CHEMICAL CRITERIA OF ANAEROBIOSIS 21 



to multiply. This method is well known to be adapted to the 

 culture of microphilic aerobes, however. 



Plant and animal tissues also reduce methylene blue in the 

 depths of liquid media. In one instance a piece of sterile guinea 

 pig kidney under mineral oil kept methylene blue decolorized 

 in its immediate neighborhood at 37°C. for 196 hours whereas 

 the control without tissue but with an equivalent depth of oil 

 was completely recolored in thirty minutes. 



Many investigators, as already noted, have referred to the 

 decolorization of methylene blue by animal and plant tissues 

 as well as by various inert substances in culture media as indicat- 

 ing anaerobic conditions therein. Of these, Zinsser, Hopkins 

 and Gilbert (1915) recognized most clearly that we have to deal 

 here with another process in addition to reduction, namely 

 adsorption. They were unable, by extraction of animal organs, 

 to secure any reducing substance whatever apart from the tissues 

 and concluded that adsorption is mainly responsible for the loss 

 of color in media containing methylene blue in the presence of 

 such agents. This conclusion was strengthened by their obser- 

 vation that heated tissues are nearly, if not quite, equal to 

 unheated tissues for this purpose. Similar observations were 

 previously made by Wrzosek (1907), Liefmann (1907), Guillemot 

 and Szczawinska (1908), and Hata (1908) but it is doubtful if 

 any of these workers appreciated the important role of adsorption. 



It is possible, as I shall show presently, to extract reducing 

 substances from both plant and animal tissues, and in this 

 important respect their action upon methylene blue differs from 

 that of inert particulate substances such as sand. 



When a small piece of potato was placed in an aqueous solution 

 of methylene blue (1 : 100,000) at room temperature, the solution 

 adjacent to the tissue lost its color within two to three hours and 

 within a few more hours the test tube showed a lightly colored 

 bluish liquid in which the potato fragment was slightly tinged 

 with blue, most prominently at its uppermost end. No recolor- 

 ation occured in such a partially decolorized solution on exposure 

 in a Petri dish nor could it be decolorized by boihng except on 

 alkalinization. In contrast, the potato fragment became mark- 



