THE CULTURE OF ANAEROBIC BACTERIA. 307 



then, with proper precautions against contamination, poured 

 on top of the inoculated medium and quickly cooled. This 

 extra layer is, as a rule, unnecessary. 



Isolated colonies can be obtained by the method just given. The 

 liquefied glucose agar or gelatin tubes are inoculated in the usual 

 manner for making- plates. The contents of the tubes are then solidi- 

 fied and an extra layer of the medium is poured into each tube. 



The method of Roux deserves mention under this head. 

 The inoculated medium is drawn up into a sterile glass-tube 

 pipette (Fig. 61), which is then sealed above and below the 

 liquid by means of a flame. A deep layer is thus obtained 

 with little or no air above. By cutting- the tube a colony 

 can be easily reached. 



Exhaustion of air. Pasteur in his studies on the bacillus 

 of malignant edema employed special tubes which were 

 connected with an air-pump, and, when a vacuum was 

 reached, they were sealed in the flame. The method has 

 been simplified by Gruber and is easy of execution. Special 

 test-tubes, with a constriction below the cotton plug 1 may 

 be obtained. After the medium is inoculated a vacuum is 

 produced in the tube which is then sealed in the flame. If 

 desired, colonies can be obtained by " rolling- " the sealed 

 tube. 



Absorption of oxygen. If a solution of pyrogallic acid is 

 rendered alkaline it will immediately become dark, then 

 brown, and finally black, due to the rapid absorption of 

 oxygen. This reaction has been utilized in a variety of 

 methods. Other chemicals may be used for absorbing oxy- 

 gen, but they have no advantage over that mentioned. 

 Buchner's method consists in placing the inoculated tube 

 inside of a larger one, which contains on the bottom some 

 pyrogallic acid. Caustic alkali is added to the acid and the 

 tube is then closed at once with a rubber stopper. 



