THE CULTURE OF ANAEROBIC ORGANISMS 61 



by ordinary plate methods certain pathogenic organisms, such 

 as b. tuberculosis, b. mallei, and the pneumococcus, when such 

 occur in conjunction with other bacteria. These grow best on 

 special media, and the first two grow so slowly that the other 

 organisms present may outgrow them, cover the whole plates, and 

 make separation difficult. The method adopted in such cases is 

 to inoculate an animal with the mixture of bacilli, wait until the 

 particular disease develops, kill the animal, and with all aseptic 

 precautions (vide p. 140) inoculate tubes of suitable media from 

 characteristic lesions situated away from the seat of inoculation, 

 e.g., from spleen in the case of b. tuberculosis, spleen or liver 

 in the case of b. mallei, and heart blood in the case of 

 pDeuniococcus. 



Separation by killing Non-spored Forms by Seat. — This is 

 a method which has a limited application. As has been said, 

 the spores of a bacterium resist heat more than the vegetative 

 forms. When a mixture contains spores of one bacterium and 

 vegetative forms of this and other bacteria, then if the mixture 

 be heated for 10 minutes at 80° C. all the vegetative forms may 

 be killed, while the spores will remain alive and will develop 

 subsequently. Several tubes of different media should be 

 inoculated and treated thus, as the success of the method is very 

 variable. The method is also often used to aid in the separation 

 of b. tetani, vide infra. 



The Principles of the Culture of Anaerobic 

 Organisms. 



All ordinary media, after preparation, may contain traces of 

 free oxygen, and will absorb more from the air on standing. 

 (1) For the growth of anaerobes this oxygen may be expelled by 

 the prolonged passing of an inert gas, such as hydrogen, through 

 the medium (liquefied if necessary). Further, the medium must 

 be kept in an atmosphere of the same gas while growth is going 

 on. (2) Media for anaerobes may be kept in contact with the 

 air, if they contain a reducing agent which does not interfere 

 with bacterial growth. Such an agent takes up any oxygen 

 which may already be in the medium, and prevents further 

 absorption. The reducing body used is generally glucose, though 

 formate of sodium may be similarly employed. The preparation 

 of such media has already been described (pp. 36, 38). In this 

 case the medium ought to be of considerable thickness. 



The Supply of Hydrogen for Anaerobic Cultures. — The gas is generated 

 in a large Kipp's apparatus from pure sulphuric acid and pure zinc. It 



