Eosin Methylene Blue (EMB) Agar : Composition, uses and colony characteristics

Eosin Methylene Blue Agar is a both selective and differential culture medium. It is selective culture medium for gram-negative bacteria (selects against gram positive bacteria) and is commonly used for the isolation and differentiation of coliforms and fecal coliforms.

EMB media assists in visual distinction Escherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera.

Escherichia coli colonies in Eosin Methylene Blue  (EMB) Agar (Note: Greenish Metallic Sheen)

Differentiation between these gram negative bacilli is based on the colony color.

  1. Colored colonies in Eosin Methylene Blue (EMB) Agar: Lactose fermenter
  2. Colorless colonies in Eosin Methylene Blue (EMB) Agar: Non lactose fermenter

The combination of the two dyes eosin and methylene blue inhibits most Gram positive bacteria but allows many Gram negative organisms to grow.

Gram negative bacteria that ferment the lactose produce acid which turns the colonies dark purple as the acid acts upon the dyes.  In addition, certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen.  Other lactose fermenters produce larger, mucoid colonies, often purple only in their center.  In EMB agar, most of the strains of E.coli colonies have a characteristic green sheen. Rapid fermentation of lactose & production of strong acids, thus rapid reduction in the pH of the EMB agar the critical factor in the formation of the green metallic sheen observed with E.coli, rapid fermentation of lactose and formation of strong acids. Lactose non-fermenters are either colorless or light lavender.

Another commonly used media for selective isolation of Gram negative rods and differentiation of the member of Enterobacteriaceae as lactose fermenter and non-lactose fermenter is MacConkey Agar. 

Composition of Eosin-Methylene Blue Agar (EMB)

  1. Enzymatic Digest of gelatin
  2. Lactose: Sugar, helps to differentiate lactose fermenter from non lactose fermenter
  3. Dipotassium Phosphate
  4. Eosin Y: Indicator
  5. Methylene Blue: pH indicator
  6. Agar

Expected colony characteristics of organisms in EMB agar (Quality control)

  1. Escherichia coli ATCC 25922: Blue-black bulls eye; may have green metallic sheen
  2. Pseudomonas aeruginosa ATCC 27853: Colorless

Uses of Eosin-Methylene blue agar

Primary purpose: Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.

  1. Eosin Methylene Blue Agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples (presence of E.coli in the river/water sample indicates possibility of fecal contamination of water so does the presence of other pathogenic enterics).
  2. EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group. For culture of Salmonella and Shigella, selective medium such as MacConkey agar and eosin-methylene blue (EMB) agar is commonly used.
    Escherichia coli colonies grow with a metallic sheen with a dark center, Aerobacter aerogenes colonies have a brown center, and nonlactose-fermenting gram-negative bacteria appear pink.