64 ME THODS OF C UL 77 VA TION OF BA C TERIA . 



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 AN^ROBIC 

 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 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. Formate of sodium may 

 be employed. The preparation of such media has already 

 been described (pp. 43-46). In this case the medium 

 ought to be of considerable thickness. 



The Supply of Hydrogen for Ancerobic Cultures. The 

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

 sulphuric acid and pure zinc. It is passed through three 

 wash bottles as in Fig. 21. In the first is placed a solu- 

 tion of lead acetate (i in 10 of water) to remove any traces 

 of sulphuretted hydrogen. In the second is placed a i in 

 10 solution of silver nitrate to remove any arsenietted 



