Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

Wynne, E. Staten; Rode, L. J.; Hayward, Abraham

doi:10.3109/10520294209105750

Cited by 6 publications

(8 citation statements)

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“…Because the differentiation of pathogenic microorganisms from nonpathogens by EMB agar was dependent on the amounts of acidic products resulting from the fermentation of lactose (7), it appeared that this mechanism might also account for the differentiation of E. coli and E. aerogenes. Results obtained with bromothymol blue as an indicator of final pH of the culture medium indicated that E. coli did produce a strongly acidic condition during growth at the expense of lactose, whereas E. aerogenes yielded a much less acidic medium.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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

Horvath

Ropp

1974

International Journal of Systematic Bacteriology

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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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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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

Horvath

Ropp

1974

International Journal of Systematic Bacteriology

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“…However, the production of red colonies by lactose fermenters and blue colonies by nonfermenters in a medium containing a mixture of these two dyes would be posible only if the dyes do not combine chemically (Wynne et al, 1942). The filter paper adsorption technique of Clifton and Madison (1931) was applied to 1 per cent aqueous solutions of acid fuchsin and methylene blue and to a mixture containing 1 per cent of each dye.…”

Section: Resultsmentioning

confidence: 99%

“…(1) Generally only colonies of lactose fermenters are colored, whereas the colorless colonies of nonfermenters tend to be masked by the diffusion of dye from fermenting colonies (Holt-Harris and Teague, 1916). Though two dyes are employed in eosin methylene blue agar, a chemical combination occurs to yield the dye methylene blue eosinate (Wynne et al, 1942), which is absorbed by fermenters only. Since methyl green resembles methylene blue in having a quinoid structure with a pentavalent nitrogen atom, a similar combination is chemically feasible in the recently devised eosin methyl green sulfite agar of Fredericq (1947).…”

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Acid Fuchsin Methylene Blue Agar: A New Differential Medium for Enteric Bacteria

Jeter

Wynne

1949

J Bacteriol

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“…In a paper by Wynne, Rode and Hayward, 1942, dealing with a study of the Levine eosin-methylene-blue agar medium, it is pointed out that if the eosin is left out, the organisms which ferment letose (Escherichia-Aerobacter) produce colorless colonies, whereas the non-fermenters (Salmonella, Eberthela and Shigella) take up the methylene blue producing deep blue colonies. On the other hand, if the methylene blue is omitted, the lactose fermenters take up the eosin, producing pink colonies whereas the non-fermenters are colorless.…”

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The Effect of Concentration of Dyes on Differentiation of Enteric Bacteria on Eosin-Methylene-Blue Agar

Levine¹

1943

J Bacteriol

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Holt-Harris and Teague introduced eosin-methylene-blue as an indicator for differentiation of the colon from the typhoid-dysentery group of bacteria in 1916. Their medium consisted of 1 per cent Witte's peptone, 0.5 per cent beef-extract (Liebig's), sodium chloride, sucrose and lactose, solidified with 1 per cent agar. The reaction was adjusted to + 0.8 per cent normal acid. The medium was cleared with egg whites, filtered, and sterilized in the Arnold for 30 minutes at 100°C. on three successive days. They recommended, as an indipator, 2 ml. of a 2 per cent yellowish eosin and 2 ml. of a 0.5 per cent methylene blue per 100 ml. of the medium. The medium was very satisfactory for its purpose, but the details of preparation made it rather cumbersome and the necessitated change of peptones after the war, coupled with resultant differences in reaction of the medium adjusted on a total acidity scale, tended towards variable results in the hands of different investigators.Levine, in 1918, suggested a simplified medium in which Difco peptone was employed as a base, di-potassium phosphate (0.2 per cent) was utilized as a buffer, to automatically adjust the reaction of the medium to a desired point, and sterilization at 15 pounds for 15 minutes in the autoclave was substituted for the more cumbersome Arnold intermittent sterilizing process. To this basal medium was added 5 ml. of a 20 per cent sterile lactose solution, 2 ml. of 2 per cent yellowish eosin, and 2 ml. of a a per cent methylene blue per 100 ml. of melted basal medium. This medium was designed primarily for the purpose of differ-* If desired, all constituents may be added prior to sterilization.

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Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

Cited by 6 publications

References 6 publications

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

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

Acid Fuchsin Methylene Blue Agar: A New Differential Medium for Enteric Bacteria

The Effect of Concentration of Dyes on Differentiation of Enteric Bacteria on Eosin-Methylene-Blue Agar

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