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
Peptic digest of animal tissue 10.000
Dipotassium phosphate 2.000
Eosin – Y 0.400
Methylene blue 0.065
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
- dissolved in 1000 ml of distilled water is 35.96 grammes.
- Mix until a suspension of uniform consistency is obtained. To completely dissolve the medium, bring it to a boil.
- By autoclaving for 15 minutes at 121 °C and 15 lbs of pressure, sterilise. AVOIDOVERHEATING
- 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.
- Add to sterilised Petri dishes.
- Plates should be warmed up to room temperature.
- Prior to inoculation, the surface of the agar has to be fully dry.
- As quickly as possible after collecting a specimen, inoculate and streak it.
- 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.
- Plates should be incubated aerobically for 18–24 hours at 35–37 °C with light protection.
- Check the colony morphology on the plates. After 24 hours, if the results are negative, repeat the incubation process.
Result Interpretation on EMB Agar
|Escherichia coli||Blue-black bull’s eye; may have a green metallic sheen|
|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.