EMB Agar: Composition, Principle, Preparation, Results, Uses

EMB Agar Overview

  • EMB agar (Eosin Methylene Blue) is a differential microbiological medium that gives a colour indication to distinguish between organisms that digest lactose (like E. coli) and those that do not. It somewhat limits the development of Gram-positive bacteria (e.g., Salmonella, Shigella).
  • Holt-Harris, Teague, and Levine are credited with creating the first EMB agar, which they later modified.
  • As a result, it combines the Levine, Holt-Harris, and Teague formulas. It comprises two carbohydrates in addition to the phosphate and peptic digest of animal tissue described by Holt-Harris and Teague.
  • In medical labs, the medium is crucial for quickly differentiating gram-negative harmful microorganisms.

Composition of EMB Agar

Ingredients                                                    Gms/liter

Peptic digest of animal tissue                        10.000

Dipotassium phosphate                                   2.000

Lactose                                                            5.000

Sucrose                                                           5.000

Eosin – Y                                                         0.400

Methylene blue                                                0.065

Agar                                                               13.500

Final pH (at 25°C): 7.2±0.2

Principle of EMB Agar

  • Eosin and methylene blue are combined in a 6:1 ratio to form EMB agar, which is what gives it its name.
  • Lactose is fermented by gram-negative bacteria, which cause the pH to decrease by producing acid. As a result, the colonies are more likely to absorb the dye. This leads the clusters to become dark purple as a result of the acid’s reaction with the pigments.
  • In addition, several lactose-fermenting bacteria produce flat, black clusters with a green metallic sheen.
  • Other larger, mucoid colonies are produced by other lactose fermenters, sometimes with a purple core.
  • On EMB agar, the majority of E. coli strain clusters show a unique green sheen. Fast generation of strong acids from the fermentation of lactose results in a rapid drop in the pH of the EMB agar, which is essential for the development of the green metallic sheen associated with E. coli.
  • By deaminating proteins, lactose non-fermenters may increase the pH. This inhibits the absorption of the colour. The colonies will be colourless.
  • The result is that lactose non-fermenters are either colourless or pale lavender.
  • Animal tissue that has been digested in the stomach provides carbon, nitrogen, and other vital growth elements.
  • Due to the fact that lactose and sucrose are fermentable carbohydrates, they serve as energy sources.
  • Methylene blue and eosin-Y are used as differential markers. The medium is buffed with phosphate.

Preparation and Method of Use of EMB Agar

  1. dissolved in 1000 ml of distilled water is 35.96 grammes.
  2. Mix until a suspension of uniform consistency is obtained. To completely dissolve the medium, bring it to a boil.
  3. By autoclaving for 15 minutes at 121 °C and 15 lbs of pressure, sterilise. AVOIDOVERHEATING
  4. To oxidise the methylene blue (i.e., to regain its blue hue) and suspend the flocculent precipitate, cool to 45–50 °C and shake the medium.
  5. Add to sterilised Petri dishes.
  6. Plates should be warmed up to room temperature.
  7. Prior to inoculation, the surface of the agar has to be fully dry.
  8. As quickly as possible after collecting a specimen, inoculate and streak it.
  9. If the item to be cultured is on a swab, isolate the swab by rolling it over a tiny space of the agar surface and then streaking it using a sterile loop.
  10. Plates should be incubated aerobically for 18–24 hours at 35–37 °C with light protection.
  11. Check the colony morphology on the plates. After 24 hours, if the results are negative, repeat the incubation process.

Result Interpretation on EMB Agar

Organisms Growth
Escherichia coli Blue-black bull’s eye; may have a green metallic sheen
Pseudomonas aeruginosa Colorless
Enterobacter aerogenes Good growth; pink, without sheen
Klebsiella pneumoniae Pink, mucoid colonies
Proteus mirabilis Luxuriant growth; colorless colonies
Salmonella Typhimurium Luxuriant growth; colorless colonies

Uses of EMB Agar

  • For the separation of gramme negative enteric bacteria from clinical and non-clinical materials, EMB Agar (Eosin Methylene Blue Agar) is advised.
  • The ability to distinguish between gram-positive and gram-negative bacteria is useful.
  • It aids in the separation and distinction of gram-negative and enteric bacilli.
  • It is used to examine the quality of the water, particularly to see whether any dangerous microbes are present.
  • It distinguishes among the bacteria that cause dysentery, typhoid, and colon disease.
  • The Salmonella and Shigella genera, as well as other nonpathogenic lactose-fermenting enteric gram-negative rods, may all be visually distinguished using EMB medium.
  • Additionally, it aids in the separation of enteric bacilli that ferment lactose from those that do not.

Limitations of EMB Agar

  • EMB Agar should be infected alongside a non-selective medium.
  • For comprehensive identification, it is advised to run biochemical, immunological, molecular, or mass spectrometry tests on colonies from pure cultures.
  • On EMB Agar, certain Shigella and Salmonella bacteria might not be able to thrive.
  • On this medium, gram-positive bacteria like yeast, enterococci, and staphylococci can grow and typically form pinpoint colonies.
  • On this medium, non-pathogenic, non-lactose-fermenting microbes can also flourish. Further biochemical tests must be carried out to separate these organisms from harmful strains.
  • To obtain effective isolation of mixed flora samples, serial inoculation could be necessary.
  • As a result, the typical green metallic sheen of E. coli may not be produced by other strains. As a result, the green metallic sheen is not a reliable indicator of E. coli.


  • https://laboratoryinfo.com/emb-agar-eosin-methylene-blue-agar/
  • https://microbeonline.com/eosin-methylene-blue-emb-agar-composition-uses-colony-characteristics/
  • http://himedialabs.com/TD/M317.pdf
  • https://bio.libretexts.org/Ancillary_Materials/Experiments/Microbiology_Labs_I/23%3A_Eosin_Methylene_Blue_Agar_(EMB)
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