Eosin Methylene Blue Agar or EMB Media Practical

Eosin Methylene Blue Agar or EMB Media Practical

Today we will see Preparation of complex media Eosin Methylene Blue Agar or EMB Media, we will also see the EMB agar composition, EMB agar preparation, result of E coli on EMB agar and Salmonella EMB agar

Eosin Methylene Blue Agar or EMB Media Practical


To prepare Eosin Methylene Blue Agar or EMB Media

  • It is useful in differentiating gram positive and gram negative bacteria.
  • It helps in the isolation and differentiation of enteric bacilli and gram negative bacilli.
  • It is used in testing the quality of water, especially in determining if the water is contaminated by harmful microorganisms. It differentiates microorganisms in the colon-typhoid-dysentery group


Eosin Methylene Blue Agar is a complex, selective, differential medium generally for gram-negative bacteria. It contains peptone, lactose, sucrose and the dyes is Eosin Y and Methylene Blue. It contains the dyes in the ratio of 6:1 and is both toxic to gram-positive microorganisms. The peptone provides a complex mixture of carbon, nitrogen and other nutritional components. The sugar are included to encourage the growth of enteric bacteria and differentiate them based on colour reactions, created when combined with dyes. The color indicator distinguishes microorganisms that are lactose fermenting and non-lactose fermenting.

The Rapid lactose fermentation by these bacteria produces acids, which lower the pH. This encourages dye absorption by the colonies, which are now colored purple-black.

Lactose non-fermenters may increase the pH by diamination of proteins. This ensures that the dye is not absorbed. The colonies will be colorless. Microorganisms that have the ability to ferment lactose have nucleated colonies with dark centers. Lactose supports coliform such as Escherichia coli and sucrose supports pathogen such as Proteus and Salmonella sp.

The purpose of the dye is two fold –

  • They inhibits the growth of gram positive organisms
  • The react with vigorous lactose fermenter and turn the growth dark purple or black. This dark growth is typically of Escherichia coli and is usually accompanied by a green metallic sheen. Other lactose fermenters are Enterobacter or Klebsiella produce colonies that can range from pink to purple. Non fermenters and sucrose fermenters typically retain their normal colour take the the coloration of the medium.

EMB agar Composition

Ingredient Amount in g/L
Peptone 10
Lactose 5
Sucrose 5
Dipotassium, PO4 2
Eosin Y 0.4
Methylene blue 0.065
Distilled water 1L
Agar 13.5
Final pH 7.1


  • Chemicals
  • PH meter
  • Glasswares like flask, glass rod
  • Autoclave
  • Laminar air flow


  1. 100 ml distilled water was taken in conical flask into which ingredients where dissolve accordingly.
  2. pH of the media was checked by pH meter before further processing.HCl or NaOH was added as per need.
  3. Digestion was done carefully before undergo autoclaving for 15 minutes at 121 °c under 15 lb/ inch2 pressure.
  4. After autoclaving media was cooled down to a certain extend and poured in plate inside Laminar Air Flow
  5. Plates were cooled to room temperature and stored to use for bacteriological work

Possible Result

Microorganisms that are gram negative, ferment the lactose, producing dark purple colonies. Some lactose fermenting microorganisms produce dark and flat colonies characterized by a distinct green metallic sheen color. Some lactose fermenting microorganisms create large mucoid colonies with a noticeable purple colon in the middle.

Most E. coli colonies have a green sheen, but sometimes they have blue-black bulls eye. On the other hand, lactose non-fermenting microorganisms are characterized by a light lavender color. Sometimes, they are colorless.


  • Media should not be discoloured as the methylene blue is a crucial components for identifying bacterial growth
  • Overeating should be avoided during digestion, as it may cause ingredient to distort.

E coli on EMB Agar

If E. coli is grown on EMB, it will give a distinctive metallic green sheen due to the metachromatic properties of the dyes, E. coli movement using flagella, and strong acid end-products of fermentation. Some species of Citrobacter and Enterobacter will also react in this way to EMB.


What is Eosin Methylene blue agar used for ?

Eosin Methylene blue agar used for gram-negative lactose fermenting and non lactose fermenting bacteria

Is eosin methylene blue agar selective or differential media?

Eosin Methylene blue agar is both the selective and differential

What is the results of E coli on emb agar?

E coli shows distinct green metallic sheen on emb agar.

What is the results of Salmonella on emb agar?

Salmonella don’t ferment lactose or produce acid, their colonies appear grey on EMB agar.

Source –

https://laboratoryinfo.com/emb-agar-eosin-methylene-blue-agar/ (1%)

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