SEPARATION OF ANAEROBES 57 



be boiled for a few minutes all the vegetative 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. (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, 37). 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 

 is passed through three wash-bottles, as in Fig. 21. In the first is 

 placed a solution of lead acetate (1 in 10 of water) to remove any traces 

 of sulphuretted hydrogen. In the second is placed a 1 in 10 solution of 

 silver nitrate to remove any arsenietted hydrogen which may be present 

 if the zinc is not quite pure. In the third is a 10 per cent solution 

 of pyrogallic acid in caustic potash solution (1 : 10) to remove any 

 traces of oxygen. The tube leading from the last bottle to the vessel 

 containing the medium ought to be sterilised by passing through a 

 Btmsen flame, and should have a small plug of cotton wool in it to filter 

 the hydrogen germ-free. 



Separation of Anaerobic Organisms. (a) By Roll-tubes. 

 A 1J inch test-tube has as much gelatin put into it as would be 

 used in the Esmarch roll- tube method. It is corked with an 

 india-rubber stopper having two tubes passing through it, as in 

 Fig. 22. The ends of the tubes are partly drawn out as shown, 



