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1.28: EMB Agar

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    Learning Objectives
    • Identify that EMB medium is both selective and differential.
    • Explain how EMB is a selective medium and how this works.
    • Explain how EMB is differential and how this works.
    • Define coliform.
    • Successfully utilize EMB medium and interpret its results.


    Eosin-Methylene Blue (EMB) Medium

    Eosin-methylene blue (EMB) medium is a selective medium and a differential medium used for isolation of Gram-negative rods in a variety of specimen types, including being used frequently in clinical laboratories. EMB is a selective medium because it inhibits Gram-positive bacterial growth. The two dyes in this medium, eosin and methylene blue, act as selective agents to inhibit Gram-positive bacteria, but allow for the growth of Gram-negative bacteria.

    EMB enables differentiation of bacterial species that are coliforms due to its dyes and its lactose content. EMB contains two types of sugars: lactose and sucrose. It is the lactose that is the key to this medium's ability to differentiate coliform bacteria. Lactose-fermenting bacteria (Escherichia coli and other coliforms) produce acid using lactose. When acids are produced by bacteria, the combination of the dyes (which serve as pH indicators in this medium) produces color variations in the colonies because of the acidity. Strong acidity produces a deep purple colony with a green metallic sheen, whereas less acidity may produce a brown-pink coloration of colony. Nonlactose fermenters appear as translucent or pink.



    coliform: A classification of bacterial species that is typically associated with animal digestive tracts and fecal contamination in the environment. These bacterial species are Gram-negative bacilli that do not produce endospores and have the ß-galactosidase gene, and can therefore break down lactose. Coliform bacteria break down lactose to produce acid and gas.

    EMB plate with E. coli shows E. coli growing on the petri plate produces a metallic green color

    Figure 1: Eosin-methylene blue ( EMB) agar plate inoculated with Escherichia coli (a Gram-negative coliform bacterium) showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose and acid production which precipitates the green metallic pigment. Image by Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada.


    Laboratory Instructions

    1. To observe the effects of EMB medium as selective and differential, aseptically conduct quadrant streak plates on three different EMB plates with:
      • Escherichia coli - a Gram-negative coliform species
      • Pseudomonas aeruginosa - a Gram-negative non-coliform species
      • Staphylococcus aureus - a Gram-positive species
    2. Invert and incubate the plates for 24-48 hours.
    3. Observe, record, and interpret the results.


    Results & Questions


    bacterial species growth (+/-) species is likely Gram (+/-) colony color(s) lactose utilization produces acid (+/-) species is a coliform (+/-)
    Escherichia coli          
    Pseudomonas aeruginosa          
    Staphylococcus aureus          
    1. Record the results and interpretations from the EMB plates in the table above.
    2. What types of bacterial species is EMB used to identify?
    3. How is EMB a selective medium?
    4. How is EMB a differential medium?
    5. Define coliform.
    6. Explain the roles of the dyes in EMB (there are two).
    7. Explain the role of lactose in EMB.


    This page titled 1.28: EMB Agar is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline.

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