THE CULTURE OF ANAEBOBTC 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 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. 



