at first as a thin, opaque, floating membrane. 

 This rapidly thickens into a white, wrinkled, or granular layer, spread- 

 ing out in all directions, and covering the entire surface of the fluid in 

 from four to six weeks. Later, portions of the membrane sink to the 



1 Th. Smith, Jour. Exp. Med., iii, 1898, 



