6 IVAN C. HALL 



as I shall show, has grown up of adding a trace of this dye to 

 the culture medium — either with or without inoculation — as a 

 criterion of anaerobiosis. 



We have akeady referred to the fundamental observations 

 of Smith (1893) (1896) on the decolorization of methylene blue 

 in alkaline solutions containing glucose or peptone under anaerobic 

 conditions induced by heating. Kabrhel (1899) and Hammerl 

 (1901) used such a solution along wdth their cultures as an 

 indication of the successful exclusion of oxygen and the latter 

 showed that the sugar might be replaced with sodium formate, 

 Fremlin (1904) found an alkaUnized methyl alcohol solution of 

 methylene blue more delicate than an aqueous solution but 

 recognized the possible inhibitory action of the volatile spii'it 

 upon bacterial growth. 



As Bernthsen has shown, commercial methylene blue is likely 

 to be a mixture with methylene azure, the latter being formed bj' 

 the action of alkalis. Underhill and Closson (1905) have given 

 methods for the purification of both, which however is not 

 necessary in using methylene blue as a criterion of anaerobiosis 

 since both compounds yield colorless leuco-bases under similar 

 conditions; furthermore methylene azure is formed from meth- 

 ylene blue under conditions of alkalinity such as obtain in the 

 test. 



EXPERIMENTAL WORK 



Decolorization — Preliminary discussion 



The writer's interest in methylene blue as a criterion of anaero- 

 biosis dates from the invention of the constricted tube and marble 

 device (Hall, 1915). It was possible to show by its use that 

 certain shipments of tubes contained 4 per cent with defective 

 bore so that they could not be used. A properly made tube con- 

 taining a methylene blue solution of certain composition, with 

 a good marble seal will not permit the return of color below the 

 marble for several days after decolorization by heating. One 

 must not fall into the error of assuming, however, that decolori- 

 zation of methylene blue necessarily indicates suitability for 

 anaerobic growth; there are many factors, aside from the reduc- 



