Isolation and Cultivation 317 



which receives an addition of two or three drops of 40 per 

 cent, formaldehyde or "formalin." After standing from 

 fifteen to thirty minutes, transfers are made to appro- 

 priate culture media, when the acid-proof organisms are 

 apt to develop, the others having been destroyed by the 

 formaldehyde. 



Blood-serum. Koch first achieved artificial cultivation 

 of the tubercle bacillus upon blood-serum, upon which 

 the bacilli are first apparent to the naked eye in about two 

 weeks, in the form of small, dry, whitish flakes, not unlike 

 fragments of chalk. These slow r ly increase in size at the 

 edges, and gradually form small scale-like masses, which 

 under the microscope are found to consist of tangled 

 masses of bacilii, many of which are in a condition of 

 involution. 



Kitasato* has published a method by which Koch was 

 able to secure the tubercle bacillus in pure culture, from 

 sputum. After carefully cleansing the mouth the patient 

 is allowed to expectorate into a sterile Petri dish. By this 

 method the contaminating bacteria from the mouth and 

 receptacle are excluded, so that the expectorated material 

 contains only bacteria present in the lungs. The material 

 is carefully washed a great many times in renewed sterile 

 distilled water until all bacteria not inclosed in the muco- 

 purulent material are removed; it is then carefully opened 

 with sterile instruments, and the culture medium glycerin 

 agar-agar or blood-serum is inoculated from the center. 

 Kitasato has been able by this method to demonstrate that 

 many of the bacilli ordinarily present in tubercular sputum 

 are dead, although they continue to stain well. 



Kitasato 's method of washing the sputum has been 

 modified and simplified by Czaplewski and Hensel f in 

 their studies of whooping-cough. Instead of washing the 

 flakes of sputum in water contained in dishes, they shook 

 them in sterile peptone water contained in test-tubes. The 

 shaking in the test-tube being so much more thorough 

 than the washing in dishes, fewer changes of the fluid are 

 necessary, three or four washings being sufficient. 



Glycerin Agar-agar. In 1887 Nocard and Roux J gave 



* "Zeitschrift fur Hygiene," Bd. xi. 



t "Centralbl. f. Bakt. u. Parasitenk.," xxn, Nos. 22 and 23, p. 643. 



f'Ann. de 1'Inst. Pasteur," 1887, No. 1. 



