84 ANAEROBIC ORGANISMS : ISOLATION AND CULTURE 



the flame at such a distance from the other 

 extremity as will allow it to be conveniently 

 held by the fingers of the left hand, and, turn- 

 ing it during the process of heating, draw it 

 out to a fine point. Pass the whole of the 

 tube through the flame a sufficient number 

 of times to ensure thorough sterilisation both 

 of the interior of the tube and of the cotton- 

 wool plug, and place it on one side to cool. 



Prepare a tube of glucose gelatine by 

 boiling it in a beaker of water for some 

 minutes in order to get rid of the free oxy- 

 gen, and by means of a finely drawn pipette 

 introduce into the medium a sufficient quan- 

 tity (three or four drops will suffice) of a strong 

 solution of sulphindigotate of soda, previously 

 sterilised by boiling in a plugged test-tube, 

 to tint it a distinct blue colour. Cool down 

 the tube of gelatine by placing it under the 

 tap until it reaches a temperature at which 

 the hand can without discomfort bear the 

 heat of the tube when it is held in it (about 

 40° C), and inoculate the medium with a trace 

 of the material to be studied.^ Thoroughly 

 incorporate this with the medium in the same 

 manner as in the case of ordinary plate cul- 

 tures. Break ofi~ the finely-drawn-out point 

 of the Vignal tube and, after passing the 

 extremity through the flame, insert it into 

 the tube of gelatine. Aspirate the air slowly 

 through the cotton-wool plug until the gela- 

 tine reaches the lower part of the constriction 

 of the upper extremity. Withdraw it from 

 the test-tube and, holding it in the right 

 hand with the forefinger pressed tightly on 

 the upper aperture, slightly elevate the other 

 extremity and seal it off in a small Bunsen 

 flame. Seal the upper end in a similar 



^ Successful culture in a Vignal tube depends upon 

 the complete isolation of the colonies. In order to 

 ensure this, it is well to make a second or even third 

 test-tube dilution before aspiration into the tube. 



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