The mechanism involved in the action of eosin-methylene blue agar (EMB) in differentiating from was investigated using prototrophic strains of these organisms and a Lac auxotroph, X-961. The final pH of the EMB agar resulting from growth of these organisms at the expense of lactose was shown to play a critical role in the differential action. An EMB complex, which formed under acidic conditions, appeared to be involved in the differential action of this medium. The molecular weight of the complex indicated an eosin to methylene blue ratio of 1 to 1. Ultraviolet and infrared spectral data indicated the occurrence of an amide bond between the dyes when complexing occurred. On this basis, a mechanism for complexing of eosin and methylene blue, under acidic conditions, was proposed.


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