18 IVAN C. HALL 



yet in the known absence of non living reducing^ agents, the 

 decolorization of methylene blue in culture media may be taken 

 as a fair indication of anaerobic growth where the conditions of 

 anaerobiosis are such as not in themselves to decolorize the dye. 

 The failure of certain streptococci to decolorize methylene blue 

 in milk as sherman and Albus (1918) found, appears to be a 

 matter of inhibition; it is interesting to note Brown's (1920) 

 observation that some of these fprms will develop in the depths 

 of agar containing decolorized methylene blue but not in the 

 colored band near the surface; contrary-wise it is possible for 

 many organisms to grow aerobically upon media colored wuth 

 methylene blue without decolorization. The possible role of 

 adsorption of methylene blue by bacterial bodies in its relation 

 to true reducing processes still remains to be investigated. 



Recoloration of methylene blue 



Whereas w^e are able only to speculate as to the basic expla- 

 nation of these various phenomena a knowledge of them enables 

 us to guage correctly the concentration of ingredients in the use 

 of methylene blue as a criterion of anaerobiosis. Such use 

 depends, as already noted, upon the recoloration of decolorized 

 methylene blue in the presence of air, and the failure of recolor- 

 ation when air is excluded. But recoloration does not occur in 

 glucose solutions in alkali stronger than N/32 in which marked 

 caramelization has occurred, nor in peptone more strongly 

 alkaline than n/16, nor in agar sufficiently alkalinized to prevent 

 solidification; neutralization of such glucose solutions permits 

 recoloration, however (Yellow + blue = green). 



As a general rule, the delicacy of methylene blue as a criterion 

 of anaerobiosis varies directly as the kind and amount of reducing 

 agent employed, and the temperature used to effect decoloriza- 

 tion, and inversely as the alkalinity of the solution. As shown 

 in table 2 those decolorized solutions last to lose their color were 

 first to regain it. In general a moderate concentration of glucose, 

 e.g., 0.5 to 2 per cent with a low concentration of alkali (n/500 

 to n/1000 NaOH) gives the best results for tests involving 



