Eosin Methylene Blue (EMB) Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology and Uses

Eosin Methylene Blue (EMB) Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology and Uses

Introduction of Eosin Methylene Blue (EMB) Agar

EMB stands for Eosin Methylene Blue and EMB agar is a differential bacterial medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment the lactose (e.g., E. coli, Klebsiella) and those that do not (e.g., Proteus, Salmonella, Shigella). This agar was originally devised by Holt Harris and Teague and further modified by Levine. The modified form is a combination of the Levine and Holt-Harris and Teague formulae which contains a peptic digest of animal tissue and phosphate as recommended by Levine and two carbohydrates as suggested by Holt-Harris and Teague. The medium is important in both clinical and non-clinical laboratories to distinguish gram-negative pathogenic bacteria in a short period of time and thus it is recommended for the isolation and differentiation of gram-negative enteric bacteria from clinical and nonclinical specimens.

Principle of Eosin Methylene Blue (EMB) Agar

Peptic digest of animal tissue (PDAT) serves as a source of carbon, nitrogen, and other essential growth nutrients while Lactose and sucrose are the sources of energy by being fermentable carbohydrates. Disodium phosphate buffers the medium and agar is the solidifying agent.  EMB agar contains two dyes, eosin and methylene blue that serve as differential indicators. Gram-negative bacteria that ferment the lactose produce acid that lowers the pH. This enhances dye absorption by the colonies and turns the colonies dark purple as the acid acts upon the dyes.  Besides, 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. Most of the strains of Escherichia coli colonies have a characteristic green sheen in this medium. Rapid fermentation of carbohydrate,  lactose, creates the production of strong acids, therefore a rapid reduction in the pH of the EMB agar is the critical factor in the formation of the green metallic sheen observed with Escherichia coli, rapid fermentation of lactose, and formation of strong acids. Lactose non-fermenters, on the other hand, may increase the pH by deamination of proteins. This clears that the dye is not absorbed and hence colonies will be colorless. Lactose non-fermenter bacteria are therefore either colorless or light lavender.

Composition of Eosin Methylene Blue (EMB) Agar

Ingredients         Gms/liter

  • Peptic digest of animal tissue:    10.0
  • Dipotassium phosphate: 2.0
  • Lactose: 5.0
  • Sucrose: 5.0
  • Eosin – Y: 0.40
  • Methylene blue: 0.065
  • Agar: 13.5
  • Final pH (at 25°C): 7.2±0.2

 Preparation of Eosin Methylene Blue (EMB) Agar

  1. Suspend 35.96 grams of the dehydrated medium in 1000 ml purified/distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes i.e. validated cycle.
  4. Cool to 45-50°C.
  5. Mix well before pouring into sterile Petri plates.
  6. Leave for drying.

 

Storage and Shelf life of MacConkey agar

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for EMB agar

  • Test specimen
  • EMB agar
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains ( E. coli, Pseudomonas aeruginosa and Staphylococcus aureus)
  • Waste bin

Procedure of Eosin Methylene Blue (EMB) Agar

  1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Inoculate and streak the specimen as soon as possible after collection.
  3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
  4. Streak for isolation with a sterile loop.
  5. Incubate plates aerobically at 35-37ºC. for 18-24 hours and protect from the light.
  6. Examine colonial characteristics.

Colony Morphology of Eosin Methylene Blue (EMB) Agar

Organisms           Growth

Escherichia coli: Blue-black bulls’ 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

Control Strains

E. coli ATCC 25922: Blue-black bulls’ eye; may have a green metallic sheen

Pseudomonas areuginosa ATCC 27853: Colorless

Staphylococcus aureus ATCC 25923: No growth

Uses of Eosin Methylene Blue (EMB) Agar

 

  1. EMB agar is recommended medium for the isolation and differentiation of gram-negative enteric bacteria from both clinical and non-clinical samples.
  2. Lactose of the medium helps in the isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.
  3. It is beneficial in differentiating gram-positive and gram-negative bacteria.
  4. It assists in the isolation and differentiation of enteric bacilli and gram-negative bacilli.
  5. It is applied in testing the quality of water, especially in determining if the water is contaminated by harmful microbes.
  6. It is also useful in visual distinction E. coli, other non-pathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera.

Limitation of EMB agar

 

  1. A non-selective medium should be inoculated together with EMB Agar.
  2. Confirmation cannot be completed using EMB agar and therefore biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
  3. Some strains of bacteria like Salmonella and Shigella may fail to grow on this agar.
  4. Even being selective, there is still a chance of growing Some gram-positive bacteria like enterococci, staphylococci, and yeast (fungus) will grow on this medium and usually form pinpoint colonies.
  5. Subculturing may be required to ensure adequate isolation of mixed flora samples.
  6. Some strains of Escherichia coli may not produce an attribute green metallic sheen; consequently, the green metallic sheen is not diagnostic for Escherichia coli.

Keynotes on Eosin Methylene Blue (EMB) Agar

  • Avoid overheating during preparation.
  •  If no growth after 24 hours, reincubate an additional 24 hours.
  • Store the EMB agar away from light to avoid photooxidation
  • Colour and Clarity of prepared medium -Reddish purple-colored, opalescent gel with a greenish cast and finely dispersed precipitate forms in Petri plates.
  • The above composition of EMB agar is Himedia formulation.

Further Readings on Eosin Methylene Blue (EMB) Agar

 

  1. https://himedialabs.com/TD/M317.pdf
  2. https://legacy.bd.com/ds/technicalCenter/inserts/L007374(10).pdf
  3. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0069
  4. https://catalog.hardydiagnostics.com/cp_prod/product/g25-emb-levine-agar-eosin-methylene-blue-for-culture-of-gram-negative-bacteria-15x100mm-plate-order-by-the-package-of-10-by-hardy-diagnostics-media-prepared
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