Eosin methylene blue

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E. coli on EMB agar

Eosin methylene blue (EMB, also known as "Levine's formulation") is a selective stain for gram-negative bacteria.[1] EMB contains dyes that are toxic to gram-positive bacteria. EMB is the selective and differential medium for coliforms. It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. A common application of this stain is in the preparation of EMB agar, a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella).[2] Organisms that ferment lactose display "nucleated colonies"—colonies with dark centers.[3]

This medium is important in medical laboratories by distinguishing pathogenic microbes in a short period of time.[4]

  • Rapid lactose fermentation produces acids, which lower the pH. This encourages dye absorption by the colonies, which are now colored purple-black.
  • Lactose non-fermenters may increase the pH by deamination of proteins. This ensures that the dye is not absorbed. The colonies will be colorless.

On EMB if E. coli is grown it will give a distinctive metallic green sheen (due to the metachromatic properties of the dyes, E. coli movement using flagella, and strong acid end-products of fermentation). Some species of Citrobacter and Enterobacter will also react this way to EMB.[5] This medium has been specifically designed to discourage the growth of gram positive bacteria.[6]

EMB contains the following ingredients: peptone, lactose, dipotassium phosphate, eosin Y (dye), methylene blue (dye), and agar.

There are also EMB agars that do not contain lactose.


  1. ^ Levine, M (1918). "Differentiation of B. coli and B. aerogenes on a simplified eosin-methylene blue agar". J Infect Dis. 23: 43–47. doi:10.1086/infdis/23.1.43.
  2. ^ "Biochemical Tests Explanation"
  3. ^ Differential Media (Levine's Formulation)
  4. ^ Bachoon, Dave S., and Wendy A. Dustman. Microbiology Laboratory Manual. Ed. Michael Stranz. Mason, OH: Cengage Learning, 2008. Exercise 8, "Selective and Differential Media for Isolation" Print.
  5. ^ EMB Agar Growth Examples
  6. ^ "Eosin-Methylene Blue Agar Plates Protocol". September 29, 2007. Archived from the original on November 30, 2011.

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