Differential Media:
Eosin Methylene Blue Agar
(Levine's Formulation)

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EMB Agar

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This is another selective-differential plating medium for the detection and isolation of gram-negative bacteria. It is especially used in the isolation of coliforms from water and food samples when streaked from selective enrichment media such as EC Broth and Brilliant Green Lactose Bile Broth. Likely coliform colonies are then subjected to the preliminary IMViC tests and then other tests (as needed) in the identification process. The following table shows the essential features of EMB Agar.

selective/differential feature ingredient(s) in EMB Agar
selective agent(s) eosin Y and methylene blue
source of amino acids which may
be deaminated (alkaline rx.)
amino acid added for detection of
decarboxylation (alkaline rx.)
fermentable sugar(s) (acid rx.) lactose (1%)
pH indicator eosin Y & methylene blue:
net acid = dark,
net alkaline = light
source from which H2S may
be produced
indicator of H2S production none

Coliform colonies will show a relatively dark color die to the acid produced from lactose fermentation. There can be a variety of colony types, but the two "classical" types of coliform colonies are often noted: Click on images for alternate and/or larger views in separate window.

  • Coli-type colonies are very dark, almost black, when observed directly against the light. By reflected light a green sheen can be seen which is due to the precipitation of methylene blue in the medium from the very high amount of acid produced from fermentation. Those which form this type of colony are methyl red-positive lactose-fermenters such as most strains of E. coli and some strains of Citrobacter.

  • Aerogenes-type colonies are less dark. Often a dark center is seen surrounded by a wide, light-colored, mucoid rim – resulting in a "fish-eye" type of colony. Those which form this type of colony are methyl red-negative lactose-fermenters which include most strains of Klebsiella and Enterobacter.

Non-lactose-fermenting colonies produce no acid from fermentation, so the lighter-colored alkaline reaction is seen. Colonies of Pseudomonas (a strictly-aerobic non-fermenter) are shown at right.

Page last modified on 3/21/04 at 8:15 PM, CST.
John Lindquist:  new homepage, complete site outline.
Department of Bacteriology, U.W.-Madison

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