CHEMICAL CRITERIA OF ANAEROBIOSIS 7 



tion of oxygen tension, in the cultivation of anaerobes. How- 

 ever, the failure of a properly balanced solution to remain decolor- 

 ized indicates a defect in the method of air exclusion proposed. 

 Decolorization of methylene blue probably occurs at a definite 

 point during the abstraction of oxygen — a point yet to be deter- 

 mined. So decolorization may indicate suitability for some 

 organisms and not for others. Methylene blue tests with 

 McLeod's (1913) plate were satisfactory yet the bacteriological 

 use of this plate in our hands was never satisfactory. Therefore, 

 while we must admit that the decolorization of methylene blue 

 solution, delicately adjusted, frequently correlates with successful 

 anaerobic cultures, it is more important to recognize the limits 

 and conditions of this test and to appreciate that the factors 

 which enter into the successful decolorization of methylene blue 

 are not necessarily common to the growth of all obligative 

 anaerobes. It should be emphasized especially that acidifica- 

 tion, probably through absorption of carbon dioxide from the 

 air, may account for a return of color to decolorized methylene 

 blue solutions and that in this case the dye cannot be bleached 

 again without re-alkaUnization. 



Essential factors in decolorization 



Preliminary experiments had to do with tests of Griibler's 

 methylene blau fiir Bacillen in tv/o culture media commonly 

 used for the cultivation of anaerobes — magnesium carbonate 

 glucose broth (Hall, 1915) and neutral (phenolphthalein) glucose 

 agar. A trace of methylene blue in either of these media is 

 easily decolorized by heating in a boiling water bath. In the 

 open air such decolorized solutions quickly recover their original 

 blue color but protected from air remain decolorized indefinitely. 

 Thus in the constricted tube filled with glucose broth the color 

 returns above but not below the marble seal; in deep glucose 

 agar the color returns to the upper layers first and gradually 

 descends. But it was noticed in certain cases of methylene blue 

 glucose broth allowed to stand for several days that heating 

 failed to decolorize the dye although it had done so originally. 

 The outcome was a series of experiments to determine the 



