20 IVAN C. HALL 



are two possible factors in the recoloration of methylene blue 

 by exposure to air, oxygen and carbon dioxide — two processes, 

 oxidation and acidification. 



Since the reaction rests unquestionably upon a quantitative 

 basis, even though we know nothing of the absolute values in 

 oxygen and carbon dioxide concerned, the volumes of test solu- 

 tion in relation to surface exposure, where time marks the progress 

 of recoloration, is of great importance in comparative tests. 

 With equal surface exposure large volumes regain their color 

 more slowly than small volumes. In all cases care has been 

 taken to use the same size tubes and identical volumes in a 

 given experiment unless otherwise stated. Differences in volume 

 between experiments account readily for certain apparent dis- 

 crepancies in actual observations of time required for recoloration. 



Application to methods of culture 



With these data at hand tests have been made of a great many 

 methods of cultivation, in which connections I gladly acknowl- 

 edge the aid of my student, Miss Margaret Eakin. Here, as in 

 the culture of anaerobic microorganisms, we have to distinguish 

 between the factors of oxygen tension reduction and of reduced 

 oxygen tension maintenance. 



We have referred already to the literature on biological reduc- 

 tion of methylene blue; to this we may add that our experiments 

 show the general possession by living cells of the property of 

 reduction. This property is common to many aerobes and 

 anaerobes, so in symbiotic mixtures, methylene blue is reduced 

 as a matter of course. With a broth culture of hay bacillus in 

 an external rubber stoppered tube and a smaller internal tube 

 containing slanted 2 per cent agar with 1 : 100,000 methylene 

 blue and n/100 NaOH analogous to the method of Salomonson 

 (1889), only partial reduction was obtained in twenty-four 

 hours at 37°C. and similar tubes of nutrient agar inoculated with 

 B. hotulinus, B. tetani, B. welchii and other obhgative anaerobes 

 failed to yield satisfactory surface growth. Failure of complete 

 decolorization here is correlated with refusal of strict anaerobes 



