Mechanism of Action of Eosin-Methylene Blue Agar in the Differentiation of Escherichia coli and Enterobacter aerogenes

Abstract

The mechanism involved in the action of eosin-methylene blue agar (EMB) in differentiating Escherichia coli from Enterobacter aerogenes was investigated using prototrophic strains of these organisms and a Lac-auxotroph, E. coli X-96 1. 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 t o methylene blue ratio of 1 t o 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.The use of eosin-methylene blue (EMB) agar as a differential medium for the colon-typhoiddysentery group has become well established since its introduction by Holt-Harris and Teague (2) in 19 16. The medium contained the dyes eosin and methylene blue, which yielded a sharp distinction between lactose-and nonlac t ose-fermenting organisms.Two years later, Levine (3) employed a modification of the original EMB medium and concluded that the modified EMB could be used t o differentiate lactose fermenters from non-lac tose ferm enters.Although the medium had been used extensively since its introduction, the mechanism of its differential action was not known. Wilson (6), in a review of differential staining by mixtures of eosin and methylene blue, called attention to a dye complex which formed under acidic conditions. It was proposed that such a complex might be responsible for the differential action of EMB agar.Wynne, Rode, and Hayward (7) assumed that the differential action of EMB was a direct function of the acid produced during fermentation. Pathogens belonging to the genera Salmonella and Shigella were compared with the nonpathogens Escherichia coli and two En terobacter species, and it was concluded that the pathogens could not ferment lactose t o produce a sufficiently low pH to result in colored colonies on EMB, whereas the nonpathogens could reduce the pH t o a level at which colored colonies resulted.The mechanism by which EMB agar differentiated E. coli and Enterobacter aerogenes remained unknown. T o demonstrate the mechanism of this differential action, the effect of pH was investigated. The molecular structure of the dye complex was determined, and a mechanism for formation of the dye complex was proposed. MATERIALS AND METHODSBacterial strains and media. The strain of E. aerogenes was supplied by M. M. Brent, and the culture of E. coli wild type was obtained from S.Harmon, both of the Department of Biology, Bowling Green State University. The culture of E. coli X-961, a lactose-negative auxo troph, was supplied by R. Curtiss I11 of Oak Ridge Laboratories, Oak Ridge, Tenn.Stock cultures were maintained on cystine Trypticase agar stabs (Difco Laboratories, Detroit, Mich.) at 15 C...

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