OXYGEN ABSORPTION METHOD 93 



absorption of oxygen along the line of puncture the tube should 

 be stoppered by a layer of liquid agar, or a mixture of vaseline 

 and paraffin as recommended by Miquel, run in over the top of the 

 medium after the inoculation has been made. A simpler, and 

 equally efficacious way, if a due amount of care in manipulation 

 is exercised, is to heat gently the test-tube by turning it in a small 

 Bunsen flame immediately above the upper level of the medium, 

 until slight liquefaction of the medium at this point occurs. The 

 subsequent solidification of this thin liquid layer will effectually 

 seal the point of entry of the needle. 



To render evident the absorption of the oxygen in the medium, 

 it is well to tint it with the sodium sulphindigotate solution, as 

 described under the head of Vignal's tubes. 



10. Oxygen absorption by superficial cultivation of a hig-hly 

 aerobic org-anism— 



Melt a tube of glucose agar or glucose gelatine (preferably 

 tinted with sodium sulphindigotate solution) in a beaker of boiling 

 water. Boil for some minutes in order to get rid of free oxygen. 

 Cool rapidly down to 40° and inoculate in the usual manner. 

 Solidification should be accelerated by placing the tube in cold 

 water in order to avoid further absorption of oxygen, A layer 

 of liquid agar charged with a cultivation of the Bacillus subtilis or 

 other strong aerobe is then poured on the top of the solidified 

 medium. The avidity with which the oxygen is taken up by the 

 bacteria in the upper layer will effectually prevent any further 

 absorption of it in the lower one. To examine, or to reculture, it 

 is only necessary to make a file mark on the glass at the point 

 required and carry the crack round the tube with a red-hot wire. 

 It is well, however, first to break the bottom of the tube in order 

 to avoid the possible sudden expulsion of the contents by gas 

 generated by the organisms. In the case of gelatine, another 

 method is to plunge the test tube for a moment into boiling water 

 so as slightly to liquefy the medium at the point of adherence 

 to the tube walls, and then, after withdrawing the cotton-wool 

 plug and flaming the tube orifice, to allow the solid medium \.o 

 fall into a sterile Petri dish, when cultivations can be made from 

 any desired point. 



II. Potato cultures in vacuo or in hydrogen — 

 Take a Roux potato-tube containing the medium and liquid 

 (as described on p. 41), and substitute for the cotton- wool plug a 



