EMB agar | Principle | Preparation | Interpretation

☰ Content :

Ⅰ. Overview

Ⅱ. Preparation / composition

Suspend the components, dehydrated powder, in water (36 grams in 1000 ml of purified/distilled water). The medium is boiled for a few seconds until the ingredients are completely dissolved. Sterilize in an autoclave at 15 lbs (121°C) pressure for 15 minutes. avoid overheating.

Cool to 45-50°C and stir the medium to oxidize the methylene blue (i.e. restore its blue color) and suspending the flocculant precipitate. Pour into Petri-dishes.

Composition EMB Agar

Ingredients Gram/liter
Animal Tissue Peptic Digest 10,000
Dipotassium Phosphate 2,000
Lactose 5,000
Sucrose 5,000
Eosin – Y 0.400
Methylene blue 0.065
Agar 13.500

Ⅲ. Principle

EMB Agar contains two dyes, eosin and methylene blue which inhibit the growth of many microorganisms Gram-positives that accompany it. These dyes serve as differential indicators in response to carbohydrate fermentation.

  • Lactose-fermenting gram-negative bacteria acidify the medium, and under acidic conditions the dyes produce a deep purple complex that is usually associated with a green metallic sheen. This metallic green sheen is an indicator of the vigorous lactose and/or sucrose fermentation capacity typical of fecal coliforms.
  • A smaller amount of acid production, which is the result of slow fermentation (by slow-fermenting lactose organisms), results in a brown-pink coloration of growth.
  • Lactose non-fermenters, such as Shigella and Salmonella, form clear, colorless or amber colonies that are easily distinguished from coliforms.

Lactose and sucrose are sources of energy by being fermentable carbohydrates.

The phosphate buffers the medium.

The distinctive metallic sheen produced by E. coli on this medium is due to acid production resulting in an amide bond between eosin and methylene blue, other coliforms not producing enough acid to cause this reaction.

 Gélose  EMB

Klebsiella in EMB Agar

Ⅳ. Results

◈ After 24 hours at 37°C aerobically, in general:

Organisms Growth
E. coli Blue-black colonies with green metallic sheen
Klebsiella Pink and mucoid colonies
Enterobacter aerogenes Good growth; pink, dull
Proteus Variable in size, blue-green to blue or salmon in color, most stumps being black in the center or over their entire area
Salmonella Colorless Colonies
Shigella Colorless Colonies
Pseudomonas Colorless Colonies
Gram positive bacteria Inhibition (partial to complete)