CHEMICAL CRITERIA OF ANAEROBIOSIS 6 



as first to use litmus to indicate acid and reduction changes 

 respectively, the last reference apparently being altogether 

 erroneous. The decolorization of indigo and methylene blue 

 in culture media were studied by Spina (1887) whose interest in 

 these dyes hinged rather upon their reduction by bacterial growth 

 though he recognized the phenomenon as occurring most vigor- 

 ously in the depths and noted the return of color on exposure 

 to the air. Kitasato and Weyl (1890) confirmed this observation 

 so far as regards sodium indigo sulphate. The decolorization of 

 all three dyes by sterile culture media under anaerobic conditions, 

 as well as by living aerobic and anaerobic cultures, was especially 

 investigated by Smith (1893) (1896) who noted the necessity of 

 some organic substance such as glucose or peptone and an 

 alkaline reaction in the case of sterile media decolorized by heat. 



I found neutral litmus solutions unaffected in color by heating 

 for twenty minutes in a boiling water bath, and the same is 

 true of litmus with 1 per cent glucose. Litmus solutions with 

 1 per cent glucose and HCl stronger than n/8 were precipitated 

 by heating and the precipitate was not redissolved on coohng; 

 weaker acid solutions were unaffected except for reddening. 

 Strong alkali n/2 to N/'32 caramelized the sugar and decolorized 

 the dye permanently; weaker solutions decolorized on boiling 

 for a few minutes and regained their origmal blue color only on 

 exposure to air. 



The recoloration of such decolorized solution of litmus, indigo 

 and methylene blue by exposure to air indicates reversible 

 reactions and constitutes the key to the use of such dyes as 

 criteria of anaerobiosis. 



McLeod (1913) cleverly utihzed the blue laboratory pencil 

 mark as a criterion of anaerobiosis upon the basis of its decolori- 

 zation in the absence of air. Some pencils fail to respond, how- 

 ever, according to my experience. 



METHYLENE BLUE AS A CRITERION OF ANAEROBIOSIS 



The most valuable and most extensively advocated chemical 

 criterion of anaerobiosis is methylene blue. Introduced as an 

 ingredient of culture media by Spina (1887), studied as an indi- 



