EMB Agar: Composition, uses and colony characteristics

Eosin Methylene Blue (EMB) agar is both selective and differential culture medium. It is a selective culture medium for Gram-negative bacteria (selects against Gram-positive bacteria) and is commonly used for the isolation and differentiation of coliforms and fecal coliforms.

EMB media assists in visual distinction Escherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera.

 Escherichia coli colonies in Eosin Methylene Blue Agar (Note: Greenish Metallic Sheen)

Escherichia coli colonies on EMB agar (Note: greenish metallic sheen)

Differentiation between these gram-negative bacilli is based on the colony color.

  1. Colored colonies in EMB agar: Lactose fermenter
  2. Colorless colonies in EMB agar: Non-lactose fermenter

The aniline dyes (eosin and methylene blue) inhibit Gram-positive and fastidious Gram-negative bacteria.

They combine to form a precipitate at acid pH, thus also serving as indicators of acid production.

Gram-negative bacteria that ferment the lactose produce acid which turns the colonies dark purple as the acid acts upon the dyes.  In addition, 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.  In EMB agar, most of the strains of E.coli colonies have a characteristic green sheen. Rapid fermentation of lactose & production of strong acids, thus a rapid reduction in the pH of the EMB agar the critical factor in the formation of the green metallic sheen observed with E.coli, rapid fermentation of lactose and formation of strong acids. Lactose non-fermenters are either colorless or light lavender.

Another commonly used media for selective isolation of Gram-negative rods and differentiation of the member of Enterobacteriaceae as lactose fermenter and non-lactose fermenter is MacConkey Agar. 

Composition of EMB agar

Ingredient  Eosin methylene blue (EMB) Agar (g/L)
Peptone 10 g
Lactose 5 g
Sucrose 5 g
Dipotassium, PO4 2 g
Agar 13.5 g
Eosin Y 0.4 g
Methylene blue 0.065 g
Distilled water 1 L
Final pH 7.1
  1. Enzymatic Digest of gelatin
  2. Lactose: Sugar, helps to differentiate lactose fermenter from the non-lactose fermenter
  3. Dipotassium Phosphate
  4. Eosin Y: Indicator
  5. Methylene blue: pH indicator
  6. Agar

Expected colony characteristics of organisms in EMB agar (Quality control)

  1. Escherichia coli ATCC 25922: Blue-black bull’s eye; may have green metallic sheen
  2. Pseudomonas aeruginosa ATCC 27853: Colorless

Uses of EMB agar

Primary purpose: Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.

  1. EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples (presence of E.coli in the river/water sample indicates the possibility of fecal contamination of water so does the presence of other pathogenic enterics).
  2. EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group. For culture of Salmonella and Shigella, selective medium such as MacConkey agar and EMB agar is commonly used.
    Escherichia coli colonies grow with a metallic sheen with a dark center, Aerobacter aerogenes colonies have a brown center, and nonlactose-fermenting gram-negative bacteria appear pink.
About Acharya Tankeshwar 460 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Good day,
    Please I need a help,I am working on water sample. What agar can I use in place of membrane feacal coliform agar in order to isolate feacal coliform.

    • In our laboratory, we first incubate the water sample in MacConkey broth (please refer to MPN method) if result comes positive, we streak it (from MacConkey broth) to EMB Agar and incubate the plate at 44.5°C.

  2. please, i am a water microbiologist and would like to know how to distinguish E.coli from Total coliform on EMB Agar……..

  3. i carried out a research titled “the common bacteria associated with the hands of primary pupils” here in a local government in Nigeria using two primary schools as a study area. the sample was collected according to Age, Gender, and Schools. the bacteria isolated includes: staphylococcus aureus, staphylococcus epidermidis, pseudomonas sp, and Escherichia coli. am confuse on how to arrange the tables in respect to Age, school, organisms isolated and the type of statistical method/ tool to use. pls help me with the table using even a sample of the table.thanks.

  4. My name is Ella Pilot and I’m doing a science fair project looking at bacteria in the soil of my school’s playground to see if it’s fecal coliform. We believe it may be because of the smell that comes during the spring run-off. We had a pond near right behind the school, that got filled in when houses were built over top, and now we get the water run-off from the houses on two of the sides of our playground.
    Could I send you the pictures of the colonies I prepared in EMB agar? I need help identifying the bacteria colonies.

    Thanks for taking the time to read this and reply.

    Ella Pilot

    • Thank you so much Akenji. In your convenience, please share how it can be made more useful to visitors.

  5. Hello i am in microbiology right now as a sophomore in high school and I am having trouble identifying what bacteria grew on the EMB agar plate.. It is gram negative and grew purple with clear in some area around the edges any clue?

    • Jessica
      EMB agar is a selective media(only Gram-negative bacteria grow in it). Lactose fermenter (such as Escherichia coli, Klebsiella spp, Citrobacter spp, Enterobacter spp), give purple color (colorless colonies by Non-lactose fermenter), but you have to use panel of biochemical tests to identify the isolate you have.

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