CHEMICAL CRITERIA OF ANAEROBIOSIS 19 



liquids; 2 per cent neutral agar, plus 1 part n:1 NaOH per 100 

 is satisfactory for tests involving solid media. 



There is a possible fallacy in the use of too weakly alkaline 

 solutions, namely, that on standing they cannot be decolorized 

 by boiling. By exposing all the seven possible combinations of 

 one, two, or all, of the thiee factors, glucose, alkali, and dye, 

 for forty-eight hours, and then adding those lacking in each of 

 six of these, it can be shown readily that only those originally 

 containing alkali deteriorate; that is, deterioration consists in 

 loss of alkaUnity. The test solution must be freshly alkalinized, 

 though the glucose methylene blue or agar methylene blue may 

 be kept as stock solutions. Loss of abihty to decolorize might 

 conceivably be attributable to acid in the glassware though I 

 have never encountered this factor knowingly. The change of 

 reaction is most reasonably attributed to absorbtion of atmos- 

 pheric carbon dioxide. An easy proof of change in reaction of 

 faintly alkaline solutions on exposure to air is afforded if one 

 heats n/1000 NaOH colored with phenolphthalein in a con- 

 stricted tube with marble seal in a bath of boiling water; this dye 

 is not affected by such heating. But on cooling for several hours 

 the color above the marble fades while that below remains. Or, 

 drawing air through such a colored solution causes it to fade, 

 through change of reaction, but if the air be washed by bubbhng 

 through several tubes of strong lye to remove CO2, with the 

 efficacy of such removal tested by passage through clear hme 

 water, the phenolphthalein test solution remains alkaline. A 

 repetition of this last experiment with a decolorized methylene 

 blue solution gives the same result, i.e., recoloration, with air 

 containing COo and air freed therefrom, except that the solution 

 recolorized with the latter continues susceptible to repeated 

 decolorization longer than with the former. This proves that 

 CO2 is not the only factor in recolorization of methylene blue 

 as it is in the change of reaction in the phenolphthalein experi- 

 ment. If the air be carefully washed in several successive 

 mixtures of alkaline pyrogallol so as to remove both carbon 

 dioxide and oxygen its passage through a decolorized methylene 

 blue solution does not cause the return of color. In short, there 



