EMB Agar Composition, Principle, Preparation, Results, Uses

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Eosin Methylene Blue (EMB), a microbiological media, is a differentiating medium that slightly inhibits the growth and color of Gram-positive bacteria. It also provides a color indicator to distinguish between organisms that ferment lactose (e.g. E.coli) and those who do not (e.g. Salmonella, Shigella). Holt-Harris, Teague, and Levine first created EMB agar.

This mixture of the Levine, Holt-Harris, and Teague formulae is called “The Medium”. It contains a peptic digestion of animal tissue and phosphate (as recommended by Levine) and two carbohydrates (as suggested by Holt–Harris or Teague. Medical laboratories use the medium to quickly distinguish gram-negative pathogenic microbes.

Principle of EMB Agar

EMB agar is distinguished by the presence of a mixture of the two dyes, eosin or methylene blue, in a ratio of 6 to 1. The pH is lowerened by Gram-negative bacteria, which ferment lactose. The acid reacts with the dyes to make the colonies darker purple. This encourages dye absorption. Certain lactose-fermenting bacteria can also produce flat, dark colonies that have a metallic sheen.


Other lactose fermenters create larger, more mucoid colonies that are often purple in the center. Most E.coli strains have a distinctive green sheen when grown on EMB agar. Rapid fermentation of lactose & formation of strong acid, hence a rapid drop in pH on EMB agar is critical for the formation of the characteristic green metallic sheen.

On the other hand, lactose nonfermenters may raise pH by deamination proteins. This prevents the dye from being absorbed. The colonies will remain colorless. The lactose nonfermenters will be either colorless or very light lavender. Peptic digest is a process that breaks down animal tissue into carbon, nitrogen, or other essential growth nutrients. Succrose and lactose are fermentable carbohydrates that provide energy. EosinY and methyleneblue are used as indicators. The medium is buffered by phosphate.

Composition of EMB Agar

Peptic digest of animal tissue10.000
Dipotassium phosphate2.000
Eosin – Y0.400
Methylene blue0.065

Final pH (at 25°C): 7.2±0.2

Preparation and Method of Use of EMB Agar

  1. 35.96 grams should be suspended in 1000 ml of distilled water.
  2. Mix until uniform suspension. To dissolve the medium completely, heat to boiling
  3. For 15 minutes, sterilize by using an autoclave at 15 lbs pressure (121degC). AVOID OVERHEATING.
  4. To oxidize the methyleneblue (i.e., cool the medium to 45-50°C) shake the container. to bring back its blue color and to suspend the flocculent.
  5. Pour into sterile Petri plates.
  6. Allow plates to heat to room temperature.
  7. Before inoculating, the agar surface must be completely dry.
  8. As soon as possible after collecting the specimen, innoculate it and streak it.
  9. If the specimen is placed on a swab for culture, roll the swab across a small area of the surface of the agar and streak it with a sterilized loop.
  10. For 18-24 hours, incubate plates at 35-37°C. Protect from light.
  11. Check plates for colonial morphology. If the results are not satisfactory after 24 hours, incubate for an additional 24 hours.

Result Interpretation on EMB Agar

Escherichia coliBlue-black bull’s eye; may have a green metallic sheen
Pseudomonas aeruginosaColorless
Enterobacter aerogenesGood growth; pink, without sheen
Klebsiella pneumoniaePink, mucoid colonies
Proteus mirabilisLuxuriant growth; colorless colonies
Salmonella TyphimuriumLuxuriant growth; colorless colonies

Uses of EMB Agar

  • EMB Agar (Eosin Methylene Blue Agar), is used to isolate and differentiate gram-negative enteric bacteria from clinical specimens and other nonclinical samples.
  • It can be used to distinguish between gram-positive and gram–negative bacteria.
  • It aids in the differentiation and isolation of enteric and gram-negative bacteria.
  • It is used to test the quality of water, particularly to determine if it is contaminated with harmful microorganisms.
  • It differentiates microorganisms in the colon-typhoid-dysentery group.
  • EMB media aids in visual differentiation Escherichia Coli, other nonpathogenic lactose fermenting enteric gram negative rods, as well as the Salmonella, Shigella genera.
  • It aids in the differentiation and isolation of non-lactose fermenting enteric bacteriailli.

Limitations of EMB Agar

  • Inoculating with EMB Agar should include a non-selective medium.
  • For complete identification, it is important to perform biochemical, immunological and molecular testing on colonies grown in pure culture.
  • EMB Agar may not grow certain strains of Salmonella or Shigella.
  • This medium can be used by some gram-positive bacteria such as yeast, enterococci, and staphylococci. They will usually form pinpoint colonies.
  • Non-pathogenic, non-lactose-fermenting organisms will also grow on this medium. These organisms must be distinguished from pathogenic strains by additional biochemical tests.
  • To ensure sufficient isolation of mixed flora, serial inoculation may need to be done.
  • Some E.coli strains may not produce the characteristic green metallic sheen.
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