68 METHODS OF CULTIVATION OF BACTERIA. 



Separation by killing Non-spored Forms by Heat. 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 boiled for a few minutes all the vege- 

 tative forms will be killed, while the spores will remain 

 alive and will develop subsequently. This method can be 

 easily tested in the case of cultivating B. subtilis from hay 

 infusion. A little chopped-up hay is placed in a flask of 

 water, which is boiled for about ten minutes. On this being 

 allowed to cool and stand, in a day or two a scum forms 

 on the surface, which is found to be a pure culture of the 

 bacillus subtilis. 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. (i) 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 inter- 

 fere 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 em- 

 ployed. The preparation of such media has already been 

 described (pp. 46-49). In this case the medium ought to 

 be of consideraSle thickness. 



The Supply of Hydrogen for Anaerobic Cultures. The 

 gas is generated in a large Kipp's apparatus from pure 



