ISOLATION OF ANAEROBIC ORGANISMS 83 



working of the combined apparatus presents no difficulty, and is 

 very effective. By turning the tap " e " horizontally, connection is 

 made between the pump and any receptacle or apparatus in which 

 it is desired to create a vacuum. By turning it vertically, con- 

 nection is at once made between the reserve of hydrogen and the 

 vacuum so caused, and this latter is at once filled by an inrush of 

 gas. By repeating the process as many successive " washings " in 

 hydrogen as desired can be made, and the receptacle can then be 

 sealed off in vacuo or in hydrogen as desired. The addition of a 

 vacuum gauge to the air pump, although not actually necessary, 

 will be found of great advantage generally, and of special assistance 

 in these successive washings in hydrogen. 



Methods of Isolation and Culture of Anaerobic Organisms^ 



i. Vigrnal's tubes— 



For gelatine cultures this method, given a certain amount of 

 dexterity in the manipulation of the blow-pipe flame or Bunsen 

 burner, is one of the most simple and at the same time most 

 efficient means of isolating or culturing anaerobic organisms, inas- 

 much as during the whole period of their growth there is ocular 

 evidence of the presence or absence of free oxygen in the culture 

 medium. 



To prepare a Vignal tube. — Take a length of thin glass tubing 

 about \ inch internal diameter and some 15 inches long. With 

 the aid of the blow-pipe flame or Bunsen burner make a constric- 

 tion at about 2 or 3 inches from one end, and insert well above 

 I it a light cotton-wool plug. Partially close the external orifice of 

 i the tube by turning the end in the flame. Place the tube again in 



^ A method by which fairly accurate information may be obtained as to 

 whether an organism under examination is {a) aerobic, or {b) anaerobic, has 

 been suggested by the Committee of Bacteriologists appointed by the American 

 Public Health Association {Reports and Papers, American Public Health 

 Association, vol. xxiii., p. 78). It consists in the inoculation of a fermentation 

 tube, of the pattern shown on Fig. 1 5 (p. "jf), filled with glucose bouillon from 

 which the free oxygen has been expelled in process of sterilisation, with the 

 organism in question. "Growth in the bulb, to which access to oxygen is 

 allowed, is aerobic, while growth in the closed branch, from which oxygen is 

 excluded, is anaerobic." Growth in both, on the other hand, will be evidence 

 in favour of the organism being a facultative anaerobe. The bubbles which 

 collect in the closed branch during sterilisation should be removed by tilting 

 the tube while the medium is still very hot ; otherwise they will again be 

 absorbed by the fluid. 



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