CULTUEE OF ANAEROBIC BACTERIA 77 



A large test-tube has as much glucose-gelatine medium 

 put into it as would be required in the ordinary roll culture. 

 It is then corked with an indiarubber stopper, through which 

 pass two angle tubes, one of which passes to the bottom 

 of the tube, both of the external limbs being plugged 

 with cotton-wool. Three or more such tubes are pre- 

 pared and sterilised in the ordinary way in the steam 

 steriliser. 



The liquid or other substance to be examined for anaerobic 

 bacteria is attenuated by dilution with ordinary melted 

 gelatine medium, as in an ordinary plate culture. The 

 prepared sterilised tubes are inoculated from these attenua- 

 tions. The tubes are placed in water at 30° C, to keep the 

 media in a molten condition. Hydrogen gas is now passed 

 through the long tube which dips into the media for about 

 15 to 30 minutes. The gas supply tubes are then sealed off 

 by means of a blowpipe flame, and the media in the tube is 

 rolled and cooled as in the ordinary Esmarch tube. The 

 gelatine is thus enclosed in an atmosphere of hydrogen, in 

 which the colonies are free to develop. When the colonies 

 have grown sufficiently, they can be subcultured into 'deep' 

 tubes of glucose-gelatine. The needle may be prevented 

 from carrying air into the stab by first melting the 

 upper half-inch of the medium. From such ' stab ' or 

 ' deep ' cultures anaerobic growths may be maintained 

 almost indefinitely by successive subcultures in similar 

 tubes. 



The supply of hydrogen gas for the above purpose is best 

 generated from a large Kipp's apparatus, using pure zinc 

 and sulphuric acid, of course, with the usual precautions to 

 prevent admixture with air. The gas, before passing to the 

 culture-tube, should be washed by passing through a wash- 

 bottle containing water. 



Perhaps the simplest method for the cultivation of the 



