88 A MANUAL OF BACTERIOLOGY 



puncture, and a well-fitting rubber cap may be applied 

 while the tube is hot. The heating expels a portion of the 

 air, and, with a well-fitting cap, creates a negative pressure 

 within the tube, so that the residual oxygen is not so 

 readily absorbed, or the tubes may be placed in a Buchner 

 apparatus (see below). 



When, however, an organism is required to grow 

 anaerobically on the surface of the medium, or in a fluid 

 medium, some other method must be adopted. 



For fluid cultures Hamilton's method is the simplest of 

 all. The fluid in the tubes is covered with a layer of olive 

 oil or, better, liquid paraffin 1-2 cm. thick, and the tubes 

 are then sterilised. The layer of oil prevents the access 

 of air. The only disadvantage is that the inoculation, or 

 the withdrawal of culture, must usually be performed with 

 a sterile glass pipette ; if a wire needle be used the 

 material is very liable to be detached in passing through 

 the oil. 



Buchner's method is that usually adopted, and consists 

 in absorbing the oxygen by means of alkali and pyrogallic 

 acid, and so cultivating in an atmosphere of nitrogen. 

 This can be carried out in two ways either in a wide- 

 mouthed bottle with well-fitting glass stopper, sufficiently 

 large to contain the test-tubes, or in a Buchner's tube. 

 For the first the inoculated culture tubes are placed in 

 the bottle, into which a few cubic centimetres of a strong 

 aqueous solution of pyrogallic acid have previously been 

 poured. By means of a thistle funnel, an equal volume of 

 20 per cent, caustic potash x or soda solution is then 

 added. As quickly as possible the thistle funnel is with- 

 drawn without mixing the solutions, and the stopper, well 



1 Thirty-two grm. of pyrogallic acid and 64 grm. of caustic potash dis- 

 solved in 100 c.c. of water will absorb 9,200 c.c. of oxygen. At the same 

 time some carbon monoxide is evolved (122-5 c.c.). The evolution of CO 

 is a minimum when the potash is in excess and only one -fifth of the theoretical 

 absorbabje amount of is absorbed, 



